Journal of Clinical Medicine Article Transcutaneous Electrical Neuromodulation of the Cervical Spinal Cord Depends Both on the Stimulation Intensity and the Degree of Voluntary Activity for Training. A Pilot Study Hatice Kumru 1,2,3, *,† , María Rodríguez-Cañón 1,4,† , Victor R. Edgerton 1,5 , Loreto García 1,2,3 , África Flores 4 , Ignasi Soriano 1,2,3 , Eloy Opisso 1,2,3 , Yury Gerasimenko 6,7,8 , Xavier Navarro 1,4 , Guillermo García-Alías 1,4,‡ and Joan Vidal 1,2,3,‡ 1 Fundación Institut Guttmann, Institut Universitari de Neurorehabilitació Adscrit a la UAB, 08916 Badalona, Spain; rodriguezcanonmaria@gmail.com (M.R.-C.); vre@ucla.edu (V.R.E.); loretogarcia@guttmann.com (L.G.); isoriano@guttmann.com (I.S.); eopisso@guttmann.com (E.O.); xavier.navarro@uab.cat (X.N.); guillermo.garcia@uab.cat (G.G.-A.); jvidal@guttmann.com (J.V.) 2 Universitat Autònoma de Barcelona, 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, 08193 Bellaterra, Spain; africa.flores@uab.cat  5 Department of Neurobiology, University of California Los Angeles, Los Angeles, CA 90095, USA 6 Pavlov Institute of Physiology, 199034 St. Petersburg, Russia; yury.gerasimenko@louisville.edu Citation: Kumru, H.; Rodríguez- 7 Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40292, USA Cañón, M.; Edgerton, V.R.; García, L.; 8 Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, KY 40292, USA Flores, Á.; Soriano, I.; Opisso, E.; * Correspondence: hkumru@guttmann.com Gerasimenko, Y.; Navarro, X.; † Equal role as first author. García-Alías, G.; et al. ‡ Equal role as senior PI. Transcutaneous Electrical Neuromodulation of the Cervical Abstract: Electrical enabling motor control (eEmc) through transcutaneous spinal cord stimulation Spinal Cord Depends Both on the offers promise in improving hand function. However, it is still unknown which stimulus intensity or Stimulation Intensity and the Degree which muscle force level could be better for this improvement. Nine healthy individuals received the of Voluntary Activity for Training. following interventions: (i) eEmc intensities at 80%, 90% and 110% of abductor pollicis brevis motor A Pilot Study. J. Clin. Med. 2021, 10, 3278. https://doi.org/10.3390/ threshold combined with hand training consisting in 100% handgrip strength; (ii) hand training jcm10153278 consisting in 100% and 50% of maximal handgrip strength combined with 90% eEmc intensity. The evaluations included box and blocks test (BBT), maximal voluntary contraction (MVC), F wave Academic Editors: Ursula persistency, F/M ratio, spinal and cortical motor evoked potentials (MEP), recruitment curves S. Hofstoetter and Karen Minassian of spinal MEP and cortical MEP and short-interval intracortical inhibition. The results showed that: (i) 90% eEmc intensity increased BBT, MVC, F wave persistency, F/M ratio and cortical MEP Received: 2 June 2021 recruitment curve; 110% eEmc intensity increased BBT, F wave persistency and cortical MEP and Accepted: 22 July 2021 recruitment curve of cortical MEP; (ii) 100% handgrip strength training significantly modulated MVC, Published: 25 July 2021 F wave persistency, F/M wave and cortical MEP recruitment curve in comparison to 50% handgrip strength. In conclusion, eEmc intensity and muscle strength during training both influence the results Publisher’s Note: MDPI stays neutral for neuromodulation at the cervical level. with regard to jurisdictional claims in published maps and institutional affil- Keywords: transcutaneous spinal cord stimulation; intensity effect; muscle strength effect; hand iations. training; neuromodulation; cervical spinal cord Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Transcutaneous spinal cord stimulation (tSCS) used as a method of electrical enabling distributed under the terms and motor control (eEmc) is a novel, non-invasive method and alone or combined with hand conditions of the Creative Commons training offers promise in improving hand function since it can modify the