The use of statistical MUNE in a multicenter clinical trial

Techniques to estimate motor unit number (MUNE) measure the number of functioning motor units in a muscle. In diseases characterized by progressive motor unit loss, such as amyotrophic lateral sclerosis (ALS), MUNE may be useful to monitor disease progression or beneficial response to treatment. As part of a multicenter, placebo-controlled, randomized, double-blind clinical trial testing the efficacy of creatine in patients with ALS, statistical MUNE was measured in 104 patients tested monthly for 6 months. The objective was to determine whether MUNE was a reliable and sensitive outcome measure in the context of a multicenter trial. Formal training and reliability testing was required for all MUNE evaluators. Testing of normal controls showed a high degree of test-retest reliability. All patient data were combined as the experimental treatment showed no efficacy. There was a 23% decline in MUNE over 6 months. The technique as employed in this trial overemphasized the presence of small motor units; this problem was partially addressed by poststudy data monitoring and censuring. Thus, MUNE can be used reliably as an outcome measure in multicenter clinical trials; specific remedies are suggested for the difficulties encountered in this study.     

Bayesian statistical MUNE method

We have developed a new method of motor unit number estimation (MUNE) for assessing diseases such as amyotrophic lateral sclerosis (ALS). We used data from the whole stimulus-response curve and then performed a Bayesian statistical analysis. The Bayesian method uses mathematical equations that express the basic elements of motor unit activation after electrical stimulation and allows for the sources of variability and uncertainty in this formulation. The Bayesian MUNE method was used to determine the most probable number of motor units in 8 normal subjects, 49 ALS subjects, and 3 subjects with progressive lower motor neuron (LMN) weakness. In normals the number of motor units was calculated to be 75-85 in hand and 40-58 in foot muscles. In ALS subjects the number of motor units per muscle was less than in normal subjects. In 17 ALS subjects and 3 subjects with LMN weakness the median, ulnar, or peroneal nerve was studied on repeated occasions over an average of 189 days (range 63-1,071) and the number of motor units progressively declined, with a half-life ranging from 62-834 days. The results of our MUNE technique were reproducible on replicate studies. A Bayesian statistical MUNE method is a new approach that can be used to study ALS patients serially for assessment and treatment trials.     

Reproducibility of statistical motor unit number estimates in amyotrophic lateral sclerosis: comparisons between size- and number-weighted modifications

Motor unit number estimations (MUNEs) can directly assess motor unit populations in muscle and quantify the degree of physiological or pathological motor unit degeneration. A high degree of reproducibility and reliability is required of any effective quantitative tool. MUNE is being increasingly applied clinically, and statistical MUNE has several advantages over alternative techniques. Nevertheless, the optimal method of applying statistical MUNE with respect to its reproducibility has not been established. We performed statistical MUNE by selecting the most compensated compound muscle action potential (CMAP) area as a test area and modified the results obtained by using the weighted mean surface-recorded motor unit potential (SMUP). MUNE measurements made in patients with amyotrophic lateral sclerosis (ALS) showed better reproducibility after incorporating the size-weighted modification. Therefore, we suggest that the size-weighted MUNE in combination with the selection of testing "neurogenically compensated" CMAP areas is a more reliable method of statistical MUNE analysis in ALS patients.     

Comparison of incremental with multipoint MUNE methods in transgenic ALS mice.

Several methods of motor unit number estimation (MUNE) are in current use. Uncertainty still exists about which is preferable and how results obtained from one method compare to another. We studied changes with MUNE over time in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS), using both incremental and multipoint methods. This mouse model of motor neuron degeneration is highly consistent, with a monotonic decline in motor neuron number starting at approximately 60 days of life. Five mice were studied four times each, starting at day 60 of life and approximately every 20 days thereafter, using both methods. Results were quite comparable for both methods, with the incremental method yielding slightly higher estimates of motor unit size, and hence smaller MUNEs. Correlations between the two methods were 0.71 for single motor unit action potential (SMUAP) amplitude and 0.95 for MUNE. In this model, therefore, both MUNE methods yield similar estimates and are equally effective at documenting progression of a lower motor neuron disorder.     

Reproducibility of motor unit number estimation in individual subjects

Although the reproducibility of motor unit number estimation (MUNE) for groups of subjects has been studied, there is little such data for individuals. Prediction intervals represent a tool to study individual MUNE reproducibility and represent the range of values expected for a future MUNE if the true number of motor units remains unchanged. MUNE was performed using the statistical method on 48 normal individuals. The prediction interval was found to be a function of the intrasubject coefficient of variation. Using a commercial manufacturer's recommended technique and software, prediction intervals were found to be so broad as to be of uncertain value. We found that by averaging two MUNE observations for each determination, and using the method of weighted averages for calculating the size of an average single motor unit potential, the intrasubject coefficient of variation was reduced from 16.48% to 8.77%, and the 90% prediction interval became sufficiently narrow to be clinically useful. False-negative rates were also lowered substantially using these techniques. Thus, simple modifications of an existing MUNE program improved the clinical utility of this program for the longitudinal study of patients in whom changes in motor unit number over time are of importance, such as those with motor neuron diseases.     

