**3. Use of Motor Unit Number Estimation (MUNE) and Macroelectromyography (Macro-EMG) in the diagnosis and management of ALS: A brief historical overview**

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 tool to quantify muscle involvement and evaluate response to therapy [28-30]. Electromyography (EMG) investigation, usually performed with concentric needle electrodes [31], plays an essential role in the diagnosis and monitoring of ALS [32-34]. Amplitude, duration, area, shape, stability on repeated discharges of 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 dener‐ vation-reinnervation process and number of functioning motor units by evaluating recruit‐ ment-activation pattern [28, 35]. In Motor Neuron Diseases (MND), standard needle electromyography often reveals evidence of chronic reinnervation (increased motor unit action potential amplitudes and duration, with reduced recruitment), eventually associated with fasciculations and signs of denervation activity in progress, but provides little information about the extent of both motor neuron loss and axonal regeneration. The supramaximal CMAP amplitude also provides little evidence of the extent of motor neuron loss and normal CMAP amplitudes might mistakenly suggest that motor neuron loss has not occurred yet [36, 37].

A particular method to evaluate the full MU is the so-called macro-EMG [38-41]. 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 study of putative therapies [33, 42] by evaluating size of individual MU [39, 43].

Among quantitative electrodiagnostic (EDX) techniques, the methodology of Motor Unit Number Estimation (MUNE) has been previously and widely employed in measuring loss of functioning MU in ALS patients [36, 44-49].

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 changes in compound motor action potential (CMAP) amplitude over time [50]. 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 [36, 51-55]. Its inter-individual and intra-individual reproducibility linearly increases as disease progresses, making this technique particularly useful in the symptomatic stage of the disease [36, 55-57].

**i.** taking into consideration simultaneously Macro-EMG and MUNE changes in

Changes in Motor Unit Loss and Axonal Regeneration Rate in...

http://dx.doi.org/10.5772/56561

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**iii.** evaluating Macro-EMG and MUNE changes both in sporadic and familiar cases

In the group of 15 symptomatic SOD-1 mutation carriers, two were found to have a point mutation in exon 4, codon 100, GAA to GGA-Glu100Gly; two were found to have a point mutation in exon 4, codon 113, ATT to ACT-Ile113Thr; five were found to have a point mutation in exon 5, codon 148, GTA to GGA-Val148Gly, and six with homozygous for aspartate to alanine mutations in codon 90 (homD90A), representing the most common SOD-1 mutation with a typical recessive fashion inheritance. Sixty ALS patients (34 males: mean age ± SD 60.0 ± 15.5 years; 26 females: mean age ± SD 62.0 ± 9.2 years) were enrolled in the study and examined basally (T0) and every 4 months (T1, T2, and T3). Fifteen of these patients are familial (SOD-1 mutation carriers, 9 males: mean age ± 1SD 46.3 ± 14.8 years; 6 females: mean age ± 1SD 49.0 ± 8.5 years). 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 females and 20 males, mean age: 57.7 ± 13.8 years) served as controls. All patients had probable or definite ALS, according to the well known criteria of the World Federation of Neurology [18]. 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 ± SD: 12.2 ± 11.0 months). Twenty-two patients presented a bulbar onset and the remaining a spinal one. As concerns symptoms and signs, among SOD-1 mutation carriers 10 have the spinal type, while only 5 patients have the bulbar type. Forty patients were in treatment with riluzole (Rilutek®, 50 mg) at a mean daily dosage

Standard macro-EMG method was applied [39]. The SFEMG recording surface was exposed 7.5 mm from the tip and the recording was made using two channels: the first one in whom the SFEMG activity was displayed (using the cannula as reference) and used to identify the MU and trigger the averaging procedure (band-pass filter for this channel: 500-10KHz); fiber density (FD) of the triggering single fibre electrode was recorded. The second channel averaged the activity from the cannula until a smooth baseline and a constant macro MUP was obtained (Filter pass-band: 5-10KHz). We measured from the averaged signal the total area between the curve and the baseline, the maximal peak-to-peak amplitude (macro-MUP) during the total sweep time of 70ms [63]. Results were expressed as individual area values from at least 20 trials. The relative macro amplitude was expressed as the obtained mean value [39]. Fibre density was expressed as number of time locked spikes obtained on the SFEMG channel [64]. In twenty-nine patients (subgroup 1: 19 males and 10 females; mean age ± 1SD: 60,0 ± 11,8 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: 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; 7 males

proximal and distal muscles in the same sample of patients;

**ii.** following all our patients with a one-year follow-up;

of 100 mg throughout the period of EDX follow-up.

(sALs and fALS).

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 assump‐ tion that variability is due solely to the number of motor units responding in an intermittent manner [58]. More recently, Shefner and colleagues proposed a new method to follow over time motor unit loss in patients with ALS [59]: nerves were stimulated at 3 specified locations and 3 increments were obtained at each location. Average single motor unit action potential (SMUP) amplitude was calculated by adding the amplitude of the third increment at each location and dividing by 9; SMUP was divided into maximum CMAP amplitude to determine the MUNE. This approach needs further validation, but has some unquestionable advantages: it's easy to perform, well tolerated by patients and specialized equipment is not necessary. Most important, by applying the multipoint method MUNE values decline rapidly in patients with ALS, although the rate of decline is similar to that obtained with the standard incremental technique.

Use of Macro-EMG is limited to muscles from which electrical activity can be elicited without any interference from other muscles [60]; 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 sensitivity each other.
