20230808-AI coding-1st round/437 – Chen 2018.txt

PMID: 29427282 DOI: 10.1007/s12640-018-9877-3
Methods
Ethical Approval
The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China). All experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Sprague-Dawley multiparous dams (n = 31) with litters containing male pups (n = 135) were purchased from Experimental Animal Center of Sun Yat-sen University, China. We only used male offspring to exclude the influence of estrogen on the biochemical data and neurocognitive functions. The pups from postnatal day 0 (P0) to P20 were housed with the dams in a 12-h:12-h light:dark cycle (light from 07:00 to 19:00), and room temperature (RT) was maintained at 21 ± 1 °C. On P21, the pups were weaned and housed 4–6 per cage in a standard environment.

Anesthesia
SD rats at P7 (weight 14–16 g) were randomly divided into the air-treated control (C group), the 1.2% sevoflurane-exposed (1.2% sevo group), and the 2.4% sevoflurane-exposed (2.4% sevo group). Rats in the 1.2% sevo group and the 2.4% sevo group were placed in a plastic container and exposed to 1.2 or 2.4% sevoflurane continuously for 6 h, using air as a carrier, with a total gas flow of 2 L min−1. A nasopharyngeal airway tube was put in their mouth to prevent apnea and hypoxia when the rats stopped moving in the container. During exposure, the temperature inside the container was maintained at 30 °C using an external heating device (NPS-A3 heating device, Midea Co., Guangdong, China) and a hot water bag on the bottom of the container with a constant temperature maintained between 30 and 35 °C. The concentrations of sevoflurane, oxygen, and carbon dioxide in the chamber were monitored by a gas monitor (Detex-Ohmeda, Louisville, CO, USA). During exposure, an investigator monitored the rats’ spontaneous respiratory frequency and skin color every 5 min to detect any apnea or hypoxia. The rats were immediately exposed to air and excluded from the experiment if these symptoms were detected. Sevoflurane administration was terminated 6 h later, and the rats were exposed only to air. When the rats were moving freely again, they were placed back into their maternal cages. Rats in the C group were exposed to the same container as the rats in the 1.2% sevo and 2.4% sevo group but were exposed to air alone for 6 h.

Arterial Blood Gas Analysis
We performed arterial blood analysis in order to exclude the influence of respiratory or metabolic disorder. The arterial blood samples from the C, 1.2% sevo, and 2.4% sevo groups were obtained from the left cardiac ventricle immediately after removal from the maternal cage (n = 5 in each group) at the end of anesthesia. They were analyzed immediately after collection using a blood gas analyzer (Gem Premier 3000, US). We analyzed the pH, arterial carbon dioxide tension (PaCO2), arterial oxygen tension (PaO2), and blood glucose levels of the arterial blood samples.

Morris Water Maze Test
On P35, the rats were tested for spatial learning and memory ability using the Morris water maze (MWM). Three groups of rats (n = 10 in each group, weight 90–100 g) were tested on the MWM, which consists of two different tests including hidden platform acquisition and a probe trial test, at P35–P40 using the Water Maze Tracking System (MT-200; Chengdu, China). A white platform (12 cm diameter) was submerged in a circular pool (160 cm in diameter, 50 cm in height) that was filled with warm water (23 ± 2 °C). The pool, located in a room with no windows, was virtually divided into four quadrants. A video camera connected to the computer running the tracking software was suspended above the pool and captured the rats’ movements for analysis. At P35, before the test, a single habituation trial was performed without the platform; in this trial, the rats were placed in the water for 120 s. In the hidden platform acquisition test, performed at P36–P39, each rat was placed, facing the wall of the pool, in one of the four quadrants and allowed to swim freely in search of the escape platform for a maximum of 120 s. The experiment was repeated with four trials per day for four consecutive days. The average escape latency time (latency to reach the platform) was measured to evaluate spatial learning ability. At P40, a probe trial test was performed by removing the platform and releasing the rats into the water for 120 s. We calculated the time spent in the quadrant that previously contained the target and the frequency of crossing the former location of the platform. The rats were dried and placed back into a heated cage after completing each test.

Western Blot Analysis
On P10 and 28, rats (n = 5 in each group at each sacrifice time point) were sacrificed by rapid decapitation, and the bilateral hippocampus areas were harvested and stored at − 80 °C until use. Protein was extracted using RIPA lysis buffer (Keygen Biotech, Nanjing, China). The amount of protein in each hippocampal tissues was measured using a protein assay kit (BCA, Pierce, Thermo, USA). Polyacrylamide-SDS gels with an equal amount of 50-μg load in each lane were electrophoresed, and the proteins transferred onto PVDF membranes (Millipore, Carrigtwohill, Ireland). The blots were blocked with 5% skim milk in Tris-buffered saline (150 mM NaCl, 0.1% TWEEN 20, 20 mM Tris, pH 7.4) for 1 h and then incubated overnight at 4 °C with anti-BDNF (1:1000, Novusbio, USA), anti-TrkB (1:800, Millipore, Ireland), anti-postsynaptic density (PSD-95) (1:2000, Abcam, England), and anti-synaptophysin (1:20,000, Abcam, England) primary antibodies. After rinsing, membranes were probed with corresponding secondary antibodies at RT for 2 h. Immunoreactive bands were detected with an enhanced chemiluminescence detection system (Bio-Rad, USA). A β-actin antibody (1:1000, ABclonal, China) was used to normalize for sample loading and transfer. The intensities of the bands were densitometrically quantified using ImageJ.

