The experiment was conducted both in vitro and in vivo. In vitro experiments, PC12 cells were received physiological and pharmacological doses of estrogen stimulation . Morphological changes of cells were observed by light microscopy, and cell proliferation was detected by Brdu incorporation and flow cytometric analysis. After PC12 cells were exposed to oxygen and glucose deprivation (OGD) for 4 hours (h), the cells were reperfused for 20 h. Cell viability was detected by MTT assay, cell damage was validated by LDH release assay, and cell apoptosis was detected by flow cytometric analysis and western blot. In in vivo experiments, 12 weeks-old female Sprague–Dawley (SD) rats were ovariectomized (OVX), and following a 10-day recovery period, the animals were subjected to a daily subcutaneous injection of different doses of E2 for 4 weeks via an injection on the back of the neck. The animals were then subjected to middle carotid artery occlusion (MCAO). After 2 h of transient occlusion, cerebral blood flow was restored by removing a nylon suture for 22 h. Finally, neurological deficits were assessed by the Garcia test, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining was utilized to evaluate infarct volume. Nissl staining was used to observe the morphologic neuronal changes in ischemic penumbra; and western blot was used to detect apoptosis in ischemic penumbra. All reagents were purchased from Sigma (St Louis, Mo, USA), except those noted to be purchased from other suppliers.
The PC12 cells were plated at a density of 3 × 105cells/well in a 6-well multiwall plate or 104 cells/well in a 96-well multiwall plate at 37°C under 5% CO2 and 95% oxygen in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin (100 μg/ml) and penicillin (100 units/mL). The cells were treated with different concentrations of E2 (Cayman, America), which were diluted in dimethyl sulfoxide (DMSO) solution (1:5000). The cells were divided into several groups: group A: negative control; group B: DMSO; group C: 10 nM E2; group D: 20 nM E2; group E: 10 μM E2; and group F: 20 μM E2. After 24 h treatment, the morphology of the cells in the 6-well multiwall plates was observed and recorded using an Olympus Microscope (Tokyo, Japan).
BrdU incorporation assay
Cell proliferation was determined by immunocytochemical assessment of BrdU incorporation into replicating DNA of living cells using the Cellomics BrdU Cell Proliferation kit (Thermo Fisher Scientific, Pittsburgh, PA). Briefly, the PC12 cells were plated at a density of 1.5 × 104 cells/well on glass coverslips in 24-well multiwells and the cells were incubated with 50 μM Brdu with different concentrations of E2, as described above. After 24 h incubation, the cells were fixed with 4% paraformaldehyde for 1 h, followed by permeabilization and blocking. After washing, the sections were probed with mouse anti-BrdU primary antibody (1:500 dilution, Sigma) overnight at 4°C, followed by FITC-conjugated donkey anti-mouse IgG (1:500 dilution, Invitrogen) at room temperature for 45 minutes (min) in the dark. Propidium iodide (PI) dye was used to label all nuclei. The sections were mounted with 50% glycerol for examination under a fluorescence microscope.
Cell cycle analysis
Cell cycle was assessed by flow cytometry, as previously described . After 24 h E2 treatment, the cells were collected by trypsinization, and centrifuged in phosphate buffered saline (PBS) twice. The cells were then fixed in pre-cooled 70% ethanol at -20°C, and stained with PI solution. DNA content was determined by flow cytometry using CellQuest Software. 10,000 events were counted for each sample (FACSCalibur, Becton–Dickinson). The percentage of cells in a particular cell cycle stage was calculated by the ModFit software (Becton–Dickinson, USA).
Oxygen and glucose deprivation and reperfusion (OGD-R)
After 24 h incubation with E2, the cells were washed twice in d-Hanks buffer and switched to d-Hanks buffer (OGD medium) with different E2 concentrations. Then the cells were switched to a modular incubator chamber. The chamber was flushed with 3 L/min of a 95% N2/5% CO2 gas mixture for 30 min at room temperature at 3 L/min. The chamber was then sealed and placed in a 37°C container. OGD was carried out for 4 h. Following the OGD, the cells were incubated with DMEM (without fetal bovine serum) with different E2 concentrations for an additional 20 h reperfusion under normal conditions.
Cell viability analysis
After OGD-R, the MTT assay was used to detect cell viability, as previously described . Briefly, MTT was dissolved in DMEM, and added to each well for incubation at a final concentration of 0.5 mg/ml at 37°C for 4 h. Then, the medium was replaced with 150 μl of DMSO. The optical density (OD) was recorded on a Universal Microplate Reader (Elx 800, Bio-TEK instruments Inc., USA) at 490 nm. Cell viability was expressed as a percentage of the control value.
