**8. Conclusion**

On examination, she had no evidence of wasting, but there were generalised fasciculations, especially in the triceps and quadriceps regions. Her tone was normal in the upper and lower limbs. She had no clinical weakness with MRC grade 5/5 power in all muscle groups, proxi‐ mally and distally. Her sensory examination was normal to touch, vibration and position. Her reflexes were all present and symmetrical. Her gait was normal, as she was able to walk on her heels and toes. She was able to perform tandem walking and Romberg's sign was negative. Nerve conduction studies performed by independent neurologist were normal, with no evidence of a large fibre peripheral neuropathy. Needle EMG studies was also normal, with no evidence of active or chronic denervation in bilateral distal and proximal muscles sampled. Initial EDB MUNE was reduced with normal APB MUNE's. Despite this, a clinical diagnosis on fALS was not made given her normal needle EMG study, and she was observed over the next 6 months. Over this time, she developed MRC grade 4/5 weakness of ankle dorsiflexion, bilaterally and her EDB MUNE dropped by 14-20%. Despite this reduction, compound muscle action potential amplitudes were maintained. Needle EMG studies were repeated and once again normal there was no spontaneous activity (fibrillation potentials) and motor unit

**Table 6.** showing progressive handgrip, dorsiflexion power, thenar and EDB MUNE and needle EMG results of Case 3 (Normal EMG refers to the absence of fasciculation and fibrillation potentials, normal motor unit potentials and

**Sept-08 Feb-09 Aug-09 Aug-10**

124 (14% increase)

123 (17% increase)

134 (38% increase)

127 (41% increase) 131

124

134

117

**Months Baseline 5 months 6 months 12 months Riluzole commenced R Handgrip** 65 70 68 65 **L Handgrip** 62 70 62 65

> 109 (13% decrease)

> 105 (23% decrease)

97 18% decrease)

90 (13% decrease)

Given her family history, a presumptive diagnosis of non-SOD 1 fALS was made and com‐ menced on Riluzole therapy. This resulted in an improvement in clinical symptoms of tiredness and fasciculations, allowing her to return to work. Her EDB MUNE improved by 34-60%, and increased further over the next year. Despite this, her treating neurologist considered this was a placebo effect and ceased Riluzole. Within 2 weeks, her generalised aches

recruitment was normal, despite the presence of weakness.

**R APB MUNE** 126

210 Current Advances in Amyotrophic Lateral Sclerosis

**L APB MUNE** 136

**R EDB MUNE** 107

**L EDB MUNE** 104

**Needle EMG** Normal

normal recruitment)

Motor neurone disease (MND) is a group of fatal, progressive neurodegenerative disorders, with an overall median survival is approximately 4.0 years from the onset of symptoms. By the time most patients with MND are aware of clinical weakness and seek review by their primary physician or neurologist, a significant proportion of motor units have already been lost. Early detection of motor neurone loss in clinically apparently unaffected muscles is therefore important to establish an early diagnosis of the condition.

Motor unit number 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. There was no detectable difference in the 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).. In addition,


there was no detectable difference in the number of EBD motor units in the group of asymp‐ tomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (EDB p>0.95), or population controls (EDB p>0.50). Symptomatic sporadic MND subjects showed a definite loss of motor units with fewer motor units compared to all other groups (p<0.001). There was no overlap between MUNE results in symptomatic and asymptomatic subjects. These results indicate that the group of asymptomatic carriers of the SOD1 mutation have no significant difference in the number of motor neurones, when compared to age and sex matched controls. All carriers had a full complement of motor neurones during the asympto‐ matic phase, indicating that mutation carriers have normal survival of motor neurones and that symptomatic MND is not the end result of a slow attrition of motor neurones. This implies that gradual pre-symptomatic loss of motor neurones does not occur in asymptomatic SOD1 mutation carriers. This supports the observation that sudden, catastrophic loss of motor neurones occurs immediately prior to the onset of symptoms and the development of the disease, rather than a gradual attrition of motor neurones over time. These results suggest that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms. This observation is an important contribution to the current understanding of the pathogenesis

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213

The statistical MUNE technique was used for the study. 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. It has been suggested that the statistical MUNE is unreliable in the presence of clinical weakness due to motor unit instability. (Shefner, 2009). The difference is that our study was performed on asymptomatic patients, without clinical

MUNE has been performed in a number of different ways over the years, each with their advantages and limitations. The choice of technique depends largely on the speed and simplicity of the technique, as well as its accuracy and reproducibility. The way the average single motor unit potential (SMUP) size is obtained distinguishes the several techniques. Most employ electrical stimulation of the motor nerve to determine the sizes of the SMUP, but a few use needle EMG. The statistical MUNE technique was used for the study. (Daube, 1995). This technique has been compared to the multiple point stimulation method and found to be more reproducible (7% vs. 12%) and faster to administer. (Lomen-Hoeth & Olney, 2000). The technique has been greatly modified since its original description, (Shefner et al, 2004), but numerous studies have shown that MUNE can be used to monitor change in ALS patients when used by experienced technicians, even though evaluator bias needs to be taken into

There have been studies performed indicating that the statistical MUNE is unreliable in the presence of clinical weakness due to motor unit instability in ALS. (Shefner et al., 2011). In our previous study, we showed that there was no detectable difference in the number of motor units in 19 SOD 1 mutation carriers as a group, compared to their 34 SOD 1 negative family controls (APB p>0.46 and EDB p>0.95), or 23 population controls (APB, p>0.70 and EDB, p>0.50). (Aggarwal & Nicholson, 2001). It also showed that MUNE may be used as a reliable

of MND. (Aggarwal & Nicholson, 2001).

weakness.

account. (Shefner et al., 2004).

