Motor unit number estimation--a Bayesian approach

All muscle contractions are dependent on the functioning of motor units. In diseases such as amyotrophic lateral sclerosis (ALS), progressive loss of motor units leads to gradual paralysis. A major difficulty in the search for a treatment for these diseases has been the lack of a reliable measure of disease progression. One possible measure would be an estimate of the number of surviving motor units. Despite over 30 years of motor unit number estimation (MUNE), all proposed methods have been met with practical and theoretical objections. Our aim is to develop a method of MUNE that overcomes these objections. We record the compound muscle action potential (CMAP) from a selected muscle in response to a graded electrical stimulation applied to the nerve. As the stimulus increases, the threshold of each motor unit is exceeded, and the size of the CMAP increases until a maximum response is obtained. However, the threshold potential required to excite an axon is not a precise value but fluctuates over a small range leading to probabilistic activation of motor units in response to a given stimulus. When the threshold ranges of motor units overlap, there may be alternation where the number of motor units that fire in response to the stimulus is variable. This means that increments in the value of the CMAP correspond to the firing of different combinations of motor units. At a fixed stimulus, variability in the CMAP, measured as variance, can be used to conduct MUNE using the "statistical" or the "Poisson" method. However, this method relies on the assumptions that the numbers of motor units that are firing probabilistically have the Poisson distribution and that all single motor unit action potentials (MUAP) have a fixed and identical size. These assumptions are not necessarily correct. We propose to develop a Bayesian statistical methodology to analyze electrophysiological data to provide an estimate of motor unit numbers. Our method of MUNE incorporates the variability of the threshold, the variability between and within single MUAPs, and baseline variability. Our model not only gives the most probable number of motor units but also provides information about both the population of units and individual units. We use Markov chain Monte Carlo to obtain information about the characteristics of individual motor units and about the population of motor units and the Bayesian information criterion for MUNE. We test our method of MUNE on three subjects. Our method provides a reproducible estimate for a patient with stable but severe ALS. In a serial study, we demonstrate a decline in the number of motor unit numbers with a patient with rapidly advancing disease. Finally, with our last patient, we show that our method has the capacity to estimate a larger number of motor units.     

A Comparison of Three Electrophysiological Methods for the Assessment of Disease Status in a Mild Spinal Muscular Atrophy Mouse Model

Objectives

There is a need for better, noninvasive quantitative biomarkers for assessing the rate of progression and possible response to therapy in spinal muscular atrophy (SMA). In this study, we compared three electrophysiological measures: compound muscle action potential (CMAP) amplitude, motor unit number estimate (MUNE), and electrical impedance myography (EIM) 50 kHz phase values in a mild mouse model of spinal muscular atrophy, the Smn1^c/c mouse.

Methods

Smn1^c/c mice (N = 11) and wild type (WT) animals (−/−, N = 13) were measured on average triweekly until approximately 1 year of age. Measurements included CMAP, EIM, and MUNE of the gastrocnemius muscle as well as weight and front paw grip strength. At the time of sacrifice at one year, additional analyses were performed on the animals including serum survival motor neuron (SMN) protein levels and muscle fiber size.

Results

Both EIM 50 kHz phase and CMAP showed strong differences between WT and SMA animals (repeated measures 2-way ANOVA, P<0.0001 for both) whereas MUNE did not. Both body weight and EIM showed differences in the trajectory over time (p<0.001 and p = 0.005, respectively). At the time of sacrifice at one year, EIM values correlated to motor neuron counts in the spinal cord and SMN levels across both groups of animals (r = 0.41, p = 0.047 and r = 0.57, p  = 0.003, respectively), while CMAP did not. Motor neuron number in Smn1^c/c mice was not significantly reduced compared to WT animals.

Conclusions

EIM appears sensitive to muscle status in this mild animal model of SMA. The lack of a reduction in MUNE or motor neuron number but reduced EIM and CMAP values support that much of the pathology in these animals is distal to the cell body, likely at the neuromuscular junction or the muscle itself.     

Physiological characteristics of motor units in the brachioradialis muscle across fatiguing low-level isometric contractions.

