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Role of Microglial Calcium Waves in Ischemic Stroke

小胶质细胞钙波在缺血性中风中的作用

基本信息

批准号：
10418797
负责人：
Petr Tvrdik
金额：
$ 16.15万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10418797
关键词：
Ablation Acute Address Affect American Animals Apoptosis Behavioral Brain Calcium Calcium Channel Calcium Oscillations Calcium Signaling Cause of Death Cell Death Cell Survival Cell physiology Cells Cerebral Ischemia Development Diffuse Disease Female Functional disorder Genetic Histologic Hypoxia Image Immune Immune system Immunoassay Infarction Inflammation Inflammatory Inflammatory Response Injury Ischemic Stroke Lead Measures Mediating Microglia Middle Cerebral Artery Occlusion Molecular Mus Necrosis Neurologic Neuronal Injury Neurons Pathology Pathway interactions Pattern Periodicals Pharmacology Phase Pilot Projects Process Public Health Recurrence Reporter Role Secondary to Spreading Cortical Depression Stroke System Techniques Testing Therapeutic Intervention Tissues Transgenic Mice Traumatic Brain Injury United States National Institutes of Health Work acute stroke base behavioral outcome brain cell brain parenchyma burden of illness calcium indicator clinical translation cohort comorbidity cytokine disability genetic approach improved improved outcome in vivo inhibitor innovation interest ischemic injury male mouse model neuroinflammation neuronal survival novel novel therapeutic intervention post stroke prevent protective effect response single cell sequencing stroke intervention stroke outcome stroke patient tool two photon microscopy two-photon

项目摘要

Abstract Ischemic stroke is a leading cause of death and severe disability in US and worldwide. Stroke-induced hypoxia promotes a cascade of pathophysiological responses that lead to necrosis in the ischemic core, and apoptosis in the hypo-perfused tissue known as penumbra. Cell death triggers an inflammatory response that contributes to secondary injury and potentially harms the neurons surviving the initial insult. Microglia are the principal immune cells in the brain parenchyma but their specific roles in secondary injury and the underlying mechanisms of induction remain unclear. We hypothesize that increased calcium signaling is a key mechanism in the acute stroke-induced microglial activation, possibly leading to increased release of proinflammatory cytokines. We have developed a mouse reporter that indicates intracellular calcium in microglial cells. In this system, we use 2- photon imaging and middle cerebral artery occlusion (MCAo) to study microglial responses to ischemic injury in vivo. We have demonstrated periodical waves of calcium activity in cortical microglia following intraarterial occlusion, consistent with patterns of cortical spreading depolarizations (CSD). We propose to test the role of these calcium transients by pharmacological inhibition of calcium influx, mediated by the calcium release- activated calcium (CRAC) channels, and by genetic ablation of CRAC channel subunits. In Aim 1, we will directly test whether the novel CRAC channel inhibitors developed by CalciMedica can reduce microglial activation, neuro-inflammation and ultimately infarct size in the mouse model of MCAo. Pharmacological effects of these blockers will be characterized with 2-photon imaging and their immune-protective effects in vivo will be evaluated by cytokine profiling. In Aim2, the CRAC channel subunits Stim1 and Stim2 will be genetically ablated in brain microglia and behavioral outcomes and infarct size after MCAo stroke will be evaluated. Stroke kills almost 130,000 Americans each year. If successful, clinical translation of this approach could help to reduce the burden of this disease. Our overreaching objective is to apply the tools and techniques assembled under this pilot study to a broader R01 project investigating CRAC-mediated calcium overload in all other brain cells during ischemic injury.

摘要缺血性卒中是美国和世界范围内死亡和严重残疾的主要原因。脑卒中缺氧促进一系列病理生理反应，导致缺血核心坏死和细胞凋亡被称为半影的低灌注组织中。细胞死亡引发炎症反应，继发性损伤，并可能损害最初损伤后存活的神经元。小胶质细胞是脑实质中的免疫细胞，但它们在继发性损伤中的特定作用及其潜在机制归纳法仍不明确。我们假设钙信号的增加是急性心肌梗死的一个关键机制，中风诱导的小胶质细胞活化，可能导致促炎细胞因子释放增加。我们已经开发了一种小鼠报告基因，可以指示小胶质细胞中的细胞内钙。在这个系统中，我们使用 2-光子成像和大脑中动脉闭塞（MCAo）研究小胶质细胞对缺血性损伤的反应 in vivo.我们已经证明了在动脉内注射后，皮层小胶质细胞中钙离子活性的周期性波动，闭塞，与皮质扩散去极化（CSD）模式一致。我们建议测试的作用，这些钙瞬变是由钙释放介导的钙内流的药理学抑制引起的，活化钙（CRAC）通道，以及通过基因消融CRAC通道亚基。在目标1中，我们直接测试CalciMedica开发的新型CRAC通道抑制剂是否可以减少小胶质细胞活化，在MCAo小鼠模型中的神经炎症和最终的梗死面积。这些药物的药理作用阻断剂将通过双光子成像进行表征，并评价其体内免疫保护作用通过细胞因子分析。在Aim2中，CRAC通道亚基Stim1和Stim2将在脑中被基因消融将评估MCAo卒中后的小胶质细胞和行为结果以及梗死面积。中风几乎每年13万美国人。如果成功，这种方法的临床转化可能有助于减轻负担这种疾病。我们的长远目标是应用这项试验性研究所收集的工具和技术到一个更广泛的R01项目，研究在缺血性脑损伤过程中所有其他脑细胞中CRAC介导的钙超载。损伤