Physiology/Pharmacology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology105 Inflammation and DrugsSunday, May 05, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 99-125/C0104-C0130Organizing Section: Physiology/PharmacologyProgram Number: 99 Poster Board Number: C0104Presentation Time: 8:30 AM - 10:15 AMZylet (loteprednol etabonate 0.5%/tobramycin 0.3%) Comparedwith Tobradex (dexamethasone 0.1%/tobramycin 0.3%) for theTreatment of Lid Inflammation in Two PopulationsTimothy L. Comstock 1 , Heleen H. DeCory 1 , Kirk Bateman 2 . 1 MedicalAffairs, Global Pharmaceuticals, Bausch & Lomb Incorporated,Rochester, NY; 2 Statistics, Bausch & Lomb Incorporated, Rochester,NY.Purpose: To compare loteprednol etabonate 0.5%/tobramycin 0.3%(LE/T) and dexamethasone 0.1%/tobramycin 0.3% (DM/T)ophthalmic suspensions for the treatment of lid inflammation acrosstwo patient populations.Methods: Two multicenter, randomized, investigator-masked,parallel-group clinical trials with similar designs were conducted. Inboth studies patients aged ≥18 years withblepharokeratoconjunctivitis in at least one eye received LE/T orDM/T administered QID for 2 weeks. The first study was conductedat clinical centers in the US; the second study at clinical centers inChina. Signs and symptoms were assessed at baseline and on days 3,8, and 15. The primary endpoint in this analysis was the change frombaseline (CFB) to day 15 in composite blepharitis severity (lidhyperemia, lid scaling or crusting, and lid margin hypertrophy, eachmeasured on a 5-pt scale). Safety outcomes included an assessmentof AEs, and changes in visual acuity (VA), biomicroscopy, andintraocular pressure (IOP).Results: A total of 273 patients (136 LE/T, 137 DM/T) and 354patients (178 LE/T, 176 DM/T) were included in the ITT populationin the US and China studies, respectively. Patients in the US studywere primarily White (>80%); all patients in the China study wereChinese. A significant CFB in blepharitis severity was seen in bothpopulations with both treatments at each follow-up visit. At day 15,the reduction in composite blepharitis severity was 68.8% for LE/Tvs. 66.7% for DM/T in the US study and 75.0% for LE/T vs. 77.4%for DM/T in the China study. Patients treated with DM/T experienceda significant increase in IOP compared to those patients treated withLE/T beginning at day 8 (US study) or Day 3 (China study)(P≥0.0339). In both studies twice as many patients in the DM/Tgroup experienced IOP increases of ≥ 5 mm Hg over baselinecompared to the LE/T group. In the US study, one patient (DM/Tgroup), while in the China study, 19 patients (6 LE/T; 13 DM/T)experienced an IOP increase of ≥10 mm Hg over baseline.Conclusions: LE/T was effective in the treatment of lid inflammationwith similar results compared to DM/T in both a primarily Whitepopulation and a Chinese population. LE/T had a better safety profilewith respect to change in IOP especially in Chinese patientsconsidered at higher risk for steroid-induced IOP.Commercial Relationships: Timothy L. Comstock, Bausch &Lomb Incorporated (E); Heleen H. DeCory, Bausch + Lomb (E);Kirk Bateman, Bausch + Lomb (E)Clinical Trial: NCT01028027, NCT00447577Program Number: 100 Poster Board Number: C0105Presentation Time: 8:30 AM - 10:15 AMInflammatory processes in branch retinal vein occlusion in miceElisa Dominguez 1, 3 , Sophie Cavallero 1, 3 , William Raoul 1, 3 , SophieLavalette 1, 3 , Michel Paques 1, 2 , Florian Sennlaub 1, 3 . 1 Institut de laVision - INSERM U 968, Paris, France; 2 CHNO des Quinze-Vingts,Paris, France; 3 UPMC UMRS 968, Paris, France.Purpose: Retinal vein occlusion (RVO) is an important cause ofblindness because of the subsequent microvascular remodelling thatmay lead to a variable combination of capillary non-perfusion andmacular edema. To date, the inflammatory processes that accompanyBRVO and its role in the microvascular remodelling have receivedlittle attention. Here, we analyzed the involvement of inflammation ina model of laser-induced BRVO in mice.Methods: A complete and permanent occlusion of a retinal vein ofC57Bl/6J Rj mice was obtained using 534nm laser. We followed invivo the development of tissue changes by funduscopy, scanninglaser ophthalmoscopy and optical coherence tomography. At differenttime points after RVO, animals were sacrificed and the expression ofvarious genes were analyzed by real-time RT-PCR. Iba1immunostaining was performed on the whole retina at 3 and 7 daysafter RVO to evaluate macrophage/microglia responses. To quantifycell proliferation, mice were intraperitoneally injected with thenucleotide analog EdU and killed at 3d and 7d after RVO.Results: Permanent occlusion of a retinal vein was obtained in alltreated eyes with minimal damage to surrounding tissue.Subsequently, dilation of the affected vein was observed. Few retinalhemorrhages were observed at 24h which slightly increased at day 3.Retinal mRNA expression of the chemokine CCL2 significantlyincreased 4h after RVO. The macrophage/microglia markers F4/80and cd11b increased at 3 days. Macrophage/microglia recruitmentwas confirmed at 3d by increased Iba1 positive cells throughout thearea of the occluded vein and persisted at 7d after RVO. Iba1-positiveperivascular macrophages were also more numerous around occludedveins up to the periphery than intact veins in the same eye or incontrol eyes. The macrophage/microglia response was still increasedat 7d after RVO but at lower levels. Seven days after RVO,proliferation occurred mainly in endothelial cells of capillariesupstream of the occluded vein.Conclusions: Our data shows that BRVO induces CCL2 expressionand perivascular and retinal macrophage recruitement concomittantlywith retinal hemorraghe and endothelial cell proliferation distal to thelaser site. The BRVO mouse model will allow us to study the role ofinflammatory processes in vascular leakage and endothelial cellproliferation.Commercial Relationships: Elisa Dominguez, None; SophieCavallero, None; William Raoul, None; Sophie Lavalette, None;Michel Paques, MerckSerono (C), Roche (C), Sanofi (C); FlorianSennlaub, NoneProgram Number: 101 Poster Board Number: C0106Presentation Time: 8:30 AM - 10:15 AMIL-12p35 Inhibits Th1 proliferation and promotes the expansionof Th17 and TregIvy M. Dambuza, Chengrong Yu, Rashid M. Mahdi, Renxi Wang,Sung-Hye Kim, Charles E. Egwuagu. Laboratory of Immunology,NEI, Bethesda, MD.Purpose: The IL-12 family cytokines have emerged as an importantregulator of host immunity and in particular T-helper lineagecommitment. It is comprised of IL-12, IL-23, IL-27, IL-35 and eachmember consists of an α (p19, p28, p35) and a β (p40, Ebi3) subunit.Although the bioactivities of each heterodimer cytokine are wellcharacterized, little is known about functions of the independent αand β subunits. In this study we addressed the effects of IL-12p35subunit in T and B cell activation and differentiation.Methods: Full-length mouse IL-12p35 was cloned and expressed inHigh Five insect cells. The secreted recombinant IL-12p35 (rIL-12p35) was purified on His-Trap columns and size exclusion HPLC©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 – Physiology/Pharmacologyloaded with vitamin A and E to modify the pro-inflammatorycytokine profile after their administration on nasal mucosa, eyelidand ocular surface. This effect could be due to the anti-inflammatorymechanism of the vitamins and also to the barrier effect of liposomeson mucosal surfaces.Commercial Relationships: Davide Scollo, None; TeresioAvitabile, None; Giulia Malaguarnera, None; Roberta Amato,None; Giuseppe Napolitano, None; Giuseppina Marrazzo, None;Michele Reibaldi, None; Antonio Longo, None; CaterinaGagliano, NoneProgram Number: 107 Poster Board Number: C0112Presentation Time: 8:30 AM - 10:15 AMSystemic factors impacting intraocular levels of vascularendothelial growth factor and interleukin-6 in diabeticretinopathySohee Jeon, Won Ki Lee. Ophthalmology, Seoul Saint Mary'shospital, Seoul, Republic of Korea.Purpose: To identify systemic factors correlating with intraocularlevels of interleukin-6 (IL-6) and vascular endothelial growth factor(VEGF) in diabetic retinopathy (DR).Methods: This study was a cross-sectional study. Forty-twoconsecutive patients (42 eyes) undergoing pars plana vitrectomy(PPV) for DR were enrolled. Aqueous humor was sampled just priorto PPV for assay of IL-6 and VEGF via multiplex cytokine array.One day before PPV, patient characteristics (age, gender, duration ofdiabetes, body mass index [BMI], systolic and diastolic bloodpressure, alcohol, and smoking status) were recorded and a number ofsystemic markers amassed, including fasting and postprandialglucose, homeostasis model assessment (HOMA)-IR, HOMA-beta,C-peptide, insulin, total cholesterol, triglycerides, high-densitylipoprotein-cholesterol, low-density lipoprotein-cholesterol,apolipoprotein (Apo)-A, Apo-B, and lipoprotein A (Lp-A).Relationships between systemic determinants and intraocularcytokine levels (IL-6 and VEGF) were analyzed by univariate andmultivariate regression.Results: Median levels of IL-6 and VEGF were 15.3 pg/mL (range,2.4-10124.5 pg/mL) and 21.1 pg/mL (range, 3.2-766.1 pg/mL),respectively. After adjustment for age, gender, duration of diabetes,and BMI, multivariate analysis showed significant association ofsmoking (p=0.002) and HOMA-IR (p=0.003) with intraocular IL-6levels, while intraocular VEGF and systemic Lp-A levels correlatedsignificantly (p=0.032).Conclusions: Insulin resistance (HOMA-IR) and smoking statusimpacted intraocular levels of IL-6, while intraocular VEGF levelswere influenced by Lp-A. An appreciation for the relationshipbetween systemic factors and intraocular level of various cytokinesmay help elucidate the complex pathophysiology of DR.Commercial Relationships: Sohee Jeon, None; Won Ki Lee,Novartis (F), Novartis (C), Bayer (C)Support: None in the Support field below.Program Number: 108 Poster Board Number: C0113Presentation Time: 8:30 AM - 10:15 AMEffects of topical indomethacin, bromfenac, and nepafenac onLPS-induced inflammationClaudio Bucolo 1 , Giuseppina Marrazzo 1 , Chiara Bianca MariaPlatania 1 , Monica Zurria 2 , Filippo Drago 1 . 1 Clinical and MolecularBiomedicine, University of Catania, Catania, Italy; 2 R&D, Alfa-Intes,Casoria, Italy.Purpose: To investigate the effects of topical nonsteroidal antiinflammatorydrugs (NSAIDs) on retinal vascular leakage, andinflammatory markers in endotoxin-induced uveitis (EIU) in rats.Methods: EIU was induced in rats by single footpad injection of LPS(350 μg/kg, Salmonella typhimurium). Animals were treatedaccording to the ARVO statement for the use of animals inophthalmology and vision research. Topical indomethacin 0.5%(Indom), bromfenac 0.09% (Yellox), and nepafenac 0.1%(Nevanac) were given before and after LPS injection (fouradministrations). Twenty-four hours after LPS injection the animalswere euthanized and retina and plasma were collected to assess PGE2and C-reactive protein (CRP) levels using ELISA. Retinal vascularleakage was assessed by Evans blue extravasation.Results: Twenty-four hours after LPS injection, significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyNOV C-ter 2.5 µg was as effective as dexamethasone 20 µg. RealtimePCR analysis of the irises and ciliary bodies showed nosignificant effects on the expression of VEGF, VEGF-R1, VEGF-R2,COX-2, Hey-1, Notch-1 and Jagged-1. IL-6 was significantlydownregulated by NOV 2.5 µg (P = 0.009). IL-1β, TNF-α, and MCP-1 were significantly downregulated but to a lesser extent.Conclusions: Anti-inflammatory effects observed for NOV C-ter, inthe endotoxin-induced uveitis model, seem to be mediated by adownregulation of pro-inflammatory cytokines, mainly IL-6, IL-1β,TNF-α and MCP-1.Commercial Relationships: Chadi Mehanna, Sisène (E); NorbertJ. Minet, Sisene SAS (E); Marie-Chritine Naud, None; CelineOlmiere, Laboratoires THEA (E); Jean-Louis Bourges, None;Francine F. Behar-Cohen, Inserm/Univesrité ParisDescartes (P)Support: Partial support from Sisene and Laboratoires THEAProgram Number: 110 Poster Board Number: C0115Presentation Time: 8:30 AM - 10:15 AMValidation of the Anterior Chamber Paracentesis Model for theScreening of Novel Ophthalmic Anti-Inflammatory DrugLaura Belen, Kortni Violette, Jennifer Brackett, Andy Whitlock. Ora,Inc., Andover, MA.Purpose: The development of novel anti-inflammatory drugs fortreatment of inflammatory conditions such as dry eye, chronicallergy, blepharitis, uveitis, or ocular trauma is complicated by thelack of a preclinical model with a robust, consistent inflammatoryresponse. The anterior chamber paracentesis model (ACP) is an acuteanimal model of ocular inflammation that involves the breakdown ofthe blood aqueous barrier and subsequent release of proteins andPGE2 into the aqueous humor (AH). PGE2 up-regulation is acommon factor of many ocular surface diseases such as dry eye,chronic allergy, and blepharitis. The purpose of this study was tobenchmark numerous classes of anti-inflammatory drugs (steroids,NSAIDs, and cyclosporine) in an ACP model to determine if it couldserve as a surrogate for ocular surface inflammation.Methods: Inflammation was induced in NZW rabbits by aspirating100 µL of AH from the anterior chamber. The extent of inflammationwas assessed 2 hours following the challenge by slit lamp and postlifebiochemical analysis of AH for protein exudates by BCA assayand PGE2 levels by ELISA. Anti-inflammatory drugs tested includedDexamethasone (Dex), Prednisolone (Pred), Keterolac, Bromfenac,and Restasis. Balanced saline was used as a vehicle control. Fouranimals (8 eyes) per treatment were dosed bilaterally four times dailyone day prior to ACP. On the day of AH collection, animals had aclinical observation and received topical treatment 3, 2, 1 and .5hours prior to the first AH collection. Animals received topicaltreatment 30, 60 and 90 minutes post first collection.Results: Only bromfenac and ketorolac significantly reduced AHprotein exudates in the NZW rabbits following ACP compared tovehicle control (p < .01). Dex, bromfenac and keterolac statisticallydecreased PGE2 levels compared to vehicle control (p < .01, p < .05,p< .05). Dex and Restasis also resulted in decreased levels of PGE2release, but the decrease was not statistically significant.Conclusions: Both steroids and NSAIDs appear to be efficacious inthis model. While not significant, Restasis had some efficacy thatmay be increased by adjusting the power of the study. The results ofthis study indicate that ACP could serve as a surrogate model forocular surface inflammation and help further novel drug discoveryfor indications such as dry eye and belpharitis.Commercial Relationships: Laura Belen, Ora, Inc. (E); KortniViolette, Ora, Inc. (E); Jennifer Brackett, Ora, Inc. (E); AndyWhitlock, Ora, Inc. (E)Program Number: 111 Poster Board Number: C0116Presentation Time: 8:30 AM - 10:15 AMComparison of effects between glucocorticoids and mapracoratafter repeat topical ocular administration in rabbits for 28 daysKathleen Krenzer, Sherwin Jiang, Ezra R. Lowe, Mary Richardson.Preclinical Development, Bausch + Lomb, Rochester, NY.Purpose: The purpose of this investigation was to assess the systemiceffects of glucocorticoids (GC) as compared to mapracorat, a newselective glucocorticoid receptor agonist (SEGRA) after repeatedtopical ocular administration for 28 days in rabbits with a 1-weekrecovery.Methods: The test articles were mapracorat (1, 3, and 6%), Maxidex(dexamethasone sodium phosphate 0.1%), PredForte (prednisoloneacetate, 1.0%), and Durezol (difluprednate, 0.05%). The controlswere the mapracorat vehicle or saline. Seven NZW rabbits/sex/groupwere used; 4/sex/group were euthanized after 28 days of dosing and3/sex/group were euthanized after a 1-week recovery. One eye ofeach animal was dosed with the respective test article QID (postoperativeregimen) and the other eye with the control. In the controlgroup, one eye was dosed with the control and the contralateral eyeremained untreated. Animals were periodically assessed for clinicalsigns, body weight, and ophthalmic findings (including intraocularpressure). At the end of the dosing period, blood samples werecollected for toxicokinetic assessment of systemic exposure. Animalswere euthanized and blood and tissues collected for clinicalchemistry and histopathology assessments.Results: No adverse ocular effects were observed for any of the testarticles. As compared to controls, significant systemic effects wereobserved in the GC-treated animals, including low body weights andeffects on the metabolic/endocrine, cardiovascular, liver, andimmune. Partial reversibility was observed after a 1-week recoveryperiod. For the mapracorat-treated groups, no effects were observedwith the 1% concentration. For the 3 and 6% groups, no meaningfuleffects were observed for cardiovascular or liver systems. Doserelatedchanges were observed for some of the metabolic (lowadrenal gland weight) and immune (low thymus weights, lowlymphocytes) systems. There was partial (adrenal gland) or full(thymus) reversibility after a 1-week recovery period. Whenobserved, however, the magnitude of the effects was to lesser degreethan those observed in the GC-treated animals. No effects wereobserved with mapracorat that were not within the known effects ofGCs.Conclusions: After 28 days of repeat ocular dosing in rabbits,mapracorat, up to 6%, demonstrates a better systemic safety profile inrabbits than observed with traditional ocular GCs.Commercial Relationships: Kathleen Krenzer, Bausch + Lomb(E); Sherwin Jiang, None; Ezra R. Lowe, Bausch & Lomb (E);Mary Richardson, Bausch and Lomb (E)Support: None.Program Number: 112 Poster Board Number: C0117Presentation Time: 8:30 AM - 10:15 AMEfficacy of Tocilizumab, an IL-6 receptor inhibitor in ocularinflammatory disorders: a pilot studyVimal Sarup 1, 3 , Anum Butt 1, 2 , Kanza Aziz 1, 2 , C. Stephen Foster 1, 3 .1 Massachusetts Eye Research & Surgery Institution, Cambridge, MA;2 Ocular Immunology & Uveitis Foundation, Cambridge, MA;3 Harvard Medical School, Boston, MA.Purpose: Tocilizumab is a Humanized Anti Interleukin 6Monoclonal antibody evaluated and used for its beneficial effects onsystemic autoimmune inflammatory disease but not for non infectiousocular inflammation. In this pilot study, tocilizumab was evaluated tostudy its effect on ocular Inflammation refractory to the extended©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 – Physiology/Pharmacologyspectrum of conventional and immunomodulatory medications.Methods: Eleven patients with non resolving non infectious ocularinflammation involving anterior segment, posterior segment or both,who had not responded completely to other medications wereevaluated. Tocilizumab was given via intravenous infusions toaugment the existing medications on a set schedule and dosage.Detailed ocular examination with slit lamp and dilated fundusexamination was done. Ocular inflammation was graded according toSUN criteria. A full spectrum of diagnostic testing includedfluorescein angiography, OCT, and Ultrasound B scan as necessary.Age, sex, race, ocular and systemic side effects were noted inaddition. Systemic side effects were evaluated by blood tests andphysical examinations by general physicians or rheumatologists.Results: Nine patients were Caucasians, eight were females, averageage was 43.8 yrs, and they had failed four systemicimmunomodulatory medications on average. There were 6 cases ofUveitis, 3 of Scleritis, 1 orbital inflammatory disease, and 1peripheral ulcerative keratitis. Eight patients responded favorably byreduction in ocular inflammation including four patients who wentinto remission. Two patients had infusion reactions with initiation oftreatment and the drug had to be discontinued. Two others hadsystemic side effects but the ocular inflammation had shownimprovement.Conclusions: These data indicate that tocilizumab improves anteriorand posterior segment ocular inflammation of varied non infectiousetiologies. It has the potential to not only stabilize but induceremission of inflammation, even though other systemic medicationsfailed or induced systemic side effects. Further double blindprospective trials are necessary to evaluate its role in ocularinflammation.Commercial Relationships: Vimal Sarup, None; Anum Butt,None; Kanza Aziz, None; C. Stephen Foster, Abbott MedicalOptics (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), Ista Pharmaceuticals (C), Lux Biosciences, Inc.(C), Lux Biosciences, Inc. (F), Novartis Pharmaceuticals Corporation(C), Novartis Pharmaceuticals Corporation (F), XOMA Ltd (C)Program Number: 113 Poster Board Number: C0118Presentation Time: 8:30 AM - 10:15 AMEvaluation of XG-102 effects in the treatment of endotoxininduceduveitis in ratsIkram El-Zaoui 1, 2 , Elodie Touchard 1, 2 , Marianne Berdugo Polak 1, 2 ,Claire Abadie 4 , Catherine Deloche 3 , Jean-Marc Combette 4 , FrancineF. Behar-Cohen 1, 3 . 1 CENTRE DE RECHERCHE DESCORDELIERS TEAM 17 UMRS 872, Paris, France; 2 Universitéparis Descartes Paris-5, Paris, France; 3 department of ophthalmology,Hotel Dieu,APHP, Paris, France; 4 Solid Drug Development.S.A,Geneve, Switzerland.Purpose: To confirm the efficacy of XG-102 (JNK Inhibitor) in thetreatment of endotoxin-induced uveitis (EIU) in rats, XG-102 doseeffectwas evaluated using different routes of administration.Methods: EIU was induced in Lewis rats by LPS injection. XG-102was administered at the time of LPS challenge either by intravenous(IV, 3.2, 35 or 355 µg/injection), intravitreal (IVT, 0.08, 0.2 or 2.2µg/eye) or sub-conjunctival (SCJ, 0.2, 1.8 or 2.2 µg/eye) injections.Controls received either the vehicle or Dexamethasone phosphateinjections. Clinical effects were scored at the peak of the disease(24hours after LPS). XG-102 targeting was controlled by Westernblottingthe phosphorylated c-Jun in the “RPE-Choroid” complex.The anti-inflammatory effects were evaluated using quantification ofcells infiltration in ocular tissues and evaluation of the expression ofinflammatory mediators(iNOS at the mRNA levels in the neuroretina.Results: XG-102 clinically demonstrated adose-dependent antiinflammatoryeffect in EIU after IV and SCJ administrations. Therespective doses of 35 µg and 1.8 µg were efficient as compared withthe vehicle-injected controls, and the highest doses, respectively 355µg and 22 µg, were as efficient as Dexamethasone. After IVTinjections, the anti-inflammatory effect of XG-102 was clinicallyevaluated similar to the corticoid’s effect with all the tested doses.Whatever the route administration tested, the lowest efficient doses ofXG-102 targeted efficiently the c-Jun N-terminal kinase; reduce cellsinfiltration in the treated eyes and iNOS expression in the retina.Conclusions: These results confirm that XG-102 has potential fortreating intraocular inflammation. The subconjunctival route will befurther investigated in humans.Commercial Relationships: Ikram El-Zaoui, None; ElodieTouchard, None; Marianne Berdugo Polak, None; Claire Abadie,None; Catherine Deloche, Solid Drug Development SA (E), XigenSA (C); Jean-Marc Combette, Xigen (C); Francine F. Behar-Cohen, Inserm/Univesrité ParisDescartes (P)Program Number: 114 Poster Board Number: C0119Presentation Time: 8:30 AM - 10:15 AMSystemic symvastatin rescues death of retinal ganglion cells fromoptic nerve injury possibly through suppression of glial NF-κBactivationSeita Morishita, Hidehiro Oku, Masahiro Tonari, Teruyo Kida,Taeko Horie, Tsunehiko Ikeda. Osaka Medical College, Takatsukishi,Japan.Purpose: To determine whether systemic simvastatin rescues deathof retinal ganglion cells (RGCs) from optic nerve injury.Methods: We studied the effect of systemic simvastatin on thesurvival of RGCs after the optic nerve of rats was crushed.Simvastatin (3.0 mg/kg/day) or its solvent (placebo) was giventhrough an osmotic mini-pump 1week prior to crushing the opticnerves. We also performed immunohistological evaluations and realtimePCR to determine the expressions of the CD68, TNF-α, andiNOS genes in the neuroinflammation of the crushed optic nerves.Results: There was a recruitment of CD68 positive cells, namelymicroglia/macrophages, at the crushed site. Assessment by real timePCR showed that mRNA levels of CD68 significantly increased aftercrushing the optic nerve, which peaked on day 5. Systemicsimvastatin significantly (P=0.002, ANOVA followed by Fisher)suppressed the increase on day 3. In addition, simvastatinsignificantly suppressed up-regulation of TNF-α and iNOS genes.The mean number (± SEM) of RGCs stained by TUJ-1 antibody was1816.3 ± 94.9/mm2 in sham operated rats (n=6), which decreased to831.4 ± 82.6/mm2 (n = 9) on day 7 after the optic nerve was crushedwith placebo treatment. This reduction was significantly (P=0.01,Scheffe) reduced to 1169.2 ± 82.2/mm2 (n = 9) with systemicsimvastatin treatment. We also found simvastatin (1.0 μM)significantly suppressed NF-κB activation and iNOS expressioncaused by TNF-α (50ng/ml) in cultured optic nerve astrocytes.Conclusions: These results suggested that systemic simvastatinsuppressed the neuro-inflammtion and rescued death of RGCs aftercrushing the optic nerves. One possible mechanism of the neuroprotectiverole is suppression of NF-κB activation of optic nerveastrocytes.Commercial Relationships: Seita Morishita, None; Hidehiro Oku,None; Masahiro Tonari, None; Teruyo Kida, None; Taeko Horie,None; Tsunehiko Ikeda, NoneProgram Number: 115 Poster Board Number: C0120Presentation 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 – Physiology/PharmacologyInfliximab Attenuates Tumor Necrosis Factor-α-InducedAlterations in Non-Pigmented Ciliary EpitheliumHiroshi Yamada 1 , Masahiko Yoneda 2 , Shingo Inaguma 3 , MasayoshiIwaki 1 , Masahiro Zako 1 . 1 Ophthalmology, Aichi Medical University,Nagakute, Japan; 2 Biochemistry and Molecular Biology, School ofNursing and Health, Aichi Prefectural University, Nagoya, Japan;3 Pathology, Aichi Medical University, Nagakute, Japan.Purpose: In human non-pigmented ciliary epithelial cells(HNPCECs) in vitro, we investigated changes in the mRNA andprotein expression levels of matrix metalloproteinases (MMPs) andtissue inhibitors of metalloproteinases (TIMPs) in the presence oftumor necrosis factor-alpha (TNF-α), and examined the effects ofinfliximab addition. Degradation of claudin-1 and occludin, andpermeability changes in HNPCECs were also evaluated.Methods: HNPCECs were cultured in the presence or absence ofTNF-α, and TNF-α-exposed cells were treated with or withoutinfliximab. We measured the expression levels of MMP-1, MMP-2,MMP-3, MMP-9, TIMP-1, and TIMP-2 in HNPCECs by real-timepolymerase chain reaction and by enzyme-linked immunosorbentassay. HNPCECs and swine ciliary body were treated with MMPs,and immunostained. Permeability of MMP-treated-HNPCECs wasmeasured using a cell-based permeability assay.Results: The expressions of MMP-1, MMP-3, and MMP-9 increasedin the presence of 10 ng/mL TNF-α, and these altered expressionlevels were reversed by the addition of infliximab. Immunostainingshowed that MMP-1, MMP-3, and MMP-9 degraded claudin-1 andoccludin, after which significant increases in HNPCEC permeabilitywere detected.Conclusions: TNF-α increased the expressions of MMPs in cellscomprising the blood-aqueous barrier (BAB). Components of thetight junctions of the BAB were degraded by MMPs, which increasedpermeability through the cells. Infliximab was effective at attenuatingthe TNF-α-induced increases in MMP expressions in cellscomprising the BAB, suggesting that such treatment may clinicallyprevent anterior uveitis.Commercial Relationships: Hiroshi Yamada, None; MasahikoYoneda, None; Shingo Inaguma, None; Masayoshi Iwaki, None;Masahiro Zako, NoneProgram Number: 116 Poster Board Number: C0121Presentation Time: 8:30 AM - 10:15 AMOcular Pharmacokinetics of 0.2% and 0.4% KetorolacTromethamine Formulated in DuraSite or DuraSite 2 DeliverySystems Compared to Acular LS in RabbitsAfshin Shafiee 1 , Lyle M. Bowman 2 , Eddie Hou 2 , Kamran Hosseini 1, 3 .1 Preclinical, InSite Vision, Alameda, CA; 2 Development, InSiteVision, Alameda, CA; 3 Clinical, InSite Vision, Alameda, CA.Purpose: To compare the ocular penetration of 0.2% and 0.4%ketorolac formulated in DuraSite or the new generation, DuraSite 2,delivery systems to Acular LS (0.4% ketorolac).Methods: The left eye of male and female rabbits (n=32/group)received either a single topical instillation of ketorolac 0.2% orketorolac 0.4% formulated in DuraSite, or DuraSite 2, or Acular LS.At predetermined timepoints (0.25, 0.5, 1, 2, 4, 6, 12, and 24 hours),4 rabbits/group/timepoint were sacrificed and the ketorolac levels inthe aqueous humor (AH) were quantified using LC-MS/MSmethodology. PK parameters (C max , T max , AUC 0.25-24h ) weredetermined.Results: Ketorolac 0.4% formulated in DuraSite 2 achieved thehighest C max (1889 ± 884 ng/mL) and AUC (6836 ng/mL*h) values,an increase of 6.9- and 4.8-fold over Acular LS which had the lowestC max (275 ± 83 ng/mL) and AUC (1424 ng/mL*h) values,respectively. Ketorolac 0.2% formulated in DuraSite 2 had C max(1077 ± 415 ng/mL) and AUC (4490 ng/mL*h) values that were 3.9-and 3.2-fold higher than Acular LS, respectively. Ketorolac 0.2% and0.4% formulated in DuraSite also provided better AHpharmacokinetics with C max values that were 2.9- and 4.4-fold higherthan Acular LS, respectively, and AUC values that were 2.3- and 4.0-fold higher than Acular LS, respectively.Conclusions: Ketorolac formulated in DuraSite markedly improveddrug delivery kinetics to the AH compared with Acular LS; the newgeneration delivery system, DuraSite 2, showed enhanced penetrationover DuraSite. DuraSite 2 formulation may allow a major reductionin the dosing regimen or lowering of the ketorolac levels in theophthalmic formulation; it may potentially lessen the side effectprofiles associated with the topical use of ketorolac.Commercial Relationships: Afshin Shafiee, InSite Vision (E); LyleM. Bowman, InSite Vision (E); Eddie Hou, InSite Vision (E);Kamran Hosseini, InSite Vision Inc. (E)Program Number: 117 Poster Board Number: C0122Presentation Time: 8:30 AM - 10:15 AMITF2357 regulates IL-10 via JAK/STAT signaling pathway toattenuate inflammation and fibrosisShyam S. Chaurasia 1, 2 , Yu-Chi Liu 1 , Alison Tan 1 , Rayne Lim 1 ,Jodhbir S. Mehta 1, 3 . 1 Tissue Engineering and Stem Cell Group,Singapore Eye Research Inst, Singapore, Singapore; 2 SignatureResearch Program in Neuroscience & Behavioral Disorder, Duke-NUS Graduate Medical School, Singapore, Singapore; 3 SingaporeNational Eye Centre, Singapore, Singapore.Purpose: To study the mechanism of action of a histone deacetylaseinhibitor, ITF2357 on fibrosis and inflammation in vitro and in vivoin a rabbit PRK model of corneal wound healing.Methods: Twenty rabbits underwent -9.0D photorefractivekeratectomy (PRK) surgery in one eye and were divided into 3groups based on post-op treatment with a single dose of saline (3days), ITF2357 (0.02% for 3 days) or MMC (0.02% for 60 sec postsurgery).Post-op clinical examination was made for 4 weeks usingslit lamp microscopy and in vivo confocal microscopy (IVCM).Cultured primary human corneal fibroblasts (pHCFs) were used tostudy the ITF2357-induced activation of IL-10 and JAK/STATsignaling pathway using specific signaling inhibitors byimmunocytochemistry, western blot and ELISA.Results: ITF2357 significantly reduced corneal haze andextracellular matrix formation on IVCM. IL-10 expression was upregulatedin the ITF2357 treated PRK corneas compared to the MMCtreated. Cultured pHCFs with ITF2357 produced elevated levels ofIL-10 in a time-dependent manner. This in turn, resulted in theactivation of pSTAT3 and downstream signaling with overexpressionof SOCS3 expression. Inhibition of recruitment of STAT3 to thereceptor complex with a specific inhibitor blocked thephosphorylation of STAT3, preventing its nuclear entry and hencedecreased IL-10 production.Conclusions: ITF2357 activates IL-10 levels via activation ofJAK/STAT signaling pathway to exhibits its anti-fibrotic and antiinflammatoryactivity in cornea wound healing.Commercial Relationships: Shyam S. Chaurasia, None; Yu-ChiLiu, None; Alison Tan, None; Rayne Lim, None; Jodhbir S.Mehta, NoneSupport: SingHealth Grants-R729/13/2010/SHF & R756/40/2010SHF to SSC; NMRC NIG- R751/35/2010 to SSC, TCR -R620/41/2008 to JSM; BMRC TCRP - R826 to SSCProgram Number: 118 Poster Board Number: C0123Presentation 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 – Physiology/PharmacologyLack of Correlation between PGE 2 levels and Blood AqueousBarrier Inhibition in a rabbit model of ocular inflammationL David Waterbury. Raven Biosolutions LLC, San Carlos, CA.Purpose: In a model of ocular inflammation, several topical NSAIDswere simultaneously tested for inhibition of PGE 2 and for bloodaqueous barrier breakdown following LPS administration. Thepurpose of the study was to determine if inhibition of PGE 2concentrations was correlated with inhibition of the breakdown of theblood aqueous barrier following the dosing with topical NSAIDs.Methods: Topical dosing of suspensions (0.1% FR122047, 0.1%trans-resveratrol) or solutions (0.1% amfenac, 0.09% bromfenac, 0.1% diclofenac, 0.4% ketorolac) of NSAIDs were administered to oneeye of New Zealand White Rabbits (n=6). Rabbits received LPS(Salmonella enterica typhimurium, 10 µg/kgm), and fluoresceinisothiocyanate-dextran (FITC-dextran), M.W. = ~70,000, 30mg/kgm, iv, at 1 hour). FITC-dextran was used to determine theblood aqueous leakage. At 2 hours after topical dosing , aqueoushumor samples were simultaneously analyzed for PGE 2 and FITCdextranconcentrations.Results: All test drugs significantly inhibited PGE 2 concentrations inthe aqueous humor. The degree on PGE 2 inhibition ranged from ~ 50to 97%, and the inhibition of FITC inhibition ranged from 7 to 98%.Amfenac was more effective in inhibiting FITC-dextranaccumulation than PGE 2 concentrations (97% vs 68% respectively).FR122047, trans-resveratrol, and diclofenac were active in loweringPGE 2 , but did not significantly affect FITC-dextran concentrations.Both ketorolac and bromfenac were equally active with 2 hourdosing, but with 12 hour pre-dosing, only ketorolac was active insuppressing FITC-dextran.Conclusions: Taking into account COX selectivity and potency,there did not appear to be a correlation between PGE 2 and FITCdextraninhibition suggesting different sites of action for both effects.Commercial Relationships: L David Waterbury, Allergan (F),Omeros (C), Syntex/Roche (P)Program Number: 119 Poster Board Number: C0124Presentation Time: 8:30 AM - 10:15 AMSuprachoroidal Microinjection of Triamcinolone Acetonide isWell Tolerated in the Albino RabbitRozemarijn S. Verhoeven 1 , Samirkumar R. Patel 1 , Karen Viaud-Quentric 2 , Florian Cacciamani 2 , Thierry Amar 2 , Benjamin Yerxa 1 .1 Clearside Biomedical, Alpharetta, GA; 2 Iris Pharma, La Gaude,France.Purpose: To evaluate ocular tolerability and toxicokinetics ofsuprachoroidal administration of triamcinolone acetonide (TA) usinga Clearside Biomedical proprietary microneedle in a GLP study in theNew Zealand White rabbit.Methods: On Day 0, rabbits (5/sex/group) were administered a singlebilateral suprachoroidal injection of vehicle, 3.2 mg or 5.2 mg of TA(Triesence®) using a 33g 750µm microneedle. Clinical observations,body weights, food and water consumption, slit lamp biomicroscopywith McDonald-Shadduck scoring, fundus evaluation, intraocularpressure assessment (IOP), electroretinography (ERG), and systemicexposure were assessed up to 17 weeks post-dose. Animals weresacrificed on Day 1 and Week 13 for macroscopic observations,ocular toxicokinetics, and ocular histopathology.Results: There were no adverse effects related to test article ormethod of administration on clinical observations, body weight, bodyweight gain, food and water consumption, or ophthalmicexaminations. No effect on ERG a- or b-wave amplitude or implicittime was noted in any animal. A mild, transient increase in IOP of 2-3 mmHg was observed in the TA groups on Days 7 and 28, whichresolved by Week 13 and was not considered adverse. Inflammatorycells and test article were observed in the suprachoroidal space ofTA-treated animals on Day 1 but not Week 13 as assessed byhistopathology. Systemic exposure to TA was minimal. TA wasobserved at high concentrations in the sclera/choroid and retina, to alesser extent in the iris/ciliary body, and was present only at lowconcentrations in the aqueous humor, lens, and vitreous.Conclusions: A single bilateral suprachoroidal injection of 3.2 or 5.2mg TA using a microneedle was well tolerated in the albino rabbit.Systemic exposure to TA was minimal, and absorption of TA into theposterior segment of the eye was observed with minimal TAexposure to the anterior segment of the eye. These data suggest thatsuprachoroidal drug delivery is well tolerated, results in distributionof TA to the sclera/choroid and retina, structures that are importanttargets for anti-inflammatories in posterior segment disease, andlimits TA exposure in the anterior segment.Commercial Relationships: Rozemarijn S. Verhoeven, ClearsideBiomedical (E); Samirkumar R. Patel, Clearside Biomedical (E),Clearside Biomedical (I), Clearside Biomedical (P); Karen Viaud-Quentric, Iris Pharma (E); Florian Cacciamani, Iris pharma (E);Thierry Amar, None; Benjamin Yerxa, Clearside Biomedical (I)Support: Georgia Research AllianceProgram Number: 120 Poster Board Number: C0125Presentation Time: 8:30 AM - 10:15 AMAssessment of Efficacy and Safety of a Novel Protocol for PulsedIntravenous Cyclophosphamide for Recalcitrant or SevereOcular Inflammatory DiseaseAna M. Suelves 1, 2 , Cheryl A. Arcinue 1, 2 , Jesús María González-Martín 4 , C. Stephen Foster 1, 3 . 1 Massachusetts Eye Research andSurgery Institution, Cambridge, MA; 2 Ocular Immunology & UveitisFoundation, Cambridge, MA; 3 Harvard Medical School, Boston,MA; 4 University of Valencia, Valencia, Spain.Purpose: To analyze the success rate of pulsed intravenous (IV)cyclophosphamide (CyP) for non-infectious ocular inflammatorydisease and to identify risk factors for failure of therapy.Methods: Retrospective, interventional, cohort study. Through acomputer search of the Massachusetts Eye Research and SurgeryInstitution’s database, we identified 65 patients (110 eyes) who weretreated with IV CyP between May 2005 and April 2012. The mainoutcomes evaluated through review of the electronic health record ofeach patient were clinical response, corticosteroid-sparing effect,recurrence rate, calculated “risk factors” for failure, visual acuity andadverse reactions.Results: Pulsed IV CyP achieved complete remission ofinflammation (for at least 2 visits) in 54 patients (84.4%). Sustainedremission of inflammation occurred in 70% of patients within 3months, 86.6% of patients within 6 months, and 91.7% within 9months. The mean time to achieving quiescence was 3.5 months. Thesuccess rate in reducing corticosteroid to prednisone 10 mg/day orless within 6 months, while maintaining control of ocularinflammation, was 89.7% (95% Confidence Interval (CI) 81.1-93.5%). The mean duration of clinical remission for those patientswho had a positive response to CyP was 32.67 months (95% CI25.91-39.43). Relapse of vasculitis was observed in 1 patient (1.5%)after completing the course of therapy. Early initiation of therapyduring the course of the disease was correlated with a lesser rate ofrecurrence (p=0.028). The most common adverse effects were nausea(29%) and transient lymphopenia (26%). The mean best-correctedvisual acuity (BCVA) improved from 0.59 ± 0.66 at baseline to 0.30± 0.54 at 6 months of follow-up (p < 0.001). The mean follow-upperiod was 31.61 ± 20.47 months.Conclusions: Pulsed IV CyP employing our protocol results in anextremely high rate of sustained complete remission in patients 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 – Physiology/Pharmacologyrecalcitrant and fulminant, vision-threatening ocular inflammatorydisorders, with an excellent safety profile in the hands of physicianstrained and skilled in the art of this therapy. The protocol makespossible tapering and discontinuing corticosteroids in most patients.Early initiation of therapy may decrease the risk of relapses.Commercial Relationships: Ana M. Suelves, None; Cheryl A.Arcinue, None; Jesús María González-Martín, None; C. StephenFoster, 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)Program Number: 121 Poster Board Number: C0126Presentation Time: 8:30 AM - 10:15 AMAcute serous retinal detachment after uncomplicated cataractsurgeryMaurizio G. Uva 1 , Antonio Longo 1 , Michele Reibaldi 1 , Mario D.Toro 1 , Vincenza Bonfiglio 1 , Faro Giuseppe 1 , Andrea Russo 1 ,Caterina Gagliano 2 , Teresio Avitabile 1 . 1 Institute of Ophtalmology,University of Catania, Catania, Italy; 2 Opthalmology, NEST(Neurovisual Science Technology), Catania, Italy.Purpose: To report some cases of acute serous retinal detachment(ASRD) after uncomplicated cataract surgery.Methods: In a retrospective study, were collected the data of patientsthat developed an ASRD after an uncomplicated phacoemulsificationwith IOL implantation.Diagnosis was made at the first post-operative day, when all patientshad a very low best corrected visual acuity (BCVA) in spite of a goodaspect of the anterior segment, without significant keratopathy andwith only trace cells in the anterior chamber; OCT revealed a serousretinal detachment with intraretinal fluid accumulation in maculararea. Patients received systemic treatment with indomethacin 20 mgQD and acetazolamide 125 mg TID, and topical indomethacin 0.1%eyedrops in addition to usual post-operative treatment (topical:tropicamide 1% BID, steroids and antibiotics QID; oral cefixime 400mg QD for 5 days).Patients were examined postoperatively at 1, 3, 7, and 30 days.BCVA and central foveal thickness (CFT), measured by a Spectraldomain Spectralis OCT, were evaluated.It was evaluated if demographics, preoperative ocular conditions,systemic diseases, parameters of surgery were related to thedevelopment of ASRD.Results: From January 2009 to June 2012, on 3900 cataract surgeryperformed, an ASRD was detected in 5 patients (3m, 2f, mean age59±9 years).Pre-operatively, mean BCVA was 0.16±0.09 decimals.At the first post-operative day, mean BCVA was 0.08±0.03 decimals,and CFT was 836±96 microns; all eyes had intraretinal fluidaccumulation and serous retinal detachment.At the following controls, BCVA improved and CFT reducedsignificantly (both ANOVA p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyassigned to 6 treatment groups (3 animals per treatment group). Asingle dose of 50µL of the dosing solution was topically administeredinto the conjunctival sac of both eyes of each animal. Animals wereeuthanized and the aqueous humor was collected at 1 hour ± 5minutes, 2 hours ± 15 minutes, 4 hours ± 15 minutes, 8 hours ± 15minutes, 12 hours ± 15 minutes, or 24 hours ± 15 minutes followingdosing. The iris/ciliary body, lens, vitreous, retina, choroid, sclera,conjunctiva, and cornea (target tissues) were also collected from eacheye for analysis. Dosing solutions were analyzed to confirmradiochemical purity; radioactive concentration of the dosingsolutions was calculated using liquid scintillation counting (LSC).Results: Radioactive residues of 14 C-Iabeled bromfenac, expressed asmean parts per million [(ppm) µg/g] was seen in all target tissues ofthe eyes, with the highest concentrations found in the cornea,conjunctiva, and sclera. The concentrations in the tissues diminishedto varying degrees over the 24 hour study period, with the exceptionof the lens, which increased insignificantly from the 1 hour timepoint. The levels detected in the lens and vitreous humor were lowand close to background levels.Conclusions: Significant penetration and measurable amounts of14 C-labeled bromfenac were detected in most ocular target tissuesover 24 hours, with highest levels in the cornea, conjunctiva, andsclera. The 14 C-low concentration bromfenac residues in oculartissues were similar to those previously reported with 0.09% 14 C-bromfenac, the currently available concentration of bromfenacophthalmic solution.Commercial Relationships: George Baklayan, Bausch & Lomb (E)Program Number: 124 Poster Board Number: C0129Presentation Time: 8:30 AM - 10:15 AMComparison of Methotrexate and Mycophenolate in theTreatment of Chronic UveitisTiffany Truong 1 , Zvi A. Kresch 1 , Sanjay R. Kedhar 1, 2 , Vicente Diaz 1 ,John V. Mauro 1 , C. Michael Samson 1, 2 . 1 NY Eye and Ear Infirmary,New York, NY; 2 New York Medical College, New York, NY.Purpose: To compare efficacy and side effects of methotrexate andmycophenolate in the treatment of chronic non-infectious uveitis.Methods: Charts of patients seen by the New York Eye & EarInfirmary Uveitis Service in 2004-2005 who were treated with eithermethotrexate or mycophenolate and had follow-up data for aminimum of three years after initiation of medication were reviewed.Statistical analysis was performed comparing aspects of efficacy andtolerability, such as ability to control inflammation, time to controlinflammation, ability to prevent vision loss, and incidence of sideeffects.Results: A total of 95 patients were included in the study. Control ofinflammation at 1, 2, and 3 years after initiating medication wasstatistically equal between the two groups, ranging from 71% to 80%with mycophenolate, and 67% to 70% with methotrexate.Mycophenolate achieved control of inflammation faster thanmethotrexate at 6 and 9 months (p=.0179, .0485 respectively), but thedrugs evened out in the long run. Visual acuity was preserved equallyin both groups. Side effects were minimal in both groups.Conclusions: Both methotrexate and mycophenolate have equalability to control inflammation and prevent vision loss in patientswith chronic uveitis who are either unresponsive or intolerant ofsteroid treatment. Mycophenolate achieves control of inflammationmore quickly in the 6 to 9 month range, but the drugs are equal at 1year and longer. Both drugs are tolerated with minimal side effects.This is the first report of a comparison of these two drugs in a cohortat a single treatment center.Commercial Relationships: Tiffany Truong, None; Zvi A. Kresch,None; Sanjay R. Kedhar, None; Vicente Diaz, None; John V.Mauro, None; C. Michael Samson, CLS Pharmaceuticals (I),PCAsso (I)Program Number: 125 Poster Board Number: C0130Presentation Time: 8:30 AM - 10:15 AMRandomized, Placebo-Controlled, Integrated Phase III ClinicalTrials of a Once Daily, Low-Concentration, Modified BromfenacOphthalmic Solution Following Cataract Surgery: Focus on Zeroto Trace Anterior Chamber InflammationJames A. Gow 1 , James D. Boyce 2 , Harvey J. Reiser 3 , Robert Berry 4 ,Jung T. Dao 5 , Simon P. Chandler 1 . 1 Bausch & Lomb, Irvine, CA;2 Orange County Ophthalmology Medical Group, Garden Grove, CA;3 Eye Care Specialists, Kingston, PA; 4 Eye Care Arkansas PA, LittleRock, AR; 5 Cornea Consultants of Arizona, Phoenix, AZ.Purpose: To evaluate in a post-hoc analysis the reduction of ocularinflammation to 0 or trace anterior chamber inflammation of lowconcentration,modified bromfenac ophthalmic solution dosed oncedaily compared to placebo following cataract surgery in 2 integratedclinical trials.Methods: Subjects undergoing unilateral cataract surgery(phacoemulsification or extracapsular cataract extraction) withposterior chamber IOL implantation were randomized to either lowconcentration,modified bromfenac ophthalmic solution (n=222) orplacebo (n=218). Once daily dosing began 1 day before cataractsurgery, continued on the day of surgery, and through post-surgeryDay 14. The proportion of subjects with trace anterior chamberinflammation, defined as a Summed Ocular Inflammation Score(SOIS) of 0-0.5 (0-5 cells in the anterior chamber and flare grade of0), was assessed at Days 1, 3, 8, and 15. Safety was assessed by theincidence and frequency of ocular and systemic adverse events, andophthalmological evaluations (visual acuity, slit lamp examination,intraocular pressure, and dilated funduscopic examination). Statisticalsignificance was determined using a Fisher’s exact test.Results: In the intent-to-treat population, subjects had a mean age of68.0 years, were predominantly Caucasian (74.8%), and included ahigher percentage of female subjects (65.2%). Baselinecharacteristics were similar across treatment groups. A significantlyhigher proportion of subjects achieved trace ocular inflammation inthe bromfenac group compared to placebo as early as Day 3 (27.9%vs. 13.8%, p=0.0008), continued on Day 8 (55.4% vs. 24.3%, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyAnanth Iyer, Rick Gallagher. RT&D, DSM, Berkeley, CA.Purpose: We report the unique properties of novel miktoarmbioresorbable poly(ether-ester) urethane (PEEsU) accessed usingcommercially available α-methoxy-ω-diol poly(ethylene glycol)(Ymer N120) in comparison with linear PEEsU starting frompoly(ethylene glycol) (PEG diol)with potential applications as oculardrug delivery systems, adhesives and sealants, scleral buckles,punctal plugs, and tissue engineering applications.Methods: Candidate PEEsU having ethylene oxide to L-lactide(EO/LLA) ratios of 0.5, 2 and 4 were prepared using a one-pot twostepsynthesis. In the first step, polyester diols were synthesized bythe ring opening polymerization (ROP) of L-lactide with theappropriate diol using stannous octoate as the catalyst. In the secondstep, the resultant polyester diol was chain extended usinghexamethylene diisocyanate (HDI) to yield the PEEsU. Thepoly(ether-ester) molecular weight buildup was verified using nuclearmagnetic resonance spectroscopy (NMR). The different PEEsU werecharacterized by gel permeation chromatography (GPC) anddifferential scanning calorimetry (DSC). Tensile properties weremeasured on dry films per ASTM D1708. The hydrolytic degradationof these polymers were followed in a pH=7.4 phosphate buffersolution at 37°C using protocols set in ISO10993-13.Results: Tensile load at break for the Ymer N120 based PEEsUranged from 18.06N for EO/LLA of 0.5 to 0.03N for EO/LLA of 2.PEG diol (PEG 1000) based PEEsU for EO/LLA of 0.5 was brittleand film tensile properties were not measurable. The hydrolyticdegradation studies were followed by GPC molecular weight dropmethod. Thus, during a 4 week incubation period, weight averagemolecular weight (Mw) of PEEsU with EO/LLA=0.5 dropped by25% while it dropped by 70% for EO/LLA = 2 during the same timeperiod. PEEsU with EO/LLA=4 completely degraded in three days.Additionally thermal profiles of the representative PEEsU weremeasured using DSC.Conclusions: We demonstrate the successful synthesis of pendantMPEG based hydrolytically degradable PEEsU. Different forms ofthe polymer such as films, gels and viscous liquids were made bycontrolling the EO/LLA ratio of the polyester segments that werethen chain extended with HDI. By controlling the EO/LA ratio in thePEEsU, the degradation times of the polymer was manipulated fromdays to weeks and may be further modified pending applicationrequirements.Commercial Relationships: Ananth Iyer, DSM (E); RickGallagher, DSM Biomedical Inc. (E)Methods: Fresh etched porous silicon film was sonicated to producepSi microparticles. pSi particles were oxidized at 800 degree C. DNRwas absorbed into the pSi particles by soaking 10 mg pSi in 300 µLof 5mg/mL DRN solution overnight at room temperature. The drugloading was quantitated by thermogravimetric analysis (TGA). TheDNR loaded pSi particles were divided into two groups: group 1 withPLGA (poly (lactic-co-glycolic acid) coating and group 2 without.For PLGA coating, DRN loaded pSi particles were allocated into10% PLGA dichloromethane solution and vortex for 20 min. Themixture was dispersed into 2% PVA (polyvinyl alcohol) aqueoussolution for evaporation of dichloromethane. PLGA coated particleswere characterized under a light microscopy for PLGA capsulation.The particles with or without PLGA were allocated each into threeclosed vial with 1.5 mL of DPBS and incubated under 37°C on amini labroller. At designated time points, 1mL supernatant wascollected and the same amount of DPBS was added back into thevial. DNR in the supernatant was quantitated using a fluorescencespectrophotometer.Results: The DNR loading into pSi particles was determined to be32.99 µg/mg. Light microscopy showed 80% pSi particles werecapsulated by PLGA and the non-capsulated pSi particles had a meandiagonal size of 75 µm (median 68.4 µm). DNR release from pSiwithout PLGA capsulation demonstrated a predicted peakconcentration of 7200 ng/mL while only 1200 ng/mL for PLGAcapsulated pSi. The DNR release profile from pSi particles wastypical first-order kinetics while a sustained release mode wasachieved through PLGA capsulation (Figure).Conclusions: PLGA capsulation can slow down DNR release frompSi particles and reduce the initial burst release as well as improvethe drug release kinetics optimized toward intravitreal drug deliveryapplication.Program Number: 1070 Poster Board Number: C0047Presentation Time: 1:00 PM - 2:45 PMPLGA capsulated porous silicon particles for sustainedintravitreal delivery of daunorubicinKaihui Nan 1 , Feiyan Ma 1 , Huiyuan Hou 1 , William R. Freeman 1 ,Michael J. Sailor 2, 3 , Lingyun Cheng 1 . 1 Department ofOphthalmology, Jacobs Retina Center at University of California,San Diego, CA; 2 Department of Chemistry and Biochemistry,University of California, San Diego, CA; 3 Department ofBioengineering, University of California, San Diego, CA.Purpose: Daunorubicin (DNR) is a FDA approved antiproliferationagent which has been used to treat proliferative vitreoretinopathy(PVR). However its narrow therapeutic window and short vitreoushalf-life limit its intraocular application. We have shown thatadsorption loading of DNR into porous silicon (pSi) particles canprovide a 2-week release in rabbit vitreous. However, DNR releasewas still fast and caused retinal toxicity. In the current study weaimed to develop a better controlled vitreous release system usingPLGA capsulizing DNR loaded pSi particles.Commercial Relationships: Kaihui Nan, None; Feiyan Ma, None;Huiyuan Hou, None; William R. Freeman, OD-OS, Inc. (C);Michael J. Sailor, Spinnaker Biosciences (I); Lingyun Cheng,Spinnaker Biosciences (C)Support: NIH Grant EY 020617 and NSF Grant DMR-1210417Program Number: 1071 Poster Board Number: C0048Presentation Time: 1:00 PM - 2:45 PMMechanism of ultrasound-mediated transscleral delivery:temporary alteration of scleral structure increases permeabilityof macromoleculesYing Chau 1, 3 , Wai-Leung Suen 1 , Jun Jiang 2 , Yan Zeng 2 , Jianan Qu 2,3 . 1 Chemical and Biomolecular Engineering, The Hong KongUniversity of Science and Technology, Hong Kong, Hong Kong;2 Department of Electronic and Computer Engineering, The HongKong University of Science and Technology, Hong Kong, Hong©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 – Physiology/PharmacologyKong; 3 Division of Biomedical Engineering, The Hong KongUniversity of Science and Technology, Hong Kong, Hong Kong.Purpose: We have proposed the use of low frequency ultrasound as anon-invasive approach to modulate the ocular barriers for transscleraldrug delivery to the posterior segment of the eye. Using thisapproach, we observed significantly enhanced delivery in vivo. Wehypothesize that ultrasound increases the porosity of scleral fibernetwork to allow improved diffusion of macromolecules. Here, weaim to understand the effect of ultrasound on sclera, the first barrierin the transscleral route of delivery.Methods: The penetration of FITC-dextran of 70kDa through rabbitsclera was measured ex vivo with and without ultrasound. Sonicationwas applied directly above sclera at pre-designated frequency andintensity. Diffusivity was calculated by fitting the penetration profile,obtained by fluorescent microscopy of cryosectioned sclera, with 1-Ddiffusion equation. The collagen network structure of sclera wasvisualized dye-free using two-photon excitation microscopy (TPEM).Pore size of sclera was estimated by Renkin’s restricted diffusionmodel and textual analysis of TPEM images.Results: Dextran penetrated deeper into ultrasound-treated sclera,confirming that the barrier function of the sclera was weakened bysonication (Figure 1a). The transscleral penetration distance increaseswith decreasing frequency, suggesting the role of cavitation.Diffusivity of dextran increased up to 8 times in sclera after lowfrequency sonication. The enhancement was temporary, with thescleral permeability restored in 3 hours (Figure 1b). TPEM imagerevealed that ultrasound disrupted the ordered alignment of collagenand increased the scleral pore size (Figure 2), agreeing with theprediction by Renkin’s model.Conclusions: Low frequency ultrasound alters the collagen networkstructure of sclera to increase the porosity, thereby enhancing thediffusion of macromolecules through the outmost barrier in thetransscleral route of delivery.Figure 1. a) Penetration distance of 70 kDa dextran in 15 minutes insclera immediately after sonication at the indicated frequency (n=3).b) Restoration of scleral permeability post ultrasound. Penetrationdistance of 70 kDa in 15 minutes in sclera at varying time lag aftersonication at 40 kHz is shown (n=3).Figure 2. Two-photon excitation microscopy image (100 μm x 100μm) of sclera before and after ultrasound treatment at 40 kHz.Commercial Relationships: Ying Chau, None; Wai-Leung Suen,None; Jun Jiang, None; Yan Zeng, None; Jianan Qu, NoneSupport: Innovation and Technology Fund and Research GrantsCouncil of Hong Kong SARProgram Number: 1072 Poster Board Number: C0049Presentation Time: 1:00 PM - 2:45 PMAn aqueous clear rapamycin topical drop for retinal deliveryKishore Cholkar 1 , Sriram Gunda 2 , Ravinder Earla 1 , Ashim K. Mitra 1 .1 University of Missouri Kansas City, Kansas City, MO; 2 PPD, Inc,Richmond, VA.Purpose: The objectives of this study are (i) to develop an aqueous,clear mixed nanomicellar formulation (MNF) of rapamycin,optimization and characterization, (ii) to determine MNF cytotoxicityand transport across ocular cells and (iii) to determine in vivo oculartissue distribution of optimized novel rapamycin loaded MNF posttopical drop administration into rabbit cul-de-sac.Methods: Polymers such as Vitamin E TPGS (D-alpha-tocopherylpolyethylene glycol 1000 succinate), octoxynol-40 and rapamycin aremixed in varying ratios to obtain an optimized formulation. Thenovel MNF was prepared by solvent evaporation technique. In vitrorelease studies were conducted with a dialysis bag method andcytotoxicity and transport studies were conducted on HCEC andARPE-19 cells. In vivo studies were conducted in New Zealand(NZW) male white rabbits.Results: Rapamycin was loaded into MNF to generate an overallloading of 2 and 4mg/mL. Optimized formulation was characterizedfor its improvement in drug loading, entrapment efficiency, size,polydispersity, surface charge, morphology and rapamycin release.Optimized MNF showed a size range of 28-35 nm and encapsulationpercentage > 95% respectively. Absence of free or unentrappedrapamycin in the MNF was confirmed by proton NMR spectroscopy.The MNF drug release was found to be sustained. Cytotoxicitystudies on HCEC and ARPE-19 cells treated with placebo andrapamycin loaded optimal MNF’s showed no significant difference incell survival relative to untreated (medium) cells. Transport studiesshowed higher rapamycin permeability. In vivo rapamycin oculartissue distribution studies show higher rapamycin concentrations(362.35 ± 56.17 ng/g tissue) in back of the eye tissues (retinachoroid)with no rapamycin detected in vitreous humor (VH).Conclusions: An optimized and characterized clear aqueous MNF ofrapamycin was prepared. The novel MNF had no cytotoxic effect onHCEC and ARPE-19 cells and had higher permeability. In vivoocular tissue distribution studies show that therapeutic levels ofrapamycin were observed in retina-choroid with no rapamycin in VH,post topical drop administration. Results indicate rapamycin loaded©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 – Physiology/PharmacologyMNF follows conjunctival-scleral pathway to reach back of the eyetissues (retina-choroid).Commercial Relationships: Kishore Cholkar, None; SriramGunda, None; Ravinder Earla, None; Ashim K. Mitra, NoneProgram Number: 1073 Poster Board Number: C0050Presentation Time: 1:00 PM - 2:45 PMCell-Encapsulated Device for Intraocular Delivery of Glial-CellDerived Neurotrophic Factor (GDNF)Francisca SY Wong 1 , Calvin CH Wong 1 , Barbara P. Chan 2 , Amy C.Lo 1, 3 . 1 Ophthalmology, The University of Hong Kong, Hong Kong,Hong Kong; 2 Mechanical Engineering, The University of HongKong, Hong Kong, Hong Kong; 3 Research Center of Heart, Brain,Hormone and Healthy Aging, The University of Hong Kong, HongKong, Hong Kong.Purpose: While GDNF is able to exert neuroprotective effects onphotoreceptor cells, successful administration of such therapeuticprotein has been hindered by short half-life and the lack of asustained drug delivery method. A cell-based immunoisolatedintraocular drug delivery device for continuous GDNF release wasdesigned. The photoreceptor rescuing effects in a rat model withinherited retinal degeneration were examined after the implantationof the gel device.Methods: HEK293 cells that overexpress GDNF were encapsulatedin a composite matrix constituted of 2mg/ml collagen and 1%alginate. The collagen-alginate gel device was intravitreally injectedinto Royal College of Surgeons rats on post-natal day 28 (P28). Ratswere randomly divided into four groups: sham, un-operated, vehiclecontrol, and treatment groups. Vitreous contents were collected forGDNF content assessment on P35 and P42. Enucleation was carriedout on P56 for histological evaluations. H&E stained paraffinsections of the retina were examined for the degree of morphologicalrescue via quantifying the outer nuclear layer photoreceptors atvarious retinal regions.Results: Significant amount of GDNF was released into the vitreousafter 7 and 14 days of device implantation. Outer nuclear layer(ONL) linings in the treatment group were better aligned at thecentral retinal regions when compared to the control groups. Increasein mean ONL cell counts were observed across the whole retina, inparticularly, the center of the inferior retina.Conclusions: Cell growth, proliferation and sustained release ofGDNF were achieved through implanting the cell-encapsulatingcollagen-alginate gel device, resulting in morphological rescue ofphotoreceptor cells in vivo. This system could potentially be appliedas a sustained drug release platform of GDNF and/or othertherapeutic proteins in various ocular conditions involving similarpathological phenotypes.Commercial Relationships: Francisca SY Wong, None; CalvinCH Wong, None; Barbara P. Chan, The University of Hong Kong(P); Amy C. Lo, NoneSupport: Seed Funding from The University of Hong KongProgram Number: 1074 Poster Board Number: C0051Presentation Time: 1:00 PM - 2:45 PMLong-lasting eye drop delivery platform for targeted oculardelivery applicationsFrank Gu 1, 2 , Shengyan Liu 1 , Lyndon W. Jones 2 . 1 ChemicalEngineering, University of Waterloo, Waterloo, ON, Canada;2 Optometry and Vision Science, University of Waterloo, Waterloo,ON, Canada.Purpose: Common topical formulations, such as eye drops orointments, suffer from low ocular bioavailability due to rapiddrainage through the naso-lacrimal duct, nearly constant dilution bytear turnover, and low drug permeability across the cornealepithelium. As a result, topical formulations are normallyadministered multiple times daily in order to achieve therapeuticefficacy, resulting in a higher potential for side effects and lowerpatient compliance. Our study aims to design nano-scaled drugcarriers to overcome the rapid clearance of current eye drop solutionsthereby improving drug retention on the corneal surface.Methods: NPs composed of poly(D,L-lactide)-b-Dextran (PLA-Dex)surface functionalized with a mucoadhesive ligand, phenylboronicacid (PBA), were developed as drug carriers. Using Cyclosporine A(CycA) as a model drug, CycA molecules were encapsulated withinPLA-Dex using emulsification. Controlled drug release andbiocompatibility studies were performed in vitro and in vivo.Results: We showed that the nanoparticle carrier can be used torelease CycA in vitro and in vivo. The average size of NPs werefound to be in the range of 25 and 28 nm. The NPs showedencapsulated up to 13.7 wt% CycA and exhibited sustained releasefor up to 5 days in vitro at a clinically relevant dose. We fine-tunedthe PBA density on the NP surface to maximize the mucin-NPinteraction without compromising the particle stability in vitro. Weshowed that the NP formulation did not significantly affect thetransparency or the solution viscosity, which improves patientcompliance. The surfaces of the nanoparticles have a defined numberof ligands capable of targeting the corneal surface, allowing drugsencapsulated in the particles to effectively circumvent the tearturnover mechanism, and significantly improving their cornealretention.Conclusions: The surface of the nanoparticle formed from a linearblock copolymer poly(D,L-lactide)-b-Dextran was modified withPBA to form a mucoadhesive nanoparticle for topical ocular drugdelivery application. The simplicity of NP design suggests it candeliver a wide range of bioactive agents including both hydrophilicand hydrophobic compounds. These results suggest the potential ofour nanoparticle approach can be used as a platform technology toprovide long-lasting delivery of therapeutic agents to treat anterioreye diseases.Commercial Relationships: Frank Gu, None; Shengyan Liu,None; 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)Support: NSERC Strategic NetworkProgram Number: 1075 Poster Board Number: C0052Presentation Time: 1:00 PM - 2:45 PMHyaluronic acid based tablet for slow release of ilomastat inglaucoma filtration surgeryAbeer Mohamed Ahmed 2, 1 , Alastair Lockwood 2, 1 , Steve Brocchini 2 ,Peng T. Khaw 1 . 1 National Institute for Health Research (NIHR)Biomedical Research Centre at Moorfields Eye Hospital NHSFoundation Trust and UCL Institute of Ophthalmology, London,United Kingdom; 2 UCL School of Pharmacy, London, UnitedKingdom.Purpose: Ilomastat is a matrix metalloproteinase inhibitor (MMPi)that has been shown to inhibit fibrosis after glaucoma filtrationsurgery (GFS) in a rabbit model of ocular fibrosis. To reduce scarringand fibrosis following glaucoma surgery, a sustained dosage formwould be advantageous that allows a prolonged local concentration ofilomastat to be maintained within the subconjunctival space.Hyaluronic acid (HA) is used in ocular medicine. HA was examinedas a matrix to fabricate a small tablet for subconjunctivalimplantation after GFS.Methods: Ilomastat (1.0 mg) was dissolved in butanol (40% w/v).©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 – Physiology/PharmacologyAn aqueous solution of hyaluronic acid (3.0 mg) was prepared. Theilomastat solutions was then added to the aqueous polymer solutionand gently mixed overnight. The mixture was freeze dried. Thelyophilised powder was pressed into a tablet using 3 mm punch anddie. The release of ilomastat from the tablet in phosphate bufferedsaline (PH 7.4) was determined at flow rate of 2.0 µL at 35.0 °C in aflow rig. The concentration of the released ilomastat was measuredusing by HPLC at 280 nm.Results: A small tablet designed for ocular implantation wassuccessfully fabricated with a dispersion of ilomastat in both linearand cross linked HA matrices. The weight of the tablet was in therange of 4.5-6.5 mg. Fabrication of the ilomastat tablet using linearHA (Healon® GV) resulted in a sustained release of ilomastat over11 days with a total ilomastat release of 83.2±1.8% (maximumilomastat concentration of 246.5±32.3 µM at tmax of 31.0±18.0 h).The release of ilomastat was more prolonged when cross linked HA(Healaflow®) was used. A total of 101.48±4.48% was released after26 days with a maximum concentration of 212.01±23.31 µM (t max4.3 h). The concentration of the released ilomastat was within thetherapeutic range (10-100 µM). The total release of ilomastat from amixture of linear and cross linked HA (1:1) was 63.3 % (maximumconcentration of ilomastat was 199.5 µM at tmax of 33.1 h) after 14days. The release of ilomastat was faster when more linear HA wasincorporated in the dispersion mixture.Conclusions: Sustained release of ilomastat was achieved by itsdispersion in cross linked HA matrix up to 28 days. The prolongedrelease of ilomastat from a dispersion of cross linked HA may besuitable for a successful sub-conjunctiva implant for improvement ofthe outcome of glaucoma filtration surgery.Commercial Relationships: Abeer Mohamed Ahmed, SteveBrocchini (WO09/063222) (P), Peng Khaw (WO09/063222) (P);Alastair Lockwood, None; Steve Brocchini, None; Peng T. Khaw,University College Moorfields (P)Support: Medical research council G801650, Fight for sight, NIHRMoorfields Biomedical Research Centre, Freemasons Grand Charityand Helen Hamlyn TrustProgram Number: 1076 Poster Board Number: C0053Presentation Time: 1:00 PM - 2:45 PMNOVEL AQUEOUS NANOMICELLAR FORMULATIONSCONTAINING FLUOCINOLONE ACETONIDE FOR THETREATMENT OF POSTERIOR UVEITISSujay Shah, Asha Patel, Sulabh Patel, Dhananjay Pal, Ashim K.Mitra. Division of Pharmaceutical Sciences, University of MissouriKansas City, Kansas City, MO.Purpose: Diseases like posterior uveitis affect the posterior segmentof the eye and can cause partial or total vision loss. Current forms oftreatments require administration of high doses to overcome staticand dynamic barriers present in the eye. Fluocinolone acetonide (FA)is a highly potent glucocorticoid therapeutic with anti-inflammatoryproperties. Current therapy involves surgical placement of intravitrealimplant of FA. This method has several disadvantages likeretinal detachment, redness, pain and discomfort - all of which canlead to patient non-compliance. Therefore, the optimum strategy is todevelop eye drops of FA. Since FA is very poorly soluble in water, itis difficult to prepare high concentration clear aqueous solution eyedrops of FA. Therefore, our aim in this study is to develop aqueousnanomicellar formulations containing FA. Vitamin E TPGS (1K), asurfactant polymer has been utilized to prepare nanomicelles. Wealso synthesized polymer with higher molecular weight of PEG(2000) (2K). This modified polymer has a very low critical micellarconcentration (CMC) which might improve the stability upon teardilution in eye.Methods: Modified TPGS was synthesized by conjugation D-αtocopherylsuccinate and mPEG having molecular weight of 2000.The product was purified by dialysis method. CMC values werecalculated using standard pyrene method. Micelles were prepared bythin film hydration technique. Box-Behnken design was used tooptimize the formulation to achieve maximum entrapment andsolubility of drug. Size and zeta potential of micelles was measured.Cytotoxicity studies were conducted on corneal and retinal cells.Results: 2K polymer was successfully synthesized with a yield of65%. The CMC value obtained was 7.28μg/ml which is significantlyless than commercially available TPGS 1K. Results showed thatsolubility of FA maybe increased up to 26 times with newlysynthesized 2K polymer. Entrapment efficiency greater than 90%was achieved with 2K polymers. Nanomicelles exhibited very smallsize (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyIgG1 application did not result in appreciable uptake into peripheralblood, suggesting that topical treatment with anti-VEGF antibodieswould not contribute to the development of systemic complications.Commercial Relationships: Stacy Hu, None; Steven B. Koevary,NoneSupport: Massachusetts Lions Eye Research FundProgram Number: 1078 Poster Board Number: C0055Presentation Time: 1:00 PM - 2:45 PMA Pilot Safety Assessment of a Topical Ocular Device(TODDD) Intended for Sustained Drug DeliveryCharles D. Leahy 1, 4 , Rodney Gutner 2 , Whittney Varney 2 , JackRulander 4 , Stephen Johnston 4 , Francis S. Lai 4 , Kathryn S. Crawford 3 ,Roger Albright 5 , Jeanne Y. Ellis 1, 4 , Edward J. Ellis 1, 4 . 1 VistaScientific LLC, Andover, MA; 2 New England College of Optometry,Boston, MA; 3 PharmOcu, Andover, MA; 4 University ofMassachusetts Lowell-Massachusetts Medical Device DevelopmentCenter, Lowell, MA; 5 Foresight Regulatory Strategies, Inc.,Wilmington, MA.Purpose: To establish safety, comfort and retention of TODDD forcontinuous wear studies. Although intended to deliver drug whileworn on the superior sclera 24/7 for several months at a time, in thisearly stage human study it was without drug and worn 12-15 hr/dayfor 1 week.Methods: An open label study evaluating safety, retention andsubjective symptoms of comfort of the TODDD was conducted at theNew England College of Optometry. At Visit 1 device was placed onone eye and subjects were instructed on insertion and removalprocedures. They then wore the single device daily (12-15 hr) for 7±2days until Visit 2. Safety evaluations on each visit included VA,keratometry, and slit-lamp grading and any fluorescein and lissaminegreen staining using the Brien Holden V. I. Grading Scales. Fit,stability and integrity of the device were observed. Subjects reportedon comfort, positioning, and ejection of the device at each visit, andeach evening after device removal via electronic diary.Results: 12 (11 females and 1 male, age 24-26) of 14 screenedsubjects were dispensed the device and all 12 completed the study.There were no significant adverse events or safety findings. Slit lampgrades after 1-4 hours and 1 week of wear showed increases of 0 or 1across all categories in either eye, and grade 0 or 1 conjunctivalstaining was reported in all subjects at all examinations, but for onesubject, who had grade 3 lissamine staining of the conjunctiva atVisit 2. There were no lid gradings above 2, and no corneal findingsabove grade 1 in any subject. In the majority of subjects at all timepoints, the device positioned on the superior sclera without excessrotation, completely under the lid, with good stability and movement.The devices remained clean and intact. At Visit 1 only mildlydecreased ocular comfort associated with some awareness of devicewas reported. At Visit 2 all but 1 subject reported comfortable wearalways or most of the time. No devices spontaneously ejected fromthe eye or were lost. Among prior contact lens wearers, most subjectsconsidered the device handling “comparable to contact lens”.Conclusions: This preliminary study indicates that the device is welltoleratedin this subject population. Retention was demonstrated andthe device produced no safety concerns after 7 days of daily wear.Continuous wear studies are planned.Commercial Relationships: Charles D. Leahy, Vista ScientificLLC (I), Vista Scientific LLC (P); Rodney Gutner, Vista ScientificLLC (F); Whittney Varney, None; Jack Rulander, Vista ScientificLLC (F); Stephen Johnston, Vista Scientific (F); Francis S. Lai,Vista Scientific (F); Kathryn S. Crawford, Vista Scientific, Inc. (C);Roger Albright, Johnson & Johnson Vision Care Inc. (C),CooperVision Inc. (C), Menicon Ltd. (C), Semprus Biosciences (C),VISTA Scientific, LLC (C); Jeanne Y. Ellis, Vista Scientific (I),Vista Scientific (P); Edward J. Ellis, Vista Scientific LLC (I), VistaScientific LLC (P)Support: NIH Grant EY013479Program Number: 1079 Poster Board Number: C0056Presentation Time: 1:00 PM - 2:45 PMPrecisely Engineered Biodegradable Intraocular Implants for theSustained Release of DexamethasoneAndres Garcia, Janet Tully, Benjamin Maynor, Benjamin Yerxa.Liquidia Technologies, Durham, NC.Purpose: The ability to fabricate biodegradable intraocular implantswith uniform size, shape and dose for the sustained delivery ofactives in multiple regions of the eye has proven elusive with currenttechnologies. The acceptance of intravitreal implants for the localizedtreatment of multiple back-of-the-eye conditions have paved the wayfor the development of a new generation of smaller intraocularimplants in the anatomically and clinically desirable, yet “hard-tomanufacture”size range of 100μm to 1,000μm. The ability toreproducibly fabricate implants in this size range opens up a windowof opportunities for the injection and localization of implants againstmultiple target tissues of the inner eye where greater spatialconstraints may exist.Methods: We report the ability to precisely fabricate 150μm x150μm x 500μm intraocular implants comprised of a blend ofmicronized dexamethasone and poly(lactic-co-glycolic) acid (PLGA)for the tunable release of active using the PRINT technology.Physicochemical characterization of the implants was performed toevaluate the overall mass uniformity range, dexamethasone contentuniformity across individual implants, and the in-vitro release ofdexamethasone from the implants in 1X PBS at 37°C.Results: Liquidia’s PRINT technology unique ability to impartcontrol over size and shape (Figure 1A) allowed for the fabrication ofdexamethasone/PLGA implants (Figure 2A) with a high degree ofmass uniformity (14μg, ±1μg) and dexamethasone content (2.2μg±0.2μg). Furthermore, PRINT implants enabled for the sustainedrelease of dexamethasone over therapeutically relevant timelines,with over 40% of the initial cargo retained in the implants after 35days in 1X PBS at 37°C.Conclusions: The PRINT technology uniquely allows for thefabrication of intraocular implants with uniform size, shape and dose.We demonstrated the ability to fabricate dexamethasone/PLGAintraocular implants in the desirable size range of 100μm to 1,000μmfor sustained release applications where anatomical constraints maycall for uniquely engineered implants.©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 – Physiology/PharmacologyFigure 1: A) SEM image of 150μm x 150μm x 500μmdexamethasone/PLGA PRNT intraocular implants, B) Close-up viewof the implant’s surface, showing micronized dexamethasoneembedded in PLGA matrix, C) In vitro dexamethasone release profilefrom PRINT implants over 35 days.Commercial Relationships: Andres Garcia, Liquidia Technologies(E), Liquidia Technologies (I); Janet Tully, Liquidia Technologies(E); Benjamin Maynor, Liquidia Technologies (E), LiquidiaTechnologies (I); Benjamin Yerxa, Liquidia (E)Program Number: 1080 Poster Board Number: C0057Presentation Time: 1:00 PM - 2:45 PMIntraocular pharmacokinetics of bevacizumab and ranibizumabin vitrectomized versus non-vitrectomized rabbit eyesJeeyun Ahn 1, 2 , Sunyoung Park 3 , Hyuncheol Kim 3, 4 , Ji Hyun Park 5 , JiYeon Park 5 , Duck Jin Hwang 1, 5 , Seong Joon Ahn 1, 5 , Yong-Kyu Kim 1,5 , Kyu Hyung Park 1, 5 , Se Joon Woo 1, 5 . 1 Department ofOphthalmology, Seoul National University College of Medicine,Seoul, Republic of Korea; 2 Department of Ophthalmology, SeoulMetropolitan Government Seoul National University BoramaeMedical Center, Seoul, Republic of Korea; 3 Department of Chemical& Biomolecular Engineering, Sogang University, Seoul, Republic ofKorea; 4 Interdisciplinary Program of integrated Biotechnology,Sogang University, Seoul, Republic of Korea; 5 Department ofOphthalmology, Seoul National University Bundang Hospital,Seongnam, Republic of Korea.Purpose: To analyze intraocular pharmacokinetic properties ofbevacizumab and ranibizumab in vitrectomized and nonvitrectomizedrabbit eyes.Methods: Twenty-five-gauge pars plana vitrectomy withoutlensectomy was performed in 35 rabbit eyes and 36 nonvitrectomizedrabbit eyes served as control. Intravitreal injection of1.25mg/0.05mL bevacizumab was performed in 17 vitrectomized(group 1-1) and 18 non-vitrectomized (group 1-2) eyes, respectively.Intravitreal injection of 0.25mg/0.025mL ranibizumab was performedin 18 vitrectomized (group 2-1) and 18 non-vitrectomized (group 2-2)eyes, respectively. Eyes were enucleated at 1 hour, 1, 2, 5, 14 and 30days after the intravitreal injections and frozen at -80°C.Bevacizumab and ranibizumab concentrations in the vitreous andaqueous humor, as well as the retina/choroid, were determined usingdirect enzyme-linked immunosorbent assay.Results: Vitreous clearance of bevacizumab and ranibizumabshowed distinct patterns, consisting of 2 phases and 1 phase,respectively. The vitreous half-life of bevacizumab and ranibizumabwere 6.99, 7.06, 2.51 and 2.75 days in vitrectomized and nonvitrectomizedeyes, respectively.Conclusions: Vitrectomy did not substantially affectpharmacokinetic properties or chorioretinal concentrations ofintravitreally injected bevacizumab or ranibizumab in rabbit eyes.Commercial Relationships: Jeeyun Ahn, None; Sunyoung Park,None; Hyuncheol Kim, None; Ji Hyun Park, None; Ji Yeon Park,None; Duck Jin Hwang, None; Seong Joon Ahn, None; Yong-KyuKim, None; Kyu Hyung Park, None; Se Joon Woo, NoneSupport: National Research Foundation of Korea (NRF) funded bythe Ministry of Education, Science and Technology (No. 2011-0009606)Program Number: 1081 Poster Board Number: C0058Presentation Time: 1:00 PM - 2:45 PMKey considerations for choice of syringes for delivery ofintravitreal therapies in enhancing safety and efficacy oftherapies injected in the eyeGAUTAM SHETTY. Specialized Drug Delivery, Unilife, YORK, PA.Purpose: Inaccuracy of dose injected into the vitreous can result inoverdosing (potentially causing increased intraocular pressure) orunderdosing (potentially reducing drug efficacy and/or increasingdrug injection frequency); this introduces unpredictability andpotential variability in clinical outcomes for treatments delivered totreat back-of-eye disorders. Inaccuracy of dose in inherent in syringesused currently in the delivery of injectable drugs into the vitreous ofthe eye for the treatment of retinal disorders. The opportunity toimprove clinical outcomes by reducing the dose volume from thecurrent 50uL dose volume administered in case of anti-VEGFtreatments would further exacerbate the inaccuracy problem. Also,syringes used to deliver off-label bevacizumab are not designed tostore the same for an extended period of time. There are currently noguidelines from PQRI on the extractable content for drugs to beinjected inside the eye. In absence of any guidelines, understandingthe extractable content of syringes is a critical consideration in thesafety of off-label bevacizumab in the treatment of retinal disorders.Methods: Key determinants of inaccuracy for delivery of microlitersize dose were identified in tuberculin syringes, that are currentlyused to deliver intravitreal drugs. A device has been developed tomitigate factors causing inaccuracy of a microliter sized dose. Doseaccuracy was assessed using gravimetric technique.Extractable compounds in tuberculin and insulin syringes using waterand isopropanol as extraction media was determined byHPLC/PDA/MS analysis.Results: Dose accuracy of 10uL dose within 2% was shown withcoefficient of variation of 3%. Extractable compounds (volatile andnon-volatile) were determined.Conclusions: It is possible to improve accuracy of dose injected intothe eye. With tools available, dose volumes injected in the eye can bereduced further to further mitigate potential risk of increase inintraocular pressure. Further research to determine toxicity ofextractable compounds from syringes used to store off-labelbecaizumab are necessary to fully understand any potential risk posedby the same.Commercial Relationships: GAUTAM SHETTY, Unilife (E),Unilife (P)Program Number: 1082 Poster Board Number: C0059Presentation Time: 1:00 PM - 2:45 PMFreeze Drying to Develop a Bevacizumab-based Tablet forOcular ImplantationGarima Sharma 1, 2 , Ashkan Khalili 2 , Sahar Awwad 1, 2 , Kiran Malik 3 ,Paul Matejtschuk 3 , Simon Gaisford 1 , Steve Brocchini 1, 2 , Peng T.©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 – Physiology/PharmacologyKhaw 2 . 1 Pharmaceutics, UCL School of Pharmacy, London, UnitedKingdom; 2 National Institute for Health Research (NIHR)Biomedical Research Centre at Moorfields Eye Hospital NHSFoundation Trust and UCL Institute of Ophthalmology, London,United Kingdom; 3 National Institute for Biological Standards andControl, Health Protection Agency, Potters Bar, United Kingdom.Purpose: Bevacizumab has shown the potential to control scarringwhen administered into the subconjunctival space followingglaucoma filtration surgery (GFS). To overcome the problem of rapidclearance, a solid dosage implantable tablet form of this antibody thatprovides prolonged release in the bleb is being developed. In an effortto optimise dose, we are examining each step used for tabletfabrication. As is often the case for any protein pharmaceuticalpresented in a solid reconstitutable form, a critical process step islyophilisation, which can cause aggregation of the protein with lossof activity. The aim of this study is to characterise the lyophilisationof the bevacizumab formulation used to fabricate the subconjunctivalimplantable tablet. Determination of the glass transition temperature(Tg’) and collapse temperature (Tc) during lyophilisation assists inthe development of a suitable freeze-drying cycle.Methods: Differential Scanning Calorimetry (DSC) was used tomeasure the Tg’ of the excipients alone and in combination with theantibody. Freeze Drying Microscopy (FDM) was used to study thecollapse temperature (Tc) for the excipients (trehalose and hyaluronicacid (HA)) and the formulation. The antibody formulation wascharacterised by size exclusion chromatography (SEC) and gelelectrophoresis.Results: DSC experiments indicate the sub-ambient glass transitiontemperature (Tg’) for bevacizumab to be -28.24°C (n=2) and that oftrehalose to be -29.36°C (n=2). The melting endotherm of HA was -23.71°C (n=2) as no Tg’ could be observed. This can be attributed tothe presence of buffer salts present in the HA formulation leading to aeutectic melt. FDM was used to monitor the progress oflyophilisation to determine the collapse temperature of theformulation (Fig. 1). The collapse temperature of the pharmaceuticalformulation of bevacizumab was found to be -36°C (n=2).Conclusions: Freeze-drying is a suitable technique to obtain a solidform of antibody that can be fabricated as a tablet. Characterisation offreeze-drying using FDM and DSC suggested that starting theprimary drying below -36 °C may avoid collapse of the cake duringscale up for freeze drying of the bevacizumab formulation.Fig. 1. Progression of freeze drying of bevacizumab formulation fortablet fabrication using FDM: A) Sample frozen at -60°C B) Sampledrying at -40°C C) Sample drying with collapse at -35°CCommercial Relationships: Garima Sharma, None; AshkanKhalili, University College London (P); Sahar Awwad, None;Kiran Malik, None; Paul Matejtschuk, None; Simon Gaisford,None; Steve Brocchini, None; Peng T. Khaw, University CollegeMoorfields (P)Support: NIHR Moorfields Biomedical Research Centre, UCLSchool of Pharmacy, Grand Charity, Helen Hamlyn Trust, Fight forSightProgram Number: 1083 Poster Board Number: C0060Presentation Time: 1:00 PM - 2:45 PMEfficiency of a new pre-loaded, microincision insertion system fora 1-piece hydrophobic-acrylic intraocular lensScott Evans 1 , Don R. Nixon 2 . 1 AMO, Santa Ana, CA; 2 NorthernOntario School of Medicine, Ontario, ON, Canada.Purpose: To evaluate the efficiency of using a pre-loaded insertionsystem for the TECNIS® 1-piece hydrophobic-acrylic IOL comparedto the standard insertion system.Methods: Forty TECNIS® 1-piece lenses were inserted into a salinebath under simulated operating room (OR) conditions, twenty (20)using a standard insertion system for the lens (UNFOLDER®Platinum inserter) and twenty (20) using a preloaded insertion system(TECNIS iTec inserter). For consistency, one investigator (DRN)performed all procedures. Time from opening the IOL package toloading the IOL into the insertion system, from IOL loaded to IOLmoved to delivery position, and time to deliver the IOL into thesaline bath were recorded. IOLs evaluated were representative of thewhole diopter range; 5 D, 20 D, and 34 D. Ease of use of the twoinserters, as well as complications related to the insertion system,were assessed.Results: There were no damaged IOLs in either group. Theprocedure time for each step and the overall combined usage timewas significantly less with the preloaded inserter compared to thestandard inserter. Time from package opening to IOL loading was 12± 2.2 sec and 35 ± 4.5 sec (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyphotographed using a digital camera with coaxial lighting and thedissolution medium was sampled for rapamycin quantitation. Thephotographs were imported into Image J and the color of the particleswas measured after thresholding of the images.Results: Hydrolytic and oxidative degradation of pSi, and release ofrapamycin from pSi caused an observable change of the pSi particlesfrom reddish to yellowish and then to transparent. The correspondingchanges in the reflected light intensity was correlated with thecumulative drug release (r=0.93, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologySupport: Grand Charity, UCL School of Pharmacy, NIHRMoorfields Biomedical Research Centre, Fight for Sight, HelenHamlyn Trust, Medical Research CouncilProgram Number: 1089 Poster Board Number: C0066Presentation Time: 1:00 PM - 2:45 PMPharmacokinetics Demonstrating Sustained DexamethasoneDelivery from a Punctum Plug in a Canine ModelAnkita Desai, Charles D. Blizzard, Michael Bassett, Amar S.Sawhney, Peter Jarrett, Michael McGrath, Arthur Driscoll. R&D,Ocular Therapeutix, Inc, Bedford, MA.Purpose: To examine the dose-dependent pharmacokinetics ofdexamethasone delivered from a biodegradable hydrogel punctumplug in a canine model.Methods: Micronized dexamethasone (Dex) was suspended in amulti-arm PEG solution at two different levels (high and low dose)and injected into small bore tubing prior to cross-linking. Fluoresceinwas conjugated into the hydrogel to aid plug visualization through thetissue using the blue light from a slit lamp. The Dex hydrogel matrixwas dried and cut into punctum plugs. The Dex plugs were insertedinto the inferior canaliculus of beagles and a subset was removedeach week for imaging. Tear fluid samples were collected at weeklyintervals and dexamethasone concentration was determined byLC/MS. The high dose was evaluated in a toxicology study and thelow dose is a clinically representative dose designed for four weeksof tapered release that was assessed in a PK study.Results: The Dex plug demonstrated a sustained drug release profilein tear fluid with a tapering effect over the treatment period.Explanted plugs (Figure Two) of the low dose formulation illustratethe drug release over time, with full drug clearance from the plug atfour weeks.Conclusions: Topical corticosteroids, such as dexamethasone, areused to treat inflammation for various ophthalmic conditionsincluding post-operative inflammation. Many therapies requiremultiple daily administrations (up to hourly for severe conditions) forthe first several weeks followed by tapering to a lesser frequency asthe inflammation subsides and the condition resolves. A single-dosedexamethasone punctum plug which biodegrades may provide a moreconvenient option to help eliminate patient compliance with stringentdosing requirements, and help ensure appropriate treatment andresolution of the condition.Commercial Relationships: Ankita Desai, Ocular Therapeutix (E);Charles D. Blizzard, Ocular Therapeutix, Inc. (E); Michael Bassett,Ocular Therapeutix (E); Amar S. Sawhney, Ocular Therapeutix (E);Peter Jarrett, Ocular Therapeutix (E); Michael McGrath, OcularTherapeutix, Inc. (E); Arthur Driscoll, Ocular Therapeutix (E)Program Number: 1090 Poster Board Number: C0067Presentation Time: 1:00 PM - 2:45 PMUse of galectin-3 fusions to extend the surface residence time ofproteins topically applied to the eyeThomas Barnes, Joseph T. Kovalchin, Allyson Masci, MichaelSchmidt, Pamela Pegman, Patricia A. Lowden, ChristianDombrowski. Eleven Biotherapeutics, Cambridge, MA.Purpose: One of the key limiting parameters in the penetration andeffectiveness of topically applied ocular therapeutics is their meanresidence time. Most of an instilled solution clears through drainage,with first-order clearance about 4 times that of bulk tear flow (~5ul/min). For protein drugs, the problem becomes more acute due tothe additional burden of reduced penetrance through the cornea andconjunctiva. Residence time might be increased however by fusing aprotein of interest to a mucosal surface binding protein, such asgalectin-3 (gal-3). Gal-3 is a small pentameric ocular surface residentprotein that cross-links O-type mucins.Methods: We used Gaussia luciferase (luc) as our test protein, fusedat its N-terminus to one or 2 copies of the carbohydrate bindingdomain of gal-3 (gal). Fusions were expressed in HEK cells andpurified by IMAC chromatography, and evaluated mucin binding byELISA and by their ocular surface half-lives on mouse eyes andrabbit corneas, using luciferase activity as the readout.Results: We compared luc, luc-gal and luc-gal-gal in a variety ofassays. Addition of gal-3 moieties to luciferase did not affect eitherthe specific or relative activities of luciferase. On ELISA platescoated with MUC1 and asialofetuin, one gal-3 copy increased thebound luciferase by 10-20-fold, and two copies increased it by 200-250-fold. Binding to isolated rabbit corneal punches after a 30-minwash was increased by 6 and 12-fold with 1 or 2 gal copies,respectively. 30 mins after topical instillation in mouse eyes, luc andluc-gal had near background luciferase activity in enucleated eyes,while luc-gal-gal was 95-fold higher. In a time-course study, luc waslost from the surface by 15 min, luc-gal by 30 min, while luc-gal-galrequired 180 min or more to reach background levels. The t1/2 ofluc-gal-gal was at least 6-fold higher than that of luc. We alsoshowed that gal3 can be readily fused to therapeutic moieties,including our novel 17kD chimeric IL-1 blocker which is the activesubstance in our clinical development product EBI-005.Conclusions: We have demonstrated that fusion of a protein to thesmall sugar-binding domain of gal-3 increases the half-life of itsocular surface residence by at least 6-fold. This might afford a meansto increase the absorption of biologics from the ocular surface,reducing either the dose required, the frequency of administration, orboth.Commercial Relationships: Thomas Barnes, ElevenBiotherapeutics (E), Eleven Biotherapeutics (P), Eleven©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 – Physiology/PharmacologyBiotherapeutics (I); Joseph T. Kovalchin, Eleven Biotherapeutics(E), Eleven Biotherapeutics (P), Eleven Biotherapeutics (I); AllysonMasci, Eleven Biotherapeutics (E); Michael Schmidt, ElevenBiotherapeutics (I), Eleven Biotherapeutics (E), ElevenBiotherapeutics (P); Pamela Pegman, Eleven Biotherapeutics (E);Patricia A. Lowden, Eleven Biotherapeutics (E); ChristianDombrowski, Eleven Biotherapeutics, Inc. (E), ElevenBiotherapeutics, Inc. (P)Program Number: 1091 Poster Board Number: C0068Presentation Time: 1:00 PM - 2:45 PMApplication of PRINT Microparticle and NanoparticleTechnology Toward Preparation of Ophthalmic SuspensionFormulations with Improved Tolerability and EfficacyBenjamin Maynor, Andres Garcia, Janet Tully, Benjamin Yerxa.Liquidia Technologies, Research Triangle Park, NC.Purpose: To use PRINT technology to produce micro andnanoparticles of controlled microstructure and nanostructure that aresuitable for the preparation of aqueous ophthalmic suspensionformulations without use of solubilizing excipients (e.g. cremaphor,oils, cyclodextrins).Methods: PRINT technology, a novel drug/excipient micromoldingapproach, was used to produce monodisperse nonspherical particlesof itraconazole, cyclosporine, and tacrolimus. Specifically, 10 microntriangular templates, 3 micron toroids, 200 nm cylindrical and 1micron cylindrical polymeric templates were used to prepare particlesof cyclosporine, tacrolimus, and itraconazole, respectively.Dissolution characteristics of itraconazole suspensions wereevaluated and compared to bulk and micronized itraconazole usingstandard dissolution test methods.Results: Monodisperse, shape-specific microparticles andnanoparticles were successfully prepared of cyclosporine, tacrolimus,and itraconazole. Characterization of these particles usingmicroscopy confirms that monodisperse populations of 10 microntriangles, 3 micron toroids, and 200 nm and 1 micron cylinders wereproduced of cyclosporine, tacrolimus, and itraconazole, respectively.The sizes and shapes of these microparticles and nanoparticles aresuitable for use in ophthalmic suspension dosage forms. Dissolutionstudies of itraconazole cylinder suspensions indicate that theseparticles dissolve faster under sink conditions than traditionalmicronized itraconazole (50% dissolution at 5 min for PRINTitraconazolecylinders vs. 15 minutes for micronized itraconazole),suggesting that itraconazole PRINT formulations may have greaterocular surface bioavailability than micronized formulations.Conclusions: We demonstrate that PRINT technology is a promisingapproach for the development of improved suspension formulationsof compounds such as cyclosporine, tacrolimus, and itraconazole.Dissolution experiments show enhanced dissolution time of theseparticles compared to traditional micronized drug formulations,without the use of excipients with poor tolerability profile.characteristics of PRINT-itraconazole microparticles and comparisonto micronized suspensions.Commercial Relationships: Benjamin Maynor, LiquidiaTechnologies (E), Liquidia Technologies (I); Andres Garcia,Liquidia Technologies (E), Liquidia Technologies (I); Janet Tully,Liquidia Technologies (E); Benjamin Yerxa, Liquidia (E)Program Number: 1092 Poster Board Number: C0069Presentation Time: 1:00 PM - 2:45 PMEvaluation of drug delivery and biocompatibility ofbiodegradable microfilms in the anterior segment of the ratmodelYan Peng 1 , Yu-Chi Liu 2, 3 , Nyein Chan Lwin 2 , Subbu S Venkatraman 1 ,Tina Wong 2, 3 , Jodhbir S. Mehta 2, 3 . 1 School of Materials Science andEngineering, Nanyang Technological University, Singapore,Singapore; 2 Singapore Eye Research Institute, Singapore, Singapore;3 Singapore National Eye Centre, Singapore, Singapore.Purpose: To develop a biodegradable, sustained-release,prednisolone acetate (PA)-loaded poly [d,l-lactide-co-ε-caprolactone](PLC) drug delivery system and to evaluate its biocompatibility,feasibility and release characteristics both in vitro and in vivo.Methods: Blank and 40% PA-loaded PLC microfilms with thicknessof 100µm and diameter of 2mm were developed and tested in vitroand in vivo. The degradation and drug release profiles of themicrofilms were evaluated in the in vitro and in vivo experiments.We further implanted the microfilms to the subconjunctival space ofthe rats (n=51). All eyes were monitored using slit lamp biomicroscopywith Hackett McDonald ocular scoring system andanterior segment optical coherence tomography. Histological studieswith Hematoxylin-Eosin, Picrosirus red staining andimmunohistochemistry analysis were performed to evaluate andcompare the presence of inflammatory and fibrotic reaction in blankand PA-loaded microfilm groups. PA concentrations in the aqueoushumor were determined by high-performance liquid chromatography(HPLC).Results: Subconjunctivally-implanted PA-loaded PLC microfilmswere able to deliver prednisolone acetate in a sustained manner over3 months, with a steady rate of 0.002mg/day in vivo. Eyes with eitherblank or PA-loaded implanted microfilms showed very minimalinflammatory response at the insertion sites and mild degree ofcollagen encapsulation around the microfilms, with significantly lessCD11c cells at 2 weeks (P = 0.001) and collagen extent at 2 and 4weeks (P = 0.001 and P = 0.002) in PA-loaded microfilm group.Desirable anterior chamber PA levels were achieved, with theconcentrations at 76.67±5.86, 69.33±2.3 and 42.67±4.1 ng/ml at 2, 4and 12 weeks, respectively.Conclusions: PA-loaded PLC microfilm displays goodbiocompatibility, feasibility and desirable sustained drug releaseprofiles. This device provides a promising alternative with greatpotential application in the treatments of ocular anterior segmentdiseases.Commercial Relationships: Yan Peng, None; Yu-Chi Liu, None;Nyein Chan Lwin, None; Subbu S Venkatraman, None; TinaWong, 61, 250,006 (P); Jodhbir S. Mehta, NoneSupport: Translational and Clinical Research (TCR) Programme(NMRC/TCR/002-SERI/2008)A) Scanning electron microscopy images of PRINT microparticlescomposed of cyclosporine and tacrolimus; B) Aqueous dissolutionProgram Number: 1093 Poster Board Number: C0070Presentation Time: 1:00 PM - 2:45 PMSafety of DuraSite vehicle as part of a topical treatment incataract surgery patientsJudith Hutcheson, Lyle M. Bowman, Kamran Hosseini. InSite Vision,Alameda, 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 – Physiology/PharmacologyPurpose: To investigate the safety of DuraSite vehicle(polycarbophil, sodium chloride, edetate disodium dihydrate, andsterile water for irrigation) in post cataract surgery patients.Methods: In a well-controlled multicenter Phase II clinical trial, 169patients were randomized into 4 different groups to investigatedifferent dosing regimens of Bromfenac in DuraSite 0.075% (ISV-303) compared with the non DuraSite based commercial Bromfenacformulation (0.09%). DuraSite was the vehicle (either with or withoutthe drug) in three arms of the study (approximately 3/4 of the totalpatients, n=127). All patients received the investigational medicationone day after cataract surgery, and self-administered the formulationb.i.d. for a period of two weeks. Slit-lamp biomicroscopy, IOPmeasurement and visual acuity test were performed on 4 clinicalvisits while ophthalmoscopy was performed on Days 1 and 29. Allpatients received a safety examination at Days 8, 15 and 30 postdosing.Results: The incidence of adverse events for all DuraSite containingarms, ISV-303 BID, ISV-303 QD and DuraSite Vehicle BID wascomparable to the active control of commercial Bromfenac 0.09%BID. No major differences were observed in BCVA, IOP, orophthalmoscopy evaluations.Conclusions: DuraSite-containing ophthalmic formulations are welltolerated for use in cataract surgery patients as shown in a wellcontrolledmulticenter clinical trial.Commercial Relationships: Judith Hutcheson, InSite Vision (E);Lyle M. Bowman, InSite Vision (E); Kamran Hosseini, InSiteVision Inc. (E)Clinical Trial: NCT01190878Program Number: 1094 Poster Board Number: C0071Presentation Time: 1:00 PM - 2:45 PMNeuroprotective effect on the retinal ganglion cells bybrimonidine loaded HSA nanoparticles in the acute optic nervecrush modelHyuncheol Kim 1 , Hyungwon Moon 1 , Kyoung Nam Kim 2 , Yu JeongKim 2 , Jin Wook Jeoung 2 , Ki Ho Park 2 . 1 Department of Chemical andBiomolecular Engineering, Sogang University, Seoul, Republic ofKorea; 2 Department of Ophthalmology, Seoul National UniversityCollege of Medicine, Seoul, Republic of Korea.Purpose: To examine the neuroprotective effect of α2-agonistbrimonidine loaded human serum albumin (HSA) nanoparticles onthe survival of retinal ganglion cells after acute optic nerve crush.Methods: Brimonidine loaded HSA nanoparticles were fabricated bydesolvation technique. The size distribution and zeta potential wereevaluated by dynamic light scattering method. The loaded amountand in vitro release rate of brimonidine from the HSA nanoparticleswere determined with the HPLC. Acute optic nerve crush wasinduced by clipping the optic nerve for 60s. After optic nerve injury,brimonidine loaded HSA nanoparticles, free HSA nanoparticles, andbalance salt solution (BSS) control were adiministered intravitreally.Retinal ganglion cell loss was evalulated by cell count in a retinal flatmount, which was visualized by the retrograde transport of rhodamindextran3000. Retinal ganglion cell loss was determined 5 days postadministration.Results: The brimonidine loaded HSA nanoparticles showed narrowsize distribution and negatively charged surface to be 152.78 +/- 51.1nm and -29.7 +/- 7.52 mV, respectively. The concentration ofbrimonidine in HSA nanoparticles was 214.12 μg/mL andconsistently released for over 5 days. In the neuroprotection analysis,compared to the uncrush retinal ganglion cells (control group), theBSS treated group described the retinal ganglion cell loss of 66.81 +/-4.34 % (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologysmooth surfaces and absence of pores for MS-A but with smallsurface pores in MS-B. The inclusion of vit-E in MS-B increased theprotein entrapment from 23.4±0.5% to 30.4±1.1%. A three-phasicsustained GDNF in-vitro release was obtained for both formulationsfor 133 days. In phase-I 37 and 33 pgGDNF/mgMS/day wereobtained for MS-A and MS-B respectively. In phase-II 144pgGDNF/mgMS/day for MS-A and 187 pgGDNF/mgMS/day forMS-B were denoted. Finally, 8 (MS-A) and 14 (MS-B)pgGDNF/mgMS/day were observed. Less retinal cell death (p250ng/mL through 7 days of treatment.Results: The average moxifloxacin levels in the tear film by LC-MS/MS ranged from 2,465 to 3,236ng/mL through day 7 as shown inFigure 1. Tear fluid concentrations were below the limit-ofquantification(

