Repeater F-waves in amyotrophic lateral sclerosis: Electrophysiologic indicators of upper or lower motor neuron involvement?

Journal Pre-proofs Repeater F-waves in amyotrophic lateral sclerosis: electrophysiologic indicators of upper or lower motor neuron involvement? Emel Oguz Akarsu, Nermin Gorkem Sirin, Elif Kocasoy Orhan, Bahar Erbas, Hava Ozlem Dede, Mehmet Baris Baslo, Halil Atilla Idrisoglu, Ali Emre Oge PII: DOI: Reference:

S1388-2457(19)31273-8 https://doi.org/10.1016/j.clinph.2019.09.030 CLINPH 2009017

To appear in:

Clinical Neurophysiology

Accepted Date:

25 September 2019

Please cite this article as: Oguz Akarsu, E., Gorkem Sirin, N., Kocasoy Orhan, E., Erbas, B., Ozlem Dede, H., Baris Baslo, M., Atilla Idrisoglu, H., Emre Oge, A., Repeater F-waves in amyotrophic lateral sclerosis: electrophysiologic indicators of upper or lower motor neuron involvement?, Clinical Neurophysiology (2019), doi: https://doi.org/10.1016/j.clinph.2019.09.030

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.

REPEATER F-WAVES IN AMYOTROPHIC LATERAL SCLEROSIS: ELECTROPHYSIOLOGIC INDICATORS OF UPPER OR LOWER MOTOR NEURON INVOLVEMENT? Emel OGUZ AKARSUa, Nermin Gorkem SIRINa, Elif KOCASOY ORHANa, Bahar ERBASa,b, Hava Ozlem DEDEa, Mehmet Baris BASLOa, Halil Atilla IDRISOGLUa, Ali Emre OGEa. a Istanbul b

University, Istanbul Faculty of Medicine, Department of Neurology, Istanbul, Turkey.

Demiroglu Bilim University, Faculty of Medicine, Department of Pharmacology, Istanbul, Turkey.

Corresponding Author: Emel OGUZ AKARSU Tel: +90 212 4142000/32575 Fax: +90 212 5334393 Email: [email protected] Address: Istanbul University Istanbul Faculty of Medicine, Department of Neurology, Capa, Fatih, Istanbul, 34390, Turkey. Running title: Repeater F-waves in ALS

1

Abstract Objective: To extract insight about the mechanism of repeater F-waves (Frep) by exploring their correlation with electrophysiologic markers of upper and lower motor neuron dysfunction in amyotrophic lateral sclerosis (ALS). Methods: The correlations of Frep parameters with clinical scores and the results of neurophysiological index (NI), MScanfit MUNE, F/M amplitude ratio (F/M%), single and paired-pulse transcranial magnetic stimulation (TMS), and triple stimulation technique (TST) studies, recorded from abductor digiti minimi (ADM) and abductor pollicis brevis (APB) muscles of 35 patients with ALS were investigated. Results: Frep parameters were correlated with NI and MScanfit MUNE in ADM muscle and F/M% in both muscles. None of the Frep parameters were correlated with clinical scores or TST and TMS measures. While the CMAP amplitudes were similar in the two recording muscles, there was a more pronounced decrease of F-wave persistence in APB, probably heralding the subsequent split hand phenomenon. Conclusion: Our findings suggest that the presence and density of Freps are primarily related to the degree of lower motor neuron loss and show no correlation with any of the relatively extensive set of parameters for upper motor neuron dysfunction. Significance: Freps are primarily related to lower motor neuron loss in ALS. Keywords: repeater F-wave; amyotrophic lateral sclerosis; triple stimulation technique; MScanFit MUNE; transcranial magnetic stimulation; split hand

Highlights: 

Repeater F-waves (Frep) seem to be related to lower motor neuron loss in ALS.



None of the transcranial magnetic stimulation measures was correlated with Frep in ALS.



Lower F-wave persistence in APB muscle may predict the subsequent split hand phenomenon.

2

1. Introduction There has been a tremendous work for electrophysiologic biomarkers in detecting and quantifying upper motor neuron (UMN) and lower motor neuron (LMN) dysfunction in amyotrophic lateral sclerosis (ALS) (Grieve et al., 2016, Huynh et al., 2019, Vucic and Rutkove, 2018). Neurophysiological index (NI) and motor unit number estimation (MUNE) methods have been widely used as potential biomarkers for LMN loss. The NI was suggested to demonstrate LMN loss in patients with ALS even in presymptomatic muscles and found to be sensitive in detecting disease progression (de Carvalho et al., 2005a, de Carvalho et al., 2003, Swash and de Carvalho, 2004, Vucic and Rutkove, 2018). Although several MUNE methods were proposed since the invention of the first technique in 1971, none was accepted as a standard method due to various limitations inherent to the technique (de Carvalho et al., 2018, Gooch et al., 2014, Gooch and Shefner, 2004). Recently, a new MUNE method, called MScanFit MUNE, was introduced by Hugh Bostock, as a fast technique depending on different principles as compared with other conventional techniques. A few studies have been published with MScanFit MUNE, which provided promising results in quantifying motor unit loss for diagnosis and follow-up of patients with ALS (Jacobsen et al., 2017, Kristensen et al., 2019, Verber et al., 2019). Conventional single and paired-pulse transcranial magnetic stimulation (TMS) methods have been studied for more than three decades and have provided information about the functions of the corticomotoneuronal system (Huynh et al., 2016, Kujirai et al., 1993, Rossini et al., 2015, Ugawa et al., 1991, Wittstock et al., 2007). However, variability of the evoked motor responses stands as a major problem for these techniques in the studies for various diseases including ALS.

One of the

solutions against this variability problem is the threshold tracking method which was recently developed for paired-pulse TMS studies and suggested to be a good biomarker in the assessment of UMN dysfunction in ALS (Vucic and Rutkove, 2018, Vucic et al., 2018). Another method to overcome this difficulty, is the triple stimulation technique (TST) introduced more than two decades ago as an objective and sensitive method for quantifying UMN involvement in patients with ALS (Magistris et al., 1998, Magistris et al., 1999). It consists of collisions that eliminate the effects of desynchronization of the descending volleys in corticospinal tract (CST) and provide a measure for the amount of conduction failure. It has been proven to improve the diagnostic sensitivity of conventional TMS and several studies revealed TST as a diagnostic and monitoring tool for ALS (Attarian et al., 2007, Furtula et al., 2013, Kleine et al., 2010, Komissarow et al., 2004, Rosler et al., 2000, Wang et al., 2019).

