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

• 批准号: 10303222
• 负责人: Petr Tvrdik
• 金额: $ 28.26万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303222
• 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/antagonist; 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

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.

抽象的 缺血性中风是美国和全球严重残疾的主要原因。中风引起的缺氧 促进一系列病理生理反应导致缺血性核心坏死，凋亡 在被称为半肿的垂直组织中。细胞死亡会触发炎症反应 继发损伤并可能损害幸存最初侮辱的神经元。小胶质细胞是主要的 脑实质中的免疫细胞，但它们在继发损伤和潜在机制中的特定作用 诱导仍然不清楚。我们假设增加的钙信号传导是急性的关键机制 中风引起的小胶质细胞激活，可能导致促炎细胞因子的释放增加。我们 已经开发了一个指示小胶质细胞中细胞内钙的小鼠报告基因。在此系统中，我们使用 2-光子成像和脑动脉闭塞（MCAO）研究小胶质细胞对缺血性损伤的反应 体内。我们已经证明了肉毒内部的皮质小胶质细胞中钙活性的周期性波 闭塞，与皮质扩散去极化（CSD）的模式一致。我们建议测试 通过药理学抑制钙涌入的这些钙瞬变，由钙释放介导 激活的钙（CRAC）通道，以及通过CRAC通道亚基的遗传消融。在AIM 1中，我们将直接 测试钙甲藻开发的新型CRAC通道抑制剂是否可以减少小胶质激活， MCAO小鼠模型中的神经炎症和最终梗死大小。这些药理作用 将用2光子成像来表征阻滞剂，并将评估其在体内的免疫保护作用 通过细胞因子分析。在AIM2中，CRAC通道亚基STIM1和STIM2将在大脑中遗传消融 将评估MCAO中风后的小胶质细胞和行为结果和梗死大小。中风几乎杀死 每年有13万美国人。如果成功，这种方法的临床翻译可以帮助减轻负担 这种疾病。我们的超越目标是应用此试点研究中组装的工具和技术 在缺血性的所有其他脑细胞中研究CRAC介导的钙过载的更广泛的R01项目 受伤。