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. Author manuscript; available in PMC: 2023 Oct 1.

Published in final edited form as: Anesth Analg. 2022 Jun 21:10.1213/ANE.0000000000006125. doi: 10.1213/ANE.0000000000006125

Dexmedetomidine Diminishes, But Does Not Prevent, Developmental Effects of Sevoflurane in Neonatal Rats

Zhengbo Yang ^†,^a, Yuanyuan Tong ^*,^a,^b, Jason O Brant ^‡, Ningtao Li ^*,^†, Ling-Sha Ju ^*, Timothy E Morey ^*, Nikolaus Gravenstein ^*,^§, Barry Setlow ^§,^∥, Jiaqiang Zhang ^†, Anatoly E Martynyuk ^*,^§

^†Department of Anesthesiology and Perioperative Medicine, People’s Hospital of Zhengzhou University, Zhengzhou, Henan, China

^*Department of Anesthesiology, University of Florida College of Medicine, Gainesville, Florida

^‡Department of Biostatistics, University of Florida College of Medicine, Gainesville, Florida.

^§McKnight Brain Institute, University of Florida College of Medicine, Gainesville, Florida.

^∥Department of Psychiatry, University of Florida College of Medicine, Gainesville, Florida.

^b

Current address: Department of Anesthesiology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China

^a

Contributed equally

AUTHOR CONTRIBUTIONS

Name: Zhengbo Yang, MD.

Contribution: This author helped with the acquisition, analysis, and discussion of behavioral data, and revising the manuscript.

Conflicts of Interest: None

Name: Yuanyuan Tong, MD.

Contribution: This author helped with the acquisition, analysis, and discussion of behavioral data, and revising the manuscript.

Conflicts of Interest: None

Name: Jason O. Brant, PhD.

Contribution: This author helped with the acquisition, analysis, and discussion of RRBS data, drafting and revising the manuscript.

Conflicts of Interest: None

Name: Ningtao Li, MD.

Contribution: This author helped with the acquisition, analysis, and discussion of EEG data, and revising the manuscript.

Conflicts of Interest: None

Name: Ling-Sha Ju, MD.

Contribution: This author helped with the acquisition, analysis, and discussion of RRBS data, with statistical analyses, drafting and revising the manuscript.

Conflicts of Interest: None

Name: Timothy E. Morey, MD.

Contribution: This author helped with the conceptualization of the study, data analysis and interpretation, drafting and revising the manuscript.

Conflicts of Interest: None

Name: Nikolaus Gravenstein, MD.

Contribution: This author helped with the conceptualization of the study, data analysis and interpretation, drafting and revising the manuscript.

Conflicts of Interest: None

Name: Barry Setlow, PhD.

Contribution: This author helped with the conceptualization of the behavioral studies, data analysis and interpretation, drafting and revising the manuscript.

Conflicts of Interest: None

Name: Jiaqiang Zhang, MD.

Contribution: This author contributed to the conceptualization and design of the study, data analysis and interpretation, drafting and revising the manuscript.

Conflicts of Interest: None

Name: Anatoly Martynyuk, PhD.

Contribution: This author was responsible for the conception and design of the study, data analysis and interpretation and writing the manuscript.

Conflicts of Interest: None

^✉

University of Florida College of Medicine, PO Box 100254, JHMHC, 1600 SW Archer Road, Gainesville, FL 32610-0254. Tel.: 352-294-8344; Fax: 352-392-7029; Address to amartynyuk@anest.ufl.edu.

PMCID: PMC9481710 NIHMSID: NIHMS1809239 PMID: 35759382

Abstract

BACKGROUND:

Sevoflurane increases neuronal excitation in neonatal rodent brains through alteration of GABA(A) receptor signaling and increases corticosterone release. These actions may contribute to mechanisms that initiate the anesthetic’s long-term neuroendocrine and neurobehavioral effects. Dexmedetomidine, a non-GABAergic α2-adrenergic receptor agonist, is likely to counteract sevoflurane-induced neuronal excitation. We investigated how dexmedetomidine pretreatment may alter the neurodevelopmental effects induced by sevoflurane in neonatal rats.

METHODS:

Postnatal day (P) 5 Sprague-Dawley male rats received dexmedetomidine (DEX; 25 μg/kg, intraperitoneal) or vehicle prior to exposure to 2.1% sevoflurane (SEVO) for 6 h (the DEX+SEVO and SEVO groups, respectively). Rats in the DEX-only group received DEX without exposure to SEVO. A subcohort of P5 rats was used for electroencephalographic and serum corticosterone measurements. The remaining rats were sequentially evaluated in the elevated plus maze on P80, prepulse inhibition of the acoustic startle response on P90, Morris water maze (MWM) starting on P100, and for corticosterone responses to physical restraint for 30 min on P120, followed by assessment of epigenomic DNA methylation patterns in the hippocampus.

