Nitrosative Stress in the Rat Retina at the Onset of Streptozotocin-Induced Diabetes.

BACKGROUND/AIMS: Nitric oxide is a multifunctional molecule that can modify proteins via nitrosylation; it can also initiate signaling cascades through the activation of soluble guanylate cyclase. Diabetic retinopathy is the leading cause of blindness, but its pathogenesis is unknown. Multiple mechanisms including oxidative-nitrosative stress have been implicated. Our main goal was to find significant changes in nitric oxide (NO) levels and determine their association with nitrosative stress in the rat retina at the onset of diabetes. METHODS: Diabetes was induced by a single intraperitoneal administration of streptozotocin. The possible nitric oxide effects on the rat retina were evaluated by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a specific marker for NO-producing neurons, detected by histochemistry performed on whole retinas and retina sections. Immunohistochemistry was also performed on retina sections for iNOS, 3-nitrotyrosine (3-NT) and glial fibrillary acidic protein (GFAP). Retinal nitric oxide levels were assessed by measuring total nitrate/nitrite concentrations. Retinal cGMP levels were determined by radioimmunoassay. Western blots for nitrotyrosine (3-NT) and oxidized proteins were performed. RESULTS: In the present study, we found increased activity of NADPH-diaphorase and iNOS immunoreactivity in the rat retina at the onset of diabetes; this increase correlated with a remarkable increase in NO levels as early as 7 days after the onset of diabetes. However, cGMP levels were not modified by diabetes, suggesting that NO did not activate its signaling cascade. Even so, Western blots revealed a progressive increase in nitrated proteins at 7 days after diabetes induction. Likewise, positive nitrotyrosine immunolabeling was observed in the photoreceptor layer, ganglion cell layer, inner nuclear layer and some Müller cell processes in the retinas of diabetic rats. In addition, levels of oxidized proteins were increased in the retina early after diabetes induction; these levels were reduced by the administration of L-NAME. In addition, stress in Müller cells was determined by immunoreactivity to the glial fibrillary acidic protein. CONCLUSIONS: Our findings indicated the occurrence of nitrosative stress at the onset of diabetes in the rat retina and emphasized the role of NO in retinal function and the pathogenesis of retinopathy.

Potential Role of Endoplasmic Reticulum Stress in Pathogenesis of Diabetic Retinopathy.

Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Multiple biochemical abnormalities have been proposed to explain the development of retinopathy, including oxidative stress. Although the existence of oxidative stress has been established in the retina from long standing diabetic animals, pathogenesis and progression of retinopathy remain unclear. In order to gain insight into the pathogenesis of diabetic retinopathy, we analyzed the levels of different oxidative stress biomarkers in the retina at early stages during the progress of streptozotocin-induced diabetes. No significant changes in glutathione content, expression of NADPH-oxidase, levels of lipid peroxidation, nor production of free radicals were observed in the retina up to 45 days of diabetes induction. Likewise, a transient decrease in aconitase activity, parallel to an increase in the superoxide dismutase activity was observed at 20 days of hyperglycemia, suggesting a high capacity of retina to maintain its redox homeostasis, at least at early stages of diabetes. Nonetheless, we found an early and time-dependent increase in the levels of oxidized proteins, which was not affected by the administration of the antioxidant quercetin. Also, positive immunoreactivity to the reticulum stress protein CHOP was found in glial Müller cells of diabetic rat retinas. These findings suggest the occurrence of endoplasmic reticulum stress as a primary event in retina pathogenesis in diabetes.