PMID: 26541582 DOI: 10.1016/j.brainres.2015.10.050
4. Experimental procedures
4.1. Animals
A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Laboratory Animal Co., Ltd., China) were used in this study. They were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 80-23, revised in 2011). All rats were maintained under standard laboratory temperature and humidity and a 12 day/night cycle (8 am/8 pm), and were allowed free access to food and water. The study was approved by the Experimental Animal Care Committee of the Fujian Medical University Union Hospital, and efforts were made to minimize the number of animals used and their suffering.

4.2. Experimental protocol
The animals were randomly allocated into 10 groups (n=24 per group; Fig. 1): (1) Sham, without hypoxia-ischemia; (2) HI/Control, received cerebral hypoxia-ischemia; (3) HI+Atractyloside (Atr), (4) HI+Cyclosporin A (CsA), treated like the control and respectively injected with Atr (10 mg/kg) and CsA (5 mg/kg); (5) HI+sevoflurane (Sev), treated like the control and received sevoflurane postconditioning; (6) HI+Sev+LY, (7) HI+Sev+L-N, (8) HI+Sev+SB, (9) HI+Sev+Atr, (10) HI+Sev+CsA, treated like the HI+Sev group and respectively injected with LY294002 (0.3 mg/kg), L-NAME (10 mg/kg), SB216763 (0.2 mg/kg), Atr (10 mg/kg), and CsA (5 mg/kg). LY294002, L-NAME, and SB216763 are specific blockers of Akt, eNOS, and GSK-3β, respectively. Atr and CsA open and close, respectively, mPTPs. In each group, the brains of the rats that received behavioral testing (from 32-days-old to 42-days-old; n=12 per group) were harvested for determination of hippocampal neuron count and morphology study, and the brains of the other 12 rats were harvested 24 h after the intervention for Western blot analysis, and study of mitochondrial permeability transition pore opening.

4.3. Cerebral HI model and sevoflurane postconditioning
The cerebral HI model was adapted from a procedure described previously (Ren et al., 2014). Briefly, the rats were anesthetized with pentobarbital sodium (0.5–1%, 40–50 mg/kg, intraperitoneal), and their left common carotid arteries were permanently ligated with a double 7–0 surgical silk; the arteries in the Sham group, however, were not ligated. A dose of 5 µL of 0.1% DMSO or drug (LY294002, L-NAME, SB216763, Atr, CsA) with 0.1% DMSO was injected into the left lateral ventricle immediately after the surgery as previously described (Satoh and Onoue, 2005). After waking, the rats were returned to their cages with the mothers for 1.5–2.5 h, and then placed in a chamber containing humidified 8% O2–92% N2 for 2 h. The air temperature in the chamber was maintained at 36.5±1 °C. The chamber was then exposed to room air for 15–20 min. For sevoflurane postconditioning, the animals were placed in a chamber containing 2.5% sevoflurane in 30% O2–70% N2 for 30 min after cerebral HI injury. After waking, the neonates were cleaned with 75% alcohol and returned to their mothers.

4.4. Novel object recognition test
The rats were evaluated with a nonspatial object recognition memory task 25 days after the intervention as described by Ennaceur and Delacour (1988) and Bruel-Jungerman et al. (2005). Briefly, for the first 3 days, after being comforted and stroked, each animal was put into an open chamber made of black plexiglas (80×80×60 cm3) for a 5 min acclimation and the test was conducted on the fourth day. Before the test, the animals received a 5 min training in the chamber containing 2 different objects (a white cube and a red cylinder) fixed at adjacent angles with a spacing of 10 cm from the field wall. Rats were put into the chamber with their backs turned towards the objects and allowed to explore the chamber freely for 5 minutes. Exploratory behavior can be identified when rats touch the objects with their noses or put their noses at places within 2 cm of the objects. To test memory storage, the white cube was kept in the chamber and the red cylinder was replaced by a blue semisphere. Exploratory time of new (T2) and old (T1) objects within 5 min was recorded and memorization ability of the rats was assessed by discrimination index: DI=T2/(T1+T2). The blue semisphere was replaced by a green prism 3 h after training, and the green prism was replaced by a yellow irregular shape 24 h after the training. The time each rat used to explore new and old objects was recorded for calculating DI. The DIs at 5 min, 3 h, and 24 h after the training (DI0 h, DI3 h, DI24 h) represent the instant, short-term, and long-term memory, respectively. Data with total exploration time less than 20 s were excluded from statistic analysis. The field was always provided with even light, and the objects and fields were cleaned with 75% ethanol after each testing.

