Neuronal nitric oxide synthase activation and peroxynitrite formation in ischemic stroke linked to neural damage

Nitric oxide (NO) is a new intercellular messenger that occurs naturally in the brain without causing overt toxicity. Yet, NO has been implicated as a mediator of cell death in cell death. One explanation is that ischemia cau ses overproduction of NO, allowing it to react with superoxide to form the potent oxidant peroxynitrite. To address this question, we used immunohisto chemistry for citrulline, a marker for NO synthase activity, and 3-nitrotyr osine, a marker for peroxynitrite formation, in mice subjected to reversibl e middle cerebral artery occlusion. We show that ischemia triggers a marked augmentation in citrulline immunoreactivity but more so in the peri-infarc t than the infarcted tissue. This increase is attributable to the activatio n of a large population (similar to 80%) of the neuronal isoform of NO synt hase (nNOS)that is catalytically inactive during basal conditions, indicati ng a tight regulation of physiological NO production in the brain. In contr ast, 3-nitrotyrosine immunoreactivity is restricted to the infarcted tissue and is not present in the peri-infarct tissue. In nNOS(Delta/Delta) mice, known to be protected against ischemia, no 3-nitrotyrosine immunoreactivity is detected. Our findings provide a cellular localization for nNOS activat ion in association with ischemic stroke and establish that NO is not likely a direct neurotoxin, whereas its conversion to peroxynitrite is associated with cell death.