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Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures
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Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures Isabel Weimera,b,* , Franz Woreka , Thomas Seegera , Horst Thiermanna , Veit-Simon Eckleb , Christian Grasshoffb , Bernd Antkowiakb,c a b c
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany Department of Anaesthesiology, Experimental Anaesthesiology Section, Eberhard-Karls-University, Waldhoernlestrasse 22, 72072 Tuebingen, Germany Werner-Reichardt-Centre for Integrative Neuroscience, Eberhard-Karls-University, 72076 Tuebingen, Germany
H I G H L I G H T S
Neuromuscular signs of OP poisoning in vivo manifest in nerve-muscle cocultures. Recovery of muscular function was evident 3 d and 7 d after intoxication with nerve agents. Cocultures may serve as test system to investigate long term effects of nerve agent intoxication.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2015 Received in revised form 31 July 2015 Accepted 4 August 2015 Available online xxx
Aside from nerve agents, various highly toxic pesticides belong to the group of organophosphorus (OP) compounds, thereby causing a large number of intoxications every year. Unfortunately, there are still shortcomings in the current treatment for OP poisoning and research on novel therapeutic options is restricted in several aspects. In this study we investigated the suitability of organotypic cocultures for pharmacological in vitro studies involving OP compounds. These slice cultures are derived from murine spinal cord and muscle tissue forming functional neuromuscular synapses, which trigger spontaneous contractions of muscle fibers. Using video microscopy to quantify muscle activity, we assessed the viability of cocultures after exposure to soman and VX, and the associated loss and recovery of neuromuscular function. Antidotal treatment was not provided. The application of nerve agents led to an almost complete loss of muscle activity. However, cell cultures regained equivalent muscular function to the control situation three and seven days after intoxication. In summary, the tested in vitro system could be a promising tool for the investigation of long term effects and therapeutic options for OP poisoning. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Self-regeneration Neuromuscular junction Organophosphorus compounds AChE inhibition Spinal cord—skeletal muscle cocultures
1. Introduction Although being outlawed by the international chemical weapons convention, the latest use of sarin against civilians in 2013 proved to be an appalling wake-up call to bring the potential threat of organophosphorus (OP) compounds back into public focus. Aside from nerve agents this group of highly toxic
Abbreviations: AChE, acetylcholinesterase; ACh, acetylcholine; ColQ-AChE, collagen-tailed form of AChE; im.post, immediate post; IMS, intermediate syndrome; IQR, interquartile range; NA, nerve agent; nAChR, nicotinic acetylcholine receptor; OP, organophosphorus; ROI, region of interest. * Corresponding author at: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany. E-mail address: [email protected] (I. Weimer).
substances includes various pesticides, which are still in regular use in most developing countries, thereby causing a large number of intoxications every year (Jeyaratnam, 1990; Jokanovic, 2009). In general, OP compounds can be absorbed via skin contact, inhalation, or ingestion depending on the physical properties of the substance and the circumstance of uptake, i.e. accidental or even intentional in case of attempted suicide. The toxicological effects of OP poisoning basically arise from the inhibition of acetylcholinesterase (AChE). As a consequence, the accumulation of acetylcholine (ACh) causes cholinergic overstimulation at muscarinic and nicotinic synapses and eventually leads to neuromuscular dysfunction and respiratory failure (de Jong, 2003; Holmstedt, 1959). Unfortunately, the commonly used antidotal treatment with atropine and oximes has considerable deficits in case of delayed administration of antidotes or if oximes
http://dx.doi.org/10.1016/j.toxlet.2015.08.004 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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generally lack efficacy as noted for specific OP compounds. The underlying problem concerning the latter is a conformational change in the OP-enzyme-complex (“aging”), eliminating the possibility of AChE reactivation, which invalidates the effect of oximes (Aldridge and Reiner, 1972; Marrs et al., 2006; Worek and Thiermann, 2013). Thus, continuing research to improve current standards and establish additional therapeutic options is mandatory, but not always a simple task considering the inherent ethical considerations and methodical limitations. In this study we investigated the suitability of organotypic cocultures prepared of murine spinal cord and skeletal muscle to perform pharmacological long-term studies involving OP compounds. During the first two weeks after preparation functional neuromuscular synapses are formed in these slice cultures stimulating spontaneous contractions of muscle fibers, which can be quantified via video microscopy. In a series of preliminary experiments oximes succeeded in restoring muscle activity in the cocultures after inhibition by VX (Drexler et al., 2011). As a second step, the recent study was designed to focus on self-regenerative processes. In the manner of a proof of concept, we intended to assess the capacities of sole auto-regeneration in this in vitro system, without pharmacological intervention. For this purpose, the viability and muscle activity of cocultures was monitored for seven days after exposure to the nerve agents soman and VX without providing antidotes.
fluoro-2-deoxyuridine, 10 mM cytosine-b–D-arabino-furanoside, 10 mM uridine) were once added to the nutrient fluid to reduce proliferation of glial cells. Spontaneous muscle contractions developed in about 70% of the cocultures during the first week ex vivo, indicating the formation of functional neuromuscular synapses innervating the muscle tissue. To make sure that major developmental steps in maturation had been passed (Avossa et al., 2003; Furlan et al., 2007; Rosato-Siri et al., 2004), video microscopic recordings were carried out between day 14 and 38 in vitro. 2.3. Experimental solutions and intoxication of slice cultures Nutrition fluid for culturing was used as standard solution throughout the experiment. For application of nerve agents, a separate amount of solution containing nerve agent (NA solution) was prepared by adding soman or VX to the nutrition fluid shortly before the start of the experiment (final concentration: soman 10 mM, VX 0.75 mM). Poisoning of cocultures was performed by incubating the slices with the NA solution for 18 min. Subsequently, the NA solution was removed and the plastic tubes were washed twice and refilled with standard nutrient fluid. In the following time the slice cultures were maintained according to standard procedures (see Section 2.2). 2.4. Video microscopic recording and quantification of muscle activity
