Evaluation of neuromuscular transmission in organophosphorus pesticide toxicity

Organophosphorus (OP) pesticide toxicity is a global health problem. Respiratory failure due to neuromuscular transmission dysfunction accounts for about 300,000 deaths annually in rural Asia. However, the clinical manifestation is complex, and described in terms of acute, intermediate, and chronic syndromes. The underlying mechanism of toxicity is still unclear. OP pesticides contain inhibitors of acetylcholinesterase (AChE), for example dimethoate, emulsified in an organic solvent, typically cyclohexanone. A hypothesized mechanism is initial excitotoxicity through inhibition of acetylcholinesterase followed by failure of neuromuscular synaptic transmission. I tested this electrophysiologically in vitro by measuring properties of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in isolated sciatic nerve/flexor digitorum brevis muscles from mice, bathed in HEPES-buffered mammalian physiological saline (MPS). Muscle action potentials were abolished with μ-conotoxin (2μM). First, we tested the effects of plasma taken from Göttingen minipigs instilled orally (isofluorane anaesthesia) with a formulated pesticide (2.5ml/kg) whose active ingredient is dimethoate dissolved in cyclohexanone. This plasma abolished evoked synaptic transmission and increased spontaneous MEPP frequency within 60-180 minutes of bath application. However plasma from minipigs instilled with dimethoate alone produced no failure of transmission. Plasma contained either pesticide or dimethoate significantly increased the half decay time of EPPs. However, pesticide-plasma also contained the metabolites omethoate (100μM) and cyclohexanol (5 mM). We found that bath application of omethoate alone caused a potent dose-dependent increase in EPP decay time. Cyclohexanol (5 mM) also increased EPP decay time but it also decreased both the excitability of axons and MEPP amplitude. In combination, omethoate and cyclohexanol produced greater disruption of neuromuscular transmission than either dimethoate or cyclohexanone, alone or in combination and this was particularly evident in isometric tension recordings, in which prolonged after-contraction and slow relaxation were observed during and immediately following tetanic stiumuation in the presence of omethoate and cyclohexanol. Voltage-clamp recordings of endplate currents (EPC) partially supported the EPP observations. Surprisingly, cyclohexanol-treated preparations showed no significant increase in EPC and MEPC decay time. However, there was some evidence of activity-dependent decline in MEPC amplitude in cyclohexanol while quantal content in these preparations showed evidence of an increase suggesting a homeostatic response in evoked transmitter release with cyclohexanol treatment. Analysis of presynaptic currents in cyclohexanol treated preparations also revealed preliminary evidence of sensitivity to cyclohexanol compared to control preparations. Finally, I tested the effects NMJ transmission of 24hr exposure to OP pesticide and its metabolites using a novel organ culture system, utilising a mouse mutant (WldS) with a slow nerve degeneration phenotype. After incubation of 24 hrs with MPS + pesticides and metabolites, these muscles showed significant reduction in function (response to nerve stimuli with EPP/action potential ± MEPPs) compared to control cultures. Together, the data indicate that failure of neuromuscular transmission by pesticide-plasma cannot be explained solely by dimethoate-mediated inhibition of acetylcholinesterase. Rather, a combination of metabolic breakdown products exerts potent, harmful presynaptic and postsynaptic effects. Either blocking the metabolic conversion of the constituents of OP pesticides, or transiently blocking their effects on receptors may therefore be an effective strategy for treatment of OP pesticide toxicity

‘Calcium bombs' as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Excitotoxicity is thought to be an important factor in the onset and progression of amyotrophic lateral sclerosis (ALS). Evidence from human and animal studies also indicates that early signs of ALS include degeneration of motor nerve terminals at neuromuscular junctions (NMJs), before degeneration of motor neuron cell bodies. Here we used a model of excitotoxicity at NMJs in isolated mouse muscle, utilizing the organophosphorus (OP) compound omethoate, which inhibits acetylcholinesterase activity. Acute exposure to omethoate (100 μM) induced prolonged motor endplate contractures in response to brief tetanic nerve stimulation at 20–50 Hz. In some muscle fibers, Fluo-4 fluorescence showed association of these contractures with explosive increases in Ca(2+) (“calcium bombs”) localized to motor endplates. Calcium bombs were strongly and selectively mitigated by increasing Mg(2+) concentration in the bathing medium from 1 to 5 mM. Overnight culture of nerve-muscle preparations from Wld(S) mice in omethoate or other OP insecticide components and their metabolites (dimethoate, cyclohexanone, and cyclohexanol) induced degeneration of NMJs. This degeneration was also strongly mitigated by increasing [Mg(2+)] from 1 to 5 mM. Thus, equivalent increases in extracellular [Mg(2+)] mitigated both post-synaptic calcium bombs and degeneration of NMJs. The data support a link between Ca(2+) and excitotoxicity at NMJs and suggest that elevating extracellular [Mg(2+)] could be an effective intervention in treatment of synaptic pathology induced by excitotoxic triggers

Donepezil inhibits neuromuscular junctional acetylcholinesterase and enhances synaptic transmission and function in isolated skeletal muscle

