Selective Anesthesia-induced Neuroinflammation in Developing Mouse Brain and Cognitive Impairment

AN estimated 6 million children undergo surgical care each year in the United States alone.1The widespread and prevalent use of anesthesia in children makes its safety a major health issue of interest2(reviewed in Sun3). Recent population studies have suggested that anesthesia and surgery could be risk factors for subsequent cognitive impairment (reviewed in Sun3). Specifically, children who have multiple exposures (e.g., three times) to anesthesia and surgery at an early age (e.g. , before age 4) are at an increased risk of developing learning disabilities4,5(reviewed in Sun3). These data suggest that children may not reach cognitive potentials compared with their peers who have not undergone anesthesia and surgery. These findings have become a major public health issue.2However, not every child develops cognitive impairment after having anesthesia and surgery, and older children may be less susceptible to this phenomenon.4Therefore, we have hypothesized that there is a multifactorial model of the cognitive impairment such that the combination of an environmental insult (precipitating factors, e.g. , selective anesthesia) with an age vulnerability (predisposing factors, e.g. , certain age groups) is needed to cause the cognitive impairment. In the present studies, we have tested this hypothesis by identifying the selective effects of anesthetics (sevoflurane vs. desflurane) and anesthesia regimen (one vs. three times) on cognitive impairment in different age groups (6 vs. 60 days) of mice.Neuroinflammation, including microglia activation and increases in the levels of proinflammatory cytokines in the brain, may lead to cognitive impairment.6–10Specifically, proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α and interleukin (IL)-6, can be released by the microglia during its activation, fueling brain inflammation and leading to cognitive impairment in humans11–14and in animals.15–17We therefore assessed the effects of different anesthetics and anesthesia regimens on the levels of IL-6 and TNF-α, and ionized calcium-binding adaptor molecule 1 (IBA1), the marker of microglia activation,18,19in the brain tissues of mice.Finally, enriched environment (EE) has been shown to improve brain function. Delayed EE mitigates anesthesia-induced learning and memory impairment in rats.20We therefore determined whether EE, which occurred immediately after the anesthesia, could attenuate the anesthesia-induced cognitive impairment in mice in the present experiments.The animal protocol was approved by the Standing Committee on Animals at Massachusetts General Hospital, Boston, Massachusetts. Postnatal day (P) 6 or P60 C57BL/6J (The Jackson Laboratory, Bar Harbor, ME) mice (both male and female) received either anesthetic sevoflurane or desflurane plus 60% oxygen (balanced with nitrogen) to maintain sufficient partial pressure of oxygen (PO2) levels during anesthesia, as performed in our previous studies.21Control groups received 60% oxygen at an identical flow rate in similar chambers. There was no significant difference in learning and memory function between the mice that received 60% oxygen and the mice that received 21% oxygen (data not shown). The anesthetic and oxygen concentrations were measured continuously (GE Datex-Ohmeda, Tewksbury, MA). The temperature of the anesthetizing chamber was controlled to maintain a 37° ± 0.5°C rectal temperature in the mice. We determined pH, and partial pressure of oxygen (PO2) and carbon dioxide (PCO2) in the neonatal mice using the methods described by Satomoto et al .22Specifically, the young mice had a quick arterial blood sampling from the femoral artery at the end of 2 h of anesthesia, and the samples were transferred into heparinized glass capillary tubes. A single sample (100 μl) was analyzed immediately after blood collection by using a blood gas analyzer (ITC, Edison, NJ). Anesthesia with 3% sevoflurane21,22for 2 h did not significantly change the values of pH, PO2, or PCO2as compared with the control group. Anesthesia with 9% desflurane for 2 h did not significantly change the values of pH, PO2, or PCO2as compared with the control group (pH, 7.33 ± 0.05 vs. 7.41 ± 0.14 for control vs. desflurane anesthesia; PO2, 174 ± 12.4 mmHg vs. 142 ± 27.0 mmHg for control vs. desflurane anesthesia; and PCO2, 48 ± 5.1 mmHg vs. 41 ± 9.9 mmHg for control vs. desflurane anesthesia). Furthermore, as compared with the control mice, the anesthetized mice did not show significant changes in behavior (e.g. , eating, drinking, general activity, and body weight) after the anesthesia. Mortality rate of mice in these studies was less than 1%. For the intervention studies, ketorolac (1 mg/kg),23one of the nonsteroidal anti-inflammatory drugs, was given to mice 30 min by means of intraperitoneal injection before each of the 3-day sevoflurane anesthesia sessions.