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ARVO 2016 Annual Meeting Abstracts 163 Corneal Wound Repair and Healing Sunday, May 01, 2016 3:15 PM–5:00 PM Exhibit/Poster Hall Poster Session Program #/Board # Range: 1248–1294/D0196–D0242 Organizing Section: Cornea Program Number: 1248 Poster Board Number: D0196 Presentation Time: 3:15 PM–5:00 PM A Surgical Cryoprobe for Targeted Transcorneal Freezing to Treat Corneal Endothelial Dysfunction Alina Akhbanbetova1, Shinichiro Nakano2, Stacy L. Littlechild1, Robert D. Young1, Madara Zvirgzdina1, Nigel J. Fullwood3, Shigeru Kinoshita4, Naoki Okumura2, Noriko Koizumi2, Andrew J. Quantock1. 1Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom; 2Biomedical Engineering, Doshisha University, 1-3 Miyakodami-Tatara, Japan; 3Biomedical and Life Sciences, Lancaster University, Lancaster, United Kingdom; 4 Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. Purpose: To examine the effects on corneal tissue of localized freezing induced by a new surgical cryoprobe. The machine was designed to remove endothelial cells from the posterior surface of the cornea in a reproducible and targeted manner to aid the treatment of corneal endothelial dysfunction. Methods: A freezing console was designed and manufactured based on the use of nitrous oxide as a cryogen. The console was connected to one of four different cryoprobes, each with a different freezing tip (1.8 mm-diameter, flat profile; 2.4 mm-diameter, flat profile; 2.4 mm-diameter concave profile; 3.4 mm-diameter, concave profile) at which temperatures below -50°C were achieved. In vitro studies were conducted on 426 porcine corneas, followed by a small number of in vivo investigations on rabbit corneas. After treatment the epithelial basement membrane, corneal stroma, and corneal endothelium were investigated by slit-lamp microscopy, ultrasound pachymetry, and light and electron microscopy. Results: In vivo and in vitro the corneal epithelium was destroyed by freezing, but the epithelial basement membrane remained intact. In vitro, reproducible corneal endothelial damage was achieved using the 3.4 mm-diameter cryoprobe tip with the concave profile. The damage occurred after a short, 3-sec freeze, and was confined to a circular region of the endothelium located directly under the surface application position of the cryoprobe tip. Corneal edema was seen in vivo 24-hrs after freeze injury and was accompanied by alterations to the arrangement of collagen fibrils, but this resolved by 10-days and 1-month concurrent with endothelial repopulation of the wound area. Conclusions: Surface corneal freezing using a 3.4 mm-diameter concave cryoprobe induces transient stromal edema, but leaves the epithelial basement membrane intact which likely aids epithelial resurfacing. Localized destruction of the endothelial monolayer was achieved in a consistent manner, and represents a potentially useful approach to help treat corneal endothelial dysfunction. Commercial Relationships: Alina Akhbanbetova, None; Shinichiro Nakano, None; Stacy L. Littlechild, None; Robert D. Young, None; Madara Zvirgzdina, None; Nigel J. Fullwood, None; Shigeru Kinoshita, None; Naoki Okumura, None; Noriko Koizumi; Andrew J. Quantock, None Support: Ser Cymru The Life Sciences Research Network Wales UK: Research Studenship Program Number: 1249 Poster Board Number: D0197 Presentation Time: 3:15 PM–5:00 PM The use of topical insulin to treat refractory neurotrophic corneal ulcers Angeline L. Wang, Eric Weinlander, Brandon Metcalf, David M. Gamm, Michael Struck. Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI. Purpose: Refractory neurotrophic corneal ulcers are clinically challenging and potentially blinding. Current research focuses on targeted therapies to improve corneal epithelial healing in these cases. Insulin has been shown to improve corneal epithelial healing in vitro and in diabetic animal models; however, clinical experience with topical insulin in patients with non-healing corneal wounds is limited. The purpose of this study is to present three cases of refractory neurotrophic corneal ulcers that were treated with topical insulin. Methods: Retrospective chart review of patients’ exam findings, medications, and procedures. Regular insulin at a concentration of 1 unit per mL of artificial tears was prescribed topically as one drop two or three times daily. Results: The three patients included a 2-year-old girl with a history of excised orbital teratoma and proptosis; a 2-year-old boy with aniridia, congenital glaucoma status post multiple glaucoma procedures, and bilateral corneal decompensation; and a 24-year-old woman with a history of herpes zoster keratoconjunctivitis. All three patients were noted to have decreased or absent corneal sensation in one eye. Each patient developed a neurotrophic corneal ulcer with associated epithelial defect and stromal thinning. The ulcers were refractory to a range of traditional treatments, up to and including surgical management with permanent tarsorrhaphy in one patient. The addition of topical insulin resulted in complete corneal reepithelialization ranging from 13 to 25 days following initiation of treatment. One patient reported increased eye irritation and redness with the treatment; no systemic side effects were noted. Conclusions: Topical insulin may be an effective treatment for refractory neurotrophic corneal ulcers. Proposed mechanisms include increased corneal epithelial cell migration and restoration of bidirectional trophic signaling through preservation of corneal nerves. Insulin has been shown to promote cell migration and closure of artificial wounds in cultured sheets of corneal epithelial cells. In diabetic mice, topical insulin appears to slow the loss of sub-basal plexus corneal nerves. Further study is needed to determine the clinical efficacy and side effect profile of topical insulin in corneal wound healing. Commercial Relationships: Angeline L. Wang, None; Eric Weinlander, None; Brandon Metcalf, None; David M. Gamm, None; Michael Struck Program Number: 1250 Poster Board Number: D0198 Presentation Time: 3:15 PM–5:00 PM The cornea wound healing properties of retinoic acid passed by the action of the LOXL4 protein Loic Blanchon1, Aureli Comptour1, Marion Rouzaire1, Corinne Belville2, Frederic Chiambaretta1, 4, Vincent Sapin1, 3. 1EA 7281 - Reitnoids Reproduction Developmental Diseases, Université d’Auvergne, Clermont-ferrand, France; 2GReD - EA7281 - Retinoids, Reproduction Developmental Diseases, Université d’Auvergne INSERM, Clermont-ferrand, France; 3Biochemistry and Molecular Biology Department, CHU Clermont-Ferrand, Clermont-ferrand, France; 4Ophthalmology, CHU Clermont-Ferrand, Clermont-ferrand, France. Purpose: Epithelial wound healing is a multistep mechanism implying a combination of molecular and cellular events. Following alkali burn traumatisms, cell migration, proliferation and These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts differentiation were demonstrated to be required to recover an intact corneal epithelium and a good visual acuity. Already studied for its pro-healing properties, vitamin A and its active derivatives (i.e. retinoic acid / RA) appear to be good candidates to better understand the cornea wound healing. After having previously demonstrated in-vitro and in-vivo the effect of RA on wound corneal epithelium by acting on cell migration, we studied here the lysyl oxidase (LOX) gene family regulated by RA and the importance of such proteins for corneal epithelium recovery Methods: HCE cells were treated by RA for LOX family members’ characterization and induction by PCR /qPCR and immunocytochemistry. Promotology experiments were done to study LOX(s) induction by RA and the RAR (Retinoic Acid Receptor) isoforms implied. Scratch assays were done after RA treatment in combination with βAPN (β-aminopropionytrile, a LOX family enzymatic inhibitor) or after transfection with siRNA against LOX family members. Cell proliferation and migration were determined after scratch assay. The right eye of 7 male CD1 per group was burned with NaOH and treated with RA and w/o βAPN 6 times/ day (d) during 7d. To evaluate wound healing, wounds areas were quantified from slit lamp photographs at 0 and 7d using imageJ Results: Among the 5 members of LOX family, only LOXL4 expression (mRNA and protein levels) is increased in HCE treated with RA from 12 to 60 hours. This regulation passed through the RARα/RXRα heterodimer fixation on a RARE-DR5 type (Retinoic Acid Response Element) located in the LOXL4 promoter. In the in-vitro model, the use of βAPN or siRNA directed against LOXL4 showed a decrease in the wound healing by inhibiting cell migration promoted by RA treatment. In the in-vivo model the use of βAPN during RA treatment also inhibits such positive action on wound healing Conclusions: We confirmed the importance of RA in cornea epithelium wound repair. Then, we demonstrated that LOXL4, a gene implied in the ECM dynamic and induced by RA, is essential for such phenomenon. This study is one of the first demonstrating a direct link between vitamin A regulated genes and cornea wound healing and could constitute a cornerstone of future clinical therapies Commercial Relationships: Loic Blanchon, None; Aureli Comptour, None; Marion Rouzaire, None; corinne Belville, None; frederic chiambaretta, None; Vincent Sapin, None Program Number: 1251 Poster Board Number: D0199 Presentation Time: 3:15 PM–5:00 PM Acceleration of EGF expression and related cell behaviors induced by travoprost were cancelled by further addition of an EGF receptor inhibitor in corneal epithelium Yukihisa Takada, Osamu Yamanaka, Takayoshi Sumioka, Yuka Okada, Shizuya Saika. Wakayama Medical University, Wakayama, Japan. Purpose: To examined the effects of epidermal growth factor (EGF) receptor inhibitor, PD168393 (PD), on corneal epithelium treated with travoprost. We previously reported that travoprost induced the proliferation of corneal epithelial cells with increased expression of EGF in vitro and in organ-culture of a mouse cornea (ARVO2015). Methods: Human corneal epithelial cells (HCE) were cultured for 24 hrs in the presence or absence of travoprost (0.04 g/l) and/ or PD (10μM). We examined expression of E-cadherin by using immunohistochemistry and cell proliferation by Alamar blue assay. Mouse eyeball was organ-cultured for 48h in the presence or absence of travoprost and/or PD. Expression of EGF, Ki67, phosphorylated (p) -beta catenin and adhesion molecules (E-cadherin, p-FAK) and signal pathway related with EGF (pErk, pSmad3) in corneal epithelium was immunohistochemically examined. Results: PD blocked travoprost promotion of cell proliferation and E-cadherin expression in the cultured cells. Promotion of expression of EGF, Ki 67, p-beta catenin, E-cadherin, p-FAK by travoprost in corneal epithelium of an organ-cultured mouse eye was counteracted by further addition of PD. Travoprost induced phosphorylation of Erk and Smad3, that was also canceled by adding PD. Conclusions: Acceleration of EGF expression and related cell behaviors, i.e., promotion of cell proliferation, adhesion molecule expression, signaling activation, by adding travoprost to corneal epithelial cell culture or organ-cultured corneal epithelium were cancelled by further addition of PD. Control of EGF signal could be a strategy to prevent travoprost-induced corneal epithelial disorder. Commercial Relationships: Yukihisa Takada; Osamu Yamanaka, None; Takayoshi Sumioka, None; Yuka Okada, None; Shizuya Saika, None Program Number: 1252 Poster Board Number: D0200 Presentation Time: 3:15 PM–5:00 PM NSAIDs causes corneal epithelial damage by inhibition of leukotriene B4 receptor 2 signaling Satoshi Iwamoto1, 2, Tomoaki Koga2, Toshiaki Okuno2, Akira Murakami1, Akira Matsuda1, Takehiko Yokomizo2. 1 Ophthalmology, Juntendo Univ School of Med, Tokyo, Japan; 2 Biochemistry, Juntendo Univ School of Medicine, Tokyo, Japan. Purpose: Compromised corneal epithelialization in patients treated with NSAIDs had been reported. However, pathophysiological mechanism of delayed corneal wound healing with non-steroidal antiinflammatory drugs (NSAIDs) usage is still not clear. In ARVO 2015, we reported that a non-canonical BLT2(Leukotriene B4 receptor 2) ligand, 12(S)-hydroxyheptadecatrienoic acid (12-HHT) accelerated corneal epithelial wound healing, and 12-HHT production is inhibited by NSAIDs eye drop. In this study, we further investigated the mechanism how 12-HHT/BLT2 pathway accelerate corneal epithelialization. Methods: Naïve corneal tissue obtained from BLT2(-/-) and congenic wild type (WT) mice was subjected to next generation sequencing (NGS) analysis to compare gene expression profiles. Human corneal epithelial cell line (HCET) overexpressed BLT2 gene was established and scratch assay experiments were carried out. Results: The expression of acta2 gene (encoding alpha-smooth muscle actin) was attenuated in the naïve cornea of BLT(-/-) mouse compared to WT mouse. In BLT2 overexpressed HCET cells, accelerated epithelial cell wound closure and increase of acta2 gene expression was observed. Conclusions: 12-HHT/BLT2 pathway accelerates corneal epithelial wound healing with increase of acta2 gene expression. Commercial Relationships: Satoshi Iwamoto, None; Tomoaki Koga, None; Toshiaki Okuno, None; Akira Murakami, None; Akira Matsuda, None; Takehiko Yokomizo Program Number: 1253 Poster Board Number: D0201 Presentation Time: 3:15 PM–5:00 PM Effects of loss of TRPV4 function on corneal epithelial wound healing in mice Yuka Okada1, Peter S. Reinach3, Masayasu Miyajima2, Shizuya Saika1. 1Ophthalmology, Wakayama Medical University, Wakayama, Japan; 2Laboratory Animal Center, Wakayama Medical University, Wakayama, Japan; 3Wenzhou Medical University, Wenzhou, China. Purpose: To determine if transient receptor potential vanilloid 4 (TRPV4), TRPV1-related component, contributes to epithelial wound healing in a mouse cornea. Growth factor expression pattern was also evaluated. TRP family consists of groups of TRPV, ankyrin These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts 1 (TRPA1) or melastatin (TRPM). We previously reported the involvement of TRPV1 and TRPA1 in corneal epithelial wound healing in mice (IOVS 2014, ARVO 2015). Methods: Imunohistochemistry was carried out to examine the expression opattern of TRPV4 in mouse cornea. Time dependent closure of a 2.0 mm diameter central corneal epithelial debridement was monitored in the right eyes of C57BL/6 (wild type, WT, n =61) and TRPV4-null (KO, n =61) mice for up to 36 h using fluorescein green staining. BrdU-labeling in WT and KO mice evaluated proliferative activity during healing. RT-PCR analyzed interleukin 6 (IL-6), substance P (SP) calcitonin gene-related peptide (CGRP), nerve growth factor (NGF) and transforming growth factor b1 (TGFb1) gene expression levels. Results: TRPV4 was detected in the basal cells of the corneal epithelium. The open wound area in the TRPV4 KO mice at 18 and 24 hr was 1.6-fold and 7.34-fold bigger than in the WT counterpart, respectively. BrdU-labeled cells were fewer in the KO healing epithelium than in the WT counterpart at 24 and 36 hr (p<0.05). Increases in CGRP, NGF and TGFb1 gene expression were smaller than those in the WT counterpart (p<0.05) whereas increases in IL-6 and SP were unaffected by the loss of TRPV4 function. Conclusions: TRPV4 mediated signaling contributes to increases in CGRP, NGF and TGFb1 gene expression and repair of a corneal epithelial debridement in mice. Commercial Relationships: Yuka Okada; Peter S. Reinach, None; Masayasu Miyajima, None; Shizuya Saika, None Program Number: 1254 Poster Board Number: D0202 Presentation Time: 3:15 PM–5:00 PM Effect of Vitamin D Receptor Knockout on Diabetic Mouse Corneal Epithelial Wound Healing Mitchell A. Watsky1, Xiaowen Lu2. 