Abstract

Editor’s Perspective
What We Already Know about This Topic
  • Exposure to environmental stressors and endocrine disruptors can induce multigenerational effects resulting in neurobehavioral and other abnormalities in the offspring

  • Early-life anesthesia exposure in rodents alters neurocognitive function in their offspring, but whether exposure of adult animals affects offspring has not been previously reported

What This Article Tells Us That Is New
  • Repeated exposures of adult rats to sevoflurane (2.1%, three times, 3 h on every second day) induce neurobehavioral abnormalities in the exposed males and in male but not female progeny

  • The neurobehavioral abnormalities in male offspring are accompanied by increased methylation and decreased expression of the potassium ion-chloride ion cotransporter Kcc2 gene that regulates neuronal chloride homeostasis, and, thereby, the functional modalities of γ-aminobutyric acid type A receptor–mediated neurotransmission

  • Sevoflurane exposure also induces hypermethylation of the Kcc2 gene in both male and female parental germ cells

  • These observations suggest that epigenetic reprogramming of parental germ cells is involved in transmitting the adverse effects of sevoflurane exposure of adult rats to their male progeny

Background

Sevoflurane administered to neonatal rats induces neurobehavioral abnormalities and epigenetic reprogramming of their germ cells; the latter can pass adverse effects of sevoflurane to future offspring. As germ cells are susceptible to reprogramming by environmental factors across the lifespan, the authors hypothesized that sevoflurane administered to adult rats could induce neurobehavioral abnormalities in future offspring, but not in the exposed rats themselves.

Methods

Sprague-Dawley rats were anesthetized with 2.1% sevoflurane for 3 h every other day between postnatal days 56 and 60. Twenty-five days later, exposed rats and nonexposed controls were mated to produce offspring.

Results

Adult male but not female offspring of exposed parents of either sex exhibited deficiencies in elevated plus maze (mean ± SD, offspring of both exposed parents vs. offspring of control parents, 35 ± 12 vs. 15 ± 15 s, P < 0.001) and prepulse inhibition of acoustic startle (offspring of both exposed parents vs. offspring of control parents, 46.504 ± 13.448 vs. 25.838 ± 22.866%, P = 0.009), and increased methylation and reduced expression of the potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) in the hypothalamus. Kcc2 was also hypermethylated in sperm and ovary of the exposed rats. Surprisingly, exposed male rats also exhibited long-term abnormalities in functioning of the hypothalamic-pituitary-gonadal and -adrenal axes, reduced expression of hypothalamic and hippocampal Kcc2, and deficiencies in elevated plus maze (sevoflurane vs. control, 40 ± 24 vs. 25 ± 12 s, P = 0.038) and prepulse inhibition of startle (sevoflurane vs. control, 39.905 ± 21.507 vs. 29.193 ± 24.263%, P < 0.050).

Conclusions

Adult sevoflurane exposure affects brain development in male offspring by epigenetically reprogramming both parental germ cells, while it induces neuroendocrine and behavioral abnormalities only in exposed males. Sex steroids may be required for mediation of the adverse effects of adult sevoflurane in exposed males.

The U.S. Food and Drug Administration recommends avoiding anesthesia in children under 3 yr old whenever possible, as it may negatively affect their brain development.1  This recommendation is based largely on an earlier body of work in animals, particularly rodents, showing that neonates are especially susceptible to lasting adverse effects of anesthesia during the first 2 postnatal weeks.2  In rodents, the first 2 weeks of life are characterized by unique physiologic properties, such as excitatory signaling through γ-aminobutyric acid type A (GABAA) receptors,3,4  a major substrate for the inhibitory (sedative) effects of γ-aminobutyric acid–ergic (GABAergic) anesthetics in the more mature brain.5–7  GABAA receptor signaling is excitatory during early life due to elevated levels of intraneuronal Cl, maintained by relatively low and high levels of the potassium ion-chloride ion cotransporter KCC2 and sodium ion-potassium ion-chloride ion cotransporter NKCC1, respectively.3,4  During the second postnatal week in rats, GABAA receptor signaling gradually becomes inhibitory, primarily due to age-dependent increases in KCC2.3,4  The magnitude of GABAA receptor excitatory signaling and the proper timing of its transition from excitatory to inhibitory are key for normal brain development, and impairments in KCC2 and shifts in GABAA receptor signaling toward excitation have been linked to neuropsychiatric disorders in both humans and animal models.8,9  It appears that exposure to GABAergic anesthetics early in life may induce developmental abnormalities at least in part by enhancing the magnitude of GABAA receptor excitatory signaling at the time of anesthesia, and by disrupting normal developmental increase in Kcc2 expression.10–12  In support of this contention, exposure of neonatal rats to GABAergic anesthetics, such as sevoflurane, causes electroencephalography-detectable epileptic seizures and multifold increases in corticosterone secretion at the time of anesthesia.12  After maturing to adulthood, these anesthetic-exposed rats have downregulated Kcc2 levels, exacerbated hypothalamic-pituitary-adrenal axis responses to stress, and behavioral abnormalities.10,11  Inhibition of NKCC1 at the time of anesthesia ameliorates acute seizure activities, as well as many of the lasting developmental effects of GABAergic anesthetics,11,12  suggesting that anesthetic-exacerbated GABAA receptor–mediated excitation in neonatal brain is an important initiating step in anesthetic-induced neurobehavioral abnormalities in the exposed animals.

Surprisingly, neonatal parental sevoflurane also impairs Kcc2 expression in the brains of future male offspring by initiating DNA methylation at the 5´ position of cytosine residues adjacent to guanines in the Kcc2 gene in parental gamete cells.10  These findings suggest that neonatal sevoflurane can induce two distinct types of adverse effects in the exposed animals—excitatory GABAA receptor signaling–dependent effects in the exposed neonatal brain and epigenetic reprogramming of their germ cells, which can pass adverse effects of neonatal parental sevoflurane to future unexposed offspring.10  Epidemiologic studies and research in animal models suggest that parental germ cells can be susceptible to reprogramming by environmental factors across the lifespan.13–15  Therefore, here we tested the hypothesis that sevoflurane can affect future offspring even when administered to adult rats. We further hypothesized that the primarily inhibitory GABAA receptor signaling in the brain of young adult rats3,16  renders them resistant to the adverse neuroendocrine and behavioral effects of sevoflurane.

Materials and Methods

Animals

All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee (Gainesville, Florida). Sprague-Dawley rats were bred at the University of Florida animal care facility. The rats were housed under controlled illumination (12-h light–dark, lights on at 7:00 am) and temperature (23 to 24°C) with free access to food and water. Within 24 h of delivery, litters were culled to 12 pups. At the age of 21 days, pups were weaned and housed in sex-matched pairs for the remainder of the study.

Treatment Groups

Male and female rats (generation F0) were randomized for treatment and breeding pairs using a randomization plan generator (http://www.randomization.com/), and the investigators were blind to group assignments. The F0 rats underwent anesthesia on postnatal days 56, 58, and 60. The rats were held in a temperature-controlled chamber to maintain body temperature at +37°C with a continuous supply of 30% oxygen in air (1.5 l/min) during anesthesia, which was induced by 6% sevoflurane for 3 min followed by 2.1% sevoflurane for 177 min for anesthesia maintenance (the Sevoflurane group). Gas monitoring was performed using a calibrated Datex side stream analyzer (Datex-Ohmeda, Finland), which sampled from the animal chamber interior. Rats in the F0 control group (Control group) were not subjected to anesthesia. A subset of the rats in the F0 Control and Sevoflurane groups was sacrificed 1 h after the last exposure to sevoflurane (or equivalent timepoint in the Control group) to collect blood and brain tissue samples to measure serum levels of corticosterone and to evaluate expression of KCC2 in the paraventricular nucleus of the hypothalamus (see fig. 1 for schematic of experimental design). The remaining Control and Sevoflurane F0 male and female rats were mated on postnatal day 85 to produce offspring (generation F1). Only rats from different litters were mated. F1 rats were categorized as the offspring of (1) control males and control females; (2) exposed males and control females; (3) control males and exposed females; and (4) exposed males and exposed females. The females were housed individually throughout the entire gestation and postpartum rearing periods.

Fig. 1.

Study design.

Fig. 1.

Study design.

Sixty-four F0 rats (32 control and 32 sevoflurane-exposed) were used as breeders to produce 122 F1 rats, which were not exposed to sevoflurane anesthesia and were subjected to animal facility rearing only. The F0 rats were sequentially evaluated in the elevated plus maze starting on postnatal day ~125 and prepulse inhibition of the acoustic startle response on postnatal day ~135 (fig. 1). A subset of these animals was sacrificed on postnatal day ~160 to collect trunk blood, brain, and testis and ovarian tissues for further analyses. The rest of these animals were physically restrained for 30 min on postnatal day ~160 to measure corticosterone responses, followed by collection of tissue samples for further analyses. The F1 rats were evaluated in the elevated plus maze starting on postnatal day ~60 and prepulse inhibition of startle on postnatal day ~70 (fig. 1). One half of these animals were sacrificed on postnatal day ~95 to collect trunk blood and brain tissue samples for further analyses, and the rest of these animals were tested for the corticosterone responses to physical restraint for 30 min on postnatal day ~95 before collecting the brain tissue samples.

Behavioral Tests

Assessment of Behavior in the Elevated Plus Maze.

The elevated plus maze studies were performed using an elevated plus maze apparatus and BIO-EPM 3C video tracking software (EB Instruments, USA) during the light phase of the dark–light cycle as previously described by our laboratory.10,12,17  If a fall occurred, the animal was removed from the study (one F0 male rat was removed from the study for this reason).

Assessment of Prepulse Inhibition of Startle.

The prepulse inhibition of startle tests were performed using a SR-Lab startle apparatus (San Diego Instruments, USA) as previously described by our laboratory.10,12,17  The percentage of prepulse inhibition of startle for each prepulse level was calculated using the following formula: % prepulse inhibition of startle = 100 x [(pulse alone) – (prepulse + pulse)]/pulse alone. Data were collected as the highest voltage during the response window amplitude.

