PMID: 23314109 PMCID: PMC3580035 DOI: 10.1097/ALN.0b013e3182834d5d
Materials and Methods
Mice Anesthesia
The protocol was approved by the Massachusetts General Hospital Standing Committee (Boston, Massachusetts) on the Use of Animals in Research and Teaching. Three-month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice. The pregnant mice were identified and then housed individually. The offspring mice were weaned 21 days after birth. Animals were kept in a temperature-controlled (22°–23°C) room under a 12-h light/dark period (light on at 7:00 AM); standard mouse chow and water were available ad libitum . At gestational day (G) 14, the pregnant mice were assigned randomly to an anesthesia group or a control group. Mice randomized to the anesthesia group received 2.5% sevoflurane in 100% oxygen for 2 h in an anesthetizing chamber. The control group received 100% oxygen at an identical flow rate for 2 h in an identical chamber as described in our previous studies.9The mice breathed spontaneously, and concentrations of anesthetic and oxygen were measured continuously (Datex-Ohmeda Inc., Tewksbury, MA). The temperature of the anesthetizing chamber was controlled to maintain rectal temperature of the animals at 37° ± 0.5°C. Mean arterial blood pressure was not measured in these mice because the same sevoflurane anesthesia was shown not to alter the values of blood pressure and blood gas in our previous studies.9Anesthesia was terminated by discontinuing sevoflurane and placing the animals in a chamber containing 100% oxygen until 20 min after return of the righting reflex. The anesthesia with 2.5% sevoflurane (approximately 1.1 minimum alveolar concentration) for 2 h in mice was used to demonstrate whether clinically relevant sevoflurane anesthesia in pregnant mice, which had been shown to induce neurotoxicity in adult mice,9could also induce neurotoxicity in fetal mice and then neurobehavioral deficits in offspring mice. Twenty pregnant mice were included in the experiments, which generated a sufficient number of fetal mice for the biochemistry studies (n = 6 per arm), and offspring mice for the biochemistry (n = 6 per arm) and behavioral studies (n = 15 per arm). Our pilot studies showed a mean difference of 1.5 (3 vs.  1.5) in platform crossing times, with an SD of 1.8 in the control group and 1.3 in the anesthesia group. From the pilot study, we also estimated a mean difference of 150% (250% vs.  100%) in interleukin (IL)-6 levels in brain tissues, with an SD of 51 in the control group and 54 in the anesthesia group. Assuming this study would have similar effect sizes, a sample size of 6 per arm for the biochemistry studies and a sample size of 15 per arm for the behavioral studies would lead to a 90% or larger power to detect the differences using two-sample Student t  test with 5% type I error.

Mouse Primary Neurons
The protocol was approved by the Massachusetts General Hospital Standing Committee on the Use of Animals in Research and Teaching. The harvest of neurons was performed as described in our previous studies.13,14Seven to 10 days after harvesting, the neurons were treated with 4.1% sevoflurane for 6 h as described in our previous studies.9The treatment with 4.1% sevoflurane for 6 h was used to determine whether the sevoflurane anesthesia, which can induce cytotoxicity,9could also reduce levels of postsynaptic density-95 (PSD-95), the marker for synapse. The IL-6 antibody (10 μg/ml) was administrated to the neurons 1 h before the sevoflurane treatment. The neurons were harvested at the end of anesthesia and were subjected to Western blot analysis.

Brain Tissue Harvest and Protein Level Quantification
Immediately after the sevoflurane anesthesia, we performed a cesarean section to extract the fetal mice and harvested their brain tissues. We also used decapitation to kill postnatal day (P) 31 offspring mice and harvested their brain tissues. Separate groups of mice were used for the Western blot analysis and the immunohistochemistry studies, respectively. For the Western blot analysis, the harvested brain tissues were homogenized on ice using immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, and 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A) as described in our previous studies.15The lysates were collected, centrifuged at 12,000 rpm for 15 min, and quantified for total proteins with bicinchoninic acid protein assay kit (Pierce Technology Co., Iselin, NJ).15 

Western Blot Analysis
Western blot analysis was performed using the methods described in our previous studies.15Whole cerebral hemispheres were used for Western blot analysis because there would be an insufficient amount of hippocampus tissues from the fetal mice for Western blot analysis. IL-6 antibody (1:1,000 dilution; Abcam, Cambridge, MA) was used to recognize IL-6 (24 kDa). PSD-95 antibody (1:1,000; Cell Signaling Technology, Danvers, MA) was used to detect PSD-95 (95 kDa). A caspase-3 antibody (1:1,000 dilution; Cell Signaling Technology) was used to recognize full-length caspase-3 (35–40 kDa) and caspase-3 fragment (17–20 kDa) resulting from cleavage at aspartate position 175. Antibody anti–β-actin (1:10,000; Sigma, St. Louis, MO) was used to detect β-actin (42 kDa). Western blot quantification was performed as described by Xie et al.  16Briefly, signal intensity was analyzed using a Bio-Rad (Hercules, CA) image program (Quantity One). We quantified the Western blots in two steps. First, we used β-actin levels to normalize (e.g. , determining the ratio of IL-6 to β-actin amount) protein levels and control for loading differences in the total protein amount. Second, we presented changes in protein levels in mice or neurons undergoing sevoflurane anesthesia as a percentage of those in the control group. One hundred percent of protein level changes refer to control levels for the purpose of comparison with experimental conditions.