**Table 7.** showing progressive handgrip, dorsiflexion power, thenar and EDB MUNE and needle EMG results of Case 4 (Normal EMG refers to the absence of fasciculation and fibrillation potentials, normal motor unit potentials and normal recruitment)

there was no detectable difference in the number of EBD motor units in the group of asymp‐ tomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (EDB p>0.95), or population controls (EDB p>0.50). Symptomatic sporadic MND subjects showed a definite loss of motor units with fewer motor units compared to all other groups (p<0.001). There was no overlap between MUNE results in symptomatic and asymptomatic subjects.

**Mar-04 Oct-04 Apr-05 Nov-05 Mar-06 Jun-06**

**Riluzole Ceased**

130 (34% increase)

147 (60% increase)

BD

124 123

130 129

100 110

107 126

113 134 126 133

106

98

(34% increase)

(45% increase)

(18% increase)

(43% increase) **Riluzole Recommenced Apr-06**

> 99 (23% decrease)

> 90 (39% decrease)

117 123

126 128

111 121

123 120

**Months Baseline 7 months 6 months 7 months 4 months 3 months**

**R Handgrip** 50 20 50 40 40 35

**L Handgrip** 40 20 40 38 40 30 **L Thenar MUNE** 131 105 105 121 118 124 **R EDB power** 5/5 4/5 4/5 4/5 4/5 4/5

> 84 (33% increase)

> 92 (56% increase)

**Needle EMG** Normal Normal Normal **Date of study Dec-06 Jul-07 Mar-08 Sept-08 May-09 Nov-09 Apr-10 Months 6 months 7 months 8 months 6 months 8 months 6 months 5 months**

**R Handgrip** 55 45 25 34 40 40 40

(30% decrease)

**L Handgrip** 50 45 25 35 45 30 32

(32% decrease)

**R EDB power** 4/5 4/5 4/5 4+/5 4/5 4+/5 4+/5

(30% decrease)

**L EDB power** 4/5 4/5 4/5 4+/5 4+/5 4+/5 4+/5

(40% decrease)

**Table 7.** showing progressive handgrip, dorsiflexion power, thenar and EDB MUNE and needle EMG results of Case 4 (Normal EMG refers to the absence of fasciculation and fibrillation potentials, normal motor unit potentials and

132 93

146 88

**Riluzole Daily** Riluzole

**L EDB power** 5/5 4/5 4/5 4/5 4/5 4/5

**Riluzole Started**

96 (36% decrease)

63 (20% decrease)

59 (14% decrease)

126 132 92

122 131 89

124 (25% increase)

117 (30% increase)

**Riluzole changes**

**R Thenar MUNE** 151

212 Current Advances in Amyotrophic Lateral Sclerosis

**R EDB MUNE** 79

**L EDB MUNE** 69

**Riluzole changes**

**R Thenar MUNE**

**L Thenar MUNE**

**R EDB MUNE**

**L EDB MUNE**

normal recruitment)

These results indicate that the group of asymptomatic carriers of the SOD1 mutation have no significant difference in the number of motor neurones, when compared to age and sex matched controls. All carriers had a full complement of motor neurones during the asympto‐ matic phase, indicating that mutation carriers have normal survival of motor neurones and that symptomatic MND is not the end result of a slow attrition of motor neurones. This implies that gradual pre-symptomatic loss of motor neurones does not occur in asymptomatic SOD1 mutation carriers. This supports the observation that sudden, catastrophic loss of motor neurones occurs immediately prior to the onset of symptoms and the development of the disease, rather than a gradual attrition of motor neurones over time. These results suggest that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms. This observation is an important contribution to the current understanding of the pathogenesis of MND. (Aggarwal & Nicholson, 2001).

The statistical MUNE technique was used for the study. 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. It has been suggested that the statistical MUNE is unreliable in the presence of clinical weakness due to motor unit instability. (Shefner, 2009). The difference is that our study was performed on asymptomatic patients, without clinical weakness.

