20230808-AI coding-1st round/4315 – Ling 2017.txt

PMID: 29042986 PMCID: PMC5639422 DOI: 10.3892/etm.2017.5004
Materials and methods
Animals According to previous observations (15), a total fo 49, male Wistar rats (14.54±1.52 g) at postnatal day 7 (P7) were selected for experimental analyses. The Wistar rats at P7 were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Rats were housed in polypropylene cages under a 12-h alternating light/dark cycle, with food and water supplied ad libitum in the institutional animal facilities. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Bengbu Medical College (Anhui, China) and performed according to the Guide for the Care and Use of Laboratory Animals (16). All efforts were made to minimize animal suffering and to reduce the number of animals used.
Anesthesia methods A total of 48 Wistar rats at P7 were randomly divided into four groups (n=12), including the 0, 2, 4 and 6-h treatment group, in which Wistar rats were exposed to 3% sevoflurane for 0, 2, 4 and 6 h, respectively. For anesthesia, Wistar rats were placed in a temperature-controlled (37±0.5°C) plexiglas anesthesia chamber. First, the rats were subject to 5% sevoflurane exposure for 30 sec, provided in a gas mixture of 5% carbon dioxide, 21% oxygen and balanced nitrogen at a flow rate of 10 l/min. Next, the rats were exposed to 3% sevoflurane for the specified time period at a rate of 1.5 l/min. During the anesthesia process, the concentrations of sevoflurane, carbon dioxide and oxygen in the gas mixture were monitored with an anesthetic gas monitor (Datex-Ohmeda S/5; GE Healthcare Life Sciences, Chicago, IL, USA). Rats were breathing spontaneously during anesthesia. Anesthesia was ended by discontinuing the anesthetics, and then rats were housed in normal conditions until 12 weeks old, at which time behavioral tests were performed.
Behavioral experiments As described in our previous study (9), three tests were conducted in sequence, including the elevated plus-maze (EPM), O-maze and Y-maze. For each behavioral test, the movement tracks of experimental rats were recorded by a video-tracking software (Any-Maze version 5.1; Stoelting Co., Wood Dale, IL, USA) and analyzed by an additional researcher who was blinded to the experimental protocols. All the test were performed during the dark phase (active period of rats) between 1 a.m. and 4 p.m. The experimental details of EPM test, O-maze and Y-maze were as described in previous studies (9,17–19). Briefly, the EPM and O-maze tests were used to assess the anxiety-like behavior in rodents, while the Y-maze test was used to investigate the immediate spatial working memory (a pattern of manifestation of cognitive function) of rodents.
Gene expression microarray analysis Rats were anaesthetized by isoflurane with an induction dosage of 4%, maintained at 2% (RuiTaibio, Beijing, China) and decapitated to obtain the hippocampus, which was then stored at −80°C until RNA extraction. Total RNA was extracted from the hippocampal tissues using a standard TRIzol reagent (catalogue no. 15596026; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as described previously (9). In brief, after the tissue was homogenized, 0.3 ml TRIzol was added to each sample. Then, 0.3 ml 100% chloroform (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was added to stratify the sample solution; transfer the aqueous phase containing the RNA to a new tube. Finally, 0.5 ml isopropanol was added to the aqueous phase to precipitate the total RNA. The quality and concentration of the RNA samples were then assessed at he absorbance ratios of A260/280 and A260/230 using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.), and samples were denatured by 2% agarose gel electrophoresis.
The whole transcription profile of all mRNAs targeted by the microarray for each sample was determined using an Affymetrix Rat Genome U34 Array (Thermo Fisher Scientific, Inc.). Sample labeling and array hybridization were performed according to the manufacturer's instructions with minor modifications. In order to analyze the gene expression, CapitalBio Corporation (Beijing, China) completed the following steps: Briefly, mRNA was purified from total RNA following the removal of rRNA using mRNA-ONLY™ Eukaryotic mRNA Isolation kit (Epicentre, Madison, WI, USA). Next, each sample was amplified and transcribed into fluorescent cDNA along the entire length of the transcripts without 3′ bias using random primers (catalogue no. 79236; Qiagen, Hilden, Germany). Subsequent to purification with an RNeasy Mini kit (Qiagen, Hilden, Germany), the labeled cDNAs were hybridized with the specific probes on the Array. The hybridized arrays were washed, fixed and scanned at 5 mm/pixel resolutions with an Agilent DNA microarray scanner (G2505C; Agilent Technologies, Inc., Santa Clara, CA, USA).

