- Email: [email protected]
Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice
Chemico-Biological Interactions 206 (2013) 555–560
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice Berthold Drexler a,⇑, Horst Thiermann b, Bernd Antkowiak a, Christian Grasshoff a a b
Department of Anaesthesiology, Experimental Anaesthesiology Section, Eberhard-Karls-University, Waldhoernlestr. 22, 72072 Tuebingen, Germany Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany
a r t i c l e
i n f o
Article history: Available online 6 July 2013 Keywords: Acetylcholine Succinylcholine Neuromuscular endplate
a b s t r a c t Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments – atropine, oximes and diazepam – are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested. The test-system evaluated in the current study showed sensitivity to succinylcholine with an EC50 as low as 1 lM thereby being close to the EC50 value in adult human patients (2.6 lM). Furthermore, action potential activity of spinal ventral horn neurons was not altered by succinylcholine. The latter observations strongly suggest that our preparation well predicts the qualitative and quantitative actions of novel drugs targeting the neuromuscular system in vivo. In summary, cocultures of spinal cord and muscle tissue seem to be a valid test-system for the development and investigation of new oximes. Moreover, practical aspects like transport over long distances to further laboratories, the opportunity to conduct longterm studies and the reduction of animal usage display further advantages of its use. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pesticide poisoning is an important clinical problem in rural regions of the developing world, killing at least 250–370,000 people every year [1]. Among pesticides, organophosphorus insecticides are the most important, being responsible for more than 2/3 of deaths due to their high toxicity and widespread use [2]. Organophosphorus insecticides and nerve agents share a common mechanism of toxic action by inhibiting acetylcholinesterase accounting for the same core treatments – atropine, oximes, and diazepam [3]. Although the core treatments are defined, high case fatalities up to 40% were reported for intoxication even with organophosphorus insecticides [2]. The high rate of fatalities points to inadequate treatment obviously due to limited understanding of the detailed mechanisms of toxicity. Atropine and oximes were introduced into
⇑ Corresponding author. Address: Experimental Anaesthesiology Section, Department of Anaesthesiology and Intensive Care, Eberhard-Karls-University, Waldhoernlestr. 22, 72072 Tuebingen, Germany. Tel.: +49 7071 298 1121; fax: +49 7071 292 5073. E-mail address: [email protected] (B. Drexler). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.06.021
clinical practice in the 1950s without randomized controlled clinical trials. As a result the ideal regimens for either therapy are a matter of discussion. Accordingly, trials on alternative interventions are hindered because the best way to apply the core treatments has not yet been established and is highly variable in clinical practice. This variability impedes the development of a widely accepted study protocol and limits the external validity of study results. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or with moderate poisoning [3]. Testing the efficacy of oximes remains a challenge since experiments in humans are unethical and achieving standardized experimental conditions in clinical trials is extremely complex, thereby bringing up the demand of an appropriate in vitro-test system. To meet the requirements of a reliable test we have lately described an in vitro-system which is easy to handle and allows for conducting toxicological long-term studies [4]. The test system consists of cocultures of spinal cord and muscle tissue. Spontaneous muscle activity is measured by video microscopy while corresponding action potential activity of spinal neurons can be quantified by extracellular electrophysiological recordings. Previous experiments
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Fig. 1. Chemical structure of the natural neurotransmitter acetylcholine and the muscle relaxant succinylcholine. Note that succinylcholine consists of two acetylcholine molecules that are linked by their acetyl groups.
demonstrated that 10 lM obidoxime sufficiently restored muscle function after induction of paralysis by the nerve agent VX [4]. However, it remained unclear whether this happened at pathophysiologically relevant concentrations of acetylcholine which remains an important issue in judging the transferability of the results into complex biological systems. To tackle this issue the current study was performed to evaluate a test system which consists of cocultures of spinal cord and muscle tissue with regard to its nerve-muscle interactions. The system is intended for the implementation of experiments with oximes under physiological and pathophysiological conditions. Pathophysiological changes in the extracellular acetylcholine-concentration are difficult to determine in vivo since the transmitter is ultra rapidly cleaved in the synaptic cleft by acetylcholinesterase. In contrast, di-acetylcholine (succinylcholine) is eliminated by hydrolysis by butyrylcholinesterases and is in clinical use as a depolarizing muscle relaxant. Succinylcholine is composed of two acetylcholine molecules (Fig. 1), linked end to end at the acetyl side, and has the potential to exert acetylcholine-like effects at nicotinic acetylcholine receptors [5]. The nicotinic acetylcholine receptors are members of a neurotransmitter-gated ion channel superfamily. Like GABAA- or glycine receptors, they are composed of five transmembrane units with a central pore. The muscle nicotinic acetylcholine receptor is present at the postsynaptic muscle membrane in the neuromuscular junction, neuronal nicotinic acetylcholine receptors are expressed both presynaptically and postsynaptically. In clinical use, application of succinylcholine as a peripheral acting muscle depolarizing drug results in fasciculation of skeletal muscle by activation of muscle-type nicotinic acetylcholine receptors without evoking symptoms at the central nervous system. The principle idea of the experiments was to validate nervemuscle cocultures as an ex vivo model system for pharmacological interventions of the cholinergic system by studying the effects of succinylcholine on nerve muscle interactions and on spontaneous action potential activity. The effects of succinylcholine in the cocultures should meet two criteria: (1) di-acetylcholine (succinylcholine) should induce a concentration-dependant depression of muscular activity at clinically relevant concentrations and (2) succinylcholine should not interact with neuronal acetylcholine receptors, which was verified by assessing the effects of the muscle relaxant on spontaneous action potential activity in the cultures.
