Our proposed study will delineate the roles of tissue oxygenation (pO2) and specific free radicals in the pathophysiology of cerebral ischemia and reperfusion. We have developed techniques to measure tissue pO2 and generation of free radicals in vivo, as a function of time in specific focal regions of the brain, using Electron Paramagnetic Resonance (EPR) spectroscopy. Using a middle cerebral artery occlusion (MCAO) model of ischemic stroke in the rat, we have obtained preliminary results showing that decreased pO2 after MCAO leads to a dramatic and unexpected increase in generation of free radicals. Normobaric hyperoxia treatment immediately after an MCAO not only increased tissue pO2, but also decreased free radical generation, contrary to common expectation. Furthermore, hyperoxia treatment during ischemia reduces infarction volume and improves the neurological function of the animal. These results demonstrate that studying the effects of tissue pO2 on free radical generation, and the resulting molecular responses, will be critically important in understanding the molecular events in free radical-induced brain injury, and in developing effective oxygen-based treatment strategies for ischemic stroke. We hypothesize that low levels of localized tissue pO2 during focal cerebral ischemia result in an increased generation of free radicals, which in turn activates deleterious molecular events, including the activation of matrix metalloproteinase (MMP) and the caspase cascade, leading to microvascular damage and cell death. To test this hypothesis, we will: 1) Determine the effect of hyperoxia treatment on tissue pO2 and free radical generation in the ischemic core, penumbra, and control areas in the MCAO model of ischemic stroke in the rat. 2) Determine the effect of hyperoxia treatment on the expression and activation of MMP-2, 3 and 9, and caspase-3, 8, and 9 following cerebral ischemia and reperfusion at the same locations where tissue pO2 and free radical generation are measured. 3) Determine the effects of increasing tissue pO2 with hyperoxia treatment on blood-brain barrier opening, neurological score, infarction volume, and edema. The proposed research will provide new insight into the mechanism of cerebral injury during ischemic stroke, and aid in the design of more effective neuroprotective strategies.
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