Acute administration of diazepam or midazolam minimally alters long-term neuropathological effects in the rat brain following acute intoxication with diisopropylfluorophosphate.

Acute intoxication with organophosphorus cholinesterase inhibitors (OPs) can trigger seizures that rapidly progress to life-threatening status epilepticus. Diazepam, long considered the standard of care for treating OP-induced seizures, is being replaced by midazolam. Whether midazolam is more effective than diazepam in mitigating the persistent effects of acute OP intoxication has not been rigorously evaluated. We compared the efficacy of diazepam vs. midazolam in preventing persistent neuropathology in adult male Sprague-Dawley rats acutely intoxicated with the OP diisopropylfluorophosphate (DFP). Subjects were administered pyridostigmine bromide (0.1 mg/kg, i.p.) 30 min prior to injection with DFP (4 mg/kg, s.c.) or vehicle (saline) followed 1 min later by atropine sulfate (2 mg/kg, i.m.) and pralidoxime (25 mg/kg, i.m.), and 40 min later by diazepam (5 mg/kg, i.p.), midazolam (0.73 mg/kg, i.m.), or vehicle. At 3 and 6 months post-exposure, neurodegeneration, reactive astrogliosis, microglial activation, and oxidative stress were assessed in multiple brain regions using quantitative immunohistochemistry. Brain mineralization was evaluated by in vivo micro-computed tomography (micro-CT). Acute DFP intoxication caused persistent neurodegeneration, neuroinflammation, and brain mineralization. Midazolam transiently mitigated neurodegeneration, and both benzodiazepines partially protected against reactive astrogliosis in a brain region-specific manner. Neither benzodiazepine attenuated microglial activation or brain mineralization. These findings indicate that neither benzodiazepine effectively protects against persistent neuropathological changes, and suggest that midazolam is not significantly better than diazepam. Overall, this study highlights the need for improved neuroprotective strategies for treating humans in the event of a chemical emergency involving OPs.

Delayed Adjunctive Treatment of Organophosphate-Induced Status Epilepticus in Rats with Phenobarbital, Memantine, or Dexmedetomidine.

Organophosphate (OP) exposure induces status epilepticus (SE), a medical emergency with high morbidity and mortality. Current standard medical countermeasures lose efficacy with time so that treatment delays, in the range of tens of minutes, result in increasingly poor outcomes. As part of the Countermeasures Against Chemical Threats Neurotherapeutics Screening Program, we previously developed a realistic model of delayed treatment of OP-induced SE using the OP diisopropyl fluorophosphate (DFP) to screen compounds for efficacy in the termination of SE and elimination of neuronal death. Male rats were implanted for electroencephalogram (EEG) recordings 7 days prior to experimentation. Rats were then exposed to DFP, and SE was induced for 60 minutes and then treated with midazolam (MDZ) plus one of three antiseizure drugs (ASDs)-phenobarbital (PHB), memantine (MEM), or dexmedetomidine (DMT)-in conjunction with antidotes. EEG was recorded for 24 hours, and brains were stained with Fluoro-Jade B for quantification of degenerating neurons. We found that PHB + MDZ induced a prolonged suppression of SE and reduced neuronal death. MEM + MDZ treatment exacerbated SE and increased mortality; however, surviving rats had fewer degenerating neurons. DMT + MDZ significantly suppressed SE with only a minimal reduction in neuronal death. These data demonstrate that delayed treatment of OP-induced SE with other ASDs, when added to MDZ, can achieve greater seizure suppression with additional reduction in degenerating neurons throughout the brain compared with MDZ alone. The effect of a drug on the severity of seizure activity did not necessarily determine the drug's effect on neuronal death under these conditions. SIGNIFICANCE STATEMENT: This study assesses the relative effectiveness of three different delayed-treatment regimens for the control of organophosphate-induced status epilepticus and reduction of subsequent neuronal death. The data demonstrate the potential for highly effective therapies despite significant treatment delay and a potential disconnect between seizure severity and neuronal death.

Antiseizure and neuroprotective effects of delayed treatment with midazolam in a rodent model of organophosphate exposure.

