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N.A. Rao, S. Ito, G.S. Wu; Peroxynitrite Generation by Resident Retinal Microglia in EAU . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4591.
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Purpose: In intraocular inflammation, retinal microglia share virtually all the surface markers with activated macrophages. However, the former can be precisely identified in-vivo by labeling with 4Di-10ASP after optic nerve axotomy. We evaluated such labeled microglia for generation of oxidant, peroxynitrite (ONOO-) at various times during the development of experimental autoimmune uveo-retinitis (EAU). Methods: Twenty-four Lewis rats were axotomized and 4 Di-10ASP was applied on the cut stump of the optic nerve. At day 28 postaxotomy, all rats were immunized with S-antigen peptide to induce EAU. Six animals each were killed on days 6, 8, 9 and 10 after the peptide injection. The enucleated globes were divided into two portions for ex-vivo retinal culture and cell isolation. Both retinal tissue and isolated cells were exposed to 50 µM dihydrorhodamine 123 in media supplemented with B27. After incubation, 14 µ sections were obtained from retinal tissue. Oxidized dihydrorhodamine, indicative of intracellular generation of ONOO- within the dye labeled cells was visualized under the confocal microscope. Also a group of 12 axotomized rats were injected with the peptide and three animals each were killed on days 6, 8, 9 and 10 post injection. Enucleated eyes were examined histologically for development of EAU. Results: Dihydrorhodamine oxidation to fluorescent rhodamine was seen by day 8 post-immunization in the dye labeled microglia. At day 9, 85% of the cells were positive for fluorescent rhodamine, and at day 10, 100% were positive. No rhodamine fluorescence was seen at day 6 post-immunization. Histologically, day 10 post S-antigen injection, eyes showed early signs of EAU. No EAU was observed in enucleated eyes of days 6 through 9 post-injection. Conclusions: Generation of ONOO- by the retinal microglia on days 8 and 9 post-immunization indicates that microglia are activated early in the phase of uveitis development and they may play a role in initiation of retinal damage leading to amplification of uveo-retinitis.
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