Biochemical and Biophysical Research Communications 593 (2022) 129e136

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Biochemical and Biophysical Research Communications

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y b b r c

Neonatal exposure to sevoﬂurane impairs preference for social novelty in C57BL/6 female mice at early-adulthood

Huayue Liu a, c, 1, Xiaowen Meng a, c, 1, Yixuan Li b, Shiwen Chen b, Yumeng Ji b, , Xin Jin a, c, * Shaoyong Song a, d, Fuhai Ji a, c, ** a Institute of Anesthesiology, Soochow University, Suzhou, 215006, PR China b Suzhou Medical College of Soochow University, Suzhou, 215123, PR China c Department of Anesthesiology, First Afﬁliated Hospital of Soochow University, Suzhou, 215006, PR China d Department of Pain Medicine, Dushu Lake Hospital Afﬁliated to Soochow University, Suzhou, 215124, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 December 2021 Accepted 8 January 2022 Available online 12 January 2022

Keywords: Sevoﬂurane Sociability Preference for social novelty Anesthesia Autism

Social interaction deﬁcit is core symptom of children with autism, owing to interaction of genetic pre- disposition and environmental toxins. Sevoﬂurane could induce neurotoxicity in developing brain in rodent models. This study aims to investigate whether sevoﬂurane anesthesia in neonatal period could impair social behaviors in male and female mice. Twenty-eight male and thirty-one female mice were randomly assigned to receive 3.0% sevoﬂurane or 60% oxygen on postnatal day 6. They were tested for social interaction behaviors at one- and two-month-old. In addition, the cortex and hippocampus of neonatal mice undergoing sevoﬂurane anesthesia were harvested for immunoblotting analysis. As a result, both male and female mice undergoing sevoﬂurane anesthesia showed strong sociability and weak preference for social novelty at juvenile age. In addition, the male mice developed normal pref- erence for social novelty at early-adulthood; However, the female mice remained weak preference for social novelty. Furthurmore, sevoﬂurane anesthesia could decrease the levels of PSD95 but not Neuroligin-1 in the hippocampus but not cortex of neonatal mice. In conclusion, sevoﬂurane anesthesia in neonatal period could disturb development of social memory and impair preference for social novelty in female mice at early-adulthood, with the potential mechanism of decreasing PSD95 expression in the hippocampus of C57BL/6 mice. © 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Children with autism spectrum disorders are largely increasing and the ratio of boys and girls is nearly 4:1 all over the world [1,2]. The core symptom of autism is social interaction deﬁcit, with the potential mechanism of genetic predisposition and environmental toxicants [3,4]. Meanwhile, some anesthetics are reported to induce neurotoxicity in the developing brain [5,6]. Sevoﬂurane, an inha- is commonly used in pediatric anesthesia. lational anesthetic,

Abbreviations: ASD, autism spectrum disorders; PND, postnatal day; PSD95,

postsynaptic density-95.

Corresponding author. Department of Anesthesiology, Suzhou, 215006, PR

China. ** Corresponding author. Department of Anesthesiology, Suzhou, 215006, PR China.

Preclinical studies suggested that neonatal anesthesia with sevo- ﬂurane could impair learning and memory in rodents [7,8]. In particular, neonatal exposure to 3% sevoﬂurane for 6 h in mice could cause learning deﬁcit in fear conditioning test and social deﬁcit in open ﬁeld cage [9]. However, it is uncertain whether sevoﬂurane could impair biological proﬁles of social afﬁliation and social memory in mice, and whether the male and female mice would behave differently.

Sevoﬂurane could produce anesthetic effect by stimulating GABA receptors and inducing an imbalance of excitatory and inhibitory neurotransmission [10]. Although anesthetists are taking advantage of sevoﬂurane for rapid onset time and short duration, pediatric patients are taking risk of sevoﬂurane-induced develop- mental neurotoxicity and behavioral abnormality. Therefore, we set out to investigate whether neonatal anesthesia with sevoﬂurane could disturb sociability and preference for social novelty in male and female mice at juvenile age and early-adulthood.

E-mail addresses: jifuhai@hotmail.com (F. Ji), jinxin@suda.edu.cn (X. Jin). 1 These authors contributed equally to this work (H. Y. Liu and X.W. Meng).

