This grant is requested to continue studies of oxidative brain injury mediated by carbon monoxide (CO) that were begun four and one-half years ago with funds provided by an R29 grant. CO poisoning is a major cause of both death and neurological morbidity world wide. Hypoxia mediated by carboxyhemoglobin does not adequately explain the development of neuropathology. We have found that CO poisoning precipitates an oxidative stress in the brain due to a cascade of events involving both the brain microvasculature and circulating blood elements. The studies proposed in this grant will investigate this cascade. We hypothesize that microvascular oxidative stress occurs initially because of peroxynitrite produced by a reaction between the superoxide radicals that are normally released by mitochondria, and nitric oxide liberated by platelets after exposure to CO. This leads to phospholipase activation in endothelial cells. Subsequent cyclooxygenase activity causes an acceleration in the oxidative stress, which stimulates P-selectin expression. Cerebral hypoperfusion, which occurs with continued CO exposure, causes hypoxia and CO binds to cytochrome oxidase. A large flux of superoxide radicals from mitochondria occurs, and' radicals reaching the vascular lumen facilitate leukocyte adherence to P-selectin. Release, of excitatory amino acids causes neuronal nitric oxide production, which also contributes to the early oxidative stress. Leukocytes become activated, attach to the endothelium via B2 integrins, and leukocyte proteases cause endothelial cell xanthine dehydrogenase to be converted to oxidase. Reduced oxygen species, transition metals, and nitric oxide interact to cause brain lipid peroxidation, and days later neuronal dysfunction becomes apparent. Proposed studies of this hypothetical cascade will involve immunohistochemical methods for demonstrating peroxynitrite mediated oxidation and microvascular P-selectin expression, and biochemical assays of myeloperoxidase, xanthine dehydrogenase, xanthine oxidase, and lipid peroxidation. Electron paramagnetic resonance spectroscopy will be used to measure nitric oxide synthesis. We will also investigate coupling between brain blood flow and metabolic changes such as mitochondrial redox state using microscopic electrodes, reflectance fluorometry and laser doppler. Modification of leukocyte B2 integrin function occurs during CO poisoning, and also presents an approach for treating CO poisoning. Impaired cGMP synthesis will be investigated as a possible mechanism for inhibition of B2 integrin function caused by both nitric oxide and hyperbaric oxygen treatment of CO-poisoned rats.
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