4.5. Morris water maze test
After the novel object recognition test, the Morris water maze was used to test spatial learning and memory (Peng et al., 2012, Jiang et al., 2004). Briefly, a black circular pool (120 cm in diameter, 50 cm in height) was filled with water (25±1 °C) to a depth of 25 cm and located in a quiet room. Chinese ink was added to make the water opaque. The water maze was conceptually divided into 4 quadrants, and a hyaline platform (10 cm in diameter) was submerged 1 cm below the surface of the water at the midpoint of the third quadrant. In the place navigation trial, each rat underwent 4 successive trials a day for 5 days for memory acquisition training, with a 15 min interval between trials for the rat to recover physically. The sequence of water-entry points differed each day, but the location of the platform was constant. Escape latency (EL) to find the platform was measured up to a maximum of 120 s. On locating the platform, the rat was left there for 15 s before the next trial. If the rats failed to locate the platform within 120 s, it was guided to the platform and allowed to stay there for 15 s. Latency and the search strategies, including straight strategy, tendency strategy, marginal strategy, and random strategy, were recorded for each trial. Twenty-four hours after the last training session, a space exploration trial was performed. The platform was removed from the pool and rats were allowed to swim freely for 60 s. Four indexes were calculated: (1) the time spent by the rats in the third quadrant in which the platform was hidden during acquisition trials; (2) the number of rats crossing exactly over the original position of the platform; (3) the search path in the target quadrant; (4) the total movement distance. Search speed was calculated by total movement distance divided by 120 cm/s. All trials were videotaped by a camera located 2 m above the water surface and computer analyzed.

4.6. Histology of left hippocampal neurons
After the behavioral studies, rats were anesthetized with pentobarbital, transcardialy perfused with 200 mL of 4 °C heparin saline solution and then with 300 mL of 4% paraformaldehyde. Left hippocampus was made into a wax block according to Paxinos–Waston methods. Continual coronal sections (4 µm in thickness) at approximately 3.3 mm caudal to bregma were obtained, and subjected to hematoxylin–eosin (HE) staining. The sections were examined by an observer blinded to the rat group assignment. Neurons microscopically showed a clear boundary, a round or an oval shape, a smooth cell membrane, basophilic cytoplasm (Nissl body), a large and round nucleus, a clear nuclear membrane and a large and round nucleolus will be defined as surviving neurons. Apoptotic neurons will not be regarded as surviving ones. Surviving neurons in pyramidal cell layer of the CA1 and CA3 regions were counted (n/mm) by two investigators blind to experimental conditions, and a count was determined by averaging the total of 5 sections.

4.7. Western blot analysis
Proteins were separated on a 12% SDS-PAGE gel, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked using 5% nonfat milk and incubated with a mouse anti-p-Akt, t-Akt, p-eNOS, t-eNOS, p-GSK-3β, and t-GSK-3β monoclonal antibody (mAb) (Cell Signaling Technology, Beverly, MA, USA) or a mouse anti-β-actin mAb (Sigma, USA). The proteins were visualized and quantified using ECL reagents (Pierce, IL, USA).

4.8. mPTP opening assay
Preparation of mitochondria was adapted from a procedure described previously (Wu et al., 2006). All procedures were carried out in the cold (0–4 °C). Hippocampal pieces were placed in isolation buffer (250 mmol/L sucrose, 210 mmol/L mannitol, 1 mmol/L K-EDTA, 10 mmol/L Tris–HCl, pH 7.4) and homogenized (10 mL buffer/g). The homogenate was immediately centrifuged at 2000g for 3 min. The supernatant was centrifuged again at 2000g for 3 min, the second supernatant was decanted and centrifuged at 12,000g for 8 min, and the resulting supernatant was decanted and resuspended in isolation buffer without K-EDTA. The suspension was centrifuged at 12,000g for 10 min and the resulting mitochondrial pellet was resuspended in the same buffer. Mitochondrial protein concentration was quantified according to the Bradford׳s method using 1 g/mL bovine serum albumin (BSA) as standard. Purity and integrity of isolated mitochondria were confirmed by neutral red-Janus green B staining (Sigma, USA). Isolated mitochondria from the hippocampus (0.5 mg protein) was resuspended in swelling buffer (71 mmol/L sucrose, 215 mmol/L mannitol, and 10 mmol/L sodium succinate in 5 mmol/L HEPES, pH 7.4) to a final volume of 2 mL, and incubated at 25 °C for 2 min. mPTP-induced mitochondrial swelling was confirmed by 5 min incubation with the strong mPTP inhibitor CsA before addition of CaCl2, and was measured with a spectrophotometer (Beckman DU800, USA) as a reduction in optical density at 540 nm (OD540) (Kristal and Brown, 1999, Baines et al., 2003).

4.9. Statistical analysis
All data were presented as mean±standard deviation (SD). For comparison between multiple groups, data were analyzed by one-way ANOVA. When a statistical difference was determined by ANOVA, the least significant difference (LSD) procedure was applied. The percentage of search strategies were examined by the Mann–Whitney method, and repetitive measure ANOVA was used to measure mean EL at different time points. Spatial probe trial data were analyzed by one-way ANOVA and principal components analysis (PCA). All analyses were performed with SPSS 13.0 for Windows, and a value of P<0.05 was considered significant.