3

Repeater F-waves (Frep), which result from backfiring of the same individual motor neurons, have been shown to exist in peripheral neuropathies, poliomyelitis, and ALS, implying that motoneuronal and/or axonal involvement are important factors for their generation (Chroni et al., 2012, Hachisuka et al., 2015, Ibrahim and el-Abd, 1997, Macleod, 1987, Zheng et al., 2016). On the other hand, F-wave abnormalities have been found in UMN diseases, suggesting the presence of altered excitability in the spinal motoneuronal pool, influenced by supraspinal or spinal inputs (Bischoff et al., 1992, Eisen and Odusote, 1979, Fisher, 1986, Schiller and Stalberg, 1978). In diseases with spasticity, like multiple sclerosis and myelopathy, both absolute and relative amplitudes of Fwaves were found to be higher than those of the controls (Argyriou et al., 2010, Bischoff et al., 1992, Eisen and Odusote, 1979). On the other hand, F-wave persistence and amplitude were reduced during acute spinal shock and at the early stages of cerebrovascular diseases, especially when consciousness was impaired (Chroni et al., 2007, Chroni et al., 2006, Drory et al., 1993, Leis et al., 1996). In ALS, being a disease affecting both UMN and LMN, cortical and peripheral mechanisms have been proposed to account for the F-wave abnormalities (Argyriou et al., 2006, Carvalho et al., 2002, Drory et al., 2001, Fang et al., 2015). An increased number of Freps in the presence of clinical UMN involvement was reported in ALS (Fang et al., 2015). In contrast, atrophied muscles which were more marked in the thenar region have been found to generate more Freps, consistent with the split hand phenomenon which occurs in the same disease (Fang et al., 2016). Overall, the mechanism of the generation of Freps is still debatable. In the present study, we aimed to investigate Freps in the thenar and hypothenar muscles of patients with ALS and their correlation with other electrophysiologic markers in order to extract some insight for the dominancy of UMN or LMN dysfunction in the mechanism of their emergence. 2. Methods 2.1 Subjects A total of 15 female and 20 male patients with ALS who were referred to our electromyography (EMG) laboratory for diagnostic examinations were included into the study. There were 28 definite, 1 probable, and 6 possible ALS cases, according to the Awaji criteria (de Carvalho et al., 2008). The mean age of the patients was 52.9±13.0 years. Eighteen healthy volunteers who were employees in our institution and their relatives (mean age 45.4±12.9 years; 8 women) were recruited as controls. There were no significant age or sex differences between the patient and the control groups.

4

All patients with ALS underwent routine electrophysiologic procedures consisting of sensory and motor conduction studies in at least one upper and lower extremities and needle EMG in at least three regions (de Carvalho et al., 2008) in order to confirm widespread anterior horn cell involvement, as well as to exclude focal or generalized neuropathies. None of the subjects had median/ulnar entrapment neuropathies in routine nerve conduction studies. Subjects with known central or peripheral nervous system involvement and medical conditions potentially affecting these structures were excluded from the study. The Revised ALS Functional Rating Scale (ALSFRS-R) and Medical Research Council Sum Score (MRC sum score) were used for the evaluation of patients’ clinical status (Cedarbaum et al., 1999, Hermans et al., 2012). The ALSFRS-R upper limb score (UL), composed of questions 4-6 in the ALSFRSR, was also calculated (Cedarbaum et al., 1999). At the time of investigation, patients with ALS had an average of 13.5±13.0 months’ disease duration, ranging between 2 and 78 months, and the mean ALSFRS-R and ALSFRS-R UL scores were 38.7±7.2 (16-47) and 9.7±2.4 (4-12), respectively. All but 5 patients with ALS had clinical UMN signs (brisk deep tendon reflexes and/or a Hoffman sign) at the studied extremity. The local ethics committee approved the study (Istanbul University, Istanbul Faculty of Medicine, number 197, 12.02.2016), and all subjects gave written informed consent. 2.2 Nerve conduction studies and F-wave parameters All recordings were obtained from the abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles on the less affected side of the patients and the non-dominant side of the controls with adhesive surface recording electrodes placed on the bellies of muscles (active) and on the metacarpophalangeal joints of the 1st and 5th fingers, respectively. Patients with compound muscle action potential (CMAP) amplitudes less than 1 mV in any muscle were excluded from the study. For the F-wave studies, a Medelec Synergy electrodiagnostic device (version 20.1.0, Natus Medical Inc.) was used and 90 consecutive supramaximal stimuli were applied with a frequency of 1 Hz on the median (for the responses from APB) and ulnar nerves (for those from ADM) at the wrist. F-waves with amplitudes lower than 40 microvolts were excluded from the measurements (Argyriou et al., 2010, Argyriou et al., 2006). Freps, defined as waves with the same latency, amplitude, and waveform were verified visually by the same examiner (EOA), (Figure 1).

5

For each nerve, F-wave persistence, minimum, mean, maximum latencies and mean and maximum peak-to-peak amplitudes of F-waves were measured. Indices related to F-waves and the NI were calculated as follows (Chroni et al., 2012, Swash and de Carvalho, 2004, Vucic and Rutkove, 2018): Mean F-wave amplitude: Sum of peak-to-peak amplitudes of F-waves/number of traces with F-waves F-wave persistence = (Number of definable F-waves per 90 stimuli/90) x 100 Frep persistence = (Number of total Freps per 90 stimuli/90) x 100 Non-repeater F-wave (Fnonrep) persistence = Number of total non-repeater F-waves per 90 stimuli/90) x 100 Index Frep = 100 x number of Frep / total number of traces with F-wave Number of RN = Number of neurons that produce Freps Index RN = 100 x number of RN / number of traces with different F-wave shapes F/M amplitude ratio (F/M%) = 100 x mean amplitude of the recorded F-waves/distal CMAP amplitude NI = [CMAP amplitude (mV) x F-wave frequency%] / distal motor latency (ms). Electrophysiologic split hand was calculated as the ratio of CMAP amplitude recorded from the APB by median nerve stimulation to the CMAP amplitude recorded from the ADM by ulnar nerve stimulation (APB/ADM amplitude ratio). Values less than 0.6 were accepted as positive for split hand (Kuwabara et al., 2008). 2.3 Single and paired-pulse TMS studies A Nicolet Viking Select EMG and two Magstim 200 magnetic stimulators connected to a Bistim module were used for single and paired-pulse TMS studies. Recording parameters were as follows: sweep speed: 10-50ms/division, sensitivity: 0.2-5mV/division, and filter settings: 2 Hz-10kHz. A round coil with an outside diameter of 12 cm was located tangentially over the vertex to record motor evoked potentials (MEP) from APB and ADM muscles during moderate voluntarily contraction. Five to eight stimuli were applied with intensities obviously higher than the motor threshold (70% to 90% of the maximal output of the stimulator). The current direction in the coil was selected as the one which produced the highest amplitude motor responses (usually clockwise for recording from the right side). The shortest latency of MEPs were measured for each muscle. Central 6

