Effect of Ischemic Preconditioning on Microglial Transcriptome

描述(由申请人提供)：NIH小型研究资助计划(R03)的提案重点在于确定缺血预适应(IPC)对小胶质细胞转录组的影响。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。对有或没有缺血预适应的整个脑组织中分离出的RNA进行的微阵列分析表明，缺血预适应和缺血(单独或顺序组合)都能诱导大规模的基因组重编程反应，这些反应在本质上是不同的(甚至是不同的)。然而，人们对细胞水平上发生的基因组变化知之甚少。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，在这一过程中起着关键作用。小胶质细胞在IPC现象中的作用尚不清楚。在这里，我们将结合一个成熟的中风体内模型(小鼠大脑中动脉阻塞/再灌注(MCAO/R)范例)和一种新的细胞分析形式(体外流式细胞分选小胶质细胞的基因组微阵列表征)。本项目需要建立和优化两种方法：(A)采用IPC范式的小鼠MCAO/R模型和(B)急性分离成年小鼠脑组织中常驻小胶质细胞的体外流式细胞仪技术。然后，我们将对从遭受IPC和/或中风(通过MCAO/R)的小鼠分离的小胶质细胞中提取的RNA进行微阵列基因组分析。这个细胞类型和疾病特异性基因组数据集的建立将允许进行本体论分析，以及识别在IPC和/或卒中后小胶质细胞中特异激活/调节的启动子元件、转录因子、信号通路和个别基因。数据集的建立还将允许在小胶质细胞特异性和(先前发表的)全脑来源的缺血预适应和/或中风的小鼠转录之间进行直接比较。我们预计，将通过发布在基因表达总览上向科学界提供的数据集，将对试图阐明IPC、神经炎症和中风的细胞和分子机制的研究人员有价值。它应该提供对缺血半影区小胶质细胞的病理生理状态的关键见解，并增强我们产生关于小胶质细胞在IPC中作用的新假说的能力。它在确定急性中风治疗干预的分子靶点方面也可能被证明是有效的。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 for a NIH Small Research Grant Program (R03) focuses on determining the effect of ischemic preconditioning (IPC) on the microglial cell transcriptome. 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. Microarray analyses on RNA isolated from whole brain tissue that has undergone ischemia with or without prior IPC has demonstrated that both IPC and ischemia (alone or in sequential combination) induce large scale genomic reprogramming responses that are each distinct (and even disparate) in nature. However, little is known about the genomic changes that occur at the cellular level. Inflammatory responses in the brain are critical in the pathophysiology of stroke. Microglia, the brain's resident tissue macrophages, are critical in this process. The role of microglia in the IPC phenomenon is unknown. Here we will combine a well-established in vivo model of stroke (the mouse middle cerebral artery occlusion/reperfusion (MCAO/R) paradigm) with a novel form of cellular analysis (genomic microarray characterization of ex vivo flow cytometrically sorted microglia). The project requires that we establish and optimize two methods: (A) mouse MCAO/R model with the IPC paradigm and (B) ex vivo flow cytometric technique for acute isolation of resident microglia from adult mouse brain tissue. We will then perform microarray genomic analysis on RNA extracted from microglia isolated from mice that have undergone IPC and/or stroke (via MCAO/R). Establishment of this cell-type- and disease- specific genomic dataset will allow for ontological analysis as well as identification of promoter elements, transcription factors, signaling pathways and individual genes that are activated/regulated specifically in microglia following IPC and/or stroke. Establishment of the dataset will also allow for direct comparison between the microglia-specific and (the previously published) whole-brain derived ischemic preconditioned and/or "stroked" mouse transcriptomes. We anticipate that the dataset, which will be made available to the scientific community through posting on the Gene Expression Omnibus, will be valuable to researchers trying to elucidate the cellular and molecular mechanisms of IPC, neuroinflammation and stroke. It should provide key insights into the pathophysiologic state of microglia in the ischemic penumbra and enhance our ability to generate novel hypotheses about the role of microglia in IPC. It may also prove effective in identifying 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.