20230808-AI coding-1st round/341 – Huang 2016.txt

PMID: 26966008 DOI: 10.1007/s12640-016-9615-7
Materials and Methods
Animal Treatment
All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 °C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water. The PND-7 male rat pups (11–14 g) were randomly assigned to ketamine-treated and control groups. In the treated group, ketamine was diluted in 0.9 % normal saline, and PND-7 rats were intraperitoneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg × 4 injections). Control rats received an equal volume of normal saline. Temperature probes were used to facilitate control of temperature at 36.5 ± 1 °C using computer-controlled heater/cooler plates integrated into the floor of the chamber. Between each injection, animals were returned to their chamber to help maintain body temperature and reduce stress.

BrdU Injections
All animals received an intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma) at a dosage of 100 mg/kg after ketamine anesthesia according to the following experimental schedule.

Experiment 1: To evaluate the effect of ketamine on the proliferation and differentiation of NSCs in the DG during the BGS, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after ketamine treatment. The animals were then anesthetized and fixed by perfusion at 24 h after each BrdU injection. The experimental protocol is described in Tables 1a and 2.
Experiment 2: To exclude the GFAP/BrdU double-positive cells that were proliferative astrocytes, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after exposure to treatment. The animals were then perfused at 3 h after each BrdU injection. The experimental protocol is detailed in Tables 1b and 2.

Experiment 3: To determine the effect of ketamine on the migration of newborn granule neurons in the DG, the PND-7 rats received three consecutive BrdU injections on PND-7, 8, and 9 after exposure to treatment. At 28 and 35 days after the last BrdU injection, the animals were anesthetized and fixed by perfusion. The experimental protocol is described in Table 1c and 2.

Cell Apoptotic Assays
Nestin/caspase-3 and GFAP/caspase-3 double-immunofluorescence staining was utilized to detect whether ketamine could induce the apoptosis of NSCs or astrocytes. At 12 h after the end of control and ketamine-anesthesia treatment, the neonatal rats were anesthetized and fixed by perfusion (n = 5 per group).

Tissue Preparation and Immunofluorescence
At the indicated time point, animals were deeply anesthetized and then transcardially perfused with 0.9 % normal saline followed by 4 % paraformaldehyde. The brains were removed, postfixed overnight in 4 % paraformaldehyde, and placed in 30 % sucrose until sunk. The coronal sections of brain were cut consecutively at a thickness of 30 μm when the hippocampus was initially exposed. The fifteenth section was taken and stored in PBS. According to the Atlas of the Developing Rat Brain and previous reports (Ashwell and Paxinos 2008; Paxinos and Watson 1986), the positions of hippocampus coronal sections selected in our study were about 2.20–2.25 mm posterior to the bregma at PND-8 rats, about 2.35–2.40 mm posterior to the bregma at PND-14 rats, about 2.50–2.55 mm posterior to the bregma at PND-21 rats, and about 2.75–2.85 mm posterior to the bregma at PND-37 and PND-44 rats, respectively.

For Nestin/BrdU, β-tubulin III/BrdU, GFAP/BrdU, and NeuN/BrdU double-immunofluorescence staining, the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37 °C and then washed three times with PBS for 5 min between each of these steps. Blocking of nonspecific epitopes with 10 % donkey serum in PBS with 0.3 % Triton-X for 2 h at room temperature preceded incubation overnight at 4 °C with the primary antibodies listed in Table 3 in PBS with 0.3 % Triton-X. On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen Carlsbad, CA) for 2 h at room temperature.
Astrocytic development was detected by using GFAP single-labeled staining. The sections were incubated overnight at 4 °C with a fluorescent antibody for the GFAP (Table 3). After three washes in PBS, sections were incubated with secondary fluorescent antibody (Invitrogen) for 2 h at room temperature.

To characterize the phenotype of cell apoptosis, brain sections were analyzed by double-labeled staining. The sections were incubated overnight at 4 °C with the appropriate primary antibodies listed in Table 3. After three washes with PBS, the sections were incubated with the suitable secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.

A skilled pathologist blinded to the study conditions examined the labeled sections using a laser scanning confocal microscope (Fluoview 1000, Olympus). The number of single- or double-positive cells in the hippocampal DG was quantified using Image-Pro Plus software.

Western Blot Analysis
Thirty and thirty-seven days after the control or ketamine-anesthesia treatment, the animals were decapitated, and the hippocampal DG tissue was dissected carefully with anatomic microscope (leica EZ4HD). The harvested hippocampal tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were centrifuged at 14,000 rpm for 15 min at 4 °C and were resolved by 12 % polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4 °C with the primary antibodies: rabbit anti-GFAP antibody (1:1000, Millipore) and GAPDH. The membranes were then incubated with appropriate secondary alkaline phosphatase-conjugated donkey anti-rabbit antibody (1:10,000, Abcam) for 1 h. The band intensity was quantified using Image J software (n = 5 per group).

Morris Water Maze Test
The hippocampal-dependent spatial memory abilities were tested by using the Morris water maze (MWM). Different set of rats were tested 2 months after administration of ketamine on PND-7. A circular, black painted pool (180 cm diameter, 50 cm deep) was filled with water to a depth of 30 cm. The water temperature was maintained at 25 ± 1 °C. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and placed in the center of the III quadrant which was determined with four starting locations called I, II, III, and IV at equal distance on the edge of the pool. During five consecutive days, the experiments were conducted in a dark and quiet laboratory, all the rats were trained four times per day, the starting positions were random for each rat. When the rat found the platform, the rat was allowed to stay on it for 30 s. If a rat did not find the platform within 120 s, the rat would be guided gently to the place and allowed to stay on it for 30 s, and the latency time to find the hidden platform was recorded as 120 s. The average time from four trials represented as the daily result for the rat. On the sixth day, the hidden platform was removed, and the rat was placed in the opposite quadrant. Rats were allowed to swim freely for 120 s. The numbers the rat swam to cross the previous platform area, and the times the rat stayed in the target quadrant within 120 s were recorded. Each animal’s path was tracked by a computerizing video system. After every trial, each rat was placed in a heater plates for 1 to 2 min until dry before being returned to its chamber. The data were analyzed using software for the MWM (Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China).

Statistical Analysis
The statistical analysis was conducted using SPSS 13.0, and the graphs were created using GraphPad Prism 5. The data were analyzed using Mann-Whitney U test. The interaction between time and group factors in a two-way ANOVA was used to analyze the difference of escape latency between rats in the control group and rats treated with ketamine in the MWM. The data are presented as the mean ± SD, and p < 0.05 was considered statistically significant.