All experimental procedures involving animals were performed in accordance with the guidelines from the National Institutes of Health and approved by the Animal Care and Use Committee of Zhengzhou University Medical School (Zhengzhou, China). A study population of 48 male New Zealand rabbits (mean body weight, 3.60±0.4 kg) was purchased from the Shanghai Laboratory Animal Center (Shanghai, China). The rabbits were randomly divided into three groups: Control, sham and SSS (n=16 per group). Rabbits in the sham and SSS groups received a high-lipid diet (1% cholesterol, 5% yolk, 5% lard and 89% basic feedstuff) for 3 months. The disease model was established according to the method described by Zhang et al (9). Briefly, the rabbits were anesthetized with 3% pentobarbital sodium (1 ml/kg), administered intravenously. The SA was exposed and ligated proximal to the origin of the VA. Sham-operated rabbits underwent all surgical procedures, with the exception of the SA occlusion.

The eyeblink experiment was conducted 30 days after the surgery. Rabbits (n=10 per group) were adapted to a Plexiglas restrainer box (Sigma-Aldrich, St. Louis, MO, USA) for a single 20-min session 3 days prior to the initiation of the experiments. The conditioned stimulus (CS) was a 1-kHz, 85-dB tone, and the unconditioned stimulus (US) was a 6-psi corneal airpuff. In the delay-conditioning paradigm, the duration of the tone was 500 msec, followed 400 msec after its onset by a 100-msec airpuff US. The CS and US co-terminated. The rabbits received 7 training sessions over 7 consecutive days. Each session consisted of 80 paired CS-US trials, and the inter-trial interval was 20–30 sec. One week after the last day of training, the rabbits received 15 trials in which only the CS was presented.

A further 18 rabbits (n=6 per group) were administered BrdU (Sigma-Aldrich) at a dosage of 100 mg/kg body weight (one intraperitoneal administration per day) for the last 3 days of the experimental period. The rabbits were sacrificed 2 h after the last injection. The rabbits were intravenously anesthetized with 3% pentobarbital sodium (1 ml/kg) and sacrificed. The obtained tissues were transcardially perfused with ice-cold 0.9% NaCl solution, followed by a freshly prepared solution of 4% paraformaldehyde in 0.1 M sodium phosphate buffer. The brains were removed and postfixed overnight, cryostat-sectioned (30 µm) in series and stored at −80°C.

For BrdU and Ki67 immunohistology, antigen retrieval was performed by incubating the slides in 10 mM sodium citrate buffer (pH 6.0) at 95–98°C for 15 min. After cooling for 1 h, the sections were rinsed with 0.1 M phosphate-buffered saline (PBS) and treated with blocking solution (3% bovine serum albumin, 5% goat serum and 3% Triton in 0.1 M PBS; Sigma-Aldrich) for 2 h at room temperature. The slides were incubated with monoclonal mouse anti-rabbit antibodies against BrdU (1:5,000; NA61-100UGCN; EMD Millipore, Billerica, MA, USA) and Ki67 (1:1,000; 12160; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. After washing three times in 0.1 M PBS with Tween-20, the sections were incubated with the Cyanine 3-conjugated secondary goat anti-mouse Alexa Fluor 488 antibodies (1:1,000; A10547; Invitrogen Life Technologies, Carlsbad, CA, USA) for 2 h at room temperature. The sections were counterstained with DAPI and mounted with an aqueous mounting medium.

Extracellular concentrations of adenosine triphosphate (ATP) and adenosine in the cerebellar cortex were evaluated using microdialysis techniques. The animals were positioned in a stereotactic frame. Prior to insertion, the probes were perfused with artificial cerebrospinal fluid (CSF), containing 147 mM NaCl, 4 mM KCl, 2.3 mM CaCl₂ and 0.9 mM MgCl₂ (pH 7.4), at a rate of 2 µl/min. In all rabbits, a 360-min stabilization period was implemented following the insertion of the microdialysis probe. The dialysate was collected every 15 min for later analysis. The concentrations of ATP and adenosine were determined using an Agilent 1200 HPLC system with a photodiode array detector (Agilent Technologies, Santa Clara, CA, USA), and separation was performed using a Hypersil base-deactivated silica C-18 reverse-phase column (column length, 300 mm; column ID, 4.6 mm; particle size, 10 µm; Thermo Scientific, Inc., Waltham, MA, USA). The mobile phase consisted of 0.1 M phosphate buffer (pH 7.0) and methanol (1%). The flow rate was 1 ml/min and the column temperature was 25°C. Ultraviolet (UV) detection was conducted at 254 nm using an Ultraviolet Detector (GD-3; Puyang Medical Instrument Co. Ltd., Nanjing, China). The concentrations of ATP and adenosine were quantified based on linear calibration.

The levels of interleukin (IL)-1β, IL-6, malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) and the activities of CuZn-superoxide dismutase (CuZn-SOD) and catalase (CAT) were determined using commercial kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China).

Data are expressed as the mean ± standard error of the mean. The differences in cell-counting results were analyzed using the non-parametric Mann-Whitney test. Repeated measures analysis of variance (ANOVA) and Fisher's protected least significant difference (LSD) multiple comparison test were used to determine differences in ATP and adenosine concentrations among the groups. All other results were compared using one-way ANOVA, followed by the LSD post hoc test for intergroup comparisons. P<0.05 was considered to indicate a statistically significant difference.

To observe the effects of SSS on the cognition of the rabbits, eyeblink experiments were conducted. No significant differences were found among the results from three groups (Fig. 1; P>0.05).

Neurogenesis in the rabbit brains was examined. Quantitative assessment revealed no significant difference in the number of BrdU- or Ki67-labeled cells in the subventricular or subgranular zones among the three groups (data not shown). Notably, a reduced number of BrdU- and Ki67-positive cells were observed in the cerebellar cortex of rabbits in the SSS group (101±21 and 85±17 cells/mm², respectively) compared with the control (178±33 and 146±25 cells/mm², respectively) and sham (198±19 and 158±21 cells/mm², respectively) groups (Fig. 2; P<0.05), suggesting that SSS exerted a negative impact on neurogenesis in the cerebellar cortex.

ATP and adenosine levels in the CSF of the cerebellar cortex were measured in vivo using microdialysis. The CSF samples were processed using HPLC, and the levels of ATP and adenosine were determined with a UV detector. Immediately following probe insertion, the ATP and adenosine levels were elevated due to the tissue damage caused by probe insertion. The initially elevated levels of ATP and adenosine declined to plateau levels within 6 h. At 6 h after probe insertion, the extracellular concentrations of ATP and adenosine in the SSS group rabbits (n=5) were 6.2±2.3 and 10.5±1.7 nM compared with the control group, respectively (Fig. 3; P<0.05). By comparison, the concentrations of ATP and adenosine in the control and sham group rabbits (n=5 per group) were 11.7±2.0 and 13.1±1.5 nM, and 13.7±2.4 and 15.9±2.1 nM, respectively (Fig. 3). Thus, reduced levels of ATP and decreased levels of adenosine were found in the cerebellar cortex, and these submicromolar differences in ATP concentration among the groups were consistent with the results of the immunohistological analyses.

No differences were detected in the levels of IL-1β, IL-6, MDA, 8-OHdG, CuZn-SOD and CAT among the three groups (Fig. 4; P>0.05).