**6.2 Statistical MUNE method**

636 Amyotrophic Lateral Sclerosis

(MUNE) originated in 1967. At the time there was no satisfactory method of assessing the extent of denervation in muscles during life. Analysis of the density of the electromyographic interference pattern during maximal effort was not quantitative, and

The principle of MUNE is that if one can measure the mean single motor unit amplitude (SMUP), it is possible to obtain an estimate of the total number of motor units in the muscle. The results achieved were comparable with estimates of alpha motor fibres obtained by

MUNE has been performed in a number of different ways, each with their advantages and limitations. (Stein & Yang, 1990). The choice of technique depends on the speed and simplicity of the technique, as well as its accuracy and reproducibility. Some methods sample a very small proportion of the number of motor units innervating a muscle (typically 10-20). The coefficient of variation associated with different methods range from 10-45%. (McComas, 1991). If the variability is too large, then the technique cannot be used to follow

The way the average single motor unit potential (SMUP) size is obtained distinguishes the several techniques available. Most employ electrical stimulation of the motor nerve to

Each method measures both the average size of the potentials generated by single motor units - single motor unit potentials (SMUP) and the size of the compound muscle action

Whereas the methods of measuring the average SMUP differ, they have common assumptions about the measurement of the supramaximal CMAP and the measurement of

i. Maximal stimulation of any peripheral motor nerve activates all the muscles innervated by that nerve distal to the point of stimulation. Therefore, measurements of the CMAP are the summation of activity from multiple muscles and the MUNE is more accurately an estimate of the number of motor units in a group of muscles rather than in a single

For example, the median CMAP recorded at abductor pollicis brevis (APB) is more correctly a "thenar MUNE", as it is a summation of the activity of APB, opponens pollicis, flexor

Extensor digitorum brevis (EDB) on the other hand, is a muscle innervated by the deep peroneal nerve. The only source of interfering muscle action potential is from extensor hallucis longus, which can be reduced by correct position of the stimulating electrodes. The muscle belly is flat in profile, eliminating deeper motor units as a cause of small potentials. The recording electrode is placed transversely across the innervation zone, resulting in a

ii. The motor unit potentials used in the calculation of the average SMUP are representative of those generated by the total population of units. All methods, select a subset of the total population of motor units, measure their sizes and calculate an

Average single motor unit potential SMUP amplitude or area.

required the full co-operation of the patient.

motor unit loss reliably over time.

the average SMUP.

muscle.

counting axons in specimens of motor nerves. (McComas, 1971).

determine the sizes of the SMUP, but a few use needle EMG.

pollicis brevis, and to a lesser extent, the lateral lumbricals.

simple biphasic negative-positive M wave.

average SMUP for that subgroup.

The motor unit number estimate is calculated by:

potential (CMAP) obtained with maximal stimulation of a motor nerve.

Maximum CMAP amplitude or area MUNE=

We used the statistical electrophysiological technique of motor unit number estimation (MUNE), (Daube, 1998), was used to estimate the number of motor units in thenar and extensor digitorum brevis muscles. The statistical method estimates the average size of SMUP's and the number of motor units in a group of muscles innervated by the nerve being stimulated, based on the normal variation of the submaximal CMAP evoked with constant stimuli. No attempt is made to identify individual motor unit potentials. The method relies on the known relation between the variance of multiple measures of step functions and the size of the individual steps when the steps have a Poisson distribution. S.D. Poisson was a French mathematician (1781-1840).

Poisson statistics are useful when the distribution arising for events occur randomly in time or when small particles are distributed randomly in space. They have been used to calculate the number of quanta released from a nerve terminal at the neuromuscular junction when the individual quanta are too small to be distinguished, as in myasthenia gravis. ( Lomen-Hoerth & Slawnych, 2003).

In pure Poisson statistics, the size of a series of measurements is multiples of the size of a single component. In a Poisson distribution there is a discrete asymmetrical distribution in which responses are found at some levels and others where there are no responses (Figure 4). (McNeil, 1996).

