Experiments were performed using Sprague-Dawley rats (male; age, 7 days; average weight 16–17 g) obtained from the Experimental Animal Center of Sun Yat-sen University, (Guangzhou, China). All interventions and post-anesthesia animal care were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the Guidelines of the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China). The use of animals in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-Sen University (approval no. 2014A-073). All efforts were made to minimize suffering and the number of animals used. The rats were maintained under a 12-h light-dark cycle (light between 07:00 and 19:00), and the room temperature was maintained at 21±1°C. Rats had ad libitum access to water and food. A total of 19 litters, 48 control and 51 treated pups were used in the present study.

Rats were anesthetized using the protocol described previously (8). Seven-day-old male rats were randomly divided into air-treated control (Con group, n=15) and sevoflurane-treated groups (Sevo group, n=15). Rats in the Sevo group were placed in a plastic container and exposed to 2.3% sevoflurane for 6 h using air as a carrier, with a total gas flow of 2 l/min. In order to maintain the body temperature of rats during sevoflurane exposure, the chamber was heated to 38°C using an external heating device (NPS-A3 heated device; Midea Group, Beijiao, China) and an internal hot water bag. The levels of sevoflurane, oxygen and carbon dioxide in the container were monitored using a gas monitor (Detex Ohmeda, Inc., Louisville, CO, USA). During exposure, the respiration frequency and the skin color of the pups was monitored by an investigator. Rats were excluded from the experiment if any signs of apnea or hypoxia were detected. Sevoflurane exposure ceased following 6 h. Upon waking from anesthesia, rats were returned to the maternal cage for 6 h before they were sacrificed. The rats in the control group were placed into the same plastic container as those for the sevoflurane group, where they were exposed to air for 6 h.

Arterial blood analysis was performed on both groups using the methods described previously (8). The blood samples were obtained from rats immediately following removal from the maternal cage (0 h, n=3 in each subgroup) and at the end of exposure (6 h, n=3 in each subgroup). Briefly, the arterial blood sample (50 µl) was rapidly obtained from the left cardiac ventricle and transferred into a heparinized glass capillary tube. Each sample was analyzed immediately following collection with a blood gas analyzer (GEM Premier 3000; Instrumentation Laboratory, Bedford, MA, USA), and the analysis was repeated >3 times. The pH, arterial carbon dioxide tension, arterial oxygen tension, and blood glucose levels in arterial blood samples were analyzed in order to exclude other factors, including acidosis, hypercapnia, hypoxemia and hypoglycemia that may induce apoptosis.

In order to determine alterations in the activity of cleaved caspase-3 in the hippocampus following sevoflurane exposure, rats from the Con and Sevo groups (n=3) were deeply anesthetized with 10% chloral hydrate (350 mg/kg, Jianglai Biology, Inc., Shanghai, China) intraperitoneally on P7 at 6 h following exposure to sevoflurane or air for 6 h, before they were perfused with 4% paraformaldehyde (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in phosphate-buffered saline (PBS; pH 7.4) through the left cardiac ventricle. The brains were dissected and placed in 4% paraformaldehyde (Sigma-Aldrich; Merck Millipore) at 4°C overnight. Following fixation, the brains were soaked in 10, 20 and 30% sucrose for an additional 24 h. A sliding microtome (Scientific Instruments, Inc., West Palm Beach, FL, USA) was used to cut the brains into coronal sections (30 µm), which were blocked with a solution containing 1% bovine serum albumin (Sigma-Aldrich; Merck Millipore) and 0.4% Triton X-100 (Sigma-Aldrich; Merck Millipore) for 2 h at room temperature. Tissue sections were subsequently incubated with a rat anti-rabbit cleaved caspase-3 primary antibody (cat. no. 9661, dilution, 1:1,500; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C for 72 h. After washing with PBS, the sections were then incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG secondary antibody (cat. no. 43413, dilution, 1:400; Sigma-Aldrich; Merck Millipore) in the dark for 2 h at room temperature. Finally, the sections were washed with PBS, mounted on gel-coated slides and observed under a fluorescence microscope (Axio Imager Z1; Carl Zeiss AG, Oberkochen, Germany). CA1 and CA3 hippocampal regions in both groups were selected for analysis by fluorescence microscopy. The images were analyzed using Image-Pro Plus software (version, 16.0; Media Cybernetics, Inc., Rockville, MD, USA), and the integral optical density (IOD) of part of the CA1 and CA3 regions in each photograph was collected. A total of 3 sections were analyzed for each rat in both experimental groups (n=3).

Total RNA was isolated from the hippocampus using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the miRNeasy Mini kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's instructions. The RNA quality and quantity were measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA), and RNA integrity was determined by gel electrophoresis.

