20230808-AI coding-1st round/435 – Li 2019.txt

PMID: 31263401 PMCID: PMC6585163 DOI: 10.3389/fncel.2019.00251
Materials and Methods
Animal Protocols
We performed all the experimental protocols according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23). The animal procedures were approved by the Animal Care and Use Committee of Xi’an Jiaotong University and designed to minimize the number and suffering of rats used. PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.

Morris Water Maze
The spatial learning and memory function of rats after ketamine exposure were tested by MWM experiments as described in a previous study (Shen et al., 2013). Specifically, PND 42–47 rats (n = 10 per group) were trained for place trials and spatial probe tests in a large tank (diameter: 150 cm, depth: 60 cm), which was filled to a depth of 32 cm of warm water (maintained around 25 ± 1°C) and divided into four quadrants. A platform (diameter: 12 cm, height: 30 cm) was placed in the center of the third quadrant (the target) and submerged approximately 2 cm beneath the water surface. We poured milk powder into the water to make the water opaque. We conducted the place trials at PND 42–46 with 4 trials daily at the same time point and performed the probe trials on PND 47 after 5 days’ training. The swimming of rats during the tests was recorded by a video tracking system installed above the tank. In place trials, rats were placed into four quadrants (spaced 20 min apart) to swim freely for a maximum of 120 s. If the rats could not find the platform within 120 s, they were allowed to stay on the platform for 20 s to observe the environment by guiding. The time for rats to reach the platform and swimming speed were recorded. In probes trials, the platform was removed and the rats were put into the first quadrant and allowed to swim for 120 s. The times of rats crossing the original platform were recorded.

Anesthetic Exposure in vivo and Tissue Preparation
The PND 7 rats, weighing 13–18 g, were housed with their mother and maintained at a temperature of 24°C in a 12 h/12 h light/dark cycle with free access to food and water. We assigned the rats randomly into three groups (28 rats from 7 nests in each group, 4 pups per nest): (i) the rats in control group received equal volume of normal saline by intraperitoneal injection as ketamine solution at corresponding time points; (ii) the rats in ketamine group received 40 mg/kg ketamine, diluted in normal saline and administrated by intraperitoneal injection (ketamine, Sigma–Aldrich Inc. St. Louis, MO, United States), the initial injection was considered to be the loading dose, 30% of it was injected at approximately 40 min intervals to maintain the anesthesia for 4 h (Lu et al., 2017); (iii) the rats in the 17β-estradiol group received 17β-estradiol (17β-estradiol, Tocris, Minneapolis, MN, United States; DMSO, Sigma–Aldrich, St Louis, MO, United States) dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 ug/ml, 100 ug/kg 17β-estradiol administered intraperitoneally 8 h, 1 h prior to and 3 h after ketamine’s initial injection (Liu et al., 2007). During anesthesia, all pups were kept on an electric blanket with the temperature set at 36.5 ± 1°C to maintain body temperature and reduce stress. We observed the respiratory rate, skin color, and body movement of rats carefully and tested the voluntary movement by clamping the pup tails. Pulse oxygen saturation (SpO2) was detected by attaching the infant pulse oximetry probes to the rat abdomen. After the anesthesia, the pups received BrdU (50 mg/kg, intraperitoneal injection) every 24 h for 7 consecutive days. On PND 14, the rats were decapitated and the brain tissues of SVZ and SGZ were harvested to detect neurogenesis.

At 12 h after anesthesia, rat pups (n = 6 per group, captured randomly) were sacrificed by decapitation. Both the brain tissue from SVZ and SGZ were isolated immediately on ice and the stored at −80°C until use for western blotting. The rats (n = 6 per group, captured randomly) were sacrificed and perfused transcardially with 0.9% saline 7 days after anesthesia, followed by cold 4% paraformaldehyde in PBS. Then the harvested brains were postfixed in 4% paraformaldehyde overnight at 4°C and dehydrated in 30% sucrose solution for 3–4 days, as we described previously (Lu et al., 2017). The brain tissue from bregma +0.2 mm to bregma −6.0 mm was the region of interest, which were cut into 16 μm coronary tissue slices by freezing microtome (SLEE, Germany). These brain slices were collected and used for future immunohistochemistry staining. The rest of the rat pups (n = 10 per group) were bred for behavior study at adulthood.

Immunohistochemistry
Immunohistochemistry was used to evaluate NSC proliferation in SVZ and SGZ by BrdU staining. Firstly, the brain slices were incubated with 2 N HCl for 30 min to denaturate the DNA at 37°C. After being incubated with 0.1 mol L−1 boric acid (pH 8.5) for 10 min at room temperature followed by three times washing with 0.1 M PBS, the slices were blocked by 2% goat serum and 0.3% Triton X-100 for 2 h at room temperature, then incubated with the mouse monoclonal anti-BrdU antibody (1:200, Abcam, United Kingdom) at 4°C overnight. The next day, after three washings with 0.1 M PBS, the slices were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies for 2 h at room temperature. BrdU-positive cells were counted within defined regions of interest in the SVZ and SGZ. In total, the mean numbers of BrdU-positive cells of six brain slices for each rat, spaced approximately 200 μm apart, were examined by the observer blindly. For each slice, five regions were captured by fluorescence microscopy (BX51, Olympus, Tokyo, Japan), and the planar area enclosed by each region was 50 × 50 μm. The edges of the captured regions were defined according to structural details to ensure the fields did not overlap (Zhang et al., 2009). The density of positive cells was presented as the total number of BrdU-positive cells in the SVZ and SGZ.

