Adult Sprague Dawley female rats were bilaterally ovariectomized one week before performance of global cerebral ischemia. At the time of ovariectomy, placebo or 17β-estradiol time-release pellets (0.025mg; 21 day release pellet) (Innovative Research of America, Sarasota, Fl) were implanted subcutaneously in the upper mid-back region under the skin. For cerebral ischemia, all animals (excepting sham control) underwent 4-vessel occlusion (4-VO) global cerebral ischemia performed as described previously (Pulsinelli and Brierley, 1979 (link); Pulsinelli and Buchan, 1988 (link); Zhang et al., 2006a (link)). Briefly, animals were anesthetized using chloral hydrate (350 mg/kg, ip), and both common carotid arteries (CCA) of the rat were separated. A silastic ligature was placed loosely around each artery without interrupting the blood flow, and the incision was then closed. Immediately after this procedure, both vertebral arteries (VA) at the level of the alar foramina were permanently occluded with the use of electrocautery. After 24 hr recovery, the bilateral CCA were exposed under light anesthesia with 3% isoflurane, and occluded with aneurysm clips to induce 10 min forebrain ischemia. Animals which lost their righting reflex within 30 seconds and whose pupils were dilated and unresponsive to light during cerebral ischemia were selected for the experiments, as this indicates successful global ischemia. The clips were then removed, and the blood flow through the arteries was confirmed before the wound was sutured. Rectal temperature was maintained at 36.5 to 37.5°C throughout the experiment with a thermal blanket. The animals of the sham group underwent identical procedures except that the CCA were simply exposed, but there were no occlusion.