PMID: 27147542 PMCID: PMC4913390 DOI: 10.1093/bja/aew064
Methods
Ethical approval
The Animal Care and Use Committee of Tokyo Medical and Dental University approved the protocol and the study was performed in accordance with relevant aspects of ARRIVE guidelines.

Experimental mice
Postnatal day five C57BL/6 mice (average body weight: 2.7 g), as a litter with their mother, were purchased from SLC (SLC Japan Inc., Shizuoka, Japan). Mice were housed under a 12-h light-dark cycle (lights on from 08:00 to 20:00), and room temperature was maintained at 21 (1°) C. The same number of pups from each litter was used for the experiments to reduce variability related to the use of different litters (total number of pups used=110). For the behavioural study, male mice were weaned at three or four weeks of age and housed four mice per cage for each experimental group. All mice had ad libitum access to water and food. Behavioural testing was carried out only on male mice to avoid potential variability caused by the oestrous cycle.16

Anaesthesia
We used sevoflurane anaesthesia protocol as previously published.3 On postnatal day six, mice were placed in a humidified chamber (180×180×200 mm) heated to 38 (1)°C, and were exposed to 3% sevoflurane in 40% oxygen for 6 h, which was delivered via a calibrated flowmeter (Shinano, Tokyo, Japan), with a total gas flow of 1 L min−1. During sevoflurane anaesthesia, all mice maintained spontaneous breathing and eventually exhibited the loss of the righting reflex.

For the experimental treatments, the pups from the same litter were randomly divided into four groups; (1) non-anaesthesia (NA group, control mice), (2) NADPH oxidase inhibitor treatment (apocynin) group where mice received intraperitoneal apocynin, 50 mg kg−1,17,18 in a total injection volume of 10 µl, (3) 3% sevoflurane exposure (SEVO group, where mice received 3% sevoflurane for 6 h), and (4) apocynin in combination with sevoflurane (apocynin+SEVO group, where mice received 3% sevoflurane for 6 h in addition to intraperitoneal apocynin, 50 mg kg−1). Apocynin powder (Abcam, Cambridge, MA, USA) was dissolved in ethanol to make a stock solution, which was then diluted with phosphate buffered saline (PBS) to the appropriate concentration for administration. As pups are very small, injection volumes were limited to 10 µl and so the percentage of alcohol in the solution needed to be sufficient to fully dissolve the apocynin powder. However, during perfusion, the abdominal tissue did not show any sign of damage 24 h after anaesthesia induction, suggesting that local ethanol toxicity was negligible. The preparation of apocynin i.p. injection was done according to previously published studies.17,18 Mice in the NA and apocynin groups received carrier gas. Mice were returned to the original litter after treatment.

Detection of superoxide
Mice were anaesthetized using i.p. pentobarbital (50 mg kg−1) and adequate anaesthesia was ascertained by lack of response to tail pinch. They were then rapidly transcardially perfused with 10 ml cold 0.1 M PBS and the brain was quickly removed and immediately frozen at −80°C. Slices, 10-μm-thick, were cut on a cryostat and mounted onto microscope slides. Concentrations of superoxide in the brain were assessed using dihydroethidium (DHE) (Sigma-Aldrich, St. Louis, MO, USA) fluorescence.19 Each slice was incubated with 10 μM DHE for 30 min at 37°C in the dark. After incubation, the sections were washed with PBS (pH 7.4) three times and analysed by fluorescence microscopy using the Zeiss LSM Image Browser (Carl Zeiss MicroImaging, Jena, Germany). Mean DHE fluorescence values of five regions of interest in the cortex layer (two for each slice) were quantified.

Histopathological evaluation
For histopathology, mice were anaesthetized as before, 18 h after treatment, and perfusion was performed. The brain was removed and immersed in 4% paraformaldehyde in PBS overnight at 4°C. Subsequently, 50-μm-thick coronal sections were cut, and then washed with PBS in 0.3% Triton X-100 for 10 min, and endogenous peroxidase activity was blocked with 1% H2O2 for 30 min. Thereafter, sections were incubated with rabbit anti-cleaved caspase-3 antibody (Cell Signaling Technology; 1:300) in PBS/Tween-20 overnight at 4°C for detecting apoptosis. Sections were incubated with peroxidase-conjugated secondary antibody (EnVision+System, Dako, Tokyo, Japan) for 2 h and washed with PBS. Then, the sections were reacted with 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. The sections were mounted on slides and counterstained with 0.5% neutral red solution. Our previous investigation3 showed that the apoptotic response to sevoflurane was robust over the whole brain.