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Michael Bassett, Ocular Therapeutix(E); Charles D. Blizzard, Ocular Therapeutix, Inc. (E); Peter K.Jarrett, Ocular Therapeutix (E); Arthur Driscoll, OcularTherapeutix (E); Ankita Desai, Ocular Therapeutix (E); DeepaMulani, Ocular Therapeutix (E); Michael McGrath, OcularTherapeutix, Inc. (E); Amar S. Sawhney, Ocular Therapeutix (E)Program Number: 1098 Poster Board Number: C0075Presentation Time: 1:00 PM - 2:45 PMDistribution And Clearance Of Microparticles AndNanoparticles In The Suprachoroidal Space After InjectionUsing Hollow Microneedles In RabbitsYoo C. Kim 1 , Mark Prausnitz 1 , Henry F. Edelhauser 2 . 1 ChemicalEngineering, Georgia Tech, Atlanta, GA; 2 Ophthalmology, EmoryUniversity Eye Center, Atlanta, GA.Purpose: The suprachoroidal space (SCS) is a virtual space betweenthe sclera and choroid tissues and has been shown to accommodate avariety of fluids and particles in rabbit and porcine eyes. Drugsdelivered in this potential space can be used to treat posteriorsegmentdiseases such as age-related macular degeneration. Thisstudy examines the distribution and clearance of microparticles andnanoparticles after administration within the SCS using a hollowmicroneedle up to 112 days following injection into eyes of NewZealand White rabbit eyes.Methods: Hollow metal microneedles 750-800 µm in length wereused to inject 50 µL of non-biodegradable, fluorescently tagged,polystyrene particles with various sizes (20 nm, 200 nm, 2 µm, 10µm) in balanced salt solution. A hollow microneedle was inserted 3mm posterior to the limbus in New Zealand White rabbit eyes.Rabbits were sacrificed 14 or 112 days after injection. The eyes weresnap frozen, dissected and imaged to visualize the spread of theparticles within the SCS followed by a homogenization process toquantify the number of particles inside the SCS by fluorescence.Results: Fourteen days after injection, the 20 nm, 200 nm, 2 µm and10 µm sized particles covered an suprachoroidal surface area of201±44 mm2, 191±25 mm2, 169±19 mm2 and 185±10 mm2,respectively. After 112 days, these particles covered an area of131±4.7 mm2, 135±1.3 mm2, 127±24 mm2 and 170±11 mm2,respectively. These particles showed a 35%, 30%, 25%, and 9%reduction in coverage area, respectively between days 14 and 112,which was statistically significant (p < 0.001). In addition, thenumber of particles decreased between days 14 and 112, as shown bya 48%, 69%, 45% and 28% reduction in fluorescent signal intensityof particles, which was statistically significant (p < 0.001).Conclusions: A single hollow microneedle was able to reliably inject50 µL of particle formulations that have a particle size up to 10 µmwithin the SCS of rabbit eyes. The particles spread over asuprachoroidal area of up to 200 mm2, which decreased by 9 - 35%between days 14 and 112. In addition, 28% - 69% of the particlesappeared to be cleared from the SCS between days 14 and 112. Weare currently determining whether this apparent clearance is due toremoval by macrophages or simply a reduction of the fluorescencesignal intensity of the particles over time.Commercial Relationships: Yoo C. Kim, None; Mark Prausnitz,Clearside Biomedical (I), Clearside Biomedical (P), ClearsideBiomedical (S); Henry F. Edelhauser, Clearside Biomedical (P),Clearside Biomedical (I), Clearside Biomedical (C)Support: NIH Grant R24 EY017404Program Number: 1099 Poster Board Number: C0076Presentation Time: 1:00 PM - 2:45 PMExamination of in vitro release profiles of bromfenac, diclofenacand nepafenac as drug candidates for sustained release NSAIDpunctum plugPeter K. Jarrett, Rami Elhayek, Sarah Guedez, Amar S. Sawhney.Ocular Therapeutix, Bedford, MA.Purpose: To examine the in vitro release profiles of threenonsteroidal anti-inflammatory drugs (NSAIDs): Bromfenac (BFc),Diclofenac (DFc) and Nepafenac (NFc) from polyethylene glycol(PEG) hydrogel punctum plugs.Methods: BFc, DFc and NFc were each suspended with equalloading in a multi-arm PEG solution and injected into small diametersilicone tubing prior to cross-linking. The hydrogel NSAID matrixconfined within the silicone tubing was cut to 5mm plugs. TheNSAID release profile was determined in PBS pH7.4 at 37°Csimulating the release from a punctum plug (PP) inserted in thecanaliculus. The release media was sampled and exchanged daily.Plugs were removed for photographic imaging to track the release ofthe drug qualitatively indicated by drug clearance from both ends ofthe plug. Percent drug release was determined using UV/Visspectrophotometry.Results: As shown in Figure 1, each NSAID was released from theplug at a rate relative to their aqueous solubility (BFc, DFc and NFchaving a water solubility of 53, 0.6 and 0.006 mg/mL respectively).The most soluble BFc was released in 7 days, DFc was released in 14days, and the least soluble NFc’s projected release was calculated atapproximately 120 days. Images of a DFc-loaded hydrogel plugshowing drug clearance over time are included within Figure 2.Conclusions: BFc, DFc and NFc each showed variable rates ofsustained release from a PEG hydrogel plug corresponding to theirwater solubility. Solubility modifiers and/or modifications to the PEGhydrogel matrix can be employed to tailor the release profile of BFc,DFc and NFc, depending on the drug load and duration of therapyrequired. Topical NSAIDs, such as BFc, DFc and NFc are used totreat post-surgical inflammation as well as various ophthalmicconditions, and typically require multiple daily dose administrationsover extended periods of time. A single dose NSAID punctum plugmay provide more consistent dosing while eliminating issues ofpatient non-compliance.©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 – Physiology/PharmacologyCommercial Relationships: Thomas Yorio, NoneProgram Number: 1277Presentation Time: 9:35 AM - 9:55 AMOpioid and Natriuretic Peptide Axis: Nature’s OcularNeuroprotectantsShahid Husain. Ophthalmology, Medical Univ of South Carolina,Charleston, SC.Commercial Relationships: Shahid Husain, NoneProgram Number: 1278Presentation Time: 9:55 AM - 10:15 AMBradykinin: A Peptide for All SeasonsNaj Sharif. Pharma Regulatory Affairs, Alcon Research Ltd, FortWorth, TX.Commercial Relationships: Naj Sharif, Alcon Research, Ltd (aNovartis Co.) (E)Commercial Relationships: Peter K. Jarrett, Ocular Therapeutix(E); Rami Elhayek, OCULAR THERAPEUTIX INC (E); SarahGuedez, Ocular Therapeutix (E); Amar S. Sawhney, OcularTherapeutix (E)216 Peptides and Polypeptides in Ocular Health and Dysfunction- MinisymposiumMonday, May 06, 2013 8:30 AM-10:15 AM618-620 MinisymposiumProgram #/Board # Range: 1274-1278Organizing Section: Physiology/PharmacologyContributing Section(s): Cornea, Glaucoma, Lens, Retinal CellBiologyProgram Number: 1274Presentation Time: 8:35 AM - 8:55 AMPeptides in the Eye: An OverviewMiguel Coca-Prados. Ophthalmology & Visual Sci, Yale UnivSchool of Medicine, New Haven, CT.Commercial Relationships: Miguel Coca-Prados, NoneProgram Number: 1275Presentation Time: 8:55 AM - 9:15 AMVEGF and PDGF: Roles in Physiology and PathologyPatricia A. D'Amore. Ophthalmology, Schepens Eye Res Inst, MassEye & Ear, Boston, MA.Commercial Relationships: Patricia A. D'Amore, Valeant (C)Program Number: 1276Presentation Time: 9:15 AM - 9:35 AMEndothelin: Friend or Foe?Thomas Yorio. North Texas Eye Research Institute andPharmacology & Neuroscience, Univ of North Texas Hlth Sci Ctr,Fort Worth, TX.255 Retina/RPE: New Drugs, Mechanisms of Action, andToxicityMonday, May 06, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 1937-1968/C0151-C0182Organizing Section: Physiology/PharmacologyContributing Section(s): Biochemistry/Molecular BiologyProgram Number: 1937 Poster Board Number: C0151Presentation Time: 11:00 AM - 12:45 PMOptimization of Cone-Directed AAV-Mediated GeneAugmentation Therapy for CNGB3-Achromatopsia by Use of theIRBP/GNAT2-Promoter and Intravitreal CNTF AdministrationConnie Y. Yeh 1, 2 , Simone Iwabe 1 , Sanford L. Boye 3 , KendraMcDaid 1 , Christine Harman 2 , Rong Wen 4 , William W. Hauswirth 3 ,Andras M. Komaromy 1, 2 , Gustavo D. Aguirre 1 . 1 School of VeterinaryMedicine, University of Pennsylvania, Philadelphia, PA; 2 College ofVeterinary Medicine, Michigan State University, East Lansing, MI;3 College of Medicine, University of Florida, Gainesville, FL;4 Bascom Palmer Eye Institute, University of Miami, Miami, FL.Purpose: AAV5-mediated cone-directed gene therapy results inrescue of cone function and day vision in canine models of CNGB3-achromatopsia. However, the treatment success rate is

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyRescue of cone function and day vision was achieved with both AAVserotypes; best results were obtained by use of ~10 12 vectorgenomes/mL (2/6 and 4/6 injected eyes responding with AAV5 andAAV8 respectively). A therapeutic effect was not obtained in allAAV-treated eyes. However, all 3 eyes treated with intravitrealCNTF 6 months following unsuccessful gene augmentation therapyshowed robust rescue of cone function; this was not the case withintravitreal PBS injection.Conclusions: Gene expression can be targeted to both canine conesubclasses and their function restored in CNGB3-achromatopsia withthe IRBP/GNAT2 promoter in either AAV5 or AAV8. It remainsunclear why some animals do not respond to AAV-mediated genereplacement therapy alone; however, treatment outcome can beenhanced by combination with intravitreal CNTF injection in eyesthat do not respond.Commercial Relationships: Connie Y. Yeh, None; Simone Iwabe,None; Sanford L. Boye, PCT/US2012/062478 (P); KendraMcDaid, None; Christine Harman, None; Rong Wen, NeurotechUSA (C); William W. Hauswirth, AGTC (I), Bionic Sight (I),AGTC (C), Syncona (C), RetroSense (C); Andras M. Komaromy,None; Gustavo D. Aguirre, NoneSupport: NIH (EY019304, EY017549, EY006855, EY018586,P30EY001583, P30EY008571, P30EY14801, RR007063), DoD(W81XWH-09-1-0674), FFB, MVRF, RPBProgram Number: 1938 Poster Board Number: C0152Presentation Time: 11:00 AM - 12:45 PMConversion to aflibercept for chronic refractory or recurrentneovascular age-related macular degenerationYoshihiro Yonekawa 1 , John B. Miller 1 , John I. Loewenstein 1 , LuciaSobrin 1 , Dean Eliott 1 , Demetrios Vavvas 1 , Joan W. Miller 1 ,Christopher M. Andreoli 1, 2 , Ivana K. Kim 1 . 1 Massachusetts Eye andEar Infirmary, Boston, MA; 2 Harvard Vanguard Medical Associates,Boston, MA.Purpose: To examine the outcomes of patients with refractory orrecurrent neovascular age-related macular degeneration who wereconverted from bevacizumab and/or ranibizumab to afliberceptintravitreal injections.Methods: This was a two-center, retrospective, interventional, noncomparativeseries. Treatment histories, visual acuity (VA), andcentral macular thickness (CMT) on spectral-domain OCT werecollected. Patients were divided into “refractory” (those withpersistent exudation despite monthly injections) or “recurrent” (thosewho required repeated injections to maintain a dry macula).Results: 102 eyes of 94 patients were included in the study. 68 wererefractory, and 34 were recurrent. A mean of 20.4 priorbevacizumab/ranibizumab injections and a mean of 3.8 afliberceptinjections were administered. Mean follow-up was 18 weeks. MeanVAs were: 20/50-1 before conversion, 20/50-2 after 1 afliberceptinjection (P = .723), and 20/50+2 after the final injection (P = .253).Subgroup analysis of refractory and recurrent cases also showedstable VA. Of the refractory cases, mean CMT improved after 1injection (P < .001) and the final injection (P < .001). Intraretinal (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyComparison of the toxicity of different drug deliverynanoparticles in RPE and photoreceptor cellsYueran Yan, Haijiang Lin, Hidetaka Matsumoto, Peggy Bouzika,Joan W. Miller, Demetrios Vavvas. Massachusetts Eye and EarInfirmary, Harvard Medical School, Boston, MA.Purpose: Multiple nanoparticles made of synthetic materials havebeen widely used as drug delivery systems. However, their toxicity tothe retina is still not clear. In this study, three different kinds ofnanoparticles made of poly lactic-co-glycolic acid (PLGA),polycaprolactone (PCL) and PEGylated polymer (PEG-P) werestudied and their toxic effects in RPE cells and photoreceptors weredetermined in vitro and in vivo.Methods: PLGA, PCL, and PEG-P nanoparticles were prepared byan oil-in-water (O/W) emulsion/solvent evaporation method.ARPE19 cells were treated with different concentrations of each ofthe three nanoparticles. The toxicity observed in each case wasdetermined at different time points by MTT (Sigma Aldrich), LDH(Promega), ATP (Promega) and TUNEL (Millipore) assays. For thein vivo study, PEG-P nanoparticles were injected into C57BL/6mouse eyes intravitreally and sub-retinally. Eyes were enucleated atday 3 or 7. TUNEL staining was used to evaluate photoreceptor celldeath. Photoreceptor cell loss was evaluated by measuring thethickness of outer nuclear layer (ONL). Microglias in ONL werequantified in retinal whole mount immunolabeled with Iba-1antibody. RPE degeneration was also assessed in RPE whole mountimmunolabeled with ZO-1 antibody.Results: Our experiments showed that there was almost no toxicityof PEG-P nanoparticles in ARPE19 cells. Most importantly, notoxicity was observed when exposing the cells to a high PEG-Pconcentration (200ug/ml) for up to 1 week. PLGA nanoparticlesshowed greater toxicity than their counterparts. At 24 hours, PLGAcaused more than 20% cell death at a concentration of 25ug/ml and50% cell death at 200ug/ml. These results were confirmed withdifferent cell death assays. The toxic effect of these nanoparticles inthe RPE cells and the photoreceptors was also determined in vivo.Conclusions: PLGA, PCL, and PEG-P nanoparticles show differenttoxicity profiles. PEG-P has the least toxicity in the RPE cells andphotoreceptors.Commercial Relationships: Yueran Yan, None; Haijiang Lin,None; Hidetaka Matsumoto, None; Peggy Bouzika, None; JoanW. Miller, Massachusetts Eye and Ear Infirmary (P), Novartis (I),Alcon (C), KalVista Pharmaceuticals (C); Demetrios Vavvas, MEEI(P), Kala pharmaceuticals (C), Roche (C), Genentech (C)Program Number: 1941 Poster Board Number: C0155Presentation Time: 11:00 AM - 12:45 PMA Wnt Inhibitor for Treatment of Diabetic RetinopathyDanyang Chen, Erica Little. Charlesson, LLC, Oklahoma City, OK.Purpose: Diabetic retinopathy is characterized by multiplepathological processes which are associated with numerousmolecular pathways, such as vascular endothelial growth factor, Wnt,inflammation and other pathways. Accumulated evidence hasdemonstrated that the Wnt pathway is responsible for diabeticretinopathy. Thus, the drugs targeting Wnt pathway would beeffective in the treatment of diabetic retinopathy.Methods: The effects of a Wnt inhibitor (CLT-010) on the Wntpathway and retinal vascular endothelial cell growth weredetermined. Dual-luciferase reporter assay and Western blotting wereperformed to evaluate the effects of CLT-010 on the Wnt signalingactivation and Wnt-responsive gene expression, respectively. MTTassay was used to examine the cell viability.Results: CLT-010 attenuated the Wnt pathway over-activation byaffecting the phosphorylation of low-density lipoprotein receptorrelatedprotein 6 and β-catenin. The compound also down-regulatedWnt3a-induced over-expression of Wnt-responsive gene andinhibited the growth of retinal vascular endothelial cells.Conclusions: CLT-010 had inhibitory effects on the over-activationof Wnt signaling, over-expression of Wnt-responsive gene andgrowth of retinal vascular endothelial cells.Commercial Relationships: Danyang Chen, Charlesson, LLC (E);Erica Little, Charlesson LLC (E)Support: NIH Grant EY019417 and NIH Grant EY021683Program Number: 1942 Poster Board Number: C0156Presentation Time: 11:00 AM - 12:45 PMImpact of Ocriplasmin Therapy in Symptomatic VitreomacularAdhesion (VMA) Patients Considered to be Clinical Candidatesfor VitrectomyBaruch D. Kuppermann. Gavin Herbert Eye Inst Dept Ophthal,University of California Irvine, Irvine, CA.Purpose: To evaluate efficacy and safety of ocriplasmin versusplacebo for pharmacologic VMA resolution in the subset of eyesconsidered to be clinical candidates for vitrectomy.Methods: The ocriplasmin phase 3 program included symptomaticpatients with OCT-confirmed VMA who were randomized to receivea single intravitreal injection of 125 µg ocriplasmin (n=464) orplacebo (n=188). Clinical criteria for consideration for vitrectomy inthis subset analysis was visual acuity (VA) of 65 ETDRS letters(20/50) or less (n=301) or full-thickness macular hole (FTMH,equivalent to stage II) (n=153) at baseline. A total of 127 patients metboth criteria. In patients without baseline FTMH we evaluated therate of VMA resolution at 28 days post-injection. In patients withbaseline FTMH, VMA resolution and FTMH closure at day 28 wereevaluated.Results: Pharmacologic VMA resolution at day 28 was observed in asignificantly larger proportion of eyes in the ocriplasmin groupcompared to placebo. Among patients with a baseline VA of 65letters or less, 33.2% of eyes treated with ocriplasmin achieved VMAresolution compared with 11.5% in the placebo group (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyProgram Number: 1943 Poster Board Number: C0157Presentation Time: 11:00 AM - 12:45 PMSCHISANDRIN B IMPROVES THE VISUAL MOTORRESPONSE AND PRESERVES PHOTORECEPTORS IN THEZEBRAFISH PDE6C CONE DYSTROPHY MUTANTYuk Fai Leung 1, 3 , Liyun Zhang 1 , Leelyn Chong 1 , Jin Cho 1 , Kam MingKo 2 . 1 Biological Sciences, Purdue University, West Lafayette, IN;2 Division of Life Science, The Hong Kong University of Science &Technology, Hong Kong, Hong Kong; 3 Biochemistry and MolecularBiology, Indiana University School of Medicine Lafayette, WestLafayette, IN.Purpose: Schisandrin B (Sch B), an active component of FructusSchisandrae, has been indicated in traditional Chinese medicine(TCM) to offer eye benefits. This study investigated the extent towhich Sch B could improve retinal degeneration in the zebrafishpde6c cone dystrophy mutant.Methods: A titration series of Sch B was first conducted to identify atreatment dosage that would activate G6PD, a known marker of SchB treatment, without affecting the gross morphology. Then, pde6cand wild-type (WT) siblings were treated with the optimized 1.875µM Sch B or equal amount of DMSO carrier from 3 days postfertilization(dpf) to 6 dpf. The treatment scheme was chosen becauseit was theorized that most TCMs play a protective role in diseasesand thus, the best chance to determine the protective effect of thetreatment would be before any observable photoreceptordegeneration at 4 dpf. The visual performance of the resulting larvaewas analyzed by optokinetic response (OKR) and visual-motorresponse (VMR), a light-induced swimming activity (Zhang et al.,Asia-Pac J Ophthalmol 2012; 1:374-383; Emran et al., J Vis Exp.2008;3(20)). The VMR in this study was optimized to detect lightinducedresponse originated from the eyes, as the VMR was mostlyabolished in the enucleated larvae. The photoreceptors in theseembryos were also analyzed by immunohistochemistry and qPCRResults: The pde6c larvae elicited a substantially reduced VMR;thus, their activity profile was very different from the WT siblings.Treating pde6c with 1.875 µM Sch B increased their VMR inresponse to the light-ON but not the light-OFF stimulus and theOKR. Interestingly, the histological analyses indicated that the rodswere preserved in the pde6c mutants and no noticeable difference inthe cones. These observations are also supported by a substantialincrease in the expression of gnat1 (rod marker) but not gnat2 (conemarker) in the qPCR results.Conclusions: Sch B might have improved the VMR of pde6cmutants by the preserving their rods. Since one of the best knownfunctions of Sch B is its antioxidant property, oxidative stress isimplied to play a role in cell death of the rods in the pde6c mutants.In addition, this study has established the utility of using VMR inzebrafish for identifying drugs that may have eye benefits.Commercial Relationships: Yuk Fai Leung, None; Liyun Zhang,None; Leelyn Chong, None; Jin Cho, None; Kam Ming Ko, NoneProgram Number: 1944 Poster Board Number: C0158Presentation Time: 11:00 AM - 12:45 PMPhase I, Randomized, Double-Masked, Vehicle-Controlled Studyto Evaluate Safety, Tolerability and Pharmacokinetics ofRecombinant Human Nerve Growth Factor Eye Drops inHealthy VolunteersRonald R. Buggage, Pier A. Ruffini, Mauro P. Ferrari. DompeS.p.A., Milan, Italy.Purpose: Nerve growth factor, an endogenous protein involved in thedifferentiation and maintenance of neurons, is a potential treatmentfor ocular neurodegenerative conditions. A clinical trial evaluatingthe safety of a topical ophthalmic formulation of recombinant humannerve growth factor (rhNGF) in normal subjects was conducted.Methods: Healthy adult volunteers with no notable ocular/medicalhistory and having normal ophthalmic and general examinations atscreening and baseline were enrolled and assigned to 1 of 3 studyphases. In Part 0, 1 eye received a single administration of rhNGF0.5, 5 or 20 µg/ml. In Part A, 1 drop of rhNGF 20, 60 or 180 µg/ml orvehicle was administered to 1 eye every 4 hours for 1 day while inPart B, the same dose regimens or vehicle was administered to 1 eyedaily for 5 days. In all phases treatment was randomized and thefellow eye received a matching vehicle regimen. Safety evaluationsincluded a complete ophthalmic exam, ECG, vital signs, urinalysisand routine labs. Plasma samples for pharmacokinetics andimmnogenicity were obtained in parts A and B.Results: Parts 0, A and Part B first cohort have been completed with45/73 subjects treated with rhNGF. Masked review of all availablesafety information reveals good tolerability to the study medicationwith no clinically significant ocular or systemic changes observed.Ocular adverse events (AEs), generally transient and mild, includedwarm feeling, pressure, hazy film, increased corneal fluoresceinstaining, blurred vision, upper eyelid burning, headache above theeyes, photophobia, itching and loss of color distinction. In 3/8subjects the AEs affected both eyes. 8 subjects reported non-ocularAEs not related to the study treatment. No serious adverse eventswere recorded.Conclusions: NGF is a promising therapy for neurotrophic keratitis,retinitis pigmentosa and optic neuropathies, ocular neurodegenerativediseases for which no effective treatments currently exist. Earlyfindings from a first in human trial evaluating a topical ophthalmicformulation of rhNGF suggest no local or systemic safety concerns.Unmasked data including pharmacokinetic analysis andimmunogenicity will be presented to complete the safety profile ofrhNGF in healthy volunteers.Commercial Relationships: Ronald R. Buggage, Dompe S.p.A.(E); Pier A. Ruffini, Dompé S.p.A. (E); Mauro P. Ferrari, Dompés.p.a. (E)Clinical Trial: NCT01744704Program Number: 1945 Poster Board Number: C0159Presentation Time: 11:00 AM - 12:45 PMAntiglycating potential of procyanidin-B2 isolated fromcinnamon bark: Prevention or treatment of diabetic ocularcomplications (cataract & retinopathy)G Bhanuprakash Reddy, Puppala Muthenna, ChandrasekharAkileshwari, Ganugula Raghu, Palla Suryanarayana. Biochemistry,National Institute of Nutrition, Hyderabad, India.Purpose: Accumulation of advanced glycation endproducts (AGE)due to non-enzymatic glycation of proteins has been implicated inseveral pathophysiologies associated with diabetic ocularcomplications. Earlier we have identified a few dietary sources suchas cinnamon that have the potential to inhibit AGE formation. In thisstudy, we have isolated and identified procyanidin-B2 as theantiglycating agent from cinnamon bark (Cinnamomum zeylanicum)and investigated its potential to prevent diabetic cataract andretinopathy in rat model.Methods: Using bioassay-guided fractionation, procyanidin-B2 wasidentified as an antiglycating agent from cinnamon and demonstratedits antiglycating potential and mechanism of action of using in vitroand ex vivo protein glycation systems. Further the effect ofprocyanidin-B2 and its dietary source (cinnamon) to prevent diabeticcataract and retinopathy was investigated using streptozotocininduceddiabetic rat model.Results: Under in vitro conditions, procyanidin-B2 inhibited 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 – Physiology/Pharmacologyprotein glycation as assessed by AGE-fluorescence, SDS-PAGE andimmunodetection of specific AGE. Further, we provided insight intothe mechanism of inhibition of protein glycation that it scavengesfree radicals directly and trapping of dicarbonyls. In addition,procyanidin-B2 inhibited the glycosylated hemoglobin formation inhuman RBC under ex vivo conditions. We also demonstrated thatfeeding of proycanidin-B2 to diabetic rats in the diet was effectiveagainst development of cataract and retinopathy in streptozotocinmainly through its antiglycating potential. Procyanidin-B2 decreasedlevels of glial fibrillary acidic protein expression and vascularendothelial growth factor and enhanced nerve growth factorexpression in diabetic retina.Conclusions: Thus, the active principle procyanidin-B2 isolated fromdietary cinnamon might be useful for the treatment and/ or preventionof diabetic ocular complications.Commercial Relationships: G Bhanuprakash Reddy, IndianCouncil of Medical Research (P); Puppala Muthenna, None;Chandrasekhar Akileshwari, None; Ganugula Raghu, None;Palla Suryanarayana, NoneSupport: Department of Biotechnology & Life Sciences ResearchBoard, IndiaProgram Number: 1946 Poster Board Number: C0160Presentation Time: 11:00 AM - 12:45 PMEffects of resveratrol, epigallocatechine gallate (EGCG) andcurcumin on the proliferation of human retinal endothelial cellsin vitroAnne F. Alex 1 , Manfred Spitznas 2 , Christian Kurts 3 , Nicole Eter 1 .1 Department of Ophthalmology, University of Muenster MedicalCenter, Muenster, Germany; 2 Department of Ophthalmology,University of Bonn Medical Center, Bonn, Germany; 3 Institutes ofMolecular Medicine and Experimental Immunology, University ofBonn Medical Center, Bonn, Germany.Purpose: Choroidal neovascularization (CNV) is a commonlyoccuring pathology in various eye diseases, e.g. in an advanced stageof age-related macular degeneration (AMD). Vascular endothelialgrowth factor (VEGF) is known to be upregulated and leads toenhanced endothelial cell growth. The polyphenols resveratrol,epigallocatechine gallate (EGCG) and curcumin haveantiproliferative and antiinflammatory effects. In this study, theeffects of these polyphenols on human retinal endothelial cells(hREC) were investigated in a cell culture model.Methods: hREC were cultured in 2% serum-containing cell culturemedium and different concentrations of resveratrol, EGCG andcurcumin were tested. Flow-cytometric analysis was performed after24, 48 and 72 hours of incubation. Absolute cell numbers werecounted, cell proliferation was measured with the Carboxyfluoresceinsuccinimidyl ester (CFSE) dilution assay and dead cells wereanalysed with the nuclear dye Hoechst 33258. Apoptotic cells couldbe distinguished with active caspase 3 staining.Results: All three polyphenols diminished absolute cell numbers andthe number of cell cycles in a dose-dependent manner compared tothe untreated control. 25 µM Resveratrol led to a significantreduction in cell cycle numbers over 48 hours of nearly 40%. EGCGalso had a strong effect on the proliferation and 25 µM reduced theaccomplished cell cycles to 0,65 (control: 0,78), with 50 µM theeffect was even stronger. Curcumin showed effects already with 5µM and these effects were enhanced with 10 µM. Only EGCG with aconcentration of 25 µM and higher slightly induced cell death,resveratrol even significantly reduced apoptosis of hREC.Conclusions: All three polyphenols reduced the absolute number ofhREC and had an inhibitory effect on the cell proliferation byreducing the number of cell cycles in a dose-dependent manner. Incomparison to previously published data on retinal pigment epithelialcells (Alex et al., 2010) the concentrations were lower to achievethese effects on hREC. In vivo, a cell specific targeting mighttherefore be achieved by dosing. Further studies are needed toevaluate effects on other retinal cells.Commercial Relationships: Anne F. Alex, None; ManfredSpitznas, None; Christian Kurts, None; Nicole Eter, Novartis (F),Bayer (R), Heidelberg Engineering (R), Sanofi Aventis (C), Allergan(C), Bausch and Lomb (C)Program Number: 1947 Poster Board Number: C0161Presentation Time: 11:00 AM - 12:45 PMCirculating miRNAs as Biomarkers of Retinal ToxicityQinghai Peng, Wenhu Huang, Walter Collette, Michelle Twamley,Shirley Aguirre, Annette John-Baptiste. Pfizer, San Diego, CA.Purpose: To examine whether circulating retinal-enrichedmicroRNAs were altered post treatment in a drug induced retinaltoxicity study in rat.Methods: Wistar Hans rats were administered a single intravitrealinjection of either a pan-CDK inhibitor (10 or 30 µg/eye) or a HSP90inhibitor (9 or 27 µg/eye). EDTA whole blood samples werecollected predose, and on days 1, 3 and 7 post dose. Retinal-enrichedmiRNAs and the liver-enriched miR-192 (used as a control) wereanalyzed by qRT-PCR. Electroretinography (ERG) was performedpredose and at end of study to assess retinal function. Eyes werecollected either on day 8 or at 2 weeks post dose and processed forhistopathologic evaluation.Results: Elevations of plasma miR-124a, miR-96, and miR-183peaked on day 3 post dose in the pan-CDKi high dosed animalsdemonstrating greater than 300X, 10X and 6X fold increasesrespectively in a time-dependent manner . The low dose group of thepan-CDKi treated animals demonstrated slight elevations of miR-124a, miR-96 and miR-183. Neither dose group in the pan-CDKitreated animals demonstrated any change in miR-192 and retinaldegeneration was confirmed by ERG and histopathology.Comparatively, the HSP90 treated group, exhibited no changes inmiRNA levels, ERG or histopathology.Conclusions: This study demonstrated plasma miR-96, miR-124a,and miR-183 are strong contenders as retinal toxicity biomarkers.Although these miRNAs need additional validation whether theytruly predict retinal toxicity or not prior to histopathology, theseresults provide promise for further test using additional retinaltoxicants.Commercial Relationships: Qinghai Peng, None; Wenhu Huang,None; Walter Collette, None; Michelle Twamley, Pfizer, Inc. (E);Shirley Aguirre, None; Annette John-Baptiste, NoneProgram Number: 1948 Poster Board Number: C0162Presentation Time: 11:00 AM - 12:45 PMComparison of intraocular retinal pigment epithelial (RPE) cellinjections in vitrectomized wild type pigsElliott H. Sohn 1, 2 , Chunhua Jiao 2 , Robert F. Mullins 2 , Woojin Jung 2 ,Stephen R. Russell 1, 2 , Edwin M. Stone 1, 2 , Budd A. Tucker 2 . 1 RetinaService, University of Iowa Hospitals & Clinics, Iowa City, IA;2 Institute for Vision Research, University of Iowa, Iowa City, IA.Purpose: Various sources of RPE cells have been proposed for cellreplacement therapy (e.g. in the subretinal space to treat geographicatrophy) and in generating models of proliferative vitreoretinopathy(PVR). In this pilot study we sought to determine the effects ofinduced pluripotent stem cell derived RPE cells (iPS-RPE) deliveredto the subretinal space and allogeneic RPE cells injected into thevitreous cavity of vitrectomized wild-type mini-pigs.Methods: 23 gauge vitrectomy was performed in 12-week-old©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 – Physiology/Pharmacologyyucatan miniature swine (n=10). Three treatment cohorts consisted ofthose given blebs of balanced salt solution alone (controls); blebsfollowed by intravitreal injection of porcine-derived GFP-positiveRPE cells (250,000 cells, RPE group); and subretinal blebs ofporcine-derived, GFP-positive iPS-RPE cells (250,000 cells, iPS-RPE group). Indirect ophthalmoscopy, fundus photography andspectral-domain-OCT were performed at weekly post-op intervals.Post-op vitreous samples were screened for inflammatory cytokinesusing a commercial cytokine array and total proteins were evaluatedon silver-stained SDS-PAGE gels for proteomic analysis.Results: Vitreous membranes were seen in all eyes in the iPS-RPEcell group but no PVR or retinal detachment was observed. Epiretinalmembranes and retinal detachment were seen in a third of eyes in theRPE group. No PVR or vitreous membranes were seen in the salinecontrols.At post-op week 3, vitreous IL-8 levels were elevated in the iPS-RPEgroup compared to the porcine RPE and saline controls (image 1);these levels were higher than those observed in post-op weeks 1 and2. IL-12 levels were greater in post-op week 3 compared to post-opweek 1 of the iPS-RPE group (image 2). TGF-beta levels were verylow or below limits of detection in all eyes.Silver staining revealed a distinct subset of bands in the vitreous ofpigs that received iPS-RPE cells. MALDI-MS of one of these bandsidentified several proteins including transthyretin and beta-2-microglobulin.Conclusions: Subretinal injection of iPS-RPE cells in wild-typemini-pigs may result in an increase in inflammatory response specificto particular cytokines. Exogenous intravitreal RPE cells may resultin a modest rate of PVR. Further research is needed to verify thesefindings.Assessment of the therapeutic value of phloroglucinol inStargardt’s diseasePhilippe Brabet 1 , David Cia 2 , Claire Vigor 3 , Nathalie Jacquemot 2 ,Benoit Lerat 1 , Laurent Guillou 1 , Celine Crauste 3 , Christian P.Hamel 1 , Joseph L. Vercauteren 3 . 1 Institute for Neurosciences ofMontpellier, INSERM U1051, Montpellier, France; 2 Laboratoire deBiophysique Neurosensorielle, UMR Inserm 1107, Clermont-Ferrand, France; 3 Institut des Biomolécules Max Mousseron(IBMM), UMR CNRS 5247, Montpellier, France.Purpose: Autosomal recessive Stargardt's disease is the mostcommon cause of macular dystrophies due to mutations in ABCA4gene. When transporter ABCA4 activity is impaired, all-trans retinal(atRAL) accumulates and triggers formation withphosphatidylethanolamine of A2-PE by carbonyl and oxidative stress.Daily photoreceptor outer segments (POS) phagocytosis by retinalpigment epithelium (RPE) leads to A2E accumulation, causing RPEdeath and progressive photoreceptor loss. Our goal is to design andselect powerful chemicals against carbonyl and oxidative stress (anti-COS).Methods: Phloroglucinol (benzene-1,3,5- triol) was first assessed forits efficacy as anti-COS. ARPE-19 cell line and rat RPE primarycultures were pre-incubated with phloroglucinol before incubationwith oxidative (H2O2 and A2E) and carbonyl (atRAL) stressors. Cellviability was measured using tetrazolium compound (MTT) assay.Alternatively, phloroglucinol was co-incubated with atRAL. Testtubeexperiments were carried out to inhibit the synthesis of A2Efrom atRAL and ethanolamine in the presence of phlorogucinol.Adducts formed between phloroglucinol and atRAL were identifiedby NMR and mass analysis.Results: Treatment of RPE cells with phloroglucinol alone does notaffect the cell viability. Exposure of RPE cells to stressors causeddose-dependent decreases in cell viability, whereas pretreatment withphloroglucinol significantly reduced the decline. Co-treatment ofRPE cells with phloroglucinol and atRAL drastically prevented RPEcell death. In the presence of atRAL and ethanolamine,phloroglucinol led to complete inhibition of A2E synthesis.Conclusions: Phloroglucinol has a dual action as anti-carbonyl stressand anti- oxidant in RPE cells. The proposed mechanism for the anticarbonylstress action is the trapping of all-trans retinal. Lowerprotection observed during pre-incubation compared to co-incubationsuggests a low bioavailability of phoroglucinol.Commercial Relationships: Philippe Brabet, None; David Cia,None; Claire Vigor, None; Nathalie Jacquemot, None; BenoitLerat, None; Laurent Guillou, None; Celine Crauste, None;Christian P. Hamel, None; Joseph L. Vercauteren, NoneSupport: This work was supported by private foundations (RetinaFrance, IRRP, UNADEV), University of Montpellier (UM1 andUM2), and Inserm.Commercial Relationships: Elliott H. Sohn, None; Chunhua Jiao,None; Robert F. Mullins, Alcon Research Ltd (F); Woojin Jung,None; Stephen R. Russell, IDx, LLC (I), IDx, LLC (P); Edwin M.Stone, None; Budd A. Tucker, NoneProgram Number: 1949 Poster Board Number: C0163Program Number: 1950 Poster Board Number: C0164Presentation Time: 11:00 AM - 12:45 PMRecurrence of macular edema after intravitreal bevacizumabinjection in eyes with macular edema secondary to retinal veinocclusionyoung gyun Kim 1 , Ji young Moon 1 , Kyung Hoon Seo 2 , Seung yongLee 3 , Eung Suk Kim 3 , Seung young Yu 2 , Hyung woo Kwak 2 . 1 Euljimedical center, Seoul, Republic of Korea; 2 Kyung Hee hospital,Seoul, Republic of Korea; 3 Eulji University Hospital, Daejeon,Republic of Korea.Purpose: The purpose of this study is to find out the incidence ofrecurred macular edema after the intravitreal bevacizumab injectionand related factors which affects the recurrence of macular edemaduring the 6 months’ follow-up.Presentation 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 – Physiology/PharmacologyMethods: Intravitreal bevacizumab injection was performed in 73eyes of 73 patients who had macular edema with RVO. Bestcorrected visual acuity (BCVA) and central macular thickness (CMT)were checked for 6 months after the injection. Recurrence wasdefined as a 30% increase in CMT after an initial decrease of CMT.Factors that differed between the recurred group and the non-recurredgroup were identified.Results: Recurrence occurred in 38 of 73 eyes (49.4%) and the meantime between injection and recurrence was 15.2 weeks. The meaninterval between the onset and the initiation of intravitrealbevacizumab injection was 3.42 weeks in the recurred group, whichshowed a significant difference compared to the non-recurredgroup(P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyPresentation Time: 11:00 AM - 12:45 PMNovel GFAP Species in Retinal GliosisJohn Wizeman, Paola Bargagna-Mohan, Sean O'Rourke, RoyceMohan. Neuroscience, University of Connecticut Health Center,Farmington, CT.Purpose: Retinal gliosis occurs after injury and is an insidiousprocess that also undermines several leading retinal diseases. Acharacteristic hallmark of gliosis is the upregulation of the type IIIintermediate filaments (IF) glial fibrillary acidic protein (GFAP) andvimentin. In injury models, the absence of GFAP and its bindingpartner vimentin has been shown to be protective. We have used theGFAP/vimentin targeting small molecule withaferin A (WFA;Bargagna-Mohan JBC 2010) as a chemical probe of GFAPexpression in an injury model.Methods: C57BL/6N, 129SvEV vimentin-deficient or wild type129SvEV mice were subjected to a corneal alkali injury (1N NaOH)followed by epithelial debridement. Mice were allowed to heal (3 to 5days), after which retinal cups were isolated and placed into explantorgan culture in medium containing DMEM/F12 +10% FBS andantibiotics. Retinal cultures were treated with either WFA or theproteasome inhibitor epoxomicin for 3 days and soluble tissueextracts (200 mM NaCl, 1% NP-40) were subjected to western blotanalysis.Results: As expected, injury increased 52kDa soluble GFAPexpression in the retina. Unexpectedly, there was injury-relatedincrease in novel high molecular weight (100-200 kDa) GFAPspecies in retinas from all mouse strains. This high molecular weightGFAP species was also susceptible to perturbation by WFA, with 50nM WFA decreasing total GFAP levels by ~2.4 fold as compared toan injured, untreated retina. Higher concentrations of WFA had animpact on polyubiquitination levels. At 1 μM WFA, the total amountof ubiquitinated species was decreased ~3.8 fold compared to theinjured, untreated retina. Treatment with epoxomicin causedaccumulation of ubiquitinated GFAP species, a change that was alsomodulated with the addition of WFA.Conclusions: Collectively, our results suggest that injury causes aninduction of soluble GFAP expression in retinal Müller gliaindicative of a novel post-translational modification. Importantly,these high molecular weight, modified GFAP species were targetedby WFA, mediated by a mechanism independent of proteasomeinhibition. The identification of the causative modification to GFAPcould lead to a deeper understanding of the mechanisms of gliosiscaused by either injury or disease. The interaction of GFAP with theubiquitin proteasome pathway remains a critical mechanism thatneeds to be characterized.Commercial Relationships: John Wizeman, None; PaolaBargagna-Mohan, UKY Research Foundation, US8283323B2 (P);Sean O'Rourke, None; Royce Mohan, University of KentuckyResearch Foundation (P)Support: NIH R01 EY016782; John A. and Florence MatternSolomon Endowed ChairProgram Number: 1954 Poster Board Number: C0168Presentation Time: 11:00 AM - 12:45 PMEffect of Intravitreal Injection of Iodoacetic Acid in Mice as aModel of Pharmacological Induced Monolateral PhotoreceptorDegenerationSarah Roesch 1 , Stephan Hesse 1 , Christine Haselier 1 , Babac A.Mazinani 1 , Gernot Roessler 1 , Christiane Pfarrer 2 , Peter Walter 1 .1 Ophthalmic clinic, University hospital RWTH, Aachen, Germany;2 University of Veterinary medicine Hannover, Anatomical Institute,Hannover, Germany.Purpose: To characterize the effect of intravitreal injection ofIodoacetic Acid (IAA) in comparison to its systemic application as ameasure to induce monolateral photoreceptor degeneration.Methods: Seven weeks old C57BL/6J mice received eitherintravitreal injections of IAA (25 animals) in different concentrations(between 0.1-1 mg/kg bodyweight (BW)) or systemic treatment[intraperitoneal (8 animals, 40-70 mg/kg BW) and intravenous (3animals, 60-70 mg/kg BW)] and were observed in the following fiveweeks using ERG, OCT, and histology.Results: Systemic application of IAA showed a mortality of 25% andtoxic systemic effects with weight loss in the first two days of 10%.Intravenous application of IAA resulted in an extinction of the ERGand a thinning of the retina in the OCT. Intraperitoneal injection ofIAA in mice showed no effect in the ERG or OCT for five weeksafter injection.Animals that received intravitreal injections showed no weight lossafter injection.Three to five days after injection the eyes developed cataracts rangingfrom 20% of cases at an IAA concentration of 0.1 mg/kg BW to100% at an IAA concentration of 0.25 mg/kg BW.Higher intravitreal IAA concentrations lead to extinguished ERGsfrom 16% of cases at concentrations of 0.2 mg/kg BW to 90% at 0.25mg/kg BW.Morphological signs of retinal degeneration after intravitrealinjection were not observed, neither in the OCT nor in the histology.Conclusions: Intravitreal injection of IAA leads to dense cataracts atlower concentrations than ERG changes occurred. ERG results mustbe interpreted carefully in the presence of induced cataracts.Morphological signs of retinal degeneration were not seen. Insummary, the intravitreal injection of IAA did not prove to be anadequate model of monocular retinal degeneration in mice.Commercial Relationships: Sarah Roesch, None; Stephan Hesse,None; Christine Haselier, None; Babac A. Mazinani, None;Gernot Roessler, None; Christiane Pfarrer, None; Peter Walter,Novartis (R), Bayer (R), Second Sight (R), Bayer (F), Novartis (F)Support: DFG WA 1472/6-1, DFG PAK 469Program Number: 1955 Poster Board Number: C0169Presentation Time: 11:00 AM - 12:45 PMPHLOROTANNIN-RICH NATURAL EXTRACT FROMBROWN SEAWEED ASCOPHYLLUM NODOSUMPREVENTS IN VITRO HIGH GLUCOSE RETINALDAMAGESMelody Dutot 1 , Roxane Fagon 1 , Patrice Rat 2 .1 Research&Development, YSLAB, Paris, France; 2 Chimie-Toxicologie Analytique et Cellulaire (EA 4463), Faculté dePharmacie, Université Paries Descartes, Paris, France.Purpose: Activators of the class III histone deacetylase SIRT1,which are also potent antioxidant molecules, are considered astherapeutics for the treatment of type 2 diabetes. In this study, weinvestigated the protective cellular effects of phlorotannin-richnatural extract from brown seaweed ascophyllum nodosum againsthigh glucose on retinal pigmented epithelial (RPE) cells and Müllerglial cells.Methods: RPE cells (ARPE-19 human cell line) and Müller cells(MIO-M1 human cell line) were incubated with normal (5mM)glucose or high (25mM) glucose for 48 hours. Reactive oxygenspecies (ROS) production, mitochondrial transmembrane potential,and SIRT1 enzymatic activity were evaluated. Phlorotannin-richnatural extract from brown seaweed ascophyllum nodosum wastested for its ability to inhibit oxidative damages and SIRT1 activityalterations induced by high glucose.Results: Phlorotannin-rich natural extract from brown seaweed©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 – Physiology/Pharmacologyascophyllum nodosum prevented glucose-induced ROSoverproduction and alterations of mitochondrial transmembranepotential. We observed a significant increase in SIRT1 activity withphlorotannin-rich natural extract from brown seaweed ascophyllumnodosum.Conclusions: Phlorotannin-rich natural extract from brown seaweedascophyllum nodosum protected retinal cells against oxidative stress,mitochondrial damages and SIRT1 activity alteration induced by highglucose. Ascophyllum nodosum represents a promising ingredient forfunctional food supplements for diabetic patients.Commercial Relationships: Melody Dutot, YSLAB (E); RoxaneFagon, None; Patrice Rat, NoneSupport: Adebiopharm ER67Program Number: 1956 Poster Board Number: C0170Presentation Time: 11:00 AM - 12:45 PMFenretinide Inhibits Ocular Neovascularization (NV) byUpregulation of Bone Morphogenic Protein-2 (BMP-2) andReduction of Inflammatory Macrophages and VEGFRebecca K. Stevens 1 , Peter A. Campochiaro 1 , Ji-kui Shen 1 , Brian C.Oveson 1 , Sean F. Hackett 1 , Nathan L. Mata 2 . 1 Ophthalmology, JohnsHopkins University, Baltimore, MD; 2 Acucela Inc., Seattle, WA.Purpose: N-(4-hydroxyphenyl) retinamide (4HPR; fenretinide), asynthetic retinoic acid derivative, is a promising chemopreventiveagent for tumors. A recent clinical trial showed that 4HPR reducedthe incidence of choroidal NV in patients with age-related maculardegeneration and geographic atrophy. We sought to investigate theeffects of 4HPR in mouse models of ocular neovascularization (NV).Methods: The effects of intraocular and systemic 4HPR were testedin mice with laser-induced choroidal NV, oxygen-induced ischemicretinopathy, and rho/VEGF transgenic mice. Levels of variousmRNAs were measured by real-time RT-PCR. The effects of 4HPRor all-trans retinoic acid (atRA) on AP1 activity was tested with adual luciferase expression assay using a pGL3-AP1 artificialpromoter construct in the RPE16 cell line.Results: Oral administration of 4HPR suppressed choroidal NV andintraocular injection of 10-7or 10-8M 4HPR significantly suppressedNV in each of the 3 mouse models. Injection of 4HPR also reducedthe influx of macrophages into ischemic retina. Unlike atRA, 4HPRdid not stimulate expression of RARs or RXRs in the retina afterintraocular injection, nor did it reduce expression of AP1-responsivegenes. In cultured RPE cells, atRA, but not 4HPR, suppressed AP1activity. After intraocular injection of 10-7or 10-8M 4HPR in micewith ischemic retinopathy, there was a significant reduction inmRNA for VEGF and CCL2, and an increase in mRNA for BMP2.Intraocular injection of recombinant BMP2 significantly suppressedischemia-induced NV.Conclusions: Systemic or intraocular 4HPR suppresses ocular NV,not through activation of RARs or RXRs and suppression of AP1, butrather by increasing expression of BMP2 and reducing inflammatorymacrophages with concomitant reduction in VEGF.Commercial Relationships: Rebecca K. Stevens, None; Peter A.Campochiaro, Advance Cell Technology (C), Aerpio (C), Elan (C),Gene Signal (C), Genentech (C), GlaxoSmithKline (C), LPath, Inc(C), Norvox (C), Regeneron (C), Genentech (F), Genzyme (F),GlaxoSmithKline (F), Oxford Biomedica (F); Ji-kui Shen, None;Brian C. Oveson, None; Sean F. Hackett, None; Nathan L. Mata,Acucela (P)Support: NIH Grant EY012609Program Number: 1957 Poster Board Number: C0171Presentation Time: 11:00 AM - 12:45 PMBlocking the necroptosis pathway decreases RPE andphotoreceptor damage induced by NaIO3Haijiang Lin, Miin Roh, Hidetaka Matsumoto, Albert H. Alhatem,Peggy Bouzika, Yusuke Murakami, Joan W. Miller, DemetriosVavvas. Ophthalmology-retina, Massachusetts Eye and Ear, HarvardMedical School, Boston, MA.Purpose: Sodium iodate (NaIO3) has been used extensively as aretinotoxin to induce RPE cell damage and degeneration ofphotoreceptors in vitro and in vivo. RIP-Kinase dependentprogrammed necrosis is an important redundant cell death pathwaythat has been shown to be involved in photoreceptor cell death. Wewanted to investigate if these pathways are actively involved in RPEand photoreceptor cell death after NaIO3 insult.Methods: ARPE-19 cells were exposed to different concentration ofNaIO3 in the presence or absence of various concentration of RIPKinhibitors ( Nec-1) or pan-caspase inhibitor (Z-VAD) individually orin combination. Cell death was determined at different time points byMTT (Sigma Aldrich), LDH (Promega), ATP (Promega) and TUNEL(Millipore) assay. C57BL/6 and RIP3-/- mice were treated with aperitoneal injection of NaIO3 and eyes were enucleated at day 3 or 7.TUNEL staining was used to evaluate photoreceptor cell death.Photoreceptor cell loss was evaluated by measuring the thickness ofouter nuclear layer (ONL). Microglias in ONL were quantified inretinal whole mount with Iba-1 antibody. RPE degeneration was alsoassessed in RPE whole mount with ZO-1 antibody.Results: NaIO3 resulted in significant cell death of ARPE-19 cells.Treatment with Nec1 resulted in better protection than treatment withzVAD (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyResults: 19 eyes (13 central [C]RVO eyes,6 branch [B]RVO eyes)of19 patients were included for analysis.At 1 month (19 eyes)meanBCVA,retinal sensitivity,and central macular thickness (CMT)improved from 0.50±0.34 LogMAR, 10.51±4.31 dB, and 762±259μm to 0.38±0.34 LogMAR, 12.28±5.06 dB, and 385±191μm,respectively.At 3 months (19 eyes) improvement of meanBCVA,retinal sensitivity,and CMT decreased (0.54±0.36LogMAR,11.62±5.05 dB, and 518±251 μm);at this time point, 2 eyeswere retreated with intravitreal dexamethasone.At 4 months (17eyes)mean BCVA,retinal sensitivity and CMT were 0.68±0.45LogMAR,8.64±4.45dB, and 671±239 μm respectively;at this timepoint, 4 eyes were retreated.At 5 months (13 eyes) mean BCVA,retinal sensitivity, and CMT were 0.83 ±0.42 LogMAR, 8.1±3.4 dB,and 670±218 μm ;at this time point, 6 eyes were retreated.Interestingly 7 eyes (4 eyes with CRVO, 3 eyes with BRVO)did notunderwent retreatment up to 6 months from first intravitrealdexamethasone;inthese eyes mean BCVA,retinal sensitivity,and CMT changed from0.46±0.29 LogMAR, 12.27±3.52 dB, and 715±219 μm (baseline,versus 0.53±0.37LogMAR, 9.48±4.53dB, and 790±286μm, in eyesthat underwent retreatment

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologysignificant reduction in the accumulation of retinyl esters in treatedmice (Fig. 1C), suggesting the transferred hRPE65 was functional.Conclusions: These results indicate that both NP and uncompactedplasmid VMD2-hRPE65-S/MAR can mediate appreciable geneexpression and improvement in an RPE-associated disease phenotypefor the life of the animal. They further highlight that nanocompactionmay not be essential for effective expression in the RPE, althoughadditional safety studies for uncompacted DNA will be an essentialstep. These outcomes establish proof-of-concept for effective nonviralgene therapy in the RPE, and provides an excellent platform fordelivery of large genes.aflibercept, and bevacizumab showed average dissociationequilibrium constant (K D ) values of 67, 9263, and 4456 pM,respectively. A much slower dissociation rate constant was obtainedfor ranibizumab than for aflibercept or bevacizumab, resulting in ahigher affinity for rhVEGF by ranibizumab. This observation wasconsistent with data from Format 2, although the extremely slowdissociation of ranibizumab from rhVEGF, and challenges in datafitting for aflibercept precluded an accurate determination of kineticsparameters for the binding of these molecules to rhVEGF. UsingFormat 3, aflibercept had a K D of 1.9 pM for rhVEGF. However thisformat was found to be unsuitable for evaluating ranibizumabbinding to rhVEGF because a significant amount of ranibizumabdissociated from the capture antibody during sample analysis.Conclusions: In a direct comparison under uniform conditions,ranibizumab had a higher binding affinity for rhVEGF thanaflibercept. The Biacore assay format profoundly affected the bindingof aflibercept to VEGF and the indirect capture format was notsuitable for evaluating ranibizumab binding to rhVEGF. This studyhighlights the importance of rigorous experimental design and carefulexecution of in vitro binding studies. In addition, it suggested thatbiological implications of the binding affinity/kinetics results need tobe interpreted within broader context including potency,pharmacokinetics and clinical efficacy data.PI-15 months improvement in the rpe65 -/- phenotype. A. Greenphotopic b-waves were significantly improved in naked (VMD2-hRPE65-S/MAR) and NP (NP-VMD2-hRPE65-S/MAR) treatedcompared to saline injected controls. Retinyl esters (B) and thefundus albipunctatus phenotype (C) were reduced compared touninjected animals. * P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyday for the following 5 months. The effect of this therapy wasevaluated in 5 patients (6 eyes). Visual acuity, metamorphopsia, andcentral retinal thickness quantified by ocular coherence tomography(OCT) were recorded at baseline and follow-up visits.Results: After a mean follow up time of 7.8 months we could receiveimprovement of visual acuity in 5 eyes, only one eye disclosed anunchanged visual acuity (mean visual acutiy rose from 0.66 beforetherapy to 0.89 at the end ot the therapeutic phase). The centralretinal sicknes was reduced during treatment and fell from a mean of568 µm to 273 µm. There were no adverse events related to theadministration of aspirin.Conclusions: Although there may have been a spontaneousimprovement without treatment, acetyl salicyl acid showedeffectiveness and has a plausible mechanism in CSCR.Commercial Relationships: Nicole Stuebiger, NoneProgram Number: 1963 Poster Board Number: C0177Presentation Time: 11:00 AM - 12:45 PMCeramide Biosynthesis Inhibition Protects the Retina from Light-Induced DegenerationMd Nawajes A. Mandal 1, 2 , Hui Chen 1, 3 , Julie-Thu Tran 1, 2 , MichaelH. Elliott 1, 2 , Richard S. Brush 1, 2 . 1 Ophthalmology, Univ ofOklahoma Hlth Sci Ctr, Oklahoma City, OK; 2 Dean McGee EyeInstitute, Oklahoma City, OK; 3 Ophthalmology, Sichuan Academy ofMedical Sciences & Sichuan Provincial People’s Hospital, ChengduCity, China.Purpose: Light-induced retinal degeneration (LIRD) in albino ratscauses apoptotic photoreceptor cell death. Ceramide, a sphingolipidmetabolite, is a second messenger for apoptosis. We tested whetherincreases in ceramide mediate photoreceptor apoptosis in lightstressedretinas and if inhibition of ceramide formation can protectthe retina from LIRD.Methods: Sprague Dawley (SD) rats were exposed to 2,700 luxwhite light for 6 h and the retinal levels of ceramide and itsintermediary metabolites were measured by GC-MS or ESI-MS/MS.Enzymes of the de novo biosynthetic and sphingomyelinase pathwaysof ceramide generation were assayed, and gene expression wasmeasured by qRT-PCR. The synthetic drug FTY720, analogous tosphingosine and known to inhibit de novo ceramide biosynthesis, wasadministered intraperitoneally before light damage. The dosage andtemporal effect of FTY720 on the retina in vivo was measured byhistological and functional analyses.Results: Retinal ceramide levels increased concurrently with theincrease of dihydroceramide and dihydrosphingosine immediatelyand at various time points after light stress, well before activeapoptosis. Light stress in retina induces ceramide generationpredominantly through the de novo pathway. Intraperitoneal (IP)injection of FTY720 (10 mg/kg) prevented ceramide generation bythe de novo pathway and protected retinal structure and function. Wedetermined that the neuroprotection of FTY720 was independent ofits immunosuppressive action.Conclusions: We conclude that ceramide increase by de novobiosynthesis mediates photoreceptor apoptosis in the LIRD modeland that inhibition of ceramide production protects the retina againstlight stress.Commercial Relationships: Md Nawajes A. Mandal, None; HuiChen, None; Julie-Thu Tran, None; Michael H. Elliott, None;Richard S. Brush, OUHSC (P)Support: NIH grants: EY022071 (NAM), EY019494 (MHE),COBRE grant RR017703, core grant EY012190 and EY021725.Grants from Foundation Fighting Blindness, and an unrestricted grantfrom Research to Prevent Blindness, Inc.Program Number: 1964 Poster Board Number: C0178Presentation Time: 11:00 AM - 12:45 PMOcriplasmin as an Adjunct to Vitrectomy for the Treatment ofPediatric Patients: Results of the MIC TrialEmmanuel Chang. Ophthalmology, Associated Retinal Consultants,Royal Oak, MI.Purpose: The ocriplasmin MIC trial was a phase 2, single-center,randomized, double-masked, placebo-controlled study to determineefficacy and safety of ocriplasmin versus placebo as a preoperativeadjunct to vitrectomy in pediatric patients.Methods: The MIC trial included pediatric vitrectomy candidates 16years or younger (including infants) with vitreous attachment to theposterior pole. Patients were randomized to receive a singleintravitreal injection of 175 µg ocriplasmin (n=16) or placebo (n=8)30-60 minutes before planned start of vitrectomy. The primary endpoint was total macular posterior vitreous detachment (PVD) asassessed by masked surgeon under an operating microscope at thebeginning of vitrectomy. Selected secondary end points includedinvestigator assessment of vitreous liquefaction at the beginning ofvitrectomy; immediate postoperative retinal reattachment status;presence of postoperative proliferative vitreoretinopathy andretinopathy of prematurity (ROP) classification by fluoresceinangiography on follow-up. Selected safety assessments includedadverse event reports and B scan ultrasonography.Results: The trial is complete with findings to be reported in early2013. Vitreous detachment status was measured on a 3-point gradescale with Grade 0 being no PVD at beginning of vitrectomy andGrade 3 being total PVD without disc attachment. Vitreousliquefaction status was measured on scale of 1 (formed gel) to 4(water consistency). Postoperative retinal reattachment status wasscored as present or absent immediately after vitrectomy.Postoperative ROP classification on follow-up was scored accordingto a 5-stage scale with Stage 1 defined by presence of a faintdemarcation line and Stage 5 by total retinal detachment.Conclusions: Findings from the MIC trial may be informative forphysicians seeking adjunct treatments to support vitrectomyoutcomes in pediatric patients.Commercial Relationships: Emmanuel Chang, NoneSupport: ThromboGenics, Inc.Clinical Trial: NCT00986362Program Number: 1965 Poster Board Number: C0179Presentation Time: 11:00 AM - 12:45 PMQuinic Acid Derivative, KZ-41 Protects Against Radiation-Induced Retinal Endothelial Cell Dysfunction: An Early - to -Late Stage Treatment of Radiation RetinopathyJordan J. Toutounchian 1 , Qiuhua Zhang 2 , Jayaprakash Pagadala 1 ,Duane D. Miller 1 , Jena J. Steinle 2 , Charles R. Yates 1 .1 Pharmaceutical Sciences, Univ of Tennessee Hlth Sci Ctr, Memphis,TN; 2 Ophthalmology, Univ of Tennessee Hlth Sci Ctr, Memphis, TN.Purpose: Radiation-induced damage to the vascularized retinalsegments of the posterior portion of the eye triggers an exuberantpro-inflammatory response resulting in leukocyte adhesion, vesselblockage, decreased oxygen supply, and retinal endothelial cell(REC) death. In this study we evaluated the effect of KZ-41 onradiation-induced leukocyte adhesion to human RECs and retinalneovascularization (RNV) in a murine oxygen-induced retinopathy(OIR) model.Methods: Leukocyte Adhesion: Human primary RECs were grownto confluence onto 75x38mm glass slides. Irradiated RECs (singledose 30 Gy; 137Cs at ~3 Gy/min) were placed into a parallel-plateflow chamber and treated with either KZ-41 (10μM) or vehicle(PBS). U937 (human monocytic) cells were then perfused over RECs©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 – Physiology/Pharmacologyand digital images were obtained every 30 min over two hours. OIR:Mouse pups (n=5/group) were exposed to 75% oxygen at post-natalday (P)7 for 5 days and then returned to normal oxygen (P12). Micereceived daily ocular administration of either KZ-41-loadednanoemulsion (100 mg/kg) or vehicle on P12-17. Eyes wereenucleated at P17 and retinal whole-mounts stained using theendothelial cell specific marker isolectin B4-594. Images wereacquired using a Nikon Eclipse 80i confocal microscope with Nikon-NIS elements software. Vaso-obliteration was determined bycomparing vascular regrowth to avascular area around the opticnerve. Image analysis was performed using Adobe Photoshop.Results: Radiation increased leukocyte-REC adhesion as early twohours post-exposure as compared to unirradiated controls (2 ± 2 vs.87 ± 18 adhered cells; P < 0.05). Whereas, KZ-41 treatment reducedleukocyte adhesion to irradiated RECs (25 ± 12 vs. 87 ± 18 adheredcells; P < 0.05). In the murine OIR model, KZ-41 reduced theavascular area by two-fold as compared to vehicle treated mice (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: SALVATORE FARO, None; TeresioAvitabile, None; Giulia Malaguarnera, None; Franco MarcoLivio, None; Maurizio G. Uva, None; maria vittoria cicinelli,None; Cristina Cassar Scalia, None; Elena Lionetti, None;Caterina Gagliano, NoneCommercial Relationships: Jennifer Le Couter, Genentech, Inc.(E); Kapil Gadkar, Genentech (E); J. Michael Elliott, Genentech(E); Thomas Lee, None; Y. Gloria Meng, Genentech Inc (E), Roche(I); Linda Zhang, None; Margaret Kenrick, Genentech (E); SailetaPrabhu, Genentech (E); Justin M. Scheer, Genentech (E)Support: Genentech, Inc.Program Number: 1968 Poster Board Number: C0182Presentation Time: 11:00 AM - 12:45 PMEFFECT OF AN ANTIOXIDANT BLEND IN DIABETICMACULAR EDEMASALVATORE FARO 1 , Teresio Avitabile 1 , Giulia Malaguarnera 2 ,Franco Marco Livio 1 , Maurizio G. Uva 1 , maria vittoria cicinelli 3 ,Cristina Cassar Scalia 1 , Elena Lionetti 1 , Caterina Gagliano 3 .1 Ophthalmology, University of Catania, Catania, Italy; 2 Clinical andMolecular Biomedicine, University of Catania, Catania, Italy;3 NEST, Neurovisual Science Technology, Catania, Italy.Purpose: To evaluate the effect of treatment with an antioxidant,vasoprotective and neuroprotective blend containing naturalcompounds on the progression of diabetic macular edema.Methods: Sixty patients with diabetes type I and II with nonproliferativeretinopathy and macular edema in early stage wereenrolled. Twenty patients (group 1) were treated with acetazolamide500 mg/die; twenty patients (group 2) were treated with anantioxidant blend containing extracts of red berries, Ginkgo Bilobaand white willow bark together with carnosine and α-lipoic acid (twotablets/die). Group 3 (twenty patients: control group) did not assumeany treatment. The observations were made at baseline, 1 and 3months after enrollment. We measured central macular thicknesswith spectral OCT. Plasma levels of glucose and glycosylatedhemoglobin (HbA1c) were also considered. Treatment doses: Group1 took acetazolamide 500 mg/die; Group 2 took antioxidant blend: 2capsules per day, one in the morning and 1 in the evening; Group 3received no treatment beside the usual antidiabetic therapy.Results: The level of plasma glucose and HbA1c were statisticallylower in the group 2 (antioxidant blend) compared to acetazolamideand control groups (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyDifferential Comparison of Aqueous HumorPhosphatidylcholines from Control and Glaucomatous DonorsAyman Aljohani, Yenifer C. Guerra, Maria C. Piqueras, Richard K.Lee, Sanjoy K. Bhattacharya. Bascom Palmer Eye Institute,University of Miami, Miami, FL.Purpose: To compare the phosphatidylcholines (PC) of the aqueoushumor (AQH) from control and primary open angle glaucoma(POAG) donors by mass spectrometric shotgun lipidomics. Ourhypothesis is that select PCs are absent in POAG AQH, which maycontribute to increased stiffness of trabecular meshwork in POAG.Methods: The human AQH samples were collected adhering totenets of declaration of Helsinki under IRB approved protocols fromcontrol and POAG donors of diverse race and both genders withmean age of 55 ±8 years (n=10 each). Controls were cataract surgerypatients. AQH PCs were extracted using the Bligh and Dyer method,re-suspended in Isoproyl, Acetonitrile solution and analyzed on aTSQ Quantum Access Max instrument in positive mode. Precursorion scan (PIS) for phosphocholines (PC; product m/z of 184) wereperformed using direct infusion. Ratiometric quantification wasperformed for m/z range of 200-1000. The PC species of control andglaucoma patients were identified using MZmine 2.9 version anddatabase from lipidmaps.org. The profiles were compared using inhousewritten softwares. Further confirmatory analyses wereperformed on a high resolution Q-exactive mass spectrometer.Results: Overall total amount of PCs in POAG normalized to proteinamounts were always less than that in controls. We found 7 and 4unique PCs in controls and POAG AQH respectively. The number ofunique PC species that showed up are 2 in control (12:0/14:1(9Z)),PC(13:0/20:5(5Z,8Z,11Z,14Z,17Z))] and only one in POAG[PC(12:0/14:1(9Z))] AQH.Conclusions: Lower amounts of phosphatidylcholines were found inPOAG compared to controls. This was confirmed by high resolutionmass spectrometry. Absence of select PC species in POAG is likelyto contribute to reduced membrane fluidity and likely result instiffness to TM cells in glaucoma.Commercial Relationships: Ayman Aljohani, None; Yenifer C.Guerra, None; Maria C. Piqueras, None; Richard K. Lee, NationalEye Institute (F); Sanjoy K. Bhattacharya, NoneSupport: Supported by an unrestricted grant from RPB to Universityof Miami, NIH grants P30EYEY014801 and R01EY016112.Program Number: 1971 Poster Board Number: C0185Presentation Time: 11:00 AM - 12:45 PMOcular Hypertension-Induced MMPs Production Within OpticNerve: A Regulatory Role of δ-Opioid-ReceptorsSudha Singh, Mohammad F. Pathan, Naseem Akhter, Melissa Nix,Shahid Husain. Ophthalmology, Medical University of SouthCarolina, Charleston, SC.Purpose: This study was designed to determine the changes in thematrix metalloproteinases (MMPs) production in optic nerve head(ONH) and purified ONH astrocytes in response to ocularhypertensionand TNF-α-induced injury, respectively. We also havedetermined if δ-opioid-receptors activation counterbalances theproduction of MMPs.Methods: Brown Norway rats were used to elevate intraocularpressure (IOP) by injecting 50 µL of 2 M hypertonic saline into thecircumferential limbal veins. Animals were treated with δ-opioidreceptoragonist, SNC-121 (1 mg/kg; i.p.), daily for 7 days. Patternelectroretinograms (PERG) and retinal ganglion cells (RGCs) in flatmountswere counted at week-6 post injury. Primary cultures ofhuman ONH astrocytes were isolated and purified byimmunopanning. Cells were treated with SNC-121 (1μM), 15minutes prior to TNF-α (25 ng/mL) treatment for 24 hours. Theexpression of MMP-1, MMP-2, and MMP-3 were determined byimmunohistochemistry and Western blotting.Results: PERG amplitudes and RGCs were reduced by 18% and28%, respectively, in ocular-hypertensive eyes. PERG deficits andreduction in RGCs in hypertensive eyes were significantly (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyin the export of GRα. Finally, nuclear shuttling of GRα to and fromthe nucleus is absolutely ATP-dependent.Commercial Relationships: Adnan Dibas, None; Abbot F. Clark,Alcon Research, Ltd. (F); Thomas Yorio, NoneSupport: EY016242Program Number: 1973 Poster Board Number: C0187Presentation Time: 11:00 AM - 12:45 PMNon-invasive Intraocular Pressure Measurements in ZebrafishD Joshua Cameron, Pinakin G. Davey. College of Optometry,Western University of Health Sciences, Pomona, CA.Purpose: The zebrafish (Danio rerio) eye shares many similaritieswith the human eye and is increasingly being used as model for eyedevelopment, disease and regeneration. A mutant zebrafish, coinedbugeye because of its grossly buphthalmic eyes, has been shown tohave some similarities with human glaucoma due to its elevatedintraocular pressure (IOP) and optic nerve pathology. Weinvestigated whether a non-invasive clinical tonometer could measureIOP in zebrafish and importantly, distinguish bugeye from wild-typefish.Methods: Adult zebrafish were anesthetized in tricaine and placed ona styrofoam bed. A moist paper towel was placed over the fish body,leaving the head and gills exposed. Tricaine solution was perfusedover the gills to keep the fish oxygenated and sedated. The fish’s eyewas brought up to the probe of a Paradigm blood flow analyzerpneumotonometer and the IOP was repeatedly measured andaveraged. The average IOP of several strains of fish were thencompared to each other using analysis of variance.Results: Bugeye eyes (n=13) were significantly larger compared toTU/TL (n=10) strains both in size and ratio to the head (p=0.006 and1.6E-06 respectively). The zebrafish quickly recover after IOPmeasurement, returning to normal activity within minutes of beingplaced in fresh water. IOP values did not significantly vary in wildtypefish over 12 months of age. Strain AB fish (n=29) had slightlyhigher IOP values compared to TU/TL, but the difference was notsignificant. Bugeye mutants had significantly elevated IOP valuescompared to wild-type fish (bugeye 25.3 mmHg; TU/TL 14.6 mmHg;p=0.01). However, IOP in bugeye zebrafish did not significantlycorrelate with eye size or eye/head ratios.Conclusions: A non-invasive pneumotonometer can be used tomeasure IOP in adult zebrafish and distinguish bugeye fish fromwild-type strains.Commercial Relationships: D Joshua Cameron, None; Pinakin G.Davey, NoneProgram Number: 1974 Poster Board Number: C0188Presentation Time: 11:00 AM - 12:45 PMCharacterization of TRPV4 expression & function in the ciliarybody & trabecular meshworkAmber M. Frye 1 , Daniel A. Ryskamp 1, 2 , Peter Barabas 1 , TundeMolnar 1 , David Krizaj 1, 2 . 1 Ophthalmology & Visual Sciences, MoranEye Institute, University of Utah School of Medicine, Salt Lake City,UT; 2 Interdepartmental Program in Neuroscience, University of Utah,Salt Lake City, UT.Purpose: Aqueous humor dynamics is regulated by balancedproduction in the ciliary body & removal through the primary(“conventional”) & secondary outflow pathways. Regulation of thevolume of trabecular meshwork (TM) cells thus directly impacts theoutflow facility & affects the amplitude of intraocular pressure. Todetermine the molecular mechanism regulating the outflow, weexamined the expression & function of theosmosensory/mechanosensitive TRPV4 cation channel in the anteriorchamber (AC) of the eye.Methods: Cryosections from C57BL6 mouse eyes & human punchesfrom trabeculectomies were immunostained with a validated TRPV4antibody (Ryskamp et al., 2011) & colabeled with the TM markeraquaporin 1. Nuclei were labeled with propidium iodide & imageswere acquired with a confocal microscope. Alternatively, TM tissueswere loaded with the calcium indicator dye fura-2 & the calciuminsensitivecell volume indicator calcein. Calcium levels [Ca2+]i andcell volume were determined using high-resolution optical imaging.To functionally map TRPV4 expression, eye sections were incubatedwith 100 nM GSK1016790A (GSK), a selective TRPV4 agonist, inthe presence of extracellular agmatine (AGB 5 mM, a cation influxmarker) and/or the TRPV4 antagonist HC067047 (HC 1 µM). Ocularslices were then fixed & immunostained for AGB. To assess theimpact of maximal TRPV4 activation on histology, 75 µM GSK wasinjected into the AC. Eye slices were processed for H&E, apoptosismarkers, metabolic markers, & EM ultrastructure.Results: TRPV4 immunoreactivity was observed confirming itsexpression in the pars plicata of the fluid producing, non-pigmentedepithelial cells lining the ciliary processes, but was absent from thepigmented epithelial cells of the ciliary body. Both mouse & humanTM were labeled by the TRPV4 antibody. The functional presence ofTRPV4 in these cells was confirmed by GSK- & HC-dependentchanges in [Ca2+]i & AGB influx. In contrast to the pronouncedproapoptotic effects of GSK in retinal cells, saturating TRPV4activation induced minimal apoptosis & histological changes withinthe ciliary body & TM cells.Conclusions: Our data provide molecular & physiological evidenceof TRPV4 localization in the ciliary body, trabecular meshwork &the posterior eye and suggest a novel mechanism that couldpotentially regulate the volume of cells within the conventionaloutflow pathway.Commercial Relationships: Amber M. Frye, None; Daniel A.Ryskamp, None; Peter Barabas, None; Tunde Molnar, None;David Krizaj, NoneSupport: NIH, DOD, FFB, University of Utah and an unrestrictedaward from RPB to the Moran Eye Institute.Program Number: 1975 Poster Board Number: C0189Presentation Time: 11:00 AM - 12:45 PMOcular Hypertension-Induced Changes in the Anterior Segmentof Cynomolgus MonkeysShenouda Yacoub, Quinn Sessums, Byron H. Li, Ganesh Prasanna.Glaucoma Research, NIBR-Ophthalmology, Fort Worth, TX.Purpose: To compare the corneal thickness (CT), corneal curvature(CC), anterior chamber depth (ACD), iridocorneal angle (ICA), andcorneal endothelial cell density (ECD), in naïve and chronic ocularhypertensive (OHT) cynomolgus monkeys.Methods: Chronic OHT of various durations (0.7-12 y) was inducedin the right eye (OD) of 52 cynomolgus monkeys by argon lasertrabeculoplasty. The left eye (OS) with normal intraocular pressure(IOP) was untreated. An additional 23 naïve animals served ascontrols. The central corneal thickness (CCT), para-central cornealthickness (PCCT), peripheral corneal thickness (PCT), CC, ACD andICA were evaluated with a Galilei Dual Scheimpflug Analyzer(GDSA, SIS, Switzerland). The ECD at central corneal area wasevaluated with a SP2000 Specular microscope (Topcon).Results: In the 23 naïve 4-yo monkeys, there were no differences inCT, CC, ACD, ICA, and ECD between OD and OS. In 20 OHT 4-yomonkeys (OHT duration in OD = 0.7 y), the differences of CT, CC,ACD and ICA between OD and OS were insignificant. However,significant ECD reduction in OD (-17.2%, P < 0.01) was detectedcompared to the OS. In a second group of 32 OHT monkeys (age = 7-16 y; OHT duration = 2-12 y), the mean CCT of OD (490 ± 23 µm)©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 – Physiology/Pharmacologywas thicker (11 µm, 2.4%, p < 0.05) than the contralateral controleyes (479 ± 20 µm). The ECD of OD was reduced (-20%, p < 0.001)compared to OS. There was no significant difference in PCCT, PCT,ACD, and ICA between OD and OS.Conclusions: Chronic OHT in monkeys caused a duration-dependentreduction in ECD in the central corneal area. Longer duration of IOPelevation correlated with a more severe reduction, concomitant with asmall increase of CCT. There was no difference in CC, ACD, andICA between OHT and normal eyes. The thickening of CCT andprogressive decrease of ECD observed in monkey eyes withincreased duration of OHT is similar to the disease progression inprimary open angle glaucoma patients.Commercial Relationships: Shenouda Yacoub, Novartis (E);Quinn Sessums, None; Byron H. Li, Novartis (E); GaneshPrasanna, Alcon Research Ltd (E), Novartis Institute of BiomedicalResearch (E)Program Number: 1976 Poster Board Number: C0190Presentation Time: 11:00 AM - 12:45 PMRegulation of Mammalian Sympathetic NeurotransmitterRelease and Intraocular Pressure by Hydrogen Sulfide Donor,GYY 4137Ankita Salvi 1 , Pratik Bankhele 1 , Jamal Jamil 1 , Ya Fatou Njie-Mbye 2 ,Madhura S. Kulkarni 2 , Sunny E. Ohia 2 , Catherine A. Opere 1 .1 Creighton University, Omaha, NE; 2 Texas Southern University,Houston, NE.Purpose: We have evidence that hydrogen sulfide (H 2 S) donors canregulate sympathetic neurotransmission and intraocular pressure(IOP) in the mammalian anterior uvea. In the present study, weinvestigated the effect of a slow releasing H 2 S donor, GYY 4137 onelectrically evoked [ 3 H]NE release in isolated superfused, bovine irisciliarybodies (ICB), in vitro and on IOP in male New Zealand Whiterabbits, in vivo.Methods: Isolated bovine ICB were incubated in oxygenated Krebssolution containing 2.5 μCi/ml of [ 3 H]NE and then prepared forneurotransmitter release studies. Release of [ 3 H]NE was elicited bytwo (S 1 and S 2 ) electrical pulses (300 d.c electrical pulses) applied 27min apart. For IOP studies, a single drop of GYY 4137 (0.1-2%) andvehicle were instilled into the right and left rabbit eyes, respectively.IOP measurements were made using a pneumatonometer (model 30classic; Reichert Ophthalmic Instruments, Depew, NY) until baselinereadings resumed.Results: GYY 4137 (1-30μΜ) attenuated field-stimulated [ 3 H]NErelease in bovine ICB in a concentration-dependent manner,achieving an inhibition of 20.8% (n=3; p0.05) on [ 3 H]NE release, they bothreversed the inhibitory action of GYY 4137 (10-30 µM) on theneurotransmitter release. GYY 4137 (0.1-2%) reduced IOP in bothtreated and untreated eyes for a duration of 7-9 h, exhibiting amaximum effect after 5-6 h. For instance, GYY 4137 (2%) elicited amaximum IOP inhibition of 27.76 % (n=5; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Arthur J. Sit, Glaukos, Corp. (F),Alcon Laboratories, Inc. (C), Allergan, Inc. (C), Glaukos, Corp. (C),Sensimed, AG (C); Nitika Arora, None; Jay W. McLaren, None;Mehrdad Malihi, None; Lilit Voskanyan, Glaukos corporation (E)Support: American Health Assistance Foundation, Glaukos Corp.,Mayo Foundation for Medical Education and Research, Research toPrevent BlindnessProgram Number: 1978 Poster Board Number: C0192Presentation Time: 11:00 AM - 12:45 PMExpansion of Schlemm’s Canal by Travoprost in HealthySubjects determined by Fourier-Domain Optical CoherenceTomographyJUNYI CHEN 1 , Haili Huang 1 , Shenghai Zhang 1 , Xueli Chen 1 ,Xinghuai Sun 1, 2 . 1 Ophthalmology, Eye & ENT Hospital of FudanUniversity, Shanghai, China; 2 State Key Laboratory of MedicalNeurobiology, Institutes of Brain Science, Fudan University,Shanghai, China.Purpose: To determine the effect of travoprost 0.004% onSchlemm’s canal (SC) in healthy human eyes using Fourier-domainoptical coherence tomography (FD-OCT).Methods: Twelve healthy volunteers were recruited for a doubleblind,placebo-controlled, randomized, and paired comparison study.Right eyes of subjects were randomly assigned to receive eithertravoprost 0.004% or placebo; the contralateral eye received the othertreatment. FD-OCT imaging of SC and measurements of intraocularpressure (IOP) were carried out beforeand at 1, 2, 4, 6, 8, 12, 24, 36,48, 60, 72 and 84 hours after eye drop instillation.Results: After instillation of travoprost eye drops, IOP graduallyreduced, and the SC lumens expanded, while those values remainedunchanged in placebo treated eyes. At 8 hours after the travoprostadministration, the mean SC area increased 90.30% and 90.20%,respectively, in nasal and temporal quadrant of the treated eyes ascompared to the placebo group. The SC area and IOP showed asimilar pattern of changes at most time points examined. Intravoprost-treated eyes, a statistically significant correlation betweenSC area and IOP is observed (r= -0.2817; p=0.0004). Measurementsof the SC area showed sufficient repeatability and reproducibility.Conclusions: SC can be noninvasively imaged and quantitativelyassessed in the living healthy human eye by FD-OCT. Travoprosttreatment leads to SC lumen expansion accompanied by a drop ofIOP in the healthy eye, likely as a result of the enhancement ofpressure sensitive trabecular meshwork outflow induced bytravoprost.Commercial Relationships: JUNYI CHEN, None; Haili Huang,None; Shenghai Zhang, None; Xueli Chen, None; Xinghuai Sun,NoneSupport: the Key Clinical Program of the Ministry of Health(2011-36),and the National Natural Science Foundation of China(NSFC81020108017)Clinical Trial: 20100375Program Number: 1979 Poster Board Number: C0193Presentation Time: 11:00 AM - 12:45 PMThinning of RNFL is correlated with declining of PhNR ERGamplitude in glaucomatous monkeysByron H. Li, Nalini V. Rangaswamy, Steven Q. Sessums, Richard L.Ornberg, Ganesh Prasanna. NIBR Ophthalmology, Novartis, FortWorth, TX.Purpose: To understand the structure-function relationship betweenretinal nerve fiber layer (RNFL) thickness and ERG in the nonhumanprimate model of glaucoma.Methods: Chronic ocular hypertension (OHT) of various durations(1.6-13 y) was induced in the right eye (OD) of 43 cynomolgusmonkeys by argon laser trabeculoplasty. The left eye (OS) wasuntreated with normal intraocular pressure (IOP). An additional 25naïve animals served as controls. The juxtapapillary RNFL thicknessin the normal and experimental eyes was evaluated with SpectralisSD-OCT (Heidelberg Engineering, Germany). The photopic ERGresponded to 8 ascending luminous intensities of brief-(≤ 5 msec) andlong-(200 msec) durations red Ganzfeld flashes on a rod-suppressingblue-adapting background were recorded with Espion ERG System(Diagnosys, MA, USA).Results: In the 25 naïve 5-yo monkeys, there was no difference inRNFL thickness and ERG amplitudes between OD and OS. In 23OHT 5-yo monkeys (OHT duration = 1.6 y), significant thinning ofOD RNFL (-16 ± 5 µm, -11%, p < 0.05) was detected compared tothe OS in the nasal-inferior (NI) section. This early structural changedid not significantly alter ERG but a trend was observed in photopicnegative response (PhNR). In a second group of 20 OHT monkeys(age = 6-17 y; OHT duration = 2-13 y), Global RNFL thickness ofOD (82 ± 4 µm) was significantly reduced (-17%, p < 0.001) thancontralateral control eyes (99 ± 2 µm). Only the ERG PhNR but notthe a- and b-waves response was significantly reduced in the OHTeyes. Brief flash stimulation was more sensitive to detect PhNRamplitude changes than long duration flash. There was a significantnegative correlation (R2=0.8304) between RNFL thickness andPhNR by comparing the difference of eyes (OS-OD) in each measurein the 43 OHT monkeys.Conclusions: Initial thinning of RNFL was detected in the NI sectionof the eyes with 1.6 y of OHT. This early structural change did notalter ERG but a trend was observed in PhNR. A longer duration(7.5±3.7 yo) of IOP elevation significantly reduced RNFL thicknessand PhNR in lasered monkey eyes with strong correlation.Progressive thinning of RNFL and reduction of PhNR amplitudesobserved in monkey eyes with different duration of chronic OHT islikely indicative of progressive loss of retinal ganglion cells and issimilar to the disease progression in POAG in humans.Commercial Relationships: Byron H. Li, Novartis (E); Nalini V.Rangaswamy, Novartis Institute for Biomedical Research (NIBR-FW) (E); Steven Q. Sessums, None; Richard L. Ornberg, AlconResearch (E), Novartis Institute for Biomedical Research (E);Ganesh Prasanna, Alcon Research Ltd (E), Novartis Institute ofBiomedical Research (E)Program Number: 1980 Poster Board Number: C0194Presentation Time: 11:00 AM - 12:45 PMIn Vivo Agreement of Intraocular Pressures SimultaneouslyMeasured Using Tonometer and Manometers Placed in Anteriorand Vitreous Cavity under General AnesthesiaHyun Seung Yang, June-Gone Kim. Department of Ophthalmology,Asan Medical Center, Seoul, Republic of Korea.Purpose: To analyze the agreement between three intraocularpressure (IOP) measurements obtained using tonometer andmanometers placed in anterior chamber (AC) and vitreous cavity(VC) in various vitreous conditions and their association with ocularproperties.Methods: Sixty nine patients who were planning to have vitreoussurgery for epiretinal membrane or macular hole (ERM and MH;n=26), vitreous hemorrhage (DMVH; n=24), or silicone oil removal(n=19) were included consecutively in this prospective study. Oneexaminer, who was blinded to the patient’s information, performed acomplete ophthalmologic examination, including the cornealthickness (CT), anterior chamber depth (ACD), and axial length(AL). Another examiner simultaneously measured IOP usingTonopen AVIA® and 2 automated transducers during the 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 – Physiology/Pharmacology(Fig. 1).Results: The mean POAIOP, ACIOP and VCIOP were 16.7±3.7mmHg, 16.7±3.8 mmHg and 16.0±4.6mmHg, respectively. Thecorrelations between POAIOP and ACIOP, between ACIOP andVCIOP, and between POAIOP and VCIOP were all very good(R=0.92, 0.65, and 0.76, respectively). However, the VCIOP wassignificantly higher compared with POAIOP or ACIOP in the ERMand MH group (by 1.4 and 1.5 mmHg, respectively), butsignificantlylower in the DMVH (by -1.0 and -1.1 mmHg, respectively) and thesilicone oil groups (by -2.8 and -2.7 mmHg, respectively) (Fig.2). Inthe multivariate analysis, only CT among ocular properties wassignificantly correlated with POAIOP (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyMethods: Sixteen eyes of 8 healthy participants (age 27-61; mean37.8 years) were studied. Variables were measured in two visits.During one study visit, IOP was measured in the sitting position bypneumatonometry, outflow facility was measured by digital Shiøtztonography, and EVP was measured by using an automatedvenomanometer, based on the pressure-chamber method. Afterbaseline measurements, participants drank 15 ml/kg of water within10 minutes. Variables were re-measured at 10 min, 30 min, and 60min after ingestion of water. During another study visit, baselineaqueous humor flow rate was determined during a one hour intervalby fluorophotometry. Participants were then asked to drink sameamount of water within 10 minutes. Aqueous humor flow was remeasuredon the intervals 0-10 minutes, 10-30 minutes, and 30-60minutes after water drinking. Aqueous humor flow rate, outflowfacility, IOP, and EVP after drinking water were compared to thesame variables at baseline by using generalized estimating equation(GEE) models. The increase in IOP after water drinking wascompared to the change in EVP by using GEE models.Results: EVP and IOP increased after water drinking by 3 mmHg at30 minutes (Table 1, see p-values in table). Outflow facility did notchange after water drinking. Aqueous humor flow decreased duringthe 10-minute interval after water drinking but was not different frombaseline during any other interval. The rise in IOP was not differentfrom the rise in EVP at 10 minutes and 30 minutes (p=0.06 and p=0.8respectively). At 60 minutes, the difference in IOP from baseline wasless than the difference in EVP from baseline (p=0.004).Conclusions: IOP rises after the water drinking test primarilybecause of the increase in EVP. Changes in outflow facility andaqueous humor flow do not contribute to the rise in IOP. Theelevation in IOP recovers earlier than the elevation in EVP after thewater drinking test.Purpose: To develop a chronic ocular hypertension model in rats byinducing foreign body reaction (FBR) and scar tissue formation inSchlemm’s canal (SC) and trabecular meshwork (TM) followinginsertion of human hair into the SC .Methods: A human hair was inserted around SC of one eye in rats;the other eye was untreated. Intraocular pressure (IOP) wasmonitored weekly for eight months with a TonoLab. Retinalfunctional changes were assessed using photopic negative response(PhNR) elicited using green Ganzfeld flashes (22.76cds/m2).Immunohistochemical analysis was performed on anterior segmentsof treated and control eyes. Sections were immunolabeled with Ilba1antibody for macrophages and stained with Trichrome collagen.Retinal flat-mounts were prepared with Brn-3a immunolabeling tocount retinal ganglion cells (RGCs).Results: Hair inserted into the rat SC produced a gradual, chronicIOP elevation lasting at least eight months. At week 31, a 25%increase in IOP was observed in the experimental eye (IOPexp eye =20 ± 1 mmHg; mean ± SEM; IOPcontralateral eye = 16 ± 1 mmHg; p= 0.039). Six months post-procedure, granuloma, increasedmacrophages and foreign body giant cells, and dense fibrosis basedon collagen staining were observed in the angle around the insertedhair of the experimental eye. The figure shows positive staining ofCD3 immunoreactivity was mainly detected in the SC and TM areaaround the inserted hair, which confirm the T cell infiltrating.Preliminary data in FBR eyes indicate a trend toward reduction inPhNR post 8 months (35 ± 2.8%, p = 0.333), indicating reducedretinal ganglion cell (RGC) activity. In retinal sections, Brn-3alabeled RGCs were reduced 26% ± 19 (P = 0.001) at six months and37 ± 16% (P = 0.001) at eight months compared to contralateral eyes.Conclusions: In rats, a FBR model produces stable, moderatelyelevated IOP which results in functional and morphological changesto the retina, probably via changes in RGC density.Commercial Relationships: Nitika Arora, None; Jay W.McLaren, None; Arthur J. Sit, Glaukos, Corp. (F), AlconLaboratories, Inc. (C), Allergan, Inc. (C), Glaukos, Corp. (C),Sensimed, AG (C)Support: American Health Assistance Foundation, Mayo Foundationfor Medical Education and Research, Research to prevent blindness(Schaub Special Scholar Award and departmental grant)Program Number: 1983 Poster Board Number: C0197Presentation Time: 11:00 AM - 12:45 PMForeign-body-reaction-induced chronic ocular hypertension inratBing Li 1 , Yu Wang 2 , Shutong Cao 2 , Olga Kraszewska 2 , RichardOrnberg 2 , Ganesh Prasanna 1 . 1 Glaucoma Research, NIBR, Novartis,Induced immune-based inflammationCommercial Relationships: Bing Li, Novartis (E); Yu Wang,Novartis, Alcon (E); Shutong Cao, nibr (E); Olga Kraszewska,Novartis (E); Richard Ornberg, Novartis Institute for BiomedicalResearch (E); Ganesh Prasanna, Alcon Research Ltd (E), NovartisInstitute of Biomedical Research (E)Program Number: 1984 Poster Board Number: C0198Presentation Time: 11:00 AM - 12:45 PMEffect of the Water Drinking Test in Subjects with PureAutonomic Failure and Normal Tension GlaucomaWilliam M. Watkins 1 , David Robertson 2 , Karen M. Joos 1 . 