Neuromuscular Transmission Failure Research Articles

Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5days post-injury (dpi); both recovered to uninjured (0dpi) level by 10dpi. At 0dpi, tibialis anterior isometric force of CKO was less than CON. At 10dpi, isometric force was reduced by half in both groups. During intermittent contractions (75Hz, 330mss-1, 120s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

Congenital myasthenic syndrome secondary to pathogenic variants in the SLC5A7 gene: report of two cases

BackgroundCongenital Myasthenic Syndromes (CMS) are rare genetic diseases, which share as a common denominator muscle fatigability due to failure of neuromuscular transmission. A distinctive clinical feature of presynaptic CMS variants caused by defects of the synthesis of acetylcholine is the association with life-threatening episodes of apnea. One of these variants is caused by mutations in the SLC5A7 gene, which encodes the sodium-dependent HC-3 high-affinity choline transporter 1 (CHT1). To our knowledge there are no published cases of this CMS type in Latin America.Case presentationWe present two cases of CHT1-CMS. Both patients were males presenting with repeated episodes of apnea, hypotonia, weakness, ptosis, mild ophthalmoparesis, and bulbar deficit. The first case also presented one isolated seizure, while the second case showed global developmental delay. Both cases, exhibited incomplete improvement with treatment with pyridostigmine.ConclusionsThis report emphasizes the broad incidence of CMS with episodic apnea caused by mutations in the SLC5A7 gene and the frequent association of this condition with serious manifestations of central nervous system involvement.

Neuromuscular junction in amyotrophic lateral sclerosis. Is there more to follow?

Background. Amyotrophic lateral sclerosis (ALS) causes progressive degeneration of upper motor neurons in the cortex, and lower motor neurons in the spine. In addition, it is unclear where motor neuron dysfunction begins and what causes motor neuron degeneration: whether it is the dying forward process or the dying back phenomenon where motor neuron degeneration begins distally at the nerve terminal or at the neuromuscular junction and progresses toward the cell body, is still a matter of debate. Materials and methods. Are there neuromuscular junction disorders in the early stages of ALS? To answer this, we described 5 patients with ALS presented at Petre Sarajishvili Institute of Neurology in 2018–2022, 3 males and 2 females aged 50–61 years. ALS diagnosis was based on clinical signs, the Gold Coast criteria, electromyography (Awaji), neuroimaging, blood and urine tests. At the early stage of the disease, only asymmetric ptosis and diplopia were noted, which did not improve on pyridostigmine or steroids. Results. We studied patients’ anamnesis, physical data, evaluated their mental, cognitive functions and neurological status. We have also interviewed family members, as it was often difficult for the patient to accurately describe the symptoms. Acetylcholine receptor antibodies were mildly positive only in one patient. Thymoma was excluded. The neurophysiological study showed only marked neuromuscular transmission failure in orbicularis oculi, there were no clinical and electromyographic signs of motor neuron damage. Conclusions. Approximately 2 years later, all five patients developed clinical and electromyographic signs of ALS. In the present study, neuromuscular junction disorders are found to play an important role in the pathogenesis of ALS and may serve as a useful early diagnostic marker.

Development of a Biosensor to Detect Venom of Malayan Krait (Bungarus candidus).

Malayan krait (Bungarus candidus) envenoming is a cause of significant morbidity and mortality in many Southeast Asian countries. If intubation and specific antivenom administration are delayed, the most significant life-threatening outcome may be the inhibition of neuromuscular transmission and subsequent respiratory failure. It is recommended that krait-envenomed victims without indications of neurotoxicity, e.g., skeletal muscle weakness or ptosis, immediately receive 10 vials of antivenom. However, the administration of excess antivenom may lead to hypersensitivity or serum sickness. Therefore, monitoring venom concentrations in patients could be used as an indicator for snake antivenom treatment. In this study, we aimed to develop a screen-printed gold electrode (SPGE) biosensor to detect B. candidus venom in experimentally envenomed rats. The gold electrodes were coated with monovalent Malayan krait IgG antivenom and used as venom detection biosensors. Electrochemical impedance spectrometry (EIS) and square wave voltammetry (SWV) measurements were performed to detect the electrical characterization between B. candidus venom and monovalent IgG antivenom in the biosensor. The EIS measurements showed increases in charge transfer resistance (Rct) following IgG immobilization and incubation with B. candidus venom solution (0.1-0.4 mg/mL); thus, the antibody was immobilized on the electrode surface and venom was successfully detected. The lowest current signal was detected by SWV measurement in rat plasma collected 30 min following B. candidus experimental envenoming, indicating the highest level of venom concentration in blood circulation (4.3 ± 0.7 µg/mL). The present study demonstrates the ability of the SPGE biosensor to detect B. candidus venom in plasma from experimentally envenomed rats. The technology obtained in this work may be developed as a detection tool for use along with the standard treatment of Malayan krait envenoming.

