In total, 84 adult female Sprague-Dawley rats (weight, 180–220 g; age, 6–7 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. All rats were individually housed in cages under a 12-h light/dark cycle (lights on at 07:00 a.m.) at a temperature of 22±2°C and a humidity of 50–65%. The rats had free access to water and rodent chow. The experimental procedures were approved by The Institutional Committee on The Care and Use of Animals of Nanjing University of Chinese Medicine. Every effort was made to minimize the number of animals used and their suffering. The experimental procedure is presented in Fig. 2.

The rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal injection; cat. no. 20021216; Sinopharm Chemical Reagent Co., Ltd.) and were then mounted on a stereotaxic frame (RWD Life Science Co., Ltd.) for implantation of the infusion cannula [guide cannula: Inner diameter (ID)=0.38 mm, outer diameter (OD)=0.56 mm; internal cannula: ID=0.36 mm, OD=0.20 mm; RWD Life Science Co., Ltd.]. The cannula was implanted toward the right lateral ventricle (A, −0.8; L, 1.5; H, 4.0) according to a rat brain atlas (21). The cannula was then anchored to the skull with dental cement and skull screws. An osmotic mini pump was implanted subcutaneously (s.c.) on the back and connected by a tube to the infusion cannula. After the surgery, rats were allowed to recover for at least 7 days in their cages.

Animals were anesthetized with sodium pentobarbital, and the bilateral ovaries were excised through flank incisions (1.5 cm inferior to the palpated rib cage; 1–2 cm from the spine). Sham surgery was performed similarly to the ovariectomy procedure but without ovary removal. After the surgery, rats were placed in their cages and allowed to recover for 7 days.

SSD (purity ≥95%; cat. no. S9321; Sigma-Aldrich; Merck KGaA) was first dissolved in 12% Tween-80 (cat. no. P4780; Sigma-Aldrich; Merck KGaA) and then diluted with 0.9% saline to a final Tween concentration of 0.5%. E2 (cat. no. E8875; Sigma-Aldrich; Merck KGaA) was dissolved in sesame oil. The non-selective estrogen receptor (ER) inhibitor ICI182780 (cat. no. 1047/1; Tocris Bioscience) was first dissolved in dimethyl sulfoxide (10 mM; cat. no. d103273; Shanghai Aladdin Biochemical Technology Co., Ltd.) and then diluted to 0.1% by volume in artificial cerebrospinal fluid (aCSF). The composition of the aCSF was: NaCl 124.0 mM, KCl 3.0 mM, NaHCO₃ 26.0 mM, MgCl₂·6H₂O 1.2 mM, NaH₂PO₄·2H₂O 1.2 mM, C₆H₁₂O₆ 10.0 mM, CaCl₂ 2.0 mM. All chemical reagents were purchased from Sigma-Aldrich (Merck KGaA).

OVX rats were randomly divided into six subgroups (n=12/subgroup): Saline subgroup, 0.9% NaCl containing 0.5% Tween-80 was administered via intragastric gavage (i.g.) to the rats at the same concentration as in the other groups for 5 weeks; O-E2 subgroup, 2 µg/kg E2 (s.c.) was administered for 5 weeks; O-SSD 1.8 subgroup, 1.8 mg/kg SSD (i.g.) was administered for 5 weeks; O-SSD 0.9 subgroup, 0.9 mg/kg SSD (i.g.) was administered for 5 weeks; O-SSD 1.8-ICI, 1.8 mg/kg SSD was administered for 5 weeks, with 500 µg/day ICI182780 (intracerebroventricular injection) co-administered for the final week; and O-SSD 0.9-ICI subgroup, 0.9 mg/kg SSD was administered for 5 weeks, with ICI182780 co-administered for the final week. The dosages of SSD (0.9 and 1.8 mg/kg) used in the current study were based on previous reports, on the basis of sufficient behavioral discrimination and an absence of significant side effects (19,22,23). Rats in the sham group (n=12) underwent no drug treatment. The experimental procedure is shown in Fig. 2. All drugs were administered with a comparable volume once per day at 8:00 a.m.

Fear conditioning tests were performed in a chamber composed of a plexiglas box (27×31×36 cm) with stainless steel grids (diameter, 5 mm; pitch, 15.7 mm) on the floor (Coulbourn Instruments). Each rat was placed in the chamber and subjected to a 2-min adaptation period followed by a 2-min preconditioning phase (without any stimulation) in which the freezing time was measured. During the conditioning period, a tone (80 dB) was presented as the conditioned stimulus for 20 sec, and then a foot shock (0.5 mA) was delivered as an unconditioned stimulus during the last 2 sec of the tone stimulus. After 10 tone-shock pairs with 60 sec interstimulus intervals (all animals exhibited >12 sec freezing in a 2-min period), the rats were returned to their cages. Context-dependent tests were performed 1 and 24 h after the conditioning, in which the freezing time was measured in the same contextual conditions, but without any stimulation, for 3 min. Freezing time (%)=freezing time/total measurement time (24).