MUNE has been performed in a number of different ways over the years, each with their advantages and limitations. The choice of technique depends largely on the speed and simplicity of the technique, as well as its accuracy and reproducibility. The way the average single motor unit potential (SMUP) size is obtained distinguishes the several techniques. Most employ electrical stimulation of the motor nerve to determine the sizes of the SMUP, but a few use needle EMG. The statistical MUNE technique was used for the study. (Daube, 1995). This technique has been compared to the multiple point stimulation method and found to be more reproducible (7% vs. 12%) and faster to administer. (Lomen-Hoeth & Olney, 2000). The technique has been greatly modified since its original description, (Shefner et al, 2004), but numerous studies have shown that MUNE can be used to monitor change in ALS patients when used by experienced technicians, even though evaluator bias needs to be taken into account. (Shefner et al., 2004).

There have been studies performed indicating that the statistical MUNE is unreliable in the presence of clinical weakness due to motor unit instability in ALS. (Shefner et al., 2011). In our previous study, we showed that there was no detectable difference in the number of motor units in 19 SOD 1 mutation carriers as a group, compared to their 34 SOD 1 negative family controls (APB p>0.46 and EDB p>0.95), or 23 population controls (APB, p>0.70 and EDB, p>0.50). (Aggarwal & Nicholson, 2001). It also showed that MUNE may be used as a reliable method of pre-symptomatic detection of motor unit loss in SOD1 mutation carriers. Following 69 SOD1 family members and population controls over a 1-year period, with thenar and EDB MUNE tests repeated every 3 to 6 months, assessed the test-retest reproducibility of the technique. The mean difference between thenar MUNE results on separate occasions in asymptomatic subjects was +/- 3.6%, with a range of 0-11.7%, and +/- 4.6%, with a range of 0-15.7% in EDB MUNE. Our test-retest correlation was high, with Pearson correlation coeffi‐ cients of 0.93 for APB MUNE and 0.78 for EDB MUNE. (Aggarwal, 2009). This indicates that there is reproducibility of our statistical MUNE technique, despite recent revisions and reservations. The results can be used as a baseline for progressive MUNE studies and any change in MUNE of greater than 5% should not be considered as a variation in measurement. This is contrary to a recent report indicating that the statistical MUNE cannot be used to detect mild to moderate motor unit loss. (Blok et al., 2010).

commencing Riluzole and recurred after discontinuing Riluzole. She also had an improvement in MUNE and subsequent decline in MUNE, which are temporally associated with the

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215

After commencing Riluzole, there was an improvement in symptoms and MUNE. It is therefore possible that if treatment is commenced prior to significant motor neurone loss, the

In case 3, MUNE dropped when treatment was ceased on two separate occasions and improved when treatment was recommenced, with the operator being unaware of the Riluzole dose

In our series, we noted an improvement in symptoms, especially a decrease in muscle fasciculations and an increase in MUNE number after commencing Riluzole. We suggest that previous trials have been performed in the symptomatic phase of the disease when 70-80% of motor units have already been lost, rather than in the pre-symptomatic phase of the disease, when the therapeutic benefit might be possible. If "treatment" is commenced prior to signifi‐ cant motor neurone loss occurring, the progression of disease may be able to slowed down. MUNE is believed to reduce because of remodelling of the motor units and in our study, the compound muscle action potential amplitudes were maintained despite a reduction in MUNE. This is because early in the disease, the rate of cell death is low. (Cheah et al., 2010). The increase in MUNE may be either due to reinnervation of the damaged muscles or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP.

In one of the cases in the study, Riluzole was commenced once she developed mild weakness. At the time, there was a slight reduction in MUNE, but conventional needle EMG examination did not fulfil the criteria to make the diagnosis of MND. In view of her strong family history and positive genetic testing, a presumed diagnosis of MND was made. Since commencing Riluzole there has been no significant reduction in her EDB MUNE over the last 2 years, and her weakness of right dorsiflexors has only progressed marginally from MRC grade 4+/5 to 4/5 power. It is possible that since "treatment" was commenced prior to the loss of a significant number of motor neurones, this may have slowed down the progression of the disease in this individual case. Early in the course of ALS, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. As the amount of intracellular damage increases, a critical threshold is reached, which overwhelms cellular homeostasis, resulting in rapid apoptosis and cell death. The increase in MUNE numbers may be either due to reinner‐ vation of the damaged muscle or repair of poorly functioning synapses, at the early stage of

Maximum isometric grip strength was also obtained using the Jamar hydraulic dynamometer with standardised (middle handle) positioning and instructions. Maximum isometric grip strength did not reduce, even when MUNE dropped, once again supporting McComas' theory that patients can maintain normal muscle twitch tension until loss of about 70-80% of motor units, before collateral reinnervation was unable to provide functional compensation. (McComas et al., 1971). Maximum isometric grip strength using the Jamar hydraulic dyna‐ mometer also does not correlate with the number of functional motor neurones in thenar group

administration of Riluzole.

changes.

progression of disease can be slowed down.

the disease, without resulting in a change in CMAP.

During the course of the study, a significant fall in motor unit number was detected in 5 of the SOD1 mutation carriers, several months before the onset of weakness and the diagnosis of motor neurone disease (MND) being made. There was no detectable loss of motor units in the other 15 SOD1 mutation carriers or in the group of SOD1 mutation negative relatives. From the study, a threshold MUNE of less than 100 was considered to imply that symptoms were imminent.

