Excitotoxicity and Inflammation

描述(申请人提供)：脑缺血后数小时甚至数天发生的延迟性神经元损伤的病理生理学还知之甚少。现在很明显，兴奋性毒性和炎症可以在缺血性神经变性的进展中同时起作用。虽然炎症细胞从周围渗出可能会导致神经元损伤，但中枢神经系统实质细胞中表达的“炎症”基因也可能起到有害的作用。大量动物实验结果表明，环氧合酶-2(COX-2)是脑缺血和/或直接注射兴奋性毒素后诱导表达最强的基因之一。COX-2也被证明在致命的大脑侮辱后，在人脑的神经元中被诱导。 在上一次/当前的赠款期间，我们的实验室是第一个在 NMDA诱导神经细胞COX-2的诱导/活性和神经细胞死亡。这一观察结果是以下建议的基础。这项为期5年的研究计划的目标是：1)充分阐明COX-2活性对体内兴奋性毒性的贡献，方法是直接在海马内注射NMDA，并采用选择性药理学方法来鉴定有效预防损伤的化合物以及阐明它们的治疗时间窗，2)利用最新的分子生物学方法确定NMDA调节COX-2表达的分子机制，以确定COX-2启动子中负责NMDA诱导COX-2mRNA转录的顺式元件及其各自的反式激活因子，以及在控制mRNA稳定性的3‘UTR区；3)通过使用化学抑制剂、反义寡核苷酸和基因敲除技术来确定COX底物释放的酶途径，以验证cPLA2介导NMDA刺激的花生四烯酸释放这一有趣而具体的假说。全 设计了三个目标来确定特定的药物以及在导致兴奋性毒性神经细胞死亡的COX-2转导级联中潜在的治疗干预的替代部位。

DESCRIPTION (provided by applicant): The pathophysiology of delayed neuronal injury which occurs hours and even days following cerebral ischemia are poorly understood. It is now apparent that excitotoxicity and inflammation can operate concurrently in the progression of ischemic neurodegeneration. While extravasation of inflammatory cells from the periphery may contribute to neuronal damage, 'inflammatory' genes expressed in parenchymal cells of the CNS can also play a deleterious role. Results from numerous animal studies indicate that cyclooxygenase-2 (COX-2) is one of the genes most strongly induced following cerebral ischemia and/or direct excitotoxin injection. COX-2 has also been shown to be induced in neurons of human brain following a lethal cerebral insult. During the last/current grant period, our laboratory was the first to establish a direct link between NMDA-induced neuronal COX-2 induction/activity and neuronal cell death. This observation serves as the basis for the following proposal. The objectives of this following 5 yr research plan of study are 1) to fully elucidate the contribution of COX-2 activity to excitotoxicity in vivo using direct intrahippocampal injections of NMDA and a selective pharmacological approach to identify compounds which effectively prevent injury as well as to elucidate their therapeutic time window, 2) to determine the molecular mechanisms by which NMDA regulates COX-2 expression using state of art molecular biological approaches to identify the cis-elements and their respective trans-activating factors in the COX-2 promoter responsible for NMDA-induced COX-2 mRNA transcription as well as in the 3' UTR region which governs mRNA stability; and 3) to identify the enzymatic pathway for COX substrate liberation by employing chemical inhibitors, antisense oligonucleotides and knockout technology to test the interesting and specific hypothesis that cPLA2 mediates NMDA-stimulated arachidonic acid release. All three aims are devised to identify specific drugs as well as alternate sites for potential therapeutic intervention in the COX-2 transduction cascade leading to excitotoxic neuronal cell death.