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 P30 or P84 mice were tested in the Morris water maze (MWM) four times per day for 7 days. Each mouse was placed in the pool to search for the platform. The starting points were random for each mouse. Mice that found the platform were allowed to stay on it for 15 s. If a mouse did not find the platform within a 90-s period, it 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 Abcam, Cambridge, MA) was used to recognize IL-6 (24 kDa). TNF-α antibody (1:1,000 dilution; Abcam) was used to recognize TNF-α (17 kDa). Western blot quantification was performed as described by Xie et al. 25Briefly, signal intensity was analyzed using the image analysis program Quantity One (Bio-Rad Laboratories, Hercules, CA). We quantified the Western blots in two steps. First, we used β-actin levels to normalize protein levels (e.g. , determining the ratio of IL-6 to β-actin amount) and control for loading differences in the total protein amount. Second, we presented protein level changes in mice undergoing 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.Mice were anesthetized with isoflurane briefly (1.4% isoflurane for 5 min) and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The anesthesia with 1.4% isoflurane for 5 min in mice provided adequate anesthesia for the perfusion procedure without causing significant changes in blood pressure and blood gas according to our previous studies.25Mouse 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. The sections were incubated with IBA1 antibody (ab5076, 1:100; Abcam) and secondary antibody (Alexa 568, 1:500; Invitrogen, Carlsbad, CA). Finally, the sections were analyzed in mounting medium under a confocal microscope with a 20× objective lens and photographs of the sections were taken. An investigator who was blind to the experimental design counted the number of IBA1-positive cells using ImageJ version 1.38 (National Institutes of Health, Bethesda, MD).The EE in the current experiment was performed as described in previous studies with modifications.26–28Specifically, an EE was created in a large cage (70 × 70 × 46 cm) that included five or six toys (e.g. , wheels, ladders, and small mazes). The mice were put in the EE every day for 2 h from P8–P30. The objects were changed two to three times per week to provide a novel and challenging environment.Data regarding biochemistry changes were expressed as mean ± SD. Data in changes of escape latency were expressed as mean ± SEM. The data for platform crossing time were not distributed normally and thus are expressed as median and interquartile range. The number of samples varied from 6–16, and the samples were distributed normally with the exception of platform crossing time (tested by normality test, data not shown). 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. The post hoc Bonferroni test was used to compare the difference in escape latency between the control and anesthesia groups in each day of the MWM. The Mann–Whitney U test was used to determine the difference between the sevoflurane and control conditions on platform crossing times. There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. Finally, a Student two-sample t test was used to determine the difference between the anesthesia and control groups in levels of IL-6, TNF-α, and IBA1 positive cells. 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.Given the that three not to anesthesia and surgery increased the risk of cognitive impairment in have an animal model in which mice were treated with sevoflurane for 2 h for 1 or days. animal model the single multiple exposures of anesthesia and to the anesthesia-induced mice were treated with 3% sevoflurane anesthesia for 2 h for from The mice were tested in the MWM from A comparison of the time that each mouse to reach the platform during reference training (escape latency) that there was a statistically significant of time and group on escape latency in the MWM between mice the control and mice the sevoflurane anesthesia two-way ANOVA with repeated The post hoc Bonferroni test that the mice that received the sevoflurane anesthesia had a escape latency than the mice the control on and A comparison of the number of times that each mouse the of the platform at the end of reference training (platform crossing times) that the sevoflurane anesthesia interquartile the platform crossing times as compared with the control interquartile P Mann–Whitney U There was no significant difference in mouse swimming between the mice in the sevoflurane anesthesia group and the mice in the control group (data not shown). These data suggest that multiple exposures of sevoflurane in young mice may cognitive impairment in the mice 1 after the findings that multiple exposures to sevoflurane in young mice cognitive we the cytokines, particularly IL-6 and TNF-α, are with cognitive therefore assessed the effects of the sevoflurane anesthesia on the levels of IL-6 and TNF-α in brain tissues of the mice. The brain tissues of the mice were harvested at the end of the anesthesia and to Western blot analysis to determine levels of IL-6 and of IL-6 that the sevoflurane anesthesia to a IL-6 as compared with the control There was no significant difference in β-actin levels between the mice that received the sevoflurane anesthesia and the mice with the control The quantification of the Western blot that the sevoflurane anesthesia increased IL-6 levels in brain tissues of the mice ± vs. ± P of TNF-α that the sevoflurane anesthesia a TNF-α as compared with the control group There was no significant difference in β-actin levels between the mice that received the sevoflurane anesthesia and the mice with the control The quantification of the Western blot that the sevoflurane anesthesia increased TNF-α levels in the brain tissues of the mice ± vs. ± P these suggest that multiple exposures of sevoflurane in young mice may proinflammatory levels in