A total of 50 male Sprague-Dawley rats (age, 8 weeks; weight, 240–270 g) were obtained from the Animal Center, Shenyang Medical University. The rats were individually maintained in sterile cages in an animal center under ~55% humidity and at 23±2°C, and were exposed to 12-h light/dark cycles. All rats were allowed free access to water and a laboratory rodent diet. The experimental procedures involving rats were conducted in accordance with the guidelines of the Care and Use of Laboratory Animals Committee China Medical University (15). The present study was approved by the Animal Ethics Committee, Dongying District People's Hospital (Dongying, China; approval. no. DSH/2017/067). The rats were separated into five groups (n=10/group): Sham group, VD group, VD + asiaticoside group, rapamycin group and rapamycin + asiaticoside group. The rats in all groups, with the exception of the sham group, were subjected to bilateral occlusion of carotid arteries (17). Chloral hydrate (10%; 300 mg/kg) anesthesia was intraperitoneally administered to the rats, after which they were fixed supine on hot pads and a ventral midline incision was made to the neck. No signs of peritonitis were observed in the rats following anesthesia with 10% chloral hydrate. Muscles on either side of the trachea were incised carefully to expose the carotid arteries. Subsequently, double ligation was performed for permanent occlusion of the arteries. The same procedure without vessel ligation was repeated in the sham group. The rapamycin and rapamycin + asiaticoside groups were injected with rapamycin (50 µl) 1 day before surgery directly into the ventricle using a catheter. Asiaticoside dissolved in physiological saline at 5 mg/kg body weight was given to treatment groups via the intragastric route as a single dose after surgery.

The spatial memory of rats was assessed using the T-maze test 28 days after surgery, using previously reported methodology (18). Each trial in the T-maze test involves two runs: A sample run and a choice run. The sample run consisted of forcing the rats to enter one of the two arms of the maze in order to obtain sugar placed in left arm, while the second arm of the maze was closed using a sliding door. During the choice run, the door was opened and rats were left to choose one of the arms freely. The time duration set between the two runs was 10 sec, and the rats entering the previously unvisited arm were rewarded. Subsequently, the time duration between the two runs was increased to 90 and 180 sec. Each session involved five trials every day and the time gap between two trials was set at 10 min. The number of corrections was taken as the number of times rats entered the arm that was previously unvisited.

The MWM test was used to assess cognitive ability of the animals 28 days after surgery (17). Briefly, the rats were given four training trials every day for 5 consecutive days. The training trials consisted of placing the rats alternately in four different quadrants of a pool of water and allowing them to locate a platform during 120 sec. The rats were then given 20 sec to rest on the platform; time taken to find the platform was counted as escape latency. The rats unable to locate the platform during the assigned duration were guided towards it and allowed to rest for 20 sec. The platform was removed from the pool on the 6th day of the probe test and rats were permitted to swim freely to locate the removed platform. The swimming activity of the rats was monitored and recorded video-graphically. The platform crossings were recorded by calculating the platform location crossed by each rat.

The rats were sacrificed using pentobarbital overdose. Subsequently, normal saline and paraformaldehyde (4%) in sodium phosphate buffer were perfused transcardially. The rat brains were dissected and then subjected to fixing in 4% paraformaldehyde at 4°C. After 3 days of fixing, the brain samples were embedded in paraffin and sliced into 2-µm sections.

The brain tissues were fixed with 2.5% glutaraldehyde in PBS at 4°C for 2 h, and then with 1% osmium tetroxide (Ph 7.4) for 2.5 h at 4°C. Subsequently, the tissues were stained with uranyl acetate (1% aqueous) solution overnight at 4°C prior to embedding in Durcupan (Sigma-Aldrich; Merck KGaA). An ultracut microtome (Leica Microsystems, Inc.) was used to cut hippocampal tissues into ~60-nm sections, which were placed on formvar-coated copper grids. The sample sections were subjected to staining for 5 min with uranyl acetate (2.5%) and lead citrate (3%) followed by examination under a 7650 transmission electron microscope (Hitachi High-Technologies Corporation).

The rats were sacrificed using pentobarbital overdose. Subsequently, normal saline and 4% paraformaldehyde in sodium phosphate buffer was perfused transcardially. The hippocampal tissues were excised and then homogenized with RIPA lysis buffer (Beyotime Institute of Biotechnology) mixed with PMSF. The lysates were centrifuged at 12,000 × g for 15 min at 4°C, followed by protein content determination using the BCA assay kit (Beyotime Institute of Biotechnology). The protein samples (30 µg) were separated by SDS-PAGE on 12% gels and were then transferred to PVDF membranes, which were blocked using 3% BSA (Sigma-Aldrich; Merck KGaA) in Tris-buffered saline for 40 min at room temperature. The membranes were probed with primary antibodies at 4°C overnight, washed with PBS and then incubated for 2 h with horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature. Detection and visualization of protein bands were performed using an enhanced chemiluminescence system (EMD Millipore) and the blots were semi-quantified by Quantity-One software version 4.6.3 (Bio-Rad Laboratories, Inc.). The primary antibodies used were: Anti-mTOR (cat. no. 2972; dilution 1:400; Cell Signaling Technology, Inc.), anti-LC3B (cat. no. 2775; dilution 1:1,000; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-mTOR (cat. no. 2971; dilution 1:250; Cell Signaling Technology, Inc.), anti-Beclin 1 (cat. no. sc-48341; dilution 1:1,000; Santa Cruz Biotechnology, Inc.) and anti-β-actin (cat. no. 4967; dilution 1:1,000, Cell Signaling Technology, Inc.).

