Specific pathogen-free Sprague Dawley rats (n=8; 1:1 male:female; weight, ~252 g) were purchased from Shanghai Super B&K Laboratory Animal Co., Ltd. (license number 2013-0016; Shanghai, China) and maintained in a temperature-controlled environment (25°C) and a humidity of 40–60% with a standard 12 h light/dark cycle and ad libitum access to food and water. All experimental procedures were approved by the ethics committee of Guizhou Medical University (Guiyang, China). A total of 18 pups from four litters were randomly divided into three groups (n=6 in each group; 1:1 male:female): i) Control group, ii) isoflurane group and iii) a C21 treatment group. Post-natal day 7 rats inhaled 1.3% isoflurane (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 3 h each day for a consecutive 3 days. Following the administration of isoflurane, rats in the C21 treatment group received 0.1 ml C21 (1 µg/kg; cat. no. C160; Sigma-Aldrich; Merck KGaA) intraperitoneally each day for a consecutive 4 days, while the rats in the isoflurane group received a similar volume of saline. Following treatment, the rats were anesthetized with 1% sodium pentobarbital (45 mg/kg; intraperitoneal injection) and decapitated. Rats weighed 10–15 g at the time of sacrifice. Fresh brain tissues were collected for flow cytometry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, while the brain tissues were fixed for transmission electron microscopy (TEM) and the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labelling (TUNEL) assay.

The cortex, hippocampus, amygdala and hypothalamus were isolated and ground. A single cell suspension was prepared following trypsinization. A metal mesh was applied to isolate the single cells from the homogenates. Cells were centrifuged at 375 × g for 2 min at 4°C, and 5×10⁵ cells were collected for flow cytometry. Apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis kit [cat. no. AP101-100-kit; Hanzhou Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China] according to the manufacturer's protocol. Following staining with Annexin V-FITC (5 µl) and PI (10 µl) together for 5 min at room temperature, cells were detected using a flow cytometer (NovoCyte 2060R, ACEA Biosciences, Inc., San Diego, CA, USA) with excitation at 488 nm and emission at 530 nm. Data were analyzed using FlowJo 10 (FlowJo LLC, Ashland, OR, USA).

Collected brain tissues were fixed in 10% formalin at 4°C for 24 h for the TUNEL assay. Cortex, hippocampus, amygdala and hypothalamus were subsequently separated and cryoprotected in 30% sucrose for 1 h at 4°C, prior to slicing into 20 µm sections with a freezing microtome. TUNEL staining (2 µl) was performed using the ApopTag In Situ Apoptosis Detection kit (cat. no. C1089; Beyotime Institute of Biotechnology, Shanghai, China) at 45°C for 2 h, following the manufacturer's protocol. Following this, 3 drops of mounting medium containing 4′,6-diamidino-2-phenylindole (cat. no. ab104139; Abcam, Cambridge, UK) was added and the slides were then covered. After staining, the sections were imaged using fluorescence microscopy (Olympus Corporation, Tokyo, Japan). Four fields in each slice were analyzed and apoptotic cells were counted.

The bilateral hippocampus was fixed in 2.5% glutaraldehyde at 4°C for 2 h, dehydrated in 70, 75, 80, 85 and 95% ethanol, embedded in paraffin, solidified, and sectioned into 70 nm slices. Following this, sections were stained with 3% uranyl acetate and 3% lead citrate for 7 min at room temperature and imaged using transmission electron microscopy (80 kV; JEM-1230; JEOL, Ltd., Tokyo, Japan).

Peroxisome proliferator-activated receptor (PPAR)-α levels were detected with a PPAR-α ELISA kit (cat. no. ml003331; MLbio, Shanghai, China) according to the manufacturer's protocol. The reagents in the kit were kept at room temperature for 30 min. A standard curve was established. Cortex, hippocampus, hypothalamus and amygdala tissues were homogenized and centrifuged at 11,000 × g for 10 min at 4°C and the supernatants were used in the ELISA. All standard samples and test samples required three duplicates. A blank control, without sample or enzyme reagent, was used. The absorbance was detected at 450 nm using a microplate reader (RT-6100; Rayto Life and Analytical Sciences Co., Ltd., Shenzen, China).

