Journal of Clinical Medicine Article Cervical Electrical Neuromodulation Effectively Enhances Hand Motor Output in Healthy Subjects by Engaging a Use-Dependent Intervention Hatice Kumru 1,2,3, *, África Flores 4 , María Rodríguez-Cañón 4 , Victor R. Edgerton 1,5 , Loreto García 1,2,3 , Jesús Benito-Penalva 1,2,3 , Xavier Navarro 1,4 , Yury Gerasimenko 6,7 , Guillermo García-Alías 1,4 and Joan Vidal 1,2,3 1 Fundación Institut Guttmann, Institut Universitari de Neurorehabilitació Adscrit a la Universitat Autònoma de Barcelona, 08916 Badalona, Spain; vre@ucla.edu (V.R.E.); loretogarcia@guttmann.com (L.G.); jbenito@guttmann.com (J.B.-P.); xavier.navarro@uab.cat (X.N.); guillermo284@gmail.com (G.G.-A.); jvidal@guttmann.com (J.V.) 2 Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 3 Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, 08916 Badalona, Spain 4 Departament de Biologia Cel·lular, Fisiologia i Immunologia & Insititute of Neuroscience, Universitat Autònoma de Barcelona, and CIBERNED, Bellaterra, 08193 Barcelona, Spain; africa.flores@uab.cat (Á.F.); rodriguezcanonmaria@gmail.com (M.R.-C.) 5 Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA 6 Pavlov Institute of Physiology, 199034 St. Petersburg, Russia; yuryg@ucla.edu 7  Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40292, USA  * Correspondence: hkumru@guttmann.com; Tel.: +34-934-977-700 Citation: Kumru, H.; Flores, Á.; Rodríguez-Cañón, M.; Edgerton, V.R.; Abstract: Electrical enabling motor control (eEmc) through transcutaneous spinal cord stimulation García, L.; Benito-Penalva, J.; is a non-invasive method that can modify the functional state of the sensory-motor system. We Navarro, X.; Gerasimenko, Y.; hypothesize that eEmc delivery, together with hand training, improves hand function in healthy García-Alías, G.; Vidal, J.et al. subjects more than either intervention alone by inducing plastic changes at spinal and cortical levels. Cervical Electrical Neuromodulation Ten voluntary participants were included in the following three interventions: (i) hand grip training, Effectively Enhances Hand Motor (ii) eEmc, and (iii) eEmc with hand training. Functional evaluation included the box and blocks Output in Healthy Subjects by test (BBT) and hand grip maximum voluntary contraction (MVC), spinal and cortical motor evoked Engaging a Use-Dependent potential (sMEP and cMEP), and resting motor thresholds (RMT), short interval intracortical inhibition Intervention. J. Clin. Med. 2021, 10, 195. https://doi.org/10.3390/ (SICI), and F wave in the abductor pollicis brevis muscle. eEmc combined with hand training retained jcm10020195 MVC and increased F wave amplitude and persistency, reduced cortical RMT and facilitated cMEP amplitude. In contrast, eEmc alone only increased F wave amplitude, whereas hand training alone Received: 12 December 2020 reduced MVC and increased cortical RMT and SICI. In conclusion, eEmc combined with hand grip Accepted: 5 January 2021 training enhanced hand motor output and induced plastic changes at spinal and cortical level in Published: 7 January 2021 healthy subjects when compared to either intervention alone. These data suggest that electrical neuromodulation changes spinal and, perhaps, supraspinal networks to a more malleable state, while Publisher’s Note: MDPI stays neu- a concomitant use-dependent mechanism drives these networks to a higher functional state. tral with regard to jurisdictional clai- ms in published maps and institutio- Keywords: transcutaneous spinal cord stimulation; hand training; combined intervention; neuro- nal affiliations. modulation; cervical spinal cord Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Electrical stimulation of the spinal cord is an emerging valuable tool in clinical practice distributed under the terms and con- for facilitating the recovery of sensory and motor function in subjects with spinal cord ditions of the Creative Commons At- injury (SCI) [1]. Outstanding clinical achievements have been recently reported showing tribution (CC BY) license (https:// that lumbar epidural stimulation facilitates the recovery of posture, stepping, and vol- creativecommons.org/licenses/by/ untary control of the lower limbs in subjects with chronic SCI [2–6]. Cervical epidural 4.0/). J. Clin. Med. 2021, 10, 195. https://doi.org/10.3390/jcm10020195 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 195 2 of 18 stimulation increased hand grip force and hand volitional control in tetraplegic patients [7]. On the other hand, recently developed non-invasive transcutaneous electrical spinal cord stimulation (electrical enabling motor control, eEmc) has demonstrated its efficacy to im- prove lower limb motor function after paralysis [8–10], as well as hand grip strength and voluntary control in tetraplegic patients [11–14]. In both stimulation conditions, the under- lying hypothesis states that the spinal sensory–motor networks above, within, and below the lesion are neuromodulated and raised into an elevated functional state that enables and amplifies voluntary motor control [11–14]. In the context of SCI rehabilitation, the term eEmc [8–10] emphasizes and distinguishes this technique from other transcutaneous electrical stimulation approaches that directly induce muscular contraction, instead of facilitating and enabling voluntary control. Since transcutaneous spinal cord stimulation (tSCS) is a non-invasive and safe method, it can be easily applied in healthy subjects and can also be employed to unravel the neurophysiological mechanisms acting on sensory and motor recovery in individuals suffering from SCI [12]. By applying high-frequency trains of stimuli (i.e., eEmc) to the thoracolumbar segments, locomotor-related neuronal networks can be neuromodulated to physiological states that enable the generation of bilateral rhythmic step-like movements with the legs placed in a gravity-neutral position in non-injured subjects [15] and in spinally injured subjects categorized as a complete SCI (ASIA A) [16]. One of the mechanisms considered to be important in that movement is mediated by activation of