**4. Our experience**

Compared with previous studies (Bromberg et al., 1993; de Carvalho et al., 2005b), our idea was taking into consideration simultaneously Macro-EMG and MUNE changes, both in proximal and distal muscles, in the same sample of patients with a one-year follow-up. Sixty-one ALS patients (34 male: mean age ± SD 60.0 ± 15.5, range 20-82 yrs; 27 female: mean age ± SD 62.0 ± 9.2 yrs, range 30-82 years), were enrolled in the study and examined basally (T0) and every 4 months (T1, T2 and T3). Macro Motor Unit Potentials (macro MUPs) were derived from Biceps Brachialis (BB) muscle; MUNE was performed both in BB and Abductor Digiti Minimi (ADM) muscles of the same side. Thirty-three healthy volunteers

(13 women and 20 men, mean age: 57.7 ± 13.8 years, range 28 - 77 years) served as controls.

How to Assess Disease's Severity and Monitor Patients with

0.888) at every time of testing (Gan and Jabre, 1992).

increase up to T3: +3.5% (T1); +15.4% (T2); +22.4% (T3) (Fig 2).

from Sartucci et al., 2011).

(+62.1%; p < 0.005).

value.

(p>0.05, Figure 5).

Amyotrophic Lateral Sclerosis: Lessons From Neurophysiology 617

Fig. 2. Time evolution of each macro EMG parameters (area, amplitude and FD) with the time, keeping in the consideration disease duration at the beginning of the observation. Sample with a disease duration of 12-24 months exhibited a more steep slope (modified

In ALS patients at T0, both Macro-MUP area and FD were above upper normal limits: macro-MUP area was 4397.6 ± 2554.9 μVms (+ 285.8%; p < 0.001), mean FD 2.01 ± 0.2

The macro EMG MUP area was abnormal in 57 (93.4%). and normal in 4 (6.6%) patients, the FD resulted increased in 55 pt. (90%) and normal in 6 (10%). Macro EMG MUP area and peak-to-peak amplitude exhibited a good correlation (Spearmann coeff. of correlation =

Macro MUPs area (*Figure* 2) resulted progressively increased at every time, especially at T3 compared with T0: Area: + 45.3% (T1); + 49.0% (T2); + 83.6% (T3); FD showed a trend to

FD resulted increased in cases with longer disease duration (*Figure 2*). Anyway, the FD was generally increased when macro EMG amplitude was also increased in the first stage of disease; after less than one year (about 8 months) they showed a large dispersion of

MUNE (*Figure 3*) in controls resulted in BB muscle 91.9 ± 18.9, with a mean step area of 2.09 ± 0.7 μV/ms and a Mean Maximal M wave of 131.9 ± 36.0 mV; for the ADM muscle 87.7 ± 14.6, with a mean step area of 1.05 ± 0.4 mV/ms and Mean Maximal M wave of 61.3 ± 21.2 mV. In ALS patients, values were behind normal limits in 56 (91.8%) and within normal limits in 5 (8.2%) in BB muscle; in 60 (98.4%) and in 1 (1.6%) in ADM muscle. Functioning MUs number progressively decreased in both muscles throughout the entire follow-up period. The Pearson's correlation coefficient was 0,61, suggesting the rate and amount of MU decrease was approximately similar in both muscles (Cuturic et al., 2005). In ALS MUNE exhibited a parallel trends in proximal and distal muscles (BB and ADM), independently of disease duration (see *Figures 3 and 4*); mean step area, instead, increased more in BB, especially in patients with longer disease duration. MUP amplitude at T0 did not show any significant difference between females and males, even if a bit higher in males

All patients had probable or definite ALS, according to the criteria of the World Federation of Neurology (Brooks et al., 2000).

The sample group of patients included cases with a disease duration from clinical onset of symptoms to the time of the first examination less than 48 months (mean ± 1SD: 12.2 ± 11.0 months); only few cases had a disease duration behind this limit (11 patients; about 14.3 %). Twenty-two patients presented a bulbar onset and the remaining a spinal one (Brooks et al*.*, 2000). Muscle strength over time was evaluated by MRC score for all muscles (0-5 grading system). Forty patients were in treatment with riluzole (Rilutek® 50 mg), at a mean daily dosage of 100 mg (50 mg BID) throughout the entire period of EDX follow-up.

