Expert consensus on the combined investigation of carpal tunnel syndrome with electrodiagnostic tests and neuromuscular ultrasound.

Expert consensus was sought to guide clinicians on the use of electrodiagnostic tests (EDX) and neuromuscular ultrasound (NMUS) in the investigation of suspected carpal tunnel syndrome (CTS). Consensus was achieved using the Delphi method via three consecutive anonymised surveys of 15 experts and was defined as rating agreement ≥ 80%. The panel agreed that combining EDX and NMUS is more informative than using each modality alone. NMUS adds value in patients with clinically suspected CTS with non-localizing or normal EDX, atypical EDX, failed CTS surgery, polyneuropathy, and CTS suspected to be secondary to structural pathology. The median nerve cross-sectional area should be measured at the site of maximal nerve enlargement, and the nerve should be scanned from mid-forearm to the palm. The group also identified those situations where the wrist-to-forearm area ratio and longitudinal scans of the median nerve should also be obtained. EDX should always be performed to quantify CTS severity and in individuals over age 70. This document is an initial step to guide clinicians on the combined investigation of CTS using EDX and NMUS, to be updated regularly with the emergence of new research.

Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin.

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066-1315 (emGFP-1066:TetC), 1093-1315 (emGFP-1093:TetC) and 1109-1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

Protocols for electrophysiological recordings and electron microscopy at C. elegans neuromuscular junction.

Release of neurotransmitters by synaptic vesicle exocytosis at presynaptic terminals is critical for neuronal communication within the nervous system. Electrophysiology and electron microscopy are powerful and complementary approaches used to evaluate the function of synaptic proteins in synaptic transmission. Here, we provide a protocol detailing the use of these two approaches at C. elegans neuromuscular junctions, including steps for worm picking and dissection, in vivo electrophysiological recording, and sample preparation for electron microscopy, followed by imaging and analysis. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021) and Li et al. (2021).

Minutes-timescale 3D isotropic imaging of entire organs at subcellular resolution by content-aware compressed-sensing light-sheet microscopy.

Rapid 3D imaging of entire organs and organisms at cellular resolution is a recurring challenge in life science. Here we report on a computational light-sheet microscopy able to achieve minute-timescale high-resolution mapping of entire macro-scale organs. Through combining a dual-side confocally-scanned Bessel light-sheet illumination which provides thinner-and-wider optical sectioning of deep tissues, with a content-aware compressed sensing (CACS) computation pipeline which further improves the contrast and resolution based on a single acquisition, our approach yields 3D images with high, isotropic spatial resolution and rapid acquisition over two-order-of-magnitude faster than conventional 3D microscopy implementations. We demonstrate the imaging of whole brain (~400 mm3), entire gastrocnemius and tibialis muscles (~200 mm3) of mouse at ultra-high throughput of 5~10 min per sample and post-improved subcellular resolution of ~ 1.5 µm (0.5-µm iso-voxel size). Various system-level cellular analyses, such as mapping cell populations at different brain sub-regions, tracing long-distance projection neurons over the entire brain, and calculating neuromuscular junction occupancy across whole muscle, are also readily accomplished by our method.

Salbutamol modifies the neuromuscular junction in a mouse model of ColQ myasthenic syndrome.

The ß-adrenergic agonists salbutamol and ephedrine have proven to be effective as therapies for human disorders of the neuromuscular junction, in particular many subsets of congenital myasthenic syndromes. However, the mechanisms underlying this clinical benefit are unknown and improved understanding of the effect of adrenergic signalling on the neuromuscular junction is essential to facilitate the development of more targeted therapies. Here, we investigated the effect of salbutamol treatment on the neuromuscular junction in the ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency. ColQ-/- mice received 7 weeks of daily salbutamol injection, and the effect on muscle strength and neuromuscular junction morphology was analysed. We show that salbutamol leads to a gradual improvement in muscle strength in ColQ-/- mice. In addition, the neuromuscular junctions of salbutamol treated mice showed significant improvements in several postsynaptic morphological defects, including increased synaptic area, acetylcholine receptor area and density, and extent of postjunctional folds. These changes occurred without alterations in skeletal muscle fibre size or type. These findings suggest that ß-adrenergic agonists lead to functional benefit in the ColQ-/- mouse and to long-term structural changes at the neuromuscular junction. These effects are primarily at the postsynaptic membrane and may lead to enhanced neuromuscular transmission.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

Muscle contractility dysfunction precedes loss of motor unit connectivity in SOD1(G93A) mice.