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O2)-hypercapnia (5% CO2) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdimax). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H2O), hypoxia-hypercapnia (∼10 cm H2O), or sigh (∼36 cm H2O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdimax was 61 and 46 cm H2O, respectively. Chloroquine decreased Pdimax by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdimax suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process.NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Neuromuscular junction transmission failure in aging and sarcopenia: The nexus of the neurological and muscular systems.

Sarcopenia, or age-related decline in muscle form and function, exerts high personal, societal, and economic burdens when untreated. Integrity and function of the neuromuscular junction (NMJ), as the nexus between the nervous and muscular systems, is critical for input and dependable neural control of muscle force generation. As such, the NMJ has long been a site of keen interest in the context of skeletal muscle function deficits during aging and in the context of sarcopenia. Historically, changes of NMJ morphology during aging have been investigated extensively but primarily in aged rodent models. Aged rodents have consistently shown features of NMJ endplate fragmentation and denervation. Yet, the presence of NMJ changes in older humans remains controversial, and conflicting findings have been reported. This review article describes the physiological processes involved in NMJ transmission, discusses the evidence that supports NMJ transmission failure as a possible contributor to sarcopenia, and speculates on the potential of targeting these defects for therapeutic development. The technical approaches that are available for assessment of NMJ transmission, whether each approach has been applied in the context of aging and sarcopenia, and the associated findings are summarized. Like morphological studies, age-related NMJ transmission deficits have primarily been studied in rodents. In preclinical studies, isolated synaptic electrophysiology recordings of endplate currentsor potentials have been mostly used, and paradoxically, have shown enhancement, rather than failure, with aging. Yet, in vivo assessment of single muscle fiber action potential generation using single fiber electromyography and nerve-stimulated muscle force measurements show evidence of NMJ failure in aged mice and rats. Together these findings suggest that endplate response enhancement may be a compensatory response to post-synaptic mechanisms of NMJ transmission failure in aged rodents. Possible, but underexplored, mechanisms of this failure are discussed including the simplification of post-synaptic folding and altered voltage-gated sodium channel clustering or function. In humans, there is limited clinical data that has selectively investigated single synaptic function in the context of aging. If sarcopenic older adults turn out to exhibit notable impairments in NMJ transmission (this has yet to be examined but based on available evidence appears to be plausible) then these NMJ transmission defects present a well-defined biological mechanism and offer a well-defined pathway for clinical implementation. Investigation of small molecules that are currently available clinically or being testing clinically in other disorders may provide a rapid route for development of interventions for older adults impacted by sarcopenia.

Motor neuron loss and neuromuscular transmission failure following chemogenetic inhibition of BDNF/TrkB signaling in adult rats

