**6. Methods**

The nitric oxide (NO) produced by nitric oxide synthase (NOS) reacts spontaneously with O2-

may also cause an increase in O2- production by inhibition of mitochondrial electron flow, resulting in further generation of peroxynitrite. This facilitates nitrosylation of tyrosine residues of critical cytosolic proteins thus injuring cells. This reaction is copper dependent. The source of free copper may be mutant SOD1, which cannot accept the ion from the copper chaperone (CCS) protein. Mutant SOD1 possibly exhibit metal mediated cytotoxicities by disrupting the intracellular homeostasis of Cu and Zn, which are potential neurotoxins.

The target proteins for nitrosylation include the neurofilament (NF) subunits, which may result in abnormal NF accumulation and subsequent disruption of the NF network and axonal transport, as there is a high neurofilament content in motor neurones. It has also been dem‐ onstrated that transgenes encoding mutant NF subunits can directly cause selective degener‐ ation and death of motor neuones. (Cleveland, 1999). Conformational changes have been described in the mutations, Ala4Val, Gly37Arg and His6Arg that may affect the rim of the

Glutamate is released from the presynaptic terminal activates the glutamate receptor on the postsynaptic cell membrane. It is then cleared from the synaptic cleft by specific glutamate transporters such as EAAT2. (Trotti et al., 1999). Astrocyte (glial cell) dysfunction may result in selective loss of EAAT2, interfering with the normal clearance of glutamate and allowing it to accumulate in the cell membrane and continue to activate the receptor. (Bruijin et al., 1997). Once activated, the glutamate receptor causes a calcium influx and a cascade of toxicity. The neurone does not have the capacity to buffer this efficiently due to a deficiency in calcium binding proteins (CBP's). This results in disturbances in mitochondrial metabolism and as a

To date, the only effective approved treatment for amyotrophic lateral sclerosis is Riluzole, (Cheah et al., 2010), which has a neuroprotective role, possibly due to pre-synaptic inhibition of glutamate release. (Doble, 1996). Treatment of human ALS patients or transgenic Cu, Zn superoxide dimutase 1 (SOD 1) mice, most commonly produce a modest but significant increase in survival. (Bensimon et al., 1994). It has also been shown to have a small beneficial

Apoptosis is characterised by a series of cellular changes leading to non-inflammatory cell death. Mitochondrial involvement in the apoptotic pathway also leads to the release of cytochrome c, an activator of the initiator caspase-9, which in turn activates caspase-3, which are executioners in the breakdown of essential cellular proteins. There is evidence that the mutant SOD1 transgene causes motor neurone death in mice through caspase-mediated programmed cell death. (Li et al., 2000). This may then be a target for inhibiting the apoptotic cascade, as it has been shown in a SOD1 transgenic mouse model that a small peptide caspase inhibitor (*zVAD-fmk*), prolonged survival after onset of disease by nearly 70%. (Kosti et al., 1997). It has also been reported that there are elevated levels of *bax* protein in MND spinal

electrostatic guidance channel coded by exon 3. (Sjalander et al., 1995).

effect on bulbar function, but not muscle strength. (Miller et al., 2007).

motor neurones, which promotes apoptosis. (Mu et al., 1996).

consequence, motor neurone cell death. (Beal, 1996).

), which nitrosylates proteins leading to damage. Excess NO

to generate peroxynitrite (ONOO-

192 Current Advances in Amyotrophic Lateral Sclerosis

(Gurney & Tomasselli, 1994).

The Department of Molecular Medicine at Concord hospital had a large database of family members with a known family history of MND, who had blood samples collected for DNA, as part of a previous linkage study. From this database, family members were contacted by telephone by the department's genetic counsellor and informed about the study.

The regional committees for Ethics in Medical Research from Central Sydney Area Health Service, Royal North Shore Hospital and Prince Charles Hospital, approved this study.