RESULTS:

Acutely, DEX depressed sevoflurane-induced electroencephalogram-detectable seizure-like activity (mean ± SEM, SEVO vs DEX+SEVO, 33.1 ± 5.3 s vs 3.9 ± 5.3 s, P < .001), but it exacerbated corticosterone release (SEVO vs DEX+SEVO, 169.935 ± 20.995 ng/mL vs 280.853 ± 40.963 ng/mL, P = .043). DEX diminished, but did not fully abolish, sevoflurane-induced corticosterone responses to restraint (Control: 11625.230 ± 877.513, SEVO: 19363.555 ± 751.325, DEX+SEVO: 15012.216 ± 901.706, DEX-only: 12497.051 ± 999.816; F_(3,31) = 16.878, P < .001) and behavioral deficiencies (time spent in the target quadrant of the MWM: Control: 31.283% ± 1.722%, SEVO: 21.888% ± 2.187%, DEX+SEVO: 28.617% ± 1.501%, DEX-only: 31.339% ± 3.087%; F_(3,67) = 3.944, P = .012) in adulthood. Of the 391 differentially methylated genes in the SEVO group, 303 genes in the DEX+SEVO group had DNA methylation patterns that were not different from those in the Control group (i.e., they were normal). DEX alone did not cause acute or long-term functional abnormalities.

CONCLUSIONS:

This study suggests that the ability of DEX to depress SEVO-induced neuronal excitation, despite increasing corticosterone release, is sufficient to weaken mechanisms leading to long-term neuroendocrine/neurobehavioral abnormalities. Furthermore, the effects of DEX may be sufficient to prevent changes in DNA methylation in the majority of genes affected by SEVO, epigenetic modifications that could predict abnormalities in a wide range of functions.

Keywords: Dexmedetomidine, Sevoflurane, Neonatal anesthesia, Corticosterone, Electroencephalogram, Stress, Behavior

INTRODUCTION

Recent clinical studies have not found neurocognitive deficits in healthy children who underwent short general anesthetic (GA) exposure (<1 h) for elective surgeries.¹ However, neurocognitive effects of GAs have been found in very sick children who were more likely to have prolonged and/or repeated GA exposures.^2,3 The exact mechanisms whereby GAs induce such long-term effects, as well as clinically viable approaches to alleviate them, are unknown.

Sevoflurane (SEVO) is a commonly used GA whose anesthetic effects are mediated by multiple molecular pathways, including enhancement of GABA type A receptor (GABA_AR)-mediated inhibition of neuronal excitability. Signaling though GABA_ARs, however, differs qualitatively during the early stages of brain development. During this period, intraneuronal levels of Cl⁻, the charge carrier through GABA_AR channels, are elevated mainly due to relatively low and high expressions of the K⁺-Cl⁻ (KCC2) Cl⁻ exporter and the Na⁺-K⁺-Cl⁻ (NKCC1) Cl⁻ importer, respectively.^4,5 The resulting transmembrane gradients of Cl⁻ support depolarizing Cl⁻ currents, which can be sufficient to activate voltage-gated Ca⁺⁺ channels and to relieve the Mg⁺⁺-block of Ca⁺⁺ permeable N-methyl-D-aspartate receptor (NMDAR) channels.⁴

SEVO, counterintuitively to its GA status, induces hyperexcitation patterns in the electroencephalograms (EEGs) of neonatal rats, including EEG-detectable epileptic seizure-like activity.^6–8 It does so not only by enhancing GABA_AR signaling, but also by increasing expression of Nkcc1 and reducing expression of Kcc2, as well as by increasing release of the steroid stress hormone corticosterone.^6–8 Corticosterone may contribute to the hyperexcitatory effects of SEVO by increasing glutamatergic excitatory signaling.^9,10 Both SEVO-altered GABA_AR signaling and corticosterone levels may be required for SEVO-induced EEG-detectable seizure-like activity and long-term neurobehavioral abnormalities.^6–8,11,12

Dexmedetomidine (DEX), an α2-adrenergic receptor agonist with sedative, anxiolytic, and anti-inflammatory effects, is increasingly used in clinical practice in combination with other GAs.^13,14 DEX may induce changes in neuronal excitation and Ca⁺⁺ influx opposite to those induced by SEVO in the neonatal brain. DEX upregulates and downregulates activity of voltage-gated K⁺ and Ca⁺⁺ channels, respectively, and it diminishes excitatory glutamatergic signaling.^15–19 In combination with evidence that DEX reduces cortisol levels in surgical patients,²⁰ these findings suggest that it might counteract the effects of SEVO. Studies of the effects of DEX on neurotoxic effects of SEVO in neonatal rats, however, have generated somewhat conflicting findings.^21–26

Here we investigated whether DEX can alleviate SEVO-induced neuronal hyperexcitation and corticosterone release at the time of exposure, as well as long-term effects of SEVO.

METHODS

Animals

All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. 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 a.m.) and temperature (23–24 °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 two per cage for the rest of the study. Experimental data in this study are from 134 male rats.