Brain derived neurotrophic factor (BDNF) signaling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. However, the specific effects of reduced BDNF/TrkB signaling on neuromuscular structure and function in adult mammals are not as well known. In this study, we employed a novel 1NMPP1 sensitive TrkBF616 rat model to evaluate the effect of 14 days inhibition of BDNF/TrkB signaling on the number and morphology of phrenic motor neurons (PhMNs) and diaphragm muscle (DIAm) neurotransmission in adult animals. We hypothesized that reduced BDNF/TrkB signaling results in PhMN loss and impaired DIAm neuromuscular transmission. Adult female and male TrkBF616 rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were labeled via intrapleural injection of Alexa-Fluor647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor488 CTB. After 14 days of treatment, the spinal cord was excised and 100 μm thick spinal sections containing PhMNs were imaged in 3D using two-channel confocal microscopy. Phrenic nerve and DIAm was excised en bloc, and the extent of neuromuscular transmission failure (NMTF) was assessed by comparing DIAm isometric forces evoked by nerve (40 Hz in 330 ms duration trains repeated each s) compared to direct muscle stimulation (40 Hz 330 ms duration train) superimposed every 15 s. Presynaptic (synaptophysin immunoreactivity) and postsynaptic (α-Bungarotoxin) compartments of neuromuscular junctions (NMJs) were labeled and imaged in 3D by confocal microscopy and the extent of pre- and postsynaptic apposition assessed. 1NMPP1-induced BDNF/TrkB inhibition reduced the total number of PhMNs by ~16%, reduced the mean PhMN somal surface areas by ~25%, reduced PhMN dendritic surface area by ~38%, impaired secondary CTB uptake by 2.5-fold and impaired neuromuscular transmission by ~33%, with no overt evidence of NMJ derangement. We conclude that inhibition of BDNF/TrkB signaling in adult TrkBF616 rats is sufficient to lead to PhMN loss, morphological degeneration, deficits in retrograde axonal transport and impaired DIAm neurotransmission. Supported by NIH: R01-AG44615 and R01-HL146114 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inducible deletion of endothelial cell EfnB2 attenuates neuromuscular regeneration in mouse skeletal muscle

Background: Acute injury of skeletal muscle disrupts myofibers, microvessels and neuromuscular junctions (NMJs). While myofiber regeneration is well-characterized, little is known of concurrent events in microvessels and motor nerves following injury. Endothelial cell [EC] ephrin B2 [EfnB2] is required for sprouting angiogenesis during embryonic development, for promoting vascularization of tumors in the adult, and for microvascular-dependent guidance of axon regeneration following nerve resection. Hypothesis: We hypothesized that following acute injury to skeletal muscle 1) EC EfnB2 is required for restoring microvascular volume; and 2) loss of EC EfnB2 would impair recovery of neuromuscular structure and function. Methods: Mice (male and female, age 3-5 months) were bred for EC-specific conditional knockout [CKO] of EfnB2 following tamoxifen injection; noninjected CKO mice served as “wildtype” controls [WT]. Following injury by local injection of the myotoxin BaCl2, the gluteus maximus [GM] muscle was studied for microvascular analysis or the tibialis anterior [TA] muscle was evaluated for recovery of neuromuscular function. Mice were studied (n=4-6/group) at 3 timepoints for vascular studies (0, 5, and 10 days post injury [dpi]) and 2 timepoints for neuromuscular structure and function (0 and 10 dpi). Results: Intravascular staining with fluorescent wheat germ agglutinin revealed a diminished microvascular area in the GM of CKO mice at 5 dpi vs. 10 dpi (means ± SE; CKO 5 dpi 35.5% ± 2.5 vs. CKO 10 dpi 49.8% ± 3.9, P&lt;0.01). No difference in vascular area was recorded in WT groups at 5 vs. 10 dpi (WT 5 dpi 45.5% ± 2.6 vs. WT 10 dpi 46.7% ± 2.2, P=0.99). Direct electrical stimulation of the TA resolved a reduction in maximal force at 10 dpi when compared to 0 dpi for both WT and CKO (WT 0 dpi 128.6 g ± 4.1 vs. WT 10 dpi 64.7 g ± 3.0, P&lt;0.0001) (CKO 0 dpi 112.7 g ± 5.0 vs. CKO 10 dpi 65.9 g ± 5.0, P&lt;0.0001). Indirect stimulation (sciatic nerve) resolved initial neuromuscular transmission failure (INMTF) reduced submaximal force in CKO but not WT (CKO 0 dpi 2.0 ± 4.3% vs. CKO 10 dpi -30.0 ± 6.5 %, P&lt;0.001), which correlated with perturbed recovery of NMJ innervation; (R2=0.328, P=0.003), endplate area; (R2=0.479, P=0.001), and nicotinic receptor clusters; (R2=0.207, P=0.025). Conclusion: Following acute injury of skeletal muscle in the adult mouse, loss of EfnB2 in ECs delays microvessel regeneration in the GM and disrupts the recovery of NMJ structure and function in the TA. Support: NIH grant F32 HL-152558 (NLJ), APS Postdoctoral Fellowship (ABM), NIH LRP (ABM), Margaret Proctor Mulligan Fellowship, MU (NLJ, SSS), NIH R01 AR078045 (DDC), School of Medicine Development Award, MU (SSS), NIH MERIT Award R37 HL-041026 (SSS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute inhibition of autophagy impairs maximal diaphragm force generation in old mice

