Targeted Mitochondrial Uncoupling in Focal Cerebral Ischemia

Mounting evidence indicates that the disruption of mitochondrial function is a critical event in the pathway leading to ischemic-reperfusion injury. Strategies directed at stabilizing mitochondria following ischemia-reperfusion are therefore being actively pursued. Mitochondrial uncouplers are a class of compounds that, by causing a "leak" of protons back into the matrix, lead to a drop in the mitochondrial membrane potential. Our preliminary data indicate that animals exposed to one such uncoupler develop 40% smaller infarct volumes, even when the compound is administered after one hour of reperfusion. Although the mechanism is not known, the predicted reduction in mitochondrial membrane potential could decrease the formation of damaging reactive oxygen species and reduce the uptake of potentially toxic levels of calcium, both which may lead to apoptosis. In this proposal, we pursue the following three specific aims: Specific Aim 1 - To determine if mitochondrial uncoupling stabilizes mitochondrial function following reversible focal cerebral ischemia. Specific Aim 2 - To determine if mitochondrial uncoupling attenuates apoptotic-related events following reversible focal cerebral ischemia. Specific Aim 3 - To determine if administration of mitochondrial uncoupling confers a long-term improvement in the functional outcome in rats undergoing reversible focal cerebral ischemia. To accomplish this we will perform oxygen consumption assays as a direct measure of mitochondrial function and fluorescent probe assays to quantify mitochondrial reactive oxygen species formation and calcium levels. We will also measure several parameters of apoptosis including cytochrome c release and activation of caspases-3 and -9. Lastly, using the Morris Water Maze and Beam Walk, we will examine the long-term functional outcome in animals treated with uncouplers following ischemia-reperfusion. We hypothesize that exposure to non-toxic doses of uncouplers will ameliorate the neuronal and behavioral sequelae of reversible cerebral ischemia through a mechanism involving preservation of mitochondrial function.