Treatment Groups

Figure 1 shows an overview of the study design. Rats were randomized into treatment groups using a randomization plan with a web-based generator, and the investigators were blinded to group assignments. The postnatal day (P) 5 rats were held in a temperature-controlled chamber with a continuous supply of 30% oxygen in air (1.5 L/min) during anesthesia, which was 6% SEVO for 3 min for anesthesia induction and then 2.1% SEVO for anesthesia maintenance, as previously described.^6–8,27 A rectal temperature probe was used to monitor and maintain body temperature at ~+37 °C. DEX [25 μg/kg, intraperitoneal injection (IP)] or vehicle (IP) was administered 30 min prior to the onset of anesthesia with SEVO (the DEX + SEVO and SEVO groups, respectively). Rats in the Control group received vehicle (IP). Rats in the DEX-only group received DEX (25 μg/kg, IP) without exposure to SEVO. The dose of DEX was chosen based on data in the literature on DEX-induced modulation of neurodevelopmental effects of neonatal anesthesia in rodents.^21–26,28

Figure 1. — Illustration of study design. See Methods for details.

One subset of P5 rats (n = 10 in the SEVO group and n = 7 in the DEX + SEVO group) were instrumented for EEG recording during a minor 12- to 15-min surgical procedure performed under isoflurane anesthesia (2.0%–2.5%). The EEG recordings lasted for 1 h of baseline activity and for another hour during SEVO sedation: 6% SEVO for 3 min for anesthesia induction and 2.1% SEVO for 57 min for anesthesia maintenance, as previously described^6–8 (see Supplemental Text 1 for more details). Immediately upon completion of EEG recordings, rats were sacrificed by decapitation and trunk blood samples were collected for measurements of serum levels of corticosterone. Another subset of P5 rats (n = 45) was used to collect trunk blood samples to measure serum levels of corticosterone after 6 h of anesthesia with SEVO or at equivalent time points in other experimental groups.

The remaining rats were sequentially evaluated in the elevated plus maze (EPM)¹¹ starting on P80, prepulse inhibition (PPI) of the acoustic startle response^11,29 on P90, Morris water maze (MWM)^12,30 starting on P100, and for corticosterone responses to physical restraint for 30 min³¹ on P120, followed by collection of the hippocampal tissue samples²⁷ for in vitro studies, as described in our publications referenced above. (see the Supplemental Text 1 for more details).

Reduced Representation Bisulfite Sequencing (RRBS)

For the RRBS analyses, genomic DNA was isolated from the hippocampal tissue of the same rats that were used for the in vivo studies (5 randomly selected rats/group). Briefly, genomic DNA was digested with MspI and size selected for fragments of 150–350 bp to enrich for CpG-rich regions of the genome. Following bisulfite conversion, the library was sequenced on the Illumina Novaseq platform. After processing, DNA regions were considered differentially methylated [differentially methylated regions (DMRs)] if they had ≥5 CpGs within 200 bp of each other, with a read depth of ≥5 reads/site/sample, a 2D-KS test p-value of ≤ .05, a false discovery rate (FDR)–corrected (Benjamini-Hochberg) MWU p-value (q-value) of ≤.01, and a difference in methylation between groups of ≥10% (absolute value). Only DMRs that fell within 15 kb of the transcription start site of a gene were analyzed (see the Supplemental Text 1 for more details).

Statistical Analyses

The primary outcomes in this study were the in vivo acute and long-term effects of SEVO and DEX. The experimental results of the in vitro mechanistic studies represent the secondary outcomes. Sample size calculations were done using SigmaPlot 14.0 software (Systat Software, Inc., San Jose, CA), assuming a range of anticipated differences in mean outcomes and standard error based on background data and past experience with similar measurements in Sprague-Dawley rats. ^{6–8,11,12,27,29,31} This analysis indicated that sample sizes of at least 15 rats/group for behavioral studies, 7 rats/group for EEG studies, and 5 rats for in vitro measurements (serum corticosterone levels and RRBS) were required to detect differences between treatment groups, with effect sizes of ≥0.8, assuming an α level of 0.05. This translated to a mean difference of 10% in percent of time spent in the open arms (EPM), percent of PPI of the startle, percent of time spent in the target quadrant (MWM), percent of total duration of EEG-detectable seizure-like activity and in vitro measurements. To account for possible attrition over the course of the 4-month study (estimated at 20% due to unexpected diseases, removal of animals from behavioral tests because of housing incidents such as cage flooding, incidents during behavioral tests such as a fall from the maze during the EPM test, and removal of outliers, among other factors, based on our previous experience with similar studies), group sizes for some comparisons were larger than those calculated from the power analysis. Statistical analyses were conducted on raw data using SigmaPlot 14.0 software, which 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 ± SEM. Boxplots were used to identify outliers. No outliers were detected that were not in the plausible range of values for the outcomes; therefore, all data were maintained in analyses. An independent t-test was used to analyze the effects of treatments on SEVO-induced EEG-detectable seizure-like activity and serum corticosterone levels after completion of EEG recordings. One-way ANOVA was used to assess differences in corticosterone levels, time spent and number of entries in the open arms of the EPM, time spent in each quadrant and number of crossings over the former platform during the MWM test. Two-way repeated ANOVA with experimental groups and time as the independent variables was run to analyze changes in serum corticosterone levels before and after the restraint. Two-way ANOVA was used to analyze the PPI data, with the treatment and prepulse intensity as independent variables, and the MWM latencies to escape data, with experimental groups and days of training as the independent variables. To assess the differences in total corticosterone concentrations before and after the restraint, the area under the curve with respect to ground [AUCg, g – ground (level of corticosterone before the restraint)] was calculated and compared across experimental groups using one-way ANOVA. Multiple pairwise comparisons were done with the Fisher LSD method. The comparisons were run as two-tailed tests, where applicable. P < .05 was considered significant.