Basal and Stress-induced Activity of the Hypothalamic-Pituitary-Adrenal Axis.

Blood samples (~300 µl) were collected using the “tail clip” method at rest and 10, 60, and 120 min after the restraint. Physical restraint was administered using rodent holders (Kent Scientific Corporation, USA). Serum corticosterone was measured using commercial ELISA kits (Cayman Chemical Company, USA) according to the manufacturer’s instructions.

Tissue Collection.

After decapitation, the trunk blood samples were collected and centrifuged at 4°C, 1,000 g (where g is the relative centrifugal force) for 15 min, and then kept at –80°C for hormone assays. The brain was removed from the skull onto ice pads. The hypothalamus was isolated by making an anterior cut at the level of the optic chiasm, a posterior coronal section anterior to the mammillary bodies, two sagittal cuts parallel to the lateral ventricles, and a dorsal horizontal cut at the level of the anterior commissure, as described previously.18  The hippocampus was isolated from the respective blocks. All brain tissue samples were placed in vials filled with RNAlater solution (Invitrogen, USA) and then stored at –80°C. Testis tissue was removed and washed with 0.9% saline before weighing. Sperm were isolated from the caudal epididymis of adult males into phosphate-buffered saline with 1% bovine serum albumin solution at 37°C using a swim-up assay. After settling for 30 min, sperm-containing supernatant was centrifuged for 5 min at 4,000 rpm. Sperm pellets were stored at –80°C. After separation from the adipose tissues, ovaries were stored at –80°C.

Hormonal Measurements.

Hormone analyses in serum samples isolated from F0 and F1 rats were performed using commercially available kits according to the manufacturer’s instructions. Serum concentrations of follicle stimulating hormone (CSB-E06869r) and luteinizing hormone (CSB-E12654r) were quantified using ELISA kits (Cusabio Technology LLC, USA). Serum testosterone and estradiol concentrations were measured using ELISA kits (582701, Cayman Chemical Company, USA, and ES180S-100, Calbiotech, USA, respectively).

Quantitative mRNA Measurements.

Levels of mRNA for Nkcc1 and Kcc2 in the hypothalamus and hippocampus, for corticotropin-releasing hormone, estrogen receptor α, estrogen receptor β, aromatase, gonadotropin-releasing hormone in the hypothalamus, and for glucocorticoid receptor in the hippocampus were analyzed via reverse transcription–polymerase chain reaction in a StepOnePlus Real-Time polymerase chain reaction System (Applied Biosystems, USA) as previously described by our laboratory.10  RNA was extracted from the samples using an RNeasy Plus Kit (Qiagen, USA), reverse-transcribed with a high-capacity complementary DNA reverse transcription kit (Bio-Rad Laboratories, USA), and then analyzed via reverse transcription–polymerase chain reaction. Taqman probes specific for the genes described at the beginning of this section were obtained from Applied Biosystems: corticotropin-releasing hormone (Rn01462137_m1), glucocorticoid receptor (Rn00561369_m1), estrogen receptor α (Rn01430446_m1), estrogen receptor β (Rn00562610_m1), aromatase (Rn00567222_m1), gonadotropin-releasing hormone (Rn00562754_m1), Nkcc1 (Rn00582505_m1), and Kcc2 (Rn00592624_m1). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA (Rn01775763_g1). Gene expression was calculated using the “double delta Ct” method for relative quantification, and data are presented as relative fold change from that of control animals.

Bisulfite Sequencing.

Genomic DNA was extracted from the sperm pellet and ovaries of adult F0 rats and from hippocampal and hypothalamic tissues of F1 rats using the DNeasy Blood and Tissue kit (Qiagen, Germany). The sodium bisulfite conversion was performed with EZ DNA Methylation kits (Zymo Research, USA) following the manufacturer’s instructions. The primers (Kcc2: forward: GATTGTAAGTGTTTTTATTATTGAGTTGTATATT; reverse: AATAAACTTTTCCCCTTTTATACCC) were designed for the bisulfite-converted DNA sequences, using previously published sequences.10  Polymerase chain reaction amplification was performed with HotStar Taq (Qiagen, Germany). Amplicons were cloned into pCR4-TOPO vector with the TOPO TA cloning kit for sequencing (Life Technologies, USA). Miniprep was performed on each positive clone using ZR Plasmid Miniprep kit (Zymo Research, USA). Sanger sequencing was done by Genewiz (USA) using M13R primers. The DNA methylation status of all 5´ position of cytosine residues adjacent to guanines sites was analyzed using Benchling Molecular Biology 2.0 Software (Benchling, USA). The bisulfite sequencing analysis of the Kcc2 DNA was performed only in F1 male progeny of control males and control females and exposed males and exposed females matings.

Immunohistochemistry.

Rats were anesthetized with sevoflurane and transcardially perfused with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were collected and fixed in the 4% paraformaldehyde overnight and then dehydrated in 30% sucrose solution in phosphate-buffered saline at 4°C for 2 days. The brains were cut into 18-µm-thick coronal sections using a cryostat. After blocking with 10% normal goat serum for 1 h at room temperature, the slices were incubated with the primary antibody (rabbit anti-KCC2, 1:500; MilliporeSigma, USA) in 10% normal goat serum at 4°C overnight. After washing with PBS for 3 × 5 min, the slices were exposed to the secondary antibody, Alexa Fluor 549 goat anti-rabbit (Invitrogen, USA). Slides were then washed with phosphate-buffered saline for 3 × 5 min and incubated for 10 min at room temperature with 4´,6-diamidine-2´-phenylindole dihydrochloride (1:1,000; Sigma, USA) in phosphate-buffered saline for 10 min. The sections were mounted and covered with a coverslip after washing with phosphate-buffered saline for 3 × 5 min. A confocal microscope (IX2-DSU spinning disk confocal fluorescent microscope system, Olympus, Japan) was used to capture the fluorescent images. The immunofluorescence intensity of the KCC2-immunoreactive cells was measured using ImageJ software (National Institutes of Health, USA). Three images from the paraventricular nucleus of the hypothalamus of each animal were taken, and the average intensity values were calculated to use for statistical analysis.

Statistical Analysis

Statistical analyses were conducted on raw data using SigmaPlot 14.0 software (Systat Software, Inc., USA), which automatically checks if a data set meets test criteria (Shapiro–Wilk for normality test and Brown–Forsythe for Equal Variance Test). Values are reported as mean ± SD. The primary outcome in this study was the neurobehavioral changes in offspring of rats exposed to sevoflurane in young adulthood. All other outcome measures were the secondary outcomes. One F0 male rat was removed from the elevated plus maze study because of a fall from the maze. Another F0 male rat in the control group was removed from the behavioral studies because of injury while mating with a female rat. Boxplots were used to identify outliers. No outliers were detected that were in not plausible range of values for the outcome (indicating measurement or recording error). Therefore, all values were maintained in analyses. An independent t test was used to analyze F0 data for serum corticosterone levels and Kcc2 expression 1 h after the last exposure to sevoflurane, elevated plus maze, testis weight, hormone levels for testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone and gene expression for corticotropin-releasing hormone, glucocorticoid receptor, estrogen receptor α, estrogen receptor β, aromatase, gonadotropin-releasing hormone, Nkcc1, and Kcc2. All comparisons were run as two-tailed tests. To analyze F1 data for elevated plus maze, testis weight, testosterone level, and gene expression for corticotropin-releasing hormone, glucocorticoid receptor, Nkcc1, and Kcc2, one-way ANOVA was used. A two-way repeated-measures ANOVA was used to analyze the prepulse inhibition of startle data, with the treatment and prepulse intensity as independent variables. A two-way repeated-measures ANOVA was used to analyze changes in serum corticosterone levels at rest and at three time points after the restraint, with experimental groups and time as the independent variables. To assess differences in total corticosterone concentrations, area under the curve with respect to baseline (levels of corticosterone at rest), was calculated and compared across experimental groups using one-way ANOVA. Two-way repeated measures ANOVA with treatment as between-subject factor and the 5´ position of cytosine residues adjacent to guanines site as within-subject factor was used to analyze data on the frequency methylation of 5´ position of cytosine residues adjacent to guanines sites in the Kcc2 gene promoter in F0 and F1 tissue samples. An independent t test was used to analyze DNA methylation level at all six 5´ position of cytosine residues adjacent to guanines sites. All multiple pairwise comparisons were done with the Holm–Sidak method. P ≤ 0.05 was considered statistically significant. Statistical details are presented in text and in figure legends. The sample sizes in this study were based on previous experience with the same experimental techniques.10,11,17  This study was not designed to detect an anesthetic × sex interaction.

Results

Systemic Abnormalities in Male Offspring of Rats Exposed to Sevoflurane in Young Adulthood

In the elevated plus maze test, there was a statistically significant between-subjects effect of young adult parental exposure to sevoflurane on time spent in open arms (F[3,58] = 8.514, P < 0.001; fig. 2A) in F1 males. Specifically, F1 male progeny of sevoflurane exposed fathers and unexposed mothers (P =0.040), unexposed fathers and exposed mothers (P < 0.001), and exposed fathers and exposed mothers (P < 0.001) spent less time in the open arms compared to F1 male offspring of control fathers and control mothers. Also, there were between-subjects effects of parental exposure to sevoflurane on number of crossings (F[3,58] = 4.657, P =0.006; fig. 2B) and distance traveled (F[3,58] = 6.288, P < 0.001; fig. 2C) during the elevated plus maze test. Only F1 male offspring of both exposed parents made fewer crossings (P =0.003) and traveled shorter distances (P =0.001). All groups of F1 females were similar in respect to time spent in open arms (fig. 2D), number of crossings (fig. 2E), and distance traveled (fig. 2F) during the elevated plus maze test.

Fig. 2.

