Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaapproaches. In the absence of DPPC, clean contact lenses were highlyhydrophilic and resisted dewetting, whereas when soaked overnightin 1 mg/ml lysozyme showed substantial dewetting. Standarddeviation across 4 samples was about 10%. In the presence of DPPC,dewetting is stabilized on the lysozyme adsorbed contact lens surface.For clean lenses, a 200% step-strain produces a low force responseindicating no attachment of cells to the lens substrate. Whereas forcontact lens soaked overnight in 1 mg/ml lysozyme solution, a highermaximum force is exhibited, indicating relatively greater celladhesion to the contact lens. This stress relaxes quickly to a newequilibrium value, likely due to the re-organization and relaxation ofthe adhered corneal cells.Conclusions: Our findings show that adsorbed lysozyme decreasesthe hydrophilicity of the lens surface leading to dewetting. Depositionof lysozyme also makes the lens surface more attractive to celladhesion.Fig1: Normalized wet area as a function of time with standarddeviation of 4 samples, for pure and lysozyme adsorbed contactlenses.Fig2: Step-strain data for uncoated and lysozyme coated contact lenswith corneal epithelial cells.Commercial Relationships: Saad Bhamla, None; Claire M.Elkins, None; David Bergsman, None; Gerald G. Fuller, NoneProgram Number: 489 Poster Board Number: B0126Presentation Time: 10:30 AM - 12:15 PMSecretory Phospholipase A2 Type IIA Levels across the WearCycle: Response to Lens Age or Indication of CL IntoleranceWalter L. Nash, Alan Landers, Manal M. Gabriel, Jennifer D. Lane,Michael S. Foster. Vision Care, Alcon, Duluth, GA.Purpose: To evaluate the difference in day one versus day twentyeightlevels of secretory phospholipase A2 Type IIA (sPLA2IIA) inthe tear envelope of symptomatic (decline in comfort over wearcycle) and asymptomatic subjects.Methods: Lenses were collected from subjects aseptically and storedat below -20 degrees C. Subjects wore the same non-ionic siliconehydrogel lens material and used the same lens care regimen. Lenseswere equilibrated to ambient temperature and rinsed in 2 mL ofphosphate buffered saline for 5 minutes with agitation. The rinsatewas subjected to a sandwich ELISA using a monoclonal antibodyspecific for sPLA2IIA. The plate was washed and anAcetylcholinesterase (ACHe); Fab’conjugate was added to the platewhich selectively binds to a different epitope on the sPLA2IIAmolecule. Finally, the enzymatic activity of ACHe was measured byadding a reagent containing the ACHe substrate and reading the plateat a wavelength of 420 nm by spectrophotometry.Results: Symptomatic and asymptomatic subjects exhibited similarlevels of sPLA2IIA at day 1 as on day 28; with symptomatic subjectsexhibiting 17.2±6.2 and 17.8±8.9 ng/lens sPLA2IIA, respectively,and asymptomatic subjects exhibiting 21.7±5.6 and 24.6±9.2 ng/lenssPLA2IIA, respectively. Comparing the amount sPLA2IIA in the tearenvelope between symptomatic and asymptomatic subjects on bothday 1 and day 28 showed no statistical differences using nonparametricMann-Whitney analysis.Conclusions: sPLA2IIA hydrolyzes fatty acids generating freearachidonic acid and lysophospholipids, the precursors of proinflammatorylipid mediators. Studies have reported elevated levelsof sPLA2IIA in the tears of intolerant contact lens wearers. Thisstudy demonstrates no changes in the level of sPLA2IIA in the tearenvelope over the course of the wear cycle in symptomatic orasymptomatic lens wearers. Our findings, suggest that the mechanismdriving comfort at the time of lens replacement may not be the sameas the mechanism driving contact lens intolerance as described in theliterature.Commercial Relationships: Walter L. Nash, ALCON, a NovartisCompany (E); Alan Landers, Alcon (E); Manal M. Gabriel, Alcon,A Novartis company (E); Jennifer D. Lane, Alcon Laboratories (E),Novartis (E); Michael S. Foster, Alcon, a Novartis Company (E)Program Number: 490 Poster Board Number: B0127Presentation Time: 10:30 AM - 12:15 PMCharacterization of contact lenses through oxygen permeability,equilibrium water content, and silicone contentScott Curtin, Michelle Seitz, Meng Ouyang, Katarina Tomic,Meredith Wiseman, Hanny Vanwersch. DSM, Berkeley, CA.Purpose: In an effort to contribute to the growing knowledge base ofmaterials properties of silicone hydrogel (“SiHy”) contact lenses andimprove material design for formulation, DSM has evaluated threeprincipal material parameters that affect oxygen permeability (Dk);amount of silicone component present, intrinsic permeability ofsilicone component, and connectivity of silicone-containing phaseMethods: The coulometric oxygen permeability (Dk) andequilibrium water content (EWC) of commercial SiHy contact lenseswere measured at DSM using methods described in ISO 18369-4:2006 and ANSI Z80.20-2010. A modified Mocon MH 2/21 wasused to measure the oxygen permeability for 5 contact lenses usingCIBA Vision O2 Optix for the Q value. The silicon and fluorinecontents of dried commercial lenses were measured by X-rayfluorescence to approximately ± 0.5 wt%.Results: CIBA Vision Air Optix 108±3.3 (110), CIBA Vision DailiesTotal One 131±4.3 (140), Acuvue Oasys 105±1.6 (105), AcuvueTrueye 114±2.1 (110), Coopervision Biofinity 128±6.1(128) withmanufacturer-claimed values in parentheses and all lenses besidesCIBA Vision lenses were reported using polarographic method . Awet blot gravimetric method was used to measure EWC for six lenseswith measured values closely matching reported values. CIBA VisionAir Optix 32.7±0.5% (33%), CIBA Vision Dailies Total One29.9±0.8% (33%), Acuvue Oasys 37.3±0% (38%), Acuvue Trueye44.5±0.4% (46%), Coopervision Biofinity 46.3±0.8% (48%), andCIBA Vision O2 Optix 30.5±0.5% (33%). Additionally, the atomicsilicon and fluorine contents of the dried lenses were measured with©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaX-ray Fluorescence.Conclusions: Dk of SiHy lenses can be understood in terms of threeprincipal material parameters: the amount of silicone componentpresent, the intrinsic permeability of silicone component, and theconnectivity of silicone-containing phase. The volume fraction of thesilicone phase is a more meaningful parameter than EWC inunderstanding Dk and it can be estimated using the EWC and atomicpercentages of Si and F. A simple morphologically basedpermeability model can describe the Dk of commercial lenses. Thisgives a framework for how to achieve sufficient Dk while minimizinguse of silicone materials by optimizing connectivity and intrinsicpermeability of silicone phase.©2012 DSM. All rights reserved.Commercial Relationships: Scott Curtin, None; Michelle Seitz,None; Meng Ouyang, None; Katarina Tomic, None; MeredithWiseman, None; Hanny Vanwersch, NoneProgram Number: 491 Poster Board Number: B0128Presentation Time: 10:30 AM - 12:15 PMEffect of lens care system on silicone hydrogel contact lenswettabilityCecile A. Maissa 1 , Michel Guillon 1 , Stephanie Wong 1 , Anna Lane 1 ,Trisha Patel 1 , Renee J. Garofalo 2 . 1 OTG Research & Consultancy,London, United Kingdom; 2 Alcon, Fort Worth, TX.Purpose: The purpose of this study was to compare the effect of therepeated usage of two different care systems (one hydrogen peroxidecleaning and disinfecting system and one PHMB containingmultipurpose system) with silicone hydrogel lenses worn on a dailywear basis for a three month period. A specific aspect of interest wasthe study of effects of the care systems on contact lens wettability.Methods: Seventy-four symptomatic contact lens wearers, currentlywearing either ACUVUE® OASYS® (n=37) or PureVision (n=37),constituted the study population. The investigation was a two-armprospective, bilateral study of three month duration to evaluate thepotential beneficial effects of CLEAR CARE® compared with renu®fresh TM . The subjects were randomized to use one of the two caresystems. The effects on contact lens wettability were assessed withthe Tearscope and reported in terms of Pre-lens Tear Film NonInvasive Break Up Time. Baseline assessments were carried out atthe dispensing visit with new contact lenses. At the two and threemonth follow-up visits the contact lenses had been worn for at leastsix hours prior to the visits, and were at least 11 days old forACUVUE® OASYS® and 25 days old for PureVision.Results: The results obtained showed that:i. Whereas no difference in contact lens wettability was observed atdispensing between the two lens care groups (Mean Median NIBUT:4.5 vs. 4.2s, p=0.518) a significantly more stable pre-lens tear filmwas observed with CLEAR CARE® than with renu® freshTM atboth the two (Mean Median NIBUT: 4.6 vs. 3.7s p=0.005) and three(Mean Median NIBUT: 5.8 vs. 4.2s p=0.028) month visits.ii. With CLEAR CARE® a significant improvement in contact lenswettability was recorded compared with their habitual contact lensesand care system at three months (Mean Median NIBUT 5.8 vs. 4.0sp0.05). However, storageof the lens in some MPSs showed more sustained wetting after 8hours wear, with similar values at (A) and (B); for OL, ReNu andComplete; for DD and MS Biotrue, Express and Complete; and forPL ReNu, Express and Complete. The OSDI questionnaire showedless discomfort after 8 hours wear (at B) with storage in all the MPSscompared to the pack solution (p< 0.02).Conclusions: The effect of all MPS on the initial wetting of a SCL(compared to that with the pack solution) was limited. However, theability to sustain wetting of a SCL over 8 hours of wear was betterwith Biotrue, Opti-Free Express and Complete. The wettingparameter of drying duration gave the closest objective measure tosubjective responses to lens wear (Figure 1and 2).*Fagehi R, Tomlinson A, Manahilov V. ARVO 2011 E-abstract:6524, 2011.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadid not substantially change over the course of the 720 minutes ofmechanical wear. Optical microscopy confirmed that the lenses stillmaintained a surface gel layer.Conclusions: A simple laboratory method for wearing contact lensesthrough mechanical agitation was developed and characterized. Evenafter 720 minutes of simulated wear, the lubricity and surface gellayers were maintained on the delefilcon A lenses.Brennan, AAO 2009.Dunn, et al., Tribology Letters, 2013.Commercial Relationships: Alan Tomlinson, Allergan (E),Allergan (R), Bausch and Lomb (C), TearLab (C), TearLab (I),Alcon, CibaVision (C), Pfizer (R), Pfizer (C); Raied Fagehi, None;Velitchko Manahilov, NoneExperimental Setup for Laboratory Wear Studies.Program Number: 493 Poster Board Number: B0130Presentation Time: 10:30 AM - 12:15 PMLaboratory Model for Wear of Contact Lenses and Effects onLens Lubricity of Surface Gel LayersW G. Sawyer 1 , Juan M. Uruena 1 , Thomas E. Angelini 1 , Alison C.Dunn 1 , John Pruitt 2 . 1 Mechanical and Aerospace Eng, University ofFlorida, Gainesville, FL; 2 Biocompatibility Projects, Alcon VisionCare Research, Johns Creek, GA.Purpose: Lubricity of contact lenses is recognized as an importantcontributor of comfort (Brennan 2009). Experiments aimed atquantifying the lubricity of lenses are traditionally measured on newlenses that have been removed from the package and either gentlyrinsed in a saline solution or tested in the packing solution directly(Dunn 2013). The purpose of this research is to establish a simple andscalable method to mechanically exercise contact lenses and simulatewear in a controlled laboratory environment, and then to performlubricity measurements on the front curve surface of the lenses atvarying time points of wear.Methods: Poly(HEMA) (60% pHEMA) hydrogels were moldedinside of transparent acrylic containers to a thickness ofapproximately 3 mm. A series of poly(HEMA) spheres of 6 mmdiameter were also molded and added to the hydrogel lined cup to apacking fraction of approximately 25%. A single contact lens(delefilcon A) was placed within the ensemble of hydrogel spheresand the container was filled with Phosphate Buffered Saline. Theentire collection was shaken at 31 Hz for various time points. Imageanalysis was used to track the hydrogel spheres and to quantify thecollision frequencies. Based on this analysis a mathematical model ofmechanical energy and tribological severity was developed andcompared to the tribological severity of 1,000 blinks per hour ofwear. Prior to friction testing lenses were examined for evidence ofwear and damage was quantified using scanning optical microscopy.Results: This delefilcon A lenses showed a characteristic frictioncoefficient of mu=0.02, with a standard deviation for eachmeasurement of approximately mu = 0.005. The friction coefficientsPlot of Friction Coefficient versus Simulated Wear Time.Commercial Relationships: W G. Sawyer, Alcon (F); Juan M.Uruena, Alcon (F); Thomas E. Angelini, alcon (F); Alison C.Dunn, Alcon (F); John Pruitt, Alcon (E)Program Number: 494 Poster Board Number: B0131Presentation Time: 10:30 AM - 12:15 PMAssessment of the relationship between contact lens coefficient offriction and subject lens comfortJami R. Kern 1 , Joseph M. Rappon 2 , Erich Bauman 2 , Ben Vaughn 3 .1 Global Medical Affairs, R&D, Alcon, Fort Worth, TX; 2 Vision CareR&D, Alcon, Fort Worth, TX; 3 Biostatistics, Rho, Chapel Hill, NC.Purpose: To examine the relationship between subjective comfortand contact lens coefficient of friction among multiple soft contactlens materials.Methods: A meta-analysis of clinical data (n=157) exploring theassociation between comfort and contact lens lubricity was conductedon 5 soft contact lens materials (delefilcon A, lotrafilcon B,balafilcon A , balafilcon A2, etafilcon A+). Subjective data forinsertion comfort, overall comfort and end of day comfort wereobtained from a database of clinical trials that included studiesconducted between 2004 and 2011. Trials were included in theanalysis unless they met exclusion criteria, including; extended or©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacontinuous lens wear, exclusively acute measures, multi-purpose lenscare utilized, contra-lateral eye study design, small sample size (n

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 497 Poster Board Number: B0134Presentation Time: 10:30 AM - 12:15 PMSelenium covalently incorporated into the polymer of contact lenscase material inhibits bacterial biofilm formationTed W. Reid 1, 2 , Phat L. Tran 1 , Thomas Mosley 2 , Courtney Jarvis 1 ,Daniel Webster 4 , Robert E. Hanes 3 , Abdul Hamood 5 . 1 Ophthal &Visual Science, Texas Tech Univ Health Sciences Ctr, Lubbock, TX;2 Selenium Ltd., Lubbock, TX; 3 Selenium Ltd., Austin, TX; 4 CellBiology, Texas Tech University Health Sciences Ctr, Lubbock, TX;5 Microbiolgy, Texas Tech University Health Sciences Ctr, Lubbock,TX.Purpose: Contact lens case bacterial biofilm formation has become amajor cause of contact lens contamination. This is a serious problemsince it has been found that bacteria grow even in the presence ofcontact lens cleaning solution. Silver as an antimicrobial has beenincorporated into contact lens cases, however, silver has severaldrawbacks. Any patient with silver or metal allergies cannot use thesecases and silver has minimal effects against Staphylococcus aureus(S. aureus), Stenotrophomonas maltophilia (S. maltophilia), anddifferent fungi. In addition, silver is expensive and has to leach out ofthe case to be active. In contrast, selenium does not have to leach outof the material to be active since it kills by the catalytic formation ofsuperoxide radicals and it is much less expensive. Thus, this projectwas carried out to test the ability of selenium, covalently incorporatedinto the polymer of contact lens case material, to inhibit biofilmformation by different bacteria.Methods: A polymer of polypropylene was produced thatincorporated organo-selenium monomers into the final polymer. Thismaterial was then injection molded. The resulting material was testedfor its ability to inhibit biofilm formation. with silver or metalallergies cannot use these cases and silver has minimal effects againstS. aureus and S. maltophilia were tested since these bacteria areresistant to killing with silver. Pseudomonas aeruginosa and Serraciamarcessans were also tested. The bacteria were allowed to grow inthe presence of the polypropylene (with or without selenium) for 24hours. The bacteria were then removed by vortexing and assayed.The bacterial concentration was determined by a colony forming unitassay (plating on agar). The biofilm was also imaged by confocallaser scanning spectroscopy and was then quantitated by COMSTATanalysis.Results: Selenium containing polypropylene showed over 7 logs(complete) inhibition against S. aureus, S. maltophilia and P.aeruginosa, and also was fully active after soaking in PBS for theequivalent of 8 weeks. S. marcessans showed 4 logs of killing.Conclusions: The results showed that selenium covalentlyincorporated into a polypropylene polymer could be injection moldedyet showed total inhibition of S. aureus S. maltophilia and P.aeruginosa biofilm and was stable to soaking for 8 weeks.Commercial Relationships: Ted W. Reid, Selenium Ltd. (I),Selenium Ltd. (P); Phat L. Tran, None; Thomas Mosley, Selenium,Ltd. (E); Courtney Jarvis, None; Daniel Webster, None; Robert E.Hanes, Selenium, Ltd. (F), Selenium, Ltd. (I), Selenium, Ltd. (E),Selenium, Ltd. (P); Abdul Hamood, NoneProgram Number: 498 Poster Board Number: B0135Presentation Time: 10:30 AM - 12:15 PMEffect of Oxidation on Binding of Fatty Acids to PureVision andAcuvue Contact LensesThomas J. Millar, Burkhardt S. Schuett. School of Science andHealth, Univ of Western Sydney, Penrith, NSW, Australia.Purpose: Contamination reduces the longevity of contact lenses.Although lipids contaminate contact lenses, it is not known if this ispreferentially by oxidised lipids. Such knowledge can be useful indeveloping cleaning solutions.Methods: A Fenton reaction was optimized and used to prepareoxidized oleic acid and linolenic acid. The degree of oxidation wasquantified by measuring peroxides, malonyldialdehyde reactivespecies and build-up of polymerized aldehydes. Based on thesemeasurements, 14 C fatty acids were oxidised to different degrees.PureVision and Acuvue contact lenses were loaded with these at35°C and lipid binding was determined by measuring the ratio ofbound to unbound radioactivity.Results: The degree of oxidation using the Fenton reaction dependedon the amount of desaturation. With time, only peroxides wereformed from oleic acid whereas linolenic acid was eventually brokendown completely. It was determined that 20h incubation with50µg/mL of lipids gave optimal binding. Acuvue lenses bound ~50%more non-oxidised lipids than PureVision lenses. There was noincrease in binding of oxidised lipids compared with non-oxidisedlipids in Acuvue lenses. There was an increase of ~50% in binding ofmildly oxidised lipids in PureVision lenses. If the lipids werestrongly oxidised then they bound less than non-oxidised lipids inboth contact lens types.Conclusions: There are differences in the ability of different contactlenses to bind oxidised lipids. It appears that mildly oxidised lipids,such as might occur in vivo can bind more strongly to some types ofcontact lenses than others. This experimental procedure provides aplatform for testing the ability of multi-purpose cleaning solutions toremove oxidised lipids.Commercial Relationships: Thomas J. Millar, Alcon (F), Allergan(F); Burkhardt S. Schuett, Alcon (F), Allergan (S)Support: AlconProgram Number: 499 Poster Board Number: B0136Presentation Time: 10:30 AM - 12:15 PMIn-Vivo Wettability of Contact Lenses Worn in a Low HumidityEnvironmental Exposure Chamber (LH-EEC) Show ComparableChanges to Traditional Field TrialsFiona Soong 1 , Jalaiah P. Varikooty 2 , Nancy J. Keir 2 , Lyndon W.Jones 2 , Piyush Patel 1 . 1 R & D, Inflamax Ressearch, Toronto, ON,Canada; 2 CCLR, University of Waterloo, Waterloo, ON, Canada.Purpose: The LH-EEC is a natural provocation research model,which tightly controls environmental variables (humidity;temperature; air flow) and is a valuable tool to study dry eye, withlittle information on its utility to evaluate contact lens (CL) wear. Thepurpose of this study was to use the LH-EEC model to observe invivoCL wettability changes and to compare these results with thosereported in typical CL field studies.Methods: Ten symptomatic CL wearers were randomized and fitwith 1-day Acuvue® Moist® (etafilcon A): CLA in one eye and 1-Day Acuvue® TruEye (narafilcon A): CLB in the contralateraleye. They were exposed to LH-EEC for 180 mins with instruction towatch a movie screen. Following CL insertion, 3 consecutivemeasures of tear osmolarity were taken with TearLab® prior to LH-EEC entry and exit. Dryness symptoms were rated from 0 (nodiscomfort) to 4 (constant discomfort), and were collected atspecified intervals throughout the chamber visit as were observationsof blink rate (#blinks/min). In-vivo wettability was graded using a 0(excellent) to 4 (extremely poor) scale with 0.25 steps.Results: After only 180 mins in the LH-EEC, there was trend ofincreasing tear osmolarity for both CLA (7.4±3.6mOsmol) and CLB(4.80±3.23mOsmol), but this was not significant (p>0.05). Drynesssymptom scores showed non-significant increase from pre to postchamber for CLA (+1.10±0.53) but a significant (p=0.001) increasefor CLB (+1.40±0.31). Blink rate significantly increased (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafrom pre-EEC rates of 42.0±4.8 blinks/min to average maximum of61.2±4.3 blinks/min. Lens wettability worsened significantly overtime for both CLA and CLB 0.58±0.18 (p=0.01) and 0.65±0.25(p=0.03) respectively. These values are comparable to changes inwettability seen after 8 hrs of wear with both study materials(Luensmann et al; Keir et al.)Conclusions: The LH-EEC exacerbates ocular symptoms and signsafter 180 mins with CL wear. Significant changes in lens wettabilitywere seen during the exposure and yielded values comparable toresults shown in traditional trials with 8 hrs of lens wear. The LH-EEC provides a way to accelerate extended day CL wear andprovides noteworthy research options for a controlled provocationstudy of CL and dry eye signs and symptoms in a shorter time course.Commercial Relationships: Fiona Soong, Inflamax Research (C);Jalaiah P. Varikooty, Alcon (F); Nancy J. Keir, TearScience (F),Alcon (F), Alcon (R), Allergan (F), Johnson & Johnson (F),CooperVision (F), Visioneering, Inc. (F); Lyndon W. Jones, Alcon(F), Alcon (R), Allergan (F), Abbott Medical Optics (R), Bausch &Lomb (R), Ciba Vision (F), Ciba Vision (R), CooperVision (F),Johnson & Johnson (F), Johnson & Johnson (R); Piyush Patel,Inflamax Research (E)Program Number: 500 Poster Board Number: B0137Presentation Time: 10:30 AM - 12:15 PMViscoelasticity and mesh-size at the surface of hydrogelscharacterized with microrheologyThomas E. Angelini, Ryan M. Nixon, Alison C. Dunn, Juan M.Uruena, John Pruitt, W G. Sawyer. MAE, University of Florida,Gainesville, FL.Purpose: Delefilcon A contact lenses contain a water gradientstructure that transitions from a low water content silicone hydrogelcore to a high water content surface gel. This work explores theproperties of the high water content surface gel, designed to mimicthe physical properties of the corneal surface, improving contact lenscomfort. The ~6 µm thick gels have an average water content thatexceeds 80%, yet the properties of the outer layer that makes directcontact with the eye are not known. This study examines the elasticand viscous moduli at the outermost surface of these soft gels tounderstand the physical interactions between contact lenses and theeye.Methods: Microrheological tests were performed on the surfaces of astandard silicone hydrogel (balafilcon A) and on water gradientcontact lenses (delefilcon A). Microspheres (0.5 micron radius) weresandwiched between 3mm sections of lens material and kept indeionized water. Video microscopy was performed at 90xmagnification, and the beads were tracked using digital imageanalysis software.Results: The mean-squared-displacement (MSD) was computed foreach particle, and averaged (N=32 for nelfilcon A; N=80 forbalafilcon A). Beads embedded in the surface gel layer of delefilconA exhibited significant motion, displacing between 50 to 100nm overtimescales below two seconds. By contrast, no detectable beadmotion above the noise threshold was observed in the balafilcon Asystem; beads moved only 10nm to 16nm over the same two secondperiod. We compute the frequency-dependent elastic and viscousmoduli, G’ and G’’ from the MSD measurement. We find moduliwith very weak frequency dependence between 10 and 100 radiansper second, and an elastic modulus that varies between 0.3 and 1 Pa.Thus, from elasticity theory of cross-linked flexible polymers, weestimate that the water content at the outermost region of thedelefilcon A surface gel layer is above 99%.Conclusions: Surface gel layers on contact lenses possess afrequency dependence similar to low concentration polymer gels.Remarkably, the modulus at the outermost surface is over 1000 timeslower than the mean elastic modulus of the entire surface gel layer.This large modulus change, however, only requires roughly 15%reduction in polymer concentration. This suggests that the polymerconcentration marginally drops near the surface as polymer chainsand crosslinks become sparse.Commercial Relationships: Thomas E. Angelini, alcon (F); RyanM. Nixon, Alcon (F); Alison C. Dunn, Alcon (F); Juan M. Uruena,Alcon (F); John Pruitt, Alcon (E); W G. Sawyer, Alcon (F)Program Number: 501 Poster Board Number: B0138Presentation Time: 10:30 AM - 12:15 PMIn vitro Uptake and Release of Natamycin Dex-b-PLANanoparticles from Silicone Hydrogel Contact Lens MaterialsChau-Minh Phan 1 , Lakshman N. Subbaraman 1 , Lyndon W. Jones 1 ,Shengyan Liu 2 , Frank Gu 2 . 1 Centre for Contact Lens Research,School of Optometry and Vision Science, University of Waterloo,Waterloo, ON, Canada; 2 Department of Chemical Engineering,University of Waterloo, Waterloo, ON, Canada.Purpose: To evaluate the uptake and release of the antifungal agentnatamycin encapsulated within poly(D,L-lactide)-dextrannanoparticles (Dex-b-PLA NPs) from model silicone hydrogelcontact lens materials.Methods: Six model contact lens materials (gel A:poly(hydroxyethyl methacrylate, pHEMA); gel B: 85% pHEMA:15% [Tris(trimethylsiloxy)silyl]-propyl methacrylate (TRIS); gel C:75% pHEMA: 25% TRIS; gel D: 85% N,N dimethylacrylamide(DMAA): 15% TRIS; gel E: 75% DMAA: 25% TRIS; gel F:DMAA) were prepared using photoinitiation. The resulting lensmaterials were incubated in two conditions: (1) natamycin dissolvedin deionized (DI) water, and (2) natamycin encapsulated within Dexb-PLANPs in 9.1 % dimethylsulfoxide (DMSO)/DI water for 7 days(d) at 25±3 oC. The release of natamycin from these materials in 2mL of unpreserved saline solution, pH 7.4 at 32±2 oC was monitoredusing UV-Visible spectrophotometry at 304 nm over 7 d. The releasesolution was replenished every 24 hours (h).Results: The uptake of natamycin by all model lens materialsincreased between 1 and 7 d (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEffects of Silicone Hydrogel Contact Lenses Applied ImmediatelyAfter Sub-Bowman’s KeratomileusisXingwu Zhong, Shaohui Gao. State Key Laboratory ofOphthalmology, Zhongshan Ophthalmic Center, Guangzhou, China.Purpose: To evaluate the effects of silicone hydrogel contact lensesapplied immediately after Sub-bowman keratomileusis.Methods: The study was based on 46 myopic patients (92consecutive eyes) who underwent bilateral Sub-BowmanKeratomileusis (SBK). Each patient wore a PureVision contact lensin one randomly-selected eye and nothing in the fellow eyeimmediately after SBK. The contact lens was removed the next day.Fluorescein staining(FL), tear film break up time(TBUT), schirmer Itest(SIT), central corneal thickness(CCT), ocular surface diseaseindex(OSDI), corneal hysteresis(CH), corneal resistance factor (CRF)and corneal flap-related complications (postoperatively) wereassessed preoperatively and 1 day (except for CH and CRF), 1 week,1 month and 3 months postoperatively.Results: FL and TBUT of treated eye were improved at 1 day and 1week postoperatively in contrast with the controlled, but significantlyaggravations were found at 1 day and 1 week after SBK comparedwith preoperative level. There was no significantly difference in SITbetween treated and controlled eye postoperatively, but both eyesshowed the same reduction contrasted with preoperation except for 1day. CCT of treated eye was thinner than controlled at 1 day aftersurgery and both decreased obviously compared with preoperatively.OSDI of treated eye was alleviated more at 1 day after SBK than thecontrolled, but a significant severity existed at any visit for both incontrast with that before surgery. There were no differences in CHand CRF between treated and controlled eye after surgery, but botheyes showed significantly decrease at any follow-up time. Noobviously differences were found in complications related to thecorneal flap between treated and controlled eye postoperatively.Conclusions: Silicone hydrogel contact lenses applied immediatelyafter SBK can effectively reduce corneal epithelium staining ,increase tear film stability , relieve corneal edema and alleviatediscomfort. However, it can’t shorten the duration of ocular surfacechanges and symptoms revalant to SBK.Commercial Relationships: Xingwu Zhong, None; Shaohui Gao,NoneSupport: National Natural Science Foundation of ChinaGrantNO.81070754 and the Fundamental Research Funds of StateKey Lab of Ophthalmology Grant NO.2010C07.Clinical Trial: under checkProgram Number: 503 Poster Board Number: B0140Presentation Time: 10:30 AM - 12:15 PMHigh levels of sIgA and exudated serum albumin in tears ofcontact lens related Dry Eye patients three months afterdiscontinuation of lens usePiera Versura 1 , Alberto Bavelloni 2 , Chiara Coslovi 1 , WilliamBlalock 3 , Marco Grillini 1 , giuseppe giannaccare 1 , Emilio C.Campos 1 . 1 Ophthalmology Unit, DIMES Department, Alma MaterStudiorum University of Bologna and S.Orsola-Malpighi TeachingHospital, Bologna, Italy; 2 Laboratory of Musculoskeletal CellBiology, Istituto Ortopedico Rizzoli, Bologna, Italy; 3 Institute ofMolecular Genetics, CNR-National Research Council of Italy,Bologna, Italy.Purpose: To evaluate discontinued contact lens (CL) wearers withparticular reference to secretory IgA (sIgA) and exudated serumalbumin in tears as markers of local inflammation.Methods: 45 CL wearers diagnosed as having dry eye (DE)according to DEWS guidelines (DEWS grade 1-3) and 25 matchednormal non-CL wearer volunteers were included in this study.Patients had discontinued the use of various types of CLs (12 RGP,33 soft disposable or frequent replacement CL-average time from lastuse 60±30 days) because of associated discomfort. OSDIquestionnaire score, Schirmer test I, Break Up Time (BUT),Lissamine green vital staining, corneal esthesiometry (Cochet-Bonnet), conjunctival scraping and imprint cytology were performed.Total tear protein content (TP), Lysozyme-C (LYS-C), Lactoferrin(LACTO) and exudated serum albumin (ALB) were evaluated(mg/ml) with the 2100 Bioanalyzer (Agilent Technology, CA, USA,P230 Lab-chip kit). Validation of kDa range for heavy chain IgAbands in each lane was carried out as previously described (Versura Pet al, Mol Vis 2012). The sIgA/LYS-C ratio was calculated as anindex of the increased activity of the IgA-producing cells in thelachrymal gland. Data were statistically evaluated and correlated withwear parameters (Pearson’s r) (significance p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornealevel of deposition was determined by computer enhanced imageanalysis. An image of each lens was converted to mean grey scale,which is a numerical representation of the degree of deposition. Thisvalue was used as the baseline for calculation for each individuallens. Each lens was re-imaged after a single cleaning regimen and the% deposition removal was calculated.Results: The image analysis demonstrated that three of the caresystems, Simplus, Advance, and Optimum removed significantlygreater amounts of lipid/protein deposits (p-value

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafound in contact lens cases of Polyquaternium/Aldox care systemscan adhere to contact lenses in relatively high numbers. This mayfacilitate their transfer into the eye and the production of cornealinfiltrates.Commercial Relationships: Ajay Kumar Vijay, Bausch+Lomb(F); Mark D. Willcox, Allergan Inc (C), Allergan Inc (R), BrienHolden Vision Institute (P), Bausch + Lomb (C), Basuch + Lomb (R)Program Number: 507 Poster Board Number: B0144Presentation Time: 10:30 AM - 12:15 PMAntimicrobial Activity of Melimine or Cathelicidin Bound toContact LensesDebarun Dutta 1, 2 , Mark D. Willcox 2 . 1 Brien Holden Vision Institute,Sydney, NSW, Australia; 2 School of Optometry and Vision Science,University of New South Wales, Sydney, NSW, Australia.Purpose: Development of antimicrobial contact lens could have thecapacity to reduce the rate of contact lens related adverse events. Thepurpose of this study was to evaluate two cationic peptides coated oncontact lenses for their activity against P. aeruginosa and S. aureus.Methods: Minimal inhibitory concentration (MIC) of two peptides,Melimine (a synthetic peptide) and Cathelicidin (LL37) wasmeasured against strains of P. aeruginosa and S. aureus. Increasingconcentrations of peptides were covalently bound to contact lenses.Antimicrobial activity against the bacteria was evaluated bymeasuring the amount of cell death compared to control lenses withno melimine or LL37.Results: MIC of LL37 against both P. aeruginosa and S. aureus was3.9µg ml -1 , whereas for Melimine against the same bacteria it was500 µg ml -1 and 250 μg ml -1 respectively. Contact lenses covalentlyreacted with 1mg ml -1 Melimine showed 0.8 log and 2.6 loginhibition against P. aeruginosa and S. aureus respectively whereasno inhibition was detected with LL37 at that concentration. Contactlenses prepared with 3mg ml -1 melimine and LL37 showed 3.1 logand 3.3 log inhibition against P. aeruginosa , 3.9 log and -0.2 loginhibition against S. aureus respectively.Conclusions: Whilst Melimine on contact lenses had activity againstboth bacterial types; covalently bound LL37 was not active against S.aureus. These differences suggest different mechanisms of actionagainst Gram-negative or Gram-positive bacteria by these twocationic peptides.Commercial Relationships: Debarun Dutta, None; Mark D.Willcox, Allergan Inc (C), Allergan Inc (R), Brien Holden VisionInstitute (P), Bausch + Lomb (C), Basuch + Lomb (R)Program Number: 508 Poster Board Number: B0145Presentation Time: 10:30 AM - 12:15 PMEstablishing a standard method for evaluating efficacy againstAcanthamoebaMonica Crary, Rhonda Walters, John Bartell, Manal M. Gabriel,Jagath Kadurugamuwa, Bradley J. Catalone. Alcon Laboratories,Fort Worth, TX.Purpose: To establish a standard method for Multipurpose ContactLens Solution (MPS) efficacy testing against Acanthamoeba byidentifying and reducing variables.Methods: Bacterized and axenic cultures of trophozoites were usedto determine if culture methods affected MPS efficacy. Cysts thatwere prepared by starvation with or without Escherichia coli weretested to determine variability in susceptibility to MPS followingbacterization. Additionally, the effect of heterogeneous cultures onMPS efficacy was determined by examining the increasing ratios ofcysts to trophozoites in axenic cultures that were harvested between3-7 days of age. Most Probable Number was used for enumeration atdifferent post-test time points to determine the earliest appropriateread date for both cell types. Other variables such as inoculumconcentrations and MPS volumes were also examined.Results: Current recommendations suggest that bacterization ofAcanthamoeba is the preferred culture method when evaluating MPSefficacy. However, residual E. coli decreases the available biocide,thereby resulting in variability correlated with bacterialconcentration. Current culture methods are limited in their ability togenerate homogeneous populations of trophozoites. Axenictrophozoite cultures can contain cysts which contribute to theobserved variability of MPS efficacy. Harvesting of trophozoites at 3days results in a more homogenous population of trophozoites ascompared to harvesting at 5 or 7 days. Another variable thatcontributes to conflicting results in the literature is the duration ofincubation prior to enumeration. These studies clearly demonstratethat the minimum incubation period for trophozoites plates is 7 days,whereas for cysts it is 14 days. Earlier time points result in incorrectenumeration. Other potential sources of variability include surface tovolume ratio of organisms and MPS.Conclusions: The maximization of a homogenous population oftrophozoites or cysts is necessary to obtain reproducible results whentesting MPS efficacy. Variability of test results is affected by thepurity of the target cell type. For maximum accuracy, enumeration ofcells should not be performed prior to the minimum incubationperiod. These identified variables have a significant effect on thereproducibility and the performance of MPS against Acanthamoeba.Commercial Relationships: Monica Crary, Alcon Labs (E);Rhonda Walters, Alcon Laboratories (E); John Bartell, Alcon Labs(E); Manal M. Gabriel, Alcon, A Novartis company (E); JagathKadurugamuwa, Alcon Labs (E); Bradley J. Catalone, Alcon, aNovartis Company (E)Program Number: 509 Poster Board Number: B0146Presentation Time: 10:30 AM - 12:15 PMRisk Factors for Contact Lens Related Microbial Keratitis inSingaporeChris Lim 1 , Nicole A. Carnt 1, 2 , Mohamed Farook 3 , Janice Lam 3 ,Jodhbir S. Mehta 3 , Donald T. Tan 3 , Fiona Stapleton 1 . 1 School ofOptometry and Vision Science, University of New South Wales,Sydney, NSW, Australia; 2 Moorfields Eye Hospital Trust, London,United Kingdom; 3 Singapore National Eye Centre, Singapore,Singapore.Purpose: Patterns of contact lens prescribing, wearer behavior andenvironmental microbiota vary across different cultures and climates,which may impact risks for microbial keratitis. This studyinvestigates independent risk factors for microbial keratitis in contactlens wearers in Singapore.Methods: Cases were contact lens wearers presenting to SingaporeNational Eye Centre with microbial keratitis between 2009-2010.Contact lens wearers attending for routine aftercare at a nearbyUniversity Clinic over the same time period were identified ascontrols. All wearers completed a previously validated questionnairedescribing contact lens wear history, hygiene and compliance habitsand demographics. Risk factors significant in univariate analysis(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadeposition using radiochemical analysis.Methods: Thirteen model lens materials were created based oncombinations of dimethylacrylamide (DMAA), hydroxyethylmethacrylate (HEMA) andmethacryloxypropyltris(trimethylsiloxy)silane (TRIS). The materialswere prepared with and without the incorporation of a hydrophilicnovel substance such as hyaluronic acid, alginate, and a siliconesurfactant. The model materials were hydrated and advancing contactangles were measured using the sessile drop technique. All materialswere incubated in a complex artificial tear solution (ATS) containingprotein, lipid, mucin and a trace amount of either 14 C-CH or 14 C-PCfor 3 and 14 days. All materials were then extracted, processed andmasses of deposition were quantified (ng/disc) using standardcalibration curves.Results: For the HEMA-based materials tested, only the materialsthat incorporated the silicone surfactant and alginate showed astatistically significant increase in wettability (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaserially diluted 10-fold in PBS and 10-μl aliquots of each dilutionwere spotted on LB agar plates. The plates were incubated at 37degrees C for 24 hours and the CFU were counted. For confocal laserscanning microscopy (CLSM), we used the S. aureus strain AH1333which carries the gene that encodes the green fluorescent protein.Results: Colony forming unit assays showed total inhibition,representing over 6 logs of Staphylococcus aureus killing on organoseleniumpolymerized hydrogels. Confocal laser scanningmicroscopy confirmed these results.Conclusions: The organo-selenium hydrogel polymer successfullyblocked the formation of a bacterial biofilm on the polymer byStaphylococcus aureus in vitro.Commercial Relationships: Phat L. Tran, None; Abdul Hamood,None; Daniel Webster, None; Courtney Jarvis, None; Robert E.Hanes, Selenium, Ltd. (F), Selenium, Ltd. (I), Selenium, Ltd. (E),Selenium, Ltd. (P); Ted W. Reid, Selenium Ltd. (I), Selenium Ltd.(P)Program Number: 515 Poster Board Number: B0152Presentation Time: 10:30 AM - 12:15 PMContact Lens Disinfecting Solution vs. Blister Pack: a SubjectiveEvaluationScott Schatz 1 , Balamurali Vasudevan 2 , Sara N. Gaib 3 , KimbalCooper 4 . 1 Appalachian College of Optometry, Grundy, VA; 2 Collegeof Optometry, Midwestern University, Glendale, AZ; 3 College ofOptometry, Midwestern University, Glendale, AZ; 4 College of HealthSciences, Midwestern University, Glendale, AZ.Purpose: To study subjectively the effect of contact lens disinfectingsolution on the ocular surface in the presence of either a hydrogel or asilicone hydrogel soft contact lens.Methods: Twenty young adult subjects were examined for this studyover 2 visits. Subjects were fit (randomized) with either a hydrogellens or silicone hydrogel lens in either eye. The lenses were presoakedovernight in either Puremoist or Revitalens contact lensdisinfecting solution or were obtained from the blister pack in arandomized process. Subjective feedback on discomfort, burning,dryness and irritation on a scale of 1 to 5 was obtained at baseline,prior to lens insertion, and after 8 hours of wear. Chi-square analysiswas performed.Results: There was a statistically significant increase in dryness afterlens insertion for Proclear and Purevision lenses taken from theblister pack , relative to baseline (p = 0.01). Following 8 hours of lenswear, there was a statistically significant increase in burningsensation relative to baseline for all Proclear lenses. This held truewhether they were obtained from the blister pack or pre-soaked inRevitalens MPDS or Puremoist MPDS (p=0.01).Conclusions: Subjectively, lenses from the blister pack producedmore subjective symptoms of dryness upon initial insertion thanthose pre-soaked in either solution. Proclear lenses produced moresubjective symptoms of burning after 8 hours of wear relative tobaseline irrespective of whether they were obtained from the blisterpack, or pre-soaked in either solution.Commercial Relationships: Scott Schatz, Abbot Medical OpticsInc (F); Balamurali Vasudevan, None; Sara N. Gaib,Bausch+Lomb (C), Alcon (R), Vistakon (R), Coopervision (R);Kimbal Cooper, NoneSupport: Study was supported by a grant from Abbott MedicalOpticsProgram Number: 516 Poster Board Number: B0153Presentation Time: 10:30 AM - 12:15 PMAntimicrobial Efficacy of New Investigational MultipurposeDisinfecting Solution and Comparison to CommerciallyAvailable Multipurpose Disinfecting SolutionsMarina Milenkovic, Nancy Brady, Anthony Lam. Corneal R&DMicrobiology, Abbott Medical Optics, Santa Ana, CA.Purpose: To evaluate antimicrobial efficacy of investigationalmultipurpose formulation against bacteria, fungi and Acanthamoebaspp. and compare it to commercially available multipurposedisinfecting solutions (MPS). Study was done according to ISO14729:2001/A.2010 standard.Methods: The multipurpose disinfecting solutions studied were -Investigational MPS-1: polyhexamethylene biguanide (PHMB) +poloxamer (PLX) and currently marketed Japan products MPS-2:polyquaternium (PQ1) + tetronic 1304, MPS-3: PHMB + poloxamine(PLA) and MPS-4: PHMB +PLX.Test organisms were: Staphylococcus aureus (ATCC 6538),Pseudomonas aeruginosa (ATCC 9027), Serratia marcescens (ATCC13880), Candida albicans (ATCC 10231), Fusarium solani (ATCC36031) and Gram-negative clinical isolates. Test solutions wereevaluated at the minimum recommended disinfection time of 4 hours.Disinfectant efficacy of MPS was also evaluated againstAcanthamoeba trophozoites.Results: After 4 hours exposure, Investigational MPS-1 and MPS-3showed >3 log kill of ISO bacteria and fungi. MPS-2 and MPS-4failed to meet stand-alone criteria for ISO bacteria and fungi. MPS-1,MPS-3 and MPS-4 showed >3 log kill against clinical isolates, whileMPS-2 showed 3 logreduction for Acanthamoeba trophozoites, while MPS-4 achieved

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaof accumulated cholesterol (0.93±0.02µg/lens, 0.95±0.01µg/lensrespectively), while lotrafilcon A and lotrafilcon B deposited thelowest amounts (0.37±0.03; 0.47±0.12). OptiFree PureMoistremoved more cholesterol than the other solutions for all lensmaterials; however, the amount of cholesterol cleaned wasstatistically significant for balafilcon A and senofilcon A lensmaterials (p=0.006 and p=0.042). Sensitive Eyes and the other MPSevaluated showed no significant effect on lipid removal (p>0.05).Conclusions: Lipid-removal efficacy varies depending on thecombination of lens material and solution. Only one MPS showed asignificant reduction of lipids for any of the lenses tested. It will bevaluable to conduct further work to determine the efficacy of MPS inremoving lipid deposits on worn lenses and how these deposits mayimpact subjective comfort.Commercial Relationships: Hendrik Walther, None; LakshmanN. Subbaraman, None; Lyndon W. Jones, Alcon (F), Alcon (R),Allergan (F), Abbott Medical Optics (R), Bausch & Lomb (R), CibaVision (F), Ciba Vision (R), CooperVision (F), Johnson & Johnson(F), Johnson & Johnson (R)Support: Alcon USAProgram Number: 518 Poster Board Number: B0155Presentation Time: 10:30 AM - 12:15 PMAntimicrobial Efficacy of Multipurpose Disinfecting Solutionsagainst Clinical Isolates after Prolonged StorageAnthony Lam, Nancy Brady, Marina Milenkovic. Corneal R&DMicrobiology, Abbott Medical Optics, Santa Ana, CA.Purpose: To compare antimicrobial efficacy of commerciallyavailable multipurpose disinfecting solutions (MPS) against Gramnegativeclinical isolates following prolonged storage in lens case inthe presence of a lens.Methods: The multipurpose disinfecting solutions studied were -MPS-1: polyquaternium (PQ1) + alexidine dihydrochloride (ALX),MPS-2: PQ1 + polyhexamethylene biguanide (PHMB), MPS-3: PQ1+ myristamidopropyl dimethylamine (ALDOX) and MPS-4: PQ1 +ALDOX + nonanoyl ethylenediaminetriacetic acid (EDTA).Test solutions were inoculated with Gram-negative clinical isolates inthe appropriate lens case, in the presence of Acuvue2 (etafilcon A)lens. Test solution efficacy was evaluated at minimum recommendedmanufacturer disinfection time of 6 hours and following prolongedstorage.Test solutions were also inoculated with Gram-negative clinicalisolates in the test tube and tested according to ISO 14729.Results: After 6 hours exposure, MPS-1 and MPS-2 showed >3 logkill against clinical isolates in a test tube, while MPS-3 and MPS-4failed stand-alone criteria. MPS-1 and MPS-2 maintained efficacy inthe lens case in the presence of Acuvue2 lens and achieved >2.5 logkill. MPS-3 and MPS-4 showed 3 log killfor all organisms tested. MPS-2 and MPS-3 failed to achieve 3 logkill for one organism tested. MPS-4 failed to achieve 3 log kill fortwo organisms tested.Following 30 days storage in a lens case, MPS-1, MPS-2 and MPS-3achieved >3 log kill for all organisms tested, while MPS-4 failed toachieve 3 log kill for two organisms tested.Conclusions: Gram-negative clinical isolates are resistant to MPS-3and MPS-4. MPS-1 and MPS-2 showed ability to reduce microbialload under worst case conditions, tested in a lens case with Acuvue2lens present.Commercial Relationships: Anthony Lam, Abbott Medical Optics(E); Nancy Brady, Abbott Medical Optics (E); Marina Milenkovic,Abbott Medical Optics (E)Program Number: 519 Poster Board Number: B0156Presentation Time: 10:30 AM - 12:15 PMDisinfection Efficacy of Rigid Gas Permeable Lens Care SystemsSuzanne F. Groemminger, Denise Callahan. Bausch & Lomb Inc,Rochester, NY.Purpose: This study compared the disinfecting efficacy of Rigid GasPermeable (RGP) lens care products.Methods: The primary disinfecting solution from each lens caresystem was evaluated using the ISO/FDA Stand Alone Procedure forDisinfecting Products. Three distinct lots of each product were testedusing the regimen time listed on the patient instructions. The systemstested using a 4 hour disinfection time included two-single bottlemultipurpose products: Boston Simplus and Unique pH, a two bottlesystem: Boston Advance Cleaner and Conditioning Solution. Thethree 3 bottle system: Lobob Optimum, was tested using a 6 hourdisinfection time.Products were challenged with 3 bacterial species, Staphylococcusaureus (Sa), Pseudomonas aeruginosa (Pa) and Serratiamarcescens(Sm) a yeast, Candida albicans (Ca) and a mold, Fusariumsolani (Fs).The primary acceptance criteria states that the number of bacteriarecovered per mL shall be reduced by a mean value of not less than3.0 logs within the minimum recommended disinfection period. Themold and yeast recovered per mL shall be reduced by a mean valueof not less than 1.0 log within the minimum disinfection time with noincrease at not less than 4 times the disinfection time.There are secondary acceptance criteria which require that there is acombined log reduction for the mean values of all three bacteria ofnot less than 5.0 logs within the recommended disinfection time. Theminimum acceptable mean log reduction for any single bacteria typeis 1.0 log. Stasis for the yeast and mold must be observed for therecommended disinfection time.Results: Disinfection efficacy results demonstrated Simplus,Advance and Unique pH passed the primary criteria, with >4.7 logreduction for all three bacterial species and >4.2 log reduction of themold. Both single bottle systems gave >4.2log reduction for theyeast, while Advance demonstrated a 1.4 log reduction. Optimumfailed to pass the primary criteria for 2 of 3 of the bacterial species(Sa-2.7 logs and Sm-0.6 logs) and also failed the secondary criteriafor yeast and mold.Conclusions: RGP care products demonstrated varying degrees ofeffectiveness to in their ability to disinfect RGP contact lenses.Boston Simplus, Boston Advance, and Unique pH all passed theprimary acceptance criteria for disinfection efficacy set forth by ISOand the FDA. Lobob Optimum had very weak activity, and failed tomeet even the secondary stand alone criteria.Commercial Relationships: Suzanne F. Groemminger, Bausch &Lomb Inc (E); Denise Callahan, Bausch and Lomb, Inc. (E)Program Number: 520 Poster Board Number: B0157Presentation Time: 10:30 AM - 12:15 PMEffect of Contact Lenses and Lens Cases on Disinfection Efficacyof Four Multipurpose Disinfection SolutionsManal M. Gabriel, Cynthia McAnally, John Bartell, Rhonda Walters,Jebree Spencer, Linda Clark, Bradley J. Catalone. Vision Care,Alcon, Fort Worth, TX.Purpose: To evaluate the effect of different contact lens materialsand cases on the disinfection efficacy of Multipurpose DisinfectionSolutions (MPDS) following the November 12, 2008 draft standard,Antimicrobial Efficacy Endpoint Methodology to DetermineCompatibility of Contact Lens Solutions, Lens Cases and HydrogelLenses for Disinfection (AEEMC).©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Brien C. David, Bausch + Lomb (E);Denise Callahan, Bausch and Lomb, Inc. (E); Julie Bair, Bausch &Lomb, Inc. (E); Susan E. Norton, Bausch and Lomb, Inc. (E)Program Number: 523 Poster Board Number: B0160Presentation Time: 10:30 AM - 12:15 PMEfficacy Of Contact Lens Solutions Against Achromobacterxylosoxidans Biofilms Using Confocal MicroscopyDavid J. McCanna, Jaclyn M. Chang, Lakshman N. Subbaraman,Lyndon W. Jones. Centre for Contact Lens Research, School ofOptometry and Vision Science, Waterloo, ON, Canada.Purpose: Biofilms of Achromobacter xylosoxidans (Ax) can developin contact lens cases. These microorganisms can attach to the contactlens and cause microbial keratitis. This study evaluated theantimicrobial efficacy of contact lens solutions against Ax biofilmsby measuring the damage to cell membranes of Ax using confocalmicroscopy.Methods: Ax biofilms were formed by incubating the bacteriaovernight on glass coverslips. The biofilms were then exposed tocontact lens solutions for four hours. Commercial contact lenssolutions evaluated contained the antimicrobials polyhexamethylenebiguanide (PHMB), polyquaternium-1 (PQ1) and alexidine (ALX),and PQ1 and Aldox (AD). After exposure, the bacteria were stainedwith SYTO 9 and propidium iodide (PI). Using a confocalmicroscope with a 488nm laser and the appropriate emission filtersthe number of cells with damaged cell membranes was determined.In addition to evaluating contact lens solutions, four concentrations ofbenzalkonium chloride (BAK) 0.05%, 0.01%, 0.005% and 0.001% inphosphate buffer saline were also evaluated to demonstrate doserelated effects at exposure times as short as 5 minutes.Results: The contact lens solution that caused the greatest damage tothe Ax cell membranes was the formulation based on PQ1-ALX. Theother formulations tested based on PHMB and PQ1 with AD causedsome of the bacteria to lose membrane integrity but did not cause asmuch damage to the bacteria cell membranes (p < 0.05) as the PQ-ALX formulation. Dose effects of the preservative BAK could beseen at 5 minutes of exposure time. BAK at 0.005% and 0.01%caused an increase in the number of cells that were permeable to PIcompared to the phosphate buffered control (48% and 62%respectively, p < 0.05). All of the Ax bacteria were permeable to PIafter exposure to 0.05% BAK.Conclusions: One of the five lens care systems tested caused asubstantial number of Ax bacteria to lose membrane integrity. Also,this method was able to detect the effect different concentrations ofBAK have on the membrane integrity of the Ax biofilm bacteria.Understanding the ability of antimicrobials to damage bacteria cellmembranes could help in the development of lens care solutions thatcan reduce and/or eliminate Ax biofilms from lens cases.Commercial Relationships: David J. McCanna, None; Jaclyn M.Chang, None; Lakshman N. Subbaraman, None; Lyndon W.Jones, Alcon (F), Alcon (R), Allergan (F), Abbott Medical Optics(R), Bausch & Lomb (R), Ciba Vision (F), Ciba Vision (R),CooperVision (F), Johnson & Johnson (F), Johnson & Johnson (R)Body mass index, peripheral corneal thickness and anteriorchamber depth in young European adults - A pilot studySven Jonuscheit 1, 2 , Michael J. Doughty 1 , Raul Martin 3 , Ana del RioSan Cristóbal 3 , Lisa J. Mackintosh 1 , David MacTaggart 1 , MichaelHiscock 1 . 1 Vision Sciences, Dept. of Life Sciences, GlasgowCaledonian University, Glasgow, United Kingdom; 2 DiabetesResearch Group, Institute for Applied Health Research, GlasgowCaledonian University, Glasgow, United Kingdom; 3 OptometryResearch Group, Instituto Universitario de Oftalmobiología Aplicada(IOBA), Universidad de Valladolid, Valladolid, Spain.Purpose: To assess the relationship of body mass index (BMI) andthe corneal thickness profile in normal white European individuals.Methods: For this pilot study, 63 eyes of 63 healthy subjects wereassessed. Following completion of an ocular and general healthquestionnaire body height and weight were measured and the BMIcalculated. Ocular assessments included habitual visual acuity, slitlampbiomicroscopy, and optical coherence tomography. Non-contactspecular microscopy was performed to rule out cornealendotheliopathy. Scheimpflug photography (Pentacam) was used toassess central, mid-peripheral and peripheral corneal thickness aswell as anterior chamber depth (ACD) at eleven locations nominally1 mm apart along the horizontal meridian. Two consecutiveScheimpflug scans were performed and the mean value used foranalyses. Descriptive statistics were generated. The associationbetween BMI, corneal thickness and ACD at central as well as offcenterlocations was assessed using univariate regression analyses.The coefficient of correlation (r) was calculated.Results: The mean [SD] age was 27 [7] years. Mean body height andbody weight were 1.69 [0.08] meters and 64.6 [11.4] kilogramsrespectively, the mean BMI was 22.7 [3.1]. Mean central cornealthickness was 558 [33] micrometers (µm). Corneal thicknessincreased progressively and asymmetrically from the corneal centerto the periphery with a significantly greater thickness at all nasallocations as compared to the respective temporal sites (P0.05).Conclusions: For this cohort of young, healthy white Europeanadults with normal-weight average BMI, peripheral corneal thicknesswas inversely related to BMI. The findings suggest the possibility ofa different corneal thickness profile in individuals with aboveaverageBMI. Further studies on the relationship between BMI,obesity and corneal parameters are indicated.Commercial Relationships: Sven Jonuscheit, Santander UK plc(F); Michael J. Doughty, None; Raul Martin, None; Ana del RioSan Cristóbal, None; Lisa J. Mackintosh, Santander UK plc (F);David MacTaggart, Santander UK PLC (F); Michael Hiscock,NoneSupport: Santander UK plc Grant127 Corneal Epithelium and Imaging ISunday, May 05, 2013 10:30 AM-12:15 PMExhibit Hall Poster SessionProgram #/Board # Range: 524-567/B0161-B0204Organizing Section: CorneaProgram Number: 524 Poster Board Number: B0161Presentation Time: 10:30 AM - 12:15 PMProgram Number: 525 Poster Board Number: B0162Presentation Time: 10:30 AM - 12:15 PMObjective Estimation for Uncertainty of Restoring CornealTopography SurfaceAnatoly Fabrikant. R&D, Abbott Medical Optics, Fremont, CA.Purpose: Corneal topography (CT) field can be restored frommeasurements data by decomposing available data into Zernikepolynomials and mapping the CT field in the desired area. Theaccuracy of such restoration depends on the measurement noise and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneanumber of available data. Here we present an algorithm for CT dataassimilation, which yields both CT map and an objective estimationof the restoration accuracy.Methods: The Kalman-Bucy technique is used to combine measuredCT heights with a priori mean and covariance of Zernike coefficients,estimated for the general population (CT data for 308 virgin eyes).This algorithm yields a statistically optimal estimate of Zernikecoefficients for the measured field and also their covariance matrix,which provides a measure of the measurement uncertainty. Theefficiency of the proposed method is demonstrated using archivedcorneal topography data from previous clinical studies.Results: For any CT measurement the proposed technique yields anestimate of Zernike amplitudes and their covariance matrix, whichresult in a reconstructed map of CT heights with no gaps within thearea (Fig.1A). The covariance matrix gives the uncertainty (std) ofCT height restoration (Fig.1B). The uncertainty is higher at the areaedges, because the restoration is based mainly on the data frominternal area. Restored field in the measurement gaps has the highestuncertainty, close to the a priori variance of the general population.Conclusions: The proposed algorithm assimilates measurement datatogether with a priori information, derived from statistics of generalpopulation, which protects the results from measurement outliers. Itrestores the CT field in the entire area and provides an objectiveestimate of measurement uncertainty, based on the measurementnoise level and the number of available data. The uncertainty mapdisplays the areas where the map is less reliable and to what extent.diabetes or history of previous refractive surgery. A control group ofhealthy patients were also included. All patients were screened forperipherial neuropathy using the MNSI questionnaire and classifieddepending on the score obtained as without, mild-moderate or severeperipherial neuropathy. Confocal microscopy was performed toobtain corneal analysis from endothelium to epithelium and fourcorneal subepithelial nerve plexus parameters were evaluated:Number of fibers, tortuosity of fibers, number of beading andbranching grade. Statistical analysis was made using Pearson and t-Student tests, a p ≤ 0.05 was considered statistically significant.Results: We included 14 patients in each group. In the study groupthe mean value of HbA1C was 9.5%. In the study group, 28.5% ofthe patients were classified as without peripheral neuropathy, 28.5%as mild-moderate peripheral neuropathy and 42.8% as severeperipheral neuropathy. The number of fibers, beadings and thebranching grade was decreased in diabetic patients compared with thecontrol group (p = 0.004, p ≤ 0.001 and p = 0.028 respectively). Thegrade of tortuosity was higher in patients with diabetes comparedwith healthy subjects with a p ≤ 0.001. There was a tendency ofprogression of corneal neuropathy with higher levels of HbA1C.Conclusions: The assessment of the corneal nerve plexus is a usefultool in the global study of the diabetic patient by establishing theabsence or presence of early neuropathic damage, even beforeclinical manifestations appear.Commercial Relationships: Juan Antonio De la Campa, None;Oscar Baca, None; Alejandro Babayan, None; Regina Velasco,None; Cristina Pacheco-Del-Valle, NoneA B Fig. 1. A - CT height measured with Atlas topographer andrestored within 6mm diameter circle. B - Estimated uncertainty ofmeasured and restored CT height (Hyperopia Sph=2.25D,Cyl=0.25D)Commercial Relationships: Anatoly Fabrikant, Abbott MedicalOptics (E)Program Number: 526 Poster Board Number: B0163Presentation Time: 10:30 AM - 12:15 PMCorneal nerve plexus condition in type 2 Diabetes Mellituspatients assessed by Confocal MicroscopyJuan Antonio De la Campa, Oscar Baca, Alejandro Babayan, ReginaVelasco, Cristina Pacheco-Del-Valle. Cornea and RefractiveSurgery, Fundacion Hospital Nuestra Señora de la Luz, Mexico,Mexico.Purpose: To correlate the metabolic status of patients with type 2Diabetes Mellitus and its relationship with corneal nerve plexusalterations assessed by Confocal Microscopy along with clinicalquestionnaires to detect peripherial diabetic neuropathy.Methods: Patients with type 2 Diabetes Mellitus within the first yearof diagnosis were included. All patients were asked for glycosylatedhemoglobin (HbA1C). Inclusion criteria included values ofglycosylated hemoglobin ≥7%. We excluded patients with diagnosisof central or peripheral neuropathy, ocular disease not related toProgram Number: 527 Poster Board Number: B0164Presentation Time: 10:30 AM - 12:15 PMNon-invasive objective metrics of Bulbar Hyperemia for ClinicalTrials EndpointsNeha Gadaria-Rathod, Kyu-In Lee, Benyamin Y. Ebrahim, Penny A.Asbell. Ophthalmology, Mount Sinai School of Med, New York, NY.Purpose: Bulbar hyperemia is a prominent feature of ocular irritationassociated with dry eye disease (DED), infection and allergy. Theaims of this study were to evaluate a modified topographer(OCULUS Keratograph 5M) as a non-invasive objective metric forevaluation of DED and ocular surface inflammation, and to evaluateits precision in grading bulbar redness and its correlation withclinician and subject grading.Methods: An IRB approved prospective study of patients presentingwith either normal eyes or external ocular disease was conducted. 39eyes of 20 patients were evaluated for bulbar redness independentlyby 2 ophthalmologists (CLS1 and CLS2), by patients’ selfassessment(SS) and by the keratograph using the R-scan software.Standardized CCLRU grading scales were used to score the bulbarredness on a scale of 0-4. To establish precision of the keratograph, 2pictures (KR1 and KR2) of the same eye were taken by the sameclinician, 2 minutes apart. The repeatability of measurements wasthen analyzed using ANOVA and the repeatability index ‘r’.Bivariate correlation analysis was done to get the Spearman’scorrelation coefficient.Results: The keratograph grading showed high repeatability; r=0.90,p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneabulbar redness. Being an objective measure, it reduces the inherentvariability of subjective assessments. Subjective assessment mayoverestimate redness due to normally present large blood vessels onthe conjunctiva that are not indicative of inflammation. Thekeratograph takes into account proportion of bulbar area occupied byvessels, number of vessels and the proportion of area occupied bythin vessels and thus reduces overestimation of hyperemia. Thus thisnovel instrument may prove to be a non-invasive, objectivebiomarker of ocular surface inflammation and thus serve as animportant endpoint in clinical trials of ocular surface disease.Table 1Commercial Relationships: Neha Gadaria-Rathod, None; Kyu-InLee, None; Benyamin Y. Ebrahim, None; Penny A. Asbell, RPS(F)Support: This study was supported in part by Research to PreventBlindness (RPB) Foundation and the Martin and Toni SosnoffFoundation.Program Number: 528 Poster Board Number: B0165Presentation Time: 10:30 AM - 12:15 PMClinical Validation of the Cassini Color LED CornealTopography (CLCT) in post penetrating keratoplasty (PKP)Ronald Ensing 1 , Fleur de Lange 2 , Harry de Vries 1 , Michel Zaal 2 ,Victor A. Sicam 1 . 1 i-Optics B.V., The Hague, Netherlands; 2 OMCZaandam, Zaandam, Netherlands.Purpose: To report results of a clinical investigation involvingcomparison of corneal aberration measurements of CLCT withconventional Placido based and Scheimpflug based topographers.Methods: 23 PKP eyes from 16 subjects (age: 58 years ± 14 years,ranging from 35 to 81 years, 9 OD and 14 OS) were measured bythree instruments: Cassini (i-Optics BV, The Hague, TheNetherlands), OPD Scan (Nidek, Gamagori, Japan) and Pentacam(Oculus, Wetzlar, Germany). Corneal aberrations (ZernikeConvention at 6 mm corneal zone) were compared. An artificial toricsurface with gold standard measurement of 2.22 Diopter was alsomeasured to assess the accuracy of the instruments. The standarddeviation of three trials for every eye measurement was used tocharacterize precision of the instrument. The measurement withmedian defocus was used for inter-instrument comparison. Thepaired student’s t-test was used on the median data to findstatistically significant differences.Results: Nidek OPD measures astigmatism of the toric surface with2.7% error while CLCT measures the toric surface with with 0.5%error. The Pentacam has a precision reaching a mean of 0.25 μm forastigmatism measurements. Both the OPD and CLCT do not exceed amean of 0.096 μm of precision in the measurement of cornealaberrations. Statistically significant differences have been foundbetween the Cassini and the OPD for astigmatism (p = 0.0292) andquadrafoil (p = 0.0281) aberrations. The spherical aberrationmeasured with Pentacam was significantly different from bothCassini and OPD (p = 0.0114 and p = 0.0194 respectively).Conclusions: The lower accuracy of the OPD in measuringrotationally non-symmetric aberrations such as astigmatism andquadrafoil, can be explained by the fact that rings are used in themeasurement. The use of rings does not measure the irregularfeatures of the cornea accurately. The poorer repeatability of thePentacam is a result of motion artifacts during acquisition. TheCassini and the OPD have comparable repeatability because it takesinstantaneous measurements and is therefore not affected by motionartifacts. Among the three instruments, the Cassini measures cornealaberrations both accurately and precisely.Commercial Relationships: Ronald Ensing, i-Optics B.V. (E);Fleur de Lange, None; Harry de Vries, i-Optics (E); Michel Zaal,i-Optics (F); Victor A. Sicam, i-Optics BV, The Hague, TheNetherlands (E), Patent/i-Optics BV, The Hague, The Netherlands(P), Patent/VU University Medical Center, Amsterdam, TheNetherlands (P)Support: The research is supported by i-Optics BV, The Hague, TheNetherlands.Program Number: 529 Poster Board Number: B0166Presentation Time: 10:30 AM - 12:15 PMTopographically Guided Corneal Cross-LinkingDavid B. Usher, Radha Pertaub, Marc D. Friedman, Ronald F.Scharf, David Muller. Avedro Inc, Waltham, MA.Purpose: To determine the feasibility of a topographically guidedcorneal cross-linking device.Methods: A proprietary corneal cross-linking device was developed.A UVA LED source illuminates a digital micromirror device (DMD).Light reflected from the DMD is projected on to a subject’s eye. Thesystem controls the configuration of the DMD’s mirrors such that anarbitrary UVA pattern can be projected on to the eye and modulatedat video rates. A digital camera is used to record views of thesubject’s eye. A Graphical User Interface allows an operator toimport topographies from a third party corneal topographer anddefine UVA irradiation patterns based on the imported topographydata set. A common reference frame between the cross-linking deviceand the topographer is established via a registration algorithm.Images of the subject’s iris exported by the topographer are comparedto images recorded by the cross-linking device’s digital camera. Iristextures and limbus boundaries visible within each dataset are used tocalculate a geometrical relationship between the two camera views.Eye motion during the application of the UVA light is accounted forby tracking the location of the eye in the cross-linking device’sdigital camera. This detected eye motion forms feedback formodulating the DMD mirrors such that the incident UVAillumination tracks relative to the eye. Eye motion, in terms of pupildisplacements, was calculated for 20 eyes from 10 subjects each over30 second periods.Results: An average frame-to-frame (60 Hz) eye motion of 23.9 um(Range: 16.6 - 38.0) was recorded across subjects. Rotations of 2.1°and 2.8° were measured for two subjects where images were recordedby both the cross-linking device and the corneal topographer.Conclusions: The proposed UVA treatment system demonstratesunique features that will be able to advance the science of cornealcross-linking. Each individual mirror of the DMD can be controlledto correct beam uniformity through irradiance calibrations. Itsflexibility allows a surgeon complete freedom when configuring theUVA dose across different areas of the cornea to a high degree ofaccuracy. The eye tracking preserves this accuracy by accounting foreye motion. The integration of the topography data allow a surgeon touse variables such as corneal elevations, power maps, k readings,corneal thickness maps, and epithelial thickness maps when creatingpatent specific cross-linking pretreatment plans and procedures.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: David B. Usher, Avedro Inc (E);Radha Pertaub, Avedro Inc (E); Marc D. Friedman, Avedro Inc(E); Ronald F. Scharf, Avedro, Inc. (E); David Muller, Avedro Inc(E)Program Number: 530 Poster Board Number: B0167Presentation Time: 10:30 AM - 12:15 PMCorneal nerve morphology: a non-invasive surrogate of nervefibre damage and repair in chemotherapy-associated peripheralneuropathyBernardo F. Sanchez Dalmau 1, 2 , Marisol Lopez Moreno 1, 2 , RubenTorres 1, 2 , Santiago Ortiz-Perez 1, 2 , Dolores Vela 3 , Elena Fraga 2 ,Pablo Villoslada 2 . 1 Institut Clinic d'Oftalmologia, HospitalClinic.Seu Maternitat, Barcelona, Spain; 2 NeuroImmunology Group,IDIBAPS, Barcelona, Spain; 3 Hematology Unit, Hospital General deGranollers, Granollers, Spain.Purpose: Chemotherapy-associated neuropathy is a cause ofdisability in patients with cancer, for diagnosis invasive techniquessuch as skin or sural nerve biopsy are used. Corneal confocalmicroscopy is a noninvasive technique that allows to assess “in-vivo”all structures of the cornea, including the corneal nerves. The purposeof the current study is to determine the ability of this technique toquantify the degeneration and regeneration of corneal nerves inpatients with peripheral neuropathy secondary to chemotherapy.Methods: Twelve patients with chemotherapy-associated neuropathy(study group) and 12 healthy volunteers were included. Laser in vivoconfocal microscopy (IVCM) of the cornea was performed in one eyeof each participant using the Heidelberg Retina Tomograph with theRostock Cornea module. By a combination of clinical assessment andsymptomatic neuropathy score an overall neuropathy score wasobtained.Results: Median age was 59 year in the study group and 57.2 years inthe control group. The average time between evaluation andcompletion of chemotherapy was 11,8 month (2-38 months). Theseverity of the neuropathy was mild in one patient (8.3%), moderatein 8 (66.6%) and severe in 3 patients (25%).The IVCM revealed areduction in nerve density and number of branching in 7 patients(58.3%) compared with 1 (8.3%) in the control group (p=0.0272).There was not a significant correlation between the sub-basal nervefindings and the severity of the neuropathy and with the time ofchemotherapy ending.Conclusions: Correlation between the chemotherapy-associatedneuropathy and sub-basal nerve morphology has been found. Giventhat IVCM is a non invasive and painless technique which allowsserial assessment of the sub-basal nerve morphology we suggest thatIVCM may be an important tool to assess nerve degeneration andregeneration and therefore become a surrogate marker in themonitoring of chemotherapy associated neuropathy.Commercial Relationships: Bernardo F. Sanchez Dalmau, None;Marisol Lopez Moreno, None; Ruben Torres, None; SantiagoOrtiz-Perez, None; Dolores Vela, None; Elena Fraga, None; PabloVilloslada, Heidelberg Engineering (C), Novartis (F), Novartis (F),Roche (C), Bionure (I), Bionure (P)Program Number: 531 Poster Board Number: B0168Presentation Time: 10:30 AM - 12:15 PMLaser In Vivo Confocal Microscopy Demonstrates a LowerDensity of Peripheral Corneal Nerve Fibers Compared to theCentral Cornea in Normal SubjectsJae Young You 1 , Bernardo M. Cavalcanti 1 , Susan Cheng 1 , MoniqueL. Trinidad 1 , Douglas Critser 3 , Amy Watts 2 , Charles D. Leahy 2 ,Christine W. Sindt 3 , Pedram Hamrah 1 . 1 Cornea and RefractiveSurgery, Massachusetts Eye and Ear Infirmary, Boston, MA;2 Contact Lens, Massachusetts Eye and Ear Infirmary, Boston, MA;3 Ophthalmology, University of Iowa, Iowa City, IA.Purpose: To quantify subbasal corneal nerve density by in vivoconfocal microscopy in the central and four peripheral quadrants ofnormal subjects.Methods: In vivo confocal microscopy (IVCM; HRT3/RCM) of thecentral cornea and 4 peripheral quadrants (superior, inferior, nasal,and temporal) was performed in 37 subjects with normal clinical slitlampexamination. Three representative images were chosen for eacharea and quantified using NeuronJ, a plugin for ImageJ (NIH). Twomasked observers measured the number and density of total nerves,main trunks and branches. Statistical analysis was performed withANOVA with Bonferroni correction to compare the differencesbetween the 5 areas. A linear regression model was applied to assessthe changes for gender and age.Results: The average age of normal subjects was 30 years, rangingfrom 19 to 69 years. The central cornea had a significantly highernumber and density of total nerves (16.2 n/frame [14.8-17.6] 95%CI;and 20067.7 µm/mm2 [18776.6-21358.8] 95%CI), main trunks (3.3[2.9-3.6]; and 8848.4 [7915.3-9781.4]), and branches (12.8 [11.4-14.2]; and 11219.3 [10152.2-12286.4]) in comparison to allperipheral areas (p0.05),including the number and density of total nerves for superior (9.2[7.8-10.6]; and 11638.1 [9834.7-13441.5]), inferior (9.2 [7.5-11.0];and 11169.3 [9080.2-13258.4]), temporal (8.9 [7.5-10.3]; and 9535.3[8452.7-10617.9]), and nasal (7.8, [6.2-9.4] and 9621.0, [7832.3-11409.8]). An inverse correlation to age (R=-0.20; p=0.017), but notfor gender (p=0.781) was shown for all nerve parameters.Conclusions: In normal eyes, IVCM shows a higher number anddensity for subbasal nerve fibers for the corneal center in comparisonto the periphery. An inverse correlation is observed for nerveparameters and age. A standardized approach is demonstrated tomeasure the subbasal plexus for future clinical studies.Commercial Relationships: Jae Young You, Alcon Research LTD(F); Bernardo M. Cavalcanti, None; Susan Cheng, None; MoniqueL. Trinidad, None; Douglas Critser, None; Amy Watts, None;Charles D. Leahy, Vista Scientific LLC (I), Vista Scientific LLC(P); Christine W. Sindt, alcon (F); Pedram Hamrah, NoneSupport: NIH K08-EY020575, New England Corneal TransplantResearch Fund, Falk Medical Research Trust, Alcon Research LTDProgram Number: 532 Poster Board Number: B0169Presentation Time: 10:30 AM - 12:15 PMRiboflavin Dosimetry in the Cornea using a KXL-II and theScheimpflug PrincipleMarc D. Friedman, David B. Usher, Radha Pertaub, David Muller.Avedro, Waltham, MA.Purpose: To determine the feasibility of a new device (KXL-II) thatcombines custom UVA illumination patterns with a Scheimpflugimaging system for measuring riboflavin (RF) diffusion rates in thecornea.Methods: A proprietary corneal imaging device was developed. AUVA LED source illuminates a digital micromirror device (DMD)and is in turn projected on to a subject’s eye. Two digital cameras aremounted ±45° to the apex of the cornea. The mirrors on the DMD areconfigured such that UVA illumination forms a series of slits. As theUVA passes through the cornea the resultant fluorescence profileschange with the cross-sectional distribution of RF. Profiles ofmultiple slits observed simultaneously map the distribution ofriboflavin in the radial direction (Figure 1). The two off axis camerasemploy a Scheimpflug principle whereby their focal planes are tiltedin order to optimize the system’s optical design in terms of focal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadepth (Figure 2). Whole porcine eyes were placed at the device’sfocal plane. Images from the side cameras were recorded at regularintervals after the application of RF solution.Results: Sequences of images recorded at regular intervals duringand after the application of RF solution showed increasedfluorescence at increased depths within the cornea. A focal planeangle of 18.2° to the apex of the eye was found to increase theuseable depth of field of the system by a factor of over 2.5.Conclusions: An advanced riboflavin dosimetry device is presented.The KXL-II system combines the use of UVA projection via a DMDwith side cameras that monitor riboflavin fluorescence in the cornea.The camera’s tilted focal plane relaxes the constraints on the systemsdepth of field. The DMD allows for rapid alternation betweenillumination profiles intended for cross-linking treatment and slitconfigurations intended for fluorescent dosimetry. With this device itmay be possible to respond and compensate to live dosimetryreadings as riboflavin is consumed and measured at various depthswithin the cornea. Real time dosimetry measurements will play asignificant role in determining the underlying mechanisms of cornealcross-linking and assist with the development of additional riboflavinformulations.Figure 1 Schematic of slit UVA illumination projected on to theCornea.topographer [Jongsma et al. OVS; 1998]. Three tasks were set. SinceESP requires instillation of fluorescein, the optimal combination ofthe fluorescein strips and the eye physiological solution that results inthe best quality of recorded images was assessed. Further, therepeatability of the instrument in measuring artificial and real eyesurfaces was studied. Finally, the new topographer was tested inclinical settings against traditional Placido disk topographer (E300,Medmont) for a range of normal, astigmatic, and highly aberratedeyes (post corneal grafts).Results: The best results in terms of highly resolved ESP imageshave been achieved with BIO GLO sterile strips (1 mg fluoresceinsodium) with Hial eye drops (0.4 mg/ml hialuronian sodium). Forartificial surfaces, the repeatability of instrument in a dynamic case,when operator manually focused the instrument, was very high whilethe accuracy of the instrument in terms of the RMS error was lessthan 10um but depended on the instrument-surface distance. Theworking distance was estimated at +/-250um from the best focalplane. For real eyes, the coverage area was routinely greater than16mm and often reached up to 20mm diameter. Single instillation offluorescein allowed acquisition from 3 to 20 measurements (taken inless than 30 second intervals). Repeatability, evaluated withrefractive spherical component over an 8 mm corneal diameter, washigh (differences less than 0.125 D). When tested against the E300topographer, ESP showed excellent repeatability for spherical power(in an 8 mm corneal diameter) but seemed to overestimate theastigmatic component, which seemed to depend on the instrument’sworking distance. For highly aberrated eyes, in situations where E300could not perform a valid measurement, the ESP was still performingwell.Conclusions: ESP has high potential clinical utility. It couldsubstitute the currently used videokeratoscopes and provide a newdiagnostic quality in those applications in which information oncorneoscleral topography is of essence.Commercial Relationships: D Robert Iskander, Eaglet Eye (F),Eaglet Eye (I)Support: Eaglet Eye - research grantFigure 2 Schematic showing side camera’s use of Sceimpflugprinciple. Focal plane intersects with measurement zone in cornea.Commercial Relationships: Marc D. Friedman, Avedro Inc (E);David B. Usher, Avedro Inc (E); Radha Pertaub, Avedro Inc (E);David Muller, Avedro Inc (E)Program Number: 533 Poster Board Number: B0170Presentation Time: 10:30 AM - 12:15 PMThe clinical utility of the Eye Surface ProfilerD Robert Iskander. Institute of Biomedical Engineering andInstrumentation, Wroclaw University of Technology, Wroclaw,Poland.Purpose: To evaluate the effectiveness of the Eye Surface Profiler inmeasuring the topography of the anterior eye surface.Methods: Eye Surface Profiler (ESP) is a newly developed corneaand sclera topographer that can measure up to 20mm of the anteriorsurface of the eye. It is an evolution of a wide field height eyeProgram Number: 534 Poster Board Number: B0171Presentation Time: 10:30 AM - 12:15 PMSubclinical Keratoconus Detection Based On PentacamScheimpflug Tomography IndicesPablo R. Ruisenor Vazquez 1, 2 , Marianella Delrivo 2 , FernandoFuentes Bonthoux 1, 2 , Tomás Pfortner 2 , Pablo Chiaradia 1 , JeremiasG. Galletti 1, 2 . 1 Ophthalmology Division, Hospital de Clínicas,University of Buenos Aires, Buenos Aires, Argentina; 2 ECOS(Clinical Ocular Studies) Laboratoy, Buenos Aires, Argentina.Purpose: To evaluate the diagnostic performance of corneal indicesprovided by the Pentacam tomograph for detecting subclinicalkeratoconus.Methods: Observational case series of 136 eyes from 136 healthysubjects in group 1 and 42 topographically-unremarkable eyes from42 keratoconic patients in group 2, evaluated with cornealtopography, aberrometry, and Scheimpflug tomography for Belin-Ambrosio D indices. For data analysis, Student's t test was used tocompare means and receiver operating characteristic (ROC) curveswere used to analyze the diagnostic performance of D indices forkeratoconus detection. The statistical significance criteria used was p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea(0.24 ± 0.07 vs 0.66 ± 0.35 µm, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaedge of the optical zone (8-9mm diameter) with future wide-fieldOCT.Commercial Relationships: Maolong Tang, Optovue Inc. (F); YanLi, Optovue, Inc. (F), Optovue, Inc. (P), Carl Zeiss Meditec, Inc. (P);David Huang, Optovue (F), Optovue (I), Optovue (P), Optovue (R),Carl Zeiss Meditec (P)Support: NIH grant EY018184Program Number: 537 Poster Board Number: B0174Presentation Time: 10:30 AM - 12:15 PMNormal Variants of Human Limbus Detected by In Vivo LaserScanning Confocal MicroscopyElfren R. Baclagon 1 , Siamak Zarei-Ghanavati 2 , Sophie X. Deng 1 .1 Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA; 2 CorneaDepartment and Eye Research Center, Mashhas University ofMedical Sciences, Mashhad, Islamic Republic of Iran.Purpose: To report normal variations of the human limbal structuresusing in vivo laser scanning confocal microscopy (LSCM).Methods: Fourteen eyes from 13 healthy individuals between theages of 22 to 94 years were included in this study. Confocal imagingof the cornea and limbus was performed using Heidelberg RetinaTomograph III Rostock Corneal Module.Results: The typical structure of the palisade of Vogt (POV) wasdetected in 57% of eyes. Four additional structures and patterns werefound in the normal limbus that had not been reported previously ineyes that did not have the POV. Whorl-like distribution of limbalepithelial cells was present in two eyes. Mixed corneal andconjunctival epithelial cells in a mosaic pattern were found at theposterior limbus in two eyes. Bright dots pattern within the normallimbal epithelial cells was observed in the wing and the basal layer infive eyes. The forth structure, “limbal lacuna” was detected in two ofthe subjects. It consisted of deep stromal lacuna filled with limbalepithelial cells.Conclusions: There are several structures variants in the normallimbus. POV may not be the only structure that harbors limbalstem/progenitor cells.Commercial Relationships: Elfren R. Baclagon, None; SiamakZarei-Ghanavati, None; Sophie X. Deng, NoneSupport: CIRM TR2-01768 and NEI 5T32EY007026-35by fungal keratitis was diagnosed with epithelial downgrowth afterundergoing 3 penetrating keratoplasties and placement of a glaucomadrainage device over a 3-year period. In case two, a 48 year-old malewith a history of acanthamoeba keratitis developed epithelialdowngrowth after undergoing two therapeutic keratoplasties over aone-year period. In case three, a 40 year-old female with a history ofperforating fungal keratitis developed epithelial downgrowth after 2therapeutic keratoplasties over a three-month period. In all threecases, IVCM revealed hyper-reflective nuclei characteristic ofepithelium and AS-OCT identified an epithelial layer at the level ofthe endothelium.Conclusions: This report provides useful images of epithelialdowngrowth from both IVCM and AS-OCT. These noninvasiveimaging modalities may potentially be more sensitive in identifyingand monitoring epithelial downgrowth than routine lightbiomicroscopy.Figure 1: A) Slit lamp photo showing diffuse epithelial downgrowth.B) IVCM showing hyper-reflective nuclei of epithelial cells at thelevel of the endothelium. C and D) AS-OCT with a hyper-reflectiveepithelial layer posteriorly and growing over the iris. The angle iscompletely sealed.Program Number: 538 Poster Board Number: B0175Presentation Time: 10:30 AM - 12:15 PMImaging of epithelial downgrowth following penetratingkeratoplasty by confocal microscopy and high-resolution opticalcoherence tomographyVivian Lien 1 , Michael Chen 1 , Dennis E. Cortes 1, 2 , Jennifer Li 1 , MarkJ. Mannis 1 . 1 Ophthalmology & Vision Science, University ofCalifornia Davis Eye Center, Sacramento, CA; 2 Ophthalmology,Pontificia Universidad Católica de Chile, Santiago, Chile.Purpose: To report the characteristics of epithelial downgrowthfollowing penetrating keratoplasty using in vivo confocal microscopy(IVCM) and high-resolution anterior segment optical coherencetomography (AS-OCT).Methods: A retrospective case review was performed of 3 eyes of 3patients that developed epithelial downgrowth after multiplepenetrating keratoplasties. IVCM images were obtained at varioustime points using the Heidelberg Retina Tomograph III RostockCornea Module (Heidelberg Engineering, Germany) and AS-OCTimages were obtained using a high-resolution spectral-domain OCT(Heidelberg Engineering, Germany). In two cases, the diagnosis wasconfirmed by histopathologic evaluation.Results: Three cases developed epithelial downgrowth. In case one, a40 year-old male with a history of a corneal laceration complicatedFigure 2: A) Slit lamp photo of diffuse epithelial downgrowth withunaffected area centrally. B) IVCM showing hyper-reflective nucleiof epithelial cells at the level of the endothelium. C) AS-OCT withhyper-reflective layer of epithelial downgrowth posteriorly withcorresponding uncompromised area centrally.Commercial Relationships: Vivian Lien, None; Michael Chen,None; Dennis E. Cortes, None; Jennifer Li, None; Mark J.Mannis, NoneProgram Number: 539 Poster Board Number: B0176Presentation Time: 10:30 AM - 12:15 PMRole of glutathione peroxidase 4 in maintaining homeostasis ofcorneal epithelial cells©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaOsamu Sakai 1, 2 , Takashi Ueta 1 , Yasuo Yanagi 1 , Shiro Amano 1 .1 Opthalmology, The University of Tokyo, School of Medicine,Tokyo, Japan; 2 Research Laboratory, Senju Pharmaceutical Co Ltd,Kobe, Japan.Purpose: Antioxidant enzymes are involved in the homeostasis ofcorneal epithelial cells. Glutathione peroxidase 4 (GPx4) is one of theantioxidant enzymes that can directly reduce lipid peroxidationcaused by oxidative stress. The purpose of the present study was toinvestigate the role of GPx4 in maintaining homeostasis of cornealepithelial cells.Methods: Simian virus 40 immortalized human corneal epithelial(HCE) cells were used. HCE cells were transfected with GPx4siRNA, and we tested for lipid oxidation, wound healing, andcytotoxicity. Lipid oxidation was confirmed by immunostaining of 4-hydroxy-2-nonenal (4-HNE). In the wound healing model, holedefects were formed in confluent HCE cells, and wound areas wereevaluated 2 days after defect formation. Evaluation of cellcytotoxicity was conducted by assay of LDH activity, staining ofannexin V/PI, immunostaining of apoptosis inducing factor (AIF),and western blot of caspase 3. In oxidative stress study, cultured HCEcells were transfected with GPx4 overexpressing plasmid and GPx4siRNA, and then were treated with hydrogen peroxide. Cytotoxicitywas evaluated by assay of LDH activity 24 hours after the treatment.Results: GPx4 knockdown caused 2.3-fold increase in the levels oflipid oxidation (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaform stable crosslinked products and is an effective crosslinkingagent for Descemet membrane fixation. Therefore, these resultsindicated that the genipin-fixed Descemet membrane may be appliedas a excellent culture carrier. The non-toxic nature crosslinkergenipin may be useful for the application on the reconstruction of theocular surface.Commercial Relationships: Lung-Kun Yeh, None; Shih-ChunHuang, NoneSupport: Chang Medical Research Project G CMRPG3A1291 ;National Science Council Grants (Taiwan) 1012314B182A056MY3Program Number: 542 Poster Board Number: B0179Presentation Time: 10:30 AM - 12:15 PMThe Role of Nrf2-Mediated Defense System in Corneal EpithelialWound HealingRyuhei Hayashi 1 , Noriko Himori 2 , Keiko Taguchi 3 , Yuki Ishikawa 1 ,Kohji Uesugi 1 , Motokazu Tsujikawa 1 , Toru Nakazawa 2 , MasayukiYamamoto 3 , Kohji Nishida 1 . 1 Ophthalmology, Osaka UniversityMedical School, Suita, Japan; 2 Ophthalmology, Tohoku universityschool of medicine, Sendai, Japan; 3 Department of MedicalBiochemistry, Tohoku university school of medicine, Sendai, Japan.Purpose: The Nrf2-mediated defense system plays a central role inprotecting cells by activating genes against these types of stress. Inthe present study, we investigated the role of the Nrf2-mediateddefense system in corneal epithelial wound healing by using Nrf2-knockout (KO) mice.Methods: The corneal epithelium of wild type (WT) and Nrf2 KOmice were removed by treatment of n-heptanol for 1 minute underanesthesia. The epithelial defect was stained with 1% fluoresceinsolution and photographed at 0, 6, 12, 18, 24, 30, 36, 48, 60, and 72 hafter epithelial debridement. Injured corneas healed at various timepoints were subjected to immunohistochemistry with Ki-67and Nrf2antibody. Telomerase-immortalized corneal epithelial cell line(C/TERT) was used for cell migration assay and siRNA experiment.Results: Nrf2 was expressed in the corneal epithelium of WT mice,but not in KO mice. Observation of wounds after 24 h of healingrevealed that healing of the corneal epithelium was significantlydelayed in the Nrf2 KO mice, while Nrf2 was activated in the cornealepithelium of WT mice. Ki-67-staining revealed that the number ofKi-67-positive proliferation cells was significantly lower in the Nrf2KO mice than in the WT mice at 24-36 h after injury; however, thesenumbers were approximately equivalent by 48 h. To clarify the roleof Nrf2 during wound healing, we performed in vitro experiments ofsiRNA for Nrf2. The result showed that Nrf2 knock-downsignificantly delayed corneal epithelial cell migration.Conclusions: This study provides evidence that the Nrf2-mediateddefense system plays a crucial role in corneal epithelial woundhealing, by regulating the cell-migration activities of cornealepithelial cells.Commercial Relationships: Ryuhei Hayashi, None; NorikoHimori, None; Keiko Taguchi, None; Yuki Ishikawa, None; KohjiUesugi, None; Motokazu Tsujikawa, Shionogi & Co. (C), DaiichiSankyo Co. (F), Daiichi Sankyo Co. (R), Santen Co. (R), AMO Co.(R); Toru Nakazawa, Kowa Company Ltd. (F), Kowa CompanyLtd. (C); Masayuki Yamamoto, None; Kohji Nishida, Alcon (C),Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F), OsakaUniversity (P)Support: Grants-in-Aid for Scientific Research from the Ministry ofEducation, Culture, Sports, Science and Technology in JapanProgram Number: 543 Poster Board Number: B0180Presentation Time: 10:30 AM - 12:15 PMIncreased fragility and acceleration of migration of cornealepithelial cells in an epiplakin deficiencyMasahide Kokado 1 , Yuka Okada 1 , Kazushi Ishikawa 2 , HiromitsuShimada 2 , Sakuhei Fujiwara 2 , Masayasu Miyajima 3 , Shizuya Saika 1 .1 Department of Ophthalmology, Wakayama Medical University,Wakayama, Japan; 2 Department of Dermatology, Oita University,Oita, Japan; 3 Laboratory animal center, Wakayama MedicalUniversity, Wakayama, Japan.Purpose: Epiplakin is one of intermediate filament-relatedcomponents. To examine fragility and mechanism of migration ofcorneal epithelial cells in an epiplakin deficiency. We previouslyreported that the loss of epiplakin facilitates healing of a defect incorneal epithelium in mice (ARVO 2009), and epiplakin knockdownby Short-interfering RNA (siRNA) was accelerated the migration ofHCEC and suppressed TGFb1 activation of p38 and JNK signals(ARVO2012).Methods: (1) We used Epiplakin KO (KO) (n=4) and wild type(WT) (n=4). The corneal surface was gently brushed with a surgicalmicro-sponge. Then ultrathin sections were cut and observed undertransmission electron microscopy. (2) We ran real-time RT-PCR forcell-cell connection-related components in RNA samples obtainedfrom KO (n=12)and WT (n=12). (3)Effect of siRNA knockdown ofEPPK on E-cadherin expression was also studied in AV40-immortalized corneal epithelial cells by using western blotting.Results: (1)Transmission electron microscopy showed that eachlayer of the epithelial cells was well maintained following brushing ina WT epithelium although partial separation between cells of thesuperficial layer. In a KO epithelium one or two layer(s) of basal orsupra-basal epithelial cells were found to be in the original positionfollowing the treatment and upper layer cells were found to beremoved. (2)Real-time RT-PCR also showed that mRNA expressionof E-cadherin was suppressed by the loss of epiplakin, while that ofdesmoglein-1 or desmoplakin-1 was not affected by epiplakin geneknockout in a mouse cornea. (3)EPPK knockdown suppressed E-cadherin expression in the cells.Conclusions: The loss of epiplakin affects the corneal epitheliumintegrity. The mechanism of acceleration of cell migration in the KOcorneal epithelium is to be further investigated, although suppressionof expression of E-cadherin in the absence of EPPK might beincluded. The possibility of the potential corneal epitheliumdisturbance in the patient who has abnormalities to EPPK wassuggested.Commercial Relationships: Masahide Kokado, None; YukaOkada, None; Kazushi Ishikawa, None; Hiromitsu Shimada,None; Sakuhei Fujiwara, None; Masayasu Miyajima, None;Shizuya Saika, NoneProgram Number: 544 Poster Board Number: B0181Presentation Time: 10:30 AM - 12:15 PMTransfer of mucosal epithelial cells and extracellular matrix by agelatin matrix technique in vitroAllen Ho 1 , Li-Fang Wang 2 . 1 Jianguo School, Taipei, Taiwan;2 National Taiwan University, Taipei, Taiwan.Purpose: Poor connection and adhesion of epithelial cells tounderlying connective tissues is the basic defect of blister disease ofconjunctiva, skin, and mouth mucosal membrane such as ocularcicatricial pemphigoid. Transplantation of extracellular matrix(ECM) along with the autologous buccal mucosal epithelial cells maymodulate the survival and subsequent proliferation of transplantedepithelial cells. Thus, we have developed a technique to transfernative ECM with human mucosal epithelial cells in vitro.Methods: Confluent monolayer human mucosal epithelial cells in theculture plate was pretreated with 0.25% edetic acid for 15 minutes©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand coated with a 800 μ layer of 20% gelatin. Patches of the mucosalepithelial cells were harvested and transferred to another cultureplate. Cell viability and the ability of the transferred cells toproliferate in culture were determined. The ECM transferred alongwith the cells was characterized by immunohistochemistry.Results: Human mucosal epithelial cells in culture can be harvestedas an organized cell patch with high efficiency (92.3 ± 4.5%) andhigh cell viability (89.5 ± 5.5%). Cells harvested from tissue cultureplates divided and became confluent within 21 days.Immunohistochemistry demonstrates that the transferred ECM alongcontains laminin and type IV collagen.Conclusions: We were able to harvest mucosal epithelial cells as anorganized monolayer from tissue culture plate along with theirintercellular extracellular matrix. This approach may provide apractical technique for isolating and transplanting cultivatedautologous epithelial cells to reconstruct the damaged ocular surfacein Steven-Johnson syndrome, chemical and thermal injury, and ocularcicatricial pemphigoid.Commercial Relationships: Allen Ho, None; Li-Fang Wang, NoneProgram Number: 545 Poster Board Number: B0182Presentation Time: 10:30 AM - 12:15 PMMedical management of limbal stem cell deficiency with antiinflammatorytherapy and tear film optimizationBryan Kim 1 , Pejman Bakhtiari 1 , Kamran Riaz 2 , Clara C. Chan 3 ,Jeffrey Welder 1 , Surendra Basti 2 , Ali R. Djalilian 1 . 1 Ophthalmology,University of Illinois Eye and Ear Infirmary, Chicago, IL;2 Ophthalmology, Northwestern University Feinberg School ofMedicine, Chicago, IL; 3 Ophthalmology, University of Toronto,Toronto, ON, Canada.Purpose: To characterize the clinical features and medicalmanagement of cases with limbal stem cell deficiency (LSCD) thatwere reversible with anti-inflammatory therapy.Methods: Retrospective case series of 23 patients (35 eyes) at 3tertiary referral centers who were seen between 2007 and 2011.These patients initially had clinical findings consistent with LSCDbut then had resolution of these findings with only medicalmanagement. Main outcome measures included comparison of ocularsurface findings during and after medical treatment and changes invisual acuity.Results: Mean patient age was 41 years (range, 18-77) with 9 malesand 14 females. Etiologies of LSCD included contact lens wear (27eyes), contact lens wear in the setting of ocular rosacea (3 eyes),benzalkonium chloride (BAK) toxicity (2 eyes), and idiopathic (3eyes). Clinical findings included progressive epitheliopathy withassociated opaque epithelium arising from the limbus, loss of limbalarchitecture, and late fluorescein staining in a wavy or whorl pattern.Extent of limbal disease involvement varied from 30 to 360 degreesof limbus, with the superior limbus as the most common site ofinvolvement (31 eyes). Medical management was initiated in allpatients who had persistent disease after 3 months of conservativemeasures (e.g. discontinuing contact lens wear). The treatmentsincluded: topical corticosteroids (11 eyes), topical cyclosporine (11eyes), topical vitamin A (6 eyes), doxycycline (3 eyes), and punctalocclusion (15 eyes). Following treatment, all 35 eyes achieved astable ocular surface and resolution of LSCD over a mean follow-upof 7.9 months (range, 2-36 months). 32 eyes experiencedimprovement in visual acuity from an initial mean log MAR of 0.363(20/46) to a post-treatment mean log MAR of 0.136 (20/27)(P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMulticentric study validation of a new molecular method forLimbal Stem Cell Deficiency diagnosis based on MUC5ACtranscript detection in corneal epithelium by reverse dot-blotstripTatiana M. Suarez-Cortes 1 , Iker Garcia 1 , Jaime Etxebarria 2 , JesusMerayo-Lloves 3 , Josep Torras 4 , Ana Boto-de-los-Bueis 5 , David Diaz-Valle 6 , Rosalia Mendez 6 , Xabier Landaluce 1 , Arantxa Acera 1 .1 Biomed R & D, Bioftalmik, Derio, Spain; 2 Department ofOphthalmology, Cruces Hospital, Plaza Cruces 12, Baracaldo, Spain;3 Fundación de Investigación Oftalmológica, Oviedo, Spain;4 Department of Ophthalmology, Hospital Clinic de Barcelona,Barcelona, Spain; 5 Department of Ophthalmology, Hospital La Paz,idiPaz, Madrid, Spain; 6 Department of Ophthalmology, HospitalClínico San Carlos, Madrid, Spain.Purpose: To validate the efficacy, accuracy and sensitivity of a PCRstripbased on reverse dot-blot for detection of MUC5AC transcriptas indicative of the presence of goblet cells in cornea of patients withLimbal Stem Cell Deficiency (LSCD), and to evaluate the correlationwith clinical diagnosis.Methods: Eighty-five corneal impression cytology (IC) samplesfrom 54 subjects were analyzed in the study: 44 clinically diagnosedLSCD corneas and 41 healthy corneas. Sixteen conjunctivalimpression cytology (IC) samples were assayed as positive control. Atotal of 101 impression cytology samples were analyzed in the study.All the corneal impression cytologies were processed by RNAextraction, retrotranscription, and analyzed by the presence of gobletcells in the cornea by the PCR-strip based on reverse blotting system(Limbokit).Results: The total IC samples analyzed indicated that 43 of 44samples clinically diagnosed as LSCD were confirmed positive forMUC5AC, 33 of 41 healthy corneas were confirmed negative forMUC5AC, 4 healthy corneas were found positive, and 4 wererendered inconclusive results. All conjunctival impression cytologiesused as positive control were confirmed MUC5A positive. The dataindicate a global correlation of 91.1%. (p=0.0001). Confirmation ofPCR results in TAE-agarose gels indicated that reverse dot-blot stripwas more sensitive for bands detection and enhances visualization ofresults. The overall sensitivity, specificity, Positive Predictive Value(PPV) and Negative Predictive Value (NPV) of Limbokit visualizedon PCR-strips, were 98%, 89%, 91% and 97% respectively.Conclusions: The PCR-strip test based on reverse blotting was foundto be a sensitive technique for detection of MUC5AC transcript incorneal epithelium. The test results correlate well with clinicaldiagnosis of characterized LSCD cases. The overall sensitivity,specificity, and positive and negative predictive values weresatisfactory for diagnostic purposes. The PCR-strip based test(Limbokit) constitutes a robust system for the detection of MUC5ACin corneal epithelium and may be used for early detection and formild cases of Limbal Stem Cell Deficiency, and also as an objectiveclinical tool for monitoring of treatments and surgical decisions.Commercial Relationships: Tatiana M. Suarez-Cortes, BioftalmikS.L. (E); Iker Garcia, Bioftalmik Applied Research (P); JaimeEtxebarria, bioftalmik (C); Jesus Merayo-Lloves, Ferrara & HijosSL (I); Josep Torras, None; Ana Boto-de-los-Bueis, None; DavidDiaz-Valle, None; Rosalia Mendez, None; Xabier Landaluce,Bioftalmik (E); Arantxa Acera, BIOFTALMIK SL (E)Support: NEOTEC Program, Grant IDI-20080118Program Number: 548 Poster Board Number: B0185Presentation Time: 10:30 AM - 12:15 PMOptimized culturing conditions for limbal epithelial cellscultivated on semi-synthetic collagen matricesCorinna Petsch 1 , Ursula Schlotzer-Schrehardt 1 , Markus Frey 2 ,Johannes Menzel-Severing 1 , Friedrich E. Kruse 1 , Bjoern O.Bachmann 1 . 1 Department of Ophthalmology, University of Erlangen-Nuremberg, Erlangen, Germany; 2 RESORBA WunversorgungGmbH & Co.KG, Nuremberg, Germany.Purpose: To optimize culturing conditions for limbal epithelial cellson variants of a semi-synthetic collagen matrices with different invivo degradation characteristics.Methods: Limbal epithelial stem cells were clonally enriched on 3T3feeder cells and subcultivated on 3 variants (two crosslinked and onenon-crosslinked variant) of a semi-synthetic type I collagen substrate(RESORBA, Germany). For clonal enrichment and subcultivation onthe collagen matrices 3 different cell culture media were evaluated:MCDB151, DMEM/F12-1 (with bovine pituitary gland extract) andDMEM/F12-2 (without bovine pituitary gland extract). After fixationcell cultures were examined concerning cell adhesion, proliferationand cellular phenotype by light and electron microscopy as well asimmunohistochemistry.Results: Immunohistochemistry as well as light and electronmicroscopy revealed no differences in adhesion, proliferation and cellsheet formation between cell cultures on either variant of the collagenmatrix. When cultured with MCDB151 or DMEM/F12-1 monolayerformation of limbal epithelial cells was seen, while the use ofDMEM/F12-2 resulted in a multilayered cell sheet.By immunohistochemistry, E-Cadherin (as a marker for adherensjunctions) and K3/12 (as a differentiation marker) were localized inall cell layers. Integrinα6 (marker for hemidesomsomes) and p63 (aputative stem cell marker) were expressed in the basal cell layer. P63was also apparent in upper layers of cells cultured on non-crosslinkedcollagen matrices.Electron microscopically hemidesmosomes were seen on cells of thebasal cell layer of cultures on either collagen substrate when culturedwith DMEM/F12-1 or DMEM/F12-2.Conclusions: Crosslinked and non-crosslinked variants of a semisyntheticcollagen matrix are suitable for the cultivation of limbalepithelial cells. The use of DMEM/F12-2 with either collagen matrixresulted in a multilayered cell sheet of cultured limbal epithelial cells.The ability to serve as a growth substrate for limbal epithelial cellsand its known biocompatibility with the cornea indicates that the usedcollagen matrices might be suitable for cultivation and transplantationof ex vivo expanded limbal epithelial cells.Commercial Relationships: Corinna Petsch, RESORBAWundversorgung GmbH &Co.KG, Nuremberg, Germany (F); UrsulaSchlotzer-Schrehardt, None; Markus Frey, RESORBA MedicalGmbH (E); Johannes Menzel-Severing, None; Friedrich E. Kruse,None; Bjoern O. Bachmann, NoneSupport: Medical Valley EMR Erlangen Project A-06, sponsored bythe Federal Ministry of Education and Resarch, Germany (BMBF)Program Number: 549 Poster Board Number: B0186Presentation Time: 10:30 AM - 12:15 PMCD45+F4/80+ immune cells are rapidly recruited to the woundedge after corneal debridement woundsGauri Tadvalkar 1 , Sonali Ghosh 1 , Ahdeah Pajoohesh-Ganji 1 , JaniceL. Walker 2 , A S. Menko 2 , Mary Ann Stepp 1 . 1 Anatomy &Regenerative Biology, George Washington University MedicalCenter, Washington, DC; 2 Pathology, Anatomy and Cell Biology,Thomas Jefferson University, Philadelphia, PA.Purpose: A population of leader cells was found to direct migrationof lens epithelial cells in response to wounding. The current studieswere carried out to determine 1) whether leader cells are involved inthe response of the corneal epithelium to injury, and 2) test thehypothesis that leader cells are immune cells.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: 1.5 mm debridement wounds were made manually in 8week old BALB/c using one of two techniques: a rotating burr (RB)or a dulled blade (DB). Mice were either sacrificed at the time ofwounding (Time 0, T0) or allowed to heal for 1 or 6 hrs. All eyeswere fixed within 5-10 min after injury. Corneas were used for wholemount immunofluorescence studies to localize CD45 (the commonleukocyte antigen), F4/80 (a monocyte marker), GL3 (an epitopepresent on γδT cells), and vimentin, at the leading edges of thewounded corneas. For each variable studied, no fewer than 3 corneasfrom 3 different mice were used.Results: CD45+ cells appear at the leading edge in the T0 corneas.T0 DB wounds recruit more F4/80+ cells compared to RB woundsand this difference persists through the 6 hr time point. Similar levelsof F4/80+ cells were present at T0 and 1 hr after wounding. Therewere several detectable vimentin+GL3+ cells present at the leadingedge at T0 and 1 hr post-injury for both RB and DB wounds. Bycontrast, after 6 hrs, GL3+ cells were absent after DB, and only rarelyseen after RB wounding.Conclusions: The rapid migration of leader cells to the wound edgefollowing mock cataract surgery in the lens is also seen in the corneaafter manual debridement wounding. The numbers of leader cellsrecruited at T0 and 6 hrs after wounding varied depending on the typeof wound. All of the leader cells are positive for CD45, with themajority being positive also for F4/80. A subpopulation of the leadercells are vimentin+GL3+ but these cells are more transient than theF4/80+ cells since they are largely absent by 6 hrs after wounding.Unlike the mouse cornea, the majority of the leader cells in thechicken lens wound model express vimentin. While the functions ofthese rapidly appearing immune cells is not clear, our studies indicatethat wound healing in chicken and mouse and in diverse tissuesincluding the lens and cornea share a similar property: rapidrecruitment of immune derived cells to the leading edge.Commercial Relationships: Gauri Tadvalkar, None; SonaliGhosh, None; Ahdeah Pajoohesh-Ganji, None; Janice L. Walker,None; A S. Menko, None; Mary Ann Stepp, NoneSupport: EYO08512 (MAS), EY021784 (ASM,MAS)Program Number: 550 Poster Board Number: B0187Presentation Time: 10:30 AM - 12:15 PMBasement Membrane Removal Reduces Inflammation in theMouse Cornea and Enhances Wound HealingSonali Pal-Ghosh 1 , Ahdeah Pajoohesh-Ganji 1 , Gauri Tadvalkar 1 ,Daniel R. Saban 2 , Mary Ann Stepp 1 . 1 Anatomy & RegenerativeBiology, George Washington University Medical Center,Washington, DC; 2 Ophthalmology and Immunology, DukeUniversity School of Medicine, Durham, NC.Purpose: An in vivo mouse model has been developed thatreproducibly induces recurrent epithelial erosions in wild-type micespontaneously within two weeks after a single 1.5 mm cornealdebridement wound using a dulled blade. Using a rotating burr ratherthan a dulled blade to create a 1.5 mm wound allows mice to healwithout developing erosions. These experiments were conducted todetermine the cause of the difference in healing outcomes after dulledblade compared to rotating burr wounds.Methods: A 1.5 mm area of corneal epithelium was removed fromthe corneal surface of adult C57BL/6 mice using either a dulled bladeor rotating burr. Corneas were allowed to heal in vivo for 0, 3, 6 hr or5 days. After sacrifice, tissues were used for immunofluorescence,QPCR, flow cytometry, and/or chemokine protein array studies. Flowexperiments were repeated three times. QPCR was performed oncontrol (n=8) and 6 hr dulled blade or rotating burr wounded (n=8)corneas. For QPCR studies, RNA isolated from epithelial cells onlyand whole dissected corneas were compared. All experiments wererepeated at least twice.Results: Data show that 1) erosions form after dulled blade but notafter rotating burr wounds, 2) the basement membrane (BMZ) is leftbehind after dulled blade wounds but is removed by the rotating burr,3) there are more monocytes (CD45+/ Ly6C hi/ Ly6G+/ CD11b hi/F480 low/ CD11c+) and γδ T cells (CD45+/GL3 hi) recruited 6 hrafter dulled blade wounds, and 4) chemokine array studies show that,at the time of wounding and 5 days later, chemokines are elevatedafter dulled blade compared to rotating burr wounds.Conclusions: Despite the fact that rotating burr wounds remove theBMZ, damage more of the subbasal nerves, and induce morecytokine mRNAs within corneal epithelial cells, they heal withoutdeveloping erosions. Improved healing after trauma is associatedwith basement membrane removal and the reduced influx ofmonocytes and γδT cells 6 hr after wounding. Data from chemokinearrays indicate more CCL8 and CCL12 and complement proteins (5aand Factor D) retained on the corneal stroma after dulled bladewounds that may account for the early increase in monocyte and γδTcells seen after dulled blade wounds.Commercial Relationships: Sonali Pal-Ghosh, None; AhdeahPajoohesh-Ganji, None; Gauri Tadvalkar, None; Daniel R.Saban, Schepens Eye Res Inst, Mass Eye and Ear, (P), ElevenBiotherapuetics (R); Mary Ann Stepp, NoneSupport: EYO08512 (MAS), EY021784 (ASM,MAS)Program Number: 551 Poster Board Number: B0188Presentation Time: 10:30 AM - 12:15 PMSchirmer Testing and Corneal Surface Findings in ChronicGraft-Versus-Host DiseaseKathleen M. Williamson 1 , Evan Allan 3 , Michelle P. Lin 2 , Michael C.Wu 1 . 1 Ophthalmology, University of Washington, Seattle, WA;2 Ophthalmology, Cleveland Clinic, Cleveland, OH; 3 Ophthalmology,University of Oklahoma, Oklahoma City, OK.Purpose: To report whether low Schirmer testing scores arepredictive of ocular surface findings in patients with chronic graftversus-hostdisease (cGVHD) diagnosed after hematopoietic celltransplantation.Methods: Retrospective study of 429 consecutive patients withcGVHD referred for ophthalmic examination between 1990 and2006. Office visit records were reviewed for extraction of dataincluding visual acuity, Schirmer testing scores, slit-lampbiomicroscopic findings (including corneal staining with fluorescein),and fundoscopic abnormalities. In this analysis, we considered therelationship between Schirmer testing scores and corneal staining.Two arbitrary cutoff values were used to define ‘low’ Schirmerscores, 5mm or less and 10mm or less. Analysis was then performedto determine if there was an association between ‘low’ scores andcorneal surface staining.Results: In our population of patients with cGVHD, Schirmer testingscores of 5 mm or less are associated with presence of cornealfluorescein staining (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 552 Poster Board Number: B0189Presentation Time: 10:30 AM - 12:15 PMBoric Acid-based Multipurpose Contact Lens Care Solutions(MPSs) Cytotoxicity Effect on Human Corneal Epithelial CellsKissaou T. Tchedre 1 , Masaki Imayasu 2 , Yuichi Hori 3 , H D.Cavanagh 4 . 1 R&D and Innovation Center, Menicon LTD, Le Mans,France; 2 R&D center, Menicon, Co. ltd, Kasugai, Japan;3 Ophthalmology, Toho University Sakura Medical Center, Sakura,Japan; 4 Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas,TX.Purpose: The purpose of this study is to determine whethercommercially available new multipurpose contact lens care solutions(MPSs) have any cytotoxicity effect on human corneal epithelial(HCE-T) cells. MPSs effect on membrane-associated mucins (MUC1and MUC16) expressions in the Rat cornea was also assessed.Membrane-associated mucins are one of the major components of theocular surface that play a vital role in the maintenance of the ocularsurface integrityMethods: HCE-T cells were treated with different concentrations ofMPS-F (1ppm PHMB, no boric acid), MPS-G (1.3ppm PHMB, 1ppmPQ-1, boric acid), MPS-H (1.6 ppm, Alexidine, 3ppm PQ-1, boricacid), MPS-I (1ppm PHMB, boric acid), and MPS-J (5ppm ALDOX,10ppm PQ-1, boric acid): 100% treatment for 30 minutes and 10%treatment for 24 hours. Cell death was measured by using aviability/cytotoxicity assay kit. Winstar Rats were also subjected toMPSs (1 drop in the right eye every 10 minutes for 1 hour). The leftEye was used as control (1 drop of PBS every 10 min for 1 hour).Cornea lysates were subsequently prepared and used for Western blotanalysis for MUC1 and MUC16.Results: The viability/cytotoxicity assay result showed that MPSscontaining boric acid induce cell death in HCE-T cells. The westernblot result showed that boric acid-based MPS down-regulatemembrane-associated mucins in the cornea while MPSs without boricacid had no effect on membrane-associated mucins.Conclusions: The concentration of boric acid used in commerciallyavailable multipurpose contact lens care solutions should be chosencarefully to avoid MPS-related ocular surface damage. Ocular surfacedamage simultaneously promotes microbial pathogens and potentiallyincreases clinical rates of infection.Commercial Relationships: Kissaou T. Tchedre, Menicon, Co. Ltd(E); Masaki Imayasu, Menicon Co., Ltd. (E); Yuichi Hori, None; HD. Cavanagh, Menicon Ltd (C)Program Number: 553 Poster Board Number: B0190Presentation Time: 10:30 AM - 12:15 PMThe Effect of Amniotic Membrane De-epithelialization Methodon its Biological Properties and Ability to Promote LimbalEpithelial Cell CultureGary Hin-Fai Yam 1 , Ting Zhang 1 , Andri K Riau 1 , Roger W.Beuerman 1 , Donald T. Tan 2 , Jodhbir S. Mehta 1, 2 . 1 Singapore EyeResearch Institute, Singapore, Singapore; 2 Singapore National EyeCenter, Singapore, Singapore.Purpose: To characterize the de-epithelialized human amnioticmembrane (HAM) and compare cell attachment and proliferationefficiencies.Methods: HAM was de-epithelialized by 20% ethanol (AHAM), 1.2U/ml Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM) and 5M urea (UHAM), respectively, followed bygentle scrapping with a #15 blade. Surface topology, extracellularmatrix (ECM) and growth factor content were characterized andcompared to intact HAM by electron microscopies (EM), atomicforce microscopy (AFM), immunohistochemistry and westernblotting. Primary human limbal epithelial cells (LEC) attachment andproliferation efficiencies were assayed. Statistical significance wascalculated by SPSS and Fisher’s Least Significant Difference test.Results: EHAM, THAM and UHAM had intact basal lamina andsmooth basement membrane surface shown under transmission andscanning EM and AFM. Cell remnants stayed on AHAM. Disruptedbasement membrane and stroma was found in DHAM.Immunostaining intensity quantification and hierarchical clusteringrevealed that ECM composition of EHAM and UHAM resembled tointact HAM. In contrast, DHAM and THAM had drastic loss of ECMand growth factor content. LEC attachment efficiency at 24 hourspost-seeding was the highest in EHAM (51% as on conventionalculture surface), followed by UHAM and AHAM. However, cellproliferation indices at day 10 of culture were similar among differentHAM substrates, suggesting repair of ECM and basement membraneby growing epithelial cells.Conclusions: Urea denudation preserved the basement membraneintegrity, ECM and growth factor composition, and had higher cellattachment and proliferation efficiencies. With its short processingtime, urea treatment offers a novel alternative for HAM deepithelialization.Commercial Relationships: Gary Hin-Fai Yam, None; TingZhang, None; Andri K Riau, None; Roger W. Beuerman, Allergan(F), SERI (P), Santen (R); Donald T. Tan, Network MedicalProducts (P), Carl Zeiss Meditec (F), Alcon Labs (F), Bausch &Lomb (F), Allergan (F), Santen (F); Jodhbir S. Mehta, NoneSupport: Singapore National Medical Research Council grants IBGand R485/34/2006Program Number: 554 Poster Board Number: B0191Presentation Time: 10:30 AM - 12:15 PMKnockdown of MUC16 Alters Tight Junctions of CornealEpithelial Cells Resulting in Decreased TransepithelialResistanceIlene K. Gipson, Sandra J. Spurr-Michaud, Ann S. Tisdale. HarvardMed Sch/Dept Ophthal, Schepens Eye Research Inst/MEEI, Boston,MA.Purpose: MUC16 is a major membrane associated mucin of thehuman corneal epithelium. We have shown that MUC16 is a barrierto rose bengal dye penetrance and pathogen adherence. We alsodemonstrated that MUC16’s cytoplasmic tail associates with actinthrough the ezrin, radixin, moesin family of actin linking proteins.Because MUC16 may be associated with actin filaments that insertinto the apical web of actin filaments that terminate in tight junctions,the purpose of this study was to determine the role of MUC16 in tightjunction formation and function as measured by transepithelialresistance (TER).Methods: An immortalized human corneal limbal epithelial cell line(HCLE) (Gipson et al, 2003) and the HCLE cell line stablytransfected with siRNA to MUC16 in which MUC16 levels werereduced by 70% and a vector control line were used. Cells werecultured for optimal mucin expression and stratification. Assayscomparing the cell lines included measurement of apical cell size,localization and mRNA levels of Z01, a tight junction protein, andTER using an Evum2 Epithelial Voltohmmeter.Results: HCLE cells knocked down (KD) for MUC16 showed asignificant 2-3 fold (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacells correlated with a significant decrease in TER (39 +3 ohms)compared to controls (127 + 5 ohms, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaClinical Trial: NCT01712022Program Number: 557 Poster Board Number: B0194Presentation Time: 10:30 AM - 12:15 PMBone Marrow derived Mesenchymal Stem Cells forReconstructing the Limbal NicheAli R. Djalilian 1 , Behrad Y. Milani 1 , Hossein M. Sagha 1 , PeimanHematti 2 . 1 Ophthalmology, Univ of Illinois at Chicago, Chicago, IL;2 Medicine, University of Wisconsin, Madison, WI.Purpose: In patients with limbal stem cell deficiency, in addition tothe loss of the limbal epithelial stem cells, the limbal niche isinvariably damaged/destroyed. Therefore, strategies are needed toreconstruct the limbal niche. Mesenchymal type cells have beenshown to be an important part of the niche. In this study, weexamined whether mesenchymal stem cells (MSC) from the bonemarrow (BM) can function as niche support cells.Methods: Human bone marrow MSCs were cultured in serumcontaining media. Primary human corneal epithelial cells were grownin KSFM before being passaged onto plates with BM-MSC. Colonyforming efficiency of epithelial cells co-cultured with BM-MSC wasexamined. Conditioned media from BM-MSC and limbal fibroblastswere compared in terms of their ability to promote epithelial growth.BM-MSC were also grown in decellularized human corneas toexamine their growth and differentiation in 3D.Results: As reported by others, BM-MSC supported colonyformation of corneal epithelial cells, which appeared to be dependenton the passage number of the BM-MSC. Pre-treatment of BM-MSCwith mitomycin C reduced their ability to support corneal epithelialcell growth as evident by the effect of conditioned media (non-MMCvs MMC treated) on corneal epithelial cells. BM-MSC proliferated in3D culture conditions in decellularized human cornea adopting akeratocyte-like phenotype.Conclusions: These results indicate that BM-MSC can support theclonal growth of corneal epithelial cells. More studies are needed todetermine the optimal growth conditions for their use as limbal nichecells.Commercial Relationships: Ali R. Djalilian, None; Behrad Y.Milani, None; Hossein M. Sagha, None; Peiman Hematti, NoneSupport: National Eye Institute of the National Institutes of Healthwith Career Development Grant K08EY017561-A1 (ARD) and CoreGrant EY01792, the Cless Family Foundation, and a CareerDevelopmental Award (ARD) and unrestricted departmental grantfrom Research to Prevent Blindness.Program Number: 558 Poster Board Number: B0195Presentation Time: 10:30 AM - 12:15 PMDecreased Phosphorylation at the Serine-2 Residue of the RNAPolymerase II in Corneal Epithelial Stem CellsSatoshi Kawasaki 1 , Katsuhiko Shinomiya 1 , Keita Aoi 2, 1 , ShigeruKinoshita 1 . 1 Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto,Japan; 2 Faculty of Life and Medical Sciences, Doshisha University,Kyotanabe, Japan.Purpose: Continuous regeneration of stem cells is known to beessential for maintaining the life-long homeostasis of any types ofcells in the body of animals. In the stem cells of ocular surfaceepithelia, several positive and negative markers have been reported.Recent studies have shown that stem cells are adapted to hypoxicconditions with slow cell cycling, low transcriptional activity, lowermitochondrial respiration, and higher glycolysis for the generation ofadenosine-5'-triphosphate (ATP). The purpose of this present studywas to report a new marker for corneal epithelial stem cells based onlow transcriptional activity.Methods: Corneal tissue samples obtained from humans, rabbits, andmice were cryo-embedded, sliced into thin sections, fixed, andimmunostained with antibodies against the phosphorylated serine-2of the RNA polymerase II (RNAP II), a marker of activatedtranscription, as well as several positive or negative markers ofcorneal epithelial stem cells such as N-cadherin, p63, and keratin 12.Results: In all of the tissue samples, a decreased level ofphosphorylation at the serine-2 residue of the RNAP II was found atthe basal cells of the limbal epithelium. Immunostaining withantibodies against the above-described established markers forcorneal epithelial stem cells proved that those cells were, in fact,corneal epithelial stem cells.Conclusions: The findings of this study show that phosphorylatedserine-2 of the RNAP II is a new negative marker for cornealepithelial stem cells. Corneal epithelial stem cells may have a generalfeature of stem cells in terms of low transcriptional activity.Commercial Relationships: Satoshi Kawasaki, None; KatsuhikoShinomiya, None; Keita Aoi, None; Shigeru Kinoshita, SenjuPharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Support: 24592675Program Number: 559 Poster Board Number: B0196Presentation Time: 10:30 AM - 12:15 PMEffect of Specular Focal Distance on Endothelial Cell CountingAccuracyJackie Hai, Vivian Xue. HAI Laboratories, Inc., Lexington, MA.Purpose: To quantify the relationship between specular focaldistance and image distortion, in order to determine a predictivemodel of cell counting error based on amount of deviance from trueendothelial cell shape and size.Methods: High resolution specular image of a standard calibrationlens was captured in focus to establish baseline pachymetry of 0μm.Subsequent images captured 30μm, 40μm, 50μm, 60μm, 70μm,80μm, 90μm and 100μm from the baseline were analyzed for changesin area caused by optical distortion. Analysis was carried out in asingle-blind trial with 10 sample counts performed for each specularimage to determine mean areas.Results: Given a true area of 10,000μm 2 for the baseline distance of0μm, a negative correlation was found to exist between focal distance(F) and mean area (A). For F=30μm, A=9666μm 2 ; F=40μm,A=9444μm 2 ; F=50μm, A=9180μm 2 ; F=60μm, A=9055μm 2 ; F=70μm,A=8869μm 2 ; F=80μm, A=8767μm 2; F=90μm, A=8694μm 2 ;F=100μm, A=8467μm 2 . Using simple linear regression, the celldensity error (E) can be extrapolated based on deviance from truearea. For F=10μm, E=1.26%; F=20μm, E=2.90%; F=30μm,E=4.58%; F=40μm, E=6.53%; F=50μm, E=8.12%; F=60μm,E=9.99%; F=70μm, E=11.92%; F=80μm, E=13.91%; F=90μm,E=15.98%; F=100μm, E=18.13%.Conclusions: Cell counting error can be considered negligible at 0-29μm, non-negligible at 30-59μm and problematic at 60-100μm offocal deviance.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSpecular images of donor cornea captured 100 micrometers out offocus, 60 micrometers out of focus, 30 micrometers out of focus, and0 micrometers out of focus (i.e. completely in focus).Table: Projected cell density counting error based on deviance fromtrue focus and true area.Commercial Relationships: Jackie Hai, HAI Laboratories, Inc. (E);Vivian Xue, HAI Laboratories, Inc. (E)Program Number: 560 Poster Board Number: B0197Presentation Time: 10:30 AM - 12:15 PMEvaluation of Terrien Marginal Degeneration Using AnteriorSegment Optical Coherence TomographyXiaoqiang Liu, Fang Wang. shanghai tenth people's hospital,Shanghai, China.Purpose: To investigate the morphologic characteristics of Terrienmarginal degeneration (TMD) using anterior segment opticalcoherence tomography (AS-OCT).Methods: Slitlamp microscopy, AS-OCT and corneal topographywere performed in ten eyes of six patients (four male, two female)with TMD.Results: Clinically, all patients demonstrated typical TMD.Unilateral isolated TMD was presented in two female patients. AS-OCT clearly demonstrated the extension of the ectatic part, thickness,and structure of the cornea in all cases. A characteristic thinned zoneof peripheral cornea was showed in all affected eyes. Hydrop in theectatic part of cornea was seen in one patient. The topographic mapsshowed irregular astigmatism in four eyes.Conclusions: The AS-OCT detects the microstructure of corneaclearly and can be used to monitor the progression of TMD.Commercial Relationships: Xiaoqiang Liu, None; Fang Wang,NoneProgram Number: 561 Poster Board Number: B0198Presentation Time: 10:30 AM - 12:15 PMHuman graft cornea imaging with full-field optical coherencetomographyWajdene Ghouali 1 , Kate Grieve 2 , Otman Sandali 1 , Elena Basli 1 ,Julien Bullet 1 , Laurent Laroche 1 , Vincent Borderie 1 . 1 CHNO desQuinze-Vingts, Paris, France; 2 Institut Langevin, Paris, France.Purpose: There is currently no efficient method for the study ofepithelium and stroma of human graft corneas. The aim of our studyis to evaluate the performance of a full-field optical coherencetomograhy system in the evaluation of human graft corneas.Methods: Our study was carried out using a full-field OCT systemfrom LLTech ®, developed for non-invasive imaging of tissuestructures in depth. Images were acquired on human donor corneas(in normal and oedematous conditions) and surgical specimens ofpathological corneas (Fuchs dystrophy, keratoconus, stromalkeratitis)Results: The Full-field OCT device from LLTech® enables threedimensional images to be obtained with ultrahigh resolution (1micron in all directions) comparable to traditional histologicalsections. This allows a precise visualisation of the cells and thedifferent structures (epithelium, Bowman’s membrane, stroma,Descemet’s membrane and endothelium) in normal corneas (figure1), but also in pathological corneas (even in the presence of anoedema), with specific lesions in each condition.Conclusions: Optical microscopy, with a detailed view of the cornealendothelium and a cell density determination, remains the « goldstandard » in the study of human cornea grafts. However, full-fieldOCT, thanks to a more complete anatomical stydy of the cornea,could be helpful in the evaluation and the selection of human corneagrafts whose quality plays a major role in the outlook of the cornealtransplant©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawere seen in the posterior stroma. In LCD, hyperreflective linear andbranching structures with poorly demarcated margins were noted inthe stroma. In PPMD, a variety of vesicular and linear abnormalitiescould be identified at the level of the endothelium. In FD, Descemet'smembrane was thickened with round hyporeflective structures(guttae) at level of the endothelium,with fibrosis and activatedkeratocytes seen in the stroma.Conclusions: AS-OCT and IVCM are non-invasive, high-resolutionimaging modalities useful in the visualization of the cornealmicrostructural changes related to corneal dystrophies. These newimaging technologies are rapidly becoming widely used instrumentsfor both research and patient care, and may be useful in elucidatingthe pathogenesis and natural course of corneal dystrophies.The figure 1 shows full-field OCT images of the cornea from the 3Ddata stack. The upper four images are en face optical slicesdescending through the sample depth: EP epithelium; BM Bowman'smembrane; ST stroma; DM+E Descemet's membrane andendothelium. The lower image is a depth slice through the corneashowing the different layers as labelled above. Scale bars show100μm.Commercial Relationships: Wajdene Ghouali, None; KateGrieve, None; Otman Sandali, None; Elena Basli, None; JulienBullet, None; Laurent Laroche, None; Vincent Borderie, NonePatient with EBMD evaluated with in vivo confocal microscopy(IVCM) and anterior segment OCT (AS-OCT).Program Number: 562 Poster Board Number: B0199Presentation Time: 10:30 AM - 12:15 PMHigh-Resolution Anterior Segment Optical CoherenceTomography and in vivo Confocal Microscopy in the Evaluationof Corneal DystrophiesDennis E. Cortes 1, 2 , Jennifer Li 1 , Michael Chen 1 , Raju Poddar 1 ,Robert J. Zawadzki 1 , John S. Werner 1 , Mark J. Mannis 1 . 1 Departmentof Ophthalmology & Vision Science, UC Davis Eye Center,Sacramento, CA; 2 Department of Ophthalmology, PontificiaUniversidad Católica de Chile, Santiago, Chile.Purpose: To describe the findings observed with high-resolutionanterior segment optical coherence tomography (AS-OCT) and invivo confocal microscopy (IVCM) in patients with cornealdystrophies.Methods: Five patients with common corneal dystrophies seen inclinical practice were evaluated with a high-resolution spectraldomainAS-OCT (Heidelberg Engineering, Germany) and IVCMusing the Heidelberg Retina Tomograph 3 Rostock Cornea Module(Heidelberg Engineering, Germany). A high-speed swept-sourceOCT (SS-OCT) prototype was also used to reconstruct the threedimensionalstructures of the cornea.Results: Patients included in this study had the following cornealdystrophies: epithelial basement membrane dystrophy (EBMD),granular cornea dystrophy (GCD), lattice cornea dystrophy (LCD),posterior polymorphous corneal dystrophy (PPMD) and Fuchs’endothelial dystrophy (FD). AS-OCT and IVCM provided preciseinformation about the pathology in the different layers of the corneain all patients. There was a significant correlation between the twoimaging modalities.In EBMD, we identified an abnormal epithelial basement membraneprotruding into the corneal epithelium and clusters of epithelial cellsin the tear film. In GCD, the majority of opacities were visualized inthe anterior two-thirds of the corneal stroma although rare opacitiesPatient with granular dystrophy evaluated with in vivo confocalmicroscopy (IVCM) and anterior segment OCT (AS-OCT)Commercial Relationships: Dennis E. Cortes, None; Jennifer Li,None; Michael Chen, None; Raju Poddar, None; Robert J.Zawadzki, None; John S. Werner, None; Mark J. Mannis, NoneSupport: NEI 014743, RPBProgram Number: 563 Poster Board Number: B0200Presentation Time: 10:30 AM - 12:15 PMAnalysis Of Early Postoperative Morphologic Features Of ClearCorneal Incisions Created With The Femtosecond CataractLaser Using Anterior Segment Optical Coherence Tomography:Comparison Of Intended Versus Achieved Wound ParametersSurendra Basti, Dilraj S. Grewal. Ophthalmology, NorthwesternUniversity Feinberg School of Medicine, Chicago, IL.Purpose: To analyze the morphology of clear corneal incisions (CCI)performed using a Femtosecond Cataract Laser (Catalys, Optimedica,Santa Clara, CA) in patients undergoing femtosecond laser-assistedcataract surgery using spectral domain anterior segment opticalcoherence tomography (AS-OCT).Methods: Morphology of clear corneal incisions created with theFemtosecond cataract laser was studied with AS-OCT. An intendedtriplanar incision was programmed into the Catalys femtosecond lasersoftware. The first plane was at 90 degree to the corneal surface andextended to 40% corneal depth, the second plane was an angledintrastromal plane and the third plane was at 45 degrees to theposterior corneal surface and reached the second plane at 70% depth.The intended incision width was 2.85 mm and length was 1.8 mm.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaAS-OCT was performed on the first postoperative day using theCirrus HD-OCT (Carl Zeiss Meditec, Dublin, CA). The clear cornealincision length, incision depth, angles of the tri-planar cornealincision, and wound gaping were measured using software calipers.The variability in wound length, depth and angle were calculated andcompared to the programmed software settings. Five architecturalfeatures were used to describe the clear corneal incisions: gaping ofthe wound at the epithelial side, gaping of the wound at theendothelial side, within wound gape, misalignment of the roof andfloor of the incision at the endothelial side and local Descemet’smembrane (DM) detachment. This study is ongoing and will include30 eyes.Results: On analysis of first post-operative day AS-OCT images ofthe initial 3 eyes undergoing femtosecond cataract extraction, 2/3eyes had endothelial side wound gape, 1/3 had epithelial wound gape,2/3 eyes had a within wound gape and 3/3 eyes had a focal DMdetachment. All three eyes had visible 3-plane profile on AS-OCT.The clear corneal incision length was within 100 microns of theintended length. The incision depth was within 8 percent of theintended depth.Conclusions: Our initial results suggest that clear corneal incisionsusing the femtosecond cataract laser were close to the intended sizeand depth. A significant proportion of eyes had marginal and stromalwound gape and focal DM detachment.Commercial Relationships: Surendra Basti, None; Dilraj S.Grewal, NoneSupport: An Unrestricted Grant From Research To PreventBlindness, Inc., New York, New YorkA: slit lamp photo of EBMD B: in-vivo confocal microscopy (IVCM)revealing linear hyperreflective structures corresponding to abnormalepithelial basement membrane extending into the corneal epitheliumC: anterior segment OCT (AS-OCT) revealing hyperreflectivematerial in the posterior epithelium and anterior stromaProgram Number: 564 Poster Board Number: B0201Presentation Time: 10:30 AM - 12:15 PMEpithelial Basement Membrane Dystrophy: A Study with in vivoConfocal Microscopy and High-Resolution Anterior SegmentOptical Coherence TomographyPeter Wu 1 , Dennis E. Cortes 1, 2 , Jennifer Li 1 , Michael Chen 1 , Mark J.Mannis 1 . 1 Ophthalmology & Vision Sciences, University ofCalifornia Davis Eye Center, Sacramento, CA; 2 Ophthalmology,Universidad Catolica de Chile, Santiago, Chile.Purpose: To report the pathological changes of patients withepithelial basement membrane dystrophy (EBMD), the most commonhereditary anterior corneal dystrophy, using in vivo confocalmicroscopy (IVCM) and high-resolution anterior segment opticalcoherence tomography (AS-OCT).Methods: Five patients with EBMD seen in clinical practice wereevaluated with a high-resolution spectral-domain AS-OCT(Heidelberg Engineering, Germany) and IVCM using the HeidelbergRetina Tomograph 3 Rostock Cornea Module (HeidelbergEngineering, Germany).Results: AS-OCT revealed hyperreflective material in the posteriorepithelium and anterior stroma. These findings were correlated withIVCM findings that showed multiple linear and curvilinearhyperreflective structures, corresponding to abnormal epithelialbasement membrane extending into the corneal epithelium.Additionally, IVCM revealed hyperreflective deposits in the anteriorstroma with signs of activation of anterior keratocytes, intraepithelialmicrocysts, and clusters of epithelial cells in the tear film.Conclusions: The acquisition of high-resolution imaging of thecornea with IVCM and AS-OCT provides new insights into themicrostructural characteristics of EBMD and may be usefulmodalities in elucidating the pathogenesis and natural course of thiscorneal dystrophy.A: slit lamp photo of EBMD B: oblique section of in-vivo confocalmicroscopy revealing an area of fibrosis at the sub-epithelial level C:anterior segment OCT (AS-OCT) demonstrating hyperreflectivematerial in the posterior epithelium and anterior stromaCommercial Relationships: Peter Wu, None; Dennis E. Cortes,None; Jennifer Li, None; Michael Chen, None; Mark J. Mannis,NoneProgram Number: 566 Poster Board Number: B0203Presentation Time: 10:30 AM - 12:15 PMStudy Of Ocular Surface impairment in children presentingongoing dry eye with MGD with Meibomian Gland AnalysisDominique Bremond-Gignac 1, 2 , Solange Milazzo 1 . 1 Ophthalmology,St Victor Center, University Hospital of Amiens, Picardie JulesVerne University, Amiens, France; 2 CNRS IRIS UMR8194, Paris VUniversity, Paris, France.Purpose: To evaluate in a retrospective study, the ocular surfaceimpairment in children presenting ongoing MGD with an analysis ofmeibomian glands. Tear film quality is conditionned by meibomiangland production of the lipid layer. Anomaly of this production inchildren can lead to ocular surface impairment and an evaluation ofMeibomian Glands is a key point of the exploration of productionfunction.Methods: Our retrospective study included 12 children (22 eyesevaluated) with a mean age 11yo, range 4 to 17yo, of two groups thatpresented at ocular consultation. Group a, 6 with ongoing MGD,group b 6 control children who had been tested with no ocular surfaceimpairment. All children underwent a Meibomian Gland Analysiswith meibography images with infrared illumination acquired withCobra system. An evaluation with Phoenix software calculated areaof loss of glands. The density of inferior eyelids Meibomian glandshad also been evaluated.Results: In group a, all children presented clinical signs of MGD dueto ocular blepharitis or dry eye. In group b no ocular signs of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaconjunctiva or ocular surface anomalies were found. In group a arealoss of meibomian gland was evaluated from 41% to 59%. Density ofglands was low. In group b area loss of meibomian gland wasevaluated from 10% to 42%. In younger children (4 to 7 yo) densityof glands was tight and lenght size shorter.Conclusions: In children, few data are known about MeibomianGland development and function. Our study is suitable in childrenwith dry eye for the evaluation of Meibomian Glands and can be auseful tool. A larger study has to be performed for a betterunderstanding of dry eye ocular surface impairment in children.Commercial Relationships: Dominique Bremond-Gignac, None;Solange Milazzo, NoneProgram Number: 567 Poster Board Number: B0204Presentation Time: 10:30 AM - 12:15 PMGender differences in ocular biometric datayoona jang 1 , Hiroshi Uozato 1, 2 , Takushi Kawamorita 1, 2 , YukoShibata 3 . 1 Department of Visual Science, Kitasato UniversityGraduate School, Sagamihara, Japan; 2 Ophthoptics and VisualSciences, Kitasato University School of Allied Health Science,Sagamihara, Japan; 3 Ophthalmology and Visual Sciences, KitasatoUniversity Graduate School, Sagamihara, Japan.Purpose: To compare the differences in ocular biometric data withregard to genderMethods: Seventy-two eyes from 38 healthy subjects (mean age =21.4 ± 2.7 years, range: 18 to 31 years) including 19 males (mean age= 22.5 ± 2.6 years) and 19 females (mean age = 20.3 ± 2.5 years)were recruited. Anterior chamber depth (ACD) and axial length (AL)were measured with IOL Master TM (Carl Zeiss Meditec, Jena,Germany). ACD was defined as the length between the center ofanterior surface of cornea and the center of anterior lens surface.Angle lambda (kappa), distance from central pupillary eccentricity tothinnest corneal thickness, corneal curvature(CR), cornealpower(CP), central corneal thickness(CCT) were measured withpachymetry with a dual scheimpflug imaging system Galilei TM(Ziemer Ophthalmic Systems, Port, Switzerland ). The statisticalsignificance of the gender differences between measurements wasevaluated by Student’s t-test.Results: The mean ACD was 3.77 ± 0.32 mm for males and 3.65 ±0.25 mm for females. Males had significantly deeper ACD thanfemales (p = 0.049). The mean angle lambda, AL and CCT were 3.15± 1.60 °, 25.31 ± 1.59 mm, 552.01 ± 30.05 μm for males and 3.86 ±1.58 °, 24.79 ± 1.15 mm, 551.81 ± 20.23 μm for females,respectively. Males had significantly larger angle lambda thanfemales (p = 0.028). There was no statistically significant differencein the AL and distance from central pupillary eccentricity to thinnestcorneal thickness in gender comparison, but there are tendencyassociated with differences gender (p = 0.056, p = 0.054). There wasno statistically significant difference in mean CR, CP and CCTbetween males and females.Conclusions: Both ACD and angle lambda had statisticallysignificant difference with gender. And, AL and distance from centralpupillary eccentricity to thinnest corneal thickness were tendencyassociated with gender difference. However, gender did not show anystatistically significant effect on cornea as CR, CP and CCT.Therefore, consider to gender would be an important factor to ocularbiometric examinations for refractive and cataract surgeries.Commercial Relationships: yoona jang, None; Hiroshi Uozato,None; Takushi Kawamorita, None; Yuko Shibata, None138 Dry Eye and Lacrimal Gland ISunday, May 05, 2013 1:00 PM-2:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 900-954/B0205-B0259Organizing Section: CorneaProgram Number: 900 Poster Board Number: B0205Presentation Time: 1:00 PM - 2:45 PMDoes Altering the Volume of an Artificial Tear Applied to theEye Influence Vision?William H. Ridder, Monisha Paripatyadar, Lisa Wahl. Basic &Visual Science, Southern California College of Optometry, Fullerton,CA.Purpose: Artificial tears (AT) are the most common treatment fordry eye. Several studies have shown that immediately after an AT isplaced on the eye vision is degraded for a short time. The purpose ofthis investigation was to determine if there is a correlation betweenthe volume of AT placed on the eye and the degradation in vision.Methods: Six normal, adult subjects took part in this project. Snellenacuity was better than 20/25 in the eye tested. The subjects had 1hour of training with the contrast sensitivity (CS) measurementtechnique before data collection. CS to a 14 cpd sine wave grating(16 ms stimulus presented at 1 sec after the blink) was continuallytracked (using a 2 AFC technique) before and after (minimum of 25minutes) an AT (Refresh Optive, Allergan, Inc.) was applied. Thevolumes of the ATs applied, randomized across visits, were 25, 45, or65 µl (Pos D Pipette, Rainin Inst.). The data for different dropvolumes were collected on separate days. The magnitude of the lossin normalized contrast sensitivity was compared across dropvolumes.Results: The average loss in normalized CS (mean ± SD) for thedrop volumes were: 0.46 ± 0.182, 0.50 ± 0.171, and 0.60 ± 0.293 for25, 45, and 65 µl, respectively. A paired t-test found a significantdifference in the CS loss between the 25 and 65 µl drop sizes (p =0.05, t = 2.46). No other comparisons were significant (p > 0.05).Conclusions: The CS decreased as the volume of the applied ATincreased. This may be the result of a greater disruption in the tearlayer as the AT drop volume increases.Commercial Relationships: William H. Ridder, None; MonishaParipatyadar, None; Lisa Wahl, NoneProgram Number: 901 Poster Board Number: B0206Presentation Time: 1:00 PM - 2:45 PMEffects of Omega-3 Fatty Acid and Hyaluronic acid Mixture EyeDrops on the Ocular Surface in Desiccating Stress inducedMurine Dry EyeZhengri Li 1 , Jung-Han Choi 2 , Hyo Seok Lee 3 , Ji-Suk Choi 4 , Han JinOh 5 , Kyung Chul Yoon 6 . 1 Chonnam National Uinversity and hospital,Gwangju, Republic of Korea; 2 Chonnam National Uinversity andhospital, Gwangju, Republic of Korea; 3 Chonnam NationalUinversity and hospital, Gwangju, Republic of Korea; 4 ChonnamNational Uinversity and hospital, Gwangju, Republic of Korea;5 Chonnam National Uinversity and hospital, Gwangju, Republic ofKorea; 6 Chonnam National Uinversity and hospital, Gwangju,Republic of Korea.Purpose: To investigate the effects of topical application of omega-3Fatty Acid (FA) and hyaluronic acid (HA) mixtures on inflammationand oxidative stress markers in a mouse model of experimental dryeye (EDE).Methods: Eye drops consisting of 0.02%, or 0.2% omega-3 FA and0.1% HA mixtures, or 0.1% HA were applied in desiccating stressinduced murine dry eye. Corneal irregularity and corneal fluoresceinstaining scores were measured at 5 and 10 days after treatment.Levels of interleukin (IL)-1β, IL-17, and interferon gamma-inducedprotein (IP)-10 were measured in the conjunctiva using a multiplex©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaimmunobead assay at 10 days. The concentrations of HEL and 4-HNE were measured with enzyme-linked immunosorbent assays inconjunctiva tissue.Results: Although all parameters were lower in the 0.02% FAmixture and HA-treated groups compared with the EDE group, theeffect was not statistically significant. However, mice treated with0.2% omega-3 FA mixture showed a significant improvement incorneal irregularity and corneal fluorescein staining compared withEDE and HA-treated mice. A significant decrease in the levels of IL-1β, IL-17, and IP-10 were observed in the 0.2% FA mixture-treatedgroup, compared with the other groups. In the 0.2% FA mixturetreatedgroups, the concentrations of HEL and 4-HNE were alsolower than the other groups.Conclusions: Topical omega-3 FA mixture eye drops can improvecorneal irregularity and corneal epithelial barrier disruption, anddecrease inflammatory cytokines and oxidative stress markers on theocular surface. These results suggest that mixture of 0.2% Omega-3FA and HA can be useful for treatment of dry eye treatment.Commercial Relationships: Zhengri Li, None; Jung-Han Choi,None; Hyo Seok Lee, None; Ji-Suk Choi, None; Han Jin Oh,None; Kyung Chul Yoon, NoneProgram Number: 902 Poster Board Number: B0207Presentation Time: 1:00 PM - 2:45 PMEvaluation of a Topical Cis-urocanic acid in a Murine CAEModel of Dry Eye DiseaseAndy Whitlock 1 , Laura Belen 1 , Jennifer Brackett 1 , Burkhard Blank 2 .1 Ora, Inc., Andover, MA; 2 Laurantis Pharma, Turku, Finland.Purpose: The goal of this study was to test the potential of Cisurocanicacid (Cis-UCA) in a topical formulation as a treatment fordry eye.Methods: The study employed a murine model of dry eye diseasethat combines of environmental and pharmacologic induction ofpathologic signs of disease. Cis-UCA was provided by the sponsor asa clear solution at concentrations of 0.0, 0.5, 1.0, and 2.5%.Restasis®, dry eye treatment (scopolamine and chamber only), andnaive (room air) controls were included in the study as well. Micewere dosed topically three times a day in both eyes for a total of 22days. Animals received 3 days of prophylactic topical treatment (noscopolamine or chamber) and 14 days of dry eye treatment (withscopolamine and chamber).Fluorescein staining evaluations wereperformed using Ora’s novel Micron III imaging system at varioustime points throughout the study. Corneal images were evaluatedusing Ora’s proprietary clinical scale as well as a custom imageanalysis algorithm.Results: 1.0%Cis-UCA was most effective in reducing cornealfluorescein staining as compared to vehicle and the dry eye treatmentcontrols. Throughout the study duration, the average staining of allthe eyes in the group was maintained at about 8 units, whereas thevehicle and dry eye treatment groups steadily increased to amaximum average staining of approximately 12 units by Day 17. Theaverage corneal staining for mice in the 1.0%Cis-UCA treatmentgroup was statistically lower than both the vehicle and the dry eyetreatment group (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaLong-Term Corneal Sensitivity after PRK determined by NoncontactGas EsthesiometryJuana Gallar 1 , Jukka A. Moilanen 2 , M Carmen Acosta 1 , Juha M.Holopainen 2 , Carlos Belmonte 1 , Timo M. Tervo 2 , Waldir Neira 2, 1 .1 Instituto de Neurociencias, Universidad Miguel Hernandez-CSIC,San Juan de Alicante, Spain; 2 Ophthalmology, University ofHelsinki, Helsinki, Finland.Purpose: To evaluate the long-term (5-12 years) evolution of cornealsensitivity to mechanical and chemical stimuli after photorefractivekeratectomy (PRK).Methods: Nineteen patients (12 male, 7 female; mean age 35.3;range: 21-55 years at the time of surgery) who underwent PRK forlow to moderate myopia (mean spherical equivalent -3.0 D; range -2.5 to -8.0 D), and 14 control individuals (5 male, 9 female; meanage: 40.3, range: 28-54 years) were examined at 1 week, and 0.5, 2, 5and at least 10 years (range 10.5-12 years) after surgery. Cornealsensitivity to mechanical (air flow between 0 and 260 ml/min) andchemical (0 to 80% CO 2 in the air, at subthreshold flow) stimulationwas tested with non-contact gas esthesiometry (original Belmonteesthesiometer: 1 week-2 years; modified Belmonte-CRCERTesthesiometer: > 5years) using 0-10 Visual Analogue Scales (VAS)and performing intensity-response curves. The sensation thresholdvalue and the slope of the intensity-response curve were calculated.Results: Corneal sensitivity to mechanical stimulation was presentbut significantly reduced 1 week after surgery when compared withcontrols (mechanical thresholds: 203±49 vs. 105±14 ml/min; slopes:0.0044 vs. 0.0219 VAS unit/ml of flow, respectively). The decreaseof corneal mechanical sensitivity was more pronounced 0.5 yearsafter PRK and remained below control values 2 and 5 yearspostoperatively (thresholds: 267±18; 260±10, and 249±13 ml/min,respectively). Corneal sensitivity to chemical stimulation was onlyslightly modified after PRK (slightly enhanced at 1 week but slightlyreduced at 0.5, 2 and 5 years). Sensitivity to both mechanical andchemical stimulation presented normal values 10 years after PRK(mechanical threshold: 91±12 ml/min, slope: 0.0218 VAS unit/ml offlow).Conclusions: Immediately hyperesthesia to chemical sensitivity isattributable to sensitization. The long-lasting reduction of cornealsensitivity to mechanical stimulation after PRK indicates that thetransduction capacity to mechanical forces of injured nerve fibers ispermanently impaired, contributing to the altered sensationsexperienced after PRK. New regenerating nociceptive corneal nervesslowly invade the denervated area, being responsible of the recoveryof mechanical and chemical sensitivity observed at 10 years afterPRK.Commercial Relationships: Juana Gallar, None; Jukka A.Moilanen, Adventus Technology Inc. (C); M Carmen Acosta,None; Juha M. Holopainen, None; Carlos Belmonte, None; TimoM. Tervo, None; Waldir Neira, NoneSupport: SAF2011-22500, and in part by BFU2008-04425,CSD2007-00023 and IPT-2011-1110-900000 (Ministerio deEconomía y Competitividad, Spain, and FEDER, EU), Evald andHilda Nissi Foundation and Finnish Eye Foundation (Finland)Program Number: 905 Poster Board Number: B0210Presentation Time: 1:00 PM - 2:45 PMThe Ocular Surface Sensory Response to Tear Film InstabilityWith and Without a Contact LensCarolyn G. Begley 1 , Jun Zhang 1 , Ping Situ 1 , Ziwei Wu 1 , Trefford L.Simpson 2 . 1 School of Optometry, Indiana University, Bloomington,IN; 2 Optometry and Vision Science, University of Waterloo,Waterloo, ON, Canada.Purpose: Tear film instability (TFI) is a core mechanism of dry eye(DEWS, 2007), but its sensory impact on the ocular surface remainspoorly understood. In this study, we test the hypothesis that TFIdirectly stimulates ocular surface sensory neurons and that wearing asoft contact lens (CL) partially blocks this stimulation.Methods: Ten adapted CL wearers participated in 2 study visits.While not wearing CLs, subjects were seated behind a slit lampbiomicroscope and were asked to keep one eye open as long aspossible (maximum blink interval=MBI) while fluorescein TFI wasmonitored and subjects simultaneously indicated the level ofdiscomfort using a “discomfort knob” (DK) potentiometer (0-10scale). The MBI procedure was repeated 10 times. Discomfort andburning sensations during and after each trial were rated using 0-10visual analogue scales (VAS). The entire procedure was repeated at asecond study visit while wearing CLs for a fixed 30 sec MBI withretroillumination used to view TFI over CLs.Results: The discomfort intensity and slope measured by the DK wassignificantly lower (paired t-test, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: In male SD rats, spontaneous blinking and eye wipebehaviors elicited by hypertonic saline (2.5 and 5.0 M) was examined2 and 8-10 weeks following unilateral removal of the infra- andexorbital lacrimal glands and in age matched controls. Furthermore,the effect of topical lidocaine (4%) on spontaneous blinking and theeffect of morphine (5 mg/kg, s.c.) on spontaneous blinking and eyewipe responses was determined in dry eye and control animals.Results: Lacrimal gland removal resulted in an increase inspontaneous blinking in the ipsilateral eye that remained consistentlyelevated over an 8-week period. Lidocaine application reducedspontaneous blinking down to control levels, whereas glycerol andartificial tear solutions reduced spontaneous blinking. The time spenteye wiping was also enhanced in response to hypertonic saline (2.5and 5.0 M) at both the 2 and 8 wk time-points. Morphine attenuatedboth spontaneous blinking and the response to hypertonic saline indry eye animals.Conclusions: These results indicate that dry eye produced bylacrimal gland removal produces hyperalgesia in the rat, as quantifiedin the eye wipe assay. In addition, spontaneous blinks in dry eyeanimals and their reduction by morphine and topical anesthesia,indicate the presence of persistent irritation elicited by the activationof corneal nociceptors.Commercial Relationships: Ian D. Meng, None; Stephen T.Barton, None; Andrew L. Twaite, NoneSupport: NIH Grant R01EY0212301 - in keeping with less hydrophobicity and no interaction of lacritinwith SDC4. Alone, SDC1 peptide1, scrambled SDC1 peptide 1 andSDC4 peptide assumed a random coil structure.Conclusions: Ligation of lacritin’s C-terminal amphiphathic alphahelix with GAGAL in SDC1 is enhanced by mutual hydrophobicitythat improves alpha helicity.Program Number: 907 Poster Board Number: B0212Presentation Time: 1:00 PM - 2:45 PMAlpha Helicity of Lacritin’s C-Terminal Syndecan-1 BindingDomain is Enhanced by Interaction with the Mutual BindingRegion in Syndecan-1Jeff Romano 1 , Jacob Irwin 2 , Inchan Kwon 2 , Gordon W. Laurie 1 . 1 CellBiology, University of Virginia, Charlottesville, VA; 2 ChemicalEngineering, University of Virginia, Charlottesville, VA.Purpose: Lacritin is a small tear protein (~18 kDa) with mitogenic,cytoprotective and prosecretory activities. When added topically torabbit eyes, lacritin promotes basal tearing. Lacritin targets thecorneal epithelial cell surface protein syndecan-1 (SDC1), a widelyexpressed heparan sulfate proteoglycan via a heparanase dependentmechanism that facilitates interaction between lacritin amino acids100-109 in its C-terminal alpha-helix and the SDC1 core proteinsequence GAGAL nestled in SDC1’s heparan sulfate rich N-terminus. Since GAGAL is hydrophobic, we wondered whether itmight influence development of lacritin’s C-terminal alpha helixnecessary for binding. Here we performed comparative circulardichroism analyses of lacritin peptide 1898 (aa 95-119) alone, and inthe presence of SDC1 peptide 1 (aa 20 - 30), scrambled SDC1peptide 1, or the equivalent region in SDC4 (SDC4 peptide).Methods: Synthetic peptides were generated by Genescript to apurity of >95%, dissolved in 10mM dodecylphosphocholine andsubjected to CD analysis in the 190-250 nm range with 0.1 nm pitch,50 nm/min scan speed, 8 second response time and a 2 nmbandwidth. A 1 mm pathlength quartz cuvette with cylindricalgeometery (Hellma) was used, stored in 2% Hellmanex prior tousage, washed vigorously 3 times with ddH2O, subsequently washedwith 100% ethanol, and then dried completely with pure N2 beforeand between samples. The spectra displayed represent the average ofat least 5 sample runs. Between sample runs, the dd H2O blank wasmonitored to ensure protein buildup in the cuvette was not occurring.Results: Lacritin amino acids 95-119 (81 µM) assumed an alphahelical structure in dodecylphosphocholine. This was enhanced bySDC1 peptide 1 (57 µM), but not by scrambled SDC1 peptide 1.SDC4 peptide was partially effective, but less so than SDC1 peptideCommercial Relationships: Jeff Romano, 7,932,227 (P), 7,648,964(P), 7,459,440 (P), 7,320,870 (P); Jacob Irwin, None; InchanKwon, None; Gordon W. Laurie, UVa Patent Foundation (F)Support: NH Grant EY018222Program Number: 908 Poster Board Number: B0213Presentation Time: 1:00 PM - 2:45 PMLacritin accelerated autophagy promotes clearance of aggregatedproteins and is dependent on stressKeith B. Zimmerman 1 , Milton F. Tyler 1 , Ningning Wang 1 , RonaldRaab 2 , Robert L. McKown 2 , Gordon W. Laurie 1 . 1 Cell Biology,University of Virginia, Charlottesville, VA; 2 Integrated Science &Technology, James Madison University, Harrisonburg, VA.Purpose: Lacritin is a natural tear protein that promotes the survivalof human corneal epithelial (HCE) cells stressed with inflammatorycytokines INFG and TNF, both of which are elevated in dry eye.Survival is dependent on lacritin stimulated autophagic flux, which islikely to remove stress-aggregated and damaged proteins. We havenow addressed this assumption by transduction of mCFP taggedHuntingtin mutant constructs Htt103Q and Htt25Q in HCE cellsstably transduced with autophagy marker LC3B double tagged withmCherry and EGFP. Htt103Q forms toxic aggregates whereasHtt25Q remains soluble, thus only Htt103Q cells are significantlystressed.Methods: Retroviral pBabe-puro-mCherry-EGFP-LC3B and pBabe-Hygro-Htt103Q-mCFP or pBabe-Hygro-Htt25Q-mCFP wereexpanded in HEK293T cells, and used to co-transduce human cornealepithelial (HCE) cells (Riken). Cells grown on glass cover slips werethen treated either with 10 nM lacritin or inactive C-25 lacritin for 10,30, 60 min. After washing, cells were fixed, rinsed, mounted andexamined in a Zeiss LMS 700 confocal microscope. Colocalization ofmCherry with mCFP was analyzed using Fiji.Results: Lacritin, but not C-25, stimulated the autophagic captureand transfer of toxic Htt103Q aggregates into autolysosomes. Captureand transfer peaked at 30 min. Transfer was monitored over time as©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEGFP (mCherry-EGFP-LC3B) was quenched by autolysosomalacidity , unlike pH insensitive mCherry. No lacritin stimulatedautophagy was evident in Htt25Q cells in keeping with the absence ofstress.Conclusions: Lacritin signaling rids cells of toxic aggregatedproteins that develop during stress by rapidly and transientlystimulating autophagy. Autolysosomal digestion products then feedinto and restore oxidative phosphorylation.Commercial Relationships: Keith B. Zimmerman, None; MiltonF. Tyler, None; Ningning Wang, None; Ronald Raab, None;Robert L. McKown, EyeRx Research, Inc. (I); Gordon W. Laurie,UVa Patent Foundation (F)Support: NH Grant EY018222Program Number: 909 Poster Board Number: B0214Presentation Time: 1:00 PM - 2:45 PMLacritin as the Primary Epithelial Survival Activity in BasalHuman Tears Reveals Novel Features About AutophagicSurvival SignalingNingning Wang 1 , Ronald Raab 2 , Robert L. McKown 2 , Cindy M.Hutnik 3 , Gordon W. Laurie 1 . 1 Cell Biology, University of Virginia,Charlottesville, VA; 2 2Integrated Science and Technology, jamesmadison university, harrisonburg, VA; 3 Depts. of Ophthalmology &Pathology, Ivey Eye Institute, London, ON, Canada.Purpose: As the fluid thought to support the survival of the avascularcorneal epithelium, tears are rich in proteins with growth and survivalcapabilities - including lacritin, a tear prosecretory mitogen thatsupports the survival of stressed corneal epithelial cells in culture,and promotes basal tearing in rabbits and monkeys. Here we askwhether tears are indeed prosurvival, if so does tear lacritincontribute, and what is the FOXO3 survival mechanism?Methods: Human corneal epithelial (HCE; Riken) cells weresensitized with INFG, and then treated with TNF in the in thepresence of normal or dry eye tears, normal tears mock or lacritinimmunodepleted, or with dry eye tears supplemented withrecombinant lacritin or negative control C-25. FOXO3 translocationserved as a readout for cell stress (nuclear) or survival (cytoplasmic).FOXO3 immunoprecipitates were used to screen for FOXO3 bindingpartners.Results: Normal and mock depleted tears, but not lacritin depletedtears promoted the survival of INFG/TNF stressed HCE cells. Tearsfrom dry eye patients supplemented lacritin promoted cell survival,but not dry eye tears alone nor C-25 supplemented dry eye tears. Wediscovered that autophagy mediator ATG101 binds FOXO3 instressed cells treated with lacritin, and that shRNA depletion ofATG101 or FOXO3 abrogates lacritin stimulated autophagy andsurvival.Conclusions: Lacritin appears to be the primary survival factor inbasal human tears. Surprisingly it is not compensated by other tearfactors when depleted. Lack of dry eye tear survival activity can berescued with recombinant lacritin, suggesting efficacy in treatinghuman dry eye. Lacritin rapidly stimulates FOXO3/ATG101 ligation.ATG101 is known to bind and stabilize the ATG13/ULK1 complexresponsible for initiating autophagy - thus explaining how lacritin sorapidly stimulates autophagy.Commercial Relationships: Ningning Wang, None; Ronald Raab,None; Robert L. McKown, EyeRx Research, Inc. (I); Cindy M.Hutnik, None; Gordon W. Laurie, UVa Patent Foundation (F)Support: EY018222, EY013143 (to GWL).Neutralization of Ocular Surface TNF-α Reduces Ocular Surfaceand Lacrimal Gland Inflammation Induced by In Vivo Dry EyeYongwoo Ji, Yu Jeong Byun, Jin Sun Kim, Eunae Jeong, Hyung KeunLee. Institute of Vision Research, Department of Ophthalmology,Yonsei University College of Medicine, Seoul, Republic of Korea.Purpose: The purposes of this study are to investigate theeffectiveness of tumor necrosis factor (TNF)-α blocker for treatmentof dry eye (DE)-induced inflammation and determine a moreeffective method to suppress lacrimal gland inflammation. Efficacyof topical vs. systemic administration of TNF-α blockers wasdetermined using a murine DE model.Methods: The TNF-α blocker HL036 was developed by modificationof the TNF receptor I. Protein purity, binding affinity, and clearanceof TNF-α was compared with etanercept. Using DE inducedC57BL/6 mice, corneal erosion, tear secretion, and goblet cell countswere measured after subcutaneous or topical treatment withetanercept or HL036. Inflammatory cytokines in cornea and lacrimalglands were determined by quantitative reverse transcriptionpolymerase chain reaction and enzyme-linked immune absorbentassay.Results: HL036 showed TNF-α binding affinity comparable toetanercept, as measured by surface plasmon resonance. HL036concentration was significantly higher in cornea and anterior segmentthan etanercept, and effectively eliminated TNF-α on ocular surfaces.Etanercept was preferentially concentrated in the posterior segment.Corneal erosion, goblet cell counts, and tear secretion were improvedonly with topically applied etanercept and HL036. Ocular surfaceinterferon-γ (IFN-γ), interleukin-6 (IL-6), IL-21, and IL-22 weresignificantly decreased by topical HL036. DE induced lacrimal glandIFN-γ and IL-6 expression was effectively suppressed by topicaletanercept, glucocorticoid, and HL036.Conclusions: Topical TNF-α blockers effectively suppressedlacrimal gland and corneal inflammation by suppressing IFN-γ, IL-21, and IL-6. Differences in TNF-α affinity, clearance, and localconcentration of blockers may account for the anti-inflammatoryeffects in different ocular regions.Program Number: 910 Poster Board Number: B0215Presentation Time: 1:00 PM - 2:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Yongwoo Ji, None; Yu Jeong Byun,None; Jin Sun Kim, None; Eunae Jeong, None; Hyung Keun Lee,NoneSupport: This work was supported by the Industrial StrategicTechnology Development Program, (Project No.: 10040233,Molecular engineering and drug development of anti-TNF antibodyfragment for local inflammatory disease) funded by the Ministry ofKnowledge Economy (MKE, Korea) and Basic Science ResearchProgram (Grant no: 2012-0001384) and Advanced Science ResearchProgram (grant no.: 2012R1A2A2A02009081) through the NationalResearch Foundation of Korea (NRF), funded by the Ministry ofEducation, Science, and Technology, Korea.Program Number: 911 Poster Board Number: B0216Presentation Time: 1:00 PM - 2:45 PMProtection of barrier function and anti-inflammatory effects ofrebamipide in human corneal epithelial cellsHiroshi Tanaka 1, 2 , Ken Fukuda 3 , Waka Ishida 3 , Yosuke Harada 3 ,Tamaki Sumi 3 , Atsuki Fukushima 3 . 1 Machida Hospital, Kochi, Japan;2 Opthalmology, Kyoto Prefectural Univ of Med, Kyoto, Japan;3 Opthalmology, Kochi Medical School, Nankoku, Japan.Purpose: Dry eye causes the tear hyperosmolality which stimulatesan inflammatory cascade and disrupts corneal barrier function.Rebamipide which has mucin secretagogue activity is used for thetreatment of dry eye in Japan. This study examined the effects ofrebamipide on barrier function and cytokine expression in a humancorneal epithelial (HCE) cell line.Methods: Barrier function of HCE cells was evaluated bymeasurement of transepithelial electrical resistance. The subcellularlocalization of the tight-junction protein zona occludens (ZO)-1 wasexamined by immunofluorescence analysis. The release of cytokineswas determined with enzyme-linked immunosorbent assays, and theintracellular abundance of cytokine mRNAs was quantitated byreverse transcription and real-time polymerase chain reactionanalysis. Degradation of the NF-κB inhibitor IκBα was detected byimmunoblot analysis.Results: Rebamipide increased barrier function of HCE cells in aconcentration-dependent manner as well as blocked both the loss ofbarrier function and the disappearance of ZO-1 from the cell surfaceinduced by tumor necrosis factor (TNF)-α or IL-1β. Rebamipide alsosuppressed cytokine-induced expression of interleukin-6 andinterleukin-8 at the mRNA and protein levels as well as inhibited theTNF-α-induced degradation of IκBα.Conclusions: Together with its mucin secretagogue activity,rebamipide is effective for the treatment of dry eye thorough themechanism by up-regulation of barrier function and antiinflammatoryeffects.Commercial Relationships: Hiroshi Tanaka, None; Ken Fukuda,None; Waka Ishida, None; Yosuke Harada, None; Tamaki Sumi,None; Atsuki Fukushima, Otsuka Pharmatheutical Co., Ltd (F)Program Number: 912 Poster Board Number: B0217Presentation Time: 1:00 PM - 2:45 PMExpression and secreting mechanism of mucins induced by DA-6034 in cultured human conjunctival epithelial cellsTae-im Kim, Hun Lee, Ji Won Jung, Eung Kweon Kim. Visionresearch institute, Department of Ophthalmology, Yonsei UniversityCollege of Medicine, Seoul, Republic of Korea.Purpose: The purpose of this study was to investigate the mechanismof ocular mucin secretion mediated by DA-6034 in cultured humanconjunctival epithelial cells.Methods: After application of 100 μM DA-6034, in cultured humanconjunctival epithelial cells, expressions of MUC1, MUC4,MUC5AC, MUC16, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptorswere measured by reverse transcription-polymerase chain reaction(RT-PCR), real-time PCR, and Western blot analysis. The effects of100 μM DA-6034 on expression of mucin-secreting cells weremeasured by the counting of Periodic Acid-Schiff (PAS) positivecells. The effects of DA-6034 on Cl- currents were measured byperforated patch clamp. The changes of Intracellular Ca2+concentrations ([Ca2+]i) mediated by 100 μM DA-6034 weremeasured by loading cultured human conjunctival epithelial cellswith fluorescent calcium indicator Fluo-4/AM and 0.05% pluronic F-127. The level of intracellular Ca2+ was monitored by fluorescencemonitoring cameraResults: RT-PCR and real-time PCR showed that DA-6034 increasedMUC1, MUC4, MUC5AC, MUC16, P2Y2, P2Y4, and P2Y6receptor gene expression. Western blot analysis showed that DA-6034 increased MUC5AC and MUC16 protein expression. DA-6034induced the expression of mucin-secreting cells as demonstrated byPAS staining. DA-6034 stimulated not only active Cl- transport butalso [Ca2+]i increases in cultured human conjunctival epithelial cells.[Ca2+]i increases were demonstrated by emitted fluorescence.Conclusions: We concluded that DA-6034 stimulated mucinproduction and secretion accompanied with the enhanced expressionof P2Y2, P2Y4 and P2Y6 receptor in cultured human conjunctivalepithelial cells. In addition, DA-6034 stimulated active Cl- transportand [Ca2+]i increases.Commercial Relationships: Tae-im Kim, None; Hun Lee, None; JiWon Jung, None; Eung Kweon Kim, NoneProgram Number: 913 Poster Board Number: B0218Presentation Time: 1:00 PM - 2:45 PMRegulators of MUC5AC gene expression in conjunctival epitheliaof dry eye patientsRosa M. Corrales, Stephen C. Pflugfelder. Ophthalmology, BaylorCollege of Medicine, Houston, TX.Purpose: T helper (Th) cytokines and transcription factors have beenfound to modulate goblet cells differentiation and morphology inmice. We investigated expression of T helper 1 (Th1) and Th2cytokine receptors and transcription factors in the conjunctivalepithelia of a dry eye population and compared them with healthysubjects.Methods: Conjunctival impression cytology (CIC) samples wereobtained from 16 dry eye patients (tear deficient with a Schirmer-1score < 5mm wetting in 5 minutes) and 13 healthy subjects. RNAwas extracted, cDNA synthesized and real-time PCR performedusing Taqman probes to evaluate expression of the following genes:MUC5AC, keratin 7 (K7), forkhead box protein A2 (FOXA2), SAMpointed domain-containing Ets transcription factor (SPDEF),Krueppel-like factor 4 (KLF4), IFN-γR, IL-13-Ra1 and IL13Ra2.The housekeeping gene hypoxanthine phosphoribosyltransferase1(HPRT-1) was amplified to normalize levels of expression. Notemplate controls and RNA were studied for evidence ofcontamination. Results were analyzed using the comparative Ctmethod using values from healthy subjects as calibrator. The studentt-test for independent samples was used to compare means betweengroups.Results: FOXA2 expression was below the level of detection. K7and MUC5AC transcripts were decreased in dry eye samples.Expression of the IL-13 signaling receptor (IL-13Rα1) and SPDEFwere unchanged, while expression of the IL-13 decoy receptor, IL-13Rα2, as well as IFN-γR and KLF4 were significantly increased inthe dry eye conjunctival epithelium.Conclusions: These results indicate that dry eye disrupts signalingpathways in the conjunctival epithelium that are involved in goblet©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaells differentiation, resulting in decreased signaling by the Th2cytokine Il-13 and increased signaling by the Th1 cytokine IFN-γ.Expression of the goblet cell transcription factor KLK4 increased.Commercial Relationships: Rosa M. Corrales, None; Stephen C.Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb(C), Baylor College of Medicine (P)Support: NIH Grant EY11915 (SCP), Research to Prevent Blindess,Oshman Foundation, William Stamps Farish Fund, HamillFoundationProgram Number: 914 Poster Board Number: B0219Presentation Time: 1:00 PM - 2:45 PMDecellularization of porcine lacrimal gland tissue fordevelopment of a lacrimal gland scaffoldKristina Spaniol 1 , Alexander Kunze 1 , Marco Metzger 2 , GerdGeerling 1 , Stefan Schrader 1 . 1 ophthalmology, university hospitalDüsseldorf, Düsseldorf, Germany; 2 tissue engineering andregenerative medicine, university hospital Würzburg, Würzburg,Germany.Purpose: In cases of severe dry eye due to lacrimal glandinsufficiency engineering of a lacrimal gland tissue construct in vitromight be a promising future treatment approach. Aim of this studywas to evaluate structure and main basement membrane componentsof native porcine lacrimal gland tissue before and after adecellularization process in order to develop an acellular scaffold forlacrimal gland regeneration.Methods: Lacrimal glands were extracted from six domestic pigsafter euthanasia. Six biological replicates were used for this study.Each gland was cut into four pieces, two of them were left native andthe other two were decellularized using sodium desoxychelate inultra-pure water over night. Tissue pieces were embedded in paraffinas well as OCT and the morphology of native and decellularizedtissue was examined histologically by hematoxylin and eosin (H&E)staining. Additionally, expression of basement membrane markers,such as laminin, collagen IV and fibronectin was evaluated byimmunostaining.Results: Histology showed an intact connective tissue matrix afterthe decellularization process. Immunohistochemistry revealed theexpression of major basement membrane components such ascollagen IV and laminin in native lacrimal gland tissue and thesecomponents were still detectable after the decellularization of thelacrimal gland tissue. Efficacy of the decellularization process wasdemonstrated by complete absence of nuclei in the lacrimal glandtissue, as assessed by DAPI-staining.Conclusions: Decellularization of lacrimal gland tissue generates anintact acellular scaffold with preserved acinar structures, containingmajor basement membrane components such as collagen IV andlaminin. An intact basement membrane is a prerequisite for essentialcellular processes like adhesion, migration as well as proliferationand therefore decellularized lacrimal gland tissue might be apromising scaffold for lacrimal gland regeneration in vitro.Commercial Relationships: Kristina Spaniol, None; AlexanderKunze, None; Marco Metzger, None; Gerd Geerling, Alcon (C),Allergan (C), Thea Pharma (C), Novagali (C), Bausch & Lomb (C),Tearlab Inc. (C); Stefan Schrader, NoneProgram Number: 915 Poster Board Number: B0220Presentation Time: 1:00 PM - 2:45 PMFunctional rabbit acinar cells cultured on decellularized lacrimalgland scaffoldHui Lin 1 , Guoying Sun 1 , Hong he 1 , Jennifer Elisseeff 1 , Samuel C.Yiu 1, 2 . 1 Wilmer Eye Institute, Johns Hopkins University, Baltimore,MD; 2 Ophthalmology, 2. King Khaled Eye Specialist Hospital,Riyadh, Saudi Arabia.Purpose: Aqueous tear-deficient dry eye is a multifactorial chronicdisorder in which the lacrimal glands fail to produce enough tears tomaintain a healthy eye surface. The treatment primarily involvespalliation using ocular surface lubricant. Construction of abioengineered lacrimal gland having functional secretory epithelialcells is a potentially promising option for providing long-term reliefto severe dye eye patients. Previously, we have successfullydecellularized the rabbit lacrimal gland to create a novel scaffold for3 dimensional culture of acinar cell. Current study investigates thesecretory function of cells seeded and cultured in decellularizedlacrimal gland tissue.Methods: NZW rabbit lacrimal glands purchased from Pel Freezewere treated with 1% sodium dodecyl sulfate or 1% Triton X-100 for36 to 40 hours for decellularization. The rabbit lacrimal gland acinarcells were isolated by enzyme mixtures of collagenase, hyaluronidaseand DNase. Subsequently, the isolated cells were layered over 5%Ficoll and centrifuged at 50g for purification. Prior to seeding, thedecellularized lacrimal gland scaffold was treated with Matrigel,fibronectin or collagen I in different groups, and with Peter’scomplete medium (PCM) in control group. The lacrimal constructswere cultured in PCM for up to 2 weeks. The live/dead assay for cellviability was performed on day 2, 7 and 14. The beta-hexosaminidasesecretion assay for assessing acinar cell function was performed onday 2 and 7. RNA was also extracted to evaluate the gene expressionof beta-hexosaminidase.Results: Majority of the lacrimal acinar cells in the decellularizedscaffold survived for at least 7 days in all groups, however, cellviability decreased at day 14. Use of extracellular matrix to treat thescaffold during the seeding process helped improve cell viability.Beta-hexosaminidase activity increased in the supernatant mediumfollowing stimulation with 100uM carbachol on day 2 and 7.Carbachol stimulation was also found to up-regulate mRNA level ofbeta-hexosaminidase in the reseeded cells.Conclusions: Isolated acinar cells successfully cultured indecellularized lacrimal gland tissue and maintained the cell viabilityand secretory function for at least 1 week. Further investigation isrequired to optimize the decellularization and seeding process toimprove the duration of cell viability and function.Commercial Relationships: Hui Lin, None; Guoying Sun, None;Hong he, None; Jennifer Elisseeff, Collagen Vitrigel (P); Samuel C.Yiu, NoneSupport: Partially supported by an unrestricted grant from Researchto Prevent Blindness, New York, NY to the Wilmer Eye InstituteProgram Number: 916 Poster Board Number: B0221Presentation Time: 1:00 PM - 2:45 PMENaC in the rabbit lacrimal gland and its changes duringSjögren’s syndrome and pregnancyChuanqing Ding, Michael Lu, Jianyan Huang. Cell &Neurobiology/Doheny Eye Institute, University of SouthernCalifornia, Los Angeles, CA.Purpose: The epithelial sodium channel (ENaC), which is comprisedof α, β, and γ subunits, plays a critical role in the control of Na+balance and has been implicated in the development and progressionof exocrine gland pathology. The aim of the present study was toinvestigate the expression of ENaC in the rabbit lacrimal gland (LG)and its potential changes in rabbits with induced autoimmunedacryoadenitis (IAD), a rabbit model of Sjögren’s syndrome, and interm-pregnant rabbits, a physiological condition that has been shownto exhibit altered LG secretion and ocular surface symptoms.Methods: Total mRNA of α, β, and γ subunits was extracted from©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawhole LG, acinar cells, and epithelial cells from various ductsegments by laser capture microdissection (LCM) for real time RT-PCR. LG lysates were processed for Western blot and cryosections ofLG were used for immunofluorescence. Goat polyclonal primaryantibodies of α, β, and γ were purchased from Santa CruzBiotechnology (Santa Cruz, CA).Results: mRNA for both α and γ was expressed in whole LG lysateswhile β was undetectable. In rabbits with IAD, the levels of mRNAfor α and γ were 20.9% and 58.9% lower (p0.05).However, we were unable to detect mRNA of any of these threesubunits in LCM samples of epithelial cells due to their extremelylow level. Western blot demonstrated bands for both α (90 kDa) and γ(85 kDa) but β was undetectable. In rabbits with IAD, densitometryanalysis showed that expression of α decreased 22% while γdecreased 26% (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Yutaka Sakurai, None; Masataka Ito,None; Yoko Karasawa, None; Yoshihiko Usui, None; TakaakiHattori, None; Hiroshi Goto, None; Masaru Takeuchi, NoneProgram Number: 919 Poster Board Number: B0224Presentation Time: 1:00 PM - 2:45 PMPotential Regulatory T Cell Mechanisms Of ImmuneSuppression In A Mouse Model Of Dry EyeKatherine S. Held 1 , Chris S. Schaumburg 1 , Jianping Gao 1 , Larry A.Wheeler 1 , Margarita Calonge 2 , Jerry Y. Niederkorn 4 , Stephen C.Pflugfelder 3 , Michael E. Stern 1 . 1 R&D, Allergan, Inc, Irvine, CA;2 IOBA, University of Valladolid, Valladolid, Spain; 3 Baylor Collegeof Medicine, Houston, TX; 4 Ophthalmology, UT SW-Med Center,Dallas, TX.Purpose: To evaluate immunosuppressive properties of inducibleand natural T regulatory (Tregs) subsets following induction ofexperimental dry eye in mice.Methods: Flow cytometry was utilized to characterize Tregs withinthe periphery during experimental dry eye to determine i) thefrequencies of inducible, iTregs, and natural, nTregs, and ii) the levelof expression markers indicating activation and the capacity forsuppression by metabolic disruption and dendritic cell immunemodulation. C57BL/6 wild-type female mice were exposed todesiccating environmental stress (DS; subcutaneous scopolamineinjections, humidity

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaaccording to two schedules: Schedule 1 (S1): to model prevention,dosing started at the beginning of housing in ICES (Day1) and lastedfor 21 days; Schedule 2 (S2): to model treatment, dosing started onDay22 after the housed mice developed dry eye and lasted untilDay35. The efficacy of the L-carnitine treatment compared to that ofthe vehicle (PBS) was tested for ocular surface integrity byfluorescein staining; corneal epithelial apoptosis by TUNEL orCaspase-3 assays; goblet cell numbers by PAS staining; andexpression of inflammatory mediators, TNF-α, IL-17, IL-6 or IL-1βusing real-time PCR, on Days 0, 14, 21 and/or 35.Results: Compared to the vehicle, eyes treated with L-carnitineshowed a significant reduction in corneal fluorescein staining on Day14, 21 and 35 for S1 (p=0.0001, 0.0001, and 0.017, respectively) andDay35 for S2 (p=0.0001); the number of TUNEL-positive apoptoticcorneal epithelial cells on Day21 for S1 (p=0.032) or Day35 for S2(p=0.005); the expression level of TNF-α, IL-17, IL-6, or IL-1β (allp0.05) among populations.In order to study the relation between genotype and phenotype, weperformed both additive and dominant gene model then did linearregression analysis, results of which indicated that EDAR V370Awas not correlated with MG density and diameter(p>0.05,Spearman).Conclusions: Genotyping revealed that difference of derived EDARallele frequency did exist among populations. Based on limited©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaphenotype data provided by LSCM, we didn’t demonstrate the impactof population difference on MG morphological pattern, noassociation was found between genotype and phenotype.Due to small sample size and other reasons, we didn't get outcomesconsistent with those in previous animal experiments. Furtherpopulation-based association studies need to be carried out tounequivocally test our hypothesis and reach a definite conclusion.FIGURE 1. Meibomian glandular acinar units imaged by LSCM.(A)Chinese; (B)Uyghur;(C)European.TABLE 1. Correlation between EDAR V370A and MGsCommercial Relationships: Yujing YANG, None; JianJiang Xu,None; Jiaxu Hong, NoneProgram Number: 924 Poster Board Number: B0229Presentation Time: 1:00 PM - 2:45 PMNotch regulation of PPAR-gamma and development ofmeibomian gland dysfunctionMindy K. Call, Katy Fischesser, Matthew O. Lunn, Winston Kao.Ophthalmology, University of Cincinnati, Cincinnati, OH.Purpose: Meibomian gland dysfunction (MGD) is a chronic, diffuseabnormality of the meibomian glands, which is characterized byterminal duct obstruction and/or qualitative/quantitative changes inglandular secretion resulting in alterations of the tear film, eyeirritation and inflammation. One particular molecule thought to playan important role in lipid secretion is peroxisome proliferatoractivated receptor gamma (PPARλ). Given the importance of PPARλin lipid metabolism, this study aims to elucidate its role in meibomiangland formation/function and the development of MGD. Thehypothesis is that loss of PPARλ will disrupt meibomian glandformation/function resulting in a hyposecretory state. In addition thisstudy will examine the role of Notch signaling in regulating PPARλexpression.Methods: Transgenic animals, utilizing both Le-cre and K14-rtTAdriver mice, were used to ablate PPARλ and Jag1 and to expressdnMAML. Animals were examined for signs of MGD/dry eye viatear solution assessment, HRT II, slit lamp, lissamine green staining,oil red O staining and lyve 1 staining. In addition, eyes were collectedat various time points to assess expression of Notch signalingcomponents. All reported research was conducted in compliance withthe ARVO Statement for the Use of Animals in Ophthalmic andVision Research and approved by the IACUC of the University ofCincinnati.Results: Loss of PPARλ resulted in abnormalities in meibomiangland morphogenesis leading to dry eye-like symptoms. Specificallythere was an increase in the number of cell layers lining each acinus,fewer and more condensed lipid droplets, “swollen” eyelids,increased protein concentration in the tears and increasedlymphangiogenesis. Inhibition of the notch signaling pathway usingdnMAML mice resulted in a significant meibomian gland defect.With a phenotype similar to what was seen in the absence of PPARλ.Loss of Jag1 did not have as profound of an effect on meibomiangland formation or function, which may be explained bycompensation by other Notch ligands.Conclusions: Together these data, suggest that PPARλ is critical forthe proper formation and function of the meibomian glands and alsodemonstrate a mouse model that can be used to study MGD. Notchsignaling also appears to be an important regulator of PPARλ.Commercial Relationships: Mindy K. Call, None; KatyFischesser, None; Matthew O. Lunn, None; Winston Kao, NoneSupport: from NIH/NEI EY013755, Research to Prevent Blindness,Ohio Lions Eye Research FoundationProgram Number: 925 Poster Board Number: B0230Presentation Time: 1:00 PM - 2:45 PMTear Meniscus Dimensions and Location of Marx’s Line inMeibomian Gland DysfunctionJason Feuerman, Stephen C. Pflugfelder. Ophthalmology, BaylorCollege of Medicine, Houston, TX.Purpose: In patients with tear film instability and meibomian glanddysfunction (MGD), Marx’s line (ML) is often displaced anteriorly.Bron, et al, suggest that ML may be formed as a result of a relativelyincreased solute concentration at the peripheral apex of the tearmeniscus, which may thus play a significant role in thepathophysiology of primary MGD. We hypothesized that tearmeniscus height, area, and length of anterior excursion along the lidmargin positively correlate with the extent of anterior migration ofML in patients with MGD.Methods: We performed a retrospective analysis of consecutivepatients with eye irritation symptoms, MGD with tear film instability,no aqueous tear deficiency, and eyelid photographs and OCTs ofsufficient quality for analysis. 32 eyes of 17 patients were included.Photographs of lissamine green-stained lower eyelids were analyzedusing the ImageJ analysis software. The furthest anterior migration ofML was measured in 3 zones: temporal, central, and nasal. For eacheye, the tear meniscus height, area, and length of anterior excursion atthe center of the lower eyelid were measured using Fourier-DomainRTVue-100 optical coherence tomography (OCT). Spearmancorrelation analysis was performed.Results: The mean eye age was 68 ± 10 years. In the temporal zone,there was a statistically significant positive correlation between thefurthest anterior migration of ML and the central tear meniscus height(r = 0.468, p = 0.007), area (r = 0.492, p = 0.004), and anteriorexcursion (r = 0.448, 0.010). In the central zone and the nasal zonethere was no significant correlation between the furthest anteriormigration of ML and any of the OCT measurements.Conclusions: In patients with symptomatic MGD, central tearmeniscus height, area, and anterior excursion positively correlatewith the furthest anterior migration of ML in the temporal zone, butnot in the central or nasal zone. A possible explanation is that agingchanges such as conjunctivochalasis, which tends to be morepronounced temporally, physically impedes lateral tear migrationleading to increased central tear pooling, while also promotinganterior excursion of the tear meniscus temporally by acting as abridge. By this process, the solute gradient mechanism couldcontribute to the initiation of MGD. MGD could also just be initiatedby increased exposure of meibomian gland orifices to tears,regardless of their osmolarity.Commercial Relationships: Jason Feuerman, None; Stephen C.Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb(C), Baylor College of Medicine (P)©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: NIH Grant EY11915 (SCP), Research to PreventBlindness, Oshman Foundation, William Stamps Farish Fund, HamillFoundationProgram Number: 926 Poster Board Number: B0231Presentation Time: 1:00 PM - 2:45 PMCommensal microflora of eyelid margins and meibomian glandfunction in normal humansHua Zhu 1, 2 , Judith L. Flanagan 1, 2 , Nisha S. Yeotikar 1 , Negar BabaeiOmali 1 , Daniel Tilia 1 , Shamil Y. Iskandar 1 , Brien A. Holden 1, 2 , EricB. Papas 1, 2 . 1 Brien Holden Vision Institute, Sydney, NSW, Australia;2 School of Optometry and Vision Science, University of New SouthWales, Sydney, NSW, Australia.Purpose: The etiology of meibomian gland dysfunction remainsunclear. The aim of our study was to assess the association betweenthe commensal microflora of the eyelid margins and the function ofthe meibomian glands in different age groups of normal subjects.Methods: 103 subjects, aged from 25 to 65 years old, with nosystematic conditions, pre-existing ocular irritation, injury orinfection, were included in the study. An ocular swab was taken fromthe lower lid margin of the left eye of each subject. Conventionalcultivation techniques were used for isolation and identification ofmicroorganisms. Meibum quality (MQ) and meibomian glandexpressibility (MGE) of the lower eyelid were graded following theguidelines of TFOS workshop on meibomian gland dysfunction.Meibography was performed on the lower eyelids using an infra-redcamera, and meibomian gland loss factors (MGLF, degrees 0 to 4)were assessed. The association between microorganism counts andmeibomian gland functional grading/s (including MQ, MGE, orMGLF) was assessed.Results: The most commonly identified microorganisms werecommensal skin bacteria including Propionibacterium species (87%),and coagulase negative staphylococci, mainly Staphylococcusepidermidis (80%). A significantly higher number of microorganisms(348 ± 231 cfu per swab) was found on eyelid swabs collected fromsubjects with an MQ grade of 4 (no meibum expressed) and an MGEgrade of 3 (no glands expressible) compared to the MQ grades 0-2 (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Keratoconus (KC) is a corneal disease, which ischaracterized by corneal thinning leading to loss of visual acuitythrough ectasia and irregular astigmatism. Clinically, the symptomsof KC can be highly variable and in part, depend on the stage of theprogression of the disorder as well as associated eye-rubbing, itching,etc. There is currently no effective management for KC, nor anyadequate biomarkers to predict the outcome of clinical severity of thedisease. Furthermore, there is considerable confusion in the fieldregarding the pathophysiology of the disease and key involvement ofinflammation. To that end, this study was designed to address thesequestions.Methods: We correlated the clinical features associated with KC in acohort of 54 patients with tear proteomic profiles. This noninterventionalstudy was approved by the Narayana Nethralaya IRB.Clinical grades and associated symptoms were graded as per theAmsler-Krumeich classification (Grade I: 32 patients, Grade II: 14patients, Grade III: 8 patients). Quantitative Proteomic analysis wascarried out using iTRAQ with nanoLC-MS/MS on 25μg total tearprotein from each patient and pooled healthy control subjects (n = 12)for comparison.Results: We observed that eye-rubbing correlated with increasingclinical grades and morbidity of KC. In total, over 1000 tear proteins(< 1% false discovery rate) were identified from the whole study.Quantitative proteomic results from a group of up-regulated anddown-regulated proteins (ratio for KC/control > 1.5, or < 0.67)revealed a positive correlation between several tear proteins (LCN1,PLA2G2A, SCGB2A1, MSLN and CRYAB) and KC clinical grades(Grade I, II and III). A negative correlation between a group of tearproteins (ALB, IGHG2 & G3, A2M, complement factors C3, C4A,C6 & H, ORM1, KNG1, PRDX1, SERPIN-F1, GSN) and KC clinicalgrades was also observed.Conclusions: These proteins could be used alone or in combinationas biomarkers for predicting the progression or severity ofkeratoconus. These proteins also shed light on the underlyingderegulation of important inflammatory and immunomodulatorypathways that may be key drivers of KC pathophysiology.Commercial Relationships: Arkasubhra Ghosh, None; RohitShetty, None; Lei Zhou, Singapore Eye Research Institute (P);Sharon D'Souza, None; Roger W. Beuerman, Allergan (F), SERI(P), Santen (R)Clinical Trial: J0002GQQProgram Number: 929 Poster Board Number: B0234Presentation Time: 1:00 PM - 2:45 PMSeveral Tear Protein Levels Of Breast Cancer Patients Differfrom Healthy Participants: A Microarray StudyKsenia Keller 1, 2 , Julia Pieter 2 , Daniel Boehm 2 , Norbert Pfeiffer 3 ,Franz H. Grus 1 . 1 Experimental Ophthalmology, University MedicalCenter, Mainz, Germany; 2 Gynecology and Obstetrics, UniversityMedical Center, Mainz, Germany; 3 Ophthalmology, UniversityMedical Center, Mainz, Germany.Purpose: Currently no molecular biomarkers for early detection ofbreast cancer (BC) are available. The explorations of cancerproteome revealed several new findings according significant levelchanges of proteins. Even in the distant body fluids like tears wereported over 20 de- or increased proteins in pooled BC and healthy(CTRL) samples in our previous study. Here we report our additionalstudy regarding individual tear protein profiling using high sensitiveantibody-microarray plattformMethods: Tear fluid was obtained from 38 women, whereas 19 ofthem were diagnosed with primary non-metastasized BC, with thehelp of Schirmer test. Tear proteins were eluted overnight with 0.1%n-dodecyl-beta-D-maltoside. Proteins were precipitated and theirconcentration was determined. 5 µg of labeled proteins from eachparticipant were incubated on microarrays with antibodies againstcomplement proteins, other immune components and high abundanttear proteins. After signal visualization a semi-quantitativecomparison of protein levels was performedResults: We detected several significantly different distributedproteins in BC and CTRL groups. For example, the interleukins IL2and IL1beta were decreased in BC samples (p=0.025 and p=0.017,respectively). Furthermore we used the artificial neuronal networks totest the discrimination ability of the putative biomarkers. Thus thebest combination of biomarkers revealed the sensitivity of 89% andspecificity of 100%. An area under the curve of 0.91 was achievedConclusions: This pilot study provides more findings to our previoustear protein profiling investigations. Obviously also in other bodyfluids besides serum and tumor microenvironment several proteinderegulations occur leading perhaps to changed signal cascades andaberrant reactions. This study also shows the immediate involvementof immune system, whereas its component levels are changed incomparison to CTRL subjects. Further explorations are ongoing forthe verifying the results in a larger study population for establishmentof putative BC biomarkersCommercial Relationships: Ksenia Keller, None; Julia Pieter,None; Daniel Boehm, None; Norbert Pfeiffer, Sensimed AG (F),Sensimed AG (R), MSD (F), MSD (R), Alcon (F), Allergan (F),Novartis (F), Novartis (R), Bayer (F), Heidelberg Engineering (F),Bausch&Lomb (F), Boehringer-Ingelheim (F), Carl Zeiss Meditech(F), Chibret (F), Nidek (F), Pfizer (F), Santen (F), Santen (R),Topcon (F), Ivantis Inc (F), Ivantis Inc (R); Franz H. Grus, NoneSupport: partly founded by MAIFORProgram Number: 930 Poster Board Number: B0235Presentation Time: 1:00 PM - 2:45 PMMethod development for analysis of tear proteins using selectedreaction monitoringSimin Masoudi 1, 2 , Ling Zhong 3 , Mark Raftery 3 , Mark D. Willcox 2 .1 Brien Holden Vision Institute, Sydney, NSW, Australia; 2 School ofOptometry and Vision Science, University of New South Wales,Sydney, NSW, Australia; 3 Bioanalytical Mass Spectrometry Facility,University of New South Wales, Sydney, NSW, Australia.Purpose: Previous studies of dry eye and contact lens (CL) relateddry eye have shown qualitative differences in lactoferrin, lysozyme,lipocalin 1, prolactin-induced protein and proline rich protein 4. Theaim of this study was to develop a method for simultaneous detectionand accurate quantification of these five proteins in human tearsusing selected reaction monitoring (SRM) mass spectrometry.Methods: Basal tears were collected from normal subjects (male=1,female=6, CL=3, No CL=4).Tears were processed as describedbefore with modifications. SRM transitions from two peptidesrepresenting each protein were selected using Skyline software thenanalyzed using nano-flow liquid chromatography mass spectrometry.Calibration standards were generated using unlabelled synthetizedpeptides diluted to a range of 1 to 1000fmol/µL in the presence of afixed amount of stable-isotopically labelled peptides (50fmol/µL).The ratios of the peak areas of the unlabelled/labelled peptides wereplotted against the concentrations of the corresponding unlabelledpeptides. The concentration of endogenous proteins (µg/µL) in tearsamples was deduced using the peak area ratio of the endogenouspeptides to labelled peptides.Results: The limits of quantification for the selected peptides werebelow 50pg/μl. The recovery of peptides from spiked digested tearswas ≥56% and the coefficient of variation values were ≤16% whichshow good precision of the method. For lactoferrin(1.20±0.77μg/μL), lysozyme (2.11±1.50μg/μL) and lipocalin-1©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea(1.751±0.99μg/μL) the findings were consistent with findings fromprevious ELISA studies. Tear levels of prolactin-induced protein(0.09± 0.06μg/μL) and proline rich 4 (0.80±0.50μg/μL) are reportedhere for the first time.Conclusions: SRM method can be used for simultaneous detectionand quantification of the five selected proteins in low volume humantear samples (2.5µl per sample) without prior purification of eachprotein component or need for antibodies.Commercial Relationships: Simin Masoudi, None; Ling Zhong,None; Mark Raftery, None; Mark D. Willcox, Allergan Inc (C),Allergan Inc (R), Brien Holden Vision Institute (P), Bausch + Lomb(C), Basuch + Lomb (R)Program Number: 931 Poster Board Number: B0236Presentation Time: 1:00 PM - 2:45 PMDry Eye and Systemic DiseasesClaudia Henrich, Esen K. Akpek. Cornea, The Wilmer Eye Instituteat Johns Hopkins, Baltimore, MD.Purpose: To evaluate the prevalence of associated inflammatorysystemic diseases in a cohort of patients with dry eye syndrome.Methods: Medical records of patients who presented to the WilmerEye Institute Ocular Surface Diseases and Dry Eye Clinic with dryeye symptomatology during a period of two years (January 2010 toDecember 2011) were reviewed retrospectively. Patients weredivided into two groups: patients with clinically significant dry eyedisease (defined as Schirmer test result without topical anesthesia≤10 mm at 5 minutes in either eye or bulbar conjunctival stainingwith lissamine green scored based on Oxford scale ≥1 in either eye)and patients with dry eye symptomatology but without the clinicalfindings.Results: A patient list of 326 patients was generated electronicallyusing a diagnostic code of ICD 375.15. Sixty two patients wereexcluded as they presented primarily with other ocular surfaceproblems and dry eye was a secondary diagnosis. Two hundred andsixty four patients with a primary diagnosis of dry eye were includedin the analysis. The majority of the patients (215/264, 81.4%) werefemale. Two hundred and seventeen (217/264; 82.2%) had clinicallysignificant dry eye. About half of the patients (121/264, 45.8%) hadan underlying inflammatory systemic disease on presentation. Onehundred and nine of them (109/121, 90.1%) had a clinicallysignificant dry eye. Thirty one patients (31/264, 11.7%) had primarySjögren Syndrome, 38 (38/264; 14.4%) had thyroid disease (eitherhypothyroidism, Graves disease or Hashimotos thyroiditis), 13(13/264, 4.9%) had rheumatoid arthritis, 42 (42/264, 15.9%) hadother rheumatic diseases.In 50 patients (50/143, 35.0%) without a previously known systemicdisease (regardless of the severity of the dry eye) a further work upwas performed based on review of systems. In 12 patients (12/50,24.0%) a diagnosis based on the work up was established: 10 patients(10/50, 20.0%) were diagnosed with thyroid eye disease, 2 patients(2/50, 4.0%) were diagnosed with Sjögren syndrome or presumedSjögren syndrome, one (1/50, 2.0%) was diagnosed with a mixedconnective tissue disease.Conclusions: Dry eye is a very common condition. Although usuallya local disease with a benign course, prevalence of systemic diseaseassociation seems to be significant. Based on clinical suspicion andreview of systems, further diagnostic testing might uncover some ofthese previously undiagnosed conditions.Commercial Relationships: Claudia Henrich, None; Esen K.Akpek, Alcon (F), Allergan (F), Baush & Lomb (C)FODE Highlights Impression Based Mucin Defects on the OcularSurface in Dry Eye Patients: Kinetics and Retrieval by TearLipocalinPo-Ting Yeh 1, 2 , Ben J. Glasgow 2, 3 , Richard Casey 2 . 1 Ophthalmology,National Taiwan University Hospital, Taipei, Taiwan;2 Ophthalmology, Jules Stein Eye Institute, David Geffen School ofMedicine at UCLA, Los Angeles, CA; 3 Pathology and LaboratoryMedicine, David Geffen School of Medicine at UCLA, Los Angeles,CA.Purpose: A fluorescent probe was used to identify mucin depletedareas on the ocular surface and test the hypothesis that tear lipocalin,retrieves lipids from the eyes of normal and dry eye subjects.Methods: Fluorescein labeled octadecyl ester, FODE, wascharacterized by mass spectrometry and absorbancespectrophotometry. The use of FODE to define mucin defects wasstudied with impression membranes under conditions that selectivelydeplete mucin. The kinetics of FODE removal from the ocularsurface was analyzed by sampling tears from control and dry eyepatients at various times. The tear protein-FODE complexes wereisolated by gel filtration and ion exchange chromatographies,monitored with absorption and fluorescent spectroscopies andanalysed by gel electrophoresis. Immunoprecipitation verified FODEcomplexed to tear lipocalin.Results: FODE exhibits an isosbestic point at 473nm, pKa of 7.5,and red shift relative to fluorescein. The low solubility of FODE inbuffer is enhanced with 1% Tween 80 and ethanol. FODE adheres tothe ocular surface of dry eye patients. FODE produced visiblestaining at the contact sites of membranes, which correlated withremoval of mucin. Despite the fact that tear lipocalin was reduced indry eye patients, FODE removal followed similar rapid exponentialdecay functions for all subjects. The majority of FODE was elutedbound to tear lipocalin.Conclusions: Tear lipocalin retrieves lipid rapidly from the humanocular surface in mild to moderate dry eye disease and controls. Withimprovements in solubility, FODE may have potential as afluorescent probe to identify mucin depleted areas.FPLC of tear samples from dry eye subjects. (●) Absorbance at 280nm (○) Fluorescence intensity (λem=511 nm). Inset left, coomasssiestainedtricine-SDS-PAGE. Lane 1, fx 4-6. Lane 2, fx 8-10. Lane 3,fx 14-16. Lane 4, fx 21-23. Lane 5, fx 26-28. Lane M, molecularweight markers. Lactoferrin (Lf), lysozyme (Ly) and tear lipocalin(TL) are indicated at left. Inset right, fluorescence of anion exchangechromatography performed on fractions 21-23 of control subjects.Fluorescence was seen only in the elution buffer (high salt) fractions.Gel filtration chromatography of control sample was nearly identicalto that of dry eye.Program Number: 932 Poster Board Number: B0237Presentation Time: 1:00 PM - 2:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFODE staining of sites where Schirmer test II was performed(arrows). Photos taken after: (A) 3 minutes (B) 5 minutes.Commercial Relationships: Po-Ting Yeh, None; Ben J. Glasgow,None; Richard Casey, NoneSupport: NIH EY11224 (BG), EY000331 (Core), NIH S10-RR023718 A (Shared Instrumentation Grant) and the Edith and LewWasserman Professorship (BG)FIGURE 1. Flowchart of the step approach for identifying riskfactors of dry eye using univariate and multivariate analysis.Program Number: 933 Poster Board Number: B0238Presentation Time: 1:00 PM - 2:45 PMFactors associated with dry eye: The Korea National Health andNutrition Examination Survey 2010Tyler Hyung Taek Rim 1 , Ji Min Ahn 2 , Sangchul Yoon 1 , Tae-im Kim 1 ,Eung Kweon Kim 1 , Kyoung Yul Seo 1 . 1 Department ofOphthalmology, Yonsei University College of Medicine, Seoul,Republic of Korea; 2 Ophthalmology, Siloam Eye Hospital, Seoul,Korea, Seoul, Republic of Korea.Purpose: To assess socio-demographic and health-related risk factorsassociated with dry eye.Methods: In 2010, a total of 5,726 randomly selected nationalrepresentative participants of the Korea National Health and NutritionExamination Survey underwent additional ophthalmologicexaminations by the Korean Ophthalmologic Society. Subjects wereasked to respond to a question of “Until now have you ever beendiagnosed before of having dry eye, either eye, by a doctor?” withpossible choices of “no” or “yes.” Independent variables weredivided into four categories: 1) socio-demographic factors, 2) healthexamination variables, 3) health behavioral risk factors and 4)variables concerning the eyes. The risk factors for dry eye wereidentified by using a multivariate logistic regression analysis.Results: The prevalence of dry eye was 8.2% (95% confidenceinterval (CI) 7.3-9.2) in subjects aged 19 or older. History of any eyesurgeries (adjusted Odds Ratio (aOR)=2.2, 95% CI, 1.7-2.8), femalesex (aOR=2.2, 95% CI, 1.5-3.3), extremely stressful status(aOR=1.8,95% CI, 1.0-3.1), and hypercholesterolemia of ≥240 mg/dL(aOR=1.3, 95% CI, 1.0-1.7) were independent risk factors for dryeye. Binge alcohol users (aOR=0.6, 95% CI, 0.5-0.9) and subjectswith occupations of farming, fishing, and forestry (aOR=0.2, 95% CI,0.1-0.4) were less likely to have dry eye compared to their referencegroups of non-drinkers and subjects with occupations inadministration, management or white-collar professionals,respectively. Among subjects who received an eye surgery, ptosissurgery (aOR = 6.2, 95% CI, 1.4-27.1), cataract surgery(aOR = 1.8,95% CI, 1.2-2.6), and refractive surgery(aOR=2.9, 95% CI, 1.8-4.5)were important risk factors.Conclusions: Ophthalmologists should be aware that pastexperiences of eye surgeries could be one of the most important riskfactors of dry eye, and should focus on treating dry eye problems insubjects who will receive or have already received an eye surgery.FIGURE 2. An association between type of eye surgery and dry eye.Commercial Relationships: Tyler Hyung Taek Rim, None; Ji MinAhn, None; Sangchul Yoon, None; Tae-im Kim, None; EungKweon Kim, None; Kyoung Yul Seo, NoneSupport: The authors state that they have no financial or conflicts ofinterest to disclose.Program Number: 934 Poster Board Number: B0239Presentation Time: 1:00 PM - 2:45 PMPrevalence of asymptomatic dry eye in patients with CollagenVascular Diseases in South IndiaRAMYA RAVINDRAN, Kalpana Suresh, Varshini Varadaraj.Ophthalmology, SRMC and RI, Chennai, India.Purpose: Dry Eye Syndrome (DES) is common in patients withcollagen vascular disorders. The purpose of this study was to identifyasymptomatic DES in patients with collagen vascular disorders inSouth India. In our part of the world, awareness regarding theimplications of late diagnosis is still lacking. The objectives were toidentify DES, classify it's severity based on the ocular surfaceintegrity and to delineate the sub set that had not undergone a priorophthalmic evaluation.Methods: A prospective, cross sectional, non interventional,observational study was conducted in a university hospital. 75patients diagnosed with various collagen vascular disorders with noocular complaints from the departments of General medicine,Pediatrics and Rheumatology formed the study group. 75 age and sexmatched controls were used for comparison. Patients with local andsystemic conditions that contribute to DES were excluded. Schirmer'stest, TBUT and rose bengal (RB) staining were performed and scoredaccording to the Van Bijsterveld scoring system. History of their lastophthalmic visit was noted. The Mann Whitney U test was used forstatistical analysis.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: In our study we noticed a female preponderance (64%). Postdiagnosis with collagen vascular disorders, 76% of the patients hadnot undergone an ophthalmic examination. 30.66% of the patients inthe study group had an abnormal Schirmers test compared to 12% ofthe controls (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMateo, None; Jose M. Herreras, None; Michael E. Stern, Allergan,Inc. (E); Maria J. Gonzalez-Garcia, NoneSupport: The present study was partially supported by (1) Junta deCastilla y León: VA145A11-2 and 04/09/VA/0158; (2) University ofValladolid FPI-UVa-2008; (3) Ministry of Economy andCompetence: PTQ-09-02-01913, CDTI: IDI-20060676, CICYT:SAF2010-15361.Program Number: 937 Poster Board Number: B0242Presentation Time: 1:00 PM - 2:45 PMReproducibility of tearfilm osmolarity measurement assessedwith electrical impedance in patients with dry eye syndrome andhealthy subjectsGerhard Garhofer 1 , Semira Kaya 1 , Johannes Nepp 2 , DoreenSchmidl 1 , Leopold Schmetterer 1, 3 . 1 Department of ClinicalPharmacology, Medical University of Vienna, Vienna, Austria;2 Department of Ophthalmology, Medical University of Vienna,Austria, Austria; 3 Department of Biomedical Engineering andPhysics, Medical University of Vienna, Austria, Austria.Purpose: Dry Eye Syndrome (DES) is a common medial conditionthat affects up to 20% of adults aged 45 years or older. Standardclinical tests are limited by their poor reproducibility and show only aweak correlation to patients complains. It has been hypothesized thatmeasurement of tear film osmolarity may be a new and promisingnon-invasive approach for the diagnosis and monitoring of treatmentsuccess. The current study investigated the short time and day-to-dayreproducibility of the Tear Lab Osmolarity system.Methods: 20 patients with DES and 20 age and sex matchedvolunteers were included in this study. Tear film osmolarity wasmeasured in both groups on three consecutive study days to calculateday-to-day reproducibility. To assess short time reproducibility ofosmolarity measurements were taken 3 times per study day. Tear filmbreak up time (BUT) and Schirmer 1 test were assessed by standardclinical tests. Coefficient of variation was calculated as a measure ofreproducibility.Results: Schirmer test and BUT was lower in patients with DEScompared to healthy subjects on all three study days (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea(OSED) score (cornea: 15 points maximum, conjunctiva: 6 pointsmaximum), Schirmer 1 test (ST1, mm) were measured, and thefactors determining OSED score were investigated by multipleregression analysis in which the parameters were chosen using thestepwise procedure.Results: The corneal OSED score was found to be determined byTMR, SG, and FBUT, and was described as: -0.363 - (3.997 x TMR)- (3.284 x SG) - (0.189 x FBUT) (R2=0.54, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: Prospective, randomized, multicenter, double-blindplacebo-controlled study were performed. A total of 65 dry eyepatients were randomly assigned to placebo (n=33) and 1% BHB eyedrop (n=32) and patients were received 6 times a day for 4 weeks.Corneal fluoresecin staining, cornea and conjuctival rose bengalstaining, tear film BUT, Schirmer test and subjective symptoms wereevaluated.Results: In the corneal rose bengal score, statistically significantimprovement was observed between the placebo and 1% BHB dropat 2 and 4 week (P < 0.05).In the corneal fluorescein staining score, significant improvementwas observed between placebo and 1% BHB eye drop at 2 week inthe patient with Schirmer ≤ 5 mm (n=38, P < 0.05). Trend toward asignificant improvement between placebo group and 1% BHB eyedrop at week 4 in the patient with Schirmer ≤ 5 mm with apparentforeign sensation (n=23, P < 0.1). There was no significant differencein tear film status and subjective symptoms between placebo and 1%BHB eye drop. All of adverse effects were mild ocular symptoms andspontaneously recovered in both group.Conclusions: These results indicate that 1% BHB is safe andeffectiveness in the treatment of ocular surface disorder in teardeficientdry eye patient.Commercial Relationships: Tetsuya Kawakita, None; EtsukoTakamura, None; Jun Shimazaki, Santen Pharmaceutical Co. (F),Otsuka Pharmaceutical Co. (F), Abott Medical Optics (F); JunkoOgawa, None; Miki Uchino, Santen Pharmaceutical Co (F); KazumiFukagawa, None; Kenichi Yoshino, None; Mari Tanaka, None;Machiko Shimmura-Tomita, None; Kazuo Tsubota, AcuFocus,Inc (C), Allergan (F), Bausch Lomb Surgical (C), Functional visualacuity meter (P), JiNS (P), Kissei (F), Kowa (F), Santen, Inc. (F),Otsuka (F), Pfizer (C), Thea (C), Echo Denki (P), Nidek (F), Ophtecs(F), Wakasa Seikatsu (F), CEPT Company (P)Support: OphtecsProgram Number: 942 Poster Board Number: B0247Presentation Time: 1:00 PM - 2:45 PMThe Korb-Blackie Lid Light TestDonald R. Korb 1, 2 , Caroline A. Blackie 1, 2 . 1 Korb Associates, Boston,MA; 2 TearScience, Morrisville, NC.Purpose: The Korb-Blackie (KB) lid light test was developed toinvestigate the possibility that apparently normal, closed, lids fail tocreate the necessary protective seal to prevent ocular surfacedessication during sleeping. The test results are correlated withsymptoms of eye discomfort upon awakening.Methods: Patients (n=148) reporting for routine eye examinationsthat met the inclusion criteria for the study were enrolled. Inclusioncriteria: willingness to participate in the study, over the age of 18, nolid or closure (e.g. lagophthalmos) abnormalities, no current ocularinflammation/disease, no ocular surgery within the last 6 months, nohistory of lid surgery. After answering a simple questionnaireregarding their eye comfort upon awakening, patients were asked torest their heads back against the head rest of a semi-reclined examchair and to close their eyes as if they were falling sleep. Atransilluminator was lightly placed against the closed outer uppereyelid of each eye. The apparently closed lids were examined for thepresence of any light emanating from the lid area between the lashes.The examiner positioned their eye level inferiorly such that they werelooking up, to optimize viewing of the designated region. The lidswere divided into three sections: temporal, central and nasal. Theamount of visible light in each section was quantified on a scale of 0 -3 where 0 = no light, 1 = minimal, 2 = moderate and 3 = severe. Eyediscomfort upon awakening was quantified on a scale of 0 - 2 whereas 0 = no discomfort, 1 = mild and 2 = significant discomfort.Results: The mean age of the patients = 53.9±16.2 years (50 males;98 females). Data for right eyes only are presented. The mean lightscore for each lid region was: temporal = 0.3±0.5; central = 1.0±1.0;nasal = 0.5±0.7, indicating the central region is the least likely toclose completely. The level of eye discomfort upon awakening wassignificantly correlated with the number of lid sections (0-3)emanating light during the KB lid-light test (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFig. 1. Comparison between fluorescence intensity and NaFconcentration from a 5-ìm thick film (filled diamonds) and theory(line). Colors corresponding to intensity values are also shown.of SIRC cell cultures treated with BAK, pure and mixed with HA,was examined.Results: Pure BAK instantaneously inserted in the lipid films(corneal or meibomian). The interaction resulted in impairedspreading and discontinuous patchy structure of the lipid films,increased surface pressure-area hysteresis and partial displacement ofthe lipids by BAK from the surface. The inclusion of HA in the filmssubphase opposed to these adverse effects and at ≥ 0.1% HA theproperties of the lipid films were maintained for the entire BAKconcentration range. Identical results were obtained with cell cultureexperiments which showed that SIRC cells maintained high viabilityat ≥ 0.1% HA.Conclusions: HA at concentration ≥ 0.1% HA was able to efficientlysuppress the adverse effects of BAK on the properties of meibomianand corneal lipid films and on the viability of SIRC cells. Thusmixtures of BAK and HA represent prospective compositions foreyedrop formulations. Surface chemistry based approach is proposedfor in vitro molecular scale characterization of pharmaceuticallyapplicable polymers and their interactions with tear film lipids.Commercial Relationships: Georgi A. Georgiev, RohtoPharmaceuticals (F), Santen (F), Menicon (F); Norihiko Yokoi,Santen Pharmaceutical (F), Otsuka Pharmaceutical (F), Kowa (P),Kissei Pharmaceutical (C), Menicon (F), Alcon Japan (F), Whitemedical (F), Nitten (F), Rohto (C), Nidek (F), Johson & Johnson (F);Slavyana Ivanova, None; Rumen Krastev, None; ZdravkoLalchev, NoneSupport: The research is funded by Santen Pharmaceutical.Fig 2. Predicted dynamic of color change for black-spot formation.Commercial Relationships: CHENG-CHUN PENG, None; BoTan, None; Meng C. Lin, TearLab Corporation (F), Allergan, Inc.(F); Clayton J. Radke, novartis corporation (F)Program Number: 944 Poster Board Number: B0249Presentation Time: 1:00 PM - 2:45 PMSurface Chemistry Study Of The Interactions Of BenzalkoniumChloride and Hyaluronic Acid With Meibomian And CornealLipidsGeorgi A. Georgiev 1 , Norihiko Yokoi 2 , Slavyana Ivanova 1 , RumenKrastev 3 , Zdravko Lalchev 1 . 1 Biochemistry, Sofia University "StKliment Ohridski", Sofia, Bulgaria; 2 Ophthalmology, KyotoPrefectural University of Medicine, Kyoto, Japan; 3 Biomaterials,NMI Naturwissenschaftliches und Medizinisches Institut an derUniversität Tübingen, Reutlingen, Germany.Purpose: Dodecyl dimethyl benzyl ammonium chloride (BAK) iscommonly used preservative in eyedrop formulations, known toimpair the integrity of the tear film lipid layer and of the cornealepithelium membranes. We studied the capability of high molecularweight (Mw 1x10+6) hyaluronic acid (HA; Santen Pharmaceutical,Osaka, Japan) to protect meibomian and corneal lipid films at theair/water interface from the adverse action of BAK.Methods: Human meibum was collected from healthy volunteers;corneal lipids were extracted from SIRC cell culture. The interactionsof BAK with meibomian and corneal lipids at the air/water interfacein presence and absence of HA in the film subphase were examinedin vitro at blink-like compression/expansion of film area byLangmuir surface balance. The sample’s lateral elasticity andcapability to compress and spread during dynamic area changes wereevaluated through the surface pressure-area isotherms and isocycles.The lipid films morphology was monitored by Brewster AngleMicroscopy. BAK concentration was kept within the clinical range of0.005-0.02%; HA was used in the range of 0.01-0.3%. The viabilityProgram Number: 945 Poster Board Number: B0250Presentation Time: 1:00 PM - 2:45 PMEfficacy of rebamipide for laser in situ keratomileusis-associateddry eyeYosai Mori, Ryohei Nejima, Ayami Masuda, Yoko Maruyama,Keiichiro Minami, Kazunori Miyata. Miyata Eye Hospital,Miyakonojo, Japan.Purpose: Dry eye syndrome is a major complication after laser insitu keratomileusis (LASIK), and is occasionally hard to improvewith an artificial tear or hyaluronic acid treatment. Rebamipide is aquinolinone derivative stimulating mucin secretion as well asincreasing goblet cells on the conjunctiva, resulting in improvementof ocular surface disorders. Aim of the study is to evaluate theefficacy of rebamipide for dry eye after LASIK.Methods: This prospective study comprised 32 eyes of 16 patientswho had LASIK-associated dry eye and had been treated with theartificial tear or hyaluronic acid eye drop. Rebamipide 2% eyedrop(Mucosta, Ohtsuka Pharmaceutical) was additionally instilled 4 timesa day for 4 weeks. Tear secretin was examined with the Schirmer testwith anesthesia before and at 4 weeks after the rebamipide treatment.Tear breakup time (BUT), fluorescein stain, and lissamine green stainwere examined before and at 1 and 4 weeks. Fluorescein staining onthe cornea was evaluated by the summation of area and densityscores (none:0 to severe:3). Lissamine green staining in theconjunctiva was scored in none:0 to full:18. Questionnaire of 14symptoms according to Ocular Surface Disease Index was performedbefore and at 4 weeks. Change in the examinations after theadditional treatment was evaluated with the Friedman's test followingScheffe ad-hoc comparison. Symptoms were compared with theWilcoxon signed-ranks test.Results: Mean tear secretin did not change beween beforerebamipide treatment (8.8 mm) and after 4 weeks (8.2 mm). BUTsignificantly increased from before (3.4 sec.) to 1 week (4.8 sec.,P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneathe rebamipide treatment (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFigure 1. Schematic of the TFLL. At eye temperature, asymmetriccrystallites (mostly phase A) are dispersed within an isotropic,viscoelastic continuous liquid. Polar lipids orient at the aqueous/lipidinterface, but orientation does not persist deep into the bulk lipidlayer. Drawing is not to scale.Commercial Relationships: Clayton J. Radke, novartis corporation(F); Liat Rosenfeld, None; Colin Cerrretani, Alcon Corporation(F); Danielle L. Lieske, None; Michael Toney, NoneSupport: novartis corporationProgram Number: 948 Poster Board Number: B0253Presentation Time: 1:00 PM - 2:45 PMBlood Flow in eye lids and Surface Temperature ofTarsalConjunctiva Decreases in Patients with ObstructiveMeibomianGland DysfunctionReiko Arita 1, 2 , Rika Shirakawa 2 , Motoko Kawashima 3 , Kouzo Itoh 1 ,Sachiko Inoue 3 , Shiro Amano 2 , Kazuo Tsubota 3 . 1 Ophthalmology,Itoh Clinic, Saitama, Japan; 2 Ophthalmology, Tokyo University,Tokyo, Japan; 3 Ophthalmology, Keio University, Tokyo, Japan.Purpose: Meibomian glands secrete lipid into the tear film, therebypreventing excessive evaporation of the tear film by forming a thinoily layer on the tear film.The surface temperature in the tarsalconjunctiva was significantly lower in patients with MGD than thosein normal controls(Arita R, et al. Arch Ophthalmol. in press). Thepurpose of this study was to investigate the blood flow in the eye lidsand the surface temperature in the tarsal conjunctiva and to examinethe correlation between the blood flow and the surface temperature inthe tarsal conjunctiva and ocular surface parameters in patients withMGD.Methods: Twenty five eyes of 25 patients (11men, 14women; mean± standard deviation of age, 74.2±10.3 years) who were diagnosed asobstructive MGD and30 eyes of30 healthy volunteers (18 men, 12women; 64.6±14.4 years) as a control group.The blood flow in the eyelid was measured with a newly developedblood flow meter using a red light. The surface temperature of thecentral cornea, andupper and lower tarsal conjunctiva was measuredusing ocular surface thermography. Changes in meibomian glandswere scored using non-contact meibography (meibo-score, 0-6). Lidmargin abnormality (0-4), superficial punctuate keratopathy (SPK)(0-9), and meibum (0-3) was scored. Tear film breakup time (BUT)was measured. This study was approved by the institutional reviewboard of Itoh clinic and adhered to the tenets of the Declaration ofHelsinki.Results: The blood flow in the eye lid was significantly lower inpatients with MGD than in normal controls (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFigure 1. Thickness h, osmolarity c and intensity I at different timesFigure 2. Color contour plots of h, c, f and I. Blue is smaller, red islarger. The thickness and intensity plots (left) are quite similar, as arethe plots for osmolarity and fluorescein concentration (right).Commercial Relationships: Richard J. Braun, None; Carolyn G.Begley, Santen, Inc. (C), ohnson & Johnson Vision Care, Inc. (C),ohnson & Johnson Vision Care, Inc. (F); Adam J. Winkeler, None;Peter E. King-Smith, None; Javed Siddique, NoneSupport: NIH Grants EY021794, EY017951, and NSF Grant1022706Program Number: 950 Poster Board Number: B0255Presentation Time: 1:00 PM - 2:45 PMEffects of Stressed Environments on the Ocular SurfaceTemperature, Lipid Layer Thickness and HeterogeneityRanjini Kottaiyan 1 , Gheorghe Salahura 1, 2 , James M. Zavislan 2, 1 ,Geunyoung Yoon 1, 2 , James V. Aquavella 1 . 1 Flaum Eye Institute,University of Rochester, Rochester, NY; 2 Center for Vision Science,The Institute of Optics, Rochester, NY.Purpose: To investigate the effects of low relative humidity (RH)and high temperature conditions on the ocular surface temperature(OST), tear lipid thickness (LT) and uniformity in normal and dry eyesubjects using thermal imaging and quantitative tearscope forimaging the lipid layer.Methods: A total of 18 eyes comprising 5 normal, 9 aqueousdeficiency (ADDE) and 4 with meibomian gland dysfunction (MGD)were included in the study. ADDE and MGD were diagnosed basedon clinical criteria. Measurements were taken with a thermal cameraand a quantitative tearscope before and after 30 minutes acclimationto three different environmental conditions in our controlledenvironmental chamber. The three conditions are 40% RH and 75 °F(condition 1), 20% RH and 75 °F (condition 2), and 40% RH and 85°F (condition 3). Subjects were asked to blink once every 5 secondsfor about 3-4 times during the measurements. Thermal data wasanalyzed to calculate the average OST in the central 9 mm of thecornea, while the average LT and uniformity were calculated fromapproximately the central one-third of the cornea, excluding thepupillary region. Changes in OST and LT were compared amongstthe different groups.Results: The average OST is 34.9 ± 0.2°C, 33.8 ± 0.3°C and 34.6 ±0.2°C in normal, ADDE and MGD subjects respectively. The averageLT is 40.4 ±8.3 nm, 57.9 ± 30 nm, and 39.6±7.8 nm in normal,ADDE and MGD subjects respectively at baseline. After 30 min ofacclimation, normal subjects did not show a significant change inOST under any of the conditions. ADDE, however, showed asignificant increase in OST by 0.6°C, 0.4°C and 0.6°C in conditions1 (p=0.01), 2 (p=0.02) and 3 (p=0.005) respectively. MGD showedan increasing trend in OST in conditions 1 and 2, but an interestingdecreasing trend in condition 3 although none of these trends showeda statistical significance. On the lipid thickness, ADDE showed anincrease in LT by 8.77 ± 10.95 nm and a decrease of 14.47 ± 29.04nm, in conditions 1 and 2 respectively. Also, MGD showed anincrease of heterogeneity by 0.18 ± 0.19 and a less relativeheterogeneity of 0.14 ± 0.23 in conditions 2 and 3 respectively.Conclusions: The dry eye groups differ in responding to differentstressed environmental conditions.This finding suggests that theeffects of the ocular stressors on the tear parameters can enhance ourability to differentiate the various dry eye symptoms.Commercial Relationships: Ranjini Kottaiyan, None; GheorgheSalahura, None; James M. Zavislan, None; Geunyoung Yoon,Bausch & Lomb (F), Johnson & Johnson (F), Allergan (C), StaarSurgical (C), CIBA Vision (F), Acufocus (C); James V. Aquavella,NoneSupport: Research to Prevent Blindness unrestircted/challenge grantProgram Number: 951 Poster Board Number: B0256Presentation Time: 1:00 PM - 2:45 PMFactors Predicting the Ocular Surface Response to DesiccatingEnvironmental StressAnastasia Alex 1, 2 , Austin Edwards 3, 2 , J. Daniel Hays 3, 2 , MichelleKerkstra 3, 2 , Amanda Shih 3, 2 , Cintia S. De Paiva 2 , Stephen C.Pflugfelder 2 . 1 University of Toledo College of Medicine, Holland,OH; 2 Baylor College of Medicine, Houston, TX; 3 Rice University,Houston, TX.Purpose: To identify factors predicting the ocular surface response toexperimental desiccating stress.Methods: The ocular surfaces of both eyes of 15 normal and 10 dryeye subjects were exposed to a controlled desiccating environment(15-25% RH and 3 L/min airflow) in goggles for 90 minutes. Eyeirritation symptoms, blink rate, tear meniscus dimensions, noninvasive(RBUT) and invasive tear break-up time, and cornealfluorescein and conjunctival lissamine green dye staining wererecorded before and after desiccating stress. Pre- and post-exposuremeasurements were compared and Spearman correlations betweenclinical parameters before and after desiccating stress werecalculated.Results: Conjunctival and corneal dye staining significantlyincreased in all subjects following 90-minute exposure to desiccatingenvironment and the magnitude of change was similar in normal anddry eye subjects, except superior cornea staining was greater in dryeye. Irritation severity in the desiccating environment was associatedwith baseline dye staining, baseline tear meniscus height and blinkrate after 45 minutes. Desiccation-induced change in cornealfluorescein staining was inversely correlated to baseline tearmeniscus width, whereas change in total ocular surface dye stainingwas inversely correlated to baseline dye staining, RBUT and tearmeniscus height and width. Blink rate from 30-90 minutes ofdesiccating environment was higher in the dry eye than normalgroup. Blink rate significantly correlated to baseline corneal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafluorescein staining and environmental induced change in corneal FLstaining.Conclusions: Ocular surface dye staining increases in response todesiccating stress. Baseline ocular surface dye staining, tear meniscusheight and blink rate predict severity of ocular surface dye stainingfollowing exposure to a desiccating environment.Commercial Relationships: Anastasia Alex, BiocentricDevelopments, LLC (P); Austin Edwards, Biocentric DevelopmentsLLC (P); J. Daniel Hays, Biocentric Developments, LLC (P);Michelle Kerkstra, Biocentric Developments, LLC (P); AmandaShih, Biocentric Developments, LLC (P); Cintia S. De Paiva, GlaxoSmith Kline (C), Baylor College of Medicine (P); Stephen C.Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb(C), Baylor College of Medicine (P)Support: NIH grants R01EY11915 (SCP), Research to PreventBlindness, The Oshman Foundation, The William Stamps FarishFund, The Hamill Foundation and Allergan, Inc.Program Number: 952 Poster Board Number: B0257Presentation Time: 1:00 PM - 2:45 PMThe Effects of Cosmetic Eye Pencil Application on the Tear Filmand Ocular SurfaceAlison Ng 1 , Katharine Evans 1 , Rachel V. North 1 , Christine Purslow 2,1 . 1 School of Optometry & Vision Sciences, Cardiff University,Cardiff, United Kingdom; 2 School of Health Professions, PlymouthUniversity, Plymouth, United Kingdom.Purpose: To investigate and compare the effects of cosmetic eyepencil on the ocular surface and tear film when applied to periorbitalskin (ELO) or the mucocutaneous junction (ELI).Methods: 24 healthy female subjects (mean±SD age: 24±5 years)underwent a 5 day washout period, refraining from all eye cosmeticuse and contact lens wear, prior to study commencement. Subjectswere supplied with pencil eyeliner (MaxFactor Kohl pencil 020Black, Procter & Gamble, UK) and randomised to either applyingELO or ELI daily for 7 consecutive days. Subjects crossed overfollowing a 1 week washout period and applied eyeliner with thealternative method.On day 1 and 7 of each method of eyeliner application, the followingclinical parameters were assessed: non-invasive tear break-up time(NITBUT) and lipid layer thickness (LLT) using a Tearscope(Keeler, Windsor, UK); bulbar redness, conjunctival and cornealstaining were graded to 0.1 increments on the Efron Grading Scaleand ocular comfort on a scale from 0 to 10 (where 10 indicatedmaximal comfort). Additionally, Ocular Surface Disease Index(OSDI) scores were compared after 7 days of each intervention.Results: There were no significant differences between the clinicalparameters after 1 day of ELO and ELI application. However, after 7days of eyeliner use, there was a clinical and statistically significantimprovement in LLT with ELI compared with ELO(wave→amorphous, p=0.032). Other values for bulbar redness,NITBUT and conjunctival staining were similar between eyelinerapplications. Subjects reported a slight decrease in comfort scoresafter 7 days of ELI compared with ELO (7.8±1.7 vs. 8.3±1.7respectively), but it was not significant (p=0.162). OSDI scores weresignificantly worse after 7 days of ELI compared with ELO(8.27±8.48 vs. 6.07±7.59 respectively, p=0.046).Conclusions: The short-term application of eye cosmetic pencilsclose to the ocular surface does not appear to be detectable clinically,but 7 consecutive days of ELI application appears to increase LLTand dry eye symptomology compared to ELO application. ELIapplication is likely to increase the contact of waxes, lipids and otheringredients from the pencil to the ocular surface compared to ELO,which may increase the lipid content of the tear film but also disruptocular surface homeostasis, accounting for the changes in symptoms.Commercial Relationships: Alison Ng, None; Katharine Evans,None; Rachel V. North, None; Christine Purslow, NoneSupport: Funding provided by School of Optometry & VisionSciences, Cardiff University, Wales, UKProgram Number: 953 Poster Board Number: B0258Presentation Time: 1:00 PM - 2:45 PMEffects of Computer Usage on Tear Film Osmolarity andPrecorneal Tear Film ThicknessStephanie Chu, Kelley Bohm, Kristin O. Chapman, Christopher E.Starr. Weill Cornell Medical College, New York City, NY.Purpose: Prolonged computer usage is a common cause of aconstellation of ocular symptoms such as irritation, dryness andfatigue collectively known as computer vision syndrome (CVS).Using novel diagnostic tools we aim to objectively quantify theeffects of computer usage on tear film dynamics.Methods: A prospective cohort study of 20 healthy volunteersubjects (40 eyes) were evaluated in the morning and again in theevening after prolonged computer usage. Outcome measures weretear osmolarity (TearLab Osmolarity System) and precorneal tearfilm thickness as measured by a modified Heidelberg ocularcomputed tomography (OCT). The average age of patients was 28.5years (range 24-35), with 60% females. Computer usage times weremeasured by an evening survey. Data was analyzed using paired twotailed t-test, Pearson co-efficient and Chi-square analysis with p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasessions during which hydrogel contact lenses were fitted to botheyes and a fluorescein solution was applied in one of three ways toone eye only; directly onto the IEL, OEL, or via a liposomal spray tothe closed lid. The lenses were harvested after 20 minutes andexamined using UV spectrophotometry. Differences in UVabsorbance over 400-600nm indicated fluorescein uptake comparedto control lenses.Results: Part 1: A significant change in the lipid layer pattern of thetear film was observed after five minutes for IEL (p=0.007), but notuntil 20 minutes for OEL (p=0.037). Tear film stability decreasedsignificantly within five minutes for IEL application (p=0.010), butnot until 20 min for OEL (p=0.045).Part 2: Contact lenses harvested after IEL application demonstratedsignificantly greater absorbance compared to control lenses(p=0.005). Where fluorescein was applied via spray or OEL, nosignificant difference in absorbance compared to controls wasobserved (p=0.098 and p=0.124, respectively). Comparing theapplication methods, absorbance following IEL was significantlyincreased compared to OEL (p=0.014) and spray (p=0.044).Conclusions: Lipid-based, water-based and liposomal solutions canmigrate across the muco-cutaneous junction when applied in closeproximity to the eye. All three applications studied showed somemigration; however application to the IEL was found to be mosteffective in allowing for migration into the tear film. Application of alipid based solution to the IEL exhibited migration that was 4 timesfaster than OEL application. This has implications for drug deliveryand cosmetic use around the eyelid margins.Commercial Relationships: Christine Purslow, None; ClareConaty, None; Alison Ng, None139 Ocular Surface and Tear FilmSunday, May 05, 2013 1:00 PM-2:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 955-978/B0260-B0283Organizing Section: CorneaProgram Number: 955 Poster Board Number: B0260Presentation Time: 1:00 PM - 2:45 PMTear Film Biomarker Profiling of Subjects with Dry Eye Diseaseby Multiplex AnalysisSuzanne Hagan 1, 2 , Alan Tomlinson 1 , Louise Madden 1 , Anne MarieClark 2 , Katherine M. Oliver 1 . 1 Vision Sciences, Glasgow CaledonianUniversity, Glasgow, United Kingdom; 2 Biological and BiomedicalSciences, Glasgow Caledonian University, Glasgow, UnitedKingdom.Purpose: Dry eye disease (DED) is a distressing disorder, commonlyassociated with ageing, contact lens wear and autoimmunesyndromes. It affects 15%-30% of the over-50s in Western and Asianpopulations and is one of the fastest-growing eye problems in thisdemographic. DED is significantly underdiagnosed and no “goldstandard” currently exists for its clinical diagnosis. Recent Multiplexstudies of tear fluids from DED subjects have implicatedinflammatory cytokines in this disorder. In this study, we investigatedtear fluids from DED and normal subjects for a panel of cytokinesusing the multiplex bead assay.Methods: This study comprised 15 DED subjects (3 males, 12females) and 20 healthy controls (2 males, 18 females). All subjectsprovided informed consent and the study adhered to the Declarationof Helsinki tenets. Tear samples were collected from the externalcanthus of open eyes, avoiding additional tear reflex. Glassmicrocaps were used to collect 1μl tears. Samples were diluted 1:50and stored at -80C until use. Tear cytokine levels were determinedwith a multiplex bead assay (R and D Systems) and quantified usinga Luminex IS200. Briefly, tear samples were incubated with specificantibody-coated beads for 3h. Washed beads were then incubatedwith biotin-labelled secondary antibodies, followed by a streptavidin-PE incubation. Standard curves of known cytokine concentrationswere used to calculate protein concentrations and data underwentanalysis by an in-house statistician.Results: Detectable levels of IL-8 (> 4.05pg/ml) were observed in12/15 DED subjects (mean 23.1pg/ml) and 16/19 normals (mean20.6pg/ml). Some variation in IL-8 levels was noted in DED subjects(4.9-83.8pg/ml), but a trend for increased IL8 in DED subjects wasobserved, compared to normals. Moreover, detectable levels of all 7inflammatory proteins (IL1β, IL2, IL6, IL8, IL17, TNF-α and IFN-γ)were observed in 2 DED subjects, a result not observed in normals.Conclusions: Increased IL8 levels in DED suggests a function inocular surface inflammation. This data confirms previous Multiplexbead studies, indicating a role for IL8 in DED. Further studies mayalso shed light on roles for the other 6 cytokines detected in 2 DEDsubjects. Moreover, this technology appears to be sensitive enough todetect low abundance proteins in minute sample sizes and thereforemay be useful in screening tear fluids for potential biomarkers ofDED.Commercial Relationships: Suzanne Hagan, Allergan (F); AlanTomlinson, Allergan (E), Allergan (R), Bausch and Lomb (C),TearLab (C), TearLab (I), Alcon, CibaVision (C), Pfizer (R), Pfizer(C); Louise Madden, None; Anne Marie Clark, None; KatherineM. Oliver, Allergan (F)Program Number: 956 Poster Board Number: B0261Presentation Time: 1:00 PM - 2:45 PMDouble rows of meibomian gland orifices observed by noninvasiveinfrared meibographyRika Shirakawa 1 , Reiko Arita 1, 2 , Shima Fukuoka 1, 3 , SatoshiYamamoto 4 , Kaori Yonehara 4 , Tsuyoshi Haraguchi 4 , Shiro Amano 1 .1 Ophthalmology, University of Tokyo, Tokyo, Japan;2 Ophthalmology, Itoh Clinic, Tokyo, Japan; 3 Ophthalmology, ToyoKyosai Hospital, Tokyo, Japan; 4 Topcon Corporation, Tokyo, Japan.Purpose: Multiple rows of meibomian gland orifices (MGOs), whichwere first reported in 1992 by Hykin and Bron, exist mainly in theupper eyelids of young people. Little is known about the morphologyof the meibomian glands and their influences on the ocular surface.We observed lid margins and meibomian glands in healthy youngadults to explore the incidence, morphology and function of doublerows of MGOs.Methods: Subjects were consecutive cases of healthy malevolunteers under 36 years of age. After obtaining written consent, wemeasured the width of the eyelid, counted the number of MGOs inthe upper and lower eyelids, obtained a fluorescein staining score,tear break-up time (BUT), meibum expressibility grade, tearmeniscus height under the slit-lamp microscopy, and performed aSchirmer tear production test. We also recorded images ofmeibomian glands by non-invasive infrared meibography, whichwere later reviewed to count the number of meibomian glands in eachlid. We scored a meiboscore (grade 0-3) according to the ratio of thearea of missing glands. “Double rows” were defined where more than4 orifices were aligned in a second row distinctly separate from theprimary row. This study was approved by the institutional reviewboard of University of Tokyo School of Medicine, and adhered to thetenets of the Declaration of Helsinki.Results: We examined 35 eyes of 35 people (all male, average age28.9±2.8). We observed double rows of MGOs in the upper eyelidsof 10 eyes (28.5%), whereas none (0%) were observed in the lowereyelids. Comparing between the double rows group (n=10) and the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasingle row group (n=25), the number of the orifices and number ofthe meibomian glands in the upper eyelids are significantly higher inthe double rows group (48.0±5.7 vs 36.3±4.9, p6 hours after a singledose. In the IL-1 mouse and ExLac rat models of dry eye disease,treatment with ENaC blockers significantly improved tear volumeand corneal fluorescein staining.Conclusions: Our studies demonstrate that ENaC inhibitors providelong-acting increases in tear volume and that restoring volume aloneis sufficient to provide significant improvements in ocular surfacehealth in models of dry eye disease. Our data provide a clear rationalefor the development of ENaC blockers to treat the underlying ocularsurface desiccation in dry eye disease.Commercial Relationships: William Thelin, Parion Sciences (E);M. Ross Johnson, Parion Sciences (I), Parion Sciences (E), ParionSciences (S); John Ansede, Parion Sciences (E); Karl Donn, ParionSciences (E); Dongfang Yu, Parion Sciences (F); Barbara Grubb,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaParion (F), Parion (I); Driss Zoukhri, Parion Sciences (F); RichardC. Boucher, Parion Sciences (C); Jose L. Boyer, Parion Sciences (E)Support: NIH Grant EY020705; NIH BrIDGs; NC BiotechnologyCenter Grant 2009-CFG-8005Program Number: 959 Poster Board Number: B0264Presentation Time: 1:00 PM - 2:45 PMVariability of tear osmolarity in dry eye patients and controlsNicole M. Fuerst 1 , Mina Massaro-Giordano 1 , Bridgette E. McCabe 1 ,Ilaria Macchi 2 , Maxwell Pistilli 1 , Gui-Shuang Ying 1 , Vatinee Y.Bunya 1 . 1 Ophthalmology, Scheie Eye Institute, Philadelphia, PA;2 Campus Biomedico University, Rome, Italy.Purpose: To examine the reproducibility and quantify the variabilityof tear osmolarity in different populations of dry eye patients andcontrols.Methods: Seventy-four eyes of thirty-seven subjects (18 Sjogren'ssyndrome, 10 blepharitis, and 9 controls) were evaluated for thevariability of measurements using the TearLab system. Controls weredefined as those with no history of symptoms or signs of dry eyedisease. 94% of Sjogren’s syndrome patients and 80% of blepharitispatients were on systemic or topical dry eye medications at the timeof enrollment. For all subjects, three consecutive osmolaritymeasurements were taken at one minute intervals in each eye toassess the within-session variability. For fifteen subjects, threemeasurements were taken at each of 3 time points throughout the day(9-10am, 12-1pm, 3-4pm) to examine the inter-session variabilityover the course of the day. The within-session and inter-sessionvariability were assessed based on the standard error of measurement(SEM), calculated from the analysis of variance.Results: Among all subjects, the within session variation for a singleosmolarity measurement was 14.4 mOsm/l (13.8 for SS, 8.8 forblepharitis, and 16.2 for controls). When the average of the threeconsecutive measurements in a single session were used, thevariability of osmolarity measurement was 8.3 mOsm/l. Betweensessionvariability was 17.2 mOsm/l for a single osmolarity measure,and 10.4 mOsm/l for an averaged measurement. The variability ofosmolarity measurements between two eyes of the same subject wasnot correlated (pearson rho=-0.05, p=0.76).Conclusions: A single measurement of tear osmolarity with theTearLab system had a standard error measurement of 14.4 mOsm/L.We did not find any correlation between eyes as far as variability ofmeasurements. Further larger studies would be helpful in studying theutility of tear osmolarity measurements in different dry eyepopulations.Commercial Relationships: Nicole M. Fuerst, None; MinaMassaro-Giordano, Tear Lab (R); Bridgette E. McCabe, None;Ilaria Macchi, None; Maxwell Pistilli, None; Gui-Shuang Ying,None; Vatinee Y. Bunya, TearLab (F)Support: Vatinee Bunya: National Eye Institute (K12-EY-015398),Research to Prevent Blindness, Mina Massaro-Giordano: Research toPrevent Blindness, Maxwell Pistilli and Gui-Shuang Ying: VisionCore Grant (P30 EY001583).Program Number: 960 Poster Board Number: B0265Presentation Time: 1:00 PM - 2:45 PMSymptom Burden of Patients with Dry Eye Disease: A FourDomain AnalysisJoelle Hallak, Sarmad H. Jassim, Sandeep Jain. Ophthalmology andVisual Sciences, University of Illinois at Chicago, Chicago, IL.Purpose: Intensity and affective interference are not measured bycurrent standardized dry eye symptom questionnaires. Wehypothesize that symptom burden of Dry Eye Disease (DED) isdetermined with greater accuracy when a 4 domain questionnaire,that measures symptom persistence, intensity, activity, as well asaffective interference, is utilized.Methods: The 4 domain DED symptom burden questionnaire wasdeveloped from well-established and validated symptom burden toolsused in other chronic diseases, such as the MD Anderson tool thatassesses pain in cancer patients. A pilot study was performed wherethe DED symptom burden questionnaire and the Ocular SurfaceDisease Index (OSDI) questionnaires were administered to 15patients. A weighted item response analysis was performed for thesymptom burden questionnaire (maximum persistence scores weremultiplied with the intensity and average activity and affective scoreswere then added to compute a total symptom burden score). TheOSDI (index) score was calculated from OSDI item responses. Tearproduction was measured by the schirmer test and correlated with thesymptom burden score and the OSDI index score.Results: Results from the OSDI questionnaire showed more equaland comparable scores between patients with significantly differentschirmer measurements than results from the symptom burdenquestionnaire. Two patients had OSDI scores of 27 with schirmerscores of 1 and 14, respectively. The symptom burden scores of thesepatients, however, differed 13 versus 34.43. A negative correlationwas shown between symptom burden scores and schirmer scoreswhereas a positive correlation was shown between OSDI scores andschirmer test scores.Conclusions: Intensity may be an essential domain to include insymptom questionnaires for DED, specifically when correlatingreported symptoms with clinical signs. A complete analysis of dryeye symptom burden includes 4 domains (persistence and intensity ofsymptoms, and intereference with activity and affect).Commercial Relationships: Joelle Hallak, None; Sarmad H.Jassim, None; Sandeep Jain, PCT/US20/51562 (P)Program Number: 961 Poster Board Number: B0266Presentation Time: 1:00 PM - 2:45 PMFrequency and Risk Factors Associated with Ocular SurfaceDisease in Patients Attending a Tertiary Care OphthalmologyCenter in Mexico CityJaime D. Martinez 1 , Nallely Ramos-Betancourt 1 , Anat Galor 2 ,Francisco Beltran 1 , Jorge Ozorno-Zarate 1 , Valeria Sánchez Huerta 1 ,Marco Antonio Torres Vera 1 , Everardo Hernandez-Quintela 1 .1 Asociación para Evitar la Ceguera en México I.A.P, Mexico,Mexico; 2 Bascom Palmer Eye Institute, Miami, FL.Purpose: The purpose of this study was to ascertain the frequencyand risk factors for ocular surface disease (OSD) among patients inMexico attending a tertiary care ophthalmology center.Methods: 200 consecutive patients seen in an ophthalmologic centerin Mexico City from October 2012 to November 2012 underwent acomprehensive examination, including measurement of tear filmbreak-up time (TBUT), fluorescein staining classified by Oxfordscheme, Schirmer test type 1 and evaluation of Meibomian GlandDysfunction (MGD). Symptoms of OSD were evaluated by theOcular Surface Disease Index (OSDI) and Dry eye Questionnaire(DEQ-5). Information on demographics, exposures, past medical andocular history, and medications was also collected. SymptomaticOSD was defined as having an OSDI score ≥23 or DEQ-5 score ≥12,clinical OSD was defined as having a Schirmer test 66©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneayears). Female gender imparted a 1.7 fold increased risk of disease asdefined by the DEQ5 (95% CI 0.92-3.31, p-value 0.09). OSD basedon clinical signs was seen at a frequency of 91%; 69% of patients hadan MGD score >1. The use of a duodenal ulcer medication was foundto be a risk factor for OSD using both the DEQ-5 and OSDIdefinitions (OR 2.8 and 13.1, p values < 0.05). Patients with diabetesmellitus had less OSD by the DEQ5 definition compared to theircounterparts without diabetes (OR 0.12, 95% CI 0.02-0.98, p-value0.047).Conclusions: This is the first study to demonstrate the frequency ofsymptomatic and clinical OSD in a tertiary care ophthalmologycenter in Mexico. The frequency of OSD in our population rangedfrom 28% using a symptomatic definition to 91% using objectivemeasures.Commercial Relationships: Jaime D. Martinez, None; NallelyRamos-Betancourt, None; Anat Galor, None; Francisco Beltran,None; Jorge Ozorno-Zarate, None; Valeria Sánchez Huerta,None; Marco Antonio Torres Vera, None; Everardo Hernandez-Quintela, NoneProgram Number: 962 Poster Board Number: B0267Presentation Time: 1:00 PM - 2:45 PMBlink and Extended Blinks in a Dry Eye PopulationAshley M. LaFond, Patrick Johnston, John D. Rodriguez, Keith J.Lane, Endri Angjeli. Ora, Inc., Andover, MA.Purpose: The tracking of spontaneous blink activity has been shownto permit an investigator to gather important information on theclinical state of a dry eye subject. Previous research has shown thatthe majority of blinks are incomplete with zero lid contact durationtime. During a complete blink, lid duration contact time may varyfrom less than 10ms to 80ms. Eyelid closures of up to multipleseconds, which we refer to as "extended blinks", may also beobserved. Here we investigate the relative incidence of incompleteand extended blinks in a dry eye population.Methods: We consider a sample population of 11 non-MGD dry eyesubjects and a control group of 10 normal subjects. Blink informationwas obtained using video analysis of each subject viewing a 10minute nature documentary. Incomplete blinks were tracked bypercent of palpebral fissure closure as ¼, ½ and 3/4. Extended blinksof multiple seconds were classified per the cutpoints(sec) = (0, 0.1,1).Results: Total number of blinks observed for all subjects was 4990(1414 normal, 3756 dry eye). Of total blinks, 50.6% were incomplete(dry eye) versus 52% (normal). Dry eye subjects were over 10 timesmore likely than normals to exhibit blinks of one second duration orlonger (2.3% vs 0.2% of total respective blinks, p=0.023). Mean lidclosure duration for dry eye subjects was 7.074 (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: TMH is 2.2±0.4(×100μm) before and is significantlyreduced to 1.3±0.3(×100μm) (59%) after CL wearing(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneameasurements are equivalent in the sense that one is the reciprocal ofthe other (a subject with N blinks over time period T has a BR of N/Tand an average IBI of T/N). As means, however, they are notequivalent since the reciprocal of mean BR does not equal the meanof IBI, and this raises the question of preference.We consider two aspects. First, if a particular blink measurement isdesired (BR or IBI), we assess the merits of normal versus gammamodels. Second, in cases where an investigator is interested in blinkfrequency but is not committed to a particular measurement, wepropose a novel method to compare the appropriateness of the twomeasurements.Methods: For a particular measurement (BR or IBI), two-meanmodels were fit assuming both normal and gamma distributions andthese were compared using the Akaike information criterion (AIC).Differences in AIC compare the relative goodness of fit of twostatistical models, the model with the lower AIC being preferred.Importantly (because normal and gamma models are not special casesof each other), models compared by AIC need not be nested.BR and IBI cannot be compared directly because comparisons of AICrequire models with identical outcomes. However, the functionalrelationship between BR and IBI permits comparison based on ageneralization of the Box-Cox transformation method. For example,estimates with outcome IBI = 1/BR using a gamma model areidentical to estimates based on BR using an inverse gamma model,and it is the latter model that provides the appropriate AIC forcomparison.Results: The gamma model was preferred to the normal model forBR by 10 units of AIC, and for IBI by 7 units of AIC. In cases whereeither measurement is allowed to represent blink frequency, BR waspreferred to IBI by 4 units of AIC (in both cases using a gammamodel).Conclusions: It is not uncommon for t-tests to be used in the analysisof BR and IBI, and while these tests are optimal under normality,they are not optimal under the superior gamma approximationindicated by this study. In addition, we have proposed a novelmethod to compare BR and IBI, in effect selecting the preferred scaleon which to measure blink frequency. In the present study BR waspreferred to IBI.Commercial Relationships: Patrick Johnston, Ora, Inc (E); LisaSmith, Ora, Inc. (E); John D. Rodriguez, Ora, Inc. (E); Keith J.Lane, Ora, Inc. (E); George W. Ousler, Ora, Inc. (E); RichardAbelson, Statistics & Data Corporation (E)Program Number: 968 Poster Board Number: B0273Presentation Time: 1:00 PM - 2:45 PMZone A (ZA), Posterior Lid Margin Vascularization: An EarlySign of Ocular Surface Disease (OSD)Alexander Gan 1 , Claudia M. Prospero Ponce 2, 1 , Andrew M. Quinn 1 ,Patricia Chevez-Barrios 2 , Alice Z. Chuang 1 , Richard W. Yee 1 . 1 TheRobert Cizik Eye Clinic.The Richard S. Ruiz Department ofOphthalmology and Visual Science. University of Texas HealthScience Center at Houston., Houston, TX; 2 Ocular Pathology,Department of Pathology and Genomic Medicine., The MethodistHospital, Houston, TX.Purpose: To analyze the histologic, clinical, and morphologicalcharacteristics of the avascular zone of the inferior posterior lidmargin and its possible clinical significance as a sign of early and latechronic inflammation in OSD.Methods: Retrospective chart review of the OSD findings and theOcular Surface Disease Index (OSDI) questionnaire were performedin 49 patients(pts), >20 yrs old seen in a tertiary care center. Inclusioncriteria: pts with a complete ocular surface evaluation includinganterior blepharitis(AB), vascularization(V) of the inferior lidmargin, meibomian gland obstruction(O) and turbidity(T). Basal teartest(BTT) and lissamine green staining(LGS) and quantification ofthe ZA was graded based on the degree of V noted on the evertedposterior inferior lid margin. Exclusion criteria: previous surgery oron topical anti-inflammatory treatment. OSDI scores were dividedinto 2 groups (normal:≤12vs dry eye:>12), ZA was divided into 2groups normal and severe and were compared to OSD signs usingchi2test. Lower lid biopsy was obtained for histology.Results: Forty-nine charts were reviewed and analyzed, 14 patientshad normal OSDI and 35 had dry eye OSDI. There was no significantstatistical difference between OSDI groups and all OSD findings[AB,p=0.08;V,p=0.8;O,p=0.05;T,p=0.7;ZA,p=0.7(77.1%dry eyeOSDI vs 85.7% normal OSDI). LGs and BTT were not statisticallydifferent between the two OSDI groups (p>0.5, p>0.1). Comparingthe ZA groups, 10 pts had normal grading, 39 pts had severe grading.No significant statistical differences were found between ZA groupsand OSD findings (AB,p=0.6;V,p=0.2;O,p=0.9;T, p=1.0). LGS andBTT were not statistically different between the ZA group. Patientswith severe ZA grading were found to have normal-mild OSDfindings (AB=84.2%, V=82.1%, O=56.4%, T= 32.4%), in contrast topatients with severe ZA grading that had severe OSD(AB=15.8%,V=18.0%,O=43.6%,T=67.6%). Histology showedinflammatory response and increased number of dilated vessels in theposterior lid margin.Conclusions: The OSDI questionnaire did not correlate with anyOSD clinical signs. ZA grading was noted to be severe even in ourcohort with mild disease. ZA vascularization may be a helpfulclinical sign of early OSD.Commercial Relationships: Alexander Gan, None; Claudia M.Prospero Ponce, None; Andrew M. Quinn, None; PatriciaChevez-Barrios, None; Alice Z. Chuang, None; Richard W. Yee,MTTI (P), Allergan (R)Program Number: 969 Poster Board Number: B0274Presentation Time: 1:00 PM - 2:45 PMIs there a relationship between ocular discomfort and circulatingplasma levels of sex hormones? Preliminary findingsBlanka Golebiowski 1 , Ulrike Hampel 1, 2 , Noor Badarudin 1 , IsabelleJalbert 1 , Michele C. Madigan 1 , Fiona Stapleton 1 . 1 School ofOptometry and Vision Science, University of New South Wales,Sydney, NSW, Australia; 2 Department of Anatomy 2, FriedrichAlexander University Erlangen Nürnberg, Erlangen, Germany.Purpose: Dry eye is a common problem especially for women postmenopause.This study explored the relationship between circulatinglevels of plasma sex hormones and ocular dry eye symptoms.Methods: A cross-sectional, single visit study was conducted. Thestudy involved a convenience sample of 74 subjects without ocularsurface disease, including 52 females (mean age 35.3±13.4years,range 18.8-70.3) and 22 males (mean age 34.2±13.8years, range20.2-75.3). Subjects completed the Dry Eye Questionnaire (DEQ5)and numerical ratings of discomfort, dryness, foreign body (FB)sensation, burning and watering. Tear osmolarity (TearLab) andvolume (Phenol Red Thread) were assessed. Venous blood wascollected and plasma concentrations of oestradiol (E2) and totaltestosterone (TT) were determined using specific Enzyme-linkedimmunosorbent assay. Associations were examined using Pearson’sor Spearman’s correlations, and differences between groups wereassessed using Independent samples t-test or Mann-Whitney U test,as appropriate.Results: Mean group E2 concentration was 65.2±50.9pg/ml infemales and 40.7±23.8pg/ml in males; TT concentration was0.49±0.29 and 4.3±1.6ng/ml respectively. All symptoms measureswere higher in females (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafemales (p=0.02); there was no difference in tear osmolarity. Infemales, increased ocular symptoms correlated with higher levels ofE2 (DEQ5 Rho=0.36, p=0.01; dryness Rho=0.36, p=0.01; FBRho=0.37, p=0.01). Higher TT in females correlated with more FBsensation (Rho=0.30, p=0.03) and lower tear volume (Rho=-0.30,p=0.04). No association was found between tear osmolarity andhormone levels in females. In males, no evidence of a relationshipbetween hormone levels and ocular symptoms or tear parameters wasapparent. Although concentrations of E2 and TT were reduced withage in females (E2 Rho=-0.36, p=0.01; TT Rho=-0.37, p=0.01), therewas no association between age and ocular symptoms in either malesor females.Conclusions: Higher circulating levels of sex steroid hormonesappear to play a role in increased symptoms of dry eye in femaleswithout ocular surface disease, but not in males. This effect does notappear to be influenced by age. More detailed analysis andexploration of co-related factors such as levels of free testosterone iswarranted to further explore these relationships in thepathophysiology of dry eye.Commercial Relationships: Blanka Golebiowski, TearLab (F);Ulrike Hampel, None; Noor Badarudin, None; Isabelle Jalbert,Blackmores Limited (F), TearLab Corporation (F); Michele C.Madigan, None; Fiona Stapleton, NoneProgram Number: 970 Poster Board Number: B0275Presentation Time: 1:00 PM - 2:45 PMCorrelation of Tear Meniscus Dimensions with ClinicalParameters of Ocular Surface Disease in Subgroups of Dry EyeCynthia I. Tung 1, 2 , Andrew F. Perin 1 , Koray Gumus 1 , Stephen C.Pflugfelder 1 . 1 Ophthalmology, Baylor College of Medicine, Houston,TX; 2 Ophthalmology, University of Texas Medical Branch,Galveston, TX.Purpose: Evaluate the relationship between tear meniscusdimensions and parameters of ocular surface disease in subcategoriesof dry eye.Methods: Prospective analysis comparing tear meniscus dimensionsto clinical ocular surface parameters was performed for 128 eyesfrom 64 subjects. Tear meniscus height (TMH) and tear meniscusarea (TMA) were measured using optical coherence tomography(Optovue). Ocular surface parameters included tear break-up time(TBUT), corneal staining, conjunctival staining, and Ocular SurfaceDisease Index (OSDI). Study groups included meibomian glanddysfunction (MGD) (n=23; OSDI>20; TBUT20; TBUT