motor conduction time (CMCT) was calculated using F-wave latencies for both muscles by the following formula: MEP-(F+M-1)/2 (Rossini et al., 2015). Resting motor thresholds (RMT), cortical and ipsilateral silent periods (CSP and ISP), and paired-pulse TMS studies were performed by recording from ADM muscle using a figure-of-eight coil with the outside diameter of 7 cm for each ring (Rossini et al., 1994, Rossini et al., 2015). The coil was positioned over the motor cortex area of ADM muscle with the handle pointing to posterolateral direction, 45 degrees away from the midsagittal line. RMT and CSP were determined as described by the International Federation of Clinical Neurophysiology (IFCN) committee (Rossini et al., 2015). CSP was studied using 140% of RMT during voluntary tonic contraction of ADM (Orhan et al., 2011). Eight rectified traces were averaged and CSP duration was measured from stimulation onset to the reappearance of voluntarily muscle activity (Orhan et al., 2011, Rossini et al., 2015). For ISP, recording electrodes were moved to the homologous muscle on the other side. ISPs were elicited using TMS of 140% RMT during voluntary tonic contraction of ipsilateral ADM with the coil at the same position as CSP. At least 16 rectified traces were averaged for each patient. The onset of ISP was detected by the time of reduction of ongoing EMG activity to one-third of its original envelope amplitude and its end was determined by the time at which the initial EMG activity was restored (Wittstock et al., 2007). Paired-pulse TMS studies were performed using conventional methods as described previously (Kujirai et al., 1993, Rossini et al., 2015). Conditioning and test stimuli were adjusted to 70% and 140% RMT, respectively (Kujirai et al., 1993, Orhan et al., 2011, Rossini et al., 2015). Interstimulus intervals (ISI) were arranged as 2 and 3 ms for short interval cortical inhibition (SICI) and 10 and 12 ms for intracortical facilitation (ICF). Paired stimuli with different ISIs were delivered randomly. The data of ISIs, 2-3 ms, and those of ISIs, 10-12 ms, were averaged for the calculations of SICI and ICF. 2.4 Triple Stimulation Technique (TST) The TST was performed by recording from both the ADM and APB using the methods described by Magistris et al. (Magistris et al., 1998, Magistris et al., 1999). Two TST responses were recorded: TSTtest and TSTcontrol. For TSTtest, three stimuli were applied with certain delays in the following order: magnetic stimulation with an overtly suprathreshold intensity over the scalp with a round coil, electrical supramaximal stimulation at the wrist to median and ulnar nerves for recording from the APB and ADM, respectively, and at the Erb’s point. The first delay between magnetic 7

stimulation at the scalp and electrical stimulation at the wrist was arranged by subtracting the latency of the CMAP elicited by stimulation at the wrist from the shortest MEP latency. The second delay between electrical stimulation at the wrist and at Erb’s point was determined as the difference between the latencies of the CMAPs elicited by stimulation at these points. The TSTcontrol study comprised three electrical stimuli, at Erb’s point, at the wrist, and again at Erb’s point, respectively. First and second delays were adjusted as the difference between the latencies of CMAPs elicited by stimulation at Erb’s point and at the wrist. TST% was calculated as the ratio of TSTtest amplitude to that of the TSTcontrol multiplied by 100. Figure 2 shows examples of TST traces obtained from a control and a patient with ALS. Reference values for TST% were obtained from the data of 23 healthy subjects in our laboratory. The lower normal limit was calculated as mean -2SD, which revealed 79.4% for median and 82.4% for ulnar nerves. TST and TMS studies could not be performed on 4 patients because of their refusal to attend TMS studies. 2.5 MSCAN FIT MUNE Stimulus response curves or CMAP scans were recorded from both the APB and ADM using previously described methods with the software on the Nikolet Viking Select EMG system (version 11.1) (Maathuis et al., 2012). A bipolar stimulator was located over the median or ulnar side of the wrist and fixed to the position where the highest response was recorded with the lowest stimulus intensity. After determining the lowest stimulus intensity to produce maximal CMAP and the minimum intensity to elicit 50 µV motor response, 500 consecutive stimuli at 2 Hz frequency in a descending order were applied to generate a CMAP scan (Maathuis et al., 2012). MScanFit MUNE was calculated from CMAP scan data, which were initially exported to Microsoft Excel by using a freeware version of the program developed by Professor Hugh Bostock (Bostock, 2016, Jacobsen et al., 2017). 2.6 Statistical Analyses Nonparametric tests were used in statistical analyses because the majority of variables were not normally distributed. The Mann-Whitney U test was performed for intergroup comparisons. Correlations of different F-wave parameters, clinical scores, CMAPs, TMS measurements, and TST parameters were analyzed using Spearman rank correlation. A receiver operating characteristics (ROC) curve was used for the discriminative power of index Frep and index RN values in both muscles. The F-wave parameters were compared between the patients with low and normal TST% using Mann-Whitney U test.

8

Patients were also divided into two groups according to Awaji criteria [patients with definite and probable (n=29) and those with possible ALS (n=6)]. A nonparametric comparison was performed for F-wave parameters between these two groups. The Statistical Package for the Social Sciences (IBM SPSS Statistics, New York, USA) Ver. 20.0 software was used for statistical evaluations and p<0.05 was accepted as the limit of significance.