OBJECTIVE: Exposure to organophosphates (OPs) and OP nerve agents (NAs) causes status epilepticus (SE) and irreversible brain damage. Rapid control of seizure activity is important to minimize neuronal injury and the resulting neurological and behavioral disorders; however, early treatment will not be possible after mass release of OPs or NAs. METHODS: We utilized a delayed-treatment model of OP exposure in adult rats by administration of diisopropyl fluorophosphate (DFP) to study the relationship between the antiseizure and neuroprotective effects of the "standard-of-care" benzodiazepine, midazolam (MDZ), when given at 30, 60, and 120 minutes after SE onset. After electroencephalography (EEG) recordings, neural damage in serial brain sections was studied with Fluoro-Jade B staining. RESULTS: MDZ-induced seizure suppression was equivalent in magnitude regardless of treatment delay (ie, seizure duration). When assessed globally (ie, normalized across 10 different brain regions) for each treatment delay, MDZ administration resulted in only nonsignificant reductions in neuronal death. However, when data for MDZ treatment were combined from all three delay times, a small but significant reduction in global neuronal death was detected when compared to vehicle treatment, which indicated that the substantive MDZ-induced seizure suppression led to only a small reduction in neuronal death. SIGNIFICANCE: In conclusion, MDZ significantly reduced DFP-induced SE intensity when treatment was delayed 30, 60, and even up to 120 minutes; however, this reduction in seizure intensity had no detectable effect on neuronal death at each individual delay time. These data show that although MDZ suppressed seizures, additional neuroprotective therapies are needed to mitigate the effects of OP exposure.

Experimental Substantiation of Inhalation Administration of Pathogenic Therapy of Toxic Convulsive Disorder for Correction of External Respiration Disorders.

We studied the dynamics of respiratory function in rats during intratracheal poisoning with diisopropyl fluorophosphate and pentylenetetrazole in doses corresponding to the LD50 in humans. The maximum of external respiration impairment was recorded in 30 min after poisoning. Administration of diazepam and atropine both separately and in combination during the development of the first signs of poisoning did not significantly affect the respiration parameters, but reduced the incidence of seizures and contributed to a decrease in the rate of animal death. Intratracheal administration of cholinolytic, ß2-adrenomimetic, or glutamate receptors antagonist promoted correction of the respiratory function. It was found that the maximum therapeutic effect in case of diisopropyl fluorophosphates poisoning was achieved after intratracheal administration of ipratropium bromide (0.086 mg/kg), salbutamol (0.086 mg/kg), and MK-801 (0.1 mg/kg), while in case of pentylenetetrazole poisoning, intratracheal administration of ipratropium bromide (0.086 mg/kg) was most effective.

Chronic Treatment with Naltrexone Prevents Memory Retention Deficits in Rats Poisoned with the Sarin Analog Diisopropylfluorophosphate (DFP) and Treated with Atropine and Pralidoxime.

Humans and rats poisoned with sarin develop chronic neurological disabilities that are not prevented with standardized antidotal therapy. We hypothesized that rats poisoned with the sarin analogue diisopropylfluorophosphate (DFP) and resuscitated with atropine and pralidoxime would have long-term memory deficits that were preventable with naltrexone treatment. Long Evans rats (250-275 g) were randomized to: DFP (N = 8): single subcutaneous (SC) injection of DFP (5 mg/kg). Treatment (N = 9): DFP (5 mg/kg) followed by chronic naltrexone (5 mg/kg/day × 12 weeks). Control (N = 12): single SC injection of isopropyl alcohol, (DFP vehicle) followed by chronic naltrexone (5 mg/kg/day). If toxicity developed after injection, antidotal therapy was initiated with atropine (2 mg/kg) and pralidoxime (25 mg/kg) and repeated as needed. After 12 weeks, rats underwent testing for place learning (acquisition) across 5 days of training using the Morris Water Maze. On day 6 a memory retention test was performed. Statistical analysis was performed using IBM SPSS Statistics. Rats receiving DFP rapidly developed toxicity requiring antidotal rescue. No differences in acquisition were seen between the DFP vs. DFP + naltrexone rats. During memory testing, DFP-poisoned rats spent significantly less time (29.4 ± 2.11 versus 38.5 ± 2.5 s, p < 0.05) and traveled less distance (267 ± 24.6 versus 370 ± 27.5 cm, p < 0.05) in the target quadrant compared to the treatment group. Treatment rats performed as well as control rats (p > 0.05) on the test for memory retention. Poisoning with DFP induced impaired memory retention. Deficits were not prevented by acute rescue with atropine and pralidoxime. Chronic naltrexone treatment led to preserved memory after DFP poisoning.

4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration.