Systemic second-generation (F1) effects of young adult parental exposure to sevoflurane. (A–F) Time spent in open arms of the elevated plus maze, number of crossing the open arms, and distance traveled by male (A–C) and female (D–F) offspring. F1 rats were categorized as the offspring of (1) control males plus control females (con-M*con-F); (2) exposed males plus control females (sevo-M*con-F); (3) control males plus exposed females (con-M*sevo-F); and (4) exposed males plus exposed females (sevo-M*sevo-F). Data are means ± SD from 15 or 16 rats per group. *P < 0.05 versus con-M*con-F. (G and H) Percentage of prepulse inhibition of startle responses at prepulse (PP) intensity of 3, 6, and 12 decibel in male (G) and female (H) offspring. Data are means ± SD from 14–16 rats per group. *P < 0.05 versus con-M*con-F. (I–L) Plots showing the respective levels of serum corticosterone across each collection point, as well as the total corticosterone responses in male (I and J) and female (K and L) offspring. Serum levels of corticosterone at rest were taken as baselines for calculations of the total corticosterone responses. Data are means ± SD from eight rats per group. Multiple pairwise comparisons were done with the Holm–Sidak method. Color coding of experimental groups in J and L is also applicable to I and K.

Fig. 2.

Systemic second-generation (F1) effects of young adult parental exposure to sevoflurane. (A–F) Time spent in open arms of the elevated plus maze, number of crossing the open arms, and distance traveled by male (A–C) and female (D–F) offspring. F1 rats were categorized as the offspring of (1) control males plus control females (con-M*con-F); (2) exposed males plus control females (sevo-M*con-F); (3) control males plus exposed females (con-M*sevo-F); and (4) exposed males plus exposed females (sevo-M*sevo-F). Data are means ± SD from 15 or 16 rats per group. *P < 0.05 versus con-M*con-F. (G and H) Percentage of prepulse inhibition of startle responses at prepulse (PP) intensity of 3, 6, and 12 decibel in male (G) and female (H) offspring. Data are means ± SD from 14–16 rats per group. *P < 0.05 versus con-M*con-F. (I–L) Plots showing the respective levels of serum corticosterone across each collection point, as well as the total corticosterone responses in male (I and J) and female (K and L) offspring. Serum levels of corticosterone at rest were taken as baselines for calculations of the total corticosterone responses. Data are means ± SD from eight rats per group. Multiple pairwise comparisons were done with the Holm–Sidak method. Color coding of experimental groups in J and L is also applicable to I and K.

There was a statistically significant effect of young adult parental sevoflurane exposure on prepulse inhibition of startle responses in F1 male rats (F[3,174] = 7.590, P < 0.001; fig. 2G). Multiple pairwise comparisons indicated that when compared to offspring of unexposed parents, only F1 males of both exposed parents (P =0.009) or those of control fathers and exposed mothers (P =0.027) exhibited reduced prepulse inhibition of startle at prepulse of 3 decibel. In contrast to F1 males, there was no treatment effect on prepulse inhibition of startle in F1 female rats (F[3,168] = 0.584, P =0.627; fig. 2H). The startle amplitudes were similar among all experimental groups of F1 male (F[3,58] = 1.991, P =0.125) and F1 female (F[3,56] = 0.514, P =0.674) rats.

Measurements of serum levels of corticosterone in the postnatal day ~95 F1 male and female rats before and after physical restraint revealed no differences among experimental groups of the same sex (F[3,84] = 0.335, P =0.800, males; fig. 2, I and J; and F[3,84] = 0.142, P =0.934, females; fig. 2, K and L).

Reduction in the Potassium Ion-Chloride Ion Cotransporter KCC2 Gene (Kcc2) Expression in Male Offspring of Rats Exposed to Sevoflurane in Young Adulthood

There were similar levels of Nkcc1 mRNA in the hypothalamus of all four treatment groups of the postnatal day ~95 F1 male rats (F[3,20] = 0.928, P =0.446; fig. 3A), but there was a between-subjects effect of parental sevoflurane exposure on hypothalamic Kcc2 mRNA levels (F[3,20] = 5.636, P =0.006; fig. 3B). Specifically, F1 male progeny of sevoflurane-exposed fathers and mothers had lower levels of Kcc2 mRNA when compared to F1 male offspring of control fathers and control mothers (P =0.012). In the hypothalamus of F1 females, there were not between-subjects effects of parental sevoflurane exposure on Nkcc1 mRNA levels (F[3,19] = 0.886, P =0.466; fig. 3C) or Kcc2 mRNA levels (F[3,20] = 0.738, P =0.542; fig. 3D).

Fig. 3.

Molecular second-generation (F1) effects of young adult parental exposure to sevoflurane. (A–H) The respective levels of sodium ion-potassium ion-chloride ion cotransporter NKCC1 gene (Nkcc1) mRNA and potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) mRNA in the hypothalamus of F1 males (A and B) and F1 females (C and D) and in the hippocampus of F1 males (E and F) and F1 females (G and H). F1 rats were categorized as the offspring of (1) control males plus control females (con-M*con-F); (2) exposed males plus control females (sevo-M*con-F); (3) control males plus exposed females (con-M*sevo-F); and (4) exposed males plus exposed females (sevo-M*sevo-F). Data normalized against control are means ± SD from 6 rats per group (n = 5, female hypothalamic Nkcc1 in con-M*con-F). *P < 0.05 versus con-M*con-F. (I–L) The levels of corticotropin-releasing hormone gene (Crh) mRNA in the hypothalamus and glucocorticoid receptor gene (Gr) mRNA in the hippocampus in F1 males (I and J) and F1 females (K and L). Data normalized against control are means ± SD from 5 or 6 rats per group. Multiple pairwise comparisons were done with the Holm–Sidak method.

Fig. 3.

Molecular second-generation (F1) effects of young adult parental exposure to sevoflurane. (A–H) The respective levels of sodium ion-potassium ion-chloride ion cotransporter NKCC1 gene (Nkcc1) mRNA and potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) mRNA in the hypothalamus of F1 males (A and B) and F1 females (C and D) and in the hippocampus of F1 males (E and F) and F1 females (G and H). F1 rats were categorized as the offspring of (1) control males plus control females (con-M*con-F); (2) exposed males plus control females (sevo-M*con-F); (3) control males plus exposed females (con-M*sevo-F); and (4) exposed males plus exposed females (sevo-M*sevo-F). Data normalized against control are means ± SD from 6 rats per group (n = 5, female hypothalamic Nkcc1 in con-M*con-F). *P < 0.05 versus con-M*con-F. (I–L) The levels of corticotropin-releasing hormone gene (Crh) mRNA in the hypothalamus and glucocorticoid receptor gene (Gr) mRNA in the hippocampus in F1 males (I and J) and F1 females (K and L). Data normalized against control are means ± SD from 5 or 6 rats per group. Multiple pairwise comparisons were done with the Holm–Sidak method.

In the hippocampus of F1 males, there were no effects of parental sevoflurane exposure on Nkcc1 mRNA levels (F[3,20] = 1.357, P =0.284; fig. 3E), but there was a significant effect on Kcc2 mRNA levels (F[3,20] = 41.375, P < 0.001; fig. 3F). Only male offspring of both exposed parents (P < 0.001) and offspring of exposed fathers and control mothers (P < 0.001) had reduced hippocampal Kcc2 mRNA levels compared to offspring of two control parents. In the hippocampus of F1 females, there were not significant between-subjects effects of parental neonatal sevoflurane exposure on Nkcc1 mRNA level (F[3,20] = 0.925, P =0.447; fig. 3G) or Kcc2 mRNA level (F[3,20] = 0.524, P =0.671; fig. 3H).

Consistent with the observations of normal levels of corticosterone (fig. 2, I and J), levels of corticotropin-releasing hormone mRNA in the hypothalamus (F[3,20] = 0.519, P =0.674; fig. 3I) and glucocorticoid receptor in the hippocampus (F[3,19] = 3.293, P =0.043; fig. 3J) of postnatal day ~95 F1 males were not different among all four treatment groups. In addition, there were no significant between-subjects effects of parental sevoflurane exposure on either hypothalamic corticotropin-releasing hormone mRNA (F[3,20] = 1.225, P =0.327; fig. 3K) or hippocampal glucocorticoid receptor mRNA levels (F[3,19] = 0.860, P =0.479; fig. 3L) in F1 female rats.

Elevated DNA Methylation of the Potassium Ion-Chloride Ion Cotransporter KCC2 Gene (Kcc2) Expression Promoter Region in Sperm and Ovary of Rats Exposed to Sevoflurane in Young Adulthood and in the Hypothalamus and Hippocampus of Their Offspring

Increased DNA methylation in the Kcc2 promoter in gamete cells of adult rats exposed to sevoflurane as neonates and in the hypothalamus and hippocampus of their male offspring has recently been described.10  We therefore sought to determine if similar effects would be observed in response to young adult exposure to sevoflurane. There was a significant effect of treatment (F[1,36] = 30.8, P < 0.001) and within-subjects effect of the 5´ position of cytosine residues adjacent to guanines site (F[5,36] = 20.066, P < 0.001) on the frequency of 5´ position of cytosine residues adjacent to guanines methylation in the Kcc2 promoter region in sperm of the postnatal day ~160 F0 rats (fig. 4A). There was also a statistically significant interaction between the 5´ position of cytosine residues adjacent to guanines site and treatment (F[5,36] = 10.036, P < 0.001). Pairwise multiple comparison analysis showed that young adult exposure to sevoflurane led to increased methylation in F0 male rats at 5´ position of cytosine residues adjacent to guanines site 1 (P < 0.001 vs. control) and 5´ position of cytosine residues adjacent to guanines site 2 (P =0.006 vs. control). Similarly, there was a significant effect of treatment (F[1,36] = 34.714, P < 0.001) and within-subjects effect of 5´ position of cytosine residues adjacent to guanines site (F[5,36] = 42.686, P < 0.001) on the frequency of 5´ position of cytosine residues adjacent to guanines site methylation in the Kcc2 gene promoter in ovarian tissue of F0 rats (fig. 4B). There was also a statistically significant interaction between 5´ position of cytosine residues adjacent to guanines site and treatment (F[5,36] = 12.343, P < 0.001). Pairwise multiple comparison analysis showed that young adult exposure to sevoflurane led to increased methylation in F0 female rats at the 5´ position of cytosine residues adjacent to guanines site 1 (P < 0.001 vs. control) and 5´ position of cytosine residues adjacent to guanines site 2 (P < 0.001 vs. control).