2. Material and methods 2.1. Drugs and chemicals Save for the horse serum (Life Technologies, Carlsbad, CA, USA), all experimental solutions and substances were purchased from Sigma–Aldrich (St. Louis, MO, USA). Soman (pinacolyl methylphosphonofluoridate) and VX (O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate) were made available by the German Ministry of Defence in Bonn. 2.2. Preparation of spinal cord—skeletal muscle cocultures All procedures were in accordance with the German law on animal experimentation and approved by the animal care committee (Eberhard-Karls-University, Tuebingen, Germany). Moreover, all efforts were made to minimize animal suffering and the number of animals used. Organotypic cocultures were prepared from embryos of pregnant wild type C57bl6J mice as described previously (Braschler et al., 1989). In brief, pregnant animals were deeply anesthetized with isoflurane and decapitated before the uterus was aseptically removed and embryos (E 15) were stored in ice-cold Gey’s balanced salt solution. Embryos were decapitated and the spinal column was separated from inner organs and limbs before it was embedded in an agar block. A vibratome (WPI, Hitchin, UK) was used to cut 300 mm-thick transverse slices. Each tissue slice, containing spinal cord and adjacent skeletal muscle, was fixed onto a glass coverslip by coagulating a drop of heparinized chicken plasma with thrombin solution. The coverslips were transferred into plastic tubes and supplemented with 750 ml nutrient fluid, which consisted of horse serum (25%), Hanks’ balanced salt solution (25%), basal medium Eagle (50%) and neuronal growth factor added to a final concentration of 10 nM. Following the roller tube technique by Gähwiler, the tissue was further maintained in a roller drum at 37 C and nutrient fluid was renewed twice a week (Gähwiler, 1981). Subsequent to preparation and to every renewal of nutrient fluid, cell cultures were incubated at 95% oxygen and 5% carbon dioxide for 1–3 h before the plastic tubes were tightly sealed again by screw caps. One day after preparation, antimitotics (10 mM 5-
For video microscopic recordings each plastic tube containing a coculture was placed on an inverted microscope (AXIO Observer. D1, Carl Zeiss Microscopy, Jena, Germany) with a mounted video camera (DMK 21AU04, The Imaging Source Europe GmbH, Bremen, Germany) connected to a laptop. After a distinctive section with active muscle fibers was identified and brightness and contrast were adjusted, muscle contractions were recorded for 180 s and stored on hard disc (file format: AVI, frame rate: 30 fps, software for digitalization: IC Capture, The Imaging Source Europe GmbH, Bremen, Germany) for offline data analysis as described previously (Drexler et al., 2011; Drexler et al., 2013). In short, a region of interest (ROI) with high-contrast borders of muscle fibers was defined, and the brightness change of pixels between subsequent video frames was quantified by an in-house developed algorithm written in Matlab (The MathWorks Inc., Natick, MA, USA). The changes in brightness are caused by the dislocation of muscle fibers during contraction and can be correlated with the frequency of the contractions. For followup recordings the ROI selected at control was re-identified visually by microscopic photographs or preceding recordings. 2.5. Experimental design and data analysis Muscle activity was assessed in two separate test series (one with soman and one with VX), each with a timeframe of one week. Video-microscopic recordings were performed at four different stages: (I) before application of nerve agents ('control' or ‘baseline activity'), (II) immediately after incubation with soman or VX and refilling the plastic tubes with standard nutrient fluid ('immediate post intoxication'), (III) three days after intoxication (‘3d’) and (IV) seven days after intoxication (‘7d’). The software programs Matlab (Math-Works, Natick, MA, USA) and Prism (GraphPad, La Jolla, CA, USA) were used for statistical analysis. Unless stated otherwise, data are presented as median and interquartile range (IQR). Differences between selected data sets within the same series were tested by the Wilcoxon matchedpairs signed rank test. To facilitate comparison between the two test series (with soman and VX) we calculated the relative activity rate by normalizing the data for each cell culture relative to its
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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baseline activity. In this case, the Mann–Whitney test was used to compare selected data sets. P values 0.05 were considered significant for all analyses. 3. Results 3.1. Spontaneous contractions of muscle fibers in organotypic cocultures before and after exposure to nerve agents This study was designed to assess whether organotypic cocultures of murine spinal cord and muscle tissue remain viable and possess the ability for auto-regeneration of muscular function after intoxication with nerve agents. For this purpose, we used a video microscopic approach to examine the effect of soman and VX on spontaneous contractions of muscle fibers. Cell cultures were obtained from 5 different preparations and displayed a wide range of baseline activities (minimum: 0.25 Hz, maximum: 2.99 Hz; n = 24), which varied not only between preparations but also between slices of the same preparation. However, previous studies with matching experimental procedures proved that no statistically significant changes in muscle activity occur after media exchange or during standard maintenance of cocultures (Drexler et al., 2011; Eckle et al., 2014). Therefore, activity changes were ascribed to the toxic effects of nerve agent treatment and subsequent auto-regeneration. In the first series of experiments with soman, the averaged baseline activity was only about half as high as in the second series with VX. But even so, differences in the relative activity rates following intoxication did not turn out as statistically significant at any point. A summary of relative activity rates and p values for the comparison of the soman and VX series is given in Table 1. 3.2. Loss and recovery of muscle activity after exposure to soman The slice cultures of the first series of experiments displayed an averaged contraction frequency of 0.75 Hz (IQR 0.49 Hz) before incubation with 10 mM soman. As expected, the application of nerve agent resulted in an inhibition of neuromuscular function manifesting as significant loss of muscle activity. The averaged contraction frequency dropped to 0.09 Hz (IQR 0.14 Hz), which constitutes merely 8.4% (IQR 26.5%) of the initial rate. Yet, three days after exposure to soman, slice cultures showed significant recovery of muscle contractions back to 0.44 Hz (IQR 0.54 Hz) and regained muscular function equivalent to the control situation after one week (0.62 Hz (IQR 0.84 Hz)). Fig. 1 shows the averaged contraction frequencies at different stages in the experiment.
Fig. 1. Averaged muscle contraction frequencies of slice cultures at different stages of the experiment with soman. Ends of boxes are defined by the 25th and 75th percentile. The in-box line represents the median and error bars display Tukey– Whiskers, which mark 1.5 times the interquartile distance or the highest/lowest data point, whichever is shorter. Significant results for selected group comparisons are indicated by asterisks (Wilcoxon matched-pairs signed rank test; *p 0.05, **p 0.01, ***p 0.001). No significant differences are noted as “ns”; im.post = immediate post.
(IQR 0.78Hz), which amounts to 21.7% (IQR 49.1%) of the initial rate. Extensive recovery was observed at day three and seven, i.e. cell cultures displayed contraction frequencies almost equivalent to the baseline activity (3d: 1.61 Hz (IQR 0.75 Hz), 7d: 1.59 Hz (IQR 1.16 Hz)). Boxplots of the contraction frequencies in the VX series are given in Fig. 2. 4. Discussion Due to persistent shortcomings in the standard treatment for OP poisoning, the research on novel therapeutic options is a matter of urgency. To overcome associated methodical obstacles like restraints for in vivo studies and the lack of valid clinical data, we tested the aptitude of nerve-muscle cocultures for
3.3. Loss and recovery of muscle activity after exposure to VX In the second series of experiments we used VX at a final concentration of 0.75 mM to intoxicate the slices. Save for the higher baseline activity (1.69 Hz (IQR 0.93 Hz)), the results were similar to the ones obtained with soman. After exposure to the nerve agent, muscle contractions dropped considerably to 0.47Hz
Fig. 2. Averaged muscle contraction frequencies of slice cultures at different stages of the experiment with VX. For further explanation, see Fig. 1.
Table 1 Comparison of relative activity rates of cocultures between the two test series with soman and VX.
