Role of endothelial nitric oxide generation and peroxynitrite formation inreperfusion injury after focal cerebral ischemia

Background and Purpose-Reperfusion injury is one of the factors that unfavo rably affects stroke outcome and shortens the window of opportunity for thr ombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfu sion have been demonstrated. Concomitant generation of these radicals can l ead to formation of the strong oxidant peroxynitrite during reperfusion. Methods-We have examined the role of NO generation and peroxynitrite format ion on reperfusion injury in a mouse model of middle cerebral artery occlus ion (2 hours) and reperfusion (22 hours). The infarct volume was assessed b y 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permea bility was evaluated by Evans blue extravasation. Nitrotyrosine formation a nd matrix metalloproteinase-9 expression were detected by immunohistochemis try. Results-Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N-omega-nitro- L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7 -nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interesti ngly, prereperfusion administration of both NOS inhibitors decreased tyrosi ne nitration (a marker of peroxynitrite toxicity) in the ischemic area. LNA treatment also significantly reduced vascular damage, as indicated by decr eased Evans blue extravasation and matrix metalloproteinase-9 expression. Conclusions-These data support the hypothesis that in addition to the detri mental action of NO formed by neuronal NOS during ischemia, NO generation a t reperfusion plays a significant role in reperfusion injury, possibly thro ugh peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect a gainst reperfusion injury suggests that the source of NO is the cerebrovasc ular compartment.