INTRODUCTION: Electrophysiological measurements are used in longitudinal clinical studies to provide insight into the progression of amyotrophic lateral sclerosis (ALS) and the relationship between muscle weakness and motor unit (MU) degeneration. Here, we used a similar longitudinal approach in the Cu/Zn superoxide dismutase (SOD1[G93A]) mouse model of ALS. METHODS: In vivo muscle contractility and MU connectivity assays were assessed longitudinally in SOD1(G93A) and wild type mice from postnatal days 35 to 119. RESULTS: In SOD1(G93A) males, muscle contractility was reduced by day 35 and preceded MU loss. Muscle contractility and motor unit reduction were delayed in SOD1(G93A) females compared with males, but, just as with males, muscle contractility reduction preceded MU loss. DISCUSSION: The longitudinal contractility and connectivity paradigm employed here provides additional insight into the SOD1(G93A) mouse model and suggests that loss of muscle contractility is an early finding that may precede loss of MUs and motor neuron death. Muscle Nerve 59:254-262, 2019.

A pilot study of neuromuscular ultrasound as a biomarker for amyotrophic lateral sclerosis.

INTRODUCTION: This study explores the reliability and responsiveness of neuromuscular ultrasound in amyotrophic lateral sclerosis (ALS). METHODS: Investigations were conducted with 10 healthy controls, 10 patients with ALS (single point in time), and 10 different patients with ALS (followed over 6 months; 4 completed follow-up). Ultrasound was used to measure the thickness of the geniohyoid, bilateral biceps/brachialis, bilateral tibialis anterior, and bilateral hemidiaphragms (at inspiration and expiration). Interrater and intrarater reliability and change in muscle thickness over 6 months were measured. RESULTS: Interrater correlation coefficients ranged between 0.80 and 0.99 in healthy controls and between 0.78 and 0.97 in patients with ALS. Intrarater correlation coefficients ranged between 0.83 and 0.98 in healthy controls. The mean percentage decline in muscle thickness over 6 months was 20.25%. DISCUSSION: Muscle ultrasound appears to be a reliable technique for measuring important muscles in patients with ALS. Larger studies with age-matched controls should be conducted to assess further the responsiveness of this biomarker in ALS. Muscle Nerve 59:181-186, 2019.

Neuromuscular ultrasound in electrically non-localizable ulnar neuropathy.

INTRODUCTION: The aim of this study was to determine the value of high-resolution ultrasound (HRUS) in patients with ulnar neuropathy whose electrophysiology displayed an axonal, non-localizing pattern. METHODS: A prospective study of patients referred to an electromyography laboratory for ulnar neuropathy was performed. Of the 56 patients with clinical and electrodiagnostic (EDx) evidence of ulnar neuropathy, 12 were identified with non-localizing electrophysiology who subsequently underwent HRUS of the ulnar nerve. RESULTS: HRUS localized the ulnar neuropathy in all patients. In 2 patients, HRUS demonstrated structural lesions not at the elbow. DISCUSSION: HRUS often adds complementary information to standard EDx studies, including ulnar neuropathy. Thus, HRUS should be employed in patients with a non-localizing ulnar neuropathy on EDx studies. Muscle Nerve 58: 655-659, 2018.

Imaging Analysis of the Neuromuscular Junction in Dystrophic Muscle.

Duchenne muscular dystrophy (DMD), caused by the absence of the protein dystrophin, is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but there is little information to determine if damage from contraction-induced injury also occurs at or near the nerve terminal axon. Interestingly, both human patients and the mouse model for DMD (the mdx mouse) present fragmented neuromuscular junction (NMJ) morphology. Studies of mdx mice have revealed presynaptic and postsynaptic abnormalities, nerve terminal discontinuity, as well as increased susceptibility of the NMJ to contraction-induced injury with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyography. Focusing on the NMJ as a contributor to functional deficits in the muscle represents a paradigm shift from the more prevalent myocentric perspectives. Further studies are needed to determine the extent to which the nerve-muscle interaction is disrupted in DMD and the role of the NMJ in the dystrophic progression. This chapter lists the tools needed for nerve terminal and NMJ structural analysis using fluorescence imaging, and provides a step-by-step outline for how to stain, image, and analyze the NMJ in skeletal muscle, with specific attention to mdx muscle.