functional state Attribution (CC BY) license (https:// of the sensory-motor system [1,2]. eEmc consists of low intensity electrical stimulation creativecommons.org/licenses/by/ applied for changing the physiological states of spinal networks to a level that it enables 4.0/). the spinal network to better respond to voluntary commands and proprioceptive inputs. J. Clin. Med. 2021, 10, 3278. https://doi.org/10.3390/jcm10153278 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 3278 2 of 16 In the literature, the different terminologies have been used for transcutaneous spinal cord stimulation: “transcutaneous spinal stimulation (TSS) [3], painless transcutaneous electrical enabling motor control (pcEmc) [4], transcutaneous electrical stimulation of the spinal cord (TESS)” [5] or “transcutaneous enabling motor control (tEmc)” [6]. Recent developments in therapeutic approaches for spinal cord injury (SCI) showed that tSCS alone [3] or a combination of spinal cord stimulation with pharmacological treatment [7] or with hand training [6,8,9] allowed improving hand motor function in individuals with SCI. It has been hypothesized that spinal electrical stimulation can change the excitability of spinal circuitry and potentially neuromodulate the spinal network to facilitate and enhance the restoration of paralyzed limb function [5–9]. Plastic changes have been also reported in the control of upper limb function in healthy subjects following eEmc alone [5] and combined with hand training [10]. It has been suggested that the mechanisms recruited by eEmc combined with physical training, although partially overlapping, may involve different and perhaps synergistic processes leading to more effective reorganization of neural circuits [10]. It was recently reported that tSCS was capable of facilitating cortically evoked muscle responses and the degree of facilitation progressively increased during the 1 s stimulation training, and was still evident 0.5 s after the end of the training in monkeys [11]. The most likely mechanism of eEmc occurs via transcutaneous tonic spinal activation by elevating spinal networks excitability [12] and may affect interneuronal pathways that generate action potentials on motoneurons within a motor pool in a more normal stochastic time frame [13]. As such, eEmc is hypothesized to potentiate the generation of postsynaptic excitatory potentials and, thus, shift the spinal motor network excitability closer to the excitation threshold. In addition, activation of back musculature under the electrodes of tSCS [11] and of sensory afferents at the level of dorsal roots or via the spinal pathways can contribute to elevating neural excitability [14,15]. According to reports in the literature, there was great variability in the intensity of eEmc applied, though most of the studies used high intensities, close to the participants’ tolerance threshold. In cervical SCI, the stimulus intensity for eEmc varied from below the resting spinal motor threshold (RMT) to an intensity adjusted to enable maximal grip strength or adjusted based on the participant’s functional task performance [5–9]. When eEmc was combined with hand training, variable protocols were used: either maxi- mal handgrip to submaximal isometric hand movement such as squeezing/grasping or standard stretching, active assistive range of motion exercises, intensive gross and fine motor skill training or the intensity that made the task easiest [6,8,9]. All these studies reported significant functional improvement of hand muscle strength and/or neurophysio- logical changes. We recently showed that even one single session of cervical eEmc is able to modify the excitability of neural networks controlling upper limb function in healthy subjects [10]. These changes strongly depend on the combination of eEmc with hand training, since improved hand grip force and increased spinal and corticospinal output were found in comparison to each intervention