the spinal central pattern generators of locomotion [17]. The degree to which this specific mechanism contributes to upper limb movements, however, remains unclear, since it may act together with other adaptations to allow the recovery of a wide range of movements that are not predominantly repetitive, such as stepping. Importantly, continuous eEmc delivery also improves hand motor performance after one single exposure from 20 min to 2 h in SCI patients [11–14]. A recent work reveals that high-frequency eEmc induces plastic changes on neuronal circuits controlling upper limb function also in intact subjects [14]. Activity-dependent plasticity is the key phenomenon thought to underlie functional recovery observed after both physical activity and stimulation-based rehabilitative ap- proaches in SCI [18–20]. However, the mechanisms recruited by physical training and by electrical stimulation, although partially overlapping, may involve different and perhaps synergistic processes leading to more effective neural circuit reorganization. However, reported studies do not address whether eEmc applied alone or together with hand training showed better clinical or neurophysiological effects in SCI patients. Indeed, the majority of the studies only applied eEmc combined with hand training [9,10,12,13]. More studies are needed to directly assess the role of use-dependent mechanisms in an attempt to optimize therapeutic efficacy of stimulation approaches and training. Here we hypothesized that the application of eEmc together with hand grip training can increase hand motor output more than either of the two interventions alone. We further hypothesized that the improved function would be the result of a synergistic reorganization of both spinal and supraspinal networks that could enable the performance of the neuromuscular unit being engaged. To answer these questions, each participant was subjected to a hand motor functional and neurophysiological assessment before and after each of three interventions (training, eEmc, and training + eEmc). To detect hand muscle strength and a manual dexterity, we used the hand grip maximum voluntary contraction (MVC) and the box and blocks test (BBT). Spinal cord excitability was assessed by F wave and by recruitment curves of spinal motor evoked potentials (sMEPs) in hand and arm muscles induced by single-pulse tSCS. Corticospinal excitability was measured by resting motor threshold (RMT) and cortical motor evoked potentials (cMEPs) in hand muscles induced by transcranial magnetic stimulation (TMS), and changes in intracortical inhibitory circuits were tested by short interval intracortical inhibition (SICI). J. Clin. Med. 2021, 10, x FOR PEER REVIEW 3 of 18 J. Clin. Med. 2021, 10, 195 3 of 18 2. Experimental Section 2. Experimental Section 2.1. Participants 2.1. Participants Thirty healthy subjects were selected for participating in this study (Consolidated Thirty healthy subjects were selected for participating in this study (Consolidated Standards of Reporting Trials (CONSORT) flow diagram on Figure 1). Recruitment of par- Standards of Reporting Trials (CONSORT) flow diagram on Figure 1). Recruitment of ticipants consisted of self-selection within the current users of our installations as well as participants consisted of self-selection within the current users of our installations as well asking the participants for referrals. Eleven healthy volunteers accepted informed consent as asking the participants for referrals. Eleven healthy volunteers accepted informed and completed inclusion criteria with no known history of neurological disorders and ac- consent and completed inclusion criteria with no known history of neurological disorders cepted to participate in the study, and ten of them (7 men, 3 women; mean age = 38 ± 11 and accepted to participate in the study, and ten of them (7 men, 3 women; mean age years, age range: 24–60 years; Table 1) completed all three interventions, and were in- = 38 ± 11 years, age range: 24–60 years; Table 1) completed all three interventions, and cluded in the data analysis (CONSORT flow diagram on Figure 1). One subject rejected to were included in the data analysis (CONSORT flow diagram on Figure 1). One subject continue rejected toatcontinue the beginning of the study at the beginning because of the studyofbecause unpleasant sensationsensation of unpleasant through through cervical electrical stimulation. Inclusion criteria were: male or female between 18 and cervical electrical stimulation. Inclusion criteria were: male or female between 18 and 65 years old without any neurological disorder and other disorder which could limit 65 years old without any neurological disorder and other disorder which could limit the experiment (uncontrolled the experiment cancer, arthritis, cancer, (uncontrolled etc.), and had written arthritis, etc.), informed consent.informed and had written Exclusionconsent. criteria were any metal implants, implanted electrical devices, medications that could Exclusion criteria were any metal implants, implanted electrical devices, medications that raise sei- zure threshold, cardiac conditions, and history of syncope or concussion with could raise seizure threshold, cardiac conditions, and history of syncope or concussion loss of con- sciousness, with loss of tinnitus, or pregnancy. consciousness, tinnitus,The study protocol or pregnancy. was approved The study by the protocol was Research approved byEth- the ics Committee of the Institute Guttmann and was conducted in accordance Research Ethics Committee of the Institute Guttmann and was conducted in accordance with the Dec- laration with theof Helsinki [21]. Declaration of Helsinki [21]. Figure 1. CONSORT 2010 flow diagram showing the number of subjects involved in each phase of the study. Figure 1. CONSORT 2010 flow diagram showing the number of subjects involved in each phase of the study. J. Clin. Med. 2021, 10, 195 4 of 18 Table 1. Demographic data from the ten subjects participating in the present study. Stimulation intensities employed during high-frequency electrical enabling motor