1 VanderbiltEye Institute, Vanderbilt University Medical Center, Nashville, TN;2 Clinical Pharmacology Division/Medicine, Vanderbilt UniversityMedical Center, Nashville, TN.Purpose: Intraocular pressure (IOP) is influenced by systemicvascular control. Pure autonomic failure (PAF) patients haveelevation of blood pressure (BP) and symptomatic improvementFort Worth, TX; 2 Retina Research, NIBR, Novartis, Fort Worth, TX. following water consumption. This study examines the effect 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 – Physiology/Pharmacologywater consumption on BP, IOP, and central choroidal thickness(ChT) in subjects with (PAF) or normal tension glaucoma (NTG).Methods: This study was approved by the Vanderbilt University IRB(ClinicalTrials.gov NCT00338065) and informed consent wasobtained from all subjects. PAF (n=10), NTG (n=10), and control(n=10) subjects were evaluated by baseline mean arterial systemicblood pressure (MAP), Goldmann and Tono-pen (ReichertTechnologies, NY) tonometry, and optical coherence tomography(OCT, Spectralis, Heidelberg, CA) central choroidal thickness (ChT).The subjects then consumed 500 ml of room-temperature waterwithin 5 minutes. MAP, tonometry, and ChT measurements wereobtained 15 minutes after water consumption. The method to obtainChT was adapted from Mwanza, et al with the Spectralis OCTenhanced depth imaging protocol.Results: Baseline MAP (mm Hg) for each group was 68+14.5 (PAF),97.7+8.2 (NTG), and 97.8+8.8(Control). After water consumption,the MAP increased to 87.1+11.2 (PAF,P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology12, 24, 48, 72 and 96 hours after administration. Compounds wereadministered topically in a 30 µL volume once into the right eye.Results: In dogs, ONO-9054 lowered IOP in a dose-dependentmanner, and the reduction of IOP persisted for at least 24 hours afteradministration. ONO-9054 at 3 µg/mL or more showed a more potentand longer-lasting reduction of IOP than latanoprost, ONO-9054 at10 µg/mL showed more potent and longer-lasting reduction of IOPthan the fixed latanoprost / timolol combination. In monkeys, ONO-9054 lowered IOP in a dose-dependent manner, and the reduction ofIOP at 3 and 10 µg/mL was observed for up to 48 hours afteradministration and IOP at 30 µg/mL persisted for up to 72 hours afteradministration. The IOP reduction of ONO-9054 at 1 µg/mL wascomparable to that of latanoprost and showed more potent andlonger-lasting IOP-reducing effects at concentrations of 3 µg/mL ormore.Conclusions: The IOP-lowering effects of the FP/EP3 agonist ONO-9054 were more potent and longer-lasting than those produced by anFP receptor agonist, a beta-adrenergic receptor antagonist and a fixedcombination FP agonist/beta-adrenergic antagonist. These resultssuggest that ONO-9054 should be considered further in clinical trialsin patients with elevated intraocular pressure due to glaucoma.Commercial Relationships: Kazufumi Nagai, ONOPHARMACEUTICAL CO.,LTD (E); Shinsaku Yamane, ONOPHARMACEUTICAL CO., LTD. (E); Kazumi Moriyuki, ONOPharmaceutical Co Ltd (E); Tomohiro Karakawa, ONOPHARMACETICAL CO., LTD (E); Shintaro Nakao, ONOPharmaceutical Co.,LTD (E); Tsutomu Shiroya, ONOPharmaceutical.Co.Ltd. (E); Yutaka Shichino, ONO PharmaceuticalCo Ltd (E)Program Number: 1987 Poster Board Number: C0201Presentation Time: 11:00 AM - 12:45 PMBAICALEIN LOWERS INTRAOCULAR PRESSURE INGERBILSChi-wai W. Do 1 , Chi-ting T. Leung 1 , Ho Lung Henry Chan 1 ,Mortimer M. Civan 2 , Chi-ho To 1 . 1 School of Optometry, The HongKong Polytechnic University, Hong Kong, Hong Kong; 2 Departmentof Physiology, University of Pennsylvania, Philadelphia, PA.Purpose: Currently, no clinical treatments are available to preventthe glaucomatous optic neuropathy except for lowering intraocularpressure. Baicalein is a common traditional Chinese medicine thathas no reported toxicity. We have previously demonstrated thatbaicalein inhibits chloride secretion and net fluid transport across theexcised ciliary epithelium, potentially reducing aqueous humorformation and intraocular pressure. The aim of this study is todetermine the changes in intraocular pressure by baicalein in livinganimals.Methods: Gerbils (sand rats) were used in the experiments. Baicaleinwas administered intra-peritoneally to the animals in the treatmentgroup once every two days for two weeks, while saline solution wasused in the control animals. Measurements of the intraocular pressurewere conducted under anesthesia with a rebound tonometer(TonoLab) prior to drug administration.Results: Intra-peritoneal injection of baicalein (100mg/kg)significantly lowered IOP in gerbils. The maximum hypotensiveeffect was achieved two days after the first injection and thenmaintained at almost the same level throughout the experimentalperiod. After two weeks, baicalein-treated animals had a lowerintraocular pressure of -3.92 ± 0.89 mmHg (N = 4, p < 0.05) ascompared to the saline-treated controls, accounting for a 23-26%reduction of intraocular pressure from the baseline measurements. Ata lower concentration of baicalein (4mg/kg), a hypotensive effect of15% was also observed (N = 5).Conclusions: Baicalein appears to function as an ocular hypotensiveagent. Its site and mode of action remain to be determined.Commercial Relationships: Chi-wai W. Do, None; Chi-ting T.Leung, None; Ho Lung Henry Chan, None; Mortimer M. Civan,None; Chi-ho To, NoneSupport: General Research Fund (B-Q34C), NIH Grant (EY13624),PolyU Niche Fund (1BB8W) and PolyU Internal Grants (A-PL45, G-YK88, G-U585 and G-U858)Program Number: 1988 Poster Board Number: C0202Presentation Time: 11:00 AM - 12:45 PMDefinition of Normal Ophthalmic Measures in the African GreenMonkeyRobin J. Goody, Wenzheng Hu, Steve D. Whittaker, Steve T. Henry,Rohn Brookes, Michael J. Struharik, Erich Hechanova, Matthew S.Lawrence. RxGen, Inc, Hamden, CT.Purpose: Clinical trials require assessment of patients to confirmsuitability for study enrollment. Similar considerations have to bemade preclinically for efficacy and safety studies, particularly inlarge animal models where smaller cohort sizes are often employed.Defining the normal range of a test species, whether ophthalmicparameters or otherwise, can facilitate study recruitment and supportevaluation of adverse or therapeutic responses to treatment. Weconducted ophthalmic examinations in a population of healthyAfrican green monkeys to define the normal range of theseparameters in this species.Methods: Ophthalmic examinations were performed on healthy adultmale and female African green monkeys. Animals were sedated withketamine and xylazine and endpoints including laser flarephotometry, pachymetry, tear film break up, corneal staining andanatomic dimensions were evaluated. The normal range was definedas mean values plus (upper value) or minus (lower values) thestandard deviation multiplied by two. All studies were conducted incompliance with the ARVO Statement for the Use of Animals inOphthalmic and Vision Research.Results: The normal range of laser flare in healthy eyes was 1.5 to10.9 photons/msec, with an average of 5.4 photons/msec. Nosignificant differences in laser flare were observed between male andfemale eyes. Central corneal thickness measures were significantlyhigher in males (471±4.7 μm) than females (453±4.5 μm, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyBK2A77: A novel non-peptide bradykinin B2 agonist lowersintraocular pressure in ocular hypertensive cynomolgus monkeysGanesh Prasanna 1 , Naj Sharif 1 , Byron H. Li 1 , Curtis Kelly 1 , ShouxiXu 1 , Linya Li 1 , Daniel Scott 1 , Rad Daly 1 , Mark Hellberg 2 , KeithCombrink 2 . 1 Glaucoma Research, Novartis Institute of BiomedicalResearch (NIBR) Ft. Worth/Alcon Research Ltd, Fort Worth, TX;2 Global Discovery Chemistry, Novartis Institute of BiomedicalResearch (NIBR) Ft Worth/Alcon Research Ltd, Fort Worth, TX.Purpose: Bradykinin (BK), a nonapeptide has been shown toregulate intraocular pressure (IOP) in different species includingrabbits and monkeys. Presently we intended to characterize the IOPlowering effects of BK2A77, a novel and selective non-peptidebradykinin B2 receptor agonist in ocular hypertensive (OHT)cynomolgus monkeys.Methods: BK2A77 was evaluated in several in-vitro efficacy andreceptor binding assays using human cloned B1 and B2 CHO cellsand in human ciliary smooth muscle (HCM) cells. Intracellularcalcium mobilization ([Ca 2+ ] i ) was assessed using FLIPR assay andprostaglandin (PG) release was measured using an EIA assay. Ocularsafety assessments including slit lamp examinations were performedin monkeys following topical ocular application of BK2A77.BK2A77 or vehicle-induced IOP changes were measured using anAlcon computerized pneumatonometer in normal and hypertensiveeyes of cynomolgus monkeys.Results: BK2A77 is a selective B2 receptor agonist with EC 50 valuesof 13 ± 5 nM (E max = 92 ± 1%; BK response was 100%) in [Ca 2+ ] iassay and 12 ± 7 nM (E max = 100 ± 14%) in the PG release assaysrespectively. BK2A77 exhibited comparable high affinity binding toB2 receptors (Ki = 3 - 10 nM) with no detectable affinity towards B1receptors. Topical ocular dosing of BK2A77 (3 μg x 3 times, 1 hourapart) to sedated cynomolgus monkeys caused no flare or cells toappear in the anterior chamber as observed up to 24h post-dose. Noother adverse effects were noted. A single topical ocular applicationof BK2A77 (0.03 - 3 μg) caused a dose-dependent IOP reduction upto 25% from baseline between 6 - 24h post-dose in the hypertensiveeyes of conscious cynomolgus monkeys. Maximal percent IOPreduction of 25% was observed at 0.9 - 3 μg doses. While themagnitude of IOP reduction with BK2A77 appeared to be similar tothat observed with travoprost, the duration of action appeared to last>24h for BK2A77.Conclusions: Unlike the issues surrounding topical ocularapplication of BK peptide, including non-selectivity against B1 andB2 receptors, poor ocular penetration and susceptibility to rapiddegradation by angiotensin converting enzyme, BK2A77 offersseveral new therapeutic advantages. BK2A77 is a selective nonpeptideB2 receptor agonist capable of robust and long lasting IOPreduction that appears to also be well tolerated following topicalocular dosing in OHT monkeys.Commercial Relationships: Ganesh Prasanna, Alcon Research Ltd(E), Novartis Institute of Biomedical Research (E); Naj Sharif,Alcon Research, Ltd (a Novartis Co.) (E); Byron H. Li, Novartis (E);Curtis Kelly, Alcon / Novartis Institutes for Biomedical Research(E); Shouxi Xu, Novartis (E); Linya Li, Alcon Labs (E); DanielScott, NIBR at ALCON (E); Rad Daly, Novartis Institute ofBiomedical Research (E); Mark Hellberg, NIBR (E), Alcon (P);Keith Combrink, NoneProgram Number: 1990 Poster Board Number: C0204Presentation Time: 11:00 AM - 12:45 PMEvaluation of Changes in Intraocular Pressure ImmediatelyAfter Intravitreal Injection of Anti-VEGF MedicationJulio C. Chora, Victor H. Gonzalez. 19204, Valley Retina Institute,McAllen, TX.Purpose: To determine changes in the intraocular pressure (IOP) inpatients receiving intravitreal injection of 4 Anti-VEGF Medication(bevacizumab, pegaptanib, ranibizumab, aflibercept).Methods: This was a retrospective chart review of eighty fiveconsecutive patients who received intravitreal Anti-VEGF medicationfor various conditions such as exudative age-related maculardegeneration, venous occlusions, proliferative diabetic retinopathyand diabetic macular edema.IOP was measured using a Reichert Tono-Pen® XL ApplanationTonometer before and 1 minute after intravitreal injection.Results: Out of the 85 patients 70% were female, and 30% weremale with an average age of 74.4 years. 50% of the patients werebeing treated for exudative age-related macular degeneration, diabeticmacular edema 27%, proliferative diabetic retinopathy 13%, and veinocclusions 10%. 56% were injected with ranibizumab, 20% withpegaptanib, 19% with bevacizumab and 5% with aflibercept.Baseline median IOP was 15 mmHg (Max Value 22 mmHg and MinValue 12 mmHg).1 minute after the injection the IOP rose from a high value of 89mmHg to a low of 15 mmHg with a median of 60 mmHg.With ranibizumab the IOP rose from a a high value of 89 mmHg to alow value of 24 mmHg with a median of 59.50 mmHg, withbevacizumab the IOP rose from a high value of 87 mmHg to a low of15 mmHg with a median of 55 mmHg, with aflibercept the IOP rosefrom a high value of 75 mmHg to a low value of 54 mmHg with amedian of 58.50 mmHg and with pegaptanib the IOP rose from ahigh value of 89 mmHg to a low value of 33 mmHg with a median of86 mmHg.Conclusions: All medications had a significant elevation of IOP withpegaptanib having the highest elevation probably related to the highervolume of pegaptanib that needs to be injected (0.09 ml).The elevation in IOP demonstrated in this study is significant enoughto justify a focused study on possible long-term side effectssecondary to this acute IOP elevation.Commercial Relationships: Julio C. Chora, None; Victor H.Gonzalez, Genetech (C), Regeneron (C), Pfizer (C), Valiant (C),Alimera (C)Program Number: 1991 Poster Board Number: C0205Presentation Time: 11:00 AM - 12:45 PMEfficacy and safety of Polyquad-preserved Travoprost in OcularHypertensives and Open Angle Glaucoma patients: an openlabel, observational, 6-month, switch studyTeresa Rolle 1 , Rachele Penna 1 , Alessandro G. Actis 1 , LuigiaScudeller 3 , Gianmaria Pasinetti 4 , Gemma C. Rossi 2 . 1 ClinPhysiopathol-Section of Opht, University of Torino, Torino, Italy;2 Eye Clinic, University of Pavia, IRCCS Policlinico San MatteoFoundation, University of Pavia, Pavia, Italy; 3 Clinical Epidemiologyand Biometric Unit, Scientific Direction, IRCCS Policlinico SanMatteo Foundation, University of Pavia, Pavia, Italy; 4 Eye Unit,Istituto Beato Palazzolo, Bergamo, Italy.Purpose: To evaluate the clinical benefit of eliminating BAK fromprostaglandin analog therapy examining the safety and efficacy ofpolyquad -preserved travoprost ophthalmic solution compared toprevious use of latanoprost monotherapy.Methods: This was an observational study. Consecutive adults withopen-angle glaucoma or ocular hypertension treated with latanoprostmonotherapy who were going to change brand therapy to the genericone, were switched to travoprost BAK-free ophthalmic solution. Allpatients were submitted to an ophthalmic examination, IOPmeasurement and ocular surface status (BUT and corneal staining)evaluation. Patients' discomfort was evaluated with the OcularSurface Disease Index (OSDI). All examinations 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 – Physiology/Pharmacologybaseline and 6 months later. Descriptive statistics were produced fordemographic and clinical characteristics of cases. Median andinterquartile range are presented for non-normally distribuitedvariables. For group comparison, parametric and non-parametric testswere used for quantitative variables and Pearson’s χ2 test forcategorical variables. All analysis refer to right eye, left eye’s dataare similar.Results: 44 patients were enrolled and treated with polyquadpreservedtravoprost once a day. TF-BUT changed from 8 [IQR 6-10]sec at baseline to 10 [IQR 8-12] sec at 6 month (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyperformed daily without the pressure decreases associated withsedation and removing the anesthesia-related risk and side effects,reducing the anesthesia-related risk to the animals.Commercial Relationships: Mark Vezina, Charles RiverLaboratories (E); Sylvie Wise, Charles River (E); Kelly Tenneson,Charles River (E), Eleven Biotherapeutics (E); Martin Bussieres,Charles River Laboratories (E), V&O Services Inc (C); TimothyBryant, Charles River Laboratories (E); Elridge Edwards, CRL (I)Program Number: 1994 Poster Board Number: C0208Presentation Time: 11:00 AM - 12:45 PMExpression of Circadian Rhythm Genes in the Mouse Iris-CiliaryBody ComplexJeffrey J. Dunmire 1 , Lauren Dalvin 1 , Rachida Bouhenni 1 , Deepak P.Edward 2, 3 . 1 Ophthalmology, Summa Health System, Akron, OH;2 Ophthalmology, King Khaled Eye Specialist Hospital, Riyadh, SaudiArabia; 3 Ophthalmology, Wilmer Eye Institute, Johns HopkinsUniversity, Baltimore, MD.Purpose: It is well known that intraocular pressure (IOP) exhibits acircadian rhythm. However, the molecular basis for this circadianpattern of IOP remains to be understood. We hypothesized that alocal circadian clock exists in the iris-ciliary body complex, whereclock gene expression may contribute to daily IOP variation. Weinvestigated the temporal and spatial expression of circadian clockgenes in iris-ciliary body of mice.Methods: C57BL/6J mice were entrained to a 12-hour light-darkcycle for two weeks and then sacrificed at Zeitgeber Times (ZT) 2, 6,10, 14, 18, and 22 (n=4 per group), where ZT 12 is the beginning ofthe dark cycle. Eyes were enucleated and either flash-frozen or fixedin 10% neutral-buffered formalin. Following dissection of iris-ciliarybody tissues, RNA was extracted and assessed by real-time PCR inan array format profiling 84 circadian clock or clock-controlledgenes. Immunohistochemistry (IHC) was performed on formalinfixed eyes to confirm protein expression and identify localization.Results: Among the 84 genes tested in the PCR array, 35 showedexpression with varying degrees of circadian oscillation. Thecircadian clock genes Bmal1, Clock, Cry1, Cry2, Per1, Per2, andPer3 were all found to be rhythmically expressed. Expression levelswere highest for Bmal1 and Clock, with peak expression between ZT2-6, and their oscillation was antiphase to Cry1, Cry2, Per1, Per2, andPer3. Other well known clock-controlled genes that showed a clearpattern of rhythmic expression included Dbp, Rev-erbA-α, Rev-erbβ,Tef, and Rorc. The amplitude of oscillation was greatest for Dbp,with a 17-fold difference in expression between ZT 10 and ZT 22.IHC showed strong staining of CLOCK and BMAL1 at ZT 14 in thenon-pigmented ciliary epithelium. Expression was also detected inthe anterior surface of the iris, but to a much lesser degree. Proteinlocalization was both nuclear and cytoplasmic, with increaseddistribution to the cytoplasm at ZT 14 compared with ZT 2.Conclusions: The components of an intrinsic circadian clock wereidentified in the mouse iris-ciliary body complex with temporalchanges in clock gene expression and immunolocalization of theirprotein products. This finding is a first step toward elucidation ofclock-controlled pathways that may be involved in aqueous humordynamics and an understanding of diurnal IOP variation.Commercial Relationships: Jeffrey J. Dunmire, None; LaurenDalvin, None; Rachida Bouhenni, None; Deepak P. Edward, NoneSupport: Summa FoundationProgram Number: 1995 Poster Board Number: C0209Presentation Time: 11:00 AM - 12:45 PMSphingosine-1-Phosphate signaling in cultured human trabecularmeshwork cellsSietse T. Braakman 1 , Kristin M. Perkumas 2 , Darryl R. Overby 1 ,David F. Woodward 3 , W Daniel Stamer 2 . 1 Bioengineering, ImperialCollege London, London, United Kingdom; 2 Ophthalmology, DukeUniversity, Durham, NC; 3 Biol Sci RD-2C, Allergan, Inc, Irvine, CA.Purpose: Sphingosine-1-Phosphate (S1P) is a bioactive lipid thatdecreases conventional outflow facility in human, porcine and mouseeyes, likely by decreasing the permeability of Schlemm’s canalendothelium (SCE). Located upstream of SCE, the trabecularmeshwork (TM) is a prime candidate for S1P synthesis. In this study,we test the hypothesis that human TM cells synthesize S1P as apossible mechanism to regulate facility. We also measure S1P levelsin human aqueous humor (AH).Methods: TM cell lines were isolated from non-glaucomatous humandonors and differentiated by allowing confluent cultures to mature for1 week in DMEM containing 10% fetal bovine serum (FBS) followedby 1 week in 1% FBS. After differentiation, medium was refreshed(1% FBS) and conditioned by the cells for 2 hours, before beingsampled. AH samples were obtained from 5 non-glaucomatouspatients during cataract surgery. Conditioned medium, fresh mediumcontaining 1% FBS, and pooled AH were analyzed for S1P andprostaglandin E2 (PGE2) using LC-MS/MS. Cell viability wasmeasured by lactate dehydrogenase (LDH) activity using acolorimetric NAD assay. Cell lysates were analyzed by western blotfor sphingosine-kinase 1 (SphK1).Results: S1P in fresh medium (1% FBS) was measured to be 3.5 nM,which decreased to 1.4±0.3nM (mean±SD, n=29) after 2h of cellconditioning. In contrast, PGE2 levels remained constant (65±22nMvs. 60nM, n=29, p=0.6). Ceramides and sphingosine, substrates forS1P synthesis, were present in conditioned media (26±9.7 and5.0±1.4nM, n=29). In AH, S1P was below the detection limit, whilePGE2 was measured to be 28.8nM. There were no signs of cell death,as measured by LDH (6.3-14.3 mIU/mL vs. 1139mIU/mL in positivecontrol). SphK1 was detected by western blot in both TM and SCcells.Conclusions: S1P was undetectable in AH and conditioned mediafrom TM, however both TM and SC cells expressed the enzymes andsubstrates necessary for S1P generation and receptors for signaling.Since S1P agonists/antagonists alter outflow facility, endogenous S1Pgeneration must be local (autocrine/paracrine), stimulated and/orshort lived.Acknowledgements: Lipidomics Core Facility MUSCCommercial Relationships: Sietse T. Braakman, None; Kristin M.Perkumas, None; Darryl R. Overby, Allergan, Inc. (F), Allergan,Inc. (C); David F. Woodward, Allergan Inc. (E); W Daniel Stamer,Allergan (F), Alcon (F), Acucela (C), Aerie (C), Cytokinetics (C)Support: Stichting Dondersfonds (STB), NIH EY022359, Researchto Prevent BlindnessProgram Number: 1996 Poster Board Number: C0210Presentation Time: 11:00 AM - 12:45 PMShear Stress Stimulation of NO release from Schlemm’s CanalCellsNicole E. Ashpole 1 , Darryl R. Overby 2 , C R. Ethier 3 , W DanielStamer 1, 4 . 1 Biomedical Engineering, Duke University, Durham, NC;2 Biomedical Engineering, Imperial College, London, UnitedKingdom; 3 Biomedical Engineering, Georgia Institute of Technology,Atlanta, GA; 4 Ophthalmology, Duke University, Durham, NC.Purpose: Nitric Oxide (NO) has important physiological effects,including increasing endothelial permeability and smooth musclerelaxation. In vascular endothelia, NO is produced by endothelial NOsynthase (eNOS), whose activity and abundance are regulated byshear stress. In Schlemm’s Canal (SC) shear stress is calculated to becomparable to those in large arteries (2-20 dynes/cm^2). Here 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 – Physiology/Pharmacologyinvestigate the relationship between NO production and shear stressin cultured human SC cells.Methods: Two strains of human SC endothelial cells isolated fromnon-glaucomatous donor eyes were seeded into Ibidi flow chambersat confluence, allowed to acclimate for at least 7 days and subjectedto continuous shear stress (0.1, 10 and 15 dynes/cm^2) for 7 days.Cell alignment was assessed using phase-contrast microscopycoupled with Cell Profiler analysis (Broad Institute). NO productionwas measured by two methods: (i) DAF-FM Fluorescence(Invitrogen) monitored using ImageJ analysis, giving a qualitativemeasure of NO concentration; and (ii) Griess Reagent reaction(Invitrogen), which measures nitrite concentration, a product of thespontaneous oxidation of NO. HUVECs were used as a positivecontrol.Results: SC cells, like HUVECs, aligned with the direction of flow, abehavior that was both time and shear-dependent. While HUVECsaligned within hours, SC cells took days. NO production by SC cellsincreased with shear stress as assayed by both DAF-FM Fluorescenceand Griess Reagent. A similar effect was seen in HUVECs (Table).Conclusions: Human SC cells respond to shear stress similar to othervascular endothelia, aligning with flow and increasing NO productionin a shear-dependent manner. When IOP increases (and SC collapses,thus increasing shear stress in SC), our data suggest that NOproduction by SC cells will increase. This could relax trabecular cellsand increase permeability of the inner wall to increase outflowfacility. It will be important to see if the shear-eNOS response iscompromised in glaucomatous SC cells.Commercial Relationships: Nicole E. Ashpole, None; Darryl R.Overby, Allergan, Inc. (F), Allergan, Inc. (C); C R. Ethier, None; WDaniel Stamer, Allergan (F), Alcon (F), Acucela (C), Aerie (C),Cytokinetics (C)Support: Research to Prevent Blindness and National Institutes ofHealth (Grant #EY017007 and #EY022359).Program Number: 1997 Poster Board Number: C0211Presentation Time: 11:00 AM - 12:45 PMPeptide and Non-Peptide Bradykinin (BK) Agonists andAntagonists Help Define Functional BK Receptors in HumanTrabecular Meshwork and Ciliary BodyNaj Sharif 1 , Parvaneh Katoli 1 , Curtis R. Kelly 1 , Linya Li 1 , ShouxiXu 1 , Yu Wang 1 , Ganesh Prasanna 1 , Keith D. Combrink 1 , MarkHellberg 1 , Shahid Husain 2 . 1 Ophthalmology Research, AlconResearch, Ltd-Novartis Institute of Biomedical Research (NIBR),Fort Worth, TX; 2 Ophthalmology, Medical University of S. Carolina,Charlston, SC.Purpose: 1) to examine bradykinin (BK) receptor system in humantrabecular meshwork (h-TM), human ciliary muscle (h-CM) andciliary process (CP) by immunohistochemistry; 2) topharmacologically characterize the associated signaling mechanismsin isolated cells from these tissues using peptide and non-peptide BKagonists (Compounds 1 & 2) and antagonists; 3) to study theintraocular pressure (IOP)-lowering effects of Compound 1 (CMPD-1)Methods: Published methods were utilized throughoutResults: Human and Cynomolgus monkey TM, CM and CPexpressed a high level of B 2 -receptor protein immunoreactivity. Inisolated h-TM, h-CM and human non-pigmented epithelial (h-NPE)cells, BK and related analogs (e.g. Lys-BK; Hyp 3 -BK) exhibited ahigh potency (EC 50 = 2-8 nM, n = 3-6), while Des-Arg 9 -BK (B 1 -receptor agonist) was much weaker (EC 50 = 4.2 µM), at stimulatingintracellular Ca 2+ ([Ca 2+ ] i ) mobilization. Two non-peptide B 2 -receptor agonists (CMPD-1 & 2) had lower potencies (CMPD-1EC 50 s = 150-276 nM; CMPD-2 EC 50 s = 25-74 nM; n = 3-29) thanBK in these assays. While BK peptides and CMPD-2 were fullagonists, CMPD-1 was a partial agonist (E max = 38% in NPE; 64% inCM; 80% in TM cells). These effects of BK, CMPD-1 & 2 wereblocked by HOE-140 (peptide B 2 -antagonist; K i = 1-8 nM; n = 4-6)and WIN-63448 (non-peptide B 2 -antagonist; K i = 157 - 450 nM, n =4-5) in all these ocular cells. While h-CM and h-TM cells respondedto BK, CMPD-1 & 2 by secreting prostaglandins (PGE 2 , PGF 2α ) (e.g.EC 50 s = 4 - 61 nM in h-TM cells, n = 3-5; CMPD-1 E max = 28%),NPE cells failed to release any PGs. The latter PG secretion effects ofthe BK agonists were also blocked by HOE-140 and WIN-63448, andwere attenuated by cyclooxygenase inhibitors (bromfenac andflurbiprofen). BK, CMPD-1 & 2 also increased secretion of Pro-MMP-3 by 1.3-1.6 fold above basal levels in h-CM cells 24 hr postincubation. CMPD-1 lowered IOP in conscious ocular hypertensiveCynomolgus monkeys (e.g. 37.7 ± 5.4% with 30 µg, 24 hrs posttopical ocular dosing).Conclusions: These data support the existence of functionally activeB 2 -receptors in h-TM, h-CM and h-NPE cells that mediate [Ca 2+ ] imobilization, PG secretion (not in NPE cells) and pro-MMP-3 release(h-CM). These data help explain the potent, efficacious, andprolonged IOP-lowering effects of CMPD-1.Commercial Relationships: Naj Sharif, Alcon Research, Ltd (aNovartis Co.) (E); Parvaneh Katoli, Alcon Labs (E); Curtis R.Kelly, Alcon / Novartis Institutes for Biomedical Research (E);Linya Li, Alcon Labs (E); Shouxi Xu, Alcon, Novartis (E); YuWang, Novartis, Alcon (E); Ganesh Prasanna, Alcon Research Ltd(E), Novartis Institute of Biomedical Research (E); Keith D.Combrink, Novartis (E); Mark Hellberg, NIBR (E), Alcon (P);Shahid Husain, NoneProgram Number: 1998 Poster Board Number: C0212Presentation Time: 11:00 AM - 12:45 PMEffect of ONO-9054 on Aqueous Humor Dynamics in MonkeysTomohiro Karakawa, Shinsaku Yamane, Kazufumi Nagai, ShintaroNakao, Tsutomu Shiroya, Yutaka Shichino. ONOPHARMACEUTICAL CO., LTD, Mishimagun, Japan.Purpose: ONO-9054 (Ono Pharmaceuticals, Osaka Japan) is a novelprodrug compound. ONO-9054 is an isopropyl ester derivative of thefree acid ONO AG-367 that has been classified as a dual FP/EP3agonist that may be effective in lowering intraocular pressure inhumans. The purpose of this study was to investigate the effect ofONO-9054 on the aqueous humor dynamics in cynomolgus monkeys.Methods: Aqueous humor flow was measured with afluorophotometer in monkeys. Ophthalmic vehicle, timolol (5000μg/mL), latanoprost (50 μg/mL) and ONO-9054 (3 μg/mL) wereadministered topically into the eye, and aqueous humor flow rate andintraocular pressure (IOP) measurements were conducted. Outflowfacility was measured by a two-level, constant-pressure perfusion©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 – Physiology/Pharmacologymethod in monkeys. Latanoprost (50 μg/mL) and ONO-9054 (3 and30 μg/mL) were administered to the eye unilaterally, and thecontralateral eye was administered ophthalmic vehicle solution.Measurements of outflow facility and IOP were conducted.Results: Timolol, latanoprost, and ONO-9054 reduced IOP. Timololdecreased the aqueous humor flow (0.93 ± 0.16 μL/min) relative tovehicle (1.88 ± 0.13 μL/min). On the other hand, ONO-9054 at 3μg/mL (2.06 ± 0.24 μL/min) and latanoprost (2.05 ± 0.08 μL/min)had no effect on aqueous humor flow. When compared with thecontralateral vehicle-treated eyes (0.62 ± 0.10 μL/min/mmHg),latanoprost had no effect on the outflow facility (0.62 ± 0.11μL/min/mmHg). Likewise, ONO-9054 at 3 μg/mL had no effect onthe outflow facility (0.56 ± 0.08 μL/min/mmHg). On the other hand,ONO-9054 at 30 μg/mL slightly increased outflow facility (0.87 ±0.09 μL/min/mmHg) relative to contralateral eyes (0.66 ± 0.08μL/min/mmHg).Conclusions: ONO-9054, a dual agonist for prostaglandin FP andEP3 receptors, did not have any effect on aqueous humor flow. Thepresent results suggest that the mechanism of IOP-lowing for ONO-9054 appears to involve an enhancement of uveoscleral outflow,similar to the mechanism reported for pure FP receptor agonists, andan effect on conventional outflow pathways.Commercial Relationships: Tomohiro Karakawa, ONOPHARMACETICAL CO., LTD (E); Shinsaku Yamane, ONOPHARMACEUTICAL CO., LTD. (E); Kazufumi Nagai, ONOPHARMACEUTICAL CO.,LTD (E); Shintaro Nakao, ONOPharmaceutical Co.,LTD (E); Tsutomu Shiroya, ONOPharmaceutical.Co.Ltd. (E); Yutaka Shichino, ONO PharmaceuticalCo Ltd (E)Program Number: 1999 Poster Board Number: C0213Presentation Time: 11:00 AM - 12:45 PMCabergoline and IOP: implications for structure-based drugdiscovery of selective dopaminergic ligandsFilippo Drago, Chiara Bianca Maria Platania, GiuseppinaMarrazzo, Gian Marco Leggio, Claudio Bucolo. Clinical andMolecular Biomedicine, University of Catania, Catania, Italy.Purpose: To investigate the role of cabergoline, an anti-Parkinsonagent, on intraocular pressure regulation using wild type (WT) andD3R knockout (KO D3R-/-) mice. Further, to assess the precise roleof dopaminergic and serotonergic systems on IOP modulation, acomputational structure-based study was also carried out.Methods: WT and KO D3R-/- C57BL6J mice were used. Both micewere used with normal eye pressure or steroid-induced ocularhypertension. All animals were treated according to the ARVOstatement for the use of animals in ophthalmic and vision research.Mice were treated with cabergoline at different concentration (0.01%,0.1%, 1%, 5%) and IOP measured by tonometer. We modeled andoptimized the structures of hD3, h5-HT1a, h5-HT2a, h5-HT2b, h5-HT2c receptors by homology modeling and by molecular dynamicsrespectively. Next we docked, using AutoDock 4.2, cabergoline intothe binding sites of these receptors, and rescored the binding modeswith DSX-score.Results: Topical application of cabergoline significantly (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyACD were temporally close to the lowest habitual IOP during theday.Conclusions: The increase in axial length at night (approximately 75µm) can be attributed to an increase in anterior chamber depth(approximately 90 µm). The increase in anterior chamber depth isindependent of any change in lens thickness. These potentially IOPmediatedchanges appear complimentary towards maintenance ofrefractive status of the eye, demonstrating inherent homeostaticmechanisms of the eye despite significant changes in the IOP.Brimonidine use does not alter the normal diurnal rhythm of ocularbiometric parameters but a more potent ocular hypotensive drug may.Commercial Relationships: Shan Fan, None; Vikas Gulati, None;Donna G. Neely, None; Nathan V. Harms, None; Carol B. Toris,Alcon (F), Allergan (F)Support: AGS MAPS grant (VG); Research to Prevent BlindnessClinical Trial: NCT01144494Program Number: 2001 Poster Board Number: C0215Presentation Time: 11:00 AM - 12:45 PMVoltage-coupled single-needle constant-pressure anteriorchamber perfusion in live miceMinHee K. Ko 1 , Aleksandr Yelenskiy 2 , Jose M. Gonzalez 1 , James C.Tan 1 . 1 Ophthalmology, University of Southern California, LosAngeles, CA; 2 School of Medicine, University of Wisconsin,Madison, WI.Purpose: To describe voltage-coupled single-needle constantpressureanterior chamber perfusion in live miceMethods: We established apparatus in which transduced pressurewas coupled to a microperfusion pump to automatically modulateflow rate to maintain a steady pressure and measure outflow facilityby constant pressure perfusion. All perfusion was performed by 35Gsingle-needle anterior chamber cannulation in anesthetized mice. Wecharacterized the following: (i) perfusion pressure stability; (ii)outflow facility; (iii) presence of “washout”; (iv) effect of differentperfusates such as phosphate buffered saline with Ca 2+ and Mg 2+ (PBSw Ca/Mg), PBS without Ca/Mg (PBS w/o Ca/Mg), and Barany’ssolution. H&E staining was performed on frozen sections followingperfusions.Results: Twenty nine live C57BL/6 mice underwent perfusion.Constant pressure was attained within seconds, stably maintained,and not significantly affected by different perfusates (p>0.05).Relationship between flow rate and pressure fit a linear function forperfusion between 15-35mmHg (R 2 >0.9). Outflow facilitydetermined by 1-level constant pressure perfusion was similarirrespective of perfusate (p>0.5): 0.0095 μl/min*mmHg for PBS wCa/Mg (n=10); 0.0123 μl/min*mmHg for PBS w/o Ca/Mg (n=10);0.0074 μl/min*mmHg for Barany’s solution (n=9). Needle resistancewas negligible relative to physiologically relevant perfusion. 2-levelconstant pressure perfusion over 2 hours showed no washoutphenomenon. Histological disruption of drainage tissue followingperfusions was not seen.Conclusions: Stable constant pressure perfusion underphysiologically relevant conditions was achieved by perfusion usingsingle-needle cannulation, which is well-suited to the tiny mouse eye.The washout phenomenon was not seen. Our methods are potentiallyapplicable to live mouse studies.Commercial Relationships: MinHee K. Ko, None; AleksandrYelenskiy, None; Jose M. Gonzalez, None; James C. Tan, NoneSupport: NIH grant EY020863, NIH EY03040, Career DevelopmentAward from Research to Prevent Blindness (JCHT), and anunrestricted grant from the Research to Prevent BlindnessProgram Number: 2002 Poster Board Number: C0216Presentation Time: 11:00 AM - 12:45 PMEffects of Nitric Oxide Donor on Outflow Facility in MiceJason Y. Chang 1 , Catherine M. Marando 1 , C R. Ethier 1, 2 , W DanielStamer 3 , Darryl R. Overby 1 . 1 Bioengineering, Imperial CollegeLondon, London, United Kingdom; 2 Biomedical Engineering,Georgia Institute of Technology, Atlanta, GA; 3 Ophthalmology,Duke University, Durham, NC.Purpose: Overexpression of endothelial nitric oxide synthase(eNOS) has been shown to increase conventional outflow facility andlower intraocular pressure (IOP) in transgenic mice (Stamer et al.IOVS 2011 52:9438). To better understand how nitric oxide (NO)contributes to the regulation of IOP in non-transgenic animals, weexamined the effects of a NO-donor on conventional outflow facilityin wild-type mice.Methods: Enucleated eyes from C57BL/6 mice (6-8 week oldfemales) were perfused at pressures of 8, 15, 20 and 25 mmHg usinga computerized system. Eyes were perfused at 35°C in a bath ofisotonic saline with Dulbecco’s phosphate buffered saline + 5.5mMglucose (DBG) supplemented with either a light-activated NO-donor(100µM S-nitroso-N-acetylpencillamine, SNAP; N = 4) or inactivedonor (100µM N-acetylpencillamine, NAP; N = 4). Eyes wereperfused either immediately after enucleation or after 4 hours storageat 4°C, with the treatments assigned randomly. The conventionaloutflow facility was defined as the slope of the linear regressionthrough the flow rate-pressure data. NO release from SNAP and NAPwas characterized using an NO-sensitive probe (WPI; ISO-NO II)that was calibrated following the manufacturer’s protocol. Lightexposure levels were measured using a luminometer (Mastech).Results: SNAP was highly light sensitive: a brief initial lightexposure (10 min; 680-800 lumens/m2) followed by darkness causeda burst of NO release, however continued light exposure led to aninitial increase followed by a rapid NO depletion. Under conditionsof initial light exposure followed by darkness, NO release from 100µM SNAP (10 mL) increased over 1 hr to reach a peak of 140 ± 20nM (mean ± SD), while NO release from 100 µM NAP wasnegligible (0 ± 4 nM). Conventional outflow facility in SNAP-treatedeyes following brief light exposure was 64% greater than in NAPtreatedeyes (0.0570±0.0147 vs. 0.0348±0.0085 µL/min/mmHg; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologytest the hypothesis that plasma membrane proteins from human TMcells are trafficked to exosomes in response to external stimuli.Methods: To monitor plasma membrane protein trafficking toexosomes, we utilized a novel experimental paradigm. Culturedhuman TM cells were serum starved and biotinylated on their cellsurface. Cells were then incubated with or without fetal bovine serum(10% v:v) for various lengths of time. Following serum stimulation,media was collected over time and exosomes were isolated by serialultracentrifugations and characterized by established techniques.Exosome proteins were separated by SDS-PAGE and biotinylatedproteins were detected with strepavadin conjugated HRP.Results: Proteins accessible to the extracellular surface of humantrabecular meshwork cells were exclusively labeled with biotin, asindicated by lack of cytoplasmic actin biotinylation . As early as 4hours after media change, biotinylated proteins were detected onexosomes released from human TM cells, regardless of the presenceof serum. Notably, we observed the appearance of several uniquebiotinylated proteins in exosomes from cells that were stimulatedwith serum. In particular, a ~65kDa, biotinylated exosome proteinwas reproducibly observed. Treatment of cells with the Ca++ionophore, ionomycin (10μM, 10 minutes), at the end of the 4 hourserum stimulation increased the abundance of the observedbiotinylated exosome proteins.Conclusions: Our data demonstrates that plasma membrane proteinsfrom human TM cells are endocytosed, trafficked to, incorporated inand are constitutively released with exosomes. Additionally, wedetected a unique set of proteins trafficked to exosomes upon ligandstimulation. These findings suggest that the endocytic pathway isvery active in TM cells and that exosomal trafficking responds toenvironmental cues. Both are potentially involved in the regulationreceptor signaling, outflow facility and thus intraocular pressure.Commercial Relationships: W M. Dismuke, None; Brian S.McKay, None; Aaditya Khatri, None; Kristin M. Perkumas,None; W Daniel Stamer, Allergan (F), Alcon (F), Acucela (C),Aerie (C), Cytokinetics (C)Support: Research to Prevent BlindnessProgram Number: 2004 Poster Board Number: C0218Presentation Time: 11:00 AM - 12:45 PMPEDF Effects on Outflow FacilityMorgan E. Rogers 1 , Iris Navarro 1 , Kristin M. Perkumas 1 , R RandAllingham 1 , Pratap Challa 1 , Craig E. Crosson 2 , W Daniel Stamer 1 .1 Duke University, Durham, NC; 2 Medical University of SouthCarolina, Charleston, SC.Purpose: Preliminary data from our laboratory show that pigmentepithelium derived factor (PEDF) increases transendothelial electricalresistance of human SC and porcine AAP endothelial monolayers in atime and dose-dependent manner. In this study, our goal was todetermine PEDF levels in human aqueous humor, and the effect ofPEDF on outflow function in enucleated mouse eyes.Methods: Aqueous humor was obtained from consented patientsduring ocular surgery. Fresh samples were immediately placed on dryice and kept frozen at -80C until an ELISA PEDF assay (Chemikine)was performed. Eyes from culled C57BL/6 mice were enucleated andcannulated for ex vivo perfusion using a computer-controlledperfusion system optimized for mouse eyes. Purified PEDF (1μg/ml)was perfused at four different pressures (4, 8, 15, 25 mmHg),measuring flow to determine outflow facility (slope of flow/pressurerelationship). Data were compared to eyes perfused with vehiclealone (negative control) and two positive controls to establish rangefor detection of changes in outflow facility: dithia PGE-1 (10 nM)and sphingosine-1-phosphate (S1P, 5μM).Results: Eighteen human aqueous humor samples were examinedand found to contain PEDF at levels ranging from 0.12-10.72 μg/mL(1.519 ± 2.492). We did not detect a relationship between PEDF leveland gender, race or donor glaucoma status. We perfused a total of 27C57BL/6 mouse eyes. Compared to vehicle-perfused controls, weobserved an 86% increase in outflow facility for dithia PGE-1 (0.035vs. 0.019 μl/min/mmHg), and a 20% decrease in outflow facility forS1P (0.017 vs. 0.022 μl/min/mmHg), similar to values previouslyreported. We perfused 7 eyes with PEDF and observed an averageoutflow facility of 0.018 μl/min/mmHg compared to 0.019μl/min/mmHg for controls. Interestingly, there appeared to be twosets of responses within our data: 4 eyes having mean outflow facilityof 0.010 ± 0.005 μl/min/mmHg, and the other 3 with mean of 0.028 ±0.001 μl/min/mmHg.Conclusions: PEDF is present in human aqueous humor atphysiologically relevant concentrations. On average PEDF did notsignificantly affect outflow facility. However, the data set wasbimodal with 4 eyes decreasing outflow facility by 50%, and theother 3 increasing outflow facility by 67%. We do not presentlyunderstand this phenomenon but have only tested one concentrationof PEDF which was effective in vitro but may be too high ex vivo.We plan to perform a full concentration-response.Commercial Relationships: Morgan E. Rogers, None; IrisNavarro, None; Kristin M. Perkumas, None; R Rand Allingham,New World Medical (C); Pratap Challa, None; Craig E. Crosson,Alimera Sciences (C), Lexicon Pharmaceuticals, Inc (R); W DanielStamer, Allergan (F), Alcon (F), Acucela (C), Aerie (C),Cytokinetics (C)Support: EY022359Program Number: 2005 Poster Board Number: C0219Presentation Time: 11:00 AM - 12:45 PMOcular hypertensive effect of pilocarpine in the anesthetized ratJeffrey W. Kiel, Alma L. Maldonado. Ophthalmology, Univ of TexasHlth Sci Ctr SA, San Antonio, TX.Purpose: A previous study by Pang et al. (Exp Eye Res 80: 207-214,2005) reported that pilocarpine tended to increase IOP in consciousrats one hour after dosing but the effect was not significant. Thepresent study sought to determine if continuous IOP measurementbefore and after application would detect a more robust and rapidresponse.Methods: In rats (n=14) anesthetized with ketamine plus xylazine,we measured femoral mean arterial pressure (MAP) and IOP bydirect cannulation, carotid blood flow (BFcar) by transit timeultrasound and heart rate (HR) by a digital cardiotachometer. Theprotocol entailed 10 min of baseline followed by 20 min ofmeasurement after topical application of pilocarpine (1%). The data(mean +/- standard error) were analyzed by paired t-test.Results: The systemic parameters were unchanged (p>0.05 for MAP,BFcar and HR) before and after pilocarpine (MAP: 96 +/- 4 Vs 92 +/-4 mmHg; BFcar: 4.2 +/- 0.3 Vs 4.0 +/- 0.3 ml/min; HR: 268 +/- 9 Vs268 +/- 9 bpm). IOP increased significantly from 12.6 +/- 0.7 to 14.8+/- 1.0 mmHg (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyfoundation for the development of novel therapeutic agents anddiagnostic tests.Commercial Relationships: Jonathan D. Nussdorf, None; JanetManalac, None; Yan Lu, None; Song Hong, None; Nicolas G.Bazan, NoneCommercial Relationships: Jeffrey W. Kiel, None; Alma L.Maldonado, NoneSupport: NIH Grant EY09702, van Heuven endowmentProgram Number: 2006 Poster Board Number: C0220Presentation Time: 11:00 AM - 12:45 PMPoly-Unsaturated Fatty Acids in Human Aqueous HumorJonathan D. Nussdorf 1, 2 , Janet Manalac 1 , Yan Lu 3 , Song Hong 3 ,Nicolas G. Bazan 3 . 1 Department of Ophthalmology, Ochsner ClinicFoundation, New Orleans, LA; 2 University of Queensland School ofMedicine, Brisbane, QLD, Australia; 3 Neuroscience Center,Louisiana State University Health Science Center, New Orleans, LA.Purpose: The purpose of this study is to determine whether lipidsderived from omega-3 and omega-6 polyunsaturated fatty acids(PUFA) are present in human aqueous humor.Methods: Undiluted aqueous humor samples were collected from 10patients who underwent either elective cataract surgery and/or nonelectiveglaucoma surgery. Samples were removed from the anteriorchamber following the initial paracenthesis and prior to any furtherintervention. The samples were immediately packed in dry ice andstored at -80 °C. The samples were analyzed in a masked fashion forthe presence of poly-unsaturated fatty acids. Lipids were extractedfrom aqueous humor by solid phase exaction, and analyzed via liquidchromatography tandem mass spectrometry (LC-MS/MS, ThermoScientific) in negative-ion mode. Deuterium-labeled prostglandin D2(PGD2-d4) and/or docosahexaenoic acid (DHA-d5) were added toeach sample as the internal standard for quantification.Results: The analysis of ten samples of aqueous humor demonstratedthe presence of the omega-6 fatty acid linoleic acid (LA), the omega-3 fatty acid-alpha linolenic acid (ALA), with its metabolites DHAand EPA. A one-way repeated measures ANOVA demonstrated asignificant difference between the concentration levels of the 8 lipidsidentified in the aqueous humor [F (7, 27) = 634.2, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyProgram Number: 2176Presentation Time: 3:00 PM - 3:15 PMMULTIFUNCTIONAL ANTIOXIDANTS PROTECT CELLSFROM MITOCHONDRIAL DYSFUNCTION AND ABETANEUROTOXICITYHiroyoshi Kawada 1 , Peter F. Kador 1, 2 . 1 Pharmaceutical Sciences,University of Nebraska Medical Center, Omaha, NE;2 Ophthalmology, University of Nebraska Medical Center, Omaha,NE.Purpose: We have synthesized orally active multifunctionalantioxidants (MFAOs) possessing distinct free radical scavengingactivity and independent metal attenuating activity and demonstratedin rats that they delay cataract formation and protect thephotoreceptor layer against light damage. Since mitochondrialdysfunction and amyloid beta (Aβ) neurotoxicity are associated withage-related retinal changes, the effect of MFAOs on these factorshave been investigated.Methods: Human neuroblastoma (SH-SY5Y) cells and retinalpigmented epithelial (RPE) cells were pre-incubated with mediumcontaining 1 mM/10 µM of select MFAOs along with their respectivenonfunctional parent compounds as controls, and clioquinol (CQ).Mitochondrial viability was assessed after 3-hr exposure tomanganese chloride with/without the presence of drugs usingHoechst 33342 and rhodamine 123 staining. The neurotoxic potentialof Zn promoted Aβ aggregation on these cells was evaluated withZinquin staining (10 µM) which visualizes the cellular levels of labileZn released from the Aβ:Zn complex by MFAOs, their respectivenonfunctional parent compounds or CQ. Cellular fluorescence wasevaluated using Zeiss confocal scanning laser microscopy (LSM).Results: The mitochondria of cells stained with rhodamine fluoresceand exposure to manganese chloride induces mitochondrialdysfunction which results in loss of fluorescent staining. Cells treatedwith MFAOs or CQ in the presence of manganese chloride retainedfluorescence while fluorescence was lost when the cells were treatedwith the nonfunctional parent compounds. Zinquin staining of cellsexposed to Aβ:Zn aggregates and MFAOs or CQ demonstrated thepresence of labile Zn suggesting that these compounds have a metalattenuating effect on the Aβ:Zn aggregate. Similar Zinquin stainingwas not observed in cells similarly exposed to the Aβ:Zn aggregatesand treated with the nonfunctional parent compounds.Conclusions: MFAOs protect both human neuroblastoma and RPEcells against manganese chloride induced mitochondrial dysfunctionand neurotoxicity of Zn promoted Aβ aggregation by releasing Znfrom the Zn-Aβ complex. Since mitochondrial dysfunction and Zn-Aβ complex are both present in age-related macular degeneration,MFAOs may have therapeutic potential.Commercial Relationships: Hiroyoshi Kawada, None; Peter F.Kador, Aventix Animal Health (C), Aventix Animal Helth (F),Aventix Animal Helth (R), Therapeutic Vision, Inc (F), TherapeuticVision, Inc (F), Therapeutic Vision, Inc. (R), Threapeutic Vision, Inc(S), US 20090105269 (P)Support: Alzheimer's Grant 20110702 and Therapeutic Vision, Inc.Program Number: 2177Presentation Time: 3:15 PM - 3:30 PMIntegrin Peptide Therapy: The First Wet AMD ExperiencePeter K. Kaiser 1 , David S. Boyer 2 , Peter A. Campochiaro 5 , Jose LuisGuerrero-Naranjo 8 , Jeffrey S. Heier 7 , Julia Kornfield 6 , Baruch D.Kuppermann 3 , Hugo Quiroz-Mercado 4 , Samantha Salinas Longoria 8 ,Shulamit Schwartz 4 . 1 Division of Ophthalmology, Cole Eye Institute,Cleveland, OH; 2 Retina Vitreous Associates, Los Angeles, CA; 3 Deptof Ophthalmology, Unversity of California, Irvine, Irvine, CA; 4 Deptof Ophthalmology, Unversity of Colorado, Denver, Denver, CO;5 Dept of Ophthalmology, Wilmer Eye Institute - Johns Hopkins,Baltimore, MD; 6 Dept of Chemical Engineering, California Instituteof Technology, Pasadena, CA; 7 Ophthalmic Consultants of Boston,Boston, MA; 8 Dept of Ophthalmology, Association Para Evitar LaCeguera, Mexico City, Mexico.Purpose: ALG-1001 is a synthetic anti-integrin oligopeptide. ALG-1001 inhibits integrin receptors in vitro and arrests aberrant bloodvessel growth in vivo meditated by integrins αvβ3, αvβ5, and α5β1 -sites that are expressed in neovascular ocular tissue in wet AMD anddiabetic retinopathy. ALG-1001 has demonstrated a statisticallysignificant reduction in CNV, ROP, and vascular permeability inmouse models conducted by Dr. Peter Campochiaro. A 15 subjecthuman phase 1 DME study demonstrated nearly 55% of the studysubjects improving 3 lines or more in BCVA with at least a 30%reduction in OCT CMT with ALG-1001 mono-therapy.The purpose of this study is to evaluate the safety and dose ranging ofintravitreal ALG-1001 in subjects with CNV due to wet AMD with aprimary endpoint of observing for any dose limiting toxicity.Methods: Key inclusion criteria included: Baseline BCVA between20/50 and 20/320 and CNV due to AMD with no previous anti-VEGF treatment within 45 days of enrollment. A combination oftreatment naïve and previously treated subjects were enrolled. Allpatients received a loading dose of 3 monthly intravitreal injectionsof either 1.5mg, 2.5mg, or 4.0mg ALG-1001 and were followed foran additional 4 months off treatment. Safety measurements includedBCVA, slit lamp, fundus exam, IOP, OCT, FA, and fundus photos.Results: To date, 15 subjects with neovascular AMD have beenenrolled in this ongoing open label safety study. There have been noSAEs reported to date. AEs have primarily been limited to injectionrelated events. While this is an ongoing study with 15 subjectsenrolled to date, there are already clear improvements in BCVA andmacular anatomy by OCT in this mono-therapy study inapproximately 40% of the study subjects with clinical benefits lastingat least 60 days off treatment after a 3 monthly injection loadingdose.Conclusions: This was the first clinical trial of ALG-1001 in wetAMD and the first clinical study evaluating the dose ranging safety inhuman subjects. To date, there has been consistency in the lack oftoxicity across all study metrics at doses up to 4.0 mg. Despite thesmall study size and open-label monotherapy dosing regimen,clinically relevant indicators of efficacy are apparent withimprovements in BCVA alongside anatomic improvements in OCTCMT that persist at least 60 days past the last intravitreal treatment.Further studies will evaluate the further efficacy of this therapy.Commercial Relationships: Peter K. Kaiser, Allegro Ophthalmics(C), Alcon (C), Novartis (C), Bayer (C), Regeneron (C), Genentech(C), Ophthotech (C); David S. Boyer, Alcon (C), Allegro (C),Allergan (C), Bayer (C), Genentech (C), Glaukos (C), GSK (C),Neurotech (C), Optos (C), Regeneron (C); Peter A. Campochiaro,Advance Cell Technology (C), Aerpio (C), Elan (C), Gene Signal(C), Genentech (C), GlaxoSmithKline (C), LPath, Inc (C), Norvox(C), Regeneron (C), Genentech (F), Genzyme (F), GlaxoSmithKline(F), Oxford Biomedica (F); Jose Luis Guerrero-Naranjo,Neurotech (F); Jeffrey S. Heier, Acucela (C), Aerpio (C), Alimera(F), Allergan (C), Bayer (C), Forsight Labs (C), Fovea (F),Genentech (C), Genzyme (C), Genentech (F), Genzyme (F),Thrombogenics (C), Sequenom (C), Notal Vision (F), Novartis (F),Ophthotech (F), Ophthotech (C), Oraya (C), Paloma (F), Regeneron(F), Regeneron (C); Julia Kornfield, Allegro Ophthalmics (C);Baruch D. Kuppermann, Alimera (C), Allegro (C), Allergan (C),Genentech (C), Glaukos (C), GSK (F), Novagali (C), Novartis (C),Ophthotech (C), Pfizer (C), Regeneron (C), Santen (C), SecondSight©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 – Physiology/Pharmacology(C), Teva (C), ThromboGenics (C); Hugo Quiroz-Mercado, AllegroPharmaceutical (C); Samantha Salinas Longoria, None; ShulamitSchwartz, NoneClinical Trial: NCT01482871Program Number: 2178Presentation Time: 3:30 PM - 3:45 PMTopical Pazopanib for the Treatment of Previously UntreatedChoroidal Neovascularization due to Age-related MacularDegenerationRishi Singh 1 , John I. Wurzelmann 2 , Li Ye 3 , Michael A. Fries 4 , JohnNorris 2 , Mohammad Hossain 4 , Trupti M. Trivedi 4 , Deborah S. Kelly 4 .1 Cole Eye Institute, Cleveland Clinic, Cleveland, OH;2 GlaxoSmithKline, Research Triangle Park, NC; 3 GlaxoSmithKline,Upper Providence, PA; 4 GlaxoSmithKline, King of Prussia, PA.Purpose: To evaluate the potential of topical ocular pazopanib, atyrosine kinase inhibitor, to reduce retinal edema and improve visualacuity (VA) in subjects with previously untreated subfoveal choroidalneovascularization (CNV) secondary to age-related maculardegeneration (AMD) over 4 weeks and to characterize thesafety/tolerability of the study drug over 12 weeks.Methods: In a multicountry Phase 2a open-label trial, 19 eligiblesubjects were treated with pazopanib eye drops 10 mg/mL (1 drop), 4times daily (QID) for 12 weeks. VA, optical coherence tomography(OCT), and safety were assessed weekly in Month 1, biweekly inMonth 2, and once in Month 3. The primary assessments were thechanges from baseline after 4 weeks of treatment in central retinalthickness (CRT) as measured by OCT, and best-corrected visualacuity (BCVA) as determined by the electronic ETDRS method.Results: There were no meaningful changes from baseline at Week 4in mean (standard deviation [SD]) CRT (38 [90] µm) or mean (SD)BCVA (0 [10] letters) (primary endpoint). Similarly, there were nomeaningful changes from baseline in any of the additional parametersmeasured by OCT, or in CNV or total lesion size as measured byfluorescein angiography. There were no obvious differences observedfor changes from baseline in BCVA or CRT between subjects withthe CFH Y402H T allele (CT and TT genotypes combined) andsubjects with the CC genotype. In the study eye, 8 of 19 subjectsexperienced 9 ocular adverse events (AEs), 1 of which was severe(macular edema due to progression of the underlying disease). Ninesubjects discontinued due to protocol-defined stopping criteria andreceived rescue medication. Nine subjects reported nonocular AEs, 1of which was severe. No deaths or serious AEs were reported.Steady-state concentrations appeared to have been reached by Week2 after administration of study drug.Conclusions: In subjects with previously untreated neovascularAMD, pazopanib eye drops 10 mg/mL QID as monotherapy did notappear to improve BCVA or decrease CRT. There was no meaningfulchange from baseline in CRT, retinal morphology, CNV size, or totallesion size. Pazopanib eye drops 10 mg/mL were generally safe andwell tolerated when instilled QID for 12 weeks.Commercial Relationships: Rishi Singh, Genentech (C), Alcon (C),Bausch and Lomb (R), Zeiss (R), Quark Pharmaceuticals, Inc. (F);John I. Wurzelmann, GlaxoSnithKline (E); Li Ye, None; MichaelA. Fries, GSK (E); John Norris, GlaxoSmithKline (E); MohammadHossain, GlaxoSmithKline (E); Trupti M. Trivedi,GlaxoSmithKline (E); Deborah S. Kelly, GlaxoSmithKline (E)Support: Clinical trial support from GSKClinical Trial: 2011-000243-24Program Number: 2179Presentation Time: 3:45 PM - 4:00 PMIKK2 Inhibition Using TPCA-1/PLGA Microspheres Attenuatesthe Laser Induced Choroidal NeovascularizationQiutang Li 1, 2 , Subhash Gaddipati 1, 2 , M. Clarke Miller 2 , John O.Trent 2, 3 , Henry J. Kaplan 1 , Qingxian Lu 1, 2 . 1 Department of Ophthaland Visual Science, University of Louisville, Louisville, KY; 2 JGBrown Cancer Center, University of Louisville, Louisville, KY;3 Department of Medicine, University of Louisville, Louisville, KY.Purpose: IKK2 is a key kinase in activation of transcriptional factorNF-kappaB that regulates multiple cellular processes includinginflammation, stress response, cell death and angiogenesis.Neovascularization is a hallmark of wet AMD. The present studyaims to assess the therapeutic effect of IKK2 inhibitor, TPCA1, onthe laser-induced choroid neovascularization (CNV) usingbiodegrable poly-lactide-co-glycolide (PLGA) microsphere deliveryvehicle.Methods: (1) The water-insoluble small molecule, TPCA-1, wasloaded into PLGA microspheres by packaging 10 mg of TPCA-1 into100 mg of PLGA, average MW 7,000-17,000 lactide:glycolide(50:50). The sphere size and TPCA-1 encapsulation efficiency weremeasured to meet the pharmaceutical applicable standard. (2) TPCA-1 release from polymers in vitro was tested by dialysis of TPCA-1-PLGA polymers (10 mg/0.5 ml PBS-Tween80) against 50 ml ofexternal media that were replaced daily. The released TPCA-1 in thedialysis medium was extracted with dichloromethane and quantifiedby HPLC. (3) In vivo releasing, tissue distribution, safety, andinflammatory response to TPCA-1 was tested on the wild typeC57BL/6 mice after bilateral retrobulbar injections of TPCA-1-PLGA (10 mg of microspheres loaded with 1 mg TPCA-1 suspendedin 100 μL PBS) and sham-loaded PLGA microspheres (10 mg ofmicrospheres suspended in 100 μL PBS). (4) The development ofCNV after laser photocoagulation in TPCA-1-PLGA treated mice andcontrols was quantified by scoring the fluorescence leakage andisolectin-B4-594 stain areas.Results: TPCA-1 encapsulation efficiency into the microspheresreached to more than 95%, with average PLGA bead size of 2microns in diameter. TPCA-1-loaded beads showed consistentcumulative drug release in vitro and in vivo for up to a month.Histologic analysis and OKR testing showed no cellular andfunctional toxicity. In addition, laser-induced choroidneovascularization was significantly attenuated by retrobulbarinjection of TPCA-1-PLGA microspheres.Conclusions: Retrobulbar injection of small molecular IKK2inhibitor, TPCA-1, delivered by biogradable PLGA microsphere, canachieve a sustained and controllable drug release into choroid/retinatissues and attenuate the laser-induced CNV development without thesystemic toxicity. Our results suggest IKK2 inhibition is aninnovative therapeutic approach for treating wet AMD.Commercial Relationships: Qiutang Li, None; SubhashGaddipati, None; M. Clarke Miller, None; John O. Trent, None;Henry J. Kaplan, None; Qingxian Lu, NoneSupport: NIH Grant EY021584Program Number: 2180Presentation Time: 4:00 PM - 4:15 PMEffects of intravitreally injected ranibizumab and aflibercept onretina and choroid of monkey eyesUlrich Schraermeyer, Sylvie Julien. Experimental VitreoretinalSurgery, Centre for Ophthalmology, Tubingen, Germany.Purpose: Because there is evidence that the Fc domain of anti-VEGFdrugs may cause unexpected effects in retinal and choroidal vessels,the effects of intravitreal ranibizumab and aflibercept on monkeyeyes were investigated.Methods: Four Cynomolgus monkeys were intravitreally injected©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 – Physiology/Pharmacologywith 0.5mg ranibizumab (Lucentis ® ) and another four with 2mgaflibercept (Eylea ® ). All eyes underwent fluorescein angiography(FA) and OCT before injection as well as before sacrifice (1 or 7days post-injection). Three untreated monkeys and one injected withaflibercept vehicle served as controls. The eyes were inspected bylight and electron microscopy and the choriocapillaris lumen widthwas measured by morphometry.Results: Neither FA nor OCT showed any effects of drug treatment.Focal thrombocyte activation, fibrin formation and microparticleswere seen in choroidal vessels 24 hours after injection of either drug,but more often with ranibizumab. Hemolysis in the choriocapillariswas observed in all eyes even vehicle-injected. In aflibercept-treatedeyes swelling of endothelial cells was observed and individual RPEcells were completely filled with hemoglobin indicating cell death(Fig.1b). Deposits on the endothelial cells of choroidal vessels weredenser after treatment with aflibercept compared to ranibizumab orcontrol. The choriocapillaris lumen width was significantly reducedafter treatment with either drug. After aflibercept, individualcapillaries collapsed completely (Fig.1a).Conclusions: OCT and FA are not suitable to detectmicroangiopathy. Both drugs reduced similarly the choriocapillarislumen. The cause of hemolysis is not clear, as it can also be inducedby fluorescein. After aflibercept treatment, microangiophathy(Fig.1a) and RPE cell death (Fig.1b) were observed. The densematerial attached on choroidal vessel walls indicates protein complexformation as observed after bevacizumab treatment 1 . Whether theseresults are related to aflibercept’s Fc domain or to othercharacteristics of the molecule remain to be investigated.1 Schraermeyer & Julien (2012) 10.1517/14712598.2012.748741[doi]Fig.1a: After aflibercept treatment endothelial cells (arrow) showmicroangiopathy and the lumen (asterisk) of the choriocapillaris iscompletely collapsed. Fig.1b: A dead RPE cell with damagedmitochondriae (arrowhead) is filled with hemoglobin (asterisk) afteraflibercept treatment. B = Bruch’s membraneCommercial Relationships: Ulrich Schraermeyer, NovartisPharma AG (F), Novartis Pharma AG (R), Acucela (C); SylvieJulien, Novartis Pharma AG (F), Novartis Pharma AG (R)Support: Novartis Pharma AGProgram Number: 2181Presentation Time: 4:15 PM - 4:30 PMDevelopment and Implementation of an ELISA to Detect “Anti-Ranibizumab” Immunity in Age-Related Macular DegenerationPatientsAaron L. Magno 1, 2 , May Lai 1, 2 , Cora Pierce 1 , Kathleen M. Davern 3 ,Matthew E. Wikstrom 4 , Thomas W. Chalberg 5 , Ian Constable 2 ,Elizabeth P. Rakoczy 1, 2 . 1 Molecular Ophthalmology, Lions EyeInstitute, Perth, WA, Australia; 2 Centre for Ophthalmology andVisual Sciences, The University of Western Australia, Perth, WA,Australia; 3 Monoclonal Antibody Facility, Western AustralianInstitute for Medical Research, Perth, WA, Australia; 4 Centre forExperimental Immunology, Lions Eye Institute, Perth, WA,Australia; 5 Avalanche Biotechnologies, San Francisco, CA.Purpose: To determine if there is a correlation between the nonresponsivenessof patients to ranibizumab and the presence of antiranibizumabantibodies in patients with exudative age-relatedmacular degeneration (AMD). In order to achieve this we havedeveloped an enzyme-linked immunosorbent assay (ELISA) to detectanti-ranibizumab antibodies in serum of patients who are respondersand non-responders to ranibizumab.Methods: The assay we developed uses a homogeneous biotindioxigeninbased bridging ELISA format. Briefly, patient serum wassimultaneously incubated with both a ranibizumab-biotin conjugateand a ranibizumab-dioxigenin conjugate. Following incubation thismixture was loaded into a streptavidin coated well and complexes ofanti-ranibizumab antibodies with the biotin-dioxigenin conjugateswas detected using a horseradish peroxidase-conjugated antidioxigeninantibody. Mice were immunised with ranibizumab togenerate a positive control for this assay. A cohort of 48 AMDpatients was recruited from the clinic at the Lions Eye Institute,Western Australia. The cohort included 8 patients participating in ananti-VEGF gene therapy study currently being conducted by ourgroup. All patients had received multiple injections of ranibizumabprior to sampling of their serum.Results: Mouse serum containing anti-ranibizumab antibodies wasused to successfully validate this assay. While the mouse serumgenerated a robust response no anti-ranibizumab antibodies weredetected in the 48 patient serum samples screened.Conclusions: In this initial limited study we have found nocorrelation between patient responsiveness to ranibizumab and antiranibizumabantibodies. However, having successfully generated arobust assay capable of detecting anti-ranibizumab antibodies withinsera this study will be expanded to 300 patients and will continue toinclude further patients from our anti-VEGF gene therapy study. Agreater understanding of anti-ranibizumab antibodies will assist in thedevelopment of guidelines to match the treatment strategies to theresponsiveness of patients.Commercial Relationships: Aaron L. Magno, AvalancheBiotechnologies, Inc. (F), Avalanche Biotechnologies, Inc. (R); MayLai, Lions Eye Institute (P); Cora Pierce, None; Kathleen M.Davern, None; Matthew E. Wikstrom, None; Thomas W.Chalberg, Avalanche Biotechnologies, Inc. (E), AvalancheBiotechnologies, Inc. (I), Avalanche Biotechnologies, Inc. (P),Avalanche Biotechnologies, Inc. (S); Ian Constable, Lions EyeInstitute (P), Avalanche Biotechnologies (C); Elizabeth P. Rakoczy,Avalanche Biotechnologies (C), Lions Eye Institute (P)Support: National Health and Medical Research council ofAustralia: 1010405312 Gene TherapyTuesday, May 07, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 2707-2746/A0151-A0190Organizing Section: Physiology/PharmacologyProgram Number: 2707 Poster Board Number: A0151Presentation Time: 8:30 AM - 10:15 AMCorrective Gene Therapy for RPGR-XLRP Rescues a CanineModel at Mid-Stage DiseaseWilliam A. Beltran 1 , Artur V. Cideciyan 2 , Alfred S. Lewin 3 , SimoneIwabe 1 , Sanford L. Boye 4 , William W. Hauswirth 4 , Samuel G.Jacobson 2 , Gustavo D. Aguirre 1 . 1 Clinical Studies, School ofVeterinary Medicine, University of Pennsylvania, Philadelphia, PA;©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 – Physiology/Pharmacology2 Ophthalmology, Scheie Eye Institute, School of Medicine,University of Pennsylvania, Philadelphia, PA; 3 Molecular Genetics &Microbiology, University of Florida, Gainesville, FL;4 Ophthalmology, University of Florida, Gainesville, FL.Purpose: Mutations in the RPGR gene are the most common causeof X-linked RP in man, a condition that is currently still incurable.However, we have recently shown in two canine models that AAVmediatedgene transfer of human RPGRORF15 cDNA can rescuephotoreceptors when delivered prior to the onset, or at an early stageof degeneration. We now investigate whether gene therapy deliveredat a more advanced stage of disease can still provide a positiveoutcome.Methods: An AAV2/5 vector construct (titer: 1.51 x 10 11 vg/ml)carrying full-length human RPGRORF15 cDNA under the control ofa hIRBP promoter was injected subretinally in three 12-wk-oldXLPRA2 dogs. At that age, there is on-going cell death and the ONLthickness is reduced by ~ 40%. In addition, one XLPRA2 dog wasinjected shortly after the onset of disease (5.1 wks of age) as an earlydisease stage control. Contra-lateral eyes were either injected withBSS, or received a similar dose of viral construct intravitreally.Photoreceptor structure and function was assessed by means of noninvasiveretinal imaging (cSLO/ SD-OCT) and ERG at 39 and 42weeks of age, respectively.Results: In vivo retinal imaging showed preserved ONL thickness inthe treated retinal areas. ERG function was greater in treated than incontrol eyes, with more than an 8-, 1.2-, and 1.6-fold difference in theamplitudes of rod, mixed rod-cone, and cone 29 Hz flicker responses,respectively. ONL thickness was better preserved in the animaltreated at 5.1 weeks than in the 3 dogs injected at 12 weeks of age,and ERG amplitudes were in average 1.6-fold higher.Conclusions: These results expand our recently published findingsby showing that a sustained and beneficial effect on photoreceptorstructure and retinal function can be achieved even when deliveringRPGR gene augmentation at a mid-stage of XLRP disease. This hasimportant translational application given that most patients are likelyto have relatively advanced disease at the time of treatment.Commercial Relationships: William A. Beltran, None; Artur V.Cideciyan, None; Alfred S. Lewin, University of Florida (P);Simone Iwabe, None; Sanford L. Boye, PCT/US2012/062478 (P);William W. Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C),Syncona (C), RetroSense (C); Samuel G. Jacobson, None; GustavoD. Aguirre, NoneSupport: NIH EY-06855, -17549, -021721, -022012, FoundationFighting Blindness, Macula Vision Research Foundation, Research toPrevent Blindness, Inc.Program Number: 2708 Poster Board Number: A0152Presentation Time: 8:30 AM - 10:15 AMRECOVERY OF VISUAL FUNCTION FOLLOWING GENETHERAPY IN A LARGE ANIMAL MODEL OF CNGA3ACHROMATOPSIAEdward Averbukh 1 , Ron Ofri 2 , Elisha Gootwine 3 , Raaya Ezra-Elia 2 ,Hen H. Honig 3 , Alexander Rosov 3 , Esther Yamin 1 , AlexeyObolensky 1 , William W. Hauswirth 4 , Eyal Banin 1 . 1 Ophthalmology,Hadassah Hebrew University Medical Center, Jerusalem, Israel;2 Koret School of Veterinary Medicine, Hebrew University ofJerusalem, Jerusalem, Israel; 3 Department of Ruminant Research,Agricultural Research Organization, the Volcani Center, Bet Dagan,Israel; 4 Department of Ophthalmology, University of Florida,Gainesville, FL.Purpose: Recently, we reported on novel hereditary dayblindness insheep caused by a mutation in the CNGA3 gene (Reicher et al.,Genomics 95:101-104, 2010). Since mutations in this gene can alsocause achromatopsia in humans, we decided to use these sheep as acone-enriched, large animal model to evaluate safety and efficacy ofCNGA3 gene therapy.Methods: The unique anatomical features of the ovine eye requireddevelopment of a surgical procedure for subretinal delivery.Subsequently, different types of Adeno-Associated Viral (AAV)vectors carrying the intact human or mouse CNGA3 gene weredelivered unilaterally into the subretinal or vitreal space of affectedsheep. Animals were electrophysiologically and behaviorallyassessed preoperatively and 2 and 6 months post-operatively. A subgroupof animals were also tested 12 months after treatment. Conefunction was measured by electroretinography (ERG) following lightadaptation (10 min., 30 cd/m2). Responses to photopic flash andflicker (10-80Hz) stimuli were recorded at 4 intensities (1-10 cd xsec/m2). Behavioral assessment included scotopic and photopic mazetesting under standardized conditions. Passage times and number ofcollisions were recorded. Age-matched normal and day-blind sheepwere similarly assessed as controls.Results: Cone function as measured by ERG was significantlydepressed in affected sheep prior to surgery. Following surgery, therewas significant improvement in eyes treated by either the human orthe mouse CNGA3 gene under control of the red-green Opsinpromoter. Behaviorally, there were no differences between dayblindand normal controls in scotopic testing, but dayblind animals failed tonavigate the maze under photopic conditons. Following delivery ofeither the human or the mouse CNGA3-carrying vector, the ability ofpreviously dayblind sheep to navigate the photopic maze withoutcollisions improved dramatically, approaching that of normalcontrols. The electrophysiological and behavioral improvement inoperated sheep persisted for at least 1 year post-op without affectingthe animals' health.Conclusions: AAV-mediated gene therapy improves cone-dependantvisual function in CNGA3 dayblind sheep, with a good safety profile.The long-term electrophysiological and behavioral improvement inthis naturally-occurring large animal model may pave the way toapplication of similar treatment in human achromatopsia patients.Commercial Relationships: Edward Averbukh, None; Ron Ofri,None; Elisha Gootwine, None; Raaya Ezra-Elia, None; Hen H.Honig, None; Alexander Rosov, None; Esther Yamin, None;Alexey Obolensky, None; William W. Hauswirth, AGTC (I),Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C); EyalBanin, NoneSupport: Israeli Ministry of Health Grant 3-00000-8290, the MaculaVision Research Fund (MVRF) and the Yedidut 1 Research GrantProgram Number: 2709 Poster Board Number: A0153Presentation Time: 8:30 AM - 10:15 AMPhase I Gene Therapy Trial in Israeli Patients with LeberCongenital Amaurosis Caused by a Founder RPE65 Mutation:Safety and Efficacy Update with Up to Two Years of Follow-upEyal Banin 1 , Alexey Obolensky 1 , Yitzhak Hemo 1 , Devora Marks-Ohana 1 , Malka Sela 1 , Esther Yamin 1 , William W. Hauswirth 2 , SamuelG. Jacobson 3 , Dror Sharon 1 . 1 Ophthalmology, Hadassah-HebrewUniv Med Ctr, Jerusalem, Israel; 2 Ophthalmology, University ofFlorida at Gainsville, Gainsville, FL; 3 Scheie Eye Institute,University of Pennsylvania, Philadelphia, PA.Purpose: Gene therapy of human patients with Leber congenitalamaurosis (LCA) due to mutations in the RPE65 gene became areality following demonstration of safety and efficacy in RPE65-mutant dog and mouse models. Our phase I clinical gene therapy trialin Israeli patients, launched in February 2010, was the fourth of itskind worldwide (NCT00821340). The purpose of this report is todescribe the results in the first three patients treated, with up to two©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 – Physiology/Pharmacologyyears of follow-up.Methods: Gene therapy was performed in three LCA patients (ages21-29 years) who harbor a homozygous splicing mutation (c.95-2A>T; IVS2-2A>T) in the RPE65 gene. Following vitrectomy,subretinal injection of an AAV2-hRPE65 viral vector carrying thenormal human RPE65 gene was carried out in one or two sites,avoiding the foveal area. Ocular and systemic safety parameters weremonitored closely, including resolution of the subretinal blebs,possible viral spread and immune response to the vector. Visualfunction and structure were evaluated repeatedly as per protocol byclinical eye exams, computerized light- and dark-adapted perimetry,Goldmann perimetry and non-invasive color, infrared, OCT andautofluorescence imaging.Results: Two years of follow-up data are available for the first twopatients, and 18 months for the third. No toxicity or complicationswere observed to date in any of the patients. Post-operative dataindicates stable visual acuity and increased sensitivity to light in thetreated regions in all patients, to varying degrees. In the first patient,up to 100-fold increases persisted through the two year exam, andinterestingly, he began to use these extra-foveal areas as his preferredlocus for fixation. The third patient also reports and objectivelyshows significant functional improvement. The treatment effect in thesecond patient was slow to occur and is less pronounced.Conclusions: Magnitude of the treatment effect varies betweenpatients, but previous studies by others as well as the present studyattest to the safety and efficacy of gene therapy for treatment ofRPE65 LCA.Commercial Relationships: Eyal Banin, None; Alexey Obolensky,None; Yitzhak Hemo, None; Devora Marks-Ohana, None; MalkaSela, None; Esther Yamin, None; William W. Hauswirth, AGTC(I), Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C);Samuel G. Jacobson, None; Dror Sharon, NoneSupport: The Macula Vision Research Foundation (MVRF), TheYedidut 1 Research Grant and NIH U10-EY13729Clinical Trial: NCT00821340Program Number: 2710 Poster Board Number: A0154Presentation Time: 8:30 AM - 10:15 AMAAV Mediated Gene Transfer Restores Retinal and VisualFunction in Lebercilin-/- (LCA5) MiceDaniel C. Chung, Jeannette Bennicelli, Adam Wojno, Nicoletta A.Commins, Robert W. Bloom, Daniel J. Bennett, Thu T. Duong, MeeraSivalingam, Arkady Lyubarsky, Jean Bennett. FM Kirby Center forMolecular Ophthalmology, Scheie Eye Institute, University ofPennsylvania Perelman School of Medicine, Philadelphia, PA.Purpose: Lebercilin, encoded by the LCA5 gene, is a ciliary proteinfound in the connecting cilia of photoreceptors in vivo and in themicrotubules, centrioles and primary cilia of mammalian cellscultured in vitro. It is widely expressed in human tissues throughoutdevelopment. Since loss of lebercilin function is associated withLeber Congenital Amsurosis (LCA), we used a gene augmentationstrategy to test the ability to correct retinal degeneration in vivo inknockout (Lca5-/-) mice.Methods: Cohorts of genotyped neonatal (P1-2) Lca5-/- micereceived intravenous injections of 15 ul ofAAV2/9.CBA.lebercillin.flag via the right palpebral vein. Someanimals were co-injected with a smaller dose of AAV2/9.eGFP to beable to monitor transduction efficiency non-invasively in vivo.Littermates were injected with AAV2/9.eGFP as control.Retinal/visual function testing was performed at 28-35 days postinjection by pupillary light response, water maze testing, andelectroretinograms. Ophthalmoscopy, fundus photography and SD-OCT imaging was used for retinal structure analysis. Animals wereeuthanized and eyes were sectioned for immunofluorescence analysisfor gene expression and retinal structure.Results: Mice injected with AAV2/9.CBA.Lebercilin.flag,showedimprovements in speed and accuracy in the water maze test, andimproved pupillary light reflexes and electroretinograms, whencompared to control animals. Ophthalmoscopy and fundus photos ofanimals receiving injection of AAV.EGFP revealed widespread GFPpositiveretinal cells. SD-OCT imaging documented an increase inouter nuclear layer (ONL) thickness in experimental versus controlanimals. Analysis of the experimental and control AAV-injected eyesshowed expression of the transgene in the inner and outer retina.Immunofluorescence analyses in AAV2/9.CBA.lebercilin.flaginjectedmice revealed flag-positive cells. The ONL was thicker inAAV2/9.CBA.Lebercilin.flag-injected mice than in untreatedlittermates.Conclusions: AAV mediated gene augmentation therapy via anintravenous route can restore retinal function in Lca5-/- mice.Improvements in retinal function were demonstrated using behavioraland physiologic testing and correlated with retinal structure andtransgene expression. The proof-of-concept data from these studieswill expedite the process of moving forward with a human clinicaltrial.Acknowledgements: In collaboration with the LCA5 consortium.Commercial Relationships: Daniel C. Chung, None; JeannetteBennicelli, None; Adam Wojno, None; Nicoletta A. Commins,None; Robert W. Bloom, None; Daniel J. Bennett, None; Thu T.Duong, None; Meera Sivalingam, Canon Inc (F); ArkadyLyubarsky, None; Jean Bennett, Gensight Biologics (S)Support: Foundation Fighting Blindness, Foundation for RetinalResearch, F.M. Kirby Foundation, Research to Prevent Blindness,Mackall Foundation Trust, Hope for Vision FoundationProgram Number: 2711 Poster Board Number: A0155Presentation Time: 8:30 AM - 10:15 AMAAV-Mediated Expression of Secreted Human CiliaryNeurotropic Factor (hCNTF) Leads to Long-Term Preservationof Cone Photoreceptors in an Autosomal Recessive Model ofRetinitis PigmentosaDaniel M. Lipinski 1 , Alun R. Barnard 1 , Mandeep S. Singh 1 , EdwardLee 2 , Robert E. MacLaren 1, 2 . 1 Nuffield Lab of Ophthalmology,University of Oxford, Oxford, United Kingdom; 2 Moorfield EyeHospital and University College London Institute of OphthalmologyBiomedical Research Centre, London, United Kingdom.Purpose: Loss of cone photoreceptors secondary to advanced roddegeneration leads to a decline of central vision in retinitispigmentosa (RP). hCNTF has previously been shown to preserveretinal neurons, but its long-term neuroprotective effect in vivoremains poorly characterised. Herein, we assess the effect of hCNTFin a rhodopsin knockout mouse model of RP with transgenicfluorescence in cone photoreceptors, allowing cone survival to bequantified longitudinally against a background of rod-specificdegeneration by fundus imaging.Methods: rAAV2/2 vector expressing secreted hCNTF was injectedintravitreally (2µl) at high (1x10^10gp/µl), medium (1x10^9gp/µl) orlow (1x10^8gp/µl) titre at postnatal week (W) 4 in Rho-/-TgOPN1-EGFP+/- mice (n=5-8 per group); the contralateral eye received 2µlPBS. Intrinsically fluorescent cones were quantified by in vivoscanning laser ophthalmoscopy (SLO) at W8, 10, 12, 15, 18, 21, 24and 30. Function was assessed by electroretinography (ERG) at W8,10, 12 and 15, and visual acuity determined at W30 by optomotorresponse (OMR) and laser speckle Doppler flowometry.Immunohistochemistry (IHC) and total RNA sequencing wasperformed at W30.©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 – Physiology/PharmacologyResults: IHC showed transduction of ganglion cells and Müller gliaafter intravitreal injection of rAAV2/2.hCNTF vector. In vivoquantification of fluorescent cones demonstrated preservation for 30weeks in high and medium titre groups (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyof promoters able to express in the retina. We further conclude thateither the computational method of MiniPromoter design, and/or thecharacterization of those promoters using single-copy site-specific inthe mouse genome, renders them unusually suitable for use in AAVand perhaps other viruses. Finally, the MiniPromoters are all human-DNA sequence and so should be particularly useful for clinicalapplications.Commercial Relationships: Elizabeth M. Simpson, PleiadesPromoter Project: S100B MiniPromoters (P), Pleiades PromoterProject: OLIG1 MiniPromoters (P), Pleiades Promoter Project:PITX3 MiniPromoters (P), Pleiades Promoter Project: CLDN5MiniPromoters (P), Pleiades Promoter Project: MKI67MiniPromoters (P), PCP2 Minipromoters (P); Charles N. de Leeuw,UBC (P); Frank M. Dyka, 61/560,437 (P), PCT/US2012/062478(P); Sanford L. Boye, PCT/US2012/062478 (P); Michelle Zhou,None; Lisa Borretta, None; Robert Holt, None; Daniel Goldowitz,None; William W. Hauswirth, AGTC (I), Bionic Sight (I), AGTC(C), Syncona (C), RetroSense (C); Wyeth Wasserman, NoneSupport: Operating - Genome Canada, Pleiades Promoter Project;Genome British Columbia, Pleiades Promoter Project, SoF, andCanEuCre; GlaxoSmithKline R&D Ltd., Pleiades Promoter Project;BC Mental Health and Addiction Services, Pleiades PromoterProject; Child and Family Research Institute, Pleiades PromoterProject; University of British Columbia (UBC) Institute of MentalHealth, Pleiades Promoter Project; UBC Office of the Vice PresidentResearch, Pleiades Promoter Project. Salary - Canadian ResearchChairs (to D.G. and E.M.S.); Canadian Institutes of Health ResearchNew Investigator Award (to W.W.W.); Canadian Institutes of HealthResearch Canada Graduate Scholarships (to C.N.d.L.); MichaelSmith Foundation for Health Research Awards (to C.N.d.L., R.A.H.,and W.W.W).Program Number: 2714 Poster Board Number: A0158Presentation Time: 8:30 AM - 10:15 AMSustained Transgene Expression with Non-viral Gene TransferFollowing Chitosan Mediated DeliveryAna Vanessa Oliveira 1, 4 , Gabriela A. Silva 2, 3 , Daniel C. Chung 4 .1 Doctoral Program in Biomedical Sciences, Department ofBiomedical Sciences and Medicine, University of Algarve, Faro,Portugal; 2 Centre for Molecular and Structural Biomedicine(CBME/IBB, LA), University of Algarve, Faro, Portugal;3 Department of Biomedical Sciences and Medicine, University ofAlgarve, Faro, Portugal; 4 F.M. Kirby Center for MolecularOphthalmology, Scheie Eye Institute, University of PennsylvaniaPerelman School of Medicine, Philadelphia, PA.Purpose: A successful ocular gene therapy strategy requires efficientgene transfer and stable transgene expression. Recently, severalstrategies have been evaluated to promote safe, site-specificintegration, long-term gene expression and the ability to mediatelarge gene transfer. One of the most promising technologies exploitsa site-specific recombinase, the phage phiC31 integrase. We aim todevelop a novel system for sustained large gene transfer by couplinghybrid polymeric vectors with phiC31-integrase to simultaneouslypromote enhanced delivery and transgene integration, thereforeobtaining sustained transgene expression.Methods: Chitosan-pDNA and chitosan/hyaluronic acid-pDNAnanoparticles were prepared using a NH3:PO4 ratio of 15:1. Particleswere produced using either pGFPattB, pCEP290attB (>8kb) with orwithout pCMVINT at a molecular ratio of 2:1. These nanoparticleswere used for pDNA encapsulation and transfection studies oncultured HEK293T cells. Co-transfection with pCMVINT was doneby delivering the two plasmids in the same particle or separately.Transgene expression was evaluated by fluorescence microscopy andwestern blot analysis.Results: Both types of particles encapsulated pDNA efficiently, evenwhen two plasmids were complexed simultaneously. Transfectionstudies indicate that transfection efficiency and transgene expressionis affected by polymer size and type of polyplex used, as well as theway integrase is delivered with the polyplexes. Transgene expressiondecreased over time, however GFP expression was still detected 14weeks after transfection. Over-expression of the large transgeneCEP290 following delivery by chitosan polyplexes was detected atleast 14 days post transfection.Conclusions: We have shown that long-term transgene expressioncan be achieved with a non-viral approach using hybrid polymerpolyplexes, as well as its ability to deliver large genes. The combinedstrategy of polymers and integrase is more efficient than nonintegrativestrategies. Long-term transgene expression will also beevaluated in vivo in mouse models of retinal pathologies, with furthertransfection optimization.Commercial Relationships: Ana Vanessa Oliveira, None;Gabriela A. Silva, None; Daniel C. Chung, NoneSupport: Fundação para a Ciência e Tecnologia (PTDC/SAU-BEB/098475/2008 to G.A.Silva, SFRH/BD/70318/2010 toA.V.Oliveira), Marie Curie Reintegration Grant (PIRG-GA-2009-249314 to G.A.Silva) under the FP7 program and Hope for Vision, FM Kirby Foundation to D.C.Chung.Program Number: 2715 Poster Board Number: A0159Presentation Time: 8:30 AM - 10:15 AMAAV-mediated Lpcat1 gene replacement therapy rescues retinaldegeneration in rd11 miceXufeng Dai 1 , Juanjuan Han 1 , Zi-Bing Jin 1 , Yan Qi 1 , Jie Li 2 , Wen-TaoDeng 2 , Bo Chang 3 , William W. Hauswirth 2 , Ji-Jing Pang 1, 2 . 1 The EyeHospital, School of Ophthalmology and Optometry, WenzhouMedical College, Wenzhou, China; 2 University of Florida,Gainesville, FL; 3 The Jackson Laboratory, Bar Harbor, ME.Purpose: Retinal degeneration 11 (rd11) mice are a newlydiscovered naturally occurring animal model with early and rapidphotoreceptor dysfunction/degeneration caused by a spontaneousmutation in the Lpcat1 gene encoding lysophosphatidylcholineacyltransferase. This study is to test whether highly efficient tyrosinemutant AAV-mediated gene replacement therapy can restore theretinal function/structure in rd11 mice.Methods: At postnatal day 14, 1 μl of AAV8 (Y733F)-smCBA-Lpcat1 vector (10 13 viral genome containing particles/ml) wasinjected subretinally into one eye of 20 rd11 mice. Uninjectedcontralateral eyes were used as controls. Ten weeks after injection,dark- and light-adapted ERGs were recorded. Then both treated andcontrol eyes were harvested for immunohistochemical andmorphological studies.Results: In treated eyes, ERG signals were recorded 10 weeks aftertreatment; both dark- and light-adapted ERG amplitudes were about70% of the normal level seen in wild type C57 mice. Dark- and lightadaptedERGs were unrecordable in untreated rd11 eyes. More thanhalf of the outer nuclear layer (ONL) was preserved in treated eyes,while only one row of ONL nuclei remained in untreated eyes.Lpcat1 expression was observed in the outer retina, particularly inphotoreceptor outer segments in treated eyes byimmunohistochemistry, but not in untreated eyes. Both M- and S-cone opsin were observed extensively in treated eyes; in contrast,cone opsin was nearly undetectable in untreated eyes.Conclusions: Tyrosine-capsid mutant AAV mediated Lpcat1expression restores/maintains rod and cone function/structure for atleast 10 weeks in the rd11 mice, a retinal degeneration model withearly and rapid photoreceptor degeneration.©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 – Physiology/PharmacologyCommercial Relationships: Xufeng Dai, None; Juanjuan Han,None; Zi-Bing Jin, None; Yan Qi, None; Jie Li, None; Wen-TaoDeng, None; Bo Chang, None; William W. Hauswirth, AGTC (I),Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C); Ji-JingPang, NoneSupport: NIH grants, EY021721, EY019943, FFB, MVRF, andRPB, Inc.Program Number: 2716 Poster Board Number: A0160Presentation Time: 8:30 AM - 10:15 AMEnhancement of AAV2-mediated retinal transduction in diabeticratsNundehui Díaz-Lezama 1 , Zhijian Wu 2 , Mayda Ramírez 1 , BibianaMoreno-Carranza 1 , Gonzalo Martinez de la Escalera 1 , Peter Colosi 2 ,Carmen Clapp 1 . 1 Instituto de Neurobiología, Universidad NacionalAutónoma de México (UNAM), Querétaro, Mexico; 2 Neurobiology,Neurodegeneration and Repair Laboratory, National Eye Institute,National Institutes of Health, Bethesda, MD.Purpose: Ocular gene therapy based on the adeno-associated virus(AAV) vector-mediated delivery of antiangiogenic molecules offersconsiderable promise for the treatment of diabetic retinopathy. AAVtype-2 (AAV2) uses cell surface heparan sulphate proteoglycans(HSPG) as the primary receptors for cell entry. HSPGs are present onretinal ganglion cells, and likely mediate vector transduction. Here,we compared the transduction of retinal ganglion cells in diabetic andnormal rats following intravitreal delivery of AAV2 vectors, andinvestigated whether such transduction correlates with the level ofHSPG expression in the respective retinas.Methods: An AAV2 CMV EGFP reporter vector was delivered byintravitreal injection (2.8e10 vg/eye) to adult non-diabetic rats(controls) and diabetic rats treated two weeks prior with a singleinjection of streptozotocin. One month after vector administration,retinal flatmounts were examined for EGFP expression, by confocalmicroscopy, and expression of the HSPGs syndecan, glypican, andperlecan, by quantitative PCR.Results: In normal rat retinas, transduction was limited to occasionalcells in the ganglion cell layer. In the diabetic rats, the transductionwas enhanced more than 4-fold in ganglion cell somas and processes.The expression of mRNAs for syndecan and glypican was elevated 2-and 1.5-fold in the diabetic rat retina, respectively, whereas that ofperlecan was not modified.Conclusions: Retinal transduction by AAV2 vectors is enhanced indiabetes and may be mediated by elevated expression of HSPGs onganglion cells. The diabetes-induced factors responsible for thesechanges warrant further study. AAV2 vectors may be desirable forgene therapeutics targeting in diabetic retinopathy.Commercial Relationships: Nundehui Díaz-Lezama, None;Zhijian Wu, None; Mayda Ramírez, None; Bibiana Moreno-Carranza, None; Gonzalo Martinez de la Escalera, None; PeterColosi, None; Carmen Clapp, NoneSupport: CONACYT grant S0008-161594 (C.C.) and National EyeInstitute, NIH, USA (P.C.).Program Number: 2717 Poster Board Number: A0161Presentation Time: 8:30 AM - 10:15 AMEx vivo gene transfer of anti-angiogenic soluble Flt-1 to cornealepithelial cells sheets suitable for ocular surface reconstructionJingbo Liu, Victor H. Guaiquil, Aihong Liu, Mark Rosenblatt.Margaret M. Dyson Vision Research Institute, Weill Cornell MedicalCollege, New York, NY.Purpose: To evaluate ex vivo gene transfer of anti-angiogenicsoluble Flt-1 (sFlt1) to corneal epithelial cell sheets transplanted in arabbit model of total limbal stem cell deficiency (TLSCD).Methods: Mouse sFlt1 was cloned into pLenti6.3/V5-DEST vectorusing the Gateway systems. The vector was cotransfected with viralpackaging plasmids into 293FT cells and the produced viruses wereharvested and titrated. Primary rabbit corneal epithelial cells (RCEC)were transduced with the lentivirus and the sFlt1 mRNA expressionand protein levels were analyzed by qRT-PCR, immunofluorescencestaining and ELISA, respectively. Functional assays using aortic ringwere performed to evaluate the inhibitory effect of sFlt1 onVEGF164 induced endothelial cell proliferation. RCEC transducedwith sFlt1 were grown as sheet on decellularized amnioticmembranes and transplanted on the rabbit corneas with TLSCD. Thebiocompatibility of the cell sheet was monitored by slit lampbiomicroscopy.Results: Lentiviruses containing msFlt1 gene were successfullyproduced. qRT-PCR analysis showed high level of expression ofmsFlt1 mRNA in the transduced RCEC. The sFlt1 protein wasdetected by immunofluorescence staining in the cytoplasm and up toone week by ELISA in the culture medium. The aortic ring assaysshowed that sFlt1 inhibits the VEGF164 induced proliferative effectof aortic endothelial cells (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologymurine EC compared to immortalized human EC. In addition, theoverall plateau of expression was highest in primary EC (~80%).Conclusions: Recombinant AAV pseudotypes differ in their tropismand transduction efficiency between murine and human corneas. Ingeneral high levels of gene expression can be obtained with severalof these pseudotypes. Characteristic slow-onset kinetics of proteinexpression have to be taken into account when applying AAVs intranslational applications, e.g. in corneal storage in eye banks.Commercial Relationships: Thomas A. Fuchsluger, None;Christian Mueller, 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: Eye Bank of America Research GrantProgram Number: 2719 Poster Board Number: A0163Presentation Time: 8:30 AM - 10:15 AMEffect of intraviteal injection of AAV9.TGFβ2 on the expressionof anterior segment and intraocular pressure in rabbitsGuilin Zhan, Chris Long, Allan Shepard, Nasreen Jacobson, RichardOrnberg, Yu Wang, Shutong Cao, Ganesh Prasanna. OphthalmologyRes./Glaucoma Res., Novartis Institutes for Biomedical Res., FortWorth, TX.Purpose: TGFβ2 levels are elevated in aqueous humor (AH) ofprimary open angle glaucoma patients and have been associated withultrastructural changes to trabecular meshwork (TM) leading toelevated intraocular pressure (IOP). This study is to explore viraltransduction and overexpression of TGFβ2 on IOP in New Zealandrabbits.Methods: Eight rabbits received unilateral intravitreal injection of50µl of AAV9-mut446-sc-smCBA-hTGFβ2 (1.25E12 vg/mL) andother four received AAV9-mut446-sc-smCBA-hGFP (6.4E12vg/mL). The injections were repeated one week following the initialinjection. IOP was measured at day 0 and once or twice per weekusing a pneumatonometer on day 6-62 post second-injection. Ocularinflammation including flare, cells and fibrin in anterior chamber andGFP expression were examined by slit lamp (SL) beginning one dayafter injection. The expression of GFP in the anterior segment ofrabbits was assessed by immunohistochemistry (IHC). Total andbioactive TGFβ2 in rabbit AH were detected by ELISA 62 days postinjection.Results: GFP expression was detected in 75% of four AAV9.GFPtreated rabbits by SL beginning 14 days post-injection and 50% ofthese injected eyes showed a positive expression of GFP at 49 and 62days post-injection by IHC in anterior segment of eye. Comparedwith uninjected (UI) eyes, total TGFβ2 in AH showed an increasingtrend (P=0.1) in AAV9.TGFβ2 (2314 ± 316 vs UI eye: 1664 ± 198pg/mL), while was significantly different in AAV9.GFP treatedgroup (2412 ± 246 vs UI eye: 1972 ± 185 pg/mL; p=0.035).Bioactive TGFβ2 levels in AAV9.TGF β2 eyes were 41 ± 9 pg/mL(UI eye: 42 ± 9 pg/mL) while that in AAV9.GFP eyes was 16 ± 8pg/mL (UI eye: 41 ± 21 pg/mL). No statistically significantdifference in total and bioactive TGFβ2 levels were found betweenAAV9.TGFβ2 and AAV9.GFP injected groups. Inflammation couldbe detected in 50% of eyes in both groups lasting for 2-3 weeks.Compared with pre-injection baseline, IOP was not significantlyincreased in either AAV9.TGFβ2 (BL: 20 ± 0.8 mmHg; Day 62: 23 ±0.5 mmHg) or AAV9.GFP treated eyes (BL: 17 ± 0.5 mmHg; Day62: 19 ± 1.5 mmHg).Conclusions: Our results indicate that AAV9.GFP can be transducedin the ocular anterior segment of rabbits. However, TGFβ2 levels inAH did not result in elevated IOP in rabbits and could be due tospecies differences and/or insufficient expression of TGFβ2 withthese vectors.Commercial Relationships: Guilin Zhan, Novartis Institution forBiomedical Research (E); Chris Long, None; Allan Shepard, None;Nasreen Jacobson, Novartis Institute Biomedical Res (E); RichardOrnberg, Novartis Institute for Biomedical Research (E); Yu Wang,None; Shutong Cao, nibr (E); Ganesh Prasanna, Alcon ResearchLtd (E), Novartis Institute of Biomedical Research (E)Program Number: 2720 Poster Board Number: A0164Presentation Time: 8:30 AM - 10:15 AMChanges in S and L/M cone opsin expression in the RPE65 dogmodel following AAV mediated gene addition therapyKnut Stieger 1 , Daniela Klein 1 , Alexandra Mendes-Madeira 2 ,Fabienne Rolling 2 , Silke Haverkamp 3 , Birgit Lorenz 1 . 