**1. Introduction**

612 Amyotrophic Lateral Sclerosis

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Amyotrophic Lateral Sclerosis (ALS) is a fatal, neurodegenerative disorder affecting upper and lower motor neurons; it's the commonest of the motor unit diseases in Europe and North America, characterized by a broad spectrum of clinical presentations mimicking vertebral stenosis, motor polyradiculoneuropathies and myopathies (Juergens et al., 1980; Swash, 2001). Striking asymmetry and selective involvement of individual groups of muscles, especially of hand and forearm, are typical early features of the disease. On average, delay from onset of symptoms to diagnosis is about 14 months and expected survival commonly ranges from months to a few years (Andersen et al., 2007).

Clinical neurophysiology in ALS plays a fundamental role both in the diagnosis of suspected disease and in the assessment of its severity and progression, offering a promising perspective to quantify muscle involvement and evaluate response to therapy (Brooks et al., 2000; Olney and Lomen-Hoerth, 2000; Beghi et al., 2002). Neuroimaging using magnetic resonance imaging (MRI), magnetic resonance spectroscopy (1HMRS), positron emission tomography (PET) and functional MRI may prove valuable results (Pohl et al., 2001), although they are complex, expensive and not always available. On the other hand, blood tests are necessary: hypoglycaemia insulinoma-related and autoimmune hyperthyroidism can be mistaken for ALS as they cause generalized muscle weakness, sometimes accompanied by fasciculations without a significant sensory impairment. Spinal fluid analysis could be helpful to rule out rare conditions closely mimicking ALS, such as meningeal infiltration with lymphoma, multifocal motor neuropathy (MMN) or a motor variant of inflammatory demyelinating neuropathy (CIDP). EMG investigation, usually performed with concentric needle electrodes (Daube et al., 2000), plays an essential role in the diagnosis and monitoring of ALS (Bromberg et al., 1993; Eisen, 2001; de Carvalho et al.,

How to Assess Disease's Severity and Monitor Patients with

**3. Methodological and technical considerations** 

2007); ten incremental steps are commonly recorded (Sartucci et al., 2007).

position (spatial coordinates have been annotated in patients schedule).

ranging from 0.75 to 0.86 (Sartucci *et al.*, 2007).

macro MUP was obtained (Filter pass-band: 5-10 kHz).

**4. Our experience** 

Amyotrophic Lateral Sclerosis: Lessons From Neurophysiology 615

The most used MUNE technique relays on manual incremental stimulation of the motor nerve, known as the McComas technique (McComas, 1995), modified by Ballantyne and Stålberg. The following test settings were used: sweep duration 50 ms, gain 2 mV/Div for M wave, 0.5 mV/Div for each step; filters 20 – 10 KHz (Keypoint Clinical Manual, 1999). The use of specific software for MUNE detects "alternation", eliminates subjectivity and the sampling of artifactually small motor units in ALS patients (McComas, 1995; Hong et al.,

Percutaneous stimuli were delivered over musculocutaneous nerve immediately below axilla, recording from BB muscles, and ulnar nerve at the wrist by recording from the ADM muscle of the same upper limb (Sartucci et al., 2007; Sartucci et al., 2011). Signals are detected with common surface electrodes, Ag/AgCl type, tapered on the cutis over the target muscles with a common muscle-belly tendon montage. In those patients who underwent follow-up after several months, each test was performed exactly on the same side with the same electrode

At least two consecutive MUNE measures are usually performed on each patient to verify the consistency of our results; when required, further estimation was made until the MUNE was clearly stable. The mean of the two or more tests was calculated (Henderson et al., 2007). The results showed an excellent reproducibility with test-retest correlation coefficients

The standard macro-EMG method is routinely applied in our patients (Stålberg, 1983). We employ a recording electrode, consisting of a modified single fibre EMG (SFEMG) electrode with the cannula Teflon insulated except for the distal 15 mm. The SFEMG recording surface is exposed 7.5 mm from the tip and the recording is made using two channels: the first one in whom the SFEMG activity is displayed (using the cannula as reference) and used to identify the MU and trigger the averaging procedure (band-pass filter for this channel: 500- 10 kHz); fiber density (FD) of the triggering single fibre electrode is recorded. The second channel averaged the activity from the cannula until a smooth baseline and a constant

Total area between the curve and the baseline, the maximal peak-to-peak amplitude (macro-MUP) during the total sweep time of 70 ms are measured (Bauermeister and Jabre, 1992). Results are expressed as individual area values from at least 20 recordings. The relative macro amplitude is expressed as the obtained mean value (Stålberg, 1983). Fibre density is expressed as number of time locked spikes obtained on the SFEMG channel (Sanders and Stålberg, 1996).

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.

2005b). Amplitude, duration, area, shape, stability on repeated discharges of motor units (MU) and activity at full effort are parameters conventionally used to evaluate disease's stage. EMG may also assess the presence of activity of the denervation-reinnervation process and number of functioning motor units by evaluating recruitment-activation pattern (Brooks et al., 2000; Finsterer and Fuglsang-Frederiksen, 2001). However, these parameters represent only indirect indicators of the number of surviving muscle fibers.

A particular method to evaluate the full MU is the so-called macro-EMG (Stålberg, 1980; Stålberg and Fawcett, 1982; Stålberg, 1983; Dengler et al., 1990). This technique provides information from a larger area of the muscle than traditional needle EMG methods. The signal is recorded by most of the fibers inside the entire MU and is often employed to follow the degree of reinnervation. That represents a quantitative technique and can be applied to follow progression and effects of putative therapies (de Carvalho et al., 2005a; de Carvalho et al., 2005b) by evaluating size of individual MU (Stålberg, 1983; Guiloff et al., 1988).