control (eEmc) are also shown. eEmc Intensity (mA) Subject Sex Age Hand Preference C3–C4 C6–C7 1 male 44 right 29 32–38 2 male 60 right 59–67 69–77 3 female 25 right 28–30 30–32 4 female 27 left 34 36 5 female 33 right 52–54 59–63 6 male 41 right 34–38 45 7 male 24 right 36–50 56–59 8 male 51 right 52–63 63–77 9 male 39 right 59 72–74 10 male 38 both 54–67 63–77 2.2. Experimental Design It was a randomized crossover study, which included three different interventions. Interventions consisted of: (i) hand grip training, (ii) eEmc, or (iii) eEmc combined with hand grip training. All participants received each intervention once with at least 1 week between each intervention. For hand functional outcomes, we assessed MVC during hand grip force and BBT. For neurophysiological assessment at spinal cord level, we evaluated F wave of abductor pollicis brevis (APB) muscle and recruitment curves of sMEPs in hand and arm muscles induced by single-pulse tSCS, applied to C3–C4 and then to C6–C7. At cortical level: RMT and cMEPs induced by TMS in hand muscles at 120% of RMT and SICI were recorded. All neurophysiological recordings were done in the dominant upper extremity muscles. The functional and neurophysiological assessments were realized at baseline, just after intervention (starting at 00 post) and one hour after the end of intervention (starting at 600 post, follow-up) to evaluate the possible short- and long-lasting aftereffects of each intervention. The duration of each experiment was around 4 h including the set up. Each complete functional and neurophysiological assessment took 30–45 min for each timepoint and was carried out in the following order: BBT, MVC, F wave, cortical RMT, cMEPs, SICI, cMEP recruitment curve, spinal RMT, and sMEP recruitment curve. 2.3. Functional Assessment 2.3.1. Maximum Voluntary Contraction during Hand Grip Strength The hydraulic hand grip dynamometer (Jamar Model 5030J1, Sammons Preston, NJ, USA) was used for MVC. Participants were asked to perform MVC for 4 s, as soon as they noticed a triggering stimulus, which was a mild electrical pulse at an intensity of 3 mA with 0.5 ms of duration delivered with ring electrodes to the fifth finger of the dominant hand. We recorded the maximal and maintained force during these 4 s in three consecutive trials, with at least one minute of rest between each trial. We registered the electromyographic (EMG) activity from the arm and hand muscles. 2.3.2. Box and Blocks Test This is a test of manual dexterity consisting of a box with a center partition [22]. The participant must pick up the maximum number of small cube-shaped blocks, one at a time, and drop them at the other side of the partition in 60 s. The score of BBT is represented by the number of blocks transported. 2.4. Neurophysiological Assessment 2.4.1. Electromyographical Recording EMG activity was recorded with a conventional EMG machine (Medelec Synergy, Oxford Instruments; Surrey, UK). After standard skin preparation, disposable adhesive J. Clin. Med. 2021, 10, 195 5 of 18 surface electrodes (outer diameter of 20 mm; Technomed) were placed over the muscle belly of the APB, abductor digiti minimi (ADM), flexor carpi radialis (FCR), extensor digitorum (ED), and biceps brachii (BB) muscles of the dominant arm, with the cathode proximal and the anode approximately 2 cm distally. The EMG signal was amplified and then filtered using a band-pass of 30 Hz–10 kHz, amplitude sensitivity of 0.1–0.5 mV and epochs of 100 ms sweep duration and recorded at a sampling rate of 50 kHz. During assessments, we ensured that the baseline EMG activity of all recorded muscles was lower than 50 µV of amplitude before delivering each single stimulus: EMG activity was checked out online and, if necessary, the subject was reminded to be relaxed. If any background EMG activity was observed after stimulus delivery, this recording was eliminated in situ and stimulation procedure was repeated. EMG signals were stored in a Synergy computer and analyzed offline with MATLAB. 2.4.2. F Wave To measure the spinal motoneuron pool excitability, F wave was recorded in APB muscle using suprathreshold electrical median nerve stimulation at the wrist level [23,24]. Both stimulus delivery and EMG signal recording were processed by a conventional EMG machine (Medelec Synergy, Oxford Instruments; Surrey, UK). Electrical stimuli of 0.5 ms of duration were delivered at 1 Hz and the electrical intensity was set at supramaximal level to induce the maximal amplitude in M wave (Mmax) (range: 17–30 mA) [24]. A minimum of 10 stimuli were delivered to calculate F wave persistency [25]. 2.4.3. Recruitment Curve of sMEPs To assess spinal cord excitability, we used monophasic rectangular 1-ms single-pulse tSCS delivered through 2 cm diameter hydrogel adhesive electrodes (axion GmbH, Ham- burg, Germany) as cathodes at C3–C4 and then at C6–C7 and two 5 × 12 cm rectangular electrodes placed symmetrically over the iliac crests as anodes. The electric intensity for spinal RMT in APB muscle of the dominant hand was determined at baseline condition in each intervention. RMT was defined as the lowest intensity that elicited sMEPs of ≥50 µV peak-to-peak amplitude at APB muscle in at least 5 out of 10 consecutive trials. Recruitment curves of sMEPs were established at gradual increasing intensities from 90% to 150% of RMT (at 10% increments, three recordings at each intensity) in all recorded muscles of the dominant hand [14,26]. 2.4.4. Transcranial Magnetic Stimulation Changes in corticospinal excitability and SICI were evaluated using a Magstim® BiStim2 TMS (Magstim Company, Whitland, Wales, UK). Subjects were seated in a chair, resting their pronated forearms on a desk in front of them and were asked to stay relaxed but awake throughout the assessments. A figure-of-eight coil was held tangentially to the scalp over the motor area of the dominant hand in the optimal position for activating the APB in a posterior-anterior current direction. The hot point for evoking the largest cMEP in APB was marked over scalp. The following parameters were measured before, after, and during follow-up of each intervention: 1. RMT, defined as the lowest intensity of TMS that evoked a cMEP of ≥50 µV peak-to-peak amplitude in the APB muscle in at least 5 of 10 consecutive trials. 