Transient Middle Cerebral Artery Occlusion (MCAO) Model and Sham Surgery

Adult, male, Sprague-Dawley rats (300 to 350 g, BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were used in this study. The rats were housed in groups of 4 at an ambient temperature of 22 ± 1 °C with a 12-h light–dark cycle according to the guidelines of the Institutional Animal Care and Use Committee (approval no. 104042801) at Chi Mei Medical Center. Pellet rat chow and tap water were available ad libitum. The rats were anesthetized with sodium pentobarbital (25 mg/kg, intraperitoneally; Sigma-Aldrich, St. Louis, MO, USA) and a mixture containing ketamine (4.4 mg/kg, intramuscularly [i.m.]; Nankuang Pharmaceutical, Taipei, Taiwan), atropine (0.02633 mg/kg, i.m.; Sintong Chemical, Taoyuan, Taiwan), and xylazine (6.77 mg/kg, i.m.; Bayer AG, Leverkusen, Germany). Experimental stroke was induced by transient MCAO as previously described.¹⁴ We have standardized the MCAO model, with stroke rats showing ≥80% reduction in regional cerebral blood flow during a 1 h occlusion period as determined by laser Doppler (OxyFlo™, Oxford Optronix Ltd., Oxford, UK). Focal cerebral ischemia was maintained for 1.5 h. The incision was closed, and 1 cm of the nylon suture was left protruding, so it could be withdrawn to allow reperfusion. After surgery, anesthesia was discontinued, and the rat was allowed free access to food and water. For sham surgery, each animal underwent the same surgical procedures without MCAO and received the same postoperative care.