BrdU Injections and Immunofluorescence
For the 5′-bromo-2-deoxyuridine (BrdU) injections, we followed the methods as previously described (Chen et al. 2015; Tozuka et al. 2005). BrdU has been described as a marker of neurogenesis and can incorporate into DNA only during the S-phase of the mitotic process (Kee et al. 2002). BrdU (Sigma, America) was dissolved in normal saline (10 mg mL−1) and injected at a dosage of 300 mg/kg. To investigate the effects of 1.2% sevoflurane on cellular proliferation, we performed a single injection of BrdU i.p. 24 h after sevoflurane exposure. Three days later, the rats were perfused, and their brains were processed for immunofluorescence. To investigate the effects of 1.2% sevoflurane on the survival of newborn cells, we performed a single injection of BrdU i.p. 24 h before sevoflurane exposure. Four weeks (28 days) after the BrdU injection, the rats were perfused, and their brains were processed for immunofluorescence.

For morphological examination, rats were deeply anesthetized with chloral hydrate at P10 and P35 (n = 5 in each group at each sacrifice time point, 80–100 g) and then transcardially perfused with 0.9% normal saline at RT followed by a fixative solution of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M PBS (pH 7.4) at 4 °C. The brains were harvested, postfixed in 4% paraformaldehyde for 8 h, and subsequently soaked in 30% sucrose until they sank. Consecutive frozen coronal sections of the hippocampus were cut at a thickness of 30 μm. Every fifth section of the consecutive sections was processed by BrdU staining. DNA was first denatured by incubation with 2 N HCl for 30 min at 37 °C followed by a 15-min wash in 0.1 M boric acid (pH 8.5), with three 10-min washes in 0.01 M PBS before each step. The sections were blocked in 3% BSA and 0.4% Triton X-100 for 2 h at RT before being incubated with primary antibody (rat anti-BrdU, 1:200, ab6326, Abcam, UK) in 1% BSA overnight at 4 °C. Then, the sections were incubated with secondary antibody (Cy3 goat anti-rat IgG, 1:200, KGAB018, Keygentee, China) for 2 h at RT. Fluorescence was detected with a fully automatic fluorescence microscope (Olympus BX63, Japan). An observer who was blinded to group assignment was responsible for counting the number of BrdU-positive cells at × 200 magnification. Total cell counts were divided by the total number of sections for analysis.

Transmission Electron Microscopy
TEM was used to assess synaptic plasticity in the hippocampus after exposure to treatment (n = 5 in each group) at P35. Twenty-eight days after exposure to treatment, the rats were perfused transcardially with 50 mL of 0.9% normal saline, followed by 50 mL of a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde (Sigma-Aldrich, G6257, USA) in 0.1 M PBS. Approximately 1 mm3 of tissue per rat was dissected from the hippocampus and fixed in 2% glutaraldehyde for 2 h at 4 °C. The tissues were rinsed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide for 2 h. Then, the tissue was rinsed with distilled water before undergoing dehydration in a graded ethanol series. Subsequently, the tissue was infiltrated overnight at 4 °C using a mixture of half acetone and half resin. The tissue was embedded in resin 24 h later and then cured fully as follows: 37 °C overnight, 45 °C for 12 h, and 60 °C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructural changes in synapses in the hippocampus were observed under TEM. Five pictures of each subregion per ultrathin section (five rats in total per group) were taken at each of two magnifications: × 13,500 and × 37,000. All pictures taken at × 13,500 magnification were used to observe the number of synapses, and all pictures taken at × 37,000 magnification were used to measure the thickness of the postsynaptic density and the width of the synaptic cleft. The number of synapses was expressed as the average number of synapses in each picture taken at × 13,500. The thickness of the postsynaptic density and the width of the synaptic were expressed as the average values for all synapses in all pictures taken at × 37,000, as described. We measured the distances using the image analysis software ImageJ.

Statistical Analysis
The results were expressed as the mean ± standard deviation (SD) for each group. The statistical tests were conducted using the computerized statistical package SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism Software version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). The arterial blood data were analyzed using Student’s t test. One-way ANOVA was used to evaluate differences in the quantities of hippocampal proteins, numbers of BrdU-positive cells and synapses, and ultrastructure parameters of synapses among groups. Unpaired t tests and two-way ANOVA were used to analyze the results of the MWM. Each experiment was performed at least three times. A value of P < 0.05 was considered statistically significant.