Fig. 4.

DNA methylation in the promoter region of potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) in parental (F0) sperm and ovary and in hypothalamus and hippocampus of their offspring (F1). (A, a–d) Bisulfite sequencing of the 5´ position of cytosine residues adjacent to guanines dinucleotides (CpG sites) in the Kcc2 gene of 10 clones from four individual sperm DNA samples isolated from control and sevoflurane-exposed F0 male rats. Heat maps show distribution of unmethylated (blue cells) and methylated (red cells) CpG sites in sperm DNA samples isolated from control (A, a) and sevoflurane-exposed (A, b) F0 male rats. Histograms showing methylation frequency at each CpG site (A, c) and DNA methylation level at all six CpG sites (A, d). The results of similar analysis of bisulfite sequencing of CpG dinucleotides in the Kcc2 gene of 10 clones from four individual ovary DNA samples isolated from sevoflurane-exposed and control F0 female rats shown in B, a–d. Color coding of experimental groups in A, d, and B, d, is also applicable to A, c, and B, c. (C and D) Shown are the respective methylation frequency at each CpG site and DNA methylation level at all six CpG sites in the Kcc2 gene of 10 clones in the hypothalamus (C, a–d) and hippocampus (D, a–d) of F1 male offspring of control sires and control dams (con-M*con-F) and of sevoflurane-exposed sires and sevoflurane-exposed dams (sevo-M*sevo-F). Data are means ± SD from 4 rats per group. *P < 0.05 versus Control (A and B) and versus con-M*con-F (C and D). Color coding of experimental groups in C, d, and D, d, is also applicable to C, c, and D, c. Multiple pairwise comparisons were done with the Holm–Sidak method.

Fig. 4.

DNA methylation in the promoter region of potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) in parental (F0) sperm and ovary and in hypothalamus and hippocampus of their offspring (F1). (A, a–d) Bisulfite sequencing of the 5´ position of cytosine residues adjacent to guanines dinucleotides (CpG sites) in the Kcc2 gene of 10 clones from four individual sperm DNA samples isolated from control and sevoflurane-exposed F0 male rats. Heat maps show distribution of unmethylated (blue cells) and methylated (red cells) CpG sites in sperm DNA samples isolated from control (A, a) and sevoflurane-exposed (A, b) F0 male rats. Histograms showing methylation frequency at each CpG site (A, c) and DNA methylation level at all six CpG sites (A, d). The results of similar analysis of bisulfite sequencing of CpG dinucleotides in the Kcc2 gene of 10 clones from four individual ovary DNA samples isolated from sevoflurane-exposed and control F0 female rats shown in B, a–d. Color coding of experimental groups in A, d, and B, d, is also applicable to A, c, and B, c. (C and D) Shown are the respective methylation frequency at each CpG site and DNA methylation level at all six CpG sites in the Kcc2 gene of 10 clones in the hypothalamus (C, a–d) and hippocampus (D, a–d) of F1 male offspring of control sires and control dams (con-M*con-F) and of sevoflurane-exposed sires and sevoflurane-exposed dams (sevo-M*sevo-F). Data are means ± SD from 4 rats per group. *P < 0.05 versus Control (A and B) and versus con-M*con-F (C and D). Color coding of experimental groups in C, d, and D, d, is also applicable to C, c, and D, c. Multiple pairwise comparisons were done with the Holm–Sidak method.

There was a between-subjects effect of parental treatment on 5´ position of cytosine residues adjacent to guanines site methylation in the promoter of Kcc2 gene in the hypothalamus of the postnatal day ~95 male offspring of both exposed parents (F[1,36] = 48.286, P < 0.001), and a within-subject effect of 5´ position of cytosine residues adjacent to guanines site (F[5,36] = 42.629, P < 0.001; fig. 4C). There was also a statistically significant interaction between 5´ position of cytosine residues adjacent to guanines site and treatment (F[5,36] = 15.886, P < 0.001). Pairwise multiple comparison analyses showed that young adult parental exposure to sevoflurane led to increased DNA methylation in the Kcc2 gene promoter in the hypothalamus of F1 male progeny of both exposed parents at 5´ position of cytosine residues adjacent to guanines site 1 (P < 0.001 vs. F1 males from the control males and control females group), 5´ position of cytosine residues adjacent to guanines site 2 (P =0.002 vs. F1 males from the control males and control females group), and 5´ position of cytosine residues adjacent to guanines site 3 (P =0.013 vs. F1 males from the control males and control females group).

There was also a between-subjects effect of parental treatment on 5´ position of cytosine residues adjacent to guanines site methylation in the promoter of Kcc2 gene in the hippocampus of male offspring of both exposed parents (F[1,36] = 21.740, P < 0.001, and within-subject effect of 5´ position of cytosine residues adjacent to guanines site (F[5,36] = 20.852, P < 0.001; fig. 4D). There was also a statistically significant interaction between 5´ position of cytosine residues adjacent to guanines site and treatment (F[5,36] = 5.268, P < 0.001). Pairwise multiple comparison analyses showed that young adult parental exposure to sevoflurane led to increased DNA methylation in the Kcc2 gene promoter in the hippocampus of F1 male progeny of both exposed parents at 5´ position of cytosine residues adjacent to guanines site 1 (P < 0.001 vs. control males and control females), 5´ position of cytosine residues adjacent to guanines site 2 (P =0.018 vs. control males and control females), and 5´ position of cytosine residues adjacent to guanines site 3 (P =0.018 vs. control males and control females).

Systemic Abnormalities in Male Rats Exposed to Sevoflurane in Young Adulthood

Unexpectedly, we found that more than 2 months after exposure to sevoflurane anesthesia, F0 male rats exhibited behavioral deficiencies. They spent less time in the open arms of the elevated plus maze (t(28) = 2.18; P =0.038; fig. 5A), but did not differ from their control counterparts in number of crossings (t(28) = 1.456; P =0.157; fig. 5B) or distance traveled (t(28) = –1.351; P =0.188; fig. 5C). In F0 females, there was no significant effect of sevoflurane on any of these elevated plus maze parameters (fig. 5, D–F).

Fig. 5.

Systemic first generation (F0) effects of young adult exposure to sevoflurane. The percentage of time spent in open arms of the elevated plus maze, number of crossing the open arms, and distance traveled by male (A–C) and female (D–F) rats. Data are means ± SD from 15 male and 16 female rats per group. (G and H) The percentage of prepulse (PP) inhibition of startle responses at PP intensity of 3, 6, and 12 decibel in male (G) and female (H) rats. Data are means ± SD from 21 males in the Control group, 22 males in the Sevoflurane group, and 16 females per group. *P < 0.05 versus Control. (I–L) The respective levels of serum corticosterone across each collection point, as well as the total corticosterone responses in male (I and J) and female (K and L) rats. Serum levels of corticosterone at rest were taken as baselines for calculations of the total corticosterone responses. Data are means ± SD from 8 rats per group. Color coding of experimental groups in J and L is also applicable to I and K. (M–O) shown are serum levels of testosterone (M), estradiol (N), and testis weight (O) in male rats. (P and Q) Serum levels of testosterone and estradiol in female rats. Data are means ± SD from 8 rats per group. Data of testis weight are from 16 rats in control group and 13 rats in sevoflurane group. *P < 0.05 versus Control.

Fig. 5.

Systemic first generation (F0) effects of young adult exposure to sevoflurane. The percentage of time spent in open arms of the elevated plus maze, number of crossing the open arms, and distance traveled by male (A–C) and female (D–F) rats. Data are means ± SD from 15 male and 16 female rats per group. (G and H) The percentage of prepulse (PP) inhibition of startle responses at PP intensity of 3, 6, and 12 decibel in male (G) and female (H) rats. Data are means ± SD from 21 males in the Control group, 22 males in the Sevoflurane group, and 16 females per group. *P < 0.05 versus Control. (I–L) The respective levels of serum corticosterone across each collection point, as well as the total corticosterone responses in male (I and J) and female (K and L) rats. Serum levels of corticosterone at rest were taken as baselines for calculations of the total corticosterone responses. Data are means ± SD from 8 rats per group. Color coding of experimental groups in J and L is also applicable to I and K. (M–O) shown are serum levels of testosterone (M), estradiol (N), and testis weight (O) in male rats. (P and Q) Serum levels of testosterone and estradiol in female rats. Data are means ± SD from 8 rats per group. Data of testis weight are from 16 rats in control group and 13 rats in sevoflurane group. *P < 0.05 versus Control.

As in the elevated plus maze task, there was a significant effect of sevoflurane exposure on prepulse inhibition of startle in adult F0 male rats (F[1,123] = 6.765; P =0.010; fig. 5G), but not in F0 female rats (F[1,60] = 0.049; P =0.827; fig. 5H). Multiple pairwise comparisons indicated that exposure to sevoflurane led to impaired prepulse inhibition of startle responses in F0 male rats at prepulse intensity of 3 decibel (P =0.042 vs. control). Startle stimuli by themselves caused similar responses in the control and sevoflurane groups of F0 male (t(41) = –0.969; P =0.338) and F0 female (t(30) = 1.465; P =0.153) rats.

