Middle Cerebral Artery Occlusion Model

Focal cerebral ischemia was modeled by occluding the left middle cerebral artery (MCAO), based on the methods described by Longa et al. [26]. Briefly, the mice were anesthetized with chloral hydrate (30 mg/kg, intraperitoneal injection). A midline neck incision was then made to expose the left common carotid artery, the external carotid artery, and the internal carotid artery, which were all then isolated and ligated. A monofilament coated with silicone rubber (Xinong, 1418A, Beijing, China) was inserted into the internal carotid artery (9–10 mm) through the common carotid artery, to the beginning of the middle cerebral artery (MCA). A laser Doppler approach was used to monitor MCA occlusion and reperfusion as we previously described [27]. For this procedure, a small incision was made in the skin overlying the temporalis muscle and a 0.7-mm flexible laser Doppler probe (model P10) was positioned on the superior portion of the temporal bone (6 mm lateral and 2 mm posterior from the bregma). One hour after the induction of ischemia, the monofilament was removed to restore blood flow. Relative cerebral blood flow had to rise to at least 50 % of preischemic levels for the mice to be included in the study and subjected to further analyses. The body temperature of the mice was maintained at 37.0 ± 0.5 °C during surgery, and the mice were kept in a well-ventilated room at 25 ± 3 °C in individual cages, with the provision of food and water, until they regained full consciousness.