The rats were sacrificed after anaesthetization with overdose of sodium pentobarbital. Transcardial perfusion was performed with normal saline (0.9%) and then with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Dissection of the brain was followed by fixing with 4% paraformaldehyde at 4°C for 3-days and then paraffin embedding. Brains were sliced into 5-µm sections followed by staining at 60°C for 45 min with 1% Toluidine Blue. Neuronal survival was determined by the examination of Nissl-positive cells in the rat hippocampus. Light microscope (model, BX53; Olympus Corporation) was used for calculation of normal neurons in the CA1 subfield at ×400 magnification. The average number of neurons was calculated in three sections and five representative fields were randomly chosen for each section.

The rats were sacrificed after anaesthetization with 200 mg/kg sodium pentobarbital intraperitoneally. Hippocampal tissues were extracted, subjected to PBS washing and then fixed in 10% neutral buffered formalin at room temperature for 45 min. The tissues were decalcified using EDTA (10%) followed by embedding in paraffin and were then sliced into 3-µm sections. The sections were subjected to H&E staining for 40 min at room temperature and were then examined under a light microscope (Olympus IX81; Olympus Corporation) at ×200 magnification.

The data are presented as the mean ± standard deviation of three experiments. Data analysis was performed using SPSS 16.0 statistical software (SPSS, Inc.). The statistical differences between groups were determined using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Changes in behaviors were significantly impaired in the VD rat model group compared with in the sham group (Fig. 2). Asiaticoside treatment of rats with VD significantly improved the changes in behaviors. In addition, rapamycin administration also mediated an impairment in spontaneously altered behaviors in the rats. Asiaticoside failed to significantly alleviate spontaneously altered behavior impairment in VD rats treated with rapamycin. These findings indicated that asiaticoside effectively improved VD-mediated changes in behavior compared with the VD group.

Escape latency exhibited a significant increase in the VD model group compared with in the sham group (P<0.05; Fig. 3). Treatment of VD rats with asiaticoside caused a significant reduction in escape latency (P<0.05). Rapamycin administration also increased escape latency. The rapamycin-mediated increase in escape latency in rats could not be significantly reduced by asiaticoside treatment.

The distance travelled, swim speed, swim time and number of platform crossings were significantly reduced for rats in the VD group compared with in the sham group (P<0.05; Fig. 4). However, asiaticoside significantly alleviated VD-mediated decreases in distance travelled, swim time and number of platform crossings (P<0.05). Administration of rapamycin also significantly reduced distance travelled, swim time and number of platform crossings (P<0.05). However, asiaticoside treatment could not significantly alleviate the rapamycin-mediated reduction in distance travelled, swim time and number of platform crossings.

Abnormalities in the hippocampal tissues of rats were detected using H&E staining (Fig. 5A) and analysis of Nissl bodies (Fig. 5B). The abnormalities were markedly evident in the hippocampus of VD rats compared with in the sham group. Neuronal apoptosis and degeneration were clearly detected in the hippocampus of VD rats. Conversely, VD-mediated hippocampal tissue damage was significantly alleviated by asiaticoside treatment of the rats (P<0.05). In addition, abnormalities were also induced in the hippocampal tissues of rats treated with rapamycin. However, no significant improvement in rapamycin-induced hippocampal tissue damage was observed following treatment with asiaticoside.

Autophagosome formation was clearly detected near the nuclei in the hippocampus of rats in the VD group, but was absent in the sham group (Fig. 6A and B). The primary autophagosome count was also increased in the hippocampus of rats with VD. Treatment of rats in the VD group with asiaticoside alleviated formation of autophagosomes and markedly suppressed the number of primary lysosomes. In rapamycin-administered rats, the numbers of autophagosomes were markedly increased. No significant reduction in autophagosome formation was observed in rapamycin-administered rats treated with asiaticoside.

The expression levels of Beclin 1 and LC3II were significantly elevated in the hippocampal tissues of rats in the VD group compared with in the sham group (Fig. 7A and B). Treatment with asiaticoside alleviated VD-mediated increases in Beclin 1 and LC3II expression in the rat hippocampal tissues. The phosphorylation of mTOR in the hippocampal tissues of rats in the VD group was markedly suppressed compared with in the sham group. Conversely, asiaticoside treatment prevented suppression of mTOR phosphorylation in the hippocampal tissues of rats with VD.

Rapamycin administration markedly suppressed activation of mTOR in rat hippocampal tissues compared with in the sham group (Fig. 8). Conversely, the protein expression levels of Beclin 1 and LC3II were markedly upregulated by rapamycin in the hippocampus of rats. The rapamycin-mediated suppression of p-mTOR, and elevation of Beclin 1 and LC3II expression in the rat hippocampus could not be alleviated by asiaticoside treatment.