new_pdfs/10.1097_ALN.0b013e3181974fa2.txt

Anesthesiology 2009; 110:628 –37

Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.

Neonatal Exposure to Sevoflurane Induces Abnormal Social Behaviors and Deficits in Fear Conditioning in Mice Maiko Satomoto, M.D.,* Yasushi Satoh, Ph.D.,† Katsuo Terui, M.D., Ph.D.,‡ Hideki Miyao, M.D., Ph.D.,§ Kunio Takishima, Ph.D.,(cid:1) Masataka Ito, M.D., Ph.D.,# Junko Imaki, M.D., Ph.D.**

Background: Neonatal exposure to anesthetics that block N- methyl-D-aspartate receptors and/or hyperactivate (cid:1)-aminobu- tyric acid type A receptor has been shown to cause neuronal degeneration in the developing brain, leading to functional deficits later in adulthood. The authors investigated whether exposure of neonatal mice to inhaled sevoflurane causes defi- cits in social behavior as well as learning disabilities.

Methods: Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 6 h. Activated cleaved caspase-3 immunohisto- chemical staining was used for detection of apoptosis. Cognitive functions were tested by pavlovian conditioned fear test. Social behavior was tested by social recognition and interaction tests. Results: Neonatal exposure to sevoflurane significantly in- creased the number of apoptotic cells in the brain immediately after anesthesia. It caused persistent learning deficits later in adulthood as evidenced by decreased freezing response in both contextual and cued fear conditioning. The social recognition test demonstrated that mice with neonatal exposure to sevoflu- rane did not develop social memory. Furthermore, these mice showed decreased interactions with a social target compared with controls in the social interaction test, indicating a social interaction deficit. The authors did not attribute these abnor- malities in social behavior to impairments of general interest in novelty or olfactory sensation, because they did not detect sig- nificant differences in the test for novel inanimate object inter- action or for olfaction.

Conclusions: This study shows that exposure of neonatal mice to inhaled sevoflurane could cause not only learning def- icits but also abnormal social behaviors resembling autism spectrum disorder.

MANY pregnant women, newborns, and infants are ex- posed to a variety of anesthetic agents to prevent pain during childbirth or for surgical procedures. Anesthetic agents sometimes have to be administered during an important period of brain growth, the brain growth spurt period, which occurs from the last 3 months of pregnancy until approximately 2 yr after birth (in hu- mans) or during the first 2 weeks after birth (in mice and rats).1– 4 To minimize risks to the fetus or neonates, it is necessary to study the effect of anesthetics not only in

terms of teratogenicity, but also on the developing ner- vous system.

Recently, it has been demonstrated that neonatal ad- ministration of anesthetics induced widespread neuro- degeneration and severe deficits in spatial learning tasks in rodents.5,6 Jevtovic-Todorovic et al.5 reported that neonatal exposure to a cocktail of anesthetics that are commonly used in pediatric surgery induced brain cell death 15 times more frequently than in control rat brains, and that these animals developed learning prob- lems later in adulthood. Fredriksson et al.6 reported that coadministration of an N-methyl-D-aspartate (NMDA) receptor antagonist with (cid:1)-aminobutyric acid type A (GABAA) receptor agonists synergistically potentiated neonatal brain cell death and resulted in functional def- icits in adult mice, although the underlying mechanism is not fully understood. The most thoroughly investi- gated drug that has NMDA antagonist and GABAA agonist property is ethanol, which induces fetal alcohol syn- drome if the fetus is exposed during the brain growth spurt.3,7 Although detrimental effects of anesthetics on cognitive function have been reported, to our knowl- edge, few studies have investigated the effects of anes- thetics on social behavior. Therefore, we designed the current study to investigate the potential risks of neona- tal exposure to anesthetics to cause social abnormalities. Sevoflurane (2,2,2-trifluoro-1-[trifluoromethyl]ethyl flu- oromethyl ether) is one of the most frequently used volatile anesthetics for induction and maintenance of general anesthesia during surgery and cesarean delivery because of its low blood gas partition coefficient and low pungency. It is especially useful for infants and children because of its properties of rapid induction and recovery together with less irritation to the airway.8 Sevoflurane has been shown to enhance GABAA receptors9 and block NMDA receptors, although more research is necessary to better characterize its effects on NMDA receptors.10 In this investigation, we studied the potential risks of neo- natal exposure to sevoflurane to cause social abnormal- ities and cognitive deficits in mice.

Postgraduate Student, # Associate Professor, ** Professor, Department of Developmental Anatomy and Regenerative Biology, † Assistant Professor, (cid:1) Pro- fessor, Department of Biochemistry, National Defense Medical College. ‡ Asso- ciate Professor, Department of Obstetric Anesthesia, § Professor and Chairman, Department of Anesthesiology, Saitama Medical Center, Saitama Medical Univer- sity, Kawagoe, Saitama, Japan. Received from the Department of Developmental Anatomy and Regenerative Biology, National Defense Medical College, Tokorozawa, Saitama, Japan. Submit- ted for publication May 24, 2008. Accepted for publication November 10, 2008. Supported in part by the Ministry of Defense of Japan, Tokyo, Japan (Drs. Satoh and Imaki). There are no financial relationships between any of the authors and any commercial organization with a vested interest in the outcome of the study.

Address correspondence to Dr. Satoh: Department of Biochemistry, National Defense Medical College, 3-2 Namiki, Tokorozawa 359-8513, Japan. ys@ ndmc.ac.jp. Information on purchasing reprints may be found at www.anesthesiology. org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.

Materials and Methods

The experiments were approved by the Committee for Animal Research at National Defense Medical College (Tokorozawa, Saitama, Japan). Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan). The animals were illuminated with a 12-h light– dark cycle (light from 07:00 to 19:00), and room tem-

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perature was maintained at 21° (cid:1) 1°C. At the age of 3 weeks, the mice were weaned and housed in groups of 4 animals in a room. Mice had ad libitum access to water and food.