Nine rats randomly selected from each group were sacrificed on the day after behavioral tests under anesthesia at 8:00-9:00 a.m. The skulls were then dissected, and the bilateral hippocampi were quickly removed and placed on ice. The hippocampal CA1 areas were rapidly extracted and cut into ~1×1×0.5 mm³ sections according to the brain atlas (21); these sections were immediately frozen in liquid nitrogen.

Samples from six rats in each group were analyzed by ELISA (cat. no. KGE014; R&D Systems, Inc.), in order to determine the content of E2 in the hippocampus, according to the manufacturer's protocol. Absorbance was measured at 450 nm with a microplate reader.

Samples from the remaining three rats in each group were used for western blot analysis. Tissue proteins were extracted with 5% RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology), and centrifuged at 13,500 × g for 15 min at 4°C, and the protein concentration was determined using a Pierce bicinchoninic protein assay kit (cat. no. 23223; Thermo Fisher Scientific Inc.). Equal amounts of protein (30 µg/lane) were separated by 7.5–12% SDS-PAGE (cat. nos. 1610171TA and 1610175TA; Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes (cat. no. IPVH00010; EMD Millipore). The membranes were incubated with TBS containing 0.05% Tween-20 (TBS-T; cat. no. SRE0031; Sigma-Aldrich; Merck KGaA) and 5% non-fat dry milk for 2 h at room temperature to block nonspecific binding and were immunoblotted at 4°C overnight with the following primary antibodies: Rabbit anti-ERα (1:1,000, cat. no. 21244-1-AP; Proteintech Group, Inc.), rabbit anti-ERβ, (1:1,000, cat. no. 14007-1-AP; Proteintech Group, Inc.), rabbit anti-GAPDH (1:1,000, cat. no. AP0063; Bioworld Technology, Inc.). After being washed by TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody goat anti-rabbit immunoglobulin G (1:5,000, cat. no. BS13278; Bioworld Technology, Inc.) at room temperature for 2 h. Subsequently, the membranes were washed in TBS-T, and were developed with an enhanced chemiluminescence kit (cat. no. NEL104001EA; PerkinElmer, Inc.). Each experiment was performed independently at least three times, and the integrated intensity was measured using Image-Pro-Plus 6.0 (Media Cybernetics, Inc.).

In each group, tissues from the remaining three rats were processed for immunohistochemistry, according to our previously described procedure (25). Briefly, the rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (cat. no. AR1068; Wuhan Boster Biological Technology, Ltd.). The brains were extracted, postfixed in 4% paraformaldehyde at 4°C for 24 h and embedded in paraffin; subsequently, samples were cut into 5-µm sections. The three sections containing the hippocampal CA1 region were incubated with 3% hydrogen peroxide (cat. no. AR1108; Wuhan Boster Biological Technology, Ltd.) at room temperature for 10 min, 10% goat serum (cat. no. ab7481; Abcam) at 37°C for 30 min. Subsequently, sections were incubated with the primary antibodies rabbit anti-ERα (1:200; cat. no. 21244-1-AP; Proteintech Group, Inc.) or rabbit anti-ERβ (1:200, cat. no. 14007-1-AP; Proteintech Group, Inc.) at 4°C overnight and the biotinylated secondary antibody goat anti-rabbit IgG (1:500, cat. no. BS13278; Bioworld Technology, Inc.) were incubated at room temperature for 30 min. The sections were subsequently counterstained with hematoxylin (0.5%; cat. no. H104302; Shanghai Aladdin Biochemical Technology Co., Ltd.) at room temperature for 3 min.

The adjacent slices were selected for Nissl staining (0.5% thionine; cat. no. 78338-22-4; Sigma-Aldrich; Merck KGaA; 37°C, 10 min), and micrographs of the hippocampal CA1 region were captured under a light microscope (Axio Vert A1; Carl Zeiss AG). Nissl-labeled neurons were calculated from two or three random fields per slice, and technicians blinded to the samples manually made the measurements. Briefly, in each Nissl-stained slide (magnification, ×400) the number of neurons was counted in a calibrator (125×125 µm) and the density (cells/mm²) was subsequently calculated. The criterion for acceptance as a neuron was clear staining of a soma and a nucleus, which were distinctly differentiated from their backgrounds (25).

Data are expressed as the means ± SEM. The significance of the differences between groups was evaluated by one-way analysis of variance (ANOVA) followed by Fisher's least significant difference post hoc test using SPSS version 16.0 (SPSS Inc.). P<0.05 was considered to indicate a statistically significant difference.

