Microglial Toll-like Receptor-4 and Ischemic Preconditioning

小胶质细胞 Toll 样受体 4 和缺血预处理

• 批准号: 7917210
• 负责人: Jonathan Weinstein
• 金额: $ 18.08万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917210
• 关键词: Adverse effects; Agonist; Antibodies; Area; Biochemical; Biological Assay; Brain; Brain Injuries; Cause of Death; Cells; Cerebral Ischemia; Chaperonin 60; Enzymes; Experimental Models; Exposure to; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Profile; Goals; Heat shock proteins; Hypoglycemia; Hypoxia; Hypoxia Inducible Factor; Immune; Immune response; In Vitro; Infarction; Inflammatory; Inflammatory Infiltrate; Inflammatory Response; Injury; Intervention; Investigation; Ischemia; Ischemic Brain Injury; Ischemic Penumbra; Ischemic Preconditioning; Knock-out; Knowledge; Lipopolysaccharides; Mediating; Mediator of activation protein; Microglia; Middle Cerebral Artery Occlusion; Modeling; Molecular; Molecular Target; Mouse Strains; Mus; Myeloid Cells; Nuclear Translocation; Outcome; Oxygen measurement, partial pressure, arterial; Pattern; Pattern recognition receptor; Peripheral; Phenotype; Process; Proteins; Regulator Genes; Relative (related person); Reperfusion Therapy; Research; Research Personnel; Research Project Grants; Resistance; Role; Signal Transduction; Sorting - Cell Movement; Source; Stimulus; Stroke; Surface Antigens; Testing; Therapeutic Intervention; Tissues; Toll-like receptors; United States; Validation; Work; acute stroke; attenuation; chemokine; cytokine; disability; in vivo; in vivo Model; insight; macrophage; microbial; mouse toll-like receptor 4; neurobehavioral; neuroinflammation; neuroprotection; novel; pathogen; preconditioning; public health relevance; research study; response; toll-like receptor 4

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.

描述（由申请人提供）： 该建议的重点是表征小胶质Toll样受体-4（TLR4）在缺血性预处理（IPC）中的作用。 IPC是一种强大的神经保护现象，其中短暂的脑缺血赋予了对随后的缺血挑战的短暂耐受性。 IPC构成的细胞和分子机制的表征是中风研究中重要且活跃的研究领域。大脑的炎症反应在中风的病理生理学中至关重要。小胶质细胞是大脑的驻留组织巨噬细胞，在此过程中至关重要。 TLR介导强大的免疫反应对包括脂多糖（LPS）在内的多种病原体相关的分子模式。由中枢神经系统中的小胶质细胞表达的TLR4直接与中风的病理生理有关。但是，小胶质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信号传导是最佳的IPC介导的神经保护作用和IPC介导的炎症反应的衰减所必需的，随后长时间的缺血（中风）所见。我们进一步假设缺氧诱导因子-11（HIF-11）是低氧张力下基因表达的中心调节剂，对于介导小胶质细胞TLR4信号对IPC的影响至关重要。为了检验这些假设，我们将首先在TLR4敲除和控制小鼠上使用良好的中风/IPC（小鼠中大脑中动脉闭塞/再灌注范式）的体内模型。我们将评估梗塞量和神经行为结果。然后，我们将使用离体流式细胞仪来表征单独的中风，单独使用IPC或IPC随后是中风的缺血性皮质中炎症性浸润和小胶质细胞表型的表征。其次，我们将对来自TLR4基因敲除和对照小鼠的原代小胶质细胞进行体外缺血实验，以表征TLR4信号传导在调节小胶质细胞对低氧/低血糖的小胶质细胞反应中的作用。第三，除全身性TLR4敲除菌株外，我们还将使用具有髓样细胞靶向的HIF-11敲低小鼠菌株来研究HIF-11在介导小胶质细胞TLR4信号对IPC的影响中的作用。这些研究的结果对于试图阐明IPC，神经炎症和中风的细胞和分子机制的研究人员将很有价值。它将提供对缺血性半胶质细胞的病理生理状态的关键见解，并确定用于急性中风治疗干预的分子靶标。 公共卫生相关性： 中风是严重长期残疾的主要原因，也是美国第三大死亡原因。目前可用于急性中风的药理学疗法的数量很少，并且受到时间限制的限制，适度的功效和严重副作用的潜力。该研究项目的一个主要目标是提高我们对中风病理生理学的机械理解。通过这样做，我们希望鉴定出新的细胞和分子靶标，以用于急性中风的治疗干预。