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaGroup, IOBA-University of Valladolid, Valladolid, Spain;2 Networking Research Center on Bioengineering, Biomaterials andNanomedicine (CIBER-BBN), Valladolid, Spain; 3 Schepens EyeResearch Institute/Massachusetts Eye and Ear, Boston, MA;4 Department of Ophthalmology, Harvard Medical School, Boston,MA.Purpose: To determine if Th1 and Th2 pro-inflammatory cytokinesaffect cultured rat conjunctival goblet cells and to measure theireffects on proliferation, intracellular calcium levels ([Ca 2+ ] i ) and highmolecular weight glycoconjugate secretion that includes MUC5AC.Methods: Rat conjunctival goblet cells were grown from tissueexplants. Passage 1 cells were cultured in supplemented RPMImedium. The expression of goblet (CK-7) and stratified squamous(CK-4) specific cell markers was analyzed by immunofluorescenceand the lectin UEA-1 was used to identify goblet cell secretoryproducts. To evaluate proliferation cells were serum starved for 24 h,treated with the cytokines IFN-γ (3ng/ml), IL-4 (10ng/ml), IL-5(10ng/ml) or IL-13 (10ng/ml) for 24 h and then stimulated with thecholinergic agonist carbachol (Cch) or the allergic mediatorhistamine for 2 h. Proliferation was measured by WST-8 assay (n=3).To measure [Ca 2+ ] i , cells were loaded with fura2 and analyzed usingInCyte Im2TM Ratio Imaging System. The effect of Cch andhistamine on [Ca 2+ ] i was studied in cells treated for 15 min or 24 hwith cytokines or buffer (n=5). High molecular weightglycoconjugate secretion was measured in supernatants fromuntreated and 24 h cytokine-treated cells using an enzyme-linkedlectin assay (n=3).Results: Rat cultured cells expressed goblet cells markers (CK-7 andsecretory products identified by UEA-1), but not stratified epithelialmarker (CK-4). The Th1 cytokine IFN-γ decreased goblet cellproliferation by 0.79 fold, whereas Th2 cytokines IL-4, IL-5 and IL-13, and histamine increased it by 1.94, 2.65, 2.89 and 2.46 foldrespectively. IFN-γ used for 15 min significantly blocked Cchmediatedincrease in [Ca 2+ ] i (p = 0.009), and IL-4 and IL-13 had thesame effect on histamine-mediated [Ca 2+ ] i increase after 15 min preincubation(p=0.006 and p=0.003, respectively). When cells wereincubated with cytokines for 24 h, only IL-13 maintained theblockade (p=0.037). IFN-γ significantly blocked Cch-inducedsecretion (p=0.002), but Th2 cytokines did not have significanteffects on histamine-induced secretion.Conclusions: Th1- (IFN-γ) and Th2- (IL-4, IL-5 and IL-13) derivedcytokines have opposite effects on proliferation and secretion fromcultured rat goblet cells. These findings could explain the differencesin goblet cell function found in inflammatory ocular surface diseases,such as dry eye and allergic conjunctivitis.Commercial Relationships: Laura Garcia-Posadas, None; DayuLi, None; Robin R. Hodges, None; Marie A. Shatos, None;Yolanda Diebold, None; Darlene A. Dartt, NoneSupport: FEDER-CICYT Grant MAT2010-20452-CO3-01 and FPIScholarship Program BES-2011-046381 and EEBB-I-12-05371(Ministry of Science and Innovation, Spain), and Regional JCyLGrant VA132A11-2. NIH EY019470.Purpose: Benzalkonium chloride (BAK) is the most commonlyfound preservative in eye drops, and has been shown to cause ocularsurface inflammation and toxicity. Lacritin is a human tearglycoprotein secreted from the lacrimal glands that has been found tobe cytoprotective. This study was designed to determine if theprosecretory and mitogenic properties of lacritin confer protection toa cultured human corneal epithelial (HCE) cell line, CRL-11515, andprimary HCE cells after exposure to the ocular preservative agentBAK.Methods: Recombinant human lacritin and negative control fragmentC-25 were cloned into intein fusion vectors, expressed in E. coli, andpurified on chitin beads and DEAE Sepharose. Metabolic curveswere established after exposure of subconfluent CRL-11515 cells toBAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II)provided a measure of autophagy in CRL-11515 cells exposed tolacritin and/or BAK.Results: BAK reduced CRL-11515 cellular metabolic activity in atime and dose dependent manner. BAK-induced cellular stress wasevident by elevated autophagy that increased with risingconcentrations of BAK compared to control (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaLacritin treatment of CRL-11515 cells rescues BAK inducedautophagy. (A) LC3-II increased in CRL-11515 cells treated withBAK (0.004%) compared to control (**P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaindividual examiner reliability suggests that experience is animportant factor in reliability.Commercial Relationships: Stephanie Cox, None; CatherineVuong, None; Lisa Jones-Jordan, None; Kelly K. Nichols, None;Jason J. Nichols, Vistakon (R), Vistakon (F), Alcon (R), Alcon (F),Bausch and Lomb (R)Support: NIH/NEI grant R01 EY015519, NIH/NEI T35 EY007088Program Number: 976 Poster Board Number: B0281Presentation Time: 1:00 PM - 2:45 PMMatrix Metalloproteinases-8, -9 and Myeloperoxidase areElevated in the Tears of Patients with Ocular CicatricialPemphigoid and Stevens-Johnson SyndromeSamer N. Arafat 1, 2 , Ana M. Suelves 3 , Sandra J. Spurr-Michaud 2 ,James Chodosh 1 , C. Stephen Foster 3 , Claes H. Dohlman 1 , Ilene K.Gipson 2 . 1 Massachusetts Eye and Ear Infirmary, Boston, MA;2 Schepens Eye Research Institute, Massachusetts Eye and EarInfirmary, Boston, MA; 3 Massachusetts Eye Research and SurgeryInstitution, Cambridge, MA.Purpose: To determine levels of matrix metalloproteinases (MMPs),tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) andinflammatory marker myeloperoxidase (MPO) in the tears of patientswith ocular cicatricial pemphigoid (OCP) and Stevens-Johnsonsyndrome (SJS).Methods: Tears were collected from 7 SJS, 38 OCP and 40 postcataract(control) eyes. 60μL of sterile balanced salt solution wasinstilled on each eye and tears washes were recovered with amicropipette. The tear concentrations of MMP-2, -3, -7, -8, -9 and -12 (ng/mL) in each eye were measured using a Fluorokine® HumanMMP MultiAnalyte Profiling (MAP) Kit. TIMP-1 and MPOconcentrations (ng/mL) were measured using a Fluorokine® humanCardiac B MAP Kit. Both assays were run on a Bio-Rad Bio-Plexanalyzer powered by Luminex® technology. Total MMP activity wasmeasured fluorometrically using an OmniMMP RED fluorogenicsubstrate. Values for all assays were standardized to the protein massloaded in each assay.Results: The concentrations (ng/μg total protein) of MMP-8, -9 andMPO were statistically higher in tears of patients with SJS (4.3 ± 2.0,10.2 ± 4.5 and 6.7 ± 2.0, respectively) than in OCP (0.4 ± 0.2, 0.9 ±0.4 and 1.5 ± 0.4, respectively), and both were significantly higherthan in control (0.01 ± 0.003, 0.06 ± 0.01 and 0.4 ± 0.1, respectively).The ratio of MMP-8/TIMP-1 and MMP-9/TIMP-1 were statisticallyhigher in SJS (7.4 ± 3.4 and 16.2 ± 6.6, respectively) than in OCP(1.1 ± 0.6 and 4.6 ± 2.5, respectively) and both were significantlyhigher than in control (0.01 ± 0.003 and 0.05 ± 0.01, respectively).The total MMP specific activity (RFU/min/μg protein) was higher inSJS (25.02 ± 10.60) than in both OCP (4.75 ± 2.73) and control (1.25± 0.87). Spearman rank correlation tests showed significantcorrelations between MMP-8 and MMP-9, and between MMP-9 andMPO across all groups. MMP-8 correlated with MPO in control andOCP patients (p < 0.0001), and to a lesser extent with SJS (p =0.066).Conclusions: Since activated neutrophils are known to be a source ofMMP-8, -9 and MPO, the high levels of these enzymes in the tears ofSJS and OCP patients and the strong correlation between MMP-8,MMP-9 with MPO suggest that inflammatory cells are the primarysource of the elevated enzymes. In addition to MMP-8 and MMP-9,MPO was found to be a marker of inflammatory ocular surfacedisease.Commercial Relationships: Samer N. Arafat, None; Ana M.Suelves, None; Sandra J. Spurr-Michaud, None; James Chodosh,Alcon (C), Allergan (C), 3-V Biosciences (C), Novabay (C); C.Stephen Foster, Abbott Medical Optics (C), Abbott Medical Optics(F), Alcon Laboratories, Inc. (C), Alcon Laboratories, Inc. (F),Allergan, Inc. (C), Allergan, Inc. (F), Eyegate Pharmaceuticals, Inc.(I), Eyegate Pharmaceuticals, Inc. (F), IOP Opthalmics (C), IstaPharmaceuticals (C), Lux Biosciences, Inc. (C), Lux Biosciences,Inc. (F), Novartis Pharmaceuticals Corporation (C), NovartisPharmaceuticals Corporation (F), XOMA Ltd (C); Claes H.Dohlman, None; Ilene K. Gipson, NoneSupport: Boston Keratoprosthesis Fund (MEEI), NEI RO1 03306 toIKGProgram Number: 977 Poster Board Number: B0282Presentation Time: 1:00 PM - 2:45 PMAutologous Serum Tears for Treatment of Dry Eye Syndrome inGraft Versus Host DiseaseRyan J. Fante, Roni M. Shtein, Shahzad I. Mian, Munira Hussain.Ophthalmology and Visual Sciences, UM Kellogg Eye Center, AnnArbor, MI.Purpose: To evaluate the effectiveness of autologous serum tears fortreatment of severe dry eye syndrome (DES) in patients with GraftVersus Host Disease (GVHD).Methods: Retrospective review of 30 eyes of 15 patients with GVHDand severe DES was performed at baseline and after initiatingtreatment with autologous serum tears. Severity of ocular surfacedisease was evaluated using the Schirmer test, fluorescein stainingand Ocular Surface Disease Index (OSDI). Paired t-tests wereperformed to compare baseline assessment to post-treatment scores.Results: The study population included 15 total patients, 7 femaleand 8 male; the average age was 54 years (s.d =9, range 40-67).Follow-up duration averaged 11 months (range 1-40), and 60% wereconcurrently using the Prosthetic Replacement of Ocular SurfaceEnvironment (PROSE) lens. Patients had an average of 3 blooddraws (s.d.=2, range: 1-8) for serum tears in this time period.There was a statistically-significant improvement in OSDI scores at 1month and final time-points compared to pre-treatment (baseline:59.7 +/- 27.0; 1 month: 42.5 +/- 22.1, p=0.03; 3 month: 49.5 +/-37.0,p=0.07; final: 40.1 +/- 26.7, p=

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: This retrospective study included 23 patients who received20% autologous serum tears (4-8 x/day), were on concurrent antiinflammatorytreatment, and were followed with serial laser IVCM.Only patients were included who did not have any change in antiinflammatoryregimen during the follow-up period. Three IVCMimages of one randomly selected eye for each patient were analyzedby two masked observers for DC density. Images were analyzed forbaseline, as well as 3 and 6 months after treatment with serum tears,and compared to 23 age-matched control subjects. Student’s t-testand ANOVA were used for analysis.Results: Nineteen female and 4 males with a mean age of 53.3 ±18.92 years (range, 19-97 years) were included. The indications forusing serum tears were dry eye disease (20 eyes) and keratoneuralgia(3 eyes). At baseline, DC density was significantly higher in patientswith dry eye disease (55.8 ± 9.9 cells/mm2, P=0.01), but not inkeratoneuralgia (40.3 ± 18.4, P=0.37), compared to the control group(19.1± 3.1). DC density increased from 53.8 ± 8.9 cells/mm2 beforetreatment, to 103.1 ± 22.4 at 3 months (191% increase, P=0.04) andto 165.1 ± 57.8 at 6 months (307% increase, P=0.06).Conclusions: Treatment with autologous serum tears may beassociated with increased density of corneal epithelial DC in patientswith concurrent anti-inflammatory treatment, demonstrating changesin corneal immunity. Additional prospective clinical trials arerequired to confirm the data and determine clinical implications.Commercial Relationships: Ahmad Kheirkhah, None; ShrutiAggarwal, None; Bernardo M. Cavalcanti, None; DeborahWitkin, None; Nadia Wong, None; Monique Trinidad, None;Candice Williams, None; Reza Dana, Allergan (C), Alcon (C),Bausch & Lomb (C), Eleven Bio (I), GSK (F), Novabay (C),Revision Optics (C), Novaliq (C), RIgel (F); Pedram Hamrah, NoneSupport: NIH K08-EY020575, Research to Prevent BlindnessCareer Development Award, Falk Medical Research Trust, NewEngland Corneal Transplant Research Fund140 Cell and Molecular Biology and Stem CellsSunday, May 05, 2013 1:00 PM-2:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 979-1023/B0284-B0328Organizing Section: CorneaProgram Number: 979 Poster Board Number: B0284Presentation Time: 1:00 PM - 2:45 PMHuman Adipose-Derived Stem Cells Support the Growth ofLimbal Stem/progenitor CellsHua Mei, Martin N. Nakatsu, SHEYLA GONZALEZ, Elfren R.Baclagon, Sophie X. Deng. Ophthalmology, Jules Stein Eye Institute,UCLA, Los Angeles, CA.Purpose: To examine whether human adipose-derived stem cells(ASCs) could support the growth of human limbal stem/progenitorcells (LSCs).Methods: Three different culture methods were tested, includingculturing the LSCs directly on the feeder cells (regular method), a 3-dimensional (D) culture system (sandwich method) and a 3-D culturesystem coated with fibrin (fibrin method). Freshly isolated limbalepithelial cells (LECs) in single cell suspension or cell sheets wereco-cultured with the feeder cells for 2 weeks. Cultured LECs wereexamined for their cell morphology, proliferation rate, p63alphabrightpopulation and expressions of putative LSC markers, ABCG2,ΔNp63, N-cadherin and keratin (K) 14, and the differentiation markerK12. LECs cultured on 3T3-J2 feeder cells using the regular methodwere used as a control.Results: Single LECs cultured on ASCs had limited proliferation anddisplayed differentiated morphology in all three culture methods.When LECs were cultured as cell sheets, the sandwich methodsupported the highest proliferation rate for ASCs, which was 4.0-foldhigher than the control (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConclusions: These findings demonstrated that periostin is a novelprotein that is mainly expressed by basal layer of human limbalepithelial cells and co-localized with p63. Periostin expression isassociated with higher proliferative capacity and less differentiationconditions in HCECs. Periostin may have a potential role inmaintaining the properties of human corneal epithelial progenitorcellsCommercial Relationships: Yangluowa Qu, None; Cheng Li,None; Wei Li, None; Zuguo Liu, Bausch&Lomb (R), Allergern (R),Alcon (R), Santen (R)Support: supported by Chinese National key sencientific researchproject 2013CB967003Program Number: 981 Poster Board Number: B0286Presentation Time: 1:00 PM - 2:45 PMDifferentiation Potential of Limbal Fibroblasts and BoneMarrow Mesenchymal Stem Cells to Corneal Epithelial CellsKishore Reddy Katikireddy, Reza Dana, Ula V. Jurkunas. SchepensEye Research Institute and Massachusetts Eye and Ear, Departmentof Ophthalmology, Harvard Medical School., Boston, MA.Purpose: It has been shown that human limbal fibroblasts (LFs) andbone marrow mesenchymal stem cells (BM MSCs) are multipotent.Specifically, a side population of stage-specific embryonic antigen-4(SSEA4)-positive LFs differentiates into a variety of cell types. Thepresent study compared the stem cell characteristics of SSEA4+ andSSEA4- LFs to those of BM MSCs, and determined their potential todifferentiate into the corneal epithelial phenotype.Methods: Human cadaveric limbal tissue (n=6) was treated withdispase to remove the epithelium and endothelium. Single cellsuspensions were obtained by digesting the stroma with 0.025%trypsin. Stem cell enrichment was performed by exposure of BMMSCs and LF cells in KnockOut ESC/iPSC medium for 12-15 days.LF and BM MSCs were sorted for SSEA4+ and SSEA4- cells byMagnetic-Activated Cell Sorting. Cell doubling time (CDT), stemcell (SC) marker analysis, and colony forming efficacy (CFE) wereperformed on sorted LF and BM MSCs. Epithelial phenotype wasachieved using induction and differentiation media. Differentiatedcells were characterized for corneal cytokeratins (CKs).Results: After separation, enriched LFs and BM MSCs, 97.4 ± 0.6%and 93.5 ± 0.7% SSEA4+ subgroups were achieved, respectively.The CDT of SSEA4+ LFs was 102 ± 1 hr and SSEA4- LFs was 58.2± 1.5 hrs (p=0.002). CDT of SSEA4+ BM MSCs was 105 ± 1 hr. andSSEA4- BM MSC was 56.3 ± 2 hrs (p=0.002). LF and BM MSCsubgroups were negative for pan-cytokeratin. After enrichment,SSEA4+ cells showed the ability to form cell aggregates, whileSSEA4- cells were mostly adherent to the culture plates. Thetranscript levels of SC markers OCT4, SOX2, Nanog and Rexo1were higher in SSEA4+ than SSEA4- groups of LF and BM MSCs(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To study the role of ocular master regulator, Pax6, in thedysregulation of keratinocyte progenitor cells (PCs) during T cellmediatedkeratoconjunctivitis sicca (KCS) with keratinizingsquamous metaplasia (SQM).Methods: Aire KO mice and immunodeficient (scid) recipients ofAire KO-derived autoreactive CD4+T cells were used as models ofautoimmune SQM. Proliferative activity was measured by the densityof Ki67+ basal cells, designated as the mitotic index (MI). Therelative length of S phase in the 4-phase cell cycle was obtained bydividing the density of BrdU+ cells (labeled by a single BrdUpulsing) by the MI. Activated PCs, transient amplifying cells (TACs),postmitotic cells (PMCs), young terminally differentiated epithelialcells (TDCs) and immune effector cells were labeled by continuousBrdU pulsing for 7 days. Label-retaining cells including slow-cyclingSCs, long-lived PMC/TDCs and memory T cells were visualizedafter a 21-day BrdU. Local adenoviral transfer of Pax6 was employedto assess Pax6’s modulatory effects on PC’s self-renewal anddifferentiation in Aire KO’s SQM tissue.Results: Compared to wild type mice, the MI of p63+ PCs increased3.1±1.2 fold in the limbus and cornea of Aire KO mice. Such anincrease in proliferation was accompanied by a 42% decrease in Pax6transcripts, a 52% decrease in scid recipients of Aire KO-derivedCD4+ T, and an 83% decrease in clonal cells of Aire KO PCscultivated on 3T3 feeders (p < 0.05, n=5 per group). BrdUincorporation kinetics revealed a 1.9-fold, 2.1-fold, and 0.5-foldlabeling index in Aire KO mice following 1-shot, 7-day pulse and 21-day chase protocols, respectively (all p < 0.05 vs WT at 1-fold).Together, these results suggested downregulation of Pax6 isassociated with an accelerated epithelial turnover and a relativelyshorter S phase in rapid cycling cells. Adenoviral induction of Pax6in Aire KO basal PCs restored the corneal phenotype.Conclusions: The downregulation of Pax6 in basal PCs during ocularautoimmune SQM is mediated by PC- and limbal niche-reactiveCD4+ T effectors. Restoration of Pax6 reverses aberrant epidermallineagecommitment suggests adjuvant Pax6 gene therapy maycompliment immunosuppressants as a novel therapeutic approach tomanage SQM disease in sicca patientsCommercial Relationships: Ying-Ting Chen, None; Nancy A.McNamara, NoneSupport: R01 EY016203-06Program Number: 984 Poster Board Number: B0289Presentation Time: 1:00 PM - 2:45 PMA Comparison Between Three Mouse Models of Corneal LimbalStem Cell DeficiencyNeda -. Afshar, Asadolah Movahedan, Ali R. Djalilian.Ophthalmology and Visual science, University of Illinois at Chicago,Chicago, IL.Purpose: Creating a mouse model that best represents the corneallimbal stem cells deficiency (LSCD)Methods: Three different methods were used to create mouse modelsof corneal LSCD in C57BL/6J mice (6 eyes per group). Group 1:Complete mechanical removal of corneal epithelium with a bluntscraper from limbus to limbus. Group 2: Complete mechanicalcorneal epithelial removal followed by heat cauterization, 360degrees all around the limbus. Group 3: Applying 0.1% sodiumhydroxide (NaOH) to the limbal area for 30 seconds using 2mm filterpaper discs, after removing the whole corneal epithelium with a bluntscraper. Slit lamp examination was performed to evaluate the degreeof fluorescein staining and corneal neovascularization compared tocontrol eyes. Eyes were removed for whole mount immunostainingfor Cytokeratin 12 and Cytokeratin 8.Results: All groups showed evidence of late fluorescein staining withneovascularization consistent with limbal stem cell deficiency. Theresults in group 1 showed more variability with varying degrees ofstaining/neovascularization and gradual recovery of corneal typeepithelium by two months in some eyes. The results were lessvariable in group 2 with more stable pattern of LSCD over time in alleyes. Group 3 demonstrated the most severe phenotype with frequenthemorrhage and significant opacification of the corneal stroma. Longterm studies with quantitative comparison is currently underway.Conclusions: Mechanical corneal epithelial removal in combinationwith thermal injury (group 2) provides a more reproducible method(compared to mechanical removal alone) and a less destructivemethod (compared to alkali injury) for developing corneal LSCD inmice.Commercial Relationships: Neda -. Afshar, None; AsadolahMovahedan, None; Ali R. Djalilian, NoneSupport: Career development grant K08EY017561-A1 to A.R.D.,Core grant EY01792 from the NIH, and by the Cless FamilyFoundation. A.R.D. is the recipient of a Career Development Awardfrom Research to Prevent Blindness.Program Number: 985 Poster Board Number: B0290Presentation Time: 1:00 PM - 2:45 PMOptimization of Ex Vivo Expansion of Limbal EpithelialProgenitors by Maintaining Native Niche Cells on DenudedAmniotic MembraneMegha A. Mahabole, Szu-Yu Chen, Scheffer C. Tseng. R&D, TissueTech Inc., Miami, FL.Purpose: Transplantation of ex vivo expanded limbal epithelialprogenitor cells (LEPC) on denuded amniotic membrane (dAM) is analternative solution for treating corneal blindness due to limbal SCdeficiency. We reported isolation and expansion of limbal niche cells(NCs) that express embryonic SC (ESC) and angiogenic markers inserum-free modified ESC medium (MESCM). We question whetherthe conventional ex vivo expansion protocol of LEPC on dAM can befurther optimized by maintaining limbal NCs by switching the culturemedium from supplemented hormonal epithelial medium (SHEM) toMESCM.Methods: A limbal cluster was obtained from each 1/12 piece of thehuman corneosclera ring and was cut 1 mm from within and beyondthe anatomic limbus and further digested in 1 mg/ml collagenase Afor 18 h at 37 degrees celsius, transferred to dAM and cultured inSHEM or MESCM. On Day 8-10, epithelial outgrowth sheets wereremoved by dispase and subjected to real-time qPCR andimmunostaining for expression of corneal markers (p63α, pax6, K12)and NC markers (FLK-1, NG2, PDGFR-B, CD31 and CD34). 1000single cells were seeded on 6-well dish containing MMC-3T3 for 12-14 days before rhodamine B staining.Results: qPCR of epithelial outgrowth on dAM at Day 8 in SHEMshowed a significant loss of corneal and ESC markers when compareto freshly collagenase-isolated limbal clusters, suggesting that theconventional ex vivo expansion method was not optimized.Nonetheless, the resultant monolayer sheets and cell size wereconsistently smaller in MESCM than in SHEM on Day 8. qPCRfurther confirmed significant upregulation of NC markers in©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaoutgrowth sheets expanded in MESCM when compare to that inSHEM on Day 10. Cytospin preparations further revealed asignificantly higher percentage of PCK-/ Vim+ cells found inMESCM (14.8%, n= 1352) than SHEM (0.6%, n=1531, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatransient amplifying (TA) progeny, we compared miRNA expressionpatterns in the stem cell-enriched limbal epithelium versus the TAcell-enriched corneal epithelium at several developmental stages.Methods: Laser capture microdissection (LCM) was used toprecisely isolate fresh populations of limbal and corneal epitheliumfrom unfixed cryosections, thereby acquiring miRNA expressionprofiles from relatively “quiescent” cells. LCM was performed onmouse limbal and corneal epithelium from postnatal (PN) mice atdays: 3, 7, 14 and 28. Total RNA was isolated from triplicate samplesand miRNA expression profiles were obtained using qRT-PCR arrays(Exiqon). We conducted loss-of-function experiments with miR-103and -107 in cultured human limbal keratinocytes (HLEKs), inconjunction with Northern, Western, and immunohistochemicalanalyses. We used luciferase reporter assays in HeLa cells to identifypotential targets of miR-103 /107.Results: miR-103 /107 were preferentially expressed in the limbalepithelium at each of the developmental time points. Northern blotanalysis revealed higher expression of miR-103 /107 in HLEKsversus human corneal epithelial keratinocytes. Treatment of HLEKswith antagomirs to either miR-103 or miR-107, resulted in an initialloss of cell-cell contacts, which yielded small, rounded cellscompared to control-treated (antago-124) HLEKs. Consistent with aloss in cell-cell contact, E cadherin (E-cad) was no longerimmunohistochemically detectable at the cell-cell junctions, 3hr postantagotreatment. A marked downregulation was noted in p120catenin and E-cad protein, essential components of adherensjunctions. NEDD9, a non-catalytic scaffolding protein that negativelyregulates localization of E-cad and promotes its degradation, wasshown to be a target of miR-103/107.Conclusions: Maintenance of cell-cell contact and communicationbetween stem cells and TA cells are crucial for stem cell nichehomeostasis. The positive role that miR-103/107 plays in E-cadherinmediatedlimbal epithelial cell-cell contacts via down-regulation ofNEDD9 helps stabilize this aspect of the stem cell niche and maintainstem cell-TA cell integrity.Commercial Relationships: Julia V. Katsnelson, None; JongKook Park, None; Wending Yang, None; Han Peng, None; RobertM. Lavker, NoneSupport: NIH Grants EY06769, EY019463Program Number: 989 Poster Board Number: B0294Presentation Time: 1:00 PM - 2:45 PMComparison of Different Limbal Epithelial Stem Cell IsolationMethods to Improve the Epithelial Sheet Quality forTransplantationSHEYLA GONZALEZ, Martin N. Nakatsu, Hua Mei, Sophie X. Deng.Jules Stein Eye Institute UCLA, Los Angeles, CA.Purpose: To investigate different preparation methods of humanlimbal stem cells (LSCs) for cultivation using dispase II, collagenaseA and the explant culture.Methods: Limbal tissues were incubated with dispase II at 37°C for2 hours (h) or with collagenase A at 37°C or 4°C for 2h or overnightto obtain limbal epithelial cell sheets. Half of the cell sheets werefurther incubated with trypsin to achieve single cell suspension. Theexplant culture was done using a 2x2 mm limbal biopsy. Limbalepithelial cells and limbal tissues were cultured on growth-arrested3T3-J2 mouse cells in SHEM5 medium. The phenotype assessmentincluded stem/progenitor morphology and expression level of LSCputative markers including ABCG2, p63α, N-cadherin andcytokeratin (K) 14, the differentiation marker K12, and the stromalcell markers vimentin (Vim) and α-smooth muscle actin by qRT-PCRand immunohistochemistry.Results: No LSC growth on 3T3-J2 cells was achieved when limbaltissues were incubated with collagenase overnight at 37°C. Amongthe other collagenase treatments tested, the most efficient wasincubation at 37°C for 2h in the incubator. This isolation methodyielded the highest K14 + Vim - progenitor cell population (86.2%) anda moderate K14 - Vim + stromal cell number (9.7%). The LSCs had thebest stem/progenitor-like morphology and the highest level ofputative LSC markers. Collagenase but not dispase isolated the entireepithelial sheet with the underneath stromal cells. When thephenotype of cultured LSCs was compared among all three culturemethods, all LSCs displayed a stem/progenitor-like morphology.However, LSCs cultured from collagenase digestion had the highestmRNA level of putative LSC markers and the highest percentage ofp63α bright cells (21.7% vs. 6% from explant and 12.7% fromdispase, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Aaron Yeung, None; AndrewHopkinson, None; Harminder S. Dua, NoneProgram Number: 991 Poster Board Number: B0296Presentation Time: 1:00 PM - 2:45 PMCharacterization of Biomaterial free Cell Sheets Cultured fromHuman Oral Mucosal Epithelial CellsMee Kum Kim 1, 2 , Joo Hee Lee 3 , Ja Young Lee 1, 2 , Ah Young Koh 2 ,Yun Hee Kim 3 , Won Ryang Wee 1, 2 , Saewha Jeon 3 . 1 Ophthalmology,Seoul National University Hospital, Seoul, Republic of Korea;2 Laboraory of Corneal Regenerative Medicine and OcularImmunology, Seoul Artificial Eye Center, Seoul, Republic of Korea;3 Cutigen Research Institute, Tego Science Inc., Seoul, Republic ofKorea.Purpose: To investigate the characteristics of biomaterial free cellsheets cultured from oral mucosal epithelial cells both in vitro and ina limbal deficient animal model.Methods: Both human oral mucosal epithelial cells and limbalepithelial cells were cultured to form cell sheets with or withoutfibrin glue for 2 weeks. The resulting sheets were detached using abuffer containing 1% dispase and transplanted to limbal deficientrabbits which had been chemically injured with lamellar limbectomy.The adhesion stability of these biomaterial-free cell sheets wereevaluated three days after transplantation. In parallel, ColonyForming Efficiency(CFE) as well as the immunohistochemistry usingantibodies specific to proliferation and differentiation of epithelialcells were performed to characterize the cell sheets.Results: CFE in Human oral mucosal epithelial cells and limbalepithelial cells were measured to be 57.5% and 14%, respectively.K1, K3, and K4 were expressed in mucosal epithelial sheets while K3and K12 were in limbal epithelial sheets. The cell sheets werecomposed of three to six layers of stratified, well differentiatedmucosal epithelial cells with 87.5% viable cells present. The in vivoadhesion stability of biomaterial-free cell sheets of oral mucosalepithelial cells was comparable to that of fibrin-based counterpart.Conclusions: Our results suggest that biomaterial free cell sheets oforal mucosal epithelial cells can be a good candidate in the treatmentof limbal deficient diseases.Commercial Relationships: Mee Kum Kim, None; Joo Hee Lee,None; Ja Young Lee, None; Ah Young Koh, None; Yun Hee Kim,None; Won Ryang Wee, None; Saewha Jeon, Tego Science Inc. (E)Program Number: 992 Poster Board Number: B0297Presentation Time: 1:00 PM - 2:45 PMHuman adipose-derived stem cells promote wound healing ofcorneal epithelial cells in vitroLadan Espandar 1 , Tomas Blanco 1 , Rose Mathew 1 , Natalie A.Afshari 2 , Bruce Bunnell 3 , Daniel R. Saban 1 . 1 Ophthalmology, DukeUniversity, Durham, NC; 2 University of California San Diego, SanDiego, CA; 3 Tulane University, New Orleans, LA.Purpose: Human adipose-derived stem cells (hASCs) have beenshown to promote wound healing in the skin, and may thus havepotential application for ocular surface regenerative therapy andwound healing. The purpose of this study is to investigate the effectof hASCs on corneal epithelial cells (CECs) using an in vitro woundhealing model.Methods: An established in vitro epithelial wound-healing modelwas utilized in which a 0.5 mm scratch was made on a monolayer ofconfluent human corneal epithelial cells (hCECs). hASCs seeded on atranswell membrane was added to determine their effect on epithelialwound healing. Controls included scratched CECs without theaddition of hASCs, or mitomycin-C-treated scratched hCECs. Woundclosure was evaluated by time-lapse inverted microscopy for aminimum of 24 hours. The average wound width and migration speedat seven random areas were assessed and quantified digitally usingMetaMorph® software.Results: Wound closure in the condition containing CECs alone tookas long as 18 hours, whereas the addition of hASCs decreased thistime significantly to 13 hours. Likewise, without hASCs the averagewound width (166.6 µm) was significantly higher than with hASCs(138.7 µm, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Tingting Qian, None; Jiaxu Hong,None; JianJiang Xu, NoneProgram Number: 994 Poster Board Number: B0299Presentation Time: 1:00 PM - 2:45 PMTranscription factor screening of limbal vs. corneal epitheliumidentifies distinct patterns for SOX-9 and peroxisomeproliferator-activated receptor gamma (PPARG)Johannes Menzel-Severing, Matthias Zenkel, Friedrich E. Kruse,Ursula Schlotzer-Schrehardt. Department of Ophthalmology,University of Erlangen-Nuremberg, Erlangen, Germany.Purpose: To identify transcriptional regulators potentially involvedin limbal epithelial stem cell homeostasis and differentiation.Methods: Limbal and central corneal epithelial specimens wereobtained from four human post-mortem donor eyes using lasercapture microdissection. RNA extracted from these specimensunderwent linear amplification (MessageAmp II, Ambion). Sampleswere screened for differential expression of stem cell transcriptionfactor genes using quantitative real-time PCR arrays(SABiosciences). Candidate genes were confirmed using specificreal-time PCR hydrolysis probe assays (Roche) andimmunohistochemistry of frozen corneal sections.Results: Of 86 genes screened, nine appeared to be differentiallyexpressed, and were therefore investigated further (FOXP2,HOXA11, KLF2, SOX9, STAT3, WRN, MYC, DACH1, PPARG).Using individual assays, increased mRNA expression in limbalspecimens was confirmed for SOX9 and PPARG.Immunohistochemistry showed nuclear localization of SOX-9 inlimbal basal cells, whereas no specific staining was seen in centralcorneal epithelium. PPARG was detected within nuclei of basalepithelial cells of conjunctiva and central cornea, whileperinuclear/cytoplasmic staining was observed in small, basal cellclusters at the limbus.Conclusions: Transcription factor SOX-9 has been suggested toepithelium and the subependymal zone. Several reports haveidentified links to Wnt signaling transducer beta-catenin, for which arole in maintenance of limbal stem cells has been proposed. PPARGis a nuclear receptor associated with differentiation pathways ofadipocytes and epithelial cells as well as growth inhibition ofcarcinoma cells. Nuclear export of PPARG is mediated byMAP2K1/MEK1, which invites to speculate about a possibleupstream activity of the MAPK-pathway in putative limbalprogenitors. Further research is warranted to elucidate the functionalinvolvement of SOX-9 and PPARG in corneal epithelial celldifferentiation, and to assess the usefulness of modulating theiractivity in the context of cell therapy.Commercial Relationships: Johannes Menzel-Severing, None;Matthias Zenkel, None; Friedrich E. Kruse, None; UrsulaSchlotzer-Schrehardt, NoneProgram Number: 995 Poster Board Number: B0300Presentation Time: 1:00 PM - 2:45 PMWnt promotes proliferation of corneal epithelial progenitor cellsin xeno-feeder free cultivationKyung-Sun Na 1, 2 , Jeewon Mok 2 , Jungmook Lyu 2 , Choun-Ki Joo 1, 2 .1 Department of Ophthalmology, The Catholic University of Korea,Seoul, Republic of Korea; 2 Catholic Institutes of Visual Science, TheCatholic University of Korea, Seoul, Republic of Korea.Purpose: This study was to establish a simple, xeno-feeder freemethod for cultivating human corneal limbal explants , and also toexplore the effect of Wnt signaling on epithelial progenitor cellproliferation in this cultivation system in vitro and in vivo.Methods: The limbal tissue explants from cadaveric donor wascultured in Isocove’s Modified Dulbecco’s Medium (IMDM) and lowcalcium Panserin 801 medium in 1:1 ratio. The outgrowing cells wereexamined to characterize with flow cytometry,immunohistochemistry, and real-time PCR (RT-PCR). Sprague-Dawley male rats after alkali injuries using 1N NaOH were used forin vivo verification, after which cultivated epithelial sheets weretransplanted. Corneal opacity, re-epithelialization, andneovasculazation was observed for 2 week, and the tissue sectionsanalyzed with hematoxylin and eosin stain (HE stain). Conditionedmedia from L cells secreting Wnt-3a (Wnt3a-L-CM) was used in thecultivation system, and morphological changes and gene expressionlevel were observed.Results: There was migration of fibroblast like stromal cells fromlimbal explants initially, and then, epithelial cells migrated andgrown on stromal cells as an autofeeder layer, which was revealed bymorphological and immunohistochemical methods. RT-PCR showedthat the expression of epithelial progenitor cells are more intensecompared to fresh limbal tissue. Side population (SP) cells weredetected 0.43 ± 0.04 % (n=5) of the primary culture. Flow cytometryresulted 49.12% of E-cadherin, 40.44% of p63, and 44.55% ofABCG2 identified in the cells from explants. Maintaining cornealtransparency without neovascularization was observed in rats aftercultivated epithelial sheets transplantation for 2 weeks. Predominantincreased tightly packed epithelial cells with Wnt3a-L-CM wereobserved compare to the control CM. ABCG2, p63, Lef1, and CK3was increased in Wnt3a-L-CM.Conclusions: This explants culture system with combining media touse stromal cells as autofeeder layer, showed to expand sufficientlimbal epithelial progenitor cells in vitro and to be transplantedrestoring transparency. Also, these findings demonstrated that Wntsignaling play an important role in the proliferation of limbalepithelial progenitor in the proposed cultivation system. This studymay have clinical impact on the expansion of corneal epithelialprogenitor cells for ocular surface reconstruction.contribute to progenitor cell regulation in hair follicle, intestinal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Kyung-Sun Na, None; Jeewon Mok,None; Jungmook Lyu, None; Choun-Ki Joo, NoneProgram Number: 996 Poster Board Number: B0301Presentation Time: 1:00 PM - 2:45 PMHuman limbal epithelium expanded in a complex medium ormedium with human serum: a comparison of morphology andcytokeratin expressionMeeta Pathak 1 , Kristiane Haug 1 , Aboulghassem Shahdadfar 1 , EliGulliksen 1 , Magnus Roeger 3 , Liv Drolsum 1 , Jon K. Slettedal 1 , BjornNicolaissen 1 , Katerina Jirsova 2 . 1 Centre for Eye Research, Dept. ofOphthalmology, Oslo University Hospital, Oslo, Norway;2 Laboratory of the Biology and Pathology of the Eye, GeneralTeaching Hospital and Charles University, Prague, Czech Republic;3 Dept. of Pathology, Oslo University Hospital Ullevål, Oslo, Norway.Purpose: We compared aspects of ultrastructural morphology andthe expression of selected cytokeratins in human limbal epitheliumexpanded in a complex medium (COM), which contains variousrecombinant growth factors, hormones, Cholera toxin and fetalbovine serum (FBS), and culture medium with human serum (HS) asthe only growth supplement (HS).Methods: Limbal biopsies retrieved from corneo-scleral rings wereplaced with the epithelium facing down on the basement membranesurface of samples of human amniotic membranes. The biopsies werecultured in parallel in either COM or HS at 37°C, 5% CO2, and 95%air. Culture medium was changed every 2-3 days and the epitheliumwas expanded for 3 weeks. Specimens were examined by lightmicroscopy, transmission electron microscopy (TEM) andimmunohistochemistry. Expression of CKs 3, 7, 12, 14, and 19 wasevaluated using fluorescence microcopy, and intensity was graded ona scale from 0 (negative) to 3 (strongly positive).Results: By TEM, cytoplasmic density and distribution ofintermediate filaments showed a similar pattern in epithelial cellsexpanded in both types of medium. In basal cells, the filaments wereloosely organized and inconspicuous apart from those associated withintercellular junctions. Clusters of linear or wavy intermediatefilaments (tonofilaments) were encountered to a varying extent in thecytoplasm of cells in the supra-basal layers.By immunohistochemistry, expression of CK3 was not detected(grade 0) in sections of expanded epithelium regardless of type ofmedium. Both COM and HS supported the expression of CK 7, 12,14 and 19, and grading of the samples for intensity of fluorescencerevealed a similar pattern in epithelium engineered in the two typesof medium. Intensity of fluorescence in sections of epithelium labeledfor CK12 and CK19 was evaluated as weak (grade 1), while theintensity in sections of epithelium labeled for CK7 and CK 14 wasevaluated as strongly positive (grade 3).Conclusions: Our findings indicate that COM and HS may equallysupport the expression of selected CKs, suggesting similar degrees ofepithelial proliferation and differentiation. Further, a difference indensity of cytoplasmic intermediate filaments between basal andsupra-basal cells indicates a maintained gradient for epithelialdifferentiation in both types of medium.Commercial Relationships: Meeta Pathak, None; Kristiane Haug,None; Aboulghassem Shahdadfar, None; Eli Gulliksen, None;Magnus Roeger, None; Liv Drolsum, None; Jon K. Slettedal,None; Bjorn Nicolaissen, None; Katerina Jirsova, NoneProgram Number: 997 Poster Board Number: B0302Presentation Time: 1:00 PM - 2:45 PMComparative gene expression analysis of human cornea limbalepithelial stem cells and differentiated corneal epitheliumGoran Petrovski 1, 2 , Reka Albert 1, 2 , Zoltan Vereb 2 , Morten C. Moe 3 ,Ole Kristoffer Olstad 4 , Andras Berta 1 , Laszlo Fesus 2 . 1 Department ofOphthalmology, University of Debrecen, Medical and Health ScienceCenter, Debrecen, Hungary; 2 Stem Cells and Eye ResearchLaboratory, Department of Biochemistry and Molecular Biology,University of Debrecen, Medical and Health Science Center,Debrecen, Hungary; 3 Department of Ophthalmology, Oslo UniversityHospital, Oslo, Norway; 4 Department of Medical Biochemistry, OsloUniversity Hospital, Oslo, Norway.Purpose: Limbal epithelial stem cells (LESCs) are responsible forcorneal epithelium regeneration. Specific molecular markers forLESCs have not been well defined. Our goal was to find new putativemarkers for these cells and to identify associated molecular pathways.Methods: Limbal tissue explants and central corneal epithelium wereharvested from cadavers (according to the Guidelines of the HelsinkiDeclaration). The explants were cultured ex vivo and expanded intoLESCs in a human serum containing medium. Genome-widemicroarray analysis was performed using Affymetrix GeneChipHuman Gene 1.0 ST Array containing more than 28,000 genetranscripts. Functional analysis using Ingenuity software was carriedout to identify pathways and molecules specific for LESCs.Results: LESCs showed upregulated expression of 10 top molecules(flavin containing monooxygenase (FMO) 1 and 2, fibronectin 1(FN1), kallikrein (KLK) 6 and 7, transcobalamin 1 (TCN1),semaphorin 3A (SEMA3A), annexin A3 (ANXA3), V-set domaincontainingT-cell activation inhibitor 1 (VTCN1) and heat shockprotein beta-8 (HSPB8), and downregulated expression of cartilageacidic protein 1 (CRTAC1), alcohol dehydrogenase class 4 mu/sigmachain (ADH7), hepatic leukemia factor (HLF), CD36, Doublecortindomain containing 5 (DCDC5), Diacylglycerol kinase beta (DGKB),protein prune homolog 2 (PRUNE2), anoctamin 4 (ANO4), deathassociated protein-like 1 (DAPL1) and carbonic anhydrase (CA6)compared to differentiated corneal epithelium (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSaghizadeh 1 . 1 Regenerative Medicine Institute, Cedars-Sinai MedicalCenter, Los Angeles, CA; 2 Ophthalmology and Cell Biology, StateUniversity of New York, Downstate Medical Center, Brooklyn, NewYork, NY; 3 University of California, Los Angeles, CA.Purpose: Human amniotic membrane (HAM) is a commonsubstratum for culturing limbal cells for future transplantation. Bestresults are obtained when amniotic epithelium is removed fromHAM. Most existing methods require long treatments and leave manycells attached to HAM. The purpose was to develop a reliable methodof HAM denuding with effective and fast removal of amnioticepithelium.Methods: Fresh HAM was mechanically separated from chorion, andcryopreserved in PBS with 10% DMSO. After thawing and washing,HAM was de-epithelialized by different methods: soaking in 0.02%EDTA in PBS at 37°C for 1 hour; EDTA followed by gentle scrapingwith electric toothbrush; EDTA followed by scraping with n-heptanolsoaked cotton tip; 125 μg/ml thermolysin in PBS at 37°C for 9 minfollowed by gentle mechanical scraping. Alternatively, HAM placedin CellCrownTM inserts was rubbed on the epithelial side for 10-30seconds with cotton tip soaked in 0.5 M NaOH and immediatelywashed in PBS. Denuded OCT-embedded HAM was cryosectionedand immunostained for typical components of limbal basementmembrane (BM), including laminin α2, γ1, and γ3 chains, α1/α2 typeIV collagen, perlecan, nidogen-2, and fibronectin. NaOH-denudedHAM was used to culture human telomerase-immortalized cornealepithelial cells, limbal cells from corneoscleral rims, and inducedpluripotent stem cells (iPSC) derived from limbal epithelial cells.Cultured cells were checked for putative stem cell marker expression(ΔNp63α, ABCG2, and keratins 14, 15, 17, and 19) byimmunostaining.Results: Control HAM was positive for all BM markers except forlaminin α2 chain that gave weak and inconsistent staining. HAM decellularizationwith EDTA or EDTA with n-heptanol left out manyadherent epithelial cells; rubbing with toothbrush produced localtears. Thermolysin and NaOH resulted in the best cell removal withcontinuous staining for all BM markers tested. However,thermolysin-treated HAM became fragile and could be easilydamaged during manipulation, which was not seen after NaOHdenuding. Corneal epithelial cell line, limbal cells, and limbalderivediPSC all grew well on NaOH-denuded HAM. Cultured cells,especially limbal cells were positive for putative stem cell markers.Conclusions: HAM de-cellularization with NaOH results in rapidand thorough amniotic cell removal, and ensures excellentpreservation of HAM structure and robust stem cell growth ondenuded HAM.Commercial Relationships: Alexander V. Ljubimov, None; DhruvSareen, None; Loren Ornelas, None; Anais Sahabian, None;David M. Hemmati, None; Chantelle A. Ghiam, None; William J.Brunken, None; Yaron S. Rabinowitz, None; Clive Svendsen,None; Mehrnoosh Saghizadeh, NoneSupport: NIH EY13431, CTSI grant UL 1RR033176, andRegenerative Medicine Institute grants.Program Number: 999 Poster Board Number: B0304Presentation Time: 1:00 PM - 2:45 PMExpansion of Human Corneal Epithelial Stem/Progenitor Cells inFeeder-Free Explant CulturesSophie X. Deng, Martin N. Nakatsu, SHEYLA GONZALEZ, Hua Mei.Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA.Purpose: To present a feeder-free culturing method of humancorneal epithelial stem/progenitor cells in vitro.Methods: Primary limbal epithelial cell (LEC) sheets isolated fromhuman sclerocorneal tissues were trypsinized to produce single LECsand co-cultured with growth arrested 3T3-J2 feeder cells for 14 days.Additionally, the outgrowth of LECs from limbal explant pieces wasalso cultured for 14 days in the presence or absence of 3T3-J2 feeder.The phenotype of the cultured LECs was assessed by their mRNAexpression level of putative stem cell markers and differentiationmarker by qRT-PCR and immunocytochemistry. The percentage ofp63 bright cells in each culture was assessed, and the cellproliferation was evaluated by the Ki67 expression and cell number.Results: We observed no significant difference in cultured LECmorphology among each culturing method. The LEC growth rateincreased over 9-fold in NF explant cultures compared to 3T3-J2explant cultures and the adjusted growth rate between NF culturesand 3T3-J2 single LEC cultures had similar yields (p>0.05). Geneexpression of putative limbal stem cell markers, ABCG2 and ΔNp63were elevated among NF cultures compared to the gold standard andexplants on 3T3-J2. We observed a 7.6-fold and 2.2-fold increase inABCG2 and ΔNp63 expression respectively when comparing NFexplant cultures to the gold standard, while there was only a 4.4-foldand 1.4-fold increase in ABCG2 and ΔNp63 expression respectivelywhen comparing 3T3-J2 explant cultures to the gold standard.However, we did observe an increase in K12 expression in NFexplant cultures when compared to the gold standard (6.2-fold).There was a large increase in Ki67 proliferation (4.0-fold) whencompared to the gold standard. There was no difference in Ki67expression between 3T3-J2 explant cultures and the gold standard.Finally, examination of p63α expression in each condition reviewedno discernable differences in the percentage of p63α bright cellsbetween the gold standard (9.0%) and NF explant cultures (10.0%),but we did see a decrease in the 3T3-J2 explant cultures (6%).Conclusions: 3T3 feeder cells may not be necessary for the growthof stem/progenitor cell population in the primary explant culture. Theexplants themselves may already contain niche factors that arerequired for the viability of corneal stem cells.Commercial Relationships: Sophie X. Deng, None; Martin N.Nakatsu, None; SHEYLA GONZALEZ, None; Hua Mei, NoneSupport: CIRM Grant TR2-01768, NIH Grant EY021797Program Number: 1000 Poster Board Number: B0305Presentation Time: 1:00 PM - 2:45 PMSox9, a determinant of hair follicle stemness, identifies a subset ofmurine basal corneal epithelial cells expressing putative stem cellmarkersRachel Sartaj 1 , Aihong Liu 1 , Elaine Fuchs 2 , Mark Rosenblatt 1 .1 Ophthalmology Department, Weill Cornell Medical College, NewYork, NY; 2 Laboratory of Mammalian Cell Biology andDevelopment, Rockefeller University, New York, NY.Purpose: To localize the expression of Sox9 in the cornea and toidentify genes co-expressed with this determinant of hair folliclestemness.Methods: Antibodies against Sox9 were used to immunostaincorneal sections from wild-type mice, and the nuclear localization ofSox9 determined by wide field and confocal microscopy.Localization of Sox9 within the cornea epithelium was confirmed byimmunostaining for GFP in Sox9-eGFP mice expressing GFP underthe control of the sox9 promoter region. Corneal epithelial cells wereisolated from Sox9-egfp mice via sequential enzymatic treatments,and the cell suspension used for FACS sorting to obtain purifiedpopulation of Sox9-expressing (GFP +) cells and Sox9 nonexpressing(GFP-) cells. RNA was separately isolated from GFP +and GFP - populations and qRT-PCR performed to determine therelative expression of putative corneal epithelial stem cell markersABCG2, p63 and N-cadherin (Cdh2) as well as the differentiationmarkers keratin-12 (Krt12) and involucrin (Ivl).©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: In the mouse cornea, Sox9 and GFP proteins were located inthe basal cells of central and limbal cornea epithelium in postnatalday 12 (P12) and adult mice (P70). The expression of Sox9 and GFPproteins are restricted to the basal layer of the corneal epithelium atP12 and increases to 2 layers as the Sox9 progeny divides from thebasal layer to one row above. Quantitative PCR experiments showeda significantly higher relative expression of known corneal epithelialstem cell markers in the Sox9- expressing cells obtained by FACSsorting. We found that there was up regulation of ABCG2 (5 fold),p63 (18 fold), and Cdh2 (3.6 fold), in the sox9 expressing cells. Incontrast, when we analyzed the corneal differentiation markers inthese cells, we found down regulation of Krt12 (5 fold) and Ivl (2.5fold).Conclusions: Sox9 may be a novel marker of corneal epithelial stemcells given its localization to basal corneal epithelial layers an its coexpressionwith other putative stem cell markers. A possible role forSox9 in mediating corneal epithelial stemness could have utility inthe diagnosis and treatment of corneal limbal stem cell deficiency.Commercial Relationships: Rachel Sartaj, None; Aihong Liu,None; Elaine Fuchs, None; Mark Rosenblatt, NoneSupport: Starr Foundation Tri-Institutional Stem Cell Initiative,NYSTEM, Research to Prevent BlindnessProgram Number: 1001 Poster Board Number: B0306Presentation Time: 1:00 PM - 2:45 PMLimbal mesenchymal stromal cells (L-MSC) displayimmunosuppressive properties across donor and speciesboundariesDamien G. Harkin 1, 2 , Laura J. Bray 2, 1 , Celena Heazlewood 3 , KerryAtkinson 3 . 1 School of Biomedical Sciences, Queensland University ofTechnology, Brisbane, QLD, Australia; 2 Queensland Eye Institute,Brisbane, QLD, Australia; 3 Mater Medical Research Institute,Brisbane, QLD, Australia.Purpose: We have evaluated the immunosuppressive properties of L-MSC with the view to using these cells in allogeneic cell therapies forcorneal disorders. We hypothesized that L-MSC cultures wouldsuppress T-cell activation, in a similar way to those established fromhuman bone marrow (BM-MSC).Methods: MSC cultures were established from the limbal stroma ofcadaveric donor eye tissue (up to 1 week postmortem) using eitherconventional serum-supplemented growth medium or a commercialserum-free medium optimized for bone marrow derived MSC(MesenCult-XF system). The MSC phenotype was examined by flowcytometry according to current and emerging markers for humanMSC. Immunosuppressive properties were assessed using a mixedlymphocyte reaction (MLR) assay, whereby the white cell fractionfrom two immunologically incompatible blood donors are culturedtogether in direct contact with growth arrested MSC. T-cell activation(proliferation) was measured by uptake of tritiated thymidine. HumanL-MSC were tested in parallel with human BM-MSC and rabbit L-MSC. Human and rabbit L-MSC were also tested for their ability tostimulate the growth of limbal epithelial (LE) cells in colonyformation assays (for both human as well as rabbit LE cells).Results: L-MSC cultures were >95% negative for CD34, CD45 andHLA-DR and positive for CD73, CD90, CD105 and HLA-ABC.Modest levels (30%) of CD146 expression were observed for L-MSCcultures grown in serum-supplemented growth medium, but not thosegrown in MesenCult-XF. All MSC cultures derived from both humanand rabbit tissue suppressed T-cell activation to varying degreesaccording to culture technique and species (MesenCult-XF >> serumfedcultures, rabbit L-MSC >> human L-MSC). All L-MSCstimulated colony formation by LE cells irrespectively of thecombination of cell species used.Conclusions: L-MSC display immunosuppressive qualities, inaddition to their established non-immunogenic cell surface markerprofile, and stimulate LE cell growth in vitro across speciesboundaries. These results support the potential use of allogeneic oreven xenogeneic L-MSC in the treatment of corneal disorders.Commercial Relationships: Damien G. Harkin, None; Laura J.Bray, None; Celena Heazlewood, None; Kerry Atkinson, OsirisTherapeutics Inc (I), Mesoblast Ptl (C)Support: Supported by NHMRC Project Grant No. 553038Program Number: 1002 Poster Board Number: B0307Presentation Time: 1:00 PM - 2:45 PMEDC/NHS cross-linked amniotic membrane preferentiallypreserves corneal epithelial progenitor cells by activating Wnt/βcatenin signalingDavid H. Ma 1 , Hung-Chi Chen 1 , Jui-Yang Lai 2 , Kevin S. Ma 1 , Lung-Kun Yeh 1 , Unique Yang 3 , Jessica Ma 1 . 1 Ophthalmology, Chang GungMemorial Hospital, Taipei, Taiwan; 2 Institute of Biochemical andBiomedical Engineering, Chang Gung University, Taipei, Taiwan;3 Cell Biology, University of California, Berkeley, Berkeley, CA.Purpose: Previously, we have shown that EDC/NHS cross-linkeddenuded amniotic membrane (CLDAM) is compatible for the growthof human limbo-corneal epithelial (HLE) cells in vitro and in vivo(Biomaterials, 2010, 31: 6647-6658), in this study we furtherinvestigate whether CLDAM preferentially preserves HLE progenitorcells and the underlying mechanism.Methods: HLE cells expanded from explants were cultured on dish(HLE/dish), on denuded AM (HLE/DAM) and on CLDAM(HLE/CLDAM). When near confluency, cell density, BrdU labelretention, and colony formation assay (CFA) were analyzed.Immunoconfocal microscopy, Western blot, and Q-PCR for keratin12, connexin 43, ABCG2, deltaNp63α, β-catenin and TCF-4 wereperformed. Finally, selective GSK3β inhibitors SB216763 orSB415286 were added to HLE/dish cultures to evaluate CFA anddeltaNp63α expression.Results: Compared with HLE/dish or HLE/DAM, HLE cells onCLDAM were more compact in morphology, expressed higher levelof p63, ABCG2, and lower level of connexin 43 and keratin 12. CFAwas highest in HLE/CLDAM, so were the nuclear expression of β-catenin and TCF-4. Addition of GSK3-β inhibitors to HLE/dishcultures not only increased CFE but also the expression of stem cellmarker p63.Conclusions: CLDAM showed tendency to better preserve HLEprogenitor cells in vitro, and Wnt/βcatenin signaling may beinvolved, possibly through the activation of p63.Commercial Relationships: David H. Ma, None; Hung-Chi Chen,None; Jui-Yang Lai, None; Kevin S. Ma, None; Lung-Kun Yeh,None; Unique Yang, None; Jessica Ma, NoneSupport: NSC99-2314-B-182A-027-MY3Program Number: 1003 Poster Board Number: B0308Presentation Time: 1:00 PM - 2:45 PMImmobilized HC-HA Preserves Limbal Niche Cell Phenotype toPrevent Limbal Epithelial Progenitor Cells from DifferentiationBo Han 1, 2 , Yingting Zhu 1 , Suzhen Zhang 1 , Scheffer C. Tseng 1 . 1 TissueTech, Miami, FL; 2 Department of Ophthalmology, Union HospitalHuazhong University of Science and Technology, Wuhan, China.Purpose: We have recently reported the success of isolating limbalniche cells (LNC) that can prevent limbal epithelial progenitor cells(LEPC) from differentiation in a reunion assay in 3-dimentional (3D)Matrigel. Amniotic membrane (AM) alone can help expand residuallimbal stem cells in vitro and in vivo. Because we have successfullyisolated heavy chain-hyaluronan complex (HC-HA) from AM, we©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawonder whether HC-HA can preserve LNC to prevent LEPC fromdifferentiation.Methods: LNC were cultured in MESCM on plastics, 3D Matrigel,or immobilized HC-HA. qRT-PCR and immunostaining were used tocompare expression of markers that have been reported for LNC.AMD 3100 was used to block SDF1/CXCR4 signaling. LEPC wereadded to LNC aggregates formed on immobilized HC-HA inMESCM and compared to the reunion assay in 3D Matrigel for 7days. qRT-PCR, Western blotting, and immunostaining were used tocharacterize expression of markers for LEPC and corneal epithelialdifferentiation.Results: LNC aggregated on 2D immobilized HC-HA at day 1 withsmall and round cells and expressed 2- to 4-fold higher ESC markerssuch as Nanog, Oct4, Rex1, and Sox2 at day 7 than plastic and 3DMatrigel. Such aggregation was mediated by 3 to 4-fold higherexpression of SDF1 and CXCR4 and could be abolished byAMD3100 added on day 0 but not day 4. Presumably because of highexpression of SDF1 and CXCR4, LNC attracted reunion with LEPCon immobilized HC-HA, similar to our recent report that LNCattracted reunion with LEPC in 3D Matrigel. We speculate that sucha reunion with LEPC will prevent LEPC from differentiation.Conclusions: Limbal niche cells form aggregates on HC-HA viaSDF1/CXCR4 signaling, express higher levels of ESC markers, andattract reunion with LEPC. Studies are underway to characterizewhether such reunion on immobilized HC-HA may prevent LEPCfrom differentiation.Commercial Relationships: Bo Han, Tissue Tech (F); YingtingZhu, Tissue Tech Inc (F), Tissue Tech Inc (E), Tissue Tech Inc (P);Suzhen Zhang, TissueTech, Inc (E); Scheffer C. Tseng, NIH, NEI(F), TissueTech, Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)Support: NIH, NEI, RO1 EY06819Program Number: 1004 Poster Board Number: B0309Presentation Time: 1:00 PM - 2:45 PMComparison of Human and Mouse Feeders, Xeno-free andStandard Media in Different Oxygen Tensions for Preservationof Human Limbal Progenitor Cells during CultureKalliopi Stasi 1 , Mina Massaro-Giordano 1 , Jiayan Huang 1 , JohnGearhart 2 . 1 Scheie Eye Institute, University of Pennsylvania,Philadelphia, PA; 2 Cell and Developmental Biology, Institute forRegenerative Medicine, University of Pennsylvania, Philadelphia,PA.Purpose: To identify culture conditions that allow preservation oflimbal stem cells in culture with human feeders and xeno-free mediafor potential clinical use.Methods: Limbal epithelial cells were isolated from 82 cadaveraldonors or 29 cataract patients with trypsin or dispase/trypsin in 13variations. Feeders used: 3T3-J2, adult or neonatal or fetal humandermal fibroblasts, lung fibroblasts MRC-5 and human limbalfibroblasts. Media used: Xeno media per Pellegrini group, MCBD151 (Schotzer-Schrehardt), or SHEM (Tseng), and Xeno-free mediawith Calcium 1.3mM, 1.05mM, 0.4mM and 0.1mM, and EGF 0, 2.5,5, 10 or 20ng/ml, under O2 20%, 14% or 5%. Statistical analysis wasperformed on outcomes: quantitative immunocytochemistry (Q-ICC)for p63α (cytospins), RT-PCR for p63α, Δp63 and K12, ColonyForming Efficiency (CFE), Holoclone Forming Efficiency,Percentage of Aborted Colonies, and number of passages. ICC: p63α,p63 (4A4), ABCG2, Bmi1, c/EBPδ, K12, K15 and MUC1 oncytospins and coverslips. Statistical analysis: ANOVA/MANOVAwith SAS 9.2Results: Isolation methods (cadaveral donors) evaluated for yield,viability and CFE. Yield was correlated with dispase (+0.58, p=0)and days in preservation (-0.52, p=0) and affected by trypsin (p=0)and days in preservation (p=.0006), viability was affected by trypsin(p=0.0269), and CFE was correlated with days in preservation (-0.47,p=0) and yield (+0.43, p=.0004) and affected by donor age (p=.0001)and trypsin (p=.04). Isolation method selected: dispase 2.4U/ml x2hrs and TLE x10 min. Multivariate analysis of effect of variables onnumber of passages (max 10) from 15 patients cultured on 3T3-J2showed significant effect of donor age (0.032, p= 0.002) and medium[SHEM (-2, p=0), Xeno-Free Ca0.01 with EGF10 (0.67, p=.04) orEGF20 (1.67, p=0)], and no effect of O2 14% vs. 20%. ANOVA of34 combinations of feeder, medium and O2 conditions wassignificant for Q-ICC (p=.007) and CFE (p=.0002) but not significantfor age, gender, feeders, medium or O2 alone. MRC-5 feeders withXeno-free Calcium 0.01mM and EGF10ng/ml at 20% O2 wereeffective in Q-ICC (x4.9 fold) and p63α PCR (x1.4 fold) compared tobaseline 3T3 feeders with Pellegrini medium.Conclusions: Xeno-Free media and human feeders may effectivelypreserve limbal progenitors in culture as measured with multipleoutcomes in head-to-head comparisons.Commercial Relationships: Kalliopi Stasi, None; Mina Massaro-Giordano, Tear Lab (R); Jiayan Huang, None; John Gearhart,NoneSupport: NIH Grant K12EY015398, Pennsylvania State0426/554248/8319, RPB-UnrestrictedProgram Number: 1005 Poster Board Number: B0310Presentation Time: 1:00 PM - 2:45 PMOutcomes of Penetrating Keratoplasty after ex vivo ExpandedAutologous Limbal Stem Cell Transplantation in HumansOliver J. Baylis 1, 2 , Hardeep S. Mudhar 4 , Majlinda Lako 1, 3 ,Francisco C. Figueiredo 2, 1 . 1 Institute of Genetic Medicine,Newcastle University, Newcastle-upon-Tyne, United Kingdom;2 Department of Ophthalmology, Royal Victoria Infirmary, NewcastleUpon Tyne, United Kingdom; 3 North East England Stem CellInstitute, Newcastle University, Newcastle Upon Tyne, UnitedKingdom; 4 National Specialist Ophthalmic Pathology Service, RoyalHallamshire Hospital, Sheffield, United Kingdom.Purpose: To study the clinical and histological outcomes of patientswho received ex vivo expanded autologous limbal stem cells (LSC)on human amniotic membrane (AM) followed by penetratingkeratoplasty (PK) for the treatment of unilateral total limbal stem celldeficiency (LSCD).Methods: Prospective, single-centre, noncomparative, interventionalcase series. Participants: 5 consecutive patients with unilateral LSCDwere treated at the Department of Ophthalmology, Royal VictoriaInfirmary, Newcastle upon Tyne, UK between April 2006 andNovember 2012. Intervention: HLA-matched PK was performed atleast 6 months after ex vivo expanded autologous LSC using ananimal and feeder free method combined with high dose topicalsteroid. Outcome measures: LSC survival was assessed by slit lampbiomicroscopy, histology of excised corneal buttons and post-PKcorneal impression cytology (IC) showing absence of goblet cells. PKsurvival, best corrected visual acuity (BCVA), patient-reportedoutcomes and complications were also recorded.Results: All patients were male with a mean age of 52 (range 20-77).Six PK were performed in 5 eyes of 5 patients, with 1 regraft. MeanLSC and PK follow up was 63 months (range 35-78) and 28 months(range 4-48) respectively. Postoperatively, satisfactory ocular surfacereconstruction was maintained in all eyes (100%), as confirmed byIC. One patient was lost to follow-up 4 months after PK. At lastexamination, BCVA improved in 4 eyes (≥6/36). Vision impairmentand pain scores improved in all patients (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea100% CK3 +ve corneal epithelial cells and presence of p63 +ve LSClikecells in the basal epithelium in all corneas. In addition, TEMrevealed a uniform stratified epithelium, typical of the normal centralcornea.Conclusions: This study demonstrates that transplantation of ex vivoexpanded autologous LSC cultured on AM followed by PK is aneffective method of reconstructing the corneal surface and restoringuseful vision in patients with unilateral total LSCD. Despite HLAmatchingPK and high dose topical steroid, graft rejection withfailure may still occur in this high risk group of patients. Furtherstudies with more patients and longer follow-up should be conducted.Commercial Relationships: Oliver J. Baylis, None; Hardeep S.Mudhar, None; Majlinda Lako, None; Francisco C. Figueiredo,NoneSupport: MRC, Newcastle Healthcare Charity UK NIHRBiomedical Research Centre for Ageing and Age-related disease andthe Newcastle upon Tyne NHS Hospitals trustProgram Number: 1006 Poster Board Number: B0311Presentation Time: 1:00 PM - 2:45 PMIntegrating human epithelial stem cells and keratocytes into atransparent, collagen based bioengineered cornea for diseasemodellingJames W. Foster 1 , Ana Maria Ionescu 1 , Roanne R. Jones 2 , RicardoM. Gouveia 1 , Che J. Connon 1 . 1 School of Chemistry, Food andPharmacy, University of Reading, Reading, United Kingdom;2 Department of Optics, University of Granada, Granada, Spain.Purpose: To demonstrate the integration of epithelial stem cells andkeratocytes into a single construct using defiend conditions. This willallow the interplay between the two cell types that form the anteriorcornea to be investigated. Since the collagen constructs are intendedto replace the principal refractive and image forming component ofthe eye, their optical quality was also studied to ensure thefunctionality of these bioengineered tissues.Methods: Epithelial stem cells and keratocytes were isolated fromcadaverous human tissue and cultivated in vitro. Keratocytes wereencapsualted within compressed collagen gels and allowed toproliferate and differentiate for 7 days before addition of limbalepithelial stem cells under differentiation media conditions.Expression levels of specific keratocyte markers ALDH-1, Lumican,Col5a1, and Keratocan were determined through PCR andfluorescent microscopy. The optical quality of resulting constructswas determined by Contrast Transfer Function (CTF), a simplemethod for the accurate quantification of contrast between adjacentobjects. Several bar patterns of alternating white and black lines withincreasing frequency (2, 4, 6, 10, 20, 31, and 61 cycles/mm) weredisplayed on a LCD and images were captured using a N90 Nikoncamera. The CTF and transparency levels were calculated usingadequate image analysis software (ImageJ). Secretion of ECMcomponents by keratocytes and epithelial cell behaviour wereexamined over 3 weeks.Results: The inclusion of cells within the gels increased transparencyby 10% to 66%, reaching a value sufficient for a collagen construct tobe used as a corneal replacement. The phenotype of the epithelialstem cell was characterized by IHC, qPCR, and flow cytometry asABCG2- and ΔNP63-positive, and CK3-negative. The epithelial stemcells differentiated to CK3-positive cells with accompanyingstratification (>4 cells thick). Keratocytes were shown to secretecol5a1, lumican, and keratocan. Keratocyte organisation showedincreased orthogonal alignment within the gels.Conclusions: Here we have recapitulated some of the complexinterplay between human epithelial and stromal cells within a definedculture system. This resulted in corneal constructs with therapeuticlevels of transparency and the desirable cell phenotypes.Commercial Relationships: James W. Foster, None; Ana MariaIonescu, None; Roanne R. Jones, None; Ricardo M. Gouveia,None; Che J. Connon, NoneProgram Number: 1007 Poster Board Number: B0312Presentation Time: 1:00 PM - 2:45 PMEvaluation of Corneal Stem/Progenitor Cells in leptin deficientmiceHiroki Ueno 1 , Takaaki Hattori 2 , Yuta Kumagai 1 , Noboru Suzuki 3 ,Satoki Ueno 1 . 1 Ophthalmology, St Marianna Univ School of Med,Kawasaki, Japan; 2 Ophthalmology, Tokyo Medical University,Tokyo, Japan; 3 Immunology and Medicine, St. Marianna UnivSchool of Med, Kawasaki, Japan.Purpose: Diabetic keratopathy (DK) remains difficult to be treated. Itcan cause corneal persistent epithelial defects, suggesting a role ofcorneal nerves in maintaining the corneal homeostasis. Leptindeficient mice which are widely accepted as an animal model of mildtype-II diabetes mellitus (DM) have sensory nerve conductiondeficits, small sensory nerve fiber neuropathy,intra-epidermal sensorynerve fiber loss. The purpose of this study is to investigate whetherputative corneal stem/progenitor cells are altered in obese mice and tounderstand the pathogenesis in obesity and diabetes mice lacking theleptin gene.Methods: 12 week-old male mice with mild type II DM (C57 6JHamob/ob mice) were assessed by beta-III tubulin (neural marker)immunostaining. Real-time polymerase chain reaction was performedto quantify expression of ATP-binding cassette subfamily G member2 (ABCG2), hairy enhancer of split 1 (Hes1), the low-affinity NGFreceptors (p75) as corneal progenitor cell markers. Keratin 19 andHes1 were assessed in type II DM mice and controls byimmunofluorescence microscopic studies.Results: Beta-III tubulin expression detected with immunostainingwas decreased in the diabetic corneas. Corneal subbasal plexus ofnerve fibers with mild type II DM were preferentially thinner and hadfewer branches compared to the normal mice. Hes1 and Keratin 19expression noted with immunostaining was diminished in corneas ofdiabetes mellitus when compared with normal corneas. Similarly,mRNA expression levels for Hes1 and p75 were decreased in corneaswith diabetes.Conclusions: Our results suggest that corneal stem/progenitor cellscould be altered in animal model of obesity and mild type II diabetesmellitus. Our data may provide novel evidence for the closeconnection between innervation and maintaining corneal progenitorcells and/or the stem cell niche in cases of mild type II diabetesmellitus and leptin deficient.Commercial Relationships: Hiroki Ueno, None; Takaaki Hattori,None; Yuta Kumagai, None; Noboru Suzuki, None; Satoki Ueno,NoneProgram Number: 1008 Poster Board Number: B0313Presentation Time: 1:00 PM - 2:45 PMRetinal Pigment Epithelial Cell Differentiation in PrimaryHuman Limbal Neurosphere CulturesSamuel McLenachan 1, 2 , Dana Zhang 1, 2 , Fred K. Chen 1, 2 . 1 OcularTissue Engineering, Lions Eye Institute, Nedlands, WA, Australia;2 Centre of Ophthalmology and Visual Science, University of WesternAustralia, Perth, WA, Australia.Purpose: The human corneoscleral limbus contains multipotent stemcells that can be isolated and cultured for clinical applications, suchas the treatment of limbal stem cell deficiency. In culture, limbalstem cells can be induced into the neural linage to produce cells©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadisplaying the characteristics of photoreceptors. Here, we haveexamined the hypothesis that retinal pigment epithelial (RPE) cellscan be produced from primary human limbal cultures.Methods: Human corneoscleral rims were incubated withcollagenase to facilitate the removal of the limbal epithelium (LE).LE was dissociated non-enzymatically and cultured in the presence ofEGF, FGF2 and Noggin to produce floating neurospheres (LiNS).LiNS were plated on a geltrex matrix to form adherent colonies. Inparallel, primary limbal cells were cultured in keratinocyte media toproduce adherent LE cell monolayers. Primary LE and LiNS cultureswere examined by microscopy and immunocytochemical analysis.Results: Two different populations of neurospheres were evident inprimary human LiNS cultures; pigmented and non-pigmented.Comparison of primary keratinocyte and neurosphere culturesrevealed the predominance of non-pigmented LiNS in cultures withstromal keratocyte contamination. Pigmented LiNS expressed theocular transcription factors PAX6, OTX2 and MITF, and containedcells expressing the RPE specific markers RPE65 and ZO1.Conclusions: Culture of human limbal epithelial stem cells in thepresence of EGF, FGF2 and Noggin leads to the induction of LiNScontaining pigmented cells expressing the RPE cell markers MITF,RPE65 and ZOI.Human limbal neurosphereglycosaminoglycans (GAGs). MPS are progressive disorders inwhich GAGs and their metabolic derivatives accumulate inlysosomes compromising cellular activity and ultimately leading tocell death. MPS VII, Sly syndrome, caused by a mutation in β-glucuronidase, manifests as hepatomegaly, skeletal dysplasia, shortstature, corneal clouding and developmental delay, due to theaccumulation of heparan sulfate (HS), dermatan sulfate andchondroitin 4,6-sulfate (CS). Current treatment regimens for MPS arenot effective for treating corneal clouding and mental development.Methods: We hypothesized that umbilical mesenchymal stem cells(UMSC) transplanted into the corneal stroma can participate in thecatabolism of GAGs, thus providing a means of cell therapy for MPS.For such, human UMSC were intrastromally transplanted intocorneas of 1, 2 and 3 month-old MPS VII mice.Results: UMSC transplantation restored the dendritic and hexagonalmorphology of host keratocytes and endothelial cells, respectively,and in vivo confocal microscopy (HRTII) revealed reduced cornealhaze. Immunohistochemistry using antibodies against HS and CSchains, as well as, LAMP2 revealed a decrease in GAG content andboth lysosomal number and size in the treated corneas to levelssimilar to that of littermate controls. Labeling UMSC intracellularcompartments prior to transplantation revealed the distribution ofUMSC exosomes throughout the corneal stroma and endothelium. Anin vitro co-culture assay between skin fibroblasts isolated fromMPSVII mice and UMSC labeled with LysoSensor demonstrated thatneutral exosomes released by the UMSC are up taken by thefibroblasts and proceed to fuse with the acidic lysosomes.Conclusions: Therefore, transplanted UMSC participate inextracellular GAG turnover and aid host keratocytes to metabolizeaccumulated GAG, suggesting that UMSC could be a goodalternative for treating corneal defects associated with MPS and othercongenital metabolic disorders. Moreover, given the simplicity of thetreatment, we suggest it as prophylactic treatment upon diagnosis inorder to avoid the development of corneal clouding.Commercial Relationships: Vivien J. Coulson-Thomas, None;Bruce Caterson, Abcam, Cambridge, UK (I), CosmoBio, Japan (I);Chia-Yang Liu, None; Winston W. Kao, NoneSupport: NIH/NEI RO1 EY021768, Research to Prevent Blindness,and Ohio Lions eye Research FoundationHuman LiNS cells immunostained for RPE65 (green) and zonusoccludin-1 (red)Commercial Relationships: Samuel McLenachan, None; DanaZhang, None; Fred K. Chen, NoneProgram Number: 1009 Poster Board Number: B0314Presentation Time: 1:00 PM - 2:45 PMTranspalntation of umbilical mesenchymal stem cells cures thecorneal defects of Mucopolysaccharidosis VII miceVivien J. Coulson-Thomas 1 , Bruce Caterson 2 , Chia-Yang Liu 1 ,Winston W. Kao 1 . 1 Ophthalmology, University of Cincinnati,Cincinnati, OH; 2 Laboratory of Connective Tissue Biology, School ofBiosciences, Cardiff University, Cardiff, United Kingdom.Purpose: Mucopolysaccharidoses (MPS) are a family of relateddisorders caused by a mutation in one of the lysosomalexoglycosidases required for the sequential degradation ofProgram Number: 1010 Poster Board Number: B0315Presentation Time: 1:00 PM - 2:45 PMDental Pulp: a Source of Stem Cells with Keratocyte PotentialMartha L. Funderburgh 1 , Fatima N. Syed-Picard 2 , Charles S. Sfeir 2 ,James L. Funderburgh 1 . 1 Department of Ophthalmology, Univ ofPittsburgh Sch of Med, Pittsburgh, PA; 2 Center for CraniofacialRegeneration, University of Pittsburgh, Pittsburgh, PA.Purpose: Blindness due to corneal stromal opacity can besuccessfully treated by allografts; however, inflammation or previousgraft rejections predict poor outcome for some allogenic tissue in thestroma. These difficult cases may benefit from autologous stem celltherapy or from autologous grafts of bioengineered tissue. Dentalpulp contains a potent stem cell population with immune-suppressiveproperties, cells that could be the autologous stem cells ideal forcorneal regeneration. The purpose of this study was to explore thepotential for dental pulp stem cells (DPSC) to adopt a keratocytephenotype.Methods: Dental pulp extracted from human molars was dispersedwith collagenase, cultured and used at passage 3. Cells expressingCXCR4 protein were isolated using MACS technology. Expressionof stem cell genes was determined using flow cytometry and qPCR.Differentiation to keratocytes was carried out on collagen gels oraligned nanofiber substrata in serum-free medium containing FGF2©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand ascorbate-2-phosphate.Results: Cultured cells from dental pulp expressing the chemokinereceptor CXCR4 were isolated by immunoaffinity. CXCR4+ DPSCcells had increased expression of pluripotent genes OCT4, NANOG,and SOX2, neural crest marker p75NTR, and genes expressed bycorneal stromal stem cells ABCG2, KIT, SIX2, PAX6, andNOTCH1. When the CXCR4+ cells were cultured on collagen gels inmedium that induced keratocyte differentiation, expression of stemcells genes was downregulated and mRNAs for several keratocytespecificproducts were upregulated, including keratocan (KERA),ALDH3A1, PTGDS and enzymes involved in keratan sulfatesynthesis beta-1,3, glucosaminyltransferase 7 (B3GNT7) and corneal6-O-glucosaminyl-sulfotransferase 6 (CHST6). The expression levelsof these genes were almost identical to those in corneal stromal stemcells, which under the same conditions produce a stroma-likeextracellular matrix.Conclusions: CXCR4 appears to be a key cell-surface marker ofstem cells with potential to differentiate in the keratocyte lineage.The availability and potency of stem cells from dental pulp makesthese cells an excellent candidate as a source of autologous stromalcells for future bioengineering or cell-based therapies for stromalblindness.Commercial Relationships: Martha L. Funderburgh, None;Fatima N. Syed-Picard, None; Charles S. Sfeir, None; James L.Funderburgh, NoneSupport: NIH Grants EY016415 (JLF), P30-EY008098,F31DE019753 (FS-P), Eye & Ear Foundation of Pittsburgh, Researchto Prevent BlindnessProgram Number: 1011 Poster Board Number: B0316Presentation Time: 1:00 PM - 2:45 PMCorneal stroma derived MSCs maintain immunosuppressionwith wound healing capacity in vitroZoltan Vereb 1 , Reka Albert 1, 2 , Morten C. Moe 4 , Laszlo Fesus 1 , EvaRajnavolgyi 3 , Andras Berta 2 , Goran Petrovski 1, 2 . 1 Department ofBiochemistry and Molecular Biology, University of Debrecen,Debrecen, Hungary; 2 Department of Ophthalmology, University ofDebrecen, Debrecen, Hungary; 3 Department of Immunology,University of Debrecen, Debrecen, Hungary; 4 Deparment ofOphthalmology, Oslo University Hospital, Oslo, Norway.Purpose: Mesenchymal stem cells (MSC) are the stromal cells ofbone marrow, but they can also be found in other tissues includingthe cornea. Our goal was to isolate and cultivate human cornealstroma MSC-like cells (CSMSCs) and study their role in immunityand wound healing.Methods: Corneal buttons were harvested from cadavers (accordingto the Guidelines of the Helsinki Declaration). The isolated stromalcells were cultured ex vivo in human serum containing medium. Theexpression of well-known MSC, hematopoietic, endothelial markersas well as high-end glycosylation products were measured byfluorescent microscopy and FACS in comparison to bone marrowderived MSCs. To investigate the stemness of CSMSCs, gene arrayanalysis and standardized in vitro differentiation assays wereperformed. The immunosuppressive function of these cells wasstudied by mitogen activated lymphocyte reaction in a co-culturewith CSMSCs. Proliferation was measured by BrDU incorporationassay. To describe the immunophenotype of the CSMSCs, the cellswere activated by TLR ligands and pro-inflammatory cytokines andthe secreted cytokines measured by ELISA. ECIS based woundhealing assay was performed to test the regenerative potential ofthese cells.Results: The cells isolated from human corneal stroma grew asmonolayers in vitro and could be maintained in culture for more than10 passages (n=6). According to the definition of the ISCT, the mostimportant MSC markers (CD73, CD90 and CD105) were highlyexpressed on the surface of CSMSCs with absence of endothelial(CD31, VEGFR2) or hematopoietic cell markers (CD34, CD45,CD69, CD133). The CSMSCs were able to differentiate into fat, boneand cartilage tissues showing the potency of the CSMSCs. Thesecells could close wounds within 24 hrs in vitro. They could suppressthe proliferation of mitogen activated peripheral blood lymphocytesand secrete suppressive cytokines upon pro-inflammatory activation,therefore, strengthening their unique immunosuppressive phenotypein inflammation.Conclusions: We demonstrate a method for isolating and cultivatingMSC-like cells from human corneal stroma. The ex vivo data suggestthat these cells may have a role in wound healing and immunologicalprocesses in the eye that can possibly be used in future treatments ofocular diseases and corneal stroma injuries.Commercial Relationships: Zoltan Vereb, None; Reka Albert,None; Morten C. Moe, None; Laszlo Fesus, None; EvaRajnavolgyi, None; Andras Berta, None; Goran Petrovski, NoneProgram Number: 1012 Poster Board Number: B0317Presentation Time: 1:00 PM - 2:45 PMEngraftment and Survival of Human Umbilical MesenchymalStem Cells in the Mouse Cornea: An ImmunofluorescentComputed Tomography StudyBehdad Kavianpour 1 , Geraint J. Parfitt 1 , Hongshan Liu 2 , Winston W.Kao 2 , Donald J. Brown 1 , Yilu Xie 1 , Mikhail Geyfman 1 , James V.Jester 1 , Jennifer Simpson 1 . 1 Gavin Herbert Eye Institute, Universityof California, Irvine, Irvine, CA; 2 Department of Ophthalmology,University of Cincinnati, Cincinnati, Ohio, OH.Purpose: While the field of ocular regenerative medicine hasadvanced dramatically, little is known about the engraftment andmigration characteristics of intra-stromal stem cell transplantation inthe cornea. To better understand intra-stromal stem cell engraftmentand migration, the purpose of this study was to use a novel imagingmodality (immunofluorescent computed tomography or ICT) tolocalize and quantify DiO-labeled human umbilical mesenchymalstem cells (hUMSCs) transplanted into mouse corneas.Methods: hUMSCs were DiO-labeled and injected (20,000cells/cornea) using a 33-gauge needle and Hamilton syringe into thecorneal stroma of twelve C57BL/6 mice. Cell survival followinginjection through the Hamilton needle and syringe was assessed bytrypan blue exclusion. At two, four and twelve weeks post-injection,mice were sacrificed and corneas were excised and fixed in 2% PFA.The corneas were then dehydrated and embedded in butyl methylmethacrylate and polymerized under UV light at 4C. Oncepolymerized, corneas were serially sectioned (2µm thick), stainedwith DAPI and imaged using a Leica DMI6000B. Corneal volumeswere then reconstructed using Amira and fiji software to quantify andcharacterize the distribution of DiO-labeled hUMSCs.Results: At 2 weeks, 594 DiO labeled hUMSCs were identified inthe stroma and migrated to occupy a volume of 1.23*107 μm3. At 4weeks, 45 DiO labeled hUMSCs were detected in the stroma thatoccupied a volume of 4.28*107 μm3. At 12 weeks, only 19 DiOlabeled hUMSCs in a stromal volume of 4.68*107 μm3 wereidentified. This suggests that the number of engrafted DiO-labeledhUMSC decreased over a 12-week period, although greater migrationwas observed over time. Cells injected through the Hamilton syringeshowed cell viability averaging 11.66%.Conclusions: ICT is a novel imaging modality that can track andquantify stem cell engraftment and migration over time. In thispreliminary study, hUMSCs appear to migrate and occupy a largervolume over time, however, total cell engraftment appeared to©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamarkedly decline based on DiO labeling. This suggests that techniquemodifications may be required to optimize intra-stromal stem cellviability. Antibody staining for markers of human stem celltransplantation and keratocyte differentiation may also aid ourunderstanding of corneal stem cell fate and verify loss of cells orlabeling.Commercial Relationships: Behdad Kavianpour, None; GeraintJ. Parfitt, None; Hongshan Liu, None; Winston W. Kao, None;Donald J. Brown, None; Yilu Xie, None; Mikhail Geyfman, None;James V. Jester, None; Jennifer Simpson, NoneSupport: NIH Grant EY022365 Mesenchymal Stem Cell TherapyFor Corneal Cystinosis; Research to Prevent Blindness, Inc;Discovery Eye Foundation; The Skirball Program in MolecularOphthalmology; The Cystinosis Research FoundationProgram Number: 1013 Poster Board Number: B0318Presentation Time: 1:00 PM - 2:45 PMInfluence of Secreted Ly6/uPAR-Related Protein-1 (Slurp1) onCorneal Stromal Fibroblast Proliferation, Interaction with theExtracellular Matrix, and MigrationShivalingappa K. Swamynathan 1, 2 , Sudha Swamynathan 1 .1 Ophthalmology, Univ Pittsburgh Sch of Med, Pittsburgh, PA; 2 CellBiology and Physiology, Univ Pittsburgh Sch of Med, Pittsburgh,PA.Purpose: Previously, we demonstrated that the secreted Ly6/uPARrelatedprotein-1 (Slurp1) is abundantly expressed in the cornea andis downregulated in diverse pro-inflammatory conditions. Here, weexamine the effects of Slurp1 on corneal stromal fibroblast cellproliferation, interaction with the extracellular matrix (ECM), andmigration, to understand the cellular basis of Slurp1 functions in thecornea.Methods: The effect of Slurp1 on corneal fibroblast behavior wasassessed in vitro by treating human telomerase reverse transcriptase(hTERT)-immortalized mouse corneal stromal cell line MK/T1 withhistidine-tagged mouse Slurp1 (His-Slurp1) produced in E. coli andpartially purified by Ni ion resin column chromatography. Effect ofHis-Slurp1 on MK/T1 cell (i) density was assessed by crystal violetstaining followed by measurement of absorbance at 590 nm, (ii)interaction with the ECM was evaluated on cell culture plates coatedwith different ECM components, and (iii) migration was assessed byin vitro gap filling assays.Results: Compared with the control, His-Slurp1-treated mousecorneal stromal fibroblast MK/T1 cells (i) density increased at aslower pace suggesting that Slurp1 inhibits cell proliferation, (ii)adhered with lower affinity to collagen-I-, collagen-IV-, vitronectinorfibronectin-coated culture plates suggesting that Slurp1 affectscell-matrix interaction, and (iii) migrated at a slower pace in gapfilling assays suggesting that Slurp1 inhibits cell migration.Conclusions: Our results demonstrate that Slurp1 inhibits cornealstromal fibroblast MK/T1 cell proliferation, cell-matrix adhesion andmigration, revealing the cellular basis for corneal functions of Slurp1.These results are consistent with the decreased expression of Slurp1in corneas exposed to pro-inflammatory conditions where the stromalfibroblasts proliferate at a higher rate, and migrate rapidly.Commercial Relationships: Shivalingappa K. Swamynathan,None; Sudha Swamynathan, NoneSupport: Department of Ophthalmology, University of PittsburghStart-up funds, Eye and Ear Foundation of Pittsburgh, Research toPrevent Blindness and NIH core grant P30 EY08098Program Number: 1014 Poster Board Number: B0319Presentation Time: 1:00 PM - 2:45 PMStromal Cells derived from Amniotic Membrane are capable toreestablish corneal opacityYonathan Garfias 1, 2 , Alejandro Navas 1 , Jessica Nieves-Hernández 1 ,Gibran A. Estua 1 , Rodrigo Bolaños-Jiménez 1 . 1 Research Unit,Institute of Ophthalmology, Mexico City, Mexico; 2 Biochemistry,Faculty of Medicine, Universidad Nacional Autónoma de México,Mexico City, Mexico.Purpose: Stromal mesenchymal stem cells are non-hematopoieticderived cells found in the bone marrow stroma such as in manystromal tissues. The amniotic membrane is an elastin and avascularfetal membrane that is in contact to the fetus. A mature amnioticmembrane possesses 20-50 x 10 mesenchymal cells. .By the other hand, there are many corneal disorders that directlyaffect the corneal limbus, driving inflammation, conjunctivalizationor neovascularization of the corneal tissue. The pronostic depends onthe injured area of the limbus where the corneal stem cells arelocalized. Although, it has recently reported that the cells derivedfrom the amniotic membrane mesenchyma are source toadipogenesis, chondrogenesis, osteogenesis and myogenesis, itsfunction as a source for regeneration of the ocular surface has notbeen studied.The aim of the present study is to determine the utility of these cellsto restablish the ocular surface in a chemical burn murine model.Methods: Mesenchymal cells were obtained from a placenta usingdispase/collagenase method. The cells were cultured andcharacterized by flow cytometry. Cellular transdifferentiation assayswere performed using conditioned media. A murine chemical burnwas performed in order to determine the efficacy of these cells torestablish the corneal clarity. Corneal histology was performed toidentify the incorporation of human mesenchymal cells in the murinecornea.Results: The cells obtained from the amniotic membranemesenchyma were capable to attach to the plastic wells showing afibroblast-like morphology. These cells presented mesenchymal stemcell markers such as CD29, CD73, CD44 and CD105, meanwhile,they were negative to CD45 and HLA-DR. Interestingly, these cellswere capable to differentiate into neurons and chondrocytes. Whenthese cells were intracamerally injected in a mouse burn model, thecorneal opacity was significantly reduced in comparison to theuntreated cornea. When the histology of the cornea was performed, itwas evident that the human amniotic membrane cells wereincorporated to the mouse cornea, reestablishing the structure of thecorneal tissue.Conclusions: The use of cells derived from the mesenchyma of theamniotic membrane is an important cell source to be used in theregenerative ophthalmology.Commercial Relationships: Yonathan Garfias, Institute ofOphthalmology (P); Alejandro Navas, None; Jessica Nieves-Hernández, Institute of Ophthalmology (P); Gibran A. Estua,None; Rodrigo Bolaños-Jiménez, NoneSupport: CONACYT 160286Program Number: 1015 Poster Board Number: B0320Presentation Time: 1:00 PM - 2:45 PMCorneal endothelial cells derived from monkey iPS cells: a shortterm evaluationShin Hatou 1 , Satoru Yoshida 1 , Kazunari Higa 2 , Hideyuki Miyashita 1 ,Emi Inagaki 1 , Erika Kimura 3 , Ryuhei Hayashi 3 , Kazuo Tsubota 1 ,Kohji Nishida 3 , Shigeto Shimmura 1 . 1 Department of Ophthalmology,Keio Univ School of Medicine, Shinjuku-ku, Japan; 2 Department ofOphthalmology, Tokyo Dental College Ichikawa General Hospital,Ichikawa, Japan; 3 Department of Ophthalmology, Osaka University,Osaka, Japan.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To evaluate the short term function of tissue-engineeredcorneal endothelial cells (TECE cells) derived from monkey inducedpluripotent stem cells (iPS cells).Methods: Cynomolgus monkey iPS cells were cultured in KSRmedium for 1 week and subsequently in N2 medium for 1 week,supplemented with TGF-beta inhibitor and BMP inhibitor. The iPSderivedneural crest cells were isolated as CD271 positive fraction bycell sorter. Next these cells were proliferated with EGF and FGF2,and subsequently medium was changed to an “endothelium-derivingmedium” including GSK-3beta inhibitor, retinoic acid and ROCKinhibitor. These cells were dispersed on collagen sheet and TECE cellsheets were obtained. The pump function attributable to Na,K-ATPase activity of TECE cell sheets was measured with an Ussingchamber, and compared with that of human corneal endothelial cellline (B4G12 cells). In vivo function of TECE was measured ascentral corneal thickness of rabbit eyes transplanted with TECE cellsheets for 8 days after surgery and compared with control eyesdeprived of endothelium.Results: Hexagonal mosaic pattern monolayer TECE cells wereobtained. Pump function of TECE was 2.34±0.46 mV, whereas thatof B4G12 cells was 1.07±0.20 mV. The corneal thickness of TECEtransplanted rabbit eyes (589.25±164.8μm) maintained significantlower corneal thickness than control eyes (1105.8±165.9μm)throughout the post-operative period.Conclusions: In vitro and short term in vivo function of monkey iPSderivedTECE were observed. Further long-term in vivo evaluation ofTECE transplantation to monkey eyes is needed.Commercial Relationships: Shin Hatou, None; Satoru Yoshida,None; Kazunari Higa, None; Hideyuki Miyashita, None; EmiInagaki, None; Erika Kimura, None; Ryuhei Hayashi, None;Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch LombSurgical (C), Functional visual acuity meter (P), JiNS (P), Kissei (F),Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), EchoDenki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPTCompany (P); Kohji Nishida, Alcon (C), Alcon (F), HOYA (F),Senju (F), Pfizer (F), Santen (F), Osaka University (P); ShigetoShimmura, NoneSupport: Highway Program for realization of regenerative medicinefrom Ministry of Education, Culture, Sports, Science andTechnology, Japan.Program Number: 1016 Poster Board Number: B0321Presentation Time: 1:00 PM - 2:45 PMBone Marrow-Derived Endothelial Progenitor Cells forTreatment of Corneal Endothelial DysfunctionYao Fu, Chunyi Shao, Xianqun Fan. Department of Ophthalmology,Ninth People’s Hospital, Medical School of Shanghai JiaotongUniversity, Shanghai, China.Purpose: To investigate the feasibility of inducing bone marrowderivedendothelial progenitor cells (BEPC) to differentiate intocorneal endothelial cells (CEC) for the treatment of cornealendothelial dysfunction.Methods: BEPC were isolated from human fetal bone marrow, andexpression of Dil-Ac-LDL, UEA-1, CD133 and CD34 wereexamined to identify the cells. BEPC were co-cultured with CEC for10 days in a transwell system with conditioned medium from CEC,and then cell transdifferentiation was examined byimmunocytofluorescence and electron microscopy. With a porcinecorneal acellular matrix as the carrier, the induced BEPC weretransplanted onto a cat’s cornea from which Descemet’s membraneand the endothelium had been stripped.Results: The induced BEPC resembled CEC in polygonal shape,expressing aquaporin-1, tightly opposed cell junctions, and neuronespecificenolase. Twenty-eight days after transplantation, thetransparency gradually returned to the corneas transplanted with theinduced BEPC on porcine corneal acellular matrix .Conclusions: Human fetal BEPC could be induced into cornealendothelial-like cells in vitro. Features of the induced BEPCindicated that they may be useful for the treatment of cornealendothelial dysfunction.Commercial Relationships: Yao Fu, None; Chunyi Shao, None;Xianqun Fan, NoneSupport: grant from the National Nature Science Foundation ofChina (81000366)Program Number: 1017 Poster Board Number: B0322Presentation Time: 1:00 PM - 2:45 PMStandardization of human corneal endothelial cell isolation andthe use of denuded amniotic membrane as a scaffold for humancorneal endothelial cellsKalpana Suresh, Tanvi Khanna, Alan M. Punnoose, Sarah Kuruvilla,Vishnu D. Narayanam, RAMYA RAVINDRAN, Varshini Varadaraj.Ophthalmology, Sri Ramachandra Universiy, Chennai, India.Purpose: To identify the best technique for complete denudation ofamniotic membrane.To standardize the isolation of human corneal endothelial cells.To use the denuded amniotic membrane as a scaffold for isolatedhuman corneal endothelial cells.Methods: Human amniotic membrane denudation was carried outusing 1.2 units/ml of Dispase II at 37degree C for 60 minutesfollowed by mechanical scraping and microscopic examination. Thiswas followed by isolation of corneal endothelial cells using humandonor cadaveric eyes unfit for surgical usage. Corneal endothelial anddescemet’s membrane sheets were peeled in a manner similar tocapsulorrhexis after corneoscleral button excision and enzymaticallydigested with 2mg/ml of collagenase II solution at 37 degree C and 5% CO2 for 2 hrs. Pre plating was done onto an uncoated culture wareto separate any attached fibroblasts from the endothelial cells whichwere then seeded onto denuded amniotic membrane in OptiMEMmedia supplemented with human epidermal growth factor, fibroblastgrowth nerve growth factor and bovine pituitary extract. The cellswere analyzed microscopically to assess if they maintained theirpolygonal morphology and subjected to RT-PCR analysis for Keratin3, neuron specific enolase, Vimentin and collagen VIII mRNAmarkers.Results: Microscopic examination of the denuded amnioticmembrane showed no epithelial cell remnants and an underlyingexposed stromal collagen. Peeling of the corneal endothelial anddescemet’s membrane gave sheets of ideal thickness exhibitingtypical cobblestone morphology. Enzymatic digestion of theharvested corneal tissue left behind acellular descemet’s sheets withthe endothelial cells seen floating individually or in tightly packedclusters with preplating aiding in a more fibroblast free endothelialcell isolation. Microscopic evaluation showed that a few isolated cellsmanaged to scaffold onto the amniotic membrane and that theymanage to retain that adhesion during subsequent mediareplacements.Conclusions: Usage of Dispase-II for enzymatic digestion ofamniotic membrane yielded a complete denudation which acted as asuccessful scaffold for harvested corneal endothelial cells. Furtherstudies can be done for endothelial cell proliferation serving as an invitro model for corneal tissue engineering studies.Commercial Relationships: Kalpana Suresh, None; TanviKhanna, None; Alan M. Punnoose, None; Sarah Kuruvilla, None;Vishnu D. Narayanam, None; RAMYA RAVINDRAN, None;Varshini Varadaraj, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 1018 Poster Board Number: B0323Presentation Time: 1:00 PM - 2:45 PMIsolation and characterization of p75NTR positive and highproliferativecorneal endothelial cells from the human cornealendotheliumSusumu Hara, Ryuhei Hayashi, Tomofumi Kageyama, MotokazuTsujikawa, Kohji Nishida. Ophthalmology, Osaka UniversityGraduate Scool of Medicine, Suita, Japan.Purpose: The corneal endothelium is believed to be developmentallyoriginated from periocular mesenchyme via neural crest. The humancorneal endothelial progenitor cells (HCEPs) have been investigatedbecause of their potential availability for the tissue regenerativemedicine. However, the existence and the properties of HCEPs havenot been elucidated yet. We attempted to isolate the HCEPs from thehuman corneal endothelium by using the specified culture system andp75 neurotrophin receptor (p75NTR).Methods: The Descemet's membranes were stripped from the humancornea, then, treated with a cell dissociated reagent. To isolate theHCEPs, the endothelial cells were seeded on the dish coated withlaminin and cultured in the serum-free media containing basicfibroblast growth factor. Expression of neural crest markers in theisolated HCEPs was examined by real-time PCR andimmunostaining.Results: The proliferating cells were appeared at around 14 daysafter the seeding, exhibited a bipolar, spindle-shaped morphology,similar to neural crest cells. Interestingly, the proliferating cellsexpressed neural crest markers, p75NTR and Sox9. The colonyforming efficiency was approximately 0.31±0.04%, showed nosignificant relation to donor ages. The proliferating cells were able toundergo passage several times in younger donors below 60 years old,and the proliferative capability was higher than that of human cornealendothelial cells cultivated by the conventional method with fetalbovine serum containing media.Conclusions: We succeeded in the isolation of HCEPs which hadhigh p75NTR expression and proliferative capability.Commercial Relationships: Susumu Hara, None; RyuheiHayashi, None; Tomofumi Kageyama, None; MotokazuTsujikawa, Shionogi & Co. (C), Daiichi Sankyo Co. (F), DaiichiSankyo Co. (R), Santen Co. (R), AMO Co. (R); Kohji Nishida,Alcon (C), Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F),Osaka University (P)Support: Grants-in-Aid for Scientific Research from the Ministry ofHealth, Labor and Welfare and from National Institute of BiomedicalInnovation in JapanProgram Number: 1019 Poster Board Number: B0324Presentation Time: 1:00 PM - 2:45 PMSpatial transcriptome of human cornea using next generationsequencerSuguru Nakagawa 1 , Tomohiko Usui 1 , Hiroki Ueda 2 , Genta Nagae 2 ,Shogo Yamamoto 2 , Satoru Yamagami 1 , Hiroyuki Aburatani 2 , ShiroAmano 1 . 1 Ophthalmology, Univ of Tokyo Sch of Med, Tokyo, Japan;2 Div of Genome Science, RCAST, University of Tokyo, RCAST,University of Tokyo, Tokyo, Japan.Purpose: Cornea consists of anatomically distinct three layers,corneal epithelium (CEp), corneal stroma (CS), corneal endothelium(CEn), that have different developmental lineages and molecularfunctions. To reveal their transcriptional program, we performedspatial gene expression analysis of human cornea using nextgeneration sequencer.Methods: 100 ng of total RNA was extracted from themacroscopically dissected tissue fractions; CEp, CS, CEn, limbalepithelium (LEp) and conjunctiva (Cj) of the human donorcorneoscleral tissue. For each sample, approximately 150 million of100-bp, paired-end reads were sequenced (HiSeq2000, Illumina) andmapped against transcriptome database using BWA aligner. Quantityof 86,817 transcripts was corrected by gene/exon length(RPKM/FPKM), and then compared using regression analysis.Results: 24,601 Transcript (28.3%) were expressed in common withall cell fractions. Global comparison of each spatial transcriptomeshowed that CS shows the closest pattern against CEn (correlationcoefficient (R^2); CEn vs. CS 0.844, vs. CEp 0.807, vs. LEp 0.811,vs. Cj 0.821). We identified the candidate transcripts significantlyupregulated in CEn against other tissue fractions; CEp (n=1,760),LEp (n=1,513), CS (n=1,482), Cj (n=1,322), respectively. Ascommonly up-regulated transcripts in the CEn, we listed up morethan 300 coding genes in addition to previously reported CEnspecificmarkers such as COL8A2, CA2, SLC4A4, CDH2.Conclusions: We successfully demonstrated the layer-specificpattern of transcriptome and identified the novel up-regulated genesin CEn. This resource information is useful for understanding thespatial difference in transcriptional program to modulate celllineages.Commercial Relationships: Suguru Nakagawa, None; TomohikoUsui, None; Hiroki Ueda, None; Genta Nagae, None; ShogoYamamoto, None; Satoru Yamagami, None; Hiroyuki Aburatani,None; Shiro Amano, Topcon (P)Program Number: 1020 Poster Board Number: B0325Presentation Time: 1:00 PM - 2:45 PMHuman cornea proteome: Identification and quantitation of theproteins of the three main layers including epithelium, stromaand endotheliumThomas Dyrlund 1 , Ebbe Toftgaard Poulsen 1 , Carsten Scavenius 1 ,Camilla Lund Nikolajsen 1 , Ida B. Thøgersen 1 , Henrik Vorum 2 , Jan J.Enghild 1 . 1 Department of Molecular Biology, University of Aarhus,Aarhus C., Denmark; 2 Department of Ophthalmology, AalborgHospital, Aarhus University Hospital, Aalborg, Denmark.Purpose: Diseases of the cornea are common and refer to conditionslike infections, injuries and genetic defects. Morphologically, manycorneal diseases affect only certain layers of the cornea and separateanalysis of the individual layers is therefore of interest to explore thebasic molecular mechanisms involved in corneal health and disease.Methods: The three main layers including the epithelium, stroma andendothelium of healthy human corneas were isolated and the proteinswere (i) separated by SDS-PAGE followed by in-gel trypsinization,(ii) in-solution digested without prior protein separation or, (iii) insolutiondigested followed by cation exchange chromatography. Theresulting peptides were separated by LC-MS/MS and analysed on aTripleTOF 5600 mass spectrometer. Proteins were identified in theSwiss-Prot database using the Mascot algorithm and quantified usingMascot Distiller. Data extraction and processing was done using MSData Miner.Results: A total of 3250 unique Swiss-Prot annotated proteins wereidentified in human corneas, 2737 in the epithelium, 1679 in thestroma and 880 in the endothelial layer. Of these, 1787 proteins havenot previously been identified in the human cornea by massspectrometry. In total, 771 proteins were quantified, 157 based on insolutiondigestion and 770 based on SDS-PAGE separation followedby in-gel digestion of excised gel pieces. Protein analysis revealedthat many of the identified proteins were human plasma proteinsinvolved in the complement system, coagulation and defence againstpathogen infections.Conclusions: The separation of human corneas into the three mainlayers combined with modern mass spectrometry provides new©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneainsight into the proteins present in the individual layers and therelative abundance in each layer. This provides a useful referencedataset when exploring basic molecular mechanisms involved incorneal diseases, many of which are restricted to a specific corneallayer.Commercial Relationships: Thomas Dyrlund, None; EbbeToftgaard Poulsen, None; Carsten Scavenius, None; CamillaLund Nikolajsen, None; Ida B. Thøgersen, None; Henrik Vorum,None; Jan J. Enghild, NoneSupport: R01 EY012712Program Number: 1021 Poster Board Number: B0326Presentation Time: 1:00 PM - 2:45 PMImmunocytochemical analysis after treatment withosmoprotective and oil containing lubricants in dry eye andrefractive surgery patientsRenata R. Loureiro, Rossen M. Hazarbassanov, Joyce L. Covre,Priscila C. Cristovam, Jeison D. Barros, Jose A. Gomes.Ophthalmology, UNIFESP, Sao Paulo, Brazil.Purpose: To evaluate immunostaining patterns of inflammation andosmoprotection markers after treatment with osmoprotectivelubricant compared to oil containing and non-osmoprotectivelubricants, in evaporative dysfunctional tear syndrome (EDTS) postrefractive surgery patients.Methods: 45 patients (74,28 % female)(Mean age ± SD, 32.5±10.35) were enrolled. Participants were randomized to receivetopical drops QID for the 1st month and BID for the following 2months of Optive®, FreshTears®, (Allergan, Inc., Irvine,California).They were divided into 2 groups, (A) 15 patients withEDTS, (B) 30 patients without EDTS who were referred to eitherLASIK (15) or PRK (15). In group A, 5 patients (10 eyes) weretreated with either Optive®, FreshTears® or Endura®, as well as 5patients (10 eyes) from group B/PRK and 5 patients (10 eyes) fromgroup B/LASIK. All patients were submitted to the following testsfor EDTS diagnose: Ocular Surface Disease Index (OSDI), patientsymptomatology questionnaire, visual acuity (VA), biomicroscopy,Schirmer I test without anesthesia, tear film osmolarity, fluoresceinbreak up time (FBUT), fluorescein and lissamine green 1% staining(Oxford grading), impression cytology (IC) andimmunocytochemistry (ICC) for an inflammation marker (HLA-DR)and L-carnitine, osmoprotective component.Results: Pre-treatment and 3 month follow-up exams are completedfor both groups. ICC of conjunctiva samples showed 42.86%positivity for HLA-DR staining, on group A and 20% for groupB/LASIK, 30% for PRK, before treatment (p=0.4896, χ2 test). Therewas lower HLA-DR staining for EDTS patients treated with Optive®and Endura® (28.11% and 35.6%). ICC for L-carnitine staining was53.33% positive for A, 22% for LASIK and10% for PRK subgroup,before treatment (p=0.041, χ2 test). L-carnitine ICC staining posttreatmentshowed high positivity for FreshTears® and Endura®groups, in contrast to a lower staining for Optive® subgroup.Conclusions: Conjunctival cells showed tendency of higherexpression of inflammation marker HLA-DR on EDTS patients, andfor L-carnitine as well, which could be reduced after osmoprotectivetherapy. Those markers could be used to detect EDTS in early stageand as prognostic tool for EDTS treatment.Commercial Relationships: Renata R. Loureiro, None; Rossen M.Hazarbassanov, None; Joyce L. Covre, None; Priscila C.Cristovam, None; Jeison D. Barros, None; Jose A. Gomes,Allergan (C), Pfizer (C), Genon (C), MSD (C)Support: None in the Support field belowClinical Trial: nct01741987Program Number: 1022 Poster Board Number: B0327Presentation Time: 1:00 PM - 2:45 PMThe Regeneration Potential of Mouse Lacrimal Gland FollowingDuct Ligation ProcedureYing Liu 1 , Tetsuya Kawakita 1 , Machiko Sugiyama 1 , MasatoshiHirayama 1 , Motoko Kawashima 1 , Yoko Ogawa 1 , Masataka Ito 2 ,Shigeto Shimmura 1 , Kazuo Tsubota 1 . 1 Ophthalmology, KeioUniversity School of Medicine, Tokyo, Japan; 2 National DefenseMedicine College, Saitama, Japan.Purpose: To observe the regeneration and proliferation potential ofadult mouse lacrimal gland (LG) following duct-ligation (DL)procedure and duct-ligation-release (DLR) procedure.Methods: Adult mice were divided into two groups. One group wassubjected to the DL of the right LG, and glands were collected at day0, 3, 7 and 14 after DL (n=3-5, at each time point). Another groupwas subjected to the DLR for 7 days and the ligation was released atday 7 of the right LG. Then the glands were collected at day 9, 12, 14and 17 after the ligation was released. Tear production and the glandweight were measured during the duct ligation / release (DL/R)procedure. Tissues were investigated by Hematein & Eosin (H&E)staining and immunohistochemistry.Results: Tear secretion of DL and DLR group at day 3, 7, 9, 12 and14, has significantly decreased compared with control (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapatients, 12 eyes), and corneal nerve morphology by confocalmicroscopy (ConfoScan 4, Nidek). A single best image showingnerves in the sub-basal nerve plexus (n = 6 patients, 8 eyes) andstromal nerves (n = 4 patients, 5 eyes) was chosen from eachappointment. Images were analyzed for sub-basal epithelial nervefiber length and tortuosity and stromal nerve trunk thickness usingNeuroLucida Software (MBF Bioscience, Williston, VT). Sub-basalepithelial nerve density was calculated by dividing the total length ofnerve fibers in each image by the area photographed. Tearproduction, central corneal sensitivity, sub-basal epithelial nervedensity and tortuosity, and stromal nerve trunk thickness werecompared before PROSE wear and after uninterrupted PROSE wearusing a paired t-test.Results: Tear production significantly decreased from 17.5 ± 5.2 mmbaseline to 13.3 ± 6.1 mm after at least 30 days of PROSE wear (p =0.00098). In contrast, central corneal sensitivity increasedsignificantly from baseline (46.3 ± 6.4 mm) after at least 30 days of 6hours or more of PROSE wear (53.8 ± 7.7 mm; p = 0.010). Neithersub-basal epithelial tortuosity (1.075 ± 0.035 vs. 1.077 ± 0.049; p =0.91), nor sub-basal nerve density (1711 ± 878 µm /mm2 vs. 1845 ±707 µm/mm2; p = 0.70) were altered by PROSE wear. Stromal nervetrunk thickness decreased significantly from an initial value of 8.5 ±2.3 µm to 4.7 ± 1.9 µm after wear (p = 0.038).Conclusions: Changes in tear production and corneal nerveparameter suggest that PROSE treatment may affect elements of thelacrimal functional unit. The PROSE device shields the ocularsurface from its normal environment and may allow for functionalchanges in the neural output with a concomitant change in tearproduction.Commercial Relationships: Yvonne Wang, None; Ryan M. StClair, None; Michelle N. Lee, None; Kimberly C. Sippel, None;Jessica Ciralsky, None; Priyanka Sood, None; Ana G. AlzagaFernandez, None; Christopher E. Starr, None; Mark Rosenblatt,NoneSupport: This investigation was supported by grant UL1TR000457of the Clinical and Translational Science Center at Weill CornellMedical College and Research to Prevent Blindness220 Immunology, Allergy, NeovascularizationMonday, May 06, 2013 8:30 AM-10:15 AMTCC 303 Paper SessionProgram #/Board # Range: 1286-1292Organizing Section: CorneaProgram Number: 1286Presentation Time: 8:30 AM - 8:45 AMLens-derived Sema3A inhibits angioblast migration andvascularization of the developing corneaPeter Y. Lwigale, Chelsey McKenna. Biochemistry and Cell Biology-MS140, Rice University, Houston, TX.Purpose: To determine the role of Sema3A during ocularvasculogenesis and formation of the avascular cornea. Given thatangioblasts and ocular blood vessels in the periocular region expressNrp1, a receptor for both Vegf (angiogenic factor) and Sema3A (antiangiogenicfactor), we hypothesized that lens-derived Sema3Aprevents angioblast migration and vascularization of the developingcornea.Methods: We identified the localization of migratory angioblasts andforming vasculature in the periocular region of Tg(tie1:H2B:eYFP)transgenic quail. We examined the expression of Vegf and Sema3Ain the lens by immunohistochemistry and quantified their mRNA byqPCR. We then blocked Sema3A signaling from the region of thepresumptive cornea by lens ablation or injection of Sema3Ainhibitory peptides. We also investigated whether addition ofSema3A would inhibit Vegf-induced vascularization of the cornea.Furthermore, we analyzed Nrp1(Sema-/-) mutant mice that lackSema/Nrp1 signaling for defects in corneal avascularity.Results: Our results show that angioblasts do not migrate into theregion of the forming cornea located between the ectoderm and lens.Both Sema3A and Vegf are present in the lens, but the levels ofSema3A transcripts are significantly higher than Vegf during corneadevelopment. Inhibition of lens Sema3A resulted in ectopicangioblast migration and vascularization of the forming cornea.Addition of Sema3A protein inhibited Vegf-induced vascularizationof the cornea. We also observed ectopic angioblasts and vasculaturein corneas of Nrp1(Sema-/-) mutant embryos.Conclusions: Together, our results clearly indicate that cornealavascularity is established early during ocular development and that,Sema3A signaling from the lens plays a crucial role in this process.Commercial Relationships: Peter Y. Lwigale, None; ChelseyMcKenna, NoneSupport: NIH Grant EY018050 and EY022158Program Number: 1287Presentation Time: 8:45 AM - 9:00 AMCornea Intravital Multiphoton Visualization of the ResidentMononuclear Phagocyte Network in AllergyTomas Blanco 1 , Matthew Kan 2 , Michael Gunn 2 , Daniel R. Saban 1, 2 .1 Ophthalmology, Duke University School of Medicine, Durham, NC;2 Department of Immunology, Duke University School of Medicine,Durham, NC.Purpose: The cornea houses an extensive network of residentmononuclear phagocytes, including macrophages, Langerhan’s cells,langerin+ dendritic cells (DC) and CD11b+ DC. Their function(s),particularly independent of recruited inflammatory monocytes, ispoorly understood. A novel mouse line was established with aCX3CR1-cre x ROSA26floxSTOPfloxGFP reporter system, whereinall progeny derived from the macrophage-dendritic cell precursor(MDP) lineage permanently express eGFP. We examined thesecorneas via intravital multiphoton microscopy in a model of ocularallergy, previously shown by our group to have cornealmanifestationsMethods: Transgenic mice were used and compared withcommercially available knock in CX3CR1 GFP/+. Mice wereimmunized systemically with OVA (100 ug) + pertussis toxin (300ng) + aluminum hydroxide (1 mg), or left naïve as a control. After 2wk, mice were challenged with an eye drop of Texas Red-conjugated(250 ug OVA). Microscopy was performed before and at variousindicated time-points after OVA challenge. A multiphotonmicroscope (900 nm emission) was used at < 5% of the laser power.3 separate (high efficiency non-descanned) detectors in the epiposition were used for 2nd harmonic generation, GFP, and TexasRed.Results: Before challenge, GFP+ cells were detectable within thesub-basal plexus, and seemed to be interconnected via GFP+membrane nanotubules (MNT). Within 1-3 hrs post challenge inimmunized mice, these sub-basal cells were found extending theirprocesses and making contact with OVA at the surface. Othermorphologically distinct GFP+ OVA+ cells appeared, which wereintraepithelial (i.e. anterior to the sub-basal plexus) and notassociated with MNT. In the stroma, GFP+ MNTs were notdetectable pre-challenge. GFP+ cells became OVA+ by 6-12 hrs postchallenge. This occurred despite the observation that ‘free form’OVA seemingly could not penetrate the ocular surface. Interestingly,however, GFP+ MNT became visible in the stroma and appeared to©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaextend upward toward the sub-basal plexus. All GFP+ cells werelargely undetectable in and around 24 hours, except for infiltratedintraepithelial cells.Conclusions: These data provide evidence to refute the notion thatresident mononuclear phagocytes in the cornea are immunologicallyinert, as well as direct evidence to suggest that such cells areinterconnected and may contribute to shaping an adaptive immuneresponse.Commercial Relationships: Tomas Blanco, None; Matthew Kan,None; Michael Gunn, None; Daniel R. Saban, Schepens Eye ResInst, Mass Eye and Ear, (P), Eleven Biotherapuetics (R)Support: R01EY021798Program Number: 1288Presentation Time: 9:00 AM - 9:15 AMIntravital Multiphoton Microscopy of Corneas and DrainingLymph Nodes Shows Increased Velocity of Dendritic Cells afterCorneal Transplantation and Directionality in Corneal AllograftsTakefumi Yamaguchi 1, 2 , Kai Hu 1, 2 , Deshea L. Harris 1, 2 , PedramHamrah 1, 2 . 1 Cornea Service and Schepens Eye Research Institute,Massachusetts Eye and Ear Infirmary, Boston, MA; 2 HarvardMedical School, Boston, MA.Purpose: The role of dendritic antigen presenting cells in cornealtransplantation has been firmly established. While their functionalcharacterization has thus far mainly relied on the analysis of ex vivostudies, there remains a clear need to investigate their behavior in thecontext of intact tissues in real-time. Here we evaluate in vivokinetics of dendritic cell (DC) in the cornea and submandibulardraining lymph nodes (dLN) after corneal transplantation andtrigeminal axotomy as a control for nerve damage model.Methods: Corneal buttons from BALB/c (allogeneic) and C57BL/6(syngeneic) mice were orthotopically grafted onto CD11c-GFP-DTR(C57BL/6 background) recipients. CD11c-GFP DCs were imaged inthe graft, peripheral recipient cornea and dLN under anesthesia usingmultiphoton microscopy 2 weeks after corneal transplantation andtrigeminal axotomy. The density, kinetics and speed of DCs werecalculated and 3D movies rendered using high performance 4Dimaging software (IMARIS).Results: The density of CD11c-GFP+ cells in grafts and recipientcorneas significantly increased after corneal transplantation andtrigeminal axotomy compared to controls (p< 0.01). CD11c-GFP+cells velocity in grafts significantly increased from 0.58 μm/min(normal, central cornea) and 1.26 (axotomy) to 1.30 (syngeneic), and1.80 (allogeneic, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaUnderstanding the Mechanism of Donor Bone Marrow DerivedDendritic Cells in Promoting Corneal Allograft Survival in theRatThomas Ritter, Oliver Treacy, Aideen Ryan, Mourice Morcos,Marese Cregg, Mikhail Nosov, Lisa O'Flynn. Medicine, Nt'l Univ ofIreland, Galway, Galway, Ireland.Purpose: To understand the mechanism of ex-vivo generated donorbone marrow derived dendritic cells (BMDCs) on promoting cornealallograft survival in the rat.Methods: BMDCs were propagated from Dark Agouti (DA) rat bonemarrow precursor cells in complete medium supplemented with ratGMCSF (5ng/ml) and IL-4 (5ng/ml). For glucocorticoid treatment ofBMDCs, dexamethasone (Dexa) (10 -6 M) was added to the culture. Afully allogeneic rat corneal transplantation model (DA to LEW) wasused for in vivo studies. Day 10 donor BMDCs +/- Dexa wereharvested and 1x10 6 cells/ml injected intravenously into recipients 7days prior to corneal transplant surgery. Graft survival anddevelopment of opacity, edema and neovascularisation weremonitored throughout the therapy. On the average day of rejectionthe immune microenvironment (cell populations and cytokinesexpressed) within the graft and the draining lymph nodes wasanalysed. Alloantibody production was analysed for all experimentalgroups by flow cytometry.Results: Ex vivo generated BMDCs have a semi-mature phenotypeand can be treated with Dexa to maintain their immature phenotype(reduction in MHC II, CD80 and CD86, n=5 p 30d, n=14 p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneausing qPCR analysis.Results: After injury, macrophage accumulation was significantlyincreased in MMP12-/- mice compared with WT mice. Analysis ofthe expression levels of several CCL chemokines demonstratedsignificantly elevated RNA and protein levels of CCL2 in Mmp12-/-corneas compared with WT corneas. The trafficking of macrophagesinto the central cornea following injury was significantly reduced bysubconjunctival injections of CCL2 blocking antibodies. Theexpression of a macrophage M1 marker (TNF-α) was increased ininjured corneas of Mmp12-/- mice while expression of a macrophageM2 marker (CD23) was reduced.Conclusions: Excessive accumulation of macrophages followingcorneal injury and an M1 macrophage phenotype favor a fibroticresponse to corneal injury. MMP12 appears to protect against afibrotic response to injury by negatively regulating CCL2 expression,decreasing macrophage accumulation, and by promoting a tissuereparative M2 macrophage phenotype.Commercial Relationships: Matilda F. Chan, None; Jeffrey Lin,None; Neeraj Ramakrishnan, None; Zena Werb, NoneSupport: NIH Grant K08EY018858necessary to validate these results for in vivo human corneal tissue.Additionally, safety aspects at high intensities must be investigated.Stiffness increase of all treatment groups compared to control group.237 Corneal Cross-linking and BiomechanicsMonday, May 06, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 1611-1645/D0246-D0280Organizing Section: CorneaProgram Number: 1611 Poster Board Number: D0246Presentation Time: 8:30 AM - 10:15 AMThe efficacy of corneal cross-linking shows a sudden decreasewith very high intensity UV-light and short treatment timeJeremy Wernli 1, 3 , Silvia Schumacher 1, 3 , Eberhard Spoerl 2 , MichaelC. Mrochen 1, 3 . 1 IROC Science to Innovation AG, Zurich,Switzerland; 2 Ophthalmology, Carl Gustav Carus University HospitalDresden, Dresden, Germany; 3 IROC Innocross AG, Zurich,Switzerland.Purpose: Standard treatment in case of progressive keratectasia isUV-triggered corneal cross-linking. For irradiances larger than 10mW/cm 2 and treatment times below 10 min the scientific proof of abiomechanical strengthening effect is insufficient. The authorsinvestigated the biomechanical strengthening of ex-vivo cornealtissue treated with irradiances between 3 mW/cm 2 and 90 mW/cm 2and illumination times from 30 minutes to 1 minute, respectively.Methods: 100 porcine eyes received riboflavin+UV treatment(constant irradiation dose of 5.4 J/cm 2 ) with different intensities andillumination times and were randomly assigned into 10 groups. Acontrol group (80 eyes) was not irradiated but underwent the sametreatment otherwise. Young’s modulus at 10% strain was determinedfor each strip after uniaxial stress-strain measurement. A Kruskall-Wallis test was used for statistical analysis.Results: A statistically significant difference (α=0.01) was foundbetween the median value of Young’s modulus of the treatmentgroups up to 45 mW/cm 2 (illumination times from 30 min to 2 min)compared to the control group. There was no statistically significantdifference between the treatment groups from 50 mW/cm 2 up to 90mW/cm 2 (illumination times of less than 2 min) and the controlgroup.Conclusions: The ex vivo results of corneal cross-linking performedin porcine corneas show that the Bunsen-Roscoe reciprocity law isonly valid for illumination intensities up to 40 to 50 mW/cm 2 andillumination times of more than 2 min. Further experiments areYoung’s modulus at 10% strain of the control and different treatmentgroups.Commercial Relationships: Jeremy Wernli, IROC Innocross AG(C); Silvia Schumacher, IROC Innocross AG (C); EberhardSpoerl, None; Michael C. Mrochen, IROC Innocross AG (I)Program Number: 1612 Poster Board Number: D0247Presentation Time: 8:30 AM - 10:15 AMCorneal Biomechanical Properties after UV Cross-linking in theRabbitMichael D. Twa 1 , Jiasong Li 2 , Ravi Kiran Manapuram 2 , Floredes M.Menodiado 2 , Salavat Aglyamov 3 , Stanislav Emelianov 3 , Kirill Larin 2 .1 College of Optometry, University of Houston, Houston, TX;2 Biomedical Engineering, University of Houston, Houston, TX;3 Biomedical Engineering, University of Texas, Austin, TX.Purpose: Elasticity imaging has been applied in other areas ofmedicine and more recently used to characterize the structuralproperties of ocular tissues. An OCT-based elastography method wasdeveloped and measurements were performed in rabbit corneal tissuebefore and after UV-riboflavin corneal cross-linking (CXL).Methods: Corneal elastography measurements were performed usinga Phase Stabilized Swept Source Optical Coherence Elastography(PhS-SSOCE) with a sensitivity of ~10 nm along with air-pulse tissuestimulation. Surface wave propagation was measured over a 6x6mmarea before and after UV-riboflavin corneal cross-linking. Tissueproperties (Young's modulus, surface wave propagation speed andsurface wave amplitude) were measured.Results: Treatment resulted in a measurable increase cornealstiffness confirmed by mechanical extensiometry (before CXL:E=1.32±0.39MPa; after CXL: E=2.34±0.91MPa). Surface waveamplitude and velocity was greatest near the excitation position(Amplitude= 993nm and Velocity=0.8±0.09m/s) and decreased fromthis point to the apex. Following cross-linking surface waveamplitudes decreased (141nm) and wave velocity increased (8.2±5.6m/s).©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConclusions: Ocular elastography can be performed using acombination of phase-sensitive OCT and air pulse stimulation. Thismethod can detect low amplitude tissue excitation, which can be usedto quantify corneal stiffness.Commercial Relationships: Michael D. Twa, None; Jiasong Li,None; Ravi Kiran Manapuram, Bioptigen Inc., (E); Floredes M.Menodiado, None; Salavat Aglyamov, None; Stanislav Emelianov,None; Kirill Larin, NoneSupport: EY022362Program Number: 1613 Poster Board Number: D0248Presentation Time: 8:30 AM - 10:15 AMThe Rigidity of Corneas before and after Corneal Cross-linking -as measured by Corvis® STSashia Bak-Nielsen, Iben Bach Pedersen, Anders Ivarsen, JesperHjortdal. Ophthalmology, Aarhus University Hospital, Aarhus,Denmark.Purpose: Corneal Cross-linking (CXL) for treating keratoconus hasbeen shown to stiffen the cornea in vitro and clinical studies havedocumented that progression of keratoconus is halted in most cases.The Corvis ST from Oculus dynamically measures cornealdeformation by Scheimpflug imaging during an air puff. The patternof deformation theoretically depends on the intraocular pressure, thecorneal thickness and the material properties of the cornea. Thepurpose of this study was to measure the possible stiffening effect ofCXL on patients with keratoconus, comparing groups with andwithout a previous CXL procedure.Methods: Thirty-seven keratoconus patients were included- 19 with untreated keratoconus- 18 with keratoconus treated with CXL 4-50 months previously(median 27 months)Furthermore 31 healthy subjects were included as a control group.Apart from being measured with Corvis ST the subjects underwent afull ophthalmic examination including Pentacam topography. ThePentacam measurements were used to stage the patients into 4 groupsbased on the severity of the keratoconus. The stiffness of the corneawas evaluated by the radius of corneal concavity at the maximumdeformation (DR) as calculated by the Corvis ST.Results: There was no significant difference in DR between theuntreated keratoconus group and the CXL treated group although DRappeared marginally larger in eyes that had underwent CXL (unpairedt-test, p>0.05, Table 1). Increasing keratoconus severity(Grades 1-4) had only a small and insignificant influence on DR(ANOVA, p>0.05). DR in untreated and CXL treated keratoconusgroups was significantly smaller compared with normal eyes.Conclusions: Eyes with keratoconus have a smaller DR as measuredby the Corvis ST, but the DR was similar in untreated and CXLtreated eyes. As the effect of CXL mainly is exerted in the anteriorpart of the corneal stroma, it can be speculated that DR is insensibleto changes in corneal material properties in the anterior stroma.Further studies of corneal deformation parameters in the samepatients before and after CXL are needed to further evaluate theCorvis ST and the effect of CXL.Table 1: Mean DR and 95% Confidence interval (CI) for keratoconuseyes (grade 1-4), CXL treated eyes (grade 1-4) and normal eyesCommercial Relationships: Sashia Bak-Nielsen, None; Iben BachPedersen, None; Anders Ivarsen, None; Jesper Hjortdal, CarlZeiss Meditec (R)Program Number: 1614 Poster Board Number: D0249Presentation Time: 8:30 AM - 10:15 AMAssociation of Ambient Solar Radiation with BiomechanicalProperties of the Cornea In an elderly population: The AlienorStudyCedric Schweitzer 1 , Cecile Delcourt 2 , Florence Malet 1 , Melanie LeGoff 2 , Jean-Francois Korobelnik 1, 2 , Marie B. Rougier 1 , Marie-NoelleDelyfer 1, 2 , Jean Francois Dartigues 2 , Pascale Barberger-gateau 2 ,Joseph Colin 1 . 1 Ophthalmology, University Hospital Pellegrin,Bordeaux, France; 2 INSERM, ISPED, Bordeaux university,bordeaux, France.Purpose: To analyze the association of ambient solar radiation (SR)on biomechanical properties of the cornea in adult patients.Methods: The ALIENOR (Antioxydants, Lipides Essentiels,Nutrition and maladies OculaiRes) Study is a population-basedepidemiological study on age-related eye diseases. In 2009-2010, 625subjects, aged 75 years or more, had an eye examination, includingintraocular pressure, central corneal thickness (CCT) measurements,and an evaluation of the biomechanical properties of the cornea usingthe ocular response analyser® (ORA®, reichert inc., USA). Meanlifetime ambient SR was estimated using residential history. Globalambient annual SR (a measure of solar energy including allwavelengths) was estimated using astronomic formulas and thestatistics of sunshine hours at each location. Then, for eachparticipant, average annual ambient SR was estimated by weightingannual ambient solar radiation at each location by the time spent atthat location. Participants were classified in 3 groups (Group 1:474.74 kJ/cm2). Corneal hysteresis (CH), corneal resistancefactor (CRF), corneal compensated intraocular pressure (IOPcc),Goldmann correlated intraocular pressure (IOPg) and CCTparameters were analyzed between SR groups, using mixed linearregression models taking into account data from both eyes and theirintra-individual correlations.Results: After adjustment for age and gender, there was a significantassociation of CH and CRF values with SR higher than 474.74kJ/cm2 (CH: -0.39mmHg, 95% confidence interval (CI): -0.75;-0.03,p=0.03/ CRF: -0.38mmHg, 95% CI: -0.74; -0.03, p=0.035), whereasthere was no significant association of CH and CRF values with SRlower than 458.53 kJ/cm2 (CH: 0.01mmHg, 95% CI: -0.34; 0.36,p=0.96/ CRF: 0.002mmHg, 95% CI: -0.35; 0.35, p=0.99). The CCTwas not significantly associated with SR ( SR 474.74: CCT: -4.25µm, 95%CI: -13.86;5.35, p=0.38).Conclusions: CH and CRF were significantly lower in subjectsexposed to high ambient solar radiation. Ambient UV exposure mightinduce histological changes of the cornea that influence itsbiomechanical properties.Commercial Relationships: Cedric Schweitzer, None; CecileDelcourt, Laboratoires Théa (F), Novartis (C), Bausch+Lomb (C);Florence Malet, None; Melanie Le Goff, None; Jean-FrancoisKorobelnik, Alcon (C), Allergan (C), Bayer (C), Carl Zeiss Meditec(C), Novartis (C), Thea (F); Marie B. Rougier, THEA (C),Bausch&Lomb (C), Allergan (C), Kemin (C); Marie-Noelle Delyfer,Thea Laboratories (F); Jean Francois Dartigues, Novartis (F),IPSEN (F); Pascale Barberger-gateau, Danone (F), Vifor Pharma(C); Joseph Colin, Alcon (C), Abbott (C), AdditionTechnology (C)Support: Laboratoires Théa, Fondation Voir et Entendre©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 1615 Poster Board Number: D0250Presentation Time: 8:30 AM - 10:15 AMEffect of UVA-Rb cross-linking on through-thickness strains incanine corneasJoel Palko 1 , Xueliang Pan 2 , Jun Liu 3, 4 . 1 Wexner Medical Center,Ohio State University, Columbus, OH; 2 Center for Biostatistics, OhioState University, Columbus, OH; 3 Deprtment of BiomedicalEngineering, Ohio State University, Columbus, OH; 4 Department ofOphthalmology, Ohio State University, Columbus, OH.Purpose: Monitoring the mechanical changes of the cornea beforeand after corneal cross linking (CXL) provides valuable insight intoCXL mechanisms and may help provide more personalized treatmentplans for this therapy in the prevention of progressive keratoconus.The purpose of this study was to measure through-thickness strains inthe cornea at physiologic IOPs before and after CXL using noninvasiveultrasound.Methods: The anterior 3/4 of paired canine corneoscleral shellsincluding a CXL treated group (n=6) and a control group (n=6) weremounted to a pressurization chamber within 10hrs of euthaniasia. TheCXL group completed a standard clinical CXL protocol usingriboflavin (Rb solution and UVA radiation (370nm, irradiance3mW/cm2). Control eyes were given an identical Rb treatmentwithout UVA irradiation. Cornea ultrasound scans (at 55 MHz) alongthe nasal-temporal (NT) and superior-inferior (SI) cross-sectionswere obtained before and after treatment as IOP was graduallyincreased from 5 mmHg to 45mmHg. Strain tracking was performedusing a previously validated method (Tang & Liu, J. Biomech. Engrg2012, 134(9)). Mean radial compressive strains and tangential tensilestrains were calculated for the anterior, middle, and posterior onethirds of the cornea thickness in the nasal-temporal (NT) andsuperior-inferior (SI) directions. Mean strains at IOPs of 10, 20, and30mmHg were compared between the CXL and control groups usingmixed linear models with repeated measures.Results: Statistically significant reductions in tensile andcompressive strains were found in the SI orientation at all three IOPsand all three layers in the CXL group (all p0.05). The anterior third appeared to have larger tensile strainreduction than the posterior layer in the CXL group.Conclusions: Ultrasound strain tracking revealed that the Rb-UVACXL procedure significantly reduced corneal strains (i.e., stiffenedthe cornea) during physiologic IOP elevation with more pronouncedeffects observed in the anterior cornea. The ability to measure andmonitor cornea strains may provide insight into the biomechanicaleffects of CXL and better define its role as a treatment for certainocular disorders.Commercial Relationships: Joel Palko, None; Xueliang Pan,None; Jun Liu, NoneSupport: NIHRO1EY020929 (JL), Ohio State University College ofMedicine (JP)Program Number: 1616 Poster Board Number: D0251Presentation Time: 8:30 AM - 10:15 AMIn Vivo Evaluation of Corneal Biomechanical Properties AfterCorneal Collagen Cross-linking TherapyRaksha Urs 1 , Harriet Lloyd 1 , Ronald H. Silverman 1, 2 .1 Ophthalmology, Columbia University Medical Center, New York,NY; 2 Frederic L. Lizzi Center for Biomedical Engineering, RiversideResearch Institute, New York, NY.Purpose: Collagen cross-linking therapy (CXL) is emerging as atreatment option for keratoconus. This procedure strengthens thebiomechanical properties of the cornea by cross-linking the collagenbonds. However, biomechanical tests, to evaluate CXL outcome,have been performed only on ex vivo tissue. In vivo, the efficacy ofthe treatment is verified by assessing vision quality. The objective ofthis project is to demonstrate an in vivo technique to determinedifference in biomechanical strength of the cornea after CXL.Methods: CXL procedure was performed on the right eyes of 6rabbits. The left eyes were used as controls. Acoustic Radiation Force(ARF) was used to assess corneal stiffness in vivo, once beforetreatment (Baseline BL) and weekly for four weeks after treatment(W1-W4). Cornea was exposed to ARF using a single elementtransducer (25 MHz central frequency; 6 mm aperture; 18 mm focallength; Panametrics V324-SU). The beam sequence consisted of 20pushing tonebursts of 400 μs duration (80% duty cycle). Imagingimpulses were interleaved in the dead time to allow the sametransducer to acquire radiofrequency data during the push mode toimage corneal displacement. Acoustic power levels were withinFDA-specified levels for ophthalmic safety. Displacement of thefront and back surfaces of the cornea were used to determine thechange in corneal thickness and strain. ARF induced strain was fit tothe Kelvin-Voigt model to determine the elastic modulus. Theaverage moduli were calculated for the six rabbits, for each of thefive time points (BL, W1-W4).Results: At the end of four weeks, ARF measurements showed anincrease of average elastic modulus by 33% in the treated eye, and3% in the control eye. Paired t-tests revealed statistically significantdifferences between treated and untreated eyes from W1-W4(p=0.0005, 0.04, 0.0007, 0.006). There was no significant differencebetween right and left eyes before treatment (p=0.95).Conclusions: Our findings demonstrate statistically significantdifferences in stiffness between control and CXL-treated rabbitcorneas in vivo based on axial stress/strain measurements obtainedusing ARF. The capacity to non-invasively monitor corneal stiffnessoffers the potential for clinical monitoring of CXL. Longer term©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneastudies will be required to elucidate the effectiveness of this noninvasivetechnique.Commercial Relationships: Raksha Urs, None; Harriet Lloyd,None; Ronald H. Silverman, NoneSupport: AIUM EER; NIH Grant EY021529; Research to PreventBlindnessProgram Number: 1617 Poster Board Number: D0252Presentation Time: 8:30 AM - 10:15 AMLong Term Follow-Up of Corneal Topographic Indices FollowingCollagen Crosslinking In Eyes with KeratoconusErick Hernández-Bogantes, Lihteh Wu, David Flikier. Instituto deCirugía Ocular, San José, Costa Rica.Purpose: To compare the pre-treatment and 5 year post-treatmentvisual acuity and corneal topographic indices in eyes withkeratoconus that were treated with corneal collagen crosslinking.Methods: An observational, uncontrolled, retrospective study of 7patients (12 eyes) with keratoconus that were treated with epitheliumoffcorneal collagen crosslinking between March and December of2007 was conducted. Pre-treatment and 5 year post-treatment anteriorcorneal surface (keratoconus index, index of vertical asymmetry,index of surface variance, central keratoconus index, minimum radiusof curvature, index of height asymmetry, index of heightdecentration) and normalized Belin-Ambrosio indices (frontelevation, back elevation, pachymetric progression, corneal thinnestpoint and the total analysis) were obtained by the Pentacamtopographer, Oculus Inc. (Wetzlar, Germany). The uncorrected visualacuity and best corrected visual acuity were also compared.Results: The 5 year follow-up showed that both central keratoconusindex (P= 0.0452) and best corrected visual acuity (P= 0.0313)improved following corneal collagen crosslinking. At 5 years offollow-up, there were no statistically significant differences betweenthe pre-operative and post-operative values of either the otheranterior surface variables or the normalized Berlin-Ambrosio indices.Conclusions: Even though the surface of the cornea does not appearto be dramatically altered after five years of corneal collagencrosslinking, improvement in visual acuity is obtained and remainsconstant.Commercial Relationships: Erick Hernández-Bogantes, None;Lihteh Wu, Heidelberg Engineering (R); David Flikier, NoneProgram Number: 1618 Poster Board Number: D0253Presentation Time: 8:30 AM - 10:15 AMComparison of corneal changes between standard andtransepithelial riboflavin-UVA crosslinking method usingmultiphoton microscopy and second harmonic imagingPraveena Gupta 1 , Best Anyama 2 , Kevin M. Wells 2 , MassoudMotamedi 3, 1 , Bernard F. Godley 1 , Gracie Vargas 3 . 1 Ophthalmology& Visual Sciences, Univ of Texas Medical Branch, Galveston, TX;2 School of Medicine, University of Texas medical Branch,Galveston, TX; 3 Center for Biomedical Engineering, Univ of TexasMedical Branch, Galveston, TX.Purpose: Corneal crosslinking procedures are offered as a treatmentfor keratoconus and corneal ectasic disorders. This study wasundertaken to investigate the comparative stromal changes after theUVA-crosslinking on a riboflavin-debrided and a riboflavintransepithelialtreated porcine cornea using multiphoton microscopyand second harmonic generation signals.Methods: Fifteen fresh pig eyes were treated using either thestandard method of riboflavin-UVA crosslinking or thetransepithelial method (riboflavin TRIS-EDTA) for 30 min atirradiance of 3mW/cm2. All corneas were then stained with a celldeath marker and processed for non-invasive multiphotonmicroscopy and second harmonic signal imaging. Data collectedwere analyzed using image J.Results: Standard CXL treatment resulted in severe loss of theclassic interwoven collagen architecture all the way up to 300 uMdepths, whereas, similar changes were noted only up to 150 uM depthin the TE method of CXL. Significantly higher numbers of deadkeratocytes were counted at all the depths in the standard CXLexposed eyes in contrast to fewer keratocyte deaths that were morepronounced only in the anterior stroma in the TE-CXL treatedcorneas (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawill be expanded to include an anisotropic, fiber-dependenthyperelastic material formulation.Figure 1: Corneal flap attached to biaxial extensometer.Commercial Relationships: William A. Eddington, Avedro Inc.(E); Marc D. Friedman, Avedro Inc (E); Evan A. Sherr, Avedro,Inc. (E); David Muller, Avedro Inc (E)Program Number: 1620 Poster Board Number: D0255Presentation Time: 8:30 AM - 10:15 AMFinite Element Analysis of Treatment of Corneal Astigmatismwith Collagen CrosslinkingIBRAHIM SEVEN 1, 2 , Abhijit Sinha Roy 1 , William J. Dupps 1, 2 .1 Ophthalmology, Cleveland Clinic Cole Eye Inst, Cleveland, OH;2 Biomedical Engineering, Cleveland Clinic Lerner Research Institute,Cleveland, OH.Purpose: To test the hypothesis that selective collagen crosslinkingcan alter corneal astigmatism and to assess the effects of treatmentorientation and pattern.Methods: 3D geometry of a patient with corneal astigmatism wasmeasured by a Scheimpflug tomography system (Pentacam v.1.61).Elevation data was fit and extrapolated with 12th order Zernikepolynomials routine which was coded in Python2.7. The extrapolateddata was imported into Solidworks (ver. 2011). The geometry wasmeshed with hexahedral elements by Trugrid (v. 2.3.3). Cornealbiomechanical properties were defined as nonlinear, isotropic andincompressible. The intra-ocular pressure was assumed as 15 mmHg.Finite element analyses were performed using Abaqus (v. 6.11).Since the in vivo (or pre-operation) geometry was pre-stressed byintra-ocular pressure, the geometry was solved for the no-loadcondition and pre-stresses were calculated. We simulated 4 differentcrosslinking treatment patterns with a stiffening factor of 2 andeffective depth of 300 um. The treatment was simulated with a bowtiepattern oriented on the steep astigmatism axis (pattern1), a bow-tiepattern oriented on the flat astigmatism axis (pattern2), a fan pattern(pattern3), and a central ellipse pattern oriented on the flatastigmatism axis (pattern4). The corneal anterior surface coordinateswere extracted at the end of the each simulation and the anteriorsurface axial curvature was calculated. The refractive index was asassumed as 1.3375.Results: Keratometry (Sim K) values in the preoperative eye (invivo) were 44.85/46.22@78. The SimK value changed to44.06/46.62@90, 44.83/45.88@45, 44.76/46.27@84,44.64/45.64@51 with pattern1, pattern2, pattern3, and pattern4treatment, respectively. The astigmatism value was 1.37in the preoperationstage. The value changed to 2.56, 1.05, 1.51, and 1.00 withpattern1, pattern2, pattern3, and pattern4 treatment, respectively.Conclusions: In this pilot study, simulated patterned collagencrosslinking had an effect on corneal astigmatism. Treatments on thesteep axis increased astigmatism, while treatments orthogonal to thesteep axis decreased astigmatism. Additional clinical geometries withregular and irregular astigmatism will be investigated and modelingFigure 1 - Comparison of different treatment patternsCommercial Relationships: IBRAHIM SEVEN, None; AbhijitSinha Roy, Carl Zeiss Meditec (F), Cleveland Clinic Innovations (P),Topcon Inc. (F); William J. Dupps, Zeimer (C), Topcon (F), Avedro(F), Carl Zeiss Meditec (F), Cleveland Clinic Innovations (P)Program Number: 1621 Poster Board Number: D0256Presentation Time: 8:30 AM - 10:15 AMCorneal geometric stress factor to evaluate response to cornealcollagen cross-linking in keratoconusRiccardo Vinciguerra 1, 2 , Cynthia J. Roberts 3 , Ashraf M. Mahmoud 3 ,Claudio Azzolini 2 , Paolo Vinciguerra 1 . 1 Opthalmology, HumanitasClinical and Research Center, Milan, Italy; 2 Dept. of Surgical andMorphological Sciences, University of Insubria, Circolo Hospital,Varese, Italy; 3 Ophthalmology and Biomedical Engineering, TheOhio State University, Columbus, OH.Purpose: To evaluate corneal stress distribution based purely ongeometry without consideration of loading via intraocular pressure,pre and post corneal collagen cross-linking (CXL) with a newtomographic parameter, “Corneal Geometric Stress Factor”.Methods: Tomographic data from four hundred and eighty subjects(323 right eyes and 340 left eyes) were collected retrospectively fromIstituto Clinico Humanitas (Rozzano, Italy) with up to 70 months pre-CXL and 60 months post-CXL. Pentacam U12 files (OculusOptikgerate GmbH, Wetzlar, Germany) were transferred to The OhioState University and processed independently using custom software.Corneal Geometric Stress Factor (CGSF) was calculated atcorresponding points from curvature and pachymetric maps to createa CGSF map. CGSF evaluates the cornea’s contribution to Hoopstress without considering the applied load (intraocular pressure) andit can be expressed as the radius of curvature over twice of thethickness (CGSF=R/2t). Cone Location and Magnitude Index(CLMI) and Flat zone Location and Magnitude Index (FLMI) wereapplied to the CGSF map to obtain maximum stress and minimumstress and to calculate the level of asymmetry in the stress pattern.Pre and post CXL regression analyses were performed.Results: Regression analysis showed a significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand curvature associated with CXL treatment induces a consequentreduction in both minimum and maximum stress as well as areduction of asymmetry in the stress distribution. IOP can modify thestress magnitude, but not change the pattern demonstrated.Commercial Relationships: Riccardo Vinciguerra, None; CynthiaJ. Roberts, Oculus Optikgerate GmbH (C), Ziemer OphthalmicSystems AG (C), Sooft Italia (R), Carl Zeiss Meditec (F); Ashraf M.Mahmoud, None; Claudio Azzolini, None; Paolo Vinciguerra,SOOFT Italia (C), Oculus Optikgerate GmbH (C), NIDEK, Japan(C), Schwind (C)Program Number: 1622 Poster Board Number: D0257Presentation Time: 8:30 AM - 10:15 AMEvaluation of the riboflavin and Ultraviolet light effect onkeratocytes cultivated in vitroJoyce L. Covre, Priscila C. Cristovam, Renata R. Loureiro, RossenM. Hazarbassanov, Mauro S. Campos, Élcio H. Sato, Jose A. Gomes.Ophthalmology, UNIFESP, Sao Paulo, Brazil.Purpose: Evaluate the riboflavin and ultraviolet light effect onhuman keratocytes cultivated in vitro.Methods: Keratocytes were obtained from human corneal rimsremnants of tissue previously used in corneal transplants at theDepartment of Ophthalmology of UNIFESP/EPM, and cultured inDMEM/F12 medium with FBS until confluence. The cell cultureswere characterized by immunofluorescence with antibodies to K3(epithelial marker), Thy 1(fibroblast marker), α-SMA(myofibroblast), Lumican and Keratocan (keratocyte markers). Thecorneal stromal cells were covered with collagen (200µL and 500µL) and 0.1% of riboflavin and were exposed to ultraviolet light(UV) for 30 minutes. After 24 hours, cytotoxicity was determined byMTT assay and cell viability was quantified by means of dye Hoechst33342/Propidium Iodide.Results: All cell cultures reached confluence around 20 days.Immunofluorescence demonstrated positive expression of keratocytemarkers (Lumican and Keratocan) and negative expression ofepithelial (K3), fibroblast (Thy 1) and myofibroblast (alpha-SMA)markers. After riboflavin and UV light exposure, cultivated cellswithout collagen layer presented higher cytotoxicity with MTT(oneway ANOVA, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaJasmyne C. Sermeno, None; Paul Russell, None; Christopher J.Murphy, Ocular Services On Demand (I), Ocular Services OnDemand (C), Platypus Technologies LLC (I), Imbed LLC (I), EyeKorLLC (I), Allergan (C), Genentech (C), Sarcode (C), Covance (C)Support: NIH KO8EY021142 (SMT), NIH R01EY019970 (CJM),and Research to Prevent Blindness (UC Davis)Program Number: 1625 Poster Board Number: D0260Presentation Time: 8:30 AM - 10:15 AMComparison of Biomechanical and Tomographic Data inSubclinical KeratoconusPaolo Vinciguerra 1 , Renato Ambrosio 3 , Mario R. Romano 1 , Isaac C.Ramos 3 , Claudio Azzolini 2 , Silvia Trazza 1 , Riccardo Vinciguerra 1, 2 .1 Ophthalmology, Istituto Clinico Humanitas, Milan, Italy; 2 Dept. ofSurgical and Morphological Sciences, University of Insubria, CircoloHospital,, Varese, Italy; 3 Instituto de Olhos Renato Ambrósio, Rio DeJaneiro, Brazil.Purpose: To evaluate the tomographic and biomechanical cornealchanges in subclinical keratoconus (KC).Methods: Five patients with very asymmetric KC wereretrospectively compared with fellow eyes without tomographicevidences of KC. Tomographic and biomechanical data wererespectively obtained with Pentacam and Corvis ST (OculusOptikgerate GmbH, Wetzlar, Germany).The eyes without tomographic evidences of KC were also comparedwith five healthy subjects pachymetry- and intra ocular pressure(IOP)-matched.From Pentacam analysis we considered: minimal pachymetry, singleand total deviation values(Dv) from Belin-Ambrosio EnhancedEctasia Display (BAD), and all topometric indexes obtained in thetopometric map.From Corvis ST analysis we considered: time (t1-2), length (l1-2)and velocity (v1-2) of first and second applanation, time (tC), peakdistance (pC), radius (rC) and deformation amplitude (dC) of highestconcavity, IOP and pachymetryResults: Comparison between KC and the fellow eyes revealedsignificant differences in single and total Dv evaluated (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaage of 18 who underwent both Orbscan and ORA testing. The ORAKMI software compares the corneal biomechanical properties of theexamined eye to a normative database and assigns the eye to one offive categories: normal, KCN suspect, mild KCN, moderate KCN,and severe KCN. Three masked MD observers (graders A, B, and C),all of whom are cornea fellowship trained, examined the Orbscantopography of each of the eyes, and classified each eye into the same5 categories. The agreement between observers’ classification andclassification by KMI, as well as agreement between the individualobservers, was calculated using overall agreement and free-marginalkappa statistic.Results: The KMI software identified 26 eyes as normal, comparedto 16, 32, and 28 eyes for graders A, B, and C. 16 eyes wereclassified as “KCN suspect” by KMI, compared to 14, 3, and 7 eyesby graders A, B, and C respectively. 3 eyes were classified as “mildKCN” by KMI, compared to 7, 5, and 6 eyes by graders A, B, and C,respectively. 1 eye was classified as “moderate KCN” by KMI,compared to 5, 4, and 3 eyes by graders A, B, and C, respectively. Noeyes were classified as “severe KCN” by KMI, compared to 4, 2, and2 eyes by graders A, B, and C, respectively.The three graders agreed with each other 55% of the time, with afree-marginal kappa of 0.448 (moderate agreement). Grader A andKMI agreed 30% overall, with a free-marginal kappa of 0.130 (slightagreement). Grader B and KMI agreed 46% overall, with a freemarginalkappa of 0.321 (fair agreement). Grader C and the KMIagreed 46% overall, with a free-marginal kappa of 0.321. KMIagreed with all 3 graders 13% of the time and with at least one grader63% of the time. The KMI did not agree with any grader 37% of thetime.Conclusions: There is only slight to fair agreement betweenobservers and the KMI, compared to moderate inter-observeragreement. The KMI alone is not well correlated with the subjectiveinterpretation of topography, and should be used in conjunction withother modalities to evaluate a possible keratoconus suspect.Commercial Relationships: Ryan A. Vasan, None; Ryan M. StClair, None; Syed A. Hussnain, None; Ana G. Alzaga Fernandez,None; Christopher E. Starr, NoneProgram Number: 1628 Poster Board Number: D0263Presentation Time: 8:30 AM - 10:15 AMInvestigation of corneal vibration during air puff deformationusing numerical approaches with clinical validationZhaolong Han 1 , Cynthia J. Roberts 2 . 1 Department of CivilEngineering, School of Naval Architecture, Ocean and CivilEngineering, Shanghai Jiao Tong University, Shanghai, China;2 Department of Ophthalmology and Department of BiomedicalEngineering,The Ohio State University, Columbus, OH.Purpose: To investigate cornea dynamics under air pulsedeformation and relate vibration amplitude to corneal elasticity.Methods: The phenomenon of in vivo cornea dynamic vibrationunder air pulse deformation is predicted using a nonlinear dynamicmodel based on a simplified kinematic differential equationdescribing corneal motion, y(t), as a function of equivalent externalforces, corneal mass (m), damping constant (c) representing theviscoelastic capacity of the cornea, and the coefficient of cornealelasticity, (k). Corneal vibration is validated clinically with twokeratoconic subjects and two normal subjects greater than 50 years ofage with acquired measurements using the CorVis ST, a Scheimpflugsystem capturing approximately 140 images of a single horizontalcorneal meridian during a 30ms air puff at greater than 4,000frames/s, as well as the Ocular Response Analyzer (ORA) to providecorneal compensated intraocular pressure (IOPcc). In addition a 3Dfinite element (FE) cornea model is constructed in the environment ofANSYS 11.0 to simulate the results produced.Results: Modeling predicts greater vibration amplitude with lowercoefficient of elasticity, as seen in Figure (a), which is validated withclinical measurements. Keratoconic subjects demonstrated greatervibration amplitude than older normal subjects. An example ofvibration data from one keratoconic subject is shown in Figure (b)which represents corneal surface motion of a single point through thetime course of the air puff. Also shown are two meshes from finiteelement modeling with high and low elasticities in Figure (c), whichdemonstrate greater depth of deformation with lower elastic modulusat consistent IOP.Conclusions: Both numerical methods and clinical measurementsshow that softer corneas demonstrate larger vibration amplitude andgreater depth of deformation compared to stiffer corneas. Futurework will investigate the influence of IOP on corneal elasticity andvibration amplitude, as well as increase the complexity of thenumerical modeling.Commercial Relationships: Zhaolong Han, None; Cynthia J.Roberts, Oculus Optikgerate GmbH (C), Ziemer OphthalmicSystems AG (C), Sooft Italia (R), Carl Zeiss Meditec (F)Program Number: 1629 Poster Board Number: D0264Presentation Time: 8:30 AM - 10:15 AMModeling corneal response to an air puff using deformation datato derive Young’s modulusKimberly M. Metzler 1 , Cynthia J. Roberts 1, 2 , Steven M. Whitaker 3 ,Michael J. Lawrence 3 , Jennifer E. Malik 1 , Jeffrey P. Bons 3 .1 Biomedical Engineering, Ohio State University, Columbus, OH;2 Opthalmology, Ohio State University, Columbus, OH; 3 Mechanicaland Aerospace Engineering, Ohio State University, Columbus, OH.Purpose: To create and validate a computational model thatdescribes the deformation characteristics of human donor corneasmounted in an artificial anterior chamber in response to an air puff.Methods: A 2-dimensional COMSOL Multiphysics model of an airjet impinging on a human donor cornea mounted in an artificialanterior chamber was created. The physical air jet was generated bythe CorVis ST, a device used clinically to evaluate deformationresponse in living corneas. This air jet was characterized with hotwire anemometry to acquire spatial flow velocity data. The hot wirewas placed at the jet exit on the CorVis, and then moved outwardwith micrometer control to distances of 3, 6, 9, 12, 15, 20, and 25 mmalong the centerline. The duration of the hot wire anemometryrecordings lasted 40 ms. Preliminary data of the temporal profileshows that the peak velocity along the centerline during the air puffas it exits the device (distance x = 0) is over 100 m/s. At distancesbetween 9 and 12 mm from nozzle of the CorVis ST, the peakvelocity reaches above 90 m/s. Accordingly, the model inlet velocityrepresenting the CorVis ST was set at 100 m/s. Corneal dimensionswere modeled by constructing an ellipse inside an 8mm sphere thatwas sectioned to have a width of 12 mm. The cornea section wasmounted onto a rigid body within the model, representing theBarron’s Artificial Anterior Chamber. Intraocular pressure wasmanipulated to be 10, 20, 30, 40, and 50 mmHg. Deformation datafrom a donor corneal-scleral rim mounted on an artificial anteriorchamber at these pressures was used to validate the model. Themodel was run iteratively at each pressure to determine the Young’s©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamodulus required to produce experimentally determineddeformations.Results: Maximum deformation amplitude for the model wasmatched to experimental deformation data within 0.01% error. TheYoung’s moduli were 1.569, 1.740, 1.899, 2.099, and 2.250 MPa forpressures of 10, 20, 30, 40, and 50 mmHg, respectively.Conclusions: This model supports the known relationship that asIOP increases, the cornea will become stiffer. Future studies willinclude developing a 3D model as well as modeling the whole globe.Image shows the model deformation at 10 mmHg intraocularpressure.to determine corneal material parameters, while reducing currentprevalent restrictions.Methods: Sound excitation (100-110 db) together with phasesensitive OCT was used to measure the profile of the naturalfrequencies (range 200-1000 Hz) in corneal tissue. The technique wastested in-vitro on 6 freshly enucleated pig and bovine eyes, usingcorneal flaps, corneal buttons and whole eyes. Different conditionswere tested (virgin, riboflavin-dextran instillation, cross-linkingCXL) to determine the effect of corneal rigidity and hydration.Changes in corneal stiffness were determined by the shift of naturalfrequencies and viscoelastic behavior by the phase lag between soundwave and corneal oscillation. Finite element (FE) models were builtto simulate the experimental observations.Results: We found an experimental shift in the first natural frequencyof 101.25±67.1 Hz between the anterior flaps of virgin and CXLcorneas; no significant shift was observed for posterior flaps (-12.5±32.0 Hz). Corneal buttons and globes confirmed a frequencyshift after cross-linking. The phase lag was sensitive to the testedfrequency and to corneal fixation (δ(flap)=4.06, δ (button)=6.19 andδ(globe)= 5.93 at 355Hz). FE-models predicted the first naturalfrequency to be strongly correlated with corneal stiffness(ΔE=+1.4MPa, Δf(globe)=+100Hz, Δf(button)=+250Hz,Δf(flap)=+150Hz). Natural frequencies of higher modes were alsosensitive to IOP, corneal curvature, thickness and density.Conclusions: OCT vibrography is a promising non-invasivetechnique for the estimation of corneal biomechanical properties,allowing the separation of corneal stiffness from other parameters.Image shows the model deformation at 50 mmHg intraocularpressure.Commercial Relationships: Kimberly M. Metzler, None; CynthiaJ. Roberts, Oculus Optikgerate GmbH (C), Ziemer OphthalmicSystems AG (C), Sooft Italia (R), Carl Zeiss Meditec (F); Steven M.Whitaker, None; Michael J. Lawrence, None; Jennifer E. Malik,None; Jeffrey P. Bons, NoneSupport: Ohio Lions Eye Research FoundationProgram Number: 1630 Poster Board Number: D0265Presentation Time: 8:30 AM - 10:15 AMOCT-Vibrography: A Novel Non-Contact Method to EstimateCorneal Biomechanical PropertiesSabine Kling 1 , Ernest Chang 2 , Giuliano Scarcelli 2 , Nandor Bekesi 1 ,Seok H. Yun 2 , Susana Marcos 1 . 1 Instituto de Optica, ConsejoSuperior de Invest Cientificas, Madrid, Spain; 2 Wellman Center,Massachusetts General Hospital, Boston, MA.Purpose: Corneal biomechanics are key for diagnosing cornealpathologies and evaluating treatments that alter corneal geometry orstiffness. Most methods to measure corneal biomechanics aredestructive, while in-vivo techniques (e.g. air-puff imaging) arebiased by corneal geometry and IOP. We developed a new techniqueShift of the first resonance frequency with increased corneal rigidity.Commercial Relationships: Sabine Kling, None; Ernest Chang,None; Giuliano Scarcelli, massachusetts general hospital (P);Nandor Bekesi, None; Seok H. Yun, Massachusetts GeneralHospital (P); Susana Marcos, Essilor (F), PCT/ES2012/070185 (P)Support: FIS2011-25637; ERC-2011 AdG-294099Program Number: 1631 Poster Board Number: D0266Presentation Time: 8:30 AM - 10:15 AMRegional Variation of Biomechanical Properties of Intact EyeGlobesAhmed Elsheikh 1, 2 , Charles Whitford 1 , Akram Joda 1 , Ahmed Abass 1 ,Fangjun Bao 4 , Paolo Rama 3 . 1 Engineering, University of Liverpool,Liverpool, United Kingdom; 2 National Institute for Health ResearchBiomedical Research Centre at Moorfields Eye Hospital NHSFoundation Trust and UCL Institute of Ophthalmology, London,United Kingdom; 3 Ophthalmology Department, San RaffaelleScientific Institute, Milan, Italy; 4 Eye Hospital, Wenzhou MedicalCollege, Wenzhou, China.Purpose: To determine the regional variation in biomechanicalproperties across the whole surface of the cornea and sclera of humanand porcine eyesMethods: Four human eyes and eight porcine eyes were tested usinga new inflation test method to determine the regional variation of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatheir stress-strain behavior. New techniques have been developed toallow the testing of intact eye globes, prevent degradation andswelling, remove internal components, and simulate the eye's supportby the surrounding soft tissue. The specimens were covered with afine-particle speckle pattern and the behavior monitored usingspatially oriented cameras. Finite element models that closelyrepresent eye topography were constructed (Fig 1) and thedeformation across eye surface used to determine regional variationof corneal and scleral stiffness within an inverse modeling procedure.Results: The results revealed consistent stiffness variation trends inboth human and porcine eyes. The stiffness (measured using tangentmodulus) was highest in the limbal region and reduced graduallyacross scleral surface until the posterior pole region and the areasurrounding the optic nerve, where the lowest stiffness was recorded,Fig 2 (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEngineering, The Ohio State University, Columbus, OH;3 Department of Biomedical Engineering, Cleveland Clinic LernerResearch Institute, Cleveland, OH.Purpose: LASIK creates a flap in the anterior corneal stroma,followed by ablation of the exposed stroma with an excimer laser. Anewer procedure, small incision lenticule extraction (SMILE), doesnot create a flap and uses a femtosecond laser to remove a lenticulehaving a shape corresponding to the programmed refractivecorrection. It is hypothesized that since a flap is avoided in SMILE,the mechanical stresses in a post-SMILE cornea more closelyapproximate those of an idealized cornea with unaltered materialproperties than in a comparable LASIK correction.Methods: Finite element models of myopic surgery using patientspecificcorneal geometry and an anisotropic, collagen fiberdependentformulation were constructed for LASIK and SMILE.Surgical parameters, the magnitude of myopic correction, flapthickness in LASIK and depth of lenticule creation in SMILE werevaried. Further, since the corneal stroma is generally stiffer in theanterior region, two sets of models with uniform and depth-dependentmaterial properties were constructed. A geometry-matched modelwith unaltered material properties from the preoperative state butwith post-operative geometry (including thickness) was used forcomparing the magnitude and distribution of von Mises stresses inSMILE and LASIK models.Results: We observed the stress distribution in the post-SMILEsimulations to be similar to that of the geometry-matched model sincemuch of the stiffer anterior stroma is preserved. In contrast, LASIKconsistently reduced the stress in the flap and increased the stress inthe residual stromal bed compared to the postoperative geometrymatchedmodel. Further, increase in the thickness of the flap or depthof lenticule creation resulted in a greater amount of increase in thestress in residual stromal bed of the LASIK model compared to theSMILE model.Conclusions: SMILE may present less biomechanical risk to theresidual bed of susceptible corneas than comparable correctionsinvolving LASIK flaps. Corrections at greater stromal depths inSMILE may be possible with less relative risk of ectasia than acomparable LASIK correction.Commercial Relationships: Abhijit Sinha Roy, Carl Zeiss Meditec(F), Cleveland Clinic Innovations (P), Topcon Inc. (F); William J.Dupps, Zeimer (C), Topcon (F), Avedro (F), Carl Zeiss Meditec (F),Cleveland Clinic Innovations (P); Cynthia J. Roberts, OculusOptikgerate GmbH (C), Ziemer Ophthalmic Systems AG (C), SooftItalia (R), Carl Zeiss Meditec (F)Program Number: 1634 Poster Board Number: D0269Presentation Time: 8:30 AM - 10:15 AMBiomechanical properties of the cornea and graft afterDescemet's stripping endothelial keratoplastySaima Qureshi 1 , Ninita H. Brown 1 , Naazli Shaikh 2, 1 .1 Ophthalmology, Howard University Hospital, Washington, DC;2 Ophthalmology, Veterans Affairs Medical Center, Orlando, FL.Purpose: To measure the biomechanical properties of the cornea andgraft after Descemet’s stripping endothelial keratoplasty (DSEK)using the ocular response analyzer (ORA) and to evaluate thecorrelation between corneal compensated intraocular pressure(IOPcc) and Goldmann applanated intraocular pressure (IOP GAT).Methods: A cross-sectional study of 7 eyes of 7 patients with Fuch’sendothelial dystrophy who underwent DSEK with at least 6 monthsof follow up. Corneal hysteresis (CH), cornea resistance factor(CRF), cornea compensated intraocular pressure (IOPcc) weremeasured using the ORA. At the same visit, IOP GAT was obtained.A comparison of post-DSEK ORA parameters to those of nondiseasedcorneas and the correlation of IOPcc to IOP GAT wereinvestigated.Results: Mean patient age was 71.85±11.48 years. The mean followup period was 25.71±12.72 months. Mean CH and CRF were9.90±1.40 mmHg and 9.61±1.16mmHg, respectively. The differenceof CH and CRF of post-DSEK corneas were not found to besignificant when compared to CH and CRF values of non-diseasedcorneas 1 (p value= 0.5745, unpaired t test). Cornea-compensatedintraocular pressure was not found to be significantly different whencompared with IOP GAT (p value= 0.3731, paired t test).Conclusions: The biomechanical properties of the cornea and graftafter DSEK are found to be within the range of values of nondiseasedcorneas. The difference between IOPcc and IOP GAT afterDSEK was not found to be significant.1 Values for non-diseased corneas: CH=10.25±1.6, n=100;CRF=10.25±1.85, n=100. Source: Montard R, Kopito R, Touzeau O,Allouch C, Letaief I, Borderie V, Laroche L. Ocular responseanalyzer: feasibility study and correlation with normal eyes. J FrOphtalmol. 2007 Dec.30(10):978-84.Commercial Relationships: Saima Qureshi, None; Ninita H.Brown, None; Naazli Shaikh, NoneProgram Number: 1635 Poster Board Number: D0270Presentation Time: 8:30 AM - 10:15 AMInfluence of Pregnancy on the Corneal Biomechanical PropertiesRoo Min Jun, Ga Eun Cho, Kyu-Ryong Choi. Dept ofOphthalmology, School of Medicine, Ewha Womans University,Seoul, Republic of Korea.Purpose: To determine the difference of corneal biomechanicalProperties and central corneal thickness (CCT) between pregnant andnon-pregnant women.Methods: This is a prospective case control study. Twenty six nonpregnantwomen and twenty six pregnant women with no ocularpathology or systemic diseases were recruited. Corneal hysteresis(CH) and the corneal resistance factor (CRF) were measured with theOcular Response Analyzer and the CCT was measured with anultrasonic pachymeter.Results: Twenty six eyes of 26 non-pregnant women (30.3 ± 3.8,range 23-38) and 26 eyes of 26 pregnant women (31.5 ± 2.9, range24-38) were included. CCT was not different between two groups(Non-pregnant vs. pregnant: 553.8 ± 26.3vs. 547.5 ± 29.4, P > 0.05).The CH was slightly lower in pregnant group but it was notstatistically significant (11.1 ± 1.7 vs. 10.7 ± 1.6, P > 0.05). On theother hand, the CRF was statistically lower in the pregnant group(10.2 ± 1.7 vs. 9.2 ± 1.4, P = 0.03).Conclusions: The CCT and CH were not statistically differentbetween pregnant and non-pregnant women. The CRF wassignificantly lower in the pregnant group. Change of hormone mayinfluence the corneal biomechanical properties in pregnant women.Commercial Relationships: Roo Min Jun, None; Ga Eun Cho,None; Kyu-Ryong Choi, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 1636 Poster Board Number: D0271Presentation Time: 8:30 AM - 10:15 AMElastic modulus of keratocytes and myofibroblasts differregardless of substratum stiffnessVijay K. Raghuanthan 1 , Peter C. Strom 1 , Sara M. Thomasy 1 , PaulRussell 1 , Christopher J. Murphy 1, 2 . 1 Dept of Surgical andRadiological Sciences, University of California Davis, Davis, CA;2 Dept of Ophthalmology and Vision Science, University of CaliforniaDavis, Davis, CA.Purpose: Injury to the corneal stroma initiates a cascade of eventsresulting in the differentiation of quiescent keratocytes (K) toactivated fibroblasts (F) and myofibroblasts (M). This transformationis critical for appropriate corneal wound healing and its dysregulationcan result in scar and haze formation. Transforming growth factor-β1(TGFβ1) is critical in differentiating keratocytes and fibroblasts tomyofibroblasts. We investigated the influence of substratum stiffnesson TGFβ1 induced K-F-M transition and measured the elasticmodulus of the different cell types.Methods: Primary rabbit corneal keratocytes were isolated andcultured in serum free media. Cells were then cultured onpolyacrylamide substrates of differing stiffness (5 or 25 kPa ) or ontissue culture plastic (TCP; >1GPa) in serum free media in theabsence or presence (10 ng/ml) of TGFβ1. Expression of α-smoothmuscle actin (αSMA) was determined by qPCR andimmunocytochemistry (ICC) to confirm myofibroblasttransformation. Keratocytes and myofibroblasts were visuallyidentified by differences in cell morphology. Keratocytes had acharacteristically stellate morphology while the myofibroblasts wereflatter and more spread out. Elastic moduli of keratocytes andmyofibroblasts on the various substrates were measured by atomicforce microscopy (AFM).Results: Overall, interaction with softer substrates inhibited genesisof the myofibroblast phenotype. In the presence of TGFβ1, 0.05). Chinese subjects had significantly longer AL(24.05±1.11mm) compared with Indian subjects (23.59±0.94mm) andCaucasian subjects (23.47±0.66mm). Indian subjects hadsignificantly higher CH (11.81±1.33mmHg) than Chinese subjects(11.25±1.37mmHg) and Caucasian subjects (11.34±1.76mmHg).Indian subjects also had significantly higher CRF(11.86±1.87mmHg) than Chinese (11.15±1.48mmHg) and Caucasiansubjects (10.71±1.87mmHg). Age had negative association with CHand CRF (p< 0.0001). CH and CRF were significantly higher infemale than in male (p< 0.0001). There was significantly negativeassociation between AL and CH (p= 0.001), but no significantassociation was found between AL and CRF (p> 0.05). CH and CRFwere significantly increased with CCT (p< 0.0001). After adjustingfor age, gender, CCT, AL and corneal curvature, there were stillsignificant ethnical difference in CH (p< 0.05) and CRF (p< 0.01).Conclusions: Different corneal biomechanical properties were foundin Chinese, Indian and Caucasian. A longitudinal study is needed todetermine the role of CH in the axial elongation.Commercial Relationships: Yin Zhi Wong, None; Andrew K.Lam, NoneProgram Number: 1638 Poster Board Number: D0273Presentation Time: 8:30 AM - 10:15 AMNovel Corneal Biomechanical Parameters in Myopes vsEmmetropesRachel Lee 1 , Robert Chang 1 , Ian Y. Wong 2 , Jimmy S. Lai 2 , Jacky W.Lee 2 , Kuldev Singh 1 . 1 Stanford University School of Medicine,Stanford, CA; 2 Hong Kong University School of Medicine, HongKong, Hong Kong.Purpose: While population-based studies have shown that myopia isa risk factor for glaucoma, the underlying basis of this correlation isunknown. We aim to identify novel corneal biomechanicalparameters that differentiate myopic from normal eyes using a noveltechnology.Methods: This prospective, cross-sectional study of 80 subjects withvarying degrees of myopia and 62 emmetropies was conducted atQueen Mary Hospital in Hong Kong. The Corvis ST device (Oculus,Wetzlar, Germany), which couples a pneumotonometer with a highspeed Scheimpflug camera, was used to measure IOP, CCT, and newbiomechanical parameters including corneal deformation amplitude,inward and outward applanation velocity, and highest concavitypeak-to-peak distance. The right eye of all subjects and controlsunderwent Corvis analysis. Myopes were subsequently categorized asbeing high (vision correction at least -9.0 D), moderate (-6.0 D to -9.0D), or mild (-3.0 D to -6.0 D) myopes. Exclusion criteria includedknown corneal disease, intraocular surgery within three monthspreceding the study, or prior history of refractive surgery.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: Significant findings included differences in outwardapplanation velocity (myope: -0.38±0.08 m/s; control: -0.33±0.05m/s; P=3.2E-5) and peak-to-peak distance (myope: 2.43±0.24 mm;control: 2.32±0.20 mm; P=3.6E-3). Interestingly, high (n=19), butnot moderate (n=25) or low (n=36), myopes exhibited statisticallysignificant differences in these two corneal biomechanical parametersas compared to controls.There was moderate correlation between IOP, CCT and visual acuitywith outward applanation velocity (R=0.43; 0.34; 0.50, respectively),and with peak-to-peak distance (R=0.40; 0.15; 0.32, respectively). Nocorrelation was found between age and outward applanation velocity(R=0.060) or peak-to-peak distance (R=0.039). Average IOP inmyopes and emmetropes were 15.2±2.2 mm Hg and 14.9±2.1 mmHg, respectively (P=4.0 E-1); average CCT in myopes andemmetropes was 554±34 μm and 554±39 μm, respectively (P=9.5 E-1).Conclusions: Myopes had a greater mean outward applanationvelocity and greater peak-to-peak distance at highest concavity thanemmetropic controls. In particular, high myopes demonstrated acorneal biomechanical profile distinct from that of emmetropes.Increased corneal deformability in high myopes, also found inglaucoma patients from another study, may indicate a relationshipbetween high myopia and glaucoma.Commercial Relationships: Rachel Lee, None; Robert Chang,None; Ian Y. Wong, bayer (C); Jimmy S. Lai, Pfizer (R), Allergan(R), Alcon (R); Jacky W. Lee, Allergan (F), Alcon (F), AMO (F);Kuldev Singh, Alcon (C), Allergan (C), Santen (C), Bausch andLomb (C), Transcend (C), Ivantis (C), Sucampo (C), iScience (C)Support: Stanford School of Medicine- Traveling Scholars GrantProgram Number: 1639 Poster Board Number: D0274Presentation Time: 8:30 AM - 10:15 AMThe relationship between anterior segment biometry and cornealbiomechanics in myopiaHetal Buckhurst 1 , Bernard Gilmartin 2 , Robert Cubbidge 2 , ManbirNagra 2 , Nicola S. Logan 2 . 1 School of Health Professions, PlymouthUniversity, Plymouth, United Kingdom; 2 School of Life and HealthSciences, Aston University, Birmingham, United Kingdom.Purpose: The Reichert Ocular Response analyser (ORA) providesdata on corneal biomechanics via measures of corneal hysteresis(CH), corneal resistance factor (CRF) and additional waveformparameters (AWPs). Utilising the Scheimplug principle the Pentacam(Oculus) provides data on the biometry of the cornea and anteriorsegment (AS). Given the premise that there is correspondencebetween the biomechanical and biometric properties of the AS weexamine the nature of the correspondence and whether it is altered inmyopia.Methods: Data were collected from adults 18-40 yrs of British-Whiteand British-South-Asian descent [MSE (D) 20 non-myopes (≥-0.50)0.64±1.38; 22 myopes (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneavalues parallelto failure of corneal hydration. It was found that duration andmetabolic control ofdiabetes did not change the biomechanical properties of cornea.Commercial Relationships: Faruk Ozturk, None; Serkan Akkaya,None; Ertugrul Can, NoneProgram Number: 1641 Poster Board Number: D0276Presentation Time: 8:30 AM - 10:15 AMCollagen Macrostructure and Corneal Shape: Lessons fromDifferent SpeciesMoritz Winkler 1 , Yilu Xie 2 , Tiffany Yuen 1 , Golroxan Shoa 1 , RobertHueter 3 , Kathy K. Svoboda 4 , Christopher J. Murphy 5 , Donald J.Brown 2 , James V. Jester 2, 1 . 1 Biomedical Engineering, University ofCalifornia, Irvine, Irvine, CA; 2 Gavin Herbert Eye Institute,University of California, Irvine, Irvine, CA; 3 Mote MarineLaboratory, Sarasota, FL; 4 Biomedical Sciences, Texas A&M HealthScience Center, Dallas, TX; 5 Department of Surgical andRadiological Sciences, University of California, Davis, Davis, CA.Purpose: The cornea plays a critical role both as a protective windowto the eye and as a refractive lens. In aquatic vertebrates, it provideslittle refractive power, while in terrestrial vertebrates corneal shapeneeds to be precisely controlled to project a focused image on theretina. Little is known about the changes in the structuralorganization of corneal collagen during evolution. The purpose ofthis study was to begin to characterize the macrostructuralorganization of corneal collagen in divergent species in order touncover basic mechanisms controlling corneal shape.Methods: Eyes from various species (fish, shark, birds, mammals)were fixed under pressure using paraformaldehyde to control postmortemswelling. Serial full-width (limbus to limbus) cross-sections(250μm thick) were cut using a vibratome. Sections were imagedusing nonlinear optical high resolution macroscopy (NLO-HRMac)of second harmonic generated (SHG) signals. 3-D images wererendered using Amira software, and collagen fiber structures werequantified with custom-written ImageJ macros.Results: In aquatic vertebrates stromal collagen macrostructureconsisted of simplified layers (stacks) of fibers that extended fromlimbus to limbus as continuous sheets of collagen, much like‘plywood’ in construction. Adjacent sheets were rotated 87°, formingorthogonal plies, with successive layers showing a continual rotationof over 360°. In birds collagen sheets were organized into distinctfibers with a uniform branching and fusing pattern similar to that ofchicken wire and presented a honeycomb appearance. Fibers in thesame plane appeared to extend from limbus to limbus, and successivelayers showed a 270° rotation through 2/3 stromal depth, very similaryet distinct from fish and shark. By contrast, collagen fiberorganization in mammals was irregular with varying degrees ofbranching depending on the species (human > dog > cat > rabbit).Mammals also lacked orthogonal rotational, nor were fibersconstrained to extend from limbus to limbus within the same plane.Conclusions: We have previously shown in the human cornea thatcollagen fiber branching and interconnectivity is associated withtissue rigidity. In this study, fiber branching was detected in corneasfrom terrestrial vertebrates suggesting that branching and increasedcorneal rigidity may play a role in the evolutionary adaptation of thecornea from a protective window to a refractive lens.Commercial Relationships: Moritz Winkler, None; Yilu Xie,None; Tiffany Yuen, None; Golroxan Shoa, None; Robert Hueter,None; Kathy K. Svoboda, None; Christopher J. Murphy, OcularServices On Demand (I), Ocular Services On Demand (C), PlatypusTechnologies LLC (I), Imbed LLC (I), EyeKor LLC (I), Allergan(C), Genentech (C), Sarcode (C), Covance (C); Donald J. Brown,None; James V. Jester, NoneSupport: NIH Grant EY018665, Research to Prevent Blindness, Inc.,Discovery Eye Foundation, and the Skirball Program in MolecularOphthalmologyProgram Number: 1642 Poster Board Number: D0277Presentation Time: 8:30 AM - 10:15 AMInter- and Intra-Lamellar Slippage of Collagen Fibrils as aPotential Mechanism of Keratoconus ProgressionMichael Koster 1 , Craig Boote 2 , Keith M. Meek 2 , Priscilla G. Fowler 3 ,Christopher A. Girkin 3 , Guenther Meschke 1 , Rafael Grytz 3 . 1 Instituefor Structural Mechanics, Ruhr University Bochum, Bochum,Germany; 2 School of Optometry and Vision Sciences, CardiffUniversity, Cardiff, United Kingdom; 3 Ophthalmology, University ofAlabama at Birmingham, Birmingham, AL.Purpose: To assess if inter- and intra-lamellar slippage of collagenfibrils may lead to progressive cone formation in keratoconus.Methods: A generic finite element model of the human eye wasgenerated that incorporates the micro-architecture of collagen fibrilsin the corneo-scleral shell. Inter- and intra-lamellar slippage wassimulated through residual strains of collagen fibrils using amicrostructure-based constitutive formulation. Progressive inter- andintra-lamellar slippage was imposed to an eccentric, 4-mm-diameterarea of the cornea while the model was subjected to normal IOP (15mmHg). Topographic results were compared to clinical observationof a keratoconus patient with an eccentric cone.Results: Increasing inter- and intra-lamellar slippage led toprogressive cone formation of the cornea. The results were in goodagreement with topographic observation of keratoconus patients witheccentric cone.Conclusions: The numerical results support the assumption thatinter- and intra-lamellar slippage of collagen fibrils may be theunderlying mechanism that leads to progressive cone formation inkeratoconus.Numerical simulation of keratoconus progression showing thedevelopment of an eccentric cone due to inter- and intra-lamellarslippage of corneal collagen fibrils.Commercial Relationships: Michael Koster, None; Craig Boote,None; Keith M. Meek, None; Priscilla G. Fowler, None;Christopher A. Girkin, SOLX (F), Heidelberg Engineering (F);Guenther Meschke, None; Rafael Grytz, NoneSupport: EyeSight Foundation of Alabama; Research to PreventBlindness Physician-Scientist AwardProgram Number: 1643 Poster Board Number: D0278Presentation Time: 8:30 AM - 10:15 AMClear Corneal Incision: Sealability of the Manual Versus LensARlaser generated Full Thickness Incision©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaE. Valas Teuma 1 , Liz Dumanoir 1 , Aissatou Barry 1 , Gary Gray 1 , G.Brock Magruder 2 , Steve Bott 1 . 1 R&D, LensAR Inc, Orlando, FL;2 LaserVue, Orlando, FL.Purpose: The human cornea has the ability to self-seal afterpenetrating incision wounds. The purpose of this work is to comparethe sealability of clear corneal incisions (CCIs) created by manualmeans to those generated by the LensAR Laser System - fs 3D (LLSfs3D).Methods: A total of 22 human donor globes were used for thisexperiment. The laser CCIs, were performed using the LLS-fs 3D.Standard three-plane CCIs were used for both the manual and laserCCIs. Post incisions, a high resolution Fourier domain opticalcoherence tomography (OCT) system was used to measure theincision geometry. After the OCT measurements, the sealability ofthe CCIs was measured using a digital pressure gauge. The Seidel testwas utilized to detect a leak of the aqueous onto the cornea. Acomputer-controlled stepper motor is used to push the plunger intothe eye. The IOP reported by the pressure gauge is recorded at thefirst sign of leakage. If the IOP reaches 500 mmHg without leakage,the test is considered complete.Results: One tailed t-tests of the sealability of the manual versuslaser CCIs, indicate that the mean IOP at which leakage occurred wasthe statistically the same for the two cases (p = 0.061). However,variability of IOP at leakage for the laser was significantly less thanfor the manual (±28 mmHg vs. ±94 mmHg). Regarding the measuredincision geometries, the geometric parameters measured for the laserincisions were always at least as good in accuracy and precision asthe manual and in several cases was superior. For example, a twotailed t-test of the mid-tunnel depth location versus target (50%)indicates that the laser incision was statistically significantly closer tothe target value than the manual incisions (mean 46% vs. 33%,p=0.26).Conclusions: The IOP at leakage of laser versus manual CCIs werestatistically the same. However, an F-test of the variance of the IOPelevation at which leakage occurred showed that the manual methodproduced incisions with statistically higher variance than those of thelaser CCIs. OCT measurements showed that the mean value of thelocation of the mid-plane of the three-plane CCI was placedsignificantly closer to the target placement for the laser versus themanual CCIs. Overall, the testing showed that the manual and lasermethods are statistically equivalent in sealability but that the lasermethod produces more consistent wound geometry.Commercial Relationships: E. Valas Teuma, Lensar Inc (E); LizDumanoir, None; Aissatou Barry, None; Gary Gray, LensAR (E);G. Brock Magruder, LensAR (C); Steve Bott, NoneProgram Number: 1644 Poster Board Number: D0279Presentation Time: 8:30 AM - 10:15 AMEffect of Intraocular Pressure on Speed-of-Sound and Thicknessin Ex Vivo Cornea in Intact GlobesHarriet Lloyd 1 , Mara Berganovsky 1 , Ronald H. Silverman 1, 2 , RakshaUrs 1 . 1 Ophthalmology, Columbia University Medical Center, NewYork, NY; 2 Frederic L. Lizzi Center for Biomedical Engineering,Riverside Research, New York, NY.Purpose: Ultrasound is regarded as the ‘gold standard’ for thedetermination of corneal thickness, but standard methods formeasuring this parameter have required determination on excisedcorneas, which is non-physiologic. We developed a means formeasurement of speed-of-sound in intact globes. Our objective was todetermine the effect of intraocular pressure (IOP) on corneal speed ofsound and thickness.Methods: We acquired high-resolution ultrasound data on four exvivo pig corneas. The eyeball was mounted in a custom apparatus,which included a sharpened, thin, flat metal surface that was insertedacross the anterior chamber. An 18 gauge needle attached to a salinedrip bag was inserted through the optic nerve. IOP was raised andlowered by raising or lowering the saline bag and monitored with adigital pressure gauge attached to the IV line. The eye and apparatuswas submerged in 20% dextran solution. Data of the cornea and ofthe metal surface on either side of the globe were obtained using asingle-element focused transducer with a center frequency of 35MHz. Pulse/echo ultrasound data was acquired at a 400 MHz samplerate. We measured the shift in the metal flat echo compared to itsexpected, interpolated position, and from this and the speed-of-soundin aqueous and the dextran solution solved for the speed-of-soundand thickness of the cornea.Results: The speed-of-sound averaged over all cases showedrelatively little dependence on intraocular pressure. At 0 mm thespeed-of-sound averaged Hg 1556 m/s, at 40 mm Hg it was 1560 m/sand upon return to 0 mmHg it was 1548 m/s. These differences werenot statistically significant. The thickness of the cornea at 0mmHgmeasured 1.01 mm, at 40 mmHg it was 0.91 mm, and it recovered to0.96mm as the pressure was gradually reduced to 0mmHg.Conclusions: IOP had little effect on the speed-of-sound in thecornea, indicating that ultrasound pachymeters would not be requiredto be recalibrated to compensate for IOP. However, as pressureincreased, the cornea stretched and became thinner, recoveringgradually and partially as IOP was decreased.Commercial Relationships: Harriet Lloyd, None; MaraBerganovsky, None; Ronald H. Silverman, None; Raksha Urs,NoneSupport: NIH Grant EY021529; AIUM EER; Research to PreventBlindnessProgram Number: 1645 Poster Board Number: D0280Presentation Time: 8:30 AM - 10:15 AMChange of corneal curvature under the open eye condition andthe slightly closed eye conditionYuko Shibata 1 , Hiroshi Uozato 1, 2 , Masakazu Hirota 1 , TakushiKawamorita 1, 2 . 1 Ophthalmology & Visual Sciences, Kitasato UnivGraduate School, Sagamihara, Japan; 2 Orthoptics and VisualSciences, Kitasato University, Sagamihara, Japan.Purpose: To assess the influence of squinted eyes, we measuredcorneal curvature under the condition of widely opened eye and thecondition of slightly closed eye.Methods: 39 eyes of 20 healthy subjects (age 21 - 42 yrs)participated in this study. Corneal curvatures of central zone (1 - 4mm as a diameter) were measured and front photo images of theeyelids and eye were obtained at the same time by an anteriorsegment imaging analyzer (Galilei, Ziemer Ophthalmic Systems,Port, Switzerland) under both a condition with the eye opened widelyand a condition with the eye closed slightly in which subjects openedtheir eyes narrowly with consciousness.Results: The group mean size of slightly closed eye was 67.0 ± 12.2% of that of widely opened eye condition (Range: 45.9 - 89.5%). Themean SimK value average of widely opened eye was 43.59 ± 1.39 Dand that of slightly closed eye was 43.44 ± 1.57 D, the mean flatSimK value of widely opened eye was 42.82 ± 1.32 D and that ofslightly closed eyes was 42.35 ± 1.84 D and the mean steep SimKvalue of widely opened eye was 44.36 ± 1.53 D and that of slightlyclosed eye was 44.52 ± 1.69 D. Significant differences were found inflat SimK value and steep SimK value between two conditions(Wilcoxon signed-ranks test, p < 0.05) but there was no significantdifference in SimK value average. The mean anterior instantaneouscurvature Mean K of widely opened eye was 43.03 ±1.30 D and thatof slightly closed eye was 42.98 ± 1.59 D, the mean anterior©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneainstantaneous curvature Flat K of opened eye was 42.23 ± 1.28 D andthat of slightly closed eye was 41.83 ± 1.63 D and the mean anteriorinstantaneous curvature Steep K of opened eye was 43.82 ± 1.42 Dand that of slightly closed eye was 44.13 ± 1.91 D. There was nosignificant difference in anterior instantaneous curvature values oftwo conditions (Wilcoxon signed-ranks test, p > 0.05).Conclusions: Data obtained in this study shows that in the slightlyclosed eye condition, cornea curvature of central zone had a littlechange to become more astigmatic. It was suggested that even in theslightly closed eye condition, corneal curvature was affected by theeyelids and tear film’s distribution.Commercial Relationships: Yuko Shibata, None; Hiroshi Uozato,None; Masakazu Hirota, None; Takushi Kawamorita, None238 Corneal EndotheliumMonday, May 06, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 1646-1698/D0281-D0333Organizing Section: CorneaContributing Section(s): Visual NeuroscienceProgram Number: 1646 Poster Board Number: D0281Presentation Time: 8:30 AM - 10:15 AMCyclosporin A inhibits cell death of Corneal Endothelial Cells byprotecting michondrial membrane potentialToshinari Funaki, Kaori Ohtomo, Masahiro Yamaguchi, AkiraMatsuda, Akira Murakami. Ophthalmology, Juntendo Univ School ofMedicine, Bunkyo-ku, Japan.Purpose: The number of corneal endothelial cells (CECs) decreaseswith age because corneal endothelium does not divide in vivo. Aftercorneal transplantation, the endothelial cell density on donor corneasrapidly declines by acute intraoperative trauma. Therefore it isimportant to protect CECs during surgery for the survival of theallograft.The corneal endothelium are mitochondria-rich cells, indicate thatCECs are metabolically active. It is also reported that cell death isassociated with reduced functioning of mitochondria.In glia cell and cardiomyocyte, Cyclosporin A (CsA) suppress celldeath by inhibition of the permeability transition pore (PTP) whichlead to stabilization of mitochondrial membrane potential as well asimmunosuppressive effect.The purpose of this study was to investigate whether CsA inhibit celldeath in human cultured corneal endothelial cells (hCECs) and mousecorneas.Methods: HCECs were stimulated with H2O2(0~1000nM) for 1hour. And whole mount cornea, endothelial cell side up fromC57BL/6 were stored DMEM serum free at 4C and then subjected tovarious concentrations of H2O2 (0-1000M) for 1 hour with/withoutvarious concentration CsA. Apoptosis were detected using Caspase3/7 (Invitrogen). Mitochondria in corneal endothelial cells werelabeled by the addition of MitoTracker Red CMXRos to the cornealcups (125 nM final concentration; Invitrogen) and maintaining for 30minutes in a 37C in a 5% CO2 incubater.Results: 1) Oxidative stress induced cell death was observed in aconcentration-dependent manner in hCECs.2) CsA decreased cell death by about 10%, and suppressed thereduction of mitochondrial membrane potential at 100nM in hCECs.Only the highest concentration of CsA (1000nM) showedcytotoxicity in hCECs.3) CsA suppressed the reduction of mitochondrial membranepotential at 100nM in whole mount mouse corneas.Conclusions: We conclude that CsA suppress the reduction ofmitochondrial membrane potential from oxidative stress, and inhibitcell death in corneal endothelial cells.Commercial Relationships: Toshinari Funaki, None; KaoriOhtomo, None; Masahiro Yamaguchi, None; Akira Matsuda,None; Akira Murakami, SEED(Japan) JP4855782 (P),SEED(Japan) JP5132958 (P)Support: Grant-in-Aid for Young Scientists B 23792005Program Number: 1647 Poster Board Number: D0282Presentation Time: 8:30 AM - 10:15 AMSuccess Isolation of Human Corneal Endothelial Cells forClinical UseJin San Choi 1, 3 , Matthew Giegengack 1, 2 , Eun Young Kim 4 , MinJeong Kim 4 , Ralph D'Agostino 5 , Gilson Khang 4 , Shay Soker 1 . 1 WakeForest Institute for Regenerative Medicine, Wake Forest UniversityHealth Sciences, Winston-Salem, NC; 2 Department ofOphthalmology, Wake Forest School of Medicine, Winston-Salem,NC; 3 Oular Systems, INC, Winston-Salem, NC; 4 BIN FusionTechnology, Chonbuk National University, Jeonju, Republic ofKorea; 5 Public Health Sciences-Department of Biostatistics, WakeForest School of Medicine, Winston-Salem, NC.Purpose: Corneal transplantation is a common transplant procedureto improve visual acuity by replacing the opaque or distorted hosttissue by clear healthy donor tissue. However, its clinical utility islimited due to a lack of donor supply with high quality corneas.Bioengineered neo-corneas, created using an expandable populationof human donor-derived corneal endothelial cells (HCECs), couldaddress this shortage. Thus, the objective of this study was toevaluate HCEC sourcing with various isolation methods, includingenzymatic digestion, culture medium components, and adhesiveproteins.Methods: HCECs were obtained from corneas with various ageddonors after endothelial keratoplasty. Under a dissection microscope,the Descemet’s membrane, including the attached cornealendothelium was stripped from the stroma and the cells were isolatedand expanded by explant culture and the use of enzymatic digestionwith enzyme such as collagenase II, dispase, or trypsin. In order toimprove the initial cell attachment, tissue culture plates were coatedwith collagen IV, fibronectin, or fibronectin-collagen combinationcoating mix (FNC) before cell plating.Results: In results, HCECs were successfully isolated from 32%(86/269) of donor corneas. Donor age and isolation methodinfluenced the characteristics of the resulting in vitro HCEC culture.Under all conditions tested, FNC-coated plates showed higher qualitycultures than other coatings tested.Conclusions: The results suggest that donor and isolation methodcharacteristics are the two factors that most directly affect the qualityof the resulting HCEC in vitro culture. These factors should guide theclinical expansion of HCECs for the generation of bioengineeredneo-corneas.Commercial Relationships: Jin San Choi, Ocular Systems, INC(E); Matthew Giegengack, None; Eun Young Kim, None; MinJeong Kim, None; Ralph D'Agostino, None; Gilson Khang, None;Shay Soker, NoneSupport: Ocular Systems,INCProgram Number: 1648 Poster Board Number: D0283Presentation Time: 8:30 AM - 10:15 AMA cell therapy approach to address corneal endothelialdysfunctionKaren Alvarez-Delfin 1 , Noelia J. Kunzevitzky 1, 2 , Alejandra D.Weisman 1 , Richard M. Merkhofer 1 , Jeffrey L. Goldberg 1, 3 . 1 BascomPalmer Eye Institute, Interdisciplinary Stem Cell Institute, University©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaof Miami, Miami, FL; 2 Emmetrope Ophthalmics, Coral Gables, FL;3 Shiley Eye Center, University of California San Diego, San Diego,CA.Purpose: The endothelium is the innermost layer of the cornea and isresponsible for maintaining transparency. Human corneal endothelialcell (HCEC) density gradually decreases with age, and their properfunction is affected by genetic diseases, such as Fuchs’ dystrophy, orsurgical trauma, ultimately leading to vision impairment. Suchendothelial dysfunction is an indication for corneal transplantation,although there is a global shortage of tissue donors. To overcome thecurrent poor donor availability, here we isolate, expand, andcharacterize HCECs in vitro as a first step toward cell therapy.Methods: HCECs were isolated from cadaveric donor corneas andcultured following the method described by Zhu and Joyce (2004). Inorder to improve cell morphology and proliferation rate, differentmedia additives and culture dish coatings were assayed. Cellularidentity was assessed by morphology, RT-PCR, andimmunohistochemistry. Viability was measured by Trypan bluestaining after cells were stored at room temperature, 4oC orcryopreserved in liquid N2.Results: Cultured HCECs often demonstrated characteristichexagonal-like morphology for 2-3 passages, reaching passage 8 inone culture. Time to reach confluence was highly influenced bydonor age, with youngest donors exhibiting higher proliferative rates.Donor disease also affected culture quality. Overnight storage ofHCECs at 4oC dramatically reduced viability, while cryopreservationmaintained higher viability. Immunohistochemistry showed thatcultured HCECs were positive for zonula occludens-1(ZO-1) andNa+K+ ATPase, common markers for HCEC. Fibroblastic-likemorphologic conversion positive for the marker α-smooth muscleactin (α-SMA) was a common feature at late passages, appearingearlier in poor quality cultures.Conclusions: The in vitro expansion of HCEC from donor corneasyields a number of suitable cells that could help treat patientsotherwise in need of cornea transplantation. Ongoing experiments areaddressing the ability of these cells to integrate into a host cornea andrestore endothelial function.Commercial Relationships: Karen Alvarez-Delfin, None; NoeliaJ. Kunzevitzky, None; Alejandra D. Weisman, None; Richard M.Merkhofer, None; Jeffrey L. Goldberg, NoneProgram Number: 1649 Poster Board Number: D0284Presentation Time: 8:30 AM - 10:15 AMComparison of early corneal peripherial endothelial cell lossfollowing femtosecond laser - assisted cataract surgery andconventional phacoemulsificationGábor L. Sándor, Ágnes I. Takács, Kinga Kranitz, Eva Juhasz, IllesKovacs, Zoltan Z. Nagy. Department of Ophthalmology, SemmelweisUniversity, Budapest, Hungary.Purpose: To compare early corneal peripherial endothelial cell lossafter femtosecond laser - assisted cataract surgery and conventionalphacoemulsification, using non-contact specular microscopy.Methods: In each group, 15 eyes (15 patients) underwent cataractsurgery using either femtosecond - laser assisted (Alcon LenSx lasersystem, femtolaser group) or conventional phacoemulsification(phaco group). All operations were performed by the same surgeonwith the same phaco machine (Infiniti, Alcon). Biometry wasperformed using Lenstar LS 900 (Haag-Streit AG) non-contactoptical low-coherence reflectometer. Scheimpflug camera (PentacamHR, Oculus Optikgerate GmbH) was used to measure nucleus density(Pentacam Nucleus Staging; PNS). Endothelial cell density wasmeasured by Specular Microscope NSP-9900 (Konan Nonco Robo)preoperatively and 1 month after surgery by a semiautomated maskedmanner. The measurements were performed at four identical pointson the peripherial area of the cornea. The mean value was used as theaverage cell density of periphery.For comparison of independent variables Mann Whitney U-test, andfor comparison of dependent variables Wilcoxon-test was used.Results: There was no statistically significant difference indemographics, PNS, axial lenght, phaco energy and effective phacotime between the two study groups. We found no significantdifference in preoperative cell density in the two groups (femtolasergroup: 2898±168/mm2, phaco group: 2799±219/mm2, p=0,1776). 1month after the surgery the cell density was slightly lower in thephaco group (2696±233/mm2) than in the femtolaser group(2833±140/mm2), but the difference was not statistically significant(p=0,0887). The peripherial endothelial cell loss was significant inboth the phaco group (4%, p=0,004) and the femtolaser group (2%,p=0,005).Conclusions: Results of this study suggest that femtosecond laserassistedcataract surgery does not significantly differ in early cornealperipherial endothelial cell loss from manual phacoemulsification.There is a smaller tendency of decrease in peripherial endothelial cellcount after femtosecond laser-assisted cataract surgery comparedwith conventional phacoemulsification, however the difference is notstatistically significant.Commercial Relationships: Gábor L. Sándor, None; Ágnes I.Takács, None; Kinga Kranitz, None; Eva Juhasz, None; IllesKovacs, None; Zoltan Z. Nagy, Alcon-LenSx Inc. (C)Program Number: 1650 Poster Board Number: D0285Presentation Time: 8:30 AM - 10:15 AMEffect of glaucoma tube-shunt position on corneal thickness andendothelial cell densityEuna Koo 1 , Jing Hou 1 , Ying Han 1 , Jeremy D. Keenan 1, 2 , Robert L.Stamper 1 , Bennie H. Jeng 1, 2 . 1 Ophthalmology, University ofCalifornia San Francisco, San Francisco, CA; 2 Proctor Foundation,University of California San Francisco, San Francisco, CA.Purpose: To investigate post-operative changes in corneal thicknessand endothelial cell density (ECD) after tube-shunt implantationMethods: Twenty-eight eyes of 24 glaucoma patients withsuperotemporal tube-shunts, but without prior corneal transplantationor previous tube-shunt placement, were evaluated at University ofCalifornia, San Francisco. Superotemporal (ST), central, andinferonasal (IN) ECD and corneal thickness were measured by noncontactspecular microscopy (CellChek XL Specular Microscope,Irvine, CA) and ultrasound pachymetry (DGH Pachette 2, Exton,PA), respectively. The angle of the tube relative to the cornea, tubelength, and distance between the tip of the tube and cornea, weremeasured with anterior segment optical coherence tomography(Visante OCT Anterior Segment Imaging, Dublin, CA). Linearregression analyses were used to assess effects of tube position onECD and corneal thickness.Results: The average time lag from tube implantation to time ofcornea and anterior segment measurements was 45±40.4 months(range 1-156 months). Mean ST, central, and IN ECD (cells/mm2)were 1649.7±800.0, 1940.1±862.0, and 1963.6±856.7, respectively.Mean ST, central, and IN corneal thicknesses (μ) were 640.6±103.6,536.3±86.8, and 650.8±83.6, respectively. ST ECD was lower thancentral ECD (P=0.0001) and IN ECD (P=0.003, paired t-test). Therewas no difference in ECD between central and IN cornea (P=0.97).ST and IN cornea were thicker than central cornea (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatube length (P=0.30) or tube-corneal distance (P=0.80).Conclusions: The presence of a tube shunt drainage device in theanterior chamber causes significant local endothelial cell losscompared to central cornea or cornea furthest away from the tube.Endothelial cells near the tube may be better preserved with tubesangled further away from the cornea, but a larger study is needed toanswer this question.Commercial Relationships: Euna Koo, None; Jing Hou, None;Ying Han, None; Jeremy D. Keenan, None; Robert L. Stamper,Transcend (C), Genentech (C); Bennie H. Jeng, NoneProgram Number: 1651 Poster Board Number: D0286Presentation Time: 8:30 AM - 10:15 AMHuman Cytomegalovirus-mediated inflammatory responses ofcorneal endothelial cellsMichiko Kandori 1 , Dai Miyazaki 1 , Keiko Yakura 1 , Yumiko Noguchi 1 ,Yukimi Yamamoto 1 , Yoshitsugu Inoue 1 , Tatsuo Suzutani 2 .1 Ophthalmology, Tottori University, Yonago, Japan; 2 Microbiology,Fukushima Medical University, Fukushima, Japan.Purpose: To determine whether human cytomegalovirus (CMV) caninfect corneal endothelial cells, and characterize inflammatoryresponses of corneal endothelial cells after infection.Methods: Clinical isolate of CMV was purified by density gradientand adsorbed to immortalized human corneal endothelial cells(HCEn) at a multiplicity of infection 0, 0.01 or 0.1. The infectivity ofCMV into HCEn cells was analyzed by the induction of immediateearly,early, and late genes of CMV, and inflammatory cytokineinduction by real-time RT-PCR. Inflammatory responses of HCEncells were characterized by protein array analysis of the supernatants.Results: Representative viral genes, including IE-1, UL-83, andglycoprotein B, were sequentially induced after CMV infection.CMV infection characteristically stimulated induction of IL-6 andIDO1 (indoleamine 2,3-dioxygenase 1) mRNA by HCEn cells.Protein array analysis showed significant induction of inflammatorymediators, immune regulators, or neurotrophic factors , includingBDNF, BLC, GDNF, PIGF, MCP-1, EGF, TGF-β1, PDGF-BB, IL-16, Leptin, MCSF, MCP-4, IP-10, IL-6, IL-13, IL-5, IL-15, GCP-2,FGF-9, IFN-γ, RANTES, Ckβ8-1, Eotaxin, MIP-3α, VEGF.Conclusions: CMV infects corneal endothelial cells, and provokesanti-viral responses, together with induction of immune regulatorymediators or neurotrophic factors. These results may give us clues toelucidate the pathogenesis of CMV endotheliitis.Commercial Relationships: Michiko Kandori, None; DaiMiyazaki, None; Keiko Yakura, None; Yumiko Noguchi, None;Yukimi Yamamoto, None; Yoshitsugu Inoue, None; TatsuoSuzutani, NoneProgram Number: 1652 Poster Board Number: D0287Presentation Time: 8:30 AM - 10:15 AMEngineering of Human Corneal Endothelial GraftsYingting Zhu, Bo Han, Szu-Yu Chen, Scheffer C. Tseng. Research,Ocular Surface Ctr and Tissue Tech, Miami, FL.Purpose: We have reported that knockdown by p120 siRNAselectively activates p120-catenin/Kaiso signaling and successfullyexpands contact-inhibited HCEC monolayers to an average size of2.1 ± 0.3 mm in diameter from 1/8 of the descemet membranestripped from the corneoscleral rim. Herein, we would like to showhow incorporation of other means might further expand themonolayer size.Methods: HCEC monolayers derived from 1/8 stripped descemetmembrane and cultured to 7 days in SHEM were treated withdifferent concentrations of p120 siRNA weekly with or without 100nM Kaiso siRNA or 5 µg/ml nocodazole, a microtubule disruptingagent, for up to 5 weeks. Before termination, cells were furthertreated with 10 µM BrdU for 4 h. Immunostaining was performed tomonitor cytolocalization of F-actin, α-catenin, β-catenin, p120catenin, N-cadherin, ZO-1, Na+/K+-ATPase, and BrdU labeling.Results: Consistent with our recent report, p120 siRNA knockdownuniquely promoted proliferation of contact-inhibited HCEC bynuclear translocation of p120 catenin to relieve repression by nuclearKaiso. Such proliferation was further enhanced to achieve an averagemonolayer size of 4.1 ± 0.3 mm in diameter (n=3, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: The optical density of the cells is measured via an MTTassay and compared to controls (cells in PBS alone) to obtain the cellviability after treatment with UVA-RF combination. Dose responsecurves are plotted against exposure time for low and high irradiancesand the time required to get 50% cell viability (=EC50) is estimatedusing a non-linear regression model and compared for high and lowirradiance treatments. Results show cell viability decreasing as afunction of increasing energy dose applied.Conclusions: This custom chamber allows for the comparison ofcytotoxicity levels of UVA-RF combination on human cornealendothelial cells while controlling the gaseous composition andtemperature of the chamber close to in vivo conditions.Commercial Relationships: Radha Pertaub, Avedro Inc (E); MarcD. Friedman, Avedro Inc (E); David Muller, Avedro Inc (E)Program Number: 1654 Poster Board Number: D0289Presentation Time: 8:30 AM - 10:15 AMA Nonsynthetic, Biological Carrier for Cultivated HumanCorneal Endothelial Cells (HCECs) for potential therapeuticpurposesJesintha Navaratnam, Eli Gulliksen, Kristine Ustgaard-Andersen,Jon K. Slettedal, Liv Drolsum, Bjorn Nicolaissen, AboulghassemShahdadfar. Center for Eye Research, Oslo University Hospital,Oslo, Norway.Purpose: The aim of our ongoing study is to establish a carrier forHCECs for therapeutic purposes. In the present study we investigatethe feasibility of using nonsynthetic, biological carrier for cultivatedHCECs.Methods: Descemet’s membrane with the attached endothelial cellswas carefully dissected from human corneas in small strips. One partharvested as non-cultured cells and the other cultivated in cornealendothelial cell growth medium for 6 weeks at 37 degree Celsiuswith 5% CO2 in a humidified atmosphere and the medium waschanged every 2-3 days. The cultivated HCECs were seeded onacellular, nonsynthetic carrier and cultivated for further 3 weeks incorneal endothelial cell expansion medium that was changed every 2-3 days. Cultivated HCECs on nonsynthetic carrier and non-culturedHCEC were comparatively analyzed by qRT-PCR, electronmicroscopy (EM) and immunohistochemistry.Results: In our study the cultivated HCECs seeded on nonsyntheticcarrier formed a stable monolayer. Our results show that thecultivated HCECs seeded on nonsynthetic carrier and the nonculturedHCECs are functional and express stem cell markers whenanalyzed by qRT-PCR. The expression levels of markers associatedwith neural crest, stem cells and corneal endothelial function (SNAI1,SNAI2, SOX9, NES, ZO-1, CX43, VADC2 and VADC3) are higherin cultivated HCECs seeded on nonsynthetic carrier compared tonon-cultured HCECs. The structure of cultivated HCECs seeded onnonsynthetic carrier on transmission EM is very similar compared toex vivo HCECs.Conclusions: The preliminary results show that nonsynthetic carrierused in this study can potentially be used in therapeutic purposes inthe future.Commercial Relationships: Jesintha Navaratnam, None; EliGulliksen, None; Kristine Ustgaard-Andersen, None; Jon K.Slettedal, None; Liv Drolsum, None; Bjorn Nicolaissen, None;Aboulghassem Shahdadfar, NoneProgram Number: 1655 Poster Board Number: D0290Presentation Time: 8:30 AM - 10:15 AMDifferences in corneal endothelial abnormalities in the centraland intermediate zones in Fuchs’ corneal dystrophyHisataka Fujimoto, Takeshi Soma, Yoshinori Oie, Shizuka Koh,Motokazu Tsujikawa, Naoyuki Maeda, Kohji Nishida. OsakaUniversity, Suita, Osaka, Japan.Purpose: Fuchs’ corneal dystrophy is a progressive cornealendothelial dystrophy that causes irreversible endothelial change andbullous keratopathy. To investigate the regional differences in theabnormality, we examined the corneal endothelium at multiple sites,including the periphery.Methods: 17 eyes of 9 patients (6 women and 3 men; 30-80 yearsold) with Fuchs’ corneal dystrophy were studied at Osaka UniversityHospital. We used a newly developed non-contact specularmicroscope (NIDEK CEM-530) to examine the intermediate zone 27degrees in the periphery from the corneal center in addition to thecenter and the peripheral 5 degrees from the center. We measured thesize of the degenerative area with guttata and the cellular density inthe residual intact area.Results: The abnormal areas in the central zone (center and 5 degreesfrom the center) represented 65.4 ± 33.7% of the total area, and thosein the 27 degrees of the peripheral zone represented 27.1 ± 34.2% ofthe total area. The results differed significantly (P < 0.001). Theabnormal areas in the 27 degrees of the peripheral zone of 16 of 17eyes were significantly (P< 0.05) larger than those in the center. Inthe 6 points 27 degrees of the peripheral zones, the abnormal areasinferiorly were significantly (P < 0.001) larger than superiorly, by theusage of z score analysisConclusions: In Fuchs’ corneal dystrophy, the corneal endothelialcells degenerate more rapidly in the central zone than theintermediate zone. This finding might offer new information tofacilitate an understanding of the disease mechanisms.Commercial Relationships: Hisataka Fujimoto, None; TakeshiSoma, HOYA corporation (R), Santen Pharmaceutical Co., Ltd (F),Otsuka Pharmaceutical Co., Ltd (F); Yoshinori Oie, Santen (F),HOYA (F); Shizuka Koh, Santen, Inc. (R), Johnson & Johnson (R),Topcon (R), Otsuka Pharmaceutical Co. (R); Motokazu Tsujikawa,Shionogi & Co. (C), Daiichi Sankyo Co. (F), Daiichi Sankyo Co. (R),Santen Co. (R), AMO Co. (R); Naoyuki Maeda, Topcon (F), Santen(R), Otsuka (R), Oculus (R), HOYA (R); Kohji Nishida, Alcon (C),Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F), OsakaUniversity (P)Program Number: 1656 Poster Board Number: D0291Presentation Time: 8:30 AM - 10:15 AMDevelopment and Characterization of Decellularized HumanCorneal Stroma as a Scaffold for Tissue EngineeringRadhika Tandon 1 , Sujata Mohanty 2 , Himi Singh 1 , Deepika Gupta 3 ,Seema Sen 4 , Seema Kashyap 4 , Amit K. Dinda 5 , Manjeet Jassal 3 ,Ashwini K. Agrawal 3 . 1 Department of Ophthalmology, Dr. RajendraPrasad Centre for Ophthalmic Sciences, All India Institute of MedicalSciences, New Delhi, India; 2 Stem Cell Facility, All India Institute ofMedical Sciences, New Delhi, India; 3 SMITA Research Labs,Department of Textile, Indian Institute of Technology, New Delhi,India; 4 Department of Ocular Pathology,Dr. Rajendra Prasad Centre©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafor Ophthalmic Sciences, All India Institute of Medical Sciences,New Delhi, India; 5 Department of Pathology, All India Institute ofMedical Sciences, New Delhi, India.Purpose: To evaluate the potential use of decellularized humancorneas as a scaffold for cultivating human corneal endothelial cells.Methods: Human corneal tissues (N=20) not suitable fortransplantation were used. Corneal endothelial cells were isolated byexplant culture method. For preparation of the scaffold, cornealepithelium and endothelium were removed mechanically andremaining corneal stroma was decellularized using enzymaticmethod.The resulting acellular matrices were then subjected toHaematoxylin-Eosin (H&E) staining to visualize cellular remnants;quantitative analysis to determine the DNA content; ScanningElectron Microscopy (SEM) for collagen fibril morphology; Alcianblue staining to analyse extracellular matrix (ECM);Immunohistochemistry for structural proteins collagen type I, II, IV,fibronectin; and cytotoxicity assay of decellularized cornea using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Tensilestrength of corneal tissues &Light transmission ratios was estimatedusing uniaxial load testing equipment &UV-visiblespectrophotometer. Native cornea served as control for all theexperiments. 10,000 human corneal endothelial cells were culturedon decellularized scaffold for 14 days and then analyzed using SEM,histology and Immunocytochemistry for ZO-1, and Na+ /K+-ATPase.Results: H&E staining demonstrated efficient elimination of cellularcomponents. Alcian blue confirmed good preservation of theextracellular matrix and major structural proteins collagen type I, IIIV & fibronectin were retained. The amount of DNA indecellularized cornea was 32+ 7.27ng/mg whereas in native cornea itwas 133+8.3ng/mg(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: EY08834Program Number: 1659 Poster Board Number: D0294Presentation Time: 8:30 AM - 10:15 AMDisappearance and reappearance of cilia of corneal endotheliumpreserved in corneal preservation mediaHidetoshi Tanioka, Katsuhiko Shinomiya, Satoshi Kawasaki, ShigeruKinoshita. Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto,Japan.Purpose: It is known that the primary cilia of cells are present incorneal endothelium. However, the primary cilia of donor cornealendothelium disappear when preserved in corneal preservation media.This study investigated the histological change of the cilia of cornealendothelium preserved in corneal preservation media.Methods: This study involved corneas obtained from Japanese whiterabbits. The corneas were preserved in Optisol-GS (Bausch andLomb, Rochester, NY) corneal preservation media at 4°C for 0, 1 and7 days. The cornea preserved for 7 days was incubated at 37°C inculture media (CnT-20; CELLnTEC Advanced Cell Systems AG,Bern, Switzerland) for 2 days. Corneal endothelia of these corneaswere assessed by immunohistochemistry with Anti-Acetylated alphaTubulin antibody and scanning electron microscopy (SEM).Results: Immediately after isolation, long process was observed bySEM on the corneal endothelium, and it showed positive for primarycilia by Anti-Acetylated alpha Tubulin antibody. No primary ciliawere observed on the corneal endothelium preserved for 1 or 7 days.After 7-days preservation, primary cilia were observed on theendothelium that underwent the 2-day incubation at 37°C.Conclusions: The findings of this study using rabbit corneas suggestthat the primary cilia of the corneal endothelium of human donorcorneas for transplantation also disappear during preserved inpreservation media and reappear after transplantation, and show thatthe existence of primary cilia is correlated with the viability ofcorneal endothelium.Commercial Relationships: Hidetoshi Tanioka, None; KatsuhikoShinomiya, None; Satoshi Kawasaki, None; Shigeru Kinoshita,Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Support: Grant-in-Aid for Scientific Research (KAKENHI)Program Number: 1660 Poster Board Number: D0295Presentation Time: 8:30 AM - 10:15 AMEndothelial loss and pachymetric change in patients undergoingpenetrating keratoplasty with 3 years of followElisa D. Alegria, Regina Velasco, Oscar Baca, Alejandro Babayan.Cornea, Hosp Nuestra Senora De La Luz IAP, Mexico, Mexico.Purpose: Identify endothelial loss and pachymetric changes inpatients operated penetrating keratoplasty at follow up 3 yearsMethods: Pre surgical endothelial count was obtained by the reportsof the LionsEye Bank Institute in Tampa, Florida.A non contact specular microscopy with a microscope Topcon SP-2000P in the operated was made in the operated eye. Specularmicroscopy was taken only with the image captured by auto focus.EEndothelial cel density was calculated by identify at minimum of 30cels . Specular microscope automatically calculates and displays theresults. We also performed and compare it with ultrasonicpachymetry Accutome Accupach V in the last visit.Results: We analize 20 eyes.with 3 years of follow-up. Endothelialloss is greater than 50% of initial basal count representing a p> 0002.We observe the values of pre-surgical pachymetric compared againstthe post surgical have a grat differenceConclusions: Endothelial loss of patients undergoing penetratingkeratoplasty with follow at three years is more than 50%. Thethickness pachymetric also shows significant variationCommercial Relationships: Elisa D. Alegria, None; ReginaVelasco, None; Oscar Baca, None; Alejandro Babayan, NoneProgram Number: 1661 Poster Board Number: D0296Presentation Time: 8:30 AM - 10:15 AMThe Resting Potential of Rat`s Corneal Endothelial CellsNassim S. Calixto 1 , Vinicius V. Oliveira 2 , Renata Fleming 2 , SebastiaoCronemberger 1 , Adalmir Dantas 2 . 1 Ophthalmology, Federal Univ ofMinas Gerais, Belo Horizonte, Brazil; 2 Ophthalmology, FederalUniversity of Rio de Janeiro, Rio de Janeiro, Brazil.Purpose: The mechanism whereby the cornea maintains itstransparency is not known. This study aims to register the presenceand to quantify the resting potential (RP) of endothelial corneal cells.To the best of our knowledge this seems to be the first work tosystematically measure and quantify the RP of endothelial cells of thecornea.Methods: We performed 30 experiments on corneal preparations ofSprague-Dawley rats (n=30). Immediately after decapitation theeyeballs were removed and sectioned near the limbus. The corneaswere transferred to a chamber and infused with Balanced SaltSolution (BSS) driven by a peristaltic pump in order to maintain theBSS flowing at a rate from 0.8 to 0.85 ml/min. The temperature in thechamber was set at 30 °C by means of a thermostatic bath. BSS iscomposed of (mmol/l): Na + 160.0; Cl - 130.0; - HCO 3 25.0; K + 5.0; -H2PO 4 3.0; Mg ++ 1.0; Ca ++ 1.0; Glucose 5.0, presenting a pH = 7.4and 305 mOsm/Kg. The presence or absence of electrical signal wasdetected by recording its voltage variations through two poreelectrodes delicately introduced into the cells and connected to aGrass polygraph. The statistical analysis was made by Graph PadPrism 6.0.Results: In the 30 experiments (30 eyes), the RP presented a mean of40.60 with a standard deviation of 7.05 and a standard error of 1.29.The graphics below illustrate the findings.Conclusions: Although many studies are still needed to understandthe role of the endothelium in the transparency of the cornea, ourresults demonstrate:1. the presence of the resting potential in the corneal endotheliumcells2. a mean of 40.60±7.05 mV for the resting potential of the rat’scorneal endothelial cells.This graph shows the mean value at the center line, the standard©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadeviation (SD) at the boxes, and the maximum and minimum values.Commercial Relationships: Nassim S. Calixto, None; Vinicius V.Oliveira, None; Renata Fleming, None; Sebastiao Cronemberger,None; Adalmir Dantas, NoneProgram Number: 1662 Poster Board Number: D0297Presentation Time: 8:30 AM - 10:15 AMWnt5a enhances cell migration through regulation of Cdc42 andRhoA pathway in human corneal endothelial cellsJeongGoo Lee 1 , Daniel Sand 1, 2 , J M. Heur 1, 2 . 1 Ophthalmology,University of Southern California, Los Angeles, CA; 2 Doheny EyeInstitute, Los Angeles, CA.Purpose: Wnt5a activates β-catenin-independent pathways forregulation of cellular functions that play crucial roles in woundhealing process in cornea including cell migration and polarity.Elucidation of Wnt5a signaling may help identify potentialtherapeutic targets for enhanced corneal wound healing; however,Wnt5a signaling in corneal endothelial cells (CEC) has not been wellcharacterized.Methods: Expression and/or activation of Frizzled-5, Cdc42, Rac1were analyzed by immunoblotting. Scratch-induced directionalmigration assay was employed to measure migratory rates. Activationof Cdc42 and Rac1 were determined by GTP pull-down assay. RhoAactivity was measured using RhoA specific G-LISA assay kit.Results: Stimulation of human CECs with Wnt5a alone resulted in50% enhancement in cell migration as measured by scratch inducedmigration assay and this effect could be completely blocked byML141 (Cdc42 inhibitor). Co-treatment of both Wnt5a and Y27632(inhibitor of Rho-associated kinase) resulted in a 62% enhancementof migration. The synergistic effects of Wnt5a and Y27632 on humanCECs migration were partially blocked by ML141 and this, in turn,was completely abolished by treatment with both ML141 and RhoAactivator. Under these conditions, activation of both Cdc42 and Rac1and inactivation of RhoA were observed in the Wnt5a treated cells.We further confirmed that activated Cdc42 negatively regulatesRhoA activity and this regulation plays important role in endothelialmigration.Conclusions: These findings suggest that Wnt5A mediated migrationin human CEC is mediated by activation of Cdc42 and inactivation ofRhoA.Commercial Relationships: JeongGoo Lee, None; Daniel Sand,None; J M. Heur, NoneSupport: Baxter Foundation, Research to Prevent Blindness, andNIH Core Grant EY03040Program Number: 1663 Poster Board Number: D0298Presentation Time: 8:30 AM - 10:15 AMInfluence Of Hydrodynamic Culture Conditions On TheExpression Of Cell Junctions Of Tissue-Engineered HumanCorneal EndotheliumOlivier Roy 1 , Isabelle Brunette 3, 4 , Stephanie Proulx 1, 2 . 1 LOEX/CUO2 Ophtalmologie, Université Laval, Quebec, QC, Canada; 3 Centre derecherche HMR, Montréal, QC, Canada; 4 Ophtalmologie, Universitéde Montréal, Montréal, QC, Canada.Purpose: This study was undertaken in order to evaluate theinfluence of fluid flow and pressure on the expression of celljunctions of tissue-engineered corneal endothelium in vitro.Methods: Cultured human corneal endothelial cells were seeded andcultured on a previously devitalized corneal stroma, then either left instandard culture conditions (static cultures) or placed in an artificialanterior chamber with a perfusion rate of of 5 ul/min and ahydrostatic pressure of 18 mm Hg (hydrodynamic cultures). A higherperfusion rate than normal (2.6 ul/min) was chosen in order tomaintain a constant perfusion pressure and allow for the additionalleak of perfusate through the trabecular meshwork into the dish. After4-6 days, corneas were photographed then fixed in 3.7%formaldehyde for histology, 2.5% glutaraldehyde for transmissionelectron microscopy or 4% paraformaldehyde forimmunofluorescence staining of F-actin and the tight junction proteinZO-1. Controls consisted of devitalized corneas that were not seededwith corneal endothelial cells, and were processed in the samemanner.Results: Macroscopically, corneas maintained in hydrodynamiccultures were more transparent than the static-cultured corneas. Thecorneal stromas in the histology cross-sections were thinner in thehydrodynamic-cultured corneas than in the static-cultured corneas.The mean (±SD) collagen spacing, calculated using transmissionelectron microscopy images, was 27 ±4 nm when corneas were underhydrodynamic culture conditions whereas spacing was 46 ±9 nmwhen corneas were left in static culture conditions. Controldevitalized corneas with no endothelium had a mean collagen spacingof 32 ±6 nm when cultured under the same hydrodynamic conditions.Hydrodynamic-cultured corneal endothelium expressed higheramounts of ZO-1 protein, as assessed by immunostaining.Conclusions: This study shows that corneal endothelial cells respondto an anterior chamber flow and pressure by improving cell junctionexpression in vitro. Ultimately, studying the effect of hydrodynamicculture will enable an improved understanding of morphogenesis andcell junction formation of the corneal endothelium.Commercial Relationships: Olivier Roy, None; Isabelle Brunette,None; Stephanie Proulx, NoneSupport: NSERC, FRQS Vision Research NetworkProgram Number: 1664 Poster Board Number: D0299Presentation Time: 8:30 AM - 10:15 AMThe Quebec Corneal Cell Bank: Update on Culture Success ofPathologic Human Corneal Endothelial Cells (2009-2012)Mathieu Theriault 1 , Olivier Roy 1 , Olivier Rochette-Drouin 1 , Marie-Claude Perron 3 , Isabelle Brunette 3, 4 , Stephanie Proulx 1, 2 .1 LOEX/CUO - Recherche, Centre de recherche du CHU, Quebec,QC, Canada; 2 Ophtalmologie, Université Laval, Quebec, QC,Canada; 3 Centre de recherche HMR, Montréal, QC, Canada;4 Ophtalmologie, Université de Montréal, Montréal, QC, Canada.Purpose: The purpose of this study was to assess the feasibility ofinitiating primary cultures of corneal endothelial cells from patientssuffering from various corneal diseases. We also evaluated whichconditions yielded the best results for culture.Methods: Consenting patients undergoing penetrating keratoplasty orDescemet’s stripping automated endothelial keratoplasty wereenrolled in this study. The cornea (or Descemet’s membrane),removed at the time of surgery, was sent to the laboratory. Uponreceipt, specimens were photographed, endothelial cells were isolatedand cultured, and Descemet’s membranes were fixed in 3.7%formaldehyde for histology. Data collected included diagnosis, age- Recherche, Centre de recherche du CHU, Quebec, QC, Canada;©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand sex of the donor, time spent in Optisol, culture success andnumber of cells obtained after a primary culture. Control healthy EyeBank corneas were processed in a similar manner.Results: A total of 194 specimens were obtained between August2009 and November 2012. Pathologies included Fuchs cornealendothelial dystrophy (FECD; n=93), pseudophakic bullouskeratoplathy (PBK; n=33), FECD+PBK (n=4) and other cornealdisorders (n=64). Overall, 56 of the 194 diseased specimens (FECDn=37; PBK n=3; FECD+PBK n=2; other n=14) and all of the 29healthy corneas successfully initiated an endothelial cell culture.Among the diseased specimens, the mean(±SD) donor age was 66.5±11.9 years for successful cultures and 68.9 ±15.2 years for theunsuccessful cultures. Time spent in Optisol was 2.7 ±1.2 and 3.2±2.6 days for successful and unsuccessful cultures, respectively. Thediseased specimens yielded 120 000 ±86 000 cells and the healthycorneas 122 000 ±55 000 cells.Conclusions: This study shows that successful corneal endothelialcell culture can be generated despite various corneal diseases. FECDallowed the highest success rate. Once culture was initiated, a similarnumber of cells was obtained from FECD, BPK and healthyspecimens. Patient sex and age, and the time spent by the specimen inOptisol did not influence success rate. The Quebec Corneal CellBank will become a useful tool for the study of various cornealendotheliopathies.Commercial Relationships: Mathieu Theriault, None; OlivierRoy, None; Olivier Rochette-Drouin, None; Marie-ClaudePerron, None; Isabelle Brunette, None; Stephanie Proulx, NoneSupport: CIHR, FRQS Vision Health Research NetworkProgram Number: 1665 Poster Board Number: D0300Presentation Time: 8:30 AM - 10:15 AMPerception of Cornea and Glaucoma Subspecialists RegardingPrevalence of Corneal Decompensation with Ex-Press ShuntPlacementShalin Shah 1 , Ngo Yen 1 , Thompson W. Hilary 2 , Jayne S. Weiss 1 .1 Department of Ophthalmology, Louisiana State University EyeCenter, LSU School of Medicine, LSU Health Sciences Center, NewOrleans, LA; 2 Biostatistics Section, School of Public Health,Louisiana State University Health Sciences Center, New Orleans,LA.Purpose: Corneal decompensation is a recognized complicationassociated with anterior chamber insertion of Ahmed, Baerveldt andMolteno (ABM) shunts. By comparison, there are no publicationsaddressing corneal decompensation after Ex-Press shunt placement.The purpose of this study was to assess the prevalence and onset ofcorneal decompensation with ABM shunts and Ex-Press shunts asperceived by cornea and glaucoma specialists.Methods: A survey was distributed to members of the CorneaSociety and Glaucoma Society with questions about frequency andonset of complications after anterior chamber placement of ABM andEx-Press shunts. The individual was requested to rank the followingside effects in order of perceived prevalence: chronic hypotony,corneal decompensation, endophthalmitis, infection, malignantglaucoma, pthisis bulbi, and retinal detachment. Time of onset tocorneal decompensation in Ex-Press shunts was compared to that ofthe ABM group.Results: 17 glaucoma subspecialists and 22 cornea subspecialistsparticipated. Corneal decompensation was listed as the mostprevalent of the seven possible complications by both subgroups (chisquare < .001). 65.0% of cornea subspecialists and 84.6% ofglaucoma subspecialists reported the risk of corneal decompensationto be higher with uncomplicated ABM placement than with Ex-Pressshunts (chi square