3. Results 3.1 Comparison of F-wave parameters between patients with ALS and controls Among the studied parameters, F-wave persistence was significantly low in both muscles of patients with ALS as compared with controls. The minimum, maximum, and mean F-wave latencies were longer in the ADM and APB muscles and mean and maximum F-wave amplitudes were significantly higher in the ADM muscle of patients with ALS than those of controls (Table 1). Persistence of Frep, index Frep, index RN, NI and F/M% were found significantly higher in patients with ALS when compared with the control group in both muscles (Table 1). ROC curve analysis revealed that Frep parameters had a good diagnostic power in discriminating patients with ALS from controls with high sensitivity and specificity (Figure 3). 3.2 Comparison of F-wave parameters in relation to split hand The APB/ADM amplitude ratio was positive in only 3 patients with ALS and there was also no significant difference between the mean CMAP amplitudes recorded from their thenar and hypothenar muscles. The persistences of F-waves and Fnonreps were significantly lower in thenar muscles when compared with the hypothenar region. As for the other Freps, there were no significant differences between the APB and ADM (Table 1). 3.3 Correlation analysis of Frep parameters with other electrophysiologic and clinical findings Both index Frep and index RN showed inverse correlations with NI and MScanFit MUNE in the ADM muscle (Figure 4). However, there was no significant correlation between these parameters in the APB muscle. Fnonrep persistence was negatively correlated with index RN and index Freps in both muscles (Figure 4). F/M% showed a positive significant correlation with the persistence of Frep and index Frep in both recording muscles, and with index RN in the ADM (Figure 4). None of the Frep parameters were correlated with RMT, CSP, ISP, mean SICI, and ICF values in ADM. TST% and CMCT did not show any significant correlation with Freps in both ADM and APB.

9

Freps also showed no significant correlation with the clinical parameters, ALSFRS-R, MRC sum scores, and ALSFRS-R UL scores. There was also no significant difference for any F-wave parameter between the two subgroups of ALS patients which were divided according to Awaji criteria (Supplementary Table 1). 3.4 Comparison of F-Wave parameters between patients having low and normal TST values Seventeen patients’ median TST% (54.8%) and 12 patients’ ulnar TST% (38.7%) were lower than the reference limits. When patients with ALS were categorized into two groups as those with low and normal TST%, there were no significant intergroup differences in relation to Frep parameters (Table 2). 4. Discussion 4.1 F-wave parameters in patients with ALS in comparison with controls The abnormal F-wave parameters found in this study, such as decreased persistence, longer latencies, and higher F amplitudes were in concordance with previous reports (Chroni et al., 2012, Fang et al., 2016, Peioglou-Harmoussi et al., 1987, Petajan, 1985). The higher mean F-wave amplitudes, that could not reach statistical significance in the APB muscle, might be caused by abnormal distribution of data, possibly related to very low F-wave persistence in this muscle (Cengiz et al., 2018). Our results on Freps revealing high Frep persistence, index Frep and index RN values with high sensitivities and specificities in differentiating the patients with ALS from controls, were also similar to those found by previous reports (Carvalho et al., 2002, Chroni et al., 2012, Milanov, 1992, Peioglou-Harmoussi et al., 1987). The inverse correlation between Fnonrep persistence and Frep indices in the thenar and hypothenar muscles also supported previous authors’ observations about the increased appearance of Freps when other neurons become unable to produce F-waves (Chroni et al., 2012). The split-hand sign is defined as preferential wasting of thenar group muscles with relative preservation of the hypothenar region, which appears as a specific feature for ALS (Cengiz et al., 2018, Eisen and Kuwabara, 2012, Kuwabara et al., 2008, Menon et al., 2014, Wilbourn, 2000). In the present study, the persistence of F-waves and other parameters related to this persistence (NI and persistence of Fnonreps) were significantly lower in the APB when compared with the ADM in the ALS group (Fang et al., 2016). However, contrary to expectations, we found no significant difference between the Frep parameters elicited in the APB and ADM. The reason for this discrepancy might be the earlier and more severe reduction of F-wave frequency in the APB muscle, which might counteract the detection of Freps (Cheah et al., 2011, de Carvalho et al., 2005b). Another factor 10

might be the exclusion criterion of our study that discarded patients with less than 1 mV CMAP amplitude in either muscle. This might have led to the exclusion of patients with more obvious splithand and the full blown picture of F-wave distribution in these patients (Fang et al., 2016). 4.2 The generation of Freps in patients with ALS Although it is well-established that Freps are highly prevalent in ALS, the mechanism of their generation is poorly understood (Argyriou et al., 2006, Chroni et al., 2012, Fang et al., 2015, Fang et al., 2016, Ibrahim and el-Abd, 1997, Peioglou-Harmoussi et al., 1987, Petajan, 1985). In the present study, these high numbers of Freps were found, in at least one of the recording muscles (ADM), to have significant negative correlations with two important parameters, NI and MScanfit MUNE, which have been stated as sensitive methods for evaluating the amount of functional motor neurons (de Carvalho et al., 2005a, de Carvalho et al., 2003, 2005b, Jacobsen et al., 2017, Swash and de Carvalho, 2004, Vucic and Rutkove, 2018). Although it is difficult to explain the reason for the absence of a significant correlation related to the findings elicited from the APB muscle, except for the abnormal distribution of data, the most conceivable reason might be the low frequency of F-waves in the thenar muscle, which also limits the use of NIs recorded from this muscle in detecting the progression of ALS (Cheah et al., 2011, de Carvalho et al., 2005b). Several studies reported that the extent of motoneuronal loss was associated with Freps in ALS (Fang et al., 2016, Ibrahim and el-Abd, 1997, Peioglou-Harmoussi et al., 1987, Petajan, 1985). Additionally, an increased frequency of Freps was reported in poliomyelitis, polyneuropathy, mononeuropathy, and radiculopathy, implying that axonal loss is responsible from Freps (Chroni et al., 2012, Hachisuka et al., 2015, Macleod, 1987, Pastore-Olmedo et al., 2009, Zheng et al., 2016). The measures, which correlated with persistence and indices of Freps in this study, seemed, at the first sight, to be derived from the same electrophysiological parameter, F-wave. However, MScanFit MUNE has a novel mathematical method in estimating motor units from a stimulus response curve and two other electrophysiologic measures are used, except F-wave persistence, in calculating the NI. The TMS parameters provide information about the membrane excitability of motor neurons and the integrity of corticomotoneural projections (Chen et al., 2008, Huynh et al., 2019, Huynh et al., 2016, Yildiz et al., 2017). To detect the effect of UMN involvement in generation of Freps, we performed correlation analyses between TMS parameters and Freps in ADM and APB. The RMT, CSP, ISP, and paired-pulse (SICI and ICF) studies were not correlated with Freps in hypothenar muscle. In addition, CMCT did not show any correlation with Freps in both ADM and APB. Conventional TMS techniques and paired-pulse TMS studies were criticized for high intersubject and intrasubject variability (Boroojerdi et al., 2000, Maeda et al., 2002). Therefore, we also compared Frep parameters with TST, which has been claimed to be an objective and sensitive method for detecting 11

