PMID: 31257533 PMCID: PMC6625379 DOI: 10.3892/mmr.2019.10397
Materials and methods
Rat HPC model All animal procedures were conducted with the approval of the Animal Care and Use Committee of Guangxi Medical University (Nanning, China). Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses. All pups were housed in a temperature-controlled room (22±1°C) with a 12-h light/dark schedule. H89 (Selleck Chemicals) and Sp-cAMP (Sigma-Aldrich; Merck KGaA) were prepared in 5 µl double-distilled water. The experimental set-up is illustrated in Fig. 2 (n=10) and the following experimental groupings were used: i) Normal saline group (NS group) received intraperitoneal injections of an equal volume of normal saline; ii) propofol group (P group) received intraperitoneal injections of 100 mg/kg propofol; iii) following the propofol treatment as in the P group, the propofol + Sp-cAMP group (P+Sp-cAMP group) received intracerebroventricular injections of 20 nmol/5 µl Sp-cAMP (a cAMP-dependent protein kinase agonist); iv) HPC+P group rats were placed in a chamber containing 8% oxygen and 92% nitrogen for 10 min, and the pups were subsequently exposed to room air for a further 10 min, and following five HPC cycles, the rats received an intraperitoneal injection of 100 mg/kg propofol; v) HPC+P +H89 group was exposed to 5 µmol/5 µl H89 [a protein kinase A (PKA) inhibitor] by intracerebroventricular injections, followed by the same protocol as in the HPC+P group; vi) the remaining pups in the two blank test groups received intracerebroventricular injections of dimethyl sulfoxide (D-ICV group) or normal saline (NS-ICV group). All pups were sacrificed according to standard protocols (100 mg/kg intraperitoneal sodium pentobarbital). Brain tissue slices were prepared for immunohistochemistry and the levels of PKA, CREB, phosopho (p)-CREB, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were evaluated by western blotting. Morphological and structural changes were evaluated by haematoxylin and eosin (H&E) staining and transmission electron microscopy.
Intraventricular injections As aforementioned, rats were anesthetized with sodium pentobarbital and centralized coordinates of anterior fontanel (x=0, y=0, z=0) using stereotaxic apparatus (Ryward Life Technology Co., Ltd.), the sterile cannula was implanted at AP-2 mm (front and posterior), MLR-1.5 mm (left and right of the midline), and H-2 mm (depth from the left ventricle, x=−1.0 mm, y=2 mm, z=0). After positioning, the skull was drilled, and then Sp-cAMP (5 µl) or H89 (5 µl) was slowly injected at rate of 0.1 µl/min. The blank groups following the same protocol with an equal volume of DMSO or normal saline.
ELISA The intracellular concentrations of adenylyl cyclase in the pups was determined by ELISA according to the instructions of the assay manufacturer (cat. no. S0026; Beyotime Institute of Biotechnology).
Western blot analysis All pups were sacrificed to harvest the brain tissue. The protein was extracted by RIPA Lysis Buffer (Beijing Solarbio Science & Technology Co., Ltd.) and protein concentration measured using a bicinchoninic acid protein assay (Biotype Biotech Co.). The mass of protein loaded per lane was 20 µl. Equal amounts of proteins were loaded onto 12% SDS-polyacrylamide gels. Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (0.22-µm pore size; EMD Millipore). The membranes were blocked using 5% bovine serum albumin (blocking buffer) for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: β-tubulin (1:2,000; cat. no. 48885), caspase-3 (1:1,000; cat. no. 48658) and cleaved caspase-3 (1:1,000; cat. no. 29034; all from Signalway Antibody) Bcl-2 (1:1,000; cat no. ab196495; Abcam), Bax (cat. no. 27727) PKA (cat. no. 5842S), CREB (cat. no. 9197S) and p-CREB (cat. no. 9198S) (all 1:1,000; from Cell Signaling Technology, Inc.), and GAPDH (1:10,000; cat. no. 10494-1-AP; Proteintech, Inc.). The membranes were washed three times with Tris-buffered saline 1% Tween-20 (TBST; pH 7.4) and then incubated in horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no. 134658; LI-COR Biosciences) for 2 h at room temperature (23–25°C) and washed three times with TBST. The bands were developed using an Odyssey infrared imaging system (LI-COR Biosciences) and evaluated using densitometric analysis (ImageJ 1.52 h, National Institutes of Health).
H&E and immunohistochemical staining Morphological and structural changes were observed by H&E staining. Tissues were fixed in 4% ice-cold paraformaldehyde at 4°C for 2 h and paraffin-embedded sections were obtained. The paraffin sections were dewaxed in xylene for 15 min and rehydrated using graded ethanol. The sections were immersed in haematoxylin for 30 sec and then subjected to antigen retrieval using 0.01 mol/l sodium citrate and incubated with 10% normal goat serum at room temperature for 30 min to block nonspecific binding, followed by incubation with the primary antibodies against PKA C and p-CREB (cat. nos. 5842S and 9198S, 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. The sections were incubated with streptavidin-horseradish peroxidase at room temperature for 30 min and then stained with 0.05% 3,3-diaminobenzidine substrate, followed by counterstaining with 1% haematoxylin at 37°C for 30 sec. The sections were observed using a microscope (Olympus BX53; Olympus Corporation) and four fields of the hippocampus were randomly selected in every section which represented the areas of interest and the positive cells were counted using Image-Pro Plus version 6.0 software (Media Cybernetics Inc.).
Electron microscopy The ultrastructures of neurocytes were observed by transmission electron microscopy (HITACHI H-7650; Hitachi, Ltd.). Briefly, 2.5% glutaraldehyde solution was perfused into the rats, and the tissues were fixed in 1% OsO4 at 4°C for 1 h, dehydrated in increasing concentrations of ethanol and embedded in Epon. Then, the samples were sectioned into semi-thin slices (1 µm) and stained with 1% uranyl acetate and 5% uranyl acetate at 37°C for 20 min. The ultrastructures of the entire mitochondria were measured by manually measuring length using Image Pro Plus (version 6.0.0.260, Media Cybernetics, Inc.).
Statistical analysis Data are presented as the mean ± standard error, and were analysed using SPSS version 17.0 (SPSS, Inc.) and GraphPad Prism 5 software (GraphPad Software Inc.). Multiple comparisons were performed using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test, as appropriate. P<0.05 was considered to indicate a statistically significant difference.