Control im.posta intoxication 3d 7d a
Soman series (n = 13)
VX series (n = 11)
Mann–Whitney test (soman vs. VX) p values
100.00% (i 0.75 Hz) 8.4% (IQR 26.5%) 69.2% (IQR 107.8%) 100.8% (IQR 121.6%)
100.00% (i 1.69 Hz) 21.7% (IQR 49.1%) 92.7% (IQR 63.5%) 71.5% (IQR 79.1%)
Not tested 0.3535 0.8167 0.4513
im.post = immediate post
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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pharmacological long-term studies. A basic requirement to validate an in vitro system for this purpose is the reflection of key symptoms manifesting after intoxication with OP compounds in vivo. In other words, exposing the cocultures to nerve agents should result in a notable loss of muscle activity due to the impairment of neuromuscular function. Consistent with a previous study (Drexler et al., 2011), this condition was fully met by the current results, since the averaged muscle contraction frequency dropped to a fractional amount of the initial value subsequent to the application of soman or VX. Theoretically, this effect could arise from two major causes, equally triggered by inhibition of AChE. Either the failure of neuromuscular transmission or a change in neuronal firing of the projecting motor nerves or ventral horn interneurons. Previous experiments revealed that the application of succinylcholine, a nicotinic ACh receptor (nAChR) agonist, resulted in almost complete loss of muscle activity without affecting neuronal firing on the spinal level (Drexler et al., 2013). Therefore, we suppose the failure of muscular function observed in the current study is most likely caused at the synaptic level, even though influences by altered neuronal activity cannot be entirely excluded at this point. The reason why it was possible to produce almost complete atony with succinylcholine but not with nerve agents lies in the specific way the two substances take effect. Succinylcholine was dissolved in the experimental fluid, which caused a steady disruption of neuromuscular transmission. The temporary exposure of cocultures to nerve agents, on the other hand, provoked an accumulation of ACh after motor neuron activity. Due to large intercellular spaces in the cultured tissue, however, ACh is washed out by the experimental fluid after a certain time of muscular inactivity, briefly re-enabling muscular function before it breaks down again after repeated accumulation of ACh and desensitization of nAChRs. Compared to succinylcholine, the application of nerve agents therefore induced a slightly less pronounced but still severe depression of muscle activity to 8.4% (soman) and 21.7% (VX) of the control value. Yet, significant rehabilitation of muscle contractions was noted three and seven days following intoxication. As we refrained from the application of antidotes, the re-establishment of muscular function implies the presence of self-regenerative processes, such as de novo synthesis and spontaneous reactivation of AChE (Blaber and Creasey, 1960). Unfortunately, the assessment of AChE activity per se was not feasible in this study due to technical obstacles and the experimental design, which is why we need to refer to previous results und published data for a quantitative correlation between AChE and muscle activity in the slice cultures. Literature provides a wide scope of AChE recovery-rates with values ranging between few hours (Yourick et al., 1991) and several weeks (Kasprzak and Salpeter, 1985). This panoply results from not only alternating variables in terms of experimental design but also the polymorphism of AChE itself. It is important to note, that AChE exists in a large variety of molecular forms that basically possess the same catalytic activity but differ, for instance, in quaternary structure, tissue specific expression and localization (Legay, 2000; Massoulie et al., 1993). At the neuromuscular junction, the major form of the enzyme is ColQ-AChE, consisting of three catalytic tetramers associated with a collagen-like tail (ColQ) (Rotundo et al., 2008). For a specific assessment of the insertion rate of newly synthetized ColQ-AChE into the neuromuscular junction, Krejci and MartinezPena used Alexa 488 Fasciculin 2 to label ColQ-AChE, and found that 30–40% of the enzyme was replaced after 48–72 h (Krejci et al., 2006; Martinez-Pena y Valenzuela and Akaaboune, 2007). This estimation seems comparable with the results of two earlier studies on the return of AChE activity after irreversible inhibition by soman. Grubic et al. reported a 50% recovery at the end plate region of the rat diaphragm within 4–5 days (Grubic et al., 1981), and Stitcher et al. calculated a half-maximal return of enzyme
activity after 80.4 17.7 h (Stitcher et al., 1977). Based on a minimum AChE activity level of 30% to guarantee neuromuscular function (Thiermann et al., 2009) and a straight correlation between AChE inhibition and acute toxicological signs (Gupta et al., 1987), these findings are in good agreement with the recovery of muscle contractions observed in our experiments. Statistically, we could not detect any significant difference between the test series with soman and VX, but even so, muscular inhibition seemed less pronounced and recovery appeared to proceed slightly faster in the cocultures treated with VX. The fact that VX caused a milder inhibition of muscle contractions than soman is likely to be the corollary of a considerably longer half-time of AChE aging (VX 36.5 h; soman 0.07 h), so AChE activity was probably less affected by the temporary exposure to VX (Worek and Thiermann, 2013). Likewise, the striking difference in half-time may account for the difference in recovery of muscle contractions. As mentioned above, this process may generally involve de novo synthesis and reactivation of AChE, but since the rapid aging of AChE eliminates the possibility for reactivation after inhibition by soman (Fleisher and Harris, 1965; Harris et al., 1971; Worek and Thiermann, 2013), enzyme activity is mainly restored by de novo synthesis and therefore probably slower in progress than with VX. However, considering the poor possibilities to reactivate AChE inhibited by not only soman but also tabun or cyclosarin (Worek et al., 2007), the particular importance of de novo synthesis falls into place. Even though we cannot discard the possibility that de novo synthesis in the cocultures involves newly formed synapses rather than AChE synthesis in the existent network, a pharmacological intervention to speed up this process would certainly be of great value for the treatment of intoxicated patients. But since therapeutic options of this kind are not in clinical use so far, this issue clearly warrants further investigation. Another therapeutic challenge of OP poisoning yet to be unraveled is the so called intermediate syndrome (IMS). It manifests as delayed reappearance of neuromuscular weakness and peripheral respiratory failure, usually occurring 24–96 h after intoxication, when acute signs of cholinergic crisis have already subsided (Karalliedde et al., 2006). To date, IMS is classified as disorder of neuromuscular junctions but even though the syndrome is considered to play a decisive part in OP related morbidity and mortality, its exact etiology remains unclear. Previously proposed mechanisms include prolonged AChE inhibition, down-regulation or desensitization of ACh receptors, failure of presynaptic ACh release, and myopathy (Abdollahi and KaramiMohajeri, 2012; Yang and Deng, 2007). With regard to in vitro studies the characteristic lag of symptoms in IMS represents a certain pitfall. To reflect this phenomenon, or rather its underlying pathophysiology, an appropriate test system should feature the relevant target structures and remain functional for at least the stated timeframe. From a technical point of view, nerve-muscle cocultures may be applicable due to the presence of functional neuromuscular synapses and the sustained viability for several days. However, it remains to be seen if IMS-related processes indeed manifest in the slices. 5. Conclusions In summary, we could demonstrate that exposing organotypic nerve-muscle cocultures to nerve agents results in a significant loss of muscle activity, which resembles the peripheral symptoms of severe OP poisoning in vivo. Even though antidotes were not applied, extensive rehabilitation of muscular function was observed three and seven days after intoxication, which indicates the presence of auto-regenerative processes like spontaneous reactivation and de novo synthesis of AChE. Moreover, the cultures