1 Department ofOphthalmology, Justus-Liebig-University Giessen, Giessen,Germany; 2 Institut UMR 1089, Institut de Recherche Thérapeutique1, Nantes, France; 3 Max Planck Institute for Brain Research,Frankfurt, Germany.Purpose: The Swedish Briard dog containing a null mutation in theRPE65 gene represents a naturally occurring animal model for earlyonset severe retinal dystrophy (EOSRD) in humans. Adenoassociatedvirus (AAV) mediated gene therapy in these dogs resultedin tremendous treatment benefits in terms of restoration of function inboth, rods and cones. However, treatment benefit in humans is lesspronounced. The aim of this study was to examine changes in coneopsin expression in RPE65 -/- dogs following gene therapy in orderto further elucidate the treatment effect.Methods: Retinae from 9 affected dogs (n=18) were used in thisstudy, four of them treated unilaterally, at the age of 7-12 months,with an AAV vector carrying the human RPE65 gene. Dogs weresacrificed at various ages, between 2 and 4.5 years. Antibodiesagainst S-opsin and L/M-opsin were used to study opsindelocalisation on cryosections and to quantify cone density onflatmount preparations.Results: Following gene therapy, opsin delocalisation was reduced inboth L/M and S cones. The total number of opsin stained S conesincreased by up to 40% in treated areas, compared to non-treatedareas in the same eye. L/M cone opsin staining did not increasefollowing gene therapy.Conclusions: The remarkable increase in S cone staining andreduced delocalisation of S cone opsin indicates a strong treatmenteffect on this photoreceptor subtype. In contrast, L/M cones seem tobenefit less efficiently from gene therapy, given only the reduction ofopsin delocalization. These results may have consequences for theanalysis of gene therapy results in humans, where central visualacuity is L/M cone dependent.Commercial Relationships: Knut Stieger, None; Daniela Klein,None; Alexandra Mendes-Madeira, None; Fabienne Rolling,None; Silke Haverkamp, None; Birgit Lorenz, Optos (F)Support: University Medical Center Giessen and Marburg GrantProgram Number: 2721 Poster Board Number: A0165Presentation Time: 8:30 AM - 10:15 AMNatural History of the CNGB3/NRL Double Knock-Out MouseWilliam W. Hauswirth, Seok-Hong Min, Sanford L. Boye, DanielKasuga, Qing Ruan, Jingfen Sun, Mai Tram Phan, Shannon E. Boye,Christine N. Kay. Dept. of Ophthalmology, University of FloridaCollege of Medicine, Gainesville, FL.Purpose: Achromatopsia (ACHM) is a congenital conephotoreceptor disorder with an incidence of 1:30,000. Cyclicnucleotide gated channel beta 3 (CNGB3) accounts for approximately50% of this autosomal recessive disease. CNGB3 knock-out (KO)mice have been successfully used as an animal model for genereplacement therapy. However, the mouse retina lacks a cone-©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 – Physiology/Pharmacologydominant macula and is comprised of less than 3% cones, comparedto cone rich human macula, making clinical translation difficult forany cone-related retinal degeneration. To address this problem of rodbiasin the mouse, we generated a cone exclusive mouse modellacking CNGB3 by crossing CNGB3 KO mice with mice lackingNeural retina leucine zipper (NRL KO) and characterized the naturalhistory out to four months postnatal.Methods: CNGB3/NRL double KO (DKO) mice were generated bycrossing NRL KO mice with CNGB3 KO lines. We followed thenatural history of CNGB3/NRL DKO mice with photopicelectroretinography (ERG) at 0.5, 1, 2, 3 and 4 months of age,respectively. Responses were elicited at four increasing lightintensities (1.25 cds/m2, 5cds/m2, 10cds/m2 and 25cds/m2). Forcomparison, ERG measurements were also recorded from agematchedCNGB3 KO and NRL KO mice (data collection ongoing).Following ERG, one animal from each age group was sacrificed andtheir retinas collected for histological analysis.Results: Photopic recordings at the highest flash intensity revealedthat cone amplitudes of CNGB3 KO mice were ~50 µV at 16 daysand remained low throughout the following 3 months. In contrast,ERG amplitudes of CNGB3/NRL DKO mice increased from ~50 µVat 16 days to 150 µV at 2 months and remained stable thereafter.There was a significant difference in the CNGB3 and CNGB3/NRLmodel at 1, 2, and 3 months (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology3 University of Oklahoma Health Sciences Center, Oklahoma City,OK; 4 University of Munich, Munich, Germany.Purpose: Deletion of neural retina leucine zipper in mice (Nrl −/− )results in the complete absence of rods and an all-cone retina. Nrl −/−mice lack a scotopic response and have an enhanced photopic ERGamplitude. Mutations in the gene encoding the alpha-subunit of thecone cyclic nucleotide-gated (CNGA3) channel cause cone functionloss in mammals and are associated with achromatopsia 2 in humans.Cnga3/Nrl double knockout (Cnga3/Nrl DKO) mice have been bredin part to mimic the cone-rich central retina in human achromatopsia2 with CNGA3 mutations. We therefore tested whether AAVmediatedCnga3 gene therapy targeting cones can restore conesystem function/structure in this model.Methods: At postnatal day 14, one μl of AAV5-IRBP/GNAT2-humanCnga3 vector (10 13 particles/ml) was injected subretinally intoone eye of 20 Cnga3/Nrl DKO mice. The other eye was uninjected ascontrol. Dark- and light-adapted ERGs were recorded from 6 weeksto 6 months after injection. Six months after treatment visual acuityand contrast sensitivity were measured optokinetically. Treated andcontrol eyes were harvested for immunohistochemical studies.Results: In treated eyes, restored light-adapted ERG waveforms wererecorded 6 weeks after injection and remained stable for at least 6months. Cone mediated ERG amplitudes were similar as those ofNrl −/− mice 6 months after treatment; light-adapted ERGs wereunrecordable from untreated eyes. Dark-adapted ERGs were absentin both eyes. Behavioral tests showed that improved visual acuity andcontrast sensitivity were found in treated eyes, compared to untreatedeyes under a light environment. In untreated eyes, cone opsin stainingwas greatly attenuated with remaining cone opsins mis-localized tothe inner retinal layers. In contrast, both M- and S-opsins werereadily observed in the outer segments of treated eyes although theseouter segments were much shorter than normal C57 BL/6J.Conclusions: AAV mediated gene replacement therapy restores conesystem function and halts cone degeneration in the Cnga3/Nrl DKOmouse, a cone dominant model. These results suggest that this modelwill be useful in testing approaches for rescuing cone function in thecone-rich macula/fovea of human achromatopsia.Commercial Relationships: Ji-Jing Pang, None; Ye Tao, None;Sanford L. Boye, PCT/US2012/062478 (P); Jie Li, None; Wen-TaoDeng, None; Xi-Qin Ding, None; Stylianos Michalakis, None;Martin Biel, None; Shannon E. Boye, None; William W.Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C),RetroSense (C)Support: NIH grants, EY021721, FFB, MVRF, and RPB, Inc.Program Number: 2724 Poster Board Number: A0168Presentation Time: 8:30 AM - 10:15 AMGene Delivery of ATP6 by A Mitochondrial Targeting SequenceModification of AAV Capsid VP2 Rescues Cells with MutatedT8993G MtDNA Responsible for Neuropathy Ataxia andRetinitis PigmentosaHuijun Yuan 1 , Hong Yu 1 , John Guy 1, 2 . 1 University of Miami MilerSchool of Medicine, Miami, FL; 2 Bascom Palmer Eye Institute,Miami, FL.Purpose: Two mitochondria diseases, neuropathy, ataxia, retinitispigmentosa (NARP) and maternally inherited Leigh's syndrome(MILS) are caused by a T8993G mutation in mtDNA encodingsubunit 6 of ATP synthase that is complex V. Our goal is to rescuethe consequences of this mutation that result in blindness and death.Methods: The human ATP6 gene was packaged into AAV serotype2 or AAV serotype 9 to which the cytochrome oxidase subunit 8(COX8) leader sequence was inserted into the VP2 capsid.Expression of ATP6 was under the control of the humanmitochondrial heavy strand promoter (HSP). NARP cybrid cells with100% mutated T8993G mitochondrial DNA were infected at differentMOI with self-complementary (sc) AAV9 or AAV2 containingscAAV-HSP-ATP6 with FLAG. After 48 hrs, high glucose mediawas exchanged for glucose free galactose media for 5 days thatforced the cells to rely on oxidative phosphorylation to produce ATP.Cell survival was assessed by the MTT assay. ATP synthesis wasmeasured using a luciferin-luciferase assay. Expression of mRNAand protein were respectively assessed by qRT-PCR,immunofluorescence and immunoblotting.Results: After infection of rAAV with scAAV-HSP-ATP6, cellsurvival increased in a dose-dependent matte with an absorbance0.305, 0.372, and 0.4055 for MOI of 0, 500, and 5,000 respectively.Relative to no infection, differences were statistically significant withP value of 0.007 and 0.0009.Relative to uninfected NARP cells, therate of ATP synthesis increased 41% (p< 0.01). Compared touninfected, infected cybrids ATP6 RNA transcripts were elevated 9fold and 34 fold with AAV2 and AAV9 infection (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyreduced the damage of cornea epithelia, leading to recovery of corneaulcer, and decreased the viral VP16 expression in cornea tissue (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Coralia C. Luna, None; BenjamintenOever, None; Guorong Li, None; Sonja Schmid, None; PratapChalla, None; David L. Epstein, None; Jianming Qiu, None;Pedro Gonzalez, NoneSupport: NEI EY01894, NEI EY016228, NIH P30 EY-005722, andResearch to Prevent BlindnessProgram Number: 2728 Poster Board Number: A0172Presentation Time: 8:30 AM - 10:15 AMLONG-TERM PROTECTION BY COMPLEMENTREGULATORY PROTEIN CRRY IN A MOUSE MODEL OFLIPOFUSCIN-MEDIATED RETINAL DEGENERATIONShanta Sarfare, Anita Kim, Zhichun Jiang, Marcia B. Lloyd, ShannanR. Eddington, Samer Habib, Dean Bok, Steven Nusinowitz, GabrielH. Travis, Roxana A. Radu. Ophthalmology, Jules Stein Eye Institute,Los Angeles, CA.Purpose: Mutations in the human ABCA4 gene cause recessiveStargardt disease (STGD1) and predispose to the development ofage-related macular degeneration (AMD). Mice with a knockoutmutation in the abca4 gene (abca4-/- mice) accumulate A2Elipofuscinin the retinal pigment epithelium (RPE) and exhibit slowphotoreceptor degeneration. Recently, we described complementactivation and increased oxidative stress in abca4-/- mice due to A2Eaccumulation. In this study we tested the hypothesis that increasingexpression of complement regulatory proteins (CRP’s) in the RPEwould protect these cells from complement attack and hence protectphotoreceptors from degenerating.Methods: We generated a recombinant adeno-associated viruscontaining mouse CRRY (rAAV8-CRRY), which we injectedsubretinally into one-month-old albino abca4-/- and BALB/c mice.Negative control mice received AAV8-null or rAAV8-GFP viruses.Fundus imaging was performed at different times following theinjections. Expression of CRRY was determined by qRT-PCR,immunoblotting and immunocytochemistry. Spectral-domain opticalcoherencetomography (sdOCT) and electroretinography (ERG) wereobtained as previously described. Visual retinoids and lipofuscinpigments were quantified by high-performance liquidchromatography.Results: At 12-months post-injection, mice that received rAAV-CRRY showed 2 to 7-fold increased expression of CRRY by qRT-PCR and quantitative immunoblotting. Fundus images of these micewere normal, suggesting no detrimental effects of the injections. Themean scotopic a-wave and b-wave ERG’s show no significantdifference between the CRRY and Null-treated eyes. We observedtwo-fold decreased A500-lipofuscin and a three-fold decreased A2Elevels in CRRY-treated abca4-/- mice at 11-months post-injection.sdOCT showed a limited maintenance in overall retinal thickness inCRRY-treated compared to Null virus-treated mice, which correlatedwith enhanced preservation of photoreceptors and increase in 11-cisretinalchromophore levels.Conclusions: Aberrant complement activation is strongly linked tothe development of AMD. For the first time, we show that inhibitingcomplement-convertase activity by gene therapy with a virus thatincreased CRRY expression may be useful in treating age-relatedretinal degenerations.Commercial Relationships: Shanta Sarfare, None; Anita Kim,None; Zhichun Jiang, None; Marcia B. Lloyd, None; Shannan R.Eddington, None; Samer Habib, None; Dean Bok, None; StevenNusinowitz, None; Gabriel H. Travis, None; Roxana A. Radu,NoneSupport: NIH/NEI 2P30 EY00331-45Program Number: 2729 Poster Board Number: A0173Presentation Time: 8:30 AM - 10:15 AMTesting siRNAs for use in a rapidly progressing model ofautosomal dominant retinitis pigmentosaBrian P. Rossmiller, Haoyu Mao, Alfred S. Lewin. Department ofMolecular Genetics and Microbiology, University of Florida,Gainesville, FL.Purpose: We are interested in characterizing and treating the rapidlydegenerating autosomal dominant retinitis pigmentosa (ADRP)mouse model, T17M RHO. Photoreceptors in this transgenic strainare highly susceptible to light damage. I hypothesize that theknockdown of transducin in rods, will reduce rod degeneration andpreserve cone function. It is, therefore, the purpose of this study todetermine if siRNA mediated knockdown of transducin can inhibitthe phototransduction cascade in rod cells preventing apoptosisresulting from exposure to bright light.Methods: gene (GNAT1). Two small hairpin RNAs (shRNAs) weredesigned to target homologous regions between mouse and dogGNAT1. Transfections were done in HEK293 cells (n=6) usingCMV-GNAT1 and H1-shRNA GNAT1 given at 1:0, 1:2, 1:4, and 1:6ratios. Using a plasmid with a control miRNA, total amount of DNAtransfected at each ratio was kept constant. The amount of transducinproduced was observed at 24 and 72 hours post transfection usingwestern blot with a mouse transducin specific antibody. The percentknockdown was then calculated using tubulin as the loading controland normalized against the control ratio which received only CMV-GNAT1 and CBA-GFP plasmids. Following the in vitro knock downassay, each shRNA was inserted into an a plasmid containing GFPunder the expression of a mouse opsin promoter and packaged intoadeno associated virus serotype 8 with a capsid modification, AAV8(Y733F).Results: siRNA GNAT1(a) showed the greatest knockdown with76%, 71.4% and 69.1% of the GNAT mRNA remaining at the 1:2,1:4 and 1:6 ratios, respectively, after 72 hours.Conclusions: We demonstrated siRNA GNAT1(a) to be a viablesiRNA for testing in T17M RHO transgenic mice. Both shRNAs (a)and (b) targeting GNAT1 are being packaged into AAV8 (Y733F) forin vivo analysis in mice. Once we verify reduction of transducin-α inthese mice, we will expose them to clinical light levels and measureretinal preservation. Since the siRNAs also target dog transducin,they will be tested in the T4R dog model of ADRP which is alsounusually sensitive to light damage.Commercial Relationships: Brian P. Rossmiller, None; HaoyuMao, None; Alfred S. Lewin, University of Florida (P)Support: T32-EY07132 Training grant, Foundation FightingBlindness grant TA-GT-0611-0526-UFL, NIH grant R24EY022012,and the Vision Core grant EY021721Program Number: 2730 Poster Board Number: A0174Presentation Time: 8:30 AM - 10:15 AMGene Therapy for CEP290-associated LCA in Patient-DerivedInduced Pluripotent Stem CellsErin R. Burnight 1 , Emily E. Kaalberg 1 , Mari E. Eyestone 1 , JeremyHoffman 1 , Christine M. Haas 1 , Robert F. Mullins 1 , Edwin M. Stone 1,2 , Budd A. Tucker 1 . 1 Ophthalmology and Visual Sciences, Universityof Iowa, Iowa City, IA; 2 HHMI Investigator, Iowa City, IA.Purpose: Mutations in the CEP290 gene are major contributors toLeber Congenital Amaurosis (LCA), the most severe form ofinherited retinal degenerative disease for which there is currently nocure. Autosomal recessive CEP290-associated LCA is a goodcandidate for gene-replacement therapy and reprogrammed somaticcell technologies provide researchers with the ability to study humandisease and therapeutic gene correction in vitro. The purpose of thisstudy was to develop lentiviral vectors containing human CEP290©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 – Physiology/Pharmacologyand to use these vectors to deliver wild type CEP290 to inducedpluripotent stem cell (iPSC)-derived photoreceptor precursor cells(PRPCs) from mice and humans with CEP290-associated LCA.Methods: Fibroblast-derived iPSCs were generated from the retinaldegenerative mouse model CEP290 rd16 and patients with molecularlyconfirmed CEP290-associated LCA. Mouse and human iPSCs weredifferentiated into photoreceptor precursor cells using our previouslydeveloped step-wise differentiation protocol. An HIV-1 lentiviralvector containing human CEP290 under the control of the CMVpromoter was packaged and used to transduce PRPCs from rd16 miceand LCA patients. Vector-derived mRNA and protein were detectedvia RT-PCR and western blot, respectively.Results: To evaluate CEP290 gene transfer we initially transduced asurrogate cell line, JK1, derived from murine testicular stromal cellsthat do not express the human CEP290 transcript. RT-PCR analysisusing human-specific primers demonstrated a dose-dependentincrease in CEP290 expression post-transduction. However, cellviability was significantly reduced in cultures transduced with thehighest dose. Using results from these pilot experiments, wedelivered a lower dose of CEP290 vector to iPSC-derived murine andhuman PRPCs. RT-PCR analysis and western blotting indicatedvector-derived expression in these cells.Conclusions: We show successful gene transfer of human CEP290to iPSC-derived photoreceptor precursor cells. Cell viability assayssuggest that overexpression of CEP290 is cytotoxic. Thus, it will beimportant to carefully titrate vector dosage when developing genereplacement strategies for this disease. This work will contribute toour overall goal of vision restoration in patients with LCA throughgene and patient-specific photoreceptor cell replacement.Commercial Relationships: Erin R. Burnight, None; Emily E.Kaalberg, None; Mari E. Eyestone, None; Jeremy Hoffman, None;Christine M. Haas, None; Robert F. Mullins, Alcon Research Ltd(F); Edwin M. Stone, None; Budd A. Tucker, NoneSupport: NIH EY022834; Grousbeck Family FoundationProgram Number: 2731 Poster Board Number: A0175Presentation Time: 8:30 AM - 10:15 AMAAV8 mediated gene therapy for corneal cystinosisDuy H. Nguyen 1 , Hongjun Du 1 , Matthew Bedell 1 , Seanna Grob 1 , JingLuo 1 , John L. Quach 1 , Peter Shaw 1 , Stephanie Cherqui 2 , KangZhang 1 . 1 Ophthalmology, University of California San Diego, LaJolla, CA; 2 Molecular and Experimental Medicine, Scripps ResearchInstitute, La Jolla, CA.Purpose: Cystinosis is a lysosomal storage disorder that results frommutations in the gene CTNS, which encodes cystinosin, thelysosomal membrane transporter for cystine. Corneal accumulationsof intracellular cystine crystals lead to debilitating ocularcomplications. The aim of this study is to deliver an adeno-associatedvirus (AAV8)-Ctns vector into the cornea of Ctns-/- mice to study theefficacy of AAV8-mediated CTNS gene transfer.Methods: 1.5 to 4.5-month old Ctns-/- mice from the CherquiLaboratory at The Scripps Research Institute and 2-month oldwildtype C57BL6 mice were used. AAV8 vectors cloned with eithergreen fluorescent protein (GFP) or CTNS were delivered by singleintrastromal corneal injections of 1.5 µl (vector titer= 2x1011) with aHamilton micro-syringe. Successful injection was gauged byobserving that ≥70% of the cornea became whitened and edematousimmediately following the injection. The non-injected contralateraleyes were used as controls. At 11 to 15 months post-injection, micewere sacrificed and eyes collected. CTNS mRNA expression wasmeasured using real-time polymerase chain reaction (q-PCR). Opticalcoherence tomography and corneal histology were used to evaluatechanges in corneal crystal content.Results: AAV8 vector had a high transduction efficiency thatresulted in high levels and uniform expression of reporter gene GFPin the cornea, starting from day 3 and lasted up to 11 months at thepoint of eye collection. At 12 to 15 months post-injection, there was asignificant difference in mRNA expression of CTNS as measured byqPCR in AAV8-Ctns eyes (n=4) when compared to that in theuntreated eyes (n=4) (2225±1102.02 fold increase vs. 138.5±241.33fold increase respectively, P-value = 0.0051).Conclusions: AAV8 is a reliable vector with high transductionefficiency for gene transfer in Ctns-/- mice, producing extendedexpression of cystinosin for 12 to 15 months and likely longer after asingle injection. Current study is aiming at characterizing cornealhistology and biochemistry in treated and untreated mice.Commercial Relationships: Duy H. Nguyen, None; Hongjun Du,None; Matthew Bedell, None; Seanna Grob, None; Jing Luo,None; John L. Quach, None; Peter Shaw, None; StephanieCherqui, None; Kang Zhang, Acucela (F), Genentech (F),Thrombogenics (F)Support: Cystinosis Research Foundation Grant, NEI/NIH Grants,Research to Prevent Blindness, BWF Clinical Scientist Award inTranslational ResearchProgram Number: 2732 Poster Board Number: A0176Presentation Time: 8:30 AM - 10:15 AMTropisms of AAV for Subretinal Delivery to the Neonatal MouseRetina and Its Application for In Vivo Rescue of the CrxKnockout RetinaSatoshi Watanabe 1, 2 , Rikako Sanuki 1 , Shinji Ueno 3 , TakahisaFurukawa 1 . 1 Laboratory for Molecular and Developmental Biology,Institute for Protein Research, Osaka University and JST, CREST,Suita, Japan; 2 Department of Molecular Genetics, Kyoto UniversityGraduate School of Medicine, Kyoto, Japan; 3 Department ofOphthalmology, Nagoya University Graduate of School of Medicine,Nagoya, Japan.Purpose: Adeno-associated virus (AAV) is well established as avehicle for in vivo gene transfer into the mammalian retina. This virusis promising not only for gene therapy of retinal diseases, but also forin vivo functional analysis of retinal genes. Previous reports haveshown that AAV can infect various cell types in the developingmouse retina. However, AAV tropism in the developing retina hasnot yet been examined in detail. Thus, we examined the tropisms ofseven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) forsubretinal delivery into the P0 mouse retina.Methods: We subretinally delivered seven AAV serotypes of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluatedthe tropisms of each serotype by its infecting degree in retinal cells.After subretinal injection of AAV into postnatal day 0 (P0) mouseretinas, various retinal cell types were efficiently transduced withdifferent AAVs. To confirm the usefulness of AAV-mediated genetransfer into the P0 mouse retina, we performed AAV-mediatedrescue of the Cone-rod homeobox gene knockout (Crx KO) mouse,which exhibits an outer segment formation defect, flatelectroretinogram (ERG) responses, and photoreceptor degeneration.We subretinally injected an AAV expressing Crx under the control ofthe Crx 2kb promoter into the neonatal Crx KO retina.Results: Photoreceptor cells were efficiently transduced withAAV2/5. Retinal cells, except for bipolar and Müller glial cells, wereefficiently transduced with AAV2/9. Horizontal and/or ganglion cellswere efficiently transduced with AAV2/1, AAV2/2, AAV2/8,AAV2/9 and AAV2/10. We showed that AAV-mediated Crxexpression significantly decreased the abnormalities of the Crx KOretina by histological and physiological analyses.Conclusions: In the current study, we report suitable AAV tropisms©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 – Physiology/Pharmacologyfor delivery into the developing mouse retina. Using AAV2/5 inphotoreceptor cells, we demonstrated the possibility of genereplacement for the developmental disorder and subsequentdegeneration of retinal photoreceptors caused by the absence of Crx.Commercial Relationships: Satoshi Watanabe, None; RikakoSanuki, None; Shinji Ueno, None; Takahisa Furukawa, NoneProgram Number: 2733 Poster Board Number: A0177Presentation Time: 8:30 AM - 10:15 AMAccessibility of Retinal Cells to Different AAV Serotypes AfterIntravitreal Injection in Old Versus New World PrimatesMaureen Neitz 1 , Anita E. Hendrickson 1 , Michael Lukason 2 , JayNeitz 1 , Daniel E. Possin 1 , Gelareh Abedi 3 , Jing Huang 1 , AbrahamScaria 2 , Sam Wadsworth 2 . 1 Ophthalmology, University ofWashington, Seattle, WA; 2 Gene Therapy, Genzyme-Sanofi,Framingham, MA; 3 Ophthalmology & Optometry, University ofTexas, San Antonio, TX.Purpose: Adeno-associated virus (AAV) is a promising vehicle forocular gene therapy but little is known about what cells types areaccessible to virus injected intravitreally (IVT) in primate. Here weevaluated this issue for AAV serotypes 2 and 5.Methods: 10 animals each received a single IVT injection containingan admixture of two AAVs: one carrying the gene for GFP the othercarrying the gene for a chimeric soluble VEGF receptor, sFLT02. Allvectors had a ubiquitous chicken beta actin promoter. For eachanimal, the same serotype, 2 or 5, was injected in both eyes but AAVinjected into the right eye had a wild type (wt) capsid, AAV injectedinto the left eye had a capsid modification (see Table 1). Macaquesreceived 1x10 10 vector genomes (vg) of each vector in 50 µL.Marmosets received 8x10 9 vg of each vector in 10 µL. GFPexpression was evaluated histologically, sFLT02 transgene productwas measured using a commercially available ELISA.Results: In macaques, sFLT02 expression was similar for wild typeAAV2 and modified AAV2 capsids, and expression with AAV2vectors was higher than for AAV5; however, AAV5 with modifiedcapsids gave stronger expression than wt AAV5 capsids. ELISAresults for sFLT02 in marmosets were inconsistent across animals forAAV5 vectors but modified AAV2 gave higher sFLT02 levels thanwt AAV2. All but one macaque eye injected with AAV2 gave strongGFP labeling of ganglion cells around the foveal pit with fewerfoveal amacrines, horizontals, bipolars and cones. Scattered labeledganglion cells and many Mueller cells were present in the mid to farperiphery. Pars plana epithelial cells were strongly labeled. Thedistribution of GFP was similar for eyes treated with wt versus capsidmodified AAV5. In marmosets, GFP labeling for eyes treated withAAV2 was similar to macaque but labeling at the fovea was muchless pronounced. AAV5 virus gave weak GFP labeling in marmosets,with little or no foveal labeling.Conclusions: IVT administration of AAV vectors transduces avariety of cell types within the eye including ganglion, amacrine,bipolar, horizontal cells, and to some extent, photoreceptors. Tropismdiffers among serotypes with AAV2 transducing more cell types thanAAV5. While the capsid modifications examined in theseexperiments did not appear to alter tropism, overall transgeneexpression level was affected.Commercial Relationships: Maureen Neitz, Genzyme (F), Alcon(F), Alcon (P); Anita E. Hendrickson, None; Michael Lukason,Genzyme (E); Jay Neitz, Alcon (F), Alcon (P); Daniel E. Possin,None; Gelareh Abedi, None; Jing Huang, None; Abraham Scaria,Genzyme Corporation (F), Genzyme Corporation (P); SamWadsworth, Genzyme (E)Support: NIH Grants EY09303, EY016861, EY01730, RPBProgram Number: 2734 Poster Board Number: A0178Presentation Time: 8:30 AM - 10:15 AMTransfer of TACSTD2 Gene into Corneal Epithelial Cells ofGelatinous Drop-Like Corneal Dystrophy and Its FunctionalExpressionToru Matsunaga 1, 2 , Koji Kitazawa 3 , Kenta Yamasaki 4 , Takao Sato 1, 2 ,Yasuo Watanabe 1, 2 , Toshinari Funaki 1 , Akira Matsuda 1 , NobuyukiEbihara 1 , Satoshi Kawasaki 3 , Akira Murakami 1 . 1 Ophthalmology,Juntendo University School of Medicine, Tokyo, Japan; 2 Reserch andDevelopment, SEED Co., Ltd., Saitama, Japan; 3 Ophthalmology,Kyoto Prefecutural University School of Medicine, Kyoto, Japan;4 Biomedical Engineering, Faculty of Life and Medical Sciences,Doshisha University, Kyoto, Japan.Purpose: Gelatinous drop-like corneal dystrophy (GDLD) is arefractory disease that deposits amyloid into the subepitheliumthrough increased intercellular permeability in the corneal epithelium.As a novel treatment for the disease, we have been investigating thetransfer of TACSTD2 gene that is responsible for the disease, into acontact lens (CL) to use as the device. For the purpose of this study,we have used corneal epithelial cells that were obtained from aGDLD patient and investigated the transfer and functional expressionof TACSTD2 gene.Methods: Corneal epithelial cells that had been obtained from aGDLD patient were immortalized with the lentiviral transduction ofthe simian virus 40 (SV40) large T antigen and human telomerasereverse transcription (hTERT) genes. CL that had phosphate groupsas its side chains was synthesized as the gene transfer device to allowit to form a complex, mediated by calcium ions, with plasmid DNAobtained through implanting pLenti6.3_V5-TOPO vector with theTACSTD2 gene (TACSTD2-V5). Using this DNA-CL, gene transferwas performed into the immortalized corneal epithelial cell line fromthe GDLD patient (imHCE-T_GDLD) cells, followed by observationfor expression of TACSTD2 as well as claudin (CLDN).Results: The imHCE-T_GDLD cells processed with DNA-CLobserved fluorescence in the cellular cytoplasm in theimmunostaining with an anti-TACSTD2 antibody. Localization ofCLDN1 and CLDN7 were also observed. Western blottingrecognized protein expression as well.©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 – Physiology/PharmacologyConclusions: Transfer into imHCE-T_GDLD cells and functionalexpression of TACSTD gene recognized through the use of DNA-CLhas indicated that gene transfer can be achieved by non-invasivetreatments.Commercial Relationships: Toru Matsunaga, SEED Co.,Ltd. (E),JP5132958 (P); Koji Kitazawa, None; Kenta Yamasaki, None;Takao Sato, SEED Co., Ltd. (E); Yasuo Watanabe, None;Toshinari Funaki, None; Akira Matsuda, None; NobuyukiEbihara, None; Satoshi Kawasaki, None; Akira Murakami,SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958 (P)Support: Health and Labour Sciences Research Grant on IntractableDiseases H23-Nanchi-Ippan-084Clinical Trial: UMIN000007228Program Number: 2735 Poster Board Number: A0179Presentation Time: 8:30 AM - 10:15 AMTransduction Profile of Anterior Chamber-injected CapsidMutated Self-complementary Adeno-Associated Virus Type 2 inRodentsBarbara Bogner 1 , Sanford L. Boye 2 , Seok-Hong Min 2 , James J.Peterson 2 , Qing Ruan 2 , Vince Chiodo 2 , Renee C. Ryals 2 , Herbert A.Reitsamer 1 , William W. Hauswirth 2 , Shannon E. Boye 2 .1 Ophthalmology/Optometry, Paracelsus Medical University,Salzburg, Austria; 2 Ophthalmology, University of Florida,Gainesville, FL.Purpose: The surface properties of the adeno-associated virus (AAV)capsid are essential for cellular tropism and efficiency of transgeneexpression. The transduction properties of AAV2-based capsidmutants have not been investigated in the anterior chamber (AC). Inthis study we investigate transduction profiles of AAV2 mutantscontaining self-complementary AAV (scAAV) genomes coding forgreen fluorescent protein (GFP) by localizing GFP in AC-injectedmice and rats. scAAV2 mutants contain single or combinations oftriple and septuple tyrosine (Y) to phenylalanine (F) mutations in thehighly conserved surface-exposed capsid tyrosine residues.Methods: AC injections were performed in C57BL/6 mice (1 µl) andWistar rats (2 µl). After general and topical anesthesia, the peripheralcornea was punctured (33G needle) anterior of the iridocorneal angle.scAAVs expressing GFP under the constitutive small CMV-chickenβ-actin (smCBA) promoter and vehicle control (BSS+0.014% Tween20) were infused slowly using a micropump. 4 weeks post-injection,animals were sacrificed and GFP-expression was evaluated byimmunohistochemistry and confocal microscopy. In Table 1experimental groups and titers for scAAVs are listed.Results: scAAV-injected eyes showed intense GFP-signals in thecorneal endothelium, the ciliary non-pigmented epithelium (NPE),the chamber angle and the iris. Depending on the scAAV mutant, theexpression pattern ranged from mosaic-like to homogenous. Both,vehicle control and control without injection revealed noimmunopositivity. Table 1 summarizes the localization andsemiquantitative evaluation of the GFP-signal in injected eyes.Conclusions: scAAVs bypass rate-limiting second-strand DNAsynthesis to obtain the transcriptionally active AAV genome. Thisresults in earlier onset of transgene expression and facilitates thetransduction of difficult-to-transduce cells (e. g. trabecularmeshwork). This study shows that mutagenesis of surface-exposedtyrosine residues affects the tropism and transduction efficiency ofscAAV2 in the AC. While single and triple mutants show enhancedtransduction efficiency, the septuple mutant reveals no improvementcompared to scAAV2 WT. These results are of interest for thedevelopment of transgene-based models to investigate pathologicalprocesses in the AC and for the development of gene-based therapiestargeted to the trabecular meshwork.Commercial Relationships: Barbara Bogner, None; Sanford L.Boye, PCT/US2012/062478 (P); Seok-Hong Min, None; James J.Peterson, None; Qing Ruan, None; Vince Chiodo, None; Renee C.Ryals, None; Herbert A. Reitsamer, None; William W.Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C),RetroSense (C); Shannon E. Boye, NoneSupport: Austrian Academy of Sciences, PMU-FFF, FuchsEndowment, Adele Rabensteiner Endowment, Lotte SchwarzEndowmentProgram Number: 2736 Poster Board Number: A0180Presentation Time: 8:30 AM - 10:15 AMGlycosidic Enzymes Enhance Retinal Transduction FollowingIntravitreal Delivery of AAV2Jasmina Cehajic-Kapetanovic 1, 2 , Annette Allen 2 , Paul N. Bishop 1 ,Robert Lucas 2 . 1 Institute of Human Development, University ofManchester, Manchester, United Kingdom; 2 Faculty of Life Sciences,University of Manchester, Manchester, United Kingdom.Purpose: At present only limited retinal transduction can be achievedfollowing intravitreal delivery of unmutated AAV vectors. Wehypothesized that the inner limiting membrane and extracellularmatrix proteoglycans act as a barrier to AAV vector entry into andmovement across the retina. In this study we investigated the effectsof enzymatic digestion of these barriers on the depth of vectorpenetration into the retina.Methods: The green fluorescent protein (GFP)-expressing AAV2vector was co-injected intravitreally into adult mice with variousglycosidic enzymes and their combinations. The efficacy of the virustransduction was evaluated by quantitative analysis of GFP positivecells in histological cross-sections, using fluorescent microscopy. Wealso analyzed safety of these treatments and retinal function usingelectroretinography.Results: Glycosidic enzymes namely Chondroitin ABC lyase,Heparinase III and Hyaluronan lyase, both singly and incombinations, led to a significant improvement in retinal transductionfollowing intravitreal delivery. Electroretinograms survived at muchhigher doses of enzymes than were needed for optimal retinaltransduction.Conclusions: AAV2-mediated retinal transduction is improved byco-injection of glycosidic enzymes into the vitreous. This approachmay allow intravitreal injection to become the preferred route for©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 – Physiology/Pharmacologydelivering gene therapy to the retina in both preclinical and clinicalsettings.Commercial Relationships: Jasmina Cehajic-Kapetanovic, None;Annette Allen, None; Paul N. Bishop, None; Robert Lucas, NoneSupport: Medical Research CouncilProgram Number: 2737 Poster Board Number: A0181Presentation Time: 8:30 AM - 10:15 AMAntisense oligonucleotide-based therapy for CEP290-associatedLCAAlejandro Garanto, Frans P. Cremers, Rob W. Collin. HumanGenetics, Radboud University Nijmegen Medical Centre, Nijmegen,Netherlands.Purpose: The most frequent genetic cause of Leber congenitalamaurosis (LCA) is an intronic change in CEP290, that results in theinsertion of a cryptic exon (exon X) into the CEP290 mRNA.Recently, we have shown promising results of antisenseoligonucleotide (AON)-based therapy for this disorder, sincetransient transfection of AONs to cultured lymphoblastoïd cells ofthese patients almost completely rescued the aberrant splicing ofCEP290 mRNA that is caused by the intronic mutation. The aim ofthis work is to further develop this therapeutic strategy, by employingother cell types, generate viral vectors that express AONs and bycharacterizing a CEP290 knock-in mouse model that carries part ofthe human CEP290 gene including the intronic mutation, specificallydesigned for this purpose.Methods: Skin biopsies from LCA patients homozygously carryingthe intronic CEP290 mutation allowed the generation of fibroblastcell lines. These fibroblasts were transfected with either naked AONsor AONs cloned into viral constructs. CEP290 transcripts wereanalyzed by RT-PCR, and CEP290 protein levels via Western blotanalysis. In addition, cilium length was assessed by immunostaining.Two knock-in models have been generated, and initiallycharacterized at transcriptional and morphological levels, by RT-PCRand immunohistochemistry. Animal work was performed accordingto the ARVO statement for the use of animals in ophthalmic andvision research.Results: Our data have proved the efficacy of the naked AONs infibroblasts, restoring the correct splicing of the transcript. We arecurrently working on the validation of these results, by measuringCEP290 protein levels and cilium length in AON-treated fibroblasts.We are also testing viral constructs to further application in cell linesand animal models. The initial characterization of the humanizedmodels at transcriptional level, has shown an unexpected splicingpattern in the humanized mice. This new splicing event results in theinsertion of a second cryptic exon (Y) in part of the CEP290transcripts. Ongoing work will determine the presence/absence of thehuman aberrant exon (X) and exon Y, as well as whether the splicingevents are tissue or mouse-strain-specific.Conclusions: Together, this work will be instrumental for thedevelopment of AON-based therapy for CEP290-associated LCA.Commercial Relationships: Alejandro Garanto, None; Frans P.Cremers, None; Rob W. Collin, Radboud University MedicalCentre (P)Support: FFB USA Individual Investigator Grant Award: TA-GT-0912-0582-RADProgram Number: 2738 Poster Board Number: A0182Presentation Time: 8:30 AM - 10:15 AMMitochondrial Gene Therapy for G11778A LHON: Safety ofHuman ND4 in Nonhuman Primates and Expression in Ex VivoHuman EyesRajeshwari D. Koilkonda 1 , David T. Tse 1 , William W. Hauswirth 2 ,Vince Chiodo 2 , Sanford L. Boye 2 , Martha Neuringer 3 , Tim Stout 3 ,John Guy 1 . 1 Ophthalmology, Bascom Palmer Eye Institute,University of Miami-Miller School of Medicine, Miami, FL;2 Department of Ophthalmology, University of Florida, College ofMedicine, Gainesville, FL; 3 Oregon Health and Science University,Beaverton, OR.Purpose: To demonstrate the safety and expression of allotopichuman ND4 to be used for the Leber’s hereditary optic neuropathygene therapy trial.Methods: We modified ND4 in the nuclear genetic code for importinto mitochondria. The protein was targeted into the organelle byATPc (P1) sequence. The gene was packaged into selfcomplementaryAAV2 packaged with triple Y-F capsid mutant(Y444F+Y500F+Y730F) VP3 then injected into rodent, nonhumanprimate and ex vivo human eyes. Expression and integration intocomplex I of rodents and ex vivo human eyes was tested byimmunohistochemistry and blue native (BN) PAGE. Three rhesusmacaques (1m, 2f, ages 3-7 yrs) were injected intravitreally (iv) withscAAV2(Y444,500,703F)-smCBA-P1ND4. The vector titer was1.23x10 11 vg/ml, and a volume of 200 μL was injected for a totaldose of 2.46x10 10 vg. We next tested for transgene expression in twonormal human eyes that were surgically removed for control ofperiocular spread of cancers. After incising the cornea, eyes wereimmersed immediately in tissue culture media. One eye received ivinjection of 120 μL of scAAV-ND4FLAG (1.0 x 10 12 vg/ml) theother 20 µL (4.0 x 10 12 vg/ml).Results: Rodents.2-D BN PAGE with the anti-FLAG antibodyreacted against infected retinal mitochondrial extracts from rats1month post injection (PI) of scAAV-ND4FLAG showed a distinctband of FLAG, absent in uninfected control retinal mitochondria.Antibodies against NDUFA9, NDUFS3, NDUFS4, NDUFB8 orNDUFA6 detected these nuclear encoded complex I subunits. HumanEyes. 3days PI, ND4FLAG co-localized with mitochondrial porin.Quantitative analysis revealed, injection of more than 10 11 vg, 84% ofRGCs expressed ND4FLAG. Nonhuman Primates. Clinicalexaminations and retinal fundus photography done at baseline, 5, 8and 15days PI revealed no clinical signs of inflammation, such ashaze or presence of inflammatory cells in the anterior chamber orvitreous and looked similar to the uninjected left eye. One animaldeveloped mild vitritis ~2m after AAV injection. OCT images of theretina and optic nerve head showed no structural changes frombaseline in all 3 animals, but cells were present in the vitreous of oneanimal.Conclusions: Expression of allotopic ND4 in the ex vivo human eye,safety of the test article in rodent and primate visual systems and thesevere irreversible loss of RGCs in LHON support clinical testing.Commercial Relationships: Rajeshwari D. Koilkonda, None;David T. Tse, None; William W. Hauswirth, AGTC (I), BionicSight (I), AGTC (C), Syncona (C), RetroSense (C); Vince Chiodo,None; Sanford L. Boye, PCT/US2012/062478 (P); MarthaNeuringer, Pfizer (F), Applied Genetic Technologies Corporation(F); Tim Stout, Clayton Foundation (P), Oxford Biomedica (C),AGTC (F), Peregrine Pharmaceuticals Inc (C), Stem Cells Inc (C);John Guy, NoneSupport: R24EY018600Program Number: 2739 Poster Board Number: A0183Presentation Time: 8:30 AM - 10:15 AMDual Adeno-associated virus vectors for large cDNA genereplacement therapy©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 – Physiology/PharmacologyFrank M. Dyka, Sanford L. Boye, Vince Chiodo, Shannon E. Boye,William W. Hauswirth. Ophthalmology, University of Florida,Gainesville, FL.Purpose: Adeno-associated virus (AAV) is emerging as the preferredvector to mediate gene replacement therapy due to its lowimmunogenicity, ability to efficiently transduce a wide variety of celltypes and demonstrated efficacy in clinical trials. One of its majordrawbacks is a relatively small packaging capacity, ~4.8 kb. Retinaldisorders such as recessive Stargardt disease and Usher syndrome 1Bare caused by mutations in large genes (ABCA4, MYO7Arespectively) that exceed this limit. Alternatively the use ofLentivirus or Adenovirus is problematic due to low transductionefficiency of mature photoreceptors or immunogenicity. After viralentry into the host cell nucleus, dual AAV vectors (cDNA splitbetween two defined vectors) or fragmented (packaging of randomlytruncated genomes) AAV vectors recombine to reconstitute fulllength cDNA. Capitalizing on this we sought to develop defined dualAAV vector systems capable of generating full length cDNAs oflarge genes in vitro and in vivo.Methods: The coding sequences of MYO7A and ABCA4 were clonedin AAV vector pairs, where one vector contains a CMV/chickenβactin (CBA) promoter and the 5’ portion of the cDNA sequence anda second vector contains the 3’ portion followed by a polyA signal.One vector pair shares a central cDNA sequence of 1.3 kb (simpleoverlap vector). Another pair utilizes splice donor and acceptor sites(trans-splicing vector) and the third pair utilizes these splice sites incombination with an efficiently recombinogenic overlappingsequence (hybrid vector). All vectors were packaged in AAV2 fortransduction of HEK293 cells (10,000 MOI) or retina after subretinalinjection into null ABCA4 or MYO7A mice. Cells were collected at3 days post infection and retina at 6 weeks post infection for analysis.Results: Co-infection of HEK293 cells with simple overlap, transsplicingor hybrid AAV vector pairs expressed full length protein invitro, all with equal or higher efficiency than that of the fragmentedvector. Of the three dual vector systems, the hybrid system was themost efficient at expressing full length protein. In-vivo analysis is ongoing.Conclusions: Large cDNAs exceeding the size limitations ofconventional AAV can be expressed with high efficiency andspecificity using optimally designed dual AAV vector systems. Ourresults suggest that a dual AAV vector system may be a viable optionfor the treatment of large gene disorders of the retina.Commercial Relationships: Frank M. Dyka, 61/560,437 (P),PCT/US2012/062478 (P); Sanford L. Boye, PCT/US2012/062478(P); Vince Chiodo, None; Shannon E. Boye, None; William W.Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C),RetroSense (C)Support: NIH grant EY021721, Foundation Fighting Blindness,Macula Vision Research Foundation, and Research to PreventBlindnessProgram Number: 2740 Poster Board Number: A0184Presentation Time: 8:30 AM - 10:15 AMA comparison of the effects of subretinal injection of scAAV2-CMV-GFP on retinal structure, visual function, and GFPexpression by two injectorsHui Li, Stephen H. Poor, Chad E. Bigelow, Vivian Choi, ShawnHanks, Joanna Vrouvlianis, Michael Maker, Steve Louie, Sha-MeiLiao, Bruce D. Jaffee. Novartis Institutes for Biomedical Research,Cambridge, MA.Purpose: To compare the effects of subretinal (SR) injection ofadeno-associated virus (AAV) expressing GFP and saline by twoscientists, using optical coherence tomography (OCT) andelectroretinography (ERG) and RPE/Choroid tissue flat mount forGFP expression.Methods: 1μl of scAAV2-CMV-GFP (5.67x108 DNase resistantparticle/μl) or saline, both mixed with sodium fluorescein wasinjected subretinally into the eyes of C57Bl/6 mice (16 eyes/treatment group/operator). Injection quality was evaluated at thetime of the procedure using a 3-level scoring system (1= nohemorrhage minimal intravitreal backflow, score 2 = minorhemorrhage and mild backflow, score 3 = moderate hemorrhage,significant backflow). Fundus images were acquired immediatelyafter the SR injection. OCT was performed 1 week post-injection andERG recordings were performed 2 weeks post-injection. RPEchoroidalflatmounts were made from ocular tissues collected 3weeks post-injection. Levels of GFP expression were quantifiedusing Axiovision software to delineate fluorescent area on the flatmounts.Results: 59 of 64 injections were scored a “1”, 3 injections werescored “2”, and 2 injections were scored “3”. Eyes with an injectionscore of 1 or 2 were used for OCT and ERG quantification. Retinalstructure 1 week post subretinal injection was overall similar by OCTexamination with a slight increase in incidence of injection sitetrauma and reduced media clarity for one operator. Photoreceptorfunction measured with a single flash intensity ERG showed anaverage drop of approximately 18% vs. uninjected controls (nostatistically significant) for both operators and injection materials. Nostatistically significant difference in function was detected betweengroups injected by each operator or between materials injected (AAVvector vs. saline). GFP expression per percentage of flatmount areafor score 1 was 74%, score 2 was 60%, and score 3 was 25%. Nosignificant difference in GFP coverage was observed betweenoperators.Conclusions: Subretinal injections disrupted retinal structure to asimilar degree for both injection materials (vector and saline). ERGsfrom injected eyes were reduced in amplitude vs. uninjected controls,but the reduction was independent of individual operator or injectionmaterial. Poor quality injections predicted a smaller area of GFPexpression.Commercial Relationships: Hui Li, None; Stephen H. Poor,Novartis Institue of Biomedical Research (E); Chad E. Bigelow,Novartis (E); Vivian Choi, Novartis Institute for BioMedicalResearch (E); Shawn Hanks, Novartis Institutes for BiomedicalResearch (E); Joanna Vrouvlianis, None; Michael Maker, NovartisInstitutes for Biomedical Research (E); Steve Louie, Novartis (E);Sha-Mei Liao, Novartis (E); Bruce D. Jaffee, Novartis (E)Program Number: 2741 Poster Board Number: A0185Presentation Time: 8:30 AM - 10:15 AMGlaucoma-GT, a novel gene therapy treatment for primary openangleglaucomaScott Ellis, Katie M. Binley, Vicky Scripps, Sharifah Iqball, Stuart M.Naylor, Kyriacos Mitrophanous. Oxford BioMedica (UK) Limited,Oxford, United Kingdom.Purpose: Glaucoma is the second leading cause of blindnessworldwide, affecting around 70 million people, over 2.5 millionpeople in the USA alone. It is characterized by irreversibledegeneration of the optic nerve, usually associated with an elevatedintraocular pressure (IOP). Prostenoid analogues such as Latanoprostare a first-line treatment for glaucoma due to their high level ofefficacy and low risk of side-effects, but fail to halt diseaseprogression in many patients due to non-adherence, which can be>50%. Surgery is often required, which is expensive and onlypartially effective. There is therefore a real medical need for a noveldrug that overcomes this issue of poor compliance.©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 – Physiology/PharmacologyInitial proof-of-concept for this approach has been demonstrated(Barraza et al, 2010), and we are now moving this into a translationalgene therapy platform for clinical evaluation. Glaucoma-GT is a genetherapy product aimed at lowering IOP via the prostenoid pathway: asingle transcorneal administration leads to the expression of humancyclooxygenase-2 (COX-2) and prostaglandin F (FP) receptor in thefront of the eye. COX-2 is a rate-limiting enzyme in the biosynthesisof prostaglandins, and both COX-2 and the FP receptor are downregulatedin glaucomatous eyes. Expression of both genes increasesboth prostaglandin 2α (PGF2α) biosynthesis and signaling, increasingaqueous outflow and reducing longterm IOP.Methods: Gene transfer following transcorneal administration ofEIAV-GFP vector was characterised in animal models. Variousvector genome configurations were assessed for the production ofPGF2α and activation of the FP receptor in in vitro assays. Furtherpreclinical studies are currently ongoing and data from these will bepresented.Results: Transcorneal injection of EIAV vector led to thetransduction of cells of the trabecular meshwork and cornealendothelium in vivo. Different configurations of expression cassettegave a broad range of PGF2α and FP activation, with CMV-COX-2-IRES-FP having the highest activity.Conclusions: We have demonstrated significant gene transferfollowing transcorneal administration of EIAV-GFP vector in animalmodels, and have optimised the therapeutic expression cassette toproduce high levels of prostaglandin 2α and enhanced FP activationin in vitro studies. These vector configurations will be assessed forIOP lowering in a relevant animal model.Commercial Relationships: Scott Ellis, Oxford BioMedica (E);Katie M. Binley, Oxford BioMedica (UK) Ltd (E); Vicky Scripps,Oxford BioMedica (E); Sharifah Iqball, Oxford BioMedica (UK)Ltd (E); Stuart M. Naylor, Oxford BioMedica (UK) Ltd (E);Kyriacos Mitrophanous, Oxford BioMedica (UK) Ltd (E)Program Number: 2742 Poster Board Number: A0186Presentation Time: 8:30 AM - 10:15 AMFunctional Biomarkers For Successful Molecular Therapy OfThe Outer Retina In Retinal DegenerationsVithiyanjali Sothilingam 1 , Marina Garcia Garrido 1 , Christina Seide 1 ,Susanne Koch 2 , Stylianos Michalakis 2 , Martin Biel 2 , NaoyukiTanimoto 1 , Mathias W. Seeliger 1 . 1 Division of Ocular Degeneration,Ctr for Ophthal Inst for Ophthalmic Rsch, Tuebingen, Germany;2 Department of Pharmacy-Centre for Drug Research, Center forIntegrated Protein Science Munich (CIPSM), Munich, Germany.Purpose: Retinal degenerations are severe and frequently blindinghuman diseases. Several current therapeutic approaches havedemonstrated long-lasting amelioration in respective disease modelsand show promise for a successful translation to human patients.Here, we focus on electroretinographic (ERG) features in theexperimental data in order to identify informative biomarkers forupcoming clinical trials.Methods: Core of this work is the electrophysiologic evaluation oftreated mouse models representing achromatopsia (Cnga3 -/- ) orretinitis pigmentosa (Cngb1 -/- ). Mice of these lines received adenoassociatedvirus (AAV)-mediated gene replacement therapy designedto express mouse CNGA3 or CNGB1 under control of respectivephotoreceptor-specific promoters (mouse S opsin or rhodopsin).Treatment success was monitored with different single flash andflicker protocols of in vivo electrophysiology. The findings werecompared to immunohistochemistry, in vivo imaging, and behavioralassays.Results: In both disease models, the therapeutic effects were welldetectable with several of the explorative ERG protocols used in thisstudy. However, the signal-to-noise ratio varied between theparticular tests, depending on the nature of the underlying disease(rod- or cone-specific origin). On this basis, options for biomarkerswere developed for potential use in human trials.Conclusions: The ERG is a very effective method to assess outerretinal functionality. We show here that specific ERG protocols arewell suited to follow therapeutic effects in retinal degenerations. Suchtests may thus serve as functional biomarkers for molecular therapyin upcoming clinical trials.Commercial Relationships: Vithiyanjali Sothilingam, None;Marina Garcia Garrido, None; Christina Seide, None; SusanneKoch, None; Stylianos Michalakis, None; Martin Biel, None;Naoyuki Tanimoto, None; Mathias W. Seeliger, NoneSupport: DFG Se837/6-2, Se837/7-1 (KFO 134), KerstanFoundation/RD-CUREProgram Number: 2743 Poster Board Number: A0187Presentation Time: 8:30 AM - 10:15 AMIn Vivo Quantification Of Photoreceptor Transduction EfficiencyUsing Novel Modified AAV CapsidsRenee C. Ryals, Christine N. Kay, Sanford L. Boye, Seok-Hong Min,Andrea E. Ayala, William W. Hauswirth, Shannon E. Boye.Ophthalmology, University of Florida, Gainesville, FL.Purpose: Because the majority of inherited retinal diseases arecaused by mutations in photoreceptor (PR)-specific genes, there isgreat need to develop PR-targeted gene therapies. Development of aless invasive surgical procedure is also warranted, particularly whenan underlying genetic defect results in fragile retina prone to furtherdamage upon surgically induced retinal detachment. Retinaldegeneration patients would benefit from vectors that possess anenhanced ability to transduce photoreceptors following intravitrealdelivery. The purpose of our study was to develop an in vivo assay toquantify the relative abilities of novel AAV vectors to transducephotoreceptors following intravitreal delivery to mouse.Methods: One month old heterozygote Rho-GFP mice receivedtrans-scleral, intravitreal injections (1.5 ul, 1E12vg/ml) of AAV2,AAV5 or AAV8-based vectors (with or without tyrosine/threoninecapsid mutations). 4 weeks post injection, mCherry expression wasvisualized by fundoscopy, retinas were dissociated and FACSanalysis was used to quantify the percentage of cells that were GFPpositive (rods), mCherry positive (any retinal cells transduced withAAV) and both GFP and mCherry positive (rods transduced byAAV). Percentage of mCherry positive photoreceptors= (% of cellsboth GFP and mCherry positive cells/% of total photoreceptors).Results: Fundoscopy and FACS showed that mCherry expressionvaried with the serotype injected. Photoreceptor transduction wasenhanced following injection with tyrosine/threonine capsid mutants.Retinas injected with AAV2 had 1.7% mCherry positive PRs, retinasinjected with AAV2 Y-F QM and AAV2 Y-F QM + T-V had 6.1%and 21.8% mCherry positive PRs, respectively.Conclusions: We have established a reliable in vivo assay for scoringthe relative photoreceptor transduction efficiencies of novel AAVvectors following delivery to the vitreous. This assay will be used todetermine the optimal serotype(s) with which to treat models ofinherited retinal disease. Furthermore, it will support development ofAAV-based clinical vectors for the treatment of various forms ofphotoreceptor-mediated inherited retinal disease following asurgically less invasive intravitreal injection.Commercial Relationships: Renee C. Ryals, None; Christine N.Kay, None; Sanford L. Boye, PCT/US2012/062478 (P); Seok-HongMin, None; Andrea E. Ayala, None; William W. Hauswirth,AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C);Shannon E. Boye, 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 – Physiology/PharmacologySupport: FFB Indivdual Investigator Award, Reseach to PreventBlindnessProgram Number: 2744 Poster Board Number: A0188Presentation Time: 8:30 AM - 10:15 AMThe LentiVector ® Gene Therapy Platform for Ocular Disease: aclinical updateKyriacos Mitrophanous, Scott Ellis, James Miskin, Katie M. Binley,Michelle Kelleher, Cherry A. Lucas, Stuart M. Naylor. OxfordBioMedia (UK) Ltd, Oxford, United Kingdom.Purpose: Oxford BioMedica has developed ocular gene therapiesusing its proprietary LentiVector ® platform which is based onrecombinant Equine Infectious Anaemia Virus (EIAV). Currently wehave three ocular therapies in clinical development: RetinoStat ® ,StarGen and UshStat ® , for the treatment of age-related maculardegeneration, Stargardt macula dystrophy and Usher Syndrome 1Brespectively. Regulatory approval in the US and France has beenreceived for RetinoStat ® and StarGen, and UshStat ® has beenapproved in the US. Phase I (RetinoStat ® ) and Phase I/IIa (StarGenand UshStat ® ) clinical evaluations are currently underway.Methods: The RetinoStat ® , StarGen and UshStat ® trials consist ofa dose-escalation phase followed by an expanded final cohort at thehighest safe and tolerated dose. Safety and signs of clinical benefitwill be assessed throughout these trials. Additionally, the secretednature of the transgenes in the RetinoStat ® trial has meant thattransgene expression from aqueous tap samples can also bequantified over time at each dose.Results: The dose-escalation phase of the RetinoStat ® trial has beencompleted. For StarGen and UshStat ® the dose-escalation phase isin progress. Subretinal administration of all three products has beenwell tolerated, causing no ocular inflammation or immune responsesin any patients. There have been no SAEs related to product. Allthree products have been safe and well tolerated thus far.Transduction of the retina following subretinal injection ofRetinoStat ® rapidly produced high levels of both transgenesdetectable in the aqueous. These levels are substantial, show dosedependence.Conclusions: Ocular gene therapies based on the LentiVector ®platform continue to show good safety following subretinal deliveryinto patients for three different ocular indications. The platform hasproved to be a highly effective delivery system for relatively largegenes into target retinal cells, resulting in stable and long-termexpression.Commercial Relationships: Kyriacos Mitrophanous, OxfordBioMedica (UK) Ltd (E); Scott Ellis, Oxford BioMedica (E); JamesMiskin, Oxford BioMedica (UK) Ltd (E); Katie M. Binley, OxfordBioMedica (UK) Ltd (E); Michelle Kelleher, Oxford BioMedica(UK) Ltd (E); Cherry A. Lucas, Oxford BIoMedica Ltd (C); StuartM. Naylor, Oxford BioMedica (UK) Ltd (E)Clinical Trial: NCT01301443Program Number: 2745 Poster Board Number: A0189Presentation Time: 8:30 AM - 10:15 AMAssessment of Intravitreal AAV-TEAD4 Isoforms in the OIRModelMatthew Hartzell, Andrew J. Stempel, Trevor J. McFarland, BinoyAppukuttan, Tim Stout. Opthalmology, Oregon Health & ScienceUniv, Portland, OR.Purpose: Purpose: The mouse model of oxygen-induced retinopathy(OIR) has been used extensively as a model of retinopathy ofprematurity to study retinal neovascularization (NV). We have shownthat the transcription factor TEAD4, and its isoforms, can influenceVEGF expression; a determining factor driving tuft formation in thismodel. The purpose of this study was to observe the effects ofTEAD4 isoforms on retinal NV within this model.Methods: Methods: Postnatal day 7 (P7) C57BL/6 mice wereinjected intravitreally with AAV-DJ containing either the TEAD41305 or 447 isoforms (n=4), AAV-DJ containing GFP (n=4) or PBS(n=4). The mice were then introduced to a 75% oxygen environmentfor five days before recovering in room air. All mice were sacrificedat P17 and eyes were enucleated. One eye from each mouse wasenucleated, fixed in 10% neutral buffered formalin and paraffinembedded. Tissue was sectioned at 5uM and sequential slides werestained for hematoxylin and eosin. Neovascular tuft nuclei weremanually counted (15 slides/eye). The retina from the contralateraleye was dissected and used for western blot analysis using anti-TEAD4 antibodies to confirm viral introduction of human isoforms.Results: Results: Western blotting confirmed the presence of theexogenous TEAD4 1305 and 447 isoforms. The AAV 447, 1305 andGFP injected eyes averaged 3.2, 1.6 and 5.1 tuft nuclei per sectionrespectively. PBS injected eyes had an average of 11.75 tuft nucleiper section.Conclusions: Discussion: Successful ocular transduction of P7 OIRmice was achieved with AAV-DJ viruses encoding the TEAD4 1305and 447 isoforms. Neovascular tuft nuclei counts suggest nostimulatory effect with the introduction of stimulatory TEAD4isoforms in this model. The presence of elevated levels of TEAD4 inthis model may influence the expression of VEGF, VEGF receptorand/or other angiogenic factors, perhaps leading to the decrease inoverall tuft formation in the treated groups.Commercial Relationships: Matthew Hartzell, None; Andrew J.Stempel, None; Trevor J. McFarland, None; Binoy Appukuttan,None; Tim Stout, Clayton Foundation (P), Oxford Biomedica (C),AGTC (F), Peregrine Pharmaceuticals Inc (C), Stem Cells Inc (C)Support: NIH;NEI;RO1 EY019042Program Number: 2746 Poster Board Number: A0190Presentation Time: 8:30 AM - 10:15 AMAAV-mediated Combination Therapy of Neurotrophic and Anti-Apoptotic Factors in a Mouse Model of Inherited RetinalDegenerationCecile Fortuny 1 , Leah C. Byrne 1 , Deniz Dalkara 2 , Trevor S. Lee 1 ,Bilge Esin Ozturk 1 . 1 HWNI, Flannery Lab, University of California,Berkeley, Berkeley, CA; 2 Institut de la Vision, Paris, France.Purpose: Many inherited retinal degenerative diseases, such asretinitis pigmentosa, result in blindness as a result of photoreceptorcell death. Since numerous different genetic mutations are involvedin these disorders, it is advantageous to develop general therapies thatpromote photoreceptor survival regardless of the underlyingmutation. In a mouse model, we investigated the therapeutic potentialof a combination cell survival therapy using intravitreal injections oftwo serotypes of adeno-associated virus (AAV) to express a secretedtrophic factor, glial-derived neurotrophic factor (GDNF) in Müllerglia, and an anti-apoptotic factor, X-linked inhibitor of apoptosisprotein (XIAP), in photoreceptors.Methods: C57BL/6 mice were co-injected intravitreally withdifferent concentrations of two engineered adeno-associated virus(AAV) variants: ShH10, an AAV6 variant which selectively targetsMüller glia, and 7m8, an AAV2 variant capable of extensivephotoreceptor transduction from the vitreous. Fluorophores mCherryand GFP were respectively expressed under the control of the CAGpromoter and the photoreceptor-specific rhodopsin promoter (Rho).Fundus fluorescence imaging was performed before eyes wereenucleated four weeks post-injection.Dark reared rd10 mice were injected intravitreally in one eye at p15©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 – Physiology/Pharmacologywith (1) 7m8.Rho.XIAP, (2) ShH10.CAG.GDNF or (3) a mix of bothXIAP/GDNF. The contralateral control eyes were sham injected withPBS. Electroretinograms (ERGs) were recorded to quantify rescue ofrod and cone-mediated responses at p45 and p55.Results: Imaging of retinal sections showed that, at a variety ofconcentration ratios, co-injection of 7m8 and ShH10 led tosimultaneous and strong expression of GFP in photoreceptors andmCherry in Müller glia. Co-injection of vectors encoding XIAP andGDNF led to a significant increase in amplitude of the ERG A-waveand B-wave.Conclusions: We showed that co-injected 7m8 and ShH10 remainedcell-specific and transduced their intended targeted cells withoutsignificant competitive inhibition. The combination therapy codeliveringXIAP and GDNF showed a significant effect in slowingthe progression of retinal degeneration in a rodent model of retinitispigmentosa and represents a promising gene therapy approach to treatinherited diseases.Commercial Relationships: Cecile Fortuny, None; Leah C. Byrne,None; Deniz Dalkara, None; Trevor S. Lee, None; Bilge EsinOzturk, NoneSupport: The Foundation Fighting Blindness333 Drug Delivery IITuesday, May 07, 2013 11:00 AM-12:45 PM618-620 Paper SessionProgram #/Board # Range: 3203-3209Organizing Section: Physiology/PharmacologyProgram Number: 3203Presentation Time: 11:00 AM - 11:15 AMDeslorelin and transferrin mono- and dual- functionalizednanomicelles for drug delivery to the anterior segment of the eyeTrivedi Ruchit 1 , Puneet Tyagi 1 , Sunil K. Vooturi 1 , Uday B. Kompella 1,2 . 1 Pharmaceutical sciences, University of Colorado AnschutzMedical Campus, Aurora, CO; 2 Ophthalmology, University ofColorado Anschutz Medical Campus, Aurora, CO.Purpose: To determine whether encapsulation in novel micellesenhances delivery of hydrophobic molecules to the anterior segmentof the eye after topical administration.Methods: Deslorelin and transferrin were conjugated with PLGApolymer using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide.Conjugation was confirmed using infrared spectroscopy (FTIR) andMALDI-TOF mass spectrometry. Micelles were formed by theaddition of conjugates in dichloromethane to water, and characterizedfor appearance, size, zeta potential, and CMC. Uptake of Nile red(NR) and pazopanib (anti-angiogenic drug) loaded micelles followingtopical administration was evaluated in an ex-vivo bovine eye modelat 5 and 60 minutes post administration using fluorescencespectroscopy for NR and mass spectroscopy for pazopanib.Results: FTIR and MALDI-TOF confirmed conjugate formation.Sizes were 89.0, 74.3, and 59.4 nm, CMCs were 2.5, 100, and2.5µg/ml, and zeta potentials were 1.0, 21.5, and 11.5 mV fordeslorelin-PLGA, transferrin-PLGA, and mixed micelles,respectively. Pink colored, uniform appearance indicated NR loadingin micelles. Pazopanib loading was 57, 83, and 70 µg/mg micelles,while NR loading was 5, 6, and 5 µg/mg micelles for deslorelin-PLGA, transferrin-PLGA, and mixed micelles, respectively. NRuptake in epithelium was 0.2, 16.1, 14.4, and 19.3%, at 5 minutes and0.2, 20.8, 20.3, and 24.9 % at 60 minutes for plain NR, deslorelin-PLGA, transferrin-PLGA, and mixed micelles, respectively. Additionof excess deslorelin and transferrin reduced epithelium uptake to0.31, 10.1, 9.49, and 11.77%, at 5 minutes and 0.25, 13.53, 14.61,and 15.01% at 60 minutes for plain NR, deslorelin-PLGA,transferrin-PLGA, and mixed micelles respectively. Pazopanibuptake in epithelium was 0.4, 14.6, 11.9, and 15.8 % at 5 minutes and1, 19, 14.9, and 18 % at 60 minutes for plain pazopanib, deslorelin-PLGA, transferrin-PLGA, and mixed micelles, respectively. Stromashowed ≤ 1% uptake for micelles and ≤ 0.03% for plain drugsuspension. Endothelium and aqueous humor uptake was negligiblefor both NR and pazopanib. Micelles increased corneal epithelialdelivery by 80-120 fold for Nile red and by 15-39 fold for pazopanib.Conclusions: Functionalized micelles are promising delivery systemsfor the delivery of poorly soluble hydrophobic drugs to the anteriorsegment of the eye.Commercial Relationships: Trivedi Ruchit, None; Puneet Tyagi,None; Sunil K. Vooturi, None; Uday B. Kompella, University ofColorado Denver (P), PCAsso Diagnostics (C), NanoTransTechnologies, Inc. (F), Univesity of Nebraska Medical Center (P)Program Number: 3204Presentation Time: 11:15 AM - 11:30 AMThe Influence of Formulation Factors on TransscleralIontophoretic Delivery of a Macromolecule in Vitro and in VivoSarah A. Molokhia 1, 2 , Kongnara Papangkorn 1 , Charlotte Butler 1 ,Donald Mix 1 , John Higuchi 1 , Balbir Brar 1 , S Kevin Li 3 , William I.Higuchi 1, 4 . 1 Aciont Inc, Salt Lake City, UT; 2 Ophthalmology, MoranEye Center, University of Utah, Salt Lake City, UT; 3 College ofPharmacy, University of Cincinnati, Cincinnati, OH; 4 College ofPharmacy, University of Utah, Salt Lake City, UT.Purpose: To study the effects of ionic strength and pH on thetransscleral anodal iontophoresis (AI) delivery of Immunoglobulin G(IgG) and to evaluate these effects on the amount and distribution ofIgG delivered into the rabbit eye.Methods: IgG formulations (MW~150 kDa, a surrogate forbevacizumab) of different ionic strengths and pH were investigated invitro (n=3 per group). In vitro AI experiments were conducted withconjunctiva + sclera membranes of New Zealand white (NZW)rabbits in a side-by-side diffusion cell with the IgG formulation in thedonor chamber and PBS in the receiver chamber. Silver and silverchloride electrodes were used as the conductive elements for theiontophoresis experiments. For the in vivo experiments, aniontophoretic applicator (Visulex-I) was applied on the rabbit eyeunder optimized formulation conditions. After 20 minutes of 4 mAAI delivery, the rabbits were sacrificed and the eyes enucleated. Theeyes were dissected into cornea, anterior chamber, vitreous,conjunctiva, sclera and choroid/retina and the tissues were analyzedfor IgG content by ELISA.Results: The in vitro permeability coefficient (P) value determinedfor conjunctiva+sclera membrane increased approximately 2.7 foldwhen decreasing the formulation ionic strength by approximately 13fold of a prior formulation. This corresponds to a 2.7 fold increase intotal amount delivered across the membrane. A decrease in theformulation pH from 6.5 to 2.5 resulted in a 40 fold decrease of the Pvalue. An increase in the total amount of IgG delivered in vivo wasalso observed at the lower ionic strength. The total amount of IgGdelivered in vivo into the eye and into the retina/choroid with ouroptimized low ionic strength formulation was approximately 302.2 ±30.0 μg and 5.2 ± 2.4 μg, respectively. The retina/choroid amount isof the same order of magnitude of levels delivered to theretina/choroid from an IVT injection of bevacizumab in rabbits(literature reported Cmax value of 94 μg/g, which is approximately15 μg).Conclusions: Electroosmosis can be compromised by a low pH butcan be enhanced by utilizing a low formulation ionic strength. An©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 – Physiology/Pharmacologyoptimized Visulex-I applicator and formulation can be expected todeliver to the eye a therapeutically relevant dose of anantibody/macromolecule such as Avastin.Commercial Relationships: Sarah A. Molokhia, Aciont Inc (C);Kongnara Papangkorn, Aciont Inc (E); Charlotte Butler, AciontInc (E); Donald Mix, Aciont, Inc (E); John Higuchi, Aciont Inc.(E); Balbir Brar, Aciont Inc (C), Atheronova Inc (C), ImprimisPharma (C); S Kevin Li, Aciont Inc (C); William I. Higuchi, AciontInc. (F)Support: NEI, Grant no. 2R44EY014772-02A1Program Number: 3205Presentation Time: 11:30 AM - 11:45 AMVersatile time-release technology reducing frequency of invasiveinjections in treating retinal diseasesChi-Chun Lai 1, 3 , Ling Yeung 2, 3 , Lan-Hsin Chuang 2, 3 , Yih-ShiouHwang 1, 3 , Chih-Chiang Tsai 4 , Po-Chun Chang 4 , Luke S.S. Guo 4, 5 ,Yun-Long Tseng 4 , Sheue-Fang Shih 4 , Keelung Hong 4, 5 .1 Ophthalmology, Chang Gung Memorial Hospital - Kaohsiung,Kwei-Shan, Taiwan; 2 Ophthalmology, Chang Gung MemorialHospital, Keelung, Taiwan; 3 Chang Gung University College ofMedicine, Kwei-Shan, Taiwan; 4 Taiwan Liposome Company, Taipei,Taiwan; 5 TLC Biopharmaceutical, Inc., california, CA.Purpose: To design a controlled release system capable of prolongeffective therapeutic agents against chronic eye diseases byintravitreal injection.Methods: In this study we report that a versatile drug deliveryplatform, namely BioSeizer, prolong effective therapeutic agentsagainst chronic ocular diseases. This technology is directly applicableto existing drug products through a modified dehydration-rehydrationvesicles (DRV) method. A small molecule drug dexamethasonesodium phosphate (DSP) or a macromolecular protein drug(bevacizumab) or both was formulated with this BioSeizer. To assessthe sustained release capability of BioSeizer, in vitro and in vivopharmacokinetic studies were done. The DSP orbevacizumab/BioSeizer were injected separately into the vitreous(IVI) of rabbits. The animals were sacrificed at each time point andthe eyes enucleated for the assessment of vitreous concentration ofdrugs. In another experiment, to assess the effectiveness of this slowrelease system, a rabbit model of blood retinal barrier (BRB)breakdown was used to test the prolong effect of IVI lipid based drugwith DSP.Results: The in vivo release of DSP or bevacizumab, can besustained in the vitreous chamber for more than four months by asingle intravitreal injection of a lipid-based formulation, with asufficient rate of drug release to maintain a vitreous concentration ofdrugs equivalent to that of commercial formulation. No toxic effectswere found. In the efficacy study, free DSP did not have a significanteffect on the blockade of BRB breakdown beyond 9 days postadministration.In contrast, the DSP/BioSeizer continued tosignificantly inhibit BRB breakdown 72 days after administration.Conclusions: These findings suggest that lipid-based drug deliverysystem can provide long-term and therapeutic concentration of drugsin vitreous.Commercial Relationships: Chi-Chun Lai, None; Ling Yeung,None; Lan-Hsin Chuang, None; Yih-Shiou Hwang, None; Chih-Chiang Tsai, Taiwan Liposome Company (E); Po-Chun Chang,Taiwan Liposome Company (E); Luke S.S. Guo, None; Yun-LongTseng, None; Sheue-Fang Shih, Taiwan Liposome Company (E);Keelung Hong, Taiwan Liposome Company (E), TLCBiopharmaceuticals, Inc. (E)Support: NSC 99-2314-B-182-020-MY3 (Taiwan)Program Number: 3206Presentation Time: 11:45 AM - 12:00 PMNoninvasive Dexamethasone Sodium Phosphate Ocular DrugDelivery System for the Treatment of Intermediate and PosteriorUveitisKongnara Papangkorn 1, 2 , Donald Mix 1 , Charlotte Butler 1 , JohnHiguchi 1 , Balbir Brar 1 , William I. Higuchi 2, 1 . 1 Aciont Inc, Salt LakeCity, UT; 2 University of Utah, Salt Lake City, UT.Purpose: To assess the efficacy and safety of dexamethasone sodiumphosphate Visulex system (DSP-V) with various dosing regimens intreating intermediate and posterior uveitis in New Zealand white(NZW) rabbit.Methods: Fifteen NZW rabbits were assigned to 5 groups accordingto DSP concentration, dosing, and Visulex application time (seetable). Uveitis was induced in NZW rabbits by subcutaneousinjections of complete Freund’s adjuvant and an IVT injection of H37RA antigen as described by Cheng, et. al. After induction, DSPtreatment was given to the right eye of each rabbit by DSP-V exceptthe control group. Efficacy and safety of DSP-V were evaluated dailyby indirect ophthalmoscopy (IO) and at the end of the study byhistopathological examinations (HE) to evaluate the pathology andinflammatory cell infiltration (ICI) into the eye tissues including bothanterior (conjunctiva, cornea, anterior chamber) and posteriorsections (vitreous, choroid, retina).Results: The control group exhibited significant inflammation in thevitreous, choroid, and retina; and slight or no inflammation in theconjunctiva, cornea, anterior chamber, and iris. All treatments withDSP-V showed effectiveness in the reduction of vitreous opacity(VO) and ICI of the vitreous, choroid, and retina. The VO of thecontrol group indicate that uveitis occurred within 24 h afterinduction and remained throughout the 29 day study. After Day 3,there was a clear differentiation between the control and the 10 and15 min treatment groups. By Day 10, the highest dosing regimen(Group 2) showed complete resolution of the vitreal inflammationand by Day 21, the lowest dosing regimen (Group 5) was alsocompletely clear. With the exception of the 8% single dose treatment,there appears to be a DSP dose-efficacy relationship. As for thesafety of DSP-V, the differences in inflammation scores between thetreatment and control groups would imply DSP-V is a safe modalityfor uveitis treatment. The HE also supports the safety aspect of DSP-V treatment.Conclusions: DSP-V containing 8% to 15% DSP administered for 5to 15 min is well tolerated and highly efficacious in this chronicintermediate and posterior uveitis model. Even a single dose of 8%DSP can suppress intermediate and posterior uveitis for 29 days.©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 – Physiology/PharmacologyConclusions: Administration site, compound properties andformulation impact ocular and systemic pharmacokinetics. Ourresults inform our understanding of R406 administration and thedesign and formulation of ocular therapeutics in general.Commercial Relationships: Kongnara Papangkorn, Aciont Inc(E); Donald Mix, Aciont, Inc (E); Charlotte Butler, Aciont Inc (E);John Higuchi, Aciont Inc. (E); Balbir Brar, Aciont Inc (C),Atheronova Inc (C), Imprimis Pharma (C); William I. Higuchi,Aciont Inc. (F)Support: NEI SBIR Grant R44EY014772Program Number: 3207Presentation Time: 12:00 PM - 12:15 PMOcular Delivery of the Novel Spleen Tyrosine Kinase InhibitorR406 for RetinoblastomaEleanor M. Pritchard 1, 2 , Fangyi Zhu 1 , Lei Yang 1 , Cori Bradley 2 ,Elizabeth Stewart 3, 2 , Jiakun Zhang 2 , Burgess B. Freeman 4 , MichaelDyer 2, 5 , R. Kip Guy 1 . 1 Chemical Biology and Therapeutics, St. JudeChildren's Research Hospital, Memphis, TN; 2 DevelopmentalNeurobiology, St. Jude Children's Research Hospital, Memphis, TN;3 Hematology/Oncology, St. Jude Children's Research Hospital,Memphis, TN; 4 Pharmaceutical Sciences, St. Jude Children'sResearch Hospital, Memphis, TN; 5 Ophthalmology, University ofTennessee Health Sciences Center, Memphis, TN.Purpose: Drug delivery to the eye remains a major challenge due tolimited blood retinal barrier (BRB) penetration and systemic sideeffects.Local administration routes represent a promising potentialsolution, but unfortunately relatively little is known about therelationship between compound properties and in vivo ocularpharmacokinetics. R406 is an inhibitor of a spleen tyrosine kinase(SYK), which was recently identified as a novel therapeutic target forretinoblastoma. Our objective was to investigate the effects offormulation, administration site and physical-chemical compoundproperties on ocular and systemic R406 pharmacokinetics.Methods: Systemic and intraocular pharmacokinetics were evaluatedin mice for various formulations of three forms of R406 with varyingcompound properties (R406 free base, R406 phenylsulfonate salt, andthe phosphate prodrug of R406, R788) after single dosesubconjunctival administration. PK of orally administered R788 wasalso characterized.Results: Tmax occurs at 0.5 hr for all formulations and routes tested,indicating rapid absorption of R406 into the eye. Localsubconjunctival delivery of R788 increases vitreous exposure anddecreases systemic exposure compared with R788 administeredorally. At comparable doses, R788 in cosolvent solution achieves amaximum vitreous concentration 5.7-fold higher than the waterinsoluble R406 free base in emulsion, which suggests aqueoussolubility impacts ocular penetration. In contrast, R788 administeredin suspension exhibits a lower vitreous AUC and higher systemicexposure than the less water soluble R406 salt administered inDMSO, which suggests that at higher doses the more water solubleprodrug diffuses more readily from the injection site into systemiccirculation.Concentration versus time plots of plasma (top) and vitreous (bottom)exposure following systemic (oral, squares) and local(subconjunctival injection, circles) administration of R788Formulation details and vitreous pharmacokinetic parameters forR406 formulations investigated including AUC (area under theconcentration-time curve), Cmax (maximum concentration) and tmax(time Cmax observed)Commercial Relationships: Eleanor M. Pritchard, None; FangyiZhu, None; Lei Yang, None; Cori Bradley, None; ElizabethStewart, None; Jiakun Zhang, None; Burgess B. Freeman, None;Michael Dyer, None; R. Kip Guy, NoneProgram Number: 3208Presentation Time: 12:15 PM - 12:30 PMIntravitreal long-lasting micelle formulation ofhexadecyloxypropyl-cidofovir (HDP-CDV) for cytomegalovirusretinitisFeiyan Ma 1 , Su-Na Lee 1 , Huiyuan Hou 1 , James Beadle 2 , William R.Freeman 1 , Karl Hostetler 2 , Lingyun Cheng 1 . 1 Ophthalmology, ShileyEye Center, UCSD, La Jolla, CA; 2 Department of Medicine, SanDiego VA Healthcare System and UC San Diego, LA JOLLA, CA.Purpose: We have previously shown that micelle formulation ofHDP-CDV had a long vitreous half-life and good ocular safety©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 – Physiology/Pharmacologyprofile. In the current study, we applied a pre-treatment strategy toevaluate the sustained therapeutic effect of hexadecyloxypropylcidofovir(HDP-CDV) against a rabbit HSV retinitis model.Methods: Twenty-seven New Zealand pigmented rabbits (27 eyes)received either an intravitreal equal molar concentrations of HDP-CDV (42μg/50μl), CDV (28 μg/50μl) or 5% dextrose (50μl) ascontrols 5 weeks or 9 weeks before HSV intravitreal inoculation.Intravitreal injection of a 5×10-5 dilution of 108TCID/mL HSV wasperformed to induce HSV retinitis. After the virus injection, therabbit eyes were monitored by indirect ophthalmoscopy and theretinitis was graded as 0.5, 1, 2, 3, and 4 as we described previouslyon day 3, day 6, day 9, and day 14 on which rabbits were sacrificedfor pathological evaluation. The retinitis scores were comparedamong the HDP-CDV, free CDV, and dextrose groups.Results: HSV retinitis in rabbits is a rapidly spreading retinitismodel. It presents on day 4 to 6 after virus inoculation and showsrapid progression between day 6 and day 9 before a complete retinitison day 14. For the 5-week pretreatment study, the retinitis scores onday 6 and day 9 were pooled and treated as a repeated measurementas ordinary scale to compare the retinitis severity among the threegroups. HDP-CDV showed significantly less retinitis than dextrosecontrols (p=0.0047), though no significant difference from CDVgroup (p=0.22). For the 9-week pretreatment study, HDP-CDV groupshowed a significantly less retinitis than CDV group (p=0.0053) anddextrose group (p=0.0097). At week 9, CDV had same severeretinitis as the dextrose injected eyes (p=0.23). On day 14 after thevirus inoculation, there were 2 eyes that developed retinitis out of 7eyes with HDP-CDV, 5 eyes out of 5 eyes with CDV, and 5 eyes outof 5 eyes with dextrose (p=0.0062).Conclusions: CDV can provide 5 weeks antiviral effect afterintravitreal injection. However, HDP-CDV can provide at least 9weeks antiviral effect following a single intravitreal injection of 42µg.Commercial Relationships: Feiyan Ma, None; Su-Na Lee, None;Huiyuan Hou, None; James Beadle, Chimerix Pharmaceuticals (I);William R. Freeman, OD-OS, Inc. (C); Karl Hostetler, None;Lingyun Cheng, Spinnaker Biosciences (C)Program Number: 3209Presentation Time: 12:30 PM - 12:45 PMCharacterization of the In Vitro Release Kinetics of Bevacizumabfrom a Biocompatible Reverse Thermal GelBritta M. Rauck 1 , Carlos A. Medina-Mendez 2 , Thomas R. Friberg 2 ,Yadong Wang 1 . 1 Bioengineering, University of Pittsburgh, Pittsburgh,PA; 2 Ophthalmology, University of Pittsburgh Medical Center,Pittsburgh, PA.Purpose: A biodegradable, biocompatible reverse thermal geldemonstrates excellent promise as an intraocular drug deliverysystem as it is minimally invasive and can sustain the release of drugssubstantially. In order to maximize the release profile, we loadedseveral doses of bevacizumab (Avastin) using multiple gelconcentrations and studied the release behavior in vitro. Moreover,we confirmed the ability of the release system to maintain thebioactivity of the released drug.Methods: We synthesized poly(ethylene glycol)-poly(serinolhexamethylene urethane) (ESHU), an amphiphilic block copolymerthat, in solution, undergoes a sol-gel transition upon heating fromroom temperature to body temperature. We performed in vitro releasestudies by injecting 10, 15 or 20% (w/v) ESHU containing 0.625,1.25, 2.5 or 5 mg of bevacizumab into a 1% solution of hyaluronicacid at 37°C. At 1, 3, and 7 days and weekly thereafter, samples wereobtained from the supernatant, and an enzyme-linked immunosorbentassay (ELISA) was used to determine the concentration ofbevacizumab released. We evaluated the bioactivity of the releaseddrug by quantifying the inhibition of tube formation using humanumbilical vein endothelial cells (HUVECs) cultured on Matrigel.Results: The release profile of bevacizumab from ESHU is bothdose- and concentration-dependent. In all experimental groups ESHUis capable of sustaining drug release in a near-linear fashion forapproximately 12 weeks. The burst release from the gel at day 1 isless than 10% of the total drug loaded. Low gel concentration and/orhigh drug loading results in a larger burst and ultimately a more rapidrelease profile. The bevacizumab that is released from the gel iscapable of inhibiting HUVEC tube formation when samples aremixed with culture media, suggesting that the released bevacizumabis bioactive, and that ESHU does not interfere with its mechanism ofaction.Conclusions: By altering drug dose and gel concentration parametersthe release profile of ESHU can be controlled. Compared to a clinicalsetting, in which bevacizumab must be injected monthly due to itsshort half-life, ESHU is capable of sustaining release overapproximately 3 months in vitro, suggesting that it may ultimately beused to reduce injection frequency, improve patient compliance andenhance therapeutic efficacy.Commercial Relationships: Britta M. Rauck, None; Carlos A.Medina-Mendez, None; Thomas R. Friberg, 13/581,518 (P);Yadong Wang, None339 AMD: New Drugs, Delivery Systems, and Mechanisms ofActionTuesday, May 07, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 3272-3299/A0123-A0150Organizing Section: Physiology/PharmacologyProgram Number: 3272 Poster Board Number: A0123Presentation Time: 11:00 AM - 12:45 PMRationale for Treating Wet AMD in Human Using an Oral PillConsisting of a VEGFR/PDGFR Inhibitor X-82Chris Liang 1 , David M. Brown 2 , Nauman A. Chaudhry 3 , Michael J.Elman 4 , Jeffrey S. Heier 5 . 1 Xcovery, LLC, West Palm Beach, FL;2 Retina Consultants of Houston, Houston, TX; 3 New England RetinaAssociates, New London, CT; 4 Elman Retina Group, Baltimore, MD;5 Ophthalmic Consultants of Boston, Boston, MA.Purpose: To analyze the pre-clinical and clinical safety and biologicactivity of X-82, a VEGFR/PDGFR inhibitor.Methods: X-82 was evaluated in vitro and via oral administration ina rat CNV model, focusing on safety and biologic activity. The safetyand pharmacokinetic properties of X-82 in cancer patients wereanalyzed to define the doses that might be safe and effective in AMDpatients.Results: VEGFR and PDGFR inhibition occurred with 80% inhibition of CNV. Oraladministration of X-82 in cancer patients was well tolerated with noGrade 2 or higher toxicities at exposures of 5191 ng.hr/ml and Cmaxof 942 nM. At 50mg once a day, the mean AUC, Cmax and Ctroughlevels were 1282 ng.hr/ml, 268 nM and 41 nM, respectively.Conclusions: Oral administration of X-82 at 50mg once a dayachieved exposures well above the concentration required to inhibitnew blood vessel formation; this level was < ¼ of the well toleratedexposure noted in cancer patients, suggesting that the dose should bewell tolerated and could be efficacious in treating wet AMD patients.A pilot clinical study of X-82 in patients with wet AMD has started.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. 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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Chris Liang, Xcovery, LLC (E); DavidM. Brown, Regeneron Pharmaceuticals, Inc. (F), RegeneronPharmaceuticals, Inc. (C), Regeneron Pharmaceuticals, Inc. (R),Bayer HealthCare (F), Bayer HealthCare (C), Bayer HealthCare (R),Genentech (C), Roche (C), Alimera (C), Alcon (C), Novartis (C),Thrombogenics (C), Genentech (F), Roche (F), Thrombogenics (F),GSK (F), Alimera (F), Alcon (F), Allergan (F), Eli Lilly (F);Nauman A. Chaudhry, Genentech (F), Regeneron Pharmaceuticals(F), Xcovery Vision (F), Lpath (F), DRCR network (F); Michael J.Elman, Genentech (C), Ohr Pharmaceuticals (I), Novartis (F),DRCRnet (F); Jeffrey S. Heier, Acucela (C), Aerpio (C), Alimera(F), Allergan (C), Bayer (C), Forsight Labs (C), Fovea (F),Genentech (C), Genzyme (C), Genentech (F), Genzyme (F),Thrombogenics (C), Sequenom (C), Notal Vision (F), Novartis (F),Ophthotech (F), Ophthotech (C), Oraya (C), Paloma (F), Regeneron(F), Regeneron (C)Clinical Trial: NCT01674569Program Number: 3273 Poster Board Number: A0124Presentation Time: 11:00 AM - 12:45 PMA Novel Endogenous Glycan Therapy For Retinal Diseases:Safety, In vitro Stability, Ocular Pharmacokinetics and BiodistributionShankar Swaminathan 1 , Huiling Li 1 , Mallika Palamoor 1 , DorababuMadhura 2 , Bernd Meibohm 2 , Monica M. Jablonski 1 .1 Ophthalmology, Hamilton Eye Inst, Univ TN HSC, Memphis, TN;2 Pharmaceutical Sciences, The University of Tennessee HealthScience Center, Memphis, TN.Purpose: Asialo tri-antennary oligosaccharide (NA3 glycan) is anovel endogenous compound which supports the proper folding ofouter segment membranes; promotes normal ultrastructure andmaintains protein expression patterns of photoreceptors and Müllercells in the absence of RPE support. Hence it is a potential newtherapeutic for atrophic AMD. Our purpose was to evaluate thesafety, in vitro stability, ocular pharmacokinetics and biodistributionof NA3 in the eye.Methods: NA3 was injected into the vitreous of NZW rabbits at twoconcentrations viz. 1 nM (MEC) and 100 nM (100XMEC) at threetime points (1d-14d). Safety was evaluated using routine clinical andlaboratory tests. NA3 was tagged with tritium. Ocularpharmacokinetics and biodistribution were estimated atpredetermined time points (2h-14d). Rabbits were sacrificed andvarious parts of the eye, multiple peripheral organs and plasma wereisolated. NA3 was quantified using scintillation counting.Pharmacokinetics was estimated by non-compartmental modeling(WinNonlin 6.2). A 2-aminobenzamide labeling method andsubsequent HILIC chromatographic separation were used to assessplasma and vitreous stability at simulated biological conditions.Results: NA3 was well tolerated by the eye. The concentration ofNA3 in various eye tissues was in the following order:vitreous>retina>sclera/choroid>aqueous humor>cornea>lens. AUC∞was maximum in the vitreous thereby providing a positiveconcentration gradient for the drug to reach the retina. Half-lives incritical eye tissues were between 40-60 h. NA3 concentrations werenegligible in peripheral organs. Radioactivity from tritiated NA3 wasexcreted via urine and feces. By using an optimized HILIC methodwith fluorescence detection, NA3 was found to be stable at 37°C inexcised vitreous over a minimum of 6 days, while it degraded withina few hours in plasma thereby providing a breakthrough targetedtherapy for atrophic AMD and other retinal diseases.Conclusions: NA3 glycan is safe for intraocular applications,degrades rapidly in plasma with minimal organ distribution, whilebeing stable in vitreous. Ocular pharmacokinetics confirms themovement of NA3 from vitreous to retina, the site of action. Withnovel formulation strategies, the residence time of NA3 would beincreased for therapeutic applications in chronic retinal diseases.Commercial Relationships: Shankar Swaminathan, None;Huiling Li, None; Mallika Palamoor, None; Dorababu Madhura,None; Bernd Meibohm, None; Monica M. Jablonski, 8,092,825 (P)Support: Research to Prevent Blindness, University of TennesseeResearch Foundation, Knights Templar Eye Foundation, Fight forSight, March of Dimes, International Retinal Research FoundationProgram Number: 3274 Poster Board Number: A0125Presentation Time: 11:00 AM - 12:45 PMA Single 700 μg Dexamethasone Intravitreal Implant (Ozurdex)Effectively Treats Complex Post-Operative Cystoid MacularEdemaDaniel F. Kiernan 1, 2 , Glenn Stoller 1 , Ken B. Carnevale 1 , Nina C.Mondoc 1 , Eric Donnenfeld 1 . 1 Ophthalmic Consultants of Long Island,Rockville Centre, NY; 2 Ophthalmology, Winthrop Hospital MedicalCenter, Mineola, NY.Purpose: Severe post-operative cystoid macular edema (CME)associated with posterior uveitis may occur after anterior (Irvine-Gasssyndrome) or posterior segment intraocular surgery and may result invisual loss. There is no consensus on the efficacy of various topical,oral, peribulbar, retrobulbar or intravitreal therapeutic optionscompared to natural history, but patients with this condition are oftenreferred to specialists for treatment consultation.Methods: Consecutive, retrospective review of patients diagnosedand treated with this condition in a multi-specialty practice. Allpatients had optical coherence tomography (OCT) and fluoresceinangiography (FA) at baseline, one week, one month and on mostrecent follow-up.Results: Six pseudophakic patients with acute, post-operative (≤8weeks post-surgery, 4 after anterior segment procedures and 2 afterposterior segment procedures with vitrectomy) CME associated withposterior uveitis and decreased vision (average logMAR VA= .996,range 20/60 - 20/400) and edema on OCT (avg central subfovealthickness [CST] = 640 μm, range 447 to >1000 μm) were treated withone 700 μg intravitreal dose of Ozurdex (Allergan Inc., Irvine CA).Within 1 week VA improved to an average of logMAR 0.619(~20/80) and avg CST decreased to 389 μm. At 1 month avglogMAR VA= 0.309, range 20/25 - 20/50 (p= 0.017 compared tobaseline), and avg CST = 291 μm, range 233-352 μm (p=0.016compared with baseline). At the most recent visit (average totalfollow-up of 18.4 weeks, range 12-21 weeks), logMAR VA and CSTwere not statistically different than at the 1-month interval. Nopatients developed serious adverse events, including vitreoushemorrhage, ocular hypertension or endophthalmitis, or developedrecurrent edema. FA demonstrated a progressive diminution of lateleakage at all post-treatment time points compared to baseline.Conclusions: A single intravitreal injection of Ozurdex may rapidlyand permanently treat severe post-operative CME associated withposterior uveitis in in both vitrectomized and non-vitrectomized eyes.This therapy may result in faster and more definitive visualrehabilitation and macular edema resolution compared to observationor other treatment options.©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 – Physiology/PharmacologyExample of a patient within this series who had maximal visualrecovery with macular edema and angiography leakage resolutionafter a single Ozurdex injection on Day 0.Commercial Relationships: Daniel F. Kiernan, Allergan (C),Allergan (R); Glenn Stoller, Lpath (C), Regeneron (F), Lpath (P),SKS (I); Ken B. Carnevale, None; Nina C. Mondoc, None; EricDonnenfeld, Allergan (C), Alcon (C), Abbott (C), Bausch & Lomb(C), Glaukos (C), Aquysis (C), Katena (C), Sarcode (C), Wavetec (C)Program Number: 3275 Poster Board Number: A0126Presentation Time: 11:00 AM - 12:45 PMRitonavir Inhibits HIF-1α Mediated VEGF Expression in RetinalPigment Epithelial CellsRamya Krishna Vadlapatla, Aswani Dutt Vadlapudi, Dhananjay Pal,Mridul Mukherji, Ashim K. Mitra. Department of PharmaceuticalSciences, University of Missouri Kansas City, Kansas City, MO.Purpose: Retinal hypoxia (oxygen deficiency) leading toangiogenesis is a major signaling mechanism underlying a number ofsight threatening diseases. One of the most important target genes ofhypoxia inducible factor (HIF) includes vascular endothelial growthfactor (VEGF). Intravitreal injections of VEGF antagonists resultedin serious complications and systemic side effects. Inhibiting thesignaling mechanism underlying neovascularization (HIF-pathway)with already approved therapeutic molecule may have promisinganti-angiogenic role with fewer side-effects. Hence, the primaryobjective of this study is to examine the expression of HIF-1α andVEGF in human retinal pigment epithelial (ARPE-19 and D407) cellstreated with ritonavir under hypoxic and normoxic conditions.Methods: ARPE-19 and D407 cells were cultured in normoxic andhypoxic conditions (3, 6 and 12 hours), alone or in the presence ofritonavir (5, 10 and 20μM). Quantitative real time polymerase chainreaction (qPCR), immunoblot analysis and sandwich ELISA wereperformed to check the RNA and protein expression levels of HIF-1αand VEGF. Further, in vitro anti-angiogenic activity of ritonavir wasalso assessed by measuring proliferation of choroid-retinalendothelial (RF/6A) cells.Results: Hypoxic exposure to 12 hours demonstrated elevated RNAexpression levels of HIF-1α and VEGF in both ARPE-19 and D407cells. Hence, this time point was chosen for subsequent studies.Presence of ritonavir in culture medium strongly inhibited theexpression of HIF-1α and VEGF in a concentration-dependentmanner in hypoxic conditions. Immunoblot analysis demonstratedthat ritonavir inhibited the protein expression of HIF-1α. Further,hypoxic exposure increased VEGF secretion while presence ofritonavir reduced this secretion, as demonstrated by an ELISA.Finally, ritonavir significantly inhibited the proliferation of RF/6Acells, demonstrating potential anti-angiogenic activity.Conclusions: This study demonstrates for the first time that ritonavircan inhibit the expression of HIF-1α and VEGF in ARPE-19 andD407 cells. This inhibition of hypoxia pathway and downstreamgenes may form the platform for indication of ritonavir in treatmentof various sight threatening diseases. The process of traditional drugdevelopment could be fastened since ritonavir is clinically approvedfor human use.Commercial Relationships: Ramya Krishna Vadlapatla, None;Aswani Dutt Vadlapudi, None; Dhananjay Pal, None; MridulMukherji, None; Ashim K. Mitra, NoneSupport: NIH Grants R01 EY09171-16 and R01 EY010659-14Program Number: 3276 Poster Board Number: A0127Presentation Time: 11:00 AM - 12:45 PMProtection of RPE cells by sulindac against oxidative damage isthrough ischemic preconditioning (IPC) and involves activationof the peroxisome proliferator activated receptor alpha (PPAR α)Arunodoy Sur 1 , Diane Baronas-Lowell 2 , Manas R. Biswal 1 , HowardM. Prentice 3 , Herbert Weissbach 2 , Janet C. Blanks 1 . 1 ComplexSystems, Florida Atlantic University, Boca Raton, FL; 2 CMBB,Florida Atlantic University, Jupiter, FL; 3 College of Medicine,Florida Atlantic University, Boca Raton, FL.Purpose: Age related macular degeneration (AMD) is the leadingcause of blindness among the elderly population and oxidative stresshas been implicated as a major cause of this ocular disorder.Previously we had reported that sulindac protects cultured RPE cells(ARPE19) against reactive oxygen species (ROS) induced stress. Thepurpose of this current study is to understand the protectivemechanism involved in protection by sulindac of RPE from oxidativestress induced by either tert-butyl hydrogen peroxide (TBHP) orultraviolet light (UVB). Protection of RPE cells against ROS wasalso observed with fenofibrate, a known agonist of PPARα. Theresults support a mechanism in which the protection of RPE cellsagainst oxidative stress is through a preconditioning mechanisminvolving activation of PPAR α.Methods: RPE cells were incubated with sulindac or fenofibrate for24 hours, following which the cells were either exposed to a range ofTBHP concentrations or 1200mj UVB light. Cell viability wasdetermined by the MTT assay and upregulation of preconditioningmarkers was detected using western blotting. In a light damagemouse model, sulindac was administered by intravitreal injection andevaluation of the retinal tissues for photoreceptor damage wasperformed.Results: The results indicate that sulindac confers >30 % protectionof cultured ARPE19 cells against oxidative damage. The sulindacprotection in vitro could be replaced by fenofibrate and was inhibitedby an antagonist of PPARα, indicating a role of PPARα in thesulindac effect. Other results indicated that the protective effect ofsulindac was through an IPC mechanism. In a preliminary in vivostudy, sulindac protected photoreceptors against photooxidativedamage in a light damage model in mice.Conclusions: Our findings indicate that preconditioning pathwaysand activation of PPARα play key roles in sulindac’s protectivemechanism. A more extensive in vivo study is planned to establishtherapeutic efficacy and other modes of administration. Implementingpharmacological preconditioning to deter oxidative stress outlinedhere represents a possible clinical approach to treat AMD and otheroxidative stress induced disorders.Commercial Relationships: Arunodoy Sur, None; Diane Baronas-Lowell, None; Manas R. Biswal, None; Howard M. Prentice,None; Herbert Weissbach, CHS Pharma (C), Florida AtlanticUniversity (P), CHS Pharma (S); Janet C. Blanks, NoneSupport: FAU Research Priority Grant (awarded to JB and HP)Program Number: 3277 Poster Board Number: A0128©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 – Physiology/PharmacologyPresentation Time: 11:00 AM - 12:45 PMNAT2 STUDY: OMEGA-3 LEVELS IN RED BLOOD CELLSMEMBRANES CORRELATES THE PREVENTIVE EFFECTEric H. Souied, Cecile Delcourt, Giuseppe Querques, BenedicteMerle, Theodore Smith, Pascale Benlian. Retina Creteil, UniversityParis Est Creteil, Creteil, France.Purpose: The NAT2 (Nutritional AMD treatment-2) study was arandomized, placebo-controlled, double-blind, parallel, comparativestudy. Our purpose was to evaluate the efficacy of docosahexaenoicacid (DHA) enriched oral supplementation in preventing exudativeage-related macular degeneration (AMD). Here we correlated the redblood cells membrane (RBCM) levels in omega-3 and the occurrenceof choroidal neovascularisation (CNV)Methods: 263 patients with early lesions of age-related maculopathyand visual acuity better than 0.4 LogMAR units in the study eye andneovascular AMD in the fellow-eye. Patients were randomlyassigned to receive either 840 mg/day DHA and 270 mg/dayeicosapentaenoic acid (EPA) from fish oil capsules or the placebo(olive oil capsules) for 3 years.Results: Time to occurrence and incidence of CNV in the study eyewere not significantly different between the DHA (19.5 ± 10.9months, 28.4%, respectively) and placebo groups (18.7 ± 10.6months, 25.6%, respectively). EPA+DHA level significantlyincreased in RBCM in the DHA group (+70%; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyand 233 number of junctions respectively. The average number ofjunctions calculated for the wells treated with medium (negativecontrol) was only 11.Conclusions: Fabrani-PEG20-Fabrani inhibited angiogenesis in vitrobetter than bevacizumab and ranibizumab. This work has thepotential to optimise new protein-based therapeutics, particularlyantibodies.Figure 1. A; SDS-PAGE gels of the disulfide bridging PEGylation ofthe Fab; Novex Bis-Tris 4-12% gel stained with colloidal blue forprotein (lanes M-2) and silver staining (lane 3) to detect impurity. M:Standard protein markers. Lane 1: purified Fab. Lanes 2: 3: purifiedFabrani-PEG20-Fabrani. B; The dissociation rate profiles forFabbeva, Fabbeva-PEG20-Fabbeva and bevacizumab using CM3chip with 53 RU VEGF at 25 0C.Commercial Relationships: Hanieh Khalili, None; SteveBrocchini, None; Peng T. Khaw, University College Moorfields (P)Support: NIHR Moorfields Biomedical Research CentreResults: Both OCT and CFM revealed the CNV size weresignificantly reduced, and FA grading showed significantly lessleakage in the ω-3-fed-mice compared with the ω-6-fed mice. Theconcentrations of the principal ω-3 LCPUFAs (EPA and DHA) weresignificantly increased in serum of ω-3-fed mice. The serum levels ofAA-derived eicosanoids were significantly reduced, whereas those ofEPA-derived- and DHA-derived eicosanoids were significantlyincreased in ω-3-fed mice. The mRNA expression and abundance ofprotein of ICAM-1 and E-selectin in the retina or choroid after CNVinduction were significantly reduced in ω-3-fed mice. The rollingvelocity of leucocytes on the combination of P-selectin and ICAM-1coated chamber was significantly greater in ω-3-fed mice. Theabundance of VEGF protein in both the retina and choroid wassignificantly reduced in ω-3-fed mice.Conclusions: We demonstrate that dietary supplementation of ω-3LCPUFAs mediate choroidal neovessel regression. A clearunderstanding of the mechanisms of these molecules in AMD couldbring a major shift in our approach to disease treatment. Currentlythere is an urgent need for safe nutritional or pharmacologicalinterventions for the treatment and ideally the prevention of AMD.Commercial Relationships: Ryoji Yanai, None; Lama Mulki,None; Kimio Takeuchi, None; John H. Sweigard, None; JunSuzuki, None; Philipp Y. Gaissert, None; Demetrios Vavvas,MEEI (P), Kala pharmaceuticals (C), Roche (C), Genentech (C);Wolf-Hagen Schunck, None; Joan W. Miller, Massachusetts Eyeand Ear Infirmary (P), Novartis (I), Alcon (C), KalVistaPharmaceuticals (C); Kip M. Connor, NoneSupport: the Massachusetts Lions Eye Research Fund, Inc., NationalInstitutes of Health (NIH) grants R01EY022084-01/S1 (KMC), andP30EY014104 (Core support at the Massachusetts Eye and EarInfirmary), and Research to Prevent Blindness Unrestricted Grant andan award from the Japan Eye Bank Association (RY).Program Number: 3280 Poster Board Number: A0131Presentation Time: 11:00 AM - 12:45 PMOmega-3 long chain polyunsaturated fatty acids promotechoroidal neovessel regressionRyoji Yanai 1 , Lama Mulki 1 , Kimio Takeuchi 1 , John H. Sweigard 1 , JunSuzuki 1 , Philipp Y. Gaissert 1 , Demetrios Vavvas 1 , Wolf-HagenSchunck 2 , Joan W. Miller 1 , Kip M. Connor 1 . 1 Ophthalmology,Massachusetts Eye & Ear Infirmary Harvard Medical School, Boston,MA; 2 Max Delbrück Center for Molecular Medicine, Berlin,Germany.Purpose: Age-related macular degeneration (AMD) is the primarycause of blindness in elderly individuals of industrialized countries.Prospective clinical studies have indicated that dietary intake ofomega (ω)-3 long-chain polyunsaturated fatty acids (LCPUFAs) mayhave a protective effect against AMD. This study characterizes theeffects of dietary intake of ω-3 and ω-6 LCPUFAs in a mouse modelof AMD.Methods: The present studies adhered to ARVO’s Statement for theUse of Animals in Ophthalmic and Vision Research. C57BL/6 micewere fed a diet enriched with either ω-3 or ω-6 LCPUFAs for 2weeks prior to choroidal neovascularization (CNV) induction byphotocoagulation using a 532-nm laser. CNV development wasevaluated by Fluorescein Angiography (FA), Optical CoherenceTomography (OCT), and choroidal flatmount (CFM). The primaryeicosanoids and their downstream metabolites were analyzed by LCMS/MS lipidomics. Expression of ICAM-1, E-selectin, and VEGF inthe retina, choroid, or laser-captured CNV were evaluated by RTPCRand immunoblot analysis. Macrophage invasion was evaluated inCx3cr1GFP/+ mice. Leukocyte rolling velocity was analyzed usingthe autoperfused microflow chamber assay.Program Number: 3281 Poster Board Number: A0132Presentation Time: 11:00 AM - 12:45 PMPreprogrammed Hematopoietic Stem Cells as a SystemicTherapy for Dry AMDMaria B. Grant 1 , Xiaoping Qi 2 , Yuanqing Yan 1 , Lynn C. Shaw 1 ,Alfred S. Lewin 3 , Michael E. Boulton 2 . 1 Pharmacology andTherapeutics, University of Florida, Gainesville, FL; 2 Anatomy andCell Biology, University of Florida, Gainesville, FL; 3 MolecularGenetics and Microbiology, University of Florida, Gainesville, FL.Purpose: Dry AMD represents 85% of AMD and is associated withretinal pigment epithelial (RPE) dysfunction and cell death for whichthere is no treatment. Previously we showed that systemic delivery ofgenetically reprogrammed hematopoietic stem cells (HSC) with theRPE differentiation marker, RPE 65, enhances repair of the RPElayer and results in recovery of visual function (VF) in the sodiumiodate model of acute RPE cell loss (Sengupta et al 2009). In thisstudy, we tested the efficacy of programming HSC in the superoxidedismutase 2 knockdown (SOD2 KD) model for dry AMD whichgenerates a defect predominantly in the RPE.Methods: SOD2 KD was achieved by subretinal injection of AAVcontaining an SOD2 ribozyme under the direction of an RPE specificpromoter (VMD2). Systemic administration of HSC infected withlentivirus expressing human RPE 65 (LV hRPE65) or infected withlentivirus expressing LacZ (LV LacZ) was undertaken at 1, 3 and 6months post induction of the AMD phenotype with SOD2 KD. ERGsand optomotor response tests were used to assess VF. Donor RPE65-HSC integration was assessed by confocal immunofluorescencemicroscopy. Histology and immunohistochemistry (IH) were used toidentify retinal histology and RPE repair.Results: LV gave a 75.5±5.5% (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyHSC and RPE65 expression was confirmed by IH. Systemicadministration of LV hRPE65 programmed mouse HSCs maintainedVF, retained retinal thickness and prevented degeneration of theretina of SOD2 KD mice. This preservation was not observed by nullor LV LacZ programmed HSC. At 1, 3 and 6 months following HSCinjections, photopic and scotopic ERGs were significantly higheronly in SOD2 KD mice administered LV hRPE65-mHSC but notmice receiving LV LacZ-mHSC, untreated HSCs or untreated SOD2KD mice. Using optomotor responses to further assess VF, at 1month post SOD KD, mice injected with LV RPE65-mHSC retainedVF to a level similar to WT mice, VF declined slightly(