The microarray assay and analysis of miRNA expression were performed according to the procedures described previously (20). Briefly, the RNA samples from the Con and Sevo groups were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon A/S, Vedbaek, Denmark) and hybridized on the miRCURY LNA™ array (version, 18.0; Exiqon A/S). Following three washes with the wash buffer kit (Exiqon A/S), the slides were scanned using the Agilent G2505C Microarray Scanner system (Agilent Technologies, Inc., Santa Clara, CA, USA), and the images were imported into the Axon GenePix Pro 6.0 software program (Axon Instruments; Molecular Devices, LLC, Sunnyvale, CA, USA) for grid alignment and data extraction. The intensity of the green signal following subtraction of background signal was calculated, and the mean intensity values for replicated spots on the same slide were ascertained to calculate the median intensity for each sample. The following median normalization method was applied to obtain normalized data: (Foreground signal-background signal)/median signal. The median signal was in the 50th percentile of miRNA intensity, and was observed to be >30 in all samples following background correction. The threshold value for significance used to define upregulation or downregulation of miRNA expression was considered to be a fold-change of >1.5. Hierarchical clustering analysis was performed to demonstrate distinguishable miRNA expression profiles among the samples. A false discovery rate correction was applied for multiple comparisons. miRNAs were selected for further investigation in the present study based on demonstrating a significant different expression pattern when compared with controls (fold-change >1.5). Target genes of differentially expressed miRNA sequences were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

In order to verify the accuracy of the microarray analysis, RT-qPCR experiments were performed to determine the expression of selected miRNAs using the procedures described in the manufacturer's protocol (Takara Biotechnology, Co., Ltd., Dalian, China). The primers (Invitrogen; Thermo Fisher Scientific, Inc.) used are shown in Table I. The RNA extracts were reverse transcribed into cDNA. In specific, the MMLV reverse transcriptase enzyme (Takara Biotechnology, Co., Ltd.) and RT primers of U6 and rno-miR-34c were used. The RT-qPCR reaction was performed at 95°C for 10 min (pre-denaturation) followed by 42 cycles of 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec. Following the reaction, a melting curve analysis from 55 to 95°C was applied to all reactions to ensure the consistency and specificity of the amplified product. The data were analyzed using the iQTM5 Optical System software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The RT-qPCR data were normalized using U6 RNA. The relative miRNA expression was determined by calculating the mean difference between the cycle thresholds of the miRNA and the U6 control for each sample [Δ quantification cycle (ΔC_q)] within each sample group, which was expressed as -ΔC_q for the relative change in expression. The fold-change in miRNA expression was determined by calculating the difference between the mean ΔC_q of the Sevo and Con groups at the same time point and hippocampal location (ΔΔC_q), and this value was expressed as the fold-change (2-^ΔΔCq) (21). The difference in cycle threshold alterations (−ΔΔC_q) was determined using a Student's two-tailed t-test, and a P<0.05 was considered to indicate a statistically significant difference.

Using the Gene Ontology project (http://www.geneontology.org/), target genes that were affected by the differentially expressed miRNAs in sevoflurane-exposed rat pups were identified. The Gene Ontology project was used to perform cluster analysis of the enriched biological themes, and enrichment scores equal to -log10 (P-value) were considered to indicate potential pathways. Higher enrichment score values indicate a low P-value, which therefore indicates that the cluster is significant.

At 6 h following exposure to sevoflurane or air for 6 h, 3 rats in each group were sacrificed by decapitation. The brain was dissected, and the hippocampus was quickly removed. The tissue samples were homogenized in 100 mg/ml radioimmunoprecipitation assay lysis buffer (Shanghai Shenergy Biocolor BioScience & Technology Company, Shanghai, China) containing 1% (v/v) phenylmethylsulfonyl fluoride (Shanghai Shenergy Biocolor BioScience & Technology Company) and centrifuged at 13,000 × g for 20 min at 4°C. The supernatant was separated, and the quantity of total protein extracted was measured using a Bio-Rad Protein assay (Bio-Rad Laboratories, Inc.). The samples were boiled, loaded at a concentration of 50 mg sample/lane and electrophoresed on a 10% SDS-PAGE gel, and transferred onto polyvinylidene fluoride membranes (Pall Life Sciences, Port Washington, NY, USA). The blotted membranes were incubated with rabbit polyclonal anti-p53 (dilution, 1:2,000; cat. no. Asp175; Cell Signaling Technology, Inc.), rabbit polyclonal anti-phosphorylated (p)-p53 (dilution, 1:3,000; cat. no. Asp175; Cell Signaling Technology, Inc.), mouse monoclonal anti-Bcl-2 (dilution, 1:1,000; cat. no. 15071; Cell Signaling Technology, Inc.), mouse monoclonal anti-Bax (dilution, 1:1,500; cat. no. 2772; Cell Signaling Technology, Inc.), mouse monoclonal anti-β-actin (dilution, 1:2,000; cat. no. sc-58673; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or rabbit polyclonal anti-cleaved caspase-3 (dilution, 1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.) antibodies overnight at room temperature. The membranes were then washed three times with PBS+0.05% Tween-20 (Sigma-Aldrich; Merck Millipore) mixture, and incubated for 2 h at room temperature with the anti-rabbit or anti-mouse IgG horseradish peroxidase-conjugated secondary antibodies (dilution, 1:2,000; cat. no. sc-2372 or sc-2377; Santa Cruz Biotechnology, Inc.). The protein expression levels were examined using the Pierce ECL Plus Western Blotting Substrate system (Pierce; Thermo Fisher Scientific, Inc.) and photographed. The optical density was measured using ImageJ software, V1.48u (National Institutes of Health, Bethesda, MD, USA). Immunoblots were performed at least three times for each hippocampus sample from each rat.