and quantifying UMN involvement (Attarian et al., 2007, Kleine et al., 2010, Komissarow et al., 2004). However, no correlation was found between TST and Frep parameters in both recording muscles. Additionally, similar Frep indices were found in patients with abnormal and normal TST%. Our findings, as a whole, suggest that the presence and density of Freps are primarily related to the degree of LMN loss and they show no correlation with a relatively extensive set of parameters for UMN dysfunction. Although these findings imply the nearly pure role of lower motor neuron involvement in the mechanisms of Freps, it must be considered that there are several descending pathways with intervening last-order interneurons in spinal segments exerting inhibitory or excitatory inputs on the anterior horn motoneuronal pool (Bannatyne et al., 2009, Brownstone and Bui, 2010, Hultborn et al., 1976). An influence of the spinal excitability changes on Freps cannot be fully excluded, because our TMS methods are mostly related to the function of primary motor cortex and CST. Another interesting finding of this study was the significant positive correlation between F/M% and Frep parameters in both muscles. As a response to progressive motor neuron loss, reinnervation intervenes for compensation and the results of these dual processes establish the diagnostic hallmarks of ALS (de Carvalho et al., 2008). The reduced number of motor neurons in generation of F-waves gives rise to greater number of Freps. On the other hand, large F-waves and giant Freps, have been implied to be related with reinnervated motor units (Argyriou et al., 2006, Drory et al., 2001, Eisen and Odusote, 1979, Fisher, 1988, Ibrahim and el-Abd, 1997). In addition to the role of these reduced and reinnervated motor neurons for explaining our findings, spinal motoneuronal hyperexcitability leading to a high tendency of the anterior horn cells to produce Freps, could not be completely ruled out in the genesis of this correlation (Argyriou et al., 2006, Drory et al., 2001, Fisher, 1988). A previous study demonstrated that the frequency of Freps is increased in patients with ALS with pyramidal signs when compared with non-pyramidal group (Fang et al., 2015). The authors divided the patient groups according to the presence or absence of pyramidal signs and did not use a quantitative tool for determining CST involvement. In our study, most of our patients had pyramidal signs and we additionally used a quantitative tool, the TST, for evaluating CST involvement. With the absence of a correlation between TST and Freps, it seems to be difficult to suggest that Freps are related to CST involvement. Peioglou-Harmoussi et al. showed that the frequency of Freps was higher in ALS compared with controls and in cervical myelo-radiculopathy compared with cervical radiculopathy, emphasizing the role of UMN dysfunction in generation of Freps (Peioglou-Harmoussi et al., 1987). However, a 12

study comparing dynamic F-waves between ALS and Hirayama disease showed similar percentages of Freps in patients with ALS and Hirayama disease in the standard neck position (Zheng et al., 2016). These controversial results might be because of different study designs and inadequate numbers of subjects. Spinal motor neuron excitability, which could contribute to generating Freps, might also change over time in the course of the disease. Longitudinal studies investigating Freps are needed to unravel this discrepancy. F-wave studies in diseases with pure UMN involvement such as multiple sclerosis and cerebrovascular disease showed an increase in F-wave persistence (Schiller and Stalberg, 1978), amplitude (Eisen and Odusote, 1979), prolonged F-wave duration (Bischoff et al., 1992), and latency (Fisher, 1986), but none of these studies investigated Freps. 4.3 F-wave parameters in relation to clinical measures According to our results, ALSFRS-R and MRC sum scores were not correlated with Frep parameters. These clinical scores provide a global functional evaluation in patients with ALS. Furthermore, in order to concentrate on the extremities where we performed our recordings, the ALSFRS-R UL score, the subscore of ALSFRS-R addressing upper extremity function, also revealed no signification. These suggest that, clinical scores are weaker in reflecting motoneuronal loss, probably due to the reinnervational compensation capacity of the motor system, and Freps might provide an earlier measure about motoneuron degeneration, like most of the electrophysiologic methods devoted to this subject (de Carvalho et al., 2018, de Carvalho et al., 2005a, de Carvalho and Swash, 2010, 2016, Jacobsen et al., 2019). 5. Conclusion Our findings, as a whole, suggest that the presence and density of Freps are primarily related to the degree of LMN loss and they show no obvious correlation with UMN dysfunction. Although these findings might imply the primary role of lower motor neuron involvement in the mechanisms of Freps, we think that the influence of the altered spinal excitability related to the dysfunction of pathways other than CST cannot be fully excluded.

Conflicts of interest None of the authors have potential conflicts of interest to be disclosed. Acknowledgements All authors specially thank Hugh Bostock, PhD for his support in MScanFit MUNE analyses and Lala Mehdikhanova, MD for her contribution in inclusion of the patients. 13

