PMID: 30372849
DOI: 10.1016/j.biopha.2018.09.111
2. Materials and methods
2.1. Reagents
The following anesthetics and substances were used: sevoflurane (Abbott, Wiesbaden, Germany), anti-Cx43, and anti-GAPDH(Sigma-Aldrich, St. Louis, MO, USA). All other reagents were purchased from Cell Signaling Technology (Boston, MA) unless otherwise specified.

2.2. Ethical approval
The present study was approved by the animal care and ethics committee of ShengJing Hospital of China Medical University (Shenyang, China) and was performed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals.

2.3. Study animals
A total of 30 Sprague –Dawley (SD) rats(10 males, 20 females), weighting 220–250 g, were purchased from Liaoning Changsheng Bio-Technology Co., Ltd. The rats were housed under a 14 : 10 constant light –dark cycle with free access to water and food for one week at room temperature (24 ± 1 °C), and then male and female rats were caged at a ratio of 1:2. The female rats were housed in individual cages when they were confirmed to be pregnant until they delivered naturally. The day of birth was noted as postnatal day 0 (P0). Postnatal day 7 (P7) male or female rat pups (sex hormones have on effect on the experimental results from 7 day to 14 day because SD rats are in their infancy in this period) weighing 14–18 g, were used in this study.

2.4. Anesthetic exposure
P7 rat pups were separated from their mothers and placed in a glass chamber (20 × 12 × 10 cm) resting in a water bath to maintain a constant environmental temperature of 38 °C. Pups from a different litter were randomly allocated to two groups. In the chamber, the rats were exposed to either 3% sevoflurane in a 30% oxygen carrier gas (balanced with nitrogen) or a carrier gas without sevoflurane for 4 h. The induction flow rates were 6 l/min for the first 5 min for induction and then 1 l/min for maintenance. The concentrations of sevoflurane, oxygen and carbon dioxide in the chamber were measured by a gas analyzer (Datex Cardiocap II, Datex-Ohmeda, Madison, WI, USA), and the rectal temperature of the pups was maintained at 37 ± 0.5 °C. The anesthetized pups were recovered in 30% oxygen for 20 min and returned to their mothers’ cages until the next procedure. For the intervention studies, we administered an inhibitor to the rats via an intraperitoneal injection 2 h before sevoflurane anesthesia. All the experiments were performed in a blinded manner.

2.5. Hippocampus harvesting and protein level quantification
At the end of anesthesia, five pups from each group were randomly selected and killed by decapitation at 6 h, 1 d, 3 d and 7 d. The hippocampus of each pup was harvested and then stored at −80 °C until use. We lysed the harvested hippocampus in ice-cold radio immuno precipitation assay (RIPA) buffer containing protease inhibitors (10 mM Tris-HCl, PH 7.4, 150 mm NaCl, 2 mM EDTA, 0.5% Nonider P-40, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A) and a phenylmethylsulfonyl fluoride solution(1 mM), as previously described [20,36]. The lysates were then collected and centrifuged at 1880018,800×g (Micro 21R, Thermo, Germany) for 30 min at 4 °C. We used a bicinchoninic acid (BCA) protein assay kit (Pierce, Iselin, NJ) to quantify the amount of protein.

2.6. Experimental protocol
Two experiments were performed. Experiment one, included two group, the sevoflurane group and the control group. After anesthetic exposure and in accordance with the above method, five of twenty P7 rat pups in each group were randomly sacrificed at 6 h, 1 d, 3 d and 7 d after the experimental intervention. The expression levels of Cx43, total and phosphorylated MAPKs, and total and phosphorylated c-Jun and c-Fos were tested with Western blots (Fig. 2).

Fig. 2
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Fig. 2. Schematic representation of the experimental protocol.

In experiment two, according to the MAPK signal defined in experiment one, one or several corresponding inhibitors were injected intraperitoneally 2 h before sevoflurane exposure. Only the ratio of phosphorylated JNK to JNK was increased in MAPK signal after sevoflurane exposure in experiment one (Fig. 4), so the JNK inhibitor SP600125 10mg/Kg was finally injected intraperitoneally 2 h before sevoflurane exposure without other inhibitors of ERK and p38. Rat pups in the sevoflurane and control groups received either an inhibitor or an equal volume of DMSO including control, control + SP600125, sevoflurane and sevoflurane + SP600125. After gas exposure, at least ten P7 rat pups (five were used for Western blots and five were used for immunohistochemical analyses) in four groups were sacrificed at 6h, 1d, 3d and 7d.