In individual cases, there was a reduction of 68% 8 months prior, 51% 4 months prior, 46% 6 months prior, 35% 10 months prior and 28% 6 months prior to the onset of weakness. Further motor unit loss occurred as weakness progressed and the diagnosis of MND being made.

We followed 3 subjects with a family history of ALS, 2 of which had a documented SOD 1 point mutation, who were commenced on Riluzole therapy when loss of motor units were detected using motor unit number estimation (MUNE), before the onset of symptoms i.e. presymptomatic phase. A reduction in sequential MUNE was shown to improve with a thera‐ peutic intervention, Riluzole. Currently, the only effective approved treatment for MND is Riluzole, which has a neuroprotective role, possibly due to pre-synaptic inhibition of glutamate release. Riluzole is an anti-glutamate agent that has been approved for the treatment of patients with amyotrophic lateral sclerosis in most countries. 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). There have been a least three large randomised trials involving hundreds of patients that have been unable to show that Riluzole is a disease altering agent nor does it have any restorative reports. (Miller et al., 2007).

Cases 1 and 2 are SOD 1 positive mutation carriers who developed non-specific symptoms of muscle fasciculations with no clinical weakness, but had reductions in MUNE on sequential testing. Needle EMG studies were "normal", with no evidence of active or chronic denervation in muscles sampled from bilateral distal and proximal muscles. Normal EMG was defined as the absence of fasciculation and fibrillation potentials, normal motor unit potentials and normal recruitment. A presumptive diagnosis of fALS was made, even though there are reports on incomplete penetrance. In case 3, one could argue that there is no clear evidence that she has fALS, even though she has a strong family history of ALS with an autosomal dominant pattern of inheritance. Her episodic weakness and fasciculations improved after commencing Riluzole and recurred after discontinuing Riluzole. She also had an improvement in MUNE and subsequent decline in MUNE, which are temporally associated with the administration of Riluzole.

method of pre-symptomatic detection of motor unit loss in SOD1 mutation carriers. Following 69 SOD1 family members and population controls over a 1-year period, with thenar and EDB MUNE tests repeated every 3 to 6 months, assessed the test-retest reproducibility of the technique. The mean difference between thenar MUNE results on separate occasions in asymptomatic subjects was +/- 3.6%, with a range of 0-11.7%, and +/- 4.6%, with a range of 0-15.7% in EDB MUNE. Our test-retest correlation was high, with Pearson correlation coeffi‐ cients of 0.93 for APB MUNE and 0.78 for EDB MUNE. (Aggarwal, 2009). This indicates that there is reproducibility of our statistical MUNE technique, despite recent revisions and reservations. The results can be used as a baseline for progressive MUNE studies and any change in MUNE of greater than 5% should not be considered as a variation in measurement. This is contrary to a recent report indicating that the statistical MUNE cannot be used to detect

During the course of the study, a significant fall in motor unit number was detected in 5 of the SOD1 mutation carriers, several months before the onset of weakness and the diagnosis of motor neurone disease (MND) being made. There was no detectable loss of motor units in the other 15 SOD1 mutation carriers or in the group of SOD1 mutation negative relatives. From the study, a threshold MUNE of less than 100 was considered to imply that symptoms were

In individual cases, there was a reduction of 68% 8 months prior, 51% 4 months prior, 46% 6 months prior, 35% 10 months prior and 28% 6 months prior to the onset of weakness. Further motor unit loss occurred as weakness progressed and the diagnosis of MND being made. We followed 3 subjects with a family history of ALS, 2 of which had a documented SOD 1 point mutation, who were commenced on Riluzole therapy when loss of motor units were detected using motor unit number estimation (MUNE), before the onset of symptoms i.e. presymptomatic phase. A reduction in sequential MUNE was shown to improve with a thera‐ peutic intervention, Riluzole. Currently, the only effective approved treatment for MND is Riluzole, which has a neuroprotective role, possibly due to pre-synaptic inhibition of glutamate release. Riluzole is an anti-glutamate agent that has been approved for the treatment of patients with amyotrophic lateral sclerosis in most countries. 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). There have been a least three large randomised trials involving hundreds of patients that have been unable to show that Riluzole is a disease altering agent nor does it have any restorative reports. (Miller et al., 2007).

Cases 1 and 2 are SOD 1 positive mutation carriers who developed non-specific symptoms of muscle fasciculations with no clinical weakness, but had reductions in MUNE on sequential testing. Needle EMG studies were "normal", with no evidence of active or chronic denervation in muscles sampled from bilateral distal and proximal muscles. Normal EMG was defined as the absence of fasciculation and fibrillation potentials, normal motor unit potentials and normal recruitment. A presumptive diagnosis of fALS was made, even though there are reports on incomplete penetrance. In case 3, one could argue that there is no clear evidence that she has fALS, even though she has a strong family history of ALS with an autosomal dominant pattern of inheritance. Her episodic weakness and fasciculations improved after

mild to moderate motor unit loss. (Blok et al., 2010).