Western blot analysis
For protein analysis using western blotting, mice were anaesthetized as before and 18 h after treatment, whole brains were quickly removed, and immediately homogenized on ice in 100 μl homogenization buffer containing 20 µM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM Na4P2O7, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). After centrifugation at 21,000×g for 30 min at 4°C, the supernatant was removed and stored at −80°C until use. The amount of protein in each sample was measured using a protein assay kit (BCA; Pierce, Rockford, IL, USA).

For sodium dodecyl sulfate polyacrylamide gel electrophoresis, brain protein homogenates were mixed with lane marker sample buffer (Pierce) and denatured for 5 min at 95°C. The samples were cooled, separated using a 10% Mini-PROTEAN TGX™ Gel (Bio-Rad, Hercules, CA, USA), and transferred onto a nitrocellulose membrane (Bio-Rad). Rabbit anti-4-hydroxynonenal (4-HNE) antibody (Abcam; 1:1000) was used to detect concentrations of the oxidative stress marker. Rabbit anti-p22phox and rabbit anti-gp91phox antibodies (Santa Cruz Biotechnology, Dallas, TX, USA; both 1:1000) were used to detect NADPH oxidase subunits. For detecting apoptosis, rabbit anti-cleaved caspase-3 and rabbit anti-cleaved poly [adenosine diphosphate-ribose] polymerase (PARP) antibodies (Cell Signaling Technology, Beverly, MA, USA; both 1:1000) were used. Rabbit anti-cytochrome c antibody (Abcam; 1:5000) was used to evaluate mitochondrial function. After blocking in 5% non-fat milk for 30 min, the membrane was incubated with primary antibody in TBS containing 0.1% Tween-20 overnight at 4°C. The membrane was then incubated with secondary antibody (Abcam; 1:10,000) for 1 h at room temperature after washing with Tris-Buffered Saline and Tween-20. Mouse β-actin antibody (Sigma-Aldrich; 1:5000) was used as the loading control. The protein bands were visualized using a chemiluminescence detection system (SuperSignal West Pico; Pierce). For quantification of the oxidative stress marker 4-HNE, the total intensity of the 4-HNE lane was compared between groups.20,21

Fear conditioning test
To assess long-term cognitive impairment induced by sevoflurane exposure, the fear conditioning test was performed.3,8 Male mice in each experimental group underwent behavioural testing in adulthood (11–13 weeks of age). The fear conditioning test was carried out in the daytime (between 09:00 and 12:00). The movement of mice was monitored using a computer-operated video tracking system. The apparatus used in this study were made by O'Hara & Co., Ltd. (Tokyo, Japan). The conditioning trial for contextual and cued fear conditioning consisted of a 6 min period followed by three conditioned stimulus–unconditioned stimulus pairings, each separated by 1 min. Each pairing was as follows: unconditioned stimulus, 0.5 mA foot shock intensity, 1 s duration; conditioned stimulus, 60 dB white noise, 20 s duration. The unconditioned stimulus was delivered during the last few seconds of the conditioned stimulus presentation. The contextual test was performed in the conditioning chamber for a five min period in the absence of white noise 24 h after conditioning. A cued test (on the same set of mice) was performed by presentation of a cue (60 dB white noise, 3 min duration) in an alternative context with distinct visual and tactile cues 48 h after conditioning. The duration of freezing time was recorded to assess fear memory. The freezing percentage during each test was compared between each group.

Statistical analysis
Data are shown as individual raw data points. Data from behavioural testing are expressed as mean (sd). DHE fluorescence and protein expression are expressed as a percentage of the control (NA group) value. Statistical analysis was performed using SPSS statistical software package (Version 18, SPSS Inc. Chicago, IL). Kruskal-Wallis omnibus test was used for comparisons of non-parametric data and behavioural data were analysed using anova and post hoc Neuman-Keuls multiple comparison test. A P-value<0.05 was considered statistically significant.