Voltage-sensitive dye imaging reveals chronic peri-infarct hyperexcitability after MCAO in mice

Plastic changes in the peri-infarct cortex are thought to play a key role in post-stroke functional recovery, yet the circuit-level mechanisms underlying such reorganization remain incompletely understood. In this study, we investigated chronic changes in perilesional cortical excitability and synaptic organization using a mouse model of middle cerebral artery occlusion (MCAO). Evoked cortical activity was assessed by voltage-sensitive dye (VSD) imaging following direct cortical stimulation. The ischemic hemisphere exhibited significantly greater response amplitude and broader spatial spread than the non-ischemic hemisphere, indicating enhanced perilesional cortical excitability. Immunohistochemical analyses focused on superficial cortical layers, the primary source of VSD signals, revealed significant increases in both excitatory (VGLUT1-positive) and inhibitory (VGAT-positive) synaptic markers, with a greater increase in excitatory inputs, resulting in a shift in the excitatory-inhibitory (E/I) balance toward excitation. Reduced NeuN-positive neuronal density was also observed, suggesting concurrent neuronal degeneration. These findings demonstrate chronic peri-infarct hyperexcitability after MCAO and suggest that altered synaptic E/I balance may contribute to persistent changes in cortical network responsiveness. This circuit-level reorganization may represent an important mechanism underlying post-stroke plasticity and a potential target for therapeutic modulation.