|Year : 2015 | Volume
| Issue : 3 | Page : 206-211
Muscle response of anticholinesterase-intoxicated rats to different therapeutic modalities
Abd-Elrahman M Elnaggar1, Omayma A Khorshid1, Amira A Labib MD 2, Inas A Harb1
1 Department of Pharmacology, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Neurophysiology, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||23-Mar-2015|
|Date of Acceptance||18-Apr-2015|
|Date of Web Publication||13-Aug-2015|
Amira A Labib
Department of Neurophysiology, Faculty of Medicine, Cairo University, Cairo, 12611
Source of Support: None, Conflict of Interest: None
Organophosphorus (OP) compound poisoning is a common, serious health problem that results in more than 250 000 deaths yearly worldwide, according to WHO estimates. Traditional use of atropine and oxime has failed to reduce the attendant morbidity and mortality.
The aim of the study was to evaluate the effects of magnesium sulfate and intravenous lipid emulsions versus the traditional standard of care - atropine and oxime - on butyrylcholineesterase enzyme (BuChE) activity and find whether there is a correlation between the enzyme level and muscle activity.
Adult female rats were used to study intermediate syndrome and a sublethal toxic dose of dimethoate (60 mg/kg) was used for induction of OP toxicity. Assessment of BuChE level, evaluation of De Bleecker score for muscle activity, and repetitive nerve stimulation test were carried out in all groups.
The mean BuChE serum level was significantly reduced in the OP-intoxicated group compared with the normal control group. Significant improvement was recorded in the groups treated with oxime and intralipid emulsion. The De Bleecker scale showed elevated scores in all treated groups, and the best result was with oxime and magnesium sulfate. Regarding electrophysiological findings, the most frequently found were repetitive firing after single nerve stimulation and decrement response after repetitive nerve stimulation.
This study highlighted possible new trends that could prove beneficial in the management of OP poisoning and in improving the degree of muscle weakness and its impact on respiratory muscles.
Keywords: intralipid emulsion, magnesium sulfate, organophosphorus poisoning, oxime, repetitive nerve stimulation
|How to cite this article:|
Elnaggar AEM, Khorshid OA, Labib AA, Harb IA. Muscle response of anticholinesterase-intoxicated rats to different therapeutic modalities. Egypt J Neurol Psychiatry Neurosurg 2015;52:206-11
|How to cite this URL:|
Elnaggar AEM, Khorshid OA, Labib AA, Harb IA. Muscle response of anticholinesterase-intoxicated rats to different therapeutic modalities. Egypt J Neurol Psychiatry Neurosurg [serial online] 2015 [cited 2017 Jun 28];52:206-11. Available from: http://www.ejnpn.eg.net/text.asp?2015/52/3/206/162047
| Introduction|| |
Organophosphorus (OP) compounds have many different uses, such as in medical treatment of myasthenia gravis, in chemical warfare, and in agriculture . Acute organophosphorus poisoning (OPP) occurs through various routes: oral, respiratory, or dermal exposure. It is characterized by a triphasic response in the form of an initial acute cholinergic phase, an intermediate syndrome (IMS) (both are associated with high mortality), and a disabling but nonlethal delayed polyneuropathy . Although many therapies for acute OPP exist, mortality rates are still high, ranging from 10 to 20% . Years after first use, the optimal application of the core treatment agents atropine, oximes, and diazepam is still unknown . The use of oximes is still relatively unclear, as they are mainly beneficial in patients poisoned moderately or in those affected by specific pesticides. Traditional use of pralidoxime with atropine failed to reduce the attendant morbidity and mortality . Administration of magnesium sulfate and its actual extent of benefit for patient management are still under evaluation . Intravenous lipid emulsions were also introduced for resuscitation of patients under toxicity of local anesthetic drugs. Their detoxification mechanism occurs through 'lipid sink' .
| Aim of work|| |
The aim of the study was to evaluate the effect of magnesium sulfate and intravenous lipid emulsions versus the traditional use of atropine and oxime, and to find whether there is a correlation between butyrylcholinesterase enzyme (BuChE) level and muscle activity.
| Materials and methods|| |
A prospective study was carried out on adult female albino rats obtained from the animal house of Cairo University. All experiments were carried out in the pharmacology department and followed the guidelines of the local committee of Ethics on Animal Experimentation, Cairo University. Rats were placed under a 12-h light/dark cycle in a temperature-controlled room (22 ± 2°C).