Transdiaphragmatic pressure (Pdi) measurements are used to determine diaphragm force generation across various motor behaviors. Previous studies in the diaphragm muscle found selective age-related changes at fast-twitch fatigable motor units including increased susceptibility to neuromuscular transmission failure and denervation. Accordingly, age-related changes at diaphragm motor units predominantly impair the ability to accomplish higher force behaviors, and these aging effects are evident prior to the onset of muscle atrophy. Recent findings suggest that autophagy plays a critical role in the development of age-related neuromuscular dysfunction; however, the possible contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. We hypothesized that inhibition of autophagy (using chloroquine) decreases diaphragm force-generating capacity (Pdi) in mice susceptible to age-related neuromuscular dysfunction. Adult male and female C57BL/6 mice at 16-28 months of age were randomized to receive a single intraperitoneal injection of 50 mg/kg of chloroquine or vehicle (n= 9 per treatment group) four hours before Pdi measurements, with the experimenter blinded. Pdi was measured during eupnea (room air breathing), exposure to hypoxia/hypercapnia (10% O 2 /5% CO 2 ), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Consistent with our hypothesis, there was an ~25% reduction in maximal Pdi in the chloroquine group (46 ± 12 cm H 2 O) compared to the vehicle control (61 ± 16 cm H 2 O; p &lt; 0.05). There was no effect of chloroquine on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. Acute inhibition of autophagy results in reduced maximal Pdi, consistent with that evident in old mice. Thus, we propose that autophagy impairment may be an important contributor to the reduced diaphragm force generation associated with aging. These findings help us further understand the mechanisms regulating age-related neuromuscular dysfunction and may serve as a foundation to develop novel treatments to mitigate aging effects. Support: NIH R01 AG057052. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Brain derived neurotrophic factor/tropomyosin related kinase B signaling impacts diaphragm neuromuscular transmission in a novel rat chemogenetic model.

The neuromuscular junction (NMJ) mediates neural control of skeletal muscle fibers. Neurotrophic signaling, specifically brain derived neurotrophic factor (BDNF) acting through its high-affinity tropomyosin related kinase B (TrkB) receptor is known to improve neuromuscular transmission. BDNF/TrkB signaling also maintains the integrity of antero- and retrograde communication between the motor neuron soma, its distal axons and pre-synaptic terminals and influences neuromuscular transmission. In this study, we employed a novel rat chemogenetic mutation (TrkB F616), in which a 1-naphthylmethyl phosphoprotein phosphatase 1 (1NMPP1) sensitive knock-in allele allowed specific, rapid and sustained inhibition of TrkB kinase activity. In adult female and male TrkB F616 rats, treatment with either 1NMPP1 (TrkB kinase inhibition) or DMSO (vehicle) was administered in drinking water for 14 days. To assess the extent of neuromuscular transmission failure (NMTF), diaphragm muscle isometric force evoked by nerve stimulation at 40 Hz (330 ms duration trains repeated each s) was compared to isometric forces evoked by superimposed direct muscle stimulation (every 15 s). Chronic TrkB kinase inhibition (1NMPP1 group) markedly worsened NMTF compared to vehicle controls. Acute BDNF treatment did not rescue NMTF in the 1NMPP1 group. Chronic TrkB kinase inhibition did not affect the apposition of pre-synaptic terminals (labeled with synaptophysin) and post-synaptic endplates (labeled with α-Bungarotoxin) at diaphragm NMJs. We conclude that inhibition of BDNF/TrkB signaling in TrkB F616 rats disrupts diaphragm neuromuscular transmission in a similar manner to TrkB F616A mice, likely via a pre-synaptic mechanism independent of axonal branch point failure.

Practical Management for Use of Eculizumab in the Treatment of Severe, Refractory, Non-Thymomatous, AChR + Generalized Myasthenia Gravis: A Systematic Review.

