Abstract
OBJECTIVE: Following ischemic stroke (IS), activated microglia activity could contribute to neuronal injury and blood-brain barrier (BBB) disruption. The upstream vascular-derived signal initiating this transition remains unclear; therefore, we investigated whether perivascular SPP1 regulates microglia-mediated synapse engulfment during IS. METHODS: Male C57BL/6 mice were assigned to sham, shSpp1, middle cerebral artery occlusion/reperfusion (MCAO/R), and MCAO/R + shSpp1 groups. Cerebral perfusion was assessed using laser speckle contrast imaging and super-resolution vascular imaging, while neuronal injury was evaluated using Nissl and TUNEL staining. Proteomic profiling of the ischemic penumbra identified regulators of microglia-mediated synaptic remodeling. Synaptic structure and glial-vascular unit (GVU) integrity were examined using transmission electron microscopy, immunofluorescence, and molecular analyses. Behavioral outcomes were assessed using the open-field, Barnes maze, rotarod, and wire-hanging tests. RESULTS: Compared with sham controls, MCAO/R mice displayed increased microglial synaptic engulfment and ultrastructural synaptic damage in the ischemic penumbra, accompanied by reduced synaptic protein expression. Proteomic analysis revealed upregulation of inflammatory and vascular-related pathways, with marked upregulation of SPP derived from perivascular macrophages. Spp1 silencing attenuated neuroinflammation, reduced infarct volume, improved cerebral perfusion, preserved GVU integrity, and alleviated behavioral deficits. Spp1 suppression also reduced microglial synaptic engulfment in vivo and restored synaptic protein and mRNA levels in vitro. CONCLUSION: Targeting perivascular SPP1 suppresses excessive microglia-mediated synaptic engulfment, preserves BBB integrity and synaptic architecture, and offers a GVU-centered therapeutic strategy for IS.