Microglial Cell Activation by Thrombin in Stroke

描述（由申请人提供）：该指导临床科学家发展（K 08）奖的建议重点是凝血酶诱导中风中的小胶质细胞活化。脑中的炎症反应在中风的病理生理学中至关重要。凝血蛋白酶凝血酶，蛋白水解激活受体（PAR）和小胶质细胞都强烈参与了这种炎症反应的初始步骤。在中风的情况下，凝血酶可以外渗穿过缺血受损的血脑屏障，激活其受体（PARs-1、PARs-3和PARs-4），并引起神经元和神经胶质细胞的广泛细胞反应。最近的数据表明，凝血酶/PAR信号的诱导也刺激小胶质细胞-大脑的免疫细胞的激活。这种激活反过来有助于启动/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表征凝血酶在体外激活小胶质细胞的细胞后果。对培养的小胶质细胞的这些实验将包括在不存在或存在缺血相关条件（低血糖、缺氧、氧-葡萄糖剥夺）的情况下量化增殖、活力、细胞因子产生和抗原谱的变化。然后，我们将使用PCR和流式细胞术技术来表征PAR表达在静止和激活的小胶质细胞在转录和翻译后水平。在目标二，我们将进行体内研究，重点是全身凝血酶抑制剂和PAR拮抗剂对各种结果参数的影响，在实验动物模型中风：大鼠大脑中动脉闭塞（MCAO）/再灌注。将使用激光多普勒血流仪表征脑血流变化并确认缺血性疾病。标准免疫组织化学技术和新建立的离体流式细胞术方法将分别用于确定MCAO/再灌注后的梗死体积和小胶质细胞活化/PAR表达的程度。还将测量神经行为结果的变化。在这两个目标中，我们还将讨论凝血酶诱导的交叉耐受和缺血预处理对卒中中小胶质细胞活化的影响。通过研究凝血酶PAR介导的小胶质细胞活化在缺血中的影响，我们希望既提高我们对中风中神经炎症机制的理解，又确定治疗干预的可能靶点。

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.