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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showed no decrease in viability within the experimental timeframe. At the bottom line, the tested in vitro system technically provides relevant target structures to examine the effects of nerve agents and their antidotes, including long term processes like de novo synthesis of AChE or the pathophysiology of IMS. In order to improve this experimental model, further studies should be performed comprising, at best, the concurrent assessment of neuronal activity and AChE activity, as well as a characterization of the nature of AChE de novo synthesis occurring in the cultured tissue. Conflicts of interest The author declares that there are no conflicts of interest. Acknowledgements The author would like to thank Claudia Holt and Ina Pappe for excellent technical assistance and processing of video microscopic raw data. This study was funded by the Bundeswehr Institute of Pharmacology and Toxicology (Munich) and the Department of Anaesthesiology, Experimental Anaesthesiology Section, Eberhard-Karls-University (Tuebingen). Additionally, this work has been supported by contract no. M SAB1 7A009 from the German Ministry of Defence. References Abdollahi, M., Karami-Mohajeri, S., 2012. A comprehensive review on experimental and clinical findings in intermediate syndrome caused by organophosphate poisoning. Toxicol. Appl. Pharmacol. 258, 309–314. Aldridge, W.N., Reiner, E., 1972. Enzyme Inhibitors as Substrates: Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland Pub. Co. Avossa, D., Rosato-Siri, M.D., Mazzarol, F., Ballerini, L., 2003. Spinal circuits formation: a study of developmentally regulated markers in organotypic cultures of embryonic mouse spinal cord. Neuroscience 122, 391–405. Blaber, L.C., Creasey, N.H., 1960. The mode of recovery of cholinesterase activity in vivo after organophosphorus poisoning. 1. Erythrocyte cholinesterase. Biochem. J. 77, 591–596. Braschler, U.F., Iannone, A., Spenger, C., Streit, J., Luscher, H.R., 1989. A modified roller tube technique for organotypic cocultures of embryonic rat spinal cord, sensory ganglia and skeletal muscle. J. Neurosci. Methods 29, 121–129. de Jong, R.H., 2003. Nerve gas terrorism: a grim challenge to anesthesiologists. Anesth. Analg. 95, 819–825 table of contents. Drexler, B., Seeger, T., Grasshoff, C., Thiermann, H., Antkowiak, B., 2011. Long-term evaluation of organophosphate toxicity and antidotal therapy in co-cultures of spinal cord and muscle tissue. Toxicol. Lett. 206, 89–93. Drexler, B., Thiermann, H., Antkowiak, B., Grasshoff, C., 2013. Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice. Chem. Biol. Interact. 206, 555–560. Eckle, V.S., Drexler, B., Grasshoff, C., Seeger, T., Thiermann, H., Antkowiak, B., 2014. Spinal cord–skeletal muscle cocultures detect muscle-relaxant action of botulinum neurotoxin A. Altex 31, 433–440. Fleisher, J.H., Harris, L.W., 1965. Dealkylation as a mechanism for aging of cholinesterase after poisoning with pinacolyl methylphosphonofluoridate. Biochem. Pharmacol. 14, 641–650.
5
Furlan, F., Taccola, G., Grandolfo, M., Guasti, L., Arcangeli, A., Nistri, A., Ballerini, L., 2007. ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord. J. Neurosci. 27, 919–928. Gähwiler, B.H., 1981. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342. Grubic, Z., Sketelj, J., Klinar, B., Brzin, M., 1981. Recovery of acetylcholinesterase in the diaphragm, brain, and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate. J. Neurochem. 37, 909–916. Gupta, R.C., Patterson, G.T., Dettbarn, W.D., 1987. Biochemical and histochemical alterations following acute soman intoxication in the rat. Toxicol. Appl. Pharmacol. 87, 393–402. Harris, L.W., Yamamura, H.I., Fleisher, J.H., 1971. De novo synthesis of acetylcholinesterase in guinea pig retina after inhibition by pinacolyl methylphosphonofluoridate. Biochem. Pharmacol. 20, 2927–2930. Holmstedt, B., 1959. Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol. Rev. 11, 567–688. Jeyaratnam, J., 1990. Acute pesticide poisoning: a major global health problem. World Health Stat. Q. (Rapport trimestriel de statistiques sanitaires mondiales) 43, 139–144. Jokanovic, M., 2009. Medical treatment of acute poisoning with organophosphorus and carbamate pesticides. Toxicol. Lett. 190, 107–115. Karalliedde, L., Baker, D., Marrs, T.C., 2006. Organophosphate-induced intermediate syndrome: aetiology and relationships with myopathy. Toxicol. Rev. 25, 1–14. Kasprzak, H., Salpeter, M.M., 1985. Recovery of acetylcholinesterase at intact neuromuscular junctions after in vivo inactivation with diisopropylfluorophosphate. J. Neurosci. Off. J. Soc. Neurosci. 5, 951–955. Krejci, E., Martinez-Pena y Valenzuela, I., Ameziane, R., Akaaboune, M., 2006. Acetylcholinesterase dynamics at the neuromuscular junction of live animals. J. Biol. Chem. 281, 10347–10354. Legay, C., 2000. Why so many forms of acetylcholinesterase? Microsc. Res. Tech. 49, 56–72. Marrs, T.C., Rice, P., Vale, J.A., 2006. The role of oximes in the treatment of nerve agent poisoning in civilian casualties. Toxicol. Rev. 25, 297–323. Martinez-Pena y Valenzuela, I., Akaaboune, M., 2007. Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice. Mol. Biol. Cell 18, 2904–2911. Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., Vallette, F.M., 1993. Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31–91. Rosato-Siri, M.D., Zoccolan, D., Furlan, F., Ballerini, L., 2004. 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, 2697–2710. Rotundo, R.L., Ruiz, C.A., Marrero, E., Kimbell, L.M., Rossi, S.G., Rosenberry, T., Darr, A., Tsoulfas, P., 2008. Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction. Chem. Biol. Interact. 175, 26–29. Stitcher, D.L., Harris, L.W., Moore, R.D., Heyl, W.C., 1977. Synthesis of cholinesterase following poisoning with irreversible anticholinesterases: effects of theophylline and N6,O2-dibutyryl adenosine 30 ,50 -monophosphate on synthesis and survival. Toxicol. Appl. Pharmacol. 41, 79–90. Thiermann, H., Zilker, T., Eyer, F., Felgenhauer, N., Eyer, P., Worek, F., 2009. Monitoring of neuromuscular transmission in organophosphate pesticidepoisoned patients. Toxicol. Lett. 191, 297–304. Worek, F., Thiermann, H., 2013. The value of novel oximes for treatment of poisoning by organophosphorus compounds. Pharmacol. Ther. 139, 249–259. Worek, F., Eyer, P., Aurbek, N., Szinicz, L., Thiermann, H., 2007. Recent advances in evaluation of oxime efficacy in nerve agent poisoning by in vitro analysis. Toxicol. Appl. Pharmacol. 219, 226–234. Yang, C.C., Deng, J.F., 2007. Intermediate syndrome following organophosphate insecticide poisoning. J. Chin. Med. Assoc. 70, 467–472. Yourick, J.J., Eklo, P.A., McCluskey, M.P., Ray, R., 1991. Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-L-glutamine. Cell Biol. Toxicol. 7, 229–237.
