MCAO Model Establishment

Rats were subjected to 2 h right middle cerebral artery occlusion (MCAO) using a modified intraluminal filament technique that was first put forward and interpreted by Koizumi et al. (15) in rats. The rats were weighed, and then administered an intra-peritoneal injection of 4% chloral hydrate (0.1 ml/10 g i.p.) as an anesthetic agent (16, 17). After the anesthesia, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) of rats were exposed through an incision in the middle on the neck. Subsequently, the proximal CCA and ECA were ligated, and an arterial clip was placed on the distal end of the CCA in order to block the blood flow to prevent bleeding when the filament was inserted. A filament with a diameter of 0.38 mm, whose tip was rounded by heating and coating with 0.01% poly-L-lysine, was inserted into the right CCA. The filament was advanced 18–20 mm further above the bifurcation until there was resistance, reaching, and occluding the ostium of the right middle cerebral artery (MCA). After that, the incision of the ICA was ligated, and the filament was secured in place for 2 h, after which the wound was closed, and the animals were allowed to awaken. Body temperature was maintained at 36.5–37.5°C using a heating pad during the procedure. Two hours post-occlusion, the filament was slowly withdrawn under anesthesia and animals were then returned to their cages for reperfusion (MCAO/R) (18, 19). Animals were assessed for functional impairment using the modified Bederson grading system to verify accurate occlusion of the middle cerebral artery when the rats were awakened (20, 21). Motor deficits were graded from 0 to 4. A score of 0 was given for no visible neurological deficits; a score of 1 was given for forelimb flexion; a score of 2 was given for contralateral weak forelimb grip (the operator places the animal on an absorbent pad and gently pulls the tail); a score of 3 was given for circling to the paretic side only when the tail was stimulated; and a score of 4 was given for spontaneous circling (20, 22, 23). Finally, animals with positive performances were included in our study.