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E Ellis, MB Grant; Inducible Nitric Oxide Synthase is Critical to Peroxynitrite/Nitrotyrosine Formation in a Galactose Induced Model of Diabetic Retinopathy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1346.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Altered endothelial production and/or bioavailability of nitric oxide (NO) results in endothelial cell dysfunction and is associated diabetes blood retinal barrier (DBRB) leakage. Inducible NOS (iNOS) and endothelial NOS (eNOS) are the major isoforms observed in vasculature and increased iNOS expression is observed in diabetic microvasculature. NO availability, however, may be modified by oxidative inactivation by excess superoxide in the vascular wall, also observed in diabetes. Peroxynitrite, a toxic reaction product of NO and superoxide, is a useful marker of oxidative stress. Utilizing eNOS and iNOS deficient mice made galactosemic by long-term administration of galactose to mimic diabetic retinopathy, we tested the hypothesis that the absence of iNOS in retinal vasculature protects against BRB dysfunction. Methods: Male wild type (C57BL/6J), eNOS-/-, and iNOS-/- mice were fed either a normal rodent diet or a diet supplemented with 30% galactose for 1-3 months. Retinas were reacted for NADH oxidase, the major source of superoxide in vascular endothelium, using cerium based cytochemical methodology. Nitrotyrosine, a marker for peroxynitrite, was localized on sections of retina using colloidal gold immunocytochemical localization. Sections were examined by transmission electron microscopy. All vessels in each section of retina were scored either positive or negative for NADH oxidase. Nitrotyrosine was quantified in 20 vessels from each eye for each mouse strain and rodent diet by counting colloidal gold particles in a standardized area. Results: Increased levels of NADH oxidase was observed in retinas of all mice fed the galactose diet (P<0.05 for all groups). Levels of nitrotyrosine immunoreactivity were elevated in retinas of wild type (3-fold P<0.01) and eNOS-/- mice (3.5- fold, P<0.01) fed the galactose diet compared to control-fed mice. iNOS-/- mice fed galactose showed low levels of nitrotyrosine, similar to control fed animals despite demonstrating increased NADH activity, and were free of BRB dysfunction. Conclusion: The data presented here support that excess NO produced by iNOS, not superoxide, is the critical component in determining bioavailability of NO in diabetic retinopathy and associated retinal pathology.
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