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand good candidates as scaffolds for corneal endothelial celltransplantation.Rabbit corneas implanted with biomembranes. (A) Chitosanmembrane caused purulent exudate, vascular congestion, and cornealopacity. (B) Collagen vitrigel remained transparent.endothelial cell counts in 1214 people: 20 HIV-positive cases and1194 HIV-negative controls. The mean endothelial cell count in theHIV-positive cases was 2608/mm2 while HIV-negative controls hada mean endothelial cell count of 2621/mm2 (standard deviation 333and 432, respectively). There was no statistically significantdifference in endothelial cell count in donors with or without HIVinfection after controlling for age (p=0.89). Donors of African andAsian descent in the HIV-negative group had lower endothelial cellcounts compared to Caucasians (as the reference group).Conclusions: Comparison of donor cornea endothelial cell countsbetween HIV-negative and HIV-positive individuals has not beenpreviously described. We did not find a statistically significantdifference in endothelial cell counts due to HIV-infection status.Since the patients were not known to be HIV-positive according totheir medical records and interview with family members, it may bethat the infection was relatively early in these patients.Commercial Relationships: John A. Gonzales, None; David C.Gritz, None; Patrick Gore, None; Roy S. Chuck, NoneThe control (A) and Vitrigel (B) biomambrane implanted eyesretained transparency with no histological inflammation. Eyesimplanted with Chitosan biomembrane (C), developed inflammatoryexudative phase process around the biomembrane and cornealthickening.Commercial Relationships: Guillermo Mendoza, None; JudithZavala, None; Marcos Garza-Madrid, None; Alejandro Tamez,None; Angel Zavala-Pompa, None; Gabriela Brito, None; Jorge A.Cortés_Ramirez, IMPI (P); Jorge E. Valdez, None; JenniferElisseeff, Collagen Vitrigel (P)Program Number: 1667 Poster Board Number: D0302Presentation Time: 8:30 AM - 10:15 AMExamination of Endothelial Cell Count in HIV-Negative andPositive DonorsJohn A. Gonzales 1 , David C. Gritz 1 , Patrick Gore 2 , Roy S. Chuck 1 .1 Department of Ophthalmology & Visual Sciences, MontefioreMedical Center, New York, NY; 2 Lions Eye Institute, SaintPetersburg, FL.Purpose: To describe endothelial cell counts in corneal donors withhuman immunodeficiency virus (HIV) compared to those withoutHIV infection.Methods: This is a retrospective, cohort comparison study drawnfrom the records of a large regional eye bank. Corneas that wereprocured which were later found to belong to a donor who was HIVpositiveduring the period from 2008-2012 were compared to corneasthat were procured from donors who were HIV-negative fromJanuary 1, 2012-July 2, 2012. Donor corneas were classified ashaving HIV-1/2 based on serologic evidence obtained from either thepresence of HIV antibodies, a positive viral nucleic acidamplification test (NAT), or both.Demographic data (age, gender, race) was also collected from thesame database for both cases and control. Phakic status was collectedat the time of slitlamp examination, which also reported in thedatabase for cases and controls.Demographic data was compared between cases and controls usingChi-squared and t-tests for categorical and continuous variables,respectively. A generalized linear model was built to include all thedemographic/categorical variables (including HIV status) as well asthe continuous dependent variable (endothelial cell count).Results: The donor cornea database during the target period includedProgram Number: 1668 Poster Board Number: D0303Presentation Time: 8:30 AM - 10:15 AMMorphological Complexity of Mouse Corneal Endothelial CellsRevealed by Mosaic AnalysisDennis M. Defoe, Whitney J. Rich, Theresa A. Harrison. BiomedicalSciences, East Tennessee State University, Johnson City, TN.Purpose: In a previous investigation, we examined how individualcorneal endothelial cells with distinct p27 gene mutations differ intheir proliferative behavior (Defoe et al., ARVO 2011). As a result ofthese experiments, we noticed unusual structural features of the cellsthat required further analysis. In the present study, we have begun toexamine the detailed morphology of single cells in situ, after markingthem by high cytosolic expression of green or red fluorescent proteins(GFP or RFP).Methods: To visualize small numbers of widely distributed cellsfilled with fluorescent label, we used mosaic analysis with doublemarkers (MADM; Zong et al., 2005). For MADM, two mouse lines,each with reciprocally chimeric transgenes consisting of partialcoding sequences for GFP and RFP, separated by an intronembeddedLoxP site, were interbred with an Hprt-Cre-expressingstrain. Following the limited occurrence of Cre-mediatedinterchromosomal recombination during mitosis, functional GFP andRFP were reconstituted and each expressed in one of the twodaughter cells. Corneas from MADM mice were fixed, flat-mountedand visualized by fluorescence confocal microscopy.Results: Individual cells filled with GFP or RFP appear multipolar,with many tapered pseudopodial processes radiating from their cellbody. Such extensions are indicative of a complex and highlyelaborate plasma membrane. Examination of rare cases where red andgreen cells lie directly adjacent to one another reveals that processesof the two cells undergo extensive interdigitation. Because of thisoverlap, individual cells are observed to cover a greater area thanmight be expected if their boundaries were mutually exclusive.Conclusions: Our results give a picture of corneal endothelial cellmorphology very different from the polygonal outlines observed afterstaining for actin filaments or intercellular junction proteins. The datamay help explain the discontinuous tight junction pattern seen in bothlight and electron micrographs. More importantly, they suggest thatcell density alone may be an insufficient indicator of the overallcondition of the endothelium in health and disease.Commercial Relationships: Dennis M. Defoe, None; Whitney J.Rich, None; Theresa A. Harrison, NoneProgram Number: 1669 Poster Board Number: D0304©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 8:30 AM - 10:15 AMSystematic and individual differences in donor corneaEndothelial Cell Density (ECD) measurements with specularmicroscopy vs. sucrose light microscopyBart T. Van Dooren 1, 2 , Ilse J. Claerhout 3, 4 , Paul G. Mulder 5 ,Elisabeth Pels 6 . 1 Opththalmology, Amphia Hospital, Breda,Netherlands; 2 Ophthalmology, Erasmus Medical Center, Rotterdam,Netherlands; 3 Cornea Bank, Ghent University Hospital, Ghent,Belgium; 4 Ophthalmology, Ghent University Hospital, Ghent,Belgium; 5 Amphia Academy, Amphia Hospital, Breda, Netherlands;6 Cornea Bank, Euro Tissue Bank, Beverwijk, Netherlands.Purpose: To evaluate systematic differences in ECD measurementsbetween non-contact specular microscopy, and sucrose assisted lightmicroscopy, in organ cultured human donor corneas in the GhentUniversity Hospital Cornea Bank.Methods: Measurements obtained between 1997 and 2011 in 1016corneas from 551 donors were analyzed. Only donor corneas withboth specular microscopy EDCs and sucrose-assisted lightmicroscopy ECDs were included.In 193 corneas, Topcon SP1000 specular microscopy ECDs werecompared to sucrose ECDs. In 167 corneas. Topcon SP2000P(Topcon Corp, Tokyo, Japan) ECDs were compared to sucroseECDs. A manual counting technique using calibrated graticules onprinted photographs was used for SP1000 and Sucrose ECDs, and acenter counting method on the instrument itself was used forSP2000P ECDs. Bland-Altman plots and estimates were used foranalysis, based on a linear mixed model analysis allowing for pairedcorneas.Results: Results are shown in figures 1 and 2.SP1000 ECDs had a mean difference of 254.5 cells/mm2 (lower)with sucrose ECDs, the limits of agreement (horizontal black lines)were + 274.3 and - 783.2.SP2000P ECDs had a mean difference of 71.2 cells/mm2 (lower)with sucrose ECDs, with limits of agreement: + 805.2 and -947.6.Unequal SD’s of two paired measurements cause a correlationbetween the sum and the mean of those two measurements (Pitman’stest). Hence the negative slope in the SP1000-sucrose ECD differencevs. mean regression line , and positive slope in the SP2000-sucroseECD difference vs. mean regression line (sloped red lines)Conclusions: Substantial systematic differences and huge individualdifferences exist between ECDs obtained with different measurementmethods.Specular microscopy in donor eyes resulted in only a minority ofcases in usable ECDs, and because of this and larger endothelial cellcounting sample sizes, sucrose ECDs remain the gold standard inECD determination in organ cultured donor corneas.Erroneous magnification calibration in SP1000 was shown to resultin a larger systematic difference with sucrose ECD.The small systematic error between SP2000P ECDs and sucroseECDs indicate that comparison of donor ECDs to in-vivo ECDs maybe justifiable, on the condition that instruments are calibratedcorrectly. Huge individual measurement differences may occur.Commercial Relationships: Bart T. Van Dooren, None; Ilse J.Claerhout, None; Paul G. Mulder, None; Elisabeth Pels, NoneProgram Number: 1670 Poster Board Number: D0305Presentation Time: 8:30 AM - 10:15 AMOxidative Stress Causes Mitochondrial Dysfunction in HumanCorneal Endothelial CellsThore Schmedt 1, 2 , Cecily E. Hamill 1, 2 , Yuming Chen 1, 2 , Ula V.Jurkunas 1, 2 . 1 Schepens Eye Research Inst, Boston, MA;2 Massachusetts Eye and Ear, Boston, MA.Purpose: Human corneal endothelial cells (HCEnCs) form a singlemonolayer of hexagonal cells that are non-proliferative in vivo andenter rapid cellular senescence in vitro, rendering them of limited usein the study of endothelial cell biology. Introduction of telomerase(hTERT) has been shown to extend the life span of HCEnCs, but it isunclear whether hTERT expression protects mitochondria againstoxidative stress. The purpose of this study was to investigate theeffect of hTERT on mitochondrial integrity and apoptosis in responseto the pro-oxidant menadione in HCEnCs.Methods: Highly uniform subpopulations of HCEnCs that exhibitedincreased proliferative capacity were isolated from a 21-year-olddonor (HCEnC-21), and hTERT was introduced creating the HCEnC-21T cell line. Cells were grown to confluence and treated with 25 and100 µM of menadione sodium bisulfite for 1 hour. Non-treated cellsserved as controls. Cell viability was measured by Trypan blueexclusion using an automatic cell counter. Mitochondrial integritywas determined using the JC-1 dye and red-to-green fluorescenceratios to detect depolarized (damaged) mitochondria.Results: Mitochondrial polarization of both cell lines decreased withincreasing concentrations of menadione. At baseline, the fluorescenceratios of in HCEnC-21T and HCEnC-21 were 2.63±0.41 and3.01±0.31, respectively. At 25 µM, the ratios dropped to 1.32±0.27 inHCEnC-21T and 1.91±0.04 in HCEnC-21. This is an averagedecrease of 49.9% (p=0.04) in HCEnC-21T and 36.5% (p=0.014) inHCEnC-21. Cell viability tended to decrease, but was notsignificantly lower after treatment with 25 µM. At 100 µM, thefluorescence ratios were found to be 0.73±0.07 in HCEnC-21T and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea0.76±0.09 in HCEnC-21. This corresponds to an average decrease of72.3% (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 8:30 AM - 10:15 AMObjective Assessment of the Corneal Endothelium in FuchsEndothelial DystrophyJay W. McLaren, Lori A. Bachman, Sanjay V. Patel. Ophthalmology,Mayo Clinic, Rochester, MN.Purpose: Assessment of the corneal endothelium in early stages ofFuchs endothelial dystrophy is subject to sampling errors because ofthe non-uniform distribution of guttae. In this study we developed astandardized method of endothelial assessment and examined thepredictive relationship between the effective endothelial cell density(ECD) and severity of Fuchs dystrophy based on subjective grades.We also examined the relationship between effective ECD andanterior corneal structural changes (backscattered light).Methods: The corneal endothelium of 51 eyes from 30 patients, withvarying degrees of Fuchs endothelial dystrophy, was examined byusing confocal microscopy (ConfoScan 4, Nidek Technologies) witha 20x non-contact objective. In 2 to 3 images that sampled the centralendothelium, local contiguous cell density was determined by using avariable frame method (2 to 177 cells in 1 to 10 patches, dependingon the distribution of guttae). The effective ECD was the product ofthe local cell density and the ratio of the sample area not covered byguttae to total sample area, which was determined by image analysis.The severity of the disease in each eye was assessed during slit-lampexamination by two examiners based on a modified Krachmer grade(1-6). In 55 eyes with Fuchs dystrophy from a second group of 30patients, the clinical grade was predicted from the effective ECD andthe regression coefficients from the first group, and compared to thesubjective clinical grade assigned by one examiner. The relationshipbetween effective ECD and backscatter from the anterior stroma,based on brightness of confocal images (40x objective, ConfoScan 4)in the second group, was examined by Pearson regression.Significance was determined by using generalized estimatingequation models.Results: The effective ECD decreased linearly with subjective grade(r=-0.93, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafurther tested these two hypotheses and a third one that SLC4A11acts as a Na + : B(OH) 4 - (Borate) cotransporter.Methods: HEK293 cells were transfected with phCMV2-HA-SLC4A11 (T) or empty vector (C). Membrane surface expressionwas confirmed by immunofluorescence and membrane proteinisolation. Forty eight hours post-transfection the cells were loadedwith BCECF or SBFI dyes to measure pH i and Na + i in CO 2 /HCO 3 - -rich (BR) or HCO 3 - -free (BF) Ringer.Results: We previously observed that the BF to BR induced pH ichange (acidification and slow recovery) was not significantlydifferent between C and T cells. This transition was accompanied bya slow Na + influx (C: 0.882±0.187 vs T: 0.897±0.152 mM Na + /min,p=0.951). A Na + -free pulse in BR produced a similar rate ofacidification in T and C (C: 0.042±0.014 vs T: 0.048±0.006 pH i /min,p=0.741). Further, the rate of alkalinization upon Na + -addition in BRwas not significantly different (C: 0.131±0.029 vs T: 0.155±0.015pH i /min, p=0.477). These results indicate that SLC4A11 does nottransport Na + : HCO 3 - . We previously reported that T cells recoverfrom NH 4 Cl pulse in BF 2.6 times faster than C cells. This recoverywas sensitive to the amiloride analogue EIPA (1 µM). A Na + -freepulse in BF induces a faster acidification in T respect to C (C:0.048±0.004 vs T: 0.074±0.009 pH i /min, p=0.039). Likewise, the rateof alkalinization recovery upon Na + -addition in BF is higher in T (C:0.151±0.023 vs T: 0.267±0.044 pH i /min, p=0.036). This recovery iscompletely inhibited by EIPA 1 µM in C and T cells. These resultsare consistent with SLC4A11 acting as a Na + : OH - cotransporter or aNa + :H + exchanger (NHE), however they do not rule out SLC4A11being an activator of endogenous NHEs as the recovery in presenceof EIPA 1 µM is not significantly different between C and T. In Tcells the presence of Borate 10 mM did not affect the Na + -freeinduced acidification rate (T: 0.097±0.018 vs T+Borate: 0.079±0.015pH i /min, p=0.465); inconsistent with SLC4A11 being a Na + : Boratetransporter.Conclusions: (1) SLC411 does not provide Na + :HCO 3 - or Na + :Borate transport; (2) SLC4A11 transports Na + :OH - ; or (3) it is not anion transporter per se but an activator of NHEs.Commercial Relationships: Diego G. Ogando, None; Supriya S.Jalimarada, None; Eranga N. Vithana, None; Joseph A. Bonanno,NoneSupport: NIH grant EY008834Program Number: 1676 Poster Board Number: D0311Presentation Time: 8:30 AM - 10:15 AMEffects of the Reliability Index called Sample Error of the cornealspecular microscopy on the repeatability of the results of theexaminationsFernando C. Abib 1, 2 , Richard Y. Hida 3 , Ricadro Holzchuh 3 .1 Anatomy, Federal University of Parana, Curitiba, Brazil; 2 Cornea,Clinica de Olhos Dr. Fernando Abib, Curitiba, Brazil;3 Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil.Purpose: To know the effects of the reliability index, called SampleError (SE), over the repeatability of the specular microscopyexaminations with different number of the counted endothelial cells.Methods: Transversal study of 122 endothelial examinations of 61patients with cataract (63.97 ± 8.15 years old). The Reliability Indexcalled SE was calculated by a specific software (Cells Analyzer(CA), Technicall, Brazil) developed to specular microscopy andconfocal microscopy. The Reliability Index of 95% and cut-offSample Error of 5% were used). Each examination of Konan noncontactNONCON ROBO® SP-8000 Specular Microscope wassubmitted to standard cell counting, even at the same image,according with the studied group: G40: 40 cells counted, G100: 100cells counted; G150: 150 cells counted, and GCA: the counted cellnumber was higher than the determined cell number by the CellsAnalyzer software in as many different images as necessary.Reliability index interpretation - 1. The planned SE 5% > thecalculated SE: The endothelial examination will be statisticallyrepresentative of the corneal endothelial cell population; 2. Theplanned SE 5% < calculated SE: The endothelial examination willnot be statistically representative of the corneal endothelial cellpopulation. The SE will be calculated and presented to G40, G100,G150 and GCA. The behavior of the range of the 95% confidenceinterval to ECD, A, CV and HEX, for each eye, comparing G40-G100, G40-G150, and G40-GCA, will be presented.Results: ECD average was 2,395.37±294.34 cells/mm2, A average423.64±51.09 µm2, CV average 0.40±0.04 and HEX average54.77±4.19%.The G40 SE was 0.157±0.031, G100 SE was 0.093±0.024, G150 SEwas 0.075±0.010, and GCA SE was 0.037±0.005. The increase of themarked cells decreases the SE and decrease the range of confidenceinterval (right and left eyes respectively): G40-G100 - ECD: 37.00%and 39.79%, A: 38.62% and 43.66%, CV:27.27% and 46.15%, HEX:36.74% and 40.46%; G40-G150 - ECD: 50.89% and 49.87%, A:52.05% and 53.33%, CV: 45.45% and 53.84%, HEX: 51.87% and52.96%; G40-GCA - ECD: 75.79% and 77.39%, A: 75.95% and77.37%, CV: 72.72% and 76.92%, HEX 75.93% and 76.71%.Conclusions: The Reliability Index called Sample Error from theCells Analyser tutorial method improved close 75% the repeatabilityof all results of the specular microscopy: ECD, A, CV and HEX.Commercial Relationships: Fernando C. Abib, Fernando C Abib(P); Richard Y. Hida, None; Ricadro Holzchuh, NoneProgram Number: 1677 Poster Board Number: D0312Presentation Time: 8:30 AM - 10:15 AMComparing Quantitative and Qualitative Indices of the DonatedCorneas Maintained in Optisol GS with Those Kept in Eusol CMozhgan Rezaei Kanavi 1, 2 , Mohammad Ali Javadi 1, 3 , TaherehChamani 3 , Pejman Fahim 3 . 1 Ophthalmic Research Center, ShahidBeheshti University of Medical Sciences, Tehran, Islamic Republicof Iran; 2 Department of Ophthalmology and Visual Sciences,University of Wisconsin, Madison, WI; 3 Central Eye Bank of Iran,Tehran, Islamic Republic of Iran.Purpose: This study was conducted to compare the quantitative andqualitative indices of the donated corneas maintained in Optisol GSwith those kept in Eusol C storage media.Methods: In an ante-grade single blind study, two corneas from eachdonor with a death to preservation time of less than 30 hours and witha minimum of “an apparent good cornea rating” were maintained incorneal storage media; randomly one in Optisol GS and the other inEusol C. Slit-lamp biomicroscopic and specular microscopicexaminations were performed on days 1 and 7. The results of thequalitative and quantitative indices and the final cornea rating wererecorded. Statistical analyses were performed to evaluate anydifferences between the two media.Results: 180 corneas from 90 donors with an age range of 29.3±22.4years were entered into the study: 90 corneas in Optisol GS and theother 90 in Eusol C. No specular images were obtainable on day 7 in5 of the corneas maintained in Optisol GS and 4 of those corneas inEusol C, but based on slit-lamp biomicroscopic examinations theywere rated fair. The endothelial cell density of the corneas preservedin Optisol GS on both day 1 and day 7 were significantly higher thanthose maintained in Eusol C (P=0.007 and P=0.046 respectively).Significant endothelial cell vacuolation was observed by day 7 butmore frequently seen in the corneas preserved in Eusol C than thosein Optisol GS (P=0.014). As the maintenance time of the donatedcorneas increased there was no significant difference noted between©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneathe cornea rating, endothelial pleomorphism, polymegathism andmean cell area, stromal edema and Descemet’s folds of the twogroups.Conclusions: This study shows the superiority of Optisol GS overEusol C in terms of maintaining a higher endothelial cell density anda lower rate of endothelial vacuolation when the maintenance timeincreases. Such superiority may be due to the presence of someantioxidant compounds in the Optisol GS. However, it seems thatEusol C can still be a suitable choice for the eye banks that have ahigh turnover rate of corneas with a short preservation time beforetransplantation.Commercial Relationships: Mozhgan Rezaei Kanavi, None;Mohammad Ali Javadi, None; Tahereh Chamani, None; PejmanFahim, NoneProgram Number: 1678 Poster Board Number: D0313Presentation Time: 8:30 AM - 10:15 AMClinical Evaluation of a Novel Method to Generate Precut Tissuefor Descemet Membrane Endothelial Keratoplasty (DMEK)Bjoern O. Bachmann 1 , Ursula Schlotzer-Schrehardt 1 , MartinBoergel 2 , Friedrich E. Kruse 1 . 1 Ophthalmology, University Erlangen,Erlangen, Germany; 2 German society for tissue transplantation,Hannover, Germany.Purpose: Descemet Membrane Endothelial Keratoplasty (DMEK) isa novel procedure for transplantation of corneal endotheliumresulting in unsurpassed improvement of postoperative visual acuity.A central issue regarding this technique is the complexity of graftpreparation which hinders its wide spread use. Therefore, theevaluation of precut tissue for DMEK is essential for making thistechnique amenable to a larger group of surgeons.Methods: Precut preparation of organ cultured donor corneas waspreformed as previously described resulting in incompletely strippedDescemet’s membranes where the central part was still attached tothe corneal stroma. Subsequent culture for up to 5 days was followedby the use of the precut grafts for DMEK in patients with Fuchs’endothelial dystrophy. Visual acuity, central corneal thickness(Pentacam®, Oculus) and endothelial cell density (CellCheckXLTM, Konan) were retrospectively analyzed during the earlypostoperative period.Results: Stripping was successfully completed in all grafts directlyprior to surgery. The average visual acuity (logMAR) improved from0.49 +/- 0.13 preoperatively to 0.23 +/- 0.12 one month after DMEK.Central preoperative corneal thickness decreased from 667 +/- 31µmpreoperatively to 555 +/- 60 µm at 1 week and 484 +/- 8 µm 4 weeksafter DMEK. Average endothelial cell density was 2496 +/- 271 cells/mm2 before and 2275 +/- 247 cells/mm2 3 days after precutpreparation (2 days before DMEK). One month after DMEKendothelial cell density further decreased to 1817 +/- 111 cell/mm2.Conclusions: Precut preparation of DMEK tissue, generated byincomplete Descemet stripping, leads to minor endothelial cell lossduring subsequent culture. The use of precut tissue for DMEK resultsin a fast visual recovery, only minor additional endothelial cell lossand a rapid decrease of corneal thickness.Commercial Relationships: Bjoern O. Bachmann, None; UrsulaSchlotzer-Schrehardt, None; Martin Boergel, None; Friedrich E.Kruse, NoneProgram Number: 1679 Poster Board Number: D0314Presentation Time: 8:30 AM - 10:15 AMChanges in Anterior Corneal Haze with Severity of FuchsEndothelial DystrophySejal Amin, Jay W. McLaren, Keith H. Baratz, Sanjay V. Patel.Ophthalmology, Mayo Clinic, Rochester, MN.Purpose: In corneas requiring endothelial keratoplasty for Fuchsendothelial corneal dystrophy (FECD), haze (corneal backscatter)from the anterior cornea is higher than normal and remains higherthan normal after keratoplasty. In this study, we examined thechanges in anterior corneal haze over a range of severity of FECD.Methods: In a cross-sectional study, 95 corneas of 70 patients withFECD (mean age, 67 years; range, 41-87 years) and 52 normalcorneas of 28 controls (mean age, 44 years; range, 21-77 years) wereexamined by slit-lamp and confocal microscopy. Clinical grade ofFECD was determined by slit-lamp examination based on thepresence and extent of guttae, and the presence or absence ofclinically evident edema (modified Krachmer grades 1-6). FECD wascategorized as mild (grades 1-2), moderate (grades 3-4), or advanced(grades 5-6). Corneas of control subjects were devoid of any centralguttae (grade 0). Corneal haze, measured from the reflected lightintensity profile of confocal microscopy (ConfoScan 4, NidekTechnologies) images, was standardized to reflectivity from a knownconcentration of a turbidity standard. Anterior corneal haze wasdefined as the mean reflectivity in a 10-percentile range of stromalthickness centered at the anterior stromal boundary. Haze wascompared between severities of FECD and normal by usinggeneralized estimating equation (GEE) models to account for anycorrelation between fellow eyes of the same subject. The correlationbetween anterior haze and grade was illustrated by the Pearsoncorrelation coefficient, and the significance was determined by GEEmodels.Results: Anterior corneal haze in FECD (1940 ± 700 scatter units[SU], n=95) was higher than normal controls (1185 ± 229 SU, n=52,p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapatients with FECD.Methods: Human corneal endothelial cells (HCECs) were culturedfrom endothelial-Descemet membrane lamellae obtained from 3FECD patients during Descemet's membrane endothelial keratoplasty(DMEK) after obtaining informed written consent from each patient.HCECs cultured from 3 individual normal donor corneas were usedas a control. The FECD-derived HCECs and the control HCECs werethen immortalized by SV40 and hTERT to produce iFECD andiHCEC cell lines, respectively. The gene expression levels of ECMcomponents were then analyzed by TaqMan® real-time PCR. Toelucidate ECM production, iFECD and iHCEC cells were cultured ina Transwell® culture system, and ECM deposition was then analyzedby hematoxylin-eosin (HE) staining and immunohistochemistry after2 weeks.Results: Real-time PCR revealed a significantly increased productionof type I collagen, type IV collagen, and fibronectin (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaAna G. Alzaga Fernandez, None; Kimberly C. Sippel, None;Jessica Ciralsky, None; Mark Rosenblatt, None; Priyanka Sood,NoneSupport: Research to Prevent BlindnessProgram Number: 1683 Poster Board Number: D0318Presentation Time: 8:30 AM - 10:15 AMClinical Manifestation and Effect of Ganciclovir Therapy forCytomegalovirus Corneal EndotheliitisTsutomu Inatomi 1 , Noriko Koizumi 2 , Yuichi Ohashi 3 , YoshitsuguInoue 4 , Manabu Mochizuki 5 , Kohji Nishida 6 . 1 Ophthalmology, KyotoPrefectural Univ of Med, Kamigyo-Ku, Japan; 2 BiomedicalEngineering Faculty of Life and Medical Sciences, DoshishaUniversity, Kyoto, Japan; 3 Ophthalmology, Ehime University Schoolof Medicine, Ehime, Japan; 4 Ophthalmology, Tottori University,Faculty of Medicine, Tottori, Japan; 5 Ophthalmology, Tokyo Medicaland Dental University Graduate School, Tokyo, Japan;6 Ophthalmology, Osaka University Graduate School of Medicine,Osaka, Japan.Purpose: To report the findings of an epidemiological surveyconducted in Japan regarding the clinical manifestations and theeffect of ganciclovir antiviral therapy for the treatment of patientswith cytomegalovirus (CMV) corneal endotheliitis.Methods: An epidemiological survey was conducted in Japan andthe clinical manifestations of 109 eyes of 106 cases diagnosed asCMV corneal endotheliitis were analyzed retrospectively.Prospective, multicenter clinical treatment was performed for 7 eyesof 7 cases. The mean patient age was 67.4±10.4 years, and the meanfollow-up period was 12 months. The combined therapy of systemicganciclovir (5mg/kg, 2 times daily) with topical 0.5% ganciclovirinstillation was applied for 2 weeks following the tapering of topicalinstillation. Clinical evaluation was performed by use of real-timepolymerase chain reaction (PCR) and slit-lamp examination.Results: Of the total 106 cases, 85 cases (80.2%) were male and 21cases (19.8%) were female. Coin-shaped lesions and linear keraticprecipitates (KPs) were observed in 70.6% and 8.3% of the cases,respectively. Clinical manifestations included corneal edema(73.4%), endothelial loss (81.7%), iritis (67.9%), and elevation ofintraocular pressure (66.1%). In the prospective study, the averageCMV viral load in aqueous humor before treatment was 8.55x104copies/ml (range: 8.3x102 - 1.55x105 copies/ml). Coin-shapedlesions or linear KPs were detected in 100% and 29% of the cases,respectively, and 85.7% of the cases showed sustained virologicresponse after the initial combined therapy. All cases showednegative by PCR with additional topical ganciclovir therapy, but 3cases (42.8%) showed the elevation of intraocular pression and 1 case(14.5%) showed the recurrence. Mean endothelial cell density (CED)(cell/mm2 ) at before and 1-month post treatment was 1160±365 and1048±440, respectively. No significant reduction of CED and noadverse effects were observed.Conclusions: Corneal endotheliitis with coin-shaped lesions or linearKPs, similar to the clinical rejection line, are major clinicalmanifestations in CMV corneal endotheliitis. The combined therapyof systemic and topical ganciclovir instillation is safe and effectivefor the treatment of patients with CMV corneal endotheliitis.Commercial Relationships: Tsutomu Inatomi, None; NorikoKoizumi, None; Yuichi Ohashi, None; Yoshitsugu Inoue, None;Manabu Mochizuki, Santen (F), Senju (F), Ohtsuka (F), Daiichi-Sankyo (F), Mitsubishi-Tanabe (F), AMO Japan (F), Alcon Japan(F); Kohji Nishida, Alcon (C), Alcon (F), HOYA (F), Senju (F),Pfizer (F), Santen (F), Osaka University (P)Support: Research on Measures for Intractable Diseases 074Program Number: 1684 Poster Board Number: D0319Presentation Time: 8:30 AM - 10:15 AMExpansion of corneal endothelial cells using biomimeticengineered substratesRachelle Palchesko 1, 3 , James L. Funderburgh 2, 3 , Adam W.Feinberg 1, 3 . 1 Biomedical Engineering, Carnegie Mellon University,Pittsburgh, PA; 2 Ophthalmology, University of Pittsburgh,Pittsburgh, PA; 3 Louis J Fox Center for Vision Restoration,University of Pittsburgh, Pittsburgh, PA.Purpose: Corneal endothelial cells (CECs) are non-proliferative invivo with minimal proliferation in vitro, making expansion of thesecells for therapeutic application difficult. Our previous work hasshown that culturing cells on a biomimetic substrate that mimics themechanical and biochemical properties of Descemet’s membraneenables the expansion of CECs >3000-fold compared to 139-fold ontissue culture polystyrene (TCPS). Here, we demonstrate that thebiomimetic substrate also maintains CEC phenotype and preventstransition to senescence or a fibroblast-like phenotype.Methods: CECs were isolated from bovine corneas and cultured onone of three different surfaces: the biomimetic substrate consisting ofcollagen type IV coated polydimethylsiloxane soft elastomer (COL4-PDMS) and two controls, TCPS and collagen type IV coated TCPS(COL4-TCPS). CECs were cultured for 8 passages andimmunofluorescently labeled at passages 1, 5, and 8 for fibronectin(FN), zonal occludins (ZO-1) and F-actin to analyze changes inextracellular matrix production, cell-cell coupling and polygonalmorphology. At these same time points qRT-PCR was used toquantify mRNA expression of COL4A2, COL8A1, and SLC4A4 asCEC markers and COL3A1 as the fibroblastic gene marker.Results: CECs cultured on the COL4-PDMS produced short,immature FN fibrils at all time points where as CECs on TCPS andCOL4-TCPS produced large fibrils at passages 5 and 8 similar tothose produced by fibroblasts. On COL4-PDMS CECs grew at higherdensity, had more continuous ZO-1 staining and maintained apolygonal morphology. Gene expression followed a similar patternwith fibroblast associated FN and COL3A1 genes higher on TCPSand COL4-TCPS and CEC associated COL8A1 and SLC4A4 genesmaintained at levels comparable to CECs in vivo on COL4-PDMS.Conclusions: We have demonstrated that a biomimetic substratewhich recapitulates the mechanical stiffness and collagen type IVcomposition of Descemet’s membrane significantly enhancesexpansion and maintains phenotypic stability of CECs in vitrocompared to TCPS and COL4-TCPS controls. Current efforts arefocused on extending this system to the expansion of human CECs toenhance its clinical relevance. The ability to expand CECs is crucialto obtain the cell numbers necessary for bioengineering a cornealendothelium suitable for implantation, a future goal of this researchproject.Commercial Relationships: Rachelle Palchesko, None; James L.Funderburgh, None; Adam W. Feinberg, Carnegie MellonUniversity (P)Support: This work was supported by the Ocular Tissue Engineeringand Regenerative Ophthalmology funding through the Louis J. FoxCenter for Vision Restoration, National Institutes of Health COREGrant P30 EY008098, EY09368 (to JLF), the Eye and EarFoundation of Pittsburgh, PA and an unrestricted Grant fromResearch to Prevent Blindness, New York, NYProgram Number: 1685 Poster Board Number: D0320Presentation Time: 8:30 AM - 10:15 AMMeganuclease Targeting HSV-1 Protects Against CornealEndothelitis ex-vivo and in-vivo©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEric E. Gabison 1, 2 , Marc Labetoulle 4 , Marine Gailledrat 5 , Jose A.Sahel 3 , Benoit Chapelier 3 . 1 Cornea Department, Fondation A. deRothschild, Paris, France; 2 Ophthalmology Department, HôpitalBichat, Paris, France; 3 Institut de la vision, Paris, France;4 Ophthalmology Department, Hôpital du Kremlin Bicêtre, Paris,France; 5 Cellectis, Paris, France.Purpose: Herpetic Keratitis is a leading cause of decreased bestcorrected visual acuity in developed and developing countries. Theaim of this study is to assess the antiviral property of a meganucleasetargeting HSV in the prevention of HSV endothelitis ex-vivo and invivo.Methods: Normal rabbit corneas were placed in organ culture andtransduced by a recombinant adeno-associated virus (rAAV)allowing constitutive expression of meganucleases targeting HSV-1genome or of a non-coding sequence. These organs were thensubmitted to infection by recombinant HSV-1 F(1) virus equippedwith a LacZ expressing cassette at M.O.I. 0.001 to 0.1%. Infectionrates for plaques or cells in endothelium were established byimmunostaining of envelope protein gD or X-gal staining after theend of first or second lytic cycle. Additionnaly, a rabbit model ofcorneal HSV endothelitis was developed. Intracamerular injection ofHSV F(1) were performed in rabbit eyes 2 days following intravitrealinjection of steroid. Corneal edema, keratic precipitates, ocularinflammation and infection rate for plaques or cells were analyzed inthis model.Results: Meganuclease targeting the ICP0 gene which encodes an E3ubiquitin ligase involved in viral reactivation and replication did notchange infection rates in the present organ culture model, but reducedthe average size of plaques in endothelium with a decrease of 27-46%. Conversely, the meganuclease directed against the major capsidprotein UL19 lowered the number and size of plaques, both beingreduced by half at M.O.I. 0.001%. Consequently, the expression of ameganuclease in endothelium, evidenced by RT-PCR, could eitherreduce infectious particle production or induce cell resistance toHSV-1. In vivo, experiments demonstrated a 30% decreased inendothelial plaque formation. The rate of corneal edema and keraticprecipitates was reduced in Megnuclease treated eyes as compared tocontroles.Conclusions: Our organ culture and in vivo model of herpeticendothelial infection are reproducible and efficient to quantify viralproliferative capacity. Meganuclease transduction confers asignificant inhibition of viral pathogenic effect. Meganuclease genetherapy targeting HSV-1 DNA may be an effective treatment toprotect against HSV endothelitisCommercial Relationships: Eric E. Gabison, None; MarcLabetoulle, None; Marine Gailledrat, Cellectis SA (E); Jose A.Sahel, UPMC/Essilor (P), Second Sight (F); Benoit Chapelier, NoneSupport: OSEO ACTIVE GRANT FRANCEProgram Number: 1686 Poster Board Number: D0321Presentation Time: 8:30 AM - 10:15 AMComparison of Endothelial Cell Density at the Central andPeripheral Regions in a DSAEK GraftHiroko Nakagawa, Tsutomu Inatomi, Shigeru Kinoshita. KyotoPrefectural Univ of Med, Kyoto, Japan.Purpose: To analyze endothelial remodeling, including the woundhealing, at the host/graft junction after Descemet’s StrippingAutomated Endothelial Keratoplasty (DSAEK), and compare thechange of corneal endothelium at the central and peripheral regionsof the graft using wide-field specular microscopy.Methods: This study involved 10 eyes of 10 patients (mean age: 72years) treated by DSAEK using internationally shipped precut donorcorneas. None of the patients had ocular complications prior tosurgery, and DSAEK was performed via the pull-through techniqueusing a Busin glide. Wide-field contact specular microscopy wasused to evaluate the alteration of endothelium at the central andtemporal peripheral corneal regions (approximately 1mm inside fromthe graft edge) at 1, 6, and 12 months after surgery. Endothelial celldensity (ECD), coefficient of variation (CV), and the frequency ofhexagonal cells (6A) were analyzed to elucidate the post-DSAEKendothelial remodeling pattern.Results: Mean regional endothelial cell density (cells/mm2±SD) inthe center / periphery at 1, 6, and 12 months postoperative were2286±409 / 1893±432, 2152±464 / 1550±284, and 2138±466 /1152±378, respectively. Peripheral ECD was statistically lower thancentral ECD at each follow-up time-point (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaeach significantly differentially expressed gene in normal andabnormal corneal endothelial cell function.Commercial Relationships: Cynthia Wang, None; Ricardo F.Frausto, None; Anthony J. Aldave, Alcon (R), Allergan (R), NIH(F), Bausch + Lomb (C), Allergan (C)Support: RPBProgram Number: 1688 Poster Board Number: D0323Presentation Time: 8:30 AM - 10:15 AMROCK inhibitor enhances adhesion and wound healing onhuman corneal endothelial cellsMichael J. Nicolas 1 , Aurélien Pipparelli 1 , Yvan Arsenijevic 1 , GillesThuret 2 , Philippe Gain 2 , Francois Majo 1 . 1 Ophtalmology, Jules-Gonin eye hospital, Lausanne, Switzerland; 2 Ophtalmology,University of St Etienne, St Etienne, France.Purpose: Recently it was reported that the ROCK inhibitor Y-27632promotes adhesion, inhibits apoptosis, increases the number ofproliferating monkey corneal endothelial cells in vitro and enhancecorneal endothelial wound healing both in vitro and in vivo. Here, weproposed to evaluate the effects of ROCK inhibitor on HCEC eitherin vitro or ex vivo, firstly to assess the potential of this compound toincrease the number of corneal graft available for the clinic andsecondly to validate the previous results obtained in animal models, astep required before potential clinical application.Methods: Using organ culture human cornea (N=34), the effect ofROCK inhibitor was evaluated either in vitro or ex vivo. Toxicity,endothelial cell density, cell proliferation, apoptosis, cellmorphometry, adhesion and wound healing process were evaluatedby live/dead assay standard cell counting method, EdU labelling,Ki67, Caspase3, Zo-1 and Actin immunostaining.Results: In our study, we demonstrated for the first time in humanendothelial cells ex vivo and in vitro, that ROCK inhibitor did notinduce any toxicity effect and did not modulate metabolism activity.Compared to animal model, ROCK inhibitor treatment did not inducehuman endothelial cell proliferation. However, ROCK inhibitorsignificantly enhances corneal endothelial cell adhesion and woundhealing.Conclusions: These results strongly suggest that ROCK inhibitor is apromising and safe compound to improve the treatment of cornealendothelial dysfunction in human. ROCK inhibitor could be apotential therapeutic strategy in order to improve adhesion oftransplanted human cultured endothelial cells. Furthermore, ROCKinhibitor treatment can increase in human the closure of endothelialcell defect.Commercial Relationships: Michael J. Nicolas, None; AurélienPipparelli, None; Yvan Arsenijevic, None; Gilles Thuret, None;Philippe Gain, None; Francois Majo, NoneProgram Number: 1689 Poster Board Number: D0324Presentation Time: 8:30 AM - 10:15 AMControlled Release of a Rho Kinase (ROCK)-Selective Inhibitorwith Polylactic Acid MicrospheresSho Koda 1, 2 , Takashi Saito 2 , Junji Kitano 1 , Naoki Okumura 1, 3 ,Shigeru Kinoshita 3 , Yasuhiko Tabata 2 , Noriko Koizumi 1, 3 .1 Biomedical Engineering, Doshisha University, Kyotanabe, Japan;2 Biomaterials, Kyoto University, Kyoto, Japan; 3 Ophthalmology,Kyoto Prefectural University of Medicine, Kyoto, Japan.Purpose: We previously reported that the Rho kinase (ROCK)inhibitor Y-27632 promoted the wound healing of corneal endothelialcells in a primate model. Thus, Y-27632 shows promise as a drug thatcan be used for the clinical treatment of corneal endothelial defects.The purpose of this present study was to design polylactic acid (PLA)microspheres that can be used for the controlled release of Y-27632in the anterior chamber.Methods: PLA microspheres incorporating Y-27632 (Y-27632-PLA)were prepared via a double-emulsion [(water in oil) in water] solventevaporation method. The in vitro release test of Y-27632-PLA wasperformed in a phosphate-buffered solution (pH7.4) at 37°C.Theamount of Y-27632 released was determined by use of highperformanceliquid chromatography (HPLC). To evaluate the toxicityof PLA microspheres without Y-27632 after they were injected intothe anterior chamber of rabbit eyes, slit-lamp examinations, andcorneal thickness and intraocular pressure measurements wereperformed up to14 days after injection, followed byimmunohistochemical analysis.Results: Y-27632 was released from PLA microspheres over 28days. The percent of Y-27632 released was 70.0, 77.9, and 98.2% at7, 14, and 28 days, respectively. The release pattern of Y-27632could be changed by altering the molecular weight of the PLA thatwas used. In all eyes injected with the PLA microspheres, normalcorneal thickness was observed, with no inflammation or elevation ofintraocular pressure. The corneal endothelial cell density and theexpression of functional protein were found to be normal.Conclusions: The findings of this study demonstrate that PLAmicrospheres are promising candidates for the controlled release ofY-27632. The Y-27632-PLA is applicable as a pharmaceutical agentfor the treatment of corneal endothelial dysfunction.Commercial Relationships: Sho Koda, None; Takashi Saito,None; Junji Kitano, None; Naoki Okumura, None; ShigeruKinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA(R); Yasuhiko Tabata, None; Noriko Koizumi, NoneSupport: The Funding Program for Next Generation World-LeadingResearchers from the Cabinet Office in Japan ( LS117) TheAdaptable and Seamless Technology Transfer Program throughTarget-driven R&D (AS2314212G)Program Number: 1690 Poster Board Number: D0325Presentation Time: 8:30 AM - 10:15 AMRho-kinase inhibitor enhances corneal endothelial cellproliferation via p27 degradationRyohei Numata 1, 2 , Naoki Okumura 1, 2 , EunDuck P. Kay 1 , MakikoNakahara 1 , Shinichiro Nakano 1 , Morio Ueno 2 , Shigeru Kinoshita 2 ,Noriko Koizumi 1 . 1 Biomedical Engineering, Doshisha University,Kyotanabe, Japan; 2 Ophthalmology, Kyoto Prefectural University ofMedicine, Kyoto, Japan.Purpose: We previously reported that Rho kinase (ROCK) inhibitorpromotes wound healing of corneal endothelial cells in an animalmodel and showed the possibility of the clinical application forcorneal endothelial deficient condition. The purpose of this study isto determine the mechanism by which ROCK inhibitor promotes cellproliferation in corneal endothelial cells.Methods: Cultivate monkey corneal endothelial cells (MCECs) wereused for this study. Cell proliferation was analyzed by two methodsin the absence or presence of 10 μM the selective ROCK inhibitor(Y-27632): BrdU ELISA assay to determine the incorporation ofBrdU into the newly synthesized DNA and CellTiter Glo assay tomeasure the numbers of viable cells. Activation of Akt andexpression of Cdc25A and p27 were analyzed by western blotting.Results: Incorporation of BrdU into the newly synthesized DNA wasdoubled in the cells treated with ROCK inhibitor when compared tothat of the control cells. Likewise, the viable cell numbers of MCECstreated with the inhibitor for 24 h or 48 h were 50% greater than thecell numbers in the absence of ROCK inhibitor. In addition to theincreased cell numbers, the ROCK inhibitor promoted cell adhesion.To elucidate the action of ROCK inhibitor in MCECs with or without©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaROCK inhibitor, phosphorylation of Akt was analyzed for 1 h up to24 h in serum-containing medium: in the absence of ROCK inhibitor,phosphorylation of Akt was gradually increased to 12 h. Unlike thecontrol pattern, phosphorylation of Akt reached maximum at 1 hfollowing treatment of cells with ROCK inhibitor, after which thelevel of phosphorylated Akt was gradually decreased. The expressionof Cdc25A, which activates Cdk2 that phosphorylates p27, wasobserved during the early time periods in the presence of ROCKinhibitor, while the control cells showed a late expression pattern ofCdc25A. Similarly, the amount of p27 was greatly reduced from 1 hfollowing treatment of cells with ROCK inhibitor, whereas thecontrol cells maintained high levels of p27 up to 12 h.Conclusions: The findings of the study demonstrate that ROCKinhibitor activates PI 3-kinase signaling which subsequentlypromotes degradation of p27 via Cdc25A pathway, thus leading tocell proliferation of CECs. These data suggest that the ROCK mightbe a useful pharmaceutical agent for corneal endothelial disease.Commercial Relationships: Ryohei Numata, None; NaokiOkumura, None; EunDuck P. Kay, None; Makiko Nakahara,None; Shinichiro Nakano, None; Morio Ueno, None; ShigeruKinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA(R); Noriko Koizumi, NoneSupport: The Adaptable and Seamless Technology TransferProgram through Target-driven R&D (AS2314212G),The FundingProgram for Next Generation World-Leading Researchers from theCabinet Office in Japan ( LS117)Program Number: 1691 Poster Board Number: D0326Presentation Time: 8:30 AM - 10:15 AMProliferation Propensity of Cultured Human Corneal EndothelialCells and Their Plasticity Dictated by CultureMicroenvironmentsMunetoyo Toda 1 , Kana Nakata 1 , kazuko asada 1 , Michio Hagiya 1 ,Morio Ueno 1 , Naoki Okumura 2 , Noriko Koizumi 2 , Junji Hamuro 1 ,Shigeru Kinoshita 1 . 1 Department of Ophthalmology, KyotoPrefectural University of Medicine, Kyoto, Japan; 2 Department ofBiomedical Engineering, Doshisha University, Kyoto, Japan.Purpose: It is well known that human corneal endothelium cells(HCECs) have poor proliferative ability under in vitro cultureconditions. The difficulty of cultivating HCECs hampers a detailedanalysis of their proliferation propensity. To detail molecularmechanisms underlying this impaired HCEC proliferation, weattempted to clarify the presence and proliferation propensity offunctionally heterogeneous subpopulations in cultured HCECs.Methods: The proliferative properties of cultured HCECs wereevaluated by BrdU assay and carboxyfluorescein succinimidyl ester(CFSE) dye dilution assay. To investigate if cultured HCECs containsubpopulations with distinct metabolic requirements, HCECs werestained with MitoTracker® Red (Life Technologies Corp., Carlsbad,CA) to evaluate their mitochondria content. Flow cytometry (FCM)using several surface markers was performed to characterize thesubpopulations.Results: Cell percentages in the G1, S, and G2/M phase of the cellcycle were determined by FCM using BrdU and 7-AAD. Thepercentage of cells in the S- and G2/M-phase was about 40%, andabout 60% of the HCECs were arrested in the G1-phase. CFSE assaydetected 2 subpopulations with different proliferative properties. Onedivided 7 times in 8 days of cultivation, while the other stopped celldivision at 3 times. We theorize that the latter entered premature cellsenescence at the early stage of cultivation, resulting in cell-cyclearrest. These results suggest that each subpopulation has uniquemetabolic turnover rates and energy requirements, as it is theorizedthat poor proliferation is tied to energy from mitochondria, not fromglucose metabolism. Moreover, FMC using MitoTracker® Reddetected different mitochondrial content in the cultured HCECs. Eachsubpopulation was stained with several cell surface markers selectedby global analysis, and we tried to characterize candidate markers forthe high-proliferative population.Conclusions: These findings show that cultured HCECs havedifferent subpopulations and provide the possibility of establishing aneffective method for culturing HCECs containing an enrichedsubpopulation with high proliferation ability.Commercial Relationships: Munetoyo Toda, None; Kana Nakata,None; kazuko asada, None; Michio Hagiya, JCR PharmaceuticalsCo., Ltd (E); Morio Ueno, None; Naoki Okumura, None; NorikoKoizumi, None; Junji Hamuro, None; Shigeru Kinoshita, SenjuPharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Program Number: 1692 Poster Board Number: D0327Presentation Time: 8:30 AM - 10:15 AMCell-injection Therapy Using Rho Kinase Inhibitor in a CornealEndothelial Dysfunction Rabbit ModelJunji Kitano 1 , Naoki Okumura 1, 2 , EunDuck P. Kay 1 , Morio Ueno 2 ,Junji Hamuro 2 , Shigeru Kinoshita 2 , Noriko Koizumi 1, 2 . 1 BiomedicalEngineering, Doshisha University, Kyotanabe, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan.Purpose: To investigate feasibility of corneal endothelialreconstruction by a cell-injection therapy using cultivated rabbitcorneal endothelial cells (RCECs) in a corneal endothelialdysfunction rabbit model and to determine the optimum cell numbersfor the cell-injection therapy.Methods: Rabbit corneal endothelium was denuded by intensivemechanical scraping. Cultivated RCECs in the presence of 100μM ofRho kinase (ROCK) inhibitor, Y-27632, were injected into theanterior chamber of the host animals at three concentrations (2x10 5cells, 5.0x10 5 cells, or 1.0x10 6 cells). The eyes of each animal werekept in the face-down position for 3 hours. Slit-lamp examinations,corneal thickness- and intraocular pressure- measurements, andimmunohistochemical analysis were performed for up to 14 days.Results: All eyes received cell-injection therapy showed improvedcorneal clarity; corneal clarity and corneal thickness recovered thefastest rate in the host animals that received cultivated RCECs at1.0x10 6 cell numbers. In all animal groups, corneal endotheliumdemonstrated the characteristic contact-inhibited monolayer withpolygonal cells that express the functional endothelial phenotypicproteins, ZO-1 and Na+/K+-ATPase. When the endothelial celldensity of the host animals was measured, the animal that received acell injection at 1.0x10 6 cells demonstrated significant high celldensity with 3296.6±365.1 cells/mm 2 , while the other two animalgroups showed 1432.0±200.8 cells/mm 2 (injected cell numbers:2.0x10 5 ) and 2252.2±204.6 cells/mm 2 (injected cell numbers: 5.0x10 5cells). None of the eyes of the experimental animals showed elevatedintraocular pressure or immunological rejection.Conclusions: The findings provide evidence that the cell-injectiontherapy using appropriate cell numbers with ROCK inhibitor enablescorneal endothelial regeneration by tissue engineering technique andmay be a useful clinical alternative for corneal transplantation.Commercial Relationships: Junji Kitano, None; Naoki Okumura,None; EunDuck P. Kay, None; Morio Ueno, None; Junji Hamuro,None; Shigeru Kinoshita, Senju Pharmaceutical Co (P), SantenPharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),AMO (R), HOYA (R); Noriko Koizumi, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: The Funding Program for Next Generation World-LeadingResearchers from the Cabinet Office in Japan ( LS117), TheHighway Program for realization of regenerative medicineProgram Number: 1693 Poster Board Number: D0328Presentation Time: 8:30 AM - 10:15 AMEffect of ROCK inhibiter on Apoptosis in Corneal EndothelialCellsAi Odajima 1, 2 , Naoki Okumura 1, 2 , EunDuck P. Kay 1 , Wen Chen 1 ,Morio Ueno 2 , Shigeru Kinoshita 2 , Noriko Koizumi 1 . 1 Department ofBiomedical Engineering, Faculty of Life and Medical Sciences,Doshisha University, Kyotanabe, Japan; 2 Department ofOphthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan.Purpose: Apoptosis is reportedly involved in pathological conditionof corneal endothelium such as Fuchs dystrophy or post keratoplasty.The purpose of this study is to investigate the feasibility of Rhokinase (ROCK) inhibitorfor modulating apoptosis in cornealendothelial cells.Methods: Monkey corneal endothelial cells (MCECs) were culturedand were then treated by UV radiation. Cell death was evaluated bytrypan blue staining.Apoptosis was evaluated by the quantity of thereleased cytochrome c and caspase 3 (western blotting), activationlevel of caspase 3 (CaspaseGlo 3/7 Assay), DNA flagmentation(TUNEL). As an ex vivo apoptosis model, rabbit corneal tissue wastreated by either UV (100 J/m 2 ) or H 2 O 2 (100 µM). Then, apoptoticcells were evaluated by Annexin Vimmunostaining after 24 hours.Results: MCECs were exposed to UV radiation ranging from 10 to1000J/m 2 , there was cell death in a dose-response manner; substantialcell death was observed from 50 J/m 2 .UV radiation caused massiverelease of cytochrome c into the cytoplasm; and cleavage of caspase3 was increased with UV radiation. TUNEL positive DNAfragmentationwas detected in the UV radiated cells.ROCK inhibitorsignificantly decreased UV induced Annexin V-positive apoptoticcorneal endothelial cellsin rabbit corneal tissue compared to thecontrol (2.1±0.8% and10.9±3.3%, respectively)(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawere successful. The DMEK grafts revealed a mean ECL of 17.5%(range 14 to 21%).Conclusions: Corneas can be prepared for DMEK surgery by eyebank technicians and post-stripping microscopy can be performed toassess graft quality. Additionally, the risk of tissue wastage istransferred while conserving costly operating room time. Increasingdemand for DMEK prepared tissue warrants further investigationsregarding optimization of preparation techniques.Commercial Relationships: Jeffrey D. Holiman, None; JuliaTalajic, None; David Davis-Boozer, None; Mark A. Terry, Bauschand Lomb Surgical (R), Alcon (R), Optovue (C)Program Number: 1696 Poster Board Number: D0331Presentation Time: 8:30 AM - 10:15 AMInterleukin-1β enhances cell migration through AP-1 and NF-κBpathway dependent FGF2 expression in human cornealendothelial cellsDaniel Sand, JeongGoo Lee, J M. Heur. Department ofOphthalmology, Keck School of Medicine of the University ofSouthern California, Los Angeles, CA.Purpose: To investigate IL-1β signaling in migration of humancorneal endothelial cells (CECs).Methods: Cell migration was measured using scratch-induceddirectional migration assay. Expression and/or activation of IRAK, PI3-kinase, p38, IKK, IκB, NF-κB, and FGF-2 were analyzed byimmunoblotting. AP-1 and NF-κB activities were measured by AP-1and NF-κB ELISA assay kit, respectively. Binding of AP-1 and NFκBto the promoter region of the FGF-2 gene was determined bychromatin immunoprecipitation.Results: Stimulation of human CECs with IL-1β activated expressionof FGF2 and resulted in enhanced cell migration. This, in turn, wasabolished by treatment with either IL-1 receptor antagonist or SU-5402, a pan FGF inhibitor. PI 3-kinase or IRAK 1/4 antagonistsdemonstrated that IRAK 1/4 activates PI 3-kinase, which in turnphosphorylates p38 and IKK α/β, leading to FGF2 expressionthrough activation of AP-1 and NF-κB in human CECs. Treatment ofIL-1β stimulated human CECs, with either AP-1 or NF-κBantagonists, decreased FGF2 expression and resulted in reduced IL-1β enhanced cell migration. Co-treatment of IL-1β stimulated humanCEC with both inhibitors completely blocked FGF2 expression andIL-1β enhanced cell migration. Chromatin immunoprecipitationassays demonstrated that AP-1 and NF-κB directly bind to the FGF2promoter following IL-1β stimulation.Conclusions: The results show that binding of IL-1β to its receptor inhuman CEC leads to parallel activation of AP-1 and NF-κBpathways, leading, in turn, to FGF2 expression and enhanced cellmigration.Commercial Relationships: Daniel Sand, None; JeongGoo Lee,None; J M. Heur, NoneSupport: Baxter Foundation, RPB, NIH Core Grant EY03040Program Number: 1697 Poster Board Number: D0332Presentation Time: 8:30 AM - 10:15 AMThe roles of TWEAK in human corneal endothelial cellsMasahiro Yamaguchi 1 , Nobuyuki Ebihara 1 , Toshinari Funaki 1 , AkiraMurakami 1 , Satoru Yamagami 2 . 1 Ophthalmology, JuntendoUniversity, Bunkyo-ku, Japan; 2 Ophthalmology, Tokyo University,Tokyo, Japan.Purpose: Tumor necrosis factor-like weak inducer of apoptosis(TWEAK), a member of TNF superfamily, has several rolesincluding angiogenesis, differentiation control, chemokineproduction, and so on. We previously reported the effectiveness ofTWEAK in corneal keratocyte (Ebihara et al, Exp Eye Res, 2009),and in retinal pigment epithelial cell (Ebihara et al, Curr Eye Res,2009). The roles of TWEAK in corneal endothelial cells wasexamined this time.Methods: (1) The expression of Fn14, receptor of TWEAK, wasexamined with human corneal endothelial cells by FACS. (2) Proteinarray and ELISA were conducted to measure TWEAK concentrationin human aqueous humor. (3) The chemokine expression in culturedhuman corneal endothelial cells (cHCECs) was examined withTWEAK or TWEAK/TFG-β. (4) Cell adhesion, proliferation, andmigration of cHCECs were investigated with adhesion, proliferationand in vitro scratch assays, in addition of TWEAK or TWEAK/TGFβ.Results: (1)Fn14 receptor was expressed on corneal endothelial cells.(2) Soluble TWEAK was slightly detected in human aqueous humor.(3) TWEAK stimulated the expression of RANTES, IL-8, MCP-1 incHCECs. TGF-β inhibited previous expression, but TGF-β withTWEAK lost the inhibition. (4) TWEAK promoted migration ofcHCECs.Conclusions: There are possibilities that an adequate concentrationof TWEAK in aqueous humor may maintain constancy of cornealendothelial cells and that excessive expression of TWEAK mayinduce inflammation.Commercial Relationships: Masahiro Yamaguchi, None;Nobuyuki Ebihara, None; Toshinari Funaki, None; AkiraMurakami, SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958(P); Satoru Yamagami, NoneProgram Number: 1698 Poster Board Number: D0333Presentation Time: 8:30 AM - 10:15 AMThe transparency transcriptome: gene expression profile ofhuman corneal endothelial cellsNoelia J. Kunzevitzky 1, 2 , Karen Alvarez-Delfin 1 , Richard M.Merkhofer 1 , Alejandra D. Weisman 1 , Jeffrey L. Goldberg 1, 3 . 1 BascomPalmer Eye Institute & Interdisciplinary Stem Cell Institute,University of Miami, Miami, FL; 2 Emmetrope Ophthalmics, CoralGables, FL; 3 Shiley Eye Center, University of California San Diego,San Diego, CA.Purpose: The corneal endothelium is a monolayer on the innercornea whose main function is to maintain corneal transparency. Lossof function due to dystrophy, trauma or genetic abnormalities leads toswelling, pain and loss of vision. Current treatment options includeDSAEK or penetrating keratoplasty—both limited by the shortage ofdonor corneas. Injection of cultured human corneal endothelial cells(HCECs) could provide a less invasive and readily available solution.The protocol for purifying and expanding HCECs in vitro fromcadaveric corneas has been extensively described, but the criteria foridentifying these cells are not definitive. In order to better define theHCECs’ identity and to ask basic cell biology questions about thesecells, we analyzed the gene expression profile of purified HCECs atdifferent numbers of passages in vitro and compared it to thetranscriptome of freshly dissected corneal layers.Methods: Human cadaveric corneas preserved in Optisol® wereprocured by the Lions Eye Institute for Transplant and Research(Tampa, FL). For some corneas, the HCECs were peeled offDescemet’s membrane and cultured. For other corneas, epithelial,stromal and endothelial layers were acutely dissected and minimallyprocessed for RNA extraction. RNA from at least 3 biologicalreplicates was independently collected, amplified and processed forhybridization to Affymetrix GeneChip® arrays. We classified probesby Gene Ontology and compared the gene expression profiles ofcultured HCECs and the three corneal layers.Results: Cultured HCECs expressed ~82% of 28,869 total probes.Further gene ontology analysis revealed the overrepresentation of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapathways related to cell survival, cytoskeletal remodeling, oxidativephosphorylation and cell adhesion, among others. We also identifieda number of HCEC-specific markers that can be verified viaimmunostaining and RT-PCR.Conclusions: We have begun to describe the transcriptome of thecornea, a tissue whose proper function is essential to maintainingvisual clarity, yet remains poorly characterized. In doing so we hopeto unveil the essential molecular players that regulate cellmorphology, survival, and proliferation and to better define HCECs’identity for further use in cell therapy.Commercial Relationships: Noelia J. Kunzevitzky, None; KarenAlvarez-Delfin, None; Richard M. Merkhofer, None; AlejandraD. Weisman, None; Jeffrey L. Goldberg, None247 Surgery: Non-Refractive and KeratoprothesisMonday, May 06, 2013 11:00 AM-12:45 PMTCC 303 Paper SessionProgram #/Board # Range: 1747-1753Organizing Section: CorneaProgram Number: 1747Presentation Time: 11:00 AM - 11:15 AMMicrostructural analysis of the cornea after Descemet membraneendothelial keratoplasty using in vivo confocal microscopyAkira Kobayashi, Hideaki Yokogawa, Natsuko Yamazaki, ToshinoriMasaki, Kazuhisa Sugiyama. Dept of Ophthalmology, KanazawaUniv Sch of Medicine, Kanazawa, Japan.Purpose: To investigate the in vivo corneal changes in patients withbullous keratopathy who underwent Descemet membrane endothelialkeratoplasty (DMEK) with the use of in vivo laser confocalmicroscopy.Methods: Five eyes of four patients (three men, one women; meanage, 61.3±9.6 years [mean ± standard deviation]) with bullouskeratopathy who had undergone successful DMEK were enrolled inthis study. In vivo laser confocal microscopy was performed beforeand one, three, and six months after DMEK. Selected confocalimages of corneal layers were evaluated qualitatively andquantitatively for the degree of haze and the density of deposits.Subepithelial haze, donor-recipient interface haze, donor-recipientinterface particles, and host stromal needle-shaped materials weregraded on a scale of 4 categories (grade 0; none, grade 1; mild, grade2; moderate, grade 3; severe) at each time point. Time trends of theoutcomes were graphically displayed and evaluated with Mantel-Haenszel trend test.Results: Preoperatively, the following were observed in all patients:slight corneal epithelial edema, moderate subepithelial haze,keratocytes in a honeycomb pattern, and tiny needle-shaped materialsin the stroma. After DMEK, moderate subepithelial haze persistedduring the follow-up period. Needle-shaped materials had a tendencyto decrease after DMEK. Most notably, donor-recipient interfacehaze and donor-recipient interface particles were barely noticeableafter DMEK as early as one month postoperatively.Conclusions: In vivo laser confocal microscopy can identifysubclinical corneal abnormalities after DMEK such as subepithelialhaze, host stromal needle-shaped materials, and minimum donorrecipientinterface haze/particles. These abnormalities seemed subtlecompared to Descemet stripping automated endothelial keratoplasty;this may explain the superior postoperative visual acuity afterDMEK. Further studies with this technology in a large number ofpatients and long-term follow up are needed to fully understand thelong-term corneal changes after DMEK.Commercial Relationships: Akira Kobayashi, None; HideakiYokogawa, None; Natsuko Yamazaki, None; Toshinori Masaki,None; Kazuhisa Sugiyama, NoneSupport: a Grant-in-Aid for Scientific Research (C) KAKENHI,Japan (No. 22591934)Program Number: 1748Presentation Time: 11:15 AM - 11:30 AMImpact of Donor Age on Endothelium-Descemet MembraneLayer Harvesting and Roll FormationAdam Bennett 1 , Shahira Rashad 2 , Donna Drury 1 , H D. Cavanagh 1 ,James P. McCulley 1 , Matthew Petroll 1 , Vinod V. Mootha 1 . 1 Univ TexSouthwestern Medical Center, Dallas, TX; 2 Alexandria Faculty ofMedicine, Alexandria, Egypt.Purpose: Descemet membrane endothelial keratoplasty (DMEK) isan alternative to Descemet’s stripping automated endothelialkeratoplasty (DSAEK) to surgically replace diseased cornealendothelium. Although graft rejection incidence has been reported tobe drastically lower in DMEK, wide adoption may be limited by twofactors. Harvesting the endothelium-Descemet membrane layer(EDM) can be difficult, and tight EDM scrolling can hinderunfolding once inserted into the patient’s eye. Anecdotally, surgeonshave noticed the use of younger donors has exacerbated these factors.We sought to correlate donor age with EDM stripping difficulty andscroll tightness.Methods: EDM scrolls were harvested by a cornea-fellowshiptrained ophthalmologist masked to donor age from 26 corneoscleralbuttons. An 11 mm partial trephination was used instead of bluntdissection for a consistent and even outer cut. 7.0 to 8.25 mm EDMscrolls were prepared using the SCUBA technique in Optisol GS.VisionBlue® (.06% trypan blue) staining was used to harvest andassess EDMs. The surgeon subjectively rated stripping difficulty on a1 to 5 scale (easiest to unable to strip) based upon DM adherence tounderlying stroma and radial tear formation. Three different methodswere used to characterize scrolling severity: scroll width, normalizedscroll surface area (scroll width × scroll length/surface area of EDM),and tendency for EDM scroll formation (referred to as scroll rating).A scroll rating of 1 corresponded to opposite ends of the EDM nottouching, 2 when EDM ends touch, 3 when the EDM forms onecomplete scroll and 4 when more than one scroll formed.Results: Mean donor age was 59 ± 14 years (15-69). Mean diameterof EDM scroll was 7.9 ± .23 mm (7.0-8.25). Stripping difficulty wasshown to be inversely correlated with donor age (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaDescemet’s Stripping Automated Endothelial Keratoplasty(DSAEK) vs Ultra-Thin DSAEK (UT-DSAEK) vs Descemet’sMembrane Endothelial Keratoplasty (DMEK)Peng Yan, Salina Teja, Kashif Baig. Ophthalmology, University ofOttawa - Eye Institute, Ottawa, ON, Canada.Purpose: To compare the surgical and visual outcomes betweenDSAEK, UT-DSAEK and DMEK as treatments for Fuch’sEndothelial Dystrophy (FED) and Pseudophakic Bullous Keratopathy(PBK).Methods: The first ten consecutive DSAEK, UT-DSAEK, andDMEK patients with either FED or PBK were reviewedretrospectively. In DSAEK, a 350um head was used for all singlepassdissections. In UT-DSAEK, donor corneas were prepared by atwo-pass microkeratome dissection. In DMEK, a trephine-peeltechnique was used to prepare the graft. Data was collected frombaseline up to 6-months follow-up, and outcomes includingintraoperative and postoperative complications, visual rehabilitation,endothelial cell density and follow-up graft thickness were compared.Results: The average age was 76, 69, and 67.5 years for DSAEK,UT-DSAEK, and DMEK respectively. All patients had previouscataract extraction and intraocular lens placement, with an equalnumber of FED and PBK presentations. Mean donor endothelial cellcount was 2597 for DSAEK, 2590 for UT-DSAEK group and 2709for DMEK. No donor tissues were lost during tissue preparation. TheDSAEK group had a mean preoperative best-corrected visual acuity(BCVA) of 20/200 with mean intraocular pressure (IOP) of17mmHg. The UT-DSAEK group had a mean preoperative BCVA of20/80 with mean IOP of 13mmHg. The DMEK group had a meanpre-operative BCVA of 20/120 with mean preoperative IOP of14mmHg. One patient in the DMEK group had a large persistentperipheral graft detachment despite 3 re-bubbling attempts andrequired a second DMEK procedure. Six-month outcomes of visualrehabilitation, endothelial cell loss, graft thickness and graft rejectionfor all patients will be available by March 2013.Conclusions: Endothelial Keratoplasty is constantly evolving, withDSAEK currently being the standard of care. Available literature hasshown the benefits of UT-DSAEK and DMEK, including lower ratesof graft rejection, faster and greater visual recovery and comparableendothelial cell loss. The difficulties with tissue preparation however,have resulted in a slower transition to these two procedures. Thiscomparison of outcomes between our first 10 consecutive patientshaving each procedure will shed light on the relative learning curveand encourage corneal surgeons to consider the benefits of providingthese advanced treatments to their patients.Commercial Relationships: Peng Yan, None; Salina Teja, None;Kashif Baig, Bausch and Lomb (F), Allergan (C), Alcon (C)Program Number: 1750Presentation Time: 11:45 AM - 12:00 PMDiamond knife assisted Deep Anterior Lamellar Keratoplasty(Dia-DALK): A new surgical technique for management ofKeratoconusRasik B. Vajpayee, Prafulla K. Maharana, Namrata Sharma. R Pcentre for Ophthalmic Sciences, All India Institute of MedicalSciences, New Delhi, India.Purpose: To evaluate outcomes of our new technique of DALK incases of KeratoconusMethods: DALK was performed in 20 Keratoconic eyes using ourtechnique of Dia-DALK. The technique involved marking the hostcornea with a 8-8.5 mm trephine and performing an intraoperativepachymetry along that mark at about 11 O'Clock. Subsequently,diamond knife set at a depth 40µ less than that of pachymetry readingwas used to make an incision of 2mm at 11 O'Clock position. Thisincision at that depth was then enlarged with the help of curvedVannas scissors circumferentially and a blunt lamellar dissector wasused to dissect and remove the overlying stromal layers radially. A0.25 mm oversized donor button whose descemet membrane hadbeen scrapped off, was sutured on the host bed. Main outcomemeasures analysed were BCVA, keratometry (Km), sphericalequivalent (SEQ) and endothelial cell density.Results: At 6 month the mean residual host thickness was 41.7±13.8µ. The mean log MAR BCVA improved significantly frompreoperative value of 1.847±0.289 to 0.2117±0.061 (p= 0.005).Theaverage Km improved from 66.5±7.5D to 45.1±1.5D (p=0.03). Themean SEQ decreased from -7.8 ±4.6D to -1.23±0.88 D (p=0.007). Asignificant decrease was seen in refractive astigmatism from 5.93±3.06D preoperatively to 3.23 ±1.14D (p=0.037). The meanendothelial cell loss was 5.241+3.6 %. No intraoperative perforationsoccurred in any of the cases.Conclusions: Our technique of Dia-DALK is safe, predictable andeffective for the mangement of Keratoconus. It has the potential tobecome an alternative to Big Bubble DALKCommercial Relationships: Rasik B. Vajpayee, None; Prafulla K.Maharana, None; Namrata Sharma, NoneProgram Number: 1751Presentation Time: 12:00 PM - 12:15 PMFemtosecond Laser-Assisted Deep Anterior LamellarKeratoplasty with Zig-zag Configuration: Initial OutcomesIjeoma Asota, Matthew Wade, John Xie, Sumit Garg, Roger F.Steinert, Marjan Farid. Gavin Herbert Eye Institute, University ofCalifornia Irvine, Irvine, CA.Purpose: To report the early refractive results and clinical outcomesof deep anterior lamellar keratoplasty (DALK) performed using thefemtosecond laser with the zig-zag incision configuration.Methods: 30 consecutive eyes underwent femtosecond laser assistedDALK with zig-zag configuration. Clinical records were reviewedretrospectively. Corrected distance visual acuity (CDVA), manifestand topographic astigmatism, and complications were reviewed.Results: In 26 eyes, a big-bubble was successfully achieved. Theremaining 4 eyes required dissection down to a very thin residualstromal bed. Postoperative follow-up ranged from 3 months (n=28) to2.5 years (n=3). At post-operative month 3, mean CDVA was 20/30(range 20/20-20/60), mean manifest astigmatism was 3.5 D (range1.25-6 D), and mean topographic astigmatism was 4.23 D (range 1.1-9.25 D). These outcomes remained stable throughout the follow upperiod. Complications included suture revision at post-operativemonth 3 for wound gape in one patient, and an episode of stromalrejection in one patient.Conclusions: Visual outcomes of femtosecond laser-assisted zig-zagDALK are similar to our results with zig-zag full thicknesspenetrating keratoplasty. In addition, this technique offers adecreased risk of endothelial rejection in healthy eyes compared topenetrating keratoplasty.Commercial Relationships: Ijeoma Asota, None; Matthew Wade,None; John Xie, None; Sumit Garg, None; Roger F. Steinert,Abbott Medical Optics (C), OptiMedica (C), ReVision Optics (C),WaveTec (C); Marjan Farid, NoneProgram Number: 1752Presentation Time: 12:15 PM - 12:30 PMCorneal Endothelial Cell Loss after Endothelial and PenetratingKeratoplasty for Endothelial DiseaseSanjay V. Patel, Keith H. Baratz, Jay W. McLaren, Lori A. Bachman,William M. Bourne. Ophthalmology, Mayo Clinic, Rochester, MN.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To compare changes in the corneal endothelium after threedifferent keratoplasty techniques for the treatment of endothelialdisease.Methods: Patients with corneal endothelial disease (predominantlyFuchs dystrophy) were enrolled in two consecutive prospectivestudies at Mayo Clinic, Rochester, MN. In a randomized controlledtrial, 28 eyes (26 patients) received penetrating keratoplasty (PK) ordeep lamellar endothelial keratoplasty (DLEK) with a 9 mm scleralincision. In a consecutive prospective observational study, 52 eyes(45 patients) received Descemet-stripping endothelial keratoplasty(DSEK) with a 5 mm scleral incision. Endothelial images wereacquired through 3 years (or 5 years for some eyes after DSEK) byusing confocal microscopy (ConfoScan 3 or 4, Nidek Technologies).Endothelial cell density (ECD) and morphology were determined bythe same masked observer by digitizing the apices of cells (HAI CellAnalysis System) in images acquired at various intervals afterkeratoplasty. Endothelial cell loss (ECL) was the percentage of cellslost from preoperative ECD of the donor tissue, as measured by theeye bank that provided the donor tissue. Endothelial variables werecompared between techniques by using generalized estimatingequation models to account for any correlation between fellow eyesof the same subject.Results: Donor diameter was 7.6 ± 0.1 mm (mean ± sd) for PK, 7.9 ±0.1 mm for DLEK, and 8.2 ± 0.3 mm for DSEK. Preoperative donorECD did not differ between treatments (PK, 2,845 ± 306 cells/mm2;DLEK, 2,749 ± 364 cells/mm2; DSEK, 2,925 ± 374 cells/mm2;p≥0.10). Mean ECL is summarized in the Table. At one month, therewas a trend toward higher ECL after DSEK than after DLEK and PK(p≥0.31, minimum detectable difference, 17% [α=0.05, β=0.20]). At24 and 36 months, ECL after DSEK was lower than it was afterDLEK (p≤0.004) and PK (p≤0.03). At 60 months, ECL after DSEKwas similar to that at 36 months after DLEK and PK. By 3 years,there were 0, 1, and 5 graft failures after PK, DLEK, and DSEK,respectively.Conclusions: Despite a trend toward higher early endothelial cellloss after DSEK, cell loss at 3 years after DSEK is lower than thatafter PK or DLEK. This difference might be related to graft diameteror to postoperative anatomic differences of the posterior cornealsurface. Continued observation is required to determine the longertermtrend in cell loss after DSEK.Purpose: Lamellar corneal grafts are now being performed forindications that previously were treated by full-thickness cornealgrafts. We examined the evidence for the success of these newlamellar procedures, with a focus on graft survival and visualoutcome.Methods: In a national register of >23,000 corneal grafts with up to25 years of annual follow-up, 2983 lamellar grafts were identified, ofwhich 42% were endokeratoplasties (posterior corneal endothelialcell grafts), 39% were traditional, peripheral lamellar keratoplasties,and 19% were deep anterior lamellar keratoplasties (DALKs).Kaplan-Meier plots were used to determine graft survival times, Coxproportional hazards regression was used for multivariate graftsurvival analysis, and visual outcomes were investigated using bestcorrectedSnellen acuity, that is, with any prescribed spectacle lens orcontact lens.Results: Kaplan-Meier graft survival at one year was 74% forendokeratoplasties, 80% for traditional lamellar procedures, and 93%for DALKs. The major indications for endokeratoplasty were Fuchs’dystrophy (47%) and bullous keratopathy (33%). Over the time frame2004-2012, penetrating corneal grafts performed for either of theseindications exhibited significantly better graft survival than didendokeratoplasties for the same indications (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To describe the ophthalmic clinical characteristics inpatients with pemphigus at an ophthalmological referral center.Methods: A retrospective chart review of patients with theimmunopathological diagnosis of pemphigus examined betweenJanuary 1, 2000, to April 1, 2010 was performed. Uncorrecteddistance visual acuity, best corrected distance visual acuity (BCVA),and ocular symptoms as well as ocular surface inflammatory andscarring changes were assessed.Results: 15 patients were identified, with a mean age of 68.27 ±14.35 years, and 80% (n = 12) were female. Extraocular involvementwas reported in 1 patient. All eyes showed cicatricial changes in theconjunctiva. Six eyes (36.7%) were classified as stage I; 12 eyes(40%), as stage II; 10 eyes (33%), as stage III; and 2 eyes (7%), asstage IV. A statistically significant association was found betweenBCVA and the severity of ocular involvement. Mean BCVAlogMAR was 1.66 (20/914), range from 0 (20/20) to 4 (NLP). Otherocular diseases were found in 8 (53.3%), systemic diseases in 10(66.7%) and the use of pemphigus inducing drugs in 10 patients(66.7%).Conclusions: This report represents the largest series of ocularinvolvement in pemphigus confirmed by immunopathology. Theclinical manifestations varied from conjunctival hyperemia to cornealscarring and perforation. There was a strong association betweenscarring changes and low BCVA. Ocular and systemic diseases aswell as the use of pemphigus inducing drugs may predispose toocular cicatricial changes observed in this series.Commercial Relationships: Patricia Chirinos-Saldaña, None;Isaac Zuñiga-Gonzalez, None; Julio C. Hernandez-Camarena,None; Alejandro Navas, None; Tito Ramirez-Luquín, None; AtzinRobles-Contreras, None; Maria C. Jimenez-Martinez, None;Arturo J. Ramirez-Miranda, Carl Zeiss Meditec (R); Enrique O.Graue-Hernández, NoneProgram Number: 2054 Poster Board Number: D0193Presentation Time: 11:00 AM - 12:45 PMThe Effect of Trigeminal Neurons on the Expression ofMaturation Markers by Bone Marrow-Derived Dendritic CellsSang-Mok Lee, William Stevenson, Kishore Reddy Katikireddy, HyunSoo Lee, Thomas H. Dohlman, Sunil K. Chauhan, Jing Hua, ZahraSadrai, Masahiro Omoto, Reza Dana. Schepens Eye ResearchInstitute, Massachusetts Eye and Ear Infirmary, Harvard MedicalSchool, Boston, MA.Purpose: The immune system interacts with the nervous system in avariety of settings. For example, several neuropeptides have beenshown to exert immunomodulatory effects on antigen-presentingcells including dendritic cells (DCs). The present study investigatedwhether or not cultivated trigeminal neurons (TGNs), which providesensory innervation to the ocular surface, can affect the expression ofmaturation markers by cultivated bone marrow-derived dendriticcells (BMDCs).Methods: Bone marrow and trigeminal ganglions were harvestedfrom C57BL/6 mice of 6-8 weeks age. After excluding red bloodcells, bone marrow cells were cultured in the presence ofgranulocyte/macrophage colony-stimulating factor (GM-CSF,20ng/ml) for 6 days to proliferate immature DCs. Loosely-adherentimmature DCs were collected and sub-cultured for 2 days in thepresence of IFN-γ (10ng/ml) without GM-CSF to induce maturation.Primary cultured TGN or culture supernatants were added to IFN-γstimulatedBMDCs to evaluate their effects on DC maturation. Theexpression levels of MHC class II (mouse IA/IE) and CD86 onCD11c+ DCs were analyzed using flow cytometry.Results: The addition of either TGN or culture supernatantsignificantly suppressed the expression of MHC class II by IFN-γstimulatedBMDC compared to IFN-γ-stimulated BMDCs withoutTGN or their supernatants (control group) (relative frequencies:79.64 ± 5.24% for control group, 45.55 ± 7.55% for TGN group (P ≤0.05), and 63.68 ± 4.86% for supernatant group (P ≤ 0.05),respectively; MFI: 31.72 ± 3.77 for control group, 19.38 ± 2.29 forTGN group (P ≤ 0.05), and 24.48 ± 0.87 for supernatant group (P ≤0.05), respectively, Mann-Whitney U test). The cell-surfaceexpression of CD86, a co-stimulatory molecule, demonstrated similarchanges to MHC class II expression as a result of TGN co-culture.Conclusions: Cultivated TGN or their secreted factors are capable ofsuppressing dendritic cell maturation.Commercial Relationships: Sang-Mok Lee, None; WilliamStevenson, None; Kishore Reddy Katikireddy, None; Hyun SooLee, None; Thomas H. Dohlman, None; Sunil K. Chauhan, None;Jing Hua, None; Zahra Sadrai, None; Masahiro Omoto, None;Reza Dana, Allergan (C), Alcon (C), Bausch & Lomb (C), ElevenBio (I), GSK (F), Novabay (C), Revision Optics (C), Novaliq (C),RIgel (F)Support: NIH Grant R01 EY20889Program Number: 2055 Poster Board Number: D0194Presentation Time: 11:00 AM - 12:45 PMEffect of Interleukin-17(IL-17) and IL-17 receptor on ocularsurface inflammationAi Yamada, Tohru Sakimoto, Akiko Ishimori, Takako Ohnishi,Satoshi Sugaya, Mitsuru Sawa. Ophthalmology, Nihon UniversitySchool of Medicine, Tokyo, Japan.Purpose: To investigate the effect of Interleukin-17(IL-17) and IL-17receptor (IL-17R) on ocular surface inflammation, we studiedwhether IL-17 stimulation can up-regulate inflammatory cytokines incorneal epithelial cells and fibroblast cells. We also analyzed the IL-17R expression in mouse alkali corneal burn model.Methods: Expression of IL-17R, IL-8, and CCL20 after stimulationby IL-17(100ng/ml) or tumor necrotizing factor(TNF)-α(50ng/ml)was investigated using human corneal epithelial cell line(HCE) andhuman corneal fibroblast cell line (HCF) by real-time reversetranscription polymerase chain reaction (RT-PCR).Unilateral eye ofcorneal alkali burn was made using filter paper presoaked in 1NNaOH using A/J mice. After the treatment, both eyes were enucleatedon day 5. Two-μm-thick corneal section was made using laserassisted microdissection method and the amount of RNA in thesamples was determined by real-time RT-PCR.Results: TNF-α stimulation increased significantly IL-17R, IL-8, andCCL20 expression in both HCE(1.7±0.2, 146.3±8.2, and15.1±1.8times) and HCF(1.8±0.2 , 98.8±8.8, and 70.0±10.3 times )compared to the non-treated control group(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMasahiro Omoto, Yiping Jin, Sunil K. Chauhan. Schepens EyeResearch Institute, Harvard Medical School, Boston, MA.Purpose: Along with their capacity for differentiating into cells ofmultiple lineages, mesenchymal stem cells (MSC) have generatedgreat interest for their ability to display unique anti-inflammatory andimmunomodulatory properties. The purpose of this study was toinvestigate whether systemically-injected host MSC can home to theinflamed transplanted cornea, suppress induction of alloimmunity,and promote allograft survival rate.Methods: MSC (CD45-CD34-SCA1+CD29+) were generated fromthe bone marrow of wild-type BALB/c or GFP+ C57BL/6 mice, and1x106 cells were intravenously injected to allografted recipients 2hafter surgery. To track the homing of GFP+ MSC (C57BL/6), cornealgrafts from BALB/c (H-2d) mice were transplanted onto C57BL/6(H-2b) recipient mice. MSC homing to the corneas was examined atday 3 post-transplantation by immunohistochemistry. To investigatethe effect of MSC on alloimmunity and graft survival, corneal graftsfrom C57BL/6 (H-2b) mice were transplanted onto BALB/c (H-2d)recipient mice, and then wild-type BALB/c MSC were injected.Frequencies of alloreactive IFNγ+ T cells were analyzed at day 14post-transplantation using the ELISPOT assay. Frequencies of matureCD11C+MHC-II+ antigen-presenting cells were analyzed by flowcytometry. Graft survival was evaluated by slit-lamp biomicroscopyweekly up to 8 weeks.Results: Intravenously injected GFP+MSC were found in abundancein the transplanted cornea, but not in the ungrafted (contralateral)cornea. The frequencies of mature CD11C+MHC-II+ antigenpresentingcells were substantially decreased in the corneas (50.2%vs. 76.7%) and draining lymph nodes (4.4% vs. 8.4%) of MSCinjectedallograft recipients compared to control group. The drainingLN of MSC-injected allograft recipients showed significantly lowerfrequencies allosensitized IFNγ-secreting T cells compared to thecontrol group (p=0.023). Allograft survival rate was significantly(~2-fold) higher (p = 0.03) in the MSC-injected recipients (80%,n=12) compared to the non-MSC injected group (40%, n =10).Conclusions: Our data demonstrate that systemically-administeredMSC specifically home to transplanted corneas and promote allograftsurvival by inhibiting APC maturation and induction of alloreactive Tcells. These data suggest that host MSC exert immunomodulatoryfunctions in corneal transplantation and may be used to prolongtransplant survival.Commercial Relationships: Masahiro Omoto, None; Yiping Jin,None; Sunil K. Chauhan, NoneProgram Number: 2057 Poster Board Number: D0196Presentation Time: 11:00 AM - 12:45 PMThe Cross-reactivity of Subsequent Corneal Allografting AfterXenocorneal Transplantation Using Decellularized PorcineLamella Against Allo-antigens in PrimatesHyuk Jin Choi 1, 2 , Jong Joo Lee 2, 3 , Mee Kum Kim 2, 3 , Won RyangWee 2, 3 , Hyun Ju Lee 3 , Ah Young Ko 3 , Jae-Il Lee 4 , Hee Jung Kang 5 .1 Ophthalmology, Healthcare System Gangnam Center, SeoulNational University Hospital, Seoul, Republic of Korea;2 Ophthalmology, Seoul National University College of Medicine,Seoul, Republic of Korea; 3 Laboratory of Corneal RegenerativeMedicine and Ocular Immunology, Seoul Artificial Eye Center,Seoul National University Hospital Biomedical Research Institute,Seoul, Republic of Korea; 4 Xenotransplantation Research Center,Seoul National University Hospital, Seoul, Republic of Korea;5 Laboratory Medicine, Hallym University College of Medicine,Anyang, Republic of Korea.Purpose: To investigate the cross-reactivity of subsequent cornealallografting after xenocorneal transplantation using decellularizedporcine lamella against allo-antigens in primates and to validate thefeasibility of decellularized porcine corneal lamella as a bridge to asubsequent corneal allograft.Methods: Five Chinese rhesus macaques, which had undergoneanterior partial thickness corneal transplantation using hypertonicsaline-treated decellularized porcine corneal lamellae in theantecedent experiments, were used as recipients for subsequent fullthicknesscorneal allografts. To determine whether sensitization ofthe recipients to xenoantigens led to cross-reactivity againstalloantigens, we compared; 1) allogeneic one way mixed lymphocytereaction (MLR) of the peripheral blood mononuclear cells (PBMCs)from the recipients with that of PBMCs from randomly selectedrhesus macaques, 2) amount of IgG antibodies which bound toPBMCs of a rhesus panel (5 monkeys) before sensitization with thatafter sensitization. Graft survival and immunologic profiles includingmemory T cell subset and donor rhesus-specific antibodies wereevaluated.Results: There was neither hyperacute nor acute rejection within amonth post-transplantation in all recipients. Notable changes in allmemory T cell subsets were not seen during first one month aftertransplantation even in two recipients which showed graft failure at49 days and 35 days post-transplantation, respectively. Alloreactivityon MLR was not different between rhesus recipients withxenocorneal grafts and naïve rhesus monkeys. In either of therecipients, IgG antibodies reactive against PBMCs from the panelwere not changed after sensitization by xenoantigens. Moreover,there was no change in donor-rhesus specific antibodies in allrecipients.Conclusions: Decellularized porcine corneal lamella seems not to becross-reactive against alloantigens and it is likely to be used as abridge before corneal allograft becomes available.Commercial Relationships: Hyuk Jin Choi, None; Jong Joo Lee,None; Mee Kum Kim, None; Won Ryang Wee, None; Hyun JuLee, None; Ah Young Ko, None; Jae-Il Lee, None; Hee JungKang, NoneSupport: Grant from the Korea Healthcare Technology R&DProject, Ministry for Health, Welfare & Family Affairs, Republic ofKorea (Project No. A040004)Program Number: 2058 Poster Board Number: D0197Presentation Time: 11:00 AM - 12:45 PMTear expression profiling of cytokine, chemokine and solublereceptor in keratoconus patientsJeewon Mok, Choun-Ki Joo. Catholic Institutes of Visual Science,Catholic Univ Korea, Seoul, Republic of Korea.Purpose: To determine whether the expression levels of cytokines,chemokines and soluble receptor in tear of keratoconus patientscontribute to the pathogenesis of keratoconusMethods: The patients with keratoconus were diagnosed based onthe following criteria: (1) symptoms of keratoconus (Munson sign,protrusion, Vogt’s striae, corneal thickness, scarring, Fleishcher ring,photokeratoscopy signs, video keratography signs, and refractiveerrors) and (2) medical histories (age, gender, contact lense wearing,eye rubbing, systemic disease, atopy, and connective tissue disease).Tears were collected from 28 keratoconus patients (56 eyes) and 30healthy subjects (60 eyes) by a polyurethane minisponges. Controlsubjects with no history of ocular disease were also enrolled. Theconcentrations of cytokines/chemokines were analyzed by Luminex200 using Human cytokine/chemokine (42 molecules), Humancytokine/chemokine Panel II (23 molecules) and Human solublecytokine receptor (14 molecules). The Median Fluorescent Intensity(MFI) was used to obtain the calculating cytokines, chemokines andsoluble receptor concentrations in tears©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: Of the 79 cytokines, chemokines and soluble receptors, wedetected 16 molecules that demonstrated a significant differences intears from Keratoconus patients. In cytokines, G-CSF (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: NIH, NEI, R44 EY017497, R43 EY021045, and RO1EY06819Program Number: 2061 Poster Board Number: D0200Presentation Time: 11:00 AM - 12:45 PMIn Vivo Administration of Interleukin-2 Increases CornealAllograft Survival through Expansion of CD4+CD25+ TRegulatory CellsMaryam Tahvildari, Parisa Emami-Naeini, Yihe Chen, MasahiroOmoto, Jing Hua, Sunil K. Chauhan, Reza Dana. Schepens EyeResearch Institute, Massachusetts Eye and Ear, Department ofOphthalmology, Harvard Medical School, Boston, MA.Purpose: T regulatory cells (Tregs) have a major role in inhibitinggrowth and differentiation of activated T cells following alloantigenstimulation, and it has been found that interleukin-2 (IL-2) is crucialin the generation and maintenance of these cells. In this study ofmurine corneal transplantation, we aimed to investigate the effect ofsystemic administration of IL-2 on corneal allograft survival.Methods: Corneal transplantation was performed on 8-10 week-oldmale BALB/c (H-2d) mice using C57BL/6 (H-2b) mice as donors.Prior to surgery, recipient mice received 3 intraperitoneal injections(once a day) of either IL-2 (1µg/20 g body weight, in 100µlphosphate buffer saline [PBS]) or PBS alone (as control). Injectionswere continued once daily for one week after surgery followed bytwice a week for another 4 weeks. Allografts were evaluated up to 5weeks post-transplantation and were scored using the standardopacity grading system (from 0 to +5). Frequencies ofCD4+CD25+Foxp3+ Tregs in the draining lymph nodes wereevaluated by flow cytometry on the day of transplantation (day 0),one week (day 7) and two weeks (day 14) afterwards. In addition, thesuppressive function of CD4+CD25+ Tregs derived from draininglymph nodes of recipient mice was evaluated one week aftertransplantation.Results: Of the six corneas transplanted in each group, 66.6% in theIL-2 treated group remained clear up to 5 weeks after surgerycompared to 33.3% in the control group; moreover, IL-2 treatmentdelayed the incidence of graft rejection with mean rejection time of32±1.4 days (mean±SD) for the IL-2 treated vs. 19.5±7.7 days for thecontrol group. Flow cytometric analysis showed significant increasesin the frequencies of CD4+CD25+Foxp3+ Tregs in the draininglymph nodes of recipient mice in the IL-2 treated group compared tothe control group at day 0 (14.54% vs. 10.25%, p=0.014), day 7(14.77% vs. 11.73%, p=0.009) and day 14 (16.15% vs. 12.41%,p=0.0007). Treg suppression assay revealed 16.05% increasedsuppression of effector T cells in the IL-2 treated group compared tothe control group one week after transplantation (p=0.01).Conclusions: According to our results, systemic administration ofIL-2 is capable of enhancing the frequencies and suppressive functionof CD4+CD25+Foxp3+ Tregs, and increases corneal allograftsurvival.Commercial Relationships: Maryam Tahvildari, None; ParisaEmami-Naeini, None; Yihe Chen, None; Masahiro Omoto, None;Jing Hua, None; Sunil K. Chauhan, None; Reza Dana, Allergan(C), Alcon (C), Bausch & Lomb (C), Eleven Bio (I), GSK (F),Novabay (C), Revision Optics (C), Novaliq (C), RIgel (F)Support: NIH Grant EY12963Program Number: 2062 Poster Board Number: D0201Presentation Time: 11:00 AM - 12:45 PMProteomic Analysis of Plasma and Mucosal Samples fromPatients with Stevens-Johnson Syndrome/Toxic EpidermalNecrolysisJulia Malalis 1 , Christine Mata 1 , Daniel Kahn 3 , Amy Lin 1 , Michael J.Mosier 2 , Charles S. Bouchard 1 , Josephine Cunanan 3 , Omer Iqbal 1, 3 ,Debra Hoppensteadt 3 , Jawed Fareed 3 . 1 Ophthalmology, LoyolaUniversity Chicago Stritch School of Medicine, Maywood, IL;2 Surgery, Loyola University Medical Center, Maywood, IL;3 Pathology, Loyola University Chicago Stritch School of Medicine,Maywood, IL.Purpose: Stevens-Johnson syndrome and toxic epidermal necrolysis(SJS/TEN) are life-threatening, immune-complex hypersensitivityreactions that affect the skin and mucous membranes, often resultingin significant ocular inflammation. The purpose of this study was todetermine and compare the proteomic and coagulation profile ofplasma and mucosal discharge samples in affected and unaffectedpatients.Methods: Following institutional review board approval, plasma andswabs from ocular, oral, and skin lesions were obtained from patientswith clinically suspected SJS/TEN (n=5). Two patients had biopsyprovenSJS/TEN. Three patients had alternative skin diagnoses notconsistent with SJS/TEN and were considered abnormal controls.Samples from normal healthy controls were also obtained. Followingcentrifugation, the plasma was frozen at -70°C and later thawed andanalyzed to determine thrombin-antithrombin complex (TAT,Dade®, Marburg, Germany), fibrinopeptide (F1.2, Dade®),plasminogen activator inhibitor-1 (PAI-1, Diagnostica Stago®,Asnieres Sur Seine, France), ZYMUPHEN platelet microparticleactivity (Hyphen® BioMed, Neuville-Sur Oise, France),HEMOCLOT protein C (Stago®), and STACHROME antithrombin(Stago®), using ELISA kits as per manufacturer’s instructions. Theswabs were immediately frozen at -70°C and later thawed. Thedischarges were isolated following addition of 0.25 ml of saline toeach swab and double centrifugation. The discharges and plasmasamples were analyzed using SELDI-TOF technique.Results: Analyses of the SJS/TEN plasma samples revealed amarked increase in the TAT complexes (6.3±5.9µg/ml), F1.2(430.4±202.4 pmol/L), platelet microparticles (13.1±9.3nM) andprotein C levels (90.5±63.4%), with a corresponding decrease in PAI-1 (53.3±18.8ng/ml) and antithrombin levels (80.7±42.4%) comparedto normal control human plasma, suggesting a procoagulant state.Protein chip array of the SJS/TEN skin and oral mucosal samplesexhibited two major peaks at 14.2 kDa and 15.6 kDa, in the samemolecular weight range as recombinant human granulysin, amolecule implicated in the pathophysiology of SJS/TEN. Thesepeaks were not present in the control group.Conclusions: Procoagulant factors and unique peaks suggestive ofgranulysin may lead to the development of targeted therapy aimed toattenuate local and systemic inflammatory processes in patients withSJS/TEN.Commercial Relationships: Julia Malalis, None; Christine Mata,None; Daniel Kahn, None; Amy Lin, None; Michael J. Mosier,None; Charles S. Bouchard, None; Josephine Cunanan, None;Omer Iqbal, None; Debra Hoppensteadt, None; Jawed Fareed,NoneSupport: Illinois Society for the Prevention of Blindness (ISPB)GrantProgram Number: 2063 Poster Board Number: D0202Presentation Time: 11:00 AM - 12:45 PMMorphologic Dendritic Immune Cells Parameters RevealDifferential Characteristics between the Central and PeripheralCornea: an In Vivo Confocal Microscopy Normative DataClara M. Colon 1 , Bernardo M. Cavalcanti 1, 2 , Shruti Aggarwal 1 ,Andrea Cruzat 1, 2 , Candice Williams 1 , Douglas Critser 4 , Amy Watts 3 ,Christine W. Sindt 4 , Pedram Hamrah 1, 2 . 1 Department of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaOphthalmology, Ocular Surface Imaging Center, Massachusetts Eye& Ear Infirmary, Harvard Medical School, Boston, MA; 2 Departmentof Ophthalmology, Cornea and Refractive Surgery Service,Massachusetts Eye & Ear Infirmary, Harvard Medical School,Boston, MA; 3 Contact Lens Service, Massachusetts Eye & EarInfirmary, Harvard Medical School, Boston, MA; 4 Department ofOphthalmology & Visual Sciences, Contact Lens Service, Universityof Iowa, Iowa City, IA.Purpose: To determine the morphology of dendritic immune cells(DC) in the central and four peripheral quadrants in corneas ofnormal subjects.Methods: Eighty-five normal non-contact lens wearers (85 eyes)were prospectively enrolled and underwent laser in vivo confocalmicroscopy (IVCM; HRT3/RCM) of the central cornea, as well asinferior, nasal, superior and temporal quadrants. Slit-lampexamination was performed to confirm lack of ocular disease.Baseline images were assessed for DC morphology and density bytwo masked observers. Morphology was assessed by number ofdendrites per cell, area of DC and DC field. Statistical analysis wasperformed with ANOVA/Bonferroni to compare the differencesbetween corneal areas and intraclass coefficient was used to assessinterobserver reproducibility.Results: The mean age of subjects was 31.4 years (range 20 to 69).IVCM revealed the presence of central (35.6±4.3 cells/mm2) andperipheral (74.1±4.5) corneal DC in all areas of all subjects. Thenumber of dendrites, area of DC, and DC field centrally were2.4±0.04 n/cell, 71.6±4.3 µm2, and 304.2±19.2 µm2 respectively.Number of dendrites (3.2±0.07 inferior, 2.9±0.03 nasal, 3.0±0.03superior, 2.9±0.03 temporal), area of DC (134.6±3.5, 115.3±3.4,117.0±3.5, 121.2±3.9), and DC field area (684.4±19.1, 586.2±19.7,587.7±17.4, 601±18.6), were found to be significantly larger in all 4peripheral quadrants (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 2066 Poster Board Number: D0205Presentation Time: 11:00 AM - 12:45 PMReduced expression of IL17 following rejection in baby ratkeratoplastyAntonia Hildebrand, Thomas Reinhard, Johannes Schwartzkopff.Ophtalmology, Universitätsklinik Freiburg, Freiburg, Germany.Purpose: An unexplained increase of graft rejections followingkeratoplasty is observed in very young patients (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSMAD2/3 nuclear translocation and substantially reducedcytoplasmic α-SMA normally upregulated by TGFβ1.Conclusions: Collectively, these results indicate that HC-HApurified from AM is responsible for AM’s anti-scarring action bymarked downregulation of TGFβ1 but upregulation of TGFβ3, whichis known to counteract TGF-β1 signaling. Furthermore, such an antiscarringeffect is more apparent under the challenge of TGF-β1 orserum, during which time HC-HA also suppresses Smad2/3signaling, and expression of α-SMA.Commercial Relationships: Fu Li, Ocular Surface Research &Education Foundation (F); Ying-Ting Zhu, Tissue Tech (F), TissueTech (E), Tissue Tech (P); Hua He, TissueTech, Inc. (E); Su-ZhenZhang, Tissue Tech, Inc (E); Scheffer C. Tseng, NIH, NEI (F),TissueTech, Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)Support: NIH, NEI, R44 EY017497, R43 EY021045, and RO1EY06819Program Number: 2069 Poster Board Number: D0208Presentation Time: 11:00 AM - 12:45 PMThe response of tear film neutrophils to occasional overnight lenswearMaud Gorbet 1, 2 , Doerte Luensmann 2 , Lyndon W. Jones 2 . 1 SystemsDesign Engineering, University of Waterloo, Waterloo, ON, Canada;2 Centre for Contact Lens Research - School of Optometry, Universityof Waterloo, Waterloo, ON, Canada.Purpose: During sleep, in the closed-environment, an influx ofpolymorphonuclear neutrophils (PMNs) occurs, which typicallyreturns to normal levels a few hours after waking. This clinical studywas conducted to investigate the response of tear film PMNs tooccasional overnight lens wear.Methods: Adapted lotrafilcon A lens wearers and non-lens wearerswere included (n=20 per group). Lenses were worn on a daily basisfor 30 days and two nights, one at the beginning and one at the end ofthe lens wear cycle (Day1 & Day30). After each of these two nights,ocular surface cells were collected immediately upon waking usingan eye-wash technique. Cells were also collected from the contactlenses. Cells were incubated with fluorescently labeled antibodiesagainst ICAM-1, Mac-1, CD66b and CD33 (degranulation membranemarkers) and CD45 (pan leukocyte-marker) with and withoutLipopolysaccharide. Flow cytometry was used to determine the levelof expression and the ratio between unstimulated and stimulatedPMNs was calculated.Results: After 8hrs of sleep, leukocyte cell count collected from theocular surface ranged from 60,000 to 2 million. There was nosignificant difference in the total number and leukocyte typecollected from lens wearers and non-lens wearers (p>0.05). Cellscollected from the lenses represented 9.4 ± 8.2 % of the total PMNcount. Cell samples collected from the contact lenses were onlyincluded for flow cytometry analysis if > 500 PMNs events wereobserved (n=7).Eye-wash collected PMNs showed a similar expression and ratiobetween the two groups for Mac-1 and ICAM-1 (p>0.05). Significantlower levels of expression of CD66b and CD33 were found in thelens wearers (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 2071 Poster Board Number: D0210Presentation Time: 11:00 AM - 12:45 PMBilateral Up-regulation of Limbal Intravascular AdhesionMolecules Mediates Cell Infiltration into the Cornea afterUnilateral Trigeminal Nerve AxotomyDeshea L. Harris 1, 2 , Takefumi Yamaguchi 2, 1 , Aslihan Turhan 2 ,Ulrich von Andrian 3 , Pedram Hamrah 2, 1 . 1 Ophthal-Harvard MedSchool, Schepens Eye Research Inst, Boston, MA; 2 Ophthal-HarvardMed School, Massachusetts Eye and Ear Infirmary, Boston, MA;3 Program in Cellular and Molecular Medicine-Children's HospitalBoston, Immune Disease Institute, Boston, MA.Purpose: We have recently demonstrated that P-selectin andMAdCAM-1 mediate the recruitment of corneal dendritic cells afterinflammation. Further, our group has demonstrated increased densityof leukocytes in unaffected contralateral eyes in unilateral cornealdiseases. The purpose of this study was to determine the neuralinvolvement of the trigeminal nerve in up-regulation of theseadhesion molecules.Methods: Six- to 8-week-old adult BALB/c mice underwenttrigeminal axotomy to transect the ophthalmic branch of thetrigeminal nerve. Normal, axotomized, contralateral and suturedinducedinflamed corneas (n=3/group) were excised on postoperativedays 3 and 7. RNA was isolated, DNAse treated, andconverted to cDNA. Real-time PCR was performed using primersagainst the integrin MAdCAM-1 and P-selectin. Beta-actin andGAPDH were used as housekeeping genes and results werecompared to normal and sutured controls. Homing of DC to thecornea was studied by immunostaining and confocal microscopy.Results: Following trigeminal axotomy, MAdCAM-1 was upregulatedboth on post-operative day 3 (1.8-fold) and day 7 (1.6-fold). P-selectin was also up-regulated at day y (1.6-fold) and day 7(1.9-fold) after axotomy. Interestingly, both MAdCAM-1 and P-selectin expression were even higher in contralateral unaffectedcorneas at day 3 (2.5-fold) and day 7 (2.8-fold) for MadCAM-1, and1.8-fold for day 3) and 2.3-fold for day 7 for P-selectin, as comparedto normal controls. The density of CD45+ cells in the center of thecornea significantly increased from 189+/-21 /mm2 to 550 +/-134 atday 3 and 979+/-45 at day 7 in the axotomized eye (P = 0.0015) andslightly increase to 247+/-25 at day 3 and 245+/-35 at day 7 in thecontralateral eye (P = 0.595).Conclusions: Our work demonstrates changes in the contralateralunaffected eyes as a response to ipsilateral trigeminal axotomy inmice. Trigeminal axotomy results in up regulation of the integrinMAdCAM-1 and P-selectin in both axotomized and in contralateralcorneas, and suggest a neural regulation of limbal intravascularadhesion molecules.Commercial Relationships: Deshea L. Harris, None; TakefumiYamaguchi, None; Aslihan Turhan, None; Ulrich von Andrian,None; Pedram Hamrah, NoneSupport: NIH K08-EY020575 (PH), Research to Prevent BlindnessCareer Development Award (PH), Falk Medical Research Trust (PH),Japanese Eye Bank (TY)Program Number: 2072 Poster Board Number: D0211Presentation Time: 11:00 AM - 12:45 PMTear Molecule Levels and Conjunctival Inflammatory GeneExpression in Atopic Keratoconjunctivitis (AKC)Amalia Enriquez-De-Salamanca 1, 2 , Erma Trias 1 , Verónica Martinez-Tottil 1 , Lidia Cocho 1 , Carmen García-Vázquez 1 , Maria J. Gonzalez-Garcia 1, 2 , Margarita Calonge 1, 2 . 1 Ocular Surface Group, IOBA-University of Valladolid, Valladolid, Spain; 2 Biomedical ResearchNetworking center in Bioengineering, Biomaterials andNanomedicine (CIBER-BBN), Valladolid, Spain.Purpose: To measure a panel of tear cytolkines/chemokines andmRNA expression of a panel of inflammatory molecules and theirreceptors in conjunctival cells in atopic keratoconjunctivitis (AKC)and to correlate their levels with AKC-related features andextraocular atopic disorders.Methods: 22 AKC patients and 18 age and sex-matched controlswere evaluated after 20 min in a controlled environment chamber(45% relative humidity, 22°C, 930 mb). Patients were medicallycontrolled and had discontinued medications for one week beforeexamination. Symptom questionnaires were answered by patients andcontrols. Unstimulated tears were collected from most symptomaticeye. Levels of 19 molecules (EGF, IL-1RA, IL-1β, IL-2, IL-4, IL-5,IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IP-10, eotaxin 1, fractalkine,IFN-γ, VEGF, TNF-α and RANTES) were measured by multiplexbead assay and correlated with clinical parameters. Conjunctivalepithelial cells were collected by impression cytology in upper bulbarconjunctiva and gene expression of 84 inflammatory molecules wasdetermined by RT2-PCR in pooled cDNA samples.Results: IL-5 tear levels were decreased (p=0.0121) in AKCcompared to controls. IL-6 and IL-1β tear levels were increased inAKC tear samples near to statistical significance (p=0.0509;p=0.0713). AKC patients with active atopic dermatitis (AD) hadincreased IFN-γ and TNF-α tear levels (p=0.0104, p=0.0176)compared to non-active AD. AKC patients with concomitant asthmahad decreased tear IL-2 (p=0.0282). Several molecule tear levelscorrelated with clinical signs. All molecules were detected, exceptIL-17A and eotaxin 1. Expression of 45 genes was detected in AKCconjunctival epithelial cells: 10 genes (IFNγ, CXCL2, CXCL3,CCL11, CCL24, CCL26, CCR2, CCR3, IL-17C, and NAMPT) were≥ 2 fold upregulated and 5 genes (CCL5, CXCR1, IL27, LTA, andLTB) were ≥ 2 fold dowregulated.Conclusions: IL-5 tear levels were decreased in AKC patientscompared to healthy individuals, probably related to the absence ofproliferative lesions, as described. Interestingly, the addition of activeAD correlated with 2 highly inflammatory molecules, IFN-γ andTNF-α, in tears. In spite of the fact that these patients had mildinflammation, the expression of several inflammatory genes wasaltered in their conjunctival cells. These results add furtherknowledge to the search for potential biomarkers in AKC.Commercial Relationships: Amalia Enriquez-De-Salamanca,Allergan Inc. (F); Erma Trias, None; Verónica Martinez-Tottil,None; Lidia Cocho, None; Carmen García-Vázquez, None; MariaJ. Gonzalez-Garcia, None; Margarita Calonge, Allergan (C)Support: SAF2010-15631, Ministry of Science and Innovation,SpainProgram Number: 2073 Poster Board Number: D0212Presentation Time: 11:00 AM - 12:45 PMMeasurement of wheat-specific tear IgE in patients with allergicconjunctivitisTatsuya Mimura. Department of Ophthalmology, Tokyo Women'sMedical University Med. Cent. East, Arakawa-ku, Japan.Purpose: The allergy to the hydrolyzed wheat in facial soap is amajor social issue in Japan. It has been reported that the mostfrequent early symptoms of allergy to hydrolyzed wheat protein insoap are allergic conjunctivitis and rhinitis, and the development ofwheat-dependent exercise-induced anaphylaxis (WDEIA) could beinduced by its long term use. We evaluated the relation between tearfluid levels of wheat-specific tear IgE and features of allergicconjunctivitis.Methods: A prospective, nonrandomized, cross-sectional study wasconducted in 103 moderate to severe cases of allergic conjunctivitis(allergic group) and 10 age- and sex-matched healthy control subjects©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea(control group). Specific IgE level for wheat was measured in tearfluid with an immunochromatographic assay (1-4) and a skin pricktest (SPT) was also performed. A severity score (0, 1, 2, or 3) wasassigned for various changes of the palpebral and bulbar conjunctiva,as well as for limbal and corneal lesions.Results: The positive rate and specific IgE score were higher in theallergic group than in the control group (71.8 % vs. 40.0% and1.9 ±0.7 vs. 1.4 ± 0.5). The prevalence of a positive SPT to wheat washigher in the allergic group than in the control group (6.8% vs. 0.0%).In the allergic group, the specific IgE score was higher in patient withpositive SPT results than in patients with negative SPT results (3.3 ±0.5 vs. 1.8 ± 0.6, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneamRNA expression were measured by quantitative real time PCR.Results: Seventy-seven percent of IFN-γKO cultures reached 70-80% confluence after 2 weeks, compared to 61% of WT C57BL/6cultures. Positive K7 and MUC5AC immunofluorescent stainingconfirmed the growth of conjunctival epithelium and goblet cells inculture. WST-1 cell viability assay showed IFN-γKO cultures had2.24 fold (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaAssociate Principal Researcher Department of Ophthalmology CheilEye Research Institute Cheil Eye HospitalCommercial Relationships: Yeoun-Hee Kim, None; Young JeungPark, None; Sun-Hee Kang, None; Jae-Chang Jung, None; KyooWon Lee, NoneProgram Number: 2079 Poster Board Number: D0218Presentation Time: 11:00 AM - 12:45 PMTopical Cyclosporine A for the Management of ChronicFollicular ConjunctivitisNatasha V. Nayak 1 , Anton M. Kolomeyer 1 , Jason S. Kim 2 , Eliott S.Kim 2 , Christina Fang 1 , David S. Chu 1, 2 . 1 Institute of Ophthalmologyand Visual Science, University of Medicine and Dentistry of NJ,Newark, NJ; 2 Metropolitan Eye Research & Surgery Institute,Palisades Park, NJ.Purpose: To describe the use of Cyclosporine A (CsA) in patientswith chronic follicular conjunctivitis (CFC).Methods: This study was a retrospective chart review at a tertiarycare center. Patients treated with topical CsA (1% emulsion) foridiopathic CFC between August 2001 and November 2012 wereincluded. Biopsies were performed. Main outcome measures includedtime to reduced inflammation, final grade of inflammation (0-4;grading performed by one physician [DSC]), ability to taper steroids,final visual acuity (VA), and reported side effects. Unless otherwisenoted, mean ± standard deviation (SD) were reported.Results: Twenty-two eyes of 13 patients (nine [69%] females; 12[92%] Caucasians; age of 49.5 ± 14.7 years) met study criteria. Two(15%) patients had an associated autoimmune disease (Sarcoidosisand Hashimoto’s thryroiditis/Sjogren’s syndrome). Mean follow-uptime was 233 days (range, 33-887 days). CsA was initiated 50.6 days± 46.9 days after diagnosis. Six (46%) patients ended CsA use after182.2 ± 95.0 days, in all cases after inflammation was controlled. Theremaining seven (54%) patients have ongoing management with CsA(currently for 278.9 ± 206.1 days). Final grade of inflammation (0.2 ±0.4) was improved compared to initial grade of inflammation (2.1 ±1.0) [two-tailed t-test, p-value < 0.0001]. Inflammation wascontrolled on average 40.1 ± 23.1 days (range, 5-84 days) afterinitiation of CsA. Eleven (85%) patients tapered and eventuallydiscontinued topical steroids 31.5 ± 27.9 days after initiation of CsAtreatment. One patient required re-initiation of topical steroids threemonths after initial discontinuation secondary to a flare up. Meaninitial log MAR VA for right and left eyes was 0.090 and 0.067,respectively, whereas mean final VA was 0.089 and 0.028. Three(23%) patients reported mild ocular irritation and/or burning;however, none discontinued the eye drops.Conclusions: Management of CFC with topical CsA resulted ininflammation control and allowed for steroid taper in the majority ofpatients without severe complications. Most patients require longtermCsA treatment.Commercial Relationships: Natasha V. Nayak, None; Anton M.Kolomeyer, None; Jason S. Kim, None; Eliott S. Kim, None;Christina Fang, None; David S. Chu, Abbott (F), Novartis (F),Santen (F), Eyegate (F), Lux Biosciences (F), Bausch & Lomb (R)Program Number: 2080 Poster Board Number: D0219Presentation Time: 11:00 AM - 12:45 PMSuppression of TLR3-Inducible Gene Expression by EP3 inConjunctival EpitheliumMayumi Ueta 1, 2 , Shuh Narumiya 3 , Shigeru Kinoshita 1 .1 Ophthalmology, Kyoto Prefectural Univ of Medicine, kyoto, Japan;2 Research Center for Inflammation and Regenerative Medicine,Faculty of Life and Medical Sciences, Doshisha University,kyotanabe, Japan; 3 Department of Pharmacology and Faculty ofMedicine, Kyoto University, kyoto, Japan.Purpose: We previously reported that prostaglandin E receptor 3(EP3) negatively regulates the eosinophilic infiltration of murineexperimental allergic conjunctivitis induced by toll-like receptor 3(TLR3), which causes reduced eosinophilic conjunctivalinflammation in TLR3/EP3 double knockout (DKO) mice, despitethe pronounced eosinophilic conjunctival inflammation in EP3 KOmice. It was also reported that EP3 was dominantly expressed inepithelial cells such as conjunctival epithelial cells, airway epithelialcells, and keratinocytes. In this study, to examine the effects of EP3against TLR3-inducible gene expression in conjunctival epithelium,we performed gene expression analysis of polyinosinic:polycytidylicacid (polyI:C)-stimulated murine conjunctival epithelium in wildtype(WT)-, EP3-, and EP3/TLR3 DKO mice.Methods: First, we examined the comprehensive effects of geneexpression in polyI:C-stimulated murine conjunctival epithelium ofWT mice using GeneChip® oligonucleotide microarrays. Then, tofind the transcript regulated by EP3, we compared the geneexpression of the transcripts, which were significantly and more than3-fold upregulated, in polyI:C-stimulated conjunctival epitheliumbetween WT and EP3 KO mice by quantitative reverse transcriptionpolymerase chain reaction (RT-PCR).Results: GeneChip® analysis showed that 131 transcripts wereupregulated more than 3-fold and that 31 transcripts were upregulatedmore than 10-fold upon polyI:C stimulation of murine for 6 hours.Quantitative RT-PCR findings confirmed that 21 of 31 transcriptswere significantly and more than 3 fold upregulated upon polyI:Cstimulation in murine conjunctival epithelium. All of those 21transcripts were found to be expressed in the polyI:C-stimulatedconjunctival epithelium of EP3 KO mice significantly stronger thanin that of WT mice. Moreover, mRNA expression of those 21transcripts were confirmed to be significantly reduced in the polyI:Cstimulatedconjunctival epithelium of EP3/TLR3 DKO mice than inthat of EP3 KO mice.Conclusions: EP3 suppressed the TLR3-inducible genes in polyI:Cstimulatedconjunctival epithelium, which causes reduced geneexpression in the conjunctival epithelium of TLR3/EP3 DKO mice,despite the pronounced gene expression in the conjunctivalepithelium of EP3 KO mice.Commercial Relationships: Mayumi Ueta, None; Shuh Narumiya,ONO Pharmaceuticals (F); Shigeru Kinoshita, Senju PharmaceuticalCo (P), Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co(C), Alcon (R), AMO (R), HOYA (R)Support: Japanese Ministry of Education, Culture, Sports, Scienceand TechnologyProgram Number: 2081 Poster Board Number: D0220Presentation Time: 11:00 AM - 12:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaModel of allergic keratoconjunctivitis - Stat6 signaling in chronicinflammationMichael Conwell 1 , Sonia DaSilva-Arnold 2 , Na Luo 1 , Wei Li 3 , JefferyTravers 2 , Yang Sun 1, 2 . 1 Ophthalmology, Glick Eye Institute,Indianapolis, IN; 2 Dermatology, Indiana University, Indianapolis, IN;3 Xiamen Eye Institute, Xiamen University, Xiamen, China.Purpose: The purpose of this study is to characterize the cornealinflammatory lesions found in the Stat6VT mouse as a model foratopic dermatitis. We hypothesize that the constitutive activation ofStat6 results in corneal neovascularization and pannus formation.Methods: SKH1-HrHr wild-type mice and mice which express anintegrally active Stat6 (Stat6VT), V547A/T548A, (N=20, 24respectively) where examined for the development ofkeratoconjunctivitis. Corneas were removed and examined by H&Eand PAS staining. Immunoblot analysis was performed withInterleukin-4, Interferon gamma, and CD3. Steroid treatment withclobetasol was performed on affected lesions.Results: Compared to wild-type, Stat6VT transgenic mice exhibitedincreased frequency of corneal neovascularization (N=24, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafacilitate a rapid lymphatic vessel memory response: a potentialimportant component of corneal defense.Commercial Relationships: Richard M. Tempero, None; Alicia L.Conner, None; Philip M. Kelley, NoneSupport: NH grants EY021571 and P20 RR018788Program Number: 2084 Poster Board Number: D0223Presentation Time: 11:00 AM - 12:45 PMThe Effects and Underlying Mechanism of Bevacizumab(Avastin) in Inhibiting Corneal Neovascularization in a RabbitClosed Eye Contact Lens Wear ModelWei-Li Chen 1 , Yan-Ming Chen 1, 2 , Fung-Rong Hu 1 . 1 Ophthalmology,National Taiwan University Hospital, Taipei, Taiwan;2 Ophthalmology, E-Da University Hospital, Kaohsiung, Taiwan.Purpose: To evaluate the therapeutic effects of early and latesubconjunctival injection of bevacizumab on the inhibition of cornealneovascularization (NV) in a rabbit closed eye contact lens (CL) wearmodel. The underlyng mechanism focusing on VEGF and VEGFreceptors were also evaluated.Methods: Corneal NV was induced by closed eye CL wear inrabbits. Group 0 included rabbits eyes without bevacizumabtreatment. Weekly subconjunctival injections of bevacizumab (5.0mg) for 1 month were started immediately (Group I) and 1 monthafter induction of corneal NV with continuous induction (Group II.Digital photographs were taken weekly. The percentage of involvedcorneal surface (PICS)/centricity/extent of corneal NV wereevaluated. Immunohistochemical staining was used to determine theintracorneal diffusion of bevacizumab in the different groups.Immunostaining with anti-CD31, α- smooth muscle actin (αSMA)and high-molecular-weight melanoma-associated antigen (HMW-MAA) was used to evaluate the formation of pericytes and smoothmuscle around the corneal NV. The expression of vascularendothelial growth factor (VEGF) and its receptors (VEGFR1,VEGFR2) was also evaluated. TUNEL staining was performed toevaluate cellular apoptosis after bevacizumab injection. In eachgroup, 6 rabbtit eyes were examined. There were 6 eyes in eachexperimental conditionsResults: Early treatment with bevacizumab (Group I) moresignificantly inhibited corneal NV than late treatment administered(Group II) after 4 weeks (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneainduced by suture placement in BALB/c mice (n=8 per group). Micethen received serum or Bevacizumab eye drops (3x daily,respectively) or a combination of both (3x daily for each treatment).After one week, corneal wholemounts were stained for blood (CD31)and lymphatic (LYVE-1) vessels.Results: In vitro, serum addition significantly increased whereasBevacizumab significantly reduced endothelial cell proliferation(BEC: serum mean m= +55.3%, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: B6LY5 mice aortic ring explants were cultured for 5 dwith the presence of 10ng/mL VEGF before TLR ligands 1-9 wereadded into culture media. Sprouting outgrowth was measured andcompared between TLR ligands treated groups and water treatedcontrol group after 5 further days of culture. Corneal angiogenesiswas induced by 3 Nylon corneal sutures on BALB/c mice beforewater, TLR3, TLR7 or TLR9 ligand was applied topically ondebrided cornea at day 0, day 2, and day 4. The corneal vesselingrowth was scored under surgical microscope after 7 days. Thevolumes of blood and lymphatic corneal vessels were quantified afterwhole mount immunofluorescence staining with CD31 and LYVE-1antibodies.Results: TLR7 and TLR9 activation suppressed vascular outgrowthfrom aortic ring explants compared to control. TLR9 engagementinhibited corneal blood vessel ingrowth after 7 d post cornealsuturing, while there was no difference between control and TLR3and TLR7 treatment groups. TLR9 activation also decreased thevolumes of blood and lymphatic corneal vessels compared to control.Conclusions: TLR9 activation could suppress aortic vessel sproutingin vitro as well as corneal hem- and lymph-angiogenesis in vivo.Commercial Relationships: Lei Liu, None; Jiahui Wu, None;Andrew D. Dick, Novartis (C), Novartis (F), GSK (F), Abbott (F)Support: Rosetrees TrustProgram Number: 2090 Poster Board Number: D0229Presentation Time: 11:00 AM - 12:45 PMSubconjunctival Cyclosporine A implants do not affect cornealneovascularisation after transplantation: results of a randomizedclinical trialClaus Cursiefen 1 , Thomas Reinhard 2 , Felix Bock 1 , Hans-UlrichProkosch 3 . 1 Dept of Ophthalmology, University of Cologne, Koln,Germany; 2 Dept. of Ophthalmology, University of Freiburg,Freiburg, Germany; 3 Medical Informatics, University of Erlangen,Erlangen, Germany.Purpose: To test whether subconjunctivally implanted CyclosporineA affects the incidence and degree of postkeratoplasty cornealneovascularisation.Methods: Prospective, randomized, controlled phase II/III clinicaltrial comprising trial sites in Germany, India and the USA. Overall,97 enrolled patients were randomized to receive either two dosages ofsubconjunctival Cyclosporine A implants (A: 36 and B: 40) orplacebo (n=21) at time of penetrating keratoplasty. The incidence anddegree of postkeratoplasty corneal neovascularisation were evaluated(LX201-01 study). A web-based image upload system wasdeveloped. Quantitative and objetive evaluation of standardizeddigital slit lamp pictures was performed using AnlysisB morphometrysofware.Results: No significant difference in the incidence and the degree ofcorneal neovascularisation was found between treatment groups andplacebo group. Mean neovascularisation was 3.2% ± 0.3 in treatmentA versus placebo (3.5% ± 0.27; p= 0.5) and 3.0% ± 0.4 and intreatment B versus placebo (3.5% ± 0.27; p= 0.3).Conclusions: Subconjunctival cyclosporine A does not affectpostkeratoplasty corneal neovascularisation.Commercial Relationships: Claus Cursiefen, Gene Signal (C),Alcon (R), Allergan (R), Bayer (R); Thomas Reinhard, None; FelixBock, None; Hans-Ulrich Prokosch, NoneClinical Trial: NCT00447187Program Number: 2091 Poster Board Number: D0230Presentation Time: 11:00 AM - 12:45 PMRole of Non-Endothelial Cells in Corneal AngiogenesisJin Zhao, Takayuki Nagasaki. Ophthalmology, Columbia University,New York, NY.Purpose: It has been suggested that growth of blood and lymphaticvessels in the cornea is assisted by non-endothelial cells in thestroma, in particular, during later stages when a vascular network isestablished by anastomosis. Supporting roles of stromal myeloid cellsand an existing nerve network have been suggested, but molecularand cellular mechanisms remain unresolved. The aim of this study isto examine evidence for the participation of stromal, non-endothelialcells during the vascular network formation.Methods: For tests of myeloid cells, four corneal angiogenesismodels in the mouse eye were used. 1) suture placement, 2) topicalapplication of ML9 (MLCK inhibitor), 3) Dstn corn1 mouse(spontaneous corneal vascularization model), 4) cornealconjunctivalization following total epithelial removal. Blood vesselgrowth was monitored in a live eye with fluorescence angiography.Cell distribution was determined by whole-mountimmunofluorescence with CD11b, CD45, CD90.2, F4/80, MHC-classII, CD31 (blood vessels) and LYVE1 (lymphatic vessels). Celldistribution was quantified in an area of vascular growth front wherea vascular network is formed by anastomosis. For a nerve guidancetest, suture was placed in the cornea of Thy1-CFP mice, and bothblood vessels (fluorescence angiography) and nerves (CFP) weretracked by time-lapse imaging in live animals.Results: Untouched, avascular corneas contained a small number ofF4/80, CD11b and MHC-class II positive cells, primarily in thelimbal area and sparsely throughout the entire cornea. CD90.2positive cell distribution had no limbal preference but was closelyassociated with sub-basal nerve plexus. After initiation ofexperimental angiogenesis, the number of cells with one or more ofmyeloid markers greatly increased within an area of vascular growth.There were frequent instances of a F4/80 positive cell being presentbetween two sprouting tips of growing vessels, appearing to bridgethem. Time-lapse imaging revealed no clear correlation betweenvessel growth and a CFP-positive nerve fiber network.Conclusions: Myeloid cells may play a supporting role in networkformation of pathological corneal vasculature either in a paracrinemanner or by means of fusion with endothelial tip cells, as reportedin developing brain. A guiding role of an existing nerve network, onthe other hand, is not supported by the data.Commercial Relationships: Jin Zhao, None; Takayuki Nagasaki,NoneSupport: Eye Surgery Fund, Research to Prevent BlindnessProgram Number: 2092 Poster Board Number: D0231Presentation Time: 11:00 AM - 12:45 PMImpaired angiogenic response in cornea by lacking TRPV1 inmiceKatsuo Tomoyose, Yuka Okada, Takayoshi Sumioka, Kumi Shirai,Shizuya Saika. Wakayama Medical University, Wakayama, Japan.Purpose: To investigate the effects of loss of Transient receptorpotential(TRP)V1 in the development of neovascularization (NV) in a cornealstroma inmice. TRP channels are polymodal receptors that are activated by ahost ofstimuli to mediate sensory transduction.Methods: Corneal NV from the limbal vessels were induced bycauterization ofthe central cornea of an eye by using disposable tool of Optemp. WTandTRPV1-null (KO) mice (n = 16 in each genotype) were used andkilled at day 3©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand 7. The eye was enucleated and processed or cryosectioning forhistologyand immunohistochemistry. To examine the expression of angiogenicgrowthfactors in vivo, centrally cauterized cornea (n = 40 in each of WT orKOgroup) was excised at day 3. Total RNA was extracted from samplesandprocessed for real-time RT-PCR for VEGF, TGFb1.Results: The development of CD31-labeled new vessels from thelimbus towardthe center of the cornea was impaired in KO mice as compared withWT mice atday 3 days, but not at day 7. Expression of mRNAs of VEGF andTGFb1 wassignificantly less in a KO cornea as compared with a WT cornea atday 3.Conclusions: TRPV1 signal is involved in the development ofcauterization-induced NV in corneal strom via modulation ofangiogenic geneexpression in the affected tissue.Commercial Relationships: Katsuo Tomoyose, None; YukaOkada, None; Takayoshi Sumioka, None; Kumi Shirai, None;Shizuya Saika, NoneProgram Number: 2093 Poster Board Number: D0232Presentation Time: 11:00 AM - 12:45 PMPhotodynamic Ablation of Lymphatic Vessels in the CorneaFranziska Bucher 1 , Yanlong Bi 2 , Claus Cursiefen 1 , Felix Bock 1 .1 Department of Ophthalmology, University of Cologne, Cologne,Germany; 2 Department of Ophthalmology, Tongji University Schoolof Medicine, Shanghai, China.Purpose: Corneal lymphangiogenesis is one of the main risk-factorsfor immune mediated allograft rejection after corneal transplantation.Whereas topical VEGF inhibitors can decrease pathologicalneovascularization, regression of existing pathological blood andlymph vessels in the cornea remains an unsolved problem.Verteporfin photodynamic therapy (PDT) is an established treatmentfor subfoveal choroidal neovascularization (CNV) in patients withage-related macular degeneration (AMD). This experiment shows forthe first time the effect of photodynamic ablation of lymphaticvessels in the cornea.Methods: Ten female BALB/c mice (aged 6-8 weeks) were used inthe mouse model for suture induced inflammatory cornealneovascularization. After two weeks the treatment group (n=5)received an injection of 2.5µl Verteporfin (2mg/ml) into the cornealstroma. Control mice (n=5) received an intrastromal injection of2.5µl PBS. After a two-hour dispersion, we performed PDT in all tenmice (t = 40s, P = 46mW). Corneas were excised 20 hours later andcorneal wholemounts were stained with CD31 and LYVE-1 to detectblood and lymphatic vessels. Hemangiogenesis andlymphangiogenesis were analyzed morphometrically by using asemiautomatic method based on the image analyzing program Cell^F.Results: Blood vessels were reduced to 78% and lymphatic vesselsdropped to 38% after PDT in the group treated with Verteporfincompared to control mice.Conclusions: Corneal PDT can be used to relatively selectively andspecifically reduce lymphangiogenesis in the cornea. Future studiesusing this new technique can investigate the role of lymph vessels ingraft rejection more in detail. In the clinic, this is a promising newpreoperative technique to “precondition” high-risk corneas prior totransplantation to reduce allograft rejection in high-risk patients.Commercial Relationships: Franziska Bucher, None; Yanlong Bi,None; Claus Cursiefen, Gene Signal (C), Alcon (R), Allergan (R),Bayer (R); Felix Bock, NoneProgram Number: 2094 Poster Board Number: D0233Presentation Time: 11:00 AM - 12:45 PMFormation and Regulation of Lymphatic Valves duringInflammationTan N. Truong 1 , Eda I. Altiok 1 , Eric Huang 1 , Tatiana Ecoiffier 1 , DonYuen 1 , Toshimitsu Uede 2 , Lu Chen 1 . 1 School of Optometry & VisionScience, University of California, Berkeley, Berkeley, CA; 2 Divisionof Molecular Immunology, Institute for Genetic Medicine, HokkaidoUniversity, Sapporo, Japan.Purpose: Lymphatic dysfunction is associated with a wide array ofdisorders from transplant rejection to cancer metastasis. To date,there is little effective treatment for lymphatic diseases. The corneaprovides an ideal tissue for lymphatic research due to its uniquefeatures. We have recently provided the first evidence showing thatlymphatic valves are formed during corneal inflammatorylymphangiogenesis (LG) via the up-regulation of integrin α9 (Truonget al. 2011). These structures play a critical role in directing properlymph flow within the vessels. The purpose of this study is to furthercharacterize the time course of valve formation in relation to vesselformation during corneal LG. In addition, integrin α9 antibodytherapy was evaluated to determine whether integrin α9 blockadewould interfere with the process of lymphatic valve formation duringinflammatory corneal LG.Methods: Our standardized suture-induced corneal inflammatory LGmurine model was used. Whole-mount corneas were harvested every2 weeks for up to 8 weeks post-suture for immunofluorescentmicroscopic analysis. Additionally, mice were randomized to receivesubconjunctival injections of either integrin α9 antibody or isotypecontrol twice a week for 2 weeks. Whole-mount corneas wereharvested for immunofluorescent microscopic analysis. Digitalimages were analyzed by NIH Image J software to quantifylymphatic vessels as well as valve distribution.Results: Corneal lymphatic vessel invasion area at 4, 6, and 8 weekswere significantly less than the peak at 2 weeks post-suture. Incontrast, the ratio of corneal lymphatic valves to vessel invasion areawas at its lowest at 2 weeks with a peak around 6 weeks post-suture.Moreover, therapeutic intervention with integrin α9 antibodysignificantly reduced the number of lymphatic valves after sutureinducedcorneal LG (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaLu Chen, Sammy Grimaldo, Tatiana Ecoiffier, Don Yuen. Center forEye Disease & Development, Program in Vision Science and Schoolof Optometry, University of California, Berkeley, CA.Purpose: MicroRNAs are a class of small non-coding RNAs thatnegatively regulate gene expression by binding to complimentarysequences of target messenger RNAs. Their roles in the cornea stillremain largely unknown. Here we present our new findings on theanti-lymphangiogenesis function of microRNA-184 in the corneaduring an inflammatory response in vivo.Methods: The murine in vivo suture placement model was used toinduce corneal inflammatory lymphangiogenesis (LG). Mice wererandomly selected to receive subconjunctival injections of eithersynthetic microRNA-184 mimics or control twice a week after theprocedure. Whole-mount corneas were sampled and stained withLYVE-1, the lymphatic marker, for immunofluorescent microscopicassays. Results were analyzed by NIH-ImageJ software.Results: Compared to the control condition, the lymphatic invasionarea was significantly reduced after the treatment of MicroRNA-184mimics in the inflamed cornea.Conclusions: MicroRNA-184 suppresses corneallymphangiogenesis. Its further investigation may provide noveltherapies for lymphatic related disorders in the cornea, such asinflammation and transplant rejection.Commercial Relationships: Lu Chen, None; Sammy Grimaldo,None; Tatiana Ecoiffier, None; Don Yuen, NoneSupport: This work is supported in part by research grants from NIHand University of California at Berkeley (LC).Program Number: 2096 Poster Board Number: D0235Presentation Time: 11:00 AM - 12:45 PMLive Imaging of Lymphatic Valve Formation after CornealTransplantationGyeong Jin Kang 1 , Tatiana Ecoiffier 1 , Young-Kwon Hong 2 , LuChen 1 . 1 Center for Eye Disease and Development, Program in VisionScience and School of Optometry, University of California, Berkeley,CA; 2 Department of Surgery, Norris Comprehensive Cancer Center,University of Southern California, Los Angeles, CA.Purpose: The lymphatic pathway is a major mediator for transplantrejection. Most recently, we have provided the first evidence onlymphatic valve formation during corneal inflammatorylymphangiogenesis (Truong et al. 2011). The purpose of this realtimein vivo study is to investigate the time course and pattern oflymphangiogenesis as well as lymphatic valve formation induced bycorneal transplantation with our newly developed live imagingsystem and Prox-1-GFP mice.Methods: Standard orthotopic corneal transplantation was performedwith Prox1-GFP mice as recipients. Corneal grafts of the same micewere continuously observed by the live imaging system forlongitudinal analysis. Prox-1 positive lymphatic vessels and valveswere evaluated at both limbal and corneal areas.Results: Prox-1 positive lymphatic vessels and valves were formedafter corneal transplantation. As corneal lymphangiogenesisproceeded, more valves were observed inside vessel cavities.Furthermore, lymphatic valvulogenesis was initiated inside limbalvessels before spreading into the central cornea.Conclusions: We have shown, for the first time, cornealtransplantation induces both lymphangiogenesis and valvulogenesisin vivo and in real time. Further investigation on this newphenomenon may reveal new mechanisms underlying transplantrejection. Since the lymphatic system plays an important role in manyother functions, this study may also offer new insights to ourunderstanding and treatment of other lymphatic related disordersoutside the eye.Commercial Relationships: Gyeong Jin Kang, None; TatianaEcoiffier, None; Young-Kwon Hong, None; Lu Chen, NoneSupport: This work is supported in part by research grants from NIHand University of California at Berkeley (LC).Program Number: 2097 Poster Board Number: D0236Presentation Time: 11:00 AM - 12:45 PMNovel Characterization of MHC-Class II Positive Cells inEmbryonic Cornea during Spontaneous Lymphatic EventsDon Yuen, Guangyu Li, Lu Chen. Center for Eye Disease andDevelopment, Program in Vision Science and School of Optometry,University of California, Berkeley, CA.Purpose: We recently reported that unlike the adult cornea which isdevoid of lymphatic vessels, the immature cornea is supplied bylymphatics which undergo spontaneous regression duringdevelopment. Since lymphatic vessels mediate antigen presenting celltrafficking during an immune response, this study is to investigatewhether the distribution of dendritic cells in immature cornea differsfrom the adult stage and how it correlates with the lymphatic events.Methods: Corneal samples of different developmental stages wereisolated from C57B6 mice and stained with a panel of specificmarkers for MHC-Class II, CD45, CD11c, and LYVE-1 forimmunofluorescent microscopic assays.Results: In contrast to the adult cornea where MHC Class II positivecells were found in the peripheral area, embryonic cornea at E16.5also exhibited these cells in the central area. The cells expressedCD45 and CD11c as well. As the cornea became mature andunderwent spontaneous lymphatic formation and regression, theMHC Class II positive cells were more restricted to the peripheralarea.Conclusions: Our findings have shown, for the first time, MHCClass II positive cells are present in the central cornea duringdevelopment. Further investigation on this new phenomenon mayprovide novel insights into corneal embryogenesis as well as thecreation and regulation of immune privilege in the cornea.Commercial Relationships: Don Yuen, None; Guangyu Li, None;Lu Chen, NoneSupport: This work is supported in part by research grants from NIHand University of California at Berkeley (LC)Program Number: 2098 Poster Board Number: D0237Presentation Time: 11:00 AM - 12:45 PMDeletion of Foxc1 and/or Foxc2 from neural crest cells leads tocorneal vascularization and anterior segment dysgenesisTsutomu Kume, Kathryn M. Schultz, Amy Sasman, Seungwoon Seo.Medicine, Northwestern Univ Sch of Med, Chicago, IL.Purpose: Foxc1 and Foxc2, members of the forkhead/Foxtranscription factor family, are critical regulators of vasculardevelopment. Foxc1 and Foxc2 are both expressed in ocularmesenchymes of neural crest origin. However, little is known aboutthe role of Foxc1 and Foxc2 in ocular development, mainly due to themid-gestation lethality of global knockout mice. Previously, we havedemonstrated that deletion of Foxc1 in neural crest-derived cells(NCC) leads to aberrant vessel growth in the normally avascularcorneas of mice, and that the effect is cell-type specific, because thecorneas of mice lacking Foxc1 expression in vascular endothelialcells remained avascular. Therefore, we investigate the role of Foxc1and Foxc2 in NCC during ocular development.Methods: To specifically delete Foxc1 and/or Foxc2 from NCC,conditional knockout mice for Foxc2 (NC-Foxc2-/-) and for Foxc1and Foxc2 (NC-Foxc1-/-; Foxc2-/-) were generated by crossingfloxed-Foxc1 and floxed-Foxc2 mice with Wnt1-cre mice.Results: Adult NCC-specific Foxc2 mutant mice exhibit corneal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaneovascularization and lymphangiogenesis. Mutant eyes exhibitabnormal thickness in the peripheral-to-central corneal stroma andlimbus (Figure 1). Interestingly, some mutant corneas havehyperplastic stroma with increased cell density. Mutant pupils aredisplaced from the normal central position. The anterior chamber isnormally formed in NC-Foxc2-/- mice, but not in NC-Foxc1-/-;Foxc2-/- mice. The severity of the ocular phenotype is dependent onthe cumulative “dose” of functional Foxc1 and Foxc2 genes andabnormalities are more extensive in NC-Foxc1-/-; Foxc2-/-. In fact,in NC-Foxc1-/-; Foxc2-/- mice the cornea is not present at all (Figure1). In the alkali-burn model, corneas of adult NC-Foxc2+/- mice andNC-Foxc1+/-; Foxc2+/- mice have increased cornealneovascularization and lymphangiogenesis compared with those ofcontrol mice (Figure 2).Conclusions: Taken together, these data suggest that the overlappingfunction of Foxc1 and Foxc2 in the neural crest plays an importantrole in maintaining corneal avascularity and development of theanterior eye segment.Program Number: 2099 Poster Board Number: D0238Presentation Time: 11:00 AM - 12:45 PMTopical Infliximab as an inhibitor of corneal hemangiogenesisand lymphangiogenesisGiulio Ferrari, Fabio Bignami, Chiara Giacomini, Paolo Rama.Ophthalmology -Cornea Unit-Eye Repair, San Raffaele ScientificInstitute, Milan, Italy.Purpose: To test whether topical application of the anti-TNF alphaantibody Infliximab can reduce lymphangiogenesis and/orhemangiogenesis in an alkali burn mouse model of cornealneovascularization.Methods: Eighteen C57BL/6 mice were induced alkali burn modelof the right cornea. Animals were then divided in two groups: group1 received topical Infliximab 10mg/ml six times a day (10ul), group 2received regular saline. After two weeks, animals were sacrificed,corneas stained for CD31 and LYVE1 to identify hemangiogenesisand lymphangiogenesis. Finally, the corneas were whole mountedand imaged. Neovascularization was calculated as neovascular area,i.e. the area of the cornea covered by vessels normalized for the totalarea of the cornea.Results: Following topical application of Infliximabhemangiogenesis decreased from 0.81±0.12 to 0.6±0.24 (26%reduction, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: Treatment of corneas with pellets containing thecombination of FGF2/LYVE-1 resulted in the outgrowth oflymphatic vessels with a mean neovascularization area of 0.12 ± 0.07mm 2 (n=20) whereas corneas with pellets containing FGF2 aloneshowed a mean neovascularization area of 0.23 ± 0.11 mm 2 (n=21) (p= 0.0008). Contrary no significant difference was found betweenVEGF-C induced lymphangiogenesis and the combination of VEGF-C/LYVE-1 induced lymphangiogenesis in the micropocket assay.Conclusions: LYVE-1 inhibited the FGF2-induced outgrowth oflymphatic vessels in a corneal micropocket assay suggesting thatLYVE-1 may bind FGF2 and interact with bFGF influencinglymphangiogenesis in vivo.Commercial Relationships: Birgit Regenfuss, None; NataliaPlatonova, None; Géraldine Miquel, None; Said Taouji, None;Eric Chevet, None; Andreas Bikfalvi, None; Claus Cursiefen,Gene Signal (C), Alcon (R), Allergan (R), Bayer (R)Support: German Research Foundation (DFG) SFB 643/B-10 andCu 47/4-1) to C.C. Agence National de la Recherche (ANR), InstitutNational du Cancer (INCA), Conseil Régional d’Aquitaine to A.B.and INCA, Association de Recherche contre le Cancer, Inserm(Avenir) to E.C.Program Number: 2101 Poster Board Number: D0240Presentation Time: 11:00 AM - 12:45 PMAnalysis of VEGFR-1 Isoforms in Pax6-deficient Mice CorneasPhillip A. Moore 1 , Peter J. Accola 1 , Barbara Artelt 1 , Megan Aarnio 1 ,James D. Lauderdale 2 . 1 Dept Small Animal Med/Surg, College VetMed Univ Georgia, Athens, GA; 2 Department of Cellular Biology,Franklin College of Arts and Sciences, The University of Georgia,Athens, GA.Purpose: To identify VEGFR-1 isoforms in the cornea of Pax6-deficient mice.Methods: The limbal region, central cornea and conjunctiva of Pax6+/- mice were compared to wild type mice for the presence ofVEGFR-1 and sVEGFR-1. Immunostaining was performed to wildtype (n=5) and Pax6 +/- mice (n= 5) with three antibodies: oneantibody to the N-terminus that detected both membrane and solubleVEGFR-1; one antibody to the C-terminus that detected themembrane bound form of VEGFR-1; and one antibody thatspecifically detected the soluble form of VEGFR-1. Immunoblottingwas performed on protein extracts prepared separately from thecornea and conjunctiva of wild type and Pax6 +/- mice.Results: Immunostaining revealed that both VEGFR-1 and sVEGFR-1 are present in the cornea of both wild type and Pax6 +/- mice.Immunoblotting revealed that both VEGFR-1 and sVEGFR-1 arepresent in the conjunctiva and central cornea of wild type and Pax6+/- mice. Interestingly, this analysis also revealed VEGFR-1 isoformsthat appeared to be specific to Pax6 +/- corneas.Conclusions: Our results are in contradiction to previous reports thatsVEGFR-1 is deficient in Pax6 +/- corneas. The presence ofsVEGFR-1 and VEGFR-1 isoforms specific to Pax6 +/- corneassuggests that a deficiency in sVEGFR-1 is not the cause for cornealneovascularization in Pax6 +/- mice.Commercial Relationships: Phillip A. Moore, None; Peter J.Accola, None; Barbara Artelt, None; Megan Aarnio, None; JamesD. Lauderdale, NoneSupport: UGA Veterinary Ophthalmology Research Fund, SharonStewart Aniridia Research Trust, Vision for Tomorrow FoundationProgram Number: 2102 Poster Board Number: D0241Presentation Time: 11:00 AM - 12:45 PMUse of Amaranthus leucocarpus lectin to determine corneal neovascularizationValeria L. Coria 1, 2 , Gibran A. Estua 1 , Alfredo Domínguez 1 , EdgarZenteno 2 , Jessica Nieves-Hernández 1 , Yonathan Garfias 1, 2 .1 Research Unit, Institute of Ophthalmology, Mexico city, Mexico;2 Biochemistry Department, Faculty of Medicine, UniversidadNacional Autónoma de México, Mexico city, Mexico.Purpose: The cornea is the interphase between the eye and theexternal environment, and is also the most powerful refractive surfacein the eye. In non-pathological conditions, the cornea is one of thefew avascular tissues from the body. The mechanism responsible forestablishing the avascular nature of the cornea is unknown; but, inseveral pathologycal situations like trauma, infection or surgicalprocedures, the stroma can be invaded by abnormal vessels(neovascularization) leading it to opacification. Theneovascularization is the creation of new vessels from preexistingones; in these abnormal vessels, the glycosylation could be aberrant.The glycosylation process, is the post translational modification inwhich saccharide residues get added to amino acid chains. Some ofthe tools used for studying neo-vessels in eye are lectins, such asGriffonia simplicifolia. Amaranthus leucocarpus (ALL) is a lectinthat recognizes specifically Galbeta1,3GalNAc carbohydratesstructures.The aim of this study was at determining Galbeta1,3GalNAcstructures in a corneal neovascularization murine modelMethods: This study was carried out in accordance with theInstitutional Animal Care and Use Committe and Vision Researchwith the ARVO statement for the Use of Animals in Ophthalmic andVision Research. All experiments were performed on 6-8 week-oldfemale BALB/c mice. Corneas were chemically burned with NaOH.Burned and healthy mice corneas were obtained, and were processedfor immunohistochemistry assays. ALL coupled to FITC was used toperform immunolabeling on corneal tissue; CD31 was used to colocatethe neovessel labeling of ALL-recognized structuresResults: The presence of ALL was exclusively observed in thechemically-burned mice corneal tissues, in contrast to the burnedtissues, there was not ALL-immunostaining in the healthy micecorneal tissues. Interestingly, ALL colocalized with CD31immunostaining in the burned and neovascularized corneal tissue.Conclusions: Amaranthus leucocarpus lectin is able to identifycorneal neo-vessels, suggesting that o-glycosylation process could bean important process in developing this aberrant corneal condition.Commercial Relationships: Valeria L. Coria, None; Gibran A.Estua, None; Alfredo Domínguez, None; Edgar Zenteno, None;Jessica Nieves-Hernández, Institute of Ophthalmology (P);Yonathan Garfias, Institute of Ophthalmology (P)Support: CONACYT 160286 and 167438Program Number: 2103 Poster Board Number: D0242Presentation Time: 11:00 AM - 12:45 PMNetrin-4 mediates corneal neovascularizationAnna-Karina Maier, Sabrina V. Klein, Norbert Kociok, AlineRiechardt, Enken Gundlach, Claudia Steger, Olaf Strauss, AntoniaM. Joussen. Charité, University Medicine Berlin, Berlin, Germany.Purpose: Netrin-4, a secreted protein, regulates neuronal andvascular growth. It is found in the basement membranes of bloodvessels. Previous results are contradictory supporting Netrin-4 aseither a pro- or anti-angiogenic factor. In the presented study weinvestigated the role of Netrin-4 in the mouse-model of sutureinduced,inflammatory corneal neovascularization.Methods: Corneal neovascularization was induced in Netrin-4-deficient (Ntn4-/-) and wild-type mice by suturing three 11-0 nylonintrastromally into the cornea. Vascularized area of cornealflatmounts were analyzed 14 days after suturing byimmunocytochemistry in conjunction withimage and statistical©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaevaluation. The mRNA levels for VEGF-A and Netrin-4 in mousecorneas were analyzed by qPCR.Results: In the wild-type mouse the netrin-4 expression wasincreased when corneal vascularization was increased by nylonsaturation. Accordingly, Ntn4 -/- showed an increased cornealvascularized area compared to that in wild-type mice after suturing.Additionally, mRNA level of VEGF-A was increasedin Ntn4-/-compared to that in wild-type mice.Conclusions: Netrin-4 probably acts as an anti-angiogenic factorthrough down-regulation of VEGF-A expression in the sutureinduced corneal neovascularization.Commercial Relationships: Anna-Karina Maier, None; SabrinaV. Klein, None; Norbert Kociok, None; Aline Riechardt, None;Enken Gundlach, None; Claudia Steger, None; Olaf Strauss,None; Antonia M. Joussen, NoneSupport: "Friedrich C. Luft" Clinical Scientist Pilot Program fundedby Volkswagen Foundation and Charité FoundationProgram Number: 2104 Poster Board Number: D0243Presentation Time: 11:00 AM - 12:45 PMSubconjunctival and intrastromal Bevacizumab to control ofcorneal neovascularization and opacificationSilvia Mendez, Maria Santiago, Elena Raposo, Rosario Touriño,Maria Teresa Rodriguez-Ares. Department of Ophthalmology,University of Santiago de Compostela (USC) Hospital Complex,Santiago de Compostela, Spain.Purpose: Bevacizumab is a recombinant humanized monoclonalantibody directed against vascular endothelial growth factor (VEGF).The purpose of this study was to evaluate the therapeutic effect ofsubconjunctival and intrastromal injection of Bevacizumab oncorneal neovascularization (CNV) and the secondary opacification.Methods: We have performed a prospective nonrandomizedinterventional study, including 15 eyes with CNV secondary to:keratoplasty due to corneal ectasia (n = 2), keratoplasty due toherpetic keratitis (n = 3), keratoplasty due to bullous keratopathy (n =2), herpetic keratopathy previously treated with amniotic membranetransplantation (n = 2), chemical burns (n = 3) and CNV afterpterygium surgery (n = 3). We have injected 2.5 mg / 0.1 ml ofBevacizumab, subconjunctival and intrastromal, in the area withCNV. We have analyzed the CNV regression by anterior segmentphothography in relation to the total corneal area. Furthermore, wehave evaluated the decrease of corneal opacity regarding lipiddeposits, corneal edema and local inflammation.Results: We have observed regression of CNV in all patients. Themean area of CNV regression was 37% with a range between 6 and86%. We have noticed improvement especially in younger vesselsand smaller caliber vessels (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: NIH Grants RO1 EY12731 and EY021152 and P30EY001931Program Number: 2106 Poster Board Number: D0245Presentation Time: 11:00 AM - 12:45 PMUV-Crosslinking for Fixation of Biosynthetic Corneal CollagenImplantsKerstin M. Wand 1 , Karin Kobuch 1 , Michael Baumann 3 , MayGriffith 2 , Mohammad M. Islam 2 , Johannes Junger 3 , Raphael T.Neuhann 1 , Chris Lohmann 1 . 1 Ophthalmology, Klinikum rechts derIsar, Technische Universität München, Munich, Germany;2 Regenerative Medicine, Linkoping University, Linkoping, Sweden;3 MLase AG, Germering München, Germany.Purpose: In times of donor organ shortage, also in cornealtransplantation, biosynthetic corneal implants as an alternative tohuman donor tissue have been presented. Previously a Phase Iclinical study demonstrated good results regarding stability andintegration. However suture related delay in epithelial closure lead tothinning of the implant, irreversible formation of haze andirregularity of the surface in these areas. Therefore an alternativesuture-free fixation method as UV-crosslinking is expected toimprove the visual outcome.Methods: We implanted different cell-free corneal implantsconsisting of recombinant human collagen type III: RHC III andRHC/MPC (methacrylphosphorylcholine).Ex vivo the biosynthetic corneal implants (350µm in depth, 6mm indiameter) were placed on the anterior cornea of porcine and rabbiteyes after performing deep anterior lamellar keratoplasty with eitherfemtosecond or excimer laser. After application of Riboflavin 0.1%solution for 5 minutes UV-crosslinking was performed at eitherstandard (3mW/cm2 for 30 minutes) or rapid procedure (18mW/cm2for 5 minutes). Thereafter the corneas were excised, fixed in PFA4%and embedded in paraffin.Crosslinking effects (thickness and diameter of implant, adhesionbetween implant and cornea) were evaluated by slitlampbiomicroscopy, OCT images and finally histologically (HE-stained/picrosirius stained sections, electronmicroscopy).Results: Before and after crosslinking the precise fitting of theimplant could be demonstrated in OCT images. This was moreaccurate in porcine eyes than in rabbit eyes, maybe due to thedifference in corneal thickness. Histologically we could provecrosslinks between implant and corneal stroma. After crosslinking thedifferent types of implants showed different degrees of shrinkage.There was no difference in the outcome between standard and rapidcrosslinking procedure.Conclusions: UV-crosslinking as a fixation method for biosyntheticcorneal implants was demonstrated to be promising. It can reducesuture-related complications as neovascularization, haze formationand surface irregularity. Biostability, integration and long termoutcome is further evaluated in in vivo animal experiments.Commercial Relationships: Kerstin M. Wand, None; KarinKobuch, None; Michael Baumann, None; May Griffith, Univ. ofOttawa - OHRI (P); Mohammad M. Islam, None; JohannesJunger, MLase AG (E); Raphael T. Neuhann, None; ChrisLohmann, NoneSupport: Euro Nanomed I-CARE, Transnational collaborativeproject260 Conjunctiva Cell BiologyMonday, May 06, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 2107-2130/D0334-D0357Organizing Section: CorneaProgram Number: 2107 Poster Board Number: D0334Presentation Time: 11:00 AM - 12:45 PMDevelopment of a conjunctival tissue substitute on the basis ofplastic compressed collagenCornelia Corinne Drechsler 1 , Alvena Kureshi 2 , Stephan Reichl 3 ,Gerd Geerling 1 , Julie T. Daniels 2 , Stefan Schrader 1 . 1 Ophthalmology,University Duesseldorf, Duesseldorf, Germany; 2 Institute ofOphthalmology, UCL - University College London, London, UnitedKingdom; 3 Institut für Pharmazeutische Technologie, TUBraunschweig, Braunschweig, Germany.Purpose: Ocular surface disorders can cause severe ocular surfaceinflammation, conjunctival scarring, fornix shortening andankyloblepharon. There is the need for the development of newmatrices for conjunctival reconstruction for this group of patients.Therefore, aim of this study was, to evaluate the suitability ofcompressed collagen as a substrate for the expansion of humanconjunctival epithelial cells in order to develop an epithelializedconjunctival substitute for fornix reconstruction.Methods: Human conjunctiva epithelial cells (HCEC) were culturedon acellular compressed collagen constructs or those containinghuman conjunctival fibroblasts (HCF). The epithelial cells wereevaluated for cytokeratin expression (CK19 /CK4) and theappearance of goblet cells. The presence of putative progenitorepithelial cell markers such as p63α, ABCG2 and CK15 was assessedby immunostaining. The proliferative capacity of the epithelial cellsafter expansion on the plastic compressed collagen gels wasevaluated by their colony forming efficiency and compared to cellscultured on amniotic membrane.Results: The results showed successful expansion of the conjunctivalepithelial cells on both the acellular compressed collagen constructsand those containing human conjunctival fibroblasts. Theimmunofluorescence staining revealed the expression of thecytokeratins such as CK19 as well as the putative progenitor cellmarkers such as CK15 and ABCG2. After expansion on the collagenconstructs, the conjunctival epithelial cell population was still able toform epithelial cell colonies, as tested by colony forming efficiencyassay.Conclusions: Plastic compressed collagen seems to be a suitablesubstrate for the expansion of human conjunctival epithelial cells.The presence of epithelial cells, positive for putative progenitor cellmarkers and the sustained proliferative capacity of the epithelial cellpopulation indicates the maintenance of conjunctival cells withprogenitor cell characteristics on the collagen gels. Therefore, anepithelialized conjunctival tissue construct on the basis ofcompressed collagen, might be a promising alternative bioartificialtissue substitute for conjunctival reconstruction.Commercial Relationships: Cornelia Corinne Drechsler, None;Alvena Kureshi, None; Stephan Reichl, None; Gerd Geerling,Alcon (C), Allergan (C), Thea Pharma (C), Novagali (C), Bausch &Lomb (C), Tearlab Inc. (C); Julie T. Daniels, TAP Biosystems (P);Stefan Schrader, NoneSupport: DFG SCHR 1210/3-1Program Number: 2108 Poster Board Number: D0335Presentation Time: 11:00 AM - 12:45 PMLymphatic Microvessel Density as a Predictive Marker for theRecurrence Time of Pterygium: A 3-year Follow-up StudyHaotian Lin 1 , Lixia Luo 1 , Shiqi Ling 2 , Wan Chen 1 , Zhaochuan Liu 1 ,Xiaojian Zhong 1 , Changrui Wu 1 , Weirong Chen 1 , Yizhi Liu 1 . 1 StateKey Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaGuangzhou, China; 2 Dept of Ophthalmology, Third AffiliatedHospital of Sun Yat-sen University, Guangzhou, China.Purpose: To investigate whether lymphatic microvessel density(LMVD) could be used as a predictive marker for the recurrence timeof pterygiaMethods: This was a prospective case series study. A total of 96patients with unilateral eye primary nasal pterygia were included. Theincluded patients were clinically evaluated to grade the severity oftheir pterygia (32 Grade 1, 29 Grade 2, and 35 Grade 3) before theyunderwent bare sclera resection with the use of mitomycin C. Excisedtissues from the 96 patients and the 10 normal nasal conjunctivaobtained from age-matched donor eyeballs (controls) wereimmunostained with LYVE-1 and CD31 monoclonal antibodies toevaluate lymphatic microvessel density (LMVD) and bloodmicrovessel density (BMVD). The included patients were followedup for 3 years or until the identification of pterygium recurrence,which was defined as fibrovascular regrowth past the limbus in apreviously compromised area. The recurrence time (RT) for apterygium was calculated, and its relationship with LMVD and/orBMVD was statistically analyzed.Results: In total, there were 24 cases of pterygium recurrence. Therecurrence rate (RR) of Grade 1 was 28.1% (9/32), Grade 2 was24.1% (7/29), and Grade 3 was 22.9% (8/35), as classified in theprimary pterygium (p>0.05); the overall RR was 25% (24/96) for allpatients during the 3-year follow-up. In the tissue analysis, there werea small number of CD31 (+), LYVE-1(-) BMVD and only a fewCD31 (weak), LYVE-1(+) LMVD in the 10 normal nasal conjunctivatissues. BMVD and LMVD increased significantly in the pterygiumtissue compared to the control tissue and were significantly correlatedwith both the width and area of pterygium in Grades 1-3 (all p values< 0.05). However, RT was not correlated with BMVD or pterygiumgrade, but LMVD was significantly and negatively correlated withRT within each group and in the total patient cohort. Furthermore, wedetermined that an LMVD greater than 20 in the surgical specimenswas predictive of pterygium recurrence.Conclusions: The LMVD of surgical specimens is an independentrisk factor and a valuable predictive factor for the recurrence time ofpterygia.Commercial Relationships: Haotian Lin, None; Lixia Luo, None;Shiqi Ling, None; Wan Chen, None; Zhaochuan Liu, None;Xiaojian Zhong, None; Changrui Wu, None; Weirong Chen,None; Yizhi Liu, NoneProgram Number: 2109 Poster Board Number: D0336Presentation Time: 11:00 AM - 12:45 PMNHE8 Participates in Electrolyte and Water Secretion inConjunctival and Lacrimal Gland EpitheliaMingwu Wang 1 , Jing Li 2 , Fayez K. Ghishan 2 , Hua Xu 2 .1 Ophthalmology, University of Arizona College of Medicine,Tucson, AZ; 2 Pediatrics, University of Arizona College of Medicine,Tucson, AZ.Purpose: NHE8 is a membrane protein in the sodium/hydrogenexchanger (NHE) family. It involves mucin and bicarbonate secretionin the intestine. But it is not known if NHE8 is expressed inconjunctival and lacrimal gland tissues, and what role it may play inmaintaining ocular surface homeostasis.Methods: The study was conducted in compliance with the Tenets ofthe Declaration of Helsinki and ARVO statement for the use ofanimals in Ophthalmic and Visual Research. Mouse conjunctival andlacrimal gland tissues were harvested for immunohistochemistry,total RNA extraction, and protein isolation. Age- and gender-matchedwild type (WT) and NHE8 knockout (KO) mice (eight in each group)were subjected to examiner-masked ocular surface evaluationsincluding tear pH measurement with narrow range pH paper, andunder anesthesia, phenol cotton thread test, tear break up time(TBUT), and corneal fluorescein staining.Results: The expression of NHE8 was confirmed at both mRNA andprotein levels in conjunctival and lacrimal gland tissues.Immunohistochemistry staining showed that NHE8 was located onthe membrane of both epithelial and goblet cells of conjunctiva. Inlacrimal gland, NHE8 is differentially expressed on cell membrane,with only certain gland acinar and ductal epithelial cells beingpositive. Tear pH value of KO mice was lower than that of WT mice(p < 0.001). KO mice also had significantly lower phenol cottonthread test, TBUT, and significantly higher corneal staining scores (p< 0.05, respectively).Conclusions: For the first time, we have demonstrated that NHE8 isinvolved in ocular surface pH regulation. Mice lacking NHE8function showed signs of dry eye. Our results shed new insights intothe understanding of dry eye syndrome, which could lead to newtherapeutic strategies.Commercial Relationships: Mingwu Wang, None; Jing Li, None;Fayez K. Ghishan, None; Hua Xu, NoneSupport: NIH R01DK073638, Unrestricted RPB grant toDepartment of Ophthalmology University of ArizonaProgram Number: 2110 Poster Board Number: D0337Presentation Time: 11:00 AM - 12:45 PMCarboxypeptidase A3 expression in intraepithelial mast cells ofchronic allergic conjunctivitisKanji Hori, Nobuyuki Ebihara, Toshinari Funaki, Kaori Ohtomo,Yosuke Asada, Akira Murakami, Akira Matsuda. Ophthalmology,Juntendo Univ School of Med, Bunkyo-ku, Japan.Purpose: Carboxypeptidase A3 (CPA3) is a protease known to beexpressed in mast cells. Preferential CPA3 expression inintraepithelial mast cells in atopic asthma was reported previously.We investigated the expression of CPA3 in the giant papillaeobtained from vernal keratoconjunctivitis (VKC) and atopickeratoconjunctivitis (AKC) patients, and compared the expressionwith known mast cell immunohistochemical markers(tryptase/chymase), and a mast cell activation marker; high affinityIgE receptor β chain (FcεRIβ).Methods: 10 giant papillae were resected for therapeutic purpose andsections were prepared as previously described. (IOVS.50:2871-2877) Anti-CPA3/anti-tryptase or anti-CPA3/anti-chymase doubleimmnunohistochemical staining was carried out by indirectimmunofluorescent method. Anti-CPA3/anti-FcεRIβ doubleimmunohistochemical staining was carried out by directimmunofluorescent method using Zenon labeling system(Invitorogen, CA).Results: CPA3-tryptase double immunostaining showed that CPA3positive staining was observed in the intraepithelial and subepithelialtryptase positive mast cells. CPA3-chymase double immunostainingshowed both CPA3+ chymase - mast cells and CPA3+ chymase+mast cells in the conjunctival epithelium of the giant papillaesamples. CPA3+ chymase+ mast cells preferentially located at nearthe epithelial-stromal borders. CPA3/ FcεRIβ double positive cellswere also observed the giant papillae samples.Conclusions: Preferential CPA3 expression in the intraepithelialmast cells of giant papillae was observed. Tryptase positive/Chymasenegative (MCT) mast cells are predominant mast cell type for CPA3expression. CPA3 expression may relate to mast cell activation ormaturation.Commercial Relationships: Kanji Hori, None; NobuyukiEbihara, None; Toshinari Funaki, None; Kaori Ohtomo, None;©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaYosuke Asada, None; Akira Murakami, SEED(Japan) JP4855782(P), SEED(Japan) JP5132958 (P); Akira Matsuda, NoneProgram Number: 2111 Poster Board Number: D0338Presentation Time: 11:00 AM - 12:45 PMTNFα is required for normal tissue repair in injured mouseconjunctivaOsamu Yamanaka 1 , Ai Kitano 1 , Yuka Okada 1 , Winston W. Kao 2 ,Shizuya Saika 1 . 1 Ophthalmology, Wakayama Medical University,Wakayama, Japan; 2 Ophthalmology, University of Cincinnati,Cincinnati, OH.Purpose: To examined the roles of TNFα, a major proinflammatorycytokine, in the process of tissue repair in mouse conjunctiva in vivoand the fibrogenic responses of of cultured human subconjunctivalfibroblasts (hSCFs) as well.Methods: A circumferential incision was made in the equatorialconjunctiva of the right eye of generally anesthetized TNFα-null(KO) and wild-type (WT) mice using scissors. At 2, 5, 7 and 10 days,the eyes were processed for histology and immunohistochemistry toevaluate the tissue repair process. Expression of type I collagen andgrowth factors was evaluated by real time PCR (RT-PCR). Theeffects of TNFα (10 ng/ml) on production of a smooth muscle actin(αSMA) and type I collagen and Smad3 phosphorylation usingcultured hSCFs with or without TGFβ1 (5 ng/ml) were also studied.Effects of TNFα on hSCFs proliferation, migration and Erk activationwere finally examined.Results: TNFα KO mice showed severe macrophage invasion,marked generation of myofibroblasts in healing subconjuncitvaltissue and upregulation of mRNAs of fibrogenic components, i. e.,TGFβ1, CTGF and Col1a2 chain in comparison to those of WT mice.In vitro experiments with cultured hSCFs showed that TNFαsuppressed the protein expression of αSMA and type I collagen, aswell as Smad3 phosphorylation, upon TGFβ1 stimulation. TNFαactivated Erk signal and enhance cell migration and proliferation.Conclusions: Expression of TNFα in situ has an essential role fornormal tissue repair in mouse conjunctiva and ablation of TNFα inmice leads to excess inflammation and tissue fibrosis. Administrationof counteracts TGFβ1/Smad3 activation, and stimulates Erkactivation and proliferation and migration of TNFα to cultured hSCFsthat may account for manifestations in the wound healing excessinflammation and fibrosis seen in TNFα null mice.Commercial Relationships: Osamu Yamanaka, None; Ai Kitano,None; Yuka Okada, None; Winston W. Kao, None; Shizuya Saika,NoneProgram Number: 2112 Poster Board Number: D0339Presentation Time: 11:00 AM - 12:45 PMSubstance P is a candidate for conjunctival epithelial cell derivedfactors inducing CCL2 expression in mast cellsSatoshi Iwamoto 1 , Satoshi Kawasaki 2 , Yosuke Asada 1 , Kanji Hori 1 ,Akira Murakami 1 , Nobuyuki Ebihara 1 , Akira Matsuda 1 . 1 JuntendoUniv School of Med, Bunkyoku, Japan; 2 Kyoto PrefectualUniversity, zyoukyoku, Japan.Purpose: We previously reported that (1) coculture between humanmast cells and conjunctival epithelial cells induce CCL2 expressionin mast cells, (2) CCL2 stimulation of mast cells could inducepiecemeal degranulation (PMD) of mast cells, and (3) increasedCCL2 expression and PMD was observed in the giant papillae ofvernal keratoconjunctivitis (VKC) patients. The CCL2 induction inmast cells was dependent on the conjunctival epithelial cell derivedfactor(s), therefore we examined the role of substance P (SP) as acandidate for the epithelial cell derived factor inducing CCL2expression in mast cells.Methods: Human cultured mast cells (LAD2) were stimulated withSP for 24hours. Realtime PCR analysis and CCL2 ELISA assay werecarried out to quantitate CCL2 expression in LAD2 cells. SP inhibitor(L-703606 oxalate salt hydrate) was added to the coculture model andCCL2 expression was also examined.Results: CCL2 mRNA expression in LAD2 cells was increased1.49±0.07 fold (mean±SD) by SP stimulation (10uM). CCL2concentration in the LAD2 culture supernatants was increased 1.69fold (from 585 pg/ml to 994 pg/ml) by SP stimulation (10uM). SPinhibitor (1nM) partially suppressed CCL2 upregulation in thecoculture model (22% reduction). CCL2 concentration in the LAD2culture supernatants was decreased 15% (from 844pg/ml to718pg/ml) by SP inhibitor (1uM).Conclusions: Our result showed that SP could be one of the possibleepithelial cell derived factor(s) that modulate CCL2 expression inmast cells.Commercial Relationships: Satoshi Iwamoto, None; SatoshiKawasaki, None; Yosuke Asada, None; Kanji Hori, None; AkiraMurakami, SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958(P); Nobuyuki Ebihara, None; Akira Matsuda, NoneProgram Number: 2113 Poster Board Number: D0340Presentation Time: 11:00 AM - 12:45 PMGiant Conjunctival Nevus: Clinical Features and Natural Coursein 32 CasesPhoebe Mellen 1, 2 , Audrey C. Regillo 1 , Swathi Kaliki 1, 3 , Sara Lally 1 ,Carol L. Shields 1 , Jerry Shields 1 . 1 Oncology Service, Wills EyeInstitute, Philadelphia, PA; 2 Jefferson Medical College, Philadelphia,PA; 3 Ocular Oncology Service, L.V. Prasad Eye Institute,Hyderabad, India.Purpose: To describe the clinical features, management, andoutcomes of giant (>10 mm diameter) conjunctival nevus.Methods: Retrospective case seriesResults: Of 618 eyes with conjunctival nevus, 32 (5%) wereclassified as giant conjunctival nevus. The mean patient age atdiagnosis was 34 years (median 33, range 2 - 75 years). A personalhistory of increase in nevus basal dimension or thickness was notedin 15 (47%) and increase in nevus color intensity in 2 cases (6%).The giant nevus involved cornea in 11 (34%), limbus in 23 (72%),bulbar conjunctiva in 31 (97%), fornix in 9 (28%), tarsus in 3 (9%),semilunar fold in 10 (31%), and caruncle in 7 (22%). The nevi hadmean diameter of 16 mm (median 13, range 10 - 40) and meanthickness of 2 mm (median 2, range 0.5 - 4). On slit lampexamination, intrinsic cysts were identified in 25 (78%), intrinsicblood vessels in 26 (81%), and feeder vessels in 22 (69%) of cases.Management included excisional biopsy with cryotherapy in 23(72%) and observation in 9 cases (28%). Amniotic membrane graftreconstruction was employed following excision in 3 cases (13%).Malignant melanoma developed within the giant nevus in 1 case after23 years of observation. Post excisional biopsy, nevus recurrence wasdetected in 4 cases (17%), pseudopterygium in 1 (4%), dry eye in 1(4%), and eyelid blepharoptosis in 1 (4%).Conclusions: In an ocular oncology practice, giant conjunctivalnevus represents 5% of conjunctival nevi. This benign tumor rarelytransforms into conjunctival melanoma (n=1, 3%). Managementalternatives include observation or wide excisional biopsy,cryotherapy, and reconstruction, possibly with amniotic membranegraft.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaClinical features and imaging with anterior segment opticalcoherence tomography (AS-OCT) of giant conjunctival nevus. (A) A16-year-old Caucasian male demonstrated a giant conjunctival nevuswith slight pigmentation superonasally, measuring 20 mm in basaldiameter and 4 mm in thickness. On AS-OCT, the intralesional cysts(B) were confirmed. (C) A 48-year-old Caucasian male showed giantconjunctival nevus superotemporally, measuring 18 mm in basaldiameter and 2 mm in thickness. On AS-OCT, the intralesional cysts(D) were confirmed.Commercial Relationships: Phoebe Mellen, None; Audrey C.Regillo, None; Swathi Kaliki, None; Sara Lally, None; Carol L.Shields, None; Jerry Shields, NoneProgram Number: 2114 Poster Board Number: D0341Presentation Time: 11:00 AM - 12:45 PMResolvin D1 Receptor Activation Counter-regulates H1 histaminereceptors in human and rat conjunctival goblet cellsRobin R. Hodges 1, 2 , Dayu Li 1, 2 , Richard B. Carozza 1, 2 , Marie A.Shatos 1, 2 , Nan Chiang 3, 4 , Charles N. Serhan 3, 4 , Darlene A. Dartt 1, 2 .1 Schepens Eye Research Institute, Massachusetts Eye and Ear,Boston, MA; 2 Department of Ophthalmology, Harvard MedicalSchool, Boston, MA; 3 Center Center for Experimental Therapeuticsand Reperfusion Injury, Harvard Institutes of Medicine, HarvardMedical School, Boston, MA; 4 Department of Anesthesiology,Preoperative, and Pain Management, Brigham and Women'sHospital, Boston, MA.Purpose: To determine the interactions between H1 histaminereceptor (H1R) and GPR32, a GPCR receptor for the proresolutionmediator, resolvin D1 (RvD1).Methods: Rat conjunctival goblet cells were grown in culture.Chinese hamster ovary (CHO) cells were transfected with humanH1R and GPR32 receptor. Intracellular [Ca 2+ ] ([Ca 2+ ] i ) was measuredin cells loaded with the calcium indicator dye fura 2.Results: In rat goblet cells, the H1R agonist histamine dimaleatesignificantly increased [Ca 2+ ] i by 327.2 ± 71.5 nM. Exposure toRvD1 significantly inhibited this response to 92.6 ± 21.5 nM. Theprotein kinase C inhibitor Ro-317549 and an inhibitory peptide of βadrenergic receptor kinase 1 (βARK1) reversed the inhibition ofRvD1 on the histamine dimaleate stimulated increase in [Ca 2+ ] i to197.2 ± 46.3 and 352.7 ± 89.6 nM, respectively. Similar results wereobtained in CHO cells transfected with human H1R and humanGPR32 receptor.Conclusions: We conclude that RvD1 uses PKC and βARK1 tocounter-regulate the H1R and inhibit its actions.Commercial Relationships: Robin R. Hodges, None; Dayu Li,None; Richard B. Carozza, None; Marie A. Shatos, None; NanChiang, None; Charles N. Serhan, Resolvyx Pharma (I), BWH-Partners (P); Darlene A. Dartt, NoneSupport: National Institutes of Health RO1-EY019470 to DAD and1P01GM095467 to CNSProgram Number: 2115 Poster Board Number: D0342Presentation Time: 11:00 AM - 12:45 PMEfficacy of rituximab in severe ocular cicatricial pemphigoidSerge Doan 1 , Sophie Stephan 1 , Catherine Prost 2 , Marina Alexandre 2 ,Isabelle Cochereau 1 , Eric E. Gabison 1 . 1 Ophthalmology, BichatHospital & A de Rothschild Foundation, Paris, France;2 Dermatology, Avicenne Hospital, Bobigny, France.Purpose: To report the efficacy of rituximab in patients with severeocular cicatricial pemphigoid (OCP).Methods: In this non controlled retrospective study, 18 patients withsevere OCP (8 males, 10 females ; median age, 73 years ; 31 eyes)were treated with rituximab, because of toxicity or resistance tocyclophosphamide (in 10 and 4 cases, respectively), or of rapidevolutivity (4 cases). The treatment consisted of 4 weeklyintravenous administrations (375 mg/m2) or 2 monthly intravenousadministrations (1 gram). This treatment was repeated in case ofpersistent conjunctival inflammation. Complete remission wasdefined as the absence of conjunctival inflammation.Results: Complete remission was obtained in 16 patients (89%)within a median period of 2.5 months (range, 1 to 6 months) after 1or 2 treatments (in 12 and 4 patients, respectively). The diseaserecurred in 5 of the 16 patients after 6 to 27 months. Completeremission was obtained after an additional rituximab treatment.Median follow up without any recurrence was 21 months (range, 12to 54 months). No serious adverse effect was noted.Conclusions: Rituximab is a potent treatment of severe OCP. It isparticularly interesting in case of resistance or contraindication tocyclophosphamide, or in rapidly evolutive forms.Commercial Relationships: Serge Doan, None; Sophie Stephan,None; Catherine Prost, None; Marina Alexandre, None; IsabelleCochereau, Laboratoires Thea (C), Laboratoires Thea (F), Allergan(C), Lux Bioscience (F); Eric E. Gabison, NoneProgram Number: 2116 Poster Board Number: D0343Presentation Time: 11:00 AM - 12:45 PMSurvival with Squamous Cell Carcinoma of the Conjunctiva inthe United States 1973-2009Aaron Hendrix 1 , Mark Hendrix 2 . 1 Georgetown Preparatory School,Bethesda, MD; 2 Rockville Eye Associates, Rockville, MD.Purpose: To delineate trends and factors affecting survival ofpatients with Squamous Cell Carcinoma of the Conjunctiva (SCCC)over the period 1979 to 2009.Methods: The National Cancer Institute Surveillance, Epidemiology,and End Results (SEER) Program Database (1973-2009) wasanalyzed using the SEER*Stat software. Cases for analysis wereidentified using histology codes for Squamous Cell Carcinoma(8070-8076,8078) and the site code for Conjunctiva (C69.0) from theInternational Classification for Diseases for Oncology Third Edition(ICDO-3). Relative survival rates were calculated using the LifeTable method and adjusted for censoring events using the Ederer IImethod. Relative survival curves were compared using the Coxproportional hazards model with the level of significance of P≤0.005.The relative risk ratio was calculated for each factor.Results: For the period 1973 to 2009, 734 cases of SCCC wereidentified in the database. 732 (99.7%) of the these were identifiedhistopathologically. 661 (90.0%) were reported by a hospital or lab.Patients were more likely to be male (573, 78.3%) than female (159,21.3%) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamales and 94.6% in females). The ten-year relative survival rate was84.9% (84.2% in males and 87.0% in females). Cox regressionanalysis with gender, race, year of diagnosis and age at diagnosis ascovariants was performed. Survival was better for females than males(p=0.006); better for whites than blacks (p=0.035); and better foryounger age (p=0.00). Year of diagnosis did not appear to affectsurvival (p=0.842).Conclusions: Analysis of data from the SEER Program databaseindicates a relatively low mortality from SCCC. Survival rates appearto be stable during the period 1973-2009. The five-year relativesurvival rates were similar to those found previously for cutaneuoussquamous cell carcinoma. Female gender, white race, and youngerage at diagnosis correlated with improved survival.Commercial Relationships: Aaron Hendrix, None; MarkHendrix, NoneProgram Number: 2117 Poster Board Number: D0344Presentation Time: 11:00 AM - 12:45 PMEffect of Cyclosporin A (Cys A) to the Matrix Metalloproteinase(MMP)-3 and MMP-13 on the Migration of Cultured PterygialFibroblast CellsYoung Jeung Park 1 , Yeoun-Hee Kim 1, 2 , Se-Hyung Lee 2 , Jae-ChangJung 2 , Kyoo Won Lee 1 . 1 Ophthalmology, Cheil eye hospital, Daegu,Republic of Korea; 2 Biology, Kyungpook National University,Daegu, Republic of Korea.Purpose: To investigate the effects of matrix metalloproteinase(MMP) 3 and MMP-13 expression in cultured human pterygialfibroblast cells according to the concentration and exposure time ofCyclosporine A (Cys A) after being scratched.Methods: Scratched-human pterygial fibroblast cells were exposedto a Cys A concentrations of 1 µg/ml (0.0001%) and 100 µg/ml(0.01%) for 3 or 10 minutes and then cells were washed with D-PBSand re-incubated or exposed to a MMP-3 specific inhibitor (MMP-3VII inhibitor) or MMP-9/13 inhibitor with serum depletion DMEM-F-12 (1:1) media for 48 hours. Cells were lysed for Western blottingor extracted mRNA for RT-PCR. Supernatant media were used toperform TCA precipitation for Western blotting. MMP-3 and MMP-13 expression of fibroblast after the stimulation were studied.Results: The cellular migrations were markedly suppressed by pretreatmentwith Cys A in a time- and dose- dependent manner on thecultured pterygial fibroblast cells (P < 0.01). Interestingly, thismigration was also blocked by MMP-3 specific inhibitor or MMP-9-13 inhibitor (P < 0.01). Furthermore, pre-treatment of Cys A reducedMMP-3 mRNA (P < 0.01) and MMP-13 protein (P < 0.02)expression level.Conclusions: Previous studies reported that the expression of severaltypes of MMPs was increased in pterygium, and Cys A can beeffective in preventing recurrences after primary pterygium surgery.Our results suggested that Cys A inhibited cell migration after beingscratched by reducing expression of MMPs. These findings revealtherapeutic potential for pterygium progression.Commercial Relationships: Young Jeung Park, None; Yeoun-HeeKim, None; Se-Hyung Lee, None; Jae-Chang Jung, None; KyooWon Lee, NoneProgram Number: 2118 Poster Board Number: D0345Presentation Time: 11:00 AM - 12:45 PMHistologic characteristics of conjunctivochalasis and itscorrelation with lymphangiectasisWoo C. Park, Jeong Bum Bae, Won Yeol Ryu. of Ophthalmology,College of Medicine, Dong-A University, Busan, Republic of Korea.Purpose: To evaluate the histologic characteristics ofconjunctivochalasis and its association with lymphangiectasis usingimpression cytology and biopsy.Methods: A prospective study was included in 14conjunctivochalasis patient who was performed excisional biopsyfrom Mar. to Nov. 2012. Dry eye evaluation test and impressioncytology were performed. Histologic evaluation was done with H&E,Verhoeff Van Gieson and D2-40 immuno stain using excisedconjunctiva.Results: Tear break-up time and Schirmer’s value was decreased.Impression cytology showed the decrease in goblet cell density andan increase in nucleoplasmic/cytoplasmic ratio. Conjunctiva fromconjunctivochalasis patients showed increase in infiltration of chronicinflammatory cells on H&E stain, and decrease in collagen densitywith degeneration of elastic fibers on Verhoeff Van Gieson stain, andD2-40 immunohistochemistry showed the dilation of subconjunctivallymphatics compared to those from the control.Conclusions: Conjunctivochalais is associated with lymphangiectasisand decrease of goblet cells density.Commercial Relationships: Woo C. Park, None; Jeong Bum Bae,None; Won Yeol Ryu, NoneClinical Trial: 12-077Program Number: 2119 Poster Board Number: D0346Presentation Time: 11:00 AM - 12:45 PMCysteinyl leukotriene receptors expression in the giant papillae ofthe chronic allergic conjunctivitisKaori Ohtomo 1 , Nobuyuki Ebihara 1 , Norihiko Yokoi 2 , SatoshiKawasaki 2 , Akira Murakami 1 , Akira Matsuda 1 . 1 Ophthalmology,Juntendo University School of Medicine, Tokyo, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan.Purpose: The cysteinyl leukotrienes (LTC4, LTD3, and LTE4) havemany biological effects, which augment allergic inflammation.Clinically, cysteinyl leukotriene receptor antagonists are widely usedfor the treatment of asthma and nasal allergy. To clarify the roles ofcysteinyl leukotrienes signals in severe chronic allergicconjunctivitis, we investigated the expression of cysteinyl leukotriene©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneareceptors (CysLTR) in the resected giant papillae.Methods: Anti-CysLTR1, CysLTR2, MBP, CD68, neutrophilelastases and tryptase immnunohistochemical staining wereperformed with the resected giant papillae obtained from AKC/VKCpatients (n=9). Realtime PCR analysis was carried out to examineCysLTR1 mRNA expression in giant papillae obtained from VKCpatients and control conjunctival tissue.Results: CysLTR1 positive infiltrating cells were observed in theepithelium and substantia propria of the giant papillae.CysLTR1/neutrophil elastase double positive cells were localized inthe substantia propria of the giant papillae. CysLTR2 positivevascular endothelial cells and CysLTR2/CD68 double positive cellswere present in the giant papillae. CysLTR1 mRNA was detected inVKC patients.Conclusions: CysLTR1 and CysLTR2 expression were observed inthe giant papillae obtained from AKC/VKC patients.Commercial Relationships: Kaori Ohtomo, None; NobuyukiEbihara, None; Norihiko Yokoi, Santen Pharmaceutical (F), OtsukaPharmaceutical (F), Kowa (P), Kissei Pharmaceutical (C), Menicon(F), Alcon Japan (F), White medical (F), Nitten (F), Rohto (C), Nidek(F), Johson & Johnson (F); Satoshi Kawasaki, None; AkiraMurakami, SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958(P); Akira Matsuda, NoneProgram Number: 2120 Poster Board Number: D0347Presentation Time: 11:00 AM - 12:45 PMConstitutive Expression of PTX3 in HC-HA Complex by HumanAmniotic Membrane CellsSu-Zhen Zhang 1 , Hua He 1 , Ying-Ting Zhu 1 , Scheffer C. Tseng 1, 2 .1 R&D, TissueTech, Inc, MIAMI, FL; 2 Ocular Surface Center,MIAMI, FL.Purpose: Pentraxin 3 (PTX3), an inducible secreted protein in mostcell types, is crucial for innate immunity, tissue inflammation, andstability of heavy chain-hyaluronan (HC-HA) complex in cumulusoophorus complex. We have reported that HC-HA purified fromhuman amniotic membrane (AM) is responsible for AM’s therapeuticactions and constitutively produced by AM cells. Furthermore,upregulation of PTX3 to suppress expression and activation of matrixmetalloproteinases 1 and 3 is involved in conjunctivochalasisfibroblasts. Herein, we further investigated whether PTX3 is also inHC-HA complex produced by AM, which is used as a surgical graftfor treating conjunctivochalasis.Methods: Expression of PTX3 was determined by immunostainingof AM tissue, and by RT-PCR and Western blot of supernatants andlysates of primary AM epithelial (AMEC) and stromal cells (AMSC)treated with or without TNF, IL-1β, or PTX3 siRNA in a low-serummedium. HC-HA complexes were isolated from AM PBS andguanidine HCl extracts by sequential CsCl ultracentrifugation in 6 Mguanidince HCl, and further characterized by Western blot with orwithout hyaluronidase (HAase) digestion.Results: Strong positive PTX3 staining was found in the compactstromal layer and the apical surface of the epithelium in the AM. Likeconjunctivochalasis and skin fibroblasts, PTX3 mRNA was expressedby resting AMEC and AMSC and further upregulated by TNF or IL-1β. Unlike strict contingence upon IL-1β in cultured human skin andconjunctivochalasis fibroblasts, expression of PTX3 protein wasconstitutive by resting AMEC and AMSC, while its secretion wasreduced by TNF and completely inhibited by IL-1β. Secretion ofPTX3 protein under stimulation of TNF was downregulated bysiRNA in conjunctivochalasis fibroblasts but not in AMEC andAMSC. PTX3 was released from HC-HA complex by HAase in bothwater-soluble and water-insoluble HC-HA complex purified fromAM.Conclusions: PTX3 is an integral component of HC-HA complexpurified from both water-soluble and water-insoluble AM extracts.Expression and secretion of PTX3 depends on pro-inflammatorycytokines in skin fibroblasts, but is constitutive in resting AMepithelial and stromal cells. Thus, HC-HA complex that containsPTX3 is uniquely present in AM and is responsible for AM’s clinicalapplications.Commercial Relationships: Su-Zhen Zhang, Tissue Tech, Inc (E);Hua He, TissueTech, Inc. (E); Ying-Ting Zhu, Tissue Tech (F),Tissue Tech (E), Tissue Tech (P); Scheffer C. Tseng, NIH, NEI (F),TissueTech, Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)Support: NIH, NEI, Grant R44 EY017497, R43 EY021045, andRO1 EY06819Program Number: 2121 Poster Board Number: D0348Presentation Time: 11:00 AM - 12:45 PMLymphatic and Blood Vessel Density in Human ConjunctivaFollowing Glaucoma SurgeryMirela Krasniqi 1 , Heidi Rassavong 1 , Rachida Bouhenni 1 , Jeffrey J.Dunmire 1 , Sami A. Al Shahwan 2 , Ibrahim Al Jadaan 2 , Hind Alkatan 2 ,Deepak P. Edward 2, 3 . 1 Ophthalmology, Summa-Health System,Akron, OH; 2 Ophthalmology, King Khaled Eye Specialist Hospital,Riyadh, Saudi Arabia; 3 Ophthalmology, Wilmer Eye Institute, JohnsHopkins University, Baltimore, MD.Purpose: To investigate the lymphatic vascular microvessel density(LVD) and the blood vascular microvessel density (MVD) and theirdistribution in excised leaking blebs after mitomycin C (MMC)trabeculectomy and normal conjunctiva specimens.Methods: LVD and MVD in normal human conjunctiva (n=7) andexcised blebs in the hypocellular stroma and peribleb tissue(conjunctiva adjacent to hypocellular bleb tissue, n=7) was evaluatedby immunohistochemistry using antibodies raised against LymphaticVessel Endothelial Receptor 1 (LYVE-1, lymphatic vessels) andCD34 (blood vessels). LVD and MVD counts were performed bylight microscopy in 5 fields at 20X magnification by three observers.Differences were determined using Mann Whitney U test (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: We demonstrated that CsA inhibited stress-inducedapoptosis in human conjunctival epithelial cells (IOBA-NHC);promoted T cell death via differential regulation of mitochondriapermeability transition pore (MPTP). This study is to investigatemolecular mechanisms of apoptotic signaling in both cell types underimmune and inflammatory stimulation.Methods: IOBA-NHC and Jurkat T cells were stimulated with aFas,PMA/aCD3, TNFa and/or IFNγ, in the presence or absence of CsA,and/or caspase inhibitors. Cell death was quantified by Annexin V/PIassay. MPTP and mitochondrial membrane potential (ΔΨm) weredetermined using calcein-cobalt technique and JC-1 staining,respectively. Mediators of the intrinsic and extrinsic apoptosispathways (Fas/FasL, Bax/Bcl-2, cytochrome c, and caspase activities)were evaluated using flow cytometry or Western blot analysis.Results: In IOBA-NHC: (1) MPTP activation was elicited by aFas,TNFa, and/or IFNγ, but significantly inhibited by CsA; (2) TNFa andIFNγ provoked Fas upregulation, Bax mitochondrial translocation ,mitochondrial membrane depolarization, cytochrome c release, andincreased cell death. Inflammation-induced IOBA-NHC cell deathwas caspase-dependent. CsA attenuated inflammation-inducedcaspase 8, 9 and 3 activation, and inhibited IOBA-NHC apoptosis. InJurkats: (1) PMA/aCD3-induced MPTP opening was not affected byCsA (1nM-10uM); (2) at subnanomolar concentration (>1nM), CsAcompletely abolished T cell IL-2 production; (3) at 10uM, CsAinduced FasL expression, enhanced caspase activation, andexacerbated activation-induced T cell apoptosis.Conclusions: The intrinsic (mitochondria-mediated) and extrinsic(death receptor-initiated) pathways are both involved in inflammatoryapoptosis of IOBA-NHC. CsA preserves conjunctival epithelial cellsby stabilization of MPTP, maintaining mitochondrial membranepotential, and inhibition of Fas, Bax and caspase activation. Incontrast, CsA elicited resting T cell death, and exacerbated activatedT cell apoptosis primarily through extrinsic apoptosis signaling withminimal effect on MPTP. The results suggest that the differentialeffects of CsA on the fate of ocular resident cells versusinflammatory T cells may contribute to its therapeutic efficacy intreating ocular inflammation and restoration of immune homeostasisof the ocular surface.Commercial Relationships: Jianping Gao, Allergan, Inc. (E);Reuben Sana, Allergan (E); Virginia L. Calder, Allergan Inc (C),Allergan Inc (F); Margarita Calonge, Allergan (C); Wanju Lee,Allergan, Inc. (E); Larry A. Wheeler, Allergan Pharm (E); MichaelE. Stern, Allergan, Inc. (E)Program Number: 2123 Poster Board Number: D0350Presentation Time: 11:00 AM - 12:45 PMImmunohistochemical alterations in cicatricial conjunctivitisassociated with Behcet’s diseaseKazunari Higa 1 , Yoshiyuki Satake 1 , Daisuke Tomida 1 , KatsuyaYamazoe 2 , Takahiko Hayashi 3 , Naoki Toriyama 1 , Jun Shimazaki 1 .1 Department of Ophthalmology, Tokyo Dental College, IchikawaGeneral Hospital, Ichikawa, Japan; 2 Department of Ophthalmology,Keio University School of Medicine, Shinjuku-ku, Japan;3 Department of Ophthalmology, Yokosuka Kyosai Hospital,Yokosuka, Japan.Purpose: To identify the pathogenesis of cicatricial conjunctivitis inBehcet’s disease and to evaluate the histopathological differencesfrom ocular cicatricial pemphigoid (OCP) .Methods: Conjunctival resection samples were obtained frompatients with OCP and Behcet’s disease during corneal oral mucosaepithelial sheet transplantation, penetrating keratoplasty or cataractsurgery. All subjects underwent tear and ocular surface examinationsincluding slit lamp microscopy, Schirmer test, tear clearance,fluorescein vital stainings. Informed consents were obtained, theprocedures were board reviewed and registered at Tokyo DentalCollege, Ichikawa Hospital. The tissues were mounted with the OCTcompound and frozen sections were stained with hematoxylin &eosin initially. Immunohistochemistry (IHC) for differentiation ofinflammatory cells including CD 3, 4 and 8, CD11c, CD68, CD66c,and alpha-SMA stainings were performed. In addition, IHCalterations of the supracorneal conjunctival tissues were studies withkeratin 4, 13 and involucrin antibodies. CD31, podoplanin IHCstainings were also performed.Results: None of the subjects had any systemic and ocular diseaseother than OCP or Behcet’s disease or drug use that would causecicatricial conjunctivitis. Prominent stainings were observedexclusively in the stromal sections of the supracorneal conjunctivaltissues for CD3, CD4, CD8, CD11c, CD68, CD66c,CD31 and alpha-SMA in Behcet’s disease. On the other hand, while conjunctivalsamples from OCP patients showed similar staining patterns toBehcet’s disease in the stromal tissues, OCP sections also showedremarkable positive staining of these markers in the epithelium.Interestingly, decreased keratin 4 and 13 (mucosal epithelium)stainings with prominent increase in involucrin staining wereobserved in conjunctival epitheliae of subjects with Behcet’s disease.Conclusions: Conjunctival ocular surface disorder in Behcet’sdisease differed from OCP in relation to presence of prominentstaining for inflammatory markers in the conjunctival stroma andincreased staining for keratinization markers in the epithelium. Ourfindings suggest a prominent role for conjunctival stromal vasculitisin Behcet’s conjunctival epithelial disease whereas concomitantepithelitis and vasculitis appear to be important in the pathogenesis ofthe conjunctival changes in OCP .Commercial Relationships: Kazunari Higa, None; YoshiyukiSatake, None; Daisuke Tomida, None; Katsuya Yamazoe, None;Takahiko Hayashi, None; Naoki Toriyama, None; Jun Shimazaki,Santen Pharmaceutical Co. (F), Otsuka Pharmaceutical Co. (F), AbottMedical Optics (F)Support: do not use n/aProgram Number: 2124 Poster Board Number: D0351Presentation Time: 11:00 AM - 12:45 PMCharacterization of the Gene Expression Profile ofConjunctivochalasisStella C. Paparizos 1 , Rachida Bouhenni 1 , Jeffrey J. Dunmire 1 , ToddL. Beyer 1 , Khaled K. Abu-Amero 2 , Deepak P. Edward 3, 4 .1 Ophthalmology, Summa Health System, Akron, OH;2 Ophthalmology, King Saud University, Riyadh, Saudi Arabia;3 Ophthalmology, King Khaled Eye Specialist Hospital, Riyadh, SaudiArabia; 4 Ophthalmology, Wilmer Eye Institute, Johns HopkinsUniversity, Baltimore, MD.Purpose: The pathogenesis of conjunctivochalasis remains unclear.We hypothesized that gene expression profiling of conjunctival tissuefrom patients with conjunctivochalasis would give us an insight intothe molecular pathways involved in its pathogenesis. To test thishypothesis we studied differential gene expression ofconjunctivochalasis tissue compared to age-matched controlconjunctival tissue with no history of ocular disease.Methods: Surgical specimens of conjunctivochalasis (n=4) and agematchedcontrol conjunctival tissue (n=4) were obtained forcomparison during conjunctivoplasty or strabismus surgery,respectively. Whole genome expression profiling using the Agilenthuman genome array slide 4x44K was performed on total RNAextracts. Differential gene expression was analyzed using GeneSpringGX 12. Genes with ≥2 fold increased or decreased expression acrossall four conjunctivochalasis samples were selected for further©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaanalyses. Gene ontology was determined using the PANTHER(Protein Analysis Through Evolutionary Relationships) analysissoftware.Results: A total of 1021 genes were found to be over-expressed, and1046 genes were found to be under-expressed in all fourconjunctivochalasis specimens compared to age-matched controlsafter excluding pseudo genes, cDNA transcripts without codedproteins, predicted proteins based on sequence homology, and geneswith hypothetical or uncharacterized proteins. The protein productsof the altered (over and under-expressed) genes are involved inmultiple signaling pathways and biological functions including:angiogenesis; apoptosis; chemokine/cytokine-mediatedinflammation; P53 signaling; and Alzheimer’s/prensilin.Conclusions: The gene expression profile of the conjunctivochalasisphenotype is complex. The up- and down-regulation of genes fromthe same pathways suggests that angiogenesis, chemokine/cytokinemediatedinflammation and apoptosis, through P53 regulation, couldbe important in the pathogenesis of conjunctivochalasis.Commercial Relationships: Stella C. Paparizos, None; RachidaBouhenni, None; Jeffrey J. Dunmire, None; Todd L. Beyer, None;Khaled K. Abu-Amero, None; Deepak P. Edward, NoneProgram Number: 2125 Poster Board Number: D0352Presentation Time: 11:00 AM - 12:45 PMTopical Cyclosporine A for the Treatment of ChronicKeratoconjunctivitisJason S. Kim 1 , Anton M. Kolomeyer 1 , Christina Fang 1 , Natasha V.Nayak 1 , Eliott Kim 1 , David S. Chu 1, 2 . 1 New Jersey Medical School-University of Medicine and Dentistry of New Jersey, Newark, NJ;2 Metropolitan Eye Research and Surgery Institute, Palisades Park,NJ.Purpose: To describe the use of Cyclosporine A (CsA) in patientswith chronic keratoconjunctivitis (KC).Methods: This study was a retrospective chart review at a tertiarycare center. Patients treated with topical CsA (1% emulsion) forvarious types of KC between August 2001 and November 2012 wereincluded. Main outcome measures included degree of inflammation,ability to taper steroids, visual acuity (VA), and reported side effects.Results: Thirty-one eyes of 17 patients (11 [65%] males and 6 [56%]females; mean age of all the patients was 47.9 ± 17.1 years) met thestudy criteria. Fourteen (82%) patients had chronic epidemic KC,some of which were dependent on topical steroids, two (12%)patients had Thygeson’s disease, and one (5.9%) patient had vernalKC. Eleven (65%) patients were receiving concomitant topicalsteroids initially and all were able to taper the dose with use of CsAafter 3.5 ± 2.5 months. Nine (53%) patients reported oculardiscomfort and burning. One (5.9%) patient discontinued CsAbecause of irritation and one (5.9%) reduced CsA dosage to 0.5%after four weeks of treatment due to burning sensation. One (5.9%)patient increased the dosage from 1% to 2% after 38 weeks toachieve better control. Inflammation was controlled in 16 (94%)patients.Conclusions: Management of KC with topical CsA resulted ininflammation control and allowed for steroid taper in the majority ofpatients without severe complications. This shows that, in most cases,CsA can be used to manage the effects of chronic KC.Commercial Relationships: Jason S. Kim, None; Anton M.Kolomeyer, None; Christina Fang, None; Natasha V. Nayak,None; Eliott Kim, None; David S. Chu, Abbott (F), Novartis (F),Santen (F), Eyegate (F), Lux Biosciences (F), Bausch & Lomb (R)Program Number: 2126 Poster Board Number: D0353Presentation Time: 11:00 AM - 12:45 PMSquamous Metaplasia May Persist after Pterygium Excision andLimbal-Conjunctival AutograftSoo Jeong Ryu 1 , Sang Beom Han 3 , Hee Kyung Yang 1 , Won RyangWee 2 , Joon Young Hyon 1 . 1 Ophthalmology, Seoul NationalUniversity Bundang Hospital, Seongnam-si, Gyeonggi-do, Republicof Korea; 2 Ophthalmology, Seoul National University Hospital, 101Daehak-ro Jongno-gu, Seoul 110-744, Republic of Korea;3 Ophthalmology, Samsung Medical Center, 81 Irwon-ro Gangnamgu,Seoul, Republic of Korea.Purpose: To investigate the changes of conjunctival epithelium at thepterygiumand donor graft sites after pterygium excision and limbal-conjunctivalautograft.Methods: This is a prospective study that included 16 eyes of 15patients. Impressioncytology specimens were obtained preoperatively and at 1, 3 and 6monthspostoperatively, at the site of the pterygium and donor graft site,respectively. In eachspecimen, changes of conjunctival epithelium were evaluated usingthe nucleus-to-cytoplasm(N/C) ratio and goblet cell density (GCD).Morphologic changes of cell andnucleus were also evaluated.Results: Preoperatively, Both N/C ratio and GCD were significantlyhigher inspecimens from pterygium site than those from graft harvest site. Atboth sites, GCDdecreased rapidly at 1 month after surgery, but showed gradualrecovery. Nosignificant difference in GCD was found between the two sites atpostoperative 1, 3and 6 months. Although there was no significant difference in theN/C ratio at 1 and 3month, pterygium site showed significantly higher N/C ratio thandonor graft site at 6months. At postoperative 6 months, changes suggesting squamousmetaplasia elongationof the cells and pyknotic changes in the nuclei-was noticed in 5samplesfrom the pterygium site, although these changes were never found atdonor graft site.Conclusions: Conjunctival epithelial metaplasia may persist evenafter pterygiumremoval and limbal reconstruction. Restoration of limbal barrierfunction may preventthe corneal involvement of the metaplastic conjunctival epithelialcells.Commercial Relationships: Soo Jeong Ryu, None; Sang BeomHan, None; Hee Kyung Yang, None; Won Ryang Wee, None;Joon Young Hyon, NoneSupport: None in the SupportProgram Number: 2127 Poster Board Number: D0354Presentation Time: 11:00 AM - 12:45 PMEffect of Nerve Growth Factor on in vitro Human PrimaryConjuntival Epithelial Cell Apoptosis Induced by HyperosmolarStressHungwon Tchah, Soon-Suk Kang, Eun-Soon Kim, Jae-yong Kim.Department of Ophthalmology, Asan Medical Center, Seoul,Republic of Korea.Purpose: To evaluate the effect of nerve growth factor (NGF) knownto be activated during inflammatory episodes of ocular diseases onapoptosis of cultured human primary conjunctival epithelial cells©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea(pHCEC). Conjunctival epithelial cells cover more than 90% of theocular surface. However, the role of conjunctival epithelial cells indry eye pathogenesis, where hyperosmolar stress may be important.Methods: For NGF transcription and production levels, RT-PCR andELISA were assessed with pHCECs in normal osmolar medium (307mOsm/L) or higher-osmolarity media, 350, 400, and 450 mOsm/L.For apoptosis analysis, pHCEC were cultured in normal- or 400mOsm/L osmolar medium with NGF neutralizing antibody orrecombinant human NGF for 6 hours and analysed by FACSCaliburflow cytometer. Bcl-xL, Bax, phospho-JNK, and cleaved caspase-3expression were detected by Western blotting. Inflammatorycytokine, IL-6 was analyzed using ELISA.Results: NGF levels in cultured pHCECs were up-regulated duringhyperosmolar conditions. Positive apoptotic cells significantly wereincreased in hyperosmolar-induced cells and NGF neutralizingantibody-added cells. When recombinant human NGF is added in 400mOsm/L osmolar medium, apoptotic cells were decreased andphospho-JNK, Bax, and cleaved caspase-3 expression were downregulatedbut Bcl-xL expression was increased. Upon hyperosmoticstress, IL-6 level increased and recombinant human NGF reduced IL-6 level.Conclusions: Our results suggest that hyperosmolarity inducesapoptosis of pHCECs by JNK signaling pathway. Up-regulated NGFunder hyperosmolar condition may contribute, at least in part, toreduce apoptosis of pHCEC and may be beneficial in recoveringconjunctival damage due to chronic hyperosmolar stress.Commercial Relationships: Hungwon Tchah, None; Soon-SukKang, None; Eun-Soon Kim, None; Jae-yong Kim, NoneSupport: NRF-2012R1A1A2003278Program Number: 2128 Poster Board Number: D0355Presentation Time: 11:00 AM - 12:45 PMUse of Evicel as a Fibrin Adhesive in Pterygium SurgeryLena Dixit 1, 2 , Todd R. Shepler 2 . 1 Baylor College of Medicine,Houston, TX; 2 University of Texas Southwestern at Austin, Austin,TX.Purpose: To evaluate Evicel as a safe and effective fibrin adhesivefor amniotic membrane graft fixation during pterygium surgery.Evicel is derived from pooled human plasma, whereas the goldstandardfibrin adhesive used in ophthalmology, Tisseel, containshuman and synthetic components. Tisseel requires extensivepreparation and handling prior to its use. Evicel may be equal ormore effective than Tisseel during pterygium surgery without thesame preparation and handling. Previous literature has shown thatEvicel’s clot integrity is mechanically superior to Tisseel’s whichmay be advantageous when fibrin glue is utilized as an adhesiverather than a hemostatic agent.Methods: Consecutive cases of 96 patients who underwent primarypterygium surgery with amniotic membrane placement from 1 weekto 8 months in the postoperative period were included in this study.Of these patients, 59 were treated with Evicel and 37 were treatedwith Tisseel to adhere an amniotic membrane graft to the sclera afterpterygium excision. Post-operative complications of recurrence,pyogenic granuloma, and conjunctival cysts were clinically assessed.Proportions of each complication were calculated and analyzed withstatistical significance at p < 0.05.Results: Mean patient age was 46 ± 12 (SD) (range 18-70) in theEvicel group, and 46 ± 11 (SD) (range 27-74) in the Tisseel group. Inthe Evicel group, 6.8% of patients developed a recurrent pterygium,6.8% developed a pyogenic granuloma, and 3.4% developed aconjunctival cyst. In the Tisseel group, 2.7% developed a recurrentpterygium, 2.7% developed a pyogenic granuloma, and 2.7%developed a conjunctival cyst. An unpaired two-sample t-testbetween proportions showed there was no difference in the rates ofpterygium recurrence (t value = 0.97, degrees of freedom = 93.9,two-tailed p value = 0.33), no difference in formation of pyogenicgranuloma (t value = 0.97, degrees of freedom = 93.9, two-tailed pvalue = 0.33), and no difference in formation of conjunctival cyst (tvalue = 0.2, degrees of freedom = 89.9, two-tailed p value = 0.84).Conclusions: There is no difference in the development ofpostoperative complications between Evicel and Tisseel when used asan adhesive during pterygium surgery. Evicel is a safe and effectivefibrin adhesive for amniotic membrane graft fixation after pterygiumexcision.Commercial Relationships: Lena Dixit, None; Todd R. Shepler,NoneProgram Number: 2129 Poster Board Number: D0356Presentation Time: 11:00 AM - 12:45 PMHLA-DR expression/mobilization on the conjunctival epithelialcells exposed to hyperosmolarity and desiccative stressChristophe Roubeix 1, 2 , Luisa Riancho 1, 2 , Christophe Baudouin 1, 3 ,Francoise Brignole-Baudouin 1, 4 . 1 Institut de la Vision, Paris, France;2 INSERM/UPMC Univ Paris 06, PARIS, France; 3 Quinze-VingtsNational Hospital of Ophthalmology, PARIS, France; 4 ParisDescartes University, Toxicology Department, PARIS, France.Purpose: To characterize the effects of two in vitro models of dryeye on the expression/mobilization of HLA-DR known as abiomarker of dry eye. We investigated the effect of hyperosmolarity(HO) and desiccation stress (DS) on the membrane expression andthe induction of HLA-DR mRNA as a first step to inflammation indry eye disease in correlation with other inflammatory biomarkerssuch as IL-6, IL-8.Methods: Hyperosmolar (HO) and desiccative stresses (DS) wereinduced on conjunctival cells (Wong-Kilbourne-derived of ChangConjunctiva, WKD). WKD were exposed either to NaCl-induced HOconditions (500mOsm) for 24h or to desiccating conditions byseeding them on transwell membranes (d: 2.4cm, pore 0.4 μm) andincubated in a drying chamber (relative humidity: 17±3%) for 6hafter cell culture medium removal from the upper side of thetranswell. We evaluated the effect of HO or DS on IL-6, IL-8 andHLA-DR mRNA expression by qRT-PCR. IL-6 and IL-8 weremeasured for each stress condition using ELISA assay. HLA-DR cellwas investigated using flow cytometry and immunofluorescenceanalyses.Results: Both HO and DS induced an increase of IL-6 and IL-8mRNA in WKD cells and a production of IL-6 and IL-8 in thesupernatant. HO and DS induced a mild increase of the HLA-DRmRNA synthesis and a translocation of the cytoplasmic HLA-DR tothe cell membrane confirmed by flow cytometry when compared tobasal conditions.Conclusions: By using these two in vitro dry eye models, weconfirmed the proinflammatory profile induced by hyperosmolar ordesiccating conditions on conjunctival cells. These conditions couldtrigger HLA-DR translocation at the cell membrane without thepresence of IFNγ released by immune cell infiltration. Theseobservations revealed the existence of a preliminary step in thechronic dry eye inflammation process.Commercial Relationships: Christophe Roubeix, laboratoiresTHEA (R); Luisa Riancho, None; Christophe Baudouin, None;Francoise Brignole-Baudouin, NoneSupport: Laboratoires THEAProgram Number: 2130 Poster Board Number: D0357Presentation Time: 11:00 AM - 12:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConjunctival Melanoacanthoma: A New Pigmented ConjunctivalEntitySaeed F. Al Wadani 1, 3 , Michael J. Mines 1, 2 , L. David Monroe 4 ,Charles Eberhart 1 . 1 Wilmer Eye Institute, Johns Hopkins University,Baltimore, MD; 2 Dept of Surgery, Ophthalmology Div, UniformedServices University, Bethesda, MD; 3 Ophthalmology, King SaudUniversity, Riyadh, Saudi Arabia; 4 Eye and Laser Institute, BocaRaton, FL.Purpose: A range of pigmented conjunctival lesions have beendescribed, but the nomenclature used to classify these tumors varies,and understanding of their underlying pathobiology is limited. Wereport the clinical and pathological features of a conjunctival lesionwith both melanocytic and epithelial components that does notclearly fit into the current classification scheme of pigmentedconjunctival entities. Rather, it has features similar to those of oralmelanoacanthoma, benign lesions which to our knowledge have notbeen previously reported on the ocular surface.Methods: Histopathological and immunohistochemical evaluation ofthe lesion was performed in the Johns Hopkins surgical pathologylaboratory using standard techniques.Results: The lesion arose on the perilimbal conjunctiva of a 48 yearold African American man as a solitary, well defined, reddishmovable plaque concerning for malignancy. On microscopicexamination, the lesion was characterized by thickened epitheliumwith downward growth of epithelial nests and strands highlighted bycytokeratin immunostains. Dense chronic inflammation was presentbelow the lesion. Diffusely admixed with the epithelial elements werean increased number of melanocytes immunoreactive for MITF,MART-1, S100 and HMB-45. Many of these appeared bland,dendritic and localized to the epithelial base, but pagetoid spread andmore compact cells were also noted. Ki67 revealed proliferation inboth the epithelial and melanocytic components.Conclusions: The microscopic features of this case do not clearlyconform to the existing classification of pigmented conjunctivallesions. It thus challenges current understanding of thepathophysiology of this class of entities, and raises questionsregarding appropriate clinical care. Interestingly, the lesion appearshighly similar to oral melanoacanthoma, a rare benign pigmentedlesion of the oral mucosa generally seen in black patients, andcharacterized by proliferation of both keratinocytes and HMB-45immunoreactive dendritic melanocytes. To our knowledge,conjunctival melanoacanthoma have not been previously reported.Melanoacanthoma are benign, but our conjunctival lesion hasfeatures which overlap with primary acquired melanosis with atypiaor melanoma in situ. Recognizing the potential of melanoacanthomato arise on the bulbar conjunctiva therefore may alter the diagnosisand treatments of patients with these lesions.Conjunctival MelanoacanthomaCommercial Relationships: Saeed F. Al Wadani, None; MichaelJ. Mines, None; L. David Monroe, None; Charles Eberhart, NoneSupport: Research to Prevent BlindnessMonday, May 06, 2013 2:45 PM-4:30 PMTCC 303 Paper SessionProgram #/Board # Range: 2189-2195Organizing Section: CorneaProgram Number: 2189Presentation Time: 2:45 PM - 3:00 PMFully Functional Bioengineered Lacrimal Gland Regeneration asan Organ Replacement Regenerative TherapyMasatoshi Hirayama 1 , Miho Ogawa 2 , Masamitsu Oshima 3 , TetsuyaKawakita 1 , Shigeto Shimmura 1 , Takashi Tsuji 2, 3 , Kazuo Tsubota 1 .1 Ophthalmology, Keio Univ School of Medicine, Tokyo, Japan;2 Organ Technologies Inc., Tokyo, Japan; 3 Research Institute forScience and Technology, Tokyo University of Science, Chiba, Japan.Purpose: Tear secreted from lacrimal gland plays a multifaceted roleto maintain a homeostatic microenvironment for ocular surface. Dryeye is an important public health problem, and it is expected todevelop a novel therapeutic treatment for the restoration of thelacrimal gland functions. Here, we report a successful fullyfunctioning lacrimal gland replacement achieved though thetransplantation of bioengineered lacrimal gland germ in adult mouse.Methods: The care and handling of animals were performed inaccordance with NIH guidelines. Protocols were approved by theAnimal Care and Use Committee. We had successfully demonstratedthe bioengineered lacrimal gland germ regeneration by organ germmethod (ARVO2012). We regenerated the bioengineered lacrimalgland germ and harderian gland germ, and transplanted them to adultlacrimal gland defect model mouse. The development ofbioengineered glands, histological structure including expression ofaquapolin-5 (AQP5), lactoferrin and lipids, tear secretion ability andocular surface protection effect were analyzed.Results: The bioengineered lacrimal gland germ and harderian glandgerm successfully achieved correct gland structure including acini,myoepithelial cells and nerve, followed by successful ductintegration. AQP5 and lactoferrin in the bioengineered lacrimalgland, and lipids in the bioengineered harderian gland werehistologically detected. The bioengineered lacrimal gland receivedappropriate neural control and had a secretion ability equivalent tothat of natural lacrimal gland. Tear from the bioengineered glandshad appropriate tear components such as lactoferrin. The ocularsurface status including fluorescein staining and corneal epithelialthickness was significantly improved in the bioengineered lacrimalgland transplantation mouse compared with that in the lacrimal glanddefect model mouse.Conclusions: We demonstrated that bioengineered lacrimal glandand harderian gland, which had the correct gland structure, couldproduce the tear followed by successful duct integration and restorethe lacrimal gland physiological functions in response to nervousstimulations, and could protect the ocular surface. This study thusrepresents a substantial advance and demonstrates the possibility ofnovel therapeutic approach for dry eye as a future organ replacementregenerative therapy.Commercial Relationships: Masatoshi Hirayama, None; MihoOgawa, None; Masamitsu Oshima, None; Tetsuya Kawakita,None; Shigeto Shimmura, None; Takashi Tsuji, None; KazuoTsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical(C), Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa(F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki(P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company(P)Support: None in the Support282 Dry Eye and Lacrimal Gland IIProgram Number: 2190©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 3:00 PM - 3:15 PMIdentification of Novel Epithelial Stem/Progenitor CellPopulation in Murine Uninjured Lacrimal GlandHelen P. Makarenkova 1 , Anastasia Gromova 1 , Dmitry Voronov 1, 2 ,Miya Yoshida 1 , Robyn Meech 3 . 1 Cell and Molecular biology, Thescripps Research Institute, La Jolla, CA; 2 Institute for InformationTransmission Problems, Russian Academy of Sciences, Moscow,Russian Federation; 3 Department of Clinical Pharmacology, FlindersUniversity, Bedford Park, SA, Australia.Purpose: The tear-deficient dry eye condition is an ocular-surfacedysfunction characterized by a lack of tear secretion from thelacrimal glands. The development of strategies to isolate andmanipulate lacrimal gland stem/progenitor cells from adult lacrimalgland tissue brings great promise for the design of cell replacementtherapies for patients with dry eye conditions.Methods: In this study we have examined murine lacrimal gland cellpopulations using ex vivo lacrimal gland cultures, FluorescenceActivated Cell Sorting (FACS), cell re-aggregation and clonalefficiency assays, immunostaining, q-RT-PCR microarrays, and invivo lacrimal gland regeneration assay.Results: Several recent studies indicate that multipotent progenitorcells are present in the lacrimal gland and are involved in lacrimalgland regeneration. Lacrimal gland epithelial lineage is representedby ductal, myoepithelial, and acinar cells that produce, modify, andsecrete the aqueous tear film layer. Analysis of stem cell-associatedtranscription factor expression in separated lacrimal gland epithelialand mesenchymal lineages showed that epithelial cells expressseveral epithelial-specific factors including Sox2, Sox6, and Sox9(stem cell markers), Foxp1 (forkhead transcription factor-1, regulatorof epithelial injury response), Six2, (Sine oculis-related homeobox),and Runx1 and 2 (runt family transcription factors). Moreover,expression of Runx1 was strongly upregulated in regeneratinglacrimal gland and this increase correlated with increase inexpression of epithelial stem/progenitor marker cytokeratin-5,suggesting that Runx1 is important for lacrimal gland regenerationand stem/progenitor cells expansion. Cell population analysis byFACS, 3D re-aggregate cultures and clonal efficiency assay showedthat c-kitdim/EpCam+/CD34-/Sca-CD45- cells expressed thepluripotency marker Oct-4 (octamer-binding transcription factor 4)and had elevated Runx1 expression and formed branched structuresexpressing E-cadherin in 3D cultures, suggesting that these cells maybe able to regenerate the epithelial component of the gland.Conclusions: These data support the existence of epithelial-specificstem/progenitor cells and a role for Runx1 in their regulation. Weanticipate that transplantation of epithelial-specific stem/progenitorcells could be used in the future to improve the function of damagedlacrimal glands.Commercial Relationships: Helen P. Makarenkova, None;Anastasia Gromova, None; Dmitry Voronov, None; MiyaYoshida, None; Robyn Meech, NoneSupport: NIH/NEI 1R21EY021292Program Number: 2191Presentation Time: 3:15 PM - 3:30 PMStem-like Cells in Serum Free In-vitro Cultures of HumanLacrimal GlandShubha Tiwari 1 , Mohammad J. Ali 1 , Murali Mohan Sagar Balla 1 ,Milind N. Naik 1 , Santosh Honavar 1 , Vijay Anand R. Palkonda 1 , GeetaK. Vemuganti 2, 1 . 1 Stem Cell Biology Lab, L V Prasad Eye Institute,Hyderabad, India; 2 School of Medical Sciences, University ofHyderabad, Hyderabad, India.Purpose: Tear film deficiency due to lacrimal gland dysfunction ordamage is an important cause of ocular morbidity. Restoration ofgland function by transplantation of autologous ex-vivo expandedstem cells located in the lacrimal gland is one of the options thatcould relieve this problem. We have previously reported the presenceof stem-like and functionally competent differentiated cells in in-vitrocultures of human lacrimal gland. The present study focuses on theformation of ‘lacrispheres’ under serum-free condition and theexpression of stemness in them.Methods: Fresh human lacrimal gland tissues (n=7) from patientsundergoing exenteration were harvested for cultures after IRBapproval. The gland was processed by enzymatic digestion using acocktail of collagenase and hyaluronidase. The isolated cells wereplated on ultralow attachment plates as group of two-three cells andfed with HepatoStim supplemented with epidermal growth factor,fibroblast growth factor and N2. The spheres were pulse-labeled withBrDU, analyzed for the expression of CD117 byimmunocytochemistry and their colony forming efficiency wasassessed on Matrigel.Results: Serum free cultures demonstrate spheres from humanlacrimal gland in-vitro within 2-3 days of plating. These spheresgrow in size over 3-14 days and can be serially passaged to generatesecondary spheres. Anti-BrDU labeling of these spheres indicate thepresence of 3.8% of high intensity cells at the periphery and about3% dull intensity cells at the center. These also show positivelabeling for CD117 and formation of clones in Matrigel (CFU 3.1%)indicating the presence of stemness.Conclusions: This is the first report on the generation of ‘lacrisphers’in human lacrimal gland cultures. It strengthens our initial reportsthat human lacrimal gland has a storehouse of stem cells that can bemaintained in-vitro and could possibly serve as potential source ofcell therapy for the regeneration of the functionally compromisedgland.Commercial Relationships: Shubha Tiwari, None; Mohammad J.Ali, None; Murali Mohan Sagar Balla, None; Milind N. Naik,None; Santosh Honavar, None; Vijay Anand R. Palkonda, None;Geeta K. Vemuganti, NoneSupport: International Atomic Energy Agency, C-Tracer, HERFProgram Number: 2192Presentation Time: 3:30 PM - 3:45 PMUmbilical mesenchymal stem cell transplantation for dry eye inthe mouse modelHongshan Liu, Yujin Zhang, Yong Yuan, Jianhua Zhang, Chia-yangLiu, Winston Kao. Ophthalmology, University of Cincinnati,Cincinnati, OH.Purpose: Dry eye is a multifactorial disease of the tear and ocularsurface that is characterized by T cell infiltration; and so far, there isno effective long-term cure for dry eye. We previously reported a bitransgenicdry eye mouse model of Krt12rtTA/tet-O-hIL-1b (IL-1βdriven by keratin 12 promoter) mimicking human dry eye afterdoxycycline induction. In present study, we used this mouse model totest the efficacy of umbilical mesenchymal stem cell (UMSC)transplantation in treating dry eye.Methods: UMSCs were transplanted into the corneal stroma andsubconjunctiva of bitransgenic mice following doxycycline-inductionfor 4 weeks and then removal of doxycycline for 12 weeks. In vivo,stereomicroscopy and HRT-II confocal microscopy were performedto evaluate the pathological change of ocular surface tissue atdifferent time points. Mice were sacrificed and ocular tissuesharvested were subjected to histology and immunofluorescencestaining to identify inflammatory and immune cell types andexpression of anti-inflammatory cytokines by UMSC.Results: HRT-II examination revealed that the intensity of cornealhaze was significantly decreased one week after UMSC©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatransplantation in comparison with un-treated corneas exhibitingprogressive corneal haze with times. Immunostaining with anti-CD4antibody showed that the infiltration of CD4+ cells was reduced inthe treated eyes. Immunostaining indicates that transplanted UMSCsare positive for interleukin-1 receptor antagonist (IL-1RA), but notIDO, PGE2, TSG-6, TSG-14, iNOS, and IL-10. To investigatewhether secreted IL-1RA is related to IL-1β stimulation, UMSCswere incubated in 10 ng/ml human IL-1β/α-MEM medium and theresults revealed an up-regulated level of IL-1RA expression.Conclusions: MSC transplantation may be a novel therapeuticstrategy for dry eye by suppressing T-cell infiltration into the ocularsurface tissues of dry eye.Commercial Relationships: Hongshan Liu, None; Yujin Zhang,None; Yong Yuan, None; Jianhua Zhang, None; Chia-yang Liu,None; Winston Kao, NoneSupport: NIH/NEI RO1 EY021768, Research to Prevent Blindness,Fight for Sight, and Ohio Lions eye Research FoundationProgram Number: 2193Presentation Time: 3:45 PM - 4:00 PMShort exposure to intense tear hyperosmolarity leads tofunctional alterations of the corneal nerves involved in tearingand/or ocular pain: Implications for dry eye diseaseHarumitsu Hirata, Michael L. Oshinsky, Nathan T. Fried. Neurology,Thomas Jefferson University, Philadelphia, PA.Purpose: The anterior eye encounters great variations in humidityand temperature in different atmospheric conditions such as desertand arctic environments that likely produce extreme hyperosmolarstress to the cornea. Two methods could be advanced to study theeffects of hyperosmolar stress on the corneal nerves. One is to followthe low dose over days or week, and the other is to apply extremelyhigh level for a short period (min-hour). This study investigated theshort-term effects of hyperosmolar stress on the corneal functions.Methods: In isoflurane-anesthetized rats, single extracellularrecordings were made from trigeminal ganglion while the cornea wasstimulated with hyperosmolar NaCl solutions, ocular dryness andtemperature changes. The corneal neurons excited by drying of thecornea (dry-sensitive corneal afferents) were studied.Results: One population of dry-sensitive corneal afferents that werealso sensitive to small temperature decrease of the corneal surfacewas greatly affected by the extreme hyperosmolar stress (1000 mOsmNaCl solutions) that was applied to ocular surface for only 30 min.Their responses to drying of the cornea, a stimulus thought to becritical for production of the tears, were significantly decreased oreven silenced after 30 min of hyperosmolar stress that lasted for atleast 3 hrs under the same conditions. Their responses to temperaturechanges of the cornea (19oC cooling and 13oC heating), on the otherhand, were greatly enhanced by the hyperosmolar stress after a 15min ocular application of 800-1000 mOsm NaCl solutions. Thesechanges in excitability of these neurons were not often reversed 15-30 min after replacing the hyperosmolar tears with the normal (~300mOsm) tears.Conclusions: A theoretical model predicted that small spots on thecorneal surface contain hyperosmolar tears as high as 900 mOsmduring the tear breakup. If this were confirmed experimentally, ourresults suggest that short period of these extreme hyperosmolarconditions of the eye is expected to produce immeasurably graveconsequence on the functions of the corneal nerves responsible fortearing and ocular pain sensation. Our results, thus, demonstrate theimportance of these extreme conditions in induction of early stages ofdry eye disease.Commercial Relationships: Harumitsu Hirata, None; Michael L.Oshinsky, None; Nathan T. Fried, NoneSupport: NIH Grant EY020667Program Number: 2194Presentation Time: 4:00 PM - 4:15 PMMice with Desiccating Stress-Induced Dry Eye DevelopTrigeminal Neuralgia, Despite Decreased Corneal Sensitivity andNerve Fiber DegenerationChris S. Schaumburg 1 , Katherine S. Held 1 , Euikyun Oh 1 , SvetiUgarte 1 , Larry A. Wheeler 1 , Margarita Calonge 2 , Jerry Y.Niederkorn 3 , Stephen C. Pflugfelder 4 , Robert I. Fox 5 , Michael E.Stern 1 . 1 Biological Sciences, Allergan, Irvine, CA; 2 IOBA, Universityof Valladolid, Valladolid, Spain; 3 Ophthalmology, UT SouthwesternMed Center, Dallas, TX; 4 Ophthalmology, Baylor College ofMedicine, Houston, TX; 5 Division of Reumatology, ScrippsMemorial Hospital, Lo Jolla, CA.Purpose: Pain is a major symptom of patients suffering from chronicDry Eye disease. To begin to understand if the immunopathogenesisof desiccating stress (DS)-induced Dry Eye is associated with a painresponse, corneal sensitivity, corneal nerve fiber density,neuropeptides and trigeminal neuralgia were evaluated over thecourse of disease.Methods: Dry Eye disease was induced by exposing femaleC57BL/6 mice to desiccating stress (DS: subcutaneous scopolamine(1.0mg/0.2ml) TID; humidity