RESULTS

Effects of DEX on SEVO-Induced Changes in Hyperexcitation Patterns and Corticosterone

SEVO caused hyperexcitation patterns in the EEGs in P5 rat pups. The total duration of EEG-detectable epileptic seizure-like activity per 1 h of anesthesia with SEVO was 33.1 ± 5.3 s. It consisted of 4.9 ± 0.75 episodes with an average episode duration of 6.1 ± 0.7 s (Figure 2A–D). Rat pups pretreated with DEX prior to SEVO exposure had shorter total durations (3.9 ± 5.3 s; t₍₁₅₎ = 4.380; P < .001), fewer episodes (0.174 ± 0.421 episodes; t₍₁₅₎ = 4.307; P < .001), and shorter durations of individual episodes (2.3 ± 1.1 s; t₍₁₅₎ = 2.965; P = .009) of EEG-detectable epileptic seizure-like activity (Figure 2A–E).

Figure 2. — Effects of dexmedetomidine (DEX) on sevoflurane (SEVO)-caused electroencephalogram (EEG)-detectable epileptic seizure-like activity and increases in serum levels of corticosterone in postnatal day (P) 5 male rats. A-E, Effect of DEX on SEVO-caused EEG-detectable epileptic seizure-like activity. A,B, Examples of EEG recordings in SEVO-anesthetized rats that received vehicle or DEX 30 min prior to initiation of SEVO anesthesia for 60 min. C-E, Histograms showing parameters of EEG-detectable epileptic seizure-like activity in P5 rats during 60-min exposure to SEVO that received vehicle (the SEVO group) or DEX (the DEX + SEVO group) as pretreatment. Data are means ± SEM from 10 rats in the SEVO group and 7 rats in the DEX + SEVO group. F, Plot showing serum levels of corticosterone in blood samples collected after completion of EEG recordings as in A-E. Data are means ± SEM from 10 rats in the SEVO group and 7 rats in the DEX + SEVO group. *P < .05, **P < .01, ***P < .001. G, The levels of serum corticosterone in trunk blood samples collected from P5 rats immediately after 6 h of anesthesia with 2.1% SEVO or at an equivalent time point in all other experimental groups (n = 11 in the Control group; n = 12 in the SEVO and DEX + SEVO groups; and n = 10 in the DEX-only group). Data are mean ± SEM. ***P < .001.

Compared to the SEVO group (169.935 ± 20.995 ng/mL), the DEX + SEVO group (280.853 ± 40.963 ng/mL) had higher levels of serum corticosterone (t₍₈₎ = −2.410; P = .043) after completion of EEG recordings (Figure 2F). There were effects of treatment on serum levels of corticosterone in trunk blood samples collected immediately after exposure to SEVO for 6 h (F_(3,41) = 39.231, P < .001; Figure 2G). When compared to the Control group (55.037 ± 9.059 ng/mL), the SEVO group (182.928 ± 19.369 ng/mL, P < .001) and the DEX + SEVO group (319.222 ± 29.271 ng/mL, P < .001) had increased serum levels of corticosterone. Importantly, the DEX + SEVO group had higher serum levels of corticosterone than the SEVO group (P < .001).

Effect of DEX on SEVO-Induced Neuroendocrine and Neurobehavioral Abnormalities in Adulthood

Similar to our previous findings,²⁷ there was a between-subjects effect of treatment on total serum levels of corticosterone after physical restraint for 30 min (Control: 11625.230 ± 877.513, SEVO: 19363.555 ± 751.325, DEX+SEVO: 15012.216 ± 901.706, DEX: 12497.051 ± 999.816; F_(3,31) = 16.878, P < .001; Figure 3A). The rats in the SEVO group had higher levels of corticosterone than all other groups (P ≤ .001). Although total serum levels of corticosterone in rats in the DEX + SEVO group were lower than those in rats in the SEVO group, they remained higher than in rats in the Control group (P = .012). There was an interaction between treatment and time of measurement of serum levels of corticosterone (F_(9,93) = 12.897, P < .001; Figure 3B). The SEVO group had higher serum levels of corticosterone at 10 min (Control: 177.801 ± 11.244 ng/mL, SEVO: 301.402 ± 41.698 ng/mL, DEX+SEVO: 233.722 ± 20.407 ng/mL, DEX: 139.004 ± 18.97 ng/mL; P < .001 vs all other groups) and at 60 min (Control: 57.738 ± 8.748 ng/mL, SEVO: 90.73 ± 9.197 ng/mL, DEX+SEVO: 71.357 ± 12.725 ng/mL, DEX: 103.643 ± 14.308 ng/mL; P = .036 vs the Control group), but not at 120 min, after the restraint. At 10 min after the restraint, the DEX + SEVO group had higher serum levels of corticosterone than the Control or DEX-only groups (P ≤ .02). The DEX-only group had similar serum levels of corticosterone at 10 min as the Control group (P = .089).

