2.3. Transient middle cerebral artery occlusion (tMCAO) model

The tMCAO model was constructed according to previously published methods [32]. Mice were anesthetized with 5 % isoflurane and the anesthesia was maintained at a level of 2 % isoflurane during the surgery. Buprenorphine (0.1 mg/kg, subcutaneously injection) was administered 1 h before surgery. Body temperature was maintained at 37 °C with a heating pad. The right external carotid artery (ECA) was isolated and a standardized nylon filament (RWD Life Science, Cat# MSMC21B120PK50, China) inserted into the origin of the right middle cerebral artery (MCA) via the right internal carotid artery (ICA). The filament was removed to allow reperfusion 60 min after surgery. Cortical cerebral blood flow (CBF) was recorded using a laser Doppler flowmeter (RFLSI III, RWD Life Science, China), with the reduction of blood flow to 20 % of the baseline being considered successful occlusion. Mice in the Sham group underwent the same procedure except for filament insertion.