A pure Poisson distribution has decreasing numbers at higher values. In Poisson distribution, the variance of these 30 measurements is equal to the size of the individual components making up each measurement. The variance can thus provide an estimate of the average size of the SMUP's.

The statistical method looks only at variance of the CMAP and does not require identification of individual components. It can be used when the sizes of SMUP's are too small to be isolated. The statistical method assumes that each motor unit has a similar size and that it is the same size each time it is activated.

Sequences of 30 submaximal stimuli are given. The inherent variability of the threshold of individual axons causes variations in the size of the CMAP. The average change in the submaximal CMAP amplitude caused by alternation (addition and subtraction of motor axons) is derived by Poisson statistics.

The occurrence of alternation with changing units that are activated does not modify the accuracy of the statistical method, because the method is a statistical measurement, a different result is found with each series of 30 stimuli. Therefore, multiple trials are needed to obtain the most accurate measurement. (Olney et al., 2000).

Experimental testing with trials of >300 stimuli has shown that repeated measurement of groups of 30 until the standard deviation of the repeated trials is <10% provides a close

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 639

Fig. 5. An initial scan of the CMAP (right) recorded from APB muscles in response to 30 sub-

maximal stimuli (x-axis) with equal increments between threshold and maximum stimulation. On the basis of the scan, 10% stimulus ranges are identified, according to an internal algorithm. The CMAP increments are shown at the top left and the eventual table of

results in the bottom left corner.

estimate of the number obtained with many more stimuli.86 Estimates of the SMUP size and of the number of motor units are also most reliable if made at multiple different stimulus intensities to test axons with different thresholds.

Fig. 4. Graphs illustrating Binomial and Poisson distribution (Reproduced from McNeil D. Statistical Methods. 1996; 184). The top graph (a) illustrates that binomial distribution resembles normal distribution with increasing sample size (n=50). The lower graph (b) illustrates that smaller values (λ=5) result in a normal distribution - Poisson distribution.

MUNE is calculated with the number weighted statistical method, where the mean SMUP amplitude at each level is multiplied by the number of motor units estimated at each level.

The steps in statistical MUNE are as follows:


estimate of the number obtained with many more stimuli.86 Estimates of the SMUP size and of the number of motor units are also most reliable if made at multiple different stimulus

Fig. 4. Graphs illustrating Binomial and Poisson distribution (Reproduced from McNeil D. Statistical Methods. 1996; 184). The top graph (a) illustrates that binomial distribution resembles normal distribution with increasing sample size (n=50). The lower graph (b) illustrates that smaller values (λ=5) result in a normal distribution - Poisson distribution. MUNE is calculated with the number weighted statistical method, where the mean SMUP amplitude at each level is multiplied by the number of motor units estimated at each

1. Recording surface electrodes are applied as for standard nerve conduction studies. 2. An initial scan of the CMAP is performed using a series of 30 submaximal stimuli at 1 Hz, increasing in equal increments to identify unusually large steps at which further

3. On the basis of the scan, three or four 10% stimulus ranges are identified, according to an internal algorithm. Usually, one range includes the smallest step and the other

4. At each intensity, groups of 30 responses are captured at a rate of 3Hz. Estimates are most reliable if 10 groups of 30 responses are recorded. To minimise patient discomfort, however, repetition is repeated until the standard error of the MUNE SMUP size is less

5. Statistical MUNE estimates the average size of SMUP's and the number of motor units in a group of muscles innervated by the nerve being stimulated, based on the normal

variation of the sub-maximal CMAP evoked with constant stimuli (Figure 6).

intensities to test axons with different thresholds.

The steps in statistical MUNE are as follows:

ranges where the steps are >15% (Figure 5).

information is required.

than 10%.

level.

Fig. 5. An initial scan of the CMAP (right) recorded from APB muscles in response to 30 submaximal stimuli (x-axis) with equal increments between threshold and maximum stimulation. On the basis of the scan, 10% stimulus ranges are identified, according to an internal algorithm. The CMAP increments are shown at the top left and the eventual table of results in the bottom left corner.

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

well tolerated.

**6.3 MUNE technique** 

surface of the skin.

performed.