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyJin-San Yoo, None; Sang Hoon Lee, None; Sang Jin Kim, DaewooPharm. (F), Pharmabcine (F)Program Number: 3284 Poster Board Number: A0135Presentation Time: 11:00 AM - 12:45 PMFunctional characterization of recombinant anti-VEGF variantsin vitroTobias Wimmer, Birgit Lorenz, Knut Stieger. Department ofOphthalmology, Justus-Liebig University Giessen, Giessen,Germany.Purpose: Most retinal neovascular disorders are caused by an upregulationof the vascular endothelial growth factor (VEGF).Especially disorders like age-related macular degeneration (AMD),diabetic retinopathy (DR) and retinal vein occlusion (RVO) aretreated with repeated injections of anti-VEGF molecules likeBevacizumab (Avastin®), Ranibizumab (Lucentis®), or Aflibercept(Eyelea®). Repeated injections of anti-VEGF molecules can beconnected to severe side effects like endophthalmitis and canrepresent a financial burden to the patients. The aim of this project isto develop an alternative strategy i.e. controlled expression of anti-VEGF molecules by the retina itself.Methods: The DNA sequences of the light and the heavy chain of theRanibizumab F(ab)-fragment including secretory leader sequenceswere cloned into pIREShrGFP1a separated by an internal ribosomalentry site (IRES) (Ra01). This construct was PCR mutated togenerate a Ranibizumab variant (Ra02), which is expressed as justone molecule containing a 6x glycine linker. HEK293 weretransfected with both constructs, presence in the culture mediumverified by western blot analysis and the concentrations measuredwith a custom made Ranibizumab/Lucentis® ELISA. VEGF bindingaffinities were determined in a cell free binding assay and thebiological activity evaluated in a HUVEC (human umbilicalendothelial cell) migration assay.Results: Ra01 and Ra02 were detected in cell culture medium. Theconcentrations were 374,1 ng/ml and 403,4 ng/ml for Ra01 and Ra02,respectively. VEGF binding affinity was significantly lower for Ra01and Ra02 compared to injectable Lucentis® for each testedconcentration. The inhibition of VEGF induced endothelial cellmigration by Ra01 and Ra02 was comparable with Lucentis® overall tested concentrations, but the maximum inhibitory effect of Ra01and Ra02 was reached at lower doses compared to Lucentis®.Conclusions: Ra01 and Ra02 can be produced in eukaryotic cells invitro and display comparable biological activities as commerciallyavailable Lucentis®. In vivo studies in human VEGF overexpressingmice are ongoing. These results are the basis for a gene-based therapyof human neovascular disorders.Commercial Relationships: Tobias Wimmer, None; BirgitLorenz, Optos (F); Knut Stieger, NoneSupport: DFG Grant Sti 597/2-1Program Number: 3285 Poster Board Number: A0136Presentation Time: 11:00 AM - 12:45 PMLong-term efficacy of ciliary muscle gene transfer of three sFlt-1variants in a rat model of laser-induced choroidalneovascularizationMohamed El Sanharawi 1, 2 , Elodie Touchard 3 , Romain Benard 3 ,Pascal Bigey 4 , Virginie Escriou 4 , Chadi Mehanna 1, 2 , Marie-ChritineNaud 1 , Jean-Claude P. Jeanny 1 , Marianne Berdugo Polak 1 , FrancineF. Behar-Cohen 1, 2 . 1 INSERM UMRS 872, Paris, France; 2 UniversitéPierre et Marie-Curie, Paris, France; 3 Eyevensys, Paris, France;4 INSERM U640, Paris, France.Purpose: Inhibition of vascular endothelial growth factor (VEGF)has become the standard of care for patients presenting wet agerelatedmacular degeneration. However, monthly intravitrealinjections are required for optimal efficacy. We have previouslyshown that electroporation enabled ciliary muscle gene transferresults in sustained protein secretion into the vitreous for up to 9months.Methods: Here we evaluated the long-term efficacy of ciliary musclegene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in arat model of laser-induced choroidal neovascularization (CNV).Fluorescein angiography (FA) was performed to evaluate vascularleakage. CNV growth was evaluated using retinal pigment epithelium(RPE)/choroid flatmount and infracyanine angiography. Intra-ocularVEGF levels were measured using ELISA. The mRNA expression ofVEGF was determined using RT-PCR in the RPE/choroidcomplexes.Results: All three sFlt-1 variants significantly diminished vascularleakage and neovascularization as measured by FA and flatmountchoroid at 3 weeks. FA and infracyanine angiography demonstratedthat inhibition of CNV was maintained for up to 6 months after genetransfer of the two shortest sFlt-1 variants. Throughout, clinicalefficacy was correlated with sustained VEGF neutralization in theocular media. Interestingly, treatment with sFlt-1 induced a 50%down-regulation of VEGF mRNA levels in the retinal pigmentepithelium and the choroid.Conclusions: We demonstrate for the first time that non-viral genetransfer can achieve a long-term reduction of VEGF levels andefficacy in the treatment of choroidal neovascularization.Commercial Relationships: Mohamed El Sanharawi, None;Elodie Touchard, None; Romain Benard, None; Pascal Bigey,INSERM (P), Université Paris Descartes (P); Virginie Escriou,None; Chadi Mehanna, Sisène (E); Marie-Chritine Naud, None;Jean-Claude P. Jeanny, None; Marianne Berdugo Polak, None;Francine F. Behar-Cohen, Inserm/Univesrité ParisDescartes (P)Program Number: 3286 Poster Board Number: A0137Presentation Time: 11:00 AM - 12:45 PMImmune-Like Complexes of Bevacizumab Bind to RetinalPigment Epithelial Cells and Endothelial Cells in vitroYang Liu 1 , Terra B. Potocky 2 , Jingtai Cao 1 , Joel Martin 2 , NicholasPapadopoulos 2 , Stanley J. Wiegand 1 . 1 Ophthalmology, RegeneronPharmaceuticals Inc, Tarrytown, NY; 2 Molecular Biology, Bioassay,& Protein Development, Regeneron Pharmaceuticals Inc, Tarrytown,NY.Purpose: The VEGF inhibitors ranibizumab (RAN), aflibercept(AFL), and bevacizumab (BEV) bind VEGF with different bindingstoichiometries. AFL binds VEGF exclusively as a 1:1 complex(MW: 155 kDa). In contrast, BEV:VEGF complexes areheterogeneous in size (~400kDa to >2,000 kDa) (IOVS 2012, E-Abstract 440), while one or two RAN molecules may bind to a singleVEGF dimer, forming 1:1 or 2:1 complexes. We compared thebinding patterns of BEV, RAN, and AFL to ARPE-19 cells andHUVEC, and investigated the mechanisms underlying differentialbinding of drug:VEGF complexes to cells.Methods: ARPE-19 cells were treated with equimolar concentrationof BEV, RAN, and AFL. Cell surface bound VEGF blockers weredetected by immunofluorescence staining. Cells and culture mediawere collected for TaqMan and ELISA of VEGF165 RNA andprotein levels. For acute binding assay, ARPE-19 cells wereincubated with serial dilutions of VEGF blocker alone, VEGFblocker +VEGF121 or VEGF165 followed by immunostaining of cellsurface bound VEGF blockers. ARPE-19 cells were incubated withserial dilutions of soluble neuropilin-1 (NRP-1) or heparin withVEGF165 and BEV. Cells were then processed as above for signalanalysis. Binding studies were conducted on HUVEC using the same©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. 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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyprotocols.Results: BEV showed a time-dependent increase in binding toARPE-19 cells coincident with the up-regulation of VEGF165 incultures. In contrast, AFL showed no specific binding, while RANexhibited very light binding to RPE cells. Acute binding of BEV toARPE-19 cells was noted in the presence of exogenous VEGF165,but not VEGF121. At the same concentrations, no staining wasobserved with AFL or RAN + VEGF165 or VEGF121. Similarpatterns of cell surface binding of BEV were noted in culturedHUVEC. Exogenous heparin blocked the binding of BEV:VEGF165complexes to ARPE-19 cells, whereas, NRP-1 blocked the binding ofBEV:VEGF165 complexes to HUVEC.Conclusions: These results show that BEV can bind to culturedARPE-19 cells and HUVEC. Acute binding of BEV:VEGF165complexes occurs at molar ratios previously shown to favor theformation of high molecular weight immune like complexes ofBEV:VEGF. Neither AFL nor RAN bind appreciably to RPE orendothelial cells under identical conditions. Binding ofBEV:VEGF165 complexes to RPE or HUVEC appears to bemediated via interactions of bound VEGF165 with lower affinitybinding partners, heparin or NRP-1.Commercial Relationships: Yang Liu, Regeneron Pharmaceuticals,INC (E); Terra B. Potocky, Regeneron Pharmaceuticals (E); JingtaiCao, Regeneron Pharmaceuticals, Inc. (E); Joel Martin, RegeneronPharmaceuticals (E); Nicholas Papadopoulos, RegeneronPharmaceuticals (E); Stanley J. Wiegand, RegeneronPharmaceuticals, Inc (E)Program Number: 3287 Poster Board Number: A0138Presentation Time: 11:00 AM - 12:45 PMA Mechanism-Based Binding Model for the Pharmacokineticsand Pharmacodynamics (PK-PD) of RN6G (PF-04382923), aHumanized Monoclonal Antibody against Amyloid β Peptides, inSubjects with Dry, Age-Related Macular Degeneration IncludingGeographic AtrophyKai H. Liao 1 , Pamela D. Garzone 2 , Sangeetha S. Bollini 2 , Philip M.Fanning 2 , Gilbert Wong 2 , Xu Meng 1 . 1 Pfizer Inc, San Diego, CA;2 Pfizer Inc, South San Francisco, CA.Purpose: RN6G (PF-04382923) is a humanized IgG2Δa monoclonalantibody that binds with high affinity to amyloid β (Aβ) peptides Aβ 1-40 and Aβ 1-42 at the free C-terminal region. It is under development astreatment for patients with geographic atrophy (GA) related to dryage-related macular degeneration (ARMD). Data from a singleascending dose (SAD) and emerging data from a multiple ascendingdose (MAD) study in subjects with dry ARMD showed distinctiveRN6G PK profiles characterized by a dose-dependent distributionphase, which cannot be fully explained by the observed PD (Aβ)concentration profiles in plasma. The objective of this analysis was todevelop and evaluate a mechanism-based predictive population PK-PD model that describes the data from these Phase I studies insubjects with early and with advanced dry ARMD.Methods: The database included 714 RN6G and 1178 Aβ 1-x (n=864from Gyros assay, n=314 from liquid chromatography-tandem massspectrometry) samples from the Phase I studies (B1181001 andB1181002). These data were simultaneously analyzed via nonlinearmixed-effects modeling with NONMEM, v 7.1.2.Results: The time course of RN6G and its interplay with Aβ 1-x weredelineated in the mechanism-based PK-PD model. By incorporatingan Aβ 1-x -rich compartment, the model was able to adequatelydescribe the dose-dependent distribution phase of RN6G, as well asthe Aβ 1-x profiles observed in plasma. The initial model from theSAD study was used to select the dosage regimen for the MADstudy. The preliminary PK/PD data from the MAD study providedverification of the predictions by the model, which was then used forthe dose regimen selection for the proof-of-concept study. Simulationresults suggest that the level of sequestration for free Aβ in plasmamight not be indicative of that in tissues with higher Aβ 1-xconcentration such as choroid or vitreous humour.Conclusions: A mechanism-based binding model was developed toadequately describe the distinctive disposition profiles of RN6G andthe observed Aβ 1-x concentration profiles in plasma. This modelingwork provided the rationale for dosage regimens for RN6G earlyclinical trials.Commercial Relationships: Kai H. Liao, Pfizer Inc (E); Pamela D.Garzone, Pfizer (E); Sangeetha S. Bollini, Pfizer (E); Philip M.Fanning, Pfizer (E); Gilbert Wong, Pfizer, Inc (E); Xu Meng, PfizerInc (E)Clinical Trial: NCT00877032Program Number: 3288 Poster Board Number: A0139Presentation Time: 11:00 AM - 12:45 PMShort Term Effect of Intravitreal Aflibercept Injection InIntraOuclar PressureKasra Attaran-Rezaei, Carmel Moazez, Clive H. Sell, Alan J.Gordon, Rahul Reddy, Henry M. Kwong, Stephen De Souza, MatthewC. Ziemianski, Belinda Shirkey, Shepard Bryan. Ophthalmology &Visual Science, Associated Retina Consultants, Phoenix, AZ.Purpose: Age-related macular degeneration (AMD) is the leadingcause of vision loss in patients older than 55 years, in the UnitedStates. The most severe vision loss occurs in the neovascular form ofAMD. Intravitreal Aflibercept injection has been tried for thetreatment of Wet form of AMD. In this study we evaluate the shorttermeffect of Intravitreal Aflibercept injections on intraocularpressure.Methods: 30 patients with Wet form of age related maculardegeneration, who have the inclusion criteria for intravitrealAflibercept injection were enrolled in the study. Aflibercept withdose of 2 mg administered by intravitreal injection every 4 weeks, forthree months. The intraocular pressure was measured before eachinjection with Tonopen (Reichert, NY, USA) in every visit.Results: 30 patient enrolled in the study, 20 female and 10 male withaverage age of 80 years. The IOP measurement were 17.35 mmhg atthe beginning of the study and 15.95, 16.00 and 14.85 mmhg in thenext three months visits. A one-way within-subjects ANOVA wasconducted with the factor being time (baseline, 1st month, 2nd monthand 3rd month and the dependent variable being intraocular pressure(IOP). ( p = 0.07).Conclusions: Aflibercept has been prescribed as a new and effectivetreatment for wet AMD. In this study we show that intarvitrealinjections of Aflibercept does not increase the intraocular pressure ina short term. We suggest that intarvitreal Aflibercept can be usesafely in patient with history of glaucoma.Commercial Relationships: Kasra Attaran-Rezaei, None; CarmelMoazez, None; Clive H. Sell, None; Alan J. Gordon, None; RahulReddy, None; Henry M. Kwong, None; Stephen De Souza, None;Matthew C. Ziemianski, None; Belinda Shirkey, None; ShepardBryan, NoneSupport: Associated retina consultant research supportProgram Number: 3289 Poster Board Number: A0140Presentation Time: 11:00 AM - 12:45 PMIn vivo imaging of fluorescent probes linked to antibodies againsthuman and rat vascular endothelial growth factor (VEGF)Johanna Meyer 1 , Alexander Cunea 1 , Pia Welker 2 , Kai Licha 2 ,Dagmar Sonntag-Bensch 1 , Steffen Schmitz-Valckenberg 1 , Frank G.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. 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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyHolz 1 . 1 Department of Ophthalmology, University of Bonn, Bonn,Germany; 2 Mivenion GmbH, Berlin, Germany.Purpose: To investigate fluorescent molecular probe linked toantibodies against VEGF for in vivo imaging of VEGF.Methods: Bevacizumab (a humanized monoclonal antibody, Roche),antiRatVEGF (a polyclonal antibody against rat VEGF164, R&DSystems) and B20-4.1.1 (a polyclonal antibody against human and ratVEGF, Genentech) were covalently attached to indocyanine green(ICG), a near-infrared dye, yielding soluble conjugates. Bindingproperties were assessed by an in vitro proliferation assay. Usingconfocal scanning laser ophthalmoscopy (cSLO), in vivo reflectanceand fluorescence imaging was performed in Dark Agouti rats that hadundergone argon laser photocoagulation to induce choroidalneovascularisations (CNV). Retinal uptake and fluorescence wererecorded following intravenous and intravitreal injection of the dyeconjugates for up to 100 days.Results: In vivo imaging before dye application showed ill-definedretinal lesions at day 7. Immediately following intravenous andintravitreal injection a strong fluorescence was visible. Twenty-fourhours following injection an accumulation of the antibody-conjugateat the site of the roundish laser lesions for all antibody-conjugateswere observed. Furthermore, multiple fluorescent spots were visibleup to 35 days following intravenous injection of Bevacizumab-ICGand for up to 60 days following intravitreal injection ofBevacizumab-ICG, B20-4.1.1-ICG and antiRatVEGF-ICG. Nofluorescent spots occurred after intravenous injection of B20-4.1.1-ICG or antiRatVEGF-ICG. Over time, a continuous decrease of thefluorescence intensity was observed for all antibody-conjugates.Conclusions: Pharmacokinetics of fluorescent-labeled bevacizumab,antiRatVEGF and B20-4.1.1 can be investigated in vivo followingintravenous and intravitreal injection. Strong accumulations in thesite of the laser lesion were observed for all antibody-conjugates.This novel molecular imaging approach of VEGF may be applicablein patients for earlier diagnosis and more refined individualized anti-VEGF therapies with the aim of optimizing functional outcomes.Commercial Relationships: Johanna Meyer, HeidelbergEngineering (F), Carl-Zeiss Meditec AG (F); Alexander Cunea, CarlZeiss Meditec (F), Heidelberg Engineering (F); Pia Welker,mivenion (E); Kai Licha, mivenion GmbH (E); Dagmar Sonntag-Bensch, Heidelberg Engineering (F), Carl-Zeiss Meditec AG (F);Steffen Schmitz-Valckenberg, Heidelberg Engineering (F), Optos(F), Carl Zeiss Meditec (F), Heidelberg Engineering (R), Genentech(C), Novartis (C), Novartis (R), Roche (R), Novartis (F); Frank G.Holz, Acucela (C), Allergan (C), Genentech (F), HeidelbergEngineering (F), Zeiss (F), Novartis (F), Novartis (C), Optos (F),Merz (C), Bayer (F), Bayer (C), Boehringer Ingelheim (C)Support: German Ministry of Education and Research, BMBF FKZ13N10285Program Number: 3290 Poster Board Number: A0141Presentation Time: 11:00 AM - 12:45 PMGeneration of Combination PDGF / VEGF-antagonist ECTdevicesVincent Ling, Arne Nystuen, Susan A. Elliott, Lisa Orecchio, BrendaDean, Konrad Kauper, Weng Tao. Biological Sciences, NeurotechUSA, Cumberland, RI.Purpose: PDGF and VEGF are potent pro-angiogenic moleculesimplicated in neovascular diseases. Anti-VEGF therapies that inhibitblood vessel growth have been successfully used in the treatment ofcertain cases of wet AMD, but with only ~30% of treated patientsgain 3 lines of vision. It is hypothesized that regression of CNV maybe improved with the addition of anti-PDGF therapies that target thepericyte cellular scaffolding surrounding blood vessels. To that end,we generated PDGFR-Fc producing cell lines and ECT devices forthe potential treatment of wet AMD. We also explored the concept ofcombination delivery of PDGFR-Fc and NT-503 VEGFR-Fc from anECT device system.Methods: PDGFR-Fc cell lines were produced by standardtransfection into NTC-200 cells, followed by by screening for highestrecombinant protein productivity via ELISA. These new NT-506PDGFR-Fc cell lines and Neurotech NT-503 VEGFR-Fc cell lineswere encapsulated by loading into 7.2 mm ECT devices eitherseparately, or in combination as a 50% mixture of each cell line.Devices were implanted into rabbits for 1 month, after which deviceexplants were assessed for secretion of PDGFR-Fc and VEGFR-Fcmolecules.Results: Stable PDGFR-Fc cell lines were produced that secreted ~15 picogram per cell per day (pcd). PDGFR-Fc / PDGF binding wasestablished by direct binding and solution-phase ELISA assays onpurified PDGFR-Fc. Bioactivity of PDGFR-Fc was demonstrated byinhibition of PDGF-based cellular migration assays. In-vivo PDGFR-Fc ECT implants exhibited sustained production of PDGFR-Fcprotein after one month, as based on explant device culture andvitreal PDGFR-Fc accumulation. Mixed-cell line ECT implants werealso effective in the simultaneous production of PDGFR-Fc andVEGFR-Fc, proportional to initial cell loading.Conclusions: Here we show the ability of ECT to sustain productionof a PDGF antagonist which may have practical application inconjunction with anti-VEGF therapy. As an initial proof-of-concept,we show that a mixed-cell line ECT implant affords simultaneousdelivery of PDGF / VEGF antagonists and may represent a potentfuture treatment modality for wet AMD.Commercial Relationships: Vincent Ling, NeurotechPharmaceuticals (E); Arne Nystuen, NeurotechUSA, Inc (E); SusanA. Elliott, Neurotech (E); Lisa Orecchio, Neurotech USA, Inc. (E);Brenda Dean, Neurotech USA, Inc (E); Konrad Kauper, NeurotechPharmaceuticals (E); Weng Tao, Neurotech (E)Program Number: 3291 Poster Board Number: A0142Presentation Time: 11:00 AM - 12:45 PMMORPHOLOGICAL STUDY OF THE GANGLION CELLLAYER OF RABBITS AFTER INTRAVITREAL INJECTIONOF MYCOPHENOLIC ACIDAndre M. Liber 1 , Rafael M. Ferraro 1 , Dora F. Ventura 1 , FranciscoMax Damico 2, 1 , Christina Joselevitch 1 . 1 Psychology Experimental,Universidade de Sao Paulo, Sao Paulo, Brazil; 2 medical school,universidade de são Paulo, São Paulo, Brazil.Purpose: To investigate the effects of different concentrations ofmycophenolic acid (MPA) injected in the vitreous of New Zealandalbino rabbits.Methods: Rabbits were anesthetized with ketamine 10% andxylazine 2%. Intravitreal injections were performed afterparacentesis. Right eyes were injected with 1 or 10 mg MPA, and lefteyes were injected with 0.1 mL of vehicle (polysorbate 80). After 30days, animal were sacrificed by intravenous injection of sodiumpentobarbital (70 mg/kg). After enucleation, retinas were isolated andfixed in 10% formalin for 48 h and kept in 4% formalin. Isolatedretinas were flattened on gelatinized slides and processed for Nisslstaining using cresyl violet as dye. Sample retinal fields werephotographed throughout the retina at 1 mm intervals with a digitalcamera attached to a microscope. Neurons in the ganglion cell layer(GCL) within each field were counted using NIH Scion Image 2.0software. The average density of cells was estimated for each retinaalong the nasotemporal and dorsoventral axes and the data from eachgroup were compared with those of the corresponding control.Results: Cell density values along the dorsoventral and nasotemporal©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 – Physiology/Pharmacologyaxes in the eyes injected with MPA did not differ from those injectedwith either salineor vehicle. Highest mean densities (±SD) in thedorsoventral axis were 4,094±1,856 (saline), 4,396±1,899 (vehicle),2,901+1,030 (MPA 1 mg) and 5,156 cells/mm2 (MPA 10 mg), andthose in the nasotemporal axis were 2,646±2,085 (saline),3,612±2,044 (vehicle), 2,678±1,329 (MPA 1 mg) and 3,145±1,709cells/mm2 in the nasotemporal axis. All values are very close to thosereported in literature for albino rabbits.Conclusions: MPA at 1 and 10 mg does not cause significantdecrease in GCL density in New Zealand albino rabbits 30 days afterintravitreal injection. Our results suggest multiple interpretations: (a)the doses of MPA used are safe to the retina, (b) the GCL recovers byday 30 post intravitreal injection, (c) damage starts more than 30 daysafter treatment, or (d) the method used is not sensitive enough tomeasure retinal toxicity.Commercial Relationships: Andre M. Liber, None; Rafael M.Ferraro, None; Dora F. Ventura, None; Francisco Max Damico,None; Christina Joselevitch, NoneProgram Number: 3292 Poster Board Number: A0143Presentation Time: 11:00 AM - 12:45 PMDevelopment and Characterization of a 3-Dimensional HumanCorneal Full Thickness Tissue ModelYulia Kaluzhny, Laurence d’Argembeau-Thornton, HelenaKandarova, Pat Hayden, Mitch Klausner. MatTek Corporation,Ashland, MA.Purpose: The FDA and other regulatory agencies require ocularirritation testing of ophthalmologic pharmaceuticals and manyconsumer products. Although widely used, animal testing haslimitations due to interspecies differences, variability, high cost, andanimal welfare issues. A highly reproducible, cost effective, humancell-based in vitro corneal tissue model would be of high utility forocular irritation testing and ophthalmic drug development.Methods: Using primary culture and state of the art tissueengineering techniques, we have developed a human corneal fullthickness (CFT) tissue model which includes epithelial, stromal, andendothelial layers. This model has been characterized in terms ofhistology and by testing a battery of test articles (TA, n=65) spanningthe range from ocular corrosives and severe irritants to non-irritants(NI). In vivo irritancy levels were obtained from a published databaseand in vitro effects were determined using the MTT tissue viabilityassay and transepithelial electrical resistance (TEER) measurements.Results: Using a tissue viability cutoff of 50%, the ocular CFT(OCFT) model was able to detect irritants with 100.0% sensitivityand non-irritants with 75.0% specificity (overall accuracy = 87.7%).In addition, the OCFT model was used to assess tissue recoveryfollowing exposure to increasing concentrations of surfactants andTA spanning the range of irritancy. Significant recovery at 24 and 48hours was observed for NI chemicals. For the same 65 TA, an exvivo assay, the Bovine Corneal Opacity and Permeability (BCOP),had 87.9%, 68.8%, and 78.5% assay sensitivity, specificity, andaccuracy, respectively.Conclusions: The OCFT model will address the needs of ophthalmicand other formulators who need to screen their products for irritancyand efficacy. Similar to other in vitro assays, high levels ofreproducibility at a low cost are anticipated. The OCFT model willfacilitate the development and testing of ophthalmic pharmaceuticalsand allow for basic studies related to corneal physiology, woundrepair, and pathologies.Histological cross-section: Formalin fixed, paraffin embedded, H&Estained cross-section of OCFT tissue model (4X). 1. Cornealepithelium (10X); 2. Stroma with embedded keratocytes; 3.Endothelial layer (20X); 4. Micorporous membrane; 5. Air-liquidinterface; 6. Culture medium fed through the membrane.Commercial Relationships: Yulia Kaluzhny, MatTek Corporation(E); Laurence d’Argembeau-Thornton, MatTek Corporation (E);Helena Kandarova, MatTek Corporation (E); Pat Hayden, MatTekCorporation (E); Mitch Klausner, MatTek Corporation (E)Support: NIH SBIR Grant # 9R44ES020074-02Program Number: 3293 Poster Board Number: A0144Presentation Time: 11:00 AM - 12:45 PMNutritional supplementation for age related maculardegeneration in ItalyIlaria Zucchiatti 1 , Maurizio B. Parodi 1 , maria vittoria cicinelli 2 ,Maria Lucia Cascavilla 1 , Francesco Fasce 1 , Matteo Prati 1 ,Francesco Bandello 1 . 1 Department of Ophthalmology, UniversityVita-Salute, Scientific Institute San Raffaele, Milan, Milano, Italy;2 University Vita Salute San Raffaele, Milan, Italy.Purpose: To evaluate the rate of adherence to nutritionalsupplementation prescription in Italian patients affected by advancedform of age-related macular degeneration (AMD).Methods: All Italian patients referred to the Department ofOphthalmology of Scientific Institute San Raffaele Hospital, in Milanfrom August to November 2012 were prospectively evaluated.Dilated funduscopic examinations were performed to classify patientsaccording to AREDS categories. Inclusion criteria were the presenceof AMD in AREDS category 3 and 4. Exclusion criteria were thepresence of other ocular disorders. Primary outcome measure was therate of adherence to the supplementation prescription.Results: 100 patients were included in the study, meeting theAREDS criteria for taking nutritional supplementation (95 patientswith AREDS category 4, and 5 with category 3). Thirty-six patients(36%) were taking oral supplementation, 33 of them (94%) assumingis 1 tablet/day.Oral supplementation was recommended by the personalophthalmologist in all patients assuming it (100%). Fifty-eightpatients (58%) declared to have had no information from theirophthalmologist, whereas fifteen (15%) declared to have partialknowledge about the effects of supplementation in AMD.Conclusions: Our data reveal that Italian patients with AREDScategory 3 and 4 have a low adherence to nutritionalsupplementation. More than 70% of patients have not beenadequately informed by their ophthalmologist regarding the potentialbenefits of oral supplementation for AMD. Italian ophthalmologistsshould stress the importance of nutritional recommendation.Commercial Relationships: Ilaria Zucchiatti, None; Maurizio B.Parodi, None; maria vittoria cicinelli, None; Maria LuciaCascavilla, None; Francesco Fasce, None; Matteo Prati, None;Francesco Bandello, ALLERGAN Inc. (S), NOVARTIS©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 – Physiology/PharmacologyPHARMACEUTICALS CORPORATION (S), FARMILA-THEA(S), BAYER SCHERING PHARMA (S), PFIZER Inc. (S), ALCONInc. (S), BAUSCH AND LOMB (S), GENENTECH Inc. (S),ALIMERA SCIENCES Inc. (S), SANOFI AVENTIS (S),THROMBOGENICS (S)Program Number: 3294 Poster Board Number: A0145Presentation Time: 11:00 AM - 12:45 PMFeasibility of Cryopreservation of the Encapsulated CellTechnology DevicesCahil McGovern 1 , John D. Duggan 3 , Crystal Cortellessa 1 , SandySherman 1 , Melissa Stiles 2 , Konrad Kauper 4 , Allyse Mazzarelli 6 , WengTao 5 . 1 Manufacturing, Neurotech, Cumberland, RI; 2 Quality Control,Neurotech, Cumberland, RI; 3 Quality Assurance, Neurotech,Cumberland, RI; 4 Engineering, Neurotech, Cumberland, RI;5 Research and Development, Neurotech, Cumberland, RI; 6 Histology,Neurotech, Cumberland, RI.Purpose: To investigate the feasibility of cryopreservation as apotential means of Encapsulated Cell Technology (ECT) devicestorage after manufacturing.Methods: An NTC-200 cell line secreting an antiangiogenic factorwas used for the encapsulation. Cells were expanded in a growthmedia, harvested, and formulated at a specific density in a serum freemedia, either with or without various concentrations of acryopreservative. Prepared cells were loaded into Neurotechophthalmic ECT devices. Control devices were kept at 37C whilecryopreserved devices were frozen at a rate of 1C/min and stored inliquid nitrogen (vapor phase) for 2 weeks. Cryopreserved deviceswere then removed from liquid nitrogen, rapidly thawed, washed in abalanced salt solution, and incubated in a serum free media for 6days. The devices were then cultured in fresh serum free media for 24hours. The condition media samples were collected and assayed forthe antiangiogenic factor by ELISA. The devices were subsequentlyanalyzed for metabolic activity using the CCK-8 assay (Dojindo) andthen subjected to either total DNA using the Hoefer DyNA Quant200 fluorometer (Pharmacia) or histological analysis using standardhematoxylin and eosin staining techniques.Results: During the pre-encapsulation culture period, the cellsmaintained a typical and consistent cuboidal morphology. Cellsencapsulated in devices in the fresh media group and cryopreservedgroup both maintained a normal morphology with a high density ofhealthy cells distributed throughout the device during the testingperiod. No deterioration of the device capsule was observed by visualinspection. Factor secretion levels, cell metabolic activity, and theoverall device cell load between fresh and cryopreserved deviceswere also comparable. Furthermore, secreted proteins with expectedmolecular weights were detected in the culture media from bothdevice groups. No differences in protein size or degradation betweenthe fresh and cryopreserved device groups were detected by Westernblot.Conclusions: These data suggested that the cryopreservation is afeasible method of extending the shelf life of ECT products. Longerterm and in vivo studies of cryopreserved devices are currently underinvestigation. Optimizing cryopreservation techniques in themanufacturing process will allow for the long term storage of ECTproduct and may greatly enhance accessibility of ECT productsworldwide.Commercial Relationships: Cahil McGovern, Neurotech (E); JohnD. Duggan, Patent U.S.S.N. 61/653,191 (P), Neurotech USA, Inc(E); Crystal Cortellessa, U.S.S.N. 61/653,191 (P), Neurotech USAInc. (E); Sandy Sherman, Neurotech USA Inc (E); Melissa Stiles,None; Konrad Kauper, Neurotech Pharmaceuticals (E); AllyseMazzarelli, Neurotech USA (E); Weng Tao, Neurotech (E)Program Number: 3295 Poster Board Number: A0146Presentation Time: 11:00 AM - 12:45 PMContinuous Intraocular Drug Delivery over 5 ½ Years: CiliaryNeurotrophic Factor (CNTF)Production by Encapsulated CellTechnology Implants Treating Patients with Retinitis Pigmentosaand Geographic AtrophyKonrad Kauper, Cahil McGovern, Paul Stabila, Sandy Sherman,Pam Heatherton, Brenda Dean, Crystal Cortellessa, Alice Lee, WengTao. Core Technology Development, Neurotech USA, Cumberland,RI.Purpose: Continuous intraocular delivery of biotherapeutics fortreatment of chronic retinal diseases remains a major hurdle for drugdevelopment. We have previously reported the pharmacokinetics ofCNTF delivered over 2 years by an intraocular encapsulated celltechnology (ECT) implant in patients with retinitis pigmentosa (RP)and geographic atrophy (GA). This is a follow-up evaluation ofpatients implanted up to a 5.5 - year period.Methods: All study patients received an ECT-CNTF implant,designated NT-501, in one eye. For the phase 1 RP (CNTF1) study,the protocol mandated explant of all patients at 6 months. For thephase 2 studies, including phase 2 GA (CNTF2), and phase 2 late andearly stage RP (CNTF3, and CNTF4), explants were optional andoccurred at 12, 18 and 24 months. Several additional patients fromthe CNTF4 study chose to be explanted at 30 (n=6), 44 (n=1), 54(n=1) and 66 (n=1) months post implant. The rate of CNTF secretionfrom the explants and the corresponding vitreous CNTF levels, ifavailable, were evaluated at each time point. Serum samples fromthese patients were evaluated for CNTF, anti-CNTF antibodies andantibodies to the encapsulated cells.Results: Cumulatively, the data demonstrates NT-501 implantsproduce CNTF continuously over a 5.5 year period. The range ofexplant CNTF production rate at each time point was statisticallyequivalent between the 0.5 year and 5.5 year implant period. Themean rates of CNTF production over this period varied betweenapproximately 1 ng/day to 2 ng/day, a rate shown to be effective inprotecting cone photoreceptors in RP patients (Talcott et el. IOVS,2011). Encapsulated cells, subjectively evaluated following H&Estaining of explant capsules, were viable and remained at a highpopulation density, similar to the pre-implant condition. CNTF, anti-CNTF antibodies and antibodies to the encapsulated cells were notdetected in the serum of patients.Conclusions: This follow-up study demonstrates that the intraocularECT implant continues to maintain a favorable pharmacokineticprofile for the treatment of chronic retinal degenerative diseaseswithout systemic exposure for over a half decade.Commercial Relationships: Konrad Kauper, NeurotechPharmaceuticals (E); Cahil McGovern, Neurotech (E); Paul Stabila,Neurotech Pharmaceuticals (E); Sandy Sherman, Neurotech USAInc (E); Pam Heatherton, None; Brenda Dean, Neurotech USA, Inc(E); Crystal Cortellessa, U.S.S.N. 61/653,191 (P), Neurotech USAInc. (E); Alice Lee, Neurotech USA, Inc. (E); Weng Tao, Neurotech(E)Program Number: 3296 Poster Board Number: A0147Presentation Time: 11:00 AM - 12:45 PMEffect of Apolipoprotein A1-mimetic peptides on cell viability ofcultured retinal pigment epithelial cellsYoko Miura 1, 2 , Ingrid Fritz 1 , Salvatore Grisanti 1 , Martin Rudolf 1 .1 Department of Ophthalmology, University of Luebeck, Luebeck,Germany; 2 Institute of Biomedical Optics, University of Luebeck,Luebeck, Germany.©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 – Physiology/PharmacologyPurpose: Previous studies of our research group have shown thatApoA-I mimetic peptides like D-4F and L-4F are able to reducepathological lipid deposits in and on top of Bruch’s membrane(Rudolf et al. ARVO 2011, 2013). Since the function of the retinalpigment epithelium (RPE) plays a central role in photoreceptor healthand function we evaluate the effect of these peptides on RPE cellviability in vitro.Methods: Third passage porcine RPE cell cultures were treated withdifferent concentrations of L-4F or its stereoisomer D-4F (0; 0.1; 10;100; 500; 1000; 2000; 4000 µg/ml) for 48 hrs. Nonfunctionalscrambled L-4F (sL-4F) served as a control. MTT assays(Dimethylthiazol diphenyltetrazolium bromide) were performed toanalyse the peptides’ cytotoxicity on RPE cells. RPE cellproliferation activity was assessed by measuring the optical density at577 nm using a photometric multiplate reader. Further test runs wereperformed in smaller concentration steps to detect the maximumtolerated dose of these peptides.Results: D-4F and L-4F showed no significant effect in MTT assayswith concentration ≤ 10 µg/ml. With higher concentrations, L-4F ≤375 µg/ml and ≤ 125 µg/ml for D-4F induced a significant increaseof optical density (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyVirgilio Morales-Canton 1 , Jans J. Fromow-Guerra 1 , SamanthaSalinas Longoria 1 , Rafael Romero Vera 1 , Michelle Widmann 2 ,Samirkumar R. Patel 2 , Benjamin Yerxa 1 . 1 The Asociación Para EvitarLa Ceguera (APEC), Mexico City, Mexico; 2 Clearside Biomedical,Alpharetta, GA.Purpose: To evaluate the safety and tolerability of a singlemicroneedle injection of bevacizumab into the suprachoroidal space(SCS) using a Clearside Biomedical proprietary microneedleMethods: Four adult patients with choroidal neovascularization(CNV), secondary to wet age-related macular degeneration (AMD),were enrolled in a phase 1, single-center, open-label study. Eachpatient provided informed consent and was screened for eligibility.Following application of topical anesthesia, the patient wasadministered a single unilateral injection of 100 µL bevacizumab(Avastin®) into the SCS using an 850 µm 33 gauge ClearsideBiomedical microneedle. The microneedle was inserted into thesclera approximately 8-12 mm posterior to the limbus in the superiortemporal quadrant. Treated patients remained in the clinic for 4 hoursfor observation and then returned multiple times for follow-up duringa 2 month period. Major safety examinations included intraocularpressure (IOP), angiograms, biomicroscopy, indirectophthalmoscopy, fundus photography, optical coherence tomography(OCT), visual acuity (VA), and assessment of painResults: Preliminary data shows that four patients were successfullydosed into the SCS which was confirmed via ophthalmoscopeimmediately following injection. A moderate level of pain wasrecorded for the administration. There were no unexpected or seriousadverse events related to bevacizumab or method of administrationon ophthalmic examinations. No negative effect on IOP was noted inany patient. No patients required rescue therapy or reinjection duringthe two months following treatmentConclusions: Preliminary data suggests that the SCS can besuccessfully and safely dosed via the Clearside Biomedicalproprietary microneedle using only topical anesthesia. These datashow that 100 µL bevacizumab can be delivered into the SCS withoutunexpected or serious adverse eventsCommercial Relationships: Virgilio Morales-Canton, ClearsideBiomedical (F); Jans J. Fromow-Guerra, None; Samantha SalinasLongoria, None; Rafael Romero Vera, None; Michelle Widmann,Clearside Biomedical, Inc. (E); Samirkumar R. Patel, ClearsideBiomedical (E), Clearside Biomedical (I), Clearside Biomedical (P);Benjamin Yerxa, Clearside Biomedical (I)369 Pharmacological Targets for Eye Disease: Present andFutureTuesday, May 07, 2013 2:45 PM-4:30 PM618-620 Paper SessionProgram #/Board # Range: 3698-3704Organizing Section: Physiology/PharmacologyProgram Number: 3698Presentation Time: 2:45 PM - 3:00 PMLocalization and Activity of Histone Deacetylases in the RetinaOday Alsarraf, Jie Fan, C. James Chou, Craig E. Crosson.Ophthalmology - Storm Eye Institute, Medical University of SouthCarolina, Charleston, SC.Purpose: The increase in histone deacetylase (HDAC) activity andresulting dysregulation of protein acetylation is an integral event inretinal degenerations associated with ischemia and ocularhypertension. The purpose of the current study is to determineactivity profiles and distribution of individual HDACs in the retina,and if the selective modulation of individual isoforms can limitretinal injury.Methods: Isolated mouse retinas were evaluated for class-I and IIHDAC activity using a class-specific fluorometric enzymatic assay.The activities of individual HDAC isoforms were determined by theaddition of selective isoform inhibitors and gene deletion. Retinallocalization and relative abundance of individual HDAC isoformswas determined by immunohistochemistry and Western blotting. Inselected knockout and wild type mice, electroretinogram (ERG)analysis was used to evaluate responses to 45-minutes of ischemicretinal injury at 7 days post-injury as compared to baseline values.Results: Although class-II HDACs were detected by Western blotanalysis in retinas from control mice, class-I HDACs accounted forall the measureable activity. Analysis of individual class-I isoformsdemonstrated that HDAC1 and 2 accounted for 34 ± 3.5% of totalactivity, and HDAC3 for the remaining 66 ± 5.0% activity.Immunolocalization demonstrated that HDAC1, 2 and 3 wereprimarily located in the cell bodies of inner nuclear and retinalganglion cell layers. However, HDAC1 and 2 also exhibited limitedexpression in the outer nuclear layer. In wild-type mice, ERG a- andb-wave amplitudes from ischemic eyes were significantly reduced by53.3 ± 6.3% and 59.8 ± 6.9%, respectively, as compared to preischemiabaseline values. In HDAC2 heterozygote knockout mice,baseline ERGs were not altered; however, ERG a- and b-waveamplitudes from ischemic eyes were significantly greater by 66.4 ±4.8% and 60.0 ± 7.6%, respectively, as compared to waveforms fromischemic eyes in wild-type mice.Conclusions: Although HDACs are detected in most retinal cellbodies, these studies provide evidence that HDAC1, 2 and 3 isoformsaccount for almost all activity and are predominantly expressed ininner retinal layers. The selective reduction in HDAC2 expression issufficient to significantly reduce ischemic retinal injury. These datasupport the idea that isoform selective HDAC inhibitors may providean efficacious strategy to protect the retina from injury.Commercial Relationships: Oday Alsarraf, None; Jie Fan, None;C. James Chou, None; Craig E. Crosson, Alimera Sciences (C),Lexicon Pharmaceuticals, Inc (R)Support: NH Grant EY021368-02Program Number: 3699Presentation Time: 3:00 PM - 3:15 PMSigma-1 Receptor Stimulation Protects Purified RGCs fromIschemic Insult through the Phosphorylation of ExtracellularSignal Regulated Kinase 1/2Brett H. Mueller 1, 2 , Yong H. Park 1, 2 , Hai-Ying Ma 1, 2 , Thomas Yorio 1,2 . 1 Pharmacology & Neuroscience, Univ of North Texas Hlth Sci Ctr,Fort Worth, TX; 2 North Texas Eye Research Institute, University ofNorth Texas Health Science Center, Fort Worth, TX.Purpose: Sigma-1 receptor activation and mitogen-activated proteinkinases (MAPKs) have been shown to have neuroprotective roles inprotecting retinal ganglion cells (RGCs) from cell death. The purposeof this study was to determine if sigma-1 receptor stimulation withpentazocine could promote neuroprotection under conditions ofischemia through the phosphorylation of extracellular signalregulated kinase (pERK)1/2.Methods: Primary RGCs were isolated from P3-P7 Sprague-Dawleyrats and purified by sequential immunopanning using a Thy 1.1antibody. RGCs were cultured for 7 days before subjecting the cellsto an ischemic insult (0.5% oxygen in glucose-free medium) for 6hours. During the ischemic insult, RGCs were treated withpentazocine (sigma-1 receptor agonist) with or without BD1047(sigma-1 receptor antagonist). In other experiments primary RGCswere treated with pentazocine, in the presence or absence ofPD98059 (MEK1 inhibitor). Cell survival/death was assessed 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 – Physiology/Pharmacologystaining with the calcein-AM/ethidium homodimer reagent. Levels ofpERK1/2, total ERK1/2, and beta tubulin expression were determinedwith immunoblotting and immunofluorescence.Results: RGCs subjected to an ischemic insult demonstrated morethan a 40% increase in cell death, compared to untreated controls.RGCs maintained under ischemia also showed a 50% decrease inexpression of pERK1/2 (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyComprehensive Analysis of Prostanoid Receptor AntagonistsEffects on Intraocular Pressure in Ocular Hypertensive MonkeysCarol B. Toris 1 , Tara L. Rudebush 1 , Stacey Wenthur 1 , David F.Woodward 2 . 1 Ophthalmology, Univ of Nebraska Medical Ctr,Omaha, NE; 2 Biological Sciences, Allergan, Inc., Irvine, CA.Purpose: Most of what is known about prostanoid receptor mediatedcontrol of IOP has been learned from studies of prostanoid agonists.Prostanoid antagonist effects have been studied largely from theperspective of confirming the pharmacological identity of prostanoidagonists in producing their effects. This study examines the IOPeffects of 8 prostanoid receptor antagonists in monkeys withunilateral laser-induced glaucoma to better understand a possible roleof endogenous prostaglandins in the regulation of IOP.Methods: Eight trained conscious monkeys were studied. BaselineIOPs were taken by pneumatonometry of both eyes at 9AM. Thestudy compound was applied topically to the glaucomatous eye at9:05. IOP measurements were repeated at 10AM, 12PM, 2PM, and4PM and daily at 9AM for the next three days. After 6 days ofwashout, another compound was applied. IOPs were repeated as atbaseline. Randomized dosing and IOP measurements continued untilall compounds were tested. Antagonists dosed at 1% were SC-51322(EP1), PF-04418948 (EP2), L-826266 (EP3), GW-627368 (EP4),BW-A868C (DP1), AGN-211336 (prostamide), AS- 604872 (FP),RO-3244019 (IP). The vehicle was 1% Polysorbate 80.Results: When compared with vehicle treatment, significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyknockout mice, indicating that its cytoprotective effect is PPARαdependent.Moreover, both FA and PPARα over-expression upregulatedIκBα, which is known to repress NFκβ signaling and thusdown-regulate NOX4, a primary source of ROS in both hypoxia andoxidative stress. Further, both FA and PPARα over-expression downregulatedNOX4 in hypoxia, suggesting that the effect is relevant in ahypoxic environment, and is a direct consequence of PPARαactivation.Conclusions: Taken together, our data have demonstrated for thefirst time that FA/PPARα have a neuroprotective effect in the retina,and moreover established that the protective effect is PPARαdependent.Further, because both FA and PPARα over-expressionactivate the IκBα/NFκβ axis and subsequently down-regulate NOX4,this event may also be PPARα-specific. We have also identified thatFA and PPARα affect IκBα down-regulation of NOX4, and wehypothesize that this may be responsible, at least in part, for itsprotective effect.Commercial Relationships: Elizabeth P. Moran, None; Lexi Ding,None; Ying Chen, None; Yang Hu, None; Yusuke Takahashi,None; Jian-Xing Ma, NoneSupport: This study was supported by NIH grants EY018659,EY012231, EY019309 and P20GM104934Presentation Time: 9:30 AM - 9:45 AMPurine Signaling in Müller Cells During Development and in theAdult Retina in Health and DiseaseAntje Grosche. Pathophysiology of Neuroglia, University of Leipzig,Leipzig, Germany.Commercial Relationships: Antje Grosche, NoneProgram Number: 4061Presentation Time: 9:45 AM - 10:00 AMPurinergic Signaling in Retinal ProcessingThomas E. Salt. Visual Neuroscience, UCL Institute ofOphthalmology, London, United Kingdom.Commercial Relationships: Thomas E. Salt, Lundbeck (F), Merck(C), Phytopharm (C), Neurexpert (I)Program Number: 4062Presentation Time: 10:00 AM - 10:15 AMPurines and Low Grade Inflammation at Either End of theRetina; ATP and Cytokines in Both RGCs and RPEClaire H. Mitchell. Anatomy and Cell Biology, University ofPennsylvania Library, Philadelphia, PA.Commercial Relationships: Claire H. Mitchell, University ofPennsylvania (P)409 Purine Signaling in the Eye: Role in Health and Disease -MinisymposiumWednesday, May 08, 2013 8:30 AM-10:15 AM618-620 MinisymposiumProgram #/Board # Range: 4056-4062Organizing Section: Physiology/PharmacologyContributing Section(s): Cornea, Retinal Cell BiologyProgram Number: 4056Presentation Time: 8:30 AM - 8:45 AMIntroduction to Purine SignalingJulie Sanderson. School of Pharmacy, University of East Anglia,Norwich, United Kingdom.Commercial Relationships: Julie Sanderson, NoneProgram Number: 4057Presentation Time: 8:45 AM - 9:00 AMPurine Signaling in the Trabecular Meshwork: A Regulator ofIntraocular PressureMortimer M. Civan. Physiology-Richards Bldg, Univ ofPennsylvania Sch of Med, Philadelphia, PA.Commercial Relationships: Mortimer M. Civan, NoneProgram Number: 4058Presentation Time: 9:00 AM - 9:15 AMPurinergic Receptors in Lacrimal Gland Health and Dry EyeDiseaseDarlene A. Dartt. Schepens Eye Research Institute, Boston, MA.Commercial Relationships: Darlene A. Dartt, NoneProgram Number: 4059Presentation Time: 9:15 AM - 9:30 AMTRPV4 and Hemichannels Cooperate to Play a Critical Role inPurine Signaling in the LensNicholas A. Delamere. Physiology, University of Arizona, Tucson,AZ.Commercial Relationships: Nicholas A. Delamere, NoneProgram Number: 4060418 Antibiotics and Corneal DiseaseWednesday, May 08, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 4284-4307/C0022-C0045Organizing Section: Physiology/PharmacologyContributing Section(s): CorneaProgram Number: 4284 Poster Board Number: C0022Presentation Time: 8:30 AM - 10:15 AMThe use of predatory prokaryotes to control human ocularpathogensRobert M. Shanks 1 , Kevin To 2 , Nicholas A. Stella 1 , Kimberly M.Brothers 1 , Regis P. Kowalski 1 , Eric G. Romanowski 1 , Daniel E.Kadouri 2 . 1 Ophthalmology, University of Pittsburgh, Pittsburgh, PA;2 Oral Biology, UMDNJ, Newark, NJ.Purpose: Antibiotic resistant, disease-causing microorganisms are anincreasing cause for concern in ocular infections. In an attempt tofind innovative approaches to eradicate bacteria that cause ocularinfections, we measured whether predatory bacteria couldsuccessfully kill clinical keratitis isolates of Pseudomonasaeruginosa and Serratia marcescens in vitro. The cytotoxic impact ofpredatory bacteria on a human corneal cell line was also assessed.Methods: The predatory bacteria used in this study wereBdellovibrio bacteriovorus, strains HD100 and 109J, and Micavibrioaeruginosavorus ARL-13. These are non-pathogenic, soil-derived,Gram-negative, obligatory parasites that feed on other Gram-negativebacteria. 10 8 CFU of fluoroquinolone-resistant and susceptible ocularisolates of P. aeruginosa and multidrug resistant strains of S.marcescens were mixed with 10 8 CFU of predatory bacteria. After 24and 48 hours, the numbers of surviving bacteria were enumerated.Human corneal limbal epithelial cells (HCLE) were challenged with~10 8 CFU of predatory bacteria or 5 x10 7 CFU of P. aeruginosa;HCLE viability was measured at 4 and 24 hours using alamar blue.Results: B. bacteriovorus predatory bacteria reduced viable counts ofthe S. marcescens isolates examined (n=9) by 2-4 logs. B.bacteriovorus and M. aeruginosavorus predatory bacteria reducedfluoroquinolone-resistant P. aeruginosa isolates (n=9) viable countsby up to 5 logs, and had mixed results with fluoroquinolone-©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 – Physiology/Pharmacologysusceptible isolates, ranging from no effect to a 4-log reduction.Although predatory bacteria were lethal to ocular pathogens, littlecytotoxic effect was observed when high doses of the predators wereexposed to a human corneal cell line.Conclusions: This work highlights the potential use of predatorybacteria as biological based agent for eradicating antibiotic-resistantocular infections.Commercial Relationships: Robert M. Shanks, Bausch and Lomb(C); Kevin To, None; Nicholas A. Stella, None; Kimberly M.Brothers, None; Regis P. Kowalski, Rempex (F); Eric G.Romanowski, Rempex (F), Allergan (F), Alcon/Novartis (F), 3-VBiosciences (F); Daniel E. Kadouri, NoneSupport: NIH Grant AI085570, Research to Prevent BlindnessCareer Development Award, NIH Grant EY08098Program Number: 4285 Poster Board Number: C0023Presentation Time: 8:30 AM - 10:15 AMSusceptibility of Stenotrophomonasmaltophilia to Antibiotics andContact Lens Multipurpose SolutionsKeizo Watanabe 1, 2 , Hua Zhu 2 , Rani Bandara 2 , Shiro Higaki 1 ,Masahiko Fukuda 1 , Yoshikazu Shimomura 1 , Mark D. Willcox 2 , BrienA. Holden 1 . 1 Ophthalmology, Kinki University Faculty of Medicine,Osakasayama, Japan; 2 Brien Holden Vision Institute, Sydney, NSW,Australia.Purpose: To determine the susceptibility of Australian ocular isolatesof S. maltophilia to antibiotics, and to various contact-lensmultipurposesolutions (MPS).Methods: A total of 40 strains of S. maltophilia isolated from eithercontact lenses or lens storage cases of contact lens wearers weretested in the study. Bacterial susceptibilities to antibiotics weredetermined using a disc diffusion test. The antibiotics tested includedtrimethoprim-sulfamethozole, tigecycline, fluoroquinolones,aminoglycosides, β-lactams, chloramphenicol, and polymyxin B.Susceptibility of test strains to seven commonly used MPS wasdetermined by using a broth microdilution method.Results: The resistant rates of isolates were 93% to imipenem, 15%to aztreonam, 13% to chloramphenicol, and 8% to cefepime. None ofthe isolates was resistant to trimethoprim-sulfamethozole, tigecycline,ceftazidime, gatifloxacin, levofloxacin, or moxifloxacin. Thesusceptibility of test strains to the test MPS varied with MIC levelsranging from 3% to 100% (full strength) MPS. The test strains wereless susceptible to the MPSs containing single disinfectant (PHMB)and the MPS containing PQ-1 and Aldox than to the three recentlyavailable MPS containing dual disinfectants (p < 0.05).Conclusions: The Australian ocular isolates of S. maltophilia remainsusceptible to trimethoprim-sulfamethozole, tigecycline, and mostfluoroquinolones, but are less susceptible to MPS containing single orcertain dual disinfectant/s.Commercial Relationships: Keizo Watanabe, None; Hua Zhu,Brien Holden Vision Institute (E); Rani Bandara, None; ShiroHigaki, None; Masahiko Fukuda, None; Yoshikazu Shimomura,None; Mark D. Willcox, Allergan Inc (C), Allergan Inc (R), BrienHolden Vision Institute (P), Bausch + Lomb (C), Basuch + Lomb(R); Brien A. Holden, Allergan (F), AMO (I)Program Number: 4286 Poster Board Number: C0024Presentation Time: 8:30 AM - 10:15 AMA Comparison of Prophylactic Antibacterial Efficacy ofBesifloxacin 0.6% versus Moxifloacin 0.5% at 1 hour and 3 daysPrior to PhacoemulsificationFrank A. Bucci. Bucci Laser Vision Institute, Wilkes-Barre, PA.Purpose: To compare antibacterial efficacy of besifloxacin (BESI)versus moxifloxacin (MOXI) on ocular surface tissues after 1 hourand 3 days of treatment respectively.Methods: 60 pts were randomized in a single center study to receiveeither BESI or MOXI for 3 days (QID) prior to cataract surgery intheir surgical eye, and 1 hour prior to their final cultures in their nonsurgicaleye, on the day of surgery. Lid and conjunctival cultureswere obtained prior to treatment in all eyes.Results: The lid cultures for the surgical eyes (3 day tx) revealed asignificant decrease in colony forming units (CFU) for both BESI(4,925+/-1,830 to 315+/-8 CFU) (p=0.01) and MOXI (1,124+/- 337to 207+/- 20 CFU)(p=0.01). However the percent of NO GROWTHlid cultures in these surgical eyes (3 day tx) for BESI increased from13 % to 44 % pre-tx to post tx, but only from 16% to 20 % for MOXIeyes. In addition, the CFUs of the lid cultures for the non-surgicaleyes (1hr tx) decreased significantly in the BESI eyes (3,217+/-1,022to 558+/-377 CFU) (p=0.02) but INCREASED for the MOXI eyes(1,781+/-325 to 2,268+/-897 CFU)(p=0.55). The percent of NOGROWTH lid cultures in these non-surgical eyes (3 day tx) for BESIincreased significantly from 10% to 52% pre-tx to post tx, butdecreased in the MOXI eyes from 10% to 0%. The conjunctivalcultures also revealed similar results with greater decreases in CFUsfavoring BESI vs. MOXI in the non surgical (1 hr tx) eyes, butstatistical significance was not reached due to smaller CFUs and largestatistical variances. The mean CFU in MRSE BESI surgical eyes (3day tx)(n=8)(7,020 to 15)(p=0.01) and non surgical eyes (1 hr tx)(n=6) (7,961 to 633)(p=0.03) decreased significantly. The mean CFUin MRSE MOXI surgical eyes (3 day tx)(n=8)(219 to 139) showed nosignificant decrease (p=.051) and the non surgical eyes (1hr tx)(n=9)showed a substantial INCREASE in CFU (1,051 to 3,290).Conclusions: : Both BESI and MOXI revealed significant reductionsin lid CFUs after 3 days of treatment. In contrast, only BESI revealedsignificant reductions in lid CFU after 1 hour of treatment. BESI alsorevealed more rapid and significant killing of MRSE. These resultssuggest that BESI has a significantly more rapid kill rate and is moreefficacious in MRSE eyes versus MOXI in a typical population of ptsundergoing cataract surgery.Commercial Relationships: Frank A. Bucci, Bausch & Lomb (F)Support: Physician initiated independent research Bausch LombClinical Trial: 01296542Program Number: 4287 Poster Board Number: C0025Presentation Time: 8:30 AM - 10:15 AMCytotoxicities of various ophthalmic antimicrobial solutions inSV40-immortalized human corneal epithelial cellsJae Lim Chung 1 , Sang Wroul Song 1 , Byung Yeop Kim 1 , Joon H. Lee 2 ,Kyoung Yul Seo 3 . 1 Konyang University Kim's Eye Hospital, Seoul,Republic of Korea; 2 Myunggok Eye Research Institute, KonyangUniversity, Seoul, Republic of Korea; 3 Shinchon Severance Hospital,Yonsei University, Seoul, Republic of Korea.Purpose: Currently high concentration ophthalmic antibiotics wereintroduced to increase the pharmacokinetic properties. Howevertoxicity is a concern in using these agents. This study evaluated thecytotoxicities of various antibiotic eyedrops on SV40-immortalizedhuman corneal epithelial cells by using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay.Methods: Tested drugs were Cravit (levofloxacin 0.5%), Cravit 1.5(levofloxacin 1.5%), Quixin (levofloxacin 0.5%, Benzalkoniumchloride (BAK) 0.005%), Iquix (levofloxacin 1.5%), Vigamox(moxifloxacin 0.5%), Moxeza (moxifloxacin 0.5%, xanthan gum),Gatiflo (gatifloxacin 0.3%), Zymaxid (gatifloxacin 0.5%, BAK0.005%), Besivance (besifloxacin, BAK 0.01%, durasite), Azasite(azithromycin 1%, BAK 0.003%, durasite), Tobrex (tobramycin0.3%, BAK 0.01%) and standard powders of each drug. MTS assay©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 – Physiology/Pharmacologywas performed after 5~120 minutes of exposure to each solution.Results: After the exposure to undiluted solutions cell viabilitieswere decreased to 9~73% from 5 minutes. At 30 minutes tobrex,zymaxid and quixin showed marked toxicity, Cravit 1.5, Iqux andVigamox showed moderate toxicity and Cravit and Gatiflo showedmild toxicity. When exposed to 10 times diluted solutions, Tobrex,Besivance, Zymaxid, Quixin and Azasite, BAK containing products,showed moderate toxicity. After the exposure to standard powder,0.6% besifloxacin, 0.5% gatifloxacin, 0.5% moxifloxacin, 1.5%levofloxacin and 0.5% levofloxacin showed toxicity results withdecreased order. However 1% azithromycin and 0.3% tobramycinshowed almost negative cytotoxicity.Conclusions: High concentration drugs didn’t show increasedcytotoxicity. The most important factor for the determination ofcytotoxicity of antibiotic solutions was the concentration of BAK.Commercial Relationships: Jae Lim Chung, None; Sang WroulSong, None; Byung Yeop Kim, None; Joon H. Lee, None; KyoungYul Seo, NoneProgram Number: 4288 Poster Board Number: C0026Presentation Time: 8:30 AM - 10:15 AMThe In Vitro Activity of Tigecycline for Clinically RelevantOcular PathogensRegis P. Kowalski, Tyler Kowalski, Eric G. Romanowski, Robert M.Shanks, Leela Raju. Ophthalmology/Microbiology, Univ ofPittsburgh, Pittsburgh, PA.Purpose: Tigecycline is a glycylcycline antibiotic that is FDAapproved for the treatment of skin and soft tissue infections,community-acquired pneumonia, and intra-abdominal infections.Previous studies of clinical isolates from these body sitesdemonstrated that tigecycline is active in vitro against isolates of anumber of Gram-positive and Gram-negative pathogens, includingmethicillin-resistant Staphylococcus aureus (MRSA), but lackssignificant potency against Pseudomonas aeruginosa to be useful fortreatment of systemic infections A topical ophthalmic formulation(RPX-978) is in development. The goal of the current study was todetermine the in vitro activity of tigecycline against multipleclinically relevant ocular pathogens.Methods: Minimum Inhibitory Concentrations (MIC) using CLSIreference methods for broth dilution were determined for 110 clinicalconjunctivitis isolates based on incidence at the Campbell Lab:Staphylococcus aureus (SA) (n=36), coagulase-negativeStaphylococcus (CNS) (14), Streptococcus pneumoniae (SP) (22),other Gram-positive bacteria (GP) (8) (2 Streptococcus viridansgroup and 6 beta-hemolytic Streptococcus species), Haemophilusspecies (HS) (20), and other Gram-negative bacteria (10) (2 Serratiamarcescens, 2 Proteus mirablis, 3 Pseudomonas aeruginosa, 1Enterobacter aerogenes, 1 Pseudomonas fluorescens, and 1 Klebsiellaspecies). In addition, MICs were performed on 26 keratitis isolates ofPseudomonas aeruginosa (PA) and 10 endophthalmitis isolates eachof MRSA, MSSA, MRCNS, and MSCNS. A total of 176 isolateswere tested.Results: : Data is expressed as (MIC50, MIC90 and Range of MICs)in μg/ml respectively. Conjunctivitis: SA (0.25, 0.5, 0.125-0.5); CNS(0.25, 0.5, 0.05-1.0); SP (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologycompared to BESI at all time periods. The mean aqueous penetrationlevels at ONE hour were BESI 49 / MOXI 489 ng/ml (p=0.001). Thetwo hour levels were BESI 55 / MOXI 623 ng/ml (p=0.001). AtFOUR hours the levels were BESI 74 / MOXI 331 ng/ml (p=0.01).At SIX hours following the last study drops the levels were BESI 47 /MOXI 329 ng/ml (p=0.005). The calculated mean AUC 1-6 wassignificantly greater for MOXI (2170.1 +/- 171.1) versus BESI(302.8 +/- 28.3) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyStaphylococcus aureus (MRSA) keratitis model.Methods: In 32 NZW rabbits, corneal epithelial defects were createdin the left eyes using an Amoils epithelial scrubber (abraded corneas),while the epithelia of the right corneas remained intact (intactcorneas) to determine if corneal abrasion affected drug efficacy byenhancing penetration. The corneas were intrastromally injected with1000 CFU of MRSA. Rabbits were separated into 4 groups (n=8): A)RPX (0.5% tigecycline), B) VAN 5%, C) normal saline (SAL), andD) no treatment (euthanized before treatment for baseline CFU). 4hafter MRSA challenge, the topical treatment regimen of one dropevery 15 minutes for 5h was initiated. After treatment, all eyes wereslit lamp examined for presentations of infection. The eyes weregraded using a 0-3 severity scale. After examination and 1h aftertreatment, the animals were euthanized and the corneas wereharvested for CFU determination. The data were non-parametricallyanalyzed.Results: The total clinical scores for RPX treatment were less thanSAL which was less than VAN (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology3 Pharmaceutical Sciences, University of Colorado, Aurora, CO;4 Pathology, University of Texas Medical Branch, Galveston, TX.Purpose: Chronic alcoholism has been associated with variouspathologies involving oxidative damage, and may be an importantand underrated risk factor for eye disease, especially since the oculartissues are constantly exposed to oxidants. Corneal thickness andstructural abnormality has been reported in chronic alcoholics,however this subject has not received due attention. Aldehydedehydrogenases (ALDHs) play a crucial role in the detoxification ofreactive and toxic lipid aldehydes, such as 4-hydroxynonenal (HNE)and thus protecting this tissue and the rest of the eye against UVlight-inducedoxidative damage. We have therefore investigated thestatus of oxidative damage, inflammation and ALDH isozymes incorneal pathology developed in mice upon chronic alcoholconsumption.Methods: To have blood alcohol levels similar to those observed inchronic alcoholics, deer mice (deficient in hepatic alcoholdehydrogenase) were fed 3.5% ethanol via Lieber-DeCarli diet for 6months while control mice were fed the same diet with equivalentcalories replaced by maltose-dextrin. Mice were euthanized and eyesexcised and fixed for morphological studies (hematoxylin and eosinstaining) and immunohistochemical studies (for ALDH1A1, ALDH2,ALDH3A1, protein-acetylated lysine adducts, protein-HNE adducts,and nitric oxide synthase staining). Stained images of cornealsections were used to measure the thickness of the middle and sideportions of the cornea.Results: Chronic ethanol treatment resulted in a dramatic increase ofthe corneal thickness of both the stroma and epithelial layer.Maximum change in the corneal thickness (in microns) occurred inthe middle portion (Control group: stroma, 59 ±7; epithelial layer, 28±3 and Alcohol-treated group: stroma, 110±2; epithelial layer, 41 ±2). Alcohol treatment led to a decrease in the expression of most ofthe ALDH isozymes with a concomitant increase in the oxidative andinflammatory markers and protein-acetylated lysine adducts, apotential marker of ethanol consumption.Conclusions: Our results suggest that impairment in detoxification oflipid aldehydes and acetaldehyde under chronic alcohol exposuremay be central to alcohol-induced changes in corneal structure andfunction.. Further studies using the ALDH knockout mice would laythe foundation for a novel therapeutic strategy of preserving cornealpathology in chronic alcoholics.Commercial Relationships: Naseem Ansari, None; Min Zhang,None; Cheng Wang, None; John Papaconstantinou, None; VasilisVasiliou, None; Bhupendra Kaphalia, NoneProgram Number: 4296 Poster Board Number: C0034Presentation Time: 8:30 AM - 10:15 AMSmall non-hydrophobic, cationic peptide with in vitro and in vivoefficacy against Gram negative bacteriaRoger W. Beuerman 1 , Shouping Liu 2 , Rajamani Lakshminarayanan 3 ,Jianguo Li 4 , Bai Yang 5 . 1 Singapore Eye Research Inst, Singapore,Singapore; 2 Singapore Eye Research Institute, Singapore, Singapore;3 Singapore Eye Research Institute, Singapore, Singapore; 4 SingaporeEye Research Institute, Singapore, Singapore; 5 Singapore EyeResearch Institute, Singapore, Singapore.Purpose: This study has tested the concept that antimicrobialpeptides must include hydrophobic residues in order to show a highlevel of bacterial killing both in vitro and in vivo.Methods: Two short 8 AA peptides were designed and synthesizedin two analogues one with two alanines (088) and one with twoglycines (099), these linear peptides were covalentluy linked throughthe C-terminal lysine. MICs were determined for resistant andsensitive strains of Pseudomonas along with time kill and labsimulations for resistance, membrane interactions with bacteriamodeled by molecular dynamics, NMR and finally a mouse model ofinfection with Pseudomonas ATCC 9027.Results: MICs using MHB dilution methods showed that the MICsfor Pseudomonas, E. coli, and K. pneumonia were 1.5μM for the 099compound and 2.73 μM for the 088 compound, but the MICs forStaphalococcus aureus were 5.9 and 4.7μM respectively. MICs weredetermined for 10 clinical isolates of Pseudomonas of which 2 strainswere DR and 1 strain MDR (gatifloxicin MICs >22-125μg/ml) whileMICS for these as well as 3 clinical E. coli were between 1.5-5.9μM/ml. Laboratory simulations of resistance using PseudomonasATCC 9027 with norfloxicin and gentamicin used as comparisonshowed that resistance was not induced, but was induced in the otherantibiotics, MICs increased by 30-140 fold. Both compounds testedon a rabbit model of corneal wound healing of a 5mm dia abrasionshowed no differences compared to PBS control. In a mouse modelof corneal infection with Pseudomonas with 106 CFU, withgatifloxicin (3mg/ml) used for comparison there was no difference ininfection control with B2088/99 at either 1 or 3mg/ml all applicationsat 5/day.Conclusions: This new family of non-natural peptides has importantcharacteristics which should be useful for the rapdily emergingproblem of antibiotic resistant strains of Gram negative bacteria,especially PseudomonasCommercial Relationships: Roger W. Beuerman, Allergan (F),SERI (P), Santen (R); Shouping Liu, None; RajamaniLakshminarayanan, None; Jianguo Li, None; Bai Yang, NoneSupport: TCR, ExploitProgram Number: 4297 Poster Board Number: C0035Presentation Time: 8:30 AM - 10:15 AMMolecular Design of Novel Membrane Targeting Antimicrobialswith Improved Membrane Selectivity Using Natural Compoundas a ScaffoldShouping Liu 1, 2 , Hanxun Zou 1 , Jun-Jie Koh 1 , Jianguo Li 3 , RajamaniLakshminarayanan 1, 2 , Roger W. Beuerman 1, 2 . 1 Singapore EyeResearch Institute, Singapore, Singapore; 2 Duke-NUS MedicalSchool, SRP Neuroscience and Behavioural Disorders, Singapore169857, Singapore, Singapore; 3 Bioinformatics Institute, Singapore138671, Singapore, Singapore.Purpose: To set up a new platform for design and prediction ofmembrane targeting small organic molecules based antimicrobialswith improved membrane selectivityMethods: This work describes how to tune the amphiphilicconformation of α-mangostin, a natural compound with ahydrophobic xanthone scaffold, to improve the antimicrobial activityand selectivity toward Gram positive bacteria. A series of xanthonederivatives were obtained by cationic modification of free hydroxylgroups of α-mangostin at C3 and C6 positions with amines groups ofdifferent pKa values.Results: The results show that the antimicrobial activities of thecationic xanthone derivatives can be generally predicted based on thepKa values of the corresponding amines. We have identified AM-0016 (3b) as the most potent compound in the series with potentantimicrobial activity with MIC values of 0.095-0.39 (µg/mL) againstGram-positive bacteria including MRSA, improved selectivity up to200, rapid time-kill in 10-30mins, avoidance of antibiotic resistanceand good biocompatibity. Biophysical studies and molecular dynamicsimulations also revealed that 3b disrupted the bacterial membrane byforming an amphiphilic conformation with the cationic groupslocated at the hydrophobic-water interface. In contrast, conjugationmoieties with low pKa value to the xanthone scaffold diminished theantimicrobial activities.©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 – Physiology/PharmacologyConclusions: A series of novel antimicrobials have been designedand prepared by cationic modifications of α-mangostin, a naturalxanthone with a planar hydrophobic core, to yield an amphiphilicstructure which improves selectivity for bacterial membranes throughthe hydrophobic-water interface perturbation. This design strategy isimportant as we provide a new approach to modify naturalcompounds to yield excellent antimicrobial properties, highselectivity and safety. This strategy could improve “hits” in thedevelopment of new antibiotics for drug-resistant pathogens.Commercial Relationships: Shouping Liu, None; Hanxun Zou,None; Jun-Jie Koh, None; Jianguo Li, None; RajamaniLakshminarayanan, None; Roger W. Beuerman, Allergan (F),SERI (P), Santen (R)Support: NMRC/NIG/1010/2010, SingaporeProgram Number: 4298 Poster Board Number: C0036Presentation Time: 8:30 AM - 10:15 AMIn vitro analysis of the effect of steroid in combination withantimicrobial on co-cultures of bacteria and fungi isolated fromkeratitisHerlinda Mejia-Lopez 1 , Lucero Y. Martínez-López 2 , AlejandroClinent-Flores 1 , Aida V. Rodríguez-Tovar 2 , Luis A. Bautista-Hernández 1 , Victo M. Bautista de Lucio 1 , María A. Martínez-Rivera 2 .1 Research Unit, Inst of Ophthal "Conde de Valenciana", Mexico City,Mexico; 2 Laboratory of Medical Mycology, Department ofMicrobiology, National School of Biological Sciences, InstitutoPolitécnico Nacional, México, Mexico.Purpose: Some of major diseases affecting the cornea are keratitisand may be of various origins including microbial, Currently, as aresult of various risk factors such as frequent use of broad spectrumantibiotics and topical steroids, the self-medication, use of contactlenses, chronic systemic diseases, immunosuppression, among others,increased frequency of keratitis. Clinical manifestations depend onthe causative agent; however, misdiagnosis generates chronicity withpossibility of multiple treatments. The aim of this study was toanalyze, in vitro, the effect of steroids in combination withantimicrobial agents on the growth of bacteria and fungi isolatedfrom keratitis, as well as their co-cultures.Methods: Aspergillus fumigatus, Fusarium solani, Staphylococcusaureus and Staphylococcus epidermidis isolated from patients withkeratitis whom received multiple treatments, were studied. Minimuminhibitory concentration of amphotericin B and itraconazole andmoxifloxacin and gatifloxacin were determinated. The effect of thedexamethasone and prednisolone on fungal or bacterial growth,individually and in co-cultures fungus-bacteria was studied also.Experiments were performed six times and analyzed by the ANOVAtest to calculate the significance with respect to the untreatedcontrols.Results: The growth of Fusarium solani was inhibited by thepresence of prednisolone at 312.5 mg/mL (p ≤ 0.005). On thecontrary, the growth of Aspergillus fumigatus was increased inpresence of dexamethasone at 2.22 ug/ml and 570.0 mg/mLconcentrations (p ≤ 0.05). There was no effect of corticosteroids ongrowth of bacteria. Moxifloxacin reduced the growth of F. solanidose-dependent manner. The antifungal combination withcorticosteroids or quinolones favors the growth of fungi. S. aureus incoculture with A. fumigatus and prednisolone favored the growth ofthis fungus.Conclusions: The cortiesteroids has dose-dependent inhibition effecton the growth of F. solani and A. fumigatus; similarly moxifloxacinon F. oxysporum. Combination of corticosteroids-antifungal as wellas corticosteroids-quinolones favors growth of both fungi. In cocultureof S. aureus with A. fumigatus treated with prednisolone, thebacterium favored the growth of the fungus.Commercial Relationships: Herlinda Mejia-Lopez, None; LuceroY. Martínez-López, None; Alejandro Clinent-Flores, None; AidaV. Rodríguez-Tovar, None; Luis A. Bautista-Hernández, None;Victo M. Bautista de Lucio, None; María A. Martínez-Rivera,NoneSupport: Private Assistance Foundation "Conde de Valenciana"Program Number: 4299 Poster Board Number: C0037Presentation Time: 8:30 AM - 10:15 AMIontophoresis transcorneal delivery technique for transepithelialcorneal collagen crosslinking with riboflavin in a rabbit modelVincent J. Soler 1, 2 , Myriam Cassagne 1, 2 , Camille Laurent 3 , AnneGalinier 4 , Pierre R. Fournie 1, 2 , Stéphane Galiacy 1 , Pierre Roy 5 ,Francois J. Malecaze 1, 2 . 1 UMRS 563, CPTP, Universite PaulSabatier, Toulouse, France; 2 Ophthalmology, Toulouse PurpanHospital, Toulouse, France; 3 Laboratory of Pathology, ToulousePurpan Hospital, Toulouse, France; 4 Department of Biochemistry,Toulouse Rangueil Hospital, Toulouse, France; 5 Hexamed, Paris,France.Purpose: To evaluate a new iontophoresis transcorneal riboflavindelivery technique for transepithelial corneal collagen crosslinking(CXL).Methods: ANIMALS: A total of 108 eyes from New Zealand whitealbino rabbits were included in this study. Corneas after riboflavinapplication by iontophoresis (n=25) were compared with corneastreated by conventional riboflavin application after deepithelialization(n=16). Then I-CXL (n=18) was compared with C-CXL (n=9) 14 days after treatment. All these groups were comparedwith specific controls (n=40).METHODS: Iontophoresis involved the administration of a newformulation of charged riboflavin (Ricrolin® +) into cornea byapplying a current of 1mA for 5 min. C-CXL was performedaccording to the Dresden protocol.MAIN OUTCOMES: Riboflavin diffusion in the eyes wasinvestigated by two-photon microscopy in corneas and by highperformanceliquid chromatography (HPLC) in corneas and aqueoushumors. Stromal collagen structure modifications were analyzedusing second harmonic generation imaging (SHG).Riboflavin diffusion in corneas was analyzed by measuring riboflavinemission at 500-550 nm with a two-photon microscope on cornealsections and riboflavin concentration in corneas and aqueous humorswas measured by HPLC.Corneal stromal modifications were evaluated by analyzing thecollagen fiber organization through SHG using a two-photonmicroscope.Results: Images from two-photon microscope showed that afteriontophoresis, riboflavin fluorescence and its diffusion throughoutcornea were similar to that observed in a conventional application. Inparallel, iontophoresis showed no more toxicity in the cornealepithelium than conventional application of riboflavin.Using HPLC, the corneal concentration of riboflavin was two-foldless after iontophoresis than after conventional application (936.2 ±312.5 ng/ml and 1708 ± 908.3 ng/ml, respectively, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Vincent J. Soler, None; MyriamCassagne, None; Camille Laurent, None; Anne Galinier, None;Pierre R. Fournie, None; Stéphane Galiacy, None; Pierre Roy,Hexamed (P); Francois J. Malecaze, NoneProgram Number: 4300 Poster Board Number: C0038Presentation Time: 8:30 AM - 10:15 AMComparative Analysis of In Vitro Biocompatibility Assays ofPolyhexamethylene Biguanide Disinfecting Contact Lens Multi-Purpose SolutionsMercedes Salvador-Silva 1 , Ling C. Huang 1 , James Cook 2 . 1 BiologyR&D, Abbott Medical Optics (AMO), Santa Ana, CA; 2 CornealProduct Development R&D, Abbott Medical Optics (AMO), SantaAna, CA.Purpose: This study evaluated the effects of polyhexamethylenebiguanide (PHMB) contact lens multi-purpose (MPS) solution on cellcytotoxicity, metabolic activity, membrane integrity, andbiocompatibility.Methods: Five MPS were used - MPS-1: polyhexamethylenebiguanide (PHMB) + poloxamer (PLX), MPS-2: PHMB + PLX,MPS-3: PHMB + poloxamine (PLA), MPS-4: PHMB +polyquaternium (PQ1) + PLA, and MPS-5: PHMB + PLX. In vitrobiocompatibility was assessed according to ISO 10993. MPS effecton colony formation rate was evaluated in V79 Chinese HamsterLung Fibroblast for 7 days. MPS were evaluated at 1.25%, 2.5%, 5%and 10% as diluted in cultured medium. Cytotoxicity and metabolicactivity were determined using alamarBlue dye. Corneal epithelialbarrier function was assessed by Zonula Occludens (ZO-1)immunohistochemistry (IHC) and trans-epithelial electrical resistance(TEER) in Simian virus transformed human corneal epithelial cellline (SV40 HCEC).Results: MPS-1 formulation was comparable to MPS-2 at 1.25% and2.5% and MPS-3, MPS-4, and MPS-5 at 5% and 10% in cellcytotoxicity. Treatment with 1ppm PHMB alone did not induce astatistically significant inhibition on V79 colony formation (n=3,p>0.05). MPS-1 at 50% and 75% was comparable to MPS-2, MPS-3,MPS-4, and better than MPS-5 at 100% in corneal barrier integrity asevaluated by ZO-1 IHC. Exposure to MPS-1 for 120 mins at 50%concentration was superior to MPS-3, MPS-4, and MPS-5 and similarto MPS-2 on maintaining SV40 HCEC viability as assessed byalamarBlue. TEER of SV40 HCEC showed that exposure to MPS-1for 120 mins at 50% concentration was better than MPS-3 but equalto MPS-2, MPS-4, and MPS-5. MPS-1 for 120 mins at 75%concentration was similar to MPS-5 and superior to MPS-3 and MPS-4 to maintain corneal cell membrane integrity (n=3-4, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologythe CRD method can detect disorder that cannot be evaluated byfluorescein staining. There was a significant reduction in the cellproliferation rate (%) in the LVFX and MFLX groups as comparedwith the control group (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology90 (respectively, 13.3+0.94 vs 10.8+1.31 at day30 and 13.7+1.05 vs11.1+1.52 at day90, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyPresentation Time: 11:00 AM - 12:45 PMSubfoveal Choroidal blood flow in senescenceJohn V. Lovasik, Helene Kergoat, Mireille Parent. School ofOptometry, University of Montreal, Montreal, QC, Canada.Purpose: The sub-foveal choroid is the sole blood supply for thecone photoreceptors within the foveal avascular zone (FAZ). Areduction in the sub-foveal choroidal blood flow (ChBF) has beenreported before the onset of age-related macular degeneration,suggesting a cause-effect relationship. However, a reduction in ChBFcan also be the consequence of a sub-clinical retinopathy in the FAZ,or increasing age per se. Thus, the interdependence among the ChBFand the structural integrity and function of the retina in the FAZremains poorly defined. In the present study we quantified the effectsof senescence on choroidal hemodynamics and its relation to centralvisual function.Methods: 20 healthy volunteers for each decade of life between 20and 80 years of age participated in the study. The best correctedSnellen visual acuity (VA) was recorded during a complete eye examfor each subject. A confocal near infrared (780 nm) Laser DopplerFlowmeter was used to record ChBF (AU), velocity (ChBVel-kHz),and volume (ChBVol-AU) in the choriocapillaris within the FAZ at25 Hz. Each subject was directed to fixate the center of the laserprobe that appeared as a small dim red spot of light. Foveation of thelaser spot was continued until a 10-20 sec interval with a constant DCoutput (indicating steady fixation) was obtained. The correspondingrecords of ChBF, ChBVel, ChBVol were cleaned of blink artifactsfor each subject and then averaged over time. The mean values for allsubjects were graphed as a function of subject age and a linearregression line drawn through each parameter to glean the effects ofage on choroidal hemodynamics. An alpha value of 0.05 was used forstatistical significance.Results: ChBVel decreased (r= -0.223, p= 0.02) while ChBVolincreased (r= 0.222, p= 0.02) with age. In contrast, the ChBFremained constant with age (r= 0.005, p> 0.05). The group averageChBF across all age groups, Mean= 0.957 ± SD= 0.405, wasassociated with a central VA of 0.9 minarc ± SD= 0.14 across allsubjects.Conclusions: An acuity of 20/20 or better can be maintained inseptuagenarians. The ChBF in the FAZ does not change between 20to 80 years of age in healthy individuals. Because the ChBF varied byalmost 100% within the 6 age groups studied, singular measurementsof the ChBF cannot distinguish patients differing by just one or twolines from the standard 20/20.Commercial Relationships: John V. Lovasik, None; HeleneKergoat, None; Mireille Parent, NoneSupport: CFI, NSERC, CIHRProgram Number: 4629 Poster Board Number: B0093Presentation Time: 11:00 AM - 12:45 PMTime-dependent intracellular pattern of Bevacizumab in RPEcellsShereen Hassan M. Aboul Naga 1, 2 , Michaela Dithmer 1 , JohannRoider 1 , Alexa K. Klettner 1 . 1 Ophthalmology, University of Kiel,University Medical Centre, Kiel, Germany; 2 Ophthalmology, Kasr AlAini, Cairo University Hospitals, Cairo, Egypt.Purpose: Bevacizumab is taken up into RPE cells and isintracellularly found for at least seven days. In this study, weinvestigate Bevacizumab uptake and intracellular localisation ofBevacizumab at different time points in RPE cells.Methods: For this study, RPE cell line Arpe19 and primary porcineRPE cells, passage 2, were used. Cells were treated once withBevacizumab and intracellular Bevacizumab was investigated aftervarious time periods (1 h - 7 d). For the detection of intracellularBevacizumab, Western blot and immunofluorescence was utilized.For intracellular localization, antibodies against Rab5 (earlyendosome), Rab7 (late endosome), Lamp2 (lysosome) andmicrotubuli were used. Actin was stained using Phalloidin.Results: Bevacizumab is abundantly found in porcine RPE cells andArpe19 cells as detected in immunofluorescence and Western blot.After 1 h and 4 h of stimulation, Bevacizumab is primarily locatedclose to the cell membrane and can partly be found in close proximityto or colocalizing with Rab 5, indicating that sections ofBevacizumab are taken up into early endosomes. During 1 day to 5days after Bevacizumab challenge, intracellular Bevacizumabdisplays a net-like pattern. After 7 d of Bevacizumab challenge, thepattern intracellular Bevacizumab displays becomes more diffuse.Colocalization with microtubule can hardly be seen, whileBevacizumab is found in close proximity to or colocalizing with actinfilaments, suggesting an actin-mediated intracellular transport.Colocalization with Lamp2 is rarely found, suggesting thatBevacizumab is not degraded in lysosomes.Conclusions: Bevacizumab displays a distinct, time dependentlocalization in RPE cells. The pattern suggests a controlledintracellular transport via actin filaments. Taken-up Bevacizumabdoes not seem to be transported into the lysosomal pathway and doesnot seem to be intracellularly degraded.Commercial Relationships: Shereen Hassan M. Aboul Naga,None; Michaela Dithmer, None; Johann Roider, Novartis (F),Bayer (F); Alexa K. Klettner, Novartis (F), Novartis (C), Novartis(R), Santen (R)Support: DFG KL 2425/2-1, DAAD (German Academic ExchangeService)Program Number: 4630 Poster Board Number: B0094Presentation Time: 11:00 AM - 12:45 PMAntagonism of PDGFRβ Inhibits Pericyte Recruitment in aMouse Model of Corneal NeovascularizationAmy Jensen 1 , Rosemarie Cepeda 1 , Michael Maker 1 , Chad E.Bigelow 1 , Joy Ghosh 1 , Guochun Li 1 , Patricia A. D'Amore 2 , GuntherSpohn 1 , Bruce D. Jaffee 1 , Sassan Azarian 1 . 1 Ophthalmology,Novartis, Cambridge, MA; 2 Ophthalmology, Schepens Eye ResearchInstitute, Boston, MA.Purpose: To explore the pathobiology of pericytes in ocular diseasessuch as diabetic retinopathy, in which pericyte loss is a hallmark.Pericytes are abluminally associated with the capillary endotheliumand contribute to vessel formation, stabilization, and maturation. Toexamine the role of PDGF in these functions, we inhibited the PDGFpathway in a mouse model of corneal neovascularization (CoNV).Methods: CoNV was induced in anesthetized C57BL/6 mice bymechanical abrasion of corneas. Animals were treated every otherday, with control antibodies or with PDGFRβ and VEGF neutralizingantibodies administered IP. To quantify pericytes andneovascularization (NV), corneal flat mounts were stained with α-NG2 and α-PECAM1 antibodies, to detect pericytes and endothelialcells respectively, and imaged by fluorescence microscopy. Pixel areawas measured using a program written in MatLab, and pericytecoverage (%) was calculated as NG2-labeled pixels/PECAM1-labeled pixels*100. NV area was measured using AxioVisionsoftware. Vessel morphology was assessed using AngioTool.Results: In unabraded mice, pericytes were associated only withperilimbal vessels as there were no corneal vessels. In the newvessels observed in the abraded mice there was an increase inpericyte area (6.2-fold), vessel area (3.6-fold), and pericyte coverage(1.6-fold) relative to normal limbal vessels in unabraded controls.Neutralization of PDGFRβ dramatically reduced pericyte coveragebut had no effect on vessel area or morphology. α-VEGF treatment©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 – Physiology/Pharmacologyresulted in reduced vessel area and altered vessel morphology.Neutralizing both PDGFRβ and VEGF did not elicit any additionaleffects on pericyte area, vessel area or morphology compared toneutralizing VEGF alone.Conclusions: Corneal abrasion resulted in NV which could beinhibited with an antibody against VEGF. The new vessels wereaccompanied by an increase in pericyte area and coverage, whichrepresents recruitment of new pericytes and can be blocked withsystemic α-PDGFRβ treatment. This model allows visualization ofboth existing and newly recruited pericytes and appears to be asuitable model for studying pericytes.Commercial Relationships: Amy Jensen, Novartis (E), Novartis(F); Rosemarie Cepeda, Novartis Institute for Biomedical ResearchInc. (F), Novartis Institute for Biomedical Research Inc. (E); MichaelMaker, Novartis Institutes for Biomedical Research (E); Chad E.Bigelow, Novartis (E); Joy Ghosh, Novartis (E); Guochun Li,Novartis (E); Patricia A. D'Amore, Valeant (C); Gunther Spohn,Novartis (E); Bruce D. Jaffee, Novartis (E); Sassan Azarian,Novartis (E)Program Number: 4631 Poster Board Number: B0095Presentation Time: 11:00 AM - 12:45 PMDynamic retinal venous oscillations are changed in diabetesmellitus type 1Konstantin E. Kotliar 1, 2 , Ines M. Lanzl 2 , Thorsten Siegmund 4 , ArnoSchmidt-Trucksaess 5, 3 . 1 Biomedical Engineering, Aachen Universityof Applied Sciences, Juelich, Germany; 2 Ophthalmology, MunichUniversity of Technology, Munich, Germany; 3 Preventive SportsMedicine, Munich University of Technology, Munich, Germany;4 Endocrinology and Diabetes, Bogenhausen Hospital, Munich,Germany; 5 Division of Sports Medicine, University of Basel, Basel,Switzerland.Purpose: We demonstrated previously that non-stimulated temporalretinal arterial and venous oscillations (pulsations and vasomotions)are changed in healthy volunteers with age and in patients withprimary open angle glaucoma. Whether this dynamic retinal vesselbehavior is altered in diabetes mellitus type 1 (DM1) is investigated.Methods: 33 untreated patients with DM1 (age 51.7±8.3 years) withno or non-proliferative retinopathy and 33 age and sex matchedmedically healthy volunteers were examined by Dynamic VesselAnalyzer (DVA, IMEDOS, Jena, Germany). Temporal changes ofvessel diameters of retinal arterial and venous vessel segments wereexamined with DVA in all subjects during 40 seconds. Oscillatorytemporal changes of vessel diameter were divided into high-frequent(period < 1,5 s) and low-frequent (period ≥ 1,5 s) and were evaluatedusing methods of mathematical signal analysis.Results: Quantitative parameters characterizing dynamic retinalvessel behavior did not show any differences in retinal arterialoscillations between DM1 patients and healthy control subjects. Inveins there was a significant difference in the rate of periodicity ofhigh-frequency (control: 0.08(0.06; 0.17), DM1: 0.12(0.08; 0.23)[median (1.quartile; 3. quartile)], p < 0.05) as well as of lowfrequencyoscillations (control: 0.16(0.10; 0.24), DM1: 0.62(0.49;1.23), p < 0.001) between the groups.Conclusions: Functional and morphological alterations in the retinalveins in DM1 with no or mild non-proliferative retinopathy areshown using a non-invasive in-vivo methodology. Changed retinalvenous behavior in the initial stages of diabetes mellitus might be anindication for alterations in the vascular endothelium and blood flowregulation in this disease.Commercial Relationships: Konstantin E. Kotliar, None; Ines M.Lanzl, allergan (C), alcon (R), pfizer (R), santen (R), novartis (R);Thorsten Siegmund, None; Arno Schmidt-Trucksaess, NoneProgram Number: 4632 Poster Board Number: B0096Presentation Time: 11:00 AM - 12:45 PMParameters related to Choroidal thicknessYasuki Ito 1 , Kazuhiro Oiwa 1 , Eiji Iwata 1 , Akiko Takahashi 1 ,Tetsuhiro Yasuma 1 , Kenichi Kawano 1 , Nobuyuki Hamajima 2 , HirokoTerasaki 1 . 