Among quantitative electrodiagnostic (EDX) techniques, the methodology of Motor Unit Number Estimation (MUNE) has been previously employed in measuring loss of functioning MU in ALS patients (McComas et al., 1971; Daube, 1995; McComas, 1995; Wang and Delwaide, 1998; Gooch and Shefner, 2004; Daube, 2006; Sartucci et al., 2007).

### **2. Know your enemy. The useful association of MUNE and macro-EMG**

MUNE is very sensitive in documenting disease progression in ALS. Some studies combining MUNE and standard electromyography showed a highly significant correlation between motor unit loss, clinical quantitative features and variations in compound motor action potential (CMAP) amplitude over time (Liu et al., 2009). That is not surprising considering their different targets; while MUNE assesses motor unit loss, changes in CMAP amplitude and duration also account for collateral reinnervation. A few longitudinal studies using MUNE in some ALS patients have been reported that MUNE decreases as the disease progresses and that MUNE is a very reliable and reproducible method in patients with ALS (Olney et al., 2000; Kwon and Lee, 2004; Boe et al., 2007; Hong et al., 2007; Sartucci et al., 2007; Sartucci et al., 2011). Its inter-individual and intra-individual reproducibility linearly increases as disease progresses, making this technique particularly useful in the symptomatic stage of the disease (Sartucci et al., 2007; Sartucci et al., 2011). However, results from MUNE might seem contradictory or not always conclusive in view of many studies were made on animals; in comparison with transgenic mice, it's worth remembering that the majority of cases of ALS are sporadic and the SOD-1 GD93A represents only about 20% of patients with hereditary ALS (Shefner et al., 2002; Zhou et al., 2007).

We routinely use the standard incremental technique, known as the McComas technique. Despite some limitations in comparison with statistical MUNE (alternation of motor unit, inability to recognize small motor units, small sample size), it is more reliable and less complex; in addiction, statistical MUNE cannot identify instable MUPs since it is based on the assumption that variability is due solely to the number of motor units responding in an intermittent manner (Shefner et al., 2007).

On the other hand, use of Macro-EMG is limited to muscles from which electrical activity can be elicited without any interference from other muscles (de Koning et al., 1988); moreover, it's difficult to perform it in the hands during the course of the disease due to the strong wasting of the intrinsic hand muscles. Because of these limitations, our twenty-years experience led us to combine the two techniques in order to improve diagnostic accuracy.

2005b). Amplitude, duration, area, shape, stability on repeated discharges of motor units (MU) and activity at full effort are parameters conventionally used to evaluate disease's stage. EMG may also assess the presence of activity of the denervation-reinnervation process and number of functioning motor units by evaluating recruitment-activation pattern (Brooks et al., 2000; Finsterer and Fuglsang-Frederiksen, 2001). However, these parameters

A particular method to evaluate the full MU is the so-called macro-EMG (Stålberg, 1980; Stålberg and Fawcett, 1982; Stålberg, 1983; Dengler et al., 1990). This technique provides information from a larger area of the muscle than traditional needle EMG methods. The signal is recorded by most of the fibers inside the entire MU and is often employed to follow the degree of reinnervation. That represents a quantitative technique and can be applied to follow progression and effects of putative therapies (de Carvalho et al., 2005a; de Carvalho

Among quantitative electrodiagnostic (EDX) techniques, the methodology of Motor Unit Number Estimation (MUNE) has been previously employed in measuring loss of functioning MU in ALS patients (McComas et al., 1971; Daube, 1995; McComas, 1995; Wang

MUNE is very sensitive in documenting disease progression in ALS. Some studies combining MUNE and standard electromyography showed a highly significant correlation between motor unit loss, clinical quantitative features and variations in compound motor action potential (CMAP) amplitude over time (Liu et al., 2009). That is not surprising considering their different targets; while MUNE assesses motor unit loss, changes in CMAP amplitude and duration also account for collateral reinnervation. A few longitudinal studies using MUNE in some ALS patients have been reported that MUNE decreases as the disease progresses and that MUNE is a very reliable and reproducible method in patients with ALS (Olney et al., 2000; Kwon and Lee, 2004; Boe et al., 2007; Hong et al., 2007; Sartucci et al., 2007; Sartucci et al., 2011). Its inter-individual and intra-individual reproducibility linearly increases as disease progresses, making this technique particularly useful in the symptomatic stage of the disease (Sartucci et al., 2007; Sartucci et al., 2011). However, results from MUNE might seem contradictory or not always conclusive in view of many studies were made on animals; in comparison with transgenic mice, it's worth remembering that the majority of cases of ALS are sporadic and the SOD-1 GD93A represents only about 20% of

We routinely use the standard incremental technique, known as the McComas technique. Despite some limitations in comparison with statistical MUNE (alternation of motor unit, inability to recognize small motor units, small sample size), it is more reliable and less complex; in addiction, statistical MUNE cannot identify instable MUPs since it is based on the assumption that variability is due solely to the number of motor units responding in an

On the other hand, use of Macro-EMG is limited to muscles from which electrical activity can be elicited without any interference from other muscles (de Koning et al., 1988); moreover, it's difficult to perform it in the hands during the course of the disease due to the strong wasting of the intrinsic hand muscles. Because of these limitations, our twenty-years experience led us to combine the two techniques in order to improve diagnostic accuracy.

et al., 2005b) by evaluating size of individual MU (Stålberg, 1983; Guiloff et al., 1988).

and Delwaide, 1998; Gooch and Shefner, 2004; Daube, 2006; Sartucci et al., 2007).

patients with hereditary ALS (Shefner et al., 2002; Zhou et al., 2007).

intermittent manner (Shefner et al., 2007).

**2. Know your enemy. The useful association of MUNE and macro-EMG** 

represent only indirect indicators of the number of surviving muscle fibers.
