20230808-AI coding-1st round/1256 – Lin 2018.txt

PMID: 29461008 PMCID: PMC5908131 DOI: 10.1111/jcmm.13524
2. MATERIALS AND METHODS
2.1. Drugs
All drugs were prepared just before use: propofol (Diprivan; AstraZeneca UK limited, Italy: jc393, 20 mL: 200 mg); 20% intralipid (2B6061; Baxter, Deerfield, IL, USA); SAHA (Selleck Chemicals LLC, Houston, TX, USA). HGN antisense was synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Senegenin (purity ≥ 98%) was purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Jiangsu province, China).

Anti‐β‐actin and anti‐rabbit IgG secondary antibody were obtained from Cell Signaling Technology (Cell Signaling Tech, MA, USA). Anti‐CREB (Phospho S133), anti‐NMDAR2B, anti‐HDAC2, antisynaptophysin, anti‐Ac‐H4K12 and anti‐Ac‐H3K14 antibodies were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti‐HGN antibody was synthesized by Kitgen Bio‐tech Co., Ltd.(Zhejiang province, China).

2.2. Animals
The protocol in this study was approved by the institutional review board of the First Affiliated Hospital of Nanchang University on the Use of Animals in Research and Teaching. All the methods in this study were performed in co‐ordination with relevant guidelines and regulations. Sprague Dawley (SD) rats were purchased from the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011‐0030; Nanchang, Jiangxi,China). The learning and memory functions of the parental rats were assessed using the Morris water maze (MWM) system before mating, so that to minimize the hereditary difference. Animals were housed separately under standard laboratory conditions with 12:12 light/dark cycle, 24 ± 1°C and had free access to tap water. Two female rats in cages with one male rat per cage for mating. Pregnancy was diagnosed by the sign of vaginal plug.

2.3. Drug treatment
On E7, pregnant rats received intravenous infusion of propofol (n = 10 dams) with the rate of 20 mg kg−1 h−1 for 4 hours, equal volume of saline (n = 10 dams) or intralipid (n = 5 dams), respectively.

Electrocardiograms, saturation of pulse oximetry (SpO2) and tail non‐invasive blood pressure were continuously monitored during maternal propofol exposure. Using heating lamp and temperature controller to monitor the rectal temperature and keep it at 37 ± 0.5°C. Arterial blood sampling from lateral caudal artery for blood gas analysis at the end of propofol anaesthesia. If the total time of SpO2 <95% and/or the systolic blood pressure <80% of the baseline in excess of 5 minutes, the pregnant rat was got rid of the study, and other pregnant rats were chosen to supply the sample size, so as to exclude the interfering effect of maternal hypotension or hypoxia on cognitive function in the pup rats.

After delivery, the offspring rats born to the same pregnant rat were randomly subdivided into the SAHA, SEN, HGNA group and their relative control groups (DMSO, NS(1) and NS(2) group, respectively; Figure ​Figure1).1). It has been proved that the acetylation level of histone in hippocampus obviously increased 2 hour after the administration of HDAC inhibitor.27 Therefore, 90 mg kg−1 SAHA (HDAC inhibitor), at a concentration of 0.6 μmol L−1 dissolved into dimethyl sulphoxide (DMSO) was injected to the offspring in SAHA group by the intraperitoneal route at 2 hours before each MWM trial. The same volume of DMSO was given to the DMSO group. Senegenin, a kind of Chinese medicine, was proved to up‐regulate the expression of NR2B mRNA and protein, thus to mitigate cognitive dysfunction.28 So, 15 mg kg−1 Senegenin and equal volume of saline were given intraperitoneally at 2 hours before each MWM trial to SEN or NS(1) groups, respectively. HGN antisense oligonucleotide (0.25 nmol μL−1, 1.5 μL) or normal saline (1.5 μL) was injected to offspring's hippocampus in HGNA or NS(2) group as previously described,18, 29 once daily for seven consecutive days before MWM trial.
2.4. Morris water maze test
Spatial learning and memory were assessed by the MWM test from post‐natal day 30 (P30) to P36 according to previously described5, 30 with SLY‐WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China). Briefly, the trials start at 9 o'clock in the morning in the MWM system with the pool was filled with water to a height of 1.0 cm above the top of a 15‐cm‐diameter platform, in the second quadrant (target quadrant), and the water maintained at 24 ± 1°C. The training trial was performed once a day for six consecutive days. In each training trial, offspring rats were placed in the water facing the wall of the pool in the third quadrant, the farthest one from the target quadrant. The animals were allowed to search for the hidden platform or for 120 seconds. They were allowed to remain on the platform for 30 seconds when they found the platform and the time for the animal to find the platform was recorded as escape latency (indicating learning ability). For those who did not find the platform within 120 seconds, the animals were gently guided to the platform and allowed to stay there for 30 seconds, and their escape latency was recorded as 120 seconds. At the end of the reference training (P37), the platform was removed. The offspring rats were allowed to perform spatial probe test (memory function test) for 120 seconds. Times across the platform (platform crossing times, indicate memory function), the swimming trail and speed were automatically recorded by the system. The mean value of the platform crossing times, escape latency and speed of the offspring born to the same pregnant rats was taken as the final results.