We hypothesized that neonatal exposure to sevoﬂurane could

https://doi.org/10.1016/j.bbrc.2022.01.022 0006-291X/© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).

H. Liu, X. Meng, Y. Li et al.

induce social interaction deﬁcit in mice. To verify this hypothesis, we ﬁrstly anesthetized C57BL/6 mice with 3.0% sevoﬂurane in 60% oxygen for 2 h on postnatal day 6. Next, we tested social behaviors of the male and female mice at one- and two-month-old. Finally, we examined the levels of Neuroligin-1 and PSD95 in the cortex and hippocampus of sevoﬂurane-exposed neonatal mice.

2. Methods

2.1. Animals

This study was approved by the Institutional Animal Care and Use Committee at Soochow University (Suzhou, Jiangsu, China). Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice. On postnatal day (PND) 21, the offspring mice were separated from dams and housed 4e5 per cage by gender. Four male and four female mice were speciﬁcally used as the stranger mice, which were trained to stay calmly in the enclosure before social interaction test. All the mice were raised with free access to food and water in a controlled environment (room temperature 21e22 (cid:1)C, 12/12 h light/dark cycle, and light on at 7 a.m.).

2.2. Anesthesia

Inc.) was employed to supply a consistent concentration of anesthetic gas. A sealed plastic box (20 L (cid:3) 20 W (cid:3) 6 H cm) was used as the anes- thetizing chamber, which was drilled with three holes for gas inﬂow, gas outﬂow and gas monitoring. An electric heater was placed underneath the anesthetizing chamber to keep neonatal mice warm during anesthesia. A gas analyzer (Datex-Ohmeda, Inc.) was applied to adjust gas concentrations. On postnatal day 6, the neonatal mice were randomly assigned into two groups. Twenty- eight mice (17 males and 11 female) received 3.0% sevoﬂurane in 60% oxygen for 2 h (Sevo), and thirty-one mice (11 males and 20 female) inhaled merely 60% oxygen for 2 h (Oxyg). Sex of each mouse and amount of each group were not identiﬁed until weaning on PND 21. These subject mice were tested for social interaction behavior at one- and two-month-old.

A retired anesthesia machine (Datex-Ohmeda,

Another battery of neonatal mice were treated with the air condition (control) or 60% oxygen (oxyg) or 3.0% sevoﬂurane in 60% oxygen (sevo) for 2 h on PND 6, and then killed for harvesting brain tissues 24 h after treatment. Sevoﬂurane anesthesia was strictly performed by the protocols of previous studies [11,12], in which all neonatal mice could spontaneously breath during general anes- thesia, and their arterial blood pressure and blood gas analysis showed within normal limits. The vapor for releasing sevoﬂurane was turned off at the end of anesthesia, and the residual anesthetic was washed out with 60% oxygen for 15 min. Finally, these pups were smeared with own bedding and sent back to their dams.

2.3. Social interaction paradigm

Social interaction test is performed with the three-chambered social box, with three chambers (40 L (cid:3) 20 W (cid:3) 22 H cm) and two enclosures (7 ID (cid:3) 15 H cm). The ﬂoor is painted grey to pro- vide a high contrast with the testing mice. Grid bars of the enclo- sure allow direct contacting between the subject and stranger mice. A novel video-tracking system was developed by hanging two video-cameras right above two enclosures. Thereby, two video- images were integrated into one with the montage effect in ANY- maze program (Stoelting Co., USA). The subject mouse initiates social interaction with the stranger mouse by nose-to-nose or nose-

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to-tail snifﬁng, thus the animal's head is tracked by the ANY-maze program.

2.4. Social interaction test

First of all, the stranger mice were transferred into behavioral room and hidden 2 m away from social apparatus. Each subject mouse was taken into behavioral room about 45 min before social interaction test. In the ﬁrst session (Habituation, 10-min), the sub- ject mouse was gently placed into the middle chamber, and allowed to freely explore in three chambers. In the second session (Socia- bility, 10-min), the subject mouse was guided into the middle chamber and transiently conﬁned there. An unfamiliar conspeciﬁc (Stranger 1) was introduced into one enclosure, the subject mouse was allowed to explore in three chambers and sniff at two enclo- sures containing Stranger 1 or not. In the third session (Preference for social novelty 10-min), the subject mouse was again conﬁned into the middle chamber. Another unfamiliar conspeciﬁc (Stranger 2) was introduced into the other enclosure, and the subject mouse was allowed to explore in three chambers and sniff at two enclo- sures containing Stranger 2 or Stranger 1. Placement of Stranger 1 on left and right side were balanced between trials, and two stranger mice were the same gender as the subject mice.