Proposed modification to data analysis for statistical motor unit number estimate

Motor unit number estimation (MUNE) is an important electrophysiological technique for quantitative measurement of motor neuron loss. Although commonly used, there is no consensus concerning the optimal procedure for statistical MUNE, particularly regarding several operator-dependent variables. To assess the variables, we analyzed 500 sequential, submaximal compound muscle action potential (CMAP) responses at three or four stimulus intensities in 10 controls and 10 patients with amyotrophic lateral sclerosis (ALS). In both controls and ALS patients, we found that posttest filtering data based on 20% or 25% windows or 2, 2.5, or 3 SD excludes <5% of data. Windows of 10% or 15% excluded <5% of data in controls but not in ALS patients. Excluding data based on +/-2 SD, the coefficient of variation for final MUNE was 12% in controls and 6% in ALS patients. Group sizes of 30 or 50 and sample sizes of 300 to 500 sequential CMAP responses per run yielded the lowest coefficient of variation. We propose that statistical MUNE data should be analyzed based on excluding data >2 SD from the mean, because this is operator independent, includes the majority of data, effectively excludes clearly outlying data, such as fasciculations or movement artifact, and has a reasonable coefficient of variation.     

Statistical motor unit number estimation: from theory to practice

Statistical motor unit number estimation (MUNE) is one of several experimental techniques used to estimate the number of lower motor neurons innervating a given muscle. All are fairly reproducible and have been applied successfully in monitoring neurogenic disease progression. Quantitating the number of lower motor neurons is important, since the compound muscle action potential (CMAP) and strength may not change as rapidly over time due to the confounding effect of reinnervation. MUNE techniques differ in the way they obtain samples of surface-recorded motor unit potentials (SMUP). Statistical MUNE is based on Poisson statistics, uses surface stimulation, and is useful in testing distal, superficial nerves. This review focuses on the theory behind the development of the technique, critiques the publications resulting from applying the technique in control and disease subjects, and discusses the future developments needed for clinical utility.     

The utility of motor unit number estimation methods versus quantitative motor unit potential analysis in diagnosis of ALS

Objective

To compare the diagnostic utility of motor unit number estimation (MUNE) methods to motor unit potential (MUP) analysis in amyotrophic lateral sclerosis (ALS).

Effect of changing data collection parameters on statistical motor unit number estimates

The effect of number of samples and selection of data for analysis on the calculation of surface motor unit potential (SMUP) size in the statistical method of motor unit number estimates (MUNE) was determined in 10 normal subjects and 10 with amyotrophic lateral sclerosis (ALS). We recorded 500 sequential compound muscle action potentials (CMAPs) at three different stable stimulus intensities (10-50% of maximal CMAP). Estimated mean SMUP sizes were calculated using Poisson statistical assumptions from the variance of 500 sequential CMAP obtained at each stimulus intensity. The results with the 500 data points were compared with smaller subsets from the same data set. The results using a range of 50-80% of the 500 data points were compared with the full 500. The effect of restricting analysis to data between 5-20% of the CMAP and to standard deviation limits was also assessed. No differences in mean SMUP size were found with stimulus intensity or use of different ranges of data. Consistency was improved with a greater sample number. Data within 5% of CMAP size gave both increased consistency and reduced mean SMUP size in many subjects, but excluded valid responses present at that stimulus intensity. These changes were more prominent in ALS patients in whom the presence of isolated SMUP responses was a striking difference from normal subjects. Noise, spurious data, and large SMUP limited the Poisson assumptions. When these factors are considered, consistent statistical MUNE can be calculated from a continuous sequence of data points. A 2 to 2.5 SD or 10% window are reasonable methods of limiting data for analysis.     

Motor unit number estimation predicts disease onset and survival in a transgenic mouse model of amyotrophic lateral sclerosis

Motor unit number estimation (MUNE) has proved useful in predicting rate of progression and survival in patients with amyotrophic lateral sclerosis (ALS). In animal models, it has demonstrated physiological effects of experimental medications that were not evident behaviorally. We sought to determine more specifically what aspects of function and survival that MUNE could predict in the G93A transgenic mouse model of ALS. Transgenic mice were examined in two distinct treatment studies, neither of which showed an effect of drug on survival, behavioral measures, or MUNE. MUNE was performed using a modification of the incremental stimulation method by stimulating the sciatic nerve at the sciatic notch, and recording with a circumferential surface electrode around the ipsilateral distal hindlimb. Both limbs were studied and the results averaged. MUNE was performed longitudinally on all animals from near onset to premorbid state. Each study was evaluated separately. For both studies, MUNE at initial study correlated significantly with behavioral determination of disease onset, and MUNE slope from initial to final study correlated significantly with disease duration, as measured from onset to time of death. However, the final MUNE value did not correlate with survival. Thus, in two studies involving animals with quite different disease courses, initial MUNE effectively predicted symptom onset and MUNE slope predicted survival. This suggests that MUNE has potential efficacy as a useful functional outcome measure in both animal and human studies of ALS.     