Previous studies reported that there is litter variability in the rate of apoptosis that occurs spontaneously in neonate mice.11 Therefore, a balanced number of con- trol and experimental animals were drawn from the same litters, so that each experimental condition had its own group of littermate controls. Only the male off- spring were used in this study. A total of 51 litters, 101 control and 103 treated pups, were used in this study.

Anesthesia Treatment Postnatal day 6 (P6) male mice were placed in an acrylic box and exposed to 3% sevoflurane or no anes- thetics for 6 h. The total gas flow was 2 l/min, using air as a carrier. During anesthetic exposure, the mice were kept warm on a plate heated to 38°C. Control and ex- perimental animals were under the same treatment and environment except that the control animals were ex- posed only to air.

Arterial Blood Gas Analysis Arterial blood analysis was performed essentially as described previously.5,12 Briefly, the pups underwent a quick arterial blood sampling from the left cardiac ven- tricle, and the samples were transferred into heparinized glass capillary tubes. A single sample (55 (cid:2)l) was ana- lyzed immediately after blood collection by blood gas analyzer (ABL800; Radiometer, Copenhagen, Denmark). Samples were obtained immediately after removal from the maternal cage (0 h) or at the end of anesthesia (6 h). At the time of blood sampling, the experiments were terminated by decapitation.

Histopathologic Studies Animals from both treatment and control groups were perfused transcardially with 0.1 M phosphate buffer con- taining 4% paraformaldehyde immediately after 6 h of sevoflurane anesthesia, and then the brains were ex- posed to immersion fixation for 24 h at 4°C. The brains were histologically analyzed using paraffin-embedded sections (5 (cid:2)m thick). For immunohistochemistry, anti– active caspase-3 antiserum (D175; Cell Signaling Tech- nology, Beverly, MA) was used at dilutions of 1:400 in antibody diluent (Dako, Glostrup, Denmark). Before to use, sections were dewaxed in xylene and hydrated using a graded series of ethanol. Antigenic retrieval was performed by immersing mounted tissue sections in 0.01 mM sodium citrate (pH 6.0) and heating in an autoclave (121°C) for 5 min. Deparaffinized sections were blocked for endogenous peroxidase activity as described previ- ously,13 followed by blocking with a nonspecific staining blocking reagent (Dako) for 1 h to reduce background staining. The sections were then incubated overnight in a humidified chamber at 4°C. Subsequently, peroxidase- conjugated secondary antibody (DAKO En Vision (cid:2) sys- tem; Dako) and 3,3-diaminobenzine-tetrachloride (DAB; Vector Laboratories, Burlingame, CA) were used accord- ing to the manufacturer’s instructions. Finally, the sec- tions were counterstained with Nissl. Activated caspase- 3–positive cells were counted by the investigator who was blinded to the treatment conditions.

transferase–mediated de- oxyuridine 5-triphosphate– biotin nick end labeling stain- ing was performed using an in situ apoptosis detection kit (ApopTag fluorescein; CHEMICON, Temecula, CA) accord- ing to the manufacturer’s protocol. Sections were counter- stained with 4=,6-diamidino-2-phenylindole (DAPI). Fluores- cein was histochemically examined with a fluorescent microscope (TE-2000E; Nikon, Tokyo, Japan) equipped with interlined charge-coupled device camera (DS-U1; Nikon).

Terminal deoxynucleotidyl

Laser Color Doppler Cerebral blood flow (CBF) was measured by a laser- Doppler blood perfusion imager (Peri Scan PIM II; Per- imed, Stockholm, Sweden). Mice were taken out of the chamber before and every hour during anesthetic treat- ment and were placed face down on the floor while being continuously exposed to sevoflurane via a tube with its opening positioned at the nose of the animals. Their head skins were peeled for scanning CBF, and data were captured using appropriate software (LDPIwin ver- sion 2.6; Lisca, Linko¨ping, Sweden). The perfusion re- sponse is presented in arbitrary perfusion units. Because the arbitrary perfusion units values are not absolute blood flow, the magnitude of the difference in perfusion was calculated as the ratio between the area of maxi- mum peak perfusion and areas of baseline perfusion. Arbitrary perfusion unit values were compared between anesthetized animals and those with mock anesthesia at baseline and at 1-h intervals for 6 h.

Preparation of Protein Extracts Mice forebrain was quickly removed and were homog- enized in four volumes of 20 mM Tris-HCl, pH 7.4, 2 mM EDTA, protease inhibitor cocktail (Complete, Roche Di- agnostics, Penzberg, Germany), and phosphatase inhib- itors (20 mM glycerophosphate, 1 mM Na3VO4, 2 mM NaF). After homogenization, a portion of each sample was immediately frozen at (cid:3)80°C. The rest of the ho- mogenate was centrifuged at 15,000g for 30 min at 4°C. The supernatant solutions were separated and stored at (cid:3)80°C until use. The amount of protein in each sample was measured using a protein assay kit (BCA; Pierce, Rockford, IL).

Western Blot Analysis The homogenate proteins were subjected to sodium do- decyl sulfate polyacrylamide gel electrophoresis. The pro-

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teins were transferred onto polyvinylidene fluoride mem- branes (Immobilon-P; Millipore, Bedford, MA). The blots were immunoreacted with anti– cleaved poly(adenosine diphosphate–ribose) polymerase (PARP; 1:1,000, rabbit polyclonal, Asp214; Cell Signaling) or anti–(cid:3)-actin (1:5,000, mouse monoclonal, AC-15; Sigma, St. Louis, MO) antibod- ies, and the protein bands were visualized by chemilumi- nescence detection system (SuperSignal West Pico; Pierce).

