Microglial Toll-like Receptor-4 and Ischemic Preconditioning

描述(由申请人提供)： 本研究旨在探讨小胶质细胞Toll样受体-4(TLR4)在缺血预适应(IPC)中的作用。IPC是一种强大的神经保护现象，短暂的脑缺血使其对随后的缺血挑战具有短暂的耐受性。表征IPC背后的细胞和分子机制是卒中研究中一个重要和活跃的研究领域。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。小胶质细胞是大脑中驻留的组织巨噬细胞，在这一过程中处于中心地位。TLRs介导对多种病原体相关分子模式的强大免疫应答，包括内毒素(LPS)。TLR4由中枢小胶质细胞表达，直接参与卒中的病理生理过程。然而，小胶质细胞TLR4信号在IPC中的作用尚不清楚。在这里，我们假设小胶质细胞TLR4信号对于IPC介导的最佳神经保护和IPC介导的随后的长期缺血(卒中)所见的炎症反应的减弱都是必需的。我们进一步推测，缺氧诱导因子-11(HIF-11)是低氧压下基因表达的中枢调节因子，在介导小胶质细胞TLR4信号对IPC的影响中起着关键作用。为了验证这些假设，我们将首先使用TLR4基因敲除小鼠和对照小鼠建立的中风/IPC(小鼠大脑中动脉阻塞/再灌注范例)体内模型。我们将评估脑梗塞体积和神经行为结果。然后，我们将使用体外流式细胞术来表征单纯卒中、单纯IPC或IPC继发卒中后缺血皮质的炎性浸润物和小胶质细胞表型。其次，我们将对培养的TLR4基因敲除小鼠和对照小鼠的小胶质细胞进行体外缺血实验，以研究TLR4信号在调节小胶质细胞对缺氧/低血糖反应中的作用。第三，除了系统的TLR4基因敲除菌株外，我们还将使用髓系细胞靶向的HIF-11基因敲除小鼠品系，在体内研究HIF-11在介导小胶质细胞TLR4信号对IPC的影响中的作用。这些研究结果对于试图阐明IPC、神经炎症和中风的细胞和分子机制的研究人员将是有价值的。它将提供对缺血半暗带小胶质细胞病理生理状态的关键见解，并为急性卒中的治疗干预确定分子靶点。 公共卫生相关性： 在美国，中风是导致长期严重残疾的首要原因，也是导致死亡的第三大原因。目前可用于急性中风的药物治疗数量很少，而且受到使用时间限制、疗效不佳和可能出现严重副作用的限制。这项研究的一个主要目标是增加我们对中风病理生理学的机械学理解。通过这样做，我们希望为急性卒中的治疗干预确定新的细胞和分子靶点。

DESCRIPTION (provided by applicant): This proposal focuses on characterizing the role of microglial toll-like receptor-4 (TLR4) in ischemic preconditioning (IPC). IPC is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Characterization of the cellular and molecular mechanisms that underlie IPC is an important and active area of investigation in stroke research. Inflammatory responses in the brain are critical in the pathophysiology of stroke. Microglia, the brain's resident tissue macrophages, are central in this process. TLRs mediate powerful immune responses to a variety of pathogen associated molecular patterns including lipopolysaccharide (LPS). TLR4, which is expressed by microglia in the CNS, has been directly implicated in the pathophysiology of stroke. However, the role of microglial TLR4 signaling in IPC is unknown. Here we hypothesize that microglial TLR4 signaling is required for both optimal IPC-mediated neuroprotection and IPC-mediated attenuation of inflammatory responses seen in subsequent prolonged ischemia (stroke). We further postulate that hypoxia-inducible factor-11 (HIF-11), a central regulator of gene expression under low oxygen tension, is critical in mediating the effects of microglial TLR4 signaling on IPC. To test these hypotheses, we will first use a well-established in vivo model of stroke/IPC (mouse middle cerebral artery occlusion/reperfusion paradigm) on TLR4 knockout and control mice. We will assess infarct volume and neurobehavioral outcome. Then we will use ex vivo flow cytometry to characterize the inflammatory infiltrate and microglial phenotype in ischemic cortex following stroke alone, IPC alone or IPC followed by stroke. Second, we will carry out in vitro ischemia experiments on cultured primary microglia from TLR4 knockout and control mice to characterize the role of TLR4 signaling in modulating the microglial response to hypoxia/hypoglycemia. Third, we will use a myeloid-cell targeted, HIF-11 knockdown mouse strain, in addition to the systemic TLR4 knockout strain, to investigate in vivo the role of HIF-11 in mediating the effects of microglial TLR4 signaling on IPC. The results of these studies will be valuable to researchers trying to elucidate the cellular and molecular mechanisms of IPC, neuroinflammation and stroke. It will provide key insights into the pathophysiologic state of microglia in the ischemic penumbra and identify molecular targets for therapeutic intervention in acute stroke. PUBLIC HEALTH RELEVANCE: Stroke is the leading cause of serious long-term disability and the third leading cause of death in the United States. Currently available pharmacologic therapies for acute stroke are few in number and limited by temporal restrictions on their use, modest efficacy and potential for serious side effects. A major goal of this research project is to increase our mechanistic understanding of stroke pathophysiology. By doing so, we hope to identify novel cellular and molecular targets for therapeutic intervention in acute stroke.