(Eberhard-Karls-University, Tuebingen, Germany) and were in accordance with the German law on animal experimentation. Organotypic cocultures of spinal cord, dorsal root ganglia and skeletal muscle were prepared from embryos of pregnant mice as described by Braschler [6]. All efforts were made to minimize both the suffering and number of animals used. In brief, pregnant animals were deeply anesthetized with isoflurane, the uterus was aseptically removed and embryos (E 13–15) were stored in ice-cold Gey’s balanced salt solution. The embryos were decapitated, and the spinal column was separated from the inner organs and the limbs. The spinal column was then embedded into an agar block and cut into 300 lm transverse slices using a vibratome. Afterward, slices including the spinal cord and skeletal muscle were placed on a coverslip and embedded in a plasma clot. The coverslips were inserted into plastic tubes, supplemented with nutrient fluid including nerve growth factor beta (Sigma–Aldrich, Taufkirchen, Germany) and incubated at 36 °C. The roller tube technique as described by Gähwiler [7] was used. After 1 day in culture, antimitotics were added to reduce proliferation of glial cells. The nutrient fluid was renewed twice a week. 2.2. Preparation and application of test solutions Succinylcholine (Sigma–Aldrich, Taufkirchen, Germany) was dissolved in artificial cerebrospinal fluid (ACSF) and filled into glass syringes. The drug containing ACSF was applied via bath perfusion using syringe pumps (ZAK, Marktheidenfeld, Germany), connected to the experimental chamber via Teflon tubing (Lee, Frankfurt, Germany). The flow rate was approximately 1 ml min 1. When switching from ACSF to succinylcholine-containing ACSF, the medium in the experimental chamber was replaced by at least 95% within 2 min. Recordings during succinylcholine treatment were started 10–12 min after change to the succinylcholine-containing perfusate. This time interval has been proven to be sufficient to adjust steady state conditions [8] as diffusion times in slice cultures are considerably shorter compared to acute slice preparations [9,10]. However, after removal of succinylcholine from the bathing solution, full recovery of muscle activity was not observed within a time window of 30 min when drug concentrations in the high micromolar concentration range were tested. 2.3. Recording of spontaneous action potential activity Extracellular network recordings were performed in a recording chamber mounted on an inverted microscope. Slices were perfused with ACSF consisting of (in mM) NaCl 120, KCl 3.3, NaH2PO4 1.13, NaHCO3 26, CaCl2 1.8, MgCl2 1.0, glucose 11, bubbled with 95% oxygen and 5% carbon dioxide. To facilitate reproducible neuronal firing patterns the spinal network activity was adjusted by addition of 5 lM bicuculline and 0.5 lM strychnine (both from Sigma– Aldrich, Taufkirchen, Germany) to the ASCF. The recording chamber consisted of a metal frame with a glass bottom and had a volume of 1.5 ml. All electrophysiological experiments were conducted at 34 °C. The ventral horn of the spinal cord was visually identified and ACSF-filled glass electrodes with a resistance of about 3–5 MO were inserted into the tissue until extracellular spike activity exceeding 100 lV in amplitude could be detected. Data were acquired on a personal computer with the digidata 1200 AD/DA interface and Clampex 10.1 software (Axon Instruments, Foster City, USA).
2. Material and methods 2.4. Analysis of electrophysiology data 2.1. Organotypic slice cultures of spinal cord and muscle tissue Wild type C57bl6J mice of both sexes were used for this study. All procedures were approved by the animal care committee
Extracellular recorded signals were filtered and counted offline using self-written quality controlled programs in Matlab 7.5 (The Mathworks, Natick, USA). Action potentials were registered and
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(a)
(b)
Fig. 2. (A) Schematic representation of the methodical approach. The main components are the cultured slice from the spinal cord (in the centre) with attached muscle fibres (bottom left corner). The contractions of the muscle fibres (represented by the arrows) are monitored by a video camera. Simultaneously, an extracellular electrode is positioned in the ventral horn of the organotypic spinal slice culture to detect neuronal activity. (B) Photo taken from an organotypic nerve-muscle coculture. As schematically shown in (A), three muscle fibres, spanning from top left to down right, can be identified. For the analysis of muscle activity a region of interest (ROI, displayed as yellow square, also drawn schematically in (A) is defined at the border of a muscle fibre. A contraction of the muscle fibre leads to a shift of the border of the muscle fibre. Left side: relaxed state of the muscle fibre. The bright border of the muscle fibre is located at the lower left edge of the ROI (see enlargement in the right upper corner of the figure.) Right side: contracted state of the muscle fibre. The bright border of the muscle fibre has shifted up- and rightwards and runs now right through the middle of the ROI from top left to down right.
average firing rate computed using an automated event detection algorithm with a threshold set approximately two times higher than the baseline noise. All parameters are shown as relative change compared to control condition. We used Student t-test for statistical testing, p values <0.05 were considered significant. All results are given as mean ± SEM.
MySonicDVD 4.5 software (both from adaptec, Milpitas, CA, USA) and stored in audio video interleave (avi) format. Three recordings of 70 s duration were performed for each condition (control, drug, wash). The temperature was kept at 34 °C.