In twenty-nine patients (subgroup 1, SG1: 19 males and 10 females; mean age ± 1SD: 60,0 ± 11,8 years; range 30-78 years; spinal/bulbar onset: 22/7; mean disease duration 29,7 months) macro EMG was repeated after 4 months (T1). Among the second subgroup, eleven patients (subgroup 2, SG2: 8 males and 3 females; mean age ± SD: 57,0 ± 12,8 years; range 30–72 years; spinal/bulbar onset: 10/1; mean disease duration 31 months) were re-tested after 8 months (T2) and in 8 (Subgroup 3, SGP3; 7 males and 1 female; mean age ± SD: 58,0 ± 13,6 years; range 31-82 years; spinal/bulbar onset: 7/1; mean disease duration 37 months) after 12 months from the first examination.

Both patients and controls gave their written informed consent prior to participation in the study that had been approved by the local ethical Committee and followed the tenets of Helsinki.

### **5. Results**

Macro-EMG in control subjects showed a mean area 1139.9 ± 182.8 μVms, a mean amplitude of 168.0 ± 63.7 μV, and a FD 1.24 ± 0.13 (a summary of results is given in *Figures 1* and *2*).

Fig. 1. Time evolution of macro-EMG FD (white columns) and of macro EMG area (gray columns; note the break and the different scale in ordinate) in ALS pt. The macro EMG area increase continuously with the time, paralleled by FD value, up to T2 (modified from Sartucci et al., 2011).

All patients had probable or definite ALS, according to the criteria of the World Federation

The sample group of patients included cases with a disease duration from clinical onset of symptoms to the time of the first examination less than 48 months (mean ± 1SD: 12.2 ± 11.0 months); only few cases had a disease duration behind this limit (11 patients; about 14.3 %). Twenty-two patients presented a bulbar onset and the remaining a spinal one (Brooks et al*.*, 2000). Muscle strength over time was evaluated by MRC score for all muscles (0-5 grading system). Forty patients were in treatment with riluzole (Rilutek® 50 mg), at a mean daily

In twenty-nine patients (subgroup 1, SG1: 19 males and 10 females; mean age ± 1SD: 60,0 ± 11,8 years; range 30-78 years; spinal/bulbar onset: 22/7; mean disease duration 29,7 months) macro EMG was repeated after 4 months (T1). Among the second subgroup, eleven patients (subgroup 2, SG2: 8 males and 3 females; mean age ± SD: 57,0 ± 12,8 years; range 30–72 years; spinal/bulbar onset: 10/1; mean disease duration 31 months) were re-tested after 8 months (T2) and in 8 (Subgroup 3, SGP3; 7 males and 1 female; mean age ± SD: 58,0 ± 13,6 years; range 31-82 years; spinal/bulbar onset: 7/1; mean disease duration 37 months) after

Both patients and controls gave their written informed consent prior to participation in the study that had been approved by the local ethical Committee and followed the tenets of

Macro-EMG in control subjects showed a mean area 1139.9 ± 182.8 μVms, a mean amplitude of 168.0 ± 63.7 μV, and a FD 1.24 ± 0.13 (a summary of results is given in *Figures 1* and *2*).

Fig. 1. Time evolution of macro-EMG FD (white columns) and of macro EMG area (gray columns; note the break and the different scale in ordinate) in ALS pt. The macro EMG area increase continuously with the time, paralleled by FD value, up to T2 (modified from

dosage of 100 mg (50 mg BID) throughout the entire period of EDX follow-up.

of Neurology (Brooks et al., 2000).

12 months from the first examination.

Helsinki.

**5. Results** 

Sartucci et al., 2011).

Fig. 2. Time evolution of each macro EMG parameters (area, amplitude and FD) with the time, keeping in the consideration disease duration at the beginning of the observation. Sample with a disease duration of 12-24 months exhibited a more steep slope (modified from Sartucci et al., 2011).

In ALS patients at T0, both Macro-MUP area and FD were above upper normal limits: macro-MUP area was 4397.6 ± 2554.9 μVms (+ 285.8%; p < 0.001), mean FD 2.01 ± 0.2 (+62.1%; p < 0.005).

The macro EMG MUP area was abnormal in 57 (93.4%). and normal in 4 (6.6%) patients, the FD resulted increased in 55 pt. (90%) and normal in 6 (10%). Macro EMG MUP area and peak-to-peak amplitude exhibited a good correlation (Spearmann coeff. of correlation = 0.888) at every time of testing (Gan and Jabre, 1992).