NSC Culture
Primary cultured NSCs were obtained from the cortex of rat at embryonic day 18–19 under sterile conditions. Briefly, the forebrain portion was isolated and placed in ice-cold Hank’s solution (without Mg2+ and Ca2+, Gibco, Carlsbad, CA, United States). The tissues were then dissociated and triturated mechanically by a fire-polished Pasteur pipette softly. After centrifugation, the isolated cells were collected and re-suspended in free-serum DMEM/F12 medium (Gibco, Carlsbad, CA, United States) which was supplemented with 2% B27 (Gibco, Carlsbad, CA, United States), 20 ng/ml EGF (Gibco, Carlsbad, CA, United States), 20 ng/ml bFGF (Gibco, Carlsbad, CA, United States), and 100 U/ml penicillin and phytomycin. Cells were cultured for 7 days to form enough neurospheres and then passaged at a density of 2 × 105 cells/ml followed by collection and dissociation as previously described by Reynolds and Weiss (Reynolds et al., 1992). Half of the medium was changed every 3 days. After the second passage, the cells were ready for future experiments. For identification assessment, the cells after passage were seeded onto 100 μg/mL poly-L-lysine-coated coverslips and cultured in differentiating medium that contained 100× N2 supplement, 100× B27 supplement, and 1% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States) in DMEM/F12 (without b-FGF) for 7 days.

Drug Exposure and Neurogenesis Analysis in vitro
The cells were assigned to the following groups: control group, ketamine group, and 17β-estradiol group. No drug treatment was added to the control group. NSCs in the ketamine group were exposed to 100 μM ketamine for 24 h. NSCs in the 17β-estradiol group were pretreated with 17β-estradiol (100 nM) for 30 min and then 100 μM of ketamine was added to the culture medium for 24 h. For proliferative analysis, NSCs were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with BrdU for the last 4 h. Following being fixed with 4% paraformaldehyde, the cells were stained with BrdU antibody (1:200, Abcam, United Kingdom) and DAPI. As for neuronal differentiation analysis, after being exposed to ketamine with or without 17β-estradiol for 24 h, the cells were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with differentiating medium for 7 days, then the cells were harvested for immunohistochemical staining. The cells were labeled with β-tubulin III antibody (1:500; Sigma-Aldrich Inc. St. Louis, MO, United States). Briefly, 5–7 randomly selected fields were captured in each coverslip, and the numbers of β-tubulin III-positive cells were counted (at least 200 cells per test case). Data were collected from three independent experiments.

Cell Apoptosis Test
We used terminal dUTP nick-end Labeling (TUNEL) assay to detect cell apoptosis. Briefly, after passage, the dissociated cells were exposed to ketamine with or without 17β-estradiol for 24 h. After the treatments, cells were fixed with 4% paraformaldehyde for 15 min. The TUNEL assay was performed according to the instruction of in situ Cell Death Detection Kit (Roche Inc. Roche, Mannheim, Germany). Data were collected from three independent experiments.

Western Blot Analysis
Brain tissues from the SVZ and SGZ of rats (n = 6 per group) at 12 h after anesthesia and cell cultures at 24 h following drug exposure were subjected to Western blot analyses as described in our previous studies (Lu et al., 2017). Briefly, the tissues were lysed by RIPA lysis buffer with protease and phosphatase inhibitors. The lysates were homogenized with an electric homogenizer and maintained on ice for 15 min. After being centrifuged for 15 min at 14000 rpm at 4°C, the supernatant was aspirated and the resulting lysates were placed in a new tube. We used the BCA protein assay kit to examine the protein concentrations. Bovine serum albumin (BSA) was used as a standard. An equal amount of the resulting lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel and the separated proteins were transferred to polyvinylidene fluoride membranes. After being blocked for 1 h at room temperature, the membranes were then incubated with appropriate dilutions of primary antibodies at 4°C overnight. The used antibodies included anti-caspase-3 (cleaved, 17 KDa, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-phosophorylated GSK-3β (p-GSK-3β, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-GSK-3β (1:1000, Cell Signal Technology Inc. Beverly, MA, United States), and anti-β-actin (1:1000, Cell Signal Technology Inc., Beverly, MA, United States). The following day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or anti-mouse) for 2 h at room temperature. After being enhanced by chemiluminescence (ECL), the signals were then exposed to X-ray films. Each band in the Western blot represented an independent experiment and at least three independent experiments were conducted. Data were expressed as the ratio to optical density (OD) values of the corresponding controls. The Western blots were quantified as described in our previous study.

Statistical Analysis
Data obtained from the study were presented as mean ± SEM. Every data point represented a mean for each animal in a single case. SigmaPlot 12.0 was used for all statistical analysis. Data were tested and then confirmed with normality and equal variance criteria. A one-way analysis of variance (ANOVA) following the post hoc Holm-Sidak method was used to analyze the differences among different groups. A two-tailed probability value P < 0.05 was considered statistically significant.