214 Current Advances in Amyotrophic Lateral Sclerosis

imminent.

After commencing Riluzole, there was an improvement in symptoms and MUNE. It is therefore possible that if treatment is commenced prior to significant motor neurone loss, the progression of disease can be slowed down.

In case 3, MUNE dropped when treatment was ceased on two separate occasions and improved when treatment was recommenced, with the operator being unaware of the Riluzole dose changes.

In our series, we noted an improvement in symptoms, especially a decrease in muscle fasciculations and an increase in MUNE number after commencing Riluzole. We suggest that previous trials have been performed in the symptomatic phase of the disease when 70-80% of motor units have already been lost, rather than in the pre-symptomatic phase of the disease, when the therapeutic benefit might be possible. If "treatment" is commenced prior to signifi‐ cant motor neurone loss occurring, the progression of disease may be able to slowed down. MUNE is believed to reduce because of remodelling of the motor units and in our study, the compound muscle action potential amplitudes were maintained despite a reduction in MUNE. This is because early in the disease, the rate of cell death is low. (Cheah et al., 2010). The increase in MUNE may be either due to reinnervation of the damaged muscles or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP.

In one of the cases in the study, Riluzole was commenced once she developed mild weakness. At the time, there was a slight reduction in MUNE, but conventional needle EMG examination did not fulfil the criteria to make the diagnosis of MND. In view of her strong family history and positive genetic testing, a presumed diagnosis of MND was made. Since commencing Riluzole there has been no significant reduction in her EDB MUNE over the last 2 years, and her weakness of right dorsiflexors has only progressed marginally from MRC grade 4+/5 to 4/5 power. It is possible that since "treatment" was commenced prior to the loss of a significant number of motor neurones, this may have slowed down the progression of the disease in this individual case. Early in the course of ALS, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. As the amount of intracellular damage increases, a critical threshold is reached, which overwhelms cellular homeostasis, resulting in rapid apoptosis and cell death. The increase in MUNE numbers may be either due to reinner‐ vation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP.

Maximum isometric grip strength was also obtained using the Jamar hydraulic dynamometer with standardised (middle handle) positioning and instructions. Maximum isometric grip strength did not reduce, even when MUNE dropped, once again supporting McComas' theory that patients can maintain normal muscle twitch tension until loss of about 70-80% of motor units, before collateral reinnervation was unable to provide functional compensation. (McComas et al., 1971). Maximum isometric grip strength using the Jamar hydraulic dyna‐ mometer also does not correlate with the number of functional motor neurones in thenar group of muscles as measured using the statistical method of MUNE, indicating that MUNE is a more sensitive test than MVIC for monitoring disease progression in MND. It has also been shown that MUNE is able to identify deterioration in functional motor units before handgrip maximal voluntary isometric contraction (MVIC).

EMG studies, the window of opportunity to influence the progression of this condition has

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We would argue that previous trials have all be performed in the symptomatic phase of the disease when 70-80% of motor units have already been lost, rather than in the pre-symptomatic phase of the disease, when the therapeutic benefit might change, as "treatment" is commenced prior to significant motor neurone loss occurring and therefore, the progression of disease can be slowed down. MUNE numbers are believed to reduce because of remodelling of the motor unit and in our study, the compound muscle action potential amplitudes (CMAP) were retained as early in the course of the disease, the rate of cell death is low. The increase in MUNE numbers may be either due to reinnervation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP. Therapies aimed at preserving motor neurones may be more feasible than trying to replace lost motor neurones. A number of treatment or preventative strategies have been suggested, such as measures to diminish SOD 1 aggregation or interactions to specifically reduce apoptosis in motor neurones. As motor neurone loss at this stage is rapid and precipitous, any potential treatment ideally should be given much earlier in SOD 1 mutation carriers. Larger randomised trials are necessary to study this question in a prospective, blinded fashion.

Even though MUNE evaluations were performed in an unblinded fashion, the statistical MUNE technique is performed with the assistance of an algorithm, which reduces operator bias. The main author has been using this technique for over 10 years and any operator bias is

Early in the course of ALS, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. As the amount of intracellular damage increases, a critical threshold is reached, which overwhelms cellular homeostasis, resulting in rapid apoptosis and cell death. (Clarke, 2001). The mutant neurones appear to function normally for decades, with weakness only occurring once apoptosis and cell death occurs due to a gradual accumulation

As motor neurone loss once it occurs is rapid and precipitous, any potential treatment will need to be given early to SOD1 mutation carriers. Once the disease progresses, resulting in functional impairment and disability, restorative treatments to replace lost motor neurones becomes less feasible. To date there have been a number of drugs which have undergone clinical trials in MND, for which there is no evidence of benefit. These include creatinine, high dose vitamin E, Gabapentin and nerve growth factors such as brain derived neurotrophic factor and insulin-like growth factor-1. If effective treatment for MND were to be developed to arrest the process of degeneration, therapies aimed at preserving functional motor neurones would be more feasible. This requires the ability to be able to identify individuals at risk of

This longitudinal study showed that it was possible to detect loss of motor neurones in the pre-symptomatic stage of MND in humans. This study provided further evidence that considerable motor neurone loss occurred just before the onset of symptoms or weakness.

unintentional and unlikely to explain the marked differences in sequential MUNE.

developing the disease, which currently are SOD1 mutation carriers.

of damage within the cell. (Kong & Xu, 1998).