2.7. Western blots
Fifty micrograms of each protein sample was separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‑PAGE); using a semidry blotting apparatus(Bio-Rad Laboratories, Munich, Germany), the proteins were electrotransferred to nitrocellulose membranes (Millipore Corp., Eschborn, Germany) and then incubated overnight at 4 °C with the appropriate primary antibodies: anti-Cx43 (1:1000; SAB4501175), anti-ERK1/2 (1:1000; 4695), anti-phospho-ERK1/2 (1:1000; 4370), anti-JNK (1:1000; 9252), anti-phospho-JNK (1:2000; 9251), anti-p38 MAPK (1:1000; 8690), anti-phospho-p38 MAPK (1:1000; 4511), anti-c-Jun (1:1000; 9165), anti-phospho-c-Jun (1:1000; 3270), anti-c-Fos (1:1000; 2250), anti-phospho-c-Fos (1:1000; 5348) and anti-GAPDH (1:1000; A9169). Then, the respective secondary antibodies conjugated to horseradish peroxidase (HRP) were added for 2 h followed by three washes. The positive reactive bands were detected by Amersham enhanced chemiluminescence (ECL) reagents. The blots were scanned using an Amersham Image 600 scanner (GE Healthcare Life Sciences), and the protein band density was quantified using ImageJ software. Protein expression levels were evaluated by the GAPDH ratio.

2.8. Immunohistochemical analysis of cleaved caspase-3
Caspase-3 positive cells were detected using immunohistochemistry (IHC). Five rats in each group were euthanized by transcardial perfusion with saline, followed immediately by 4% paraformaldehyde at 6 h, 1d, 3d and 7d. Then, the whole brains were harvested, postfixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3.5um-thick sections. These tissue sections were then baked, deparaffinized, rehydrated, and quenched of endogenous peroxides. A primary antibody against activated caspase-3 (1:200 dilution, catalog no. 9662; Cell Signaling Technology) was applied and incubated at 4 °C overnight followed by a 40-min incubation with a biotinylated goat antirabbit antibody (1:500 dilution; Santa Cruz Biotechnology). Hippocampal CA1 region, a region of the brain vital for memory formation, was colorized with diaminobenzidine solution for 8 min and counterstained with hematoxylin. The sections were observed using an E100 microscope (Nikon Corporation, Japan, 400× magnification), with 3 randomly chosen fields imaged per slide. One slide per animal was prepared and counted in five rats. Caspase-3 positive cells were counted manually in each hippocampal slide vision.

2.9. Morris water maze (MWM)
To assess neurodevelopmental outcomes in adolescence, particularly learning and memory functions, twenty-four rats from four groups including control, control + SP600125, sevoflurane and sevoflurane + SP600125, were subjected to the MWM after 28 d (six rats in each group), as previously described. Briefly, a circular pool (1.6 m diameter, 60 cm height) was used for the water maze, and a submerged platform (10 cm diameter, 2 cm below the surface of the water) was located at a fixed position in the pool. The water temperature was set at 23 ± 1 °C. Escape latency trials were conducted once per day for five consecutive days. In the trials, the rats were trained to swim to and locate the hidden platform. After every trial, each mouse was placed in a holding cage under a hair dryer for 5 min to dry before returning to its regular cage. The time spent finding the hidden platform and the swimming distance before reaching the platform were recorded. After the escape latency trials, the platform was removed, and the rats were allowed to swim freely for 90 s; the number of times that the former platform was crossed was determined. The entire behavioral test was recorded and analyzed using an Noldus Ethovision XT video analysis system (Netherland).

Each rat was placed on the platform in the center of the MWM for 30 s and, then released into the water from an assigned release point. The rat was allowed to swim for 90 s or until it landed on the platform. If the rat failed to reach the platform within 90 s, it was placed on the platform for an additional 10 s. The swimming distance and the time required to reach the platform were recorded using video tracking and analyzed by MWM software. After the MWM test, all twenty-four rats were sacrificed without biochemical analysis.

2.10. Statistical analysis
Data was analyzed using GraphPad Prism 6 software (version 6.0; Graphpad Software, Inc.). Statistical significance was determined by Two-way ANOVA followed by Tukey multiple comparison tests as appropriate. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) in the MWM. At least three individual trials were performed for each experiment and data represented as mean ± SEM. P <  0.05 was considered statistically significant. Specific p values are indicated in figure legends.