PMID: 29731813 PMCID: PMC5920718 DOI: 10.3892/etm.2018.5950
Materials and methods
Animal model All experiments were performed according to the guidelines of the Guide for the Care and Use of Laboratory Animals (22) and were approved by the Institutional Animal Care and Use Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University (Shanghai, China). A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China). They were housed in polypropylene cages (5 or 6 animals per cage) and kept at a 12 h light-dark cycle at room temperature (21–24°C) in 55% humidity for 7 days prior to testing. All animals had free access to food and water.
Experimental protocols There were two experimental protocols used based on the sevoflurane concentration used in previous studies (23,24) and 1.3 and 2.6% sevoflurane was used in the present study. For protocol one, 36 mice were randomly assigned into 3 groups with 12 mice in each group: The 2.6 and 1.3% sevoflurane groups and the control group (exposed to 30% O2). Following exposure to sevoflurane or O2 for 6 h, the mice from all 3 groups were sacrificed by intraperitoneal injection of 1.5% pentobarbital sodium (375 mg/kg) (Dalian Idery Biotechnology Co., Ltd., Dalian, China). Hippocampal tissue samples from these mice were collected to measure the expression of caspase-3 using immunohistochemistry, the cleavage of PARP by western blotting, and levels of BDNF, Ntrk2, pro-BDNF, p75NTR and PKB/Akt by ELISA. To evaluate whether hypoxia and respiratory depression occurred in mice during anesthesia, blood gas analysis was performed in another 78 mice, which were randomly assigned into 3 groups: 2.6% sevoflurane (n=36), 1.3% sevoflurane (n=36) and control (n=6) groups. The mice in the 1.3 and 2.6% sevoflurane groups were divided into subgroups based on the length of time they were exposed to sevoflurane (1, 2, 3, 4, 5 and 6 h), with 6 mice in each subgroup.
For protocol two, a total of 60 mice were randomly assigned into 3 groups with 20 mice in each group: 2.6, 1.3% sevoflurane and control groups. Following exposure to sevoflurane for 4 weeks, the MWM test was performed in half of the mice in each group. The MWM test was conducted on the remaining mice at week 12.

Sevoflurane exposure As stated in a previous study (25), animals were placed in a temperature-controlled (37–38°C) transparent anesthetic chamber that was connected to an anesthetic gas monitor (Datex-Ohmeda S/5, Datex-Ohmeda; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). For mice in the 1.3 and 2.6% sevoflurane groups, mixed gas (5% sevoflurane and 30% O2) was pre-aerated at a flow rate of 10 l/min until the concentration of sevoflurane reached 5% in the chamber and prior to placing mice in the chamber. Subsequently, these mice were placed into the chamber immediately. Following maintenance of 5% sevoflurane for 30 sec, mice were exposed to 1.3 or 2.6% sevoflurane for the indicated time periods (1–6 h), during which 30% O2 was continually gassed into the chamber at a flow rate of 3 l/min. For mice in the control group, 30% O2 alone was aerated into the chamber for 6 h, with a flow rate of 3 l/min.
Blood gas analysis The mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (50 mg/kg). Then blood samples (0.2 ml) were obtained from the left ventricle by cardiac puncture, after which the mice were sacrificed by intraperitoneal injection of 1.5% sodium pentobarbital (375 mg/kg). The partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2) and arterial oxygen saturation (SaO2) were detected using a portable blood gas analyzer (OPTI Medical Systems Inc., Roswell, GA, USA).
Tissue sample collection Following sevoflurane exposure, all the mice were sacrificed by intraperitoneal injection of 1.5% pentobarbital sodium (375 mg/kg). The brain was then rapidly removed and the complete hippocampus was dissected. Hippocampal tissue samples were stored at −80°C prior to use in laboratory experiments.
Immunohistochemistry The hippocampal tissues were fixed overnight in 4% paraformaldehyde at 4°C. The hippocampal slices (5-µm-thick) were subsequently prepared using a vibrating tissue slicer (Campden Instruments, Ltd., Loughborough, UK). Immunohistochemical staining was performed as previously described (26,27). Briefly, slices were incubated with hydrogen peroxide in methanol to block endogenous peroxidase activity and 10% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology, Haimen, China) to reduce non-specific antibody binding prior to immunohistochemical staining. Slices were then incubated with a rabbit anti-caspase-3 antibody (1:200; cat. no. AC033; Beyotime Institute of Biotechnology) at 4°C for 12 h, followed by three washes with PBS. Subsequently, these slices were incubated with secondary antibody (1:4,000; cat. no. A0562; biotinylated goat anti-rabbit antibody; Beyotime Institute of Biotechnology) for 30 min at 37°C. Following washing with PBS, immunoreactivity was visualized using the streptavidin-peroxidase complex and 3,3′-diaminobenzidine (both from Beyotime Institute of Biotechnology). A DM5000B light microscope (Leica Microsystems GmBH, Wetzlar, Germany) was used to observe and collect images. The image analysis software Image Pro Plus version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to count the number of caspase-3 positive cells.