brain leading to cognitive we whether a single to sevoflurane anesthesia in young mice could cognitive impairment and of brain IL-6 and TNF-α in mice. The mice were treated with 3% sevoflurane anesthesia for 2 h at The mice were tested in the MWM from there was a statistically significant of time and group on escape latency in the MWM between the mice in the control group and the mice in the sevoflurane anesthesia the mice that received sevoflurane anesthesia had than escape latency than the mice with the control on There was no significant difference in the platform crossing times between the mice in the control group and the mice in the sevoflurane anesthesia group Western blot analysis that the anesthesia with 3% sevoflurane for 2 h for 1 day at did not levels of IL-6 or TNF-α in the brain tissues of the mice. These data suggest that a single of sevoflurane anesthesia in young mice may not or cognitive impairment in the has been that sevoflurane can and desflurane we assessed the effects of multiple exposures of 9% desflurane to 3% on the function of learning and memory and brain levels of proinflammatory cytokines in mice. The mice were treated with 9% desflurane anesthesia for 2 h for from The mice were tested in the MWM from MWM studies that the desflurane anesthesia did not escape latency and did not platform crossing times as compared with the control group the desflurane anesthesia did not IL-6 or TNF-α levels in the brain tissues of the mice. These data suggest that desflurane may not lead to cognitive impairment and in the developing is a of vulnerability for the developing brain, as the brain period, in to and in to this period, the brain is susceptible to developing older children may have less risk of developing the cognitive impairment anesthesia and therefore whether the multiple exposures of sevoflurane anesthesia could cognitive impairment and in mice. The mice were treated with 3% sevoflurane for 2 h for at The mice were tested in the MWM 1 after anesthesia MWM studies that the sevoflurane anesthesia did not escape latency and did not platform crossing times in the mice. the sevoflurane anesthesia did not IL-6 or TNF-α levels in the brain tissues of the an in proinflammatory cytokines and microglia therefore assessed and compared the effects of the multiple exposures of sevoflurane on microglia activation in the between young and mice. image analysis that the anesthesia with 3% sevoflurane for 2 h for increased the of IBA1-positive the marker of microglia compared with the control group in the of young not mice of the that the sevoflurane anesthesia increased the of IBA1-positive cells in the of young ± vs. ± P not mice ± vs. ± P not ANOVA that there was a significant and that young age the sevoflurane increases in the of IBA1-positive cells these data suggest that multiple exposures of sevoflurane may which increases in the levels of proinflammatory cytokines and microglia activation, leading to cognitive impairment in young mice developing not mice the increases in the of IBA1-positive cells at 1 after anesthesia in the of young mice at data not shown). these data suggest that the may leading to cognitive has been shown to improve brain therefore whether EE could the cognitive impairment in young mice. The mice were treated with 3% sevoflurane for 2 h for the mice were to either EE or environment from at Finally, the mice were tested in the MWM from ANOVA with repeated that there was a statistically significant of time and group on escape latency of MWM between mice the sevoflurane anesthesia plus and those the sevoflurane anesthesia plus EE The post hoc Bonferroni test that the mice that received sevoflurane plus EE had escape latency as compared with the mice that received sevoflurane plus at and There was no significant difference in platform crossing times between the mice that received sevoflurane plus and the mice that received sevoflurane plus EE ANOVA with repeated that there was no statistically significant of time and group on escape latency of MWM between mice the control plus and those the control plus EE not There was no significant difference in platform crossing times between the mice the control plus and the mice the control plus EE Finally, EE did not the of IBA1-positive cells in the mouse (data not shown). These suggest that EE may the cognitive impairment in young mice, which is by the from a that a EE after anesthesia) can attenuate the learning and memory impairment in tested at after the nonsteroidal anti-inflammatory ketorolac the cognitive impairment Specifically, the sevoflurane anesthesia did not the escape latency of the MWM and did not platform crossing times as compared with control in the mice with These data suggest the of the and cognitive is the used anesthetic in We found that anesthesia with 3% sevoflurane for 2 h for in young mice cognitive impairment at a time we found that anesthesia with 3% sevoflurane for 2 h for 1 day in young mice did not cognitive impairment These suggest the of anesthesia-induced cognitive impairment such that anesthesia (e.g. , multiple may These findings the that three not to anesthesia and surgery increased the risk of cognitive impairment in suggest that anesthesia may to the cognitive impairment in children anesthesia and anesthesia with 9% desflurane with 3% for 2 h for in young mice did not cognitive impairment in the mice These findings may suggest that desflurane could a of insult