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Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures Isabel Weimera,b,* , Franz Woreka , Thomas Seegera , Horst Thiermanna , Veit-Simon Eckleb , Christian Grasshoffb , Bernd Antkowiakb,c a b c
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany Department of Anaesthesiology, Experimental Anaesthesiology Section, Eberhard-Karls-University, Waldhoernlestrasse 22, 72072 Tuebingen, Germany Werner-Reichardt-Centre for Integrative Neuroscience, Eberhard-Karls-University, 72076 Tuebingen, Germany
H I G H L I G H T S
Neuromuscular signs of OP poisoning in vivo manifest in nerve-muscle cocultures. Recovery of muscular function was evident 3 d and 7 d after intoxication with nerve agents. Cocultures may serve as test system to investigate long term effects of nerve agent intoxication.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2015 Received in revised form 31 July 2015 Accepted 4 August 2015 Available online xxx
Aside from nerve agents, various highly toxic pesticides belong to the group of organophosphorus (OP) compounds, thereby causing a large number of intoxications every year. Unfortunately, there are still shortcomings in the current treatment for OP poisoning and research on novel therapeutic options is restricted in several aspects. In this study we investigated the suitability of organotypic cocultures for pharmacological in vitro studies involving OP compounds. These slice cultures are derived from murine spinal cord and muscle tissue forming functional neuromuscular synapses, which trigger spontaneous contractions of muscle fibers. Using video microscopy to quantify muscle activity, we assessed the viability of cocultures after exposure to soman and VX, and the associated loss and recovery of neuromuscular function. Antidotal treatment was not provided. The application of nerve agents led to an almost complete loss of muscle activity. However, cell cultures regained equivalent muscular function to the control situation three and seven days after intoxication. In summary, the tested in vitro system could be a promising tool for the investigation of long term effects and therapeutic options for OP poisoning. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Self-regeneration Neuromuscular junction Organophosphorus compounds AChE inhibition Spinal cord—skeletal muscle cocultures
1. Introduction Although being outlawed by the international chemical weapons convention, the latest use of sarin against civilians in 2013 proved to be an appalling wake-up call to bring the potential threat of organophosphorus (OP) compounds back into public focus. Aside from nerve agents this group of highly toxic
Abbreviations: AChE, acetylcholinesterase; ACh, acetylcholine; ColQ-AChE, collagen-tailed form of AChE; im.post, immediate post; IMS, intermediate syndrome; IQR, interquartile range; NA, nerve agent; nAChR, nicotinic acetylcholine receptor; OP, organophosphorus; ROI, region of interest. * Corresponding author at: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany. E-mail address: [email protected] (I. Weimer).
substances includes various pesticides, which are still in regular use in most developing countries, thereby causing a large number of intoxications every year (Jeyaratnam, 1990; Jokanovic, 2009). In general, OP compounds can be absorbed via skin contact, inhalation, or ingestion depending on the physical properties of the substance and the circumstance of uptake, i.e. accidental or even intentional in case of attempted suicide. The toxicological effects of OP poisoning basically arise from the inhibition of acetylcholinesterase (AChE). As a consequence, the accumulation of acetylcholine (ACh) causes cholinergic overstimulation at muscarinic and nicotinic synapses and eventually leads to neuromuscular dysfunction and respiratory failure (de Jong, 2003; Holmstedt, 1959). Unfortunately, the commonly used antidotal treatment with atropine and oximes has considerable deficits in case of delayed administration of antidotes or if oximes
http://dx.doi.org/10.1016/j.toxlet.2015.08.004 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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generally lack efficacy as noted for specific OP compounds. The underlying problem concerning the latter is a conformational change in the OP-enzyme-complex (“aging”), eliminating the possibility of AChE reactivation, which invalidates the effect of oximes (Aldridge and Reiner, 1972; Marrs et al., 2006; Worek and Thiermann, 2013). Thus, continuing research to improve current standards and establish additional therapeutic options is mandatory, but not always a simple task considering the inherent ethical considerations and methodical limitations. In this study we investigated the suitability of organotypic cocultures prepared of murine spinal cord and skeletal muscle to perform pharmacological long-term studies involving OP compounds. During the first two weeks after preparation functional neuromuscular synapses are formed in these slice cultures stimulating spontaneous contractions of muscle fibers, which can be quantified via video microscopy. In a series of preliminary experiments oximes succeeded in restoring muscle activity in the cocultures after inhibition by VX (Drexler et al., 2011). As a second step, the recent study was designed to focus on self-regenerative processes. In the manner of a proof of concept, we intended to assess the capacities of sole auto-regeneration in this in vitro system, without pharmacological intervention. For this purpose, the viability and muscle activity of cocultures was monitored for seven days after exposure to the nerve agents soman and VX without providing antidotes.
fluoro-2-deoxyuridine, 10 mM cytosine-b–D-arabino-furanoside, 10 mM uridine) were once added to the nutrient fluid to reduce proliferation of glial cells. Spontaneous muscle contractions developed in about 70% of the cocultures during the first week ex vivo, indicating the formation of functional neuromuscular synapses innervating the muscle tissue. To make sure that major developmental steps in maturation had been passed (Avossa et al., 2003; Furlan et al., 2007; Rosato-Siri et al., 2004), video microscopic recordings were carried out between day 14 and 38 in vitro. 2.3. Experimental solutions and intoxication of slice cultures Nutrition fluid for culturing was used as standard solution throughout the experiment. For application of nerve agents, a separate amount of solution containing nerve agent (NA solution) was prepared by adding soman or VX to the nutrition fluid shortly before the start of the experiment (final concentration: soman 10 mM, VX 0.75 mM). Poisoning of cocultures was performed by incubating the slices with the NA solution for 18 min. Subsequently, the NA solution was removed and the plastic tubes were washed twice and refilled with standard nutrient fluid. In the following time the slice cultures were maintained according to standard procedures (see Section 2.2). 2.4. Video microscopic recording and quantification of muscle activity
