BIOCHEMICAL EVOLUTION OF ISCHEMIC BRAIN DAMAGE

这项建议的目的是确定生物化学变化， 在缺血性脑损伤中起关键作用。 我们的主要努力是 针对乳酸中毒在细胞损伤中的作用。 我们假设 大脑pH值（和/或乳酸盐）有一个明显的阈值， 即使组织被破坏， 再灌注。 因此，本建议的具体目标是：（i）确定 通过将组织pH和乳酸盐相关联来确定是否存在这样的阈值 直接与能量状态，（ii）定量的pH值的临界水平 和乳酸在大脑的各个区域，（iii）以确定代谢 使用组织代谢物和酶的测量的抑制位点 活动，和（iv）修改乳酸中毒的阈值水平， 限制乳酸盐的产生，并通过增加 组织. 将采用两种小鼠脑中的局灶性缺血模型：第一， 大脑中动脉闭塞，第二， 颈动脉合并全身缺氧。 这两种模式都有 在我们的实验室开发和表征，适用于 短暂局灶性缺血和延长恢复期（1周）的研究。 代谢物水平、pH值的区域变化（使用肌酸激酶 平衡），组织酶将在显微镜下测定 （10-50纳克），使用定量微量化学技术， 组织. 此外，生化改变将直接相关 冷冻大脑邻近切片出现组织学变化。 最后， 外源性试剂，如Tris缓冲液或二氯乙酸盐，将用于 改善缺血性乳酸中毒

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.