Myasthenia gravis (MG) is a rare autoimmune disorder caused by specific autoantibodies at the neuromuscular junction. MG is classified by the antigen specificity of these antibodies. Acetylcholine receptor (AChR) antibodies are the most common type (74–88%), followed by anti-muscle specific kinase (MuSK) and other antibodies. While all these antibodies lead to neuromuscular transmission failure, the immuno-pathogenic mechanisms are distinct. Complement activation is a primary driver of AChR antibody-positive MG (AChR+ MG) pathogenesis. This leads to the formation of the membrane attack complex and destruction of AChR receptors and the postsynaptic membrane resulting in impaired neurotransmission and muscle weakness characteristic of MG. Broad-based immune-suppressants like corticosteroids are effective in controlling MG; however, their long-term use can be associated with significant adverse effects. Advances in translational research have led to the development of more directed therapeutic agents that are likely to alter the future of MG treatment. Eculizumab is a humanized monoclonal antibody that inhibits the cleavage of complement protein C5 and is approved for use in generalized MG. In this review, we discuss the pathophysiology of MG; the therapeutic efficacy and tolerability of eculizumab, as well as the practical guidelines for its use in MG; future studies exploring the role of eculizumab in different stages and subtypes of MG subtypes; the optimal duration of therapy and its discontinuation; the characterization of non-responder patients; and the use of biomarkers for monitoring therapy are highlighted. Based on the pathophysiologic mechanisms, emerging therapies and new therapeutic targets are also reviewed.

HLA-DQB1*05:02, *05:03, and *03:01 alleles as risk factors for myasthenia gravis in a Spanish cohort.

Myasthenia gravis (MG) is a very heterogenic chronic autoimmune disease caused by the failure of neuromuscular transmission. The HLA gene complex has conventionally been recognized as its main genetic risk and phenotype modifying factor. Our aim was to investigate the prevalence of HLA class I and II alleles and to identify possible risk factors for sporadic MG in a Spanish cohort. We designed a clinical case-control study comparing HLA alleles and haplotype frequencies in a cohort of 234 patients with sporadic autoimmune MG with data from a group of 492 randomly selected healthy subjects. Using a high-resolution next-generation sequencing (NGS)-based HLA genotyping assay, we investigated the contribution of HLA genotypes and haplotypes in the resulting phenotype, especially, the age at onset, sex, onset MGFA class, thymic histopathology, and serological status. We found that the DQB1*05:02 and DQB1*05:03 alleles could be novel risk factors for Spanish MG cases. The HLA alleles A*01:01, B*08:01, DRB1*03:01, DRB1*14:54, and DQB1*02:01 were also risk factors for the disease. DQB1*03:01 acted as a risk factor for EOMG in women with AChR-positive antibodies and thymus hyperplasia. Additionally, several alleles were identified as potential phenotype-modifying factors that could exert a protective effect: HLA-B*35:08, DRB1*13:01, and DQB1*06:03 in MG; HLA-A*24:02 in women and DRB1*07:01 and DQB1*02:02 for early onset. HLA-C*07:01 and haplotype A1-B8-C7-DR3-DQ2 were associated with an early-onset phenotype.

The Safety Factor for Neuromuscular Transmission: Effects of Dimethylsulphoxide, Cannabinoids and Synaptic Homeostasis.

BackgroundIn myasthenia gravis, impaired postsynaptic sensitivity to acetylcholine results in failure of neuromuscular transmission and fatiguing muscle weakness.ObjectiveDevelop an ex vivo muscle contraction assay to test cannabinoids and other substances that might act on the myasthenic neuromuscular junction to restore control of the muscle.MethodsTubocurarine was added to an ex vivo, mouse phrenic nerve-hemidiaphragm muscle preparation to reduce acetylcholine sensitivity. This produced a myasthenia-like decrement in twitch force during a train of 10 nerve impulses (3 / sec). Endplate potential (EPP) recordings were used to confirm and extend the findings.ResultsSurprisingly, addition to the bath of dimethylsulphoxide (DMSO), at concentrations as low as 0.1%(v/v), partially reversed the decrement in nerve-evoked force. Intracellular electrophysiology, conducted in the presence of tubocurarine, showed that DMSO increased the amplitudes of both the spontaneous miniature EPP (MEPP) and the (nerve-evoked) EPP. In the absence of tubocurarine (synaptic potentials at physiological levels), an adaptive fall in quantal content negated the DMSO-induced rise in EPP amplitude. The effects of cannabinoid receptor agonists (solubilized with DMSO) in the contraction assay do not support their further exploration as useful therapeutic agents for myasthenia gravis. CP 55,940 (a dual agonist for cannabinoid receptor types 1 and 2) reversed the beneficial effects of DMSO.Conclusions:We demonstrate a powerful effect of DMSO upon quantal amplitude that might mislead pharmacological studies of synaptic function wherever DMSO is used as a drug vehicle. Our results also show that compounds targeting impaired neuromuscular transmission should be tested under myasthenic-like conditions, so as to avoid confounding effects of synaptic homeostasis.

