Sevoflurane Anesthesia in Pregnant Mice Induces Neurotoxicity in Fetal and Offspring Mice

ANESTHESIA neurotoxicity in the developing brain has been investigated in animals and in humans and has become a major health issue of interest to both the medical community1and the public.2Anesthesia and surgery may induce neurodevelopmental impairment and cognitive dysfunction in children (reviewed in Sun3). In preclinical studies, anesthesia has been shown to induce neurotoxicity and learning and memory impairment in young animals4(reviewed in Creeley and Olney5).Each year, over 75,000 pregnant women in the United States have nonobstetric surgery and fetal intervention procedures under anesthesia.6Anesthesia neurotoxicity in the developing brain could occur in the fetus because (1) brain development starts as early as the second trimester of pregnancy; (2) anesthesia can induce neurotoxicity in both adult and young mice, and most general anesthetics are lipophilic and thus cross the placenta easily; and (3) uterine exposure to ethanol, valproic acid, and the anesthetic isoflurane have been shown to induce behavioral abnormalities in adulthood7(reviewed in Reitman and Flood8). It remains largely to be determined, however, whether anesthesia in pregnant mice can induce (1) neurotoxicity in fetal mice (the developing brain) and (2) neurotoxicity and learning and memory impairment in offspring mice after birth.Sevoflurane is currently the most commonly used inhalation anesthetic. Previous studies have shown that anesthesia with 2.5% sevoflurane for 2 h can induce neurotoxicity in the brain tissues of adult (5-month-old) mice without statistically significant alteration in the values of blood pressure and blood gas.9We therefore determined whether the same sevoflurane anesthesia in pregnant mice could induce neurotoxicity and learning and memory impairment in fetal and offspring mice. Finally, we investigated whether environmental enrichment (EE), a complex living milieu that has been shown to improve learning and memory,10–12could ameliorate the sevoflurane-induced detrimental effects.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.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.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).15Western 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).15Immunohistochemistry 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).17A 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.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.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 and Finally, the test was used to determine the difference between the sevoflurane and control conditions on platform crossing of were statistically software version Inc., was used to analyze the pregnant mice were treated with 2.5% sevoflurane anesthesia for 2 h or under the control at The mice delivered offspring mice at and the offspring mice were tested in the MWM from P31 to of the time that each mouse to a platform during reference training (escape latency) showed that there was a statistically significant interaction between time and group based on escape latency in the MWM between mice the control and mice that were sevoflurane anesthesia = two-way ANOVA with repeated of the number of times that each mouse the of an platform at the end of reference training (platform crossing that there was a difference in the platform crossing times between the control and the sevoflurane anesthesia = There was no statistically significant difference in mouse swimming between the sevoflurane anesthesia and the control group not shown). these data that sevoflurane anesthesia in pregnant mice may induce learning and memory impairment in offspring that the sevoflurane anesthesia in pregnant mice can induce learning and memory impairment in offspring mice, we the of sevoflurane anesthesia on the levels of and caspase-3 the neurotoxicity which may of learning and memory pregnant mice received anesthesia with 2.5% sevoflurane for 2 h or the control at We harvested the brain tissues of the fetal mice at the end of the and these tissues were subjected to Western blot analysis. of IL-6 showed that the sevoflurane anesthesia IL-6 as with the control There was no significant difference in β-actin levels between the control and the sevoflurane anesthesia. of the Western blot showed that the sevoflurane anesthesia IL-6 levels in the brain tissues of fetal mice as with the control ± vs. ± = we investigated the of the sevoflurane