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References: Argyriou AA, Karanasios P, Makridou A, Makris N. F-wave characteristics as surrogate markers of spasticity in patients with secondary progressive multiple sclerosis. J Clin Neurophysiol 2010;27:120-5. Argyriou AA, Polychronopoulos P, Talelli P, Chroni E. F wave study in amyotrophic lateral sclerosis: assessment of balance between upper and lower motor neuron involvement. Clin Neurophysiol 2006;117:1260-5. Attarian S, Verschueren A, Pouget J. Magnetic stimulation including the triple-stimulation technique in amyotrophic lateral sclerosis. Muscle Nerve 2007;36:55-61. Bannatyne BA, Liu TT, Hammar I, Stecina K, Jankowska E, Maxwell DJ. Excitatory and inhibitory intermediate zone interneurons in pathways from feline group I and II afferents: differences in axonal projections and input. J Physiol 2009;587:379-99. Bischoff C, Schoenle P, Conrad B. Increased F -wave duration in patients with spasticity. Electromyogr Clin Neurophysiol 1992;32:449-53. Boroojerdi B, Kopylev L, Battaglia F, Facchini S, Ziemann U, Muellbacher W, et al. Reproducibility of intracortical inhibition and facilitation using the paired-pulse paradigm. Muscle Nerve 2000;23:1594-7. Bostock H. Estimating motor unit numbers from a CMAP scan. Muscle Nerve 2016;53:889-96. Brownstone RM, Bui TV. Spinal interneurons providing input to the final common path during locomotion. Prog Brain Res 2010;187:81-95. Cedarbaum JM, Stambler N, Malta E, Fuller C, Hilt D, Thurmond B, et al. The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of respiratory function. J Neurol Sci 1999;169:13-21. Cengiz B, Mercan M, Kuruoğlu R. Spinal excitability changes do not influence the mechanisms of split-hand syndrome in amyotrophic lateral sclerosis. Muscle Nerve 2018;58:503-8. Cheah BC, Vucic S, Krishnan AV, Boland RA, Kiernan MC. Neurophysiological index as a biomarker for ALS progression: validity of mixed effects models. Amyotroph Lateral Scler 2011;12:33-8. Chen R, Cros D, Curra A, Di Lazzaro V, Lefaucheur J-P, Magistris MR, et al. The clinical diagnostic utility of transcranial magnetic stimulation: report of an IFCN committee. Clin Neurophysiol 2008;119:504-32. Chroni E, Argyriou AA, Katsoulas G, Polychronopoulos P. Ulnar F wave generation assessed within 3 days after the onset of stroke in patients with relatively preserved level of consciousness. Clin Neurol Neurosurg 2007;109:27-31. Chroni E, Katsoulas G, Argyriou AA, Sakellaropoulos GC, Polychronopoulos P, Nikiforidis G. Level of consciousness as a conditioning factor of F wave generation in stroke patients. Clin Neurophysiol 2006;117:315-9. Chroni E, Tendero IS, Punga AR, Stålberg E. Usefulness of assessing repeater F-waves in routine studies. Muscle Nerve 2012;45:477-85. de Carvalho M, Barkhaus PE, Nandedkar SD, Swash M. Motor unit number estimation (MUNE): Where are we now? Clin Neurophysiol 2018;129:1507-16.

14

de Carvalho M, Chio A, Dengler R, Hecht M, Weber M, Swash M. Neurophysiological measures in amyotrophic lateral sclerosis: markers of progression in clinical trials. Amyotroph Lateral Scler 2005;6:17-28. de Carvalho M, Dengler R, Eisen A, England JD, Kaji R, Kimura J, et al. Electrodiagnostic criteria for diagnosis of ALS. Clin Neurophysiol 2008;119:497-503. de Carvalho M, Scotto M, Lopes A, Swash M. F-waves and the corticospinal lesion in amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2002;3:131-6. de Carvalho M, Scotto M, Lopes A, Swash M. Clinical and neurophysiological evaluation of progression in amyotrophic lateral sclerosis. Muscle Nerve 2003;28:630-3. de Carvalho M, Scotto M, Lopes A, Swash M. Quantitating progression in ALS. Neurology 2005;64:1783-5. de Carvalho M, Swash M. Sensitivity of electrophysiological tests for upper and lower motor neuron dysfunction in ALS: a six-month longitudinal study. Muscle Nerve 2010;41:208-11. de Carvalho M, Swash M. Lower motor neuron dysfunction in ALS. Clin Neurophysiol 2016;127:267081. Drory VE, Kovach I, Groozman GB. Electrophysiologic evaluation of upper motor neuron involvement in amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2001;2:147-52. Drory VE, Neufeld MY, Korczyn AD. F-wave characteristics following acute and chronic upper motor neuron lesions. Electromyogr Clin Neurophysiol 1993;33:441-6. Eisen A, Kuwabara S. The split hand syndrome in amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 2012;83:399-403. Eisen A, Odusote K. Amplitude of the F wave: a potential means of documenting spasticity. Neurology 1979;29:1306-9. Fang J, Cui L-Y, Liu M-S, Guan Y-Z, Li X-G, Cui B, et al. F wave study in amyotrophic lateral sclerosis: assessment of segmental motoneuronal dysfunction. Chin Medical J(Engl) 2015;128:1738-42. Fang J, Cui L, Liu M, Guan Y, Li X, Li D, et al. Differences in Dysfunction of Thenar and Hypothenar Motoneurons in Amyotrophic Lateral Sclerosis. Front Hum Neurosci 2016;10:99. Fisher MA. F response latencies and durations in upper motor neuron syndromes. Electromyograph Clin Neurophysiol 1986;26:327-32. Fisher MA. F/M ratios in polyneuropathy and spastic hyperreflexia. Muscle Nerve 1988;11:217-22. Furtula J, Johnsen B, Frandsen J, Rodell A, Christensen PB, Pugdahl K, et al. Upper motor neuron involvement in amyotrophic lateral sclerosis evaluated by triple stimulation technique and diffusion tensor MRI. J Neurol 2013;260:1535-44. Gooch CL, Doherty TJ, Chan KM, Bromberg MB, Lewis RA, Stashuk DW, et al. Motor unit number estimation: a technology and literature review. Muscle Nerve 2014;50:884-93. Gooch CL, Shefner J. ALS surrogate markers. MUNE. Amyotroph Lateral Scler Other Motor Neuron Disord 2004;5:104-7. Grieve SM, Menon P, Korgaonkar MS, Gomes L, Foster S, Kiernan MC, et al. Potential structural and functional biomarkers of upper motor neuron dysfunction in ALS. Amyotroph Lateral Scler Frontotemporal Degener 2016;17:85-92. Hachisuka A, Komori T, Abe T, Hachisuka K. Repeater F-waves are signs of motor unit pathology in polio survivors. Muscle Nerve 2015;51:680-5. Hermans G, Clerckx B, Vanhullebusch T, Segers J, Vanpee G, Robbeets C, et al. Interobserver agreement of medical research council sum-score and handgrip strength in the intensive care unit. Muscle Nerve 2012;45:18-25. Hultborn H, Illert M, Santini M. Convergence on interneurones mediating the reciprocal Ia inhibition of motoneurones. III. Effects from supraspinal pathways. Acta Physiol Scand 1976;96:368-91. Huynh W, Dharmadasa T, Vucic S, Kiernan MC. Functional Biomarkers for Amyotrophic Lateral Sclerosis. Front Neurol 2019;9:1141. Huynh W, Simon NG, Grosskreutz J, Turner MR, Vucic S, Kiernan MC. Assessment of the upper motor neuron in amyotrophic lateral sclerosis. Clin Neurophysiol 2016;127:2643-60. 15

