Recent studies have demonstrated that oxygen radicals such as superoxide, hydroxyl and nitric oxide are involved in neuronal cell death following cerebral ischemia and reperfusion. Other studies have demonstrated that mitochondrial dysfunction may play a major role in determining neuronal death through the apoptosis pathways after cerebral ischemia and reperfusion. Numerous cell culture studies have suggested that cytochrome C release from mitochondria initiates a cell death program through apoptosis. We have demonstrated that mitochondrial release of cytocbrome c (but not cytochrome oxidase) occurs in neurons prior to DNA fragmentation after transient focal cerebral ischemia. We also demonstrated that cytochrome C release is associated with oxidative stress, since its release is exacerbated in mice that are deficient in mitochondrial manganese superoxide dismutase (MnSOD [SOD2]), whereas cytochrome C release is significantly reduced in mice that over express cytosolic human copper/zinc (CuZn) SOD (SOD 1) activity. However, it is not clear whether oxidative stress affects upstream (signaling) pathways of cytochrome C release and downstream caspase(s) activation following transient focal cerebral ischemia. Our hypothesis is that oxidative stress induced by cerebral ischemia and reperfusion is involved in neuronal cell death through a """"""""mitochondrial cytochrome C-dependent neuronal apoptosis pathway,"""""""" that is initiated by cytochrome C release followed by activation of caspases and subsequent DNA fragmentation. It is our aim to test this hypothesis using transgenic (Tg), knockout and double Tgf knockout mice.
Our Specific Aims are: 1) To elucidate the role of excitotoxicity in oxidative signaling of mitochondrial cytochrome C release after transient focal cerebral ischemia in mice. 2) To elucidate the role of nitric oxide on oxidative signaling in cytochrome C release and subsequent neuronal apoptosis after cerebral ischemia and reperfusion. 3) To elucidate the role of oxidative stress on mitochondrial dysfunction in cytochrome C-dependent neuronal apoptosis after cerebral ischemia and reperfusion. 4) To elucidate the interplay and compartmentalization of oxidative stress on cytochrome C release, pro- and antiapoptotic protein expression and caspase activation in double Tgf knockout mice after cerebral ischemia and reperfusion. We believe these are unique and fresh approaches that will provide insights into the oxidative mechanisms involved in the pathogenesis of necrosis and apoptosis following cerebral ischemia and reperfusion.
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