Behavioral Studies As described previously for CBF and histopathologic stud- ies, some sets of mice for behavioral studies were exposed to 3% or 0% sevoflurane for 6 h at P6. They were allowed to mature, and at the appropriate ages, sevoflurane and control mice underwent behavioral tests, namely, open- field, elevated plus-maze, Y-maze, fear conditioning, social recognition, social interaction, olfactory, and novelty tests. The movement of each mouse was monitored and analyzed using a computer-operated video tracking system (SMART, Barcelona, Spain). In the tasks using apparatus with arms, arm entry was counted when all four legs of the animal entered each arm. The apparatus was cleaned after each trial. All apparatus used in this study were made by O’Hara & Co., Ltd. (Tokyo, Japan).

Open-field Test. Emotional responses to a novel en- vironment were measured by an open-field test using 8-week-old mice, by a previously described method.14 Activity was measured as the total distance traveled (meters) in 10 min.

Elevated Plus-maze Test. The elevated plus-maze test was performed as previously described.14 The elevated plus maze consisted of two open arms (25 (cid:4) 5 cm) and two enclosed arms being elevated to a height of 50 cm above the floor. Normally, mice prefer a closed environ- ment to an open area. Mouse behavior was recorded during a 10-min test period. The percentage of time spent in the open arms was used as an index of anxiety- like behavior. Mice used for the test were aged 8 weeks. Spontaneous Alternation in the Y-maze Test. This study was performed as previously described.14 This study allowed us to assess spatial working memory. The symmetrical Y maze made of acrylic consists of three arms (25 (cid:4) 5 cm) separated by 120° with 15-cm-high transparent walls. Each mouse was placed in the center of the Y maze, and the mouse was allowed to freely explore the maze for 8 min. The sequence and the total number of arms entered were recorded. The percentage of alternation is the number of triads containing entries into all three arms divided by the maximum possible number of alternations (total number of arm entries minus 2) (cid:4) 100. Mice used for the test were aged 11 weeks. The motion of the animals was manually recorded.

Fear Conditioning Test. This is a simple and sensi- tive test of hippocampal-dependent and hippocampal- independent learning as previously described.14 Briefly,

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the conditioning trial for contextual and cued fear con- ditioning consisted of a 5-min exploration period fol- lowed by three conditioned stimulus– unconditioned stimulus pairings separated by 1 min each: uncondi- tioned stimulus, 1 mA foot shock intensity, 1 s duration; conditioned stimulus, 80 db white noise, 20 s duration; unconditioned stimulus was delivered during the last seconds of conditioned stimulus presentation. A contex- tual test was performed in the conditioning chamber for 5 min in the absence of white noise at 24 h after condi- tioning. A cued test (for the same set of mice) was performed by presentation of a cue (80 db white noise, 3 min duration) in alternative context with distinct visual and tactile cues. The rate of freezing response (absence of movement in any parts of the body during 1 s) was scored automatically and used to measure fear memory. The test was performed on mice of two different age groups: 8 weeks or between 14 and 17 weeks.

Social Recognition Test. Social recognition test was conducted as described previously.15 We transferred 18- week-old mice from group to individual housing for 7 days before testing to permit establishment of a home cage territory. Testing began when a stimulus female mouse was introduced into the home cage of each male mouse for 1-min confrontation. At the end of the 1-min trial, the stimulus animal was removed and returned to an individual cage. This sequence was repeated for four trials with 10-min intertrial intervals, and each stimulus was introduced to the same male resident in all four trials. In a fifth trial, another stimulus mouse was intro- duced to a resident male mouse.

Social Interaction Test. Caged social interaction for social versus inanimate targets was performed in an open field using two cylinder cages allowing olfactory and minimal tactile interaction as described previo- usly.16 The cylinder cages were 10 cm in height, with a bottom diameter of 9 cm and bars spaced 7 mm apart. Olfactory Test. Fifteen-week-old mice were habitu- ated to the flavor of a novel food (blueberry cheese) for 3 days before testing. On the fourth day, after 24 h of food deprivation, a piece of blueberry cheese was buried under 2 cm of bedding in a clean cage. The mice were placed in the cage, and the time required to find the food was measured manually.

Novelty Test. Activity was measured as the total du- ration of interaction with an inanimate novel object (red tube) in 10 min.

The same set of mice underwent social recognition (at 19 weeks of age), social interaction (at 14 weeks of age), olfactory (at 15 weeks of age), and novelty (at 15 weeks of age) tests. In other analyses, each test was conducted with a new set of animals.

Statistical Analysis Statistical analysis was performed using Statview soft- ware (SAS, Cary, NC). Comparisons of the means of two

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Table 1. Arterial Blood Gas Analysis

Arterial Blood Gas

Time, h

n

pH

PacO2, mmHg

PaO2, mmHg

SaO2, %

Sham operation

3% Sevoflurane

0 6 0 6

8 8 8 8

7.40 (cid:1) 0.09 7.38 (cid:1) 0.04 7.32 (cid:1) 0.08 7.46 (cid:1) 0.06

26.0 (cid:1) 4.6 29.4 (cid:1) 6.2 26.9 (cid:1) 4.6 27.5 (cid:1) 6.9

100.2 (cid:1) 5.7 95.6 (cid:1) 10.0 98.5 (cid:1) 6.9 80.9 (cid:1) 4.8

95.8 (cid:1) 1.1 96.1 (cid:1) 1.0 95.4 (cid:1) 1.1 95.4 (cid:1) 0.8

Neonatal exposure to sevoflurane does not induce significant cardiorespiratory dysfunction. Analysis of arterial blood gas revealed no significant differences in any of the measured parameters between mice exposed for 6 h to sevoflurane and control (sham operation) exposed to air for 6 h (t test, all P values (cid:5) 0.05). PaCO2

(cid:6) arterial carbon dioxide tension; PaO2

(cid:6) arterial oxygen tension; SaO2

(cid:6) arterial oxygen saturation.

groups were performed using the Student t test. In the Y-maze task, comparisons of group performance relative to random levels were performed using the one-sample t test. Data of the social recognition task were analyzed by repeated-measures two-way analysis of variance. Values are presented as mean (cid:1) SEM.