All individuals participated without knowledge of their mutation status and on the under‐ standing that this would not be revealed to them. Subjects were also aware that the results obtained from the study would not be available to them and that the information would only be used for research purposes. New consents were obtained from all individuals who partici‐ pated in the study. The neurologist performing the MUNE studies also had no knowledge of their mutation status. The mutation status was only used in the final analysis of results. Subsequently, they were divided into "SOD1 negative family controls" and "asymptomatic SOD1 mutation carriers".

In addition, studies were also carried out on normal individuals, such as department techni‐ cians, spouses of SOD1 family members and individuals from the general population who attended MND support meeting and had an interest in helping to advance research into MND. This group was used as "population controls", to test the validity and reproducibility of the MUNE technique used.

Sporadic MND subjects were also initially studied once the MUNE technique had been validated to demonstrate that the MUNE technique used was able to detect a loss of motor neurones, when present. These were used as "positive controls".

#### **6.1. Motor unit number estimation**

Motor unit number estimation (MUNE) estimates the number of functioning lower motor neurones innervating a muscle or a group of muscles and is a measure of the primary patho‐ logic process of motor neurone loss. The concept of motor unit number estimation (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 interfer‐ ence pattern during maximal effort was not quantitative, and required the full co-operation of the patient.

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

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 motor unit loss reliably over time.

recorded. This can result in an underestimation of the mean SMUP size, as it may appear that there are 7 or 8 motor units when there are only 2 or 3 present, which in

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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

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

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,

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

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

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

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

turn results in an over-estimation of the MUNE.

**6.2. Statistical MUNE method**

(1781-1840).

1996).

SMUP's.

size each time it is activated.

axons) is derived by Poisson statistics.

the most accurate measurement. (Olney et al., 2000).

& Slawnych, 2003).

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

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

The motor unit number estimate is calculated by:

MUNE= Maximum CMAP amplitude (or area) Average single motor unit potential (SMUP) amplitude or area.

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

**i.** Maximal stimulation of any peripheral motor nerve activates all the muscles inner‐ vated 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 muscle.

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

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 simple biphasic negative-positive M wave.


recorded. This can result in an underestimation of the mean SMUP size, as it may appear that there are 7 or 8 motor units when there are only 2 or 3 present, which in turn results in an over-estimation of the MUNE.

#### **6.2. Statistical MUNE method**

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 motor unit loss reliably

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 potential

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

**i.** Maximal stimulation of any peripheral motor nerve activates all the muscles inner‐

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 pollicis

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 simple

**ii.** The motor unit potentials used in the calculation of the average SMUP are represen‐

**iii.** Finally, there is a phenomenon caused "alternation". This refers to fluctuations in the

tative 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

CMAP amplitude of the same motor unit with similar stimulation intensities. The thresholds of the first few motor axons excited are not sufficiently separate from one another, so that when graded increases in the stimulus intensity occur, the motor axons excited often overlap and add more than one SMUP to the CMAP being

vated 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

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

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

Average single motor unit potential (SMUP) amplitude or area.

The motor unit number estimate is calculated by:

MUNE= Maximum CMAP amplitude (or area)

in a single muscle.

biphasic negative-positive M wave.

SMUP for that subgroup.

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

over time.

194 Current Advances in Amyotrophic Lateral Sclerosis

SMUP.

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 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.

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:


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).

accessible distal muscles. The electrical activity can be recorded without interference, and in

**Figure 4.** 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

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Self-adhesive surface recording electrodes (G1) were placed transversely across the innerva‐ tion 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 case of EDB, the muscle belly is flat.

table of results in the bottom left corner.

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

#### **6.3. MUNE Technique**

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

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 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

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.

**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 infor‐

**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 ranges

**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

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

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

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

variation of the sub-maximal CMAP evoked with constant stimuli (Figure 5).

test-retest reproducibility (+/-2.8%). The method was quick to use and well tolerated.

**1.** Recording surface electrodes are applied as for standard nerve conduction studies.

to test axons with different thresholds.

196 Current Advances in Amyotrophic Lateral Sclerosis

mation is required.

than 10%.

**6.3. MUNE Technique**

The steps in statistical MUNE are as follows:

where the steps are >15% (Figure 4).