WS3.7. Repetitive Nerve Stimulation Testing

Repetitive nerve stimulation (RNS) is the most frequently used electrodiagnostic test to diagnose neuromuscular junction disorders. Although seemingly simple, attention to technique is essential to avoid technical errors. Stimulation at low rates (2–5 Hz) (LF-RNS) depletes acetylcholine stores at diseased motor endplates, causing failure of neuromuscular transmission. A decrement of at least 10% to trains of 2-3 Hz stimulation at rest or immediately after a period of maximal muscle contraction (“activation”) is the usual criterion of abnormality; 5% can be used if technical artifacts can be excluded. Take care to deliver adequate stimulation and prevent movement of the tested muscle throughout nerve stimulation; peripheral muscles should be kept warm during testing. RNS is reportedly abnormal in up to 90% of generalized myasthenia gravis (MG) patients and in up to 60% of ocular myasthenia – sensitivity is strongly dependent on the muscles selected for testing. RNS is more often abnormal in proximal, facial and clinically weak muscles; even so, RNS is frequently normal in mild or purely ocular disease. For maximal diagnostic yield, test several muscles, including those that are weak. The characteristic findings in Lambert-Eaton myasthenia (LEM) are: small CMAPs at rest, a decrement to LF-RNS, and marked facilitation. In MG, activation for 30–60 seconds is used to unmask a mild decrement. In LEM, activation is used to demonstrate facilitation; the simplest procedure is to elicit a single CMAP from the rested muscle, have the patient contract the muscle for 10 seconds, then immediately elicit a single CMAP. Facilitation of at least 60% is evidence of a pre-synaptic disorder, but may not be seen in all muscles. A decrement in at least one hand muscle is the most frequent RNS abnormality in LEM and may be seen even when CMAPs are not small and diagnostic facilitation cannot be demonstrated. In MG the decrement is usually greatest in the 4th or 5th response and is less during subsequent responses, producing a so-called ‘‘U-shaped” pattern. A continually progressive decrement is usually seen in LEM. The pattern in MuSK MG resembles that in LEM.

Impact of obstructive sleep apnea on neuromuscular transmission- a descriptive study

ABSTRACT Objective: Obstructive sleep apnea (OSA) is a sleep disorder accompanied by intermittent hypoxia. Neuromuscular transmission (NT) is known to be disturbed under chronic hypoxia. In this descriptive study, it has been aimed to test NT under intermittent hypoxia in OSA. Methods: Thirty-nine newly diagnosed OSA patients without any comorbidities or conditions that alter NT were included in the study. Jitter analysis was performed using a concentric needle electrode. Results: The mean jitter value of 39 OSA patients was 25.9 ± 3.7 μs. When compared to the mean reference jitter values, patients in the present study had significantly higher jitter (p < 0.001). Seven (17.9%) patients met the electrophysiological criteria for NT failure. Conclusion: The authors propose that intermittent hypoxia can be the trigger for NT failure in OSA. The interaction between increased oxidative stress and disturbed mitochondrial functions may also contribute.

Myasthenia gravis and COVID-19: a systematic review of case reports and case series