Figure 3. — Dexmedetomidine (DEX) administered prior to exposure to sevoflurane (SEVO) on postnatal day (P) 5 decreased SEVO-induced neuroendocrine and neurobehavioral abnormalities in adulthood. A,B, The total serum corticosterone responses (A) and the respective levels of corticosterone before physical restraint for 30 min and at 10, 60, and 120 min after the restraint (B). Data are means ± SEM from 9 rats/group (n = 8, the DEX + SEVO group). C,D Percent of time spent in open arms and percent of entries to the open arms of the EPM. Data are means ± SEM from 18 rats/group (n = 17, the DEX-only group). E, Histogram showing the percent of PPI of the startle responses at a prepulse intensity of 3 dB (PP3), 6 dB (PP6), and 12 dB (PP12). Data are mean ± SEM from 18 rats/group. F, Plots showing the values of escape latencies during the 5-day training period. G,H, Histograms showing the time spent in each quadrant (G) and the number of times the rat crossed the previous location of the escape platform (H). Data are means ± SEM from 18 rats/group. *P < .05, **P < .01, ***P < .001; ⁺P < .05 vs all the other groups; ^#P < .05 vs the Control group; ^&P < .05 vs the DEX-only group. Color coding in Figure 3A,C,D,H is applicable to the entire figure.

The treatment during the early postnatal period affected the time that rats spent in the open arms of the EPM in adulthood (Control: 20.462% ± 3.561%, SEVO: 9.508% ± 1.745%, DEX+SEVO: 22.433% ± 3.323%, DEX: 18.734% ± 0.796%; F_(3,67) = 3.257, P = .027; Figure 3C). The rats in the SEVO group spent less time in the open arms of the EPM than all other groups (P ≤ .046). The rats in the DEX-only group (P = .705) and the DEX + SEVO group (P = .662) were not different from the rats in the Control groups in terms of time spent in the open arms of the EPM (Figure 3C). There was no treatment effect on percent of entries to the open arms of the EPM (F_(3,67) = 0.592, P = .623; Figure 3D).

There was a treatment × prepulse intensity interaction in the PPI of the acoustic startle response (F_(6,136) = 2.973; P = .009; Figure 3E). Rats in the SEVO group exhibited impaired PPI of the startle responses at a prepulse intensity of 3 dB (Control: 38.357% ± 3.343%, SEVO: 16.293% ± 7.583%, DEX+SEVO: 34.487% ± 3.754%, DEX: 40.542% ± 3.766%; P = .001 vs all other groups). Pretreatment with DEX normalized the PPI of startle responses at a prepulse intensity of 3 dB (P = .056 vs the Control group), but it further exacerbated impairment in the PPI of the startle at a prepulse intensity of 6 dB (Control: 58.874% ± 2.679%, SEVO: 48.943% ± 3.527%, DEX+SEVO: 44.897% ± 3.811%, DEX: 57.945% ± 3.357%; P ≤ .027 vs the Control and DEX-only groups).

The MWM test showed a significant within-subjects effect of training day (F_(4,272) = 74.806, P < .001; Figure 3E) and effect of treatment on the escape latencies across the 5-day training period (F_(3,272) = 5.267; P = .003; Figure 3F). The rats in the SEVO group spent a longer time locating the escape platform on day 3 (Control: 22.3 ± 1.9 s, SEVO: 29.2 ± 2.5 s, DEX+SEVO: 26.2 ± 2.4 s, DEX: 26.3 ± 2.1 s; P = .032 vs the Control group), day 4 (Control: 21.6 ± 2.1 s, SEVO: 28.5 ± 2.1 s, DEX+SEVO: 25.0 ± 2.1 s, DEX: 19.1 ± 1.6 s; P ≤ .033 vs the DEX-only and Control groups), and day 5 (Control: 18.7 ± 1.7 s, SEVO: 28.8 ± 2.9 s, DEX+SEVO: 19.8 ± 2.0 s, DEX: 19.6 ± 2.4 s; P ≤ .006 vs all other groups). There was an effect of treatment on time spent in the target quadrant (Control: 31.283% ± 1.722%, SEVO: 21.888% ± 2.187%, DEX+SEVO: 28.617% ± 1.501%, DEX: 31.339% ± 3.087%; F_(3,67) = 3.944, P = .012; Figure 3G) and times crossing over the platform (Control: 2.6 ± 0. 4, SEVO: 1.2 ± 0.2, DEX+SEVO: 1.7 ± 0.3, DEX: 2.3 ± 0.4; F_(3,68) = 4.263, P = .008; Figure 3H).