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneause of a high resolution MRM approach to develop assays forbiologically important tear proteins.Methods: Human tear samples were collected from 1000 consentingpatients with no eye complaints (411 male, 589 female, average age55.5 years, SD 14.5 years) using the Schirmer tear test strips andpooled into a single global control sample. Quantification of proteinsis carried out by selecting “signature” peptides derived by trypsindigestion of the target protein. A 2-hour nanoLC-MS/MS run wasused to separate the tryptic peptides and perform quantitation ofhuman tear proteins in HR-MRM mode. Samples were analyzed intriplicate. Twenty-one high abundant proteins were further accessedfor signal reproducibility.Results: Fifty-three peptide assays that represent 51 high andintermediate abundant tear proteins were developed. All assaysshowed consistent retention time with a coefficient of variation (CV)of less than 2%. As for peak area, 17 out of the 21 assays showed areproducible peak area with CV less than 20%. Some wellcharacterized tear proteins, lacritin, mammaglobin-B, S100A4,S100A8, and prolactin inducible protein (PIP) showed the highestreproducibility of peak area, with CV less than 2%.Conclusions: These multiplexed MRM-based assays show greatpromise to be further developed for biomarker validation in humantear samples.Commercial Relationships: Lei Zhou, Singapore Eye ResearchInstitute (P); Louis Tong, None; Roger W. Beuerman, Allergan (F),SERI (P), Santen (R)Support: CG from NMRC, Singapore, Core facility funding fromSingHealth Foundation283 EndotheliumMonday, May 06, 2013 2:45 PM-4:30 PMTCC 304 Paper SessionProgram #/Board # Range: 2196-2202Organizing Section: CorneaProgram Number: 2196Presentation Time: 2:45 PM - 3:00 PMDecline in DJ-1 Leads to Decreased Nuclear Translocation ofNrf2 and Results in p53-mediated Apoptosis of Human CornealEndothelial CellsCailing Liu, Yuming Chen, Ula V. Jurkunas. Schepens/MassachusettsEye and Ear, Department of Ophthalmology, Harvard MedicalSchool, Boston, MA.Purpose: Dowregulation of DJ-1 and decreased nuclear localizationof nuclear factor erythroid-derived 2-like 2 (Nrf2) has been detectedin Fuchs endothelial corneal dystrophy endothelium. DJ-1 has beenreported to stabilize Nrf2, which binds to antioxidant responseelement in the nucleus to protect cells from apoptosis. In this study,we investigated the effect of DJ-1 downregulation on Nrf2 nucleartranslocation, Nrf2-associated protein regulation, and cornealendothelial cell susceptibility to oxidative stress.Methods: An immortalized normal human corneal endothelial cellline (HCECi) was transfectedwith 50 nM of siRNA specific to the human DJ-1 gene usingLipofectamine. Scrambled siRNA was used as control. Nrf2 and p53levels in nuclear and cytosolic extracts were evaluated by westernblotting. Nrf2-associated proteins such as Cul3 and Keap1 weredetected by immunoprecipitation (IP) with anti-Nrf2 antibody,followed by western blotting with target antibodies. Oxidative stresswas induced by exposing HCECi cells to a UVA broadband lampwith the fluence of 10 J/cm 2 . Cell pellets were harvested for westernblotting with anti-caspase 3 and p53 antibodies, while supernatantswere collected for a cell viability assay.Results: Despite similar levels of cytoplasmic Nrf2, nuclear Nrf2protein levels decreased by 2.2-fold (p=0.04) in DJ-1 siRNA-treatedHCECi cells as compared to scrambled siRNA-treated cells. IPstudies detected Nrf2 association with Cul3 and Keap1 with anincrease in Cul3-Nrf2 complex in DJ-1 siRNA-treated cells relativeto controls. UVA irradiation led to an 8.5% increase in cell death inDJ-1 siRNA-treated cells as compared to controls. Moreover,downregulation of DJ-1 led to a 16.4% increase in active caspase 3levels. This effect was augmented by UVA treatment, which led to a28.6% increase in caspase 3 as compared to controls. Downregulationof DJ-1 resulted in an increase in cytoplasmic p53 levels, while UVAirradiation resulted in a 17.7% increase in total p53 levels in DJ-1siRNA treated cells as compared to controls.Conclusions: Downregulation of DJ-1 impairs nuclear translocationof Nrf2 potentially by targeting Nrf2 for degradation through Cul3and Keap1 pathways. Decline in DJ-1 levels leads to heightened cellsusceptibility to UVA-induced apoptosis and activates p53-dependentpathway in corneal endothelium.Commercial Relationships: Cailing Liu, None; Yuming Chen,None; Ula V. Jurkunas, 61/482,769 (P), Altheos (C)Support: NIH/NEI Grant R01 EY20581 (UVJ), a Research toPrevent Blindness Award (UVJ)Program Number: 2197Presentation Time: 3:00 PM - 3:15 PMMicroRNA Analysis in Fuchs Endothelial Corneal DystrophyMario Matthaei 1, 2 , Jianfei Hu 1 , Laura Kallay 1 , Claus Cursiefen 2 ,Jiang Qian 1 , Albert S. Jun 1 . 1 Anterior Segment / Cornea, Wilmer EyeInst, Johns Hopkins Univ, Baltimore, MD; 2 Department ofOphthalmology, University of Cologne, Cologne, Germany.Purpose: MicroRNAs (miRNAs) are a class of endogenousnoncoding RNA which posttranscriptionally modulate geneexpression during development and disease. Our study investigatedthe differential miRNA expression pattern in human FuchsEndothelial Corneal Dystrophy (FECD) compared to normalendothelium.Methods: Total RNA was extracted from corneal endothelial cells ofFECD eyes (n=6) and age-matched normal autopsy globes (n=6). Theexpression of 754 well characterized miRNA sequences was analyzedin preamplified cDNA samples using OpenArray plate technology onthe QuantStudio 12K Flex system for high-throughput real-timequantification and individual targets were validated by Taqman qPCRassays.Results: We detected differential expression of at least 37microRNAs using global normalization and applying a fold-changeof 2 and p=0.05 as cut-off values. Of these 37 miRNAs, 32 showeddownregulation whereas 5 miRNAs were upregulated. MiRNA targetgenes were predicted and further analyzed by gene ontologyassessment.Conclusions: We present the first endothelial cell microRNA profilein FECD and normal endothelial samples and identify dysregulatedmiRNA expression in FECD.Commercial Relationships: Mario Matthaei, None; Jianfei Hu,None; Laura Kallay, None; Claus Cursiefen, Gene Signal (C),Alcon (R), Allergan (R), Bayer (R); Jiang Qian, None; Albert S.Jun, Johns Hopkins University (P)Support: Deutsche Forschungsgemeinschaft (DFG MA 5110/2-1 toM.M.), Richard Lindstrom/Eye Bank Association of AmericaResearch Grant (to M.M.), National Institutes of Health (EY019874to A.S.J.), Research to Prevent Blindness (to Wilmer Eye Institute)Program Number: 2198©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 3:15 PM - 3:30 PMIdentification of New markers of Human Corneal EndothelialCellsJodhbir S. Mehta 1 , Adrian Cheong 2 , Gary S. Peh 1 , William Sun 2 .1 Cornea Refractive Tissue Engineering, SNEC / SERI, Singapore,Singapore; 2 Experimental Therapeutic Unit, A Star, Singapore,Singapore.Purpose: There are no specific markers of human corneal endothelialcells (HCEC). Currently, the identification of HCEC in culture reliesmainly on expression of pump or tight junction markers.The aim ofthis study is to describe new HCEC markers.Methods: Isolated human corneal DM-HCEC and remaining stromafrom corneal donors were homogenized individually for RNAsequencing. RNA-seq libraries were prepared using AB protocol fornon-barcoded libraries and SOLiD fragment library for barcodedlibrary. The results were processed using ABI Bioscope. Geneexpression was measured by counting the number of reads mappinguniquely to both strands of each gene footprint. Results were verifiedby quantitative PCR, BD lyoplate screening, immunofluorescenceand flow cytometry using cultivated primary HCEC isolated andgrown to the third passage.Results: RNA-seq showed 5 genes that were over expressed in theHCEC-DM compared to stromal fibroblasts, GPC4, CNTN6,SLC9A7, PVRL3, and SLC4A4. The resulting over-expression ofthese genes was confirmed on comparing qPCR data from culturedHCEC and stromal fibroblasts. BD lyoplate identified CD104 andCD200 as potential markers. On immuno-histochemistry anti-GPC4,anti-CD200 and anti-SLC4A cell-surface antibodies clearly stainedthe the endothelial layer specifically. In a population of pre-CMFDAlabelled stromal fibroblasts and HCEC, mixed in a 1:1ratiofluorescent-activated cell sorting (FACS) showed a 96% recovery ofHCEC when sorted with anti-GPC4 and a 79.8% with anti-CD200.Conclusions: By RNA sequencing verified on qPCR andimmunohistochemistry we have identified two novel cell surfaceantigens on human corneal endothelial cells. These markers may beused to aid cell purification during harvesting or in the identificationof cultured HCEC to ensure quality control of cultured cells.Commercial Relationships: Jodhbir S. Mehta, None; AdrianCheong, None; Gary S. Peh, Singapore Eye Research Institute (P);William Sun, NoneSupport: NMRC TCRP 2011Program Number: 2199Presentation Time: 3:30 PM - 3:45 PMTight Junction Transmembrane Protein Claudin SubtypeExpression and Distribution in Human Corneal EndotheliumEmi Inagaki, Shin Hatou, Satoru Yoshida, Hideyuki Miyashita, KazuoTsubota, Shigeto Shimmura. Ophthalmology, Keio University,Tokyo, Japan.Purpose: The primary function of corneal endothelium is to maintaincorneal transparency by regulating corneal hydration and nutritionmodulated by barrier and metabolic pump function. However, themolecular mechanism of its barrier function is still relativelyunknown. Claudins, recently identified main components of tightjunctions,are a family of four-transmembrane-spanning proteins. Todate, 24 subtype of claudins have been identified in human.Combination of claudin subtypes may contribute not only to thetightness of tight junction strands but also ion-selective channels. Inthis study, we investigated the expression and pattern of claudins inin vivo human cornea.Methods: The experiments in this paper used remained corneal tissuesupplied from USA eye bank after the central buttons were used forcorneal transplantation. We stripped corneal endothelium withDescemet membrane and corneal epithelium from corneal stroma.Reverse transcription-polymerase (RT-PCR) was performed toevaluate that subtypes of claudins expressed in corneal endothelium,stroma and endothelium. Then, immunohistochemistry wasperformed for claudins positively expressed in RT-PCR, to confirmwhether they would express lateral side of corneal endothelium.Results: Transcripts for claudin-1, -2, -3, -4,-7, -10b, -11,-12,-14, -15, -22, -23, and -24 were identified in human corneal endothelium inRT-PCR. Claudin-1, -2, -3, -4, -7 , -11, -12, -14, -15, -22, -23, and -24 expression in corneal endothelium was common to cornealepithelium, and claudin-1, -2,-3,-4, -7, -11,-12,-14, -15, -22,-23,and -24 was common to corneal stroma. Among the claudin subtypesexpressed in corneal endothelium, immunohistochemistry revealedthe expression of claudin-1,-2, -4, -7, -10 antibodies showed bandsthat correspond to the junctional complex. Claudin-11 was alsoexpressed in corneal endothelium, however, the expression was notcontinuous but in a dotlike pattern along cell junctions.Conclusions: Claudin-1, -2, -4,-7, -10b and -11 are expressed incorneal endothelium. This combination of claudin subtype maycontribute to the uniqueness of barrier integrity and ion sensitivity incorneal endothelium.Commercial Relationships: Emi Inagaki, None; Shin Hatou,None; Satoru Yoshida, None; Hideyuki Miyashita, None; KazuoTsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical(C), Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa(F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki(P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company(P); Shigeto Shimmura, NoneProgram Number: 2200Presentation Time: 3:45 PM - 4:00 PMCorneal endothelial cells provide evidence of accelerated cellularsenescence associated with HIV infection: a case-control studySophia Pathai 1, 2 , Stephen D. Lawn 3, 2 , Paul G. Shiels 5 , Helen A.Weiss 4 , Colin Cook 6 , Robin Wood 2 , Clare E. Gilbert 1 . 1 InternationalCentre for Eye Health, London School of Hygiene & TropicalMedicine, London, United Kingdom; 2 Desmond Tutu HIV Centre,University of Cape Town, Cape Town, South Africa; 3 Dept ofClinical Research, London School of Hygiene & Tropical Medicine,London, United Kingdom; 4 MRC Tropical Epidemiology Group,London School of Hygiene & Tropical Medicine, London, UnitedKingdom; 5 Dept of Epigenetics, Institute of Cancer Sciences,University of Glasgow, Glasgow, United Kingdom; 6 Dept ofOphthalmology, University of Cape Town, Cape Town, South Africa.Purpose: Cellular senescence may be a key factor in HIV-relatedpremature biological aging. We assessed features of the cornealendothelium that are known to be associated with biological aging,and cellular senescence markers in HIV-infected adults.Methods: Case-control study of 242 HIV-infected adults and 249matched controls. Using specular microscopy, the cornealendothelium was assessed for features of aging (low endothelial celldensity [ECD], high variation in cell size, and low hexagonalityindex). Data were analysed by multivariable regression. CDKN2A (acell senescence mediator) and 8-hydroxy-2′-deoxyguanosine (anoxidative DNA damage marker) were measured in peripheral bloodleukocytes.Results: The median age of both groups was 40 years. Among HIVinfectedadults, 88% were receiving antiretroviral therapy (ART);their median CD4 count was 468 cells/μL. HIV infection wasassociated with increased odds of variation in cell size (OR=1.67;95%CI: 1.00-2.78, p=0.04). Among HIV-infected participants, lowECD was independently associated with current CD4 count

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaART with undetectable viral load, CDKN2A expression and 8-OHDG levels were higher in those with accelerated aging, asreflected by lower ECD.Conclusions: The corneal endothelium shows features consistentwith HIV-related accelerated senescence, especially among thosewith poor immune recovery.Commercial Relationships: Sophia Pathai, None; Stephen D.Lawn, None; Paul G. Shiels, Pathfinder Cell Therapy (F), SanofiAventis (C); Helen A. Weiss, None; Colin Cook, None; RobinWood, None; Clare E. Gilbert, NoneSupport: Wellcome Trust 090354/Z/09/ZProgram Number: 2201Presentation Time: 4:00 PM - 4:15 PMEfficacy and Safety Evaluation of Cell-Injection Therapy usingCultivated Human Corneal Endothelial CellsNoriko Koizumi 1, 2 , Naoki Okumura 1, 2 , Takashi Shiina 3 , ShingoSuzuki 3 , Shinichiro Nakamura 4 , Yuji Sakamoto 1 , Kenta Yamasaki 1 ,Morio Ueno 2 , Junji Hamuro 2 , Shigeru Kinoshita 2 . 1 BiomedicalEngineering, Doshisha University, Kyotanabe City, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan; 3 Molecular Life Sciences, Tokai University School ofMedicine, Isehara, Japan; 4 Research Center for Animal Life Science,Shiga University of Medical Science, Otsu, Japan.Purpose: To investigate the efficacy and safety of cornealendothelial reconstruction by a cell-injection therapy using cultivatedhuman corneal endothelial cells (HCECs) in a corneal endothelialdysfunction monkey model.Methods: The corneal endothelium of 8 eyes of 8 monkeys wasintensively scraped off up to the peripheral area to create a cornealendothelial dysfunction model. A 5.0 x 10 5 amount of cultivatedHCECs, labeled by fluorescein marker DiI, was then injected into theanterior chamber of those 8 eyes. Slit-lamp examinations andintraocular pressure (IOP) measurements of the cell-injected eyeswere then performed on postoperative day 1 through day 14.Fourteen days after cell-injection, 31 primary organs were harvestedfrom each animal after euthanasia and were set aside for histologicalexamination. To examine the ectopic deposition of donor HCECssystemically, tissue sections obtained from those 31 organs wereexamined by fluorescein microscopy to search for DiI-positive donorcells. Genomic DNA was then extracted from tissue samples of 31organs and examined by polymerase chain reaction (PCR) usinghuman-specific primers to elucidate if it was contaminated with anydonor-HCEC-derived DNA.Results: Seven days after cell-injection, 6 of the 8 eyes (75%)recovered corneal clarity. No undesirable accumulation of HCECs inocular tissue, elevation of IOP, or systemic side-effects was observed.Histological examination of the 31 organ-tissue samples revealed notumor genesis or inflammatory response. Moreover, PCRexamination of those samples revealed no genomic DNA derivedfrom the donor HCECs.Conclusions: The findings of this present study indicate that cellinjectiontherapy using cultivated HCECs is a safe and effectiveprocedure, and one that might be clinically applicable for thetreatment of corneal endothelial dysfunction.Commercial Relationships: Noriko Koizumi, None; NaokiOkumura, None; Takashi Shiina, None; Shingo Suzuki, None;Shinichiro Nakamura, None; Yuji Sakamoto, None; KentaYamasaki, None; Morio Ueno, None; Junji Hamuro, None;Shigeru Kinoshita, Senju Pharmaceutical Co (P), SantenPharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),AMO (R), HOYA (R)Support: The Funding Program for Next Generation World-LeadingResearchers from the Cabinet Office in Japan ( LS117)Program Number: 2202Presentation Time: 4:15 PM - 4:30 PMActivation of the Rho/ROCK Signaling Pathway in the Apoptosisof Corneal Endothelial CellsNaoki Okumura 1, 2 , Ai Odajima 1, 2 , EunDuck P. Kay 1 , Wen Chen 1 ,Morio Ueno 2 , Junji Hamuro 2 , Shigeru Kinoshita 2 , Noriko Koizumi 1 .1 Biomedical Engineering, Doshisha University, Kyotanabe, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan.Purpose: In the pathological condition of corneal endotheliumassociated with Fuchs’ dystrophy or post keratoplasty, apoptosis isknown to be involved. We previously reported that Rho kinase(ROCK)-inhibitor Y-27632 suppresses the apoptosis of culturedcorneal endothelial cells (CECs). The purpose of this present studywas to evaluate the involvement of the Rho/ROCK signaling pathwayin the apoptosis of CECs and the effect of ROCK inhibitor onmodulating apoptosis.Methods: Monkey corneal endothelial cells (MCECs) were culturedand then exposed to ultra-violet (UV) radiation (100J/m 2 ) to induceapoptosis. To elucidate the involvement of the Rho/ROCK signalingpathway in apoptosis, RhoA-GTPase, myosin light chain (MLC)phosphorylation, and the cleavage of caspase 3 and ROCK 1 wasevaluated by western blotting. Anti-blebbing effect of Y-27632 wasevaluated after exposure to UV radiation by time-lapse phase contrastmicroscopy, and actin and MLC phosphorylation were evaluated byimmunostaining. Annexin V staining was employed to confirm theanti-apoptotic effect of Y-27632.Results: UV radiation caused cell death in MCECs in a dosedependentmanner; a significant cell death was observed at100J/m 2 and higher dosages. Active GTPase pull-down assay showedthat UV radiation activated RhoA of the MCECs. MLCphosphorylation and the cleavage of caspase 3 and ROCK 1 wereincreased by UV radiation. Compared to the control, Y-27632-treatedMCECs exposed to UV radiation showed significantly decreasedmembrane blebbing (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaExhibit Hall Poster SessionProgram #/Board # Range: 2558-2608/D0358-D0408Organizing Section: CorneaContributing Section(s): Anatomy/PathologyProgram Number: 2558 Poster Board Number: D0358Presentation Time: 2:45 PM - 4:30 PMEffects of smoking on corneal healing timeJacquelyn Jetton 1 , Donald Stone 1 , Yoonsang Kim 2 . 1 Ophthalmology,University of Oklahoma - Dean McGee Eye Institute, OklahomaCity, OK; 2 Biostatistics and Epidemiology, University of OklahomaCollege of Public Health, Oklahoma City, OK.Purpose: To determine the effects of smoking on healing of cornealabrasions and keratitis.Methods: A retrospective study of corneal abrasion and cornealulcer. Primary outcome measured is time to epithelial closurecompared between smokers and nonsmokers. Comorbidities studiedwere neurotrophic corneas, glaucoma, previous PK, atopy andsjogren’s/rheumatoid arthritis (RA). The data were analyzed usingKaplan Meier curves and Proportional Hazards models.Results: Eighty seven patients with corneal abrasions met inclusioncriteria. Sixty six percent of the patients were nonsmokers and 33%were smokers. Mean healing time was 4.8 days in nonsmokers and5.86 days in smokers (p-value 0.01). A Hazards Ratio (HR)

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaalso performed using the conditioned media. Quantification of theeffect was done in a masked fashion. All data was analyzed forstatistical significance using Prism statistical software.Results: Increased HIF-1 expression was seen at 4,6, and 8 hourspost-ONOO- exposure (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 2:45 PM - 4:30 PMOverexpression of SIRT1 promotes high glucose-attenuatedcorneal epithelial wound healing via p53 regulation of theIGFBP3/IGF-1R/AKT pathwayYe Wang, Lixin Xie. Shandong Eye Institute, Qingdao, China.Purpose: Diabetes is one of the risk factors that lead to cornealkeratopathy and the deficiency in epithelium wound healing isrelatively common in persons with diabetes mellitus, leading tocorneal ulceration and subsequent vision loss. This study investigatedthe promotive effects of SIRT1 on high glucose-attenuated cornealepithelial wound healing via p53 regulation of the IGFBP3/IGF-1R/AKT pathway.Methods: Effects of high glucose on SIRT expression were assessedin primary human corneal epithelial cells and corneas fromIns2Akita/+mice by Western blotting. SIRT1 was activated byectopic expression. Effects of p53 as a key regulational factor andtargets AKT pathway in response to high glucose induced woundingwere investigated in corneal epithelial cells. Effects ofoverexpression of SIRT1 promotes high glucose-attenuated cornealepithelial wound healing via p53 regulation of the IGFBP3/IGF-1R/AKT pathway were investigated in corneal epithelial cells andIns2Akita/+mice.Results: High glucose induces downregulation of SIRT1 andupregulation of p53 acetylation in primary human corneal epithelialcells (CECs) and corneas from Ins2Akita/+mice. Treatment withPifithrin-α (PFT-α) dramatically decreased total p53 and acetylatedp53 expression and AKT pathway was activated in CECs. Acetyl-p53was also increased by the histone deacetylase (HDAC) class I/IIinhibitor trichostatin A (TSA) and AKT pathway was inactivated.This post-translational modifications of the p53 protein was alsoinvolved in response to high glucose induced wounding in CECs,suggesting p53 as a key regulational factor and targets AKT pathwayin corneal epithelium wound healing. Furthermore, SIRT1overexpression completely promotes high glucose-attenuated woundhealing in CECs and corneas from Ins2Akita/+mice with the downregulationof IGFBP3 protein. The molecular mechanism by whichSIRT1 promotes corneal epithelial wound healing appeared toinvolve the enhancement of IGFBP3/IGF-1/AKT pathway throughdeacetylation of p53.Conclusions: These results will provide a valuable information intothe mechanisms underlying corneal epithelial cells wound healingwith diabetes mellitus. This study also suggest a protective role ofSIRT1 in the pathogenesis of diabetic keratopathy and the regulationof SIRT1 as a possible target to attenuate high glucose-attenuatedcorneal epithelial wound healing.Commercial Relationships: Ye Wang, None; Lixin Xie, NoneProgram Number: 2564 Poster Board Number: D0364Presentation Time: 2:45 PM - 4:30 PMConditional Deletion of Notch1 in Mouse Corneal EpitheliumLeads to the Loss of the Epithelial Barrier FunctionAsadolah Movahedan 1 , Neda -. Afshar 1 , Hossein M. Sagha 1 , RobertM. Lavker 2 , Ali R. Djalilian 1 . 1 Ophthalmology & Visual Sciences,Univ of Illinois at Chicago, Chicago, IL; 2 Dermatology,Northwesrtern University, Chicago, IL.Purpose: To investigate the mechanisms that lead to keratinizationafter conditional deletion of Notch1 in the corneal epithelium.Methods: Notch1 was conditionally deleted in 3 month old Notch1flox/flox, K14-Cre-ERT +/- mice using intra-peritoneal injection of 1mg/day 4-hydroxy-tamoxifen(4-OHT) for 5 days. Eyes were seriallymonitored after 4-OHT treatment by slit lamp examination andfluorescein staining. Aqueous tear measurement was performed usingphenol red thread test. Barrier function was also assessed by thepenetration of sulfo-NHS-LC-Biotin. Immuno-fluorescence stainingfor ZO-1 was used to assess tight junctions in corneal epithelial cellscultures isolated from conditional Notch1-/- and wildtype mice.Results: Slit lamp examination after tamoxifen injection indicatedprogressive increase in corneal fluorescein staining before the onsetof corneal opacification and keratinization. Sulfo-NHS-LC-Biotinpenetrated into the corneas confirming the barrier impairment. Themean aqueous measurement for Notch1 KO group with evidence ofcorneal keratinization was significantly higher than wildtype mice(7.45 ± 2.3 mm vs 3.66 ± 1.4 mm) (p= 0.002) indicating increasedlacrimal production. Histologically, progressive loss of meibomianglands was observed in the Notch1 KO mice (as described byVauclair et al before), however this did not correlate with theepithelial barrier changes. There was no apparent difference in thegoblet cells between conditional Notch1 KO and wildtype mice. In acalcium switch experiment in vitro Notch1 -/- cells demonstrateddelayed membrane localization of ZO-1 compared to wildtype cells.Conclusions: Conditional deletion of Notch1 in corneal epitheliumleads to an impairment in the epithelial barrier in part due to impairedtight junction formation. These findings highlight the role of Notch1in epithelial differentiation and suggests that intrinsic defects in theepithelial barrier may be a contributing factor to the development ofsquamous metaplasia and inflammatory keratinization in these mice.Commercial Relationships: Asadolah Movahedan, None; Neda -.Afshar, None; Hossein M. Sagha, None; Robert M. Lavker, None;Ali R. Djalilian, NoneSupport: K08EY017561-A1 grant to A.R.D., a core grant EY01792from the National Institutes of Health, and by the Cless FamilyFoundation. A.R.D. is the recipient of a Career Development Awardfrom Research to Prevent BlindnessProgram Number: 2565 Poster Board Number: D0365Presentation Time: 2:45 PM - 4:30 PMWnt2 and beta-catenin signaling are important for cornealwound healing in diabetic ratsHAIJING SUN, Feng Wang, Ilham Bettahi, Fushin X. Yu. Waynestate university, Detroit, MI.Purpose: Delayed healing of the corneal epithelial defect may beassociated with sight-threatening complications. To understand themechanisms underlying delayed epithelial wound healing in diabeticcorneas, we investigated Wnt signaling pathway and its function inhealing CECs of normal and type I diabetes mellitus (DM) ratcorneas.Methods: To create an epithelial wound, corneal epithelial cellsmarked by 5 mm trephine were removed and collected fromstreptozotocin (STZ) and age-matched Sprague-Dawley rats. Woundhealing was monitored by fluorescein staining. At 40 hours postwounding, Wnt2 expression was determined by RT-PCR, Westernblot analysis. The distribution of Wnt2 and beta-catenin in rat corneaswas examined by Immunofluorescence staining.Results: Our genome-wide cDNA array results showed that theexpressions of multiple Wnts were altered in response to wounding.Among these Wnts, Wnt2 expression was further downregulated indiabetic corneal epithelium. RT- and realtime PCR confirmed thedifferential expression pattern of Wnt2. Both Western blotting andimmunohistochemistry revealed that Wnt2 expression was downregulatedin migrating epithelial sheet of normal cornea and almostundetectable in DM cornea. Moreover, beta-catenin, a Wnt2downstream signaling protein, was also shown an altered distributionin healing corneas.Conclusions: Our results suggest Wnt signaling is important forcorneal epithelial wound healing and that downregulation of Wnt2©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamay be responsible, in part, for the delayed epithelial wound closurein diabetic rat corneas.Commercial Relationships: HAIJING SUN, None; Feng Wang,None; Ilham Bettahi, None; Fushin X. Yu, NoneSupport: R01 EY017960Program Number: 2566 Poster Board Number: D0366Presentation Time: 2:45 PM - 4:30 PMClinical Factors Can Predict the Outcome of AutologousCultivated Limbal Epithelial TransplantationAnupam Bagdi 1 , Sayan Basu 1 , Hasnat Ali 2 , Virender S. Sangwan 1 .1 Cornea, L V Prasad Eye Institute, Hyderabad, India; 2 Biostatisticsand Clinical Epidemiology, L V Prasad Eye Institute, Hyderabad,India.Purpose: It is difficult to prognosticate the outcome of stem cellbased therapy for ocular surface disease because the risk factorspredisposing to failure of surgery are not clearly known.To addressthis issue,this study aimed to identify the clinical Indicatorsassociated with failure of autologous cultivated limbal epithelialtransplantation for the treatment of Limbal Stem Cell Deficiency(LSCD).Methods: This retrospective study included 526 eyes of patientssuffering from unilateral LSCD following ocular surface burns whounderwent autologous ex-vivo cultivated limbal epithelialtransplantation between 2001 and 2011. This procedure involvedobtaining a one-clock hour wide limbal biopsy from the healthyfellow eye and cultivating the limbal epithelial cells on a deepithelisedhuman amniotic membrane graft for 10-14 days. Aconfluent epithelial sheet along with the amniotic membrane graftwas then transplanted onto the affected eye after clearing away thepathologic tissue covering the cornea. Post-operatively success oftransplantation was defined as a completely epithelized, avascularand clinically stable corneal surface. A multivariate analysis wasperformed using multiple logistic regression to analyze the strengthof association between the pre-operative, intra-operative and postoperativeclinical factors and recurrence of LSCD.Results: Of the 526 eyes, the ocular surface was successfullyrestored in 292 (55.5%) eyes at a mean follow-up of 1.4 years.Among all the pre-, intra- and post-operative clinical factors thatwere assessed: presence of symblepharon (OR=1.2, P< 0.001),previous ocular surgeries (OR= 1.3, P= 0.02), pediatric age group(OR=1.4, P= 0.04) and simultaneous keratoplasty (OR=3.2, P= 0.02)were found to be associated with greater chances of failure ofautologous cultivated limbal epithelial transplantation.Conclusions: Patients with extensive symblepharon, patientspreviously having undergone multiple ocular reconstructiveprocedures, children and patients requiring keratoplasty along withautologous limbal transplantation are at higher risk of recurrence ofLSCD and need to be counseled accordingly.Commercial Relationships: Anupam Bagdi, None; Sayan Basu,None; Hasnat Ali, None; Virender S. Sangwan, NoneSupport: Partnership grant from Champalimaud Foundation, Lisbon,Portugal; Hyderabad Eye Research Foundation, Hyderabad, India;Department of Biotechlonology, New Delhi, India(BT/01/COE/06/02/10)Program Number: 2567 Poster Board Number: D0367Presentation Time: 2:45 PM - 4:30 PMUtilizing contact lenses as carriers for human corneal limbalepithelial and induced pluripotent stem cellsNir Erdinest, Abraham Solomon. Ophthalmology, Hadassah HebrewUniv Med Ctr, Jerusalem, Israel.Purpose: To delineate the best technique for culturing and carryinghuman corneal limbal epithelial cells and induced pluripotent stem(iPS) cells on contact lenses (CLs) for the purpose of celltransplantation for limbal stem cell deficiency (LSCD).Methods: Limbal explant sections from corneoscleral rims,remaining from corneal transplantation, or induced pluripotent stem(iPS) cells were placed on the inner aspect of 5 types of CLs coatedwith or without 0.1% gelatin. The CLs used included lotrafilcon A(Air Optix Night & Day® Aqua), vifilcon A (Focus Monthly®),lotrafilcon A (Focus Night & Day® Aqua), etafilcon A (1-DayAcuvue®) and nelfilcon A (Focus Dailies®). Plastic culture platesand the human amniotic membrane were used, respectively, asgrowth platform controls for the CLs. Comparison between thegrowth patterns on the different CLs and the gelatin coating wasmade with regard to epithelial proliferation rate and epithelial cellmorphology utilizing light and electron microscopy.Results: All the growth platforms yield monolayers culture cells withuniform epithelial cell morphology. There were marked differencesin growth patterns between the different CLs. The explants placed onsilicone hydrogel CL lotrafilcon A (CIBA Vision Corporation,Duluth, GA, USA) showed the fastest proliferative and migratoryrates, with CLs covered with gelatin. Explants applied on lotrafilconA were tightly attached to it, and growth was detected first on day 4.Lotrafilcon A (Air Optix Night & Day® Aqua) reached 91.2±8.5%confluence by day 16±2, and Lotrafilcon A (Focus Night & Day®)reached 93.7±6.2% confluence by day 14±3. iPS cells which wereseeded on CLs were attached to lotrafilcon A by day 3. Cellsexamined under light microscopy were morphologically similar tocorneal epithelium grown on 6 wells culture plate and amnioticmembrane. Electron microscopy revealed cell-to-cell links andmultiple cell layers. Microvilli and cell projections were also evidentand most probably served as anchorage points on the CLs surface.Conclusions: Silicone hydrogel CL lotrafilcon A was advantageousover the other types of CLs for culturing and carrying corneal limbalepithelial and iPS cells. These CLs may be used as possible carriersfor cultured human ocular surface epithelial cell sheets as a part of acell therapy strategy in LSCD.Commercial Relationships: Nir Erdinest, None; AbrahamSolomon, NoneProgram Number: 2568 Poster Board Number: D0368Presentation Time: 2:45 PM - 4:30 PMLumican Is Required for Epithelium Migration during Healingof Corneal Epithelium DebridementJianhua Zhang, Vivien J. Coulson-Thomas, Yong Yuan, OsamuYamanaka, Hongshan Liu, Winston W. Kao. Ophthalmology,University of Cincinnati, Cincinnati, OH.Purpose: Purpose: Lumican, a member of small leucine richproteoglycan (SLRP), is a matrikine besides serving as anextracellular matrix ECM component and regulator of collagenfibrillogenesis. Previous studies have shown that lumican promoteshealing of corneal epithelial debridement via enhanced epithelial cellproliferation. The current study attempts to determine whetherlumican plays an additional role in promoting corneal wound healingby enhancing epithelial cell migration.Methods: Methods: Adult lumican null (Lum-/-), hemizygous(Lum+/-) and wild type (Lum+/+) mice were anesthetized andsubjected to circumvent corneal epithelium debridement (2 mm indiameter) with Agerbrush®. Corneas were isolated at differentintervals (0 through 6 h) and subjected to whole mount IF in order tocharacterize the epithelial cell migration by analyzing the expressionand localization of cytoskeletal and adherens components, e.g.,paxillin, etc. The role of lumican on stress fiber and filopodia©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaformation were confirmed in vitro using a scratched wound model ofcultured human telomerase-immortalized corneal epithelial (HTCE)cells.Results: Results: Whole mount staining with phalloidin revealed adecrease in the migrated corneal epithelial sheet in Lum-/- mice, from10 and 105 µm in Lum+/+ mice to 0 and 5 µm in Lum-/- mice after 1and 6 h, respectively. Immunofluorescence staining revealed highexpression levels of paxillin throughout the epithelium with densestaining at the wound edge in Lum+/+ mice, however, Lum-/- micepresented very low expression levels of paxilin and a subtle increasein paxilin expression was restricted to the immediate wound edge.Moreover, paxilin staining revealed cellular projections in epithelialcells at the wound edge which were polarized into the wounded areasolely in the Lum+/+ mice. The in vitro scratch wound assay ofHTCE cells revealed that the addition of solely lumican to cellsmaintained in basic medium (in the absence of growth factors)enhanced stress fiber formation with filopodia projected to thewounded area to the same extent as that observed in completemedium.Conclusions: Discussion: Our observation suggests that lumicanplays a critical role in promoting wound healing via both theenhancement of cell locomotion and proliferation. Further studies areneeded to determine the molecular and cellular mechanism by whichlumican promotes wound healing.Commercial Relationships: Jianhua Zhang, None; Vivien J.Coulson-Thomas, None; Yong Yuan, None; Osamu Yamanaka,None; Hongshan Liu, None; Winston W. Kao, NoneSupport: NIH/NEI RO-1 EY011845, Research to Prevent Blindness,Ohio Lions Eye Research FoundationProgram Number: 2569 Poster Board Number: D0369Presentation Time: 2:45 PM - 4:30 PMIn Keratoconus are Epithelial and Stromal Changes Correlated?Colton Heinrich 1 , Andrew P. Kemp 1 , Jessica H. Mathew 1 , JohnGoosey 2, 1 , Jan P. Bergmanson 1 . 1 College of Optometry, Universityof Houston, Houston, TX; 2 Ophthalmology, Houston Eye Associates,Houston, TX.Purpose: The purpose of this study was to quantify histopathologicalchanges to anterior limiting lamina (ALL) and epithelial basementmembrane (BM) peripheral to Fleischer’s ring in keratoconic (Kc)corneas, to determine if a correlation exists between these changes,and to search peripheral stroma for histopathological signs ofprominent nerves.Methods: Nine surgically removed Kc corneal buttons and twocontrol corneas were preserved and processed for light andtransmission electron microscopy (TEM) using an establishedprotocol. Five digital pictures per cornea were obtained from bothALL and BM with the Tecnai G 12 twin TEM at 4200x and 16500xrespectively. Two measurements were taken per micrograph using amillimeter rule and adjusted to scale. Average values were calculatedand compared.Results: The average ALL thickness for Kc was 7.841µm(Range=5.68 - 10.22µm) and for control was 9.80µm (Range=9.70 -9.90µm). The average BM thickness was 0.4311µm (Range=0.19 -0.69µm) and was 0.17µm (Range=0.16 - 0.19µm) for Kc and controlrespectively. No correlation was found between the thickening of theBM and the thinning of the ALL (r=-0.2). Abnormal nerve fiberswith thickened basement membranes were found within the Kcperipheral stromal tissue.Conclusions: The thinned ALL and thickened BM supportedprevious reports of being characteristics in patients with Kc, not onlycentrally but also peripherally. However, the lack of correlationbetween the abnormalities of the ALL and BM provided evidencethat these pathological processes are independent of each other.Stromal nerve fibers observed in the stroma were abnormal and mayexplain the loss of corneal sensitivity noted in Kc patients but thesealterations appeared not to correlate well with their distinctprominence as seen clinically.Commercial Relationships: Colton Heinrich, None; Andrew P.Kemp, None; Jessica H. Mathew, None; John Goosey, None; JanP. Bergmanson, Contamac (F)Support: NEI/NIH Core Grant P30 EY07551; NEI/NIH TrainingGrant T35007088Program Number: 2570 Poster Board Number: D0370Presentation Time: 2:45 PM - 4:30 PMExpression of the neural stem cell marker Hes3 in the rodent andhuman ocular surfaceMatina Economopoulou 1 , Jimmy Masjkur 2 , Frederick Raiskup 1 , MikeKarl 3 , Richard H. Funk 4 , Triantafyllos Chavakis 2 , Lutz E. Pillunat 1 ,Andreas Androutsellis-Theotokis 2 . 1 Ophthalmology, University ClinicDresden, Dresden, Germany; 2 Department of Internal Medicine(MK3), University Clinic Dresden, Dresden, Germany; 3 Center forRegenerative Therapies Dresden, Dresden, Germany; 4 Department ofAnatomy, University Clinic dresden, Dresden, Germany.Purpose: Hairy and Enhancer of Split 3 (Hes3) is a recentlyestablished biomarker of neural stem cells in the fetal and adult brainof rodents and primates, including humans. Expression of Hes3persists during quiescence, allowing the detection of nonproliferatingstem cells and precursor cells. Hes3 also identifiesputative cancer stem cells in aggressive brain tumors, rendering it auseful biomarker of both normal and cancerous neural stem cells. Inthis work we address whether Hes3 can also be found on the ocularsurface.Methods: Adult mouse and human eye tissue was prepared forimmunohistochemical detection of Hes3 and other markers.Physiological human tissue was obtained from a Cornea bank andpterygium samples were obtained from pterygium excision surgeryafter patient consent.Results: The adult rodent and human eye, as well as pterygiumsamples, contain a population of cells expressing Hes3. In the humaneye, Hes3+ cells are found predominantly in the limbus where theyphysically associate with blood vessels.Conclusions: Hes3, a recent biomarker of neural stem cells in theadult brain and spinal cord, as well as in brain tumors, is alsoexpressed in the human limbus and in pterygium. Hes3+ cells in theeye also show similarities to those in the brain, in that they maintaintight physical associations with blood vessels. These results suggestthat limbal Hes3+ cells may have precursor properties, and that acommon mechanism may regulate Hes3+ cells in the brain and theeye.Commercial Relationships: Matina Economopoulou, None;Jimmy Masjkur, None; Frederick Raiskup, None; Mike Karl,None; Richard H. Funk, None; Triantafyllos Chavakis, None;Lutz E. Pillunat, None; Andreas Androutsellis-Theotokis, NoneSupport: Else Kroener-Fresenius-Stiftung and the Centre forRegenerative Therapies Dresden (CRTD) to AATProgram Number: 2571 Poster Board Number: D0371Presentation Time: 2:45 PM - 4:30 PMCorneal condition and anterior segment pathology in patientswith bullous keratopathyElena Adjievska, Petja I. Vassileva, Nikolay Surchev, TatyanaHergeldzhieva-Fileva. University Eye Hospital "Prof. Pashev", Sofia,Bulgaria.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To evaluate the corneal and anterior chamber condition inpatients with bullous keratopathy who underwent penetratingkeratoplaty (PK).Methods: A retrospective review of clinical records of allconsecutive patients with bullous keratopathy, referred for PK for theperiod January 2005 - December 2010, was performed. Preoperativestatus was analyzed with an emphasis on corneal condition andanterior segment abnormalities- presence and extent of cornealneovascularization, ocilar surface disorders, iris injuries withdistorted pupil, anterior synechia and subluxated intraocular lens.Results: Bullous keratophaty was present in 54 of all 141 patientswho underwent PK. Forty one of the patients had pseudophakickeratopathy, 6 pts were with aphakic and 3 with phakic keratopathy.One patient had graft failure, 1 pts- posttraumatic endothelialdecompensation and in 2 of them Fuchs endothelial decompensationwas found. Ocular surface abnormalities were diagnosed in 51 of thepatients (94%) . In 30 of the cases (56%) deep and superficial cornealneovasclarization was observed. Other pathological findingsincluded: painful epithelial bullae in 39cases (72%), subepithelial andstromal fibrosis in 18 patients (33%). In most patients all pathologicalsings were present but with different severity. Cornea was notneovascularizated and there was no fibrosis in only 5 cases. Theocular surface was not compromised in 3 of the patients. Anothermarker of advanced pathology was visual acuity: BCVA between0,05 and 0,1 in 1 patient (2%), light perception to 0,05 in 26 pts(47%) and hand movement to light perceptiont in the rest of the pts.Conclusions: The analysis of patients with bullous keratopathydemonstrated that advanced disease and severely affected corneal andanterior segment anatomy were present in all of the cases but withdifferent severity. In all patients from our studied group thepathological changes excluded the option for lamellar graftingtechniques, and PK remained the only treatment possibility. Mainreasons for the accumulation of such a pool of challenging patientswith delayed treatment are unreasonably prolonged conservativetherapy of postoperative complications and lack of donor tissue.Addressing these issues could allow the use of lamellar graftingtechniques, applicable only in the initial stages of bullouskeratophaty. At present PK remains the main therapeutic option forour patient pool.Commercial Relationships: Elena Adjievska, None; Petja I.Vassileva, None; Nikolay Surchev, None; Tatyana Hergeldzhieva-Fileva, NoneProgram Number: 2572 Poster Board Number: D0372Presentation Time: 2:45 PM - 4:30 PMHO-2 knockdown delays wound healing in Human CornealEpithelial (HCE) cells by altering the signaling of EGFR andFAK mediated pathwayAdna Halilovic, Daohong Lin, Gregory Joseph, Brian Shkolnik,Michal L. Schwartzman. Pharmacology, New York Medical College,Valhalla, NY.Purpose: Heme oxygenase (HO) represents an intrinsiccytoprotective and anti-inflammatory system. Inhibition of HOactivity significantly impairs wound healing in human cornealepithelial (HCE) cells. We have shown that HO-2 knockdown in vitroattenuates corneal wound healing and that HO-2 null mice display anaberrant corneal wound healing following injury. In this study, weinvestigated the mechanisms that may contribute to HO-2cytoprotective role in the corneal epithelium.Methods: HCE cells were stably transfected with lentivirus HO-2shRNA or non-target shRNA plasmids. Modified Boyden Chamberassay was used to study the migration of HO-2 knockdown cells.Flow cytometry cell cycle analysis was done using propidium iodidestaining. Western blot was performed to evaluate the expression ofproteins involved in migration and proliferation in HO-2 shRNA andnon-target shRNA cells.Results: HO-2 knockdown, using virus-mediated shRNA, impairedHCE wound healing by 35% and 25% at 24h and 48h after injury,respectively. Furthermore, HO-2 knockdown inhibited basal andEGF(10ng/mL)-stimulated HCE migration by 50% and 40%,respectively and was associated with a 35% decrease in p-Akt and45% decrease in p-EGFR levels. Flow cytometric analysis showedthat EGF-stimulated cell cycle progression was attenuated in HO-2knockdown cells. Immunofluorescence of HO-2shRNA and controltransfected cells revealed a cytoplasmic distribution of p-FAK. Uponinjury, control cells displayed typical p-FAK as focal adhesion pointsin the membrane, whereas in HO-2 knockdown cells p-FAK had nofocal distribution.Conclusions: Knockdown of HO-2 results in attenuated cornealepithelial wound healing. Deficiency of HO-2 impairs wound healingby a mechanism that involves alterations in ERK, Akt and FAKsignaling pathways, all of which have been shown to play animportant role in growth factor-mediated cell migration andproliferation following injury.Commercial Relationships: Adna Halilovic, None; Daohong Lin,None; Gregory Joseph, None; Brian Shkolnik, None; Michal L.Schwartzman, NoneSupport: NIH Grant EY06513 (MLS)Program Number: 2573 Poster Board Number: D0373Presentation Time: 2:45 PM - 4:30 PMDecreased Incidence of Perioperative Corneal Injuries Followingan Ophthalmology Educational Initiative For AnesthesiaProviders at an Academic Medical CenterAmanda L. Ely, Ingrid U. Scott, Tabassum F. Ali, Denise Kerchner,David Liang, Michael Wilkinson. Ophthalmology, Penn StateHershey Ophthalmology, Hershey, PA.Purpose: To investigate whether an ophthalmology educationalinitiative directed toward anesthesia providers can decrease theincidence of perioperative corneal injury at an academic medicalcenter.Methods: Prospective chart review of consults placed forperioperative eye pain resulting from corneal injury between July 1,2011 and January 4, 2012 (including patient and surgical risk factorsfor corneal injury as reported in the literature). An ophthalmologyeducational initiative, including a Grand Rounds lecture anddistribution of educational materials regarding perioperative ocularcare, was provided to anesthesia providers on January 5, 2012 basedon the Mayo Clinic Model (Martin et al., Anesthesiology 2009).Beginning on January 5, 2012, an email was sent to the attending andresident physician of any patient who experienced a perioperativecorneal injury; the email notified the physicians of the corneal injuryand provided information to the physicians on how such injuries maybe prevented in the future. Prospective chart review of consults(identical to the pre-initiative data collection) was then performedbetween January 6, 2012 through June 30, 2012. The rates ofperioperative corneal injury pre- versus post-initiative werecompared.Results: The rate of perioperative corneal injury was 0.35% beforethe educational initiative compared with 0.18% following theinitiative (p=0.04). The proportions of corneal injuries that wereassociated with risk factors reported in the literature for such injurieswere not significantly different pre- versus post-initiative except forlength of surgical case. Pre-initiative a higher proportion of cornealinjuries occurred after a surgery

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConclusions: An ophthalmology educational initiative directedtoward anesthesia providers was associated with a significantdecrease in the incidence of perioperative corneal injury. Thisinformation may be helpful for other medical centers in order toemphasize the potential impact of educational initiatives to preventperioperative corneal injury.Commercial Relationships: Amanda L. Ely, None; Ingrid U.Scott, None; Tabassum F. Ali, None; Denise Kerchner, None;David Liang, None; Michael Wilkinson, NoneSupport: n/aProgram Number: 2574 Poster Board Number: D0374Presentation Time: 2:45 PM - 4:30 PMMOLECULAR IDENTIFICATION AND FUNCTIONALCHARACTERIZATION OF THE VITAMIN CTRANSPORTERS EXPRESSED BY HUMAN CORNEALEPITHELIAL CELLS (HCEC)Varun Khurana, Aswani Dutt Vadlapudi, Ramya Krishna Vadlapatla,Dhananjay Pal, Ashim K. Mitra. Pharmaceutical Sciences, Universityof Missouri Kansas City, Kansas City, MO.Purpose: The objective of this research was to functionallycharacterize the Sodium Dependent Vitamin C Transporter (SVCT)in human corneal epithelial cells (HCEC). This nutrient transportercan be targeted to improve the delivery of drugs that get effluxed outby the efflux transporters (P-gp, MRP and BCRP).Methods: Uptake was performed with human corneal epithelial cells(HCEC). Uptake of [14C] ascorbic acid ([14C] AA) was studied inthe absence and presence of excess unlabelled AA, anion transporterinhibitors. Effect of pH, sodium and temperature on the uptake of[14C] AA was also studied. Concentration dependency studies(0.1µM-1000µM) were performed. Role of cellular protein kinasemediated pathways and Ca2+/CaM mediated pathways on theregulation of AA uptake has also been studied. Reverse transcriptionpolymerase chain reaction (RT-PCR) for SVCT was carried out onRNA isolated from HCEC.Results: Uptake of [14C] AA was found to be saturable with a K¬mof 144.87 µM and Vmax of 38.33 pmol/mg protein/min. Uptake of[14C] AA was pH, sodium, energy and temperature dependent. Themembrane transporter was also under the regulation of cellularprotein kinase C (PKC), protein kinase A (PKA), Ca2+/CaMmediated pathways and metabolic inhibitors. Uptake of [14C] AAwas significantly inhibited in the presence of different concentrationsof cold L- ascorbic acid and D-Iso ascorbic acid. The observedpermeability values for transepithelial transport were 2.29±0.56×10-4, 2.07±0.45×10-4, 1.49±0.38×10-4, 1.8±0.24×10-4 and1.57±0.66×10-4 cm/s for [14C] AA, L-AA (100µM), L-AA(500µM),D-Iso AA(100µM) and D-Iso AA (500µM) respectively. Ethidiumbromide staining of the gel showed a major 626-bp bandcorresponding with the amplified SVCT2 cDNA.Conclusions: Sodium Dependent Vitamin C Transporter (SVCT)which plays an important role in the uptake of ascorbic acid has beenidentified and functionally characterized in HCE cell line. Thisnutrient transporter can be targeted to improve the delivery of drugsthat acts as substrate for the efflux transporters (P-gp, MRP andBCRP). The study also provides useful information on the substratespecificity of this carrier system. This study suggests that HCE cellsexpress the hSVCT2 system and may serve as a useful in vitroexperimental model to study the permeability of ascorbic acidconjugated prodrugs.Commercial Relationships: Varun Khurana, None; Aswani DuttVadlapudi, None; Ramya Krishna Vadlapatla, None; DhananjayPal, None; Ashim K. Mitra, NoneSupport: NIH R01EY09171-16 and NIH R01EY010659-14Program Number: 2575 Poster Board Number: D0375Presentation Time: 2:45 PM - 4:30 PMAn animal model for epithelial downgrowthJessica E. Weinstein, Matthew J. Weiss, Jeffrey L. Goldberg.Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami,FL.Purpose: Epithelial downgrowth is a rare but devastatingcomplication of penetrating trauma or surgery. It can result in visionloss, intractable glaucoma or corneal decompensation. Treatmentmethods are usually non-curative and are often followed byrecurrence. An animal model of epithelial downgrowth is essentialfor the development of future diagnostics and therapies, but to datethere are few options. Here we developed a rat model of epithelialdowngrowth.Methods: Mouse corneal epithelial cells (MCECs) were injected intothe anterior chamber of Sprague-Dawley rats and observed over 4weeks. Anterior chamber ocular coherence tomography (OCT) andintra-ocular pressure (IOP) measurements were done at baseline, 2and 4 weeks. In each rat, the contralateral eye was used as a control.OCT measurements of corneal thickness were analyzed usingMatLab software. Rat eyes were fixed by immersion for 2-3 hourswith 4% PFA and then embedded in OCT Tissue-Tek and frozen.Whole eyes were sectioned. Half the representative samples werestained for H&E and the rest were stained withimmunohistochemistry with corneal epithelial cell marker antibodies.Results: H&E staining showed presence of membranes andadditional cells in the anterior chamber. DAPI-stained nuclei wereevident in the membranes in the experimental eyes when compared tocontrol eyes. Anterior chamber OCT showed thicker corneas inexperimental eyes compared to control eyes. IOPs were notsignificantly elevated in experimental eyes compared to control eyes.Conclusions: A novel rat model was developed for epithelialdowngrowth which mimicked some features of the typical humandisease. Further experiments with longer time-points are planned toinvestigate whether IOP increases eventually. Development of thisepithelial downgrowth model may lead to new opportunities fordiagnostic and treatment development.Commercial Relationships: Jessica E. Weinstein, None; MatthewJ. Weiss, ASCRS Foundation Grant (F); Jeffrey L. Goldberg, NoneSupport: RPB Medical Student Fellowship; ASCRS GrantProgram Number: 2576 Poster Board Number: D0376Presentation Time: 2:45 PM - 4:30 PMDescriptive Study of Ocular Surface changes in Patients withCongenital GlaucomaSimone Finzi, Ruth M. Santo, Monique Matsuda, Ernst W. Oltrogge,Marcio H. Mendes, Fernando E. Naves, Rodrigo P. Azevedo,Frederico Lazar, Bruno C. Cardoso, Alberto Betinjane.Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil.Purpose: Recent studies show a relationship between POAG andlacrimal dysfunction. Several factors may lead to alteration of theocular surface (OS) in patients with primary congenital glaucoma(PCG). The purpouse of this study is to elaborate a clinicaldescription of the ocular surface (OS) in patients with GCPMethods: After informed consent, 34 patients diagnosed with PCGwere included in the study. Ophthalmologic examination wasperformed, and clinical evaluation of OS. The patients weresubmitted to a questionnaire to evaluate symptoms of dry eyeResults: We examined 34 patients (60 eyes) diagnosed with PCG to17.97 ± 7.91 years. 17 patients without use of eyedrop for at least 1year and 17 patients using eye drops, these 9 patients (64%) using 3or more drops. The patients had chronic use of eye drops with a mean©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaof 6.07 ± 7.56 years. All patients underwent surgery. In the interview,18 patients (52%) complained of dry eye. On examination it wasobserved: IOP OD: 14.62 ± 5.72 mmHg, OS: 12.55 ± 14.16 mmHg;pachymetry: OD: 567.37 ± 87.04 μ, OS: 555.92 ± 51.76 μ; BiometryOD : 25.80 ± 4.27 mm, OS: 23.99 ± 2.31 mm. In assessing the OSresulted: Schirmer Test OD = 25.93 ± 10.44 mm, OS: 23.28 ± 11.84mm; BUT: OD = 11.26 ± 4.50 s, OS = 11.76 ± 11.76 s , LacrimalMeniscus: OD = 0.82 ± 1.15 mm, OE: 0.77 ± 0.93 mm, no patienthad a significant positive staining Rose Bengal.Conclusions: Despite PCG is a rare disease and this study has aheterogeneous sample, our patients expressed mild symptoms of dryeye, but without clinical signs consistent with lacrimal dysfunction.Subjective evaluation of the questionnaire is controverted in children,but other factors beyond the chronic use of eye drops, asbuphthalmos, lagophthalmos and anatomic alterations from surgery,may contribute to the symptoms of the disease in this patients sampleCommercial Relationships: Simone Finzi, None; Ruth M. Santo,None; Monique Matsuda, None; Ernst W. Oltrogge, None; MarcioH. Mendes, None; Fernando E. Naves, None; Rodrigo P. Azevedo,None; Frederico Lazar, None; Bruno C. Cardoso, None; AlbertoBetinjane, NoneProgram Number: 2577 Poster Board Number: D0377Presentation Time: 2:45 PM - 4:30 PMLacking Muc16 affects intra-epithelial differentiation and woundhealing in corneal epithelium in miceShizuya Saika 1 , Kumi Shirai 1 , Yuka Okada 1 , Masayasu Miyajima 2 ,Dong-Joo Cheon 3 , Richard R. Behringer 3 . 1 Ophthalmology,Wakayama Medical University, Wakayama, Japan; 2 Animal Center,Wakayama Medical University, Wakayama, Japan; 3 Genetics,University of Texas M.D. Anderson Cancer Center, Texas, TX.Purpose: A membrane-bound mucin, Muc16, is expressed inconjunctiva in mice. We examined the effects of the loss ofconjunctival Muc16 in the structure and behavior of cornealepithelium in mice.Methods: (1) Histology at light and electron microscopic levels andimmunohistochemistry of the corneal epithelium of 26 wild-type(WT) and 16 Muc16-null (KO) mice was performed. Shape of thenuclei in basal and suprabasal later was observed to know thedifferentiation of suprabasal cells. TUNEL stain and BrdU-evaluationof cell proliferation were also included. (2) Healing of a round defectin corneal epithelum was evaluated in WT (n = 24) and KO (n = 24)mice at intervals of healing up to 30 hrs.Results: 1) Light and electron microscopies showed an impairmentof differentiation of suprabasal cells. The nuclei of the suprabasalcells exhibited a more round shape in a KO mouse as compared withthem in a WT epithelium. Keratin 14 was detected in both basal andsuprabasal cells in a KO epithelium, while restricted to the basal cellsin a WT mouse. Cell proliferation was more marked in a KO mousethan in 1 WT mouse. TUNEL-labeled cells were not obsderved inboth WT and KO mice. (2) healing of a round defect in cornealepithelium was significantly promoted by lacking Muc16.Conclusions: Cell proliferation enhancement and the presence ofkeratin 14-positive cells with a relatively round nuclei in thesuprabasal layer suggests the acceleration of intraepithelial cellturnover in mice lacking Muc16 in conjunctiva. The loss of Muc16indirectly affected wound healing (migration) in corneal epitheliumin mice.Commercial Relationships: Shizuya Saika, None; Kumi Shirai,None; Yuka Okada, None; Masayasu Miyajima, None; Dong-JooCheon, None; Richard R. Behringer, NoneProgram Number: 2578 Poster Board Number: D0378Presentation Time: 2:45 PM - 4:30 PMLongitudinal Assessment of Neuropathy in Diabetes using novelophthalmic MARKers (LANDMark): Baseline findingsNathan Efron 1, 2 , Nicola Pritchard 1, 2 , Katie Edwards 1, 2 , Geoff P.Sampson 1, 2 , Anthony Russell 3, 4 , Ioannis N. Petropoulos 4, 5 , UazmanAlam 4, 5 , Hassan Fadavi 4, 5 , Mitra Tavakoli 4, 5 , Rayaz A. Malik 4, 5 .1 Institute of Health & Biomedical Innovation, Queensland Universityof Technology, Kelvin Grove, QLD, Australia; 2 School of Optometryand Vision Science, Queensland University of Technology, KelvinGrove, QLD, Australia; 3 Department of Diabetes and Endocrinology,Princess Alexandra Hospital, Woolloongabba, QLD, Australia;4 School of Medicine, University of Queensland, St Lucia, QLD,Australia; 5 Centre for Endocrinology and Diabetes, Institute ofHuman Development, University of Manchester, Manchester, UnitedKingdom.Purpose: Over the past decade, corneal nerve morphology andcorneal sensation threshold have been explored as potential surrogatemarkers for the evaluation of diabetic neuropathy. We present thebaseline findings of a Longitudinal Assessment of Neuropathy inDiabetes using novel ophthalmic Markers (LANDMark).Methods: The LANDMark Study is a 5-year, two-site, naturalhistory (observational) study of individuals with Type 1 diabetesstratified into those with (T1W) and without (T1WO) neuropathyaccording to the Toronto criteria, and control subjects. All studyparticipants undergo detailed annual assessment of neuropathyincluding corneal nerve parameters measured using corneal confocalmicroscopy and corneal sensitivity measured using non-contactcorneal esthesiometry.Results: 396 eligible individuals (208 in Brisbane and 188 inManchester) were assessed: 76 T1W, 166 T1WO and 154 controls.Corneal sensation threshold (mbars) was significantly higher in T1W(1.0 ± 1.1) than T1WO (0.7 ± 0.7) and controls (0.6 ± 0.4) (P=0.002);post-hoc analysis (PHA) revealed no difference between T1WO andcontrols (Tukey HSD, P=0.502). Corneal nerve fiber length(mm/mm 2 ) (CNFL) was lower in T1W (13.8 ± 6.4) than T1WO (19.1± 5.8) and controls (23.2 ± 6.3) (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Nathan Efron, None; NicolaPritchard, None; Katie Edwards, None; Geoff P. Sampson, None;Anthony Russell, None; Ioannis N. Petropoulos, None; UazmanAlam, None; Hassan Fadavi, None; Mitra Tavakoli, None; RayazA. Malik, NoneSupport: Juvenile Diabetes Research Foundation (Grant 27-2007-878)Program Number: 2579 Poster Board Number: D0379Presentation Time: 2:45 PM - 4:30 PMExpression of Olfactory Receptor Genes on Mouse OcularSurfaceVladlen Z. Slepak 1 , Alexey Pronin 1 , Konstantin Levay 1 , YaohongTan 2 , Dmitry Velmeshev 3 , Mohammad Faghihi 3 , Valery I.Shestopalov 2 . 1 Molecular and Cellular Pharmacology, University ofMiami, Miami, FL; 2 Opthalmology, University of Miami, Miami, FL;3 Psychiatry, University of Miami, Miami, FL.Purpose: Ocular surface is continuously exposed to tear componentsas well as xenobiotics and commensal and pathogenicmicroorganisms. To advance our understanding how the outer eyeinteracts with its environment, we asked which cellular receptors areexpressed on the ocular surface, focusing on identification of genesencoding G protein-coupled receptors (GPCRs).Methods: Total RNA was isolated from mouse cornea andconjunctiva and subjected to next-generation sequencing usingIllumina platform. The data was analyzed by CuffLinks and TopHatsoftware packages. Gene expression was confirmed by RT-PCRusing total or poly-adenylated RNA obtained from independentcornea and conjunctiva preparations. Cellular localization oftranscripts was analyzed by in situ hybridization using RNAscopetechnology on paraffin-embedded slices of mouse eyes.Results: We performed directional RNA deep sequencing, utilizingEpicenter library preparation kit to generate more than 45 millionreads representing over 10,000 transcripts. We identified more than150 GPCR gene transcripts, of which more than 100 were putativeolfactory receptors (Olfr’s). Our RT-PCR analyses confirmed thepresence of several GPCR and G protein transcripts, including the Gprotein associated with olfaction, Golf. In contrast, a number ofcontrol genes, such as insulin or rhodopsin did no show significantexpression. In situ hybridization showed that mRNA for Olfr558 waspresent primarily in basal epithelial cells of cornea and conjunctiva.Conclusions: Several species of mRNA encoding putative olfactoryreceptors are expressed on the mouse OS.Commercial Relationships: Vladlen Z. Slepak, None; AlexeyPronin, None; Konstantin Levay, None; Yaohong Tan, None;Dmitry Velmeshev, None; Mohammad Faghihi, None; Valery I.Shestopalov, NoneSupport: NIH grants RO1EY018666 (VZS) , R01EY017991 (VIS)Program Number: 2580 Poster Board Number: D0380Presentation Time: 2:45 PM - 4:30 PMNeuropeptide from trigeminal nerve promotes the stratificationof human corneal epithelial cellsJi-Ae Ko, Chihiro Ohki, Taiichiro Chikama, Yoshiaki Kiuchi.Department of Ophthalmology, Hiroshima Univ Grad Sch of BiomedSci, Hiroshima, Japan.Purpose: We have previously shown that the presence of establishedneural cells (PC12) resulted in promotion of stratification in HCEcells using coculture system. We have now examined the keyfactors(s) released from neural cells on stratification of HCE cellswith trigeminal nerve culture.Methods: Trigeminal nerve cells and simian virus 40-transformedhuman corneal epithelial (HCE) cells were cultured on opposite sidesof a collagen vitrigel membrane. Next, HCE cells were cultured aloneand stimulated with several neuropeptides from trigeminal nerve. Theobservation for stratification of HCE cells were examined byimmunofluorescence analysis with of tight junctional proteins. Also,the expression of junctional proteins in HCE cells was examined byRT-PCR and immunoblot analyses.Results: The presence of trigeminal nerve cells resulted in promotionof stratification in HCE cells. Furthermore, released substance P andCGRP from trigeminal nerve also effected in promotion ofstratification of HCE cells.Conclusions: These results suggest that neuropeptides released fromtrigeminal neural cells may play an important role in the regulation ofintercellular communication between corneal epithelial cells as wellas in the maintenance of corneal structure and function.Commercial Relationships: Ji-Ae Ko, None; Chihiro Ohki, None;Taiichiro Chikama, None; Yoshiaki Kiuchi, NoneProgram Number: 2581 Poster Board Number: D0381Presentation Time: 2:45 PM - 4:30 PMRole of PININ in the Regulation of Alternative Splicing of LongNon-coding RNAsJeong-Hoon Joo, Stephen P. Sugrue. Anatomy and Cell Biology,University of Florida, Gainesville, FL.Purpose: GG-H Whole Transcriptome Array analysis suggestedinvolvement of PININ (PNN) in the alternative splicing of multiplelong non-coding RNAs (lncRNAs). To further investigate PNN’s rolein the regulation of the alternative splicing of lncRNAs in a cornealepithelial context, we performed detailed analyses for the detectionand identification of alternatively spliced lncRNAs.Methods: Total RNA was isolated from PNN knockdown humancorneal epithelial (HCET) cells or Pnn-deficient mouse cornea, andsubjected to RT-PCR assays. Quantification of alternatively splicedlncRNAs was performed by ChemiDocTM XRS+ and Image LabSoftware Version 4.0. Detection of alternatively spliced lncRNAswas achieved through in situ hybridization with variant-specific RNAprobes on human cornea sections.Results: The sequence analyses and quantification of splice variantsof candidate lncRNAs, such as RP11-322M19.1 and LOC100505761,clearly demonstrated complex configuration of their splicing changesand significant impact of PNN on the process. Knockdown of PNN inHCET cells led to the specific alterations in the inclusion of multiplecassette exons as well as in the usage of alternative splice sites inRP11-322M19.1 and LOC100505761, resulting in the considerablenet changes in the ratio between splice variants. Our analysis alsouncovered PNN’s impact on the transcript levels of several lncRNAsincluding Linc00085 and HAS2-AS1 (Hyaluronic acid synthase 2-antisense RNA 1). Interestingly, a mouse ortholog of HAS2-AS1,Has2as, clearly exhibited a differential splicing pattern among threemajor splice variants in Pnn-deficient mouse cornea. Sincehyaluronic acid has been closely implicated in the formation ofpersistent lens stalk, which has been consistently observed in Pnn-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadeficient mouse embryos, it will be of great relevance to investigatethe mechanism underlying Pnn’s impact on the alternative splicing ofHas2as. Finally, in situ hybridization analyses revealed the presenceof two splice variants of RP11-295G20.2, one of PNN-regulatedlncRNAs, in the nuclei of corneal epithelial cells, but not in thestromal cells of human cornea, suggesting their potential roles incorneal epithelial cells.Conclusions: The data strongly suggest PNN’s role in the alternativesplicing of a specific subset of lncRNAs that might have significantimpact on the corneal epithelium. (NIH Grant R01 EY007883, P30EY021721)Commercial Relationships: Jeong-Hoon Joo, None; Stephen P.Sugrue, NoneSupport: NIH Grant R01 EY007883, P30 EY021721Program Number: 2582 Poster Board Number: D0382Presentation Time: 2:45 PM - 4:30 PMLocalisation of Yap/Taz in corneal epithelia: a marker ofmechano-sensitivity and role in epithelial homeostasisChe J. Connon, Roanne R. Jones, James W. Foster. School ofChemistry, Food and Pharmacy, University of Reading, Reading,United Kingdom.Purpose: To investigate the cellular location of the nucleartranscription factor Yap/Taz in limbal and central corneal epithelialcells and relate this expression to changes in corneal stiffnesscentripetally across the ocular surface.Yap/Taz has recently been found to be an important regulator ofmechanotransduction. We believe that changes in substrate stiffnessoccur across the cornea and that a centripetal stiffness gradient drivesboth differentiation and migration of epithelial cells form the limbustowards the central cornea forming an important structuralcomponent of ocular surface homeostasis.Methods: The localisation of Yap/Taz, CK3, CK14 and ZO-1 acrossthe ocular surface of bovine corneas was examined byimmunohistochemistry. Limbal stem cells were isolated form freshbovine corneas and expanded upon type I collagen gels of differingstiffness for 14 days. The localisation of Yap/Taz, CK3, CK14 andZO-1 was examined by immunohistochemistry within these cornealconstructs. Furthermore, the transcription levels of Yap/Taz, CK3and ABCG2 were quantified by QPCR.Results: Across the healthy bovine cornea Yap/Taz waspredominately expressed cytoplasmically within the limbus, where asin central corneal epithelial cells Yap/Taz was retained within thenucleus. Isolated limbal epithelial cells expanded upon the morecompliant collagen gels showed significantly less gene expression ofYap/Taz, which was predominately cytoplasimic at the protein level,whereas more nuclear expression was seen within epithelial cellsexpanded upon the stiffer collagen gels. This corresponded with morecells expressing cytokeratin 3 and ZO-1 and less cytokeratin 14 andABCG2 at the gene and protein level.Conclusions: The nuclear to cytoplasmic expression ratio of Yap/Tazbetween limbal and central epithelial cells supports the notion of acentripetal stiffness gradient across the corneal surface. Thesuitability of YAP/Taz as a marker of mechanosensitivity (substratestiffness) in corneal epithelial cells was successfully demonstrated byuse of collagen gels as cell substrates with differing stiffness. Thepresence of a centripetal stiffness gradient across the cornea is likelyto underpin new directions in corneal wound healing and ourunderstanding of ocular surface homeostasis.Commercial Relationships: Che J. Connon, None; Roanne R.Jones, None; James W. Foster, NoneSupport: Medical Research Council G0900877/1Program Number: 2583 Poster Board Number: D0383Presentation Time: 2:45 PM - 4:30 PMVitamin D Receptor Knockout Affects Mouse Corneal TightJunctionsMitchell A. Watsky, Rodolfo A. Elizondo, Zhaohong Yin. Physiology,Univ of Tennessee Health Sci Ctr, Memphis, TN.Purpose: Vitamin D is typically known to be produced in the skinand activated through successive hydroxylation steps in the liver andkidneys. Our lab has recently determined that vitamin D can also beproduced in the cornea and can influence the barrier function of thecorneal epithelium. The purpose of this study was to determine ifproteins associated with corneal tight junctions are affected byvitamin D receptor (VDR) knockout and if the junctions themselvesare altered as observed using transmission electron microscopy(TEM).Methods: Wild type 2.5 month-old C57BL/6 mice along withheterozygous (+/-) and homozygous (-/-) VDR null mice (JacksonLabs) were used in this study. Total RNA was obtained from freshlyisolated mouse cornea epithelium. Real-time PCR was used toquantify mRNA levels of the tight junction-associated proteinsoccludin and ZO-1. 2ug total RNA was used for the subsequentsynthesis of cDNA using the ThermoScript RT-PCR system for firststrandsynthesis at 25°C 10min, 50°C 50min, and was inactivated byheating to 85°C for 5 min. Equal amounts of cDNA were applied forPCR amplification in quadruplicate at a final volume of 25 μl of 2XRT2 Real-Time SYBR Green master mix. Amplification wasperformed for 95°C 10min, followed by 40 cycles at 94°C 15 s and60°C 60 s. Quantitative values were obtained from the thresholdcycle value (Ct), which is the point where a significant increase offluorescence is first detected. GAPDH was used as an internal RNAcontrol, and each sample was normalized on the basis of its GAPDHgene content (ΔCt). Transmission electron microscopy (TEM) wasused to examine tight junction structures in the cornea epithelium andendothelium.Results: Occludin levels in -/- mice were significantly lower thanwild type and +/- mice. ZO-1 was significantly lower in -/- versuswild type mice. TEM demonstrated significant reductions inepithelial and endothelial tight junction structures in -/- corneasversus those from wild type mice.Conclusions: VDR ablation results in significant loss of tightjunction-associated proteins and tight junction structures in mousecorneas.Commercial Relationships: Mitchell A. Watsky, None; Rodolfo A.Elizondo, None; Zhaohong Yin, NoneSupport: NIH Grant EY021747Program Number: 2584 Poster Board Number: D0384Presentation Time: 2:45 PM - 4:30 PMPharmacological Analysis of Epidermal Growth Factor ReceptorLigands on Corneal Epithelial CellsBrian P. Ceresa 1, 2 , Joanne L. Peterson 2 . 1 Pharmacology andToxicology, University of Louisville, Louisville, KY; 2 Cell Biology,University of Oklahoma HSC, Oklahoma City, OK.Purpose: An active epidermal growth factor receptor (EGFR) is bothnecessary and sufficient for promoting the homeostasis of the cornealepithelium. However, stimulation of the EGFR with epidermalgrowth factor (EGF) has not been an effective strategy foraccelerating the repair of damaged corneal epithelium. Sincesynthetic EGFR ligands are not available, we have examined the roleof endogenous EGFR ligands in promoting corneal epithelial cellgrowth and migration. These findings will aid in developingalternative strategies for stimulating EGFR activity that will furtherour understanding of the progression and reversal of diseases of the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacorneal epithelium, such as corneal erosions.Methods: The endogenous EGFR ligands were tested for their abilityto induced EGFR phosphorylation and promote in vitro cornealwound healing, cell migration, and proliferation using immortalizedcorneal epithelial cells (hTCEpi cells).Results: All EGFR ligands can enhance the phosphorylation of theEGFR. Despite comparable levels of receptor phosphorylation, theligands produced quantitatively different in vitro wound healing.Pharmacologically, betacellulin is the most potent and efficaciousmediator of in vitro corneal epithelial wound healing.Conclusions: In the absence of synthetic EGFR ligands to restore ormaintain corneal epithelial homeostasis, we have examined theability of other ligands to execute this function. Regarding efficacy inpromoting in vitro corneal epithelial wound healing, betacellulin >HB-EGF > EGF. These data indicate alternative ligands should beconsidered as mediators wound healing/homeostasis in vivo.Commercial Relationships: Brian P. Ceresa, None; Joanne L.Peterson, NoneSupport: NIH Grant EY021497Program Number: 2585 Poster Board Number: D0385Presentation Time: 2:45 PM - 4:30 PMThe Anti-inflammatory Effects of Resolvin-D1 on HumanCorneal Epithelial CellsAbraham Solomon, Nir Erdinest. Ophthalmology, Hadassah-HebrewUniv Med Ctr, Jerusalem, Israel.Purpose: To evaluate the anti-inflammatory effects of Resolvin-D1(RV-D1) and its mode of action on human corneal epithelial (HCE)cells in vitro.Methods: Cultured HCE cells were incubated for different periodsand at several concentrations of RV-D1. Oleic acid (OA) andDexamethasone (DM) served as negative and positive controls,respectively. Cells were stimulated with polyriboinosinic :polyribocytidylic acid (poly I:C). The protein contents and mRNAexpression levels of Tumor Necrosis Factor-α (TNF-α), Interleukin(IL)-6, IL-1β and IL-8 were evaluated with multiplex fluorescentbead immunoassay (FBI) and with real time-PCR, respectively. Theexpression of Inhibitory Factor-κBα (I-κBα) was evaluated with realtime-PCR.Results: The protein contents of the pro-inflammatory cytokinesTNF-α, IL-6, IL-1β and IL-8 were significantly increased afterstimulation with Poly I:C. After incubation of the cells with RV-D1at concentration of 1µM, the protein content of TNF-α decreased to20.76±9.3% (p < 0.05), IL-6 to 43.54±14.16% (p < 0.001), IL-1β to46.73±15.93% (p > 0.05) and IL-8 to 51.15±13.01% (p < 0.05),compared with stimulation with poly I:C alone. Similar results weredemonstrated for the mRNA levels of each of these cytokines. Theanti-inflammatory effects of RV-D1 were comparable to those ofDM. The effects of RV-D1 on Poly I:C stimulated HCE cells weredose dependant for the reduction in protein contents of TNF-α, IL-6,IL-1β and IL-8. The decrease in the mRNA expression of TNF-α, IL-6, IL-1β and IL-8 was shown to be related to a decrease in theexpression of I-κBα.Conclusions: RV-D1 may serve as a potent anti-inflammatory agentin ocular surface inflammation, as evaluated in cultured HCE. Theanti-inflammatory effects of RV-D1 are comparable to those ofcorticosteroids, and are mediated through NF-κB signal transduction.Commercial Relationships: Abraham Solomon, None; NirErdinest, NoneProgram Number: 2586 Poster Board Number: D0386Presentation Time: 2:45 PM - 4:30 PMNon-invasive corneal examination of individuals with PANK2mutationMarine Hovakimyan 1 , Karen Falke 1 , Rudolf F. Guthoff 1 , Susanne A.Schneider 2 , Hanna Zimmermann 3 , Alexander U. Brandt 3 ,Friedemann Paul 3 , Jens Wuerfel 3 , Petr Dusek 4 , Oliver Stachs 1 .1 Department of Ophthalmology, University of Rostock, Rostock,Germany; 2 Department of Neurology, University of Kiel, Kiel,Germany; 3 NeuroCure, Charité - University Medicine Berlin, Berlin,Germany; 4 Department of Neurology and Center of ClinicalNeuroscience, Charles University, Prague, Czech Republic.Purpose: Pantothenate kinase-associated neurodegeneration is aprogressive autosomal recessive disorder characterized by early onsetneurological symptoms and iron deposition in the brain, particularlyin globus pallidus. This is a very rare condition caused by mutationsin the gene encoding pantothenate kinase 2 (PANK2), which isrequired for the phosphorylation of pantothenic acid and in theformation of coenzyme A. The heterozygotes were not reported tomanifest any significant abnormalities. While the retinal involvementis well-accepted, there are no data in the literature dealing with thecorneal alterations in this condition. The present study aimed to findany abnormalities in the corneal microstructure in patients withPANK2 mutations.Methods: Patients, homozygous for the PANK2 mutation andasymptomatic heterozygous gene mutation carriers have beenincluded in this preliminary study. The corneal examination wasperformed using in vivo confocal laser-scanning microscopy(CLSM). The corneal sensation was measured with Cochet-Bonnetesthesiometer.Results: All examined corneae displayed a physiological sensation inCochet-Bonnet esthesiometry. Both, in homozygous andheterozygous individuals the in vivo CLSM revealed a normalepithelium with very good distinguishable cell layers. The nerves insubbasal nerve plexus (SBP) revealed normal density and ordinarystructure and distribution in both phenotypes. The number of antigenpresenting cells, dendritic cells, was increased in the homozygousphenotype. Keratocytes nuclei appeared overall as bright ovalobjects, distributed through the entire stroma with a decreasingdensity in anterior-posterior direction. Intrastromal hyperreflectivemicrodots occurred in the anterior stroma of all investigated subjects.The most striking finding of our study was the abundant presence ofvery small hyperreflective particles at the level of Bowman’smembrane, which were found only in homozygotes, but not in theheterozygous gene carriers.Conclusions: This is the first study addressing the corneal features inindividuals with PANK2 mutations. Here, we could show that despiteabsence of clinical signs, the cornea is involved in the pathology ofdisease. The nature and role of the particles observed at theBowman’s membrane has to be appreciated in experimental studies,involving PANK2-mutant animals.Commercial Relationships: Marine Hovakimyan, None; KarenFalke, None; Rudolf F. Guthoff, None; Susanne A. Schneider,None; Hanna Zimmermann, None; Alexander U. Brandt, None;Friedemann Paul, None; Jens Wuerfel, Novartis (R); Petr Dusek,None; Oliver Stachs, NoneProgram Number: 2587 Poster Board Number: D0387Presentation Time: 2:45 PM - 4:30 PMForme Fruste Keratoconus Detection by Pattern Analysis ofCorneal, Epithelial, and Stromal Thickness Maps with OpticalCoherence TomographyYan Li 1 , Ou Tan 1 , Robert Brass 2 , Jack L. Weiss 3 , David Huang 1 .1 Ophthalmology, Oregon Health and Science University, Portland,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaOR; 2 Brass Eye Center, Latham, NY; 3 Gordon & Weiss VisionInstitute, San Diego, CA.Purpose: To screen forme fruste keratoconus (FFK) by analyzingcorneal, epithelial and stromal thickness map patterns with opticalcoherence tomography (OCT).Methods: A 26,000 Hz Fourier-domain OCT system (RTVue CAM,Optovue, Inc.) with 5 µm axial resolution was used. A“Pachymetry+Cpwr” scan pattern (6 mm scan diameter, 8 radials,1024 axial-scans each, repeat 5 times) centered on the pupil was usedto image the cornea of normal and FFK eyes. A computer algorithmwas developed to automatically calculate the corneal, epithelial andstromal thickness maps. The pattern map was defined as the thicknessmap divided by the average thickness of the map. The normalsubjects were divided into the training and evaluation groups. TheOCT maps of the training group were averaged and normalized toserve as the normal average pattern maps. The corneal, epithelial andstromal thickness map pattern standard deviation (PSD) values werecalculated by root-mean-square of the difference between theindividual pattern maps and the normal average pattern maps.Diagnostic accuracy was evaluated by the area under the receiveroperating characteristic curve (AROC) for distinguishing FFK eyesfrom normal eyes (evaluation group).Results: From the normal group, 108 eyes of 57 subjects wereassigned for training (defining the normal pattern maps) and 42 eyesof 22 subjects were used for AROC evaluation. The PSD values forstromal, corneal, and epithelial thickness maps were all significantly(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaIntravital Imaging of the Cellular Dynamics of LysM-PositiveCells in a Corneal Suture Mouse ModelAyaka Koga 1, 2 , Mayumi Ueta 2, 3 , Ryuki Minamiyama 1, 2 , MasaruIshii 4 , Noriko Koizumi 1, 2 , Shigeru Kinoshita 2 . 1 BiomedicalEngineering, Doshisha University, Kyotanabe, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan; 3 Research Center for Inflammation and RegenerativeMedicine, Doshisha University, Kyotanabe, Japan; 4 Laboratory ofBiological Imaging, WPI-Immunology Frontier Research Center,Osaka University, Suita, Japan.Purpose: Intravital imaging is a technique used to analyze cellularand molecular dynamics stereoscopically and time-dependently in anin vivo animal model. In LysM-eGFP mice [gene-targeted miceexpressing enhanced green fluorescent protein (EGFP) under thecontrol of the endogenous lysozyme M promoter], myeloidgranulocyte cells are labeled with GFP in vivo, and the resultingendogenous neutrophils are brightly labeled. The purpose of thispresent study was to analyze the dynamics of LysM-positivegranulocytes (neutrophils) in vivo in a LysM-eGFP corneal suturemouse model using intravital imaging.Methods: A 1-mm 10-1 nylon suture was surgically inserted throughall layers in the center of the cornea of a LysM-eGFP mouse.Dynamics of the LysM-positive granulocytes were then analyzed invivo using intravital imaging.Results: In the unsutured normal cornea (0 hours) of the LysM-eGFPmouse model, LysM-positive granulocytes were present in thecorneal stroma of only the limbal region. At 2 hours after insertion ofthe corneal suture, LysM-positive granulocytes migrated from thelimbal capillary loop to the cornea and conjunctiva, and reached thecorneal suture at 6 hours after insertion. The number of LysMpositivegranulocytes then increased and peaked at 48 hours afterinsertion of the suture. At 72 hours after insertion, infiltration of theLysM-positive granulocytes continued, yet decreased.Conclusions: The findings of this study show that neutrophilsmigrated from the limbal capillary loop and infiltrated to the cornealsuture. Through the use of intravital imaging, the cellular dynamicsthat underlie various physiological and pathological conditions of thecornea were able to be elucidated.Commercial Relationships: Ayaka Koga, None; Mayumi Ueta,None; Ryuki Minamiyama, None; Masaru Ishii, None; NorikoKoizumi, None; Shigeru Kinoshita, Senju Pharmaceutical Co (P),Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon(R), AMO (R), HOYA (R)vessels, the epithelial thickness of the cornea at the limbus and theepithelial thickness at the conjunctiva were quantified (ImageJ, NIHImage). Quantitative differences were analyzed with repeatedmeasure ANOVA.Results: Figures 1A & B show examples of a median filtered OCTimage of the superior and inferior limbus. The outermost lowreflective layer corresponds to the epithelium with conjunctivalvessels. In the layers beneath, episcleral vessels, scleral tissue andapparent deeper drainage channels are visible. For the upper andlower limbus, mean (±SD) corneal epithelial thickness were108±11μm and 93±18μm, and mean conjunctival epithelial thicknesswas 44±10μm and 42±6μm, respectively. The average vesseldiameter for the superior episclera and conjunctiva were 34±7μm and13±1μm. The corresponding diameters for the inferior limbus were26±8μm and 12±2μm.The corneal epithelium at the limbus was thicker than theconjunctival epithelium (LOC p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaIn vivo corneal confocal microscopy (IVCM) and ocular surfaceassessments in patients with mechanical microkeratome vsfemtosecond laser-assisted LASIKMunira Hussain, Jonathan Greene, Nilesh Raval, Matthew Brumm,Shahzad I. Mian, Roni M. Shtein. Ophthalmology and VisualSciences, University of Michigan, Ann Arbor, MI.Purpose: To compare subbasal corneal nerve density by in vivoconfocal microscopy (IVCM) and clinical outcomes 7 to 9 years afterlaser in situ keratomileusis (LASIK) with flap creation bymicrokeratome (MK) or femtosecond laser (FL)Methods: Prospective, comparative, IRB approved study of 46 eyeswhich underwent LASIK, with MK (n=20) and FL (n=26), averageof 8 years ago. Sub-basal corneal nerve density by IVCM, tearproduction by Schirmer test, corneal fluorescein and conjunctivallissamine green staining, tear break up time (TBUT), tear osmolarity,central corneal thickness by ultrasound pachymetry, corneal sensationusing Cochet-Bonnet esthesiometry and Ocular Surface DiseaseIndex (OSDI) were assessed in all subjects. Two-tailed t-tests wereperformed to compare subjects with MK and FL.Results: There were more females than males in the MK group (90%vs 46%, p=0.029) and there was a trend of younger patients in theMK vs FL group (50 yrs vs 55 yrs, p=0.069). Patients in the FL grouphad decreased corneal sub-basal nerve density (7.96 mm/mm2 vs11.40 mm/mm2, p=0.011) and increased corneal sensitivity (5.5 mmvs 4.4 mm, p=0.013) compared to the MK group. There was nosignificant correlation between corneal nerve density and cornealsensitivity (r=-0.23). Patients who underwent LASIK with FL hadsignificantly thinner corneas (486 µm vs 537 µm, p=0.0005) thanthose who had MK LASIK. There were no significant differences inOSDI scores (p=0.097), tear osmolarity (p=0.292), tear production(p=0.208), TBUT (p=0.081), lissamine (p=0.532) or fluoresceinstaining (p=0.853) between the 2 groups.Conclusions: Our preliminary results indicate that there may not belong-term differences in standard measures of ocular surface health(tear osmolarity, Schirmer, ocular surface stain, OSDI) betweenpatients who had the two techniques of LASIK flap creation. Theredo appear to be differences in subbasal nerve density, in cornealthickness and in corneal sensitivity. Further research is needed tounderstand why these differences are observed.Commercial Relationships: Munira Hussain, None; JonathanGreene, None; Nilesh Raval, None; Matthew Brumm, None;Shahzad I. Mian, None; Roni M. Shtein, NoneProgram Number: 2592 Poster Board Number: D0392Presentation Time: 2:45 PM - 4:30 PMIn Vivo Ocular Imaging of the Anterior Segment of the Canineusing High-Resolution Optical Coherence Tomography andConfocal MicroscopyAnn R. Strom 1 , Dennis E. Cortes 2 , Sara M. Thomasy 1 , Philip H.Kass 3 , Vijaykrishna K. Raghuanthan 1 , Mark J. Mannis 2 , ChristopherJ. Murphy 1, 2 . 1 Department of Surgical and Radiological Sciences,University of California, Davis, Davis, CA; 2 Department ofOphthalmology & Vision Science, University of California, Davis,CA; 3 Department of Population Health and Reproduction, Universityof California, Davis, CA.Purpose: To compare normative data for the dog cornea andconjunctiva obtained using high-resolution Fourier-domain opticalcoherence tomography (FD-OCT), in vivo confocal microscopy(ICVM) and ultrasound pachymetry (US). To determine in vivoretinal thickness for the dog, using FD-OCT.Methods: The cornea and conjunctiva of 16 eyes from 8 healthyyoung female intact Beagles were imaged using FD-OCT and IVCM.Central, dorsal paraxial and dorsal perilimbal corneal thickness wasalso measured using US. Quantitative measures for epithelialthickness of the cornea and conjunctiva (FD-OCT), full thickness ofthe cornea (FD-OCT and US) and central retinal thickness (FD-OCT)were determined.Results: The cornea and the corneal epithelium was thinnestcentrally and thickest peripherally. Central and perilimbal cornealthickness as measured by FD-OCT was 497.5 ± 30.3 μm vs. 646.1 ±60.4 μm, respectively. Using FD-OCT, central corneal, perilimbalcorneal and conjunctival epithelial thickness was 52.4 ± 7.25 μm,69.1 ± 7.9 μm and 42.98 ± 6.0 μm, respectively. Central retinalthickness as determined by FD-OCT was 199.4 ± 14.4 μm. IVCMenabled detailed visualization of all corneal elements andquantification of epithelial, nerve fiber, stromal and endothelial celldensities. In agreement with the literature, US pachymetry yieldedsimilar though predictably significantly higher values (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacircumferential distribution (i.e. temporal-nasal, superior-inferior) ofmacrophage-nerve fibre interactions.Conclusions: This study is the first to highlight a direct physicalassociation between nerves and resident immune cells in the murinecornea. These normal data may serve as the basis for future studiesinto the propagation of innate immune responses as well as havingimplications in the study of neurotrophic keratitis and peripheralneuropathy.Commercial Relationships: Yashar Seyed-Razavi, None; Holly R.Chinnery, None; Paul G. McMenamin, NoneSupport: NH&MRC Project Grant (#572709)Program Number: 2594 Poster Board Number: D0394Presentation Time: 2:45 PM - 4:30 PMCORNEAL CURVATURE VARIATIONS EARLY AFTERTRABECULECTOMYLaura Landi, Donatella Musetti, Alessandro Bagnis, MarinaPapadia, Riccardo Scotto, Carlo Enrico Traverso. University EyeClinic, Genova, Italy.Purpose: To examine the immediate effect of trabeculectomy oncorneal curvature.Methods: Cross-sectional study. Eleven eyes from 11 patients (6men, 5 females) with primary open angle glaucoma (POAG) wereincluded in this study. All patients underwent glaucoma surgerybecause of uncontrolled intraocular pressure (IOP) despite maximalmedical therapy. Nine eyes underwent standard trabeculectomy,while an Ex-press device was implanted under a sclera flap in 2 eyes.In all cases mitomycin C (MMC) was applied intra-operatively. Theday before surgery and the first day postoperatively, the cornealcurvature of each patient was evaluated by videokeratoscopy (TMS-4, Tomey, Nagoya, Japan). Student’s t-test and Pearson correlationwere used for statistical analysis.Results: Mean age was 64.9 ± 13.6 years (mean ± SD, range: 36 to80 years). Mean IOP was 27.8 ± 9.1 mmHg preoperatively and 16.6 ±10.5 mmHg in the first day postoperatively. Mean preoperative andpostoperative astigmatism values were 1.04 ± 0.77 and 4.63 ± 3.86diopters respectively. The mean Surface Asimmetry Index (SAI) was0.78 ± 0.29 preoperatively and 1.39 ± 0.39 postoperatively.Statistically significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadifferences between manifest refraction and the three imagingmodalities.Conclusions: Compared with topography and Scheimpflugphotography, OCT better reflects the change in spherical equivalentcorneal refractive change due to LASIK and has less variability. Forastigmatic change, all three modalities were statistically differentfrom the manifest change. However, OCT had the highest correlationand was the only modality with a mean difference less than 0.25 D.OCT provides an improved method to characterize spherical andastigmatic corneal refractive power - as would be needed for cataractsurgery - in the difficult to measure post LASIK population.[1] RP McNabb, F LaRocca, S Farsiu, AN Kuo, and JA Izatt,Biomed. Opt. Express 3, 2050-2065 (2012).[2] N.S Jaffe and HM Clayman, Trans Am Acad OphthalmolOtolaryngol 79, OP615-OP630 (1975)Table 1:Change after LASIK between MRx and 3 imagingmodalities.Commercial Relationships: Ryan P. McNabb, Bioptigen, Inc. (P);Sina Farsiu, Duke University (P); Sandra Stinnett, None; JosephA. Izatt, Bioptigen, Inc. (I), Bioptigen, Inc. (P), Bioptigen, Inc. (S);Anthony N. Kuo, Bioptigen (P)Support: NIH EY020001, EY021522; Coulter TranslationalPartnership Award; Research to Prevent BlindnessProgram Number: 2597 Poster Board Number: D0397Presentation Time: 2:45 PM - 4:30 PMConfocal microscopy: New tool for the follow-up of conjunctivalintraepithelial neoplasiaVitor S. Maduro 1 , Luisa Vieira 1 , Ana Magriço 1 , Arnaldo Santos 1 ,Manuela Martins 2 . 1 Ophthalmology Cornea, CHL-ZC, Lisboa,Portugal; 2 Pathology Department, CHL-ZC, Lisboa, Portugal.Purpose: To analyze the contribution of in vivo confocal microscopyfor the diagnosis and follow-up of conjunctival intraepithelialneoplasiaMethods: Retrospective case series of 5 patients with unilateralconjunctival intraepithelial neoplasia (CIN) submitted to surgery plusadjuvant therapy in a follow-up period of 12 months. All patientswere evaluated before and after surgery with slit-lamp photographyand confocal microscopy (Heidelberg Retina Tomograph II, RostockCornea Module). The authors did a comparative analysis of slit-lampphotographs, confocal microscopy images and histologicalexamination photographs of the same lesions.Results: Five patients (4 males, 1 female) with a mean age of 79,6years, were enrolled in this study. Three were identified as high-gradeintraepithelial neoplasia and two as carcinoma in situ. Thehistological findings correlated well with those seen by confocalmicroscopy: epithelial disorganization with acanthosis, parakeratosis,cellular pleomorphism, high cellular and nuclear reflectivity, highnuclear to cytoplasmic ratio and sometimes binucleation. The lesionis well demarcated and the subbasal corneal nerves were notvisualized in areas affected by CIN. Confocal microscopy hasidentified 1 relapse and was useful to monitor the treatment response.Conclusions: In vivo confocal microscopy may be important notonly in the diagnosis of CIN, but also detecting relapses andmonitoring the treatment response, in a relative non-invasive manner.Commercial Relationships: Vitor S. Maduro, None; Luisa Vieira,None; Ana Magriço, None; Arnaldo Santos, None; ManuelaMartins, NoneProgram Number: 2598 Poster Board Number: D0398Presentation Time: 2:45 PM - 4:30 PMPhenotypic characterization of Reis-Bücklers corneal dystrophyQingfeng Liang 1 , Xuguang Sun 1 , Zhiqiang Pan 1 , Antoine Labbe 1, 2 .1 ophthalmology, Beijing Institute of Ophthalmology,Beijing TongrenEye Center, Beijing Tongren Hospital, Capital Medical University,Beijing, China; 2 Ophthalmology, Quinze-Vingts National EyeCenter, Paris and Versailles Saint-Quentin-en-Yvelines University,Versailles, France., Pairs, France.Purpose: To determine the phenotype of Reis-Bücklers cornealdystrophy (RBCD) using biomicroscopy, in vivo confocalmicroscopy (IVCM), anterior segment optical coherence tomography(AS-OCT), and light and electron microscopy.Methods: Five patients from a Chinese family with clinical RBCDand TGFBI gene mutations were evaluated. All patients underwentcomplete ophthalmogic examinations including slit-lampbiomicroscopy, IVCM and AS-OCT analysis. After penetratingkeratoplasty, corneal buttons were examined under light andtransmission electron microscopy.Results: Slit-lamp biomicroscopy showed irregularly ring-shapedand geographic grey-white opacities arranged symmetrically at or justnear the level of Bowman's membrane in all affected eyes. UsingIVCM, Bowman’s membrane was replaced by a granular andirregular hyper-reflective material without any shadows. In theanterior stroma next to the Bowman’s membrane, amyloid-likehyper-reflective material was also observed. AS-OC showed highlyreflective deposits concentrated primarily at the level of Bowman'smembrane. Analysis of corneal buttons under light microscopyrevealed that Bowman's membrane was replaced by a granular tissue,which extended into the anterior corneal stroma. Using electronicmicroscopy, rod-shaped bodies were observed within this granulartissue.Conclusions: Although genetic analysis might facilitate precisediagnosis and corneal dystrophy classification, these results suggestthat IVCM and AS-OCT may be a useful adjunct to slit lampbiomicroscopy for the diagnosis and management of RBCD.Commercial Relationships: Qingfeng Liang, None; Xuguang Sun,None; Zhiqiang Pan, None; Antoine Labbe, NoneProgram Number: 2599 Poster Board Number: D0399Presentation Time: 2:45 PM - 4:30 PMIn Vivo Morphology of the Limbal Palisades of Vogt Correlateswith Progressive Stem Cell Deficiency in Aniridia-RelatedKeratopathyTor P. Utheim 1, 2 , Neil S. Lagali 3 , Ulla Eden 3 , Xiangjun Chen 2 , RuthRiise 4 , Anette Dellby 3 , Per Fagerholm 3 . 1 Avdeling for medisinskbiokjemi, Oslo University Hospital, Oslo, Norway; 2 SynsLaserKirurgi AS, Oslo, Norway; 3 Department of Clinical and ExperimentalMedicine, Faculty of Health Sciences Linköping University,Linköping, Sweden; 4 Department of Ophthalmology, InnlandetHospital, Elverum, Norway.Purpose: To investigate morphologic alterations in the limbalpalisades of Vogt in a cohort of Norwegian patients with congenitalaniridia.Methods: All subjects were examined bilaterally by slit lampbiomicroscopy, and the limbal palisades of Vogt were examined©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamicroscopically by laser-scanning confocal microscopy (IVCM). Slitlamp biomicroscopy was used to grade the stage of aniridia-relatedkeratopathy (ARK) based on degree of conjunctivalization of theperipheral and central cornea. By IVCM, specific limbal morphologyof epithelial cells, palisade ridges and projections was assessed.Results: Two eyes were found without clinical signs of ARK, and inthese eyes, the limbal palisades had a normal-appearing morphology.In the earliest stages of ARK where conjunctiva extended over thelimbal barrier, recognizable microscopic remnants of palisadestructures were apparent, but no normal-appearing palisade structureswere visible by IVCM. Limbal epithelial cells lost a regular mosaicand conjunctival-type epithelium invaded the limbal space. In all eyeswith later stages of ARK, conjunctivalization of the periphery orentire cornea was observed clinically, while no palisade structureswere present by IVCM. In a control group, normal limbal palisadesof Vogt structures were observed in the superior limbus in all eyes,but in five eyes in the inferior limbus normal palisades structureswere replaced by abnormal features.Conclusions: There appears to be a strong relationship of themicroscopic appearance of the limbal palisades of Vogt in aniridiaand the corresponding clinical degree of conjunctivalization of thecornea, which lends support to the theory that the palisades of Vogtrepresents a stem cell niche. IVCM could be a useful tool to assessearly morphologic changes that accompany stem cell deficiency.Commercial Relationships: Tor P. Utheim, None; Neil S. Lagali,None; Ulla Eden, None; Xiangjun Chen, None; Ruth Riise, None;Anette Dellby, None; Per Fagerholm, NoneSupport: Crown Princess Margareta’s Foundation for the VisuallyImpaired, Carmen and Bertil Regnérs Foundation for Research in EyeDisease, County Council of Östergötland, and Aniridia NorwayProgram Number: 2600 Poster Board Number: D0400Presentation Time: 2:45 PM - 4:30 PMLaser In Vivo Confocal Microscopy Demonstrates Diminishmentof Subbasal Nerve Plexus in Early Stage Fuchs EndothelialCorneal DystrophyShruti Aggarwal, Bernardo M. Cavalcanti, Andrea Cruzat, LauraRegali, Ula V. Jurkunas, Pedram Hamrah. Cornea, Ophthalmology,Massachusetts Eye& Ear Infirmary, Boston, MA.Purpose: To evaluate corneal sensation and subbasal nerve plexuschanges using laser in vivo confocal microscopy (IVCM) in early andlate stage Fuchs endothelial corneal dystrophy (FECD).Methods: A prospective, cross sectional study was conducted and 33FECD patients (33 eyes) were included. FECD patients wereclassified into early (without edema) and late stage (with edema).Fourteen eyes with pseudophakic bullous keratopathy (PBK) wereused as positive controls (edematous cornea) and 17 normal agematchedcontrols eyes as negative controls. IVCM (HRT3/RCM) wasperformed in the central cornea. Three representative images wereevaluated by masked 2 observers for density and number of subbasalnerves (NeuronJ), and presence of dendritic immunecells(DC)(ImageJ). Central corneal sensation was assessed byCochet-Bonnet esthesiometer.Results: Eyes with FECD and PBK showed significantly diminishedsubbasal nerves, including total nerve length (11.5±1.3 and2.8±0.7mm/mm2, respectively;p=0.001) and total number of nerves(8.8±1.1 and 2.2±0.4 n/frame;p=0.001), as compared to controls(23.3±8.1 mm/mm2 and 25.9±5.3 n/frame). Decreased nervescorresponded (R = 0.32) to diminished sensation in both FECD(4.9±0.2cm;p=0.045) and PBK (3.6±0.6;p=0.001) as compared tocontrols (5.9±0.04). Both early stage (n=22) and late stage FECD(n=11) showed significant reduction in total nerve density (13.1±1.4;9.9±1.2, respectively;) and number (8.2±2.5; 6.5±2.1), compared tocontrols (p100cells/mm2).Conclusions: IVCM demonstrates profound diminishment ofsubbasal nerves in both FECD and PBK, correlating to decreasedsensation. Interestingly, even early-stage FECD patients haddecreased subbasal nerves. Further, increased DC were found inpatients with FECD, demonstrating subclinical inflammation. Thedata suggests that reduction in subbasal nerves and inflammation maypotentially play a role in FECD. Additional studies are required toinvestigate whether subbasal nerve alterations are directly caused bydecreased endothelial cell density, or potentially lead to loss ofendothelial cells.Commercial Relationships: Shruti Aggarwal, None; Bernardo M.Cavalcanti, None; Andrea Cruzat, None; Laura Regali, None; UlaV. Jurkunas, 61/482,769 (P), Altheos (C); Pedram Hamrah, NoneSupport: Funding: NIH K08-EY020575, Research to PreventBlindness Career Development Award, Falk Medical Research Trust,New England Corneal Transplant Research FundProgram Number: 2601 Poster Board Number: D0401Presentation Time: 2:45 PM - 4:30 PMNew Diagnostic Parameters in Staging Limbal Stem CellDeficiency Using In Vivo Laser Scanning Confocal MicroscopyEric H. Chan 1 , Martin N. Nakatsu 2 , Sophie X. Deng 2 . 1 David GeffenSchool of Medicine at UCLA, Los Angeles, CA; 2 Cornea Division,Jules Stein Eye Institute, Los Angeles, CA.Purpose: To investigate the changes in corneal cytology andepithelium in different stages of LSCD using in vivo confocalmicroscopy (ICM) to establish a more accurate diagnostic algorithm.Methods: This was a prospective study, in which a total of 33 eyesdiagnosed clinically with LSCD were selected for ICM examinationand analyzed retrospectively. Of those 33 eyes, 20 eyes alsounderwent impression cytology. Ten normal eyes were included ascontrols for ICM examination. Confocal imaging of the centralcornea, the superior, nasal, inferior, and temporal limbus werecollected using the Heidelberg Retina Tomograph III RostockCorneal Module. Epithelial layer thickness, basal cell density, andbasal cell diameter were measured in images demonstrating clearepithelial cell morphology. Statistical analysis was evaluated usingANOVA, Kruskal-Wallis tests, and two-tailed T-tests.Results: Imaging of epithelial cell morphology in LSCD eyesrevealed prominent nuclei, loss of distinct cell borders, and generalloss of basal cells with and without preservation of subbasal nerves.In normal eyes, the average epithelial thickness measured by ICMwas 47.8 µm, 70.0 µm, 60.2 µm, 58.6 µm, and 62.2 µm in the centralcornea, inferior limbus, nasal limbus, superior limbus, and temporallimbus, respectively. When compared to respective normal limbuslocations, the epithelial thickness in the limbus affected by LSCDdecreased by 27% and 45% in early stage and intermediate stagedisease, respectively. Differences between early and intermediatechanges in epithelial thickness of the limbus were statisticallysignificant (p=0.04). Nine of the 20 eyes were positive for gobletcells on impression cytology. Goblet cells if present were foundirrespective of LSCD stage.Conclusions: Our data show that the limbal epithelium becomesprogressively thinner in advanced LSCD. Additionally, presence ofgoblet cells is not a reliable diagnostic marker of the early andintermediate stages of LSCD.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Eric H. Chan, None; Martin N.Nakatsu, None; Sophie X. Deng, NoneProgram Number: 2602 Poster Board Number: D0402Presentation Time: 2:45 PM - 4:30 PMIncidence and type of higher order corneal aberrations in thecataract populationNeil M. Vyas 1 , Bonnie A. Henderson 2 . 1 Ophthalmology, BostonUniversity, Boston, MA; 2 Ophthalmic Consultants of Boston, Boston,MA.Purpose: Corneal higher order aberrations (HOA) are commonculprits in symptoms such as glare, halos, night vision disturbances,and decreased contrast sensitivity. These can become even moreapparent after cataract surgery with presbyopia correcting multifocalintraocular lenses. Evaluation of HOAs has been proposed asscreening tool to predict which patients may be dissatisfied afterimplantation of these lenses. This project compares the incidence andtypes of higher order aberrations in patients presenting for cataractextraction.Methods: Retrospective case series of consecutive patients whopresented for cataract extraction. Patients underwent topographicaland higher order corneal wavefront mapping with the Nidek OPD 2.Age, gender, amounts and types of corneal higher order aberrationswere recorded in both eyes.Results: 200 eyes (100 patients) were evaluated. Trefoil was noted tohave the highest incidence and highest Z-coefficient values across allage groups. It accounted for more of the total higher order aberrationsthan coma or quatrefoil. This was also noted to be consistent betweeneyes. A correlation between increasing age and increasing totalamounts of quatrefoil was also observed. The average values forcoma were similar between eyes across all age groups.Conclusions: The total amount of corneal HOA, especiallyquatrefoil, increased with age. The most common HOA and highestZernike coefficient in patients undergoing cataract surgery wastrefoil. Knowing the average amount of different HOAs may beuseful when evaluating the HOA maps of preoperative patients whoare choosing multifocal intraocular lenses in order to better predictpost operative satisfaction. Given the large variation of errorsbetween eyes, each eye should be evaluated separately and successfuloutcome in one eye does not necessarily mean similar success can beanticipated in the fellow eye.Commercial Relationships: Neil M. Vyas, None; Bonnie A.Henderson, Alcon (C), Bausch and Lomb (C)Program Number: 2603 Poster Board Number: D0403Presentation Time: 2:45 PM - 4:30 PMComparison of Dual Scheimpflug imaging parameters in eyeswith forme fruste keratoconus, keratoconus and low and highametropiaMaria A. Henriquez, Luis Izquierdo, Harumi M. Moreyra.Ophthalmology, Oftalmo-Salud, Lima, Peru.Purpose: To evaluate the efficacy of several parameters obtainedfrom Dual Scheimpflug Analyzer to discriminate between formefruste keratoconus, keratoconus and low and high ametropia.Methods: This prospective study included 382 eyes (100 eyes withlow ametropia, 50 with high cylinder, 29 with high myopia, 41 formefruste keratoconus and 162 keratoconus). Scheimpflug imaging usingthe Galilei Dual Scheimpflug Analyzer (Ziemer) was performed and30 parameters derived from pachymetry, curvature, anterior andposterior elevation maps, corneal wavefront high order aberrations,asphericity and others was recorded. Receiver operating characteristiccurves (ROC) was used and logistic regression analysis was used toevaluate the sensitivity and specificity of the parameters in aconstructed model.Results: There were statistically significant difference between lowametropic eyes and high cylinder group in the anterior and posteriorelevation at the thinnest point of the cornea (p < 0.001 both). Therewere not statistically significant differences between the lowametropic eye and high myopic group in pachymetric and elevationvariables (p > .05). Logistic regression analysis showed that acombination of corneal power, thickness, elevation, and cornealaberrations had a 100% sensitivity and specificity discriminatingbetween low ametropic eyes and keratoconus.Conclusions: Combined analysis of anterior and posterior cornealpower, thickness, elevation and corneal aberrations effectivelydiscriminated between ectatic corneas and normal corneas. Highastigmatism group showed greater posterior elevation values thanhigh myopic group and eyes with low refraction.Commercial Relationships: Maria A. Henriquez, None; LuisIzquierdo, None; Harumi M. Moreyra, NoneSupport: None in the Support field belowProgram Number: 2604 Poster Board Number: D0404Presentation Time: 2:45 PM - 4:30 PMStructural changes in the retina and cornea during diabeticneuropathyMaxwell Stem 1 , Munira Hussain 1 , Melody Chan 2 , Stephanie J. Chiu 2 ,Pratul Srinivasan 2 , Jeffrey M. Sundstrom 1 , Thomas W. Gardner 1 ,Sina Farsiu 2 , Rodica Pop-Busui 1 , Roni M. Shtein 1 . 1 Ophthalmologyand Visual Sciences, University of Michigan, Ann Arbor, MI;2 Biomedical Engineering, Duke University, Durham, NC.Purpose: To examine the relationship between neuroretinal thicknessand corneal nerve fiber length (CNFL) relative to diabetic neuropathy(DN) status in patients with diabetes mellitus (DM)Methods: In this cross-sectional study, we examined 25 diabeticpatients without DN, 10 patients with mild DN, 8 patients with severeDN, and 9 controls without diabetes. DN status was assigned basedon a combination of clinical symptoms, signs, and neurophysiologictesting. Patients underwent optical coherence tomography (OCT)imaging of the retina, in vivo confocal microscopy (IVCM) of thecorneal sub-basal nerve plexus, and nerve conduction velocity (NCV)testing. DOCTRAP software was used to segment inner retinal layerboundaries. Post-hoc analysis of the OCT and IVCM images wasperformed to quantify the average thickness of each retinal layer andthe average CNFL. ANOVA was used to assess for differences inretinal thickness, CNFL, and NCV among the subjects. Pearsoncorrelations were used to evaluate the relationship between NCV andthe retinal and corneal parameters.Results: All 25 diabetic patients without DN had type 1 DM, and theremaining patients with DN had type 2 DM. There were nosignificant differences in average retinal nerve fiber layer, ganglioncell/inner plexiform layer, inner nuclear layer, outer plexiform layer,outer nuclear layer/inner segment, outer segment, retinal pigmentepithelial, or total retinal thicknesses among the 4 groups. Patientswith type 2 DM and severe DN had reduced corneal nerves (CNFL ±SD = 12.5 ± 6.1 mm/mm 2 ) compared to controls (20.7 ± 2.2mm/mm 2 ) (p=0.009). Persons with type 1 DM without DN also hadreduced CNFL (15.1 ± 4.7 mm/mm 2 ) relative to controls (p=0.033).Peroneal NCV was reduced only in severe DN (mean velocity ± SD =32.4 ± 4.1 m/s) relative to controls (45.8 ± 6.9 m/s) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacontrols. This suggests that IVCM of the corneal sub-basal nerveplexus may be a more sensitive indicator of peripheral nervedegeneration in type 1 vs. type 2 DM.Commercial Relationships: Maxwell Stem, None; MuniraHussain, None; Melody Chan, None; Stephanie J. Chiu, DukeUniversity (P); Pratul Srinivasan, None; Jeffrey M. Sundstrom,None; Thomas W. Gardner, Kalvista (C), Aerpio (C), Akebia (C),Penn State University (P); Sina Farsiu, Duke University (P); RodicaPop-Busui, None; Roni M. Shtein, NoneSupport: This research was supported by the Michigan DiabetesResearch & Training Center funded by NIH P60DK020572 from theNational Institute of Diabetes and Digestive and Kidney Diseases(RMS); American Diabetes Association-Merck (MS); JuvenileDiabetes Research Foundation (TWG); Taubman Institute (TWG);Research to Prevent Blindness (TWG); NIH Grant 1R01EY022691-01 (SF); NIH Grant R01 HL102334 (RP)Program Number: 2605 Poster Board Number: D0405Presentation Time: 2:45 PM - 4:30 PMRepeatability of peripheral (near-limbal) corneal thicknessmeasurements of Pentacam Scheimpflug corneal topographyRaul Martin 1 , Sven Jonuscheit 2, 3 , Michael J. Doughty 2 , Ana del RioSan Cristóbal 1 , Lisa J. Mackintosh 2 , David MacTaggart 2 , MichaelHiscock 2 . 1 Optometry Research Group, IOBA Eye Institute,Valladolid, Spain; 2 Vision Sciences, Department of Life Sciences,Glasgow Caledonian University, Glasgow, United Kingdom;3 Diabetes Research Group, Institute for Applied Health Research,Glasgow Caledonian University, Glasgow, United Kingdom.Purpose: To assess the repeatability of peripheral corneal thickness(CT) measurements along the horizontal meridian as close to thelimbus as possible in healthy eyes with Scheimpflug topographyPentacam.Methods: CT was measured in 63 corneas of 63 healthy subjects ateleven corneal locations nominally 1 mm apart along the horizontalmeridian with Scheimpflug topography Pentacam. Two consecutiveScheimpflug scans were performed in quick succession to determinethe repeatability of the pachymetry values measured in each corneallocation up to 5 mm away from the corneal apex. Ocular assessmentsalso included habitual visual acuity, slit-lamp biomicroscopy, andoptical coherence tomography. To rule out corneal endotheliopathynon-contact specular microscopy was performed. Descriptivestatistics were generated. The repeatability was determined byassessing absolute differences between measurement 1 and 2. Therelative difference was then calculated and expressed as the ratio ofthe absolute difference to the central CT. A variant of Bland-Altmananalysis was carried out to determine the effect of the overallmagnitude of CT on the differences between pachymetrymeasurements.Results: The mean [SD] age was 27 [7] years. CT increasedprogressively and asymmetrically from the corneal center to theperiphery. Mean CT (µm) at the temporal 5 mm location was 771[49] and 823 [53] at the corresponding nasal location; central CT was558 [33]. Peripheral CT repeatability, based on absolute differences(microns) between 2 measurements was 2.41 [13.41] temporally and-1.03 [15.39] nasally, both at 5 mm away from the corneal apex.These values decreased to just 0.27 [5.33] for central CT. Thisdifference in repeatability between peripheral and central locationswas not statistically significant (p=0.918 Kruskal-Wallis ANOVA,nasal and temporal). Regression analyses indicate that differencesbetween measurements (repeatability) were not dependent on theabsolute thickness values (p=0.978; r= 0.10).Conclusions: Pentacam shows excellent repeatability not only forcentral pachymetry measurements near the corneal apex, but also forperipheral measurements close to the corneal limbus 5 mm awayfrom the corneal centre in normal eyes. The results provide evidenceof the utility of Pentacam assessment of the peripheral cornea inextended contact lens wear and surgical procedures.Commercial Relationships: Raul Martin, None; Sven Jonuscheit,Santander UK plc (F); Michael J. Doughty, None; Ana del Rio SanCristóbal, None; Lisa J. Mackintosh, Santander UK plc (F); DavidMacTaggart, Santander UK PLC (F); Michael Hiscock, NoneSupport: Jose Castillejo Program, Spanish Ministry of EducationProgram Number: 2606 Poster Board Number: D0406Presentation Time: 2:45 PM - 4:30 PMComparison of corneal shape and ocular aberrations in eyes withforme fruste keratoconus to that of normal corneaRyotaro Ueki 1, 2 , Naoyuki Maeda 1 , Mutsumi Fuchihata 1 , ShizukaKoh 1 , Takashi Kitaoka 2 , Kohji Nishida 1 . 1 Ophthalmology, OsakaUniv School of Medicine, Osaka-Shi, Japan; 2 Ophthalmology,Nagasaki Univ School of Medicine, Nagasaki-Shi, Japan.Purpose: Forme fruste keratoconus (FFK) is defined as a corneawhich has no abnormal findings by slit-lamp examinations andPlacido-based corneal topography, and is the fellow eye of a patientwith clinical keratoconus. FFK has recently gained a lot of attentionbecause it is the borderline condition between keratoconus suspectand normal cornea. The purpose of this study was to compare thecorneal shape and ocular aberrations of eyes with FFK to that ofnormal corneas.Methods: We studied 38 eyes with FFK and 50 eyes with normalcorneas. The corneal thickness and location of thinnest point, Belin-Ambrósio enhanced ectasia Display (BAD) parameters, Df, Db, Dp,Dt, Da, and D, and corneal coma aberrations (anterior, posterior andtotal cornea) for a 4 mm diameter were obtained with a rotatingScheimpflug camera (Pentacam HR, OCULUS Optikgeräte GmbH,Germany). The ocular spherical aberration (C4,0) and comaaberration (C3,1 and C3,-1) for a 4 mm diameter were obtained by anaberrometer (KR-1W, Topcon Corp., Japan). Multiple logisticregression analyses and the receiver operating characteristic (ROC)analyses were performed on these data to determine whether the twogroups can be differentiated.Results: Multiple logistic regression analyses showed the BADparameter D, the anterior corneal coma, and the total corneal comawere significant variables for differentiating FFK group from thenormal group (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Equilibrium distribution ratio of fluorescein between a/hand stroma, which is an important parameter required for estimationof corneal endothelial permeability in vivo, is affected by pH andadsorption to macromolecules. To examine the stromalmicroenvironment, we have established a confocal scanningmicrofluorometer (CSMF) for measurement of fluorescence lifetimeof fluorescein across the cornea.Methods: A custom-built CSMF was interfaced with components forlifetime measurements in the time domain based on time-correlatedsingle photon counting (TCSPC). The excitation source was apicosecond pulsed laser (Becker & Hickl, Berlin, Germany, BDL-445-SMC) operated at a repetition rate of 50 MHz with the outputpower at the focal plane set to 1.4 μW. The emission photons (>530nm) were detected by a high-speed hybrid photon counter (HPM-100-40) with a resolution of 120 ps. The detector output was coupledto a TCSPC module (SPC-130-EM, Becker & Hickl) with referencederived from the laser pulses. The decay functions were analyzed bynon-linear least squares using the SPCM Imaging software. Depthscanning was performed with a nano-positioning scanning assembly(M126-DG; PI Polytec) coupled to the microscope body. Freshporcine eye balls were mounted underneath the objective (40x W;0.75 NA Zeiss), and fluorescein was loaded into the cornea byscratching the epithelium.Results: The instrument response function, obtained using RoseBengal, which is reported to have a lifetime of 16 ps, indicated a fullwidth at half maximum of 150 ps. The fluorescence decay curve forfluorescein (1 μM in PBS at pH = 8.0), which could be fitted to amono-exponential decay, yielded a lifetime of 3.99 ns. Increasing thefluorescein concentration 10-fold did not affect the lifetime. Lifetimeof fluorescein across the stroma, ~30 min after instillation of 10 μM,varied from 3.6 to 3.9 ns at the anterior and posterior stroma,respectively. The depth resolution of all the measurements was 12μm.Conclusions: Previously, intensity measurements have shownsignificant uneven equilibrium distribution of fluorescein across thestroma, with the ratio of fluorescence emission between aqueoushumor and stroma increasing from 3 at the anterior stroma to 2 at theposterior. However, a significantly higher decrease in the lifetime offluorescein in the anterior stroma indicates lower pH and/or increasedalbumin in the anterior stroma.Commercial Relationships: Yueren Wang, None; Sven Peters,None; Martin Hammer, None; Yiran Jiang, None; Tom Kemerly,None; Uday B. Kompella, University of Colorado Denver (P),PCAsso Diagnostics (C), NanoTrans Technologies, Inc. (F),Univesity of Nebraska Medical Center (P); Sangly P. Srinivas, NoneProgram Number: 2608 Poster Board Number: D0408Presentation Time: 2:45 PM - 4:30 PMAnalysis of Interocular Ocular Surface Aberrations UsingSurface AberrometryVaradharajan Jayakumar, Natalie Hutchings, VasudevanLakshminarayanan, Lyndon W. Jones. Optometry and VisionScience, University of Waterloo, Waterloo, ON, Canada.Purpose: The purpose of this study was to evaluate the inter-ocularcharacteristics of the ocular surface aberrations using surfaceaberrometry.Methods: The sample comprised ten participants with non-invasivetear breakup time (TBUT) between 5.38s and 7.82s (mean OD 6.94s;mean OS 6.24s). Measurements of dynamic anterior surfaceaberrations and corneal topography were obtained in both eyes of allparticipants using the Topcon CA200F corneal analyzer (25 fps).Anterior surface aberration measurements were obtained for two, 15second open eye intervals. Surface aberrations coefficients wereobtained for a 6mm pupil diameter.Analysis of the data was done using the R statistical program andStatistica. The raw RMS and the coefficient for vertical prism weresmoothed using a running mean procedure (R: CATools; k=11). Asegmented linear regression (R: Segmented) was fitted to the smoothdata. The highest positive slope for the linear segments and the timesafter the start of the blink epoch at which this occurred (thebreakpoint) were determined. The breakpoint is assumed tocorrespond with the timepoint at which the ocular surface starts tolose regularity.Results: The highest positive slope for the RMS was, on average,higher in the second eye measured (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaexamine the association between DED and determinants.Results: Of the 3824 females 367 (9.6%) were assigned as a case ofDED, and 3171 (82.9%) as a control. The prevalence increased everyage decade, from 2.7% in 20 to 30 years, to 20.0% in 80 years andolder. 794 (20.8%) had dry eye symptoms in the preceding threemonths, which was also more prevalent with increasing age.Determinants that were significantly associated with DED were age,contact lens wear, asthma, eczema, allergy, cataract surgery,rheumatoid arthritis, osteoarthritis, migraine and stroke. The highesteffect sizes were found with depression (odds ratio (OR) 1.67 (95%CI 1.27-2.19)), irritable bowel syndrome (OR 2.24 (95% CI 1.76-2.85)) and chronic widespread pain syndrome (OR 2.13 (95% CI1.42-3.18) (all p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Thirugnana Vijmasi, None; FeelingYu Ting Chen, None; Robert L. McKown, EyeRx Research, Inc.(I); Gordon W. Laurie, UVa Patent Foundation (F); Nancy A.McNamara, NoneSupport: NIH Grant EY016203; NIH Grant EY02162Program Number: 2668Presentation Time: 9:15 AM - 9:30 AMBridging ocular therapeutics and contact lenses via thermoresponsiveprotein polymersWan Wang 1 , Pu Shi 1 , Suhaas Aluri 1 , Pang-Yu Hsueh 1 , Maria C.Edman 1 , Denise S. Ryan 6 , Robert L. McKown 5 , Sarah F. Hamm-Alvarez 1, 3 , Gordon W. Laurie 4 , John A. MacKay 1, 2 . 1 Department ofPharmacology and Pharmaceutical Sciences, University of SouthernCalifornia, Los Angeles, CA; 2 Department of BiomedicalEngineering, University of Southern California, Los Angeles, CA;3 Keck School of Medicine, University of Southern California, LosAngeles, CA; 4 University of Virginia, Charlottesville, VA;5 Department of Integrated Science and Technology, James MadisonUniversity, Harrisonburg, VA; 6 US Army Warfighter Refractive SurgResearch Ctr, Fort Belvoir, VA.Purpose: To influence the bioavailability of the prosecretory mitogenlacritin, we fused it with elastin-derived protein polymers, elastin-likepolypeptides (ELPs) that have a range of assembly properties. ELPsreversibly phase separated onto Proclear TM contact lens and mayprovide an alternative route for delivery of protein therapeutics to theocular surface system.Methods: Lac-ELP genes were expressed in E. coli and purifiedutilizing Inverse Transition Cycling (ITC) followed by size exclusionchromatography. Phase behavior and nanoparticle formation offusion proteins were characterized by optical density, Dynamic LightScattering (DLS), TEM and Cryo-TEM. In vitro activities were testedusing rabbit lacrimal gland acinar cells (LGACs) and SV40-immortalized human corneal epithelial cells (HCE-T). In vitroProclear TM contact lens retention was characterized by fluorescencedetection.Results: A novel fusion protein library based on recombinant humanlacritin and elastin-like-polypeptides (ELPs) was designed as apotential therapeutic for the ocular surface. The Lacritin-ELP fusionproteins imparted thermo-sensitive assembly of micron-sizedcoacervates or 130-140nm diameter micelles depending on thesequence of their ELP tag. Exogenous Lac-ELPs promoted β-hexosaminidase secretion from rabbit lacrimal gland acinar cells(LGACs); evoked Ca 2+ wave propagation across Human CornealEpithelial Cells (HCE-T) and were taken up by both LGACs andHCE-T cells. ELPs reversibly attached onto Proclear TM contact lensand the total retention time was temperature and Tt dependent.Conclusions: Exploration of thermo-responsive prosecretorymitogen lacritin-ELPs may provide an alternative approach to adjusttheir ocular retention and interaction with target cellular receptor.Moreover, biocompatible elastin-like polypeptides (ELPs) appear toprovide a reversible, temperature dependent bridge between potentialocular therapeutics and other polymers used on the ocular surface.Commercial Relationships: Wan Wang, University of SouthernCalifornia (P); Pu Shi, None; Suhaas Aluri, None; Pang-Yu Hsueh,University of Southern California (P); Maria C. Edman, Universityof Southern California (P); Denise S. Ryan, None; Robert L.McKown, EyeRx Research, Inc. (I); Sarah F. Hamm-Alvarez,None; Gordon W. Laurie, UVa Patent Foundation (F); John A.MacKay, University of Southern California (P)Support: USC School of Pharmacy, NIH GRANT EY011386, theUSC Whittier foundation and the USAMRAA/TATRC VRP HAD11262019Program Number: 2669Presentation Time: 9:30 AM - 9:45 AMLifitegrast 5.0% Ophthalmic Solution Reduces Ocular SurfaceStaining and Improves Symptoms in Patients with Dry EyeDisease: Results of a Phase 3 Study (OPUS-1)Charles P. Semba 1 , Gail L. Torkildsen 2 , Francis A. D'Ambrosio 3 ,John Lonsdale 4 , Eugene B. McLaurin 5 , Richard A. Eiferman 6 ,Kathryn S. Kennedy 7 , John D. Sheppard 8 . 1 Dept. Ophthalmology andVisual Sciences, SARcode Bioscience, Inc., Brisbane, CA; 2 AndoverEye Associates, Andover, MA; 3 D'Ambrosio Eye Care, Lancaster,MA; 4 Central Maine Eye Care, Lewiston, ME; 5 Total Eye Care,Memphis, TN; 6 University of Louisville, Louisville, KY;7 Independent Biostatistical Consultants, Tempe, AZ; 8 EasternVirginia Medical School, Norfolk, VA.Purpose: To report the results of a pivotal study comparing lifitegrastto placebo in subjects with dry eye disease (DED). DED is associatedwith ocular surface inflammation leading to symptoms of discomfort.Lifitegrast is an investigational drug targeting the integrinlymphocyte antigen-1 (LFA-1) and inhibits binding to its cognateligand, intracellular adhesion molecule-1 (ICAM-1) by serving as anICAM-1 decoy. LFA-1/ICAM-1 interactions are key steps regulatingT-cell mediated inflammation.Methods: A randomized, double-masked, placebo-controlled studyenrolled 588 dry eye subjects randomized 1:1 to lifitegrast or placebo(vehicle) administered BID for 84 days. Eligibility included no activelid margin disease, STT ≥ 1 and ≤ 10 mm, and worsening offluorescein staining and symptoms upon exposure to a controlledadverse environment (CAE). Corneal fluorescein / lissaminestaining parameters (Ora) and symptoms (visual analog scale[dryness, burning/stinging, photophobia, FB sensation, pain, blurredvision, itching], ocular discomfort (Ora)) were collected at baseline,Day 14, 42, and 84.Results: Lifitegrast demonstrated superiority to placebo in reducinginferior (P=0.0007), superior (P=0.0392), and total corneal staining(P=0.0148), baseline to Day 84. This was accompanied by significantimprovements in nasal (P=0.0039), temporal (P=0.0936) and total(P=0.0285) lissamine staining, baseline to Day 84. Onset of actionwas observed as early as Day 14 and maintained through Day 84. Thechief medical complaints of dryness and discomfort weresignificantly improved at Day 84 (P=0.0291, P=0.0273, respectively).There were no serious ocular adverse events (AE). The most commonocular AE were instillation site complaints (e.g., irritation) upon theinitial dose of lifitegrast at Day 0. No significant changes wereobserved in IOP, BCVA, slit-lamp, dilated fundoscopy, and cornealsensitivity. The most common non-ocular AE was dysgeusia (taste)in ∼13% of lifitegrast subjects.Conclusions: Lifitegrast 5.0% ophthalmic solution demonstratedsuperiority to placebo in reduction of corneal fluorescein andconjunctival lissamine staining - key clinical parameters of dry eyedisease; reductions in staining were associated with significantimprovements in key symptoms. Lifitegrast appears safe and welltoleratedwhen administered BID for 84 days.Commercial Relationships: Charles P. Semba, SARcodeBioscience, Inc. (E); Gail L. Torkildsen, Ora (C), Ora (R), AndoverEye Associates (C); Francis A. D'Ambrosio, None; John Lonsdale,Sarcode (F), Seikagaku Corporation (F), CTD Development america,Inc. (F); Eugene B. McLaurin, None; Richard A. Eiferman,Sarcode (F); Kathryn S. Kennedy, SARcode Bioscience, Inc. (C);John D. Sheppard, Alcon (C), Allergan (C), Abbott (C), Bausch &Lomb (C), EyeGate (C), Lux Biosciences (C), SarCode (C), ScienceBased Health (C), 1-800-Doctors (C), Merck (C), Lacrisciences (C),©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaOcucure (C), EyeRx Research (C), SarCode, EyeRx, Ocucure,Lacrisciences (I)Support: Study supported by SARcode Bioscience,Inc.Clinical Trial: NCT01421498Program Number: 2670Presentation Time: 9:45 AM - 10:00 AMEffects of L-carnitine, Erythritol and Betaine on InflammatoryMarkers in Primary Human Corneal Epithelial Cells Exposed toHyperosmotic StressXia Hua 1, 2 , Zhitao Su 1, 3 , Ruzhi Deng 1, 3 , Jing Lin 1 , De-Quan Li 1 ,Stephen C. Pflugfelder 1 . 1 Ophthalmology, Baylor College ofMedicine, Houston, TX; 2 Ophthalmology, Tianjin Eye Hospital,Tianjin, China; 3 Ophthalmology, School of Optometry andOphthalmology, Wenzhou, China.Purpose: Hyperosmolarity has been recognized to be a proinflammatorystress in the pathogenesis of dry eye disease. This studywas to explore the suppressive effects of osmoprotectants onproduction of pro-inflammatory mediators in primary human cornealepithelial cells (HCECs) exposed to hyperosmotic stress.Methods: Primary HCECs were established from donor limbaltissue. The cultures in iso-osmolar medium (312 mOsM) wereswitched to hyperosmotic media (400-500 mOsM) by adding 50-90mM NaCl, with or without prior incubation of differentconcentrations (2, 10 or 20mM) of L-carnitine, erythritol or betaine.The mRNA expression by HCECs treated for 4 hours was determinedby RT-qPCR. The protein production in the conditioned media fromcultures treated for 24 hours was evaluated by ELISA.Results: Hyperosmotic stress (400, 450 or 500 mOsM) significantlystimulated the mRNA expression of pro-inflammatory cytokines,TNF-α (3.2 to 16.2 fold), IL-1β (2.2 to 3.5 fold) and IL-6 (3.1 to 7.3fold), and chemokines, IL-8 (3.1 to 4.9 fold), CCL2 (6.1 to 15.3 fold)and CCL20 (2.4 to 4.1 fold) in HCECs, mostly in an osmolaritydependent fashion. Interestingly, the stimulated these proinflammatorymarkers was significantly but differentially suppressedby L-carnitine, erythritol or betaine. L-carnitine appeared to have thegreatest inhibitory effects, and down-regulated 54-77%, respectively,of the stimulated mRNA levels of TNF-α (down from 12.3 to 5.7fold), IL-1β (2.2 to 0.9 fold), IL-6 (7.3 to 2.9 fold), IL-8 (4.6 to 2.0fold), CCL2 (15.3 to 3.5 fold) and CCL20 (4.1 to 1.5 fold) by HCECsexposed to 450 mOsM. The stimulated production of these markersby hyperosmotic stress and the suppressive effects of L-carnitine,erythritol and betaine were further confirmed at protein levels byELISA. L-carnitine suppressed 49-79%, respectively, of thestimulated protein levels of TNF-α (down from 81.3 to 17.4 pg/ml),IL-1β (56.9 to 29.2 pg/ml), IL-6 (12.8 to 4.6 ng/ml), and IL-8 (21.2 to10.9 ng/ml) by HCECs exposed to 450 mOsM.Conclusions: Our findings demonstrate that hyperosmotic stressstimulates the expression and production of proinflammatorymediators in HCECs. L-carnitine, erythritol and betaine serve asosmoprotectants that suppress the inflammatory responses in HCECsexposed to hyperosmotic stress. L-carnitine has the bestosmoprotectant effect among the three.Commercial Relationships: Xia Hua, None; Zhitao Su, None;Ruzhi Deng, None; Jing Lin, None; De-Quan Li, None; Stephen C.Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb(C), Baylor College of Medicine (P)Support: NIH Grant EY119515 (SCP), Core Grant for VisionResearch EY002520 , Allergan Inc., Research to Prevent Blindness,Oshman Foundation, William Satmps Farish FundProgram Number: 2671Presentation Time: 10:00 AM - 10:15 AMAge-related changes in the meibomian gland revealed byImmunofluorescent Computed TomographyGeraint J. Parfitt, Yilu Xie, Donald J. Brown, James V. Jester. GavinHerbert Eye Institute, University of California, Irvine, Irvine, CA.Purpose: Recent studies suggest that age-related meibomian gland(MG) dysfunction is defined by acinar atrophy and a decrease in cellproliferation and gland volume. We have developed a novel method,immunofluorescent computed tomography (ICT), which enables 3-Dquantification of multiple antigens in large tissue volumes to validateand expand on these findings in the mouse MG.Methods: Lower eyelids from a 5month and 2year old mouse wereexcised and fixed in 2% PFA for 24hrs. Lids were embedded inbutyl-methyl methacrylate and polymerised under UV light at 4 o C for20hrs before serially sectioning at 2µm intervals for 1mm. Both datasets were then sequentially immunostained for the cytokeratins 1, 5and 6 and the cell proliferation marker Ki67, prior to mounting withDAPI. Image acquisition was carried out using a 20x/0.75NAobjective on a Leica DMI6000B microscope and alignment and 3-Dreconstruction was performed using Amira software. Finally,quantification of gland volume, cell density and Ki67+ nuclei wascarried out using ImageJ.Results: ICT of 3 young and 3 old mouse MGs confirmed acinaratrophy and revealed a mean gland volume loss of 77% with age.Furthermore, ICT visualised an elaborate ductal system in the youngmouse MG that undergoes truncation of epithelial tissue with age anda subsequent decrease in the proximity of the gland to the Obicularismuscle. 3-D reconstructions also show that, in some cases, the orificemay narrow due to hyper-keratinisation. Interestingly, 3-D renderingof K5 and K6 reveals acini in younger MGs that largely consist ofepithelial cells, suggesting dynamic acini turnover. ICTquantification of cell density and proliferation in both young andaged MGs showed no statistically significant differences in celldensity (p = 0.28) and proliferation (p = 0.9) even though the totalcell and proliferation counts throughout the MG are reduced withaging (75% and 73% loss, respectively).Conclusions: ICT is an emerging and powerful technique to visualisemultiple cell populations in large volumes at high resolution thatenables us to better understand MG structure and function. Thecorrelation of MG atrophy with decreases in cell and proliferationcounts suggests a potential deficiency in the adult stem cellpopulation and their differentiation. Further analysis with ICT mayallow us to localise and quantify putative stem cells to understand thematuration process of the MG.Commercial Relationships: Geraint J. Parfitt, None; Yilu Xie,None; Donald J. Brown, None; James V. Jester, NoneSupport: Research to Prevent Blindness, Inc. Discovery EyeFoundation, NIH/NEI EY021510. The Skirball Program in MolecularOphthalmology322 Corneal Surgery Non-refractive ITuesday, May 07, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 3066-3101/D0001-D0036Organizing Section: CorneaProgram Number: 3066 Poster Board Number: D0001Presentation Time: 8:30 AM - 10:15 AMA quantitative approach to evaluate the overall quality of corneaused in a comparative study between two hypothermic storagemediaMohit N. Parekh 1 , Gianni Salvalaio 2 , Stefano Ferrari 1 , Marie-ClaudeAmoureux 3 , Cecile Albrecht 3 , Denis Fortier 3 , Diego Ponzin 1, 2 .©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea1 Epithelial Stem Cell Research Center, The Veneto Eye BankFoundation, Venice, Italy; 2 Eye Bank, The Veneto Eye BankFoundation, Venice, Italy; 3 Eurobio, Paris, France.Purpose: To investigate and validate a new evaluation technique forcalculating the overall quality (OQ) of the donor cornea and to checkits efficacy in a comparative study for corneas preserved in Optisol-GS and Cornea Cold®.Methods: 24 pairs of corneas suitable for research with intactepithelial layer and good endothelial morphology were selected. 12Right and 12 left corneas and vice versa were placed in each mediumfor a 4 week comparative study. The study was divided into 4 phaseswhere phase 1 was open and the rest were blinded (maskedobservers). Endothelium was stained with trypan blue and cells werecounted under a light microscope with a grid to check the mortalityand endothelial cell density (ECD) manually. The ECD and themortality were incorporated together to measure the viableendothelial cell density (VECD). Difference between epithelial andendothelial layer (using a grid on the knob) was evaluatedmicroscopically for thickness measurement which was alsoconfirmed using OCT. A transparency device was used forcalculating the degree of transparency. Epithelium was stained usingtrypan blue to check the integrity, morphology and viability. All theabove subjective parameters were converted to numerical values(range) for determining the OQ of the cornea. Statistical analysis wasused to confirm the significant difference.Results: The conversion to numerical values helped to evaluate thequality of corneas at different time intervals of preservation indifferent media. Students t-test showed statistically better results(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapatients with FED compared to PBK at 6 months post DSAEK.However, there was no difference in eventual contrast sensitivitybetween the 2 groups 2 years post DSAEK.Commercial Relationships: Fiona Pin Miao P. Lim, None;Marcus Ang, None; Hla M. Htoon, None; Donald T. Tan, NetworkMedical Products (P), Carl Zeiss Meditec (F), Alcon Labs (F),Bausch & Lomb (F), Allergan (F), Santen (F); Jodhbir S. Mehta,NoneProgram Number: 3069 Poster Board Number: D0004Presentation Time: 8:30 AM - 10:15 AMComparison of Dehydration Kinetics of Organ Cultured HumanCorneas by Different Deswelling Media using Optical CoherenceTomographyMartin Hermel, Sabine Salla, Nicole Hamsley, Peter Walter, Anne C.Rieck. Dept of Ophthalmology and Aachen Cornea Bank, RWTHAachen University, Aachen, Germany.Purpose: Human donor corneas experience swelling while in organculture, and require dehydration by placing in hyperosmolar culturemedia prior to grafting. In this study we compared the deswellingkinetics of different media by measuring central corneal thickess(CCT) over time using optical coherence tomography (OCT).Methods: The study was conducted in accordance with theDeclaration of Helsinki. Ethical approval was obtained. Thirty-threehuman donor corneas not suitable for transplantation were stored inMEM + antibiotics + 2% fetal calf serum (FCS) for 15 up to 28 daysand were then randomly assigned to four groups. Dehydration wasinduced by using serum-containing deswelling media with dextran(BK2 [“Biochrom K2”, Biochrom, n=8], CJ [“CorneaJet”, gift ofEuroBio, n=9] or DM [MEM + antibiotics + 2% FCS + 5% dextran500, n=8]), or a serum-free medium with an undisclosed deswellingcompound (SA3, [“StemAlpha 3”, gift of Stemalpha, n=8]). CCT wasmeasured by OCT (Heidelberg Engineering) before deswelling andafter 1, 2, 3, 6, 12, 24, 48, 72 and 144 hours. Data are presented asmean±SD. Dehydration kinetics was analysed via the nonlinearplatform in JMP10 Pro.Results: Prior to deswelling, CCT was 1123±195µm, 1157±74µm,1133±177µm and 1229±152µm in the BK2, CJ, DM and SA3 groups,respectively. Minimal corneal thickness was obtained after 12h inSA3 (702±207µm), 24h in BK2 (615±99µm) and 48h in CJ(599±37µm) and DM (625±125µm) groups. Final CCT after 144hwas 672±114µm, 626±57µm, 666±147µm and 929±149µm in theBK2, CJ, DM and SA3 groups, respectively. The deswelling kineticswas best modelled as a biexponential function (overall r 2 =0,554) withdecay rates of the media differing at the α=0.05 level.Conclusions: All media displayed dehydration of the corneal stromaand reduction of CCT.SA3 displayed a marked increase in CCT after 12h, possibly due todegradation of the proprietary deswelling agent, and should thus notbe used in conjunction with other serum-containing culture media.All dextran containing media obtained minimal CCT values after 24to 48h deswelling, with a slight subsequent CCT increase in BK2 andDM, which may reflect dextran degradation. Grafting of organcultured corneas should be performed after 24 to 48h deswelling.Commercial Relationships: Martin Hermel, EuroBio (F),StemAlpha (F); Sabine Salla, None; Nicole Hamsley, None; PeterWalter, Novartis (R), Bayer (R), Second Sight (R), Bayer (F),Novartis (F); Anne C. Rieck, NoneProgram Number: 3070 Poster Board Number: D0005Presentation Time: 8:30 AM - 10:15 AMThe influence of donor factors on suitability of corneas forpenetrating keratoplastyJohn Armitage 1 , Mark N. Jones 2 , Isaac Zambrano 3 , Fiona M.Carley 3 , Derek M. Tole 1 . 1 Clinical Sciences, Univ of Bristol-BristolEye Hosp, Bristol, United Kingdom; 2 NHS Blood and Transplant,Bristol, United Kingdom; 3 Manchester Royal Eye Hospital,Manchester, United Kingdom.Purpose: Analysis of the influence of donor and recipient factors onfive-year graft survival for validation of the quality standards appliedin the CTS Eye Banks in Bristol and Manchester.Methods: Corneas stored by the CTS Eye Banks between April 1999and March 2005 were included in the study. A logistic regressionanalysis was carried out to determine the influence of donor factorson the suitability of corneas for penetrating keratoplasty (PK).Survival data were analysed by univariate methods (Kaplan-Meiersurvival) and multiple regression (Cox proportional hazards), asappropriate for influence of donor factors on 5-year graft survival.Results: Suitability for PK (n=7107). Donor age (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaby measuring the area of NV, the level of opacification and thecornea thickness. The area of NV from the limbus into the graft wasmeasured by using corneal fluorescein angiography (FA) and digitalmeasurements. Persistence of opacification was assessed by slit-lampmicroscopy and graded on a 0 to 4+ scale. Cornea thickness andcornea astigmatism was measured by ocular coherence topography(OCT). At sacrifice (day 28) the cornea was fixed for histologicalanalysis of neovascularization, inflammation, and rejection.Results: Corneal neovascularization, opacification, cornea thicknesswas significantly higher in the interrupted suture group compared tothe continuous suture group. (p = 0.006 or less). All corneas in thecontinuous suture group exhibited minimal neovascularization andgenerally presented with ≤ 1 opacification at each time point studied.A single running suture results in less postoperative astigmatismcompared with the interrupted sutures technique.Conclusions: Corneas within the interrupted suture group wereassociated with a significant increased level of neovascularization,opacification and astigmatism followed by a higher rate of rejection.The suturing method appears to be a factor leading to the rate ofcornea rejection in a rabbit PK model and may be correlated tohuman surgical outcomes.Commercial Relationships: Maria A. Parker, None; Trevor J.McFarland, None; Winston Chamberlain, None; Scott Ellis,Oxford BioMedica (E); Kyriacos Mitrophanous, Oxford BioMedica(UK) Ltd (E); Tim Stout, Clayton Foundation (P), OxfordBiomedica (C), AGTC (F), Peregrine Pharmaceuticals Inc (C), StemCells Inc (C)Program Number: 3072 Poster Board Number: D0007Presentation Time: 8:30 AM - 10:15 AMMost frequent indications for keratoplasty at 2 academic centersover a 6-year periodKimberly Hsu 1 , Shu-Hong Chang 4, 1 , Whitney Brothers 2 , Sean L.Edelstein 2 , Hugo Y. Hsu 3, 2 , George J. Harocopos 1 . 1 Ophthalmology,Washington University in St. Louis, St. Louis, MO; 2 Ophthalmology,Saint Louis University, St. Louis, MO; 3 Ophthalmology, Universityof Southern California, Los Angeles, CA; 4 Ophthalmology,University of Washington, Seattle, WA.Purpose: To determine the most frequent indications for keratoplastyfrom 2002-2007 at 2 academic centers and to compare this data to themost common indications for keratoplasty nation-wide over this sametime period.Methods: A retrospective review was performed for the indicationsfor keratoplasty from 2002-2007 at Washington University in St.Louis (WUSTL) and St. Louis University (SLU). A pathologydatabase search was used to find all pathology reports of keratoplastyspecimens. The patients’ age, sex, right or left eye, type ofkeratoplasty, indication for keratoplasty, and ocular comorbiditieswere recorded. A total of 855 and 110 eyes underwent keratoplasty atWUSTL and SLU, respectively, during this time period. Statisticalreports were obtained from the Eye Bank Association of America(EBAA) for the corresponding years 2002-2007. The indications forkeratoplasty were reviewed and averaged for these 6 years andcompared to the data from our academic centers.Results: At WUSTL, the most common indication for keratoplastywas graft failure at 31.6%. This was followed by Fuchs cornealdystrophy at 22.1%, pseudophakic and aphakic bullous keratopathy at17.6%, and keratoconus at 6.9%. At SLU, the most commonindications for keratoplasty were PBK/ABK at 34.4% and graftfailure at 32.7%. This was followed by Fuchs corneal dystrophy(10%) and keratoconus (6.4%). Averaging the 2002-2007 EBAAdata, the most common indication for keratoplasty nation-wide wasPBK/ABK at 19.9%. This was followed by keratoconus at 15.9% andthen Fuchs dystrophy at 13.3%. Re-grafts accounted for only 13.2%of keratoplasties performed during this time period nation-wide.Conclusions: In our review of the indications for keratoplasties attwo academic centers in St. Louis, we found re-grafts to account forapproximately one third of all keratoplasties, which was more thandouble the proportion in the national data during this time period. Wepostulate that these higher percentages for re-grafting represent areferral bias of complex patients to academic centers who ultimatelyrequire multiple keratoplasties.Commercial Relationships: Kimberly Hsu, None; Shu-HongChang, None; Whitney Brothers, None; Sean L. Edelstein, None;Hugo Y. Hsu, Bausch & Lomb (R); George J. Harocopos, NoneProgram Number: 3073 Poster Board Number: D0008Presentation Time: 8:30 AM - 10:15 AMOutcome of Combined Penetrating Keratoplasty andVitreoretinal Surgery using Temporary EckardtKeratoprosthesis and Analysis for Factors affecting CornealAllograft SurvivalDae Seung Lee 1 , Jang won Heo 1 , Mee Kum Kim 1 , Hyuk Jin Choi 2 ,Won Ryang Wee 1 , Joo Youn Oh 1 . 1 Ophthalmology, Seoul NationalUniversity, Seoul, Republic of Korea; 2 Seoul National UniversityHospital Healthcare System Gangnam Center, Seoul, Republic ofKorea.Purpose: To evaluate the outcome of combined penetratingkeratoplasty and vitreoretinal surgery using temporary Eckardtkeratoprosthesis and to analyze the factors affecting the cornealallograft survival.Methods: We reviewed medical records of 11 patients whounderwent combined corneal allotransplantation and pars planavitrectomy using temporary Eckardt keratoprosthesis. Primaryoutcome measure was the survival rate of corneal grafts. Patientdemographics, clinical features of the cornea and retina such asdisease entities and preoperative status, or surgical methods werecompared between the group with graft rejection and the group withgraft survival.Results: The overall graft survival rate was 45.5 % (5/11 eyes).Retinal surgery was successful in 90.9 % (10/11 eyes). Rejectionoccurred in 5 patients (45.5 %, 5/11 eyes) with the mean survivaltime of 82.8 days during a follow up of 579 days. Four of 5 cases hadthe graft rejection within 2 months after surgery, and one patient hadrejection at 7 months. The presence of inflammation orvascularization in the cornea before surgery was a significant factoraffecting the occurrence of graft rejection (p value = 0.006). Activeinflammation was present preoperatively in the recipient cornea in allpatients with rejection. Corneal neovascularization was present in therecipient cornea in 4 of 5 patients with rejection. In contrast, therewas no inflammation or vascularization in patients without rejection.The patient age, the presence of glaucoma, entities of corneal andretinal diseases, types of retinal surgery including the use of siliconeoil or gas tamponade had no significant correlation with the graftsurvival or rejection.Conclusions: Combined corneal allotransplantation and pars planavitrectomy using temporary Eckardt keratoprosthesis allowed forsuccessful surgical intervention of retinal diseases in patients withcoexisting corneal opacity. Although the retinal outcome wasexcellent in 90.9 %, corneal allografts survived in 45.5%, largelydepending on the preoperative status of the cornea including thepresence of active inflammation or vascularization.Commercial Relationships: Dae Seung Lee, None; Jang won Heo,None; Mee Kum Kim, None; Hyuk Jin Choi, None; Won RyangWee, None; Joo Youn Oh, NoneSupport: None in the Support field below©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 3074 Poster Board Number: D0009Presentation Time: 8:30 AM - 10:15 AMComparison Of Graft Survival Following PenetratingKeratoplasty And Descemet’s Stripping Endothelial KeratoplastyIn Medically And Surgically Treated Glaucoma PatientsGeorge C. Papachristou, David S. Greenfield, Terrence P. O'Brien,Joyce C. Schiffman, Wei Shi, Shawn M. Iverson. Ophthalmology,Bascom Palmer Eye Institute, Palm Beach Gardens, FL.Purpose: To compare corneal graft survival rate after primarypenetrating keratoplasty (PK) and Descemet’s stripping endothelialkeratoplasty (DSEK) in patients with medically and surgically treatedglaucoma.Methods: A retrospective review of patients who underwent primarycorneal transplant surgery (PK or DSEK) at Bascom Palmer EyeInstitute with pre-existing glaucoma from January 1, 2005 toDecember 31, 2010. Inclusion criteria consisted of patients withglaucomatous optic nerve damage and visual field loss, age ≥ 18,corneal decompensation requiring corneal transplant surgery, and ≥6months of follow-up. Only the first eye per patient was selected.Graft failure was defined as >3 months of corneal edema or opacity,resulting in loss of optical clarity correctable only by surgery.Corneal graft survival was calculated using Kaplan-Meier survivalanalysis and risk factors were evaluated using Cox proportionalhazards analysis.Results: A total of 332 corneal transplants (261 PK, 97 DSEK) wereperformed during the study period. Fifty-five eyes (28 PK, 27 DSEK)from 55 patients (mean age 73±13 yrs in PK and 76±10 in the DSEKgroup, p =0.32) met the enrollment criteria and were selected forreview. Glaucoma diagnoses consisted of POAG 28(51%), PXFG12(22%), CACG 7(13%), uveitic glaucoma 4(7%), NTG 2(4%), andothers 2(4%). The 1, 3, and 5-year graft survival rates in the PKgroup (87%, 47%, and 47%) were compared with the DSEK group(57%, 36%, and 24%) and found to be significantly higher at 1 year(p=0.009) but lost significance at subsequent follow-up (p=0.18).Among eyes which received glaucoma drainage implants (n=20), the1, 3, and 5-year survival rates were 63%, 26%, and 26%,respectively, in the eyes with anterior chamber implants (n=15)versus 100%, 100%, and 100%, respectively, in those with pars planaplacement (n=5, p=0.04).Conclusions: Though patients with pre-existing glaucoma had atrend towards higher graft survival in the PK compared to the DSEKgroup; this difference was not found to be statistically significant.Tube placement in the anterior chamber is a significant risk factor forgraft failure.Commercial Relationships: George C. Papachristou, None; DavidS. Greenfield, National Eye Institute (R), Carl Zeiss Meditec (R),Optovue (R), Heidelberg Engineering (R), Allergan (C), Alcon (C),Merz (C), Quark (C), SOLX (C), Biometric Imaging (C), Senju (C);Terrence P. O'Brien, None; Joyce C. Schiffman, None; Wei Shi,None; Shawn M. Iverson, NoneSupport: P30EY014801 University of Miami Core Grant: AnUnrestricted Grant from Research to Prevent Blindness, New York,New YorkProgram Number: 3075 Poster Board Number: D0010Presentation Time: 8:30 AM - 10:15 AMSelf-retained amniotic membrane for high-risk penetratingkeratoplasty - one-year resultsPho Nguyen 1 , Ramya N. Swamy 1 , Kelly Rue 1 , J M. Heur 1 , Samuel C.Yiu 2, 3 . 1 Doheny Eye Inst, USC / Keck School of Medicine, LosAngeles, CA; 2 Wilmer Eye Inst, Johns Hopkins Univ, Baltimore,MD; 3 King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia.Purpose: To evaluate the role of self-retained cryopreserved humanamniotic membrane (hAM) in penetrating keratoplasty (PKP) graftsurvival.Methods: A retrospective noncomparative interventional case seriesof 58 cases of high-risk PKP with concurrent placement of a selfretainedcryopreserved hAM device (ProKera®) at a tertiary eyecenter from January 2009 to July 2010. Outcome measure was graftsurvival. All surgeries were performed by one surgeon (SCY).Results: Average age was 66.7 ± 17.2 years and 54% were males. 51eye were pseudophakic and one aphakic. 27 eyes were glaucomatous;24 of these had glaucoma drainage implants and 2 hadendocyclophotocoagulation performed. 12 patients had PKP for thefirst time and 46 had repeat PKP (average number of prior PKP 1.63± 1.1, range: 1-5). High-risk factors included repeat PKP (79.3%),corneal neovascularization (51.7%), preexisting glaucoma (46.6%),and anterior synechiae (37.9%). Both First Transplant and RepeatTransplant groups had similar survival rates until 6 months aftertransplant. At year 1, the First Transplant group appeared to havebetter survival (67%), compared to that of the Repeat Transplant(43%). This advantage, however, did not reach statistical significance(hazard ratio 0.562, log-rank test p = 0.207). Eyes with 3+ high-riskfactors had higher failure (odds ratio = 5.81, p = 0.003).Conclusions: Compared to literature, the benefits of hAM inprolongation of graft survival were indeterminate in our study.However, this study has many limitations, e.g. retrospective study ofa high-risk population with lack of control and many confounders.Further studies are recommended to provide definitive information onobjective and subjective outcomes.Commercial Relationships: Pho Nguyen, None; Ramya N.Swamy, None; Kelly Rue, None; J M. Heur, None; Samuel C. Yiu,NoneSupport: NEI core grant EY03040 and an unrestricted grant fromResearch to Prevent Blindness. Pho Nguyen is supported by the HeedOphthalmic Foundation and the Fletcher Jones Foundation.Program Number: 3076 Poster Board Number: D0011Presentation Time: 8:30 AM - 10:15 AMAnterior Chamber Depth and Penetrating Keratoplasty GraftSurvivalChristine N. Pham Lagler, Wuqaas M. Munir. Ophthalmology,Boston University School of Medicine, Boston Medical Center,Boston, MA.Purpose: To examine the association between pre-operative anteriorchamber depth (ACD) and penetrating keratoplasty graft survival inpatients with prior or concomitant intraocular lens placement.Methods: This retrospective study examined cases of penetratingkeratoplasty with prior or concomitant intraocular lens placementperformed by a single surgeon (WMM) between 2006 and 2011.Cases with missing ACD measurements or immediate post-operativegraft complications were excluded. Logistic regression analysis wasthen performed to determine if there was a statistically significant©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaassociation between pre-operative ACD and graft failure. Graftfailure was defined as irreversible visually compromising cornealedema or opacification at the time of last follow-up. ACDmeasurements were obtained by manual or immersion A-scanultrasonography.Results: Thirty-one eyes in 28 patients were identified as meetinginitial study criteria. Ten eyes were subsequently excluded, with 21eyes remaining. Age ranged from 50-81 years, averaging 68.17 years.There were 9 males and 11 females. There were 10 right eyes and 11left eyes. Duration of follow-up ranged from 3-60 months, averaging8 months.There were 4 cases of graft failure identified (4/21, 19%), including 1re-graft. ACD in these cases ranged from 1.47-3.67mm, averaging2.48mm. ACD in cases without failure ranged from 2.16-5.48mm,averaging 3.21mm. Logistic regression analysis did not show astatistically significant relationship between ACD and graft failure (p= 0.19, CI = 0.029-2.01).Conclusions: While limited by a small sample size, our studysuggests that there is no significant relationship between preoperativeanterior chamber depth and penetrating keratoplasty graftsurvival.Commercial Relationships: Christine N. Pham Lagler, None;Wuqaas M. Munir, NoneProgram Number: 3077 Poster Board Number: D0012Presentation Time: 8:30 AM - 10:15 AMA new anvil profile in femtosecond laser assisted penetratingkeratoplastyLuca Menabuoni 1 , Ivo Lenzetti 1 , Annalisa Canovetti 1 , AlexMalandrini 1 , Francesca Rossi 2 , Roberto Pini 2 . 1 U O Oculistica,Ospedale Misericordia and Dolce, Prato, Italy; 2 Institute of AppliedPhysics "Nello Carrara", Italian National Research council, SestoFiorentino (FI), Italy.Purpose: To describe and assess the use of a new anvil-liketrephination pattern in penetrating keratoplasty (PK) assisted byfemtosecond laser.Methods: 30 eyes underwent anvil PK. An Intralase FemtosecondLaser 150 KHz (iFS150TM, Abbott Medical Optics -AMO, SantaAna, CA, USA) was used to create anvil shaped penetrating cuts onboth donor and recipient corneas. Diode laser welding procedure wasperformed in order to improve the healing process. All patients wereevaluated for corrected distance visual acuity, pachimetry,topography and endothelial cell density.Results: All surgery were successful and without any intraoperativecomplications. This profile enables a safe and easy to performsuturing procedure, with an immediate closure effect evidencedduring surgery. The large interface between donor and recipienttissue supports the laser welding procedure. A 6 months follow upstudy showed that the anvil shaped flap provides a better visualacuity recovery and a reduction in the number of rejection. Meanpost-operative BSCVA (logMAR, mean ± SD) was 0.50 ± 0.24 at 1month, 0.32±0.19 at 3 months, 0.19±0.14 at 6 months. Meanpachimetry was 537 ± 56 µm at 1 month, 550±77 µm at 3 months and528±70 µm at 6 months. Mean preoperatory endothelial cell densitywas 2300, while postoperative results are 1945±363 at 1 month,1881±401 at 3 months and 1781±393 at 6 months.Conclusions: Use of anvil trephination profile was effective and safeto perform PK. Short term visual results and refractive results areencouraging compared with those of conventional PK studies. Longerterm follow-up and comparative studies are necessary to determineprecisely advantages of this technique.An anvile-like trephination profile in a human patient (6 months p.o.)Commercial Relationships: Luca Menabuoni, None; Ivo Lenzetti,None; Annalisa Canovetti, None; Alex Malandrini, None;Francesca Rossi, None; Roberto Pini, NoneProgram Number: 3078 Poster Board Number: D0013Presentation Time: 8:30 AM - 10:15 AMSutureless Penetrating Keratoplasty Using a Femtosecond Laser.A Laboratory ApproachAshley Behrens 1, 2 , Hind M. Alkatan 2 , Emad Badawy 2 , FarhanAlanazi 2 , Nasser Alsabaani 2 , ABDULMAJED ALJAETHEN 2 .1 Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, MD;2 Ophthalmology, King Khaled Eye Specialist Hospital, Riyadh, SaudiArabia.Purpose: To demonstrate the feasibility of a new form of penetratingkeratoplasty with a lamellar component in an experimental animaland human cadaver eyes. Graft dislocation test and bursting pressurewere used to evaluate stability.Methods: 25 freshly enucleated sheep cadaver eyes and 4 humancadaver eyes not suitable for corneal transplantation were used in thestudy. A divergent angle trephination (from surface to endothelium)using the Intralase FS (Abbot Medical Optics, IL, USA) was planned.Eyes were placed in a special holder and bursting pressure aftertransplantation was measured using Tonopen (Reichert, Depew, NY).Donor corneas measured 7.3 mm, recipients 7.0 mm. Aftermechanical tests, globes were fixated in formalin for HE and PASstaining.Results: No major difficulties were observed during the procedure.The insertion of the donor discs was relatively straightforward,through the planned self-sealing incisions of 4 mm. Total pressureresisted after bursting pressure tests was above 75 mmHg in all cases.With manipulation of the graft with cyclodialysis spatula, donorswere easily centered in recipient beds. At histology, selected eyesshowed a good apposition of the wound, despite some graft shrinkagein some areas induced by the fixation process. A 45 degree cut anglewas confirmed in pathology slides. Endothelial cells looked favorablyhealthy in the animal eyes evaluated.Conclusions: A potential new method for corneal transplantationpreserving an intact corneal surface was demonstrated. Graft stabilityand wound apposition seemed acceptable in our model. Although anex vivo study cannot demonstrate the events occurring in realpatients, the biomechanical feasibility of the procedure seems to beproven with this study.Commercial Relationships: Ashley Behrens, None; Hind M.Alkatan, None; Emad Badawy, None; Farhan Alanazi, None;Nasser Alsabaani, None; ABDULMAJED ALJAETHEN, NoneSupport: Al-Amin Medical InstrumentsProgram Number: 3079 Poster Board Number: D0014©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 8:30 AM - 10:15 AMDescemet’s Membrane Endothelial Keratoplasty (DMEK) :Over-stripping the graft bed promotes donor adherenceMichael D. Straiko, Mark A. Terry, Julia Talajic, David Davis-Boozer. Ophthalmology, Devers Eye Institute, Portland, OR.Purpose: It has been previously reported that avoiding overlap of thedonor DMEK graft with the host Descemet’s membrane (DM) maypromote graft adherence. Based on this suggestion, we altered oursurgical technique and analyzed our results for a difference in the rateof re-bubbling procedures and primary graft failure (PGF).Methods: This study is a retrospective review of a non-randomizedsurgical intervention. 52 DMEK cases were performed for Fuchs’dystrophy eyes. 28 eyes were identified where the host was strippedsmaller than the graft resulting in overlap of the graft and the hostDM. 24 eyes were identified where the host was purposely strippedwider than the size of the graft to avoid overlap of the DMEK graftand host DM. The incidence of graft replacement and frequency ofre-bubbling was compared between the 2 groups.Results: 15 of 28 (53.5%) grafts that overlapped with the host DMrequired rebubbling. This is significantly more than the 6/24 (25%)grafts without overlap of the host DM that required rebubbling(p=0.036). There was no significant difference in the rate of PGF.There was a 10.7% PGF rate for the overlap group and 8.3% for thenon-overlap group (p=1.0).Conclusions: Avoiding overlap of the DMEK graft with the host DMappears to promote adherence of the graft and decrease the rate ofrebubbling procedures. A prospective, randomized study with agreater sample size would be advantageous to further validate thisfinding.Commercial Relationships: Michael D. Straiko, None; Mark A.Terry, Bausch and Lomb Surgical (R), Alcon (R), Optovue (C);Julia Talajic, None; David Davis-Boozer, NoneProgram Number: 3080 Poster Board Number: D0015Presentation Time: 8:30 AM - 10:15 AMPachymetry Assisted Lamellar Keratoplasty for Corneal EctasiaJulio C. Hernandez-Camarena 1 , Victor M. Boullosa 1 , AlejandroNavas 1 , Arturo J. Ramirez-Miranda 1 , Cesar Carriazo 2 , Enrique O.Graue-Hernández 1 . 1 Cornea and Refractive Surgery, Instituto deOftalmología "Conde de Valenciana", Mexico City, Mexico;2 Anterior Segment and Refractive Surgery, Carriazo ScientificOrganization, Barranquilla, Colombia.Purpose: To evaluate the structural, refractive and visual outcomesof pachymetry assisted lamellar keratoplasty in patients with cornealectasia.Methods: Prospective, non-randomized consecutive case series.Patients with corneal ectasia who underwent unilateral surgery usingcustom pachymetry assisted lamellar keratoplasty (PALK) wereevaluated. Host corneas were treated using transepithelial excimerlaser ablation planned to leave 100µm of stromal residual bed. Donorcornea endothelium and Descemet membrane were removedmechanically and then secured to the host cornea with 16 interrupted10-0 nylon sutures. Eyes were examined preoperatively, day 1, month1, 3, 6 and 12 postoperatively. Outcome measures were correcteddistance visual acuity (CDVA), manifest and keratometric refraction,endothelial rejection episodes, conversion to penetrating keratoplasty(PKP), corneal thickness and endothelial cell density (ECD).Results: Five patients with keratoconus grade III and IV (Amsler-Krumeich) and one patient with post-LASIK ectasia were treatedwith PALK. Average follow up was 3.8 months. The meanpostoperative CDVA was LogMAR 0.44 (±0.37)(p=0.02). The meanpostoperative manifest spherical equivalent was -3.00±1.01 (p=0.01)and the mean postoperative keratometric reading was 41.51±2.60(p=0.01). Postoperative central corneal thickness improved to689.16±29.64 (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Descemet’s stripping automated endothelial keratoplasty(DSAEK) has replaced penetrating keratoplasty as the treatment ofchoice for corneal endothelial dysfunction. Surface quality of thedonor button plays an important role in determining the visualoutcome following DSAEK. This study measured and compared thesurface roughness of endothelial grafts dissected with a femtosecondlaser (FS) and two mechanical microkeratomes (MKs).Methods: Endothelial buttons were harvested from 18 humancorneas unsuitable for transplantation with the Intralase FS60 laser(Abbott Medical Optics, Santa Anna, Ca.; n=4) and the Moria ALTK(Moria, Antony, France; n=6) or the Gebauer SLc MKs (GebauerMedizintechnik, Neuhausen, Germany; n=8). Following dissectionthe endothelial buttons were fixed in 3.0% glutaraldehyde andexamined using a confocal profiler (Sensofar PLu 2300, Terrassa,Spain) and environmental scanning electron microscopy (ESEM)(FEI-Quanta 600; Hillsboro, OR). Linear mixed model analysis wasused to quantify the difference in surface roughness between thedifferent dissection techniques.Results: Confocal profiling was able to obtain quantitative surfaceroughness measurements and high resolution 3-dimensionalreconstructions of the central 2x1.5 mm2 area of the dissectedbuttons. ESEM evaluation allowed wide field analysis at lowermagnification which corresponded well with the 3-dimensionalconfocal reconstructions. Surface roughness of FS dissected buttons(RMS =1.89 µm) was significantly higher compared with the Moria(RMS=1.05 µm) and Gebauer MKs (RMS=0.92 µm) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaLudwig M. Heindl, None; Claus Cursiefen, Gene Signal (C), Alcon(R), Allergan (R), Bayer (R)Support: Deutsche Forschungsgemeinschaft (STE 1928/2-1, Cu47/4-1), Koeln Fortune, Ruth und Helmut Lingen StiftungProgram Number: 3085 Poster Board Number: D0020Presentation Time: 8:30 AM - 10:15 AMIn Vivo Confocal Microscopy to Detect the Wound HealingProcess after DSAEK and nDSAEKJen-Pin Sun, Wei-Li Chen, Ying-Han Lin, Chien-Tzu Peng, Fung-Rong Hu. Ophthalmology, National Taiwan University Hospital,Taipei, Taiwan.Purpose: To evaluate the wound healing process after DSAEK andnDSAEK in an animal model.Methods: Six DSAEK and six nDSAEK were performed on NewZealand white rabbit eyes. In vivo confocal microscopy was used toevaluate the interface changes at 1 week, 2weeks, 1month, 2 monthsand 3 months after operations.Ultrasond pachymetry was used to measure corneal thickness. Atcertain time points, tissue sections was examined by hematoxylin andeosin (H&E) staining, immunohistochemical staining, TUNEL(Terminal deoxynucleotidyl transferase-mediated dUTP Nick EndLabeling ) staining and transmission electron microscopy (TEM).Results: 1.The interface haziness of DSAEK and nDSAEKdiminished gradually, and nDSAEK has greater interface opacitythan DSAEK at all time points examined.2.There was no significant differences of total corneal thicknessbetween DSAEK and nDSAEK at all time points.3.Tissue section using H&E staining showed preserved interfacerecipient Descemet’s membrane and decreased endothelial celldensity three months after nDSAEK.4.Transmission electron microscopy revealed morphologicallychanged interface recipient endothelial cell in nDSAEK three monthsafter operation.5.The interface recipient endothelial cell density in nDSAEKgradually decreased and underwent apoptosis in the follow-up period.Conclusions: NDSAEK has similar surgical results as DSAEK in allparameters measured in this study. However, the interface hazinesswas greater than DSAEK, and recipient endothelial cell at theinterface underwent apoptosis during the whole observationalprocess.External eye photograph at different time points after nDSAEK. (A)2 days after operation. (B) 1 week after operation. (C) 2 weeks afteroperation. (D) 1 month after operation.H&E staining showed well attached graft and preserved interfacerecipient Descemet’s membrane (arrows) 3 months afternDSAEK.(RS : recipient stroma; GS: graft stroma)Commercial Relationships: Jen-Pin Sun, None; Wei-Li Chen,None; Ying-Han Lin, None; Chien-Tzu Peng, None; Fung-RongHu, NoneProgram Number: 3086 Poster Board Number: D0021Presentation Time: 8:30 AM - 10:15 AMReproducibility of graft preparations in Descemet membraneendothelial keratoplastyKathrin Rössler, Ursula Schlotzer-Schrehardt, Bjoern O. Bachmann,Friedrich E. Kruse. Department of Ophthalmology, University ofErlangen-Nürnberg, Erlangen, Germany.Purpose: To assess the reproducibility of manual graft preparationand to evaluate the incidence rate and nature of structural anomaliesof Descemet’s membrane (DM) preventing successful graftpreparation in Descemet membrane endothelial keratoplasty(DMEK).Methods: In a prospective single-center nonrandomized consecutivecase series, 350 corneoscleral buttons from donors aged 18 to 95years stored in Optisol-GS or Dulbecco's Modified Eagle’sMedium were used for DMEK surgery in 343 consecutive patientswith Fuchs’ endothelial dystrophy. Residual endothelial cell-DMcomplexesobtained after successful DM stripping for DMEK andwhole donor corneas obtained after unsuccessful DM stripping wereexamined by transmission electron microscopy andimmunohistochemistry.Results: Uneventful manual separation of DM resulting in a regularand smooth cleavage plane was achieved in 335/350 (95.7%) ofdonor corneas by use of a previously established bimanualsubmerged preparation technique. Although 8/350 (2.3%) of donorcorneas showed focal adhesions of DM to the corneal stroma anddeveloped isolated tears during stripping, preparation of the graftcould be successfully completed. However, 7/350 (2.0%) of donorcorneas showed extremely strong adhesion and multiple tears of DMpreventing successful preparation of the graft. These specimensrevealed either ultrastructural (peg-like interlockings) or biochemicalabnormalities (increased staining intensities for adhesiveglycoproteins) along the DM-stroma interface.Conclusions: Using an appropriate technique, manual preparation ofgrafts for DMEK with reproducible tissue qualities is possible in thevast majority of donor corneas. Tissue loss rate can be reduced toabout 2% of donor corneas, but cannot be eliminated completely dueto interindividual structural and biochemical aberrations of the DMstromainterface preventing DM stripping and graft preparation.Commercial Relationships: Kathrin Rössler, None; UrsulaSchlotzer-Schrehardt, None; Bjoern O. Bachmann, None;Friedrich E. Kruse, NoneProgram Number: 3087 Poster Board Number: D0022Presentation Time: 8:30 AM - 10:15 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaLow-energy and high-frequency femtosecond laser in theproduction of donor corneal lamellaeGustavo Victor 1 , Walton Nosé 3 , Sidney J. Sousa 2 , Milton R. Alves 1 .1 Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil;2 Ophthalmology, University of Sao Paulo - FMRP, Ribeirao Preto,Brazil; 3 Ophthalmology, Federal University of Sao Paulo, Sao Paulo,Brazil.Purpose: This study evaluated the efficacy and reliability of a lowenergyfemtosecond laser with a high repetition rate in themanufacture of deep anterior and endothelial corneal lamellaeMethods: This is a prospective and laboratory investigation. Twentyfivehuman corneal buttons were cut tangentially to provide thickanterior lamellae (diameter, 10 mm; thickness, 500 µm). The cutswere made using an LDV® femtosecond laser in a Ziemer® anteriorchamber. For a better edge, the lamellae were trephined with an 8-mm trephine (Katena®). The center thicknesses of the whole corneasand of the anterior lamellae were measured using a Mitutoyo®thickness gauge with an accuracy of 0.001 mmResults: The thicknesses of the 25 corneas ranged from 500 to 705µm (mean, 584 ± 51 µm). The thicknesses of the anterior lamellaeranged from 420 to 480 µm (mean, 455 ± 12.7 µm). The thicknessesof the posterior lamellae ranged from 30 to 250 µm (mean, 129 ±52.8 µm). There were no discrepancies between the observed andexpected diameters of the lamellae, and all cuts were perfectly round.The lamellar interfaces appeared regular by surgical microscopy.There were no cases of inter-lamellar adhesionConclusions: The LDV® femtosecond laser appears to be a safe andreliable instrument for cutting deep anterior lamellae from donorcorneoscleral buttons. Even the worst-case scenario for the expectedcut precision for the whole population of donated corneas would onlybe about ± 32 µmCommercial Relationships: Gustavo Victor, None; Walton Nosé,None; Sidney J. Sousa, None; Milton R. Alves, NoneSupport: FAPESPProgram Number: 3088 Poster Board Number: D0023Presentation Time: 8:30 AM - 10:15 AMTears of post-LASIK corneal donor tissue during surgeonperformeddonor graft preparation for DSAEKDrew Davis 1 , Peter A. Karth 1 , Christopher Croasdale 2, 3 , Steven B.Koenig 1 . 1 Medical College of Wisconsin, Wauwatosa, IN;2 Ophthalmology, Davis Duehr Dean Clinic, Madison, WI;3 University of Wisconsin School of Medicine and Public Health,Madison, WI.Purpose: To describe four cases by two different surgeons in whichtears or tissue damage occurred during surgeon-performedpreparation of the posterior lamellar corneal lenticule in post-laserassisted in situ keratomileusis (LASIK) corneal donor tissue forDescemet stripping automated endothelial keratoplasty (DSAEK).Methods: In this retrospective case series, all donor corneal tissueshad previously undergone LASIK surgery. In all cases, a Moria®artificial anterior chamber maintainer (Moria, Doyleston, PA) and aMoria® ALTK microkeratome (300-um-depth) was used by thesurgeon to separate the lamellar corneal tissue.Results: In all four cases, the post-LASIK corneal donor tissues weretorn or damaged during preparation. In two cases, large tears of theposterior lamella occurred during surgeon preparation of the donorlenticule with a microkeratome, and the tissue was deemed unusable.One case was subsequently canceled immediately prior to plannedDSAEK; in the second case, an additional donor cornea was availableand was successfully transplanted. In the third and fourth cases, thetissue was damaged but was deemed usable. A button-holeperforation occurred in the anterior lamellar cap of the third case, anda linear paracentral ridge was inadvertantly created on the posteriorlamella of the donor tissue in the fourth case.Conclusions: While published literature supports use of post-LASIKdonor corneal tissue in DSAEK, we believe that our casesdemonstrate a need for caution in surgeon preparation of post-LASIKdonor corneal tissue. To avoid tissue waste and cancelled procedures,we recommend post-LASIK donor tissue be prepared in thelaboratory prior to the planned procedure. As more potential cornealdonors undergo LASIK each year, effective preparation of post-LASIK donor corneal tissue will become an increasingly importantconsideration.Commercial Relationships: Drew Davis, None; Peter A. Karth,None; Christopher Croasdale, None; Steven B. Koenig, NoneProgram Number: 3089 Poster Board Number: D0024Presentation Time: 8:30 AM - 10:15 AMDSAEK for Corneal Decompensation After Ex-Press ShuntSurgery -A Case SeriesJayne S. Weiss 1 , Shalin Shah 1 , Hilary Thompson 2 , Christina Bovone 3 .1 Department of Ophthalmology, LSU, New Orleans, LA;2 Biostatistics Section, School of Public Health, LSUHSC, NewOrleans, LA; 3 Villa Igea Hospital, Forli, Italy.Purpose: Corneal decompensation is a known risk of anteriorchamber implantation of older glaucoma shunts (Ahmed, Baerveldt,Molteno). There is little literature discussing this association of thisrisk with Ex-press shunts. The purpose of the study was to determinewhether the Ex-Press shunt implantation could be associated withdevelopment of subsequent corneal decompensation and whetherDSAEK surgery could be successfully performed in such cases.Methods: A retrospective case review from one corneal surgeon ofall cases of DSAEK performed since 2005 that had prior Ex-Pressshunt surgery. Ocular history including number of surgicalprocedures, intraocular pressure prior to and after Ex-Press shunt,best corrected visually acuity (BCVA) before Ex-Press shunt surgery,before and after DSAEK, and surgical complications were reviewed.Results: The average age and average intraocular pressures (IOP)prior to of Ex-Press shunt placement was 63.8 years +/- 3.4 standarddeviation (SD) and 24.60 mmHg +/- .40 SD respectively. BCVAprior to Ex-Press shunt placement was log MAR 0.64 +/-.16 SD. Theaverage post-operative IOP after Ex-Press shunt was 16.80 mmHg+/- 1.0 SD. The mean number of surgeries prior to DSAEK was 2.8+/- 1.64. 4 of the 5 patients with corneal decompensation after Ex-Press shunt placement had one or more prior filtering procedures witha mean of 1.75 +/- .83. One of these patients also had 2 prior failedEx-Press shunts. Vision decreased to count fingers in all patientsbefore DSAEK. The average age at which significant cornealdecompensation was identified and DSAEK was performed was 66.0years +/- 3.2 SD. with average time of onset of corneal edema afterEx-Press shunt of 2.2 years +/- .44 SD. The average BCVA afterDSAEK was .78 +/- .18 SD with decreased vision resulting fromoptic nerve damage in all cases. All corneas remained clear. IOP wascontrolled in 4 patients, 1 patient required cyclocrytherapy for IOPcontrol.Conclusions: This is the first case series documenting cornealdecompensation after Ex-Press shunt placement. Long term follow upof patients who have had Ex-Press shunt surgery is needed todetermine if the shunt device itself increases the risk of cornealdecompensation. This case series demonstrates that DSAEK surgerycan be successful in treatment of corneal decompensation after Ex-Press valve placement.Commercial Relationships: Jayne S. Weiss, None; Shalin Shah,None; Hilary Thompson, None; Christina Bovone, NoneSupport: Lions Eye Foundation, Research to Prevent Blindness©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 3090 Poster Board Number: D0025Presentation Time: 8:30 AM - 10:15 AMMorphological study of posterior corneal grafts obtained forDSAEK comparing two different harvesting techniquesJuan A. Duran, Elío Díez-Feijóo. Instituto de Oftalmología, BasqueCountry University, Bilbao, Spain.Purpose: To compare morphological aspects (stromal surface, OCTprofile and posterior curvature) of corneal grafts for DSAEK,obtained by two different techniques: manual lamellar dissection andautomatic microkeratome cut.Methods: Three donor corneas were prepared by manual lamellardissection using a 6/0 nylon thread. The technique was describedpreviously (ARVO 2012 poster A177). The other group consisted inthree pre-cut corneas obtained from our eye bank. An automaticmicrokeratome (Moria) was employed. All six grafts were used forDSAEK. Macroscopic examination of the stromal surface was madeon the anterior button. OCT scan (Visante) and posterior curvature(Atlas topography) were obtained in all patients at the third monthpost-operative period.Results: We observed shape differences in the grafts among the twogroups. Smother surface was observed in the group harvested bymanual dissection. This group show also a regular thicknessthroughout the button, whereas grafts obtained by automaticmicrokeratome technique were not regular, being thicker at the edges.This finding correlates with the increased curvature in this group ofcorneas when implanted.Conclusions: Posterior corneal grafts obtained by lamellar dissectionfor DSAEK have a smother stromal surface and a homogeneousthickness when compared with buttons prepared with microkeratome.Commercial Relationships: Juan A. Duran, None; Elío Díez-Feijóo, NoneProgram Number: 3091 Poster Board Number: D0026Presentation Time: 8:30 AM - 10:15 AMDSAEK: Endothelial cell loss is greater for Pseudophakic BullousKeratopathy than for Fuchs’ DystrophyJulia Talajic 1 , Mark A. Terry 1 , Michael D. Straiko 1 , Asem A.Alqudah 1 , David Davis-Boozer 2 . 1 Devers Eye Institute, Portland, OR;2 Lions VisionGift, Portland, OR.Purpose: To determine whether Fuchs’ endothelial dystrophy eyesand postsurgical bullous keratopathy (PBK) eyes have differing ratesof endothelial cell loss and longterm graft survival followingDescemet’s stripping automated endothelial keratoplasty (DSAEK).Methods: A review of 854 cases of DSAEK was performed tocompare the survival rate and donor endothelial cell loss betweeneyes with a pre-operative diagnosis of PBK (n=106) versus that ofFuchs’endothelial dystrophy (n=748). Cell loss at 6, 12, and 24months post-operatively was compared with student t-tests. Longtermsurvival was compared with Chi Square test.Results: The mean cell loss for Fuchs’ versus PBK was 25% versus30% at 6 months (p=.012), 25% versus 30% at 1 year (p=.078), and26% versus 40% at 2 years (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneagraft failure.Methods: Retrospective cohort study of 195 DSAEK graftsperformed at a tertiary medical center between January 2007 andAugust 2012.Results: 71 grafts were performed in eyes that had required surgicalor medical management of elevated intraocular pressure prior toDSAEK. 22 were performed in eyes that had previous glaucomasurgery including trabeculectomy and/or tube implant. One graft wasperformed concomitantly with a tube implant. 11 eyes withpreexisting glaucoma required glaucoma valve implant within 24months after DSAEK as compared to only one eye withoutpreexisting glaucoma (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaImmediate postoperative local Descemet membrane detachmentsand interface fluid pockets after DMEK vary in location anddegree as shown by optical coherence tomographyCarolin Le Blanc, Sebastian E. Siebelmann, Ludwig M. Heindl, ClausCursiefen, Philipp Steven. Ophthalmology, University of Cologne,Cologne, Germany.Purpose: Replacement of diseased endothelium by Descemetmembrane endothelial keratoplasty (DMEK) has been demonstratedto effectively treat patients with corneal endothelial diseases.Subtle clinical undetectable residual interface fluid may be present atthe end of surgery, possibly representing a risk for post-operativeentire graft detachment or limitation in visual restoration.To detect residual clinically undetectable interface fluid and graftdetachment early after DMEKMethods: 10 patients (2 male/8 female) undergoing DMEK surgeryat the Department of Ophthalmology, University of Cologne wereexamined 3-4 hours, 5-7 hours and 7-9 hours after DMEK surgeryusing both a time-domain OCT (SL-OCT, Heidelberg Engineering,Heidelberg, Germany) and a spectral-domain OCT (Spectralis,Heidelberg Engineering, Heidelberg, Germany). Parameters includedpresence, localization and potential reduction of graft detachment.Results: In all patients and at all time points localized graftdetachment and subtle clinically undetectable interface fluid wasobserved. Graft detachments were localized at different positions ofthe cornea in between the examinations and featured differentdegrees. Detachments were better visualized by spectral domain OCTthan by time domain OCT.Conclusions: In the first hours after DMEK surgery, interface fluidand graft detachment is present in all cases despite nearly completeanterior chamber air-fill. Inconsistent localization of detachments isthought to be caused by interface fluid shift due to air bubblemovement within the eye. These findings suggest considerable tissuerearrangements early after DMEK despite nearly complete anteriorchamber air filling.Commercial Relationships: Carolin Le Blanc, None; Sebastian E.Siebelmann, Bauch & Lomb (F); Ludwig M. Heindl, None; ClausCursiefen, Gene Signal (C), Alcon (R), Allergan (R), Bayer (R);Philipp Steven, Novaliq (R), Allergan (C), Bausch & Lomb (C),MSD (C), Deutsche Chefaro (C)Support: Deutsche Forschungsgemeinschaft DFG (STE1928/2-1,CU ), Koeln Fortune Medical Faculty University of Cologne, Ruthund Helmut Lingen StiftungProgram Number: 3097 Poster Board Number: D0032Presentation Time: 8:30 AM - 10:15 AMThe comparative analysis of clinical outcome in penetratingkeratoplasty in use of domestic or imported cornea from United-States in South KoreaJa Young Lee 1, 2 , Mee Kum Kim 1, 2 , Joo Youn Oh 1, 2 , Hyuk Jin Choi 1, 2 ,Won Ryang Wee 1, 2 . 1 Seoul National University Hospital, Seoul,Republic of Korea; 2 Laboraory of Corneal Regenerative Medicineand Ocular Immunology, Seoul Artificial Eye Center, Seoul,Republic of Korea.Purpose: To analyze the graft survival and clinical outcomes ofpenetrating keratoplasty by comparing domestic donor cornea withimported donor cornea in the change of endothelial cell profiles andthe rate of complications in Korea.Methods: Medical records of 236 eyes of 211 patients who wereavailable for at least 1 year after penetrating keratoplasty (PKP)between November 2004 and August 2011 at our hospital wereretrospectively reviewed. Patients were divided 2 groups according totypes of donor cornea(domestic;108 eyes, imported from Sightlife,Seattle; 128 eyes) and further subdivided by the combination of thecataract surgery. Characteristics of the donor corneas (donor age,time taken from death to preservation and death to transplant) werecompared between in domestic and imported cornea. Post-operativebest corrected visual acuity (BCVA), combined surgeries,complication rate, change of endothelial cell profiles and survivalanalysis of the grafts were evaluated.Results: Among patients, 108 eyes had PKP by domestic cornea and128 eyes had by imported cornea. Mean time taken from the donor tocorneal preservation was 8.9 and 8.0 hours in domestic and importedgroup, without statistical difference. Imported group (119.6 hours)took significantly longer time from death of donor to transplant thandomestic group (52.3 hours). Mean endothelial cell density wastemporarily higher in imported group, and then it was not differentfrom each other over time until 5 years. In addition, there was nodifference in complication rates and graft survival time between thosegroups, regardless of the combination of cataract surgery.Conclusions: Although long-term journey was taken from the Unitedstates to the Korea in imported cornea, imported cornea is likely tobring comparable clinical outcomes to domestic cornea.Commercial Relationships: Ja Young Lee, None; Mee Kum Kim,None; Joo Youn Oh, None; Hyuk Jin Choi, None; Won RyangWee, NoneProgram Number: 3098 Poster Board Number: D0033Presentation Time: 8:30 AM - 10:15 AMOutcomes of Thin Lenticule Descemet Stripping AutomatedEndothelial Keratoplasty using a Single-Pass MicrokeratometechniquePrafulla K. Maharana, Namrata Sharma, Rasik B. Vajpayee.Opthalmology, R.P.Centre, AIIMS, New Delhi, India.Purpose: To evaluate the safety and efficacy of thin lenticuledescemet stripping automated endothelial keratoplasty (DSAEK)using a single-pass Microkeratome techniqueMethods: Ten cases of endothelial disorder [4-pseudophakic bullouskeratopathy, 3- fuch’s endothelial dystrophy, 3- congenital hereditaryendothelial dystrophy (CHED), and 1-regraft] underwent theprocedure. Intraoperative pachymetry was done for all donor tissuesmounted on artificial chamber. Donor lenticule was prepared with asingle pass Carriazo Barraquer microkeratome (Moria, Antony,France) with the cutting head depth of 400µm. Sutureless DSAEKwas performed in all cases through a 3.5mm corneoscleral tunnel andusing Busin glide for graft insertion. The main outcome measureswere best corrected visual acuity (BCVA) and graft thickness (withanterior segment optical coherence tomography).Results: At 6 month the mean log MAR BCVA was 0.19±0.11. Themean donor lenticule thickness was 132.63 ±39.52µm. The meanintraoperative endothelial cell loss was 26.5±1.4%.No perforationoccurred during donor preparation. No significant intraoperative orpostoperative complication was noted in any of the cases. Nodifficulty in graft manipulation was encountered during surgery.Conclusions: The technique of single pass thin lenticule DSAEK issafe and effective without any intraoperative complications. Itprovides optimal visual outcome and may be an alternative to doublepass microkeratome technique.Commercial Relationships: Prafulla K. Maharana, None;Namrata Sharma, None; Rasik B. Vajpayee, NoneProgram Number: 3099 Poster Board Number: D0034Presentation Time: 8:30 AM - 10:15 AMClinical outcome of repeat penetrating keratoplastyMargareta Claesson 1 , John Armitage 2 . 1 Ophthalmology, SahlgrenskaUniversity Hospital, Mölndal, Sweden; 2 Clinical Sciences, Universityof Bristol, Bristol, United Kingdom.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: The aim of this study was to compare the clinical outcomeof regrafts with first grafts.Methods: Two-year outcome data were obtained from the SwedishCornea Transplant Register for patients undergoing penetratingkeratoplasty (PK) between 2001and 2008. The survival and visualoutcome of regrafts with the original diagnoses of keratoconus,Fuchs’ endothelial dystrophy (FED) or bullous keratopathy (BK)were compared with first grafts for the same diagnoses by univariateand logistic regression methods.Results: The failure rate for grafts in keratoconus increased threefoldin regrafts compared with first grafts (i.e., 17% vs. 6%, p=0.002)doubled in FED regrafts (33% vs. 15%, p=0.001). In BK, the failurerate was already high in first grafts and the increase in failure ofregrafts was minimal (p=0.9). Visual acuity was also worse inregrafts compared to first grafts, mainly in the keratoconus and FEDpatients. In the keratoconus group, visual acuity with preferredcorrection was ≥0.5 in 69% of first grafts compared with only 55% inregrafts (p=001). In FED 52% of first grafts but only 19% of regraftsachieved VA ≥0.5, (p=0.001). The visual outcome of regrafts in BKwas poor but little different from first grafts where fewer than 20%achieved VA ≥0.5.Conclusions: This analysis confirmed the poorer survival of regraftswhere the original indication was keratoconus or FED. In addition,visual outcome was also worse than in first grafts. However, theoutcomes for regrafts in BK were similar to first grafts.Commercial Relationships: Margareta Claesson, None; JohnArmitage, NoneSupport: Sahlgrenska University research grantProgram Number: 3100 Poster Board Number: D0035Presentation Time: 8:30 AM - 10:15 AMFemtosecond Laser "Mini-Bubble" Deep Lamellar Dissection forDALK and DSAEKRoger F. Steinert, Marjan Farid, Sumit Garg, Matthew Wade.Ophthalmology, Gavin Herbert Eye Institute, UC Irvine, Irvine, CA.Purpose: To better define the parameters of femtosecond laser deepanterior lamellar dissection that result in a smooth bed for either deepanterior lamellar keratoplasty (DALK) or for preparation of donormaterial in thin stroma descemet stripping automated endothelialkeratoplasty (DSAEK)Methods: 10 fresh human corneal scleral donors were mounted onartificial anterior chambers and exposed to femtosecond laserdissections 30-50 microns anterior to Descemet membrane using aniFS 150 kHz laser (Abbott Medical Optics, Santa Ana, CA).Variables studied were spot separation, pattern of laser scanning, andpulse energy. Key outcomes were 1) ease of tissue separation; 2)gross inspection of the tissue bed; 3) spectral domain opticalcoherence tomography (OCT); and 4) trypan blue/alizarin redstaining of the endothelium.Results: Separation of the tissue plane was easiest with acombination of close spot separation (4x4 microns) and high pulseenergy (3.5 microjoules). The smoothest bed was obtained with widepulse spacing (10x10 microns); low pulse energy just higher thanthreshold (0.4 5 to 0.6 microjoules) and 8 alternating raster and spiralpasses, rotated 45 degrees between each pair of passes. OCT revealedstromal separation either at or slightly anterior to Descemetmembrane. Staining showed no evidence of endothelial injury.Conclusions: Wide pulse spacing and very low pulse energydelivered in multiple passes results in a smoother stromal plane in thedeep cornea, compared to tight spot spacing and higher pulse energylevels. This "mini-bubble" technique may be useful in both DALKand in donor tissue preparation for DSAEK.Commercial Relationships: Roger F. Steinert, Abbott MedicalOptics (C), OptiMedica (C), ReVision Optics (C), WaveTec (C);Marjan Farid, None; Sumit Garg, None; Matthew Wade, NoneSupport: Departmental development grant, Research to PreventBlindness; Discovery Eye FoundationProgram Number: 3101 Poster Board Number: D0036Presentation Time: 8:30 AM - 10:15 AMCorneal thickness after posterior lamellar keratoplastyAnders Ivarsen, Jesper Hjortdal, Nicolaj Aagaard, J.ChristianHedegaard, Henrik Sejersen, Christina Møller. Ophthalmology,Aarhus University Hospital, Aarhus, Denmark.Purpose: Descemet’s stripping automated keratoplasty (DSAEK) hasbecome the most frequently performed treatment for visuallysignificant endothelial dystrophy. The surgery causes a transientincrease in recipient corneal hydration; however, it remains unknownwhether structural changes in the recipient cornea also occur.Methods: Seventy-six patients treated for endothelial dystrophy werefollowed with routine clinical examination for at least one year afterDSAEK. At all visits, central corneal thickness (CCT) wasdetermined with a Haag-Streit optical pachymeter. The difference inCCT from 3 days to 1 year after sugery (ΔCCT = CCT 3days -CCT 1year ) was calculated as a crude measure of postoperative cornealedema. At their latest visit, patients were examined with spectraldomain OCT (SD-OCT; Heidelberg Spectralis with anterior segmentmodule) to determine the central thickness of the recipient cornea(recipient corneal thickness; RCT). SD-OCT was also used todetermine CCT in a group of normal corneas.Results: From 1 to 6 years after DSAEK, RCT averaged 490 ± 29μm, which was significantly less than the CCT of 531 ± 18 μm thatwas observed with SD-OCT in normal corneas (p < 0.001). RCTmeasured 487 ± 28 μm after one year (n = 43), 491 ± 29 μm after 2years (n = 24), and 505 ± 26 μm after 3 to 6 years (n = 9), with aslight but significant increase over time (Pearson’s corr; r 2 = 0.06; p =0.03). Correlating RCT with ΔCCT also showed a significantcorrelation between postoperative corneal edema and RCT, withmore edema causing the recipient cornea to become thinner over time(r 2 = 0.11; p = 0.006).Conclusions: In patients treated with DSAEK for endothelialdystrophy we found corneas to be thinner than normal once thepostoperative edema had resolved.The correlation between postoperativeedema and RCT may suggest that wash-out of stromalground substances induce the thinning, with the subsequent gradualincrease in thickness being caused by resynthesis of extracellularmaterial. However, the long-lasting nature of the observed changescould also suggest underlying structural abnormalities in endothelialdystrophy.Commercial Relationships: Anders Ivarsen, None; JesperHjortdal, Carl Zeiss Meditec (R); Nicolaj Aagaard, None;J.Christian Hedegaard, None; Henrik Sejersen, None; ChristinaMøller, None323 Corneal Surgery RefractiveTuesday, May 07, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 3102-3147/D0037-D0082Organizing Section: CorneaProgram Number: 3102 Poster Board Number: D0037Presentation Time: 8:30 AM - 10:15 AMRole of Subconjunctival Bevacizumab in Post-PterygiumExcision Management©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSonia B. Dhoot 1 , Howard Guan 2 , Keith Tokuhara 3 . 1 Ophthalmology,University of Missouri Kansas City, Kansas City, MO;2 Ophthalmology, Loma Linda Medical Center, Loma Linda, CA;3 Ophthalmology, Arrowhead Regional Medical Center, Colton, CA.Purpose: The exact pathogenesis of pterygium formation isunknown, but previous research has demonstrated that variousgrowth factors, including VEGF, may play a role. The use of VEGFinhibitors bevacizumab and ranibizumab is poorly understood ininhibiting pterygium growth. The objective of this study was tocompare the effects of wound healing and recurrence rates inpostoperative bevacizumab versus pterygium excision alone.Methods: This was a prospective trial. Thirty-one patients with aprimary pterygium of at least 2 mm in size and without any previousocular surgery were included. Seventeen patients received 5mgpostoperative bevacizumab on postoperative weeks two and six,while fourteen patients received no bevacizumab. Outcome measuresincluded best corrected visual acuity, intraocular pressure, recurrence,and any sight threatening complications at two weeks, two months,and six months postoperatively.Results: Six patients were lost to follow-up, four of which wereassigned to the bevacizumab group. Of the remaining thirteenpatients in the bevacizumab group, four had recurrence of thepterygium (30.7%). Two of the remaining twelve in the control grouphad a recurrence (16.7%).Conclusions: Bevacizumab does not improve recurrence rates forpterygia when used as an adjunctive therapy postoperatively. It mayeven cause increased rates of recurrence, although further studies areneeded before arriving at this conclusion.Figure 1. A. Preoperative photograph of a 4mm nasal pterygium ofthe left eye. B. Recurrence of pterygium 2 months after surgery in apatient who received postoperative bevacizumab.Figure 1. A. Preoperative photograph of a 4mm nasal pterygium ofthe left eye. B. Recurrence of pterygium 2 months after surgery in apatient who received postoperative bevacizumab.Commercial Relationships: Sonia B. Dhoot, None; Howard Guan,None; Keith Tokuhara, NoneClinical Trial: NCT01736449Program Number: 3103 Poster Board Number: D0038Presentation Time: 8:30 AM - 10:15 AMThe effect of pterygium surgery on corneal astigmatismClinton J. Duncan, Sarah Logan, Kent L. Anderson. Ophthalmology,University of Texas Health Science Center at San Antonio, SanAntonio, TX.Purpose: Quantify the effect pterygium surgery has on cornealastigmatism and identify factors that may predispose patients to havelesser or greater astigmatism after pterygium surgery. Calculate thesuccess rate of pterygium surgery by looking at recurrence rate andidentify those factors predisposing to recurrence.Methods: An IRB approved retrospective chart review of all TexasDiabetes Institute (TDI) patients that underwent pterygium surgery atthe University Hospital System (UHS) and the Medical Arts andResearch Center (MARC) over a 3 year period was completed. Sex,age, race, and pterygium details including eye (left vs right), side(nasal vs temporal), primary vs recurrent, and size were analyzed.Surgery details including duration, surgical technique, medications(pre-, intra-, post-), surgeon(s), and site were also included inanalysis. Pre- and post-operative simulated topography and refractionwere compared to determine statistical significance. Factorsassociated with changes in astigmatism and recurrence rates wereanalyzed.Results: Review and analysis was completed on records from 92eyes from 82 patients. The total mean pre-operative (3.46, S.D. 0.62)and mean post-operative (1.99, S.D. 1.85) simulated cornealastigmatism values were significantly different (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaHispanic ethnicity (16/45 vs 0/6) was associated with higherrecurrence rates. Younger age (37.8 vs 45.1, p= 0.0630) was alsoassociated with recurrence. Use of sutures was not found to correlatewith recurrence rate (4/15 vs 12/36, p= 0.7485). Size of thepterygium was not associated with higher recurrence rate. Variationsin the dose, frequency, and duration of steroid use did not differbetween recurrence and non-recurrence groups.Conclusions: Resident recurrence rates and complications werecomparable to other reports within the literature. The two major riskfactors associated with recurrence within this study were Hispanicethnicity and younger age. Differences in the vigor of theinflammatory response within these groups may underlie thedifference in risk. However, the post-operative management withanti-inflammatory medication, including initial frequency, week offirst taper, and total duration, was not found to be significantlydifferent between these two groups. Additionally, the use ofabsorbable sutures, which might enhance the inflammatory response,did not affect the risk of recurrence. Intraoperative use of MMC forinitial excisions was rare in this academic setting, but has been usedfor many excisions of recurrent pterygium. Further investigation ofMMC for initial excisions in these high-risk groups may bewarranted.Commercial Relationships: Adam Sise, None; Tanuj Banker,None; Wellington Chang, None; Sasikala Pillai, NoneProgram Number: 3105 Poster Board Number: D0040Presentation Time: 8:30 AM - 10:15 AMComparison of fibrin glue and autologous blood for conjunctivalautograft fixation in pterygium surgerySalina Teja, Sophie Boucher, Kashif Baig. Ophthalmology,University of Ottawa Eye Institute, Ottawa, ON, Canada.Purpose: Our purpose is to investigate the use of a novel technique,graft fixation with autologous blood, by comparing its anatomic andvisual outcomes to those obtained with fibrin glue in pterygiumexcision surgery.Methods: This is a retrospective comparative case series of patientswith a primary nasal pterygium who underwent excision. All patientshad a conjunctival autograft from the superior bulbar conjunctiva tocover the scleral bed. 17 patients had fixation of the autograft withautologous blood (AB) and 17 patients had fixation with fibrin glue(FG). Data was collected up to 6-months post operatively andincluded; surgical cost and time, conjunctival graft stability andhealing, visual acuity and pterygium recurrence. Outcomes werecompared between the two groups and assessed for statisticalsignificance with a paired student t test.Results: Of our 34 patients, 15 were female and 18 were male with amean age of 52. The mean size of conjunctival autograft was 36mm2in the AB group and 42mm2 in the FG group. There were no intraoperativecomplications in either group. Surgical costs differed ineach group: the FG group incurred the cost of the fibrin glue whereasthe AB group had the additional costs associated with an extra 15minutes of procedure time. At 1-month post-operatively, 4 patients inthe AB group had lost their graft compared to zero in the FG group,showing greater stability in the FG group. Mild graft displacementwas seen in 3 patients in each group. The visual acuity was stable inboth groups. There was no incidence of pterygium recurrence ineither group. 6-month follow up outcomes will be analyzed byFebruary 2013.Conclusions: Pterygium recurrence rates vary due to many factorsincluding surgical technique. The conjunctival autograft fixated withautologous blood has been shown to be safe and effective but its roleis not well established. The fibrin glue technique is widely used butposes a risk of hypersensitivity reactions, discomfort, scarring andinfection. Our preliminary results show that fixation with autologousblood produces similar visual and pterygium recurrence outcomes tofibrin glue fixation, however seems to have less stable grafts atfollow up. Our comparison of the efficacy and stability of theconjunctival autograft between the two techniques at 6 month followup will help to further establish the role of autologous blood inpterygium surgery.Commercial Relationships: Salina Teja, None; Sophie Boucher,None; Kashif Baig, Bausch and Lomb (F), Allergan (C), Alcon (C)Support: University of Ottawa Medical Research FundProgram Number: 3106 Poster Board Number: D0041Presentation Time: 8:30 AM - 10:15 AMExpression of the pro-inflammatory molecule RAGE in humanpterygiaElia J. Duh 1 , Samar A. Al-Swailem 2 , Zhenhua Xu 1 , Samuel C. Yiu 1, 2 ,Lijuan Wu 1 . 1 Ophthalmology, Johns Hopkins Wilmer Eye Inst,Baltimore, MD; 2 King Khaled Eye Specialist Hospital, Riyadh, SaudiArabia.Purpose: Inflammation is associated with development and growthof pterygia, including promotion of angiogenesis. The importantinflammatory mediators in pterygia are continuing to be defined.RAGE, the Receptor for Advanced Glycation Endproducts, is knownto be a strong pro-inflammatory molecule expressed in vascularendothelium and other cell types. In this study, we examined RAGEexpression and immunolocalization in human pterygium and normalconjunctival tissue.Methods: Pterygium specimens were obtained during surgery at theKing Khaled Eye Specialist Hospital (KKESH). In the same patients,conjunctiva were obtained from the autograft during surgery. Tissuespecimens were formalin-fixed and paraffin-embedded. Tissuesections were analyzed by immunohistochemistry with anti-RAGEantibody. Expression and localization of RAGE were evaluated inpterygium and corresponding conjunctiva.Results: In both pterygium tissue and corresponding conjunctiva,there was expression of RAGE in the epithelium. In addition, othercell types exhibited expression, notably vascular endothelial cells aswell as fibroblasts and possibly macrophages. There was distention ofendothelial cells in pterygium specimens with associated prominentstaining of RAGE in pterygium specimens as compared to normalconjunctiva.Conclusions: Our data indicate the expression of RAGE in bothpterygial tissues and conjunctiva. The prominent expression ofRAGE in vascular endothelium in pterygia suggest a possible role ofthis molecule in promoting the inflammatory process in pterygiumprogression.Commercial Relationships: Elia J. Duh, None; Samar A. Al-Swailem, None; Zhenhua Xu, None; Samuel C. Yiu, None; LijuanWu, NoneSupport: This work was supported by funds from Research toPrevent Blindness and a KKESH/JHU-WEI grant.Program Number: 3107 Poster Board Number: D0042Presentation Time: 8:30 AM - 10:15 AMEffect of Porcine Chondrocyte Derived Extracellular Matrix onthe Pterygium in Mouse ModelKyeong Hwan Kim 1, 2 , Hye Sook Lee 2 , Jae Wook Yang 1, 2 .1 Department of Ophthalmology, Inje University College of Medicine,Busan, Republic of Korea; 2 Ocular Neovascular Research Center,Inje University Busan Paik Hospital, Busan, Republic of Korea.Purpose: To investigate the effect of porcine chondrocyte derivedextracellular matrix (PCDECM) on the pterygial tissue growth inmouse model.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: Human pterygial epithelial cells were isolated and culturedfrom the specimens during the surgical removal (IRB No. : 12128).Cultured cells were stained with pan-CK, CK3/2p, vimentin, mucin-1, and CK13 for the confirmation of pterygial epithelial cells.PCDECM were supplied from the another laboratory (RegenprimeCo. Ltd., Gyeonggi-do, South Korea), which was manufactured withthe method described elsewhere. To establish pterygium murinemodel, 10,000 cells in 10 microliters of PBS were injected to thenasal subconjunctival space in both eyes of athymic nude mice.PCDECM dissolved in PBS (25 mg/mL, 10 microliters) were injectedto the nasal subconjunctival space in the right eye 7, 10 and 14 daysafter epithelial cell injection (ECM group), and PBS (10 microliters)were injected to the same area of the opposite eye as same scheduleas the left eye (control group). Image analysis with the photographwas performed using ImageJ® to compare the lesion size.Results: Isolated pterygial cells were positive for pan-CK, CK3/2p,vimentin and mucin-1, and negative for CK13 underimmunofluorescence microscopy. Pterygial lesion in mouse modelwas confirmed 7 days after subconjunctival injection of humalpterygial epithelial cells. There was no significant difference in lesionsize baseline (7 days after epithelial cell injection; 25.6% in ECMgroup vs. 24.9% in control group, expressed as the ratio of lesion areato entire cornea). On the day 17 after epithelial cell injection, thelesion size compared to the entire cornea was increased to 35.4% incontrol group, however, ECM group showed less change in size as26.4% (9.8 % point and 0.8 % point increase from the baseline,respectively).Conclusions: Our findings suggest that PCDECM seems to suppresspterygial epithelial cell growth and it could be used as a promisingmaterial for the noninvasive treatment of pterygium.Commercial Relationships: Kyeong Hwan Kim, None; Hye SookLee, None; Jae Wook Yang, NoneSupport: Grant of the Korea Healthcare Technology R&D Project,Ministry of Health & Welfare Affairs, Republic of Korea. (grantnumber A120006 and A120078)Program Number: 3108 Poster Board Number: D0043Presentation Time: 8:30 AM - 10:15 AMLinear systems analysis of Pre and Post Operative ZernikeCoefficients in LASIK SurgeryDaniel R. Neal 1 , Thomas D. Raymond 1 , Amelia Saliba 2 , Jill V.Treyes 2 . 1 Research and Development, AMO WaveFront Sciences,LLC, Albuquerque, NM; 2 Research and Development, AMODevelopment Inc, Milpitas, CA.Purpose: Improvements in LASIK outcomes will require advancedanalyses to reveal correlations between preoperative measurementsand systematic variations in outcomes. We postulate that pairwiseand multivariate analyses could be used effectively to identifycorrectable systematic errors in LASIK outcomes.Methods: This retrospective study analyzed the pre and postoperative aberrometry and keratometry data on 120 eyes of 63patients that underwent normal LASIK surgery using a 193 nmexcimer laser equipped with iris registration. Pre- and post-surgerymeasurements were taken with a combined aberrometer and cornealtopographer instrument. Wavefront measurements were expressed inZernike polynomials and resized to 4 mm analysis pupil. JMP 8.0software was used to analyze the third to fourth order Zernikecoefficients for both pairwise and multivariate correlation andstatistical significance.Results: Statistically significant correlations were observed betweenpre operative and post operative wavefront coefficients. Figure 1illustrates the pre to post operative multivariate correlations for theOD eyes; statistical significance for pairwise correlations are denotedwith asterisks. Note that the correlation coefficients can exceed 40%.Both low and high order terms interact to affect the outcome. Thesedata support the hypothesis that systematic errors in LASIKoutcomes can be identified through these analyses.Conclusions: That many of the correlations are statisticallysignificant affords the hope that improvements in treatment planningcan eliminate many of these errors thereby enhancing the quality ofLASIK outcomes.Figure 1 The multivariate correlation between pre and post operativeZernike coefficients for wavefront aberration are shown above;asterisks denote correlations that are statistically significant. Thesedata show the well known correlation between pre operative sphericalequivalent (Z4) and post operative spherical aberration (Z12) as wellas many other correlated terms.Commercial Relationships: Daniel R. Neal, Abbott Medical Optics(E), Abbott Medical Optics/US20120172854A1 (P); Thomas D.Raymond, Abbott Medical Optics (E), Thomas D. Raymond (S);Amelia Saliba, AMO (E), Abbott (I); Jill V. Treyes, AMODevelopment (E)Support: Internally funded by AMOClinical Trial: NCT01220466Program Number: 3109 Poster Board Number: D0044Presentation Time: 8:30 AM - 10:15 AMA novel model of digitized clinical validation of femtosecondLASIK flap diameter and opaque bubble layer (OBL) incidenceIoanna Kontari 1 , George Asimellis 1 , A. John Kanellopoulos 1, 2 .1 LaserVision.gr Eye Institute, Athens, Greece; 2 NYU MedicalSchool, New York, NY.Purpose: The development of digital, quantitative and accurateanalysis of LASIK flap diameter and OBL occurrence.Methods: Flaps from 100 consecutive myopic and hyperopic LASIKpatients, treated with the Alcon-WaveLight FS200 femtosecond andEX500 excimer lasers were digitally recorded and processed. Flapdimensions and OBL extent was evaluated utilizing a newlydevelopeddigital procedure with a commercially available imageprocessing software. Specifically, the flap creation report imageprovided by the FS200 internal system was analyzed digitally andcalibrated on a scale converting pixels to measurable length in mm.In a similar fashion, the OBL occurrence was measured as apercentage of the actual flap surface area.Results: For the myopic cases, with planned flap diameter 8.00 (n =20) and 8.50 mm (n = 60), a small negative bias was discovered (-0.15 mm and -0.12 mm, respectively) with very small size fluctuation(diameter stdev = ± 0.04 and ± 0.03 mm, respectively). For thehyperopic cases, with planned flap diameter 9.50 mm (n = 20), asmall positive bias was discovered (+ 0.06 mm), again with verysmall size variation (diameter stdev < 0.009 mm). Of the flaps©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneastudied, the majority (> 50%) had negligible or no OBL, and 20%less than 10% of the total flap area. The remaining had OBL to up of20% of the total flap area. In all cases the flap was ‘delayed’, that isdeveloping in areas already processed by the femtosecond laser, thusnot interfering with the flap creation.Conclusions: Τhis highly accurate, novel validation digital techniqueconfirms the high reproducibility in flap dimensions and smalloccurrence of OBL in both myopic and hyperopic LASIK cases withthe FS200 femtosecond laser.Commercial Relationships: Ioanna Kontari, None; GeorgeAsimellis, None; A. John Kanellopoulos, Alcon Laboratories, Inc.(C), Avedro (C), Bausch and Lomb (C), Ocular Therapeutix (C)Program Number: 3110 Poster Board Number: D0045Presentation Time: 8:30 AM - 10:15 AMThe effect of procedure room temperature and humidity onLASIK outcomesMichael I. Seider 1 , Stephen D. McLeod 1 , Travis Porco 1 , Steven C.Schallhorn 1, 2 . 1 Ophthalmology, University of California, SanFrancisco, San Francisco, CA; 2 Optical Express, Inc., Glasgow,United Kingdom.Purpose: To determine if procedure room temperature and humidityduring LASIK affects refractive outcomes in a very large patientsample.Methods: Patients aged 18 to 75 years old who underwent LASIK atan Optical Express, Inc. location in their United Kingdom and Irelandcenters from January 1, 2008 to June 30, 2011 who met inclusioncriteria (including post-operative refractive goal of plano) wereincluded. Patient age, gender, pre- and one month post-LASIKmanifest refraction and flap creation technique were recorded as wellas the ambient temperature and humidity during LASIK. Effect sizedetermination, in addition to univariate and multivariate analysis wasperformed to characterize the relationships between LASIKprocedure room temperature and humidity and post-operativerefractive outcome.Results: 202,394 eyes of 105,712 patients were included. Whenconsidering all eyes in our population, an increase of one degreeCelsius during LASIK was associated with a 0.003 diopter morehyperopic refraction one month post-operatively (P=0.0094) and anincrease in one percent humidity was associated with a 0.0004 moremyopic refraction (P0.05). The central K values decrease by a mean of1.35 ± 3.1 and 0.41 ± 3.5 respectively (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaYishan Qian. Ophthalmology, Eye and ENT hospital FudanUniversity, Shanghai, China.Purpose: To investigate the characteristics of corneal epithelialpachymetry map in patients wearing orthokeratology lens (OK lens)using the automatically segmentation function of SD-OCT.Methods: 60 patients who had been fitted with myopic OK lens forat least one night were included in the study (mean age, 10.6±2.38years) and 11 patients without any history of contact lens served ascontrols. Patients were divided into 6 groups according to theduration of orthokeratology lens wearing. An SD-OCT (RTVue SD-OCT, Optovue, Inc, CA) with a pachymetry module was used tomeasure the central 6mm corneal epithelial topography. Theepithelial thickness of the central 3 mm, the average thickness of the3 to 5mm ring (Peripheral 1, P1), and the average thickness of the 5to 6mm ring (Peripheral 2, P2) were recorded and compared withinindividual groups. Munnerlyn’s formula was used to model theexpected change in refractive error based on measured changes intopographical corneal epithelial thickness.Results: The epithelial thickness of the central cornea wassignificantly reduced in all ortho-k groups. Except for group one, nosignificantly difference in the epithelial thickness of 3 to 5mm (P1)was found between the ortho-k group and the control one. For P2, theepithelial thickness increased in group2 to 5, but only group 3reached significant level. The refractive change predicted byMunnerlyn’s formula based on changes in topographical cornealepithelial thickness was much less than the measured refractivechanges found in all groups.Conclusions: Epithelial pachymetry map automatically generated bythe SD OCT could provide object and comprehensive information ofepithelial change caused by overnight wearing of OK lens. OK lenscan cause significant thinning of central corneal epithelium andthickening of midperipheral corneal epithelium. Changes induced byovernight orthokeratology can not be wholly explained by theinduced changes in corneal epithelial thickness. Change in stromalthickness or the bending of the corneal tissue may also play a role.Presentation Time: 8:30 AM - 10:15 AMPatient Satisfaction and Quality of Vision after Wavefrontguided(WFG) vs. Wavefront-optimized (WFO) LASIKRose K. Sia 1 , Richard D. Stutzman 2 , Joseph F. Pasternak 2 , Denise S.Ryan 1 , Jennifer B. Eaddy 1 , Lorie A. Logan 1 , Lamarr Peppers 1 ,Edward W. Trudo 1 , Kraig S. Bower 3 . 1 US Army WarfighterRefractive Surg Research Ctr, Fort Belvoir Community Hospital, FortBelvoir, VA; 2 Ophthalmology, Walter Reed National MilitaryMedical Center, Bethesda, MD; 3 The Wilmer Eye Institute, JohnsHopkins University, Baltimore, MD.Purpose: To compare higher order aberration (HOA) root meansquare (RMS) and patient satisfaction of postoperative vision afterWFG vs. WFO LASIK.Methods: Participants randomized to receive WFG LASIK (VISXStar S4, Abbott Medical Optics) or WFO LASIK (WavelightAllegretto Wave Eye-Q, Alcon Surgical) underwent testing todetermine their HOA RMS preoperatively and at 6 months (M)postoperatively. RMS HOA were analyzed at four different pupilsizes (4, 5, 6, and 7mm) using Complete Ophthalmic AnalysisSystem (COAS, Abbott Medical Optics). A repeated measuresanalysis of variance (RM-ANOVA) was used to compare WFG vs.WFO LASIK HOA RMS at each pupil size over time. Participantsresponded to a questionnaire preoperatively and 6M postoperatively.A Mann-Whitney test was used to compare patient satisfaction ofpostoperative vision. A p-value

