**6. Conclusions and unanswered questions**

Our study design was a prospective study to evaluate ongoing denervation/reinnervation process. Main aim was to objectively measure the extent of MU loss and the accompanying changes in innervation pattern during the time in ALS patients (Stålberg, 1983; Jabre, 1991), and therefore is often impossible to perform it in a hand muscle in the course of ALS due to the strong wasting of the intrinsic hand muscles; consequently to evaluate distal time disease evolution and its behaviour compared with proximal district we had to use only MUNE.

Area and amplitude of the Macro-MUP reflect number and size of muscle fibers in the motor unit (Schwartz et al., 1976; Stålberg et al., 1976). MUNE instead is the ideal tool for the assessment of disease in which primary defect is MU loss (Strong et al., 1988; Gooch and Shefner, 2004; Daube, 2006; Sartucci et al., 2007).

ALS is featured by repetitive cycles of denervation/reinnervation and the mechanism lead to a variation of fibre density within a given motor unit (Stålberg, 1983; de Carvalho et al., 2005a; de Carvalho et al., 2005b). If this rearrangement is interrupted by new processes of denervation, following further motor neuron loss, this will lead to areas of grouped atrophy and loss of muscle fibers. Reinnervation process are strictly interwoven with lower motor neuron loss; quantization and tracking of MU loss with simultaneously gauging countervailing collateral dynamic innervation may be assessed by combining MUNE and macro-EMG (Gooch and Shefner, 2004; Pouget, 2006). The macro-EMG gives a global view of the MU. First, the physical length of the electrode (15 mm), cover the entire diameter of an average sized MU; the large electrode surface suppresses the contribution of the closest action potentials and favours the relative influence of slow components so including distant fibers (Sanders and Stålberg, 1996).

Macro-EMG parameters in controls were in agreement with data of others authors (Stålberg and Fawcett, 1982; Stålberg, 1983; Jabre, 1991). Both macro-MUP area, amplitude and FD were beyond upper normal limits, as expected, in ALS (Bauermeister and Jabre, 1992; Gan and Jabre, 1992). Macro-EMG parameters progressively increased, at least in the first eight months compared with baseline as proved by coefficient of correlation at each time displaying a progressive increment of correlation up to 8 months, suggesting the process of MU rearrangement begins to fail after 8 months of disease course. Also when macro EMG area and amplitude were increased, FD was parallely increased.

The time elapsed from disease onset plays a fundamental role, since patients included with a diseases duration between 12 and 24 months showed largest changes in Macro EMG features, suggesting a higher efficiency of compensatory mechanisms at least in early stages of disease. Evidence of some MU loss at baseline compared with controls and its trend over time, together with a broader mean step area, yields novel insights into the pathophysiology of MU loss and its relationship to motor function in patients with ALS (Daube et al., 2000; Sartucci et al., 2007). Fluctuation of MU estimates between separate time could suggest reversible motoneurons dysfunction (Gooch and Shefner, 2004). The coefficient of correlation for MUNE – macro EMG mean area regression line was not significant (= - 0.17) in BB muscle, suggesting that both processes go on in some way independently. In more advanced stages, a decline of the strength of the surviving MUs, especially those with higher thresholds, seems to contribute to the progressive muscle weakness, in addition to both corticospinal degeneration and reduction in motoneurons

Our study design was a prospective study to evaluate ongoing denervation/reinnervation process. Main aim was to objectively measure the extent of MU loss and the accompanying changes in innervation pattern during the time in ALS patients (Stålberg, 1983; Jabre, 1991), and therefore is often impossible to perform it in a hand muscle in the course of ALS due to the strong wasting of the intrinsic hand muscles; consequently to evaluate distal time disease evolution and its behaviour compared with proximal district we had to use only

Area and amplitude of the Macro-MUP reflect number and size of muscle fibers in the motor unit (Schwartz et al., 1976; Stålberg et al., 1976). MUNE instead is the ideal tool for the assessment of disease in which primary defect is MU loss (Strong et al., 1988; Gooch and

ALS is featured by repetitive cycles of denervation/reinnervation and the mechanism lead to a variation of fibre density within a given motor unit (Stålberg, 1983; de Carvalho et al., 2005a; de Carvalho et al., 2005b). If this rearrangement is interrupted by new processes of denervation, following further motor neuron loss, this will lead to areas of grouped atrophy and loss of muscle fibers. Reinnervation process are strictly interwoven with lower motor neuron loss; quantization and tracking of MU loss with simultaneously gauging countervailing collateral dynamic innervation may be assessed by combining MUNE and macro-EMG (Gooch and Shefner, 2004; Pouget, 2006). The macro-EMG gives a global view of the MU. First, the physical length of the electrode (15 mm), cover the entire diameter of an average sized MU; the large electrode surface suppresses the contribution of the closest action potentials and favours the relative influence of slow components so including distant