tested alone [10]. The intervention consisted in eEmc at an intensity of 90% the RMT at the cervical spinal cord and handgrip training at maximum voluntary contraction (MVC) [10]. However, it remains unknown if the intensity of electrical stimulation for eEmc and the level of hand grip strength in such combined strategy can affect motor strength and/or functional outcome, and if plastic changes occur at spinal or cortical level. Thus, the objectives of this study were to test: (1) the effect of different electrical stimulation intensities for eEmc combined with the maximum force of hand grip during training, and (2) the effect of different hand grip strength during training combined with 90% of spinal RMT electrical stimulation for eEmc on hand function and spinal cord and cortical excitability. We hypothesized that eEmc at higher stimulus intensity applied at two sites of cervical spinal cord combined with a higher level of hand grip strength can enhance motor strength and functional outcome and eventually modulate spinal or cortical neural circuits more than lower stimulus intensity and lower hand grip strength during training. sites of cervical spinal cord combined with a higher level of hand grip strength can en- hance motor strength and functional outcome and eventually modulate spinal or cortical J. Clin. Med. 2021, 10, 3278 neural circuits more than lower stimulus intensity and lower hand grip strength during 3 of 16 training. 2. Experimental Section 2. Experimental Section 2.1. Study Design 2.1. Study Design Nine healthy volunteer subjects (age range 25–60 years; mean age 39.8 ± 11.1 years; 3 Nine healthy volunteer subjects (age range 25–60 years; mean age 39.8 ± 11.1 years; females and 6 males) participated in the study (Table 1). The inclusion criteria were: age 3 females and 6 males) participated in the study (Table 1). The inclusion criteria were: between 18 and 65 years, without any neurological disorder and uncontrolled disease, age between 18 and 65 years, without any neurological disorder and uncontrolled disease, which which could could limit limit the the experiment, experiment, and and given given written written informed informed consent. consent. Exclusion Exclusion criteria criteria were were any implanted metallic or electrical devices, and pregnancy. The study protocol any implanted metallic or electrical devices, and pregnancy. The study protocol was was approved approved byby the the Research Research Ethics Ethics Committee Committee of of the the Institute Institute Guttmann Guttmann and and was was conducted conducted in in accordance accordance with with the the Declaration Declaration of of Helsinki. Helsinki. Table Table 1. Demographic Demographicdata dataofofhealthy healthyindividuals individualsand andcurrent intensities current used intensities forfor used cervical stimulation cervical at C3-C4 stimulation andand at C3-C4 at C6- at C7 level. C6-C7 level. eEmc Intensity Applied during Stimulation (mA) eEmc Intensity Applied during Stimulation (mA) C3-C4 C6-C7 C3-C4 C6-C7 % eEmc + 100% MVC %eEmc + 100%MVC 90%eEmc + 50% 90%eEmc + 50% MVC Subject Sex Age Hand 80% % eEmc 90%+ 100% 110%MVC 90%eEmc + %eEmc 80% + 100%MVC 90% 110% 90%eEmc MVC + 1 M Subject Sex 44 Age R Hand 32 80% 29 90% 40 110% 50% 29 MVC 80% 34 90%38 44 110% 50% MVC 38 2 M 60 R 69 59 77 63 72 72 90 72 1 M 44 R 32 29 40 29 34 38 44 38 32 FM 25 60 R R 27 69 30 59 33 77 2963 7232 7230 9040 7232 43 FF 27 25 L R 32 27 34 30 44 33 3429 3236 3036 4046 3241 54 FF 33 27 R L 37 32 54 34 53 44 4734 3648 3663 4662 4158 65 MF 41 33 R R 27 37 36 54 37 53 3147 4838 6345 6242 5834 76 MM 51 41 R R 51 27 52 36 75 37 6331 3861 4563 4290 3481 87 MM 39 51 R R 48 51 59 52 77 75 4963 6156 6372 9088 8159 98 MM 38 39 B R 45 48 67 59 73 77 6149 5653 7277 8875 5985 9 M 38 B 45 67 73 61 53 77 75 85 M: male; F: female; L: left; R: right; B: both. M: male; F: female; L: left; R: right; B: both. This study was performed in 2 parts. In the first