Male F0 rats had higher total corticosterone responses to physical restraint on postnatal day ~160 when compared to their control counterparts (t(14) = –6.209; P < 0.001; fig. 5, I and J). This increase was due to higher levels of corticosterone at 10 min (P < 0.001 vs. control) and 60 min (P =0.036 vs. control) after restraint, as serum levels of corticosterone before the restraint (P =0.736 vs. control) and 120 min (P =0.787 vs. control) postrestraint were not different in control and sevoflurane-exposed rats (fig. 5I). There was no difference in serum corticosterone levels between control and sevoflurane-exposed F0 female rats (fig. 5, K and L).

During collection of sperm for the DNA methylation studies, we incidentally found that sevoflurane-exposed F0 males had reduced testis weight (t(27) = 4.494; P < 0.001; fig. 5N) more than 3 months after sevoflurane exposure. Counterintuitively, their serum levels of testosterone were increased (t(14) = –2.839; P =0.013; fig. 5M), although their serum levels of estradiol were normal (t(14) = 0.703; P =0.494; fig. 5O). F0 female rats in the sevoflurane and control groups were not different with respect to serum levels of testosterone (P =0.743; fig. 5P) or estradiol (P =0.600; fig. 5Q).

Reduction in the Potassium Ion-Chloride Ion Cotransporter KCC2 Expression in Male Rats 1 h after the Last Exposure to Sevoflurane

Our current and recently published findings10  indicate that rats exposed to sevoflurane in young adulthood and in the early postnatal period develop comparable systemic abnormalities. Given that sevoflurane acts as a stressor in neonatal rats and initiates developmental abnormalities, at least in part, by potentiating excitatory GABAA receptor signaling,10–12,17  here we tested whether sevoflurane can induce a stress-like19–22  reduction in KCC2 expression in young adult rats. To measure acute effects of sevoflurane, rats were exposed to 2.1% sevoflurane anesthesia for 3 h on postnatal day 56, postnatal day 58, and postnatal day 60. Brain tissue and trunk blood samples were collected 1 h after sevoflurane anesthesia on postnatal day 60. Consistent with the stress-like effects of sevoflurane reported in previous work,10–12,17  the exposed male and female rats had increased serum levels of corticosterone compared to controls (t(6) = –3.313; P = 0.016, males, fig. 6A; and t(8) = –3.949; P = 0.004, females; fig. 6B). Despite similar increases in corticosterone levels in sevoflurane-exposed males and females, immunofluorescence evaluations of KCC2 expression in the paraventricular nucleus of the hypothalamus region found reductions in KCC2 level in male (t(6) = 3.343; P = 0.016), but not female (t(6) = 0.773; P = 0.469), F0 rats (fig. 6, C–F).

Fig. 6.

Acute effects of young adult sevoflurane exposure. (A and B) The respective levels of serum corticosterone in male (A) and female (B) rats. Data are means ± SD (n = 4 and n = 5 per treatment group in males and females, respectively). *P < 0.05 versus Control. (C–F) Representative confocal images and quantitative analysis of the potassium ion-chloride ion cotransporter KCC2 immunoreactivity in the paraventricular nucleus (PVN) of the hypothalamus of the control and sevoflurane-exposed male (C and E) and female (D and F) rats. (E) Representative confocal images of the PVN from the control and sevoflurane-exposed male rats, immunostained for 4´,6-diamidine-2´-phenylindole dihydrochloride (DAPI; left; blue) and KCC2 (middle; red); the merge column shows colocalized images (right; red and blue). The arrowheads indicate the cells shown in the boxed areas at higher magnifications. The KCC2 immunoreactivity, located on the periphery of the neurons (red color), decreased in the PVN neurons from the sevoflurane-exposed male rats. Similar representative confocal images of the PVN from the control and sevoflurane-exposed female rats shown in (F). Scale bars, 50 µm. The sevoflurane-exposed males, but not sevoflurane-exposed females, had reduced KCC2 expression (*P < 0.05 vs. Control; C and E). Data are means ± SD (n = 4 rats per group).

Fig. 6.

Acute effects of young adult sevoflurane exposure. (A and B) The respective levels of serum corticosterone in male (A) and female (B) rats. Data are means ± SD (n = 4 and n = 5 per treatment group in males and females, respectively). *P < 0.05 versus Control. (C–F) Representative confocal images and quantitative analysis of the potassium ion-chloride ion cotransporter KCC2 immunoreactivity in the paraventricular nucleus (PVN) of the hypothalamus of the control and sevoflurane-exposed male (C and E) and female (D and F) rats. (E) Representative confocal images of the PVN from the control and sevoflurane-exposed male rats, immunostained for 4´,6-diamidine-2´-phenylindole dihydrochloride (DAPI; left; blue) and KCC2 (middle; red); the merge column shows colocalized images (right; red and blue). The arrowheads indicate the cells shown in the boxed areas at higher magnifications. The KCC2 immunoreactivity, located on the periphery of the neurons (red color), decreased in the PVN neurons from the sevoflurane-exposed male rats. Similar representative confocal images of the PVN from the control and sevoflurane-exposed female rats shown in (F). Scale bars, 50 µm. The sevoflurane-exposed males, but not sevoflurane-exposed females, had reduced KCC2 expression (*P < 0.05 vs. Control; C and E). Data are means ± SD (n = 4 rats per group).

Abnormalities in the Hypothalamic-Pituitary-Gonadal and Hypothalamic-Pituitary-Adrenal Axes in Male Rats Exposed to Sevoflurane in Young Adulthood

Acute KCC2 and long-term systemic effects of young adult sevoflurane in the exposed males only suggest an involvement of sex steroids. To test whether sevoflurane-induced increases in serum testosterone levels involve changes in functioning of the entire hypothalamic-pituitary-gonadal axis, we measured the expression of gonadotropin-releasing hormone in the hypothalamus and serum levels of follicle-stimulating hormone and luteinizing hormone. Consistent with the finding that sevoflurane increased systemic levels of testosterone in postnatal day ~160 F0 male rats, these same rats also had increased hypothalamic levels of gonadotropin-releasing hormone mRNA (t(10) = –2.519; P =0.030; fig. 7A) and serum levels of luteinizing hormone (t(14) = –4.932; P < 0.001; fig. 7B), while serum levels of follicle-stimulating hormone (t(10) = 1.026; P =0.329; fig. 7C) were not different from those in F0 control male rats. There were no treatment effects on the hypothalamic levels of gonadotropin-releasing hormone mRNA (fig. 7D) or serum levels of luteinizing hormone (fig. 7E) and follicle-stimulating hormone (fig. 7F) in F0 female rats.

Fig. 7.

Molecular first generation (F0) effects of young adult exposure to sevoflurane. (A–F) The levels of hypothalamic gonadotropin-releasing hormone (Gnrh) mRNA, serum levels of luteinizing hormone and follicle-stimulating hormone of male (A–C) and female (D–F) rats. Data are means ± SD from 8 rats per group (n = 6 rats per group in follicle-stimulating hormone). *P < 0.05 versus Control. (G–L) shown are levels of aromatase mRNA, estrogen receptor α gene (Erα) mRNA, and estrogen receptor β gene (Erβ) mRNA in the hypothalamus of male (G–I) and female (J–L) rats. Data normalized against control are means ± SD from 6 rats per group. *P < 0.05 versus Control. (M–P) Shown are the respective levels of hypothalamic corticotropin-releasing hormone gene (Gnrh) mRNA and hippocampal glucocorticoid receptor gene (Gr) mRNA in males (M and N) and females (O and P). Data normalized against control are means ± SD from 6 rats per group (n = 8, male corticotropin-releasing hormone; n = 5 in the Control group and n = 4 in the Sevoflurane group in female glucocorticoid receptor). *P < 0.05 versus Control.

Fig. 7.

Molecular first generation (F0) effects of young adult exposure to sevoflurane. (A–F) The levels of hypothalamic gonadotropin-releasing hormone (Gnrh) mRNA, serum levels of luteinizing hormone and follicle-stimulating hormone of male (A–C) and female (D–F) rats. Data are means ± SD from 8 rats per group (n = 6 rats per group in follicle-stimulating hormone). *P < 0.05 versus Control. (G–L) shown are levels of aromatase mRNA, estrogen receptor α gene (Erα) mRNA, and estrogen receptor β gene (Erβ) mRNA in the hypothalamus of male (G–I) and female (J–L) rats. Data normalized against control are means ± SD from 6 rats per group. *P < 0.05 versus Control. (M–P) Shown are the respective levels of hypothalamic corticotropin-releasing hormone gene (Gnrh) mRNA and hippocampal glucocorticoid receptor gene (Gr) mRNA in males (M and N) and females (O and P). Data normalized against control are means ± SD from 6 rats per group (n = 8, male corticotropin-releasing hormone; n = 5 in the Control group and n = 4 in the Sevoflurane group in female glucocorticoid receptor). *P < 0.05 versus Control.

The elevated testosterone may modulate the hypothalamic-pituitary-adrenal axis responses to stress through estrogen receptors after testosterone aromatization to estradiol in the brain. One such mechanism includes modulation by estradiol of the glucocorticoid receptor–mediated negative feedback effect of corticosterone on the hypothalamic-pituitary-adrenal axis activity. Estradiol can both inhibit and enhance the negative feedback effects of glucocorticoids by activating estrogen receptor α and estrogen receptor β, respectively.23,24  In further support of involvement of testosterone and estradiol in exacerbated hypothalamic-pituitary-adrenal axis responses to stress, we found that the hypothalamic levels of aromatase mRNA were increased in F0 sevoflurane-exposed males (t(10) = –4.333; P =0.002; fig. 7G). In addition, the F0 male rats exposed to sevoflurane had increased and decreased hypothalamic levels of estrogen receptor α mRNA (t(10) = –5.144; P < 0.001; fig. 7H) and estrogen receptor β mRNA (t(10) = 3.156; P =0.010; fig. 7I), respectively. Again, consistent with the normal hypothalamic-pituitary-adrenal axis responses to stress in the exposed F0 females, the increase in the hypothalamic aromatase mRNA in sevoflurane-exposed F0 female rats did not achieve statistical significance (t(10) = –1.878; P =0.090; fig. 7J). Moreover, in the F0 females, the hypothalamic levels of estrogen receptor α mRNA (fig. 7K) were not different between Sevoflurane and Control groups, while those of estrogen receptor β mRNA were slightly, though significantly increased in the Sevoflurane group (t(10) = –2.521; P =0.030; fig. 7L) when compared to the Control group.