Sociability is characteristic of the mouse taking more time snifﬁng its conspeciﬁc mouse compared with an inanimate object. Preference for social novelty is characteristic of the mouse taking more time snifﬁng an unfamiliar mouse compared with a familiar one. Four parameters were measured for judging social choice, including 1) time snifﬁng at the enclosure, 2) number of sniffs, 3) time exploring in the chamber, and 4) number of entries. Snifﬁng time at the enclosure was primary outcome, number of sniffs at the enclosure and time exploring in the chamber were secondary outcomes. In social interaction test, “at the enclosure” is deﬁned as the head of mouse entering an area about 3 cm around the enclo- sure, as described in similar social study [13]. And “in the chamber” is deﬁned as the head of mouse entering into the chamber.

2.5.

Immunoblotting analysis

The brain tissues of neonatal mice were harvested on dry ice at 24 h after treatment. Next, the cortex and hippocampus were ho- mogenized on ice using the immunoprecipitation buffer plus pro- tease inhibitor. And then, the lysates were centrifuged at 15,000 rpm for 30 min at 4 (cid:1)C. After that, the lysates were quan- tiﬁed for total protein by the bicinchoninic acid (BCA) protein assay kit (MultiSciences Biotech Co., Ltd. Cat: PQ0012, Lot: A91041). Finally, western blot was performed by the protocols to analyze protein levels in cortex and hippocampus. Neuroligin-1 antibody (1:1000; Santa Cruz Biotechnology, Inc.) was used to detect neuroligin-1 (101 kDa). PSD-95 antibody (1:1000; Cell Signaling Technology, Inc.) was used to detect PSD-95 (95 kDa). Antieb-actin (1:5000; Sigma) was used to detect b-actin (42 kDa).

2.6. Statistical analysis

Data were expressed as Mean ± SD. Statistical analyses were performed by using GraphPad Prism 5.0 (San Diego, USA). Data representing social behavior of testing mice were normally distributed by Kolmogorov-Smirnov test. Data of each mouse from the left or right side were mutually exclusive, and two-tailed paired t-test was used to determine side preference, which was supported by other social studies [14,15]. Student's t-test was used to assess differences in the levels of Neuroligin-1 and PSD95 expression in cortex and hippocampus of mice. P values less than 0.05 (*), 0.01 (**) and 0.001 (***) were considered statistically signiﬁcant.

H. Liu, X. Meng, Y. Li et al.

3. Results

3.1. Both male and female subject mice show strong sociability and weak preference for social novelty at one-month-old, and sevoﬂurane anesthesia on postnatal day 6 could not inﬂuence the biologic proﬁles of social afﬁliation and social memory in the juvenile mice

The subject mice undergoing anesthesia with 3.0% sevoﬂurane in 60% oxygen on postnatal day 6, both males and females, showed strong sociability at one-month-old, as proved by taking more time (Fig. 1A, 183.3 ± 51.6 vs 73.6 ± 32.6 s, P < 0.0001 Male; 250.7 ± 72.7 vs 71.1 ± 33.3 s, P ¼ 0.0002 Female) approaching the stranger 1 mouse, snifﬁng more frequently (Fig. 1B, 53.6 ± 21.1 vs 22.9 ± 10.6 , P < 0.0001 Male; 49.0 ± 15.9 vs 21.8 ± 9.4, P ¼ 0.0024 Female) at the enclosure containing stranger 1, and spending more time exploring (Fig. 1C, 348.3 ± 56.7 vs 170.6 ± 47.4 s, P < 0.0001 Male; 377.5 ± 71.4 vs 161.5 ± 58.8 s, P ¼ 0.0002 Female) in the chamber with stranger 1, as compared with the empty side. Collectively, the male and fe- male mice, exposed to sevoﬂurane in the neonatal period, showed the well-developed social afﬁliation at the juvenile age.