part, we studied the effect of different This study was performed in 2 parts. In the first part, we studied the effect of different electrical stimulation intensities for eEmc calculated from the spinal RMT of abductor pol- electrical stimulation intensities for eEmc calculated from the spinal RMT of abductor licis brevis (APB) muscle combined with the maximum hand grip strength during train- pollicis brevis (APB) muscle combined with the maximum hand grip strength during ing. In the second part, we studied the effect of different handgrip strength levels during training. In the second part, we studied the effect of different handgrip strength levels training combined with 90% spinal RMT of stimulus intensity for eEmc (Figure 1). during training combined with 90% spinal RMT of stimulus intensity for eEmc (Figure 1). Figure 1. Schematic representation of the experiment conditions, order of intervention and the evaluation time points Figure 1. Schematic during each representation experiment, of the and and the functional experiment conditions, motor strength order of assessment andintervention and the assessments neurophysiological evaluation time points performed. during each experiment, and the functional and motor strength assessment and neurophysiological assessments per- formed. 2.2. Interventions The study included a total of four interventions, tested in two parts separated at least one week apart. All interventions combined eEmc with hand training. In the first part, we studied the effect of three electrical eEmc stimulation at intensity 80%, 90% and 110% of spinal RMT of the APB muscle, combined with the maximum hand grip strength (100% of J. Clin. Med. 2021, 10, 3278 4 of 16 MVC). In the second part, we studied two different levels of hand grip strength for training, 50% and 100% of MVC, combined with eEmc at stimulus intensity of 90% of spinal RMT. Each intervention consisted of trains of 20 s of concomitant eEmc stimulation and hand training, followed by 80 s of rest, for 30 min. The subject alternated the two hands during eEmc, resulting in nine hand-training attempts for each hand and 18 in total during the whole intervention [10]. The eEmc was delivered through two circular hydrogel adhesive electrodes (2 cm diameter, Axion GmbH, Leonberg, Germany) placed along the midline over spinous pro- cesses C3-C4 and C6-C7. eEmc was delivered using biphasic rectangular 1 ms pulses at a frequency of 30 Hz, with each pulse filled with a carrier frequency of 10 kHz [10]. For eEmc, we used two channels of a five-channel current-controlled stimulator of Biostim-5 stimulator (Cosyma Inc., Moscow, Russia) with two pairs of anode and cathode. Each chan- nel was set up independently. Stimulation of the first channel was delivered by a cathode at C3-C4 level and stimulation of the second channel by a cathode at C6-C7, with anodes placed at the iliac crests. The hand training consisted of holding the hand grip dynamometer and maintaining a hand grip strength of 100% of MVC for all stimulus intensities of eEmc in the first part. In the second part, either 100% (100% MVC) or 50% (50% MVC) of hand grip strength during hand training was combined with eEmc at 90% of stimulus intensity. 2.3. Functional and Motor Strength Assesment of the Healthy Participants The evaluation protocol consisted of two hand functional outcomes, the Box and Block test (BBT) and the measurement of hand grip strength during a MVC with a dy- namometer. Neurophysiological assessment was made on the dominant hand and arm muscles. The dominant hand was determined according to the Edinburgh inventory [16]. F-wave and spinal motor evoked potentials (spinal MEPs) in response to single-pulse C3-C4 and C6-C7 tSCS were recorded in the APB muscle to study the spinal cord excitabil- ity. At the cortical level, using transcranial magnetic stimulation (TMS), we determined the cortical RMT of APB muscle, and recorded cortical motor evoked potentials (cortical MEPs), short-interval intracortical inhibition (SICI) and recruitment curve of cortical MEPs. The neurophysiological studies were performed with an EMG machine (Medelec Synergy, Oxford Instruments; Surrey, England). The evaluation protocol was repeated at three time points: before (PRE), just after (POST) and one hour after finishing the intervention (FOLLOW) in each subject and experimental study (Figure 1). The total duration of each experiment was around four hours. 