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConclusions: Scanning acoustic microscopy is a novel tool forexamining the biomechanical properties of human corneal tissue. Itwas used successfully to assess corneal stiffness after applyingcollagen cross-linking. Similar results were observed in corneastreated with low and high intensity cross-linking protocols.Table 1. Patient satisfaction and quality of vision questionnaireresults.Commercial Relationships: Rose K. Sia, None; Richard D.Stutzman, None; Joseph F. Pasternak, None; Denise S. Ryan,None; Jennifer B. Eaddy, None; Lorie A. Logan, None; LamarrPeppers, None; Edward W. Trudo, None; Kraig S. Bower, NoneSupport: Dept of Defense W81XWH-09-2-0018Clinical Trial: NCT01097525Program Number: 3115 Poster Board Number: D0050Presentation Time: 8:30 AM - 10:15 AMBiomechanical changes in Human Corneas after Low and HighIntensity Collagen Cross-Linking Treatment measured usingScanning Acoustic MicroscopyIthar M. Beshtawi 1 , Riaz Akhtar 2 , Chantal Hillarby 3 , ClareO'Donnell 1, 4 , Xuegen Zhao 5 , Arun Brahma 6 , Fiona M. Carley 6 , BrianDerby 5 , Hema Radhakrishnan 1 . 1 Faculty of Life Sciences, TheUniversity of Manchester, Manchester, United Kingdom; 2 School ofEngineering, University of Liverpool, Liverpool, United Kingdom;3 School of Clinical and Laboratory Science, The University ofManchester, Manchester, United Kingdom; 4 Optegra, Manchester,United Kingdom; 5 School of Materials, The University ofManchester, Manchester, United Kingdom; 6 Manchester Royal EyeHospital, Manchester, United Kingdom.Purpose: To assess and compare the biomechanical properties ofpostmortem human corneas after low and high intensity collagencross-linking using scanning acoustic microscopy (SAM).Methods: Two groups of five human corneal pairs were included inthe study. In group (A), five corneas were treated with low intensitycross-linking (epithelium removed, 0.1% riboflavin applied for 30minutes following which UV-A irradiation (365 nm, 3mW/cm 2 ) wasapplied for 30 minutes alongside the riboflavin solution) Thecontralateral cornea was exposed to riboflavin only, and served as acontrol. In group (B), five corneas were treated with high-intensitycollagen cross-linking (epithelium removed, riboflavin 0.1% solutionapplication for 30 minutes prior to UV-A irradiation (365 nm,9mW/cm 2 ) for 10 minutes, while the riboflavin was re-applied. Thecontralateral cornea was exposed to riboflavin only, and served as acontrol. The biomechanical properties of all corneas were testedusing SAM.Results: In the low intensity cross-linked corneas (group A), thespeed of sound of the treated corneas was 1674.51 ± 21.38 ms -1anteriorly and 1598.24 ± 27.83 ms -1 posteriorly, while it was 1593.96± 22.51 ms -1 anteriorly and posteriorly 1578.64 ± 18.83 ms -1 in theuntreated corneas. In the high-intensity cross-linked corneas (groupB), the speed of sound of the treated corneas was 1663.55 ± 20.16ms -1 anteriorly and 1593.17 ± 20.30 ms -1 posteriorly, while it was1582.38 ± 15.30 ms -1 anteriorly and 1564.12 ± 18.46 ms -1 posteriorlyin the untreated corneas. The speed of sound is directly proportionalto the stiffness of the tissue. Therefore the results indicate thatcorneal stiffness changes are similar in the low and high intensitycross-linking groups.The anterior and posterior (200 x 200) µm portion of low intensitycross-linked (A and B, respectively) and untreated (C and Drespectively) corneal sections.The anterior and posterior (200 x 200) µm portion of high intensitycross-linked (A and B, respectively) and untreated (C and Drespectively) corneal sections.Commercial Relationships: Ithar M. Beshtawi, None; RiazAkhtar, None; Chantal Hillarby, None; Clare O'Donnell, None;Xuegen Zhao, None; Arun Brahma, None; Fiona M. Carley,None; Brian Derby, None; Hema Radhakrishnan, NoneProgram Number: 3116 Poster Board Number: D0051Presentation Time: 8:30 AM - 10:15 AMInflammatory and lacrimal gland proteins in the tear film afterfemtosecond LASIKAndrea Petznick 1 , Lei Zhou 1, 3 , Roger W. Beuerman 1, 3 , Siew KwanKoh 1 , Louis Tong 2, 1 , Jodhbir S. Mehta 2, 1 . 1 Ocular Surface ResearchGroup, Singapore Eye Research Institute, Singapore, Singapore;2 Singapore National Eye Centre, Singapore, Singapore; 3 Duke-NUSGraduate Medical School, Singapore, Singapore.Purpose: Dry eye is a major side effect of LASIK. It has not beenclear if these complaints are caused by inflammatory changes orregenerative corneal nerve changes. A proteomics study of tears wascarried out with patients who underwent femtosecond LASIK toaddress two questions: 1) Is LASIK-associated dry eye a result ofinflammation? 2) Does the newer femtosecond platform VisuMaxusing smaller flap, higher repetition rate, lower laser pulse energy anddifferent corneal suction reduce adverse ocular surface changes?Methods: In a prospective contra-lateral paired eye study, cornealflaps of 22 patients were created by either the VisuMax or IntraLaselaser. Tear samples were collected before surgery, and at 1 week and3 months postoperatively using Schirmer’s strips. A panel of dry eye©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaprotein markers were analyzed using iTRAQ (isobaric tagging forrelative and absolute quantitation) mass spectrometry. Tear proteinratios were calculated relative to preoperative protein levels atbaseline.Results: There was little change in the inflammatory ocular surfacecondition, but lacrimal gland secretions were affected. The proinflammatoryassociated protein S100A9 was downregulated while α-1-acid glycoprotein 1 (ORM1) was upregulated in IntraLase at 1week postoperatively. The lacrimal gland proteins, lipocalin-1 andlysozyme showed lower ratios in IntraLase and VisuMax, whileprolactin-inducible protein (PIP) and lactoferrin were dowregulatedonly in IntraLase at 1 week postoperatively. Downregulation oflipocalin-1, PIP and lactoferrin was observed in IntraLase 3 monthsafter surgery.A comparison between lasers showed that IntraLase affected proteinratios significantly more. At 1 week postoperatively, α-enolase(ENO1) ratio was greater in IntraLase as compared to VisuMax(1.324 vs. 0.940, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneameasurements for patients with laser refractive surgery issystematically reduced with its size reduction linearly related torefractive error, ablation optical zone and corneal eccentricity. TheHartmann-Shack system is limited in measuring post-operativewavefront aberrations for whole pupil area, and therefore it providesunderestimates of wavefront aberrations for post-operative eyes.Commercial Relationships: Ying Wu, None; Ji C. He, None;Xingtao Zhou, None; Renyuan Chu, NoneProgram Number: 3119 Poster Board Number: D0054Presentation Time: 8:30 AM - 10:15 AMCorrelation of Temperature and Humidity to the Incidence ofLASIK Flap Striae In A Very High Volume Refractive SurgeryCenterNeema Nayeb-Hashemi 1 , Ronald R. Krueger 1 , Minoru Tomita 2 .1 Ophthalmology, Cleveland Clinic Foundation Cole Eye Institute,Cleveland, OH; 2 Ophthalmology, Shinagawa LASIK Center, Tokyo,Japan.Purpose: To correlate temperature and humidity to the monthlyincidence of flap striae after LASIK performed by experiencedsurgeons at a single center between June 2007 and April 2012Methods: Data on all LASIK cases performed at the ShinagawaLASIK Center in Tokyo between June 2007 and April 2012 wasreviewed and organized by month for total LASIK cases as well asthe total number of striae requiring flap realignments. Statisticalanalyses were then performed to determine any significantdifferences in incidence by month, season, and year. Using data fromthe Japan Meteorological Agency, average monthly humidities andtemperatures for the same time were obtained and compared tomonthly realignment rates.Results: A total of 614,340 eyes had LASIK surgery of which 5,244developed striae requiring realignment, a cumulative incidence of0.85%. The average number of LASIK cases per month ranged from8,959 to 12,028, while the average flap striae per month ranged from300 to 534. There was a significant decline in the total number ofLASIK cases and macrostriae in 2011 (p.05). Averaged for all years, themonthly incidence ranged from 0.657% to 1.006%. The lowestincidences were in the summer months (June-August). Subgroupanalysis demonstrated no difference in the incidence of macrostriaebetween years, however there was a notable decline in the incidenceof macrostriae for the summer season (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatreatment planning, when treatment target adjustment is applied tocompensate for these aberrations. The adjustments may be derivedstatistically for each site, surgeon, or tool (see Fig. 2).The site-specific SA0 can be readjusted such that the adjusted scatterplot will become more compact with increased correlation and R2values (compare Fig.2 B and C). This allows a better fit for thenomogram adjustment as well as more precise modeling of the corneahealing.Conclusions: Flap-induced aberrations for LASIK treatments maysubstantially contribute to the surgery outcome. The magnitude ofaberrations is different for different surgeons, sites, or tools, anshould be estimated for each surgeon individually. Data for differentsurgeons may be combined for subsequent analysis and adjustments.mean spherical refraction increased by +2.21D; both statisticallysignificantly. However, neither achieved the full targeted correctionof +3.80D.Conclusions: Corneal thickness changes measured at pupil centersignificantly increased for the inlay eyes, reflecting the additivenature of the procedure. However, the cornea did not thicken to thefull added thickness of the inlay, possibly due to epithelial thinningand potential sources of image misalignments. Significant steepeningof the anterior corneal surfaces showed positive results for thecorneal shape changes induced by the inlay implantation. Thesechanges in the corneal shape were highly correlated with the visualoutcome, although, the corneal power slightly underestimated therefraction change.Commercial Relationships: Eon Kim, None; Klaus Ehrmann,None; Jukka A. Moilanen, Adventus Technology Inc. (C); JenniferD. Choo, Adventus Technologies (C); Sylvie Franz, AdventusTechnology (E), Adventus Technology (F), Brien Holden VisionInstitute (C)Fig. 2. Induced SA vs. pre-op SE trend lines for two surgeons (A),regression for measured SA data (B), and regression for the dataadjusted by the surgeon-specific .Commercial Relationships: Stan Bentow, Abbott Medical Optics(E); Anatoly Fabrikant, Abbott Medical Optics (E); Guang-mingG. Dai, Abbott Medical Optics (E)Program Number: 3121 Poster Board Number: D0056Presentation Time: 8:30 AM - 10:15 AMCorrelation between the changes in corneal power and visualoutcome following inlay implantationEon Kim 1, 2 , Klaus Ehrmann 2, 3 , Jukka A. Moilanen 4 , Jennifer D.Choo 4 , Sylvie Franz 3, 4 . 1 Vision CRC, Sydney, NSW, Australia;2 School of Optometry and Vision Science, University of New SouthWales, Sydney, NSW, Australia; 3 Brien Holden Vision Institute,Sydney, NSW, Australia; 4 Adventus Technology, Inc, Irvine, CA.Purpose: To determine the correlation between the changes in thecorneal power and the visual outcome following inlay implantation tocorrect hyperopia.Methods: Five subjects underwent corneal inlay implantation ontheir non-dominant eye. Topography maps using Pentacam HR weretaken to obtain corneal thickness, best-fit sphere radii of anterior andposterior corneal surfaces and corneal power, prior, and at 5 timepoints up to two months post-operative. The visual outcome usingstandard subjective techniques was measured which data were thenconverted into power vector coordinates (M, J0, J45).Results: Corneal thickness measured at pupil center significantlyincreased by (mean ± SD) 22.5 ± 19.4 µm at two months postoperativetime point. The mean anterior corneal radius of curvaturesignificantly steepened by 0.21 ± 0.12 mm and there were nostatistically significant changes for the posterior corneal radius ofcurvature. The mean corneal power increased by +1.86D and theProgram Number: 3122 Poster Board Number: D0057Presentation Time: 8:30 AM - 10:15 AMEvaluation of femtosecond laser-assisted combined Descemet’sstripping automated endothelial keratoplasty (DSAEK) andastigmatic keratotomy (AK) surgeryKathy M. Tran 1 , Silas Wang 1 , Ioanna Kontari 2 , George Asimellis 2 , A.John Kanellopoulos 1, 2 . 1 New York Univ School of Med, New York,NY; 2 LaserVision.gr Eye Institute, Athens, Greece.Purpose: To evaluate the safety, efficacy, and clinical parameters incombined clear cornea DSAEK and AK surgery performed with theLenSx femtosecond laser (Alcon, Fort Worth, TX).Methods: 15 eyes of 13 consecutive patients who underwentcombined DSAEK and AK were evaluated pre-operatively and 6months post-operatively for: age, uncorrected distance visual acuity(UDVA), corrected distance visual acuity (CDVA), refraction,cylinder (C), topographic cylinder change (TCc), endothelial cellcount (ECC), and possible complications. The 3 host cornea incisionsof 3.5 mm, 1.2 mm, and 1.2 mm, along with 2 partial thicknessarcuate incisions at the 6mm optical zone, were performed by thelaser. The arcuate incisions were adjusted manually at the slit lamp at3 months.Results: The mean age was 68 years and mean values pre-operativelyand post-operatively were respectively: UDVA: 20/400 and 20/40,CDVA: 20/100 and 20/25, C: -2.25 D and -0.55 D. The mean TCcwas -2.45D. No complications were experienced during the follow-upperiod.Conclusions: This novel bladeless femtosecond laser-assisted clearcornea combined DSAEK and AK surgery appears to be safe andeffective in facilitating reproducible wound creation and cornealastigmatic reduction.Commercial Relationships: Kathy M. Tran, None; Silas Wang,None; Ioanna Kontari, None; George Asimellis, None; A. JohnKanellopoulos, Alcon Laboratories, Inc. (C), Avedro (C), Bauschand Lomb (C), Ocular Therapeutix (C)Program Number: 3123 Poster Board Number: D0058Presentation Time: 8:30 AM - 10:15 AMPhotorefractive keratectomy after deep anterior lamellarkeratoplastyPichaporn Artornsombudh, Yakov Goldich, Noa Avni-Zauberman,Uri Elbaz, Setareh Ziai, David Rootman. Ophthalmology, Universityof Toronto, Toronto, ON, Canada.Purpose: To report the efficacy and safety of photorefractivekeratectomy (PRK) for the treatment of residual refractive error©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneafollowing deep anterior lamellar keratoplasty (DALK).Methods: All patients that had PRK for residual myopia, hyperopiaand astigmatism following DALK between January 2008 to October2012 were included in this study. Mitomycin C (MMC) was appliedin all cases. The uncorrected distance visual acuity (UDVA),corrected distance visual acuity (CDVA), manifest refraction, cornealtopography, early and late complications were assessed.Results: The study included 13 eyes of 13 patients. Nine patientswere male and four patients were female. Mean age was 41.3 ± 18.4years (Mean ± SD). The indications for DALK were: keratoconus (7eyes), post-infectious scar (4 eyes), post-laser scar (1 eye), and postradial keratotomy (1 eye). Six patients underwent intralase enabledDALK (IE-DALK). The mean period between the DALK and PRKwas 27.4 ±12.7 months. The preoperative UDVA was 0.88 ± 0.53logMAR. The preoperative mean CDVA was 0.20 ± 0.18 logMARand the mean spherical equivalence (SE) was -2.65 ± 5.00 diopters(D). The mean follow up after PRK was 12.5 ± 11.7 months. At thefinal visit, the mean UDVA was improved to 0.23 ± 0.25 logMAR (P= 0.001). The mean CDVA was 0.17 ± 0.22 logMAR (P = 0.57), andthe mean final SE was -0.05 ± 1.41 D (P = 0.069). Astigmatismtended to be decreased from -3.27 ± 1.76 D preoperatively to -0.94 ±0.49 D postoperatively (P=0.001). UDVA improved more than twolines in 11 eyes (84.6%). One eye (7.7%) had one line improvementof UDVA and one eye (7.7%) had no improvement. All eyes hadcomplete epithelization within 5 days after procedure. Mild cornealhaze without significant visual reduction was presented in 4 eyes(30.8%). One eye had corneal epithelial rejection at 6 months afterPRK with MMC which resolved after treatment with topical steroids.Conclusions: Using PRK with MMC for the treatment of residualrefractive error after DALK lead to improvement in uncorrectedvisual acuity and can be considered as an acceptable treatmentmodality to improve quality of vision following DALK.Commercial Relationships: Pichaporn Artornsombudh, None;Yakov Goldich, None; Noa Avni-Zauberman, None; Uri Elbaz,None; Setareh Ziai, None; David Rootman, AMO (F)Program Number: 3124 Poster Board Number: D0059Presentation Time: 8:30 AM - 10:15 AMComparison of the Ocular Response Analyzer and the Belin-Ambrósio Ectasia Display for Detecting Eyes at High Risk ofDeveloping Ectasia After Refractive SurgeryMaria Eugenia Vola Ravina 1 , Renato Lisboa 1 , Patricia Schimchak 3 ,Kody J. Kishi 2 , Natalie A. Afshari 1 , David J. Schanzlin 4 .1 Ophthalmology, University of San Diego California, La Jolla, CA;2 University of Calgary, Calgary, AB, Canada; 3 Cornea, CETAO,Montevideo, Uruguay; 4 Gordon-Weiss-Schanzlin, La Jolla, CA.Purpose: To compare diagnostic accuracies of the Ocular ResponseAnalyzer (ORA) and the Belin-Ambrósio Ectasia Display (BAD)with the Randleman Ectasia Risk Score (RS) in eyes undergoingrefractive surgery.Methods: Two hundred and forty-nine eyes of 136 myopic patients,with no previous ocular surgery evaluated for refractive surgery wereincluded in the study. Before surgery, all eyes underwent a clinicalexamination and evaluation with Pentacam (Oculus, Wetzlar,Germany) and ORA (Reichert, Buffalo, NY). We calculate the RS forall eyes as if a laser in situ keratomileusis with a hypothetical 150 μmflap would be performed. Eyes with a RS ≥ 4 were considered tohave a high risk of post-operative ectasia. Parameters included in theanalysis from the ORA were keratoconus match index (KMI), cornealhysteresis (CH), and corneal resistance factor (CRF). Parametersfrom the BAD provided by the Pentacam included in the analysiswere anterior elevation change (DF), posterior elevation (DF),pachymetric progression (DP), absolute thinnest point (DT),displacement of the thinnest point (DY), and combined index (D).These “D values” represent the standard deviation from an internaldatabase of healthy eyes. Areas under the receiver operatingcharacteristic curves (AUC) were calculated to summarize thediagnostic accuracy of each parameter. Logistic regression wasperformed for the best parameter in order to access the magnitudeand direction of the association with the RS.Results: Eighty-seven eyes with a RS ≥ 4 were included in the groupwith a high risk of ectasia, whereas 162 eyes with a RS < 4 wereincluded in the control group. The ORA parameter with the largestAUC was the CRF (0.68 ± 0.05), followed by CH (0.65 ± 0.05) andKMI (0.64 ± 0.04). The BAD parameter with the largest AUC wasthe DT (0.80 ± 0.04), followed by the D (0.64 ± 0.05), and the DY(0.62 ± 0.05). DT provided by the BAD performed significantlybetter than CRF provided by the ORA (0.80 vs. 0.68; P = 0.003).Each 1 mmHg of decrease in the CRF was associated with a 46%increase in the odds of presenting a high risk in the RS.Conclusions: The absolute thinnest point provided by the BAD fromthe Pentacam performed better than the CRF from the ORA indetecting patients at a high risk of postoperative corneal ectasia.Larger studies may help to confirm these preliminary findings.Commercial Relationships: Maria Eugenia Vola Ravina, None;Renato Lisboa, None; Patricia Schimchak, None; Kody J. Kishi,None; Natalie A. Afshari, None; David J. Schanzlin, RefocusGroup (C), Oasis Medical (C)Program Number: 3125 Poster Board Number: D0060Presentation Time: 8:30 AM - 10:15 AMVisual Performance Comparison of Wavefront-optimized andWavefront-guided Laser in situ keratomileusis (LASIK)Kraig S. Bower 1 , Lamarr Peppers 2 , Rose K. Sia 2 , Richard D.Stutzman 3 , Joseph F. Pasternak 3 , Denise S. Ryan 2 , Edward W.Trudo 2 . 1 Ophthalmology, Wilmer Eye Institute, Lutherville, MD; 2 USArmy Warfighter Refractive Surg Research Ctr, Fort BelvoirCommunity Hospital, Fort Belvoir, VA; 3 Ophthalmology, WalterReed National Military Medical Center, Bethesda, MD.Purpose: To compare visual acuity and contrast sensitivity resultsafter wavefront-guided (WFG) and wavefront-optimized (WFO)LASIK.Methods: This was a prospective randomized study that comparedvisual acuity (VA) and contrast sensitivity (CS) before and aftereither WFG (n=18) or WFO (n=17) LASIK. Subjects were matchedfor preoperative refractive error (mean spherical equivalent = -3.28±1.33D) and age (31.6 ±8.1 years) WFG surgeries were performedusing the VISX Star S4 (Abbott Medical Optics) and WFO surgerieswith the Wavelight Allegretto Wave Eye-Q (Alcon Surgical).Corneal flaps were created using the Intralase femtosecond lasersystem (Abbott Medical Optics). Best corrected VA and small letter(20/25) CS were measured with the Super Vision back-illuminatedletter chart (PrecisionVision®; PV) and low luminance, night visionassessed with the PV 25% Low Contrast Chart viewed through a darkgreen night vision filter. Measurements were obtained at baseline andat 1, 3 and 6 months postoperatively. Repeated measures ANOVAwas used to compare WFO vs. WFG LASIK over time.Results: There was no significant difference in ablation depth (WFG:49.9 ±11.9µ; WFO 57.6 ±21.7µ; p=0.20). All subjects retainedexcellent high contrast VA (20/16) with no difference between WFGand WFO, while CS improved slightly in the WFG group (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaWFO LASIK in terms of night vision performance and low contrastacuity.(1:5,970). This is similar or lower than previous reports (1:1,000 to1:5,000). While there were relatively few microbial cases, severalassociations were observed and include male gender (1:4,429),preoperative myopia (1:5,501), undergoing a surface ablationprocedure (1:3,161) and the use of a mechanical keratome for theLASIK flap creation (1:3,938).Commercial Relationships: Steven C. Schallhorn, AMO (C),Allergan (C); Julie M. Schallhorn, None; Adenay Padilla, NoneTable 1. Acuity and Contrast means± SD. P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMalvern, PA) was performed before surgery and one month after thesame, in order to obtain the postsurgical corneal tissue ablation bysubtraction. All surgeries were performed by different surgeons usingthe same microkeratome (M2 single use microkeratome, Moria Inc.,Doylestown, PA). The corneal ultrasonic pachymetry was takencentrally by the same technician. Measurement was performed oneach eye nine times and mean was reported.Results: Data from 26 patients (52 eyes) who underwent LASIKwere available for the initial analysis. We analyzed a total of 19patients (36 eyes). Seven patients were excluded because they did notcome to follow-up visits and two eyes were excluded because ofsurgical complications. The mean age of the analyzed subjects was25.89 years (range 20-42 years). The mean predicted corneal tissueablation depth was 49.11 μm (standard deviation [SD], 19.60 μm)and the mean postsurgical corneal tissue ablation depth was 61.64 μm(SD, 28.07 μm). The mean preoperative spherical equivalent was -4.07 D (SD, 1.92 D) and the mean postoperative spherical equivalentwas 0.18 D (SD, 0.73 D). We analyzed the variables under study witht test (p< 0.001).Conclusions: The postsurgical corneal tissue ablation depth wasgreater than the predicted corneal tissue ablation depth determined bythe platform used in this study, and it should be considered byrefractive surgeons in patients with thin corneas in order to avoidresidual beds of less than 300 μm.Commercial Relationships: Juan A. Curiel, None; CristinaPacheco-Del-Valle, None; Oscar Baca, None; Alejandro Babayan,None; Regina Velasco, NoneProgram Number: 3129 Poster Board Number: D0064Presentation Time: 8:30 AM - 10:15 AMBilateral Implantation of Hydrogel Corneal Inlays in HyperopicPresbyopesAdam J. Roy 1 , Alan J. Lang 1 , Tonya Porter 1 , Keith Holliday 1 , GuruSharma 1 , Arturo Chayet 2 , Edna Favela 1 , Enrique Barragan 3 , SandraGomez 3 . 1 R & D, ReVision Optics, Inc., Lake Forest, CA; 2 CodetVision Institute, Tijuana, Mexico; 3 Laser Ocular Hidalgo, Monterrey,Mexico.Purpose: To provide hyperopic, presbyopic patients with improvednear, intermediate, and distance vision using bilateral hydrogelcorneal inlays.Methods: Twenty-two hyperopic (mean pre-op spherical equivalent(MRSE): 0.99 D (non-dominant eye) and 0.97 D (dominant eye)[+0.25 to +1.75 D]), presbyopic (mean pre-op reading addrequirement: 1.89 D (both eyes) [+1.50 to +2.25 D]) subjects wereimplanted with 2.0 mm diameter hydrogel corneal inlays (ReVisionOptics, Inc.)* under a femtosecond corneal flap, in the non-dominanteye, followed for 3 to 6 months, and then implanted with a like inlayin the dominant eye. The Optec® 6500 Vision Tester was used torecord visual acuities. Ability to perform everyday tasks (five tasksfor each of three distance ranges) without additional visual aid wasascertained using a questionnaire. The study was performed inconformance with an IRB-approved protocol.Results: The non-dominant (NONDOM) and dominant (DOM) eyesin the same subject responded similarly to the corneal inlay. In botheyes measured separately, uncorrected visual acuity (UCVA) hadimproved significantly at 3 months, by an average of 5 lines of near,3 lines of intermediate, and 1 line of distance vision. Near andintermediate vision improved in 100% of the eyes. 86% of implantedNONDOM eyes and 95% of DOM eyes achieved 20/25 or better nearUCVA compared to 0% preoperatively. 77% of implantedNONDOM eyes and 86% of DOM eyes achieved 20/25 or betterintermediate UCVA compared to 0% preoperatively. Both groupshad an average improvement of 1 line distance UCVA though twosubjects lost one line of distance vision in the NONDOM eye. Theaverage distance UCVA for each eye was 20/21. At 3 months afterthe second inlay, binocularly, 95% of subjects achieved 20/25 orbetter near UCVA and intermediate UCVA, and 100% of subjectsachieved 20/20 or better distance UCVA. There was a significantimprovement in task performance and patient satisfaction withbilateral inlays in comparison to pre-op. At 3 months after the 2ndimplantation, 95% of subjects were satisfied or very satisfied withtheir overall vision.Conclusions: Bilateral implantation of the ReVision Optics cornealinlay is potentially an effective tool to improve near, intermediate,and distance vision in hyperopic presbyopes up to +1.75 D MRSE.*CAUTION: Investigational device. Limited by Federal (UnitedStates) law to investigational use.Commercial Relationships: Adam J. Roy, Revision Optics (E);Alan J. Lang, ReVision Optics (E); Tonya Porter, ReVision Optics(E); Keith Holliday, ReVision Optics (E); Guru Sharma, None;Arturo Chayet, None; Edna Favela, Revision Optics (E); EnriqueBarragan, revision optics (C); Sandra Gomez, Revision Optics (C)Program Number: 3130 Poster Board Number: D0065Presentation Time: 8:30 AM - 10:15 AMNear Functional Range of a Near Center Hydrogel Corneal Inlayin Presbyopic SubjectsAlan J. Lang 1 , Adam J. Roy 1 , Keith Holliday 1 , Tonya Porter 1 , GuruSharma 1 , Arturo Chayet 2 , Edna Favela 1 , Enrique Barragan 3 , SandraGomez 3 . 1 R & D, ReVision Optics, Lake Forest, CA; 2 Codet VisionInstitute, Tijuana, Mexico; 3 Laser Ocular Hidalgo, Monterrey,Mexico.Purpose: Explain with clinical data and ray-trace simulations how anintracorneal inlay provides a functional near range, independent ofage (Add) and improves intermediate VA.Methods: 192 emmetropes / low-hyperopes (MRSE: -0.50 to +1.75D), presbyopic (Add 1.50 to +2.50 D), subjects were implanted witha 2 mm diameter hydrogel corneal inlay (RaindropTM Near VisionInlay, ReVision Optics)*, in the non-dominant eye. The Optec® 6500recorded visual acuities. Everyday task performance (five tasks eachdistance) was ascertained via self-questionnaire. The studies wereeach conducted according to an IRB-approved protocol. Zemaxsimulated letter charts yielded near range for fixed VA levels, as afunction of pupil size and spectacle defocus, using the inlay effectderived from post-pre wavefront difference maps in a finite eyemodel. The inlay effect ranged from -4 D at pupil center, returning tothe unaltered anterior cornea at 4 mm diameter.Results: The mean preop uncorrected near visual acuity (UCNVA) islogMAR 0.51 and by 3 months mean UCNVA is logMAR 0.06(p 6points: p 2levels: p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapower ≥ 2.5 D improves near task performance and VA, regardless ofage (Add), with high patient satisfaction. The small myopic shift ofdistance Rx potentially improves intermediate VA.*CAUTION: Investigational device. Limited by Federal (UnitedStates) law to investigational use.Commercial Relationships: Alan J. Lang, ReVision Optics (E);Adam J. Roy, Revision Optics (E); Keith Holliday, ReVision Optics(E); Tonya Porter, ReVision Optics (E); Guru Sharma, None;Arturo Chayet, None; Edna Favela, Revision Optics (E); EnriqueBarragan, revision optics (C); Sandra Gomez, Revision Optics (C)Program Number: 3131 Poster Board Number: D0066Presentation Time: 8:30 AM - 10:15 AMLonger-term Stability of Refractive Corneal Lenticule ExtractionProcedures Compared with LASIKJesper Hjortdal, Iben Bach Pedersen, Anders Ivarsen.Ophthalmology, Aarhus University Hospital, Aarhus, Denmark.Purpose: Short-term follow-up studies have shown that thepredictability, efficacy, and safety of all-femtosecond laser basedcorneal refractive procedures for correction of myopia are similar toLASIK. The purpose of the present clinical quality control study wasto evaluate the longer-term stability of these procedures.Methods: 90 patients operated on both eyes with femtosecondLASIK (LASIK), femtosecond lenticule extraction (FLEX), or smallincision lenticule extraction (SMILE) participated in a followexamination more than one year after surgery. All patients had beentreated for moderate to high myopia (-11.00 to -4.50 D of sphericalequivalent refraction) in both eyes. 3 months after surgery and at thefollow-up visit more than one year after surgery, uncorrected visualacuity (UCVA), manifest refraction, and best corrected visual acuity(BCVA) was measured and changes in refractions, UCVA, andBCVA were calculated.Results: The average preoperative spherical correction was -7.00 D,cylinder was -0.62 D, and spherical equivalent correction (SEQ) was-7.28 D. There were no significant differences in refractions orBCVA between the three groups before surgery. Post-operativerefractive results at 3 months after surgery are shown in Table 1.Changes in UCVA, BCVA, and SEQ were small from 3 months tomore than one year of surgery (Table 2). ANOVA tests revealed nosignificant change in SEQ over the longer-term period after the 3types of surgery, and there was no significant difference betweengroups. UCVA improved significantly over the longer-term period ineyes treated with FLEX and SMILE, but not after LASIK. Post hoctests showed that eyes operated with SMILE improved significantlymore than eyes treated with LASIK. BCVA improved in all groupsfrom 3 months to more than one year after surgery. Post hoc tests didnot reveal any significant difference between the treatments.Conclusions: Spherical equivalent refraction was stable from 3months after surgery. Uncorrected and best spectacle corrected visualacuity improved from 3 months to years after surgery. The longertermstability of the recently introduced all-femtosecond laser basedprocedures seems similar to femtosecond-LASIK, and uncorrectedvisual acuity may even improve more over time in eyes operated withSMILE compared with LASIK.Table 1. 3 months Post-OP. Mean (SD)Table 2. Longer-term changes. Mean (SD)Commercial Relationships: Jesper Hjortdal, Carl Zeiss Meditec(R); Iben Bach Pedersen, None; Anders Ivarsen, NoneProgram Number: 3132 Poster Board Number: D0067Presentation Time: 8:30 AM - 10:15 AMModeling of Human Refractions for Refractive SurgeryGuang-ming G. Dai. R & D, Abbott Medical Optics, Milpitas, CA.Purpose: To investigate the statistical distribution of sphere andcylinder for refractive surgical candidates.Methods: Based on population statistics for 393,139,704 eyes in a2006 national survey and a refractive surgery patient database for8,246,757 eyes performed in the US until 2006, a statistical analysiswas performed. For refractive sphere, a normal distribution is areasonable measure for virgin eyes. For refractive surgicalcandidates, however, a Rayleigh distribution better represents thestatistics as emmetropic patients do not seek refractive surgery(except for presbyopes). For refractive cylinder, a normal distributionis a reasonable measure for both the virgin eyes and refractivesurgical candidates. These statistical models were used to generaterandom eyes for scientific research or commercial use.Results: One million random normal eyes and one million refractivesurgical eyes were generated based on the proposed algorithms.Roughly 79% eyes are myopic, 13% are hyperopic and 7% are mixedastigmatic. The statistics of the simulated eyes follow the statisticsassumed. This allows a “real-world” testing, or a “simulated clinicaltrial” for the development of new treatment algorithms, orverification and validation of to-be-released treatment softwareproducts, greatly speeds up the process and improves the efficiencyfor product development in a commercial environment. It may alsobe used in the vision research when a large number of eyes arerequired for statistical analysis.Conclusions: The refractions of human eyes (sphere and cylinder)follow the proposed statistical distribution based on a large nationaloptometric database. Simulation based on the statistics provides auseful means for vision research and commercial use.Commercial Relationships: Guang-ming G. Dai, Abbott MedicalOptics (E)Program Number: 3133 Poster Board Number: D0068Presentation Time: 8:30 AM - 10:15 AMEvaluation of Limbal Ultrastructure in Twelve Year and TwentyeightYear Post-operative Keratoconic CorneasErin Dooley 1 , Steven Gardner 1 , Sally Hayes 1 , Jonathan Harris 1 , KimNielsen 3 , Jesper Hjortdal 3 , Thomas Sorensen 2 , Nicholas Terrill 2 ,Craig Boote 1 , Keith M. Meek 1 . 1 Biophysics, Cardiff UniversitySchool of Optometry and Vision Sciences, Cardiff, United Kingdom;2 Diamond Light Source, Didcot, United Kingdom; 3 Department ofOphthalmology Aarhus University Hospital, Arhus, Denmark.Purpose: To use X-ray scattering techniques to analyse peripheralcorneal and limbal, tissue from a patient with advanced keratoconouswho underwent bilateral penetrating keratoplasty twelve and twentyeightyears prior.Methods: Peripheral cornea/limbal tissue of the enucleated corneaswas evaluated using small and wide angle X-ray scatteringtechniques at synchrotron stations I22 and I02 Diamond LightSource, Didcot, UK. The corneas were wrapped in clingfilm toprevent dehydration and placed in a purpose built Perspex and Mylarchamber during x-ray exposure. The corneas were sampled at 0.5 mmintervals in a 29 x 29 mm grid (small angle) and at 0.25 mm intervalsin a 60 x 67 and 60 x 63 grids (wide angle). Normal human corneaswere also scanned in the same manner as controls. Wide-anglepatterns were used to quantify collagen orientation and small-anglepatterns to measure fibril diameter and spacing.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: In both KC corneas, small angle analysis demonstrated adisordered ring of collagen fibril spacing in the peripheralcornea/limbus where an additional equatorial peak was evidentconsistently at ~80 and ~120 nm. This double peak, which indicatestwo populations of fibrils each with a different interfibril spacing,was observed only slightly in the corresponding limbal region of thenormal tissue ~5mm from centre. In addition, both corneasdemonstrated regions of peripheral corneal opacity which appeared tocorrespond with additional high scatter peaks ~140 nm. Wide angleanalysis of the KC peripheral tissue demonstrated unique concentricrings of preferential collagen orientation which were not observed inthe normal corneas.Conclusions: There were clear and evident changes in the cornealultrastructure in both keratoconus specimens. The causes of thechanges are unknown and it is not possible to conclude if they are aresult of keratoconus pathology or have occurred during the periodfollowing corneal implant. However, this is the first demonstration ofstructural changes in the periphery and limbus of the keratoconuscornea.Commercial Relationships: Erin Dooley, None; Steven Gardner,None; Sally Hayes, None; Jonathan Harris, None; Kim Nielsen,None; Jesper Hjortdal, Carl Zeiss Meditec (R); Thomas Sorensen,None; Nicholas Terrill, None; Craig Boote, None; Keith M. Meek,NoneSupport: Medical Reserach CouncilProgram Number: 3134 Poster Board Number: D0069Presentation Time: 8:30 AM - 10:15 AMEvaluate the safety and efectiveness of the AcrySof Phakic Anglesupported intraocular Lens for the correction of high myopiaMireya G. Arellano. Fundacion Hospital Nuestra Señora de la Luz,Distrito Federal, Mexico.Purpose: To evaluate the safety and effectiveness of the AcrySofCachet Phakic angle-supported Intraocular Lens (pIOL) for thecorrection of high myopia.Methods: Retrospective and prospective study including patientswith high myopia who had implantation of the Acrysoft Cachet pIOL(Alcon Laboratories, Inc., Fort Worth, TX). The outcome measuresincluded uncorrected visual acuity (UCVA), best corrected visualacuity (BCVA), endothelial cell density was measured withTOPCON specular microscope SP-2000P (taking 5 measures at thecenter of the cornea and 25 contiguous cells) , contrast sensitivity(SWCT chart) and adverse effects. The statistical study was madewith Wilcoxon test.Results: We included evaluated 14 eyes of 9 patients with ages from22 to 53 years, mean 34.1 years; 66.6% were female and 33.3% male.The mean follow up period was 29 months (range 9 to 38 months),with a mean preoperative spherical equivalent of -14.39, rangingfrom -10.25 to -19.25. The mean postoperative spherical equivalentwas -0.5, ranging from -3.25 to +0.62. The mean postoperativeUCVA was 20/30 (LogMar 0.17) or better and the BCVA 20/25 (logMar 0.098 or better. The contrast sensivity was made with CDVA,considering the low, medium or high spatial frequency, mean spatialfrequencies were 6.28, 5.07 and 3.78 cycle per degree respectively.The change of the mean preoperative endothelial cell density from2763.4 cells to a mean postoperative cell density of 2626.9, wasstatistically significant (p=0.013). Only one patient was steroidhiperreactor, which was normalized after it was suspended. None ofthe patients showed IOP raised in time or other adverse effects.Conclusions: AcrySof Cachet showed an effective alternative for thecorrection of high myopia, it is consistent with other publishedreports of phakic IOLs, where the UCVA and BCVA were excellent.None of the patients had significant adverse effects. Howeverendothelial cell loss over time was statistically significant, which hasbeen attributed to the proximity of anterior chamber pIOL to theendothelium, the surgery itself and physiology decrease with aging.An important variable to consider is that the procedure was made inour Hospital which has residents in training. Additional follow-upwill be necessary to elucidate the long-term effectsCommercial Relationships: Mireya G. Arellano, NoneProgram Number: 3135 Poster Board Number: D0070Presentation Time: 8:30 AM - 10:15 AMPatient Satisfaction and Quality of Vision after Wavefrontguided(WFG) vs. Wavefront-optimized (WFO) Photorefractivekeratectomy (PRK)Denise S. Ryan 1 , Rose K. Sia 1 , Richard D. Stutzman 2 , Joseph F.Pasternak 2 , Lamarr Peppers 1 , Jennifer B. Eaddy 1 , Lorie A. Logan 1 ,Edward W. Trudo 1 , Kraig S. Bower 3 . 1 US Army WarfighterRefractive Surg Research Ctr, Fort Belvoir Community Hospital, FortBelvoir, VA; 2 Ophthalmology, Walter Reed National MilitaryMedical Center, Bethesda, MD; 3 The Wilmer Eye Institute, JohnsHopkins University, Baltimore, MD.Purpose: To compare higher order aberration (HOA) root meansquare (RMS) and patient satisfaction of postoperative vision afterWFG vs. WFO PRK.Methods: Participants randomized to receive WFG PRK (VISX StarS4, Abbott Medical Optics) or WFO PRK (Wavelight AllegrettoWave Eye-Q, Alcon Surgical) underwent testing to determine theirHOA RMS preoperatively and at 6 months (M) postoperatively. RMSHOA were analyzed at four different pupil sizes (4, 5, 6, and 7mm)using Complete Ophthalmic Analysis System (COAS, AbbottMedical Optics). A repeated measures analysis of variance (RM-ANOVA) was used to compare WFG vs. WFO PRK HOA RMS ateach pupil size over time. Participants responded to a questionnairepreoperatively and 6M postoperatively. A Mann-Whitney test wasused to compare patient satisfaction of postoperative vision. A p-value

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFigure 1. WFG vs. WFO PRK higher order aberrations (HOA) rootmean square (RMS)Response Analyser. Furthermore 30 healthy persons between 20-30years were measured as a control group.Results: There were no significant differences in IOPcc between the4 groups. As DA and DR correlated linearly with CCT, DA and DRwere adjusted to a corneal thickness of 476 µm in all groups.DR in all refractive groups was significantly lower than the controlgroup, but no significant differences were found between therefractive groups (Table 1). LASIK had a significantly lower DA,when compared with SMILE and the control group. There was nosignificant difference in DA between SMILE and the control group(Table 2).Conclusions: The low DR in all refractive groups indicates that laserrefractive surgery results in higher corneal elasticity despite adjustingfor differences in CCT. The significantly lower DA in LASIK couldbe a result of stress-stiffening appearing in untouched collagens. Asmore collagens are left untouched after flap-free laser treatments(SMILE), the stress on each collagen fiber becomes less. A DA moresimilar to the control group may support, that SMILE is a morebiomechanically neutral corneal laser refractive procedure.Table 1. Patient satisfaction and quality of vision questionnaireresults.Commercial Relationships: Denise S. Ryan, None; Rose K. Sia,None; Richard D. Stutzman, None; Joseph F. Pasternak, None;Lamarr Peppers, None; Jennifer B. Eaddy, None; Lorie A. Logan,None; Edward W. Trudo, None; Kraig S. Bower, NoneSupport: Dept of Defense W81XWH-09-2-0018Clinical Trial: NCT01097525Program Number: 3136 Poster Board Number: D0071Presentation Time: 8:30 AM - 10:15 AMComparing the Corneal Biomechanical Stability after LASIK,ReLEx FLEx and ReLEx SMILE with Ultra High Speed Camera(Corvis® ST)Iben Bach Pedersen, Sashia Bak-Nielsen, Anders Ivarsen, JesperHjortdal. Ophthalmology, Aarhus University Hospital, Aarhus,Denmark.Purpose: Laser refractive surgery involves removal of corneal tissue.Modern flap-free laser treatments of the cornea (SMILE) mayweaken the cornea to a lesser extent than flap based treatments (FLExand LASIK). With the new device, Corvis ST from Oculus, it ispossible to measure high-speed pictures of the corneal deformationduring an air-pulse. With the deformation amplitude (DA) and thedeformation radius (DR) at highest concavity, changes in the cornealrigidity after laser refractive surgery may be compared.Methods: Patients treated for high myopia (-9,5 to -4,5 D) more thanone year ago were invited for a follow up examination. Eighty-sevenpatients operated on both eyes participated, and were divided into 3refractive groups:Laser-Assisted In Situ Keratomileusis, LASIK (30 patients)Femtosecond Lenticule Extraction, ReLEx-FLEx (30 patients)Small Incision Lenticule Extraction, ReLEx-SMILE (27 patients)The average uncorrected visual acuity, best corrected visual acuityand IOP was similar in all refractive groups. With Corvis ST, thecentral corneal thickness (CCT), DA and DR was measured, andcorneal corrected IOP (IOPcc) was measured with the OcularTable 1: Mean DR with 95% CI.Table 2: Mean DA with 95% CI.Commercial Relationships: Iben Bach Pedersen, None; SashiaBak-Nielsen, None; Anders Ivarsen, None; Jesper Hjortdal, CarlZeiss Meditec (R)Program Number: 3137 Poster Board Number: D0072Presentation Time: 8:30 AM - 10:15 AMFemtosecond (FS) Laser Techniques to Facilitate Deep AnteriorLamellar Keratoplasty (DALK)Perry S. Binder 1, 2 , Roger F. Steinert 1, 2 , James E. Hill 2 , Michael A.Campos 2 . 1 Gavin Herbert Dept of Ophthal, Univ of California, IrvineCA, San Diego, CA; 2 Abbott Medical Optics Inc., Santa Ana, CA.Purpose: To develop and study femtosecond laser techniques andsettings that facilitate deep stromal resections for DALK.Methods: Three approches in human eye bank eyes were used tocreate smooth beds for DALK using a 150 kHz FS laser: Procedure1) Debulking (N=12) removing 300 µm @ 9.0 mm (3x3 spot/line SL,0.6 µJ) followed by 100 µm resection (2x2 or 3x3 SL, 0.6 µJ);Procedure 2) Deep single or double raster pass (N=3) @ 7.0 mmdiameter, 8x8 SL, 0.3 µJ); Procedure 3) Multiple raster passes usingtwo spiral and then 2 raster patterns using the same patient interface,followed by a raster pattern with a side cut (N= 8) forcing the FSproducedgases to dissect Descemet’s membrane (DM) away fromthe stroma (7.0 mm diameter, 8x8 SL, 900 side cut 1.6 µJ, 2.1 µJraster). The corneas were then fixed in glutaraldehye and prepped forLM, SEM, and TEM.Results: Procedure 1 created a bed as smooth as more superficial FSlaser dissections, but left a significant thickness of posterior stroma inplace. Procedure 2 removed more stroma than #1, but it was not as©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasmooth and still left significant stroma behind. Procedure 3 removedthe most stroma and in two cases left an almost bare DM behind, butthe edges of the resection were rougher than the other procedures.One case of perforation of DM occurred. There was no morphologicdamage to the endothelium.Conclusions: FS laser-created deep lamellar beds are not normally assmooth as < 120µm flaps, but the debulking and single or doubleraster passes can create smooth beds. The multiple raster passtechnique, dubbed the “Little Bubble Technique” was able to bareDM and remove the most stroma, but has the risk of perforation ofDM similar to the “Big Bubble Technique”. It appears to have a morepredictable depth resection and does not require mechanical airinjection. Modifications of SL, energy settings, side cut angles andside cut energy may be more predictable in baring DM.Commercial Relationships: Perry S. Binder, Abbott MedicalOptics inc. (C); Roger F. Steinert, Abbott Medical Optics (C),OptiMedica (C), ReVision Optics (C), WaveTec (C); James E. Hill,Abbott Medical Optics (E); Michael A. Campos, Abbott MedicalOptics (E)Program Number: 3138 Poster Board Number: D0073Presentation Time: 8:30 AM - 10:15 AMRefractive surgery evaluation and risk of corneal ectasia, interrateragreement and comparison to a new software: the SCOREanalyzerFlorence Cabot 1 , Sonia H. Yoo 1 , Alain Saad 2 , George Kymionis 3 , AnaPaula Canto 1 , Damien Gatinel 2 . 1 Ophthalmology, Bascom PalmerEye Institute, Miami, FL; 2 Ophthalmology, Rothschild Foundation,Paris, France; 3 Ophthalmology, University of Crete, Heraklion,Greece.Purpose: To assess the level of agreement between 5 refractivesurgeons and a new software in detecting corneal ectasia risk duringrefractive surgery evaluation.Methods: Retrospective multicenter study including 168 eyes of 84patients. Three attending ophthalmologists subspecializing inrefractive surgery and 2 cornea fellows reviewed 168 Orbscan(Bausch and Lomb, Rochester, NY) topographic quadmaps extractedfrom the Bascom Palmer Eye Institute refractive evaluation database.They rated the maps and determined whether a refractive surgery(Laser in situ keratomileusis [LASIK], or Photorefractivekeratectomy [PRK]) was advisable or not. Topographic maps weredivided in 3 different groups according to the followingclassification: LASIK or PRK (group 1), PRK only (group 2), Nosurgery (group 3). Only topographic indices were used to classify themaps. Age, daily activities, family history, symmetry between botheyes and refraction were not taken into account to rate the maps. Thesame maps were also screened by a new corneal ectasia risk detectionsoftware combined with the Orbscan: the SCORE analyzer(Technnolas Perfect Vision). Multirater kappa statistics were used toassess inter-rater agreement.Results: 43.1% and 58.9% of topographic maps were classified ingroup 1 ; 37.1% and 2.9% in group 3 by cornea fellows. 75,6% ,46.2% and 55% of topographic maps were classified in group 1 ;13.2% , 18.7% and 1.8% in group 3 by subspecialized attendingophthalmologists. 57.7% of topographic maps were classified ingroup 1 and 9.6% in group 3 by the SCORE analyzer (TechnolasPerfect Vision, Munich, Germany). Inter-rater agreement was slightto fair compared with that expected by chance: the overall rate ofagreement was 56% and the fixed marginal kappa coefficient was0.24.Conclusions: The inter-rater agreement between experiencedrefractive surgeons with different backgrounds is relatively low.Using the SCORE analyzer as an objective assessment in determiningthe patient’s candidacy for refractive surgery is valuable. Age,refraction, symmetry between eyes and family history are otherfactors that may be incorporated into the SCORE analysis in thefuture to make the assessment more robust.Commercial Relationships: Florence Cabot, None; Sonia H. Yoo,Alcon Labs (C), Carl Zeiss Meditec (C), Bausch and Lomb (C),Trascend Medical (C), Allergan (C), Optimedica (C), University ofMiami (P); Alain Saad, Acufocus (C); George Kymionis, None;Ana Paula Canto, None; Damien Gatinel, Technolas (C)Program Number: 3139 Poster Board Number: D0074Presentation Time: 8:30 AM - 10:15 AMThe effect of high order aberrations on the accuracy of wavefrontablations using LASEK on high myopesJeff G. Grigsby 1, 3 , Kathy Vasquez 3 , Michael Tschoepe 4, 3 , Thomas R.Walters 5, 3 , Billy Cook 2 , Robert G. Sheets 2 , Dennis K. Neely 2 .1 Redwine Research, LLC, Midland, TX; 2 VisionHealth Specialties,Midland, TX; 3 Eye LASIK Midland, Midland, TX; 4 RealEyes VisionCenter, New Braunfels, TX; 5 Eye LASIK Austin, Austin, TX.Purpose: LASEK is used to correct myopia, hyperopia, astigmatismand high order aberrations (HOA) such as coma, trefoil and sphericalaberration. It is unclear how these pre-operative HOA affect theaccuracy of the final refractive result in LASEK. Additionally,Teus,et al., 2007 proposed that LASIK nomograms may overcorrecthigh myopes receiving LASEK.Methods: A retrospective analysis of 113 eyes of 69 patients whohad LASEK performed between 2005-2010 using either a standard orCustomvue© ablation was performed. Subjects had at least -6.00diopters of sphere pre-operatively. The standard ablation grouputilizing physician adjustments included 63 eyes averaging -7.49 Dsphere and average astigmatism of -1.41 D. The Customvue©ablation group utilizing physician adjustments included 50 eyesaveraging -6.56 D sphere and average astigmatism of -0.63D. Theuse of intra-operative mitomycin C was slightly higher in thestandard vs. the Customvue© group (74% vs 54%), but was notdifferent between the pre-operative < 0.1000μ normalized polarZernicke coefficient (NPZE) and the > 0.1000μ NPZE Customvue©spherical aberration groups. Refractions were performed at 1, 3, 6and 12 mths post-operatively.Results: The mean spherical equivalent post-operative refraction wasnot significantly different between the two groups (standard groupmean +0.07 D, std. deviation 0.62 D; Customvue© +0.15 D, 0.58 D).When pre-operative physician adjustments were removed from finalCustomvue© refractive results, 32% of Customvue LASEK eyeswould have had a final result of ≧ ±0.50 D. 23 of the 50 eyes had apre-operative positive spherical aberration >0.1000μ NPZE. Withouta physician adjustment these 23 eyes would have been responsible for69% of the eyes with post-operative refractions ≥ ±0.50 D. Themeans were similar between the eyes with pre-operative sphericalaberration < 0.1000μ NPZE and those > 0.1000μ NPZE (-0.0136,0.0413 D), but the variances were significantly different (0.1388 vs.0.5681 D, p 0.1000μ NPZE puts the Customvue© patient athigher risk of enhancement unless adequately compensated with anappropriate physician adjustment.Commercial Relationships: Jeff G. Grigsby, None; KathyVasquez, None; Michael Tschoepe, None; Thomas R. Walters,None; Billy Cook, None; Robert G. Sheets, None; Dennis K. Neely,None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 3140 Poster Board Number: D0075Presentation Time: 8:30 AM - 10:15 AMMonocular refractive surgery in patients with anisometropiaAlejandro Tamez, Julio C. Hernandez-Camarena, Juan F. Lozano-Ramirez, Guillermo Mendoza, Alejandro Rodriguez Garcia, Jorge E.Valdez. Tecnologico de Monterrey, Monterrey, Mexico.Purpose: To investigate the efficacy and safety of LASIK for thecorrection of anisometropia inadult patients.Methods: A retrospective, case series. From a random sample of1190 patients from the cornea and refractive service we found 14adult patients that underwent monocular LASIK/PRK foranisometropia. We evaluated the preoperative and postoperativerefractive error, spherical equivalent (SE), uncorrected visual acuity(UCVA) and best corrected visual acuity (BCVA). Snellen visualacuity measurements were converted to LogMAR for statisticalpurposes.Results: The mean age was 33.2(±12.2) years. The averagepreoperative SE in the treated eyes was -1.96(±3.69), the average SEof the untreated eye was 0.25(±0.30). Preoperative UCVA was0.99(±0.34) and average preoperative BCVA was 0.26(±0.15). 13patients had LASIK and only one patient had PRK with an averagefollow-up time of 15.8(rank 1-74) months. The average postoperativeSE decreased to -0.24(±0.33)(p=0.11). Four patients (28.6%) gained1 line of vision, 1 (7.14%) patient gained 2 lines of vision, 1 patientlost 1 line of vision (PRK patient), the rest remained unchangedcompared to preoperative BCVA. There was a significant differencebetween UCVA pre [0.99(±0.34)] and postoperative [0.24(±0.13)](p0.05).At postoperative month 12, there were no statistically significantdifferences between the groups with respect to 5% and 25% contrastsensitivity (WFG vs. WFO: 0.23 ± 0.16 vs. 0.26 ± 0.16; 0.23 ± 0.16vs. 0.22 ± 0.08 logMAR; P=0.78, P=0.74, respectively), and all of thesubjective symptoms evaluated on the questionnaire (all P>0.05).Conclusions: There were no differences at 12 months in quality ofvision outcomes between WF guided and WF optimized LASIKusing the WaveLight® Allegretto Eye-Q 400 Hz excimer laserplatform in myopic patients with or without astigmatism.Commercial Relationships: Christopher Sáles, None; Edward E.Manche, Seros Medical, LLC (I), Calhoun Vision, Inc (I), GersonLehrman (C), Best Doctors (C)Clinical Trial: NCT01138189Program Number: 3142 Poster Board Number: D0077Presentation Time: 8:30 AM - 10:15 AMTear menisci and corneal subbasal nerve density in patients afterLaser In Situ KeratomileusisLiang Hu, Wenjia Xie, Dong Zhang, Jia Chen, Fan Lu. School ofOptometry & Ophthalmology, Wenzhou Medical College, Wenzhou,China.Purpose: To investigate the relationship between tear menisci andcorneal subbasal nerve density in patients after Laser In SituKeratomileusis (LASIK).Methods: Thirty-two low to moderate myopic eyes of 16 patientswere enrolled. The height (H) and area (A) of upper (U) and lower(L) tear menisci (TM), UTMH, UTMA, LTMH, and LTMA, weremeasured by real-time anterior segment OCT before and at 1 week, 1month, 3 months after surgery. Central, temporal and nasal cornealsubbasal nerve densities were measured by corneal confocalmicroscopy before and at 1 month, 3 months after surgery.Results: Tear menisci parameters and corneal nerve densities alldecreased significantly postoperatively (post hoc, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadensities. The amount of tear secretion after LASIK might depend onresidual corneal nerves at 1 month rather than corneal nerve recovery.Commercial Relationships: Liang Hu, None; Wenjia Xie, None;Dong Zhang, None; Jia Chen, None; Fan Lu, NoneProgram Number: 3143 Poster Board Number: D0078Presentation Time: 8:30 AM - 10:15 AMMicrokeratome-related complications in the first 10,000 lasikmarcos garcia 1 , Breno Barth 1, 3 , Walton Nosé 2 , Breno M. Azevedo 3 ,Gustavo Victor 1 , Milton R. Alves 1 . 1 Ophthalmology, University ofSao Paulo, Sao Paulo, Brazil; 2 Ophthalmology, Federal University ofSao Paulo, Sao Paulo, Brazil; 3 Potiguar University, Natal, Brazil.Purpose: To evaluate the incidence of microkeratome-relatedcomplications in making corneal flap in the first 10,000 surgeriesperformed by the same surgeonMethods: A series of cases of intervention, involving 5,387 patients(10,000 eyes) underwent LASIK surgery by the same surgeonbetween June 1998 and March 2010, at the same place andequipment. Patients who had complications related to themicrokeratome were selected for the study. The incidence ofcomplications related to making the corneal flap was calculated, aswell as the incidences of these complications in each1000 surgeriesResults: In 10,000 surgeries, there were 53 complications that led todiscontinuation of the LASIK procedure (0.53%). The incidence ofcomplications with the microkeratome was 1 in 188.6 surgeries(0.53%). In early 1000 the incidence of surgical complications was 1in 83.3 surgeries (1.2%). And the surgery number 6001-10000, theincidence of complication was 1 in 289.9 surgeries (0.36%)Conclusions: This study shows that there is a learning curve forusing the microkeratome and the incidence of complicationsassociated with making the corneal flap decreases as the surgeongains more experience. Complications were more frequent in the first1000 surgeries (1.2%), decreasing considerably and progressivelythereafterCommercial Relationships: marcos garcia, None; Breno Barth,None; Walton Nosé, None; Breno M. Azevedo, None; GustavoVictor, None; Milton R. Alves, NoneProgram Number: 3144 Poster Board Number: D0079Presentation Time: 8:30 AM - 10:15 AMLaser in situ keratomileusis following the implantation of anangle-supported phakic intraocular lensCRISTINA FERNANDEZ-VIGO ESCRIBANO 1 , Ana MacarroMerino 2 , José Ignacio Fernández-Vigo 3 , José Fernández-VigoLópez 2 . 1 CENTRO DE OFTALMOLOGIA BARRAQUER,BARCELONA, Spain; 2 CIOA UNIVERSIDAD DEEXTREMADURA, BADAJOZ, Spain; 3 HOSPITAL CLINICO SANCARLOS, MADRID, Spain.Purpose: Evaluate the safety, effectiveness, predictability, andstability of the combination of an angle-supported phakic intraocularlens (pIOL) implantation and laser-assisted in situ keratomileusis(LASIK) for correcting high myopia and astigmatism.Methods: 22 eyes of 14 patients with a preoperative sphericalequivalent (SE) between -7.00- and -19.75-diopters (D) were studied.Implantation of an angle-supported pIOL Acrysof Cachet® was doneas the first surgery. LASIK was performed at 3 months after pIOLsurgery, once stability of topography and refraction were proved.Main outcome measures were uncorrected visual acuity (UCVA),best-corrected visual acuity (BCVA), refraction, applanationtonometry and corneal endothelial study (cell density and coefficientof variation), with a minimum follow-up of 3 months after LASIK.Results: The mean SE refraction decreases from -11.06±3.25 Dbefore pIOL implantation to -0.68±0.66 D 3 months after pIOLsurgery and to -0.15±0.33 D 3 months after LASIK. Snellen DecimalUCVA was 0.53±0.16 3 months after pIOL surgery, increasing to0.84±0.20 3 months after LASIK. There was an increase in 20/40 orbetter UCVA from 66.66% after pIOL surgery to 95.23% afterLASIK addition. At the final follow-up, SE was within ±0.50 D ofemmetropia in 16 eyes (72.72%) and within ±1.00 Din 19 eyes(100%).The mean endothelial cell counts after 3 months after LASIKsuggest that no corneal endothelial damage was produced by LASIKitself.Conclusions: Combine an angle-supported pIOL implantation andLASIK appears to be safe, effective, predictable and a stableprocedure to correct eyes with high myopia with astigmatism.Commercial Relationships: CRISTINA FERNANDEZ-VIGOESCRIBANO, None; Ana Macarro Merino, None; José IgnacioFernández-Vigo, None; José Fernández-Vigo López, NoneProgram Number: 3145 Poster Board Number: D0080Presentation Time: 8:30 AM - 10:15 AMIdentification of microorganisms in water samples used inrefractive surgery facilitiesFrancisco B. Silva 1 , Cristina V. Niero 4 , Christiane L. Nogueira 2 ,Camilla P. Uzam 4 , James D. Lima Junior 2 , Fernando P. Pinto 3 ,Antonia Maria O. Machado 3 , Sylvia C. Leão 2 , Denise Freitas 1 , AnaLuisa Hofling-Lima 1 . 1 Ophthalmology, Federal University of SãoPaulo, São Paulo, Brazil; 2 Microbiology, Imunology andParasitology, Federal University of São Paulo, São Paulo, Brazil;3 Central laboratory, São Paulo Hospital, São Paulo, Brazil; 4 BiologySciences, Diadema Campus, Federal University of São Paulo, SãoPaulo, Brazil.Purpose: To identify the presence of microorganisms in differentsystems of water delivery to steamer devices used in sanitisationroutine procedure for laser in-situ keratomileusis in three differentrefractive surgery centersMethods: Environmental samples of water were collected after aroutine activity in three different refractive surgery centers, that usedthe same model of steamer. Two centers used tap water thatunderwent filtration and distillation, and was stored in non sterilegallons. Water samples were collected from the tap, filter hose,distillation outlet and collector, and storage gallons. The third centerused sterile distilled water and a water sample was collected from thewater bag. Samples from the steamer reservoir, hose and condensedwater from the steamer outlet were collected from all devices. Allsamples were then vacuum filtered through 0.45 µm nylonmembranes (Millipore), decontaminated by cetylpyridinium chloride0,05%(CPC) and plated on Middlebrook 7H10-OADC agar, 7H10-OADC-Panta and Löwenstein-Jensen medium. Acid-fast bacillidetected in culture were identified by PCR-Restriction EnzymeAnalysis of hsp65 gene (PRA-hsp65) and typed by Pulsed Field GelElectrophoresis (PFGE)-DraI. Other bacteria were identified byPhoenix Automated System.Results: In two centers, microorganisms were identified in differentsamples of water. In the first center, Mycobacterium chelonae wasisolated from the distilled water storage gallons and steamerreservoir. Mycobacterium mucogenicum was isolated from tap water.All M. chelonae isolated colonies showed the same PFGE pattern,confirming that a single strain was present in the distilled water andstorage gallons. Other aerobic bacteria were isolated from the storagegallons and steamer reservoir. In the second center, M. chelonae wasisolated in samples from the tap and filter hose, and other aerobicbacteria were found in water samples from the distillation outlet andcollector, storage gallons, steamer reservoir and hose. Samplescollected from the third center, that used sterile distilled water,showed no evidence of microorganisms growth after cultures.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConclusions: Our results show that the use of distilled tap water insanitisation care of surgical instruments could be a potential source ofcontamination. The methodology applied to this study wasappropriate to identify aerobic, anaerobic and mycobacteria.Commercial Relationships: Francisco B. Silva, None; Cristina V.Niero, None; Christiane L. Nogueira, None; Camilla P. Uzam,None; James D. Lima Junior, FAPESP (F); Fernando P. Pinto,None; Antonia Maria O. Machado, None; Sylvia C. Leão, None;Denise Freitas, None; Ana Luisa Hofling-Lima, NoneProgram Number: 3146 Poster Board Number: D0081Presentation Time: 8:30 AM - 10:15 AMComparison of Optical Quality in Low Myopic and ModerateMyopic Patients Operated With PRK and FemtoLASIKEric Perez-Campagne, Hana Landoulsi, Damien Gatinel. Cataract &Refractive Surgery, Rothschild Foundation, Paris, France.Purpose: The purpose of this study is to compare objectively thequality of vision assessed by double-pass imaging in low myopic andmoderate myopic patients operated with PRK and FemtoLASIK.Methods: Prospective comparative study of patients who underwentbilateral refractive surgery. Inclusion criteria were:

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea3 is a herpes virus entry mediator. Incubation of HCLE cells withcorneal transmembrane mucin isolates, but not with albumin—aprotein control lacking affinity for galectin-3—decreased viralinfectivity. Competition binding assays revealed that HSV-1 failed toelute the biological counter-receptor MUC16 from galectin-3 affinitycolumns.Conclusions: Our results suggest that HSV-1 can infect cornealepithelial cells using galectin-3, a component of the ocular surfacebarrier, as a cellular receptor. Transmembrane mucins in cornealepithelial cells prevent infection by limiting herpesvirus access togalectin-3 on the apical glycocalyx.Commercial Relationships: Pablo Argueso, None; JeromeMauris, None; Ashley M. Woodward, NoneSupport: NIH Grant EY014847Program Number: 3218Presentation Time: 11:15 AM - 11:30 AMIdentification and Validation of PNN-regulated Splicing Eventsin Human Corneal Epithelial CellsStephen P. Sugrue, Jeong-Hoon Joo. Anatomy & Cell Biology,University of Florida, Gainesville, FL.Purpose: Conditional inactivation of Pnn in developing mousecorneal epithelium resulted in severe disruption in epithelialdifferentiation. Since PNN is found associated with a large number ofsplicing proteins and exerts influence on splice site selection inminigenes, we identified PNN-regulated splicing events in a cornealepithelial context and explored the relevance of PNN’s impact onalternative splicing of its target genes.Methods: Isoform-specific RT-PCR assays were performed oncontrol and PNN knockdown human corneal epithelial (HCET) cells.The level of alternatively spliced transcripts was quantified byChemiDocTM XRS+ and Image Lab Software Version 4.0.Results: PNN knockdown in HCET cells resulted in the increasedinclusion of entire intron 11 of FOXJ3, which leads to prematuretranslational termination and most likely nonsense-mediated decay(NMD) of alternatively spliced FOXJ3 transcripts. On the other hand,retained intron 9 of a transcription factor FAM50A upon PNNknockdown is not expected to cause a frame shift nor earlytermination, thus predicted to add 49 amino acids to FAM50A,highlighting the complexity of splicing-dependent mRNA qualitycontrol mechanism and the importance of precise regulation ofsplicing events. While an alternative cassette exon (24a) of a guaninenucleotide exchange factor, ECT2, is found to be a PNN-silencedexon, inclusion of intron 9 in GLT8D1 is determined to be enhancedby PNN. Most interestingly, our study clearly indicated PNN’sinvolvement in the regulation of alternative splicing of two essentialcomponents of the γ-secretase protein complex, which plays a centralrole in Alzheimer’s disease and Notch signaling pathway.Knockdown of PNN promotes inclusion of introns 3 and 15 inPSENEN (PEN-2) and NCSTN (nicastrin) transcripts respectively.Since Notch signaling has been shown to play a key role in thecorneal epithelial differentiation and maintenance, our findings onPNN’s involvement in the alternative splicing of two majorcomponents of γ-secretase protein complex may provide a valuableinsight not only to the PNN’s functional mechanism but also to themany aspects of corneal epithelial biology.Conclusions: Our study identifies an exciting panel of alternativelyspliced transcripts to be explored for their biological significance incorneal epithelial development and maintenance. (NIH Grant R01EY007883, P30 EY021721)Commercial Relationships: Stephen P. Sugrue, None; Jeong-Hoon Joo, NoneSupport: R01 EY007883, P30 EY021721Program Number: 3219Presentation Time: 11:30 AM - 11:45 AMSuture placement on the mouse cornea induces K8 and Muc5acexpression within K12+ corneal epithelial cellsMary Ann Stepp 1 , Gauri Tadvalkar 1 , Victor L. Perez 4 , Yaohong Tan 4 ,Sonali Ghosh 1 , James D. Zieske 3 , Albert Lee 2 , Vickery E. Trinkaus-Randall 2 , Ahdeah Pajoohesh-Ganji 1 . 1 Anatomy & RegenerativeBiology, George Washington University, Washington, DC;2 Biochemistry, Boston University School of Medicine, Boston, MA;3 Ophthalmology, Harvard/Schepen's Eye Research Institute, Boston,MA; 4 Ophthalmology, Bascom Palmer Eye Institute, Miami, FL.Purpose: The corneal epithelium is maintained by a stem cellpopulation that is bipotential and can give rise to both cornealepithelial cells and corneal goblet cells which are generated uponinjuries close to the limbus (Pajoohesh-Ganji, et al., Stem Cells,30:2032-2043, 2012). To investigate whether corneal goblet cellprogenitors are present in the central mouse cornea, we triggered animmune response by placing sutures in the central cornea and thenevaluated the corneal epithelium for the presence of corneal gobletcells.Methods: Adult BALB/c (5) and C57BL6 (4) mouse corneas weresutured as described for corneal transplantation studies studies (Danaand Streilein, IOVS, 37:2485-2494, 1996) and were allowed torespond to the sutures for 14 days. Sutures were classified as low orhigh risk depending upon the length of the suture thread left incontact with the corneal epithelium. After 14 days, mice weresacrificed, eyes fixed, and corneas processed for whole mountimmunofluorescence to localize K12, K8, and Muc5ac within thecorneal epithelium. Corneas were stained with the sutures in place.Results: Data show that sutures induce expression of goblet cellmarkers including K8 and Muc5ac within K12 positive cornealepithelial cells within 14 days after sutures are placed in the cornea inboth BALB/c and C57BL6 mice but the upregulation is more robustin BALB/c compared to C57BL6 mice. Similar upregulation is seenfor low and high risk sutures.Conclusions: The data obtained support the fact that the immuneresponse of the mouse cornea to a suture can induce the conversion ofcorneal epithelial and goblet progenitor cells located in the centralcornea into goblet cells. Since the corneas were not debrided, it isunlikely that the cells that generate the goblet cells migrate from thelimbus. We have shown previously that wounding the cornea near thelimbus upregulates corneal goblet cell differentiation on the ocularsurface. Additional studies are underway to determine whether theseevents can occur in rat corneas in vitro in organ culture and in humancorneal equivalentsCommercial Relationships: Mary Ann Stepp, None; GauriTadvalkar, None; Victor L. Perez, Alcon (C), Bausch & Lomb (C),Genentech (C), Cleveland Clinic Foundation (P), Alcon (F), Alcon(R); Yaohong Tan, None; Sonali Ghosh, None; James D. Zieske,None; Albert Lee, None; Vickery E. Trinkaus-Randall, None;Ahdeah Pajoohesh-Ganji, NoneSupport: RO1 EY008512 (MAS), RO1 EY005665 (JDZ). RO1EY06000 (VTR), R01 EY018624-04 (VLP), RPB Physician-ScientistAward (VLP), P30 EY014801 (VLP), Research to Prevent BlindnessUnrestricted Grant (VLP)Program Number: 3220Presentation Time: 11:45 AM - 12:00 PMCorneal Confocal Microscopy detects neuropathy beforeretinopathy and nephropathy in children with Type 1 Diabetes:A Preliminary Study©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMitra Tavakoli, Rayaz A. Malik. Centre for Endocrinology &Diabetes, Institute of Human Development, University ofManchester, Manchester, United Kingdom.Purpose: Early detection and prevention of long-term complicationsby maintaining good metabolic control is the key goal of paediatricdiabetes management. Whilst we have readily available diagnostictechniques for retinopathy (fundus photography) and nephropathy(UAER), there is no equally sensitive measure for diabeticneuropathy. The aim of the present study was to assess the utility ofin vivo corneal confocal microscopy (IVCCM) in identifying earlynerve damage in children with T1DM.Methods: 25 children with type 1 diabetes mellitus (average age:13±1 yrs; average duration of diabetes 8 years) with no evidence ofretinopathy or microalbuminuria and 10 aged matched controlsubjects underwent assessment with IVCCM (HRT III) to quantifycorneal nerve fibre density (NFD), branch density (NBD) and length(NFL). Each examination took ~ 6 minutes and no child reported thatCCM was uncomfortable.Results: There was a significant reduction in NFD (no/mm2) (32.5 ±1.9 v 41.1±1.8, P=0.007), NBD (no/mm2) (50.6 ± 4.5 v 72.5 ± 6.5,P=0.008) and NFL (mm/mm2) (20.6 ± 0.9 v 29.4 ±1.3, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaan elongated and interconnected fibroblastic morphology, and asignificant increase in cellular light scattering was measured. Thisstromal haze gradually decreased as wound healing progressed.Conclusions: Overall, this modified system allows high resolution 3-D image stacks from the full thickness rabbit cornea to be obtainedduring in vivo wound healing. These datasets can be used forinteractive visualization of corneal cell layers, measurement of sublayerthickness, assessment of cell morphology and connectivity, andestimation of stromal backscatter (haze) during wound healing.Commercial Relationships: Matthew Petroll, None; Daniela B.Hagenasr, None; H D. Cavanagh, Menicon Ltd (C); Danielle M.Robertson, NoneSupport: NIH Grants EY013322 and EY020799, and Research toPrevent Blindness, Inc.Program Number: 3223Presentation Time: 12:30 PM - 12:45 PMOptical coherence tomography (OCT) combined withvideokeratography to detect ‘early’ keratoconus - a new“pachymetry/asymmetry” index to quantify disease severityYaron S. Rabinowitz, Yelena Bykhovskaya, Xiaohui Li, Ana Laura C.Canedo. Cornea Genetic Eye Institute, Beverly Hills, CA.Purpose: To develop parameters using a combination of opticalcoherence tomography (OCT) and videokeratography to ‘early’detect keratoconus.Setting: Study was performed at the Cornea Genetic Eye Institute inBeverly Hills, CA.Methods: We studied the following groups: 180 normal eyes, 46 eyewith moderate keratoconus, 54 eyes with early keratoconus, 7 eyeswith ‘forme fruste’ keratoconus and 16 keratoconus ‘suspects’. Weperformed videokeratography, wavefront analysis and measured OCTindices to determine which combination was the most sensitive forseparating all study groups.Results: A combination of videokeratography and OCT indices (I-Svalue and Minimum pachymetry) was statistically the mostsignificant in separating keratoconus groups from normals (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: Expression of the TRA-1-60 antigen was investigated incorneo-limbal specimens from 10 human donor eyes using light andelectron microscopic immunohistochemistry. TRA-1-60 mRNAexpression was analyzed after laser capture microdissection of basallimbal epithelial cell clusters and limbal stromal cells, respectively,and pre-amplification of RNA by real-time PCR. In addition,antibodies against putative stem/progenitor markers, includingABCG2, p63alpha, cytokeratin 15, N-Cadherin, Sox-9, and Oct-4, aswell as against laminin-1, Melan A, CD11c, and CD45 were used indouble labelling experiments.Results: The TRA-1-60 epitope was abundantly expressed on thesurface of a subset of mesenchymal cells in the anterior limbal stromadisplaying a well-defined demarcation to the peripheral cornealstroma. The TRA-1-60 positive cells cumulated adjacent to limbalbasal epithelial cell clusters expressing putative stem/progenitormarkers and established intimate contact with epithelial cells bynumerous cell processes. In addition, few TRA-1-60 positive cellswere found to be integrated within the basal epithelial layer overlyingthe epithelial basement membrane. Dual staining showed that allTRA-1-60 positive cells co-expressed vimentin, but were negative forMelan A, CD11c, and CD45 excluding their nature as intraepithelialmelanocytes, dendritic cells or lymphocytes. Moreover, TRA-1-60staining was also associated with perivascular cells of anteriormostlimbal blood vessels. Real-time PCR confirmed differentialexpression of TRA-1-60 in limbal stromal cells compared to cornealstromal cells.Conclusions: The unequivocal presence of pluripotent cells withinsubepithelial stroma, blood vessel walls, and basal epithelium at thehuman limbus provides new insights into the constitution of thelimbal niche and its interaction with limbal epithelial stem/progenitorcells. These cells may have a great potential in corneal regenerationand wound healing.Commercial Relationships: Friedrich E. Kruse, None; NareshPolisetti, None; Johannes Menzel-Severing, None; UrsulaSchlotzer-Schrehardt, NoneProgram Number: 3226Presentation Time: 11:30 AM - 11:45 AMDonor MSCs expressing MHC class II molecule trigger immuneresponses in an animal model of lacrimal gland chronic graftversus host diseaseYoko Ogawa, Shigeto Shimmura, Saori Yaguchi, Takaaki Inaba,Kazuo Tsubota. Department of Ophthalmology, Keio Univ School ofMedicine, Shinjuku-Ku, Japan.Purpose: To examine using a cGVHD mouse model whether donorMSCs expressing MHC class II have a potential to trigger lacrimalgland cGVHD.Methods: We chose a MHC-compatible, minor antigen (miHA)-incompatible model of cGVHD involving bone marrowtransplantation (WBMT) with 8 week-old donor B10.D2 (H-2d) miceand recipient BALB/c mice (H-2d). We first modified the cGVHDmodel by co-transplanting prospectively isolated HSCs withprospectively isolated MSCs. This model closely mimics clinicalfindings of patients suffering from cGVHD. We examined lacrimalgland inflammation and fibrosis, expression of MHC class II onMSCs, T cell subsets and cytokine production in peripheral bloodafter the co-transplantation.Results: We found that serum levels of IL-6 increased in recipientmice transplanted with mismatched MSCs starting at 3 weeks aftertransplantation. This coincided with the appearance of lacrimal glandinflammation and fibrosis and MSC-derived cells in the peripheralblood of recipients starting at 3 weeks after transplantation andgradually increasing up to 7 weeks. Interestingly, these MSC-derivedcells in the peripheral blood expressed MHC class IIantigens.Flowcytometry revealed that both MSCs and T cells frommismatched MSC-transplanted recipients produce IL-6. ProliferatedT cells are predominantly recipient derived CD4+ T cells. MHC classII molecule was expressed in a subset of MSC cells, but thefrequency was significantly increased after co-culture with recipientT cells.Conclusions: These data suggest that mismatched MSCs and host Tcells stimulate each other via the MHC class II molecule. Our resultsshow that donor MSCs expressing MJHC class II molecule triggerimmune responses in cGVHD related dry eye, challenging currentparadigms on the pathogenesis of the disease.Commercial Relationships: Yoko Ogawa, None; ShigetoShimmura, None; Saori Yaguchi, None; Takaaki Inaba, None;Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch LombSurgical (C), Functional visual acuity meter (P), JiNS (P), Kissei (F),Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), EchoDenki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPTCompany (P)Support: Japanese Ministry of Education, Science, Sports andCulture# 23592590Program Number: 3227Presentation Time: 11:45 AM - 12:00 PMIntegral Analysis of Gene Signatures and MicroRNA Expressionof Cultured Human Corneal Endothelial Cells in Relation toTheir Functions, Cell Senescence, Epithelial-MesenchymalTransition, and FibrosisKazuko Asada 1 , Munetoyo Toda 1 , Michio Hagiya 1 , Kana Nakata 1 ,Morio Ueno 1 , Naoki Okumura 2 , Noriko Koizumi 2 , Junji Hamuro 1 ,Shigeru Kinoshita 1 . 1 Department of Ophthalmology, KyotoPrefectural University of Medicine, Kyoto, Japan; 2 Department ofBiomedical Engineering, Doshisha University, Kyotanabe, Japan.Purpose: Premature cell senescence in cultured human cornealendothelial cells (HCECs) is partly due to epithelial-mesenchymaltransition (EMT), a phenotypic switch in which HCECs undergophenotypic changes resulting in dampened cell propagation. In thisstudy, we investigated and compared HCEC gene-signature featuresbetween HCECs cultured with or without EMT-like phase transitionto understand the molecular aspects underlying the difficulty inpropagating HCECs in culture.Methods: Gene expression signatures of cultured HCECs with nosign of EMT and those with EMT-like phenotypic changes werecompared by use of polymerase chain reaction (PCR) array assay.Due to increasing evidence indicating that microRNA (miR)regulates gene functions, we also integrally analyzed miR expressionin corneas from distinct donors and cultured HCECs without EMTliketransition.Results: Fresh donor HCECs elicited completely distinct genesignatures from those of corneal epithelial cells from the same tissues[correlation coefficient (CC), 0.58~0.62]. Of interest, miR profileswere analogous with those from epithelium (CC, 0.95). Irrespectiveof the presence of EMT-like transition during culture, mRNA-basedgene signatures similar to those of fresh tissue were revealed (CC,0.7~0.8), while those of miR were far distinct (CC, 0.25~0.50). Thisimplicates the crucial roles of miR in regulating the functional genesof HCECs. The expression profile of miR varied dramaticallybetween donors 20 and 60 years of age. In the integral PCR arrayassay of gene signatures relating to EMT-like transition, fibrosisrevealed a significant decrease in the expression of distinct genes.Moreover, many senescence- and cell-cycle-related genes weredownregulated in the cultured HCECs with EMT-like transition.Conclusions: Gene-signature features of HCECs and corneal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaepithelium cells from fresh donor tissues were quite distinct, yetsimilar in miR expression. HCEC mRNA expression remained stableduring culture, while HCEC miR expression varied widely duringculture, thus indicating the fragility of miR expression. EMT-liketransition during culture triggered significant changes of miR traits.These findings will hopefully open a new pathway towards efficientin vitro propagation of HCECs.Commercial Relationships: Kazuko Asada, None; MunetoyoToda, None; Michio Hagiya, JCR Pharmaceuticals Co., Ltd (E);Kana Nakata, None; Morio Ueno, None; Naoki Okumura, None;Noriko Koizumi, None; Junji Hamuro, None; Shigeru Kinoshita,Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Program Number: 3228Presentation Time: 12:00 PM - 12:15 PMCXCR4 Expression Marks Cells in Limbal Stroma with thePotential to Differentiate to KeratocytesJames L. Funderburgh, Andrew Hertsenberg, Martha L.Funderburgh, Kira L. Lathrop, Yiqin Du. Ophthalmology, Univ ofPittsburgh School of Medicine, Pittsburgh, PA.Purpose: Mesenchymal cells from limbal stroma exhibit stem cellproperties and can regenerate stroma in vivo and vitro. Recentpublications showed stromal cells associating with limbal epithelialcells to express the chemokine receptor CXCR4. The study examinedthe hypothesis that stromal CXCR4 expression allows selection ofstromal stem cells with a high potential for corneal regeneration.Methods: Limbal stromal cells were recovered from human cornealrims by collagenase digestion and expanded in culture at clonaldensity in a low-serum medium containing cholera toxin(PMID:22078813). Gene expression patterns were determined byqPCR. Protein expression was assayed by flow cytometry. CXCR4+cells were isolated with MACS immunoadsorption or by FACS.Keratocyte phenotype was determined by gene and extracellularmatrix expression after culture on collagen or aligned nanofibersubstrata. Immunostaining was carried out on tissue wholemountsand sections using immunofluorescent detection with confocalmicroscopy.Results: Strong staining for CXCR4 protein was limited to smallpopulations of limbal cells subjacent to the epithelial basementmembrane. In uncultured digests and primary cultures, CXCR4positive cells amounted to one third of limbal cells in flow cytometry,but in central stroma less than 1% total cells were CXCR4+. Afterpassage, the proportion of CXCR4+ in cultured limbal cellsdecreased, particularly in confluent cultures. Separated CXCR4+cells expressed pluripotent markers Oct4, SOX2, NANOG, andneural crest marker p75NTR. Under differentiation conditions,CXCR4+ cells exhibited enhanced expression of keratocyte genesand matrix compared to CXCR4- cells.Conclusions: CXCR4 is a cell surface protein the abundance ofwhich correlates well with the potential of cells to differentiation tokeratocytes in vitro. This correlation differs from other widely knownmesenchymal stem cell markers and thereby provides a novel anduseful tool in isolating and enriching populations of stromal stemcells for use in corneal bioengineering and direct-cell based cornealtherapy.Commercial Relationships: James L. Funderburgh, None;Andrew Hertsenberg, None; Martha L. Funderburgh, None; KiraL. Lathrop, PCT/US2012/027268 (P); Yiqin Du, NoneSupport: NIH Grants EY016415, P30-EY008098, Eye & EarFoundation of Pittsburgh, Research to Prevent BlindnessProgram Number: 3229Presentation Time: 12:15 PM - 12:30 PMmiRNA EXPRESSION PROFILING IN CENTRAL ANDLIMBAL DIABETIC AND NORMAL HUMAN CORNEASUSING DEEP SEQUENCINGMehrnoosh Saghizadeh 1 , Jordan Brown 2 , Alexander V. Ljubimov 1, 3 ,Vincent A. Funari 2, 3 . 1 Surgery/Ophthalmology, Cedars-Sinai MedicalCenter, Los Angeles, CA; 2 Genomic Core, Cedars-Sinai MedicalCenter, Los Angeles, CA; 3 3David Geffen School of Medicine,University of California Los Angeles, Los Angeles, CA.Purpose: To identify and characterize miRNA expression by deepsequencing analysis in central and limbal parts of normal and diabetichuman corneas.Methods: Total RNA was extracted from age-matched humanautopsy normal (n=10) and diabetic (n=12) 8.5 mm-trephined centralcorneas and their limbi using mirVana miRNA Ambion kit. From 44total RNA samples that passed quality control standards small RNAs(22-28 bp) were gel isolated and miRNA libraries were preparedusing Illumina small RNA Sample Preparation Kit according to themanufacturer’s protocol. Samples were multiplexed and pooled intotwo lanes for a 35 bp sequencing run. Upon completion ofsequencing, FASTQ files were converted to FASTA files, andadapters were trimmed from each sequencing. FASTA files forsequencing were parsed by sample and aligned using BLAST with90% similarity to miRBase v19. Student’s t-test was performed foreach of four comparisons: normal central vs. normal limbus, normalcentral vs. diabetic central, normal limbus vs. diabetic limbus, anddiabetic central vs. diabetic limbus. For each comparison, significantmiRNAs were identified (fold change > 2, p-value < 0.05). Aknowledge base (Ingenuity Pathway Architect) was searched forbiochemical pathways that could be regulated by differentialmiRNAs.Results: A number of miRNAs with significant differential expressedin diabetic vs. normal corneas and in central vs. limbal parts werequantitatively identified; some were not present in the mirbase 19.Comparison of normal central corneas vs. normal limbi and diabeticcentral corneas vs. diabetic limbi yielded a few differential miRNAspecies. Many more miRNAs had differential expression betweennormal vs. diabetic central corneas (90) and especially, normal vs.diabetic limbi (141). In the central corneas, about equal numbers ofmiRNAs were either up- or downregulated in normal vs. diabetictissue. However, in the diabetic limbus, 133 miRNAs weredownregulated compared to normal, with only 8 upregulated.Conclusions: The results are consistent with previously observedsignificant changes in the expression of stem cell markers in diabeticlimbal epithelial cells. Abnormal miRNA expression may be animportant mechanism of diabetic changes affecting corneal epithelialstem cell compartment and wound healing. Manipulating miRNAlevels in diabetic corneas could potentially help alleviate symptomsof diabetic keratopathy.Commercial Relationships: Mehrnoosh Saghizadeh, None;Jordan Brown, None; Alexander V. Ljubimov, None; Vincent A.Funari, NoneSupport: NIH R21 EY022771, Cedars-Sinai Department of Surgery,and Regenerative Medicine Institute.Program Number: 3230Presentation Time: 12:30 PM - 12:45 PMStandardized cultivation and transplantation of limbal stem cellgrafts: Results of a phase I/II clinical trialNadia Zakaria 1, 2 , Tine Possemiers 1, 2 , Inge Leysen 1 , Jos J. Rozema 1 ,Carina Koppen 1 , Zwi Berneman 2 , Marie-Jose B. Tassignon 1 .1 Ophthalmology, Antwerp University Hospital, Antwerp, Belgium;©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea2 Center for Cell Therapy and Regenerative Medicine, AntwerpUniversity Hospital, Antwerp, Belgium.Purpose: To describe the results of a phase I/II clinical trial forstandardized, non-xenogenic, cultivation and “no touch” surgicaltransplantation of limbal stem cell grafts.Methods: 18 eyes of 18 patients were transplanted with eitherautologous (n=15) or allogenic (n=3) limbo-amnion composite graftsthat were generated using a standardized culture protocol free ofxenogenic culture products and transplanted using a standardized “notouch” surgical technique. In vitro cellular outgrowth and phenotypeof the limbo-amnion composite graft was assessed prior totransplantation. The clinical outcome measures investigated were:corneal neovascularization, central corneal opacity, pain,photophobia and visual acuity pre and post transplantation.Results: Limbal epithelial cells showed an average outgrowth of14.2mm ±3.7mm by day 14. The majority of the cells displayed aprogenitor phenotype: p63 bright, CK14, desmoglein, ABCG2 brightand CK3/12 dim protein expression. The transplant recipients werefollowed up for a mean of 22 months (range 4-43 months). 12 out ofthe 18 transplant recipients were graded successful (12 hadanatomical success and 7 also attained some degree of functionalsuccess), giving an overall success rate of 67%. We did not see asignificant reduction in pain, photophobia or central corneal opacityfor the patient group post transplant. However, the ocular surfacephotographs for pre- and post stem cell transplantation, showed asignificant (p=0.007) reduction in the percentage area of cornealneovascularization [Fig.1].Conclusions: We have been able to show that our standardized,xenogenic free culture system and “no touch” surgical technique hasoutcome measures comparable to other clinical studies. Thistechnique has the added advantage of being free from animalcontaminants such as mouse feeder layers and foetal bovine serum.Improved functional success is attained once penetrating keratoplastyis performed following successful stem cell grafting.Program #/Board # Range: 3456-3481/D0083-D0108Organizing Section: CorneaProgram Number: 3456 Poster Board Number: D0083Presentation Time: 11:00 AM - 12:45 PMA Prospective Trial Evaluating Scleral Rebound TonometryShuchi B. Patel, Sara L. Duke, Andrew Logeman. Ophthalmology,University of Chicago, Maywood, IL.Purpose: Glaucoma is known to occur in about 75% of patientsfollowing a keratoprosthesis, but accurate pressure readings tomonitor for progression are not possible. Thus we sought todetermine if a predictable relationship exists between Goldmannapplanation tonometry (GAT) and scleral rebound tonometry (RT) toprovide an accurate and reliable assessment of intraocular pressure(IOP) via scleral measurements.Methods: A prospective non-randomized trial of individuals 18 yearsof age and older. Each had his/her IOP measured by GAT, nextcorneal RT then scleral RT on the inferotemporal sclera. Thepatient’s age, gender, refractive error, central corneal thickness(CCT), axial length (AL) and phakic status were recorded. Pearson’scorrelation and multivariate regression were used for statisticalanalysis.Results: 116 eyes from 59 patients (37-90 years old) have beenexamined to date. Mean GAT IOP was 15.91 mmHg (SD 4.13), meancorneal RT was 14.50 mmHg (SD 4.24) and mean scleral RT was48.84 (SD 21.41). Mean spherical equivalent refraction (SE) was -0.21 D (SD 2.05), mean CCT was 547.68 µm (SD 45.65), mean ALwas 24.06 mm (SD 1.21). 89 eyes were phakic and 27 werepseudophakic. Pearson analysis reveals a strong positive correlationbetween GAT and corneal RT (0.77) but weak positive correlationbetween GAT and scleral RT (0.22) as well as corneal RT and scleralRT (0.22). This trend persists when phakic and pseudophakic eyesare evaluated independently. In the final multiple regression model(Table 1) to evaluate the association of GAT, CCT, AL and SE withscleral RT, only CCT in pseudophakic eyes was found to have asignificant positive correlation (p=0.04).Conclusions: Scleral RT shows IOP measurements that areconsistently higher than corneal IOP measurements by roughly 33-34mmHg. Corneal RT correlates well to the gold standard of IOPmeasurement, GAT; unfortunately, scleral RT measurements havepoor correlation to corneal measurements independent of phakicstatus. Analysis of scleral RT with relation to GAT, CCT, AL and SEconcurrently fails to reveal a statistically significant regression modelin either phakic or pseudophakic eyes. Our study reveals that sceralRT does not provide accurate and reliable IOP measurements.Fig 1. Eyes with total limbal stem cell deficiency before (A, E) &after (C, G) limbal stem cell transplantation within the softwareprogram for corneal neovascularization (CNV) mapping: (B, F, D,H). There was a significant reduction in %area of CNV post limbalstem cell transplantation (I) (**p= 0.007), but no significant decreasein the degree of corneal opacification post stem cell transplant (J).Commercial Relationships: Nadia Zakaria, None; TinePossemiers, Aeon Astron Europe B.V. (F); Inge Leysen, None; JosJ. Rozema, None; Carina Koppen, None; Zwi Berneman, None;Marie-Jose B. Tassignon, NoneSupport: IWT-TBM 90868Clinical Trial: NCT00845117345 Corneal Surgery Non-refractive II and KeratoprosthesisTuesday, May 07, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionFinal Multivariate Regression Model Assessing the RelationshipBetween Scleral Rebound Tonometry and Ophthalmic VariablesCommercial Relationships: Shuchi B. Patel, None; Sara L. Duke,None; Andrew Logeman, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 3457 Poster Board Number: D0084Presentation Time: 11:00 AM - 12:45 PMLimbal and Scleral Pneumatonometer versus ManometricReading in Cadaver Eyes with Type 1 Boston KeratoprosthesisJanet M. Lim, Genie Bang, Thasarat S. Vajaranant, Ahmad A. Aref,Maria S. Cortina, Jose De la Cruz. Illinois Eye and Ear Infirmary,UIC Department of Ophthalmology and Visual Sciences, Chicago,IL.Purpose: The use of keratoprostheses (KPro) to restore vision in eyeswith corneal opacities has become increasingly popular. However,since glaucoma remains a major visual limiting factor in many ofthese eyes, it is important to be able to accurately measure and followintraocular pressure (IOP). In this study, we assessed the differenceand correlation between manometric IOP and pneumatonometrymeasurements on the sclera and limbus in cadaver eyes with KPros.Methods: Two cadaver eyes were acquired from the Illinois Eye-Bank, and a Type 1 Boston KPro was implanted. The manometricIOP was varied between 5 to 40 mm Hg (in 5 mm Hg increments),and the pneumatonometer was used to measure IOP in 4 quadrants;superotemporal (ST), inferotemporal (IT), inferonasal (IN), andsuperonasal (SN). For every 5 mm Hg increment in IOP, 4measurements were taken at each quadrant at the scleral limbus (SL)and 4 measurements on the sclera, 2 mm away from the limbus (S2),yielding 60 data points at each area of the globe. Paired t-test andPearson’s correlation were used for the analysis.Results: The difference between the measured IOP usingpneumatonometry and manometric IOP (Table 1) was lowest in theSL ST measurement (2.0 ± 3.9 mm Hg) and highest in the S2 INmeasurement (10.8 ± 6.0 mm Hg). When measurements between 5 to25 mm Hg and 30 to 40 mm Hg were compared, there was a decreasein the standard deviation in the SL SN measurements (3.38 and 2.98,respectively, p=0.004). All other areas showed an increase in thestandard deviation as the IOP was raised. The difference betweenmeasured IOP and manometric pressure readings were on averagehigher for S2 measurements than SL measurements (2.8 ± 5.0 mmHg vs. 7.8 ± 5.5 mm Hg, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapostoperative vision of at least 20/200, 8 had preexisting severeretinal disease or glaucoma, which limited visual prognosis. Only 1of the 9 patients with potential for at least 20/200 vision failed toachieve it, due to a dense retroprosthetic membrane. Of those 15patients over the age of 65, 10 (66.7%) achieved at least 20/200vision within the 3 month postoperative period. 100% of the 27patients retained the keratoprosthesis at the 3 month postoperativeperiod.Conclusions: Our study demonstrates excellent short term deviceretention. A majority of the patients achieved at least 20/200 visionwithin the 3 month postoperative period. The Boston type 1keratoprosthesis remains a viable option for salvaging vision in olderpatients with corneal disease in which traditional keratoplasty wouldcarry a poor prognosis.Commercial Relationships: Kristine Lo, None; Kathryn Colby,Novartis (S); James Chodosh, Alcon (C), Allergan (C), 3-VBiosciences (C), Novabay (C)Support: Research to Prevent Blindness, Boston Kpro FundProgram Number: 3460 Poster Board Number: D0087Presentation Time: 11:00 AM - 12:45 PMThe Boston Keratoprosthesis Type I in Mucous MembranePemphigoidSotiria Palioura 1 , Bryan Kim 2 , Claes H. Dohlman 1 , James Chodosh 1 .1 Ophthalmology, Massachusetts Eye & Ear Infirm, Boston, MA;2 Ophthalmology, University of Illinois at Chicago, Chicago, IL.Purpose: To evaluate the use of the Boston keratoprosthesis type Iimplantation in patients with mucous membrane pemphigoid througha retrospective, interventional case series.Methods: Retrospective review of 8 eyes of 8 patients with severeocular surface disease and corneal blindness due to mucousmembrane pemphigoid who underwent Boston keratoprosthesis typeI implantation at the Massachusetts Eye and Ear Infirmary fromJanuary 1, 2000 through December 31, 2009. Data on preoperative,operative, and postoperative findings were collected. The mainoutcome measures analyzed were best-corrected visual acuity,keratoprosthesis retention, and postoperative complications. Theoutcomes were compared with those reported previously on a similargroup of patients with mucous membrane pemphigoid whounderwent keratoprosthesis type II implantation.[1]Results: The mean age of patients was 71.3 years (range, 55 to 94years), and the mean duration of their disease was 6.1 years (range,1.7 to 11.4 years). Visual acuity after surgery improved to 20/200 orbetter in 6 eyes (75%) and to 20/40 or better in 3 eyes (37.5%). Onlyone out of 6 eyes (16.6%) was able to maintain visual acuity of20/200 or better over a mean follow up period of 3.2 years. Inpatients with mucous membrane pemphigoid that underwent Bostonkeratoprosthesis type II implantation, 4 out of 13 eyes (31%)maintained visual acuity of 20/200 or better over a mean follow upperiod of 3.3 years.[1] Five of the 8 Boston keratoprosthesis type Idevices (62.5%) were extruded or had to be replaced during a meanfollow up time of 1.7+/-1.7 years, whereas 6 out of the 15 type IIdevices (40%) were previously reported to require re-implantation orrepair over a mean follow up time of 3.5+/-1.7 years.[1] Loss ofvision to worse than 20/200 during the follow up period was due tokeratoprosthesis type I extrusion, end-stage glaucoma, and retinal orchoroidal detachment.Conclusions: The clinical outcomes of the Boston keratoprosthesistype I in mucous membrane pemphigoid are guarded and, as judgedfrom the literature, less favorable than those of Bostonkeratoprosthesis type II for the same disease.Reference:1. Pujari, S., Siddique, S.S., Dohlman, C.H., Chodosh, J. (2011) TheBoston Keratoprosthesis Type II: The Massachusetts Eye and EarInfirmary Experience. Cornea 30, 1298-1303.Commercial Relationships: Sotiria Palioura, None; Bryan Kim,None; Claes H. Dohlman, None; James Chodosh, Alcon (C),Allergan (C), 3-V Biosciences (C), Novabay (C)Support: Supported in part by an unrestricted grant to theDepartment of Ophthalmology, Harvard Medical School, fromResearch to Prevent Blindness, Inc., NY, NY.Program Number: 3461 Poster Board Number: D0088Presentation Time: 11:00 AM - 12:45 PMProsthetic Replacement of the Ocular Surface Ecosystem(PROSE) for Visual Rehabilitation in Patients with CornealEctasiaNadeem H. Fatteh, Karen Deloss, Christopher T. Hood.Ophthalmology, University of Michigan, Ann Arbor, MI.Purpose: To describe the utility of the PROSE device for visualrehabilitation in patients with corneal ectasia.Methods: We performed a single-center retrospective chart review of46 eyes of 28 patients with ectasia fit for the PROSE device between2010 and 2012. All patients had unsatisfactory vision and hadexhausted previous nonsurgical options for visual correction.Topographic indices from the Atlas corneal topographer, change invisual acuity, and achievement of satisfactory fit were recorded.Results: 42 eyes of 26 patients had keratoconus or pellucid marginaldegeneration, and 4 eyes of 2 patients had ectasia after LASIK. Meanage was 42.0 ±13.8 years (range 16 - 69), with 8 females (29%) and20 males (71%). All patients had previously tried rigid gas-permeablelens fitting but could not achieve satisfactory fit or visual acuity.Additionally, 7 patients had tried and failed a piggyback lens, 9patients a hybrid lens, and 3 patients a commercially available sclerallens.According to the Amsler-Krumeich classification for severity ofkeratoconus, 1 eye was stage 1, 14 eyes were stage 2, 10 eyes werestage 3 eyes and 21 eyes were stage 4. The steepest simulatedkeratometry value averaged 52.57 ± 6.26 diopters (D) and the meandifference between steepest and flattest simulated keratometry was6.25 ± 4.12 D. All eyes were successfully fit with the PROSE device,achieving a comfortable fit with improved subjective visual acuity.Best-corrected acuity improved from 0.438 logMAR (Snellenequivalent 20/55) to 0.062 logMAR (Snellen equivalent 20/23) withthe PROSE device (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: 1) To evaluate the association between phakic status andthe risk of postoperative cystoid macular edema (CME) afterimplantation of Boston Keratoprosthesis Type I (Kpro). 2) Toevaluate the association between iris-backplate touch (IBPT) onanterior segment optical coherence tomography (ASOCT) and therisk of postoperative CME after Kpro.Methods: Retrospective non-interventional chart review. All patientswho underwent Kpro type I implantation at the Illinois Eye and EarInfirmary from 2007-2012 were reviewed. Patients were included inthe study if they had at least one postoperative macular OCT and onepostoperative ASOCT. Patients were excluded for poor qualityimages and aniridia. For all patients who met inclusion criteria, datawere obtained including phakic status, pre-op and post-op visualacuity, time to final follow up, presence of CME on macular OCTand presence of IBPT on ASOCT. Two-tailed Fisher’s exact test wasthen performed to evaluate the statistical significance of phakic statusand IBPT on postoperative CME.Results: One hundred and three Kpro were implanted into 95 eyes of94 patients over a 5 year period. Fifty-eight eyes met inclusioncriteria. Seven eyes were excluded for poor image quality andaniridia. Of the remaining 51 eyes, 21 (41.1%) had CME onpostoperative macular OCT and 9 (17.6%) were pseudophakic. OnFisher’s Exact Test there was a statistically significant positivecorrelation between implantation of pseudophakic-typekeratoprosthesis and presence of postoperative CME (p = 0.023). Onreview of ASOCT, 23 (45.1%) had evidence of IBPT. There was nosignificant correlation found between IBPT and postoperative CMEon Fisher’s exact test (p = 0.167).Conclusions: CME is a frequent postoperative complication afterKpro implantation and can contribute to poor visual acuity outcomes.Patients with pseudophakic Kpros may be at higher risk forpostoperative CME. Although IBPT may be a source of chronicinflammation in these patients, its presence on ASOCT is not a riskfactor for postoperative CME.Commercial Relationships: Kaitlyn M. Wallace, None; Joshua H.Hou, None; Randee C. Miller, None; Clement C. Chow, None;Jose De la Cruz, alcon (C), amo (C); Felix Y. Chau, None; MariaS. Cortina, NoneProgram Number: 3463 Poster Board Number: D0090Presentation Time: 11:00 AM - 12:45 PMIn vivo evaluation of microbial infection on surfacefunctionalized titanium as artificial cornea candidate materialXiao-Wei Tan 1 , Rajamani Lakshminarayanan 1 , Gwendoline Goh 1 ,Melina Setiawan 1 , Shouping Liu 1 , Roger W. Beuerman 1, 2 , Donald T.Tan 3, 4 , Jodhbir S. Mehta 1, 3 . 1 Singapore Eye Research Institute,Singapore, Singapore; 2 Yong Loo Lin School of Medicine, NationalUniversity of Singapore, Singapore, Singapore; 3 Singapore NationalEye Centre, Singapore, Singapore; 4 Department of Clinical Sciences,Duke-NUS Graduate Medical School, Singapore, Singapore.Purpose: Artificial keratoprosthesis skirts are prone tomicrobiological infection after implantation. Previous studies haveshown that coating titanium surface with antimicrobial peptide(AMP), human beta-defensin analogue SESB2V, could improve thebactericidal effect of the titanium alloy in an in vitro environment.Here we aimed to test the bactericidal effect of the functionalizedtitanium oxide (TiO2) with a rabbit cornea infection model.Methods: SESB2V AMP was bound onto the surface of titaniumoxide via crosslinking with polydopamine. A corneal stroma pocketwas created by a femtosecond laser assisted LASIK surgery. TiO2discs were inserted into the pocket through a small corneal incision.After 1 week of wound healing, 50 ul S.aureus (1X103 CFU/ml)were injected into the pocket right above the TiO2 inserts. Theimplanted corneas were compared with normal and sham-operatedcorneas through slit lamp observation and anterior segment opticalcoherence tomography (AS-OCT). After 2 days of infection, rabbitcornea tissue was collected for haematoxylin and eosin (H&E)staining. Inflammatory response was also evaluated by staining withCD11b and MMP9 antibodiesResults: There were less incidence of corneal infection and lessextent of infection on rabbit corneas with SESB2V coated implantscompared to those corneas with non-coated implant. Histologicalanalysis also revealed that less inflammatory cells were found in thecornea pocket tissue with the AMP coated TiO2 discs compared tothose with non-coated TiO2 discs.Conclusions: SESB2V AMP significantly improved the bactericidaleffect of TiO2 discs in vivo, which is a potential candidatebiomaterial for artificial cornea skirt. This would further expand theusage of TiO2 in the development of keratoprosthesis device.Commercial Relationships: Xiao-Wei Tan, None; RajamaniLakshminarayanan, None; Gwendoline Goh, None; MelinaSetiawan, None; Shouping Liu, None; Roger W. Beuerman,Allergan (F), SERI (P), Santen (R); Donald T. Tan, NetworkMedical Products (P), Carl Zeiss Meditec (F), Alcon Labs (F),Bausch & Lomb (F), Allergan (F), Santen (F); Jodhbir S. Mehta,NoneSupport: SHF/FG488S/2010Program Number: 3464 Poster Board Number: D0091Presentation Time: 11:00 AM - 12:45 PMBandage CL fitting characteristics and complications in patientswith Boston Type I keratoprosthesis surgeryEllen Shorter, Charlotte E. Joslin, Timothy T. McMahon, Jose De laCruz, Maria S. Cortina. University of Illinois at Chicago, Chicago,IL.Purpose: To describe bandage contact lens fitting characteristics andcomplications in patients that have undergone Boston Type Ikeratoprosthesis surgery at the University of Illinois at Chicago(UIC). Proper bandage lens fit is critical to lens retention and isnecessary to protect the ocular surface from complications related tocorneal desiccation.Methods: Medical records data was abstracted and analyzed amongpatients who underwent Boston Type I keratoprosthesis surgery atUIC between January 1, 2007 and June 1, 2012.Results: 76 eyes of 71 patients were identified who underwentsurgery at UIC with a minimum of 6 months of follow-up. Presurgicalindications included non-inflammatory (38 eyes), limbalstem cell deficiency (LSCD) (20 eyes), chemical/thermal burn (14eyes) and autoimmune disease (4 eyes). Forty-three percent achievedvisual acuity of 20/200 or better compared to 3% before surgery.Bandage lens loss occurred between visits in 38% of patients anddeposits were noted in 20%. Lens loss was more common in theLSCD and chemical burn group while lens deposits were mostcommon in the autoimmune group.The final bandage lens incorporated a power change in 38% while30% remained in the same fitting parameters as the initial postsurgicallens. Sagittal depth was increased by decreasing the basecurve or increasing the diameter in 37%. Frequent replacementsilicone hydrogel lenses were used to re-fit 22% of patients. Hybridcontact lenses were fit in 7% of patients due to frequent lens loss orvisually significant deposits despite refitting.Of the eyes with corneal melt requiring repeat keratoprosthesis orkeratoprosthesis removal, 62% had problems with bandage lens loss.Corneal melt occurred in 8% of the non-inflammatory group, 35% inthe LSCD group, 21% in the chemical and thermal burn group and inno eyes in the autoimmune group. At last follow-up, 91% retained a©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneakeratoprosthesis device.Conclusions: Well-fit bandage contact lenses are essential to lensretention and success in patients who have undergone Boston Type Ikeratoprosthesis surgery. Contact lens over-refraction is important inall patients as best-corrected vision improved with a refractive powerchange in 38% of our patients.Commercial Relationships: Ellen Shorter, None; Charlotte E.Joslin, None; Timothy T. McMahon, Alcon (R); Jose De la Cruz,alcon (C), amo (C); Maria S. Cortina, NoneProgram Number: 3465 Poster Board Number: D0092Presentation Time: 11:00 AM - 12:45 PMKeratoprosthesis Imaging Using Anterior Segment OpticalCoherence Tomography to Detect Graft Host Defects in theClinic and in the Operating RoomMark N. Welch, Michael Banitt, Audina M. Berrocal, Victor L. Perez.Ophthalmology, Bascom Palmer Eye Institute, Miami, FL.Purpose: Anterior segment optical coherence tomography (AS-OCT)was used to diagnose subclinical defects in the graft host junction ofpatients with keratoprostheses.Methods: Noncomparative observational case series: two patientswith questionable thinning of the donor corneal tissue were imagedwith AS-OCT and determined to have abnormal gaps between thedonor tissue and the keratoprosthesis. Tissue thinning was alsoobserved.Results: The AS-OCT allows for imaging of the abnormal positionof a keratoprosthesis. The first case was a seidel negative adult whowas imaged in the clinic and determined to have thinning of thedonor tissue which required replacement of the keratoprosthesis. Thesecond case was a child in the operating room undergoing an examunder anesthesia (EUA). A non-rhegmatogenous retinal detachmentwas found with chorioretinal folds. After repair of the retinaldetachment, an AS-OCT was performed and a large gap was notedbetween the keratoprosthesis and the donor tissue of this seidelnegative patient. This image led to the appropriate replacement of thekeratoprosthesis.Conclusions: High resolution AS-OCT images provide non-contact,cross-sectional images of a keratoprosthesis and can detect whetherthe prosthesis and donor tissue are in appropriate position. Theimages can also detect donor tissue thinning. This is helpful, becausethinning of donor tissue can be subclinical. Depending on the deviceused, images can be obtained with upright positioning in the clinic orwith supine positioning in the operating room. These images assistwith clinical decisions. It may also be helpful to obtain AS-OCTimages on all pediatric patients with a keratoprosthesis at every EUA.AS-OCT image of a keratoprosthesis in poor contact with donorcorneal tissue in the the other eye of the same patient.AS-OCT image of an intact keratoprosthesis in a two year old maleCommercial Relationships: Mark N. Welch, None; MichaelBanitt, None; Audina M. Berrocal, thrombogenics (C), genentech(C); Victor L. Perez, Alcon (C), Bausch & Lomb (C), Genentech(C), Cleveland Clinic Foundation (P), Alcon (F), Alcon (R)Program Number: 3466 Poster Board Number: D0093Presentation Time: 11:00 AM - 12:45 PMAnterior Segment Optical Coherence Tomography in Patientswith Boston Type I Keratoprosthesis Allows Early Detection andMonitoring of Corneal and Scleral MeltsDavid Sola-Del Valle 1 , Bernardo M. Cavalcanti 1, 2 , Andrea Cruzat 1, 2 ,Claes H. Dohlman 1 , Pedram Hamrah 1, 2 . 1 Ophthalmology - Cornea &Refractive Surgery Service, Massachusetts Eye & Ear Infirmary,Harvard Medical School Department of Ophthalmology, Boston,MA; 2 Ophthalmology: Ocular Surface Imaging Center,Massachusetts Eye & Ear Infirmary, Harvard Medical SchoolDepartment of Ophthalmology, Boston, MA.Purpose: To quantify corneal/scleral thickness in Boston type Ikeratoprosthesis (KPro) patients using spectral domain anteriorsegment optical coherence tomography (AS-OCT) for early detectionand monitoring of corneal and scleral melting.Methods: This retrospective study included 10 eyes of 10 patientswho underwent implantation of Boston type I KPros at theMassachusetts Eye and Ear Infirmary, Boston, MA. Cornealthickness in the four cardinal quadrants around the KPro optic stemas well as potential spaces (gaps) and areas of melting were measuredserially using non-contact AS-OCT (RTVue OCT, Optovue Inc.,Fremont, CA). The findings were correlated with findingsdocumented by slit-lamp examination. The Mann-Whitney test wasused to compare data.Results: Ten patients (5 men; 5 women) with a mean age 65.7 ± 13.6years were included. Four eyes had polymethylmethacrylate(PMMA) back plates, 5 had titanium, and 1 had consecutive PMMAand Titanium back plates. Six eyes had gaps between the KPro stemand the donor cornea with an average size of 117 ± 86 μm. Threeeyes were noted to have early corneal melting with an residualstromal bed of 461 ± 84 μm, and 1 was noted to have scleral meltingwith average residual sclera of 200 ± 163 μm. The size of the gapsaccording to type of back plate was similar (PMMA, 88 ± 65 μm vs.Titanium, 110 ± 71 μm; p-value 0.11), but the average distancebetween optic and residual cornea in patients with early cornealmelting was larger with PMMA back plates (346 ± 125 μm vs. 204 ±44 μm; p-value

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamelting. Early identification of corneal/scleral thinning and meltingin Boston type I KPro patients allows for early intervention and maylead to improved outcomes.Commercial Relationships: David Sola-Del Valle, None;Bernardo M. Cavalcanti, None; Andrea Cruzat, None; Claes H.Dohlman, None; Pedram Hamrah, NoneSupport: Keratoprosthesis Manufacturing Fund, NIH K08-EY020575, Research to Prevent Blindness Career DevelopmentAward, Falk Medical Research TrustProgram Number: 3467 Poster Board Number: D0094Presentation Time: 11:00 AM - 12:45 PMScanning Laser Ophthalmoscope Microperimetry through theBoston KeratoprosthesisRony R. Sayegh, Claes H. Dohlman, Mary Lou Jackson.Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA.Purpose: Scanning laser ophthalmoscope (SLO) microperimetry isuseful in accurately mapping macular function. We explore thefeasibility and potential use of this method in eyes implanted with aBoston keratoprosthesis.Methods: Retrospective review of eyes with a type I Bostonkeratoprosthesis tested with OPKO SLO microperimetry. Bestcorrectedvisual acuity, contrast sensitivity and complete slit-lampand posterior segment examination findings were noted. Visual fieldand ocular coherence tomography results were collected whenavailable.Results: Five eyes were included in this study. Mean age was 66years (range: 54 to 82). Mean LogMAR best-corrected visual acuitywas 0.76 (range: 0.30 to 1.20). Contrast sensitivity was decreased inall eyes at a mean of 0.90 log units (range: 0.45 to 1.5). SLOmicroperimetry could be performed on all 5 eyes. Accurate functionalmapping of the macula and preferred retinal locus was obtained in allcases and was not affected by media opacities or decreased contrastsensitivity. The results correlated well with the findings on Goldmannperimetry and OCT in cases in which these were available.Conclusions: SLO microperimetry through the Bostonkeratoprosthesis is possible. It is a good adjunct to standard perimetrywith involvement of central fixation for accurate macular mapping. Itmay also allow better assessment of macular pathology unveiled byclearing of the visual axis with a keratoprosthesis.Commercial Relationships: Rony R. Sayegh, None; Claes H.Dohlman, None; Mary Lou Jackson, Nonehistologic appearance (H&E staining) and presence of inflammation(immunolocalization of CD45 positive cells) in rabbit corneal tissues50 days after an intralamellar implant of the modified Ti. Surfacecharacterization of the modified Ti included assessment by scanningelectron microscopy (SEM), X-ray diffraction crystallography(XRD), Fourier transform infrared spectroscopy (FTIR) andchemical/color stability was determined after exposure to 70%alcohol for three months.Results: Blue and brown coloration of Ti was achieved byanodization at 33 and 13.4 volts, respectively with a current supply of3 Amps, (Figure 1). The in vitro biocompatibility assays showed nosignificant differences in cell proliferation, cytotoxicity or migrationbetween HCLE cells co-cultured with surface modified or nonmodifiedTi (p> 0.745 for all group comparisons. Analysis of cornealtissues that had harbored the Ti implants showed normal cellappearance, and lack of CD45 or TUNEL positive cells. SEMshowed the presence of a nano porous surface and similarcrystallographic prints were observed with XRD and FTIR for thenon-coated and the modified Ti, (Figure 2). Three months exposureto alcohol had no effect on the color or color homogeneity of theoxidized surface.Conclusions: Ti backplate coloring was achieved by altering thevoltage of the electrochemical anodization and controlling thethickness of the Ti oxidation. In vitro and in vivo results suggest thatthe modified Ti is equally biocompatible and as safe as the standardnon-coated Ti. The color modification of the BKpro may improve thecosmesis and acceptance of the KPro by patients.*Supported by The Boston Keratoprosthesis Fund at theMassachusetts Eye and Ear Infirmary.Program Number: 3468 Poster Board Number: D0095Presentation Time: 11:00 AM - 12:45 PMAssessment of Titanium Modification for Coloring the Backplateof Boston KeratoprosthesisEleftherios I. Paschalis 1, 2 , James Chodosh 1 , Sandra J. Spurr-Michaud 2 , Andrea Cruzat 1 , Allyson Tauber 1 , Irmgard Behlau 1 , IleneK. Gipson 2 , Claes H. Dohlman 1 . 1 Ophthalmology, Massachusetts Eyeand Ear Infirmary - Harvard Medical School, Boston, MA;2 Ophthalmology, Schepens Eye Research Institute - Harvard MedicalSchool, Boston, MA.Purpose: Recent use of a titanium (Ti) backplate has improved thedesign and biocompatibility of the Boston Keratoprosthesis (BKPro).Titanium’s shiny metallic appearance, however, makes itcosmetically less favorable. The purpose of this study was to developand test a coloring, surface modification of Ti.Methods: Ti coloring was achieved electrochemically by controlledanodization of the Ti surface, which creates an oxide film on the Ti.Biocompatibility was assessed in vitro by co-culture of the modifiedTi with human corneal limbal epithelial cells (HCLE) followed byassays of cell proliferation, cytotoxicity and migration, and in vivo byCommercial Relationships: Eleftherios I. Paschalis, None; JamesChodosh, Alcon (C), Allergan (C), 3-V Biosciences (C), Novabay(C); Sandra J. Spurr-Michaud, None; Andrea Cruzat, None;Allyson Tauber, None; Irmgard Behlau, None; Ilene K. Gipson,None; Claes H. Dohlman, NoneProgram Number: 3469 Poster Board Number: D0096Presentation Time: 11:00 AM - 12:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFemto-second-laser and eximer-laser assisted preparation ofbiosynthetic corneal collagen donor implants and the recipientbedRaphael T. Neuhann 1 , Kerstin M. Wand 1 , Karin Kobuch 1 , MichaelBaumann 4 , May Griffith 2 , Mohammad M. Islam 2 , Johannes Junger 4 ,Roland Ritter 3 , Chris Lohmann 1 . 1 Ophthalmology, Klinkum Rechtsder Isar, Munich, Germany; 2 Regenerative Medicine, LinkopingUniversity, Linkoping, Sweden; 3 Technolas Perfect Vision, Munich,Germany; 4 MLase AG, Germering, Germany.Purpose: The goal was to achieve a setup that allows precise dockingof the laser as well as easy handling of the recipient and donorcorneas for an ALK (anterior lamellar keratoplasty) usingbiosynthetic corneal collagen implants. Before starting in vivo trailsin the rabbit eye, the trial setup was tested ex vivo on porcine andrabbit eyes. Furthermore the diameter, depth and surface of therecipient bed as well as the diameter and thickness of the biosyntheticcornea were examined.Methods: We used two different laser platforms on eyes of twodifferent species (porcine eyes and rabbit eyes). One was the VictusFemto-laser-System (Technolas Perfect Vision, Germany) and aPrototype Excimer Laser (M-Lase, Germany). On both lasers thebiosynthetic donor tissue was cut first (Diameter 600µm). The donortissue (thickness 350µm) was placed on a porcine eye to facilitateplacement under the laser aperture. The recipient porcine and rabbiteyes were treated with the same parameters (Diameter 600µm, depth350µm).In case of the eximer laser two specially designed aluminum maskshad to be used to achieve the desired diameter and shape of the cuts.The diameter, depth(stromal bed), thickness (donor tissue) andsurface structure were evaluated histologically (paraffin embedded,HE staining) and by OCT Imaging as well as electronmikroskopy.Results: Both laser systems allowed precise cutting of thebiosynthetic corneal implants as well as the donor tissue. Theexcimer laser treatment provided a smoother surface structureregarding the stromal bed, but the treatment duration wassignificantly longer than the treatment duration of the femto-secondlaser laser.Conclusions: Both laser platforms offer precise cutting options of thedonor and recipient tissue. Although both treatment setups in the exvivo animal trials showed promising results, the in vivo setup(presented separately) might be a greater challenge regardingpositioning, docking and cutting parameters.Commercial Relationships: Raphael T. Neuhann, None; KerstinM. Wand, None; Karin Kobuch, None; Michael Baumann, None;May Griffith, Univ. of Ottawa - OHRI (P); Mohammad M. Islam,None; Johannes Junger, MLase AG (E); Roland Ritter, TechnolasPerfect Vision (E); Chris Lohmann, NoneSupport: Euro Nanomed I-CARE, Transnational collaborativeprojectProgram Number: 3470 Poster Board Number: D0097Presentation Time: 11:00 AM - 12:45 PMImaging of Surface Defects and Biofilm Formation of ExtrudedKeratoprostheses Using Confocal MicroscopyHeather A. Durkee 1, 4 , Darlene Miller 2 , Victor L. Perez 2 , YohSawatari 3 , Aleksandra V. Rachitskaya 2 , Audina M. Berrocal 2 ,Eduardo C. Alfonso 2 , Jean-Marie A. Parel 1, 2 . 1 OphthalmicBiophysics Center, Bascom Palmer Eye Institute, University ofMiami Miller School of Medicine, Miami, FL; 2 Department ofOphthalmology, Bascom Palmer Eye Institute, University of MiamiMiller School of Medicine, Miami, FL; 3 Department of MaxillofacialSurgery, University of Miami Miller School of Medicine, Miami, FL;4 Department of Biomedical Engineering, College of Engineering,University of Miami, Coral Gables, FL.Purpose: The goal of the study was to examine surface structure andbiofilm deposits on keratoprostheses (KPro) that extruded frompatients secondary to Streptococcal Endophthalmitis. Confocalmicroscopy studies allow for fresh cultures in contrast to biofilmdetection using SEM which was presented previously (De La Cruz etal, IOVS Meeting abstracts 2011 52:345).Methods: Three keratoprostheses (2 Boston Type I and 1 ModifiedOsteo Odonto Keratoprothesis (MOOKP)) that extruded due toStreptococcal Endophthalmitis were stained with BacLightLive/Dead fluorescence assay and imaged with a Leica 5PS confocalmicroscope with 63x objective lens. Surface features of thekeratoprostheses were visualized using bright field illumination. Thebiofilm and bacteria distribution across the entire KPro wereevaluated using volumetric image sets that were captured fromdifferent regions of the keratoprostheses.Results: Biofilm deposits were present on all three of the KPros (seeFigure). Bacteria were found in higher quantities in areas of the opticwith surface scratches. Surface features corresponding to machiningmarks were found on the 2 Boston KPros and surface scratches onboth of the optical surfaces of all KPros.Conclusions: Surface irregularities on the KPros created duringfabrication allowed for more bacterial and biofilm growth. Improvingsurface quality of the implants might reduce the areas where bacteriaand biofilm can attach to the keratoprothesis.Figure 1: Image A (left) bright field of scratches on cylindrical faceof MOOKP (right) live dead stain of same section, biofilm depositsappear in dome-like shapes; Image B (left) bright field of deepscratch in cylindrical face of MOOKP, (right) bacteria deposit increvices of scratch; Image C (left) bright field of center anterior faceof KPro#1, machining marks can been identified as concentric rings(right) organic matter deposited on anterior face of optic; Image Dunidentified mark on posterior face of optic (inside anterior chamber)Commercial Relationships: Heather A. Durkee, None; DarleneMiller, None; Victor L. Perez, Alcon (C), Bausch & Lomb (C),Genentech (C), Cleveland Clinic Foundation (P), Alcon (F), Alcon(R); Yoh Sawatari, University of MIami (P); Aleksandra V.Rachitskaya, None; Audina M. Berrocal, thrombogenics (C),genentech (C); Eduardo C. Alfonso, Bio Tissue (C); Jean-Marie A.Parel, CROMA (F), InnFocus (F), Abeamed (F), University ofMiami (P)Support: USAMRMC Department of Defense W81XWH-09-1-0674; Florida Lions Eye Bank; NIH Center Grant P30EY14801; anunrestricted grant from Research to Prevent Blindness; Henri andFlore Lesieur Foundation (JMP)Program Number: 3471 Poster Board Number: D0098Presentation Time: 11:00 AM - 12:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaImprovement of the water tightness of a monoblockeratoprosthesis with the use of a surgical bio-glue. Preliminaryresults in dogsPierre F. Isard 1 , Marielle Mentek 2 , Thomas Dulaurent 1 . 1 CentreHospitalier Vétérinaire Saint-Martin, Saint-Martin Bellevue, France;2 INSERM U1042, Grenoble, France.Purpose: The mid- and long-term water tightness is a key point inthe keratoprosthesis (KP) implantation procedure. Majorcomplications such as athalamia and endophtalmitis may be linked toincomplete water tightness. The use of a surgical bio-glue may be asatisfactory solution to prevent the risk of aqueous humor leakage.Methods: Patients: 5 dogs were presented for vision loss of cornealorigin, secondary to an immune-mediated chronic superficialkeratitis.Keratoprosthesis: KPro-LID® is a full thickness monobloc PMMAKP, with 4 retrocorneal fixation sites and without colonisable skirt.Surgical bio-glue: GRF® was originally manufactured for vascularsurgery. It is composed of gelatin/resorcin (adhesive property) andformaldehyde/glutaraldehyde (hardening property).Surgical procedure: aAfter 6 months, no water-tightness defect wasobserved and no abnormal tissue reaction was identified, secondaryto the use of GRF glue. 0.2 mm depth x 5.5 mm diameter superficialkeratectomy was performed in the central cornea. Around thekeratectomy site and at the same depth, the superficial cornea wasundermined over 360°. The dissected cornea was cut over 300°, 2mm in front of the limbus and parallel to it. The rim was reflectedand a complete penetrating, 5.5 mm diameter central keratectomywas performed. After filling the anterior chamber with viscomaterial,the keratectomy was enlarged with a 3 mm full-thicknessradial incision. The KP was implanted and anchored to the cornea bypassing 4 U-shaped sutures through the retrocorneal fixation sites.The corneal radial incision was sutured and the GRF glue wasapplied over the uncovered corneal surface, up to the edge of the KP.The superficial rim was repositioned and the circular peripheralincision was sutured. The dogs were re-evaluated at 8 days, 15 daysand then monthly for 6 months.Results: After 6 months, no water-tightness defect was observed andno abnormal tissue reaction was identified, secondary to the use ofGRF glue.Conclusions: The GRF glue improved the water-tightness of theKPro-LID in dogs. The ease of use and safety of this bio-glue make itan important step of the KP implantation surgical procedure.Implanted KP after retrocorneal and GRF glue (arrow) fixation.GRF glue addition with a needle (25G) after retrocorneal fixaton.Commercial Relationships: Pierre F. Isard, None; MarielleMentek, None; Thomas Dulaurent, NoneProgram Number: 3472 Poster Board Number: D0099Presentation Time: 11:00 AM - 12:45 PMAgreement among Transpalpebral, Transcleral and TactileIntraocular Pressure Measurements in Eyes with Type 1 BostonKeratoprosthesisJessica L. Liu, Thasarat S. Vajaranant, Maria S. Cortina, Jacob T.Wilensky. Glaucoma, University of Illinois at Chicago, Chicago, IL.Purpose: The use of keratoprostheses (KPro) to restore vision in eyeswith corneal opacities has become increasingly popular in the lastfive years. Intraocular pressure (IOP) is a cardinal measurementemployed in glaucoma management. This presents a major problemsince glaucoma remains a major visual limiting factor in eyes withKPro and most forms of tonometry require an intact cornea. Thepurpose of this study is to determine if transpalpebral IOPmeasurement can be an alternative method of measuring IOP andyield valuable data in eyes with KPro.Methods: We retrospectively reviewed IOP measurements inpatients who had received Type 1 Boston KPro, and their IOP wereestimated by three different methods during routine visits to theircorneal surgeon. The surgeon estimated the IOP range tactilely bypalpation of the globe. A pneumatonometer (Model 30 Classic;Mentor, BioRad, Santa Ana, California, USA) was used to measureIOP by placing the tonometer tip on the sclera peripherally to thecontact lens in the inferotemporal quadrant. The Diaton tonometer(BiCOM, Inc., Long Beach, NY, USA) was used to obtain valuesthrough the upper lid in accordance with the instructions by themanufacturer. An average of two Diaton IOP measurements was usedin the analysis. Since the tactile IOP were recorded as a range ratherthan a definite number, we computed the percent agreement, thepercentage of eyes in which pneumatometer or Diaton IOPs werewithin 2 mmHg of the tactile IOP range. Two-tailed t-test was used tocompare the mean of pneumatonometer and Diaton IOPmeasurements.Results: The analysis included 23 eyes of 20 patients. Thepercentage agreement was 85% between tactile range andpnematonometer IOPs, and 95% between tactile range and DiatonIOPs. Pneumatonometer consistently yielded higher IOP values,compared to Diaton (p = 0.04). The overall IOP mean ± SD was 17.2± 6 mmHg for pneumatonometer and 13.8 ± 5 mmHg for Diatontonometer.Conclusions: The presence of KPro did not appear to interfere withIOP with Diaton tonometer, and Diaton tonometer yielded IOPreadings that were similar to those obtained by palpation. Scleralpneumotonometry yielded values that were consistently higher thantactile estimates and Diaton IOP. In addition to routine IOP estimatesby palpation, transcleral and transpalpebral IOP measurements can beconsidered to monitor patients with KPro.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Jessica L. Liu, None; Thasarat S.Vajaranant, None; Maria S. Cortina, None; Jacob T. Wilensky,NoneSupport: Carson Gabriel Fund, Research to Prevent BlindnessProgram Number: 3473 Poster Board Number: D0100Presentation Time: 11:00 AM - 12:45 PMRetinal Detachments in Eyes After Boston Keratoprosthesis Type1Sachin Jain, Clement C. Chow, Jennifer I. Lim, Lawrence J. Ulanski,Jose De la Cruz, Maria S. Cortina, Felix Y. Chau. Illinois Eye andEar Infirmary. UIC Department of Ophthalmology and VisualSciences, University of Illinois, Chicago, IL.Purpose: To report the frequency, timing, visual significance,contributing factors, and management of retinal detachment (RD)after successful Boston Keratoprosthesis Type I (KPro) implantation.Methods: A retrospective chart review was conducted of consecutiveKPro implantations at a single institution over a 5 year period.Preoperative and postoperative best-corrected visual acuity (BCVA),ocular co-morbidities, anatomic outcomes, surgical interventions, andcomplications were analyzed.Results: One hundred and three KPros were implanted into 95 eyesof 94 patients over a 5 year period. Fourteen of the 95 eyes (14.7%)developed RDs during a median follow-up of 27.5 months (range 12-58). Pre-KPro diagnoses included HSV keratitis, chemical burn,aniridia, Peters anomaly, Fuchs’ dystrophy, congenital glaucoma,end-stage glaucoma and limbal stem cell deficiency. Ten of 14 eyeshad concomitant surgery, including intraocular lens removal (6), parsplana vitrectomy (PPVx, 5), silicone oil fill (2 for hypotony), Ahmedtube placement (2), pupilloplasty (1) and tarsorrhaphy(1). Themedian time from KPro placement to occurrence of RD was 9months (range 0.5-40 months). Thirteen of 14 (92.9%) eyesunderwent PPVx; only 3 eyes required KPro removal during RDrepair. BCVA prior to KPro surgery was 1.91±0.28 (logarithm ofminimum angle of resolution ± standard deviation). BCVA improvedafter KPro to 1.23±0.58 (p 20/400 at final follow up.Conclusions: RD is a significant complication after KPro surgeryoccurring in 14.7% of 95 eyes. The visual acuity gain after KProsurgery is lost when a retinal detachment occurs, despite goodanatomic outcomes in a majority of eyes (77%) after RD repair.Various ocular coexisting co-morbidities and post-KProcomplications contributed to RDs and decreased visual acuity.Commercial Relationships: Sachin Jain, None; Clement C. Chow,None; Jennifer I. Lim, QLT (F), Genentech (R), Regeneron (R);Lawrence J. Ulanski, Allergan (C); Jose De la Cruz, alcon (C),amo (C); Maria S. Cortina, None; Felix Y. Chau, NoneProgram Number: 3474 Poster Board Number: D0101Presentation Time: 11:00 AM - 12:45 PMImplantation of Biosynthetic Collagen III Corneal Implants invivo in Rabbit Eyes: Surgical Technique and ClinicalPerformanceKarin Kobuch 1 , Raphael T. Neuhann 1 , Kerstin Wand 1 , JohannesJunger 3 , Michael Baumann 3 , Roland Ritter 4 , Mohammad M. Islam 2 ,May Griffith 2 , Chris Lohmann 1 . 1 Ophthalmology, TechnischeUniversität Muenchen, Klinikum rechts der Isar, Munich, Germany;2 Regenerative Medicine, University of Linkoeping, Linkoeping,Sweden; 3 MLase, Munich, Germany; 4 Technolas PV, Munich,Germany.Purpose: To assess laser assisted implantation , sutureless fixation byUV crosslinking and clinical performance of biosynthetic collagen IIIimplants in vivo in rabbit eyes after deep anterior lamellarkeratoplasty (DALK).Methods: Corneal implants from recombinant human collagen III(RHC III or RHC/MPC, 300µm thickness, diameter 6mm) wereplaced on the anterior cornea of rabbit eyes (n=12) after performingDALK with either femtosecond (Victus, Technolas PV Munich,Germany) or excimer laser (MLase Germering, Germany). Implantswere prepared in a corresponding design. After application ofRiboflavin 0.1% for 5 minutes UV-crosslinking was performedaccording to a rapid procedure (18mW/cm2 for 5 minutes).Thereafter a soft bandage contact lens was placed and a tarsoraphywas performed. Postoperatively the rabbits were observed over twoor six weeks by OCT and slitlamp biomicroscopy. Finally the eyeswere examined histologically (HE-stained/ picrosirius stainedsections, electronmicroscopy).Results: Femtosecond laser assisted surgery enabled a 3D cut designfor fixation of the implant. Both laser cuts showed excellent surfaceproperties within the corneal bed and implant. Certain types ofimplants showed minor shrinkage after crosslinking, resulting inperipheral missmatch and reduced adhesion. Good corneal adhesionand fixation of the implant could be achieved in areas, where implantand corneal bed were in close contact intraoperatively and aftercrosslinking, demonstrated by OCT and histology. Loss of implantduring follow up occured in 4 cases due to intra- and postoperativecomplications (laser n=2, opening of tarsoraphy n=2). Cornealneovascularisation was observed around the implant in 3 cases, butnever within the implant. Histology showed good integration of theimplant, reepithelisation and repopularisation with keratocytes.Conclusions: Laser assisted surgery and sutureless fixation by UVcrosslinking show an excellent potential to optimize the surgicalprocedure and minimize complications for implantation of collagenIII biosynthetic corneal implants. The procedure may be morecomplicated in rabbit eyes due to anatomical differences like reducedcorneal thickness and different corneal curvature. The parameters forlaser application will have to be further adapted to guarantee exactfitting and contact of implant and recipient cornea.Commercial Relationships: Karin Kobuch, None; Raphael T.Neuhann, None; Kerstin Wand, None; Johannes Junger, MLaseAG (E); Michael Baumann, None; Roland Ritter, TechnolasPerfect Vision (E); Mohammad M. Islam, None; May Griffith,Univ. of Ottawa - OHRI (P); Chris Lohmann, NoneSupport: EURO Nanomed I-CareProgram Number: 3475 Poster Board Number: D0102Presentation Time: 11:00 AM - 12:45 PMThe effect of the presence of preoperative silicone oil, absence ofprior corneal surgery, and postoperative scleral contact lens useon Boston keratoprosthesis outcomesKareem Moussa 1 , John Petrowski 1 , Natalie A. Afshari 2 . 1 Duke EyeCenter, Duke University, Durham, NC; 2 Shiley Eye Center,University of California, San Diego, La Jolla, CA.Purpose: To report outcomes of the Boston keratoprosthesis in eyeswith a preoperative history of silicone oil, eyes with no prior cornealsurgery, as well as eyes that received a scleral contact lens©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapostoperatively.Methods: Retrospective chart review. Preoperative, intraoperative,and postoperative data were collected from a total of 17 eyes from 16patients who received a Boston keratoprosthesis and had previoussilicone oil, absence of prior corneal surgery, or postoperative sclerallens use.Results: 17 eyes from 16 patients were included in the study. Foureyes (24%) had silicone oil prior to keratoprosthesis placement, teneyes (59%) had no prior history of corneal surgery, and four eyes(24%) received a Jupiter scleral contact lens following the operation.In the four eyes that had silicone oil prior to keratoprosthesisplacement, preoperative visual acuity ranged from light perception tohand motion. All four eyes (100%) developed retroprosthesismembranes postoperatively, three of which (75%) required surgicalexcision. Only one eye (25%) attained visual acuity ≥20/200 at afollow-up of 29 months. In the ten eyes with no prior history ofcorneal surgery, preoperative visual acuity ranged from lightperception to 20/400. Postoperatively, eight eyes (80%) attainedvisual acuity ≥20/200 at an average follow-up of 22.8 months. In thefour eyes that received a scleral contact lens (SCL) following theoperation, preoperative visual acuity ranged from hand motion to20/400. Postoperatively, all four eyes (100%) attained visual acuity≥20/200 at an average follow-up of 11.7 months.Conclusions: Implantation of a Boston keratoprosthesis in eyes thatpreviously had silicone oil should be considered with caution as theseeyes may have poor postoperative visual acuity and may developdense retroprosthesis membranes. The Boston keratoprosthesis hasfavorable visual acuity outcomes in eyes without a prior history ofcorneal surgery. Postoperative use of scleral contact lenses in patientswith keratoprosthesis is well-tolerated.Commercial Relationships: Kareem Moussa, None; JohnPetrowski, None; Natalie A. Afshari, NoneProgram Number: 3476 Poster Board Number: D0103Presentation Time: 11:00 AM - 12:45 PMEvaluation Of The Interface Between Keratoprosthesis andDonor Cornea By Anterior Segment OCTRiccardo Scotto, Marina Papadia, Alessandro Bagnis, Carlo EnricoTraverso. Ophthalmology, DiNOGMI, University of Genoa, Genova,Italy.Purpose: The synthetic Keratoprosthesis can restore visual functionin patients affected by corneal blindness that are poor candidates forkeratoplasty. Advances in medical treatment have resulted in amarked improvement in retention rates and visual outcomes. AnteriorSegment (AS) OCT imaging provides additional information aboutthe status of the prosthesis and the donor graft cornea.Methods: AS-OCT imaging was performed at every follow-up visitto analyze prosthesis structure, iris position and interface betweendevice and donor cornea.Results: Five eyes of 5 patients implanted with the Boston type Ikeratoprosthesis, between 2008 and 2011, were included in thisretrospective analysis. Mean corneal thickness near the prosthesiswas 626 ±34 μm (range 549-694 μm) in the nasal sector, and 643 ±59μm in the temporal sector (range 571-657 μm).Conclusions: Spectral-domain AS-OCT allows high resolutionimaging of the keratoprosthesis device-donor cornea interface,precise visualization of the iris profile and quantification of the iridocornealangle. This imaging technique, able to provide highresolutionimages of the anterior segment, can help identify the riskfor keratoprosthesis-related complications, such as corneal meltingand angle closure glaucoma.Commercial Relationships: Riccardo Scotto, None; MarinaPapadia, None; Alessandro Bagnis, None; Carlo Enrico Traverso,MSD (F), Alcon (F), Santen (F), Thea (F), Allergan (F)Program Number: 3477 Poster Board Number: D0104Presentation Time: 11:00 AM - 12:45 PMChondro-ocular graft transfer:: An alternative to allografttransplantation?David Myung 1 , Christopher N. Ta 1 , Edward Yung 2 , Curtis W. Frank 3 .1 Ophthalmology, Stanford University School of Medicine, Byers EyeInstitute, Palo Alto, CA; 2 Santa Clara Valley Medical Center, SantaClara, CA; 3 Chemical Engineering, Stanford University, Stanford,CA.Purpose: To evaluate the use of cartilage as a potential corneal andscleral graft material in order to possibly increase donor tissueavailability worldwide, as well as to explore a new avenue towardcorneal tissue replacement in cases of repeat allograft rejectionthrough chondro-kerato autograft transfer.Methods: Cartilage specimens were harvested from animals fromvarious anatomic locations, and cut into circular discs. Glycerol wasused to dehydrate the samples to varying water content levels. Allspecimens were weighed and measured before and after dehydration.Specimens were then crosslinked at various levels of hydration usingriboflavin-5-phosphate at UV light. The dimensional stability andwater content of the crosslinked samples were then measured. Bothcrosslinked and uncrosslinked, and hydrated and dehydratedspecimens were then processed for histological evaluation andcompared to corneal tissue.Results: Dehydration of cartilage using glycerol renders it lessopaque and more transparent. The effect of crosslinking on thedimensional stability of cartilage tissue were analyzed both in termsof water content changes, transparency, and histological morphology.Conclusions: Much of the developing world lacks access to donorcorneal and scleral tissue. Chondral allografts or xenografts to replacethese tissues could be aninexpensive solution to meet this worldwide clinical need. Inaddition, chondro-kerato autograft transfer may provide a way todeliver viable corneal replacement tissue to the eyes of patients whohave suffered from repeat allograft failures.Commercial Relationships: David Myung, None; Christopher N.Ta, Stanford University (P); Edward Yung, None; Curtis W.Frank, NoneProgram Number: 3478 Poster Board Number: D0105Presentation Time: 11:00 AM - 12:45 PMPolydopamine-coated and polyethylene glycol-impregnatedcorneas as tissue carriers for the Boston KeratoprosthesisSara Bozorg 1 , Kyung Jae Jeong 2 , Samer N. Arafat 1 , Daniel S.Kohane 2 , Claes H. Dohlman 1 . 1 Department of Ophthalmology,Massachusetts Eye and Ear Infirmary, Boston, MA; 2 Department ofAnesthesiology, Division of Critical Care Medicine, Children'sHospital Boston, Harvard Medical School, Boston, MA.Purpose: To assess the integration and resistance to enzymaticdegradation of porcine corneas impregnated with polyethylene glycol(PEG) or coated with polydopamine (PDA). The aim is to be able touse each modified tissue as a carrier for the Boston Keratoprosthesis.Methods: Porcine corneas were lyophilized then rehydrated in eitherA) phosphate buffer saline (PBS), B) PEG solution (10% w/v) forone day then cross-linked with ultraviolet light, C) dopamine solution(2mg/mL in Tris buffer, pH 8.7) for one day, which polymerized(polydopamine, PDA) and coated the corneas. (n = 15 trephinedbuttons in each group). Integration of PEG hydrogel with cornea wasevaluated by scanning electrom microscopy (SEM) as well as by©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneameasuring the compressive moduli. Resistance of the corneas toenzymatic degradation was evaluated by exposure to clostridiumcollagenase solution.Results: Porcine corneas coated with PDA (Group C) showedsignificantly longer degradation time (>91h) than both control(5.0±0.7 h) and PEG treated groups (26±2.7 h). Mechanical strengthtesting revealed a difference between both PEG and PDA treatedgroups compared to the control group.Conclusions: PEG- and PDA-modified corneas showed a markedlyincreased resistance to enzymatic degradation. The creation of amussel inspired PDA coating on corneas may increase the strengthand effectiveness of keratoprosthesis carrier tissue. In addition, PDAmodifiedcorneas turn black which would reduce glare - a majorproblem in many keratoprosthesis cases (aniridia, etc.). Further invivo studies are needed to assess the viability and safety of thesemodified corneas.PMMA structures. Image projections created via confocalmicroscopy show that the depth of cell growth in Gas FoamedPHEMA-PMMA was greater than that observed on Salt PHEMA-PMMA. Day 7 image projections of Gas Foamed PHEMA-PMMAshow viable cells at depths of approximately 100 µm below thesurface on which cells were seeded. For Salt PHEMA-PMMA, thedepth of cell growth is less pronounced at day 7; however, cellproliferation data confirms that Salt PHEMA-PMMA structures arecytocompatible. SEM and μCT data indicate that both structures havea high density of pores. Among the two structures, Gas FoamedPHEMA-PMMA appeared to have the higher pore interconnectivity.For Gas Foamed PHEMA-PMMA, elastic modulus (E) and ultimatetensile strength (UTS) are 4081 ± 808 kPa and 263 ± 66 kPa,respectively. For Salt PHEMA-PMMA, E and UTS are 678 ± 72 kPaand 125 ± 25 kPa, respectively.Conclusions: Pore architecture, mechanical stability, andcytocompatibility are vital design parameters for KPros. PorousPHEMA-PMMA is cytocompatible. Increased pore interconnectivityappears to allow greater cell growth into the body of porousPHEMA-PMMA structures. The polymers appear to be strongenough to be sutured and to maintain their structures under ocularforces as host tissue integrates. KPros made with porous PHEMA-PMMA may provide additional options for patients for whom donorcorneas are inappropriate or inaccessible.Polydopamine modified cornea as carrier for the BostonKeratoprosthesis.Commercial Relationships: Sara Bozorg, None; Kyung Jae Jeong,None; Samer N. Arafat, None; Daniel S. Kohane, None; Claes H.Dohlman, NoneProgram Number: 3479 Poster Board Number: D0106Presentation Time: 11:00 AM - 12:45 PMDesigning a Novel Porous Keratoprosthesis to Promote CorneaCell IngrowthAmelia L. Zellander 1 , Richard A. Gemeinhart 2 , Behrad Milani 3 , AliR. Djalilian 3 , Mohsen Makhsous 4 , Michael Cho 1 . 1 Bioengineering,University of Illinois at Chicago, Chicago, IL; 2 BiopharmaceuticalSciences, University of Illinois at Chicago, Chicago, IL;3 Ophthalmology and Visual Sciences, University of Illinois atChicago, Chicago, IL; 4 Physical Medicine and Human MovementSciences, Northwestern University, Chicago, IL.Purpose: Limited donor cornea supplies and cornea transplantrejection necessitate the development of safe and effectivekeratoprostheses (KPros). This study evaluates cell growth into novelporous polymers that could be used in a corneal replacement device.Methods: Porous Salt PHEMA-PMMA was composed of 10% v/vmethyl methacrylate (MMA), 45% v/v 2-hydroxyethyl methacrylate(HEMA), and 0.07 M sodium chloride. Porous Gas FoamedPHEMA-PMMA was composed of 20% v/v MMA and 40% v/vHEMA. Prior to the introduction of human corneal fibroblasts(HCFs), polymer samples were coated with collagen type I. Cellproliferation and viability were assessed using AlamarBlue and LiveDead Cell Viability assays (Invitrogen), respectively. The structuresof the porous PHEMA-PMMA samples were evaluated usingscanning electron microscopy (SEM) and micro-computedtomography (μCT). Mechanical properties were evaluated in tension.Results: A high level of cell viability was observed on PHEMA-SEM ImagesCommercial Relationships: Amelia L. Zellander, Tebios (F);Richard A. Gemeinhart, None; Behrad Milani, None; Ali R.Djalilian, None; Mohsen Makhsous, None; Michael Cho, NoneSupport: Office of Naval Research N00014-06-1-0100Program Number: 3480 Poster Board Number: D0107Presentation Time: 11:00 AM - 12:45 PMA fish scale-derived scaffold for corneal reconstructionT H. van Essen 1 , Sarah J. Sparks 2 , Lisanne van Zijl 2 , Greg Chen 3 ,Chien C. Lin 3 , Horng J. Lai 3 , Gregorius P. Luyten 1 , Abdoelwaheb ElGhalbzouri 2 , Martine J. Jager 1 . 1 Ophthalmology, Leiden UnivMedical Center, Leiden, Netherlands; 2 Dermatology, Leiden UnivMedical Center, Leiden, Netherlands; 3 Research, Aeon AstronEurope B.V., Leiden, Netherlands.Purpose: A natural occurring, easy obtainable fish scale-derivedcollagen scaffold (FSCS) has been developed for reconstructing thecornea. This FSCS could form a cheap and simple alternative tocurrent keratoprostheses. We assessed in vitro whether the FSCS canbe repopulated by epithelium and fibroblasts. In addition, weevaluated cell adhesion of the FSCS and measured its opticalproperties.Methods: The light scattering and transmission values of the FSCS, adecalcified and decellularized layered build collagen type Iextracellular matrix, were measured. In addition, human cornealepithelial cells (HCECs) and human corneal stromal cells were coculturedwith the FSCS. Dispase and laser treatments were used tooptimize entrance for stromal cells into the FSCS. The cytotoxicity ofFSCS was assessed using an MTT assay. The effect of FSCS oncellular repopulation, morphology, phenotype, and adhesion were©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaanalyzed using an EdU proliferation assay, histology, andimmunhistochemical stainings.Results: The FSCS had a light scattering value of log(s) =1.62 anddirect light transmission of 90%. Human stromal cells and HCECsproliferated on the FSCS. HCECs were positive for differentiationmarker CK3/12, several adhesion markers and deposited laminin.Treatment with dispase II and laser generated horizontal tunnelsallowed stromal cells to extend their cytoplasma in between thecreated spaces.Conclusions: This easy obtainable, natural occurring FSCSdemonstrates a reasonable optical clarity, is able to sustain (primary)corneal epithelium and seem to facilitate attachment for stromal cells.These properties highlight its potential as candidate for cornealreconstruction and as alternative to current keratoprostheses. Moreresearch on cellular adhesion and reduction of light scattering will beperformed.Commercial Relationships: T H. van Essen, Aeon Astron EuropeB.V. (F); Sarah J. Sparks, Aeon Astron Europe BV (E); Lisannevan Zijl, Aeon Astron Europe BV (E); Greg Chen, None; Chien C.Lin, None; Horng J. Lai, Aeon Astron Europe BV (E); GregoriusP. Luyten, None; Abdoelwaheb El Ghalbzouri, None; Martine J.Jager, Aeon Astron (F), Agentschap.nl (F)Support: Agentschap.nl (Ministry of Economic Affairs)manual technique, it exceeded 69°C which is higher than bone’sdenaturation temperature.Conclusions: There was no difference in the effectiveness of the 3bonding agents at 13 months. A dynamic stress test will be necessaryto differentiate between the bonding agents. The µ-mill keeps thetemperature below the denaturation temperature and thus is safer thanthe manual technique. These two new observations can improve theMOOKP surgery technique.Acknowledgments: The authors acknowledge the scientificcontributions of Giancarlo Falcinelli and Andres Bernal.Program Number: 3481 Poster Board Number: D0108Presentation Time: 11:00 AM - 12:45 PMIMPROVEMENTS IN THE MODIFIED OSTEO-ODONTOKERATOPROSTHESIS (MOOKP) SURGERY TECHNIQUEMariela C. Aguilar 1 , Yoh Sawatari 2 , Alex Gonzalez 1 , William Lee 1 ,Cornelis J. Rowaan 1 , Deepa Sathiah 2 , Darlene Miller 3 , Victor L.Perez 3 , Eduardo C. Alfonso 3 , Jean-Marie A. Parel 1 . 1 OphthalmicBiophysics Center, Department of Ophthalmology, Bascom PalmerEye Institute, University of Miami Miller School of Medicine,Miami, FL; 2 Oral Maxillofacial Division, Department of Surgery,University of Miami Miller School of Medicine, Miami, FL;3 Department of Ophthalmology, Bascom Palmer Eye Institute,University of Miami Miller School of Medicine, Miami, FL.Purpose: To assess the long-term effectiveness of 3 bonding agentsthat are periodically subjected to high static pressures on the opticalcylinder face mimicking traumatic ocular incidents, and to develop anautoclavable µ-milling device designed to contour and drill theMOOKP lamina in an efficient and precise manner.Methods: An instrument was fabricated to stabilize the lamina andapply direct pressure to the cemented optical cylinder. The strengthsof cyanoacrylate adhesive (Histoacryl), a 2-part bone cement(Stryker), and the dental acrylic (Jet) were compared. 4 laminas peragent preserved at 36°C in 30cc of BSS with antibiotic (gatifloxacin2µg/cc) and antifungal (amphotericin B 0.2mg/cc) for >12 monthswere subjected monthly to static forces ranging from 200g, 500g, and1kg (1kg force = 200mmHg IOP equivalent). The force range wasexpanded to include 2kg and 3kg at month 13. An autoclavable µ-mill was developed to adapt a commercially available Stryker drill;using extracted canines, laminas were fashioned while monitoringinternal temperatures via two thermocouples.Results: All 12 laminas resisted the applied static forcescorresponding to pressures of 2, 5 and 10kg/cm 2 and the 30kg/cm 2applied at month 13. The µ-mill performance was tested on resectedcadaveric lamina specimens. Planar surfaces are better than 50µm,perpendicularity is better than 1 degree; drilling departure fromovality is less than 10µm assuring excellent fit with the opticalcylinder. The µ-mill was shown to be as efficacious as the manualtechnique, as it took less than 10 min to create a lamina. Moreover,the canine’s canal temperature never exceeded 38°C whereas with theInstrument fabricated to stabilize the lamina and apply direct pressureto the cemented optical cylinder.Autoclavable µ-milling device.Commercial Relationships: Mariela C. Aguilar, None; YohSawatari, University of MIami (P); Alex Gonzalez, None; WilliamLee, None; Cornelis J. Rowaan, None; Deepa Sathiah, None;Darlene Miller, None; Victor L. Perez, Alcon (C), Bausch & Lomb(C), Genentech (C), Cleveland Clinic Foundation (P), Alcon (F),Alcon (R); Eduardo C. Alfonso, Bio Tissue (C); Jean-Marie A.Parel, CROMA (F), InnFocus (F), Abeamed (F), University ofMiami (P)Support: DOD Grant DAMD-W81XWH-09-1-0675; NIHR01EY018624-04, NIH Center Grant P30EY14801, Florida LionsEye Bank, RPB Physician Scientist Grant (VLP), Henri and FloreLesieur Foundation (JMP).371 Refractive SurgeryTuesday, May 07, 2013 2:45 PM-4:30 PMTCC 303 Paper SessionProgram #/Board # Range: 3711-3717Organizing Section: CorneaProgram Number: 3711Presentation Time: 2:45 PM - 3:00 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaIn Vivo Confocal Microscopy Demonstrates a Profound Increasein Immune Dendritic Cells and Decrease in Corneal Nerves inPatients with Post-Refractive Surgery KeratoneuralgiaYureeda Qazi 1 , Shruti Aggarwal 1 , Bernardo M. Cavalcanti 1 , AndreaCruzat 1 , Leslie J. Wu 2 , Perry Rosenthal 2 , Pedram Hamrah 1, 3 .1 Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA;2 Boston Foundation for Sight, Needham, MA; 3 Immune DiseaseInstitute, Harvard Medical School, Boston, MA.Purpose: To analyze subbasal corneal nerve and immune cellchanges in post-refractive surgery corneal pain patients, and theircorrelation to clinical signs and symptoms.Methods: Laser in vivo confocal microscopy (IVCM) images of 17patients with keratoneuralgia after refractive surgery (LASIK: n=28eyes; PRK: n=5 eyes), and 62 controls (n=62 eyes), were analyzedretrospectively by two masked observers for subbasal nerves, andimmune dendritic (DC) and inflammatory cells (IC). Keratoneuralgiawas confirmed by the presence of pain with PROSE devices and lackof a response to proparacaine. Detailed history, symptom assessment(Ocular Surface Disease Index [OSDI] questionnaire) and a clinicalexamination (slit-lamp biomicroscopy) were obtained.Results: Patients had constant pain and photophobia with sensitivityto air (94%) and chemical fumes (47%). Although minimal cornealfluorescein staining was noted, a significant loss in lengths of totalsubbasal nerves compared to controls (12.8±4.7 vs. 24.3±3.9mm/mm2, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasamples are needed. Despite the fact that the refraction was nearplano, 5 eyes lost lines of CDVA due to haze.Commercial Relationships: Karla P. Lopez, None; Gabriela L.Pagano, None; Alejandro Navas, None; Tito Ramirez-Luquín,None; Arturo J. Ramirez-Miranda, Carl Zeiss Meditec (R);Enrique O. Graue-Hernández, NoneProgram Number: 3714Presentation Time: 3:30 PM - 3:45 PMDetermination of the excimer laser ablation rate in the porcinecornea after corneal collagen cross-linking (CXL)Olivier Richoz 1 , Samuel Arba Mosquera 2 , Thomas Magnago 2 ,Farhad Hafezi 1, 3 . 1 Ophthalmology, Geneva University Hospital,Geneva, Switzerland; 2 R&D, Eye Tech Solutions, Kleinostheim,Germany; 3 Ophthalmology, Doheny Eye Institute, Keck School ofMedicine, Los Angeles, CA.Purpose: A combination of collagen cross-linking (CXL) andcustomized surface ablation, performed sequentially, may be apromising means to correct for part of the irregular astigmatism inkeratoconus and postoperative ectasia. Accordingly, surgeons will beconfronted with patients that will present with previously crosslinkedcorneas requiring excimer laser ablation. Currently, theablation rate per pulse in a cross-linked cornea is unknown, leading topotential inaccuracies in the amount of ablated tissue andpostoperative result.Methods: The excimer laser ablation rate of porcine corneas wasanalyzed using optic coherence pachymetry (OCP) ex vivo. Corneaswere de-epithelialized and soaked with riboflavin 0.1% solution for20 minutes. Riboflavin was washed off the corneal surface andcorneas were cross-linked with 18 mW/cm2 for 5 minutes (CCL-365Vario). Excimer laser ablation was performed (Schwind AMARIS750S) to a total depth of 200 µm in four consecutive steps of 50 µmeach. Controls were treated similarly, but were not irradiated withUV-A. 20 eyes were examined in each experimental condition.Results: The following depth-dependent differences were obtained:the first ablation from 0-50 µm microns showed no significantdifferences in ablation depth between cross-linked corneas andcontrols. For the three consecutive stromal ablations in deep stroma,we observed significantly less ablation in the cross-linked corneawhen compared to non-cross-linked controls: 10.9 µm less for 50 - 99µm (p=0.0001), 7.4 µm less for 100 - 149 µm (p=0.0003), and 7.2µm less for 150 - 199 µm (p=0.003). Statistical analysis wasperformed using the Kruskal-Wallis test.Conclusions: Following CXL, the excimer laser ablation rate in thecornea is significantly lower than in the untreated cornea and seemsto be depth-dependent. Adaptation of the ablation rate anddevelopment of a nomogram for surface ablation in cross-linkedectatic corneas will be important to help improving best spectaclecorrectedvisual acuity.Program Number: 3716Presentation Time: 4:00 PM - 4:15 PMLaser thermal load induces characteristic changes in the cornealsurface including asphericityCommercial Relationships: Olivier Richoz, None; Samuel ArbaSean J. McCafferty, Jim T. Schwiegerling. Arizona Eye Consultants,Mosquera, SCHWIND eye-tech-solutions (E), SCHWIND eye-tech-Tucson, AZ.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.solutions (P); Thomas Magnago, Schwind eye-tech-solutions (E);Farhad Hafezi, Schwind (F), Ziemer (F), PCT/CH 2012/000090 (P)Program Number: 3715Presentation Time: 3:45 PM - 4:00 PMEnhanced Screening for Ectasia Susceptibility among LASIKCandidatesRenato Ambrosio Jr 1, 2 , Isaac C. Ramos 1 , Ana Laura C. Canedo 1 ,Allan Luz 1, 2 , Rosane Correa 1 , Frederico P. Guerrra 1 .1 Ophthalmology, Rio de Janeiro Cornel Tomography andBiomechanics Study Group, Rio de Janeiro, Brazil; 2 Ophthalmology,Federal University of Sao Paulo, Sao Paulo, Brazil.Purpose: To develop objective methods to detect preoperativeectasia risk (susceptibility) among LASIK candidates consideringclinical data, front surface curvature (topometric) data, and 3-Dpachymetric and elevation (tomographic) data.Methods: A retrospective nonrandomized study involved 23 eyesthat developed ectasia after LASIK and 266 eyes with stable LASIKoutcomes (minimal follow up of 12 months). Preoperative clinicaldata and Oculus Pentacam data were available for all cases. ClassicERSS (Ectasia Risk Score System) was calculated based on age,spherical equivalent, residual stromal bed, central thickness, andsubjective classification of corneal topography (front surface axialcurvature map). Front surface curvature (topometric) andtomographic (thickness profile and front/back elevation) indices wereassessed. Non parametric Mann-Whitney’s test was performed toassess differences between the groups. Different combinations thatbest distinguished ectasia and stable LASIK groups were createdusing Fisher's linear discriminant analysis (LDA) based on clinicalparameters plus topometric data, and on clinical parameters plustomographic data. The area under the ROC curve (AUC) werecalculated for each LDA functions with pairwise comparisons.Results: Statistically significant differences were found among thegroups for all tested parameters (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Examine excimer laser thermal load as the elusive etiologyfor corneal asphericity.Methods: Cadaveric porcine eyes were pressurized and stabilized forprocessing and imaging. Both a scanning excimer laser and a CO2laser were used to delivered a uniform disk of radiant energy to theexposed corneal stromal surface. Thermal load was determined bymeasuring corneal surface temperature during irradiation. Cornealprofilometry was measured with broad-band optical interferometrybefore and after laser irradiation. Photomicrographs of the stromalsurface were taken before and after irradiation and the images weresuperimposed to examine changes in positional marks examiningmechanical alterations in the stromal surface.Results: Uniform scanning excimer laser ablation to corneal stromaproduces a significant central steepening and peripheral flattening inthe central 3mm diameter. Q-values, measuring asphericity in thecentral 2mm of the cornea, were significantly lower pre-ablation thanpost ablation (-5.03+/-4.01vs.-52.4+/-18.7,respectively). Surfaceroughness increased significantly following ablation. The central2mm of the stromal surface contracted by 2.21%+/-0.80% at asustained temperature of 5C. Isolated thermal load from uniform CO2laser irradiation without ablation also produces central cornealsteepening and paracentral flattening in the central 3mm diameter. Q-values, measuring asphericity in the central 2mm of the corneaincreased significantly and it was correlated with the temperature rise(R2=0.767). Surface roughness increased significantly and was alsocorrelated with temperature rise (R2=0.851). The central stromalsurface contracted and underwent characteristic morphologic changeswith the applied thermal load which correlated well with thetemperature rise (R2=0.818).Conclusions: : The thermal load created by laser irradiation creates acharacteristic spectrum of morphologic changes on the porcinecorneal stromal surface which correlates to the temperature rise and isnot seen inorganic, isotropic material. The highly similar surfacechanges seen with both lasers are likely indicative of temperatureinduced transverse collagen fibril contraction and stress redistribution.Refractive procedures which produce significant thermalload should be cognizant of these morphological changes.Corneal thermal response:BeforeCommercial Relationships: Sean J. McCafferty, None; Jim T.Schwiegerling, Alcon Laboratories (F), Wavetec (F), Visioneering(C)Program Number: 3717Presentation Time: 4:15 PM - 4:30 PMEffect of small aperture intra-corneal inlay on peripheral kineticvisual fieldsEric T. Brooker, Abhiram S. Vilupuru, George O. Waring. AcuFocus,Irvine, CA.Purpose: The KAMRA intra-corneal inlay (AcuFocus, Inc.)alleviates the symptoms of presbyopia by extending the depth offocus through small aperture optics. Following monocularimplantation over a patient's coaxially sighted corneal reflex, theopaque inlay, which has an overall diameter of 3.8mm and a centralaperture of 1.6mm, only allows central light rays to reach the retina,therefore increasing the eyes depth of focus. The main objective ofthis study was to evaluate visual acuity and the extent of the visualfield following implantation of a small aperture corneal inlay.Methods: Four subjects were implanted monocularly with the inlayin their non-dominant eye. Visual acuity and pupil size were recordedin conjunction with the visual field testing. Automated Goldmannkinetic perimetry was performed in both implanted and nonimplantedeyes using the HAAG-STREIT Octopus 900. Goldmannsize III4e targets moving at a speed of 5o/sec were presented along16 isopters spanning the full extent of the visual field. Total area aswell as extent of the field in superior, inferior, nasal and temporaldirections was calculated. The data is presented as mean ± sd andstatistical comparisons were performed using Student's t test.Results: UCDVA at time of study was 0.035 ± 0.11 logMAR in theimplant eye and -0.065 ± 0.07 logMAR in the non-implant eye.UCNVA was 0.01 ± 0.13 logMAR in the implant eye and 0.60 ± 0.05logMAR in the non-implant eye. Mesopic pupil size for implant andnon-implant eye was 5.5 ± 0.7 and 5.5 ± 1.1 mm respectively. Totalarea of the visual field in the implant eye was 12825 ± 2080 deg2compared to 12321 ± 1383 deg2 in the non-implanted eye (p = 0.25).Extent of the visual field in the implant and non-implanted eyes was,superiorly (47.5 ± 12.5, 48 ± 11 deg), inferiorly (63.5 ± 1.3, 64.5 ±0.6 deg), nasally (58.8 ± 7.4, 56.5 ± 3.3 deg) and temporally (83.3 ±4, 83.5 ± 4 deg).Conclusions: Implantation of a small aperture intra-corneal inlayimproved UCNVA while maintaining UCDVA. The inlay did notdecrease the extent of visual field as demonstrated by the lack ofdifference in total area and extent of kinetic visual field whencomparing the implanted and non-implanted eyes.Commercial Relationships: Eric T. Brooker, AcuFocus, Inc. (C);Abhiram S. Vilupuru, AcuFocus (E); George O. Waring,AcuFocus (C)378 Corneal Wound Repair, Transparency ITuesday, May 07, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 3865-3913/D0109-D0157Organizing Section: CorneaAfterProgram Number: 3865 Poster Board Number: D0109Presentation Time: 2:45 PM - 4:30 PMInhibition of Kir4.1 (KCNJ10) by miR-205 stimulatesTGFA/EGF release in human corneal epithelial cellsDaohong Lin, Adna Halilovic, Sherin Thomas, Kemeng Wang, PengYue, Lars Bellner. Pharmacology, New York Medical College,Valhalla, NY.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To test the hypothesis that miR-205 plays a key role instimulation of growth factors release, TGFA and EGF, in woundedcorneal epithelial cell by targeting 3-UTR of KCNJ10, therebyinhibiting the K channel activity which leads to Ca2+ influx andactivating EGFR/ERK phosphorylation.Methods: The expression level of miR-205 and KCNJ10 wereexamined by stem-loop qRT-PCR and western blot in cultured HCEcells. The wound-healing assay was used to examine the recoveryrate of HCE cells. TGFA and EGF in the media, an index as growthfactor secreted by HCE, were measured by ELISA. We used dualluciferase activity as a report gene to assess the effect of miR-205 onKCNJ10 expression. To study the role of miR-205 and KCNJ10 inregulation of HCE function, the HCE cells were transfected withmiR-205 mimic or KCNJ10 siRNA. The patch-clamp technique wasused to measure the K currents in HCE cells.Results: Mechanic scratching HCE cells stimulated the expression ofmiR-205 by 50% within 1 hour and decreased the expression ofKCNJ10 within 24 hours. Application of miR-205 antagomersignificantly delayed the healing process of HCE, an effect waspartially revered by inhibiting K channels with BaCl2. Moreover,inhibition of kcnj10 channels with shRNA also partially restored thedelayed healing by miR-205 antagomer. The possibility that increasein miR-205 in wounded HCE cells facilitates the cell regeneration byinhibiting inward rectifier K channels was also confirmed by thefinding that miR-205 decreased luciferase report gene activity in cellstransfected with 3’UTR of KCNJ10. Moreover, Western blot andpatch-clamp experiments demonstrated that suppression of theendogenous mir-205 expression enhanced the expression in KCNJ10but not KCNJ16 in plasma membrane and inward K currents in HCEcells by 150%. The possibility that inhibition of K channels by miR-205 may stimulate growth factor release through a depolarizationinducedCa2+ signaling is also suggested by the finding thatinhibition of KCNJ10 by siRNA increased TGFA/EGF release intomedia through upregulation of EGFR/ERK pathway.Conclusions: We conclude that miR-205 is one of the importantfactors stimulating healing of wounded HCE cell by inhibitingKCNJ10 inwardly-rectifying K channels thereby enhancingTGFA/EGF release and facilitating the repair process.Commercial Relationships: Daohong Lin, None; Adna Halilovic,None; Sherin Thomas, None; Kemeng Wang, None; Peng Yue,None; Lars Bellner, NoneSupport: AHA 11SDG7360052Program Number: 3866 Poster Board Number: D0110Presentation Time: 2:45 PM - 4:30 PMEffects of In-vivo Application of Cold Atmospheric Plasma onCorneal Wound Healing in New Zealand White RabbitsRashed Alhabshan 1 , David Belyea 1 , Mary Ann Stepp 1, 2 , JeffreyBarratt 1 , Sanjeev Grewal 1 , Alexey Shashurin 3 , Michael Keidar 3 .1 Department of Ophthalmology, George Washington University,Washington, DC; 2 Department of Anatomy and RegenerativeBiology, George Washington University, Washington, DC;3 Department of Mechanical and Aerospace Engineering, GeorgeWashington University, Washington, DC.Purpose: Cold Atmospheric Plasma (CAP) has been shown to reducecorneal infection but little is known about the impact of CAP onhealthy corneal tissues and their ability to respond to injuries. Toexamine the effect of Cold Atmospheric Plasma (CAP) on woundhealing after corneal epithelial and basement membrane ablation inNew Zealand white rabbits.Methods: Twelve New Zealand white rabbits were assigned into twogroups, Group A (7 rabbits) and Group B (5 rabbits). Five rabbitsfrom each group underwent surgical intervention to the right eye, a 6mm central corneal ablation to the epithelium and stroma using anAlger brush with 0.5 mm burr. After ablation, all rabbits in group Areceived 120s application of CAP. Two rabbits in Group A receivedCAP application without ablation. Eyes monitored for haze,inflammation, and reepithelialization with slit lamp examination. 24hours after ablation, two corneas were harvested from each group.The 20th day, the remaining corneas were harvested, fixed informalin and stained with H&E. In addition, immunofluorescencemicroscopy was performed to assess scar formation using antibodiesagainst fibronectin and αsmooth muscle actin.Results: Corneal reepithelialization on day 1 showed that rabbits ingroup A (treated) had an average epithelial defect of 9.25 mm2 andgroup B (untreated) had a defect of 12.05 mm2, not a statisticallysignificant difference. H & E stained sections showed the expectedresponses of stromal fibroblasts to debridement injury in both groups.Epithelial thickness and stromal cell counts on both groups 20 daysafter injury showed no significant statistical differences. At 24 hoursand 20 days after injury, analyses show that CAP-treatment increasedfibronectin deposition in the anterior stroma and αSMA localizationwithin stromal fibroblasts; quantitative analysis ofimmunofluorescence data will be needed to determine whether thesedifferences are significant.Conclusions: CAP treatment following corneal injury does notinterfere with rate of wound closure or induce increasedinflammation. CAP treatment did affect corneal tissues since αSMAand fibronectin localization within stromal fibroblasts appears toincrease slightly after CAP treatment regardless of whether corneashave been wounded. More studies are needed to evaluate thepotential effects of CAP on the cornea and its possible applications inthe field of Ophthalmology.Commercial Relationships: Rashed Alhabshan, None; DavidBelyea, None; Mary Ann Stepp, None; Jeffrey Barratt, None;Sanjeev Grewal, None; Alexey Shashurin, None; Michael Keidar,NoneSupport: "GWU MFA Award"; "NIH RO1 EY08512 to MAS"; "TheMansour F Armaly Glaucoma Research Fund (fund # ET11161)"Program Number: 3867 Poster Board Number: D0111Presentation Time: 2:45 PM - 4:30 PMCorneal optical density during Hyperbaric Oxygen TherapyKnut Evanger 1 , Guro Vaagbø 2 , Einar Thorsen 2, 3 , Olav H. Haugen 1, 4 .1 Institute of Clinical Medicine, University of Bergen, Bergen,Norway; 2 Hyperbaric Medicine Unit, Dept. of OccupationalMedicine, Haukeland University Hospital, Bergen, Norway; 3 Instituteof Medicine, University of Bergen, Bergen, Norway; 4 Department ofOphthalmology, Haukeland University Hospital, Bergen, Norway.Purpose: To evaluate the influence of hyperbaric oxygen therapy onthe anterior corneal optical density and the refractive index. Little isknown about the effect on the anterior corneal surface when the eyeis exposed to enhanced oxygen concentration and pressure.Methods: 16 patients (32 eyes) were receiving 100% oxygen at apressure of 240 kPa in a monoplace chamber for 90 min daily.Anterior corneal optical density was measured with the densitometryprogram of the Pentacam Scheimpflug imaging system (OculusOptikgeräte GmbH) at baseline and repeated after completed 19 daysof treatment. The 180-degree rotating Scheimpflug camera capturesoptical density data according to the light scattering intensity of theanterior cornea. The density measurement values are standardizedfrom 0 (no clouding) to 100 (tissue completely opaque). TheGladstone-Dale constant was calculated at baseline by using theGladestone-Dale formula, the mean anterior corneal optical densityvalue, and a refractive index of 1.376 of the normal human cornea.Results: Mean anterior corneal optical density and refractive index©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawere 26.05 ± 1.48 and 1.376 (normal human cornea) at baseline, and26.43 ± 1.48 and 1.381 ± 0.02 after 19 days of treatment (p < 0.15).The resultant Gladestone-Dale constant was 0.014. No change incorneal transparency was revealed by the biomicroscope slitlamp.Conclusions: No significant change in anterior corneal opticaldensity and refractive index were present after the hyperbaric oxygentherapy, only a small increase from baseline appeared.Commercial Relationships: Knut Evanger, None; Guro Vaagbø,None; Einar Thorsen, None; Olav H. Haugen, NoneClinical Trial: Project no: 3.2009.736Program Number: 3868 Poster Board Number: D0112Presentation Time: 2:45 PM - 4:30 PMOsteo-odonto-keratoprosthesis (OOKP) and the testing of threedifferent adhesives for bonding bovine teeth with optical PMMAcylinderKristin Weisshuhn 1 , Isabelle Berg 2 , Daniel Tinner 3 , Christoph Kunz 2 ,Michael M. Bornstein 4 , Markus Steineck 5 , Konrad Hille 6 , DavidGoldblum 1 . 1 Ophthalmology, University Hospital Basel, Basel,Switzerland; 2 Maxillofacial Surgery, University Hospital Basel,Basel, Switzerland; 3 Practice of Dentistry and Reconstruction, Basel,Switzerland; 4 Oral Surgery and Stomatology, School of DentalMedicine, University Bern, Bern, Switzerland; 5 Dentistry, UniversityBasel, Basel, Switzerland; 6 Ophthalmology, Ortenau ClinicOffenburg, Offenburg, Germany.Purpose: The preparation of the lamina in osteo-odontokeratoprosthesis(OOKP) is complex and its longevity and watertightness important. Until now only acrylic bone cements have beenused for bonding the optical cylinder into the dentin of the tooth. Ouraim was to evaluate different dental adhesives for OOKPpreparations.Methods: Specimens of bovine teeth were produced by preparing 1.5mm thick slices with holes of 3.5 mm in diameter in the dentin of thetooth. Every group (n=10 per group) was luted with a differentadhesive: Classic Poly-(Methyl Methacrylat) bone cement, auniversal resin cement and a glass ionomer cement. Every specimenof each group underwent force measurement completed with auniaxial traction machine.Results: The highest mean force was measured for Poly-(MethylMethacrylat) bone cement (128.2 N), followed by universal resincement (127.9 N) with no statistically significant difference. Glassionomer cement showed significant lower force values (78.1 N).Conclusions: Excellent bond strength combined with the easyapplication was found for universal resin cement, which mightbecome an alternative to acrylic bone cement in preparing an OOKP.Commercial Relationships: Kristin Weisshuhn, None; IsabelleBerg, None; Daniel Tinner, None; Christoph Kunz, None; MichaelM. Bornstein, None; Markus Steineck, None; Konrad Hille, None;David Goldblum, NoneProgram Number: 3869 Poster Board Number: D0113Presentation Time: 2:45 PM - 4:30 PMDifference in ocular damage by 40 and 95 GHz exposure torabbit eyeMasami Kojima 1, 2 , Nailia Hasanova 1 , Hiroshi Sasaki 1 , KazuyukiSasaki 1 . 1 Vis Res for Environmtl Hlth/Med Res Inst, Kanazawa MedUniv, Kahoku, Japan; 2 Medical Chemistry, Nursing school ofKanazawa Medical University, Kahoku, Japan.Purpose: Millimeter waves (MMW) are prevalent in high-speedwireless communication, automobile collision prevention systemsand high-resolution radar imaging. We examined frequency (40 and95 GHz) dependent differences of corneal damage in rabbit eye.Methods: Pigmented rabbits (N=48, Dutch, 13-16 week-old) wereexposed unilaterally to 95 GHz MMW at 200 mW/cm 2 for 6 or 10minutes, and 40 GHz MMW at 200, 300, 400 mW/cm 2 for 6 min bylens antenna. Systemic anesthesia during exposure and ocularexamination was induced by medetomidine hydrochloride (0.5mg/kg). Ocular changes were evaluated by slit-lamp. Corneal surfacetemperature during exposure was recorded by thermograph camera(R300, NEC Avio). Microencapsulated thermochromic liquid crystal(MTLC) injected into the anterior chamber prior to exposure withcolor change recorded by video camera during exposure revealedheat transport.Results: Corneal surface temperature following 95 GHz 200mW/cm 2 , 6 and 10 min exposure was 42.6±2.1 degrees Celsius (C)and 43.2±1.3 degrees C, respectively (NS). Representative oculardamage were diffuse corneal epithelial cell damage immediately afterexposure, corneal epithelial defect and corneal edema 1 day afterexposure. Exposure for 10 min caused more severe cornealepithelium defect (12/12 eyes) than for 6 min (4/7 eyes). Maximumcorneal surface temperatures by 40 GHz at 200, 300 and 400mW/cm 2 were 41.4±1.1, 42.5±1.1, and 45.5±2.1 degrees C,respectively. Exposure for 6 min to 40 GHz at 200 mW/cm 2 causedtransient corneal diffuse damage (3/6 eyes) and at 300 mW/cm 2diffuse corneal epithelial damage (6/6 eyes). Diffuse cornealepithelial cell damage occurred immediately following exposure at400 mW/cm 2 (3/5 eyes), and corneal epithelial defect (2/4 eyes) andcorneal edema (2/4) 1 day after. Morphological evaluation showed 95GHz at 200 mW/cm 2 and 40 GHz at 400 mW/cm 2 , for 6 min, causedsimilar damage. Corneal surface penetration depth of 40 and 95 GHzwas 0.59 and 0.31 mm, respectively. MTLC analysis revealed that 40GHz exposure transported heat to iris and lens by aqueous humorconvection within 20 sec, compared to 60 sec by 95 GHz (Fig).Conclusions: Ocular heat effects by 40 and 95 GHz MMW differedwith 95 GHz being more severe. MMW penetration depth, heattransport, and dissipation from cornea play important roles in oculardamage.Aqueous humor convection by 40 or 95 GHz exposureCommercial Relationships: Masami Kojima, None; NailiaHasanova, None; Hiroshi Sasaki, None; Kazuyuki Sasaki, NoneSupport: The Committee to Promote Research on the PotentialBiological Effects of Electromagnetic Fields, Ministry of InternalAffairs and Communications, JapanProgram Number: 3870 Poster Board Number: D0114Presentation Time: 2:45 PM - 4:30 PMRetinoids improvements during the treatment by humanamniotic membrane of corneal alkali burns in a mouse modelNicolas Bonnin 1, 2 , Loic Blanchon 2 , Corinne Belville 2, 3 , GeorgesSouteyrand 1 , Frederic Chiambaretta 1, 2 , Vincent Sapin 2 .1 Ophthalmology, Gabriel Montpied Hospital, Clermont-Ferrand,France; 2 R2D2 EA7281, Auvergne University, Clermont-Ferrand,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFrance; 3 GRED INSERM 1103, Auvergne University, Clermont-Ferrand, France.Purpose: The aim of this study is to establish the effects of a humanamniotic membrane (HAM) treatment by retinoids (active derivativesof vitamin A); just before its graft on a corneal alkali burn using amouse model.Methods: This protocol was approved by the regional ethiccommittee (CEMEA Auvergne). Thirty eyes of male mice,dispatched into 5 groups: the first group was untreated, the othersreceived a treatment by HAM grafts prepared according to fourdifferent conditions and one control: non cultivated (control group),cultivated during 24 hours in a standard culture medium, cultivated ina culture medium containing all-trans retinoic acid (ATRA) dissolvedin dimethylsulfoxide (DMSO) and cultivated in a culture mediumcontaining DMSO. Induction of HAM by ATRA was assessed byPCR quantification of RARβ (already published as a gene induced byretinoids). The photographs evaluated corneal re-epithelialization,histopathology evaluated ulceration depth, immunohistochemicalanalysis and signal quantification numbered the expression of MMP9and VEGF.Results: For the ulcers surface, we noted a significant differencebetween the groups HAM cultivated during 24 hours without or withATRA compared to the untreated group; and any significantdifference between the group HAM cultivated in a mediumcontaining DMSO and the untreated group. Concerning the ulcersdepth, there was a significant difference between the group treated byHAM cultivated 24 hours with ATRA compared to the untreatedgroup. Immunohistochemistry assays showed that MMP 9 and VEGFexpression were decreased in the groups treated with HAM cultivatedduring 24 hours with ATRA compared to the other groups.Conclusions: The positive effects of HAM culture show itsbiological effect more than its mechanical effect. DMSO appears toreduce the effect of ATRA, implying quickly the use of a new nontoxicvector for clinical applications. Nevertheless, our results clearlyshowed that the induction of MAH by ATRA appears to have abeneficial effect on the ulcers depth and the expression of MMP 9and VEGF. Our preliminary results are really promising in order topropose a positive engineering of HAM applied for wound healinggraft on cornea in the next future.Commercial Relationships: Nicolas Bonnin, None; LoicBlanchon, None; Corinne Belville, None; Georges Souteyrand,None; Frederic Chiambaretta, None; Vincent Sapin, NoneProgram Number: 3871 Poster Board Number: D0115Presentation Time: 2:45 PM - 4:30 PMExamination of Corneal Anatomy in Attempted Ninety PercentDepth Suture Repair of Traumatic Corneal Lacerations, UtilizingPost-Operative High Resolution Anterior Segment OCT ImagingAlice C. Lorch, Peter Veldman. Ophthalmology, Massachusetts Eye& Ear Infirm, Boston, MA.Purpose: To assess suture placement and the anatomic restoration ofcorneal anatomy in attempted 90 percent depth (partial thickness)suture repair of corneal lacerations, utilizing post-operative anteriorsegment OCT imaging.Methods: Five patients with attempted 90 percent suture depth repairof traumatic corneal lacerations were examined with high resolutionanterior segment OCT following open globe repair. Included patientssustained Zone 1 open globe injuries that were repaired by the MEEItrauma service using standard partial thickness suturing technique.Imaging of the cornea utilizing high-resolution anterior segment OCTwas performed cross-sectionally over each corneal suture within oneweek of repair. Primary outcomes included depth of each cornealsuture (%) on each side of the wound, endothelial misalignment ateach suture (microns) and difference in corneal thickness betweeneach side of the wound (%) in instances of this misalignment.Results: Anterior segment OCT of corneal sutures in one pilotpatient revealed a wide range of depths from 50% to full thickness. Inone illustrative pass, the corneal suture depth was 55% on one sideand 42% on the other side of the wound, with 135 microns ofmisalignment of the endothelium. The cornea thickness on the side ofthe wound with stromal exposure was 9% increased. Analysis of allincluded patients in this study is pending.Conclusions: Despite the high frequency of corneal laceration repairsafter trauma, there are no studies examining the post-operativeanatomy of attempted partial thickness sutured corneal repair. Thisstudy serves to establish a baseline regarding the actual depth of thesesutures in a series of patients with traumatic corneal lacerations. Italso serves to examine the degree of endothelial misalignment, andsubsequent edema due to stromal exposure, in these attempted partialthickness closures. This is significant because corneal edema afterlaceration repair can hinder post-operative monitoring and treatmentof posterior segment pathology and may lead to penetratingkeratoplasty or keratoprosthesis to facilitate timely retinal surgerywhen indicated. This study is the first to utilize high resolutionimaging techniques to establish these baseline characteristics ofattempted partial thickness cornea repair in ocular trauma.Commercial Relationships: Alice C. Lorch, None; Peter Veldman,NoneProgram Number: 3872 Poster Board Number: D0116Presentation Time: 2:45 PM - 4:30 PMTransglutaminase-2 Knockdown Effect Epithelial-MesenchymalTransition during Corneal Wound HealingAihua Hou 1 , Louis Tong 1, 2 . 1 Singapore Eye Research Institute,Singapore, Singapore; 2 Singapore National Eye Center, Singapore,Singapore.Purpose: Transglutaminase-2 (TGM-2) is a multifunctional crosslinkingenzyme involved in wound healing. Cornea wound closurewas delayed in TGM-2-/- knockout mice. Also, Knockdown ofTGM-2 reduced epithelial cell adhesion and migration. Epithelialmesenchymaltransition (EMT) was suggested to be involved inwound healing in cornea epithelial cells. This study aimed toinvestigate whether TGM2 defect perturbs EMT during cornealwound healing.Methods: The central cornea of anesthetized mice was marked by atrephine, and the epithelium was peeled off using forceps under adissecting microscope. Mouse eyes were harvest at different timepoints and embedded in OCT, and then were sectioned with cryostatat 8µM thickness. Immunofluorescent staining was performed onmouse eye sections using anti-Slug, anti-Twist and anti-β-Cateninantibodies.Results: In TGM2+/+ mice, EMT marker Slug was observed 4hrsfollowing corneal abrasion, while in TGM2-/- mice, slug wasobserved at 24hours. Interestingly, in both TGM2+/+ and TGM2-/-mice, Slug was expressed only in the nuclei of cells at basal layer ofthe impaired epithelium. By immunostaining, at both naive andwounded cornea epithelium, TGM2 +/+ mice had stronger β-cateninexpression than TGM2-/- mice. In TGM2+/+ mice, strong β-Cateninstaining was observed at the advancing edge of corneal epithelium 4hrs after abrasion, this phenomenon was absent at the wound edge inTGM2-/- mice. Weak Twist staining was observed in both naive andwounded corneal epithelium, but no obvious difference betweenTGM2+/+ and TGM2-/- mice.Conclusions: TGM-2 knockdown delayed Slug expression inwounded corneal epithelium, and also reduced β-Catenin expression.These findings may suggest perturbation of EMT, which has©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaimplications for wound response. The therapeutic significance is inthe modulation of wound healing via TGM-2 in ocular surfacediseases.Commercial Relationships: Aihua Hou, None; Louis Tong, NoneSupport: This research is supported by the Singapore NationalResearch Foundation under its NMRC/TCR/002-SERI/2008 andadministered by the Singapore Ministry of Health’s National MedicalResearch Council.Program Number: 3873 Poster Board Number: D0117Presentation Time: 2:45 PM - 4:30 PMOcular Phenotype and Therapeutic Interventions in theEctodermal Dysplasia Keratitis-Ichthyosis-Deafness SyndromeDavid J. Armstrong 1 , Maeve Lagan 1 , Janet E. Sinton 2 , Stephen B.Kaye 3 , Colin E. Willoughby 4 . 1 Department of Ophthalmology, RoyalVictoria Hospital, Belfast, United Kingdom; 2 Department ofOphthalmology, Altnagelvin Area Hospital, Londonderry, UnitedKingdom; 3 St Paul's Eye Unit, Royal Liverpool Hospital, Liverpool,United Kingdom; 4 Centre for Vision and Vascular Science, RoyalVictoria Hospital, Belfast, United Kingdom.Purpose: The purpose of the study was to report the ocularmanifestations, the clinical course, and the therapeutic managementof four patients with KID syndrome who had a molecular diagnosis.Methods: Four patients with KID syndrome (KID ; MIM148210)from across the UK were recruited for a general and ocularexamination and GJB2 (Cx26) mutational analysis. The ocularassessment included best corrected visual acuity, slit lamp biomicroscopyand ocular surface assessment. Mutational analysis of thecoding region of GJB2 (Cx26) was performed by bidirectionalSanger sequencing. Specific therapeutic interventions using oralketaconazole were performed in 2 patients supplemented with subconjunctivalbevacizumab injection in one patient.One patientreceived both sub conjunctival and intracorneal bevacizumab.Results: All four affected individuals had the characteristic systemicfeatures of KID syndrome. Main ophthalmic features werevascularising keratopathy, ocular surface disease, hyperkeratotic lidlesions, recurrent epithelial defects and corneal stromal scarring.Each patient was found to have missense mutation in KID syndrome,which results in substitution of aspartic acid with asparagine at codon50 (p.D50N). In one patient, multiple surgical procedures, includingsuperficial keratectomies and lamellar keratoplasty, failed to preventsevere visual loss. In contrast, oral therapy with ketaconzolestabilised the corneal and skin disease in two other patients with KIDsyndrome. The patient who underwent intracorneal bevacizumabinjection showed a marked reduction in corneal vascularisationfollowing a single application.Conclusions: KID syndrome is a rare ectodermal dysplasia causedby heterozygous mutations in GJB2 (Cx26) with a severe,progressive vascularising keratopathy. It has been suggested that oralketoconazole therapy may play a role in the prevention of KIDsyndrome associated corneal disease, in contrast to surgicalprocedures which have a high failure rate. Oral ketaconazole therapyoffers benefit in stabilising the corneal and skin disease and furtherstudies in a larger series of patients is warranted. The role of subconjunctivalbevacizumab has not been established, however theintracorneal injection of bevacizumab appears to have localisedbenefit with decreased corneal vascularisation in one patient.Commercial Relationships: David J. Armstrong, None; MaeveLagan, None; Janet E. Sinton, None; Stephen B. Kaye, None;Colin E. Willoughby, NoneSupport: The Ciaran Buckley TrustProgram Number: 3874 Poster Board Number: D0118Presentation Time: 2:45 PM - 4:30 PMNovel Therapeutic Strategies for Corneal TraumaAlex Cohen 1, 2 , Timothy M. Boyce 2 , Xiaowu Gu 2, 1 , Michael H.Elliott 1, 2 . 1 Ophthalmology, Dean McGee Eye Institute, OklahomaCity, OK; 2 Ophthalmology, University of Oklahoma, Oklahoma City,OK.Purpose: To evaluate the role of caveolin-1 (Cav-1) in cornealwound healing.Methods: Immunofluorescence microscopy was performed uponhuman and mouse corneas to identify the pattern of Cav-1expression. Next, primary corneal epithelial cells were prepared frommouse corneas and in vivo wound healing assays were performed. Inaddition, Wild-type (WT) and Cav-1 knockout (KO) mice weresubjected to in vivo corneal epithelial and stromal wounds. Somecorneas were prepared for immunofluorescence microscopy while therate and pattern of wound healing was observed in others.Results: Cav-1 is preferentially expressed in the basal epithelium ofthe limbus of human and mouse corneas. This location is where thecorneal stem cells (CSC) reside. The next experiments showed thatCav-1 immuno-reactivity is increased at the leading edge of the cellsresponsible for closing an epithelial wound both in vitro and in vivo.Next, a 100% epithelial defect was created in the eyes of WT andCav-1 KO mice. Here we found that the Cav-1 KO mouse corneashealed at a faster rate than their WT counterparts. At 96h followingthe wound, the Cav-1 KO corneas were almost completely healed,while the WT mice showed a 50% or greater epithelial defect. Thiswas confirmed in vitro using primary WT and Cav-1 KO culturedcorneal epithelial cells. At 24 hours after the initial wound, the Cav-1KO wound was approximately 80% closed while the WT wound wasonly about 30% closed. Next, mice were subjected to a deep, stromalwound with a diamond burr. The mice were allowed to heal andmonitored for a period of 4 weeks. At the end of this time period thecorneas were imaged using a digital slit lamp camera and we foundthat the corneas of WT mice showed dense central scar formationwhile those of Cav-1 KO were essentially clear.Conclusions: Cav-1 is necessary for proper corneal epithelial woundhealing in vivo and there is compelling evidence for a role in stromalscar formation as well.Commercial Relationships: Alex Cohen, None; Timothy M.Boyce, None; Xiaowu Gu, None; Michael H. Elliott, NoneSupport: Oklahoma Center for Adult Stem Cell ResearchProgram Number: 3875 Poster Board Number: D0119Presentation Time: 2:45 PM - 4:30 PMImpaired Wound Healing In Corneal Stroma Of A Nitric OxideSynthase Type II -deficient MouseTakayoshi Sumioka 1 , Yuka Okada 1 , Norihito Fujita 1 , MasayasuMiyajima 2 , Shizuya Saika 1 . 1 Ophthalmology, Wakayama MedicalUniversity, Wakayama, Japan; 2 Laboratory Animal Center,Wakayama Medical University, Wakayama, Japan.Purpose: To investigate the effects of loss of nitric oxide synthasetype II (NOS2) in the healing of an injured corneal stroma in mice.Cell culture experiments were also conducted to clarify the effects oflacking NOS2 on ocular fibroblast gene expression.Methods: The effect of lacking NOS2 on corneal stroma woundhealing following an incision injury was evaluated by using NOS2-null (KO) mice. Histological, immunohistochemical and mRNAexpression analyses were performed. Ocular fibroblasts from wildtype (WT) and KO mice were used to study the role of NOS2 onfibrogenic gene expression.Results: Healing of an incision injury in corneal stroma was delayedwith less invasion of macrophages and reduction in expression ofaSMA and fibronectin in a KO mouse as compared with a WT©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamouse. In vitro experiments showed that the loss of NOS2suppressed mRNA expression of TGFb1 and collagen Ia1, but notMCP-1, in ocular fibroblasts. Being inconsistent with the in vivofinding, lacking NOS2 upregulated a-smooth muscle actin mRNA insimple ocular fibroblast culture.Conclusions: NOS2 is required for macrophage invasion andprimary healing of injured corneal stroma folowing incision injury.Appearance of myofibroblasts in the injured corneal stroma wassuppressed by lacking NOS2 in vivo, although the loss of NOS2promoted myofibroblast generation in vitro in monolayer culture.Commercial Relationships: Takayoshi Sumioka, None; YukaOkada, None; Norihito Fujita, None; Masayasu Miyajima, None;Shizuya Saika, NoneProgram Number: 3876 Poster Board Number: D0120Presentation Time: 2:45 PM - 4:30 PMMethod Development for Evaluating Clear Corneal CataractWound IntegrityArthur Driscoll 1 , Suzanne LaScalza 2 , Peter K. Jarrett 1 , MichaelMcGrath 1 , Michael Bassett 1 , Amar S. Sawhney 1 . 1 R&D, OcularTherapeutix, Bedford, MA; 2 Clinical Affairs, Ocular Therapeutix,Bedford, MA.Purpose: To develop a quantitative method simulating forces an eyemay experience during patient manipulation post-clear cornealcataract surgery.Methods: A Dontrix Gauge (GAC International Inc., Bohemia,NY)(Figure 1a), which is a spring gauge used to apply and measureforces in orthodontics, was modified to have a 3 mm atraumatic tip.The resulting Calibrated Force Gauge (CFG) allows application ofcalibrated and quantifiable force to the eye in 0.25 ounce increments(Figure 1b). After in-vitro and in-vivo evaluations, an initial clinicalstudy was conducted using the CFG to apply one ounce of forcetemporal to the limbus in healthy volunteers to examine intraocularpressure (IOP) changes and compare the findings with those in theliterature. After confirming the force simulating ocular surfacemanipulation, the CFG was used to challenge clear corneal incisions(CCIs) which were stromally hydrated (Study 2) or closed with 10-0Nylon suture (3-1-1 with buried knot)(Study 3); the CFG was placed0.5 mm temporal to the incision.Results: In the first study, mean baseline IOP was 17.5 mmHg androse to 43.4 mmHg upon applying 1.0 ounce of force using the CFG.Literature states that application of light and firm digital forces on theeye equate to IOP of 27 and 58 mmHg respectively. As a result, 1.0ounce of force appeared adequate to simulate eye touching, rubbing,or forced blinking. When the CFG was used to challenge CCIs closedwith stromal hydration in Study 2 (n=30), 66.7% of wounds leakedwith ≤ 1.0 ounces of force. In the suture group (Study 3), woundswere challenged prior to and after application of the suture. OnlyCCIs which leaked on the initial challenge were sutured andchallenged with the CFG a second time. Out of these wounds, 23.7%leaked with application of ≤ 1.0 ounces of force (Figure 2).Conclusions: Applying one ounce of force proximal to a CCIappears to be a clinically relevant assessment of wound integrity andpropensity to leak under patient manipulation. Reports in theliterature suggest that wounds which leak have a 44-fold increasedrisk for endophthalmitis. This risk is hypothesized to be related towound gape allowing fluids to travel between the ocular surface andanterior chamber. If a wound leaks under CFG manipulation aftercataract surgery, it may be an indication that the incision architectureis not adequate and closure is necessary to prevent postoperativewound leaks.Commercial Relationships: Arthur Driscoll, Ocular Therapeutix(E); Suzanne LaScalza, Ocular Therapeutix (E); Peter K. Jarrett,Ocular Therapeutix (E); Michael McGrath, Ocular Therapeutix, Inc.(E); Michael Bassett, Ocular Therapeutix (E); Amar S. Sawhney,Ocular Therapeutix (E)Program Number: 3877 Poster Board Number: D0121Presentation Time: 2:45 PM - 4:30 PMEffects of Transforming Growth Factor Beta isoforms (TGFβs)On Diabetic Corneal Wound HealingIlham Bettahi, Feng Wang, HAIJING SUN, Fu-shin X Yu.Ophthalmology, Wayne state university, Detroite, MI.Purpose: Background: Pathological conditions such as diabetesmellitus delay corneal epithelial wound healing frequently,potentially resulting in persistent corneal defects and recurrentcorneal erosion. Transforming growth factor Beta (TGFβs) are keymediators of wound healing. To explore the role(s) of TGFβ isoforms(1, 2 and 3) in corneal epithelial wound healing, we assessed theeffect of TGFβ on epithelial wound closure in cultured porcinecornea in the presence of normal or high glucose concentrations.Methods: Methods: Genome-wide cDNA array was performed usingepithelial cells isolated from normal and diabetic corneas prior to and40 h post epithelial debridement wound. The array results wereverified using RT- and realtime-PCR and immuniohistochemistry. Toassess the effects of TGFβ isforms on corneal epithelial woundhealing, wounded porcine corneas were cultured in high glucose (25mM) and treated with recombinant TGFβ1-3.Results: Results: The expressions of TGFβ1-3 were elevated inresponse to wounding and this elevation for TGFβ3 is dampened indiabetic rat corneas. cDNA array analysis revealed several TGFβtargeted genes were also downregulated in diabetic healing CECs.Immunohistochemistry showed that TGFβ3 is highly expressed inentire migrating epithelial sheet in non-diabetic corneas while only afew cells at the leading edge in diabetic cornea. In cultured corneas,TGFβ3, but not TGFβ1, accelerated high glucose delayed epithelialwound closure.Conclusions: Conclusion: In type II diabetic corneas, the expressionof TGFβ3 but not TGFβ1 response to wound is suppressed byhyperglycemia and TGFβ3, an antifibrosis factor, might be used totreat delayed wound healing in the cornea and potentially in the skinof diabetic patients.Commercial Relationships: Ilham Bettahi, None; Feng Wang,None; HAIJING SUN, None; Fu-shin X Yu, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSupport: NIH grants R01 EY017960, EY010869, Midwest EyeBankProgram Number: 3878 Poster Board Number: D0122Presentation Time: 2:45 PM - 4:30 PMNanoparticle Vectored siRNAs Reduce Profibrotic GeneExpression in Wounded Rabbit CorneasSriniwas Sriram 1 , Paulette M. Robinson 2 , Alfred S. Lewin 3 , GregoryS. Schultz 2 . 1 Biomedical Engineering, University of Florida,Gainesville, FL; 2 Ob/Gyn and Opthalmology, University of Florida,Gainesville, FL; 3 Molecular Genetics and Microbiology, Universityof Florida, Gainesville, FL.Purpose: Corneal haze remains a major problem following injury orrefractive surgery. We tested the efficiency of a commerciallyavailable histidine-lysine peptide nanoparticle formulation to deliversiRNAs to a laser ablated cornea, to reduce pro-fibrotic mRNA, andto reduce corneal haze in a rabbit model.Methods: To test the efficacy of siRNA delivery, excimer laserablated rabbit corneas were topically dosed with fluorescently labeledscrambled siRNA sequences complexed with polymeric nanocarriers(22.5 uM). Excised corneas were maintained in organ culture for 8hours then fixed, sectioned, and analyzed via fluorescent microscopy.To test the efficacy of an anti-fibrotic formulation of siRNAsdelivered with these nanoparticles, corneas of nine rabbits wereablated with an excimer laser. One eye was treated with nanoparticlescomplexed with a triple combination of siRNA that targeted majorscarring genes (TGFβ1, TGFβR2 and CTGF), while the other eyereceived empty nanoparticles as a control. To test the molecularefficacy of the siRNAs, corneas of 3 rabbits were excised 24 hourspost-wounding, RNA was extracted and quantified by RT-qPCR.Knockdown percentages of the target genes were compared to thepaired control eyes. To determine therapeutic effect, corneas of 6rabbits were assessed for levels of haze on days 14 and 15 usingdigital photographs, in vivo confocal microscopy, and were gradedfor haze using the standard 0-4 semiquantitative scale.Results: Tissue sections from organ cultured corneas analyzed byconfocal microscopy showed high levels of fluorescence in all layersof the treated cornea when compared to untreated controls. In antifibrotictreated rabbit corneas, the siRNA triple combinationproduced an average knockdown of 57% for TGFB1, 25 % forTGFBR2 and 24% for CTGF. One rabbit had a maximumknockdown of 80% for TGFB1, 57% for TGFBR2 and 46% forCTGF, indicating the siRNA triple combination was effectivelydelivered to the cornea in this animal. Results from haze grading onday 14 showed a decrease in haze formation in three out of the sixtreated rabbits.Conclusions: These results indicate histidine-lysine peptidenanoparticles were effective in delivering siRNAs to all cell layers ofexcimer ablated corneas. With optimization of the dose and deliveryof the triple siRNA combination, this gene targeted therapy may bean innovative treatment to reduce scar formation.Commercial Relationships: Sriniwas Sriram, None; Paulette M.Robinson, None; Alfred S. Lewin, University of Florida (P);Gregory S. Schultz, NoneSupport: US Department of Defense W81XWH-10-1-0917Program Number: 3879 Poster Board Number: D0123Presentation Time: 2:45 PM - 4:30 PMAloe vera: An in-vitro study of effects on corneal wound closureand collagenase activityElizabeth Curto 1 , Amber Labelle 2 , Heather L. Chandler 1 . 1 The OhioState University, Columbus, OH; 2 University of Illinois Urbana-Champaign, Champaign, IL.Purpose: To evaluate the in vitro effects of an aloe vera solution on1) the viability and wound healing response of corneal cells, and 2)the ability to alter collagenase and gelatinase activity.Methods: Primary cultures of corneal epithelial cells and fibroblastswere prepared from grossly normal enucleated canine globes andtreated with an aloe solution (doses ranging from 0.0 - 2mg/mL).Cellular viability was evaluated using a colorimetric assay. A cornealwound healing model was used to quantify cellular ingrowth across adefect made on the confluent surface. Anti-collagenase and antigelatinaseactivity was evaluated by incubating a bacterialcollagenase/gelatinase with aloe solution (doses ranging from 0.0 -500µg/mL) and comparing outcome measures to a generalmetalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum(doses ranging from 0.0 - 100%).Results: None of the concentrations of aloe solution testedsignificantly affected viability of corneal epithelial cells orfibroblasts. Concentrations ≥175µg/mL significantly (p≤0.001)slowed the rate of corneal fibroblast wound closure while aloeconcentrations

