CO POISONING IN THE CONTEXT OF A REPERFUSION INJURY

这笔资金是为了继续研究氧化性脑损伤而申请的。 由一氧化碳(CO)调节，开始于四年半 由29卢比的赠款提供的资金。一氧化碳中毒是一个主要原因 世界范围内的死亡和神经疾病。低氧由以下因素介导 碳氧血红蛋白不能很好地解释 神经病理学。我们已经发现，一氧化碳中毒会使一种氧化 由于涉及大脑和大脑的一系列事件而引起的大脑压力 微血管和循环血液成分。建议进行的研究 这笔拨款将调查这一连串的事件。我们假设 微血管氧化应激最初是由于过氧亚硝酸根引起的 由超氧自由基之间的反应产生，这些自由基通常是 线粒体释放，血小板释放一氧化氮 接触一氧化碳。这导致内皮细胞中磷脂酶的激活 细胞。随后的环氧合酶活性会导致细胞内 氧化应激，刺激P-选择素的表达。大脑 持续接触一氧化碳时出现的低灌注率会导致缺氧 CO与细胞色素氧化酶结合。大量的超氧自由基通量 从线粒体开始，自由基到达血管管腔 促进白细胞与P-选择素的黏附。兴奋性的释放 氨基酸引起神经元一氧化氮的产生，这也是 导致了早期的氧化应激。白细胞被激活， 通过B2整合素与内皮细胞附着，白细胞蛋白水解酶引起 内皮细胞黄嘌呤脱氢酶转化为氧化酶。 还原的氧物种、过渡金属和一氧化氮相互作用 导致大脑脂质过氧化，并在几天后导致神经元功能障碍 变得显而易见。对这一假设性级联的拟议研究将 应用免疫组织化学方法显示过氧亚硝酸根 介导氧化和微血管P-选择素表达，以及 髓过氧化物酶、黄嘌呤脱氢酶、黄嘌呤的生化测定 氧化酶和脂质过氧化作用。电子顺磁共振 光谱学将用于测量一氧化氮的合成。我们还将 研究脑血流和代谢变化之间的耦合 作为线粒体氧化还原状态的显微电极，反射率 荧光法和激光多谱勒。白细胞B2整合素的修饰 功能发生在一氧化碳中毒过程中，并提出了一种方法 治疗一氧化碳中毒。受损的cGMP合成将被调查为 B_2整合素功能受抑制的可能机制 一氧化氮和高压氧对一氧化碳中毒大鼠的治疗作用。

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.