However, the subject mice undergoing anesthesia with 3.0% sevoﬂurane in 60% oxygen on postnatal day 6, either males or fe- males, showed weak preference for social novelty at one-month- old, as evidenced by taking no more time (Fig. 1E, 105.2 ± 50.7 vs 151.8 ± 72.3 s, P ¼ 0.068 Male; 139.6 ± 55.8 vs 171.5 ± 68.1 s, P ¼ 0.3189 Female) approaching the stranger 2 mouse, snifﬁng no more frequently (Fig. 1F, 31.0 ± 13.0 vs 37.2 ± 14.4, P ¼ 0.3085 Male; 33.1 ± 11.8 vs 36.2 ± 7.0, P ¼ 0.4993 Female) at the enclosure containing stranger 2, or spending no more time exploring (Fig. 1G, 229.8 ± 90.3 vs 271.9 ± 80.2 s, P ¼ 0.3126 Male; 246.2 ± 54.3 vs 282.8 ± 63.1 s, P ¼ 0.3063 Female) in the chamber with stranger 2, as compared with the stranger 1 side. Together, the male and female mice, exposed to sevoﬂurane in neonatal period, showed the undevel- oped social memory at the juvenile age.

3.2. The male subject mice, but not the female, showed normal preference for social novelty at two-month-old, and sevoﬂurane anesthesia on postnatal day 6 could impair the development of social memory of female mice at the early-adulthood

The subject mice undergoing anesthesia with 3.0% sevoﬂurane in 60% oxygen on postnatal day 6, both males and females, showed strong sociability at two-month-old, as proved by taking more time (Fig. 2A, 196.2 ± 50.8 vs. 59.3 ± 24.3 s, P < 0.0001 Male; 189.9 ± 60.4 vs. 58.4 ± 23.8 s, P ¼ 0.0028 Female) approaching the stranger 1 mouse, snifﬁng more frequently (Fig. 2B, 66.5 ± 17.3 vs. 28.4 ± 10.8, P < 0.0001 Male; 58.3 ± 13.6 vs. 30.4 ± 6.9, P ¼ 0.0051 Female) at the enclosure containing stranger 1, and spending more time exploring (Fig. 2C, 347.4 ± 55.6 vs. 160.0 ± 43.1 s, P < 0.0001 Male; 324.6 ± 65.2 vs. 175.8 ± 43.8 s, P ¼ 0.008 Female) in the chamber with stranger 1, as compared with the empty side. Collectively, the male and female mice, exposed to sevoﬂurane in the neonatal period, showed the sustained social afﬁliation at the early- adulthood.

Meanwhile, the male subject mice, but not the females, under- going anesthesia with 3.0% sevoﬂurane in 60% oxygen on postnatal day 6 showed normal preference for social novelty at two-month- old, as evidenced by taking more time (Fig. 2E, 102.0 ± 42.0 vs. 146.6 ± 59.3 s, P ¼ 0.0463 Male; 101.5 ± 34.7 vs. 138.0 ± 49.9 s, P ¼ 0.2462 Female) approaching the stranger 2 mouse, snifﬁng more frequently (Fig. 2F, 37.1 ± 12.2 vs. 53.1 ± 15.0, P ¼ 0.0102 Male; 40.3 ± 10.3 vs. 53.1 ± 26.0, P ¼ 0.3074 Female) at the enclosure con- taining stranger 2, or spending more time exploring (Fig. 2G, 211.2 ± 58.3 vs. 284.3 ± 59.9 s, P ¼ 0.0254 Male; 228.9 ± 65.3 vs. 269.8 ±

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64.3 s, P ¼ 0.4307 Female) in the chamber with stranger 2, as compared with the stranger 1 side. Together, the male mice exposed to sevoﬂurane in the neonatal period showed the already- developed social memory at the early-adulthood, but the female mice remained undeveloped social memory at the early-adulthood.