(Aggarwal, 2009).

been missed.

This confirms McComas' observation that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units occurs. (McComas, 1971). This suggests that handgrip MVIC is not as sensitive as thenar MUNE for monitoring disease progression, as it is unable to detect early motor neurone loss due to the presence of compensatory mechanisms. The surviving motor neurones enlarge their territo‐ ries, through collateral sprouting (reinnervation) until late in the disease, when collateral reinnervation is no longer able to provide full functional compensation. Thenar MUNE however does examine all of the motor units that are involved in handgrip MVIC, as forearm flexors and ulnar-innervated muscles are involved in the generation of handgrip MVIC. It also confirms Felice's study which showed that in patients with MND, 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).

This study also shows that there can be substantial loss in MUNE and still have an essentially normal EMG with minimal signs of acute denervation or motor unit potential remodelling, as one would expected that at a minimum, the muscles with transiently reduced MUNE numbers should have reduced recruitment during EMG studies.

As there is no corroboration with needle EMG in the pre-symptomatic stage of the disease in this study, this requires a paradigm shift in the traditional concept that needle EMG is the "gold" standard for the diagnosis of ALS. McComas showed that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units, before collateral reinnervation was unable to provide functional compensation (McComas, 1971). The function of motor neurons remains normal because the remaining motor units incorporate more muscle fibres by collateral sprouting. This should result in larger motor unit potentials, firing at higher rates with fewer motor units active i.e. reduced recruitment. Traditional neurologists and neurophysiologists will find it hard to understand physiologi‐ cally how there can be substantial loss in MUNE and still have normal EMG with no signs of acute denervation or motor unit potential remodelling.

As MUNE is a measure of the primary pathologic process of motor neurone loss and can identify that the number of motor units are reduced, even in the presence of a non-diagnostic needle EMG. Needle electromyography may reveal evidence of chronic reinnervation, but provides little direct evidence to the extent of motor neurone and axonal loss. These cases clearly indicate that loss of motor neurones is detectable in the pre-symptomatic phase, which is detectable before significant needle EMG changes of pathology. In addition, compound muscle action potential amplitudes were maintained, despite a significant reduction in MUNE. Even though some may argue that a reduction in MUNE cannot be used to support the diagnosis fALS, our previous study, suggests that once changes start to occur on conventional EMG studies, the window of opportunity to influence the progression of this condition has been missed.

of muscles as measured using the statistical method of MUNE, indicating that MUNE is a more sensitive test than MVIC for monitoring disease progression in MND. It has also been shown that MUNE is able to identify deterioration in functional motor units before handgrip maximal

This confirms McComas' observation that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units occurs. (McComas, 1971). This suggests that handgrip MVIC is not as sensitive as thenar MUNE for monitoring disease progression, as it is unable to detect early motor neurone loss due to the presence of compensatory mechanisms. The surviving motor neurones enlarge their territo‐ ries, through collateral sprouting (reinnervation) until late in the disease, when collateral reinnervation is no longer able to provide full functional compensation. Thenar MUNE however does examine all of the motor units that are involved in handgrip MVIC, as forearm flexors and ulnar-innervated muscles are involved in the generation of handgrip MVIC. It also confirms Felice's study which showed that in patients with MND, 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

This study also shows that there can be substantial loss in MUNE and still have an essentially normal EMG with minimal signs of acute denervation or motor unit potential remodelling, as one would expected that at a minimum, the muscles with transiently reduced MUNE numbers

As there is no corroboration with needle EMG in the pre-symptomatic stage of the disease in this study, this requires a paradigm shift in the traditional concept that needle EMG is the "gold" standard for the diagnosis of ALS. McComas showed that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units, before collateral reinnervation was unable to provide functional compensation (McComas, 1971). The function of motor neurons remains normal because the remaining motor units incorporate more muscle fibres by collateral sprouting. This should result in larger motor unit potentials, firing at higher rates with fewer motor units active i.e. reduced recruitment. Traditional neurologists and neurophysiologists will find it hard to understand physiologi‐ cally how there can be substantial loss in MUNE and still have normal EMG with no signs of

As MUNE is a measure of the primary pathologic process of motor neurone loss and can identify that the number of motor units are reduced, even in the presence of a non-diagnostic needle EMG. Needle electromyography may reveal evidence of chronic reinnervation, but provides little direct evidence to the extent of motor neurone and axonal loss. These cases clearly indicate that loss of motor neurones is detectable in the pre-symptomatic phase, which is detectable before significant needle EMG changes of pathology. In addition, compound muscle action potential amplitudes were maintained, despite a significant reduction in MUNE. Even though some may argue that a reduction in MUNE cannot be used to support the diagnosis fALS, our previous study, suggests that once changes start to occur on conventional

voluntary isometric contraction (MVIC).