In agreement with the exacerbated corticosterone responses to stress in sevoflurane-exposed F0 male rats at postnatal day ~160, these rats had increased hypothalamic corticotropin-releasing hormone mRNA levels 2 h after the restraint (t(14) = –3.181; P =0.007; fig. 7M), as well as reduced levels of glucocorticoid receptor mRNA in the hippocampus (t(10) = 2.493; P =0.032; fig. 7N). Neither hypothalamic corticotropin-releasing hormone mRNA levels (fig. 7O) nor hippocampal glucocorticoid receptor mRNA levels (fig. 7P) were different in sevoflurane-exposed and control F0 female rats.

Finally, considering the controlling roles of GABAA receptor signaling in hypothalamic gonadotropin-releasing hormone and corticotropin-releasing hormone neuronal activity, it is plausible that sevoflurane initiates enhancements of gonadotropin-releasing hormone and corticotropin-releasing hormone neuronal activity and, hence, the entire reproductive and stress axes, by downregulating Kcc2 expression and rendering GABAA receptor signaling less inhibitory or even excitatory. To assess whether exposure of young adult rats to sevoflurane led to persistent alterations in expression of Cl transporters, brain hypothalamic and hippocampal tissue samples were collected more than 3 months after exposure (postnatal day ~160). The F0 male rats from the Sevoflurane group had normal Nkcc1 mRNA levels (t(10) = 1.065; P =0.312; fig 8A), but decreased Kcc2 mRNA levels (t(10) = 2.273; P =0.046; fig. 8B) in the hypothalamus. In contrast, the F0 female rats from the Sevoflurane group had unaltered levels of both Nkcc1 mRNA (t(10) = 0.155; P =0.880; fig. 8C) and Kcc2 mRNA (t(10) = 1.346; P =0.208; fig. 8D) in the hypothalamus. In the hippocampus of F0 male rats from the Sevoflurane group, Kcc2 mRNA levels, but not Nkcc1 mRNA levels, were significantly reduced (t(10) = 2.387, P =0.038, fig. 8, E and F). The hippocampal mRNA levels for Nkcc1 and Kcc2 were similar in control and sevoflurane-exposed F0 female rats (fig. 8, G and H).

Fig. 8.

Effects of sevoflurane exposure on hypothalamic and hippocampal potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) and sodium ion-potassium ion-chloride ion cotransporter NKCC1 gene (Nkcc1) mRNA in F0 rats. (A–H) Shown are the respective levels of Nkcc1 mRNA and Kcc2 mRNA in the hypothalamus of F0 males (A and B) and F0 females (C and D) and in the hippocampus of F0 males (E and F) and F0 females (G and H). Data normalized against control are means ± SD from 6 rats per group (n = 5, female hippocampus in sevoflurane group). *P < 0.05 versus Control.

Fig. 8.

Effects of sevoflurane exposure on hypothalamic and hippocampal potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) and sodium ion-potassium ion-chloride ion cotransporter NKCC1 gene (Nkcc1) mRNA in F0 rats. (A–H) Shown are the respective levels of Nkcc1 mRNA and Kcc2 mRNA in the hypothalamus of F0 males (A and B) and F0 females (C and D) and in the hippocampus of F0 males (E and F) and F0 females (G and H). Data normalized against control are means ± SD from 6 rats per group (n = 5, female hippocampus in sevoflurane group). *P < 0.05 versus Control.

Discussion

The results of these experiments show that sevoflurane administered to young adult rats of either sex can induce abnormalities in their male offspring, as well as in the exposed male rats themselves. The reduction in Kcc2 expression in the hypothalamus and hippocampus of the F1 male progeny of the exposed parents, as well as hypermethylation of the Kcc2 promoter in F0 male and female gamete cells and F1 male hypothalamus and hippocampus, support the involvement of epigenetic mechanisms in transmitting adverse effects of sevoflurane exposure in young adult rats to the next generation. The findings that both the physiology and behavior of sevoflurane-exposed F0 females were normal, while their ovarian Kcc2 promoter was hypermethylated and male progeny of exposed F0 females exhibited neurobehavioral abnormalities, suggest that sevoflurane affects somatic and germ cells via different mechanisms.

Intergenerational Effects of Young Adult Sevoflurane

Human and animal studies provide evidence that exposure to alcohol, endocrine disruptors, and stress can affect future generations.10,12–15,25–27  Surprisingly, the heritable effects of general anesthetics are poorly studied, despite the fact that anesthetic agents share molecular mechanisms of action with alcohol and may act as both environmental stressors and endocrine disruptors.10–12,17,27–29  Anecdotal observations point to the possibility of heritable effects of anesthesia and surgery in humans.30  Some evidence of heritable effects of general anesthetics comes from clinical surveys indicating that anesthesia care providers may have altered female:male offspring ratios.31–35  Importantly, sex ratio effects have been linked to alcohol, stress, and endocrine disruptors as well.36–38  Our recently published10  and current findings support the possibility that sevoflurane can induce intergenerational effects. Obviously, sevoflurane-induced epigenetic modulation of Kcc2 in F0 gamete cells and F1 male brain may contribute to the intergenerational effects of young adult sevoflurane exposure, but it is unlikely that sevoflurane-induced changes in Kcc2 are the only mediating mechanism. Indeed, this study found that male progeny of exposed dams and control sires had normal Kcc2 mRNA levels in the hypothalamus and hippocampus but exhibited deficiencies in the elevated plus maze and prepulse inhibition of startle behavioral tests. It is possible that Kcc2 DNA methylation in gamete cells affects other epigenetic mechanisms that program offspring brain development, and/or that multiple genes are independently epigenetically modified by sevoflurane in F0 gamete cells and F1 brain. Furthermore, the epigenetic effects of GABAergic anesthetics may not be limited to DNA methylation, as we and others recently reported experimental evidence for involvement of not only DNA methylation,10,39  but also histone acetylation,40,41  in adverse effects of neonatal sevoflurane exposure in rats.

Despite different anesthesia regimens in our recent study of intergenerational effects of neonatal anesthesia10  and the young adult anesthesia in the current study, similarities in the intergenerational effects of sevoflurane outweighed differences between the two models. These findings are also consistent with those reported by Rodgers et al. that 42-day exposure of male rats to stress either throughout puberty or in adulthood leads to similar blunted hypothalamic-pituitary-adrenal axis responses in their progeny.14  Moreover, repeated exposure of 11-week-old male mice to the anesthetic agent enflurane negatively affected their offspring’s behavior.42  The susceptibility of germ cell maturation to sevoflurane over an extended period of the lifespan, from the early postnatal period10  through young adulthood (this study), suggests that the gamete cells at different stages of maturation may be susceptible to epigenetic modifications initiated by sevoflurane. It will be important in future studies to analyze epigenetic effects of parental exposure to anesthetics in F0 oocytes, which represent only a minor part of total ovarian tissue that was tested in this study.

Irrespective of whether fathers, mothers, or both parents were exposed to sevoflurane as young adults, only male F1 progeny exhibited epigenetic, gene expression, and systemic abnormalities. Given that Kcc2 was hypermethylated in both male and female gamete cells in F0 rats, it is plausible that parental sevoflurane initiates mechanisms leading to disruption of embryonic DNA methylation reprogramming in F1 males, but not in F1 females, resulting in affected F1 males but normal F1 females. Our findings that the exposed but physiologically unaffected dams, similar to exposed and affected sires, pass deleterious effects of sevoflurane to unexposed male offspring raise the possibility that male offspring may be affected even when anesthesia level or duration is not sufficient to induce significant abnormalities in the exposed parents.

Adverse Effects of Young Adult Sevoflurane in the Exposed Rats

The long-term adverse effects of general anesthesia in early childhood and at advanced ages are a widely recognized health-related concern and the subject of extensive clinical and laboratory research.43  Investigations of such effects in young adults in their prime reproductive age, however, are relatively scarce. Several studies have assessed effects of isoflurane in young adult rats, primarily using young adult rats as comparisons to other age groups. Isoflurane administered to postnatal day 60 rats affected progenitor proliferation and improved spatial memory in one study and had no effect in other one.44,45  Crosby et al. also observed improvement in spatial memory in rats anesthetized with 1.2% isoflurane–70% nitrous oxide at 6 months of age.46  Aside from the fact that two studies found long-term effects of isoflurane in young adult rats, different isoflurane concentrations and exposure regimens make it difficult to compare the effects across these studies. Such comparison is even more problematic with our current findings in F0 males, given that we tested not only a distinct anesthesia regimen, but also a different anesthetic agent. Clearly, further research is needed to elucidate the full range of long-term effects of young adult sevoflurane anesthesia and mechanisms that mediate such effects.

