Animals have been positioned in the Kopf little animal stereotaxic frame using the incisor bar lowered .mm below horizontal zero. A stainless steel cannula was lowered stereotaxically to the ideal lateral ventricle to a place defined through the following coordinates mm posterior to bregma mm lateral to bregma, ?mm under the skull surface in accordance with the atlas of Paxinos and Watson Histological evaluation Neuronal cell reduction was assessed by histological examination of toluidine blue stained brain sections at the degree with the dorsal hippocampus from motor vehicle and estradiol infused animals killed at days immediately after ischemia. Animals have been deeply anesthetized with pentobarbital , blood was collected by cardiac puncture for assay of plasmaestradiol ranges and perfused transcardially with ice cold paraformaldehyde in PBS . Brains were eliminated and immersed in fixative . Coronal sections were cut with the degree of the dorsal hippocampus with an electronic cryostat , and just about every fourth area was collected and stained with toluidine blue.
The quantity of surviving pyramidal neurons per m length of the medial CA pyramidal cell layer was counted bilaterally in sections per animal as described underneath a light microscope at magnification. Cell counts through the suitable and left hippocampus on each and every of the four sections had been averaged this content to provide a single worth for each animal Serum estradiol assay Tubes containing full blood were positioned on ice and centrifuged at g for min. Serum was collected and stored right up until analyzed. Serum hormone ranges have been measured by fluoroimmunoassay by using the DELPHIA estradiol assay . All assays had been performed in duplicate, and also the imply value was reported. The sensitivity of detection is pg ml. The interand intra assay coefficients of variance are . and respectively Western blot evaluation For quantification of protein abundance during the hippocampal CA, Western blot examination was performed as described . In brief, experimental and sham animals had been deeply anesthetized with pentobarbital , blood was collected by cardiac puncture for assay of plasma estradiol amounts and killed by decapitation at , and h soon after reperfusion.
Hippocampi were quickly dissected, and transverse slices of dorsal hippocampus were cut by using a Mcllwain tissue chopper. The CA was swiftly micro dissected, positioned in ice cold saline supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail and homogenized in lysis buffer containing HEPES , MgCl , EGTA , dithiothreitol , sucrose , protease inhibitor Vinflunine cocktail and phosphatase inhibitor cocktail . Part of the sample from each animal was used to isolate cytosolic fraction by differential centrifugation. Proteins from whole cell lysates and cytosolic fractions were separated by SDS Webpage and subjected to Western blot examination.