2.6. Quantification of muscle activity 2.5. Recording and analysis of muscle activity data Muscle contractions in nerve-muscle-cultures are controlled by acetylcholine-releasing motor neurons that project to muscle fibres [11,12]. Muscle movements were monitored before and during succinylcholine-exposure by a video camera (Hamamatsu C-2400, Hamamatsu Photonics, Herrsching, Germany), digitized for further analysis using an AVC-2310 A/D converter and
Muscle activity was quantified offline using a Hewlett Packard Z800 computer (Picturetools, Hamburg, Germany). First, the regions of interest (ROI) were defined by visual inspection. Care was taken that these ROI contained high-contrast borders of muscle fibres which were dislocated in the course of muscle contractions. Due to the muscle movements, the brightness of pixels (8 bit resolution) located within the ROI changed with time. These changes well correlated with the intensity and frequency of muscle
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contractions [4]. An in-house developed quality controlled algorithm was used for quantifying the changes in brightness between subsequent video frames. Software for analyzing the data was written in Matlab 7.1. (The MatWorks Inc., Natick, MA, USA). The recording of neuronal action potential firing and the analysis of muscle activity is schematically shown in Fig. 2A and B. For statistical testing the statistic toolbox of Matlab (The MatWorks Inc., Natick, MA, USA) was used, p values smaller than 0.05 were considered as significant.
A
3. Results 3.1. Effects of succinylcholine on muscle activity of organotypic spinal nerve-muscle cocultures The first set of experiments was composed to assess the first criteria mentioned in the introduction, namely the induction of a concentration-dependant depression of muscular activity at clinically relevant concentrations. For this purpose 52 nerve-muscle cocultures were studied. The averaged muscle activity under control conditions was 0.86 ± 0.09 Hz. The actions of succinylcholine were tested in a concentration range from 1 nM to 1 mM. No significant effects on muscular activity of nerve-muscle cocultures were observed up to a concentration of 100 nM. Though, at concentrations starting from 1 lM succinylcholine dramatically reduced the muscle activity of nerve-muscle cocultures, showing almost complete atony at 3 lM (1 lM succinylcholine 28.90 ± 0.06%; 3 lM 12.46 ± 0.05% and 10 lM 0.04 ± 0.02% of control activity, respectively). The concentration–response curve of succinylcholine in organotypic nerve-muscle cocultures is displayed in Fig. 3. From these data the EC50 of succinylcholine in organotypic nerve-muscle cocultures can be estimated as slightly smaller than 1 lM in vitro. As the concentration–response curve proved to be quite steep in the range of around 1 lM a detailed analysis of the effects of succinylcholine was performed (Fig. 4A and B). It turned out that succinylcholine was most effective in depressing spontaneous muscular activity within the concentration range of 0.1 and 1.0 lM especially in cultures with high basal activity.
B
Fig. 4. (A) Actions of small concentrations of succinylcholine on the muscular activity of nerve-muscle cocultures. The scatter plot of single experiments illustrates the effect of small concentrations of succinylcholine (100 nM, given as cross and 1 lM, given as square) on the muscular activity of organotypic cultures. In case of the muscular activity being not changed by succinylcholine, the symbol is depicted on the diagonal line. In case of a depression by succinylcholine, the symbol is plotted below and on the right of the diagonal line. In the opposite case, the symbol is located above and left of the diagonal line (increased muscular activity in the presence of small concentrations of succinylcholine). In approximately two thirds of all nerve-muscle cocultures small concentrations of succinylcholine (0.1 and 1 lM) lead to a depression of muscular activity. (B) The actions of succinylcholine at low concentrations are depending on the spontaneous activity of the nerve-muscle cocultures. Cocultures were divided into three groups according to their spontaneous basal activity (<0.5 Hz, 0.5–0.7 Hz and >0.7 Hz). The muscular activity in the presence of succinylcholine (0.1 and 1.0 lM) was normalized to control condition. In the group with the lowest basal activity succinylcholine slightly increased muscular activity (n = 12, n.s.). However, in the group with the highest basal activity succinylcholine led to a depression of muscular activity (n = 12, p < 0.02). Taken together succinylcholine induced a depression of muscular activity especially in preparations with high spontaneous muscular activity.
3.2. Succinylcholine does not change spontaneous action potential firing activity in organotypic slices
Fig. 3. Actions of succinylcholine on muscular activity. The concentration–response curve of succinylcholine is given as median including 95%-confidence interval. At a concentration of 1 lM succinylcholine leads to a significant depression of the muscular activity in nerve-muscle cocultures. However, at 3 lM succinylcholine there is an almost complete depression of muscular activity which means that the concentration–response-curve of succinylcholine in organotypic nerve-muscle cocultures is very steep.
In a following set of experiments 97 extracellular recordings in spinal ventral horn interneurons were performed to assess the effects of succinylcholine on the spontaneous action potential firing rate. The recordings were conducted in organotypic nerve-muscle cocultures at an age between 17 and 27 days ex vivo. The averaged action potential firing rate under control conditions was 34.2 ± 3.2 Hz. Representative traces of the multi-unit action potential firing activity in the absence and presence of succinylcholine
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(1 lM) are depicted in Fig. 5A. The typical firing pattern of cultured spinal neurons was composed of phases of high action potential firing (bursts of action potentials) which are separated by phases of low activity (Fig. 5A). The actions of succinylcholine on neuronal activity of spinal neurons were tested over a broad concentration range. Succinylcholine, from 1 nM to 1 mM did not induce systematic changes in neuronal activity (Fig. 5B). Therefore it can be concluded that the actions of succinylcholine on muscular activity are not mediated via changes of the neuronal activity in the cocultures.