BACKGROUND AND PURPOSE: Donepezil, a piperidine inhibitor of acetylcholinesterase (AChE) prescribed for treatment of Alzheimer's disease, has adverse neuromuscular effects in humans, including requirement for higher concentrations of non‐depolarising neuromuscular blockers during surgery. Here, we examined the effects of donepezil on synaptic transmission at neuromuscular junctions (NMJs) in isolated nerve‐muscle preparations from mice. EXPERIMENTAL APPROACH: We measured effects of therapeutic concentrations of donepezil (10 nM to 1 μM) on AChE enzymic activity, muscle force responses to repetitive stimulation, and spontaneous and evoked endplate potentials (EPPs) recorded intracellularly from flexor digitorum brevis muscles from CD01 or C57BlWld(S) mice. KEY RESULTS: Donepezil inhibited muscle AChE with an approximate IC(50) of 30 nM. Tetanic stimulation in sub‐micromolar concentrations of donepezil prolonged post‐tetanic muscle contractions. Preliminary Fluo4‐imaging indicated an association of these contractions with an increase and slow decay of intracellular Ca(2+) transients at motor endplates. Donepezil prolonged spontaneous miniature EPP (MEPP) decay time constants by about 65% and extended evoked EPP duration almost threefold. The mean frequency of spontaneous MEPPs was unaffected but the incidence of ‘giant’ MEPPs (gMEPPs), some exceeding 10 mV in amplitude, was increased. Neither mean MEPP amplitude (excluding gMEPPs), mean EPP amplitude, quantal content or synaptic depression during repetitive stimulation were significantly altered by concentrations of donepezil up to 1 μM. CONCLUSION AND IMPLICATIONS: Adverse neuromuscular signs associated with donepezil therapy, including relative insensitivity to neuromuscular blockers, are probably due to inhibition of AChE at NMJs, prolonging the action of ACh on postsynaptic nicotinic acetylcholine receptors but without substantively impairing evoked ACh release

Cutaneous Leishmaniasis, Sri Lanka

Cutaneous leishmaniasis (CL) is an emerging disease in Sri Lanka. Of 116 patients with clinical symptoms suggestive of CL, 86 were confirmed positive for Leishmania donovani. Most patients had single dry lesions, usually on the face. Patients were from 5 of the 7 agroclimatic zones in Sri Lanka

“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Excitotoxicity is thought to be an important factor in the onset and progression of amyotrophic lateral sclerosis (ALS). Evidence from human and animal studies also indicates that early signs of ALS include degeneration of motor nerve terminals at neuromuscular junctions (NMJs), before degeneration of motor neuron cell bodies. Here we used a model of excitotoxicity at NMJs in isolated mouse muscle, utilizing the organophosphorus (OP) compound omethoate, which inhibits acetylcholinesterase activity. Acute exposure to omethoate (100 μM) induced prolonged motor endplate contractures in response to brief tetanic nerve stimulation at 20–50 Hz. In some muscle fibers, Fluo-4 fluorescence showed association of these contractures with explosive increases in Ca2+ (“calcium bombs”) localized to motor endplates. Calcium bombs were strongly and selectively mitigated by increasing Mg2+ concentration in the bathing medium from 1 to 5 mM. Overnight culture of nerve-muscle preparations from WldS mice in omethoate or other OP insecticide components and their metabolites (dimethoate, cyclohexanone, and cyclohexanol) induced degeneration of NMJs. This degeneration was also strongly mitigated by increasing [Mg2+] from 1 to 5 mM. Thus, equivalent increases in extracellular [Mg2+] mitigated both post-synaptic calcium bombs and degeneration of NMJs. The data support a link between Ca2+ and excitotoxicity at NMJs and suggest that elevating extracellular [Mg2+] could be an effective intervention in treatment of synaptic pathology induced by excitotoxic triggers

The construction and evaluation of a device for mechanomyography in anaesthetized Göttingen minipigs

OBJECTIVE: To devise a method for assessing evoked muscle strength on nerve stimulation [mechanomyography (MMG)] in the anaesthetized minipig. STUDY DESIGN: Prospective observational. ANIMALS: Sixty male Göttingen minipigs weighing 10.5–26.0 kg. METHODS: After cadaveric studies, a limb fixation device was constructed which allowed the twitch responses of the pelvic limb digital extensor muscles to be measured by force-displacement transduction in response to supramaximal train-of-four (TOF) stimulation of the common peroneal nerve. The device was tested in 60 minipigs weighing 10.5–26.0 kg positioned in dorsal recumbency. RESULTS: The technique recorded the MMG of the common peroneal-pelvic limb digital extensor nerve-muscle unit for up to 12 hours during which twitch height remained constant in 18 animals in which single twitch duration was <300–500 ms. In 42, in which twitch duration was >300–500 ms, 2 Hz nerve stimulation caused progressive baseline elevation (reverse fade) necessitating a modified signal capture method for TOF ratio (TOFR) computation. However, T1 was unaffected. The mean (range) of the TOFR in pigs with reverse fade was 1.2 (1.1–1.3). CONCLUSIONS AND CLINICAL RELEVANCE: The technique allowed MMG recording in unparalysed pigs in response to TOF nerve stimulation and revealed a hitherto unreported complication of MMG monitoring using TOF in animals: reverse fade. This complicated TOFR calculation