The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was used to carry out the research (Fluri et al., 2015 (link)). Rats were anesthetized with pentobarbital sodium (40 mg/kg) by intraperitoneal injection and the pain reflex was detected by the Randall-Selitto deep pressure test (calipers applied to the hind paw of the rat) in the perioperative period. Surgery was performed after the pain reflex disappeared. Briefly, the rat underwent a neck incision, exposing the right external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA). After ligation of the distal ECA and proximal CCA, we clipped the ICA and made a small cut at the distal end of the CCA ligation. A thread was inserted (0.38–0.40 mm; MSRC40B200PK50, Shen Zhen RWD Life Science Co., Ltd., Shenzhen, China) with a thick head to approximately 16–20 mm and fixed. After 2 h, the thread tail was pulled out but the head was retained to restore blood circulation. The skin incision was then sutured. Sham rats underwent the same procedure but without occlusion. A successful model could be judged by Horner syndrome in the left eye when the rats awakened, bending of its right forelimb on lifting the tail, and the ability to move in a circle as they moved autonomously on the ground. Rats with massive bleeding, subarachnoid hemorrhage, and premature death/drop-out were excluded after cerebral ischemia–reperfusion injury (Feng C. et al., 2020 (link)). A total of 198 SD rats were operated on, in this experiment, of which 155 were finally included in the experiment and 16 were excluded due to unsuccessful molding; the mortality rate was 13.64%.
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