LDH release assay
After OGD-R, cytotoxicity was quantitatively assessed by measuring the activity of LDH released from the damaged cells into the culture medium. Briefly, cells were treated with 0.5% Triton X-100, and the media which contained detached cells were collected and centrifuged. The supernatant was used for the assay of LDH activity. The enzyme activity was determined by using an assay kit according to the manufacturer’s instructions. LDH leakage was expressed as the percentage of the total LDH activity (LDH in the medium + LDH in the cells), according to the equation LDH released (%) = (LDH activity in the medium/total LDH activity) × 100. Cultures under normal conditions (control group) represent basal LDH release.
Flow cytometric analysis
After OGD-R, cell apoptosis was assayed by flow cytometry, as previously described . Briefly,the cells were washed with 1 × annexin V-FITC binding buffer prior to staining with annexin V-FITC and PI for 15 min at room temperature in the dark. The stained cells were immediately analyzed using flow cytometry. Apoptotic and necrotic cells were quantitated by annexin V binding and PI uptake. The annexin V-FITC+/PI– cell populations were considered to represent apoptotic cells.
One hundred and twenty female adult SD rats weighing 200–220 g were obtained from the Laboratory Animal Center of the Fourth Military Medical University. The animals were maintained under a 12:12-h light–dark cycle and 25°C temperature. The experimental procedures in this study were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University, P. R. China.
OVX and estrogen replacement
The animals were randomly divided into five groups (n = 24): group A: sham control; group B: rats with OVX without E2 replacement; group C: rats with OVX and 6 μg/kg E2 replacement group; group D: rats with OVX and 20 μg/kg E2 replacement group; and group E: rats with OVX and 50 μg/kg E2 replacement group. OVX was adopted by dorsolateral incisions, as previously described . The animals in the sham group were subjected to the same operation; however, their ovaries were kept intact. Five days following OVX, vaginal smears were taken for 5 days before estrogen replacement therapy to confirm OVX and cessation of the estrous cycle . Then these animals received daily subcutaneous injection of different doses of E2 (diluted in sesame oil solution) on the back of the neck for four weeks. The dose of estrogen replacement was based on previous studies  and the results of our pilot study.
Four weeks after HRT, MCAO was conducted, as previously described . The animals within the sham group were confirmed in diestrus by vaginal smears before MCAO. After 2 h of transient occlusion, cerebral blood flow was restored by removing a nylon suture for 22 h. Physiological parameters included rectal temperature, blood pressure, heart rate, blood gas, and glucose were monitored, as previously described .
The neurological deficit was determined according to the Garcia Test , as shown in Additional file 1: Table S1.
Detection of serum estrogen level
The levels of serum estrogen were detected to confirm HRT. Briefly, after neurological evaluation, the animals were anesthetized with an overdose of pentobarbital sodium. The blood was collected from the ophthalmic artery. Serum estradiol was measured by EIA kit (Cayman Chemical, Ann Arbor, MI).
Nissl staining was applied to observe morphologic changes in cells within the ischemic penumbra after MCAO. After neurological evaluation, the brains (n = 5) were perfused with cold 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) (pH 7.4). Post-fixation, the brains were taken out and cryoprotected in 20% sucrose and 30% sucrose solution. After the brains were washed in cold water, 14 μm thick sections were prepared using the Leica CM1900 frozen slicer. Then the frozen sections were stained with 0.1% cresyl violet for 20 min, rinsed with PBS, dehydrated by graded alcohol, transparent by xylene, and mounted with neutral gum. The sections were observed using light microscopy.
Assessment of infarct volume
Infarct volume was assessed by TTC staining (n = 13), as previously described . Briefly, after the blood was collected, the rats were decapitated. The brain was rapidly removed and cooled in ice-cold saline for 10 min. Coronal sections (2 mm) were cut and immersed in 2% TTC at 37°C for 30 min, and then transferred to 4% paraformaldehyde in 0.01 M PBS (pH 7.4) for 24 h fixation. The brain slices were photographed using a digital camera (Canon ixus 220HS). The unstained areas were defined as infarcts, and were measured using Photoshop CS3. The infarct volume was calculated by measuring the unstained area in each slice, and multiplying the area by the slice thickness (2 mm). Then the total volume was assessed by summing the area from all six slices.
The expression of pro-apoptotic protein caspase-3 in PC12 cells and ischemic penumbra (n = 6) was detected by Western blotting. In brief, soluble lysates of PC12 cells or penumbra were mixed with sample buffer and NuPAGE reducing agent. Extracted proteins were separated by 12% SDS-PAGE and were then electrically transferred to polyvinylidene difluoride membranes. Afterward, the membranes were blocked in 5% non-fat dry milk diluted in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. Western blots were probed with rabbit anti-caspase-3 antibodies overnight at 4°C. The membranes were then incubated with an IRDye secondary anti-rabbit antibody for 1 h. Protein bands were visualized using the LI-COR Odyssey System.
Data are presented as means ± SEMs. Differences among the groups were analyzed by one way ANOVA. Differences between two groups were analyzed by two-tailed Student’s t-test with SPSS 11.5. A P-value of < 0.05 was considered statistically significant.