2. Material and methods 2.1. Drugs and chemicals Save for the horse serum (Life Technologies, Carlsbad, CA, USA), all experimental solutions and substances were purchased from Sigma–Aldrich (St. Louis, MO, USA). Soman (pinacolyl methylphosphonofluoridate) and VX (O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate) were made available by the German Ministry of Defence in Bonn. 2.2. Preparation of spinal cord—skeletal muscle cocultures All procedures were in accordance with the German law on animal experimentation and approved by the animal care committee (Eberhard-Karls-University, Tuebingen, Germany). Moreover, all efforts were made to minimize animal suffering and the number of animals used. Organotypic cocultures were prepared from embryos of pregnant wild type C57bl6J mice as described previously (Braschler et al., 1989). In brief, pregnant animals were deeply anesthetized with isoflurane and decapitated before the uterus was aseptically removed and embryos (E 15) were stored in ice-cold Gey’s balanced salt solution. Embryos were decapitated and the spinal column was separated from inner organs and limbs before it was embedded in an agar block. A vibratome (WPI, Hitchin, UK) was used to cut 300 mm-thick transverse slices. Each tissue slice, containing spinal cord and adjacent skeletal muscle, was fixed onto a glass coverslip by coagulating a drop of heparinized chicken plasma with thrombin solution. The coverslips were transferred into plastic tubes and supplemented with 750 ml nutrient fluid, which consisted of horse serum (25%), Hanks’ balanced salt solution (25%), basal medium Eagle (50%) and neuronal growth factor added to a final concentration of 10 nM. Following the roller tube technique by Gähwiler, the tissue was further maintained in a roller drum at 37 C and nutrient fluid was renewed twice a week (Gähwiler, 1981). Subsequent to preparation and to every renewal of nutrient fluid, cell cultures were incubated at 95% oxygen and 5% carbon dioxide for 1–3 h before the plastic tubes were tightly sealed again by screw caps. One day after preparation, antimitotics (10 mM 5-
For video microscopic recordings each plastic tube containing a coculture was placed on an inverted microscope (AXIO Observer. D1, Carl Zeiss Microscopy, Jena, Germany) with a mounted video camera (DMK 21AU04, The Imaging Source Europe GmbH, Bremen, Germany) connected to a laptop. After a distinctive section with active muscle fibers was identified and brightness and contrast were adjusted, muscle contractions were recorded for 180 s and stored on hard disc (file format: AVI, frame rate: 30 fps, software for digitalization: IC Capture, The Imaging Source Europe GmbH, Bremen, Germany) for offline data analysis as described previously (Drexler et al., 2011; Drexler et al., 2013). In short, a region of interest (ROI) with high-contrast borders of muscle fibers was defined, and the brightness change of pixels between subsequent video frames was quantified by an in-house developed algorithm written in Matlab (The MathWorks Inc., Natick, MA, USA). The changes in brightness are caused by the dislocation of muscle fibers during contraction and can be correlated with the frequency of the contractions. For followup recordings the ROI selected at control was re-identified visually by microscopic photographs or preceding recordings. 2.5. Experimental design and data analysis Muscle activity was assessed in two separate test series (one with soman and one with VX), each with a timeframe of one week. Video-microscopic recordings were performed at four different stages: (I) before application of nerve agents ('control' or ‘baseline activity'), (II) immediately after incubation with soman or VX and refilling the plastic tubes with standard nutrient fluid ('immediate post intoxication'), (III) three days after intoxication (‘3d’) and (IV) seven days after intoxication (‘7d’). The software programs Matlab (Math-Works, Natick, MA, USA) and Prism (GraphPad, La Jolla, CA, USA) were used for statistical analysis. Unless stated otherwise, data are presented as median and interquartile range (IQR). Differences between selected data sets within the same series were tested by the Wilcoxon matchedpairs signed rank test. To facilitate comparison between the two test series (with soman and VX) we calculated the relative activity rate by normalizing the data for each cell culture relative to its
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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baseline activity. In this case, the Mann–Whitney test was used to compare selected data sets. P values 0.05 were considered significant for all analyses. 3. Results 3.1. Spontaneous contractions of muscle fibers in organotypic cocultures before and after exposure to nerve agents This study was designed to assess whether organotypic cocultures of murine spinal cord and muscle tissue remain viable and possess the ability for auto-regeneration of muscular function after intoxication with nerve agents. For this purpose, we used a video microscopic approach to examine the effect of soman and VX on spontaneous contractions of muscle fibers. Cell cultures were obtained from 5 different preparations and displayed a wide range of baseline activities (minimum: 0.25 Hz, maximum: 2.99 Hz; n = 24), which varied not only between preparations but also between slices of the same preparation. However, previous studies with matching experimental procedures proved that no statistically significant changes in muscle activity occur after media exchange or during standard maintenance of cocultures (Drexler et al., 2011; Eckle et al., 2014). Therefore, activity changes were ascribed to the toxic effects of nerve agent treatment and subsequent auto-regeneration. In the first series of experiments with soman, the averaged baseline activity was only about half as high as in the second series with VX. But even so, differences in the relative activity rates following intoxication did not turn out as statistically significant at any point. A summary of relative activity rates and p values for the comparison of the soman and VX series is given in Table 1. 3.2. Loss and recovery of muscle activity after exposure to soman The slice cultures of the first series of experiments displayed an averaged contraction frequency of 0.75 Hz (IQR 0.49 Hz) before incubation with 10 mM soman. As expected, the application of nerve agent resulted in an inhibition of neuromuscular function manifesting as significant loss of muscle activity. The averaged contraction frequency dropped to 0.09 Hz (IQR 0.14 Hz), which constitutes merely 8.4% (IQR 26.5%) of the initial rate. Yet, three days after exposure to soman, slice cultures showed significant recovery of muscle contractions back to 0.44 Hz (IQR 0.54 Hz) and regained muscular function equivalent to the control situation after one week (0.62 Hz (IQR 0.84 Hz)). Fig. 1 shows the averaged contraction frequencies at different stages in the experiment.
Fig. 1. Averaged muscle contraction frequencies of slice cultures at different stages of the experiment with soman. Ends of boxes are defined by the 25th and 75th percentile. The in-box line represents the median and error bars display Tukey– Whiskers, which mark 1.5 times the interquartile distance or the highest/lowest data point, whichever is shorter. Significant results for selected group comparisons are indicated by asterisks (Wilcoxon matched-pairs signed rank test; *p 0.05, **p 0.01, ***p 0.001). No significant differences are noted as “ns”; im.post = immediate post.
(IQR 0.78Hz), which amounts to 21.7% (IQR 49.1%) of the initial rate. Extensive recovery was observed at day three and seven, i.e. cell cultures displayed contraction frequencies almost equivalent to the baseline activity (3d: 1.61 Hz (IQR 0.75 Hz), 7d: 1.59 Hz (IQR 1.16 Hz)). Boxplots of the contraction frequencies in the VX series are given in Fig. 2. 4. Discussion Due to persistent shortcomings in the standard treatment for OP poisoning, the research on novel therapeutic options is a matter of urgency. To overcome associated methodical obstacles like restraints for in vivo studies and the lack of valid clinical data, we tested the aptitude of nerve-muscle cocultures for
3.3. Loss and recovery of muscle activity after exposure to VX In the second series of experiments we used VX at a final concentration of 0.75 mM to intoxicate the slices. Save for the higher baseline activity (1.69 Hz (IQR 0.93 Hz)), the results were similar to the ones obtained with soman. After exposure to the nerve agent, muscle contractions dropped considerably to 0.47Hz
Fig. 2. Averaged muscle contraction frequencies of slice cultures at different stages of the experiment with VX. For further explanation, see Fig. 1.
Table 1 Comparison of relative activity rates of cocultures between the two test series with soman and VX.