The purpose of this study was to determine (i) if decomposition-based quantitative electromyography (DQEMG) could detect changes in motor unit potential (MUP) morphology and motor unit (MU) firing pattern statistics associated with muscle fatigue, (ii) if any detected changes are correlated with surface electromyographic (SEMG) signs of fatigue, and (iii) if significant fatigue-dependent changes are repeatable within individuals. Mean MU firing rates and the morphology of MUPs detected using needle and surface electrodes during constant-torque isometric contractions held until exhaustion were investigated in the brachioradialis (BR) muscle in 10 healthy volunteers (mean age=28.6 yr, SD+/-3.9). Time dependant changes were investigated using an analysis of variance with normalized time as a main effect. Partial correlation coefficients were computed using a repeated measures analysis of covariance to determine if changes in MU firing rates, needle-detected MUPs and surface-detected MUPs (SMUPs) were related to changes in SEMG signal amplitude and frequency parameters. Intraclass correlation coefficients (ICCs) were used to determine the within-subject repeatability of changes in MU firing rates, and MUP and SMUP parameters. Significant decreases in mean MU firing rates were found along with significant increases in various duration and area related parameters in both MUPs and SMUPs across the fatiguing contraction. The SEMG signal demonstrated the expected changes with fatigue: an increase in amplitude and a decrease in frequency content. SEMG amplitude was significantly positively correlated with SMUP peak-to-peak voltage (r=0.85, p<0.05), and SMUP area (r=0.86, p<0.05). Mean power frequency was significantly negatively correlated with SMUP negative peak duration (r=-0.74, p<0.05). The significant time-dependent changes were reliably observed (ICCs were 0.94 for MUP peak to peak amplitude, 0.97 for MUP area and 0.95 for MUP area to amplitude ratio, 0.95 for SMUP peak-to-peak voltage, 0.83 for SMUP area, 0.99 for SMUP negative peak amplitude and 0.88 for SMUP negative peak area). The decreases in mean MU firing rates measured along with the increases in amplitude, duration and area parameters of MUPs and SMUPs and their partial correlation with SEMG amplitude during submaximal fatiguing contractions of the BR, suggest that recruitment is a main cause of increased SEMG amplitude parameters with fatigue. We conclude that DQEMG can be effectively and reliably used to detect changes in physiological characteristics of MUs that accompany fatigue.     

Spatial analysis of motor unit potentials of the rat medial gastrocnemius

The spatial analysis of the potentials of single motor units of the rat medial gastrocnemius muscle evoked by stimulation of the fibres of split ventral roots was carried out with a bipolar electrode moving in the direction perpendicular to the longitudinal axis of the muscle fibres. During this movement of the electrode a variability was observed in the time of the biphasic potential from its maximum to minimum, and in the peak-to-peak amplitude of these potentials. The potentials recorded outside the territory of the motor unit had a lower amplitude in relation to the potentials from the territory of the unit. This made localization of the motor unit on the cross-section of the muscle possible. Differences in the duration of the potential from maximal to minimal amplitude (maximum-minimum amplitude time--M-MAT) of each investigated motor unit from successive recording sites reflected the number of fibres contributing to the action potential and the distance of the recording surface of the electrode from the zone of the motor end-plates of this motor unit. The greatest diameter of the territory of the observed motor units reached 2.5 mm.     

Motor unit number estimation and quantitative needle electromyography in stroke patients

ObjectiveTo evaluate the effect of upper motor neuron damage upon motor units’ function by means of two separate and supplementary electrophysiological methods.MethodsThe abductor digiti minimi muscle of the non-paretic and the paretic side was studied in forty-six stroke patients with (a) motor unit number estimation (MUNE) – adapted multiple point stimulation method and (b) computerized quantitative needle electromyography (EMG) assessing the configuration of voluntary recruited motor unit potentials. Main outcome comparisons were focused on differences between non-paretic and paretic side.ResultsOn the affected hands mean MUNE value was significantly lower and mean area of the surface recorded single motor unit potentials was significantly larger than the corresponding ones on the non-paretic hands. EMG findings did not reveal remarkable differences between the two sides. Neither severity nor chronicity of stroke was related to MUNE or EMG parameters.DiscussionMUNE results, which suggested reduced motor unit numbers in stroke patients, in conjunction with the normal EMG features in these same muscles has given rise to different interpretations. In a clinical setting, reinnervation type changes in the EMG similar to that occurring in neuronopathies or axonal neuropathies should not be expected in muscles with central neurogenic lesion.     

