Investigation of Ongoing Denervation and Reinnervation in Amyotrophic Lateral Sclerosis by Using Concentric Needle Electrode with Single Fiber Electromyography Method

Abstract

Introduction:

The aim of this study is to demonstrate the conduction disturbance at the neuromuscular junction in a cranial muscle by measuring jitter with a concentric needle (CN) electrode in the diagnosis of Amyotrophic Lateral Sclerosis (ALS) and to investigate the utility of evaluating the peak number as an ongoing reinnervation marker.

Method:

Twelve patients diagnosed with ALS were included in this study. Single fiber electromyography (SFEMG) was performed using a CN electrode during the voluntary contraction of the right extensor digitorum communis (EDC) and left frontalis muscles.

Results:

In SFEMG from the right EDC muscle, the mean jitter value was high in all of them. The average jitter calculated in EDC muscles was 57.76±24.17 μs. The mean jitter value in the frontal muscles was 28.91±10.21 μs. In all patients, the number of CN electrode peaks was more than 4 in the EDC muscle and above 4 in 91.67% of the frontal muscle.

Conclusion:

Detection of high jitter in SFEMG examination indicates that the examined muscle undergoes a denervation-reinnervation process in the case of increased peak number values. When such a determination is made in the extremity muscles, it becomes important for the diagnosis of ALS.

INTRODUCTION

Motor neuron disease is a group of neurodegenerative diseases that affect the upper motor neuron (UMN) and / or lower motor neuron (LMN) and it is characterized by motor neuron death. Amyotrophic Lateral Sclerosis (ALS), the most common classical motor neuron disease (MND), progresses with the involvement of upper and lower motor neurons together (1).

Diagnosis in ALS is made with history, clinical, neurophysiological evaluations, and the exclusion of other causes. Various diagnostic criteria and algorithms have been created for the early diagnosis of ALS. The most recently developed “Revised El Escorial Diagnostic Criteria” and “Awaji Algorithm” say that LMN involvement requires the presence of active and chronic denervation as EMG evidence (2,3). In recent studies, “Gold Coast Criteria” started to become prominent in early diagnosis. In the study of Pugdahl et al., the sensitivity was reported as 88.2% (4).

Detection of UMN involvement in ALS is based on clinical findings. Electrophysiological studies are of great importance in detecting LMN involvement. Electrophysiological demonstration of LMN involvement in the diagnosis of ALS disease is provided by nerve conduction studies and needle electromyography, and progression can be followed by various Motor Unit Number Estimation (MUNE) methods. With the motor neuron death, the muscle fibers that remain denerved may be reinnervated by collateral sprouting from the axon of surviving motor neurons. The process continues as denervation and reinnervation, and survival is determined according to this process. The terminal axon sprouts formed by collateral sprouting and the newly formed motor endplates are immature. This situation decreases the safety factor of the neuromuscular junction, and the active, ongoing denervation-the reinnervation process, as in ALS, may cause impairment of tests evaluating nerve-muscle conduction.

Highlights

• Early diagnosis is important in Amyotrophic Lateral Sclerosis (ALS).

• It can be used in the differential diagnosis of high jitter ALS in single fiber EMG

• Increased number of peaks can be valuable for the early diagnosis of ALS.

SFEMG, basically measures the variability of the time elapsed during the formation of the action potential on the muscle fiber from one generation of the motor unit that sustains the voluntary muscle to the next. One of the single fiber potentials is selected as the trigger and the temporal variation of the other potential (slave potential) from one ignition of the motor unit to the next is calculated. This instability is called “jitter”. Jitter occurs due to the variation in transition time between the terminal branch of the motor axon and the point where the action potential on the muscle is recorded. This variability may result from motor axon terminal branches (reinnervation), neuromuscular junction (neuromuscular junction diseases), and conduction properties of the muscle membrane (myopathies). In SFEMG, the instability of neuromuscular conduction and the degree of collateral sprouting-mediated reinnervation are measured with the evaluation of “fiber density”, which is another parameter, with “jitter” examination. In SFEMG, abnormally increased jitter, block in neuromuscular transmission and increased fiber density are the reflection of early reinnervation and collateral sprouting (5). In recent years, SFEMG has been started to be performed with CN electrode since it costs less and it has an advantage of being disposable (6). Since the recording surface area of the CN electrode is relatively large, for jitter measurement, the lower frequency filter should be raised and the CN with the smallest recording surface should be preferred. Even though the potentials recorded with CN electrodes are not visibly distinguishable from single fiber potentials, they may consist of synchronized and almost simultaneous action potentials from multiple muscle fibers. Therefore, the CN cannot be used to measure the fiber density due to the larger recording surface radius of the electrode. The peaks observed through the CN electrode contain more muscle fibers. However, changes in the number of peaks recorded by the concentric electrode may indirectly reflect changes in fiber density and asynchronous conduction in the motor unit.

