Microglial P2Y12 Drives Chronic Cerebral Hypoperfusion via Ca 2+ Activity–Mediated Neuroinflammation and Vascular Injury

BACKGROUND: Chronic cerebral hypoperfusion is a major driver of vascular cognitive impairment (VCI), but the role of microglial purinergic signaling in chronic cerebral hypoperfusion–induced neurovascular dysfunction remains undefined. This hypothesis-testing study with a novel research premise aimed to address this knowledge gap by clarifying whether microglial P2Y12 (purinergic receptor P2Y12), a microglia-enriched G-protein–coupled receptor, mediates VCI pathogenesis through regulation of microglial calcium signaling and microglia-vascular crosstalk under chronic cerebral hypoperfusion conditions. METHODS: VCI was induced in adult male mice by asymmetrical bilateral carotid artery stenosis. Microglia-specific P2Y12 knockout mice were generated using P2Y12 flox/flox :CX3CR1 (CX3C chemokine receptor 1) CreER mice. Cognitive function was assessed by object location recognition, 3-chamber social interaction, and Morris water maze tests. Cerebral blood flow was measured by laser speckle contrast imaging and functional ultrasound. Neuroinflammation, microvascular integrity, and perivascular microglial Ca 2+ activity were evaluated by immunofluorescence, flow cytometry, and 2-photon GCaMP6f (the fast variant of the genetically encoded calcium indicator GCaMP6) imaging. The clinically approved L-type Ca 2+ channel blocker nimodipine was administered starting 42 days postasymmetrical bilateral carotid artery stenosis. RESULTS: All experimental animals exhibited consistent baseline physiological and behavioral status without intergroup differences throughout the procedure. Chronic cerebral hypoperfusion induced persistent upregulation of P2Y12 in hippocampal microglia. Microglial P2Y12 genetic ablation effectively ameliorated hypoperfusion-triggered cognitive impairment, hippocampal atrophy, and neuronal loss, preserved cerebral perfusion, and inhibited sustained microglial inflammatory activation and pathological microglia-microvessel interaction. Mechanistically, P2Y12 signaling mediated aberrant ATP-triggered Ca 2+ hyperactivity in perivascular microglia, and this pathological phenotype was eliminated by microglial P2Y12 deletion. Pharmacological intervention with nimodipine recapitulated the protective effects of genetic P2Y12 inhibition, normalizing abnormal microglial calcium activity, alleviating neuroinflammation and disruptive microglia-vascular crosstalk, maintaining cerebral structural and perfusion homeostasis, and rescuing cognitive dysfunction in model mice, while exerting no adverse effects on control animals. CONCLUSIONS: The microglial P2Y12/Ca 2+ axis is a critical pathogenic driver of chronic cerebral hypoperfusion–induced VCI, linking sustained microglial activation to neuroinflammation and cerebral microvascular injury. Pharmacological inhibition of this axis with nimodipine recapitulates the benefits of genetic P2Y12 deletion, identifying a clinically translatable therapeutic strategy for VCI treatment. All conclusions are fully supported by the comprehensive behavioral, imaging, and molecular experimental results obtained in this study.