Peroxynitritc mediated oxidative injury in optic nerve ischemia
PURPOSE To investigate the role of peroxynitrite mediated oxidative injury in the pathogenesis of ocular ischemia. Peroxynitrite. formed by the reaction of Superoxide and nitric oxide, nitrates tyrosine. Nitrotyrosine can be localized in tissue specimens using antibodies against nitrotyrosine. MATERIAL and METHODS Ocular ishemia and reperfusion injury was induced in rats by ligation of the left internal and external carotid arteries. After 30 minutes the ligatures were removed. Animals were sacrificed 1 and 24 hours later. For detection of ischemia, the eyes and attached optic nerves were excised and immersed in 1% 2.3,5-triphenyltetrazolium chloride (TTC) for 30 minutes. The tissue was fixed in 4% paraformaldehyde overnight then processed for cryomicrotomy. Cryosections were immunolabeled with a rabbit polyclonal antibody against nitrotyrosine. followed by a goat antirabbit antibody conjugated to 5 nm colloidal gold, then silver enhanced. Controls consisted of the secondary antibody without the primary nitrotyrosine antibody and the primary nitrotyrosine antibody incubated with nitrotyrosine. Unstained sections were examined by light microscopy for double labeling with TTC and nitrotyrosine reaction products. RESULTS Nitrotyrosine inununogold heavily labeled the retina and optic nerve of the ischemic left eyes that also showed absence of TTC staining. In contrast, the nonischemic right eyes showed TTC staining of glial cells of the optic nerve and neuronal and glial cells of the retina, with some light imunogoid staining for nitrotyrosine. Controls with the primary antibody omitted from the immunostaining protocol showed no inununostaining. CONCLUSIONS Peroxynitrite may play a role in the pathogenesis of oxidative injury to ischémie optic nerve and retina.
Bhatti, MT; Guy, J; Greenwald, D; Day, AL; Ye, Z; Beckman, JS
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