anesthesia in pregnant mice on levels of the marker of in the brain tissues of the fetal mice. of PSD-95 showed that the sevoflurane anesthesia in pregnant mice PSD-95 in the Western blot as with the control of the Western blot showed that the sevoflurane anesthesia in pregnant mice PSD-95 levels in the brain tissues of fetal mice as with the control ± vs. ± = Finally, we the of sevoflurane anesthesia in pregnant mice on caspase-3 in the brain tissues of fetal mice. showed that the sevoflurane anesthesia in pregnant mice levels of caspase-3 fragment without statistically significant changes in the levels of full-length caspase-3 in the brain tissues of fetal mice The quantification of the Western based on the ratio of caspase-3 fragment to full-length that the sevoflurane anesthesia in pregnant mice caspase-3 as with the control ± vs. ± = these that anesthesia with 2.5% sevoflurane for 2 h in pregnant mice may induce in a in marker and caspase-3 in fetal mice, which may then lead to learning and memory that sevoflurane anesthesia may neurotoxicity in fetal mice and learning and memory impairment in offspring mice at a time (e.g. , we the of the sevoflurane anesthesia on levels of in the hippocampus of P31 mice. analysis showed that the sevoflurane anesthesia levels of the the hippocampus of P31 mice of the immunohistochemistry image showed that the sevoflurane anesthesia levels of synaptophysin ± vs. ± = that the sevoflurane anesthesia in pregnant mice may induce at a time (e.g. , to learning and memory that the sevoflurane anesthesia IL-6 levels and PSD-95 levels in brain tissues of fetal mice at we then determined their in mouse primary with 4.1% sevoflurane for 6 h PSD-95 levels in mouse primary neurons as with the control The treatment with sevoflurane PSD-95 levels as with the control but IL-6 antibody the sevoflurane-induced in PSD-95 by PSD-95 the treatment of sevoflurane plus IL-6 antibody the treatment of sevoflurane plus saline of the Western blot showed that the sevoflurane treatment PSD-95 levels ± vs. ± = and IL-6 antibody the sevoflurane-induced in PSD-95 levels ± vs. 20 ± = Two-way ANOVA that there was an interaction between IL-6 antibody and and that IL-6 antibody the sevoflurane-induced in PSD-95 levels = that the sevoflurane-induced in PSD-95 level may be on the sevoflurane-induced in IL-6 IL-6 antibody also PSD-95 levels in the primary neurons has been shown to improve learning and we therefore whether EE can ameliorate the sevoflurane-induced learning and memory Two-way ANOVA with repeated analysis showed that there was a statistically significant interaction between time and group based on escape latency between mice sevoflurane anesthesia plus standard and sevoflurane anesthesia plus and EE the sevoflurane-induced in escape latency of mice swimming in the MWM = anesthesia plus EE also the platform crossing times of mice in the MWM as with sevoflurane anesthesia plus = EE did not alter escape latency or platform crossing times of mice swimming in the MWM Two-way ANOVA with repeated analysis showed that there was no statistically significant interaction between time and group based on escape latency between mice the control plus and control plus EE in the MWM = there was a statistically significant group effect based on escape latency between mice the control plus and control plus EE = the swimming of the mice in the MWM between of these conditions were not not shown). these data that EE may ameliorate the learning and memory impairment in the offspring mice that is by the sevoflurane anesthesia in the pregnant mice. are with the that EE cognitive that EE can ameliorate the sevoflurane-induced learning and memory and is the with cognitive dysfunction and determined the of EE on the sevoflurane-induced of IL-6 and marker PSD-95 and synaptophysin levels in the brain tissues of offspring mice. IL-6 showed that sevoflurane anesthesia in pregnant mice IL-6 levels in the brain tissues of P31 offspring mice and that EE the The quantification of the Western blot that the sevoflurane anesthesia IL-6 levels ± vs. ± = EE the sevoflurane-induced in IL-6 levels ± vs. ± = There was no significant difference in IL-6 levels between the control plus and control plus EE of PSD-95 showed that sevoflurane anesthesia in pregnant mice PSD-95 levels in the brain tissues of P31 offspring mice, and EE the sevoflurane-induced in PSD-95 levels in the brain tissues of offspring mice at P31 ± for sevoflurane plus EE vs. ± for sevoflurane plus