3.3. Neonatal exposure to 3.0% sevoﬂurane in 60% oxygen, as well as inhalation of 60% oxygen, decreased PSD95 levels in the hippocampus, but not the cortex, of neonatal mice

In the cortex (Fig. 3A) and the hippocampus (Fig. 3B) of neonatal mice, the immunoblotting displayed that anesthesia with 3.0% sevoﬂurane (lanes 9-12) and inhalation of 60% oxygen (lanes 5-8) did not change the levels of bands representing Neuroligin-1 expression, as compared with the control (lanes 1-4), Quantiﬁca- tion of Western blot, based on the ratio of Neuroligin-1 levels to b- Actin levels, did not show that neonatal exposure to sevoﬂurane change the levels of Neuroligin-1 in the cortex (Fig. 3C, 100.0±33.1 vs. 92.7±14.0, P¼0.6975 grey) or the hippocampus (Fig. 3D, 100.0±19.3 vs. 84.8±20.0, P¼0.3155 grey) of the neonatal mice, as compared with the control (white) .

The immunoblotting displayed that anesthesia with 3.0% sevo- ﬂurane (lanes 9-12) and inhalation of 60% oxygen (lanes 5-8) did not change the levels of bands representing PSD95 expression in the cortex (Fig. 3E) of neonatal mice, as compared with the control (lanes 1-4). Quantiﬁcation of Western blot did not show that neonatal exposure to sevoﬂurane change the levels of PSD95 in the cortex (Fig. 3G, 100.0±23.4 vs. 120.1±27.9, P¼0.3113 grey), as compared with the control (white). However, the immunoblotting displayed that anesthesia with 3.0% sevoﬂurane (lanes 9-12) and inhalation of 60% oxygen (lanes 5-8) decreased the levels of bands representing PSD95 expression in the hippocampus (Fig. 3F) of neonatal mice, as compared with the control (lanes 1-4). Quanti- ﬁcation of Western blot decreased that neonatal exposure to sev- oﬂurane change the levels of PSD95 in the cortex (Fig. 3H, 100.0±35.9 vs. 49.2±10.8, P¼0.035 grey), as compared with the control (white).

4. Discussions

This study was to explore whether neonatal anesthesia with sevoﬂurane could induce social interaction deﬁcit in C57BL/6 mice, and we performed the experiment with several important con- siderations and preparations. Firstly, we conducted inhalational anesthesia with 3.0% sevoﬂurane in 60% oxygen for 2 h as in similar studies [16e18], and this regimen of anesthesia in neonatal mice was designed to mimic clinical anesthesia in pediatric patients. Secondly, we employed the three-chambered social paradigm to assess social behaviors of the subject mice [19e21] which could reﬂect two biological proﬁles of sociability and preference for social novelty [22]. Thirdly, the subject mice were arranged for social interaction tests at one- and two-month-old, as the juvenile age and early-adulthood were considered to be two critical periods of brain development in human [13,23]. Finally, we tested social interaction behaviors of male and female mice respectively, in or- der to investigate interaction effects of anesthesia and sex on social behaviors of the mice. Thereby, these results were adequate to determine whether neonatal exposure to sevoﬂurane could induce social interaction deﬁcit in C57BL/6 mice.

Our data suggested that both male and female mice, in the three-chambered social test, showed strong sociability and weak preference for social novelty at one-month-old, indicating the robust social afﬁliation with the conspeciﬁc and underdevelopment of social memory at the juvenile age. However, the male mice un- dergoing either sevoﬂurane anesthesia or oxygen control displayed

