Sham operated rats were subjected to exactly the same anesthesia and surgical treatments as animals subjected to world wide ischemia, except that the carotid arteries weren’t occluded. In most instances, anesthesia was discontinued just after initiation of carotid artery occlusion. The anesthesia was initiated again right after the non disturbing aneurism clips were removed and maintained before the icv treatments were complete. Altogether, animals were under anesthesia 5 min before carotid artery occlusion and again for approximately 15 min buy AG-1478 beginning just after reperfusion to inject drugs. Human body temperature was monitored and maintained at 0. 5 C having a rectal thermistor and warmth lamp until recovery from anesthesia. The following were excluded in the study: animals that failed to show total loss of the righting reflex and pupilar dilation, animals that displayed apparent behavioral manifestations, and animals with loss of more than 20% of body weight by 1 week after ischemia. Ninety three rats were put through worldwide ischemia. There were 3 deaths due to respiratory arrest, 9 other rats were excluded from the study because they did not show neurological symptoms of ischemia. Halothane anesthetized animals were injected with 50 ug of estradiol or vehicle in 5 ul of saline by unilateral injection into the right lateral ventricle at a flow rate of 5 ul/ minute just after reperfusion. Some animals were injected together with the PI3K inhibitor LY294002 or vehicle soon after estradiol Gene expression or vehicle injection and again 12 h later. Intracerebroventricular injections of 75% DMSO have no apparent harmful consequences. Animals were situated in a Kopf small animal stereotaxic frame with the incisor bar lowered 0. 4mm below horizontal zero. A stainless-steel cannula was diminished stereotaxically into the right lateral ventricle to a situation described by these coordinates: 0. 92mm posterior to bregma, 1. 2mm lateral to bregma, 3. 6mm below the skull surface according to the atlas of Paxinos and Watson. Neuronal cell loss was assessed by histological study of toluidine blue stained brain sections at the amount of the dorsal hippocampus from car and estradiol infused animals killed at seven days after ischemia. Animals were deeply anesthetized with pentobarbital, blood was obtained by cardiac puncture for assay of plasmaestradiol levels and perfused transcardially with snow cold4%paraformaldehyde in PBS. Brains were removed and immersed in fixative. Coronal sections were cut at the level of the dorsal hippocampus with an electric cryostat, and every fourth section was collected and stained with toluidine blue. How many remaining pyramidal neurons per 250 um period of the medial CA1 pyramidal cell layer was measured bilaterally in 4 sections per animal as described under a microscope at 40 magnification.