The quantification of Western blot was based not only on the images presented in figures but also on the images not presented in the figures to have adequate effect size (e.g. , n = 6 in biochemistry studies).15 

Immunohistochemistry
Immunohistochemistry was performed using the methods described in our previous studies.17P31 offspring mice were anesthetized with sevoflurane briefly (2.5% sevoflurane for 4 min) and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1M phosphate buffer at pH 7.4. The anesthesia with 2.5% sevoflurane for 4 min in mice provided adequate anesthesia for the perfusion procedure without causing statistically significant changes in blood pressure and blood gas according to our previous studies.9Mouse brain tissues were removed and kept at 4°C in paraformaldehyde. Five-micron frozen sections from the mouse brain hemispheres were used for the immunohistochemistry staining.17The sections were incubated with the primary antibody synaptophysin (1:500; Sigma) dissolved in 1% bovine serum albumin in phosphate-buffered saline at 4°C overnight. The next day, the sections were exposed to secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG [H+L]; Invitrogen, Grand Island, NY). Finally, the sections were wet mounted and viewed immediately using a fluorescence microscope (60×). We used the mouse hippocampus in the studies of immunohistochemistry density quantification to determine whether sevoflurane anesthesia can induce neurotoxicity in the hippocampus. The photographs were taken and an investigator who was blind to the experimental design counted the density of synaptophysin using ImageJ version 1.38 (National Institutes of Health, Bethesda, MD).17 

Morris Water Maze
A round steel pool, 150 cm in diameter and 60 cm in height, was filled with water to a height of 1.0 cm above the top of a 10-cm diameter platform. The pool was covered with a black curtain and was located in an isolated room with four visual cues on the wall of the pool. Water was kept at 20°C and opacified with titanium dioxide. The P31 offspring mice were tested in the Morris water maze (MWM) four times per day for 7 days. Each of the mice was put in the pool to search for the platform, and the starting points were random for each mouse. When the mouse found the platform, the mouse was allowed to stay on it for 15 s. If a mouse did not find the platform within a 90-s period, the mouse was gently guided to the platform and allowed to stay on it for 15 s. A video tracking system recorded the swimming motions of the animals, and the data were analyzed using motion-detection software for the MWM (Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China). At the end of the reference training (P37), the platform was removed from the pool and the mouse was placed in the opposite quadrant. Mice were allowed to swim for 90 s and the times the mouse swam to cross the platform area was recorded (platform crossing times). Mouse body temperature was maintained by active heating as described by Bianchi et al .18Specifically, after every trial, each mouse was placed in a holding cage under a heat lamp for 1 to 2 min until dry before being returned to its regular cage.

Environmental Enrichment
The EE in the current experiment was created in a large cage (70 × 70 × 46 cm) that included five or six toys (e.g ., wheels, ladders, and small mazes) as described in previous studies, with modification.10,11The pregnant mice were put in the EE every day for 2 h before delivery. The pregnant mice delivered offspring mice at G21. Then, the mother and the babies were put in the EE again every day for 2 h from P4 to P30. The objects were changed two to three times per week to provide newness and challenge.

Statistical Analysis
The nature of the hypothesis testing was two-tailed. Data were expressed as mean ± SD. The data for platform crossing time were not distributed normally and thus were expressed as median and interquartile range (IQR). The number of samples varied from 6–15, and the samples were distributed normally, with the exception of platform crossing time (tested by normality test, data not shown). Two-way ANOVA was used to determine the interaction of IL-6 antibody and sevoflurane treatment, and the interaction of EE and sevoflurane anesthesia. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) between mice in the control group and mice treated with anesthesia in the MWM. Multiple comparisons in escape latency of MWM were adjusted using the Bonferroni method (with seven tests and a threshold of 0.05/7 = 0.0071). There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. The Student two-sample t  test was used to determine the difference between the sevoflurane and control conditions on levels of IL-6, PSD-95, and synaptophysin. Finally, the Mann–Whitney U  test was used to determine the difference between the sevoflurane and control conditions on platform crossing times. Values of P < 0.05 were considered statistically significant. SAS software version 9.2 (SAS Institute Inc., Cary, NC) was used to analyze the data.