Among all groups (sham, O-saline, O-E2, O-SSD 1.8, O-SSD 0.9, O-SSD 1.8-ICI and O-SSD 0.9-ICI), the mean freezing fraction prior to conditioning exhibited no significant differences (F_6,77=0.47, P=0.65, one-way ANOVA; Fig. 3). Compared with the sham operation group, ovariectomy significantly shortened the freezing time (P<0.01, O-saline vs. sham; Fig. 3); this finding is in agreement with results from a previous study (26). However, the freezing time was markedly prolonged in OVX animals after E2 administration at 1 or 24 h (P<0.05 or P<0.01, O-E2 vs. O-saline; Fig. 3), thus indicating that the memory loss in OVX rats was due to an estrogen deficit. Notably, treatment with SSD (SSD 1.8 and SSD 0.9) exerted a similar effect to E2 administration, which lasted for at least 24 h despite slight attenuation (Fig. 3).

Intracerebroventricular administration of ICI182780 markedly blocked the increase in freezing time in the O-SSD 1.8 group at 1 h (P<0.05 O-SSD 1.8-ICI vs. O-SSD 1.8) and 24 h (P<0.05, O-SSD 1.8-ICI vs. O-SSD 1.8; Fig. 3), and there was also a clear tendency toward reduced freezing in the O-SSD 0.9-ICI group (Fig. 3). These results indicated that ERs may be involved in SSD-mediated effects.

The E2 levels in the hippocampus were significantly lower in OVX rats compared with in sham-operated animals (P<0.01, O-saline vs. sham; Fig. 4), whereas this decrease was reversed following E2 administration (P<0.05, O-E2 vs. O-saline; Fig. 4). However, SSD treatment (O-SSD 1.8 or O-SSD 0.9) exhibited no influence on E2 levels (P=0.46 and P=0.82, respectively, for O-SSD 1.8 or O-SSD 0.9 vs. O-saline; Fig. 4). In addition, the ER inhibitor ICI182780 did not influence E2 levels (P=0.22, O-SSD 1.8-ICI vs. O-SSD 1.8; P=0.59, O-SSD 0.9-ICI vs. O-SSD 0.9; Fig. 4). These results suggested that the SSD-mediated improvement in memory deficit in OVX rats may not be attributed to E2 alterations in the hippocampus.

As shown in Fig. 5, ERα and ERβ were widely expressed in hippocampal neurons; this has also been reported by previous studies (27–29). ERα was present in both the extranuclear (cytoplasmic regions) and intranuclear sites of the neurons, with prominent extranuclear expression (Fig. 5Aa). In addition, ERβ was mainly expressed in the extranuclear sites (Fig. 5Bb), which is in line with previous reports (30–32). The staining intensities for ERs exhibited obvious differences among the groups. Western blotting was conducted for semi-quantitative analysis of ER expression (Fig. 5C). ERα exhibited a significant downregulation in the hippocampus of OVX rats (P<0.01, O-saline vs. sham; Fig. 5D), whereas E2 supplementation substantially reversed this downregulation (P<0.01, O-E2 vs. O-saline; Fig. 5D). Notably, ERα expression in the hippocampus was also significantly enhanced by SSD treatment in the O-SSD 1.8 (P<0.01, O-SSD 1.8 vs. O-saline; Fig. 5D) and O-SSD 0.9 groups (P<0.05, O-SSD 0.9 vs. O-saline; Fig. 5D) of OVX rats. Conversely, ERβ expression exhibited no significant differences among these groups (sham, O-saline, O-E2, O-SSD 1.8 and O-SSD 0.9) by one-way ANOVA (F_4,10=0.75, P=0.58; Fig. 5D). These findings indicated that SSD activated ERα expression in the hippocampus of OVX rats. Furthermore, ERα and ERβ expression in the O-SSD groups was markedly inhibited by ICI182780 intervention (P<0.01, O-SSD 1.8-ICI vs. O-SSD 1.8 and O-SSD 0.9-ICI vs. O-SSD 0.9; Fig. 5D), confirming that the inhibitor ICI182780 had suppressive effects on ERα and ERβ.

Given that neuron number conclusively influences neural functioning in the nervous system, this study aimed to determine whether SSD-induced behavioral improvement in OVX rats may be associated with changes in neuron number. As shown in Fig. 6A-H, the number of neurons in the hippocampus exhibited no significant alterations among the groups (sham, O-saline, O-E2, O-SSD 1.8, O-SSD 0.9, O-SSD 1.8-ICI and O-SSD 0.9-ICI; F_6,119=1.429, P=0.209; Fig. 6H), thus suggesting that SSD-mediated improvements in memory deficits in OVX rats were not attributed to changes in the number of neurons.