2. Mean amplitude of cMEPs using single-pulse TMS at 120% of RMT of ABP in five recordings. 3. SICI using paired-pulse TMS with a subthreshold conditioning stimulus (80% of RMT) and a suprathreshold test stimulus (120% of RMT) at interstimulus interval of 2 ms in 5 recordings without background activity (between 1 to 5 recordings for each subject were rejected because of background activity) [27]. 4. Recruitment curves were obtained at increasing intensities from 90% to 150% of RMT of APB, at 10% increments (three recordings at each intensity). The absence of baseline activation was verified before carrying out each of the abovementioned recordings. J. Clin. Med. 2021, 10, 195 6 of 18 2.5. Interventions The study consisted of three interventions: (1) hand training, (2) eEmc, (3) eEmc combined with hand training. There was at least 1 week between each experiment. Hand training protocol was adapted from previous studies [12] and consisted in grasping a hand grip dynamometer at maximum maintainable contraction for 20 s, followed by an 80-s resting period. This was repeated alternating right and left hands for 30 min (9 times per hand). Thus, the total duration of contractions for each treatment was 3 min for each hand and a total of 9 min of maximum effort for all three treatments for each muscle group. Mean sustained force was registered for each contraction. In all cases, the subjects were instructed and closely monitored to assure that a neutral wrist position and a 90◦ angle of the elbow was maintained while performing MVC with hand grip (Figure 2a). A second condition consisted of delivering eEmc in the same time pattern (20 s of stimulation followed by an 80-s resting period for 30 min) in absence of hand training. eEmc was carried out with the transcutaneous electrical stimulator BioStim-5 (Cosyma Inc., Moscow, Russia). Previous reports indicate that applying stimulation simultaneously at two sites within the cervical area is consistently more effective than a single stimulation site [12]. Thus, we delivered eEmc simultaneously at two sites along the midline between spinous processes C3–C4 and C6–C7 during the corresponding intervention period. Regarding the possible mechanisms involved, it is critical to recognize that the intensity of stimulation at each spinal level was set at 90% of RMT induced by single-pulse tSCS at APB muscle of the dominant hand (range: ~30–80 mA, Table 1). Stimulation was continuously delivered using 2 cm diameter hydrogel adhesive electrodes (axion GmbH, Hamburg, Germany) as cathodes and two 5 × 12 cm rectangular electrodes placed symmetrically over the iliac crests as anodes. eEmc employed biphasic rectangular 1-ms pulses, each one filled with a carrier frequency of 10 kHz (i.e., each 1-ms pulse was composed of ten 0.1-ms biphasic rectangular pulses), that were delivered at a frequency of 30 Hz [13,17,28]. During stimulation, the subjects reported a non-painful but uncomfortable tingling sensation down the arms and at the site of stimulation, with some associated tonic contraction of paraspinal and posterior neck muscles. The last condition was eEmc combined with hand training. The subjects performed maximum sustainable contractions and simultaneously received eEmc, alternating right and left hands for 30 min. The abovementioned tonic contraction of neck muscles during stimulation did not interfere with performance of repeated grip contractions, since force levels achieved in the dynamometer during this intervention were similar to those achieved during hand training without stimulation and participants reported no impediments to perform the training. 2.6. Data Analysis and Statistics For each assessment, we calculated the mean ± standard error measurement (SEM) at baseline, right after and sixty minutes after intervention (00 post and 600 post intervention respectively) in each condition. We calculated the % changes according to baseline for each timepoint evaluation when appropriate. BBT, cMEP, SICI, and cMEP slopes could be easily compared between interventions without this normalization, so absolute values were preserved. The analysis of these outcome measures revealed the same effects if normalized. For analyzing EMG activity during MVC, the area under the curve (AUC) in EMG of each muscle was calculated from the beginning of EMG signal during 4 s of MVC. For the F wave analysis, we measured the peak-to-peak amplitude and for Mmax we measured the maximum amplitude of M wave. F wave was considered if peak-to-peak amplitude was at least 1% of M wave (Mmax) amplitude and F persistence was calculated by dividing the number of F responses by the number of stimuli from 10 recordings. The maximum amplitude of F was normalized to Mmax amplitude to obtain Fmax/Mmax ratio. For recruitment curve of sMEPs and cMEPs, we measured the peak-to-peak MEP amplitude (µV) induced in each recording of all muscles and then we calculated the mean amplitude of MEPs from three recordings for each intensity for each subject and for each J. Clin. Med. 2021, 10, 195 7 of 18 experimental condition. To detect the possible influence of each intervention and counteract the masking effect of interindividual variability, recruitment curves obtained at 00 post and 600 post intervention were normalized to baseline recruitment curves (which were considered as 0% change for all intensities) registered for each session and each subject. As a measure of corticospinal excitability, in addition, slope of non-normalized recruitment curves was calculated through linear regression from 90% to 150% RMT intensities, an interval that fitted linear distribution in all conditions and muscles [29]. For calculating SICI percentage, averaged peak-to-peak amplitude of the conditioned cMEP (obtained after the conditioning stimulus of 80% RMT with 2 ms separation from conditioned stimulus of 120% RMT) was expressed as a percentage of the averaged ampli- tude of the test cMEP (obtained at supramaximal 120% RMT stimulus): % = (conditioned cMEP/test cMEP) × 100. Data were expressed as mean ± SEM. Shapiro–Wilk test was used to assess whether data were normally distributed or not. All sets of data fulfilled normal distribution re- quirement except F wave persistence, cortical RMT, SICI, and cMEP slope. For normally distributed data, repeated measures two-way analysis of variance (ANOVA) was used. For post-hoc analysis, we used Tukey’s test. For not normally distributed data, Friedman’s analysis was used followed by post-hoc Dunn’s test. Least squares regression was applied as linear regression method to calculate slope of sMEP and cMEP recruitment curves. Significance level was set at p < 0.05 in all cases. 