Neuromuscular ultrasound in patients with carpal tunnel syndrome and normal nerve conduction studies.

INTRODUCTION: Nerve conduction studies (NCS) are sensitive for carpal tunnel syndrome (CTS), but a small proportion of patients with clinical CTS have normal NCS. This retrospective study was designed to assess the neuromuscular ultrasound findings in a group of CTS patients. METHODS: The electronic medical record was reviewed by a neurologist to identify patients who had a diagnosis of CTS with normal NCS, including either mixed median-ulnar comparison or transcarpal sensory studies, and complete neuromuscular ultrasound evaluation for CTS. RESULTS: Fourteen individuals (22 wrists) met all criteria. A total of 92.3% had median nerve cross-sectional area enlargement at the wrist (mean 16.3 mm2 ), 100% had increased wrist-to-forearm median nerve area ratio (mean 2.4), 82.4% had decreased median nerve echogenicity, 75.0% had decreased median nerve mobility, and 7.1% had increased median nerve vascularity. CONCLUSION: A large proportion of patients with clinical CTS but normal NCS have abnormal neuromuscular ultrasound findings. Muscle Nerve 55: 913-915, 2017.

FDG-PET detects nonuniform muscle activity in the lower body during human gait.

INTRODUCTION: Nonuniform muscle activity has been partially explained by anatomically defined neuromuscular compartments. The purpose of this study was to investigate the uniformity of skeletal muscle activity during walking. METHODS: Eight participants walked at a self-selected speed, and muscle activity was quantified using [18 F]-fluorodeoxyglucose positron emission tomography imaging. Seventeen muscles were divided into 10 equal length sections, and within muscle activity was compared. RESULTS: Nonuniform activity was detected in 12 of 17 muscles (ƒ > 4.074; P < 0.046), which included both uni- and multi-articular muscles. Greater proximal activity was detected in 6 muscles (P < 0.049), and greater distal versus medial activity was found in the iliopsoas (P < 0.042). CONCLUSIONS: Nonuniform muscle activity is likely related to recruitment of motor units located within separate neuromuscular compartments. These findings indicate that neuromuscular compartments are recruited selectively to allow for efficient energy transfer, and these patterns may be task-dependent. Muscle Nerve 54: 959-966, 2016.

A randomized trial of diagnostic ultrasound to improve outcomes in focal neuropathies.

INTRODUCTION: Neuromuscular ultrasound is valid, reliable, and accurate, but it is not known whether combining it with electrodiagnostic studies leads to better outcomes in individuals with focal neuropathies. METHODS: One hundred twenty individuals with focal neuropathy, based on history, examination, and electrodiagnosis, were enrolled in this study. All patients underwent neuromuscular ultrasound and were randomized to either have their ultrasound results sent to the referring physician or not have them sent. Outcomes were assessed at 6 months by evaluators blinded to group assignment. RESULTS: The Overall Disability Sum Score and 7 of 8 domains of the Medical Outcomes Study 36-item Short-Form Health Survey (SF-36) showed more improvement in the "report sent" group, although only the general health perception domain was significant (P = 0.005). CONCLUSIONS: Most 6-month outcomes did not reach statistical significance between the 2 groups. However, the "report sent" group had trends toward better outcomes, with significance being reached in the general health perception domain of the SF-36.

Quantitative neuromuscular ultrasound in intensive care unit-acquired weakness: A systematic review.

Intensive care unit-acquired weakness (ICU-AW) causes significant morbidity and impairment in critically ill patients. Recent advances in neuromuscular ultrasound (NMUS) allow evaluation of neuromuscular pathology early in critical illness. Here we review application of ultrasound in ICU-AW. MEDLINE-indexed articles were searched for terms relevant to ultrasound and critical illness. Two reviewers evaluated the resulting abstracts (n = 218) and completed full-text review (n = 13). Twelve studies and 1 case report were included. Ten studies evaluated muscle thickness or cross-sectional area (CSA): 8 reported a decrease, and 2 reported no change. Two studies reported preservation of muscle thickness in response to neuromuscular electrical stimulation, and 1 found no preservation. One study found decreases in gray-scale standard deviation, but no change in echogenicity. One study described increases in echogenicity and fasciculations. Ultrasound reliability in ICU-AW is not fully established. Further investigation is needed to identify ultrasound measures that reliably predict clinical, electrodiagnostic, and pathologic findings of ICU-AW.