DNA Methylation Patterns in the Hippocampus and Modification by Pretreatment With DEX

When compared to the Control group, rats in the SEVO group had 407 DMRs in 391 genes [differentially methylated genes (DMGs)] (Figure 4A). Of the 391 DMGs in the SEVO group, rats in the DEX + SEVO group had 54 genes whose 55 DMRs had the same genomic coordinates and direction change in DNA methylation (the common DMGs with overlapping DMRs). The SEVO and DEX + SEVO groups shared an additional 34 genes whose 36 DMRs did not overlap by genomic coordinates (the common DMGs with not overlapping DMRs). These findings indicate that of the 391 DMGs in the SEVO group, 303 DMGs were normalized in the DEX + SEVO group, i.e., these DMGs in the DEX + SEVO group were not different from those in the Control group (unique DMGs in the SEVO group) (Figure 4B). The complete list of all DMGs in the SEVO group is also provided in Supplemental Table 1. The unique DMGs are potential candidates to mediate the SEVO-induced neuroendocrine and neurobehavioral abnormalities and/or changes in other functions not tested in this study. Therefore, we more closely analyzed the unique DMGs (Figure 4C–F).

Figure 4. — Dexmedetomidine (DEX) administered prior to exposure to sevoflurane (SEVO) on postnatal day (P) 5, prevented SEVO-induced changes in DNA methylation patterns in the majority, but not all, hippocampal genes, as detected in tissue samples isolated from P≥130 rats. A, Heatmap depicting 407 differentially methylated regions (DMRs) in 391 genes in the SEVO group compared to Control group. Each DMR is presented (rows) with the change in methylation scaled (from −2 to 2) across all samples (columns) so that the mean is depicted in black, hypermethylated DMRs (278) in red, and hypomethylated DMRs (129) in cyan. B, Volcano plot showing three categories of DMRs in the SEVO group: 1) 55 DMRs that shared genomic coordinates with DMRs in the DEX+SEVO group (red dots); 2) 36 DMRs that did not overlap by genomic coordinates with DMRs in the DEX+SEVO group (green dots); 3) 316 DMRs that in the DEX+SEVO group were not different from the same DNA regions in the Control group (DMRs unique to the SEVO group). Horizontal dashed red line and vertical dashed red lines separate the non-significant DMRs (grey dots). Symbols of genes with the most significant unique DMRs are shown in the figure. The numbers of genes in each category of DMRs in the SEVO group are summarized in a pie chart. C, A chord plot of representative enriched GO terms and associated genes. The colored boxes next to gene symbols indicate the change in methylation. D, PCoA plot of enriched GO terms (colored circles) associated with genes with unique DMRs. Circles with the same color represent GO terms within the same parent term; the distance between circles represents the similarity between GO terms and the size of the circles represents the enrichment score. E, A plot depicting the analyses of gene-disease interactions for genes with unique DMRs and diseases from the Comparative Toxicogenomics Database (CTD). The resulting enrichment scores for the top enriched diseases are shown. F, Inference scores for the DMR-containing genes associated with autism spectrum disorders (ASDs) and attention-deficit/hyperactivity disorder (ADHD) are plotted as a heatmap (only genes with inference scores in the 95^th percentile are shown).

The overrepresentation analysis of the unique DMGs resulted in 128 enriched gene ontology (GO) terms. These 128 GO terms covered a wide array of biological processes ranging from nervous system development and function to regulation of stress responses, metabolic processes, vasculature development, and blood circulation. In addition, several of the enriched GO terms were associated with molecular processes, which are the main substrates for the actions of GAs to induce sedation. Examples of such GO terms include ‘cation transport’, ‘anterograde trans-synaptic signaling’, ‘regulation of ion transmembrane transporter activity’, ‘excitatory synapse assembly’, and others. Selected enriched GO terms are shown in Figure 4C. The complete list of the enriched GO terms and the respective DMGs is provided in Supplemental Table 2.

Of the 128 enriched GO terms for the unique DMGs, semantic similarity scores were calculated for 122 enriched GO terms, using GOSemSim³² via rrvgo.³³ Six enriched GO terms did not have GO term relationships for the Rn6 genome; therefore, a similarity score could not be calculated and these terms were excluded from the semantic similarity calculations. These 122 terms were grouped using hierarchal clustering with complete linkage of semantic similarity scores. The resulting 11 groups were plotted in a principal coordinate analysis (PCoA) plot (Figure 4D). All term groups and individual enriched GO terms in each group are provided in Supplemental Table 3.

The unique DMGs were analyzed for interactions with diseases using the Comparative Toxicogenomics Database (CTD) querier R package.^34,35 The topmost enriched disease categories, based on enrichment score, were ‘Nervous System Disease’, ‘Neoplasms’, and ‘Mental Disorders’. The enrichment scores [-log10(p-value)] for some of these disease categories are illustrated in Figure 4E. The inference scores for the unique DMGs associated with autism spectrum disorders and attention-deficit/hyperactivity disorder, which were part of the ‘Mental Disorders’ category, are shown in Figure 4F. The results of the unique DMG-disease interaction analysis are provided in Supplemental Table 4.