Progression of multiple innervation of reinnervated rat muscle treated with bilobalide]

During motor nerve regeneration a transitory polyinnervation of muscle cells occurs, which represents a phase of rearrangement of the recovered innervation. Bilobalide, a terpene extrated from Ginkgo biloba leaves, was proposed to affect some aspects of nervous system development and regeneration. In this work, influence of bilobalide on polyinnervation in reinnervated extensor digitorum longus muscle was studied, through electrophysiological and histological techniques. The muscle was denervated crushing the sciatic nerve and it was examined at 1 or 2 months after denervation. The polyinnervated muscle cells in controls reached 24% at 1 month and thus the percentage decreased. In muscles of bilobalide treated rats the number of polyinnervated cells was decreased at both times.     

Staircase potentiation in isolated frog skeletal muscle: power spectral analysis of the evoked compound muscle action potential

1. The mechanical and electrophysiological effects of repetitive, low-frequency electrical stimulation on paired sartorii muscles from small male frogs have been investigated, in vitro. 2. Stimulation for 90 sec at 5 Hz resulted in a progressive rise (staircase) than fall (fatigue) in peak twitch tension. 3. The root mean square amplitude, peak-to-peak amplitude, conduction velocity and mean power frequency of evoked compound muscle action potentials (CMAPs) decreased over the stimulation period. 4. Results suggest that alterations in the shape of the CMAP during repetitive stimulation may contribute to the staircase phenomenon.     

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology¹. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 °C is maintained by a heating plate and continuously measured by a rectal thermo probe². Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes³ we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.     

Multichannel thin-film electrode for intramuscular electromyographic recordings.

It is currently not possible to record electromyographic (EMG) signals from many locations concurrently inside the muscle in a single wire electrode system. We developed a thin-film wire electrode system for multichannel intramuscular EMG recordings. The system was fabricated using a micromachining process, with a silicon wafer as production platform for polyimide-based electrodes. In the current prototype, the flexible polymer structure is 220 microm wide, 10 microm thick, and 1.5 cm long, and it has eight circular platinum-platinum chloride recording sites of 40-microm diameter distributed along the front and back surfaces with 1,500-microm intersite spacing. The system prototype was tested in six experiments where the electrode was implanted into the medial head of the gastrocnemius muscle of rabbits, perpendicular to the pennation angle of the muscle fibers. Asynchronous motor unit activity was induced by eliciting the withdrawal reflex or sequential crushes of the sciatic nerve using a pair of forceps. Sixty-seven motor units were identified from these recordings. In the bandwidth 200 Hz to 5 kHz, the peak-to-peak amplitude of the action potentials of the detected motor units was 75 +/- 12 muV and the root mean square of the noise was 1.6 +/- 0.4 muV. The noise level and amplitude of the action potentials were similar for measures separated by up to 40 min. The experimental tests demonstrated that thin film is a promising technology for a new type of flexible-wire intramuscular EMG recording system with multiple detection sites.     

Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

Acute nerve stretch and the compound motor action potential

In this paper, the acute changes in the compound motor action potential (CMAP) during mechanical stretch were studied in hamster sciatic nerve and compared to the changes that occur during compression.In response to stretch, the nerve physically broke when a mean force of 331 gm (3.3 N) was applied while the CMAP disappeared at an average stretch force of 73 gm (0.73 N). There were 5 primary measures of the CMAP used to describe the changes during the experiment: the normalized peak to peak amplitude, the normalized area under the curve (AUC), the normalized duration, the normalized velocity and the normalized velocity corrected for the additional path length the impulses travel when the nerve is stretched. Each of these measures was shown to contain information not available in the others.During stretch, the earliest change is a reduction in conduction velocity followed at higher stretch forces by declines in the amplitude of the CMAP. This is associated with the appearance of spontaneous EMG activity. With stretch forces < 40 gm (0.40 N), there is evidence of increased excitability since the corrected velocities increase above baseline values. In addition, there is a remarkable increase in the peak to peak amplitude of the CMAP after recovery from stretch < 40 gm, often to 20% above baseline.Multiple means of predicting when a change in the CMAP suggests a significant stretch are discussed and it is clear that a multifactorial approach using both velocity and amplitude parameters is important. In the case of pure compression, it is only the amplitude of the CMAP that is critical in predicting which changes in the CMAP are associated with significant compression.     