1Cellular Biology & Anatomy, Augusta University, Augusta, GA; 2Cellular Biology & Anatomy, Augusta University, Augusta, GA. Purpose: Diabetes is a significant complicating factor in numerous pathological conditions, and diabetic keratopathy effects approximately 70% of all diabetics. Our recent studies determined that vitamin D receptor (VDR) knockout adversely affects corneal wound healing. This study was designed to investigate VDR knockout effects on corneal epithelial wound healing of diabetic mice. Methods: The low dose streptozotocin injection method was used to induce diabetes mellitus in VDR heterozygous (HET) and VDR knockout (KO) mice along with their wildtype (WT) littermates. Corneal epithelium wound healing experiments were carried out in these mice. Mice were anesthetized and 2 mm central wounds were made using an Alger brush. Wound closure was measured at 0, 12, 20, 28, 36, 48, 60, and 72 h after wounding (measurements stopped if the wound was completely healed) by photographing fluoresceinstained corneas and digitally outlining the wound margins and calculating the wound areas using Olympus CellSens Dimension software. Mice were anesthetized with isoflurane administered through an anesthesia vaporizer for cornea photography. Results: Wound areas versus time were plotted and a linear regression was run on the data. Healing rates were equated to the regression slope. A rate of -0.018 mm/h was measured in 6-week diabetic duration KO mice versus a rate of -0.05 mm/h in 6-week WT diabetic mice. A rate of -0.021 mm/h was measured in 8-week duration diabetic KO mice versus a rate of -0.046 mm/h in 8-week WT diabetic mice. KO diabetic mice of 6- and 8-week diabetes duration had significantly slower healing rates than WT diabetic mice. There was no significant difference in the healing rate of 4-week diabetic KO mice versus 4-week diabetic WT mice. Conclusions: KO diabetic mice have significantly slower healing rates than the WT diabetic mice of 6- and 8-week diabetes duration. Vitamin D appears to exert a significant influence early in the development of diabetic keratopathy. Commercial Relationships: Mitchell A. Watsky, None; Xiaowen Lu, None Support: National Institutes of Health (NIH) National Eye Institute (NEI) Grant R01EY021747 Program Number: 1255 Poster Board Number: D0203 Presentation Time: 3:15 PM–5:00 PM Role of SKQ1 On Corneal Wound Healing: An In Vitro Model Using Human Corneal Epithelial Cell Culture Model Yi Wei1, Penny A. Asbell1, Natalia Perekhvatova2, Maxim Skulachev2, Anton Petrov2, Boris Chernyak3. 1Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY; 2Mitotech S. A., 42, rue de la Vallee, Luxembourg; 3Belozersky Institute Of Physico-Chemical Biology, Moscow State University, Moscow, Russian Federation. Purpose: SKQ1 (Visomitin) is a novel mitochondrial-targeted antioxidant that holds promise for treatment of ocular surface diseases. The goal of this study is to determine if SKQ1 plays a role in corneal wound healing process. Methods: Human corneal limbus epithelial (HCLE) cell cultures were pretreated for 1 hour with varying concentrations of SKQ1 (0400 nM). A single-streak wound was then created and fresh medium containing SKQ1at concentrations corresponding to the amounts for pretreatment were in place. Photographs were taken at indicated timepoints until wound closure and the resulting images were analyzed with Image-J. SKQ1 cytotoxicity was determined by a standard MTT assay. HCLE cell proliferation was evaluated by treating single cells with varying concentrations of SKQ1 for 6 days and quantified by MTT assays. HCLE cell migration was evaluated after wounds treated with SKQ1 and 10 μM pp38 kinase inhibitor SB203580 for 10 hours. Results: SKQ1 concentrations up to 250 nM showed no toxicity. Much higher concentrations were needed to produce cytotoxicity. In comparison to the no-SKQ1 control, addition of 50 nM SKQ1 significantly increased their wound healing rates by 4, 9 and 9%, corresponding to 4, 8 and 12 hours of SKQ1 treatment. Furthermore, as low as 25 nM SKQ1 doubled the cell proliferation rates. Finally, addition of SB203580 completely abolished the stimulated wound healing by SKQ1. Conclusions: SKQ1 were shown to significantly enhance the corneal epithelial wound healing process likely through enhancement of cell proliferation and migration. The data provide support for SKQ1 as a promising new therapeutically strategy for treatment of corneal epithelial wounds and damages. Commercial Relationships: Yi Wei, None; Penny A. Asbell; Natalia Perekhvatova, Mitotech, S. A.; Maxim Skulachev, Mitotech S.A.; Anton Petrov, Mitotech, S. A.; Boris chernyak, Belozersky Institute Of Physico-Chemical Biology, Moscow State University, Moscow, Russia (C) Support: Funded in partial by a research grant from Mitotech SA Pharmaceuticals, the Martin & Toni Sosnoff Foundation and Research to Prevent Blindness (RPB) These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1256 Poster Board Number: D0204 Presentation Time: 3:15 PM–5:00 PM Cell patterning in native and newly formed corneal ECM post lamellar keratectomy Pouriska Kivanany1, Byron Weiss2, Elias Choi2, Kevin Lam3, Nihan Yönet-Tanyeri4, David Schmidtke3, Matthew Petroll1. 1 Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX; 2University of Texas Southwestern Medical School, University of Texas Southwestern Medical Center, Dallas, TX; 3Department of Bioengineering, University of Texas at Dallas, Richardson, TX; 4Department of Biomedical Engineering, Istanbul Medipol University, Istanbul, Turkey. Purpose: The organized arrangement of the collagen lamellae plays a key role in corneal transparency. Following injury to the cornea, quiescent corneal keratocytes transform into fibroblasts and myofibroblasts. We have previously shown that following freezeinjury, migrating corneal fibroblasts align parallel to the stromal lamellae. In this study, we compare cell and extracellular matrix (ECM) patterning within and on top of the stroma following lamellar keratectomy (LK). Methods: LK was performed on New Zealand White Rabbits. From 1 to 60 days after injury, rabbits were monitored using in vivo confocal microscopy and sacrificed to further investigate cell and matrix patterning. The corneal tissue was fixed and labeled with Alexa Fluor 488 phalloidin in situ, and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging. Cell patterning was also assessed in vitro, by plating corneal keratocytes on top of aligned fibrillar collagen substrates that were generated using a microfluidics approach. Results: Immediately following LK, cell death occurred in the native corneal stroma directly beneath the area of injury. At 7 and 21 days after LK, confocal and SHG results indicated that the alignment of fibroblasts repopulating this region was highly correlated with the orientation of the collagen lamellae. In contrast, cells on top of the native corneal stroma were randomly arranged, contained more prominent stress fibers, and secreted fibrotic ECM. Alpha smooth muscle actin (α-SMA) was also detected in this region, indicating the presence of myofibroblasts. At 60 days, cells and matrix on top of the stroma were co-aligned into lamellar-like structures. Corneal haze peaked at 21 days after LK, and returned to near baseline levels by 60 days. In culture studies, corneal keratocytes stimulated with platelet derived growth factor aligned parallel to unidirectional collagen fibers. Conclusions: Based on these results, we hypothesize that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after corneal injury in the rabbit. Initially, cells on top of the native stroma align randomly and produce a disorganized ECM, since they do not have topographic cues to guide them. Over time, cells remodel this fibrotic ECM to produce a lamellar structure that is similar to the native corneal stroma. Commercial Relationships: Pouriska Kivanany, None; Byron Weiss, None; Elias Choi, None; Kevin Lam, None; Nihan Yönet-Tanyeri, None; David Schmidtke, None; Matthew Petroll, None Support: NIH Grants R01 EY013322 and P30 EY020799, and Research to Prevent Blindness, Inc. Program Number: 1257 Poster Board Number: D0205 Presentation Time: 3:15 PM–5:00 PM Transient and Selective Ingrowth of Lymphatic Vessels into the Cornea after Incision Injury Deniz Hos1, Jens Horstmann1, Sebastian E. Siebelmann1, Franziska Bucher1, Philipp Steven1, Felix Bock1, Reza Dana2, Claus Cursiefen1. 1Ophthalmology, University of Cologne, Cologne, Germany; 2Schepens Eye Research Institute, Boston, MA. Purpose: Corneal lymphangiogenesis contributes to several ocular pathologies such as dry eye, ocular allergy and corneal graft rejection. However, a physiological role for corneal lymphangiogenesis, e.g. during corneal wound healing, has not been described so far. Therefore, aim of this study was to evaluate whether corneal lymphangiogenesis occurs during the physiological healing course after a perforating corneal incision injury. Methods: A central perforating corneal incision was performed in C57BL/6 mice. Afterwards, corneal opacity and edema were scored clinically and analyzed by optical coherence tomography (OCT). In order to investigate their relationship to corneal hem- and lymphangiogenesis, blood and lymphatic vessels were analyzed in whole mounts stained with CD31 and LYVE-1. Real-time PCR was performed to analyze gene expression of the lymphangiogenic ligands VEGF-C and VEGF-D and the corresponding receptor VEGFR-3. Results: Injured corneas developed opacity and edema, detectable as an increase of central corneal thickness (CCT), which peaked in the first two weeks after incision injury (mean CCT in uninjured: 90.1µm; after 1 week: 147.9µm, p<0.01; after 2 weeks: 165.6µm, p<0.01) and then gradually decreased until corneas became clear after 4 weeks (mean CCT after 4 weeks: 125.4µm, p>0.05). In addition, incision injury resulted in selective ingrowth of lymphatic, but not blood vessels, into the cornea. Corneal lymphangiogenesis peaked within the first two weeks (mean lymphvascularized area in uninjured: 2.09%; after 1 week: 7.35%, p<0.001; after 2 weeks: 6.87%, p<0.01) and then regressed (mean lymphvascularized area after 4 weeks: 3.45%, p>0.05). Furthermore, ingrowth of corneal lymphatic vessels was accompanied by upregulated gene expression of VEGF-C (x2.5 after 1 week; x2.0 after 2 weeks; x1.7 after 4 weeks; all p-values <0.01), VEGF-D (x2.0 after 1 week, p<0.01; x1.0 after 2 weeks, p>0.05; x1.1 after 4 weeks, p>0.05) and VEGFR-3 (x14.4 after 1 week, p<0.001; x1.7 after 2 weeks, p<0.05; x3.4 after 4 weeks, p<0.01). Conclusions: A central perforating incision injury leads to transient and selective ingrowth of lymphatic vessels into the cornea, which points to a putative physiological role of lymphangiogenesis during corneal wound healing. Commercial Relationships: Deniz Hos, None; Jens Horstmann, None; Sebastian E. Siebelmann, None; Franziska Bucher, None; Philipp Steven, None; Felix Bock, None; Reza Dana, None; Claus Cursiefen, None Support: German Research Foundation FOR2240 “(Lymph) angiogenesis and cellular immunity in inflammatory diseases of the eye” (HO 5556/1-1); EU COST BM1302; Gerok-Program, University of Cologne Program Number: 1258 Poster Board Number: D0206 Presentation Time: 3:15 PM–5:00 PM LncRNA Expression Profile and Bioinformatic Analyses of LncRNA-gene Network in Corneal Epithelial Wound Healing Dongsheng Yan, Qiongjie Cao, Xiaoting Zhao, Dewei Peng. Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou Zhejiang, China. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Purpose: Long non-coding RNAs (LncRNAs) are a novel class of non-protein coding transcripts longer than 200 nucleotides. Recent studies demonstrated that lncRNAs are essential in a wide range of physiological and pathological processes. The roles of lncRNAs in corneal epithelial wound healing, however, remains unknown. In the present study, we profile their involvement in corneal epithelial renewal and develop a lncRNA-gene network that affects wound healing outcome. Methods: Mouse corneal epithelial cell layers were scratch wounded and harvested. Total RNA was extracted. We used the Affymetrix Microarray Platform to analyze differentially expressed lncRNAs during mouse corneal epithelial wound healing. Real time RTPCR analyses were performed to validate the microarray findings. Bioinformatic analyses predicted the target genes of candidate lncRNAs and their involved signaling pathways. Results: Microarray analysis indicated that 1500 lncRNAs were significantly downregulated whereas 1253 others were markedly upregulated during corneal wound healing (fold change>2). The expression of 8 lncRNAs was successfully confirmed by Realtime RT-PCR. Bioinformatic analyses revealed involvement of a complex lncRNA-gene network during corneal epithelial wound healing. Conclusions: Our results revealed differentially expressed lncRNA patterns during corneal epithelial wound healing and a complex lncRNA-gene network that is modulated by these lncRNAs. Functional analysis will be performed to investigate the roles of specific lncRNAs in corneal epithelial wound healing. Commercial Relationships: Dongsheng Yan, None; Qiongjie Cao, None; Xiaoting Zhao, None; Dewei Peng, None Support: National Natural Science Foundation of China Grant 81170821, 973 Project (2012CB722303) from the Ministry of Science and Technology of China, and Science Foundation of Wenzhou Medical University QTJ11020. Program Number: 1259 Poster Board Number: D0207 Presentation Time: 3:15 PM–5:00 PM Copper promotes lysyl oxidase activity in corneal cells and tissues Santosh K. Muddana1, Hironori Uehara1, Dallas Shi1, Faisal Ahmed1, Ramesh Rallabandi2, Balamurali K. Ambati1. 1Opthamology, Moran Eye Center, Salt Lake City, UT; 2Chemistry, University of Utah, Salt Lake City, UT. Purpose: Keratoconus is a corneal disorder that is a leading cause of corneal transplant, and due to corneal biomechanical weakness secondary to insufficient collagen cross-linking defects. Mutations in lysyl oxidase-1 (loxl1), which is responsible to collagen cross linking, have been identified in keratoconus patients. LOXL1 requires copper as a co-enzyme. In this study, we examined whether copper treatment can enhance collagen cross-linking in corneal cells and tissues. Methods: Human keratoconus corneas were obtained from the patient undergoing corneal transplantation. Human healthy corneas were obtained from Utah Lion Eye Bank. Each cornea was bisected and one half was incubated in 0.0016 mg/ml copper sulfate (CuSo4) Optisol for 1 week at 25°C and the other half was incubated in normal optisol. The Cornea samples were washed with PBS and powdered in liquid nitrogen and pulverized with a Freezer Mill. Cornea samples were demineralized with 0.5 M EDTA in 0.05 M Tris buffer, pH 7.4, for 48 h at 4C. The insoluble fraction was washed twice with distilled water by centrifuging at 5000rpm at 47C for 20 min and lyophilized. The lyophilized sample was hydrolyzed with 6 N HCl for 24 h at 110°C. The hydrolysate was neutralized with 2.5 N NaOH and dissolved in distilled water. Analysis was done for Lysinonorleucine (LNL) and Histidinohydroxylysinonorleucine (HHL) separately by HPLC. Corneal stroma cells from normal and keratoconus cornea were cultured and treated with and without 0.0016mg/mL CuSO4. Then, the conditioned culture medium was subjected to a peroxidasecoupled fluorometric assay for lox activity assay. Results: HPLC demonstrated an increase in LNL and HHL in copper treated keratoconus corneas, while their levels were not detected in control-treated keratoconus cornea. Loxl1 activity was also increased with copper treatment in both normal and keratoconus corneal stroma cells. Conclusions: CuSO4 treatment increase lysyl oxidase activity and dramatic increase in LNL and HHL collage crosslinking. This may indicate a role for copper therapy in keratoconus. Commercial Relationships: Santosh K. Muddana, None; Hironori Uehara; Dallas Shi, None; Faisal Ahmed, None; Ramesh Rallabandi, None; Balamurali K. Ambati, None Support: EY017950 Program Number: 1260 Poster Board Number: D0208 Presentation Time: 3:15 PM–5:00 PM The role of blepharitis in the healing course of corneal ulcers Cynthia I. Tung1, James Reidy2, Ruth M. Mattern2, Sangita P. Patel2, 3. 