The GABAA receptor/testosterone/aromatase/estradiol/KCC2 pathway may be a key mediator of sevoflurane’s long-term neuroendocrine effects in F0 male rats. Because many gonadotropin-releasing hormone neurons are excited by GABAA receptor signaling even under basal conditions,47,48  sevoflurane may initially stimulate gonadotropin-releasing hormone neuronal activity and, hence, the entire reproductive axis. Male-specific factors may be required for sevoflurane’s actions to acutely reduce KCC2 expression, as similar increases in corticosterone levels in sevoflurane-exposed female rats were not accompanied by a reduction in KCC2 expression. The GABAA receptor/testosterone/aromatase/estradiol/KCC2 pathway in F0 male rats may function as a system with a positive feedback effect leading to a persistently upregulated hypothalamic-pituitary-gonadal axis. Sevoflurane-induced increases in systemic testosterone and in brain aromatase expression may lead to reductions in Kcc2 expression, and in turn to diminished inhibitory or increased stimulatory control of gonadotropin-releasing hormone neurons by KCC2/GABAA receptor signaling. The plausibility of this scenario is supported by literature demonstrating that estradiol increases expression of aromatase and decreases expression of KCC2 in the brain.49,50  Interestingly, consistent with our findings, Galanopoulou and Moshé found that estradiol reduced KCC2 expression in males only.50 

The GABAA receptor/testosterone/aromatase/estradiol/KCC2 pathway is also likely to be involved in dysregulated (exacerbated) hypothalamic-pituitary-adrenal axis responses to stress and stress-dependent behavioral abnormalities. One such mechanism includes modulation by estradiol of the glucocorticoid receptor–mediated negative feedback effect of corticosterone on hypothalamic-pituitary-adrenal axis activity. Estradiol can both inhibit and enhance the negative feedback effects of glucocorticoids by activating estrogen receptor α and estrogen receptor β, respectively.23,24  In support of this mechanism are our findings that hypothalamic levels of aromatase and estrogen receptor α mRNA were elevated, whereas hypothalamic levels of estrogen receptor β mRNA and hippocampal levels of glucocorticoid receptor mRNA were downregulated. Higher estradiol concentrations are thought to contribute to higher corticosterone at rest or after stress exposure in adult female rats when compared to their male counterparts.51–53  Hence, our findings of exacerbated corticosterone responses to stress in F0 males, but not F0 females, suggest that exposure to sevoflurane in young adulthood induces a persistent transformation of the male stress response to a more “female-like” form. Upregulated expression of hypothalamic aromatase and resulting elevated levels of estradiol may also explain why in sevoflurane-exposed males, elevated levels of testosterone were accompanied by exacerbated hypothalamic-pituitary-adrenal axis responses to stress, because it was previously shown that gonadectomy of male rats elevated, while androgen replacement blunted the corticosterone response to stress.54  The persistent downregulation of Kcc2 expression and resulting GABAA receptor depolarizing and stimulatory signaling in the hypothalamic paraventricular nucleus are likely to further contribute to dysregulated stress responses and behavioral abnormalities by impairing fundamental mechanisms of hypothalamic-pituitary-adrenal axis functioning; e.g., the neuroactive steroid and GABAA receptor negative feedback–based mechanism of desensitization to stress.55  Finally, it is plausible that the negative feedback effect of elevated testosterone contributed to the reduced testis weight in exposed F0 males.

In conclusion, our results demonstrate that parental exposure to sevoflurane in young adulthood epigenetically reprograms germ cells, leading to neurobehavioral abnormalities in adult male progeny. These findings also provide evidence that sevoflurane administered even in young adulthood induces neurobehavioral deficits, profound alterations in the hypothalamic-pituitary-gonadal axis, and dysregulated hypothalamic-pituitary-adrenal axis responses to stress in exposed male rats. Exposed young adult female rats exhibited no long-term physiologic or behavioral abnormalities, but together with the exposed males, passed the adverse effects of sevoflurane exposure to their unexposed male offspring. These differential effects of sevoflurane exposure in males and females could suggest that distinct mechanisms mediate the somatic and germ cell effects of young adult sevoflurane exposure. It is important to note, however, that the current study was not powered to detect sex differences, and hence it will be important in future work to conduct direct comparisons between males and females.

Acknowledgments

The authors would like to thank Terrie Vasilopoulos, Ph.D., Department of Anesthesiology, University of Florida College of Medicine, Gainesville, Florida, for her advice and help with the statistical analyses.

Research Support

Supported by the National Institutes of Health, Bethesda, Maryland (grant Nos. R01NS091542 and R01NS091542-S, to Dr. Martynyuk), the Escher Autism Fund (San Jose, California; to Dr. Martynyuk), the I. Heermann Anesthesia Foundation (Gainesville, Florida; to Dr. Ju), and the Jerome H. Modell, M.D., F.A.H.A., Endowed Professorship, Gainesville, Florida (to Dr. Gravenstein).

Competing Interests

The authors declare no competing interests.