to the concentrations at the synapse in spite of an equilibrium time of about 30 min. This has been previously demonstrated by Benkwitz et al. who showed that diffusion of the intravenous anaesthetic etomidate into brain slices requires approximately an hour to reach 80% equilibration at a typical recording depth of 100 lm [10]. Another possibility for evaluating pharmacologic effects on nicotinic receptors is the use of oocytes which express human muscular nicotinic acetylcholine receptors. Jonsson et al. found that succinylcholine concentration-dependently activated the muscle-type nicotinic acetylcholine receptor with an EC50 value of 10.8 lM [5]. The drawback of oocytes is that they do not represent the neuromuscular junction and are therefore an inadequate tool for testing interactions of organophosphate compounds and oximes. Thus the first demand, the sensitivity to succinylcholine is met by our test-system. The second requirement is the insensitivity of the spontaneous interneuronal activity to succinylcholine. As it has been demonstrated by Jonsson et al. in the oocyte preparation, succinylcholine does not activate the neuronal nicotinic acetylcholine receptors [5] and this finding is resembled in the cocultures of spinal cord and muscle tissue. In a preceding study we have demonstrated that cocultures of spinal cord and muscle tissue are a valuable test-system for investigating the effects of the nerve agent VX on muscle activity. While VX reduced muscle activity as expected the subsequent application of obidoxime at a clinical relevant concentration resulted in a restoration of muscle function [4]. A special feature of cocultures of spinal cord and muscle tissue is that the restoration of muscle activation took place after 24 h of antidotal treatment and remained constant over an observation period of approximately one week. This expanded time scale identifies the greatest advantage of the study system, the potential to conduct long-term studies. However it was unclear whether this activation-restoration of muscle-action happened in a test-system where the sensitivity of nicotinic acetylcholine receptors corresponds to that reported for humans. This could be confirmed by the results of the current study showing a sensitivity of muscle activity to succinylcholine as reported for adult patients previously. In general cocultures of spinal cord and muscle tissue seem to be an excellent test-system for the development and investigation of new oximes, since practical aspects like transport over long distances to further laboratories, the opportunity to conduct long-term studies and the reduction of animal usage display further advantages of its use.
4. Discussion
References
B
Fig. 5. (A) Original recording of multi-unit action potential firing from an organotypic nerve-muscle coculture. The typical firing pattern of ventral horn neurons ex vivo consists of bursts of action potentials separated by phases of very low neuronal activity. In the presence of 1 lM succinylcholine the neuronal activity does not show apparent changes. (B) Summary of electrophysiological data. The graph displays the normalized firing rate (1.0 corresponds to the activity at control conditions) in the presence of increasing concentrations of succinylcholine ranging from 1 nM to 1 mM. Note that there is no systematic change in neuronal action potential firing in the presence of the drug.
The potency of succinylcholine in humans has been determined in adult patients previously. Roy et al. investigated the concentration-effect relation of succinylcholine during propofol anaesthesia by measuring plasma concentrations of succinylcholine after its injection to characterize the front-end kinetics [13]. The calculated molar EC50 value in these adult human patients was 2.6 lM. The test-system evaluated in the current study showed a comparable sensitivity to succinylcholine with an EC50 as low as 1 lM. Further test-systems which have been involved in the admission of oximes are less sensitive to succinylcholine. In particular the mouse phrenic nerve-hemidiaphragm model was used in several studies [14– 16]. Advantages of the phrenic nerve-hemidiaphragm preparation are similarities with human neuromuscular junctions [17] and that it contains the diaphragm as an important muscle of respiration [18]. The sensitivity of the phrenic nerve-hemidiaphragm preparation to succinylcholine is approximately 10-fold lower, as indicated by an EC50 of 21.3 lM for adult rats [18]. However, this relatively high EC50 value can be put into perspective since the concentrations in the bath perfusion medium of the phrenic nerve-hemidiaphragm preparation is probably higher compared
[1] D. Gunnell, M. Eddleston, M.R. Phillips, F. Konradsen, The global distribution of fatal pesticide self-poisoning: systematic review, BMC Public Health 7 (2007) 357. [2] M. Eddleston, Patterns and problems of deliberate self-poisoning in the developing world, QJM 93 (2000) 715–731. [3] M. Eddleston, N.A. Buckley, P. Eyer, A.H. Dawson, Management of acute organophosphorus pesticide poisoning, Lancet 371 (2008) 597–607. [4] B. Drexler, T. Seeger, C. Grasshoff, H. Thiermann, B. Antkowiak, Long-term evaluation of organophosphate toxicity and antidotal therapy in cocultures of spinal cord and muscle tissue, Toxicol. Lett. 206 (2011) 89–93. [5] M. Jonsson, M. Dabrowski, D.A. Gurley, O. Larsson, E.C. Johnson, B.B. Fredholm, L.I. Eriksson, Activation and inhibition of human muscular and neuronal nicotinic acetylcholine receptors by succinylcholine, Anesthesiology 104 (2006) 724–733. [6] U.F. Braschler, A. Iannone, C. Spenger, J. Streit, H.-R. Lüscher, A modified roller tube technique for organotypic cocultures of embryonic rat spinal cord, sensory ganglia and skeletal muscle, J. Neurosci. Methods 29 (1989) 121–129. [7] B.H. Gähwiler, Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods 4 (1981) 329–342. [8] B. Antkowiak, Different actions of general anaesthetics on the firing patterns of neocortical neurons mediated by the GABAA receptor, Anesthesiology 91 (1999) 500–511. [9] J.A. Gredell, P.A. Turnquist, M.B. MacIver, R.A. Pearce, Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro, Br. J. Anaesth. 93 (2004) 810– 817.