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

**Figure 4.** 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.

accessible distal muscles. The electrical activity can be recorded without interference, and in the case of EDB, the muscle belly is flat.

Self-adhesive surface recording electrodes (G1) were placed transversely across the innerva‐ tion 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

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 generally

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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

To assess the test-retest reproducibility of the technique, SOD1 family members and popula‐ tion 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 Spearman

All results were entered into a database and analysed using a standard statistical software package (SPSS 9.05 for Windows). For the initial part of the study, the MUNE results from asymptomatic SOD1 mutation carriers were grouped together. Although different mutations in SOD1 have different effects on the progression of the disease once symptoms occur, these

Motor unit estimates in carriers were compared to age and sex matched family controls without the SOD1 mutation, and sporadic (non-SOD1) MND patients. To determine whether groups had different numbers of motor units, an unpaired t-test was used. Although there were some outlying results, the distributions were not sufficiently skewed to contradict the use of the t-

The group of asymptomatic SOD1 mutation carriers were followed over the next 2 to 5 years, depending on the volunteers' motivation, both clinically and by MUNE. Results were com‐ pared to their initial baseline MUNE and the date of the study when this reduction was first

It has been suggested that the traditional neurological examination is inadequate for docu‐ menting motor performance impairment with reliability. (Hanten et al., 1999). Generally, manual motor testing used in a standard neurological motor examination does not allow objective documentation of change in performance, as it may be influenced by the patient's history and progress. Major changes are apparent, but subtle changes are difficult to determine

There are a number of methods that have been developed to quantify maximal voluntary isometric contraction (MVIC). It has been proposed that this is a clinically useful, reliable,

different mutations do not influence on the age of onset of symptoms.67

test. Statistical significance was accepted at a p-value of <0.05.

**6.4. Maximal voluntary isometric contraction testing**

detected, was used as the date when motor neurone loss commenced.

by performing EDB MUNE. In these subjects, only thenar MUNE's were performed.

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

correlation coefficients.

with accuracy.

**Figure 5.** At each intensity level (runs 1-4), groups of 30 responses are captured at a rate of 3Hz. The CMAP ampli‐ tudes 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 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 surface of the skin.

Initially, bilateral thenar and EDB MUNE's were obtained from all subjects. After the repro‐ ducibility 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 generally less than 20mA with duration of 0.05-0.1ms.

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 performed.

To assess the test-retest reproducibility of the technique, SOD1 family members and popula‐ tion 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 Spearman correlation coefficients.

All results were entered into a database and analysed using a standard statistical software package (SPSS 9.05 for Windows). For the initial part of the study, the MUNE results from asymptomatic SOD1 mutation carriers were grouped together. Although different mutations in SOD1 have different effects on the progression of the disease once symptoms occur, these different mutations do not influence on the age of onset of symptoms.67

Motor unit estimates in carriers were compared to age and sex matched family controls without the SOD1 mutation, and sporadic (non-SOD1) MND patients. To determine whether groups had different numbers of motor units, an unpaired t-test was used. Although there were some outlying results, the distributions were not sufficiently skewed to contradict the use of the ttest. Statistical significance was accepted at a p-value of <0.05.

The group of asymptomatic SOD1 mutation carriers were followed over the next 2 to 5 years, depending on the volunteers' motivation, both clinically and by MUNE. Results were com‐ pared to their initial baseline MUNE and the date of the study when this reduction was first detected, was used as the date when motor neurone loss commenced.

#### **6.4. Maximal voluntary isometric contraction testing**

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 surface of the skin.

**Figure 5.** At each intensity level (runs 1-4), groups of 30 responses are captured at a rate of 3Hz. The CMAP ampli‐ tudes 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.

198 Current Advances in Amyotrophic Lateral Sclerosis

Initially, bilateral thenar and EDB MUNE's were obtained from all subjects. After the repro‐ ducibility phase of the study, generally only right-sided studies were performed. Once a It has been suggested that the traditional neurological examination is inadequate for docu‐ menting motor performance impairment with reliability. (Hanten et al., 1999). Generally, manual motor testing used in a standard neurological motor examination does not allow objective documentation of change in performance, as it may be influenced by the patient's history and progress. Major changes are apparent, but subtle changes are difficult to determine with accuracy.