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Fig. 1. The juvenile mice anesthetized with 3.0% sevoﬂurane in 60% oxygen on PND 6, either male or female, show strong sociability but weak preference for social novelty at one-month-old. In the sociability test, the subject mice in either group, take more time snifﬁng the stranger 1 mouse (A), sniff more frequently at the enclosure containing stranger 1 (B), and spend more time exploring in the chamber with stranger 1 (C), as compared to the empty side. In the test of social novelty preference, the subject mice in either group, take no more time snifﬁng the stranger 2 mouse (E), sniff no more frequently at the enclosure containing stranger 2 (F), or spend no more time exploring in the chamber with stranger 2 (G), as compared to the stranger 1 side. (D, H) There are not signiﬁcant differences between two sides in the number of entries into the chamber. Data are expressed as Mean ± SD. N ¼ 11 Oxygen and 17 Sevoﬂurane for males, 20 Oxygen and 11 Sevoﬂurane for females. Paired t-test, two-side. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 2. The early-adult female mice, anesthetized with 3.0% sevoﬂurane in 60% oxygen on PND 6, show strong sociability but weak preference for social novelty at two- month-old. In the sociability test, the subject mice in either group, take more time snifﬁng the stranger 1 mouse (A), sniff more frequently at the enclosure containing stranger 1 (B), and spend more time exploring in the chamber with stranger 1 (C), as compared to the empty side. In the test of social novelty preference, the male mice take more time snifﬁng the stranger 2 mouse (E), sniff more frequently at the enclosure containing stranger 2 (F), and spend more time exploring in the chamber with stranger 2 (G), as compared to the stranger 1 side. However, the female mice undergoing neonatal anesthesia with sevoﬂurane show no signiﬁcant difference (E, F and G, right) between the stranger 2 and stranger 1 side. (D, H) There are not signiﬁcant differences between two sides in the number of entries into the chamber. Data are expressed as Mean ± SD. N ¼ 11 Oxygen and 15 Sevoﬂurane for males, 12 Oxygen and 7 Sevoﬂurane for females. Paired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 3. Neonatal exposure to 3.0% sevoﬂurane in 60% oxygen or only 60% oxygen decrease the level of PSD95 in hippocampus, but not cortex, of the female mice. Western blot analysis displays that neither oxygen (lanes 5e8) nor sevoﬂurane (lanes 9e12) decreases the level of Neuroligin-1 in either cortex (A) or hippocampus (B) of the female mice, as compared to the control condition (lanes 1e4). Quantiﬁcation of Western blot shows that neither oxygen (black bar) nor sevoﬂurane (grey bar) decreases the level of Neuroligin-1 in either cortex (C) or hippocampus (D), as compared to the control condition (white bar). Meanwhile, western blot analysis displays that both oxygen (lanes 5e8) and sevoﬂurane (lanes 9e12) decreases the level of PSD95 in hippocampus (F) but not cortex (E) of the female mice, as compared to the control condition (lanes 1e4). Quantiﬁcation of Western blot shows that either oxygen (black bar) or sevoﬂurane (grey bar) decreases the level of PSD95 in hippocampus (H) but not cortex (G), as compared to the control condition (white bar). N ¼ 4 in each, Student's t-test. *P < 0.05.

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normal preference for social novelty at two-month-old, indicating the well-development of social memory at the early-adulthood. Meanwhile, the female mice undergoing neonatal exposure to sevoﬂurane remained very weak preference for social novelty, indicating the developmental retardation of social memory at the early-adulthood. Taken together, these results demonstrated that sevoﬂurane anesthesia in neonatal period could impair preference for social novelty of the female mice at the early-adulthood. In addition, the developmental neurotoxicity of sevoﬂurane anes- thesia could be partial to female but not male, and neurobehavioral abnormality could be partial to social memory but not social afﬁliation.

Three-chambered social paradigm was widely used for social interaction test in mouse model, which could examine sociability and preference for social novelty [15,24]. Sociability was deﬁned as the propensity of testing mice to spend more time exploring in the chamber containing Stranger 1 than in the empty chamber [14,23]. Preference for social novelty was deﬁned as the propensity of testing mice to spend more time exploring in the chamber con- taining Stranger 2 than in the chamber containing Stranger 1 [24,25]. Because the testing mouse might take too much time wandering and self-grooming, the time exploring in the chamber could not totally represent its real choice [26]. Therefore, we measured time snifﬁng at the enclosure, as the primary outcome, for accessing social choice of mice. Meanwhile, time exploring in the chamber and number of sniffs at the enclosure were used as the secondary outcomes. Besides, number of entries to side chamber was considered to be an internal control of general activities.

