Cyclooxygenase-2 Regulation of Blood-Brain Barrier Opening in Ischemic Stroke

描述(申请人提供)：神经炎症导致缺血性中风的进行性损害。缺血性卒中后24-48小时血脑屏障(BBB)的破坏是导致神经元死亡的病理机制之一。迫切需要确定限制中风后继发性损害的药理策略。环氧合酶(COX)-2/前列腺素E2(PGE2)通路积极参与炎症反应，加重最初的缺血性脑损伤。我们的长期目标是了解如何操纵COX-2/PGE2通路来阻断缺血性卒中后的进行性损伤级联反应。本项目的总体目标是确定COX-2/PGE2的升高如何在缺血性卒中中介导血脑屏障损伤和神经元死亡。我们的中心假设是，抑制COX-2/PGE2通路可以通过减少基质金属蛋白酶-3/-9的产生来预防血脑屏障损伤和神经元死亡。提出这项研究的基本原理是，了解COX-2/PGE2对中风的急性(有害)和晚期(可能有益)影响的机制，应该为通过阻断COX-2/PGE2途径的药物进行更有选择性和更有效的治疗干预确定新的靶点。具体目的1：确定COX-2介导的基质金属蛋白酶-3/-9表达/活性增加和缺血后血脑屏障损伤的下游效应因子。本实验验证了COX-2衍生的前列腺素E_2通过激活特异性EP受体(S)，在局灶性脑缺血损伤中诱导基质金属蛋白酶-3/-9的产生和血脑屏障破坏的假说。我们将利用体内药理学方法在已建立的缺血性中风大鼠模型中进行研究。这将与EP受体和基质金属蛋白酶-3/-9的免疫组织化学、免疫印迹和生化分析相结合。具体目的#2：确定调节COX-2/PGE2途径的药物的治疗时间窗和长期效果。这一目的的实验验证了这一假设，即缺血后使用阻断缺血诱导的PGE2形成/信号转导的药物提供神经保护，而不干扰恢复的后期阶段。我们将利用体内药理学方法，结合创新的核磁共振技术和一系列测试来评估神经功能。MRI方法将与免疫组织化学分析相结合，以测量神经生成和血管生成。我们期望这项研究将对COX-2衍生的PGE2及其EP受体在缺血性卒中后的神经炎症过程中的作用提供重要的知识。我们还期望确定针对COX-2/PGE2的药物给药的临床相关治疗时间窗。这些结果预计将产生重要的积极影响，因为它们将确定新的和亟需的方法，以减少中风引起的血脑屏障损害的破坏性后果。

DESCRIPTION (provided by applicant): Neuroinflammation leads to progressive damage in ischemic stroke. Disruption of the blood-brain barrier (BBB) occurring at 24-48 h following ischemic stroke is a pathological mechanism that contributes to neuronal death. There is an urgent need to identify pharmacological strategies that limit the secondary damage following stroke. The cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) pathway is actively involved in inflammatory events that exacerbate initial ischemic brain injury. Our long-term goal is to understand how the COX-2/PGE2 pathway can be manipulated to block the progressive injury cascade following ischemic stroke. The overall objective of this project is to determine how increased COX-2/PGE2 mediates BBB damage and neuronal death in ischemic stroke. Our central hypothesis is that inhibition of the COX-2/PGE2 pathway protects against BBB damage and neuronal death by reducing MMP-3/-9 production. The rationale for the proposed research is that understanding the mechanisms involved in acute (deleterious) and late (possibly beneficial) effects of COX-2/PGE2 in stroke should identify novel targets for a more selective and effective therapeutic intervention with drugs blocking the COX-2/PGE2 pathway. Specific Aim #1: To identify the downstream effectors of COX-2-mediated increase in MMP-3/-9 expression/activity and damage to the BBB after ischemia. Experiments in this aim test the hypothesis that COX-2-derived PGE2 induces MMP-3/-9 production and BBB breakdown through activation of specific EP receptor(s) in focal ischemic brain injury. We will utilize an in vivo pharmacological approach in a well- established rat model of ischemic stroke. This will be coupled with immunohistochemical, immunoblotting and biochemical analyses of EP receptors and MMP-3/-9. Specific Aim #2: To determine the therapeutic time window of protection and long-term effects of drugs modulating the COX-2/PGE2 pathway. Experiments in this aim test the hypothesis is that post- ischemic treatment with agents blocking ischemia-induced PGE2 formation/signaling provides neuroprotection without interfering in the late recovery phase. We will utilize an in vivo pharmacological approach together with innovative MRI techniques and a battery of tests to evaluate neurological function. The MRI approach will be coupled with immunohistochemical analyses to measure neurogenesis and angiogenesis. It is our expectation that this research will provide significant knowledge of the contribution of COX-2- derived PGE2 and its EP receptors to the neuroinflammatory process that follows ischemic stroke. We also expect to identify a clinically relevant therapeutic time window for the administration of drugs targeting COX- 2/PGE2. Such results would be expected to have an important positive impact, since they would identify novel and much-needed approaches to reduce the devastating consequences of stroke-induced BBB damage.