Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity.

Introduction: Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery. Methods and results: Mice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size. Discussion: In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.

Antidotal treatment of botulism in rats by continuous infusion with 3,4-diaminopyridine.

Botulinum neurotoxins (BoNTs) are highly potent, select agent toxins that inhibit neurotransmitter release at motor nerve terminals, causing muscle paralysis and death by asphyxiation. Other than post-exposure prophylaxis with antitoxin, the only treatment option for symptomatic botulism is intubation and supportive care until recovery, which can require weeks or longer. In previous studies, we reported the FDA-approved drug 3,4-diaminopyridine (3,4-DAP) reverses early botulism symptoms and prolongs survival in lethally intoxicated mice. However, the symptomatic benefits of 3,4-DAP are limited by its rapid clearance. Here we investigated whether 3,4-DAP could sustain symptomatic benefits throughout the full course of respiratory paralysis in lethally intoxicated rats. First, we confirmed serial injections of 3,4-DAP stabilized toxic signs and prolonged survival in rats challenged with 2.5 LD50 BoNT/A. Rebound of toxic signs and death occurred within hours after the final 3,4-DAP treatment, consistent with the short half-life of 3,4-DAP in rats. Based on these data, we next investigated whether the therapeutic benefits of 3,4-DAP could be sustained throughout the course of botulism by continuous infusion. To ensure administration of 3,4-DAP at clinically relevant doses, three infusion dose rates (0.5, 1.0 and 1.5 mg/kg∙h) were identified that produced steady-state serum levels of 3,4-DAP consistent with clinical dosing. We then compared dose-dependent effects of 3,4-DAP on toxic signs and survival in rats intoxicated with 2.5 LD50 BoNT/A. In contrast to saline vehicle, which resulted in 100% mortality, infusion of 3,4-DAP at ≥ 1.0 mg/kg∙h from 1 to 14 d after intoxication produced 94.4% survival and full resolution of toxic signs, without rebound of toxic signs after infusion was stopped. In contrast, withdrawal of 3,4-DAP infusion at 5 d resulted in re-emergence of toxic sign and death within 12 h, confirming antidotal outcomes require sustained 3,4-DAP treatment for longer than 5 d after intoxication. We exploited this novel survival model of lethal botulism to explore neurophysiological parameters of diaphragm paralysis and recovery. While neurotransmission was nearly eliminated at 5 d, neurotransmission was significantly improved at 21 d in 3,4-DAP-infused survivors, although still depressed compared to naïve rats. 3,4-DAP is the first small molecule to reverse systemic paralysis and promote survival in animal models of botulism, thereby meeting a critical treatment need that is not addressed by post-exposure prophylaxis with conventional antitoxin. These data contribute to a growing body of evidence supporting the use of 3,4-DAP to treat clinical botulism.

Refractory and Super-Refractory Status Epilepticus in Nerve Agent-Poisoned Rats Following Application of Standard Clinical Treatment Guidelines.

Nerve agents (NAs) induce a severe cholinergic crisis that can lead to status epilepticus (SE). Current guidelines for treatment of NA-induced SE only include prehospital benzodiazepines, which may not fully resolve this life-threatening condition. This study examined the efficacy of general clinical protocols for treatment of SE in the specific context of NA poisoning in adult male rats. Treatment with both intramuscular and intravenous benzodiazepines was entirely insufficient to control SE. Second line intervention with valproate (VPA) initially terminated SE in 35% of rats, but seizures always returned. Phenobarbital (PHB) was more effective, with SE terminating in 56% of rats and 19% of rats remaining seizure-free for at least 24 h. The majority of rats demonstrated refractory SE (RSE) and required treatment with a continuous third-line anesthetic. Both ketamine (KET) and propofol (PRO) led to high levels of mortality, and nearly all rats on these therapies had breakthrough seizure activity, demonstrating super-refractory SE (SRSE). For the small subset of rats in which SE was fully resolved, significant improvements over controls were observed in recovery metrics, behavioral assays, and brain pathology. Together these data suggest that NA-induced SE is particularly severe, but aggressive treatment in the intensive care setting can lead to positive functional outcomes for casualties.

Functional basis for dose-dependent antagonism of rat and rabbit neuromuscular transmission by the bis-pyridinium oxime MMB4.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Corneal Endothelial Cell Toxicity Determines Long-Term Outcome After Ocular Exposure to Sulfur Mustard Vapor.

PURPOSE: Ocular exposure to sulfur mustard (SM) vapor causes acute loss of corneal endothelial cells (CECs). Persistent corneal endothelial pathologies are observed in eyes that do not recover from SM exposure, suggesting that endothelial toxicity contributes to mustard gas keratopathy (MGK). Here, we evaluated the contributions of endothelial loss to acute and chronic corneal injuries in SM-exposed eyes. METHODS: Rabbit eyes were exposed in vivo to equivalent doses of SM using 9-, 11-, or 14-mm vapor caps. The effects of exposure area on corneal injury progression were longitudinally evaluated over 12 weeks using clinical evaluations. The effects of exposure area on CEC morphology, endothelial and epithelial ultrastructure, and endothelial barrier function were determined from 1 day to 12 weeks. RESULTS: SM exposure caused loss of CECs and failure of endothelial barrier integrity at 1 day, independent of exposure cap size. By 3 weeks, eyes exposed with the 14-mm vapor cap exhibited increased corneal permeability, repopulation of the endothelium by cells with fibroblastic morphology, and abnormal deposition of extracellular matrix. Eyes exposed with 9- or 11-mm vapor caps exhibited transient symptoms of injury that fully resolved, with the rate of recovery correlated with cap size. CONCLUSIONS: The nonlinear correlation between endothelial lesion size and probability of developing MGK suggests that the CEC loss is a determinative factor for emergence of MGK. These studies illustrate the importance of endothelial repair in preventing MGK. Furthermore, they exclude chemical modification of basement membrane as a mechanistic cause of recurrent epithelial erosions in MGK eyes.