Adult (3-month old) Sprague Dawley female rats were bilaterally ovariectomized. Placebo or 17β-estradiol (E2) Alzet minipumps (0.025mg; 14-21 day release) were implanted subcutaneously in the upper mid-back region under the skin at the time of ovariectomy (Immediate, Imm) and global cerebral ischemia (GCI) performed one week later. In some animals, long-term E2 deprivation was performed in which the animals were ovariectomized and 10 weeks later (10W), placebo or E2 minipumps were implanted and one week later GCI was performed (see Supplemental Figure 1). The dose of E2 used produces serum E2 levels of ∼10 pg/ml, which represents physiological low Diestrus I levels of E2 (Q. G. Zhang et al., 2008 (link)). For GCI, all animals (except sham control) underwent 4-vessel occlusion (4-VO) GCI performed as described previously (W. A. Pulsinelli and J. B. Brierley, 1979 (link); W. A. Pulsinelli and A. M. Buchan, 1988 (link); Q. G. Zhang et al., 2006a (link); Q. G. Zhang et al., 2008 (link)). Briefly, 24h after electrocautery of the vertebral arteries, the common carotid arteries (CCAs) were 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 GCI were selected for the experiments, as this indicates successful GCI. 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 CCAs were simply exposed, but not occluded.