Thirty female albino rats, weighing 190-220 g, were divided into five groups: (a) normal (control, N); (b) OP-intoxicated rats; (c) atropine and oxime-treated OP-intoxicated rats; (d) atropine, oxime, and intravenous lipid emulsion-treated OP-intoxicated rats; and (e): atropine, oxime, and magnesium sulfate-treated OP-intoxicated rats.
The used drugs and chemicals were dimethoate, atropine sulfate, obidoxime, intralipid emulsion, magnesium sulfate, urethane (for induction of anesthesia), and cholinesterase kit.
Assessment parameters: (a) measurement of serum BuChE level: this was carried out using the cholinesterase kit, PCB260, wherein the increase in absorbance in the unit time at 405 nm is proportional to the activity of the butyrylcholinesterase in the sample. (b) Assessment of muscle strength of the rat: this was carried out using the following:
- Scoring criteria of De Bleecker et al. :muscle activity after painful stimulation of the tail was determined by grades:
- 0: Normal mobility.
- 1: Ataxic gait due to hind limb weakness.
- 2: Stretch movement only after painful stimulation of the tail.
- 3: No voluntary movement even after painful stimulation of the tail.
- Muscle assessment by repetitive nerve stimulation: This was carried out to demonstrate the degree of affection by performing in-vivo electromyography in rats poisoned with an organophosphate - namely, dimethoate. The animals underwent serial electromyography, together with single and repetitive nerve stimulation.
- Single stimulation of the sciatic nerve: intensity 4 mA, frequency 3 Hz, and pulse duration 1 ms.
- Train of impulses at 3 Hz: 20 impulses of sciatic nerve at intensity 4 mA, frequency 3 Hz, and pulse duration 1 ms.
- Muscle exercise for 2 min: continuous sciatic nerve stimulation at intensity 4 mA, frequency 3 Hz, and pulse duration 1 ms for 2 min.
- The ratio of the amplitudes of the ninth to the first compound muscle action potential (CMAP) (9 : 1 ratio): the decremental response is characterized by a progressive fall in the amplitude of the successive CMAPs elicited by a train of supramaximal electrical stimuli delivered to the motor nerve.
The 9:1 ratio is arbitrarily taken as an objective marker of severity of the decremental response [Figure 1]. It represents a desensitization type of neuromuscular blockade. It correlates with the presence of clinically recognizable IMS and can be considered as an electrodiagnostic marker for it .
The following devices were used: PowerLab Data Acquisition [Model number: PowerLab 4/30 with Labchart Pro (Product ML866/P); AD Instruments, Australia], Bridge amplifier, Electrical stimulator units, Stimulator HC Leads, Isometric force transducer, Fixing unit for the animal, and heater table [Figure 2].
All of the obtained data were compiled in tables and statistically analyzed. The treated and control groups were compared using the EPI cal test, the Student t-test, and analysis of variance. P values less than or equal to 0.05 were considered significant.
| Results|| |
The mean BuChE serum level was recorded for each rat group. Significant difference was seen between groups 3, 4, and 5 (P ≤ 0.05) [Figure 3].
|Figure 3: The mean serum level of butyrylcholineesterase enzyme (BuChE). *(P ≤ 0.05). G1: control, N group; G2: OP-intoxicated group; G3: atropine+oxime-treated OP-intoxicated group; G4: atropine+oxime+intralipid emulsion-treated OP-intoxicated group; G5: atropine+oxime+magnesium sulfate-treated OP-intoxicated group. OP, organophosphor us.|
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The mean De Bleecker score for muscle activity was recorded for each rat group [Table 1].
