Anesthesiology 2007; 106:1147–55 Copyright © 2007, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Cholinergic Modulation of Sevoflurane Potency in Cortical and Spinal Networks In Vitro Christian Grasshoff, M.D.,* Berthold Drexler, M.D.,* Harald Hentschke, Ph.D.,† Horst Thiermann, M.D.,‡ Bernd Antkowiak, Ph.D.§ Background: Victims of organophosphate intoxication with dred thousand casualties every year.6 Scenarios of mass injury cholinergic crisis may have need for sedation and anesthesia, such as the terrorist attack in Tokyo in 1995 with the nerve but little is known about how anesthetics work in these pa- agent sarin occur rarely. However, when they become reality, tients. Recent studies suggest that cholinergic stimulation im- pairs ␥-aminobutyric acid type A (GABA ) receptor function. victims are likely to suffer not only from intoxication but also A 1,7 Because GABAA receptors are major targets of general anesthet- from physical trauma. These subjects may have to undergo Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/106/6/1147/364219/0000542-200706000-00015.pdf by guest on 01 October 2021 ics, the authors investigated interactions between acetylcholine surgical interventions, raising the need for general anesthesia.8 and sevoflurane in spinal and cortical networks. So far, little is known about how general anesthetics act Methods: Cultured spinal and cortical tissue slices were ob- in patients afflicted with cholinergic crisis. This problem is tained from embryonic and newborn mice. Drug effects were assessed by extracellular voltage recordings of spontaneous of relevance because there is clear evidence in the litera- ␥ action potential activity. ture that -aminobutyric acid type A (GABAA) receptor– Results: Sevoflurane caused a concentration-dependent de- mediated inhibition, a molecular mechanism that is in- ؍ crease in spontaneous action potential firing in spinal (EC50 volved in mediating the sedative and hypnotic actions of .mM) slices 0.01 ؎ 0.29 ؍ mM) and cortical (EC 0.02 ؎ 0.17 50 most clinically used anesthetics, is hampered by cholin- Acetylcholine elevated neuronal excitation in both prepara- tions and diminished the potency of sevoflurane in reducing ergic stimulation: In rat cerebral cortical slices, it was dem- action potential firing in cortical but not in spinal slices. This onstrated that acetylcholine reduces ␥-aminobutyric acid brain region-specific decrease in sevoflurane potency was mim- (GABA) release from presynaptic terminals by activating 9 icked by the specific GABAA receptor antagonist bicuculline, muscarinic receptors. Furthermore, a recent study has suggesting that (1) GABA receptors are major molecular targets A shown that GABA release is also decreased via nicotinic for sevoflurane in the cortex but not in the spinal cord and (2) receptors.10 Besides these presynaptic actions of acetylcho- acetylcholine impairs the efficacy of GABAA receptor–mediated inhibition. The latter hypothesis was supported by the finding line, further mechanisms may come into play, rendering that acetylcholine reduced the potency of etomidate in depress- GABAA receptor–mediated inhibition ineffective. Excessive ing cortical and spinal neurons. neuronal activity, as observed during cholinergic crisis, Conclusions: The authors raise the question whether cholin- shifts the equilibrium potential for chloride ions toward ergic overstimulation decreases the efficacy of GABAA receptor function in patients with organophosphate intoxication, more positive values, thereby reducing the amplitude of 11,12 thereby compromising anesthetic effects that are mediated pre- GABAA receptor–mediated synaptic events. In addi- dominantly via these receptors such as sedation and hypnosis. tion, extracellular accumulation of GABA may desensitize synaptically located GABAA receptors and, as a conse- MANY potent insecticides and nerve agents belong to the quence, depress synaptic GABAergic transmission. family of organophosphorus compounds. These highly toxic We have previously shown that volatile anesthetics chemicals act by blocking acetylcholinesterase activity, such as halothane, isoflurane, and enflurane depress neu- thereby causing a potentially life-threatening cholinergic crisis ronal activity in cortical networks in vivo and in vitro by hallmarked by centrally mediated symptoms such as general- potentiating GABAA receptor–mediated synaptic inhibi- ized convulsions, respiratory failure, and cardiovascular insta- tion at concentrations causing sedation and hypno- bility.1–3 Furthermore, peripheral symptoms including rhinor- sis.13,14 Assuming that actions on the molecular and rhea, hypersalivation, bronchoconstriction, and network level are linked causally, how does a cholin- 1,4,5 