The protective potential of three groups of genes will be studied in ischemia-like injury of brain injury of brain cells from cortex, hippocampus, and striatum, in primary culture. First an antioxidant strategy will be tested by over-expressing CuZn superoxide dismutase (SOD1) using herpes virus and adenoviral vectors to achieve rapid expression in neurons and astrocytes, and retroviral vectors for prolonged, stable expression in astrocytes. Whether acute expression can provide protection will be tested. If this is not protective, the effect of prolonged stable expression and the use of bicistronic vectors, to rapidly express both SOD and a downstream antioxidant vectors, to rapidly express both SOD and a downstream antioxidant enzyme, will be tested. We will test for protective effects, as well as for induction of other antioxidant enzymes. Whether protection correlates with induction of other antioxidant enzymes will be determined. Under conditions where protections seen the extent of oxygen radical production, lipid peroxidation and changes in level of glutathione will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined, as well as the time window in which expression can still protect, since it is important to develop therapeutic strategies that are effective after insults. Second, we will study the ability of Bcl-2 expression to protect in the same injury paradigms. Oxidative status and the time window in which Bcl-2 can protect will be determined. Third we will study the ability of the inducible heat shock protein 70 (HSP70) to protect from these injury again analyzing the time window during which this gene can protect and whether it blocks apoptotic or necrotic cell death. Primary cultures are particularly useful for analyzing mechanisms of ischemic brain injury and mechanisms of protection at the cellular level. Primary cultures of neurons and glial cells and pure astrocyte cultures will be mad3e from hippocampus and striatum. Results will be compared with parallel studies carried out on primary cultures from neocortex. In addition, astrocyte cultures will be produced from transgenic mice made with different gene dosages of SOD1 or MnSOD (SOD2), including knockouts lacking these genes, to determine the importance of these enzymes for astrocyte survival of ischemia-like insults. These studies will provide fresh insight into possible mechanisms of protection by three candidate genes for anti- ischemic gene therapy tested in three brain regions.
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