1 Head and Neck Surgery, MD Anderson Cancer Center, Houston, TX; 2Ophthalmology, University at Buffalo, Buffalo, NY; 3Research Service, VA Western New York Healthcare System, Buffalo, NY. Purpose: Blepharitis is a known risk factor for the development of infectious keratitis, but the relationship between blepharitis and the healing course of corneal ulcers has not been defined. We performed a retrospective clinical study to determine the time to re-epithelialization of corneal ulcers in the presence and absence of blepharitis. We hypothesized that the presence of blepharitis would prolong the time to re-epithelialization of corneal ulcers. Methods: A retrospective chart review was performed for 23 corneal ulcer patients that were diagnosed and treated over a 3 and a half year period (December 2011 to June 2015) by three ophthalmologists at a university eye institute. Two groups were studied: one with presence of blepharitis (n=11) and one with absence of blepharitis (n=12) on clinical slit lamp exam. Inclusion criteria were ICD-9 diagnosis codes for corneal ulcers (370.0, 370.01, 370.03). Exclusion criteria included neurotrophic, viral, fungal, acanthamoebic ulcers, ulcers greater than 6mm in longest diameter on presentation, association with uveitis or endophthalmitis, and history of corneal surgery or significant ocular surface disease. Number of days were recorded from the first visit where corneal ulcer was noted to the first visit re-epithelialization was noted. Student’s t-test and Pearson’s correlation coefficient were used for statistical analysis. Results: In the blepharitis group, time to re-epithelialization of the corneal ulcer was significantly longer (19.3±15.1 days), compared to the non-blepharitis group (7.3±4.6 days) (p=0.02). Median days to re-epithelialization were 13 and 6, respectively. In correlating time to re-epithelialization with age, the non-blepharitis group showed a weak positive correlation (R = +0.23), while the blepharitis group showed almost no correlation (R = +0.081). Conclusions: We conclude that blepharitis may create an environment of ocular surface inflammation that significantly delays the healing of corneal ulcers. Commercial Relationships: Cynthia I. Tung; James Reidy, None; Ruth M. Mattern, None; Sangita P. Patel, None Support: RPB unrestricted grant These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1261 Poster Board Number: D0209 Presentation Time: 3:15 PM–5:00 PM Conjunctival resection for peripheral ulcerative keratitis (PUK) Ore-oluwa C. Erikitola, Lona Jawaheer, Kanna Ramaesh, Deepa Anijeet. Tennet’s Institute of Ophthalmology, NHS Greater Glasgow and Clyde, Glasgow, United Kingdom. Purpose: Conjunctival resection as management strategy for PUK, though described 40 years ago, is relatively underutilized. Conjunctiva adjacent to PUK has been shown to be populated by inflammatory cells and cytokines. We describe a case series where conjunctival resection was used early on in the course of the disease, as a minimally invasive adjuvant treatment. Methods: This is a retrospective review of 3 eyes of 3 patients with PUK who had conjunctival resection. Aetiology, clinical presentation, treatment, clinical course and stability after conjunctival resection were analysed. Results: Mean age of two male and one female patient was 68.7 years (range: 60-76 years). All patients had unilateral disease. Aetiologies included one case each of chronic recurrent blepharokeratitis (case 1), rheumatoid arthritis (case 2) and ANCA positive connective tissue disease (case 3). Extent of ulceration was 2 clock hours in case 1 and 6 clock hours in cases 2 and 3. Depth of thinning was 30 % in case 1 and 80% in cases 2 and 3. Cases 2 and 3 with connective tissue disease had systemic prednisolone in a dose of 50- 60 mg for less than 2 weeks. All patients had topical steroids (dexamethasone 0.1%) up to a frequency of 6 times a day for less than 2 weeks. All patients also received 2 hourly topical lubricants, as well as anti-metalloproteinase (tetracycline). Patients underwent excision and recession of adjacent limbal conjunctiva (limbal conjunctivectomy) as conservative measures failed to make any significant improvement in clinical course. Conservative measures were continued post operatively (tapering systemic and topical steroids, continuing topical lubricants and tetracycline derivative) in all 3 patients. Ulcerative component healed within 2 weeks. In each case, surgical treatment was successful in faster healing and tapering of systemic steroids. No recurrences were reported at 4-12month follow up. Conclusions: Conjunctival resection can be an effective and simple adjuvant therapeutic modality in the treatment of PUK. Removing adjacent para-limbal source of immune trigger can aid in the quicker resolution of PUK, with reduced requirement for systemic immunosuppression with its harmful side effects. Larger comparative case studies will be needed to confirm this small scale clinical observation. Figure 1: Case 2: a) Before and b)16 days after conjunctival resection Figure 2: a) Before and b) 5 days after conjunctival resection Commercial Relationships: Ore-oluwa C. Erikitola, None; Lona Jawaheer; Kanna Ramaesh, None; Deepa Anijeet, None Program Number: 1262 Poster Board Number: D0210 Presentation Time: 3:15 PM–5:00 PM Suture Retention Test as a novel Method for Scaffold Characterization in Ophthalmology Florian Küng1, 2, Piotr Stafiej1, 2, Dirk W. Schubert2, Friedrich E. Kruse1, Thomas A. Fuchsluger1. 1Department of Ophtalmology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; 2Institute of Polymer Materials, FriedrichAlexander-Universität Erlangen-Nürnberg, Erlangen, Germany. Purpose: In order to attach a scaffold on a patient sutures are commonly used. The quantity describing the resistance of a scaffold against the pull-out of a suture is called ’suture retention strength’. This quantity is commonly tested for tubular cardial scaffolds. A novel approach to modify the testing procedure was performed in order to adjust the suture retention test for characterizing ophthalmological scaffolds. Methods: Polycaprolactone (PCL) is dissolved in chloroform and cast into petri dishes in order to generate films by solution casting. The amount of PCL was varied in order to cast films of different thickness. The thickness of each film was measured in a circular pattern with a gauge. 21 round samples of a diameter of 14 mm were cut out of each film according to a circular pattern. The samples were sutured using a Vicryl Polyglactin 910 4-0 suture. The suture was tied to a loop. The loop was secured with a bolt and more than 50% of the sample was clamped in a tensile tester. Using a constant deformation rate, force was applied on the sample until the suture was pulled out. In order to generate benchmark data from a material currently applied in ophthalmology 20 samples of amniotic membrane were tested under the same conditions. Results: PCL films of 0.2 ± 0.02 g, 0.4 ± 0.02 g, 0.6 ± 0.02 g and 0.8 ± 0.02 g were cast. The thickness of the films was between 7 µm and 150 µm. Suture retention strength, elongation at the maximum force and the elongation at rapture were determined for each sample. The total mean of the PCL data of the suture retention strength is 1.85 ± 1.21 N while the mean of the elongation at maximum force is 4.09 ± 1.26 mm and the mean of the elongation rapture is 6.75 ± 1.54 mm. The amniotic membrane with a thickness of 51 ± 8 µm showed a suture retention strength of 0.20 ± 0.06 N at an elongation of 5.38 ± 1.51 mm and a total elongation of 6.95 ± 1.59 mm. Conclusions: It is possible to measure samples on the scale of an ophthalmological scaffold in a suture retention test. The ’suture retention test’ is a reliable option to characterize the rigidity of scaffolds used in ophthalmology. By variation of input parameters first dependencies of the suture retention strength were revealed. Using the experimental data, a first comparison between the results of the amniotic membrane and PCL is accessible. Commercial Relationships: Florian Küng, None; Piotr Stafiej, None; Dirk W. Schubert, None; Friedrich E. Kruse, None; Thomas A. Fuchsluger, None These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1263 Poster Board Number: D0211 Presentation Time: 3:15 PM–5:00 PM Comparative results of cell culture of human corneal epithelial cells (HCE) and human corneal keratocytes (HCK) on electrospun nanofiber matrices of Polycaprolactone blended with Poly(glycerol sebacate) and chitosan Piotr Stafiej1, 2, Florian Küng1, 2, Daniel Thieme1, Marta Czugala1, Dirk W. Schubert2, Friedrich E. Kruse1, Thomas A. Fuchsluger1. 1 Department of Ophthalmology, University of Erlangen-Nurnberg, Erlangen, Germany; 2Institute of Polymer Materials, University of Erlangen-Nurnberg, Erlangen, Germany. Purpose: We have previously shown that Polycaprolactone (PCL)/ Poly(glycerol sebacate) (PGS) nanofiber matrices show properties for ocular surface reconstruction and the possibility to introduce linker groups for drug attachment. To evaluate the biocompatibility of these materials, we now evaluated the biological activity of two human corneal cell types on these matrices. Methods: The nanofiber matrices were electrospun in different blends of PCL with PGS and with chitosan. Electrospun fiber meshes with either random orientation or aligned have been collected on a substrate and cut into circular samples with a diameter of 14 mm. After rinsing the samples in PBS buffer overnight and sterilization by UV-Light the samples were put into 24-well-Plates and incubated with HCE and HCK cells for 8 days. Wells with PCL foil were used as controls. During cultivation biological activities were measured for each sample after 2, after 5 and after 8 days by cell proliferation assay (WST-8). After the incubation time the samples were prepared for confocal microscopy and pictures have been taken to check the cell morphology. (Results shown as random: Mean ± SD / aligned: Mean ± SD) (AU=Absorbance Units) Results: HCE and HCK cells could be cultured on each of the materials, PCL alone, PCL blended with PGS and PCL blended with chitosan. On aligned fiber meshes an orientation of the HCE and the HCK cells in direction of the fibers can be detected on confocal microscopic pictures by checking the growth direction of the F-Actin fibers. In contrast the cells grew random on the random fiber meshes. The bioactivity of the HCE cells was lowest on PCL chitosane [0,006 ± 0,003 AU / 0,046 ± 0,02 AU] and similar on the fibers with PGS [0,175 ± 0,12 AU / 0,078 ± 0,03 AU] and PCL alone [0,149 ± 0,07 AU / 0,078 ± 0,01 AU], while the bioactivity for the HCK cells was low on each material. Conclusions: Blending PCL with PGS shows good results for culturing HCE cells, wether further research has to be conducted on the chitosane material. Combined with the good alignment of the cells to the oriented fiber meshes the blend of PCL with PGS shows the best attributes for the further research with the goal to produce active surfaces for the tissue engineering while further research on the blend of PCL with chitosane is needed. Commercial Relationships: Piotr Stafiej, None; Florian Küng; Daniel Thieme, None; Marta Czugala, None; Dirk W. Schubert, None; Friedrich E. Kruse, None; Thomas A. Fuchsluger, None Program Number: 1264 Poster Board Number: D0212 Presentation Time: 3:15 PM–5:00 PM Soluble Epoxide Hydrolase Inhibition Ameliorates Diabetic Neurotrophic Keratopathy and Accelerates Delayed Epithelial Wound Healing in the Diabetic Mouse Corneas Haijing Sun1, Jiaoyue Hu2, Wei Li2, Fushin X. Yu1, 2. 1Ophthalmology, Wayne state university, DETROIT, MI; 2Eye Institute, Xiamen University, Xiamen, China. Purpose: Soluble epoxide hydrolase (encoded by Ephx2) rapidly hydrolyzes biologically active epoxyeicosatrienoic acids into the less biologically active metabolites, dihydroxyeicostrienoic acids and has been linked to cardiovascular diseases. This study was aimed to assess the adverse effects of hyperglycemia-induced expression of EPHX2 in pathogenesis of diabetic keratopathy and its reverse in EPHX2 deficient mice or by pharmacological inhibition. Methods: C57BL/6 Ephx2 knockout mice were induced to develop diabetes by intraperitoneal injection of streptozotocin (STZ). The expression of EPHX2 detected by cDNA array was verified by immunohistochemistry. EPHX2 levels and enzymatic activities were assessed using Western blotting and sEH activity assay. The normal and diabetic mice were subjected to epithelium debridement wound and allowed to healing for indicated times. Sensory nerve regeneration post epithelium wounding was assessed with whole mount confocal microscopy. Results: EPHX2 was only expressed in corneal epithelia of diabetic corneas and its levels increased with the duration of hyperglycemia in C57BL/6 mice. The levels of EPHX2 and sEH activities inversely correlated to the rate of epithelial wound closure. Moreover, EPHX2 inhibitor significantly accelerated epithelial wound closure in diabetic corneas. EPHX2 knockout diabetic mice have increased rate of epithelial wound closure enhanced delayed nerve regeneration, similar to the wild-type B6 mice. Conclusions: Our data showed that EPHX2 expression increases in diabetic corneas and the elevated EPHX2 plays a detrimental role in epithelial wound repaired. Targeting EPHX2 pharmacologically may represent a new approach for treating diabetic keratopathy. Commercial Relationships: Haijing Sun, None; Jiaoyue Hu, None; Wei Li, None; Fushin X. Yu, None Support: NIH grants EY010869 and EY017960 Program Number: 1265 Poster Board Number: D0213 Presentation Time: 3:15 PM–5:00 PM Human growth hormone released from a biocompatible hyaluronic acid biomaterial modulates wound healing in an in vivo corneal chemical burn model Gina L. Griffith1, Barbara M. Wirostko3, 2, Hee-Kyoung Lee3, 2, Anthony J. Johnson4, 1, David O. Zamora1. 1Ocular Trauma, United States Army Institute for Surgical Research, San Antonio, TX; 2 Moran Eye Center, University of Utah, Salt Lake City, UT; 3Jade Theraputics, Inc., Salt Lake City, UT; 4San Antonio Military Medical Center, San Antonio, TX. Purpose: Hyaluronic acid, a ubiquitously expressed polysaccharide, is of great interest in the bioengineering and regenerative medicine communities for use as an off-the-shelf biomaterial. In this study, cross-linked carboxymethylated hyaluronic acid (CMHA-S)-based film strips were utilized to provide a sustained release of recombinant human growth hormone (rHGH) and facilitate the repair and regeneration of damaged ocular tissues. Our purpose was to test the hypothesis that CMHA-S strips, with and without loaded rHGH, are biocompatible in vivo and can be securely and safely retained in the eye for the treatment of corneal epithelial chemical burns. Methods: The nictitating membranes of 30 New Zealand white rabbits (~3.0 kg) were removed 3 wks prior to wound creation and strip placement. Burns 5.5 mm in diameter were created by placing a circular filter paper soaked in 1N NaOH centrally onto the cornea for 30 seconds. Wounds were immediately rinsed with sterile buffered saline, and the eye evaluated using the McDonald-Shadduck ophthalmic exam and fluorescein staining. Animals were randomly grouped (n=5 per group) for treatment with control CMHA-S strips or with CMHA-S strips containing 50 or 150 µg/strip of rHGH. At one and two weeks post strip placement, eyes were evaluated by slit lamp and in vivo confocal microscopy. Corneal histology was performed using H&E and Masson’s Trichrome stain. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Results: Upon re-hydration, CMHA-S strips exhibited swelling to yield a clear soft oblong strip of ~4 mm wide by ~15 mm long, able to be manipulated with forceps and placed into the lower eye cul-desac. All strips were retained in the eye for a minimum of 96 hrs, with a maximum retention time of 14 days post placement. Wounds treated with rHGH loaded films exhibited an increase in wound closure compared to those treated with unloaded strips. Loaded and unloaded strips were biocompatible and did not reveal any pathological effects to the eye or surrounding tissues clinically or on histopathology. Conclusions: CMHA-S film strips are biocompatible and easily retained in the ocular cul-de-sac. Furthermore, when compared to unloaded strips, the rHGH-loaded strips are capable of modulating the re-epithelialization of acute corneal burns. These results advance the overall efforts to develop the first FDA-approved ocular pharmaceutical indicated for corneal wound healing. Commercial Relationships: Gina L. Griffith, None; Barbara M. Wirostko, Jade Theraputics, Inc. (P), Jade Theraputics, Inc., Jade Theraputics, Inc. (I), Jade Theraputics, Inc. (F); HeeKyoung Lee; Anthony J. Johnson, None; David O. Zamora, None Program Number: 1266 Poster Board Number: D0214 Presentation Time: 3:15 PM–5:00 PM Sphingolipids and TGF-β signaling in the human cornea Sarah E. Nicholas, Tyler Rowsey, Megan Stiles, Sufiya Khanam, Nawajes A. Mandal, Dimitrios Karamichos. Ophthalmology, University of Oklahoma Health Sciences Center, Midwest city, OK. Purpose: Corneal fibrosis leaves the cornea opaque and can result in partial or complete vision loss for which the only current treatment is corneal transplantation. Recently sphingolipids (SPL) have been linked to corneal fibrosis and found to be modulated by the transforming growth factor-β (TGF-β) pathway. The aim of this study is to dissect the role of sphingolipids in corneal fibrosis and their signaling mechanisms related to the three TGF-β isoforms. Methods: Healthy human corneal fibroblasts (HCFs) were cultured in EMEM with 10% FBS and 0.5mM 2-O-α-D-glucopyranosyl-Lascorbic acid in 3D constructs and allowed to grow for 4 weeks in the presence of 0.1ng/mL TGF-β1, TGF-β2, and TGF-β3. Cultures without any growth factors served as Controls. After 4 weeks the cultures were examined for the expression of sphingolipid specific pathway signaling using Real-Time PCR and Western Blot. Results: We investigated all five sphingosine-1-phosphate receptors 1 through 5. TGF-β1 and TGF-β3 led to significant upregulation of sphingosine-1-phosphate receptor 3 (S1PR3) when compared to Control HCFs. TGF-β2 did not modulate S1PR3. TGF-β2, on the other hand, led to significant upregulation of sphingosine kinase 1 (SphK1), while TGF-β3 resulted in upregulation of SphK2. Conclusions: Overall, we have drawn some intriguing signaling cross talk between sphingolipid molecules and TGF-β isoforms in human corneal fibroblasts. This study suggests that TGF-β3 driven upregulation of S1PR3 may represent a novel target in regulating corneal fibrosis. Future studies will dissect the signaling mechanism further. Commercial Relationships: Sarah E. Nicholas, None; Tyler Rowsey, None; Megan Stiles, None; Sufiya khanam, None; Nawajes A. Mandal, None; Dimitrios Karamichos, None Support: EY025256 Program Number: 1267 Poster Board Number: D0215 Presentation Time: 3:15 PM–5:00 PM Fibroblast-epithelial cell interactions upregulate the expression of galectin-3 in cornea Pablo Argueso1, 2, Andrea Cruzat1, 2, Jerome Mauris1, 2. 1Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, MA; 2 Department of Ophthalmology, Harvard Medical School, Boston, MA. Purpose: Direct intercellular contacts between stromal and epithelial cells are involved in a number of functions critical to the development and repair of the cornea. We recently identified a crucial role for the carbohydrate-binding protein galectin-3 in promoting matrix metalloproteinase 9 (MMP9) in epithelial cells through its interaction with the cell surface receptor CD147. The goal of this study was to determine whether the interaction of fibroblasts with epithelial cells regulates the expression and function of galectin-3. Methods: Primary human corneal fibroblasts and immortalized human corneal-limbal epithelial cells were maintained in DMEM medium supplemented with 10% serum and keratinocyte serumfree medium, respectively. The presence of galectin-3 and CD147 in whole cell lysates was quantified by Western blot. Affinity chromatography was performed using a sepharose-conjugated galectin-3 column. MMP9 secretion was determined by gel zymography. Results: Both fibroblasts and epithelial cells produced galectin-3. Epithelial cells expressed the highly glycosylated forms of CD147, known to induce MMP production, whereas fibroblasts expressed glycoforms of lower molecular weight. CD147 from epithelial and fibroblast origin bound to galectin-3 in a galactose-dependent manner, however, exogenous galectin-3 failed to induce MMPs in fibroblasts, in contrast to epithelial cells. Co-culture of fibroblasts with epithelial cells resulted in an upregulation of galectin-3, concomitant with an enhanced expression of CD147 and MMP9. The increase in MMP9 was time-dependent, reaching its maximum at 24-48 h, and was directly related to the number of fibroblasts in culture. Conclusions: Our results indicate that galectin-3 expression is dependent on the interaction between epithelial and stromal cells, and suggest that sustained upregulation of galectin-3 may potentially lead to stromal matrix degradation and therefore may be clinically relevant to corneal disease. Commercial Relationships: Pablo Argueso, None; Andrea Cruzat, None; Jerome Mauris, None Support: Supported by NIH/NEI R01EY024031 (PA) and BostonKPro research fund (AC) Program Number: 1268 Poster Board Number: D0216 Presentation Time: 3:15 PM–5:00 PM Development of a large animal ex vivo corneal fibrosis model for translational research Todd L. Marlo3, Elizabeth A. Giuliano3, Ajay Sharma3, 2, Rajiv R. Mohan2, 1. 1Veterinary Medicine and Surgery, Biomedical Sciences, Veterinary Pathology, and Mason Eye Institute, University of Missouri, Columbia, MO; 2Harry S. Truman Memorial Veterans Hospital, Columbia, MO; 3Veterinary Medicine & Surgery, University of Missouri, Columbia, MO. Purpose: Currently there is no ex vivo model of the equine cornea. We sought to determine if the equine cornea is suitable as an ex vivo model. Specifically, to assess the equine cornea’s extracellular matrix and cellularity after 7 days using two different culture techniques (an air/liquid interface and immersion system) to determine the best ex vivo equine corneal model. Our hypothesis is that the air/liquid interface model would be superior to the immersion model system. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Methods: Equine corneas with 2 mm of perilimbal sclera (n=14) are freshly harvested from horses undergoing humane euthanasia and free of anterior segment disease. One scleral-corneal ring from each horse is randomly placed in the air/liquid interface organ culture system with the contralateral scleral-corneal ring being placed in the immersion condition organ culture system for 7 days. All scleralcorneal rings were evaluated using serial daily gross photography, histology, RT-PCR and TUNEL assay. Freshly harvested healthy equine corneas were utilized as controls for all evaluated parameters. Results: Scleral-corneal rings placed in immersion condition had complete loss of corneal transparency on gross photography by 7 days, showed a significant level (p<0.05) of stromal disorganization, significantly increased (p<0.05) αSMA levels on RT-PCR, and apoptosis on TUNEL assay when compared to controls. The airliquid corneas had weak stromal disorganization on histopathologic examination and were not significantly different from normal equine corneal controls on any other evaluated parameter therefore proving our hypothesis. Conclusions: The air-liquid culture condition maintained equine cornea’s cellular extra cellular matrix and preserved corneal transparency. Conversely, the immersion system resulted in near complete degradation of the normal equine corneal architecture after 7 days in culture. The air-liquid interface system is a viable option to maintain a normal equine cornea in an ex-vivo setting for translational studies. Commercial Relationships: Todd L. Marlo; Elizabeth A. Giuliano, None; Ajay Sharma, None; Rajiv R. Mohan, None Support: Mainly from VAF grant VAF2015-03 (TM); University of Missouri Phi Zeta grant (TM); Ruth M. Kraeuchi Missouri Endowment Chair Ophthalmology Fund (RRM); Partially from the RO1EY17294 National Eye Institute, NIH, Bethesda, Maryland, USA and 1l01BX00035701 Veterian Health Affairs, Washington DC, USA grants Program Number: 1269 Poster Board Number: D0217 Presentation Time: 3:15 PM–5:00 PM Accelerated healing of corneal endothelial lesions induced by engineered FGF-1 derivatives Ralph Bradshaw1, Michael Blaber2, Amuthakannan Subramanian1, David Ornitz3, David Eveleth1. 1Research, Trefoil Therapeutics, San Diego, CA; 2Florida State University, Tallahassee, FL; 3Washington University, St. Louis, MO. Purpose: Purpose: Engineered versions of FGF-1 are known to be potent stimulators of dissociated corneal endothelial cell proliferation in vitro. These experiments tested the ability of eFGF-1s to accelerate the healing of corneal endothelial lesions in an organ culture model Methods: Methods: The eFGF-1s TTHX1001 and TTHX1114 was generated via site-directed mutagenesis and the enhanced stability and potency evaluated via isothermal equilibrium denaturation and X-ray crystallography. Stimulation of FGF receptors was evaluated in BAF3 cells. Mitogenic potency was tested using NIH 3T3 and primary human corneal endothelial cell cultures. Healing of corneal endothelial lesions was evaluated in an organ culture system using trypan blue quantification of the lesion areas. Results: Results: Both TTHX1001 and TTHX1114 have enhanced thermodynamic stability relative to the wild type FGF-1 and stimulate FGF receptors at lower concentrations. Both potently stimulated the proliferation of human corneal endothelial cells in culture, with TTHX1114 approximately 100-fold more potent than wild type FGF1. These eFGFs also accelerated the healing of lesions in the corneal endothelium of rabbit corneas in organ culture. The increased potency of TTHX1001 and TTHX1114 was not proportional to the degree of thermodynamic stabilization. Conclusions: Conclusions: Engineered FGFs can potently accelerate the healing of corneal endothelial lesions making these agents potential therapeutics for corneal endothelial dystrophies. Commercial Relationships: Ralph Bradshaw, Trefoil Therapeutics; Michael Blaber, Trefoil Therapeutics (C); Amuthakannan Subramanian, Trefoil Therapeutics; David Ornitz, Trefoil Therapeutics (C); David Eveleth, Trefoil Therapeutics Program Number: 1270 Poster Board Number: D0218 Presentation Time: 3:15 PM–5:00 PM Insight Into the Corneal Wound Healing Response: Transcriptomic and Metabolomic Analysis of Corneal Epithelial Cells Challenged by Bacteria Kimberly Brothers, Stephen Harvey, Nicholas Stella, Robert M. Shanks. Ophthalmology, University of Pittsburgh, Pittsburgh, PA. Purpose: The cornea is an essential transparent barrier that protects the eye from exposure to microbes on a daily basis. Wound healing is critical for maintaining this protective barrier. We recently demonstrated that certain bacteria prevent corneal wound healing in vitro and ex vivo. With the contact lens-associated keratitis pathogen Serratia marcescens, LPS was responsible for this inhibition. This study sought to determine the impact of LPS-containing bacterial secretomes on the host response of corneal epithelial cells in vitro. Methods: Growth medium (mock) and sterile bacterial culture filtrates (secretomes) were added to human corneal limbal epithelial (HCLE) cells. HCLE supernatants from mock and secretome treated cells were collected 24 hours post challenge and analyzed by cytokine array, ELISA, microarray, and metabolomic analysis. Contents of challenged HCLE cells were also metabolomically analyzed. To identify the role of TLR4 in S. marcescens inhibition of wound healing, HCLEs were independently treated with OxPAPC and CLI-095 inhibitors and tested in cell migration assays. Results: HCLEs treated with S. marcescens secretomes resulted in increased IL-1α, IL-1β, GM-CSF, and IL-6 as determined by cytokine array, ELISA for IL-1 β, and microarray. Metabolomics from secretome treated HCLEs had elevated levels of phosphoethanolamine, a phosphorylated amino alcohol associated with autophagy, lipid signaling, and apoptosis. TLR4 inhibition of HCLE cells did not prevent S. marcescens secretome inhibition of corneal wound healing, suggesting this inhibition is TLR4 independent. Conclusions: Using a combination of transcriptomics and metabolomics this study provides insight into the impact of bacteria on human corneal epithelial cells. Together these data support when HCLEs are treated with S. marcescens secretomes, there is an increase in pro-inflammatory markers and small molecules consistent with activation of the autophagy pathway correlating with altering epithelial cell wound healing. Commercial Relationships: Kimberly Brothers, None; Stephen Harvey, None; Nicholas Stella, None; Robert M. Shanks, None Support: NH grant F32EY024785 These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1271 Poster Board Number: D0219 Presentation Time: 3:15 PM–5:00 PM Evaluation of a therapeutic anti-TNF-α drug delivery system for ocular alkali burns Chengxin Zhou1, 3, Marie-Claude Robert4, 5, Fengyang Lei1, 3, Vassiliki Kapoulea1, 3, James Chodosh2, 3, Claes H. Dohlman2, 3, Eleftherios I. Paschalis1, 3. 