Results

Neonatal Exposure to Sevoflurane Did Not Induce Significant Disturbance in Ventilation, Oxygenation, or CBF To examine the effect of neonatal exposure to sevoflu- rane, we exposed P6 mice to 3% sevoflurane for 6 h. Hypoxia is a known cause of neuronal cell death.17 To assess the adequacy of ventilation and oxygenation, we examined the blood gas data in mice during the anesthe- sia. Control samples were obtained from pups exposed to air during the same period. We found that pH, arterial carbon dioxide tension, arterial oxygen tension, and arterial oxygen saturation did not differ significantly from sham control (table 1). These results, together with

the fact that pups looked pink throughout the 6 h of gas exposure, led us to conclude that it was unlikely that apoptosis in this protocol was caused by hypoxia/ hypoventilation.

Further, to assess the adequacy of cerebral perfu- sion, we measured CBF during anesthesia using a laser-Doppler blood perfusion imager. Control mice were exposed to air for corresponding period of the sevoflurane treated mice. Anesthesia treatment with sevoflurane did not affect CBF compared with control mice at any point during the 6 h of anesthesia (figs. 1A and B).

Neonatal Exposure to Sevoflurane Induced Extensive Apoptotic Neurodegeneration Sevoflurane anesthesia significantly increased cleaved caspase-3 apoptosis in the mice immediately after expo- sure (table 2 and figs. 2– 4). Figures 2B and D showed that the increased apoptosis was most robust in the caudate/putamen, retrosplenial cortex, dorsal hip- pocampal commissure, and neocortex in the brains of pups with sevoflurane exposure. In other sections, thal-

A

Fig. 1. Neonatal exposure to sevoflurane (Sevo) did not induce hypoperfusion of the brain. (A) Representative images of laser color Doppler for cerebral blood flow in a control mouse (upper panel) and in a sevoflurane exposed mouse (lower panel). The degree of perfusion is shown by the color code, with red repre- senting high perfusion and blue repre- senting lower perfusion. No different pat- terns of cerebral blood flow were observed between control and anesthe- tized mice throughout the 6-h exposure period. (B) Time course of cerebral blood flow measured by laser color Doppler during 6 h of 3% sevoflurane administra- tion. The degree of perfusion is pre- sented in arbitrary perfusion units. There was no difference between sevoflurane and control groups during the 6-h period (control, n (cid:2) 3; 3% sevoflurane, n (cid:2) 4). Scale bar: 5 mm.

B

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Table 2. Brain Regions in Which Sevoflurane-induced Neurodegeneration Was Heavily Concentrated

Brain Region

Severity, Fold Increase

CA1 (hippocampus) CA3 (hippocampus) Dentate gyrus Dorsal hippocampal commissure Frontal cortex Temporal cortex Amygdala Caudate/putamen Mammillary complex Retrosplenial cortex Subiculum Pontine nuclei Inferior colliculus Thalamus

4.8 29.7 32.5 65.2 66.7 148.0 51.0 47.7 21.1 166.5 121.5 38.8 32.1 376.7

Severity of damage is expressed as the fold increase (i.e., how many times greater) in the density of degenerating neurons labeled by activated cleaved caspase-3 immunohistochemical staining in the sevoflurane-treated brain (n (cid:6) 6 mice) compared with the rate of degeneration in the same region of control brain (n (cid:6) 6 mice).

amus, subiculum, inferior colliculus, and pontine nuclei were also shown to be damaged severely (figs. 3B and C). Figure 4B indicated that severe neuronal damage occurred in the extrahippocampal circuit, which is be- lieved to be important for mediating learning and mem- ory functions,18 including dorsal hippocampal commis- sure as well as thalamus and retrosplenial cortex. Apoptosis were also observed in amygdala (fig. 4D), hippocampus (fig. 4F), frontal cortex (fig. 4H), and mam- millary complex (fig. 4J). These data indicated that the apoptotic response to sevoflurane was robust and fol- lowed a pattern that was characteristic of the pattern reported for other anesthetic drugs or ethanol.18

It was reported that caspase-3 cleavage may occur under a condition that is a nonapoptotic event,19 raising questions about the reliability of cleaved caspase-3 im- munostaining for detecting cell death. Therefore, to ver- ify that the cleaved caspase-3 immunoreactivity repre- sent authentic apoptosis, we also performed terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate– biotin nick end labeling as another inde- pendent measure of apoptotic cell death. We found the same pattern of staining as observed by cleaved caspase-3 staining (figs. 4L, N, P, and R).

Further, to verify that the previously described immu- nohistochemically detectable reactivity represented au- thentic apoptosis and to quantify the apoptosis re- sponse, we examined cortex extracts from control and sevoflurane-treated pups by Western blot analysis using antibody specific for cleaved PARP. PARP is one of the main cleavage targets of caspase-3 in vivo, and the cleav- age is readily detected in many apoptosis model.20 West- ern immunoblotting with anticleaved PARP antibody de- tected immunoreactivity in sevoflurane-exposed pup

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Fig. 2. The apoptotic response to sevoflurane (Sevo) was robust in pup brain. Light microscopic views of the mouse brain after exposure to room air for 6 h (A and C) and to 3% sevoflurane for 6 h (B and D). Sections were immunochemically stained to reveal caspase-3 activation (A–D). Black dots represent caspase- 3–positive cells, which indicate apoptosis. A substantially higher density of cleaved caspase-3–positive profile is present in sevoflurane-treated brain. cp (cid:2) caudate/putamen; dhc (cid:2) dorsal hippocampal commissure; rs (cid:2) retrosplenial cortex. Scale bars: 1 mm.

extracts, whereas the band was under detection level in control pub brain extracts (fig. 4S).