Control im.posta intoxication 3d 7d a
Soman series (n = 13)
VX series (n = 11)
Mann–Whitney test (soman vs. VX) p values
100.00% (i 0.75 Hz) 8.4% (IQR 26.5%) 69.2% (IQR 107.8%) 100.8% (IQR 121.6%)
100.00% (i 1.69 Hz) 21.7% (IQR 49.1%) 92.7% (IQR 63.5%) 71.5% (IQR 79.1%)
Not tested 0.3535 0.8167 0.4513
im.post = immediate post
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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pharmacological long-term studies. A basic requirement to validate an in vitro system for this purpose is the reflection of key symptoms manifesting after intoxication with OP compounds in vivo. In other words, exposing the cocultures to nerve agents should result in a notable loss of muscle activity due to the impairment of neuromuscular function. Consistent with a previous study (Drexler et al., 2011), this condition was fully met by the current results, since the averaged muscle contraction frequency dropped to a fractional amount of the initial value subsequent to the application of soman or VX. Theoretically, this effect could arise from two major causes, equally triggered by inhibition of AChE. Either the failure of neuromuscular transmission or a change in neuronal firing of the projecting motor nerves or ventral horn interneurons. Previous experiments revealed that the application of succinylcholine, a nicotinic ACh receptor (nAChR) agonist, resulted in almost complete loss of muscle activity without affecting neuronal firing on the spinal level (Drexler et al., 2013). Therefore, we suppose the failure of muscular function observed in the current study is most likely caused at the synaptic level, even though influences by altered neuronal activity cannot be entirely excluded at this point. The reason why it was possible to produce almost complete atony with succinylcholine but not with nerve agents lies in the specific way the two substances take effect. Succinylcholine was dissolved in the experimental fluid, which caused a steady disruption of neuromuscular transmission. The temporary exposure of cocultures to nerve agents, on the other hand, provoked an accumulation of ACh after motor neuron activity. Due to large intercellular spaces in the cultured tissue, however, ACh is washed out by the experimental fluid after a certain time of muscular inactivity, briefly re-enabling muscular function before it breaks down again after repeated accumulation of ACh and desensitization of nAChRs. Compared to succinylcholine, the application of nerve agents therefore induced a slightly less pronounced but still severe depression of muscle activity to 8.4% (soman) and 21.7% (VX) of the control value. Yet, significant rehabilitation of muscle contractions was noted three and seven days following intoxication. As we refrained from the application of antidotes, the re-establishment of muscular function implies the presence of self-regenerative processes, such as de novo synthesis and spontaneous reactivation of AChE (Blaber and Creasey, 1960). Unfortunately, the assessment of AChE activity per se was not feasible in this study due to technical obstacles and the experimental design, which is why we need to refer to previous results und published data for a quantitative correlation between AChE and muscle activity in the slice cultures. Literature provides a wide scope of AChE recovery-rates with values ranging between few hours (Yourick et al., 1991) and several weeks (Kasprzak and Salpeter, 1985). This panoply results from not only alternating variables in terms of experimental design but also the polymorphism of AChE itself. It is important to note, that AChE exists in a large variety of molecular forms that basically possess the same catalytic activity but differ, for instance, in quaternary structure, tissue specific expression and localization (Legay, 2000; Massoulie et al., 1993). At the neuromuscular junction, the major form of the enzyme is ColQ-AChE, consisting of three catalytic tetramers associated with a collagen-like tail (ColQ) (Rotundo et al., 2008). For a specific assessment of the insertion rate of newly synthetized ColQ-AChE into the neuromuscular junction, Krejci and MartinezPena used Alexa 488 Fasciculin 2 to label ColQ-AChE, and found that 30–40% of the enzyme was replaced after 48–72 h (Krejci et al., 2006; Martinez-Pena y Valenzuela and Akaaboune, 2007). This estimation seems comparable with the results of two earlier studies on the return of AChE activity after irreversible inhibition by soman. Grubic et al. reported a 50% recovery at the end plate region of the rat diaphragm within 4–5 days (Grubic et al., 1981), and Stitcher et al. calculated a half-maximal return of enzyme
activity after 80.4 17.7 h (Stitcher et al., 1977). Based on a minimum AChE activity level of 30% to guarantee neuromuscular function (Thiermann et al., 2009) and a straight correlation between AChE inhibition and acute toxicological signs (Gupta et al., 1987), these findings are in good agreement with the recovery of muscle contractions observed in our experiments. Statistically, we could not detect any significant difference between the test series with soman and VX, but even so, muscular inhibition seemed less pronounced and recovery appeared to proceed slightly faster in the cocultures treated with VX. The fact that VX caused a milder inhibition of muscle contractions than soman is likely to be the corollary of a considerably longer half-time of AChE aging (VX 36.5 h; soman 0.07 h), so AChE activity was probably less affected by the temporary exposure to VX (Worek and Thiermann, 2013). Likewise, the striking difference in half-time may account for the difference in recovery of muscle contractions. As mentioned above, this process may generally involve de novo synthesis and reactivation of AChE, but since the rapid aging of AChE eliminates the possibility for reactivation after inhibition by soman (Fleisher and Harris, 1965; Harris et al., 1971; Worek and Thiermann, 2013), enzyme activity is mainly restored by de novo synthesis and therefore probably slower in progress than with VX. However, considering the poor possibilities to reactivate AChE inhibited by not only soman but also tabun or cyclosarin (Worek et al., 2007), the particular importance of de novo synthesis falls into place. Even though we cannot discard the possibility that de novo synthesis in the cocultures involves newly formed synapses rather than AChE synthesis in the existent network, a pharmacological intervention to speed up this process would certainly be of great value for the treatment of intoxicated patients. But since therapeutic options of this kind are not in clinical use so far, this issue clearly warrants further investigation. Another therapeutic challenge of OP poisoning yet to be unraveled is the so called intermediate syndrome (IMS). It manifests as delayed reappearance of neuromuscular weakness and peripheral respiratory failure, usually occurring 24–96 h after intoxication, when acute signs of cholinergic crisis have already subsided (Karalliedde et al., 2006). To date, IMS is classified as disorder of neuromuscular junctions but even though the syndrome is considered to play a decisive part in OP related morbidity and mortality, its exact etiology remains unclear. Previously proposed mechanisms include prolonged AChE inhibition, down-regulation or desensitization of ACh receptors, failure of presynaptic ACh release, and myopathy (Abdollahi and KaramiMohajeri, 2012; Yang and Deng, 2007). With regard to in vitro studies the characteristic lag of symptoms in IMS represents a certain pitfall. To reflect this phenomenon, or rather its underlying pathophysiology, an appropriate test system should feature the relevant target structures and remain functional for at least the stated timeframe. From a technical point of view, nerve-muscle cocultures may be applicable due to the presence of functional neuromuscular synapses and the sustained viability for several days. However, it remains to be seen if IMS-related processes indeed manifest in the slices. 5. Conclusions In summary, we could demonstrate that exposing organotypic nerve-muscle cocultures to nerve agents results in a significant loss of muscle activity, which resembles the peripheral symptoms of severe OP poisoning in vivo. Even though antidotes were not applied, extensive rehabilitation of muscular function was observed three and seven days after intoxication, which indicates the presence of auto-regenerative processes like spontaneous reactivation and de novo synthesis of AChE. Moreover, the cultures
