The study protocol was approved by the Ethics Committee of Jinling Hospital, Nanjing University and the experiments were performed in accordance with the Guideline for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. Male Sprague-Dawley rats aged from P5 to P55 days were used for sevoflurane anesthesia and subsequent analyses (Figure 1B). The pups after birth were housed with the dam until weaning in the P21. All rats were housed in a standard condition under a 12:12 hour light/dark cycle with lights from 7:00 to 19:00, with the room temperature of 23±1℃, and free access to food and water.
We used three different sets of rats for the following experiments. The first set of rats (N=36) were used to study the neurotoxicity of sevoflurane (the 0, 4, 8 and 24 hours after the end of last anesthesia) and the suitable dose of Bix (Bix-01294, 2-(Hexahydro-4-methyl-1H-1, 4-diazepin-1-yl)-6, 7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine trihydrochloride) (Cayman, Ann Arbor, MI, USA) to improve the detrimental effect of sevoflurane exposure. The second set of rats (N=36) were used to study the effect of Bix on the sevoflurane anesthesia at P8 and P35. The third set of rats (N=32) were used for behavioral tests. The second and third sets of rats were equally randomized into one of the four groups: control + vehicle (Con + Veh) group, control + Bix (Con + Bix) group, sevoflurane + vehicle (Sev + Veh) group, or sevoflurane + Bix (Sev + Bix) group.
Sevoflurane Anesthesia and Drug Treatment
Anesthesia was induced with 6% sevoflurane by 100% of 2 L/minute O2 for 3 minutes and maintained with 3% sevoflurane by 100% of 2 L/minute O2 for 1 hour and 57 minutes in a thermostatic chamber daily for three successive days from P5 to P7. Previously, we have shown that this anesthesia protocol could cause the neurobehavioral abnormalities without respiratory depression (6). The rats breathed spontaneously, and concentrations of anesthetic and oxygen were measured continuously using a calibrated Datex side stream analyser that sampled from the interior of the chamber. Temperature of the rats was maintained at 37±0.5℃ during anesthesia by heating lamps and heating pads. The pups returned to their mother cage after recovery from anesthesia. Accordingly, Bix was diluted with vehicle (normal saline) to final 0.2 mg/ml and subcutaneously injected with 0.25, 0.5, and 1 mg/kg 15 minutes before each sevoflurane exposure, respectively.
All behavioral tests were conducted in a sound-isolated room with the instruments (Shanghai Softmaze Information Technology Co. Ltd., Shanghai, China). The rats used for behavioral tests were not analyzed in further biochemical experiments.
Open Field (OF) Test
At P35, the rat was placed into the OF apparatus (XR-XZ301, 100 cm × 100 cm × 40 cm black plastic box) for 10 minutes. The behaviors of each rat were traced by a camera for further analysis. At the end of each test, the surface of the arena was thoroughly cleaned with 75% alcohol to avoid the presence of olfactory cue.
Fear Conditioning (FC) Test
The FC test was performed by using the FC paradigm (XR-XC404, 30 cm × 30 cm × 45 cm soundproof chamber) at P39-41. The FC test was a simple and sensitive test of hippocampus-dependent and hippocampus-independent memory function. During the training, the rat was allowed to explore the training chamber for 180 seconds. Within the last 30 seconds, the rat was subjected to a tone (30 seconds, 70 dB, 3000 HZ) with a terminating 2 seconds electrical foot-shock (0.5 mA), followed by an additional 30 seconds without any stimulation. The rat was placed in the training chamber for 5 minutes (context association testing, without tone or foot-shock) after 24 hours. Then, the cue conditioning test was conducted on the next day in a novel training chamber with a radically changed context. A 3 minutes exploration of the new context was followed by 3 minutes of tone presentation (70 dB, 3000 HZ, without foot-shock). Fear response was reflected by the time of freezing.
