Microglial Cell Activation by Thrombin in Stroke

描述(由申请人提供)：这份指导临床科学家发展奖(K08)的建议重点是凝血酶在中风中诱导小胶质细胞激活。脑部的炎症反应在中风的病理生理学中起着至关重要的作用。凝血酶、凝血酶、蛋白水解性激活受体(PARs)和小胶质细胞都与炎症反应的初始步骤密切相关。在中风的情况下，凝血酶可以通过缺血损害的血脑屏障外渗，激活其受体(PARS-1、-3和-4)，并从神经元和胶质细胞引发广泛的细胞反应。最近的数据表明，凝血酶/PAR信号的诱导也刺激了小胶质细胞的激活，小胶质细胞是大脑的免疫细胞。这种激活反过来又有助于启动/协调大脑对缺血的炎症反应。对于这些拟议的研究，我们的第一个具体目标将是在体外表征凝血酶激活小胶质细胞的细胞后果。在培养的小胶质细胞上的这些实验将包括在没有或存在与缺血相关的条件(低血糖、低氧、缺糖)的情况下，量化增殖、活性、细胞因子分泌和抗原谱的变化。然后，我们将使用聚合酶链式反应和流式细胞仪技术来表征静止和激活的小胶质细胞在转录和翻译后水平上的PAR表达。在第二个目标中，我们将开展体内研究，重点研究全身性凝血酶抑制剂和PAR拮抗剂对实验性卒中动物模型：大鼠大脑中动脉闭塞/再灌注的各种结局参数的影响。激光多谱勒血流计将用于表征脑血流的变化和确认缺血情况。标准的免疫组织化学技术和新建立的体外流式细胞术方法将分别用于确定MCAO/再灌注后的脑梗塞体积和小胶质细胞活化/PAR表达的程度。神经行为结果的变化也将被测量。在这两个目标中，我们还将探讨凝血酶诱导的交叉耐受和缺血预适应对卒中小胶质细胞激活的影响。通过研究凝血酶PAR介导的小胶质细胞活化在脑缺血中的作用，我们希望提高我们对卒中神经炎症机制的认识，并找出可能的治疗干预靶点。

DESCRIPTION (provided by applicant): This Mentored Clinical Scientist Development (K08) Award proposal focuses on thrombin's induction of microglial cell activation in stroke. Inflammatory responses in the brain are critical in the pathophysiology of stroke. The coagulation proteinase thrombin, proteolytically activated receptors (PARs) and microglial cells have all been strongly implicated in the initial steps of this inflammatory response. In the setting of stroke, thrombin can extravasate across an ischemia-compromised blood-brain barrier, activate its receptors (PARs-1, -3 and -4) and elicit a wide range of cellular responses from neurons and glia. Recent data suggests that induction of thrombin/PAR signaling also stimulates activation of microglia - the immune cells of the brain. This activation in turn helps initiate/orchestrate the brain's inflammatory response to ischemia. For these proposed studies, our first Specific Aim will be to characterize the cellular consequences of microglial activation by thrombin in vitro. These experiments on cultured microglia will include quantifying changes in proliferation, viability, cytokine elaboration and antigenic profile in the absence or presence of ischemia-related conditions (hypoglycemia, hypoxia, oxygen-glucose deprivation). We will then use PCR and flow cytometric techniques to characterize PAR expression in quiescent and activated microglia at both the transcriptional and post-translational levels. In Aim two, we will carry out in vivo studies focusing on the effects of systemic thrombin inhibitors and PAR antagonists on a variety of outcome parameters in an experimental animal paradigm for stroke: rat middle cerebral artery occlusion (MCAO)/reperfusion. Laser doppler flowmetry will be used to characterize changes in cerebral blood flow and confirm ischemic conditions. Standard immunohistochemical techniques and a newly established ex-vivo flow cytometric method will be used to determine infarct volume and extent of microglial activation/PAR expression, respectively following MCAO/reperfusion. Changes in neuro-behavioral outcomes will also be measured. In both Aims we will also address the impact of thrombin-induce cross-tolerance and ischemic pre-conditioning on microglial activation in stroke. By studying the impact of thrombin's PAR-mediated activation of microglia in ischemia we hope to both improve our understanding of the mechanism of neuro-inflammation in stroke and identify possible targets for therapeutic intervention.