216 Current Advances in Amyotrophic Lateral Sclerosis

Medical Research Council manual muscle testing. (Felice, 1997).

should have reduced recruitment during EMG studies.

acute denervation or motor unit potential remodelling.

We would argue that previous trials have all be performed in the symptomatic phase of the disease when 70-80% of motor units have already been lost, rather than in the pre-symptomatic phase of the disease, when the therapeutic benefit might change, as "treatment" is commenced prior to significant motor neurone loss occurring and therefore, the progression of disease can be slowed down. MUNE numbers are believed to reduce because of remodelling of the motor unit and in our study, the compound muscle action potential amplitudes (CMAP) were retained as early in the course of the disease, the rate of cell death is low. The increase in MUNE numbers may be either due to reinnervation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP.

Therapies aimed at preserving motor neurones may be more feasible than trying to replace lost motor neurones. A number of treatment or preventative strategies have been suggested, such as measures to diminish SOD 1 aggregation or interactions to specifically reduce apoptosis in motor neurones. As motor neurone loss at this stage is rapid and precipitous, any potential treatment ideally should be given much earlier in SOD 1 mutation carriers. Larger randomised trials are necessary to study this question in a prospective, blinded fashion.

Even though MUNE evaluations were performed in an unblinded fashion, the statistical MUNE technique is performed with the assistance of an algorithm, which reduces operator bias. The main author has been using this technique for over 10 years and any operator bias is unintentional and unlikely to explain the marked differences in sequential MUNE.

Early in the course of ALS, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. As the amount of intracellular damage increases, a critical threshold is reached, which overwhelms cellular homeostasis, resulting in rapid apoptosis and cell death. (Clarke, 2001). The mutant neurones appear to function normally for decades, with weakness only occurring once apoptosis and cell death occurs due to a gradual accumulation of damage within the cell. (Kong & Xu, 1998).

As motor neurone loss once it occurs is rapid and precipitous, any potential treatment will need to be given early to SOD1 mutation carriers. Once the disease progresses, resulting in functional impairment and disability, restorative treatments to replace lost motor neurones becomes less feasible. To date there have been a number of drugs which have undergone clinical trials in MND, for which there is no evidence of benefit. These include creatinine, high dose vitamin E, Gabapentin and nerve growth factors such as brain derived neurotrophic factor and insulin-like growth factor-1. If effective treatment for MND were to be developed to arrest the process of degeneration, therapies aimed at preserving functional motor neurones would be more feasible. This requires the ability to be able to identify individuals at risk of developing the disease, which currently are SOD1 mutation carriers.

This longitudinal study showed that it was possible to detect loss of motor neurones in the pre-symptomatic stage of MND in humans. This study provided further evidence that considerable motor neurone loss occurred just before the onset of symptoms or weakness. (Aggarwal, 2009).

This study indicates that SOD1 mutation carriers have normal survival of motor neurones, with as carriers had a full complement of motor neurones during the asymptomatic phase. Significant pre-symptomatic loss of motor neurones did not occur in asymptomatic SOD1 mutation carriers. Sudden and widespread motor neurone death occurs at the time develop‐ ment of the symptomatic symptoms, rather than life-long motor neurone loss. Sudden, catastrophic and multifocal loss of motor neurons occurs immediately prior to the onset of symptoms and the development of MND. This suggests that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms, rather than life-long motor neurone loss. Also, if the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS and develop treatments for sporadic MND. The mutant SOD1 protein itself cannot be the trigger, as it is constantly expressed. There may however be a gradual accumulation of a toxic product, possibly SOD1, which has changed into a new toxic conformation or aggregate, resulting in neuronal damage. The possibility of an individual neuron undergoing apoptosis increases as damage accumulates. This cumula‐ tive damage may be due to oxidative stress, resulting in disruption of the cellular structure and function.

this stage is rapid and precipitous, any potential treatment will need to be given much earlier

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

http://dx.doi.org/10.5772/56559

219

Determining the mechanism by which mutations in the Cu/Zn superoxide dismutase (SOD1) gene triggers the destruction of motor neurones causing MND remains unknown. At present, the favoured hypothesis is that the mutation causes disease as a result of a toxic gain of function by the mutant SOD1 provoking selective neurotoxicity, probably disrupting the intracellular homeostasis of copper and/or protein aggregation. However, as the amount of intracellular damage increases, the chance that a cell will die also increases. This cumulative damage may be due to oxidative stress, in which an imbalance between the production of reactive oxygen species and cellular antioxidant mechanisms results in chemical modifications of macromole‐ cules, thereby disrupting cellular structure and function. It is possible that the high metabolic activity in motor neurones, combined with the toxic oxidative properties of the mutant SOD1, causes massive mitochondrial vacuolation in motor neurones, resulting in degeneration, earlier than other neurones, triggering the onset of weakness. Prominent cytoplasmic intra‐ cellular inclusions in motor neurones and within astrocytes surrounding them developed by the onset of clinical disease and in some cases represented the first pathological sign of disease. These aggregates increased in number as the disease progressed. This indicates that the mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurones and this damage triggers the functional decline of motor neurones and the clinical onset of symptoms. The absence of motor neurone death in the early stages of the disease indicates that the majority

Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than replacing lost motor neurones. If the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS. At this stage, detecting the onset of motor neurone loss in asymptomatic individuals will identify those who may benefit from early institution of an active management program to improve their quality of life, until more effective treatment modalities become available for this devastating condi‐ tion This observation is an important contribution to the current understanding of the pathogenesis of MND, as it shows that motor neurone disease does not seem to be the end result of slow attrition of motor neurones. MUNE may be able to be used as a method of presymptomatic testing of individuals who on genetic testing are SOD1 mutation carriers. Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than

Prof. Garth Nicholson who introduced me to research into motor neurone disease and his continuing support. Prof. David Burke and Assoc. Prof. Alastair Corbett for their professional guidance and Prof. Jasper Daube for his technical assistance regarding the technique used in

of motor neurones could be rescued after early clinical diagnosis.

in SOD1 mutation carriers.

replacing lost motor neurones.

**Acknowledgements**

Neurofilament heavy polypeptide (NF-H) is an abundant stable cytoplasmic protein located in neuronal cells in large axons and may be used as a cell type marker. Abnormal accumulation of NF-H in motor neurones is associated with ALS, but it is unclear to what extent these contribute to human disease. Analysis of blood serum markers looking for increased levels of NF-H was not performed in this study, but would be interesting to be done in the future to the compare levels of NF-H in the carriers.

The results of this study indicate that the risk of cell death probably remains constant through‐ out life of the neurone and that cell death occurs randomly in time and is independent of that of any other neurone. This suggests a "one-hit" biochemical phenomenon in which the mutation imposes an abnormal mutant steady state on the neurone and a single catastrophic event randomly initiates cell death and apoptosis. Early in the course of MND, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. The delay in clinical onset was thought to reflect the gradual accumulation of damage within the neurones, as a result of the mutation, which ultimately overwhelms cellular homeostasis leading to cell death. The living mutant neurons function very well for years or decades but the probability that an individual neurone undergoes apoptosis increases as damage accumulates within it. A mutant neurone in an older patient will have accumulated a greater amount of damage and will therefore be more likely to die than in a younger patient. Consequently, early in the course of disease, the chance of a cell containing a sufficient amount of damage to initiate apoptosis is small, and the rate of cell loss is correspondingly low. The mutant neurones appear to function normally for decades, with weakness only occurring once apoptosis and cell death occurs due to a gradual accumulation of damage within the cell. Therapies aimed at preserving motor neurones may be more feasible than trying to replace lost motor neurones. A number of treatment or preventative strategies arise, such as measures to diminish SOD1 aggregation or interactions to specifically reduced apoptosis in motor neurones. As motor neurone loss at this stage is rapid and precipitous, any potential treatment will need to be given much earlier in SOD1 mutation carriers.

Determining the mechanism by which mutations in the Cu/Zn superoxide dismutase (SOD1) gene triggers the destruction of motor neurones causing MND remains unknown. At present, the favoured hypothesis is that the mutation causes disease as a result of a toxic gain of function by the mutant SOD1 provoking selective neurotoxicity, probably disrupting the intracellular homeostasis of copper and/or protein aggregation. However, as the amount of intracellular damage increases, the chance that a cell will die also increases. This cumulative damage may be due to oxidative stress, in which an imbalance between the production of reactive oxygen species and cellular antioxidant mechanisms results in chemical modifications of macromole‐ cules, thereby disrupting cellular structure and function. It is possible that the high metabolic activity in motor neurones, combined with the toxic oxidative properties of the mutant SOD1, causes massive mitochondrial vacuolation in motor neurones, resulting in degeneration, earlier than other neurones, triggering the onset of weakness. Prominent cytoplasmic intra‐ cellular inclusions in motor neurones and within astrocytes surrounding them developed by the onset of clinical disease and in some cases represented the first pathological sign of disease. These aggregates increased in number as the disease progressed. This indicates that the mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurones and this damage triggers the functional decline of motor neurones and the clinical onset of symptoms. The absence of motor neurone death in the early stages of the disease indicates that the majority of motor neurones could be rescued after early clinical diagnosis.

Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than replacing lost motor neurones. If the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS. At this stage, detecting the onset of motor neurone loss in asymptomatic individuals will identify those who may benefit from early institution of an active management program to improve their quality of life, until more effective treatment modalities become available for this devastating condi‐ tion This observation is an important contribution to the current understanding of the pathogenesis of MND, as it shows that motor neurone disease does not seem to be the end result of slow attrition of motor neurones. MUNE may be able to be used as a method of presymptomatic testing of individuals who on genetic testing are SOD1 mutation carriers. Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than replacing lost motor neurones.