1Department of Ophthalmology, Schepens Eye Research Institute-Massachusetts Eye and Ear, Boston, MA; 2 Massachusetts Eye and Ear, Boston, MA; 3Harvard Medical School, Boston, MA; 4Department of Ophthalmology, Universite de Montreal, Montreal, QC, Canada; 5Centre Hospitalier de l’Universite de Montreal, Hopital Notre-Dame, Montreal, QC, Canada. Purpose: Ocular burns cause corneal inflammation, neovascularization (CNV) and scarring. We have shown that tumor necrosis factor alpha (TNF-α) is upregulated after burn and that prompt TNF-α inhibition improves corneal wound healing. However, systemic administration of TNF-α inhibitors can cause significant adverse events, while topical administration is limited by poor corneal bioavailability and the need for frequent drug application. This study was designed to test a novel drug delivery system (DDS) for sustained-release of TNF-α inhibitor to the ocular surface. We used a rabbit ocular alkali burn model to assess the efficacy of the DDS in terms of reduction of corneal CNV, opacity and improvement of corneal wound healing. Methods: DDS was prepared using porous PDMS/PVA composite fabrication that was loaded with 80μg infliximab. Evaluation was performed in 6 Dutch-belted pigmented rabbits that received ocular alkali burn with 2N NaOH. Three rabbits received subconjunctival implantation of anti-TNF-α DDS and 3 sham DDS immediately after the burn. Rabbits were followed with photography for 3 months and analyzed for CNV, opacity and re-epithelialization. Inflammation was assessed using CD45 antibody in tissue sections. Results: Rabbits treated with anti-TNF-α DDS achieved complete corneal re-epithelialization within 63.6 ± 7.5 days of burn, reduced CNV (0.13 ± 0.05 % of cornea area) and corneal opacity (central score = 2.17 ± 0.85; peripheral score = 0.33 ± 0.41 in a 0-4 scale), while sham DDS treated rabbits exhibited persistent epithelial defect until the end of the study (92 days), increased CNV (0.31 ± 0.14 % of cornea area, p<0.05) and corneal opacity (central score = 3.5 ± 0.57, p>0.05; peripheral score = 2.44 ± 1.41, p<0.05). Anti-TNF-α DDS was well tolerated. The number of CD45+ cells in anti-TNF-α DDS treated eyes was significantly lower (mean=4322 cells/ cornea) compared to sham DDS eyes (mean=17049 cells/ cornea, p<0.05). Infliximab was still present in the DDS 3 months after implantation, evident by anti-human IgG immunolocalization. Conclusions: Sustained topical delivery of anti-TNF-α for the treatment of ocular alkali burn is feasible using the described DDS. Local anti-TNF-α therapy suppresses corneal inflammation and CNV, and improves wound healing. The described DDS may be beneficial to a variety of ocular surface diseases amenable to biologic therapy. Commercial Relationships: Chengxin Zhou, None; MarieClaude Robert, None; Fengyang Lei; Vassiliki Kapoulea, None; James Chodosh, None; Claes H. Dohlman, None; Eleftherios I. Paschalis, None Support: Boston Keratoprosthesis Research Fund, Massachusetts Eye and Ear, Boston, MA Program Number: 1272 Poster Board Number: D0220 Presentation Time: 3:15 PM–5:00 PM Relaxin 2 augments tissue growth factor β effects on keratocytes Ulrike Hampel1, 2, Detlev Holland3, Friedrich P. Paulsen1. 1 Department of Anatomy II, Friedrich Alexander University medical faculty, Erlangen, Germany; 2Ophthalmology, Johannes Gutenberg University, Mainz, Germany; 3Bellvue Klinik, Kiel, Germany. Purpose: We previously showed that the pregnancy hormone relaxin 2 increases corneal epithelial wound healing by influencing migration and proliferation. To investigate the influence of relaxin 2 on keratocytes and its potential effect on the stroma. Methods: Primary keratocytes were isolated and cultured from transplant residuals of donor corneae. Keratocytes were stimulated with 1 nM relaxin and in combination with 3 ng/ml tissue growth factor β (TGFβ) for 48 hours. Differentiation of keratocytes to myofibroblasts was investigated by smooth muscle actin (SMA) expression with the help of western blot analysis and concomitant contractility by collagen gel contraction assays. Lumigan, collagens 1A and 1B mRNA expressions were evaluated by real-time RT-PCR and collagen production was measured in supernatants by sircol soluble collagen assay. Results: Relaxin 2 alone does not influence the SMA expression, but 10 nM relaxin 2 in combination with TGFβ increases the SMA expression compared to TGFβ alone. Relaxin 2 does not further increases TGFβ-induced contractility of keratocytes. TGFβ-elevated collagen 1A expression was reduced by relaxin 2, whereas collagen 1B and lumigan expression was not influenced by relaxin 2. Total collagen content measured in supernatants was not influenced by relaxin 2. Conclusions: Case reports of corneal decompensation during pregnancy accuse elevated relaxin levels. Our findings do not support this theory. Relaxin 2 alone does not influence keratocytes. However, in combination with TGFβ relaxin 2 can increase wound healing processes of the corneal stroma. Commercial Relationships: Ulrike Hampel, None; Detlev Holland, None; Friedrich P. Paulsen, None Support: DFG HA 6344/2-1 Program Number: 1273 Poster Board Number: D0221 Presentation Time: 3:15 PM–5:00 PM Targeting alpha v integrins promotes regenerative healing in the cornea Stephanie Gillespie, Audrey M. Bernstein. Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY. Purpose: Scarring and fibrotic disease result from the persistence of myofibroblasts characterized by high cell-surface expression of alpha v integrins, which activate the fibrotic factor TGFβ leading to pathological cell adhesion and fibrotic matrix secretion. We propose that increasing ubiquitin-mediated intracellular degradation of alpha v integrins prevents scarring and induces regenerative healing. Methods: Experiments were performed in supplemented serumfree media on collagen. Transient transfection in primary human corneal cells (HCFs) with USP10 or control cDNA was performed using Amaxa Nucleofection. Standard lentiviral infection using immortalized htert-HCFs was used to generate a USP10 overexpressing cell line. TGFβ activity was measured by coculturing USP10 cells with htert-SMAD-luciferase/GFP reporter cells and Bright Glo Luciferase Reagent. Detection of a-SMA and FN-EDA was by RT-PCR, Western blot, and immunocytochemistry. Porcine corneas, control or wounded by keratectomy, were treated with control or USP10 siRNA and cultured for 2 weeks prior to histological examination for fibrotic markers. USP10 deubiqiuintion of integrin β-chains was examined by immunoprecipitation of These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts integrins in Ubiquitin-FLAG overexpressing htert-HCFs and Western blot for FLAG. Results: Through genetic screening (RNA seq), we determined that corneal stromal myofibroblasts overexpressed a subset of deubiquitinating enzymes (DUBs), which remove ubiquitin from proteins, saving them from degradation. We found that overexpression of the DUB, USP10 produced the fibrotic profile: a) significantly increased αv/β5/β3/β1 protein levels (2.0-3.9 fold), b) activated TGFβ (70% increase), c) increased FN-EDA and alpha smooth muscle actin (α-SMA) gene (1.4-2.1 fold) and protein expression (2.6-5.1 fold), as well as promoting organization of FNEDA fibrils and α-SMA stress fibers. Silencing USP10 in corneal organ culture prevented induction of fibrotic markers and promoted regenerative healing in the epithelium and stroma. Mechanistically, we found that USP10 deubiquitinated integrin β-chains leading to decreased degradation and increased recycling to the cell surface. Conclusions: This novel mechanism puts DUB expression at the head of the cascade that regulates alpha v integrin cell-surface abundance and fibrosis, and suggests USP10 as a novel anti-fibrotic target. USP10 overexpression induces an increase in FN-EDA (2.7 fold) compared to control. Commercial Relationships: Stephanie Gillespie, None; Audrey M. Bernstein, None Support: This work was supported by The Research to Prevent Blindness and NIH-NEI R01 EY024942 and T32 GM 062754 (July 2013-July 2015). Program Number: 1274 Poster Board Number: D0222 Presentation Time: 3:15 PM–5:00 PM Alterations in corneal nerves in DIO mice may explain changes in focal adhesion proteins and migration Vickery E. Trinkaus-Randall, Jenna Meyer, Martin Minns, Gregory Teicher, Celeste Rich. Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, MA. Purpose: The P2X7 purinergic receptor is localized in the corneal epithelium with a distinct apico-basal polarity. Upon injury, P2X7 expression becomes diffuse at the wound margin and decreases away from the wound. Inhibitors of the receptor not only attenuate migration but alter the cytoskeletal rearrangements that are part of wound healing. DIO mice (pre-diabetic model for Type II diabetes) exhibit altered levels of the receptor. Our goal was to examine the corneal nerves in the DIO and control mice and ask if alterations are associated with compromised cell migration and wound repair. Methods: Experiments were performed using in vivo, ex vivo organ culture and in vitro models. DIO and WT mice were sacrificed at 15 and 7.5 weeks after initiation of respective diets. Corneas were removed and either fixed or maintained in organ culture after epithelial debridement to monitor wound closure. A human corneal epithelial cell line was used to examine cytoskeletal rearrangements after injury. Results: Corneas were stained for Tuj1 and P2X7. Nine tiles of the corneas were imaged separately from the apical surface through the stromal nerves and stitched into a composite image. Compression of z-stacks revealed a striking lack of nerves in the DIO mice. In the control epithelium the nerves were concentrated in the basal region and only 20% of the nerves were detected in the most apical region. In the DIO corneas there was a 75% reduction in basal nerves and a 30% reduction in apical nerves. The decrease may explain the lack of the prototypical whorl detected in control. Analysis of the central stroma revealed a 2.3 fold greater number of Tuj1 positive nerves in the DIO that exhibited different morphologies. P2X7 was detected along the corneal nerves. While P2X7 mRNA is elevated there was no detectable difference in localization of P2X7 in the unwounded epithelium. After injury in the DIO corneas the change in P2X7 localization detected previously was absent. Additional experiments demonstrated that inhibition of P2X7 decreased turnover of focal adhesion clusters and altered organization of the cytoskeleton at the leading edge. Conclusions: The high fat, high calorie diet of DIO mice results in a lack of corneal sensory nerves and a lack of change in localization of P2X7 at the wound margin, indicating the importance of P2X7 in regulating proper early signaling events after corneal epithelial injury. Commercial Relationships: Vickery E. Trinkaus-Randall, None; Jenna Meyer, None; Martin Minns, None; Gregory Teicher, None; Celeste Rich Support: NIH Grants RO1 EY06000 and R21EY024392, the Massachusetts Lions FOundation and the New England Corneal Transplant Fund Program Number: 1275 Poster Board Number: D0223 Presentation Time: 3:15 PM–5:00 PM Pathophysiological analysis of corneas 20-years post-Radial Keratotomy shows a lack of corneal remodelling I-Ping Loh1, David Lockington2, Charles N. McGhee1, Trevor Sherwin1. 1Department of Ophthalmology, Faculty of Medical and Health Science, The University of Auckland, AUCKLAND, New Zealand; 2University of Glasgow, Glasgow, United Kingdom. Purpose: Radial Keratotomy (RK) was once a surgical option for altering refractive power by making radial incisions on the cornea. However this procedure was often beset with complications and side effects and is now considered highly controversial. A rare opportunity enabled the study of the long term pathophysiological changes in a pair of RK corneas 20 years after the surgery was performed. Methods: This pair of post-RK corneas was studied histologically with Masson’s Trichrome and immunohistochemically with antibodies that targeting the cellular and structural proteins including integrin α3β1, cytokeratin 3/12 and laminin. Results: Slit lamp examination and dark field microscopy identified 12 RK incisions in each cornea. Histological examination showed that the incisions reached a depth of up to 90% of the stroma. Masson’s Trichrome staining highlighted epithelial plugs at all of the 12 sites of radial incisions that disrupted Bowman’s membrane and extended into the anterior stromal region. Immunohistochemical labelling using integrin α3β1 showed the epithelial plugs were of basal epithelial origin. New basement membrane material appeared appeared to be deposited in the epithelial plug forming an acellular cystic zone inside the plug. The epithelium at the incision sites appeared thickened and indented. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Labelling of keratocyte nuclei revealed that fibrotic cells appeared to have invaded and remained within the plane of the incision. However a lack of keratocyte migration subsequent to this apparent migration left a cellular exclusion zone in the stroma which extended ~100 microns either side of the incision. These cellular rearrangements still persisted 20 years after surgery. Conclusions: This study demonstrates that cellular rearrangements following radial keratotomy leaves the stroma with a significant cellular exclusion zone indicating a lack of corneal remodelling over a period of 20 years. It also caused the disruption of the Bowman’s membrane and formation of cellular plugs in the anterior stroma that are of epithelial origin. These pathological processes may explain why RK surgery was often beset with complications and this data could usefully inform corneal healing processes following more modern procedures such as relaxing incisions. Commercial Relationships: I-Ping Loh, None; David Lockington, None; Charles N. McGhee; Trevor Sherwin, None Support: Auckland Medical Research Foundation Grant 1111010; Save Sight Society Grant 3622588 Program Number: 1276 Poster Board Number: D0224 Presentation Time: 3:15 PM–5:00 PM Discovery of FetuinA and global signalling/remodelling modules driving myofibroblast differentiation during corneal wound healing in patients Arkasubhra Ghosh1, 2, Krishnatej Nishtala1, Dhananjay Kumar1, Rohit Shetty3. 1GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India; 2Singapore Eye Research Institute, Singapore, Singapore; 3Cornea and refractive services, Narayana Nethralaya, Bangalore, India. Purpose: Stromal cell differentiation is an essential process in corneal wound healing. Using quantitative proteomics, we attempt to discover alterations in global protein networks during activation of quiescent keratocytes to active fibroblasts and myofibroblasts. Methods: Human corneal fibroblasts(HCF) were transformed into keratocytes(KT) by culturing in serum free media for 96h. HCF treated with 1ng/ml TGFβ every 24h for 5 days differentiated to myofibroblasts(MYO). The cell types were identified by specific expression of marker genes Aldh3(KT), Thy-1(HCF) and αSMA(MYO). Cell lysates and patient tear samples were subjected to quantitative proteomic analysis using iTRAQ labelled tandem LCMS/MS. Tear samples were collected after approval from Institutional Ethics Committee and written, informed consent from corneal wound patients(n=6) and age-matched healthy controls(n=6). Results: Over 700 proteins were identified with 95% confidence and filtered by >1.5-fold iTRAQ ratio and p<0.05. PANTHER analysis showed cytoskeletal regulation, protein assembly and integrin signalling pathway most significantly altered. A novel TGFβ regulatory protein FetuinA was discovered significantly elevated in MYO compared to KT suggesting a global role in repair phenotype. Actin cytoskeleton regulators COF1, PROF were lower in KT compared to HCF whereas ACTN1, MYL12A, MHC9 were elevated in MYO. Chaperones GRP78, Endoplasmin, HSP90A/B HSP70, etc were also increased in MYO. Differentiation and focal adhesion proteins FN, TLN1, ZYX, FLNA etc were upregulated in HCF and MYO. These proteins were further validated by immunoblotting. Proteomic analysis of tears from patients undergoing corneal wound healing demonstrated elevated FetuinA and cytoskeletal proteins, recapitulating observations from in vitro model. Conclusions: We report a unique set of differentiation factors for wound healing primarily in cytoskeletal and chaperone signalling modules. We uncover FetuinA as highly elevated in myofibroblasts and corneal wound patient tears indicating its critical role in regulating proper healing by modulating TGF-beta function. The data illustrates distinct interactive protein networks that reveal a specific response to TGFβ (from wound or exogenous addition) mediated differentiation process. These novel proteins can serve as drug targets to treat corneal scarring. Commercial Relationships: Arkasubhra Ghosh, None; Krishnatej Nishtala, None; Dhananjay Kumar, None; rohit shetty, None Support: Narayana Nethralaya Foundation Program Number: 1277 Poster Board Number: D0225 Presentation Time: 3:15 PM–5:00 PM Downstream signal pathway of epidermal growth factor receptor on the in vitro corneal wound healing model Masataka Ito1, Yoko Karasawa2, Masaru Takeuchi2. 