Upon collection of signal, technical quality control was performed using dChip version 2005 (Affymetrix; Thermo Fisher Scientific, Inc.) with the default settings. Expression data were normalized by quantile normalization and the robust multichip average algorithm, as previously described (20). Probe-level files were generated following normalization. According to the fold change (FC) analysis (FC >2.0) and false discovery rate (FDR) analysis (FDR <0.05), differentially expressed genes were identified through FC filtering according to the predetermined P-value threshold for significant differences (set at P<0.05).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was reversely transcribed into cDNA using a PrimeScript RT Reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) following the manufacturer's instructions, as previously described (21). Next, qPCR was performed using a SYBR Green PCR kit (Takara Biotechnology Co., Ltd.) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR conditions included an initial step at 95°C for 5 min, followed by 40 cycles of annealing and extension, prior to quantification at 95°C for 15 sec and 60°C for 30 sec. Each cDNA sample was analyzed in triplicate, in a final volume of 25 µl, containing 1 µl cDNA, 400 nM of the forward and reverse gene-specific primers (1 µl), 12.5 µl 2x SYBR Green master mix (catalogue no. 639676; Takara Biotechnology Co., Ltd.) and 10.5 µl distilled water. The relative gene expression level was quantified based on the cycle threshold values (22) and normalized to the reference gene, which was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences used were listed as follows: KIF2A: F 5′-ATTTTCTCTCATTGACCTGGCTG-3′, R 5′-ACTCCTTGAGTGCTAAAAGGC-3′; RYBP: F 5′-CGACCAGGCCAAAAAGACAAG-3′, R 5′-CACATCGCAGATGCTGCAT-3′; DOCK7: F 5′-CCATCTGGAAGCGCCTTTG-3′, R 5′-ACGATGATCTCTAGCGTGTCT-3′; CDC40: F 5′-CTCTAGCTGCTTCGTATGGCT-3′, R 5′-CAAGTGCATGAGAGAGTCCGC-3′; PTBP3: F 5′-CCAGCCATTGGATTTCCTCAA-3′, R 5′-AAAAAGCCCATGTGGTGTGATA-3′; NRTN: F 5′-GGGCTACACGTCGGATGAG-3′, R 5′-CCAGGTCGTAGATGCGGATG-3′; TMEM205: F 5′-CACTTGCTGGTCTTGTCTGGT-3′, R 5′-GGAGACGTGAAAATAGACTGGG-3′; SLC39A3: F 5′-GGTGGCGTATTCCTGGCTAC-3′, R 5′-CTGCTCCACGAACACAGTGA-3′; CDCA3: F 5′-GAGTAGCAGACCCTCGTTCAC-3′, R 5′-TCTCTACCTGAATAGGAGTGCG-3′; KIF1C: F 5′-AGTGTGGGTTTGTGTGTATGAG-3′, R 5′-CCAGCATCGCACCATGTAGA-3′; CAS P3: F 5′-ATGGAGAACAACAAAACCTCAGT-3′, R 5′-TTGCTCCCATGTATGGTCTTTAC-3′; GAP DH: F 5′-AGGTCGGTGTGAACGGATTTG3′, R 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
Immunohistochemical assay Following sacrifice, the entire rat brain was rapidly removed, washed with phosphate-buffered saline, incubated for at least 48 h in 4% paraformaldehyde (Sigma-Aldrich; Merck, Darmstadt, Germany) and embedded in paraffin. Next, the paraffin-embedded tissues were sectioned into 4-µm slices, and sections with the hippocampus structure were used for immunohistochemical analyses. Slices were incubated with rabbit anti-caspase-3 primary antibody (Catalogue no. AC030; Beyotime Institute of Biotechnology; 1:200) at 4°C overnight, then incubated with biotinylated anti-rabbit secondary antibody (catalogue no. A0277; Beyotime Institute of Biotechnology; 1:1,000) for 30 min at 37°C, and immunoreactivity was then visualized by addition of a streptavidin-peroxidase complex and 3,3′-diaminobenzidine (both from Beyotime Institute of Biotechnology). Counterstaining was performed with hematoxylin (Zhongshan Golden Bridge, Beijing, China). Subsequent to each incubation step, slices were washed with Tris-buffered saline/Tween 20 three times for 5 min each. All images were captured using an Axioskop fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).
Western blotting The preparation of hippocampal tissues for protein extraction was performed as described in previous studies (23,24). Briefly, total proteins were extracted using the radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). The hippocampus tissue proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to a nitrocellulose membrane. After blocking with 5% non-fat milk for 1 h at room temperature and being washed three times (10 min each) using 1x TBST, the membrane was incubated with primary antibodies at 4°C overnight and then with horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology) at room temperature for 2 h. The primary antibodies used were as follows: Rabbit anti-cleaved-poly (ADP-ribose) polymerase (PARP) antibody (catalogue no. AP102; 1:200) and rat anti-GAPDH antibody (catalogue no. AG019; 1:5,000; both from Beyotime Institute of Biotechnology). Subsequently, the target proteins were visualized by an enhanced chemiluminescence method (catalogue no. W1001, Promega Corporation, Madison, WI, USA) and analyzed with the Gel Image Documentation System (Wealtec Corp., Sparks, NV, USA). The relative level of PARP was normalized to that of GAPDH, as presented by band intensity.
Statistical analysis All data are presented as the mean ± standard error of the mean, and all statistical analyses were conducted using GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) and SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA) software. For behavioral tests and RT-qPCR results, one-way analysis of variance (ANOVA) was applied to compare intergroup differences with Bonferroni post hoc tests. The correlation analysis was performed using Pearson's correlation coefficients. For western blotting and immunohistochemistry results, two-tailed Student's t-test was applied for comparison. A P-value of <0.05 was considered to indicate differences that were statistically significant. Any additional experimental data not provided in the current study are indicated by ‘data not shown’ and can be obtained upon request.