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[10] C. Benkwitz, M. Liao, M.J. Laster, J.M. Sonner, E.I. Eger, R.A. Pearce, Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies, Anesthesiology 106 (2007) 114–123. [11] V. Magloire, J. Streit, Intrinsic activity and positive feedback in motor circuits in organotypic spinal cord slice cultures, Eur. J. Neurosci. 30 (2009) 1487–1497. [12] M.D. Rosato-Siri, D. Zoccolan, F. Furlan, L. Ballerini, Interneurone bursts are spontaneously associated with muscle contractions only during early phases of mouse spinal network development: a study in organotypic cultures, Eur. J. Neurosci. 20 (2004) 2697–2710. [13] J.J. Roy, F. Donati, D. Boismenu, F. Varin, Concentration-effect relation of succinylcholine chloride during propofol anesthesia, Anesthesiology 97 (2002) 1082–1092. [14] J.E. Tattersall, Ion channel blockade by oximes and recovery of diaphragm muscle from soman poisoning in vitro, Br. J. Pharmacol. 108 (1993) 1006–1015.
[15] T. Seeger, F. Worek, L. Szinicz, H. Thiermann, Reevaluation of indirect field stimulation technique to demonstrate oxime effectiveness in OP-poisoning in muscles in vitro, Toxicology 233 (2007) 209–213. [16] H. Thiermann, P. Eyer, F. Worek, Muscle force and acetylcholinesterase activity in mouse hemidiaphragms exposed to paraoxon and treated by oximes in vitro, Toxicology 272 (2010) 46–51. [17] T. Seeger, M. Eichhorn, M. Lindner, K.V. Niessen, J.E. Tattersall, C.M. Timperley, M. Bird, A.C. Green, H. Thiermann, F. Worek, Restoration of soman-blocked neuromuscular transmission in human and rat muscle by the bispyridinium non-oxime MB327 in vitro, Toxicology 294 (2012) 80– 84. [18] L.P. Fortier, R. Robitaille, F. Donati, Increased sensitivity to depolarization and nondepolarizing neuromuscular blocking agents in young rat hemidiaphragms, Anesthesiology 95 (2001) 478–484.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice Berthold Drexler a,⇑, Horst Thiermann b, Bernd Antkowiak a, Christian Grasshoff a a b
Department of Anaesthesiology, Experimental Anaesthesiology Section, Eberhard-Karls-University, Waldhoernlestr. 22, 72072 Tuebingen, Germany Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany
a r t i c l e
i n f o
Article history: Available online 6 July 2013 Keywords: Acetylcholine Succinylcholine Neuromuscular endplate
a b s t r a c t Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments – atropine, oximes and diazepam – are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested. The test-system evaluated in the current study showed sensitivity to succinylcholine with an EC50 as low as 1 lM thereby being close to the EC50 value in adult human patients (2.6 lM). Furthermore, action potential activity of spinal ventral horn neurons was not altered by succinylcholine. The latter observations strongly suggest that our preparation well predicts the qualitative and quantitative actions of novel drugs targeting the neuromuscular system in vivo. In summary, cocultures of spinal cord and muscle tissue seem to be a valid test-system for the development and investigation of new oximes. Moreover, practical aspects like transport over long distances to further laboratories, the opportunity to conduct longterm studies and the reduction of animal usage display further advantages of its use. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pesticide poisoning is an important clinical problem in rural regions of the developing world, killing at least 250–370,000 people every year [1]. Among pesticides, organophosphorus insecticides are the most important, being responsible for more than 2/3 of deaths due to their high toxicity and widespread use [2]. Organophosphorus insecticides and nerve agents share a common mechanism of toxic action by inhibiting acetylcholinesterase accounting for the same core treatments – atropine, oximes, and diazepam [3]. Although the core treatments are defined, high case fatalities up to 40% were reported for intoxication even with organophosphorus insecticides [2]. The high rate of fatalities points to inadequate treatment obviously due to limited understanding of the detailed mechanisms of toxicity. Atropine and oximes were introduced into
⇑ Corresponding author. Address: Experimental Anaesthesiology Section, Department of Anaesthesiology and Intensive Care, Eberhard-Karls-University, Waldhoernlestr. 22, 72072 Tuebingen, Germany. Tel.: +49 7071 298 1121; fax: +49 7071 292 5073. E-mail address: [email protected] (B. Drexler). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.06.021
clinical practice in the 1950s without randomized controlled clinical trials. As a result the ideal regimens for either therapy are a matter of discussion. Accordingly, trials on alternative interventions are hindered because the best way to apply the core treatments has not yet been established and is highly variable in clinical practice. This variability impedes the development of a widely accepted study protocol and limits the external validity of study results. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or with moderate poisoning [3]. Testing the efficacy of oximes remains a challenge since experiments in humans are unethical and achieving standardized experimental conditions in clinical trials is extremely complex, thereby bringing up the demand of an appropriate in vitro-test system. To meet the requirements of a reliable test we have lately described an in vitro-system which is easy to handle and allows for conducting toxicological long-term studies [4]. The test system consists of cocultures of spinal cord and muscle tissue. Spontaneous muscle activity is measured by video microscopy while corresponding action potential activity of spinal neurons can be quantified by extracellular electrophysiological recordings. Previous experiments
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B. Drexler et al. / Chemico-Biological Interactions 206 (2013) 555–560
Fig. 1. Chemical structure of the natural neurotransmitter acetylcholine and the muscle relaxant succinylcholine. Note that succinylcholine consists of two acetylcholine molecules that are linked by their acetyl groups.
