Excitotoxicity and Inflammation

描述（由申请人提供）：脑缺血后数小时甚至数天发生的迟发性神经元损伤的病理生理学知之甚少。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。虽然炎性细胞从外周外渗可能导致神经元损伤，但在CNS实质细胞中表达的“炎性”基因也可发挥有害作用。大量动物实验结果表明，环氧合酶-2（考克斯-2）是脑缺血和/或直接注射兴奋毒素后最强烈诱导的基因之一。考克斯-2也显示在致命的脑损伤后在人脑神经元中被诱导。 在上一个/目前的资助期内，我们的实验室是第一个建立直接联系的实验室， NMDA诱导的神经元考克斯-2诱导/活性和神经元细胞死亡。这一意见是以下建议的基础。该后续5年研究计划的目的是：1）使用直接海马内注射NMDA和选择性药理学方法充分阐明考克斯-2活性对体内兴奋性毒性的贡献，以鉴定有效防止损伤的化合物以及阐明其治疗时间窗，2）确定NMDA调节考克斯-2表达的分子机制，使用现有技术的分子生物学方法鉴定负责NMDA-2的考克斯-2启动子中的顺式元件及其各自的反式激活因子。诱导考克斯-2 mRNA转录以及控制mRNA稳定性的3' UTR区;和3）通过使用化学抑制剂、反义寡核苷酸和敲除技术来鉴定考克斯底物释放的酶途径，以测试cPLA 2介导NMDA刺激的花生四烯酸释放的有趣和特异性假设。所有 设计了三个目的以鉴定特异性药物以及在导致兴奋毒性神经元细胞死亡的考克斯-2转导级联中潜在治疗干预的替代位点。

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.