Progressive motor neuron loss in ALS leads to denervation in muscle fibers. Reinnervation is achieved by collateral sprouting to compensate for denervated muscle fibers. In needle EMG, fibrillation and positive sharp waves observed at rest are denervation and MUP and recruitment – interference pattern changes are also evidence of reinnervation (7). The determinant for MUP amplitude in needle EMG is the ratio of muscle fibers located close to the needle. In normal physiology, muscle fibers innervating from the same motor neuron are dispersed into the muscle in a mosaic form, and fibers belonging to the same motor unit are rarely found near the needle. However, as most of the fibers detected near the needle by collateral sprouting are innervated from the same motor unit, MUP amplitude also increases. Phase number increases in both neurogenic and myogenic processes (8–10).

When typical denervation and reinnervation findings are detected in routine needle EMG, it is doubtless that the examined muscle and related myotome are involved in the pathophysiological process. However, advanced EMG methods such as SFEMG may be useful in diagnosing cases where the involvement is milder. Except from its use in diagnostic studies of neuromuscular junction diseases, SFEMG has also been studied in muscle diseases associated with denervation and reinnervation. Before MUP pathologies become evident, reinnervation begins within the motor unit area. There are two dimensions of detecting reinnervation with SFEMG at this micro level: The first is the high jitter that will be detected due to the low immature endplate safety factor formed by collateral sprouting in the reinnervation fixation of SFEMG. High jitter can be found in the presence of newly developed immature axon sprouts, nascent motor endplates, and regenerated muscle fibers, if past and reinnervated (8,11-13).

In this study, the “SFEMG test” was applied via the CN electrode during the voluntary mild contraction of the right EDC muscle and the left frontalis muscle to twelve ALS patients. The peak number was examined to evaluate the properties of the obtained MUPs, and the jitter values in the EDC and Frontalis muscles, and the fiber density.

METHOD

Twelve patients who were followed up with a definite diagnosis of Amyotrophic Lateral Sclerosis according to Awaji criteria were included in the study. Informed consent was obtained from all participants.

The study was conducted between February 2019-February 2020 at Istanbul University, Istanbul Medical School, Department of Neurology, Electrodiagnostic Neurology Department, Electromyography Laboratory. İstanbul University İstanbul Medical Faculty Ethics Committee approved the study (26.10.2018 / 45103048).

Nerve Conduction Studies

Median, ulnar, sural and superficial fibular sensory nerve conduction examinations and, median, ulnar, tibial and fibular motor nerve conduction examinations were performed in all of the participants.

In sensory nerve conduction examinations, the screen sweep rate was set to 1 ms / div, sensitivity to 10 μV / division, and the amplifier filter setting to 10 Hz – 2 kHz.

In motor nerve conduction examinations, the screen sweep speed was set to 5 ms / div., sensitivity to 5 mV/div. and the amplifier filter to 5 Hz – 10 kHz. Bipolar superficial electrodes were used for recording and stimulation.

Conventional Needle Electromyography Test Standards

Needle electromyography with disposable CN electrodes (37 mm × 0.46–26G, Spes Medica, Disposable Concentric Needle Electrode Genova, Italy) was performed on upper and lower extremities, bulbar and/or trunk muscles. In needle EMG examinations, sensitivity for spontaneous activity was set to 50 μV/div., Sensitivity to 200 μV/div. Sensitivity for MUP configuration to 1 mV/div., for the interference pattern, the amplifier filter setting was 5 Hz – 10 kHz. The screen sweep rate was 10 ms/div. for spontaneous activity and MUP configuration, 20 ms/div. for the interference pattern.