Many organophosphorous esters synthesized for applications in industry, agriculture, or warfare irreversibly inhibit acetylcholinesterase, and acute poisoning with these compounds causes life-threatening cholinergic overstimulation. Following classical emergency treatment with atropine, an oxime, and a benzodiazepine, surviving victims often suffer brain neurodegeneration. Currently, there is no pharmacological treatment to prevent this brain injury. Here we show that a cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) ameliorates the damage caused by diisopropylfluorophosphate (DFP) in the hippocampal area CA1. DFP has been frequently used as a surrogate for the warfare nerve agent sarin. In rats, DFP is lethal at the dose used to cause brain damage. Therefore, to observe brain damage in survivors, the death rate was reduced by pre-administration of the peripherally acting antidotes pyridostigmine and methyl atropine or its analog ipratropium. Pyridostigmine bromide, methyl atropine nitrate, and ipratropium bromide were dissolved in saline and injected intramuscularly at 0.1mg/kg, 20mg/kg, and 23mg/kg, respectively. DFP (9mg/kg) dissolved in cold water was injected intraperitoneally. 4R (6mg/kg) dissolved in DMSO was injected subcutaneously, either 1h before or 5 or 24h after DFP. Neurodegeneration was assessed with Fluoro-Jade B and amino cupric silver staining; neuroinflammation was measured by the expression of nestin, a marker of activated astrocytes. Forty-eight hours after DFP administration, 4R decreased the number of dead neurons by half when injected before or after DFP. 4R also significantly decreased the number of activated astrocytes. These data suggest that 4R is a promising new drug that could change the therapeutic paradigm for acute poisoning with organophosphorous compounds by the implementation of a second-stage intervention after the classical countermeasure treatment.

[Specific features of the anticonvulsant effect of memantine in mice intoxicated with a model organic phosphate].

The effect of memantine administration has been studied on the model of mice poisoning with an anticholinesterase compound. It is established that the memantine action is due to its influence on the cholinesterase activity in the brain, blood plasma, and erythrocytes in addition to its NMDA-blocking action. Memantine promotes oxime-induced erythrocyte enzyme reactivation on the model of mice poisoning with anticholinesterase compound (0.8 LD50).

Pro-2-PAM therapy for central and peripheral cholinesterases.

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.

Post-intoxication vaccination for protection of neurons against the toxicity of nerve agents.

Nerve agents are highly toxic organophosphates (OPs) that can cause severe damage to the central and peripheral nervous systems. The central nervous system insult results in seizures and neuronal death. The glutamatergic system apparently contributes to the neuropathology. Using a model of OP intoxication causing death of retinal ganglion cells in the mouse eye, we show here that intoxication is exacerbated if the mice are devoid of mature T cells. The retinal neurons could be protected from these effects by vaccination, 7 days before or immediately after intoxication, with the copolymer glatiramer acetate (Cop-1), recently found to limit the usual consequences of an acute glutamate insult to the eye. These findings underlie a new therapeutic approach to protection against OP intoxication, based on the rationale that boosting of the adaptive immunity recruited at the site of intoxication helps the local cellular machinery such as resident microglia to withstand the neurotoxic effects.

The treatment of delayed polyneuropathy induced by diisopropylfluorophosphate in hens.

This study was undertaken to examine the influence of atropine, oximes and benzodiazepine on organophosphate-induced delayed polyneuropathy (OPIDP) in hens, which were poisoned with diisopropylfluorophosphate (DFP). The birds were treated with a standard neuropathic dose of DFP (1.1 mg/kg, s.c.), which produced typical signs of OPIDP. The development of OPIDP was observed within the followings 22 days. All drugs were given subcutaneously (s.c.), intramuscularly (i.m.) or intraperitoneally (i.p.), 20 min before the poison. The results obtained have shown that atropine (20 mg/kg, i.p.) only in combination with oxime TMB-4 (15 mg/kg, i.m.) produced significant improvement of OPIDP symptoms in comparison with positive control. Clinical signs and symptoms of OPIDP in the group which was treated with atropine (20 mg/kg, i.p.), TMB-4 (15 mg/kg, i.m.) and midazolam (2.5 mg/kg, i.m.) were more improved than that in the presence of a combination of atropine and TMB-4. The results of these experiments have shown that it is possible to prevent the development of DFP-induced OPIDP in hens by treatment with atropine and TMB-4 or atropine, TMB-4 and midazolam when given before DFP.

Phosphotriesterase, pralidoxime-2-chloride (2-PAM) and eptastigmine treatments and their combinations in DFP intoxication.