The data are presented as the mean ± standard deviation. Statistical analysis was performed using the two-tailed Student's t-test and the SPSS software program (version, 16.0; SPSS, Inc., Chicago, IL, USA). All data in the study represent at least three independent experiments for 3 samples in each experiment. P<0.05 was considered to indicate a statistically significant difference.

As shown in Table II, no significant alterations were observed between the control group and the sevoflurane group for pH, oxygen tension, carbon dioxide tension and glucose levels. During sevoflurane and air exposure, the skin color of the rat pups remained pink (data not shown).

In order to determine the effect of sevoflurane exposure on apoptosis in hippocampal tissues, immunohistochemistry assay analysis was performed to measure the level of cleaved caspase-3 (red regions; Fig. 1A and B) and the IOD of CA1 and CA3 regions. As illustrated in Fig. 1C, the IODs in the CA1 and CA3 regions were significantly higher in the sevoflurane group when compared with the control group (P=0.001).

Microarray analysis indicated that 688 miRNAs were expressed in the hippocampus of rats at 7 days of age. Following normalization of the signal intensities to the miRNA expression levels, miR-124-3p, miR-9a-3p and miR-125b-5p were observed to be expressed at the highest levels (Fig. 2). Compared with the control group, the expression levels of 266 miRNAs in the hippocampus of the sevoflurane group were altered. Among these 266 miRNAs, 8 miRNAs (miR-3559-5p, miR-92a-2-5p, miR-214-5p, miR-382-3p, miR-181b-1-3p, miR-101a-5p, miR-130b-5p, and miR-188-3p) were significantly upregulated, and one miRNA (miR34-c) was significantly downregulated following sevoflurane exposure (P=0.001; Fig. 3A).

In order to validate the microarray results, RT-qPCR analysis was performed to confirm the expression of the nine identified miRNAs. As demonstrated in Fig. 3B, RT-qPCR analysis confirmed that, miR-3559-5p, miR-92a-2-5p, miR-214-5p, miR-382-3p, miR-181b-1-3p, miR-101a-5p, miR-130b-5p and miR-188-3p expression levels were increased, whereas miR34-c expression was significantly reduced following sevoflurane exposure.

Target genes of differentially expression miRNA sequences were identified using KEGG pathway analysis (http://www.genome.jp/kegg/). All pathways that demonstrated a P-value of <0.05 are listed in Table III. The identified pathways included mineral absorption, tuberculosis, the p53 signaling pathway, glycolysis/gluconeogenesis, drug metabolism-cytochrome P450, metabolism of cytochrome by cytochrome P450, antigen processing and presentation, and pyrimidine metabolism associated genes. Among these pathways, only miR-34c was directly associated with the p53 signaling pathway. This suggests that the miR-34c and the p53 pathway may be involved in mediating sevoflurane-induced apoptosis in the hippocampus of developing rat brains.

Western blot analysis was performed to assess the effect of sevoflurane on the targets of the p53 signaling pathway in the hippocampus. As shown in Fig. 4, sevoflurane exposure significantly increased the level of the p-p53 protein (P=0.03; Fig. 4A) and Bax (P=0.001; Fig. 4B), and significantly decreased the level of Bcl-2 protein (P=0.001; Fig. 4B) in the hippocampus of neonate rat brains when compared with the control rat brains. In addition, sevoflurane exposure significantly increased the expression of cleaved caspase-3 when compared with the control group (P<0.05; Fig. 4C).