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

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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.