The protective action of i.v. administered eptastigmine, an organophosphate hydrolase (phosphotriesterase), or pralidoxime-2-chloride (2-PAM) and their combination in acute diisopropylfluorophosphate (DFP) intoxication were evaluated in mice. The mice received the physostigmine derivative, eptastigmine (0.9 mg/kg body wt, i.v.), 10 min prior to the i.p. injection of DFP (1.8 mg/kg body wt). Phosphotriesterase (66 micromol/min x ml/g and 6 microg/g body wt) or 2-PAM (30 mg/kg body wt) were given i.v. 30 min after DFP. The animals also received atropine sc (37.5 mg/kg body wt) immediately after DFP. The cholinesterase (ChE) activities were not protected or reactivated by 2-PAM alone. The ChE activities in brain and plasma were protected by phosphotriesterase. Eptastigmine alone assisted the recovery of the brain ChE activities. Also the combination of eptastigmine-phosphotriesterase protected the brain enzymes. It did not, however, provide any additional protection compared with phosphotriesterase-treatment on its own. In brain, the combination of eptastigmine with 2-PAM resulted in partly restored enzyme activities 24 hr after DFP exposure. In plasma, eptastigmine did not prevent the inhibition of ChE by DFP. However, when it was combined with phosphotriesterase, it significantly promoted the recovery of plasma ChE activity. In lung and in erythrocytes, the various combinations of antidotes caused only minor changes in the ChE activities.

Eptastigmine-phosphotriesterase combination in DFP intoxication.

A novel therapy against organophosphate exposure, the combination of a carbamate eptastigmine and an organophosphate hydrolase (phosphotriesterase) was studied in mice against diisopropylfluorophosphate (DFP) (1.75 mg/kg) exposure. Mice received eptastigmine (0.9 mg/kg; iv) 10 min prior to the ip injection of DFP. Phosphotriesterase (83 U/g body weight) was injected iv 10 min after DFP. Eptastigmine (1.5 mg/kg; iv) inhibited the acetylcholinesterase activities in brain and erythrocytes for a longer time than physostigmine. Eptastigmine caused only minor changes in the behavior and activity of the animals, whereas physostigmine clearly reduced their activity for about 30 min. The eptastigmine pretreatment clearly supplemented the protective effect of phosphotriesterase against DFP: the plasma butyrylcholinesterase activity was doubled and the activity recovered faster than in animals treated with phosphotriesterase alone. In lung, butyrylcholinesterase activity was initially lower after eptastigmine-phosphotriesterase than phosphotriesterase treatment alone. However, the activity returned 24 hr later to normal in eptastigmine-phosphotriesterase-treated groups. With phosphotriesterase only, it recovered only to 75% of the control level. Presumably eptastigmine, by preventing the binding of DFP to cholinesterases, caused an elevation of free DFP levels in body fluids and promoted phosphotriesterase hydrolysis of DFP.

Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors.

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2-4 months) and aged (22-24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [3H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [3H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.

Acute and subacute organophosphate poisoning in the rat.

The intermediate syndrome in organophosphate poisoning is clinically characterized by weakness in the territory of cranial nerves, weakness of respiratory, neck and proximal limb muscles, and depressed deep tendon reflexes. It occurs between the acute cholinergic crisis and the usual onset of organophosphate-induced delayed neurotoxicity. The weakness has been ascribed to muscle fiber necrosis. Fenthion has been the most common cause. This study assesses the occurrence of the necrotizing myopathy in rats in relation to the clinical course and the acetylcholinesterase (AChE) inhibition after poisoning with organophosphates representative for each of the major types of organophosphate-related neurotoxicity. Marked differences are noted in the duration of cholinergic symptoms and of AChE inhibition after either paraoxon and mipafox, or fenthion poisoning. The necrotizing myopathy begins shortly after the initial decline in AChE activity with all organophosphates studied. Maximal muscle involvement occurs within the first 2 days of the poisoning with all organophosphates studied. The myopathy is not aggravated by a further decline in AChE activity in fenthion poisoning. Our data argues against the monophasic necrotizing myopathy being the cause of the intermediate syndrome, and is suggestive of persistent AChE inhibition being involved.

Prevention of organophosphate intoxication with presynaptic cholinergic blockers.

