A total of 96 adult male Sprague-Dawley rats (age, 8–9 weeks; weight, 350–450 g) were obtained from Shenyang Military Region General Hospital Laboratory Animal Center [Shenyang, China; license no. SCXK (Liao) 2012-00022012-0002]. Animals were housed at a constant temperature (22±1°C), with 50% relative humidity and a12-h light/dark cycle. The rats had access to food and autoclaved water ad libitum. All animal procedures were approved by the Animal Experiments Ethics Committee of the General Hospital of Shenyang Military Region (Shenyang, China).

CPB surgery was performed as previously reported (7), with minor modifications. Briefly, rats received an intraperitoneal (i.p.) injection of 10% chloral hydrate (300 mg/kg; Shanghai Ziyuan Pharmaceutical Co., Ltd., Shanghai, China) for anesthesia. Photopic oral intubation was performed using a 16 G intravenous (i.v.) catheter, and animals were mechanically ventilated with a small animal ventilator (settings: Frequency, 60 beats/min; tidal volume, 3 ml/kg; inspiratory to expiratory ratio, 1:1.5) connected to a monitor to observe the heart rate, oxygen saturation and rectal temperature of the rats. During surgery, anesthesia was maintained with i.v. injection of pipecuronium bromide (0.1 mg/kg; Hangzhou Minsheng Pharmaceutical Co., Ltd., Hangzhou, China).

The puncture site was sterilized with iodophor (Shandong Lierkang Disinfection Technology Co., Ltd., Dezhou, China), followed by exposure and puncture of the vein. Right femoral vein catheterization (24G) was performed to open the fluid path, which was transfused with 6% hydroxyethyl starch (Guangdong Jiabao Pharmaceutical Co., Ltd., Qingyuan, China) and connected to a microinfusion pump. The left femoral artery was catheterized (22G) and used to monitor blood pressure. Coccygeal artery catheterization (22G) and right internal jugular vein catheterization (18G) were performed to drain blood for CPB. The drainage tube, a homemade blood storage device, a constant peristaltic pump (Baoding Longer Precision Pump Co., Ltd., Baoding, China), silicone tubing (internal diameter, 4 mm) and a rat membrane oxygenator (Guangdong Kewei Medical Instrument Co. Ltd., Dongguan, China) were installed between the two puncture sites to establish the CPB circuit. Heparin sodium (300 IU/kg; Shenyang Haitong Pharmaceutical Co., Ltd., Shenyang, China) was injected into the left femoral vein once the activated clotting time reached 480 sec.

CPB was performed with the membrane oxygenator to supply oxygen. The low-flow CPB velocity was 35 ml/kg/min, which was later increased to 100–120 ml/kg/min at full-flow bypass. To prevent air embolism, 1–2 ml of blood was retained in the blood storage device. Mean arterial pressure was maintained at >60 mmHg, partial CO2 pressure at 35–45 mmHg, base excess at −3-3 mmol/l mmHg, pH at 7.35–7.45 and hematocrit at >0.25. Rats were treated with 2–20 µg/100 g epinephrine hydrochloride (Wuhan Grand Pharmaceutical Group Co., Ltd., Wuhan, China) and fluids during surgery to maintain a stable circulation.

Rats were randomly divided into four groups (n=24/group): i) The Sham group (S group), in which intubation and mechanical ventilation were performed in the right femoral artery only and the right internal jugular vein was catheterized without bypass; ii) the CPB surgery group (C group), which received the CPB surgery aforementioned; iii) the α7nAchR agonist group (P group), which received an i.p. injection of the α7nAchR agonist PHA568487 (0.8 mg/kg; Tocris Bioscience; Bio-Techne, Minneapolis, MN, USA) 30 min prior to CPB establishment; and iv) the PHA568487 + α7nAchR antagonist group (M group), which were also pretreated with PHA568487 (0.8 mg/kg) for 30 min, followed by i.p. injection of the α7nAchR antagonist methyllycaconitine (MLA; 6 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). CPB surgery was performed 60 min after MLA injection.

