20230808-AI coding-1st round/3879 – Chen 2020.txt

PMID: 32271540 DOI: 10.1021/acschemneuro.0c00106
Materials and Methods
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Animals and Treatments
Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study. The mice were bred and maintained in the animal care facility following the standard rearing conditions of 12 h light and 12 h dark. All mouse studies were performed following the guidelines established by the Institutional Animal Care and Use Committee in Quanzhou First Hospital Affiliated to Fujian Medical University (QFH2017jb43i).
BRL-50481 (Tocris Bioscience, Bristol, United Kingdom) was dissolved in 2.5% dimethyl sulfoxide (Sigma, St. Louis, MO) with 0.9% NaCl and injected intraperitoneally into pups before subjecting them to sevoflurane, with a vehicle injection as control. Thirty minutes later, the injected pups were put into a semiclosed chamber and exposed to 3% sevoflurane for 4 h. After exposure, pups were returned to the parents’ cages and monitored for health status until the following tests.
The pups were randomly divided into five groups as follows:
Sham: vehicle intraperitoneal injection;
Control: 5 mg/kg BRL-50481 intraperitoneal injection;
B0: Sevoflurane anesthesia, vehicle intraperitoneal injection;
B1: Sevoflurane anesthesia, 1 mg/kg BRL-50481 intraperitoneal injection;
B5: Sevoflurane anesthesia, 5 mg/kg BRL-50481 intraperitoneal injection.
Each group contained 10 pups.
Morris Water Maze Test and Analysis
The spatial memory ability of control and treated mice was determined using the Morris water maze test developed by Richard Morris. (31) In brief, a 160 cm diameter and 60 cm high circular tank was filled with water at 30 cm high. The water temperature was maintained at 22 °C. A 12 cm diameter circular platform was submerged 1 cm below the water surface in the center of one of the four virtual quadrants. (32)
The control and treated mice were trained four times per day for 6 days. The mouse was released into the water, and it navigated to reach the platform. The maximum swimming time of the tested mouse was 80 s. If the mouse could escape to the refuge within 60 s, the delay to find the platform time was recorded as 60 s. Mice were allowed to stay on the platform for 15 s, and then they were sent to their cages under a heat lamp to maintain their core temperature. The escape latency was recorded by a tracking system, and data were analyzed using ViewPoint video tracking system (ViewPoint Behavior Technology, Civrieux, France). Three daily trials were averaged for each animal. (32)
Immunohistochemistry (IHC) Analyses
Mice were euthanized and perfused with cold phosphate-buffered saline and 4% paraformaldehyde immediately. The brains were fixed with 4% paraformaldehyde overnight and then cryoprotected by immersion in 30% sucrose at 4 °C for 48 h. Coronal sections (25 μm) were cut using a manual rotary microtome (Leica, Wetzlar, Germany).
The caspase-3 IHC staining was performed as previously described. (33) The cleaved caspase-3 antibody (ab13847) was purchased from Abcam (Cambridge, MA).
Immunoblotting Analyses
Frozen hippocampus homogenates were lysed using radioimmunoprecipitation buffer (Bioequip, Shanghai, China). The samples were subjected to immunoblotting analysis as described previously. (33) The pCREB (Ser133, #9198, 1:1000 dilution) and CREB (#9197, 1:2000 dilution) primary antibodies were ordered from Cell Signaling Technology (Danvers, MA), and the internal control β-actin antibody was ordered from Abcam (ab8226, 1:2000 dilution).
cAMP Concentration Assay
cAMP levels were measured using the mouse cAMP ELISA kit (ab133051, Biocompare, South San Francisco, CA) following the manufacturer’s instructions.
Statistical Analysis
Statistical analyses were carried out by using the SPSS 11.0 package. Differences between groups were analyzed using analysis of variance (ANOVA) or two-sample t test with Bonferroni correction. All data represent mean ± standard deviation (SD). Statistical significance thresholds were set at *P < 0.05.