1 Ophthalmology, Nagoya University Graduate School ofMedicine,, Nagoya, Japan; 2 Department of Preventive Medicine,Nagoya University Graduate School of Medicine,, Nagoya, Japan.Purpose: The choroidal thickness has been reported to besignificantly correlated with age and axial length. In addition, thechoroid in eyes with central serous chorioretinopathy (CSC) has beenreported to be thicker. Because smoking is a risk factor of CSC, wetested the hypothesis that the choroid will be thicker in smokers. Wealso examined whether the choroidal thickness was affected by bodyweight.Methods: A total of 495 subjects (197 men, 298 women) who were≥40-years-of-age and were in the Comprehensive HealthExamination Program (Yakumo Study) in 2011 were studied. Allwere examined by optical coherence tomography, fundusphotography, and axial length measurements. The choroidal thicknesswas measured in the OCT images, and the relationship with theocular parameters, bodyweight, sex, and smoking were determined.Results: The average choroidal thickness was 222.3 ± 90.7 µm. Thechoroidal thickness was significantly correlated with age (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyor TUNEL staining. Inflammation was investigated by counting thenumber of leukocyte adherent to the blood vessels (or retinalleukostasis). The expression of inflammatory and BRB makers wasdetermined by real-time quantitative PCR and/or immunofluorescentstaining.Results: Vascular leakage was significantly increased in Akitadiabetic mice but was not in the Akita.PGFKO diabetic micecompared to their respective non-diabetic littermates. Retinalcapillary degeneration was significantly increased in the Akitadiabetic mice as compared with the wild type (WT) non-diabeticmice or the Akita.PGFKO diabetic mice. Apoptotic cell death wassignificantly increased in the Akita diabetic mice as compared withthe Akita.PGFKO diabetic mice. Similarly, blockade of VEGFR1 byMF1 significantly inhibited diabetes-caused BRB breakdown, cellapoptosis and retinal leukostasis. Furthermore, in diabetic conditions,the expression of inflammatory markers, such as ICAM-1 and IL-1αwere significantly suppressed by blockade of PGF/VEGFR1signaling. In contrast, the expression of BRB markers, such as E-cadherin and ZO-1, were significantly elevated due to its blockade.Conclusions: The results suggested PGF/VEGFR1 signaling isinvolved in the adverse complications during DR and is a potentialtarget in the treatment of diabetes complications, such as diabeticmacular edema.Commercial Relationships: Hu Huang, None; Da'KuawnJohnson, None; Dorothy Kim, None; Gerard A. Lutty, NoneSupport: NIH Grant EY017164Program Number: 4634 Poster Board Number: B0098Presentation Time: 11:00 AM - 12:45 PMComputational Model of Oxygen Transport in Retina and OpticNerveDavid Bragason 1 , Einar Stefánsson 1, 2 . 1 Ophthalmology, LandspitaliUniversity Hospital, Reykjavik, Iceland; 2 Medicine, University ofIceland, Reykjavik, Iceland.Purpose: In order to increase our understanding of oxygen saturationpatterns observed in retinal vessels, we propose a computationalmodel to describe oxygen transport and its dependence on local,systemic and extrinsic factors, such as vessel width, blood flow,illumination and oxygen supplementation.Methods: A mathematical model of arteriole-venule pairs andsurrounding tissue was developed. Coupled non-linear partialdifferential equations describing convection, diffusion and interactionof oxygen with hemoglobin and oxygen-consuming tissue are solvednumerically. Taking into account non-uniform blood-flow andhematocrit profiles, longitudinal and radial oxygen saturation andpartial pressure gradients are calculated. The results are comparedwith measurements obtained with the Oxymap retinal oximeter(Oxymap ehf., Reykjavik, Iceland), as well as with data previouslypublished by researchers using other methods.Results: Oxygen saturation gradients along major retinal vesselsreflect oxygen consumption of perivascular tissue, with a smallercomponent due to countercurrent exchange between closely spacedvessels. Our model predicts longitudinal saturation gradientsconsistent with those measured in retinal oximetry. Oxygenpenetrates by diffusion into a perivascular tissue layer comparable inthickness to the capillary-free zone. A gradient of 1 - 4 % insaturation is predicted in central retinal vessels along the optic nerve.The model predicts reduced oxygen saturation with decreased bloodflow. It helps explain a distribution width of 5 - 10 % in saturation inshort segments of retinal vessels and the variability in saturationobserved between normal eyes. According to the model, the 3 %increase in retinal arteriolar saturation observed in darkness can beaccounted for by increased blood flow in the dark. Diffusion currentsof oxygen in vitreous close to major retinal arterioles are predicted tobe approx. 10 -6 ml O 2 /cm 2 /sec, compatible with previously publishedresults, obtained with polarographic methods.Conclusions: Our model predicts retinal vessel oxygen saturationpatterns that are consistent with those observed in retinal oximetryand helps us gain a quantitative understanding of some aspects ofoxygen transport in the retina and optic nerve.Saturation profile in central retinal vessels along 10 mm in opticnerveCommercial Relationships: David Bragason, None; EinarStefánsson, Oxymap ehf (P), Oxymap ehf (I), Oculis ehf (P), Oculisehf (I), Risk ehf (I), Acta Ophthalmologica (E)Support: Helga Jonsdottir and Sigurlidi Kristjansson Memorial FundProgram Number: 4635 Poster Board Number: B0099Presentation Time: 11:00 AM - 12:45 PMIncreased Retinal Vascular Tortuosity in Obstructive SleepApneaAmir Mohsenin 1 , Vahid Mohsenin 2 , Ron A. Adelman 1 . 1 Department ofOphthalmology, Yale University School of Med, New Haven, CT;2 Yale Center for Sleep Medicine, Yale University School ofMedicine, New Haven, CT.Purpose: Obstructive sleep apnea (OSA) is a highly prevalentdisorder with significant vascular morbidity and mortality. Affectedpatients are subjected to intermittent hypoxemia, hypercapnia, arterialblood pressure surges and increased intracranial pressure duringsleep. We sought to examine the effects of OSA on retinalvasculature tortuosity.Methods: A pilot retrospective chart review was conductedidentifying patients with and without OSA who had undergonefundus photography. 7 control subjects and 9 subjects with OSA wereanalyzed. Measurements were taken of the superior and inferiortemporal retinal artery and vein starting at the optic disc rim to thecrossing point of 2 circles centered on the optic disc with diametersof 5 disc diameters (DD) and 10DD. Retinal vascular tortuosity (tau)was assessed by calculating the arc length/chord length of each vesselsegment as per previously published methods.Results: Patients with OSA have significant increases in arterial andvenous tortuosity when measured at the 10DD length. At 5DD, therewas no statistically sig difference in arterial tortuosity between thetwo groups. Venular tortuosity was significantly increased at the 5DDmark in patients with OSA.Conclusions: These findings demonstrate an association betweenobstructive sleep apnea and increased retinal vessel tortuosity. Alarger study will be necessary to examine the prevalence andsignificance of this vascular abnormality in OSA.Commercial Relationships: Amir Mohsenin, None; VahidMohsenin, None; Ron A. Adelman, 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 – Physiology/PharmacologySupport: Leir Foundation. Newman's Own Foundation.Program Number: 4636 Poster Board Number: B0100Presentation Time: 11:00 AM - 12:45 PMOxygen saturation in retinal hemorrhagesOlafur Palsson 1, 2 , Sveinn H. Hardarson 1, 2 , Thorunn S. Eliasdottir 2 ,Einar Stefánsson 1, 2 . 1 Department of Ophthalmology, LandspítaliUniversity Hospital, Reykjavik, Iceland; 2 University of Iceland,Reykjavik, Iceland.Purpose: To develop and test a method to measure oxygen saturationof extravasated hemoglobin in retinal tissue in disorders such ascentral retinal vein occlusion (CRVO).Methods: The spectrophotometric retinal oximeter (Oxymap ehf,Reykjavik, Iceland) simultaneously delivers two monochromaticimages of the same area of the fundus, one which is is sensitive tooxygen saturation (600nm) and one which is not (570nm). The lightabsorbance of retinal hemorrhages was measured at these twowavelengths in 7 CRVO patients. The light absorbance was used toestimate oxygen saturation in the hemorrhages. Variability betweenmeasurements of areas within the same retinal hemorrhage wasdetermined. The mean oxygen saturation in retinal vessels wasmeasured with automated software.Results: The mean saturation in the retinal hemorrhages was58±18% (mean±SD, n=7). The mean saturation in retinal venules inthe same patients was 33±14% (p=0.03). Standard deviation betweenrepeated measurements on the same hemorrhage in the same imagewas 14.6% (saturation percentage).Conclusions: The results suggest that measurement of oxygensaturation of extravascular hemoglobin in retinal tissues is possible.Saturation values in hemorrhages are between saturation values inarterioles and venules. The oxygen saturation can be correlated toPO2 and therefore might be an indicator of the oxygenation of thetissue.Commercial Relationships: Olafur Palsson, None; Sveinn H.Hardarson, Oxymap ehf. (F), Oxymap ehf. (I), Oxymap ehf. (C),Oxymap ehf. (R), Patent no. 7774036 (P), Patent application13/377,749 (P), Optos plc. (F); Thorunn S. Eliasdottir, None; EinarStefánsson, Oxymap ehf (P), Oxymap ehf (I), Oculis ehf (P), Oculisehf (I), Risk ehf (I), Acta Ophthalmologica (E)Support: The Icelandic Center for Research (Rannís), TheUniversity of Iceland Research Fund, The Landspítali UniversityHospital Research FundProgram Number: 4637 Poster Board Number: B0101Presentation Time: 11:00 AM - 12:45 PMAssessment of Total Retinal Blood Flow under SystemicHypercapnia and HypocapniaAyda M. Shahidi 1 , Sunni R. Patel 1 , John G. Flanagan 1, 3 , Ou Tan 2 ,David Huang 2 , Christopher Hudson 1, 3 . 1 Ophthalmology & VisionScience, University Health Network, Toronto, ON, Canada;2 Ophthalmology, Oregon Health and Science University, Portland,OR; 3 Optometry & Vision Science, University of Waterloo,Waterloo, ON, Canada.Purpose: To investigate the effect of change in systemic partialpressure of CO2 on total retinal blood flow (TRBF) as measured byDoppler Fourier-domain optical coherence tomography (OCT)Methods: TRBF scans were captured in nine healthy individuals(mean age ± standard deviation: 27±4, 6 males) using the RTVueOCT double ring blood flow protocol. Measurements were capturedduring homeostatic PETCO2 levels, hypercapnia (+5/+10/+15 mmHgPETCO2), back to baseline and hypocapnia (-5/-10/-15 mmHgPETCO2) using a custom-designed computer controlled gas blender(RespirAct) with a sequential gas delivery rebreathing system. Theorder for hyper- and hypo-capnia conditions was randomized.Repeated measure analysis of variance (reANOVA) and Tukey’spost-hoc analysis were used to compare Doppler OCT measurementsamongst breathing conditions. The effect of end-tidal CO2 on theoutcomes was investigated using regression models.Results: TRBF (45.9 ± 10.9, vs 60.9 ± 12.1 µl/min), superior RBF(24.0 ± 9.3 vs 33.8 ± 9.5 µl/min) , arterial (14.1 ± 2.7 vs 19.9 ± 4.5mm/s) and venous (12.5 ± 1.7 vs 15.6 ± 1.3 mm/s) velocities weresignificantly different between baseline measurements and extremehypercapnia (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyon ETDRS chart as opposed to a combined treatment with selectivelaser photocoagulation. A larger sample and further follow-up areneeded.Commercial Relationships: Blanca C. Flores, None; Victor H.Gonzalez, Genetech (C), Regeneron (C), Pfizer (C), Valiant (C),Alimera (C); Roberto Diaz-Rohena, NoneClinical Trial: NCT01486771Program Number: 4639 Poster Board Number: B0103Presentation Time: 11:00 AM - 12:45 PMPersistent hyaloid vessels counteract insufficient retinal perfusionin the mouse eyeChristina Seide, Marina Garcia Garrido, Vithiyanjali Sothilingam,Naoyuki Tanimoto, Susanne C. Beck, Mathias W. Seeliger. Div ofOcular Neurodegeneration, Ctr for Ophthal Inst for Ophth Rsrch,Tuebingen, Germany.Purpose: Persistence of embryonic vitreal vasculature beyond thetime of its physiologic regression during ocular development wasobserved in a number of mouse models where retinal vascularizationis impaired. Here, we evaluate the nature and distribution ofpersistent hyaloid vessels in relation to the topography of retinalperfusion defectsMethods: In vivo imaging was performed with a HeidelbergEngineering HRA I using native confocal scanning-laserophthalmoscopy (cSLO) as well as fluorescein (FA) and indocyaninegreen angiography (ICGA). Mouse models with insufficiencies inretinal vascularization already starting during development wereselected for this study. The setting of different confocal planesallowed a selective analysis of the distribution of hyaloid and retinalsurface vasculature. The topography of vascular alterations of retinalvessels was then correlated with the distribution of vitreal vascularpersistence.Results: The combination of FA and ICGA revealed a detailedoverview of the distribution of retinal vessels and their state ofperfusion. Retinal regions with a reduced or lacking circulation couldoften be identified via a leakage of fluorescein indicating enhancedvascular ultrafiltration due to VEGF upregulation in dependenttissues. In all cases studied, areas of insufficient retinal perfusioncolocalized with persistent hyaloid vasculature. The strongestinhibition of hyaloid vessel regression was present in mice with atotal lack of parts of the retinal vasculature.Conclusions: The physiology of the regression of vitreal vesselsduring development is not entirely understood. Some aspects suggesta more or less fixed time window for this process, in particular withrespect to the apoptotic death of vascular cells and their removal fromthe vitreous. On the other hand, we show here that insufficient retinalperfusion, presumably via factors like VEGF, clearly leads to a localpersistence of hyaloid vasculature, which in turn improves thecompromised perfusion at least for some time. This finding may helpto develop symptomatic therapeutic concepts in respective diseaseslike ROP.Commercial Relationships: Christina Seide, None; MarinaGarcia Garrido, None; Vithiyanjali Sothilingam, None; NaoyukiTanimoto, None; Susanne C. Beck, None; Mathias W. Seeliger,NoneSupport: DFG Se837/6-2; GIF 1127-155.2/2010Program Number: 4640 Poster Board Number: B0104Presentation Time: 11:00 AM - 12:45 PMRetinal Vessel Diameter at High AltitudeGabriel Willmann 1 , Andreas Schatz 1 , M Dominik Fischer 1, 3 , KaiSchommer 2 , Eberhart Zrenner 1 , Karl-Ulrich Bartz-Schmidt 1 , FlorianGekeler 1 . 1 Centre for Ophthalmology, University of Tübingen,Tübingen, Germany; 2 Department of Sports Medicine, UniversityHospital Heidelberg, Heidelberg, Germany; 3 Nuffield Laboratory ofOphthalmology, University of Oxford, Oxford, United Kingdom.Purpose: This study aimed to quantify the impact of acute highaltitude exposure on retinal vessel diameter and to assess possiblecorrelations to symptoms of acute mountain sickness (AMS) and highaltitude headache (HAH). This work is related to the Tuebingen HighAltitude Ophthalmology (THAO) study.Methods: VesselMap 1 analyzer (Imedos Systems, Germany) wasused to quantify changes of retinal vessel diameter within one diopterdistance of the papilla in 18 healthy subjects during acute highaltitude exposure to 4559 m compared to baseline recordings (341 m)using infrared fundus images obtained from a Spectralis® device(Heidelberg Engineering, Germany). Intra-individual differenceswere calculated using ANOVA with a significance level of p < 0.05.Pearson’s correlation was used to assess a possible linkage betweenretinal vessel diameter and scores of AMS and HAH.Results: Analysis of intra-individual differences revealed asignificant (p < 0.05) increase of mean arterial (MAD; increasedMADaltitude = 13.6 μm) and venous diameter (MVD; increasedMVDaltitude = 26.7 μm) at high altitude in healthy subjectscompared to baseline recordings. Average arterial and vein diametersat baseline and high altitude were: MADbaseline = 122.72±14.78 μmvs. MADaltitude = 136.36±19.84 μm; MVDbaseline = 148.02±15.32μm vs. MVDaltitude = 171.74±22.09 μm; mean±sd) Changes werecompletely reversible upon descend. Pearson’s coefficient showedneither a correlation between increased retinal vessel diameter andAMS (MAD vs. AMS-c score: r = 0.02, p = 0.95; MVD vs. AMS-cscore: r = -0.17, p = 0.51) nor with HAH (MAD vs. headacheAMS-cscore: r = -0.17, p = 0.50; MVD vs. headacheAMS-c score: r = -0.10,p = 0.71).Conclusions: A significant increase in central retinal vessels for botharteries and veins occurs in response to acute exposure to highaltitude in healthy subjects. This may be attributed to the physiologicresponse to the effects of hypoxia during acute high altitude exposurein non-acclimatized subjects. The missing correlation of retinal vesseldiameter and symptoms of AMS or HAH is of special interest asrestricted cerebral and retinal venous outflow is currently debated tobe associated with a greater headache burden in response to highaltitude hypoxia. Our findings may provide a novel basis for thisdebate.Commercial Relationships: Gabriel Willmann, None; AndreasSchatz, None; M Dominik Fischer, None; Kai Schommer, None;Eberhart Zrenner, Retina Implant AG (F), Retina Implant AG (I),Retina Implant AG (C), Retina Implant AG (P), QLT Inc (C),Servier, Paris (C), Steinbeis GmbH&CoKG, Stuttgart (I), SteinbeisGmbH&CoKG, Stuttgart (C), Neurotech, USA (C), Pfizer, USA (C);Karl-Ulrich Bartz-Schmidt, Retina Implant (P); Florian Gekeler,Retina Implant AG (F), Okuvision GmbH (F), Retina Implant AG(C), Retina Implant AG (P)Program Number: 4641 Poster Board Number: B0105Presentation Time: 11:00 AM - 12:45 PMRetinal and Cerebral Oxygenation Response to Graded HypoxiaSunni R. Patel 1 , Christopher Hudson 2, 3 , Ayda M. Shahidi 1 , SusithKulasekara 2 , Joseph Fisher 4 , John G. Flanagan 2, 3 , W Alan Mutch 5 .1 Ophthalmology and Vision Sciences, Toronto Western Hospital,Toronto, ON, Canada; 2 Department of Ophthalmology and VisionScience, University of Toronto, Toronto, ON, Canada; 3 School ofOptometry and Vision Science, University of Waterloo, Toronto, ON,Canada; 4 School of Physiology, University of Toronto, Toronto, ON,Canada; 5 Perioperative Medicine and Anaesthesiology, University ofManitoba, Winnepeg, MB, 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 – Physiology/PharmacologyPurpose: To determine whether major retinal vessel oxygensaturation (SO2) and frontal cortex tissue oxygenation (StO2)changes with controlled manipulation of inhaled oxygen (ETO2)occur alongside one another.Methods: Non-invasive hyperspectral retinal imaging (Photon HRC,Photon etc, QC, Canada) and cortical near-infrared spectroscopy(FORE-SIGHT Cerebral Oximeter, CasMed, CT, USA)measurements were acquired in 11 healthy volunteers (29±4 yrs,8M). Continuous readings were acquired during normoxia and gradedhypoxic conditions (i.e., end-tidal [ET] O2 80mmHg, 60mmHg and50mmHg), whilst end-tidal CO2 tensions were stabilized athomeostatic baseline values, using a computer-controlled sequentialgas blender system (RespirAct, Thornhill Research, ON, Canada).Results: There was a marked decrease in retinal artery SO2(Friedman analysis; p=0.006) and cerebral StO2 (p=0.001) duringhypoxia which was most pronounced during ETO2 50mmHg (95±8%vs. 77±10% and 70±3% vs. 61±3%).Conclusions: This novel preliminary study demonstrated asignificant decrease in SO2 and StO2 of healthy individuals thatoccur concurrently during controlled isocapnic hypoxia.Commercial Relationships: Sunni R. Patel, None; ChristopherHudson, Opovue Inc (F); Ayda M. Shahidi, None; SusithKulasekara, None; Joseph Fisher, Thornhill Research Inc. (I),thornhill Research Inc (E); John G. Flanagan, HeidelbergEngineering (C), Heidelberg Engineering (R), HeidlebergEngineering (F), Carl Zeiss Meditec (C), Carl Zeiss Meditiec (R),Carl Zeiss Meditiec (R), Alcon Pharmaceuticals (R), AlconPharmaceuticals (R), Optovue Inc (F), Optovue Inc (F), Photon etc(F), Photon etc (F); W Alan Mutch, NoneSupport: ORF - REProgram Number: 4642 Poster Board Number: B0106Presentation Time: 11:00 AM - 12:45 PM3-D Computer-Automated Threshold Amsler Grid to QuantifyRetinal Deficits Before and After Standard Treatment of WetAge-related Macular DegenerationKristie Lin 1 , Wolfgang Fink 2, 3 , Sami Kamjoo 1 , Michael Davis 1 , TomChang 1 . 1 Retina Institute of California, Arcadia, CA; 2 Physics,Mathematics and Astronomy, California Institute of Technology,Pasadena, CA; 3 Biomedical Engineering, University of Arizona,Tuscon, AZ.Purpose: To characterize and quantify disease activity and responseto treatment of wet Age-related Macular Degeneration (AMD) withstandard intravitreal anti-VEGF agents using 3-D Computer-Automated Threshold Amsler Grid (3D-CTAG) as a functionalassessment of macular function over time.Methods: 10 eyes in 10 patients with wet AMD underwentquantification of central field abnormalities with 3D-CTAG (Fink &Sadun, JBO 2004) before and one week after treatment with standardintravitreal anti-VEGF agents including bevacizumab (Avastin,Genentech), ranibizumab (Lucentis, Genentech), and aflibercept(Eyelea, Regeneron). Quantitative analysis of 3D visual fieldabnormalities included: Lost Area Grade (LAG = scotoma area ratioas a function of contrast sensitivity), Preserved Area Grade (PAG =intact visual field area ratio as a function of contrast sensitivity), andHill-of-Vision Volume Loss (HVL = ratio of not seen vs. totalnumber of Amsler grid points).Results: Wet AMD patients exhibited a central defect at low vs. alarger central defect at higher contrast sensitivity levels. Wedemonstrated the quantification and improvement of macularstructure and function using 3D-CTAG before and after standardtreatment of wet AMD: LAG, PAG, and HVL indices showedmarked improvement following treatment, which correlated withregression of the choroidal neovascular membrane complex after useof standard anti-VEGF agents. Overall, 3D-CTAG provided easyclinical access to measuring and assessing the functional health of theretina in vivo to quantify change over time.Conclusions: 3D-CTAG is a direct, non-invasive, and easy-to-usefunctional retinal imaging technology. It allows characterization andquantification of longitudinal changes in macular function before andafter treatment of patients with wet AMD. 3D-CTAG may proveuseful as a screening tool in wet AMD and other central maculardiseases like geographic atrophy due to dry AMD. As newertherapeutics are developed, the distinctive and quantitativemeasurements offered by 3D-CTAG may become an increasinglyimportant marker to characterize AMD, monitor disease severity, andprovide quantitative outcome measures of therapy. Objective indices(e.g., LAG, PAG, HVL) delivered by 3D-CTAG may serve as usefuladjunctive measures in clinical trials guided by functional outcomes.Commercial Relationships: Kristie Lin, Janssen PharmaceuticalCompanies (C), Thrombogenics (C), Sequenom (C); Wolfgang Fink,University of Arizona (P), California Institute of Technology (P);Sami Kamjoo, None; Michael Davis, Sequenom (C), Johnson andJohnson Research and Development (C), Synergetics (C), Allergan(C), Citi Financial Group (C); Tom Chang, NoneClinical Trial: PRO00007677Program Number: 4643 Poster Board Number: B0107Presentation Time: 11:00 AM - 12:45 PMTreatment of retinal capillary hemangioblastoma using inhibitorsof the HIF pathwayMridul Mukherji, Divya Teja Vavilala, Prakash Swami, VKChaithanya Ponnaluri. Pharmacy - Pharmaceutical Sci, Univ ofMissouri - Kansas City, Kansas City, MO.Purpose: Retinal capillary hemangioblastoma (RCH, aka bloodvessel tumor) is observed in patients with mutations in the VHLtumor suppressor gene. This disease is a multisystem tumorsyndrome where the mutated VHL protein (pVHL) does not bind anddegrade the regulatory α-subunits of the hypoxia-inducible factoreven under normoxic conditions. This results in overexpression ofHIF-dependent pro-angiogenic factors. As a result,hemangioblastomas of retina, CNS, and kidney are highly vascular innature. All current therapies for RCH have significant limitations andside-effects; e.g., photocoagulation and cryotherapies cause tissuedestruction and inflammation, while anti-VEGF therapies hadminimal detectable beneficial effects. This lack of efficacy of anti-VEGF therapies is possibly due to overexpression of other proangiogenicfactors (e.g. EPO, PDGF, etc.) in RCH. Thus, thereremains a critical need for the development of an effective agent forthe treatment of RCH. Since inhibition of the HIF pathway issufficient to suppress tumor growth by mutated VHL cells in mousexenografts, it offers an attractive target to treat RCH.Methods: Four renal cell carcinoma derived cell lines (RCC4, T314,PRC3 and WT8) and two human retinal pigment epithelial cells(D407 and ARPE19) were used to evaluate the expression of cancerstem cell (CSM) and angiogenic markers. Honokiol, digoxin anddoxorubicin were used as HIF inhibitors in cell culture model.Inhibition of CSM and angiogenic marker expression by these smallmolecules was evaluated using qPCR, western blot analysis andELISA.Results: RCC4 and PRC3 cells which lack VHL showedupregulation of HIF pathway, thus induction of CSM and proangiogenicgenes as compared to T314 and WT8 (both havefunctional pVHL), respectively. In D407 and ARPE19 cells HIF isstabilized under hypoxic conditions, thereby mimicking VHL diseasephenotype, showing induction of CSM and pro-angiogenic 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 – Physiology/PharmacologyHonokiol, digoxin and doxorubicin significantly inhibited the HIFpathway, thus lowering the induction of CSM and pro-angiogenicgenes in these cell lines.Conclusions: Loss of VHL in RCH leads to activation of HIFpathway and thus induces expression of CSM and pro-angiogenicgenes. Small molecule inhibitors of HIF pathway like honokiol,digoxin and doxorubicin lowered expression of CSM and proangiogenicgenes, and thus presents a novel therapeutic strategy forRCH treatment.Commercial Relationships: Mridul Mukherji, None; Divya TejaVavilala, None; Prakash Swami, None; VK ChaithanyaPonnaluri, NoneProgram Number: 4644 Poster Board Number: B0108Presentation Time: 11:00 AM - 12:45 PMShort-term Effects of Cocoa on Retinal Reactivity in Individualswith Prediabetes and Type 2 DiabetesMary E. Lott 1 , Alicia Johns 1 , Joshua Oman 1 , Julia E. Slocomb 2 ,Michael Herr 1 , Robert A. Gabbay 3 , David A. Quillen 4 , KerstinBettermann 2 . 1 Heart and Vascular Institute, Penn State Milton SHershey Med Ctr, Hershey, PA; 2 Neurology, Penn State Milton SHershey Med Ctr, Hershey, PA; 3 Endocrinology, Penn State Milton SHershey Med Ctr, Hershey, PA; 4 Opthalmology, Penn State Milton SHershey Med Ctr, Hershey, PA.Purpose: To examine the effects of short term cocoa flavanolingestion (i.e. seven days) on retinal blood vessel endothelial functionin individuals at different disease stages of diabetes. We hypothesizethat short term ingestion of high flavanol cocoa compared to aplacebo will be associated with enhanced vascular reactivity. We alsohypothesize that the degree of vascular response to cocoa will likelybe greater in groups without structural vessel damage (i.e. controlsand prediabetics).Methods: Individuals with prediabetes (n=5), type 2 diabetes (n=5)and healthy controls (n=5) were recruited. Subjects’ ages ranged from40 to 71 years. Using a double blinded, randomized crossover design,this pilot study had subjects consume a high flavanol cocoa (13grams of pure cocoa) vs. placebo (0 grams of pure cocoa) drink forseven days with a one week washout period. Retinal blood vesselvasoreactivity was measured using a flickering light stimulus(Dynamic Vessel Analyzer, Imedos, Jena, Germany). Fasting glucoseand insulin were also measured.Results: High flavanol cocoa ingestion led to an increase in peakretinal vein vasodilation to flickering light in prediabetic individuals(pre 2.32% ± 0.59% vs. post 4.52% ± 0.75%, P=0.04). Similar effectswere shown in the retinal artery but did not reach significance (pre1.71% ± 0.82% vs. post 3.18% ± 0.80%, P=0.24). There was a trendfor a small improvement the retinal vein with the placebo drink (pre2.34% ± 0.60% vs. post 3.08% ± 0.33%, P=0.08) without any changein the artery diameter (pre 1.45% ± 0.72% vs. post 1.59% ± 0.29%,P=0.82). There were no significant diameter changes in the control ortype 2 diabetic groups to high flavanol cocoa or placebo. Althoughthe drinks did not alter fasting glucose and insulin in the prediabeticor control groups, fasting glucose slightly increased in individualswith type 2 diabetes (135 to 154 mg/dl; P=0.01).Conclusions: Short term high flavanol cocoa appears to improveretinal endothelial function in individuals with prediabetes within oneweek. However, a larger sample size is needed to confirm thesefindings and a longer duration to potentially see improvements intype 2 diabetic individuals.Commercial Relationships: Mary E. Lott, None; Alicia Johns,None; Joshua Oman, None; Julia E. Slocomb, None; MichaelHerr, None; Robert A. Gabbay, None; David A. Quillen, None;Kerstin Bettermann, NoneSupport: Funded by Barsumian Trust Grant; Product supplied byThe Hershey CompanyProgram Number: 4645 Poster Board Number: B0109Presentation Time: 11:00 AM - 12:45 PMAltered Vascular Microenvironment by Bevacizumab in DiabeticFibrovascular MembraneShintaro Nakao 1 , Keijiro Ishikawa 1 , Shigeo Yoshida 1 , Ri-ichiroKohno 1 , Masanori Miyazaki 1 , Hiroshi Enaida 1 , Toshihiro Kono 2 ,Tatsuro Ishibashi 1 . 1 Ophthalmology, Kyushu University, Fukuoka,Japan; 2 Ophthalmology, Fukuoka University, Fukuoka, Japan.Purpose: The purpose of this study was to evaluate the impact ofintravitreal bevacizumab (IVB) on 3 cellular components (vascularendothelial cells, pericytes, and myofibroblasts) of the vascularmicroenvironment in fibrovascular membranes (FVMs) ofproliferative diabetic retinopathy (PDR) patients.Methods: Immunohistological studies with Abs of CD34, α-SMA,and TGF-β were performed on 20 surgical specimens obtained duringa pars plana vitrectomy from 8 IVB-treated eyes, while 12 remaineduntreated. Four different indexes of vascular phenotype (vasculararea, vascular major axis, CD34(+) endothelial area, and blood vesseldensity), and α-SMA expression in vascular and stromal componentswere quantitatively-analyzed.Results: The intraluminal area of blood vessels, CD34(+) endothelialarea, and the blood vessel density in IVB-treated FVMs weresignificantly less than in untreated FVMs. The number of CD34(+)blood vessels in IVB-treated FVMs was similar to in untreatedFVMs. IVB could not affect vascular as well as stromal αSMA(+)area significantly. However, the ratio of vascular αSMA(+)area/CD34(+) area was significantly higher in IVB-treated FVMsthan in untreated FVMs. TGF-β expression could be observed in theIVB-treated FVM.Conclusions: IVB might primarily affect blood vessels, and theeffects on pericytes and myofibroblasts might be secondary. IVBtreatment regulates vascular microenvironment by the contraction ofblood vessels, the increasing pericyte ratio, and TGF-β expression inFVMs of PDR patients.Commercial Relationships: Shintaro Nakao, None; KeijiroIshikawa, None; Shigeo Yoshida, None; Ri-ichiro Kohno, None;Masanori Miyazaki, None; Hiroshi Enaida, None; ToshihiroKono, None; Tatsuro Ishibashi, NoneProgram Number: 4646 Poster Board Number: B0110Presentation Time: 11:00 AM - 12:45 PMRetinal Oxygenation During Intravitreal Treatment for CentralRetinal Vein OcclusionSindri Traustason, Morten D. de La Cour, Michael Larsen. Dept ofOphthalmology,Glostrup Univ Hosp, Copenhagen University,Glostrup, Denmark.Purpose: Intravitreal ranibizumab has in recent years becomestandard of care treatment for macular edema in central retinal veinocclusion. While the treatment generally reduces macular edema andimproves visual acuity, cases of increased retinal ischemia have beenreported. The purpose of this study was to investigate the effect ofintravitreal anti-VEGF on retinal oxygenation in patients with centralretinal vein occlusion.Methods: Patients with central retinal vein occlusion in one eye wereincluded in a prospective single-group observational study. Includedpatients received treatment according to standard clinical procedureand were followed over a period of six month. All patients receivedthree initial monthly injections of intravitreal ranibizumab (Lucentis,Novartis), followed by a three month follow-up period, during whichinjections were given as needed.©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 – Physiology/PharmacologyAt each visit, patients underwent examinations which included bestcorrected visual acuity, central macular thickness with SD-OCT andretinal oximetry.Results: At baseline, retinal venous oxygen saturation was reduced inthe affected eye, compared to fellow eye (35 ± 14% and to 57 ± 9%,respectively, mean ± SD, p = 3.1 x 10 -5 , paired t-test).Retinal venous oxygen saturation and BCVA increased and macularthickness decreases during ranibizumab treatment. No significantchanges were found in retinal arterial saturation (Fig. 1).Conclusions: Our results indicate that retinal venous saturation isincreased during treatment with intravitreal ranibizumab and do notpoint towards increased retinal ischemia.Changes in retinal oximetry, best corrected visual acuity (BCVA) onthe ETDRS scale and central retinal thickness (OCT) from baseline tomonth 4 and 7. P-values indicate results from one sample t-tests(μ=0).Commercial Relationships: Sindri Traustason, Novartis (F),Oxymap (R); Morten D. de La Cour, Alcon (C), Novartis (C);Michael Larsen, NoneSupport: Novartis, Th. Elmquist’s Fund for Eye Research, VeluxFoundation, Carl and Nicoline Larsen's Fund, The Danish Eye HealthSociety, Synoptik FoundationClinical Trial: NCT01360385Program Number: 4647 Poster Board Number: B0111Presentation Time: 11:00 AM - 12:45 PMRationale for Bimonthly Ranibizumab and QuarterlyAflibercept. A Drug and Disease Assessment Model in Wet AgerelatedMacular DegenerationDaniele Veritti 1 , Gianluca Gorni 2 , Laura Perissin 3 , Paolo Lanzetta 1 .1 Department of Ophthalmology, University of Udine, Udine, Italy;2 Department of Mathematics and Computer Science, University ofUdine, Udine, Italy; 3 Department of Biomedical Sciences andTechnologies, University of Udine, Udine, Italy.Purpose: A drug and disease assessment model was used to evaluatethe impact of different treatment regimens on intraocular ranibizumaband aflibercept concentration and free vascular endothelial growthfactor (VEGF) proportion.Methods: A time-dependent mathematical model using WolframMathematica software was developed on the 12-month data fromPIER, MONT BLANC, CATT, VIEW. Pharmacokinetic and affinitydata for ranibizumab and aflibercept were obtained from publishedreports. Two alternative regimens with bimonthly ranibizumab andquarterly aflibercept were simulated.Results: The mathematical model showed good correlation betweenclinical trial data and intraocular VEGF proportion. In the fixedmonthly 0.5 mg ranibizumab regimen that has been evaluated inMARINA, ANCHOR and CATT trials highest free VEGF levels staybelow 1/100,000 of total VEGF. Following the quarterlyadministration of 0.5 mg ranibizumab (PIER study), highest freeVEGF levels reach the 60% of total VEGF. In the individualizedregimen studied in the MONT BLANC trial free VEGF proportionreaches 100% during the maintenance phase. The aggressiveindividualized regimen with 0.5 mg ranibizumab studied in theCATT trial with a simulated even distribution of injections maintainsfree VEGF levels constantly below 0.5% of total VEGF. Simulationsof the two alternative regimens suggest that such treatmentapproaches maintain the free VEGF proportion under thresholdlevels. Free VEGF proportion remains stably below 0.5% and 0.1%with bimonthly ranibizumab and quarterly aflibercept respectively.Conclusions: Fixed bimonthly ranibizumab or quarterly afliberceptregimens may result in visual acuity improvement with reducedburden over treatment strategies applied in clinical trials.Commercial Relationships: Daniele Veritti, None; GianlucaGorni, None; Laura Perissin, None; Paolo Lanzetta, Alimera (C),Allergan (C), Bayer (C), Novartis (C), Novartis (R), Roche (C),Iridex (P)Program Number: 4648 Poster Board Number: B0112Presentation Time: 11:00 AM - 12:45 PMRetinal Vein Occlusions in Young Patients: Visual Outcomes andAssociated Systemic Risk FactorsNoureen Khan 1, 2 , Michael M. Lai 1, 2 . 1 Dept. of Ophthalmology,Georgetown University/Washington Hospital Center, Washington,DC; 2 The Retina Group of Washington, Chevy Chase, MD.Purpose: To assess the visual outcomes in patients forty years of ageand younger with retinal vein occlusion and to identify associatedsystemic risk factors.Methods: This is a retrospective, comparative, consecutive caseseries. Fifty eyes from 50 patients were diagnosed with branchedretinal vein occlusion (BRVO), hemi-central retinal vein occlusion(HRVO) or central retinal vein occlusion (CRVO) that presented inpatients 40 years or younger from 1997 to 2002. Risk factors(hypertension, hyperlipidemia, diabetes, obesity, glaucoma, smoking,OCP use, hypercoagulable lab abnormalities, and final visual acuity(VA) were recorded and compared.Results: Final visual acuity (VA) found that 46% had improvedvisual outcomes. Seventy eight percent ended up with at least 20/60vision or better. Patients with good baseline vision (20/60 or better)were found to have statistically significant better final VA comparedto patients with baseline vision of less than 20/60 (P=

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyProgram Number: 4649 Poster Board Number: B0113Presentation Time: 11:00 AM - 12:45 PMEffects of Prostacyclin on Isolated Porcine Retinal Arterioles:Cross-Talk between Nitric Oxide and ProstacyclinShinji Ono, Taiji Nagaoka, Tsuneaki Omae, Takayuki Kamiya,Akitoshi Yoshida. Ophthalmology, Asahikawa Med University,Asahikawa, Japan.Purpose: To investigate the vasomotor activity of prostacyclin(PGI2) on porcine retinal arterioles.Methods: Porcine retinal arterioles were isolated, cannulated, andpressurized without flow in vitro. Changes in diameter were recordedusing videomicroscopic techniques. To confirm the role of theendothelium, we compared the responses before and after endothelialremoval. To examine the involvement of endothelium-derivedrelaxing factor nitric oxide (NO), we assessed the responses in thepresence of NO synthase inhibitor NG-nitro-L-arginine methyl ester(L-NAME).Results: The retinal arterioles dilated in a concentration-dependentmanner in response to PGI2. This vasodilation decreasedsignificantly after the endothelium was removed. L-NAMEsignificantly inhibited PGI2-induced vasodilation comparable todenudation.Conclusions: PGI2 elicits vasodilation of the retinal arteriolesmediated not only by the vascular smooth muscle but also theendothelium, suggesting that an endothelium-dependent componentof PGI2-induced vasodilation may be involved because of productionof NO. We speculated that cross-talk between NO and PGI2 mayexist in the retinal circulation.Commercial Relationships: Shinji Ono, Kaken PharmaceuticalCo.,Ltd. (F); Taiji Nagaoka, None; Tsuneaki Omae, None;Takayuki Kamiya, None; Akitoshi Yoshida, NoneProgram Number: 4650 Poster Board Number: B0114Presentation Time: 11:00 AM - 12:45 PMPerifoveal Microvasculature in Human Eyes with VascularComorbiditiesGeoffrey Chan 1, 2 , Chandra Bala 1, 2 , Paula Yu 1, 2 , William Morgan 1 ,Ian L. McAllister 1 , Stephen J. Cringle 1, 2 , Yu Dao-Yi 1, 2 . 1 Centre forOphthalmology and Visual Science, Perth, WA, Australia;2 Australian Research Council Centre of Excellence in VisionScience, Canberra, ACT, Australia.Purpose: The microvasculature change which precedes thedevelopment of clinically manifest ocular disease has not beenclarified. This study delineates the morphological characteristics ofthe perifoveal microvasculature in patients with vascularcomorbidities.Methods: Comparisons were made between 11 human eyes frompatients with vascular comorbidities and 17 healthy control eyes. Alleyes were absent of clinically evident ocular disease.Microcannulation and targeted perfusion techniques were used tolabel the retinal microvasculature. Retinae were then flat mountedand the peripapillary region 2mm nasal to the fovea imaged usingconfocal laser scanning microscopy. Two- and three-dimensionalimage reconstructions were used to perform quantitativemeasurements of individual capillary networks within the perifovea.Parameters measured included capillary diameter, capillary loop area,capillary density and capillary surface area. Comparisons were madebetween normal eyes we previously studied and those from patientswith vascular comorbidities.Results: Capillary diameter was increased in all layers of the retinain patients with vascular comorbidities with the exception of thenerve fibre layer capillary network. Capillary loop area was reducedin all layers of the retina in patients with vascular comorbiditiesexcept the deep capillary network of the inner nuclear layer.Capillary density was reduced within the nerve fibre layer of eyeswith vascular comorbidities. There was no difference in the relativeoccupied capillary surface area between healthy and diseased eyes.Conclusions: Morphometric differences in retinal capillary networksare present between healthy patients and those with vascularcomorbidities. Understanding the capillary changes that precedeclinical ocular disease may be useful for defining the pathogenesis ofretinal vascular disease.Commercial Relationships: Geoffrey Chan, None; Chandra Bala,None; Paula Yu, None; William Morgan, National Health andMedical Research Council (F); Ian L. McAllister, None; Stephen J.Cringle, None; Yu Dao-Yi, NoneProgram Number: 4651 Poster Board Number: B0115Presentation Time: 11:00 AM - 12:45 PMFlavonoid-rich dark chocolate improves endothelial function inretinal vesselsNaim Terai, Alexandra Gedenk, Eberhard Spoerl, Richard P.Stodtmeister. Ophthalmology, University of Dresden, Dresden,Germany.Purpose: As a sign of autoregulation retinal arterioles and venolesdilate in response to flicker stimulation. This response can beattributed to vascular endothelial function. It has been shown that incoronary arteries the endothelial-dependent vasomotion improvessignificantly after flavonoid-rich dark chocolate. Aim of the presentstudy was to investigate whether dark chocolate leads to animprovement of retinal endothelial function in glaucoma patients andage-matched healthy subjects using the Dynamic Vessel Analyzer(DVA,Imedos, Jena, Germany).Methods: Subjects: Patients with primary open-angle glaucoma (GL)n=18; m/f=5/13, control subjects (CS) n=18 m/f=7/1; Age GL: 65±6(arithmet mean±s, years); CS: 64±7 (p=0.72). RR prior DVA:GL141/82±18/9 mmHg; CS: 133/79±14/11 (p=0.17/0.22). Visualfield: GL: Mean defect: -17.9,-2.1,2.7 db (Minimum, median,maximum); PSD: 1.1, 2.4, 15.6.The patients and controls were assigned to dark or white chocolate(C) by randomisation with forced equal distribution. The number ineach of the four groups was 9. Examination: Following pupil dilationmeasurement of blood pressure, intraocular pressure, blood glucosewas done followed by DVA. This instrument consists of a funduscamera, video equipment and computer-aided process control.Recording procedure: Online measurement of the diameter of aretinal arteriolar and a venolar vessel segment, 50s baseline, threeperiods of 20s flicker stimulation and 80s follow- up registration.Three registrations were averaged.The change of the vessel diameter following flicker stimulationbefore and 2 hours after chocolate ingestion was measured. Statistics:Eight paired Wilcoxon tests with Bonferroni-Holm correction. H0:Vessel dilation (DIL)before=DILafter. H1:DILafter>DILbefore. Onesided 5% sigificance level p=0.1.Results: GL: DIL of arterioles before and after comsuption of darkCor whiteC didn't differ. Lowest p=0.41. CS: DIL of the arteriolesdidn't differ significantly. Lowest p=0.56. DIL of venules beforedarkC =1.9, 3.1, 4.4; after darkC=1.9, 5.2, 6.9; p=0.08. DIL of veinsbefore whiteC=2.4, 2.8, 5.6; after whiteC=1.4, 3.0, 5.8; p=0.95.Conclusions: In GL patients retinal vessel autoregulation remainedunchanged by darkC or whiteC. In our control subjects, however, theextent of the autoregulatory response to flicker stimulation improvedstatistically significantly by darkC but not by whiteC which containsno flavonoids.©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 – Physiology/PharmacologyCommercial Relationships: Naim Terai, None; AlexandraGedenk, None; Eberhard Spoerl, None; Richard P. Stodtmeister,Novartis (F)Program Number: 4652 Poster Board Number: B0116Presentation Time: 11:00 AM - 12:45 PMInhibition of retinal neovascularization by luteolin viasuppression of VEGF expression and VEGFR signaling pathwaySung Wook Park 1, 2 , Chang Sik Cho 1 , Hyoung Oh Jun 1 , Nam HeeRyu 3 , Jin Hyoung Kim 1 , Young S. Yu 1 , Jin Sook Kim 3 , Jeong HunKim 1, 2 . 1 FARB, Department of Ophthalmology, Seoul NationalUniversity Hospital, Seoul, Republic of Korea; 2 Biomedical Sciences,Seoul National University College of Medicine, Seoul, Republic ofKorea; 3 Diabetic Complications Research Center, Division ofTraditional Korean Medicine Integrated Research, Korea Institute ofOriental Medicine, Daejeon, Republic of Korea.Purpose: This study was to investigate the anti-angiogenic effect ofluteolin against reactive oxygen species (ROS) induced retinalneovascularizationMethods: The toxicity of luteolin was evaluated through modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay inhuman retinal microvascular endothelial cells (HRMECs) as well asterminal deoxynucleotidyl transferase dUTP nick-end labelingstaining in the retina of C57BL/6J. After intravitreal injection ofluteolin in the mouse model of ROP, retinal neovascularization wasexamined by fluorescence angiography and vessel counting. Antiangiogenicactivity of luteolin was evaluated by VEGF-inducedmigration and tube formation assay. The effect of luteolin on t-BHinducedROS production was measured with 2’7’-dichlorofluoresceindiacetate. The effect of luteolin on t-BH induced and hypoxiainducedVEGF transcription and expression were evaluated by RT-PCR and Western blot, respectively.Results: Luteolin never affected the viability of HRMECs up to 10μM, where luteolin never induce any structural change in all retinallayers. Luteolin inhibited retinal neovascularization in the mousemodel of ROP. Moreover, VEGF-induced migration and tubeformation were significantly decreased by co-treatment of luteolin.Luteolin attenuated VEGF transcription via blockade of t-BH inducedROS production. Luteolin suppressed hypoxia-induced VEGFexpression via attenuating hypoxia inducible factor 1 α expression.Conclusions: Our results suggest that luteolin could be a potent antiangiogenicagent for retinal neovascularization, which is related toanti-oxidative activity to block ROS production and to subsequentlysuppress VEGF expression and the pro-angiogenic effect of VEGFCommercial Relationships: Sung Wook Park, None; Chang SikCho, None; Hyoung Oh Jun, None; Nam Hee Ryu, None; JinHyoung Kim, None; Young S. Yu, None; Jin Sook Kim, None;Jeong Hun Kim, NoneSupport: the Bio-Signal Analysis Technology Innovation Programof MEST/NRF, Republic of Korea (2011-0027723), Mid-CareerResearcher Program, Republic of Korea (2011-0017910) ofMEST/NRF, grant from the Korea Institute of Oriental Medicine(KIOM) (K11040)Program Number: 4653 Poster Board Number: B0117Presentation Time: 11:00 AM - 12:45 PMPlasma VEGF-levels are variable in patients with persistent renaldysfunction and diabetes: possible implications and risks forophthalmic anti-VEGF therapyMarkus van der Giet, Mirjam Schuchardt, Nicole Pruefer, JasminPruefer. Med. Klinik - SP Nephrology, Charite - UniversitätsmedizinBerlin, Berlin, Germany.Purpose: Ophthalmic anti-VEGF therapies are used commonlywithout monitoring of systemic risks and their intraocular applicationmight cause unexpected effects to systemic VEGF-dependentregulatory mechanisms. Our study aims at quantifying systemicVEGF-levels in situations commonly found in the target populationof ocular anti-VEGFsMethods: Blood was drawn form 30 healthy controls (age 28-78years), 30 patients with type I or II diabetes without renal dysfunction(24-84 years, DM), 30 patients with chronic kidney disease (CKD)stage III-V (incl. hemodialysis patients) and 30 patients with type I/IIdiabetes and renal dysfunction (CKD III-V, CKD/DM). VEGF A-Dwere quantified using 96-well ELISA (Cusabio, Wuhan, China)according to manufacturer’s protocol. VEGF levels were quantifiedby comparison to standard concentration curves; all values are givenas means.Results: Mean plasma VEGF A levels were 85.8 pg/ml in healthycontrols and 77.6 pg/ml in DM patients. These levels weremoderately elevated in CKD (129.92pg/ml) and CKD/DM (111.9pg/ml) patients (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyby combination with Placental Growth Factor (PlGF) inhibitionin the Oxygen induced Retinopathy (OIR) model in miceEunice Cheung, Ivan B. Lobov, George D. Yancopoulos, Stanley J.Wiegand. Ophthalmology, Regeneron Pharmaceuticals, Inc.,Tarrytown, NY.Purpose: Vascular endothelial growth factor A (VEGF-A) signalingthrough VEGF receptor 1 (VEGFR1) and VEGFR2 regulates bothphysiological and pathological angiogenesis [Luttun et al., NatureMed. 2002, 8:831-840]. We have reported that genetic deletion orinhibition of the VEGFR1 ligand, PlGF, reduced vasoobliteration andneovascularization in OIR [IOVS 2009; 50:E-Abstract 2943; IOVS2010; 51:E-Abstract 4486]. We also demonstrated that inhibition ofeither PlGF or VEGFR1 could improve normal blood vessel regrowthand also lessen neovascularizations [IOVS 2011; 52:E-Abstract6064]. In this study, we administered DC101, a VEGFR2 blockingantibody, alone or in combination with a PlGF blocking antibodyafter vessel loss was complete, to evaluate the effects of selectiveblockade of activity of VEGFR2 alone, or in combination withinhibition of the VEGFR1 ligand, PlGF, in the murine model of OIR.Methods: Mice were place in a hyperoxic environment (75% O2) atpostnatal day 6 (P6) and returned to room air at P11. Pups wereinjected systemically with 25mg/kg DC101 or a control protein (Fc)at P12. Left eyes of DC101 treated pups were injected intravitreally(IVT) with 5ug of a polyclonal Goat anti-Mouse PlGF-2 or a controlIgG (Normal Goat IgG) at P13. Retinal flatmounts were collected atP16 and endothelial cells stained with FITC-labeled Grifforniasimplicifolia lectin I and anti-NG2, a pericyte marker.Results: At P16, pathological neovascularizations worsened in micetreated with DC101 relative to controls. In contrast, IVTadministration of PlGF antibody in mice treated with DC101produced significant improvement of pathological neovascularizationrelative to animals treated with DC101 and control IgG. Noappreciable differences were noted among groups in the extent of theresidual avascular area on P16.Conclusions: This study demonstrates that selective pharmacologicalneutralization of VEGFR2 is ineffective in ameliorating pathologicalneovascularization in OIR, while co-incident neutralization of theVEGFR1 ligand, PlGF results in significant amelioration ofpathological neovascularization.Thus, this study further supports thehypothesis that inhibition of VEGFR1 ligand, such as PlGF, can playan important role in the inhibition of pathological angiogenesis.Commercial Relationships: Eunice Cheung, RegeneronPharmaceuticals (E); Ivan B. Lobov, Regeneron Pharmaceuticals,Inc. (E); George D. Yancopoulos, Regeneron Pharmaceuticals (E),Regeneron Pharmaceuticals (I), Regeneron Pharmaceuticals (P);Stanley J. Wiegand, Regeneron Pharmaceuticals, Inc (E)Support: None in the Support fieldProgram Number: 4655 Poster Board Number: B0119Presentation Time: 11:00 AM - 12:45 PMTreatment of proliferative idiopathic macular telangiectasia Type2 with intravitreal bevacizumabRanjit Sandhu, Robin D. Hamilton. Medical Retina, Moorfields EyeHospital, London, United Kingdom.Purpose: Idiopathic macular telangiectasia Type 2 is bilateral andcharacterised by retinal opacification, vascular telangiectasia, rightangledvenules, intraretinal crystalline deposits, foveal thinning,retinal pigment epithelial hypertrophy and rarely, choroidalneovascularisation (proliferative form).The purpose of this study was to determine the effect of treatmentwith intravitreal bevacizumab on retinal thickness and visual acuityin the proliferative form of MacTel Type 2 in a small case series.Methods: A retrospective review of clinical notes of patients withidiopathic macular telangiectasia Type 2 complicated by choroidalneovascularisation based on fundus fluorescein angiography whoreceived treatment with intravitreal bevacizumab injections wascarried out. All patients had Snellen visual acuity testing, fundusfluorescein angiography and optical coherence tomography (OCT) atbaseline. The patients were treated until the choroidal neovascularmembrane (CNV) showed no signs of activity based on the OCTfindings. Visual acuity and central macular thickness were recordedat final follow-up visit.Results: Ten eyes of seven patients, of which two were males andeight females, were included. The average age was 60 years ± 5 yearsand the average follow-up was 14 months ± 12 months (range, 2-34months). The visual acuity was 0.6 ± 0.5 logMAR at baseline and 0.6logMAR ± 0.4 logMAR at final follow-up. The central macularthickness was 223µ ± 41µ at baseline and 229µ ± 53µ at final followup.The average number of bevacizumab injections administeredwere 3 (range, 1-8). There was no improvement in the visual acuityor central macular thickness. The CNV resolved in all ten eyes ofseven patients. One patient did not require bevacizumab as the CNVregressed spontaneously.Conclusions: Although the use of intravitreal bevacizumab in theproliferative form of idiopathic macular telangiectasia Type 2 did notimprove the visual acuity or reduce the central macular thickness, itresulted in the regression of the CNV. As the probability ofprogression from absence to presence of an IS/OS PR break has beenrecently reported to be as high as 72% after 5 years by the MacTelStudy group and CNV complicating MacTel posing a further threat tocentral visual loss, treatment with intravitreal bevacizumab preventsfurther visual loss in this potentially sight threatening condition.Commercial Relationships: Ranjit Sandhu, None; Robin D.Hamilton, Novartis (R), Bayer (R), Ellex (R), Valon (R)Program Number: 4656 Poster Board Number: B0120Presentation Time: 11:00 AM - 12:45 PMComputational model of blood flow through the choriocapillarishighlights marked heterogeneity of blood flowMoussa A. Zouache 1, 2 , Philip J. Luthert 1, 2 , Ian Eames 3 . 1 Institute ofOphthalmology, University College London, London, UnitedKingdom; 2 NIHR Biomedical Research Centre in Ophthalmology,London, United Kingdom; 3 Department of Mechanical Engineering,University College London, London, United Kingdom.Purpose: The choroid is a pigmented vascular layer located betweenthe retina and the sclera. It supplies the outer retina with oxygen andnutrients through a dense, one-layered network of capillaries calledthe choriocapillaris. The blood flow through the choriocapillaris isremarkably high. A discrete lobular segmentation of the plexus isobserved in the macula and the posterior fundus. The aim of thisstudy was to investigate, theoretically, the heterogeneity of bloodrenewal within the choriocapillary plexus over the macular 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 – Physiology/Pharmacologyand posterior pole.Methods: A computational model informed by the angioarchitectureof the choriocapillaris as described in the literature and as revealed inwhole mounts stained for CD31, a vascular marker, was built in orderto simulate blood flow within choriocapillary lobules. Using a simplegeometry, analytical and numerical solutions for the flow wereobtained.Results: The computer model included the spacing and distributionof arterioles and venules connecting with the plexus and derivedparameters including the pressure distribution and velocity fields.The model revealed significant heterogeneities in blood renewal overthe vascular network. The number of regions of minimal bloodrenewal was shown to equate to the number of arterioles and venulessupplying each choriocapillary lobule.Conclusions: The choriocapillary plexus displays significantheterogeneities in blood renewal resulting from the intrinsicanatomical and functional organization of the plexus. This impliesthat there may be areas of critical perfusion even given the very highblood flow rates within the choriocapillaris as a whole. Observationsmay have relevance for the pathogenesis of degenerative diseasessuch as macular degeneration. Furthermore, this model provides thebasis for a more comprehensive model of the pathophysiology of theback of the eye.Commercial Relationships: Moussa A. Zouache, None; Philip J.Luthert, None; Ian Eames, NoneSupport: Special Trustees of Moorfields Eye Hospital andUniversity College London Impact Studentship SchemeProgram Number: 4657 Poster Board Number: B0121Presentation Time: 11:00 AM - 12:45 PMPeriocular injection of a broad spectrum antiangiogenic therapyregresses choroidal neovascularizationOfra Benny, Zai-Long Chi, Lauren Bazinet, Robert J. D'Amato.Boston Children's Hospital, Harvard Medical School, Boston, MA.Purpose: Current therapies with intravitreal injections of antivascularendothelial growth factor (VEGF) agents for intraocularneovascularization suppress neovascularization and vessel leakage.However, nearly half of AMD patients do not respond to this therapy,and those that do require injections repeatedly and chronically,poising them for clinical ocular complications. The purpose of ourstudy is to develop a less invasive, local treatment that can safely beadministered chronically, as a means of simultaneously improvingthe potency of treatment and reducing the burden for patients withAMD.Methods: Lodamin is a polyethylenglycol-poly lactic acid (PEG-PLA) conjugate of the antiangiogenic drug TNP-470, a Methionineaminopeptidase 2 (MetAp2) inhibitor and selective cytostatic agent ofendothelial cells. A laser-induced CNV model was used in C57/Blmice, with 6 lesions inflicted per retina. Average CNV size wasmeasured following isolectin IB4 staining of retinal vessels.Results: Lodamin was administered as a single retrobulbar orperibulbar injection (100ug per eye) on the day of laser treatment. Byday 7, 42% inhibition and 34% inhibition in CNV size was recorded,respectively. Additionally, experiments were performed withestablished CNV lesions where treatment was given on Day 7 postlaser.In these cases, Lodamin was able to regress CNV by 45% asmeasured on day 14 post CNV induction.Conclusions: We have previously demonstrated Lodamin to be apotent broad-spectrum antiangiogenic drug in multiple angiogenicmodels. Lodamin was effective in treating CNV when administeredorally. However in the interest of reducing systemic exposure, localadministration (intravitreal) was explored, yielding positive results.We now show that Lodamin is also effective when given byperiocular administration, thus minimizing the risks clinicallyassociated with intravitreal injection. Lodamin’s ability to regresspreexisting neovascularization together with its improved deliverymay change the paradigm of patient therapy and improve currentdisease treatment.Commercial Relationships: Ofra Benny, None; Zai-Long Chi,None; Lauren Bazinet, None; Robert J. D'Amato, BostonChildrens Hospital (P)Program Number: 4658 Poster Board Number: B0122Presentation Time: 11:00 AM - 12:45 PMEfficacy and safety of subconjunctival bevacizumab for recurrentpterygiumLarissa S. Stival 1 , Anelise M. Lago 1 , Marisa N. Figueiredo 1 , RicardoG. Bittar 2 , Marcia L. Machado 1 , Joao J. Nassaralla 2 . 1 Cornea andRefractive Surgery, Instituto de Olhos de Goiania, Goiania, Brazil;2 Retina and Vitreous, IOG, Goiania, Brazil.Purpose: To evaluate clinical outcomes and complications ofsubconjunctival bevacizumab (Avastin) in patients with recurrentpterygium.Methods: This prospective clinical trial included 36 patients (36eyes), who underwent pterygium surgery and who were diagnosedwith recurrent pterygium. All pacientes received subconjunctivalapplication of bevacizumab (0,5ml). Pterygium vascularity andthickness was graded according Tan's rating. The size of thepterygium (measured in mm) was recorded from baseline to 8 weeksafter injection. Treatment-related complications and adverse eventswere reported. The main outcome of measurements was the change insize, vascularity and thickness.Results: There were 18 males (50%) and 18 females (50%) of 36patients with a mean age of 58,75 years (SD 10,98 years). 30.6% ofpacients had recurrent pterygium in both eyes, being 47.2% in the lefteye and 22.2% in the right eye. 44.4% of patients were from Goiânia(Brazil) and 55.6% from other cities. More than half of patients(58.3%) had a family history of pterygium. There was a significantdifference in the size of pterygium at different intervals (P < 0.05)and the mean surface area was reduced. 66.7% of patients presentedhyposphagma on the 2nd day after the subconjunctival application,decreasing to 30.6% by day 7 and finally no patient after 1 month.Most patients (69.4%) had improvement of irritative symptomswithin 2 days, 88.9% after 7 days and 97.2% after 1 month.Conclusions: Subconjuctival bevacizumab injection is useful inmanagement of patients with recurrent pterygium without significantlocal or systemic adverse effects.Commercial Relationships: Larissa S. Stival, None; Anelise M.Lago, None; Marisa N. Figueiredo, None; Ricardo G. Bittar,None; Marcia L. Machado, None; Joao J. Nassaralla, NoneClinical Trial: NCT01744756Program Number: 4659 Poster Board Number: B0123Presentation Time: 11:00 AM - 12:45 PMA Treat and Extend Regimen Using Anti-VEGF Therapies forCentral Retinal Vein OcclusionChristopher J. Brady, Rayan Alshareef, Andre J. Witkin, Carl D.Regillo. Wills Eye Institute, Jefferson Univ School of Medicine,Philadelphia, PA.Purpose: With the FDA approval of ranibizumab and aflibercept formacular edema due to central retinal vein occlusion (CRVO), a newstandard of care has been adopted for this condition. The initialclinical trials were designed using a monthly treatment approachfollowed by as-needed injections. A treat and extend (T&E) approachhas been described in the treatment of neovascular age-relatedmacular degeneration (ARMD), and is being increasingly adopted.©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 – Physiology/PharmacologySuch an approach has the potential to reduce the burden of treatmenton patients and physicians while maintaining effectiveness in thetreatment of CRVO.Methods: Nine physicians who routinely adopt a T&E extendapproach were identified. A retrospective analysis of their patientswith CRVO treated with either bevacizumab or ranibizumab for atleast six months was undertaken. Change in Snellen visual acuity andcentral macular thickness (CMT) were analyzed. Adherence to aT&E protocol was also analyzed.Results: Sixty-six patients were identified, 25 of whom weretreatment-naïve, and thus included in the analysis. The mean age was73.6 years, and 63% were female. Sixty-seven percent reportedhypertension and 14.8% reported diabetes mellitus. Mean duration ofsymptoms prior to therapy was 131 days. After 6 months follow-up,mean visual acuity improved to 20/80 from 20/200+1. Mean CMTimproved 229.4 microns (from 652 to 422 microns).Conclusions: There are several theoretical advantages to a T&Eregimen for CRVO compared with monthly and as needed strategiesincluding fewer office visits and fewer injections, and therefore lowerrisk of side effects including endophthalmitis. Compared withARMD, T&E for CRVO appears to be less successful. This may bedue to differences in pathophysiology, or perhaps demographics thathinder adherence to a strict treatment protocol. Given the logisticalbenefits and reduced risk profile, an initial attempt at T&E with a lowthreshold to maintain therapy at fixed interval is reasonable.Commercial Relationships: Christopher J. Brady, None; RayanAlshareef, None; Andre J. Witkin, None; Carl D. Regillo,Genentech (C), Regeneron (C), Alcon (C), Thrombogenics (F), GSK(F), ACT (F)Program Number: 4660 Poster Board Number: B0124Presentation Time: 11:00 AM - 12:45 PMIn vitro studies on the antiangiogenic effects of PigmentEpithelium Derived Factor and SomatostatinAnna Salas Torras, Andrea R. Carvalho, Ibane Abasolo, Miguel A.Zapata, Laura N. Distefano, Simo Schwartz, Jose Garcia-Arumi.Ophthalmology, Vall d'Hebron Research Inst, Barcelona, Spain.Purpose: Pigment Epithelium Derived Factor (PEDF) andSomatostatin (SST) have been demonstrated to be two of theprincipal natural antiangiogenic and anti-VEGF factors present in theretina. Both peptides are promising tools to be used in differentstrategies for a variety of retinal disorders affecting the endothelialcompartment such as PDR and wet AMD.The aim of this study is to evaluate the antiangiogenic andantiproliferative properties of PEDF and SST in several in vitromodels, as a preliminary proof-of-concept assay before testing theirefficacy in vivo.Methods: To test the antiangiogenic properties of PEDF and SST invitro, two cell types were used: the ARPE-19 cell line derived fromthe retinal pigment epithelium, and a primary culture of humanumbilical vein endothelial cells (HUVECs), as a model of vascularendothelium. Both are the main cell types affected in proliferativeeye diseases.First we determined the expression of SST receptors (SSTR) inHUVECs and ARPE-19 by RT-PCR. PEDF receptors could not beverified, as they are still not identified. Antiproliferative action ofPEDF and SST were tested in HUVEC and ARPE-19 cells by MTTassay after 24 and 48 h of incubation. In addition, HUVECs wereused as an endothelial model to perform migration and tubuleformation assays under VEGF treatment, in order to test theantiangiogenic activity of PEDF and SST.Results: Using RT-PCR, ARPE-19 cells were found to expressSSTR-1, -2, -3 and -5, while HUVEC cells mainly express SSTR-1and -2. In proliferation assays, SST was found to significantly inhibitARPE-19 proliferation up to a 40% at 10-4M, (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologypredictable intervals for re-treatment.This series is, to the best of our knowledge, one of first to reportclinical experience with sequential use of anti-VEGF agents andOzurdex. Further prospective, controlled studies need to beperformed to better determine efficacy, duration of effect, and sideeffects.Commercial Relationships: Evangelia Papavasileiou, None;Andre Grixti, None; Vineeth Kumar, None; Som Prasad,Thrombogenics (C), Bausch & Lomb (C), Alcon (R), Novartis (R),Bayer (C), Nidek (C), Allergan (R)Program Number: 4662 Poster Board Number: B0126Presentation Time: 11:00 AM - 12:45 PMShort Term Ocular Response of retinal blood flow to intravitrealinjection of bevacizumab (Avastin○R) treatment in eyes withcentral retinal vein occlusionSeung W. Lee. Ophthalmology, Dongguk Unv, Gyeongju Hosp,Gyeongju, Republic of Korea.Purpose: To study the effect of intravitreal bevacizumab (1.25mg)injection (IVBI) on the retinal blood flow and the retinal vesselcaliber in patients with central retinal vein occlusion (CRVO).Methods: We conducted a retrospective, uncontrolled study of 26patients. Twelve eyes underwent IVBI and were considered to be thestudy group. The control group composed of 14 eyes, which wereobserved at least 3 months before retinal photocoagulation.Depending on the clinical requirements, all eyes in the study grouphad at least one IVBI. Retinal arteriovenous transit time (RAVTT)and visual acuity were used to monitor the evolution of CRVO, andfollow up. The diameter of the retinal vessel diameter was measuredwith using the software available on the IMAGEnet program beforethe first IVT injection and 30 days after the third injection.Results: Initially, the mean RAVTT was 23.0 ± 6.1 seconds in thestudy group and 28.2 ± 5.4 seconds in the control group. Threemonths later, RAVTT improved to 17.1 ± 2.9 seconds in the studygroup, but did not change to 28.4 ± 6.5 second in the control group.The change in RAVTT was statistically significant, only in the studygroup (p= 0.003). In the study group, the significant vasoconstrictionwas observed in the retinal vein (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyp = 0.23). The CVs for V were 12.1 ± 4.9% in the arterioles and 14.5± 6.6% in the venules with FD-OCT and 11.9 ± 5.6% in the arteriolesand 11.7 ± 5.7% in the venules with the bidirectional LDV apparatus.There were no significant differences in CVs obtained by twomethods (arterioles, p = 0.55; venules, p = 0.49).Conclusions: The current study showed that the newly developedDoppler FD-OCT enables accurate and reproducible measurementsof the blood velocity in the retinal arterioles and venules in humans.Commercial Relationships: Akitoshi Yoshida, None; TaijiNagaoka, None; Tomofumi Tani, None; Eiichi Sato, None;Takafumi Yoshioka, None; Kenji Sogawa, None; SeigoNakabayashi, NoneProgram Number: 4665 Poster Board Number: B0129Presentation Time: 11:00 AM - 12:45 PMMechanisms of retinal venous pulsation inferred from diameterwaveforms analysisFabrice Moret 1 , Wolf Lagreze 2 , Charlotte M. Poloschek 1, 2 , MichaelBach 1 . 1 Sect. Visual Function and Electrophys, Eye Hospital,University of Freiburg, Freiburg, Germany; 2 Sect.Neuroophthalmology, Eye Hospital, University of Freiburg, Freiburg,Germany.Purpose: The retinal venous outflow resistance is a candidateelement in the etiology of glaucoma. The cardiac pulsation,especially its pressure waveform, can characterize the nature ofresistive elements in vascular segments. Expanding on recent datacollected to investigate the spontaneous venous pulsation's time ofcollapse, we examine the pulsation waveform in young healthysubjects and test explanatory hypotheses.Methods: We analyzed fundus movies collected in 12 subjects.Arterial lateral displacement and venous diameter waveforms weremeasured near the disk on sequences with and without imageprocessing improvement. We then reviewed the various mechanismsproposed so far and assessed each of them with our data. Proposedmechanisms include: the "classical view", a pulse transfer through themicrocirculation, a spontaneous pulsation, a Venturi effect, acommon adventitial sheath segment, the "constant inflow/variableoutflow" hypothesis, and an intracranial pressure interaction.Results: Venous pulsations show different amplitudes andwaveforms, suggesting more than one mechanism at play. None ofthe previous mechanisms alone fully explains the measuredwaveforms. We propose two mechanisms: 1. For waveforms showinga damped-like version of the arterial pressure: initially (and wellestablished),the pulsation of the choroidal volume governed by aWindkessel effect leads to an intraocular pressure pulsation of similarwaveform, then this pulsation is transferred to the retinal circulationupstream of the measurement sites. 2. For venous diameterwaveforms matching the arterial pressure waveforms: the pressurepulsation of the central retinal artery is transferred to the centralretinal vein in a non-extensible common adventitial sheath segmentin the optic nerve.Conclusions: The waveform of the retinal venous pulsation obtainedfrom fundus movies informs on the nature of the venous outflowresistance in ways not matched by an analysis of the amplitude alone.It would now be of interest as of whether quantitative changes areobserved in glaucoma.Commercial Relationships: Fabrice Moret, None; Wolf Lagreze,Merz (C), Allergan (C); Charlotte M. Poloschek, None; MichaelBach, NoneSupport: Deutsche Forschungsgemeinschaft BA877/19-2Program Number: 4666 Poster Board Number: B0130Presentation Time: 11:00 AM - 12:45 PMEffect of Nitric Oxide on Increased Retinal Blood Flow inResponse to Flicker Stimuli in CatsTakafumi Yoshioka, Taiji Nagaoka, Akitoshi Yoshida.Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.Purpose: To investigate the role of nitric oxide (NO) in the increasein retinal blood flow (RBF) during flicker stimuli.Methods: Twelve adult cats were used in this study. After anesthesiawas induced with sevoflurane in each animal, laser Dopplervelocimetry (LDV) was used to measure the vessel diameter (D) andblood velocity (V) simultaneously and calculate the RBF in thesecond-order retinal arterioles during flicker stimulation. In the firstseries of studies, the flicker frequencies and light intensities rangedfrom 2 to 64 Hz and 300 to 3000 lux, respectively. The durations ofdark adaptation and flicker stimulation ranged from 0 to 30 minutesand 60 to 300 seconds, respectively. In the second series of studies,we injected phosphate-buffered saline (PBS) or NG-nitro-L-argininemethylester (L-NAME) into the vitreous and measured the D, V, andRBF during flicker stimuli 2 hours later.Results: Flicker stimulations from 2 to 32 Hz increased the RBF inthe retinal arterioles. In contrast, flicker stimulation with 64 Hz didnot affect the RBF. At 16 Hz, the increase in RBF was enhanced inresponse to light intensities of 300~3,000 lux, dark adaptation timesof 0~30 minutes, and flicker stimulation times of 60~180 seconds. At16 Hz, 3,000 lux, 30 minutes of dark adaptation, and 180 seconds ofstimulation time, flicker stimulation resulted in a 51% increase inRBF over baseline in the PBS group (n=6) and an 11% increase inthe L-NAME group (n=6), a difference that was significant(P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Tomofumi Tani, None; TaijiNagaoka, None; Takafumi Yoshioka, None; Akitoshi Yoshida,NoneProgram Number: 4668 Poster Board Number: B0132Presentation Time: 11:00 AM - 12:45 PMMeasurement of Retinal Blood Flow Using a Newly DevelopedDoppler Fourier-Domain Optical Coherence TomographyInstruments in CatsTaiji Nagaoka, Tomofumi Tani, Eiichi Sato, Takafumi Yoshioka,Kenji Sogawa, Seigo Nakabayashi, Akitoshi Yoshida.Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.Purpose: To report the reproducibility of retinal blood flowmeasurements by using a newly developed Doppler Fourier-domainoptical coherence tomography (FD-OCT) instrument in anesthetizedcats.Methods: Four cats (2.5-5.0 kg) were anesthetized using a mixture ofsevoflurane and room air. We measured retinal blood flow by usingboth the FD-OCT and the bidirectional LDV (Canon Laser BloodFlowmeter, Canon Co., Japan) which was customized for cats. Thereproducibility of retinal blood flow measurements was assessed bycalculating the coefficients of variation (CV) for repeatedmeasurements (5X) of retinal vessel diameter, time-averagecenterline blood velocity, and blood flow (RBF) at temporal retinalarterioles and venules. In addition, we examined the changes inretinal blood flow in arterioles in response to systemic inhalation of100% pure oxygen (hyperoxia) by using both instruments.Results: The averaged values of CV (mean ± SD) for RBF were9.7% ± 3.8% and 8.2% ± 3.5% in arterioles and 10.3% ± 0.8% and7.4% ± 0.9 % in venules with LDV and FD-OCT, respectively (p =0.34 and p = 0.18). After 10 minutes of hyperoxia, RBF decreased by-43.8 ± 11.2% and -43.9 ± 7.4% with LDV and FD-OCT,respectively. There was no significant change in RBF in response tohyperoxia between LDV and FD-OCT (p = 0.87).Conclusions: The current study revealed that newly developed FD-OCT enable the accurate and reproducible measurements of bloodvelocity in retinal arterioles and venules in in vivo cats models.Commercial Relationships: Taiji Nagaoka, None; TomofumiTani, None; Eiichi Sato, None; Takafumi Yoshioka, None; KenjiSogawa, None; Seigo Nakabayashi, None; Akitoshi Yoshida, Nonetargeting (measurement diameter about 60 μm). In vivo validationconsisted of: (1) Study of linearity of the instrument with a rotatingdiffusing wheel. (2) Measurement reproducibility during a singleexperimental session (n = 5 rats, 1 hour, 4 measurements of 5-minduration) by calculation of intra-individual Variation Coefficients(VC) and Intra-class Correlation Coefficient (ICC). (3) Blood Flow(BF), velocity (Vel) and volume (Vol) (mean ± SD) were evaluatedin response to 100% oxygen breathing (n = 8 rats).Results: Vel varied linearly with the rotating speed (R2=0.9806)while Vol did not change (standard deviation/mean = 2.9%). Shorttermreproducibility of retinal arterial Vel shows strong agreement(ICC=0.9, IC 95%=0.7-0.99), associated with low intra-individualVC (7.8 ± 6.6 %). After 2 minutes of 100% oxygen breathing, retinalarterial Vel was significant decreased by 17.0 ± 13.7 % (p=0.012),and optic nerve head BF by 11.0 ± 9.7 % (p=0.018).Conclusions: This new LDF device allows repeatable measurementsof ocular blood flow in small animals. Rat optic nerve head andretinal blood flow measurements are of crucial interest inphysiological and pathophysiological studies. This prototype will beused to investigate various animal models (diabetes mellitus,glaucoma, vascular disorders) and for therapeutic evaluation.Optical scheme of the laser Doppler flowmeter prototype (LDF),based on the Schlieren arrangement of the laser delivery anddetection system, and rat fundus image.Program Number: 4669 Poster Board Number: B0133Presentation Time: 11:00 AM - 12:45 PMDevelopment of a prototype of laser Doppler flowmeter for themeasurement of blood flow of retinal vessels and optic nerve headtissue in small animals (rats)Marielle Mentek 1 , Christophe Chiquet 3, 1 , Frederic Truffer 2 , MarioBernabei 2 , Benjamin Mottet 3 , Jean-Paul Romanet 3 , Diane Godin-Ribuot 1 , Martial Geiser 2 . 1 INSERM U1042, Grenoble, France;2 Institute of system engineering, University of Applied Sciences ofWestern Switzerland, Sion, Switzerland; 3 Ophthalmologydepartment, CHU Grenoble, Joseph Fourier University, Grenoble,France.Purpose: The aim of the present study was to validate a new laserDoppler flowmeter (LDF) device, specifically designed to measureretinal and optic nerve head blood flow in the rat. We investigated thereproducibility of blood flow parameters measurements on opticnerve head and retinal arteries in Wistar rats along with the effects ofhyperoxia.Methods: Rats were immobilized on a modified stereotactic table,which allows free rotation of the instrument around the eye, in orderto aim the probing beam on the fundus. The fundus is illuminatedwith a green light and an image is captured with a CCD for easyTypical rat posture for ocular LDF recording. LDF is held on ahorizontal movable stereotatic table with a special mechanical devicepermitting three-dimensional movements (θ,φ,z) and micrometricpositioning of the probing beam on the rat's fundus.Commercial Relationships: Marielle Mentek, None; ChristopheChiquet, None; Frederic Truffer, None; Mario Bernabei, None;Benjamin Mottet, None; Jean-Paul Romanet, None; Diane Godin-Ribuot, None; Martial Geiser, 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 – Physiology/PharmacologySupport: Germaine de StaëlProgram Number: 4670 Poster Board Number: B0134Presentation Time: 11:00 AM - 12:45 PMRetinal blood velocity significantly decreased in rhegmatogenousretinal detachment and recovered after surgical treatment inboth artery and veinHirofumi Kinoshita, Kiyoshi Suzuma, Masafumi Uematsu, RyotaroUeki, Takashi Kitaoka. Nagasaki Univ School of Medicine,Nagasaki, Japan.Purpose: To evaluate retinal blood velocity by laser speckleflowgraphy (LSFG) in rhegmatogenous retinal detachment (RRD)patients before and after vitrectomy surgery.Methods: Subjects included 36 patients (36 eyes) who hadundergone successful vitrectomy surgery for unilateral RRD fromMay 2010 to March 2011. Preoperative, and 1- and 3-monthpostoperative retinal blood velocities were measured. We determinedmean retinal blood velocity via LSFG measurements of the mean blurrate (MBR) of the major vessels in the optic disc, retinal artery andretinal vein beside the optic disc corresponds to detachment area.When we investigated correlation between the preoperative MBR andthe postoperative MBR in this study, we calculated the values as thepercent of the preoperative MBR.Results: 18 males and 18 females patients (mean age 56.3 ± 10.6years, ranging from 32 to 74) with RRD were included in this study.All of the patients underwent 23-gauge pars plana vitrectomy, whichincluded SF6 gas tamponade. The average preoperative visual acuity,which was expressed as the LogMAR, was 0.62 ± 0.76. Significantimprovement of the LogMAR was noted at 3 months after thesurgery (0.10 ± 0.28; P < 0.0001). After surgery, average MBR of themajor vessels in the optic disc of the affected eyes graduallyimproved, and the average 3-month postoperative MBR (130.43 ±36.18%; P < 0.01) significantly increased as compared to the averagepreoperative MBR. Moreover, 1-month and 3-month postoperativeMBR of retinal artery in detachment area were 141.1 ± 32.7% and144.1 ± 30.2%, respectively as compared to preoperative MBR ofretinal artery in detachment area. 1-month and 3-month postoperativeMBR of retinal vein in detachment area were also increased to 117.5± 18.8% and 125.4 ± 20.4%, respectively as compared topreoperative MBR of retinal vein in detachment area.Conclusions: LSFG results showed that the mean retinal bloodvelocity in the optic disc, both retinal artery and vein in detachmentarea recovered after successful vitrectomy surgery for RRD patients.Commercial Relationships: Hirofumi Kinoshita, None; KiyoshiSuzuma, None; Masafumi Uematsu, None; Ryotaro Ueki, None;Takashi Kitaoka, NoneProgram Number: 4671 Poster Board Number: B0135Presentation Time: 11:00 AM - 12:45 PMOcular rigidity as estimated based on measurement of pulseamplitude using pneumotonometry and fundus pulse isdependent on intraocular pressureAnton B. Hommer 1, 2 , Gerhard Garhofer 2 , Leopold Schmetterer 2 .1 Krankenhaus Hera Vienna, Vienna, Austria; 2 Dep.of. ClinicalPharmacology, University Clinic, Vienna, Austria.Purpose: Evidence has accumulated that the biomechanicalproperties of the optic nerve head and the sclera play a role in thepathophysiology of glaucoma. In this study, the hypothesis was testedthat abnormal ocular structural stiffness as estimated based onmeasurements of intraocular pressure amplitude and ocular funduspulsation amplitude is dependent on intraocular pressure.Methods: Onehundredsixtyeight healthy subjects aged between 18and 45 years were included in this study. The ocular pulse amplitudeand pulsatile ocular blood flow were assessed withpneumotonometry. The fundus pulsation amplitude was measured byusing laser interferometry. Based on the Friedenwald equation, acoefficient of ocular rigidity (E1) was calculated relating pulseamplitude to fundus pulsation amplitudeResults: The ocular fundus pulsation amplitude was 4.21 ± 0.99 μm,the ocular pulse amplitude was 3.07 ± 0.61 mmHg. This resulted in acalculated factor E1 of 0.047 ± 0.007 AU. Multiple regressionanalysis revealed that fundus pulsation amplitude and pulseamplitude were dependent on pulse pressure amplitude (p < 0.001)whereas E1 was dependent on intraocular pressure (p < 0.001).Conclusions: The present study indicates that ocular rigidity isdependent on intraocular pressure.Commercial Relationships: Anton B. Hommer, Allergan (R),Allergan (C), Alcon (R), Santen (R), Merck (R); Gerhard Garhofer,None; Leopold Schmetterer, NoneProgram Number: 4672 Poster Board Number: B0136Presentation Time: 11:00 AM - 12:45 PMSphingosine 1-phosphate elicits constriction of isolated porcineretinal arteriolesTakayuki Kamiya, Taiji Nagaoka, Tsuneaki Omae, Shinji Ono,Akitoshi Yoshida. Asahikawa Medical University, Asahikawa, Japan.Purpose: Sphingosine 1-phosphate (S1P), a member of a largefamily of lipid metabolites called sphingolipids, induces a widevariety of biologic responses, i.e., immune responses, inflammatoryprocesses, organ perfusion, and regulation of vascular tone indifferent organs through various high-affinity G-protein-coupledreceptors (S1PR) 1-5. However, few reports have addressed the effectof S1P on the retinal circulation. We examined the effect of S1P todetermine the signaling mechanisms involved in the retinalmicrovasculature.Methods: Porcine retinal arterioles (internal diameter, 70-100 µm)were isolated, cannulated, and pressurized (55 cmH2O) without flowin this in vitro study. Videomicroscopic techniques were used torecord the changes in diameter in response to S1P.Results: The retinal arterioles were constricted in a dose-dependentmanner (1 nM-10 µM) in response to S1P. This vasoconstrictiveresponse to S1P was not attenuated after removal of the endothelium.Blockade of S1PR2 by the S1PR2 antagonist JTE-013 abolished thevasoconstrictive response to S1P, whereas the S1PR1 antagonist(compound 5) and the S1PR3 antagonist (suramin) did not affect thevasoconstrictive response. The PKC inhibitor chelerythrine and storeoperatedcalcium channels (SOCE) blocker (2-APB) significantly(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology5 Cardiovascular Medicine, The Ohio State University, Columbus,OH.Purpose: Intraocular pressure (IOP) is pulsatile in nature due to thefilling of the ocular blood vessels with each ventricular contraction.Ocular pulse amplitude (OPA) is defined as the difference betweenmaximum and minimum deflections in this signal. Average values ofOPA in healthy subjects range from 1-4mmHg. Normal appearanceof the waveform includes regular cycles corresponding to theheartbeat. The purpose of the current investigation was to determinethe source of an irregular waveform of extreme values.Methods: Ocular pressure waveforms were digitally recorded fromboth eyes of a subject being examined during training to use thePASCAL Dynamic Contour Tonometer (DCT), with custom softwareprovided by the manufacturer (Ziemer, Port, Switzerland). It wasnoted that the waveform had an unusual shape consistent with anearly ventricular contraction every other beat, as seen in Figure 1. Itwas also noted that ocular pulse amplitude (OPA) was >9mmHg,which is extraordinarily high. The subject presented for a thoroughexamination by a cardiologist.Results: The subject was determined to be in ventricular bigeminy,as seen in Figure 2, which was the source of the irregular appearanceof the OPA waveform. In addition, it was determined that he hadaortic insufficiency which explained the extreme value for OPA thatcorresponded to bounding carotid pulses via palpation. Afterreplacement of the aortic valve, the bigeminy resolved and the ocularpulse waveform became regular in appearance with an OPA of 1.6-2.0mmHgConclusions: The ocular pulse waveform is a direct reflection ofhemodynamics. Evaluating this waveform may provide an additionalopportunity for screening subjects for cardiovascular abnormalities.Wednesday, May 08, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 5043-5075/B0001-B0033Organizing Section: Physiology/PharmacologyProgram Number: 5043 Poster Board Number: B0001Presentation Time: 2:45 PM - 4:30 PMA minimally invasive and localised intra-scleral delivery of drugloadedthermoresponsive polymeric implants to treat posteriorocular diseasesHannah L. McMillan, Steven J. Fallows, Thakur Raghu R. Singh,David S. Jones. School of Pharmacy, Queen's University Belfast,Belfast, United Kingdom.Purpose: This study evaluates minimally invasive hollowmicroneedles (HMNs) to deliver thermoresponsive-based intrascleralimplants for sustained drug release.Methods: Gel formulations containing thermo-responsive polymeri.e. poloxamer, loaded with fluorescein sodium (FNa), were evaluatedfor rheological analysis, syringeability through HMNs & penetrationforces of HMNs into rabbit sclera. Visualisation of gel-based implantformation in sclera and scleral recovery were also determined, usingoptical coherence tomography (OCT). Ex vivo FNa release followingintrascleral delivery of gels was tested in Franz-diffusion cells.HMNs were fabricated from different hypodermic needles (i.e. 26G,29G & 30G) that were adjusted to different heights (i.e., 400, 500 or600 µm).Results: Gelation temperatures of poloxomer formulations rangedfrom 20.8 - 30.7°C, which showed Newtonian behaviour at 20°C andpseudoplastic (shear-thinning) behaviour at 37°C. The maximumforce and work required in expelling gels from HMNs increased withpoloxamer concentration, with volume of gel expelled (30, 50 or 100µL) and with decrease in needle aperture (26G to 30G). Neverthelesslow force (i.e., 0.123 - 2.021 N) & work (0.139 - 6.000 Ns) wasrequired across all variables tested. Forces required to penetrate thescleral tissue correlated well with increasing needle size and depth ofpenetration (equator; 0.549 - 1.159 N, anterior; 0.710 - 1.346 N,posterior; 0.905 - 1.589 N). OCT studies revealed that HMNs hassuccessfully able to penetrate to the required depth and allowedintrascleral injection of 50 µL of gel. Recovery of the sclerafollowing injection correlated well with penetration depth. FNarelease was 80.88- 87.56%, 70.33 - 75.02% and 52.86 - 60.63%,respectively after 24 h, when 50 µL of FNa-poloxamer gels wereinjected at a depth of 400 µm, 500 µm and 600 µm, intrasclerally.Conclusions: This study has indicated the potential for sustaineddelivery when using thermo-responsive polymers injectedintrasclerally via HMNs and also scleral recovery and implantvisualization. Results suggest that this method may offer a minimallyinvasive means of treating posterior segment diseases.Commercial Relationships: Jean B. Kassem, None; Steven E.Katz, None; Cynthia J. Roberts, Oculus Optikgerate GmbH (C),Ziemer Ophthalmic Systems AG (C), Sooft Italia (R), Carl ZeissMeditec (F); Ashraf M. Mahmoud, None; Robert H. Small, Ziemer(F); Subha V. Raman, None468 Drug Delivery IIIOCT images of 50 µL (red) injected to a depth of 400 µm at 0 h (a), 1h (b) and 2 h (c). Dashed yellow shows empty space in sclera createdvia injection and its subsequent closure.©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 – Physiology/PharmacologyCommercial Relationships: Hannah L. McMillan, None; Steven J.Fallows, Wellcome Trust (F); Thakur Raghu R. Singh, None;David S. Jones, NoneSupport: Wellcome Trust Biomedical Vacation Scholarship 2012Program Number: 5044 Poster Board Number: B0002Presentation Time: 2:45 PM - 4:30 PMHindered convective transport of nanoparticles andmacromolecules in the vitreous humorAnita N. Penkova 1, 2 , Komsan Rattanakijsuntorn 1 , Yang Tang 2 , RexMoats 2, 3 , Michael R. Robinson 4, 2 , Susan S. Lee 4, 2 , Satwindar S.Sadhal 1, 2 . 1 Aerospace & Mechanical Engineering, University ofSouthern California, Los Angeles, CA; 2 SAIRC, Saban ResearchCenter, Children’s Hospital Los Angeles, Los Angeles, CA;3 Biomedical Engineering, University of Southern California, LosAngeles, CA; 4 Global Pharmaceutical Sciences, Allergan, Inc, Irvine,CA.Purpose: 1. To investigate the extent of transport enhancement ofmacromolecules and nanoparticles due to convective flow of water inthe vitreous humor.2. To establish deviation from convective transport predictions byforced-flow experiments and comparison with conventional theorycalculations.3. To propose corrections to convective transport to allow for largeparticlehindrance in the vitreous humor.Methods: Fresh ex vivo whole bovine eyes were individuallyinjected with two gadolinium-based contrast agents [Gd-Albumin,and 30 nm gadolinium-based particles (Gado CELLTrackTM,Biopal, Worcester, MA)]. After imaging the eye, the contrast-agentbolus was subjected to a high rate of water flow with a syringe pump.The water (0.9% saline solution) was injected at the top of the bovineeye at 100 μl/min, and was allowed to drain through small slits cut atthe bottom of the eyeball. After one hour of pumping, the eye wasimaged again and the contrast-agent distribution was obtained. Fortheoretical comparison the convective transport was also evaluated bynumerical modeling based on the conventional convection-diffusionequation.Results: The experimentally measured convective transport of Gd-Albumin and 30 nm nanoparticles was found to be somewhat slowerthan the carrier (0.9% saline solution). The conventional convectiondiffusionequation with the u.▽c term for convective transport seemsto over-predict the convection rate, indicating some degree ofhindrance to convection for nanoparticles and Gd-Albumin.Conclusions: It has been claimed in several theoretically-basedinvestigations that convection significantly enhances the transport oflarge particles and macromolecules in the vitreous humor. However,current investigations have raised questions about the validity ofusing the unhindered convective term u.▽c which does notdiscriminate between the different solutes. We have hypothesizedthat large molecules, when subjected to high rate of water flow in thevitreous humor, will experience resistance, depending on therespective permeabilities of the injected surrogate solute. We areproposing that the usual convection term be adjusted to allow for thefiltration effect on the larger particles in the form (1-σ)u.▽c withimportant implications for computational modeling.Commercial Relationships: Anita N. Penkova, Allergan, Inc (F);Komsan Rattanakijsuntorn, None; Yang Tang, None; Rex Moats,Allergan (F); Michael R. Robinson, Allergan (I); Susan S. Lee,Allergan, Inc. (E); Satwindar S. Sadhal, Allergan, Inc. (F)Support: This work has been supported with a Research Grant byAllergan, Inc.Presentation Time: 2:45 PM - 4:30 PMSlow-release intraocular drug delivery by injectable PEAmicrofibrils (DSM, NL)Gabriele Thumann 1 , George Mihov 2 , Jens Thies 2 , Anja Kemp 2 ,Katharina Morawa 1 , Martina Kropp 1 . 1 Department ofOphthalmology, University of Geneva, Geneva, Switzerland; 2 DSM,Geleen, Netherlands.Purpose: Ophthalmic drug therapy of the posterior segment requiresthat effective concentrations of drug reach the target tissue, a goalcomplicated by limited penetration, short half-life of many drugs anddifficult access to the posterior segment. Generally drug delivery tothe posterior segment of the eye is accomplished by intravitreal (ivt)injection, often requiring frequent injections, e.g. AMD treatmentwith ranibizumab or aflibercept. Continuous drug delivery systemswould be useful to avoid frequent administration and to deliver thedrug at a more physiological concentration. Here we report propertiesof biodegradable amino acid polyester amide (PEA) polymers (DSM,NL) that can deliver a spectrum of drugs in a sustained, zero-orderbehavior and degrade via an enzyme-mediated pathway.Methods: Deformability and in vitro degradation of the PEA fibrils(120-300 µm in diameter) was analyzed by incubation at 37o C inPBS, in α-chymotrypsin, in human, rabbit and bovine vitreous. Invivo biocompatibility and degradation was analyzed bysubconjunctival (sc) and ivt implantation in normal and VEGFtreatedrabbits that simulate blood-retinal barrier breakdown. Drugrelease by single PEA fibrils loaded with 10% dexamethasone wasanalyzed in vitro.Results: During the first 2 hours the fibrils decreased in length 57%in PBS, 62% in rabbit vitreous and 59% in human vitreous; thedecrease in length was accompanied by an increase in diameter.Degradation by chymotrypsin depended on the amino acidcomposition of the PEA. Degradation in normal vitreous was slightbut increased in vitreous from VEGF-treated rabbits. Sc and ivt PEAfibrils were well tolerated with no evidence of inflammation, retinaldamage or changes in IOP. After an initial peak dexamethasone wasreleased at a constant rate of 0.05-1.0 µg/ml for 140 days, whereasPLGA 75/25 did not release significant amounts of dexamethasoneduring the first 120 days, followed by 90% release from day 120 today 140.Conclusions: PEA polymers (DSM, NL) are well tolerated in theocular environment, degrade slowly intravitreally and can be loadedwith drugs that are released with zero-order kinetics over a period ofmonths. Since PEAs are well tolerated in the ocular environment andcan be manufactured to be loaded with different classes of drugs,these materials would be ideal for sustained delivery of drugs to theposterior segment.Commercial Relationships: Gabriele Thumann, DSM (F); GeorgeMihov, DSM (E); Jens Thies, None; Anja Kemp, DSM (E);Katharina Morawa, DSM Biomedical (F); Martina Kropp, DSM(F)Support: DSMProgram Number: 5046 Poster Board Number: B0004Presentation Time: 2:45 PM - 4:30 PMFucoidan but not Mannan inhibits Bevacizumab uptakeindependently of phagocytosis and reduces VEGF expression inthe RPEMichaela Dithmer 1 , Tim Meyer 2, 3 , Elisabeth Richert 1 , JohannRoider 1 , Alexa K. Klettner 1 . 