BIOCHEMICAL EVOLUTION OF ISCHEMIC BRAIN DAMAGE

这项建议的目标是确定 在缺血性脑损伤中起关键作用。我们的主要努力是 针对乳胶沉着症在细胞损伤中的作用。我们假设 大脑的pH值(和/或乳酸)有一个敏锐的阈值，在这个阈值上 高能磷酸盐的恢复被阻止，即使组织被 再灌流。因此，这项提案的具体目标是：(一)确定 通过将组织的pH值和乳酸相关联来确定是否存在这样的阈值 直接与能量状态有关，(Ii)量化pH值的临界水平 和乳酸在大脑的不同区域，(Iii)测定代谢 通过组织代谢物和酶的测定确定抑制部位 活动；以及(4)通过以下方式修改乳杆菌病的门槛水平 限制乳酸盐生产，增加乳酸菌的缓冲能力 组织。 将采用两种小鼠局灶性脑缺血模型：第一， 大脑中动脉闭塞2例，闭塞1例 颈动脉合并全身性缺氧。这两款车型都有 已在我们的实验室开发和表征，适用于 短暂性局灶性脑缺血和延长恢复的研究(1周)。 代谢物水平、pH(使用肌酸激酶)的局部变化 平衡)，组织酶将在显微样本中进行测定 (10-50 ng)在冷冻干燥中使用定量微量化学技术 组织。此外，生化变化将直接相关。 冰冻脑组织的相邻切片有组织学改变。最后， 外源性试剂，如Tris Buffer或二氯乙酸酯，将用于 修改缺血型乳胶沉着症。

The objective of this proposal is to identify biochemical alterations that play a critical role in ischemic brain damage. Our primary effort is directed at the role of lactacidosis in cellular damage. We hypothesize that there is a sharp threshold for brain pH (and/or lactate), at which restoration of high-energy phosphates is prevented even if the tissue is reperfused. Thus, the specific aims of this proposal are (i) to determine whether such a threshold exists by correlating tissue pH and lactate directly with energy state, (ii) to quantitate the critical levels of pH and lactate in various regions of brain, (iii) to determine the metabolic sites of inhibition using measurements of tissue metabolites and enzyme activities, and (iv) to modify the threshold level of lactacidosis by restricting lactate production and by increasing the buffer capacity of the tissue. Two models of focal ischemia in mouse brain will be employed: first, occlusion of the middle cerebral artery and second, occlusion of one carotid artery combined with systemic hypoxia. Both of these models have been developed and characterized in our laboratory and are suitable for the investigation of transient focal ischemia and extended recovery (1 week). Regional alterations of metabolite levels, pH (using the creatine kinase equilibrium), and tissue enzymes will be determined in microscopic samples (10-50 ng) using quantitative microchemical techniques in freeze-dried tissue. Further, the biochemical alterations will be correlated directly with histologic change in adjacent sections of frozen brain. Finally, exogenous agents, such as Tris buffer or dichloroacetate, will be used to modify the ischemic lactacidosis.