demonstrated that 10 lM obidoxime sufficiently restored muscle function after induction of paralysis by the nerve agent VX [4]. However, it remained unclear whether this happened at pathophysiologically relevant concentrations of acetylcholine which remains an important issue in judging the transferability of the results into complex biological systems. To tackle this issue the current study was performed to evaluate a test system which consists of cocultures of spinal cord and muscle tissue with regard to its nerve-muscle interactions. The system is intended for the implementation of experiments with oximes under physiological and pathophysiological conditions. Pathophysiological changes in the extracellular acetylcholine-concentration are difficult to determine in vivo since the transmitter is ultra rapidly cleaved in the synaptic cleft by acetylcholinesterase. In contrast, di-acetylcholine (succinylcholine) is eliminated by hydrolysis by butyrylcholinesterases and is in clinical use as a depolarizing muscle relaxant. Succinylcholine is composed of two acetylcholine molecules (Fig. 1), linked end to end at the acetyl side, and has the potential to exert acetylcholine-like effects at nicotinic acetylcholine receptors [5]. The nicotinic acetylcholine receptors are members of a neurotransmitter-gated ion channel superfamily. Like GABAA- or glycine receptors, they are composed of five transmembrane units with a central pore. The muscle nicotinic acetylcholine receptor is present at the postsynaptic muscle membrane in the neuromuscular junction, neuronal nicotinic acetylcholine receptors are expressed both presynaptically and postsynaptically. In clinical use, application of succinylcholine as a peripheral acting muscle depolarizing drug results in fasciculation of skeletal muscle by activation of muscle-type nicotinic acetylcholine receptors without evoking symptoms at the central nervous system. The principle idea of the experiments was to validate nervemuscle cocultures as an ex vivo model system for pharmacological interventions of the cholinergic system by studying the effects of succinylcholine on nerve muscle interactions and on spontaneous action potential activity. The effects of succinylcholine in the cocultures should meet two criteria: (1) di-acetylcholine (succinylcholine) should induce a concentration-dependant depression of muscular activity at clinically relevant concentrations and (2) succinylcholine should not interact with neuronal acetylcholine receptors, which was verified by assessing the effects of the muscle relaxant on spontaneous action potential activity in the cultures.
(Eberhard-Karls-University, Tuebingen, Germany) and were in accordance with the German law on animal experimentation. Organotypic cocultures of spinal cord, dorsal root ganglia and skeletal muscle were prepared from embryos of pregnant mice as described by Braschler [6]. All efforts were made to minimize both the suffering and number of animals used. In brief, pregnant animals were deeply anesthetized with isoflurane, the uterus was aseptically removed and embryos (E 13–15) were stored in ice-cold Gey’s balanced salt solution. The embryos were decapitated, and the spinal column was separated from the inner organs and the limbs. The spinal column was then embedded into an agar block and cut into 300 lm transverse slices using a vibratome. Afterward, slices including the spinal cord and skeletal muscle were placed on a coverslip and embedded in a plasma clot. The coverslips were inserted into plastic tubes, supplemented with nutrient fluid including nerve growth factor beta (Sigma–Aldrich, Taufkirchen, Germany) and incubated at 36 °C. The roller tube technique as described by Gähwiler [7] was used. After 1 day in culture, antimitotics were added to reduce proliferation of glial cells. The nutrient fluid was renewed twice a week. 2.2. Preparation and application of test solutions Succinylcholine (Sigma–Aldrich, Taufkirchen, Germany) was dissolved in artificial cerebrospinal fluid (ACSF) and filled into glass syringes. The drug containing ACSF was applied via bath perfusion using syringe pumps (ZAK, Marktheidenfeld, Germany), connected to the experimental chamber via Teflon tubing (Lee, Frankfurt, Germany). The flow rate was approximately 1 ml min 1. When switching from ACSF to succinylcholine-containing ACSF, the medium in the experimental chamber was replaced by at least 95% within 2 min. Recordings during succinylcholine treatment were started 10–12 min after change to the succinylcholine-containing perfusate. This time interval has been proven to be sufficient to adjust steady state conditions [8] as diffusion times in slice cultures are considerably shorter compared to acute slice preparations [9,10]. However, after removal of succinylcholine from the bathing solution, full recovery of muscle activity was not observed within a time window of 30 min when drug concentrations in the high micromolar concentration range were tested. 2.3. Recording of spontaneous action potential activity Extracellular network recordings were performed in a recording chamber mounted on an inverted microscope. Slices were perfused with ACSF consisting of (in mM) NaCl 120, KCl 3.3, NaH2PO4 1.13, NaHCO3 26, CaCl2 1.8, MgCl2 1.0, glucose 11, bubbled with 95% oxygen and 5% carbon dioxide. To facilitate reproducible neuronal firing patterns the spinal network activity was adjusted by addition of 5 lM bicuculline and 0.5 lM strychnine (both from Sigma– Aldrich, Taufkirchen, Germany) to the ASCF. The recording chamber consisted of a metal frame with a glass bottom and had a volume of 1.5 ml. All electrophysiological experiments were conducted at 34 °C. The ventral horn of the spinal cord was visually identified and ACSF-filled glass electrodes with a resistance of about 3–5 MO were inserted into the tissue until extracellular spike activity exceeding 100 lV in amplitude could be detected. Data were acquired on a personal computer with the digidata 1200 AD/DA interface and Clampex 10.1 software (Axon Instruments, Foster City, USA).
