CELLULAR AND MOLECULAR MECHANISMS INVOLVED IN INJURY FOLLOWING GLOBAL ISCHEMIA

我们实验室的最新发现表明，缺血后 体温过低揭示了一系列发生在 全脑缺血后明显延长的时间范围。这一范式 将使我们能够研究细胞和分子机制 全脑缺血后早期和迟发性神经元损伤。初步 项目中总结的数据表明，兴奋性毒性是一种重要的 参与急性和迟发性缺血后损伤的过程。 拟议的协议旨在确定涉及的关键事件 在这个过程中。通过使用原位杂交，免疫组织化学， 和定量的Northern和Western印迹，我们将确定 短暂性全脑缺血是否会导致心肌细胞的改变 谷氨酸受体亚基的配置，导致异常 增强神经元对正常亚致死水平的 谷氨酸。缺血后低温可能会延缓这一过程，或揭开面纱 发展出更细微的继发性损伤过程。这些措施包括 细胞外谷氨酸或喹啉酸的激增，产生 氧自由基和/或一氧化氮，所有这些都可以参与 再灌注期晚期的兴奋性毒性作用。通过使用 微透析时，我们将评估是否会出现继发性兴奋性激增 在再灌流后期会产生氨基酸或羟基自由基。 并确定它们是否与迟发性损伤有关 进程。通过使用形态和生化程序，我们将 确定短暂性全脑缺血是否导致表达延迟 诱导型一氧化氮合酶，导致一氧化氮激增 在再灌流后期可能会产生有害的物质。 由于继发性神经元损伤的过程也可能涉及 控制细胞死亡和死亡的特定基因的表达 缓慢发展过程的发展，如程序化细胞 死亡，我们将使用DNA梯形技术的原位末端标记 为了检测凋亡型DNA片段化是否先于 迟发性神经元损伤。我们还将利用mrna的差异。 表征以下新基因表达的展示方法 缺血，并评估缺血后亚低温对这些细胞的影响 改变。这些研究将提供新的数据，使我们能够 更好地了解兴奋性毒性在缺血神经元中的作用 损坏。重要的是，确定迟发性神经元损伤的机制 脑缺血后可能使我们制定一种药理策略 可以用来提供永久保护的。

Recent findings from our laboratory demonstrate that postischemic hypothermia unveils a spectrum of injury processes occurring over a markedly extended time frame following global ischemia. This paradigm will enable us to study cellular and molecular mechanisms involved in early and delayed neuronal injury following global ischemia. Preliminary data summarized in the project implicate excitotoxicity as an important process involved in the acute as well as delayed postischemic injury. The proposed protocols are targeted at identifying key events involved in this process. By using in situ hybridization, immunohistochemistry, and quantitative Northern and Western blotting, we will determine whether transient global ischemia leads to alteration in the configuration of glutamate receptor subunits, resulting in abnormally enhanced susceptibility of neurons to normally sub-lethal levels of glutamate. Postischemic hypothermia may delay this process, or unmask the development of more subtle secondary injury processes. These include a surge in extracellular glutamate or quinolinic acid, generation of oxygen radicals and/or nitric oxide, all of which can participate in the excitotoxic process during the late reperfusion period. By using microdialysis, we will assess whether a secondary surge in excitatory amino acids or hydroxyl radicals occurs during the late reperfusion phase and determine whether they are linked to the delayed injury process. By using morphological and biochemical procedures, we will ascertain whether transient global ischemia leads to delayed expression of inducible nitric oxide synthase, resulting in a surge in NO production which can be detrimental during the late reperfusion phase. Since the process of secondary neuronal damage may also involve the expression of specific genes which control the onset of cell death and the development of a slowly progressive process such as programmed cell death, we will use end-labeling in situ with DNA laddering techniques to detect whether DNA fragmentation of the apoptotic type precedes the delayed neuronal damage. We will also make use of the mRNA differential display method to characterize the expression of novel genes following ischemia, and assess the effects of postischemic hypothermia on these changes. These studies will provide new data which will enable us to understand better the role of excitotoxicity in ischemic neuronal damage. Importantly, identifying mechanisms of delayed neuronal injury following ischemia may enable us to develop a pharmacological strategy that can be employed to provide permanent protection.