2.4. Animal Model of Global Cerebral Ischemia, Experimental Design, and Treatment

Transient forebrain ischemia was induced in adult male Wistar rats (10–12 weeks, Charles River) by the standard four-vessel occlusion model (4VO) described previously [28,29]. Briefly, both vertebral arteries were irreversibly occluded by electrocoagulation under anesthesia, with a mixture of atropine, ketamine, and diazepam (0.25, 62.5, and 5 mg/kg, respectively) delivered through intraperitoneal injection. After 24 h, ischemia was induced by carotid occlusion with atraumatic clips, for 15 min, and then the clips were removed from the carotid arteries to allow reperfusion. Body temperature was maintained at 37 °C during the surgical procedure. The animals were studied after 5 days of reperfusion (R5d) after which they were sacrificed. Sham control animals were prepared in the same way, without carotid occlusion. We performed a power analysis (http://www.biomath.info/power/ttest.htm) to determine the sample size. Significance level and statistical power were set at 0.05 and 0.8 (80%), respectively, which afforded a sample size of <6 subjects per group. The treatments were administered with concealed allocation, and in randomized order, according to a computer-generated randomization program. Ischemic animals were treated with a vehicle solution (saline/ethanol, 90:10 vol/vol) or nitrone ISQ-201, dissolved in a vehicle solution through a single intravenous tail injection, when the carotid arteries were unclamped for reperfusion. In therapeutic window experiments, the animals were treated once at the reperfusion onset or at the set reperfusion time points, after ischemia. For the administration, at 6 h and 48 h of reperfusion after ischemia, the animals were anesthetized with isoflurane for 2–3 min and 5 min, respectively, prior to the treatment administration. An independent researcher prepared the treatments for each animal, according to the randomization schedule. Data of each experimental group were normalized to each vehicle control group. All procedures associated with animal experiments were approved by the Animal Care Ethics Committee of the Hospital Universitario Ramón y Cajal (Madrid, Spain, code no. 06/2015) and were performed according to the ARRIVE guidelines.