2.5. Brain hippocampus harvest
The day after the MWM test, rats were anaesthetized with isoflurane and killed by cervical dislocation. Hippocampus tissues were harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at −80°C (for Western blot analyses) or immersed in 4% paraformaldehyde (for immunofluorescence assay).

2.6. Western blot analysis
The hippocampus (n = 6, with three male and three female offspring rats from each group) were homogenized on ice in lysis buffer containing a protease inhibitors cocktail. Protein concentration was determined by the bicinchoninic acid protein assay kit. Protein samples (20 μg) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a PVDF membrane. The membranes were blocked by non‐fat dry milk buffer for 1.5 h and then incubated overnight at 4°C with antihistone H3 (acetyl K14) (1:2000), antihistone H4 (acetyl K12) (1:10000), anti‐NMDAR2B (1:1000), anti‐HGN (1:1000), antisynaptophysin (1:10000) and anti‐β‐actin (1:2000), respectively. Thereafter, the membranes were washed three times with TBS‐T buffer for 15 minutes and incubated with the horseradish peroxidase (HRP)‐conjugated secondary antibody for 2 hours at room temperature. The immune complexes were washed three times with TBS‐T buffer and detected using the ECL system (Millipore Corporation, MA, USA). The images of Western blot products were collected and analysed by ImageJ 1.50i (Wayne Rasband, National Institutes of Health, USA). The density of observed protein band was normalized to that of β‐actin in the same sample. The results of offspring from all the other group were then normalized to the average values of normal saline control offspring (control+NS group) in the same Western blot. The mean expression level of all of the offspring born to the same mother rat in the same group was calculated as the final expression level of the observed proteins.

2.7. Immunofluorescence staining
Immunofluorescence staining was used to assess HDAC2 and phospho‐CREB in the hippocampus of offspring rats after the MWM test. Hippocampus from offspring rats (n = 6, with three male and three female offspring rats from each group) were fixated in paraformaldehyde. Five‐μm frozen sections of the hippocampus were used for the immunofluorescence staining. The sections were incubated with anti‐HDAC2 (1:300) and anti‐CREB (1:100) dissolved in 1% bovine serum albumin in phosphate‐buffered saline at 4°C overnight. Then, the sections were incubated with fluorescent‐conjugated anti‐rabbit secondary antibody (1:300) for 1 hour in the dark at room temperature. Negative control sections were incubated with PBS as a substitute for primary antibody. Finally, the sections were wet mounted and viewed immediately using a inverted fluorescence microscope (200×) (Olympus, Japan). The target protein was red, and nuclei were blue. The proteins of HDAC2 and p‐CREB were excited by the green light, while the DAPI was performed by UV blue light. All images were recorded at 10 × 20× (Exp Acq‐700mmm, Offset Acq‐1, Gain Acq‐1, Gamma Acq‐300). The density of HDCA2 and p‐CREB staining was conducted on the images using Image‐Pro Plus 6.0 (Media Cybernetics Inc., USA). The images were converted it into black and white pictures. After intensity calibration, hippocampal CA1 area was chosen to analyse and the integrated optical density (IOD) was measured. IOD/Area was calculated as the protein expression level.

2.8. Statistical analysis
All analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data from escape latency in the MWM test were subjected to a repeated measures two‐way analysis of variance (RM two‐way ANOVA) and were followed by least significant difference t (LSD‐t) analysis when a significant overall between‐subject factor was found (P < 0.05). Data from Western blot and immunofluorescence staining results were subjected to one‐way ANOVA analysis. All data well provided for any of the variables. The LSD t test was used to determine the difference between groups. Statistical significance was declared at P < .05.