neuromuscular block are commonly observed. Suicidal ergic-induced suppression of GABAA receptor function and accidental poisoning by organophosphates is a wide- affect the potency of general anesthetics in depressing spread problem in the developing world, causing several hun- neuronal activity? In the current study, interactions be- tween acetylcholine and sevoflurane were analyzed be- * Research Assistant, † Assistant Professor, § Professor of Experimental Anes- cause volatile anesthetics were recommended for main- thesiology, Experimental Anesthesiology Section, Department of Anesthesiology, tenance of anesthesia in nerve agent–intoxicated University of Tuebingen, Tuebingen, Germany. ‡ Postdoctoral University Lec- 1 turer of Pharmacology and Toxicology, GAF Institute of Pharmacology and patients. Experiments were conducted in organotypic Toxicology, Munich, Germany. slice cultures derived from the neocortex and spinal Received from the Experimental Anesthesiology Section, Department of An- cord because these neuronal microcircuits are important esthesiology and Intensive Care, Eberhard-Karls-University, Tuebingen, Germany. Submitted for publication September 25, 2006. Accepted for publication Febru- substrates for producing major components of general ary 9, 2007. Supported in part by contract No. M SAB1 3A014 from the German anesthesia such as amnesia, hypnosis, and immobili- Ministry of Defense, Munich, Germany. 14–20 Address correspondence to Dr. Grasshoff: Experimental Anesthesiology Sec- ty. The results indicate that cholinergic stimulation tion, Department of Anesthesiology and Intensive Care, Eberhard-Karls-Univer- reverses the depressant effects of sevoflurane by differ- sity, Schaffhausenstr. 113, D-72072 Tuebingen, Germany. [email protected]. Individual article reprints may be pur- ent mechanisms in cortical compared with spinal net- chased through the Journal Web site, www.anesthesiology.org. works (1) by increasing neuronal excitability in both Anesthesiology, V 106, No 6, Jun 2007 1147 1148 GRASSHOFF ET AL. brain regions and (2) by decreasing anesthetic potency required to suppress movement in response to noxious stim- in cortical networks. ulation in 50% of subjects. We used the EC50 values for general anesthesia proposed by Franks and Lieb.30 Therefore, we assumed that 1 MAC corresponds to an aqueous concentra- 26 Materials and Methods tion of 0.35 mM sevoflurane. Sevoflurane was administered via bath perfusion using Spinal Slice Cultures gastight syringe pumps (ZAK Medicine Technique, Markt- All procedures were approved by the animal care com- heidenfeld, Germany), which were connected to the ex- mittee (Eberhard-Karls-University, Tuebingen, Germany) perimental chamber via Teflon tubing (Lee, Frankfurt, Ger- and were in accord with the German law on animal many). The flow rate was approximately 1 ml/min. To experimentation. Spinal cord slices were prepared from ensure steady state conditions, recordings during anes- embryos of pregnant 129/SvJ mice (days 14 and 15) Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/106/6/1147/364219/0000542-200706000-00015.pdf by guest on 01 October 2021 thetic treatment were conducted 10–15 min after starting according to the method described by Braschler et al.21 the perfusate change. The calibration of the recording Neocortical slices were obtained from 2- to 5-day-old system was performed as previously reported.31 129/SvJ mice as previously reported.22,23 Excised slices were placed on a coverslip and embedded Data Analysis in a plasma clot (Sigma, Taufkirchen, Germany). The cov- Data were bandpass filtered between 3 and 10 kHz as erslips were transferred into plastic tubes containing 0.75 acquired on a personal computer using the Digidata ml of nutrient fluid and incubated with 95% oxygen and 5% 1200 analog-to-digital/digital-to-analog interface (Axon carbon dioxide at 36.0°C for 1–2 h. One hundred milliliters Instruments, Union City, CA). Records were in addition of nutrient fluid consisted of 25 ml horse serum (Invitro- stored on a Sony data recorder PC 204A (Racal Elek- gen, Karlsruhe, Germany), 25 ml Hanks’ Balanced Salt So- tronik, Bergisch Gladbach, Germany). Further analysis lution (Sigma), and 50 ml Basal Medium Eagle (Sigma). For was performed using self-written software in OriginPro spinal slices, nutrient fluid included 10 nM Neuronal version 7 (OriginLab Corporation, Northampton, MA) Growth Factor (Sigma). The roller tube technique was used and MATLAB 6.5 (The MathWorks Inc., Natick, MA). to culture the tissue.24 After 1 day in culture, antimitotics Data analysis was conducted as described previously.26 (10 ␮M 5-fluoro-2-deoxyuridine, 10 ␮M cytosine-b-d-arabino- After close inspection of the data, a threshold was set furanoside,