Role of environmental stressors in determining the developmental outcome of neonatal anesthesia

Abstract

Background: The majority of studies evaluating neurocognition in humans who had procedures under anesthesia early in life found long-term deficits even though the typical anesthesia duration normalized to the human life span is much shorter than that shown to induce developmental abnormalities in rodents. Therefore, we studied whether subsequent environmental stressors contribute to deficiencies programmed by a brief neonatal etomidate exposure.

Methods: Postnatal days (P) 4, 5, or 6, Sprague-Dawley rats, pretreated with vehicle or the Na⁺-K⁺-2Cl^- (NKCC1) inhibitor, bumetanide, received two injections of etomidate resulting in anesthesia for 2h. To simulate stress after anesthesia, the animals were exposed to a single maternal separation for 3h at P10. 3-7days after exposure to etomidate the rats had increased hypothalamic NKCC1 mRNA and corticotropin releasing hormone (CRH) mRNA and decreased K⁺-2Cl^- (KCC2) mRNA levels with greater changes in males. In rats neonatally exposed to both etomidate and maternal separation, these abnormalities persisted into adulthood. These animals also exhibited extended corticosterone responses to restraint stress with increases in total plasma corticosterone more robust in males, as well as behavioral abnormalities. Pretreatment with the NKCC1 inhibitor ameliorated most of these effects.

Conclusions: Post-anesthesia stressors may exacerbate/unmask neurodevelopmental abnormalities even after a relatively short anesthetic with etomidate, leading to dysregulated stress response systems and neurobehavioral deficiencies in adulthood. Amelioration by bumetanide suggests a mechanistic role for etomidate-enhanced gamma-aminobutyric acid type A receptor-mediated depolarization in initiating long-lasting alterations in gene expression that are further potentiated by subsequent maternal separation.

Keywords: Behavior; Developing brain; Environmental factor; Etomidate; Maternal separation; Stress.

Figure 1

Anesthesia with etomidate for 2 h at postnatal days (P) 4, 5 or 6 increased hypothalamic levels of Na⁺-K⁺-2Cl⁻ (NKCC1) mRNA, and reduced hypothalamic levels of K⁺-2Cl⁻ (KCC2) mRNA in male and female rat pups. Shown are the respective NKCC1 and KCC2 mRNA levels (A–H) and the resulting NKCC1/KCC2 mRNA ratios (I–L). Etomidate increased hypothalamic NKCC1 mRNA levels in male and female pups 3–7 days after the anesthetic when compared to that of control pups (P < 0.001) and bumetanide-pretreated etomidate-anesthetized pups (P = 0.012), whereas the increase immediately after anesthesia was significant only compared to that of control pups (P = 0.034). Bumetanide alleviated the effects of etomidate on the NKCC1/KCC2 mRNA ratio 3–7 days after exposure to the anesthetic in males (P <0.001 vs Etomidate (ET), but P = 0.07 vs Control), but not in females (P = 0.016 vs Control, and P = 0.886 vs ET). Data normalized against Control are means ± SEM from 5 rats per treatment group. *P < 0.05 vs. Control, and Bumetanide (BU) + ET; ^#P < 0.05 vs. Control; ^@P < 0.001 vs. female ET group.

Figure 2

Anesthesia with etomidate for 2 h at postnatal days (P) 4, 5 or 6 increased hypothalamic levels of corticotropin releasing hormone (CRH) mRNA in male and female rat pups with greater increases in males. These effects were alleviated by pretreatment with bumetanide prior to anesthesia with etomidate. The brain hypothalamus tissue samples were collected ~2 h after the onset of anesthesia with etomidate (P4–P6) or 3–7 days later (P9–P11) for qRT-PCR analyses. Shown are the levels of CRH mRNA at P4–P6 (A–B) and P9–P11 (C–D). Bumetanide alleviated the effects of etomidate in males (P = 0.89 vs Control, and P < 0.001 vs Etomidate (ET)) but not in females (P = 0.999 vs Control, and P = 0.14 vs ET). Data normalized against control are means ± SEM from 5 rats per treatment group. *P < 0.05 vs. Control, and BU + ET; ^@P < 0.05 vs. female ET group.

Figure 3

Anesthesia with etomidate for 2 h at postnatal days (P) 4, 5 or 6 followed by maternal separation for 3 h at P10 lead to increased hypothalamic levels of Na⁺-K⁺-2Cl⁻ (NKCC1) mRNA reduced hypothalamic levels of K⁺-2Cl⁻ (KCC2) mRNA in adult male and female rats. These effects were alleviated by pretreatment with bumetanide prior to anesthesia with etomidate. Shown are the respective levels of NKCC1 mRNA (A,B) and KCC2 mRNA (C,D), and the resulting NKCC1/KCC2 mRNA ratios (E,F). Bumetanide prevented the increases in the hypothalamic NKCC1/KCC2 mRNA ratios in males (P < 0.001 vs Etomidate (ET) + Maternal Separation (SEP), and P = 0.506 vs Control) and females (P < 0.001 vs ET + SEP, but P=0.0012 vs Control). Data normalized against control are means ± SEM from 5 rats per treatment group. *P < 0.05 vs. all treatment groups in males and vs. Control, and Bumetanide (BU) + ET + SEP in females; ^#P < 0.05 vs. all groups, except ET; ^@P < 0.05 vs. female ET + SEP group.

Figure 4

Anesthesia with etomidate for 2 h at postnatal days (P) 4, 5 or 6 followed by maternal separation for 3 h at P10 lead to increased hypothalamic levels of corticotropin releasing hormone (CRH) mRNA in adult male and female rats, with greater effects in male rats. Shown are the respective levels of CRH mRNA in male (A) and female (B) rats. Data normalized against control are means ± SEM from 5 rats per treatment group. *P < 0.05 vs. all treatment groups except Etomidate (ET) only group; ^#P < 0.05 vs. Control group; ^@P = 0.004 vs. female ET + Maternal separation (SEP) group.

Figure 5

Anesthesia with etomidate for 2 h at postnatal days (P) 4, 5 or 6 followed by maternal separation for 3 h at P10 lead to increased serum levels of corticosterone after physical restraint for 30 min in adult male and female rats. These effects were alleviated by pretreatment with bumetanide prior to anesthesia with etomidate. Shown are the respective levels of serum corticosterone in male (A) and female (B) rats across each collection point, as well as the total corticosterone response (AUCg in male (C) and female (D) rats. Morning measurements were taken as baselines for calculations of the total corticosterone responses. Data are means ± SEM from 9 male rats per treatment group and from 6 female rats per treatment group, except the Bumetanide (BU) + Etomidate (ET) + Maternal separation (SEP) group, which had 7 female rats. *P<0.05 vs. Control, SEP, and BU + ET + SEP groups. ^#P<0.05 vs. SEP, and BU + ET + SEP groups.

Figure 6

Anesthesia with etomidate for 2 h at postnatal days (P) 4, 5 or 6 followed by maternal separation for 3 h at P10 lead to reduced time spent in open arms of the elevated plus maze (EPM) in adult male, but not female rats, an effect that was alleviated by pretreatment with bumetanide prior to anesthesia with etomidate. The EPM tests were performed at P~60. Shown are % of time spent in open arms of the EPM and distance traveled by male (A,B) and female (C,D) rats. Data are means ± SEM from 14–16 male and 14–17 female rats per treatment group. ^#P < 0.05 vs. Control, and Bumetanide + Etomidate (ET) + Maternal separation (SEP) groups.