Rat Model of Focal Cerebral Ischemia

All animal experiments were carried out according to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.¹⁴ Male Sprague Dawley rats, weighing 300-330 g (Beijing Vital River Experimental Animal Science and Technology Co, Ltd, Beijing, China), were anesthetized by inhaling 4% isoflurane in the O₂ mixture on a warming pad at 37°C. Anesthesia was maintained with 2.5% isoflurane. The right middle cerebral artery occlusion (MCAO) was established by inserting a silicone coated nylon filament (φ = 0.028 cm) as previously described.¹⁵ Generally, under the operating microscope, the right external carotid artery (ECA) and internal carotid artery (ICA) were exposed through a midline incision. Nylon filaments were inserted from the ECA into the ICA and gently advanced approximately 20 mm until resistance indicating that the tip of the filament had passed through and blocked the origin of the middle cerebral artery. Reperfusion was accomplished by removing the filament at 1.5 hours after MCAO. Sham-operated animals also underwent the same process, but sutures did not exceed the ICA bifurcation.