**6.3 MUNE technique**

640 Amyotrophic Lateral Sclerosis

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.

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

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 surface of the skin.

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 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 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 Spearman correlation coefficients.

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

3. 20 asymptomatic (pre-clinical) SOD1 mutation carriers (test group),

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

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

subjects were divided into four test groups.

2. 32 SOD1 negative (normal) family controls;

**7. Results** 

**7.1 Demographics** 

1. 24 population controls;

glu100gly;

ile113thr;

val148gly;

val148ile.

**SOD1 Negative Family** 

with a range of 16 to 73 years of age.

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 643

A total of eighty-eight (88) subjects (45 males and 43 females) gave informed consent. The

a. 5 subjects with point mutation in exon 4, codon 100, GAA to GGA, Glu to Gly) –

b. 5 subjects with point mutation in exon 4, codon 113, ATT to ACT, Ile to Thr) –

c. 5 subjects with point mutation in exon 5; codon 148, GTA to GGA, Val to Gly) –

d. 5 subjects with point mutation in exon 5, codon 148, GTA to GGA, Val to Ile)

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

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

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 7).

> **Thenar (APB) muscle Cases MUNE (Range)**

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

**Population Controls** 24 148 (115-254)

**Controls** 32 138 (106-198) **SOD1 Mutation Carriers** 20 144 (109–199) **Sporadic MND patients** 12 45 (5–84)

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

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 t-test. 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 compared 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**

It has been suggested that the traditional neurological examination is inadequate for documenting 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).

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).