In the sociability tests, both male and female mice, either the control or the sevoﬂurane-exposed, took much more time snifﬁng Stranger 1 compared with the empty enclosure at one- and two- month-old. These results suggested that 1) social afﬁliation with conspeciﬁc could be a stable and basic instinct of mice, and 2) neonatal exposure to sevoﬂurane could not impair the sociability of male and female mice. In the social novelty preference tests, both male and female mice, either the control or the sevoﬂurane- exposed, did not take more time snifﬁng Stranger 2 compared with Stranger 1 at one-month-old, and the male but not female mice preferred Stanger 2 to Stranger 1 at two-month-old. These results suggested that 1) social memory of the mice could be un- stable and superior neurocognitive function of mice [22,24,25], and 2) neonatal exposure to sevoﬂurane could selectively impair pref- erence for social novelty of female, but not male, mice at the early- adulthood.

In this study, the male mice showed normal preference for novel conspeciﬁc at the early-adulthood, rather than at the juvenile age and this phenomenon suggested that social recognition memory was age-dependent in subject mice [26e28]. Meanwhile, the fe- male mice undergoing sevoﬂurane anesthesia, but not the control, showed weak preference for novel conspeciﬁc at the early- adulthood, and this abnormality suggested that sevoﬂurane- induced neurotoxicity could disturb neurodevelopmental process of social memory in female mice. In clinical phenotypes, children diagnosed as autism spectrum disorders are gender-biased, with more boys outweighing girls. We had no idea of the sexual discrepancy between social deﬁcit in human being and social memory impairment in mouse model, and the potential explana- tion should be based on the development of body and brain. In terms of biological dimorphism, the boys could lag behind the girls in mental and physical development in adolescence, while the fe- male mice should weigh less than male mice in juvenile and early- adulthood.

It was reported that sevoﬂurane anesthesia could impair learning and memory by inducing neuronal apoptosis and inhib- iting synapse plasticity [11,29,30], which were in accordance with

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disturbance of social memory in female mice undergoing sevo- ﬂurane anesthesia in our study. Next, we performed immunoblot- ing analysis to determine whether sevoﬂurane anesthesia in neonatal mice could affect the expression of Neuroligin-1 and PSD95 in the brain tissues. As a result, immunobloting analysis suggested that sevoﬂurane could not change the levels of Neuroligin-1 in the cortex and hippocampus of neonatal mice. However, we found that sevoﬂurane could decrease the levels of PSD95 in the hippocampus of neonatal mice. This result suggested the sevoﬂurane-induced neurotoxicity in developing brain of neonatal mice, although the detailed signaling pathways and neuropathologic mechanisms were unclear at this moment. Chung et al. reported that neonatal exposure to sevoﬂurane could cause the long-term memory impairment in C57BL/6J mice but not autism-like features [7], indicating the complication of social behavior and recognition memory in mice. In future study, we will explore the neuropathologic mechanisms of social memory impairment, and assess the inconformity of normal preference for social novlety in the oxygen-controlled mice with decreased levels of PSD95 in the hippocampus of neonatal mice.

limitations. Firstly, we found the impairment of social memory in female mice undergoing sevo- ﬂurane anesthesia, and then performed immunobloting analysis in brain tissue of sex-mixed mice, because it was difﬁcult to distin- guish male and female mice in neonatal period. Secondly, we had only anesthetized neonatal mice with 3% sevoﬂurane for 2 h, and this regimen mimicking clinical anesthesia could be close to lower- limit. In future, we will anesthetize neonatal mice with 3% sevo- ﬂurane for 6 h or 2 h for three times, to explore the accumulative impact of sevoﬂurane-induced neurotoxicity on social behaviors in mice. Thirdly, we performed 3.0% sevoﬂurane anesthesia in 60% oxygen in neonatal mice in animal study, but we had no idea of whether 60% oxygen itself could produce negative effects in developing brain. In future, we will determine the effect of 60% oxygen on social interaction behaviors of mice.

This study has several

In conclusion, C57BL/6 mice have two biological proﬁles of so- ciability and preference for social novelty, which were dominated by robust social afﬁliation and gradually-developed social memory. Neonatal exposure to sevoﬂurane could not impair sociability and preference for social novelty in male mice; however it could disturb preference for social novelty in female mice, with the potential mechanism of decreasing PSD95 levels in the hippocampus.

Declaration of competing interest

The authors declare no competing ﬁnancial interests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82001126 to S.Y.S, 82072130 and 81873925 to F.H.J), Natural Science Foundation of Jiangsu Province (BK20191171 to F.H.J).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.bbrc.2022.01.022.

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