3. Results 3.1. Hand Motor Output Figure 2a shows a general setting for MVC assessment. MVC at baseline was 30.6 ± 0.9 kg. This value was consistent between sessions (within-subject variation coefficient was 10.9 ± 1.2%), although inter-subject variability was considerable (between-subjects variation coefficient was 28.7 ± 3.0%). Thus, all values were normalized to baseline for further analysis. Repeated- measures ANOVA showed that intervention (F(2, 18) = 11.2, p < 0.001) and timepoint (F(2, 18) = 3.67, p < 0.05) factors, as well as their interaction (F(4, 36) = 8.77, p < 0.001) had a significant effect in MVC. MVC tended to increase following eEmc combined with training and did not change after eEmc alone, whereas training alone reduced MVC significantly after intervention during follow-up (p < 0.001 according to Tukey’s post hoc). The percentage of MVC vs. baseline levels was bigger after eEmc with training compared to training alone at 00 and 600 post intervention (p < 0.001 for each comparison) and also compared to eEmc intervention alone (p < 0.01 for each comparison) (Figure 2b). The EMG activity of APB and ADM changed significantly according to two-way ANOVA (F(1, 52) = 16.43, p < 0.001 and F(1, 52) = 6.77, p < 0.01, respectively) (Figure 2c,d). There was a significant interaction between intervention and timepoint factors for APB and ADM muscles (F(4, 36) = 3.36, p < 0.05 and F(4, 36) = 4.34, p < 0.01, respectively). Tukey’s test showed that the training condition significantly reduced EMG activity of APB (at 00 and 600 post training) and ADM (at 600 post intervention) (p < 0.05). Moreover, EMG activity in APB and ADM was significantly lower following training or eEmc alone than following eEmc with hand training at both 00 and 600 post-intervention (p < 0.05 at least for each comparison) (Figure 2c,d). The EMG of FCU, ED, or BB muscles did not change significantly by intervention or timepoint according to two-way repeated measures ANOVA. Figure 2e shows a representative EMG of APB muscle (subject #5) corresponding to MVC trials during “training” and “eEmc with training” interventions. This figure depicts similar EMG activity levels at basal conditions before these two interventions, but higher EMG activity at both 00 post and 600 after the combined intervention, an effect that becomes obvious when calculating the difference in EMG activity between “eEmc with training” and “training” condition. trials during “training” and “eEmc with training” interventions. This figure depicts simi- lar EMG activity levels at basal conditions before these two interventions, but higher EMG J. Clin. Med. 2021, 10, 195 8 of 18 activity at both 0′ post and 60′ after the combined intervention, an effect that becomes obvious when calculating the difference in EMG activity between “eEmc with training” and “training” condition. (a) (b) (c) (d) (e) Figure 2. Maximal Figure 2. Maximalvoluntary voluntarycontraction contraction(MVC) (MVC)during hand during gripgrip hand strength. (a) (a) strength. Picture showing Picture general settinggeneral showing and position setting for andhand gripfor position MVC hand assessment. grip MVC (b) Percentage assessment. (b)of MVC vs. of Percentage baseline levelsMVC at 0 min (0′ post) and vs. baseline levels60atmin (60′ (0 0 min post) after 0 post) each and 60 intervention. min (600 post)Significant after eachdifferences between intervention. interventions:  p < 0.05,  p < 0.01,  p < 0.001. (c,d) Percentage of area under the curve Significant differences between interventions: F p < 0.05, FF p < 0.01, FFF p < 0.001. (AUC) of electromyographic (EMG) activity vs. baseline levels from APB (c) and from ADM (d). (c,d) Percentage of area under the curve (AUC) of electromyographic (EMG) activity Significant differences between interventions:  p < 0.05,  p < 0.01,  p < 0.001. (e) Repre- vs. baseline sentative APB musclelevels EMGfromregistry APB (c) and from (obtained ADM from (d). #5) subject Significant differences corresponding betweenvolun- to maximum F p 0.05, FF p < 0.01, FFF p tary contraction (MVC) trials during training and eEmc with training interventions atmuscle interventions: < < 0.001. (e) Representative APB basal, 0′ and EMG registry (obtained from subject #5) corresponding to maximum voluntary contraction (MVC) trials during training and eEmc with training interventions at basal, 00 and 600 after intervention. Upper panels correspond to rectified values, and lower panels show difference between registers of both interventions for better difference appreciation. APB, abductor pollicis brevis; ADM, abductor digiti minimi. J. Clin. Med. 2021, 10, x FOR PEER REVIEW 9 of 18 60′ after intervention. Upper panels correspond to rectified values, and lower panels show differ- ence between registers of both interventions for better difference appreciation. APB, abductor pol- 60′ licisafter intervention. brevis; Upperdigiti ADM, abductor panels correspond to rectified values, and lower panels show differ- minimi. J. Clin. Med. 2021, 10, 195 9 of 18 ence between registers of both interventions for better difference appreciation. APB, abductor pol- licis brevis; ADM, abductor digiti minimi. Manual dexterity was assessed with the BBT in order to detect possible alterations induced by power grip training and/or eEmc-based interventions. Two-way ANOVA did