Arterial and venous blood samples were collected prior to CPB (T0), upon completion of CPB surgery (T1), 2 h post-CPB (T2) and 6 h post-CPB (T3); subsequently, rats were sacrificed with 2% pentobarbital sodium (40 mg/kg by i.p. injection; Merck Sharp & Dohme, Shanghai, China). The systemic circulation system of the rats was infused with saline (250–400 ml), and the whole brain was collected on the ice and divided into two halves along the median sagittal line. The hippocampus was isolated from each of the two halves, the right half was fixed in 4% paraformaldehyde (PFA) at room temperature for 24 h, and the left side was stored at −80°C for western blot analysis. Sera were separated by centrifugation at 1,000 × g for 10 min at 4°C, and stored at −80°C.

Fixed hippocampal tissues were gradually dehydrated with ethanol and embedded in paraffin. Paraffin blocks were subsequently sectioned (5 µm) and stained with a Hematoxylin & Eosin (H&E) staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Double-blind evaluation of hippocampal injury was performed by two expert pathologists. Images of the histopathological examination were captured by a light microscope (Olympus Corporation, Tokyo, Japan) at ×400 magnification.

A terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) Assay kit (Shanghai Fusheng Industrial Co., Ltd., Shanghai, China) was used, according to the manufacturer's protocol, to determine the effects of α7nAchR agonist treatment on apoptosis in the fixed and mounted hippocampal sections. DAPI was used as a nuclear stain, with sections stained with 100 ng/ml DAPI for 5 min. Apoptotic rates were examined and images captured using a light microscope (Olympus Corporation, Japan) at a magnification of ×400, and the densitometric scanning was finally analyzed by using the MetaMorph BX41 Image Analysis System (Olympus Corporation, Japan). A total of 5 images were captured randomly for each section at ×400 magnification and integral optical density was calculated using Microscopic Image Analyzer (MetaMorph BX41 Image Analysis System). Percentages of TUNEL-positive cells above untreated controls were calculated as follows: %apoptosis = (number of TUNEL-positive cells / number of total cells) × 100.

To further determine the effects of α7nAchR on apoptosis in the hippocampus, expression levels of the cellular apoptosis maker Caspase 3 was examined by immunohistochemical analysis. Briefly, dimethylbenzene was used to remove the paraffin from the hippocampal sections, followed by immersion in distilled water. Subsequently, antigen retrieval was conducted by placing the slides in a microwave in 10 mmol/l citrate buffer, pH 6.0, for 15 min. The slides were washed with 0.01 mmol/l PBS (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) every 5 min for 3 times, followed by incubating in TBS + 0.3% H₂O₂ + 0.1% saponin at room temperature for 15 min to block the endogenous peroxidase. The slides were blocked with goat serum (Sigma-Aldrich; Merck KGaA) in TBS + 0.1% saponin for 20 min at room temperature, followed by incubating with polyclonal rabbit anti-Caspase 3 (1:300; ab13847; Abcam, Cambridge, UK) overnight at 4°C. The slides were incubated with biotin-conjugated secondary antibody (1:2,000; ab6720; Abcam) for 30 min, and 3,3′-diaminobenzidine stain (8 min at room temperature) was used to visualize Caspase 3 expression in the hippocampus. Images of Caspase 3 expression were captured with a light microscope (Olympus, Japan) at a magnification of 400x. A total of 5 images were captured randomly for each section at ×400 magnification and integral optical density was calculated using Microscopic Image Analyzer (MetaMorph BX41 Image Analysis System).

