In primary neuronal cultures, N-methyl-D-aspartate (NMDA) neurotoxicity, and in vitro model of cerebral ischemia, is mediated, in part, by nitric oxide (NO). Despite numerous studies implicating NO in glutamate mediated neurotoxicity, there is evidence that NO may play no role in NMDA neurotoxicity and may in fact be neuroprotective. NO may be neuroprotective by nitrosylating and inactivating the NMDA receptor. NO s double edge sword may be related to it s redox state, with the NO radical being toxic and the nitrosonium ion being neuroprotective. In order to better understand the role of NO in a variety of physiologic processes in which NO has been implicated to regulate including glutamate neurotoxicity, the applicants have generated mice carrying a selective mutation in the neuronal NOS gene by targeted deletion in embryonic stem cells. Utilizing the NOS knockout mice and their wild type controls, a series of experiments are proposed to clarify the potential mechanisms and involvement of NO in neurotoxicity and to identify alternative pathways of toxicity. Neuronal damage due to cerebral ischemia may occur through excess NO production. Combined oxygen-glucose deprivation in neuronal cultures as well as excitatory amino acid administration will be used as in vitro model of cerebral ischemia. The susceptibility to neuronal injury will be evaluated in neuronal NOS knockouts and compared to wild type controls. Experiments will be performed to determine whether targeted deletion of neuronal NOS leads to additional changes that might account for the resistance to glutamate mediated toxicity. The distribution and density of glutamate receptors will be evaluated. 45Ca2+ accumulation will be examined in response to excitatory amino acids in neuronal NOS knockouts and compared to wild type controls. NADPH diaphorase or NOS neurons are resistant to NMDA type neurotoxicity and are highly susceptible to kainate and quisqualate neurotoxicity. NOS and NADPH diaphorase neuronal colocalize with somatostatin and NPY. The NOS knockouts possess normal NPY and somatostatin neurons. As such the susceptibility of these neurons to NMDA, kainate and quisqualate will be examined in NOS knockouts versus wild type controls to determine whether the susceptibility of these neurons is altered in NOS knockouts. Additionally, the distribution of glutamate receptors will be examined on NOS neurons to evaluate whether the differential expression of glutamate receptors accounts for differential susceptibility to toxicity. Other potential pathways of neurotoxicity exist as NOS inhibitors are only partially protective. The neuronal NOS knockouts provide a unique opportunity to examine other potential pathways of glutamate neurotoxicity without the influence of NO. As such, glutamate neurotoxicity and oxygen-glucose deprivation in neuronal cultures will be examined in the neuronal NOS knockouts and compared to wild type controls after the administration of a variety of inhibitors of other potential pathways of neurotoxicity. These agents will include scavengers of the superoxide anion, and free radicals. In addition, the effects of phospholipase A2 inhibitors, cyclooxygenase inhibitors, and lipoxygenase inhibitors will be examined.
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