Transient middle cerebral artery occlusion (MCAO) model and animal treatment

Rats were under isoflurane anesthesia. We exposed the right middle cerebral artery (MCA) through a cranial burr hole, which was located approximately 2.5 mm lateral and 2.0 mm posterior to the bregma. The blood flow of MCA was monitored using a Laser Doppler flowmetry (Moor Instruments Inc., Wilmington, USA). The blood flow of MCA was above 500 min/div before MCAO procedure. We then exposed both the right common carotid artery (CCA) and internal carotid artery (ICA) via a neck midline incision, followed by the ligation of the pterygopalatine artery proximal to its branch. We then inserted a 3–0 nylon filament suture coated with poly-L-lysine (Sigma-Aldrich, Shanghai, China) into the right external carotid artery through the CCA and moved it up to the ICA at a distance of 20–25 mm to block the right MCA. As a result, the blood flow of MCA was decreased to less than 100 min/div (>80%). Blood flow from the right MCA was blocked for 15 minutes, followed by removing the suture for reperfusion (a return to >7% of the baseline within 10 min of suture withdrawal). Sham-operated rats received the same surgery process without the MCAO. The treatment with L655,708 (α5-GABA_AR inverse agonist, 0, 1, or 5 mg/kg) was initiated at post-MCAO days 3 and 7. Rats received intraperitoneal injections of vehicle (i.e., saline) or L655,708 once daily for 4 consecutive days. Sham-operated rats received no treatment, and a separate group of MCAO rats received vehicle injections once daily for 4 consecutive days starting at post-MCAO day 1.