Nitric oxide (NO) is a radical gas that is both a molecular mediator and a cytotoxin. Recent data indicate that NO may be involved in cerebral ischemic damage. NO is synthesized by constitutive endothelial or neuronal nitric oxide synthase (cNOS) and by an inducible NOS (iNOS) expressed in selected cells during inflammation. Because iNOS produces a large amount of NO, iNOS induction is thought to be responsible for cytotoxicity in many cell systems and could participate in the mechanisms of cerebral ischemia. In the present application the investigator proposes to test the hypothesis that iNOS is induced after cerebral ischemia and that sustained NO production by iNOS contributes to the late stages of tissue damage. Molecular and cellular biological techniques will be used, in conjunction with methods for assessing tissue outcome, to study iNOS induction after cerebral ischemia and to begin to define its pathogenic role. Focal cerebral ischemia will be produced by middle cerebral artery occlusion in rats or mice. In the first aim, it will be determined whether iNOS enzymatic activity is induced in the ischemic brain. In the second aim, the molecular basis for the induction of iNOS activity will be studied. In particular, reverse transcription-polymerase chain reaction will be used to determine whether iNOS mRNA is expressed after ischemia. In the third aim, the cell type in which iNOS protein is expressed and the topography of iNOS expression will be determined. In the fourth aim, it will be established whether iNOS induction contributes to cerebral ischemic damage. First, it will be determined whether aminoguanidine, a relatively selective iNOS inhibitor, reduces ischemic damage. Second, antisense oligonucleotides will be used to determine whether downregulation of iNOS protein expression reduces infarct size. In the fifth aim, the investigator will investigate the molecular mechanisms of iNOS induction. Knockout mice with a null mutation of the transcription factor interferon-gamma regulatory factor 1 (IRF-1) do not express iNOS during inflammation, indicating that IRF-1 is required for iNOS expression. These mutant mice will be used to test the hypothesis that IRF-1 is also required for iNOS expression in cerebral ischemia. These studies will explore aspects of the pathophysiology of cerebral ischemia that have not thus far been investigated. Preliminary data indicate that this is a most promising area of research that may lead to new avenues for the treatment of cerebral ischemia.
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