The role of CD11c+ microglia in post-ischemic stroke recovery

项目总结/摘要 目前对缺血性中风患者的治疗选择允许血液再灌注到大脑，但失败了。 以解决神经缺陷，这会导致中风幸存者长期的身体和认知障碍。 因此，改善脑恢复和解决神经功能缺损的新治疗策略是至关重要的。 needed.神经元死亡诱导表达促炎介质的小胶质细胞活化，导致 缺血性中风后脑损伤的加重。因此，靶向小胶质细胞被认为可以改善 中风后的脑损伤然而，最近的研究表明，小胶质细胞的消除增加了大小 脑梗死和脑梗死后暂时性大脑中动脉闭塞小鼠的神经功能缺损 （tMCAO），用于缺血性中风研究的动物模型。这些数据表明， 小胶质细胞对缺血性损伤的反应，或小胶质细胞从神经毒性到神经毒性的暂时表型转变， 在缺血性损伤后随时间的神经保护作用。本研究的目的是研究CD 11 c- 介导的信号增强小胶质细胞的这些不同功能或表型转变，并确定 CD 11 c+小胶质细胞在缺血性卒中后脑恢复中的潜在神经保护作用CD 11 c是一种 整合素α链蛋白，其广泛用作常规树突状细胞（cDC）的定义标志物。 CD 11 c调节cDC的细胞粘附、迁移和吞噬活性。CD 11 c介导的信号传导的作用 缺血性中风后小胶质细胞的变化尚不清楚。转录组分析表明，CD 11 c+小胶质细胞可能 在神经损伤后起保护作用，但缺乏支持这一观点的实验证据。 我们在Ccr 2 +/RFP x CD 11 c-EYFP小鼠中进行了tMCAO，并观察到CD 11 c + tMCAO后大脑中的小胶质细胞。我们的初步数据显示：1）CD 11 c+小胶质细胞主要是 tMCAO后72小时出现在脑梗死边缘。这些小胶质细胞主要共同- 表达激活标志物IBA-1; 2）CD 11 c + IBA-1+小胶质细胞迁移并维持在 tMCAO后14天的胶质瘢痕;和3）在第14天，CD 11 c + IBA-1-小胶质细胞亚群出现在tMCAO后14天， 大脑的梗塞区域。在目的1中，我们假设小胶质细胞中的CD 11 c表达促进脑恢复 通过引导小胶质细胞迁移到梗死核心，并增强其清除死细胞的能力。我们 将使用CD 11 c-EYFP报告基因小鼠与CD 11 c缺陷小鼠杂交来解决这一假设。的作用 小胶质细胞中的CD 11 c在促进缺血性卒中后的脑恢复中的作用将通过单因素试验进一步确定。 细胞RNA测序。在目标2中，我们假设慢性期CD 11 c+小胶质细胞的消除 缺血性损伤后增加脑梗塞并加重神经功能缺损。我们将检验这一假设 使用骨髓嵌合体策略，其中tMCAO将在CD 11 c+小胶质细胞被 选择性消融。阐明小胶质细胞从促炎性转变为 神经保护可以帮助开发新的策略，促进缺血性中风后的神经恢复。

Project Summary/Abstract Current treatment options for patients experienced ischemic stroke allow blood reperfusion to the brain but fails to resolve the neurological deficits, which cause long-term physical and cognitive disabilities in stroke survivors. Thus, novel therapeutic strategies for improving brain recovery and resolving neurological deficits are critically needed. Neuronal death induces the activation of microglia that express proinflammatory mediators, leading to the exacerbation of brain damage following ischemic stroke. Thus, targeting microglia was thought to ameliorate brain injury post-stroke. However, recent studies have shown that the elimination of microglia increases the size of brain infarct and worsens the neurological deficits of mice following transient middle cerebral artery occlusion (tMCAO), an animal model for ischemic stroke studies. These data suggest a functional heterogeneity of microglia in response to ischemic insults, or a temporal phenotypic shift of microglia from neurotoxic to neuroprotective over time following ischemic injury. The goal of this proposal is to investigate whether the CD11c- mediated signaling potentiates these diverse functions or phenotypical shift in microglia, and to determine the potential neuroprotective role of CD11c+ microglia in brain recovery following ischemic stroke. CD11c is an integrin alpha chain protein that is widely used as a defining marker for conventional dendritic cells (cDCs). CD11c regulates cell adhesion, migration, and phagocytic activity of cDCs. The role of CD11c-mediated signaling in microglia following ischemic stroke is not known. Transcriptome analysis suggests that CD11c+ microglia may play a protective role following neurological insults, but experimental evidence to support this notion is lacking. We performed tMCAO in Ccr2+/RFP x CD11c-EYFP mice, and observed a substantial increase of CD11c+ microglia in the brain following tMCAO. Our preliminary data have shown that 1) CD11c+ microglia mainly appeared at the border of infract of the brain 72 hours following tMCAO. These microglia predominately co- expressed the activation marker IBA-1; 2) CD11c+ IBA-1+ microglia migrated to and were maintained within the glial scar 14 days following tMCAO; and 3) On day 14, a subset of CD11c+ IBA-1- microglia appears at the peri- infarct area of the brain. In Aim 1, we hypothesize that CD11c expression in microglia promotes brain recovery by directing the migration of microglia to the infarct core, and enhances their ability to eliminate dead cells. We will address this hypothesis using CD11c-EYFP reporter mice crossed with CD11c-deficient mice. The role of CD11c in microglia in promoting brain recovery following ischemic stroke will be further determined by single- cell RNA sequencing. In Aim 2, we hypothesize that the elimination of CD11c+ microglia at the chronic phase following ischemic injury increases brain infarct and exacerbates neurological deficits. We will test this hypothesis using a bone marrow chimera strategy, in which tMCAO will be performed in mice that CD11c+ microglia are selectively ablated. Elucidating the mechanisms by which microglia switch from proinflammatory to neuroprotective can help develop novel strategies that promote neurological recovery following ischemic stroke.