1 Ophthalmology, University of Kiel,University Medical Center, Kiel, Germany; 2 Immunology, Universityof Kiel, University Medical Center, Kiel, Germany; 3 MedizinischeKlinik I, Charite-Campus Benjamin Franklin, Berlin, Germany.Program Number: 5045 Poster Board Number: B0003©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 – Physiology/PharmacologyPurpose: We have previously shown that Bevacizumab is taken up inRPE cells and stored for at least seven days. In this study, we furtherinvestigate the mechanisms of uptake, focussing on the influence ofsugar moieties.Methods: For the experiments, Arpe19 cells and primary porcineRPE cells of passage 2 and 3 were used. Cells were treated with 250µg/ml Bevacizumab and evaluated after different time intervals (1 h -7 d). Fucoidan or Mannan, respectively, was applied at aconcentration of 100 µg/ml. Bevacizumab uptake was evaluated withimmunofluorescence. Phagocytosis was measured in a phagocytosisassay using opsonized FITC latex beads. VEGF expression wasdetermined in Western blot.Results: Fucoidan, but not Mannan, strongly reduces Bevacizumabuptake in RPE cells. Phagocytotic ability was not compromised byeither Fucoidan or Mannan. Fucoidan, but not Mannan, significantlyreduces VEGF165 and VEGF121 expression in Western blot.Conclusions: Fucoidan inhibits Bevacizumab uptake, which is notmediated via phagocytosis but may be connected to the reduction ofVEGF expression in RPE cells by Fucoidan. This may hint towardsuptake mechanism that involves immunocomplex binding.Commercial Relationships: Michaela Dithmer, None; Tim Meyer,None; Elisabeth Richert, None; Johann Roider, Novartis (F),Bayer (F); Alexa K. Klettner, Novartis (F), Novartis (C), Novartis(R), Santen (R)Support: DFG KL2425/2-1Program Number: 5047 Poster Board Number: B0005Presentation Time: 2:45 PM - 4:30 PMIn Vivo Pharmacokinetics of Injectable, Intravitreal, Sustained-Release Latanoprost FormulationsWilliam S. White, Mae Hu, Glenn Huang, Tan Pham, FainaKarasina, Aaron Lee, Maria Gorlina, Vernon Wong. IconBioscience, Inc, Sunnyvale, CA.Purpose: To study the in vivo pharmacokinetics of injectable,intravitreal, sustained-release latanoprost formulationsMethods: In vivo anterior chamber drug levels were measured inNew Zealand White rabbits receiving single doses of one of twoformulations of sustained-release latanoprost administered byintravitreal injection. Aqueous humor samples were obtained weeklyand analyzed for latanoprost acid concentrations using highperformanceliquid chromatography with mass spectrometry.Results: Sustained levels of latanoprost acid in the aqueous humorwere demonstrated for 3 to 7 months in animals injected with singledoses of sustained-release latanoprost formulations. The sustainedreleaseformulation containing 559 µg of latanoprost demonstratedsustained levels of anterior chamber latanoprost acid which decreasedto an average latanoprost acid aqueous humor level of 17.5 pg/mL onday 84. The sustained-release formulation containing 1118 µg oflatanoprost demonstrated sustained anterior chamber levels oflatanoprost acid which decreased to an average latanoprost acidaqueous humor level of 19.1 pg/mL on day 231.Conclusions: In vivo anterior chamber levels of latanoprost acidwere sustained for up to 7 months and were well-tolerated in rabbitsreceiving sustained-release latanoprost formulations injectedintravitreally.Commercial Relationships: William S. White, Icon Bioscience (E);Mae Hu, Icon Bioscience, Inc (E), Icon Bioscience, Inc (I); GlennHuang, Icon Bioscience, Inc. (P), Icon Bioscience, Inc. (E), IconBioscience, Inc. (I); Tan Pham, Icon Bioscience Inc (E), IconBioscience Inc (P); Faina Karasina, Icon Bioscience Inc (I), IconBioscience Inc (E); Aaron Lee, Icon Bioscience Inc (C), IconBioscience Inc (R); Maria Gorlina, None; Vernon Wong, IconBioscience Inc (E)Program Number: 5048 Poster Board Number: B0006Presentation Time: 2:45 PM - 4:30 PMHystem, a bio-absorbable protein delivery polymer: safety,tolerability and efficacy in a rabbit corneal debridement modelMaryJane Rafii 1 , Barbara M. Wirostko 1, 2 , Liliana Werner 2 , NickMamalis 2 , Thomas Zarembinski 4 , Stacy Pritt 3 , Glenwood G. Gum 3 .1 Ophthalmics, Jade Therapeutics, Salt Lake City, UT;2 Ophthalmology, University of Utah, SLC, UT; 3 Absorption Systems,SD, CA; 4 BioTime, Almeda, CA.Purpose: HyStemTM (BioTime, CA), a biodegradable hyaluronicacid (HA)-based polymer shown to promote wound healing, can beused for local, ocular sustained delivery of proteins. Safety,tolerability, and efficacy of this polymer, alone and in combinationwith recombinant human growth hormone (rhGH) to acceleratewound healing, was evaluated in a rabbit corneal debridement model(CDM). rhGH was selected based on its ability to activate growthfactors, e.g., Insulin-like Growth Factor and Epidermal GrowthFactor, that have been shown to be involved in corneal reepithelialization.Methods: To assess the tolerability of HyStem cross-linked withglutathione (GSSG); non-cross-linked HyStem, HyStem/GSSG, andRingers lactate (RL) (control) were applied four times a day (QID)for 4 days topically in a CDM using New Zealand rabbits (NZR)(N=3, 2 eyes/arm). Twice daily, slit lamp exams with photos wereemployed to evaluate healing. Tissues were harvested on day 5 andhistopathology was performed. To evaluate the efficacy ofHyStem/GSSG/rhGH in accelerating wound healing, a validatedNZR CDM model (N=11) was used. All 22 eyes received topicaldexamethasone (dex) QID for 7 days, alone (control; n=8 eyes), withHyStem/GSSG BID (n=4 eyes), or with HyStem/GSSG/rhGH BID (4μg/50 μL drop; n=10 eyes). Healing was assessed via daily slit lampphotos. Tissues were harvested on day 7 and histopathology wasperformed. Time to complete healing and daily % healing werecompared across groups.Results: Tolerability study revealed that HyStem and HyStem/GSSGwere well tolerated, with a trend for faster return to normal histologyvs. RL. In the efficacy study, HyStem/GSSG andHyStem/GSSG/rhGH yielded excellent safety and tolerability:histopathology was normal, with no inflammation or angiogenesis. Inspite of the small sample size, an efficacy trend was seen with a fasterrate to complete defect closure in the HyStem/GSSG/rhGH group asearly as Day 5. By Day 6, 80% of eyes with HyStem/GSSG/rhGHwere completely healed vs. 50% of HyStem/GSSG vs. 38% of dexalone.Conclusions: HyStem can deliver, rhGH, such that an efficacy signalfor faster corneal wound healing is demonstrated. HyStem andHystem/GSSG/rhGH were well tolerated in vivo, with normalhistopathology. HyStem is a viable polymer to deliver proteins, e.g.,rhGH, for corneal defects that have impaired healing.Commercial Relationships: MaryJane Rafii, Jade Therapeutics(P), Jade Therapeutics (S), Jade Therapeutics (I), Pfizer Inc. (C),Regenron (C); Barbara M. Wirostko, Jade Therapeutics (P), JadeTherapeutics (I), Jade Therapeutics (E), Altheos Inc. (C), Merck (C),SKS Ocular (C), USTAR (F); Liliana Werner, Aaren Scientific (F),Abbott Medical Optics (F), Advanced Vision Science (F), AlconLaboratories (F), Anew Optics (F), Bausch & Lomb Surgical (F),Calhoun Vision (F), Innovia (F), MRI Research (C), Powervision(C), Rayner Intraocular Lenses (F), Visiogen (C); Nick Mamalis,Abbott Medical Optics (F), Alcon Laboratories, Inc (F), Allergan (F),Anew (C), Bausch & Lomb (F), Calhoun Vision, Inc (F), NuViiew,Inc (F), OpticaMedica (C), Powervision (F); Thomas Zarembinski,BioTime, Inc. (E); Stacy Pritt, Jade Therapeutics (E), Absorption©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 – Physiology/PharmacologySystems (E), Amakem (E); Glenwood G. Gum, Jade Therapeutics(F)Support: The Utah Science Technology and Research initiative atthe University of Utah unrestricted TCIP grantProgram Number: 5049 Poster Board Number: B0007Presentation Time: 2:45 PM - 4:30 PMDelivery of Human Growth Hormone via DSM’s Poly(esteramide)Julien Bérard 1 , John Zupancich 1 , Audrey Hecka 1 , George Mihov 1 ,Sarah Reiver 1 , Jens Thies 1 , Kenneth A. Messier 1 , Barbara M.Wirostko 2 , MaryJane Rafii 2 . 1 DSM, Geleen, Netherlands; 2 JadeTherapeutics, LLC, Salt Lake City, UT.Purpose: Fibrillar, degradable polymer-based constructs for thelocal, sustained delivery of recombinant human growth hormone(rhGH) were generated. The safety, tolerability and possible efficacyof subconjunctivally placed devices was evaluated in a rabbitdebridement model. rhGH was selected based on an ability to upregulateand modulate various growth factors (i.e., insulin growthfactor, epidermal growth factor) that have been shown to be involvedin corneal re-epithelialization.Methods: rhGH-loaded, multi-layer constructs were prepared viaencapsulation of solid protein within poly(ester amide) (PEA)matrices through use of an innovative film formation and assemblyprocess. Constructs were subsequently shaped into fibers withdimensions that facilitate passage through a 27 gauge needle.Construct in vitro release performance was evaluated over a period of30 days and the bioactivity of released rhGH confirmed via aproliferative cell assay. In vivo experiments compared PEA/rhGH(~12ug/device) and PEA (i.e., blank) constructs placedsubconjunctival, perilimbal to BSS and topical rhGH (100ug/ml)delivered four times daily in a standardized rabbit corneal epithelialdebridement model (N=9, 18 eyes). Safety, tolerability, and efficacywere assessed daily and histopathology was performed on Day 7.Time to complete healing and daily percent healing was comparedacross arms over 7 days.Results: In vitro results demonstrate that over 90% of rhGHencapsulated within early generation PEA fibers is released over thecourse of 3 days. Advances in multi-layer construct design reveal thepotential to sustain the release of rhGH for multiple weeks. In vivoresults showed PEA/rhGH and PEA constructs were well tolerated,with histopathology on all eyes revealing normal healing with noinflammation or angiogenesis. An efficacy signal was difficult toascertain due to the small number of animals utilized in the currentstudy, and the rapid healing that occurs in normal healthy rabbits.Conclusions: Subconjunctivally placed rhGH-loaded PEA fiberswere well tolerated in vivo and no histopathologic concerns wereidentified. In order to test efficacy, a preclinical model of impairedwound healing, such as an alkali burn, should be utilized. Studyresults indicate that PEA could be a viable polymer for the sustaineddelivery of proteins to the eye.Commercial Relationships: Julien Bérard, DSM (E); JohnZupancich, DSM (E); Audrey Hecka, DSM (E); George Mihov,DSM (E); Sarah Reiver, DSM (E); Jens Thies, None; Kenneth A.Messier, DSM Biomedical (E); Barbara M. Wirostko, JadeTherapeutics (P), Jade Therapeutics (I), Jade Therapeutics (E),Altheos Inc. (C), Merck (C), SKS Ocular (C), USTAR (F);MaryJane Rafii, Jade Therapeutics (P), Jade Therapeutics (S), JadeTherapeutics (I), Pfizer Inc. (C), Regenron (C)Program Number: 5050 Poster Board Number: B0008Presentation Time: 2:45 PM - 4:30 PMDesign of a Non-Invasive Core-shell Nanoparticulate DrugDelivery System for Posterior Part of the EyeBinapani Mahaling, Dhirendra S. Katti. Biological Sciences andBioengineering, Indian Institute of Technology Kanpur, Kanpur,India.Purpose: Providing drug molecules to the posterior part of the eye intherapeutic concentration and for adequate durations is difficultbecause of barrier properties of different layers of the eye. Thedifferential permeability towards hydrophilic or hydrophobicmolecules and surface charge of different layers of the eye furthercomplicates the matter. Hence, the purpose of this study was todevelop a noninvasive core-shell nanoparticle-based drug deliverysystem for targeting the posterior part of the eye. It was hypothesizedthat a nanoparticulate system with a hydrophobic core and ahydrophilic shell can potentially overcome the barriers due to thepreferential permeability of hydrophilic molecules in the outer andhydrophobic molecules in the inner layers of the eye.Methods: Nanoparticle cores of poly(lactic acid) (PLA) were coatedwith a chitosan (CHI) shell. CHI was chosen due to hydrophilicnature and its potential to overcome the tear fluid barrier because ofits positive charge, bioadhesive and permeability enhancingproperties. PLA and coumarin loaded PLA nanoparticles weresynthesized by emulsion solvent evaporation method and modifiedwith chitosan by adsorption followed by chemical crosslinking.Coumarin loaded PLA and PLA-CHI formulations were administeredas eye drops onto the eye of C57BL/6J mice with PBS, PLA andPLA-CHI nanoparticles as controls. At predetermined time pointsmice were euthanized. The eyes were then enucleated, fixed,sectioned and analyzed under a fluorescence microscope.Results: Fluorescence was detected in coumarin loaded PLA andPLA-CHI particles in retina indicating the penetration ofnanoparticles across the layers up to the retina, whereas, the controlgroups showed no fluorescence in retina. The levels of fluorescenceobserved for PLA-CHI core-shell nanoparticles was higher than thatof PLA nanoparticles at the retina indicating the possible role ofPLA-CHI in enhancing penetration.Conclusions: These results suggest that PLA-CHI core-shellnanoparticles show potential to be developed as a non-invasive drugdelivery system for posterior part of the eye.Commercial Relationships: Binapani Mahaling, None; DhirendraS. Katti, NoneSupport: Indian Council of Medical ResearchProgram Number: 5051 Poster Board Number: B0009Presentation Time: 2:45 PM - 4:30 PMDevelopment and characterization of silicone pressure sensitiveadhesive episcleral implantHe Wen, S Kevin Li. University of Cincinnati, Cincinnati, OH.Purpose: Ocular implants provide prolonged delivery of therapeuticagents to the eye, minimize systemic drug exposure, and reduce theneed of multiple intraocular drug injections. Episcleral implants areocular drug delivery systems that prolong drug delivery through thetransscleral route. Materials currently available for constructingocular implants are limited. Silicone pressure sensitive adhesives(PSA) are non-toxic and highly compressible, have high cohesivestrength, and offer good adhesion to biological substrates. Thismaterial can form a polymer-drug matrix that improves drugcompactibility and allows prolonged drug release at low polymercontent. The objectives of this study were to develop and characterizea silicone PSA episcleral implant system for transscleral delivery ofsmall molecules and macromolecules.Methods: Dexamethasone, atenolol, and bovine serum albumin(BSA) were selected as the model drugs. Silicone PSA 7-4302 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 – Physiology/Pharmacologythe polymer used to construct the episcleral implant. The implant hadan average diameter of 3.5 mm and thickness of 0.7 mm. In vitrodrug release experiments were conducted with implants of varyingpolymer to drug mass ratios in a well-stirred vial system.Results: The silicone PSA episcleral implants provided sustaineddrug release that could last for over one month in the present study invitro. Drug release from the implants followed a dissolutioncontrolled release mechanism with the higher water solubility drugshowing faster release rate than the low solubility drug. Increasingpolymer content in the implant led to a significant decrease in thedrug release rate.Conclusions: Drug release from the silicone PSA episcleral implantis influenced by the solubility of the drug and the polymer content inthe implant. The episcleral implant demonstrates the ability toprovide drug release for an extended period of time, and therefore hasthe potential to offer safe and effective treatment to chronic oculardiseases via the transscleral route.Commercial Relationships: He Wen, None; S Kevin Li, Aciont Inc(C)Program Number: 5052 Poster Board Number: B0010Presentation Time: 2:45 PM - 4:30 PMOcular injection characteristics and in vitro release profiles oflipophilic dye using thermogel PLGA-PE-PLGA as a sustainedreleasedrug delivery deviceEva Abarca, Page M. Potter, Jacklyn H. Salmon, Brian C. Gilger.North Carolina State University, Raleigh, NC.Purpose: Thermosensitive biodegradable gels may be useful forsustained delivery of drugsintravitreally (IVT) or into the suprachoroidal space (SCS). Thepurpose of this study is to determine if a thermogel polymer canimprove release kinetics of lipophilic carbocyanine dye (DiI) and toevaluate if these polymers can be effectively injected into the eye.Methods: Release kinetics of DiI from poly-(d,l-lactic acid-coglycolicacid)-polyethylene glycol (PLGA-PEG-PLGA) wasdetermined in vitro. Liquid 20% PLGA-PEG-PLGA (200 µl at 2°C)was added to glass vials, either alone or mixed with 0.1 mg DiIcrystals, 0.1 mg DiI dissolved in ethanol, or 0.1 mg DiI dissolved inethanol with 2% hyaluronic acid (HA). A 5th group had no PLGA-PEG-PLGA, just 0.1 mg DiI crystals. The vials were then maintainedat 37°C allowing gels to form,1 ml of PBS was added and dailysamples were collected for analysis by spectrofluoroscopy for 21days. To characterize ocular injection and gel formation, 12normothermic ex-vivo pig eyes were injected with liquid thermogel(2°C) with 0.1 mg DiI. Six eyes were injected into the SCS using a33 gauge 750 depth microneedle, whereas 6 eyes were injected IVT.High-frequency (50Mhz) or 20 Mhz ultrasound was used to imageand to evaluate the effect and distension in the SCS or vitreous,respectively.Results: Release of DiI from thermogel was significantly higher thanDiI crystals over the first 7 days. Area under the curve (AUC) wassignificantly higher (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyDexamethasone Sustained Delivery From PolyesteramideMicrospheres For Intraocular Administration. Influence OfSterilizationVanessa Andres-Guerrero 1 , Mengmeng Zong 2 , George Mihov 2 ,Aylvin A. Dias 2 , Rocio Herrero-Vanrell 1 . 1 PharmaceuticalTechnology, Faculty of Pharmacy/Complutense University, Madrid,Spain; 2 DSM, Geleen, Netherlands.Purpose: Amino acid based polyesteramides (PEAs) are a newfamily of biodegradable polymeric materials based on α-amino acids,aliphatic dicarboxylic acids and aliphatic α-ω diols. The aim of thecurrent study was to determine the effect of a sterilization dose of γ-irradiation (25kGy) on the release profile of dexamethasone (DX)-loaded PEA microspheres (Ms), to evaluate the use of these systemsas carriers for controlled intraocular drug delivery.Methods: DX-loaded Ms were prepared following an emulsionsolventevaporation technique. Ms were characterized by particle size(dynamic light scattering), morphology (scanning electronmicroscopy) and DX-encapsulation efficiency. In vitro release studieswere carried out for 24 days by suspending the Ms (5mg) in 1.5ml ofPBS (pH 7.4, 37C) isotonized with NaCl. At pre-set times (1h, 24hand twice a week for 24 days) the release medium was collected andDX levels were quantified by HPLC. Freeze-dried DX-loaded Mswere sterilized with a 60Co radiation source (25 kGy). Sterilized Mswere characterized following the same procedures described above.Results: PEA Ms were spherical and had smooth surface with sizesranging between 20-40 µm in all cases. The encapsulation efficiencywas 85.14±0.99% for non-sterilized DX- Ms and 85.28±0.81% forsterilized DX-Ms (180.99±2.39 µg DX/mg Ms and 181.57±2.76 µgDX/mg Ms, respectively). The DX release profile of non-sterilizedMs had an initial burst of 17.69±0.58 µg DX/mg Ms released in thefirst 24 h, similar to the one obtained for sterilized Ms (17.28±0.44µg DX/mg Ms). A progressive sustained release of DX was observedfor the following days. At day 24, the cumulative release of DX was80.86±2.40 µg DX/mg Ms for non-sterilized Ms and 79.45±4.74 µgDX/mg Ms for sterilized Ms (45.27±1.11% and 43.80±3.28% of theencapsulated DX, respectively).Conclusions: PEA Ms developed are capable of providing asustained release of DX for at least 24 days. Non significantdifferences were found between sterilized and non-sterilized MS.Gamma-radiation had no significant effect on the release profile ofthe microspheres. Biodegradable PEA Ms are potentially useful todevelop new controlled drug delivery systems for treating ophthalmicdiseases affecting the back of the eye.Commercial Relationships: Vanessa Andres-Guerrero, None;Mengmeng Zong, DSM (E); George Mihov, DSM (E); Aylvin A.Dias, DSM Biomedical (E); Rocio Herrero-Vanrell, NoneSupport: PANOPTES (project number 246180) under the 7thResearch Framework Program of the European Union.Program Number: 5055 Poster Board Number: B0013Presentation Time: 2:45 PM - 4:30 PMPharmacodynamic study of Intravitreal liposomal doxorubicinby matrix-assisted laser desorption/ionization-mass spectrometry(MALDI/MS)Hsi-Kung Kuo, Yi-Hao Chen, Pei-Chang Wu. Ophthalmology,Kaohsiung Chang Gung Memorial Hospital and Chang GungUniversity College of Medicine, Kaohsiung, Taiwan.Purpose: to investigate the pharmacodynamics of doxorubicin andliposomal doxorubicin (Lipo-Dox) after intravitreal injection.Methods: Pigmented rabbits were used. One eye acceptedintravitreal injection of doxorubicin or Lipo-Dox (10 μg/ml). Anothereye was injected with 0.1 ml BSS as the control. To evaluate thepharmacodynamic change of the drugs, the animals were sacrificedon the day 1, 3, 5, 7 and 14 after the injection. The vitreous contentsand retina extracts were prepared for LC/MS/MS and MALDI/MSstudy. On the matrix-assisted laser desorption/ionization-imagingmass spectrometry (MALDI/IMS) group, the full eye sections areprepared from cryo-section.Results: Pharmacodynamic study by MALDI/MS showed liposomaldoxorubicin existed in the vitreous for at least 14 days with thehighest density on day 7. The result is compatible with the slowlyreleased property of liposomal drugs. To study the drug distributionwith MALDI/IMS was failed due to background interference.Conclusions: Intravitreal injection of liposomal doxorubicin canobtain a sustained drug concentration for at least 14 days with thepeak on day7.Commercial Relationships: Hsi-Kung Kuo, None; Yi-Hao Chen,None; Pei-Chang Wu, NoneSupport: CMRPG891281 from Chang-Gung Memorial Hospital,TaiwanProgram Number: 5056 Poster Board Number: B0014Presentation Time: 2:45 PM - 4:30 PMCavernous Sinus as Ocular Pharmacokinetic Compartment forBetter Understanding of Posterior Segment and ContralateralEye Drug AvailabilityMuhammad Abdulrazik. Ophthal/Innovative Interventions, EastJerusalem Biomedical Institute, East Jerusalem, Occupied PalestinianTerritory.Purpose: To study the possibility that the drainage of drug loadedocular blood to the cavernous sinus is coupled with returned drugtransfer in the opposite direction, not through the systemiccirculation.Methods: Texas-Red labeled dextran (40-kDa) solution (2.5 mg/ml)was injected into the sub-conjunctiva of the rat eye. Eyes wereenucleated and intracranial optic nerves, up to the chiasm, wereharvested. Tissues were snap-frozen and processed for visualizationby fluorescence microscopy. In an extension of previous brimonidineocular pharmacokinetic study, whole-tissue brimonidine incontralateral eye tissues was quantified at 5 minutes after singletopical instillation of 50µl of 3H-radiolabeled brimonidine solution(0.2%) to the right eye of albino rabbits.Results: In the area of the optic nerve head, dextran was heavilyloaded in episcleral and periocular veins but absent in the posteriorciliary artery. In the sampled intracranial segment of the optic nerve,just before the junction with the optic-chiasm, nerve axons were freeof dextran while pial and sheath veins were draining dextran towardsthe cavernous sinus. Whole-tissue brimonidine calculation was basedon the evidenced drug concentration per gram tissue and whole-tissueweight. The sum of brimonidine accumulation at 5 min. post dosingfor all tissues of the contralateral eye was 151.08±39.21 ng. Theestimated amount of brimonidine that could have entered the eye viablood circulation in first 5 minutes post dosing (67.3±26.8 ng) wascalculated by using previously reported value of blood flow perminute into the rabbit eye and the evidenced brimonidineconcentration in the systemic blood in current study.Conclusions: The dextran study has shown the accumulation ofdextran in the cavernous sinus area of the rat by non-systemicallymediated vascular drainage. The evidenced brimonidineaccumulation in the contralateral rabbit eye (151.08±39.21 ng) wassignificantly higher than highest possible amount of brimonidine(67.3±26.8 ng) that could be extracted from the systemic blood bycontralateral eye tissues, suggesting that contralateral eyebrimonidine accumulation represents in part returned drug transferfrom the cavernous sinus toward the eye via non-systemic vascular©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 – Physiology/Pharmacologyconnections. Further studies are needed to support the integration ofthe cavernous sinus in ocular pharmacokinetic modeling.Commercial Relationships: Muhammad Abdulrazik, NoneProgram Number: 5057 Poster Board Number: B0015Presentation Time: 2:45 PM - 4:30 PMInfluence of Physicochemical Properties on Drug Delivery acrossSclera into Choroid-RetinaAyumi Yoshimatsu 1 , Chiho Yabuta 1 , Akira Ohtori 1, 2 , MitsuyoshiAzuma 1 . 1 Senju Laboratory of Ocular Sciences, Senju PharmaceuticalCo., Ltd., Kobe, Japan; 2 Laboratory of Ocular Drug Delivery System,Kyushu Institute of Technology, Fukuoka, Japan.Purpose: Intravitreal injections are currently used to deliver drugs tothe retina and other tissues in the back of eye. Topical instillation ofophthalmic drugs would be more comfortable for patients and lowerthe risk of infection. Reports indicate that topical instillation of somecompounds indeed diffuse from the posterior periocular tissues acrosssclera and reach the posterior choroid-retina. The specificphysicochemical properties of such drugs would be important fortheir permeability, diffusivity and partitioning into ocular tissues, butsuch data are limited. Thus, the purpose of the present study was todetermine the influence of physicochemical properties of selecteddrugs on their passage across sclera into choroid-retina.Methods: Model compounds were selected to range in molecularweight (MW) from 300 to 10,000 and included a wide range oflipophilicity. Sclera and sclera-choroid-retina were excised fromrabbit globes, and were mounted side-by-side in Ussing diffusionchambers. Model compounds in buffer were added to the donorchamber adjacent to sclera, while buffer solution alone was present inthe receptor chamber. Permeation rates, time lags, and permeabilityacross sclera or sclera-choroid-retina preparations were determinedby pharmacokinetics modeling. Diffusion and partition coefficientswere then calculated based on a bilayer membrane model.Results: Cumulative transport of drug across sclera and sclerachoroid-retinaincreased as drug concentration in donor solutionincreased except for compound of MW 10,000. Permeability wasnegatively correlated with MW in sclera and sclera-choroid-retina.Drug diffusivity in sclera and choroid-retina decreased as MWincreased, and diffusion coefficients in choroid-retina were 3 to 10times less than that in sclera. Partition coefficients into choroid-retinaincreased as lipophilicity increased. This relationship was notobserved for sclera.Conclusions: Optimal modification of ocular drugs used for topicalinstillation needs to decrease MW and to balance solubility withlipophilicity.Commercial Relationships: Ayumi Yoshimatsu, SenjuPharmaceutical Co., Ltd. (E); Chiho Yabuta, Senju PharmaceuticalCo., Ltd. (E); Akira Ohtori, SENJU PHARMACEUTICAL CO.,LTD. (E); Mitsuyoshi Azuma, Senju Pharmaceutical Co., Ltd. (E)allogeneic rat corneal grafts after penetrating keratoplasty (PK).Methods: A total of 48 PK were performed using Fisher rats(allogeneic groups) and Lewis rats (syngeneic group) as donors andLewis rats as recipients: group 1 (n=12), syngeneic control; group 2(n=12), allogeneic control without treatment; group 3 (n=12),allogeneic grafts with subconjunctivally-implanted PA-loadedmicrofilm treatment; group 4 (n=12): allogeneic grafts with 1% PAeye drops treatment. All grafts were evaluated for 28 days by ascoring rejection index (RI), assessing graft opacity, edema andneovascularization by slit lamp biomicroscopy and anterior segmentoptical coherence tomography (ASOCT). Time to rejection wasanalyzed with Kaplan-Meier survival analysis. PA concentrations inthe aqueous humor were measured using high-performance liquidchromatography. Histopathological and immunohistochemicalstaining were also performed.Results: The PA-loaded microfilms achieved a sustained and steadyrelease at a rate of 7.0ug/day, with a consistent drug concentration of207-209 ng/ml in the aqueous. The mean survival was >28 days ingroup 1, 9.9±0.8 days in group 2, 26.8±2.7 days in group 3 (P =0.023 as compared with group 2), and 26.4±3.4 days in group 4 (P =0.027 as compared with group 2). Statistically significant decrease inCD4+, CD8+, CD163+, CD11c+, CD 25+ and CD54+ cellinfiltration in group 3 as compared with group 2 was observed onimmunohistochemistry (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyFigure 2: Kaplan-Meier analysis of the rejection-free graft survivalCommercial Relationships: Yu-Chi Liu, None; Yan Peng, None;Nyein Chan Lwin, None; Subbu S Venkatraman, None; TinaWong, 61, 250,006 (P); Jodhbir S. Mehta, NoneSupport: Translational and Clinical Research (TCR) Programme(NMRC/TCR/002-SERI/2008)Program Number: 5059 Poster Board Number: B0017Presentation Time: 2:45 PM - 4:30 PMTreatment of Acute Posterior Uveitis by Injection ofTriamcinolone Acetonide into the Suprachoroidal Space UsingMicroneedlesBrian C. Gilger 1 , Eva M. Abarca 1 , Jacklyn H. Salmon 1 , SamirkumarR. Patel 2 . 1 Clinical Sciences, North Carolina State University,Raleigh, NC; 2 Clearside Biomedical, Alpharetta, GA.Purpose: To compare effects of microneedle injection oftriamcinolone acetonide (TA) into the suprachoroidal space (SCS)versus intravitreal (IVT) TA injection in a model of acute posterioruveitis.Methods: Twenty weanling pigs had BSS or lipopolysaccharide(LPS) IVT injection followed 24 hours later with SCS or IVTinjection of 0.2 mg or 2.0 mg TA. SCS injections were made usingpurpose-designed microneedles and 27G needles were used for IVTinjections. Clinical ocular inflammatory scores (CIS) and intraocularpressure measurements (IOP) were collected daily, whileelectroretinography, optical coherence tomography (OCT), and widefieldocular fundus photography were performed on -1, 0, and 3 daysafter treatment. Pigs were then euthanized, aqueous and vitreoushumor collected for cell counts and protein levels, and the eyes wereprocessed for histopathology.Results: SCS TA injection using microneedles was simple, effective,and not associated with adverse effects, elevated IOP, or toxicity.Cumulative mean CIS of eyes receiving 0.2 and 2.0mg SCS TA werenot significantly different than either control (non-uveitis induced)eyes or eyes receiving 2.0mg IVT TA at all examination times.Cumulative mean CIS, however, of 0.2mg IVT TA was notsignificantly less than LPS injected, vehicle treated eyes. OCT vitrealcellular infiltrate was significantly lower after treatment with both 0.2and 2.0 mg SCS TA than eyes treated with vehicle but were notsignificantly different than eyes treated with IVT TA. Histologicinflammatory suppression after 0.2 and 2.0mg SCS TA was similar toeyes treated with 2.0mg IVT TA. Mean vitreal cell counts and proteinconcentrations were not significantly different between eyes injectedwith 0.2mg SCS TA and 0.2 and 2.0mg IVT TA. Furthermore, 2.0mgSCS TA mean cell counts and protein concentrations were notsignificantly different than negative control eyes.Conclusions: Results from this study suggest that delivery of TA tothe SCS provides effective control of acute posterior uveitis in amodel that is similar in anatomy, size, and retinal vascular pattern tothe human eye. There were no adverse effects, increased IOP, orevidence of procedural or drug toxicity following injection of TA intothe SCS using microneedles in porcine eyes. This study supports thefurther evaluation of the SCS as a site of drug delivery to ocularposterior segment.Commercial Relationships: Brian C. Gilger, Clearside (F),Allergan (F); Eva M. Abarca, Clearside (F); Jacklyn H. Salmon,Clearside (F); Samirkumar R. Patel, Clearside Biomedical (E),Clearside Biomedical (I), Clearside Biomedical (P)Support: Georgia Research AllianceProgram Number: 5060 Poster Board Number: B0018Presentation Time: 2:45 PM - 4:30 PMOcular and Systemic Pharmacokinetics of a PDE4 InhibitorFollowing Topical Administration (Eyedrop) in Male Dutch-Belted RabbitsDavid C. Gale 1 , Caroline J. Sychterz 2 , Ciara Rodgers 2 , SherryWang 2 , Tom Wilde 2 , Anu Shilpa Krishnatry 2 , Harma Ellens 2 .1 Ophthalmology DPU, GlaxoSmithKline, King of Prussia, PA; 2 PTSDMPK-UM, GlaxoSmithKline, King of Prussia, PA.Purpose: To evaluate the ocular and systemic pharmacokinetics of aphosphodiesterase 4 (PDE4) inhibitor following topical eyedropadministration in male Dutch-Belted rabbits.Methods: All studies were conducted according to the GSK Policyon the Care, Welfare and Treatment of Laboratory Animals afterreview by the GSK Institutional Animal Care and Use Committeeand in compliance with the ARVO Statement on the Use of Animalsin Ophthalmic and Visual Research. Male Dutch-Belted rabbits weretopically dosed with a potent PDE4 inhibitor (GSK907188, IC50

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyefficacious concentrations in the cornea, conjunctiva and lacrimalgland.Commercial Relationships: David C. Gale, GlaxoSmithKline (E);Caroline J. Sychterz, GlaxoSmithKline (E); Ciara Rodgers,GlaxoSmithKline (E); Sherry Wang, GlaxoSmithKline (E); TomWilde, GlaxoSmithKline (E); Anu Shilpa Krishnatry,GlaxoSmithkline (E); Harma Ellens, GlaxoSmit5hKline (E)Program Number: 5061 Poster Board Number: B0019Presentation Time: 2:45 PM - 4:30 PMAqueous humor concentration of Bromfenac 0.09% (Bromday)compared with Bromfenac in DuraSite 0.075% (Bromsite) incataract patients undergoing phacoemulsification after 3 daysdosingKamran Hosseini 1 , Judith Hutcheson 1 , Lyle M. Bowman 2 . 1 Clinical,InSite Vision Inc, Alameda, CA; 2 Development, InSite Vision,Alameda, CA.Purpose: To compare the aqueous humor penetration of twodifferent formulations of Bromfenac, namely commercial Bromday(0.09%) with Bromfenac in Durasite (0.075%).Methods: A multi center, double-masked study randomized 60patients requiring cataract extraction. Patients were to receive one oftwo treatment arms. Once a day dosing of Bromday and Bromfenacin Durasite was instructed for 2 day prior to surgery. Patients wereinstructed to instill the last (third) dose in the morning of surgery.After completion of the paracentesis site, aqueous humor wascollected through the peripheral clear cornea with a 30-gauge needle.Following collection, aqueous samples were frozen and stored priorto analysis. Drug concentrations were analyzed by a validated liquidchromatography tandem mass spectrometry method from extractedtissue samples using an internal standard. The comparison of meansbetween groups was performed using an F-test.Results: The average aqueous humor concentration of Bromfenac inDurasite 0.075% was 2 times the concentration of Bromday 0.09%.Bromfenac in Durasite achieved a mean peak aqueous humorconcentration of 49.33 compared with 23.64 ng/ml for Bromday.Conclusions: Bromfenac in Durasite 0.075% achieved significantlygreater aqueous humor concentrations when compared to Bromfenac0.09% in patients undergoing phacoemulsification.Commercial Relationships: Kamran Hosseini, InSite Vision Inc.(E); Judith Hutcheson, InSite Vision (E); Lyle M. Bowman, InSiteVision (E)Clinical Trial: NCT01387464Program Number: 5062 Poster Board Number: B0020Presentation Time: 2:45 PM - 4:30 PMComparison of the Total Amount of Triamcinolone AcetonideDelivered Via Suprachoroidal or Intravitreal AdministrationBrian Burke, Rozemarijn S. Verhoeven, Samirkumar R. Patel.Clearside Biomedical, Raleigh, NC.Purpose: The purpose of this study was to compare the total amountof triamcinolone acetonide (TA) delivered into a pig eye wheninjected into the suprachoroidal space using a Clearside Biomedicalproprietary microneedle or into the vitreous using a standard 30gauge needle.Methods: Whole pig cadaver eyes (Sioux-Preme Packing)enucleated within 24 hours after death were used for all injections.Intravitreal and suprachoroidal injections of TA were performedusing Triesence® (Alcon Labs). Intravitreal injections wereperformed using a 30 g needle (Becton-Dickinson) andsuprachoroidal injections were performed using a ClearsideBiomedical proprietary microneedle. 1 mL syringes (Becton-Dickinson) were loaded with the required amount of Triesence® ateach of the three volumes assessed: 50, 100, and 150 µL (2, 4, and 6mg, respectively). The residual amount of TA present in thesyringe/needle assembly after injection was determined by RP-HPLC. The total amount of TA delivered to the eye for each dosevolume was determined as the difference in the total amount loadedinto a syringe before injection into the pig eye versus the residualamount of TA recovered from the syringe/needle assembly afterinjection.Results: Average total dose administered following 50, 100 and 150µL TA injected into the suprachoroidal space ranged from 86-92% ofthe target dose level, while average total dose administered following50 and 100 µL TA injected into the vitreous ranged from 88-89%.Virtually no difference was observed between the two routes ofadministration and needles for each volume.Conclusions: The target dose level of TA can be consistentlydelivered into the SCS using a microneedle or into the vitreous usinga 30 g needle. Total amount of TA delivered was similar between thetwo administration routes.Commercial Relationships: Brian Burke, Clearside Biomedical(E); Rozemarijn S. Verhoeven, Clearside Biomedical (E);Samirkumar R. Patel, Clearside Biomedical (E), ClearsideBiomedical (I), Clearside Biomedical (P)Program Number: 5063 Poster Board Number: B0021Presentation Time: 2:45 PM - 4:30 PMSuprachoroidal Microinjection Delivers TriamcinoloneAcetonide to Therapeutically-Relevant Posterior OcularStructures and Limits Exposure in the Anterior SegmentHenry F. Edelhauser 1, 2 , Samirkumar R. Patel 2 , Carol Meschter 3 ,Robin Dean 3 , Kendall Powell 4 , Rozemarijn S. Verhoeven 2 .1 Ophthalmology, Emory Univ Eye Center, Atlanta, GA; 2 ClearsideBiomedical, Alpharetta, GA; 3 Comparative Biosciences, Sunnyvale,CA; 4 Tandem Labs, Durham, NC.Purpose: To evaluate the ocular and systemic PK of triamcinoloneacetonide (TA) in the New Zealand White rabbit followingintravitreal (IVT) injection or administration into the suprachoroidalspace (SCS) using a Clearside Biomedical proprietary microneedle.Methods: On Day 0, male rabbits (5/group) received a singlebilateral administration of 4 mg TA (100 µL Triesence®) via SCSinjection using a 33g 750µm microneedle or IVT injection using astandard 30g needle. Clinical observations, body weights, andintraocular pressure (IOP) were assessed up to 13 weeks post-dose.Plasma and ocular matrixes (aqueous humor (AH), lens, iris/ciliarybody (ICB), vitreous humor (VH), sclera/choroid (SC), and retina)were sampled on Days 1, 14, 28, 56, and 91. Plasma (LLOQ 0.5ng/mL) and ocular matrixes (LLOQ 2 - 15 ng/mL) were analyzedusing LC-MS/MS, and resulting data were assessed fornoncompartmental PK parameters.Results: Preliminary data shows that there were no observed adverseeffects related to treatment or method of administration. TA inplasma peaked on Day 1 at 4 ng/mL in both groups, and TA wasquantifiable in all ocular matrixes through Day 91. Following SCSTA, TA was observed (in decreasing order) in SC>retina>VH>ICB>lens>AH. SCS TA Cmax and AUC (area under theconcentration curve) was increased in SC (Cmax: 11-fold, AUC: 11-fold) compared with IVT TA. SCS and IVT TA retina Cmax andAUC were roughly equivalent, but SCS TA peaked more quickly(Day 1) compared with IVT TA (Day 14). Following IVT TA, TAwas observed in VH>ICB>retina>lens>SC>AH. IVT TA Cmax andAUC was increased in AH (Cmax: 755-fold, AUC: 715-fold), lens(Cmax: 290-fold, AUC: 682-fold), ICB (Cmax: 24-fold, AUC: 44-fold) and VH (Cmax: 4-fold, AUC: 52-fold) compared with SCS TA.Conclusions: Preliminary data suggest that both IVT and SCS TA©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 – Physiology/Pharmacologywere well tolerated in the albino rabbit and systemic exposure wasminimal by either route. These data show that SCS TA is absorbed atmuch greater proportions into the clear/choroid and retina, while IVTTA distributes throughout the eye, indicating that SCS administrationusing a microneedle is a targeted approach for delivering TA totherapeutically-relevant ocular structures of posterior segment diseaseand limiting anterior segment exposure.Commercial Relationships: Henry F. Edelhauser, ClearsideBiomedical (P), Clearside Biomedical (I), Clearside Biomedical (C);Samirkumar R. Patel, Clearside Biomedical (E), ClearsideBiomedical (I), Clearside Biomedical (P); Carol Meschter, None;Robin Dean, None; Kendall Powell, None; Rozemarijn S.Verhoeven, Clearside Biomedical (E)Program Number: 5064 Poster Board Number: B0022Presentation Time: 2:45 PM - 4:30 PMProtective Effects of Transscleral Drug Delivery Device AgainstPhotoreceptor Cell Death in S334ter Rhodopsin Mutant RatsNobuhiro Nagai 1 , Hirokazu Kaji 2 , Hideyuki Onami 1 , TakuyaYamada 2 , Yuki Katsukura 1 , Yumi Ishikawa 1 , Matsuhiko Nishizawa 2 ,Yukihiko Mashima 3 , Toshiaki Abe 1 . 1 Graduate School of Medicine,Tohoku University, Sendai, Japan; 2 Graduate School of Engineering,Tohoku University, Sendai, Japan; 3 R-Tech Ueno, Tokyo, Japan.Purpose: To evaluate the protective effects of a transscleral drugdelivery device that can release unoprostone isopropyl (UNO) in acontrolled release manner against photoreceptor cell death in S334terrhodopsin mutant rats.Methods: The device consists of a reservoir, controlled-releasecover, and drug formulations, which were made of photopolymeizedpoly(ethyleneglycol) dimethacrylate that partially containstri(ethyleneglycol) dimethacrylate. These parts were fabricated via amicrofabrication technique that used an AutoCAD design. UNO, aprostanoid for antiglaucoma eyedrops marketed in Japan, was loadedin the device. High-performance liquid chromatography was used toevaluate the release amount of UNO. After the devices were placedonto the sclera of eyes in 2 weeks old S334ter rats, flashelectroretinograms were recorded. Histological examinations wereperfomred to evaluate the thickness of the outer nuclear layer.Results: UNO was released with zero-ordered kinetics from thedevice. Electroretinographic amplitudes of the a- and b-wavesincreased significantly in rats treated with UNO-loaded devicescompared with saline-loaded devices. The outer nuclear layerthickness was thinned in the group treated with saline-loaded devices,but the group treated with UNO-loaded devices suppressed the retinaldegeneration.Conclusions: Transscleral UNO delivery device protected againstphotoreceptor cell death in S334ter rhodopsin mutant rats. The devicemay offer a less-invasive method of drug delivery to achievesustained release of medications for intravitreal drug delivery and thetreatment of various retinal diseases.Commercial Relationships: Nobuhiro Nagai, R-Tech Ueno (F);Hirokazu Kaji, None; Hideyuki Onami, None; Takuya Yamada,None; Yuki Katsukura, None; Yumi Ishikawa, None; MatsuhikoNishizawa, None; Yukihiko Mashima, R-Tech Ueno (E); ToshiakiAbe, R-TECH UENO, LTD. (F)Support: Grant-in-Aid for Young Scientists (A) from the MEXT,Japan, Health Labour Sciences Research Grant from the MHLW,JapanProgram Number: 5065 Poster Board Number: B0023Presentation Time: 2:45 PM - 4:30 PMIn vitro cytotoxicity screening and pharmacokinetic modeling: atool in the development of ocular drug delivery systemsEva Tuominen 1 , George Mihov 2 , Mengmeng Zong 2 , Sanjay Sarkhel 1 ,Aylvin A. Dias 2 , Arto Urtti 1 . 1 Centre for Drug Research, Faculty ofPharmacy, University of Helsinki, Helsinki, Finland; 2 DSM, Geleen,Netherlands.Purpose: The aim was to combine in vitro cytotoxicity screeningassays and ocular pharmacokinetic modeling in order to aid thedevelopment of safe ocular drug delivery systems. Polyesteramide(PEA) is a potential polymer platform for controlled release systemsof ocular drugs. Cytotoxicity of hydrolytic degradation products ofbiodegradable polyesteramide were tested using this combinationmethod.Methods: A cell based cytotoxicity screening platform has beendesigned for testing of polymers, associated particles and degradationproducts of polymers. Human retinal pigment epithelial cell line(ARPE-19) was used as the test cell line. A broad concentration rangeof the materials was used in the tests. Degradation products of PEAwere tested with MTT assay and, polyethylene imine (PEI) and poly-L-lysine (PLL) were used as controls. ARPE-19 cells on a 96-wellplate were incubated for 5 hours with different concentrations of thepolymers (0.0001 - 5 mg/ml). Kinetic ocular modelling was carriedout using Stella software (ISEE systems 9.0). Kinetic simulationmodel was built to predict the intravitreal concentrations of the PEAdegradation products and control polymers. In the model, theintravitreal polymer dose was set at 10 mg, the dissolution took placein sink conditions, and the degradation rate of free PEA was varied inthe simulations.Results: The PEA degradation products did not show any signs oftoxicity in the MTT assay at test concentrations (0.0001 - 5 mg/ml),but the control polymers showed toxicity (PEI: IC 50 = 6.3 ± 1.0µg/ml; PLL: IC 50 = 48.7 ± 11.0 µg/ml). For PEA borne materials thepharmacokinetic model predicted a maximum total concentrationrange of 0.2 - 4 mg/ml, for PEI 1.4 - 6 mg/ml and PLL 1.2 - 6 mg/ml.Conclusions: Combination of kinetic modeling and cellular toxicitytesting indicates ocular toxicity of PLL and PEI. PEA and itsdegradation are predicted to be safe after intravitreal administration.Cytotoxicity screening and pharmacokinetic simulations are apromising tool in the development of ocular drug delivery systems.Commercial Relationships: Eva Tuominen, None; George Mihov,DSM (E); Mengmeng Zong, DSM (E); Sanjay Sarkhel, None;Aylvin A. Dias, DSM Biomedical (E); Arto Urtti, NoneSupport: PANOPTES (EU-FP7 246180)Program Number: 5066 Poster Board Number: B0024Presentation Time: 2:45 PM - 4:30 PMRetinal Safety and Efficacy of a Dexamethasone BiodegradableImplant to Treat Macular Edema Associated to Retinal VeinOcclusion: A Phase I/II Clinical TrialRubens C. Siqueira 1 , Renato B. Cunha 1 , Andre Messias 1 , Armando S.Cunha 2 , Silvia Fialho 2 , Rodrigo Jorge 1 . 1 Retina, Sao PauloUniversity, Sao Jose do Rio Preto, Brazil; 2 Pharmacology, MinasGerais Federal University, Belo Horizonte, Brazil.Purpose: To evaluate the safety and efficacy of a biodegradableimplant containing 350 µg of dexamethasone (DDS-25 gauge) for thetreatment of macular edema associated to retinal vein occlusion(vME)Methods: Prospective, nonrandomized, open-label, phase I/II clinicaltrial, including 10 patients (n=10 eyes) with chronic vME, showingETDRS best-correct visual acuity (BCVA) of 20/50 or worse.Evaluations included BCVA, spectral-domain optical coherencetomography (OCT - Spectralis Heidelberg Engineering) fordetermination of central macular thickness (CMT), full-fieldelectroretinography (ISCEV standard ERG), kinetic visual field(Octopus 900), and fluorescein and indocyanine green angiography.©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 – Physiology/PharmacologyEvaluations were performed at baseline, and 1, 4, 12 and 24 weeksafter intravitreal DDS-25 insertion.Results: The mean ± SE (range) CMT at baseline was 461.2 ± 41.3μm (288 to 701 µm), and 439.6 ± 40.4 µm (259 to 631 µm), 442.5 ±44.6 µm (255 to 632 µm), 354.6 ± 31.2 µm (228 to 537 µm), 316.5 ±26.4 µm (226 to 441 µm) at 1, 4, 12, and 24 weeks respectively.Showing a significant improvement of 144.7 ± 46.0 µm at 24 weeks(P=0.0059; ANOVA). BCVA improved significantly in 0.14 ± 0.06logMAR (7 ETDRS letters) at 24 weeks (P=0.0248), with 6 patientsimproving between 1 to 4 ETRS lines. Mean rod b-wave amplitudewas 265.4 ± 30.4 µV at baseline and 255.6 ± 32.6 µV at 4 weeks(P=0.375), and no significant changes were observed for any ERGparameters, visual fields or angiography during follow-up. Relatedadverse events included no significant IOP elevation of 3.2 ± 1.6mmHg (P=0.071) at week 24.Conclusions: Our data suggests that the DDS-25 is safe and efficientfor the treatment of vME in a short follow up time (6 months). Alarger prospective randomized study is warranted to confirm thesepreliminary findings.Commercial Relationships: Rubens C. Siqueira, None; Renato B.Cunha, None; Andre Messias, None; Armando S. Cunha, None;Silvia Fialho, None; Rodrigo Jorge, NoneClinical Trial: NCT01662518Program Number: 5067 Poster Board Number: B0025Presentation Time: 2:45 PM - 4:30 PMTherapeutic effect of stealth-type polymeric nanoparticles withencapsulated cyclosporine A on experimental autoimmuneuveoretinitisTsutomu Sakai 1 , Kana Kuroyanagi 1 , Tsutomu Ishihara 2 , KiichiroOkano 1 , Hiroshi Tsuneoka 1 . 1 Ophthalmology, Jikei Univ School ofMedicine, Setagaya-ku, Japan; 2 Chemical biology and appliedchemistry, Nihon university college of engineering, Kooriyama,Japan.Purpose: The therapeutic effects of cyclosporine A encapsulated inbiocompatible and biodegradable blended nanoparticles of poly(lactic acid) (PLA) homopolymers and PEG-block-PLA copolymers(stealth nanocyclosporine) were examined in an experimentalautoimmune uveoretinitis (EAU) model in Lewis rats.Methods: EAU was induced by S-antigen peptide in Lewis rats.Accumulation of systemically administered Cy7-labeled stealthnanoparticles in inflamed eyes of rats with EAU was assessed usingin vivo fluorescence imaging. And the therapeutic effect of stealthnanocyclosporine or saline on EAU was examined. The eyes wereobtained 7 days after the treatment and the histological score wasdetermined. using pathological findings. The expression ofinflammatory cytokines including IL-6, IL-17, and VEGF wasdetermined immunohistochemically.Results: Cy7-stealth nanoparticles accumulated in inflamed eyes ofrats with EAU and remained in situ for a 3-day period. Systemicallyadministered stealth nanocyclosporine reduced the clinical scores ofrats with EAU within 1 day and maintained the effect for 2 weeks.This treatment also decreased the histological scores and theexpression of inflammatory cytokines in the retina of EAU.Conclusions: The strong therapeutic benefit on EAU obtained withthe stealth nanocyclosporine may have been due to prolonged bloodcirculation and targeting to the inflamed uvea and retina, in additionto sustained release in situ.Commercial Relationships: Tsutomu Sakai, None; KanaKuroyanagi, None; Tsutomu Ishihara, None; Kiichiro Okano,None; Hiroshi Tsuneoka, NoneProgram Number: 5068 Poster Board Number: B0026Presentation Time: 2:45 PM - 4:30 PMDevelopment of an in vitro pharmacokinetic model of the humaneyeSahar Awwad 1, 2 , Alastair Lockwood 1, 2 , Abeer Mohamed Ahmed 1, 2 ,Garima Sharma 1, 2 , Ashkan Khalili 2 , Steve Brocchini 1, 2 , Peng T.Khaw 2 . 1 UCL School of Pharmacy, London, United Kingdom;2 National Institute for Health Research (NIHR) Biomedical ResearchCentre at Moorfields Eye Hospital NHS Foundation Trust and UCLInstitute of Ophthalmology, London, United Kingdom.Purpose: Pharmacokinetics remains a major challenge in drugdevelopment for the posterior segment. Accurate evaluation andoptimisation of vitreous drug distribution is hindered by differencesbetween humans and animal platforms. Furthermore, measurementsat serial time points necessitate use of many animals, which has bothcost and ethical implications. We aimed to design an in vitro flowmodel that would mimic posterior segment fluid dynamics, and aidthe determination of vitreous drug distribution. Similar to the designof simulated gastric solutions to mimic dissolution within theintestinal tract, we are also focused on developing a simulatedvitreous solution that can be used in early preclinical studies.Methods: Several prototype chambers (~4.2 mL internal volume )were fabricated incorporating both a model anterior and posteriorsegment. These were filled with simulated vitreous (a polymericcombination of hyaluronic acid and agar) replicating the viscosityand rheology of human vitreous. Aqueous flow input ports and anoutput sampling port were used to provide fluid flow by introducing a2.0 μl/min flow rate with phosphate buffered saline (pH 7.4) from theanterior segment at room temperature (25°C). Dexamethasonesodium phosphate (1.0 mg) was solubilised in distilled water (1.0 ml)and this solution (50 μl) was injected into the model. Samples (100μl) were taken over 5 days at 24 hourly intervals from differentanatomical regions. Analysis was performed using High PerformanceLiquid Chromatography (HPLC) at 240 nm.Results: The viscosity properties of the hyaluronic acid-agarcombination were found to be similar to that reported for humaneyes. This solution is thought to be a reliable simulation of in vivoviscosity that can occur. A concentration of 1.03 mg/ml (±0.06mg/ml) dexamethasone sodium phosphate was injected into themodel and sampled for 5 days. It was sampled at various points fromthe model and release kinetics showed it was within the therapeuticrange (10-100 uM).Conclusions: This simple dynamic posterior segment model may aidthe pharmacokinetic study of ocular drugs in early preclinicaldevelopment. Further studies to incorporate saccadic movements,different forms of dexamethasone, temperature conditions and invivo-in vitro correlations are required including testing the releasekinetics of other anti-inflammatory and anti-fibrotic drugs.Commercial Relationships: Sahar Awwad, None; AlastairLockwood, None; Abeer Mohamed Ahmed, Steve Brocchini(WO09/063222) (P), Peng Khaw (WO09/063222) (P); GarimaSharma, None; Ashkan Khalili, University College London (P);Steve Brocchini, None; Peng T. Khaw, University CollegeMoorfields (P)Support: NIHR Moorfields Biomedical Research Centre, UCLSchool of Pharmacy, Grand Charity, Helen Hamlyn Trust, Fight forSightProgram Number: 5069 Poster Board Number: B0027Presentation Time: 2:45 PM - 4:30 PMTunable sustained intravitreal drug delivery system fordaunorubicin using oxidized porous siliconHuiyuan Hou 1 , Alejandra Nieto 2 , Feiyan Ma 1 , Su-Na Lee 1 , KaihuiNan 1 , William R. Freeman 1 , Michael J. Sailor 2, 3 , Lingyun Cheng 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 – Physiology/Pharmacology1 Department of Ophthalmology, Shiley Eye Center, UCSD, SanDiego, CA; 2 Department of Chemistry and Biochemistry, UCSD, SanDiego, CA; 3 Department of Bioengineering, UCSD, San Diego, CA.Purpose: Daunorubicin (DNR) is a potent therapeutic agent forunwanted proliferative ocular diseases. However, it has a narrowtherapeutic window and short vitreous half-life. We have previouslyshown that covalent bonding DNR to functionalized porous silicon(pSi) can extend DNR vitreous stay from days to months. Currentstudy is to investigate the feasibility and capacity of regulating DNRrelease by quantitatively altering nano pore size of this uniquedelivery system.Methods: Porous silicon microparticles were prepared byelectrochemical etches followed by ultrasonication. Three differentetching parameters were used to acquire three nano-pore sizes (15nm,26nm, and 43nm). pSi particles were oxidized at 800°C and in vitrodegradation was examined at 37°C in a closed system by quantitatesilicon using ICP-OES. For in vivo drug release study, the oxidizedpSi particles were further silanized for daunorubicin loading. DNRwas loaded into the pSi particles by covalent attaching. Threemilligrams of each type of DNR loaded particle was intravitreallyinjected into 4 rabbits eyes. Only one eye of each animal was used.After the injection, the eyes were examined at day 1, 3, 7 and 14 byslit lamp biomicroscopy, indirect ophthalmoscopy, and tonometry.All rabbits were sacrificed at day 14 and vitreous was dissected out.The supernatant of the vitreous was subjected to HPLC/MS/MS fordaunorubicin quantitation.Results: The 32-day in vitro degradation studies showed thatdegradation rate of pSi was associated with pore sizes; with the largerpore being faster degraded. The mean degradation rate of pSi with46nm pores was significantly larger than that of the other two poresizes (44.2 vs 25.7 or 21.2 ug/mL, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology(E); Josh Rowe, Allergan (E); Mohammad R. Kazemi, None; JieShen, Allergan (E)Program Number: 5071 Poster Board Number: B0029Presentation Time: 2:45 PM - 4:30 PMOptimization of In-vitro DiffER model to study/compare theeffect of Formulations on Cross-Scleral transport of poorlyaqueous soluble drugThomas E. Rowe 1 , Kenneth W. Reed 2, 1 , Tuhin Bhowmik 1 .1 Encompass Pharmaceutical Services, Norcross, GA;2 Pharmaceutical Sciences, Belmont University, Nashville, TN.Purpose: Ophthalmic drugs intended for the treatment of back of theeye disorders often suffers the limitation of cross-scleral penetrationto achieve therapeutic efficacy. The purpose of this study was tooptimize the Diffusion-erosion in-vitro model (DiffER) to understandthe behavior of the drug diffusion/ transport from the formulation intothe sclera and out to the receptor media. This method will help us topre-screen various formulations at an early Formulation &Development Phase to indicate the possibility of extending theformulation to be tested on animal models.Methods: Frozen mature rabbit scleras were thawed in diffusionmedia and placed on specially adapted spherical diffusion Franz Cellsand dosed with 0.3 mg (about 2 drops) of active in three differentformulations (emulsion, aqueous suspension, and paraffinsuspension). For the dynamic experiments the pre corneal layer wasinitially flushed with PBS at an increased flow rate immediately postdosageto simulate reflex tear flow then reduced to a basal flow ratefor the remaining duration of the experiment. To evaluate drugretention and extent of absorption characteristics of each formulation,eroded solution from the pre-corneal layer was collected at intervalsfor analysis. Receiver chamber solution was also sampled at selectedintervals. Analysis of samples were performed via HPLCResults: The results from the DiffER experiment were contrastingand varied from formulation to formulation. The order of drugtransport in the formulation was quantified and permeability coefficientswere calculated.Conclusions: The DiffER may be efficiently utilized as an in-vitrotool to discern the rate of drug transport through optic tissuesbetween different drug formulations.Commercial Relationships: Thomas E. Rowe, Encompass (E);Kenneth W. Reed, Encompass (C); Tuhin Bhowmik, NoneProgram Number: 5072 Poster Board Number: B0030Presentation Time: 2:45 PM - 4:30 PMOcular bioavailability of brimonidine 0.1% BID in non-humanprimatesChinatsu Tosha, Sherri Decker, Guadalupe Ruiz, Octavio Avalos,Werhner Orilla, Larry Gruber, Ton Lin, Alexandra S. Almazan,Daniel W. Gil, James A. Burke. Biological Science, Allergan, Inc.,Irvine, CA.Purpose: To determine whether topically applied Alphagan 0.1%BID in non-human primates would deliver sufficient brimonidine tothe central retina to activate α2-adrenergic receptors for aneuroprotective effect.Methods: Alphagan 0.1% (Allergan, Inc., Irvine, CA) wasadministered to one or both eyes of six female cynomolgus monkeysevery 12 hours (BID; 9 eyes) for four weeks. Three contralateral eyesserved as controls and received placebo. Animals were euthanized 2hours following the final dose. Then, ocular tissues from anteriorsegments (aqueous humor (AH), iris and ciliary body (CB)) and fromposterior segments (conjunctiva, peripheral sclera, vitreous humor,and tissues from a central 8 mm punch biopsy: neurosensory retina,underlying RPE/choroid, and sclera) were collected and assayed forbrimonidine concentrations. The brimonidine plasma concentrationwas also analyzed to examine the possible systemic transfer of thedrug.Results: The brimonidine concentrations in anterior segments were189 ± 132 nM (AH), 29147 ± 7526 nM (iris) and 322560 ± 73384nM (CB). The concentrations in posterior segments were 11062 ±6027 nM (conjunctiva), 9565.4 ± 3927 nM (peripheral sclera), 47 ±11 nM (vitreous), 122 ± 26 nM (central retina), 3,352 ± 1,017 nM(RPE/choroid) and 1,994 ± 465 nM (sclera). The plasmaconcentration of brimonidine in the bilaterally treated monkeys was0.49 ± 0.43 nM. The retinal brimonidine concentrations in controluntreated eyes were 16% of that of treated eyes. The brimonidineconcentrations in anterior segments of control eyes were 29 ± 44 nM(AH), 1123 ± 523 nM (iris) and 15436 ± 10596 nM (CB). Theconcentrations in posterior segments of control eyes were 1808 ±1675 nM (conjunctiva), 807 ± 428 nM (peripheral sclera), 807 ± 428nM (vitreous), 807 ± 428 nM (central retina), 985 ± 319 nM(RPE/choroid) and 327 ± 295 nM (sclera).Conclusions: This study indicates that Alphagan 0.1% BID deliverspharmacologically sufficient brimonidine to the central retina of nonhumanprimates. The distribution of plasma and ocular tissuebrimonidine concentrations suggests that delivery to the retina wasvia the periocular pathway; not via systemic transfer. The strongbonding of brimonidine to ocular melanin may allow a long retentionof brimonidine in the pigmented ocular tissues after the topicaladministration. Melanin in choroid and RPE may play a major role infacilitating the delivery of brimonidine to the central retina.Commercial Relationships: Chinatsu Tosha, Allergan, Inc. (E);Sherri Decker, Allergan (E); Guadalupe Ruiz, Allergan Inc. (E);Octavio Avalos, Allergan (E); Werhner Orilla, Allergan, Inc. (F),Allergan, Inc. (I), Allergan, Inc. (E), Allergan, Inc. (P); LarryGruber, None; Ton Lin, Allergan Inc (E); Alexandra S. Almazan,Allergan (E); Daniel W. Gil, Allergan (E); James A. Burke,Allergan, Inc (E)Program Number: 5073 Poster Board Number: B0031Presentation Time: 2:45 PM - 4:30 PMSustained Delivery of Prostaglandin from Drug-ContainingDepots Using Ocular Rings in BeaglesKathryn S. Crawford 1 , Jeanne Y. Ellis 2 , Jack Rulander 3 , StephenJohnston 3 , Francis S. Lai 3 , Edward J. Ellis 2 , Charles D. Leahy 2 .1 PharmOcu, Andover, MA; 2 Vista Scientific, LLC, Andover, MA;3 Massachusetts Medical Device Development Center, University ofMassachusetts, Lowell, MA.Purpose: To evaluate the efficacy of sustained-release latanoprostfrom depots in a topical ophthalmic drug delivery device(TODDD).Methods: The right eyes of 8 ocular normotensive adult beagle dogswere fitted with ocular ring devices, each containing 2 latanoprostdrugdepots (cylindrical cores, 600 μg latanoprost). The depots werematched in volume and surface release area to those of a humanconfigureddevice. A device with blank depots containing nolatanoprost was placed on the right eye of 1 additional animal. Allleft eyes remained untreated. Clinical slit-lamp exams wereperformed pre-placement on Day 1 and post-placement on Days 1, 8and 17. Daily observations were performed to assess the presence ofthe device (retention) and any ocular abnormalities. Intraocularpressure (IOP) and pupil diameter were measured pre- and postplacementon Day 1, and on Days 4, 8, and 16. Plasma samples werecollected on Day 1 prior to, and approximately 4 hours after deviceinsertion, and on Day 8. Tear samples were collected on Day 16 bySchirmer strip in the lower cul-de-sac. Tear and plasma samples wereanalyzed by LC/MS/MS for latanoprost and latanoprost acid. 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 – Physiology/Pharmacologylower limit of quantitation was 0.5 ng/mL.Results: IOP reduction in the treated eye compared to the control eyewas approximately 3 mmHg on Day 4 (n=6) and Day 8 (n=4), and 7mmHg on Day 16 (n=3) in the dogs that had retained the latanoprostloadeddevices, representing a 37% reduction in normotensive IOPfrom baseline. There was no effect on IOP in the animal wearing theplacebo device. The IOP returned to baseline levels in all eyes afterremoval of the devices. A range of 25 - 215 ng/mL latanoprost wasrecovered from the tears of animals wearing the drug devices. Nolatanoprost was detected in plasma or in the tear samples fromuntreated eyes.Conclusions: Long-term retention of this ring device configurationand dimensions in this species was not achieved, but this studydemonstrates the therapeutic feasibility of the sustained-release (nonring)TODDD configured for human ocular dimensions. In vitroanalysis of the worn devices and of the extended latanoprost releaseprofile from new devices relative to tear levels measured at Day 16indicates that sustained IOP-lowering could be achieved over a 2-3month time period.Commercial Relationships: Kathryn S. Crawford, VistaScientific, Inc. (C); Jeanne Y. Ellis, Vista Scientific (I), VistaScientific (P); Jack Rulander, Vista Scientific LLC (F); StephenJohnston, Vista Scientific (F); Francis S. Lai, Vista Scientific (F);Edward J. Ellis, Vista Scientific LLC (I), Vista Scientific LLC (P);Charles D. Leahy, Vista Scientific LLC (I), Vista Scientific LLC (P)Support: NIH Grant R44EY013479Conclusions: Release of model drugs metformin and ciprofloxacinshows that this material is suitable for prolonged release of bothhydrophobic and hydrophilic molecules. The data provides a basis forpredicting polymer & drug formulations that will produce a desiredrelease profile for an intra-vitreal device. The analysis method can beextrapolated to examine and predict release from potentially anybiomaterial formulation. Cytotoxicity results support the use of thematerials intravitreally.Figure 1. Significant difference in release between hydrophobicciprofloxacin and hydrophilic metformin exists.Commercial Relationships: Ivana Postic, None; HeatherSheardown, Alcon (F), Alimera Sciences (F)Program Number: 5074 Poster Board Number: B0032Presentation Time: 2:45 PM - 4:30 PMEvaluating & Predicting Drug Release from an ImplantableBiomaterialIvana Postic, Heather Sheardown. Chemical Engineering, McMasterUniversity, Hamilton, ON, Canada.Purpose: Drug delivery from siloxane-based materials is attractivedue to its inherent properties and biocompatibility. The workdescribes a siloxane-based matrix with polyethylene glycol (PEG)additive, developed to act as an intra-vitreal implant for release oftherapeutics to the posterior segment of the eye. The effects ofvarious constituents on the release rate were examined in order todevelop predictable, long-term, controlled drug release.Methods: PEG (1-10 wt%, 500-20,000MW) was blended with liquidPDMS (Sylgard® 184 Kit) and 5-10wt% of drug (metforminhydrochloride or ciprofloxacin). Platinum curing agent was added tothe entire mixture and cured at 70°C. Quarter-inch discs werepunched out and drug release (37°C, 100rpm) into 1ml of posphatebuffered saline was measured using UV spectroscopy. ProMVsoftware was used to apply multi-variate methods (ProSensus-Ancaster,Canada). Cytotoxicity testing was performed by incubatingPEG/PDMS discs for 48 hours with aRPE19, a retinal pigmentepithelium cell line. Cellular metabolism was measured viafluorescence from PrestoBlue reagent.Results: Standard analysis showed significant differences in releasebetween the two drug types (Figure 1). Release of hydrophobicciprofloxacin showed promise for very long-term release (less than5% released at 17 days). Similar to the release of metformin, thehydrophilicity of the material could be adjusted up to 10wt% and500MW without significantly affecting the release rate from that ofthe control. Increasing the concentration of ciprofloxacin from 5wt%to 10wt% allowed for even further prolonged release. Analysis usinglatent variable methods indicates good fit and predictability of releasewithin the experiments conducted. The drug delivery devices showgenerally low cytotoxicity when cultured in the presence of aRPE19cells.Program Number: 5075 Poster Board Number: B0033Presentation Time: 2:45 PM - 4:30 PMA hollow metal microneedle reduce reflux in intravitrealinjectionsSungHo Lee 1 , Dongkyu Lee 2 , Yong Song You 2 , Soon Hyun Kim 2 ,Chang Yeol Lee 3 , Hyungil Jung 3 , Sung Jin Lee 4 , Oh Woong Kwon 2 .1 R&D Center, Lumieye Genetics Co., Ltd., Seoul, Republic of Korea;2 Retina center, NUNE Eye Hospital, Seoul, Republic of Korea;3 Biotechnology, Yonsei University, Seoul, Republic of Korea;4 Ophthalmology, Soonchunhyang University Hospital, Seoul,Republic of Korea.Purpose: The study aimed to evaluate the reduction of reflux inintravitreal injections using a minimally invasive hollow metalmicroneedle.Methods: Rabbit twenty eyes undergoing intravitreal injection wereallocated into two groups to compare the vitreal reflux after injectionof 0.02 ml of 0.1% sodium fluorescin (Alcon) using a 30G needle anda hollow metal microneedle (five millimeter long, hundred outerdiameter, fifty inner diameter and fifteen degree beveled angle). Theamount of drug reflux was estimated by measuring amount offluorescin in an absorbent cotton swab and the width ofsubconjunctival fluorescence staining area.Results: The mean measured reflux of volume was statistically lesswith hollow metal microneedle (0.27 SD ± 0.25 ng for swab absorbedfluorescin; 3.4mm SD ± 0.5 for width of subconjunctivalfluorescence staining area) than undergoing the 30G needle injection(1.51 SD ± 0.63 ng for swab absorbed fluorescin; 8.2 mm SD ± 0.7for width of subconjunctival fluorescence staining area) .Conclusions: Analyses of vitreal reflux showed that hollow metalmicroneedle prevent vitreal reflux drugs after intravitreal injectionthan 30G needle. Moreover, microneedle provide accuracies in drugadministration, lesser scleral damage and the risk of complicationafter repeat injections.Commercial Relationships: SungHo Lee, None; Dongkyu Lee,None; Yong Song You, None; Soon Hyun Kim, None; Chang Yeol©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 – Physiology/PharmacologyLee, None; Hyungil Jung, None; Sung Jin Lee, None; Oh WoongKwon, None504 Anterior Eye: Physiology and PharmacologyThursday, May 09, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 5399-5417/A0043-A0061Organizing Section: Physiology/PharmacologyContributing Section(s): Anatomy/PathologyProgram Number: 5399 Poster Board Number: A0043Presentation Time: 8:30 AM - 10:15 AMInduction of MAPK Phosphatase-1 by the Novel SelectiveGlucocorticoid Receptor Agonist Mapracorat SuppressesInflammatory Signaling Pathways in Human Corneal EpithelialCellsMegan E. Cavet, Karen L. Harrington, Thomas R. Vollmer, MaryRichardson, Jin-Zhong Zhang. Pharmaceutical R&D, Bausch &Lomb, Rochester, NY.Purpose: Prior studies demonstrated that mapracorat, a novelselective glucocorticoid receptor agonist (SEGRA), potently inhibitsanti-inflammatory cytokine release in human corneal epithelial cells(HCEpiC). The aim of this study was to determine the contribution ofan endogenous mitogen activated protein kinase (MAPK) regulator,MAPK phosphatase-1 (MKP-1), to mapracorat’s anti-inflammatoryeffects in HCEpiC.Methods: HCEpiC were challenged with IL-β or hyperosmolarity(480 mOsm) and the levels of MKP-1 protein and MAPKphosphorylation in the presence of mapracorat were determined byWestern blotting. A range of mapracorat doses were tested anddexamethasone (DEX) was used as the control. The effects of theMKP-1 inhibitor triptolide on IL-β- and 480 mOsm-inducedinflammatory signaling in the presence of mapracorat was alsodetermined.Results: Mapracorat alone did not stimulate the expression of MKP-1 in HCEpiC. IL-1β or 480 mOsm media significantly increasedMKP-1 expression over basal levels. Following IL-β or hyperosmolarchallenge, mapracorat further significantly increased MKP-1 proteinlevels at least 2 fold, with a peak expression at 1 h with IL-1β and 2-4h with 480 mOsm media. The effects were not dose-dependent withinthe tested mapracorat concentration range (30 - 300 nM). Themagnitude of increase in MKP-1 was comparable to that observedwith DEX. Concomitantly to the increase in MKP-1, mapracorat alsosignificantly reduced the levels of phosphorylated p38 and JNKMAPKs induced by IL-1β or hyperosmolarity. Triptolide-inducedMKP-1 inhibition reversed the inhibitory effects of mapracorat on IL-1β or 480 mOsm-induced MAPK signaling.Conclusions: These data suggest that mapracorat exerts its antiinflammatoryeffects, at least in part, by increasing the expression ofthe MAPK deactivator MKP-1 in human corneal epithelial cells.Commercial Relationships: Megan E. Cavet, Bausch + Lomb (E);Karen L. Harrington, Bausch + Lomb (E); Thomas R. Vollmer,Bausch + Lomb (E); Mary Richardson, Bausch and Lomb (E); Jin-Zhong Zhang, Bausch & Lomb, Inc (E)Program Number: 5400 Poster Board Number: A0044Presentation Time: 8:30 AM - 10:15 AMPirfenidone nanoparticles improve healing and prevent cornealscarring following alkali burnSarbani Hazra 1 , Sushovan Chowdhury 2 , Rajdeep Guha 2 , TrivediRuchit 3 , Uday B. Kompella 3 , Aditya Konar 2 . 1 Veterinary Surgery &Radiology, WBUAFS, Kolkata, India; 2 Animal Facilities, IndianInstitute of Chemical Biology, Kolkata, India; 3 Department ofPharmaceutical Sciences, University of Colorado Anschutz MedicalCampus, Aurora, CO.Purpose: This study was designed to evaluate the effects ofpirfenidone nanoparticles on corneal reepithelization and scarring,major clinical challenges after alkali burn.Methods: Effect of pirfenidone on collagen I and α-SMA synthesisfrom TGFβ induced primary corneal fibroblast cells was assessed byimmunocytochemistry and immunoblotting. Pirfenidone loaded poly(lactide-co-glycolide) (PLGA) nanoparticles were prepared,characterized and their delivery in primary corneal fibroblast cellswas examined. Alkali burn was induced in one eye of SpragueDawley rats (n=18) followed by once daily topical treatment withPBS (n=6), free pirfenidone (n=6), or pirfenidone nanoparticles (n=6)containing equivalent amount of pirfenidone. Corneal reepithelizationwas assessed daily by flourescein dye test, absence ofstained area indicated complete re-epithelization and the time forcomplete re-epithelization was determined. Corneal haze wasassessed daily for 7 days under slit lamp microscope and gradedusing standard method (Fantes et al. 1990). After 7 days, collagen Ideposition in the superficial layer of cornea was examined byimmunohistochemistry.Results: Pirfenidone prevented (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyand inflammatory response after corneal injury. In light microscopicfindings, the corneal thickness was increased and manypolymorphonuclear leukocytes infiltrated in the corneal stroma in thecontrol group. In immunohistochemical study, in sulindac-liposometreated group, the F4/80 positive cells were detected lower thancontrol group (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyage effect in human vitreous humor relative to rabbit, dog andmonkey vitreous humor.Methods: Latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinorprostaglandinF2α-1-isopropyl ester) was used as a prototypesubstrate to monitor esterase metabolism. Human vitreous humor wasobtained from individual donors ranging in age from 67 to 86 years.Young and old vitreous humor was obtained from rabbit (2years old), dog (2-5 and >5 years old) and monkey (3-4 and > 5 yearsold). Metabolite formation was measured following incubation at37°C of 500 μL vitreous humor and 6 μM latanoprost with timepointstaken at 0, 10, 20, 30 and 60 minutes. Liquid chromatography tandemmass spectrometry (LC-MS/MS) was used to quantify latanoprostdisappearance and latanoprost acid formation.Results: Latanoprost was metabolized to latanoprost acid in humanand animal vitreous humor indicating esterases are active in vitreoushumor. Rates of esterase metabolism were highest in human followedby rabbit, followed by monkey, followed by dog. Esterasemetabolism appeared to decrease with increasing age except in rabbit.Latanoprost was stable when incubated in buffer for 60 minutes at37°C.Conclusions: Esterase mediated latanoprost metabolism is active inhuman and animal vitreous humor. Rate of esterase metabolism washighest in human as compared to all animals tested. Among theanimal species tested rates of esterase metabolism in rabbit are mostsimilar to human. Esterase metabolism tended to decrease with age.Commercial Relationships: Mayssa Attar, Allergan (E); Jie Shen,Allergan (E); Moon S. Kim, Allergan (E); Que-Chau Radojicic,Allergan (E)Program Number: 5405 Poster Board Number: A0049Presentation Time: 8:30 AM - 10:15 AMBicarbonate sensitive soluble adenylyl cyclase (sAC) generatesthe cAMP in aqueous humor (AH)Yong Lee 1 , Yong Lee 1 , Lavoisier Ramos-Espiritu 3 , Lihua Y.Marmorstein 1 , Jochen Buck 3 , Lonny Levin 3 , Alan D. Marmorstein 1, 2 .1 Ophthalmology and Vision Science, University of Arizona, Tucson,AZ; 2 Physiology, University of Arizona, Tucson, AZ;3 Pharmacology, Weill Cornell College of Medicine, New York, NY.Purpose: Recently we demonstrated that sAC a bicarbonate sensitiveenzyme that catalyzes the conversion of ATP to cAMP plays a role inregulating outflow facility in mice. sAC is highly expressed in ciliarybody but cold not be detected in drainage tissues implying acommunicative pathway linking the cilliary body to regulation ofoutflow facility. In this study we aimed to determine whether cAMPis present in the AH of mice and whether it is generated by sAC.Methods: AH was collected from the eyes of 3-6-month old sACknock-out (KO) mice or WT litermates by cannulation of the anteriorchamber with borosilicate glass microneedles. AH collection wasperformed between 2:00 PM and 4:00 PM to avoid the influence ofcircadian dynamics on our measurements. AH from both eyes of fourmice were pooled and cAMP concentration determined using acommercial ELISA kit (Bio-Rad).Results: Levels of cAMP in AH of sAC KO mice were 0.37 ± 0.59pmol/ml (mean±sd, n=3) significantly different (p < 0.05) from the2.67 ± 1.33 pmol/ml (mean±sd, n=3) measured in litermate controls.Conclusions: cAMP levels in sAC KO mice were reduced ~7.25fold. These findings suggest that cAMP in AH is generated primarilyby sAC. Further studies are necessary to understand the effects ofvarying cAMP in AH on outflow facility.Commercial Relationships: Yong Lee, None; Yong Lee, None;Lavoisier Ramos-Espiritu, None; Lihua Y. Marmorstein, None;Jochen Buck, None; Lonny Levin, CEP Biotech (I), CEP Biotech(P); Alan D. Marmorstein, NoneSupport: NIH EY021153 and an unrestricted grant from ResearchTo Prevent BlindnessProgram Number: 5406 Poster Board Number: A0050Presentation Time: 8:30 AM - 10:15 AMBeneficial Assessment of A Combined Tropicamide and VariousOxime Treatments Against Miosis and Visual DysfunctionFollowing Ocular Exposure to the Nerve Agent SarinXxx Xxxx. Xxxxxx, Xxxxxx, MD.Purpose: Eye exposure to the organophosphorus irreversibleacetylcholinesterase inhibitor sarin results in long-term miosis (areduction of at least 50% of pupil width) and reduction in visualfunction. Anti-cholinergic drugs, such as atropine, are used topicallyin order to counter these effects and obtain symptomatic relief.Unfortunately, such compounds attenuate ocular discomfort at theexpense of producing mydriasis and partial cycloplegia symptoms,which may worsen visual performance. This study was aimed to testbeneficial drugs in contradicting the sarin-induced miosis and visualimpairment, which will minimally affect vision.Methods: Male Pigmented Long-Evans rats were topically exposedto sarin (0.2-1 μg), and 20 min later were topically treated. Pupilswere illuminated with an infrared spotlight and images were digitallyrecorded with a computerized infrared-capable video camera, thusmeasuring pupil width. Pupil width was determined 15 min -8 hfollowing exposure and treatment. Visual function assessment wasperformed using the Cued Morris Water Maze task, 15-35 minfollowing sarin exposure and treatment. In this version, cuednavigation involves finding a goal location by approaching a singlecue that marks the visible goal.Results: Rats exposed topically to various sarin doses showed adose-dependent miosis, which partially recovered within 4-8 h.Oxime treatments with or without the anti-cholinergic drugtropicamide differentially improved the sarin induced miosis and theresulting impairment in visual performance.Conclusions: The miotic as well as the visual defects observed,following topical sarin exposure are contradicted to various extent bythe treatments used.Commercial Relationships: Xxx Xxxx, NoneProgram Number: 5407 Poster Board Number: A0051Presentation Time: 8:30 AM - 10:15 AMDexamethasone Modifies Viability of Mitomycin C-TreatedTenon’s FibroblastsShu-Wen Chang 1, 2 , Wei-Ting Ho 1 , Tsan-Chi Chen 1 , San-Fong Chou 3 .1 Department of Ophthalmology, Far Eastern Memorial Hospital, NewTaipei City, Taiwan; 2 Department of Ophthalmology, NationalTaiwan University Hospital, Taipei, Taiwan; 3 Department of MedicalResearch, Far Eastern Memorial Hospital, New Taipei City, Taiwan.Purpose: To investigate whether dexamethasone modifies the effectof mitomycin C (MMC) in human Tenon’s capsule fibroblasts(HTFs) and to explore its molecular mechanism.Methods: HTFs were treated with MMC for 5 minutes and incubatedin DMEM with 10 μM dexamethasone (DEX). Viability of thetreated HTFs was analyzed by WST-1 assay. The amount of IL-8section in untreated, MMC-treated, or peroxisome proliferatoractivatedreceptor gamma (PPARγ)-silenced HTFs was determinedby enzyme-linked immunosorbent assay. Expression of PPARγ andthe apoptotic proteins was examined by immunoblotting.Results: Recombinant IL-8 noticeably suppressed HTF cellproliferation in a dose-dependent manner. MMC treatmentsignificantly upregulated IL-8 secretion and inhibited cellproliferation in HTFs. Both effects were reversed by DEXincubation. DEX upregulated PPARγ and Bcl-xL in untreated HTFs©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 – Physiology/Pharmacologyand MMC-treated HTFs at 1 day and 2 days of incubation,respectively. PPARγ silencing reduced Bcl-xL expression andenhanced IL-8 secretion. However, MMC treatment and/or DEXincubation did not alter the expression of two apoptotic indicators,PARP-1 and pro-caspase-3.Conclusions: DEX reversed the MMC-inhibited HTF cellproliferation and diminished the MMC-upregulated IL-8 secretion viaupregulating the PPARγ expression (Figure 1).Conclusions: Our data show that TRPV1 activates without forming ahigher order oligomer and that a subset of channels exhibit lateralmobility as they gate. Although the mechanism by which TRPV1activity and TRPV1 mobility are coupled and the role of mobilitychanges in cell signaling remain to be determined, our datademonstrate the power of single-molecule measurements to revealaspects of signaling not observable in macroscopic experiments.Given previous work indicating that TRPV1 is part of a signalingcomplex including the TrkA receptor for nerve growth factor andPI3K, our data suggest that dynamic localization of these complexesin response to TRPV1 agonists may constitute a new form ofregulation of local signaling.Commercial Relationships: Eric Senning, None; Sharona E.Gordon, NoneSupport: NH grant EY01730, EY017564Figure 1. Scheme depicting reverse regulation of dexamethasonein MMC-induced cell death of HTFs.Commercial Relationships: Shu-Wen Chang, None; Wei-TingHo, None; Tsan-Chi Chen, None; San-Fong Chou, NoneProgram Number: 5408 Poster Board Number: A0052Presentation Time: 8:30 AM - 10:15 AMTRPV1 and corneal PainEric Senning, Sharona E. Gordon. Physiology and Biophysics,University of Washington, Seattle, WA.Purpose: Current analgesic treatment for corneal damage orfollowing ophthalmic surgery falls short of desired goals. Onepromising approach is the temporary deactivation of TRPV1(transient receptor potential vanilloid 1) expressing nociceptors byadministration of the TRPV1 agonist resiniferatoxin. Here weconsider the molecular pathways of TRPV1 regulation.Inflammatory signals increase the excitability of TRPV1-expressingnociceptors, at least in part, by increasing the number TRPV1channels in the plasma membrane (PM) of nociceptors. However, thedynamics of TRPV1 in the PM have not been studied. Wehypothesize that TRPV1 properties are heterogeneous within a celland that channels are actively regulated as a function of local activity.Methods: We used total internal reflection fluorescence (TIRF)microscopy to explore the dynamics of single TRPV1 molecules inliving cells. Combining TIRF microscopy with whole-cell patchclamp of isolated mouse sensory neurons and HEK293T/17 cellsallowed us to image both the localization of single TRPV1 moleculeswithin cells (TRPV1-eGFP) and their activity (capsaicin-activatedfluorescent “sparklets” at site of Ca 2+ influx).Results: A number of surprising findings came out of our singlemoleculeexperiments: (1) TRPV1 channels in isolated sensoryneurons and in cultured cells are mobile, with a range of D eff from0.01 to 0.2 µm 2 /s; (2) a given Ca 2+ sparklet arises from exactly onechannel, with two-state fluorescence reflecting capsaicin-activatedgating; (3) photobleaching and fluorescence intensity analysisindicate that TRPV1 channels do not cluster in the PM; and (4) thelateral mobility of TRPV1 in the plasma membrane decreased as aProgram Number: 5409 Poster Board Number: A0053Presentation Time: 8:30 AM - 10:15 AMDifferent Roles of Carbonic Anhydrase in Human vs. BovineCorneal Endothelial TransportThomas M. Malikowski 1 , Michael E. Duffey 2 , Sangita P. Patel 3, 4 .1 School of Medicine & Biomedical Sciences, University at Buffalo,Buffalo, NY; 2 Physiology & Biophysics, University at Buffalo,Buffalo, NY; 3 Ophthalmology, SUNY Eye Institute, University atBuffalo, Buffalo, NY; 4 Research Service, VAWNYHS, Buffalo, NY.Purpose: Regulation of fluid homeostasis in the cornea is critical formaintaining corneal clarity. Buffering by carbonic anhydrases plays acentral role in rabbit and bovine models of corneal endothelial fluidtransport. Topical carbonic anhydrase inhibitors (CAIs) in rabbitsresult in decreased fluid efflux measured by increases in cornealthickness. However, routine use of topical and systemic CAIs forglaucoma in humans does not increase corneal thickness. Thehypothesis tested here is that there are species differences in the roleof carbonic anhydrases in corneal endothelial transport.Methods: Protocols were approved by the R&D Committee (VAMC,Buffalo, NY). Bovine eyes were obtained from local abattoirs. Deidentifiedhuman corneas not suitable for transplant were obtainedfrom the local eye bank. Corneal endothelial transport was measuredusing the short-circuit current (I sc ) technique. The recording solutionwas (in mM): 111.6 NaCl, 29 NaHCO 3 , 4.8 KCl, 1.0 CaCl 2 , 0.8MgCl 2 , 0.9 NaH 2 PO 4 , 10 HEPES, 5 glucose, bubbled with 5% CO 2 /95% air, pH 7.5. CAIs were added (500 µM acetazolamide, 100 µMethoxzolamide, 100 µM dorzolamide, or 100 µM brinzolamide) andI sc measured. Water and DMSO were used as controls. Drug effectwas the percentage change of total I sc .Results: All CAIs generated significant (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Thomas M. Malikowski, None;Michael E. Duffey, None; Sangita P. Patel, Alcon ResearchInstitute (F)Support: Alcon Research Institute Young Investigator Grant (SPP).Funded in part by Ralph Hochstetter Medical Research Fund in honorof Dr. Henry C. and Bertha H. Buswell (SPP). University at Buffalo,School of Medicine, Summer Research Fellowship (TMM).Unrestricted Grant, Research to Prevent Blindness, NY, NY.Program Number: 5410 Poster Board Number: A0054Presentation Time: 8:30 AM - 10:15 AMROCK Inhibitor Y-27632 Enhances the Mucin Secretion fromGoblet Cells Derived from Limbal Stem CellsWon Seok Choi 1 , Eui Sang Chung 2 , Tae-Young Chung 2 , Joon YoungHyon 3, 4 , Won Ryang Wee 3 , Young Joo Shin 1 . 1 Ophthalmology,Hallym University College of Medicine, SEOUL, Republic of Korea;2 Ophthalmology, Sungkyunkwan University School of Medicine,SEOUL, Republic of Korea; 3 Ophthalmology, Seoul NationalUniversity College of Medicine, SEOUL, Republic of Korea;4 Ophthalmology, Seoul National University Bundang Hospital,SEOUL, Republic of Korea.Purpose: To investigate the effect of Rho-associated coiled kinaseinhibitor (ROCK inhibitor) Y-27632 on Goblet cells derived fromcorneal limbal stem cells.Methods: Goblet cells were derived from human limbal stem cellsand cultured. The cells were treated with ROCK inhibitor. Periodicacid-Schiff (PAS) staining and immunofluorescence staining ofABCG2, CK-7 and MUC5AC was performed. Cell proliferation ratewas measured with BrdU labeling assay.Results: The cultured cells were stained with ABCG2 and CK-7.Goblet cell derived from limbal stem cells showed mucin secretionby PAS staining and MUC5AC staining. Cell proliferation rateincreased with ROCK inhibitor.Conclusions: Goblet cells were differentiated from limbal stem cells.ROCK inhibitor enhanced mucin secretion and proliferation rate inGoblet cells derived from limbal stem cells.Commercial Relationships: Won Seok Choi, None; Eui SangChung, None; Tae-Young Chung, None; Joon Young Hyon, None;Won Ryang Wee, None; Young Joo Shin, NoneProgram Number: 5411 Poster Board Number: A0055Presentation Time: 8:30 AM - 10:15 AMImpact of Acute Exposure to High Altitude on Anterior ChamberGeometryM Dominik Fischer 1, 2 , Gabriel Willmann 1 , Andreas Schatz 1 , KaiSchommer 3 , Ahmad Zhour 1 , Eberhart Zrenner 1 , Karl-Ulrich Bartz-Schmidt 1 , Florian Gekeler 1 . 1 Centre for Ophthalmology, UniversityHospital Tuebingen, Tuebingen, Germany; 2 Nuffield Laboratory ofOphthalmology, University of Oxford, Oxford, United Kingdom;3 Department of Sports Medicine, Medical Clinic, University HospitalHeidelberg, Heildelberg, Germany.Purpose: This study aimed to quantify the impact of acute exposureto high altitude on central corneal thickness and the geometry of theanterior chamber angle. This work is related to the Tuebingen HighAltitude Ophthalmology (THAO) study.Methods: Anterior segment spectral domain optical coherencetomography was used to quantify changes of central cornealthickness, anterior chamber angle and angle opening distance in 14healthy subjects between baseline recordings (341 m) and duringacute exposure to high altitude (4559 m).Results: Detailed longitudinal analysis revealed highly significant (p< 0.0001) increased central corneal thickness (CCT) in healthysubjects during acute altitude exposure (CCTbaseline = 517.53±28.28μm vs. CCTaltitude = 539.87±31.28 μm; mean±sd). This change wascompletely reversible upon descend and no subject demonstratedpersisting structural or functional sequels. Geometric measures of theanterior chamber angle remained consistent with no significantchanges in angle opening distance (AOD) at 500 μm (AODbaseline =695.96±190.00 μm vs. AODaltitude = 673.71±179.59 μm; p = 0.52)and stable measurements of anterior chamber angle (ACA) in degree(ACAbaseline = 37.85±6.53 vs. ACAaltitude = 36.29±5.81 μm; p =0.34).Conclusions: Significant changes of CCT occur in response to acuteexposure to high altitude in healthy control subjects. This might bedue to decreased atmospheric pressure and consequently decreasedblood oxygen saturation (SpO2) in non-acclimatized subjects andconstitute a mild corneal edema formation. Interestingly, AOD at 500μm and ACA remained stable during the acute challenge to hypoxicconditions at high altitude. This is the first time a quantitativeapproach has been used to assess changes of the anterior segmentduring acute, non-acclimatized high altitude exposure. As such, itmight provide a basis for the debate on changes of intraocularpressure during exposure to high altitude.Commercial Relationships: M Dominik Fischer, None; GabrielWillmann, None; Andreas Schatz, None; Kai Schommer, None;Ahmad Zhour, None; Eberhart Zrenner, Retina Implant AG (F),Retina Implant AG (I), Retina Implant AG (C), Retina Implant AG(P), QLT Inc (C), Servier, Paris (C), Steinbeis GmbH&CoKG,Stuttgart (I), Steinbeis GmbH&CoKG, Stuttgart (C), Neurotech, USA(C), Pfizer, USA (C); Karl-Ulrich Bartz-Schmidt, Retina Implant(P); Florian Gekeler, Retina Implant AG (F), Okuvision GmbH (F),Retina Implant AG (C), Retina Implant AG (P)Program Number: 5412 Poster Board Number: A0056Presentation Time: 8:30 AM - 10:15 AMEvaluation of Feature Detectors and Descriptors on the IrisSandro I. De Zanet 1, 2 , Michael Rueegsegger 1, 2 , Tobias Rudolph 1, 2 ,Sebastian Wolf 2 , Jens H. Kowal 1, 2 . 1 Ophthalmic TechnologiesARTORG Center, University of Bern, Bern, Switzerland;2 Department of Ophthalmology, University of Bern, Bern,Switzerland.Purpose: Characterizing and matching human iris patterns isessential for video tracking in ophthalmic surgery as well as in irisrecognition. This study presents an evaluation of feature detectionand description algorithms. The evaluation is based on a database of19 eyes captured in the infrared spectrum.Methods: Repeatability and precision/recall were measured forcombinations of state-of-the-art feature detectors and descriptors:SURF, SIFT, FAST, STAR and BRISK as detectors and SURF,SIFT, FREAK, BRISK and BRIEF as descriptors. The algorithmswere tested with images of the iris in different geometric andphotometric transformations. A ground truth was provided by usingmanually segmented and non-rigidly registered irides and maskingout reflections. Images were acquired from 40 human irides. The eyeswere illuminated by infrared LEDs and captured by a camera via abeam splitter on a slit lamp microscope. To fixate the gaze of thepatients yellow LEDs were used to indicate different gaze angles.Images where a good ground truth could not be found were discardedwhich results in 19 testable irides.Results: The most accurate detector/descriptor combination has beenfound to be STAR/SU-BRISK which features fast detection incombination with fast matching through the Hamming distance.Especially the SU-BRISK descriptor has proven to be the mostdistinctive independent from the used detector. Generally orientationandscale-variant descriptors performed far better than their invariantcounterparts. Additionally binary descriptors based on intensity©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 – Physiology/Pharmacologycomparisons like BRIEF, BRISK or FREAK yield a morediscriminatory descriptor than real value based vectors. Through theevaluation of detected point distributions it is evident that the regionfrom the collarette to the pupil border is the are with the mostfeatures. Gaze change of 15° proved to decrease the matchingaccuracy. However, decreasing the contrast with less intensive lightimpaired accuracy more than gaze change. The area under theprecision-vs-recall-curve shows a range of 0.01 to 0.71 whichindicates the importance of isolating the best configuration for irisimages.Conclusions: In this study a good candidate for real-time featurematchinghas been found which can be used in torsional eye trackingused in beam therapy, namely the STAR detector with the scale- androtation-variant BRISK descriptor.film (TF)).Results: In-vivo: median lid margin thickness was 1.8mm (LL) and1.9mm (UL), but not significant different (p=0.258). Lissamine greendrops stayed unaltered in all subjects. Median OB grade was 3. TheLL margin tightened in blinks by 1.2mm (median) but this was notrelated to OB (r=-0.25; p=0.220). Median TMD ratio (TMD almostclosed eye / TMD opened eye) was 1.8 indicating an increasingseparation of the upper eyelid margin from the cornea. This smalleffect was significantly correlated to OB (r=0.875; p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyCommercial Relationships: Ta-Ching Chen, None; Fung-RongHu, NoneProgram Number: 5415 Poster Board Number: A0059Presentation Time: 8:30 AM - 10:15 AMGrowth factor secretion in human keratocyte cultures followingphotodynamic inactivation (PDI)Tanja Stachon 1 , Jiong Wang 1, 2 , Achim Langenbucher 3 , BertholdSeitz 1 , Nora Szentmáry 1 . 1 Department of Ophthalmology, SaarlandUniversity Hospital, Homburg/Saar, Germany; 2 Department ofOphthalmology, Renmin Hospital of Wuhan University, Wuhan,China; 3 Experimental Ophthalmology, Saarland University,Homburg/Saar, Germany.Purpose: Photodynamic inactivation (PDI) may be an alternativetreatment option of infectious keratitis, with increasing resistance ofmicroorganisms to antibiotics. In previous studies we determinedviability, apoptosis, proliferation, CD34 and α-smooth actinexpression of keratocytes following PDI. The purpose of our presentstudy was to assess the secretion of KGF, VEGF, TGFβ1, HGF andFGFb in human keratocytes following PDI, in vitro.Methods: Primary human keratocytes were isolated by digestion incollagenase A (1 mg/ml) from human corneal buttons, and cultured inDMEM/Ham’s F12 medium supplemented with 10% fetal calfserum. Five and twenty-four hours after PDI (100 nM chlorine e6,illumination 13 minutes at 670 nm), the release of growth factors wasdetermined using enzyme-linked immunosorbent assay (ELISA). Theprotein concentration of the cells was measured with Bradford Assay.Results: Five hours following PDI, the secretion of HGF was 0.43pg/µg protein, and FGFb expression was 3.47 pg/µg protein. Thesecretion of HGF decreased (p < 0.01) and the release of FGFbincreased significantly (p < 0.0001) compared to controls. At thistime point TGFβ1 was not detectable and VEGF and KGF secretionwas not significantly different from control cultures. Twenty-fourhours after illumination expression of none of the growth factors wassignificantly different compared to controls. Treatment only withillumination or Ce6 did not show changes in the expression of growthfactors at any time point.Conclusions: Five hours after PDI, HGF expression decreases andFGFb expression increases in keratocyte cell cultures. However, 24hours after treatment growth factor secretion seems to be normalized.The altered secretion of HGF and FGFb may play a role in activationof keratocytes and wound healing response after PDI of the cornea.Commercial Relationships: Tanja Stachon, None; Jiong Wang,None; Achim Langenbucher, None; Berthold Seitz, None; NoraSzentmáry, NoneProgram Number: 5416 Poster Board Number: A0060Presentation Time: 8:30 AM - 10:15 AMEfficacy of a novel synthetic topical tetrapeptide on elicitinganalgesia subsequent to experimentally induced chemical cornealinjuryBruce I. Gaynes, Michael Russo, David Goldmeier. Ophthalmology,Loyola University Chicago, Maywood, IL.Purpose: To ascertain analgesic action of a novel synthetictetrapeptide (SIS-ZEP04) analogue in reducing pain in a rat model ofexperimentally induced chemical corneal injury.Methods: Eight adult Sprague Dawley rats were utilized for study.Following approval of the local animal use committee, animalsunderwent treatment with 20 µL of 0.01 mg/mL of tetrapeptide in0.01% ethanol vehicle twice daily in the right eye for 14 consecutivedays. A negative control was employed in the fellow eye. Efficacy ofthe peptide as an analgesic was ascertained by the capsaicin eyeirritant test comprised of a dilute 0.02% solution of capsaicin.Capsaicin administration to both eyes was performed at baseline, day7 and day 14. The time required for resolution of blepharospasm, eyewiping and squinting following administration of capsaicin wasrecorded as a surrogate indicator of analgesic efficacy in relation tothe control eye.Results: Mean time (seconds) for recovery from capsaicin inducedirritation in both right and left eyes at baseline was 38.0 +/- 7.8 and52.2 +/- 9.8 seconds respectively. Following SIS-ZEP04 treatment(day 14) mean time for capsaicin induced irritation recovery was12.75 +/-5.6 and 24.4 +/- 14.69 seconds for right and left eyesrespectively. A statistically significant reduction in mean time tocapsaicin recovery was found for both right and left eyes (paired oneway t test, p=0.0125 and 0.0036 respectively, alpha=0.5). Cochet-Bonnet aesthesiometry measurements did not deviate from baselinelevels at study conclusion.Conclusions: The exogneous application of a synthetic tetrapeptideappears to demonstrate significant efficacy in reducing ocular painand modifying pathways of nociception following experimentalchemical ocular injury. As analgesic action was apparent in bothtreated and control eyes, it is unclear what effect, if any, the vehicleexerted in minimizing ocular pain or rather if a central action inducedby systemic absorption of the peptide is in place. Moreover, theanalgesic action appears to occur without reduction in cornealsensitivity. Further study is required to clarify the mechanism bywhich this peptide exerts apparent analgesia in the mammalian eye.Commercial Relationships: Bruce I. Gaynes, Shulov Institute ofScience Ltd (P), Shulov Institute of Science Ltd (F); Michael Russo,None; David Goldmeier, NoneSupport: Shulov Institute of Science Ltd. Rehovot, IsraelProgram Number: 5417 Poster Board Number: A0061Presentation Time: 8:30 AM - 10:15 AMEnantiomeric Separation, Ophthalmic Formulations andMydriatic Activity Oo Cyclopentolate HydrochlorideDanilo Aleo 1 , Sergio Mangiafico 1 , Maria G. Saita 1 , Barbara Melilli 1 ,Melina G. Cro 1 , Sebastiano Mangiafico 1 , Nicola D'Antona 2 ,Giovanni Nicolosi 2 . 