PMID: 24061597 DOI: 10.1097/ALN.0000435846.28299.e7
Materials and Methods
Animals
All experiments were conducted according to the institutional ethical guidelines for animal experiments of the National Defense Medical College and were approved by the Committee for Animal Research at National Defense Medical College (Tokorozawa, Saitama, Japan). Inbred C57BL/6 mice were used in this study and maintained as described previously.5 

Anesthesia and Hydrogen Treatment
Sevoflurane anesthesia was carried out as described previously.5  In brief, on postnatal day 6 (P6), pups were placed in a humid chamber immediately after removal of mice from the maternal cage. A 3% concentration of sevoflurane was administered in 30% oxygen as the carrier gas. Control mice were exposed to 30% oxygen. Hydrogen gas (1.3%) was supplied as described previously.30  Total gas flow rate was 2 l/min.

Mouse Study Design
In each experiment, siblings from the same litter were randomly allocated into one of the following groups so that each group was balanced on littermate. No obvious differences (e.g., body size and weight) were observed within the litters, and there was no significant difference in mean body weight among the groups (data not shown).

Survival rate of delivered pups: control, sevoflurane, and sevoflurane + hydrogen groups (n = 17–19 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.

Pup exchange test: control and sevoflurane groups (n = 6 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.

Behavioral studies: control, sevoflurane, and sevoflurane + hydrogen groups (n = 10–11 dams for each group); the primary outcome measure was latencies for pup retrieval; in the pup retrieval test, a minimum biologically important difference was set at a 30% increase from the baseline level in the control group.

Hormonal assay: control, hydrogen, sevoflurane, and sevoflurane + hydrogen groups (n = 4–5 dams for each group); a minimum biologically important difference was set as 30% decrease from the baseline level in the control group.

Immunohistochemical study: control and sevoflurane groups (n = 5 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.

In total, we prepared 160 female pups, which received anesthesia or hydrogen treatment at P6 (55 of control, 56 of sevoflurane, 40 of sevoflurane + hydrogen, and 9 of hydrogen groups). Among them, eight pups with sevoflurane and one pup with sevoflurane + hydrogen died during the treatment. Then, these siblings from the same litter were reunited and cohoused till the experiment (mice were similarly caged and housed in all groups). At 3 weeks of age, mice were weaned and allowed to further mature. At 7–9 weeks of age, female mice were mated with healthy males that had not been exposed to any anesthetic. Among them, 23 female mice did not get pregnant (eight of control, six of sevoflurane, five of sevoflurane + hydrogen, and four of hydrogen groups) and 1 control mouse died due to failure of delivery. These mice were excluded from the final analysis. Thus, for first delivery experiments, we used 46 control dams, 42 sevoflurane-treated dams, 34 sevoflurane + hydrogen–treated dams, and 5 hydrogen-treated dams. These mice were allocated as described above (1–5 in this section).

Among them, some dams were further analyzed for behavioral studies of parous dams: the same sets of mice for behavioral studies in first-time delivery were reused in behavioral studies in second-time (parous) delivery (control: 7 for survival rate and 11 for behavioral studies; sevoflurane-treated: 8 for survival rate and 10 for behavioral studies).

For paternal study experiments, 26 age-matched male mice were either subjected to anesthesia (n = 13) or control (n = 13) treatment at P6 (no mice died during the treatment). Siblings from the same litter were allocated into each group almost equally (i.e., groups were balanced on littermate).

Oxytocin and Vasopressin Assay
Plasma concentrations of oxytocin and vasopressin in dams at 10 weeks of age were examined by enzyme-linked immunosorbent assay using commercially available kits (Oxytocin enzyme-linked immunosorbent assay kit and arg8-Vasopressin enzyme-linked immunosorbent assay kit; Enzo Life Sciences, Farmingdale, NY). Assays were performed according to the manufacturer’s instructions. Blood samples were collected from the inferior vena cava within 6 h after parturition.

Immunohistochemical Study
Immunohistochemical studies using the anti-c-Fos antibody (rabbit polyclonal; sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) were performed as previously described.30  Samples were obtained within 6 h after parturition. The numbers of immunoreactive cells were counted by an observer blinded to the groups.

Behavioral Studies
On the morning of parturition, maternal behaviors were examined. Maternal behavioral studies using first-time mothers were performed at 10–12 weeks of age. The same sets of female mice were reused in the maternal behavioral studies for second-time (parous) mothers: those mice were mated again at 19–25 weeks of age, and maternal behaviors were examined at 22–28 weeks of age. Paternal behavioral studies using male mice were performed at 11 weeks of age. Survival rate (percentage of the number of pups at the indicated day compared with that at birth) was recorded until P6. In each experiment, observation was made by the same observer who was blinded to the groups. All apparatus used in this study was made by O’Hara & CO., LTD. (Tokyo, Japan).

Evaluation of Maternal Behavior
Pregnant females were individually housed for a few days before parturition and examined for maternal behavior on the morning of parturition. The number of pups with milk in their digestive tract and that of poorly cleaned pups (with placenta, amniotic membrane, or umbilical cords) was recorded on that day. Nest quality was also evaluated at the same time using the score system described previously31  with some modifications: grade 3, shaped like a deep hollow surrounded by high banks; grade 2, a hollow with medium-height banks; grade 1, flat with low banks, but still discrete; grade 0, no depression in bedding with no banks. Each new dam was also evaluated for time spent crouching over pups and the percentage of newborns scattered for 20 min with minimal disturbance as described previously.32  The percentage of scattered pups was expressed as a percentage with respect to time. We calculated the percentage of scatter as follows for each pup: (duration of scatter/total time observed (20 min) × 100). We then calculated the average for each group. These evaluations were carried out before the pup retrieval test.

Pup Retrieval Test
The pup retrieval test was performed essentially as described previously.14  Before the test, pups were separated from dams for 30 min. At the beginning, each mouse was put in one corner of a cage and three of her pups were placed in different corners of the same cage. The cages were continuously observed for 10 min with minimal disturbance. Latencies to sniff a pup for the first time and to return each pup to the nest were evaluated.

Evaluation of Parental Behavior
Parental behavior of virgin male mice toward pups was evaluated for 20 min. At the beginning, each mouse was put in one corner of a cage and three new born pups were placed in different corners of the same cage as described in the pup retrieval test. Latencies to sniff a pup for the first time and the numbers of males which committed attacks toward pups were evaluated. If any of the pups was attacked during the test, all pups were removed immediately and this subject was considered as “attack.”

Pup Exchange Test
The pup exchange test was conducted as described previously with some modifications.14  Pups born to a female dam couple (a dam with sevoflurane exposure at P6 and a control), which were born on the same day, were exchanged within 12 h after delivery. The number of surviving pups was evaluated for 6 days after birth.

Olfactory Test
The olfactory test was conducted as described previously.3 

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Comparisons of the means of each group were performed using Student t test, one-way ANOVA followed by Bonferroni post hoc test, and two-way ANOVA followed by Bonferroni post hoc test. Comparisons of the survival rate until P6 were performed using a log-rank (Mantel-Cox) test. We did not exclude any data in this study. P values of less than 0.05 were considered statistically significant. Values are presented as the mean ± SEM in bar graphs.