Single motor unit variability with threshold stimulation in patients with amyotrophic lateral sclerosis and normal subjects

Repetitive nerve stimulation often shows responses with an abnormal decrement in patients with amyotrophic lateral sclerosis (ALS), suggesting instability of the neuromuscular junction; however, the pathophysiology and response characteristics of this instability are not clear. We evaluated response variability of 47 single motor units from 16 patients with ALS and 51 units from 10 normal subjects, acquired by delivering threshold stimuli sporadically at 0.5 HZ or less. In addition, in 46 other different single motor units obtained from 21 patients with ALS, variability was studied at both 1- and 3-HZ stimulation rates. Motor units from patients with ALS were significantly more variable than those from normal subjects, even when their larger amplitude was accounted for. This increased variability was not rate dependent. Response variability is a critical measure in the statistical method of motor unit number estimation and is attributed to variability in the number of units activated; the fact that variability of single motor units varies with disease state may be a potentially confounding factor in the application of the technique.     

Motor unit number estimation: A technology and literature review

Introduction: Numerous methods for motor unit number estimation (MUNE) have been developed. The objective of this article is to summarize and compare the major methods and the available data regarding their reproducibility, validity, application, refinement, and utility. Methods: Using specified search criteria, a systematic review of the literature was performed. Reproducibility, normative data, application to specific diseases and conditions, technical refinements, and practicality were compiled into a comprehensive database and analyzed. Results: The most commonly reported MUNE methods are the incremental, multiple‐point stimulation, spike‐triggered averaging, and statistical methods. All have established normative data sets and high reproducibility. MUNE provides quantitative assessments of motor neuron loss and has been applied successfully to the study of many clinical conditions, including amyotrophic lateral sclerosis and normal aging. Conclusions: MUNE is an important research technique in human subjects, providing important data regarding motor unit populations and motor unit loss over time. Muscle Nerve 50 : 884–893, 2014     

Motor unit number estimates and quantitative motor unit analysis in healthy subjects and patients with amyotrophic lateral sclerosis

Limitations associated with global measures of function in patients with amyotrophic lateral sclerosis (ALS) and the qualitative nature of needle electromyography have stimulated the development of alternate means of monitoring disease severity and progression in ALS. Thus, the objective of this study was to examine the ability of one these techniques, decomposition-based quantitative electromyography (DQEMG), to obtain electrophysiological data, including motor unit number estimates (MUNEs), from a group of patients with ALS. The first dorsal interosseous and biceps brachii muscles were studied in 10 healthy subjects and 9 patients with ALS. Following the acquisition of a maximum M wave, needle- and surface-detected EMGs were collected simultaneously during 30-second contractions performed at 10% of the maximum voluntary contraction force to obtain motor unit potential (MUP) trains. DQEMG was then used to extract the surface-detected MUP associated with each MUP train, the mean size of which was divided into the maximum M wave to obtain a MUNE. The results suggest that quantitative electrophysiological data obtained using DQEMG are representative of the pathophysiological changes in the lower motor system in ALS patients, supporting its use in studies documenting the natural history and progression of the disease.     

Effect of small motor unit potentials on the motor unit number estimate

Small surface motor unit potentials (S-MUPs) may have a negative influence on the variability of the motor unit number estimate (MUNE). According to published consensus criteria S-MUPs with a negative peak amplitude smaller than 10 muV should be omitted. The effect of omitting small S-MUPs on the MUNE was evaluated using a simulation model. The model incorporated a healthy and amyotrophic lateral sclerosis (ALS) distribution formed with real S-MUPs. Using a random drawing process the MUNE was calculated with and without small S-MUPs. In the healthy population 27% of all S-MUPs were small. MUNE determined without these S-MUPs was marginally less variable. However, MUNE values dropped about 24% at a sample size of 20. In ALS, only 12% of the total population of 130 S-MUPs were small. MUNE dropped about 12% without the small S-MUPs. By omitting small S-MUPs the differences between the healthy and ALS distributions become smaller. Therefore, incorporating small S-MUPs in the estimate is suggested.     