General Behavior Was Normal in Mice with Neonatal Exposure to Sevoflurane To examine responses to a novel environment, mice with neonatal exposure to sevoflurane were assayed in an open-field test. These mice did not differ from control animals in their exploratory behavior (fig. 5A; t test, t (cid:6) 1.24, P (cid:5) 0.05). To study whether anxiety-related behav- ior of mice with neonatal exposure to sevoflurane was affected, mice underwent an elevated plus-maze test. Anxiety-related behavior was assessed by the percentage of time spent in the open arms of the test equipment. Anesthetized mice did not differ significantly in the per- centage of time spent in the open arms (fig. 5B; t test, t (cid:6) 0.76, P (cid:5) 0.05). These results indicate that the emotional state of mice with neonatal exposure to sevoflurane did not differ grossly from controls under the conditions of this study.

Further, to examine whether exposure of the develop- ing brain to sevoflurane was associated with changes in

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being no difference compared with control (fig. 5C; t test, t (cid:6) 1.20, P (cid:5) 0.05). This result suggests that spatial working memory was not affected by exposure of the developing brain to sevoflurane.

Neonatal Exposure to Sevoflurane Induced Deficits in Contextual and Cued Fear Conditioning To assess the influence of neonatal exposure to sevoflurane on long-term memory, mice underwent con- textual/cued fear conditioning. In this paradigm, mice learn to associate previous neutral auditory cues and the apparatus (context) with electric foot shock in a single training session, such that robust long-term memory was established for an experimental context (hippocampus dependent) and an auditory cue (hippocampus indepen- dent).21,22 Long-term memory was assessed based on the freezing reaction of the mice in response to the context or the conditioned cue. The freezing response to the same context in mice with neonatal exposure to sevoflu- rane was reduced significantly compared with controls after a 24-h retention delay at 8 weeks of age (fig. 6A; t test, t (cid:6) 3.10, P (cid:7) 0.01). The response of mice with sevoflurane to the cued fear conditioning was also re- duced significantly after a 48-h retention delay compared with control mice (fig. 6B; t test, t (cid:6) 3.16, P (cid:7) 0.01). It was reported that exposure of infant mice to ethanol induced neuroapoptosis and subsequent memory im- pairments that were very severe at P30 and less severe at P75.18 This result provided evidence favoring the inter- pretation that recovery of some type of learning func- tions might occur in later adulthood in ethanol-treated mice. Therefore, further to examine whether the neona- tal exposure to sevoflurane causes permanent neurocog- nitive deficits in mice and how it evolves over time, we undertook an assessment of hippocampal function at later time point using another set of mice.

Fig. 3. Thalamus and other regions were also severely damaged after neonatal exposure to sevoflurane (Sevo). Sagittal (A and B) and coronal (C) views of the mouse brain after exposure to room air for 6 h (A) and to 3% sevoflurane for 6 h (B and C) as described in figure 2. A substantially higher density of cleaved caspase-3–positive profile is present in thalamus (th) as well as subiculum (s), inferior colliculus (ic), and pontine nuclei (pn). rs (cid:2) retrosplenial cortex. Scale bars: 1 mm.

spatial working memory, mice underwent a Y-maze spontaneous alternation task. Working memory refers to a cognitive function that provides concurrent temporary storage and manipulation of the information necessary for complex cognitive tasks. This test examines whether mice remember the position of the arm selected in the preceding choice. Mice with and without sevoflurane exposure performed this task with 64.3 (cid:1) 7.3% and 60.0 (cid:1) 8.3% correct choices, respectively, which were well above the expected results of random choices (ran- dom choice (cid:6) 50%; one-sample t test, P (cid:7) 0.05), there

We found that neonatal exposure to sevoflurane caused deficits in the fear conditioning test at 14 –17 weeks of age similar to the results at 8 weeks. The freezing response of mice with sevoflurane was reduced significantly in contextual tests compared with that of controls after a 24-h retention delay (fig. 6C; t test, t (cid:6) 3.48, P (cid:7) 0.01). The freezing response of sevoflurane- exposed mice to cued fear was also reduced significantly compared with that of controls after a 48-h retention delay at 14 –17 weeks of age (fig. 6D; t test, t (cid:6) 2.11, P (cid:7) 0.05). These results strongly suggested that exposure of P6 mice to sevoflurane caused hippocampal-depen- dent and -independent neurocognitive deficits that per- sisted for relatively long time periods (at least from 8 to 14 –17 weeks of age) of the mice’s lifespan.

Neonatal Exposure to Sevoflurane Induced Abnormal Social Interaction Mice are a social species and exhibit social interaction behavior.23 Therefore, we investigated whether mice

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with neonatal exposure to sevoflurane display abnormal social behaviors. First, we investigated social memory, which depends predominantly on olfactory cues. This ability is needed for social familiarity and can be identi- fied as a consistent decrease in olfactory investigation during repeated encounters with a female in the social recognition test. Control mice showed a significant de- cline in the time spent in investigating a female with subsequent presentation of the same female in trials 3 and 4, as compared with trial 1 (fig. 7A). This decrease was not due to a general decline in olfactory investiga-

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Fig. 4. Inhaled sevoflurane increased ap- optosis in several regions of the brain. Light microscopic views of the mouse brain after exposure to room air for 6 h (A, C, E, G, and I) and to 3% sevoflurane (Sevo) for 6 h (B, D, F, H, and J). Higher density of cleaved caspase-3–positive profile was present in extrahippocampal circuit (A and B), amygdala (C and D), hippocampus (E and F), frontal cortex (G and H), and mammillary complex (I and J). Scale bars: 1 mm in A and B, 50 (cid:3)m in C–J, and 100 (cid:3)m in I and J. (K–R) Termi- nal deoxynucleotidyl transferase–medi- ated deoxyuridine 5-triphosphate– biotin nick end labeling (TUNEL; L, N, P, and R) showed similar pattern of neuroapopto- sis to cleaved caspase-3 staining. Sections stained with 4=,6-dia- were midino-2-phenylindole (DAPI; K, M, O, and Q). Representative images of cortex (K, L, O, and P) and caudate (M, N, Q, and R) are shown. Scale bars: 500 (cid:3)m. (S) Poly(adenosine diphosphate–ribose) poly- merase (PARP) was cleaved after neonatal exposure to sevoflurane. Protein extracts of control (exposure to room air for 6 h) and sevoflurane-exposed cortex were prepared and analyzed for cleaved PARP immunoreac- tivity on Western blot. Representative blot from three independent results (from three pairs of pups) was shown. (cid:4)-Actin reactivity was used as a protein loading control. dhc (cid:2) dorsal hippocampal commissure; rs (cid:2) retro- splenial cortex; th (cid:2) thalamus.