DISCUSSION

In this study, DEX weakened the ability of SEVO to cause EEG-detectable epileptic seizure-like activity; however, it exacerbated SEVO-increased corticosterone levels in neonatal rats at the time of exposure. Pretreatment with DEX also alleviated, but did not fully prevent, neuroendocrine and neurobehavioral abnormalities induced by neonatal exposure to SEVO and epigenomic changes in the majority of affected hippocampal genes. DEX alone, without SEVO anesthesia, did not affect any of the measured parameters.

Collectively, the findings of this study suggest that multiple molecular mechanisms are involved in mediating DEX-induced modulation of developmental effects of SEVO. It is plausible that DEX induces its therapeutic effects by normalizing the Ca⁺⁺ influx enhanced during SEVO-induced EEG-detectable epileptic seizures. It may do so through modulation of its own molecular targets, such as activation of hyperpolarizing K⁺ currents and inhibition of voltage-gated Ca⁺⁺ channels and excitatory glutamatergic signaling.^15–19 It has been proposed that DEX may indirectly facilitate the activity of GABAergic interneurons in the ventrolateral preoptic nucleus through suppression of noradrenergic inhibitory projections from the locus coeruleus.³⁶ The parvocellular paraventricular nucleus neurons that control the HPA axis activity receive extensive GABAergic innervation from multiple brain regions.³⁷ Therefore, if DEX similarly disinhibits GABAergic neurons in the HPA axis, such disinhibition in concert with the SEVO-induced shift toward more excitatory (and greater) GABAergic signaling⁸ may contribute to the observed DEX-induced exacerbation of SEVO-caused corticosterone release.³⁸ In indirect support of such a mechanism, DEX alone did not increase corticosterone release at the time of exposure, but it potentiated the increase in corticosterone release caused by SEVO. This may also be a reason that DEX diminished, but did not fully prevent, SEVO-induced long-term abnormalities.

Our findings that DEX alone had no effect on corticosterone secretion agree with some clinical studies that DEX did not reduce cortisol secretion when administered as bolus injections.²⁰ Interestingly, DEX increased serum glucocorticoid levels in rabbits that received a combination of DEX and ketamine,³⁹ which is more in line with our findings that DEX exacerbated SEVO-increased corticosterone secretion in neonatal rats. In future studies it will be important to test whether the ability of DEX to potentiate a SEVO-caused increase in corticosterone secretion is specific to the early postnatal period in rodents, the age when GABA_AR signaling can be less inhibitory or even excitatory, especially in the presence of SEVO.⁸

Parallels can be drawn between our findings and those of other laboratories on acute and long-term effects of DEX, both alone and in the presence of SEVO. Similar to our findings of a lack of acute and long-term adverse effects of DEX, DEX by itself even at high cumulative doses induced no or only slight neuronal apoptosis when compared to clinically relevant doses of SEVO.^21,23 Also, DEX was safe when administered alone in terms of induction of long-term abnormalities, and it reduced neurobehavioral effects of SEVO.^25,26 Perez-Zoghbi et al. found that a low dose of DEX reduced apoptotic cell death induced by an anesthetic dose of SEVO, but it had no effects when coadministered with a subanesthetic dose of SEVO.^21,22 In contrast, Lee et al. reported that DEX exacerbated neuronal injury caused by this dose of SEVO.²³ Both groups reported a higher degree of neuronal apoptosis in neonatal rats when subanesthetic doses of SEVO were supplemented with higher doses of DEX.^22,24 These findings, along with our observations of exacerbated stress-like response in the presence of the two agents, point to the possibility that the combination of SEVO and DEX may induce acute neurotoxic effects by acting via similar molecular mechanisms. One such mechanism may be alterations in GABA_AR signaling, as discussed above.

GAs, in particular SEVO, are polyvalent agents known to modulate a variety of biological processes, some of which may not be directly involved in mediating GA-induced sedation. Abnormalities in many of these processes, however, have the potential to result in lasting health-related consequences.⁴⁰ Such a possibility is supported by our findings of epigenomic changes in multiple genes induced by neonatal exposure to SEVO. Such epigenomic changes may not only underlie deficiencies in the development and functioning of the central nervous system, but they may also be linked to regulation of stress responses, metabolic processes, vasculature development, and blood circulation, among others.

Future mechanistic studies will be needed to determine whether the changes we found in DNA methylation marks lead to transcriptional, translational, and functional abnormalities. Future mechanistic studies will also be needed to assess whether DEX-induced prevention of epigenomic changes produced by SEVO is required for the therapeutic functional effects of DEX in neonatal rats anesthetized with SEVO. Additionally, future mechanistic studies will be needed to elucidate the roles of differentially methylated genes that were common to the SEVO and DEX + SEVO groups, as well as differentially methylated genes that were unique to the DEX + SEVO group in the remaining functional abnormalities observed in that group. It will be important to test whether early-life exposure to SEVO is associated with an increased rate of disease states predicted by the epigenomic changes found in this study.