Myasthenia gravis (MG) is an autoimmune disease involving neuromuscular transmission and possible respiratory failure when concomitant with COVID-19. The aim of this study was to analyze the need for ventilatory support (VS), length of hospital stay (LOS) and mortality in patients diagnosed with MG and COVID-19. In this systematic review, PubMed, SciELO, LILACS, MEDLINE and IBECS databases were searched for primary studies published from January 2010 to March 2021, with no language restrictions. Fourteen eligible studies were identified. The main factor associated with the need for VS was the use of antibiotics other than azithromycin (AZM) for the treatment of COVID-19 (RR 1.60; 95% CI 1.20–2.91; p = 0.009). Patients who used hydroxychloroquine (HCQ) and AZM had almost twice the risk of needing invasive ventilatory support (IVS) (RR 1.94; 95% CI 1.07-3.52; p = 0.16). There were nonsignificant trends towards less need for IVS in patients who used intravenous immunoglobulin (IVIg) and corticosteroid therapy (RR 0.54; 95% CI 0.09–3.26; p = 0.60). There was a trend towards shorter LOS in patients who received therapy with IVIg and corticosteroid therapy [8 (5 - 8) vs 19 (12.2–23.7); p = 0.007]. 10.3% (n = 4/39) died and 100% did not use IVIg or IVIg and prednisone. There was a non-significant trend towards higher mortality in patients who used AZM (RR 2.55; 95% CI 0.26–30.02; p = 0.60). IVIg and corticotherapy presented themselves as a favorable alternative in relation to the outcomes.KEY WORDS: Coronavirus infections; length of stay; Myasthenia gravis; Respiratory insufficiency.

Congenital Disruption of Glycinergic Signaling Results in Fewer Phrenic Motor Neurons and Impaired Diaphragm Neuromotor Control

Glycine (Gly) is a key inhibitory neurotransmitter at phrenic motor neurons (PhMN). In development, Gly is excitatory, transitioning to inhibitory action in the postnatal period. In adult conditions such as amyotrophic lateral sclerosis (ALS), excessive excitatory signaling onto motor neurons results in excitotoxic motor neuron death. However, it is not known how an imbalance of neurotransmission early in development impacts PhMNs at maturity. This is a knowledge gap, as some congenital hypertonia conditions exhibit an imbalance in neurotransmitter signaling, with these individuals suffering significant morbidity and mortality from respiratory disorders. Using an animal model of congenital hypertonia (spa mouse [B6.Cg-Glrbspa/J] with a Gly receptor mutation), we evaluated PhMN and motor unit physiology. Similar to previous work in other Gly mutants, we hypothesized that spa mice will have excessive developmental PhMN loss with preserved PhMNs having a smaller somal size compared to wildtype (WT) mice. We also hypothesized that fewer PhMNs in spa mice will result in an increased diaphragm (DIAm) innervation ratio (number of DIAm fibers innervated by a PhMN) and increased DIAm neuromuscular transmission failure (NMTF). Spa and WT mice from our colony, ages 6 to 16 weeks, underwent unilateral retrograde labeling of PhMNs via rhodamine phrenic nerve dip. After three days, mice were euthanized, perfused with 4% paraformaldehyde, cervical spinal cord excised and processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging. Absolute PhMN counts were obtained using mosaic images of adjacent serial sections. Somal size measurements (i.e. surface area) were obtained using ImageJ. DIAm force was determined from DIAm strip preparations stretched to optimal length, and force was evoked by phrenic nerve stimulation at 40 Hz in 330 ms duration trains repeated each s (33% duty cycle) for 120 s. To assess NMTF, force evoked by phrenic nerve stimulation was compared to force evoked by direct DIAm stimulation superimposed every 15 s. Total DIAm fiber number was estimated in H&E stained cross-sections. Spa mice had 30% fewer PhMNs compared to WT (P=0.005) with somal size being 22% smaller in spa mice (P=0.019). Spa mice had 20% NMTF during the initial stimulus train compared to 8% in WT mice with spa mice having 82% NMTF and WT having 40% NMTF at end of stimulation. Spa mice had a 39% greater innervation ratio of DIAm motor units compared to WT. Developmental PhMN loss and somal size is significantly disrupted in congenital hypertonia due to impaired Gly signaling and may impact performance of high-force expulsive DIAm activities. In addition, the increased innervation ratio of DIAm motor units in spa mice, likely contributes to the NMTF due to branch point failure. Overall, this results in less effective airway defense and straining behaviors in congenital hypertonia. Our next step is to identify crucial developmental milestones when excessive PhMN loss occurs and the impact on large versus small PhMNs.