References

References
1.
FDA Drug Safety Communication: FDA approves label changes for use of general anesthetic and sedation drugs in young children
US Food and Drug Administration
,
April 24, 2017
Available at: https://www.fda.gov/Drugs/DrugSafety/ucm554634.htm. Accessed April 27, 2017.
2.
Stratmann
G
: .
Review article: Neurotoxicity of anesthetic drugs in the developing brain.
Anesth Analg
.
2011
;
113
:
1170
9
3.
Ben-Ari
Y
: .
The GABA excitatory/inhibitory developmental sequence: A personal journey.
Neuroscience
.
2014
;
279
:
187
219
4.
Khazipov
R
,
Valeeva
G
,
Khalilov
I
: .
Depolarizing GABA and developmental epilepsies.
CNS Neurosci Ther
.
2015
;
21
:
83
91
5.
Antkowiak
B
,
Rudolph
U
: .
New insights in the systemic and molecular underpinnings of general anesthetic actions mediated by γ-aminobutyric acid A receptors.
Curr Opin Anaesthesiol
.
2016
;
29
:
447
53
6.
Kotani
N
,
Akaike
N
: .
The effects of volatile anesthetics on synaptic and extrasynaptic GABA-induced neurotransmission.
Brain Res Bull
.
2013
;
93
:
69
79
7.
Khan
KS
,
Hayes
I
,
Buggy
DJ
: .
Pharmacology of anaesthetic agents II: Inhalation anaesthetic agents.
Cont Educ Anaesth Critical Care Pain
.
2014
;
14
:
106
11
8.
Lemonnier
E
,
Villeneuve
N
,
Sonie
S
,
Serret
S
,
Rosier
A
,
Roue
M
,
Brosset
P
,
Viellard
M
,
Bernoux
D
,
Rondeau
S
,
Thummler
S
,
Ravel
D
,
Ben-Ari
Y
: .
Effects of bumetanide on neurobehavioral function in children and adolescents with autism spectrum disorders.
Transl Psychiatry
.
2017
;
7
:
e1056
9.
Tang
X
,
Kim
J
,
Zhou
L
,
Wengert
E
,
Zhang
L
,
Wu
Z
,
Carromeu
C
,
Muotri
AR
,
Marchetto
MC
,
Gage
FH
,
Chen
G
: .
KCC2 rescues functional deficits in human neurons derived from patients with Rett syndrome.
Proc Natl Acad Sci USA
.
2016
;
113
:
751
6
10.
Ju
LS
,
Yang
JJ
,
Morey
TE
,
Gravenstein
N
,
Seubert
CN
,
Resnick
JL
,
Zhang
JQ
,
Martynyuk
AE
: .
Role of epigenetic mechanisms in transmitting the effects of neonatal sevoflurane exposure to the next generation of male, but not female, rats.
Br J Anaesth
.
2018
;
121
:
406
16
11.
Ju
LS
,
Yang
JJ
,
Gravenstein
N
,
Seubert
CN
,
Morey
TE
,
Sumners
C
,
Vasilopoulos
T
,
Yang
JJ
,
Martynyuk
AE
: .
Role of environmental stressors in determining the developmental outcome of neonatal anesthesia.
Psychoneuroendocrinology
.
2017
;
81
:
96
104
12.
Zhang
J
,
Xu
C
,
Puentes
DL
,
Seubert
CN
,
Gravenstein
N
,
Martynyuk
AE
: .
Role of steroids in hyperexcitatory adverse and anesthetic effects of sevoflurane in neonatal rats.
Neuroendocrinology
.
2016
;
103
:
440
51
13.
Murphy
SK
,
Itchon-Ramos
N
,
Visco
Z
,
Huang
Z
,
Grenier
C
,
Schrott
R
,
Acharya
K
,
Boudreau
MH
,
Price
TM
,
Raburn
DJ
,
Corcoran
DL
,
Lucas
JE
,
Mitchell
JT
,
McClernon
FJ
,
Cauley
M
,
Hall
BJ
,
Levin
ED
,
Kollins
SH
: .
Cannabinoid exposure and altered DNA methylation in rat and human sperm.
Epigenetics
.
2018
;
13
:
1208
21
14.
Rodgers
AB
,
Morgan
CP
,
Bronson
SL
,
Revello
S
,
Bale
TL
: .
Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation.
J Neurosci
.
2013
;
33
:
9003
12
15.
Yehuda
R
,
Daskalakis
NP
,
Lehrner
A
,
Desarnaud
F
,
Bader
HN
,
Makotkine
I
,
Flory
JD
,
Bierer
LM
,
Meaney
MJ
: .
Influences of maternal and paternal PTSD on epigenetic regulation of the glucocorticoid receptor gene in Holocaust survivor offspring.
Am J Psychiatry
.
2014
;
171
:
872
80
16.
Dehorter
N
,
Vinay
L
,
Hammond
C
,
Ben-Ari
Y
: .
Timing of developmental sequences in different brain structures: Physiological and pathological implications.
Eur J Neurosci
.
2012
;
35
:
1846
56
17.
Xu
C
,
Tan
S
,
Zhang
J
,
Seubert
CN
,
Gravenstein
N
,
Sumners
C
,
Vasilopoulos
T
,
Martynyuk
AE
: .
Anesthesia with sevoflurane in neonatal rats: Developmental neuroendocrine abnormalities and alleviating effects of the corticosteroid and Cl(-) importer antagonists.
Psychoneuroendocrinology
.
2015
;
60
:
173
81
18.
Quennell
JH
,
Howell
CS
,
Roa
J
,
Augustine
RA
,
Grattan
DR
,
Anderson
GM
: .
Leptin deficiency and diet-induced obesity reduce hypothalamic kisspeptin expression in mice.
Endocrinology
.
2011
;
152
:
1541
50
19.
Ostroumov
A
,
Thomas
AM
,
Kimmey
BA
,
Karsch
JS
,
Doyon
WM
,
Dani
JA
: .
Stress increases ethanol self-administration via a shift toward excitatory GABA signaling in the ventral tegmental area.
Neuron
.
2016
;
92
:
493
504
20.
Hewitt
SA
,
Wamsteeker
JI
,
Kurz
EU
,
Bains
JS
: .
Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis.
Nat Neurosci
.
2009
;
12
:
438
43
21.
Miller
S
,
Maguire
J
: .
Deficits in KCC2 and activation of the HPA axis lead to depression like behavior following social defeat.
Horm Stud
.
2014
;
2
:
2
22.
Tsukahara
T
,
Masuhara
M
,
Iwai
H
,
Sonomura
T
,
Sato
T
: .
The effect of repeated stress on KCC2 and NKCC1 immunoreactivity in the hippocampus of female mice.
Data Brief
.
2016
;
6
:
521
5
23.
Weiser
MJ
,
Handa
RJ
: .
Estrogen impairs glucocorticoid dependent negative feedback on the hypothalamic-pituitary-adrenal axis via estrogen receptor alpha within the hypothalamus.
Neuroscience
.
2009
;
159
:
883
95
24.
Weiser
MJ
,
Foradori
CD
,
Handa
RJ
: .
Estrogen receptor beta activation prevents glucocorticoid receptor-dependent effects of the central nucleus of the amygdala on behavior and neuroendocrine function.
Brain Res
.
2010
;
1336
:
78
88
25.
Curtis
SW
,
Conneely
KN
,
Marder
ME
,
Terrell
ML
,
Marcus
M
,
Smith
AK
: .
Intergenerational effects of endocrine-disrupting compounds: A review of the Michigan polybrominated biphenyl registry.
Epigenomics
.
2018
;
10
:
845
58
26.
Hill
SY
,
Tessner
KD
,
McDermott
MD
: .
Psychopathology in offspring from families of alcohol dependent female probands: A prospective study.
J Psychiatr Res
.
2011
;
45
:
285
94
27.
Morgan
CP
,
Chan
JC
,
Bale
TL
: .
Driving the next generation: Paternal lifetime experiences transmitted via extracellular vesicles and their small RNA cargo.
Biol Psychiatry
.
2019
;
85
:
164
71
28.
Ren
LQ
,
Sun
XX
,
Guan
Y
: .
Effects of sevoflurane or propofol combined with remifentanil anesthesia on clinical efficacy and stress response in pregnant women with pregnancy-induced hypertension.
Eur Rev Med Pharmacol Sci
.
2018
;
22
:
1825
9
29.
Das
W
,
Bhattacharya
S
,
Ghosh
S
,
Saha
S
,
Mallik
S
,
Pal
S
: .
Comparison between general anesthesia and spinal anesthesia in attenuation of stress response in laparoscopic cholecystectomy: A randomized prospective trial.
Saudi J Anaesth
.
2015
;
9
:
184
8
30.
Escher
J
: .
Bugs in the program: Can pregnancy drugs and smoking disturb molecular reprogramming of the fetal germline, increasing heritable risk for autism and neurodevelopmental disorders?
Environ Epigenet
.
2018
;
4
:
dvy001
31.
Wyatt
R
,
Wilson
AM
: .
Children of anaesthetists.
Br Med J
.
1973
;
1
:
675
32.
Gupta
D
,
Kaminski
E
,
McKelvey
G
,
Wang
H
: .
Firstborn offspring sex ratio is skewed towards female offspring in anesthesia care providers: A questionnaire-based nationwide study from United States.
J Anaesthesiol Clin Pharmacol
.
2013
;
29
:
221
7
33.
Nagella
AB
,
Ravishankar
M
,
Hemanth Kumar
VR
: .
Anaesthesia practice and reproductive outcomes: Facts unveiled.
Indian J Anaesth
.
2015
;
59
:
706
14
34.
Nagella
AB
,
Ravishankar
M
,
Hemanth Kumar
VR
: .
Anaesthesia practice and reproductive outcomes: Facts unveiled.
Indian J Anaesth
.
2016
;
60
:
225
35.
Gupta
D
: .
Firstborn female offsprings are significantly more common among Indian anaesthesiologists as compared to national child sex ratio.
Indian J Anaesth
.
2016
;
60
:
224
36.
Dickinson
H
,
Parker
L
: .
Do alcohol and lead change the sex ratio?
J Theor Biol
.
1994
;
169
:
313
5
37.
James
WH
: .
Offspring sex ratios at birth as markers of paternal endocrine disruption.
Environ Res
.
2006
;
100
:
77
85
38.
Navara
KJ
: .
Programming of offspring sex ratios by maternal stress in humans: Assessment of physiological mechanisms using a comparative approach.
J Comp Physiol B
.
2010
;
180
:
785
96
39.
Ju
LS
,
Jia
M
,
Sun
J
,
Sun
XR
,
Zhang
H
,
Ji
MH
,
Yang
JJ
,
Wang
ZY
: .
Hypermethylation of hippocampal synaptic plasticity-related genes is involved in neonatal sevoflurane exposure-induced cognitive impairments in rats.
Neurotox Res
.
2016
;
29
:
243
55
40.
Jia
M
,
Liu
WX
,
Yang
JJ
,
Xu
N
,
Xie
ZM
,
Ju
LS
,
Ji
MH
,
Martynyuk
AE
,
Yang
JJ
: .
Role of histone acetylation in long-term neurobehavioral effects of neonatal exposure to sevoflurane in rats.
Neurobiol Dis
.
2016
;
91
:
209
20
41.
Dalla Massara
L
,
Osuru
HP
,
Oklopcic
A
,
Milanovic
D
,
Joksimovic
SM
,
Caputo
V
,
DiGruccio
MR
,
Ori
C
,
Wang
G
,
Todorovic
SM
,
Jevtovic-Todorovic
V
: .
General anesthesia causes epigenetic histone modulation of c-Fos and brain-derived neurotrophic factor, target genes important for neuronal development in the immature rat hippocampus.
Anesthesiology
.
2016
;
124
:
1311
27
42.
Tang
CK
,
Chalon
J
,
Markham
JP
,
Ramanathan
S
,
Turndorf
H
: .
Exposure of sires to enflurane affects learning function of murine progeny.
Anesth Analg
.
1984
;
63
:
729
30
43.
Vutskits
L
,
Xie
Z
: .
Lasting impact of general anaesthesia on the brain: Mechanisms and relevance.
Nat Rev Neurosci
.
2016
;
17
:
705
17
44.
Stratmann
G
,
Sall
JW
,
May
LD
,
Bell
JS
,
Magnusson
KR
,
Rau
V
,
Visrodia
KH
,
Alvi
RS
,
Ku
B
,
Lee
MT
,
Dai
R
: .
Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats.
Anesthesiology
.
2009
;
110
:
834
48
45.
Zhu
C
,
Gao
J
,
Karlsson
N
,
Li
Q
,
Zhang
Y
,
Huang
Z
,
Li
H
,
Kuhn
HG
,
Blomgren
K
: .
Isoflurane anesthesia induced persistent, progressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents.
J Cereb Blood Flow Metab
.
2010
;
30
:
1017
30
46.
Crosby
C
,
Culley
DJ
,
Baxter
MG
,
Yukhananov
R
,
Crosby
G
: .
Spatial memory performance 2 weeks after general anesthesia in adult rats.
Anesth Analg
.
2005
;
101
:
1389
92
47.
DeFazio
RA
,
Heger
S
,
Ojeda
SR
,
Moenter
SM
: .
Activation of A-type gamma-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons.
Mol Endocrinol
.
2002
;
16
:
2872
91
48.
Herbison
AE
,
Moenter
SM
: .
Depolarising and hyperpolarising actions of GABA(A) receptor activation on gonadotrophin-releasing hormone neurones: Towards an emerging consensus.
J Neuroendocrinol
.
2011
;
23
:
557
69
49.
Nugent
BM
,
Wright
CL
,
Shetty
AC
,
Hodes
GE
,
Lenz
KM
,
Mahurkar
A
,
Russo
SJ
,
Devine
SE
,
McCarthy
MM
: .
Brain feminization requires active repression of masculinization via DNA methylation.
Nat Neurosci
.
2015
;
18
:
690
7
50.
Galanopoulou
AS
,
Moshé
SL
: .
Role of sex hormones in the sexually dimorphic expression of KCC2 in rat substantia nigra.
Exp Neurol
.
2003
;
184
:
1003
9
51.
Weinstock
M
,
Razin
M
,
Schorer-Apelbaum
D
,
Men
D
,
McCarty
R
: .
Gender differences in sympathoadrenal activity in rats at rest and in response to footshock stress.
Int J Dev Neurosci
.
1998
;
16
:
289
95
52.
Figueiredo
HF
,
Ulrich-Lai
YM
,
Choi
DC
,
Herman
JP
: .
Estrogen potentiates adrenocortical responses to stress in female rats.
Am J Physiol Endocrinol Metab
.
2007
;
292
:
E1173
82
53.
Iwasaki-Sekino
A
,
Mano-Otagiri
A
,
Ohata
H
,
Yamauchi
N
,
Shibasaki
T
: .
Gender differences in corticotropin and corticosterone secretion and corticotropin-releasing factor mRNA expression in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala in response to footshock stress or psychological stress in rats.
Psychoneuroendocrinology
.
2009
;
34
:
226
37
54.
Handa
RJ
,
Nunley
KM
,
Lorens
SA
,
Louie
JP
,
McGivern
RF
,
Bollnow
MR
: .
Androgen regulation of adrenocorticotropin and corticosterone secretion in the male rat following novelty and foot shock stressors.
Physiol Behav
.
1994
;
55
:
117
24
55.
Camille Melón
L
,
Maguire
J
: .
GABAergic regulation of the HPA and HPG axes and the impact of stress on reproductive function.
J Steroid Biochem Mol Biol
.
2016
;
160
:
196
203