Loss of contractile pericytes and their impaired calcium dynamics exacerbate brain ischemic stroke of awake mice in acute and chronic phases

Ischemic stroke frequently results in persistent neurovascular uncoupling, whereby neuronal activity fails to evoke appropriate microvascular responses despite restoration of upstream blood flow. The cellular mechanisms governing this dysfunction along the arteriolar-capillary continuum remain poorly understood. Using two-photon microscopy and laser speckle imaging in awake Acta2-GCaMP8 mice subjected to transient middle cerebral artery occlusion, we examined calcium signaling and contractile function of vascular smooth muscle cells, precapillary sphincters (PS), and contractile pericytes during stroke progression. During acute phase, PSs exhibited pronounced calcium elevations and strong constriction, amplifying downstream capillary constriction through spatially localized calcium signaling. Following reperfusion, excessive calcium elevations persisted without proportional diameter changes, indicating calcium signaling dysregulation and early uncoupling between mural cell calcium dynamics and vascular responses. In the chronic phase, ischemia induced PS-associated loss of contractile pericytes, leading to capillary dilation and sustained impairment of neurovascular coupling. Although pericyte coverage and calcium signaling partially recovered from second week post stroke, whisker-evoked vascular responses and calcium sensitivity remained compromised. At the network level, blood flow responses became spatially heterogeneous, with pericyte-lost regions exhibiting prolonged hyporesponsiveness during functional hyperemia. These findings identify PSs as key regulators of ischemia-induced microvascular dysfunction and highlight mural cell calcium dysregulation and pericyte loss as drivers of persistent neurovascular uncoupling after stroke.