2.3.1. Box and Block Test (BBT) The BBT was used to assess hand dexterity. The participant was seated in front of a box split in two halves, the one on the preferred side full of small cube-shaped blocks [17]. The subject had to move the blocks one by one to the other side, crossing the middle line, during 60 s. The total number of moved blocks was the BBT score. 2.3.2. Maximum Voluntary Contraction (MVC) during Hand Grip The participant was seated in front of a table, with the dominant forearm resting on it in neutral position, holding a dynamometer (Jamar Model 5030J1, Sammons Preston, NJ, USA) with the hand. For trigger signal, we used a mild electric stimulus (3 mA intensity with pulse width 0.5 ms) delivered by means of ring electrodes around the fifth finger. Following the trigger signal, the participant had to perform a MVC and maintain it during 4 s; the strength was measured in kilograms (kg) in three consecutive trials, with at least one minute rest between them. 2.4. Neurophysiological Assesment Disposable adhesive surface electrodes (outer diameter 20 mm; Technomed, Maas- tricht Airport, Netherlands) were placed over the muscle belly of the APB, abductor digiti J. Clin. Med. 2021, 10, 3278 5 of 16 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 after standard skin preparation. The EMG signal was amplified, filtered using band-pass of 30 Hz–10 kHz, and epochs of 100 ms sweep duration at amplitude sensitivity of 0.1–0.5 mV recorded at a sampling rate of 50 kHz. EMG activity was visualized online, with the subject relaxing her/his arm and hand. If any background EMG activity was observed after stimulus delivery, the recording was discarded, and the stimulation was repeated. Offline analysis was performed with MATLAB. 2.4.1. F Wave Persistency and F/M Wave Ratio Suprathreshold electrical stimulation (pulse width 0.5 ms) of the median nerve at the wrist was used to elicit the maximum M wave and F wave in the APB muscle for at least 15 recordings. We assessed spinal motoneuron excitability using F-wave persistency and the Fmax/Mmax ratio (F/M ratio) in the APB muscle. 2.4.2. Spinal MEP Stimulation was delivered using two hydrogel adhesive disk electrodes (20 mm diam- eter; Axion GmbH, Germany) as cathodes along the midline between spinous processes C3-C4 and C6-C7. The anodes were two adhesive rectangular electrodes (5 × 12 cm) placed symmetrically over the iliac crest. Monophasic rectangular single pulses of 1 ms duration were delivered from a Biostim-5 stimulator (Cosyma Inc., Moscow, Russia). The spinal RMT was calculated as the lowest intensity of electrical stimulation applied first at C3-C4 and then at C6-C7 that evoked a spinal MEP of ≥50 µV peak-to-peak amplitude in the APB muscle in at least 5 of 10 consecutive trials. Then, the recruitment of spinal MEPs on all the muscles tested was recorded for each stimulation site and time point. Recruitment curves of spinal MEPs were derived from responses to gradually increasing stimulation intensity from 90% to 150% of the baseline spinal RMT at 10% increments, with three repetitions at each intensity. 2.4.3. Transcranial Magnetic Stimulation (TMS) A Magstim® BiStim2 TMS (Magstim Company, Whitland, Wales, UK) apparatus was used. 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 was defined as a point over the scalp where the largest amplitude cortical MEP in APB muscle was recorded. 