Vocal training mitigates age-related changes within the vocal mechanism in old rats.

Aging affects voice production and is associated with reduced communicative ability and quality of life. Voice therapy is a critical component of treatment, but its effects on neuromuscular mechanisms are unknown. The ultrasonic vocalizations (USVs) of rats can be used to test the effects of aging and voice use on the laryngeal neuromuscular system. This study tested the hypothesis that age-related changes in the USVs of rats and laryngeal neuromuscular junctions can be reversed through vocal exercise. Young and old rats were trained for 8 weeks to increase their USVs and were compared with a no intervention group pre- and post-treatment. USV acoustics and aspects of neuromuscular junction (NMJ) morphology were measured in the thyroarytenoid muscle. Vocal training reduced or eliminated some age differences found in both USVs and NMJs. We conclude that vocal exercise may assist in mitigating age-related changes in voice characteristics and underlying neuromuscular adaptations.

Isometric contractions reduce plantar flexor moment, Achilles tendon stiffness, and neuromuscular activity but remove the subsequent effects of stretch.

The effects of isometric contractions and passive stretching on muscle-tendon mechanics and muscle activity were studied in 16 healthy human volunteers. First, peak concentric and passive ankle joint moment data were recorded on an isokinetic dynamometer with electromyographic monitoring of the triceps surae; real-time motion analysis of the lower leg and ultrasound imaging of the Achilles-medial gastrocnemius muscle-tendon junction were simultaneously conducted. Second, the subjects performed six 8-s maximal voluntary isometric contractions (MVICs) before repeating the passive and active trials. Although there was no decrease in isometric joint moment after MVICs, peak concentric moment was significantly reduced (11.5%, P < 0.01). This was accompanied by, and correlated with (r = 0.90, P < 0.01), significant reductions in peak triceps surae electromyographic amplitude (21.0%, P < 0.01). Achilles tendon stiffness (10.9%, P < 0.01) and passive joint moment (4.9%, P < 0.01) were also significantly reduced. Third, the subjects performed three 60-s static plantar flexor stretches before being retested 2 and 30 min after stretch. The stretch protocol caused no significant change in any measure. At 30 min after stretching, significant recovery in concentric moment and muscle activity was detected at dorsiflexed joint angles, while Achilles tendon stiffness and passive joint moment remained significantly reduced. These data show that the performance of MVICs interrupts the normal stretch-induced losses in active and passive plantar flexor joint moment and neuromuscular activity, largely because concentric strength and tendon properties were already affected. Importantly, the decrease in Achilles tendon stiffness remained 30 min later, which may be an important etiological factor for muscle-tendon strain injury risk.

[Ultrastructural analysis of mouse levator auris longus muscle intoxicated in vivo by botulinum neurotoxin type A]. / Análisis ultraestructural del músculo levator auris longus de ratón intoxicado in vivo por la neurotoxina botulínica tipo A.

We studied the short and long term ultrastructural changes produced by botulinum neurotoxin type A injected in vivo, at a sublethal dose, in mouse levator auris longus muscle. The neurotoxin had a temporary effect on nerve terminals which consisted in a generalized paralysis, that affected the following features of the neuromuscular sample's morphology: size of the nerve terminals, vesicle population, mitochondrial appearance, Schwann cell's morphology, development and distribution of post-synaptic membrane folds, and nuclear morphology of the different elements of the motor end plate. Besides, the amount of endomysial connective tissue was significantly greater compared to non-intoxicated cases, and these changes were more notorious during the first couple of weeks. 20 to 25 days after the injection, during the recovery phase, we observed nerve terminals with a variable appearance: some completely degenerated, enveloped by Schwann cell processes, and new contacts characterized ultrastructurally for their small size, scarce vesicles, partially enveloped by Schwann cells, early myelinized axons and barely developed synaptic folds. Sixty days after the injection, the axon terminal recovered its normal appearance: synaptic vesicles filled the axon's cytoplasm, and the mitochondria showed normal appearing cristae and electronic densities. We conclude that botulinum neurotoxin type A produces changes related to denervation of the nerve terminals and affects the motor end plate components. Schwann cells play an important role both in the morphofuntional recovery of nerve terminals and in their degradation.