Macro-EMG parameters in controls were in agreement with data of others authors (Stålberg and Fawcett, 1982; Stålberg, 1983; Jabre, 1991). Both macro-MUP area, amplitude and FD were beyond upper normal limits, as expected, in ALS (Bauermeister and Jabre, 1992; Gan and Jabre, 1992). Macro-EMG parameters progressively increased, at least in the first eight months compared with baseline as proved by coefficient of correlation at each time displaying a progressive increment of correlation up to 8 months, suggesting the process of MU rearrangement begins to fail after 8 months of disease course. Also when macro EMG area and amplitude were increased, FD was

The time elapsed from disease onset plays a fundamental role, since patients included with a diseases duration between 12 and 24 months showed largest changes in Macro EMG features, suggesting a higher efficiency of compensatory mechanisms at least in early stages of disease. Evidence of some MU loss at baseline compared with controls and its trend over time, together with a broader mean step area, yields novel insights into the pathophysiology of MU loss and its relationship to motor function in patients with ALS (Daube et al., 2000; Sartucci et al., 2007). Fluctuation of MU estimates between separate time could suggest reversible motoneurons dysfunction (Gooch and Shefner, 2004). The coefficient of correlation for MUNE – macro EMG mean area regression line was not significant (= - 0.17) in BB muscle, suggesting that both processes go on in some way independently. In more advanced stages, a decline of the strength of the surviving MUs, especially those with higher thresholds, seems to contribute to the progressive muscle weakness, in addition to both corticospinal degeneration and reduction in motoneurons

**6. Conclusions and unanswered questions** 

Shefner, 2004; Daube, 2006; Sartucci et al., 2007).

fibers (Sanders and Stålberg, 1996).

parallely increased.

MUNE.

number (Dengler et al., 1990). Our study also showed a significant correlation between MRC scores and EDX measurements throughout the whole course of the disease only for ADM muscle. The absence of a significant correlation between MUNE and MRC values (p > 0.05) for BB could confirm the specificity of EDX investigations to track over time changes in muscle MU features and number. Muscle strength seems to decline more linearly than MUNE values: that could be explained, as recently suggested by Liu et al. (2009) with the persistence of a small proportion of lower motor neurons long-term surviving.

### **6.1 Gender and amyotrophic lateral sclerosis. Lessons from motor unit estimation**

Another interesting result is about gender differences (*Figure 5*); in fact, some studies have reported a significant male predominance until the sixth decade of life and an older average age at onset for females, sometimes explained with a possible protective effect of estrogen. In our experience, MUP amplitude at T0 did not show any significant difference between females and males, even if a bit higher in males: MUP amplitudes were 86.9 ± 21.2 µV and 84.1 ± 17.5 µV for the biceps brachii and abductor digiti minimi muscle, respectively, in females, 90.7 ± 17.3 µV and 88.2 ± 16.8 µV in males (p>0.05). This is only a trend, as gender don't influence motor unit loss neither corresponding decline in MRC values over time. The lack of significant differences between females and males in both spinal and bulbar form, as emerged from our sample, is consistent with results reported by Hegedus (Hegedus et al., 2009): the antioxidant effects of estrogens and their proved role in preventing glutamaterelated toxicity *in vitro* (Kruman et al., 1999; Nakamizo et al., 2000) could not delay both the early retraction of nerve terminals from neuromuscular end-plates and the dying-back of the axons during asymptomatic phase *in vivo*, as well as the denervation/reinnervation process in later stages. However, there is a substantial lack of studies describing the contribution of gender in progression of ALS; that's likely due to the discrepancy between humans patients and animal models, in terms of disease and presymptomatic phase duration, absence of sensitive biological markers and different pathogenesis (sporadic vs. SOD1-related; Zhou et al., 2007).

### **6.2 MUNE and Macro-EMG in evaluating response to treatment**

Our investigation was aimed to evaluate also the EDX effects of one of the most common drug employed in the ALS, riluzole (Leigh et al., 2003), on the fundamentals process of ALS: the primary process of motorneurons loss and denervation, and the secondary process of reinnervation. Riluzole is a benzothiazole derivative with a wide range of effects on glutamate pathways including inhibition of presynaptic glutamate release; it is relatively safe and well tolerated. Prescription of riluzole is restricted to patients with probable or definite ALS. At the moment, there is no convincing evidence that treatment at 100 mg daily is associated with a significant increase in survival (Miller et al., 2007); its effects on quality of life and survival are weak especially in older patients (over 75 years), in those with bulbar onset and at more advanced stages (Miller et al., 2003). We did not detect any significant electrophysiological difference between patient intaking the drug and those who didn't (see *Figure 6*), but considering the high attrition rate it's quite difficult to draw any conclusion about the effect of pharmacological treatments on neurophysiological parameters. Future studies are then required to solve this dilemma.

How to Assess Disease's Severity and Monitor Patients with

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Fig. 6. Effects of Riluzole on the macro EMG and MUNE parameters with the time, in patients intaking (filled circle) or not (empty circle) the drug (modified from Sartucci et al., 2011).