Morris Water Maze (MWM) Test
The MWM task was carried out at P49–55. In the spatial discrimination trials, the rat was placed in a round tank (XR-XM101, diameter 120 cm, height 50 cm) filled with opaque water containing an escape platform (height 22 cm) submerged 2 cm below the surface of water. The platform remained in the same location relative to the four visual cues in the tank. The rat received four swims per day for six consecutive days using a 15 seconds interval on platform between swims. Every training session, the rat was placed carefully in the water next to the maze wall and allowed to swim until finding the platform or for 60 second (whichever came first). The time taken to climb the platform (latency) was recorded. In the probe trials (on the seventh day), the rat was placed in the tank for 60 seconds during which the escape platform was removed from the tank, the time spent in swimming in the target quadrant and the swimming route were recorded. The water temperature was maintained at 22±1℃. After finished the per day swimming task, the rat was removed from the tank, dried with a clean towel, and placed in a holding box that is warmed with a temperature-controlled heating pad placed beneath it.
The Western blot analysis was performed previously (17). The rat was sacrificed by 2% sodium pentobarbital (70 mg/kg, intraperitoneal [i.p.]) at the indicated time points ( 0, 4, 8, and 24 hours [P8] after the end of last sevoflurane exposure and P35), and the hippocampus was dissected, frozen, and stored at -80℃. On the day of analysis, the brain tissues were allowed to equilibrate to a temperature of 4℃. Tissue protein was collected by using the protein extraction kit (Nanjing KeyGEN Biotech, Nanjing, China). The protein concentrations were determined using the QuantiPro BCA Assay kit (Nanjing KeyGEN Biotech, Nanjing, China). Protein extracts (70 μg protein/lane) were separated using polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes with an electrophoretic transfer system. Membranes were blocked with 5% nonfat- dried milk in tris-buffered saline tween (TBST) for 1 hour and then incubated with the primary antibodies: anti-rabbit cleaved caspase-3 (polyclonal, #9661, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit-G9a (monoclonal, #3306, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit H3K9me2 (monoclonal, #4658, 1:1000, Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit β-actin (polyclonal, #YM3214, 1:3000, ImmunoWay, New Jersey, USA) overnight in a room at 4℃. After thorough washing, membranes were incubated in 5% nonfat-dried milk in TBST with the secondary antibody (goat anti-rabbit, #AP132P, 1:5000, Millipore, Darmstadt, Germany) for 2 hours at room temperature. Enhanced chemiluminescent and the Image Quant Software (Syngene, Cambridge, United Kingdom) were used to visualize and quantitate the immunoreactivity, respectively.
The immunohistochemistry was performed as we described previously (6). Briefly, the rats were anesthetize with 2% sodium pentobarbital (70 mg/kg, i.p.) at P8, and perfused with phosphate buffered saline (PBS), followed by freshly prepared 4% paraformaldehyde. Sections of paraffin-embedded tissue (4 μm) were cut and mounted on Vectabond adhesive-coated slides (RM2016, Shanghai Leica Instrument Co. Ltd., Shanghai, China). Sections were deparaffinized in xylene and rehydrated in ethanol to water, then were washed in PBS, and incubated with the primary antibody anti-rabbit cleaved caspase-3 (polyclonal, #9661, 1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4℃ for 24 hours. The sections were washed three times for 5 minutes with PBS and incubated in the secondary antibody (goat anti-rabbit, #AP132P, 1:500, Millipore) for 50 minutes, following three washes in PBS and staining using the 3, 3'-diaminobenzidine chromogenic liquid. The cells of brown were considered to be positive. An investigator who blinded to the treatment conditions counted the number of cleaved caspase-3+ cells by using the Image Quant Software.
The statistical analyses were performed using the Prism software (GraphPad, San Diego, CA, USA). The data were presented as the mean ± standard error of the mean (SEM) and statistically analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA with Bonferroni's post hoc test. Pearson's rank correlation coefficient method was used for correlation analysis. In all of the comparisons, P<0.05 was considered to indicate statistical significance.