ImmunoStroke: Microglial responses controlling post-stroke angiogenesis and microvascular integrity

该项目研究了小胶质细胞通过促进微血管完整性和抑制白细胞浸润来保护脑微血管的假设，这些机制至少部分依赖于ApoE。将通过将小鼠暴露于短暂的大脑中动脉细丝闭塞（tMCAO）（一种临床相关的缺血/再灌注损伤模型）来研究这一概念。伴随地，在Cx 3cr 1CreER-hM 3Dq DREADD（设计者受体仅由设计者药物激活）小鼠中，小胶质细胞活化将被集落刺激因子-1受体（CSF 1 R）抑制剂阻断或被氯氮平N-氧化物刺激，或通过将TREM 2抗体4D 9递送至野生型小鼠。使用Cx 3cr 1creERT 2ApoEflox/flox小鼠的小胶质细胞ApoE选择性缺陷，我们将阐明小胶质细胞ApoE介导微血管和脑实质反应的作用。内皮选择性转录组分析将解开微血管基因表达的签名，响应小胶质细胞失活或过度激活。同时，将使用体内磁共振成像（MRI）/磁粒子成像（MPI）和离体3D光片荧光显微镜（LSFM）的组合研究微血管重塑和血管生成。将通过荧光激活细胞扫描（FACS）研究脑炎症反应。利用Slco 1c 1-icre/ERT 21 Mrks Kdrtm 2Sato小鼠，选择性缺乏血管内皮生长因子受体-2（VEGFR 2）的脑微血管内皮细胞，我们将研究缺血后血管生成对脑实质重塑和神经恢复的贡献。我们的首要假设是，当小胶质细胞反应被激活和血管生成被促进时，微血管完整性的保存将免疫平衡向抗炎状态转移，从而实现长期神经元存活、成功的脑重塑和神经恢复，而小胶质细胞失活有利于促进继发性神经退行性过程的微血管促炎状态。

This project examines the hypothesis that microglia protect the cerebral microvasculature by promoting microvascular integrity and inhibiting leukocyte infiltration in the brain via mechanisms, which are at least partly ApoE-dependent. This concept will be investigated by exposing mice to transient filament middle cerebral artery occlusion (tMCAO), a clinically relevant ischemia/ reperfusion injury model. Concomitantly, microglial activation will either be blocked with colony-stimulating factor-1 receptor (CSF1R) inhibitors or stimulated by clozapine N-oxide in Cx3cr1CreER-hM3Dq DREADD (designer receptors exclusively activated by designer drugs) mice, or by delivery of the TREM2 antibody 4D9 to wildtype mice. Using Cx3cr1creERT2ApoEflox/flox mice selectively deficient for ApoE in microglial cells, we will elucidate the role of microglial ApoE in mediating microvascular and brain parenchymal responses. Endothelial-selective transcriptome analyses will unravel microvascular gene expression signatures in response to microglial deactivation or overactivation. In parallel, microvascular remodeling and angiogenesis will be studied using a combination of in vivo magnetic resonance imaging (MRI)/ magnetic particle imaging (MPI) and ex vivo 3D light-sheet fluorescence microscopy (LSFM). Brain inflammatory responses will be studied by fluorescence-activated cell scanning (FACS). Utilizing Slco1c1-icre/ERT21Mrks Kdrtm2Sato mice, selectively deficient for vascular endothelial growth factor receptor-2 (VEGFR2) in cerebral microvascular endothelial cells, we will study the contribution of post-ischemic angiogenesis to brain parenchymal remodeling and neurological recovery. Our overarching hypothesis is that when microglial responses are activated and angiogenesis is boosted, the preservation of microvascular integrity shifts the immune balance toward an anti-inflammatory state, enabling long-term neuronal survival, successful brain remodeling, and neurological recovery, while microglial deactivation favors a microvascular proinflammatory state promoting secondary neurodegenerative processes.