PMID: 29434807 PMCID: PMC5776508 DOI: 10.3892/etm.2017.5651
Materials and methods
Animals The current study was approved by the Animal Care Committee at Sun Yat-sen University (Guangzhou, China) and performed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals (27). A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029). The pups were housed in the same cage as their mothers and were kept under temperature-controlled environmental conditions (26°C) on a 14:10 constant light-dark cycle until P7. The mother mice had free access to food and water. The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17). The control mice were exposed to normal air for 6 h under the same condition (n=12). The concentrations of anesthetic gas, oxygen and carbon dioxide in the chamber were measured using a gas analyzer (Datex-Ohmeda; GE Healthcare, Chicago, IL, USA). All animals were sacrificed 2 h following termination of sevoflurane/oxygen exposure and their cortices were used for western blotting (sevoflurane group, n=6; control group, n=6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (sevoflurane group, n=6; control group, n=6).
Tissue preparation Half of the mice in each group were used for western blotting and half of the mice for TUNEL studies. For western blotting, mouse pups were anaesthetized by inhaling 3% of sevoflurane until loss of the righting reflex (LORR), which indicated the mice had lost consciousness. Then the mice were sacrificed by decapitation. Cortices were isolated immediately on ice and then stored at −80°C until use. For TUNEL studies, mouse pups were sacrificed by inhaling 3% of sevoflurane until LORR and perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at 4°C. Their brains were post-fixed in the same fixative for 48 h at 4°C, and then paraffin embedded and sectioned into 6-µm-thick sections. As described in previous studies (15,16,25), at least three sections in the same plane of the hippocampus for each animal were selected to detect cells that exhibited positive TUNEL staining; all sections used in TUNEL were 100 µm apart and the sections were according to Figures 129–131 in the Atlas of the Developing Mouse Brain (28).
Western blotting Western blotting was performed as previously described (15,16,25). Briefly, the protein concentration in each sample was determined using a BCA protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Sample proteins (40 µg/lane) were separated on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h. Membranes were subsequently incubated at 4°C overnight with the following primary antibodies: Anti-cleaved caspase-3 (cat no. 9664) at 1:2,000 dilution, anti-α-fodrin (which contain SBDP145 and SBDP120 fragments; cat no. 2122) at 1:2,000 dilution, anti-phosphorylated-(p)-JNK (cat no. 4668) at 1:2,000 dilution, anti-JNK (cat no. 9252) at 1:2,000 dilution, anti-p-ERK1/2 (cat no. 4376) at 1:1,000 dilution, anti-ERK1/2 (cat no. 4695) at 1:1,000 dilution, anti-p-P38 (cat no. 4631) at 1:1,000 dilution, anti-P38 (cat no. 9212) at 1:1,000 dilution, anti-p-CREB (cat no. 9198) at 1:1,000 dilution, anti-p-nuclear factor-κB (NF-κB) (cat no. 3033) at 1:1,000 dilution, anti-p-Akt (Ser 473) (cat no. 4060) at 1:2,000 dilution, anti-Akt (cat no. 4685) at 1:5,000 dilution, anti-p-GSK-3β (Ser 9) (cat no. 5558) at 1:2,000 dilution, anti-GSK-3β (cat no. 9315) at 1:2,000 dilution, anti-p-CRMP-2 (Thr 514) (cat no. 9397) at 1:2,000 dilution, anti-CRMP-2 (cat no. 9393) at 1:2,000 dilution and anti-β-actin (cat no. 3700) at 1:2,000 dilution (all Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-p-GSK-3β (Ty 216) (cat no. ab75745; Abcam, Cambridge, USA) at 1:2,000 dilution. The membranes were washed with TBST three times and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, cat no. A0216; goat anti-rabbit IgG, cat no. A0208; 1:2,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The membranes were washed with TBST three times and visualized using an enhanced chemiluminescence detection system (cat no. 34580; Thermo Fisher Scientific, Inc.). Images were scanned using an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of p-ERK1/2, p-JNK, p-p38, p-Akt, p-GSK-3 and p-CRMP-2 were normalized to the bands of total ERK1/2, JNK, p38, Akt, GSK-3β and CRMP-2 from the same samples. The band signals of the other proteins were normalized to those of β-actin and the results in each group were normalized to that of the corresponding control group.
TUNEL assay TUNEL was performed following a previously described protocol (15,16). A Dead End™ fluorometric TUNEL system (Promega Corporation, Madison, WI, USA) was used and staining following the manufacturer's protocol. Briefly, TUNEL labeling was conducted with a mix of 45 µl equilibration buffer, 5 µl nucleotide mix and 1 µl recombinant terminal deoxynucleotidyl transferase (rTdT) enzyme in a humidified, lucifugal chamber for 1 h at 37°C, and then Hoechst 33258 (H-33258; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to stain nuclei for 10 min at room temperature. The sections were protected by anti-Fade solution and mounted on glass coverslips with clear nail polish sealing the edges. Slides were protected from direct light during the experiment. The images of TUNEL positive cells in the retrosplenial cortex (RS), frontal cortex (FC) and parietal association cortex (PtA) areas were acquired by Ti-S inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) and analyzed using NIS-Elements Basic Research imaging processing and analysis software (version 3.0; Nikon Corporation). The density of TUNEL positive cells in the three cortical regions was calculated by dividing the number of TUNEL positive cells by the area of that brain region.
Statistical analysis Sample size was calculated using PASS 11 software (NCSS, LLC, Kaysville, UT, USA) to achieve 80% power at a significance level of P<0.05. All data were determined to be normally distributed using the Shapiro-Wilk test and had no significant heterogeneity of variance as detected by Levene's test. GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used to conduct all statistical analyses. Data were presented as mean ± standard deviation and were analyzed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.