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 test. 2.4.4. Neurophysiological Parameters We recorded the following parameters pre and post intervention (1) the BBT score to evaluate hand dexterity and (2) the MVC for handgrip strength. To evaluate spinal cord excitability changes: (1) F wave persistency and F/M wave ratio; (2) recruitment curve of spinal MEP. To evaluate cortical excitability changes: (1) cortical RMT, defined as the lowest intensity of TMS that evoked a cortical MEP of ≥50 µV peak-to-peak amplitude in the APB muscle in at least 5 of 10 consecutive trials; (2) mean amplitude of cortical MEPs using single-pulse TMS at 120% of cortical RMT of ABP in 5 recordings; (3) short intracortical inhibition (SICI) using paired-pulse TMS with a subthreshold conditioning stimulus (80% of cortical RMT) and a suprathreshold test stimulus (120% of cortical RMT) at interstimulus interval of 2 ms in 5 recordings without background activity [18]; (4) recruitment curves of cortical MEP obtained at increasing intensities from 90% to 150% of cortical RMT of APB, at 10% increments (three recordings at each intensity). The absence of baseline EMG activity was verified before carrying out each of the recordings; between 1 to 6 recordings for each subject were rejected because of background activity. J. Clin. Med. 2021, 10, 3278 6 of 16 2.5. Data Analysis and Statistics All data are expressed as the mean ± standard error of the mean (SEM), at baseline (PRE), just after (POST) and sixty minutes after finishing the intervention (FOLLOW) in each condition. We calculated the % changes with respect to baseline for each time point evaluation when appropriate. In the analysis of the recruitment curves, differences between pre vs. post intervention, and vs. follow were calculated. BBT was calculated just from one trial and MVC (hand muscle strength) was calculated as the mean of 3 trials. For the F wave, the peak-to-peak amplitude and for Mmax, the maximum amplitude of the M wave was measured. F wave was considered if peak-to-peak amplitude was at least 1% of M wave (Mmax) amplitude. F wave persistence was calculated by dividing the number of F responses by the number of stimuli from 15 recordings. The maximum amplitude of F wave was normalized to Mmax amplitude to obtain Fmax/Mmax ratio. For recruitment curves of spinal MEPs and cortical MEPs, we measured the peak- to-peak MEP amplitude in each recording, and then calculated the mean amplitude of MEPs from 3 recordings for each intensity and for each subject and for each experimental condition. Recruitment curves obtained at post and follow-up were normalized calculating the amplitude difference according to pre intervention recruitment curves values. For SICI, averaged peak-to-peak amplitude of the conditioned MEP (obtained after the conditioning stimulus of 80% cortical RMT) was expressed as a percentage of the average amplitude of the test MEP (obtained at supramaximal 120% cortical RMT stimulus), according to % = (conditioned MEP/test MEP) * 100. Shapiro–Wilk test was used to assess if data were normally distributed, and spheric- ity was evaluated with Mauchly’s test. Greenhouse–Geisser correction was used when the assumption of sphericity was violated (p < 0.05). All data was normally distributed, and a two-way repeated measures ANOVA (RM-ANOVA) was used for all statistical analyses to study the main effects of intervention and time on the functional and motor strength assessment and neurophysiological assessments for the raw and the normalized data. Interaction effect was also studied to determine if the interaction between interven- tion and time factors could affect the dependent variable. For significant interaction in the normalized data, time point and intervention simple effects were calculated and shown in results and figures. Time point multiple comparisons were performed with the Dunnett test (comparison between baseline vs. post and baseline vs. follow-up). The intervention multiple comparisons were compared with the Tukey test for the first part of the study (three interventions) and with Šidák test for the second part (two interventions). Signifi- cance level was set at p < 0.05 in all cases. Posterior estimation effect size η2 was calculated (0.01, 0.06, >0.14 as small, medium and large effect respectively). 