Mice were anesthetized with 2% isoflurane (RWD, China), and a midline cervical incision was made to expose the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The CCA was ligated distally, and two ligatures were placed on the ECA, one at its distal end and the other at the ECA-ICA bifurcation. A silicon-coated filament (RWD, China) was inserted into the ICA through a small incision in the ECA to occlude the middle cerebral artery. After 2 hours, the filament was removed to initiate reperfusion. Rectal temperature was maintained at 37°C throughout surgery. Sham-operated mice underwent identical procedures without filament insertion.