lschemic tolerance is the genome's own endogenous protective response to ischemic stress. The goal of this application is to discover the genes and gene pathways regulating this protection and to do so with a goal of providing new therapeutic strategies for stroke. We will be guided by genes discovered in tolerance models developed in project 1 and genes further characterized as neuroprotective in vitro in project 2. We will then directly test Candidate gene products and candidate neuroprotective pathways in mice by use of routine models over-expressing a candidate gene, in knockouts or by altering the expression of the gene product of interest. We will use mouse lines available to us or create new models if required as we have done previously. Thus we plan to: 1) Determine whether increased expression or presence of candidate neuroprotective genes identified in Projects 1 and 2 using in vitro and in vivo models of preconditioning, improves neuronal survival in ischemia in vivo. 2) Determine whether decreased expression or inhibition of function of candidate neuroprotective genes, identified in Projects 1 and 2 using in vitro and in vivo models of preconditioning, reduces tolerance and neuronal survival in ischemia in vivo. 3) Determine which genes are altered in tolerance in the absence of neuroprotective genes to elucidate the function of novel genes in ischemic preconditioning. We will begin with a novel inflammatory pathway surrounding the osteopontin gene, which was revealed in our initial microarray screening. By a combined analysis of genes discovered in multiple models in vivo and in vitro in projects 1 and 2 our goal is to discover gene sets which share common promoter elements and reveal the common restricted pathways regulating tolerance to ischemia in brain. Knowledge of such pathways should be broadly relevant to injury states across organs and beyond ischemia.