Transient Global Ischemia
Transient global ischemia was induced by using the 4-VO method with minor modifications (Pulsinelli et al., 1982; Han et al., 2016). Briefly, rats were anesthetized by intraperitoneal (i.p.) injection of 400 mg/kg chloral hydrate (Fluka, Buchs, Switzerland). Body temperature was maintained at 37.0 ± 0.2°C during surgery using heating pads. The bilateral vertebral arteries were exposed and permanently electrocauterized. Both common carotid arteries (CCAs) were occluded with carotid artery clips for 15 min, and blood flow was monitored at a point in the skull (bregma: −2.0 mm, midline: 5.0 mm) with a probe connecting a laser-Doppler flowmetry device (MoorVMS-LDF2, Moor Instruments, Devon, UK) before, during and after ischemia. The ischemic model was considered successful when the cerebral blood flow decreased to 10%–15% of pre-ischemic levels and returned to the baseline (100 ± 10%) after release of the carotid artery clips. Sham animals underwent all aspects of handling and surgery without occlusion of the vertebral and CCAs.