May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Peroxynitrite Generation by Resident Retinal Microglia in EAU
Author Affiliations & Notes
  • N.A. Rao
    Department of Ophthalmology, Doheny Eye Institute, Los Angeles, CA, United States
  • S. Ito
    Department of Ophthalmology, Doheny Eye Institute, Los Angeles, CA, United States
  • G.S. Wu
    Department of Ophthalmology, Doheny Eye Institute, Los Angeles, CA, United States
  • Footnotes
    Commercial Relationships  N.A. Rao, None; S. Ito, None; G.S. Wu, None.
  • Footnotes
    Support  NIH Grant EY13253
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4591. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : 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.

Keywords: chorioretinitis • oxidation/oxidative or free radical damage • retina 
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