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneain HaCaT cells.The excipients did not interfere with effects of BMP-7 and also didnot affect cell viability.Conclusions: BMP-7 with excipients has the ability to reducefibrosis and also the excipients contributed to the stabilization ofBMP-7. As such, it seems possible that this technology can beapplied to eye drops and become a new innovative treatment inpharmaceutical markets.Commercial Relationships: Jin-Wook Jang, None; Hansoo Kim,None; Chan-Young Cho, None; Jinkuk Kim, None; Ju-WoongJang, None; Young-Sik Kim, None; Ynag-je Cho, NoneSupport: 10037842 (Ministry of Knowledge Economy, Repulic ofKorea)Program Number: 3881 Poster Board Number: D0125Presentation Time: 2:45 PM - 4:30 PMGalectin-3 Enhanced Epithelialization in Explanted MonkeyCorneas with Alkali BurnAtsuko Fujii 1, 2 , Thomas R. Shearer 2 , Mitsuyoshi Azuma 1, 2 .1 Laboratory of Ocular Sciences, Senju Pharmaceutical Co, Ltd.,Beaverton, OR; 2 Department of Integrative Biosciences, OregonHealth & Science University, Portland, OR.Purpose: Following an injury leading to the loss of the cornealepithelium, the remaining epithelial cells immediately attempt toclose the defect. Poor healing of epithelial wounds is a major clinicalproblem, leading to persistent epithelial defects and ulceration. Wepreviously showed that carbohydrate-binding protein galectin-3 (Gal-3) enhanced wound closure in explanted monkey corneas laceratedby n-heptanol. Alkali injuries of the eye often cause extensivedamage to the cornea due to rapid penetration and damage to deeperocular structures. The purpose of the present experiment was to studyGal-3 in the more severe alkali burn model, and to compare results tothe n-heptanol model.Methods: An alkali burn was created by a 60 sec application of a 7.5mm diameter filter disc soaked in 1N sodium hydroxide onto thecentral cornea of enucleated monkey eyes. The corneas were thenexcised, incubated for various times with or without recombinantGal-3, and stained with 1% sodium fluorescein. Corneal woundclosure was quantified by digital image analysis.Results: After an alkali burn, the corneal wound area became smallerin a time-dependent manner. Exogenous recombinant Gal-3 enhancedthis wound closure to the same extent as in corneas wounded with n-heptanol. However, data suggested that the rates of healing weredifferent in the two models.Conclusions: Exogenous Gal-3 showed a beneficial effect on closureof wounds caused by either alkali or n-heptanol. In both cases, thismay be because Gal-3 binds to ECMs such as laminin and collagenand promotes lamellipodia formation by cross-linking to α3 integrin.Although initial cytokines released from local cells may be differentin our two chemical models, the results indicate that Gal-3 may be acandidate drug to enhance epithelialization in human cornea damagedby variable causes.Dr. Shearer receives a research contract and consulting fees from, andDr. Azuma and Ms. Fujii are employees of, Senju Pharmaceutical Co.Ltd.Commercial Relationships: Atsuko Fujii, Senju PharmaceuticalCo., Ltd. (E); Thomas R. Shearer, Senju Pharmaceutical Co., Ltd.(F), Senju Pharmaceutical Co., Ltd. (C); Mitsuyoshi Azuma, SenjuPharmaceutical Co., Ltd. (E)Program Number: 3882 Poster Board Number: D0126Presentation Time: 2:45 PM - 4:30 PMMicroRNA-182 Inhibits Human Corneal Epithelial CellProliferation and MigrationDongsheng Yan 1, 2 , Xiaoyan Chen 1, 2 , Jiao Wang 1, 2 , Lili Tu 1, 2 .1 School of Optometry and Ophthalmology, Wenzhou MedicalCollege, Wenzhou, China; 2 State Key Laboratory Cultivation Baseand Key Laboratory of Vision Science, Ministry of Health of P. R.China, Zhejiang Provincial Key Laboratory of Ophthalmology andOptometry, Wenzhou, China.Purpose: MicroRNAs (miRNAs) are endogenous short (~22)nucleotide RNAs which inhibit protein translation through binding totarget mRNAs. Recent studies have demonstrated that miR-182 canregulate tumor cell proliferation and migration. The role of miR-182in corneal wound healing, however, remains unclear. In the presentstudy, we investigated the function of miR-182 in human cornealepithelial cells.Methods: Realtime RT-PCR was performed to detect the expressionof miR-182 in mouse corneal epithelium during wound healingprocess. Human corneal epithelial cells were transfected with miR-34a. MTS and wound-healing assay was carried out to evaluate theeffect of miR-182 on human corneal epithelial cell proliferation andmigration, respectively. The expression of c-Met protein wasdetermined by Western blotting.Results: miR-182 was downregulated during corneal wound healingprocess. Transfection of miR-182 into human corneal epithelial cellsled to a significant decrease in cell proliferation and migration. miR-182 downregulated the expression of c-Met by Western blot analysis.Conclusions: Our results demonstrated that miR-182 inhibitedhuman corneal epithelial cell proliferation and migration bydownregulation of c-Met. This indicates that miR-182 may play animportant role in corneal wound healing process.Commercial Relationships: Dongsheng Yan, None; XiaoyanChen, None; Jiao Wang, None; Lili Tu, NoneSupport: National Natural Science Foundation of China(81071682&81272286)Program Number: 3883 Poster Board Number: D0127Presentation Time: 2:45 PM - 4:30 PMThe effects of IL-6 receptor blockade on gene expressions inexperimental corneal alkali burnSatoshi Sugaya, Tohru Sakimoto, Ai Yamada, Takako Ohnishi, AkikoIshimori, Mitsuru Sawa. Nihon University school of medicine,Tokyo, Japan.Purpose: Using experimental corneal alkali burn model, wepreviously reported the suppressive effect of topical instillation ofanti-Interleukin-6 receptor (IL-6R) antibody on infiltrations ofinflammatory cells. Using this model, we investigated the geneexpressions of inflammation-related factors and chemotactic factors.Methods: Unilateral eye of corneal alkali burn was made using afilter paper dipped in 1 N NaOH solution using BALB/c mice. Threeeyes of 3 mice were treated by topical instillation of anti-IL-6Rantibody solution (MR16-1, 2 µg/µL) from day 5 to day 28 afterwounding. (MR16-1 group)(from day 5 to day 14; 3 times/day, fromday 15 to day 28; 2 times/day) The control group (3 eyes of 3 mice,PBS group) underwent topical 0.01M PBS (pH 7.4) solution. Themice were euthanatized on day 28 and 2-μm-thick sections weremade. Corneal stroma section was made using laser capturemicrodissection system, and total RNA in the specimens wasdetermined by quantitative PCR array method (Mouse Th17Response Array, QIAGEN).Results: In MR16-1 group, MMP (matrix metalloproteinase)-13(6.39±3.54; mean ± SD) expression was decreased by six timescompared with that in PBS group. The chemotactic factors ofmacrophage, MCP (monocyte chemotactic protein)-1 (5.83±1.95)©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand CCL22 (2.78±0.43), were decreased in MR16-1 group comparedwith those in PBS group. In MR16-1 group, IL-17RE, the receptorfor IL-17, was higher two times values than that in PBS group(2.37±0.15).Conclusions: Anti-inflammatory effect of topical instillation of anti-IL-6R antibody could be caused by the suppression of chemokinesand MMP. Because IL-17 producing cells are induced by IL-6signaling, the up-regulation of IL-17R by IL-6R blockade may havethe relationship with IL-6R signal transduction.Commercial Relationships: Satoshi Sugaya, None; TohruSakimoto, None; Ai Yamada, None; Takako Ohnishi, None;Akiko Ishimori, None; Mitsuru Sawa, HOYA Co. (F), SantenPharmaceutical Co. (F)Program Number: 3884 Poster Board Number: D0128Presentation Time: 2:45 PM - 4:30 PMCD11b+GR1+ Myeloid Cells Promote Trigeminal GanglionNeurite Growth: Implications for Corneal Nerve RegenerationSonal Gandhi, Joy Sarkar, Wallace Chamon, Shweta Chaudhary,Sapna Tibrewal, Yong-Soo Byun, Sarmad H. Jassim, AbhishekSharma, Neil Mohindra, Sandeep Jain. Ophthalmology and VisualSciences, University of Illinois at Chicago, Chicago, IL.Purpose: It is becoming increasingly clear that the nervous andinflammatory (myeloid) systems display considerable overlap in theirmolecular and cellular repertoire. Bone marrow-derived YFP+ cellsinfiltrate the cornea during nerve regeneration in thy1-YFP mouse.We characterized these cells and determined whether they promotetrigeminal ganglion (TG) cell neurite growth.Methods: Excimer laser annular keratectomy was performed in thy1-YFP+ mice and corneas were imaged to visualize regenerating nervesand infiltrating cells. Bone marrow cells (BMCs) were harvestedfrom naive thy1-YFP mice and a flow cytometry analyzer was usedto assess the expression levels of cell surface markers in the totalBMC population. Compartmental cultures of dissociated TG cellswere performed and sorted YFP+ cells (>95% pure) were co-culturedin the side compartments with neurites to determine theirneurotrophic effect.Results: Following annular keratectomy, YFP+ cells infiltrate thecornea and localize adjacent to transected nerves. These are bonemarrow-derived cells that share surface markers(CD11b+Gr1+Ly6C+Ly6G-F4/80low) with monocytic myeloidderivedsuppressor cells (MDSCs); thus, we call them YFP-MDSCs.They are CD11c, CD3e and MHC-II negative. YFP-MDSCssignificantly increase the growth of TG neurites and make physicalcontact with neurites. When cultured in a transwell with TG neurites,YFP-MDSCs express neurotrophins and secrete NGF and BDNF inthe conditioned media.Conclusions: CD11b+Gr1+ myeloid cells promote neurite growth inTG cells. These cells infiltrate the cornea after nerve transectionsurgery and interact with the regenerating nerves; thus they maypromote reinnervation by their neurotrophic action. One mechanismby which these cells promote neurite growth is by secretion ofneurotrophins.Commercial Relationships: Sonal Gandhi, None; Joy Sarkar,None; Wallace Chamon, None; Shweta Chaudhary, None; SapnaTibrewal, None; Yong-Soo Byun, None; Sarmad H. Jassim, None;Abhishek Sharma, None; Neil Mohindra, None; Sandeep Jain,PCT/US20/51562 (P)Support: NIH Grant EY018874Program Number: 3885 Poster Board Number: D0129Presentation Time: 2:45 PM - 4:30 PMSemaphorin 7a actions on nerves and myeloid cells in the corneapromote nerve regeneration, thus linking neuronal and myeloidsystemsSarmad H. Jassim, Abed Namavari, Shweta Chaudhary, SapnaTibrewal, Yong-Soo Byun, Sonal Gandhi, Neil Mohindra, Hyun Lee,Joy Sarkar, Sandeep Jain. Ophthalmology and Visual Sciences,University of Illinois at Chicago, Chicago, IL.Purpose: We determined Sema7a localization and quantity in naivecorneas and during nerve regeneration after lamellar flap surgery. Wealso performed structure-function analyses of Sema7a to determinewhether peptides that span RGD integrin-binding or RTS disintegrinmotifs selectively influence neuroregeneration.Methods: Immunolocalization and Western blot analyses wereperformed to evaluate the abundance of Sema7a in naive corneas andcorneas undergoing nerve regeneration after lamellar corneal surgeryin thy1-YFP+ neurofluorescent mice. We used compartmentalcultures of dissociated trigeminal ganglion cells to determine theeffect of Sema7a exposure on neurite outgrowth in vitro. Finally, aSema7a pellet was implanted under the corneal flap after lamellartransection surgery to determine the neuronal and inflammatoryeffects of Sema7a supplementation in vivo. We synthesized N-acetylated, C-amidated RGD- or RTS-containing peptides derivedfrom the Sema7a sequence and determined the number of dissociatedTG cells that adhered to collagen coated culture plates whenincubated with the peptides.Results: Sema7a is expressed in the cornea, mainly concentrated inthe epithelium with less expression in the stroma. Corneal Sema7aexpression increases after nerve transecting lamellar surgery and islocalized near the regenerating nerve fronds. Sema7a induces neuritegrowth in vitro as potently as NGF. Exposure of trigeminal neuritesto Sema7a (20 nM) in the side compartment significantly increasedneurite length. The implanted Sema7a pellet significantly increasedYFP+ cell (CD11b+GR1+ immature myeloid derived suppressorcells) influx into the cornea as well as increased corneal nerve length.In contrast to 7A-RGD peptide, 7A-RTS peptide does not stimulateneurite growth but it reduces adhesion of neurons to collagen surface.Conclusions: Sema7a is constitutively expressed in the cornea andpotently stimulates nerve regeneration and influx of immaturemyeloid cell. Sema7a peptides spanning integrin-binding motifs(RGD or RTS) selectively influence neuroregeneration. This immunesemaphorin links nerve regeneration and myeloid systems in thecornea.Commercial Relationships: Sarmad H. Jassim, None; AbedNamavari, None; Shweta Chaudhary, None; Sapna Tibrewal,None; Yong-Soo Byun, None; Sonal Gandhi, None; NeilMohindra, None; Hyun Lee, None; Joy Sarkar, None; SandeepJain, PCT/US20/51562 (P)Support: NIH Grant EY018874Program Number: 3886 Poster Board Number: D0130Presentation Time: 2:45 PM - 4:30 PMThe Role of Heat Shock Protein 27 and Signal TransductionPathway of its Phosphorylation in Corneal Epithelial WoundHealingJae Yong Kim, Soon-Suk Kang, In Seok Song, Eun-Soon Kim,Myoung Joon Kim, Hungwon Tchah. Ophthalmology, University ofUlsan College of Medicine, Asan Medical Center, Seoul, Republic ofKorea.Purpose: To investigate the role of heat shock protein 27 (HSP27)and signal transduction pathway of its phosphorylation in the woundhealing of cultured corneal epithelial cells.Methods: study 1) The scramble small interfering RNA (siRNA) andsiRNA against the HSP27 were created. The cultured corneal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaepithelial cells were divided into two groups: scramble siRNA-treatedgroup vs. siRNA-HSP27-treated group. Scratch-induced directionalwounding assay and flow cytometry were performed to know the roleof HSP27. Study 2) the cultured corneal epithelial cells werewounded by the scratch. As time passed, the expression ofphosphorylated and non-phosphorylated HSP27, p38 mitogenactivatedprotein kinase (MAPK), and MAPK/Erk were evaluatedusing the Western blotting assay.Results: the siRNA against the HSP27 effectively blocked theexpression of non-phosphorylated HSP27. In scratch-induceddirectional wounding assay, siRNA-HSP27-treated group showed thedelayed epithelial migration. Flow cytometry showed siRNA-HSP27-treated group had more apoptotic cell death. In Western blotting, thep38 MAPK was immediately phosphorylated, folling the HSP27expression after epithelial wounding.Conclusions: The role of HSP27 in corneal epithelial wound healingcan be anti-apoptosis as well as migration. The p38 MAPK can beinvolved in HSP27 phosphorylation in corneal epithelial woundhealing.human keratocytes. The sterility of each PRGF-Endoret samplestored for different time points was also analyzed.Results: Several morphogens including PDGF-AB, VEGF, EGF andalso Vitamin A showed no significant variations along the studyperiod. On the other hand, Fibronectin and TGF-β1 concentrationshowed significant reduction among fresh and freezing storagesamples. Osmolarity of samples stored at - 20 degree Celsiusincreased significantly with regard to fresh samples, although theosmolarity values of samples stored at -20 degree Celsius was withinosmolar range tolerated by the cornea. No differences were observedon proliferation and migration of keratocytes at any time of the studyusing the different PRGF-Endoret samples. Sterility analysisperformed on each eye drops samples stored at different timesshowed no microbiological contamination.Conclusions: Biological activity of the PRGF-Endoret eye drops ispreserved after storage under freezing conditions for 3 months. Allfactors excluding TGF-β1 and fibronectin were maintained constant.Assuming this, we conclude that PRGF-Endorert eye drops maintaintheir biological stability for at least 3 months.Commercial Relationships: Gorka Orive, BTI BiotechnologyInstitute (E); Francisco Jose Muruzabal, Biotechnology Institute(E); Ander Pino, BTI Biotechnology Institute (E); Eduardo Anitua,Biotechnology Institute (P)Support: CEYEC (n0 CEN-20091021) project, which has beensupported by the Centre for Industrial Technological Development(CDTI) in the fifth edition of the CENIT program.Commercial Relationships: Jae Yong Kim, None; Soon-SukKang, None; In Seok Song, None; Eun-Soon Kim, None; MyoungJoon Kim, None; Hungwon Tchah, NoneSupport: Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by the Ministry ofEducation, Science and Technology (MEST) (NRF-2010-0025662)and a grant (2012-464) from the Asan Institute for Life Sciences,Seoul, Korea.Program Number: 3887 Poster Board Number: D0131Presentation Time: 2:45 PM - 4:30 PMBiological stability of Plasma Rich in Growth Factors (PRGF-Endoret) eye drops after 3 months of storageGorka Orive, Francisco Jose Muruzabal, Ander Pino, EduardoAnitua. Biotechnology Institute, Vitoria, Spain.Purpose: PRGF-Endoret eye drops are used for the treatment of awide range of ocular surface diseases. The objective of this study wasto evaluate whether PRGF-Endoret eye drops maintain theircharacteristics and their biological activity after 3 months stored at -20 degree Celsius.Methods: Blood from ten healthy donors was collected, centrifugedand Plasma Rich in Growth Factors (PRGF-Endoret) was preparedavoiding the buffy coat. The volume obtained was aliquoted to beused fresh or after storage at - 20 degree Celsius for 15, 30 and 60days. Osmolarity, vitamin A, fibronectin and several growth factorslike PDGF-AB, VEGF, EGF and TGF-β1 were quantified on freshsamples and after storage for 15, 30 and 60 days at - 20 degreeCelsius. The proliferative and migratory potential of PRGF-Endoreteye drops after different storage conditions were assayed on primaryProgram Number: 3888 Poster Board Number: D0132Presentation Time: 2:45 PM - 4:30 PMProteome study of keratocytes after PRGF-Endoret treatmentFrancisco Jose Muruzabal, Gorka Orive, Eduardo Anitua.Biotechnology Institute, Vitoria, Spain.Purpose: Plasma rich in growth factors (PRGF®-Endoret®) is anautologous technology that contains a pool of proteins specificallyaddressed for wound healing and tissue regeneration. Treatment withPRGF-Endoret eye drops stimulates those cells close to damage areato proliferate, migrate and differentiate with the aim of promoting theregeneration of injured cornea. The aim of this study was tocharacterize the proteins and signaling pathways involved in thekeratocyte activation after PRGF-Endoret treatment.Methods: Blood from healthy donors was collected, centrifuged andPlasma Rich in Growth Factors (PRGF-Endoret) was preparedavoiding the buffy coat. Primary human keratocytes were incubatedwith PRGF-Endoret eye drops at different times of treatment,collected and analyzed using a proteomic approach that combines 2-DE followed by MALDI-TOF/TOF.Results: The deregulated proteins obtained at different time pointswere grouped into families and networks according to gene ontology.The proteomic analysis of human keratocytes treated with PRGF-Endoret revealed a wide range of deregulated proteins. These proteinsare mainly associated with signaling pathways related to theregulation of cell death, proliferation, cytoskeletal and motorproteins, and wound healing, among others.Conclusions: A wide range of proteins and signaling pathways werederegulated after PRGF-Endoret treatment. We found an enrichmentof both proteins and protein families specifically involved in tissueregeneration and wound healing.Commercial Relationships: Francisco Jose Muruzabal,Biotechnology Institute (E); Gorka Orive, BTI BiotechnologyInstitute (E); Eduardo Anitua, Biotechnology Institute (P)Support: CEYEC (n0 CEN-20091021) project, which has beensupported by the Centre for Industrial Technological Development(CDTI) in the fifth edition of the CENIT program.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 3889 Poster Board Number: D0133Presentation Time: 2:45 PM - 4:30 PMWound healing in rabbit corneas after flapless refractivelenticule extraction with a novel 345nm ultraviolet femtosecondlaserChristian M. Hammer 1 , Johannes Menzel-Severing 1 , CorinnaPetsch 1 , Naresh Polisetti 1 , Bjoern O. Bachmann 1 , Jörg Klenke 2 ,Katrin Skerl 2 , Christian Wüllner 2 , Christof Donitzky 2 , Friedrich E.Kruse 1 . 1 Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany; 2 WaveLight GmbH (Alcon),Erlangen, Germany.Purpose: To preclinically characterize corneal wound healing inNew Zealand White (NZW) rabbits after flapless refractive lenticuleextraction (FLE) with a novel 345nm ultraviolet femtosecond laser(UV-FSL) developed by Alcon-WaveLight.Methods: A total of 20 NZW rabbits was included in this study. Oneeye received FLE while the contralateral eye served as untreatedcontrol. The lenticules (-5dpt) had a diameter of 5.5mm and were cutwith 80nJ and a spot separation of 4µmx4µm and were removed viatwo extraction canals. Four animals were sacrificed after 48 hours, 1week, 2 weeks, 4 weeks and 3 months, respectively. Lenticular bedsand extraction canals were prepared for standard histology andfluorescence microscopy. To investigate cell death rate, proliferation,myofibroblastic transdifferentiation of keratocytes and inflammation,TUNEL assay (Roche) as well as immunostaining for Ki67, αSMAand CD11b was performed, respectively, on sagittal sections ofkryoembedded specimens and subjected to qualitative andquantitative analysis.Results: Standard histological analysis revealed a zone of keratocytedepletion of approximately 50µm thickness at the extraction site.Epithelial and endothelial cells appeared unharmed. At the sites ofepithelial incision, a callus of epithelial cells formed. At 48h,TUNEL-assay analysis showed pronounced staining of keratocytesnear the extraction site (159.9±18.4cells/mm), which steadilydecreased to 74.9±19.8cells/mm at 1 week and 5.7±4.8 cells/mm at 2weeks. At 4 weeks, no TUNEL-positive keratocytes were detectedanymore. Marked Ki67 staining of keratocytes was evident at 48h(10.0±3.8 cells/mm), which had decreased conspicuously at one week(5.2±1.7 cells/mm) and 2 weeks (0.4±0.5cells/mm). At 4 weeks, noKi67 staining was found anymore. The corneal stroma was free ofαSMA- and CD11b-positive cells at all times. Only in closeproximity to the epithelial callus, some isolated keratocytes wereαSMA-positive and very few CD11b-positive cells were observed.Conclusions: Preclinical in-vivo assessment of corneal woundhealing after application of the novel 345nm UV-FSL showedpromising results after FLE in NZW rabbits. Together with thebenign development of cell death rate and keratocyte proliferation,the absence of inflammation and transdifferentiated keratocytesadvocates an initiation of the clinical phase.Commercial Relationships: Christian M. Hammer, None;Johannes Menzel-Severing, None; Corinna Petsch, RESORBAWundversorgung GmbH &Co.KG, Nuremberg, Germany (F);Naresh Polisetti, None; Bjoern O. Bachmann, None; Jörg Klenke,WaveLight GmbH (E); Katrin Skerl, WaveLight GmbH (E);Christian Wüllner, WaveLight GmbH (E); Christof Donitzky,WaveLight GmbH (E), WaveLIght GmbH (P); Friedrich E. Kruse,NoneSupport: German Federal Ministry of Ecucation and Research,01EX1011AProgram Number: 3890 Poster Board Number: D0134Presentation Time: 2:45 PM - 4:30 PMEffect of fat-derived mesenchymal stem cells on the cornealalkalie burn injury in dogsYoung Sam Kwon. Veterinary Surgery, Kyungpook NationalUniversity, Daegu, Republic of Korea.Purpose: This study was performed to evaluate the effect of topicalapplication of fat-derived mesenchymal stem cells (FDMSCs) oncorneal alkalie burn injury in dogs.Methods: The corneal alkalie burn was induced by applying a filterpaper soaked in NaOH on the cornea for 60 seconds, including upperlimbus. Ten dogs were divided into two groups: control group,vehicle-treated; MSC group, FDMSC-treated. Corneal opacity,epithelial defects and neovascularization were evaluated on days 1, 3,7, 10, and 14. Hematoxylin and eosin (H&E), Masson’s trichrome(MT) and immunohisochemical staining were performed to evaluategeneral histopathology, collagen fiber architectures, numbers of nitricoxide synthase (iNOS), caspase-3, tumor necrosis factor-α (TNF-α),interleukin(IL)-1β and CD105-positive cells. Real-time PCR wasperformed to measure the mRNA expressions of IL-1β, IL-6, IL-10,TNF-α and transforming growth factor-β (TGF-β).Results: We observed that MSCs improved the corneal recovery bydecreasing corneal opacity and epithelial defects. The epitheliumthickness, cornea damaged depth and areas, the number ofinflammatory cells and vessels, and the numbers of iNOS, caspase-3,TNF-α and IL-1β-positive cells were significantly decreased in theMSC group. But, the number of CD105-positive cells wassignificantly higher in the MSC group than the control group. Inaddition, the mRNA expression of TNF-α was significantly decreasedand IL-6, IL-10 and TGF-β were significantly increased in the MSCgroup.Conclusions: Our study suggests that topical application of FDMSCsmay stimulate the healing of alkalie-induced corneal injury in dogs.Commercial Relationships: Young Sam Kwon, NoneSupport: NRF 2010-0007523Program Number: 3891 Poster Board Number: D0135Presentation Time: 2:45 PM - 4:30 PMOptical coherence tomography analysis of hydrofluoric aciddecontamination of human cornea by mannitol solutionRicardo Nose 1, 2 , Fabio B. Daga 1 , Adriana S. Forseto 2 , GustavoVictor 2 , Sidney J. Sousa 3 , Walton Nose 2 , Niro Kasahara 1 . 1 Santa Casade Sao Paulo, Sao Paulo, Brazil; 2 Eye Clinic Day Hospital, SaoPaulo, Brazil; 3 Ophtalmoogy, Faculdade de Medicina Universidadede São Paulo de Ribeirão Preto, São Paulo, Brazil.Purpose: Purpose: to evaluate the efficacy of mannitol solution asdecontamination agent on the chemical burn of the human cornea.Methods: Methods: eight donor corneas from an eye bank wereexposed to 25 μL of 2.5% hydrofluoric acid (HF) solution on a filterpaper for 20 s. Three eyes were rinsed with 1000 ml of mannitol 20%for 15 min immediately after removal of the filter paper, 3 other wererinsed with tap water (1000 ml for 15 min) and two eyes were notrinsed. Microstructural changes were monitored in the time domainby OCT imaging for 90 min.Results: Results: tap water reduced the penetration depth toapproximately half the thickness of the cornea at 15 min; scatteringwithin the anterior cornea was higher than that for the unrinsed eye.With mannitol, no increased scattering is observed in the posteriorpart of the corneal stroma within a time period of 1 h after rinsing.OCT images revealed low-scattering intensity within the anteriorstroma at the end of the rinsing period.Conclusions: Conclusion: in eye bank human corneas, mannitol is anefficient agent to decontaminate HF burn.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Ricardo Nose, None; Fabio B. Daga,None; Adriana S. Forseto, None; Gustavo Victor, None; Sidney J.Sousa, None; Walton Nose, None; Niro Kasahara, NoneProgram Number: 3892 Poster Board Number: D0136Presentation Time: 2:45 PM - 4:30 PMEffect of human albumin in combination of blood derivatives richin growth factors in the wound healing capability of cornealepithelial cellsNoelia Andollo 1 , Vanesa Freire 1, 4 , Arturo E. Grau 3 , JaimeEtxebarria 1, 5 , Juan A. Duran 2, 4 , Maria-Celia Morales 5 . 1 CellBiology And Histology, University of The Basque Country,BioCruces Health Research Institute, Leioa, Spain; 2 Ophthalmology,University of the Basque Country, BioCruces Health ResearchInstitute, Leioa, Spain; 3 Hospital Sótero del Rio, Santiago, Chile; 4 R& D Dept., Instituto Clínico-Quirúrgico de Oftalmología, Bilbao,Spain; 5 University Hospital of Cruces, BioCruces Health ResearchInstitute, Barakaldo, Spain.Purpose: Blood derivatives rich in growth factors are goodpromoters of corneal wound healing. Our aim is to test in vitro thecapability of human albumin as a synergic compound combined witha blood derivative rich in growth factors to improve the healing rateof corneal epithelial cells.Methods: In vitro proliferation and wound healing experiments wereperformed using the human corneal epithelial HCE cell line. Westudied the following treatments: 1) 20% Serum of Plasma Rich inGrowth Factors (s-PRGF) 2) 50% s-PRGF 3) 10% Human serumalbumin (albumin) 4) 20% s-PRGF+10% albumin 5) 50% s-PRGF+10% albumin 6) 10% FBS and 7) 1% bovine serum albumin(BSA) as control. To manufacture s-PRGF whole blood was collectedby venipuncture from healthy volunteers.Results: The results show that human albumin induces highlysignificant higher proliferation of HCE cells than any dose of s-PRGFafter 72 hours of treatment. Moreover, no significant differences arefound among human albumin, 20% s-PRGF+albumin and 50% s-PRGF+albumin. Conversely, 20% and 50% s-PRGF treatmentsimprove cell migration with respect to the rest of the treatments, withstatistically significant differences. Treatments of albumin, 20% s-PRGF+albumin and 50% s-PRGF+albumin do not show significantdifferences among them.Conclusions: Human albumin by itself induces higher proliferationof epithelial corneal cells but lower cell migration, compared with theeffect of the blood derivative s-PRGF. In addition, combination ofboth does not improve proliferation and affects negatively migration.Commercial Relationships: Noelia Andollo, None; Vanesa Freire,None; Arturo E. Grau, None; Jaime Etxebarria, bioftalmik (C);Juan A. Duran, None; Maria-Celia Morales, NoneProgram Number: 3893 Poster Board Number: D0137Presentation Time: 2:45 PM - 4:30 PMCryopreservation Preserves the Structural Integrity, BiochemicalComponents and Biologic Function of Amniotic MembraneTissueEk Kia Tan 1, 2 , Marissa T. Cooke 3 , Christian Mandrycky 3 , Hua He 1, 2 ,Julie O'Connell 4 , Todd C. McDevitt 3, 5 , Scheffer C. Tseng 1, 2 . 1 OcularSurface Research and Education Foundation, Miami, FL; 2 Researchand Development, TissueTech, Inc., Miami, FL; 3 Wallace H. CoulterDepartment of Biomedical Engineering, Georgia Institute ofTechnology and Emory University, Atlanta, GA; 4 Amniox Medical,Marietta, GA; 5 Parker H. Petit Institute for Bioengineering andBioscience, Georgia Institute of Technology, Atlanta, GA.Purpose: The therapeutic potential of amniotic membrane (AM) hasbeen examined in a variety of clinical indications, particularly inophthalmology where AM has been used extensively for indicationssuch as pterygium, conjuntivochalasis, and corneal defects. To date,there has been no comprehensive study comparing cryopreservedamniotic membrane processed with the CryoTek method and freshAM.Methods: Fresh AM, either thin or thick, and fresh amniochorionwere compared to CryoTek processed thin and thick AM andamniochorion. Histological properties were assessed by hematoxylinand eosin, Masson’s Trichrome, and Safranin O staining.Biochemical properties were measured by contents of protein,albumin, and hyaluronan (HA) with their molecular weight spectrum.Functional assays compared macrophage viability and proliferation,and human corneal fibroblast TGF-β1 induction.Results: Histochemical staining confirmed that the cryopreservationprocess did not alter the tissue architecture or collagen andglycosaminoglycan content. Biochemically, cryopreservation reducedtotal protein and human serum albumin contents, but retained highmolecular weight hyaluronan species including HC-HA complex,known to exert anti-inflammatory and anti-scarring effects. Bothfresh and cryopreserved AM water-soluble extracts similarlysuppressed viability and proliferation of RAW264.7 macrophages,and dose-dependently inhibited the TGF-β1 promoter activity inhuman corneal fibroblasts.Conclusions: These results collectively indicate thatcryopreservation by the CryoTek method effectively preserveshistological, biochemical and functional components of the AMtissue.Commercial Relationships: Ek Kia Tan, TissueTech, Inc. (E);Marissa T. Cooke, None; Christian Mandrycky, None; Hua He,TissueTech, Inc. (E); Julie O'Connell, None; Todd C. McDevitt,Amniox Medical (C); Scheffer C. Tseng, NIH, NEI (F), TissueTech,Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)Support: Research Alliance Venture Lab Grant #398, Atlanta, GAand a research grant from TissueTech, Inc., Miami, FL.Program Number: 3894 Poster Board Number: D0138Presentation Time: 2:45 PM - 4:30 PMBiological Differences between Cryopreserved and DehydratedAmniotic Membrane Tissue GraftsLorraine S. Chua 1, 2 , Marissa T. Cooke 3 , Christian Mandrycky 3 , EkKia Tan 1, 2 , Julie O'Connell 4 , Scheffer C. Tseng 1, 2 , Todd C.McDevitt 3, 5 . 1 Ocular Surface Research & Education Foundation,Miami, FL; 2 Research & Development, TissueTech, Inc., Miami, FL;3 Wallace H. Coulter Department of Biomedical Engineering, GeorgiaInstitute of Technology and Emory University, Atlanta, GA;4 Amniox Medical, Marietta, GA; 5 Parker H. Petit Institute forBioengineering and Bioscience, Georgia Institute of Technology,Atlanta, GA.Purpose: Amniotic membrane (AM) processing methods candramatically impair both the structural integrity, and biologicalactivity, of critical matrix cell signaling factors essential for theintended use of the product. To analyze the effect of different AMavailable commercially for the ophthalmology market on conservingthe therapeutic potential of the tissue, we compared cryopreserved(CryoTek) and dehydrated (Purion®) processed tissue grafts inphysical and biochemical assays.Methods: Cryopreserved thin (CT-Thin) and thick (CT-Thick)tissues were compared to dehydrated, (EF) and (AF) AM tissuegrafts. Structural properties of cryopreserved and dehydrated AMwere assessed by histological staining while biochemical propertieswere measured in soluble tissue extracts by comparing hyaluronan(HA) content and molecular weight (MW) spectrum; and criticalproteoglycan (HC-HA) and protein (PTX3) signaling factors.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: Histochemical staining demonstrated dehydrated tissueshaving a more compact extracellular matrix compared to thecryopreserved tissues even after the prescribed hydration duration.HA quantity was highest in CT-Thick, and although content wasrelatively similar in CT-Thin and EF/AF, the MW analysis revealedall cryopreserved samples contained high MW HA, while thedehydrated samples contained low MW HA. Essential signalingproteins, HC-HA and PTX3 detected by western blots were present incryopreserved samples but were either compromised, or completelyabsent, in dehydrated tissues.Conclusions: The cryopreservation process better preserves thestructural integrity and biochemistry of AM tissue grafts incomparison to dehydrated grafts, and suggests the therapeutic benefitof dehydrated AM may be compromised as a result.Commercial Relationships: Lorraine S. Chua, TissueTech Inc.(E); Marissa T. Cooke, None; Christian Mandrycky, None; EkKia Tan, TissueTech, Inc. (E); Julie O'Connell, None; Scheffer C.Tseng, NIH, NEI (F), TissueTech, Inc. (F), TissueTech, Inc. (E),TissueTech, Inc. (P); Todd C. McDevitt, Amniox Medical (C)Support: Research Alliance Venture Lab Grant #398, Atlanta, GAand a research grant from TissueTech, Inc., Miami, FL.Program Number: 3895 Poster Board Number: D0139Presentation Time: 2:45 PM - 4:30 PMCovalently immobilized epidermal growth factor acceleratesproliferation of human corneal epithelial cellsShin Ae Park 1 , Vijaykrishna K. Raghuanthan 1 , Sara M. Thomasy 1 ,Christopher J. Murphy 1, 2 . 1 Department of Surgical and RadiologicalSciences, UC Davis, Davis, CA; 2 Department of Ophthalmology &Vision Science, UC Davis, CA, CA.Purpose: Corneal wound healing involves the coordination of anumber of complex processes including cell migration, cellproliferation, re-stratification, as well as matrix deposition and tissueremodeling. Cell migration and proliferation are greatly influencedby the presence of growth factors and delivery of cytoactive factorsin a corneal wound remains a difficult challenge. Here, weinvestigated an in vitro model for the covalent immobilization ofepidermal growth factor (EGF) to enhance corneal epithelial cellproliferation.Methods: Unmodified glass coverslips or those modified with thiosilaneor propyl silane were used as stimulants of a corneal woundbed. Disulfide bonds were reduced to expose sulfhydryl (-SH) groupsusing 10μM tris(2-carboxyethyl)phosphine (TCEP). Humanrecombinant EGF, modified with a heterobifunctional crosslinker(sulfo-SMCC) was covalently linked to the -SH groups of the silane.The cytoactivity of the covalently limmobilized EGF wasinvestigated by measuring proliferation of human immortalizedcorneal epithelial cells (hTCEpi) using the MTT assay over days.Cytotoxicity of the reducing agent, TCEP, was also determined byMTT assay over a differential dose range (1 nM to 1 mM).Results: Proliferation of hTCEpi cells was significantly greater (>2fold) when EGF was covalently linked to the surface in comparisonwith the non treated control groups (p < 0.05). TCEP was determinedto be non-toxic for doses 0.05).Conclusions: Our data strongly demonstrate that TCEP canpotentially be used as a safe reducing agent for covalentimmobilization of cytoactive factors. Importantly, we successfullydemonstrate that covalent immobilization of EGF (30 ng) potentlyenhances proliferation of hTCEpi cells in vitro. Covalent integrationof EGF into corneal wounds, is a unique strategy to promote reepithelializationusing significantly less cyctoactive factor and dosefrequency than topical application.Commercial Relationships: Shin Ae Park, None; Vijaykrishna K.Raghuanthan, None; Sara M. Thomasy, None; Christopher J.Murphy, Ocular Services On Demand (I), Ocular Services OnDemand (C), Platypus Technologies LLC (I), Imbed LLC (I), EyeKorLLC (I), Allergan (C), Genentech (C), Sarcode (C), Covance (C)Support: NIH Grant 1K08EY021142Program Number: 3896 Poster Board Number: D0140Presentation Time: 2:45 PM - 4:30 PMSubstrate topography enhances corneal epithelial cell electrotaxisBrian Reid 1 , Jing Gao 1 , Vijaykrishna K. Raghuanthan 2 , PaulRussell 2 , Christopher J. Murphy 2, 3 , Min Zhao 1, 3 . 1 Dermatology,University of California, Davis, Davis, CA; 2 Surgical andRadiological Sciences, University of California, Davis, Davis, CA;3 Ophthalmology and Vision Science, University of California, Davis,Davis, CA.Purpose: Corneal epithelial cells (CECs) attach to the underlyingstroma via a 3D extracellular matrix basement membrane (BM) thatpossesses intrinsic biophysical (topography, stiffness) andbiochemical cues (chemoattractants, growth factors, soluble factors).Cellular behavior can be significantly modulated by these factorsduring wound healing. An important yet grossly under-studied factorin corneal wound healing is the role of electric fields (EFs). Corneawounds generate significant EFs which cells respond to bydirectional migration. Here, for the first time, we investigated theimpact of simultaneous presentation of topographic cues and EFs onCEC migration.Methods: Immortalized human CECs were cultured on unpatterned,stochastically patterned (mimicking the stochastic arrangement on theBM) or anisotropically patterned polymeric substrates (biomimeticscale 400 to 4000nm pitches; pitch = ridge width + groove width)coated with fibronectin. EFs (0-150 mV/mm) were applied to cellsacross an electrotaxis chamber for 3h. Single cell migration, cellmigration as a monolayer across a simulated wound, and alterationsin the gene expression of focal adhesion kinase (FAK), andcytoskeletal regulator ROCK1 were determined.Results: In the absence of an EF, cells migrated faster on stochasticsubstrates than on flat surfaces (21.14µm/h vs. 18.44µm/hrespectively, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 3897 Poster Board Number: D0141Presentation Time: 2:45 PM - 4:30 PMRole of Soluble Vimentin in Regulating the ERK Pathway duringCorneal FibrosisPaola Bargagna-Mohan, Royce Mohan. Neuroscience, Univ ofConnecticut Health Center, Farmington, CT.Purpose: Soluble vimentin (sVim) along with its cytoskeletalfilamentous form drives corneal fibrosis via promotion of cellproliferation (Bargagna-Mohan JBC 2012). We have investigatedwhether sVim acts as a signaling chaperone that mediatesextracellular-signal-regulated kinase 1/2 (ERK1/2) activity bycontrolling its nuclear expression since MAPK/ERK signaling isknown to affect corneal repair. In this study, we employed geneticand chemical biology approaches to investigate the role of sVimpERKaxis in the healing cornea.Methods: Primary cultures of rabbit corneal fibroblasts (RbCFs)were employed for cell proliferation studies. Cells were serumstarved for 48h, trypsinized and then allowed to re-attach to cultureplates for 2h in presence of 10% serum. To control the amount ofsVim available in culture, cells were treated at the time of platingwith different doses of Withaferin A (WFA). Cytoplasmic andnuclear extracts were collected and expression of phosphorylated-ERK (p-ERK) was analyzed at different time points by western blotanalysis. Cells were also immunostained for p-ERK andphosphorylated-Vim (p-Vim). In vivo experiments were performedusing a model of corneal alkali burn injury in Vim+/+ and Vim-/-mice. WFA was peritoneally injected (2 mg/kg/d) every day for 2weeks and eyes were collected for immunohistochemistry andwestern blot analysis.Results: In proliferating RbCFs a time-dependent translocation of p-ERK from the cytoplasm into the nucleus occurred by 1h postspreadingand WFA treatment strongly inhibited this transition bymaintaining cytoplasmic p-ERK levels 10-fold higher than its nuclearlevels. At 24h post-plating, control cells displayed a well-extendedcytoskeleton staining for p-Vim and strong nuclear p-ERKexpression, whereas in WFA-treated cells p-ERK remainedperinuclear and co-localized with p-Vim. Our in vivo data revealedthat alkali injury induced p-ERK expression strongly in Vim+/+corneas and WFA downregulated this at d14 post-injury.Interestingly, injured Vim-/- corneas displayed lower levels of p-ERK expression similar to that of Vim+/+- mice treated with WFA.Conclusions: sVim engagement occurs with stimulation of theERK1/2 pathway, which WFA potently antagonizes throughreduction of nuclear pERK1/2 complexes. This mechanism may alsoexplain lower pERK levels when vimentin is genetically abrogated invivo.Commercial Relationships: Paola Bargagna-Mohan, UKYResearch Foundation, US8283323B2 (P); Royce Mohan, Universityof Kentucky Research Foundation (P)Support: RO1 Grant EY016782, John A. and Florence MatternSolomon Endowed ChairProgram Number: 3898 Poster Board Number: D0142Presentation Time: 2:45 PM - 4:30 PMDysregulated Heme Oxygenase-Ferritin System in PterygiumPathogenesisLars Bellner 1 , Timothy P. Fox 1 , Katherine H. Gotlinger 1 , Michael W.Dunn 2 , Tatyana Milman 3 , Gerald W. Zaidman 2 , Michal L.Schwartzman 1, 2 . 1 Pharmacology, New York Medical College,Valhalla, NY; 2 Ophthalmology, New York Medical College,Valhalla, NY; 3 New York Eye & Ear Infirmary, New York, NY.Purpose: Pterygium is an ocular surface disease of humans attributedto chronic ultraviolet-B exposure. It involves invasive centripetalgrowth with associated inflammation and neovascularisation.Cyclooxygenases (COX), lipoxygenases (LOX) and cytochromeP450 monooxygenases (CYP) derived eicosanoids have beenimplicated in ocular surface inflammation and neovascularization.These eicosanoids are subjected to regulation by enzymes such asheme oxygenases (HO) and Ferritin.Methods: Quantitative PCR and LC-MS/MS based lipidomics wereperformed on pterygium from patients undergoing surgical removalof pterygium. Control tissues consisted of donor corneal grafts. Inaddition, LC-MS/MS based lipidomics was performed on tearscollected from patients prior to surgery.Results: mRNA of HO-2, the constitutive HO isoform, wasupregulated by 40% in pterygium as compared to control tissue,while the mRNA level of the inducible form, HO-1, wasdownregulated by more than 50%. Ferritin light and heavy chainmRNA expression levels were 60% and 30% lower in pterygium ascompared to control. CYP4B1 mRNA levels were 2-fold higher inpterygium compared to control. Lipidomic analysis indicated adoubling in the level of COX-derived PGE 2 and TxB 2 in pterygiumas compared to control. Among LOX-derived metabolites, the antiinflammatory15-HETE levels were significantly reduced inpterygium (79.3±48.11 pg/mg protein) as compared to control(586.2±213.5 pg/mg protein), whereas pro-inflammatory LOX andCYP4B1-derived 12-HETE levels were 10-fold higher in pterygium(2768±832.3 pg/mg protein) compared to control (231.4±87.35pg/mg protein). PGE 2 and the HETEs were also present in tears frompatients with pterygium, but were not detected in tears from healthyvolunteers.Conclusions: We believe a dysfunctional HO-Ferritin system leadsto increased mRNA levels of CYP4B1 and increased production ofthe LOX- and COX-derived proinflammatory lipid mediators, thuscontributing to the pathogenesis of pterygium. Moreover, the lowerexpression levels of ferritin have the potential of increasingintracellular iron levels, contributing to oxidative stress and could bea major cause of the iron-deposition often observed along the leadingedge of the pterygium, Stocker’s line.Commercial Relationships: Lars Bellner, None; Timothy P. Fox,None; Katherine H. Gotlinger, None; Michael W. Dunn, None;Tatyana Milman, None; Gerald W. Zaidman, None; Michal L.Schwartzman, NoneProgram Number: 3899 Poster Board Number: D0143Presentation Time: 2:45 PM - 4:30 PMCorneal Fibrosis Associated with Trauma and Infection UsingMouse ModelsHong-Yuan Zhu 1, 2 , Jennifer P. Ng 1 , Shuhaida Salleh 1 , Thet T. Aung 1 ,Roger W. Beuerman 1 . 1 Singapore Eye Research Institute, Singapore,Singapore; 2 Department of Ophthalmology, Daqing People'sHospital, The Fifth Affiliated Hospital of Harbin Medical University,Daqing, China.Purpose: Fibrosis is a common outcome of inappropriate tissuerepair and responsible for corneal blindness often as the aftermath ofinfections or trauma. The goal of this study was to understand therelationship between fibrosis and inflammation following infection ortrauma. To this end mouse models of fibrosis associated withinfection and trauma were developed.Methods: A mouse model with corneal fibrosis triggered byinfection (IF) (Pseudomonas aeruginosa) or anterior keratectomy(AK) was used. Real Time PCR was used to examine the expressionof αSMA, HMGB1, RAGE and S100A8/9 in the isolated corneastroma at postoperative times (PO) 6h, 16h, 2, 7 and 14 days (D). Amultiplex cytokine assay was used to examine the expression ofIFNγ, IL6, MIP2, MIP1a, MIP1b, GCSF, VEGF and RANTES in the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaisolated cornea stroma and tears at PO 2, 7, 14D and 4 weeks (W).Ten mice were used for each time point.Results: RT-PCR of stroma after IF or AK, compared with normalstroma (N), showed that RAGE and S100A8/9 increased and peakedat PO16h (RAGE: 2.5 fold increased after AK and 5.6 fold after IF;S100A8:134 fold after AK and 95 fold after IF; S100A9:145 foldafter AK and 97 fold after IF; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Histatins are naturally occurring oral peptides produced byhumans and non-human primates that demonstrate direct antiinfectiveactivity, potent anti-inflammatory properties, and stimulateepithelial wound healing in several tissue and organ culture systems.Isolation of histatin and its topical use for corneal wound healing hassuggested the potential for significantly accelerated wound healing.To evaluate and quantify this potential, the effect of histatin on theocular surface of New Zealand White rabbits was evaluated in arandomized, blinded, placebo-controlled study.Methods: Eight (8) mm circular, central corneal epithelial defectswere created in the right eye of 12 rabbits. Rabbits were subsequentlyrandomized into 3 treatment groups, with 4 animals in each group:“10”: 10ug/ml histatin; “0.1”: 0.10 ug/ml histatin; “Veh”: vehiclecontrol (excipient without active agent). Each group was treated withthe respective dose 3 times per day until complete healing of theepithelium occurred. All investigators were masked to the therapeutictreatment given to the rabbits. Periodic evaluation of corneal woundhealing was performed twice daily by fluorescein staining and slitlamp biomicrophotography with subsequent computerized areadetermination of the size of the defect. Statistical analysis was doneto determine differences in healing rates between the groups.Results: Rabbits treated with 10 ug/ml histatin resolved by 48 hourspost wounding, while those treated with 0.10 ug/ml histatin resolvedby 54 hours and those treated with vehicle did not resolve until 72hours. Wound areas at 48 hours: 10: 0.035 ± 0.071 mm 2 ; 0.1: 2.664 ±3.083 mm 2 ; Veh: 13.599 ± 20.183 mm 2 . Mean healing rate in theactive groups were higher than the vehicle control..Conclusions: This study demonstrates the potential role of histatin inwound healing and supports the further evaluation of the efficacy ofhistatin for corneal wound healing and exploration of its benefits in avariety of traumatic, infectious and other ocular surface disorders.Commercial Relationships: Seth P. Epstein, Rapid PathogenScreening, Inc (F); Neha Gadaria-Rathod, None; KarenFernandez, Rapid Pathogen Screening, Inc. (F); Penny A. Asbell,RPS (F)Support: Supported in part by Rapid Pathogen Screening, Inc.,Sarasota, FL as well as by unrestricted grants from The Martin andToni Sosnoff Foundation, New York, New York and Research toPrevent Blindness.Program Number: 3903 Poster Board Number: D0147Presentation Time: 2:45 PM - 4:30 PMCorneal Wound Healing Model in New Zealand White Rabbitsfor Evaluating Persistent Corneal Epithelial DefectsGlenwood G. Gum 1 , Barbara M. Wirostko 2 , MaryJane Rafii 2 , StacyPritt 1 , Damian Gutierrez 1 . 1 Absorption Systems, San Diego, CA;2 Jade Therapeudics, Salt Lake CIty, UT.Purpose: To develop a delayed wound healing model for evaluatingcorneal wound healing capabilities for persistent corneal epithelialdefects (PCED) as in the cases of chronic ocular infections, severedry eye, neurotrophic/diabetic keratitis, chemical and exposure toblast traumas as in a military theater. Most preclinical epithelialdefect models heal very rapidly thus the impact on wound healing isdifficult to determine.Methods: Animals were anesthetized with ketamine and xylazinegiven intramuscularly (IM). The epithelial defect was created in thecenter of the cornea with an 8.5 mm Camellin LASEK alcohol well.A 20% ethyl alcohol solution was applied for 2.5 minutes. The ocularsurface outside the Camellin well was irrigated with balance saltsolution (BSS) during alcohol application. The alcohol solution wasremoved and cornea was irrigated with BSS. The epithelium wasremoved (to the stromal region of the cornea) by scraping with Bard-Parker blade #15. Buprenorphine was given as postoperativeanalgesia.In order to evaluate the healing mechanism of a PCED condition, andsince steroids have ability to delay healing, all groups were dosedwith Dexmaethasone (DEX). Recombinant human growth hormone(rHGH) was chosen as a positive control; rHGH has been shown toaccelerate wound healing. BSS served as a negative control. Allanimals started treatment on Day 1 post surgery. Each test group wasdosed topically with DEX (50µL) QID within 8 hrs. The rHGH-DEX(n=5) and BSS-DEX (n=4) were dosed topically QID (50µL) at least30 min. after the DEX treatment. One group consisted of DEXtreatment only (n=4). Clinical ophthalmic examinations, includingslit lamp biomicroscopy and fluorescein staining, were performedtwice daily on days 2, 3, 4, and once on day 5. Percentage of cornealwound healing was evaluated and compared across treatment groups.The groups were compared statistically and histopathologically.Results: Day 2 through day 5, the DEX group was inhibited incorneal wound healing rate. DEX-BSS and DEX-rHGH were similaron day 2. By day 3 to day 4 the DEX-rHGH was significantly fasterin corneal wound healing rate when compared to the DEX-BSS. Byday 4 PM and day 5 both DEX-rHGH and DEX-BSS were similar.Conclusions: This model can offer the opportunity for evaluatingpharmacological agents and drug delivery systems that can promotecorneal wound healing for PCED conditions.Commercial Relationships: Glenwood G. Gum, Jade Therapeutics(F); Barbara M. Wirostko, Jade Therapeutics (P), Jade Therapeutics(I), Jade Therapeutics (E), Altheos Inc. (C), Merck (C), SKS Ocular(C), USTAR (F); MaryJane Rafii, Jade Therapeutics (P), JadeTherapeutics (S), Jade Therapeutics (I), Pfizer Inc. (C), Regenron(C); Stacy Pritt, Jade Therapeutics (E), Absorption Systems (E),Amakem (E); Damian Gutierrez, NoneSupport: USTAR grantProgram Number: 3904 Poster Board Number: D0148Presentation Time: 2:45 PM - 4:30 PMNew medical device for chronic corneal ulcers healingBeatrice Cochener, Sabine Derrien. Ophthalmologie, CHU De Brest,Brest, France.Purpose: To report the cases of a series of patients suffering fromchronic corneal ulcers resistant to conventional therapies and treatedwith a new ophthalmologic solution based on ReGeneraTing Agenttechnology (RGTA, Cacicol®). RGTA is a bioengineered analog ofextracellular matrix components (heparan sulfates) which promotetissue regeneration.We report a series of 20 eyes treated with this innovative solution.Methods: We conducted a monocenter opened prospective study. Allof the 20 eyes had chronic corneal ulcers, except one who had asevere confluent keratitis. Ulcers were essentially chronic, and 8patients had already received one or several amniotic membranegrafts or, without success. Two ulcers were due to basic chemicalburns, one was associated with neurotrophic keratitis, and two wereulcers with herpetic kerititis. All patients were previously treated withclassical lachrymal substitutes, some of them also with topicalcyclosporine, corticoids, and/or A vitamin ocular ointment. The meanvertical and horizontal ulcer diameters were, respectively, 2.2 and 2.8mm (range: 1-6.5 mm). Patients were treated with Cacicol® at a doseregimen of one drop daily every 2 or 3 days for one to two monthsdepending on healing.Results: Complete healing was observed for 12 patients, i.e. a curerate of 69 %. For other patients, 3 failures and one improvementwithout complete healing were reported. However a large variationwas noted in the time for recovery of corneal integrity going fromfew days to few weeks. No correlation has been established betweenetiology, depth of ulcer and potential of regeneration. In case of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasevere ulcer and when a positive effect is observed at 1 month, aweekly drop is continued up to the complete healing.Conclusions: Cacicol® appears as a new interesting healing eyedrops in the context of severe chronic corneal ulcers resistant toconventional therapies. Its efficacy remains to be proven inrandomized double-blind studies.Commercial Relationships: Beatrice Cochener, Thea (C), Alcon(C), Novagali Santen (R), Bausch and Lomb (R), Physiol (R); SabineDerrien, NoneProgram Number: 3905 Poster Board Number: D0149Presentation Time: 2:45 PM - 4:30 PMInduction of heat shock protein 70 ameliorates ultravioletinducedphotokeratitis in miceNobuyoshi Kitaichi 1, 2 , Anton Lennikov 3 , Satoru Kase 3 , KousukeNoda 3 , Yukihiro Horie 3 , Shigeaki Ohno 2 , Susumu Ishida 3 .1 Ophthalmology, Health Sciences University of Hokkaido, Sapporo,Japan; 2 Ocular Inflammation and Immunology, Hokkaido UniveristyGraduate School of Medicine, Sapporo, Japan; 3 Ophthalmology,Hokkaido Univeristy Graduate School of Medicine, Sapporo, Japan.Purpose: Background and Puropose: Acute ultraviolet (UV) Bexposure causes photokeratitis and induces apoptosis in corneal cells.Geranylgeranylacetone (GGA) is an acyclic polyisoprenoid thatinduces the expression of heat shock protein (HSP) 70, a solubleintracellular protein expressed in various tissues, including the eyes.HSPs function as intracellular chaperones, protecting cells againstvarious stress conditions. Though the right part of the GGAmolecular structure is similar to that of vitamin K, this cannot fullyexplain the effects of GGA on various tissues. Also, the relationshipbetween HSP70 expression and corneal damage is not known. In thepresent study, we examined whether the induction of HSP70 hastherapeutic effects on UV-photokeratitis in mice.Methods: Methods: C57BL/6 mice were divided into four groups,GGA-treated (500 mg/kg/mouse) and UVB-exposed (400mJ/cm 2 ),GGA-untreated and UVB-exposed, GGA-treated but not UVBexposed,and naïve. Eyeballs were collected 24 hours afterirradiation, and corneas were stained with hematoxylin-eosin (H&E),TUNEL, anti-HSP70, and phospho-(serine/threonine) Akt substrateantibody.Results: Results: The irradiated corneal epithelium was significantlythicker in the eyes of mice treated with GGA as compared with thosegiven vehicle alone (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneanerve and CD45+ cells were quantified. Pull down experiments usingGAL3-conjugated agarose beads were performed to determine ifTrkA binds to GAL3.Results: Robust neurite outgrowth was seen in OTG culturesincubated with NGF, as compared to cultures in media alone(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamustard (NM) to determine whether hydroxamate compoundsfacilitate recovery. NM is a vesicating agent that causes separation atthe epithelial-stromal junction, and can lead to sloughing of theepithelium 1-2 days after exposure. Separation of the cell layersinvolves, in part, cleavage of hemidesmosomal collagen XVII byADAM17. Hydroxamates inhibit ADAM17 via binding to theenzyme’s catalytic zinc. Our goal was to evaluate two hydroxamatesfor their ability to inhibit ADAM17 and to explore other mechanismsfor how they favor epithelial-stromal integrity after injury.Methods: Rabbit corneas in organ culture were exposed to 100 nmolNM (in 10 ul) for 2 hr. Corneas were then washed, medium waschanged, and 3 nmol of each hydroxamate (in 20 ul) was applied toNM-exposed corneas 4 times over the course of 22 hrs. Thehydroxamate compounds used as therapies were olvanil OH(NDH4409) and retro olvanil 8 (NDH4417).Results: Without hydroxamate treatment, ADAM17 was activated inthe cornea immediately upon exposure to nitrogen mustard (NM).Activation persisted for a minimum of 24 hr post exposure,correlating with epithelial-stromal separation. With the application ofeither hydroxamate, epithelial sloughing was reduced at 24 hr postexposure, and the overall histology of the basement membrane zonewas greatly improved. Surprisingly, the improvement of histology didnot correlate with the ability of the hydroxamates to inhibitADAM17. NDH4417 was a much better inhibitor than NDH 4409 inactivity assays. Since matrix metalloproteinases (MMPs) also containcatalytic Zn ions, both NDH4417 and NDH4409 were tested for theirability to reduce MMP9 levels, and both were found to be veryeffective. Next, to investigate an upstream step in MMP activation,the expression of the MMP inducer, EMMPRIN, was evaluated.EMMPRIN was found to be upregulated by mustard exposure. BothNDH4409 and NDH4417 treatment after NM exposure reducedEMMPRIN expression levels to values lower than unexposedcontrols, despite that EMMPRIN is not known to contain a zincmoiety.Conclusions: The hydroxamates employed not only reduce theactivity of ADAM17 and MMPs, but they also lower MMP levels byattenuating expression of the MMP inducer, EMMPRIN. Themechanism behind this latter activity is as yet unknown.Commercial Relationships: Marion K. Gordon, None; Andrea D.Rodrigues, None; Rita A. Hahn, None; Donald R. Gerecke, None;Kathy K. Svoboda, None; Ned D. Heindel, NoneSupport: NIH NEI R01 EY0090576; NIH NIAMSD U54AR055073; NIH NIEHS P30 ES005022Program Number: 3911 Poster Board Number: D0155Presentation Time: 2:45 PM - 4:30 PMAn intracellular degradation pathway implicated in cornealscarringAudrey Bernstein, Lingyan Wang, Stephanie R. Gillespie.Ophthalmology, Mount Sinai School of Medicine, New York, NY.Purpose: Scarring in the cornea can lead to vision loss. Corneal hazeand stromal fibrosis is the result of the persistence of myofibroblasts(Mfs), which excessively contract tissue and secrete fibroticextracellular matrix. We recently reported that corneal Mfs arecharacterized by cell surface accumulation of integrin αvβ5 resultingfrom decreased integrin degradation. An agnostic screening approach(RNA-seq) was used to identify possible molecular mechanisms.Methods: Methods: Primary human and porcine corneal fibroblastswere cultured. Gene expression was quantified by RNA-seq, alphasmoothmuscle actin (α-SMA) and integrin expression by Westernblotting and RT-PCR, and protein complex formation by co-IP.Transfection of cDNA was by Nucleofection. Mfs were detected withantibody to α-SMA. The EDA-type of fibronectin (FN-EDA) wasimaged by confocal microscopy and quantified with Leica software.A recycling assay quantified recycling of integrins. Porcine corneas(wounded by keratectomy and control) were cultured for 2 weeksprior to histological examination. USP10 expression was quantifiedwith Image J software.Results: RNA-seq screening revealed that Mfs have increased geneexpression of a subset of de-ubiquitinases (Dubs). Dubs removeubiquitin from proteins, saving them from degradation therebystabilizing protein levels. We found that the Dub, ubiquitin specificpeptidase 10 (USP10), controls integrin αvβ5 protein levels and isincreased in Mfs. This fits our recent finding that Mf differentiationoccurs when integrin αvβ5 ubiquitination and degradation is reduced.Furthermore, over-expression of USP10 cDNA led to a 3-foldincrease in integrin αvβ5 protein expression (with no increase inRNA expression) and a 70% increase in recycling of integrin αvβ5 tothe cell surface (because of reduced degradation). USP10 overexpressionalso induced fibrotic proteins, a 2-fold increase in α-SMA,a 4.4-fold increase in FN-EDA with organization of extracellular FN-EDA when USP10 is over-expressed but not in control. Co-IPconfirmed that USP10 is in a complex with integrin αvβ5. Finally,keratectomy generated stromal Mfs with a 6.8-fold increase in USP10expression.Conclusions: Mfs have increased USP10 expression andexperimental USP10 over-expression induced fibrotic markers.Cellular degradation systems and USP10 in particular may be a novelanti-scarring target.Commercial Relationships: Audrey Bernstein, None; LingyanWang, None; Stephanie R. Gillespie, NoneSupport: This research was supported by NIH-NEI Grant EY09414,NEI Core Grant P30-EY01867, and a Research to Prevent Blindnessgrant. The authors acknowledge use of human tissues provided by theNational Disease Research Interchange (NDRI), with support fromNIH grant 5U42RR006042. Microscopy was performed at theMSSM-Microscopy Shared Resource Facility, supported withfunding from NIH-NCI shared resources grant (5R24CA095823-04),a NSF Major Research Instrumentation grant (DBI-9724504), and aNIH shared instrumentation grant (1S10RR09145-01).Program Number: 3912 Poster Board Number: D0156Presentation Time: 2:45 PM - 4:30 PMMast Cells and the Inflammatory Response to Corneal EpithelialAbrasionAlan R. Burns 1, 2 , Qiong Liu 2 , Zhijie Li 2 , Clifton W. Smith 2 . 1 Collegeof Optometry, University Eye Institute, Houston, TX;2 Pediatrics/Leukocyte Biology, Baylor College of Medicine,Houston, TX.Purpose: Inflammation is beneficial to corneal epithelial and nerveregeneration following abrasion injury. Numerous studies havedocumented a regulatory role for mast cells in inflammation, tissueregeneration and wound healing. The mast cell contribution toinflammation and wound healing after epithelial abrasion isunknown.Methods: Anesthetized adult wildtype C57BL/6 mice or mast celldeficient mice (c-kit-/-) received a 2.0 mm diameter central cornealepithelial abrasion using a golf-club spud. To inhibit mast celldegranulation, wildtype mice were pretreated with cromoglycate(orally, topically or intraperitoneally) or ketotifen (topically). For allmice, topical application of fluorescein was used to evaluate the rateof epithelial wound closure. Excised corneal whole mounts wereprepared for immunofluorescence microscopy and used to evaluatechanges in limbal vessel diameter, numbers of extravasated plateletsand neutrophils and numbers of dividing epithelial cells.Results: Mast cells were abundant near limbal vessels in wildtype©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamice, but rarely seen in c-kit-/- mice. In wildtype mice, epithelialabrasion induced an acute inflammatory response with early (3h) andsustained (out to 30h) dilation in the limbal vessels, and significantneutrophil and platelet extravasation. Mice deficient in mast cells (ckit-/-) exhibited early vessel dilation (3h), but this response was notsustained. These mice also showed diminished platelet and neutrophilrecruitment which was accompanied by delayed (6h) epithelialwound closure and significant blunting of basal epithelial celldivision (60% less at 18h post-injury). Neutrophil recruitment waspartially restored in mast cell deficient mice that received asubconjunctival injection of cultured wildtype mast cells 4 days priorto epithelial abrasion. Experiments treating wildtype mice withcromolyn or ketotifen gave results consistent with those in c-kit -/-mice.Conclusions: Collectively, the data suggest mast cells play a pivotalrole in corneal inflammation and wound healing. Specifically, mastcells are needed for sustained limbal vessel dilation and coordinatedneutrophil and platelet recruitment. In the absence of mast cells ortheir degranulation products, the diminished leukocyte recruitmentlikely accounts for the observed reduction in the rate of cornealwound closure and the number of dividing epithelial cells.Commercial Relationships: Alan R. Burns, None; Qiong Liu,None; Zhijie Li, None; Clifton W. Smith, NoneSupport: NIH Grants EY07551, EY017120, EY018239 and NationalNatural Science Foundation of China grants 39970250, 30772387and 81070703.Program Number: 3913 Poster Board Number: D0157Presentation Time: 2:45 PM - 4:30 PMChanges in Mouse Corneal Epithelial Innervation After InjuryLanny Shulman 1 , Samuel D. Hanlon 1 , Paul T. Landry 1 , Clifton W.Smith 2 , Alan R. Burns 1, 2 . 1 College of Optometry, University ofHouston, Houston, TX; 2 Department of Leukocyte Biology, BaylorCollege of Medicine, Houston, TX.Purpose: Corneal epithelial abrasion is associated with nerve injuryand while nerve regeneration in the mouse model can occur quickly,with nerve density recovering by 6 weeks post-injury, the pattern ofthe subbasal nerve plexus is abnormal. Whether nerve regenerationinvolves a change in the number of epithelial leashes and theirdistribution is unclear. An epithelial leash is defined as a group ofsubbasal nerves originating from a deeper stromal nerve. The purposeof this study is to provide a more detailed analysis of epithelial leashnumber and distribution following a central epithelial cornealabrasion.Methods: Anesthetized adult C57BL/6 mice ages 8-10 weeksreceived 2.0 mm wide central corneal epithelial abrasions with anAlger brush. Uninjured age-matched mice served as controls. Micewere euthanized at 2, 4, and 6 weeks post-injury and corneas wereexcised, fixed and incubated in neuronal specific β-III Tubulin taggedwith phycoerythrin; DAPI staining was used to identify cell nuclei.Using a DeltaVision fluorescence microscope, corneal wholemountswere partitioned into three concentric circular zones: a central 1600μm diameter zone, a middle zone extending approximately 800 μmand a peripheral 480 μm zone. Epithelial leash number anddistribution were evaluated using Image J software and a customMatlab program.Results: In the uninjured cornea, epithelial leashes were generallyconfined to the middle (72 ±12) and peripheral zones (17 ±3); veryfew leashes were detected in the central zone (6 ±4). At 2 weeks postinjury,epithelial leash counts in the central zone increased 4-6 foldover age-matched controls and remained elevated at 4 and 6 weekspost-injury. In contrast, there was no significant change in thenumber of leashes in the middle and peripheral zones at 2, 4 or 6weeks post-injury.Conclusions: The data suggest that following a central epithelialabrasion, the greatest increase in epithelial leashes occurs in thecentral zone, which prior to injury had very few leashes. The de novoappearance of epithelial leashes in the central zone contributes to theabnormal patterning of the regenerating subbasal nerve plexus.Commercial Relationships: Lanny Shulman, None; Samuel D.Hanlon, None; Paul T. Landry, None; Clifton W. Smith, None;Alan R. Burns, NoneSupport: NH Grant EY07551; NH Grant EY017120; NH GrantEY018239411 Keratoconus and BiomechanicsWednesday, May 08, 2013 8:30 AM-10:15 AMTCC 303 Paper SessionProgram #/Board # Range: 4069-4075Organizing Section: CorneaProgram Number: 4069Presentation Time: 8:30 AM - 8:45 AMMutations in the zinc finger protein gene, ZNF469 contribute tothe pathogenesis of keratoconusAndrea L. Vincent 1, 2 , Charlotte Jordan 1, 2 , Bryan Hay 1 , Amanda J.Richards 1 , Charles N. McGhee 1, 2 . 1 Ophthalmology, New ZealandNational Eye Centre, University of Auckland, Auckland, NewZealand; 2 Eye Department, Greenlane Clinical Centre, AucklandDistrict Health Board, Auckland, New Zealand.Purpose: Mutations in the zinc finger protein gene ZNF469 areidentified as one genetic cause for the recessive disorder BrittleCornea syndrome, characterised by spontaneous corneal perforations.GWAS studies have also implicated this gene as a determinant forcentral corneal thickness(CCT). We investigated the geneticcontribution of ZNF469 in a cohort of patients with keratoconus -both familial and sporadic.Methods: Patients with keratoconus were identified and recruitedfrom the University of Auckland special eye clinic, and the EyeDepartment, Auckland District Health Board. If a family history ofkeratoconus was present, family members were recruited andexamined where possible. Following informed consent, allindividuals underwent comprehensive anterior segment examinationincluding corneal topography (Orbscan, Pentacam) and axial length,and a biological sample (venous blood, saliva) collected for DNAextraction.Mutational analysis of both exons of ZNF469 was undertaken usingpolymerase chain reaction, bidirectional Sanger sequencing, and highresolution melting analysis.4 changes detected in the familial cases (Polynesian) were screenedin an ethnically matched population. For each observed change,bioinformatic databases of exome variation were used to determinepresence and frequency, and protein prediction software to determinepathogenicity.Results: Of the 43 probands, at least one probable disease causingvariant was detected in ZNF469 in 20 (46%), and in 5, two variantswere observed (11.6% - 4 compound heterozygotes, 1 homozygote).Fourteen non-synonymous missense SNPs were observed, 6 of whichwere not previously documented in any population-based geneticvariant database. For the sporadic cases, 12/32 had one change, and3/32, 2 changes. Of the familial cases 3/11 probands had one change,and 2 of the 11 had 2 changes, although only heterozygous changessegregated with disease.Conclusions: Rare missense mutations in ZNF469, predicted to bepathogenic, are found heterozygously at a frequency of 46% in a©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneakeratoconus population. ZNF469 is shown to be associated withCCT, and likely to play a role in the synthesis and/or organization ofcorneal collagen fibres. The findings in this cohort suggest thepathogenic changes observed either genetically predispose towards a“thin” cornea, which then becomes keratoconic, or are directlypathogenic.Commercial Relationships: Andrea L. Vincent, OphthalmicResearch Institute of Australia (S); Charlotte Jordan, None; BryanHay, None; Amanda J. Richards, None; Charles N. McGhee, Codatherapeutics (C)Support: Save Sight Society New Zealand, Maurice and PhyllisPaykel Trust, PBRF University of Auckland. Aucland MedicalResearch FoundationProgram Number: 4070Presentation Time: 8:45 AM - 9:00 AMMatrix Metalloproteinase-9 drives disease progression ofKeratoconusashwini ranganath, Rohit Shetty, Sharon D'Souza, KareeshmaWadia, Debashish Das, Arkasubhra Ghosh. cornea and refractivesurgery, Narayana Nethralaya eye hospital, Bangalore, India.Purpose: Keratoconus is a corneal thinning disorder of unknownetiology for which no drugs or therapies are available currently.Furthermore, the exact cause of stromal thinning is not clear. Matrixmetalloproteinases are upregulated during matrix remodeling andcause degradation of various types of collagen. MMP9 has beendemonstrated to degrade typeI and IV collagen fibers in cancer. Thisactivity is regulated by cytokine Interleukin 6(IL6). We thereforeasked whether MMP9 is upregulation in keratoconus correlates toincreasing stages of disease and depends on abnormal IL6 levels. Wequantified levels of MMP9 and IL6 in tear films of keratoconuspatients and correlated with severity of disease. To validate ourhypothesis we investigated effect of topical CsA(cyclosporineA)treatment in keratoconus as it has been demonstrated to inhibitMMP9 and has been approved for treatment of allergic eye disease.Methods: 64 eyes with keratoconus of varying severity wereincluded. This study and on label use od CsA to treat associated withkeratoconus was approved by Narayana Nethralaya IRB.Keratoconus was graded based on topographic criteria ofKeratoconus Severity Index using steep K into 56D. Tear samples were collected from lower fornix withcapillary tubes. Levels of MMP-9 and IL6 in each sample weremeasured by ELISA and were correlated with severity ofkeratoconus. 10 patients with progressive keratoconus were treatedwith topical CsA 0.05% and topography was analyzed pre and posttreatment (6 months and 1 year) to evaluate stability of keratoconus.Results: Mean MMP9 (Normal 3.9-8.3 ng/ml) and IL6 (normal 1-4.1) respectively were 42.45 and 1.03 in 56 D group.Of 10 patients treated with CsA, 8 showed stabilization ofkeratoconus, and 6 had flattening of keratometry ranging from 0.5-1.7D, 6 months to 1 year post treatment.Conclusions: Significantly higher levels of MMP9 and marginallyhigher levels of IL6 were detected in tear samples of keratoconuseyes, and their levels positively correlated with severity ofkeratoconus. Inhibiting MMP9 locally in cornea using CsA halteddisease progression and even reduced the disease in few cases. Thissuggests MMP9 is an important factor driving keratoconuspathophysiology. Also, topical CsA may be an effective diseasemodifying agent in keratoconus management.Commercial Relationships: ashwini ranganath, None; RohitShetty, None; Sharon D'Souza, None; Kareeshma Wadia, None;Debashish Das, None; Arkasubhra Ghosh, NoneClinical Trial: J0002GQQProgram Number: 4071Presentation Time: 9:00 AM - 9:15 AMNovel proteins and metabolites for the identification ofKeratoconus diseaseDimitrios Karamichos 1, 2 , Jesper Hjortdal 4 , Audrey E. Hutcheon 1, 2 ,John M. Asara 3 , James D. Zieske 1, 2 . 1 Schepens Eye ResearchInstitute/MEE, Boston, MA; 2 Department of Ophthalmology,Harvard Medical School, Boston, MA; 3 Division of SignalTransduction/Mass Spectrometry Core, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, MA; 4 Departmentof Ophthalmology, Aarhus University Hospital, Aarhus C, Denmark.Purpose: Keratoconus (KC) is a degenerative disorder of the corneawhere structural changes cause it to thin, protrude and assume a moreconical shape. Prevalence of the disease ranges from 4-600/100,000people and can result in severe vision loss. The exact cause isuncertain, and patients with advanced KC require surgery to maintainvision. One of the major clinical problems with treating the disease isthere are no methods for early detection. Also, there currently are nomodels available to investigate and understand the root causes of thedisease.Methods: In vitro, human corneal fibroblasts (HCF), keratocytes,and keratoconus cells (HKC) were isolated and cultured. Cells werestimulated with a stable Vitamin C (VitC) derivative for 4 weeks,allowing them to secrete a self-assembled matrix. In vivo, humantears were collected from healthy and KC individuals. All sampleswere processed for metabolomic and proteomic analyses using LC-MS/MS. In vitro samples were also processed for indirectimmunofluorescenceand transmission electron microscopy.Results: We identified more than 250 different metabolites of which>50 were differentially regulated between groups. Two of them,Lactate and Arginine, have been previously linked to corneal edemaand thinning. In vitro, Lactate levels were elevated 4 fold in HKCswhen compared to keratocytes and 2 fold when compared to HCFs;however, Arginine levels were significantly reduced in both HCFsand HKCs as compared to keratocytes. In addition, Glutathione levelswere reduced when compared to keratocytes, significantly in HCFsand 2 fold in HKCs. In vivo, these metabolites were regulatedsimilarly, Lactate levels increased and Arginine and Glutathionedecreased in KC patients. We also identified more than 200 proteinsin human tears, some of which may serve as new KC defect markers(i.e. Gross cystic disease fluid protein 15 (GCDFP-15) decreased by 2fold and Lactate dehydrogenase isozyme (LDH) was significantlyelevated in KC patients). Critically, our proteomics andmetabolomics agree and are cross-validating since LDH convertspyruvate to lactate.Conclusions: Overall, we have developed a novel in vitro model thatallows for the study of proteins and metabolites, both in vitro and invivo, which may help understand the root problem of KC and mayallow for identification of markers of KC disease.Commercial Relationships: Dimitrios Karamichos, None; JesperHjortdal, Carl Zeiss Meditec (R); Audrey E. Hutcheon, None;John M. Asara, None; James D. Zieske, NoneSupport: NIH Grant EY020886, NIH Grant EY03790 (Core) andHMS/HSDM Alice J. Adler Fellowship: The Eleanor and MilesShore 50th Anniversary Fellowships for Scholars in MedicineProgram Number: 4072Presentation Time: 9:15 AM - 9:30 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEvaluation of transcorneal iontophoresis of riboflavin for cornealcollagen cross-linkingAlejandro Arboleda 1, 2 , Laura Kowalczuk 3 , Michèle Savoldelli 4 ,Christophe Klein 3 , Sophia Ladraa 3 , Jean-Marie A. Parel 1, 4 , FrancineF. Behar-Cohen 3, 4 . 1 Ophthalmic Biophysics Center, Dept. ofOphthalmology, Bascom Palmer Eye Institute, University of MiamiMiller School of Medicine, Miami, FL; 2 Dept. of BiomedicalEngineering, University of Miami College of Engineering, CoralGables, FL; 3 INSERM UMRS872: Physiopathology of OcularDiseases: Therapeutic Innovations, Centre de Recherche desCordeliers, Pierre et Marie Curie University, Paris DescartesUniversity, Paris, France; 4 AP-HP Hôtel-Dieu, Dept. ofOphthalmology, Sorbonne Paris Cité, Paris Descartes University,Faculty of Medicine, Paris, France.Purpose: To determine whether two riboflavin solutions can bedelivered into the cornea through iontophoresis prior to collagencross-linking. Two conditions, epithelium-on and epithelium-off,were compared to determine which allowed for the most diffusion ofriboflavin throughout the cornea.Methods: A Coulomb-controlled iontophoresis (CCI) generator anda corneal probe were used to deliver either 0.1% riboflavindextranT5000or 0.1% riboflavin-phosphate into the eyes of 8-weekoldLewis rats under either epithelium-on (epi-on) or epithelium-off(epi-off) conditions. Within 30 minutes of CCI (1.77 mA/cm 2 for 4minutes), corneas were flat-mounted for fluorescence imaging usinga confocal microscope (LSM 710, Zeiss). The distribution ofriboflavin in the cornea was evaluated by recording the fluorescenceintensity. Aqueous humors (Aq.H) were also collected to assayriboflavin concentration by fluorometry using a multilab counter(Wallac Victor 1420, Perkin Elmer).Results: Iontophoresis delivered both riboflavin solutions throughoutthe whole cornea for the epi-off condition. For the epi-on case, onlyriboflavin-phosphate was successfully delivered to the cornea (Figure1). Corneal imaging and Aq.H assay both demonstrated thatriboflavin-phosphate delivery is more efficient than riboflavindextrandelivery. The riboflavin concentrations in Aq.H were: epi-onriboflavin-phosphate, 0.92 µg/mL; epi-on riboflavin-dextran, 0.55µg/mL; epi-off riboflavin-phosphate, 13.6 µg/mL; and epi-offriboflavin-dextran, 4.73 µg/mL.Conclusions: Iontophoresis can be used to efficiently deliverriboflavin into the cornea. The epi-off condition enhances diffusionof riboflavin into the stroma and aqueous humor. Transcornealdelivery is only possible with riboflavin-phosphate. The efficiency ofCXL through an intact epithelium, which absorbs most UV radiation,remains to be demonstrated.Figure 1: White shows riboflavin fluorescence: the whiter, the higherthe riboflavin concentration (A) Riboflavin-dextran diffusion in thecornea for the epi-off condition (B) Riboflavin-dextran diffusion inthe cornea for the epi-on condition (C) Riboflavin-phosphatediffusion in the cornea for the epi-off condition (D) Riboflavinphosphatediffusion in the cornea for the epi-on conditionCommercial Relationships: Alejandro Arboleda, None; LauraKowalczuk, None; Michèle Savoldelli, None; Christophe Klein,None; Sophia Ladraa, None; Jean-Marie A. Parel, CROMA (F),InnFocus (F), Abeamed (F), University of Miami (P); Francine F.Behar-Cohen, Inserm/Univesrité ParisDescartes (P)Support: Agence Nationale de la Recherche Grant ANR-11-RPIB-020, Florida Lions Eye Bank, NIH Center Grant P30EY14801,Research to Prevent Blindness, Florida-Georgia Louis StokesAlliance for Minority Participation, Henri and Flore LesieurFoundation (JMP)Program Number: 4073Presentation Time: 9:30 AM - 9:45 AMCorneal biomechanical properties in corneal collagen crosslinking(CXL) at high fluencesArthur Hammer 1 , Olivier Richoz 1 , David Tabibian 1 , FlorenceHoogewoud 1 , Farhad Hafezi 1, 2 . 1 Ophthalmology, Geneva UniversityHospitals, Geneva, Switzerland; 2 Ophthalmology, Doheny EyeInstitute, Keck school of medicine USC, Los Angeles, CA.Purpose: The current intensity/irradiation profile for collagen crosslinking(3 mW/cm2 for 30 minutes) has been in clinical use since1999 in a multitude of studies. Lately, different irradiation profileshave emerged, with the intention to lower irradiation time whilemaintaining the total energy dose (Bunsen-Roscoe law). Little isknown whether these modified irradiation profiles will lead to thesame increase in biomechanical stiffness observed in the originalprotocol.Methods: We investigated the biomechanical properties in ex vivoporcine corneas at 3 (n = 10), 9 (n = 10), and 18 (N = 12) mW/cm2for various time periods (30, 10, 5 minutes), while maintaining thetotal energy dose identical (Bunsen-Roscoe law). Prior to irradiation,riboflavin 0.1% was applied on the de-epithelialized cornea for 20minutes. Controls (n = 11) were treated similarly, but without UV-Airradiation. Stress-strain measurements were performed using anextensometer (Zwick Roell, Model ZO.05, Zwick GmbH & Co. KG,Ulm, Germany).Results: We observed a decrease in Young’s modulus withincreasing UV-A intensity (fluence). Young’s modulus at 4 %, 6 %, 8% and 10 % strain at 3 mW/cm2 was 1.66 N, 0.75 N, 0.44 N and 0.29N respectively. At 9 mW/cm2, we measured 1.52 N, 0.7 N, 0.39 Nand 0.24 N, whereas at 18 mW/cm2, we detected 1.2 N, 0.57 N, 0.33N and 0.21 N for 18 mW. Controls showed 0.72 N, 0.37 N, 0.37 Nand 0.16 N.Conclusions: The increase in biomechanical strength of the porcinecornea following CXL diminishes with increasing fluence/decreasingirradiation time, even if the total energy dose is maintained.The generation of chemical bonds during cross-linking is an oxygendependentprocess that depends on intra-stromal oxygenconcentration. Oxygen diffusion capacity is limited within the stromaand the increased oxygen consumption at higher fluence/lowerirradiation time might become a limiting factor for cross-linking,leading to the observed decrease in treatment efficacy.Commercial Relationships: Arthur Hammer, None; OlivierRichoz, None; David Tabibian, None; Florence Hoogewoud, None;Farhad Hafezi, Schwind (F), Ziemer (F), PCT/CH 2012/000090 (P)Program Number: 4074©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 9:45 AM - 10:00 AMSoluble collagen protects the corneal fibrils during riboflavincrosslinkingMarcos Garza-Madrid 1, 2 , Jennifer Elisseeff 1 . 1 Translational TissueEngineering Center, Wilmer Eye Institute, Johns Hopkins School ofMedicine, Baltimore, MD; 2 Ophthalmology and Visual SciencesResearch Chair, School of Medicine and Health Sciences,Tecnológico de Monterrey, Monterrey, Mexico.Purpose: Cornea crosslinking with riboflavin and UV-A is a novelprocedure able to stop the progression of keratoconus. Our aim is tocharacterize and prevent the fibrillar damage associated with thisprocedure.Methods: Fresh bovine corneas were obtained from a localslaughterhouse. Six mm biopsies, devoid of epithelium andendothelium were treated with a Riboflavin isotonic solution with orwithout soluble collagen. The tissue was then crosslinked using UV-A light. Hydration was maintained by adding crosslinking solutionperiodically. Samples were then either processed for TEM oranalyzed through DSC to identify the temperature at which themolecular structure breaks down. Fibril counting was performedmanually on a minimum of 180 fibrils per group.Results: After 30 minutes of crosslinking the denaturationtemperature of the corneas increased from 66.77±0.92°C to70.94±1.72°C (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea419 Dry Eye and Lacrimal Gland IVWednesday, May 08, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 4308-4362/C0046-C0100Organizing Section: CorneaProgram Number: 4308 Poster Board Number: C0046Presentation Time: 8:30 AM - 10:15 AMTear specific potential protein biomarker identification by 2D-DIGE based proteomics in Dry eye syndrome associated withRheumatoid ArthritisNarayanasamy Angayarkanni 1 , Saijyothi Venkata Aluru 2 , UtpalTatu 3 , Shweta Agarwal 4 , Bhaskar Srinivasan 5 , Geetha KrishnanIyer 6 , Sivakumar M. Rajappa 7 , Umashankar Vetrivel 8 , PremaPadmanabhan 9 . 1 Biochemistry and Cell Biology, Vision ResearchFoundation, Chennai, India; 2 Biochemistry and Cell Biology, VisionResearch Foundation, Chennai, India; 3 Biochemistry Dept, IndianInstitute of Science, Bangalore, India; 4 Ocular Surface Clinic,Medical Research Foundation, Chennai, India; 5 Ocular SurfaceClinic, Medical Research Foundation, Chennai, India; 6 OcularSurface Clinic, Medical Research Foundation, Chennai, India;7 Cerebrovascular and Vasculitis Research Foundation, Chennai,India; 8 Centre for Bioinformatics, Vision Research Foundation,Chennai, India; 9 Cornea Services, Medical Research Foundation,Chennai, India.Purpose: Dry eye syndrome (DES) is a disorder of the tear andocular surface. The tear film dynamics maintain normal cornealfunction. Sjogrens syndrome (SS) secondary to Rhematoid Arthritis(RA) is an autoimmune disease affecting the extracellular glands likelacrimal, salivary and therefore DES is more prevalent in RA. Thisstudy aimed to identify potential biomarkers in tear in DES secondaryto RA (RA-DES) based on 2D-based DIGE analysis compared toprimary sjogrens (PS-DES), non sjogrens (NS-DES) and healthycontrols.Methods: As part of a prospective case-control study after IRBapproval DES cases were recruited in the ocular surface clinic basedon detailed dry eye work up and the report from rheumatoid clinic.Tear specimen were collected using schirmer strips from 102 DEScases (mean age: 45 ± 3 y) that included RA-DES, PS-DES, and NS-DES along with 56 age matched controls (mean age: 43 ± 12 y).Tearproteins were extracted using 8M urea buffer, subjected to 2D-Differential gel electrophoresis (DIGE). While pooled sampleanalysis was done by 2D-DIGE, independent samples were run by2D. Differentially expressed proteins showing more than 2 foldvariation were identified based on biological variance analysis (BVA)and extended discriminate analysis (EDA) using Decyder software.These peptide spots were then detected by nano ESI-LC-MS/MSanalysis. Functional annotation was done using DAVID annotationtool.Results: Of the 5 down regulated proteins in DES-RA,Lactotransferrin isoform 1 precursor showed down regulation in allthe cases, SHC transforming 1 isoform in 63% cases, and the restnamely Ribonuclease protein subunit 20, Protocadherin andheterogeneous nuclear ribonucleoprotein Q isoform 6 showed downregulation in more than 80% cases. Three proteins were up regulatednamely, Ecto-ADP ribosyltranferase 5 precursor, Rho-related GTPbindingprotein and RhoJ precursor as seen in 80% cases. Functionalannotation of proteins specific to DES-RA, revealed that theseproteins have roles in regulation, antimicrobial activity, immune,metabolic and cellular processes.Conclusions: The study revealed characteristic proteins that aredifferentially expressed in the tears of sjogrens syndrome secondaryto RA. Further validation based on clinical correlations has to bedone before characterising them as biomarkers.Commercial Relationships: Narayanasamy Angayarkanni, None;Saijyothi Venkata Aluru, None; Utpal Tatu, None; ShwetaAgarwal, None; Bhaskar Srinivasan, None; Geetha KrishnanIyer, None; Sivakumar M. Rajappa, None; Umashankar Vetrivel,None; Prema Padmanabhan, NoneSupport: Department of Biotechnology, Govt of IndiaProgram Number: 4309 Poster Board Number: C0047Presentation Time: 8:30 AM - 10:15 AMThe Correlation of Muc-16 as compared to Dry Eye ClinicalEndpoints in Dry Eye and Normal SubjectsMichael Watson, Keith J. Lane, Andy Whitlock, George W. Ousler.Ora, Inc., Andover, MA.Purpose: Muc-16 is one of the primary membrane-associated mucinsthat form the protective glycocalix on the ocular surface. Previousstudies have shown that the extracellular domain of this glycoproteinis cleaved and released in the tear film of dry eye patients. Cleavageof Muc-16 has also been demonstrated in response to localinflammation. The purpose of this study was to confirm the upregulationof soluble Muc-16 (CA-125) in the tear film of dry eyepatients, and explore any correlations with the clinical signs of dryeye.Methods: Tears from a total of 49 eyes from 34 subjects werecollected and included in the analysis (8 normal and 26 dry eye). Acommercially available Muc-16 (CA-125) ELISA kit was used toquantify the amount of Muc-16 in each sample. Muc-16 levels ineach tear sample were normalized to the total protein concentrationdetermined for each sample. Clinical endpoints including Schirmer’stest, fluorescein staining, and tear film breakup time (TFBUT) wereassessed for both dry eye and normal patients.Results: Muc-16 values were calculated in terms of Muc-16 units (U)per microgram (µg) of total protein in a given tear sample. There wasa statistically significant inverse correlation between Muc-16 andShirmer’s and TFBUT for all subjects. Total staining, cornealstaining, and conjunctival staining showed statistically significantpositive correlations with Muc-16 for all subjects.Conclusions: The results of this study conclude that as the signs ofdry eye worsen, soluble Muc-16 values increase. Additional studiesare required to confirm this hypothesis and should ensure that equalnumbers of age-matched males and females are sampled that are bothsymptomatic and asymptomatic for dry eye.Summarizes the correlation of Muc-16 as compared to all analyzeddry eye signs. P-values are displayed for correlations that aresignificant with its subsequent coefficient of determination (R 2 ).©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Michael Watson, Ora, Inc. (E); KeithJ. Lane, Ora, Inc. (E); Andy Whitlock, Ora, Inc. (E); George W.Ousler, Ora, Inc. (E)Program Number: 4310 Poster Board Number: C0048Presentation Time: 8:30 AM - 10:15 AMThe Use of Soluble Muc-16 (CA-125) as a Clinically RelevantBiomarker and Endpoint in a Mouse Model of Dry EyeJennifer Brackett, Laura Belen, Kortni Violette, Andy Whitlock. Ora,Inc., Andover, MA.Purpose: Inflammation is a significant part of Dry Eye Disease(DED), but no clear consensus between clinical and pre-clinicalinflammatory endpoints has yet been established. Muc-16 is one ofthe primary membrane-associated mucins that form the protectiveglycocalyx on the ocular surface. Previous studies have shown thatthe extracellular domain of this glycoprotein is cleaved and releasedin the tear film of dry eye patients. This cleavage of Muc-16 has alsobeen demonstrated in other systemic inflammatory conditions, suchas ovarian cancer, for which soluble Muc-16 (CA-125) is abiomarker. The purpose of this study was to confirm the increase ofsoluble Muc-16 in the tear film of mice following Ora’sscopolamine/Controlled-Adverse-Environment (CAE) challenge.Methods: Female C57/BL6 mice were subjected to Ora’s optimizeddry eye model for 13 days or left untreated in normal housingconditions (naïve controls). Corneal staining was assessed on days 0,4, 8, and 12 using a modified Micron III imaging system to assess thecorneal damage due to the CAE challenge. The extent of staining wasassessed using Ora’s proprietary grading scale along with an imageanalysis algorithm. Tear washes were collected and analyzed fromboth treatment groups 24 hours following the final exam. Tear levelsof soluble Muc-16 were determined by ELISA for each sample. Muc-16 levels were normalized to the total protein concentrationdetermined for each sample via BCA assay.Results: Corneal staining was significantly increased (p < 0.0001) indry eye challenged mice compared to naïve controls by day 4 of thechallenge and remained elevated throughout the entire study. Tearwash analysis at the conclusion of the challenge revealed a significantincrease (p < 0.0001) of soluble Muc-16 in challenged micecompared to naïve controls. There was also a significant correlation(p= 0.0051, r 2 = 0.361) between the final corneal staining score andMuc-16 levels.Conclusions: The study confirms that soluble Muc-16 (CA-125)release into the tear film following periods of prolonged dry eyestress is conserved between the mouse model and the human clinicalcondition. This further validates the scopolamine/CAE mouse modelas a clinically relevant model for studying novel treatments for dryeye disease.Commercial Relationships: Jennifer Brackett, Ora, Inc. (E);Laura Belen, Ora, Inc. (E); Kortni Violette, Ora, Inc. (E); AndyWhitlock, Ora, Inc. (E)Program Number: 4311 Poster Board Number: C0049Presentation Time: 8:30 AM - 10:15 AMMegalin and Cubilin Levels are Altered in Tear-ProducingGlands of Vitamin D Receptor Null MiceXiaowen Lu, Mitchell A. Watsky. Department of Physiology,University of Tennessee Health Science Center, Memphis, TN.Purpose: Vitamin D is known to be produced in the skin andactivated in the liver and kidneys. Megalin and cubilin areendocytosis -associated proteins believed to act as Vit D transporters.Our lab has recently determined that Vit D can also be produced inthe cornea and is found in tear, aqueous humor, and vitreous humorof rabbits. Dietary Vit D supplementation results in increased Vit Dconcentrations in the plasma and in the tear and aqueous humor. Thepurpose of this study was to determine if megalin or cubilin levels arealtered in Vit D receptor (VDR) null mice and if feeding them aspecial diet rich in calcium, P, and lactose reverses VDR-inducedchanges.Methods: Real-time PCR was used to explore the level of mRNA ofmegalin and cubilin in mouse Harderian glands, which secrete ahighly lipid-based product into tear. Glands were isolated from wildtype C57BL/6 mice (+/+) and from heterozygous (+/-) andhomozygous (-/-) VDR null mice (Jackson Labs) that were 1 and 2.5months old. A subset of 2.5 mo (-/-) mice were fed a replenishmentdiet consisting of high Ca, P, and lactose. mRNA was isolated andcDNA was synthesized at 55 °C for 60 min, and reverse transcriptasewas inactivated by heating to 85 °C for 5 min. Equal amounts ofcDNA were applied for PCR amplification in triplicate in thethermocycler using the LightCycler® 480 System and UPL probes.The amplification was normalized by reference to that of theribosomal protein S19 gene. PCR conditions were set to 95°C for 10min initialization, followed by 40 cycles at 95 °C for 10 sec, 60 °Cfor 30 sec, 72 °C for 10 sec, followed by cool down to 40 °C for 30sec.Results: Megalin mRNA levels were decreased in VDR -/- mice at 1mo and were further decreased in 2.5 mo mice. VDR +/- mice hadlower levels at 1 mo but no change at 2.5 mo. The special dietresulted in an increase in megalin levels as compared to the 2.5 momice that was lower than control levels. Cubilin mRNA levels werelower in +/- and -/- mice at both time points with no effect of thespecial diet. 2.5 mo -/- mice had lower levels than 1 mo mice.Conclusions: VDR knockout lowers both megalin and cubilinmRNA levels in mouse Harderian gland. The special diet resulted inan increase in megalin mRNA levels but not cubilin, indicating thatcubilin is more dependent on VDR receptor activity.Commercial Relationships: Xiaowen Lu, None; Mitchell A.Watsky, NoneSupport: NIH Grant EY021747Program Number: 4312 Poster Board Number: C0050Presentation Time: 8:30 AM - 10:15 AMChanges in corneal nerve morphology and epithelial woundhealing after prolonged ocular dryness induced by lacrimal glandablationKamila Mizerska 1 , Nicolas Cuenca 2 , Carolina Luna 1 , SusanaQuirce 1 , Laura Fernandez-Sanchez 2 , Illes Kovacs 3 , M CarmenAcosta 1 , Carlos Belmonte 1 , Juana Gallar 1 . 1 Instituto deNeurociencias, Universidad Miguel Hernandez-CSIC, San Juan deAlicante, Spain; 2 Departamento de Fisiología, Genética yMicrobiología, Universidad de Alicante, Alicante, Spain;3 Department of Ophthalmology, Semmelweis University, Budapest,Hungary.Purpose: To analyze the morphological changes of corneal nervesand corneal epithelial wound healing at different times (1-8 months)after surgical removal of the lacrimal gland in guinea-pigs.Methods: The right exorbital lacrimal gland was surgically removedin anesthetized animals thereby inducing a chronic tear flowreduction (dry eye); 1, 2, 3, 4 and 8 months after surgery, eyes werefixed and cryoprotected. Whole-mount corneas were incubated withneuronal class III β-tubulin antibody and Alexa fluor 488. A set ofcorneas was incubated in ABC. Corneas of 6 non-operated guineapigs served as control. Corneal epithelial wound healing was studied1 and 6 months after surgery in 15 dry eyes and in 5 control eyes.Epithelium debridation was performed with a 2mm-diameter piece ofpaper soaked in n-heptanol. Lesions were stained with fluoresceinand photographed regularly until complete closure. Images were©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaanalyzed with image processing software. Epithelial migration rate(EMR, in µm/h) and estimated time of healing (ETH, in hours) werecalculated.Results: Density and length of subbasal nerves decreasedsignificantly after 1 month in dry eyes compared to control eyes.Subbasal nerves appeared less branched and the number of epithelialnerve terminals was significantly reduced. These effects were moreprominent 2 months after induction of eye dryness. 4-8 months aftertearing deficiency, density and length of subbasal corneal nerves hadrecovered values close to control, although nerve architecture was notfully normal. EMR was significantly decreased and ETH wassignificantly increased 1 and 6 months after surgery (Table 1),although at month 6th, ETH was partly recovered.Conclusions: The changes in corneal subbasal nerve architecture 1-2months after lacrimal gland removal suggest that nerve damagedevelops shortly after induction of reduced tearing, leading to aneurotrophic slowdown of epithelial wound healing. At longer times,regeneration of corneal nerves appears to restore in part the woundhealing capabilities of the normal corneal epithelium.Commercial Relationships: Kamila Mizerska, None; NicolasCuenca, Universidad de Alicante (P); Carolina Luna, None; SusanaQuirce, None; Laura Fernandez-Sanchez, None; Illes Kovacs,None; M Carmen Acosta, None; Carlos Belmonte, None; JuanaGallar, NoneSupport: SAF2011-22500, CSD2007-00023, IPT-2011-1110-900000 and BFU2008-04425, and in part by BFU2012-36845 andRETICS RD12/0034/0010 (Ministerio de Ciencia e Innovación,Spain, and FEDER, EU)Program Number: 4313 Poster Board Number: C0051Presentation Time: 8:30 AM - 10:15 AMIsolation and characterization of progenitor cells from intactrabbit lacrimal glandHong He 1 , Guoying Sun 1 , Hui Lin 1 , Marie A. Shatos 2 , Darlene A.Dartt 2 , Samuel C. Yiu 1, 3 . 1 Wilmer Eye Institute, Baltimore, MD;2 Shepens Eye Research Institute, Boston, MA; 3 King Khaled EyeSpecialist Hospital, Riyadh, Saudi Arabia.Purpose: Lacrimal gland dysfunction is believed to be the singlemost important factor resulting in aqueous-deficient dry eye.Regeneration of the lacrimal gland using progenitor cells maypotentially restore lacrimal gland function and therefore improve theocular surface health. Previous studies have documented the role ofprogenitor cells in the repair of murine lacrimal glands. Since rabbitis a promising animal model for further lacrimal gland study (due tobetter accessibility to its ocular surface), it is important to studywhether the progenitor cells also exist in rabbit LGs. The purpose ofthis study is to investigate the presence of progenitor cells in intactadult rabbit LGs, and if they could be isolated and expand in vitro.Methods: Frozen intact rabbit LG sections were made. While acinarand duct cells were identified by their respective cellularmorphology, myoepithelial cells were identified byimmunohistochemistry (IHC) using antibody against alpha-smoothmuscle actin (α-SMA). The presence of progenitor cells wasdetermined using IHC by exploring for the expression of selectedstem cell markers - △Np63, ABCG2, Pax6, vimentin. Immature cellswere isolated from LGs using enzyme digestion and mechanicalseparation and subsequently grown in keratinocyte growth medium(KGM) without fetal bovine serum. The expression of selected stemcell markers and α-SMA in immature cells from passage 0 (P0) topassage 2 (P2) was examined by immunocytochemistry (ICC).Results: In the intact adult rabbit LGs, some myoepithlial and ductcells expressed stem cell markers - △Np63, ABCG2, Pax6 andvimentin. Isolated immature cells were found to successfully passageand expand in serum-free KGM, and express stem cell markers -△Np63, ABCG2, Pax6 and vimentin - in a subpopulation from P0 toP2. In addition, some isolated immature cells expressing stem cellmarkers were also found to be labeled with α-SMA.Conclusions: We conclude that progenitor cells exist in intact adultrabbit LGs and can be isolated in vitro. Our results also indicate thatthe progenitor cells may not only be myoepithelial cells but also ductcells.Commercial Relationships: Hong He, None; Guoying Sun, None;Hui Lin, None; Marie A. Shatos, None; Darlene A. Dartt, None;Samuel C. Yiu, NoneProgram Number: 4314 Poster Board Number: C0052Presentation Time: 8:30 AM - 10:15 AMAlterations of Tear Functions and Ocular Surface EpithelialDifferentiation in the SOD-1 Knock- out MouseMURAT DOGRU 1, 2 , Takashi Kojima 2, 1 , Taeko Nagata 2, 1 , AyakoIgarashi 1, 2 , Kazunari Higa 1, 2 , Yoshiyuki Satake 1 , Seika Shimazaki 1 ,Shimizu Takahiko 3 , Kazuo Tsubota 2, 1 , Jun Shimazaki 1 .1 Ophthalmology, Tokyo Dental College, Ichikawa, Japan;2 Ophthalmology, Keio University School of Medicine, Tokyo, Japan;3 Institute of Aging, Chiba University School of Medicine, Chiba,Japan.Purpose: Purpose: SOD-1 knock out mouse has been reported to be amodel for age related dry eye disease. We investigated the alterationsin the tear function and conjunctival ocular surface epithelialdifferentiation in the Sod1-/- in comparison to the wild type mice.Methods: Methods: Ten eyes of 5 Sod1-/- male mice withC57BL/background and 10 eyes of 5 C57BL6 strain wild-type malemice were examined at 10 and 50 weeks in this study. Tear filmstability and corneal epithelial damage was evaluated by fluoresceinand Rose Bengal stainings. Anterior segment photography wascarried out at 10 to 50 weeks. Aqueous tear quantity was measuredwith phenol-red-impregnated cotton threads without anesthesia.Animals were sacrificed and the whole globe specimens underwentPAS and SPDEF(SAM pointed domain containing ets transcriptionfactor) immunohistichemistry staining. Quantitative Real Time-PCRfor conjunctival muc 5AC mRNA and SPDEF expression was alsoperformed. All studies were performed in accordance with the ARVOStatement for the Use of Animals in Ophthalmic and VisionResearch. Statistical analysis was performed by using t test andANOVA. A p value

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: MURAT DOGRU, OtsukaPharmaceuticals (F); Takashi Kojima, None; Taeko Nagata, None;Ayako Igarashi, None; Kazunari Higa, None; Yoshiyuki Satake,None; Seika Shimazaki, None; Shimizu Takahiko, None; KazuoTsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical(C), Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa(F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki(P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company(P); Jun Shimazaki, Santen Pharmaceutical Co. (F), OtsukaPharmaceutical Co. (F), Abott Medical Optics (F)Support: Otsuka PharmaceuticalsProgram Number: 4315 Poster Board Number: C0053Presentation Time: 8:30 AM - 10:15 AML-carnitine, Erythritol and Betaine Suppress the Production andActivity of Matrix Metalloproteinases in Primary HumanCorneal Epithelial Cells Exposed to Hyperosmotic StressRuzhi Deng 1, 2 , Zhitao Su 1, 2 , Jing Lin 1 , Xia Hua 1 , De-Quan Li 1 ,Stephen C. Pflugfelder 1 . 1 Department of Ophthalmology, BaylorCollege of Medicine, Houston, TX; 2 Optometry and Ophthalmology,Wenzhou Medical College, Wenzhou, China.Purpose: Hyperosmolarity has been recognized to be a proinflammatorystress in the pathogenesis of dry eye disease. This studyinvestigated the suppressive effect of osmoprotectants (L-carnitine,erythritol and betaine) on the production and activity of matrixmetalloproteinases (MMPs) in primary human corneal epithelial cells(HCECs) exposed to hyperosmotic stress.Methods: Primary HCECs were established from fresh donor limbaltissue explants. The cultures in iso-osmolar medium (312 mOsM)were switched to hyperosmotic media (400-500 mOsM) by adding50-90 mM NaCl, with or without prior incubation with differentconcentrations (2, 10 or 20mM) of L-carnitine, erythritol or betaine.The mRNA expression of MMPs by HCECs treated for 4 hours wasdetermined by reverse transcription and quantitative real time PCR.Their protein production and activity in the conditioned media fromcultures treated for 24-48 hours were evaluated by zymography,immunofluorescent staining, ELISA and activity assays.Results: Hyperosmotic stress (400, 450 or 500 mOsM) significantlystimulated the mRNA expression of collagenase MMP13 (3.1 to 7.5fold), gelatinase MMP9 (1.7 to 2.5 fold), stromelysin MMP3 (2.2 to4.1 fold) and matrilysin MMP7 (2.3 to 4.5 fold), mostly in anosmolarity dependent fashion. Interestingly, the stimulatedexpression of these MMPs was significantly, but differentiallysuppressed by L-carnitine, erythritol or betaine. L-carnitine appearedto have the greatest inhibitory effects, and down-regulated 52-78%,respectively, of the stimulated mRNA levels of MMP13 (down from7.0 to 3.1 fold), MMP9 (2.5 to 1.2 fold), MMP3 (4.1 to 0.9 fold), andMMP7 (4.5 to 1.5 fold) by HCECs exposed to 450 mOsM. Thestimulated production and activity of these MMPs by hyperosmoticstress and the suppressive effects of L-carnitine, erythritol andbetaine were further confirmed at protein levels by gelatin and caseingel zymography, immunofluorescent staining, ELISA and/or activityassays, respectively.Conclusions: Our findings demonstrate that hyperosmotic stressstimulates the expression, production and activity of MMPs inHCECs. L-carnitine, erythritol and betaine serve as osmoprotectantsthat suppress the production and activity of MMPs in HCECsexposed to hyperosmotic stress. L-carnitine shows the best inhibitoryeffect among the three.Commercial Relationships: Ruzhi Deng, None; Zhitao Su, None;Jing Lin, None; Xia Hua, None; De-Quan Li, None; Stephen C.Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb(C), Baylor College of Medicine (P)Support: NIH Grants EY11915(SCP) and Core Grant for VisionResarch EY002520., Allergan Inc., Research to Prevent Blindness.,Oshman Foundation,. William Stamps Farish Fund.Program Number: 4316 Poster Board Number: C0054Presentation Time: 8:30 AM - 10:15 AMSuppressive effects of 17β-estradiol on immortalized humanmeibomian gland epithelial cellsWendy R. Kam, David A. Sullivan. Schepens Eye Research Institute,Massachusetts Eye and Ear, Harvard Medical School, Boston, MA.Purpose: Previous studies have shown that 17β-estradiol (E 2 )significantly decreases adenylate cyclase activity in bone cells,thereby attenuating the cyclic AMP response to stimulatory factors(e.g. the secretagogue forskolin). It is possible that this inhibitoryeffect on signal transduction represents one mechanism by whichestrogens suppress sebaceous gland epithelial cell function. If so,such an E 2 influence on the meibomian gland, which is a largesebaceous gland, could account for the increased incidence of dry eyedisease in women taking estrogen replacement therapy. Our objectivewas to determine whether E 2 attenuates secretagogue-induced cAMPaccumulation in immortalized human meibomian gland epithelialcells (iHMGEC). As part of these studies, we also evaluated whetherE 2 modulates iHMGEC proliferation.Methods: Immortalized human meibomian gland epithelial cellswere cultured in medium free of serum and phenol red prior totreatment. For cAMP analysis, cells were treated with E 2 (100 pM),forskolin (100 µM), the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), or buffer for 10 minutes prior toquantification of intracellular cAMP with a colorimetric enzymelinkedimmunoassay. For proliferation studies, cells exposed to E 2 orbuffer for 1, 3, and 5 days were counted with a hemocytometer.Results: Estrogen treatment caused a significant decrease insecretagogue-induced cAMP accumulation in iHMGEC. Thishormonal effect was observed after cellular exposure to eitherforskolin or IBMX. In contrast, in the absence of E 2 , forskolin andIBMX elicited rapid intracellular cAMP accumulation. Treatment ofiHMGEC with E 2 for 5 days caused a slight but significant decreasein proliferation.Conclusions: Our research demonstrates that E 2 may suppress bothcAMP signaling in, and the proliferation of, iHMGEC. Themechanisms underlying these estrogen effects, as well as theirpossible role in promoting dry eye, remain to be clarified.Commercial Relationships: Wendy R. Kam, None; David A.Sullivan, Forest Research Institute (C), Horus Pharma (R), TearLab(S), Lubris (I), Santen (R)Support: NIH grant EY05612 and the Margaret S. Sinon Scholar inOcular Surface Research fundProgram Number: 4317 Poster Board Number: C0055Presentation Time: 8:30 AM - 10:15 AMImpact of azithromycin on lipid accumulation in immortalizedhuman meibomian gland epithelial cellsYang Liu, Wendy R. Kam, Juan Ding, David A. Sullivan. SchepensEye Research Institute, Massachusetts Eye and Ear, Harvard MedicalSchool, Boston, MA.Purpose: Meibomian gland dysfunction(MGD) is believed to be theleading cause of dry eye disease(DED) in the world, and afflicts tensof millions Americans. Perhaps the most common MGD treatment inthe USA is the off-label use of topical azithromycin.The efficacy ofthis macrolide antibiotic has been ascribed to its anti-inflammatoryand antibacterial actions. However, the mechanism by whichazithromycin acts on the meibomian gland is unknown. Wehypothesize that azithromycin promotes lipid accumulation in human©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneameibomian gland epithelial cells (HMGEC), given that macrolidesare known to stimulate lipid dynamics in other cell types. Our goalwas to test this hypothesis.Methods: Immortalized (i) HMGEC (5x10 4 /well) were incubatedovernight on 4-well chamber slides in keratinocyte serum-freemedium containing epidermal growth factor (EGF; 5 ng/ml) andbovine pituitary extract (50 µg/ml). Following this period cells werecultured in DMEM/F12 medium containing 10% fetal bovine serum(with or without charcoal-stripping) and EGF (10 ng/ml), in thepresence or absence of 10µg/ml azithromycin. Cellularmorphological appearance was recorded, and cells were stained withLipidTox for neutral lipid assessment, on culture days 1, 3, 5 and 7.Results: Our results show that azithromycin induces a striking, timedependentaccumulation of lipid in iHMGEC. Within 3 days ofazithromycin exposure, the number, size and staining intensity ofintracellular lipid-containing vesicles had markedly increased, ascompared to those of vehicle-treated control cells. This azithromycineffect on lipids appeared to become maximal at days 5 to 7 of thestudy. Evaluation of cellular morphology indicated that azithromycinmay promote terminal maturation of iHMGEC, given that vesicleaccumulation was often followed by a cell break-up and vesiclerelease.Conclusions: Our findings support our hypothesis that azithromycinstimulates the accrual of lipid-containing vesicles in iHMGEC. Thisazithromycin effect may be paralleled by a cellular maturation and aholocrine-like secretion.Commercial Relationships: Yang Liu, None; Wendy R. Kam,None; Juan Ding, None; David A. Sullivan, Forest ResearchInstitute (C), Horus Pharma (R), TearLab (S), Lubris (I), Santen (R)Support: Supported by NIH grant EY05612 and the Margaret S.Sinon Scholar in Ocular Surface Research fundProgram Number: 4318 Poster Board Number: C0056Presentation Time: 8:30 AM - 10:15 AMA Comparative Evaluation of Lipid-based Formulations for Dry-Eye Therapy using a Corneal Epithelial Cell Desiccation Modeland Physicochemical MeasurementsParamita Sarkar, Zora Marlowe, Amy Walsh, Brian Glass, TammyKleiber, Megan E. Cavet, Karen L. Harrington, Stephen Davio.R&D, Bausch & Lomb, Rochester, NY.Purpose: The inclusion of both aqueous and lipid components in asingle formulation to target multiple layers of the tear-film is a newtrend in dry eye therapy. This study determined the potentialbeneficial effects of novel (SG1, SG2 and AG2) and marketed(Systane Balance,SB and Refresh Optive Advanced, ROA) lipidbasedOTC products on the tear film and ocular surface usingmultiple physicochemical and corneal epithelial cell models.Methods: Viscosity was measured on a Brookfield viscometer usinga cone and plate attachment. Rheological characterization was doneon a TA AR2000 instrument. Surface tension measurements andwettability assessments were done using a Kruss-K 100 tensiometerand captive bubble method with Acuvue Oasys lenses respectively.Destabilization of formulations in simulated tear fluid (STF), releaseof lipids and their impact on a simulated tear film lipid layer (TFLL)were evaluated using a Lumisizer complemented by lipid layercompressibility studies using a Langmuir trough. Protection ofhuman corneal epithelial cells from desiccation-induced death wasevaluated using cells pre-treated with formulation and then subjectingthem to desiccation, after which cell viability was measured.Results: The viscosities of SB and ROA were around 3 cps & 12 cpsrespectively. Only SB showed shear-thinning property. The threenovel formulations had viscosities above 50 cps and were markedlyshear-thinning. All formulations had low surface tension and goodwettability due to the presence of surfactants. SB and ROA did notappear to destabilize upon STF dilution or release their componentlipids to improve the compressibility of the TFLL. SG1, SG2 andAG2 showed destabilization by STF and release of lipid and additionto TFLL. All three also provided protection to cells exposed todesiccating conditions whereas SB & ROA had minimal effect.Conclusions: Three novel dry eye formulations and two OTCproducts containing lipid and aqueous components showed markeddifferences in physicochemical properties and efficacy in an in vitromodel for desiccation-induced corneal cell death. Clinical impact ofthese differences should be evaluated.Commercial Relationships: Paramita Sarkar, Bausch and Lomb(E); Zora Marlowe, Bausch & Lomb (E); Amy Walsh, None; BrianGlass, Bausch + Lomb, Inc. (E); Tammy Kleiber, Bausch + Lomb(E); Megan E. Cavet, Bausch + Lomb (E); Karen L. Harrington,Bausch + Lomb (E); Stephen Davio, Bausch & Lomb (E)Program Number: 4319 Poster Board Number: C0057Presentation Time: 8:30 AM - 10:15 AMEarly Clinical Development of EBI-005, a Potent Interleukin-1(IL-1) Receptor-1 (R1) Blocker for Topical Ocular Treatment ofDry Eye Disease (DED)Michael H. Goldstein 1, 2 , Gregory Zarbis-Papastoitsis 2 , KathrynGolden 2 , Siddhartha Chowdhury 2 , Cameron Wheeler 2 , Gary N.Foulks 3 , Joseph T. Kovalchin 2 , Jennifer Agahigian 2 , Eric S. Furfine 2 .1 Ophthalmology, New England Eye Center, Boston, MA; 2 ElevenBiotherapeutics, Cambridge, MA; 3 Ophthalmology, University ofLouisville, Louisville, KY.Purpose: Herein we describe a stable formulation of EBI-005, anovel, potent IL-1R1 inhibitor that was optimized to treat ocularsurface disorders, such as DED. In this Phase 1 safety study, EBI-005was administered topically to the eyes of healthy volunteers. Multiplelines of evidence indicate that IL-1R1 blockade is therapeuticallyactive in reducing the signs and symptoms of DED, including a Phase2 clinical study using a prototype topical ocular IL-1R1 blocker. Insupport of this and other clinical studies, EBI-005 was well-toleratedin six-week mouse and rabbit GLP toxicology studies.Methods: In a double-masked, placebo-controlled study, healthyvolunteers were administered one drop of placebo, 5 or 20 mg/mLEBI-005 three times in one day. Each dose cohort contained eightsubjects, six were randomized to receive drug and two randomized toplacebo. Subjects receiving EBI-005 were administered drug in onlyone eye, the second eye received placebo. Safety assessmentsincluded: adverse event reporting, visual acuity (best correctedvision), slit lamp examination, intraocular pressure, dilated fundusexamination, ocular surface sensitivity (Cochet-Bonnetesthesiometry), corneal pachymetry, ocular surface microbiology,and serum laboratory testing. Subjects were confined to a phase oneunit for 4 days. A safety committee made up of the principalinvestigator, medical director and independent safety monitorreviewed the clinical data prior to escalating to the 20 mg/ml dose.Results: Administration of one drop of 5 or 20 mg/mL EBI-005 threetimes over one day was generally well-tolerated in healthy volunteersand adverse events were not substantially different than placebo.Visual acuity, slit lamp examination, ocular surface sensitivity andocular surface microbiology were not significantly changed afteradministration of EBI-005.Conclusions: Stable formulations of 5 and 20 mg/mL EBI-005 werewell-tolerated in healthy volunteers when administered three times ina day. These results supported the start of an ongoing six-week studydesigned to assess the safety and biological activity of EBI-005 inpatients with DED.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Michael H. Goldstein, ElevenBiotherapeutics (C), Eleven Biotherapeutics (R); Gregory Zarbis-Papastoitsis, Eleven Biotherapeutics (E); Kathryn Golden, ElevenBiotherapeutics (E), Eleven Biotherapeutics (P); SiddharthaChowdhury, BioBridges (C); Cameron Wheeler, ElevenBiotherapeutics (E); Gary N. Foulks, Eleven Biotherapeutics, Inc(C), Lexitas Pharma, Inc (C), Bausch and Lomb, Inc (C), Santen, Inc(C), Insite Pharmaceutical, Inc (C), Rtech Ueno, Inc (C), TearLab,Inc (C), Acucela, Inc (C), Merck, Inc (C), SarCode, Inc (C); JosephT. Kovalchin, Eleven Biotherapeutics (E), Eleven Biotherapeutics(P), Eleven Biotherapeutics (I); Jennifer Agahigian, ElevenBiotherapeutics (E); Eric S. Furfine, Eleven Biotherapeutics Inc (E)Clinical Trial: NCT01748578Program Number: 4320 Poster Board Number: C0058Presentation Time: 8:30 AM - 10:15 AMPreclinical Development of EBI-005: a Potent Interleukin-1 (IL-1) Receptor-1 (R1) Blocker for Topical Ocular Administrationwas Safe in GLP Toxicology Studies and Active in a MouseModel of Dry Eye Disease (DED)Eric S. Furfine 1 , Kathy Collins 1 , David Escobar 3 , Christian Li 2 ,Patricia A. Lowden 1 , Allyson Masci 1 , Jesse Milling 1 , KellyTenneson 2 , Gary Wolfe 4 , Joseph T. Kovalchin 1 . 1 R&D, ElevenBiotherapeutics, Cambridge, MA; 2 Charles River Laboratories,Senneville, QC, Canada; 3 Intertek, San Diego, CA; 4 Gary WolfeToxicology LLC, Herndon, VA.Purpose: Herein we describe the results of non-clinical INDenablingstudies of a stable formulation of EBI-005, a novel, potent,protein IL-1R1 inhibitor optimized to treat ocular surfaceinflammatory disorders. Multiple lines of evidence indicate thattopical IL-1R1 blockade is therapeutically active in reducing signsand symptoms of DED, including a Phase 2 clinical study using aprototype IL-1R1 blocker. EBI-005 is more effective thancyclosporine, the current standard of care, in a mouse model of DED.Methods: GLP studies in mice and rabbits assessed the topical ocularand systemic toxicology of EBI-005. Animals were treated with EBI-005 four times daily for six weeks (plus a two-week recovery phase)by topical ocular administration or once daily subcutaneously.Toxicokinetics and immunogenicity were assessed. Separately, EBI-005, twice daily, was compared to positive and negative controlmolecules in a mouse model of DED that induced signs of the diseaseusing a low humidity chamber.Results: EBI-005 was well-tolerated in mouse and rabbit GLPtoxicology studies where maximum tolerated doses were not reached.Ocular toxicity in rabbits was limited to minor irritation and rednessof the conjunctiva that was associated with minor ocularmononuclear cell infiltrations. Mice treated with EBI-005 had notreatment-related ocular findings. Drug-related effects fromsubcutaneous administration were limited minor findings at theinjection site. Systemic drug concentrations after ocularadministration were low or undetectable in mice and rabbits. Ocularconcentrations were not detectable by 12 hours after the lastadministration in rabbits. Despite the appearance of anti-drugantibodies, systemic active drug levels did not decrease in themajority of animals treated subcutaneously. In a mouse model ofDED, EBI-005 was more effective than the prototype IL-1R1blocker, anakinra, and a control for non-specific protein load, mouseserum albumin. EBI-005 was active at doses as low as 0.1 mg/mLand maximally active at 10 mg/mL.Conclusions: EBI-005 was well-tolerated in mouse and rabbittoxicology studies and highly effective in a mouse model of DED.These results support the start of an ongoing six-week study designedto assess the safety and biological activity of EBI-005 in patients withDED.Commercial Relationships: Eric S. Furfine, ElevenBiotherapeutics Inc (E); Kathy Collins, Eleven Biotherapeutics (E);David Escobar, None; Christian Li, None; Patricia A. Lowden,Eleven Biotherapeutics (E); Allyson Masci, Eleven Biotherapeutics(E); Jesse Milling, Eleven Biotherapuetics (E); Kelly Tenneson,Charles River (E), Eleven Biotherapeutics (E); Gary Wolfe, ElevenBiotherapeutics (C); Joseph T. Kovalchin, Eleven Biotherapeutics(E), Eleven Biotherapeutics (P), Eleven Biotherapeutics (I)Program Number: 4321 Poster Board Number: C0059Presentation Time: 8:30 AM - 10:15 AMSoft steroid topical treatment for moderate to severe dry eye:pulse vs tapered therapyEdoardo Villani, Cesare Pirondini, Francesco Viola, RobertoRatiglia. UO Oculistica, Univ of Milan Fnd IRCCS Ca' GrandaOMP, Milan, Italy.Purpose: To review the efficacy and side effects of 2 differenttopical corticosteroid schemes (pulse vs tapered) for treatment ofmoderate to severe dry eye over a period of 12 months.Methods: The medical records of all patients treated with loteprednoletabonate 0.5% for moderate to severe dry eye from 2007 to 2012 atthe Ocular Surface Research Center of Fondazione IRCCSPoliclinico of Milan were reviewed. Patients were treated with pulse(q.i.d. for 2 weeks) or tapered (t.i.d. for 1 week, b.i.d. for 2 weeks,q.d. for 4 weeks, every other day for 8 weeks) therapy. Topicalcorticosteroid treatment was repeated when needed.We included patients with at least 12 months of follow-up. Exclusioncriteria were lymphoma, AIDS, sarcoidosis, diabetes mellitus,dystrophies or infections of the ocular surface, systemic drugs withknown ocular surface toxicity, contact lens wear, previousophthalmic surgery, and topical treatments other than loteprednol andartificial tears.Main outcome measures, evaluated over the 12 months period,included: the mean difference compared to baseline of OSDI score,BUT, corneal fluorescein and conjunctival lissamine green stainingscores (CLEK scheme), the frequency of instillation of artificial tears,the total number of steroidal drops instilled, and the steroid-relatedside effects.Results: We included 90 patients, 28 treated with pulse scheme and62 treated with tapered scheme. Baseline data showed no significantdifferences between the 2 groups. OSDI score showed a significanthigher decrement in the tapered group compared to the pulse one(mean annual decrement compared to baseline: 27±8 vs 20±6;P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaComparison of Three Commercially Available Tear SubstitutesDesigned for Evaporative Dry Eye TreatmentCharles G. Connor, Raelyn A. Ottenbreit, Laura K. Schroeder, JeffC. Rabin, Srihari Narayanan. Rosenberg School of Optometry,University of the Incarnate Word, San Antonio, TX.Purpose: Topical rewetting agents still constitute the basis of dry eyetherapy. A high prevalence of evaporative dry eyes has generated theneed for a lipid component in rewetting agents. Our study comparesthe short term effect of 3 commercially available rewetting agentsdesigned for evaporative dry eye patients based on Non-invasive tearbreakup time (NITBUT) and contrast sensitivity.Methods: Twenty four subjects (14 female, 10 male, ave. age25.33+/-2.8 yrs) were divided into two groups based on the Effronscale for Meibomian gland dysfunction (MGD). A score of 1+ orgreater was used for inclusion in the MGD group (14 subjects). Basedon OSDI score the MGD subjects were in the mild symptomrange.The other 10 subjects served as control. Baseline measures ofVA, contrast sensitivity, OSDI and NITBUT were performed on allsubjects. Then, one drop of each of the 3 agents tested was instilled.The agents tested were Allergan Optive, Alcon Systane Balance andOcusoft Retaine. NITBUT was determined at 1.5,10,30,45 and 60minutes after instillation. Contrast was remeasured right afterinstillation. The 3 agents were tested on all subjects in 3 separatevisits.Results: A 2-way repeated measures ANOVA failed to revealsignificant differences in V.A or C.S between the two groups. Butthere was a slight improvement (25%) in contrast after dropinstillation for all agents tested.Mean NITBUT for the MGD group atbaseline with Allergan Optive was 8.4, 13.5 , 9.3 , 9.1 , 8.4 and 7.7sec (at the time points mentioned above in order) while the controlNITBUT were 9.1,10.5,9.2,9.1,8.5 and 7.7sec. The results for AlconSystane Balance were:7.7,10.7,10.6, 9.2, 8.7 and 8.4 for the MGDgroup while the control NITBUT were: 9.4,10.4,9.2,9.0,11.1 and 7.9.The results for Ocusoft Retaine were: 7.6, 9.9, 8.6, 9.5, 8.0 and 8.4for the MGD group while the control NITBUT were: 6.1,7.8, 6.2, 7.4,8.6 and 5.4 sec. Subjects preferred Optive (83%) in terms of dropcomfort, followed by Balance (62.5%) and Retaine (58%). Thelargest initial effect on the NITBUT was 5.1 seconds improvementwith Optive 90 seconds after instillation.Conclusions: Our data suggests that all 3 agents had effects thatpersisted for at least 45 minutes before a return to baseline NITBUT.Overall the NITBUT data suggests that any of these products wouldbe a reasonable choice for mild evaporative dry eye comfort in the 1hour range.Commercial Relationships: Charles G. Connor, None; Raelyn A.Ottenbreit, None; Laura K. Schroeder, None; Jeff C. Rabin,None; Srihari Narayanan, NoneProgram Number: 4323 Poster Board Number: C0061Presentation Time: 8:30 AM - 10:15 AMPlasmacytoid Dendritic Cells are the Primary Source of IFN-αDuring the Immunopathogenesis of Desiccating Stress-InducedDry Eye DiseaseMichael E. Stern 1 , Chris S. Schaumburg 1 , Jianping Gao 1 , Annie M.Ratanapinta 1 , Virginia L. Calder 2 , Larry A. Wheeler 1 , Jerry Y.Niederkorn 3 , Stephen C. Pflugfelder 4 , Bruce Beutler 5 , Argyrios N.Theofilopoulos 6 . 1 Biological Sciences, Allergan, Inc, Irvine, CA;2 Ophthalmology, University College London, London, UnitedKingdom; 3 Ophthalmology, University of Texas SouthwesternMedical School, Dallas, TX; 4 Ophthalmology, Baylor UniversityCollege of Medicine, Houston, TX; 5 Center for the Genetics of HostDefense, University of Texas Southwestern Medical School, Dallas,TX; 6 Immunology & Microbial Science, Scripps Research Institute,La Jolla, CA.Purpose: Plasmacytoid dendritic cells (pDC) and derivative type Iinterferons (IFN-α/β) are important mediators of antiviral immunityand have also been implicated in autoimmunity. The contribution ofTLR7/9 signaling in pDCs was evaluated during theimmunopathogenesis of experimental Dry Eye.Methods: Female C57BL/6 wild type mice, feeble (Slc15a4 mutants)and IFNAR1-/- mice were exposed to desiccating environmentalstress (DS; subcutaneous scopolamine injections; humidity

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaaveraged. Significant differences in expression levels (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatreatment of ocular surface inflammation associated with DED and/orother inflammatory conditions.Commercial Relationships: Yi Wei, None; Pengcheng Li, None;Jun Zou, None; Seth P. Epstein, Rapid Pathogen Screening, Inc (F);Neha Gadaria-Rathod, None; Penny A. Asbell, RPS (F)Program Number: 4327 Poster Board Number: C0065Presentation Time: 8:30 AM - 10:15 AMNew recombinant hyaluronic acid for eye care and ophthalmicdrug deliveryBirgit Lundskov Fuhlendorff, Khadija Schwach-Abdellaoui, FannyLongin, Corinne Eenschooten. Novozymes Biopharma DKA/S,Bagsvaerd, Denmark.Purpose: Whether to enhance hydration and lubrication of the eye,promote wound healing, or extend drug residence time, the benefitsof incorporating hyaluronic acid (HA) in ophthalmic formulations arewell documented. However, as the delicate structure of the eyerequires highly pure and safe components, the demand for HAs withvery low impurity levels is increasing. Novozymes Biopharmaaddresses this need with a new biosynthetic HA obtained by thefermentation of the non-pathogenic strain Bacillus subtilis. Unlikepreviously established production methods, their Bacillus-basedprocess does not employ any animal-derived raw materials or organicsolvents and yields an extremely pure HA product characterized byvery low protein and endotoxin levels. In this work, we demonstrateadvantages of using Bacillus-derived HA for eye care and ophthalmicdrug delivery.Methods: Corneal tolerance was evaluated by confocal laserscanning ophthalmoscopy. HA compatibility with diclofenac wasassessed by monitoring HA molecular weight using GPC combinedwith triple-angle light scattering and refractive index detection (RID)and by following diclofenac stability by HPLC (UV 280 nm). HAstability during autoclaving was studied by monitoring HA molecularweight by SEC-MALS combined with RID and dynamic viscositybefore and after autoclaving.Results: Results show that repeated applications of Bacillus-derivedHA-containing formulations onto the cornea of rabbit eyes do notinduce corneal lesions. Good compatibility profiles between this HAand diclofenac were observed in model ophthalmic formulations asevidenced by the excellent retention of both HA molecular weightand diclofenac integrity over 84 weeks. Furthermore, BacillusderivedHA demonstrates superior stability during autoclaving whencompared to a competitor HA product.Conclusions: The high purity and biocompatibility of Novozymes’HA combined with its compatibility with ophthalmic drugs such asdiclofenac and its superior stability during autoclaving makes thisnovel HA an ingredient of choice for formulations intended for eyecare and ophthalmic drug delivery.Commercial Relationships: Birgit Lundskov Fuhlendorff, None;Khadija Schwach-Abdellaoui, None; Fanny Longin, NovozymesA/S (E); Corinne Eenschooten, NoneProgram Number: 4328 Poster Board Number: C0066Presentation Time: 8:30 AM - 10:15 AMPreliminary Studies Of Liposomal Formulation Containing AnOmega-3 Fatty Acid For Dry Eye TherapyMarta Vicario de la Torre 1 , Omar Avelino Cruz 1 , Maria CaballoGonzalez 1 , Beatriz de las Heras 2 , Jose M. Benitez-del-Castillo 3 ,Rocio Herrero-Vanrell 1 , Irene T. Molina-Martínez 1 . 1 Pharmacy andPharmaceutical Technology, Faculty of Pharmacy, ComplutenseUniversity, Madrid, Spain; 2 Pharmacology, Faculty of Pharmacy,Complutense University, Madrid, Spain; 3 Unidad Superficie eInflamación Ocular, Instituto de Investigacaión Sanitaria, HospitalClínico San Carlos, Madrid, Spain.Purpose: Dry Eye is a highly frequent disease affecting the ocularsurface. Artificial tears are directed to minimize signs and symptoms.Nevertheless the underlying inflammation is not effectively treatedand simultaneous antiinflammatory therapy is usually required. It hasbeen shown that omega (ω)-3 fatty acids have anti-inflammatoryproperties and they have successfully employed in dry eye therapy.The aim of this work was to develop and characterize a formulationwith tear film characteristics based on ω-3 fatty acid containingliposomes for dry eye disease treatment.Methods: Liposomes were prepared by organic solvent evaporationtechnique which had been modified by our research group. Briefly, aω-3 fatty acid is incorporated with the lipid components(phosphatidylcholine, cholesterol and α-tocopherol) of liposomalvesicles in a ratio 10:0.6 (phosphatidylcholine: ω-3 fatty acid).Hypotonic aqueous solution containing trehalose is employed todisperse liposomes. Finally, a bioadhesive polymer, hyaluronic acid0.2%, was also added. Formulation was analyzed in terms of size, pHand osmolarity. Tolerance studies were performed in vitro on ahuman corneal cell line (Human Inmortalized-Limbal EpithelialCells; HCLE) based on the MTT method. Liposomal formulation wasexposed to corneal cells for 15 minutes (short term exposures) and 1and 4 hours (simulating chronic treatments).Results: ω-3 fatty acid-loaded liposomes resulted in a size of115.1±8.8 nm (unimodal size distribution). The liposomalformulation containing the ω-3 fatty acid showed a neutral pH. Theformulation was hypotonic and resulted in 207.4±1.3 mOsm.Viability values were higher than 80% in the human corneal cell lineat the three different time points studied.Conclusions: This liposomal formulation containing the ω-3 fattyacid was suitable for topical ophthalmic application. Further studiesare required to analyze the antiinflammatory effects of theformulation on the ocular surface for dry eye therapy.Commercial Relationships: Marta Vicario de la Torre, None;Omar Avelino Cruz, None; Maria Caballo Gonzalez, None;Beatriz de las Heras, None; Jose M. Benitez-del-Castillo, None;Rocio Herrero-Vanrell, None; Irene T. Molina-Martínez, NoneSupport: Research Group UCM 920415 (GR 35/10-A), FISPI10/00645 and FIS PI10/00993, RETICS RD 07/0062Program Number: 4329 Poster Board Number: C0067Presentation Time: 8:30 AM - 10:15 AMClinical Comparison of Lipid-based and Aqueous Lubricant EyedropsPeter A. Simmons, Cindy Carlisle, Genming Shi, Joseph G. Vehige.Clinical Research, Allergan, Inc, Irvine, CA.Purpose: Recent research has indicated that a majority of dry eyepatients have an evaporative component to the etiology of theircondition. Lipid-containing eye drops have been proposed as a meansto reduce the rate of evaporation of the tear film, to provide betterprotection of the ocular surface from drying and the resultinghyperosmotic stress. In the past, lipid-containing eye drops have beenassociated with patient discomfort, irritation, and blur. This clinicalstudy compared a new lipid-containing eye drop designed to reducethese issues, with aqueous eye drops.Methods: A total of 315 subjects with signs and symptoms of dryeye were enrolled in this multicenter, investigator-masked,randomized clinical trial. They received 1 of 4 eye drop formulations:A unit-dose investigational formula containing lipid, a marketed unitdoseaqueous drop (Refresh Optive Sensitive), a marketed multidoselipid-containing eye drop (Refresh Optive Advanced), or a marketedmultidose aqueous drop (Refresh Optive), in a ratio of 2:2:1:1,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornearespectively. Subjects were instructed to instill 1 to 2 drops of theassigned study product in each eye as needed, but at least 2 timesdaily, for 30 days. The endpoints investigated were changes frombaseline in Ocular Surface Disease Index (OSDI), tear breakup time(TBUT) and ocular surface staining at the Day 30 visit, as well asadverse events, comfort and acceptability.Results: All groups showed improvement from baseline in OSDI atDay 30 (p1 year after bonemarrow transplantation. These patients tend to have severe symptomscoincident with extensive corneal epitheliopathy. However, Schirmerscores may vary significantly, and do not always correlate with otherdisease parameters, and thus should not be the principal diagnostic orprognostic parameter in assessing these patients.Commercial Relationships: Hasanain Shikari, None; Ujwala S.Saboo, None; Reza Dana, Allergan (C), Alcon (C), Bausch & Lomb(C), Eleven Bio (I), GSK (F), Novabay (C), Revision Optics (C),Novaliq (C), RIgel (F)Support: NIH-EY19098Program Number: 4332 Poster Board Number: C0070Presentation Time: 8:30 AM - 10:15 AMRelationships among Tear Film Stability, Tear Osmolarity,Corneal Staining History, and Dryness SymptomsThao N. Yeh 1 , Nina Tran 1 , Andrew D. Graham 1 , Harry M. Green 2 ,Meng C. Lin 1, 2 . 1 Clinical Research Center, School of Optometry,University of California, Berkeley, Berkeley, CA; 2 School ofOptometry, University of California, Berkeley, Berkeley, CA.Purpose: To explore the relationships among tear osmolarity, tearfilm stability, corneal staining history, and dryness symptoms in soft©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacontact lens wearers and non-wearers.Methods: This observational study recruited 101 non-contact lens(NCL) and experienced soft contact lens (CL) wearers betweenJanuary and September 2010. The CL group included “stainers” and“non-stainers”. Stainers had a history of inferior corneal dehydrationstaining (DHS) at a minimum of 50% of their previous visits to theClinical Research Center (CRC) and non-stainers presented withDHS at a maximum of 20% of their previous visits to the CRC, overa minimum of three visits. A dry eye questionnaire was administeredand ocular parameter measurements including non-invasive tearbreakup time (NITBUT), invasive tear breakup time (ITBUT), tearosmolarity, and corneal staining score were collected at a singlemorning visit. Room temperature and humidity were measured at thecommencement of each visit. The TearLab Osmolarity System(TearLab Corporation, San Diego, CA, USA) was used to measuretear osmolarity. NITBUT was analyzed on the natural log scale tobetter approximate normality.Results: Ninety nine subjects (69 females, 30 males; 64 non-Asians,35 Asians; 59 NCL, 40 CL) with a median age of 22 years (range 18-67 years) completed the study. Multivariate linear mixed effectsmodeling showed that tear hyperosmolarity was significantlyassociated with shorter NITBUT (p=0.002) but not with cornealstaining history or dryness symptoms. Additionally, shorter NITBUTwas significantly associated with positive corneal staining history(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMedical College, Wenzhou, China; 3 Ophthalmology, Hangzhou FirstPeople’s Hospital, Hangzhou, China.Purpose: Dry eye syndrome is a chronic inflammation of the ocularsurface due to lack of sufficient lubrication and moisture. As a result,an individual suffering from the dry eye syndrome will experienceuncomfortable symptoms of dryness, itchiness, and a burningsensation in the eyes. The goal of this study was to develop asensitive biomarker to quantitatively evaluate the severity in dry eyesby imaging limbal capillary perfusion and calculating the velocity ofblood flow.Methods: A retinal function imager (RFI, Optical Imaging Ltd,Rehovot, Israel) was used to capture reflectance changes as afunction of time under stroboscopic illumination. The system wasfurther adapted from its original use of retinal imaging to beingutilized in anterior surface imaging. Hemoglobin in red blood cellswas used as an intrinsic motion-contrast agent in the generation ofdetailed noninvasive capillary-perfusion maps (nCPMs) and thecalculation of the velocity of blood flow. Nine healthy subjects (3males and 6 females, age 29.8 ± 10.4 years) and nine previouslydiagnosed dry eye patients (4 males and 5 females, age 51.0 ± 17.5years) were recruited. The temporal conjunctivas of the patient’s righteyes were imaged with the RFI device. The velocity of blood flow inarteries and veins was measured at selected vessel segments in thetemporal conjunctivas.Results: The nCPMs showed capillaries in exquisite detail on thelimbal region and the limbal region appeared as a random distributionof branching, overlapping, and crossing vessels (Fig. 1A). Thevelocity of blood flow (Fig. 1B) in conjunctival arteries was 0.71 ±0.09 (mean ± SD, mm/s) and 0.80 ± 0.11 mm/s, for normal and dryeye patients, respectively (p < 0.05). The blood flow velocities inconjunctival veins were 0.69 ± 0.08 mm/s and 0.81 ± 0.16 mm/s, fornormal and dry eye patients, respectively (p < 0.05). There were nosignificant differences in blood flow velocities between arteries andveins in each group (P > 0.05).Conclusions: This pilot study demonstrated for the first time thatblood flow velocity of the conjunctiva may be altered in dry eyepatients, which might be a possible biomarker for evaluating dry eyeseverity and treatment efficacy. This potential biomarker may alsoprovide a window into dry eye causes and pave the way for morespecific tracking methods of dry eye endpoints in humans for thepurpose of new drug approval.Center Grant P30 EY014801, Research to Prevent Blindness (RPB),Department of Defense (DOD- Grant#W81XWH-09-1-0675).Program Number: 4336 Poster Board Number: C0074Presentation Time: 8:30 AM - 10:15 AMReal World Analytical Performance of the TearLab OsmolaritySystem with an Enhanced Temperature SensorBenjamin D. Sullivan, Stephen G. Zmina, Matthew G. Zmina,Michael S. Berg. TearLab Corp, San Diego, CA.Purpose: Tear film instability is an inherent feature of dry eyedisease. The blink-to-blink variability that characterizes the disease,but disappears in normal subjects or treated patients, has causedmany to confuse expected eye-to-eye differences for analyticalvariation of the TearLab Osmolarity System. The purpose of thisstudy was to report real world performance of first generation andrecently updated TearLab Osmolarity System, in order to quantify therelative difference between analytical and biological variability.Methods: Prior to FDA CLIA waiver, 113 TearLab systems wereinstalled under the moderately complex program, which requiredeach doctor to be independently audited by state inspectors to ensurethat the systems matched manufacturer claims. Sites performed 20Normal and 20 High Control tests on one day, followed by tensequential days of tests at each level, with expected results of 297mOsm/L and 338 mOsm/L at each level. For the enhanced systems,240 High Control tests were performed on three units at temperaturesof 20°C, 23°C, 25°C and 30°C using three batches of test cards at onesite.Results: First generation controls averaged 295.4 ± 4.0 mOsm/L and338.6 ± 4.8 mOsm/L respectively for the 20/20 tests, with an averageCV from the sites of 1.4% and 1.6% at each level. Over ten days, thecontrols averaged 294.1 ± 4.8 mOsm/L and 336.6 ± 5.2 mOsm/Lrespectively, with an average CV of 1.6% and 1.6% at each level.Enhanced systems reported a mean osmolarity of 340.3 ± 3.5mOsm/L, with an average CV of 0.8% across each temperature onHigh Controls. Using these standard deviations, a Gaussian errormodel suggests that 1.6±0.4 tests are needed to produce ameasurement within 3 mOsm/L (< 1% error) of the true value,validating the requirement that both eyes should be tested as part of astandard TearLab osmolarity examination, and that more than twotests give little additional information.Conclusions: Preliminary results of the enhanced TearLab systemsuggest it meets or exceeds the precision of the first generationsystem. The analytical precision of the TearLab Osmolarity System isa minor component of the expected variability in dry eye disease, andeye-to-eye difference can be considered a clinical indicator of dry eyerather than the result of analytical imprecision.Commercial Relationships: Benjamin D. Sullivan, TearLab, Corp.(I), TearLab, Corp. (E), US7017394 (P); Stephen G. Zmina,TearLab, Corp. (I), TearLab, Corp. (E), US8020433 (P); Matthew G.Zmina, TearLab, Corp. (E), TearLab, Corp. (I); Michael S. Berg,TearLab Corporation (I), TearLab Corporation (E)Support: TearLab, Corp.Commercial Relationships: Jianhua Wang, NIH (F), RPB (F);Hong Jiang, NIH (F); Aizhu Tao, None; Delia DeBuc, NIH/NEI-EY020607 (F), NIH Center Core Grant P30EY014801 (F), NIHR01EY020607S (F), Department of Defense (DOD-Grant#W81XWH-09-1-0675) (F), US 61/139,082 (P); Yilei Shao,None; Jianguang Zhong, None; Sandra Pineda, NoneSupport: Supported by research grants in part from the NIHEY021012, EY021336, R01EY020607 and R01EY020607S, NIHProgram Number: 4337 Poster Board Number: C0075Presentation Time: 8:30 AM - 10:15 AMEvaluation of the Validity of the Diagnostic Criteria for Dry Eyein Japan Using Subjective Symptoms: Osaka StudyNorihiko Yokoi 1 , Miki Uchino 2 , Yuichi Uchino 2 , MURAT DOGRU 2 ,Motoko Kawashima 2 , Aoi Komuro 1 , Yukiko Sonomura 1 , HiroakiKato 1 , Kazuo Tsubota 2 , Shigeru Kinoshita 1 . 1 Ophthalmology, KyotoPrefectural University of Medicine, Kyoto, Japan; 2 Ophthalmology,Keio University School of Medicine, Tokyo, Japan.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To evaluate the validity of the diagnostic criteria for dryeye using subjective symptom scores.Methods: This study involved 672 Japanese young and middle-agedoffice workers who use visual display terminals (VDT). The subjectscompleted questionnaires sent to them by e-mail designed to detectdry eye diagnosis and risk factors. Dry eye tests, including theSchirmer test, tear film breakup time (BUT), and fluorescein andlissamine green staining were then performed. In the questionnaire,the subjects answered the following questions in regard to subjectivesymptoms (each given a maximum of 4 points depending onseverity): sensitivity of light, dryness, discomfort, redness, heavyeyelids, itchiness, blurred vision, tearing, foreign body sensation,discharge, pain, and eye fatigue. The Japanese dry eye diagnosticcriteria, revised in 2006, was followed in this study.Results: Of the 672 subjects, 561 (374 males, 187 females)completed the questionnaire (response rate: 83.5%). Sixty-fivesubjects (11.6%) were diagnosed as definite dry eye disease (DED),303 subjects (54.0%) were diagnosed as suspected DED, and 193subjects (34.4%) were diagnosed as non-DED. The mean subjectivesymptom scores of participants with suspected DED (2.05±0.42) andnon-DED (1.63±0.38) were significantly less than definite DED(2.19±0.40) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaopaque, few particles, low viscosity; 2: opaque or many particles,medium viscosity; 3: opaque, solid (toothpaste); 4: not expressible.Results: 166 patients were studied. 77 patients were classified asATD, EDE or Mixed. EDE only was 67.5% (52/77), ATD only24.6% (19/77) and Mixed was 7.8% (6/77). Within ATD, 76%(19/25) was pure ATD and 24% (6/25) was Mixed. For EDE, 89.6%(52/58) was pure EDE and 10.3% (6/58) was Mixed.Out of the total number of patients, 34.9% (58/166) were classified asEDE with Grade 1 or higher, while 65.1% (108/166) had a normalquality of expressed meibum. Within the EDE category, 67.2%(39/58) were Grade 1.0 to 1.9, 22.4% (13/58) were Grade 2.0 to 2.9,6.9% (4/58) were Grade 3.0 to 3.9 and 3.4% (2/58) were Grade 4.0.Conclusions: Mixed category of EDE and ATD was the smallestsub-type, while EDE formed the largest, followed by ATD. Withinthe EDE/MGD classification, about 2/3rds was classified as mild.Less than 5% was classified as non-expressible. According to theMGD workshop, the descriptors for meibum expressed wouldclassify the majority of this cross section as subclinical MGD tosymptomatic minimal MGD.Environment (CAE SM ) on 2 visits, separated by 1-week of BIDdosing with artificial tears. Eligible patients were assigned 1:1:1 tostudy medications (1% MIM-D3, 5% MIM-D3, placebo) and dosedBID for 28 days. Patients recorded morning and evening symptomsin daily diaries. Efficacy measures were FCS post-CAE at Day 28and patient diary data over the 28-day treatment period. A post-hocanalysis based on duration of DED was performed. Subgroups werecreated based on the length of time that the patient had reportedexperiencing dry eye symptoms: 1-5 years, 5-10 years, or >10 years.FCS post-CAE and diary data were evaluated in these subgroups.Results: Patients who reported having DED for 5-10 yearsexperienced a significant reduction in inferior and total FCS post-CAE, whereas no significant improvements were observed in thepatients who reported having DED for 1-5 years or >10 years. Thepost-CAE staining scores were significantly lower in the 1% MIM-D3 group in inferior (P=0.028) and total cornea (P=0.047) comparedto placebo. Patients who reported having DED for 5-10 years alsoexperienced significant improvements for ocular dryness (P=0.029)and grittiness (P=0.035) symptoms in the 1% MIM-D3 groupcompared to placebo.Conclusions: These post-hoc data support an association between theduration of DED and the response to MIM-D3 for the reduction inboth signs and symptoms. Adjusting for disease duration, patientswith reported DED for 5-10 years had a significant treatment effectwith 1% MIM-D3 in reducing both FCS and diary symptoms ofdryness and grittiness.Commercial Relationships: Karen Meerovitch, MimetogenPharmaceuticals, Inc. (E), Mimetogen Pharmaceuticals, Inc. (P);Teresa Lama, Mimetogen Pharmaceuticals (E), MimetogenPharmaceuticals (P); Debra A. Schaumberg, Pfizer, Inc. (F), Pfizer,Inc. (C), Mimetogen (C), SARcode Bioscience (C), ElevenBiotherapeutics (C)Clinical Trial: NCT01257607Prevalence of ATD, MGD and Mixed categories of dry eyeCommercial Relationships: Milton M. Hom, Allergan (F), Bauschand Lomb (F), AMO (F), SARcode (C), TearScience (C); Justin T.Kwan, NoneProgram Number: 4340 Poster Board Number: C0078Presentation Time: 8:30 AM - 10:15 AMRelating Improvements in Signs and Symptoms to Duration ofDry Eye Disease (DED) after Treatment with MIM-D3Ophthalmic SolutionsKaren Meerovitch 1, 3 , Teresa Lama 1 , Debra A. Schaumberg 2, 4 .1 Mimetogen Pharmaceuticals, Montreal, QC, Canada; 2 Division ofPreventive Medicine, Brigham & Women's Hospital, HarvardMedical School, Boston, MA; 3 Department of Pharmacology andTherapeutics, McGill University, Montreal, QC, Canada;4 Department of Ophthalmology and Visual Sciences, University ofUtah School of Medicine, Salt Lake City, UT.Purpose: To explore the clinical outcomes of signs and symptomsafter 28 days of treatment with 1% and 5% MIM-D3 ophthalmicsolutions as a function of patient reported duration of DED.Methods: A 2-center, randomized, double-masked, placebocontrolledPhase 2 study was conducted in 150 dry eye patients. Keyeligibility criteria included OraCalibra scores of ≥ 4, ≥ 2 and ≥ 2for fluorescein corneal staining (FCS), lissamine green staining, andone symptom from the 4-Symptom Questionnaire, respectively.Additionally, patients had to demonstrate an exacerbation of FCS andocular discomfort after exposure to a Controlled AdverseProgram Number: 4341 Poster Board Number: C0079Presentation Time: 8:30 AM - 10:15 AMAutomated Detection and Enumeration of Corneal SuperficialPunctate KeratitisJohn D. Rodriguez 1 , Patrick Johnston 1 , Keith J. Lane 1 , George W.Ousler 2 . 1 R & D, Ora, Inc., Andover, MA; 2 Ora, Inc., Andover, MA.Purpose: The evaluation of corneal superficial punctate keratitis(SPK) is an important primary endpoint for dry eye clinical trials.However, staining grading remains a challenging task, as gradersmust manually divide corneal regions by eye. Moreover, the requiredenumeration of SPK is necessarily constrained due to practical limitsand can be visually difficult to resolve with sufficient sensitivity. Theuse of software to automate and standardize this process allows foractual enumeration of SPK to be made quickly and accurately. Addedsensitivity when compared to traditional clinician based scales maybe realized by implementing a continuous staining score based on thedistribution of the SPK data. To test the effectiveness of this method,we investigate SPK in a dry eye population.Methods: A sample population of 665 dry eye subjects wasconsidered. Inferior and central corneal fluorescein images wereobtained for each eye using high resolution digital photography. Theimages were processed using a custom software program developedusing open source computer vision libraries. The softwareautomatically selects the relevant corneal area of interest andenumerates SPK lesions and their cumulative surface area on thecornea. Finally, the original images were visually verified withimages output by the program with highlights of identified SPK.Results: The mean number of SPK detected in the inferior cornealregion was 33.73 and 17.63 in the central region. SPK areas (as a©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapercent of surface area) for the inferior and central corneal regionswere 0.236% (SD:50.04) and 0.182% (SD:37.34), respectively.Visual confirmation showed close qualitative agreement withsoftware results. Processing of all 2660 images requiredapproximately 60 minutes. Computed log SPK number of a randomlyselected subset of 54 images showed a 92% correlation with a clinicalgrader assessment using the Ora staining scale (0-4).Conclusions: The results show that extensive and accurateinformation may be efficiently obtained with the software basedapproach. We note that mean SPK numbers and the stained area arehigher in the inferior then the central corneal region, although therespective geometric areas are the same. The automated selection ofthe relevant region of interest and enumeration of up to severalhundred SPK quickly and efficiently allows for precise and accuratequantification of corneal desiccation.Commercial Relationships: John D. Rodriguez, Ora, Inc. (E);Patrick Johnston, Ora, Inc (E); Keith J. Lane, Ora, Inc. (E);George W. Ousler, Ora, Inc. (E)Program Number: 4342 Poster Board Number: C0080Presentation Time: 8:30 AM - 10:15 AMOcular Surface Disease prevalence in Glaucoma patients in aHigh Referral Ophthalmology Center in Mexico CityNallely Ramos-Betancourt 1 , Jaime D. Martinez 1 , Francisco Beltran 1 ,Jorge Ozorno-Zarate 1 , Cristina G. Isida Llerandi 2 , Jesus Jimenez-Roman 2 , Felix Gil Carrasco 2 , Manuel Ramirez 1 , EverardoHernandez-Quintela 1 . 1 Cornea, Asociacion Para Evitar la Ceguera,Mexico City, Mexico; 2 Glaucoma, Asociación Para Evitar la Cegueraen México, Mexico City, Mexico.Purpose: To determine the ocular surface disease (OSD) prevalencein glaucoma patients and risk factors.Methods: A cross-sectional study was designed. Consecutiverecruitment of patients was made in the Glaucoma department fromoctober to november 2012. Patients older than 18 years old, usingantiglaucomatous topical medication were included. They underwentophthalmological examination which comprised: Ocular SurfaceDisease Index (OSDI) questionnaire, tear film break up time(TFBUT), ocular surface staining with fluorescein, Schirmer I testwith anesthesia, and the presence of Meibomian gland dysfunction.Additionaly, the following risk factors were evaluated: age, sex,number and type of antiglaucomatous topical medications, time ofusage, ocular surgery history.Results: 123 patients were recruited, 87 patients (71.55%) werefemales. The mean age was 67.85 +/- 13.26 years (range, 21-90). TheOSD prevalence was 51.21% by OSDI (15.44% mild, 12.1%moderate and 23.5% severe). 104 patients (84.55%) had abnormalTFBUT (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapresent and functional in the lacrimal gland, if they exhibit sexspecificdifferences, and if a DHA-rich diet can confer protectiveactions against DE developmentMethods: DE was induced using a model of desiccating stress andscopolamine. Mice were placed on a DHA-rich or DHA-deficient dietfor 4 weeks before DE induction. Schirmer’s test was used to assesstear levels. PMN levels were quantified using myeloperoxidase assay,lipid mediator formation by LC/MS/MS-based lipidomics, and geneexpression via QPCR. Leukocyte populations were assessed by IHCResults: Lacrimal glands highly express inflammatory lipid circuits(COX, 15-LOX), which are regulated by desiccating stress. Femalemice exhibited an amplified inflammatory response compared tomales, evidenced by a marked early infiltration of PMNs, increasedpro-inflammatory PGE2, and decreased pro-resolving LXA4. Micethat received the DHA-rich diet had higher tear production (0.9mm)compared to their DHA-poor diet counterparts (0.3mm), suggestinggreater protection against desiccating stress. DHA-rich micecontained greater levels of DHA and EPA, and lower levels of ω-6PUFA AA. Levels of PGE2 were downregulated 2.4-fold and proresolvinglipid mediators were upregulated in mice on the enrichedDHA dietConclusions: These results provide the first evidence that lipidautacoid circuits are expressed and functional in vivo in lacrimalglands. There is a sex-specific difference in the inflammatoryresponse to desiccating stress. Dietary DHA in females shifts levelsof lipid mediators to resemble males and may confer protectionagainst disease progression by downregulating the immuneregulatory COX/PGE2 pathway, a primary target of corticosteroidsand NSAIDsCommercial Relationships: Kyle M. Hu, None; Samantha B.Wang, None; Erick Lu, None; Patrick J. Salveson, None; YuanGao, None; Karsten Gronert, NoneSupport: EY022208 and Sjogren's Syndrome FoundationProgram Number: 4345 Poster Board Number: C0083Presentation Time: 8:30 AM - 10:15 AMAnalysis of Electrolyte Composition of Precorneal Tear Film inNormal Dogs and Horses and its Comparative Values in Serumand PlasmaGil Ben-Shlomo, LaTisha N. Taylor. Veterinary Clinical Sciences,College of Veterinary Medicine, Iowa State University, Ames, IA.Purpose: Serum and plasma are being used for the treatment ofseveral eye diseases including keratomalacia and Keratoconjunctivitissicca. The goals of this study was to analyze the electrolyte contentfound in equine and canine tears and compare it to the electrolytesfound in serum and plasma obtained by utilizing two differentanticoagulants.Methods: Nine horses (17 eyes) and 9 dogs (18 eyes) were used inthis study. An eye examination including slit lamp biomicroscopy,fundoscopy, and Schirmer tear test (STT) was performed. All eyeswere free of ocular disease. All horses were sedated with 0.5 mg/kgof xylazine (Lloyd Laboratories, Shenandoah, IA) intravenously. Nochemical restraint was needed for the dogs. Tears and blood werecollected from all animals. Blood was collected for serum in tubeswith no anticoagulants; plasma was made by using two differentanticoagulants: Citrate Phosphate Dextrose (CPD) and heparin.Results: Most of the electrolyte values in tears were statisticallydifferent than electrolyte values in serum and plasma in both dogsand horses. In both species, potassium and chloride levels weresignificantly (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 4347 Poster Board Number: C0085Presentation Time: 8:30 AM - 10:15 AMImpact of dry eye on reading in a population based sample of theelderly: the Salisbury Eye EvaluationSuzanne W. van Landingham, Sheila K. West, Esen K. Akpek, BeatrizE. Munoz, Pradeep Y. Ramulu. Wilmer Eye Institute, Johns HopkinsUniversity, Baltimore, MD.Purpose: To determine if dry eye is associated with perceived orobjective reading deficits.Methods: A cross-sectional study. Dry eye symptoms were identifiedbased on a positive response to any of six symptom questions, whileclinically significant dry eye was identified based on the presence ofdry eye symptoms and a positive Schirmer or Rose Bengal test ineither eye. Spoken reading speed was measured using short textpassages. Subjects self-reported if they did not read, or had difficultyreading, newsprint.Results: Multivariable models showed no difference in reading speedbetween subjects with and without dry eye symptoms (β=-0.55 wpm,95% confidence interval [CI]=-6.8-5.1, p=0.85), or between subjectswith clinically significant dry eye and subjects without dry eye signsor symptoms (β=-5 wpm, 95% CI=-16 to 5, p=0.35). Multivariablemodeling also showed that, compared to asymptomatic subjects,subjects with dry eye symptoms were more likely to report readingdifficulty (OR=1.7, 95% CI 1.3-2.4, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneahad significantly higher triglyceride levels and VLDL levels (bothp

