Animals and grouping
Fifty male Sprague‑Dawley rats (8-week old) weighing 240‑270 g were obtained from the Animal Center belonging to Shenyang Medical University, China. The rats were kept in sterile cages individually in animal center under ~ 55% humidity and at 23 ± 2˚C and were exposed to 12 h light/dark cycles. All rats were allowed to access water and laboratory rodent diet freely. The experimental procedures involving rats were conducted in accordance with the guidelines of Care and Use of laboratory animals Committee China Medical University. The approval for study was given by Ethics Committee of China Medical University. The rats were separated into five groups: sham operation (Sham), Vascular dementia alone (VD), VD + asiaticoside, rapamycin alone and rapamycin + asiaticoside groups. The rats in four groups except sham group were subjected to bilateral occlusion of carotid arteries (2-VO) (Xing et al. 2016). Chloral hydrate anaesthesia was intraperitoneally given to the rats followed by fixed supine on hot pads with a ventral midline incision in the neck. Muscles on either side of the trachea were incised carefully to expose the carotid arteries. Then double ligation was performed for permanent occlusion of the arteries. The same procedure except vessel ligation was repeated in the sham rat group. The rapamycin and rapamycin + asiaticoside groups were injected rapamycin (50 µl) one day before surgery directly into the ventricle using catheter. Asiaticoside at 5 mg/kg body weight was given to treatment groups via intragastric route as single dose after surgery.
Behavioral assessment using T‑maze tests
The spatial memory of rats was assessed using T‑maze test after 28th day of surgery using previously reported methodology (Deacon and Rawlins 2006). Each of the trial in T‑maze test involved two runs-one sample and second choice run. Sample run consisted of forcing the rats to enter any of the two arms for getting sugar while second arm of maze was closed using a sliding door. During choice run the door was opened and rats were let to choose any of the arms freely. The time duration set between the two runs was of 10 sec and the rats entering unvisited arm were rewarded. Subsequently the time duration between the two runs were increased to 90 and 180 sec. Each session involved five trials every day and the time gap between two trials was set to be 10 min. The count of corrections was taken as the number of times rats entered the arm which was previously unvisited.
Morris water maze (MWM) test
The MWM test was used for assessment of cognitive ability of the animals after 28 days of surgery (Xing et al. 2016). Briefly, the rats were given four trials of training session every day for five consecutive days. The training trials consisted of placing the rats alternately in four different quadrants of water pool and allowing them to locate the platform during 120 sec. The rats were then given 20 sec to rest on the platform and time taken to find the platform was counted as escape latency. The rats unable to locate the platform during assigned time duration were guided towards it and allowed to rest for 20 sec. The platforms in the water pool were removed on 6th day of probe test and rats were permitted to swim freely for locating 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.
Analysis of neuronal survival
The rats were sacrificed after anaesthetization with 200 mg/kg doses of sodium pentobarbital via intraperitoneal route. Then normal saline and subsequently paraformaldehyde (4%) in sodium phosphate buffer was perfused transcardially. The rat brains were dissected and then subjected to fixing in paraformaldehyde (4%) at a temperature of 4˚C. After 3-days of fixing, the brain samples were paraffin embedded followed by slicing into 2‑µm sections. The sections were dyed by treatment with Toluidine Blue (1%) for 5 min at 60˚C. The sections were examined under light microscope (model, BX53; Olympus Corporation) for calculation of viable neurons in five randomly selected fields.
Transmission electron microscopy
The brain tissues were fixed for 2 h on treatment with glutaraldehyde (2.5%) in PBS at 4˚C and then with osmium tetroxide (1%; pH 7.4) for 2.5 h. The tissues were dyed using uranyl acetate (1% aqueous) solution overnight at 4˚C prior to embedding in Durcopan (Sigma‑Aldrich, Merck KGaA). Ultracut microtome (Leica Microsystems, Inc.) was used for cutting of hippocampus into ~ 60 mm sections which were put on formvar‑coated copper grids. The sample sections were subjected to dyeing with uranyl acetate and lead citrate followed by examination under 7650 transmission electron microscopes (Hitachi High‑Technologies Corporation).
The rats were sacrificed after anaesthetization with 200 mg/kg doses of sodium pentobarbital via intraperitoneal route. Then normal saline and subsequently paraformaldehyde (4%) in sodium phosphate buffer was perfused transcardially. The hippocampus tissues were excised and then homogenized on treatment with RIPA lysis buffer (Beyotime Institute of Biotechnology) mixed with PMSF. Centrifugation of lysate at 4˚C for 15 min at 12,000 x g was followed by protein content determination using BCA assay kit (Beyotime Institute of Biotechnology). The protein samples (30 µg) were isolated on 12% SDS‑PAGE and then transferred to PVDF membranes which were blocked using SBA (3%) in TBS for 40 min at room temperature. The membrane were probed with primary antibodies at 4˚C for overnight, washed with PBS and then incubated for 2 h with horseradish‑peroxidase conjugated secondary antibodies (Cell Signaling Technology, Inc.). Detection and visualization of protein bands was performed using enhanced chemiluminescence system (EMD Millipore) and quantification by Quantity‑One software version 4.6.3 (Bio‑Rad Laboratories, Inc.). The primary antibodies used were: anti-mTOR, anti-LC3B, anti-p-mTOR, anti-Beclin-1 and anti-β‑actin (Cell Signaling Technology, Inc.).
Histological analysis using Hematoxylin–Eosin (HE) staining
The tissue sections from hippocampi were perfused and subsequently fixed at 4 °C in phosphate buffer at 7.4pH. Then thin slices of hippocampi were cut using a microtome followed by rehydration in gradient sucrose solution. The sections were subsequently embedded in OCT (Tissue-Tek, Miles) and sectioned at 25 lm (Leica CM 1850, Leica Instruments). The slices after HE-staining were examined under a microscope at 200 × magnification.
The presented data are the mean ± standard deviations of three experiments. The data analysis was made by SPSS 16.0 statistical software (SPSS, Inc.). The statistical differences between groups were determined using One‑Way ANOVA followed by Bonferroni tests and student’s t-test. The differences at P < 0.05 were taken significant statistically.