in the developing brain, which studies to determine whether desflurane is a anesthetic for The findings have a system to the difference between sevoflurane and desflurane in brain in the to in and to in susceptible to including those by therefore compared the effects of the sevoflurane anesthesia on cognitive function between young and mice, and found that the anesthesia with 3% sevoflurane for 2 h for did not lead to cognitive impairment in mice. These findings are by the from the studies that early to may lead to an increased risk of developing cognitive impairment in from the animal studies that isoflurane anesthesia cognitive impairment in young including proinflammatory and microglia activation, is with cognitive impairment in and cytokines, particularly TNF-α, IL-6, and can be released by the microglia during its activation, fueling brain inflammation and leading to cognitive in cytokines can microglia activation in brain leading to the of the proinflammatory proinflammatory cytokines found that anesthesia with 3% sevoflurane for 2 h for in young mice was to the levels of proinflammatory IL-6 and TNF-α in brain tissues and the of IBA1-positive the marker of microglia activation, in the of the mice at The anesthesia with 3% sevoflurane for 2 h for 1 day in mice and 3% sevoflurane for 2 h for in mice 4) did not levels of IL-6, TNF-α, or IBA1 positive cells the brain tissues of the mice. these data suggest that the multiple exposures of sevoflurane anesthesia in young mice may cause cognitive impairment The findings that the sevoflurane anesthesia increased the of IBA1-positive cells in the of young mice not mice suggest that mice at different may have different to by which anesthesia to be have been shown to of is with increased levels of proinflammatory activation of we that anesthetics can levels to of TNF-α and IL-6 and the cytokines microglia activation to proinflammatory cytokines, leading to 9% desflurane for 2 h for in mice did not the levels of IL-6 and TNF-α in the brain tissues These different effects between sevoflurane and desflurane are with the previous findings that sevoflurane can and desflurane by which sevoflurane and desflurane have different effects on and cognitive impairment to be studies suggest that sevoflurane may the Massachusetts General and Medical in studies, we may compare the effects of sevoflurane and desflurane anesthesia on the to determine the of different effects of sevoflurane and desflurane on and cognitive there was no significant in the of IBA1-positive cells in the mouse at P30 (data not the time the mice cognitive impairment These suggest that the may not to cause the cognitive impairment. the findings suggest that there be (e.g. , between the and the cognitive impairment. studies to test this hypothesis are the current studies, we have not found that EE can the of IBA1-positive cells in the mouse it is that EE the cognitive impairment the EE may improve brain function (e.g. , of and current findings that EE can the cognitive impairment suggest that the sevoflurane anesthesia could and leading to cognitive impairment. These findings have a system for to studies to determine the (e.g. , and of the cognitive impairment. studies, these findings may that susceptible children have exposures to EE after anesthesia and that can the anesthesia-induced in the developing is therefore that surgery and factors may the anesthesia-induced in mouse developing brain and cognitive impairment. studies suggest that there is no significant difference in the of cognitive between surgery with general anesthesia and surgery without it or in et al. and et al. it is to both and animal studies to determine the of anesthesia and surgery on and cognitive has First, we did not the effects of anesthesia on of cognitive impairment (e.g. , and on learning and memory function it is the major of cognitive impairment. the current data have suggested that multiple exposures to sevoflurane anesthesia in young mice may cause and cognitive which to the of the anesthesia-induced cognitive impairment and of the anesthesia-induced Second, we assessed the effects of anesthesia in mice. is whether anesthesia-induced and cognitive impairment can in young mice and the developing brain at studies an of the effects of different and anesthesia regimens on varied of young mice (e.g. , and to test the hypothesis that the developing brain is susceptible to anesthesia it is whether the of 3% sevoflurane is to that of 9% desflurane in mice. We 3% sevoflurane and 9% desflurane in the current the of sevoflurane and desflurane in children is and brain and blood cytokines could not be at the time the were the desflurane anesthesia and anesthesia with sevoflurane for 1 day increased brain levels of cytokines cognitive the anesthesia with sevoflurane for increased the number of IBA1-positive cells in mouse Finally, anti-inflammatory with ketorolac the cognitive impairment in the mice. these findings the that at to the cognitive we have found that the of anesthetics and anesthesia regimens are with and The findings suggest the combination of an environmental insult , factors such as multiple exposures to a with age vulnerability , factors such as young a in cognitive impairment. Finally, EE and could be used to and anesthesia-induced cognitive impairment. These findings studies to anesthesia in the developing brain, leading to anesthesia care and for children who could be to brain