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaage of 53.11±16.01; while the normal control group composed 21person(42 eyes), including 7 male(14 eyes),14 female(28 eyes),withthe average age of 47.29±17.27. We used Oculus Keratograph whichcould permit automatic determination of the tear film break-up timeand point, providing a graphic representation referred to as a Tear-Map, which showed the location and size of the tear film break-uppoint according to the time(Figure 1 right). The different coloursdepended on different tear film break-up time(Figure 1 left).Not onlythe location of tear film break-up point,dry eye objective symptomscore, tear film break-up time, but corneal fluorescein staining,Schirmer I test were performed on the dry eye patients and normalperson. And subsequently, assess the difference between two groups.Results: According to the quadrant sequence of supernasal,inferonasal ,infratemporal ,supertemporal, the respective occurrenceproportion of first tear film break-up point, emerging in differentquadrants, of dry eye group was: 8 eyes taking the proportion of15.4%,19 eyes of 36.5%,10 eyes of 19.2%,15 eyes of 28.8% and ofnormal group was: 8 eyes taking the proportion of 19%,15 eyes of35.7%,14 eyes of 33.3% and 5 eyes of 11.9% respectively(Table 1).It was the inferonasal quadrant that took up the maximum ration offirst tear film break-up point and the fluorescein was more likelystained in infero-pupil area in corneal fluorescein staining test of thetwo groups(Table 2), yet no significant difference were foundbetween two groups in neither the location of first tear film break-uppoint nor the corneal fluorescein staining. (chi-squaretest,X 2 =5.13,P>0.05;X 2 =0.43,P>0.05)Conclusions: Oculus Keratograph could be feasibly used to observethe tear film break-up time and point, and it provided that the firsttear film break-up point most frequently occurred in inferonasalquadrant.Program Number: 4353 Poster Board Number: C0091Presentation Time: 8:30 AM - 10:15 AMAllgrove Syndrome: complex eye involvement and firstevaluation by laser scanning confocal microscopyAngelica Dipinto 1 , Paolo Fogagnolo 2 , Davide Allegrini 1 , MaurizioDigiuni 1 , Luca Migliavacca 1 , Chiara Olga Pierrottet 1 , LauraOttobelli 1 , Olga Oneta 1 , Luca M. Rossetti 1 . 1 Eye Clinic, San PaoloHospital, Milan, Italy; 2 G.B. Bietti Foundation IRCCS, Rome, Italy.Purpose: Allgrove Syndrome (AS or triple-A Syndrome) is a rareautosomal recessive disorder, caused by mutations in the AAAS geneon band 12q13. It is characterized by: alacrima, achalasia, adrenalinsufficiency and, occasionally, autonomic dysfunction. We reportthe ophthalmic examination of two affected siblings: a 16 years oldfemale (HH) and a 20 years old male (HI).Methods: HH and HI underwent: complete ophthalmic examination,visual evoked responses (VER), electroretinogram (ERG), opticalcoherence tomography (OCT) of the optic nerve head and retina,Heidelberg retina tomograph II (HRT II) of the optic disc. Weexamined HI’s cornea and Meibomian Glands (MGs) by laserscanning confocal microscopy (LSCM) HRT II Corneal RostockModule. We also performed corneal LSCM on 8 age-matched controlsubjects.Results: HH’s slit-lamp examination revealed a superficialkeratopathy in both eyes (OU). Schirmer I test was 0 mm in OU.VER showed a mild bilateral sofference. Optic disc HRT was normal.OCT showed a decrease of retinal nerve fiber layer thickness (RNFL)temporally in OU. HI had corneal inferior conjunctivalization andsuperficial punctate keratopathy. Schirmer test was 0 mm in the righteye (OD) and 2 mm in the left eye (OS). VER revealed a mild leftsofference. Inferior and temporal RNFL thinning was shown byOCT, OS worse than OD, whereas HRT was borderline in OU. BothHH and HI showed a red-green color deficiency, normal OCT maculaand ERG. HI was also investigated by LSCM. Nerve fibers’ numberper frame was reduced in HI (3.5±0.7) compared with controls(6.9±0.6). HI showed an important infiltration of Langerhans cellsbeneath the epithelium, absent in controls. LSCM of HI’s MGsshowed a density of 101.56±5.98 glands per square millimeter and adiameter of 62.14±20.59 micrometers. MGs were characterized by:light gray color secretions, high presence of punctate reflectingelements in the interstice and inhomogeneous appearance of acinarwalls.Conclusions: To the best of our knowledge, this is the most accuratedescription of ocular involvement in AS, a rare disease associatedwith complex, and only partially understood, ophthalmic findings.Commercial Relationships: Dan Wu, None; Jiaxu Hong, None;JianJiang Xu, NoneNerve fibers in a patient affected by AS©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMGs in a patient affected by ASCommercial Relationships: Angelica Dipinto, None; PaoloFogagnolo, None; Davide Allegrini, None; Maurizio Digiuni,None; Luca Migliavacca, None; Chiara Olga Pierrottet, None;Laura Ottobelli, None; Olga Oneta, None; Luca M. Rossetti, NoneProgram Number: 4354 Poster Board Number: C0092Presentation Time: 8:30 AM - 10:15 AMHow Does Increasing Ocular Surface Stimulation AffectBlinking?Ziwei Wu 1 , Carolyn G. Begley 1 , Ping Situ 1 , Adam J. Winkeler 1 , JunZhang 1 , Trefford L. Simpson 2 . 1 Optometry, Indiana University,Bloomington, IN; 2 Optometry and Vision Science, University ofWaterloo, Waterloo, ON, Canada.Purpose: Despite the importance of the blink, its control by ocularsurface sensory input remains controversial. In this study, we testedthe hypothesis that increasing ocular surface stimulation will lead to asignificantly increased blink rate (SIBR) that we defined as a blinkrate (BR) ≥ 2 standard errors from baseline.Methods: To control attention, 10 subjects played a video game(with the screen viewed from 20 cm) while also seated behind a slitlamp biomicroscope. Air flow (AF) was directed toward the centralcornea (15mm distance, temperature= 24 Celsius) through a 0.5mmdiameter tube. Using an ascending method of limits, the AFproducing an approximate SIBR was estimated and then, using amethod of constant stimuli 0, 0.25, 0.5, 0.75, 1 and 1.25 multiples ofthis estimated AF were presented 3 times each (in random order).BR, interblink interval (IBI) and AF were recorded simultaneouslyand custom MATLAB programs determined the SIBR for eachsubject.Results: Mean (±SD) airflow to produce a SIBR was134.9±63.9ml/min (range: 65.5 to 248.8ml/min). There was a linearcorrelation between AF and BR or IBI (Pearson’s correlationcoefficient, r= 0.939 and -0.987, respectively). The baseline (AF= 0)BR was 16.5±6.0 blinks/min and the five tested AFs these were20.3±5.9, 25.8±5.0, 30.0±8.3, 47.0±20.3 and 67.4±27.3 blinks/min.The baseline IBI was 4.5±2.1sec and at the five AFs these were3.5±1.3, 2.6±0.5, 2.4±0.9, 1.6±0.5 and 1.1±0.4sec. Figure 1demonstrates the SIBR for one subject.Conclusions: These results support the hypothesis that AFstimulation of the ocular surface leads to a SIBR when mentalconcentration is controlled, and that the increasing stimulationproduces a linear dose response at the levels of AF tested. Thismethod may allow examination of individual differences in the ocularsurface sensory response to stimulation, which may cause thevariation in SIBR among subjects.Figure 1: SIBR for one subject. The continuous line representschanges in AF from 0 to 174ml/min and each vertical line in thebottom trace represents a blink over the 4 min trial.Commercial Relationships: Ziwei Wu, None; Carolyn G. Begley,Santen, Inc. (C), ohnson & Johnson Vision Care, Inc. (C), ohnson &Johnson Vision Care, Inc. (F); Ping Situ, None; Adam J. Winkeler,None; Jun Zhang, None; Trefford L. Simpson, NoneSupport: National Eye Institute R01EY021794Program Number: 4355 Poster Board Number: C0093Presentation Time: 8:30 AM - 10:15 AMDo Tear Film Thinning Rates Vary Locally?Adam J. Winkeler 1 , Carolyn G. Begley 1 , Richard J. Braun 2 , RobertWelch 1 . 1 School of Optometry, Indiana University, Bloomington, IN;2 Mathematics, University of Delaware, Newark, DE.Purpose: The purpose of this study was to determine whether tearthinning rates differ within and outside areas of tear film break-up(TBU) using fluorescein (FL) imaging corroborated by a secondimaging method, retroillumination (RI).Methods: Mydriacyl (1.0%) and proparicaine (0.5%) were instilledinto the eyes of 6 subjects, followed by 2μl of 2% FL. Subjects keptone eye open as long as possible while a modified slit-lampbiomicroscope simultaneously imaged the tear film by FL and RI.Two areas of the tear film, superior and inferior to the pupil, weresystematically selected for analysis. TFT rates inside and outside ofareas of TBU were calculated over time at each location using theslope of a linear least squares fit of the square root of FL intensity toaccount for FL quenching (Nichols et al, 20120). Surface profilesobtained from FL intensity were correlated with integrated RIintensity for corroboration of changes in slope. TFT estimated inµm/min by assuming a 3µm starting tear film thickness.Results: FL intensity decreased in all subjects over time, reaching aminimum within areas of TBU, suggesting full thickness breaks. TheTFT rates for 2 subjects with very stable tears and minor to no TBUwere 0.0063±0.0018 units/sec (Mean ± SD). The other 4 subjectsdeveloped variable and sometimes extensive TBU with an overallaverage TFT rate of 0.0775±0.0055 units/sec, almost 10x faster thansubjects with stable tears. A subject with immediate TBU after theblink had an overall average TFT rate of 0.1477±0.0424 units/sec.The average TFT inside areas of TBU was 0.1038±0.0444 units/seccompared to 0.0576±0.0565 units/sec just outside and adjacent toTBU. This corresponded to estimated thinning rates of 2.913±1.327µm/min inside TBU areas and 1.150±1.481 µm/min in adjacent areas.The local tear film surface profiles calculated from FL intensity inareas of TBU showed increasing correlation with changes inintegrated RI image intensity as TBU developed.Conclusions: TFT varied markedly within and outside of areas ofTBU, whereas TFT showed slow even declines in a stable tear film.Our results show that spatially local differences in TFT within areasof TBU persist long after visible upward movement of the tear filmceased. Our results suggest that, if evaporation is the mainmechanism accounting for TFT, it may vary locally across the tearfilm during TBU.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Adam J. Winkeler, None; Carolyn G.Begley, Santen, Inc. (C), ohnson & Johnson Vision Care, Inc. (C),ohnson & Johnson Vision Care, Inc. (F); Richard J. Braun, None;Robert Welch, NoneSupport: NIH Grant 1R01EY021794-01Program Number: 4356 Poster Board Number: C0094Presentation Time: 8:30 AM - 10:15 AMLongitudinal Mixed Effect OSDI Model in a Select Dry EyePatient Population after Acupuncture TreatmentVanessa Bowlin 1 , Sandeep S. Samudre 1 , Deepinder K. Dhaliwal 2 .1 Clinical Research, Eastern Virginia Eye Institute, Chesapeake, VA;2 School of Medicine, University of Pittsburgh, Pittsburgh, VA.Purpose: Keratoconjunctivitis sicca (KCS) is a commonmultifactorial disorder that negatively impacts a patient’s quality oflife, quality of vision, and ocular surface. Prior studies havedocumented beneficial effects of acupuncture for treatment of KCS.Modeling this behavior could facilitate better understanding ofacupuncture mechanism of action and KCS. This retrospective studyevaluates the ocular surface disease index (OSDI) in dry eye patientswho received acupuncture treatment.Methods: In this retrospective study, OSDI data from 49 dry eyepatients randomly assigned to either true or sham acupuncturetreatment group was re-evaluated. Participants had received twotreatments and were followed at regular intervals for period of 6months. Linear mixed effect model included: Treatment (tx), actualtime (atime), square of actual time (atime^2), centered age (agec),sex, and the following interaction terms: tx*atime, agec*atime,agec*atime^2, sex*atime, sex*atime^2. Potential confounders suchas age and sex were also included in the model.Results: After 6 months, OSDI was significantly reduced from abaseline of 34 ± 3 to 16 ± 4 (p= 0.01). The final linear mixed modelequation for OSDI = 29.39 - 7.39*tx + 0.530*agec + 35.50*sex + (-0.292 + 0.0858*tx + 0.133*sex + 0.00849*agec)*atime + (0.00103 -0.00118*sex - 0.00005*agec)*atime^2 + random slope of atime +random error. This equation also reported an immediate and rapidimprovement in OSDI scores in patients receiving true acupuncturetreatment. No significant differences in OSDI were observed aftersham acupuncture treatment (p=0.09). Also, there were no adverseeffects reported.Conclusions: The study demonstrated that acupuncture improvessymptoms in dry eye sufferers evaluated by OSDI. It is possible tomodel dry eye behavior using a linear mixed effect model. Modelingthis behavior facilitates understanding of KCS and acupuncture,which can be a useful complement to KCS treatment modalities.Commercial Relationships: Vanessa Bowlin, None; Sandeep S.Samudre, TechniSight Inc. (I); Deepinder K. Dhaliwal, NoneProgram Number: 4357 Poster Board Number: C0095Presentation Time: 8:30 AM - 10:15 AMThe relationship of depression and anxiety with dry eye subtypesLouis Tong 1, 3 , Li-Yue Hong 4 , Ryan Lee 4 , Yang Zhao 4 , Sharon C.Sung 2 , Peggy P. Chiang 4 . 1 Cornea and External Eye Disease Service,Singapore National Eye Ctr, Singapore, Singapore; 2 Office ofClinical Science, Duke-NUS graduate medical school, Singapore,Singapore; 3 Ophthalmology, Yong Loo Lin School of Medicine,National University of Singapore, Singapore, Singapore; 4 SingaporeEye Research Institute, Singapore, Singapore.Purpose: Dry eye syndrome is a common, major health conditionwith association with systemic conditions such as depression andanxiety. We aim to use two previously validated questionnaires toevaluate depression and anxiety in ambulatory eye care setting, anddetermine if specific dry eye subtypes are associated with theseconditions.Methods: This is a prospective cross sectional study performed onEnglish literate patients at the Singapore National Eye Center.Participants were tasked to complete 2 interviewer-assistedquestionnaires: HADS, which screens for anxiety and depression, andCESD, which screens for depression. All participants underwent adry eye symptom questionnaire, tear break up time assessment,corneal fluorescein dye staining, Schirmer’s test, as well as asocioeconomic questionnaire assessment. Patients were classifiedinto evaporative, aqueous deficient or mixed types or symptomaticcategories based on their response to questions and the above testresults.Results: Ninety-six people, mean age 54.5 year (SD: 10.8), of which35.9% men, were recruited. The mean score for CESD was 12.1 (9.8)with 28 subjects (31.5%) having a score of above 16 (previouslypublished threshold). In the HADS the mean score for depressionassociatedquestions was 3.6 (3.6) with 13 subjects (14.1%) having ascore >=8 whereas mean score for the anxiety-associated questionswas 5.3 (3.6) with 24 subjects (26.1%) having a score >=8. AbnormalCESD was significantly associated with symptomatic aqueousdeficient and evaporative dry eyes, mixed dry eyes, and symptomaticmixed dry eyes (odds ratios range from 3.4 - 4.0). The anxiety scoresfrom HADS were significantly associated with symptomaticevaporative and symptomatic mixed dry eyes (odds ratios range (3.04- 3.2), but the HADS depression scores were not associated with anydry eye subtypes.Conclusions: The CESD and HADS were able to detect suspectswith depression and anxiety respectively. Specific types of dry eyepatients, particularly symptomatic ones, showed different tendenciesfor depression and anxiety. The results of these studies haveimplications for the management of chronic dry eye patients as wellas healthcare planning.Commercial Relationships: Louis Tong, None; Li-Yue Hong,None; Ryan Lee, None; Yang Zhao, None; Sharon C. Sung, None;Peggy P. Chiang, NoneSupport: This research is supported by the Singapore NationalResearch Foundation under its clinician scientist awardNMRC/CSA/013/2009 and administered by the Singapore Ministryof Health’s National Medical Research Council. This research issupported by the Singapore Ministry of Health’s National MedicalResearch Council under its individual research grantNMRC/1206/2009 and center grant NMRC/CG/SERI/2010.Program Number: 4358 Poster Board Number: C0096Presentation Time: 8:30 AM - 10:15 AMThe effect of tear lipid biochemistry on tear evaporation rateduring contact lens wearAthira Rohit 1, 2 , Simon H. Brown 3, 1 , Mark D. Willcox 2, 1 , FionaStapleton 1, 2 . 1 Optometry, Brien Holden Vision Institute, Sydney,NSW, Australia; 2 Optometry, School of Optometry and VisionScience, Sydney, NSW, Australia; 3 School of Health Sciences,University of Wollongong, Wollongong, NSW, Australia.Purpose: To understand the association of lipid biochemistry on lipidlayer function during short term contact lens wear.Methods: Fifteen participants with healthy eyes and no prior contactlens wear were recruited. The sample size was calculated based on apilot study of evaporation rates with a standard deviation of 5.6 g/m 2 hto detect a difference between groups at 0.5% significance level with80% power. The study was conducted in two randomized stageswithout and with contact lens wear (Ciba Vision, Focus Dailies, -0.50D).Tear evaporation rate from both eyes was measured using theVapoMeter (Delfin Technologies, Finland) and basal tears werecollected. Tear evaporation rate and tear collection were performed at©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneathe end of 6 to 8 hours of contact lens wear or similar time with nolens wear. Differences in tear evaporation rate between lens wear andno lens wear were compared using a paired t-test and associationsbetween evaporation rate and concentration of phospholipids in tearfilm lipid layer were assessed using a Pearson correlation coefficient.Results: The mean pre-corneal evaporation rate was 94.3±43.3 g/m 2 hand this was significantly lower (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneabiomarkers.Methods: 62 patients, defined by Schirmer I test as normal or ADdry eye, participated in the study. NS tear samples were collected bymicropipette and stored in PBS-antiprotease buffer at -80°C prior toBioRad 27-Plex polystyrene bead-based cytokine assay on a Luminex200 system. At the same patient visit, 8 conjunctival impressioncytology (CIC) specimens were collected from each eye, RNAextracted by Qiagen RNEasy Plus Minikit, and real-time qPCRconducted on TaqMan 384-well low density array cards in 96 geneformat.Results: NS Tear G-CSF, while not the strongest discriminatorbetween normal and AD groups, showed the largest number ofsignificant positive correlations with conjunctival expression ofbiomarkers, including pro-inflammatory cytokines, cytokinereceptors, G-protein coupled receptors, proteases, transcriptionfactors and calcium-binding proteins. Tear G-CSF also correlatedstrongly with conjunctival G-CSF receptor expression, other CSFreceptors and most strongly with IL-1β (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornearesults of the test.Results: During the forward translation step analysis, the OSD-IICdecided to change 2 adjetives for better understanding, this adjectivewas included in items 1a, 2a, and 3a. The backward translation wasalmost identical to the original questionnaire in English. In thecognitive interviews, 28 respondents did not have any difficulty whileresponding the 5-items of DEQ-5 and the consultants did notsuggested amendments of words or phrases.Conclusions: This study was able to provide an appropriatetranslation of the DEQ-5 questionnaire.Commercial Relationships: Everardo Hernandez-Quintela, None;Nallely Ramos-Betancourt, None; Jaime D. Martinez, None;Concepcion Santacruz Valdes, None; Francisco Beltran, None;Cecilia Ramírez-Assad, None; Elsa M. Mora Juarez, None;Alejandro Babayan, None433 Stroma Keratocytes, Development and DystrophiesWednesday, May 08, 2013 11:00 AM-12:45 PMTCC 303 Paper SessionProgram #/Board # Range: 4555-4561Organizing Section: CorneaContributing Section(s): CorneaProgram Number: 4555Presentation Time: 11:00 AM - 11:15 AMWnt/b-catenin canonical signaling in corneal keratocytesregulates epithelium stratification through repressingBmp4/Mapk pathwayYujin Zhang 1 , Lung-Kun Yeh 2 , Winston W. Kao 1 , Chia-Yang Liu 1 .1 Ophthalmology, University of Cincinnati School of Med, Cincinnati,OH; 2 Ophthalmology, Chang-Gung Memorial Hospital, Linko,Taiwan.Purpose: Mesenchyme-epithelial interaction plays pivotal roles oncorneal morphogenesis during development. Our previous datashowed that loss of β-catenin (Ctnnb1) in corneal stromal keratocytes(Ctnnb1cskΔ/cskΔ) triggers precocious epithelium stratification inmice. In this study, attempts were made to explore the mechanismscontrolling this aberrant developmental event in the absence ofCtnnb1.Methods: To ablate Lrp5 and Lrp6 gene in keratocytes,KR/TC/Lrp5f/f/Lrp6f/f tetratransgenic mice were fed Dox-chow fromembryonic 12.5 (E12.5) to postnatal day 0 (P0) or from P0 to P10.Embryos and neonates were collected and subjected to histology andimmunohistochemistry examination. Primary corneal stromafibroblasts from the Ctnnb1f/f mice were cultured and infected byAd-cre or Ad-EGFP virus. Expression profiles of cytokines aftervirus infection were determined by cDNA micro-array, RT-PCR andwestern blotting. MAPK signaling was examined in HTCE treatedwith BMP4 protein. Specimens from WT mice treated with BMP4protein from P0 to P10 by subcutaneous injection were subjected toimmunohistochemistry examination.Results: Lrp5cskΔ/cskΔ/Lrp6cskΔ/cskΔ double knock-out micemanifested precocious corneal epithelium stratification similar to thephenotype observed in Ctnnb1cskΔ/cskΔ mice, while singleLrp5cskΔ/cskΔ or Lrp6cskΔ/cskΔ mice were normal. Interestingly,BMP4-treated neonates displayed phenotype resemblingLrp5cskΔ/cskΔ/Lrp6cskΔ/cskΔ and Ctnnb1cskΔ/cskΔ mice.Moreover, SEM showed the collagen fibrils and stromal cells arebetter organized in neonates of aforementioned mutant and BMP4-treated mice. Cytokine cDNA array screening showed that Bmp4 wasup-regulated in cultured corneal fibroblasts from Ctnnb1cskΔ/cskΔmice. Furthermore, MAPK pathway was activated via ERK in HTCEcell by BMP4 treatment.Conclusions: The precocious corneal epithelium stratificationobserved in Ctnnb1cskΔ/cskΔ and Lrp5cskΔ/cskΔ/Lrp6cskΔ/cskΔmutants may result from the up-regulated expression of BMP4 indeveloping corneal stroma, which enhanced MAPK signaling incorneal epithelial, leading to precocious epithelium stratification. Ourdata supports the notion that cross-talk between Wnt/β-catenin/Bmp4axis (in stroma) and MAPK signaling (in epithelium) play criticalroles in corneal morphogenesis during development.Commercial Relationships: Yujin Zhang, None; Lung-Kun Yeh,None; Winston W. Kao, None; Chia-Yang Liu, NoneSupport: NIH/NEI EY013755, Research to Prevent Blindness, OhioLions Eye Research FoundationProgram Number: 4556Presentation Time: 11:15 AM - 11:30 AMPeptide Amphiphiles as Versatile Substrates for Oriented CellAdhesion and Proliferation of Human Cornea StromalKeratocytesRicardo M. Gouveia, Valeria Castelletto, Ian Hamley, Che J.Connon. School of Chemistry, Food and Pharmacy, University ofReading, Reading, United Kingdom.Purpose: To develop novel bioactive surfaces able to supportadhesion and proliferation of human corneal stroma keratocytes(HCSK) in vitro whilst emulating the cell’s in vivo phenotype. Thisincludes maintaining proper cell morphology, the expression ofspecific markers such as keratocan, lumican, and decorin, as well asthe aligned deposition of collagen type-I. To this purpose, wedeveloped peptide amphiphile (PA) molecules that self-assemble intobioactive, stable nanostructures. PAs composed by a hexadecyl lipidchain attached to a peptide headgroup comprising the integrinbindingmotifs RGD or RGDS, or the negatively charged ETTESsequence were designed and tested for their nanostructure,biocompatibility, and cell adhesion properties.Methods: The bulk properties of RGD, RGDS, ETTES, and ofbinary PA systems consisting of RGD(S) mixed with diluent ETTESwere studied by transmission electron microscopy, small-angle X-rayscattering, and X-ray diffraction. In addition, fluorescencespectroscopy was used to determine the critical concentration for PAself-assembly (c.a.c.) in water. PA solutions at 1×10 -2 -10 -4 M and atvarious RGD(S):ETTES molar ratios were dried and used as coatingsto enhance HCSK adhesion, viability, and proliferation. Furthermore,the effect of these PA substrates on the expression of HCSK markerswas evaluated by QPCR.Results: Above their c.a.c. (>0.01% w/v), PAs formed well definedtapes, with bilayer structures and β-sheet features. When used ascoating substrates, PAs containing the RGD(S) motifs specificallypromoted integrin-dependent adhesion and proliferation of HCSKswithout significantly altering the expression patterns of analysedkeratocyte markers. However, no adhesion was observed withETTES coating alone. Optimal adhesion and maximal cellproliferation was achieved with 1.25×10 -3 M RGDS:ETTES at 13:87(mol/mol) ratio. This binary system enhanced adhesion 1.4-foldrelatively to substrates composed of only the RGD or RGDSmolecules, suggesting that spacing between RGD(S) motifs promotescell adhesion, whilst epitope crowding impairs it, possibly due to acharge effect.Conclusions: Self-assembling nanostructures formed by co-assemblyof RGD(S)-displaying PAs may constitute a versatile tool for cornealtissue engineering through modulation of HCSK adhesion andproliferation.Commercial Relationships: Ricardo M. Gouveia, None; ValeriaCastelletto, None; Ian Hamley, None; Che J. Connon, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 4557Presentation Time: 11:30 AM - 11:45 AMLimbal Biopsy As a Source of Stem Cells for Autologous StromalCell-based TherapySayan Basu 1, 2 , Martha L. Funderburgh 1 , James L. Funderburgh 1 .1 Ophthalmology, University of Pittsburgh School of Medicine,Pittsburgh, PA; 2 Cornea, L V Prasad Eye Institute, Hyderabad, India.Purpose: Mesenchymal stem cells have been identified in thesuperficial stromal layers of the peripheral cornea and limbus. Thisstudy addresses the hypothesis that human stromal stem cells can beisolated from clinically replicable limbal biopsies and cultivated inconditions suitable for potentially autologous cell-based therapy.Methods: One clock hour wide superficial stromal biopsies wereobtained from the limbal region of human corneo-scleral rims.Stromal mesenchymal cells were isolated by collagenase digestion ofthe limbal fragment, with or without epithelial removal by dispase,and cultured in media containing 2% bovine or human serum. At thethird passage stromal cells were evaluated for: a) stem cell geneexpression; b) clonal growth; c) sphere formation; d) expression ofkeratocytes marker genes in differentiation conditions; and e)organization of collagen lamellae on aligned nanofiber substratum.Results: Collagenase digestion without epithelial removal was themost efficient method of isolating stromal cells, which readilyexpanded in media containing human serum. Epithelial cells werelost from the cultures by serial passage. P3 stromal cells had highclonogenecity compared to human fibroblasts, readily formed spheresand expressed stem cell gene markers (ABCG2, BMI1, CXCR4,Nestin, NOTCH1, Oct4 and SSEA4). The stromal cells culturedusing both bovine and human serum demonstrated similar ability todifferentiate into functional keratocytes, characterized byupregulation of keratocyte gene markers (ALDH, AQPR1, B3GnT7,CD34, CHST6, Keratocan and PTGDS) and generation of a multilayeredmatrix of aligned collagen fibers.Conclusions: Mesenchymal stem cells can be isolated from tinysuperficial biopsies of the human limbal stroma and expanded in vitrousing potentially autologous culture conditions. The ability of thesestromal stem cells to differentiate into functional keratocytes opensthe possibility of therapeutic strategies to cure blindness resultingfrom corneal stromal diseases with patient derived-cells isolatedusing autologous, xenobiotic-free conditions.Commercial Relationships: Sayan Basu, None; Martha L.Funderburgh, None; James L. Funderburgh, NoneSupport: NIH grants EY016415 and P30-EY008098, Research toPrevent Blindness Inc., Louis J. Fox Center for Vision Restoration-UPMC Eye CenterProgram Number: 4558Presentation Time: 11:45 AM - 12:00 PMMass Spectrometric - Based Proteomic Analysis of TGFBICorneal DystrophiesAarika Menees 1 , Surendra Dasari 2 , Ahmet Dogan 2 , Sanjay V. Patel 1 ,Keith H. Baratz 1 , Jason D. Theis 2 , Julie A. Vrana 2 , Diva R. Salomao 2,1 . 1 Ophthalmology, Mayo Clinic, Rochester, MN; 2 LaboratoryMedicine and Pathology, Mayo Clinic, Rochester, MN.Purpose: To identify the composition of corneal deposits at aproteomic level in patients with granular dystrophy type I and latticedystrophy.Methods: In a retrospective study, we identified archived penetratingkeratoplasty specimens collected between January, 1904, andDecember, 2011, consisting of formalin-fixed paraffin-embeddedtissue from patients with granular dystrophy type I (GD) or latticedystrophy (LD). The protein deposits of interest, and the surroundingnormal stroma, were dissected with laser micro-dissection (LMD).Resulting proteins were denatured with sonication and digested intopeptides by using trypsin. The peptides were subjected to liquidchromatography-based tandem mass spectrometry (LC-MS/MS).Resulting spectra (MS/MS) were identified with MyriMatch searchengine configured to search for known mutations in the TGFBI geneassociated with corneal dystrophies. Independently, the MS/MS weresearched with DirecTag-TagRecon software configured to look forunanticipated mutations. The peptide identifications were filteredwith IDPicker software to a false discovery rate of 2%.Results: We analyzed 9 specimens from 7 patients; four patientswere female (5 specimens), and three were male (4 specimens) withages at the time of penetrating keratoplasty ranging from 29 to 80years. Four patients had granular dystrophy type I (6 specimens), and3 patients had lattice dystrophy (3 specimens), with the diagnosesconfirmed by histopathology and histochemical stains. For all cases,we detected a higher concentration of TGFBI in the corneal depositscompared to the adjacent normal stroma dissected from the samespecimen and compared to a normal control cornea. The latticedystrophy cases showed amyloid deposits that were rich inapolipoprotein E and serum amyloid P-component protein in additionto TGFBI. The granular dystrophy type I specimens showed calciumbinding proteins of the S100 family. We detected a knownArg124His mutation in TGFBI gene in one case of GD. We alsodetected four novel mutations in the TGFBI protein(Cys85Gly/Gly87Ala, Asp397Ala, His451Asp, and Ala481Cys) thathave strong MS/MS evidence but need Sanger sequencing validation.Conclusions: LMD-LC-MS/MS technique represents a new methodto profile the protein content of corneal dystrophy deposits and todetect novel mutations. Our proteomic findings support a distinctpathogeneses for granular and lattice dystrophies.Commercial Relationships: Aarika Menees, None; SurendraDasari, None; Ahmet Dogan, None; Sanjay V. Patel, None; KeithH. Baratz, Assessing the likelihood of developing Fuchs CornealDystrophy (P); Jason D. Theis, None; Julie A. Vrana, None; DivaR. Salomao, NoneSupport: Research to Prevent Blindness, Mayo FoundationProgram Number: 4559Presentation Time: 12:00 PM - 12:15 PMInvestigation of Gene Therapy Using Immortalized Cells Derivedfrom a Gelatinous Drop-Like Corneal Dystrophy PatientKoji Kitazawa 1 , Satoshi Kawasaki 1 , Keita Aoi 3, 1 , KatsuhikoShinomiya 1 , Akira Matsuda 2 , Toshinari Funaki 2 , Mina Nakatsukasa 1 ,Junji Hamuro 1 , Akira Murakami 2 , Shigeru Kinoshita 1 . 1 KyotoPrefectural Univ of Med, Kyoto, Japan; 2 Juntendo University, Tokyo,Japan; 3 Doshisya University, Kyoto, Japan.Purpose: Gelatinous drop-like corneal dystrophy (GDLD) is anautosomal recessive inheritance disease caused by biallelic loss-offunctionmutations within the tumor-associated calcium signaltransducer 2 (TACSTD2) gene. We previously established twoimmortalized cell lines via the lentiviral introduction of the SV40large T antigen and hTERT genes into the corneal and conjunctivalepithelial cells of GDLD patients. The purpose of this present studywas to assess whether or not a gene therapy using these two cellslines is effective for the treatment of GDLD patients.Methods: The lentivirus vector harboring the wild-type TACSTD2gene was produced and transduced to the two immortalized cell linesdescribed above. Epithelial barrier function of the two cell lines wasinvestigated by means of trans-epithelial resistance (TER) analysisand dye permeabilization testing using fluorescein in order to assesswhether or not the transduction of the wild-type TACSTD2 geneactually normalizes the disease situation of GDLD cornea.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: The transduction efficiency of the wild-type TACSTD2gene to the immortalized cell lines was approximately 80%. TER ofthe immortalized cell lines was increased after the transduction of thewild-type TACSTD2 gene. The permeability of fluorescein in theimmortalized cell lines was decreased after the transduction of thewild-type TACSTD2 gene.Conclusions: The findings of this study show that transduction of thewild-type TACSTD2 gene normalizes the disease situation of GDLDcorneas to some extent, thus indicating that gene therapy may proveto be a promising treatment for GDLD.Commercial Relationships: Koji Kitazawa, None; SatoshiKawasaki, None; Keita Aoi, None; Katsuhiko Shinomiya, None;Akira Matsuda, None; Toshinari Funaki, None; MinaNakatsukasa, None; Junji Hamuro, None; Akira Murakami,SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958 (P); ShigeruKinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Support: H23-Nanchi-Ippan-084 from the Japanese Ministry ofHealth, Labour and Welfare.Program Number: 4560Presentation Time: 12:15 PM - 12:30 PMA Family-based Investigation of the Role of TCF4 TrinucleotideRepeat Expansion in Fuchs Endothelial Corneal Dystrophy(FECD)Keith H. Baratz 1 , Ross A. Aleff 2 , Yi-Ju Li 3 , Malinda L. Butz 4 , SimonG. Gregory 5 , Gordon K. Klintworth 6 , W. Edward Highsmith 4 , NatalieA. Afshari 7 , Eric D. Wieben 2 . 1 Ophthalmology, Mayo Clinic,Rochester, MN; 2 BIochemistry and Molecular Biology, Mayo Clinic,Rochester, MN; 3 Biostatistics and BioInformatics, Center for HumanGenetics, Duke University Medical Center, Durham, NC;4 Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN;5 Medicine, Molecular Genetics and Microbiology, Duke UniversityMedical Center, Durham, NC; 6 Ophthalmology, Duke UniversityMedical Center, Durham, NC; 7 Ophthalmology, University ofCalifornia, San Diego, San Diego, CA.Purpose: Previous studies of unrelated FECD subjects andunaffected controls revealed a strong association between thepresence of more than 50 repeats of the trinucleotide TGC in the thirdintron of the transcription factor 4 (TCF4) gene and the incidence ofFECD. To evaluate the role of this triplet repeat in the inheritance ofthe disease, we examined whether this trinucleotide repeat expansionin TCF4 segregates with the incidence of FECD in families.Methods: The corneas of FECD probands and their family memberswere graded by using a modified Krachmer scale (grade 0 - 6).Leukocyte-derived DNA was evaluated by fluorescence based shorttandem repeat (STR) assays to calculate the TGC repeat length ofboth alleles in all affected and unaffected participants. Southern blotanalysis was used to interrogate the repeat status in all DNA samplesthat had only a single allele by STR analysis. A total of 30participants (33-91 years) from 11 families were included.Results: The size of the trinucleotide repeat in patients with FECDranged between 12 and 89 TGC repeats. Expansion above 50 repeatstracked with the disease in six of eleven families. In one additionalfamily, 2 of 4 participants had FECD and repeat expansions of 59 and62 TGC repeats, one member (48 yrs.) had equivocal disease withexpansion (grade 1; 59 repeats), while the fourth family member (53yrs.) had TGC expansion but no evidence of disease (gr. 0; 59repeats). In the remaining four families, no repeat expansions abovethe 50 repeat threshold for disease association were identified in anyof the seven affected (≥ gr. 2) and one equivocally affected (gr. 1)family members. Among families with expanded TGC repeats, thevariation in repeat size was between 0 and 3 repeats in parent-childtransmission and between 0 and 6 repeats between siblings.Conclusions: Moderate expansions of the TGC repeat in TCF4predicted FECD status in 6 of 11 FECD families. In a subset ofFECD families, there was no expansion of this trinucleotide repeat.Thus, the repeat status may be useful and necessary in stratifyingpatients in further genetic studies of FECD and in studies of diseasepathophysiology using FECD-affected tissue. In this small cohort, theexpansion size varied minimally within families.Commercial Relationships: Keith H. Baratz, Assessing thelikelihood of developing Fuchs Corneal Dystrophy (P); Ross A.Aleff, Mayo Foundation (P); Yi-Ju Li, None; Malinda L. Butz,None; Simon G. Gregory, None; Gordon K. Klintworth, None; W.Edward Highsmith, None; Natalie A. Afshari, None; Eric D.Wieben, Assessing the likelihood of developing Fuchs CornealDystrophy (P)Support: NIH Grant UL1 RR024150; Research to PreventBlindness, N.Y.; and the Mayo FoundationProgram Number: 4561Presentation Time: 12:30 PM - 12:45 PMAGBL1 implicated in the pathogenesis of late-onset FCD andinteracts with TCF4John D. Gottsch 1 , Shivakumar Vasanth 2 , Nicholas Katsanis 2 , S. AmerRiazuddin 1 . 1 Wilmer Eye Institute, Johns Hopkins University Schoolof Medicine, Baltimore, MD; 2 Center for Human Disease Modeling,Duke University Medical Center, Durham, NC.Purpose: Fuchs corneal dystrophy (FCD) is a genetic disorder of thecorneal endothelium and a leading cause of corneal transplantation inthe United States. The following study was undertaken to investigatethe causality of FCD in a large family.Methods: A large familial case of FCD was ascertained with nineaffected and six unaffected individuals in three generations. Agenome-wide linkage scan was completed with an Affymetrix SNPgenotyping array. Two-point Lod scores were calculated and allregions with Lod scores >1 were confirmed by closely-spacedfluorescently-labeled short tandem repeat (STR) markers. Nextgenerationsequencing of captured exons in the critical region wasemployed to identify the causal allele(s) responsible for thephenotype. Immunohistochemical (IHC) analyses were performed oncorneal sections to investigate the expression in the cornealendothelium and a co-immunoprecipitation (Co-IP) approach wasused to investigate protein-protein interactions.Results: The genome-wide linkage analyses identified two causalloci, present on chromosomes 3p and 15q that were confirmed bygenotyping closely-spaced fluorescently-labeled STR markers.Alleles at these loci were not sufficient to independently localize thecausal phenotype; however, taken together alleles at these two locicould explain the causality and the severity associated with thephenotype. Subsequently, we identified a premature termination in acytosolic carboxypeptidase termed CCP4 also known as AGBL1present within the critical interval of 15q. This gene was identifiedpreviously in a serial analysis of gene expression (SAGE) of thecorneal endothelium. The premature termination variant was notpresent in 384 ethnically matched control chromosomes.Subsequently, we identified two independent sporadic cases thatharbored the same premature termination mutation and a secondmissense variation in three unrelated sporadic cases. IHC analysesidentified AGBL1 expression in the corneal endothelium and co-IPexperiments confirmed that AGBL1 and TCF4 proteins can bind.Conclusions: We identify two novel FCD loci and implicate AGBL1in the pathogenesis of late-onset FCD. Our data provides firstevidence of physical interactions between two late-onset FCD genes.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaWe are investigating the functional significance of AGBL1-TCF4protein-protein interactions in late-onset FCD.Commercial Relationships: John D. Gottsch, None; ShivakumarVasanth, None; Nicholas Katsanis, None; S. Amer Riazuddin,NoneSupport: This study was supported in part by National Eye InstituteGrant R01EY016835 (JDG)441 Corneal Development, Differentiation, Dystrophies, GeneticsWednesday, May 08, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 4718-4742/C0101-C0125Organizing Section: CorneaProgram Number: 4718 Poster Board Number: C0101Presentation Time: 11:00 AM - 12:45 PMRole of Shp2 Protein Phosphatase in Mouse Corneal EpitheliumStratificationChia-Yang Liu 1 , Gracia Ng 1 , Lung-Kun Yeh 2, 1 , Hongshan Liu 1 , YujinZhang 1 , Winston W. Kao 1 . 1 Ophthalmology, Univ of Cincinnati,Cincinnati, OH; 2 Department of Ophthalmology, Chang-GungMemorial Hospital, Linko, Taiwan.Purpose: Proper stratification is essential for the homeostasis ofstratified epithelia. The cellular and molecular mechanisms by whichthis process is initiated and maintained have not been clearlyidentified. Present study aims at elucidating the roles of Shp2 onsurface epithelium stratification.Methods: Shp2 was ablated in stratified epithelia of triple transgenicKrt14-rtTA/tet-O-Cre/Shp2f/f mice fed with doxycycline (Dox) chowat different embryonic and postnatal ages. In another series ofexperiment, epithelial debridement was performed in adult mice.Excised mouse eyes from experimental mice were subjected tohistology and immunohistochemistry examination.Results: When Dox induction began prior to eyelid closure, theneonates of Shp2 mutant dies within 24 hours after birth with eyelidopen. When Dox was given after E16.5, Shp2 mutants were bornwith normal eyelid development but the mutant died shortly afterP21. Corneal epithelium anomaly was prominent in that it onlyconsisted of 1-2 layers in comparison to 5-6 cell layers seen inheterozygote littermate. Interestingly, the morphology of skin,conjunctiva and lacrimal and Meibomian glands appeared unaffected.Cessation of Dox induction at P21 allowed the mutant micelive to adulthood and corneal epithelium resumed 5-6 cell layers.Similarly, corneal epithelium was reduced to 1-2 cell layers in mutantmice fed Dox from P21 to P42. Furthermore, the re-stratificationduring wound healing was compromised when corneal epithelialdebridement was generated in the adult Shp2 mutant mice. We foundthat loss of Shp2 from corneal epithelium caused significant decreasein cell proliferation but no effect on apoptosis or corneal-typedifferentiation. Strikingly, the numbers of desmosome andhemidesmosome dramatically reduced in Shp2 mutant corneas.Immunostaining revealed that laminin-b1 and E-cadherin were downregulatedin Shp2 mutant corneas as compared to those of controlmice. Noted that the cellular and molecular deficiencies concerningcorneal epithelial stratification are reversible upon Dox induction isceased, suggesting that epithelial progenitor (stem) cells are notaffected in corneas of Shp2 mutant mice.Conclusions: Our data suggest that Shp2’s role in sustaining cellproliferation and strengthening a tissue’s foundation is essential forproper epithelial stratification during corneal morphogenesis andhomeostasis.Commercial Relationships: Chia-Yang Liu, None; Gracia Ng,None; Lung-Kun Yeh, None; Hongshan Liu, None; Yujin Zhang,None; Winston W. Kao, NoneSupport: NIH/EY13755, NIH/EY21501,Research to PreventBlindness, Ohio Lions Eye Research FoundationProgram Number: 4719 Poster Board Number: C0102Presentation Time: 11:00 AM - 12:45 PMThe homeodomain transcription factor PITX2 is required toestablish correct cell lineages and angiogenic privilege in thedeveloping corneaPhilip J. Gage, Amanda L. Zacharias, Chen Kuang. Ophthal & VisScience, Univ Michigan-Kellogg Eye Ctr, Ann Arbor, MI.Purpose: Vision depends critically on normal corneal development,including correct specification of the epithelium (from surfaceectoderm), stroma and endothelium (from mesenchyme), andestablishment of an avascular environment (angiogenic privilege).Phenotypes of humans and mice with heterozygous mutations havesuggested PITX2 as an important mediator of these processes butanterior segment morphogenesis in global and tissue-specific Pitx2knockout mice is blocked prior to initiation of corneal development.Therefore, we utilized our conditional Pitx2 allele together with atamoxifen-inducible Cre recombinase to generate temporal knockoutanimals (Pitx2-tko) and assess gene function in the developingcornea.Methods: Appropriate strains were crossed to generate control andconditional mutant embryos. Timed pregnant females were injectedwith a single dose of tamoxifen early in corneal development totemporally ablate Pitx2 in the mutant embryos.Immunohistochemistry or RNA in situ hybridization was employedto assess gene expression.Results: Ablation of Pitx2 at e11.5 rescues the early block to anteriorsegment development but significantly disrupts formation of all threecorneal layers. Gene expression is altered in all three layers of Pitx2-tko corneas and the surface ectoderm and mesenchyme appear toadopt a conjunctival fate based on ectopic expression on specificmarker expression, suggesting that PITX2 is required cellautonomouslyfor specification of the stroma and endothelium andnon-cell autonomously for specification of the epithelium. Themutant corneal mesenchyme is highly vascularized, indicating thatPITX2 is required to establish angiogenic privilege. Dkk2 expressionis not maintained in mutant eyes and there is a correspondingincrease in canonical Wnt signaling activity. Vegfa and Pdgfaexpression are also increased.Conclusions: Collectively, these results suggest that PITX2-mediated suppression of canonical Wnt signaling may be animportant mechanism required to correctly specify cell lineages andestablish angiogenic privilege during corneal development. We aretesting this hypothesis further by activating canonical Wnt signalingactivity in the corneal ectoderm and mesenchyme independently ofmutations in Pitx2.Commercial Relationships: Philip J. Gage, None; Amanda L.Zacharias, None; Chen Kuang, NoneSupport: EY014126Program Number: 4720 Poster Board Number: C0103Presentation Time: 11:00 AM - 12:45 PMExpression patterns of angiogenic factors correlate with vascularpatterning of the developing anterior eyeSam Kwiatkowski, Ravi P. Munjaal, Peter Y. Lwigale. Biochemsitryand Cell Biology, Rice University, Houston, TX.Purpose: To determine the expression patterns of angiogenic factorsduring anterior eye development. The anterior eye is composed of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneahighly vascularized tissue which surrounds the avascular cornea. Wehypothesize that angiogenic factors are expressed in patterns thatsuggest their potential role during pericorneal vasculogenesis andcorneal development.Methods: Reverse Transcription PCR (RT-PCR) and RNA in situhybridization were used to identify spatiotemporal expressionpatterns of proangiogenic and antiangiogenic factors during anterioreye development and vascularization of embryonic day (E)3, E5, andE7 chick embryos.Results: Our data show that secreted proangiogenic andantiangiogenic factors are expressed in developing tissues of theanterior eye including the lens, optic cup, and presumptive iris.Assayed factors represent several gene families known to affectangiogenesis, such as, VEGF, FGF, PDGF, sFlt1, semaphorins, andnetrins. Most of these genes are broadly expressed by E3 and thenbecome localized to specific regions of developing tissues in theanterior eye by E7. Receptors for angiogenic factors were localized toangioblasts and forming ocular blood vessels.Conclusions: The expression patterns of the proangiogenic andantiangiogenic factors suggest their potential roles in vascularpatterning of the developing anterior eye. Our results provide insightinto the mechanisms regulating the development of ocular bloodvessels and formation of corneal avascularity.Commercial Relationships: Sam Kwiatkowski, None; Ravi P.Munjaal, None; Peter Y. Lwigale, NoneSupport: NIH EY022158Program Number: 4721 Poster Board Number: C0104Presentation Time: 11:00 AM - 12:45 PMRole of Netrin-4 and Laminin β2 and γ3 Chains in CornealDevelopment and MaintenanceJeremiah Martino 1, 2 , Galina Bachay 1, 2 , Michael A. Dattilo 1, 2 ,Douglas R. Lazzaro 1, 2 , William J. Brunken 1, 2 . 1 Ophthalmology andCell Biology, SUNY Downstate Medical Ctr, Brooklyn, NY; 2 SUNYEye Institute, Brooklyn, NY.Purpose: Laminins and netrins are extracellular matrix (ECM)molecules that regulate cell proliferation, migration and neuralguidance. Our previous work has shown that these molecules areexpressed in the corneal basement membranes and their expression isaltered in human disease. Our goal is to understand the role that thesemolecules play in corneal development and disease. To this end, weare continuing our characterization of the corneal phenotype in micelacking the Ntn4, LamB2, or LamC3 genes.Methods: Epithelial cell proliferation and differentiation, cornealinnervation, and endothelial cell tight-junction formation wereassayed via immunofluorescence (IF) in whole cornea and radialsection. Key developmental time-points were assayed in wild-type(WT), LamB2-/-, LamC3-/-, double-knockout LamB2-/-LamC3-/-,and Ntn4-/- mice. Cell proliferation and innervation were quantifiedin P20 and P33/34 corneas.Results: Although corneal development was grossly normal in allgenotypes, alterations in the proliferative behavior of epithelial cells,patterning of the sub-basal neural plexus and endothelial cell tightjunctionformation were observed. Epithelial proliferation, assayedwith phospho-histone H3 IF, demonstrated increased proliferation inthe LamB2-/-, LamC3-/-, double-knockout LamB2-/-LamC3-/- vs.age-matched WT mice. Keratin12 IF data showed no differencesbetween knockout and WT mice, indicating a normal cornealepithelial differentiation. Corneal innervation, assayed by axonlabeling with neurotubulin IF (TuJ-1) was aberrant. Increases innerve branching and pericorneal ring thickness at the level of the subbasalplexus was observed in P20 Ntn4-/- mice vs. WT littermates.Neural guidance at gestational day 15 (E15) was not altered in Ntn4-/- and LamC3-/- mice, demonstrating that neural pathfinding islargely intact. Lastly, endothelial cell tight-junction assembly,assessed by ZO-1 and β-catenin IF, was profoundly disrupted in P25LamB2-/- and P33 LamC3-/- and Ntn4-/- compared to age-matchedWT mice.Conclusions: Netrin-4 and laminin β2 and γ3 chains are importantcomponents of corneal basement membranes. Genetic deletion ofthese molecules disrupts epithelial and endothelial cell behavior aswell as the branching pattern of the sub-basal neural plexus. Ongoingstudies will determine the role of these molecules in corneal woundhealing or post-surgical recovery.Commercial Relationships: Jeremiah Martino, None; GalinaBachay, None; Michael A. Dattilo, None; Douglas R. Lazzaro,None; William J. Brunken, NoneSupport: NEI Grant EY12676; and Research to Prevent BlindnessChallenge Grant - OphthalmologyProgram Number: 4722 Poster Board Number: C0105Presentation Time: 11:00 AM - 12:45 PMOcular Manifestations of X-linked Reticulate PigmentaryDisorderKruti Dajee, H D. Cavanagh, Nick Hogan, Linder A. Baker, AndrewR. Zinn, Vinod V. Mootha. Ophthalmology, University of TexasSouthwestern Medical Center, Dallas, TX.Purpose: X-linked Reticulate Pigmentary Disorder is a rare geneticdisorder described in only 4 families to date. Initially described in aCanadian family, male members were noted to have diffuse reticulatepigmentation of skin and corneal dyskeratosis with significantphotophobia. Other systemic manifestations include hypohidrosis,upswept frontal hairlines, flared eyebrows, and recurrent pulmonary,genitourinary and gastrointestinal abnormalities. We assessed aTexan family comprising two brothers affected with this conditionpreviously linked to a 4.9 Mb region of chr X. Our purpose was todefine the ocular phenotype, delineate course of this disease, anddocument response to treatment.Methods: Members of the Texas family were enlisted in the study;subjects had a slit lamp examination and exam under anesthesia givensignificant photophobia. Skin and urethral mucosal biopsies wereperformed on subject 1.Results: Subjects were the only affected members of their familywith corneal and systemic manifestations. An affected sister (carrierof trait) only had isolated cutaneous findings. Photophobia onset wasnoted at age 2 and of corneal opacities around age 5. Subject 1(Figure 1) underwent 10 superficial keratectomies with excimerphototherapeutic keratectomy over a 6-year period. Exam underanesthesia revealed bilateral elevated grey corneal opacities with mildanterior and mid-stromal vessels in both subjects. Subject 1 also hadpatchy anterior stromal scarring and secondary lipid depositionnotably absent in Subject 2 (Figure 2), who has had no surgicaltreatments to date. The iris, lens, and dilated fundus examinationswith indirect ophthalmoscopy were otherwise unremarkable. Slitlamp exam revealed a normal tear lake and Schirmer’s testing at 5minutes with anesthesia was elevated indicative of reflex tearing.Tissue biopsy results of urethral mucosa revealed granulation tissuewith squamous metaplasia, hyperkeratosis, and parakeratosis. Punchbiopsy specimen of skin demonstrated basket weave stratum corneumand acanthotic epidermis with necrotic keratinocytes.Conclusions: Our study revealed that corneal dyskeratosis in X-linked reticulate pigmentary disorder results in significant visiondisability and photophobia secondary to elevated anterior cornealopacities. Phototherapeutic keratectomy was associated withrecurrence of lesions within a few months and worsening of stromalscarring.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaThis single nucleotide deletion is predicted to result in a frameshiftmutation and premature stop codon.Conclusions: Although elevated IOP is not a feature of MGC1, theseeyes may be more prone to secondary glaucoma. Our reported novelmutation in CHRLD1 expands the mutational spectrum of thisdisorder. Our family further establishes that the disorder isgenetically homogenous as the seven other families recently reportedalso all had mutations in CHRDL1.Figure 1Figure 2Commercial Relationships: Kruti Dajee, None; H D. Cavanagh,Menicon Ltd (C); Nick Hogan, None; Linder A. Baker, None;Andrew R. Zinn, None; Vinod V. Mootha, NoneSupport: Research to Prevent BlindnessSlit lamp photo of aphakic right eye with enlarged corneal diameterand patchy iris stromal defects.Program Number: 4723 Poster Board Number: C0106Presentation Time: 11:00 AM - 12:45 PMNovel CHRLD1 Mutation and Secondary Glaucoma in Patientwith X-Linked MegalocorneaXin Gong, Jess T. Whitson, Vinod V. Mootha. Ophthalmology, UTSouthwestern Medical Center, Dallas, TX.Purpose: X-linked megalocornea (MGC1) is a bilateraldevelopmental disorder of the anterior segment with enlarged, thincorneas,extremely deep anterior chambers (AC), and normalintraocular pressures (IOPs). Associated ocular features includemosaic corneal degeneration, iris atrophy/pigment dispersion, lensdislocation, and cataract. MGC1 has been recently been mapped tothe Chordin-like 1 gene (CHRDL1) encoding for ventropin. A 63year-old male with MGC1 presented to the Cornea Service 5 monthsafter secondary placement of iris fixated posterior chamberintraocular lens (IOL). Our purpose is to present the clinical course ofthis patient with MGC1 and genotyping results in his family.Methods: We performed a retrospective chart review to describeocular findings and clinical course of subject with MGC1. PCRamplification and traditional Sanger sequencing the 11 coding exonsof CHRDL1 were performed on genomic DNA from proband andsiblings.Results: Slit lamp findings of our subject included bilateral, enlargedcorneas with mosaic degeneration, extremely deep AC, iris transilluminationdefects, and increased pigment in irido-corneal angles.Central corneal pachymetery measured 498 µm OD and 517 µm OS.The patient had had cataract extraction 20 years previously and hadbeen left aphakic OU. Five months before presentation, the patientunderwent secondary placement of an iris-fixated IOL in OS. IOPswere 13 mmHg OD and 24 mmHg OS despite 3 glaucomamedications. Right optic nerve had a 0.15 and left had 0.8 cup/discratios respectively. Four months after explantation of IOL, the patientunderwent placement of a Baerveldt glaucoma implant for refractoryglaucoma. A novel CHRDL1 mutation was found in exon 10 ofproband (chrX 109,811,419 (hg19), c.1097delT, p.Leu366ArgfsX7).Left eye with iris fixated IOL. Note corneal mosaic degenerationcentrally.Commercial Relationships: Xin Gong, None; Jess T. Whitson,Alcon (R), Allergan (R), Merck (R); Vinod V. Mootha, NoneSupport: Research to Prevent Blindness; National Eye InstituteEY022161Program Number: 4724 Poster Board Number: C0107Presentation Time: 11:00 AM - 12:45 PMAn intronic TCF4 tri-nucleotide repeat expansion associated withFuchs Corneal DystrophyS. Amer Riazuddin 1 , Briana C. Gapsis 1 , Nicholas Katsanis 2 , John D.Gottsch 1 . 1 The Wilmer Eye Institute, Johns Hopkins Univ Sch ofMed, Baltimore, MD; 2 Center for Human Disease Modeling, DukeUniversity Medical Center, Durham, NC.Purpose: We previously localized three large familial cases to FCD2on chromosome 18q. Subsequently, rs613872, an intronic SNPresiding within the FCD2 critical interval was reported to beassociated with FCD phenotype. Interestingly, the linkagedisequilibrium region has been shown to harbor a tri-nucleotiderepeat present just upstream of TCF4 isoform (NM_001243231). Thefollowing study was undertaken to examine the possibility that thistrinucleotide repeat is associated with late-onset FCD.Methods: Human ocular tissue was obtained from Tissue BankInternational and the corneal endothelium was stripped of theDescemets membrane. Total RNA was extracted to synthesize cDNAand Taqman probes were used to examine the expression of TCF4 incorneal endothelium. We examined the repeat length in FCD patientsusing fluorescent labeled-STR markers that could detect repeatlengths up to 150 repeats using genomic DNAs of FCD affectedindividuals and unaffected controls. The tri-nucleotide repeat regionwas amplified, the PCR products were resolved on ABI 3100 genetic©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaanalyzer and analyzed using GeneMapper software. The distributionof the alleles and Fisher exact test were performed to test the allelicassociations using Plink algorithms.Results: We first examined the expression of TCF4 and our analysessuggest that the TCF4 is expressed in the corneal endothelium. Wenext examined the repeat length in FCD affected individuals andunaffected controls. Our results suggest that majority (212/288) of theFCD patients harbor a single allele within the normal physiologicalrange of the polymorphic repeat with the second allele undetectablewithin our measurable range. In sharp contrast, the second allele wasundetectable in only a small percentage (75/288) of controls subjects.We examined the distribution of these alleles in FCD affectedindividuals and unaffected controls and found that absence of thesecond allele is significantly associated with late-onset FCD.Interestingly, the absence of second allele segregated with diseasephenotype in the two large familial cases of FCD.Conclusions: The tri-nucleotide repeat expansion represents anothermarker that is significantly associated with FCD. We are currentlyinvestigating the possibility that a particular length and/or a specificrange of the expansion may be responsible for the Fuchs CornealDystrophy.Commercial Relationships: S. Amer Riazuddin, None; Briana C.Gapsis, None; Nicholas Katsanis, None; John D. Gottsch, NoneSupport: This study was supported by the National Eye InstituteGrant R01EY016835 (JDG)Program Number: 4725 Poster Board Number: C0108Presentation Time: 11:00 AM - 12:45 PMAn Investigation of Mitochondrial Haplogroups in FuchsEndothelial Corneal DystrophyYi-Ju Li 1, 2 , Mollie A. Minear 2 , Jacqueline Rimmler 2 , ElmerBalajonda 3 , Michael A. Hauser 2 , R Rand Allingham 3 , Gordon K.Klintworth 3, 4 , Simon G. Gregory 2 , Natalie A. Afshari 5, 3 .1 Biostatistics and Bioinformatics, Duke University Medical Center,Durham, NC; 2 Center for Human Genetics, Medicine, DukeUniversity Medical Center, Durham, NC; 3 Duke University EyeCenter, Duke University Medical Center, Durham, NC; 4 Pathology,Duke University Medical Center, Durham, NC; 5 Shiley Eye Center,University of California San Diego, San Diego, CA.Purpose: To investigate whether European mitochondrial DNA(mtDNA) haplogroups contribute to the susceptibility of Fuchsendothelial corneal dystrophy (FECD). Oxidative stress has beenimplicated in the pathogenesis of FECD, which suggests the potentialrole of mitochondria in FECD. Although mtDNA haplogroups havebeen well-established for each ethnicity, no studies have examinedthe mtDNA variation in FECD.Methods: 529 patients with FECD and 463 controls, all Caucasians,were studied. All FECD patients had guttata grading ≥ 2 by modifiedKrachmer. Controls had a normal corneal exam and age at theenrollment ≥ 50. Nine common European mtDNA haplogroups werepredefined by 10 single nucleotide polymorphisms (SNPs) in thecoding and control regions of the mtDNA genome. We genotypedthese 10 SNPs using custom-designed TaqMan® allelicdiscrimination assays. A logistic regression model with age andgender as covariates was used to test each mtDNA SNP andhaplogroup using the full dataset, and also in a subset of highergraded FECD cases [Grade3+] (grade ≥ 3, N=457). For thehaplogroup analysis, we compared each haplogroup to the mostcommon European haplogroup H. Secondary analyses wereconducted to investigate the interaction between haplogroups and theSNP rs613872 in TCF4, a known risk variant for FECD, and betweenhaplogroup and current smoking status. Since only limited smokingstatus data were available in cases (N=234), a case-only model wasapplied.Results: Three SNPs (rs3021089, rs2853826, and rs34301918)showed nominal significance in both full dataset (min p=0.05) andGrade3+ subset (min p=0.03). On average, 39.7% of subjects havehaplogroup H. Subjects with haplogroup I (3.53%) showedsignificant decrease in risk of FECD comparing to those withhaplogroup H in both full (p=0.024, odds ratio [OR]=0.42, 95%confidence interval [CI] 0.20-0.89) and Grade3+ (p=0.017, OR=0.37,95%CI=0.16-0.84) datasets. None of the mtDNA SNPs andhaplogroups interact with rs613872 in TCF4 (min p=0.26), or withcurrent smoking status (min p=0.08).Conclusions: Our data show that mtDNA haplogroup I confers asignificant protective effect on FECD risk. . We found that the role ofTCF4 in FECD is independent to the mtDNA haplogroup. While nosignificant results were obtained for smoking status, more data areneeded to confirm the current finding. Our study presents animportant step in understanding the effect of mtDNA in FECD.Commercial Relationships: Yi-Ju Li, None; Mollie A. Minear,None; Jacqueline Rimmler, None; Elmer Balajonda, None;Michael A. Hauser, None; R Rand Allingham, New World Medical(C); Gordon K. Klintworth, None; Simon G. Gregory, None;Natalie A. Afshari, NoneSupport: EY016514Program Number: 4726 Poster Board Number: C0109Presentation Time: 11:00 AM - 12:45 PMImpaired mitochondrial membrane potential in Fuchsendothelial corneal dystrophyCecily E. Hamill 1, 2 , Thore Schmedt 1, 2 , Yuming Chen 1, 2 , Ula V.Jurkunas 1, 2 . 1 Massachusetts Eye and Ear Infirmary, Boston, MA;2 Schepens Eye Research Institute, Boston, MA.Purpose: Fuchs Endothelial Corneal Dystrophy (FECD) causesendothelial cell loss via apoptosis and possibly loss of mitochondrialfunction. The purpose of this study was to compare the alterations inmitochondrial membrane potential (MMP) in response to proapoptoticagent straurosporine (STS), between normal endotheliumand FECD-affected endothelial cells.Methods: Normal and FECD endothelial cell lines (HCECi andFECDi, respectively) were exposed to a dose of STS between 0.1 and5.0 µM. Caspase-3 activity was measured using rhodamine 110 bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide). Withincreasing caspase 3 activity, there is increased fluorescence asmeasured on a plate reader. Mitochondrial damage was assessed bymeasuring uptake of JC-1 (5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcar-bocyanine iodide), which accumulates innegatively charged healthy mitochondria to form aggregates that emitred fluorescence at 590 nm. When MMP is lost, the dye does notaccumulate in the mitochondria and emits green fluorescence at 530nm. The fluorescence was measured on a plate reader, and the ratio ofred to green was calculated. The decrease in the fluorescence ratiofrom baseline indicates a decline in MMP, thus mitochondrialdamage.Results: Initially, corneal endothelial cells were exposed to STS,which caused an increase in capase-3 activity (p=0.005, ANOVA).Likewise, STS caused a decrease in fluorescence ratio in a dosedependent manner in HCECi (p=0.03, ANOVA) and FECDi (p=0.02,ANOVA). When compared to HCECi, FECDi showed a 3-folddecrease in fluorescence ratio at baseline (p=0.001) and after 1.0 µMexposure to STS (p=0.02). There was a 2.5-fold decrease influorescence ratio after STS exposure of 0.1 µM (p=0.01), 0.5 µM(p=0.02), and 5.0 µM (p=0.03) in FECDi as compared to HCECi.Conclusions: STS-induced apoptosis correlated with loss of MMP incorneal endothelium. Lower MMP in FECDi as compared to HCECi©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaat baseline suggests presence of mitochondrial dysfunction indiseased cells. With exposure to the pro-apoptotic agent, FECDi cellswere more susceptible to mitochondrial damage as compared tonormal endothelium. Therefore, mitochondrial dysfunction maycontribute to cell loss seen in FECD.Commercial Relationships: Cecily E. Hamill, None; ThoreSchmedt, None; Yuming Chen, None; Ula V. Jurkunas,61/482,769 (P), Altheos (C)Support: National Institutes of Health/National Eye InstituteR01EY020581, Research to Prevent Blindness, Cures for Kids FundProgram Number: 4727 Poster Board Number: C0110Presentation Time: 11:00 AM - 12:45 PMAbnormal extracellular matrix production in corneal endothelialcells from patients with late-onset Fuchs corneal dystrophyJulia M. Wessel, Matthias Zenkel, Ursula Schlotzer-Schrehardt,Bjoern O. Bachmann, Theofilos Tourtas, Friedrich E. Kruse.Department of Ophthalmology, University of Erlangen-Nuremberg,Erlangen, Germany.Purpose: To gain a better understanding of the molecular pathologicmechanisms underlying abnormal extracellular matrix production(ECM) in Fuchs corneal dystrophy (FCD).Methods: Human corneal endothelial-Descemet membranespecimens were obtained from patients with late-onset FCD duringDMEK surgery (n=10). To exclude unspecific findings due toendothelial damage specimens from patients with pseudophakicbullous keratopathy (PBK) were differentially analysed (n=3).Normal age-matched endothelial specimens from donor eyes (n=3)and eyes which had to be enucleated because of posterior uvealmalignant melanoma (n=3) were used as controls. Gene expressionprofiles of FCD and control specimens were compared using the“Human Extracellular Matrix and Adhesion Molecule” PCR array(RT2 Profiler PCR Array, Qiagen). Differentially expressed mRNAswere quantified (n=3) in FCD, control and PBK specimens byspecific real-time PCR assays (universal probe library, Roche).Various extracellular matrix proteins were localized in cryosectionsof corneal specimens from FCD, PBK and control eyes byimmunohistochemistry. Informed consent has been obtained from thepatients and the study has been approved by the local ethicscommittee.Results: PCR array analysis revealed a significant upregulation (upto 20-fold; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneathe heterozygous state that was not identified in either unaffectedparent. The proband of the second family was also diagnosed withACC and PPCD. Karyotype analysis revealed a partial duplicationwithin a region of chromosome 17 that has previously beenassociated with ACC. ZEB1 sequencing identified a novel deletion(c.1913-1914delCA; p.(Ser638Cysfs*5)) present in the heterozygousstate, which was also identified in the proband’s affected mother.Conclusions: Posterior polymorphous corneal dystrophy haspreviously been described in association with a number of otherocular and extraocular abnormalities. We report the first associationof PPCD with a developmental abnormality of the brain, in this casePPCD3 associated with agenesis of the corpus callosum.Commercial Relationships: Michelle Jang, None; Ashley N.Roldan, None; Ricardo F. Frausto, None; Anthony J. Aldave,Alcon (R), Allergan (R), NIH (F), Bausch + Lomb (C), Allergan (C)Support: RPBProgram Number: 4730 Poster Board Number: C0113Presentation Time: 11:00 AM - 12:45 PMPosterior Polymorphous Corneal Dystrophy is Associated withSteep Corneal CurvatureLydia Ann, Ricardo F. Frausto, Fei Yu, Catherine K. Nguyen,Anthony J. Aldave. Jules Stein Eye Institute (UCLA), Los Angeles,CA.Purpose: To demonstrate whether posterior polymorphous cornealdystrophy (PPCD) associated and not associated with mutations inthe zinc finger E-box binding homeobox 1 (ZEB1) gene ischaracterized by steep corneal curvatures.Methods: Slit lamp biomicroscopic examination and cornealtopographic imaging were performed for all available patients withposterior polymorphous corneal dystrophy and unaffected familymembers. Steep corneal curvatures were defined as averagekeratometry values greater than 48.0D in each eye. Eyes in whichpenetrating keratoplasty was performed prior to topographic imagingwere excluded. Saliva or blood was collected from each individualfor isolation of genomic DNA and automated sequencing of the ZEB1gene.Results: Corneal topographic imaging and ZEB1 screening wereperformed for 38 individuals (27 affected and 11 unaffected) from 23families with PPCD. Seven of the 27 affected individuals were foundto have mutations in the ZEB1 coding region, while screening of thepromoter and coding regions in the remaining 20 individuals failed toreveal a mutation in the ZEB1 coding region. Ten of the 38individuals (26.3%) were measured as having average keratometryvalues greater than 48.0D in each eye: 10/27 (37.0%) individualswith PPCD (6/7 individuals with ZEB1 mutations (85.7%) and 4/20individuals with PPCD but without ZEB1 mutations (20.0%)) and0/11 unaffected individuals (Fisher’s exact test: p=0.037 forUnaffected vs. affected; p=0.0042 for ZEB1 mutation vs. withoutZEB1 mutation). The mean keratometry value averaged for both eyesof affected individuals measured 48.2D as compared to 44.1D forunaffected family members (t-test p value = 0.029). The affectedindividuals with ZEB1 mutations demonstrated a mean keratometryvalue averaged for both eyes of 53.3D, as compared to affectedindividuals without ZEB1 mutations (46.5D) (t-test: p value =0.0044).Conclusions: While PPCD is considered a corneal endothelialdystrophy, the identification of abnormally steep corneal curvaturesin 25-30% of affected individuals, and in 85-90% of affectedindividuals with ZEB1 mutations, indicates that ZEB1 may play a rolein corneal stromal development and/or physiology as well.Commercial Relationships: Lydia Ann, None; Ricardo F.Frausto, None; Fei Yu, None; Catherine K. Nguyen, None;Anthony J. Aldave, Alcon (R), Allergan (R), NIH (F), Bausch +Lomb (C), Allergan (C)Support: RPBProgram Number: 4731 Poster Board Number: C0114Presentation Time: 11:00 AM - 12:45 PMIdentification of Genetic Variant Candidates for PosteriorPolymorphous Corneal Dystrophy 1 utilizing Next-GenerationSequencingRicardo F. Frausto, Jonathan Han, Anthony J. Aldave. Doris Stein,Cornea Division, Jules Stein Eye Institute, UCLA, Los Angeles, CA.Purpose: To identify the genetic basis of posterior polymorphouscorneal dystrophy 1 (PPCD1) by whole-region capture andsubsequent next-generation sequencing (NGS) of the 14.5MbpPPCD1 locus on 20p12.1-q11.21.Methods: Slit-lamp examination was performed on 29 members of amultigenerational PPCD pedigree. Peripheral blood samples werecollected from each family member, and extracted genomic DNAsamples from selected affected and unaffected individuals were usedfor NGS of the captured PPCD1 locus. Alignment of the paired-endreads was performed using the Bowtie2 algorithm, while singlenucleotide variant (SNV) detection was performed using SAMtoolsmpileup, within the Partek Flow software. A list of variants identifiedwithin coding regions of the PPCD1 locus (bounded by the D20S182and D20S195 genetic markers) was generated using the PartekGenomic Suite software. To determine the sensitivity and specificityof NGS at two different coverage levels in identifying SNVs in thePPCD1 locus, the identified variants in two unaffected individualwere compared to those previously identified in the PPCD1 locuswith Sanger sequencing.Results: Eleven individuals were classified as affected, while 18individuals had an unremarkable ocular examination and wereclassified as unaffected. NGS of the captured PPCD1 locus wasperformed on genomic DNA from 4 affected and 4 unaffectedindividuals from this pedigree. Analysis of the sequencing datarevealed 39 coding region SNVs present only in the affected samples.Twenty-six of the 39 SNVs were predicted to produce synonymousamino acid substitutions, while the remaining 13 were predicted toproduce missense substitutions. Comparing variants that werepreviously identified using Sanger sequencing to NGS resultsdemonstrated a sensitivity of 93.6% for NGS (132/141) at ≥5xcoverage and 92.9% (131/141) at ≥10x coverage. The specificity ofNGS for identification of SNVs was found to be 100% at both ≥5x or≥10x coverage.Conclusions: NGS provides a cost-effective, rapid means ofresequencing a candidate gene interval to identify coding regionvariants. In comparison to Sanger sequencing, NGS provides highsensitivity and specificity, with the advantage of high-throughputanalysis.Commercial Relationships: Ricardo F. Frausto, None; JonathanHan, None; Anthony J. Aldave, Alcon (R), Allergan (R), NIH (F),Bausch + Lomb (C), Allergan (C)Support: RPB, Oppenheimer Eye Research GrantProgram Number: 4732 Poster Board Number: C0115Presentation Time: 11:00 AM - 12:45 PMExclusion of Pathogenic Promoter Region Variants andIdentification of Novel Nonsense Mutations in ZEB1 in PosteriorPolymorphous Corneal DystrophyPejman Bakhtiari, Ricardo F. Frausto, Ashley N. Roldan, CynthiaWang, Anthony J. Aldave. Jules Stein Eye Institue, Los Angeles, CA.Purpose: To report the identification of four novel nonsensemutations in the ZEB1 gene and exclusion of promoter region©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamutations in individuals without ZEB1 coding region mutations inposterior polymorphous corneal dystrophy (PPCD).Methods: Slit lamp examination and DNA collection was performedfor individuals diagnosed with PPCD, and when available, affectedand unaffected family members. Genomic DNA prepared fromperipheral blood leukocytes and buccal epithelial cells underwentPCR amplification and automated sequencing of the ZEB1 gene andpromoter region.Results: Ten unrelated individuals with PPCD were identified andgenomic DNA was collected from each. ZEB1 mutations wereidentified in 5 of the 10 probands, four of which were novel:p.Ala150fsX36 (spontaneous), p.Arg230fsX7, p.Cys638fsX5 andp.Gly1039fsX6. Screening of the ZEB1 promoter region in 28 PPCDprobands without a ZEB1 coding region mutation identified only twoknown SNPs whose frequency in the affected probands did not differsignificantly from that in the general population.Conclusions: We report four novel frame-shift mutations, oneconfirmed to be spontaneous, in the ZEB1 gene associated withPPCD, bringing the total number of pathogenic mutations to 23, andthe percentage of PPCD associated with ZEB1 mutations to 32%.The absence of ZEB1 promoter region mutations in probands withouta ZEB1 coding region mutation indicates that other genetic loci, suchas PPCD1, are responsible for the majority of cases of PPCD.Commercial Relationships: Pejman Bakhtiari, None; Ricardo F.Frausto, None; Ashley N. Roldan, None; Cynthia Wang, None;Anthony J. Aldave, Alcon (R), Allergan (R), NIH (F), Bausch +Lomb (C), Allergan (C)Program Number: 4733 Poster Board Number: C0116Presentation Time: 11:00 AM - 12:45 PMIdentification by whole-exome next-generation sequencing ofcoding region mutations as candidates for posterior amorphouscorneal dystrophyJonathan Han, Ricardo F. Frausto, Michelle J. Kim, Anthony J.Aldave. Doris Stein, Cornea Division, Jules Stein Eye Institute, LosAngeles, CA.Purpose: To identify the causative coding region variant of posterioramorphous corneal dystrophy (PACD) by whole-exome sequencingof the 12q21.33-q23.1 PACD locus.Methods: Slit-lamp examination was performed on 54 members of amultigenerational pedigree with PACD to determine their affectedstatus. Peripheral blood samples were collected from each familymember, and extracted genomic DNA from selected affected andunaffected individuals were used for whole-exome sequencing. Boththe alignment of the paired-end reads and the variant discovery wereconducted within the Partek Flow software. A list of candidatevariants comprising those identified within the PACD locus (boundedby the D12S1812 and D12S1051 genetic markers) was generatedusing the Partek Genomic Suite software. Twenty-eight variants werethen compared to previous Sanger sequencing results using aspreadsheet application to determine the number of false positivesand false negatives at two different coverage thresholds.Results: Fifteen individuals were diagnosed as affected based oncharacteristic clinical features. Whole-exome sequencing wasperformed on genomic DNA from 5 affected and 1 unaffectedindividuals. Analysis of the sequencing data revealed 12 singlenucleotide variants (SNV) within 9 genes that were present in each ofthe affected samples but not in the unaffected sample. Six of the 12SNVs were coding region variants. In addition, two indels wereidentified that were present only in the affected individuals: a 3’UTRinsertion and a coding region deletion. By comparing variantspreviously identified in the same 5 affected and 1 unaffectedindividuals using Sanger sequencing to those identified with wholeexomesequencing, 23/30 variants (23.3% false negative rate) wereidentified at a coverage threshold 10 and 27/30 variants (10.0% falsenegative rate) were identified at a coverage threshold of 5. No falsepositives occurred at either threshold.Conclusions: We report one of the initial applications of nextgenerationresequencing technology to resequence a candidate regionpreviously associated with a corneal dystrophy. The relatively lownumber of identified variants presents a reasonable cohort ofcandidate variants that will be confirmed by Sanger sequencing,followed by determination of segregation in the remaining 10affected and 38 unaffected individuals.Commercial Relationships: Jonathan Han, None; Ricardo F.Frausto, None; Michelle J. Kim, None; Anthony J. Aldave, Alcon(R), Allergan (R), NIH (F), Bausch + Lomb (C), Allergan (C)Support: RPBProgram Number: 4734 Poster Board Number: C0117Presentation Time: 11:00 AM - 12:45 PMLattice corneal dystrophy, type 1 (LCD1): an epithelial orstromal entity ?Walter Lisch 1 , Berthold Seitz 2 . 1 Ophthalmology, Johannes GutenbergUniversity Mainz, Mainz, Germany; 2 Ophthalmology, SaarlandUniversity, Homburg/Saar, Germany.Purpose: There are controversial reports that lattice cornealdystrophy, type 1 (LCD1) is of epithelial or stromal origin. The aimof this study is to evaluate this question.Methods: We observed ten eyes of five LCD1 patients afterpenetrating keratoplasty (PKP) on both eyes with a follow-up of 8-14years post-op. A slit-lamp photo-documentation of all patients indirect and indirect illumination was performed with dilated pupil.Results: All ten corneal transplants of the LCD1-patients showedsubepithelial diffuse opacities of different severity, beginning after 3-4 years postoperatively, that were often combined with cornealerosions and consecutive pain. In none of our patients, we were ableto disclose any signs of lattice formation in form of gray lines whichrun obliquely from the surface to the midstroma in directillumination. In retroillumination, no lattice opacity units in form oftranslucent and refractile lines were visible.Conclusions: The transforming growth factor beta-induced (TGFBI)gene, that is also responsible for LCD1, is expressed above all by thecorneal epithelial cells but also by the keratocytes. We interpret thesuperficial, diffuse LCD1 opacities on the graft as the product of theepithelial cells, whereas the non-occurrence of lattice lines as long as14 years postoperatively as an indirect sign that the lattice lines arethe product of the keratocytes. We know, that stromal cornealdystrophies such as macular corneal dystrophy may not recur beforedecades on the graft due to the very slow transformation of transplantkeratocytes into pathological host keratocytes. Thus, LCD1 seems torepresent an epithelial-stromal entity, because both, the epithelialcells and keratocytes are pathophysiologically involved.Commercial Relationships: Walter Lisch, None; Berthold Seitz,NoneProgram Number: 4735 Poster Board Number: C0118Presentation Time: 11:00 AM - 12:45 PMProteolytic processing in lattice corneal dystrophyEbbe Toftgaard Poulsen 1 , Kasper Runager 1 , Michael W. Risør 1 , IdaB. Thøgersen 1 , Thomas Dyrlund 1 , Line R. Thomsen 1 , Gordon K.Klintworth 2 , Jan J. Enghild 1 . 1 Department of Molecular Biology,Aarhus University, Aarhus C, Denmark; 2 Departments of Pathologyand Ophthalmology, Duke University, Durham, NC.Purpose: Transforming growth factor beta-induced protein(TGFBIp) is a major component of the human corneal proteome.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMore than 40 different mutations, in the TBFBIp gene lead to varioustypes of protein aggregates in the cornea including both amyloiddeposits as well as non-amyloid deposits. Here we investigate theprotein composition of two related lattice corneal dystrophy casesand investigate the role of the serine protease HtrA1 in thepathogenesis of this disease.Methods: The amyloid corneal deposits from the two lattice cornealdystrophy cases were isolated from paraffin embedded tissues bylaser capture microdissection. The collected material was treated withtrypsin and analyzed by tandem mass spectrometry. Data weresearched against the Swissprot database using the Mascot algorithmand processed using MS Data Miner software. Spectral counting ofTGFBIp peptides was performed to expose any viabilities in in vivoTGFBIp processing. Furthermore, different genotypes of recombinantexpressed TGFBIp was subjected to proteolysis by the serineprotease HtrA1 and analyzed with SDS-PAGE and MS.Results: Using Exponentially Modified Protein Abundance Index themost abundant proteins in the corneal deposits were compared tohealthy corneal tissue. The amyloid deposits revealed accumulationof serum amyloid p component, clusterin, apolipoproteins A-IV andE and HtrA1. Further, spectral counting of TGFBIp peptidessuggested an increased in vivo processing of TGFBIp associated withthe amyloid deposits compared to healthy tissue. In vitro HtrA1proteolysis of different TGFBIp genotypes showed preference foramyloid forming genotypes as seen in lattice corneal dystrophy.Conclusions: The newly obtained insight into the plaque proteomeassociated with corneal dystrophies has provided new knowledge thatmay help illuminate the mechanism leading to these diseases.Commercial Relationships: Ebbe Toftgaard Poulsen, None;Kasper Runager, None; Michael W. Risør, None; Ida B.Thøgersen, None; Thomas Dyrlund, None; Line R. Thomsen,None; Gordon K. Klintworth, None; Jan J. Enghild, NoneSupport: NH Grant (EY012712) and The Danish National ResearchFoundationProgram Number: 4736 Poster Board Number: C0119Presentation Time: 11:00 AM - 12:45 PMBenzalkonium chloride accelerates amyloid fibril formation incorneal dystrophies in vitroYuichi Kaji 1, 2 , Hisashi Yagi 2 , Yusuke Kato 2 , Yuji Goto 2 , TetsuroOshika 1 . 1 Ophthalmology, University of Tsukuba, Ibaraki, Japan;2 Laboratory of Protein Folding, Institute for Protein Research OsakaUniversity, Osaka, Japan.Purpose: Corneal dystrophies are genetic disorders resulting inprogressive corneal clouding due to amyloid fibril formation derivedfrom the transforming growth factor β-induced (TGFBI) gene.Amyloid fibril formation is influenced by the presence of solventsand surfactants such as sodium dodecyl sulfate (SDS). In the presentstudy, we aimed to reveal the role of benzalkonium chloride (BAC)and SDS in amyloid fibril formation of TGFBI-derived peptides invitro.Methods: Various concentrations of BAC or SDS were added tosolutions of synthetic peptides corresponding to wild-type, Avellinocorneal dystrophy, and lattice corneal dystrophy. The time course ofthe amount of formed amyloid fibrils in the solution wasquantitatively measured using thioflavin T. In addition, the seedingeffect of amyloid fibril formation was evaluated in the presence ofBAC and SDS.Results: For all synthetic peptides, BAC and SDS acceleratedamyloid fibril formation in both de novo and seeding models at 0.01to 0.5 mM and 0.1 to 1.5 mM, respectively. BAC accelerated amyloidfibril formation in the in vitro models of corneal dystrophies.Conclusions: The result indicates that most of the eye dropscontaining BAC may deteriorate corneal dystrophies. Eye drops thatdo not contain BAC would be preferred for patients with cornealdystrophiesCommercial Relationships: Yuichi Kaji, None; Hisashi Yagi,None; Yusuke Kato, None; Yuji Goto, None; Tetsuro Oshika,NoneSupport: This work was supported by the Ministry of Education,Science, Sports, and Culture, Grant for Scientific Research,24370067 (2012-2015), and 24592618 (2012-2015), Japan.Program Number: 4737 Poster Board Number: C0120Presentation Time: 11:00 AM - 12:45 PMExploring the mechanism underlying the protein aggregation instromal corneal dystrophies caused by amyloidogenic and nonamyloidogenicmutants of TGFBIpElavazhagan Murugan 1 , Rajamani Lakshminarayanan 1 , Roger W.Beuerman 1, 2 , Shyam S. Chaurasia 1 , Jodhbir S. Mehta 1, 2 . 1 Tissueengineering and stem cell research group, Singapore Eye ResearchInstitute, Singapore, Singapore; 2 Singapore National Eye Centre,Singapore National Eye Centre, Singapore, Singapore.Purpose: Corneal dystrophies (CD) are a group of inherited disorderscaused by the deposition of proteins in various layers of the cornea.Most of the CDs in the stromal layer of the cornea have beenattributed to the mutations in the transforming growth factor induced(TGFBI) gene with a high propensity in the 4thFAS1 (fasciclin-like)domain of the protein (TGFBIp). Though these mutants exhibitdistinct clinical phenotypes as as fibrillar, non-fibrillar and combinedforms, little or no information is available on their structural andfunctional differences. To understand the mechanism underlying thepathology, we chose representatives from an amyloid (H572R) and anon-amyloid (R555W) phenotype, expressed and purified the 4thFAS1 domains of these proteins and examined them alongwith thewild-type (WT) TGFBIp under various biophysical and biochemicalconditions.Methods: The 4th FAS1 domains of the mutants and the WTTGFBIp were expressed and purified. The ability of the mutants toform oligomers was examined at various conditions of pH andtemperature using circular dichroism spectroscopy. The structuralstabilities of the mutants were analyzed using Urea denaturationstudies. The morphologies of the oligomers formed by heating themutants were studied using electron microscopy.Results: While the WT TGFBIp did not show any conversion insecondary structure, the mutant proteins R555W and H572Rexhibited a clear pH dependent irreversible conversion to oligomersin acidic conditions when heated. While there is a clear conversion atpH 5.5, there is no conversion at pH 7.0. The oligomers formed at pH5.5 were also stable when they were resuspended in pH 7.0. Thestability of the WT and the mutants were tested using Ureadenaturation studies at various pH conditions. Examination usingelectron microscopy also showed that the oligomers of theamyloidogenic H572R were bigger in size with differentmorphologies compared to the non-amyloidogenic R555Woligomers.Conclusions: The mutants clearly show that there is a pH dependentconversion in their secondary structures which leads to their differentmodes of aggregation and hence their distinct pathologies. Theoligomers of the amyloidogenic and non-amyloidogenic mutantsshow differences in their morphologies.Commercial Relationships: Elavazhagan Murugan, None;Rajamani Lakshminarayanan, None; Roger W. Beuerman,Allergan (F), SERI (P), Santen (R); Shyam S. Chaurasia, None;Jodhbir S. Mehta, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 4738 Poster Board Number: C0121Presentation Time: 11:00 AM - 12:45 PMInsights into the molecular mechanisms of TGFBIp aggregationin corneal dystrophiesKasper Runager 1, 2 , Jarl Underhaug 1 , Charlotte S. Sørensen 1, 2 ,Henrik Karring 3 , Gordon K. Klintworth 4 , Niels C. Nielsen 1 , Jan J.Enghild 2, 1 . 1 Center for Insoluble Protein Structures, AarhusUniversity, Aarhus, Denmark; 2 Department of Molecular Biologyand Genetics, Aarhus University, Aarhus, Denmark; 3 Institute ofChemical Engineering, University of Southern Denmark, Odense M,Denmark; 4 Departments of Pathology and Ophthalmology, DukeUniversity Medical Center, Durham, NC.Purpose: Transforming growth factor-β induced protein (TGFBIp) isone of the most abundant proteins in the human cornea. With morethan 40 different disease-causing mutations it is an extremelyfascinating protein from a structural and biophysical point of view.The TGFBI-linked corneal dystrophies are characterized by anaccumulation of misfolded TGFBIp in the sub-epithelial and stromalregions of the cornea. These aggregates have been found to be ofeither amorphous (granular corneal dystrophies) or amyloid (latticecorneal dystrophies) phenotypes, suggesting that differentmechanisms of aggregation are involved. In order to obtain insightinto these mechanisms, we undertook a biophysical and structuralstudy of native TGFBIp as well as of a number of disease-causingmutants.Methods: Using transverse urea-gradient gel electrophoresis andcircular dichroism spectroscopy we determined the relative stabilitiesof WT and mutant TGFBIp and of the fourth FAS1 domain (FAS1-4)of TGFBIp. Limited proteolysis was used to identify the core regionof A546T FAS1-4 fibrils. Nuclear magnetic resonance spectroscopywas used to determine the three-dimensional structures of WT andmutant FAS1-4.Results: Our results demonstrate that mutations in the FAS1-4domain lead to significant alterations in the stability of nativeTGFBIp. Significantly, we find that this behavior is mirrored whenstudying the FAS1-4 domain in isolation suggesting that the FAS1-4domain can be used as a model for TGFBIp stability and aggregation.We observe a decreased susceptibility towards proteolyticdegradation in the R555W mutant compared to WT. A solutionstructure of WT and R555W FAS1-4 shows that this is likely due todecreased flexibility in the region around W555. We further showthat conditions promoting electrostatic interactions increase theaggregation propensity of the R555W mutant.For the amyloid fibril-forming mutant A546T we have identified aprotease resistant core (the fibril core), which is essential for thedense assembly of protein material characteristic for amyloid fibers.Conclusions: Our newly gained insight into the structures of mutantFAS1-4 gives us important clues to the mechanisms of aggregationresponsible for TGFBIp deposition in the cornea. In turn, we believethat this will prove valuable in rational design of compounds toinhibit TGFBIp aggregation in vivo.Commercial Relationships: Kasper Runager, None; JarlUnderhaug, None; Charlotte S. Sørensen, None; Henrik Karring,None; Gordon K. Klintworth, None; Niels C. Nielsen, None; Jan J.Enghild, NoneSupport: NIH grant R01 EY012712Program Number: 4739 Poster Board Number: C0122Presentation Time: 11:00 AM - 12:45 PMAn inducible transgenic mouse model of TGFBI-related cornealstromal dystrophyAndrew J. Huang 1 , Chia-Yang Liu 2 , Yujin Zhang 2 , Hongshan Liu 2 .1 Ophthalmology & Visual Sciences, Washington Univ School ofMed, St Louis, MO; 2 Ophthalmology, University of Cincinnati,Cincinnati, OH.Purpose: Mutations of transforming growth factor-beta inducibleprotein (TGFBI) have been associated with various corneal epithelialor stromal dystrophies. The pathogenic implications of mutantTGFBI for these dystrophies have been elusive, partially due to thelack of animal models specific for TGFBI-related corneal dystrophy.Herein, we report a doxycycline-inducible transgenic mouse model ofcorneal stromal dystrophy caused by R124H mutation of TGFBI,which is associated with granular corneal dystrophy II (GCD 2) orAvellino corneal dystrophy (ACD).Methods: Human TGFBI cDNA with R124H mutation was used tomake the transgenic mouse line (TRE-TGFBI R124H ). The functionalTRE-TGFBI R124H lines were crossed with K14rtTA to create bitransgenicmice, K14rtTA/TRE-TGFBI R124H (K14/TGFBI R124H ), inwhich TGFBI R124H was expressed in K14+ cells by feeding thepregnant mice with doxycycline-containing chow right after mating(E0). HRT-II in vivo microscopy was used to scan mouse corneas for3D reconstruction of the TGFBI R124H corneal deposits. RT-PCR andWestern blots were performed to analyze TGFBI R124H expression.Results: RT-PCR and Western blots confirmed the expression ofTGFBI R124H in the doxycycline-fed bi-transgenic mice. Cornealopacities were noticed in the K14/TGFBI R124H mice on postnatal day42 and not in the TRE-TGFBI R124H single transgenic littermates.Serial HRT-II scannings confirmed the presence of progressivestromal opacity as well as multiple protein aggregates in the corneaof K14/TGFBI R124H mice.Conclusions: We have generated an inducible transgenic mousemodel of TGFBI R124H -related corneal stromal dystrophy. Its clinicalphenotype is in resemblance of GCD 2. Such a mouse model mayprovide better insight to the pathogenesis of TGFBI-related cornealdystrophies.Commercial Relationships: Andrew J. Huang, None; Chia-YangLiu, None; Yujin Zhang, None; Hongshan Liu, NoneSupport: NIH Grant EY17609Program Number: 4740 Poster Board Number: C0123Presentation Time: 11:00 AM - 12:45 PMIdentification of TGFBI gene mutations in Polish patients withcorneal dystrophiesMonika Udziela 1 , Monika Oldak 2, 1 , Jacek P. Szaflik 1 , AnetaFederowicz 2 , Radoslaw B. Maksym 2 , Rafal Ploski 3 , Jerzy Szaflik 1 .1 Department of Ophthalmology, Medical University of Warsaw,Warsaw, Poland; 2 Department of Histology and Embryology,Medical University of Warsaw, Warsaw, Poland; 3 Department ofMedical Genetics, Medical University of Warsaw, Warsaw, Poland.Purpose: To report the clinical and molecular findings in Polishpatients with corneal dystrophies caused by TGFBI gene mutations.Methods: Patients with clinically diagnosed corneal dystrophies(n=26; 18 unrelated families) participated in the study. Cornealphenotypes were assessed by slit lamp and confocal microscopy invivo. Genomic DNA was obtained from blood samples and exons 4,11-14, known to contain mutation hot spots, were PCR amplified andsequenced on both strands.Results: Molecular genetic testing revealed a heterozygous R555W(exon 12) mutation in fifteen (9 families) patients diagnosed withgranular corneal dystrophy type I (GCD type I). In one patient aheterozygous R124H (exon 4) mutation, typical for granular-latticecorneal dystrophy (GCD type II) was found. In three patients (2families) clinically diagnosed of having Reis-Buecklers cornealdystrophy a heterozygous R124L mutation, commonly found inpatients with this type of corneal dystrophy, was identified (GCDtype III). In two patients (2 families) a heterozygous R555Q (exon©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea12) mutation typical for Thiel-Behnke corneal dystrophy wasdetected. Heterozygous R124C (exon 4), T538R (exon 12) andH626R (exon 14) mutations were identified, respectively, in threepatients (3 families) diagnosed with lattice corneal dystrophy (LCDtype I). In two patients from one family presenting a phenotype ofgranular-lattice corneal dystrophy the R124C mutation, typicallyidentified in patients with LCD type I, was found.Conclusions: In the analyzed group of Polish patients with cornealdystrophies TGFBI gene mutations are located exclusively in themajor mutational hotspots. Noteworthy, patients with GCD type I,representing the most predominant type of corneal dystrophy in theanalyzed group (9/18 families, 50%), were all carriers of the sameR555W mutation. The results of our study indicate that a relativelystraightforward molecular analysis can be a practical use in diagnosisof these conditions and associated genetic counseling.Commercial Relationships: Monika Udziela, None; MonikaOldak, None; Jacek P. Szaflik, None; Aneta Federowicz, None;Radoslaw B. Maksym, None; Rafal Ploski, None; Jerzy Szaflik,NoneProgram Number: 4741 Poster Board Number: C0124Presentation Time: 11:00 AM - 12:45 PMThe degree of exacerbated interfacial opacity after LASIK inGranular Corneal Dystrophy type 2 is related with the width ofarea of granular deposits made before procedureEung Kweon Kim, Tyler Hyung Taek Rim, Hong Seok Kim, Tae-imKim. Ophthalmology, Yonsei Univ College of Medicine, Seoul,Republic of Korea.Purpose: To find out the relationship between the degree ofexacerbated interfacial opacity after laser in situ keratomileusis(LASIK) and typical granular or linear deposit areas in GranularCorneal Dystrophy type 2 (GCD2).Methods: We retrospectively reviewed the records and slit-lampphotographs of 119 patients with GCD2 who underwent LASIK. Allpatients received LASIK once before and were diagnosed as beingheterozygous for GCD2 by DNA analysis from peripheral bloodlater. We evaluated the area of each type of lesions using inForm®(Perkin Elmer, Inc, Waltham, MA). We designed grades as 5 stepsbased on the density and width of the area of interfacial opacities. Weinvestigated the areas of granular and linear lesions as potentialfactors associated with grade.Results: Granular lesion was located in sub-Bowman’s layer level,and linear lesion was located in deep stromal level. These lesionswere considered as pre-opacified lesions before LASIK. The visualacuity gradually decreased as the grade of interfacial opacityincreased (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadetermined to play a key role in the formation of scar tissue.Connective tissue growth factor (CTGF) is a fibrogenic cytokine thatis a downstream mediator of many of the fibrotic actions of TGF- β.In this study, we determined the microRNAs (miRs) involved incorneal wound healing. We further examine the effect of miR-133bon the expression of CTGF, TGF-β, smooth muscle actin (SMA) andcollagen rabbit corneal fibroblast (RbCF).Methods: Laser ablated mouse corneas were collected at 0 and 2days post ablation. RNA was collected from the corneas and sampleswere analyzed using the mouse cell differentiation & developmentmiRNA PCR Array (QIAgen). The ability of miR-133b to target the3’ UTR of TGF-β and CTGF was tested using a luciferase assay inRbCF. PCR was used to determine the effect of miR-133b on CTGF,TGF-β, SMA and collagen in RbCF. Finally, a migration assay wasused to determine the effect of miR-133b on RbCF migration.Results: Two days after ablation, there were 37 out of 86 miRNAswith significant expression fold changes. Specifically, mir-133b hadthe greatest fold decrease at -14.33 and mir-22 had the greatest foldincrease of 7.16. The 3’UTR region of CTGF was targeted by premiR-133bas indicated by a significant decrease of 38% (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaTHE EFFECT OF LOCAL ROCK-INHIBITION ONCORNEAL SCARRING AFTER ALKALI BURN INJURYDavine Sijnave 1 , Karolien P. Hollanders 1 , Tine Van Bergen 1 , SarahVan de Velde 1 , Evelien Vandewalle 1 , Lieve K. Moons 2 , Dirk Leysen 3 ,Ingeborg Stalmans 1 . 1 Lab of Ophthalmology, KULeuven, Leuven,Belgium; 2 Department of Biology, KULeuven, Leuven, Belgium;3 Amakem Therapeutics, Diepenbeek, Belgium.Purpose: The aim of this study was to investigate the efficacy of alocal ROCK-inhibitor AMA0526 (Amakem NV) on corneal scarringinduced by alkali burn in an in vivo mouse model.Methods: Male Swiss mice (aged 6-8 weeks) were divided randomlyinto 3 groups after chemical cauterization of the cornea by alkali.Topical treatment was initiated after the injury and given once daily.The first group received 0.1% of the local ROCK-inhibitorAMA0526 versus Vehicle (experimental group 1), the second groupwas treated with bevacizumab (Avastin; anti-VEGF) versusAMA0526 (experimental group 2) and the third group was used ascontrol and received no treatment. Corneal opacity and cornealneovascularization were graded every other day according to a 0-4scoring. Blood vessel formation, collagen deposition and macrophagecorneal infiltration after injury were assessed and compared byimmunohistochemistry on day 7 and 14 after alkali burn.Results: Both corneal opacity and neovascularization were reducedafter AMA0526 treatment compared to the vehicle treated eye(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaperformed using qPCR.Results: Gene expression of SP and NK-1 R in human cornealfibroblasts was verified in the primary cultures. The presence of SPwas also confirmed at the protein level using immunocytochemistry.Western blot and immunocytochemistry showed that the full lengthisoform of NK-1 R is the dominant isoform. Anti-Fas treatmentinduced apoptosis in the cells, and SP augmented this response, asseen by an increase in activated apoptotic enzymes. Treating cellswith SP also lead to a decreased amount of phosphorylated/activatedAkt. The effects of SP were reduced with a specific NK-1 Rantagonist.Conclusions: SP is produced by human corneal fibroblasts. Anti-Fastreatment induces apoptosis in these cells and SP augments thisresponse through a NK-1 R specific pathway, by reducingphosphorylation of Akt. This suggests that SP may play a role in theapoptosis occurring in corneal wound healing.Commercial Relationships: Peter Boman, None; Sandrine LeRoux, None; Patrik Danielson, NoneSupport: Swedish Society of Medicine, Cronqvist Foundation, KMAFoundation, Ögonfonden FoundationProgram Number: 5228 Poster Board Number: C0147Presentation Time: 2:45 PM - 4:30 PMTGF-β1 modulates the functional expression of the NK-1receptor in human corneal fibroblastsSandrine LE ROUX, Peter Boman, Patrik Danielson. Anatomy, Dept.of Integrative Medical Biology, Anatomy, Umeå University, Umeå,Sweden.Purpose: Transforming growth factor beta 1 (TGF-β1) is a cytokineinvolved in a variety of processes, such as differentiation offibroblasts into myofibroblasts. TGF-β1 has also been shown to delaythe internalization of the Neurokinin-1 receptor (NK-1 R) after itsactivation by its ligand, the neuropeptide substance P (SP).NK-1 R comprises two naturally occurring variants, a full-length anda truncated form, triggering different cellular responses. SP has beenshown to be involved in important events in the cornea essential forthe proper organization of collagen fibers and the transparency of thestroma. An impaired signaling through NK-1 R could thus disturb theextracellular matrix and impact the vision quality.We hypothesize that TGF-β1 modulates the expression pattern andfunction of the NK-1 R in human corneal fibroblasts. The purpose ofthis study was to test that hypothesis.Methods: Cultures of primary corneal fibroblasts were set-up withcells derived from healthy corneas, obtained from donatedtransplantation graft leftovers. Immunocytochemistry for the TGF-βreceptor type I (TGF-βRI) and NK-1 R was performed. Geneexpression was assessed with qPCR. Western blot was used toconfirm the gene expression results at the protein level.Results: Expression of TGF-βRI was confirmed on corneal stromalderived fibroblasts. Treating the fibroblasts with TGF-β1significantly reduced the expression of the NK-1 R gene.Furthermore, immunocytochemistry for NK-1 R showed that the fulllengthversion of the receptor is reduced after treatment by TGF-β1(figure).Conclusions: TGF-β1 downregulates the gene expression of NK-1 Rin human corneal fibroblasts, and the results also suggest that itdisturbs the signaling pathways triggered by the receptor by reducingthe full-length version of it, which might explain the delay ininternalization after activation by SP seen with TGF-β1 treatment.Immunostaining of human corneal fibroblasts for the inner loop(green; c, f) and the C-terminal (red; b, e) of the NK-1 receptor.Merged pictures in a (of b and c) and d (of e and f), with DAPI addedfor demonstrating nuclei (bluish). The inner loop is found in both thefull-length and the truncated isoform, whereas the C-terminal is onlypresent in the full-length receptor. Untreated cells (a-c) showoverlapping immunostainings, whereas cells treated with TGF-β1 (df)show reduced staining for the full-length isoform.Commercial Relationships: Sandrine LE ROUX, None; PeterBoman, None; Patrik Danielson, NoneSupport: Swedish Society of Medicine; Cronqvist Foundation; KMAFoundation; Ögonfonden FoundationProgram Number: 5229 Poster Board Number: C0148Presentation Time: 2:45 PM - 4:30 PMA Long-Lasting Scar in Murine CorneaAndrew Hertsenberg, Sayan Basu, Yiqin Du, James L. Funderburgh.Ophthalmology, University of Pittsburgh School of Medicine,Pittsburgh, PA.Purpose: Penetrating keratoplasty is a successful therapy for cornealscarring but, due do graft failure and declining donor tissueavailability, new treatment methods for corneal scarring and haze areunder investigation. Animal models of long-term scarring areimportant tools to assess these new approaches. The purpose of thisstudy is to evaluate a penetrating murine corneal injury model forlong-term changes in stromal transparency and stromal connectivetissue that replicate those typical of human corneal scarring.Methods: A wound was created by surgical removal of a 1 mm fullthickness button from C57B6 mouse corneas. Corneas were imagedby optical coherence tomography (OCT) 48 hr after wounding andweekly for 2 months. Average pixel intensity for each OCT stack wasmeasured using ImageJ. After 2 months, eyes were enucleated andfixed. Corneal collagen was examined with single harmonicgenerated (SHG) images by 2-photon microscopy. Corneas weredigested in chondroitinase ABC and stained as whole mounts withantibodies to collagen III, biglycan, chondroitin-4-sulfate, anddecorin. Birefringence was analyzed by viewing the mounted corneasthrough a cross polarizing filters.Results: Two months after wounding, polarized light microscopyrevealed a regular, patterned collagen birefringence in unwoundedcorneas, but a markedly irregular pattern in wounded area. 2-photonimages confirmed the loss of collagen organization in the scar.Immunohistochemistry revealed localized concentrations of collagenIII and biglycan at the site of the wound. OCT images documentedcorneal thickening and a distinct increase in light scatter intensity inthe wound region persisting at least 2 months after wounding.Conclusions: Previous studies showed the transient appearance offibroblasts and myofibroblasts in penetrating murine corneal wounds,however, presence and persistence of non-transparent scar tissue has©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneanot been documented. This study demonstrated light scatter, haze,disruption in collagen organization, and accumulation of extracellularmatrix components typical of human corneal scars to persist in thecentral cornea for at least 2 months after wounding. The mouse is anideal animal model due to its low cost, genetic manipulability, andrelative homology to humans. As such, a mouse model that closelyresembles corneal scarring would expedite the search for analternative treatment to corneal haze in humans.Commercial Relationships: Andrew Hertsenberg, None; SayanBasu, None; Yiqin Du, None; James L. Funderburgh, NoneSupport: NIH Grants EY016415, P30-EY008098, Eye & EarFoundation of Pittsburgh, Research to Prevent BlindnessProgram Number: 5230 Poster Board Number: C0149Presentation Time: 2:45 PM - 4:30 PMAUTOLOGOUS PLATELET RICH PLASMA THERAPY INRESISTANT CORNEAL ULCERSYakup Aksoy 1 , Uzeyir Erdem 2 . 1 Ophthalmology, Gülhane MilitaryMedical Academy Haydarpasa Education Hospital, istanbul, Turkey;2 Ophthalmology, Gülhane Military Medical Faculty, Ankara, Turkey.Purpose: To assess the results and the application procedures of theplatelet rich plasma therapy which is a novel option in resistantcorneal ulcers?Methods: We performed limited debridement after harvestingadequate samples from the margin of the lesions for culture and HSVPCR in nine eyes of the eight corneal ulcer sterile patients who had ahistory of resistant corneal ulcer one or more year inconclusive trialsof various therapies. After the debridement all eyes administeredautologous platelet rich plasma (8 drops/day).Results: Corneal ulcers of all cases were healed in about 4,3(±2,3)weeks. Visual acuity improvement was 4,1(±2,4) lines. In one case,epithelial defect relapsed after six months but, healed in 3 weeksagain using the same protocol. we also applied wet PTK in 4 eyesafter the treatment period.Conclusions: We found that autologous platelet rich plasma therapyis effective in resistant corneal ulcers as an adjunctive therapy.Further investigations should be done in wider and different caseseries to prove its benefits in resistant corneal ulcers and also indifferent clinical applications.Commercial Relationships: Yakup Aksoy, None; Uzeyir Erdem,NoneProgram Number: 5231 Poster Board Number: C0150Presentation Time: 2:45 PM - 4:30 PMInitial Evidence that Immune Cell Infiltrates are CandidateSources of Post-Surface Ablation Stromal RougheningDaniel J. Gibson, Gregory S. Schultz. Obstetrics and Gynecology,University of Florida, Gainesville, FL.Purpose: To investigate potential cellular sources of previouslyobserved post-surgical roughening of the stroma in surface ablatedcorneas.Methods: New Zealand White rabbits were anesthetized andreceived bi-lateral 6.0mm x 125µm deep excimer phototherapeutickeratectomy wounds. Rabbits were euthanized daily to generate atime line of wound healing up until re-epithelialization. Immediatelypost-euthanization, the corneas were excised and fixed overnight. Thefixed tissues were paraffin embedded, sectioned, and stained withH&E. The histological time course was grossly analyzed by lightmicroscopy, with an emphasis on finding non-epithelial cells enteringthe wounded stromal surface.Results: Based on nuclear morphology, there were many neutrophilspresent in the anterior stroma with the highest concentration being inregions which were not yet re-epithelialized. Several of theneutrophils were observed in proximity with stromal fibroblasts.Several cellular masses were observed entering the wounded stromaahead of the migrating epithelium with nuclei shaped like neutrophilsor another class of granulocyte. The cells were either still round andon the surface or beginning to flatten and enter the stroma. In at leastone sample, a neutrophil was present in front of an epithelial frontwhich had entered the stroma.Conclusions: Neutrophils are in the right place and at the right timeto be considered as the source of post-surgical, biologically induced,stromal roughening. Hypothetically, the proteolytic activity of theseinfiltrating cells and the tissue displaced by them as they migrate intothe stroma creates “tunnels” which can subsequently be entered bythe migrating epithelium. Additionally, the crudely apparent cell-cellinteraction between the neutrophils and fibroblasts may have a role inactivation of the fibroblasts. Given that topical steroids have beenreported to be reduce haze and that they are potent neutralizers ofneutrophil activities, it may be through a combination of reducedstromal roughening and reduced fibroblast interactions. Additionalwork is needed to validate this proposed mechanism of action, but theobservations reported herein strongly suggest that the neutrophils areat least responsible for biologically induced roughening of the stromafollowing surface ablation surgery.Commercial Relationships: Daniel J. Gibson, None; Gregory S.Schultz, NoneSupport: NEI R01-EY05587Program Number: 5232 Poster Board Number: C0151Presentation Time: 2:45 PM - 4:30 PMOutcomes of Traumatic Dehiscence of Penetrating Keratoplastyversus Severe Open-Globe InjuryTherese Peron, Maria A. Woodward, Roni M. Shtein. Department ofOphthalmology, University of Michigan, Ann Arbor, MI.Purpose: To compare outcomes of patients with traumaticdehiscence of penetrating keratoplasty (PK) to those with severe openglobe (OG) injuries.Methods: A retrospective review of 160 consecutive patients withsurgical repair of traumatic open globe injuries at a single center from2006 to 2012 was conducted. Of those, 36 patients underwent repairof dehisced PK, 16 of which had adequate data to calculate theOcular Trauma Score (OTS). OTS was calculated for the remainingnon-PK open globes, resulting in 34 eyes with OTS of similarseverity. Ophthalmic and demographic factors were analyzed using t-tests, Pearson correlation coefficients, chi squared tests, and Fisher’sexact test.Results: There was a trend of patients with dehisced PK being olderthan the OTS-matched patients with OG injuries (60 vs.48; p=0.06).In the overall cohort, older age correlated weakly with lower OTS (r=-0.4, p=0.008). There was no correlation between sex and OTS(p=0.20). Associated lens injury at the time of initial trauma wasmore common in the PK group (p=0.03), but was not significantlyassociated with poor OTS (p=0.18). The extent of the ocularlaceration was not significantly different between the groups(p=0.22). Of the 40 patients with postoperative follow-up, 10required subsequent enucleation - 1 from the PK group and 9 fromthe OG group (p=0.12). Of the remaining 30 patients, 67% requiredsurgery beyond the initial repair. Additional surgeries were similarlylikely in both groups (p=1.00). Two patients developedendophthalmitis, 1 from each group. Patients with dehisced PKrequired subsequent cornea surgery more often than those with OGinjuries (p=0.0025), but had similar rates for subsequent retina,glaucoma, and lens surgeries (p=0.12, p=0.55, p=0.23, respectively).The mean final visual acuity of the PK group was 20/400 versus20/100 in the OG group, but this was not statistically significant©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea(p=0.13).Conclusions: In this study, patients with traumatic dehiscence of PKwere compared to those with OG injuries with similar Ocular TraumaScores. Both groups had poor visual outcomes, often requiredmultiple surgeries, and in some cases resulted in enucleation. Nosignificant differences in outcomes were detected between patientswith dehisced PK wounds versus those with severe OG injuries.Commercial Relationships: Therese Peron, None; Maria A.Woodward, None; Roni M. Shtein, NoneProgram Number: 5233 Poster Board Number: C0152Presentation Time: 2:45 PM - 4:30 PMProteomic analysis of the interaction Fusarium solani -Staphylococcus epidermidis isolated from human corneal ulcersMariana Ortiz-Casas 1 , Aida V. Rodríguez-Tovar 2 , Juan Pablo Reyes-Grajeda 3 , Herlinda Mejia-Lopez 1 , María A. Martínez-Rivera 2 , LuisA. Bautista-Hernández 1 , Carolina Gaona-Juárez 1 , Nadia Luz López-Espinosa 1 , Victor M. Bautista 1 . 1 Microbiology and Proteomics,Institute of Opthalmology "Conde de Valenciana", Mexico City,Mexico; 2 Medical Mycology, Escuela Nacional de CienciasBiológicas, IPN, Mexico City, Mexico; 3 Medical Proteomics Unit,Instituto Nacional de Medicina Genómica, Mexico City, Mexico.Purpose: To study the proteomic profile of co-culture of Fusariumsolani and Staphylococcus epidermidis in comparison with monocultures.Methods: Fusarium solani and Staphylococcus epidermidis wereisolated from human corneal ulcers. The microorganisms werecharacterized by microbiological and molecular technics. Monoculturesand co-cultures were established in Müller-Hinton Agar.Plates containing 72-h biomass were processed to obtain the cellularextract; proteins were quantified and purified before twodymensionalelectrophoresis (2D) was performed. The proteomicprofiles were analyzed by the software Dymension 2 to determine thedifferential expression of the co-culture compared with the monocultures.The spots that showed differential expression wereidentified by mass spectrometry using MALDI-TOF 4800 (ABsciex).Results: The fungi growth was lower in the co-cultures than inmonocultures (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFigure 1. Fibril alignment in rH collagen vitrigels (Bar=500 nm)cornea (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaBOOH did not improve with 0.1% AMX administration vs. controlunless the cells were pre-treated with AMX. In more chronic cornealstress, AMX pre-treatment may provide some benefit.uptake/clearance of apoptotic neutrophils.Conclusions: Estrogen orchestrates the inflammatory leukocyteresponse in the cornea, namely by regulating macrophagephagocytosis of apoptotic neutrophils. An essential function forhealthy inflammation is limiting the innate immune response, thedysregulation of which can lead to activation of the adaptive immuneresponse and subsequent chronic disease. This has potentialramifications in the cornea, where chronic inflammation can causeblindness or lead to autoimmune diseases.Commercial Relationships: Samantha B. Wang, None; Kyle M.Hu, None; Yuning Wang, None; David W. Lin, None; JonathanJong, None; Jeremy Lai, None; Karsten Gronert, NoneSupport: EY022208Photographic comparison of mechanical "scratch" healing of AMXtreatedHCE cells vs. controlCommercial Relationships: David V. Dudok, None; KevinCheung, None; Hong Liu, None; Luca Vedovelli, None; EmilianoGhinelli, None; Ken Kenyon, None; Sunil Parapuram, None;Cindy M. Hutnik, NoneSupport: Pilot Fund Grant - Department of Ophthalmology, WesternUniversity CanadaProgram Number: 5237 Poster Board Number: C0156Presentation Time: 2:45 PM - 4:30 PMSex-Specific Differences in the Corneal Inflammatory ReparativeResponse via Estrogen Modulation of PhagocytosisSamantha B. Wang, Kyle M. Hu, Yuning Wang, David W. Lin,Jonathan Jong, Jeremy Lai, Karsten Gronert. Vision ScienceProgram, University of California, Berkeley, Oakland, CA.Purpose: Clearance of apoptotic neutrophils from tissues bymacrophages is a crucial and necessary component of inflammatoryresolution. We previously demonstrated sex-specific differences inself-resolving corneal wound healing responses, which correlatedwith sex-specific differences in macrophage phenotypes. We alsoestablished that estrogen down-regulates intrinsic pro-resolving lipidmediators via ERβ. Pro-resolving lipid mediators such as lipoxin A4regulate macrophage phagocytic capacity. Hence, we investigated ifthere is a sex-specific difference in macrophage phagocytosis and ifthis essential housekeeping function is regulated by estrogen.Methods: Age-matched male and female mice underwent fullcorneal epithelial abrasion. Bone-marrow derived macrophages wereused for in vitro studies. Neutrophils were collected from theperitoneum following zymosan A injection and allowed to apoptosebefore introduction to macrophages. Macrophage phagocytic capacitywas measured using myeloperoxidase assay. Flow cytometry was runto determine macrophage phenotype.Results: Following epithelial abrasion, there was a sex-specificdifference in leukocyte dynamics (i.e. neutrophil recruitment andclearance) in the cornea. Males had higher levels of M2-typemacrophages in the healing corneas compared to their femalecounterparts; however, estrogen did not alter macrophagepolarization into either an M1 or M2 subtype in vitro. In contrastestrogen regulated macrophage function by inhibiting the proresolvingactions of lipoxin A4 (48% inhibition of phagocytosis).Estrogen also regulated both neutrophil apoptosis and macrophage476 Corneal Stroma and KeratocytesWednesday, May 08, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 5238-5258/C0157-C0177Organizing Section: CorneaProgram Number: 5238 Poster Board Number: C0157Presentation Time: 2:45 PM - 4:30 PMKeratocyte-Keratocyte Translamellar Connectivity in the MouseCornea is Revealed using a Novel 3-D Ultrastructural ApproachSamuel D. Hanlon 1 , Nancy C. Shenoi 1 , Paul T. Harris 1 , Paul T.Landry 1 , Ali R. Behzad 2 , Evelyn S. Brown 1 , Margaret M. Gondo 1 ,Alan R. Burns 1, 3 . 1 Research, Univ of Houston College of Optometry,Houston, TX; 2 Imaging and Characterization Core Lab, KingAbdullah University of Science and Technology, Thuwal, SaudiArabia; 3 Leukocyte Biology, Baylor College of Medicine, Houston,TX.Purpose: Like the human cornea, mouse stromal keratocytes liebetween the collagen lamellae and make extensive lateral connectionswith one another forming layers essentially parallel with the cornealsurface. This parallel intralamellar network arrangement is importantto the keratocytes as it allows for cell-cell communication via gapjunctions. The extent to which keratocytes form transverse (i.e.,translamellar) connections is unclear. The purpose of the presentstudy was to use a novel imaging strategy to reconstruct theultrastructural 3-D arrangement of the keratocyte network andspecifically evaluate the distribution of translamellar keratocytekeratocyteconnections.Methods: Corneas from C57BL/6 mice between the ages of 8-12weeks were fixed, heavy metal contrasted and embedded in resinblocks for transverse serial block-face sectioning using a Gatan3view microtome system mounted in an FEI Quanta FEG 200scanning electron microscope. Stacks of 375-700 serial Z images (at100 nm intervals; XYdimensions 28x28 um) were obtained from thestroma in the limbus, paralimbus, and central cornea. Amira 5.2software was used to segment keratocytes for 3-D imagereconstruction.Results: Segmented volumes revealed extensive intralamellar contactbetween keratocytes. Translamellar contact was rare in the centralcornea region with only one translamellar contact observed in a totalof five separate reconstructions. In the paralimbus and limbusregions, translamellar contacts were more common (2.7x10 5 ±1.3x10 5and 6.6x10 5 ±2.2x10 5 per mm 3 , respectively) suggesting there may beas many as 1.2x10 5 keratocyte-keratocyte translamellar contacts in asingle mouse cornea (est. 0.4mm 3 ).Conclusions: Collectively, the 3-D data obtained by serial block-facesectioning show for the first time that in the mouse, interlamellarkeratocyte layers oriented parallel to the corneal surface are indeed©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornealinked by translamellar keratocyte-keratocyte connections, but thisoccurs predominantly at the peripheral cornea. We propose a modelwhereby keratocytes form a single contiguous 3-D network, ratherthan a series of independent parallel networks, with a higher degreeof translamellar connectivity than previously appreciated.Commercial Relationships: Samuel D. Hanlon, None; Nancy C.Shenoi, None; Paul T. Harris, None; Paul T. Landry, None; Ali R.Behzad, None; Evelyn S. Brown, None; Margaret M. Gondo,None; Alan R. Burns, NoneSupport: NIH Grant EY017120, NIH Grant EY07551Program Number: 5239 Poster Board Number: C0158Presentation Time: 2:45 PM - 4:30 PMEffect of Serum Clot Activator on KeratocytesJi-Eun Lee 1 , Seung Uk Lee 2 , Jong Soo Lee 1 . 1 Ophthalmology, PusanNational University, Pusan, Republic of Korea; 2 Ophthalmology,Kosin University, Pusan, Republic of Korea.Purpose: To evaluate the effect of serum clot activator which couldbe used for making autologous serum eye drop on humankeratocytes.Methods: Cultured human corneal keratocytes were exposed to 10,20, and 30 % SiO2 for 24, 48, and 72 hours, MTT-based calorimetricassay was performed to determine the survival rate of keratocytes andlactate dehydrogenase (LDH) leakage assay to assess thecytotoxicity. Apoptotic response was evaluated with flow cytometricanalysis and fluorescence staining with Annexin V and propiodiumiodide. Cellular morphology was evaluated by inverted phasecontrastlight microscopy and electron microscopy.Results: The survival rate of human keratocytes and cytotoxicityshowed the concentration and time dependent response andsignificant response was found after treating with 30% SiO2 for 72hours. Apoptosis developed in flow cytometry and apoptotic cellswere demonstrated in fluorescent micrograph after treating with 30%SiO2 for 48 hours but cells were non viable after 72 hours. Humankeratocytes were more detached from the bottom of the dish anddamaged cells have degenerative changes like microvillidisappearance, vacuoles formation and chromatin of the nuclearremnant condensed along the nuclear periphery after treating with30% SiO2 for 72 hours.Conclusions: SiO2, the serum clot activator, showed the toxicity onhuman keratocytes at the concentration of 30% after 72 hourstreament. So blood should be extracted with tubes without clotactivator during making the autologous serum eye drop forpreventing the possible cytotoxicity on cornea.Commercial Relationships: Ji-Eun Lee, None; Seung Uk Lee,None; Jong Soo Lee, NoneProgram Number: 5240 Poster Board Number: C0159Presentation Time: 2:45 PM - 4:30 PMCo-cultures of Human Corneal Epithelium and Self-assembledKeratocyte and Fibroblast MatrixAudrey E. Hutcheon 1, 2 , Dimitrios Karamichos 1, 2 , Xiaoqing Q. Guo 1,2 , James D. Zieske 1, 2 . 1 Schepens Eye Research Institute/MEE,Boston, MA; 2 Department of Ophthalmology, Harvard MedicalSchool, Boston, MA.Purpose: During corneal wound repair the epithelium switches frominteracting with a mature matrix to a temporary matrix that ispartially assembled by wound-healing fibroblasts. The goal of thecurrent investigation was to develop and examine models that mimicthe interaction of epithelium interacting with a keratocyte-assembledmatrix versus a fibroblast-assembled matrix.Methods: Stromal cells were isolated from corneal explants byplacing tissue in either 1 or 10% FBS in DMEM. After culture andpassaging, the 1% (keratocyte-like) or 10% (fibroblast-like) cellswere plated in Transwell dishes with stabilized Vitamin C (VitC) ±0.1ng/ml of TGF-β3 and maintained in either 1 or 10% FBS inDMEM for 3 weeks. Immortalized human corneal-limbal epithelialcells (HCLE) were then placed atop the self-assembled matrix andcultured for 4 days; after which, the cultures were airlifted andmaintained for an additional 1 or 2 weeks. The co-cultures wereprocessed and examined by transmission electron microscopy (TEM)and indirect-immunofluorescence (IF) for morphology, extracellularmatrix components and fibrotic markers. Antibodies againstcollagens I, III, and V, thrombospondin-1 (TSP-1), cellularfibronectin (cFN), and smooth muscle actin (SMA) were examined.Results: Cells isolated with 1% serum but cultured without T3 didnot assemble a matrix. As observed by TEM, the morphology of thestromal cells isolated using 1% serum had a dendritic appearance,which is characteristic of keratocytes; whereas, the 10% serum cellswere spindle shaped, a fibroblastic characteristic. This distinctdifference in cell shape was also noted after IF with SMA, which wasfound to be present in both the keratocyte-like and fibroblast-likematrices. Collagens I, III, and V localization were similar in allcultures. Most interestingly, cFN and TSP-1 expression was greatlyreduced in the keratocyte-like co-cultures compared to the fibroblastlikeco-cultures.Conclusions: We have developed models that allow for thecomparison of the interaction of human corneal epithelial cells withkeratocyte-like and fibroblast-like self-assembled matrices. Thefibroblast-like matrix appears to stimulate wound-healing responses(as indicated by TSP-1 and cFN expression) to a greater extent thanthe keratocyte-like matrix.Commercial Relationships: Audrey E. Hutcheon, None; DimitriosKaramichos, None; Xiaoqing Q. Guo, None; James D. Zieske,NoneSupport: NIH Grants EY005665, EY020886, EY03790 (Core) andDOD W81XWH-11-1-0477Program Number: 5241 Poster Board Number: C0160Presentation Time: 2:45 PM - 4:30 PMRNAi Gene Silencing Of TGF-beta Signaling: A PowerfulApproach To Control Corneal FibrosisJason T. Rodier, Ajay Sharma, Ashish Tandon, Audra Stallard, RajivR. Mohan. Mason Eye Institute, University of Missouri-Columbia,Columbia, MO.Purpose: Transforming growth factor β (TGFβ) promotes keratocytetransdifferentiation to myofibroblast and cause corneal fibrosis invivo. Our siRNA studies demonstrated that TGFβ primarily usesSMAD signaling for this transformation in the cornea. The presentstudy tested the hypothesis that SMAD-2, SMAD-3 or SMAD-4 genesilencing is a novel approach to treat corneal scarring using an invitro model. We quantified the potency of SMAD gene silencing toabrogate TGFβ pathology and myofibroblast formation usingSMADs siRNA, and (2) examined whether RNA inference (RNAi) orshort-hairpin RNA (shRNA) is a better modality to achieve sustainedSMAD gene silencing in the cornea for gene therapy using an in vitromodel.Methods: : Human donor corneas were used to obtain human cornalefibroblasts (HCF). TGFβ1 (5ng/ml) was used to induce HCFtransformation myofibroblasts under serum-free conditions.Commercial pre-validated siRNA specific for SMADs were used.The shRNA and RNAi sequences specific for SMAD-2, SMAD-3and SMAD-4 were designed using RNAi software, and cloned intomammalian expression vectors to generated SMAD-shRNA/RNAivector constructs. Lipofectamine-2000 was used for transfection.Quantitative real-time PCR, western blotting and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaimmunohistochemistry measured the levels of profibrotic genes[alpha smooth muscle actin (SMA), f-actin, tenascin, collagens] inSMAD-transfected and un-transfected samples.Results: HCF exposed to TGFβ1 showed significant SMA+ cells(80-90%, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadisturbance. Corneal stromal fibroblasts are the main source ofCCL11/eotaxin-1. To know the role of alarmins from damagedcorneal epithelial cells in tissue eosinophilia, we have now examinedthe effects of the supernatant derived from necrotic corneal epithelialcell and Th2 cytokines on corneal fibroblasts.Methods: Necrosis of cultured human corneal epithelial cells wasinduced by repeated freezing and thawing. The amount ofchemokines released into culture medium by human cornealfibroblasts was determined by enzyme-linked immunosorbent assay,surface expression of adhesion molecules on the cultured cells wasmeasured with a whole-cell enzyme-linked immunosorbent assay,and the intracellular abundance of these molecules mRNA wasquantitated by reverse transcription and real-time polymerase chainreaction analysis. Signaling by the transcription factors NF-κB wasevaluated by immunoblot and immunofluorescence analyses.Results: The supernatant from necrotic corneal epithelial cellsinduced a low level of CCL11 release by corneal fibroblasts, as didTh2 cytokines, IL-4 and IL-13. However, the combination of thesupernatant from necrotic corneal epithelial cells and IL-4 or IL-13induced a marked synergistic increase in CCL11 release. Theabundance of CCL11 mRNA in the fibroblasts was also increased ina synergistic manner by costimulation with the supernatant fromnecrotic corneal epithelial cells and IL-4 or IL-13. In addition, thecombination of the supernatant from necrotic corneal epithelial cellsand IL-4 or IL-13 synergistically increased the expression of VCAM-1 in corneal fibroblasts. The supernatant from necrotic cornealepithelial cells activated NF-κB in corneal fibroblasts, and inhibitorsof NF-κB and IL-1 receptor antagonist attenuated CCL11 release andVCAM-1 expression induced by the supernatant from necroticcorneal epithelial cells in combination with IL-4 or IL-13.Conclusions: The interaction between IL-1 from necrotic cornealepithelial cells and Th2 cytokines may play an important role intissue eosinophilia associated with ocular allergic diseases.Commercial Relationships: Ken Fukuda, None; Hiroshi Tanaka,None; Waka Ishida, None; Yosuke Harada, None; Tamaki Sumi,None; Akira Matsuda, None; Nobuyuki Ebihara, None; AtsukiFukushima, Otsuka Pharmatheutical Co., Ltd (F)Program Number: 5245 Poster Board Number: C0164Presentation Time: 2:45 PM - 4:30 PMMitomycin C Suppresses Gene Splicing in Corneal FibroblastsTsan-Chi Chen 1 , Shu-Wen Chang 1, 2 , Chih-Chieh Lee 1 , Han-FangTeng 1 . 1 Department of Ophthalmology, Far Eastern MemorialHospital, New Taipei City, Taiwan; 2 Department of Ophthalmology,National Taiwan University Hospital, Taipei, Taiwan.Purpose: To investigate the molecular mechanism of how mitomycinC (MMC) modulates the gene expression via accumulation ofFibulin-1 (FBLN1) in nucleus of human corneal fibroblasts (HCFs ).Methods: HCFs were treated with MMC at 0.2 mg/ml for 5 minutes.FBLN1 and survival motor neuron 1 (SMN1) were cloned in thelentivirus-based expression vectors with green fluorescent protein(GFP) and red fluorescent protein (RFP), respectively, and expressedin HCFs by pseudovirus infection system. Distribution of FBLN1 andSMN1 was observed by confocal microscopy afterimmunofluorescent staining for their endogenous expression oroverexpression of GFP-tagged FBLN1 and RFP-tagged SMN1.Expression and association of FBLN1 and SMN1 in cytosol andnucleus was assayed by co-immunoprecipitation and immunoblottingafter nuclear extraction. Expression level of pre-mRNA and splicedmRNA was determined by real-time PCR.Results: MMC reduced cytosolic FBLN1 expression, but enhancednuclear FBLN1 expression in HCFs. The fluorescent images showedthat FBLN1 accumulated in nucleus of MMC-treated HCFs and colocalizeswith SMN1 (Figure 1). However, the coimmunoprecipitationresults revealed that FBLN1 might indirectlybind to SMN1. Silence of FBLN1 in MMC-treated HCFs resulted indecreasing both accumulation in nuclear FBLN1 and the pre-mRNAlevel of focal adhesion kinase (FAK), but increasing the splicedmRNA level of FAK.Conclusions: Our findings first showed that FBLN1 translocated intocell nucleus after MMC treatment. FBLN1 might play a novel role asa suppressor in SMN1-dependent pre-mRNA splicing.Figure 1. Colocalization of FBLN1 and SMN1 in nucleus ofMMC-treated HCFs. HCFs at 3 days post MMC treatment werecoimmunostained with DAPI (blue) for cell nucleus and the specificantibodies for FBLN1 (red) and SMN1 (green), respectively.Commercial Relationships: Tsan-Chi Chen, None; Shu-WenChang, None; Chih-Chieh Lee, None; Han-Fang Teng, NoneSupport: NSC101-2320-B-418-002Clinical Trial: FEMH-IRB-101002-FProgram Number: 5246 Poster Board Number: C0165Presentation Time: 2:45 PM - 4:30 PMThe anti-fibrotic Halofuginone regulates the expression of Egr-1in human corneal fibroblastsChing Yuan, Megan Twite, Elizabeth F. Nelson. Ophthalmology andVisual Neurosciences, University of Minnesota, Minneapolis, MN.Purpose: Transcriptional factor early growth response-1 (Egr-1)plays important roles in the pathogenesis of fibrosis. Unlikeregulation of other transcription factors, which rely on posttranslationalmodifications (i.e., phosphorylation), regulation of Egr-1signaling is mediated via its biosynthesis and by interaction withregulatory molecules, such as Nab2. We have previouslydemonstrated the anti-fibrotic efficacy of halofuginone, a compoundderived from herbal alkaloids, in cultured human corneal fibroblasts(Nelson et al., Molecular Vision 2012). In the current study, weidentified Egr-1 as the downstream effector of halofuginone.Methods: Human corneal fibroblasts were extracted from donorcorneas provided by Minnesota Lions Eye Bank. To delineate theprofibrotic roles of Egr-1 in the corneal fibroblasts, Egr-1 transgeneas well as an Egr-1-specific DNA enzyme (ED5) were transfectedinto cells to either constitutively overexpress or suppress Egr-1expression. Human corneal fibroblast cells were treated withhalofuginone and TGFβ-1 to investigate the expression profile ofEgr-1, Nab2, and other fibrosis-related genes using qRT-PCR,Western blot, and immunohistochemistry. The transactivation ofcollagen type I gene by Egr-1 after halofuginone treatment wasevaluated using (1) a reporter plasmid containing the luciferase genedriven by the collagen type I promoter, and (2) promotertranscriptionfactor pull down assay.Results: Our results showed that overexpression or knockdown of©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEgr-1 modulated the expression of fibrotic markers, including alphasmooth muscle actin, and fibronectin in human corneal fibroblasts.Egr-1 also bound directly to the collagen type I promoter andfacilitated the production of collagen. Halofuginone significantlyreduced the expression of Egr-1 in a dose- and time-dependentmanner according to the Western blot and qRT-PCR results.Immunostaining experiments revealed that the fast nucleartranslocation of Egr-1 induced by TGFβ-1 was also abolished withhalofuginone treatment.Conclusions: The current study confirms the profibrotic role of Egr-1 in human corneal fibroblasts. Egr-1 signaling is among thedivergent fibrotic pathways affected by halofuginone, and may serveas a promising target for anti-fibrotic applications in the cornea.Commercial Relationships: Ching Yuan, None; Megan Twite,None; Elizabeth F. Nelson, NoneSupport: NIH Grant EY019552; Research to Prevent Blindness;Minnesota Medical FoundationProgram Number: 5247 Poster Board Number: C0166Presentation Time: 2:45 PM - 4:30 PMEffect of TGF-β1 and 3 on protein expression in human cornealfibroblastsXiaoqing Q. Guo 1, 2 , Audrey E. Hutcheon 1, 2 , James D. Zieske 1, 2 .1 Schepens Eye Research Institute/MEE, Boston, MA; 2 Department ofOphthalmology, Harvard Medical School, Boston, MA.Purpose: One of the intriguing puzzles of TGF-β signaling is howTGF-β1 and 2 (T1 and T2, respectively) stimulate fibrosis, whileTGF-β3 (T3) appears to stimulate regeneration. Previously, we haveused RNA arrays to compare the effect of T1 and T3 on humancorneal fibroblasts (HCF). In the current experiments, we examinedprotein expression of genes determined by the arrays to be altered byT1 or T3.Methods: Human corneal fibroblasts cultured in 10% FBS in DMEMwere exposed to stabilized Vitamin C (VitC) with/without 2ng/ml T1or T3. HCF were harvested on day 3 and protein was extracted withRIPA buffer plus protease inhibitors. Equal amount of protein wasloaded onto 4-20% gradient gels. Blots were probed with antibodiesagainst collagen I, Smad 3, Smad 7, and thrombospondin-1 (TSP-1).Band intensities were quantified with ImageJ software (v.1.45s). Atleast four samples were analyzed per condition.Results: When compared to VitC only samples (control), both T1and T3 caused a significant increase in collagen I (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadetermined with various assays.Results: Treatment with TSA and the Nrf2-ARE activator resulted inincreased inhibition of the TGF-β-induced myofibroblastdifferentiation as compared with treatment with DPI or NAC.Furthermore, TSA also decreased cellular ROS and H2O2accumulation induced by TGF-β, whereas it elevated intracellularGSH level and cellular total antioxidant capacity. In addition, TSAinduced Nrf2 nuclear translocation and up-regulated the expression ofNrf2-ARE downstream antioxidant genes, whereas Nrf2 knockdownby RNA interference blocked the inhibition of TSA on myofibroblastdifferentiation.Conclusions: This study provides the first evidence implicating thatTSA inihibits TGF-β-induced ROS accumulation and myofibroblastdifferentiation via enhanced Nrf2-ARE signaling.Commercial Relationships: Qingjun Zhou, None; Lingling Yang,None; Mingli Qu, NoneSupport: National Natural Science Foundation of China 81170816Program Number: 5250 Poster Board Number: C0169Presentation Time: 2:45 PM - 4:30 PMExploring cell plasticity: the corneal keratocyte and beyondCarol A. Greene, Trevor Sherwin, Colin R. Green. Ophthalmology,University of Auckland, Auckland, New Zealand.Purpose: Corneal keratocytes have a remarkable ability to healwounds in the cornea throughout life and also exhibit a high level ofcell plasticity. However, their differentiative potential is not limitedto repair phenotypes. It has been shown in our laboratory that it ispossible to induce human and rat stromal keratocytes to expressneuron specific proteins by adding specific growth factors to theculture medium. The purpose of this study was to carry out ex vivoand in vivo experiments in an effort to uncover the extent of cornealkeratocyte plasticity. Furthermore, to explore and compare thepotential of other cell types to exhibit similar cell plasticity.Methods: Tissue and cell culture was used for in vitro experiments.Immunohistochemistry and RT PCR were used to investigate proteinand gene expression respectively. Confocal laser scanningmicroscopy was used for imaging cells and tissue slices.Results: Keratocytes obtained from adult humans and rats expressedNestin, NF-200, and MAP2 when cultured in a neuronaldifferentiation medium containing EGF, FGF and IGF-1. RT PCRrevealed up regulation of GAD1, SYN1, SOX2, SOX10 andNOTCH1. Subsequent in vivo studies also confirmed the expressionof Nestin, MASH1, and NF200. Adult rat xiphosternum derivedchondrocytes cultured in the same neurogenic media expressed NF-200, MAP2, GAP43 and β-III-tubulin. Adult human cornealkeratocytes when cultured for 3 weeks in a chondrogenicdifferentiation medium containing TGF-β3 and dexamethasoneformed spheres that produced Collagen Type II.Conclusions: Apart from producing neuron specific proteins such asNF-200 and MAP2, the genes SOX2, SOX10 and NOTCH1 whichare associated with neurogenesis were upregulated in corneal slicescultured in the neurogenic medium. Also, the expression of genessuch as GAD1 and SYN1 which are associated with neurotransmittersynthesis and synapse formation indicate that the neuronal cellsproduced might be functional. Similar to corneal stroma, cartilagefrom adult rats contained cells that acquire a neuronal phenotype andexpress neuron specific proteins. Corneal keratocytes also have thepotential to produce the cartilage specific protein Collagen II in cellculture and ex vivo slice culture when exposed to conditions whichfavour chondrogenic differentiation. These findings will have animpact on aspects of tissue regeneration research as well as ourcurrent understanding of adult cell plasticity.Commercial Relationships: Carol A. Greene, None; TrevorSherwin, None; Colin R. Green, NoneProgram Number: 5251 Poster Board Number: C0170Presentation Time: 2:45 PM - 4:30 PMReprogramming Genes Are Expressed during SpheroidalCulture of Corneal Stromal CellsYong-Soo Byun, Lisette Yco, Brittany Shaheen, Abhishek Sharma,Shweta Chaudhary, Sonal Gandhi, Sarmad H. Jassim, Joy Sarkar,Sapna Tibrewal, Sandeep Jain. Ophthalmology and Visual Sciences,University of Illinois at Chicago, Chicago, IL.Purpose: Corneal stromal cells transform to precursor cells inspheroidal culture. We determined whether reprogramming genes areexpressed during this transformation.Methods: Stromal cells were isolated from murine corneas bysequential collagenase digestion and adherent culture to generate apopulation of mixed stromal cell phenotypes. After trypsinization,spheroidal culture was performed by seeding dissociated stromal cellsonto ultra-low attachment plates containing serum-free mesenchymalstem cell culture medium. Spheroids were analyzed for expression ofreprogramming genes. Spheroids in culture were induced todifferentiate to keratocytes, fibroblasts, myofibroblasts, neural cells,adipocytes, and osteocytes using the appropriate medium.Results: Sphere formation occurred in ultra-low attachment platesbut not in adherent culture of stromal cells. Of the essentialreprogramming genes, Oct4 and Sox2 were upregulated in thespheroids, but not c-Myc or Klf4. Myc genes were differentiallyregulated; c-Myc was downregulated and N-Myc was upregulated.Forced differentiation of spheroids reverted them to keratocytes,fibroblasts, and myofibroblasts and transformed them to neural cellsbut not to adipocytes or osteocytes.Conclusions: In spheroidal culture, adherent corneal stromal cellstransform to a less differentiated precursor form, possibly due toupregulation of essential reprogramming genes. This finding suggeststhat terminally differentiated stromal cells possess inherent plasticityand multilineage potential that can be activated.Commercial Relationships: Yong-Soo Byun, None; Lisette Yco,None; Brittany Shaheen, None; Abhishek Sharma, None; ShwetaChaudhary, None; Sonal Gandhi, None; Sarmad H. Jassim, None;Joy Sarkar, None; Sapna Tibrewal, None; Sandeep Jain,PCT/US20/51562 (P)Support: NIH Grant EY018874Program Number: 5252 Poster Board Number: C0171Presentation Time: 2:45 PM - 4:30 PMTissue-engineered corneal stromal substitutes transplanted in theliving feline model: biocompatibility, ultrastructure andperformanceMarie Boulze Pankert 1, 2 , Benjamin Goyer 3 , Myriam Bareille 2 ,Kanwarpal Singh 2 , Stephanie Proulx 3, 5 , Isabelle Brunette 2, 4 .1 Departement d'ophtalmologie, Centre Hospitalier Universitaire del'Université de la Mediterranée, Marseille, France; 2 Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada; 3 CentreLOEX de l’Université Laval, Génie tissulaire et régénération - Centrede recherche FRQS du Centre Hospitalier Universitaire, Quebec, QC,Canada; 4 Department of Ophthalmology, University of Montreal,Montreal, QC, Canada; 5 Département d’ophtalmologie et d’otorhino-laryngologie,Faculté de médecine, Université Laval, Quebec,QC, Canada.Purpose: Tissue engineering of a corneal stroma is a promisingalternative to overcome the limitations of lamellar cornealreplacement with eye bank human corneas. We have demonstratedthe feasibility of engineering all three corneal layers. The aim of this©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneastudy was to evaluate the in vivo functionality, ultrastructure andbiocompatibility of allogeneic and xenogeneic tissue-engineered (TE)stromal grafts in the feline model.Methods: Human and feline keratocytes were isolated from normalcorneas and cultured to tissue engineer a 6 sheet-stromal substituteusing a self-assembly approach. Eight healthy animals underwent twointra-stromal grafts in one eye and the controlateral eye was used as acontrol. Animals were followed during 4 months after surgery, withslit lamp ophthalmic examination, intraocular pressure measurementand in vivo optical coherent tomography (Thorlabs®). Histology andtransmission electron microscopy (TEM) were performed on allcorneas at 4 months.Results: 16 grafts were performed. The average graft transparencyscore (0 (opaque) to 4 (crystal clear) scale) was 3.3±0.4 on Day 1 and3.9±0.2 on Day 37, which was similar to that of normal controls. Theminimal intraocular inflammation (cells and flare) observed in alleyes on Day 1 entirely resolved on Day 10. The mean graft thicknessdecreased with edema resorption (Day 3: 44.5±8 µm; Day 114:31.4±5 µm). Intraocular pressure remained unchanged (Preop:11.8±3.6; Postop: 11.7±3.0 mmHg). The grafts did not attract cornealvessels. Mean corneal endothelial cell counts remained stable (Preop:2506±77 cells/mm2; Postop: 2482±70 cells/mm2). Histology showednicely integrated grafts, with undisturbed host corneal epithelium,stroma, and endothelium. TEM confirmed the normal aspect of thekeratocytes, found in greater number in the grafts, and the absence ofinflammatory cells. Spacing between the collagen fibers of the TEstromawas 33.55±7.21 nm prior to transplantation, due to edema,and 25.90±4.68 nm 4 months after transplantation, with a regulararrangement similar to that of normal native stromas. All results werestatistically significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Manuel Ramirez, None; EverardoHernandez-Quintela, NoneProgram Number: 5255 Poster Board Number: C0174Presentation Time: 2:45 PM - 4:30 PMInterclass Synergistic Effects of Small Leucine-RichProteoglycans in Regulating Collagen Fibrillogenesis DuringCorneal DevelopmentShoujun Chen 1 , Shukti Chakravarti 2 , David E. Birk 1 . 1 Department ofMolecular Pharmacology and Physiology, University of SouthFlorida, Tampa, FL; 2 Department of Medicine, Johns HopkinsUniversity, Baltimore, MD.Purpose: Small leucine-rich proteoglycans (SLRPs) are critical forthe regulation of stromal fibrillogenesis and corneal transparency.Class I and II SLRPs are enriched in the corneal stroma. These twofamilies bind to different sites on collagen fibrils. The purpose of thisstudy is to investigate cooperative interclass interactions in theregulation of fibrillogenesis in corneal stromal assembly.Methods: Compound Lum -/- /Bgn -/- mice were generated by crossbreedingthe single null mice. All experiments conformed to theARVO statement for the Use of Animals in Ophthalmic and VisionResearch. Stromal fibrillogenesis in this mouse model was analyzedusing in vivo confocal microscopy, immunohistochemistry, electronmicroscopy and molecular/biochemical approaches.Results: The phenotype of compound null mice was compared withwild type mice, Lum -/- mice, and Bgn -/- mice. The wild type mice andBgn -/- mice were phenotypically normal. Lum -/- mice exhibited cloudycorneas, while the opacity in corneas of compound Lum -/- /Bgn -/- micewas significantly greater. Both anterior and posterior light scatteringwere significantly increased in compound Lum -/- /Bgn -/- micecompared to Lum -/- mice as demonstrated by in vivo confocalmicroscopy. Disorganized thinner lamellae were observed usingelectron microscopy in the compound null mice compared to Lum -/-mice. These disorganized lamellae contain disorganized collagenfibrils with increased numbers of irregular, large diameter fibrils.These irregular collagen fibrils are heterogeneous in contour. Thedisorganized collagen fibrils also were observed at P10 when bothLum -/- mice and compound null mice have similar diameter collagenfibrils. Immunoreactivities for lumican or biglycan were not detectedin the corneas of compound Lum -/- /Bgn -/- mice as expected. Theimmuno-relativities of collagens I and V as well as other SLRPsincluding keratocan, fibromodulin and decorin were comparablebetween Lum -/- mice and compound null mice.Conclusions: The data indicate biglycan and lumican havesynergistic effects in regulating collagen fibrillogenesis duringcorneal stromal assembly. They demonstrate cooperative modulationof both linear and lateral fibril growth during early development andprovide the foundation for the regular collagen fibril organization incorneal stroma associated with transparency.Commercial Relationships: Shoujun Chen, None; ShuktiChakravarti, None; David E. Birk, NoneSupport: EY05129 and AR44745Program Number: 5256 Poster Board Number: C0175Presentation Time: 2:45 PM - 4:30 PMCollagen XII regulates collagen fibril packing, lamellarorganization, stromal architecture and corneal functionChinda Hemmavanh 1 , Edgar M. Espana 1, 2 , David E. Birk 1 .1 Molecular Pharmacology and Physiology, USF Morsani College ofMedicine, Tampa, FL; 2 Ophthalmology, University of South Florida,Tampa, FL.Purpose: Collagen XII is a fibril associated collagen (FACIT) thathas been hypothesized to regulate stromal organization andextracellular matrix interactions. Regulated fibril assembly, packing,and organization into orthogonal lamellae are vital for cornealfunction. The purpose of this study is to elucidate the role(s) ofcollagen XII in the regulation of fibril assembly and organization aswell as stromal structure which is required for corneal function.Methods: The role of collagen XII in the cornea was studied usingwild type and Col12a1-/- mice. Collagen XII expression patternswere analyzed using immuno-chemical, biochemical and molecularapproaches. Lamellae and fibril organization were analyzed usingtransmission electron microscopy in P30 corneas (n=3). Cornealthickness was measured at P30 and P60 using optical coherencetomography (n=6).Results: Collagen XII was homogeneously localized throughout thecorneal stroma in postnatal and mature corneas (P4-P90). Expressionfrom P4-P30 was relatively constant during this period with a slightdecrease in collagen XII expression at P90. Stromal organization wasdisrupted in the absence of collagen XII. Lamellar organization wasdisrupted in the Col12a1-/- compared to wild type corneas. Individuallamellae were disorganized with an increased density of fibrils in theCol12a1-/- corneas compared to controls. Mature Col12a1-/- corneaswere significantly thicker than the wild type controls. At P30, theCol12a1-/- corneal thickness was 132.2 ± 9.2μm (mean ± sd)compared to 101.4 ± 3.8μm for the wild type corneas. The P60thickness values were 117.4 ± 9.5μm and 93.8 ± 4.9μm for Col12a1-/- and wild type corneas respectively.Conclusions: Collagen XII is present in the corneal stroma duringcritical stages of postnatal development. The disruption of lamellarstructure and organization in Col12a1-/- corneas supports animportant regulatory role for collagen XII in higher orderextracellular matrix assembly, i.e., the organization of fibrils intowell ordered orthogonal lamellae. The data also support a role forcollagen XII in fibril packing with decreased interfibrillar spacingobserved in the collagen XII-null corneas. In addition, the dataindicate that the regulation of lamellar assembly has a role indetermination of corneal thickness. Ultimately collagen XII regulateslamellar organization, stromal integrity, and corneal function.Commercial Relationships: Chinda Hemmavanh, None; EdgarM. Espana, None; David E. Birk, NoneSupport: NEI Grant EY05129Program Number: 5257 Poster Board Number: C0176Presentation Time: 2:45 PM - 4:30 PMExtracellular matrix characterization of the acellular gammairradiatedcorneaJemin J. Chae 1 , Joshep S. Choi 1 , Walter Stark 2 , Jennifer Elisseeff 1 .1 Biomedical Engineering, Johns Hopkins University, Baltimore, MD;2 Ophthalmology, Johns Hopkins University, Baltimore, MD.Purpose: To assess and investigate the extracellular matrix (ECM) ofthe human acellular gamma-irradiated cornea (AGC) with respect tostructural, physical, mechanical and biological properties.Methods: The properties of the AGC and the equivalent fresh humancornea (FHC) were evaluated. The structure of corneas wereobserved by transmission electron microscopy (TEM) and lightmicroscopy. The physical and mechanical properties of ECM wereevaluated by differential scanning calorimetry (DSC), compressivemodulus testing and light transmission testing. To assess thebiological properties, a cell migration test with human keratocytesand a DNA content analysis were performed.Results: The structural properties of the AGC were generally similarto those of the FHC but the density of collagen fibers in the AGC waslower than that of the HFC and some cell debris was observed. Thesefindings were also supported by light microscopy. The AGC showeda higher light transmission rate, which corresponds with clarity,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacompared to the FHC across the entire range of the visible lightspectrum even though there was no statistical significance. In thecompressive modulus test, very similar values of young’s moduluswere found between the AGC (25.1 ± 5.8 kPa) and the HFC (24.4 ±6.4 kPa). However, in the DSC analysis, the transitional temperature(Tm), which shows matrix organization, of the AGC wassignificantly lower (61.7 ± 1.1C) than that of the HFC (65.7 ± 1.8 C).The DNA content was significantly decreased in the AGC (1.05 ±0.22 µg / mg), when compared to the HFC (1.90 ± 0.11 µg /mg). Inthe cell migration test, more keratocytes were able to migrate throughthe AGC (317± 38.7/mm2) than through the HFC (176.4±48.2/mm2).Conclusions: In this study, we found the AGC is similar to the HFCwith respect to mechanical properties and transparency. In addition,the AGCs allow for more keratocyte migration and include less DNAcontent than HFCs, properties that are potentially beneficial incorneal substitutes. The gamma-irradiation reduces the density ofcollagen fibers and changes the thermal properties of melting.However, the effects of gamma irradiation are not great enough toalter the mechanical properties of corneal ECM. This promisingcorneal substitute could be used in various applications of ophthalmicpractice including corneal transplantation, which may not need viableendothelial cells, as well as in glaucoma patch grafting.Commercial Relationships: Jemin J. Chae, None; Joshep S. Choi,None; Walter Stark, VueCare (C); Jennifer Elisseeff, CollagenVitrigel (P)Support: Frederick N. Griffith Foundation for the Advancement ofTransplantationProgram Number: 5258 Poster Board Number: C0177Presentation Time: 2:45 PM - 4:30 PMStructural changes in the anterior corneal stroma of bullouskeratopathy patients after endothelial keratoplastyNaoyuki Morishige, Yukiko Morita, Naoyuki Yamada, Koh-heiSonoda. Dept of Ophthalmology, Yamaguchi Univ Grad Sch of Med,Ube, Japan.Purpose: We have previously described the presence of subepithelialfibrosis or transdifferentiation of keratocytes into fibroblasts ormyofibroblasts in the cornea of individuals with bullous keratopathy.We have now examined the anterior corneal stroma for structuralchanges in bullous keratopathy patients after endothelial keratoplasty.Methods: Twenty-one individuals who underwent unilateralendothelial keratoplasty (DSAEK) at Yamaguchi University Hospitalbetween November 2007 and July 2012 were enrolled to this clinicalstudy. The subjects were divided into two groups on the basis ofwhether the preoperative duration of stromal edema was less than 12months (group A: three men and seven women, with a mean age of74.6 years) or at least 12 months (group B: five men and six women,with a mean age of 77.9 years). The structure of the anterior stromawas examined with an in vivo laser confocal microscope (HeidelbergRetina Tomography II-Rostock Corneal Module, HeidelbergEngineering) at various times after surgery.Results: In vivo confocal microscopy revealed anterior stromalscattering (ASS), subepithelial fibrosis, or fibroblasticmyofibroblastictransdifferentiation of keratocytes in the studysubjects. Subepithelial fibrosis was detected in 3 of the 10 cases(30.0%) and 10 of the 11 cases (90.9%) in groups A and B,respectively, before surgery, in 2 (22.2%) and 8 (80.0%) cases,respectively, at 3 months, and in 1 (11.1%) and 5 (55.6%) cases,respectively, at 6 months. Fibroblastic-myofibroblastictransdifferentiation was detected in 1 (10.0%) and 8 (72.7%) cases ingroups A and B, respectively, before surgery, in 0 and 3 (30.0%)cases, respectively, at 3 months, and in 0 and 1 (11.1%) case,respectively, at 6 months. ASS was detected in 10 (100%) and 11(91.6%) cases in groups A and B, respectively, before surgery, in 3(33.3%) and 6 (60.0%) cases, respectively, at 3 months and in 1(11.1%) and 6 (66.7%) cases, respectively, at 6 months.Conclusions: Changes in anterior stromal structure similar to thoseapparent in bullous keratopathy patients before surgery were detectedby in vivo confocal microscopy in such patients after DSAEK.Subepithelial fibrosis and ASS persisted for more than 6 months in asubstantial proportion of individuals with a preoperative duration ofstromal edema of less than 12 months.Commercial Relationships: Naoyuki Morishige, None; YukikoMorita, None; Naoyuki Yamada, None; Koh-hei Sonoda, None477 Corneal Cross-linking and KeratoconusWednesday, May 08, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 5259-5305/C0178-C0224Organizing Section: CorneaProgram Number: 5259 Poster Board Number: C0178Presentation Time: 2:45 PM - 4:30 PMMDV1224 A NEW RIBOFLAVIN FORMULATION FORTRANS-EPITHELIAL CROSS-LINKINGSergio Mangiafico, Danilo Aleo. R&D, Medivis, Catania, Italy.Purpose: The most common procedure for corneal cross-linkingrequires the use of an aqueous solution of Riboflavin-5-phosphate(RFP) instilled in denuded cornea. The aim of our study was thedevelopment of an aqueous new ophthalmic formulation based on anew Riboflavin complex (MDV1224) able to ensure the cornealtrans-epithelial penetration of the active ingredient that would ensurea correct corneal cross-linking.Methods: Trans-corneal permeability of Riboflavin in MDV1224was evaluated by using the Franz diffusion cells (excised porcinecornea) at 37°C, for 360 minutes compared to RFP. The donorchamber contained the MDV1224 or RFP. The receiver chambercontained an isotonic phosphate buffer saline solution (pH 7.4).Samples were collected from the receiver chamber every 60 min andquantitatively analyzed by high performance liquid chromatography(HPLC). The permeation parameters (e.g.: the trans-cornealpermeability, cm/sec) were calculated by plotting the amounts(µg/cm2) of Riboflavin in MDV1224 or RFP permeated through theporcine corneal cells over time (minutes). Riboflavin stability in theMDV1224 was also monitored for six months in dark conditions asrequested by the ICH recommendation and compared to RFP.Results: Trans-corneal permeability of MDV1224 (45.7x10-6cm/sec)was almost tenfold greater compared to that of RFP (4.8x10-6cm/sec). After six months, stability data at 40°C and 25°C showedhigh degradation values for both Riboflavin in MDV1224 and RFPformulations, while at 2-8°C Riboflavin in MDV1224 and RFPshowed only 3% of degradation.Conclusions: The new Riboflavin complex in the MDV1224formulation was able to increase the porcine corneal penetration by852% vs the commercial RFP. In vivo studies are needed to confirmthat the higher delivery of MDV1224 Riboflavin vs RFP is able toguarantee a correct corneal cross-linking after instillation of theMDV1224 in the intact human cornea.Commercial Relationships: Sergio Mangiafico, Medivis (E);Danilo Aleo, Medivis (E)Program Number: 5260 Poster Board Number: C0179Presentation Time: 2:45 PM - 4:30 PMCorneal sensitivity changes following cross-linking forprogressive lower stage keratoconus©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaBelquiz A. Nassaralla 1 , Anelise M. Lago 1 , Larissa Rossana S. Stival 1 ,Joao J. Nassaralla 2 . 1 Cataract Cornea & Refractive Surgery, Institutode Olhos de Goiania, Goiania, Brazil; 2 Retina and Vitreous, Institutode Olhos de Goiania, Goiania, Brazil.Purpose: To evaluate corneal sensitivity changes following cornealcross-linking (CXL) in patients with progressive lower stagekeratoconus.Methods: Thirty eight eyes of 19 patients (11 women, 8 men) wereincluded in a prospective, nonrandomized clinical study. Meanpatient age was 22 years (range, 18-26 years). Inclusion criteria werelow stage bilateral progressive keratoconus, transparent cornea andthinnest corneal thickness ≥ 440 µm. Using the Cochet-Bonnetesthesiometer central corneal sensitivity was measuredpreoperatively, after 7 days, and once a month after surgery untilrecovery of the baseline preoperative level. Normal levels of centralcorneal sensitivity were considered above 40mm.Results: Corneal sensitivity gradually returned to preoperative levelsin all treated eyes. Mean central corneal sensitivity was 52.2, 24.0,38.2, 42.4, 50.0, and 52.4 mm before surgery, at 7 days, and at 1, 2, 3and 4 months after surgery, respectively. Normal levels of cornealsensation, but not baseline preoperative levels, were noted 2 monthsafter surgery. Preoperative levels were observed 3 months aftersurgery.Conclusions: Our results suggest that after CXL for progressivelower stage keratoconus, central corneal sensitivity is decreased foras long as 3 months.Commercial Relationships: Belquiz A. Nassaralla, None; AneliseM. Lago, None; Larissa Rossana S. Stival, None; Joao J.Nassaralla, NoneClinical Trial: NCT01743443Program Number: 5261 Poster Board Number: C0180Presentation Time: 2:45 PM - 4:30 PMImpact of corneal cross-linking on drug penetration in humansChristoph Tappeiner, Markus Tschopp, Kaspar Schuerch, Beatrice E.Frueh. Department of Ophthalmology, Inselspital, UniversityHospital of Bern, Bern, Switzerland.Purpose: To analyze the influence of corneal cross-linking (CXL)with ultraviolet-A and riboflavin on drug permeability using topicallyapplied pilocarpine.Methods: Keratoconus patients (n=10) undergoing standard CXLprocedure with ultraviolet-A (5.4 J/cm2, 30 minutes) and riboflavinon one eye were included in the study. Pupillary diameter measuredbefore and every 3 minutes for 30 minutes after the topicalapplication of one drop of pilocarpine was used as an indirectmeasure for corneal permeability. Pupillary diameter was measuredwith an infrared pupillometer device (Colvard, Oasis Glendora, CA)before (baseline) and 4 months after CXL.Results: Prior to pilocarpine application no significant difference inpupillary diameter was found before CXL and 4 months later(p>0.05). Mean decrease of pupillary diameter 30 minutes after theapplication of pilocarpine was significantly higher at baselinecompared to 4 months follow-up examination (mean decrease of 4.3vs. 3.5 mm; p=0.03).Conclusions: CXL reduces corneal permeability for pilocarpine inhumans.Commercial Relationships: Christoph Tappeiner, None; MarkusTschopp, None; Kaspar Schuerch, None; Beatrice E. Frueh, NoneProgram Number: 5262 Poster Board Number: C0181Presentation Time: 2:45 PM - 4:30 PMApparent progression in children after corneal cross-linking forkeratoconusBeatrice E. Frueh, Christoph Tappeiner. Ophthalmology, Univ ofBern Inselspital, Bern, Switzerland.Purpose: To analyze progression after corneal cross-linking (CXL)in children with keratoconus.Methods: Retrospective evaluation of tomographies (Pentacam) andtopographies (TMS) of prospectively collected data after CXL inchildren younger than 18 years. Examinations were conducted priorto surgery at regular intervals during the first postoperative year, andat 2, 3, and 4 years after the procedure. Twenty-five patients (33eyes) were included in the study. Mean follow-up was 27.3 months,and the minimum follow-up 1 year. Mean age at the time of surgerywas 14.9 years. Progression was defined as an increase in Kmax(Pentacam) of at least one diopter (D) in 1 year.Results: 669 KMax comparisons were made, resulting in theidentification of 4 cases of progression. In one case, the keratoconuswas extremely advanced prior to CXL (Kmax 78.2D before surgeryand 79.3D at 1 year). One case showed marked steepening of 3.4D inthe Pentacam between 3 and 4 years after CXL, but the TMSparameters were unchanged. Because of this discrepancy, thePentacam exam was repeated and showed that Kmax was actuallystable, i.e. no progression after all (50.8D at 3 years and 50.7D at 4years). Two children with active limbal vernal keratoconjunctivitisworsened dramatically (46.4D at 1 year and 48.3D at 2 years; 53.6Dat 1 year and 54.9D at 2 years). This progression was also seen intopography. After resolution of the limbal inflammation, the Kmaxvalues returned to 46.3D and 54.2D, respectively.Conclusions: Our results confirms that CXL is very effective instabilizing keratoconus in children. True progression after CXL couldonly be verified in 1 out of 33 eyes, but that eye had alreadyprogressed to such an extreme extent prior to CXL, that it wasprobaby unrealistic to expect that CXL could arrest progression atsuch a late stage. Further, in assessing possible progression, the useof 2 different measuring devices can help detect discrepancies andthus prevent false conclusions. Moreover, limbal vernal changes canpresent a clinical picture of progression. However, this is actually apseudo-progression, that can be reversed with anti-inflammatorytreatment.Commercial Relationships: Beatrice E. Frueh, None; ChristophTappeiner, NoneProgram Number: 5263 Poster Board Number: C0182©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 2:45 PM - 4:30 PMCorneal Endothelial Cell Density Following TransepithelialCollagen Cross-LinkingMichael W. Raciti 1 , Randy J. Epstein 1, 2 , Parag Majmudar 1, 2 , WilliamTrattler 3 . 1 Rush University Medical Center, Chicago, IL; 2 ChicagoCornea Consultants, Chicago, IL; 3 Center For Excellence In EyeCare, Miami, FL.Purpose: To evaluate the change in corneal endothelial cell densityafter transepithelial collagen cross-linking (CXL) for the treatment ofcorneal ectasia.Methods: Transepithelial CXL was performed using riboflavin 0.1%followed by UV-A light for 30 minutes in patients with keratoconus,pellucid degeneration, or LASIK-induced ectasia. Only eyes withcorneal thickness greater than or equal to 400µm were treated.Endothelial cell counts were measured pre-CXL and at least 6 monthspost-CXL using noncontact specular microscopy.Results: Seventy three eyes were evaluated. The mean cornealendothelial cell density was 2522 +/- 513 cells/mm2 pre-CXL and2674 +/- 452 cells/mm2 post-CXL (P=0.063).Conclusions: This study showed no significant change in cornealendothelial cell density after transepithelial CXL byriboflavin/ultraviolet-A treatment for corneal ectasia.Commercial Relationships: Michael W. Raciti, None; Randy J.Epstein, alcon (C), bausch and lomb (C), tear science (C); ParagMajmudar, None; William Trattler, CXLO (I), CXLUSA (C),CURVERIGHT (I)Program Number: 5264 Poster Board Number: C0183Presentation Time: 2:45 PM - 4:30 PMConcurrent vs Sequential Corneal Collagen Crosslinking andIntacs® for Keratoconus and Corneal EctasiaSteven A. Greenstein 1, 2 , Peter S. Hersh 2, 1 . 1 Ophthalmology, UMDNJ- New Jersey Medical School, Newark, NJ; 2 Cornea and Laser EyeInstitute - Hersh Vision Group, Teaneck, NJ.Purpose: To determine the effect of concurrent vs sequential cornealcollagen crosslinking (CXL) and Intacs® on visual and topographicoutcomes in patients with keratoconus (kc) and corneal ectasia.Methods: 41 eyes with KC and ectasia were analyzed in aprospective randomized control clinical trial. All patients wereinitially treated with symmetric 350µm Intacs® segments (AdditionTechnology Inc, Illinois, USA). Following this Intacs® procedurepatients were randomized into 2 groups. 1 group received standardCXL immediately following the Intacs® procedure (concurrentgroup, n=23), and the second group received the identical CXLtreatment 3 months after the initial Intacs® procedure (sequentialgroup, n=18). All outcomes were analyzed 1 year after CXL therapy.The outcomes analyzed included uncorrected (UCVA) and bestcorrected (BCVA) visual acuity, and maximum (Kmax), flat (Kf),steep (Ks), and average (Kavg) keratometry as measured by thePentacam (Oculus Inc, Wetzlar, Germany).Results: Visually, UCVA significantly improved from logMAR0.96±0.31 to 0.82±0.31 (p=0.03), and 1.03±0.19 to 0.85±0.31(p=0.01), and BCVA changed from 0.36±0.17 to 0.34±0.21, and0.29±0.23 to 0.21±0.15, in the concurrent and sequential grouprespectively; although these latter changes were not statisticallysignificant (pconcurrent=0.7, psequential=0.06). Topographically, inthe concurrent group, Kmax, Kavg, Kf, Ks, changed from 61.9±8.2Dto 61.0±7.8D (p=0.15), 51.5±5.2D to 50.1±5.0D (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: David Tabibian, None; OlivierRichoz, None; Eberhard Spoerl, None; Arthur Hammer, None;Florence Hoogewoud, None; Farhad Hafezi, Schwind (F), Ziemer(F), PCT/CH 2012/000090 (P)Program Number: 5266 Poster Board Number: C0185Presentation Time: 2:45 PM - 4:30 PMCorneal collagen crosslinking in young patients: One-year resultsMarcony R. Santhiago 2, 1 , Mario L. Monteiro 1 , Haroldo Moraes 1 ,Rodrigo Espindola 1 , Ramon C. Ghanem 1 , Marcelo Netto 1 .1 Ophthalmology, Federal University of Rio de Janeiro, Rio deJaneiro, Brazil; 2 Cataract and Refractive Surgery - Ophthalmology,Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.Purpose: To analyze one-year outcomes of epithelium-off cornealcollagen crosslinking (CXL) in young patients (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaobtained in the suspension containing riboflavin and 1% fluorescein.Conclusions: Fluorescein absorbs UV-A in a significant andconcentration-depending manner. The addition of fluorescein led to asignificant reduction in the killing-rate of S. aureus in vitro. Ourresults indicate that fluorescein competes riboflavin for theabsorption of UV-A during CXL, limiting the therapeutic effect. Inconsequence, preoperative staining of the ulcus or infiltrate at the slitlampusing fluorescein must be avoided prior to CXL, since it maysignificantly reduce the therapeutic effect of CXL.Commercial Relationships: Farhad Hafezi, Schwind (F), Ziemer(F), PCT/CH 2012/000090 (P); Zisis Gatzioufas, None; ArthurHammer, None; David Tabibian, None; Olivier Richoz, NoneProgram Number: 5269 Poster Board Number: C0188Presentation Time: 2:45 PM - 4:30 PMIn Vivo Ultrasound-enhanced Penetration Of Topical RiboflavinInto The Corneal StromaRicardo Lamy, Elliot Chan, Hui Zhang, Vasant Salgaonkar, Chris J.Diederich, Jay M. Stewart. Ophthalmology, University of California,San Francisco, San Francisco, CA.Purpose: To determine whether ultrasound treatment can promotethe permeation of topical riboflavin into the corneal stroma in vivo.Methods: New Zealand white rabbits were used for the study.Treated eyes had a cup with riboflavin 0.1% placed over the centralcornea and were treated with ultrasound 1 W/cm2 at 880 Khz for 6minutes followed by the removal of the cup and the application oftwo drops of riboflavin solution every 3 minutes for 39 minutes. Inthe control eyes (no ultrasound) two drops of riboflavin solution wereapplied every 3 minutes for 45 minutes. Animals were thensacrificed, corneas were excised, and confocal microscopy wasperformed to detect the presence of riboflavin in the cornea.Results: Fluorescence intensity was significantly higher in the treatedcorneas (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaBella, None; José Ignacio Fernández-Vigo, None; Jose M. Benitezdel-Castillo,NoneSupport: None in the Support field belowProgram Number: 5272 Poster Board Number: C0191Presentation Time: 2:45 PM - 4:30 PMCorneal Deformation Characteristics and IOP before and afterCollagen CrosslinkingCynthia J. Roberts, Ashraf M. Mahmoud, Richard G. Lembach,Thomas F. Mauger. Department of Ophthalmology and Departmentof Biomedical Engineering, The Ohio State University, Columbus,OH.Purpose: To evaluate in vivo corneal deformation characteristics andmeasures of intraocular pressure (IOP) before and after cornealcollagen crosslinking (CXL).Methods: 26 eyes of 26 subjects with keratoconus or post-refractivesurgery ectasia had CXL treatment (Tx), and had pre and 6 monthpost CXL data from Goldmann Applanation Tonometry (GAT),PASCAL Dynamic Contour Tonometry (DCT), Ocularl ResponseAnalyzer (ORA) for Goldman-correlated IOPg, corneal compensatedIOPcc, corneal hysteresis, CH and corneal resistance factor, CRF, aswell as CorVis ST, for inward (T1) and outward (T2) applanationtimes, pachymetry (pach), and inward (Vin) velocity. 16 fellow eyeswho had data from the same devices at the same times were controls(C). Custom software for ORA produced pressures at inward (P1)and outward (P2) applanations, applanation times (Time1,Time2),maximum pressure, (Pmax) and time, (Tpmax). Custom software forCorVis image edges produced radius of curvature at highestconcavity, (rad-curveHC) and time, (TimeHC). Data were comparedwith t-tests.Results: Significant differences between Tx and C groups werefound in IOPg (Tx: +1.2±2.2mmHg; C: -1.1±1.4mmHg; p=.0004),IOPcc (Tx: +1.4±3.5mmHg; C: -1.3±1.9mmHg; p=.0020), P1(Tx:+7.5±17.3mmHg; C: -5.8±15.3mmHg; p=.0155), P2(Tx:+10.8±20.7mmHg; C: -8.3±10.9mmHg; p=.0003), Pmax (Tx:+25.4±59.6mmHg; C: -8.6±24.2mmHg.0138; p=), Tpmax (Tx:+.24±.74ms; C: -.11±.29ms.0375; ), Time1(Tx: +.17±.36ms; C: -.13±1.9ms; p=.0085), Pach(Tx: -71±52µ; C: -21±44µ; p=.0029), andVin(Tx: -.005±.063ms; C: +.035±.039ms; p=.0301). In the ORA, thedifference in pre to post-CXL Time2 minus Time1 was significant inC (+.21±.32ms; p=.0161), but not in Tx. In the CorVis, the differencein pre to post-CXL T2-T1, was significant in Tx(-1.11±.99ms;p0.05). UCVA (-0.09 logmar units) and BCVA (-0.07 logmarunits) improved statistically (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaproduced the following behavior: 20% Dextran (B) and distilledwater (D) containing formulations led to approximately 60% and30% corneal thinning, respectively; HPMC containing solutions (A,E) resulted in less than 10% corneal thinning; hypotonic salinesolution resulted in 10% corneal swelling.Conclusions: Various applications of CXL require management ofcorneal hydration. The removal or disturbance of corneal epitheliumalters the fluid balance of the cornea, typically causing swelling.Composition and formulation of CXL solutions can predictablymanage corneal hydration.Commercial Relationships: Evan A. Sherr, Avedro, Inc. (E); PavelKamaev, Avedro (E); Sara Rood-Ojalvo, Avedro Inc. (E); Marc D.Friedman, Avedro Inc (E); David Muller, Avedro Inc (E)Program Number: 5275 Poster Board Number: C0194Presentation Time: 2:45 PM - 4:30 PMAssessment the long term effect of corneal cross-linking therapyon corneal ectasia in progressive keratoconusIlles Kovacs, Kinga Kranitz, Andrea Gyenes, Gábor László L.Sándor, Eva Juhasz, Lorant Dienes, Janos Nemeth, Zoltan Z. Nagy.Department of Ophthalmology, Semmelweis University, Budapest,Hungary.Purpose: To evaluate stabilizing effect of cross-linking (CXL)therapy on corneal ectasia in progressive keratoconus withScheimpflug imaging.Methods: In this prospective study corneal CXL was performed in22 progressive keratoconus eyes (CXL group) and 28 clinically nonprogressivekeratoconus eyes served as controls (control group).Anterior keratometry readings at the steepest meridian (Ks), thinnestcorneal thickness (ThCT) and posterior elevation values at thethinnest point of the cornea (PE) were measured using Scheimpflugcameraat baseline and 12-25 months after enrolment. Coxproportional regression model was used to evaluate long term effectof CXL on keratoconus progression taken into account the withinsubjectfluctuations of measurements.Results: At baseline, higher Ks (CXL: 49.87±3.62 D vs. control:46.76±3.25 D; p=0.002), higher PE (CXL: 27.05±17.13 µm vs.control: 14.36±12.54 µm; p=.006) and lower ThCT values (CXL:461.52±24.43 µm vs. control: 497.64±31.68 µm; p0.05) and only 2 of 22 cases showed progressionin keratometry values beyond within-subject fluctuation. In thecontrol group marginally significant increase was measured in meankeratometry (46.90 ± 3.33 D; p=0.06) and keratometry valuesincreased beyond within-subject fluctuation in 11 of 28 cases. TheCox model showed, that after CXL treatment the ratio of furtherkeratometry progression was 0.09 (95% CL: 0.02 - 0.57; p=0.01)compared to controls after adjustment for initial ThCT.Conclusions: CXL decreased the incidence of progression inadvanced keratoconus by 91% after a long term follow-upindependently from initial corneal pachymetry. Steep keratometry isa sensitive parameter to monitor stabilizing effect of corneal crosslinkingtherapy.Commercial Relationships: Illes Kovacs, None; Kinga Kranitz,None; Andrea Gyenes, None; Gábor László L. Sándor, None; EvaJuhasz, None; Lorant Dienes, None; Janos Nemeth, None; ZoltanZ. Nagy, Alcon-LenSx Inc. (C)Program Number: 5276 Poster Board Number: C0195Presentation Time: 2:45 PM - 4:30 PMUse of Zonal Km vs Point Kmax for Analysis of Corneal CrosslinkingPentacam TopographyGrace Lytle 1 , Marc D. Friedman 1 , Peter S. Hersh 2 , David Muller 1 .1 Avedro Inc, Waltham, MA; 2 Hersh Vision Group, Cornea andLASER EYE Institute, Teaneck, NJ.Purpose: To determine whether analysis of topography in a 2 or 3mm zone centered over Kmax provides a more sensitive measure ofchange in corneal curvature in eyes with keratoconus followingcorneal cross-linking than the use of a point Kmax value.Methods: The Pentacam software module (Version 6.03r19, OculusInc.) was used to retrospectively analyze a randomly selected subsetof 28 eyes with a diagnosis of keratoconus treated with corneal crosslinkingas part of a clinical trial in 2008 through 2009. Cross-linkingwas performed by removing the corneal epithelium and thenpretreating the cornea for 30 minutes with 0.1% RiboflavinOphthalmic Solution to saturate the corneal tissue with the riboflavinphotosensitizer. The cornea was then irradiated with UVA (365 nm)at 3 mW/cm 2 for 30 minutes for a total radiant exposure of 5.4 J/cm 2 .Pentacam scans were obtained pre-operatively, and at 1, 3, 6 and 12months following corneal cross-linking. Kmax values and theircorresponding coordinates were identified by the software and wererecorded for each eye at each time point. The Pentacam softwaremodule was used to manually define a 3mm zone centered over thecoordinates of the Kmax value in each eye at each time point onsagittal curvature maps. This procedure was repeated for a 2mm zonecentered over the same coordinates. A number of software generatedparameters from this analysis were recorded, including the mean(Km) of the values contained within this zone. A statistical analysisof these values was conducted.Results: Analysis of the 3mm zonal Km using a two tailed t-testdemonstrated statistically significant improvement in sagittalcurvature from baseline at 3 months (P< 0.15, 85% confidenceinterval). Improvement in Kmax and 2mm zonal Km were notsignificant at this time point, (P=0.635 and P=0.229). All threemeasurements were statistically significant at 12 months (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaKinga Kranitz, Illes Kovacs, Andrea Gyenes, Gábor László L.Sándor, Eva Juhasz, Lorant Dienes, Janos Nemeth, Zoltan Z. Nagy.Department of Ophthalmology, Semmelweis University, Budapest,Hungary.Purpose: To evaluate characteristics of corneal flattening after crosslinking(CXL) therapy in progressive keratoconus with Scheimpflugimaging.Methods: In this prospective study corneal CXL was performed in23 progressive keratoconus eyes (CXL group) and 15 nonprogressivekeratoconus eyes served as controls (control group).Posterior elevation values (PE) were measured at the thinnest point ofthe cornea, at the corneal vertex, 2 and 3 mm paracentrally (superior,inferior, temporal and nasal from the corneal vertex) by fixed spherereference surface with Scheimpflug-camera (Pentacam: OculusOptikgeräte GmbH) before and 1 year after CXL.Student t-test on paired samples, and multivariable regressionanalysis using general estimating equation model were applied forstatistical comparisons of corneal changes after CXL therapy.Results: Thinnest corneal thickness and PE values measured at thethinnest point of the cornea decreased significantly in the CXL group(from 457.78±23.81µm to 432.52±30.29 μm, P.05) in the CXL group. All PE values remained stable in controleyes (P>.05).Change in PE values significantly correlated with initial ThCT in theCXL group (r=0.48 P=0.02).General estimating equation model also showed that CXL treatmentsignificantly decreased PE only at the thinnest point of the cornea(with a mean 6.2 µm, P0.05 for all variables). Forstage II, the mean pre-CXL UDVA was 1.20±0.64 logMar, whichwas changed to 0.72±0.33 logMar after CXL (p=0.03). The meanpre-CXL steepest meridian K and apical K were 53.26±2.33 D and61.97±5.64 D respectively. They were significantly decreased to52.912±.20 D and 60.804±.69 D respectively (p=0.03, p=0.013,respectively). The mean pre-CXL and post-CXL CDVA, flattestmeridian K, and average K did not show any significant difference(p>0.05 for all variables). For stage III, the mean pre-CXL flattestmeridian K, steepest meridian K, average K and apical K weresignificantly decreased from 53.21±1.16 D, 58.40±2.66 D,55.661±.62 D, 66.05±3.81 D to 52.48±1.30 D, 57.262±.75 D,54.851±.78 D, 65.15±3.35 D after CXL respectively (p=0.001,p=0.001, p=0.001, p=0.001). There were no significant changebetween the pre and post-CXL UDVA and CDVA (p>0.05 for bothvariables).Conclusions: One year postoperatively, the effects of CXL on visionwas more significant in early and moderate stages of keratoconus,while the topographic improvement was more prominent in advancedstage.Commercial Relationships: Nurullah Cagil, None; Ozge Sarac,None; Emine Akcay, None; Nagihan Ugurlu, None; Erol Can,None; Gamze Can, NoneProgram Number: 5279 Poster Board Number: C0198Presentation Time: 2:45 PM - 4:30 PMShorter UVA Light Exposure Protocol for Crosslinking inKeratoconus in Children Without Progression CriteriaAndres Codriansky 1, 2 , Maria Carolina Aravena 1, 2 , Marcelo Coria 1, 2 .1 Cornea and Refractive Surgery, Hospital Sotero del Rio / IOPA,Santiago, Chile; 2 Cataract Surgery, Hospital Sotero del Rio, Santiago,Chile.Purpose: To report the safety and efficacy of a shorter UVA lightexposure crosslinking treatment Protocol in children withkeratoconus without progression criteria.Methods: Case series of patients with keratoconus who wereassumed to be progressive based on age of presentation.Patients were treated with crosslinking procedure as follows: 7mmcentral epithelium mechanical debridement, instillation of isotonicriboflavin 0,1% with dextran 20% solution every 3 minutes for 30minutes, verification of riboflavin in the anterior chamber wasperformed with a slit lamp microscope, then exposure to 3mw/cm2UVA Light for 20 minutes while instilling riboflavin drops every 3minutes. Patients were patched and seen the day after, at day 3, 7, 30,3- 6 months, at one year and then accordingly to the physiciancriteria. Clinical charts were review in a retrospective manner.Results: 10 eyes of 7 patients, 1 woman, 6 men, mean age 15,8 years,(range12 to 18), mean follow up 18,9 months (range: 9 to 33months).No adverse events nor drop in best corrected visual acuity werereported.Topography remained stable, with a mean flattening of the steepestKeratometry of 0,12 D and a mean steepening of the mean K of 0,2D.There were 3 cases of treatment failure, defined as steepening on themean Keratometry of 1D or more during follow up.Conclusions: Shorter UVA light exposure protocol seems to be safein Children who were assumed to be progressing. However, it mightnot be as effective as the standard 30 minute protocol, since we had a30% failure rate in this group of patients.A randomize, prospective, clinical trial with more patients isnecessary to draw more definitive conclusions.Commercial Relationships: Andres Codriansky, None; MariaCarolina Aravena, None; Marcelo Coria, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 5280 Poster Board Number: C0199Presentation Time: 2:45 PM - 4:30 PMPapain Digestion Method for Analysis of Cross-linking inCorneal FlapsSara Rood-Ojalvo, Pavel Kamaev, Marc D. Friedman, David Muller.Avedro Inc., Waltham, MA.Purpose: To evaluate the use of papain digestion andspectrofluorometer analysis as a means to quantify the amount ofcross-linking in porcine corneal flaps that have undergone variousUVA-RF based corneal cross-linking protocols.Methods: Fresh whole porcine eyes were obtained

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapropanediol (HMPD), 2-bromo-2-nitro-1,3-propanediol(BP=bronopol), genipin (GP), glyceraldehyde (GC),paraformaldehyde (PA-polymeric), and glutaraldehyde (GApolymeric).The cell types were primary bovine corneal endothelium,human skin fibroblasts (from ATCC), immortalized human cornealepithelium (HCEC - gift from Dr. Peter Reinach), and immortalizedhuman retinal pigment epithelial cells (ARPE-19). The cells weregrown in planar culture to confluence and exposed to each agent(0.001mM to 10mM) for 24 hours followed by a 48 hour recoveryphase. Toxicity thresholds were then determined using the trypanblue exclusion method.Results: The results are summarized in Table 1. Overall, the toxicitylevels varied between cross-linking agents by a factor of 100x withthe least toxic being NE, glyceraldehyde, and NP. MNPD, HNPD,polymeric PA, and polymeric GA showed intermediate toxicity. Themost toxic agents were GP and BP. Toxicity levels were generally inagreement between cell types although in some cases there was asmuch as a 10x difference for a given compound when comparingbetween cell types.Conclusions: Regarding cell toxicity, there are significantdifferences between topical cross-linking compounds. In addition, fora given compound, toxicity can vary by cell type. Such differencesshould be considered when selecting agents to use for topicaltherapeutic corneoscleral tissue cross-linking.thickness was less than 400 microns hypotonic riboflavin wasadministered until the cornea swelled beyond 400 microns. Bothstudy groups then received 30 minutes exposure to UV light (365micron wavelength, irradiance 3mW/cm2). During UV exposure,eyes received continued riboflavin application, the formulation ofwhich was assigned by 2 study arms: 35 patients receivedriboflavin/dextran and 28 patients received hypotonic riboflavinevery 2 minutes for the duration of UV exposure. Pachymetry wasmeasured by ultrasound before the corneal epithelium was removed,after initial riboflavin loading prior to UV light exposure, and afterUV light exposure. At least 5 pachymetry measurements were takenat each time point and the lowest used for analysis. Corneal thicknessmeasurements were then analyzed between both arms of the study.Results: The average initial pachymetry, pachymetry after 30minutes riboflavin loading, and final pachymetry after UV light was436.83, 429.89 and 301.80, respectively, in the riboflavin/dextrangroup, compared to 443.07, 432.29 and 341.06, respectively, in thehypotonic group. The difference in corneal thickness between the twogroups was statistically significant when taken after UVadministration. The p values between both study arms comparing theinitial pachymetry, pachymetry after loading, and final pachymetryafter UV light was 0.67, 0.77 and 0.0002, respectively.Conclusions: The cornea thins substantially during the collagencrosslinking procedure. The use of hypotonic riboflavin rather thanriboflavin/dextran during UV administration decreases the amount ofcorneal thinning during the procedure 24%, from thinning of 135microns to 102 microns. This difference in corneal thinning may haveboth safety and efficacy implications for the CXL procedure.Commercial Relationships: Elan A. Rosenblat, None; AlexanderL. Sachs, None; Steven A. Greenstein, None; Peter S. Hersh,Avedro (C), Addition Technology (F)Support: Research to Prevent BlindnessClinical Trial: NCT01152541NE= 2-nitroethanol GC= glyceraldehyde NP= 2-nitro-1-propanolMNPD= nitrodiol HNPD= nitrotriol PA= paraformaldehyde GA=glutaraldehyde GP= genipin BP= bronopolCommercial Relationships: Mi Jung Kim, None; Quan Wen,None; Quan V. Hoang, None; Steve L. Trokel, None; David C.Paik, 12/517,382 and PCT/US2007/025126 (P)Support: NIH Grant EY020495Clinical Trial: noProgram Number: 5283 Poster Board Number: C0202Presentation Time: 2:45 PM - 4:30 PMCorneal Thickness Changes during Collagen Crosslinking usingRiboflavin/Dextran or Hypotonic RiboflavinElan A. Rosenblat 1, 2 , Alexander L. Sachs 2 , Steven A. Greenstein 1, 2 ,Peter S. Hersh 2, 1 . 1 Department of Ophthalmology, Inst ofOphthalmology and Visual Science, Newark, NJ; 2 Cornea and LaserEye Inst- Hersh Vision Group, Teaneck, NJ.Purpose: To compare the central corneal thickness between the useof riboflavin 0.1% in 20% dextran solution versus riboflavin 0.1% inwater (hypotonic) during ultraviolet light exposure in the collagencrosslinking procedure for keratoconus and corneal ectasia.Methods: 63 eyes with keratoconus and corneal ectasia wereanalyzed in a prospective randomized control clinical trial. Both armsof the study received pretreatment with riboflavin 0.1% in 20%dextran solution; one drop was administered every 2 minutes for 30Program Number: 5284 Poster Board Number: C0203Presentation Time: 2:45 PM - 4:30 PMAliphatic β-nitroalcohols for therapeutic corneoscleral tissuecross-linking: catalytic studiesDavid C. Paik, Quan Wen, Mi Jung Kim, Quan V. Hoang, Stephen L.Trokel. Ophthalmology, Columbia University, New York, NY.Purpose: As riboflavin photochemical corneal cross-linkingcontinues to gain a foothold in clinical Ophthalmology, developmentof alternative methods that neither require ultraviolet irradiation norpainful de-epithelialization continue to be an attractive goal. β-nitroalcohols (BNAs) have favorable safety profiles and function asformaldehyde donors via a reverse Henry reaction (rHr) to cross-linkcollagenous tissues. Because the reaction is reversible, catalyticmechanisms for the forward reaction were tested for tissue crosslinkingvia the reverse reaction.Methods: Using bovine sclera as a collagenous tissue substrate(8x5mm tissue pieces), 60 min incubations (37oC) of the higher ordernitroalcohols (1-5mM) 2-methyl-2-nitro-1,3-propanediol (MNPD), 2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD), and 2-bromo-2-nitro-1,3-propanediol (bronopol=BP) [a compound widely used incosmetics, etc.] were carried out with a variety of potential catalysts.Both NaH2PO4/Na2HPO4 and HCO3 buffers (pH 6.5-9.5) weretested. In addition, three enzymes (D-aminoacylase,transglutaminase, hydroxynitrile lyase from Arabidopsis thaliana)and salmon testes DNA were tested at pH 7.4. Cross-linking effectswere evaluated using thermal shrinkage temperature (Ts) analysis aspreviously described.Results: Ts values for uncross-linked control tissues were aspreviously reported (65.3oC). The BNA cross-linking reactionminutes after the corneal epithelium was removed. If the corneal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadisplays a strong pH dependency, with enhanced tissue cross-linkingeffects occurring as the pH is raised from 6.5 to 9.5. As compared tocontrol tissue, the Ts using HNPD was shifted 1.5oC at pH 8.3, 3oCat pH 8.4, 3.8oC at pH 8.5, and 5.4oC at pH 9.5, indicating a pHdependent cross-linking effect. Similar shifts were observed usingboth MNPD and BP. Because Ts shifts of 2-2.5oC are observed usingthe standard riboflavin photochemical cross-linking technique, thisdegree of Ts shift represents our target effect. The type of buffer useddid not have a bearing on the pH dependent catalytic effect. Whenreacting at pH 7.4, 37oC, for 1hr, there were no discernible catalyticeffects using the three enzymes or DNA.Conclusions: Because the cornea can tolerate a pH up to 8.5 withoutsustaining damage, and the tear film has a fairly limited bufferingcapacity, modulating the pH of a BNA corneal cross-linking solutioncould provide a useful way to rapidly strengthen corneal tissue (i.e.within minutes).Commercial Relationships: David C. Paik, 12/517,382 andPCT/US2007/025126 (P); Quan Wen, None; Mi Jung Kim, None;Quan V. Hoang, None; Stephen L. Trokel, Avedro (C)Support: NIH Grant EY020495Program Number: 5285 Poster Board Number: C0204Presentation Time: 2:45 PM - 4:30 PMANALYSIS OF THICKNESS OF CORNEAS FROM EYEBANKS AFTER APPLICATION OF ISOSMOLARRIBOFLAVIN AND HIPOSMOLAR RIBOFLAVINLuiz Felipe L. Moraes 2, 1 , Luciene Barbosa 2 , Monique Macedo 2 ,Tatiana Rayes 2 . 1 Ophthalmology, Federal University of Pernambuco,Recife, Brazil; 2 Ophthalmology, Sorocaba Eye Bank, Sorocaba,Brazil.Purpose: Compare thickness of corneas from eye banks with200mOsm and 400mOsm riboflavin.Methods: Corneas placed in artificial chamber had pachymetry withepithelium, without epithelium and 30 minutes after application ofanything (group 1-control); isosmolar (0,1% 400mOsm) riboflavin(group 2); hiposmolar (0,1% 200mOsm) riboflavin (group 3) andsaline solution (group 4).Results: 72 corneas were used; average prior pachymetry was 592μmand 548,8μm after removal of the epithelium. Group 1, 2 and 3 haddecrease in thickness and group 4 had an increase. The averagedecrease in group 2 was 68,95μm, more than twice the average ofgroup 3 (31,45μm).Conclusions: Hiposmolar riboflavin prevents the decrease inthickness when compared to isoosmolar riboflavin.Commercial Relationships: Luiz Felipe L. Moraes, None; LucieneBarbosa, None; Monique Macedo, None; Tatiana Rayes, NoneProgram Number: 5286 Poster Board Number: C0205Presentation Time: 2:45 PM - 4:30 PMCrosslinking Corneal Collagen using Rose Bengal and GreenLightIrene E. Kochevar 1 , Thomas Gisel 1 , Erol E. Verter 1 , GiulianoScarcelli 1 , Seok H. Yun 1 , Robert H. Webb 1 , Robert W. Redmond 1 ,Samir Melki 2 . 1 Wellman Center for Photomedicine, MassachusettsGeneral Hospital, Boston, MA; 2 Boston Eye Group, Boston, MA.Purpose: We have previously shown that Rose Bengal (RB) plusgreen light markedly stiffens the corneal stroma by a rapid collagencrosslinking process that is not toxic to keratocytes. The goals of thisstudy were to determine the spatial localization of crosslinks, identifymechanisms and evaluate treatment safety.Methods: RB (0.1% in PBS) was applied to de-epithelialized rabbitcornea ex vivo for 2 min. The diffusion distance of RB into thestroma was evaluated on frozen sections by fluorescence microscopy.After irradiation of RB-stained cornea with green (532 nm) light froma KTP laser (10 min, 150 J/cm 2 ), the spatial distribution of increasedelastic modulus was determined by Brillouin scattering microscopyand the average stromal stiffness was measured by uniaxialtensiometry. The availability of oxygen in the stroma for thecrosslinking mechanism was evaluated by absorption spectroscopyduring the irradiation. The irradiance and fluence on the retina of amodel eye was measured using a newly designed light deliverysystem and compared to the ANSI standard damage thresholds.Results: RB penetration into the stroma was limited to ~120 µmfrom the anterior surface and did not increase over time. Consistentwith this distribution, Brillouin microscopy demonstrated increasedelastic modulus localized to the most anterior 120 µm of the stroma.Photo-crosslinking increased the average stromal stiffness by fourfoldcompared to untreated control cornea. Irradiation of RB-stainedcornea in the absence of oxygen shifted the absorption maximumfrom 562 nm to 520 nm, a signature of photoproducts formed onlyunder anoxic conditions. During photo-crosslinking the absorptionmaximum did not change suggesting an oxygen-dependentcrosslinking mechanism. The irradiance and fluence at the retina of amodel eye during the treatment were at least 20-fold below the ANSIdamage threshold for thermal or photochemical damage.Conclusions: The shallow penetration of RB into corneal stroma andformation of crosslinks within 120 µm of the anterior surface suggestthat thin corneas of keratoconus or other ectatic cornea conditions canbe treated. Photo-crosslinking rapidly and substantially increasescornea stiffness using safe retinal exposure levels of green light.Commercial Relationships: Irene E. Kochevar, Aura MedSystems(P); Thomas Gisel, None; Erol E. Verter, None; GiulianoScarcelli, massachusetts general hospital (P); Seok H. Yun,Massachusetts General Hospital (P); Robert H. Webb, None;Robert W. Redmond, Aura Medsystems Inc (C); Samir Melki,NoneProgram Number: 5287 Poster Board Number: C0206Presentation Time: 2:45 PM - 4:30 PMCollagen Crosslinking By Infrared Femtosecond Pulses In ExvivoCorneasJuan M. Bueno, Harilaos S. Ginis, Raquel Palacios, AlexandrosPennos, Pablo Artal. Laboratorio de Optica, Universidad de Murcia,Murcia, Spain.Purpose: Collagen cross-linking (CXL) with ultraviolet (UV) A lightand riboflavin technique has been reported to increase cornealstiffness to slow down keratoconus progression. With UV CXLtreatment, irradiation is not localized and the penetration depthdepends on the attenuation of the UV light propagating through thecornea due to absorption within the riboflavin. This fact may lead toendothelial cell loss, especially in thin corneas. The purpose of thiswork was to determine if femtosecond (fs) infrared pulses couldinduce localized CXL effects through a 2-photon process on ex-vivoporcine corneas.Methods: A custom-made multiphoton microscope was used toirradiate a corneal volume and subsequently record multiphotonimages of the stroma. After de-epithelization, the eye was immersedinto a solution containing 0.125% riboflavin and 20% dextran during45 minutes. A set of control eyes were only immersed into 20%dextran solution in order to maintain their normal hydration. An area90×90×50 μm 3 within the cornea (depth location 125 μm, scanningstep 2 μm) was irradiated with the 760-nm fs-laser for 60 minutes inboth control and fs-CXL treated eyes.Results: During irradiation, multiphoton tomographic (XZ or YZ)images of the corneal stroma were adquired every 10 minutes forboth control and fs-CXL-treated corneas. For the former the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneanonlinear signal presented a maximum close to the Bowman’s layerand a uniform decrease with depth typical of a non-irradiated cornea.On the opposite, fs-CXL corneas exhibited a marked increase innonlinear signal at the treated volume with no effect on nearbycorneal layers, presumably indicative of localized changes incollagen distribution. This was revealed by means of the regular XYmultiphoton images at different depth positions throughout the entirestroma.Conclusions: This study demonstrates the feasibility of usinginfrared fs-laser pulses to perform well-localized 3-dimensional CXLtreatments within the cornea. The technique has been proven to beprecise with surrounded regions hardly affected and no thermaleffects. The combination of both surgical and monitoring functionsinto a unique fs-laser system has the potential to be a tool in clinicalenvironments and could be the basis to establish a new techniquecapable of monitoring CXL procedures in real-time.Commercial Relationships: Juan M. Bueno, None; Harilaos S.Ginis, Universidad de Murcia (P); Raquel Palacios, None;Alexandros Pennos, None; Pablo Artal, AMO (C), Voptica SL (P),Voptica SL (I), AMO (F), Calhoun Vision (F), Calhoun Vision (C),AcuFocus (C)Support: Ministerio de Educación y Ciencia, Spain (grants FIS2010-14926 and CSD2007-00013) and Fundación Séneca, Región deMurcia, Spain (grant 4524/GERM/06).Program Number: 5288 Poster Board Number: C0207Presentation Time: 2:45 PM - 4:30 PMAccelerated corneal cross-linking with pulsed lightPavel Kamaev, William A. Eddington, Sara Rood-Ojalvo, Marc D.Friedman, David Muller. Research, Avedro, Waltham, MA.Purpose: Corneal collagen cross-linking (CXL) with riboflavin (RF)strengthens corneas affected by keratoconus. This treatment involvesirradiation with continuous ultraviolet-A (UVA) which deoxygenatesthe cornea and makes keratocytes more susceptible to theaction of light. In order to avoid this self-limiting factor of CXL, wepropose to apply pulsed UVA to allow the tissue to re-oxygenate andto increase efficiency of CXL.Methods: Porcine eye globes were shipped overnight on ice from anabattoir (SiouxPreme, Sioux City, IA). The corneas were then deepithelializedand 0.1% riboflavin-5’-phosphate solution was appliedtopically during 20 min at 37°C. Eyes were placed in a beaker filledwith oxygen and irradiated for 3 min continuously or for 6 minuteswith pulsed UVA light (365 nm, 1.5 sec on / 1.5 sec off) at 30mW/cm2 using a shutter (Sutter Instrument, Novato, CA). Cornealflaps (200 µm thick) were excised from the eyes using an Intralasefemtosecond laser (Abbot Medical Optics, Santa Ana, CA). The flapswere placed into a biaxial extensometer (CellScale Biotester 5000,Waterloo, ON), using biorake attachments with 5 tines spanning awidth of 3mm. Each sample was stretched at a constant rate of 4µm/s in saline at 37°C until sample failure. The flaps were thenwashed with distilled water, dried in a vacuum and digested at 65°Cwith 2.5 units/ml of papain in 1x PBS (pH 7.4) containing 2 mM L-cysteine and 2 mM EDTA. Fluorescence of the papain digests wasrecorded at 450 nm in a QM-40 Spectrofluorometer (PhotonTechnology Int., London, Ontario, Canada) using excitation of 360nm.Results: Continuous irradiation of the corneal tissue in presence ofRF resulted in a quick depletion of dissolved oxygen. This happensvia the process of generation of reactive oxygen species (includingsinglet oxygen) and following cross-linking, mediated by freeradicals. With pulsed fractionation of UVA radiation, cross-linkingefficiency is improved by allowing the re-diffusion of oxygen duringpauses in exposure.Conclusions: Our work confirmed that re-oxygenation of corneaduring UVA pulsation increases efficiency of the CXL. From aclinical perspective, accelerated CXL with pulsed light would lead toa reduced dose for the same amount of corneal stiffening andtherefore a better safety profile.Commercial Relationships: Pavel Kamaev, Avedro (E); WilliamA. Eddington, Avedro Inc. (E); Sara Rood-Ojalvo, Avedro Inc. (E);Marc D. Friedman, Avedro Inc (E); David Muller, Avedro Inc (E)Program Number: 5289 Poster Board Number: C0208Presentation Time: 2:45 PM - 4:30 PMLong term follow-up of stiffening of rabbit corneas by WST11using near infrared lightArie L. Marcovich 1, 2 , Alexander Brandis 1 , Ilan Feine 3 , Iddo Pinkas 1 ,Ruth Goldschmidt 1 , Vyacheslav Kalchenko 4 , Daniel H. Wagner 5 ,Yoram Salomon 3 , Avigdor Scherz 1 . 1 Plant Sciences, WeizmannInstitute of Science, Rehovot, Israel; 2 Ophthalmology, KaplanMedical Center, Rehovot, Israel; 3 Biological Regulation, WeizmannInstitute of Science, Rehovot, Israel; 4 Veterinary Resources,Weizmann Institute of Science, Rehovot, Israel; 5 Materials andInterfaces, Weizmann Institue of Science, Los Angeles, CA.Purpose: To evaluate the efficacy and safety of photochemicalcorneal stiffening by Pd- bacteriochlorin 13’-(2-sulfoethyl)-amidedipotassium salt (WST11) and near infrared (NIR) illumination,using rabbit eye models.Methods: Corneas of living rabbits were pretreated topically with 2.5mg/ml WST11 in saline or in 20% dextran T-500 for 20 minutes, andilluminated with a NIR diode laser (755 nm, 10 mW/cm2) for 30minutes. Corneas of untreated fellow eyes served as controls. Therabbits were sacrificed after one month and 4 months. Corneal stripsof 4 mm width were excised. Ultimate stress and Young’s modulusmeasurements were performed using a biomechanical tester.Comparative studies were performed with standardriboflavin/ultraviolet-A light (UVA) treatment. Histology, electronspin resonance, fluorescence microscopy and optical coherencetomography (OCT) were used to evaluate treatment effects.Results: WST11/NIR treatment significantly increased cornealstiffness following treatment, compared to untreated contralateraleyes. After 1 month the ultimate stress and Young’s modulus oftreated corneas increased by 113%, and 80% respectively. WST11 in20% dextran T-500/NIR had similar stiffening results, but markedlyreduced post-treatment edema and time of epithelial healingcompared to WST11 without dextran. After 4 months, treated corneasmaintained biomechanical stiffness values measured at 1 month posttreatment. OCT demonstrated a demarcation line in the anterior thirdof the corneal stroma. Histology showed a reduction in the keratocytepopulation in the anterior half of the corneal stroma, without damageto the endothelium. Electron spin resonance demonstrated thatWST11/NIR generates hydroxyl and superoxide radicals but nosinglet oxygen in the cornea. These results, in particular whencombined with additional optical spectroscopy techniques, suggest anovel mechanism for corneal stiffening that has not been so faranticipated in connection with current stiffening approaches.Conclusions: Treatment of rabbit corneas, with thebacteriochlorophyll derivative WST11 and NIR illumination,increased their biomechanical strength through mechanism that doesnot involve singlet oxygen. WST11 in 20% dextran T-500/NIRtreatment showed less adverse effects. This treatment may have apotential for clinical use in keratoconus and corneal ectasia.Commercial Relationships: Arie L. Marcovich, Yeda Researchand Development Company, Weizmann Institute of Science (P);Alexander Brandis, Yeda Research and Development CompanyLtd., Weizmann Institute of Science (P); Ilan Feine, None; Iddo©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPinkas, None; Ruth Goldschmidt, None; Vyacheslav Kalchenko,None; Daniel H. Wagner, Weizmann Institute (P); YoramSalomon, Yeda (P); Avigdor Scherz, Yeda, Weizmann Inst scie (P)Support: Gurwin FoundationProgram Number: 5290 Poster Board Number: C0209Presentation Time: 2:45 PM - 4:30 PMAntimicrobial efficacy of riboflavin / UV-A collagen cross-linkingat different fluences in vitroFlorence Hoogewoud, Olivier Richoz, Farhad Hafezi. Ophtalmology,University Hospital of Geneva, Geneva, Switzerland.Purpose: Infectious corneal ulceration is a common cause of visualloss. The final outcome is influenced by early diagnosis andtraditionally by intensive treatment with topical antibiotics. UV-Aactivated riboflavin (corneal collagen crosslinking (CXL) representsa new potential therapeutic approach. Its mechanism of action isclosely linked to the abundant generation of free radicals. Generationof free radicals is an intensity- and time-dependent process. Thestudies performed so far have all used the standard protocol (3mW/cm2 during 30 min) that had been established for progressivekeratoconus. Our study investigates the antimicrobial efficacy ofCXL using three different intensities and time regimen whilemaintaining the same total energy dose.Methods: A porous polymer 300 μm thick, containing 96% of water,not toxic for the bacteria and not UV-absorbing was used as a supportfor the experiment. Two solutions of 106 bacteria, one of methicillinresistantStaphylococcus aureus and one of Pseudomonas aeruginosa,combined with 0.1% Riboflavin solution were instilled in thepolymer. Three different protocols of UV-irradiation (365nm) usingthe same energy (5.4 mJ) at different time and power settings weretested (3 mW/cm2 during 30 min; 9 mW/cm2 during 10 min; 18mW/cm2 during 5 min). After irradiation, the solution was culturedon a blood agar plate and the CFU (colony forming units) werecounted after 24h of incubation. Controls without riboflavin and withriboflavin but without irradiation were performed.Results: At the three different intensities, the number of bacteria wasreduced by 2 log(10) scales compared with both control groups. Nosignificant differences were detected between the three protocols.Conclusions: The bactericidal rate was comparable in all threeprotocols tested suggesting that in clinical practice the time-sparingprotocol (18 mW/cm2 during 5 min) should provide as good resultsas the standard protocol (3 mW/cm2 during 30 min). Clinical trialswill have to confirm this hypothesis.Commercial Relationships: Florence Hoogewoud, None; OlivierRichoz, None; Farhad Hafezi, Schwind (F), Ziemer (F), PCT/CH2012/000090 (P)Program Number: 5291 Poster Board Number: C0210Presentation Time: 2:45 PM - 4:30 PMCombination of Intracorneal ring segments (Ferrara and Intacts)in the care of patients with KeratoconusRoberto Albertazzi 1 , leonardo ferlini 1 , Luciano D. Perrone 1 , DanielM. Perrone 1 , Guillermo Rao 1 , José F. Alfonso 2, 3 , Jesus Merayo-Lloves 2, 3 . 1 centro de ojos quilmes, Quilmes, Argentina; 2 Fundaciónde Investigacion Oftalmologica - Instituto Oftalmológico Fernández-Vega, oviedo, Spain; 3 University of Oviedo, oviedo, Spain.Purpose: Outcome Analyisis of a combination of Intracorneal ringsegment (ICRS) both Ferrara and Intacts, for tectonic and refractivecorrection of paracentral keratoconus.Methods: 43 eyes of 33 patients with contact lens intolerance orprogressive paracentral keratoconus underwent ICRS surgery andwere followed for 1 at least 6 months. Patients were divided in twogroups acording with the coincidence of axis of astigmatism andcoma as Group I, +- 30° coincident, croissant shape and Group II,non coincident (30-75°), duck shape. Surgery were performedmanually and ICRS were implanted at 6 mm of the optical axis: 150°Ferrara ICRS were placed temporal-inferiorly and 90° intacs werelocated nasal-superiory. Tectonic outcome were evaluated as areduction of the keratometric (K) values: K minimal (Kmin) and Kmaximal (Kmax)).Visual outcome were measured as improve inuncorrected distance visual acuity (UDVA), best corrected visualacuity (CDVA), and refractive error (miopia and astigmatism)redcution (decimal scale). Data were processed with R-programinglanguage for descriptive ans inference statistical análisis análisisResults: There were a significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneato the ocular surface 5 times per day from 3 weeks to 12 weeks ofage. Other 7 mice (group P) were instilled normal physiologicalsaline eye drops. We took photographs of their cornea every week,and compared the frequency of occurrance of keratoconus-likecorneal lesion up to 24 weeks of age.Results: The frequency of keratoconus-like lesion was 28.6% at 6thweek, 57.1% at 7th week, 71.4% at 9th week, 85.7% at 16th week,and stable thereafter in the group P, and 14.3% at 6th week, 28.6% at10th week, 42.9% at 12th week, 57.1% at 16th week, and notincreased thereafter in the group H. The frequency of keratoconuslikelesion was significantly higher in group P from 6th to 9th weekof age (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawith a well-known distance between the electrodes. Two corneastorage media were used: Eusol-C and phosphate buffered salinepH=7.4.The 100-400 μm specimens containing the middle layers of cornealstroma were examined. The 9 specimens of healthy, control corneasfrom Eye Bank were obtained with second microkeratom sectionduring Ultra Thin-DSAEK. The 12 specimens of keratoconus corneaswere manually prepared from corneas removed during penetratingkeratoplasty.Results: The fitting procedure of a theoretical function to theexperimental data allowed us to determine up to three relaxationregions. The narrow peak near 1MHz can be successfully traced bythe Debye relaxation function. This peak is probably responsible forthe dipolar polarization of bound water to collagen molecules as wellas for the polarization of side polar groups in polypeptide chains.Two low frequency relaxation regions are related to diffusiveprocesses with different structural distance parameters, i.e. describingthe spatial arrangement of collagen fibrils in cornea. Peak amplitudeswere increasing upon rising electric field applied from 0.05V to 1V.Comparison between the dielectric responses for normal andkeratoconus corneas showed the significant shift of the ionic peakstowards lower frequencies ωn→ωk, with the ωn/ωk ~2. The observedshift in peak position may correspond to the increase in the averagestructural distance of different molecular compartments present inkeratoconus cornea.Conclusions: It has been shown that the method can be utilized tofollow relative changes in the spatial molecular structure ofkeratoconus corneas with respect to the normal tissue. It is alsoconsidered that the method have a great potential to be used forstructural analysis of biological systems in vivo, which are oftencharacterized by high water content and the presence of multiplekinds of ionsCommercial Relationships: Dorota Tarnawska, None; AntoniKocot, None; Maria Jastrzebska, None; Edward Wylegala, None;Alicja Ratuszna, NoneProgram Number: 5296 Poster Board Number: C0215Presentation Time: 2:45 PM - 4:30 PMCorrelation between keratoconus progression and two corneatopometric parameters: the regularity of the pachymetric map(RPM) and the Index of height decentration (IHD)George Asimellis, Ioanna Kontari, A. John Kanellopoulos. Research,LaserVision.gr Eye Institute, Athens, Greece.Purpose: to determine the specificity and sensitivity of keratoconusprogression measured with these two parameters the regularity of thepachymetric map (RPM) and the Index of height decentration (IHD).Methods: Sequential topometric measurements spaced by sixmonths, utilizing the Pentacam device (Scheimpflug imaging) wereconducted in 100 keratoconic eyes. The indices RPM and IHD werecorrelated with keratometry, UDVA, CDVA and minimalpachymetry with 6 years follow-up.Results: Distortion of the RPM and increase of the IHD appeared tobe the first markers of KCN progression in all cases, preceding by anaverage of 18 (± 4.5) months the UDVA, CDVA and keratometricchanges of over 1 diopter.Conclusions: Monitoring RPM and IHD may be a useful earlierdiagnostic tool for ectasia progression and postoperative LASIKstability. It may serve as a new definition of ectasia progression.Commercial Relationships: George Asimellis, None; IoannaKontari, None; A. John Kanellopoulos, Alcon Laboratories, Inc.(C), Avedro (C), Bausch and Lomb (C), Ocular Therapeutix (C)Program Number: 5297 Poster Board Number: C0216Presentation Time: 2:45 PM - 4:30 PMComparison of three-dimensional optical coherence tomographyparameters for early keratoconus diagnosisShinichi Fukuda 1 , Sujin Hoshi 1 , Masahiro Yamanari 2, 3 , SimoneBeheregaray 1 , Takahiro Hiraoka 1 , Yoshiaki Yasuno 2 , TetsuroOshika 1 . 1 Ophthalmology, Tsukuba University, Tsukuba, Japan;2 Computational Optics Group, Tsukuba University, Tsukuba, Japan;3 Tomey Corporation, Nagoya, Japan.Purpose: To investigate changes in keratometric, elevation,topographic, regular and irregular astigmatism, and pachymetricparameters in keratoconus, keratoconus suspect, and normal subjectsusing three-dimensional (3D) corneal and anterior segment opticalcoherence tomography (CAS-OCT), and rotating Scheimpflugcamera combined with Placido topography system (Scheimpflugcamera with topography). Also, to compare discriminative ability ofthese parameters.Methods: Forty eyes of 27 patients with keratoconus, 10 eyes of 10patients with keratoconus suspect, and 77 eye of 77 normal subjects.The keratometric parameters (anterior and posterior steep K, flat K,and Average K), anterior and posterior elevation, topographicparameters, anterior and posterior regular and irregular astigmatism(spherical, asymmetry, regular, and higher order astigmatism), andpachymetric parameters (five pachymetric parameters werecalculated from the region inside the 2-5 mm diameter: Minimum,Minimum-Median, Inferior-Superior, Inferotemporal Superonasal,and the vertical thinnest location of the cornea) were measured using3D CAS-OCT and Scheimpflug camera with topography. The areaunder the receiver-operating curve (AROC) of keratometric,elevation, topographic, regular and irregular astigmatism, andpachymetric parameters.Results: The keratometric parameters, anterior and posteriorelevation, anterior and posterior regular and irregular astigmatism,pachymetric parameters showed good discrimination between normaland keratoconic eyes in both 3D CAS-OCT and Scheimpflug camerawith topography. The AROC of all posterior parameters were higherthan 0.92 and tended to be larger than anterior segment parameters.The AROC value of Inferior-Superior, posterior elevation, andposterior asymmetry were 0.940, 0.909, and 0.906 in earlykeratoconus discrimination analysis.Conclusions: The keratometric parameters, anterior and posteriorelevation, anterior and posterior regular and irregular astigmatism,and pachymetric parameters can effectively discriminate keratoconus.Inferior-Superior, posterior elevation, and posterior asymmetryshowed best predictive accuracy in early keratoconus discriminationanalysis.Commercial Relationships: Shinichi Fukuda, None; Sujin Hoshi,None; Masahiro Yamanari, Tomey Corporation (E); SimoneBeheregaray, None; Takahiro Hiraoka, None; Yoshiaki Yasuno,Topcon Corp. (F), Tomey Corp. (F), Tomey Corp. (P); TetsuroOshika, NoneSupport: Supported in part by Grants-in-Aid 22390320 for ScientificResearch from the Ministry of Education, Culture, Sports, Scienceand Technology, Japan.Program Number: 5298 Poster Board Number: C0217Presentation Time: 2:45 PM - 4:30 PMDecreased corneal sensitivity to chemical and thermal stimuli inkeratoconus patientsLorant Dienes 1 , Kinga Kranitz 1 , Zoltan Z. Nagy 1 , Janos Nemeth 1 , MCarmen Acosta 2 , Juana Gallar 2 , Carlos Belmonte 2 , Illes Kovacs 1 .1 Ophthalmology, Semmelweis University, Budapest, Hungary;2 Instituto de Neurociencias, Univ Miguel Hernandez-CSIC, Alicante,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.Spain.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To investigate corneal sensitivity to selective mechanical,chemical, and thermal (heat and cold) stimulation in patients withkeratoconus.Methods: Corneal sensitivity to different modalities of stimulus wasdetermined in one randomized eye in 17 patients with mild ormoderate keratoconus (KC) and in 15 healthy subjects (control).Mechanical, chemical, and thermal (hot and cold) thresholds weredetermined at the center of the cornea using a Belmonte's gasesthesiometer. Patients with a history of contact lens wear wereexcluded from the study. All patients completed a questionnaire toassess dry-eye disease symptoms (ocular surface disease index -OSDI).Results: There was no significant difference in age, gender andOSDI between the KC and the control group. Mean corneal thresholdsensitivity values to chemical stimulation (KC: 33.22 ± 0.77 % CO2vs. control: 30.82 ± 0.21 % CO2; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapatients tended significantly to have keratoplasty done at 20-30 years,while female patients at more than 30 years of age.Conclusions: In this hospital-based KC study, the severity is highwith an early onset and more rapid progress to advanced stage at ayounger age. Female patients had more sever disease. These may berelated to genetic and / or environmental factors. The results haveimplications for KC screening in Saudi Arabia (and probably nearbyMiddle-Eastern countries), to improve early detection andmanagment.Commercial Relationships: Samar A. Al-Swailem, None; SamuelC. Yiu, None; Abdullah Al-Assiri, None; Nasira Asghar, None;Albaraa Al-Qassimi, NoneProgram Number: 5301 Poster Board Number: C0220Presentation Time: 2:45 PM - 4:30 PMIncreased expression of secreted frizzled-related protein-1(SFRP-1) and microtubule-associated protein light chain 3 (LC3)in keratoconusOmer Iqbal, George Fisher, Samir Vira, Daniel Kahn, Daneyal Syed,Jawed Fareed, Charles S. Bouchard. Ophthalmology, Loyola,Maywood, IL.Purpose: To study the expression of secreted frizzled-related protein-1 (SFRP-1) and Light Chain 3 (LC3), an autophagy marker, inkeratoconus and to determine the proteomic profile of normal andkeratoconic epithelial and stromal layers by ProteinChip® usingSELDI-TOF-MSMethods: Under an IRB approved protocol, surgically discarded andde-identified normal donor (n=10) and keratoconus corneas (n=10)were obtained. A segment of the cornea was fixed in formalin forimmunohistochemical staining. From the remaining cornea theendothelium-Descemets membrane was removed. The endotheliumfree cornea was soaked in prewarmed 20mM EDTA solution for 30minutes and forceps were used to strip the epithelial and the stromallayers. The samples were homogenized and the proteinconcentrations adjusted to 1mg/ml. The homogenates were analyzedusing SELDI-TOF-MS. The formalin fixed paraffin-embeddedcorneal samples were cut into sections and mounted on slides. SFRP-1 antibody and LC3 antibody were used to performimmunohistochemical staining.Results: The proteomic profile showed absence of peaks in the 20-150 kDa range in the keratoconic epithelium. However, there weredistinct peaks in keratoconus at 10.8, 12.7, 13.1, 14.6, and 15.8 kDawhich were absent in normal samples. High expressivity of SFRP-1and LC3 was observed in the keratoconus corneas. There alsoappeared to be a correlation between the expression of SFRP1 andLC3 in keratoconus tissues. Low expressivity of SFRP1 resulted inlow expressivity of LC3 while medium-high expressivity of SFRP1resulted in medium to high expressivity of LC3.Conclusions: Increased expression of SFRP-1 and LC3 was observedin Keratoconus cornea. Keratocyte autophagy associated withKeratoconus may play a role in the pathogenesis of Keratoconus.Commercial Relationships: Omer Iqbal, None; George Fisher,None; Samir Vira, None; Daniel Kahn, None; Daneyal Syed,None; Jawed Fareed, None; Charles S. Bouchard, NoneSupport: Illinois Society for the Prevention of BlindnessProgram Number: 5302 Poster Board Number: C0221Presentation Time: 2:45 PM - 4:30 PMIL1B promoter polymorphisms are associated with keratoconusin a Japanese populationTakenori Mikami 1, 2 , Akira Meguro 1 , Takeshi Teshigawara 2 , MasakiTakeuchi 1 , Misaki Ishioka 1, 3 , Miki Iwasaki 3 , Kazumi Fukagawa 3 ,Kenji Konomi 4 , Jun Shimazaki 4 , Nobuhisa Mizuki 1 . 1 Ophthalmologyand Visual Science, Yokohama City University Graduate School ofMedicine, Yokohama, Japan; 2 Yokosuka Chuoh Eye Clinic,Yokosuka, Japan; 3 Ryogoku Eye Clinic, Tokyo, Japan; 4 Departmentof Ophthalmology, Ichikawa General Hospital, Tokyo DentalCollege, Ichikawa, Japan.Purpose: Previous study reported that the polymorphisms ofinterleukin 1 alpha (IL1A) and IL1B gene regions were associatedwith keratoconus in a Korean population. In this study, weinvestigated whether the IL1A and IL1B polymorphisms areassociated with keratoconus in a Japanese population.Methods: One hundred and sixty-nine Japanese patients withkeratoconus and 390 Japanese healthy controls were recruited. Wegenotyped one IL1A single-nucleotide polymorphism (SNP)(rs2071376) and two IL1B SNPs (rs1143627 and rs16944), and thefrequencies of alleles, genotypes, and haplotypes were comparedbetween cases and controls.Results: The significant associations were observed for rs1143627 (-31 T>C) and rs16944 (-511 C>T) in the IL1B promoter region, andthe T allele of rs1143627 and the C allele of rs16944 had anincreased risk of keratoconus (P = 0.014, OR = 1.38 and P = 0.033,OR = 1.33, respectively). The TT genotype of rs1143627 wasassociated with an increased risk for keratoconus (P = 0.033, OR =1.52). The CC genotype of rs16944 also showed an increased risk forkeratoconus, but this did not reach statistical significance (P = 0.058,OR = 1.45). For rs2071376 in IL1A, there were no significantdifferences of allele and genotype frequencies between cases andcontrols. With regard to haplotypic diversity, the haplotype createdby the T allele of rs1143627 and the C allele of rs16944 had a 1.72-fold increased risk of keratoconus (P = 4.0 × 10-5).Conclusions: Our results replicate associations reported recently in aKorean population. This suggests that the IL1B gene play animportant role in the development of keratoconus through geneticpolymorphisms.Commercial Relationships: Takenori Mikami, None; AkiraMeguro, None; Takeshi Teshigawara, None; Masaki Takeuchi,None; Misaki Ishioka, None; Miki Iwasaki, None; KazumiFukagawa, None; Kenji Konomi, None; Jun Shimazaki, SantenPharmaceutical Co. (F), Otsuka Pharmaceutical Co. (F), AbottMedical Optics (F); Nobuhisa Mizuki, NoneSupport: None in the SupportProgram Number: 5303 Poster Board Number: C0222Presentation Time: 2:45 PM - 4:30 PMKeratoconus Gene Mapping: Candidate Genes in the 14q11.2homozygous regionGovindasamy Kumaramanickavel 1 , Venkata Ramana Anandula 1 ,Vedam Ramprasad 3 , Nalla Thambi Jeyabalan 1 , Arkasubhra Ghosh 1 ,Rohit Shetty 2 . 1 Basic Science Research, Narayana Nethralaya,Bangalore, India; 2 Cornea, Narayana Nethralaya, Bangalore, India;3 Spinco-Biotech, Chennai, India.Purpose: Keratoconus (KC) is the most common corneal dystrophywith a prevalence rate of 0.05% of people in the USA, even though itis classified under rare diseases. Indian subcontinent has a four foldgreater incidence of the disease. The precise etiopathogenesis of KCis unclear. However, strong family history and large autosomaldominant and few recessive pedigrees' linkage with the disorder hasshown that genetics is one of the leading causes for KC. This study isto map the gene that is causative of the disease in a consanguineousautosomal recessive family.Methods: The family members underwent detailed ophthalmicevaluation including corneal topography. The members of theconsanguineous family were bled for DNA with all ethicalconsiderations. The samples were run on genomewide Affymetrix©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSNP 6.0 GeneChip and the data were analyzed using theHomozygosity Mapper software. Further bioinformatic analyses ofthe chromosomal region identified was done by using genome-widestudy tools on the UCSC genome browser.Results: Homozygosity mapping reveals the causative region in thisfamily on chromosome 14q11.2 and associated with the SNPrs3811259. Genome browser shows the SNP centeromeric to TCRalphagene (TCRA). The region is linked to inflammatory boweldisease (IBD) and also to several transcription factor and histonedeacetylase binding sites such as FOXA1 (estrogen receptor), CTCF(11-zinc finger protein) etc and histone modifier binding sites likeHDAC2, p300 etc. A few keratoconus related candidate genes likeAPEX1 (multifunctional DNA repair enzyme), CIDE-B (cell deathinducingDFFA-like effector b) are in this region.Conclusions: There are the few candidate genes for KC in thehomozygously linked 14q11.2 region. IBD could be indicative of aninflammatory pathway involvement in the KC disease pathogenesis.Furthermore, the presence of prevalence of transcription factorbinding sites as per genome-wide ChIP-seq data and histonemodification marks suggests this region to be transcriptionally active.Interestingly, APEX1, a DNA repair gene and CIDE-B, which has arole in apoptosis, is highly expressed in the KC cornea indicating theinvolvement in non-canonical roles. Thus, this chromosomal regionmay have the key causative role gene for KC etiopathogenesis.Commercial Relationships: Govindasamy Kumaramanickavel,None; Venkata Ramana Anandula, None; Vedam Ramprasad,None; Nalla Thambi Jeyabalan, None; Arkasubhra Ghosh, None;Rohit Shetty, NoneSupport: Narayana Nethralaya FoundationProgram Number: 5304 Poster Board Number: C0223Presentation Time: 2:45 PM - 4:30 PMHomozygosity Mapping of Keratoconus to 12p13.1 region: TheIdeal Candidate GenesVenkata Ramana Anandula 1 , Venkata Ramana Anandula 1 , VedamRamprasad 2 , Nalla Thambi Jeyabalan 1 , Rohit Shetty 1 , ArkasubhraGhosh 1 , Govindasamy Kumaramanickavel 1 . 1 Basic Science Research,Narayana Nethralaya, Bangalore, India; 2 Advanced Genomics,Spinco Biotech, Chennai, India.Purpose: Kertoconus (KC) a non-inflammatory disease results inprogressively thinning of the cornea resulting to a conical shapecausing refractive errors and visual disturbances. Linkage analyses onautosomal dominant and recessive families have identified variousloci (16q22.3-23.1, 20q12, 3p14-q13, 5q14.3-q21.1, 15q 22.33-24.2,17p13, regions on chromosomes 9, 4, 11, 12, 14) for KC, though sofar no gene has been identified. Aim of this study is to map the genefor KC autosomal recessive consanguineous family of Asian Indianorigin.Methods: After ophthalmic evaluation including corneal topographyand blood samples were collected for DNA analysis from aconsanguineous KC family. Gene mapping was done usingAffymetrix SNP 6.0 GeneChip using Homozygosity Mappersoftware. Using genome-wide study tools on the UCSC genomebrowser, we did further bioinformatic analyses of the chromosomalregion identified.Results: Homozygosity data showed the susceptible chromosomeregion 12p13.1 associated with the SNP rs1544671. The region islinked to important disorders such as retinal cone dystrophy andinflammatory bowel disease (IBD). Genome browser analysis showsthe SNP centeromeric to Ras related and estrogen regulated growthinhibitor (RERG) and Ras-related small GTP-binding proteins(ARHGDIB). However, genomewide ChIP-seq and ChIA-Petanalyses demonstrate few transcription factor binding sites in thisarea including CTCF and ER-alpha.Conclusions: The homozygosity linked 12p13.1 region is rich ingenes where the IBD could be suggestive of an inflammatorypathway involvement in the KC disease pathogenesis. IBD andretinal cone dystrophy could be indicative of an alternate pathwayinvolvement in the KC disease pathogenesis. Although this regionshows limited transcriptional activity, the presence of importantgrowth inhibitory genes such as RERG, which has been shown to betumor suppressors, may have important roles in KC pathophysiology.Sequencing the region should identify the causative gene in thisfamily.Commercial Relationships: Venkata Ramana Anandula, None;Venkata Ramana Anandula, None; Vedam Ramprasad, None;Nalla Thambi Jeyabalan, None; Rohit Shetty, None; ArkasubhraGhosh, None; Govindasamy Kumaramanickavel, NoneProgram Number: 5305 Poster Board Number: C0224Presentation Time: 2:45 PM - 4:30 PMUse of the polygenic model to predict risk of keratoconus usingGWAS dataXiaohui Li 1, 2 , Yelena Bykhovskaya 2, 3 , Talin Haritunians 1 , DavidSiscovick 4 , Anthony J. Aldave 5 , Loretta B. Szczotka-Flynn 6 , Sudha K.Iyengar 7 , Jerome I. Rotter 1 , Kent Taylor 1 , Yaron S. Rabinowitz 3 .1 Medical Genetics, Cedars-Sinai Medical Center, Los Angeles, CA;2 Cornea Genetic Eye Institute, Cedars-Sinai Medical Center, LosAngeles, CA; 3 Regenerative Medicine Institute, Cedars-SinaiMedical Center, Los Angeles, CA; 4 Cardiovascular Health ResearchUnit, University of Washington, Seattle, WA; 5 The Jules Stein EyeInstitute, University of California Los Angeles, Los Angeles, CA;6 Ophthalmology & Visual Sciences, Case Western ReserveUniversity, Cleveland, OH; 7 Epidemiology & Biostatistics, CaseWestern Reserve University, Cleveland, OH.Purpose: The genetic contribution of individual SNPs to keratoconussusceptibility is usually modest and cannot be identified without largesample sizes. Thus, to begin to estimate the genetic contribution tokeratoconus, we used polygenic modeling to generate genetic scores,utilizing combination of SNPs each with a small genetic effect,estimated from our genome-wide association study (GWAS)discovery cohort and tested in our GWAS replication cohort.Methods: A discovery panel of 222 Caucasian keratoconus patientsand 3324 controls was genotyped using the Illumina 370K beadchip.Selected SNPs identified by GWAS at p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneacorrected genome-wide significance of p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornearisks patients, and relatively low complications in high risk patients.Late HSV/VZV stromal inflammation can affect flap integrity.Commercial Relationships: Heidrun Gollogly, None; LeoMaguire, NoneSupport: Research to Prevent Blindness, Mayo FoundationProgram Number: 5420 Poster Board Number: A0119Presentation Time: 8:30 AM - 10:15 AMCharacterization of patients with ocular chronic graft-versushostdisease and evaluation of a new grading scaleChristiane Blecha 1 , Daniel Wolff 2 , David A. Maerker 1 , Horst Helbig 1 ,Tina Dietrich-Ntoukas 1 . 1 Ophthalmology, University HospitalRegensburg, Regensburg, Germany; 2 Hematology and Oncology,University Hospital Regensburg, Regensburg, Germany.Purpose: Ocular chronic graft-versus-host disease (cGVHD) is oneof the most frequent complications after allogeneic hematopoieticstem cell transplantation (alloHSCT). It often leads to reduced qualityof life due to severe ocular discomfort or even vision loss in severecases. Purpose of the study was to characterize patients with ocularcGVHD.Methods: All patients with ocular symptoms in the HSCT clinicwere examined ophthalmologically. Complete ocular examinationwas performed and subjective symptoms of keratoconjunctivitis sicca(KCS) assessed. Diagnosis of cGVHD was based on the NIHconsensus criteria for cGVHD. A new grading scale was applied,which was proposed by the Consensus Conference on ClinicalPractice in cGVHD (Dietrich-Ntoukas et al., 2011). Statisticalanalyses were performed using SPSS Version 20 (IBM SPSS,Armonk, NY, USA).Results: 35 patients (male n=25, female n=10, mean age 50 years)with ocular cGVHD were included. 95% of the patients suffered froma cGVHD of other organs, mostly of oral mucosa (62%) and the skin(62%). 94% of the patients showed blepharitis, 91% conjunctivalmanifestations. 71% suffered from corneal involvement (punctatekeratopathy (97%), filamentary keratitis (26%), corneal scarring(23%), corneal erosion (17%), corneal ulcer (12%)). The new gradingscale revealed inflammatory activity in all patients (mild (26%),moderate (43%) or severe (31%)). Results of the NIH scoring system:0% score 0 (no symptoms); 0% score 1 (mild symptoms of dry eye orasymptomatic signs of KCS); 43% score 2 (moderate dry eyesymptoms partially affecting activities of daily living, requiring drops> 3x per day or punctual plugs, without vision impairment); 57%score 3 (severe dry eye symptoms significantly affecting dailyactivities or loss of vision because of KCS). All 35 patients usedartificial tears, 29 used topical cyclosporine and 7 autologous serumeye drops.Conclusions: Patients with ocular cGVHD frequently have severeocular surface disease based on impaired function of the lacrimalgland but also of the conjunctiva and the lids, mostly associated withongoing inflammatory activity. The latter is currently not covered bythe NIH grading. Ophthalmological assessment of patients afteralloHSCT including lids, conjunctiva and inflammatory activity isnecessary to confirm the diagnosis, to optimize the therapy and toprevent irreversible complications.Commercial Relationships: Christiane Blecha, None; DanielWolff, None; David A. Maerker, None; Horst Helbig, None; TinaDietrich-Ntoukas, NoneProgram Number: 5421 Poster Board Number: A0120Presentation Time: 8:30 AM - 10:15 AMProliferative Gain of Function of Stromal Fibroblasts via SDF-1in Pathogenesis of PterygiaKyoung Woo Kim, Soo Hyun Park, Jae Chan Kim. Department ofOphthalmology, Chung-Ang University Hospital, Seoul, Republic ofKorea.Purpose: Biochemical signaling via Stromal cell-derived factor 1(SDF-1) and its receptor, Chemokine receptor type 4 (CXCR4) hadinitially attracted attention as a recruiter of progenitor cells to woundfor organ repair. Paradoxically, however, SDF-1/CXCR4 signaling isimplicated in proliferation and pheno-transdifferentiation offibroblasts in tumor. With viewing pterygia as exaggerated woundand mimicry of tumor, we investigate the involvement of SDF-1expression in pathogenesis of pterygia.Methods: In cultured stromal fibroblasts of pterygia (74 eyes) andnormal conjunctiva (24 eyes), the expression patterns of SDF-1 andCXCR4 were analyzed according to normal or pterygium, recurrencyand grade based on pterygial body morphology (T1-T3) orvascularity (V1-V3). To reveal the SDF-1-triggered possible phenotransdifferentiationof pterygial fibroblasts, the expression of α-SMA,which is the marker of myofibroblasts was co-analyzed paired withSDF-1. Then, the expression level of α-SMA was determined afterdownregulation of SDF-1 in fibroblasts by siRNA. The mRNAexpression was evaluated by RT-PCR, and the proteins were detectedby Western blot analysis, immunohisto- and immunocytochemistry.Results: Expression of SDF-1 was significantly higher in recurrent,T3 or V3 grade pterygia at both mRNA and protein levels. However,there was no significant difference in CXCR4 expression. Inimmunohistochemistry, SDF-1 was highly expressed at the stromallayer in recurrent or T3 pterygia, and at perivascular area especiallyin V3 pterygia. Expression of SDF-1 was positively correlated withthat of CXCR4 or α-SMA. Furthermore, the siRNA-SDF-1 reducedthe expression level of α-SMA.Conclusions: Proliferative gain of function of pterygial fibroblast viaSDF-1 expression is possibly associated with pathogenesis ofpterygia, especially in determination of recurrence, stromal fleshnessand vascularity. This suggests that SDF-1 can be an attractive newtherapeutic target to stabilize pterygia proliferation.Commercial Relationships: Kyoung Woo Kim, None; Soo HyunPark, None; Jae Chan Kim, NoneProgram Number: 5422 Poster Board Number: A0121Presentation Time: 8:30 AM - 10:15 AMBehaviour of Conjunctival Goblet Cells after allogenicHematopoietic Stem Cell TransplantationAlexandra B. Knoll 1 , Eva Jakob 1 , Karin Brandauer 1 , Bianca C.Dobner 1 , Ute Hegenbart 3 , Peter Dreger 3 , Gerd Auffarth 2 , FriederikeMackensen 1 . 1 Interdisciplinary Uveitis Center, University HospitalHeidelberg, Heidelberg, Germany; 2 Ophthalmology, UniversityHospital Heidelberg, Heidelberg, Germany; 3 Heamatology,University Hospital Heidelberg, Heidelberg, Germany.Purpose: The ocular Graft-versus-Host-Disease (oGvHD) is afrequent sequelae after hematopoietic stem cell transplantation(HSCT). We hypothesized that a decrease of goblet cells in theconjunctival epithelium can serve as predictive marker for anemerging oGvHD. To assess goblet cell density we compared themore invasive impression cytology (IC) with the less invasive in vivoconfocal microscopy (IVCM) (Figure A,B).Methods: Patients who were listed for HSCT were examined beforeand 100 days after the transplantation. The examination included aquestionnaire, slit lamp examination, Schirmer Test II, impressioncytology and confocal microscopy of the conjunctiva with theHeidelberg Retina Tomograph with the Rostock Cornea Modulattachment (HRT-RCM). At least three specimen/pictures of eachpatient were assessed for goblet cell density and means werecalculated. The Approval of the Ethical Review Board Heidelberg©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaand informed consent was obtained.Results: 44 patients were included (mean age 56 years). 18 patientswere not re-examined after HSCT (4 died, 2 were pre final, 2 werenot transplanted, 10 repeatedly cancelled their follow-up examinationdue to poor health). Mean time to follow-up examination was 147days after HSCT. Goblet cell density before and after HSCT of allpatients with evaluable pre-and post HSCT -examination was52.6±7.5 vs. 43.9±4.1 cells/mm 2 (Mean±SEM, p=0.46, n=8,wilcoxon’s signed rank) assessed with IC and 62.7±3.4 vs. 61.6 ±4.3cells/mm 2 (p=0.8, n=24) assessed with IVCM. 8 healthy subjectswere examined via IVCM as control (mean age 38 years), meangoblet cell density was 74.8±4.3 cells/mm 2 .Conclusions: IVCM showed goblet cell numbers in similarmagnitude to the standard method IC, we therefore conclude thatIVCM can also reliably be used. Conjunctival goblet cells did notshow a significant decrease after HSCT. The time interval to followupexamination may have been too short as most patients still wereon immunosuppressants and only 2 had signs of oGvHD. Patientswho present with oGvHD symptoms will be re-examined for gobletcell density in our clinic.Figure A: Conjunctival Goblet Cells (arrows), Impression CytologyFigure B: Conjunctival Goblet Cells (arrows), In Vivo ConfoalMicroscopyCommercial Relationships: Alexandra B. Knoll, None; EvaJakob, None; Karin Brandauer, None; Bianca C. Dobner, None;Ute Hegenbart, None; Peter Dreger, None; Gerd Auffarth, None;Friederike Mackensen, Abbott (F), Heidelberg Engineering (F),Heidelberg Engineering (R)Program Number: 5423 Poster Board Number: A0122Presentation Time: 8:30 AM - 10:15 AMDifferences among signs and symptoms of dry eye amongpatients with Sjogren's syndrome, blepharitis, or no ocularsurface diseaseBridgette E. McCabe 1 , Mina Massaro-Giordano 1 , Nicole M. Fuerst 1 ,Maxwell Pistilli 1 , Gui-Shuang Ying 1 , Ilaria Macchi 2, 1 , RebeccaSalvo 1 , Vatinee Y. Bunya 1 . 1 Scheie Eye Institute- Ophthalmology,University of Pennsylvania, Philadelphia, PA; 2 Campus BiomedicoRome, Rome, Italy.Purpose: To examine the differences in dry eye clinical signs andsymptoms among patients with Sjogren’s syndrome, blepharitis, andcontrols.Methods: Seventy-four eyes of thirty-seven subjects (18 Sjogren’ssyndrome, 10 blepharitis, and 9 controls) were evaluated. 94% ofSjogren's syndrome patients and 80% of blepharitis patients were onsystemic or topical medications at the time of enrollment. Symptomswere assessed using the Ocular Surface Disease Index (OSDI),Symptom Assessment in Dry Eye (SANDE), and Visual FunctionQuestionnaire-25 (VFQ-25) questionnaires. Tear film breakup time(TBUT), ocular surface staining, meibomian gland evaluation andSchirmer tests were performed on both eyes. Tear osmolarity wasmeasured using the TearLab Osmolarity system.The comparison of dry eye symptoms and measures among the threegroups of subjects were made using analysis of variance, and thecorrelation among eye symptoms and measures was estimated withPearson’s correlation coefficient (r).Results: We found a significant difference among groups in meanvalues for OSDI (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: Lacrimal glands were removed from wildtype rats andmice. One part of the tissue was used for cell culture, the other partwas embedded for immunostaining. Cultivated cells and lacrimalgland tissue was evaluated for the expression of mesenchymalprogenitor cell markers, such as, nestin, thy 1, PDGFR-β, CD146,SMA, desmin, vimentin, GFAP, musashi-1, CD34 and pancytokeratin.Results: Cells expanded from lacrimal gland tissue explants showeda fibroblast-like cell shape and noticeable intracellular lipid droplets.After eight days of culture a subpopulation of cells showed signs ofdifferentiation into myofibroblasts by demonstrating typical stressfibers. Expression of mesenchymal stem cell markers thy-1, PDGFRβ,SMA, desmin and vimentin, musashi-1 and CD146 wasdemonstrated by immunostaining. Cells appeared to be negative forCD34. Furthermore cells positive for GFAP and nestin could bedetected in lacrimal gland tissue sections.Conclusions: Murine and rat lacrimal gland cells can be successfullyisolated and expanded in vitro using explant culture techniques. Theoutgrowing cell population shows characteristics of mesenchymalstem cells, however more studies are needed to evaluate thedifferentiation potential and regenerative ability of this cellpopulation.Commercial Relationships: Mathias Roth, None; AlexanderKunze, None; Gerd Geerling, Alcon (C), Allergan (C), TheaPharma (C), Novagali (C), Bausch & Lomb (C), Tearlab Inc. (C);Stefan Schrader, NoneProgram Number: 5425 Poster Board Number: A0124Presentation Time: 8:30 AM - 10:15 AMLong-Term Outcomes of Patients with Ocular Surface Stem CellTransplant: 20 years follow-upKunal Suri, Stephen C. Kaufman. Ophthalmology and VisualNeurosciences, University of Minnesota, Minneapolis, MN.Purpose: To evaluate the long-term outcomes and complications ofocular surface stem cell transplant (OSST).Methods: A retrospective observational case series of patients whohad OSST at the University of Minnesota Medical Center between1990 to 2000, performed by a single surgeon. Patients with minimumone-year follow-up after surgery were included. Demographic data,indication for surgery, further procedures, success rates,complications, duration of immunosuppression, and follow-up timewere recorded.Results: There were a total of 20 eyes of 15 patients with mean age48.8 ± 15.9 years (25-86 years) at the time of surgery. There were60% males and 33% had bilateral surgery. The indication for surgerywas chemical injury in 8 eyes (40%), aniridia in 7 eyes (35%),iatrogenic limbal stem cell deficiency (LSCD) in 2 eyes (10%),Sjögren’s syndrome in 2 eyes (10%) and Stevens-Johnson Syndrome(SJS) in 1 eye (5%). Total keratolimbal allograft (KLAL) wasperformed in 80% eyes (16/20), sectoral KLAL in 15% eyes (3/20)and conjunctival limbal allograft (CLAL) in 5% eyes (1/20).Glaucoma was associated pre-operatively in 55% eyes. Five eyes hadprevious keratoplasty and 2 eyes had keratoplasty with OSST. Meantime to keratoplasty after OSST was 12.6±16.2 months (2.5-57months). Repeat keratoplasty was required in 70.5% of eyes (12/17).Mean number of keratoplasties required after OSST was 1.95±1.3 (0-5). Mean follow-up was 148±59.1 months (19-251 months) and meanduration of systemic immunosuppression was 107.6±60.3 (6-186)months in 14 eyes. Stable ocular surface was seen in 66.6% eyes(2/3) after sectoral KLAL, 23.1% eyes (3/13) after total KLAL onsystemic immunosuppressive therapy and 33% eyes (1/3) after totalKLAL without systemic immunosuppressive therapy. At last visit,10% eyes (2/20) had had Boston Keratoprosthesis (K-Pro) surgery.Repeat OSST was required in 30% eyes (6/20). Complicationsincluded recurrent epithelial defect in 14 eyes (70%), one or moreepisodes of graft rejection in 13 eyes (65%), increased intraocularpressure that required placement of tube shunt in 9 eyes (45%),infectious keratitis in 5 eyes (25%) and descemetocele in 2 eyes(10%).Conclusions: Long-term follow-up after total OSST with systemicimmunosuppression showed graft failure in 77% eyes. Multipleocular surgeries were required in 70% eyes. Recurrent epithelialdefect was the most common complication occurring in 70% eyes.Commercial Relationships: Kunal Suri, None; Stephen C.Kaufman, IOP (C)Support: Unrestricted grant from Research to Prevent BlindnessProgram Number: 5426 Poster Board Number: A0125Presentation Time: 8:30 AM - 10:15 AMMild-moderate dry eye patients exhibit ocular surfacetemperature lower than normal subjectsgiuseppe giannaccare, Piera Versura, Marco Grillini, Emilio C.Campos. Ophthalmology Unit, DIMES Department, Alma MaterStudiorum University of Bologna and S.Orsola-Malpighi TeachingHospital, Bologna, Italy.Purpose: To measure ocular surface temperature (OST) in mildmoderatedry eye (DE) patients and to correlate values with diseaseseverityMethods: 30 patients diagnosed as having mild-moderate DEaccording to DEWS guidelines (DEWS grade 1 and 2, 15 patientseach) and 30 normal subjects were included in this prospectivecontrolled study. Exclusion criteria were: history of atopy, allergicdiseases, infectious surface diseases, chemical/thermal injury.Dynamic infrared non-contact thermal imaging (Tomey TG 1000,Nagoya, Japan; Emmeciquattro, Parma, Italy) was used to measureOST in 3 circular (4 mm diameter) regions: central cornea (CC),nasal conjunctiva (NC) and temporal conjunctiva (TC). Theprocedure was repeated 3 times during a single session by 2examiners. OST was measured immediately after blinking at eyeopening (T0) and after 10 second of sustained eye opening (T1); thedifference between the two values (Δ) was calculated. Discomfortsubjective symptoms were evaluated with OSDI questionnaire.Schirmer test I, Break up Time (BUT), Lissamine green vitalstaining, conjunctival scraping cytology and determination ofexudated serum albumin in tears were performed in all subjects. OSTand Δ values recorded in all regions were correlated (Pearson’s r)with the tests. Data were statistically evaluated by applying Student’st test, Mann-Whitney and Wilcoxon’s tests, when appropriate,significance p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: giuseppe giannaccare, None; PieraVersura, None; Marco Grillini, None; Emilio C. Campos, NoneSupport: This work was partially supported by a grant fromFondazione Cassa di Risparmio di Bologna and RFO ex-60% fundsfrom University of Bologna to ECCProgram Number: 5427 Poster Board Number: A0126Presentation Time: 8:30 AM - 10:15 AMOcular Surface Changes in Glaucomatous Patients Treated withand without Preservatives beta-BlockersSerena Telani 1 , Michele M. Iester 1 , Paolo Frezzotti 2 , Michele Figus 3 ,Paolo Fogagnolo 4 , Andrea Perdicchi 5 , Antonio Ferreras 6 , CarloEnrico Traverso 1 . 1 University Eye Clinic, Genova, Italy; 2 UniversityEye Clinic, Siena, Italy; 3 University Eye Clinic, Pisa, Italy;4 University Eye Clinic, Milano, Italy; 5 University Eye Clinic, LaSapienza, Rome, Italy; 6 Univerisity Eye Clinic, Zaragoza, Spain.Purpose: To determine whether there were ocular surface changes inglaucomatous patients treated with preservatives beta-blockers whoswitched to preservative-free beta-blockers.Methods: This was prospective and longitudinal study. 132 patientswith POAG treated with a preserved beta blocker were enrolled. Allthe patients underwent perimetric and gonioscopic examination,complete ophthalmologic examination, IOP measurements,evaluation of ocular surface, Schirmer test, blood pressure and heartrate at baseline and 1-3 months after changing medical treatment to apreservative-free timolol 0.1% (Timogel 0.1, Thea). At baseline andat the end of the study, all patients underwent the Ocular SurfaceDisease Index (OSDI) questionnaire. Data were analyzed by t testwhen the distribution of the data was normal, by Mann-Whitneywhen the distribution was not-normal.Results: No significant difference was found for IOP beforeswitching from preserved beta-blockers to preservative-free ones. Nosignificant difference was found in blood pressure and heart rate.However a statistically significant difference was found for abnormalfluorescein staining of cornea and conjunctiva, eyelid erythema,conjunctival hyperaemia and follicular hyperplasia. A significantdifference was found for break-up time (from 9.38 ± 4.7 sec atbaseline to 10.64 ± 4.7 after 3 months) and Schirmer’s test (from 12.9± 5.96 mm at baseline to 14.2 ± 5.87 mm after 3 months). OSDIquestionnaire showed that the patient improved the dryness andforeign body sensation.Conclusions: Preservative-free timolol 0.1 treatment maintained IOPat the same level of the other beta-blockers, but it was better toleratedin patients having signs or symptoms while on preserved betablockers. Preservative-free treatment improved the quality of lifereducing dryness, hyperaemia, follicular hyperplasia and foreignbody sensation.Commercial Relationships: Serena Telani, None; Michele M.Iester, None; Paolo Frezzotti, None; Michele Figus, None; PaoloFogagnolo, None; Andrea Perdicchi, None; Antonio Ferreras,Alcon Laboratories, Inc (R), Allergan, Inc (R), Carl Zeiss Meditec(C), Heidelberg Engineering (F), Instituto Salud Carlos III (F),Novartis (R), Oculus, Inc (F); Carlo Enrico Traverso, MSD (F),Alcon (F), Santen (F), Thea (F), Allergan (F)Program Number: 5428 Poster Board Number: A0127Presentation Time: 8:30 AM - 10:15 AMOcular surface findings associated to clinical signs ofhyperandrogenism assessed by an innovative videodermoscopicmethodIlaria Macchi 1 , Laura Guccione 2 , Flavio Mantelli 1 , AlessandraMicera 3 , Costanzo G. Moretti 2 , Stefano Bonini 1 . 1 ophthalmology,campus biomedico unversity, Rome, Italy; 2 Endocrinology, SanGiovanni Calibita Fatebenefratelli Hospital, Rome, Italy; 3 IRCCS GBBietti, Rome, Italy.Purpose: To evaluate whether ocular surface parameters in patientswith Polycystic Ovary Syndrome (PCOS), the most commonendocrine disorder in women of reproductive age, correlate with theseverity of hirsutism, the main sign of hyperandrogenism. In fact,cornea, conjunctiva, meibomian and lacrimal glands express sexhormone receptors as well as the skin, and represent, therefore,potential androgen targets.Methods: Four PCOS patients were evaluated by videodermoscopyand ophtalmic examination at baseline (T0) and after 12 months(T12) of treatment with systemic anti-androgen therapy(Bicalutamide 50mg/die). Four healthy age-matched females wereused as controls.The ophtalmic evaluation included slit lamp examination, lacrimalfunction tests (BUT, Schirmer I), and conjunctival impressioncytology for the analysis of goblet cell by PAS and immuno-staining.The skin evaluation consisted in the collection of photos andcalculation of the videodermoscopic index (VI) at 2 representativeandrogen-dependent areas of hair growth (chin and linea alba) using anew videodermoscopic software (Alfadoc, Medical Hi-Tek). Ocularsurface data were correlated statistically to the VI.Results: All women included in this study had PCOS with clinicallyrelevant hirsutism. PCOS patients had a reduction of BUT(4.9±2.2sec vs. 9.1±1.3sec in the controls, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea25 questionnaire as well as duration of follow-up are reported.Friedman test with Dunn’s post-hoc test for multiple comparisonswas used for statistical analysis.Results: 35 males and 51 females with history of SJS/TENS weretreated. Most common reported etiologies for SJS/TENS werepenicillins (n=15), lamotrigine (n=11) and ibuprofen (n=6). Mean ageat time of treatment was 31.12 years. The median visual acuity at theinitial visit was 20/60 (0.48 logMAR), while the visual acuity atcompletion of customization was 20/25 (0.096 logMAR, p0.05, in comparison with visual acuity at the completionof customization) . There was a significant improvement in the visualfunction of the patients based on the NEI-VFQ 25 questionnaire (47.7points at baseline vs 71 points at 6 months, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Hung-Chi Chen, None; Chien-TingChen, None; Hung-Ta Chen, None; Yih-Shiou Hwang, None;Hsin-Chiung Lin, None; David H. Ma, NoneProgram Number: 5432 Poster Board Number: A0131Presentation Time: 8:30 AM - 10:15 AMAntibacterial Activity of Lysozyme in Hyperosomolar ConditionsPoonam Mudgil, John Whitehall. School of Medicine, University ofWestern Sydney, Penrith, NSW, Australia.Purpose: Hyperosmolarity is one of the core mechanisms of dry eye.Its various effects on the overall dynamics of the ocular surface stillneed to be understood. Our previous research showed thathyperosmolarity does not affect stability of the lipid layer or the tearfilm. The aim of this study was to investigate the effect ofhyperosmolarity on the activity of lysozyme, the most abundantantibacterial tear protein involved in ocular surface defence, in a tearlike solution in-vitro against Staphylococcus aureus, a bacterialisolate in ocular infection.Methods: An artificial tear (AT) solution with osmolarity nearnormal (305 mOsm/Kg) and 1.5 times salt concentrationhyperosmolar (HAT) solution (478 mOsm/Kg) were prepared.Lysozyme stock solutions were prepared in AT and HAT,respectively. Overnight culture of Staphylococcus aureus wasinoculated in the artificial tear solution in the presence of twolysozyme solutions at concentrations of lysozyme normally found intears. Standard lysozyme activity assay was performedspectrophotometrically at OD 450 . Micrococcus luteus was used as acontrol for the lysozyme activity assay. After the assay, bacterialculture was diluted and plated on LB agar plates to observe viablecounts.Results: Spectrophotometric measurements showed greater decreasein optical density of the bacterial culture indicating higher lysozymeactivity in HAT as compared with AT. Viable count resultscorroborated this by showing less number of bacterial colonies inHAT as compared with AT indicating that lysozyme was more activeand killed more bacteria in HAT.Conclusions: Hyperosmolarity has no adverse effect on theantibacterial activity of lysozyme that is normally involved in ocularsurface defence. To fully understand the role of hyperosmolarity onthe ocular surface defence, future research will involve studying moreantimicrobial proteins and ocular pathogens in hyperosmolarconditions.Commercial Relationships: Poonam Mudgil, None; JohnWhitehall, NoneProgram Number: 5433 Poster Board Number: A0132Presentation Time: 8:30 AM - 10:15 AMTreatment of Ocular Surface Failure Due to Limbal Stem CellDeficiency by Cultivated Limbal Epithelial Cell Transplantation(CLET)Beatriz Eugenia E. Ramirez Villagran 1 , M. F. Pastor 3 , Jose M.Herreras 1, 2 , Inmaculada Pérez 1 , Javier García-Sancho 3 , AnaSanchez 3 , Margarita Calonge 1, 2 . 1 Ocular Surface Group, IOBA-University of Valladolid, Valladolid, Spain; 2 CIBER-BBN(Biomedical Research Networking Center in Bioengineering,Biomaterials and Nanomedicine), Spain, Spain; 3 IBGM-University ofValladolid, Valladolid, Spain.Purpose: To evaluate the efficacy of cultivated limbal epithelialtransplantation (CLET), performed under the new Europeanregulations, for the management of ocular surface failure due tolimbal epithelial stem cell deficiency syndrome (LSCD).Methods: CLET was performed in 20 eyes (19 patients) sufferingLSCD, autologous in 11 (55%) cases and allogenic in 9 (45%) eyes.In vitro expanded limbal stem cells (in a clean room facilityfollowing GMP rules) were transplanted on amniotic membrane aftersuperficial keratectomy. LSCD etiologies were: 7 chemical burns, 5iatrogenic (post-multiple surgeries), 4 immune-based inflammation(Stevens Johnson’s syndrome and ocular pemphigoid), 2 post-chronicinfections, and 2 congenital aniridia. Outcome end-points, evaluatedafter one year follow-up were: 1] Primary: 1.1) improvement onvisual-related aspects of quality of life (National Eye Institute VisualFunctioning 25 questionnaire, NEI-VF25) and symptoms (OSDIquestionnaire); and 1.2) presence of a more corneal-like phenotype incentral cornea evaluated clinically (absence of epithelial breakdownand improvement of epithelial opacity) and by in vivo confocalmicroscopy (CM); 2] Secondary: 2.1) visual acuity improvement ≥ 2lines; 2.2) amelioration of superficial vascularization.Results: The two primary outcomes were achieved by 17 (85%)eyes; of these 17 eyes, 12 (60% of the total number) also gainedvision, 3 eyes underwent successful keratoplasty, and 2 eyes had noprevious potential to gain vision with no further interventions. NEI-VF25 questionnaire improved from 62.84+22.39 to 70.56+19.57(p=0.0011) and OSDI decreased from 47.91+25.97 to 36.12+25.16(p=0.0010). Of the 3 failed eyes, 2 had autologous CLET (chemicalburns) and 1 eye had allogeneic CLET (Stevens Johnson’s). By CM,central corneal epithelium was cornea-like phenotype in 18 eyes (13cornea phenotype, 5 mixed phenotype) and it was conjunctival-likeepithelium in 2 eyes. Neovascularization was slightly affected andreduced in only 5 cases.Conclusions: CLET significantly improved the quality of cornealepithelium in patients with LSCD. The subsequent improvement insymptoms increased quality of life. These results confirm othersstating that CLET is an alternative for ocular surface failure due toLSCD although still insufficient to eliminate corneal vascularization.Commercial Relationships: Beatriz Eugenia E. RamirezVillagran, None; M. F. Pastor, None; Jose M. Herreras, None;Inmaculada Pérez, None; Javier García-Sancho, None; AnaSanchez, None; Margarita Calonge, Allergan (C)Program Number: 5434 Poster Board Number: A0133Presentation Time: 8:30 AM - 10:15 AMPrevention, Prophylaxis, and Treatment of Ocular SurfaceDisease in a Tertiary Care Inpatient Setting: Identifying Patientsat RiskJesse T. McCann, Irina Belinsky, Lisa Park. NYU Medical Center,New York, NY.Purpose: The development of exposure keratopathy and bacterialkeratitis in the inpatient population can be reduced throughprevention. Interdisciplinary care approaches and order sets can beestablished for ocular protection once risk factors and treatmenttrends are analyzed.Methods: A retrospective review of the medical records frominpatient ophthalmology consults at a large tertiary-care teachinghospital was performed from a period of March 2011 to September2012. 41 patients were identified who met the criteria for inclusion inthis study. Risk factors including neurologic status,sedation/intubation, lagophthalmos, exposure, inflammation,oncologic status, and infection were identified. The severity of theocular surface disease was graded on a six-point scale. The patients’pre-consult eye care regimen and post-consult eye care regimen wereanalyzed.Results: A total of 60 eyes from 41 patients consulted as inpatientsfor ocular surface disease were studied. The average patient age was49. Of the patients who were able to perform visual acuity testing,best-corrected visual acuity in the affected eye was 20/32 at the timeof the initial consult. Risk factors for corneal injury in the inpatient©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapopulation included prior neurologic deficit (present in 73.17% ofpatients), 65.85% had anatomical factors affecting lid closure.46.34% of patients had been intubated or in an intensive care unitduring their admission. Of the cases examined, 36.85% had preconsultcorneal care regimens. Mean grading of ocular surfacedisease was 3.12 ± 1.83, ranging from trace punctate epithelialerosions and/or chemosis (Grade 1) to macro-epithelial defect (Grade3) and bacterial keratitis (Grade 6). After ophthalmic evaluation,82.93% of patients were prescribed artificial tears, 68.29% wereprescribed ocular ointment, and 36.59% were prescribed topicalantibiotics. Eyelid taping was advised in 9.76% of the cases studied.Conclusions: By establishing risk factors for ocular surface injurychecklists and prophylaxis programs can be established. Routineprescriptions for ocular protection, including eyelid taping and theuse of ocular lubricants, can help reduce the burden of ocular surfaceinjury in the inpatient population.Commercial Relationships: Jesse T. McCann, None; IrinaBelinsky, None; Lisa Park, NoneProgram Number: 5435 Poster Board Number: A0134Presentation Time: 8:30 AM - 10:15 AMRisk Factors of Severe Acanthamoeba KeratitisMachiko Shimmura-Tomita, Hiroko Takano, Nozomi Kinoshita,Fumihiko Toyoda, Ayumi Ota, Akihiro Kakehashi. Ophthalmology,Jichi Medical University Saitama Medical Center, Saitama, Japan.Purpose: To determine risk factors for severe acanthamoeba keratitisby comparing severe cases with good prognosis cases.Methods: We reviewed medical records of 9 cases of acanthamoebakeratitis (4 males and 5 females) referred to Jichi Medical UniversitySaitama Medical Center between May 2005 and December 2011. Themean age of cases was 29, and the mean follow up period was 450days. One eye that required therapeutic keratoplasty due to corneaperforation and 3 eyes requiring optical keratoplasty were included inthe severe group. Five eyes which had good prognosis and a bestcorrected visual acuity of 1.2 on last visit were classified as mildgroup. We compared the time required for diagnosis, visual acuity onfirst visit, the history of steroid eye drops use, cornea scraping timesand clinical findings.Results: The duration between onset and diagnosis of acanthamoebakeratitis was 330 days in 1 case of severe group, and in all of theother cases was within 30 days. Best corrected visual acuity was0.01-0.8 in severe group (mean 0.32), and 0.15-1.2 in mild group(mean 0.56). A history of steroid eye drops use was found in 4 eyes(3 with 0.1% betamethasone, 1 with 0.1% fluorometholone) of severegroup (100%), and 3 eyes (all with 0.1% fluorometholone) of mildgroup (60%). Mean number of cornea scraping was 13.8 times insevere group, and 5.6 times in mild group. Keratoprecipitates werefound in all severe group eyes during follow up period. One case ofsevere group was diagnosed with diabetes mellitus at initialexamination. We detected Staphylococcus aureus by palpebralconjunctiva culture in 1 case of the severe group which was resistantto topical antibiotics.Conclusions: The use of corticosteroids and the presence ofkeratoprecipitates are possible risk factors of severe acanthamoebakeratitis. Attention is also required in patients with comorbiditiessuch as diabetes mellitus and bacterial infection.Commercial Relationships: Machiko Shimmura-Tomita, None;Hiroko Takano, None; Nozomi Kinoshita, None; FumihikoToyoda, None; Ayumi Ota, None; Akihiro Kakehashi, NoneProgram Number: 5436 Poster Board Number: A0135Presentation Time: 8:30 AM - 10:15 AMEvaluation of different applicator-systems for autologous serumeye-dropsAlexander Kunze, Kristina Spaniol, Gerd Geerling. Ophthalmology,University Hospital Duesseldorf, Duesseldorf, Germany.Purpose: Autologous serum eye-drops (ESAT) are an efficienttherapy for many diseases of the ocular surface, askeratoconjunctivitis sicca or persistent epithelial defects. Apart fromconventional eye drop bottles recently new applicator-systems areavailable for the production of autologous serum eye drops in aclosed system without the need of a health care clean room. Aim ofthis study was to compare these systems concerning costs, efficiencyand user-friendliness.Methods: Conventional 5 ml eye drop bottles (distribution: Co.Zscheile and Klinger), fusible tube-segments (Co. MacoPharma) andpharmaceutical one-way phials (Co. Meise) were evaluated. Thethree systems were used independently by healthy probands (n=10),by patients with restricted ability in applying eye drops (n=1) and byvisually impaired probands (n=10). Data concerning the userfriendlinessof each system was collected on the basis of aquestionnaire. The individual costs of the different systems includingfabrication were determined.Results: There was a considerable cost differential between theevaluated systems: the price for a three-month dose (half-year dose)was 667,55 € (1011,80 €) for the one-way phials, 494,30 € (559,70 €)for the eye drop bottles and 359,20 € (385,10 €) for the tubesegments.The probands rated the usage of the eye drop bottles andthe usage of the one-way phials to be similarly easy. Patients withimpairments of the hands rated the usage of the tube-segments to bemost difficult. Handling of the cutable tube-segments was ratedsimilarly poor by probands of all groups. In knowlegde of the prizefor each system 67% of the probands choose the conventional eyedrop bottles.Conclusions: Patients with diseases of the ocular surface mostlyneed a protracted topical therapy. Additionally they often havemanual or visual impairments. In our study conventional eye dropbottles were preferred above the two new systems regarding price andhandling. Cutable tube-segments display a low priced system but theyare not applicable for impaired persons. Even in knowledge of thelow price probands did not choose this applicator system. The onewayphials apply comfortable handling but they are not cost-efficientenough.Commercial Relationships: Alexander Kunze, None; KristinaSpaniol, None; Gerd Geerling, Alcon (C), Allergan (C), TheaPharma (C), Novagali (C), Bausch & Lomb (C), Tearlab Inc. (C)Program Number: 5437 Poster Board Number: A0136Presentation Time: 8:30 AM - 10:15 AMStandardized Quantification of Inflammatory Biomarker HLA-DR for Multicenter Clinical Trials of Ocular Surface DiseasePenny A. Asbell 1 , Seth P. Epstein 1 , Neha Gadaria-Rathod 1 , Yi Wei 1 ,Maureen G. Maguire 2 . 1 Ophthalmology, Mount Sinai School ofMedicine, New York, NY; 2 Ophthalmology, University ofPennsylvania, Philadelphia, PA.Purpose: To establish a standardized methodology for determiningthe relative amount of HLA-DR on ocular surface cells for use as aminimally invasive objective metric for large scale multicenterclinical trials and translational research studies of ocular surfacedisease.Methods: To establish a Standard Operating Procedure (SOP) forutilizing HLA-DR expression as a biomarker of inflammation of theocular surface the following was done: duplicate cell samples fromimpression cytology (IC) samples of both normal and dry eye (DE)individuals were collected and split to assess repeatability. To©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadetermine storage capability, one duplicate was stained immediatelyand the other after 30 days. To demonstrate the feasibility of the useof the SOP for a multicenter clinical trial, clinicians from out-of-statewere trained to collect IC samples which were shipped to New Yorkfor processing and analysis by a masked independent observer. ICand HLA-DR quantification were incorporated into a maskedrandomized clinical trial of DE.Results: The validity/viability of the SOPs was established: 1)sufficient numbers of cells can be collected via IC (average: 6,677 ±6,302 cells/filter (normal: 8,511 ± 7,743, n = 114, range: 1,389 −101,031; DED: 5,414 ± 5,128, n = 252, range: 1,032 − 58,892). 2)relative biomarker expression quantified in samples isprecise/repeatable (mean difference: −0.12%, p = 0.23; 95% limits ofagreement for splits: −0.82%, +0.58%); 3) personnel at distant sitescan be taught to collect, store and ship samples; 4) samples can bestored until processing can be performed without affecting results[mean difference for HLA-DR%: 0.31% (ie, Day 30 values were onaverage higher by 0.31%; p = 0.11); 95% limits of agreement were(−0.91%, +1.53%)]; 5) a large number of masked samples can bereliably tracked.Conclusions: We demonstrated the repeatability and effects ofstorage, ability to train/gather samples from distant sites and thefeasibility of use in a randomized clinical trial. HLA-DR expressionas determined from IC samples can serve as a minimally invasiveobjective metric of inflammation for efficacy and mechanism ofaction in randomized clinical trials of ocular surface disease.Commercial Relationships: Penny A. Asbell, RPS (F); Seth P.Epstein, Rapid Pathogen Screening, Inc (F); Neha Gadaria-Rathod,None; Yi Wei, None; Maureen G. Maguire, InspirePharmaceuticals (F), Amakem (F), IDx LLC (F), Merck (C)Support: Supported in part by the National Eye Institute (R34-EY017626-01) as well as by The Martin and Toni SosnoffFoundation, New York, New York.Program Number: 5438 Poster Board Number: A0137Presentation Time: 8:30 AM - 10:15 AMCytokine tear film expression in patients with primary andrecurrent pterygiumVictor M. Bautista, Nayeli Rangel-Acosta, Nadia Luz López-Espinosa, Angel Nava-Castañeda. Microbiology and OcularProteomics, Inst de Ophthal Conde de Valenciana, Mexico, Mexico.Purpose: Pterygium is an overgrowth of fibrovascular tissue, oftenwith a wing-like appearance, from the conjuctiva over the cornea.Although the pathogenesis of pterygium is not clearly understood,certain findings concerning common features in pterygium andneoplasia have been proposed, raising the possibility that a pterygiumis a neoplastic-like growth disorder. There is much debate surroundthe pathogenesis of pterygium and a number of theories have beenput forward including genetic instability, cellular proliferation,inflammatory influence, degeneration of connective tissue,angiogenesis, aberrant apoptosis or wound healing process and stemcell dysfunction. Treatment of pterygia entails its surgical excision,however in some cases they aggressively recur. The aim of this studywas to analyze the cytokine tear film expression in patients withprimary and recurrent pterygium.Methods: Tear samples from patient diagnosed with primary andrecurrent pterygium, and tears from patients without pterygiumdiagnostics were taken as a control were analyzed. Cytokine proteinarrays were performed to know relative units of 36 cytokines, withthe Profiler Array Membrane (R&D Systems, Minneapolis, USA)according to the manufacturer instructions. Densitometric analysis ofthe dot blot was performed with Diversity and GeneTools. Weconsidered cytokines with differential expression more or less thantwo times.Results: When we analyzed cytokine tear film expression amongprimary pterygium patients and controls, we found that GROa,Sicam-1, IL-8, IL-16, I-TAC, MCP-1 and MIP-1b wereoverexpressed; meanwhile IP-10, MIF and Serpin E1 weredownregulated. Cytokine tear film expression comparison amongcurrent pterigion patients and controls showed that C5/C5a, IL-16,IL-17, IL-32a and IP-10 were overexpressed; none cytokine showeddownregulation. Finally, cytokine tear film expression comparisonamong recurrent and primary pterygium exhibited a greatoverexpression of IP-10 and MIF, and MCP-1 was downregulated.Conclusions: Cytokine tear film expression in primary pterygiumsuggests an inflammatory response mediated by chemokines. Inrecurrent pterygium tear film, results showed an overexpression ofinflammatory cytokines. Comparison among primary and recurrentpterygium exhibited a MIF and IP-10 overexpression, suggesting achronic inflammatory process.Commercial Relationships: Victor M. Bautista, None; NayeliRangel-Acosta, None; Nadia Luz López-Espinosa, None; AngelNava-Castañeda, NoneProgram Number: 5439 Poster Board Number: A0138Presentation Time: 8:30 AM - 10:15 AMEffectiveness and safety of intralesional injection of 0.01 mgMitomycin C one month prior to bare sclera excision forpterygium treatmentBrian Tieu, Manuj Kapur. UTMB, Galveston, TX.Purpose: Mitomycin C (MMC) is an anti-metabolite withradiomimetic properties that is used as an adjuvant in pterygiumexcisions to reduce recurrence; however, it can cause many sideeffects particularly at high doses. Previous studies have shown thatinjection of 0.15 to 0.2 mg/mL MMC one month prior to bare scleraexcision results in low recurrence rates, but may result in somecomplications. The purpose of this study was to determine if thelowest effective concentration of 0.1 mg/ml MMC injectedintralesionally can maintain low recurrence rates and be potentiallysafer.Methods: A retrospective case review was performed of patientswho underwent bare sclera pterygium excision one month afterintralesional injection of 0.01 mg MMC. The cases were evaluatedfor complications including dellen, persistent corneal epithelial orconjunctival defect, intraocular pressure spikes, scleral thinning, andrecurrence of disease, which was defined as re-growth offibrovascular tissue greater than 1 mm past the limbus. Surgicalspecimens also were reviewed for significant changes.Results: Eleven patients underwent this procedure and were followedpost-operatively for at least 6 months. No recurrences have beennoted nor major complications. Histopathological analysis of theexcised tissue showed less vascularization.Conclusions: Intralesional injection of a lower dose of MMC onemonth prior to bare sclera excision may be as or more effective thanhigher doses, but possibly safer overall with exposure to less of theanti-metabolite.Commercial Relationships: Brian Tieu, None; Manuj Kapur,NoneProgram Number: 5440 Poster Board Number: A0139Presentation Time: 8:30 AM - 10:15 AMExtended Soft Bandage Contact Lenses Therapy for OcularGraft-Versus-Host DiseaseYichen Sun 1, 4 , Yoshihiro Inamoto 3 , Joseph P. Sheehan 1 , Peng Li 2 ,Ruikang K. Wang 1, 2 , Stephanie Lee 3 , Tueng T. Shen 1, 2 .1 Ophthalmology, University of Washington, Seattle, WA;©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea2 Bioengineering, University of Washington, Seattle, WA; 3 FredHutchinson Cancer Research Center, Seattle, WA; 4 Graduate Instituteof Clinical Medicine, National Taiwan University, Taipei, Taiwan.Purpose: Graft-versus-Host Disease (GVHD) is a majorcomplication of allogenic tissue/hematopoietic stem celltransplantation. 60% of GVHD patients have ocular involvementwith significant compromise in quality of life due to ocular symptoms(such as severe photophobia, pain and decreased visual acuity). Wereport the outcome of extended soft bandage contact lens (BCL)treatment for symptomatic relief and ocular surface protection inGVHD patients.Methods: Prospective Phase II clinical trial. IRB approval of theclinical protocol was first obtained. Patients (age 18-99) withdiagnosis of chronic GVHD as defined by the NIH criteria and ocularsymptoms of NIH eye score 2 or greater are selected for the studywith written informed consent. Extended soft bandage contact lenseswere applied to the GVHD-affected eyes with antibiotic coverageduring the two week period. Ocular exam, anterior segment OCT(AS-OCT) and patient survey are obtained. Patients are followed forone month. Clinical outcomes, such as visual acuity, cornealpresentations (abrasion, punctate epithelial erosion and filament) arecorrelated with symptomatic survey findings as well as OCTfindings.Results: 20 patients with ocular GVHD will be included in the study.Ocular manifestations of GVHD observed by clinical exam are alsocharacterized by AS-OCT. First six including patients are all shownwith improving symptoms/signs and without any occurrence ofcomplications. Symptomatic changes with the BCL therapy arecorrelated with ocular exams as well as AS-OCT.Conclusions: BCL can offer significant symptomatic relief forpatients with ocular GVHD. AS-OCT can be a feasible method tocharacterize pathological changes related to ocular GVHD. Inaddition, wearing of extended soft bandage contact lens can diminishthe attrition of cornea from eyelid and provide symptomatic relief forpatients with ocular GVHD.Commercial Relationships: Yichen Sun, None; YoshihiroInamoto, None; Joseph P. Sheehan, None; Peng Li, None;Ruikang K. Wang, National Institutes of Health (F), W.H. CoulterFoundation Translational Research Partnership Program (F),Research to prevent blindness (F), Oregon Health & ScienceUniversity (P), University of Washington (P); Stephanie Lee, None;Tueng T. Shen, NoneSupport: CA163438Clinical Trial: NCT01616056Program Number: 5441 Poster Board Number: A0140Presentation Time: 8:30 AM - 10:15 AMThree-dimensional volumetric reconstruction of the mousediabetic corneaDanielle M. Robertson 1 , Matthew Petroll 1 , Meifang Zhu 1 , VindhyaKoppaka 1, 2 . 1 Ophthalmology, Univ Texas Southwestern Med Ctr,Dallas, TX; 2 Pharmacy, University of Colorado Denver, Denver, CO.Purpose: Mouse models of diabetes are routinely used to investigatethe effects of hyperglycemia in vivo. The purpose of this project wasto investigate the use of three-dimensional volumetric imaging of thesubbasal nerve plexus and associated corneal epithelial nerve fibersto allow for quantitative assessment of corneal nerve changes in adiabetic mouse model; and to correlate alterations in corneal nerveswith total and sublayer thickness changes in vivo.Methods: Type I diabetes was induced in male C57BL/6 mice usingthe Animal Models of Diabetic Complications Consortium high dosestreptozotocin (150 mg/kg body weight) protocol. Mice wereevaluated for corneal nerve and epithelial changes at 6 and 12 weeks.Age-matched vehicle treated mice were used as controls. Serumglucose levels and body weight were assessed at 6 and 12 weeks. Aserum glucose level greater than 300 mg/dl was diagnostic ofdiabetes. Total corneal and sublayer thickness changes were assessedusing quantitative three-dimensional in vivo confocal microscopy(q3D-IVCM). Corneas were then excised and stained with anti-betatubulinIII and labeled with a FITC-conjugated secondary antibody.Nuclei were counter-stained with propidium iodide. Whole mountcorneas were imaged on a laser scanning confocal microscope. Imagestacks were reconstructed three-dimensionally and analyzed usingMetaMorph and IMARIS software.Results: Using q3D-IVCM, mean corneal thickness in control micewas 111.5 ± 11.2 µm. Sublayer thickness values were 73.2 ± 5.2 µmand 38.3 ± 6.5 µm, for the stroma and epithelium, respectively.Volumetric reconstruction of corneal nerves demonstrated thatepithelial nerve fibers extending from the subbasal plexus turn andcourse horizontally between superficial epithelial cell layers prior totermination with occasional branching and anastomoses detected. InSTZ-treated mice, significant changes in serum glucose levels andbody weight were evident. Alterations in corneal epithelial nervesand the subbasal nerve plexus were seen as early as 6 weeks.Conclusions: Three-dimensional volumetric reconstruction of themouse cornea allows for quantitative assessment of corneal tissue andnerve changes and represents a novel technique for assessingdiabetic-induced changes in the mouse cornea.Commercial Relationships: Danielle M. Robertson, None;Matthew Petroll, None; Meifang Zhu, None; Vindhya Koppaka,NoneSupport: NIH RO1 EY018219 (DMR), NIH P30 EY020799,OneSight Research Foundation, Dallas, TX (DMR), and a CareerDevelopment Award (DMR) and an unrestricted grant from Researchto Prevent Blindness, Inc., New York, NYProgram Number: 5442 Poster Board Number: A0141Presentation Time: 8:30 AM - 10:15 AMRat Exorbital Lacrimal Gland Ducts Have Gastrin ReceptorsMortimer Lorber 1 , Eva C. Permaul 2 , Supti Sen 2 , Deborah Berry 2 .1 Pharmacology & Physiology, Georgetown Univ School of Medicine,Washington, DC; 2 Lombardi Comprehensive Cancer Center,Georgetown Univ School of Medicine, Washington, DC.Purpose: In ARVO 2009, we showed that these ducts bind thecommon C-terminal pentapeptide of both Gastrin andCholecystokinin, indicating that lacrimal ducts bind one or both ofthem. This abstract reports that they bind gastrin. The possibility thatthey also bind cholecystokinin has not yet been studied.Methods: One exorbital lacrimal gland was excised from four femaleadult rats and fixed in buffered formalin. Immunohistochemistry wasperformed using a rabbit polyclonal antibody against the gastrinreceptor, CCKBR. This demonstrated reactivity against rat tissues. A1:500 dilution in phosphate buffered saline, pH 7.4 with 1% bovineserum albumin and 0.025% sodium azide, was made.The antibodywas by Alamone Labs in Jerusalem. The positive control was the ratstomach whose HCl producing parietal cells were stained. Thenegative control omitted the primary antibody.Results: In all specimens essentially all nuclei of the intercalated,intra- and interlobular, and collecting ducts were strongly positive.Their cytoplasm was negative. The nuclei and cytoplasms of acinarand myoepithelial cells were negative. The lumens of some of thelarger ducts had nuclear and cytoplasmic debris originating fromductal cytoplasmic blebs and the turnover of duct nuclei. Thismaterial enters the tears and passes through the nasolacrimal ducts tomix with nasopharyngeal secretions to enter the alimentary tract.Conclusions: The duct nuclei of rat exorbital lacrimal glands contain©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneathe specific receptor for gastrin. Just as gastrin affects gastricsecretion might it affect how the llacrimal ducts modify the acinarsecretions they receive?Commercial Relationships: Mortimer Lorber, None; Eva C.Permaul, None; Supti Sen, None; Deborah Berry, NoneProgram Number: 5443 Poster Board Number: A0142Presentation Time: 8:30 AM - 10:15 AMChanges in corneal innervations after herpes simplex virus type 1(HSV-1) latency established with different reactivationphenotypesJiucheng He 1, 2 , Richard Cosby 1, 2 , James M. Hill 1, 2 , Haydee E.Bazan 1, 2 . 1 Ophthalmology & Neuroscience Ctr, LSU Health SciencesCenter, New Orleans, LA; 2 LSU Eye Center, LSU Health SciencesCenter, New Orleans, LA.Purpose: HSV-1 is a neurotrophic virus that establishes latency insensory ganglia. Recurrent herpetic stromal keratitis (HSK) is themost common cause of infectious blindness in developed countries.Clinically, patients with HSK often have decreased cornealsensitivity, which is strongly correlated with decreased cornealinnervation after HSV infection. However, the exact pathogenesisremains unclear. Here we used a rabbit model infected with highphenotypic reactivators as well as recombinant HSV-1 with deletionsto study their effect on corneal innervation after latency wasestablished.Methods: Adult NEZ rabbits were inoculated with 50 µl (200,000-300,000 PFUs) of HSV-1 McKrae , 17 Syn+ or recombinant mutantswith gK deletion or an immediately early protein 0 (ICP0) deletion.The rabbits were sacrificed and the corneas removed between 124 to125 days post-infection. After fixation, the corneas were stained withanti-βIII tublin antibody. Images were acquired with a fluorescencemicroscope in time-lapse mode and merged together to build a wholeview of the corneal nerve architecture. Corneal subbasal nervedensity was calculated on the basis of the whole mount view of thecentral area. Differences between the HSV infected eyes, ascompared with normal control, were analyzed by ANOVA.Results: In comparison with the normal corneas, all the HSVinfectedcorneas showed significant decrease in corneal nervedensity. The corneas infected with ICP0 and gK deletion showedmild to moderate nerve damage while those infected with 17Syn+and McKrae were seriously damaged. In the ICP0 deletion eyes, therewere reduced subbasal nerve bundles, but most of the corneas kept anormal stromal network; in gK deletion eyes, both epithelial andstromal nerves were involved. Corneas infected with 17Syn+ andMcKrae displayed destroyed nerve structures and formation of a scartissue in the central cornea, in which only a few nerve fibers could bedetected. Around the scar tissue, there were some newly-regeneratednerves. In addition, a dense infiltration of inflammatory cells wasdetected.Conclusions: The results suggest that HSV infection seriouslydamages corneal innervations and persist after four months ofinfection. Reduction of axonal transport (by gk deletion) or virusreplication (by ICP0 deletion) significantly attenuates the nervedamage induced by HSV-1.Commercial Relationships: Jiucheng He, None; Richard Cosby,None; James M. Hill, None; Haydee E. Bazan, NoneSupport: NIH R019465Program Number: 5444 Poster Board Number: A0143Presentation Time: 8:30 AM - 10:15 AMAssessment of the Eyeprim® Device for Conjunctival Impressionand PCRPierre Roy 1 , Hervé Groux 2 , Francoise Cottrez 2 , Laurene Protat 1 .1 OPIA Technologies SAS, Paris, France; 2 IMMUNOSEARCH,Grasse, France.Purpose: To evaluate the use of Conjunctival impression (CI)EYEPRIM device, that previously demonstrated increased cellcollection and reproducibility than the current CI method, for themeasurement of 3 biomarkers HLA-DR, CCR4, CCR5 of the ocularsurface by quantitative Polymerase Chain Reaction (qPCR) followingdifferent extraction methods.Methods: CI specimens of the upper bulbar conjunctiva werecollected with EYEPRIM from 4 healthy volunteers in right eye (RE)and left eye (LE), without anaesthesia.Specimens were transferred in tubes containing 1ml of differentextraction media: Group 1 in Phenol/guanidine-based Qiazol LysisReagent (QIAGEN) vortexed 1min, Group 2 in Qiagen RLT softbuffer medium vortexed 1min and groups 3 & 4 in RLT buffermedium with steel beads (5mm dia.). Group 3 was agitated softly andgroup 4 transferred into the tissue lyser II (Qiagen) for 2 cycles of1.5min. RE and LE of each volunteer were processed with the sameextraction protocol to assess the reproducibility of the results.The mRNA was extracted using the RNeasy kit (Qiagen) andprocessed by qPCR with HLA-DR, CCR4 and CCR5 specificprimers.Results: For all the extraction protocols, enough ARNm material wascollected to process the 3 biomarkers. Group 1 QIAzol Lysis Reagent+ vortex, gave the best results with an average of 300ng of ARNmcollected, followed by Group 3. Group 2 was the least efficientmethod. Group 4 gave intermediate results.qPCR gave 4 different profiles of biomarkers for each volunteer: Firstvolunteer had little HLA-DR, no CCR4 and no CCR5. second hadlow expression level of all 3 biomarkers. third had highest levels ofHLA-DR and CCR5 compared with the others, last had only CCR5.None expressed significant levels of CCR4.Conclusions: EYEPRIM is an effective sampling tool, compatiblewith biomarker analysis by qPCR. This feasibility study showed avery good reproducibility of the samples, and different profiles ofconjunctiva, characterized by biomarkers, were found. Furtherclinical studies with characterized pathological patients should becompleted to identify the pathological threshold of those biomarkers,in order to confirm the potential use of EYEPRIM for the diagnosisof ocular surface disorders. Special attention should be paid to theextraction protocol.Commercial Relationships: Pierre Roy, OPIA Technologies (P);Hervé Groux, None; Francoise Cottrez, ImmunoSearch (E);Laurene Protat, Opia Technologies (E)Program Number: 5445 Poster Board Number: A0144Presentation Time: 8:30 AM - 10:15 AMClassification of the ocular surface manifestations in patientswith Stevens-Johnson SyndromeTais H. Wakamatsu, Myrna S. Santos, Telma P. Barreiro, Charles C.Farias, Flavio E. Hirai, Jose A. Gomes. Ophthalmology, FederalUniversity of São Paulo, Sao Paulo, Brazil.Purpose: To evaluate and grade the extent and severity of ocularsurface manifestations in patients with Stevens-Johnson Syndrome(SJS).Methods: Thirty four eyes of 22 patients with SJS that underwentocular surface surgery and twenty nine eyes of 19 patients with SJSwithout previous ocular surface surgery were studied. Ocular surfacemanifestations were categorized as corneal, conjunctival, eyelidcomplications and presence of dry eye disease and 9 componentswere evaluated and graded on a scale from 0 to 3 according to theirseverity. This grading system comprises of corneal (epitheliopathy,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaopacity and limbal stem cell deficiency), conjuntival (inflammationand cicatrization), eyelid (keratinization and eye lashes alterations)complications and dry eye status (Shirmer Test and cornealcicatrization). Interobserver agreement in grading the severity of SJSocular surface findings was evaluated.Results: The proposed grading system for assessing ocular surfacemanifestations demonstrated a moderate agreement. When eachfeature was analyzed separately the data shows a strong agreementfor limbal stem cell deficiency (kappa=0.67) and corneal opacity(kappa=0.75) and moderate agreement for conjunctival inflammation(kappa=0.40), conjunctival cicatrization (kappa=0.54), corneal andconjunctival keratinization (kappa=0.52), eyelid keratinization(kappa=0.42), eye lashes alterations (kappa=0.47). The most severelyaffected complication components were limbal stem cell deficiency(26 eyes; 41,2%) and dry eye (Shirmer Test - 15 eyes; 23,8%). Theseverity of corneal, conjunctival and dry eye disease was significantlycorrelated with logarithm of the minimum angle of resolution(logMAR) visual acuity.Conclusions: The authors describe a new classification for gradingthe severity of ocular surface findings in patients with SJS. Thisgrading system provides a more objective method for evaluating SJSpatients and may be adapted for use in the analysis of surgeryindications.Commercial Relationships: Tais H. Wakamatsu, None; Myrna S.Santos, None; Telma P. Barreiro, None; Charles C. Farias, None;Flavio E. Hirai, None; Jose A. Gomes, Allergan (C), Pfizer (C),Genon (C), MSD (C)Program Number: 5446 Poster Board Number: A0145Presentation Time: 8:30 AM - 10:15 AMOcular surface in Sjögren’s syndromeHayette Rebika 1 , Sihem Lazreg 2 . 1 CHU Clermont Ferrand, ClermontFerrand, France; 2 Cabinet Dr Lazreg, Blida, Algeria.Purpose: Sjögren syndrome is a chronic inflammatory disordercharacterized by exocrine gland dysfunction and a systemic course.Lymphocytic infiltration of the lacrimal and salivary glands results inthe classic sicca complex characterized by dry eyes and dry mouth.This prospective study aimed to describe the ocular and biologicalfeatures of patients with Sjögren syndrome in an Algerian population.Methods: Prospective study of 78 patients with dry eye syndrome,examined between September 2010 and November 2012. All patientsunderwent complete ophthalmological examination, including aSchirmer test, fluorescein and lissamine green staining, immuneserum assessment and salivary gland biopsy. In addition to theirtreatment prescribed by the rheumatologist, they were treated withlacrimal substitutes and punctual plugs.Results: The population was principally females (n = 75). The meanage was 45.4+/- 12.6 years old.At the first visit, the patients presented with mean Oxford and OSDIscores of 6 ±1.8 and 76.2 ± 10.2 respectively. The mean Schirmer testwas 4.5 ± 1.5. Furthermore, 40% of patients presented withfilamentous keratitis. The compromised ocular surface induced 5cases of blindness (in both eyes in one case and in one eye in 4cases).Association with rheumatoid arthritis was found in 42% of cases. TheSSG was primary in 1/3 of patients. SSA and SSB antibodies weredetected respectively in 85% and 89% of patients.In these compromised patients, punctual plugs insertion led to adecrease of about 50% on the frequency of use of lacrimalsubstitutes.Conclusions: In Algerian population, Sjögren’s syndrome is aserious disease, with an impact on the quality of life due to cornealinvolvement that is often very severe and sometimes irreversible. Inspite of correct systemic treatment (plaquenil, corticosteroids,pilocarpine), the ocular surface alterations occur.In Sjogren’s disease, symptoms of dry eye are most often associatedwith an deleterious impact on vision-related quality of life.Commercial Relationships: Hayette Rebika, None; Sihem Lazreg,NoneProgram Number: 5447 Poster Board Number: A0146Presentation Time: 8:30 AM - 10:15 AMTreatment of Ocular cGVHD with PRP: a confocal microscopystudyMarco Marenco 1 , emanuele gerace 1 , Andrea Maria Plateroti 1 ,Caterina Colica 1 , Giancarlo Ferrazza 2 , Lidia De Felice 2 , Enzo M.Vingolo 1 , Magda Gharbiya 1 , Rocco Plateroti 1 . 1 Ophthalmology,Sapienza University of Roma, Rome, Italy; 2 Hematology, SapienzaUniversity of Roma, Rome, Italy.Purpose: Graft versus host disease (GVHD) is a sequela of allogenichematopoietic stem cell transplantion (allo-SCT) which results from adonor-origin cellular response against host tissues. It involves thecornea, the conjunctiva, the lacrimal glands, the lids and the scleraand quality of life can be seriously impacted.The aims of the treatment is the resolution of the inflammation andthe maintaining of the integrity of the ocular surface.The aim of our study is to verify the efficacy of platet rich plasma eyedrop in patients with cGVHD ocular manifestations.Methods: 20 patients treated with one drop 5 time a day for 3 month.Patients underwent a completed ocular exam, a rated subjectiveassesment and a confocal microscopy (Confoscan 3). Preparation ofPRP: 10 ml of platelet rich plasma (PRP) were obtained from 16 mlof autologous peripheral blood into two REGEN RTHT tubes aftercentrifugation 8 minutes at 1500xg. It’s possible to variate PRPvolume transferring in another tube then draw off 2 ml supernatantafter centrifugation and homogenize by inversion of the tube. In thisway the platelet number can reach twice or three times greater thanperipheral values. Platelet gel was performed adding PRP 5 ml ofautologous serum plus 2 ml of Ca-gluconate.Results: 15 out of 20 patients achieved an important subjectiveimprovement after 3 months therapy; a better sub-epithelial nervusplex trophism was demonstrated in these microscopy with a cleardisminution of keratocytic activation. 3 patients showed symptomaticimprovements just during the PRP administration. The last 2 patientsdid not show any improvement, although a decrease of inflammatorycells was verified with confocal microscopy exam.Conclusions: This study proves that PRP is an effective oculartherapy which improved our patients quality of life and their cornealtrophism, even after the conclusion of therapy. Finally, it seems thatPRP play a modulation role on inflammatory cells of ocular surface.Commercial Relationships: Marco Marenco, None; emanuelegerace, None; Andrea Maria Plateroti, None; Caterina Colica,None; Giancarlo Ferrazza, None; Lidia De Felice, None; Enzo M.Vingolo, None; Magda Gharbiya, None; Rocco Plateroti, NoneProgram Number: 5448 Poster Board Number: A0147Presentation Time: 8:30 AM - 10:15 AMAngiogenin is a Requirement for Active Stromal tissueFormation and Recurrence of PterygiumJae Chan Kim, Kyoung Woo Kim, Soo Hyun Park, Yeoun Sook Chun,Sung Wook Wee. Department of Ophthalmology, Chung-AngUniversity Hospital, Seoul, Republic of Korea.Purpose: Angiogenin is originally identified as a tumor angiogenicfactor, and its biological activity is extended to stimulating cellmigration and proliferation, and more recently to promoting cellsurvival. Because angiogenesis and cell proliferation are well known©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaprerequisites for tissue formation of pterygium, we investigated theexpression profiles of angiogenin as a novel factor related withpathogenesis of pterygium.Methods: In cultured stromal fibroblasts and surface epithelial cellsof pterygia (42 eyes) and normal conjunctiva (12 eyes) as a controlgroup, angiogenin expression was analyzed by RT-PCR at mRNAlevel and by Western blotting at protein level. Immunohistochemistrywas used to assess the localization of angiogenin-expressing cells inpterygia. The pattern of expression in cultured cells and excisedtissues was comparatively evaluated according to representativeclinical indices based on recurrency, grade based on pterygial bodymorphology (T1-T3) or vascularity (V1-V3), and disease activity.Results: Angiogenin expression was higher in the stromal fibroblaststhan epithelial cells. In cultured fibroblasts of pterygia, the recurred,T3 grade or active (recurred or occurred within recent 6 months)pterygia revealed significantly higher expression than the others atboth mRNA and protein levels. In immunohistochemistry,angiogenin was strongly expressed in stromal layer of active andrecurred pterygia with T3 grade, especially at the perivascular area incontrast to scarce expression in normal conjunctiva. Pterygia withhigh vascularity had a tendency of relatively higher expression ofangiogenin.Conclusions: Angiogenin expressed in pterygia might contribute toactive stromal tissue formation with angiogenesis and furtherrecurrence. This newly uncovered finding suggests the possible effectof angiogenin to stimulate the stromal fibroblasts to proliferate inpathogenesis of pterygium.Commercial Relationships: Jae Chan Kim, None; Kyoung WooKim, None; Soo Hyun Park, None; Yeoun Sook Chun, None; SungWook Wee, NoneProgram Number: 5449 Poster Board Number: A0148Presentation Time: 8:30 AM - 10:15 AMA Plaque Method for Direct and Sensitive Enumeration ofSurviving Acanthamoeba During Biocidal Efficacy and RegimenTesting of Contact Lens Disinfection SolutionsShawn C. Lynch, Joseph G. Carr, Christopher Kovacs, Matthew A.Dehmler, Todd Bassage, Wolfgang Haas, Timothy W. Morris. Bausch& Lomb, Rochester, NY.Purpose: Currently, there are no established methods or performancecriteria for testing disinfection efficacy of contact lens solutionsagainst amoeba that can cause sight-threatening keratitis. Here wesought to develop robust, reproducible procedures for conductingstandardized biocidal efficacy and regimen testing againstAcanthamoeba castellanii using a direct plaque enumeration method.Methods: A. castellanii ATCC 50370 trophozoites were cultured inchemically defined antibiotic-free medium (AC6) prior to testingagainst polyaminopropyl biguanide/polyquaternium or alexidine/polyquaternium based solutions. For biocidal assays test solutionswere inoculated and held for 4 or 24 hr at which time aliquots wereremoved, neutralized, and serially diluted in AC6 medium containingneutralizers (AC6N). For regimen testing, amoeba in organic soilwere inoculated on both sides of contact lens for 5 minutes, followedby treatments with test and control solutions (10 sec. rub, 5 sec. rinseper side, 4 hr soak in lens cases). For both assays, test solutions,amoeba recovered from lenses, and control suspensions of amoebawere neutralized with AC6N, added to standardized suspensions of E.coli plus molten top agar, and overlaid onto nutrient-limited agarplates. Plates were incubated at 28°C and monitored daily for up to 7days to enumerate plaque forming units (pfu) within the E. coli lawn.Results: Biocidal activity showed similar results forpolyaminopropyl biguanide/polyquaternium andalexidine/polyquaternium based solutions with log reduction valuesranging from 1.3 - 2.1 at 4 hr to 3.8 - 4.5 at 24 hr. In comparison, thenegative control solution exhibited only