Spearman correlation coefficients.

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 641

The statistical technique of estimating the size of the SMUP was performed using proprietary software on a Nicolet Viking IV electromyography machine. This technique uses direct stimulation of the motor nerve. The low frequency filter was set at 2 Hz and the high frequency filter at 5 kHz. The gain for extensor digitorum brevis was set at 2 mV/div and for abductor pollicis brevis studies at 5 mV/div. The sweep speed was 2 ms/div. This method had excellent test-retest reproducibility (+/-2.8%). The method was quick to use and

This technique has been greatly modified since its original description, but numerous studies have shown that MUNE can change systematically in ALS patients when used by experienced technicians, even though evaluator bias needs to be taken into account. (Shefner et al., 2004). The statistical MUNE method has also been shown to be unreliable in the

Our study however was performed on asymptomatic patients, without clinical weakness.

Motor unit numbers were estimated in abductor pollicis brevis (resulting in a thenar MUNE) and the extensor digitorum brevis (EDB) muscle. These muscles were used, as both are easily accessible distal muscles. The electrical activity can be recorded without

Self-adhesive surface recording electrodes (G1) were placed transversely across the innervation zone of each muscle, resulting in a simple biphasic negative-positive M wave, with G2 placed over a bony prominence. The deep peroneal nerve was stimulated just above the ankle and the median nerve at the wrist with a surface stimulator. This was performed by strapping the stimulating electrode onto the surface of the skin, at the point where the threshold of the nerve to electrical stimulation was at its' lowest. A hand-held stimulator was not used, as reproducibility is enhanced when the stimulating electrodes are fixed to the

Initially, bilateral thenar and EDB MUNE's were obtained from all subjects. After the reproducibility phase of the study, generally only right-sided studies were performed. Once a reduction in MUNE was identified, bilateral studies were once again performed on selected subjects. The protocol was also modified depending on the subjects' tolerance to the procedure. Median nerve stimulation at the wrist for thenar MUNE was generally well tolerated by most subjects, as the stimulation intensity required to obtain an adequate response was

Peroneal nerve stimulation required for EDB MUNE resulted in slightly more discomfort, as the nerve is located further away from the surface of the skin. The stimulus intensity required, in some cases was up to 50-80mA with duration of between 0.1-0.3ms. Some subjects indicated that they were unwilling to continue to participate in the study due to the discomfort caused by performing EDB MUNE. In these subjects, only thenar MUNE's were

To assess the test-retest reproducibility of the technique, SOD1 family members and population controls were followed over a 1-year period, with thenar and EDB MUNE tests repeated every 3 to 6 months. The difference between MUNE results from the first and second study, and if possible, first and third studies were divided by the MUNE of the first study, and expressed as a percentage change. The results were analysed using Pearson and

presence of clinical weakness due to motor unit instability. (Shefner, 2009).

interference, and in the case of EDB, the muscle belly is flat.

generally less than 20mA with duration of 0.05-0.1ms.

Fig. 6. At each intensity level (runs 1-4), groups of 30 responses are captured at a rate of 3Hz. The CMAP amplitudes are shown at the top left, with the histogram of results at the top right. The thenar MUNE results from repeated trials are shown in the bottom left table.

The statistical technique of estimating the size of the SMUP was performed using proprietary software on a Nicolet Viking IV electromyography machine. This technique uses direct stimulation of the motor nerve. The low frequency filter was set at 2 Hz and the high frequency filter at 5 kHz. The gain for extensor digitorum brevis was set at 2 mV/div and for abductor pollicis brevis studies at 5 mV/div. The sweep speed was 2 ms/div. This method had excellent test-retest reproducibility (+/-2.8%). The method was quick to use and well tolerated.

This technique has been greatly modified since its original description, but numerous studies have shown that MUNE can change systematically in ALS patients when used by experienced technicians, even though evaluator bias needs to be taken into account. (Shefner et al., 2004). The statistical MUNE method has also been shown to be unreliable in the presence of clinical weakness due to motor unit instability. (Shefner, 2009).

Our study however was performed on asymptomatic patients, without clinical weakness.