vs. ± for control plus = There was a level of PSD-95 in the control plus EE as with the control plus showed that sevoflurane anesthesia in pregnant mice synaptophysin levels in the brain tissues of P31 offspring mice as with the control ± vs. ± = and EE the sevoflurane-induced in synaptophysin levels in the brain tissues of offspring mice at P31 ± for sevoflurane plus vs. ± for sevoflurane plus = these that EE may the sevoflurane-induced and to of the sevoflurane-induced learning and memory and of anesthesia in the developing brain its a major health issue of in Sun3). has become a of with the that anesthesia and surgery may induce neurodevelopmental impairment in children and that anesthetics are in young animals (reviewed in Sun3). pregnant women in the United States have nonobstetric surgery and fetal intervention procedures under anesthesia each therefore determined whether anesthesia with sevoflurane in pregnant mice could induce detrimental in fetal mice and offspring mice. We sevoflurane in the studies because sevoflurane is currently the most commonly used inhalation sevoflurane be the of isoflurane in pregnant mice on behavioral changes in offspring mice have been anesthesia in pregnant mice learning and memory impairment in offspring mice at P31 The same sevoflurane anesthesia neurotoxicity as by the levels of levels of marker and caspase-3 in the brain tissues of fetal mice The sevoflurane anesthesia in pregnant mice also IL-6 levels and levels of PSD-95 and synaptophysin in the brain tissues of P31 offspring mice IL-6 can be by the during their and to cognitive cognitive medical and is a postsynaptic of PSD-95 has been shown to be with in number or a of the and impairment of learning and in and In the in studies, IL-6 antibody the sevoflurane-induced in PSD-95 which that the sevoflurane-induced in IL-6 levels may lead to in PSD-95 these data that sevoflurane may (e.g. , in IL-6 which a in to learning and memory studies, of whether can the sevoflurane-induced and impairment of learning and are to test this antibody PSD-95 levels in the primary neurons could be to IL-6 antibody only the with IL-6 (e.g. , a in PSD-95 In the of IL-6 however, the IL-6 antibody may have The of these to be sevoflurane anesthesia caspase-3 in IL-6 and a in PSD-95 levels 2 h after the anesthesia in the brain tissues of fetal mice, which in the brain tissues of adult mice data that fetal mice be to neurotoxicity adult by which anesthetics induce to be have been shown to of is with levels of of the to the it to the of studies determining whether anesthetics can levels in neurons and to of (e.g. , the of interaction and may in and brain EE has also been shown to improve learning and memory found that EE the sevoflurane-induced learning and memory and the sevoflurane-induced in IL-6 levels and in that EE may the sevoflurane-induced and to of the sevoflurane-induced impairment of learning and study has First, we did not determine the (e.g. , of sevoflurane anesthesia on learning and memory however, the current were to the of sevoflurane anesthesia on behavioral changes (e.g. , learning and memory and the (e.g. , in IL-6 and Second, we on only in the because IL-6 has been shown to to learning and memory anesthesia in pregnant mice may also induce changes (e.g. , in the brain tissues of fetal mice with and to be investigated in is whether the anesthesia to the clinically cognitive or whether the for is a marker for factors that in or the of anesthesia, we determine whether anesthesia can induce and learning and memory in young mice. Our preclinical mouse be used to determine whether anesthesia can induce detrimental (e.g. , learning and memory and in young animals to the and to as and with have been shown to the neurotoxicity and neurobehavioral studies may also whether factors (e.g. , and can neurotoxicity and neurobehavioral clinically relevant sevoflurane anesthesia in pregnant mice can induce in IL-6 levels and in marker PSD-95 and caspase-3 in the brain tissues of fetal mice. The same sevoflurane anesthesia in pregnant mice also detrimental in marker PSD-95 and and impairment of learning and memory in offspring mice at 31 days after birth. that sevoflurane anesthesia in pregnant mice may induce and to learning and memory Finally, EE may be to the sevoflurane-induced learning and memory impairment by the sevoflurane-induced and in anesthesia neurotoxicity in the developing of Massachusetts General Hospital and Medical for and in the data analysis of the