In conclusion, we demonstrated that despite exacerbating SEVO-caused corticosterone secretion at the time of exposure, DEX attenuated the ability of SEVO to cause hyperexcitation patterns in the EEGs of neonatal rats. DEX diminished, but did not fully prevent, SEVO-induced long-term neuroendocrine and behavioral abnormalities. It deterred DNA methylation changes in many (though not all) SEVO-affected hippocampal genes. Our findings of changes in DNA methylation patterns in a wide range of genes in rats neonatally exposed to SEVO predict that SEVO may reduce resilience to disease states that extend beyond neurocognitive functions. Our findings also support the notion that DEX, if administered using the regimen tested in this study, may be used as an adjuvant to improve the safety profile of SEVO in neonatal rodents, although it is not sufficient to fully deter SEVO-induced abnormalities.

Supplementary Material

Supplemental Table 1

List of hippocampal differentially methylated genes (DMGs) in the SEVO group.

NIHMS1809239-supplement-Supplemental_Table_1.xlsx^{(198.1KB, xlsx)}

Supplemental Table 2

List of the enriched GO terms ranked by their q-values and the respective differentially methylated genes (DMGs).

NIHMS1809239-supplement-Supplemental_Table_2.xlsx^{(302.1KB, xlsx)}

Supplemental Table 4

Detailed table of gene-disease interactions from Comparative Toxicogenomics Database (CTD).

NIHMS1809239-supplement-Supplemental_Table_4.xlsx^{(74.4KB, xlsx)}

Supplemental Text 1

Experimental Methods

NIHMS1809239-supplement-Supplemental_Text_1.docx^{(42.5KB, docx)}

Supplemental Table 3

Detailed results of the semantic similarity reduction of GO terms.

NIHMS1809239-supplement-Supplemental_Table_3.xlsx^{(18.8KB, xlsx)}

KEY POINTS.

Question: Does dexmedetomidine pretreatment prior to sevoflurane exposure in neonatal rats alleviate acute sevoflurane-induced neuronal hyperexcitation and corticosterone release and mitigate long-term neurodevelopmental effects?
Findings: Dexmedetomidine reduced and exacerbated sevoflurane’s acute hyperexcitatory and stress-like effects, respectively, partially alleviated sevoflurane-induced long-term neuroendocrine and neurobehavioral abnormalities, and normalized DNA methylation patterns in the majority of sevoflurane-affected genes.
Meaning: Our findings: 1) may help to explain why a combination of dexmedetomidine and sevoflurane, but not dexmedetomidine alone, induces acute adverse effects and 2) predict that long-term effects of neonatal exposure to sevoflurane may extend from neurocognitive deficiencies to abnormalities in a wide range of functions.

Funding:

Supported in part by Supported in part by the Natural Science Foundation of China (No. 82071217 and 81771149 to JZ), the National Institutes of Health (R01NS091542, R01NS091542-S, and R56HD102898 to AEM), the I. Heermann Anesthesia Foundation (L-SJ), and the Jerome H. Modell, M.D., F.A.H.A. Endowed Professorship, Gainesville, Florida (N.G.).

GLOSSARY OF TERMS

AUCg: area under the curve with respect to ground
CTD: Comparative Toxicogenomics Database
DEX: dexmedetomidine
DMG: differentially methylated genes
DMR: differentially methylated regions
EEG: electroencephalogram
EPM: elevated plus maze
FDR: false discovery rate
GA: general anesthetic
GABA_AR: GABA type A receptor
GO: gene ontology
HPA: hypothalamic-pituitary-adrenal
IP: intraperitoneal
KCC2: K⁺-Cl⁻
NKCC1: Na⁺-K⁺-Cl⁻
MWM: Morris water maze
NMDAR: N-methyl-D-aspartate receptor
P: postnatal day
PCoA: principal coordinate analysis
PPI: prepulse inhibition
RRBS: reduced representation bisulfite sequencing
SEVO: sevoflurane

Footnotes

Conflicts of interest: None.

Clinical trial number and registry URL: N/A

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Table 1

List of hippocampal differentially methylated genes (DMGs) in the SEVO group.

NIHMS1809239-supplement-Supplemental_Table_1.xlsx^{(198.1KB, xlsx)}

Supplemental Table 2

List of the enriched GO terms ranked by their q-values and the respective differentially methylated genes (DMGs).

NIHMS1809239-supplement-Supplemental_Table_2.xlsx^{(302.1KB, xlsx)}

Supplemental Table 4

Detailed table of gene-disease interactions from Comparative Toxicogenomics Database (CTD).

NIHMS1809239-supplement-Supplemental_Table_4.xlsx^{(74.4KB, xlsx)}

Supplemental Text 1

Experimental Methods

NIHMS1809239-supplement-Supplemental_Text_1.docx^{(42.5KB, docx)}

Supplemental Table 3

Detailed results of the semantic similarity reduction of GO terms.

NIHMS1809239-supplement-Supplemental_Table_3.xlsx^{(18.8KB, xlsx)}

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