Ibrahim IK, el-Abd MA. Giant repeater F-wave in patients with anterior horn cell disorders: Role of motor unit size. Am J Phy Med Rehabil 1997;76:281-7. Jacobsen AB, Bostock H, Fuglsang-Frederiksen A, Duez L, Beniczky S, Møller AT, et al. Reproducibility, and sensitivity to motor unit loss in amyotrophic lateral sclerosis, of a novel MUNE method: MScanFit MUNE. Clin Neurophysiol 2017;128:1380-8. Jacobsen AB, Bostock H, Tankisi H. Following disease progression in motor neuron disorders with 3 motor unit number estimation methods. Muscle Nerve 2019;59:82-7. Kleine BU, Schelhaas HJ, van Elswijk G, de Rijk MC, Stegeman DF, Zwarts MJ. Prospective, blind study of the triple stimulation technique in the diagnosis of ALS. Amyotroph Lateral Scler 2010;11:67-75. Komissarow L, Rollnik JD, Bogdanova D, Krampfl K, Khabirov F, Kossev A, et al. Triple stimulation technique (TST) in amyotrophic lateral sclerosis. Clin Neurophysiol 2004;115:356-60. Kristensen RS, Bostock H, Tan SV, Witt A, Fuglsang-Frederiksen A, Qerama E, et al. MScanFit motor unit number estimation (MScan) and muscle velocity recovery cycle recordings in amyotrophic lateral sclerosis patients. Clin Neurophysiol 2019;130:1280-88. Kujirai T, Caramia MD, Rothwell JC, Day BL, Thompson PD, Ferbert A, et al. Corticocortical inhibition in human motor cortex. J Physiol 1993;471:501-19. Kuwabara S, Sonoo M, Komori T, Shimizu T, Hirashima F, Inaba A, et al. Dissociated small hand muscle atrophy in amyotrophic lateral sclerosis: frequency, extent, and specificity. Muscle Nerve 2008;37:426-30. Leis AA, Kronenberg MF, Stetkarova I, Paske WC, Stokic DS. Spinal motoneuron excitability after acute spinal cord injury in humans. Neurology 1996;47:231-7. Maathuis EM, Henderson RD, Drenthen J, Hutchinson NM, Daube JR, Blok JH, et al. Optimal stimulation settings for CMAP scan registrations. J Brachial Plex Peripher Nerve Inj 2012;7:4. Macleod WN. Repeater F waves A comparison of sensitivity with sensory antidromic wrist-to-palm latency and distal motor latency in the diagnosis of carpal tunnel syndrome. Neurology 1987;37:773-8. Maeda F, Gangitano M, Thall M, Pascual-Leone A. Inter-and intra-individual variability of paired-pulse curves with transcranial magnetic stimulation (TMS). Clin Neurophysiol 2002;113:376-82. Magistris MR, Rösler K, Truffert A, Myers J. Transcranial stimulation excites virtually all motor neurons supplying the target muscle. A demonstration and a method improving the study of motor evoked potentials. Brain 1998;121:437-50. Magistris MR, Rösler KM, Truffert A, Landis T, Hess CW. A clinical study of motor evoked potentials using a triple stimulation technique. Brain 1999;122:265-79. Menon P, Kiernan MC, Vucic S. ALS pathophysiology: insights from the split-hand phenomenon. Clin Neurophysiol 2014;125:186-93. Milanov IG. F-wave for assessment of segmental motoneurone excitability. Electromyograph Clin Neurophysiol 1992;32:11-5. Orhan EK, Yayla V, Cebeci Z, Baslo MB, Ovalı T, Öge AE. Excitability changes at brainstem and cortical levels in blind subjects. Clin Neurophysiol 2011;122:1827-33. Pastore-Olmedo C, Gonzalez O, Geijo-Barrientos E. A study of F-waves in patients with unilateral lumbosacral radiculopathy. Eur Jour Neurol 2009;16:1233-9. Peioglou-Harmoussi S, Fawcett P, Howel D, Barwick D. F-response frequency in motor neuron disease and cervical spondylosis. J Neurol Neurosurg Psychiatry 1987;50:593-9. Petajan JH. F-waves in neurogenic atrophy. Muscle Nerve1985;8:690-6. Rosler KM, Truffert A, Hess CW, Magistris MR. Quantification of upper motor neuron loss in amyotrophic lateral sclerosis. Clin Neurophysiol 2000;111:2208-18. Rossini PM, Barker A, Berardelli A, Caramia M, Caruso G, Cracco R, et al. Non-invasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalograph Clin Neurophysiol 1994;91:79-92. 16

Rossini PM, Burke D, Chen R, Cohen L, Daskalakis Z, Di Iorio R, et al. Non-invasive electrical and magnetic stimulation of the brain, spinal cord, roots and peripheral nerves: basic principles and procedures for routine clinical and research application. An updated report from an IFCN Committee. Clin Neurophysiol 2015;126:1071-107. Schiller H, Stalberg E. F responses studied with single fibre EMG in normal subjects and spastic patients. J Neurol Neurosurg Psychiatry 1978;41:45-53. Swash M, de Carvalho M. The neurophysiological index in ALS. Amyotroph Lateral Scler Other Motor Neuron Disord 2004;5:108-10. Ugawa Y, Day BL, Rothwell JC, Thompson PD, Merton PA, Marsden CD. Modulation of motor cortical excitability by electrical stimulation over the cerebellum in man. J Physiol 1991;441:57-72. Verber NS, Shepheard SR, Sassani M, McDonough HE, Moore SA, Alix JJ, et al. Biomarkers in motor neuron disease: a state of the art review. Front Neurol 2019;10:291. Vucic S, Rutkove SB. Neurophysiological biomarkers in amyotrophic lateral sclerosis. Curr Opin Neurol 2018;31:640-7. Vucic S, van den Bos M, Menon P, Howells J, Dharmadasa T, Kiernan MC. Utility of threshold tracking transcranial magnetic stimulation in ALS. Clin Neurophysiol Pract 2018;3:164-72. Wang Y, Wang H, Cui LY. Triple Stimulation Technique in Amyotrophic Lateral Sclerosis. J Clin Neurophysiol 2019;36:87-92. Wilbourn AJ. The "split hand syndrome". Muscle Nerve 2000;23:138. Wittstock M, Wolters A, Benecke R. Transcallosal inhibition in amyotrophic lateral sclerosis. Clin Neurophysiol 2007;118:301-7. Yildiz FG, Bekircan-Kurt CE, Varli K. Single-pulse transcranial magnetic stimulation in amyotrophic lateral sclerosis: experiences of a single institution. Neurol Sci Neurophysiol 2017;34:70-5. Zheng C, Zhu Y, Yang S, Lu F, Jin X, Weber R, et al. A study of dynamic F-waves in juvenile spinal muscular atrophy of the distal upper extremity (Hirayama disease). J Neurol Sci 2016;367:298-304.