Macro MUPs area (*Figure* 2) resulted progressively increased at every time, especially at T3 compared with T0: Area: + 45.3% (T1); + 49.0% (T2); + 83.6% (T3); FD showed a trend to increase up to T3: +3.5% (T1); +15.4% (T2); +22.4% (T3) (Fig 2).

FD resulted increased in cases with longer disease duration (*Figure 2*). Anyway, the FD was generally increased when macro EMG amplitude was also increased in the first stage of disease; after less than one year (about 8 months) they showed a large dispersion of value.

MUNE (*Figure 3*) in controls resulted in BB muscle 91.9 ± 18.9, with a mean step area of 2.09 ± 0.7 μV/ms and a Mean Maximal M wave of 131.9 ± 36.0 mV; for the ADM muscle 87.7 ± 14.6, with a mean step area of 1.05 ± 0.4 mV/ms and Mean Maximal M wave of 61.3 ± 21.2 mV. In ALS patients, values were behind normal limits in 56 (91.8%) and within normal limits in 5 (8.2%) in BB muscle; in 60 (98.4%) and in 1 (1.6%) in ADM muscle. Functioning MUs number progressively decreased in both muscles throughout the entire follow-up period. The Pearson's correlation coefficient was 0,61, suggesting the rate and amount of MU decrease was approximately similar in both muscles (Cuturic et al., 2005). In ALS MUNE exhibited a parallel trends in proximal and distal muscles (BB and ADM), independently of disease duration (see *Figures 3 and 4*); mean step area, instead, increased more in BB, especially in patients with longer disease duration. MUP amplitude at T0 did not show any significant difference between females and males, even if a bit higher in males (p>0.05, Figure 5).

How to Assess Disease's Severity and Monitor Patients with

Amyotrophic Lateral Sclerosis: Lessons From Neurophysiology 619

Fig. 5. MUP amplitude at T0 did not show any significant difference between females and males, both in spinal and bulbar form, even if a bit higher in males (p>0.05; Sartucci et al.,

All main macro-EMG parameters (area, amplitude and FD), as well as MUNE features (number of MUPs and mean step area either in the BB and ADM), did not disclose any significant difference between patients intaking the drug for both disease type (spinal or bulbar) at any time during the follow-up period (*Figure* 6). As concerns as Macro-EMG area, the difference in the mean values among the different levels of treatment is not great enough to exclude the possibility that the difference is just due to random sampling variability. There is not a statistically significant difference between riluzole vs. control (p = 0.321), as

personal data).

**5.1 Correlation between macro-MUP and MUNE** 

confirmed by FD measures over time (p = 0.588).

Fig. 3. Histogram showing MUNE values in both BB (gray columns) and ADM (white columns) muscles at every time of measurement. The trends is similar even if more evident in ADM (modified from Sartucci et al., 2011).

Fig. 4. MUNE values in both BB and ADM muscles at every time of evaluation in pt. with different disease duration and their mean value (filled circles) (modified from Sartucci et al., 2011).

Fig. 3. Histogram showing MUNE values in both BB (gray columns) and ADM (white columns) muscles at every time of measurement. The trends is similar even if more evident

Fig. 4. MUNE values in both BB and ADM muscles at every time of evaluation in pt. with different disease duration and their mean value (filled circles) (modified from Sartucci et al.,

in ADM (modified from Sartucci et al., 2011).

2011).

Fig. 5. MUP amplitude at T0 did not show any significant difference between females and males, both in spinal and bulbar form, even if a bit higher in males (p>0.05; Sartucci et al., personal data).

### **5.1 Correlation between macro-MUP and MUNE**

All main macro-EMG parameters (area, amplitude and FD), as well as MUNE features (number of MUPs and mean step area either in the BB and ADM), did not disclose any significant difference between patients intaking the drug for both disease type (spinal or bulbar) at any time during the follow-up period (*Figure* 6). As concerns as Macro-EMG area, the difference in the mean values among the different levels of treatment is not great enough to exclude the possibility that the difference is just due to random sampling variability. There is not a statistically significant difference between riluzole vs. control (p = 0.321), as confirmed by FD measures over time (p = 0.588).

How to Assess Disease's Severity and Monitor Patients with

surviving.

al., 2007).

Amyotrophic Lateral Sclerosis: Lessons From Neurophysiology 621

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

**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

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

studies are then required to solve this dilemma.

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
