Microglial Toll-like Receptor-4 and Ischemic Preconditioning

描述（由申请人提供）： 该提案的重点是表征小胶质细胞Toll样受体4（TLR 4）在缺血预处理（IPC）中的作用。IPC是一种强有力的神经保护现象，其中短暂的脑缺血赋予对随后的缺血激发的短暂耐受。表征IPC的细胞和分子机制是卒中研究中一个重要而活跃的研究领域。脑中的炎症反应在中风的病理生理学中至关重要。小胶质细胞，大脑的常驻组织巨噬细胞，在这个过程中是中心。TLR介导对多种病原体相关分子模式（包括脂多糖（LPS））的强大免疫应答。由CNS中的小胶质细胞表达的TLR 4直接涉及中风的病理生理学。然而，小胶质细胞TLR 4信号转导在IPC中的作用尚不清楚。在这里，我们假设，小胶质细胞TLR 4信号是必需的最佳IPC介导的神经保护和IPC介导的炎症反应的衰减在随后的长期缺血（中风）。我们进一步假设，低氧诱导因子-11（HIF-11），在低氧张力下基因表达的中央调节器，是介导小胶质细胞TLR 4信号转导对IPC的影响的关键。为了测试这些假设，我们将首先使用TLR 4敲除小鼠和对照小鼠的中风/IPC（小鼠大脑中动脉闭塞/再灌注范例）的良好建立的体内模型。我们将评估梗死体积和神经行为结果。然后，我们将使用离体流式细胞术来表征单独卒中、单独IPC或IPC伴卒中后缺血皮质中的炎性浸润和小胶质细胞表型。第二，我们将进行体外缺血实验，培养的原代小胶质细胞从TLR 4基因敲除和对照小鼠的特点TLR 4信号在调节小胶质细胞对缺氧/低血糖反应的作用。第三，我们将使用髓样细胞靶向，HIF-11敲除小鼠品系，除了全身TLR 4敲除品系，在体内研究HIF-11介导的小胶质细胞TLR 4信号转导对IPC的影响的作用。这些研究结果将有助于研究者阐明IPC、神经炎症和卒中的细胞和分子机制。它将为缺血半暗带中小胶质细胞的病理生理状态提供关键的见解，并确定急性卒中治疗干预的分子靶点。 公共卫生相关性： 在美国，中风是导致严重长期残疾的主要原因，也是第三大死亡原因。目前可用于急性卒中的药物治疗数量很少，并且受到其使用时间限制、适度疗效和严重副作用的可能性的限制。本研究项目的主要目标是增加我们对中风病理生理机制的理解。通过这样做，我们希望确定新的细胞和分子靶点，用于急性卒中的治疗干预。

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.