Manual Manual dexterity dexterity was wasassessed assessed with the theBBT in inorder to to detect possible alterations not show an effect of intervention (F(2,with 18) = 2.62, BBT p = 0.10) order neither detect possible timepoint alterations  intervention induced by power induced by(Fpower grip training and/or eEmc-based interventions. Two-way ANOVA did interaction (4, 36) = grip 0.206,training and/or p = 0.93). However,eEmc-based there was interventions. a significantTwo-way timepointANOVA did effect (F (2, not not show show an an effect effect of of intervention intervention (F (F (2, 18) = 2.62, p = 0.10) neither timepoint  intervention = 2.62, p = 0.10) neither timepoint × intervention 18) = 43.5, p < 0.001) with increasing number (2, 18) of boxes, being significantly higher just after interaction interaction(F(F(4, 36) = 0.206, p = 0.93). However, there was a significant timepoint effect (F(2, intervention (0′) and (4, 36) duringp = = 0.206, 0.93). However, follow-up (60′) in there was a significant comparison timepointwhen to basal evaluation effect 18) = 43.5, p < 0.001) with increasing number of boxes, being significantly higher just after (F equally = 43.5, p < 0.001) with increasing number (2, 18) considering all interventions (p < 0.001) (Figure 3). of boxes, being significantly higher just intervention (0′) and 0 during follow-up (60′) in 0 comparison to after intervention (0 ) and during follow-up (60 ) in comparison to basal evaluation when basal evaluation when equally equallyconsidering consideringall allinterventions interventions(p (p<<0.001) 0.001)(Figure (Figure3). 3). Figure 3. Number of moved blocks in the box and blocks test (BBT) for hand training, eEmc, or combination of bothof Number interventions movedblocks right blocks after intervention (0′test post) and for during follow-up training,(60′ post) Figure3.3. Number Figure in comparison of moved to basal ininthe thebox evaluation. Significant box andblocks and blocks differences (BBT) test (BBT) between hand for hand timepoints training, according eEmc, toeEmc, Tukey’s or or combination of both interventions right after intervention (0 0 post) and during follow-up (60 0 post) combination of both interventions multiple comparisons (consideringright after intervention all intervention groups(0′ as post) and *** a whole): during follow-up (60′ post) p < 0.001. inincomparison comparisontotobasal basalevaluation. evaluation.Significant Significantdifferences differencesbetween betweentimepoints timepointsaccording accordingtotoTukey’s Tukey’s multiplecomparisons multiple comparisons(considering (consideringall allintervention interventiongroups groupsasasaawhole): ***pp<<0.001. whole):*** 0.001. 3.2. Spinal Cord Excitability 3.2.Spinal 3.2. Spinal F wave Cord Cordwas Excitability used to detect excitability of anterior horn motoneurons in the spinal Excitability cord. FThere was aused significant effect of intervention over F motoneurons wave persistence (Friedman’s F wave was used to wave was todetect detectexcitability ofof excitability anterior anteriorhorn horn motoneurons in the in spinal cord. the spinal test = 5.84, There was df 3.30, p = 0.05) a significant effect(Figure 4a). Dunn’s of intervention overpost-hoc comparisons F wave persistence revealed an (Friedman’s testoverall = 5.84, cord. There was a significant effect of intervention over F wave persistence (Friedman’s increase df 3.30, pin= F0.05) persistence after (Figure 4a). eEmc post-hoc Dunn’s combined with training comparisons compared revealed to training an overall increasealone in F test = 5.84, df 3.30, p = 0.05) (Figure 4a). Dunn’s post-hoc comparisons revealed an overall that almost reached statistical significance (p = 0.053). The intervention factor persistence after eEmc combined with training compared to training alone that almost reached had a nearly increase in F persistence after eEmc combined with training compared to training alone significant impact on (pF =wave statistical significance amplitude 0.053). (F(2, 18) =factor The intervention 3.10,had p =a0.07). nearlyPost hoc comparisons significant impact on F that almost reached statistical significance (p = 0.053). The intervention factor had a nearly showed that eEmc wave amplitude delivery, (F(2, with 18) = 3.10, p =or without 0.07). Post hand training, increased hoc comparisons showed Fthatwave eEmc amplitude delivery, significant impact on F wave amplitude (F(2, 18) = 3.10, p = 0.07). Post hoc comparisons when with orcompared without hand to training training,alone (p < 0.05), increased F waveparticularly amplitude whenat 0′ post intervention compared to training(Figure alone showed that eEmc delivery, with or without hand training, increased F wave amplitude (p < 0.05), particularly at 00 post intervention (Figure 4b,c). 4b,c). when compared to training alone (p < 0.05), particularly at 0′ post intervention (Figure 4b,c). (a) (b) Figure 4. Cont. (a) (b) J. Clin. Med. 2021, 10, 195 10 of 18 J. Clin. Med. 2021, 10, x FOR PEER REVIEW 10 of 18 (c) Figure 4. Figure 4. Variables VariablesofofFFwaves wavesininAPBAPB byby median median nerve nerve stimulation. stimulation. (a) (a) F wave F wave persistency. persistency. Signif- Significant icant difference between eEmc with training and training alone (regardless of the difference between eEmc with training and training alone (regardless of the timepoint): p = 0.05. timepoint): p= 0.05. Data expressed as median ± interquartile range. (b) Averaged maximum amplitude Data expressed as median ± interquartile range. (b) Averaged maximum amplitude of F wave for of F wave for each condition and timepoint. Significant differences vs. baseline: + p < 0.05 (for training + eEmc), each condition and timepoint. Significant differences vs. baseline: + p < 0.05 (for training + eEmc), ++ p < 0.01 (for eEmc alone), and between interventions:  p < 0.05. (c) Representative APB muscle ++ p < 0.01 (for eEmc alone), and between interventions: F p < 0.05. (c) Representative APB muscle registers (obtained from subject #7) corresponding to ten consecutive median nerve stimulations registers