counter

tion, because presentation of a novel female during trial 5 resulted in a similar amount of investigation as trial 1 with the original female. In contrast, mice with neonatal exposure to sevoflurane showed high levels of sustained investigation at each encounter with the same female and the same level of investigation when presented with a new female at trial 5, significantly different from the response of controls (analysis of variance, F (cid:6) 14.51, P (cid:7) 0.001 [between control and sevoflurane administra- tion]; F (cid:6) 28.34, P (cid:7) 0.0001 [between trials]; F (cid:6) 16.64, P (cid:7) 0.0001 [interaction between trials and sevoflurane

Fig. 5. Behavioral effects of neonatal sevoflu- rane exposure were assessed by the open- field test (total distance traveled in 10 min; control, n (cid:2) 10; sevoflurane, n (cid:2) 11; A), ele- vated plus-maze test (percentage of time spent in open arms; control, n (cid:2) 18; sevoflu- rane, n (cid:2) 20; B), and Y-maze test (percentage of correct alternation response; control, n (cid:2) 10; sevoflurane, n (cid:2) 9; C). No significant dif- ferences were observed between mice with neonatal sevoflurane exposure and controls in these tests.

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Fig. 6. Neonatal exposure of mice to sevoflurane (Sevo) induced impaired memory performance in both contextual and cued tests. (A and B) Contextual and cued tests at 8 weeks of age. (A) Freezing response was measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 24 h after conditioning (control, n (cid:2) 8; sevoflurane, n (cid:2) 8). (B) Freezing response (for the same set of mice as in A) was measured in an alternative context without auditory cue (basal freezing after conditioning) or with cue 2 days after conditioning. (C and D) Contextual and cued tests for another set of mice at a different age (14 –17 weeks of age) as in A and B (control, n (cid:2) 9; sevoflurane, n (cid:2) 9). For all figures, asterisks represent statistical difference (* P < 0.05, ** P < 0.01).

administration]; fig. 7A). These data suggest that mice with neonatal exposure to sevoflurane do not develop social memory.

In a test for social versus inanimate preference, control mice spent significantly more time interacting with the social target than with the inanimate target (fig. 7B; t test, t (cid:6) 7.30, P (cid:7) 0.0001). In contrast, mice with neonatal exposure to sevoflurane spent a similar amount of time interacting with both targets (fig. 7B; t test, t (cid:6) 1.77, P (cid:5) 0.05). Furthermore, mice with neonatal exposure to sevoflurane exhibited decreased interaction with a social target compared with controls (fig. 7B; t test, t (cid:6) 2.38, P (cid:7) 0.05), indicating a social interaction deficit. We did not attribute the abnormalities in social recognition and inter- action to impairment in general interest in novelty or olfac- tory sensation, because we did not detect significant differ- ences between groups in tests for novel inanimate object interaction (fig. 7C; t test, t (cid:6) 0.21, P (cid:5) 0.05) or for olfaction (fig. 7D; t test, t (cid:6) 0.12, P (cid:5) 0.05). Therefore, it can be concluded that mice with neonatal exposure to sevoflurane demonstrated deficits in social behavior.

Discussion

This study showed that single administration of sevoflurane to neonatal mice caused a significant in-

Fig. 7. Neonatal exposure to sevoflurane (Sevo) induced abnor- mal social behavior in adulthood. (A) Olfactory investigations in mice with neonatal exposure to sevoflurane were used for social recognition test. Social memory by male mice was mea- sured as the difference in anogenital investigation. Data depict the amount of time allocated to investigating the same female during each of four successive 1-min trials. A fifth trial depicts the response to a new female. Asterisks represent statistical differences (*** P < 0.001) between each trial compared with the first trial (control, n (cid:2) 17; sevoflurane, n (cid:2) 18). (B) When exposed to caged social and inanimate targets (social interac- tion test) in an open field, control mice showed a normal preference for the social target over an inanimate target, whereas the difference of interaction time between both targets was not significant in mice with neonatal exposure to sevoflu- rane. Furthermore, mice with neonatal exposure to sevoflurane spent significantly less time interacting with the social target compared with controls (control, n (cid:2) 17; sevoflurane, n (cid:2) 18). Asterisks represent statistical differences (* P < 0.05, *** P < 0.001). (C) Time spent interacting with a novel inanimate object was not significantly affected by neonatal exposure to sevoflu- rane (control, n (cid:2) 17; sevoflurane, n (cid:2) 18). (D) Mice with neonatal exposure to sevoflurane did not show significant dif- ferences from controls in latency to find a buried treat after overnight food deprivation (control, n (cid:2) 17; sevoflurane, n (cid:2) 18).

crease in neuroapoptosis in the brain compared with littermate controls exposed only to air. Consistent with other recent evidence that apoptotic neurodegeneration can be induced by exposure during the brain growth spurt to drugs that block NMDA receptors and/or hyper- activate GABAA receptors,5,6,18 neonatal exposure to sevoflurane was shown to induce widespread apoptosis in several major brain regions, leading to impaired learn- ing later in adulthood. This finding for sevoflurane is consistent with recent finding by Johnson et al.,24 who found that a neonatal exposure to isoflurane triggers a significant neuroapoptosis response in the mouse brain. Our results of fear conditioning strongly suggested that exposure of P6 mice to sevoflurane caused learning deficits, although we could not rule out the possibility of the effects of sevoflurane on sensitization despite true conditioning to the auditory cue. Furthermore, this

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study also showed that exposure of neonatal mice to inhaled sevoflurane caused deficits in social behavior. To our knowledge, this is the first study to show that single administration of sevoflurane, which is a commonly used anesthetic in pediatric surgery throughout the world, causes a robust neuroapoptosis response in infant mouse brain and behavioral deficits in both cognitive and social spheres. The minimum alveolar anesthetic concentra- tion that prevents purposeful movement in response to supramaximal noxious stimulation in 50% of animals (minimum alveolar concentration) of sevoflurane in hu- man neonates is 3.3 (cid:1) 0.2%.8 Therefore, the concentra- tion of sevoflurane (3%) used in this mice study would be comparable to clinically used ranges.