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004
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showed no decrease in viability within the experimental timeframe. At the bottom line, the tested in vitro system technically provides relevant target structures to examine the effects of nerve agents and their antidotes, including long term processes like de novo synthesis of AChE or the pathophysiology of IMS. In order to improve this experimental model, further studies should be performed comprising, at best, the concurrent assessment of neuronal activity and AChE activity, as well as a characterization of the nature of AChE de novo synthesis occurring in the cultured tissue. Conflicts of interest The author declares that there are no conflicts of interest. Acknowledgements The author would like to thank Claudia Holt and Ina Pappe for excellent technical assistance and processing of video microscopic raw data. This study was funded by the Bundeswehr Institute of Pharmacology and Toxicology (Munich) and the Department of Anaesthesiology, Experimental Anaesthesiology Section, Eberhard-Karls-University (Tuebingen). Additionally, this work has been supported by contract no. M SAB1 7A009 from the German Ministry of Defence. References Abdollahi, M., Karami-Mohajeri, S., 2012. A comprehensive review on experimental and clinical findings in intermediate syndrome caused by organophosphate poisoning. Toxicol. Appl. Pharmacol. 258, 309–314. Aldridge, W.N., Reiner, E., 1972. Enzyme Inhibitors as Substrates: Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland Pub. Co. Avossa, D., Rosato-Siri, M.D., Mazzarol, F., Ballerini, L., 2003. Spinal circuits formation: a study of developmentally regulated markers in organotypic cultures of embryonic mouse spinal cord. Neuroscience 122, 391–405. Blaber, L.C., Creasey, N.H., 1960. The mode of recovery of cholinesterase activity in vivo after organophosphorus poisoning. 1. Erythrocyte cholinesterase. Biochem. J. 77, 591–596. Braschler, U.F., Iannone, A., Spenger, C., Streit, J., Luscher, H.R., 1989. A modified roller tube technique for organotypic cocultures of embryonic rat spinal cord, sensory ganglia and skeletal muscle. J. Neurosci. Methods 29, 121–129. de Jong, R.H., 2003. Nerve gas terrorism: a grim challenge to anesthesiologists. Anesth. Analg. 95, 819–825 table of contents. Drexler, B., Seeger, T., Grasshoff, C., Thiermann, H., Antkowiak, B., 2011. Long-term evaluation of organophosphate toxicity and antidotal therapy in co-cultures of spinal cord and muscle tissue. Toxicol. Lett. 206, 89–93. Drexler, B., Thiermann, H., Antkowiak, B., Grasshoff, C., 2013. Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice. Chem. Biol. Interact. 206, 555–560. Eckle, V.S., Drexler, B., Grasshoff, C., Seeger, T., Thiermann, H., Antkowiak, B., 2014. Spinal cord–skeletal muscle cocultures detect muscle-relaxant action of botulinum neurotoxin A. Altex 31, 433–440. Fleisher, J.H., Harris, L.W., 1965. Dealkylation as a mechanism for aging of cholinesterase after poisoning with pinacolyl methylphosphonofluoridate. Biochem. Pharmacol. 14, 641–650.
5
Furlan, F., Taccola, G., Grandolfo, M., Guasti, L., Arcangeli, A., Nistri, A., Ballerini, L., 2007. ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord. J. Neurosci. 27, 919–928. Gähwiler, B.H., 1981. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342. Grubic, Z., Sketelj, J., Klinar, B., Brzin, M., 1981. Recovery of acetylcholinesterase in the diaphragm, brain, and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate. J. Neurochem. 37, 909–916. Gupta, R.C., Patterson, G.T., Dettbarn, W.D., 1987. Biochemical and histochemical alterations following acute soman intoxication in the rat. Toxicol. Appl. Pharmacol. 87, 393–402. Harris, L.W., Yamamura, H.I., Fleisher, J.H., 1971. De novo synthesis of acetylcholinesterase in guinea pig retina after inhibition by pinacolyl methylphosphonofluoridate. Biochem. Pharmacol. 20, 2927–2930. Holmstedt, B., 1959. Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol. Rev. 11, 567–688. Jeyaratnam, J., 1990. Acute pesticide poisoning: a major global health problem. World Health Stat. Q. (Rapport trimestriel de statistiques sanitaires mondiales) 43, 139–144. Jokanovic, M., 2009. Medical treatment of acute poisoning with organophosphorus and carbamate pesticides. Toxicol. Lett. 190, 107–115. Karalliedde, L., Baker, D., Marrs, T.C., 2006. Organophosphate-induced intermediate syndrome: aetiology and relationships with myopathy. Toxicol. Rev. 25, 1–14. Kasprzak, H., Salpeter, M.M., 1985. Recovery of acetylcholinesterase at intact neuromuscular junctions after in vivo inactivation with diisopropylfluorophosphate. J. Neurosci. Off. J. Soc. Neurosci. 5, 951–955. Krejci, E., Martinez-Pena y Valenzuela, I., Ameziane, R., Akaaboune, M., 2006. Acetylcholinesterase dynamics at the neuromuscular junction of live animals. J. Biol. Chem. 281, 10347–10354. Legay, C., 2000. Why so many forms of acetylcholinesterase? Microsc. Res. Tech. 49, 56–72. Marrs, T.C., Rice, P., Vale, J.A., 2006. The role of oximes in the treatment of nerve agent poisoning in civilian casualties. Toxicol. Rev. 25, 297–323. Martinez-Pena y Valenzuela, I., Akaaboune, M., 2007. Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice. Mol. Biol. Cell 18, 2904–2911. Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., Vallette, F.M., 1993. Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31–91. Rosato-Siri, M.D., Zoccolan, D., Furlan, F., Ballerini, L., 2004. 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, 2697–2710. Rotundo, R.L., Ruiz, C.A., Marrero, E., Kimbell, L.M., Rossi, S.G., Rosenberry, T., Darr, A., Tsoulfas, P., 2008. Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction. Chem. Biol. Interact. 175, 26–29. Stitcher, D.L., Harris, L.W., Moore, R.D., Heyl, W.C., 1977. Synthesis of cholinesterase following poisoning with irreversible anticholinesterases: effects of theophylline and N6,O2-dibutyryl adenosine 30 ,50 -monophosphate on synthesis and survival. Toxicol. Appl. Pharmacol. 41, 79–90. Thiermann, H., Zilker, T., Eyer, F., Felgenhauer, N., Eyer, P., Worek, F., 2009. Monitoring of neuromuscular transmission in organophosphate pesticidepoisoned patients. Toxicol. Lett. 191, 297–304. Worek, F., Thiermann, H., 2013. The value of novel oximes for treatment of poisoning by organophosphorus compounds. Pharmacol. Ther. 139, 249–259. Worek, F., Eyer, P., Aurbek, N., Szinicz, L., Thiermann, H., 2007. Recent advances in evaluation of oxime efficacy in nerve agent poisoning by in vitro analysis. Toxicol. Appl. Pharmacol. 219, 226–234. Yang, C.C., Deng, J.F., 2007. Intermediate syndrome following organophosphate insecticide poisoning. J. Chin. Med. Assoc. 70, 467–472. Yourick, J.J., Eklo, P.A., McCluskey, M.P., Ray, R., 1991. Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-L-glutamine. Cell Biol. Toxicol. 7, 229–237.
Please cite this article in press as: I. Weimer, et al., Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord—skeletal muscle cocultures, Toxicol. Lett. (2015), http://dx.doi.org/10.1016/j.toxlet.2015.08.004