Motor neurons loss in Parkinson Disease: An electrophysiological study (MUNE)

The aim of this study was to evaluate the involvement of a peripheral motor neuron in Parkinson Disease (PD) using the motor unit number estimation (MUNE) method, which reflects motor unit loss in motor neuron diseases. Multipoint incremental MUNE method was calculated in abductor pollicis brevis (APB) and abductor digiti minimi (ADM) in forty one (41) patients with PD and forty five (45) healthy volunteers. From the analysis, the MUNE of APB was lower in PD than in the control group, especially in the sub-group aged 60 years or older. MUNE was negatively correlated with the age of patients for APB, but not with the duration of the disease and advancement of PD. The loss of motor units in sporadic Parkinson's disease revealed by multipoint incremental MUNE method is considered a sign of lower motor neuron involvement, however, loss of motor neurons is slight and does not manifest equally in all muscles . Thus, the results from this experiment should be treated with concern, as it could be a landmark for further experiments.     

1998 ISEK Congress Keynote Lecture: Motor units: how many, how large, what kind?

There are now at least nine methods for motor unit number estimation (MUNE) in living human muscles. All methods are based on the comparison of an average single motor unit potential (or twitch) with the response of the whole muscle. Such estimations have been performed for proximal and distal muscles of the arm and leg in healthy subjects and in patients with various neuromuscular disorders. In healthy subjects there is a loss of motor units which is most evident in distal muscles and after the age of 60 years. Substantial losses of motor units have been measured in patients with ALS, post-polio symptoms, and diabetic peripheral neuropathies. In contrast, normal MUNEs have been found in approximately half of patients with persisting obstetric brachial palsies. The sizes of motor units show considerable variations within the same muscle and also between muscles; very large units are usually present in severe partial denervation. Although many motor unit properties are largely governed by motoneurons, some exhibit less plasticity in humans than in other mammals.     

Neuromuscular degenerative effects of Ankaferd Blood Stopper® in mouse sciatic nerve model

Purpose: Ankaferd Blood Stopper^® (ABS), a licenced medicinal herbal extract, is commonly used as an effective topical haemostatic agent. This study is designed to investigate whether topical ABS application may cause peripheral nerve degeneration and neuromuscular dysfunction in a mouse sciatic nerve model.

Methods: Twenty mice were randomly divided into two groups; an ABS treated experimental group and a saline-treated control group. Left sciatic nerves were treated with 0.3?ml of ABS in the experimental group and 0.3?ml of sterile saline in the control group for 5?min. Peripheral nerve degeneration and neuromuscular dysfunction were evaluated by behavioural tests, electrophysiological analysis and weight ratio comparison of target muscles.

Results: The motor function, assessed by the sciatic function index, was significantly impaired in ABS-treated animals as compared to the animals treated with saline. Motor coordination, evaluated with the rotarod test, was significantly decreased (–42%) in ABS-treated animals compared to the saline-treated animals. The degree of pain, assessed by the reaction latency to thermal stimuli (hot-plate test), was significantly prolonged (313%) in ABS-treated mice when compared to the saline-treated mice. ABS-treated mice showed a significant reduction in motor nerve conduction velocity (MNCV) (–52%) and the compound muscle action potential (CMAP) (–47%); however, it significantly prolonged onset latency (23%). The gastrocnemius muscles weight ratio of the ABS group was considerably lower than that of the control group.

Conclusions: These findings demonstrate that ABS triggers peripheral nerve degeneration and functional impairment and, thus promotes a deterioration of sciatic nerves.     

Comparison of force and EMG measures in normal and reinnervated tibialis anterior muscles of the rat.

The relationship between motor unit force and the recorded voltage produced by activated muscle unit fibres (electromyogram, EMG) was examined in normal and reinnervated rat tibialis anterior muscles. The number, cross-sectional area, and radial distance from the recording electrode of muscle fibres in a given unit, obtained directly from a sample of glycogen-depleted motor units, were analysed in relation to the magnitude of the EMG signal produced by that unit. EMG peak to peak amplitude and area varied as approximately the square root of twitch force in both normal and reinnervated units. Furthermore, the EMG amplitude increased approximately as the total cross-sectional area of the motor unit (number of muscle fibres x the average cross-sectional area of the fibres) and inversely with approximately the square root of the distance of fibres from the recording electrodes on the surface of the muscle.     