showing (obtained fromatsubject M and F wave 0′ after#7) in corresponding to ten each intervention. consecutive Light median grey registers nerve Fstimulations highlight waves with showing M and F wave at 0 0 after in each intervention. Light grey registers highlight F waves with maximum amplitude. # Symbol indicates registers where no F wave presence was considered. APB, maximum amplitude. abductor pollicis # Symbol indicates registers where no F wave presence was considered. APB, brevis. abductor pollicis brevis. Excitability of spinal networks was tested by single-pulse tSCS at cervical level. The RMTExcitability of spinalwas in APB at baseline networks of 51.0was ± 2.9tested mA atbyC3–C4 single-pulse tSCS and 60.8 at mA ± 3.5 cervical level. The at C6–C7. The RMT in APB at baseline was of 51.0 ± 2.9 mA at C3–C4 and 60.8 ± 3.5 mA at recruitment curve of sMEP from upper extremity muscles did not show any significant C6–C7. The recruitment curve changes in any of sMEP muscle from at any upperand intensity extremity muscles did (p in any intervention not show(Table > 0.05) any significant S1). changes in any muscle at any intensity and in any intervention (p > 0.05) (Table S1). 3.3. Corticospinal Excitability and Intracortical Inhibition 3.3. Corticospinal Excitability and Intracortical Inhibition The RMT in APB at basal conditions was 37.9 ± 1.2% of maximum TMS intensity. The RMT in APB at basal conditions was 37.9 ± 1.2% of maximum TMS intensity. RMT RMT was significantly affected by timepoint in the training intervention (Friedman’s test was significantly affected by timepoint in the training intervention (Friedman’s test = 7.93, = 7.93, df 3.10, p < 0.05) and by intervention at 60′ post (Friedman’s test = 8.06, df 3.10, p < df 3.10, p < 0.05) and by intervention at 600 post (Friedman’s test = 8.06, df 3.10, p < 0.05) 0.05) (Figure (Figure 5a). Dunn’s 5a). Dunn’s post-hoc post-hoc comparisons comparisons confirmed confirmed that higher that higher RMT was RMTfound was found 60′ 600 after after training (p < 0.05). eEmc with training significantly reduced RMT training (p < 0.05). eEmc with training significantly reduced RMT when compared to training when compared to training alone at this timepoint (p < 0.05). Corticospinal excitability alone at this timepoint (p < 0.05). Corticospinal excitability measured by cMEP amplitude measured by cMEP amplitude obtainedRMTatstimulus 120% RMT stimulus did not interventions differ between (F(2,interventions (F(2, 18) = obtained at 120% did not differ between 18) = 0.25, p = 0.77) or 0.25, p = 0.77) or between timepoints (F(2, 18) = 1.55, p = 0.23) (Figure 5b). between timepoints (F(2, 18) = 1.55, p = 0.23) (Figure 5b). SICI was SICI was significantly affected by significantly affected by timepoint timepoint in in the the training training intervention (Friedman’s intervention (Friedman’s test = 7.40, df 3.10, p < 0.05). Dunn’s post hoc analysis revealed that hand test = 7.40, df 3.10, p < 0.05). Dunn’s post hoc analysis revealed that hand training induced training induced higher inhibition (i.e., smaller conditioned cMEP amplitude) 0at 0′ post-intervention higher inhibition (i.e., smaller conditioned cMEP amplitude) at 0 post-intervention (p < 0.05) (p < 0.05) compared compared to baseline, to baseline, with nowith nosignificant other other significant comparisons comparisons (Figure 5c). (Figure 5c). The recruitment curves of cMEP at baseline did not significantly differ between inter- ventions in any muscle and appear represented as a single pool in Figure 6a, which shows APB data. Analysis of APB muscle recruitment curves normalized to baseline showed signif- icant intervention × timepoint interaction after intervention (F(12, 108) = 3.59, p < 0.001) and during follow-up (F(12, 108) = 1.58, p < 0.05). Specifically, eEmc with training enhanced cMEP amplitude significantly in comparison to training and eEmc alone at high TMS-stimulation intensities (1.4× and 1.5× RMT at 00 post-intervention and 1.3× and 1.5× RMT at 600 post- intervention; Figure 6b,c). Corticospinal excitability was also quantified by calculating the slope of non-normalized absolute recruitment curves, which also resulted in a significant effect of intervention (Friedman’s test = 8.08, df 3.30, p < 0.05), with higher slopes for APB after eEmc with training compared to training or eEmc alone at 00 and 600 post-intervention, according to (a) Dunn’s multiple comparisons(b) (Figure 6d, Table S2). There were no significant (c) differences for slopes analyzed for other muscles (Table S2). training alone at this timepoint (p < 0.05). Corticospinal excitability measured by cMEP amplitude obtained at 120% RMT stimulus did not differ between interventions (F(2, 18) = 0.25, p = 0.77) or between timepoints (F(2, 18) = 1.55, p = 0.23) (Figure 5b). SICI was significantly affected by timepoint in the training intervention (Friedman’s J. Clin. Med. 2021, 10, x FOR PEER REVIEW 11 of 18 test = 7.40, df 3.10, p < 0.05). Dunn’s post hoc analysis revealed that hand training induced J.J. Clin. Med. 2021, 10, x FOR Clin. Med. 2021, 10, 195 PEER REVIEW 11 11 of of 18 18 higher inhibition (i.e., smaller conditioned cMEP amplitude) at 0′ post-intervention (p < 0.05) compared to baseline, with no other significant comparisons (Figure 5c). Figure 5. Effect of hand training, eEmc and eEmc + training on the corticospinal excitability and SICI. (a) Percentage of Figure 5. Effect of hand training, eEmc and eEmc + training on the corticospinal excitability and SICI. (a) Percentage of resting motor threshold (RMT) vs. baseline levels in APB at 0 min (0′ post) and 60′ min (60′ post) after intervention. Signif- resting motor threshold (RMT) vs. baseline levels in APB at 0 min (0′ post) and 60′ min (60′ post) after intervention. Signif- icant differences between interventions:  p < 0.05, (training vs. eEmc + training), and in comparison to baseline: # p < 0.05 icant differences between interventions:  p < 0.05, (training vs. eEmc + training), and in comparison to baseline: # p < 0.05 (for training). (b) Effect of training, eEmc and eEmc + training on