An essential role for orexins in emergence from general anesthesia

Abstract

The neural mechanisms through which the state of anesthesia arises and dissipates remain unknown. One common belief is that emergence from anesthesia is the inverse process of induction, brought about by elimination of anesthetic drugs from their CNS site(s) of action. Anesthetic-induced unconsciousness may result from specific interactions of anesthetics with the neural circuits regulating sleep and wakefulness. Orexinergic agonists and antagonists have the potential to alter the stability of the anesthetized state. In this report, we refine the role of the endogenous orexin system in impacting emergence from, but not entry into the anesthetized state, and in doing so, we distinguish mechanisms of induction from those of emergence. We demonstrate that isoflurane and sevoflurane, two commonly used general anesthetics, inhibit c-Fos expression in orexinergic but not adjacent melanin-concentrating hormone (MCH) neurons; suggesting that wake-active orexinergic neurons are inhibited by these anesthetics. Genetic ablation of orexinergic neurons, which causes acquired murine narcolepsy, delays emergence from anesthesia, without changing anesthetic induction. Pharmacologic studies with a selective orexin-1 receptor antagonist confirm a specific orexin effect on anesthetic emergence without an associated change in induction. We conclude that there are important differences in the neural substrates mediating induction and emergence. These findings support the concept that emergence depends, in part, on recruitment and stabilization of wake-active regions of brain.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Specific inactivation of orexinergic neurons in wild-type mice by exposure to anesthetizing doses of isoflurane and sevoflurane. Coronal sections through the perifornical hypothalamus depict c-Fos staining (red nuclei) in orexinergic neurons (green cytoplasm, A–C) or in MCH neurons (green cytoplasm, D and E) prepared from adjacent sections of perifornical hypothalamus. (A) Nonanesthetized oxygen control mouse. (B) Isoflurane-anesthetized mouse. (C) Sevoflurane-anesthetized mouse. (D) Nonanesthetized oxygen control mouse. (E) Isoflurane-anesthetized mouse. (F) Bar graphs summarizing c-Fos expression in both neuronal populations. Arrows depict examples of double-positive neurons, and arrowheads mark MCH or orexinergic neurons that lack c-Fos expression. (Scale bar: A–E, 100 μm.) Insets D-1 and E-1 show higher magnification of MCH-positive, c-Fos-negative neurons, and Insets D-2, D-3, E-2, and E-3 show higher-power views of strong or weak c-Fos signals above background, which were all scored as c-Fos positive. (Scale bar: Insets, 20 μm.) All bar graphs reveal mean ± SEM counts. Cell counts were analyzed by ANOVA with post hoc Bonferroni correction for multiple comparisons. *, P < 0.05; ***, P < 0.001; both relative to nonanesthetized oxygen control group.

Fig. 2.

Dose–response curves demonstrate equivalent sensitivity to induction of anesthesia despite genetic or pharmacologic impairment of orexin signaling. y axis depicts the fraction of mice that have lost their righting reflex as a function of the log10 of increasing concentrations of isoflurane or sevoflurane. (A) The ED50, also known as the minimum alveolar concentration at which half the mice lose their righting reflex (MACLORR), and Hill slopes are equivalent for wild-type mice (open triangles) and orexin/ataxin-3 mice (filled circles) after stepwise increases in isoflurane (black symbols) or sevoflurane (gray symbols). Best-fit sigmoidal dose–response curves are shown for wild-type mice (dashed lines) and for orexin/ataxin-3 mice (solid lines). (B) Equivalent isoflurane dose–response for mice treated with SB-334867-A (open circles) or vehicle (black triangles). Best-fit sigmoidal dose–response curves are shown for vehicle-treated (dashed lines) and for SB-334867-A-treated mice (solid lines). (C) Pharmacodynamic assessment demonstrating identical onset of isoflurane anesthesia in mice treated with vehicle or increasing doses of SB-334867-A.

Fig. 3.

Genetic and pharmacologic inhibition of orexin signaling delays emergence from anesthesia. Emergence from anesthesia was determined by the time elapsed from discontinuation of an anesthetic until the return of the righting reflex. (A) Relative to wild-type sibling controls, emergence times for orexin/ataxin-3 mice are significantly longer for isoflurane (shown in black) and for sevoflurane (shown in gray). Two-way ANOVA followed by Bonferroni testing. (B) Emergence times for wild-type C57BL/6J mice (in minutes) after a 2-h exposure to 1.25% isoflurane and i.p. injections of vehicle, 5 mg/kg SB-334867-A, or 20 mg/kg SB-334867-A. ANOVA followed by Bonferroni corrections. All data are expressed as mean ± SEM. ***, P < 0.001; *, P < 0.05.

Fig. 4.

Emergence by EEG and EMG criteria in wild-type mice while orexin/ataxin-3 siblings remain anesthetized. At 9.8 ± 2.2 min after discontinuation of the isoflurane, wild-type mice exhibit EEG and EMG evidence of emergence from anesthesia as depicted by the top two tracings that show the abrupt return of low-amplitude, fast-frequency EEG (tracing 1), along with concurrent EMG tone (tracing 2). Emergence occurs with a similar appearance in orexin/ataxin-3 mice but is significantly delayed at 19.2 ± 2.9 min.