17

Figure Legends: Figure 1. F-waves recorded from abductor digiti minimi (ADM) muscle of a patient with ALS (a) and a control case (b). Repeater F-waves (Freps) are marked by symbols in the patient. An individual symbol is used for each repeater neuron; asterisk (*), dagger (†), double dagger (‡), number sign (#). Please note that there are 4 repeater neurons in this patient. In (a), reduced F-wave persistence is also seen while there is a full persistence without ant Freps in (b). Figure 2. Traces obtained using the triple stimulation technique (TST) from a healthy control (a) and a patient with ALS (b). A1 and A2 traces were recorded by stimulating ulnar nerve at wrist and at Erb’s point, respectively. Motor evoked potential elicited by transcranial magnetic stimulation is shown in trace A3. In A4 and A5, TSTtest and TSTcontrol responses are marked by an arrow and dashed arrow, respectively. A clear reduction in TSTtest response can be seen in a patient with ALS (b). Figure 3. ROC curves of index Frep and index RN in ALS and control. The table, underneath the curves, provides cut-off values, sensitivities, specificities, and area under curve values. Index Frep, index of repeater F-waves; Index RN, index of repeater neurons. Figure 4. Correlation graphs between repeater F-wave and other electrophysiologic parameters NI, neurophysiologic index; MUNE, motor unit number estimation; index Frep, index of repeater Fwaves; Index RN, index of repeater neurons; Fnonrep persistence, persistence of non-repeater Fwaves; Frep persistence, persistence of repeater F-waves; F/M%, F/M amplitude ratio; ADM, abductor digiti minimi; APB, abductor pollicis previs

18

Table 1 Mean data and comparison of electrophysiological parameters obtained from thenar and hypothenar muscles in ALS patients and healthy controls. Median-ALS (n:35)

Ulnar-ALS (n:35)

Median- HC (n:18)

Ulnar- HC (n:18)

p (ALS vs HC)

P(median vs ulnar) (ALS)

Median Ulnar CMAP amp (mV) 6.7±2.7 6.9±1.9 10.7±2.1 10.1±1.7 <0.001 <0.001 0.507 Persistence of F57.1±31.3 87.4±17.9 90.5±10.0 95.7±6.9 0.001 0.031 <0.001 waves Fmin latency (ms) 24.7±2.8 25.4±2.7 22.9±1.8 22.9±2.3 0.014 0.003 0.371 Fmax latency (ms) 34.1±4.9 34.0±5.8 29.8±5.4 29.7±2.7 0.001 <0.001 0.577 Fmean latency (ms) 28.9±2.8 28.8±2.2 25.8±1.8 26.0±2.2 <0.001 <0.001 0.857 Fmean amp (μV) 358.1±297.6 368.1±171.3 270.5±101.9 242.5±101.8 0.513 0.008 0.164 Fmax amp (μV) 893.3±590.2 975.4±369.5 990.6±367.8 723.6±308.8 0.152 0.020 0.164 Index RN 22.1±19.2 14.4±11.8 3.2±3.2 1.8±1.7 <0.001 <0.001 0.066 Persistence of Freps 25.7±19.1 28.7±21.9 7.9±8.6 3.5±3.4 <0.001 <0.001 0.683 Persistence of 33.1±25.1 58.7±27.0 82.6±12.7 92.2±8.5 <0.001 <0.001 <0.001 Fnonreps Index Frep 47.2±25.7 34.5±25.3 8.8±9.2 3.8±3.7 <0.001 <0.001 0.43 NI 127.5±114.0 230.5±97.0 331.5±110.7 390.7±67.3 <0.001 <0.001 <0.001 F/M amp (%) 6.0±5.0 5.9±3.9 2.5±0.9 2.5±1.1 0.005 <0.001 0.657 ALS, amyotrophic lateral sclerosis; HC, healthy controls; CMAP, compound muscle action potential; NI, neurophysiologic index; RN, repeater neuron; Frep, repeater F-wave; Fnonrep, nonrepeater F-wave; amp, amplitude

19

Table 2. Comparison of repeater F wave parameters in patients with low and normal TST results.

CMAPamp (milivolt) Total persistence Persistence of Freps Persistence of Fnonreps Index Frep Index RN F/M%

Median Normal TST% Low TST% (n=14) (n=17) 7.66±2.77 5.70±2.65 65.24±28.18 52.68±32.19 28.02±18.35 24.38±20.18 37.22±27.07 31.60±23.04

Ulnar Normal TST% (n=19) 6.75±1.75 87.19±18.37 23.63±14.12 63.57±26.39

Low TST% (n=12) 7.36±2.43 87.04±20.19 33.33±25.03 53.70±23.81

Median 0.084 0.377 0.473 0.637

0.589 0.921 0.412 0.236

48.98±27.04 24.55±23.93 6.26±6.06

30.73±22.45 13.77±11.77 5.99±2.62

37.25±24.03 14.51±11.40 6.02±5.73

0.667 0.448 0.525

0.412 0.704 0.326

44.44±26.18 19.24±16.42 6.20±4.50

p

Ulnar

TST, triple stimulation technique; CMAP, compound muscle action potential; Freps, repeater F-waves; Fnonreps, non-repeater F-waves; RN, repeater neuron; F/M%; F/M amplitude ratio.

20

21

22

23

24