PMID: 26898457 DOI: 10.1016/j.biopha.2016.01.034
2. Materials and methods
2.1. Animals
Because peak anesthesia-induced neurodegeneration in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study. The housing and treatment of the animals were in accordance with the National Institutes of Health guidelines for animal experimentation and approved by the institutional animal care and use committee. The animals were kept on a 12-h light/dark cycle (light from 7 am to 7 pm) with room temperature (23 ± 1 °C).

2.2. Sevoflurane exposure
Rat pups were separated from their mothers for acclimatization prior to sevoflurane exposure. Pups from the same litter were randomly allocated to three different groups. Totally, ninety PND7 rats were included in this study (n = 30 for each group). Rats in the control group received 100% oxygen for 6 h in a chamber at 37 °C. Rats in the other two groups were exposed to either 2% sevoflurane (SEVOFRANE®, Osaka, Japan) for 3 h (Sevo1 group) or 3% sevoflurane for 6 h (Sevo2 group) under 100% oxygen in the same chamber at 37 °C as described previously [13]. The concentration of sevoflurane in the chamber was monitored and maintained by a vaporizer as we described previously [23]. The gas flow to the chamber was 2 l/min. We chose these treatments because 3 h exposure to 2% seveflurane more closely approximates typical general pediatric anesthetic episodes for anesthesia maintenance [16] and 6 h exposure to 3% sevoflurane can cause neuronal apoptosis in developing animals [11], [12], [14], [15].

2.3. Arterial blood gas analysis
To determine adequacy of ventilation and oxygenation，arterial blood samples (n = 6) were obtained from the left cardiac ventricle in each group at the end of anesthesia, and the samples were immediately analyzed by a blood gas analyzer (Radiometer, ABL800, Denmark). We compared the pH, pO2, pCO2, oxygen saturation (sO2), and the concentrations of blood glucose (Glu), lactic acid (Lac) and bicarbonate (HCO3−) among the groups. Animals were killed by lethal injection of pentobarbital at the time of blood sampling.

2.4. Analysis of apoptotic levels
2.4.1. TUNEL assay of brain
Twenty-four hours after sevoflurane exposure, six rats from each group (n = 6) were anesthetized with sodium pentobarbital and the brains were perfused, fixed, dehydrated and made into paraffin sections (5 μm), as described previously [24]. Apoptotic cells in the brain sections were detected by TUNEL Assay using the FragEL™ DNA Fragmentation Detection Kit (Merck, Darmstadt, Germany), according to the manufacturer’s protocol. Briefly, brain sections were permeabilized with proteinase K (20 μg/ml) at room temperature for 20 min. Endogenous peroxidase was inactivated by 3% H2O2. Specimens were incubated for 1.5 h with terminal deoxynucleotidyl transferase (TdT) labelling reaction mixture, and apoptotic cells were visualized with 3,3′-diaminobenzidine (DAB), and normal nuclei were counterstained with methyl green. Because the cerebral cortex reaches peak vulnerability to anesthetics at PND7 and the hippocampus is closely related to learning and memory [25], the number of apoptotic neurons in the frontal cortex and the CA1 region of the hippocampus was quantified. We selected two random viewing fields (400×) per region (frontal cortex and CA1) from one brain section per animal for analysis in a double blinded manner.

2.4.2. Western blot
Apoptosis was also assessed using western blot to quantify cleaved caspase-3 (Cl-Csp3) in all groups (n = 6). Briefly, tissue samples of the frontal cortex and CA1 region were collected from three groups twenty-four hours after sevoflurane exposure. Tissues were lysed in a buffer containing a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) and homogenated. The homogenate was centrifuged and the supernatant was collected for further analysis. Protein concentrations were measured by BCA Protein Assay Kit (Novagen, San Diego, CA, USA). Equal amounts of protein were boiled in loading buffer (Beyotime, Beijing, China) and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose, and the blots were probed overnight with anti-cleaved caspase-3 (1:200, Millipore, Darmstadt, Germany) and β-actin antibodies (1:500, internal standard, Santa Cruz, San Diego, CA, USA) at 4 °C. Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, San Diego, CA, USA) and ECL reagent (Pierce, Rockford, IL, USA). Quantitative analysis of Cl-Csp3 was normalized to β-actin using the Quantity One software.

2.5. Neurologic assessment
2.5.1. Morris water maze
To assess neurodevelopmental outcomes, particularly the learning and memory functions of juveniles, rats from all groups were subjected to Morris water maze after reaching 6 weeks of age (n = 12), as previously described [24]. Briefly, a circular pool (1.6 m diameter, 60 cm height) was used for the water maze, and a submerged platform (10 cm diameter, 2 cm below the surface of the water) was located at a fixed position in the pool. The water temperature was set at 23 ± 1 °C. Probe trials were conducted twice a day for five consecutive days. In the trials, rats were trained to swim to and locate the hidden platform. The time spent in finding the hidden platform and the swimming distance before reaching the platform were recorded. After the probe trials, the platform was removed, and the rats were allowed to swim freely for 120 s: the number of times that the former platform was crossed and the percentage of time spent in the target quadrant were determined. The entire behavioral test was recorded and analyzed using a MS-type Water Maze Video analysis system (Chengdu Instrument Ltd., Chengdu, China). Finally, to investigate cognitive function during development, the passive avoidance test was performed at 3 months.

2.5.2. Passive avoidance test
The passive avoidance test was performed as previously described [26]. The apparatus used for the passive avoidance test included a behavioral stimulation controller and a video shuttle box (Chengdu Instrument Ltd., Chengdu, China). The test relies the natural preference of rats for darkness. Briefly, on the first trial day, the rats were placed in the illuminated compartment after 2 min of habituation to the dark compartment and allowed to re-enter the dark compartment. On the following day, an electric foot shock was delivered through the grid floor of the dark compartment after the rats entered. Twenty-four hours later, the retention of passive avoidance was determined by comparing the time elapsed prior to re-entry into the dark compartment with the arbitrary maximum time of 180 s.

2.6. Statistical analysis
All data are expressed as the mean ± SEM. SAS 9.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis. One-way ANOVA was used to determine statistically significant differences between the three groups, and Tukey’s post hoc analysis was performed to correct for multiple comparisons when applicable. Statistical significance was accepted as P < 0.05.