E.B. Price 1,3 , P. Bu 2,3 , P. Sethupathi 5 , E.B. Stubbs, Jr. 2,3 , J.I. Perlman 1,3,4

1. INTRODUCTION Age-related macular degeneration (AMD) is the leading cause of visual impairment and blindness in developed countries. Approximately 1.7 million Americans have some form of AMD, and approximately 100,000 are blind from AMD. Presently, treatment for advanced AMD is very limited. The light-induced damage model is used to study the mechanism and treatment strategies for this disease. Studies have shown that the omentum, a pad of fatty tissue found in the abdomen, may have cells that help damaged tissues repair. We studied whether omental cell transplantation helps repair retinal damage by isolating and injecting these cells intraperitoneally into mice that had undergone light-induced retinal injury. SUMMARY CONCLUSIONS • Intense light exposure markedly reduced retinal function in BALB/c mice. • Intense light exposure also caused thinning of the outer retinal layers. • Injection of omental stromal cells provided some level of protection against light-induced retinal injury. Light-induced retinal injury causes a significant reduction in scotopic ERG responses. Although a significant difference was not noted on histological analysis, intraperitoneal injection of activated OSCs was found to partially protect against a net reduction of retinal function, as indicated by the differences in ERG amplitudes as compared to control eyes. These preliminary findings show that omental cell transplantation may possess therapeutic potential in the treatment of retinal disease. METHODS Electroretinography (ERG):Retinal function was evaluated in mice before and 6 days after retinal light exposure. Mice were dark-adapted overnight, anesthetized with ketamine (80mg/kg) and xylazine (8mg/kg) and their pupils were dilated. ERGs were recorded with a stainless steel wire electrode loop (LKC UTAS-E 3000) contacting the corneal surface through a layer of 1% methylcellulose, as we have previously reported (Bu 2010). Platinum needle electrodes placed in the cheek and tail served as reference and ground leads, respectively. A single-flash stimulus was presented in order of increasing luminance across a 4-log unit stimulus range (-0.00037 to +140 cd sec/m2) with an interstimulus interval of 1 minute in duration. The body temperature of anesthetized mice was maintained at 37 ºC with a heating pad. Isolation of omental cells from activated mice omentum: Male BALB/c albino mice were injected intraperitoneally with 2.5 ml of polydextran particle slurry (Biogel P–60, 120 μM; Biorad Laboratories, Richmond, CA, USA; 1:1 in PBS). At 1 week post-polydextran injection, the mice were euthanized by CO2 chamber. The activated omentums were harvested aseptically. Omental Stromal cells (OSCs) were isolated via collagenase I digestion. Immediately following light-induced retinal injury, mice were injected intraperitoneally with 4 x 106 OSCs or an equal volume of sterile saline. Light Exposure: Mice were exposed to 6000 lux of white light for 4 hours in a dedicated exposure device with circline fluorescent light bulbs. Before light exposure, mice were dark adapted for 12 hours. The pupils were dilated with 2.5% phenylephrine and 1% tropicamide just before light exposure. Histology and Morphometry: 6 days after retinal light exposure. anesthetized mice were sacrificed. Enucleated eyes were fixed in phosphate-buffered (pH 7.4) 2.5% glutaraldehyde-2% paraformaldehyde solution and embedded in epoxy resin. Retina sections (1 µm) were cut and stained with toluidine blue. REFERENCES Bu P, Basith B, Stubbs EB, Jr., Perlman JI. 2010. Granulocyte colony-stimulating factor facilitates recovery of retinal function following retinal ischemic injury. Exp Eye Res 91:104-6 Oishi A, Otani A, Sasahara M, Kojima H, Nakamura H, et al. 2008. Granulocyte colony-stimulating factor protects retinal photoreceptor cells against light-induced damage. Invest Ophthalmol Vis Sci 49:5629-35 Ritter MR, Banin E, Moreno SK, Aguilar E, Dorrell MI, Friedlander M. 2006. Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J Clin Invest 116:3266-76 Singh AK, Patel J, Litbarg NO, Gudehithlu KP, Sethupathi P, et al. 2008. Stromal cells cultured from omentum express pluripotent markers, produce high amounts of VEGF, and engraft to injured sites. Cell Tissue Res 332:81-8 Normal retina (no light) Light-exposed and vehicle-treated Light-exposed and OSCs-treated Activated Omental Stromal Cells Protect Against Light-Induced Retinal Injury E.B. Price1,3, P. Bu2,3, P. Sethupathi5, E.B. Stubbs, Jr.2,3, J.I. Perlman1,3,4 Surgery1 and Research Services2, Edward Hines, Jr. VA Hospital, Hines, IL; Departments of Ophthalmology3, Pathology4, and Microbiology and Immunology5, Loyola University Medical Center, Maywood, IL RESULTS Vehicle OSCs Pre Pre Post Post Figure 3. Photoinjury device Figure 1.Effect of omental stromal cell treatment on retinal function following light exposure. (Upper panels) ERG recordings from BALB/c mice treated with vehicle (0.9% saline) or OSCs (4 X 106) as indicated. Tracings shown were obtained prior to (pre) and 6 day after (post) light exposure. (Lower panels) Quantitative changes in ERG a- and b-wave amplitudes as indicated (n = 4 per group). Intraperitoneal injection of activated OSCs partially preserves retinal function following light exposure. Figure 2.Retinal morphology following light exposure. Shown are representative toluidine blue-stained retinal sections from naïve, vehicle-, and OSCs-treated (4 X 106) BALB/c mice. Light exposure produced a thinning of the outer retinal layers. No significance was found between treated and vehicle groups. Acknowledgements: This work was supported by Illinois Society for The Prevention of Blindness and The Richard A. Perritt Charitable Foundation. The authors have no commercial relationships to report.