Single stimulation given to the sciatic nerve (frequency 3 Hz, electrical current 4 mA, and pulse duration 1 ms) evoked different responses whether or not with repetitive firing [Table 2].
|Table 2: Showing responses of single nerve stimulation of the sciatic nerve in different groups|
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A train of 20 repetitive electrical stimuli delivered to the sciatic nerve resulted in normal percentage inhibition for both second and fourth muscle responses (did not exceed 10% inhibition from the first response) in the normal group, versus decrement, decrement-increment, increment, and fade responses in all treated groups [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9] and [Figure 10]. The train of repetitive electrical stimuli was given before and after 2 min of muscle exercise and were evoked by frequency 3 Hz stimulation, electrical current 4 mA, and pulse duration 1 ms.
|Figure 4: A train of 20 electrical stimuli before exercise elicited decrement– increment response in the organophosphorus-intoxicated group; decreasing amplitude more prominent at the fourth respon se.|
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|Figure 5: Decrement response evoked by a train of 20 repetitive electrical stumuli for the sciatic nerve after exercise (2 min gastrocnemius fatigue) in the organophosphorus-intoxicated gro up.|
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|Figure 6: Fade response evoked by a train of 20 repetitive electrical stimuli for the sciatic nerve after exercise (2 min gastrocnemius fatigue) in the organophosphorus-intoxicated gro up.|
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|Figure 7: A train of 20 stimuli of sciatic nerve evoked increment response before exercise in the atropine+oxime-treated organophosphorusintoxicated gro up.|
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|Figure 8: A train of 20 stimuli of sciatic nerve evo ked decrement– increment response before exercise in the atropine+oxime+intralipid-treated organophosphorus-intoxicated gro up.|
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|Figure 9: A train of 20 stimuli of sciatic nerve after exercise (2 min gastrocnemius fatigue) evoked decrement response in the atropine+oxime+intralipidtreated organophosphorus-intoxicated group. Black arrows show both the first and ninth responses.|
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|Figure 10: A train of 20 stimuli for sciatic nerve before exercise evoked increment response in the atropine+oxime+magnesium sulfate-treated organophosphorus-intoxicated gro up.|
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The marker of severity of the decrement response '9:1 ratio' was determined after exercise in all of the treated rat groups. Significant difference was seen only between group 5 treated with atropine, oxime, and magnesium sulfate and group 2 of the OP compound-intoxicated group (P ≤ 0.05) [Figure 11].
|Figure 11: The ratio of the amplitudes of the ninth compound muscle action potential (CMAP) to the first CMAP (9:1 ratio). *Significant difference compared with the organophosphorus (OP)-intoxicated group (P ≤ 0.05). G1: control, N group; G2: OP-intoxicated group; G3: atropine+oxime-treated OP-intoxicated group; G4: atropine+oxime+intralipid emulsion-treated OP-intoxicated group; G5: atropine+oxime+magnesium sulfate-treated OP-intoxicated gro up.|
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| Discussion|| |
The IMS following OP insecticide poisoning was first described in the mid-1980s. The syndrome comprised characteristic symptoms and signs occurring after apparent recovery from the acute cholinergic syndrome. The IMS occurs in ∼20% of patients following oral exposure to OP pesticides . BuChE is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies OP poisons . The present study showed significant improvement in the mean level of BuChE in rat groups treated with oxime and intralipid emulsion in comparison with the OP-intoxicated group. Jayawardane et al.  assessed BuChE levels in 59 acute chlorpyrifos-poisoned patients. There was a statistically significant difference between the extent of inhibition of BuChE activity between those who developed IMS spectrum disorder and those who did not. BuChE inhibition can be a marker of exposure and its level may be important in predicting severity.
In the present study, repetitive nerve stimulation in the OP-intoxicated group showed that the most frequent electrophysiological finding was repetitive firing after single nerve stimulation and decremental response after repetitive nerve stimulation. Repetitive fade and severe decrement were also noted in some rats. In accordance with the present study, Maselli and Soliven  studied acute organophosphate intoxication through in-vivo and in-vitro electrophysiologic studies in rats injected with diisopropylfluorophosphate. The intoxicated rats showed weakness, repetitive CMAPs in response to a single stimulus, and decremental response to repetitive nerve stimulation that was most pronounced at the second CMAP. Further, Karalliedde et al.  reported that electrophysiological studies following OPP have revealed three characteristic phenomena: repetitive firing following a single stimulus, gradual reduction in twitch height or CMAP followed by an increase in repetitive stimulation (decrement-increment response), and continued reduction in twitch height or CMAP with repetitive simulation (decrement response).
The role of oximes in human OPP is not clear. Several new therapies have been studied, but results are inconclusive .
Singh et al.  concluded that continuous 2-PAM infusion along with aggressive atropinization after initial decontamination improves the outcome but not the duration of mechanical ventilation in severely intoxicated patients with OP compounds.
