Preparation of rat models of cerebral ischemia/reperfusion

A total of 96 healthy, clean, male Sprague-Dawley rats aged 6–7 weeks and weighing 280–300 g were provided by the Hebei Provincial Experimental Animal Center of China (license SCXK (Ji) 2003-1-003). The rats were housed at 25 ± 3°C and humidity of 55 ± 10%, and were allowed free access to food and water. The experiment was approved by the Animal Ethics Committee of Henan University of Traditional Chinese Medicine in China. The 96 rats were equally and randomly divided into sham operation, model, mobilization, high-, moderate-, low-dose RIPTFs groups.

In all groups, except the sham operation group, rats were intraperitoneally anesthetized with 10% chloral hydrate 0.3 mL/100 g (Tianjin Municipality Kemi’ou Chemical Reagents Development Center, Tianjin, China) after fasting for 12 hours. The rats were fixed on the operation table in a supine position. A median incision was made on the neck. According to the method of Kitagawa et al. (1991), a nylon thread (approximately 0.30 mm in diameter) coated with 1 mm of silicone rubber on the head end was inserted into proximal end of the middle cerebral artery through the left common carotid artery to a depth of 18–20 mm to block the blood supply of the middle cerebral artery for 2 hours. The nylon thread was then taken out, and the left middle cerebral artery was occluded. Intraoperative temperature was maintained at 23–24°C. In the sham operation group, middle cerebral artery was exposed under anesthesia but had no embolism. Rats were scored at 24 hours after ischemia and reperfusion in accordance with the Longa's method (Longa et al., 1989): 0 points, no nerve damage symptoms; 1 point, cannot fully extend right forepaw; 2 points, circling to the right; 3 points, dumping to the opposite side; 4 points, cannot walk by itself, loss of consciousness; 5 points, death. The rats scoring 1 point or above were considered successful ischemic models.