Motor unit number estimation as a complementary test to routine electromyography in the diagnosis of amyotrophic lateral sclerosis

Electromyographic (EMG) abnormalities that reveal denervation and reinnervation caused by lower motor neuron degeneration do not reflect the number of motor units that determines muscle strength. Consequently, motor unit activity potential (MUAP) parameters do not reflect muscle dysfunction.The aim of the study was to compare the value of motor unit number estimation (MUNE) and MUAP parameters as indicators of clinical muscle dysfunction in patients with amyotrophic lateral sclerosis (ALS), and to analyze the role of MUNE as a supplement to the EMG criteria for the diagnosis of ALS.In 25 patients with ALS, MUNE by the multipoint incremental method in the abductor digiti minimi (ADM) and quantitative EMG in the first dorsal interosseous (FDI) were obtained. The Medical Research Council (MRC) scale was used to evaluate clinical muscle dysfunction. A strong correlation between the number of motor units evaluated by MUNE and ADM clinical function by the MRC scale was found (P < 0.001). An increased value of surface-detected single motor action potential was associated with a decreased MRC score for ADM (P < 0.1). No relation was found between MUAP parameters in FDI and MRC scores. Our data support the value of the MUNE method for the detection of motor unit loss in ALS, and it could be postulated that MUNE studies may be considered complementary tests for ALS in a future revision of ALS criteria.     

Electrophysiologic identification and evaluation of stylohyoid and posterior digastricus muscle complex.

PURPOSE: To identify the function of stylohyoid and posterior digastricus (STH-PD) muscle complex by the EMG techniques. METHODS: Unaffected sides of the faces of 30 patients with facial paralysis or hemifacial spasm were investigated. A concentric needle electrode was inserted to the STH-PD muscle complex and another concentric needle electrode was inserted to the orbicularis oris (OO) muscle. Simultaneous recording were obtained from two muscles using electrical stimulation (ES) (in 25 cases) and magnetic coil stimulation (MS) (in 15 cases); and both in 10 cases. Afterwards, the function of STH-PD was studied such as whistling, lip pursing, swallowing, jaw opening and closing. RESULTS: (1) The motor latency of compound muscle action potential (CMAP) of the STH-PD muscle was shorter than that of OO. (2) When the facial nerve was stimulated more distally than the stylomastoid foramen, the CMAP elicited from the STH-PD muscle complex immediately disappeared. (3) Ipsilateral MS was able to elicit the motor evoked potential (MEP) from STH-PD either at intracranially (half of cases) or at the extracranially. While OO muscle was always stimulated intracranially by MS. (4) The STH-PD muscle complex could not be basically recruited by the mimicry except lip pursing. The main recruitment were provided by swallowing and jaw opening. Cortical MS were facilitated during swallowing (5) Late reflex responses appeared in the STH-PD muscle complex during infraorbital-trigeminal and facial nerve ES. CONCLUSION: The STH-PD muscle complex is identified electrophysiologically. Although it is innervated by the facial nerve, its functions are mainly related with jaw opening and oropharyngeal swallowing. However, it is activated by the lip pursing.     

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.     

Does quantitative EMG differ myotonic dystrophy type 2 and type 1?

Genetic testing is considered the only reliable diagnostic approach in myotonic dystrophy. However it has recently been reported that a considerable number of patients with genetically proven types of the disease have unusual phenotypic presentation. The aim of our study was to evaluate motor unit reorganization reflected by various electrophysiological abnormalities in myotonic dystrophies and to compare findings between type 1 (DM 1) and type 2 myotonic dystrophy (DM2). Quantitative electromyography (EMG) recordings in 63 patients (33 with DM1 and 30 with DM2) from the biceps brachii (BB), rectus femoris (RF), first dorsal interosseus (FDI), and tibialis anterior (TA) muscles were analyzed. Mean amplitude and size index (SI) of motor unit potentials recorded in TA and RF muscles, mean potential duration in TA, and mean SI and the number of outliers with amplitude above the normal range in BB were significantly increased in DM2 as compared to DM1. Myotonic discharges were recorded more frequently in DM1 than in DM2. EMG findings significantly differ between DM1 and DM2. The presence of high amplitude potentials in lower limb muscles in DM2 patients, atypical for myogenic muscle lesions, could be explained by muscle fiber hypertrophy observed in muscle biopsies.