Thiermann et al.  studied the effect of the clinically used oximes obidoxime and pralidoxime. The presented data strongly supported the administration of appropriately dosed oximes, preferably obidoxime, in paraoxon-poisoned patients. However, there are reports from developed countries wherein administration of oximes at recommended doses and within 2 h of ingestion of OP insecticide did not prevent the onset of IMS .
In the present work, combination of magnesium sulfate with oxime had an add-on effect in reducing muscle weakness of the gastrocnemius, as 9 : 1 ratio showed significant improvement in the atropine+oxime+magnesium sulfate-treated group. In contrast to the present work, in a randomized clinical study 18 patients with OPP were administered magnesium sulfate (4 g every 6 hourly for 48 h, intravenous) or placebo, in addition to atropine and pralidoxime. No significant difference in clinical and electrophysiological outcomes was observed .
Most OP pesticides are highly fat soluble. Therefore, intravenous lipid emulsions have potential clinical applications in OPP. Zhou et al.  postulated that the combination of intravenous lipid emulsions and charcoal hemoperfusion could be used to cure severe organophosphate poisoning. In the present study, intralipid emulsions proved efficacious in reducing muscle weakness through decreased percentage inhibition of muscle for both second and fourth muscle responses before exercise, and this was more evident in the fourth response. Muscle response changed from decrement to decrement-increment. Muscle response type after exercise changed to decrement response, but compared with the OP-intoxicated group the mean percentage inhibition after exercise decreased from 73 ± 17 to 36 ± 6%. This change was statistically significant. In addition, in the present study, intravenous lipid emulsion administration significantly improved the De Bleeker score.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Kumar S, Sudhakar F, Venkateswarlu B. Current review on organophosphorus poisoning. Arch Appl Sci Res 2010; 2
Kamanyire R, Karalliedde L. Organophosphate toxicity and occupational exposure. Occup Med (Lond) 2004; 54
Dunn C, Bird SB, Gaspari R. Intralipid fat emulsion decreases respiratory failure in a rat model of parathion exposure. Acad Emerg Med 2012; 19
Eddleston M, Buckley NA, Eyer P, Dawson AH. Management of acute organophosphorus pesticide poisoning. Lancet 2008; 371
Sivagnanam S. Potential therapeutic agents in the management of organophosphorus poisoning. Crit Care 2002; 6
Pajoumand A, Shadnia S, Rezaie A, Abdi M, Abdollahi M. Benefits of magnesium sulfate in the management of acute human poisoning by organophosphorus insecticides. Hum Exp Toxicol 2004; 23
Zhou Y, Zhan C, Li Y, Zhong Q, Pan H, Yang G. Intravenous lipid emulsions combine extracorporeal blood purification: a novel therapeutic strategy for severe organophosphate poisoning. Med Hypotheses 2010; 74
De Bleecker JL, De Reuck JL, Willems JL. Neurological aspects of organophosphate poisoning. Clin Neurol Neurosurg 1992; 94
Singh G, Khurana D. Neurology of acute organophosphate poisoning. Neurol India 2009; 57
Karalliedde L, Baker D, Marrs TC. Organophosphate-induced intermediate syndrome: aetiology and relationships with myopathy. Toxicol Rev 2006; 25
Masson P, Lockridge O. Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior. Arch Biochem Biophys 2010; 494
Jayawardane P, Eyer P, Thiermann H, Senanayake N, Buckley N, Dawson A. Butyrylcholinesterase inhibition and the development of intermediate syndrome following acute organophosphate poisoning. Hum Exp. Scientific Congress of Asia Pacific Association of Medical Toxicology. Penang, Malaysia; 12-14 November 2011.
Maselli RA, Soliven BC. Analysis of the organophosphate-induced electromyographic response to repetitive nerve stimulation: paradoxical response to edrophonium and d-tubocurarine. Muscle Nerve 1991; 14
Singh S, Chaudhry D, Behera D, Gupta D, Jindal SK. Aggressive atropinisation and continuous pralidoxime (2-PAM) infusion in patients with severe organophosphate poisoning: experience of a north west Indian hospital. Hum Exp Toxicol 2001; 20
Thiermann H, Eyer P, Worek F. Muscle force and acetylcholinesterase activity in mouse hemidiaphragms exposed to paraoxon and treated by oximes in vitro. Toxicology 2010; 272
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2]