Single Fiber Electromyography Test Standards

Single fiber electromyography test standards was applied using a CN electrode (25 mm×0.30–30G, Spes Medica, Disposable Concentric Needle Electrode Genova, Italy) during the voluntary mild to moderate contraction in the right EDC muscle and left frontalis muscle.

The amplifier low frequency filter (LFF) is set to 1 kHz, the high frequency filter (HFF) is set to 10 kHz. Sensitivity was selected as 0.2 mV / div. and screen sweep speed was 0.5 ms/div.

For the jitter analysis, at least 10 individual jitter and at least 60 consecutive traces were obtained for each individual jitter (14).

For individual jitter, pathological high jitter was accepted as ≥35 μs for EDC muscle, 28 μs for frontalis muscle and average pathological jitter as ≥43 μs for EDC muscle and ≥38 μs for frontalis muscle (15).

According to Stalberg’s measurement method for Fiber density measurement, a single fiber electrode is randomly placed and a location with a muscle fiber action potential is recorded with maximum amplitude is searched. The number of single muscle fiber action potentials belonging to the same motor unit is counted provided that their amplitude exceeds 200/ uV and has a sharp contour (16). Stalberg took the fiber density as an average of 1.4–1.5 in adults, the potential number of fibers recorded with these criteria (7). In this study, the amplitude and sharpness condition for fiber density was not sought, and the peaks of the potentials used for jitter measurement were counted at the location where the potentials were recorded. Ertaş M, et al. found the peak number to be 1.46±0.6 in the study performed by concentric needle electrode and neuromuscular jitter analysis (6). The same values for the number of peaks were accepted as standard.

Statistical Analysis

Statistical Analysis was applied using “SPSS version 21” software. Descriptive statistics for electrophysiological parameters (age, gender, nerve conduction value, jitter and spike value) as well as demographic characteristics were made in the volunteer patient group and the results were presented in tables. Differences in mean jitter between different groups were analyzed by an independent sample t-test. P<0.05 was considered statistically significant.

RESULTS

Demographic Data

The ages of the patients included in the study were between 42–80 (mean age: 62.25±11.12) and the patient group consisted of 4 (33.3%) female and 8 (66.7%) male individuals.

Electrophysiological Examination

Nerve Conduction – EMG Findings Results

Electrophysiological examination of the sensory nerve conduction of the patients were within normal limits for age. No conduction block was observed in motor conduction studies and CMAP amplitudes recorded from atrophic muscles were low.

Electromyography examination showed varying degrees of fibrillation, positive spike and fasciculation potentials in the extremity muscles at rest in all patients. As extra-extremity muscle, m.genioglossus, m.trapezius, m.tongue, m.sternocleiodeomasteoideus and m.rectus abdominis muscles were examined as either single or multiple. Six of the patients had m. genioglossus, 3 had m.tongue, 1 had m. sternocleiodeomasteoideus, 2 had m.trapezius and 2 had fibrillation, positive spike and or fasciculation potentials as spontaneous denervation findings in rectus abdominis muscle. Among all the patients, patients with number 3 and 11 had signs of denervation in multiple extra-extremity muscles. In our patient number 2, diffuse spontaneous denervation was observed in all four extremities in the examination. Spontaneous denervation was not observed in the extra-extremity muscle. High-amplitude long-term motor unit potentials and reducing were observed during muscle contraction.

Single fiber EMG Results

The mean jitter value calculated in the frontal muscle was 28.91±10.21 (18.73–52) while the numbers were 57.76±24.17 right EDC muscle (34.96–111.68) (Table 1 and 2). When the jitter values of two muscles were compared, no statistically significant difference was found (p=0.233). Considering the averages, the high jitter rate for the frontal muscle was above the normal value, which should be the mean jitter in 6 of 12 patients (Figure 1). In the EDC muscle, the calculated mean jitters of all 12 patients were pathologically high.

Table 1.

