| Glutamate is established as an important mediator of ischemic brain injury by mechanisms which remain poorly defined. Nitric oxide (NO) is produced following activation of NMDA receptors by glutamate and has been implicated as a downstream mediator of glutamate toxicity Mice deficient in neuronal NO synthase (nNOS Delta/Delta) have reduced infarcts following occlusion of the middle cerebral artery (MCAo), but it is unclear how NO, that occurs naturally in the brain for years without causing damage, becomes toxic after minutes of ischemia. One explanation is that ischemia. causes NO to react with superoxide to form the potent oxidant peroxynitrite. To address this, immunohistocheimstry for citrulline, a marker for no synthase activity and 3 nitrotyrosine, a marker for peroxynitrite formation, were employed in mice subjected to reversible MCAo. During basal conditions, only 25% of nNOS-containing neurons are citrulline immunoreactive (cit-IR), indicating a tight regulation of physiological NO production in the brain. Ischemia activates the remaining, "quiescent" nNOS-neurons to markedly increase the cit-IR in the infarcted tissue, but even more so in peri-infarct tissue. By contrast, 3-nitrotyrosine immunoreactivity is restricted to the infarcted we, tissue, and is not present in the peri-infarct tissue. In nNOSDelta/Delta mice, no 3-nitrotyrosine immunoreactivity is detected. Our findings provide a cellular localization for nNOS activation 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. How peroxynitrite causes cell death is unclear One candidate pathway is that peroxynitrite damages DNA leading to activation of the nuclear enzyme, poly (ADp-ribose) polymerase (PARP), which catalyzes attachment of ADP-ribose units from NAD to nuclear proteins following DNA damage. Excessive activation of PARP can deplete NAD and ATP leading to cell death by energy depletion. To study this pathway the response of PARP deficient mice to ischemia was studied. Cerebral cortical cultures from them mice am profoundly resistant to glutamate-NO-mediated ischemic insults in vitro and these mice display a marked reduction (80%) in infarct volume after reversible MCAo. These results provide compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia. |