2. Material and methods 2.4. Analysis of electrophysiology data 2.1. Organotypic slice cultures of spinal cord and muscle tissue Wild type C57bl6J mice of both sexes were used for this study. All procedures were approved by the animal care committee
Extracellular recorded signals were filtered and counted offline using self-written quality controlled programs in Matlab 7.5 (The Mathworks, Natick, USA). Action potentials were registered and
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(a)
(b)
Fig. 2. (A) Schematic representation of the methodical approach. The main components are the cultured slice from the spinal cord (in the centre) with attached muscle fibres (bottom left corner). The contractions of the muscle fibres (represented by the arrows) are monitored by a video camera. Simultaneously, an extracellular electrode is positioned in the ventral horn of the organotypic spinal slice culture to detect neuronal activity. (B) Photo taken from an organotypic nerve-muscle coculture. As schematically shown in (A), three muscle fibres, spanning from top left to down right, can be identified. For the analysis of muscle activity a region of interest (ROI, displayed as yellow square, also drawn schematically in (A) is defined at the border of a muscle fibre. A contraction of the muscle fibre leads to a shift of the border of the muscle fibre. Left side: relaxed state of the muscle fibre. The bright border of the muscle fibre is located at the lower left edge of the ROI (see enlargement in the right upper corner of the figure.) Right side: contracted state of the muscle fibre. The bright border of the muscle fibre has shifted up- and rightwards and runs now right through the middle of the ROI from top left to down right.
average firing rate computed using an automated event detection algorithm with a threshold set approximately two times higher than the baseline noise. All parameters are shown as relative change compared to control condition. We used Student t-test for statistical testing, p values <0.05 were considered significant. All results are given as mean ± SEM.
MySonicDVD 4.5 software (both from adaptec, Milpitas, CA, USA) and stored in audio video interleave (avi) format. Three recordings of 70 s duration were performed for each condition (control, drug, wash). The temperature was kept at 34 °C.
2.6. Quantification of muscle activity 2.5. Recording and analysis of muscle activity data Muscle contractions in nerve-muscle-cultures are controlled by acetylcholine-releasing motor neurons that project to muscle fibres [11,12]. Muscle movements were monitored before and during succinylcholine-exposure by a video camera (Hamamatsu C-2400, Hamamatsu Photonics, Herrsching, Germany), digitized for further analysis using an AVC-2310 A/D converter and
Muscle activity was quantified offline using a Hewlett Packard Z800 computer (Picturetools, Hamburg, Germany). First, the regions of interest (ROI) were defined by visual inspection. Care was taken that these ROI contained high-contrast borders of muscle fibres which were dislocated in the course of muscle contractions. Due to the muscle movements, the brightness of pixels (8 bit resolution) located within the ROI changed with time. These changes well correlated with the intensity and frequency of muscle
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contractions [4]. An in-house developed quality controlled algorithm was used for quantifying the changes in brightness between subsequent video frames. Software for analyzing the data was written in Matlab 7.1. (The MatWorks Inc., Natick, MA, USA). The recording of neuronal action potential firing and the analysis of muscle activity is schematically shown in Fig. 2A and B. For statistical testing the statistic toolbox of Matlab (The MatWorks Inc., Natick, MA, USA) was used, p values smaller than 0.05 were considered as significant.
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3. Results 3.1. Effects of succinylcholine on muscle activity of organotypic spinal nerve-muscle cocultures The first set of experiments was composed to assess the first criteria mentioned in the introduction, namely the induction of a concentration-dependant depression of muscular activity at clinically relevant concentrations. For this purpose 52 nerve-muscle cocultures were studied. The averaged muscle activity under control conditions was 0.86 ± 0.09 Hz. The actions of succinylcholine were tested in a concentration range from 1 nM to 1 mM. No significant effects on muscular activity of nerve-muscle cocultures were observed up to a concentration of 100 nM. Though, at concentrations starting from 1 lM succinylcholine dramatically reduced the muscle activity of nerve-muscle cocultures, showing almost complete atony at 3 lM (1 lM succinylcholine 28.90 ± 0.06%; 3 lM 12.46 ± 0.05% and 10 lM 0.04 ± 0.02% of control activity, respectively). The concentration–response curve of succinylcholine in organotypic nerve-muscle cocultures is displayed in Fig. 3. From these data the EC50 of succinylcholine in organotypic nerve-muscle cocultures can be estimated as slightly smaller than 1 lM in vitro. As the concentration–response curve proved to be quite steep in the range of around 1 lM a detailed analysis of the effects of succinylcholine was performed (Fig. 4A and B). It turned out that succinylcholine was most effective in depressing spontaneous muscular activity within the concentration range of 0.1 and 1.0 lM especially in cultures with high basal activity.
B
Fig. 4. (A) Actions of small concentrations of succinylcholine on the muscular activity of nerve-muscle cocultures. The scatter plot of single experiments illustrates the effect of small concentrations of succinylcholine (100 nM, given as cross and 1 lM, given as square) on the muscular activity of organotypic cultures. In case of the muscular activity being not changed by succinylcholine, the symbol is depicted on the diagonal line. In case of a depression by succinylcholine, the symbol is plotted below and on the right of the diagonal line. In the opposite case, the symbol is located above and left of the diagonal line (increased muscular activity in the presence of small concentrations of succinylcholine). In approximately two thirds of all nerve-muscle cocultures small concentrations of succinylcholine (0.1 and 1 lM) lead to a depression of muscular activity. (B) The actions of succinylcholine at low concentrations are depending on the spontaneous activity of the nerve-muscle cocultures. Cocultures were divided into three groups according to their spontaneous basal activity (<0.5 Hz, 0.5–0.7 Hz and >0.7 Hz). The muscular activity in the presence of succinylcholine (0.1 and 1.0 lM) was normalized to control condition. In the group with the lowest basal activity succinylcholine slightly increased muscular activity (n = 12, n.s.). However, in the group with the highest basal activity succinylcholine led to a depression of muscular activity (n = 12, p < 0.02). Taken together succinylcholine induced a depression of muscular activity especially in preparations with high spontaneous muscular activity.
