PMID: 33756347 DOI: 10.1016/j.bbrc.2021.03.063
2. Methods and materials
2.1. Animals
This study was approved by the Institutional Animal Care and Use Committee of Soochow University. Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China). One male and four female mice were housed per cage for breeding the offspring mice. Six pregnant mice on gestational day 14 were randomly assigned to receive either 2.5% sevoflurane in 100% oxygen or just 100% oxygen as the control. Their offspring mice were correspondingly assigned as the testing mice. Several pregnant mice without any treatment were chosen to produce the offspring mice as the stranger mice. The pups were fostered by their own dams till weaning on postnatal day 21. All mice were raised in a controlled condition (21–22 °C, 12 h light/dark cycle, light on at 7 a.m.), with access to standard mouse chow and water ad libitum.

2.2. Maternal anesthesia
A clinically-retired anesthesia machine was used to supply one-way gas flow. A transparent plastic box (20 L × 20 W × 6 H cm) was used as the anesthetizing chamber, with three holes for gas inflow, gas outflow, and gas monitoring. A heating-pad was placed underneath the anesthetizing chamber to keep mice warm during anesthesia. Sevoflurane anesthesia on pregnant mice in this study was strictly performed by the protocols of previous study [10], in which arterial blood pressure and blood gas analysis were demonstrated within normal limits. The pregnant mice retained spontaneous respiration during inhalational anesthesia. Sevoflurane was washed out with pure oxygen for 15 min, and the pregnant mice with right reflex were put back to home cages.

2.3. Social apparatus
The three-chambered social box (40L × 60W × 22H cm) with two enclosures (7D × 15H cm) was used for social interaction test (Fig. 1A–C). An improved video-tracking system programed by ANY-maze (Stoelting Co., USA) was used to capture the movement of mouse. Given the testing mouse initiates social approaching to the stranger mouse by nose-to-nose or nose-to-tail sniffing (Fig. 1D), the animal’s head was tracked by the video-tracking system. Four behavioral parameters was automatically measured by ANY-maze program, including the time sniffing at the enclosure and number of sniffs at the enclosure, the time exploring in the side-chamber and number of entries into side chamber. Specifically, “at the enclosure” is defined as the mouse head entering an area of 3 cm around the enclosure.


2.4. Social interaction test
The offspring mice (N = 17 Control, 9 males and 8 females; N = 14 Sevoflurane, 9 males and 5 females) were tested at one- and two-month-old (i.e., the juvenile and early-adult age). In advance, the testing mouse was housed single for 1-h isolation in the behavioral room. The stranger mice were the identical background, same gender and similar age as the testing mouse, and they had exactly no contact before. Social interaction test is composed of three 10-min sessions of habituation, sociability, and preference for social novelty. Firstly, the testing mouse was allowed to freely explore in social box with two doorways opening. Next, an unfamiliar conspecific (Stranger 1) was introduced into one enclosure, and the testing mouse was allowed to sniff the stranger 1 or explore the empty enclosure (Fig. 1E, G). After that, another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the testing mouse was allowed to sniff the stranger 1 and stranger 2 (Fig. 1F, H). Placement of the stranger 1 on the left or right side was systematically altered between trials, and social apparatus was cleaned after each trial to minimize olfactory disturbance.

2.5. Statistical analysis
Data were shown as Mean ± SD. Graphpad Prism 5.0 software (San Diego, USA) was used for statistical analyses. Social data obtained from the left and right side were mutually exclusive in each 10-min session and they were normally distributed by Kolmogorov-Smirnov tests. Therefore, two-tailed paired t-test was used to compare the side preferences (stranger 1 vs. the opposite). Two-way repeated measures (RM) ANOVAs were used to analyze the interaction effects of treatment (control or sevoflurane) × side (stranger 1 or the opposite). Based on the preliminary study, a sample size of more than 5 (sociability) and 13 (preference for social novelty) could lead to a 90% power to detect a difference in side preference with 5% type I error. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.