Serum expression levels (in 100 µl) of S100β, TNF-α and IL-6 were determined by ELISA kits (S100β, JM-E10007507; TNF-α, JM-E10009363; IL-6, JM-E10004387; TSZ Biosciences, San Francisco, CA, USA), according to the manufacturer's protocol. Optical density was measured at 450 nm using a Spectra Max M5 Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

The frozen hippocampal tissues (100 mg) were ground with a glass homogenizer and subsequently homogenized with Radioimmunoprecipitation Assay Buffer (1 ml; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) using an IKA T10 homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany), followed by centrifugation at 12,000 × g for 15 min at 4°C. The supernatant was collected and protein quantification was performed by bicinchoninic acid assay, and equal amounts of protein lysate (40 µg) were separated by 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes in transfer buffer [12 mM Tris base, 96 mM glycine (pH 8.3) and 15% methanol]. Membranes were blocked for 2 h in TBS + 0.5% Tween-20 (TBST) with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature and subsequently probed with polyclonal rabbit anti-Akt (1:500; ab8805; Abcam), polyclonal rabbit anti-GSK3β (1:1,000; ab115774; Abcam), monoclonal rabbit anti-p-Akt (1:500; 13038; Cell Signaling Technology, Inc. Danvers, MA, USA), polyclonal rabbit anti-p-GSK3β ser9 (1:500; ab131097; Abcam), polyclonal rabbit anti-Caspase 3 (1:300; ab13847; Abcam) or monoclonal rabbit anti-β-actin antibody (1:100; 8457; CST, USA) overnight at 4°C. Membranes were washed with TBST buffer three times, followed by incubating with monoclonal goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:4,000; HS101; Beijing TransGen Biotech Co., Ltd., Beijing, China) for 1 h at room temperature. ECL chemiluminescence was used to detect protein expression levels, which were visualized by scanning densitometry (170–8070 Molecular Imager ChemiDoc XRS System; Bio-Rad Laboratories, Inc. Hercules, CA, USA) using ImageJ Software (version 1.37; National Institutes of Health, Bethesda, MD, USA).

Quantitative data were expressed as the mean ± standard deviation. Statistical analyses were performed with GraphPad Prism software, (version 6.00; GraphPad Software, Inc., La Jolla, CA, USA). Multiple comparisons were analyzed with one-way analysis of variance, followed by an appropriate multiple comparison test (Tukey's procedure). P<0.05 was considered to indicate a statistically significant difference.

To determine the protective effects of α7nAchR agonist on the morphological alterations of the hippocampus, sections were evaluated at 6 h post-CPB, the T3 time point, by H&E staining. There was no detectable morphological damage to the hippocampal tissues in the S group (Fig. 1A), whereas clear cellular degeneration and abnormal cell arrangements were observed in the samples of CPB-injured rats (Fig. 1B), which indicated that the rat model of cerebral injury caused by CPB was successfully established. Following pretreatment with the α7nAchR agonist, only a slight morphological change was observed in the P group as compared to those in the C group (Fig. 1C), which suggested that the α7nAchR agonist may have alleviated the pathological injury of the CPB-injured rats; however, the typical vacuolated degenerations in hippocampal neurons were observed in those co-treated with α7nAchR antagonist (Fig. 1D), indicating the protective effects of α7nAchR agonist may be inhibited by MLA treatment. These results suggested that activation of α7nAchR may alleviate CPB-induced pathological injury.

To determine the effects of α7nAchR agonist treatment on apoptosis in hippocampal neurons, the T3 sections were also evaluated by TUNEL staining. Compared with the control neurons in the S group, the neurons in the C group exhibited typical signs of apoptosis (Fig. 2A); neuronal apoptosis appeared to be lower in the P and M groups when compared with the C group (Fig. 2A). To further determine the effects of the α7nAchR agonist on hippocampal neuron apoptosis, the integrated OD average of apoptosis positive area was quantified in captured images from all experimental groups. Compared with the S group, apoptosis was significantly increased in CPB-injured rats in groups C, P and M, which suggested that hippocampal cell apoptosis may be induced following CPB surgery. Notably, a lower rate of neuronal apoptosis was observed in rats pretreated with the α7nAchR agonist compared with the C group (P<0.05; Fig. 2B); however, apoptosis was significantly increased in rats co-treated with the α7nAchR antagonist compared with the P group (Fig. 2B). These results indicated that CPB-induced apoptosis of hippocampal neurons may be effectively reduced by pretreatment with the α7nAchR agonist.