In Vivo cardiovascular neuromuscular preparation of rats was used to study the prophylaxis and mechanism of diisopropyl fluorophosphate (DFP) intoxication by presynaptic cholinolytic agents (ganglionic blocking agents), including hexamethonium, trimethaphan and mecamylamine. The lethal action of DFP was partially or completely prevented by pretreatment of rats with hexamethonium (10 mg/kg), trimethaphan (80 mg/kg) and mecamylamine (30 mg/kg). Combined use of these drugs with 2-PAM (100 mg/kg) improved further the prophylaxis of DFP lethality. The mechanism of prophylaxis could be due to the reduction of acetylcholine turnover and accumulation at the synaptic cleft as hemicholinium prevented the DFP intoxication efficiently. In all cases, DFP caused cardiovascular suppression before neuromuscular blockade indicating that the cardiovascular system is much more sensitive than the neuromuscular junction to DFP intoxication.

Altered modulation by excitatory amino acids of cortical phosphatidylinositol system stimulated by carbachol in rats poisoned by an anti-cholinesterase compound, diisopropyl fluorophosphate.

The effects of glutamate and N-methyl aspartate (NMDA) on carbachol-induced inositol phosphate (IP) accumulation were evaluated in slices of the cerebral cortex of rats treated with diisopropyl fluorophosphate (DFP) for 2 weeks. This induced an about 75% inhibition of cholinesterases. The IP accumulation induced by carbachol (expressed as ratio stimulated/basal IP content) was lower in DFP rats than in controls when incorporation of [3H]-myoinositol into membrane phospholipids and their hydrolysis were measured (no washing step between labeling and hydrolytic incubation). There were no differences in carbachol induced IP accumulation between control and DFP rats when only phosphoinositide hydrolysis was determined (hydrolytic incubation of prelabeled washed slices). When both incorporation of [3H]-myoinositol and the hydrolysis were measured, 0.5 mM glutamate and 0.1 mM NMDA caused a significant, about 40%, decrease of carbachol-induced IP accumulation in control rats; the inhibitory effects of glutamate and NMDA were not significant in DFP rats. When only hydrolytic IP accumulation by carbachol was studied, the inhibitory effects of glutamate and NMDA were very similar in control and DFP rats. Additional experiments on inositol phospholipid synthesis showed a significantly lesser [3H]-myoinositol incorporation (by about 30%) in DFP rats. This may explain the differences between the results obtained by the two methods. The overall data suggest that the attenuation of glutamate and NMDA effects in DFP-rats depends on a decrease of carbachol-induced IP accumulation or phosphoinositide synthesis rather than on the EAA specific action.

Route-specific cardiorespiratory and neuromuscular changes following organophosphorous poisoning in rats.

Organophosphorous compounds cause a variety of physiological effects. The effect of various routes of administration of these compounds on the pathophysiology is still an open question. The present study addresses the effect of two organophosphorous compounds, namely diisopropyl fluorophosphate and methylisopropyl phosphonofluoridate, given by two different routes (i.v. and s.c.), on physiological parameters in anaesthetized and artificially ventilated rats. Both organophosphorous compounds produced a dose-dependent rise in blood pressure, bradycardia and twitch potentiation after i.v. administration. These effects are mediated by muscarinic as well as alpha-adrenoreceptors and are not affected by adrenalectomy. However, after s.c. administration, both organophosphorous compounds produced a dose-dependent transient hypotension and bradycardia, mediated by muscarinic receptors, without affecting other physiological parameters. The results indicate the route-specific effects of these compounds on the cholinergic-catecholaminergic interactions in rats.

Analysis of the organophosphate-induced electromyographic response to repetitive nerve stimulation: paradoxical response to edrophonium and D-tubocurarine.

We studied the mechanism underlying acute organophosphate intoxication (OPI) through in-vivo and in-vitro electrophysiologic studies in rats injected with diisopropylfluorophosphate. Intoxicated rats showed weakness, repetitive compound muscle action potentials (CMAPs) in response to a single stimulus, and decremental response to repetitive nerve stimulation that was most pronounced at the second CMAP. The decrement was worsened with the administration of edrophonium, and was completely reversed by D-tubocurarine. In-vitro microelectrode studies showed no reduction in the amplitude of miniature endplate potentials (MEPPs) or in the quantal content of end-plate potentials (EPPs). However, the half-decay times of MEPPs and EPPs were significantly prolonged. Trains of stimuli induced sustained end-plate depolarization via a "staircase phenomenon" of summation of prolonged EPPs, which was enhanced by edrophonium and abolished by D-tubocurarine. These results indicate that sustained end-plate depolarization can directly account for the decrement and weakness in acute OPI.