Right extensor digitorum communis muscle single fiber jitter value

Patient	Potential couple	Jitter (min-max)	Mean ± Standard Deviation
1	20	28.10–220.00	114.68±47.62
2	31	11.10–102.00	42.74±22.33
3	26	16.80–74.10	39.07±16.66
4	17	19.90–186.00	47.30±37.60
5	17	38.50–181.00	80.41±39.32
6	19	12.30–178.00	58.42±41.36
7	18	17.90–87.10	34.96±21.12
8	17	39.80–161.00	77.76±33.10
9	20	13.80–149.00	38.46±34.96
10	17	24.60–98.10	53.42±23.72
11	19	13.40–137.00	35.19±27.41
12	21	22.10–220.00	70.69±51.63

Table 2.

Frontal muscle single fiber jitter values

Patient Number	Fiber Count	Jitter (min-max)	Mean ± Standard Deviation
1	21	6.20–93.20	27.51±19.51
2	24	7.60–71.50	19.07±13.43
3	35	8.90–32.50	19.53±6.10
4	27	14.40–110.00	39.11±27.03
5	19	16.10–79.80	39.25±17.43
6	26	1.71–122.00	28.41±26.15
7	23	8.60–65.90	31.20±14.85
8	22	10.60–34.50	18.73±6.40
9	21	7.60–32.90	19.56±6.62
10	21	11.70–85.90	28.04±16.12
11	19	14.00–204.00	52.00±42.47
12	22	12.20–52.20	24.49±10.78

Figure 1.

Frontal muscle single fiber EMG high jitter superimpose.

Peak numbers were high both for frontal muscle and EDC (Figure 2 and 3). The mean number of peaks in the EDC muscle was found to be 4.92±1.45 (2–8). The mean number of peaks in the frontal muscle was 4.71±1.42 (2–12) in all patients and above 4 in 91.67% of 11 of 12 patients. A peak number below 4 was observed in only one patient, and the peak number in this patient was 3.96±0.88 at the upper limit (Table 3).

Figure 2.

Frontal muscle single fiber EMG increased peak number.

Figure 3.

Frontal muscle single fiber EMG high jitter superimpose.

Table 3.

Right extensor digitorum communis and frontal muscle single fiber peak numbers

	EDC	EDC	Frontal	Frontal
Patient No	Min-max	Mean ± Standard Deviation	Min-max	Mean ± Standard Deviation
1	3–6	4.15±0.93	3–12	5.67±2.85
2	2–8	4.87±1.65	3–7	5.12±1.39
3	3–6	4.31±1.12	2–7	5.11±1.32
4	4–7	5.65±1.17	3–7	5.30±0.91
5	2–7	4.82±1.51	3–6	4.63±0.76
6	3–6	5.21±0.85	2–6	4.23±1.11
7	3–5	4.17±0.86	2–5	3.96±0.88
8	2–6	4.29±1.53	2–7	4.91±1.34
9	3–7	5.10±1.55	3–6	4.81±1.03
10	4–8	5.88±1.58	2–6	4.19±1.70
11	2–6	4.16±1.21	2–7	4.68±1.45
12	4–8	6.14±1.35	2–6	4.04±1.09

DISCUSSION

In diseases affecting anterior horn cells, loss of innervation is partially compensated by peripheral sprouting from intact motor units. Single fiber electromyography is a method that shows the fine structure of the parameters of MUP that we measure in routine practice. In fact the measurable parameters of MUP can show that reinnervation has started within the motor unit area, without being pathologically noticeable. In models where the sole collateral sprouting mechanism is used for reinnervation, such as ALS, the earliest electrophysiological finding of this compensatory effort is an increase in fiber density (17). As the reinnervation becomes stronger, the fiber density will also increase and reach its maximum level which is limited to the registration area of the needle. In this study, we aimed to demonstrate neuromuscular junction conduction disruption in an extra-extremity muscle by measuring jitter with a concentric needle electrode and to investigate the usability of evaluation with peak number as an indicator of ongoing reinnervation.

In our study, the mean of the jitter value calculated in SFEMG of the right EDC muscle was 57.76±24.17(34.96–114.68). Mean jitter value was high in all patients. The pathological jitter rate in the measured muscle was also more than 10%, and it introduced that the muscle was undergoing reinnervation. Cui et al., in their SFEMG examination applied during voluntary muscle on EDC muscle of 78 ALS patients, in the study where >55 μs was accepted as significant jitter, the mean jitter was 80.2 μs and the results were abnormal in all patients (18).