A longitudinal study comparing thenar motor unit number estimates to other quantitative tests in patients with amyotrophic lateral sclerosis

The following data were obtained on 21 amyotrophic lateral sclerosis (ALS) patients, aged 36–76 years (mean: 58 years), at baseline and months 4, 8, and 12: thenar motor unit number estimate (MUNE) using multiple point stimulation, mean thenar surface-recorded motor unit action potential negative-peak area, thenar compound muscle action potential amplitude, isometric hand grip strength, total Medical Research Council (MRC) manual muscle testing score, Appel ALS rating scale, and forced vital capacity (FVC). The absolute mean rate of change per month was significantly greater (P < 0.01) for MUNE values than for MRC and FVC values in the 21 ALS patients. In a subset of patients (n = 6) with slowly progressive disease, the absolute mean rate of change per month was significantly greater (P < 0.01) for MUNE values than for all other test values. In addition, MUNE values were the most sensitive index for documenting changes in disease progression over time. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 179–185, 1997.     

Proximal-distal motor unit number estimation-dynamic changes in patients with amyotrophic lateral sclerosis A one-year follow-up

BACKGROUND:Amyotrophic lateral sclerosis (ALS) is the most common of all the motor neuron diseases and the absence of a biologic marker has made both diagnosis and tracking evolution of the disease difficult, Electrodiagnostic tests play a fundamental role in quantifying pathological changes in the motor unit pool.OBJECTIVE:We assessed distal-proximal Motor Unit (MU) loss and changes using the method of motor unit number estimation (MUNE).DESIGN, TIME AND SETTING:A case-control study was performed at the Department of Neuroscience, Pisa University Medical School, Italy from December 1999 to November 2009. PARTICIPANTS:A total of 50 ALS patients were recruited, 30 males:mean age (59.6 ± 13.3) years; 20 females:mean age (63.9 ± 11.7) years; range (30-82) years; all patients had probable or definite ALS. Thirty healthy volunteers were recruited from department staffs, including 20 males and 10 females; mean age (57.7 ± 13.8) years served as controls.METHODS:MUNE was performed for both the biceps brachii and abductor digiti minimi muscles of the same side. The technique used relayed substantially on manual incremental stimulation of the motor nerve, known as the McComas technique (50 ms sweep duration, a gain of 2 mV/Div for M wave, 0.5 mV/Div for each step; filters 10-20 kHz).MAIN OUTCOME MEASURES:MUNE results were measured.RESULTS:Functioning MU numbers, measured by MUNE, decreased in the biceps brachii and abductor digiti minimi muscles over the entire one-year follow-up period (one assessment every three months) compared with baseline determination, the rate of MU decrease was similar in both muscles, but steeper distally.CONCLUSION:MUNE is a feasible method for ALS patients both proximally and distally to track changes over time in muscle MUs during the disease's evolution.     

Motor unit number estimation in the rat tail using a modified multipoint stimulation technique

Motor unit number estimation (MUNE) of the rodent hindlimb has been used mainly for following the progression of motor neuron disorders. By performing MUNE in the tail, however, progression of axonal neuropathy could also be assessed, as both proximal and distal regions would be available for study. In this investigation, three raters performed a modified multipoint stimulation MUNE technique in the tails of 14 healthy adult rats. The technique was straightforward to perform, with a relatively narrow range of motor unit number estimates of 40 ± 16 (standard deviation) for the proximal tail and 21 ± 11 for the distal tail. Intrarater reliability coefficients were 0.31 (P = 0.033) and 0.32 (P = 0.028) for the proximal and distal tail, respectively. Interrater reliability coefficients were 0.22 (P = 0.086) and 0.44 (P = 0.004). These reliability assessments, along with the relatively low motor unit estimates and narrow range of values, support the idea that rat tail MUNE may have utility in the evaluation of rodent models of neuromuscular disease, including length‐dependent neuropathy. Muscle Nerve 40: 115–121, 2009     

Effect of recording window and stimulation variables on the statistical technique of motor unit number estimation.

A variety of methods are used for the selection of recording window sizes and stimulation current levels for statistical motor unit number estimation (MUNE). This study compares different recording window sizes and stimulation current levels within those windows in the same subjects to determine the effect on MUNE value and reproducibility. Four recording windows of 10% size were compared with four of 5%, with the stimulation current set in the lower quarter, middle half, and upper quarter of the recording window. MUNE for stimulation current set in the lower quarter of the window was 81 (62-103) for 10% recording windows and 120 (108-135) for 5% recording windows, and 91 (61-123) and 133 (120-154) for stimulation current set in the middle half. Increasing the recording window size from 5 to 10% lowers the MUNE value in controls, but tends to improve reproducibility; and setting the stimulation current in the lower quarter of the window, changes the MUNE value minimally, while tending to improve further reproducibility. Excellent reproducibility of MUNE was obtained when applied to a pilot group of 10 amyotrophic lateral sclerosis patients. Based on this study, we conclude that the ideal method for statistical motor unit estimation involves using 10% recording windows and setting the stimulation current in the lower quarter of the recording window.