Most anesthetics act via NMDA and/or GABAA recep- tors. It was demonstrated that neonatal coadministration of an NMDA antagonist and a GABAA agonist was much more detrimental than either of these used alone.5,6 Although the precise mechanism for this is yet to be understood, this evidence suggested that more severe neurodegeneration was induced when both NMDA and GABAA receptors were simultaneously altered in the developing brain. Therefore, an anesthetic that has NMDA antagonist and GABAA agonist properties would be of concern when administered to the developing brain.

Autism spectrum disorders (ASDs) are a group of com- mon neuropsychiatric disorders characterized primarily by impairments in social, communicative, and behavioral functioning,25 of unknown mechanism. Epidemiologic studies have shown that the prevalence of ASDs is 3– 6 per 1,000 children. If neonatal exposure to anesthetics induces deficits in social behavior, a causal link between ASDs and neonatal exposure to anesthetics could be suggested, because deficits in social behaviors are a core feature of ASDs. Our findings revealed that neonatal exposure to sevoflurane induced deficits in social mem- interaction in mice. These tests were ory and social thought to be core paradigms to test autistic behavior in mice and have been used to measure autistic behavior in other ASD models as well.16,17,26,27 This study is the first to indicate the potential risk of general anesthetics to induce disturbances in social behaviors that resembles those seen in ASDs.

However, there is a caveat that the relevance of these mouse findings to the human situation is unknown and requires clarification. It is too early to say whether anes- thetics have the same effect in humans. There may be species differences in the detrimental effects of anes- thetic agents on the developing brain. Physicians could reduce any potential risks by limiting the duration of anesthetic administration in neonates.

In any case, the current results suggest the potential hazards of neonatal sevoflurane exposure in causing so- cial behavioral alterations. We would like to insist on the need for further research to determine whether a corre-

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lation exists between anesthesia exposure during devel- opment and ASDs in human populations. What would be the potential association between the sevoflurane-in- duced apoptosis and sevoflurane-induced changes in so- cial behaviors? Several authors have proposed that glu- tamate and GABAergic system disturbance in cortical network in ASDs may be characterized by an imbalance between excitation and inhibition in neuronal net- works.28,29 Such a change may lead to hyperexcitability or unstable neuronal networks, which may alter oscilla- tory rhythms in brain.30,31 The excitatory/inhibitory bal- ance in cortical networks may be controlled by the relative numbers and activities of glutamatergic and GABAergic neurons.29 Indeed, reduced GABAergic inhi- bition by mutations of genes encoding subunits of the GABAA receptors is associated with ASDs.28,29,32 Neural loss by a drug that may violate NMDA and GABAA recep- tors in the critical period, seen in the current study, might interfere with the developmental mechanisms pat- terning the balance between excitation and inhibition system and cause ASD-like behaviors.

Our results showed that neuronal degeneration was particularly severe in several of the specific brain regions that comprise the extrahippocampal circuit, which is believed to be important for mediating learning and memory functions. This pattern was similar to the pat- tern for neonatal exposure to ethanol, which has NMDA antagonist and GABAA agonist properties.18 These pat- tern of brain damage and subsequent learning deficits are described in ethanol-treated mice.18 In this regard, it is noteworthy that there is evidence that prenatal expo- sure to ethanol may be a factor in social difficulties in humans.33 Further including electrophysi- ologic study, will be needed to address the molecular mechanisms that explain the relevance between neona- tal exposure to sevoflurane and deficits in social behav- ior. It would also be necessary to study whether other drugs that cause neuroapoptosis in the critical period would induce deficits in social behavior.

research,

The authors thank Kyoko Takeuchi, Ph.D. (Assistant Professor), Kenji Miura, Ph.D. (Lecturer), Kazuyo Kuroki (Technician, all from the Department of Devel- opmental Anatomy and Regenerative Biology, National Defense Medical College, Tokorozawa, Saitama, Japan), Kunihito Takahashi, Ph.D., (Senior Researcher, Hakuju Institute for Health Science Co. Ltd., Tokyo, Japan), and Tatsuyo Hara- sawa (Technician, Central Research Laboratory, National Defense Medical Col- lege) for the participation in this study; and Kouichi Fukuda, Ph.D. (Associate Professor, Center for Laboratory Animal Science, National Defense Medical Col- lege), for the assistance in animal administration.

References

1. Bayer SA, Altman J, Russo RJ, Zhang X: Timetables of neurogenesis in the human brain based on experimentally determined patterns in the rat. Neurotoxi- cology 1993; 14:83–144

2. Rice D, Barone S Jr: Critical periods of vulnerability for the developing nervous system: Evidence from humans and animal models. Environ Health Perspect 2000; 108:511–33

3. Olney JW, Tenkova T, Dikranian K, Qin YQ, Labruyere J, Ikonomidou C: Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain. Brain Res Dev Brain Res 2002; 133:115–26

D o w n o a d e d

l

f r o m h

t t

p

: / /

p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d

.