3.2. Succinylcholine does not change spontaneous action potential firing activity in organotypic slices
Fig. 3. Actions of succinylcholine on muscular activity. The concentration–response curve of succinylcholine is given as median including 95%-confidence interval. At a concentration of 1 lM succinylcholine leads to a significant depression of the muscular activity in nerve-muscle cocultures. However, at 3 lM succinylcholine there is an almost complete depression of muscular activity which means that the concentration–response-curve of succinylcholine in organotypic nerve-muscle cocultures is very steep.
In a following set of experiments 97 extracellular recordings in spinal ventral horn interneurons were performed to assess the effects of succinylcholine on the spontaneous action potential firing rate. The recordings were conducted in organotypic nerve-muscle cocultures at an age between 17 and 27 days ex vivo. The averaged action potential firing rate under control conditions was 34.2 ± 3.2 Hz. Representative traces of the multi-unit action potential firing activity in the absence and presence of succinylcholine
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(1 lM) are depicted in Fig. 5A. The typical firing pattern of cultured spinal neurons was composed of phases of high action potential firing (bursts of action potentials) which are separated by phases of low activity (Fig. 5A). The actions of succinylcholine on neuronal activity of spinal neurons were tested over a broad concentration range. Succinylcholine, from 1 nM to 1 mM did not induce systematic changes in neuronal activity (Fig. 5B). Therefore it can be concluded that the actions of succinylcholine on muscular activity are not mediated via changes of the neuronal activity in the cocultures.
to the concentrations at the synapse in spite of an equilibrium time of about 30 min. This has been previously demonstrated by Benkwitz et al. who showed that diffusion of the intravenous anaesthetic etomidate into brain slices requires approximately an hour to reach 80% equilibration at a typical recording depth of 100 lm [10]. Another possibility for evaluating pharmacologic effects on nicotinic receptors is the use of oocytes which express human muscular nicotinic acetylcholine receptors. Jonsson et al. found that succinylcholine concentration-dependently activated the muscle-type nicotinic acetylcholine receptor with an EC50 value of 10.8 lM [5]. The drawback of oocytes is that they do not represent the neuromuscular junction and are therefore an inadequate tool for testing interactions of organophosphate compounds and oximes. Thus the first demand, the sensitivity to succinylcholine is met by our test-system. The second requirement is the insensitivity of the spontaneous interneuronal activity to succinylcholine. As it has been demonstrated by Jonsson et al. in the oocyte preparation, succinylcholine does not activate the neuronal nicotinic acetylcholine receptors [5] and this finding is resembled in the cocultures of spinal cord and muscle tissue. In a preceding study we have demonstrated that cocultures of spinal cord and muscle tissue are a valuable test-system for investigating the effects of the nerve agent VX on muscle activity. While VX reduced muscle activity as expected the subsequent application of obidoxime at a clinical relevant concentration resulted in a restoration of muscle function [4]. A special feature of cocultures of spinal cord and muscle tissue is that the restoration of muscle activation took place after 24 h of antidotal treatment and remained constant over an observation period of approximately one week. This expanded time scale identifies the greatest advantage of the study system, the potential to conduct long-term studies. However it was unclear whether this activation-restoration of muscle-action happened in a test-system where the sensitivity of nicotinic acetylcholine receptors corresponds to that reported for humans. This could be confirmed by the results of the current study showing a sensitivity of muscle activity to succinylcholine as reported for adult patients previously. In general cocultures of spinal cord and muscle tissue seem to be an excellent test-system for the development and investigation of new oximes, since practical aspects like transport over long distances to further laboratories, the opportunity to conduct long-term studies and the reduction of animal usage display further advantages of its use.
4. Discussion
References
B
Fig. 5. (A) Original recording of multi-unit action potential firing from an organotypic nerve-muscle coculture. The typical firing pattern of ventral horn neurons ex vivo consists of bursts of action potentials separated by phases of very low neuronal activity. In the presence of 1 lM succinylcholine the neuronal activity does not show apparent changes. (B) Summary of electrophysiological data. The graph displays the normalized firing rate (1.0 corresponds to the activity at control conditions) in the presence of increasing concentrations of succinylcholine ranging from 1 nM to 1 mM. Note that there is no systematic change in neuronal action potential firing in the presence of the drug.
The potency of succinylcholine in humans has been determined in adult patients previously. Roy et al. investigated the concentration-effect relation of succinylcholine during propofol anaesthesia by measuring plasma concentrations of succinylcholine after its injection to characterize the front-end kinetics [13]. The calculated molar EC50 value in these adult human patients was 2.6 lM. The test-system evaluated in the current study showed a comparable sensitivity to succinylcholine with an EC50 as low as 1 lM. Further test-systems which have been involved in the admission of oximes are less sensitive to succinylcholine. In particular the mouse phrenic nerve-hemidiaphragm model was used in several studies [14– 16]. Advantages of the phrenic nerve-hemidiaphragm preparation are similarities with human neuromuscular junctions [17] and that it contains the diaphragm as an important muscle of respiration [18]. The sensitivity of the phrenic nerve-hemidiaphragm preparation to succinylcholine is approximately 10-fold lower, as indicated by an EC50 of 21.3 lM for adult rats [18]. However, this relatively high EC50 value can be put into perspective since the concentrations in the bath perfusion medium of the phrenic nerve-hemidiaphragm preparation is probably higher compared
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