Cornea - ARVO

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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.
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