Molecular Mechanisms of Ischemia Reperfusion Brain Injury

描述（由申请人提供）：在心脏骤停（CA）和使用高氧和常氧通气复苏后，致死前神经化学变化与神经学结局和神经病理学的比较强烈暗示了线粒体蛋白的氧化修饰和延迟性、选择性神经细胞死亡病因学中的相关生物能量功能障碍。这些研究结果还质疑CA后患者不分青红皂白地使用100%吸入性O2（FiO 2），并建议修改现有复苏指南可能会显着改善神经功能结局。我们的主要目标是通过最大限度地减少氧化应激和最大限度地提高脑能量代谢来降低CA后的神经系统发病率和死亡率。我们的具体目标是测试以下假设，重点是年轻和老年动物的氧化性脑损伤的线粒体机制，并使用血氧饱和度为基础的调整FiO 2，实际用于院外CA优化神经功能的结果。 1.通过将缺血后血红蛋白O2饱和度维持在94 - 98%，可最大限度地减少脑缺血后的氧化性脑损伤和神经功能损害。 2.复苏后的脑过度氧合损害了神经和组织病理学结果，这是脑能量代谢受损、迟发性神经元Ca 2+失调以及促凋亡蛋白的表达和亚细胞再分布加剧的结果。 3.由于氧化应激介导的线粒体功能障碍减少，使用适度的缺氧后氧合优化体外缺氧和再氧合后的神经元存活。4.老年动物对复苏后过度氧合导致的氧化应激加剧、细胞死亡和神经功能损伤很敏感。 方法包括在全脑缺血模型中使用成熟和老年动物，使用原代神经元培养物的细胞死亡模型，测量线粒体Ca 2+转运、膜电位和脑线粒体和神经元的反应性O2物质的产生，硝基酪氨酸变化的免疫组织化学和免疫印迹分析以及代谢和凋亡蛋白的水平和细胞内分布，13 C NMR光谱分析缺血后大脑和暴露于压力的神经元原代培养物中代谢的改变，以及神经功能缺损的短期和长期测试。

DESCRIPTION (provided by applicant): Comparisons of pre-lethal neurochemical alterations to neurologic outcome and neuropathology following cardiac arrest (CA) and resuscitation using hyperoxic and normoxic ventilation strongly implicate oxidative modification to mitochondrial proteins and associated bioenergetic dysfunction in the etiology of delayed, selective neural cell death. These findings also question the indiscriminate use of 100% ventilatory O2 (FiO2) with patients following CA and suggest that modification of existing resuscitation guidelines may significantly improve neurologic outcome. Our primary goal is to reduce neurologic morbidity and mortality following CA through by minimizing oxidative stress and maximizing cerebral energy metabolism. Our specific aims are to test the following hypotheses focusing on mitochondrial mechanisms of oxidative brain injury in young and aged animals, and on optimizing neurologic outcome using oximetry-based adjustments to FiO2 that are practical for use in out-of-hospital CA. 1. Oxidative brain injury and neurologic impairment following cerebral ischemia are minimized by maintaining postischemic hemoglobin O2 saturation at 94 - 98%. 2. Post-resuscitative cerebral hyper-oxygenation worsens neurologic and histopathologic outcome as a consequence of impaired cerebral energy metabolism, delayed neuronal Ca2+ dysregulation, and exacerbated expression and subcellular redistribution of pro-apoptotic proteins. 3. Neuronal survival following in vitro hypoxia and re-oxygenation is optimized using moderate post-hypoxic oxygenation, due to reduced oxidative stress-mediated mitochondrial dysfunction. 4. Aged animals are sensitive to exacerbation of oxidative stress, cell death, and neurologic impairment by post-resuscitative hyper-oxygenation. Methods of approach include the use of mature and aged animals in models of global cerebral ischemia, models of cell death using primary neuronal cultures, measurements of mitochondrial Ca2+ transport, membrane potential, and production of reactive O2 species with brain mitochondria and neurons, immunohistochemical and immunoblot analysis of changes in nitrotyrosine and the levels and intracellular distribution of metabolic and apoptotic proteins, 13C NMR spectroscopic analysis of altered metabolism in the post-ischemic brain and in primary cultures of neurons exposed to stress, and short- and long-term tests of neurologic impairment.