Permanent focal cerebral ischemia was produced by endovascular occlusion of the left middle cerebral artery (MCAO) as described previously [15 (link)]. Briefly, animals were anesthetized by intraperitoneal injection of pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan). Body temperature was maintained at 36.5–37.5 °C throughout surgery. After a midline neck incision, the left common carotid artery was isolated under a microscope and ligated with a 4–0 silk suture (Ethicon, Issy-Les-Moulineaux, France). The external and internal carotid arteries were temporarily ligated with a 4–0 silk suture. An arteriotomy was performed proximal to the bifurcation of the common carotid artery. A silicone-coated nylon monofilament (40 mm long, 0.26 mm diameter, Beijing Sunbio Biotech, China) was introduced through the arteriotomy and advanced into the internal carotid artery up to a distance of 18–20 mm to occlude the origin of the middle cerebral artery. Four hours after this procedure, the rats were reanesthetized and the middle cerebral artery blood flow was restored by withdrawing the nylon monofilament. After surgery, the rats were returned to their home cages and maintained at 30 °C with free access to food and water.