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Lynne S. Gearinger, Bausch & Lomb,Inc. (E); Christine M. Sanfilippo, Bausch & Lomb, Inc. (E); LeningZhang, Bausch + Lomb (E); Wolfgang Haas, Bausch & Lomb, Inc.(E); Timothy L. Comstock, Bausch & Lomb Incorporated (E);Timothy W. Morris, Bausch & Lomb, Inc. (E)Clinical Trial: NCT01175590Program Number: 5451 Poster Board Number: A0150Presentation Time: 8:30 AM - 10:15 AMA Parallel-Group Study Evaluating the Safety and Efficacy ofBrimonidineTartrate 0.025% Ophthalmic Solution in aPopulation of Adult and Geriatric SubjectsGerald Horn 1 , Matt J. Chapin 2 , Paul J. Gomes 2 . 1 Eye Therapeutics,Schaumburg, IL; 2 Ora, Inc, Andover, MA.Purpose: Vasoconstrictors are currently the most widely usedophthalmic drop. Longer lasting, more effective agents withimproved safety and reduced rebound are needed.Methods: This study employed a single-center, double-masked,randomized, vehicle - controlled, parallel-group design to evaluatethe safety and efficacy of brimonidine tartrate 0.025% ophthalmicsolution versus vehicle in a population (N=57) of adult (> 40 years ofage) and geriatric (> 65 years of age) subjects dosed QID for 4weeks. Subjects were randomized without age stratification to receiveeither brimonidine or vehicle. Following instillation of testcompound, subjects scored the drop for comfort (0-10 scale)immediately, and again 1 and 2 minutes after instillation. Ocularredness was then scored by the investigator at 5, 15, 30, 60, 90, 120,180, and 240 minutes after medication instillation (0-4 scale, .5 unitincrements). Subjects were then sent home with instructions to recordocular redness scores before and 2 minutes after each of the 4 dailydoses. In addition, they were instructed that the interval betweendoses should be no less than 3.5 hours. Diaries were collected after 2weeks and again after 4 weeks.Results: Subjects reported a high degree of comfort with both thebrimonidine drop (mean comfort 0.7±1.48 for all scores) and thevehicle (0.4±1.24). Adverse events were minimal in both groups. Inthe time course of redness scores recorded after the 1st instillation,the brimonidine-treated group were both clinically and statisticallysuperior to the vehicle treated group at all time points. Thedifferences between pre- and post-instillation readings for thebrimonidine group diary scores were also significant, while those ofthe vehicle group were not.Conclusions: Brimonidine 0.025% was effective in reducing ocularredness both in office assessments by a clinician and in subjectivediary assessments. Onset of action was rapid (within 5 minutes), andfor 4 weeks in an adult population aged 40 years and older (includinggeriatrics) was tolerable and safe. The drops were very comfortable,and there is no indication of any redness rebound or drug-inducedhypersensitivity issues.Commercial Relationships: Gerald Horn, Alpha Synergy Corp (I),Alpha Synergy Corp (P), Alpha Synergy Corp (S); Matt J. Chapin,Ora, Inc. (E); Paul J. Gomes, Ora, Inc. (E)Clinical Trial: NCT01675609Program Number: 5452 Poster Board Number: A0151Presentation Time: 8:30 AM - 10:15 AMEvolution in post transplant patients with Herpetic Keratitis inHospital Fundacion Nuestra Señora de la Luzcristina C. fernandez 1 , Cristina Pacheco-Del-Valle 2 , AlejandroBabayan 3 , Oscar Baca 4 , Regina Velasco 5 . 1 cornea, HospitalFundacion Nuestra Señora de la Luz, Distrito Federal, Mexico;2 cornea, Hospital Fundacion Nuestra Señora de la Luz, DistritoFederal, Mexico; 3 cornea, Hospital Fundacion Nuestra Señora de laLuz, Distrito Federal, Mexico; 4 cornea, Hospital Fundacion NuestraSeñora de la Luz, Distrito Federal, Mexico; 5 cornea, HospitalFundacion Nuestra Señora de la Luz, Distrito Federal, Mexico.Purpose: The Herpes Simplex Virus is a common cause of cornealpathology, being the leading cause of infectious blindness corneal indeveloped countries. The recurrence rate is high. Periodic reactivationsin the cornea are particularly important because thecumulative effect of the reinfection can lead to scarring orperforation, being an etiologic diagnosis common in patients who areundergoing penetrating keratoplasty. The objective of this study wasto assess the evolution in patients with herpetic keratitis penetratingkeratoplasty and identify the factors that could influence the cornealrejection.Methods: An observational retrospective study that reviewed therecords of 36 patients in the Fundacion Hospital NuestraSeñora de laLuz was conducted. Factors were analyzed such as indication of thesurgical procedure, procedure, treatment pre surgical and postsurgicalwith acyclovir dosage and administration time, date in whichthe surgery was carried out, presence or not of activity at the time ofsurgery, outcome 3 years specifically the presence of rejection,failure or success at the time of the last review.Results: From December 2009 to December 2011 we reviewed 36records of 36 patients who underwent penetrating keratoplasty forherpetic keratitis. The patient age at surgery was 35.05 years, 22 werewomen and 13 men, 4 patients received preoperative treatment withacyclovir, 32 did not. The average treatment dose was 400 mg every12 hours and the average time was 8.5 months. As pos operativetreatment 22 received acyclovir and 14 did not, being theprophylactic dose of 200 mg every 12 hours average and thetherapeutic dose of 400 mg every 12 hours, with an average of 9.63months, 14 patients had no rejection and 22 had at least one episode ,9 of which came to failure in average 21.88 months after surgery, 6of these 9 cases were treated with pre operative acyclovir and 3 werenot.Conclusions: We conclude that the population with herpetic keratitiscan gain Visual benefits with a penetrating keratoplasty. Howeverthere are factors that can affect the prognosis and that should be takeninto account for best results.Commercial Relationships: cristina C. fernandez, None; CristinaPacheco-Del-Valle, None; Alejandro Babayan, None; Oscar Baca,None; Regina Velasco, NoneSupport: none in the support fieldthe duration of action ≥ 4 hours. QID dosing of brimonidine 0.025%©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea506 Contact Lens IIThursday, May 09, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 5453-5490/A0152-A0189Organizing Section: CorneaContributing Section(s): Visual Psychophysics / PhysiologicalOpticsProgram Number: 5453 Poster Board Number: A0152Presentation Time: 8:30 AM - 10:15 AMCorneal confocal and topographical changes with use of rigidcontact lens in keratoconus patients following collagen crosslinkingSri Vatsa Sehra, Jeewan S. Titiyal, Radhika Tandon, NamrataSharma, Rajesh Sinha. Dr Rajendra Prasad Centre for OphthalmicSciences, All India Institute of Medical Sciences, New Delhi, India.Purpose: To assess corneal confocal and topographical changes withuse of rigid gas permeable contact lens (CL) in keratoconus (KC)patients following collagen cross linking (CXL).Methods: A prospective comparative case series was performed inwhich three groups of eyes with primary KC were recruited: Group 1(CXL-CL), 26 eyes which were newly fitted with CL three monthsafter CXL; Group 2 (CXL-SL), 21 eyes with CXL done three monthsback, not fitted with CL; Group 3 (KC-CL), 25 eyes that had neverundergone CXL and were newly fitted with CL. All eyes werefollowed up for six months after recruitment. Confoscan CS4(NIDEK, Gamagori, Japan), Pentacam (Oculus, Lynnwood, WA,USA), Videokeratography (Atlas 9000, Carl Zeiss Meditec, Dublin,CA, USA) and Specular endothelial microscopy (EM 1000, Tomeycorp, Nagoya, Japan) were performed in all eyes at baseline and atone; three and six months follow up.Results: CXL-CL and KC-CL showed increase in the superficialepithelial cell size, decrease in basal epithelial cell density anddecrease in the sub-basal nerve plexus fibre count and branchingcount over follow up; whereas CXL-SL showed decrease insuperficial epithelial cell sizes, increase in basal epithelial density andregeneration of sub-basal nerve plexus (Table 1). CXL-CL had aregression of 0.93D in mean keratometry and 1.99D in maximumkeratometry (P=0.00 in both); no significant keratometric flatteningwas seen in other groups (Figure 1).Uncorrected visual acuity (UCVA) improved only in CXL-CL from0.97 ± 0.25 logarithm of the minimum angle of resolution (logMAR)units at baseline to logMAR 0.86 ± 0.30 at 6 months (P=0.00). CLusers had better BCVA (P=0.00) than patients using spectacles. Overrefractionshowed a myopic shift of 0.37D in CXL-CL (P=0.00), nosignificant change in over-refraction was seen in KC-CL.Conclusions: CL use after CXL is associated with changes in cornealepithelium and delayed recovery of corneal sub-basal nerve plexus onconfocal microscopy. It is also associated with significant corneaflattening and improvement in UCVA.Confocal microscopy epithelial and sub-basal nerve plexusparameters.Pentacam subtraction maps. a) CXL-CL patient showing centralflattening after 6 months follow up. b) CXL-SL patient showingminimal change after 6 months follow up. c) KC-CL patient showinginferior and central steepening after 6 months follow up.Commercial Relationships: Sri Vatsa Sehra, None; Jeewan S.Titiyal, None; Radhika Tandon, None; Namrata Sharma, None;Rajesh Sinha, NoneProgram Number: 5454 Poster Board Number: A0153Presentation Time: 8:30 AM - 10:15 AMIs the Use of an Integrating Sphere Really Desirable WhenMeasuring Contact Lens Transmittance?Claude J. Giasson 1, 2 , Corinne Deschênes 1 , Vasile Diaconu 1 . 1 Schoolof Optometry, University of Montreal, Montreal, QC, Canada;2 LOEX, CHA, Quebec, QC, Canada.Purpose: The standard indicates that contact lens transmittanceshould be measured with an integrating sphere (IS) in order toinclude the scatter (or indirect transmittance). To compare contactlens transmittance obtained with and without an integrating sphereand to evaluate how much light is lost in measurements done with anIS.Methods: After a baseline percent transmittance measurement of aquartz chamber filled with saline, the total and direct percent©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatransmittance of the chamber with a contact lens (Encore 100) wererespectively measured 4 times with and without an integrating spherewith a scanning spectrophotometer (Cary 5000, Varian) in the rangeof 200 to 800 nm. In other experiments, relative light levels weremeasured with and without the measuring chamber with and withoutthe IS in order to estimate how much of the signal was lost upon theaddition of the measuring chamber. Differences between total anddirect transmittance for contact lenses were tested for significance at550nm with an independent t test using the SPSS 17.0 software forWindows.Results: There were no significant differences at 550 nm between thetotal (100.8 ± 8.5%) and direct transmittance (98.3 ± 0.2%) whentested with an independent t test. The standard deviations of totaltransmittance measurement were consistently larger than the onesobtained without the integrating sphere. At 550 nm, the ratio of lightintensities measured in the absence of the chamber filled with salineto the one measured with the chamber with saline represented 46% ofthe signal when the IS was in position and 92% when it was not.Conclusions: The use of an integrating sphere when measuring thetransmittance of a contact lens allows to include the diffusion of thecontact lens in the measurement. However, in our system, the lightintensity is drastically decreased by the simple addition of a wet cellin front of an integrating sphere that reduce the signal and mayincrease the noise.Commercial Relationships: Claude J. Giasson, None; CorinneDeschênes, None; Vasile Diaconu, NoneProgram Number: 5455 Poster Board Number: A0154Presentation Time: 8:30 AM - 10:15 AMPeripheral Defocus with Spherical and Bifocal Soft ContactLensesDavid A. Berntsen, Carl E. Kramer. College of Optometry,University of Houston, Houston, TX.Purpose: Peripheral myopic defocus has been hypothesized to slowthe progression of myopia. We describe peripheral defocus whenmyopic eyes are corrected with spherical and center-distancemultifocal soft contact lenses while looking at distance and nearobjects.Methods: Twenty-five subjects with spherical contact lens-correctedrefractive error of -0.50 to -6.00 DS participated. A modified openfieldautorefractor was used to measure refractive error of the righteye while wearing a spherical soft contact lens (Biofinity) and acenter-distance multifocal soft contact lens with a +2.50-D add(Biofinity Multifocal "D" Lens). Measurements were made centrallyand along the horizontal meridian at ±20°, ±30° and ±40° from theline of sight both at near (30-cm demand) and at distance (undercycloplegia). Lens type and measurement starting location wererandomized. Repeated-measures ANOVAs with post-hoc t-tests wereused for analysis.Results: The mean (±SD) age and central spherical equivalentrefractive error were 23.8 ± 1.3 years (range: 22 to 27 years) and -3.62 ± 1.56 DS, respectively. At distance, mean spherical equivalentrefractive error (defocus) when wearing contact lenses wassignificantly more hyperopic with the spherical lens than with themultifocal lens (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaVisual acuities pre and post PROSE fitting represented as logarithmof the minimal angle of resolution (LogMAR). All patients showedan average pre-PROSE acuity of 0.791 ± 0.68 and post-PROSEacuity of 0.151 ± 0.33 (p < 0.0001).stationary contact area is clearly demonstrated in Fig 2. At a slidingspeed of V=1,000 μm/s the friction coefficient after 30 cycles isbelow μ=0.02 for the migrating contact area and approaches μ=0.25for stationary contact. As sliding speed is reduced to V=300 μm/sboth experiments give similar friction values.Conclusions: The hypothesis that friction coefficient is dependentupon the motion of the contact area was confirmed. We propose thatwater content of the gel is maintained in migrating contacts overshort time scales as occurs in the on-eye situation for the eyelidmoving over a contact lens, and that this provides a more relevantmeasurement of lubricity of a contact lens.Brennan, AAO Annual Meeting, 2009Roba, et al., Tribology Letters, 2011Dunn, et al., Tribology Letters, 2013Chen, et al., Journal of Biomechanical Engineering-Transactions ofthe Asme, 2007Ocular Surface Disease Index (OSDI) scores pre and post PROSEtreatment. All patients showed an average pre-PROSE OSDI score of58.42 ± 46.22 and post-PROSE OSDI score of 18.99 ± 17.93 (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaHyd n=59, Hydrogel DD n=170). Change in Contact Lens Dry EyeQuestionnaire-8 (CLDEQ-8) score, hours of comfortable wear, selfratingof ease of use and compliance with instructions for wear weretested with t-tests or Wilcoxon Signed Rank tests by habitual lensgroup and new lens brand.Results: The CLDEQ-8 score improved significantly for alltreatment arms (Baseline mean ranged from 11.6±6.2 for HydDDhabitual SCLs to 15.9±7.1 for Reusable Hyd) at all visits except forthe Hyd to HydDD group at the 4 month visit (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaResults: Median right and left eye investigator grades were 2.0 andinterquartile ranges were 0.5 and 1.0 respectively. There were nodifferences between eyes (Wilcoxon matched pairs p=0.30). Anumber of image processing metrics were significantly associatedwith LWE grade: these included area, convexity and staining regionthickness (Spearman rho all at least 0.61, all p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Previous studies indicate that single lens replacements ortime of day do not influence end of day (EOD) contact lens comfort,suggesting that length of wear may be a driving factor in ocularcomfort. This study investigated the impact of lens-free intervals ofvarying lengths on EOD comfort.Methods: 25 symptomatic lens wearers (comfortable wear time6.3±1.6hrs) participated in this randomised, cross-over studyinvolving nine individual 12-hour days: 1 lens-free (spectaclewearing) assessment day and 8 lens wear days. On each lens wearday, lenses were worn bilaterally in 2-hour intervals, separated bylens-free (recovery) intervals of 0, 30, 60 or 80 minutes (repeatedthroughout the day). For each 2-hour lens wear interval, a new pair oflenses was worn. Ocular comfort was rated before and after the firstlens insertion, and after each 2-hour lens interval, using a 0-100visual analogue scale (0= extremely uncomfortable and 100 = verycomfortable). This study involved 2 different types of siliconehydrogel lenses, and the order of lens type and length of recoveryperiod was randomised. Participants were unaware of the true studypurpose and that a new lens pair was used for each lens wear interval.Results: A continuous decrease in comfort with wearing time wasobserved on all lens wear days, independent of recovery periods orlens type. There were no differences in EOD comfort scores betweenlens wear days for lens 1 (Mean range 48.9±17.8 to 56.0±19.8,p>0.42) or lens 2 (Mean range 53.7±15.9 to 58.8±13.9, p>0.9), andratings were significantly lower compared to the lens-free assessmentday (70.8±16.2) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaastigmatism 60% of the time and Vision benefits 50% of the time.The health benefit of a DD contact lens for astigmatism waspresented 75% of the time. Doctors also presented conveniencebenefits and comfort benefits of a daily disposable contact lens forastigmatism 60% of the time and Vision benefits 50% of the time. 9out of 10 patients were willing to trial a DD contact lens forastigmatism based on doctor recommendation.Conclusions: This identifies a significant opportunity to fit newpatients and to upgrade more reusable contact lens wearers byproviding a strong recommendation and tying the benefits of DDcontact lenses for astigmatism to specific patient needs - whether itbe convenience, comfort, vision or health.Commercial Relationships: W L. Ball, Vistakon (E)Program Number: 5465 Poster Board Number: A0164Presentation Time: 8:30 AM - 10:15 AMVisual Correction for Irregular Corneas with Scleral LensesMuriel Schornack, Cherie B. Nau, Jeff Pyle, Sanjay V. Patel.Ophthalmology, Mayo Clinic, Rochester, MN.Purpose: Scleral rigid gas permeable lenses can optically neutralizecorneal surface irregularity in patients with primary corneal ectasia,corneal scarring, or post-surgical irregularity. In this study, wereviewed the outcomes of commercially available Jupiter sclerallenses for corneal irregularity.Methods: In a retrospective study, we identified 137 eyes of 96patients that were fit with Jupiter scleral lenses (Visionary Optics,Front Royal, VA, and Essilor Contact Lens, Inc., Dallas, TX)between June 2006 and November 2011 for primary or secondarycorneal irregularity. Specific indications for which scleral lenses wereprescribed, prior modes of optical correction, topographiccharacteristics, visual acuity prior to and after lens fitting, details ofthe fitting process, and clinical outcomes were recorded. Visualacuity with habitual correction before scleral lens wear was comparedto that with scleral lenses by using generalized estimating equationmodels to account for possible correlation between fellow eyes of thesame subject.Results: Mean age of patients was 46 years (range 11-83 years).Scleral lenses were prescribed for visual rehabilitation in patientswith keratoconus/pellucid marginal corneal degeneration (68 eyes),penetrating keratoplasty (27 eyes), post-refractive surgery (27 eyes),scarring after keratitis (7 eyes), Terrien marginal degeneration (3eyes), ocular trauma (2 eyes), monocular diplopia (2 eyes), andcongenital corneal defect (1 eye). Most patients had attempted towear corneal rigid gas permeable lenses, hybrid lenses, or piggybacklens systems prior to commencing scleral lens wear. The fittingprocess required an average of 3 visits (range 2-7), and an average of1.6 lenses (range 1-7) were ordered per eye. All patients wore lensessuccessfully at the conclusion of the fitting process. Mediansimulated steep K was 49.00 D (range 35.25-69.00 D), median flat Kwas 43.50 D (range 31.87-54.87 D), and median reference sphere was45.86 D (range 38.6-57.5 D). Median visual acuity improved from0.39 log MAR (Snellen equivalent, 20/49) with habitual correction to0.10 log MAR (Snellen equivalent, 20/25) after scleral lens wear(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadependent activity of adsorbed lysozyme on six different contact lensmaterials during the first minute and up to one week of interactionwith the material surface. Total activity of extracted lysozyme, totalin-situ activity and the activity of the outer surface layer of sorbedlysozyme were determined using the two different techniques.Micrococcal cellular interaction with surface-adsorbed lysozyme wasimaged using confocal microscopy.Results: The differences between total extracted activities, total insituactivities and surface activities were both measurable andmaterial specific. In most cases the total extracted activity > total insituactivity > surface activity. After one week, etafilcon A had thehighest extracted activity at 137μg/lens, followed by omafilcon A,balafilcon A, comfilcon A, senofilcon A, and lotrafilcon B at 27.4μg,2.9μg, 2.0μg, 0.5μg, and 0.3μg respectively. Subsequent removal ofadhered micrococcal cells was greatest on balafilcon A, which hadthe highest surface activity, and lowest on lotrafilcon B, which hadthe lowest surface activity.Conclusions: This study measured and made direct comparisonsbetween two established techniques for measuring the activity ofadsorbed lysozyme. Both techniques demonstrate material dependentdifferences in activity. While individual extraction activitiesdemonstrate the activity of underlying layers of lysozyme orlysozyme within the matrix of the material, in-situ measurementsallow conclusions to be drawn about only the biologically relevantlysozyme, and surface activity measurements reveal the activity ofjust the outer surface of lysozyme.Commercial Relationships: Brad Hall, None; Chau-Minh Phan,None; Lakshman N. Subbaraman, None; Lyndon W. Jones, Alcon(F), Alcon (R), Allergan (F), Abbott Medical Optics (R), Bausch &Lomb (R), Ciba Vision (F), Ciba Vision (R), CooperVision (F),Johnson & Johnson (F), Johnson & Johnson (R); James A. Forrest,NoneSupport: NSERCProgram Number: 5468 Poster Board Number: A0167Presentation Time: 8:30 AM - 10:15 AMPRG4 as a Natural Boundary Lubricant for Commercial SiliconeHydrogel Contact LensesMichael L. Samsom 1 , Amanda Chan 1 , Lyndon W. Jones 2 , Tannin A.Schmidt 1 . 1 Biomedical Engineering, University of Calgary, Calgary,AB, Canada; 2 School of Optometry, University of Waterloo,Waterloo, ON, Canada.Purpose: The mucin-like glycoprotein proteoglycan 4 (PRG4) isexpressed and present at the epithelium of the ocular surface. Studiesindicate PRG4 reduces friction at ocular biointerfaces, and that PRG4reduces friction in vivo during blinking by acting as a naturalboundary lubricant. Friction may influence, and contribute to, contactlens (CL) discomfort. As such, supplementation with naturallyoccurring lubricants during CL wear could combat heightenedfriction and potential CL discomfort. Therefore, the objective of thisstudy was to determine whether PRG4 is an effective in vitroboundary lubricant at a human cornea - CL biointerface.Methods: Fresh human corneas were obtained from the AlbertaLions Eye Bank. Commercially available silicone hydrogel CL werestudied: Acuvue TrueEye® (TE); Acuvue Oasys® (OAS);Acuvue2® (AC2); AirOptix® (AO). Tissues and lenses weremounted on a BOSE ELF3200 biomechanical testing machine withcustom sample holders, forming a cornea-CL biointerface. Thesesurfaces were articulated against each other at effective slidingvelocities ranging from 0.3-30 mm/s under loads of 8-25 kPa. SalineTest: Sequential testing of the 4 lenses in saline was used to compareeach lens materials friction against the cornea (order: AC2, AO, TE,OAS; n=4). PRG4 Test: TE and OAS were tested in saline, thensoaked in 300 ug/mL PRG4 for 1h and tested. (Order: TE, TE-PRG4,OAS, OAS-PRG4, n=3; OAS, OAS-PRG4, TE, TE-PRG4, n=4).Results: Saline Test: Kinetic friction coefficients were relativelyinvariant with sliding velocity. Kinetic friction coefficients (averagedover all speeds) in saline appeared similar for the different CL tested:TE (0.13±0.03, mean±SEM), AC2 (0.16±0.05), OAS (0.18±0.04) andAO (0.23±0.09). PRG4 Tests: PRG4 functioned as an effectivefriction-reducing boundary lubricant for both TE and OAS lenses.Kinetic friction values were significantly lower in PRG4 for bothlenses (TE-PRG4 0.12±0.02); OAS-PRG4 (0.10±0.02), comparedtheir respective Saline controls (TE 0.16±0.03, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornealenses, sufficient time must be allowed for the lens to reach a stableposition before assessing the depth of the post-lens fluid reservoir. Ifa lens fit is assessed prematurely, the subsequent lens settling couldgreatly reduce reservoir volume and increase contact with the cornea,and this would limit the value of using these lenses in patients withsevere ocular surface disease.Commercial Relationships: Cherie B. Nau, None; MurielSchornack, NoneSupport: Research to Prevent Blindness and Mayo FoundationProgram Number: 5470 Poster Board Number: A0169Presentation Time: 8:30 AM - 10:15 AMMultivariate Statistical Analysis Examining and Modeling DrugRelease from Contact Lens MaterialsFrances Lasowski, Giuliano Guidi, Heather Sheardown. ChemicalEngineering, McMaster University, Hamilton, ON, Canada.Purpose: Topical administration of eye drops remains the mostprevalent method of delivering drugs to the eye. However significantloss and potential systemic side effects necessitates a more effectivedrug delivery method. Contact lenses represent an attractive option asan alternative vehicle for wide range of therapeutics. Namely, the useof a model contact lens delivery system for the ocular drugs timololmaleate, roscovitine and atropine were investigated. The effects ofdrug loading, material composition and presence of wetting agentswere analyzed to further understand the drug-hydrogel interactionsthat govern release kinetics and critical material properties. However,due to the complex nature of these interactions, single variableanalysis of the data is insufficient to predict of future trends.Methods: Model lens materials were based on combinations ofdimethylacrylamide (DMA), hydroxyethyl methacrylate (HEMA)and methacryloxypropyltris(trimethylsiloxy)silane (TRIS). Thematerials were prepared with and without roscovitine (0.5 wt%),timolol maleate (0.5 wt%) and atropine (0.5 wt% & 1.5 wt%). Otherscontained hyaluronic acid at 0.1 wt%. Release studies wereperformed into PBS solutions using UV- spectroscopy and HPLC toquantify release of each drug. Swelling, extraction and contact anglestudies were done to characterize the materials. This information wascompiled and inputted into ProMV, a multivariate analysis toolprovided by Prosensus, Inc. This allowed for examination by bothPCA (principle component analysis) and PLS (projection to latentstructures).Results: Single variable analysis trends include greater total drugrelease from HEMA/TRIS materials than DMA/TRIS materials,greater release of timolol and atropine than roscovitine, and greaterrelease with HA present. The figure shows sample release kineticsfrom various materials at different drug loadings. Despite thesetrends, it is difficult to see trends bridging all drug loadings andmaterial compositions. With multivariate analysis, it is possible to seethe design space and predict compositions and drugs that yield thepreferred release kinetics and properties.Conclusions: Contact lenses provide a feasible method to deliver avariety of drugs to various ocular tissues. The use of multivariatestatistical analysis provides better modeling and creates a predictivemodel allowing optimization of future materials.Commercial Relationships: Frances Lasowski, None; GiulianoGuidi, None; Heather Sheardown, Alcon (F), Alimera Sciences (F)Support: NSERC 20/20 Ophthalmic Materials NetworkProgram Number: 5471 Poster Board Number: A0170Presentation Time: 8:30 AM - 10:15 AMA study of the feasibility of fitting custom designed contact lensesto guinea pigs, guided by AS-OCT imaging and cornealtopographyYue Liu 1 , Zhi Chen 2 , Christine F. Wildsoet 1 . 1 VIsion Science, Univ ofCA, Berkeley Sch of Optometry, Berkeley, CA; 2 Department ofOphthalmology & Vision Science, Eye & ENT hospital,FudanUniversity, Shanghai, China.Purpose: To examine the efficacy and reliability of anterior segmentOCT (AS-OCT) in guiding specialty contact lens (CL) fitting inguinea pig eyes. Small-scale clinical studies suggesting thatorthokeratology (Ortho-K) is effective in controlling myopiaprogression motivated this study, with our longer term goal being touse the myopic guinea pig model to better understand the mechanismunderlying myopia retardation with Ortho-K.Methods: Six guinea pigs underwent unilateral Ortho-K fitting withtheir contralateral eyes serving as controls. Corneal topography,refraction, biometric axial ocular parameters were assessed to aid inthe design of the Ortho-K lenses; measurements were made everyday, starting on the day of initial lens fitting until the cornealreshaping effect has stabilized. The anterior segment was assessedwith and without fluorescein staining by slit lamp biomicroscopy andphotographically recorded; AS-OCT imaging was used to monitorcorneal integrity, CL stability, and the progress of corneal reshaping.Results: On average, extended wear (EW) Ortho-K lenses couldinduce more than 6 diopters of central corneal flattening andcorresponding paracentral steepening without significantly affectingcorneal integrity and/or the stability of the lens fitting. There was lesscorneal fluorescein staining in the eyes with EW Ortho-K lenses,comparing to the non-lens wearing eye. AS-OCT imagingsignificantly reduced the frequency of lens reordering.Conclusions: The combination of AS-OCT imaging and cornealtopography provided the essential information for designing andfitting Ortho-K lenses to this animal model. The EW OrthoKmodality applied to guinea pig eyes allows for corneal reshapingsimilar to its clinical application. Further study of Ortho-K in thisapplication may yield important insight into the mechanismunderlying its myopia control effect.Commercial Relationships: Yue Liu, Coopervision (F); Zhi Chen,None; Christine F. Wildsoet, NoneSupport: NIH R01 EY12392, UC-Coopervision Discovery GrantProgram Number: 5472 Poster Board Number: A0171Presentation Time: 8:30 AM - 10:15 AMInvestigation of Latanoprost Release from Contact LensMaterials Using in vitro Cell Models©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSaman Mohammadi 1 , Lyndon W. Jones 2, 3 , Maud Gorbet 1, 3 . 1 SystemsDesign Engineering, University of Waterloo, Waterloo, ON, Canada;2 School of Optometry, University of Waterloo, Waterloo, ON,Canada; 3 Centre for Contact Lens Research, University of Waterloo,Waterloo, ON, Canada.Purpose: Drug delivering contact lenses (CL) may enhance bothdrug delivery efficacy and patient compliance with the drug regimen.Although in vivo studies are required to prove efficacy, in vitrocorneal models can prove valuable to compare release profiles andidentify the most promising candidates for in vivo studies. This studyinvestigated the uptake and delivery of Latanoprost by commerciallyavailable CL using three differing in vitro cell models.Methods: The in vitro models used diffusion through a polyethyleneterephthalate (PET) filter only (no cells), a PET filter with amonolayer of human corneal epithelial cells (HCECs) and a PETfilter with stratified HCECs, all submerged in keratinocyte serumfreemedium. Four CL materials (balafilcon A; galyfilcon A;senofilcon A; omafilcon A), were soaked for 24hrs in one of the twoforms of a glaucoma drug, Latanoprost (50 μg/ml) and LatanoprostFree-Acid (50 μg/ml). The lenses were then placed on the variousmodels (n=3) and diffusion of the drug through the models wasmeasured after 1, 3, 6 and 24 hours. To compare release profiles withserum-free medium, a drug release in phosphate buffered saline(PBS) with the “no cell” model was performed over 24hrs.Results: Over a 24hr period, drug release in PBS was notsignificantly different from serum-free medium- However, the releasein presence of cells was higher (e.g., for galyfilcon A, latanoprostrelease in presence of cells was significantly different from no cellsmodel, p=0.014). Similar differences were observed for other contactlens types except for the conventional hydrogel omafilcon A. Forboth drugs, similar release profiles were observed for monolayer andstratified cell culture models. In contrast, the latanoprost free-acidrelease in the no-cell model was higher (p=0.038) due to highersolubility of the free acid form of the drug in medium. In a monolayermodel with paraformaldehyde-treated cells, a release similar to nocells model was observed, further highlighting the importance ofmetabolically active cells.Conclusions: Latanoprost is a lipophilic prodrug in which the esterbond in the α-chain can be metabolized by cells into an alcohol andthe latanoprost free-acid (active drug) to increase bioavailability. Ourresults highlight the importance that in vitro experimental models andthe presence of cells may have on determining the release profile of adrug delivery material.Commercial Relationships: Saman Mohammadi, None; LyndonW. Jones, Alcon (F), Alcon (R), Allergan (F), Abbott Medical Optics(R), Bausch & Lomb (R), Ciba Vision (F), Ciba Vision (R),CooperVision (F), Johnson & Johnson (F), Johnson & Johnson (R);Maud Gorbet, CIBA Vision/Alcon (F)Support: CHRP (collaborative program between NSERC and CIHR)Program Number: 5473 Poster Board Number: A0172Presentation Time: 8:30 AM - 10:15 AMOcular Signs and Symptoms in Contact Lens Wearers in aControlled Low Humidity Environmental Exposure Chamber(LH-EEC), A Natural Provocation Research ModelPiyush Patel 1 , Fiona Soong 1 , Jalaiah P. Varikooty 2 , Nancy J. Keir 2 ,Lyndon W. Jones 2 . 1 Inflamax Research, Mississauga, ON, Canada;2 CCLR, University of Waterloo, Waterloo, ON, Canada.Purpose: To evaluate the effects of contact lens (CL) wear on theocular surface under controlled conditions of low humidity andairflow, to assess the validity of a natural provocation research modelfor the study of dry eye and contact lenses.Methods: The LH-EEC is validated to maintain uniform low relativehumidity (10±3%) and comfortable room temperatures which mimicnatural arid outdoor/indoor environs. Ten symptomatic CL wearersdiscontinued CL wear and used Refresh PLUS® tears t.i.d for 48hrsprior to LH-EEC Visit. Upon return to the clinic, subjects wererandomly fit with 1-day Acuvue® Moist® (etafilcon A): CLA in oneeye and 1-Day Acuvue® TruEye (narafilcon A): CLB in thecontralateral eye. They were then exposed to LH-EEC for 180 minswith visual tasking throughout. Total Ocular Symptom Scores(TOSS) (dryness+grittiness+burning/stinging+itchiness) were rated0-4 (maximum score of 16) at entry and at set intervals within theEEC. Tear Break Up Time (TBUT), fluorescein corneal staining andlissamine green conjunctival staining were collected pre and post LH-EEC using Efron scale (0-4) on the NEI grid for the cornea and nasal& temporal conjunctival zones.Results: After 180 mins in LH-EEC, mean increase from baselinewas CLA=+1.4±1.2 and CLB=+1.0±0.7 for corneal staining andCLA=+1.30±0.78 and CLB=+1.20±0.72 for conjunctival staining.The mean reduction in TBUT (secs) was CLA=-0.3±0.2 and CLB=-0.8±0.7. Symptoms escalated throughout the exposure period withTOSS increasing from baseline for both lenses (CLA=+2.4±1.7 andCLB of +3.30±1.16), with the mean change for CLB beingsignificant (p=0.02). Similarly, the dryness symptom score showed atrend to increase for CLA (+1.1±0.5) and a significant increase(p=0.001) for CLB (+1.40±0.31).Conclusions: The LH-EEC Contact Lens Dry Eye model exacerbatesocular symptoms and signs while controlling all environmentalvariables. After only 180 mins of exposure, signs indicative of ocularsurface desiccation and a parallel increase in dry eye symptoms weremeasurable. The LH-EEC model provides a valuable clinical researchoption for the study of CL and dry eye which provides rapidsymptoms and signs typically found in longer periods of in-eye wearoutside the LH-EEC.Commercial Relationships: Piyush Patel, Inflamax Research (E);Fiona Soong, Inflamax Research (C); Jalaiah P. Varikooty, Alcon(F); Nancy J. Keir, TearScience (F), Alcon (F), Alcon (R), Allergan(F), Johnson & Johnson (F), CooperVision (F), Visioneering, Inc.(F); Lyndon W. Jones, Alcon (F), Alcon (R), Allergan (F), AbbottMedical Optics (R), Bausch & Lomb (R), Ciba Vision (F), CibaVision (R), CooperVision (F), Johnson & Johnson (F), Johnson &Johnson (R)Program Number: 5474 Poster Board Number: A0173Presentation Time: 8:30 AM - 10:15 AMCorrection of infant aphakia after cataract surgery with rigidgas-permeable contact lensesAnja K. Gruenert 1 , Michael Klueppel 3 , Juergen Hausser 4 , ThomasReinhard 2 , Rainer Sundmacher 1 , Tanja Guthoff 1 , Thomas A.Fuchsluger 1 , Gerd Geerling 1 . 1 Department of Ophthalmology,Heinrich-Heine-University, Duesseldorf, Germany; 2 Department ofOphthalmology, University of Freiburg Hospital, Freiburg, Germany;3 Practice, Moers, Germany; 4 Practice, Duesseldorf, Germany.Purpose: To evaluate the visual outcome of aphakic infants treatedwith rigid gas permeable contact lenses following surgery ofcongenital cataract. Furthermore to investigate the safety andviability of rigid contact lens correction in children.Methods: We performed a retrospective analysis of infants whounderwent cataract surgery and were subsequently treated with rigidgas permeable contact lenses between 1987 and 2011 (n=75). Theinfants were divided into four prognostic groups: bilateral aphakia(Group I), monolateral aphakia with early (Group II) or late (GroupIII) surgery and aphakia with additional ocular pathologies (GroupIV). The outcome was evaluated in terms of visual acuity, refractivepower, keratometric astigmatism, compliance with the wear of the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornealenses and development of strabism.Results: The toleration of rigid contact lenses was extremely well.After a short training period most parents didn’t have any difficultiesin manipulating the lens. The visual outcome was dependent onmonolateral or bilateral cases, the age at the time of surgery andadditional pathologies. Infants with aphakia on both sides (Group I)achieved visual acuities up to 1.0, whereas in monolateral casesdevelopment of amblyopia was frequent. The functional results werebetter with early cataract surgery (Group II) than with late surgery(Group III). In Group IV the prognosis was very limited due tofurther pathologies. As hyperopia decreases during infancy the powerof rigid contact lenses had to be adjusted frequently.Conclusions: To our knowledge, at this time this is the largest groupof patients analysed. Rigid gas-permeable contact lenses are veryefficient in improving visual outcome of aphakic children. Provided agood compliance and collaboration with the parents, rigid gaspermeable contact lenses represent a preferential alternative tointraocular lens implantation after extraction of congenital cataract.Commercial Relationships: Anja K. Gruenert, None; MichaelKlueppel, None; Juergen Hausser, None; Thomas Reinhard,None; Rainer Sundmacher, None; Tanja Guthoff, None; ThomasA. Fuchsluger, None; Gerd Geerling, Alcon (C), Allergan (C), TheaPharma (C), Novagali (C), Bausch & Lomb (C), Tearlab Inc. (C)Program Number: 5475 Poster Board Number: A0174Presentation Time: 8:30 AM - 10:15 AMThe evaluation of Lid Wiper Epitheliopathy in contact lenswearers in a Controlled Low Humidity Environmental ExposureChamberLyndon W. Jones 1 , Jalaiah P. Varikooty 1 , Nancy J. Keir 1 , FionaSoong 2 , Piyush Patel 2 . 1 CCLR-School of Optometry, University ofWaterloo, Waterloo, ON, Canada; 2 Inflamax Research, Mississauga,ON, Canada.Purpose: To measure the clinical grades of lid wiper epitheliopathy(LWE) in contact lens (CL) wearers before and after exposure toconditions of temperature, low humidity and air flow in a controlledlow humidity environmental exposure chamber (LH-EEC).Methods: In this double-masked, feasibility study, 10 symptomaticCL wearers were randomized to contralateral lens wear withnarafilcon A and etafilcon A lenses. CL wear was discontinued 48 hprior to assessments and Refresh Plus® artificial tears (ATs) wereinstilled t.i.d. in both eyes. For LWE measures, the upper (UL) andlower lid (LL) margins were stained with sodium fluorescein andlissamine green dyes using an optimized technique to detect LWE.LWE was graded on a 0-3 scale (Korb et al. Eye Contact Lens, 2005)at Baseline, prior to CL insertion and entry into the LH-EEC. In theLH-EEC, subjects were exposed to controlled temperature of22±3°C, relative humidity of 10±3% and an air velocity of approx.5ft/sec for 180 min. Upon exit, CLs were removed and ATs wereinstilled every 15 min for 120 min. LWE was evaluated after postchamberexposure (PC), PC+30min, PC+90min and PC+120min.Analyses were undertaken using Statistica.Results: After 180 min in the LH-EEC, mean LWE grades in theupper lid increased from Baseline to PC and were 1.25 to 2.23 fornarafilcon A and 1.18 to 1.93 for etafilcon A. In the lower lid, itchanged from 1.00 to 2.48 for narafilcon A and 0.90 to 2.03 foretafilcon A (Wilcoxon matched pairs, all p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaTable 1: Claimed and Determined Dk Values of 9 CommerciallyAvailable Contact Lenses using Lens Stacking and Single-lens withVarious PowersCommercial Relationships: X Michael Liu, Bausch & Lomb, Inc.(E); George L. Oltean, Bausch and Lomb (E)Program Number: 5477 Poster Board Number: A0176Presentation Time: 8:30 AM - 10:15 AMThe Large-Scale Epidemiological Study on the Prescription ofContact Lenses in Japan -The Result from Analyzingapproximately 330,000 Eyes of Japanese Subjects-Eiichi Okada 1 , Nobuhisa Mizuki 2 , Tatsukata Kawagoe 2 , MaiNagasaki 1 , Naomi Matsunaga 3 , Masao Yoshida 3 . 1 Okada Eye Clinic,Yokohama, Japan; 2 Department of Ophthalmology, Yokohama CityUniversity, School of Medicine, Yokohama, Japan; 3 Department ofPublic Health, Kyorin University, School of Medicine, Mitaka,Japan.Purpose: In Japan there is an abundance of research regarding thenumber of people using contact lenses and types of lenses consumed,however, there are only a few analysis studies of Japanese subjectsregarding sex, age and refractive levels of diopter (D) based on thenumber of eyes which contact lenses are prescribed. Therefore wemade a five-year large-scale epidemiological study.Methods: The subjects for this research were 204,975 eyes from103,001 men and women aged 9-96 who visited Okada Eye Clinic inYokohama, Japan, for the prescription of contact lenses betweenJanuary 2007 and December 2011, excluding eyes withorthokeratology, cataract operated eyes and cases of changesexceeding ±5D in a year. The total number of the prescriptionsamounted to 332,469 eyes.Results: Analyzing the number of eyes which contact lenses wereprescribed by sex and age, the groups aged 20-24 showed the highestratio (19.22% in men and 17.58% in women), followed by the groupsaged 25-29 (17.53% in men and 15.60% in women) and the groupsaged 15-19 (16.46% in men and 15.29% in women). Viewing it bysex and refractive levels, the groups with refractive level of -2.75 to -4.5D showed the highest ratio (40.76% in men and 40.97% inwomen), followed by the groups of -4.75 to -6.5D (25.87% in menand 24.51% in women) and the groups of -0.75 to -2.5D (20.89% inmen and 23.19% in women). In addition, the ratio of hyperopia≥+0.75D was less than 1% (0.27% in men and 0.84% in women),however, the ratio of myopia ≤-6.75D was about 10% (11.71% inmen and 9.79% in women), and the ratio ≤-8.75D was about 2.5%(2.85% in men and 2.24% in women).Conclusions: The results of this survey revealed that the number ofeyes prescribed to contact lenses was the highest in the groups aged15-29 (approximately 50%) and -2.75 to -4.5D was most prescribed(approximately 40%) in both men and women. Although the numberof the eyes in hyperopia ≥+0.75D in women was more than in men, itmyopia ≤-6.75D in men was more than in women and it showedabout 10% in both men and women.Commercial Relationships: Eiichi Okada, None; NobuhisaMizuki, None; Tatsukata Kawagoe, None; Mai Nagasaki, None;Naomi Matsunaga, None; Masao Yoshida, NoneSupport: None in the SupportProgram Number: 5478 Poster Board Number: A0177Presentation Time: 8:30 AM - 10:15 AMComparative study of refractive change between glasses andcontact lenses users -5 years prospective study againstapproximately 273 thousand Japanese eyes-Masao Yoshida 1 , Nobuhisa Mizuki 2 , Tatsukata Kawagoe 2 , MaiNagasaki 3 , Naomi Matsunaga 1 , Eiichi Okada 3 . 1 Department of PublicHealth, Kyorin University School of Medicine, Mitaka, Japan;2 Department of Ophthalmology, Yokohama City University Schoolof Medicine, Yokohama, Japan; 3 Okada Eye Clinic, Yokohama,Japan.Purpose: There were reports analyzing prevalence rate of refractiveerror such as myopia and hyperopia by cross-sectional studies, butonly few analyzed by prospective studies. Moreover, using glassesand contact lenses are the mainstream of correcting refractive error,but it is not clear if there is a difference in the change of refraction ofthe refractive power after using these two vision correcting devices.So the purpose of this study is to compare the change in refractionbetween glasses users and contact lenses users by a large scaleepidemiological study based on a 5 years prospective study againstapproximately 273 thousand eyes of Japanese.Methods: The subject of this study is 13,977 eyes ranging in agefrom 4 to 88 years who attended Okada Eye Clinic in Yokohama,Japan for the prescription of glasses, and 273,042 eyes ranging in theage of 10 to 91 years who attended the clinic for the prescription ofcontact lenses. The subjects using orthokeratology lens, with cataractoperated eyes, and who had a change in power larger than ±5Dwithin a year while the 5 years prospective study was excluded.Results: As a result of analysis of covariance among the 12 agegroups, the change in refractions towards myopia was significantly(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaYing Jiang 1 , Michael Jacobs 2 , Saralee Bajaksouzian 2 , Altreisha N.Foster 2 , Sara M. Debanne 1 , Roger Bielefeld 3 , Matt Garvey 3 ,Sangeetha Raghupathy BSOptom 4 , Jami R. Kern 5 , Loretta B.Szczotka-Flynn 4 . 1 Department of Epidemiology & Biostatistics, casewestern reserve university, Cleveland, OH; 2 Department ofPathology, case western reserve university, Cleveland, OH; 3 Divisionof Information Technology Services, case western reserve university,Cleveland, OH; 4 Department of Ophthalmology & Visual Sciencesand University Hospitals Eye Institute, University Hospitals CaseMedical Center, Cleveland, OH; 5 Alcon Laboratories, Fort Worth,TX.Purpose: Substantial microbial bioburden on contact lenses duringextended wear and lid margins during daily wear have shown to berisk factors for the development of contact lens associated cornealinflammatory events (CIEs). This study assessed risk factorsassociated with substantial microbial bioburden of lids, cases, andsilicone hydrogel contact lenses when worn daily wearMethods: 218 patients were enrolled in the Daily Wear CornealInfiltrative Event study, fit to lotrafilcon A contact lenses,randomized to use either a preserved multipurpose solution (MPS) ora peroxide care system, and followed for 1 year. Lenses, lids, casesand transport saline were cultured at selected visits and considered tohave substantial microbial bioburden when they harbored high levelsof commensal or pathogenic organisms based on established criteria.Univariate and multivariate logistic regression analyses wereconducted at the person level to examine which demographic andsolution covariates were associated with significant bioburden at eachlocationResults: Univariate analyses revealed current or past smokers (vs.never-smokers), clerical occupations, and solution type wereassociated with a greater risk of microbial bioburden on lenses, cases,or both. Neither gender, age, nor healthcare occupations wereassociated with significant bioburden in any of the locationsexamined; additionally, neither solution type nor other demographicfactors were associated with lid bioburden or saline contamination. Inmultivariate analyses, clerical (vs. non-clerical) occupations hadsignificantly greater risk of microbial contamination on lenses (OR =2.7 (95% Confidence Interval (CI) 1.04-6.8)) and cases (OR = 3.4(95% CI 1.14-10.0)). Solution type was associated with microbialbioburden in cases (adjusted OR for the peroxide system = 7.5 (95%CI 3.8-15.1)) but not on lenses, lids or transport salineConclusions: Clerical occupations were associated with increasedmicrobial bioburden of contact lenses and cases during daily wear useof silicone hydrogel lenses. Although a hydrogen peroxide caresolution (compared to a MPS) was associated with increased lenscase bioburden, this association was not found with bioburden onlids, lenses, or in transport saline and case contamination was not arisk factor for CIEs in this studyCommercial Relationships: Ying Jiang, Alcon (F), Vistakon (F);Michael Jacobs, Alcon (F), Vistakon (F); Saralee Bajaksouzian,None; Altreisha N. Foster, None; Sara M. Debanne, None; RogerBielefeld, None; Matt Garvey, None; Sangeetha RaghupathyBSOptom, None; Jami R. Kern, Alcon (E); Loretta B. Szczotka-Flynn, Alcon Laboratories (F), Alcon Laboratories (R), Vistakon (F),Bausch & Lomb (R)Support: Alcon LaboratoriesClinical Trial: NCT00937105Program Number: 5480 Poster Board Number: A0179Presentation Time: 8:30 AM - 10:15 AMAn Examination of the Effects of Evaporation on AntimicrobialEfficacy of Contact Lens Care SolutionsNancy Brady, Marina Milenkovic, Anthony Lam. CornealMicrobiology, Abbott Medical Optics, Santa Ana, CA.Purpose: Partial evaporation of contact lens care solutions, as canoccur from failure to cap the contact lens case as directed, may resultin loss of antimicrobial efficacy of the solution and lead to contactlens-related eye infections. This study evaluated the impact of partialevaporation on the antimicrobial efficacy of several contact lens caresolutions when tested according to ISO 14729.Methods: The solutions studied were - Investigational MPS-1:polyhexamethylene biguanide (PHMB) + poloxamer (PLX) andcurrently marketed Japan products MPS-2: polyquaternium (PQ1) +tetronic 1304 MPS-3: PHMB + poloxamine (PLA) and MPS-4:PHMB +PLX.Solutions were evaporated under a stream of air to 2x and 4xconcentrations and challenged with Pseudomonas aeruginosa (ATCC9027), Serratia marcescens (ATCC 13880), Staphylococcus aureus(ATCC 6538), Candida albicans (ATCC 10231) and Fusarium solani(ATCC 36031). The assay was performed according to the standalonetest outlined in ISO 14729:2001/A.2010. The test solutionswere evaluated at 4 hours and compared to the non-evaporatedproduct. The solutions were assessed for their ability to meet thecriteria of 3 log reduction of the bacteria and 1 log reduction of fungiat the minimum time interval.Results: After 4 hrs of inocula exposure, Investigational MPS-1 metcriteria when non-evaporated and at the 2X and 4x evaporation level.MPS-2 failed to meet criteria when non-evaporated (for S. aureus andCandida) and also failed at the 2x and 4x levels (for S. marcescens, S.aureus, Candida and Fusarium). MPS-3 met criteria when nonevaporatedand 2x level but failed criteria at 4x (for Candida). MPS-4failed to meet criteria when non-evaporated (for S. aureus andCandida) and also failed criteria at the 2x and 4x (for S. marcescens,S. aureus and Candida).Conclusions: The study demonstrates that, after partial evaporationup to 4X, modelling the potential error of not capping a lens casecorrectly, MPS solutions can lose significant disinfection activity.The solutions that failed to meet stand-alone criteria when nonevaporatedshowed more pronounced loss after evaporation. Of thesolutions tested, only MPS-1 retained its full efficacy of stand-alonedisinfection activity after being evaporated to a 4x level.Commercial Relationships: Nancy Brady, Abbott Medical Optics(E); Marina Milenkovic, Abbott Medical Optics (E); Anthony Lam,Abbott Medical Optics (E)Program Number: 5481 Poster Board Number: A0180Presentation Time: 8:30 AM - 10:15 AMEvaluation of an auto-refractor for over-refraction withmultifocal contact lenses patientsAnna Giner 2 , Mikel Aldaba 2 , Montserrat Arjona 1 , Jaume Pujol 1 .1 CD6 - Optica i optometria, Polithechnical University of Catalonia,Terrassa, Spain; 2 CD6, Polithechnical University of Catalonia,Terrassa, Spain.Purpose: To evaluate the utility of an auto-refractor for multifocalcontact lenses over-refractionMethods: Non-cyclopegic distance refractive error was measured inpatients wearing multifocal contact lenses by means of the GrandSeiko Auto Ref/Keratometer WAM-5500 auto-refractometer andcompared with subjective measurements. Three commercialmultifocal contact lenses were evaluated: Air Optix Multifocal, CibaVision (refractive bi-aspheric lens with near vision in the centre),Proclear Multifocal, CooperVision (refractive lens with asphericcentre with near vision in it), Acuvue Oasys Multifocal, Johnson &Johnson (refractive lens with a multicurve design). 30 eyes of 15healthy adults were measured in the study, with a mean ± SD in age©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaof 27.67 ± 1.86 years (range: 25 to 30 years), subjective sphericalrefraction of -2.43 ± 3.56 D (+2.50 to -9.50 D), subjective astigmaticrefraction of -0.48 ± 0.44 D (0 to -1.25 D), best corrected visualacuity in the logMAR scale of -0.21 ± 0.07 (-0.1 to -0.34). The resultsof over-refraction were evaluated in terms of M, J0 and J45.Results: The mean difference between auto-refractometer andsubjective spherical over-refraction was 0.52 ± 0.37D (range: +1.08to +0.02D) for the Air Optix lens, 0.62 ± 0.43D (range: +0.94 to+0.32D) for the Proclear lens and -0.15 ± 0.11D (range: +0.07 to -0.46D) for the Acuvue Oasys lens. The mean differences inastigmatic over-refraction in terms of J0 and J45 were -0.04 ± 0.03D(range: +0.32 to -0.55) and -0.05 ± 0.04D (range: +0.15 to -0.31D)for the Air Optix lens, 0.17 ± 0.07D (range: +0.22 to +0.12D) and0.05 ± 0.04D (range: +0.42 to -0.33D) for the Proclear lens and -0.23± 0.17D (range: +0.03 to -0.50D) and -0.05 ± 0.03D (range: +0.17 to-0.29D) for the Acuvue Oasys lens.Conclusions: We have measured the over-refraction in multifocalcontact lenses users with an auto-refractometer and compared withsubjective over-refraction. The results from our measurementshighlighted a good agreement between subjective and objectivevalues in both spherical and cylindrical refractions, although somediscrepancies were found on patients with high refractive errors.Thus, we can conclude that auto-refractor is an useful tool for overrefractionin patients wearing multifocal contact lenses although caremust be taken in highly myopic or hyperopic users.Commercial Relationships: Anna Giner, None; Mikel Aldaba,None; Montserrat Arjona, None; Jaume Pujol, Visiometrics (P)Support: "Ministerio de Economia y Competitividad" Spain (BES-2012-054777)Program Number: 5482 Poster Board Number: A0181Presentation Time: 8:30 AM - 10:15 AMEvaluation of the effect of soft contact lens edge shape onconjunctival epitheliumDorota H. Szczesna-Iskander 1 , D Robert Iskander 2 . 1 Institute ofPhysics, Wroclaw University of Technology, Wroclaw, Poland;2 INSTITUTE OF BIOMEDICAL ENGINEERING ANDINSTRUMENTATION, Wroclaw University of Technology,Wroclaw, Poland.Purpose: To characterize soft contact lens edge profile effect onconjunctival epithelium using optical coherence tomography.Methods: Nine regular contact lens wearers were included in thestudy. 4 types of soft contact lenses were considered (3 monthly, 1fortnightly, power ranged from -0.5 to -5.0D.). Subjectssimultaneously wore two types of lenses (eyes were randomized). Aweek break was set between the 1st and the 2nd pair. All lensesevidenced acceptable fit. The inferior limbus area was imaged bySOCT Copernicus 40 minutes after lens application and after 2 weeksof daily wear. A novel method of differential analysis, overcomingproblems associated with optical distortions, was applied to acquiredimages to evaluate the effect of contact lens edge shape onconjunctival epithelium. In particular, the difference in conjunctivaldeformation angle (Δθ) and the difference in the normalized contactlens imprint area (ΔS) were chosen for the statistical analyses whichincluded standard descriptive statistics and the two-sided rank sumtest for medians (Wilcoxon).Results: The largest difference in conjunctival topography betweenvisits was experienced in SiHy lens with a round edge profile (groupaverage±SD and median for Δθ: 21.1±8.4° and 18.7° and for ΔS:7.0±2.4% and 5.4%). The Si-Hy lens with two-sided edge profileresulted in Δθ: 10.8±1.1°, ΔS: 3.6±4.6%; and medians of 9.8° and5.4%, respectively. The smallest difference in the conjunctivaldeformation angle was observed in Si-Hy angle edged lens (Δθ:0.2±3.1°; 3.0°, ΔS: 2.3±3.8%; 5.3%) while the difference in thenormalized contact lens imprint area was negative (showingimprovement) in Hy round edged lens (Δθ: 7.4±5.8°; 0.2°, ΔS: -6.7±4.2%; -9.0%). Statistically significant differences in Δθ wereachieved between angle edged and round edged Si-Hy lenses(p=0.046), and angle edged and two-sided edged lenses (p=0.001).ΔS showed significant difference between round edged Si-Hy and Hylens (p=0.021).Conclusions: The study indicates that the soft contact lens edgeshape plays little role in the presumable conjunctival epitheliumimprint and suggests that other lens parameters, such as the materialproperties, may play a more significant role in this phenomenon.Commercial Relationships: Dorota H. Szczesna-Iskander, None;D Robert Iskander, Eaglet Eye (F), Eaglet Eye (I)Support: POMOST/2012-5/8/0072Program Number: 5483 Poster Board Number: A0182Presentation Time: 8:30 AM - 10:15 AMThe Cause of Midday Visual Fogging in Scleral Gas PermeableLens WearersAnna McKinney 1 , William L. Miller 1 , Norman E. Leach 1 , CristinaPolizzi 1 , Eef van der Worp 2 , Jan P. Bergmanson 1 . 1 University ofHouston College of Optometry, Houston, TX; 2 Maastricht University,Maastricht, Netherlands.Purpose: Scleral gas permeable contact lens (SGP) patients mayreport midday foggy vision, necessitating lens removal. The presentstudy attempts to determine causative factors.Methods: Fifteen SGP wearers were enrolled after obtaininginformed consent and divided into two groups - those able to wearlenses 8 hours per day or more (uninterrupted) and those that have toremove lenses temporarily before 8 hours (interrupted). The TexasEye Research and Technology Center Dry Eye Questionnaire (DEQ)was completed and lens fit was assessed with biomicroscopy andVisante OCT imaging. Tear exchange was measured using afluorophotometer (Ocumetrics FM-2 Fluorotron). Two microliters ofhigh molecular weight fluorescein (FITC Dextran) was added to thelens bowl and then carefully inserted. Fluorescence was measuredimmediately and every 10 minutes for 1 hour. The amount offluorescein present after 60 minutes (T60) was compared betweenpatients.Results: Of the 15 patients, 5 (33%) were interrupted wearersaveraging 4.45 hours. Wearing time for uninterrupted patients (67%)averaged 11.75 hours. Uninterrupted wearers had an average DEQscore of 28±22 while the interrupted wearer’s averaged 54±11. Therewas a moderate correlation between DEQ scores and average dailywear times (r = -0.528). Sixty percent of both groups exhibited analignment fit. However, 80% of interrupted wearers exhibited a tightfitting edge compared to 40% of uninterrupted wearers. The averagecorneal vault for uninterrupted lens wearers was 0.29±0.24 mm whileinterrupted wearers averaged 0.71±0.44 mm. There was a moderatecorrelation between corneal vault and average daily wear times (r = -0.509). Fluorophotometry was conducted on 12 of the 15 patients. Allpatients had residual fluorescein remaining in the post-lens tear film.The T60 for the uninterrupted wearers was slightly lower than in theinterrupted wearers (0.6671; 0.7539; p=0.23). However, the tearexchange decay rate was not statistically different.Conclusions: Foggy vision, occurring in 33% of SGP lens wearers inthis study, is a multifactorial phenomenon unique to these devices.Important factors include a predisposition to dry eye and significantlygreater central corneal vault combined with lens edge tightness. Thisstudy documents, for the first time, post lens tear exchange in SGPlens wearers. Foggy vision is best countered by reassessment of lensedge fit and adjusting corneal vault.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Anna McKinney, None; William L.Miller, Contamac (F); Norman E. Leach, None; Cristina Polizzi,None; Eef van der Worp, Contamac (F); Jan P. Bergmanson,Contamac (F)Support: NIH/NEI grant T35EY00708 and Contamac USProgram Number: 5484 Poster Board Number: A0183Presentation Time: 8:30 AM - 10:15 AMDesigning Multifocal Contact Lenses using a Novel Through-Focus Image Quality Metric Highly Correlated with ClinicalVisual AcuityNishant Mohan, Amanda C. Kingston, Ian G. Cox. Bausch and Lomb,Rochester, NY.Purpose: The aim of this study was to validate the design process ofmultifocal contact lenses using a new through-focus image qualitymetric shown to correlate highly with clinical visual acuity.Methods: Individual eye models were created in commerciallyavailable optical design software (Zemax TM Bellevue, WA) andshown to correlate highly with baseline clinical through-focus visualacuity (R 2 = 0.85). For validation of predictability, 64 advancedpresbyopic eye models were used to predict through-focus imagequality of (5) multifocal contact lens designs. In Zemax TM , each ofthe (5) designs were inserted onto the anterior surface of the corneaand an over-refraction was done for each eye. Defocus wasminimized and the closest 0.25D lens was used to obtain peak imagequality at distance. Through-focus predicted logMAR wasdetermined for each eye model with each of the (5) multifocal lensesin place. To compare directly with clinical measurements of visualacuity, image quality with the contact lens on eye was subtractedfrom baseline values representing the individual eye's acuity withoutany corrective lens in place. This gives predicted normalizedlogMAR acuity. The same (7) object distances were used forcomputer modeling as in the actual clinical study. Normalized visualacuity for 24 mature presbyopes, tested monocularly, was measuredwith each of the (5) manufactured multifocals on-eye. Correlation ofclinical and predicted through-focus normalized acuity wasdetermined.Results: At all object distances, each multifocal had less than oneline difference on average between predicted and clinical normalizedlogMAR acuity. The Zemax TM models with predicted logMAR hadhigh correlation with through-focus visual acuity from the clinic (R 2between 0.90 and 0.97) for all multifocal lens designs.Conclusions: When multifocal contact lens designs were modeled onindividual computer eyes and normalized predicted logMAR wasused to determine how lenses would perform clinically, a highcorrelation was found between the computer database and averageclinical acuity results with all (5) multifocal contact lenses. Thisdesign process is highly effective in predicting on-eye performance.With this high level of predictability more design options can beexplored in the computer to optimize though-focus performancebefore a lens is manufactured and tested clinically.Commercial Relationships: Nishant Mohan, Bausch + Lomb (E);Amanda C. Kingston, Bausch & Lomb (E); Ian G. Cox, Bausch +Lomb (E)Program Number: 5485 Poster Board Number: A0184Presentation Time: 8:30 AM - 10:15 AMProsthetic Replacement of the Ocular Surface Ecosystem(PROSE) Treatment: A Partner Clinic ExperienceKira L. Segal 1 , Michelle N. Lee 2 , Kristin O. Chapman 2 , MarkRosenblatt 2 , Kimberly C. Sippel 2 , Christopher E. Starr 2 , JessicaCiralsky 2 . 1 Medicine, Memorial Sloan Kettering Cancer Center, NewYork, NY; 2 Ophthalmology, NewYork-Presbyterian, New York, NY.Purpose: PURPOSE: To evaluate demographics, clinical course, andoutcomes of patients who received the PROSE device at a partnerclinic.Methods: METHODS: Retrospective chart review of 37 consecutivepatients (66 eyes) fit with the PROSE device.Results: RESULTS: Indications for PROSE treatment includedectasia in 17 eyes (25.8%) and ocular surface disease in 49 eyes(74.2%). The average follow-up time was 4.76 months (2.4-9.35).Visual acuity improved with the PROSE device a statisticallysignificant amount (p=0.002). All patients had improved ocularsymptoms and decreased reliance on topical lubricants.Conclusions: CONCLUSIONS: PROSE is an effective treatmentoption for many patients with complex corneal diseases.Commercial Relationships: Kira L. Segal, None; Michelle N. Lee,None; Kristin O. Chapman, None; Mark Rosenblatt, None;Kimberly C. Sippel, None; Christopher E. Starr, None; JessicaCiralsky, NoneProgram Number: 5486 Poster Board Number: A0185Presentation Time: 8:30 AM - 10:15 AMEffect of Static and Non-Static in-vitro Techniques on LipidPenetration Into SiHy Contact LensesJean T. Jacob 1, 2 , Rebecca Frederick 1, 2 , Caitlin Tucker 1, 3 , LeaannLove 1, 4 . 1 Department of Ophthalmology, LSU Health SciencesCenter, New Orleans, LA; 2 Biomedical Engineering, TulaneUniversity, New Orleans, LA; 3 University of New Orleans, NewOrleans, LA; 4 Xavier University, New Orleans, LA.Purpose: We investigated the effect of replenishing the artificial tearfluid (ATF) over the study period on the lipid penetration profile intosilicone hydrogel lenses.Methods: We compared the lipid biofouling penetration profiles offive contact lens materials after 12 hours of exposure to static ATFdaily wear conditions and daily wear conditions where the ATF wasreplaced hourly using fluorescently-labeled phosphatidylcholine andcholesterol. Lipid penetration differences between the central 6 mmand the peripheral edges of the lenses were imaged with confocalmicroscopy.Results: The non-static method allowed for more adsorption andpenetration of both phosphatidylcholine and cholesterol into alllenses types tested (p=0.0001). There was approximately a 2-foldincrease in the amount of cholesterol found in all the lenses with thenon-static method as compared to the static. While there was also anincrease in the amount of PC within all lenses with the non-staticmethod compared to the static method is was not as significant as thecholesterol. The non-static method also showed more significantdifferences between the lipid penetration profiles for the lens typescompared to the static method. The non-static method showed moresurface penetration of PC in the periphery of the lens as compared tothe center for Acuvue Oasys, Biofinity and PureVision 2 than AirOptix and AirOptix Aqua which was not seen with the static method.However, the penetration profile for cholesterol did not changesignificantly between the non-static and the static methods for any ofthe lenses tested.Conclusions: The non-static method provided better differentialanalysis of lipid penetration into contact lens materials than the staticmethod. Specific differences based on lipid type and material surface©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneastructure were able to be identified that were not seen with the staticmethod. Future studies on contact lenses biofouling should use nonstaticmethodologies to more accurately mimic the in vivoenvironment.Commercial Relationships: Jean T. Jacob, 4,865,601 (P),5,282,851 (P); Rebecca Frederick, None; Caitlin Tucker, None;Leaann Love, NoneSupport: Unrestricted grant Research to Prevent BlindnessProgram Number: 5487 Poster Board Number: A0186Presentation Time: 8:30 AM - 10:15 AMVisual Performance and Optical Quality with New SiliconehydrogelSoft Contact Lenses for KeratoconusAsaki Suzaki 1 , Naoyuki Maeda 2 , Mutsumi Fuchihata 2 , Shizuka Koh 2 ,Kohji Nishida 2 , Takashi Fujikado 1 . 1 Applied Visual Science, OsakaUniversity Graduate School of Medicine, Suita, Japan;2 Ophthalmology, Osaka University Graduate School of Medicine,Suita, Japan.Purpose: To study the visual performances of a novel siliconehydrogelsoft contact lens (Si-SCL), which was designed to correct arefractive vertical asymmetry in keratoconic eyes.Methods: Fifty eyes of 37 subjects with mild keratoconus wererecruited for the study. Measurements included corneal anterioroptical coherence tomography (SS-1000, Tomey), wavefrontaberrometry (KR-1W, Topcon), subjective refraction, visual acuity(VA), visual clarity with visual analog scale (VAS). Study lenseswere made by molding method and were consisted of 6 types, inwhich asymmetrical power distribution (2D, 4D, 6D, 8D, 10D, 12D)was created to correct vertical irregular astigmatism. Based on thetopography and aberrometry data, the type of lenses was selected andfitted to the subjects. Evaluation of the on-eye performance of thelens was carried out, including wavefront aberrometry (4mm pupil,root mean square), over-refraction, VA, visual clarity with VAS.Results: Monocular visual performance with a Si-SCL improvedfrom -0.03±0.13 to -0.08±0.08 LogMAR in VA (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To evaluate the difference between day one versus daytwenty-eight levels of eight strongly-adherent contact lens-adsorbedprotein analytes (lysozyme, lactoferrin, lipocalin, free κ/λ Ig lightchain, IgA heavy chain, monomeric IgA, secretory component, andtotal protein) among a group of non-ionic silicone hydrogel contactlens wearers.Methods: Lenses were collected from subjects aseptically and storedat below -20 degrees C. All subjects wore the same non-ionic siliconehydrogel lens material and used the same lens care regimen. Lenseswere equilibrated to ambient temperature and rinsed in 2 mL ofphosphate buffered saline for 5 minutes with agitation. The lens wasextracted in SDS/urea buffer. The extraction buffer was concentrated,labeled with a fluorimetric dye, and subjected to microfluidicelectrophoresis.Results: Total strongly-adsorbed protein decreased between wearfrom day-one to day-twenty eight, 1.64 to 1.22 µg/lens (p=0.004,Mann-Whitney), respectively. Among the other resolved proteinslysozyme, lactoferrin, lipocalin, κ/λ Ig light chain, and monomericIgA also exhibited statistically-significant decreases over the wearcycle (p0.05).Accommodative lag increased for higher stimulus levels for all 3types of contact lenses. Ocular aberrations were not significantlydifferent between these 3 contact lens designs at each of the differentviewing distances (p>0.05). In addition, optical aberrations did notsignificantly differ between different viewing distances for any ofthese lenses (p>0.05).Conclusions: There was no statistically significant difference inaccommodative response between the three different low-add powermultifocal contact lenses. Optical aberrations did not differ betweenlenses at different viewing distances.Commercial Relationships: Balamurali Vasudevan, None; SaraN. Gaib, Bausch+Lomb (C), Alcon (R), Vistakon (R), Coopervision(R)532 Corneal Wound Repair and HealingThursday, May 09, 2013 10:30 AM-12:15 PMTCC 303 Paper SessionProgram #/Board # Range: 5974-5980Organizing Section: CorneaProgram Number: 5974Presentation Time: 10:30 AM - 10:45 AMEffect of Advanced Glycation End Products on apoptosis ofHuman Corneal Epithelial CellsXinyi Wu. Ophthal QiLu Hosp/Ophthal, Shandong University, Jinan,Shandong, China.Purpose: To investigate the effects of Advanced Glycation EndProducts (AGE) on apoptosis in human corneal epithelial cells andthe underlying mechanisms in vitro.Methods: Telomerase-immortalized human corneal epithelial cells(THCEs) were treated with 0-400 μg/ml AGE-modified bovine serumalbumin (BSA) for various times. THCEs apoptosis was analyzed byannexin-V-FITC and propidium iodide (PI) stain. The production ofreactive oxygen species (ROS) was measured with 2', 7'-dichlorofluorescein diacetate (DCFH-DA) dye assay and imaged onlaser scanning confocal fluorescence microscope. NADPH oxidaseactivity was examined by lucigenin-enhanced chemiluminescenceassay. The protein levels of cytochrome c, caspase-3, Bax, Bcl-2,p47phox, p67phox, phosphorylated/total proteins of ERK, JNK, andp38 MAPK were determined by Western blot.Results: Treatment of THCEs with AGE-BSA resulted in activationof the caspase 3, increase of Bax synthesis and the release ofcytochrome c, whereas, the expression of anti-apoptosis factor Bcl-2were decreased markedly. AGE-BSA induced THCEs apoptosis wasaccompanied by production of ROS. AGE-BSA-induced apoptosiswas inhibited by pretreatment with ROS quencher N-acetylcysteine(NAC). AGE-BSA activates NADPH oxidase through translocationof p47phox and p67phox, cytosolic subunits of NADPH oxidase, tothe cell membrane. Furthermore, inhibition of NADPH oxidase by itsspecific inhibitors (DPI or apocynin) significantly prevented AGE-BSA induced ROS production and subsequent apoptosis. We alsofound that AGE-BSA stimulation activated JNK and p38 MAPK.JNK inhibitor SP600125 and p38 inhibitor SB203580 effectivelyblocked AGE-BSA-induced apoptosis. In addition, inhibition of ROSgeneration with NAC completely blocked phosphorylation of JNKand p38 MAPK induced by AGE-BSA.Conclusions: AGE-BSA induced THCEs apoptosis via ROSgeneration and JNK/p38 MAPK pathway activation, providing a newmechanism for AGE-induced cell apoptosis in human cornealepithelial cells. The results suggest that regulation of ROS productionand JNK/p38 MAPK pathway may be a new strategy for preventionof AGE-induced corneal epithelial diseases.Commercial Relationships: Xinyi Wu, NoneSupport: Natural Science Foundation of China Grant (81271716)©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 5975Presentation Time: 10:45 AM - 11:00 AMType III Intermediate Filament Overexpression in the Genesis ofCorneal FibrosisRoyce Mohan 1 , Linda Cauley 2 , Paola Bargagna-Mohan 1 .1 Neuroscience, University of Connecticut Health Center HealthCenter, Farmington, CT; 2 Immunology, University of ConnecticutHealth Center, Farmington, CT.Purpose: The type III intermediate filament (IF) vimentin is highlyexpressed in the cornea during fibrosis and was recently shown to betherapeutic target for corneal fibrosis (Bargagna-Mohan, JBC 2012).We used bone marrow transplantation (BMT) studies along withwithaferin A, a vimentin/desmin-targeting small molecule probe, toinvestigate the myofibroblast-mediated genesis of corneal fibrosis.Methods: Mice 129Svev wild type (WT) and 129 Svev vimentindeficient(Vim KO) were subjected to corneal alkali injury withepithelial debridement. BMT from WT donors to Vim KO recipientmice and vice versa was performed and mice subjected to cornealinjury. Mice were treated with vehicle or WFA at 2 mg/kg/d byintraperitoneal injection for different periods (1 week to 1 month),and corneas analyzed for opacity till 9 months post-injury usingbiomicroscopy and computer imaging colorimetric technique. Oculartissues were analyzed using immunohistochemistry (IHC) andwestern blotting (WB) for fibrotic markers (vimentin, desmin, Skp2,alpha smooth muscle actin and p27Kip1)Results: We previously reported that Vim KO mice recover fromcorneal injury with attenuated fibrosis, which was mimicked by WFAtreatment in WT mice. Further improvement in corneal clarity byWFA in Vim KO was attributed to desmin downregulation. OurBMT experiments confirm that vimentin overexpression in cornealmyofibroblasts (BM recipients from Vim KO) drive fibrosis, whichwas comparable with injured WT mice not subjected to BMT. Thisresult was also corroborated in Vim KO mice (BM recipients fromWT) that showed improved corneal clarity similar to injured Vim KOmice not subjected to BMT. In our longitudinal study, WFAtreatment for 1 month with assessment at 9 months post-injuryshowed significant improvement in corneal clarity in WT recipientscompared to vehicle treated mice (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaVickery E. Trinkaus-Randall, Albert Lee, Kelsey Derricks, MatthewNugent. Ophthalmology L904, Boston University Sch of Med,Boston, MA.Purpose: Damage to corneal nerves can originate from traumaincluding surgery, infection and chemical injury. Previously wedemonstrated that injured neuronal and epithelial cells release ATPand induce a rapid response that is detected by imaging Ca2+mobilization. Our immediate goal was to understand how neuronalepithelialsignaling was altered under environmental stress.Methods: Cell cultures and organ cultures were used to determinecell communication.To perform the experiments we used a combination of a confocal(Carl Zeiss inverted LSM 700 and 510) and a multiphoton/confocalmicroscope system (Carl Zeiss upright LSM 710 NLO). Ca2+mobilization was monitored in cell-cultures and in corneas fromThy1-GFP-rats. To analyze the response to stimuli we developed acustom MATLAB software script to analyze calcium flux movies.The script can individually identify cells, normalize each calciumsignal value to the cell’s individual background, and elucidate themagnitude of response, and clusters of coordinated cells.Results: In intact corneas from Thy1-GFP-rats, the fluorescentsensory neurons were detected passing through the basal lamina andthe thin apical processes entered the basal epithelium labeled with aCa2+ indicator dye. In cultured cells the ATP stimulated responsegenerated by epithelial wound medium generated a rapid wave thatwas inhibited using Apyrase, a nucleotidase. In contrast media frominjured neuronal cells elicited both the rapid wave and a secondslower wave that was present in multiple cell clusters.Communication between cells was delineated with custom MATLABcell clustering analysis. The neuronal wound media was dissectedusing ionotropic (N-methyl-D-aspartate (NMDA) receptor inhibitorsand Apyrase where the latter abrogated the first peak but left theoscillatory glutamatergic response, while NMDA inhibitorsdiminished only the secondary response. Factors released by neuronalcells facilitated directed epithelial cell migration. When cells weresubjected to neuronal media under environmental stress there was adecrease in Ca2+ mobilization and cell clusters. In addition there wasa change in localization of an NMDA receptor (NR1).Conclusions: These results suggest that two separate modes of Ca2+mobilization mediate cell communication and provide insight intomechanisms that corneal nerves and epithelia signal to each other inresponse to injury.Commercial Relationships: Vickery E. Trinkaus-Randall, None;Albert Lee, None; Kelsey Derricks, None; Matthew Nugent, NoneSupport: NIH EY06000, Mass Lions Eye Research Fund, NewEngland Corneal Transplant FundProgram Number: 5978Presentation Time: 11:30 AM - 11:45 AMCiliary neurotrophic Factor accelerates corneal NerveRegeneration in a Murine ModelRudolf F. Guthoff, Maria Reichard, Marine Hovakimyan, OliverStachs. Ophthalmology, University of Rostock, Rostock, Germany.Purpose: The aim of this study was the in vivo examination of themouse subbasal nerve fibre plexus (SBP) during regenerationprocess. Particularly, investigations addressed the regenerationcapabilities of the injured SBP, and the influence of local ciliaryneurotrophic factor (CNTF) application on the regeneration process.We have created a simple yet effective and well-tolerated injurymodel designed to influence a distinct corneal nerve area.Methods: Twelve-week-old BALB/c mice were included in thisstudy. A circular incision through corneal epithelium and anteriorstroma was generated with a custom-made guided trephine system tocut the nerves in SBP. The surgery was performed unilaterally.Animals were subdivided in 2 groups, one of them becoming CNTFeye drops 3 times daily. In vivo confocal laser scanning microscopy(Heidelberg Retina Tomograph/Rostock Cornea Module) was used tocharacterize the SBP before and up to 8 weeks after surgery. Nervefibre density (NFD) was determined with the semi-automatic nervetracing program NeuronJ.Results: NFD was reduced in both groups 24 hours after circularcorneal incision. The regeneration of subbasal nerve fibres was basedon sprouting out of stromal nerves within the cut and on regrowth ofsubbasal nerves over the scar from outside the cut. The statisticallysignificant NFD reduction was observed for up to 3 weekspostoperatively in both groups, with and without CNTF, whencompared to intact controls. The neuroprotective influence of CNTFeye drops was observed as early as 1 week after injury. At that timepoint the CNTF group displayed 38.39 % of baseline NFD, comparedwith only 13.02% in the group without CNTF.The difference between both groups remained significant for up to 4weeks postoperatively.Eight week postoperatively NFD displayed 92.49% and 88.65% ofbaseline in the groups with and without CNTF-treatment,respectively.Conclusions: Uneventful healing of SBP could be demonstrated aftersurgical dissection in BALB/c murine corneas. The regenerationprocess was significantly accelerated by topical application of CNTF.Commercial Relationships: Rudolf F. Guthoff, None; MariaReichard, None; Marine Hovakimyan, None; Oliver Stachs, NoneProgram Number: 5979Presentation Time: 11:45 AM - 12:00 PMThe Mechanotransducers YAP and TAZ are Modulated bySubstratum Compliance in Corneal Stromal Cells and duringCorneal Wound HealingChristopher J. Murphy 1, 2 , Sara M. Thomasy 1 , Vijaykrishna K.Raghuanthan 1 , Christopher M. Reilly 3 , Paul Russell 1 . 1 Surgical andRadiological Sciences, School of Veterinary Medicine, University ofCalifornia, Davis, Davis, CA; 2 Ophthalmology & Vision Science,School of Medicine, University of California, Davis, Davis, CA;3 Pathology, Immunology & Microbiology, School of VeterinaryMedicine, University of California, Davis, Davis, CA.Purpose: Biophysical cues profoundly influence corneal cellularbehavior including processes integral to corneal wound healing suchas migration and proliferation. Mechanotransduction or thetranslation of external biomechanical cues into internal biochemicalevents remains poorly understood in corneal cells. Yes-associatedprotein (YAP) and transcriptional coactivator with PDZ-bindingmotif (TAZ) have been identified as cellular mechanotransducers.Thus, the purposes of this study were 1) to determine the effects ofsubstratum stiffness on expression of YAP/TAZ in human cornealfibroblasts (HCF) and myofibroblasts and 2) to determine the effectsof corneal wounding on expression and localization of YAP/TAZ.Methods: Primary HCFs were cultured on polyacrylamide substratesof differing stiffness (10 or 85 kPa) or on tissue culture plastic (TCP;>1GPa) in serum-containing media with 0 or 10 ng/ml TGFβ-1.Expression of YAP, TAZ and α-smooth muscle actin (αSMA) toconfirm fibroblast to myofibroblast transition was determined byqPCR. Rabbits underwent epithelial debridement only or incombination with phototherapeuic keratectomy (PTK; 6 mmdiameter, 100 μm deep) on the right eye (OD); the left eye (OS)served as a control. Rabbits were euthanized 6 days later and theglobes were removed and fixed in formalin. Immunohistochemistryfor YAP/TAZ was performed.Results: In the presence of 10 ng/ml TGFβ-1, TAZ expression was©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasignificantly greater on the more compliant substrates in comparisonto TCP. Interestingly, αSMA was markedly decreased on the 10 kPasubstrate versus 85 kPa or TCP. In normal cornea, YAP/TAZ werepredominantly localized to the epithelial cell cytoplasm andminimally detected in normal stroma. Following PTK, YAP/TAZwere markedly localized in the epithelial cell nuclei and robustexpression in anterior stromal cells was observed. Similar resultswere observed with epithelial debridement only except the depth ofeffect on anterior stromal cells was much less.Conclusions: Substratum compliance markedly alters TGFβ-1induced transformation of fibroblasts to myofibroblasts with stiffersubstrates promoting transformation and decreasing TAZ expression.YAP/TAZ expression and localization are markedly altered duringcorneal wound healing. Further studies are required to determine therole of YAP/TAZ in corneal cellular mechanotransduction andwound healing.Commercial Relationships: Christopher J. Murphy, OcularServices On Demand (I), Ocular Services On Demand (C), PlatypusTechnologies LLC (I), Imbed LLC (I), EyeKor LLC (I), Allergan(C), Genentech (C), Sarcode (C), Covance (C); Sara M. Thomasy,None; Vijaykrishna K. Raghuanthan, None; Christopher M.Reilly, None; Paul Russell, NoneSupport: NIH R01EY019970 (CJM), NIH R01EY01634 (CJM),NIH KO8EY021142 (SMT), and Research to Prevent Blindness (UCDavis)Program Number: 5980Presentation Time: 12:00 PM - 12:15 PMAnti-inflammatory therapy changes fate of alkali burned corneasassociated with dry eyeCintia S. De Paiva, Flavia S. Pelegrino, Eugene A. Volpe, Stephen C.Pflugfelder. Ophthalmology, Baylor College of Medicine, Houston,TX.Purpose: The purpose of this study was to investigate the effects ofanti-inflammatory therapy in a concomitant dry eye and cornealalkali burn murine model.Methods: C57BL/6 mice were subjected to unilateral alkali ocularburn (OB) without or with concomitant desiccating stress (DS) for 2or 5 days (D). A separate group of mice that received both OB andDS were topically treated either with 2µL Dexamethasone, 2uL ofDoxycycline or vehicle (balanced salt solution). Mice were observeddaily for appearance of corneal perforation. Mice were euthanizedafter 2 or 5 days and eyes and adnexa were collected for histology.Whole corneas were collected and lysed for gene expression.Quantitative real time PCR was performed to measure expression ofinflammation cytokines and matrix metalloproteinases (MMPs).Results: Eyes subjected to OB+DS had 20% sterile cornealperforation rate as soon as 1 day after the initiation of the insultswhich increased to 80%-90% by 5 days; significantly increasedMMP-9 immunoreactivity and activity were noted compared to OBand DS controls. Significantly increased MMP-1,-3,-8,-9, -13, IL-1βand IL-6 mRNA transcripts in cornea were found early at day 2 inOB+DS compared to OB alone. OB+DS eyes treated withDoxycycline or Dexamethasone had significant lower rate of ocularperforation (10% at 2D, 20% at 5D and 0% at 2 and 5D,respectively), and significantly decreased MMP-1,-3, -8, -9, IL-1βand IL-6 mRNA transcripts in cornea compared to vehicle-treatedeyes.Conclusions: Concomitant dry eye and alkali burn leads to ocularperforation accompanied by cytokine and MMP storm in the cornea.Early treatment with anti-inflammatory therapy is critical inpreserving eye integrity and decreasing IL-1β, IL-6 and MMPs.Commercial Relationships: Cintia S. De Paiva, Glaxo Smith Kline(C), Baylor College of Medicine (P); Flavia S. Pelegrino, None;Eugene A. Volpe, None; Stephen C. Pflugfelder, Allergan (C),Glaxo Smith Kline (C), Bausch and Lomb (C), Baylor College ofMedicine (P)Support: DOD W81XWH-12-1-0616 (CSDP), NIH Grant EY11915(SCP), RPB, Oshman Foundation, William Stamps Farish Fund andHamill Foundation.535 Dry Eye and Lacrimal Gland VThursday, May 09, 2013 10:30 AM-12:15 PMExhibit Hall Poster SessionProgram #/Board # Range: 5999-6053/A0062-A0116Organizing Section: CorneaContributing Section(s): Visual Psychophysics / PhysiologicalOpticsProgram Number: 5999 Poster Board Number: A0062Presentation Time: 10:30 AM - 12:15 PMToll-like Receptor Agonists Stimulate Matrix Metalloproteinase-9 Production in Ocular Surface CellsRachel L. Redfern, Kendall Stout, Karen Dionne. College ofOptometry, University of Houston, Houston, TX.Purpose: Dry eye syndrome (DES) is a chronic multifactorialinflammatory condition that can lead to corneal ulceration andchronic ocular discomfort. The pathogenesis of DES is not fullyunderstood and toll-like receptors (TLRs) may be in part responsiblefor the production of dry eye associated proinflammatory cytokinesand matrix metalloproteinases (MMPs) that lead to ocular surfacepathology. Previously we have shown that topical application of aTLR agonist cocktail to the ocular surface in mice with experimentaldry eye leads to corneal ulceration, yet the mechanism is unknown.This study examined the hypothesis that TLR agonists stimulate theproduction of MMP-9 in human ocular surface cells.Methods: Primary human corneal epithelial cells (HCEC) and SV40HCEC were treated with either IL-1β, TLR6/2 (FSL), TLR3(POLYI:C), TLR4 (LPS), TLR5 (Flagellin) or TLR9 (ODN2006)agonists or the vehicle control for 48 hrs. Following treatment, thecell culture media was collected to detect MMP-9 protein secretion.Total RNA was extracted from cell culture lysates. Real-time PCRand ELISA were used to quantitate MMP-9 mRNA and proteinrespectively. A statistically significant change (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Rachel L. Redfern, None; KendallStout, None; Karen Dionne, NoneSupport: NIH Grants EY018113 (RLR), EY007551 (UHCO) andEY007088 (KS)Program Number: 6000 Poster Board Number: A0063Presentation Time: 10:30 AM - 12:15 PMCorneal Epithelial Cell Protective and Wettability-enhancingProperties of Hyaluronic acid + PEG 8000 in an Artificial TearProductStephen Davio, Megan E. Cavet, Karen L. Harrington, Amy Walsh,Zora Marlowe, Brian Glass, Paramita Sarkar. Pharmaceutical R&D,Bausch & Lomb, Rochester, NY.Purpose: Artelac Rebalance® (“product”) is an artificial tear productwhich combines hyaluronic acid (HA) and a large molecular weightpolyethylene glycol, PEG-8000. We conducted in vitro studies toprobe the effect of this unique combination on corneal epithelial cellsurvival under desiccating stress. We also evaluated wettabilityaspects of the formulation using physical measurements.Methods: Corneal cell survivability under desiccating stress wasevaluated using human corneal epithelial cells (HCEpiC) pre-treatedwith saline (control) or 50% product +/- HA and +/- PEG 8000 for 10min followed by desiccation for 20 min. The percentage of livingcells was determined by staining with calcein using the LIVE/DEADassay kit (Invitrogen). Wettability of the formulation was assessed forproduct +/- HA and +/- PEG 8000 by measuring surface tension(Kruss K-100) and by measuring the effect of the formulation oncontact angle in a captive bubble apparatus (FTA 1000 + FTA32video 2.0 software) using a silicone hydrogel contact lens as modelfor the cornea. Contact angles were measured in the presence of theproduct, and after removal of product and replacement with saline.Results: Corneal cells exposed to product show significantly highersurvival to desiccating stress than cells exposed to saline (68 and72% vs. 5%, respectively). Removal of HA from the formulationdrops cell survivability to that of the saline control. Removal of PEG-8000 has no effect on cell survivability which remains equivalent tointact formulation. Surface tension (ST) measurements on the intactformulation show 64 mN/m which is a decrease from that of water,72 mN/m. This effect is entirely associated with PEG 8000. Contactangle measurements using the captive bubble apparatus show theproduct enhances the wettability of a model surface which persistsafter replacement of the product with saline. This enhancedwettability and the residual effect are both due exclusively to PEG8000.Conclusions: The in vitro and physical measurements demonstratecomplementary properties of HA and PEG 8000 in the artificial tearproduct. HA is demonstrated to enhance survival in corneal cellsunder desiccating stress. PEG 8000 demonstrates a consistentdecrease in surface tension and an ability to enhance the wettabilityof a model surface which persists after removal of the product.Commercial Relationships: Stephen Davio, Bausch & Lomb (E);Megan E. Cavet, Bausch + Lomb (E); Karen L. Harrington,Bausch + Lomb (E); Amy Walsh, None; Zora Marlowe, Bausch &Lomb (E); Brian Glass, Bausch + Lomb, Inc. (E); Paramita Sarkar,Bausch and Lomb (E)Program Number: 6001 Poster Board Number: A0064Presentation Time: 10:30 AM - 12:15 PMChange of Tear Secretion in the Lacrimal Glands of High-FatDiet-Fed C57BL/6J MiceTakaaki Inaba, Motoko Kawashima, Akiko Ito, Mitsuhiro Watanabe,Ken Shinmura, Tetsuya Kawakita, Kazuo Tsubota. keio university,Tokyo, Japan.Purpose: Aging is one of the most important factors for thepathogenesis of dry eye disease. Metabolic syndrome acceleratesaging. Although several studies have reported that diabetic patientshave an elevated incidence of dry eye disease, the mechanismresponsible for dry eyes disease remains unclear.Methods: We investigated the secretion and metabolic changesoccurring at 6 months in response to a high-fat diet (HFD).Results: Tear secretion was decreased by the HFD compared tonormal diet at 6 months. Additionally, inflammatory cell infiltrationincreased in the lacrimal glands of the HFD-fed mice.We alsoinvestigated the molecular mechanisms involved includingmetabolism and aging genes.Conclusions: The results indicate that aging accelerated by the HFDis related to dry eye.Commercial Relationships: Takaaki Inaba, None; MotokoKawashima, Santen Pharmaceutical Co., Ltd (F); Akiko Ito, None;Mitsuhiro Watanabe, None; Ken Shinmura, None; TetsuyaKawakita, None; Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F),Bausch Lomb Surgical (C), Functional visual acuity meter (P), JiNS(P), Kissei (F), Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C),Thea (C), Echo Denki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu(F), CEPT Company (P)Program Number: 6002 Poster Board Number: A0065Presentation Time: 10:30 AM - 12:15 PMComparison of two rodent models of dry eye induced byScopolaminePierre-Paul Elena 1 , Nicolas Cimbolini 1 , Sophie Antonelli 1 , LaurenceFeraille 1 , Stefano Barabino 2 , Philippe Margaron 1 . 1 Iris Pharma, LaGaude, France; 2 Clinica Oculistica, Genova, Italy.Purpose: Dry eye syndrome is a relatively common disease withmultifactorial causes. It is necessary to have an experimental modelto test and select therapeutic candidates for this disease.Here we compare two experimental models using scopolamine, atropane alkaloid drug with muscarinic antagonist effects, to suppresslacrymation and induce dry eye symptoms.Methods: The first experimental model consisted in inducing dry eyein albino rats by systemic and continuous delivery of scopolamine(20mg/day) via osmotic pumps implanted subcutaneously on Day 1.Animals were divided in three groups of five rats: In the first groupthe pumps delivered saline solution. The second and the third groupsthe pumps delivered 20 mg/day of scopolamine solution over 21days.The second experimental model consisted in placing pigmented micein a controlled environmental chamber (relative humidity 55%,temperature 20-22°C) without transdermal scopolamineadministration. The second and the third groups were placed in thecontrolled environmental chamber with transdermal scopolamineadministration.For both models, the first two groups received saline instillation andthe third group, 0,05% cyclosporine eye drops.Tear production was measured with the phenol red thread (PRT) testand the corneal defects were examined by slit-lamp observation.These examinations were preformed at baseline and on day 7 for themouse and rat models, and on days 14 et 21 only for rat model inboth eyes.Results: Symptoms of dry eye, including decrease in tear secretion,appearance of corneal defects and a rise in the inflammatory markers©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneainvestigated, were observed in both rat and mouse models. Moreoverthese symptoms decreased after treatment with topical cyclosporine,a drug used in dry eye.Conclusions: Scopolamine treated rats and combination ofscopalamine treatment and controlled environment in mice, aretherefore valuable models to mimic human dry eye syndrome.Commercial Relationships: Pierre-Paul Elena, None; NicolasCimbolini, None; Sophie Antonelli, None; Laurence Feraille,None; Stefano Barabino, None; Philippe Margaron, Iris Pharma(E)Program Number: 6003 Poster Board Number: A0066Presentation Time: 10:30 AM - 12:15 PMThe Composition of Fatty Acids in Human MeibumTomo Suzuki 1, 2 , Sayaka Kamada 1, 2 , Tetsuya Tajika 3 , SatoshiFujiwara 4 , Shigeru Kinoshita 1 . 1 Ophthalmology, Kyoto PrefecturalUniversity of Medicine, Kyoto, Japan; 2 Ophthalmology, Kyoto CityHospital, Kyoto, Japan; 3 Senju Pharmaceutical Co. LTD., Osaka,Japan; 4 Shimadzu Techno-Research, INC., Kyoto, Japan.Purpose: The lipid analysis of human meibomian gland secretion(meibum) has been conducted by many research groups in an attemptto elucidate the pathogenesis of dry eye and meibomian glanddysfunction. However, due to difficulties associated with thecollection of pure uncontaminated meibum and precise lipid analysis,the related data reported in previous studies have varied. The purposeof this present study was to reevaluate the methods of meibumcollection and lipid analysis, and to more precisely elucidate thecomposition of fatty acids in human meibum.Methods: This study involved 4 healthy volunteer subjects (2 malesand 2 females) ranging between 30 and 36 years of age. After a warmcompression of the subjects’ eyes by use of a Panasonic electronicwarm compression device (40°C, 10 minutes) on one day and withoutwarm compression on another day, a spatula was used to obtainmeibum from the eyes of each subject after a gentle squeezing of theeyelid margin by use of a meibomian gland compressor. Eachmeibum-sample collection was performed by use of a spatula thatwas thoroughly washed in high-grade organic solvents and then airdriedin order to avoid any possible lipid contamination. The obtainedmeibum was transmethylated, and then analyzed by use of gaschromatography-mass spectrometry (GC-MS).Results: The composition of fatty acids in the meibum samples wasfound to be similar between with and without warm compression, aswell as between the male and female subjects. In the meibumsamples, unsaturated fatty acids (monounsaturated: 52.0-56.5%;polyunsaturated: 3.6-3.9%) and branched fatty acids (34.7-37.6%)were found to be the major components, whereas saturated fatty acids(2.3-2.9%) were found to be only minor components. Moreover,those results were reproducibly obtained.Conclusions: The findings of this present study show that humanmeibum predominantly contains unsaturated- and branched fattyacids, which are different results from those reported in previousstudies.Commercial Relationships: Tomo Suzuki, Senju PharmaceuticalCo. (F); Sayaka Kamada, None; Tetsuya Tajika, SenjuPharmaceutical, Co., Ltd. (E); Satoshi Fujiwara, None; ShigeruKinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Support: The grant from Japanese Ministry of EducationProgram Number: 6004 Poster Board Number: A0067Presentation Time: 10:30 AM - 12:15 PMTopical atorvastatin for the treatment of dry eye associated withblepharitisStephanie L. Watson 1 , Kenneth G. Ooi 1, 2 , Frank A. Billson 1 , DenisWakefield 3 . 1 Ophthalmology, Save Sight Institute, University ofSydney, Sydney, NSW, NSW, Australia; 2 Department ofOphthalmology, Prince of Wales Hospital, Sydney, NSW, NSW,Australia; 3 Department of Pathology, School of Medical Sciences,University of New South Wales, Sydney, NSW, NSW, Australia.Purpose: To investigate topical Atorvostatin, a novel ocular therapy,for the treatment of patients with dry eye associated with blepharitis.Methods: 10 patients with symptoms and signs of dry eye andblepharitis were enrolled in a prospective pilot study of topicalAtorvostatin therapy over 1 month. The primary outcome measurewas corneal fluorescein staining.Results: An improvement in corneal fluorescein staining in thetreated eye by more than one point from baseline to completion of thetrial at week four was found in 9 of 10 patients (p = 0.007). Topicalstatin significantly improved the blepharitis score (p = 0.011) and thetear film break-up time (p = 0.005). Subjective dry eye symptomsimproved (p = 0.005). There were no serious or irreversible sideeffects.Conclusions: Topical statins are a potential therapy for patients withdry eye and blepharitis. They can be easily manufactured and couldbe widely available. It may also be of benefit in other dry eyesyndromes.Commercial Relationships: Stephanie L. Watson, Sydnovate (P);Kenneth G. Ooi, Sydnovate (P), New South Innovations (P); FrankA. Billson, None; Denis Wakefield, NoneSupport: Save Sight Institute GrantClinical Trial: 07/032Program Number: 6005 Poster Board Number: A0068Presentation Time: 10:30 AM - 12:15 PMOptical quality after instillation of three different eye drops fordry eyeShizuka Koh 1 , Naoyuki Maeda 1 , Chikako Ikeda 1, 2 , Yoshihiro Takai 1,2 , Hisataka Fujimoto 1 , Yoshinori Oie 1 , Takeshi Soma 1 , MotokazuTsujikawa 1 , Kohji Nishida 1 . 1 Ophthalmology, Osaka UniversityGraduate School of Medicine, Suita, Japan; 2 Research &Development, Rohto Pharmaceutical CO.,LTD, Kyoto, Japan.Purpose: To investigate the effect of 3% diquafosol ophthalmicsolution, 0.3% sodium hyaluronate ophthalmic solution, and 2%rebamipide ophthalmic suspension on forward light scattering andocular higher-order aberration (HOA).Methods: We evaluated forward light scattering and ocular HOAsbefore, immediately, 5, and 10 minutes after instillation of 3 differenteye drops in 7 eyes of 7 normal subjects (mean age, 30.1 ± 4.9 yearsold). Forward light scattering was quantified with the C-Quant straylight meter. (Oculus Optikgerate GmbH, Germany). HOAs wereobjectively measured for a 4-mm pupil using newly developedaberrometer (Topcon, Tokyo, Japan). The obtained aberration datawere analyzed in the central 4-mm diameter for total HOAs up to thesixth-order Zernike polynomials.Results: Significant change in forward light scattering was notedafter instillation of 2% rebamipide ophthalmic suspension and 3%diquafosol ophthalmic solution (P=0.020, 0.023, one way repeatedANOVA, respectively). Increased stray light immediately afterrebamipide ophthalmic suspension instillation recovered to thebaseline level thereafter (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea3% diquafosol ophthalmic solution (P=0.478) and after instillation of2% rebamipide ophthalmic suspension. (P=0.234).Conclusions: Quantitative serial measurement of forward lightscattering and HOAs showed that the temporal reduction in opticalquality may be mainly attributed to increased forward light scatteringafter instillation of 2% rebamipide ophthalmic suspension and 3%diquafosol ophthalmic solution, and increased HOAs after instillationof high viscosity 0.3% sodium hyaluronate ophthalmic solution innormal subjects.Commercial Relationships: Shizuka Koh, Santen, Inc. (R),Johnson & Johnson (R), Topcon (R), Otsuka Pharmaceutical Co. (R);Naoyuki Maeda, Topcon (F), Santen (R), Otsuka (R), Oculus (R),HOYA (R); Chikako Ikeda, Rohto pharmaceutical CO.,LTD. (E);Yoshihiro Takai, Rohto Pharmaceutical Co. Ltd (E); HisatakaFujimoto, None; Yoshinori Oie, Santen (F), HOYA (F); TakeshiSoma, HOYA corporation (R), Santen Pharmaceutical Co., Ltd (F),Otsuka Pharmaceutical Co., Ltd (F); Motokazu Tsujikawa, Shionogi& Co. (C), Daiichi Sankyo Co. (F), Daiichi Sankyo Co. (R), SantenCo. (R), AMO Co. (R); Kohji Nishida, Alcon (C), Alcon (F), HOYA(F), Senju (F), Pfizer (F), Santen (F), Osaka University (P)Support: Grants-in-Aid for Scientific Research from the JapaneseMinistry of Education, Culture, Sports, Science and Technology.(No. 22791659)Program Number: 6006 Poster Board Number: A0069Presentation Time: 10:30 AM - 12:15 PMEffects of diquafosol sodium eye drops on tear film stabilitySeika Den 1 , Hiroyuki Iseda 1 , MURAT DOGRU 1, 2 , Jun Shimazaki 1, 2 .1 Ophthalmology, Tokyo Dental College, Chiba, Japan;2 Ophthalmology, Keio University School of Medicine, Tokyo, Japan.Purpose: To investigate the effects of diquafosol sodium eye drops(DQS), a purinergic P2Y2 receptor agonist, on tear film stability inpatients with unstable tear film.Methods: Two prospective studies were conducted. One was anexploratory non-randomized trial on 39 eyes with dry eye symptomsand short tear film break-up time (BUT), but without epithelialdamage. Schirmer’s test value was not considered as an inclusion orexclusion criterion. Changes in questionnaire and Visual AnalogScale (VAS) scores for symptoms, BUT, Schirmer’s value, andfluorescein staining scores of the cornea and conjunctiva werestudied for 3 months. The other was a randomized clinical trial (RCT)of DQS and artificial tears in 17 eyes with short BUT. Eyes withdecreased Schirmer’s values (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaFigure 2. Molecular structure of lamellar sandwich.Commercial Relationships: Peter E. King-Smith, None; MelissaD. Bailey, None; Richard J. Braun, NoneSupport: NIH Grant EY17951 and NSF 1022706Program Number: 6008 Poster Board Number: A0071Presentation Time: 10:30 AM - 12:15 PMObservation of Spreading and Mechanical Properties of the TearFilm Lipid Layer Using A High Performance Video-BasedInterferometry SystemDaniel R. Powell, Peter E. King-Smith, Heather L. Chandler. VisionScience, Ohio State University, Columbus, OH.Purpose: Interferometric-based imaging systems have demonstratedtheir usefulness in obtaining clinically-relevant information from thepreocular tear film since the 1980’s. Camera resolution and imageblur secondary to movement of the tear film lipid layer (TFLL) areamong several limitations with these devices. The introduction of ahigh performance video-based interferometer (HPVI) can provideinformation on the spreading and mechanical characteristics of theTFLL not found in current imaging systems and includes a color keyto indicate TFLL thickness.Methods: The HPVI is capable of imaging a 6-mm diameter area ofthe pre-corneal tear film over a preselected time frame (40 sec). Avideo camera records images at 68 frames/sec at a resolution of1400x1100 pixels and used in conjunction with a stroboscopic lightsource (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneawere women. Five percent of the patients were contact lens users.Mean McMonnies Dry Eye Index Score was 14, the Ocular SurfaceDisease Index (OSDI) was 16 and the Mean Dry Eye Severity Level(DESL) was 2.1 (range, 0-3). Eighty-five percent reported dryness,75% soreness, 56% itching, 75% grittiness and 27% burningsensation in their eyes. Light sensitivity was mentioned by 73% ofthe patients, while 36 % reported mouth dryness. Thirtynine percenthad a tear meniscus height

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaquestionnaire to assess dryness symptoms. Lipid layer thickness(LLT) was assessed using the Tearscope Plus (Keeler) based on theappearance of the lipid layer. Non-invasive tear break up time(NITBUT) was assessed. The lower lid (LL) and upper lid (UL) of allsubjects were everted and the MGs imaged using the infra-red (IR)camera of the Keratograph 4 (OCULUS). A MG drop-out score(MGDS) due to complete or partial gland loss of both lids wasobtained via digital analysis of the images using ImageJ software.Results: 100% of the SS participants reported ocular and oral drynesssymptoms in the AECC questionnaire. The SS group recorded ahigher OSDI score (40.0±21.0 vs 1.7±1.7; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaClinico San Carlos, Madrid, Spain; 2 Ophthalmology, 2UniversityClinic Hospital, Düsseldorf, Germany.Purpose: To evaluate the thickness of the lipid layer of the tear filmin healthy subjects with a new interferometry device (Lipiview®)before and after instillation of various commercially available tearsubstitutes with lipid composition.Methods: Ten healthy subjects (20 eyes) were included in arandomized, double-masked study to participate in a three-day visitevaluation. Before, subjects completed a dry eye questionnaire(ocular surface disease index-OSDI-) to evaluate symptoms of dryeye. Only normal individuals, defined as a Schirmer test resultwithout anaesthesia above 10 mm/5min and Tear Break Up Time(TBUT) above 7s were included. A semiautomated interferometre(Lipiview® system), was used to measure the precorneal tear lipidlayer thickness followed by measurement of the non-invasive breakup time (NITBUT) with Oculus Keratograph® 5M. Then one of threelipid emulsion eye drops/spray (which main components containedwere lipid carbomer, rizin oil and lecithin soy respectively) wasapplied while the fellow eye received sodium hyaluronate, sodiumcarboxymethylcellulose or saline spray solution for negative control.In randomised sequence and after one week of wash-out betweenmeasurements, the 2nd and again one week later a 3rd emulsion wasalso studied. Measurements were performed at baseline and at 15, 30,60, and 120 minutes after instillation of the drops or application ofthe spray.Results: Tear lipid layer thickness significantly increased in allsubjects after lipid emulsion instillation. The emulsion with lipidcarbomer showed a significant increase at 15 and 30 time points frombaseline (p=0.012 and p=0.01). The emulsion with rizin oil alsosignificantly raised lipid layer thickness for 15 and 30 minutes(p=0.009 and p=0.002) and showed good correlation with NIBUT(p=0.001), as this parameter also increased from baseline at the sametime points. Lecithin soy spray significantly increased the lipidconcentration at 30 minutes only (p=0.006).Conclusions: Lipid emulsion eye drops significantly increase thelipid layer thickness of the tear film measured with Lipiview® inhealthy subjects. This effect lasts for only 30 minutes afterinstillation. The lipid layer thickness correlated well with the NIBUT.Commercial Relationships: Lara Borrego, None; Federico Saenz-Frances, None; David Finis, Oculus (R); Jose M. Benitez-del-Castillo, None; Gerd Geerling, Alcon (C), Allergan (C), TheaPharma (C), Novagali (C), Bausch & Lomb (C), Tearlab Inc. (C)Program Number: 6016 Poster Board Number: A0079Presentation Time: 10:30 AM - 12:15 PMBenzalkonium chloride-induced rat dry eye model mimicshyperosmolarity in tear volume deficient dry eye diseaseDavi L. Marques 1 , Monica Alves 1, 2 , Carolina Modulo 1 , LeonardoMalki 1 , Peter Reinach 1 , Eduardo M. Rocha 1 . 1 Universidade de SãoPaulo, Ribeirão Preto, Brazil; 2 Pontific Catholic University ofCampinas, Campinas, Brazil.Purpose: Chronic use of antiglaucomatous eye drops withbenzalkonium chloride (BAK) leads to ocular surface disease andtear film hyperosmolarity. In rodent BAK dry eye disease (DED)models, it is unclear if BAK induces hyperosmolarity. We show inrats that the BAK DED model also entails development of tear filmhyperosmolarity since tear volume collection is sufficient to monitorosmolarity changes.Methods: Unanesthesized male rats (n=10) were treated twice dailywith PBS containing 0.2% BAK in the right eye, whereas the left eyeserved as a control. After 7 days, tear film osmolarity was measuredwith the TearLab Osmolarity System (Ocusense). Phenol Red Threadtest (PRT) evaluated tear fluid rates in 15 sec whereas vital stainingmonitored ocular surface integrity. Observers evaluated stainingmagnitude by assigning arbitrary values of from 1-to 18. Lightmicroscopy evaluated corneal epithelial thickness.Results: Tear flows were 7.6 ± 2.01 mm in controls and 3.40 ± 0.67mm in BAK treated eyes (p=0.03). Lissamine staining was 1.0 ± 0.77in control and 8.40 ± 1.36 in BAK group (p=0.01); and fluoresceinwas 2.20 ±0.96 in control and 9.80 ±1.49 in BAK group. Controlosmolarity was 284.0 ± 3.29 mOsm and with BAK 306 ±4.09 mOsm(p=0.01). Corneal epithelial thickness decreased by day 7 relative tothat in the control group. Seven days after suspending BAKapplication, all changes recovered to their control values.Conclusions: The rat BAK DED model is novel since increases intear film osmolarity were measurable. This model is relevant sincethese rises mimic those identified in tear volume deficient patientsafflicted with DED.Commercial Relationships: Davi L. Marques, None; MonicaAlves, None; Carolina Modulo, None; Leonardo Malki, None;Peter Reinach, None; Eduardo M. Rocha, NoneSupport: Fundação de Amparo à Pesquisa do Estado de São Paulo -FAPESPProgram Number: 6017 Poster Board Number: A0080Presentation Time: 10:30 AM - 12:15 PMDebridement of the Lower Lid Margin and Line of Marx iseffective in increasing meibomian gland function and patientcomfortCaroline A. Blackie 1, 2 , Donald R. Korb 1, 2 . 1 TearScience, Morrisville,NC; 2 Korb Associates, Boston, MA.Purpose: The purpose of this study was to evaluate whethermechanical debridement of the Line of Marx (LOM) and the lowerlid margin would improve meibomian gland function and reduce dryeye symptoms.Methods: 16 symptomatic subjects undergoing treatment forevaporative dry eye (age range: 26-81 yrs, mean: 55.9 +/- 15.0 yrs)who also evidenced anteroplacement and a thickened LOM, wereenrolled and consented according to the tenets of the Declaration ofHelsinki. Exclusion criteria included active ocularinfection/inflammation, ocular surgery within the previous 6 months,lid abnormalities other than those related to meibomian glanddysfunction and/or normal age related lid margin changes. Prior todebridement the LOM was stained with lissamine green (OdysseyMedical, TN). The entire width of the keratinized lower lid marginwas debrided, followed by debridement of the stained LOM using astainless steel, foreign body, golf club spud (Akorn Ophthalmics, IL).Meibomian gland function and symptoms were assessed pre andapproximately 1-month post lid margin debridement. No new oradditional treatment was permitted during the 1-month postdebridement period. Using standardized diagnostic expression withthe meibomian gland evaluator, meibomian gland function wasevaluated along the full length of the lower lid margin. Symptomswere evaluated with the SPEED questionnaire (maximum score =28). Only data for the right eye is reported.Results: There was a significant decrease in symptoms and increasein the number of functional meibomian glands as a result of thedebridement of the lid margin and LOM. Symptoms: baseline meanpre-debridement = 13.4 +/- 4.6 and 1 month post-debridement = 10.5+/- 3.7 (p < 0.0001, paired t-test). Number of functional meibomianglands: baseline mean pre-debridement = 2.5 +/- 1.2 and 1 monthpost-debridement = 3.7 +/- 1.4 (p = 0.0007, paired t-test).Conclusions: The results indicate that debridement of the lower lidmargin and LOM provides significant symptom relief and improvesmeibomian gland function. Thus, lid margin debridement should begiven further consideration in the context of age related lid margin©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneachanges, and for the prevention and management of meibomian glanddysfunction and dry eye symptoms.Commercial Relationships: Caroline A. Blackie, TearScience (E);Donald R. Korb, TearScience (F)Program Number: 6018 Poster Board Number: A0081Presentation Time: 10:30 AM - 12:15 PMThe effect of increased periocular humidity on lipid layerthickness and ocular comfort of symptomatic contact lenswearersSamuel Kim 1 , Caroline A. Blackie 1, 2 , Donald R. Korb 1, 2 .1 TearScience, Morrisville, NC; 2 Korb Associates, Boston, MA.Purpose: To determine whether increasing periocular humiditywould 1) increase the thickness of the tear film lipid layer over thefront surface of a soft hydrogel contact lens, 2) increase wearingcomfort for symptomatic lens wearers.Methods: Symptomatic soft contact lens wearing patients wererecruited from a single practice in MA. All patients (n = 25; 5 males,17 females) had worn their lenses for a minimum of 2 hours and amaximum of 5 hours prior to examination. Inclusion criteria:minimum contact lens wear of twice a week for 5 or more hours at atime; minimum symptom score when wearing the lenses = 3 on ascale of 1-5 (1 = asymptomatic, 5 = intolerable discomfort).Exclusion criteria: all toric lenses; all lenses of over 7.75D, age over50 years, keratoconus. At three time points during the study(immediately prior to wearing goggles, after 20 mins of continuousgoggle wear and 15 minutes after the goggles were removed) patientswere required to fill out a symptom questionnaire to assess theirocular comfort. The lipid layer thickness (LLT) was observed andsemiquanitatively graded (in 30 nm increments) using aninterferometer immediately following each symptom assessment.Results: The mean age of the patients was 37.9+/-10.4 years. Afterthe goggles had been worn for 20 mins, 88% reported an increase intheir lens wearing comfort of at least one grade and 72 % evidencedan increase in LLT of at least 30nm (1 grade). After the goggles hadbeen removed for 15 mins, only 12% maintained a comfortimprovement of at least one grade; 8% reported feeling 1 grade worsethan baseline, and only 8% maintained the increase in LLT of at leastone grade (30nm); 12% evidenced a decrease in LLT of 30nm frombaseline.Conclusions: Increasing the periocular humidity results in anincrease in the LLT of the tear film over the front surface of thecontact lens and simultaneously improves lens-wearing comfort.Presumably, the increased LLT is due to decreased evaporation,which results in a thicker overall tear layer, improved protection ofthe ocular surface, improved hydrodynamic lubrication for the lidwiper and increased contact lens wearing comfort.Commercial Relationships: Samuel Kim, TearScience (E);Caroline A. Blackie, TearScience (E); Donald R. Korb,TearScience (F)Program Number: 6019 Poster Board Number: A0082Presentation Time: 10:30 AM - 12:15 PMThe Avian Membrana Nictitans: Anatomy and FunctionCharles S. Schobert, Leandro B. Teixeira, Richard R. Dubielzig. Deptof Pathobiological Sciences, UW-Madison School of Vet Med,Madison, WI.Purpose: To document the anatomy and function of the avianmebrana nictitans (MN) using gross dissection, light and electronmicroscopy. The MN is located at the medial canthus and it is used toclean, moisten, and protect the cornea by making extremely rapidexcursions over the surface. We document the special anatomicfeatures involved in MN function in birds.Methods: Globes and attached nictitans were submitted to COPLOWfrom a Bald Eagle, Haliaeetus leucocephalus, and a Mallard duck,Anas platyrhynchos. The eagle globe was dissected to show the pathof the pyramidalis tendon as it travels from the pyramidalis muscleanchored on the posterior sclera to the ventral MN where contractionpulls the MN over the corneal surface. The duck head was sectionedin the frontal plane making step sections to document the anatomy.The MN of the eagle was examined by light microscopy andtransmission electron microscopy (TEM).Results: We show the anatomy of the pyramidalis muscle and tendonand the support of the tendon by a sling from the quadratus muscle asit swings around the optic nerve. The quadratus is also anchored onthe posterior sclera superiorly in the vertical plane. Histology of theeagle MN shows a collagenous stroma with significant elastin andrichly endowed with nerves. The bulbar epithelium of the MNundulates and mucin is entrapped in a unique cellular process calledthe feather epithelium. The duck MN does not have featherepithelium possibly because the tear film would be under water,precluding the need for other MN structures. On TEM theconjunctival epithelium on the bulbar surface of the third eyelidpresented two layers of short columnar cells. The superficial cellspresented a centrally located villous projection with multipleperpendicular secondary projections producing a “fish spine”arrangement. The core of the villous projections containedlongitudinally arranged cytoskeletal elements. The remaining surfacewas rich with microvillar projections.Conclusions: The avian MN functions to clean, moisten, anddistribute tears to the corneal surface. Understanding the anatomy andfunction of the MN helps to understand one aspect of enhanced visualfunction in birds.Commercial Relationships: Charles S. Schobert, None; LeandroB. Teixeira, None; Richard R. Dubielzig, OSOD, LLC (I), Allergan(C)Program Number: 6020 Poster Board Number: A0083Presentation Time: 10:30 AM - 12:15 PMPrevalence of incomplete blinking in dry eye patients evaluatedwith tear film interferometryJoanne Shen. Mayo Clinic in Arizona, Scottsdale, AZ.Purpose: To describe the prevalence of incomplete blinking in dryeye patients evaluated with Lipiview® tear film interferometry.Methods: 72 dry eye patients' medical records and Lipiview® datawere retrospectively examined between March and November 2012at Mayo Clinic in Arizona. Demographics were recorded. Videoanalysis and data output from Lipiview® testing were evaluated.Results: The average patient's age was 67 years, with median age of70 years. The group included 61 women (84%) and 11 men. Lipidtear film thickness was measured in Interferometric Color Units(ICU). Normal ICU is greater than 40. The average ICU was 63, withmedian of 56. Video analysis of the 143 eyes which had completetesting revealed that 82% of the eyes (118/143) were observed tohave incomplete or partial blinking. Of the 118 incomplete blinkingeyes, 16% (19/118) had abnormal ICU. Of the 25 complete blinkingeyes, 32% (8/25) had abnormal ICU. Analysis of variance did notreveal statistical significance between these two groups of patients(p=0.36).Conclusions: Incomplete blinking is frequently found in patientswith dry eye syndrome undergoing Lipiview® interferometric lipidtear film analysis. Incomplete blinking may be a significantcontributor to dry eye syndrome and is caused by neurogenic,post/surgical, mechanical, contact lens wear, or prolonged computeruse. However, in this study incomplete blinking did not correlate withdecreased lipid tear film thickness. This study is limited by its small©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapopulation and retrospective nature. Further prospective controlledstudies are needed to validate the findings. In summary, Lipiview®testing can increase recognition of incomplete blinking which canguide treatment of dry eye syndrome.Commercial Relationships: Joanne Shen, NoneProgram Number: 6021 Poster Board Number: A0084Presentation Time: 10:30 AM - 12:15 PMExpression of proinflammatory cytokines in tears of aqueousdeficientdry eyesYan Wang, Jun Xiang, JianJiang Xu. Ophthalmology, Eye and ENTHospital of Fudan University, Shanghai, China.Purpose: To evaluate the expression of different cytokines in tears ofSjögren Syndrome dry eye (SSDE), Non-Sjögren syndrome dry eye(NSSDE) and healthy controls.Methods: Twenty SSDE patients with mean age 47±12.9 years, 20NSSDE patients with mean age 45.2±5.9 years and 20 gender andage matched healthy controls were recruited in this study. Schirmer 1test, tear film break-up time (TBUT), cornea fluorescein staining (FS)were examined. Tear samples were taken from using a 10-ul capillarytube to analyze inflammatory cytokine levels. Expression of IL-1β,TNF-α,IL-6,IL-8,IL-10 and IL-12 were measured by cytometricbead array (BD Biosciences, Pharmingen). Impression cytologyspecimens were obtained and PAS staining was performed toevaluate the goblet cell density.Results: The Schirmer 1 test value, TBUT and FS scores wereobviously worse in NSSDE and SSDE patients comparison with thehealthy controls, specially in the SSDE group. Goblet cell densitywas 1230.24±653cells/mm2 in healthy controls, 706±453cells/mm2in NSSDE group and 340±248cells/mm2 in NSSDE group. Nosignificant difference were found in the expression of IL-6,TNF-α,IL-10,IL-12 in tears of three groups. However a significantly increasedexpression of IL-1β and IL-8 in tears of SSDE group were examined(P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneato validate the Current Symptoms Questionnaire (CSQ) and examineits relationship to habitual symptoms through a meta-analyticalapproach.Methods: Baseline CSQ data (no intervention) from normal and dryeye subjects in 12 studies that took place between 2002 and 2011were compared to habitual symptoms concurrently measured by theDry Eye Questionnaire (DEQ). Receiver-Operating Characteristic(ROC) curve analysis was used to compare CSQ scores against theDEQ-5, a validated short form of the DEQ comprising a subset offive DEQ questions, and a prior clinical diagnosis of dry eye. Raschanalysis was used to determine some psychometric properties of theCSQ.Results: There were correlations between current and habitualsymptoms of discomfort and dryness with respect to their frequencyand intensity (Spearman r ranged 0.40-0.57, all p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadepressive symptoms evaluation (78%). In 2011, the mean age of the1526 participants was 64.66 ± 9.47 years (50-91) and 900 werewomen (59%). Among them, 244 (16%) had dry eye disease and 142(9.3%) had depression. The ZDS was significantly correlated to dryeye symptoms (r=0.097; P Optive® > Systane Ultra®, with Opticol® evaporatingalmost instantly. Thealoz® was the only drop that did not precipitateor crystallize up to 48h, suggesting it is more stable than the othertested drops.Commercial Relationships: Constanca Coelho, Thea (F);Fernanda Azancoth, Thea (F)Support: Thea has funded all the reagents used in the submittedwork.Program Number: 6027 Poster Board Number: A0090Presentation Time: 10:30 AM - 12:15 PMAnterior segment OCT study of the pre-corneal film and itsrelationship with dry-eye patient`s symptoms and quantitativetests of the tear filmMaurizio Fossarello, Pietro E. Napoli, Giovanni M. Satta, FrancoCoronella. Ophthalmology, University of Cagliari, Cagliari, Italy.Purpose: To analyze the relationship among patient`s symptoms, thestability of the pre-corneal film as assessed by anterior segment OCT,and the "common" lacrimal tests such as: Schirmer`s test 1, tearbreak-up time (TBUT), and ocular surface staining.Methods: We examined a sample of 30 patients referred for dry eyesymptoms by means of McMonnies questionnaire, Schirmer`s test 1,TBUT, Oxford scheme for grading ocular surface staining. Tthedynamic distribution of the pre-corneal film was studied by usinganterior segment OCT (Carl Zeiss Meditec Cirrus HD OCT), and itwas quantitatively evaluated on the appearance of a double line of thetear film. OCT examination was performed before and after theinstillation of artificial tears up to the 5th minute. The kappa statisticwas used to assess the agreement between tests. All examinationswere conducted in the same conditions of temperature, brightness,humidity and time of the day.Results: Our population of dry-eye patients showed 3 levels ofseverity on the basis of subjective symptoms: mild, intermediate andsevere. A good association was shown between these 3 levels ofsymptoms and the OCT pattern of dynamic distribution of the precornealfilm: the tear film double line was reduced in height(Sperman's rho = 0.83; p=0.005), and the tear film showed a reducedresidence-time on the corneal surface (Sperman's rho = 0.65; p=0.01).This association between subjective patient`s symptoms and OCTpattern of the pre-corneal film was observed also in cases where theresults of the "common" objective tests resulted normal (kappa=0.78,p=0.03).Conclusions: The analysis by anterior segment OCT of the precornealfilm can reveal abnormalities of the dynamic pattern of tearsthat may explain the patient's symptoms, even when the commonlacrimal tests appear to be normal. The dynamic pattern of tears onthe cornea is most likely due to the adhesive properties of the ocularsurface that cannot be investigated by classical tests.Commercial Relationships: Maurizio Fossarello, None; Pietro E.Napoli, None; Giovanni M. Satta, None; Franco Coronella, NoneProgram Number: 6028 Poster Board Number: A0091Presentation Time: 10:30 AM - 12:15 PMEvaluation of the Standard Patient Evaluation of Eye Dryness(SPEED) QuestionnaireNancy J. Keir 1 , William Ngo 1 , Ping Situ 5 , Donald R. Korb 3, 4 ,Caroline A. Blackie 3, 4 , Trefford L. Simpson 2 . 1 School of Optometry-CCLR, University of Waterloo, Waterloo, ON, Canada; 2 School ofOptometry and Vision Science, University of Waterloo, Waterloo,ON, Canada; 3 TearScience, Morrisville, NC; 4 Korb Associates,Boston, MA; 5 School of Optometry, Indiana University,Bloomington, IN.Purpose: To evaluate the performance of the Standard PatientEvaluation of Eye Dryness (SPEED) questionnaire by assessing itsdimensionality, repeatability, validity, and by comparing it to four©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaexisting dry eye questionnaires.Methods: A total of 50 subjects, 30 symptomatic and 20asymptomatic, as determined using the Ocular Surface Disease Index(OSDI) were enrolled. All subjects completed 5 different dry eyequestionnaires (SPEED, OSDI, DEQ, McMonnies and SESoD) in arandom order on two separate visits. Clinical measurements wereobtained on the first visit. Concordance correlation coefficient wasused to determine repeatability, Principal Component, Factor andRasch analyses were used to determine dimensionality, and thecomparison of SPEED scores to dry eye diagnosis defined by theOSDI (primarily using receiver-operator characteristic (ROC) curveanalysis) was used to determine validity.Results: The SPEED questionnaire was found to be uni-dimensionalusing Rasch analysis. Three principal components (dryness, burning,soreness/fatigue) were identified and SPEED scores between visitCCC was 0.923 (upper and lower 95% CI 0.868 to 0.955). The areaunder the ROC was 0.928. The only clinical measures that correlated“well” with SPEED scores were corneal staining (p < 0.05),meibomian gland score (MGS) (p < 0.05) and meibomian glandsyielding liquid secretion score (MGYLS) (p < 0.05).Conclusions: These results indicate that the SPEED questionnaire isa valid and repeatable instrument for measurement of dry eyesymptoms. The correlation of the SPEED score with clinicalmeasures of meibomian gland function suggests potential additionalclinical value for the diagnosis and/or management of meibomiangland dysfunction.Commercial Relationships: Nancy J. Keir, TearScience (F), Alcon(F), Alcon (R), Allergan (F), Johnson & Johnson (F), CooperVision(F), Visioneering, Inc. (F); William Ngo, None; Ping Situ, None;Donald R. Korb, TearScience (F); Caroline A. Blackie,TearScience (E); Trefford L. Simpson, NoneSupport: TearScienceProgram Number: 6029 Poster Board Number: A0092Presentation Time: 10:30 AM - 12:15 PMInhibition of Matrix Metalloproteinases by Mapracorat, a NovelSelective Glucocorticoid Receptor Agonist (SEGRA), in HumanCorneal Epithelial CellsThomas R. Vollmer, Karen L. Harrington, Jin-Zhong Zhang, MaryRichardson, Megan E. Cavet. Preclinical Pharmacology, Bausch +Lomb, Rochester, NY.Purpose: Dry eye disease is known to involve an increase in multiplematrix metalloproteinases (MMPs), particularly MMP-9, at the ocularsurface. Increased MMP-9 levels are associated with cornealepithelial barrier dysfunction that causes ocular irritation and visualdysfunction in dry eye disease. The aim of this study was todetermine the effect of mapracorat on MMP levels in human cornealepithelial cells (HCEpiC) exposed to hyperosmolarity and IL-1β.Methods: HCEpiC were challenged with 1 ng/ml IL-1β or 480mOsm hyperosmolar media (by the addition of sucrose) in thepresence of mapracorat or dexamethasone, both at 6 doses (1 - 300nM). After 24 h, culture medium was collected and evaluated forMMP release [MMP-1 MMP-2, MMP-3, MMP-9, MMP-10, MMP-12, and MMP-13] by Luminex. Dose response data were fitted to a 4parameter logistic equation to determine IC50 and efficacy.Results: IL-1β increased levels of all measured MMPs in theconditioned medium, while hyperosmolarity increased release of 4out of the 7 measured MMPs (MMP-1, 3, 9 and 13). Both IL-1β-(MMP-1, 2, 3, 9, 10, 12 and 13) and hyperosmolarity-induced(MMP-1, 3, 9, and 13) levels of MMPs were inhibited by mapracorat.Calculated IC50 values for inhibition of IL-1β and hyperosmolarityinducedMMPs for mapracorat were below 10 nM and were notstatistically significant from dexamethasone IC50 values. Mapracoratwas fully efficacious (>80% for the majority of MMPs) at reducingIL-1β and hyperosmolarity-induced cytokine release and nostatistically significant differences were identified when comparingMMP levels with those following dexamethasone treatment.Conclusions: Mapracorat potently reduced IL-1β andhyperosmolarity-induced MMP release to a similar extent asdexamethasone in HCEpiC. These data, together with mapracorat’sability to potently inhibit pro-inflammatory cytokines, and propensityfor an improved side effect profile as compared to traditional steroidswarrant investigation of this novel drug as a clinically effectivetreatment for dry eye disease.Commercial Relationships: Thomas R. Vollmer, Bausch + Lomb(E); Karen L. Harrington, Bausch + Lomb (E); Jin-Zhong Zhang,Bausch & Lomb, Inc (E); Mary Richardson, Bausch and Lomb (E);Megan E. Cavet, Bausch + Lomb (E)Program Number: 6030 Poster Board Number: A0093Presentation Time: 10:30 AM - 12:15 PMHistopathologic changes in punctal stenosisGary J. Lelli 1 , Alexander D. Port 3 , Yao-Tseng Chen 2 .1 Ophthalmology, Weill Cornell Medical College, New York, NY;2 Pathology and Laboratory Medicine, Weill Cornell Medical College,New York, NY; 3 Weill Cornell Medical College, New York, NY.Purpose: To describe the pathologic changes in punctal stenosis byreporting the histopathologic findings in a series of punctoplastyspecimens.Methods: Observational retrospective chart review. Electronic healthrecords of all patients having punctoplasty over a two-year period atan academic oculoplastic practice were examined. All patients whoserecords included pathology reports were entered into a database.Results: 24 patients, representing 30 eyes, had pathology records inthe EHR. Patients were 75% female, and had an average age of 65(19-88). Associated conditions included blepharitis (71%), dry eyesyndrome or Meibomian gland dysfunction (63%).Histopathologicexamination demonstrated chronic inflammation in 11 eyes (36.7%),fibrosis in 7 eyes (23.3%), chronic inflammation and fibrosis in 4eyes (13.3%), squamous metaplasia in 3 eyes (10%), normalconjunctival mucosa in 3 eyes (10%), and Acintomyces Israeliicanaliculitis in 2 eyes (6.7%).Conclusions: Nearly all histopathologic specimens revealed findingsconsistent with inflammation, fibrosis, or both. These findingsprovide evidence to support the hypothesis that the many etiologiccauses of punctal stenosis are linked by a common pathophysiologicmechanism involving inflammation.Representative histopathology findings in 3-snip punctoplastyspecimens. (A) Chronic inflammation with mainly lymphocyticinfiltrates in the conjunctival epithelium and underlying tissue.Magnification 400X©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaRepresentative histopathology findings in 3-snip punctoplastyspecimens. (B) Densely fibrotic collagenous scar tissue with no orminimal residual inflammation.Magnification 200XCommercial Relationships: Gary J. Lelli, None; Alexander D.Port, None; Yao-Tseng Chen, NoneProgram Number: 6031 Poster Board Number: A0094Presentation Time: 10:30 AM - 12:15 PMHyperosmolar dryness stress on 3D HCE model: a new tool forpre-clinical assessment of tear substitutesBarbara De Servi 1 , Celine Olmiere 2 , Marisa Meloni 1 . 1 VitroScreen,Milan, Italy; 2 Laboratoires Thea, Clermont-Ferrand, France.Purpose: Dryness stress model has already been established in 3Dhuman corneal epithelium cell culture (3D HCE). In order to bettermimic Tear Dysfunction Syndrome, a new stress model includingboth physical dryness stress and hyper-osmolarity has beendevelopped in 3D HCE. Relevance and predictability of this modelwere verified with reference tear substitutes.Methods: The 3D HCE tissues were transferred in controlledconditions (R.H.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneasevere dry eye disease (including Graft vs Host Disease) in a doublemasked,vehicle-controlled, physician-sponsored Phase 2 clinicaltrial. The study’s hypothesis is that Tβ4, in its role as a modulator ofcorneal surface healing and inflammation, may be able to promotehealing of the corneal surface allowing for more conventionalmodalities to take over and maintain a smooth and regular ocularsurface.Methods: Nine patients with severe dry eye (18 eyes) were treatedwith Tβ4 drops (0.1%) or vehicle control six times daily over aperiod of 28 days. They were evaluated upon entering the study aftera two week washout period as follows: Study Day 1 (randomizationand first day of treatment), 7, 14, 21, 28 (End of Treatment), and 56(follow-up). Dry eye sign and symptom assessments, such as oculardiscomfort (using the OSDI questionnaire) and corneal fluoresceinstaining (using the NEI workshop grading system), were evaluated atvarious time points throughout the study.Results: Statistically significant differences in sign and symptomassessments, such as ocular discomfort and corneal fluoresceinstaining, were seen at various time points throughout the study. Ofparticular note at Day 56, the follow-up period, were the differencesbetween Tβ4 and vehicle control. The Tβ4-treated group (12 eyes)had a 35.1% reduction of ocular discomfort compared to vehiclecontrol (6 eyes) (p=0.0141), and a 59.1% reduction of total cornealfluorescein staining compared to vehicle control (p=0.0108). Otherimprovements seen in the Tβ4-treated patients included tear filmbreakup time and increased tear volume production.Conclusions: Tβ4 eye drops were found to be safe and well-toleratedand met key efficacy objectives with statistically significant sign andsymptom improvements, compared to vehicle control, at various timeintervals, including 28 days post-treatment. These positive resultssuggest that further clinical trials are warranted and should aim toassess Tβ4’s efficacy and optimal dosing regimens in the setting ofsevere dry eye disease.Commercial Relationships: Gabriel Sosne, RegeneRxBiopharmaceuticals (C); Steven P. Dunn, None; David Crockford,None; Chaesik Kim, None; Elizabeth Dixon, NoneClinical Trial: NCT01393132Program Number: 6034 Poster Board Number: A0097Presentation Time: 10:30 AM - 12:15 PMBiomarkers in the tear fluid during chemical ocular injury in therabbit modelXxxx Xxxxxx. Xxxxxx, Xxxxxx, MD.Purpose: Analysis of biomarkers in the tear fluid enables continuousfollow-up during ocular pathologies. The sight threatening sulfurmustard (SM) induced ocular injury presents specific clinicalsymptoms for each injury stage. While the partially healed acutephase is characterized by erosions and severe inflammation, thepermanent delayed pathology that is developed only in part of theeyes, is clinically expressed by epithelial defects andneovascularization (NV). The mechanisms underlying this injury arestill in research and treatment is not sufficient. Aiming to improve thetherapeutic strategies, we focused on finding biomarkers in the tearfluid that point out towards the pathological processes during theinjury.Methods: Rabbit eyes were exposed to SM vapor and a clinicalfollow-up was carried out up to 4 weeks. Tear fluid and cornealsamples were collected at different time points and MMP-2 andMMP-9 activities were measured by zymography and VEGF, IL-8,IL-6, TNFα and IL-1β levels were measured by ELISA.Results: Typical SM-induced ocular injury was developed, includingan acute phase that subsided within a week in all of the exposed eyes,followed by a permanent delayed pathology in 50%-80% of the eyes,beginning at 2w. In the tear fluid elevation in MMP-9 activity and IL-8 level was seen. While MMP-9 activity remained high throughoutthe follow-up period in all of the exposed eyes, IL-8 levelscorresponded with the severity of the clinical symptoms.Interestingly, relatively higher IL-8 levels in tear fluid were noticedin future neovascularized eyes before NV appearance, indicating arole in prediction of the delayed pathology. IL-8 level and MMP-9activity were elevated in the corneal tissue as well, indicating apossible source for these proteins in the tear fluid.Conclusions: Biomarkers in the tear fluid are a sensitive tool formonitoring pathological mechanisms during the dynamic course ofthe ocular injury. These biomarkers can rationalize novel therapeuticstrategies and serve as a tool for treatment evaluation. Moreover, IL-8level in the tear fluid at the early stages of the injury may beindicative for the future injury outcome and the need for a preventivetherapy.Commercial Relationships: Xxxx Xxxxxx, NoneProgram Number: 6035 Poster Board Number: A0098Presentation Time: 10:30 AM - 12:15 PMCharacterization of cultivated Human Oral Mucosal EpithelialCells (HOMEs) in an autologous culture mediumCamilla Gram, Dag Krohn-Hansen, Eli Gulliksen, Kristiane Haug,Jon K. Slettedal, Morten C. Moe, Liv Drolsum, Bjorn Nicolaissen,Aboulghassem Shahdadfar. Department of Ophthalmology, OsloUniversity Hospital Oslo, Norway, Center for Eye Research, Oslo,Norway.Purpose: The aim of this study is to culture human oral mucosal cells(HOMEs) for reconstruction of human ocular surface. In the presentstudy we characterize two different cell populations expanded inmedium supplemented with autologous serum.Methods: Small biopsy of human oral mucosal epithelium washarvested and after enzymatic treatment cultured in mediumsupplemented with autologous serum for 3 weeks at 37 degreesCelsius with 5% CO2 in a humidified atmosphere and the mediumwas changed every 3-4 days. Two different cell populations werethen isolated and analyzed by qRT-PCR, electron microscopy (EM)and immunohistochemistry.Results: In our study, two isolated populations (Pa and Pb) weresuccessfully proliferated in medium supplemented with autologousserum compared with fetal bovine serum (FBS) containing medium.The levels of markers associated with proliferation, stemness, celljunction, and functional ocular surface such as KI67, ABCG2, P63,SOX9, CK19, CK3, CX43, E-CAD, and OCLN were differentiallyexpressed in these two Pa and Pb populations, but in the Papopulation stemness and cell junction related markers were extremelyhigher expressed compared with the Pb population.Conclusions: The preliminary results of this study show thatexpanded human oral mucosal cells (HOMEs) in our culture systemgenerates a committed cell population that can potentially be used inreconstraction of human ocular surface.Commercial Relationships: Camilla Gram, None; Dag Krohn-Hansen, None; Eli Gulliksen, None; Kristiane Haug, None; Jon K.Slettedal, None; Morten C. Moe, None; Liv Drolsum, None; BjornNicolaissen, None; Aboulghassem Shahdadfar, NoneProgram Number: 6036 Poster Board Number: A0099Presentation Time: 10:30 AM - 12:15 PMEffects of lens care solutions on dynamic interfacial properties ofhuman tear lipidsTatyana F. Svitova, Meng C. Lin. Optometry School, Univ ofCalifornia, Berkeley, Berkeley, CA.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: To study interfacial dynamics and rheological properties ofhuman tear lipids extracted from Schirmer strips (SSL) and effects ofthree lens-care solutions (LCS) on SSL’s interfacial behaviors.Methods: SSL samples were collected from 6 healthy subjects.Sessile bubble tensiometry was used to study interfacial properties ofSSL, LCS or a mixed film (SSL+LCS) at 22 ° C. SSL was depositedon an air bubble to form 100±20 nm-thick films. SSL films weresubjected to expansion-compression cycles with the film areachanging from ~5 to 50 mm 2 . Two ml of LCS (Baush&LombBioTrue [BT], Alcon PureMoist [PM], or AMO Revitalens [RL]) wasinjected into a cell (LCS:Model Tear Electrolytes(MTE)=1:12 v:v)and equilibrated without or with SSL film for 2 hours. The dynamicrheological properties of the films were assessed. Then MTE waspumped through the cell to remove LCS from bulk and SSL dynamicproperties were re-evaluated.Results: Equilibrium surface tension (EST), elasticity modulus (E)and relaxation times (τ) of SSL alone were 22±2.1 mN/m,10.7-14.8mN/m and 80-150 s, correspondingly. EST for LCS alone was40.3±1.5 for BT, 33.4±1.0 for PM, and 30.1±0.8 mN/m for RL. E ofall three LCS was 5 -8.5 mN/m while τ was 33-55 s. After washout,EST of LCS films increased to 43±1.2, 35.1±1.1, and 32.1±0.8 mN/mfor BT, PM and RL, suggesting irreversible adsorption of some LCScomponents. For mixed SSL+LCS films, EST remained unchanged(22±2.1 mN/m). For all mixed films, E was 0.2-0.5 mN/m and τ was50-60 s; the shape of surface pressure vs. area per microgram (π-A)iso-cycles was altered dramatically. The hysteresis Δπ (Δπ=π max -π min )was initially 48mN/m and diminished to 25 mN/m for BT and RL,and to 21.5 mN/m for PM-treated SSL. These changes persisted afterLCS washout for >24 hours, implying binding interactions betweenSSL and some components of LCS.Conclusions: Some components of LCS adsorb and bind irreversiblyto thick SSL films and make these gel-like SSL films less viscous andless elastic, resembling condensed-liquid films. Clinical implicationsof these findings suggest a possibility of tear lipids filmdestabilization upon exposure to LCS.Commercial Relationships: Tatyana F. Svitova, None; Meng C.Lin, TearLab Corporation (F), Allergan, Inc. (F)Program Number: 6037 Poster Board Number: A0100Presentation Time: 10:30 AM - 12:15 PMEfficacy of long-term treatment with diquafosol sodium for dryeye due to laser in situ keratomileusisRyohei Nejima, Yosai Mori, Ayami Masuda, Yoko Maruyama,Keiichiro Minami, Kazunori Miyata. Miyata Eye Hospital,Miyakonojyo, Japan.Purpose: To evaluate the efficacy of long-term treatment with 3%diquafosol solution (DQS), that is a P2Y2 receptor agonist facilitatingmucin secretion from the conjunctival goblet cells, for chronic dryeye after laser in situ keratomileusis (LASIK).Methods: This prospective clinical study comprising 18 eyes (9patients) to which DQS was additionally instilled 4 times a day. Tearsecretin, tear break-up time (BUT), superficial punctate keratitis(SPK), and conjunctivitis sicca were examined before and at 1,3, 6, 9and 12 months after treatment. Tear secretin was examined with theSchirmer test with anesthesia before and after the DQS treatment.And also, Tear breakup time (BUT), fluorescein stain, and lissaminegreen stain were examined before and after the DQS treatment. Aquestionnaire survey regarding 14 symptoms was also assessedbefore treatment and at 12 months after. The changes in BUT andstaining scores between before and after DQS treatment wereexamined using Kruskal-Wallis test following the Steel-Dwassmultiple comparison. Changes in the tear secretion and eachsymptom in the questionnaire were compared using the Wilcoxonsigned-ranks test. P 0.05). The mean values of the Tear Break-up time, Tear thinningtime, Schirmer 1 test, and the questionnaire were significantlydifferent compared to the normal group (p < 0.05).Conclusions: The stability and the quality of the tear film in the dryeye subjects were reduced with time resulting in an increase in theOcular aberrations and hence could affect the image quality of theeye.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaon mild temperature (particularly cool). Since subjects appear, rather,to collapse the 2 physiologically plausible pain dimensions into asingle discomfort dimension, perhaps more attention could be paid tounderstanding subjects' interpretations of the actual terms used whenmeasuring ocular surface discomfort before these words are used,untested, in scales.Mean HORMS for Normal EyesMean HORMS for Dry EyesCommercial Relationships: Johan Hedström, None; BaskarTheagarayan, NoneProgram Number: 6039 Poster Board Number: A0102Presentation Time: 10:30 AM - 12:15 PMMultidimensional scaling of words used to characterize ocularsurface discomfortTrefford L. Simpson, Yunwei Feng. School of Optometry & VisionScience, University of Waterloo, Waterloo, ON, Canada.Purpose: To use multidimensional scaling (MDS) to determine thenumber of dimensions required to account for the dissimilaritybetween typical adverbs used to characterize how the eyes feel andthen position these words in this multidimensional space.Methods: 20 subjects rated the dissimilarity of all pairs of 22“typical” adverbs that completed the phrase "My eyes are/feel...", Forexample, these adverbs included burning, scratchy, cold and OK. All231 pairwise combinations of these words were rated by subjectsusing magnitude estimation on a scale of “exactly the same” (0) to“completely different” (100). For each subject the order of pairs wasrandomized and from these ratings, dissimilarity matrices wereformed. These matrices were in turn analysed using a number ofMDS algorithms, in particular SMACOF because it also provided ajackknife error estimation for the solution. Data were analysed usingR.Results: Scree plots showed that 2 dimensions account for most ofthe data variability. Regardless of the method used the results wereconsistent and are illustrated in the Figure. The first dimension is a"discomfort" dimension with most of the words connotingunpleasantness generally clustered in the same region of the solutionspace (circles/words on the left in the fig.) and the "comfortable"words at the other end of this dimension (on the right). The seconddimension is thermal, with warm (highest in the fig.) and cool (lowestin the fig.) at opposite ends.Conclusions: The results are somewhat surprising since currentphysiological and psychophysical evidence would suggest at least 3dimensions, one based on words characterizing mechanical (painful)Commercial Relationships: Trefford L. Simpson, None; YunweiFeng, NoneSupport: NSERC Canada, Operating GrantProgram Number: 6040 Poster Board Number: A0103Presentation Time: 10:30 AM - 12:15 PMChanges of Higher Order Aberration depending on OcularSurface Indicators analyzed by Continuous Measurement afterPhacoemusificationSi-Hwan Choi, Hyung-Bin Lim. chungnam national universityhospital, department of ophthalmology, daejeon, south korea,Daejeon, Republic of Korea.Purpose: To analyze the changes of higher order aberration(HOA)measured serially by KR-1W wavefront at every second for 10seconds between before and after phacoemulsification and evaluatethe relationships with the ocular surface indicatorsMethods: Corneal Root-mean-square(RMS) of HOA was measuredon 47 eyes of 30 patients at preoperatively and postoperatively 2weeks, 4weeks, 6 weeks, 8 weeks by KR-1W using continuousmeasurement mode. We analyzed the relationships between thechanges of corneal HOA and ocular surface indicators such as tearbreak up time, Schirmer test, and superficial punctate keratitisResults: The values of corneal RMS at 1 second and 10 seconds afterblinking (0.190±0.067 /0.278±0.192) was highly increased in 2weeks after surgery(0.417±0.306)/0.422±0.294), and significantlydecreased in 8 weeks after surgery to the similar level before surgery.The difference of HOA between 1 second and 10 seconds afterblinking was increased after surgery in all periods, but it was notstatistically significant. The values of corneal RMS at 1 second and10 seconds and difference between them were not significant(p>0.05) in groups divided depending on the presence of preoperativesuperficial punctate keratitis, 5 seconds on BUT and 10 mm onSchirmer testConclusions: The changes of corneal RMS after phacoemulsificationwas statistically significant, but those depending on ocular surfaceindicators were not significant. The phacoemulsification is moresignificant factor in postoperative corneal RMS than ocular surfaceindicators.Commercial Relationships: Si-Hwan Choi, None; Hyung-BinLim, Noneattributes, one for chemical (also painful) descriptors and one based Program Number: 6041 Poster Board Number: A0104©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 10:30 AM - 12:15 PMHyperosmolar conditions Increase benzalkonium chloride (BAK)toxicity in an in vitro cornea wound healing model and an in vivodry eye mouse modelHong Liang 1, 2 , Christophe Baudouin 1, 2 , Philippe Daull 3 , Jean-Sebastien Garrigue 3 , Francoise Brignole-Baudouin 1, 2 .1 Ophthalmology-Hosp Paris, CHNO Des Quinze-Vights, Paris,France; 2 INSERM, UMR_S968,, Vision Institute, Paris, France;3 Novagali Pharma, Evry, France.Purpose: Cytotoxic effects of benzalkonium chloride (BAK) werereported to increase in hyperosmolar conditions (HO) on conjunctivalepithelial cells in vitro with the characteristics of apoptosis andoxidative stress. In order to better understand the influences of HOand BAK on the ocular surface, we tested these two conditions in acorneal wound healing assay using human corneal epithelial cells(HCE) (1), and in a mouse model of dry eye (2).Methods: (1) A wound was established by scraping manuallythrough a confluent HCE layer. Cytotoxicity, cell migration, andproliferation were analyzed after a 30mn-exposure to phosphatebufferedsaline (PBS, negative control), 0.02%BAK in normal(275mOsm/L) and in three different hyperosmolar conditions: 356-406-505mOsm/L. Immunohistology was performed for F-actincytoskeleton and proliferation marker Ki-67. (2) A dry eye modelwas induced in mice by topical administration twice a day (D) for 5Dof 0.2%BAK and 0.1%BAK in 275mOsm/L solution and in HO(505mOsm/L). We evaluated the ocular surface using Phenol redthread tear- (PRT), tear break-up time- (BUT), fluorescein- tests andin vivo confocal microscopy (IVCM). Immunohistology wasperformed to assess MUC5AC and CD45-positive cells.Results: (1) Corneal healing was delayed in 0.02%BAK, this delayincreasing in a HO-dependent manner with a F-actin disorganizationand a decrease of Ki-67+ cells. (2) 0.1%BAK in normal osmolarconditions did not induce any dry eye sign while in HO, it decreasedPRT and BUTs and increased corneal fluorescein scores. By IVCM,we found inflammatory cell infiltrations in the cornea.Immunohistology revealed the loss of MUC5AC+ cells with anincrease of CD45+ inflammatory cells in hyperosmolar 0.1%BACtreatedmouse conjunctival fornix.Conclusions: These in vivo and in vitro studies, showed the impactson the ocular surface of the association of two different stresses thatcould be commonly found in glaucoma patients treated withpreserved eyedrops, hyperosmolar conditions and BAK.Hyperosmolarity induced by BAK, potentiatess in turn BAK toxicity,thus impairing the corneal healing process and the dry eye conditions.This study highlights once more the importance of avoiding BAK inlong-term treated patients and in patients with an ocular surfacedisease.Commercial Relationships: Hong Liang, Novagali Pharma andInstitut de la vision (F); Christophe Baudouin, None; PhilippeDaull, Novagali Pharma (E); Jean-Sebastien Garrigue, NovagaliPharma (E); Francoise Brignole-Baudouin, NoneProgram Number: 6042 Poster Board Number: A0105Presentation Time: 10:30 AM - 12:15 PMInternal Standard Dilution Indicator Protocol (ISDIP): A NewerMethod for the Quantitative Assessment in Tear LipidomicStudiesANJU SIROHIWAL 1 , Jeewan S. Titiyal 1 , Sat P. Garg 1 , Seema Sen 1, 2 ,Sundararajan Baskar Singh 1, 3 , Supriyo Ghose 1 , ThirumurthyVelpandian 1, 4 . 1 Ophthalmology, All India Institute of MedicalSciences, New Delhi, India; 2 Ocular Pathology, All India IInstitute ofMedical Sciences, New Delhi, India; 3 Biophysics, All India IInstituteof Medical Sciences, New Delhi, India; 4 Ocular Pharmacology andPharmacy, All India IInstitute of Medical Sciences, New Delhi, India.Purpose: Sampling tear in patients suffering from tear deficiencydisorders for the quantitative assessment of proteomic or lipidomicstudies is challenging task due to the sampling errors due to lowvolume of aqueous phase. This study developed and validated inhealthy human volunteers, a new tear collection method ISDIP for itsutilisation in patients with dry eye.Methods: The study protocol was approved by the StandingInstitutional Human Ethics Committee, AIIMS and was followedHelsinki. Informed consent was obtained from all volunteers. Bothinternal standard (IS) and decoy marker for validation were selectedfrom the approved chemicals for ocular use. Tear samples werecollected from healthy volunteers using 3 different protocols;Standard capillary, Schirmer’s strip and ISDIP methods. All thevolunteers were subjected for TBUT and Schirmer’s test 1 score.ISDIP was used 50 µl of IS (0.005% w/v with 1.4% PVA) instilledtopically and instructed to volunteers to close their eye slowly androtate them to give enough time for tear extraction. Tear sampleswere collected after 15sec by sterilized fire polished glassmicrocapillary from outer fornix. 20µl decoy marker (ofloxacin) wasinstilled topically 5min prior to tear collection. Decoy marker and ISlevels were analyzed by LC-MS/MS in tears. The samples collectedby ISDIP from healthy eyes were subjected for the selected polar andnon-polar lipid quantification.Results: Tear was collected from healthy volunteers (13 male and 8female with mean age 29.15±5.49 and 25.87±3.56yrs respectively).Tear decoy marker level were found to be 10.6 ± 4.4 μg/ml by ISDIPin healthy volunteers and were found to be higher than other twomethods employed. The concentration of decoy marker was found tobe significant Pearson correlation (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaimproved fluorophotometry technique to the Schirmer’s test toevaluate correlations of these measures with traditional signs andsymptoms of dry eye disease (staining, ocular discomfort).Methods: The following endpoints were assessed and correlationscalculated on data from 11 subjects with DED: demographics,symptoms, quality of life (QoL), tear film break up time, Schirmer’stest scores (ST), improved tear fluorophotometry (F), fluoresceinstaining, corneal sensitivity, lid redness, and meibomian glandmeasurements. This fluorophotometry method varies from previouslyreported procedures through alterations in fluorescien drop volume,fluorescien drop concentration, and scan initiation in relation to thesubject’s blink.Results: Fluorophotometry scores correlated well with both signs andsymptoms of dry eye in this study, while Schirmer’s test did notcorrelate with any signs or symptoms measured. Higher flow rates(tear turnover rates) were associated with lower ocular discomfortscores and lower fluorescein staining (R=-0.81, p=0.005 for oculardiscomfort. R=-0.17, p=0.05 for central staining).Conclusions: Results suggest that when using this modifiedmethodology, fluorophotometry may be a better tool for measuringaqueous secretion than the Schirmer’s test, based on superiorcorrelations with traditional signs and symptoms of dry eye disease.Improving the methodology for performing fluorophotometryassessments is important to ensuring the greatest possible accuracy ofthe measurement.Commercial Relationships: Colleen Heckley, Ora, Inc. (E); EndriAngjeli, Ora, Inc. (E); Keith J. Lane, Ora, Inc. (E); Canice Ahearn,Ora Inc (E); Donna L. Welch, Ora, inc. (E); George W. Ousler,Ora, Inc. (E)Program Number: 6044 Poster Board Number: A0107Presentation Time: 10:30 AM - 12:15 PMEffects of controlled heat delivery to the eyelid margin & eyelidhygiene on symptomatology and the tear film in MGDMichel Guillon 1, 2 , Cecile A. Maissa 1 , Stephanie Wong 1 , Anna Lane 1 ,Benjamin Bossard 1 . 1 OTG Research & Consultancy, London, UnitedKingdom; 2 School of Life and Health Sciences, Aston University,Birmingham, United Kingdom.Purpose: The International Workshop on Meibomian GlandDysfunction has identified heat delivery to the eyelid margin andeyelid massage as the first step in managing MGD of any severity. Adifficulty in the past has been to deliver controlled heat to the eyelidmargin area. The objective of the investigation was therefore to testthe recommendation put forward by assessing the efficacy of thecombined use of controlled heat and eyelid massage.Methods: Two sequential devices were used: EyeGiene® (toproduce controlled heat to the eyelid margins) and Supranettes eyecleaning wipes (to deliver eyelid massage): twice a day for threeweeks and once a day for the reminder of the three monthmanagement. The population was eleven dry eye sufferers with MGDdefined by a positive OSDI score (Total score≥ 13) and at least Grade2 eyelid margin anomalies. The primary endpoints were subjectiveresponse with the OSDI, tear film stability measured with theTearscope and tear film lipid composition measured by HPLC.Results: The results showed that:i. The average OSDI score decreased from “severe” pre-treatment(Total=39.5) to “moderate” at the three week (Total=24.8; p=0.031)and three month (Total=26.6; p=0.038) follow-up. At three monthsthe incidence of severe cases was halved (73% vs. 36%) and theincidence of “asymptomatic” cases nearly one in five (0% vs. 18%).ii. Tear film stability increased significantly between pre-treatment(Med 7.7sec) and the three week (Med 12.7sec; p=0.045) and threemonth (Med 11.3; p=0.004) follow-up.iii. Total tear film lipid concentration increased significantly frompre-treatment (3.3µg/µl) to the three week (5.8 µg/µl; p=0.079) andthree month (7.4 µg/µl; p=0.032) follow-up.iv. Tear film lipid composition was altered during the study period.After three months concentrations of fatty acids (12.6% vs. 7.2%;p=0.033) and cholesterol (1.4% vs. 0.9%; p=0.038) decreased andconcentrations of wax ester/cholesterol ester (85.4% vs. 90.6%;p=0.039) and triglycerides (0.5% vs. 1.3%; p=0.013) increased.Conclusions: The study demonstrated the efficacy of the combineduse of controlled heat and mechanical massage in reducing symptomsin MGD. Further, the results suggest that this controlled managementaffect the meibomian glands as the tear film lipid concentrationincreased, and its composition was modified leading to improved tearfilm stability.Commercial Relationships: Michel Guillon, Johnson & JohnsonVision Care (C), Alcon (C), Allergan (C), OTG Research &Consultancy (F), Wipes / Optometric technology Group Ltd (P),Contact Lens Anterior Eye (S), Eye and Contact lens (S),International society for Contact Lens Research (S), Tear Film andOcular Surface (S), Alcon (F); Cecile A. Maissa, Alcon (F),Optometric Technology Group (E), Alcon (C), Allergan (C), JJVCI(C), Member of CLDW Tear film subcommittee (Tear Film & OcularSurface Society) (S), Wipes/Optometric Technology Group Ltd (P);Stephanie Wong, Optometric Technology Group (E), Alcon (F);Anna Lane, Optometric Technology Group (E), Alcon (F);Benjamin Bossard, Optometric Technology Group (E)Support: OTG Research & ConsultancyProgram Number: 6045 Poster Board Number: A0108Presentation Time: 10:30 AM - 12:15 PMA Software-based Approach for the Analysis of LissamineStaining of the ConjunctivaKeith J. Lane, John D. Rodriguez, Endri Angjeli, George W. Ousler.Clinical R & D, Ora, Inc., Andover, MA.Purpose: The evaluation of vital staining in the dry eye subject is ofprimary importance in clinical trials. While the most prevalentstaining methodology may be corneal fluoresecien staining, lissaminestaining is a valuable tool for the evaluation of conjunctival tissue.This abstract extends previous work on software detection of cornealstaining using fluorescein, where pixel intensity variations alone arerequired for enumerating corneal superficial punctate keratitis (SPK).Analysis of lissamine images, however, entails several additionalcomplications. For example, variations in image hue, edge analysis,and shape analysis must be calculated to detect staining blobs andremove false positives due to dye pooling in tissue folds. We applythis method to a sample dry eye population.Methods: High resolution digital images were obtained of the nasal,temporal, and inferior conjunctival tissue using lissamine dye in apopulation of seven mild to moderate dry eye subjects. Ten imageswere selected and graded using the Ora Calibra dry eye stainingscale (0-4) by a clinician based on image quality and representing amaximal range of staining severity. Each image was processed usingsoftware with the number of isolated stained regions, and theirrespective surface areas were calculated. Clinical grade wascompared to the log of the number of staining areas, as well as thelog of the total stained area.Results: The mean number of isolated stained regions was 73.7, andthe mean staining area (as a percent of surface area) was 0.3049.Correlation of log region number with clinical grade was 87% (91%versus log region area).Conclusions: A software approach to conjunctival grading canefficiently and accurately extract clinical information from alissamine image. The relative importance of staining area may be due©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneato the differing specificity of lissamine vital staining versusfluorescein.Commercial Relationships: Keith J. Lane, Ora, Inc. (E); John D.Rodriguez, Ora, Inc. (E); Endri Angjeli, Ora, Inc. (E); George W.Ousler, Ora, Inc. (E)Program Number: 6046 Poster Board Number: A0109Presentation Time: 10:30 AM - 12:15 PMSurgical Model for Evaporative Loss Dry Eye Model in the NewZealand White RabbitAnthony J. Johnson 1, 3 , Patricia Buttke 3 , Irene E. Kochevar 2 , Heuy-Ching H. Wang 1 , Steven R. Cora 1 , Sheri L. DeMartelaere 3 . 1 OcularTrauma Research, USAISR, Fort Sam Houston, TX; 2 WellmanCenter for Photomedicine, Massachusetts General Hospital, Boston,MA; 3 SAUSHEC Ophthalmology, San Antonio Military MedicalCenter, Fort Sam Houston, TX.Purpose: Patients with severe facial burns often suffer indirectdamage to their eyes. Burn wound contracture of the periocular skincauses cicatricial ectropion resulting in ocular exposure. Skin graftsare often required, but may be insufficient if deeper structures, suchas the periocular muscles, are injured and the protective blink reflexis lost. With loss of the blink reflex the patient quickly developsexposure keratitis. No current treatment adequately addresses thesevere keratitis that these patients develop. We established thisevaporative loss dry eye model that simulates exposure keratopathyresulting from cicatricial ectropion to assist in the development ofnovel therapies for this condition.Methods: Nine white rabbits were included in this study. The righteye of each rabbit was subjected to a 1.5 cm upper and lower lidblepharoplasty, in addition to excision of the nictitating membrane.The left eyes were untreated to serve as controls. Clinicalexamination included fluorescein staining and serial photography ondays 3, 5, 7, 14, 21 and 28. Rabbits were sacrificed on day 28 and thecornea and conjunctiva were evaluated by histopathology.Results: Compared with untreated controls, surgically treated rabbitsshowed significant changes in fluorescein scores on days 5, 7, 14, and28. Clinical examination revealed ocular surface defects ranging fromthe development of punctate epithelial erosions to corneal abrasion,beginning on day 7, with corneal ulceration developing in the mostsevere cases by week 3. Histopathological results revealed epitheliuminfiltrated by heterophilic inflammation with the underlying cornealstroma demonstrating heretophilic, lymphoplasmacytic inflammation,fibrosis and neovascularization.Conclusions: The findings of this study demonstrate that upper andlower lid blepharoplasty combined with excision of the nictitatingmembrane is an excellent surgical model of evaporative dry eye.Damage to the cornea and conjunctiva manifesting as punctateepithelial erosions and corneal ulceration is consistent with clinicalobservations of burn patients with dry eye resulting from cicitricialectropion. Future studies utilizing this model are planned to testmultiple strategies to protect the ocular surface from evaporation dueto ocular exposure.Commercial Relationships: Anthony J. Johnson, None; PatriciaButtke, None; Irene E. Kochevar, Aura MedSystems (P); Heuy-Ching H. Wang, None; Steven R. Cora, None; Sheri L.DeMartelaere, NoneSupport: Department of Defense Congressional Directed MedicalResearch ProgramProgram Number: 6047 Poster Board Number: A0110Presentation Time: 10:30 AM - 12:15 PMClinical Correlations Among Dry Eye Tests, Physical AndMetabolic Findings In Diabetes Mellitus PatientsPeter S. Reinach 1, 2 , Jacqueline F. Faustino 2 , Monica Alves 2 , DaniloRibeiro 2 , Jayter S. Paula 2 , Eduardo M. Rocha 2 . 1 Biological Sciences,SUNY College of Optometry, New York, NY; 2 Oftalmologia,Otorrinolaringologia e Cirurgia de Cabeça e Pescoço., Universidadede São Paulo, Ribeirão Preto, Ribeirao Preto, Brazil.Purpose: To determine in diabetes mellitus (DM) patients, withoutproliferative retinopathy, if there are specific sets of ocular surfacesigns and/or body mass indices as well as clinical laboratory resultsthat correlate with dry eye disease (DED) diagnosis.Methods: Ocular surface dye staining and tear film osmolarity(TFOsm) evaluation were performed (TearLab Osmometer). Theseresults were tested for correlation with measurements of either: a)body mass index; (BMI); b) thoracic and abdominal circumference(TC, AC, respectively); c) triceps crease width or; d) blood glycatedhemoglobin (HbA1c) level and hyperglycemia.Results: In 27 DM patients, 55.+- 19 years old, 20 were DM2whereas 7 others were DM1. Out of both groups, 85% werediagnosed with DED (DM1 and 2 in 75% and 90%, respectively).Most common ocular DED signs were: TFBUT (56%) and tearhyperosmolarity (52%). The most relevant systemic correlations inDM2 patients were between: number of systemic medications or ACand elevated TFOsm (r=0.48, p= 0.044; r=-0.724, p=0.0007,respectively), Ocular Surface Disease Index score with: a) TFBUTand AC (r=0.64, p=0.0044; r=0.5, p=0.033, respectively); b)lissamine green and fluorescein staining (r=0.739, p= 0.0005).However, neither HbA1c,BMI nor triceps crease width correlatedwith any of the ocular surface or DED parameters.Conclusions: DM1 and 2 highly correlate with DED. In DM2,abdominal fat accumulation correlates best with tear filmhyperosmolarity and ocular surface integrity damage. Suchcorrelations in DM2 patients suggest that specific metabolic changesmay promote DED development.Commercial Relationships: Peter S. Reinach, None; Jacqueline F.Faustino, None; Monica Alves, None; Danilo Ribeiro, None;Jayter S. Paula, None; Eduardo M. Rocha, NoneSupport: FAPESPProgram Number: 6048 Poster Board Number: A0111Presentation Time: 10:30 AM - 12:15 PMThe Influence of Punctal Occlusion on Osmolarity in Dry EyeSubjectsDavid C. Eldridge, Michael S. Berg, Benjamin D. Sullivan, WilliamD. Townsend. TearLab Corp, Bixby, OK.Purpose: Dry eye disease is a common and major source of disabilityand its onset may be triggered or modified by exposure to systemicdrugs, contact lens wear, ocular surgery and adverse environmentaland work conditions. Tear hyperosmolarity is central to the dry eyedisease pathogenesis, and may serve as a valuable marker in themonitoring of disease severity. This study is designed to determinethe variation in tear osmolarity over time using TearLab Osmometryin DED subjects being treated with punctal occlusion with punctalplugs.Methods: Ten subjects (9F/1M, age=54.3±15.8 years), with tearosmolarity in one eye greater than 311 mOsm/L, were tested for tearosmolarity, symptoms (VAS), an eyelid exam under slit lamp,fluorescein staining of the cornea and conjunctiva, followed by ameibomian dysfunction assessment. Subjects refrained fromadministering any topical drug within two hours of measurement, andwere excluded if they had any clinically significant eyelid deformity,previous ocular disease including infections, active allergy, LASIK orPRK surgery within one year of the study, abnormality ofnasolacrimal drainage, previous punctal plugs or cauterization,diagnosis of a systemic disease or a change in chronic systemic©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamedication known to affect tear production. Subjects were evaluatedat three visits, including the initial screening, at intervals ofapproximately two weeks over the course of approximately 30 days.Research was conducted in compliance with the Declaration ofHelsinki, all subjects completed informed consent prior to the study.Results: Tear osmolarity (316.9±6.2 to 304.8±16.0, p = 0.039),conjunctival staining (1.6±0.8 to 0.7±0.8, p=0.027), symptomfrequency (4.8±2.2 to 2.0±1.5, p=0.004) and symptom severity(5.1±1.2 to 1.9±1.4, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaConclusions: This study was able to provide an appropriatetranslation of the OSDI questionnaire.Commercial Relationships: Francisco Beltran, None; NallelyRamos-Betancourt, None; Jaime D. Martinez, None; ConcepcionSantacruz Valdes, None; Alejandro Babayan, None; CeciliaRamírez-Assad, None; Elsa M. Mora Juarez, None; EverardoHernandez-Quintela, NoneProgram Number: 6051 Poster Board Number: A0114Presentation Time: 10:30 AM - 12:15 PMAnterior Segment Optical Coherence Tomography changes ofocular surface after topical anti-inflammatory therapy in dryeyesPietro E. Napoli, Maurizio Fossarello, Franco Coronella, GiovanniM. Satta. Eye Clinic, University of Cagliari, Cagliari, Italy.Purpose: To evaluate the effects of therapy with topicalcorticosteroid and hypo-osmotic artificial tears on the viscoelasticproperties of the ocular surface and lacrimal turnover in dry eyes byanterior segment Optical Coherence Tomography (AS-OCT).Methods: We evaluated the ocular surface of 40 patients withvarying severity of dry eyes by AS-OCT. The OCT imaging wasenhanced by using two different eye drops containing: a)carboxymethylcellulose 0.5%, glycerol 0.9%, to study theviscoelastic properties of the ocular surface, and b) mineral oil, toanalyze the lacrimal turn-over. All patients were assigned to aregimen of fluorometholone (1 drop four times a day) and hypoosmoticsaline drops (1 drop four times a day) for 10 days. Thefollowing tests were performed before and after the therapy:McMonnies questionnaire, tear break-up time (TBUT), Schirmer 1,Oxford scheme for grading ocular surface staining, and AS-OCT. Allexaminations were conducted in the same conditions of temperature,brightness, humidity and time of the day. The paired t-test was usedto compare data before and after therapy.Results: The viscoelastic properties of the tear film, as assessed byAS-OCT, appeared significantly modified after anti-inflammatorytherapy: there was a higher adhesion of artificial tears on the ocularsurface (p=0.005), and a faster turnover of the tear film (p=0.01).Conclusions: The OCT is a useful tool to monitor the dynamicchanges of the tear film in the dry eye syndrome and to evaluate thebenefit of therapy.The OCT imaging was enhanced by using an eye drop containingcarboxymethylcellulose 0.5% and glycerol 0.9%.This methodallowed us to study the changes of the visco-elastic properties of theocular surface.The OCT imaging was enhanced by using an eye drop containingmineral oil to to analyze the lacrimal turn-over.Commercial Relationships: Pietro E. Napoli, None; MaurizioFossarello, None; Franco Coronella, None; Giovanni M. Satta,NoneProgram Number: 6052 Poster Board Number: A0115Presentation Time: 10:30 AM - 12:15 PMUse of Artificial Tears vs Cold Compresses for the Treatment ofDry EyeAndrew A. Kao, Robert Latkany. Ophthalmology, The New York Eyeand Ear Infirmary, New York City, NY.Purpose: The purpose of this study is to compare the use of artificialtears and cool compresses for the treatment of dry eye syndrome.Methods: A total of 30 consecutive patients with dry eye wereenrolled. Symptoms were evaluated using the Ocular Surface DiseaseIndex (OSDI) score. The level of meibomian gland dysfunction(MGD), epithelial erosions, tear break-up time (TBUT), andSchirmer testing were recorded. The patients were randomized to twogroups. One was instructed to use artificial tears 3 times per day forone month, then switch to cold compresses, 3 times a day for 30seconds at a time, for one month. The OSDI score and clinicalexamination were re-evaluated at one and two month follow-upvisits. The second group was given the same instructions, except thatthey started with cold compresses for the first month and switched toartificial tears for the second month. At the final visit patients wereasked which treatment regimen they preferred.Results: Cold compresses were found to be a comparable treatmentalternative to artificial tears for dry eye patients.Conclusions: Cold compresses may be a viable alternative toartificial tears for dry eye patients. Many patients with chronic dryeyes have coexisting inflammatory conditions such as meibomiangland disease and allergic conjunctivitis that may be a source of theireye symptoms. Cold compresses appear to counter some of theinflammation. Additionally, they will be a more natural and lessexpensive alternative to over the counter treatment options.Commercial Relationships: Andrew A. Kao, None; RobertLatkany, Alcon (E), Allergan (E)Clinical Trial: NYEE12.28Program Number: 6053 Poster Board Number: A0116Presentation Time: 10:30 AM - 12:15 PMLong-Term Effects of Cataract Surgery on Tear Film ParametersAnat Galor 1, 2 , Vincent D. Venincasa 1 , William J. Feuer 2 , HermesFlorez 1, 4 , David J. Lee 3 . 1 Miami Veterans Affairs Medical Center,Miami, FL; 2 Ophthalmology, Bascom Palmer, Miami, FL;3 Department of Epidemiology and Public Health, University ofMiami, Miami, FL; 4 Department of Endocrinology and Geriatrics,University of Miami, Miami, FL.Purpose: To examine the differences in tear film parameters morethan 3 months post-surgery in eyes with cataract surgery versus eyeswithout cataract surgery.Methods: 29 patients seen at the Miami Veterans Affairs MedicalCenter (VAMC) who had cataract surgery by phacoemulsification inone eye more than 3 months prior to the study date and had no historyof surgical intervention in their fellow eye. Tear film parameters weremeasured in both eyes and compared using McNemar tests fordichotomous variables and paired and single sample t-tests forcontinuous variables.Results: Mean patient age was 73 (standard deviation (SD): 11); 26patients (90%) identified themselves as white and 10 (26%) asHispanic. The mean number of days between surgery and this studywas 952 (SD: 1109). There were no statistical differences between©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneathe surgical eye and the non-surgical eye with respect to any of themeasured tear film parameters. Confidence intervals around thesedifferences were narrow enough to exclude a substantial effect ofcataract surgery. The elapsed time between cataract surgery andmeasurement of the tear parameters did not appear to affect thedifference in parameters between the two eyes.Conclusions: We found that eyes that had cataract surgery more than3 months prior to testing had no differences in their tear filmparameters compared to eyes without a history of surgery.Commercial Relationships: Anat Galor, None; Vincent D.Venincasa, None; William J. Feuer, Abbott Medical optics (F),New World Medical (F); Hermes Florez, None; David J. Lee, NoneSupport: VA career development award©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.