the supramaximal cMEP amplitude (120% transcranial (for training). (b) Effect of training, eEmc and eEmc + training on the supramaximal cMEP amplitude (120% transcranial magnetic stimulation (TMS)) according to RMT. (c) SICI: (average of conditioned cMEP) × 100/ (average of test cMEP) magnetic stimulation (TMS)) according to RMT. (c) SICI: (average of conditioned cMEP) × 100/ (average of test cMEP) recorded at APB muscle and obtained at 0′ and 60′ after each intervention. # p < 0.05 for training at 0′ post in comparison recorded at APB muscle and obtained at 0′ and 60′ after each intervention. # p < 0.05 for training at 0′ post in comparison to baseline. APB, abductor pollicis brevis; RMT, resting motor threshold; cMEP, cortically-induced motor evoked poten- to baseline. APB, abductor pollicis brevis; RMT, resting motor threshold; cMEP, cortically-induced motor evoked poten- tial; SICI, short-interval intracortical inhibition. tial; SICI, short-interval intracortical inhibition. The recruitment curves of cMEP at baseline did not significantly differ between in- The recruitment curves of cMEP at baseline did not significantly differ between in- terventions in any muscle and appear represented as a single pool in Figure 6a, which terventions in any muscle and appear represented as a single pool in Figure 6a, which shows APB data. Analysis of APB muscle recruitment curves normalized to baseline shows APB data. Analysis of APB muscle recruitment curves normalized to baseline (a) showed significant intervention (b)  timepoint interaction after intervention (c) (F(12, 108) = 3.59, p showed significant intervention × timepoint interaction after intervention (F(12, 108) = 3.59, p < 0.001) and during follow-up (F(12, 108) = 1.58, p < 0.05). Specifically, eEmc with training < 0.001)eEmc Figure 5. Effect of hand training, and and during eEmcfollow-up + training(Fon(12,the = 1.58, p < 0.05). 108) corticospinal Specifically, excitability eEmc and SICI. with training (a) Percentage enhanced cMEP amplitude significantly 0in comparison to training and eEmc alone at high enhanced cMEP amplitude significantly in comparison 0 to training 0 and of resting motor threshold (RMT) vs. baseline levels in APB at 0 min (0 post) and 60 min (60 post) after intervention. eEmc alone at high TMS-stimulation intensities (1.4× and 1.5× RMT at 0′ post-intervention and 1.3× and 1.5× TMS-stimulation intensities (1.4× and 1.5× RMT at 0′ post-intervention Significant differences between interventions: F p < 0.05, (training vs. eEmc + training), and in comparison to baseline: and 1.3× and 1.5× RMT at 60′ post-intervention; Figure 6b,c). Corticospinal excitability was also quantified # p < 0.05 (for training). (b)RMT Effectatof60′ post-intervention; training, eEmc and eEmc Figure 6b,c).on + training Corticospinal the supramaximalexcitability was also quantified cMEP amplitude (120% by calculating the slope of non-normalized absolute recruitment curves, which also re- by calculating transcranial magnetic stimulation the slope (TMS)) according to of non-normalized RMT. (c) SICI: (average absolute recruitment of conditioned cMEP)curves, which also × 100/ (average of re- sulted in a significant effect of intervention (Friedman’s test = 8.08, df 3.30, p < 00.05), with test cMEP) recorded at APB muscle and obtained at 0 and 60 after each intervention. # p < 0.05 for training at 0 postwith sulted in a significant effect 0 of intervention 0 (Friedman’s test = 8.08, df 3.30, p < 0.05), higher slopes for APB after eEmc with training compared to training or eEmc alone at 0′ higher in comparison to baseline. APB, slopes pollicis abductor for APB after RMT, brevis; eEmcresting with training compared motor threshold; to training cMEP, or eEmc alone cortically-induced motor at 0′ and 60′ post-intervention, according to Dunn’s multiple comparisons (Figure 6d, Table and 60′ post-intervention, evoked potential; SICI, short-interval intracortical inhibition.according to Dunn’s multiple comparisons (Figure 6d, Table S2). There were no significant differences for slopes analyzed for other muscles (Table S2). S2). There were no significant differences for slopes analyzed for other muscles (Table S2). (a) (b) (c) (d) (a) (b) (c) (d) (e) (e) Figure6.6.Effect Figure Effectof ofinterventions interventionson oncortical corticalrecruitment recruitmentcurve. curve.(a) (a)Pooled PooledAPBAPBrecruitment recruitmentcurvescurvesobtained obtainedatatbaseline baselinefor for Figure 6. Effect of interventions on cortical recruitment curve. (a) Pooled APB recruitment curves obtained at baseline for each subject each subject subject and and intervention. and intervention. intervention. (b,c) (b,c) Increase (b,c) Increase Increase or or decrease or decrease decrease (in(in millivolt (in millivolt (mV); millivolt (mV); indicated (mV); indicated indicated as as Δ) in ∆) in as Δ) APB in APB recruitment APB recruitment recruitment curvescurves curves withwith with each respect to baselineatat000′(b) (b)and and 60′ 0 (c)postpost intervention.Fp p<<0.05, 0.05, pp<< 0.01,FFF  p <0.001 0.001training trainingvs. vs.training training + respect respect to baseline 0′ and 6060′ (c) intervention.  p < 0.05, FF (c) post intervention.  p < 0.01,  pp << 0.001 training vs. training ++ eEmc;  p < 0.05,  p < 0.001 eEmc vs. training + eEmc. (d) Slope of APB cMEP recruitment curves. Indicated slopes eEmc;  p < 0.05,  pp < eEmc; < 0.001 eEmc vs. training + (d) Slope + eEmc. (d) Slope of APB cMEP recruitment curves. Indicated slopes correspond to the slope of the linear regression line through stimulation intensities (in %RMT) and cMEP values (in mV), correspond correspond to to the the slope slope of of the the linear linear regression regression line line through through stimulation stimulation intensities intensities (in (in %RMT) %RMT) andand cMEP cMEP values values (in (in mV), mV), multiplied ×1000 to facilitate data visualization.  p < 0.05,  p < 0.01, between interventions at 0′ post and 60′ post. (e) multiplied multiplied ×1000 ×1000 to facilitate to facilitate data visualization. data visualization.  p < 0.05,  F p