1 R&D, Medivis, Catania, Italy; 2 Istituto ChimicoBiomolecolare, CNR, Catania, Italy.Purpose: Cyclopentolate Hydrochloride (CYP) is widely used asmydriatic and cycloplegic agent. The product is marketed in theracemic form, an equimolar mixture of the two enantiomers (+) and (-) Cyclopentolate. The aim of our study was the development of anenantio-separation methodology, the preparation of two ophthalmicformulation based on (+) CYP (MDV-D) and (-) CYP (MDV-L) aswell as the evaluation of their mydriatic activity in rabbits.Methods: Optical resolution of the racemic mixture was performedvia diasteromic salt formation using (2R,3R) O-O’-di-p- toluoyltartaricacid (DPTTA) as resolving agent. A microemulsion was usedto formulate and to deliver 1% of (+) and 1% of (-) CYP. NineAlbino rabbits were used to control mydriatic effect of MDV-D,MDV-L, and of the commercial Cyclopentolate racemic solution(Ciclolux, Allergan Inc.). The pupils were filmed during 35 minuteswith a video camera connected to an operation microscope, and themean pupil diameters were measured from the video recordings.Results: A diastereoisomeric salt containing an enantiomeric excess(E.E 90%) of (-) CyP (-) DPTTA has been achieved using (2R,3R) O-O’-di-p- toluoyl-tartaric acid (DPTTA). This salt was then purified byre-crystallization from ethanol (99% purity). (-) CYP (-) DPTTA wasdissolved in a 0,4N HCl and extracted with Methyl tert-Butyl Ether(MTBE). Evaporation of aqueous phase gives (-) CyP (100% [α] 20D= -32,8). The mother liquor containing (+)CyP (-)DPTTA wasextracted with MTBE and evaporation of aqueous phase gives (+)CYP (100% [α] 20D = +32,8). 1% of (+) CYP (MDV-D) and 1% 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 – Physiology/Pharmacology(-) CYP (MDV-L) microemulsion were instilled on the ocular surfaceof rabbits and the mydriatic effect of both formulations wasperformed. MDV-D and MDV-L showed the same effect on pupilsize when compared each other or with Ciclolux.Conclusions: The comparison between the ophthalmic formulationsof the two enantiomers (MDV-D and MDV-L) with one another andin comparison to the commercial formulation of the racemic product(Ciclolux) showed no statistically significant differences in theirmydriatic activity. Studies on the known side effects in humans ofCyclopentolate enantiomers (dizziness and mental confusion,impaired coordination of movements, etc…) are in progress.Commercial Relationships: Danilo Aleo, Medivis (E); SergioMangiafico, Medivis (E); Maria G. Saita, Medivis (E); BarbaraMelilli, Medivis (E); Melina G. Cro, Medivis (E); SebastianoMangiafico, medivis (E); Nicola D'Antona, None; GiovanniNicolosi, None530 Gene Therapy and DeliveryThursday, May 09, 2013 10:30 AM-12:15 PM618-620 Paper SessionProgram #/Board # Range: 5963-5969Organizing Section: Physiology/PharmacologyProgram Number: 5963Presentation Time: 10:30 AM - 10:45 AMOral delivery of bioencapsulated myelin basic protein ameliorateamyloid burden and RGC loss in transgenic mouse model ofAlzheimer’s diseaseQiuhong Li 1 , Amrisha Verma 1 , Ping Zhu 1 , Pollob K. Shil 1 , NehaKohli 2 , Donevan Westerveld 2 , Henry Daniell 2 . 1 Ophthalmology,University of Florida, Gainesville, FL; 2 University of Central Florida,Orlando, FL.Purpose: Increased deposition of amyloid beta 42 (Aβ42) isassociated with RGC apoptosis and retinal structural and functionalimpairments. Myelin basic protein (MBP), a major structural proteinof CNS, also possesses intrinsic protease activity capable ofdegrading Aβ amyloid, binds Aβ amyloid and inhibit Aβ fibrilformation. We aimed to investigate the protective effect of oraldelivery of bioencapsulated MBP fused with the transmucosal carriercholera toxin B subunit (CTB) in triple transgenic (3xTg) mousemodel for Alzheimer's disease (AD).Methods: CTB-MBP fusion protein was expressed in chloroplasts oftobacco. CTB-GFP expressed from chloroplasts of tobacco was alsoused as control. The 3xTg AD mice (12-14 months old) were fedwith CTB-MBP bioencapsulated in plant cells for 3 months. Aβ42aggregates were evaluated by ELISA and western blotting offractionated brain and retinal proteins, as well asimmunofluorescence of frozen sections using antibodies specific toAβ42. Retinal morphology, apoptosis and RGC density wereevaluated from fixed sections.Results: Orally delivered CTB- GFP bioencapsulated in plant cellswas detected in brains and retinae of healthy mice. Brain Aβ levelswere reduced in 3xTgAD mice fed with bioencapsulated CTB-MBP,especially the Aβ-42 insoluble fraction. The amyloid plaque intensitywas reduced in a concentration dependent manner by CTB-MBPincubation with human AD and 3xTgAD mice brain sections. CTB-MBP oral delivery reduced Aβ-42 accumulation in retinae andprevented loss of retinal ganglion cells. Lyophilization of leavesincreased CTB-MBP concentration by 17-fold and facilitated longterm storage at room temperature in capsules.Conclusions: Orally delivered CTB-MBP was able to reduce Aβ42aggregates in the brain and retina and prevent RGC loss in the aged3xTg AD mouse. Bioencapsulation protected fusion protein fromacids and enzymes in the digestive system and CTB fusion facilitateduptake by target cells including brain and retina, thus this technologyprovides a novel, more efficient, environmentally friendly and costeffectivedelivery of therapeutic proteins free of human or animalpathogens to treat neurodegenerative diseases.Commercial Relationships: Qiuhong Li, None; Amrisha Verma,None; Ping Zhu, None; Pollob K. Shil, None; Neha Kohli, None;Donevan Westerveld, None; Henry Daniell, NoneSupport: American Diabetes Association, American HeartAssociation, Research to Prevent Blindness, NIH grants EY021752and EY021721 to Li; NIH R01 HL 109442 and NIH R01 HL 107904,Bill and Melinda Gates Foundation Global Health grant OPP1031406 and the Juvenile Diabetes Research Foundation grant 17-2011-286 to Dr. Henry DaniellProgram Number: 5964Presentation Time: 10:45 AM - 11:00 AMTreatment of Patients with Leber Congenital Amaurosis Type 2with an AAV Vector Expressing RPE65Tim Stout 1 , Richard G. Weleber 1 , Maureen McBride 1 , David J.Wilson 1 , Dawn Peters 1 , Margaret R. Humphries 3 , Terence R. Flotte 3 ,Lauren J. Jensen 1 , Andreas Lauer 1 , Jeffrey D. Chulay 2 .1 Ophthalmology, Casey Eye Institute-OHSU, Portland, OR; 2 AGTCInc, Alachua, FL; 3 School of Medicine, University of Massachusetts,Worcester, MA.Purpose: To evaluate the safety and efficacy of rAAV2-CB-hRPE65in patients with Leber congenital amaurosis (LCA) caused bymutations in the RPE65 gene.Methods: Twelve patients (aged 6-39y) with RPE65 mutations weretreated with a single, unilateral subretinal dose of 1.8 x 1011 or 5.4 x1011 viral genomes in 450 μL. Serial postoperative examinationsincluded measurements of acuity, static and kinetic perimetry, opticalcoherence tomography, fundus photography, luminance sensitivityand visual quality-of-life function.Results: All subjects tolerated the surgery and study agentadministration well without significant or unexpected complications.No instances of persistent subretinal fluid or ocular inflammatorydisease were observed. Visual acuities were transiently depressed inthe treated eye of all patients during the first 1 to 2 weeks aftersurgery, but returned to baseline or better in all of but two patients todate. Improvements in visual acuity after treatment were seen in thefour youngest patients (age 6 to 11 years - with better baseline visualacuities of 40 to 62 ETDRS letters) with increases ranging from 6 to12.5 ETDRS letters in the treated eye. For the three subjects withvisual acuity of 20 to 31 ETDRS letters, one had a 2.5 letter increaseand two had a 6.5 or 12 letter decrease in the treated eye. GATE totaland central 30O hill of vision analysis trended towards improvementin treated eyes when compared to baseline values. The five subjectswith the poorest baseline visual acuity (0 or 1 ETDRS letters) hadlittle or no change in their visual acuity over time, but for the foursubjects followed for at least 6 months three had a small butstatistically significant increase in kinetic perimetry visual field areawith the V4e target in the treated eye compared to baseline. Animprovement in visual functioning and quality of life was noted bymost patients.Conclusions: Treatment of LCA2 patients with rAAV2-CB-hRPE65is safe and appears effective. The greatest improvements in visualacuity were observed in younger patients who presented with betterbaseline visual acuity.Commercial Relationships: Tim Stout, Clayton Foundation (P),Oxford Biomedica (C), AGTC (F), Peregrine Pharmaceuticals Inc(C), Stem Cells Inc (C); Richard G. Weleber, AGTC (C), VFMA©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 – Physiology/Pharmacologypatent application (P), Pfizer (C), Oxford Biomedica (F); MaureenMcBride, None; David J. Wilson, None; Dawn Peters, None;Margaret R. Humphries, None; Terence R. Flotte, None; LaurenJ. Jensen, None; Andreas Lauer, Oxford Biomedica (F), Acucela(F), NIH (F); Jeffrey D. Chulay, AGTC (E)Support: AGTC Clinical Trial Support, Foundation FightingBlindness ORDC Center Grant (Weleber, C-CL-0711-0534-OHSU01)Clinical Trial: NCT00749957Program Number: 5965Presentation Time: 11:00 AM - 11:15 AMSustained Therapeutic Reversal of Canine Bestrophinopathywith Gene Therapy using Recombinant AAV2Karina E. Guziewicz 1 , Andras M. Komaromy 2 , Simone Iwabe 1 , ArturV. Cideciyan 3 , Emily V. Dutrow 1 , Barbara Zangerl 4 , William A.Beltran 1 , Samuel G. Jacobson 3 , William W. Hauswirth 5 , Gustavo D.Aguirre 1 . 1 Clinical Studies, University of Pennsylvania, Philadelphia,PA; 2 Department of Small Animal Clinical Sciences, Michigan StateUniversity, East Lansing, MI; 3 Scheie Eye Institute, University ofPennsylvania, Philadelphia, PA; 4 Centre for Eye Health, Universityof New South Wales, Kensington, NSW, Australia; 5 Department ofOphthalmology, University of Florida, Gainesville, FL.Purpose: Canine multifocal retinopathy (cmr), a spontaneous animalmodel of BEST1-associated retinopathies in man, recapitulates thespectrum of clinical and molecular features observed in humanbestrophinopathies, and is an important translational model for thedevelopment and testing of therapeutic strategies. We havepreviously shown that rAAV2-mediated BEST1 gene deliverycontrolled by human VMD2 promoter (hVMD2) specifically targetsRPE cells, and is well tolerated in the wild-type canine retina. Theaim of these studies was to assess the safety, efficiency andtherapeutic potential of rAAV2-mediated BEST1 transgeneexpression in cmr-affected dogs.Methods: Thirteen cmr-affected dogs carrying R25X, R25X/P463fsor P463fs mutations in BEST1 and exhibiting bilateral focal ormultifocal lesions were subretinally injected with rAAV2 expressingeither canine (0.136-1.59x10 11 vg/ml) or human (7.64-8.82x10 11 vg/ml) BEST1 regulated by the hVMD2 promoter. Eachtreated and control (non-injected or BSS-injected) eye was monitoredclinically and imaged serially in vivo using cSLO/SD-OCT. Formorphological studies, treated and control tissue samples werecollected at 1 - 15 months post injection (p.i.).Results: In all cases, the transient retinal detachment associated withvector delivery completely resolved within 24h p.i. Based on the SD-OCT analyses, some lesions within the treated regions disappeared asearly as 8 weeks and all resolved within 3 months after gene therapy,and the treated areas remained asymptomatic thereafter. In cases withadvanced disease, hyperfluorescent FAF signals were still detectableand likely localized to RPE. The untreated regions of the treated eyeor the contralateral control eye remained unchanged or developedlesions. Specific Best1 immunolabeling was detected only within thetreated area, and comparable efficacy was noted with canine andhuman cDNAs. Both the in vivo imaging and immunohistochemicalevaluation revealed no apparent adverse effects in RPE or retinasecondary to the BEST1 gene augmentation therapy.Conclusions: rAAV2-mediated BEST1 gene augmentation therapyshows great potential to reverse characteristic BEST1 lesions andreverse pathology in cmr models up to 15 months p.i., and carries alarge translational promise as a first specific-treatment for humanbestrophinopathies.Commercial Relationships: Karina E. Guziewicz, None; AndrasM. Komaromy, None; Simone Iwabe, None; Artur V. Cideciyan,None; Emily V. Dutrow, None; Barbara Zangerl, None; WilliamA. Beltran, None; Samuel G. Jacobson, None; William W.Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C),RetroSense (C); Gustavo D. Aguirre, NoneSupport: FFB, MVRF, NEI/NIH EY06855, EY17549, Van SlounFund, Hope for VisionProgram Number: 5966Presentation Time: 11:15 AM - 11:30 AMVertical Gene Transfer of Mutant Human G11778A ND4 in NextGeneration Mito-MiceHong Yu, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti,John Guy. Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami,Miami, FL.Purpose: To describe vertical gene transfer of the mutant humanND4 (hmutND4) bred from founder mice generated by mitochondriatargeting sequence AAV infection of mouse embryonic stem cells.Methods: hmutND4 with a FLAG epitope and mitochondrialencoded mCherry under control of a mitochondrial promoter waspackaged into mito-targeted AAV2 containing the COX8 leadersequence inserted into the VP2 capsid, then microinjected into themouse blastocyst. Retina, brain, optic nerve, heart, liver and skeletalmuscle were assessed by PCR, sequencing, two-dimensional bluenative polyacrylamide gel electrophoresis (2D BN-PAGE), complex Iactivity and histopathology. Visual function was monitored by serialpattern electroretinography (PERG), retinal structure by spectraldomain optical coherence tomography(SD-OCT) and mitochondrialgene expression by confocal laser scanning ophthalmoscopy (CLSO)of mCherry fluorescence.Results: 60 transgenic mice were generated that contained varyingexpression of in vivo mCherry fluorescence. Three females with thehighest levels of mCherry expression in the retina were mated withmales of the same strain and produced a total of 137 pups over 4generations. Red fluorescent particles were seen in the RGC layerand optic nerve head in 77% of mice, and cells with fluorescenceincreased as mice aged suggesting replication of the transgene.hmutND4 was detected by PCR in all tissues from the F0 foundermice to their F1 to F4 progeny. Characteristic of mitochondrialheteroplasmy, transgene levels differed significantly between tissues(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyVenu Talla, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti,John Guy. Ophthalmology, Bascom Palmer Eye Institute, Universityof Miami, Miller School of Medicine, Miami, FL.Purpose: To rescue visual loss and optic neuropathy in theexperimental autoimmune encephalomyelitis (EAE) mouse modelusing gene therapy with the mtHSP70 chaperone responsible forunfolding and import of most proteins into the mitochondria.Methods: EAE was induced in female DBA/1J (n=20) mice bysubdermal injection of 0.1 ml homologous spinal cord emulsion inCFA. Ten mice were rescued by intravitreal injection of ssAAVmtHSP70-Flag,10 EAE and 10 unsensitized mice injected withscAAV-Cox8-mCherry served as controls. Visual function wasassessed by pattern electroretinograms (PERG). High resolutionspectral domain OCT evaluated the thickness of the inner plexiformlayer + nerve fiber layers at 1, 3 and 6 months post injection (MPI).All mice were euthanized 6MPI. Retinas and ONs were dissected forhistological and ultrastructural evaluation. Expression ofmtHSP70Flag in the retina and ONs was evaluated 15d PI by RT-PCR, immunofluorescence (IF) and western blotting (WB).Results: IF revealed a typical punctate and perinuclear expression ofFlag-HSP70 which colocalized with mitochondrial porin and thy1.2labeled RGCs. RT-PCR and WB confirmed HSP70 expression in theretina and ON. PERG amplitude at 3M and 6MPI showed 42% and45% reduction in EAE-mCherry compared to control mCherry(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyof a vector solution at 6.1010 vector genome/mL. The primary safetyendpoints are evaluated by biomicroscopy, laser flare meter, fundusphotography, fluorescein angiography, OCT and a tolerancequestionnaire. Secondary efficacy endpoints are evaluated by globalERG, multifocal ERG, visual field, near and far visual acuity, colorvision test, pupillometry, microperimetry, visual mobility test,functional MRI and fundus autofluorescence.Results: Patients, 20 to 42 years old, were treated between october2011 and september 2012. The injected volume was between 200 μLand 770 μL of the vector solution. No adverse effects or ocularinflammation are noticed one year to 2 months after subretinalinjection. EDTRS score change on average of 5,8 letters aftersubretinal injection (0,15 Log MAR). A decrease of the nystagmus isnoticed in all patients. A decrease of the moving time in the mobilitytest in scotopic brightness is found with the treated eye. Visual fieldmodifications in the subretinal area are noticed in patients.Conclusions: The preliminary data of this AAV4 gene therapyclinical trial in patients with rpe65 retinal degeneration suggested agood tolerance and a functional efficency. This results may beconfirmed by the pursuit of the follow up of the first six patients andby thedata analysis of next included three patients.Commercial Relationships: Guylene Le Meur, None; PierreLebranchu, None; Yann Péréon, None; Sebastien Schmitt, None;Stéphane Bézieau, None; Philippe Moullier, None; FabienneRolling, None; Michel Weber, NoneSupport: PHRC07-09KClinical Trial: NCT01496040545 Retina: Physiology and PharmacologyThursday, May 09, 2013 10:30 AM-12:15 PMExhibit Hall Poster SessionProgram #/Board # Range: 6314-6365/D0185-D0236Organizing Section: Physiology/PharmacologyContributing Section(s): Biochemistry/Molecular Biology, VisualNeuroscienceProgram Number: 6314 Poster Board Number: D0185Presentation Time: 10:30 AM - 12:15 PMFunctional Expression Of TRPV Channels In The RetinalEndotheliumJennifer E. McNaughten, Mary McGahon, Graham McGeown,Timothy M. Curtis. Queens University Belfast, Belfast, UnitedKingdom.Purpose: Transient receptor potential vanilloid (TRPV) channels arepart of a superfamily of non-selective cation channels with a vastrange of physiological functions. Our aim was to identify thefunctional TRPV channel subtypes expressed in the retinalendothelium.Methods: Retinal microvascular endothelial cells (RMECs) werecultured from bovine arterioles. Fura-2-based Ca2+ microfluorimetrywas used to test for the functional expression of TRPV1, V2, V3 andV4 in RMECs using a selection of agonists and antagonists.Results: The ultrapotent TRPV1 agonist resiniferatoxin(10nM)caused a transient rise in calcium in 50% of cells tested. Theseresponses were abolished in the presence of two different TRPV1antagonists, capsazepine(5uM) and AMG9810(100nM). Functionalexpression of TRPV2 was evident from calcium peaks in response toΔ9-THC(10uM). The responses were reduced after incubation withtranilast(75uM), a TRPV2 inhibitor. Carvacrol(100uM) providedevidence for the presence of TRPV3 in RMECs causing consistantcalcium responses across a number of cells. Both tranilast andcarvacrol were used in the presence of the TRPA1 antagonist HC-030031(10uM). The well known TRPV4 channel was tested for usingGSKA(100nM) and 4αPDD(1uM). Both elicited transient rises incalcium. The specific TRPV4 antagonist HC067047(1uM) blocksresponses caused by GSKA. The aforementioned TRP channels weretested for in EGTA(1mM) buffered calcium free solution. Underthese conditions none of the agonists initiated a rise in calciumsuggesting the responses were mediated by calcium influx from theextracellular medium.Conclusions: This study provides evidence for the functionalexpression of TRPV1-V4. This warrants further research to elucidatethe physiological significance of TRP channels in endothelial cellfunction.Commercial Relationships: Jennifer E. McNaughten, None; MaryMcGahon, None; Graham McGeown, None; Timothy M. Curtis,NoneSupport: Department for Employment and LearningProgram Number: 6315 Poster Board Number: D0186Presentation Time: 10:30 AM - 12:15 PMFurther Studies On The Role Of Arachidonic Acid MetabolitesIn The Regulation Of Potassium-Induced [ 3 H]D-AspartateRelease From Isolated Bovine Retinae By 5-epi-5-F3t-IsoprostaneJamal Jamil 1 , Pratik Bankhele 1 , Ankita Salvi 1 , Thierry Durand 3 , JeanGalano 3 , Alexandre Guy 3 , Ya Fatou Njie-Mbye 2 , Sunny E. Ohia 2 ,Catherine A. Opere 1 . 1 Creighton.edu, Omaha, NE; 2 Texas SouthernUniversity, Houston, TX; 3 Universities of Montpellier I and II,Montpellier cedex, France.Purpose: We have evidence that eicosapentanoic acid (EPA)-derivedF 3 -isoprostane (F 3 -IsoP), 5-epi-5-F 3 t-IsoP inhibits excitatory aminoacid neurotransmitter release in bovine retina. In the present study,we investigated the role of arachidonic acid metabolites in theinhibitory action of 5-epi-5-F3t-IsoP on K + -induced [ 3 H]D-aspartaterelease from bovine retina, in vitro.Methods: Isolated neural retina were incubated in oxygenated Krebssolution containing 200 nM of [ 3 H]D-aspartate and then prepared forstudies of neurotransmitter release. Release of [ 3 H]D-aspartate wasevoked by K + (50 mM) stimuli applied at 90 mins (S 1 ) and at 108mins (S 2 ) after the onset of superfusion. F 3 -IsoP was added 8 min.before S 1 while antagonists were present before and during S 1 and S 2 .Results: 5-epi-5-F 3t -IsoP (0.1 nM - 0.1 µM) elicited an inhibitoryaction on K + -evoked [ 3 H]D-aspartate release in a concentrationdependentmanner, achieving a maximum inhibition of 46.9% at 0.1µM (IC 30 of 1 nM). Pretreatment of retinal tissues with thecyclooxygenase (COX) enzyme inhibitor, flurbiprofen (3 µM)unmasked a biphasic action, being inhibitory at lower (0.1 pM-10pM)and stimulatory at higher (0.1nM-0.1µM) concentrations of the IsoP.All the antagonists used exhibited no effect on K + -induced [ 3 H]Daspartaterelease. Similarly, SC 19220 (1 µM; EP 1 ) and AH 6809 (10µM; EP 1-3 /DP 1 ) had no effect on 5-epi-5-F 3t -IsoP (0.1 pM)-inducedinhibition of the neurotransmitter release. On the contrary, otherreceptor antagonists, BAY-u3405 (10 µM; TP/DP), SQ 29548 (10µM; TP) and ozagrel (10 µM; Tx-synthase) reversed the stimulatoryaction of the F 3 -IsoP (0.1 µM) on neurotransmitter release.Conclusions: The EPA-metabolite, 5-epi-5-F 3t -IsoP attenuates K + -induced [ 3 H]D-aspartate release via COX enzyme-dependentmechanisms. Furthermore, the presence of COX unmasks a TPdependentstimulatory action of this F 3 -IsoP on K + -induced [ 3 H]Daspartaterelease.Commercial Relationships: Jamal Jamil, None; Pratik Bankhele,None; Ankita Salvi, None; Thierry Durand, None; Jean Galano,None; Alexandre Guy, None; Ya Fatou Njie-Mbye, None; SunnyE. Ohia, None; Catherine A. Opere, 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 – Physiology/PharmacologyProgram Number: 6316 Poster Board Number: D0187Presentation Time: 10:30 AM - 12:15 PMProtective effects of an PPAR-γ agonist on retinalischemia/reperfusion injury in ratsXiaoyan Zhang, Yi-qin Xiao, Yu Zhang, Jiaying zhang, Wen Ye.Ophthalmology, Huashan Hospital affiliated to Shanghai FudanUniversity, Shanghai, China.Purpose: To investigate the protective effects of an PPAR-γ agonistin the rat retina after ischemia/reperfusion (I/R) injury.Methods: Retinal ischemia was induced in rats by increasing theintraocular pressure to 110mmHg for 60 minutes. The PPAR-γagonist was delivered by periocular injection and intraperitonealinjection before I/R. Seven days after I/R injury, retinal damage wasquantified by measuring the thickness of the retina, the functionalchanges of VEP and ERG, and the RGC number. The expression ofGFAP, NF-κB p65 in the retina was determined by western blot, realtimepolymerase chain reaction (PCR), and immunohistochemistry.Results: I/R caused severe disruption of the retinal construction andintegrity. In I/R group without treatment the number of RGCs wasreduced by 53%. The PPAR-γ agonist either delivered by periocularinjection or by intraperitoneal injection preserved the thickness of theretina after I/R, and the survival of RGC was 77% and 74%respectively. Pretreated with the PPAR-γ agonist also attenuate thedestruction of VEP and ERG caused by I/R. The amplitudes of theERG b-waves and the VEP P1-N2 component were significantlylower in the I/R group than in the groups pretreated with the PPAR-γagonist (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyProgram Number: 6318 Poster Board Number: D0189Presentation Time: 10:30 AM - 12:15 PMEvaluation of homocysteine and its metabolic cofactors inpatients with non proliferative and proliferative diabeticretinopathyGiulia Malaguarnera 1 , Caterina Gagliano 1 , Mario D. Toro 1 , FilippoDrago 2 , Teresio Avitabile 1 . 1 Department of Ophthalmology,university of Catania, Catania, Italy; 2 Department of ClinicalBiomedicine, university of Catania, Catania, Italy.Purpose: Homocysteine, a well-known inducer of vascularendothelial cell damage has been associated with extracellular matrixchanges. Many studies demonstrated that high levels of thisaminoacid in diabetic patients increases significantly the risk of thedevelopment of this pathology. This study has been undertaken toinvestigate the role of homocysteine and its cofactors during theprogression of the diabetic retinopathy.Methods: We measured the plasma levels of homocysteine, folicacid, vitamin B6 and vitamin B12 in 113 diabetic type 2 patients withnon proliferative retinopathy (NPDR), 52 with proliferative diabeticretinopathy (PDR) and 50 healthy subjects used as control group.Results: We found higher plasma levels of homocysteine in NPDRgroup compared to the control group (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyinvestigating intravitreous injection of carbonic anhydrase inhibitorsand pharmacotherapeautics for diabetic macular edema.Commercial Relationships: Armin Afshar, None; Rama Jager,NoneSupport: Illinois Society for the Prevention of BlindnessProgram Number: 6321 Poster Board Number: D0192Presentation Time: 10:30 AM - 12:15 PMQuality of fixation in major depressive disorder and the influenceof antipsychotic medicationSerena Fragiotta 1 , Pier Luigi Grenga 2 , Daniela Domanico 2 ,Alessandro Cutini 1 , Stefano Valente 1 , Vittoria De Rosa 1 , Enzo M.Vingolo 1 . 1 University of Rome La Sapienza, Latina, Italy;2 S.M.Goretti Hospital, latina, Italy.Purpose: To evaluate retinal function and fixation stability in majordepressive disorder (MDD) and to investigate the influence ofantipsychotic therapy on quality of fixation, using the MP-1microperimeter (Nidek Technologies).Methods: 28 patients with MDD (62.5±12.51 years) with logMARacuity ≤ 0.04 and 30 matched healthy subjects (HS) (63.62± 14.14years) with logMAR acuity ≤ 0.0 were enrolled. According to theDiagnostic and Statistical Manual of Mental Disorders 4th edition(DSM-IV), single (296.2) or recurrent episode (296.3) of MDD wereincluded. Patients with any other diagnosed mental illness or oculardisease were excluded. Retinal sensitivity, fixation stability, fixationpoints within 2 and 4 degree, and bivariate contour ellipse area(BCEA) were obtained from the MP-1 in MDD and HS. Moreoverpatients were divided according to therapy: 12 patients with (groupA) and 16 without (group B) atypical antipsychotic drugs. MeanBCEA (deg2) was normalized by logarithmic transformation(Shapiro-Wilk test, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologytreated animals were divided into three groups of 4 rats. These wereintraperitoneally injected with: 1) Suramin, 2) PPADS, 3) Suramin +PPADS at 9 and 26 weeks of diabetes in each group. Four remainingdiabetic animals received no treatment. Twelve non-diabetic rats withthe same age of diabetics were used as a control group. Animals weresacrificed after 38 weeks of diabetes. Retinas were analyzed byWestern Blot using a primary antibody against P2X2.Results: Protein expression of P2X2 was much higher in diabeticswithout treatment than in controls. Diabetic rats treated withSuramin, PPADS and the combined compound had a significantlylower expression of P2X2 than that found in diabetic animals withouttreatment.Conclusions: P2X2 receptor seems to play a role in diabeticretinopathy development. This purinergic receptor could be a targetfor the treatment of this disease.Commercial Relationships: Juan E. Gallo, EP2186529 B1 (P);Jorge Mancini, None; Gustavo A. Ortiz, None; Juan O. Croxatto,NoneProgram Number: 6324 Poster Board Number: D0195Presentation Time: 10:30 AM - 12:15 PMIntracellular pH of Retinal Ganglion Cells During SystemicHypoxiaVerleen K. McSween, Suresh Viswanathan, Joseph A. Bonanno,Stephen A. Burns, Shimin Li. School of Optometry, IndianaUniversity, Bloomington, IN.Purpose: Previously we showed that intracellular pH of retinalganglion cells (RGCs) decreases as a result of acute elevation ofintraocular pressure (IOP) (McSween et al. ARVO 2012). Thepurpose of the current study was to provide confirmatory data byexamining other perturbations, such as systemic hypoxia, that shouldalso affect RGC pH.Methods: RGCs were retrograde labeled in anesthetized adult maleBrown Norway rats by superior colliculus injection of 10kD AlexaFluor 790 dextran and the pH sensitive dye 2'-7'-bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF) dextran (30 mg/ml, 20 µl). Retinalfluorescence was measured with a confocal scanning laserophthalmoscope (HRA2, Heidelberg Engineering®) before, duringand after breathing 10% oxygen (90% nitrogen) for 4 minutes via anose cone at 2 weeks following dye injections. The retinal imageswere processed using custom MATLAB software and thefluorescence of individual cells was analyzed using the MetaMorph®image analysis software.Results: The fluorescence intensity of 517 double labeled cells wastracked. The intensity of Alexa790 labeling was not significantlydifferent before, during and after 10% oxygen breathing. Theintensity of BCECF labeling reduced by 19+11% during 10% oxygenbreathing relative to breathing room air (pGYY 4137. The substrate for endogenous H 2 S production, L-cysteinewas most potent, eliciting a maximum inhibitory action of 54.2%(n=4; p0.05) on K + -induced[ 3 H]D-aspartate release, they both reversed the inhibitory action ofGYY 4137 (10 μΜ) and L-cysteine (1-10 µM) on theneurotransmitter release. Furthermore, the nitric oxide (NO) synthaseinhibitor, L-NAME (300 µM) reversed the inhibitory action of GYY4137 (1-10 µM) on the excitatory neurotransmitter release.Conclusions: K ATP -channels, NO pathway and in situ production ofH 2 S contribute to the inhibitory action of H 2 S-producing compoundson excitatory neurotransmitter release in isolated bovine retina.Commercial Relationships: Pratik Bankhele, None; Jamal Jamil,None; Ankita Salvi, None; Ya Fatou Njie-Mbye, None; MadhuraS. Kulkarni, None; Sunny E. Ohia, None; Catherine A. Opere,NoneProgram Number: 6326 Poster Board Number: D0197Presentation Time: 10:30 AM - 12:15 PMAsymmetrical vitreous status in unilateral epimacular membraneobserved by swept-source optical coherence tomographyDanjie Li, Hirotaka Itakura, Shoji Kishi. Ophthalmology,Department of Ophthalmology, Gunma University School ofMedicine,, Maebashi, Japan.Purpose: To investigate the vitreous condition in unilateralidiopathic epimacular membrane (IEM) using swept-source opticalcoherent tomography (SS-OCT)Methods: We prospectively examined the vitreous status of 12consecutive cases of unilateral IEM using slit-lamp biomicroscopyand SS-OCT (Topcon, Japan). Best-corrected visual acuity of 12 IEMeyes were 1.2 in 4 eyes, 0.4 was in 5 eyes, and 0.5, 0.6, 0.8 in oneeye, respectively, and 1.2 in all normal fellow eyes. The meanrefractive was -4 diopter in IEM eyes and -3.5 in the fellow eyes.OCT images were obtained with a horizontal and a vertical 12 mmscan through the fovea in both eyes. We compared the prevalence ofPVD between affected and normal fellow eyes.Results: All 12 IEM eyes had complete PVD with Weiss ring on slitlampbiomicroscopy. SS-OCT confirmed no vitreous structure 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 – Physiology/Pharmacologyepiretinal membrane on the macula. Complete PVD was not observedin 12 fellow eyes. SS-OCT revealed the “posterior precorticalvitreous pocket” on the macular area in all 12 normal fellow eyes.Because of the pocket, premacular vitreous cortex was present as amembrane separated from the gel. Premacular vitreous cortex wasslightly detached in the perifoveal area in 10 eyes but attached at thedisc.Conclusions: SS-OCT enabled to depict the specific vitreouscharacteristics on the macula. The posterior wall of the “posteriorprecortical vitreous pocket” seems to serve a basic structure of IEM.Commercial Relationships: Danjie Li, None; Hirotaka Itakura,None; Shoji Kishi, NoneSupport: None in the SupportProgram Number: 6327 Poster Board Number: D0198Presentation Time: 10:30 AM - 12:15 PMThe Retina of the Cognitively Impaired Beagle Dog inAlzheimer’s DiseaseLaura Emptage 1 , Bill Milgram 3, 5 , Howard Dobson 3, 4 , ZoyaLeonenko 1, 6 , Melanie C. Campbell 1, 2 . 1 Physics and Astronomy,University of Waterloo, Waterloo, ON, Canada; 2 School ofOptometry and Vision Science, University of Waterloo, Waterloo,ON, Canada; 3 CanCog Technologies, Fergus, ON, Canada;4 University of Guelph, Guelph, ON, Canada; 5 University of Torontoat Scarborough, Scarborough, ON, Canada; 6 Biology, University ofWaterloo, Waterloo, ON, Canada.Purpose: Beagle dogs develop a form of Alzheimer’s disease (AD),Cognitive Dysfunction Syndrome, with symptoms similar to thedisease in humans. We wished to determine the differences inexpression of amyloid β between the retinas of cognitively impaireddogs and cognitively normal dogs; to characterize deposits and tocompare the results to data previously obtained from retinas ofhumans with AD. Amyloid β is known to over express within thecerebrum of patients and dogs with AD, but the sensitivity of amyloidβ deposits to fluorescent dyes is reported to differ in dog and humanbrains. Retinal amyloid β deposits would potentially allow objective,longitudinal tracking of AD in this species.Methods: Cognitive testing on these dogs included a battery of nonverbal cognitive function tests. Eyes were enucleated from twocognitively impaired and two normal dogs, euthanized for medicalreasons unrelated to this study, then fixed in formalin. Postdissection, pieces of the retinas were flat mounted and stained withThioflavin-S or Cucurmin and then studied using combinedfluorescence and atomic force microscopy (AFM). Amyloid βdeposits were expected close to the anterior surface of the flatmounted retinas of those with AD as found by our group previouslyin retinas from humans with AD. Areas of interest located byfluorescence were imaged with AFM at the nanoscale to characterizethe amyloid β deposits.Results: Retinas of the cognitively impaired dogs had depositsstained with both Curcumin and Thioflavin-S (on separate pieces ofretina). Amyloid β within the dog retina showed similarconformations to those in retinas from humans with AD, being eitheramorphous, “donut” like, or fibular. Amyloid β deposits were morenumerous and larger in size within the retina of the more cognitivelyimpaired dog. All retinas from control eyes were negative forfluorescent markers in both Thioflavin-S and Curcumin, and showedAFM images morphologically similar to human age matched controlretinas.Conclusions: Amyloid β deposits in retinas of cognitively impaireddogs have striking similarities in size, shape and location to amyloidβ deposits in the retinas of humans with AD. Staining withThioflavin-S was consistent, unlike reported inconsistent stainingwithin the dog cerebrum. These results strengthen the utility of thebeagle dog as a naturally occurring model of Alzheimer’s disease.Commercial Relationships: Laura Emptage, None; Bill Milgram,CanCog Technologies (E); Howard Dobson, CanCog Technologies(E); Zoya Leonenko, None; Melanie C. Campbell, CanCogTechnology (F), University of Waterloo (P)Support: NSERC and CIHR Canada, CFI, University of WaterlooProgram Number: 6328 Poster Board Number: D0199Presentation Time: 10:30 AM - 12:15 PMAcid Sphingomyelinase: a novel target for ischemia-inducedretinal degenerationJie Fan 1 , Bill X. Wu 2 , Craig E. Crosson 1 . 1 Ophthalmology-Storm EyeInst, Medical Univ of South Carolina, Charleston, SC; 2 Microbiology& Immunology, Medical University of South Carolina, Charleston,SC.Purpose: Acid sphingomyelinase (ASM) is an enzyme that catalyzesthe hydrolysis of sphingomyelin to the production of ceramide, whichis an important modulator of inflammatory cytokine and apoptoticsignaling. However, the role(s) of ASM in retinal neuronaldegeneration have not been investigated. The purpose of this study isto investigate whether sphingolipid-signaling is involved in theretinal ischemic injury, and whether suppression of ASM canameliorate the ischemic effect in the retina.Methods: Retinal ischemic injury was induced by elevatingintraocular pressure to 120mmHg for 45 minutes. Sphingolipidspecies that mediate the ischemia induced stress-signaling wereidentified by liquid chromatography-mass spectrometry 24 hoursfollowing ischemic injury. To assess if reducing ASM expression canprotect the retina from ischemic injury, ASM+/- mice and wild-type(WT) littermates were evaluated for changes in retinal function andmorphology by electroretinogram (ERG), and microscopicexamination at 7 days post ischemia.Results: The levels of ceramide and sphingosine displayedsignificant increases in ischemic retinas when compared withcontralateral retinas. Functional retinal assessment revealed thatischemic injury in WT mice exhibited significantly decreased a- andb-wave amplitudes by 48±11% and 54±9% of baseline levels,respectively. In ASM+/- mice, the ERG a-and b-waves were reducedby 35±6% and 29±6% of baseline levels, respectively. Althoughischemic injury was detected in ASM+/- mice, it was significantlyless than that measured in WT mice. Morphometric analysis ofischemic eyes from WT mice demonstrated a 17% decreases inoverall retina thickness and a significant loss (43% ) of cell bodies inthe retinal ganglion cell layer, when compared to contralateral eyes.However, in ASM+/- mice, the ischemic injury did not produce anysignificant decrease in overall retina thickness. In addition, only 13%of ganglion cell body loss was observed in ischemic eyes fromASM+/- mice.Conclusions: These data provided evidence that sphingolipidmetabolites play important roles in ischemic retinal injury. The ERGand retinal morphology results demonstrated that the reduction ofASM expression can partially protect the retina from retinal ischemicinjury. Hence, Inhibition of ASM may present new opportunities forthe treatment of retinal ischemic disorders.Commercial Relationships: Jie Fan, None; Bill X. Wu, None;Craig E. Crosson, Alimera Sciences (C), Lexicon Pharmaceuticals,Inc (R)Support: NIH grant EY021368, Research to Prevent Blindness(RPB)Program Number: 6329 Poster Board Number: D0200Presentation Time: 10:30 AM - 12:15 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 – Physiology/PharmacologyRetinal Expression of α-1-Antitrypsin (A1AT) in diabetic andnon-diabetic ratsGustavo A. Ortiz 1, 3 , Jorge Mancini 1, 3 , Eduardo Chuluyan 2 , Juan E.Gallo 1, 3 . 1 Ciencias Biomédicas, Universidad Austral, Pilar,Argentina; 2 Farmacología, Universidad de Buenos Aires, BuenosAires, Argentina; 3 Nanomedicine & Vision Group, UniversidadAustral, Pilar, Argentina.Purpose: Early stages of diabetic retinopathy are characterized byinflammatory changes. The role of several endogenous antiinflammatorymolecules in this disease is unknown. We aimed atevaluating the expression of A1AT in the retina of diabetic and nondiabeticrats.Methods: Five male Wistar rats of 250 g were treated with an IPinjection of 45mg/kg of streptozotocin (STZ). Animals with glycemialevels above 200 mg/dl were included in the study. Five non-diabeticrats were included as a control group. Animals were sacrificed after16 weeks of diabetes. Cross sections of retinas were analyzed byimmunohistochemistry using a primary antibody against A1AT.Protein expression was also analysed by western blot.Results: Retinas of diabetic and control rats showedimmunoreactivity of A1AT in the fiber layer, outer plexiform layerand in the external segment of photoreceptors. Higher staining wasseen in diabetics. Small vessels were only stained among diabeticanimals. No significant differences of protein expression of A1ATwere observed between groups by westernblot.Conclusions: An increased immunohistochemical staining of A1ATin diabetic rats and a similar protein expression between control anddiabetic retinas might suggest a relative insufficient quantity ofA1AT among diabetics after 16 weeks of disease. Studies in animalswith longer duration of diabetes are being conducted to furtherevaluate the role of A1AT in diabetic retinopathy.Commercial Relationships: Gustavo A. Ortiz, None; JorgeMancini, None; Eduardo Chuluyan, None; Juan E. Gallo,EP2186529 B1 (P)Support: ANPCyT (Agencia Nacional de promoción Científica yTecnológica), CONICET (Consejo Nacional de InvestigacionesCientíficas y Tecnológicas)Program Number: 6330 Poster Board Number: D0201Presentation Time: 10:30 AM - 12:15 PMDietary profile and vitamin A intake in patients with StargardtDisease and Retinitis PigmentosaAlba Miele 1 , Andrea Sodi 1 , Giacomo Abbruzzese 1 , Vittoria Murro 1 ,Francesco Sofi 3, 2 , Francesca Cesari 2 , Anna Maria Gori 2 , RosannaAbbate 2 , Alessandro Casini 3 , Ugo Menchini 1 . 1 Specialistic SurgicalSciences, Eye Clinic, University Of Florence, Italy, Florence, Italy,Italy; 2 Medical and Surgical Critical Care, University Of Florence,Florence, Italy; 3 Agency of Nutrition, University Of Florence,Florence, Italy.Purpose: Stargardt Diasease (STGD) and Retinits Pigmentosa (RP)are genetic-based relevant ocular diseases which may be affected, inopposite way, by vitamin A intake. Indeed, for STGD patients, thevitamin A intake from diet is recommended to be as low as possiblewhile, in RP patients, vitamin A is usually recommended to besupplemented. Despite these clinical indications, however, no data onnutritional habits of these patients are available. The purpose of thisstudy was to evaluate the dietary habits and nutritional intake ofvitamin A in patients with STGD and of RP, in order to seek forpossible dietary modifications.Methods: Dietary habits, vitamin A intake and clinical evaluationswere performed in 24 patients with STGD [12 M, 12 F; median age:34 years (range: 13-64)], and in 56 patients with RP [23 M, 33 F;median age: 45 years (range: 14-85)].Results: The median intake of vitamin A was 771.1 (range: 266.2-3863.2) mg/day in STGD patients and 871.2 (146.8-7935.8) mg/dayin RP patients. According to the recommended daily intakeguidelines for the Italian population, we documented in only 6 out of24 (25%) STGD patients a daily intake of vitamin A within therecommended range (600-700 mg/day) while 14/24 (58.3%) reporteda high daily intake (>700 mg/day). With regard to RP, 4/56 (7.1%)reported to be within the recommended range and 37/56 (66.1%)were above the recommended daily intake. Interestingly, STGDpatients with low vitamin A intake (600 mg/day). On the otherhand, RP patients with high vitamin A intake (>700 mg/day) showeda higher, albeit not significant, age of onset of the disease (30.8 ± 5.7years) vs. those reporting low vitamin A intake (25.5 ± 9.0 years).Conclusions: Our data reported that, despite information on theimportance of diet and vitamin A intake, a high proportion of patientswith STGD and RP do not follow recommendations for daily intakeof vitamin A through the diet. These preliminary data suggest a needof nutritional screening in STGD and RP patients.Commercial Relationships: Alba Miele, None; Andrea Sodi,None; Giacomo Abbruzzese, None; Vittoria Murro, None;Francesco Sofi, None; Francesca Cesari, None; Anna Maria Gori,None; Rosanna Abbate, None; Alessandro Casini, None; UgoMenchini, NoneProgram Number: 6331 Poster Board Number: D0202Presentation Time: 10:30 AM - 12:15 PMHypothermia protects retinal ganglion cells against ischemiaMaximilian Schultheiss, Matthias Blak, Tanja Dorfi, JohannaHofmann, Karl-Ulrich Bartz-Schmidt, Sven Schnichels, Martin S.Spitzer. Department of Ophthalmology, University of Tuebingen,Tuebingen, Germany.Purpose: Hypothermia has been shown to be neuroprotective in thetherapy of ischemic stroke. Furthermore the retina is easily accessiblefor inducing hypothermia in an acute onset of ischemia like duringcentral retinal artery occlusion. By using a cooled irrigation solutionduring pars plana vitrectomy the retina could be easily cooled downfor a certain timespan. The best temperature for cooling the retina isso far unknown. To investigate potential neuroprotective therapieslike hypothermia we developed an easy-to use chamber for 6-wellplates with inserts for organotypic cultures.Methods: To determine the optimal neuroprotective temperature weincubated retinas at 20, 30 and 37°C for 75 minutes under ischemicconditions. Hypothermia was induced for 4 hours (h) and theischemia chamber was adjusted to the desired temperature before theexperiments. For inducing ischemia the chamber was streamed withN2 for 5 minutes, then the chamber was immediately sealed and theretinas were incubated for the rest of the designated time of ischemia.Then the 6-well plate was removed from the chamber and left under asterile bench with no lid for 2 minutes to adjust the air in the wellplate to normal conditions. Afterwards the 6-well plate was incubatedat the desired temperature for 4 hours. Next the 6-well plates wereincubated for 24 and 48 h in an incubator under standard conditions.For comparison other organotypic cultures were treated with 1 mMglutamate instead of ischemia but underwent the same hypothermiaprotocol. To analyze the amount of RGCs and apoptotic RGCs, theretinas were frozen and processed for cutting. RGCsimmunohistology was performed with a Brn3a-antibody. Apoptoticcells were visualized via TUNEL-staining and overall cell amount viaDAPI -staining. Cells were counted manually.Results: With 20°C and 30°C hypothermia we observed a survival-©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 – Physiology/Pharmacologyrate that was up to 1.76x higher than in 37°C incubated ischemiccultures. For the glutamate treated probe the survival rate with 20°Cfor 4 h was the same as with untreated control samples 48 h afteronset of the experiments. Furthermore the amount of TUNELpositivecells was extremely reduced under hypothermic conditions.Conclusions: In conclusion, hypothermia is neuroprotective to retinalganglion cells against ischemia. Therefore, by a cooling the retinaduring pars plana vitrectomy the tolerance time against ischemia orglutamate stress could be increased.Commercial Relationships: Maximilian Schultheiss, None;Matthias Blak, None; Tanja Dorfi, None; Johanna Hofmann,None; Karl-Ulrich Bartz-Schmidt, Retina Implant (P); SvenSchnichels, None; Martin S. Spitzer, NoneProgram Number: 6332 Poster Board Number: D0203Presentation Time: 10:30 AM - 12:15 PMEffect of Storage Temperature and Antioxidant Concentrationon Catecholamine StabilityRandolph D. Glickman 1 , J. Christian Berry 1, 2 , Neeru C. Kumar 1 .1 Dept of Ophthalmology, Univ of Texas Hlth Sci Ctr SA, SanAntonio, TX; 2 Medical School, Baylor College of Medicine,Houston, TX.Purpose: Disturbances of dopamine metabolism may be involved inseveral retinal disorders. Accurate measurement of dopamine and itsmetabolites is complicated by the lability of these compounds due toauto-oxidation. In order to support research in this area, we carriedout a systematic study of the stability during storage for up to oneweek of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as 3,4-dihydroxybenzylamine (DHBA, a compound used as an internalstandard in catecholamine analysis), as a function of storagetemperature and the presence or absence of ascorbic acid as aprotective antioxidant.Methods: The reagents used in this study were obtained fromcommercial vendors. The test samples consisted of mixtures of DA,DOPAC, and DHBA, 50 ng/ml each, diluted in PBS from freshlyprepared stock solutions. Three groups of samples were each tested at25°C (RT), 4°C, and -75°C. Each group contained four samples with0, 1, 10, and 40 µg/ml ascorbic acid added, respectively. Theconcentration of the analytes was measured daily using HPLC withelectrochemical detection at a working potential of +460 mV.Results: After one day at RT in the absence of AA, there was a lossof ~90% of the DA, and nearly all of the DOPAC. At one day storedat 4°C, the corresponding loss was 80% of DA and 40% of DOPAC,and at -75°C only 15% of DA and 12.5% of DOPAC degraded afterone day. After six days of storage at RT, about 95% of the DA and99% of the DOPAC had degraded, while at -75°C only 23% of theDA and 30% of the DOPAC were lost. Addition of AA improved thestability of DA and DOPAC in a concentration-dependent manner atRT and 4°C, with maximal protection at 40 µg/ml of AA: over 64%of the DA and 58% of DOPAC remained at 6 days of storage. At -75°C, after six days of storage, all concentrations of AA providedexcellent protection, with virtually no DA loss, and only 15-20% lossof DOPAC. DHBA was slightly more stable than the othercompounds, and was completely protected by AA plus cold storage.Conclusions: Our observations show that catecholamine-containingsamples are sufficiently stable at -75°C that addition of AA is notnecessary, at least for storage periods of up to 1 week. In the case oflonger storage periods, addition of 1 µg/ml AA is recommended foroptimal stability of these labile compounds, particularly DOPAC.Commercial Relationships: Randolph D. Glickman, None; J.Christian Berry, None; Neeru C. Kumar, NoneSupport: Leonard and Shirley Sterling Endowment for BiochemistryResearch in the Department of Ophthalmlogy at the UTHSCSAProgram Number: 6333 Poster Board Number: D0204Presentation Time: 10:30 AM - 12:15 PMEstablishment of a retinal ischemia organ culture modelSven Schnichels, Matthias Blak, Tanja Dorfi, Johanna Hofmann,Karl-Ulrich Bartz-Schmidt, Focke Ziemssen, Maximilian Schultheiss,Martin S. Spitzer. University Eye Hosp Tuebingen, Centre forOphthalmology Tuebingen, Tuebingen, Germany.Purpose: Ischemia plays an important role in several ophthalmologicdiseases. To investigate neuroprotective agents and therapies againstthese diseases we developed an easy-to use chamber for 6-well plateswith inserts for organotypic cultures. We decided to use organotypiccultures, because in-vivo models or primary cultures are very timeconsuming,expensive and several therapies or agents cannot betested in these models.Methods: We incubated retinas at 37°C for different durations (45,60, 75, 90 and 120 minutes) under ischemic conditions. Briefly, thechamber was streamed with N2 for 5 minutes, then the chamber wasimmediately sealed and the retinas were incubated for the rest of thedesignated time. After the incubation the 6-well plate was adjusted tonormal air conditions and incubated for 24, 48 or 72h in an incubatorunder standard conditions. To analyze the amount of RGCsimmunohistology was performed with a Brna3a-antibody. Apoptoticcells were visualized via TUNEL-staining and overall cell amount viaDAPI-staining. Furthermore, Western-Blot analyses with GFAP- andThy-1-antibodies were performed. Moreover, OCT measurements ofthe organ cultures were performed for up to one week. Additionally,comparisons with retinas treated with 0.5mM and 1mM glutamatewere performed.Results: A time- and ischemia duration-dependant decrease in theamount of RGCs after 24, 48 or 72 h was observed. Moreover, theamount of TUNEL-positive RGCs was also ischemia duration- andtime-dependant. The damage to the RGCs through 75 minutes ofischemia was comparable to the amount of damage by 1mMglutamate incubation for 24h (20.27 vs. 19.69) and 48h (13.41 vs.14.41). In contrast, in glutamate treated retinas, only few apoptoticRGCs were found. The thickness of the retina significantly decreasedischemia duration- and time-dependant as observed with OCTmeasurement.Conclusions: We successfully established a cheap, reliable,reproducible, ease-to-use organotypic culture model for retinalischemia. Any therapy can now be tested under ischemic organotypicconditions. We selected 75 minutes of ischemia for further studies,because approximately 50% of the RGC died compared to the controlafter 48 h. Moreover, the RGC-loss after 75 minutes of ischemia iscomparable to the loss with 1mM glutamate. Results of aneuroprotective treatment with our chamber are shown on anotherposter from our group (Schultheiss et al.).Commercial Relationships: Sven Schnichels, None; MatthiasBlak, None; Tanja Dorfi, None; Johanna Hofmann, None; Karl-Ulrich Bartz-Schmidt, Retina Implant (P); Focke Ziemssen, None;Maximilian Schultheiss, None; Martin S. Spitzer, NoneProgram Number: 6334 Poster Board Number: D0205Presentation Time: 10:30 AM - 12:15 PMEFFECTS OF PROPOFOL OVER CIRCULAR RETINALSPREADING DEPRESSIONVinicius V. Oliveira 1 , Renata Fleming 1 , Nassim S. Calixto 2 , SebastiaoCronemberger 2 , Adalmir Dantas 1 . 1 Biophysics Institute, FederalUniv of Rio de Janeiro, Rio de Janeiro, Brazil; 2 Ophthalmology,Federal Univ of Minas Gerais, Belo Horizonte, Brazil.©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 – Physiology/PharmacologyPurpose: Spread depression (SD) was first identified on retina byGouras (1958). SD is widely associated with neuronal damage. Whileit spreads through retina, electrical and intrinsic optical signs can bemeasured. The circulating spreading depression is a unique model inwhich once the first stimulus is done, it continuously propagatesthrough the tissue.Propofol is rapidly acting water-insoluble non-barbiturate anestheticagent that is widely used as an intravenous sedative-hypnotic agent.Recently it was used over cortical SD.The main purpose of this paper is to identify if propofol was able toreduce, or even stop the spreading phenomena.Methods: We performed 30 experiments on retinal of White Leghornchicks. Retinas were transferred to a chamber and infused withRinger solution (RS). A circular cut was made in the center of theretina, in order to create a peripheral trail through which the SDcirculated. The presence or absence of SD was detected by recordingits concomitant slow voltage variations (SVV) through two poreelectrodes. The retina was firstly infused with RS and mechanicallystimulated by a sharpened tungsten wire, triggering a SD in twoopposite directions. MgSO4 blocked one of these branches. After 5laps, the infusion was changed to RS containing 56.1μM, 224.4μM e561μM of propofol. Values obtained were divided by the first valueobserved with RS. Graphs and statistical analysis were made withGraph Pad Prism 6.0, using Bonferroni`s Multiple Comparison Test.Results: Our data demonstrated that propofol reduces the amplitudeof the negative potential shift (SVV graph) - p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyAdditionally, data provide clues that SNC-121-mediated retinaneuroprotection is mediated via inhibition of multiple pathways. Dataalso indicated that nitric oxide-dependent pathway is one of thepotential targets of SNC-121-mediated retina neuroprotection.Commercial Relationships: Yasir Abdul, None; Shahid Husain,NoneSupport: NIH/NEI grant EY019081Program Number: 6336 Poster Board Number: D0207Presentation Time: 10:30 AM - 12:15 PMPerinatal hypoxia-ischemia impairs retinal ganglion cell functionand optokinetic behavior in a rat modelNikolay P. Akimov, Rene C. Renteria. Department of Physiology,University of Texas Health Science Center at San Antonio, SanAntonio, TX.Purpose: Most vision impairment after perinatal hypoxia-ischemia(periHI) is thought to be due to cortical damage. However, retinalblood supply is also impaired, and data from animal models suggestsretinal damage. Here, we examined two main issues in developmentand function of the visual system in a model of periHI: 1) whetheroptokinetic head-tracking (OKT)—a head-turn behavior to driftinggratings that depends on subcortical retinal input but does not requirethe visual cortex—is impaired, and 2) whether function of the outputneurons of the retina, the retinal ganglion cells (RGCs), is altered.Methods: We performed unilateral carotid cauterization (“ipsi-side”vs. opposite “contra-side”) followed by 8% oxygen for 2 hrs onpostnatal day (P)2 and P7 Long-Evans outbred rats (Harlan). In OKTbehavior, each direction is independently driven by one eye,unaffected by loss of input from the other eye. OKT thresholds ofcontrast (at 0.103 cyc/deg, “CT”) and spatial frequency (at max.contrast, “SPFT”) were determined at P30-35 for both horizontaldirections. RGC parameters were determined using a multi-electrodearray to record extracellular light-evoked spiking at P30-35. ON andOFF RGC receptive fields (RFs) were mapped using white-noisecheckerboards. Latency was the position of the main peak of thetemporal linear filter. A light square covering the array was variedsinusoidally at 18 contrasts ranging from 2.4% to 92% and presentedat 3.75 Hz to find RGC CT. Next, 10 frequencies from 0.75 Hz to12.5 Hz at 50% contrast were used to find RGC optimal frequency.Results: For P2 and P7 surgeries, both SPFT and CT were worse forthe ipsi-side compared to controls. For the contra-side, only CT wasworse; SPFT was unaffected. The P2 group was more impaired thanthe P7 group only for ipsi-side SPFT. For both the P2 and P7 groups,SPFT but not CT was significantly worse for the ipsi-side comparedto the contra-side of the same rats. In the P7 surgery group, ON butnot OFF RGC latencies were longer than in controls. RF diameterswere unchanged, and preliminary evidence indicates contrastthresholds and optimal frequencies were also unchanged.Conclusions: Because ON RGC latency and subcortical OKT wereimpaired, periHI may damage retinal circuits and possibly theaccessory optic nuclei. The accessibility of the eye may offer newtreatment opportunities to preserve vision in periHI patients.Commercial Relationships: Nikolay P. Akimov, None; Rene C.Renteria, NoneSupport: Knights Templar Eye Foundation Career-Starter GrantProgram Number: 6337 Poster Board Number: D0208Presentation Time: 10:30 AM - 12:15 PMThe effect of RGS9 overexpression on flicker responses of mousebipolar cellsChristopher Fortenbach 1 , Christopher Kessler 1 , Marie E. Burns 1, 2 .1 Center for Neuroscience and Department of Cell Biology andHuman Anatomy, University of California at Davis, Davis, CA;2 Ophthalmology & Vision Science, University of California, Davis,Davis, CA.Purpose: In retinal photoreceptors, changes in illumination result inchanges in membrane current and voltage that are largely determinedby the reaction rates of outer segment signal transductionmechanisms. In contrast, more downstream measures of visualfunction, including rod-driven ERG recordings and behavioral tests,suggest that phototransduction does not rate-limit performance of therod pathway (Umino et al., 2012). To investigate the time course ofsignaling at the rod synapse, we have combined electroretinoraphy(ERG) and bipolar cell recordings in mice with alteredphototransduction kinetics to better understand the mechanisms thatlimit the temporal resolution at the second stage of the rod pathway.Methods: Mice were dark-adapted overnight prior to the experiment.Corneal ERGs, rod suction electrode, and whole cell recordings fromOn and Off bipolar cells in retinal slices were elicited by calibratedflashes, steps, and sinusoidal flickering stimuli. Light-evokedresponses were subjected to Fourier analysis to generate powerspectra and normalized to the peak response amplitude forcomparison.Results: In the presence of a background light that suppressed ~40%of the rod a-wave amplitude, the normalized rod ERG flickerresponse showed frequency tuning, with a peak at 8-10 Hz. In wholecell recordings, the majority of Off bipolar cells and many On bipolarcells showed similar frequency tuning, optimally signaling at 8Hzwhen presented with a dim flickering stimulus (mean ~10R*/rod/s).In mice overexpressing the RGS9 complex, this increase in powerwas more dramatic. Both the rods and the rod-driven bipolar cellsfrom RGS9-overexpressing mice showed greater modulation athigher frequency flicker than those of wild-type mice.Conclusions: Both ERG and single cell recordings are consistentwith behavioral measures of frequency tuning in the rod pathway.The overexpression of RGS9 and the resulting acceleration of thephotoresponse recovery in rods exaggerates this tuning, improvingthe ability of bipolar cells to follow higher frequency flicker.Commercial Relationships: Christopher Fortenbach, None;Christopher Kessler, None; Marie E. Burns, NoneSupport: NIH Grants R01-EY014047 (MEB)Program Number: 6338 Poster Board Number: D0209Presentation Time: 10:30 AM - 12:15 PMC-peptide injection had synergitic effect to precultured isletstransplant in prevention of diabetic retinopathyChunzhi Dou 1, 2 , Stanley Y. Wu 3 , Xuewen Zhang 5 , Tiehua Dou 6 , GuShuyan 4 . 1 Ophthalmology, Emory University, Suwanee, GA;2 Biology, Georgia University System, Lawrenceville, GA;3 Department of Surgery, Norman Bethune Medical School, JilinUniversity, Changchun, China; 4 Department of Ophthalmology,Norman Bethune Medical School, Jilin University, Changchun,China; 5 Endocrinology, Dehui County Hospital, Dehui, China;6 Clinical Chemistry, Genova Diagnostics, Duluth, GA.Purpose: The previous studies have shown that C-peptide and VEGFpre-cultured islets transplant delayed diabetic retinopathydevelopment. The current study is to further investigate biologicalfunctions of C-peptide through in vivo administration as adjuncttherapy for islets transplant.Methods: Diabetic rat model was made by intraperitoneal injectionof Streptozotocin (STZ) as described previously. One week after theinjection, islets from donor rats were isolated and transplanted underthe kidney capsule. Recipient rats were divided into three groups.Group P rats received media cultured islets transplantation and C-peptide injections after the transplant. Group T rats received C-peptide pre-cultured islets transplantation and C-peptide injections©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 – Physiology/Pharmacologyafter the transplant. The control group rats received media culturedislets transplantation without any further treatment. Blood glucoseand body weight were monitored daily after transplant. HbA1C wasmonitored weekly. Diabetic neuropathy was evaluated with tail flicktest every 10 days. At the end of study, the transplanted islets wereretrieved for immunohistochemistry study; and eyes were enucleatedfor diabetic retinopathy evaluation.Results: The body weight, blood glucose and HbA1C were very wellcontrolled and stable in group T rats through the whole study course.Rats in group P had initial 3 days normal glucose and body weight,gradually body weight decreased and blood glucose increased, until 3weeks after the transplantation blood glucose started to drop andclose to normal. HbA1C was slightly elevated. The control rats hadnormal blood glucose and body weight during the initial 3 days andcontinuously decreased body weight and elevated blood glucose withabnormal HBA1C level. In terms of diabetic complications, bothgroup P and group T rats had no proliferative diabetic retinopathydevelopment. Two rats in group P developed mild diabeticretinopathy. In the control group, however, one rat developed PDR,three rats developed moderate to severe NPDR, 5 rats developed mildNPDR.Conclusions: This study indicated that C-peptide administrationduring post transplant only had synergistic effect to the C-peptidepre-cultured islet transplant. The mechanism is possibly throughimproving blood flow, sensitizing glucose receptor and intra-cellularsignal transduction pathway.Commercial Relationships: Chunzhi Dou, None; Stanley Y. Wu,None; Xuewen Zhang, None; Tiehua Dou, None; Gu Shuyan,NoneSupport: Partially supported by Xianmin Meng Foundationpoints during the two-hour follow-up period. Mean systolic BP inGroup P and Group D was 124.9 ±16.2 and 114.6 ±14.5, respectively(p =0.005), and mean diastolic BP was 73.6 ±12.5 and 66.2 ±10.8,respectively (p =0.002). There was no difference in PACU HR andRR. Oxygen saturation did not drop below 92% at any time.Conclusions: In the 76 patients recruited, dex provided adequatesedation, patient and surgeon satisfaction, and hemodynamicstability, with no difference in incidence of adverse effects comparedto prop. In fact, Group D shows a significantly higher surgeonsatisfaction at 10 min and level of sedation as measured by the BIS.The only significant hemodynamic parameters between groups werein systolic and diastolic BP in the PACU. There were not anyclinically significant events that warranted use of rescue medicationsfor bradycardia or hypotension. No patient experienced postoperativenausea or vomiting. The mean patient and surgeonsatisfaction was between good and excellent in both groups.Program Number: 6339 Poster Board Number: D0210Presentation Time: 10:30 AM - 12:15 PMComparison of Dexmedetomidine vs. Propofol in VitreoretinalSurgery Under Local BlockLinda Y. Huang 1 , Anuradha Patel 2 , Marianne Antoniello 2 , CatherineSchoenberg 2 , Dennis Grech 2 , Amy Davidow 2 , Tian Xia 2 , NeelakshiBhagat 1 . 1 Ophthalmology and Visual Sciences, UMDNJ-New JerseyMedical School, Newark, NJ; 2 Anesthesiology, UMDNJ-New JerseyMedical School, Newark, NJ.Purpose: To compare the efficacy of dexmedetomidine (dex) vs.propofol (prop) in vitreoretinal surgery under local block.Methods: An IRB approved double-masked, prospective randomizedstudy. Enrollment criteria include subjects between ages 18 and 65years, ASA 1-3, with good liver and renal function. Procedures wereperformed at UMDNJ's outpatient surgery center by the samesurgeon (NB) under retrobulbar block administered using sub-Tenonapproach. Patients are randomized into group P (prop) and group D(dex). Group P receives a bolus of 1 mg/kg of prop intravenously(IV) followed by a 25-100 ug/kg/min infusion. Group D receives abolus of 0.5 ug/kg of dex IV followed by a 0.2-0.7 ug/kg/hr infusion.T-test and Mann-Whitney test are used for statistical analysis.Results: 76 patients have been enrolled; 39 - group P, 37 - group Dwith one patient excluded due to claustrophobia when draped andsubsequent conversion to general anesthesia. Comparisons ofparameters are outlined in the table below.Intraoperative blood pressure (BP) and respiratory rates (RR) weresimilar in both groups, with Group P having significantly loweraverage systolic BP at 5 and 10 minutes (min) and higher average RRat 15 min. Average heart rates (HR) for all patients were between 49and 95 beats per min.In the Post-Anesthesia Recovery Unit (PACU), Group D hadstatistically significant decreased systolic and diastolic BPs at all timeCommercial Relationships: Linda Y. Huang, None; AnuradhaPatel, Hospira Inc. (F); Marianne Antoniello, None; CatherineSchoenberg, None; Dennis Grech, None; Amy Davidow, None;Tian Xia, None; Neelakshi Bhagat, NoneSupport: Humira, Inc.Clinical Trial: NCT01001429Program Number: 6340 Poster Board Number: D0211Presentation Time: 10:30 AM - 12:15 PMSitagliptin prevents blood-retinal barrier breakdown,inflammation and neuronal cell death in the retina of type 1diabetic animalsAndreia Gonçalves 1 , Ermelindo C. Leal 2 , Artur Paiva 3 , Carlos F.Ribeiro 1 , Flávio Reis 1 , Antonio F. Ambrosio 2, 4 , Rosa Fernandes 1 .1 Lab Pharmacology and Experimental Therapeutics, IBILI - Facultyof Medicine, University of Coimbra, Coimbra, Portugal; 2 Centre forNeuroscience and Cell Biology, University of Coimbra, Coimbra,Portugal; 3 Histocompatibility Centre of Coimbra, Coimbra UniversityHospitals, Coimbra, Portugal; 4 Centre of Ophthalmology and VisionSciences, IBILI - Faculty of Medicine, University of Coimbra,Coimbra, Portugal.Purpose: Diabetic retinopathy, the main microvascular complicationof diabetes, is a leading cause of vision loss and blindness. A novelclass of oral antidiabetic agents, the dipeptidyl peptidase type 4(DPP-IV) inhibitors, has shown to improve glycemic control byenhancing the levels of active incretin hormones, which in turn©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 – Physiology/Pharmacologyincrease insulin secretion in patients with type 2 diabetes (T2D). Theaim of this work was to evaluate whether sitagliptin can exertprotective effects in the retina by a mechanism independent of insulinsecretion, in a type 1 diabetes (T1D) animal model.Methods: Two weeks after streptozotocin-induced diabetes, the ratswere orally treated with sitagliptin (5 mg/kg/day) for two weeks.Glucose, HbA1c and insulin levels were evaluated in serum or totalblood. The blood-retinal barrier (BRB) breakdown was evaluatedusing Evans blue. The activity of DPP-IV was assessed using afluorogenic substrate. The content and/or distribution of tightjunction (TJ) proteins (occludin, claudin-5 and ZO-1), DPP-IV/CD26, IL-1β, TNF-α and Bax was evaluated by western blottingand/or immunohistochemistry. Retinal cell apoptosis was assessed bythe TUNEL assay. The number of CD34+ cells present in thecirculation was assessed by flow cytometry.Results: Sitagliptin treatment had no effect on weight, glucose,HbA1c or insulin levels in diabetic animals. However, it preventedthe increase in the activity and content of DPP-IV/CD26 induced bydiabetes in serum and retina. Sitagliptin also prevented the increase inBRB permeability and inhibited the alterations in the subcellulardistribution of TJ proteins induced by diabetes. Furthermore,sitagliptin was able to prevent the increase in IL-1β, TNF-α, Bax andTUNEL-positive cells in the retinas of diabetic animals. Treatmentwith sitagliptin prevented the decrease in the number of CD34+ cellsin the peripheral circulation of diabetic animals.Conclusions: In conclusion, sitagliptin inhibits the BRB breakdownin a T1D animal model, by a mechanism independent ofnormalization of glycemia, by preventing changes in TJ organization.Sitagliptin also exerted protective effects against inflammation andpro-apoptotic state in the retina of diabetic rats. Altogether, theseresults suggest that sitagliptin might be envisaged to be used in T1Dand T2D patients, to prevent or delay the development of diabeticretinopathy.Commercial Relationships: Andreia Gonçalves, None; ErmelindoC. Leal, None; Artur Paiva, None; Carlos F. Ribeiro, None; FlávioReis, None; Antonio F. Ambrosio, None; Rosa Fernandes, NoneSupport: PhD studentship - EFSD/GSK ProgrammeProgram Number: 6341 Poster Board Number: D0212Presentation Time: 10:30 AM - 12:15 PMInhibition of the diabetes-induced increased TGF-β signaling inretinal vessels leads to an abnormal endothelial phenotypeZeina Dagher, Joseph Vaz, Michael Goodridge, ChiaraGerhardinger, Mara Lorenzi. Schepens Eye Research Institute,Massachusetts Eye & Ear, Boston, MA.Purpose: To learn whether the increased TGF-β signaling induced bydiabetes in retinal vessels contributes to microangiopathy and is acandidate target for interventions. We had detected an increase inTGF-β signaling in rat retinal vessels after 3 months of diabetes(Gerhardinger et al 2009) and the known roles of TGF-β in vascularhomeostasis and pathologies make the cytokine a prime potentialcontributor to diabetic microangiopathy.Methods: TGF-β signaling and gene expression were evaluated infresh retinal vessels isolated from rats with 3 months ofstreptozotocin-diabetes untreated or treated with SM16, a selectiveinhibitor of the type I TGF-β receptor ALK5. SM16 was given for 3weeks mixed with the chow to deliver a dose of 6 mg/Kg bw/day(Anscher et al 2008). Diabetic rats received insulin to preventcatabolism. SM16-treated and untreated nondiabetic rats were used ascontrols. Smad2/3 phosphorylation and protein levels were measuredby Western blot, gene expression by PCR Array (Rat EndothelialCell Biology RT2 ProfilerTM) and RealTime PCR.Results: The retinal vessel preparations were similarly enriched inendothelial markers --at least 8-fold over the whole retina-- in allexperimental groups. In rats with 3.5 months of diabetes and HbA1cdouble the control values, only three genes changed their expressionlevel more than 2 fold with P < 0.05 when compared to control rats:endothelin 2; fibroblast growth factor 2; and placental growth factor(PGF), a VEGF family member that enhances vascular permeability.Treatment with SM16 prevented the diabetes-induced excessSmad2/3 phosphorylation without lowering the basal, and preventedPGF overexpression. In addition, SM16 changed the expression ofseveral genes that had not been altered by diabetes alone, and indirections that would make the endothelium pro-inflammatory,thrombogenic, permeable, and prone to apoptosis (increasedmonocyte chemoattractant protein-1; and decreased tissue factorpathway inhibitor, thrombomodulin, occludin, VE-cadherin, Tie-2,and VEGF A). The above SM16 effects were not observed in normalrats treated with SM16.Conclusions: The effects of SM16 on the retinal vessels of diabeticrats were not mere drug toxicity. Rather, they suggest that the smallincrease in TGF-β signaling induced by diabetes has a protective rolefor the vascular endothelium.Commercial Relationships: Zeina Dagher, None; Joseph Vaz,None; Michael Goodridge, None; Chiara Gerhardinger, None;Mara Lorenzi, NoneSupport: EY017637Program Number: 6342 Poster Board Number: D0213Presentation Time: 10:30 AM - 12:15 PMExpression of 2PK+ leak channels in ganglion cells of the mouseretinaSteven Hughes, Russell G. Foster, Stuart N. Peirson, Mark W.Hankins. Nuffield Department of Clinical Neurosciences, Universityof Oxford, Oxford, United Kingdom.Purpose: Tandem pore domain potassium leak channels (2PK+)perform essential roles in setting resting membrane potential andlevels of cellular excitability. To date the expression and functionalrole of 2PK+ channels in retinal ganglion cells (RGCs) of the retinahas not been investigated. The aims of this study are to determine thelevels of 2PK+ channel expression present in the mouse retina, andgain an improved understanding of the mechanisms that regulateresting membrane potential and cellular excitability of RGCs.Methods: qPCR and ICC analysis were used to investigate theexpression and distribution of 2PK+ channels in the mouse retina.Double labeling with retinal cell markers was used to confirm theexpression of 2PK+ channels in specific cell types of the retina,including subtypes of RGCs. Whole cell patch clamp recordings fromdissociated retinal cell cultures were used to record 2PK+ leakcurrents present in RGCs.Results: We confirm that multiple members of the 2PK+ channelfamily are expressed in the mouse retina. Based on qPCR analysis,TWIK-1, TRAAK, TASK-1 and TRESK are expressed at the highestlevels, with lower expression detected for TWIK-2, TREK-1 andTASK-3. ICC analysis confirms the widespread expression of 2PK+channels in the mouse retina, with TWIK-1, TWIK-2, TASK-1 andTREK-1 detected in Brn3a positive retinal ganglion cells and alsowithin different subtypes of melanopsin expressing pRGCs. Usingwhole cell electrophysiology we confirm the presence of multiple2PK+ type leak currents in RGCs.Conclusions: We conclude that multiple members of the 2PK+family of K+ leak channels are expressed in the mouse retina, andspecifically within retinal ganglion cells. The distinct patterns ofexpression observed are consistent with the differential expression ofthese ion channels within distinct subtypes of retinal ganglion cells,including subtypes of pRGCs. It is likely therefore that these ion©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 – Physiology/Pharmacologychannels contribute to the different electrophysiological properties ofRGC subtypes, and offer a range of mechanisms to influence theresting membrane potential and cellular excitability of these cells.The role of 2PK+ channels in neuroprotection is of significantinterest and offers new potential targets for the treatment of retinaldisease.quantitative RT-PCR, Western blot and immunohistochemistry.Phosphorylation of all isoforms p38MAPK (Thr180/Tyr182) and GR(S-211) was further evaluated. Apoptosis was confirmed byimmunolocalization of active CASPASE-3 and the subsequentcleavage of poly (ADP-ribose) polymerase (PARP) followingintravitreal injection of triamcinolone acetonide (IVTA), in an earlydiabetic rat model (26 days after induction of diabetes).Results: : IVTA significantly down-regulated mRNA expression ofCaspase 3. Activation of CASPASE-3, the subsequent cleavage ofPARP-1 and phosphorylation of p38MAPK, induced by diabetes,were attenuated by IVTA treatment, concomitant with the activationby phosphorylation of the glucocorticoid receptor (GR S-211).Conclusions: IVTA exerts neural protective effects on retinalneurons. Inhibition of the p38MAPK pathway and activation of GRplay a critical anti¬apoptotic role in retinal neurons of diabetesfollowing IVTA treatment. Both the anti-inflammatory and antiapoptoticeffects of glucocorticoids may be mediated throughinhibition of the p38MAPK pathway in diabetic retinopathy.Commercial Relationships: Xinyuan Zhang, None; Bob Bao,None; Mark C. Gillies, Novartis (R), Pfizer (R), Allergan (F), Bayer(F)Expression of TREK-1 (red) in the mouse retinaExpression of TASK-1(red) in Brn3a positive RGCs (green) of themouse retinaCommercial Relationships: Steven Hughes, None; Russell G.Foster, None; Stuart N. Peirson, None; Mark W. Hankins, NoneSupport: Wellcome Trust Program GrantProgram Number: 6343 Poster Board Number: D0214Presentation Time: 10:30 AM - 12:15 PMGlucocorticoids Inhibit p38MAPK Activation and NeuronalApoptosis in Early Diabetic RetinopathyXinyuan Zhang 1 , Bob Bao 2 , Mark C. Gillies 3 . 1 Tongren Eye Center,Beijing Tongren Hospital, Beijing, China; 2 Pathology, The Universityof Sydney, Sydney, NSW, Australia; 3 Ophthalmology, TheUniversity of Sydney, Sydney, NSW, Australia.Purpose: Intravitreal glucocorticoids and anti-vascular endothelialgrowth factor (VEGF) therapies are novel strategies for the treatmentof advanced diabetic retinopathy, a condition with inflammatory andneuropathic elements. In contrast with anti-VEGF therapy,glucocorticoids may also exert neuroprotective effects. Howglucocorticoids protect retinal neurons is unknown. The aims of thestudy are to investigate the anti-apoptotic actions of glucocorticoidson diabetic retinal neurons, and characterize the signaling cascadeinvolved.Methods: The regulation of gene expression of the four p38 mitogenactivatedprotein kinase (MAPK) isoforms (α, β, δ and γ) and theglucocorticoid receptor (GR) in the retinas was evaluated, usingProgram Number: 6344 Poster Board Number: D0215Presentation Time: 10:30 AM - 12:15 PMThe molecular mechanisms of store-operated calcium entry inMüller gliaTunde Molnar 1 , Amber M. Frye 1 , Peter Barabas 1 , Daniel A.Ryskamp 2, 1 , David Krizaj 1, 3 . 1 Ophthalmology and Visual Sciences,Moran Eye Institution, University of Utah School of Medicine, SaltLake City, UT; 2 Interdepartmental Program in Neuroscience,University of Utah, Salt Lake City, UT; 3 Department of Physiology,University of Utah, Salt Lake City, UT.Purpose: Müller cells play a key function in retinal volumeregulation, metabolism, glutamate recycling and pathology. Here wecharacterize a fundamental Ca 2+ signaling pathway in Müllerastroglia, determine its molecular mechanism and investigate itscontribution to Müller gliosis induced by ocular hypertension.Methods: Optical imaging from fura-2 -loaded cells and whole-cellpatch-clamp recording were performed in dissociated mouse Müllercells isolated from wild type and Trpc1/Trpc3 (TRPC1/3 -/- ) doubleknockout mice. Store-operated calcium signals were evoked byprolonged depletion of endoplasmic reticulum (ER) Ca 2+ stores inCa 2+ -free saline supplemented with cyclopiazonic acid (CPA).Intraocular pressure (IOP) was elevated for 7 and 14 days byinjection of microbeads into the anterior chamber of wild type andTRPC1/3 -/- mice. Müller cell reactivity was assessed with an anti-GFAP antibody.Results: Depletion of ER Ca 2+ stores induced marked store-operatedcalcium entry (SOCE) (568 ± 159 nM over the baseline of 232 ± 36nM; N=17 cells) which was manifested as [Ca 2+ ] i overshootsfollowing re-exposure of cells to the inward-directed calciumgradient. Induction of SOCE was associated with intracellular Ca 2+waves that propagated from the endfoot to the apical regions ofMüller cells. Both SOCE and Ca 2+ wave propagation weresuppressed by a mix of SOC channel inhibitors 2-APB, SKF96365and Gd 3+ . Likewise, depletion of ER stores induced a prominentinward Ca 2+ /Na + current (~ 50 pA) in the presence of TEA and Cs + .The depletion-evoked current was antagonized by 2-APB, SKF96365and Gd 3+ . The amplitude of SOCE signals was significantlydiminished in mice lacking TRPC1 and 3 isoforms (299 ± 94 nM;N=14 cells; P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyConclusions: We found that SOCE mediates significant Ca 2+ influxin mouse Müller cells mainly through TRPC1 and/or TRPC3channels. The enhanced expression of GFAP in glaucomatousTRPC1/3 -/- retinas indicates that calcium signals associated withTRPC1 and TRPC3 channels modulate the induction and/ormaintenance of Müller gliosis in the diseased retina.Commercial Relationships: Tunde Molnar, None; Amber M.Frye, None; Peter Barabas, None; Daniel A. Ryskamp, None;David Krizaj, NoneSupport: Knights Templar Eye Foundation, NIH, Department ofDefense, FFB, University of Utah and an unrestricted award fromRPB to the Moran Eye InstituteProgram Number: 6345 Poster Board Number: D0216Presentation Time: 10:30 AM - 12:15 PMContribution of GPCRs and NADPH oxidase to increasedgeneration of superoxide by retinal photoreceptor cells inelevated glucoseYunpeng Du 1 , Megan Cramer 1 , Krzysztof Palczewski 2 , Timothy S.Kern 1, 3 . 1 Medicine, Case Western Reserve University, Cleveland,OH; 2 Pharmacology, Case Western Reserve University, Cleveland,OH; 3 Stokes Veterans Administration Hospital, Cleveland, OH.Purpose: Oxidative stress is believed to play a significant role in thedevelopment of diabetic retinopathy, and we have found that theoxidative stress in diabetes is especially pronounced inphotoreceptors. We are investigating the mechanisms by whichelevated glucose increases generation of the superoxide anion byphotoreceptors. These studies focused initially on the contribution ofNADPH oxidase and its activation by GPCR-activated pathways.Methods: Initial studies are conducted in vitro using 661W cells(derived from photoreceptors) as a test system. Superoxide releasewas assayed using luciginen at normal (5mM) and high (30 mM)glucose.Results: Incubation of the cells in 30mM glucose for 4 days resultedin a significant increase in superoxide generation. Pharmacologicinhibition of the alpha-1 adrenergic receptor with Doxazosin,phospholipase C with U73122, Ca2+ release with the membranepermeable inhibitor of IP3-induced Ca2+ release (2-aminoethyldiphenylborinate), or NADPH oxidase with apocynin eachsignificantly inhibited the increase in superoxide production causedby elevated glucose.Conclusions: These findings suggest that the induction of superoxidegeneration by photoreceptors in elevated glucose could be mediatedthrough a signaling cascade involving GPCRs, PLC/IP3/Ca2+signaling, and NADPH oxidase.Commercial Relationships: Yunpeng Du, None; Megan Cramer,None; Krzysztof Palczewski, QLT Inc (F), Polgenix Inc (E), VisumInc (P), Amegen Inc (F); Timothy S. Kern, Bausch & Lomb (F),PamLab (F)Support: EY00300 and VA Merit grantProgram Number: 6346 Poster Board Number: D0217Presentation Time: 10:30 AM - 12:15 PMPro-NGF induces endothelial cell death via the P75 neurotrophinreceptor (P75NTR) in a mouse model of oxygen-inducedretinopathy (OIR)Nicholas Sitaras 1, 2 , H. Uri Saragovi 4 , Sylvain Chemtob 3, 2 ,Przemyslaw Sapieha 2 . 1 Pharmacology, Maisonneuve-RosemontHospital, Montréal-Est, QC, Canada; 2 Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, QC, Canada; 3 Pharmacology, CHUSainte-Justine, Montreal, QC, Canada; 4 Pharmacology andTherapeutics, Lady Davis Institute, Montreal, QC, Canada.Purpose: Proliferative Retinopathies (PRs) are the major cause ofblindness in working-age and pediatric populations. These diseasesare characterized by an initial microvascular dropout followed by adisparate compensatory albeit excessive and pathologicalvascularization leading to blinding tractional retinal detachment.Neurotrophins, or the lack thereof, have previously been shown tocontribute to the pathogenesis of PRs. Particularly, brain-derivedneurotrophic factor (BDNF) and nerve growth factor (NGF) caneither stimulate or inhibit angiogenesis in the retina in bothdevelopmental and pathological contexts. This discrepancy may beaccounted by the presence of neurotrophic precursor forms thatpreferentially bind to the low-affinity P75 neurotrophin receptor(P75NTR). Here we explored the expression patterns of neurotrophinprecursors and their low-affinity receptor, P75NTR and determinedtheir role in the PRs.Methods: P75NTR and pro-NGF expression were measured in wholemouse retinas by western blot and immunohistochemistry. Retinalangiogenesis was appreciated following in vivo intravitreal injectionsof selective antagonists of pro-NGF or P75NTR in C57Bl6 miceexposed to a model of oxygen-induced retinopathy (OIR) whichloosely mimics PRs (75% O2 from post natal day 7 [P7] to P12).Expression profiles of pro- and anti-angiogenic genes were assessedin whole retinas following treatment with antagonist injection.Results: Our data demonstrates preferential expression of P75NTR inMüller glia and astrocytes. In OIR, P75NTR levels increased slightlyduring the neovascularization (NV) phase (P12-P17), whereasaccumulation of pro-NGF was detected during vaso-obliteration(VO) and during NV. Inhibition of P75NTR during hyperoxicexposure significantly reduced VO at P12. This reduction in VOsecondary to inhibition of P75NTR during the hyperoxic phase wasassociated with a decrease in tumor-necrosis factor-α (TNF-α). Incontrast, antagonism of P75NTR did not impede/exacerbatepathological neovascularization (NV).Conclusions: This study highlights the novel role of P75NTR in aretinal vasculopathy. Induction of pro-NGF during the early phases ofhyperoxia-induced vaso-obliteration, activates P75NTR and provokesendothelial cell death. These results introduce potential therapeutictargets, namely P75NTR or pro-NGF, for the treatment of PRs.Commercial Relationships: Nicholas Sitaras, None; H. UriSaragovi, McGill University (P); Sylvain Chemtob, None;Przemyslaw Sapieha, NoneSupport: FRSQProgram Number: 6347 Poster Board Number: D0218Presentation Time: 10:30 AM - 12:15 PMInner nuclear layer microcystic changes in optic nerve atrophy: aprospective studyBenjamin Wolff, Vivien Vasseur, Chrysanthi Basdekidou, Jose A.Sahel, Catherine Vignal, Martine Mauget-Faÿsse. Professor SahelDept, Fondation ophthalmologic Rothschild, Paris, France.Purpose: Optic atrophy constitutes the final stage in the evolution ofoptic neuropathy. The aim of this study is to demonstrate the utilityof Spectral Domain OCT (OCT-SD) in the visualisation of hyporeflective microcystic changes (or retinal pseudocysts) in the internalnuclear layer (INL) in several cases of advanced optic atrophy.Methods: Patients with a diagnosis of optic nerve atrophy wereprospectively studied between October 2011 and July 2012. Allpatients underwent a complete neuro-ophthalmologic assessment. Aretinal nerve fibres (RNFL) analysis of the optic head and of themacula was performed in each patient using the OCT-SD togetherwith a morphological analysis of the macula.Results: 201 eyes of 131 patients have been analysed. Inner nuclearlayer pseudocysts were observed in 77 eyes (38% of cases). 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 – Physiology/Pharmacologymajority of cases optic atrophy was the result of hereditaryneuropathy, glaucoma or inflammatory neuropathy. The evolution ofthese optic neuropathies varied from 1 to 18 years (average: 5,75years). Visual acuity ranged from 1/20 to 12/10 (average: 5/10). Thesex ratio was 0,5 and patients’ average age was 37 years old. Themicrocystic changes were visualized as numerous hypo reflectivelesions located in the INL. The infrared images revealed a hyporeflective cockade corresponding to the pseudo cysts location in allcases. Fluorescein angiography (performed in 10% of cases)confirmed the absence of macular vascular leakage.Conclusions: We report the presence of microcystic changes, orpseudocystic, lesions always associated to severe optic nerve fiberloss. The reason why pseudocystic lesions develop within the retinais not well understood. They might constitute the translation ofdegeneration in relation to severe optic nerve fiber loss. Recognizingthese pseudo cysts is crucial as they may be confused with a cystoidmacular edema. Their prognostic value and their implication in thetherapeutic process need to be further evaluated.Commercial Relationships: Benjamin Wolff, None; VivienVasseur, None; Chrysanthi Basdekidou, None; Jose A. Sahel,UPMC/Essilor (P), Second Sight (F); Catherine Vignal, None;Martine Mauget-Faÿsse, BAYER (R), NOVARTIS (R)Program Number: 6348 Poster Board Number: D0219Presentation Time: 10:30 AM - 12:15 PMAdrenergic but not purinergic receptors affect ion transportacross the mouse retinal pigment epitheliumThor Eysteinsson, Sunna B. Skarphedinsdottir, Sighvatur SaevarArnason. Physiology, University of Iceland, Reykjavik, Iceland.Purpose: To develop procedures to examine the effects of adrenergicand purinergic receptors on ion transport across the mouse retinalpigment epithelium (RPE), as measured by the RPE short-circuitcurrent (I SC ) and transepithelial resistance (TER).Methods: Sheets of RPE from normal mice with the retina attachedwere mounted in Ussing chambers with a small aperture of 0.031 cm 2(EasyMount, Physiological Instruments Inc.). An aerated Krebssolution kept at 38°C was placed on each side of the tissue. The I SCand the TER were recorded with electrodes placed on each side of thetissue and connected to a voltage clamp apparatus. All drugs wereapplied for 30 min on both apical and basolateral sides.Results: The mean initial I SC (n=7) was -17,6 ± 4,5 μA/cm 2 , with theapical side negative, and TER was 82,8 ± 7,6 Ohm*cm 2 . The mouseRPE preparations remained stable for 2-3 hours. A low [Cl - ] in theKrebs reduced the I SC . Reducing [K + ] on the apical side decreased theI SC , an effect that was inhibited by 1 mM BaCl 2 . The Na-K-Clcotransporter blocker bumetanide (100 µM) reduced the I SC from -16.1 ± 5.2 to -14.5 ± 5.4 μA/cm 2 (P = 0.005). The Na-K-ATPaseblocker ouabain (1 mM) induced a biphasic response, first anincrease in the I SC to -17.4 ± 2.6 μA/cm 2 in 6 minutes, and then adecrease in 30 minutes to to -7,74 ± 4,45 μA/cm 2 , which was 36% ofI SC levels before applying ouabain (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologygenes expression. Immunohistochemistry were used to measurechanges in the levels of cytokines and reactive oxygen species(ROS). Electrophoretic mobility shift assay (EMSA) was performedto evaluate the effect of chitosan on NF-κB activation in the retina ofrats.Results: Pretreatment with chitosan dose dependently inhibited thedamage of the retina caused by IR injury. On the 7th postoperativeday, the amplitudes of the ERG b-waves were significantly higher inthe treatment group than in the control group. Chitosan suppressedthe expression of NF-κB in the ischemia retina and the production ofcytokines and ROS. EMSA demonstrated chitosan inhibited NF-κBactivation in the retina of rats wit IR injury.Conclusions: Chitosan attenuates retinal damage in IR injury byblocking NF-κB activation and NF-κB associated inflammatorycytokines production. Therefore, chitosan may provide a noveltherapeutic strategy for the treatment of retinal ischemia reperfusioninjury.Commercial Relationships: Sheng-Li Cho, None; Chang-HaoYang, NoneProgram Number: 6351 Poster Board Number: D0222Presentation Time: 10:30 AM - 12:15 PMWater-dispersible hesperetin prevents ganglion cell loss in theretina after retinal ischemia reperfusion injuryAkito Shimouchi 1 , Harumasa Yokota 1 , Taiji Nagaoka 1 , Shinji Ono 1 ,Hiroko Takumi 2 , S.Priya Narayanan 3 , Ruth B. Caldwell 3, 4 , AkitoshiYoshida 1 . 1 Ophthalmology, Asahikawa Medical University,Asahikawa, Japan; 2 Institute of Health Science, Ezaki Glico Co., Ltd,Osaka, Japan; 3 Vascular Biology Center, Medical College ofGeorgia, Augusta, Georgia; 4 VA Medical Center, Augusta, Georgia.Purpose: The citrus flavonoid hesperetin (Hpt) has been shown toimprove retinal blood flow and enhance recovery of retinal functionafter ischemic injury in rats. The aim of this study was to determinewhether water-dispersible Hpt could prevent degeneration ofganglion cell neurons in a mouse model of retinal ischemiareperfusion (I/R) injury and to examine the mechanisms of itsactions.Methods: Retinal I/R injury was induced in C57BL/6 mice byincreasing the intraocular pressure (IOP) to 110 mmHg for 40minutes followed by reperfusion. The contralateral eyes served ascontrols. The mice received daily intraperitoneal injection with eithernormal saline (NS, 0.3ml/) or Hpt (200mg/kg/) until sacrifice. Themice were sacrificed at 6h, 24h and 7 days after I/R. The wholemount retinas were immunostained with NeuN antibody to detectsurviving neurons in the ganglion cell layer at 7 days after I/R.Western blotting was performed to examine the expression ofmolecules that initiate or promote apoptosis at 6h after I/R.Results: I/R resulted in a 37% reduction in the number of ganglioncells compared to the fellow eyes in the mice treated with NS,whereas the number of NeuN positive cells in the mice treated withHpt was reduced by only 5% (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/PharmacologyPurpose: Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor through glutamate or otheragonists allows influx of cations including calcium, potassium andsodium. The purpose of this study was to investigate theneuroprotective role of the AMPA receptor in-vitro, in purifiedretinal ganglion cells (RGCs) and retinal mixed culture lacking RGCs(mixed retinal culture), by assessing the phosphorylation of cAMPresponse element-binding protein (p-CREB) stimulated by calciuminflux.Methods: Purified rat RGCs were isolated from P3-P7 Sprague-Dawley rats and cultured by a double immunopanning techniqueusing an antibody to Thy 1.1. The residual neurons in the retinafollowing RGC isolation (supernates of the panning procedure) wereused as the mixed retinal culture (lacking RGCs). Calcium imagingwas used to identify the functionality of the AMPA receptors andselectivity of the AMPA agonist. RGCs and mixed retinal neuronswere cultured for 7 days before AMPA treatment. Followingtreatment with AMPA for 6 hours, proteins were extracted andwestern blot analysis was carried out to determine changes inexpression of the p-CREB and p-ERK1/2, which were normalized tototal CREB, total ERK1/2, and beta tubulin.Results: AMPA receptors were stimulated through administration ofAMPA (100μM), which depolarized the purified RGCs and increasedintracellular calcium. The AMPA mediated calcium ion influx wassignificantly attenuated by approximately 87.8% (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacologyretina was superfused for 15 minutes with a modified Ringer solution(RS) added to concentrations of BT: 0, 100, 300, 1000, 2200 and4000 mM. Afterwards, the retina was superfused with RS, twice. Wealso measured the time interval of the SD passage through theelectrodes in order to obtain its velocity.Results: Western blotting showed the presence of an α2a- adrenergicreceptor in the retinal tissue. Immunohistochemistry demonstratedthat α2a - adrenergic receptors are present in Müller cells (Figure 1).The electrophysiological studies demonstrated a significant decreaseof the voltage shift at 4000mM (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology(DF) sodium 0.1%, in this model.Methods: Male Dutch-belted rabbits (2.0-2.5 kg) were used. ConA(5 μg/eye) or vehicle (sterile saline) was injected intravitreally tounilateral eye of the anesthetized rabbits. Rabbits were topicallyapplied with 50 μL of BF 0.1%, NF 0.1% or DF 0.1% three times aday on days -1, 0, 1, 2 relative to ConA injection (day 0). Three daysafter ConA injection, rabbits were euthanized and the vitreous bodieswere isolated. The protein concentration in the vitreous body wasmeasured by BCA methods to assess BRB breakdown.Results: The vitreous protein concentrations in ConA -injectedrabbits was 2117 ± 400 μg/mL (mean ± SD, n=6), and significantlyincreased compared with that in vehicle-injected rabbits (1307 ± 501μg/mL (n=6)). The vitreous protein concentrations of BF 0.1%, NF0.1%, and DF 0.1% groups were 1474 ± 574 (n=6), 1674 ± 452(n=6), and 1800 ± 351 (n=7) μg/mL respectively. BF 0.1%significantly suppressed BRB breakdown in this model (p