Molecular Mechanisms of Ischemia Reperfusion Brain Injury

描述(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果和神经病理学的比较强烈地表明，线粒体蛋白质的氧化修饰和相关的生物能量障碍在延迟性、选择性神经细胞死亡的病因中具有重要意义。这些发现还对CA后患者不分青红皂白地使用100%呼吸机氧气(FIO2)提出了质疑，并表明修改现有的复苏指南可能会显著改善神经预后。我们的主要目标是通过最小化氧化应激和最大化大脑能量代谢来降低CA术后神经系统的发病率和死亡率。我们的具体目标是测试以下假说，重点是年轻和老年动物氧化性脑损伤的线粒体机制，以及使用基于血氧计的FiO2调节优化神经学结果，这些调节适用于院外CA。 1.将脑缺血后的血氧饱和度维持在94-98%，可最大限度地减少脑缺血后的氧化性脑损伤和神经损伤。 2.脑复苏后高氧可导致脑组织能量代谢受损，延迟性神经元钙调节失调，促进促凋亡蛋白的表达和亚细胞再分布，从而加重神经功能和组织病理转归。 3.由于氧化应激导致线粒体功能障碍的减少，在体外低氧和复氧后，适度的低氧后给氧可以优化神经元的存活率。4.老年动物对复苏后高氧所致的氧化应激加剧、细胞死亡和神经功能损害十分敏感。 方法包括使用成熟和老年动物建立全脑缺血模型，使用原代神经元培养建立细胞死亡模型，测量线粒体钙转运、膜电位和与脑线粒体和神经元产生的反应性氧物种，免疫组织化学和免疫印迹分析硝基酪氨酸的变化以及代谢和凋亡蛋白的水平和细胞内分布，13C核磁共振波谱分析缺血后脑和暴露于应激的神经元原代培养中的代谢变化，以及神经损伤的短期和长期测试。

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.