Seven day old (postnatal day 7, PND7) neonatal Swiss CD-1 mouse pups (Adult male and female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd), weighing 8 ± 0.4g, were used in this study. The ethical committee of Qingdao Agricultural University approved all of the procedures described in this study.
All protocols used in this study were approved by the laboratory animal use ethical committee of Qingdao Agricultural University (approval number #QDAU 4322). In addition, all protocols using live animals followed internationally approved guidelines for the use of animals in research (National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC: The National Academies Press. https://doi.org/10.17226/5140).
Anesthetic Neurotoxicity Model
A total of 28 7PND mice were divided into four groups: Dex15+iso (intraperitoneal injection of 15mg/kg Dex (dexmedetomidine)), Dex20+iso (intraperitoneal injection of 20mg/kg Dex), Dex25+iso (intraperitoneal injection of 25mg/kg Dex), and the control group (intraperitoneal injection of 20 mg/kg normal saline). After injection, the mouse pups were placed in a small box (15cm×15cm×15cm) and anesthetized using 2% isoflurane (Iso) in air for 2h. The concentration of isoflurane was monitored by a probe put in the box. The interior temperature of the experimental box was maintained at 30℃. After the 2h anesthesia period, the pups were returned to their mother.
Morris water maze
When 21-day-old mice were tested in the Morris water maze (MWM), which is used to assess spatial learning abilities in rodents. The maze requires subjects to remember spatial cues to navigate in an open swimming tank to find a submerged escape platform (Vorhees and Williams 2006). An overhead camera was used to record the swim path of the mice. The water temperature was maintained at 25℃, and a sufficient amount of nontoxic white paint was added to the water in the tank to make it opaque and render the submerged platform virtually invisible. An automated tracking system (Xinruan, Shanghai, China) was used to analyze the swim path of each mouse and calculate the escape latencies. Four positions around the perimeter of the maze, arbitrarily indicated as N, S, E, and W, were used as the initiation points where the mice were released at the beginning of each trial. The order in which the starting points was random, and each starting point was used only once during each session. Once the mouse located the platform, it was allowed to remain there for 30 s before being removed from the tank. During the training period, if a mouse failed to locate the platform in 2min, the mouse was picked up when it was in the middle portion of the swim path and placed on the platform for 15s. Each mouse was trained 7 times by releasing the mouse into the water from the same position as the first trial. After training, each mouse was subjected to the probe trial.
Neuronal Cell Cultures
Neurons were isolated from anesthetized PND 4 mouse hippocampi. The mice were gently restrained and decapitated for neurons preparation as approved by the ethical committee of Qingdao Agricultural University. Both left and right hippocampi were dissected on ice and digested using 0.25% trypsin in D-Hanks medium. Neurons were isolated from a total of 10-14 neonatal 4-day-old mouse pups, combined, then cultured at 37℃ in 5% CO2 for 3-4 days in Neurobasal-A medium, supplemented with 2% B27, 1% penicillin, and streptomycin. The cultured neurons were divided into three groups: a 1.5% Iso group, a 2% Iso group, and a control group (treated with normal saline). The isoflurane or normal saline were added to the culture medium, respectively (These results are shown in the supplemental material). After evaluating the toxic effect of the different concentrations of isoflurane, the 2% isoflurane exposure was chosen to use in the experiments that included exposure to Dex. The Dex experiments were divided into four groups, dex25+iso （25mg/ml of dex was added to the culture medium）, dex20+iso (20mg/ml of dex was added to the culture medium), dex15+iso (15mg/ml of dex was added to the culture medium), and a control group (nothing was added to the culture medium). Then the neurons were cultured at 37℃ in 5% CO2 and 2% isoflurane for 4 days.
Protein Extraction and Western blot
Neuronal proteins were isolated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 10% acrylamide gels (Solarbio, Beijing China). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, Massachusetts, USA) using electroblotting. Then the membranes with the transferred proteins were blocked in phosphate-buffered saline with 0.1% Tween (PBST) containing 10% bovine serum albumin (BSA) at room temperature for 2h. The membranes were incubated for 2h in Tris-buffered saline with 0.1% Tween (TBST) with primary antibodies, including rabbit anti-BDNF (1:800), rabbit anti-RhoA (1:1000), rabbit anti-Caspase-3 (1:1000), and rabbit anti-GAPDH (1:1000) (Boster, Wuhan, China ). After three washes for 10 min each in TBST, the membranes were incubated for 1h at 37℃ with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG and diluted at 1:800 (Beyotime, Beijing, China). Then the membranes were washed three times in TBST and processed using the enhanced chemiluminescence (ECL) detection system. An antibody to GAPDH was used as the loading control. All experiments were repeated three times. The expression level of proteins was calculated as an average of the densities per band area from each group.
Cytoskeletal Depolymerization Quantification
Actin from neurons was labeled with 200mg/ml Alexa-488-phalloidin prior to observation. The neurons were fixed for 20 min with 4% paraformaldehyde at room temperature and washed three times in PBS. The neurons were treated for 20min with 0.3% Triton X-100, then washed 3 times in PBS for 5min each. Then the neurons were stained for 30min in the dark, using phalloidin labeled with rhodamine, and washed three times in PBS for 5min each. Finally, the stained neurons were incubated with DAPI (4’,6-diamidino-2-phenylindole) for 5min at room temperature and imaged under a fluorescence microscope (Nikon Eclipse 50i, Japan).
Results from this study were given as the mean ± SD. Analysis of variance (ANOVA) was used to analyze the data. Significant differences were set at p <0.05. Statistical analyses were carried out using SPSS23.0. GraphPad Prism 5.0 software was used to plot the graphs. Data for the latency time to find the hidden platform in the Morris water maze were analyzed using a two-way repeated-measures ANOVA. All assays were carried out independently and in triplicate.