mRNA was extracted from rat cortex, hippocampus, hypothalamus and amygdala using a TRIzol^® assay kit (Thermo Fisher Scientific, Inc.). The mRNA was transcribed into cDNA using a PrimeScript™ RT-PCR Kit (cat. no. DRR014A; Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol (25°C for 9 min, 37°C for 121 min and 85°C for 5 min). SYBR Green (cat. no. HY-K0501; MedChemExpress, Monmouth Junction, NJ, USA) was used to detect the expression level of Bcl-2 using cDNA as a template. qPCR was performed using the following thermocycling conditions: Initial denaturation at 95°C for 10 min; followed by 36 cycles at 95°C for 14 sec and 58°C for 1 min. The 2^−ΔΔCq method was used to quantify the results as previously described (19), and relative expression was normalized to GAPDH. The primer sequences are listed in Table I.

Homogenates from the cortex, hippocampus, amygdala and hypothalamus were obtained from each group and lysed using an illustra triplePrep Kit (cat. no. 28-9425-44; GE Healthcare Life Sciences). The protein levels were detected using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 and 5% fat-free milk for 2 h at room temperature, and were subsequently incubated overnight at 4°C with rabbit antibodies against Bcl-2 (1:1,500; cat. no. bs-0032R; BIOSS, Beijing, China) and GAPDH (cat. no. A007; 1:1,000; ABclonal Biotech Co., Ltd., Woburn, MA, USA). Following this, membranes were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000; cat. no. TA140003; OriGene Technologies, Inc., Beijing, China) for 2 h at room temperature. A chemiluminescent substrate detection reagent (cat. no. RPN2133; GE Healthcare Life Sciences) was applied to assist with staining. The target band was analyzed using Quantity One version 1.4.6 (Bio-Rad Laboratories, Inc.) for densitometric analysis.

The data are expressed as the mean ± standard deviation and were statistically analyzed using SPSS 19 (IBM Corp., Armonk, NY, USA). One-way analysis of variance followed by Newman-Keuls post-hoc test was applied to determine statistical significance. All experiments were repeated six times. P<0.05 was considered to indicate a statistically significant difference.

The TUNEL assay demonstrated that postnatal administration of isoflurane elicited marked apoptosis in the cortex, hippocampus, amygdala and hypothalamus. By contrast, C21 treatment appeared to reduce apoptosis in these four regions (Fig. 1). The apoptotic rates in each group were calculated (Fig. 2). In the control groups, the apoptotic rates were 1.23% (cortex), 1.86% (hippocampus), 2.43% (hypothalamus) and 1.40% (amygdala). The apoptotic rates in the isoflurane group were 5.53% (cortex), 5.24% (hippocampus), 5.05% (hypothalamus) and 4.28% (amygdala). The apoptotic rates in the C21+Isoflurane group were 2.87% (cortex), 3.05% (hippocampus), 3.15% (hypothalamus) and 3.29% (amygdala).

Apoptosis was further confirmed by flow cytometry. As shown in Fig. 3, approximately 20% of cells were apoptotic in the isoflurane group, which was significantly reduced by C21 treatment (~8%).

TEM further confirmed that isoflurane induced apoptosis in the hippocampus, which was ameliorated by C21 treatment (Fig. 4). The nuclei of the cells in the control group were round and transparent. The nuclei of the cells in the isoflurane group were shrunken, and condensed chromatin was evident. A marked reduction in synapse numbers was observed in the isoflurane group, but not in the control group. By contrast, the nuclear shrinkage and decrease in synapse number was less prominent in the C21 group.

PPAR-α levels in several brain regions were measured using ELISA. As shown in Fig. 5, isoflurane exposure significantly reduced PPAR-α levels in the four selected regions, compared with the control. By contrast, C21 treatment significantly elevated PPAR-α levels compared with the isoflurane group.

Bcl-2 expression in different regions was detected at both the mRNA and protein level. Fig. 6 shows that, isoflurane exposure significantly reduced Bcl-2 mRNA levels in the four selected regions. Additionally, C21 treatment significantly elevated Bcl-2 mRNA expression. Western blot analysis also demonstrated that isoflurane exposure significantly reduced Bcl-2 protein expression in the four selected regions. Furthermore, C21 treatment significantly increased Bcl-2 protein expression.