/

i

l

l

f /

1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d

/

/

/

/

f

b y g u e s t

o n 3 0 M a r c h 2 0 2 1

SEVOFLURANE NEUROTOXICITY IN THE DEVELOPING BRAIN

637

4. Dobbing J, Sands J: Comparative aspects of the brain growth spurt. Early

Hum Dev 1979; 3:79–83

5. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–82

6. Fredriksson A, Ponte´n E, Gordh T, Eriksson P: Neonatal exposure to a combination of N-methyl-D-aspartate and (cid:1)-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behav- ioral deficits. ANESTHESIOLOGY 2007; 107:427–36

7. Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Ho¨rster F, Tenkova T, Dikranian K, Olney JW: Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 2000; 287: 1056–60

M, Lang D, Speer A, Olney JW, Ikonomidou C: Apoptotic neurodegeneration following trauma is markedly enhanced in the immature brain. Ann Neurol 1999; 45:724–35

18. Wozniak DF, Hartman RE, Boyle MP, Vogt SK, Brooks AR, Tenkova T, Young C, Olney JW, Muglia LJ: Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juve- niles followed by progressive functional recovery in adults. Neurobiol Dis 2004; 17:403–14

19. Rosado JA, Lopez JJ, Gomez-Arteta E, Redondo PC, Salido GM, Pariente JA: Early caspase-3 activation independent of apoptosis is required for cellular func- tion. J Cell Physiol 2006; 209:142–52

20. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 1994; 371:346–7

8. Lerman J, Sikich N, Kleinman S, Yentis S: The pharmacology of sevoflurane

21. Kim JJ, Fanselow MS: Modality-specific retrograde amnesia of fear. Science

in infants and children. ANESTHESIOLOGY 1994; 80:814–24

1992; 256:675–7

9. Nishikawa K, Harrison NL: The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: Effects of TM2 mutations in the alpha and beta subunits. ANESTHESIOLOGY 2003; 99:678–84

22. Phillips RG, LeDoux JE: Differential contribution of amygdala and hip- pocampus to cued and contextual fear conditioning. Behav Neurosci 1992; 106:274–85

10. Hollmann MW, Liu HT, Hoenemann CW, Liu WH, Durieux ME: Modulation of NMDA receptor function by ketamine and magnesium, part II: interactions with volatile anesthetics. Anesth Analg 2001; 92:1182–91

11. Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW: Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain. Br J Pharmacol 2005; 146:189–97

12. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V: General anesthesia acti- vates BDNF-dependent neuroapoptosis in the developing rat brain. Apoptosis 2006; 11:1603–15

13. Ito M, Nakashima M, Tsuchida N, Imaki J, Yoshioka M: Histogenesis of the intravitreal membrane and secondary vitreous in the mouse. Invest Ophthalmol Vis Sci 2007; 48:1923–30

14. Satoh Y, Endo S, Ikeda T, Yamada K, Ito M, Kuroki M, Hiramoto T, Imamura O, Kobayashi Y, Watanabe Y, Itohara S, Takishima K: Extracellular signal-regulated kinase 2 (ERK2) knockdown mice show deficits in long-term memory; Erk2 has a specific function in learning and memory. J Neurosci 2007; 27:10765–76

23. Murcia CL, Gulden F, Herrup K: A question of balance: A proposal for new

mouse models of autism. Int J Dev Neurosci 2005; 23:265–75

24. Johnson SA, Young C, Olney JW: Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 2008; 20:21–8

25. American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, 4th ed. Washington, DC, American Psychiatric Association, 2000

26. Moretti P, Bouwknecht JA, Teague R, Paylor R, Zoghbi HY: Abnormalities of social interactions and home-cage behavior in a mouse model of Rett syn- drome. Hum Mol Genet 2005; 14:205–20

27. Lijam N, Paylor R, McDonald MP, Crawley JN, Deng CX, Herrup K, Stevens KE, Maccaferri G, McBain CJ, Sussman DJ, Wynshaw-Boris A: Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell 1997; 90:895– 905

28. Polleux F, Lauder JM: Toward a developmental neurobiology of autism.

Ment Retard Dev Disabil Res Rev 2004; 10:303–17

29. Rubenstein JL, Merzenich MM: Model of autism: Increased ratio of excita-

15. Jin D, Liu HX, Hirai H, Torashima T, Nagai T, Lopatina O, Shnayder NA, Yamada K, Noda M, Seike T, Fujita K, Takasawa S, Yokoyama S, Koizumi K, Shiraishi Y, Tanaka S, Hashii M, Yoshihara T, Higashida K, Islam MS, Yamada N, Hayashi K, Noguchi N, Kato I, Okamoto H, Matsushima A, Salmina A, Munesue T, Shimizu N, Mochida S, Asano M, Higashida H: CD38 is critical for social behavior by regulating oxytocin secretion. Nature 2007; 446:41–5

tion/inhibition in key neural systems. Genes Brain Behav 2003; 5:255–67

30. Uhlhaas PJ, Singer W: Neural synchrony in brain disorders: Relevance for

cognitive dysfunctions and pathophysiology. Neuron 2006; 52:155–68

31. Buzsa´ki G, Draguhn A: Neuronal oscillations in cortical networks. Science

2004; 304:1926–9

32. Hussman JP: Suppressed GABAergic inhibition as a common factor in

16. Kwon CH, Luikart BW, Powell CM, Zhou J, Matheny SA, Zhang W, Li Y, Baker SJ, Parada LF: Pten regulates neuronal arborization and social interaction in mice. Neuron 2006; 50:377–88

17. Bittigau P, Sifringer M, Pohl D, Stadthaus D, Ishimaru M, Shimizu H, Ikeda

suspected etiologies of autism. Autism Dev Disord 2001; 2:247–8

33. Bishop S, Gahagan S, Lord C: Re-examining the core features of autism: A comparison of autism spectrum disorder and fetal alcohol spectrum disorder. J Child Psychol Psychiatry 2007; 48:1111–21 /

33. Bishop S, Gahagan S, Lord C: Re-examining the core features of autism: A comparison of autism spectrum disorder and fetal alcohol spectrum disorder. J Child Psychol Psychiatry 2007; 48:1111–21 /

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