Western blotting The preparation of hippocampus protein extraction was performed as previously described (28,29). Total proteins were extracted with radioimmunoprecipitation assay buffer [1% Triton X-100, 50 mM Tris, (pH 7.4), 150 mM NaCl, M, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA and 1% sodium deoxycholate]. Following 13,000 × g centrifugation at 4°C for 20 min, the supernatant was used for western blotting (30,31). The BCA method was used to assay protein concentrations. In brief, hippocampal tissue proteins were separated by 10% SDS polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat powdered milk for 1 h at 25°C. The proteins were probed with rabbit anti-PARP antibodies (1:200, cat. no. AP102) or rat anti-GAPDH antibodies (1:5,000, cat. no. AG019) overnight at 4°C. Then, goat anti-rabbit (1:4,000; cat. no. A0208) or goat anti-rat (1:4,000; cat. no. A0192) horseradish peroxidase-conjugated secondary antibodies were used for 2 h incubation at room temperature (all from Beyotime Institute of Biotechnology). Proteins were visualized by an enhanced chemiluminescence method and analyzed with the Dolphin-Doc Plus Gel Documentation system (version 1141002; Wealtec Corp., Sparks, NV, USA). This procedure was repeated twice for all 3 groups. The relative level of PARP was presented as the band intensity and normalized to the corresponding band intensities of GAPDH.
ELISA The method of hippocampus protein extraction mentioned above was also used for ELISA. The levels of BDNF, Ntrk2, pro-BDNF, p75NTR and PKB/Akt were measured using an ELISA kit (cat. no. EK0312; Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Briefly, protein samples were added to the enzyme label plate and incubated for 1.5 h at 37°C. Next, the biotin-labeled antibodies were added for 1 h incubation at 37°C. Following washing, 30 min incubation with avidin peroxidase complex was conducted at 37°C. Color was developed using 3,3′,5,5′-tetramethylbenzidine following 20 min incubation at 37°C. Following reaction termination with a ‘stop’ solution, the products were measured at 450 nm using a microplate spectrophotometer (Spectramax 190; Molecular Devices LLC, Sunnyvale, CA, USA). All samples were assayed in duplicate and the readings were normalized to the amount of standard protein.
Behavioral studies Prior to the MWM test, mice received 2 min of touch for 5 days to avoid the fear to touch during the test. The MWM test was performed as previously described (32,33), with minor modifications. The round pool (diameter, 122 cm) was filled with warm water, made opaque by the addition of titanium dioxide and an escape platform was placed in the northwest quadrant and hidden 0.5 cm below the surface of the water. The MWM test was performed on 7 consecutive days (6 days for training and 1 day for the probe test). Briefly, mice received 4 training sessions daily for 6 consecutive days. Each trial began from a different point and ended when the mice found the platform. The time from beginning to end was considered to be the time of escape latency. If mice could not find the platform within 90 sec, the time of escape latency was recorded as 90 sec. If mice found the platform within 90 sec, the real time from beginning to end was recorded as the time of escape latency. The swim rate during training was also recorded. On day 7, the probe test was performed by allowing the mice to swim for 60 sec in the absence of the platform. During 60 sec swimming, the time spent in the northwest quadrant and platform site crossovers was recorded and analyzed using the MWM JLBehv-FCS video analysis system (DigBehv-MG; Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China).
Statistical analysis All data are presented as the mean ± standard error of the mean. A repeated measures analysis of variance (ANOVA) was used to measure the differences within groups over time. Meanwhile, one-way ANOVA was applied for comparison among groups (2.6, 1.3% sevoflurane and control groups), followed by Student Newman-Keuls post hoc test. The correlation between the swim rate and time of escape latency was identified using the Pearson Correlation coefficient. For all the analysis, P<0.05 was used to indicate a statistically significant difference. Additionally, SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) was used for the analysis of the present study.