Salidroside prevents cognitive impairment induced by chronic cerebral hypoperfusion in rats

Experimental animals

A total of 36 male Sprague–Dawley rats (mean weight, 240 ± 20 g; supplied by the Experimental Animal Centre, The Fourth Military Medical University, Xi’an, China) were assigned to one of three groups (n = 12 per group): (i) sham group (sham operated rats treated with normal saline); (ii) 2-VO group (2-VO rats treated with normal saline); (iii) 2-VO salidroside group (2-VO rats treated with salidroside [Yi-Le Pharmaceutical Co. Ltd., Shaanxi, China]). Rats were housed (n = 6 per cage) at a temperature of 22 ± 2℃, with a 12-h light/12-h dark cycle and free access to food and water. Daily 20 mg/ml salidroside intraperitoneal injection (1 ml/kg per day) or vehicle (normal saline) was started on the day before surgery and continued until the termination of the experiment (34 days postoperatively). Salidroside dose was determined based on an earlier study ¹³ and our preliminary experiments. During the last 7 days of the experiment (days 28–34 postoperatively), spatial learning and memory were assessed 1 h after drug administration. The experimental procedure is shown in Figure 1. The research protocol was approved by the Institutional Animal Ethics Committee of The Fourth Military Medical University, Xi’an, China and the study protocol complies with international rules and policies. OPEN IN VIEWER

Surgical procedures

Surgery was carried out as described. ¹⁴ Briefly, under 320 mg/kg chloral hydrate intraperitoneal anaesthesia, a ventral midline incision was made. The bilateral common carotid arteries were exposed and gently separated from the carotid sheath and vagus nerve. In rats assigned to the 2-VO and 2-VO salidroside groups, the two arteries were ligated with a 5-0 silk suture. Rats in the sham-operated groups received the same operation but the carotid arteries were not ligated. During the surgery, the body temperature of the rats was maintained at 37.5 ± 0.5℃ until the rats recovered to thermal homeostasis.

Morris water maze test

The Morris water maze test was carried out daily between day 28 and 34 after surgery, using described methods ¹⁵ with slight modifications. The apparatus comprised a circular tank (diameter 120 cm, height 50 cm) filled with water (temperature 22 ± 1℃, depth 25 cm), which was made opaque with milk so that the rats were unable to see the underwater platform. The tank was divided into four quadrants and was surrounded by several prominent cues to spatial co-ordinates, including items such as a shelf, several posters and illumination lights. Rats were trained to locate a hidden platform (diameter 10 cm), which was centred at one of the quadrants (target quadrant) and submerged ∼2 cm below the surface of the water. The swimming paths of the rats were monitored by a computerized video tracking system and swimming velocity was measured during each test.

During the training period (5 consecutive days) rats were given eight trials per day to find the hidden platform using four different starting points (equally spaced around the perimeter of the tank). All four starting points were used once in a clockwise sequence. Rats were given 60 s to find the platform and sit on it for 30 s. Rats that failed to find the location within the given time were guided to the platform and were allowed to stay on it for 30 s. Then they were picked up and placed in the water for the next trial. The time taken to find the platform (escape latency) was measured, and the mean over the eight trials was calculated. After the eighth trial, all rats were towel-dried and placed in their home cages. Investigators who conducted the Morris water test were blind to the treatment groups.

A probe test, in which the hidden platform was removed, was conducted on the day before the first training and the day after the last training. Rats were placed in the water at the opposite point to the target quadrant and were allowed to swim freely for 120 s in the tank. The percentage of time in the target quadrant was recorded as an assessment of spatial memory.

Electrophysiological recordings

The right cerebral hemisphere of six rats from each group was used for this analysis. Rats were sacrificed via isoflurane anaesthesia, the brain was rapidly removed, and 2 transverse hippocampal slices (400 -µm thick) were prepared from each rat. Slices were cut with a vibratome in ice-cold artificial cerebrospinal fluid (ACSF) solution (126 mM sodium chloride, 2.5 mM potassium chloride, 2.4 mM calcium chloride, 1.2 mM magnesium chloride, 1.2 mM monosodium phosphate, 19 mM sodium bicarbonate and 11 mM glucose, gassed with 95% oxygen and 5% carbon dioxide (pH 7.35–7.45). The slices were incubated in 95% oxygen/5% carbon dioxide-equilibrated ACSF at 34℃ for ≥1 h. Each slice was then placed on the recording chamber and submerged in continuously flowing ACSF (2 ml/min) gassed with 95% oxygen and 5% carbon dioxide (pH 7.35–7.45), and heated to 34℃.

Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 stratum radiatum using glass micropipettes with 2–4 MΩ electrical resistance and filled with 3 M sodium chloride. Schaffer collateral fibres were stimulated with bipolar stimulation electrodes (tip separation 300 µm). Stimulations were generated by a Master-8 pulse generator and a stimulus isolator (A.M.P.I, Jerusalem, Israel). Recorded electrical signals were amplified with an Axopatch™ 700B amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA) filtered at 3 kHz. Data were acquired using a computer equipped with the Digidata® 1440A acquisition system (Molecular Devices Corporation) and analysed using pCLAMP™ version 10.2 software (Molecular Devices Corporation).

The fEPSP was evoked by a square-wave pulse (100 μs duration, 0.05 Hz). Prior to the experiment, an input/output curve was made to determine the stimulus intensity required to produce a half-maximal response. All subsequent stimuli were delivered at this intensity. Baseline responses were recorded for 30 min after stable baseline responses were obtained, and the mean of 12 consecutive fEPSPs was calculated. Long-term potentiation (LTP) was induced by the application of high-frequency stimulation (HFS) (twice for 1 s at 100 Hz, applied with a 20-s interval). After HFS, stimulation resumed at 0.05 Hz and recordings were taken for another hour. The slope of evoked fEPSP was normalized to the mean fEPSP measured during the baseline recording period. Results were expressed as mean ± SD of percentages relative to control baseline.

Western blotting

The left cerebral hemisphere of six rats from each group was used for this analysis. Hippocampus homogenates were prepared in radio-immunoprecipitation assay lysis buffer, containing 50 mM Tris (pH 7.4), 150 mM sodium chloride, 1% Triton X™-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium fluoride, 5 mM ethylenediaminetetra-acetic acid (pH 8.0), 1 mM sodium orthovanadate (Beyotime Inc., Jiangsu, China), and the protease inhibitor, phenylmethylsulphonyl fluoride (1 mM). Protein concentrations were determined using the Pierce™ BCA® Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of total protein (20 µg per lane) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes. Nonspecific binding was blocked by incubation with 3% bovine serum albumin in Tris-buffered saline containing Tween 20 (TBST) for 1 h. Next, the membranes were incubated at 4℃ overnight with rabbit anti-rat Bcl-2 (1 : 400 dilution), rabbit anti-rat Bax (1 : 1000 dilution), or rabbit anti-rat active caspase-3 (1 : 500 dilution) antibodies (Abcam, Cambridge, UK). After washing three times with TBST, the membranes were incubated with goat anti-rabbit IgG-horseradish peroxidase (1 : 2000 dilution; Zhongshan Goldenbridge Ltd., Beijing, China) for 2 h at room temperature. Mouse anti-rat monoclonal antibody against β-actin (Sigma–Aldrich, St Louis, MO, USA) was used as the loading control. Enhanced chemiluminescence (Thermo Scientific) was used to develop the membrane. Relative intensities of the bands were quantified by densitometric analysis.

Histology and immunofluorescence

Morphological analysis was performed after the probe trials as described. ¹⁶ A total of six rats from each group were anaesthetized with 320 mg/kg chloral hydrate intraperitoneal injection and perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde, in 0.1 M phosphate buffer (pH 7.4). The brains were immediately removed, immersed in 4% paraformaldehyde for 24 h, and cryoprotected in a 30% sucrose solution. Coronal sections were cut at 25 -µm thickness by a frozen-stage microtome. Finally, the sections were rinsed in 0.01 M phosphate-buffered saline (PBS) and mounted onto gelatin-coated glass slides. Sections were labelled using immunofluorescence. After blocking for 1 h at 37℃ with 10% goat serum and 0.3% Triton X™-100 in PBS, sections were incubated with mouse monoclonal anti-NeuN antibody (a neuron-specific nuclear protein) (1 : 1000 dilution; Chemicon, Temecula, CA, USA.) at 4℃ overnight, followed by incubation with goat antimouse immunoglobulin G conjugated with Cy3 (1 : 400 dilution; Abcam) for 2 h at room temperature. Sections were observed via confocal laser scanning microscope (FluoView™ FV1000; Olympus, Tokyo, Japan). Digital images were captured using FluoView™ software (Olympus).

Statistical analyses

Data were presented as mean ± SD. Morris water maze escape latencies and fEPSP slopes were analysed using two-way analysis of variance (ANOVA) with repeated measures, followed by the Tukey–Kramer test. Other data were analysed using one-way ANOVA followed by Duncan multiple group comparison. Statistical analysis was performed using SPSS® version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values <0.05 were considered statistically significant.