In the light of the inhibition of α7nAchR agonist on apoptosis of hippocampal neurons, the protein expression levels of Caspase 3, a key downstream inducer of apoptosis (20), was evaluated by western blot assay. In the T0 and T1 tissue specimen, no significant differences were detected in Caspase 3 expression between any of the groups, which suggested that apoptosis was not induced at this period in time. Conversely, tissues at T2 and T3 exhibited increased Caspase 3 expression in the CPB-injured rats in groups C, P and M compared with expression in the S group rats (P<0.05; Fig. 3A and B), which implied that apoptosis was activated 3–6 h post-CPB surgery. Caspase 3 expression was significantly decreased in P group rats following pretreatment with α7nAchR agonist compared with the C group (P<0.05), whereas this effect was reversed in M group rats co-treated with the α7nAchR antagonist (P<0.05; Fig. 3A and B).

To confirm the location of Caspase 3 expression in the hippocampus, immunohistochemical analysis was used to determine the expression at T2, as the Caspase 3 expression reached a peak in the CPB-injured rats at T2 according to the western blotting data aforementioned. Caspase 3 expression was detected in the neurons of hippocampus (Fig. 3C), and Caspase 3 expression was significantly inhibited in the P group compared with the C group (P<0.05; Fig. 3D), which was consistent with western blotting results. Therefore, these results indicated that the α7nAchR agonist may effectively inhibit apoptosis in hippocampal neurons, which may partly be accomplished by suppressing the expression of Caspase 3.

Serum expression levels of S100β, TNF-α and IL-6 were measured to evaluate the inflammatory response in rats with CPB injury. Compared with the control S group, rats in the CPB groups C, P and M exhibited significantly increased levels of S100β, TNFα and IL6 at experimental time points T1-T3 (P<0.05; Fig. 4A-C, respectively), which was considered as an indicator of serious cerebral injury. The levels of S100β, TNFα and IL6 were significantly decreased rats in the P group following pretreatment with 7nAchR agonist compared with the expression levels in CPB model rats in the C group at the T1-T3 experimental time points (P<0.05); however, rats in the M group exhibited an increase in serum expression levels compared with the P group (P<0.05; Fig. 4A-C). The apparent improvement of inflammation suggested that α7nAchR agonist pretreatment may have a beneficial effect on anti-inflammatory systems of the rat model of CPB.

To further explore the underlying mechanisms by which α7nAchR agonist alleviated the cerebral injuries caused by CPB, Akt/GSK3β pathway activation was examined to determine the protective effects of α7nAchR agonist on the CPB-injured rats, as the Akt/GSK3β pathway was previously identified as a significant cell survival pathway (21). CPB rats in the C group exhibited a significant increase in the expression of p-Akt and p-GSK3β compared with expression levels in the S group (P<0.05; Fig. 5); whereas the expression levels of p-Akt and p-GSK3β were significantly increased in the P group following pretreatment with the α7nAchR agonist, compared with the S group and C group, which suggested that the α7nAchR agonist may promote the phosphorylation of Akt and GSK3β. Rats in the M group that were co-treated with the α7nAchR antagonist exhibited a significant decrease in p-Akt and p-GSK3β expression levels compared with the P group (Fig. 5). By contrast, no significant differences in the expression levels of total Akt and total GSK3β were identified between the groups (Fig. 5), which implied that the α7nAchR agonist did not affect the expressions of Akt and GSK3β. Therefore, these results indicated that the α7nAchR agonist may effectively upregulate the activation of the Akt/GSK3β signaling pathway in the CPB-injured rats.