There are a number of methods that have been developed to quantify maximal voluntary isometric contraction (MVIC). It has been proposed that this is a clinically useful, reliable, reproducible, time efficient and quantitative measure for monitoring disease progression in MND. (Hoagland et al., 1997). This would be surprising, given that in a slowly progressive denervating process, patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units occurs. (McComas, 1971).

The methods used to quantify maximal voluntary isometric contraction have included an electronic strain-gauge tensiometer and a hand-held Jamar hydraulic dynamometer. In this study, maximum bilateral isometric grip strength was obtained using the Jamar hydraulic dynamometer to determine whether this correlated with the number of functional motor neurones in the thenar group of muscles, as measured by MUNE. Standardised (middle handle) positioning and instructions were given to all subjects. Handgrip force was measured with subjects in the sitting position and with the arm flexed at 90 degrees. Two trials were performed on each hand, and the best result used for analysis. This method was used as previous studies of grip strength reliability showed that there was no significant difference in reliability between one attempt, the mean score of two or three attempts, or the highest score of three attempts. (Hamilton et al., 1994).

**4.** 12 sporadic symptomatic MND patients (positive controls).

**Table 1.** Thenar (APB) motor unit number estimates (MUNE number represents mean MUNE).

**7.2. Motor units in asymptomatic FALS (SOD1) carriers**

Statistical significance was accepted at a p-value of <0.05.

carriers, 136 with a range of 111 - 187.

a range of 16 to 73 years of age.

There was no statistically significant difference in age distribution between these groups, with

**Population Controls** 24 148 (115-254) **SOD1 Negative Family Controls** 32 138 (106-198) **SOD1 Mutation Carriers** 20 144 (109–199) **Sporadic MND patients** 12 45 (5–84)

**Thenar (APB) muscle**

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201

**Cases MUNE (Range)**

The Role of the Statistical Method of Motor Unit Number Estimation (MUNE) to…

For the initial part of the study, the baseline MUNE results were grouped together and the means of the groups were compared. The initial aim of the study was to determine if MND was due to a slow gradual attrition of motor neurones over time. If this were the case, the group of asymptomatic SOD1 mutation carriers, would be expected to have a reduced number of motor units, indicating the presence of pre-clinical motor neurone loss. Motor unit estimates in the group of asymptomatic SOD1 mutation carriers were compared to age and sex matched family controls without the SOD1 mutation, and sporadic (non-SOD1) MND patients. To determine whether groups had different numbers of motor units, an unpaired t-test was used.

The numbers of motor units in the groups of population controls, SOD1 negative family controls and asymptomatic SOD1 mutation carriers were similar. In population controls the mean thenar MUNE was 148 with a range of 115 - 254, in SOD1 negative family controls was 138 with a range of 106 - 198 and in asymptomatic SOD1 mutation carriers, 144 with a range of 109 - 199. There was no detectable difference in the mean number of thenar motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (thenar p>0.46), or population controls (thenar p>0.70) (Table 1 and Figure 6).

In population controls the mean EDB MUNE was 138 with a range of 119 - 169, in SOD1 negative family controls was 134 with a range of 107 - 180 and in asymptomatic SOD1 mutation

Once again, there was no detectable difference in the mean number of EBD motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (EDB p>0.95), or population controls (EDB p>0.50) (Table 2 and Figure 7).

Clinical neurological examination was performed, with power of thumb abduction, finger flexion and finger abduction measured according to the Medical Research Council (MRC) grading system and compared to thenar (APB) MUNE.

Felice showed that in twenty one MND patients, changes in thenar MUNE was the most sensitive outcome measure for following disease progression, when compared to other quantitative tests, such as CMAP, isometric grip strength, forced vital capacity and Medical Research Council manual muscle testing. (Felice, 1997).