Colonic Neuromuscular Transmission Failure in Female Rats after Spinal Cord Injury

Introduction Along with lost mobility, spinal cord injury (SCI) impacts visceral control of the autonomic nervous system; eliciting critical challenges to quality of life. Gastrointestinal (GI) complaints in the SCI population include decreased colonic motility and subsequent disruptions in evacuation. Although SCI damages the central nervous system, the enteric nervous system located intrinsic to the GI tract is not damaged by the initial trauma. The enteric neuromuscular control of colonic slow-wave propagation is a balance of excitatory and inhibitory inputs to smooth muscle cells that are recorded electrophysiologically as junction potentials (EJPs and IJPs, respectively). IJPs consist of a fast (purinergic; fIJP) and subsequent slow (nitrergic; sIJP) component. Our previous research has demonstrated diminished EJPs and sIJPs in male SCI rats. The higher prevalence of GI abnormalities in females underscores the differential control of motility in health and disease. This study aims to expand our mechanistic understanding of colonic dysmotility in female rat models of acute SCI. Methods Female rats received a surgical control or severe T3 contusion SCI. After 3 days recovery, ex vivo distal colon samples of the circular and longitudinal smooth muscle were dissected for electrophysiological recording and immunohistochemical analysis. For electrophysiology experiments, smooth muscle cells were recorded in response to pharmacologic treatments that isolate the EJPs and IJP components. Results Our data reveals that, in contrast to comparable regions of male rats’ distal colon, female rats have minimal sIJP (i.e., nitrergic) responses. With no significant difference between acute control and SCI responses, this suggests that female neuromuscular transmission is less dependent on nitrergic signaling. Control female sIJPs are significantly smaller than control male rats and resemble the sIJPs in SCI male rats. The resting membrane potential and fIJP component appears unchanged by SCI, with values consistent to male rats. Conclusion All of the current data illustrates sex-differences in colonic physiology and pathophysiology after SCI. Addressing gender differences would improve clinical post-injury care for these individuals. Significance To date, the mechanism for colon dysmotility after SCI remains uncertain and this project provides novel insight to female neurogenic bowel. The colonic neuromuscular junction is the final common pathway for proper motor function and abnormalities have implications for peripheral and central neurostimulation therapies. Our observed differences from neuromuscular transmission in the male rat, reinforces the importance of developing gender specific treatment. Future studies will evaluate female EJP dysfunction and specific agonist pharmacodynamics.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Diaphragm neuromuscular transmission failure in a mouse model of an early-onset neuromotor disorder.

The spa transgenic mouse displays spasticity and hypertonia that develops during the early postnatal period, with motor impairments that are remarkably similar to symptoms of human cerebral palsy. Previously, we observed that spa mice have fewer phrenic motor neurons innervating the diaphragm muscle (DIAm). We hypothesize that spa mice exhibit increased susceptibility to neuromuscular transmission failure (NMTF) due to an expanded innervation ratio. We retrogradely labeled phrenic motor neurons with rhodamine and imaged them in horizontal sections (70 µm) using confocal microscopy. Phrenic nerve-DIAm strip preparations from wild type and spa mice were stretched to optimal length, and force was evoked by phrenic nerve stimulation at 10, 40, or 75 Hz in 330-ms duration trains repeated each second (33% duty cycle) across a 120-s period. To assess NMTF, force evoked by phrenic nerve stimulation was compared to force evoked by direct DIAm stimulation superimposed every 15 s. Total DIAm fiber number was estimated in hematoxylin and eosin-stained strips. Compared to wild type, spa mice had over twofold greater NMTF during the first stimulus train that persisted throughout the 120 s period of repetitive activation. In both wild type and spa mice, NMTF was stimulation-frequency dependent. There was no difference in neuromuscular junction morphology or the total number of DIAm fibers between wild type and spa mice, however, there was an increase innervation ratio (39%) in spa mice. We conclude that early-onset developmental neuromotor disorders impair the efficacy of DIAm neuromuscular transmission, likely to contribute to respiratory complications.NEW & NOTEWORTHY Individuals with motor control deficits, including cerebral palsy (CP) often have respiratory impairments. Glycine-receptor mutant spa mice have early-onset hypertonia, and limb motor impairments, similar to individuals with CP. We hypothesized that in the diaphragm of spa mice, disruption of glycinergic inputs to MNs would result in increased phrenic-DIAm neuromuscular transmission failure. Pathophysiologic abnormalities in neuromuscular transmission may contribute to respiratory dysfunction in conditions where early developmental MN loss or motor control deficits are apparent.