Single fiber electromyography can also be performed on extra-extremity muscles such as paraspinal, trapezius and tongue, and can be used to distinguish diseases such as cervical spondylosis, narrow canal, myelomalacia, which are most likely to be confused with ALS, from ALS. In a study evaluating the comparison of jitter increase and fiber density with SFEMG electrode in ALS patients with and without cervical spondylosis, a similar increase was observed in both groups in jitter and fiber density. Based on this, it has been suggested that the increase in fiber density and jitter is caused by ALS (19).

In our study, the left frontal muscle was examined as the extra-extremity muscle in this study. The mean jitter value calculated in the frontal muscle is 28.91±10.21 (18.73–52.). In our results, it was above the accepted value of ≥38 μs for the frontalis muscle in half of the patients.

The earliest and most sensitive method used for evaluation of reinnervation is the increase in fiber density. Fiber density is calculated by counting the activity of other muscle fibers belonging to the same motor unit, triggered in a temporal locked manner together with the trigger potential in SFEMG performed during voluntary muscle (20). In models such as ALS were the only collateral sprouting mechanism is used for reinnervation, the earliest electrophysiological sign of this compensatory effort is the increase in fiber density (16,21). As the reinnervation increases, the fiber density will increase and reach the maximum level which is limited to the registry area of the needle. The main difference between SFEMG electrodes and CN electrodes is the areas of the recording surfaces. It has a larger surface than the SFN electrode and contains synchronized action potentials (22,23). Each motor unit can have one or more groups. The properties of observed MUPs are caused by the changes in the filter settings and reflect the action potential of several muscle fibers. During the denervation and reinnervation process, the immature motor nerve terminal is transmitted asynchronously by disrupting synchronous conduction at the neuromuscular junction. This may cause a change in the waveform of MUP and an increase in the number of peaks. Also, fiber density increases due to collateral reinnervation and more muscle fibers in the same area can thus be recorded by CN electrodes. Although the peak number obtained with CN electrodes is from muscle fiber groups, not the actual number of muscle fibers in the recording area, changes in the peak number may have the ability to indirectly reflect changes in fiber density and asynchronous conduction in the motor unit (24).

In cases where neuromuscular junction conduction is impaired such as MG, jitter increases. When the motor endplate transition is disrupted, some action potentials are lost as a result of conduction blocking. This variation in the MUP form was calculated by Stalberg E and Sono M using special software and named jiggle (25). Jiggle calculation was based on simulation studies such as alignment of waveforms, selection of reference point and calculation of artificial factors affecting the shape of the MUP. (25,26). In diseases with denervation-reinnervation such as ALS, significant variability in the form of MUP can be observed in the early stages of visual reinnervation. Jiggle affects the number of peaks, and the number of peaks has increased in cases where there is a jiggle.

In our study, the mean “peak number”, which can be considered as the equivalent of the fiber density parameter for the CN electrode, was found to be 4.92±1.45 (2–8) in the EDC muscle, where single fiber-like potentials were recorded with the CN electrode. This value is above 1.4 described for the SFEMG electrode and can be considered as an indicator of reinnervation during the ALS process. When the number of peaks in the frontal muscle is evaluated individually of 280 muscle fibers, the peak number is on mean 4.71±1.42 (2–12) in all patients and above 4 in 91.67% of 11 patients. Only one patient had a peak number of less than 4, and the peak number of this patient was 3.96±0.88 at the upper limit.

In a study by Liu et al., the fiber density with a single fiber electrode and the peak number with CN electrode were found to be higher in ALS patients compared to healthy individuals. In healthy controls, the peak number was within normal limits (24).

In conclusion, the SFEMG performed during voluntary muscle contraction using a CN electrode with a narrow recording surface can show that the muscle under examination is undergoing the reinnervation process with both high jitter values and high peak number. This determination is important for muscles that do not show significant MUP changes in routine needle EMG examination. In fact, detection of this finding in extra-extremity muscles becomes even more important for the differential diagnosis of ALS.