1Developmental Anatomy, National Defense Med College, Tokorozawa, Japan; 2 Ophthalmology, National Defense Med College, Tokorozawa, Japan. Purpose: We have previously reported that, in the in vitro corneal wound healing model using mouse corneal epithelial cell line, epidermal growth factor (EGF) promoted re-epithelialization of denuded area, and that the re-epithelialization did not depend on cell proliferation. The purpose of this study was to investigate which downstream signal pathway, MAP kinase pathway or phosphatidyl inositol 3 (PI3) kinase pathway, is involved in this reepithelialization. Methods: Corneal epithelial cell line TKE-2 was cultured and multilayered cultures were scratch-wounded with P10 pipette tip. Either EGF receptor kinase inhibitor AG1478 (2.5μM), MAP kinase kinase inhibitor PD08059 (20μM), PI3 kinase inhibitor LY294002 (50μM), or Wortmannin (5μM) was added to the culture and decrease of denuded area was evaluated. ImageJ software was used for the measurement of the area. Cell proliferation was evaluated by BrdU uptake and cell death by colorimetric analyses of lactate dehydrogenase in proliferating cell cultures. Results: When EGF receptor kinase inhibitor AG1478 was added, re-epithelialization was completely inhibited, while inhibition of re-epithelialization by MAP kinase kinase inhibitor PD08059 was mild. Similar to AG1478, two PI3 kinase inhibitors LY294002 and Wortmannin inhibited re-epithelialization completely. Under the administration of these inhibitors to proliferating cells, BrdU uptake was suppressed compared with control, while levels of cell death were not significantly different. Conclusions: For the re-epithelialization of denuded area, PI3 kinase pathway is the main pathway of the downstream of EGF receptor. Commercial Relationships: Masataka Ito, None; Yoko Karasawa, None; Masaru Takeuchi, None Support: Grants–in–aid for Scientific Research #26462673 Program Number: 1278 Poster Board Number: D0226 Presentation Time: 3:15 PM–5:00 PM Intraluminal Microtubule Acetylation Regulates Ocular Development and Corneal Repair Lining Cao1, Zhenjie Xu3, 5, Bo Liu2, Ying Yu3, Marcel Alavi1, Jill Helms2, Matilda F. Chan1, 4, Peter Marinkovich5, Zena Werb3. 1 Department of Ophthalmology, UCSF Medical School, San Francisco, CA, USA, San Francisco, CA; 2Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford School of Medicine, Stanford, CA, USA, Stanford, CA; 3Department of Anatomy, UCSF Medical School, San Francisco, CA; 4Francis I. Proctor Foundation, University of California, San Francisco, CA; 5Department of Dermatology, Stanford University School of Medicine, Stanford, CA. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Purpose: Microtubules (MTs) are dynamic polymers that have remained the subject of intense investigation for their roles in cell division, migration and polarity. Acetylated microtubules are present in virtually all ocular developmental processes but their roles in normal ocular physiology are unknown. Our study examined the role of tubulin acetyltransferase, Mec17, in ocular development and corneal repair. Methods: Mec17-/- mice were generated by replacing a 9.2 kB fragment of the Mec17 gene with a LacZ-neomycin reporter. Whole eyes of littermate WT and Mec17-/- mice at age E14.5 were processed for H&E staining. Mec17 expression was detected by β-galactosidase staining and acetylated a-tubulin (Ac-MT) and ZO-1 expression were analyzed by immunofluorescence staining. Corneal epithelial and endothelial injuries were induced by mechanical (scratch) and chemical injury (alkali) respectively. Results: Mec17-/- mice at E14.5 demonstrate abnormal development of major ocular structures including the cornea, lens, retina, and optic nerve with incomplete penetrance. Wild-type adult mice express Mec17 and in the cornea (all layers), retina (GCL, OPN and IS/OS layers), and optic nerve. Acetylated alpha-tubulin is expressed in the retina (GCL, IPL, OPL and IS/OS layers) and optic nerve. Following corneal epithelial injury, Mec17-/- mice showed delayed epithelial closure at 8 and 24 hours post-injury compared with littermate WT mice. Following corneal alkali injury, WT corneal endothelium displayed regular ZO-1 staining and dot-like acetylated a-Tubulin, but Mec17-/- corneas displayed disorganized ZO-1 staining and lacked acetylated a-Tubulin staining. Conclusions: Mec17 has an important role in normal ocular development and is highly expressed in the adult cornea, retina, and optic nerve. Lack of Mec17 expression results in abnormal corneal epithelial and endothelial repair. These results establish an essential function of Mec17 in normal ocular developmental and physiologic processes. Commercial Relationships: Lining Cao, None; Zhenjie Xu, None; Bo Liu, None; Ying Yu, None; Marcel Alavi, None; Jill Helms, None; Matilda F. Chan, None; Peter Marinkovich; Zena Werb, None Support: Research Evaluation and Allocation Committee (REAC) Award Program Number: 1279 Poster Board Number: D0227 Presentation Time: 3:15 PM–5:00 PM Effects of Platelet-rich Plasma on Canine Corneal Fibroblasts in LPS induced Inflammatory Condition Young Sam Kwon. Department of Veterinary Surgery, Kyungpook National University, College of Veterinary Medicine, Daegu, Korea (the Republic of). Purpose: This experiment was performed to assess the antiinflammatory and proliferative effects of platelet-rich plasma (PRP) on the canine corneal fibroblasts. Methods: To examine PRP effects, the inflammation of corneal fibroblasts was induced by lipopolysaccharide (LPS). The groups were divided into three groups: control group, DMEM containing 10% fetal bovine serum; LPS group, culture medium with LPStreated group; LPS-PRP group, culture medium with LPS and 5% PRP-treated group. The effect of PRP on the proliferation and migration was examined in the inflammatory in vitro condition. The mRNA expressions of the IL-1β, IL-6, IL-10, TNF-α, collagen type 1, MMP-9 and α-SMA were evaluated using RT-PCR. Results: The 3% or more PRP groups significantly enhanced proliferation compared to control group in proliferation assay. The migratory capacity of canine corneal fibroblasts was stimulated with 5% or more PRP group. RT-PCR results showed significant higher levels of IL-6, collagen type I, MMP-9 and α-SMA compared to LPS group. Conclusions: The results of this study suggest that application of PRP on canine corneal fibroblasts may reduce the LPS-induced inflammation and enhance corneal stromal matrix remodeling. Commercial Relationships: Young Sam Kwon Program Number: 1280 Poster Board Number: D0228 Presentation Time: 3:15 PM–5:00 PM sd-rxRNA®: Self-Delivering RNAi Compounds Show Potential for Corneal Indications Following Topical Application Michael Byrne, Melissa Maxwell, Richard Looby, Katherine Holton, James Cardia, Lyn Libertine, Pamela A. Pavco, Karen Bulock. OPHTHALMOLOGY, RXi Pharmaceuticals, Marlborough, MA. Purpose: sd-rxRNA® are stable oligonucleotides that have features of RNAi and antisense and result in spontaneous cellular uptake. When dosed by intravitreal injection sd-rxRNAs are taken up by all cell layers of the retina within 24 hours and result in dose-dependent, target-specific mRNA reduction for as long 3 weeks. RXI-109 is an sd-rxRNA, targeting connective tissue growth factor (CTGF), being evaluated in a Phase 1/2 clinical trial for subretinal fibrosis associated with wet AMD. When RXI-109 is dosed by intravitreal injection to non-human primate, dose-dependent protein reduction is noted in the retina and the cornea for at least 1 week. Current ocular clinical focus is to reduce the progression of scarring in the back of the eye. Scarring is also a major concern for many front of the eye indications, specifically those involving the cornea. Here we present new data highlighting topical delivery of sd-rxRNA to the cornea, administered as eye drops or formulated in a thermoreversible gel, in the presence of an epithelial injury. Methods: An 8 mm epithelial wound was created on the cornea of rabbits. sd-rxRNA was dosed topically 4 times a day for 1 day as eye drops or in a thermo-reversible gel. A second group of animals with an intact epithelial layer of the cornea were exposed to the same dosing regimen. At 24 and 48 hours post application, whole eyes were processed for histological evaluation of cornea cellular uptake of sd-rxRNA. Results: Macroscopically, compound was visible in the cornea in the injured cornea group at 24 hours post dose. The intensity was less at 48 hours. Microscopically, fluorescent sd-rxRNA was visible through all layers of the cornea in the presence of an epithelial injury at 24 hours when formulated in the thermo-reversible gel, and through the majority of layers when administered as drops. Cellular delivery continued to be detectable at 48 hours. No cellular uptake was visible in the cornea when the epithelial layer was intact. Conclusions: sd-rxRNA compounds are stable compounds requiring no additional delivery vehicle to be taken up by cells. RXI-109, a CTGF-targeting sd-rxRNA being developed to reduce scarring, is in a Phase 1/2 clinical trial for subretinal fibrosis associated with late-stage wet AMD. Here we provide evidence of the potential of the sd-rxRNA platform for development of topical therapeutics for front of the eye indications. Commercial Relationships: Michael Byrne, RXi Pharmaceuticals; Melissa Maxwell, RXi Pharmaceuticals; Richard Looby, RXi Pharmaceuticals; Katherine Holton, RXi Pharmaceuticals; James Cardia, RXi Pharmaceuticals; Lyn Libertine, RXi Pharmaceuticals; Pamela A. Pavco, RXi Pharmaceuticals; Karen Bulock, RXi Pharmaceuticals These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1281 Poster Board Number: D0229 Presentation Time: 3:15 PM–5:00 PM Role of Epidermal Growth Factor Receptor and Src-Family Kinase Activation in Human Corneal Epithelial Wound Healing Hasan M. Bashir1, Hiroshi Maeno2, Christine Marshall2, Claire H. Park1, Richu Raju1, John T. Seykora2, Vivian Lee1. 1 Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; 2Department of Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. Purpose: Corneal epithelial wounds are a serious global concern that can lead to deeper corneal scarring and blindness. Our previous study suggests activation of epidermal growth factor receptor (EGFR) and Src-family kinases (SFK) may play a role in corneal epithelial wound healing in murine derived corneal epithelial cells. To further understand the role of the EGFR/SFK pathway in wound healing, wound assays using immortalized human corneal epithelial cells and primary human corneal epithelial cells were examined in the presence and absence of tyrosine kinase inhibitors. Using live cell imaging and Western blot analysis, cellular events were correlated to cell migration rates. Methods: Monolayers derived from 2.040 pRSV-T ATCC (2.040) immortalized cells and primary human corneal epithelial cells (HCEC) were established and subsequently wounded. Cells were incubated with small molecule tyrosine kinase inhibitors or vehicle at various concentrations in pairs. Wound assays were either imaged with a live cell imaging system and analyzed with Image J software, or collected as protein lysates 24 hours after wounding for Western blot analysis. Results: Cells incubated with tyrosine kinase inhibitors showed marked inhibition of cell migration into the wound gaps 24 hours post wounding at all concentrations tested, while cells with vehicle only demonstrated near complete resolution of the wound area. There was a strong correlation between cell migration and activation of EGFR and SFKs. Treatment with tyrosine kinase inhibitors inhibited cell migration and this correlated with decreased levels of activated EGFR and SFK in wounded 2.040 and HCEC. Conclusions: Enhanced corneal epithelial wound healing was previously observed in murine derived corneal epithelial cells with increased levels of activated EGFR and SFK. The introduction of small molecule tyrosine kinase inhibitors inhibited the capacity of wounded human corneal epithelial cells to migrate and close wounds, paralleling decreased activation of EGFR and SFK on Western blot. Results from this study suggest a critical role for EGFR and SFK activation in human corneal epithelial wound healing with small molecule tyrosine kinase inhibitors potentiating EGFR/SFK dependent signaling. Further studies to enhance human corneal epithelial wound healing through increased activation of EGFR and SFK should be conducted. Commercial Relationships: Hasan M. Bashir, None; Hiroshi Maeno, None; Christine Marshall, None; Claire H. Park, None; Richu Raju, None; John T. Seykora; Vivian Lee, None Support: 1K08EY025742-01, K12EY015398, Research to Prevent Blindness Unrestricted Departmental Grant Purpose: Determine the correlation between the ultrastructure of the epithelial basement membrane (EBM) and the presence/absence of corneal myofibroblasts in scarred corneas after excimer laser surgery, incisional trauma, and bacterial infection Methods: Rabbits were divided into 3 groups according to the mechanism of injury: (1) -9D PRK (VISX Star S4 IR laser); (2) partial-thickness (~300μm depth) corneal incisions; (3) P. aeruginosa corneal ulcer (~6x6mm). Fellow eyes were used as unwounded controls. Immunohistochemical analysis was used to detect the myofibroblast marker α-SMA and transmission electron microscopy at 30,000X was performed to determine whether the lamina lucida (LL) and lamina densa (LD) was present Results: Group 1 had high numbers of α-SMA+ cells in the anterior stroma at 1 month after surgery and the EBM was irregular and lacked typical LL/LD morphology. At 3 months, there were no αSMA+ cells and the EBM was fully regenerated and indistinguishable from control corneas. Group 2 had opacity at the incisions at 1, 2, and 3 months after surgery. None of these corneas had α-SMA+ cells and the EBM overlying the incisions was fully regenerated at all time points. Group 3 had large numbers of α-SMA+ cells from the anterior to posterior stroma at 1 month after infection and had no evidence of LL/LD overlying the corneal opacity (Figure) Conclusions: Defective EBM correlated with the presence of corneal myofibroblasts in scarred corneas after high correction PRK or pseudomonas aeruginosa keratitis, which supports the hypothesis that generation and persistence of corneal myofibroblasts is regulated by the EBM after corneal injuries. Program Number: 1282 Poster Board Number: D0230 Presentation Time: 3:15 PM–5:00 PM Ultrastructural evidence of defective regeneration of the epithelial basement membrane as a major factor in the generation and persistence of corneal myofibroblasts after corneal injury in rabbits Gustavo K. Marino, Abirami Santhanam, K P Connie Tam, Steven E. Wilson. Ophthalmology, Cleveland Clinic, Cleveland, OH. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Immunohistochemistry for alpha-smooth muscle actin in central rabbit corneas: (A) Unwounded control; (C) -9D PRK at 1 month after surgery; (E) Partial-thickness corneal incision at 1 month after surgery; (G) Cornea at 1 month after a treated pseudomonas aeruginosa corneal ulcer (400X mag); and Transmission electron microscopy of the anterior central cornea of rabbits: (B) Unwounded control; (D) -9D PRK at 1 month after surgery; (F) Partial-thickness corneal incision at 1 month after surgery; (H) Cornea at 1 month after a treated pseudomonas aeruginosa corneal ulcer (30,000X mag). Arrowheads=a-SMA+ cells; arrows=lamina densa; “X”=absence of lamina lucida/lamina densa. Commercial Relationships: Gustavo K. Marino, None; Abirami Santhanam, None; K P Connie Tam; Steven E. Wilson, None Support: Supported by EY10056, EY015638 and Research to Prevent Blindness, New York, NY Program Number: 1283 Poster Board Number: D0231 Presentation Time: 3:15 PM–5:00 PM Desmin is a Novel Druggable Regulator of Corneal Fibrosis Alexandra Pietraszkiewicz, Christopher Hampton, Ling Lei, Paola Bargagna-Mohan, Royce Mohan. Neuroscience, University of Connecticut Health Center, Southington, CT. Purpose: The intermediate filaments (IFs) vimentin and desmin are coordinately upregulated in myofibroblasts during corneal fibrosis and their downregulation by the potent IF-targeting drug withaferin A (WFA) is protective against fibrosis and haze (JBC 2012, 287: 989- 1006). In this study, we have employed mice deficient for desmin to unravel how desmin contributes to corneal fibrosis. Methods: Wild-type (WT) 129Svev litter mates, desmin hemizygous (Des+/-) and homozygous (Des-/-) knockout mice were employed. Mice were anesthetized by intraperitoneal injection of ketamine and xylazine, and corneas were anesthetized with proparacain eye drop. A 1 μl drop of dilute 0.15 M NaOH was applied for 1 minute, the cornea was washed with saline solution, and the corneal and limbal epithelium was removed by scraping with a blunt Tooke corneal knife. After 7, 14 or 30 days of recovery, mouse corneas were assessed for corneal opacity and mice sacrificed. The enucleated eyes were either cryosectioned for immunohistochemistry (IHC) or corneas dissected and subjected to western blot (WB) analysis. In other studies, injured Des-/- mice were also treated with vehicle or 2 mg/kg/d WFA for 14 days. Results: We discovered that the injured corneas of Des-/- mice like those of WT mice lose corneal transparency and become fibrotic. Surprisingly, Des+/- corneas were protected against corneal fibrosis and revealed absence of a-smooth muscle actin and vimentin upregulation in repairing stromal cells. Recently, we showed that vimentin becomes phosphorylated at serine 38 residue (pSer38Vim) and the expression of this biomarker is correlated with corneal fibrosis (PlosOne 2015, 10(7):e0133399). Therefore, we investigated the expression of pSer38Vim in corneas of desmin-deficient mice. We discovered that the healing Des+/- corneas, unlike fibrotic Des/or WT corneas, show very low expression levels of pSer38Vim. The expression of pSer38vim was also altered in Des-/- corneas by treatment with WFA. Conclusions: Vimentin and desmin play overlapping roles in wound repair and fibrosis, however, depending of gene dosage, the degree of desmin loss can have protective or pathological consequences. Our studies, for the first time, illuminates that the phosphorylation status of vimentin is also governed by desmin during wound repair, and that targeting of pSer38vim by WFA in desmin deficiency can prevent corneal fibrosis. Commercial Relationships: Alexandra Pietraszkiewicz, None; Christopher Hampton, None; Ling Lei, None; Paola Bargagna-Mohan; Royce Mohan, University of Kentucky Research Foundation (P) Support: NIH Grant EY016782 Program Number: 1284 Poster Board Number: D0232 Presentation Time: 3:15 PM–5:00 PM A Novel Vimentin-Targeting Probe for Imaging Ocular Fibrosis Royce Mohan1, Santosh Keshipeddy2, Dennis Wright2, Paola Bargagna-Mohan1. 1Neuroscience, University of Connecticut Health Center, Farmington, CT; 2Pharmaceutical Sciences, University of Connecticut, Storrs, CT. Purpose: Vimentin is a binding target for the small molecule withaferin A (WFA). Here we have synthesized a novel WFA fluorescent analog to investigate the dynamic motility of vimentin filaments in cell cultures and fibrotic expression of vimentin in vivo. Methods: WFA was synthetically appended with a fluorescent tag to develop the imaging probe WFA-BODIPY-FL. In cell labeling assays, WFA-BODIPY-FL was added for 1 h, washed and cultured for 18 h. Cells were fixed and counter stained with antibody against serine 38 phosphorylated vimentin (pSer38vim). In cell spreading assays, rabbit corneal fibroblasts were trypsinized and plated on cover slips. After 1 h, WFA-BODIPY-FL (100 nM to 1 μM) was added to the cells for 5 min in serum-free medium, cells washed and allowed to spread. Serial images were collected every 5 seconds for 10 mins. For in vivo studies, 129Svev mice were subjected to the alkali injury model (JBC 2012, 287:989-1006) to promote corneal fibrosis. At These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts 14 days post injury mice were injected with a single intraperitoneal injection of WFA-BODIPY-FL and sacrificed after 1 h. Cryosections from mouse eyes were fixed and stained with antibody against vimentin. Results: We demonstrate that WFA-BODIPY-FL labels vimentin intracellularly efficiently at 250 nM. Cells labeled for 18 h showed WFA-BODIPY-FL fluorescence overlapped with antibody staining for pSer38vim. In cell spreading assays, depolymerized soluble vimentin shows an abundance of fluorescence around the perinuclear region. Short squiggle- and dot-like vimentin structures showed bidirectional movement and increasingly extended towards the cell periphery. Some dot-like structures moved at rapid speeds of 0.5 micrometers/sec corroborating data from published transfection studies that used hybrid vimentin-green fluorescent protein expression. Finally, in injured mice injected with WFA-BODIPY-FL we found intense fluorescence in corneal myofibroblasts that costained for vimentin. Normal uninjured mice injected with WFABODIPY-FL did not show this intense green fluorescence in the corneal keratocytes. Conclusions: We have developed a first-in-class in vivo imaging probe for studying the dynamic organization of vimentin filaments. WFA-BODIPY-FL is also bioavailable in mice and shows remarkable localization of myofibroblasts in fibrotic corneas. This biomarkerimaging reagent will be very useful as a diagnostic tool in ophthalmic applications for disease and injury-related fibrosis. Commercial Relationships: Royce Mohan; Santosh Keshipeddy, None; Dennis Wright, None; Paola Bargagna-Mohan, University of Kentucky Research Foundation (P) Support: NIH Grant EY016782 Program Number: 1285 Poster Board Number: D0233 Presentation Time: 3:15 PM–5:00 PM Differential Effects of Topical Mitomycin C and Steroid on Wound Healing and Optics after Photorefractive Keratectomy in Cats Holly B. Hindman, Margaret DeMagistris, Christine Callan, Thurma McDaniel, Krystel R. Huxlin. Ophthalmology, Univ of Rochester, Rochester, NY. Purpose: To assess the differential effects of topically applied agents on myofibroblast transformation and optical outcomes following photorefractive keratectomy (PRK) in a cat model. Methods: 26 eyes of 13 cats underwent -10 D myopic PRK. Cats were then assigned to 1 of 3 treatment groups: control, Mitomycin C (MMC – 0.02% for 2 mins) and topical steroid (BID for 2 wks). Corneal thickness and reflectivity were measured using optical coherence tomography (OCT) and wavefront aberrations were quantified using a Hartmann-Shack aberrometer. Cats were sacrificed for histology at 2, 4, and 12 wks post-PRK. Corneas were excised, fixed and stained immunohistochemically for alpha-smooth muscle actin (α-SMA). Results: 2 weeks after PRK, significant differences were noted with respect to stromal (KW=9.576, P<0.01) and epithelial thickness changes (KS=7917, P<0.05) between groups. Control and steroidtreated eyes had increased stromal thickness and decreased epithelial thickness relative to MMC-treated eyes. MMC treatment also resulted in a thinner band of α-SMA staining and lower stromal reflectivity 2 wks post-operatively than in control and steroid-treated eyes. By 4 wks post-PRK, there was no detectable α-SMA in MMC-treated eyes, whereas control and steroid-treated eyes continued to express it. By 12 wks post-PRK, stromal reflectivity in MMC-treated eyes (but not control or steroid-treated eyes) returned back to baseline. By 12 wks post-PRK, there were also significant differences in the amount of defocus change induced by PRK between the 3 groups (KW=9.269, P<0.0005, D=7.75, P< 0.01), with MMC eyes exhibiting the greatest treatment effect (defocus change: -5.45 ± 0.17µm relative to baseline). Steroid-treated eyes showed moderate regression (defocus change: -3.30 ± 0.35 µm), while control eyes had the most severe regression (defocus change: -2.04±0.36 µm). However, by 12 wks, no significant differences in induced higher order root mean square (HORMS) existed between groups (control: +0.39 ± 0.34 µm, steroid: +0.60 ± 0.15 µm, MMC: +0.91 ± 0.17 µm; KW = 2.35, P=0.34). Conclusions: Relative to steroid-treated and control eyes, MMC treatment was associated with more favorable wound healing responses and optical outcomes after PRK. This included a decrease in the amount and length of α-SMA expression, greater epithelial stability, and less myopic regression. Commercial Relationships: Holly B. Hindman, None; Margaret DeMagistris, None; Christine Callan, None; Thurma McDaniel, None; Krystel R. Huxlin, None Support: NIH 2R01EY015836-11, Research to Prevent Blindness Program Number: 1286 Poster Board Number: D0234 Presentation Time: 3:15 PM–5:00 PM Characterization of a quantitative model of corneal inflammation, wound healing, and fibrosis in the rabbit David Culp1, Elizabeth Schaefer2, Maria-Grazia Spiga1, Aliah Hackney1, Nicholas Smith1, Brian C. Gilger2. 1Powered Research, RTP, NC; 2Clinical Sciences, North Carolina State University, Raleigh, NC. Purpose: To develop and characterize a sensitive and translatable model of corneal wound healing and fibrosis in the New Zealand White Rabbit. Methods: An 8 mm central corneal defect of approximately 10-15% stromal depth was created using a corneal trephine and lamellar resection. Dexamethasone (DEX) or balanced salt saline (BSS) were administered topically 4 times a day for 28 days after keratectomy. Hackett-McDonald (HM) ocular inflammatory scores and area of fluorescein positive staining to evaluate re-epithelialization were evaluated every 12 hours for 5 days, then weekly through 28 days. Stromal fibrosis, haze, and corneal thickness were measured by SDOCT (Envisu, Bioptigen) and histopathology at 12, 24 and 48 hours and on days 7 and 28. Results: Cumulative HM scores were significantly lower in eyes treated with DEX compared to BSS for the first 4 days after surgery; however, area of fluorescein retention (re-epithelialization of the wound) was higher in DEX compared to BSS-treated eyes through 7 days and time to complete re-epithelization was longer by 3 days in DEX vs BSS eyes. On SD-OCT, corneal opacity scores on day 7 were similar between DEX and BSS treated eyes, but by day 28 there was a 60% reduction in stromal fibrosis in DEX treated eyes. Histologic scores in the anterior segment and cornea were consistently lower in DEX treated eyes on both 7 and 28 days after keratectomy. Conclusions: Using this corneal wound healing model, we demonstrated that topical DEX inhibited ocular inflammation, corneal fibrosis, and opacity compared to BSS. This keratectomy model allows quantitative assessment of specific anti-inflammatory and wound healing therapies, and can be used in pre-clinical development to provide effective proof of concept of novel compounds or delivery methods. Commercial Relationships: David Culp, Powered Research; Elizabeth Schaefer, Powered Research (C); Maria-Grazia Spiga, Powered Reserach; Aliah Hackney, Powered Research; Nicholas Smith, Powered Reserach; Brian C. Gilger, Powered Research (C) These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1287 Poster Board Number: D0235 Presentation Time: 3:15 PM–5:00 PM A novel ex vivo model of equine corneal wound healing Rita Wehrman, Joseph Haynes, Gil Ben-Shlomo. Veterinary Clinical Sciences, Iowa State University, Ames, IA. Purpose: To develop an ex vivo model of equine corneal epithelial wound healing while maintaining normal corneal anatomy. Methods: Equine corneas were harvested within two hours of humane euthanasia (for reasons unrelated to this study) and immediately processed. Corneoscleral rims were excised 2mm posterior to the limbus. The axial cornea was wounded with filter paper soaked in 1N NaOH and epithelial ulceration confirmed employing fluorescein stain. Corneas were subsequently cultured using an air-liquid interface model in media supplemented with 10% fetal bovine serum, 10ng/ml human growth factor and 5ug/ml bovine pancreatic insulin. The rocker was set to bathe the cornea 8 times per minute to simulate normal horse blinking. Corneas were stained with fluorescein and imaged every other day to assess wound healing. Ulcer size was analyzed using an image processing program and corneas were submitted for histologic evaluation when fluorescein negative. Results: All corneas healed within 4 days (96 hours) of epithelial ulceration. Histologically, corneas maintained normal architecture including viable epithelium and minimal stromal edema. Conclusions: Air-liquid interface with media bathing is an effective ex vivo model of equine corneal wound healing. Commercial Relationships: Rita Wehrman, None; Joseph Haynes; Gil Ben-Shlomo, None Program Number: 1288 Poster Board Number: D0236 Presentation Time: 3:15 PM–5:00 PM Neuropathic changes in corneal nerve fibers after abrasion injury Sue Aicher, Clayton Hudson, Sam Hermes, Deborah Hegarty. Physiology & Pharmacology, Oregon Health & Science University, Portland, OR. Purpose: Alterations in ocular sensation occur in patients with dry eye disease as well as those who have undergone vision correction surgery. Pain experienced by these patients may indicate a neuropathic pain condition. We tested the hypothesis that neuropathic changes in corneal nerve fibers underlie changes in ocular sensation using a corneal abrasion injury in rats. These basic science studies will determine the relationship between changes in corneal fiber density and neurochemical content and ocular sensation after a corneal injury. Methods: A unilateral central corneal injury was made using topical application of heptanol in male Sprague-Dawley rats. At time points after corneal abrasion injury, stereotypic nociceptive eye wipe responses to ocular application of noxious menthol were measured in either the abraded eye or the non-abraded control eye. At the conclusion of behavioral testing, rats were euthanized and perfused with aldehydes. The corneas were removed and processed for immunohistochemistry for beta-tubulin and nociceptive molecular markers. Corneal fibers were visualized with confocal microscopy and analyzed using Imaris software. T-tests were used for statistical analyses. Results: Rats tested in the injured eye 24 hours after abrasion had a significantly higher average number of eye wipe responses to ocular application of noxious menthol (13.8 ± 0.8 (SEM) eye wipes; n=4) as compared to rats tested on the uninjured side (7.7 ± 0.9 (SEM) eye wipes; n=3; t-test, p=0.002). Paradoxically, abraded corneas from the 24 hour time point had dramatically reduced corneal fiber density. One week post-injury, rats tested on the injured side (9.3 ± 0.5 (SEM) eye wipes; n=4) did not show any significant differences from rats tested on the uninjured side (9.0 ± 2.3 (SEM) eye wipes, n=4). While corneal fiber density showed evidence of recovery at one week, it was reduced compared to uninjured corneas. Conclusions: Abrasion injury reduces corneal fiber density; however this reduction does not appear to be directly correlated to behavioral responses to noxious corneal stimulation. We believe that the neurochemical content of corneal fibers will be a better predictor of pain sensitivity at the corneal surface than overall density. These findings will provide insights into the dynamics of pain transmission in the cornea and will deepen our understanding of the neuropathic changes that occur in corneal nerve fibers after injury. Commercial Relationships: Sue Aicher; Clayton Hudson, None; Sam Hermes, None; Deborah Hegarty, None Support: OHSU Presidential Bridge Funding Program Number: 1289 Poster Board Number: D0237 Presentation Time: 3:15 PM–5:00 PM Modulation of corneal wound healing in the mouse using pre-surgical fasting Matthew Petroll, Pouriska Kivanany. Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas, TX. Purpose: Long-term dietary restriction (DR), defined as reduced food intake without malnutrition, has been shown to extend lifespan in rodents, as well as provide increased resistance to many forms of acute stress. Long-term DR has also been shown to alter proliferation and fibrosis during dermal wound healing. Increased longevity has been shown to require long-term DR. However, recent studies have established that short-term, water only fasting is sufficient to promote many stress resistance responses. In this study, we investigate the effect of short-term fasting on corneal wound healing in the mouse. Methods: A transcorneal freeze injury (FI) model was used on ten C57/BL6 mice. Animals were either fed ad libitum (5 mice) or underwent 2 days of water only fasting (5 mice) prior to receiving a 1 mm diameter FI in the center of the left eye. After FI, all mice were fed ad libitum. Wound healing was assessed from 3 to 28 days after injury using 3-D in vivo confocal microscopy with a custom modified HRT-Rostock Corneal Module. At 7 days, a subset of corneas was fixed in situ, labeled for f-actin and nuclei, and imaged using confocal fluorescence microscopy. Results: The epithelium was resurfaced at 3 days after FI in both fasted and non-fasted animals. In animals fed ad libitum, activated stromal cells were observed in the anterior stroma 3 days after FI. By 7 days, activated cells were present throughout the full thickness of the corneal stroma. Short-term fasting inhibited the initial wound healing response, as indicated by a lack of activated stromal cells 3 days after injury. At 7 days, stromal cell activation appeared similar between fasted and non-fasted animals. F-actin labeling confirmed that stromal cells expressed intracellular stress fibers at 7 days after FI in both fasted and non-fasted animals. Stromal cell activation was reduced at 14 and 21 days after injury in both fasted and non-fasted animals. Conclusions: These initial results suggest that short term fasting reduces and/or delays the initial corneal stromal wound healing response in the mouse. We hypothesize that the transient nature of this response is due to the fact that the mice were refed at the time of injury. We hypothesize that by using DR both before and after injury, a sustained impact on wound healing could be achieved. Commercial Relationships: Matthew Petroll, None; Pouriska Kivanany, None Support: NIH Grants R21 EY025057 and P30 EY020799, and Research to Prevent Blindness, Inc. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1290 Poster Board Number: D0238 Presentation Time: 3:15 PM–5:00 PM Inhibitor of Differentiation Gene: Expression, Localization and Role in Human Cornea Rajiv R. Mohan1, 2, Brandie R Morgan1, 3, Govindaraj Anumanthan1, 3, Ajay Sharma1, 3, Shyam Chaurasia3, Prashant R. Sinha1, 3, Frank G. Rieger1. 1Harry S. Truman Veterans Hospital, Columbia, MO; 2Veterinary Medicine & Surgery, Biomedical Sciences, Veterinary Pathology, and Mason Eye Institute, University of Missouri, Columbia, MO; 3Veterinary Medicine & Surgery, University of Missouri, Columbia, MO. Purpose: Inhibitor of differentiation (Id) genes are DNA-binding transcription factors and shown to regulate cell proliferation, migration, angiogenesis and fibrosis. The expression and role Id genes in the cornea are still unknown. We sought to characterize the expression of Id proteins and their interactions with the profibrotic cytokine transforming growth factor β1 (TGFβ1) and anti-fibrotic cytokine bone morphogenic protein 7 (BMP7) in human cornea. Methods: Human donor corneas procured from Eye Bank were used. Id proteins were localized in human corneal sections using immunofluorescence. Primary cultures of human corneal fibroblasts (HCF) were established and treated with either TGFβ1 (5ng/ml) or BMP7 (10ng/ml) for 24 hrs in serum free medium. Expression of Id’s in response to TGFβ1, BMP7 and TGFβ1+BMP7 was measured with qPCR and immunoblotting. Results: All three major cell types of human cornea expressed Id1-4 genes. Id1 and Id2 proteins were ubiquitously expressed in epithelial cells and stromal keratocytes in the human cornea with Id1 predominantly in basal epithelial cells. The Ids were differentially regulated with TGFβ1 and BMP7 in a time dependent manner. Id proteins are exclusively expressed in human corneal fibroblasts but not in human corneal myofibroblasts. TGFβ1 and BMP7 regulated Id gene expression at different time points. Treatment of TGFβ1 to HCF showed a significant increase in Id1, Id2 and Id4 at earlier time point (2h; Id1, p<0.001; Id2, p<0.01 and Id4, p<0.001) followed by a decrease in mRNA expression at late time points (12h, 24h, and 48h). Contrary to this, BMP7 treatment showed a gradual increase in the expression of Id1 (24h p<0.005; 48h p<0.001), Id2 (24h p<0.01) and Id3 (48h p<0.001) in a time-dependent manner. Combined treatment of TGFβ1+BMP7 to HCFs showed a significant increase in Id1, Id3 and Id4 expression. Conclusions: Id genes are expressed in human cornea and play vital role in corneal wound healing and fibrosis development by regulating TGFβ1 and BMP7 activities. Id genes can be a target for restoring and maintaining corneal transparency. Commercial Relationships: Rajiv R. Mohan; Brandie R Morgan, None; Govindaraj Anumanthan, None; Ajay Sharma, None; Shyam Chaurasia, None; Prashant R. Sinha, None; Frank G. Rieger, None Support: 2RO1EY17294 National Eye Institute, NIH, Bethesda and 1I01BX000357-05 Veteran Health Affairs, Washington DC Program Number: 1291 Poster Board Number: D0239 Presentation Time: 3:15 PM–5:00 PM The Therapeutic Utility of Decorin Eye Drops for the Prevention of Corneal Scarring Maryam Esmaeili1, Zubair Ahmed1, Saaeha Rauz2, Liam M. Grover3, Ann Logan1. 1Neurotrauma, University of Birmingham, Birmingham, United Kingdom; 2Opthalmology, University of Birmingham, Birmingham, United Kingdom; 3Biochemical Engineering, University of Birmingham, Birmingham, United Kingdom. Purpose: Corneal opacity is a leading cause of worldwide blindness. The availability of drugs to treat established corneal scars or the prevention of scar formation following trauma, is limited. We have previously shown that Decorin, a naturally occurring antagonist of TGF-beta, creates a permissive microenvironment that enables healing with reduction of pro-fibrotic cascades. In this study, we examined the use of Decorin eye drops as an early stage intervention to reduce visually significant corneal scar formation in a corneal injury animal model. Methods: Decorin was delivered as a single 40µL drop onto the corneal surface of anaesthetised adult Sprague-Dawley rats and penetration into the cornea and aqueous humour (AqH) was assessed at 0, 30, 60 min by immunohistochemistry and ELISA, respectively. Eyedrop deliverable levels of Decorin were compared with those present endogenously in human AqH. An ex-vivo rat corneal-scarring alkali burn model was established by exposing the central cornea to filter paper saturated with 0.5M NaOH to varying time points (15, 30 and 60 s) followed by histological analysis of tissue for corneal scarring. The topical application of Decorin was evaluated. Results: Decorin penetrated the rat cornea into the anterior segment of the eye. While Decorin levels in corneal tissue increased over time, concentrations in the AqH decreased (10 min, 12600pg/ mL (±3382pg/mL (SEM); n=3) to 60 min, 4600pg/mL (±3494pg/ mL (SEM); n=3)). This level was comparable to physiological concentrations of Decorin in human AqH (1904pg/mL (± 360pg/ mL (SEM); n=10). Topical Decorin is able to prevent corneal scar formation in our ex vivo rat corneal-alkali injury model compared to eyes that were not treated with Decorin. Conclusions: Topical application of Decorin is effective in reducing corneal scar formation during acute alkali injury and is likely to be therapeutically useful in the prevention of corneal scarring. Anteriorsegment penetration of Decorin may afford a novel anti-fibrotic strategy for intraocular scarring processes. Commercial Relationships: Maryam Esmaeili, None; Zubair Ahmed, None; Saaeha Rauz, None; Liam M. Grover, None; Ann Logan, None Support: Wellcome Trust These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Program Number: 1292 Poster Board Number: D0240 Presentation Time: 3:15 PM–5:00 PM Subbasal Axon Density is Reduced and Reinnervation After Injury Delayed in the Syndecan-1 Null Mouse Cornea Mary Ann Stepp, Gauri Tadvalkar, Raymond Hakh, Sonali Pal-Ghosh. Anatomy and Regenerative Biology, George Washington University, Washington, DC. Purpose: The heparan sulfate proteoglycan syndecan is required for proper growth cone formation in C. elegans; although syndecan-1 is upregulated in wounded sensory axons in the mouse, how loss of syndecan-1 impacts subbasal sensory nerve (SBN) innervation and reinnervation in the syndecan-1 null mouse cornea is not known. Methods: The axon density of SBNs was quantified in unwounded as well as 1.5 mm trephine-only and debridement wounded corneas in wt and sdc1 null mice from 1-28 days after injury. For each wound type and time point a minimum of 5 corneas were assessed. SBN density was quantified in whole mount corneas stained to localize β3 tubulin. Confocal images were acquired and axon density quantified using the Sholl analysis. Significance was assessed by ANOVA. Results: SBN axon density in the center and periphery of unwounded sdc1 null mice is reduced to 80% and 58% that of the control levels. The SBNs in trephine-only (crush) wounded sdc1 null corneas reinnervated slower over time than those in wt mice; in addition, after small wounds, SBNs reinnervated significantly slower in sdc1 null mice compared to wt mice after both wound types. Conclusions: In unwounded sdc1 null mice, SBN density is reduced compared to wt mice. However, the vortex is retained. The SBNs of sdc1 null mice reinnervate slower after trephine only and debridement wounds. Previous studies have shown that reepithelialization of corneal debridement wounds of sdc1 null mice is slower and that they develop fewer recurrent erosions. These results show that sdc1 homologs (sdc2, sdc3, and sdc4) in the mouse likely compensate for the loss of sdc1 in the development and localization of SBNs in the mouse cornea. Commercial Relationships: Mary Ann Stepp; Gauri Tadvalkar, None; Raymond Hakh, None; Sonali Pal-Ghosh, None Support: EY08512, EY021784, and EY023106 Program Number: 1293 Poster Board Number: D0241 Presentation Time: 3:15 PM–5:00 PM Particulated extracellular matrix as immunomodulators for corneal wound healing Hongbo Yin1, 2, Qiaozhi Lu2, Xiaokun Wang2, Jennifer Elisseeff2. 1 Department of Opthalmology, West China hospital, Baltimore, MD; 2Translational Tissue Engineering Center, Wilmer eye institute, Baltimore, MD. Purpose: Extracellular matrix (ECM) is essential during wound repair, as it provides the structural integrity and regulates cellular functions via the actions of cytokines and growth factors. Corneal wound healing is a complex process that involves ECM remodeling and immunomodulation. Here we reported the effects of administering particulated decellularized ECM (pdECM) from porcine lymph nodes in reducing corneal scarring after manual superficial keratectomy (MSK). Methods: Porcine lymph nodes were decellularized, lyophilized and cryomilled to obtain microparticles. Young white rabbits were used as the animal model and fibrin glue (FG) was administered on wounded corneas as the standard treatment. For each rabbit, MSK was performed on one eye to remove 150 μm of anterior cornea and the other cornea was left unwounded as the healthy control. The animals were divided into three groups: FG, FG encapsulated with pdECM and no treatment. Photos of healthy and wounded corneal were taken regularly to examine the epithelial healing and cornea haze. Corneal samples were harvested at both one week and one month. Real-time PCR and immunohistochemistry were performed to evaluate the wound healing outcomes and scar formation. Results: The reepithelialization was complete within 5 days in all three groups. Treated groups had significantly reduced corneal haze scores compared to untreated corneas, and the addition of pdECM had even better effect than FG alone. pdECM was able to even further decrease the expression of both inflammatory factors (MMP9 and TNFα) at early stage (one week) and fibrosis-related genes (collagen I, TGFβ1 and CTGF) at the later stage (one month) compared to the FG group. pdECM could better maintain the corneal thickness and the normal morphology of epithelium as seen in H&E staining as well. Immunohistochemistry with CD11b demonstrated fewer macrophages infiltrations with pdECM, which also significantly downregulated the αSMA expression in regenerated corneal stroma. Conclusions: pdECM is promising in modulating corneal wound healing after MSK via reducing the expression of proinflammatory cytokines and regulating the migration of immune cells during the wound healing process. pdECMplay a key role in modulating the regenerative process and is potentially a new therapeutic in affiliating the scarlesshealing after corneal surgery. Commercial Relationships: Hongbo Yin; Qiaozhi Lu, None; Xiaokun Wang, None; Jennifer Elisseeff, None Program Number: 1294 Poster Board Number: D0242 Presentation Time: 3:15 PM–5:00 PM Chemical burn induced stromal demarcation line Koby Brosh. ophthalmology, Shaare zedek medical center, Jerusalem, Israel. Purpose: A stromal demarcation line is a well known sign after collagen cross-linking (CXL)1-5. It has been proposed that this line is the transition zone between cellular and acellular stroma and thus might reveal the depth of photochemical changes in the corneal stroma3-4. We report two cases of a similar demarcation line following chemical alkali burns. To the best of our knowledge, this is the first report of a stromal demarcation line following a chemical burn. Methods: Two cases of chemical burn induced stromal demarcation line are presented. Frequent AS-OCT follow-up was used in order to demonstrate the demarcation line and its characteristics. Results: Two patients arrived at the emergency room following an ocular alkali burn. At presentation both had total corneal erosion, corneal edema and limbal ischemia. Twelve to fifteen days later, a stromal line was apparent by both slit lamp examination and anterior segment OCT (AS-OCT). The stromal demarcation lines disappeared approximately three months after the injury. Conclusions: A stromal demarcation line may appear not only after a CXL but also after a chemical burn. The line depth may be associated with the severity of the injury, and therefore, may have prognostic significance. Patients with chemical burns should be examined for evidence of a stromal line in the corneal stroma. Stromal demarcation line (white arrows) and CCT demonstrated by horizontal OCT through the midline of the right cornea of patient 1. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Stromal demarcation line (white arrows) and CCT demonstrated by horizontal OCT through the midline of the cornea (OD) of patient 2. Commercial Relationships: koby Brosh, None These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record.