Ischemic tolerance is a powerful cerebroprotective mechanism by which a sublethal injury protects from a subsequent lethal ischemic insult. Studies over the last funding period have revealed that the inducible or "immunological" isoform of nitric oxide synthase (iNOS; NOS II) is essential for the development of the ischemic tolerance induced by systemic administration of the proinflammatory mediator lipopolysaccharide. However, the mechanisms by which iNOS participates in ischemic preconditioning have not been defined. Preconditioning induces cellular and molecular changes that are thought to ultimately increase the resistance of brain pells to ischemia. However, preservation of brain tissue depends not only on protection of neurons and glia, but also on safeguarding cerebral blood vessels. Therefore, it is conceivable that both "cytoprotective" and "vasoprotective" mechanisms contribute to the development of ischemic tolerance. The goal of this renewal application is to test the hypothesis that INOS-derived NO contributes to ischemic tolerance by preserving post-ischemic vascular function (vasoprotection). as well as increasing the resistance of brain cells to ischemia (cvtoprotection). First, we will determine whether preconditioning with lipopolysaccharide leads to better preservation of cerebral perfusion after middle cerebral artery occlusion in mice (vasoprotection). Second, we will test the hypothesis that iNOS-derived NO is responsible for the vasoprotective component of preconditioning. Third, we will use a model of NMDA-mediated neocortical injury to test the hypothesis that iNOS is also involved in the tolerance to excitotoxicity induced by lipopolysaccharide (cytoprotection). Fourth, considering that NO exerts many of its actions through peroxynitrite, its reaction product with superoxide, we will test the hypothesis that reactive oxygen species produced by NADPH oxidase are needed for the cytoprotective and vasoprotective effects of iNOS-derived NO. The proposed studies will provide insight into poorly understood aspects of the mechanisms of ischemic preconditioning that may have important implications for the prevention and treatment of ischemic stroke.