3. Results Demographic data of healthy individuals and used intensities for eEmc are given in Table 1. All volunteers finished the four experimental interventions with some discomfort sensation subjectively reported but not quantified, especially for the higher intensities during cervical stimulation, on the neck around the electrodes and slight extension of the neck because of cervical paravertebral muscle contraction. 3.1. Effects of eEmc Intensity Raw data of functional and motor strength and neurophysiological assessment during the different electrical stimulus intensity for eEmc, in the first part of the study, are shown in Table 2. J. Clin. Med. 2021, 10, 3278 7 of 16 Table 2. Raw data and statistics of functional and motor strength and neurophysiological assessment during the electrical stimulus intensity for eEmc at 80%, 90% and 110% combined with maximum (100%) handgrip strength during hand training. Time 80% 90% 110% F (DFn, DFd) p Value η2 Pre 74.1 ± 3.0 72.8 ± 3.1 71.2 ± 2.7 Ftime (2, 16) = 17.32 <0.0001 0.033 BBT (blocks number) Post 74.6 ± 3.1 75.7 ± 3.5 74.4 ± 2.9 Finterven. (2, 16) = 0.16 0.855 0.002 Foll. 74.6 ± 3.2 77.4 ± 3.4 78.0 ± 2.7 Finterac. (4, 32) = 3.49 0.018 0.014 Pre 37.8 ± 8.3 31.3 ± 11.3 36.9 ± 7.6 Ftime (2, 16) = 5.87 0.012 0.002 MVC Grip Force (kg) Post 35.1 ± 7.6 32.7 ± 11.0 35.5 ± 8.7 Finterven. (2, 16) = 3.59 0.052 0.040 Foll. 35.8 ± 9.2 33.6 ± 9.8 36.4 ± 7.5 Finterac. (4, 32) = 2.71 0.048 0.009 Pre 65.4 ± 7.9 48.8 ± 9.1 71.5 ± 6.5 Ftime (2, 16) = 0.33 0.725 0.002 F wave Persistency Post 59.3 ± 8.5 60.2 ± 8.2 59.7 ± 8.0 Finterven. (2, 16) = 3.33 0.062 0.019 Foll. 59.9 ± 9.5 57.9 ± 9.2 60.2 ± 10.1 Finterac. (4, 32) = 2.92 0.031 0.031 Pre 13,296.3 ± 1754.9 16,829.0 ± 1863.2 16,844.5 ± 1204.9 Ftime (2, 16) = 1.77 0.202 0.018 Mmax wave (microV) Post 12,752.8 ± 1427.5 15,485.3 ± 1937.6 16,234.6 ± 1579.3 Finterven. (2, 16) = 4.85 0.023 0.119 Foll. 13,697.6 ± 1726.9 17,153.6 ± 1889.8 18,452.1 ± 970.3 Finterac. (4, 32) = 0.29 0.882 0.003 Pre 5.5 ± 0.8 3.7 ± 0.8 4.2 ± 0.6 Ftime (1.92, 15.33) = 0.15 0.851 0.001 Ratio Fmax/Mmax Post 4.6 ± 0.7 5.0 ± 0.8 4.5 ± 0.5 Finterven. (1.38, 11.03) = 0.55 0.530 0.008 Foll. 4.2 ± 0.6 4.3 ± 0.7 5.4 ± 0.6 Finterac. (1.83, 14.64) = 1.93 0.082 0.069 Pre 38.7 ± 2.3 39.1 ± 2.6 39.1 ± 2.0 Ftime (1.85, 14.81) = 1.46 0.263 0.002 RMT TMS Post 37.8 ± 2.4 37.8 ± 2.4 39.3 ± 2.5 Finterven. (1.89, 15.14) = 0.21 0.805 0.002 Foll. 38.2 ± 2.4 39.1 ± 2.6 38.4 ± 2.0 Finterac. (1.91,15.31) = 1.46 0.263 0.003 Pre 13.1 ± 3.7 13.8 ± 2.9 8.3 ± 1.2 Ftime (1.13, 9.05) = 0.93 0.374 0.021 SICI in APB (%) Post 10.8 ± 2.6 12.4 ± 4.2 7.1 ± 2.6 Finterven. (1.51, 12.07) = 0.14 0.816 0.003 Follow 13.2 ± 4.2 16.7 ± 4.7 11.2 ± 4.1 Finterac. (1.90, 15.21) = 0.34 0.708 0.010 Pre 0.6 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 Ftime (2, 16) = 3.43 0.058 0.091 Cortical MEP at 120% Post 1.1 ± 0.2 0.9 ± 0.1 1.1 ± 0.2 Finterven. (2, 16) = 5.25 0.018 0.056 RMT in APB (mV) Foll. 0.8 ± 0.1 0.7 ± 0.1 1.6 ± 0.3 Finterac. (4, 32) = 4.99 0.003 0.113 Stimulus Intensity 80% 90% 110% 0.9 −0.002 ± 0.08 0.08 ± 0.11 0.04 ± 0.08 1 0.06 ± 0.20 0.13 ± 0.37 −0.04 ± 0.47 1.1 −0.08 ± 0.68 0.14 ± 0.80 0.16 ± 0.77 Fintensity (6, 48) = 10.69 <0.001 0.121 TMS recruitment: diff 1.2 0.004 ± 0.71 0.37 ± 1.20 0.68 ± 1.14 Finterven. (2, 16) = 0.74 0.491 0.030 post-pre (mV) 1.3 0.62 ± 0.96 0.07 ± 1.02 0.12 ± 1.00 Finterac. (12, 96) = 2.17 0.019 0.079 1.4 0.124 ± 0.90 0.91 ± 1.21 1.03 ± 0.71 1.5 0.14 ± 0.77 1.17 ± 1.48 1.34 ± 0.64 0.9 −0.01 ± 0.06 −0.02 ± 0.03 0.03 ± 0.09 1 0.05 ± 0.28 −0.01 ± 0.25 0.17 ± 0.80 1.1 −0.16 ± 0.46 −0.10 ± 0.43 0.43 ± 1.18 Fintensity (6, 48) = 4.09 0.002 0.078 TMS recruitment: diff 1.2 0.17 ± 0.61 0.20 ± 0.59 0.88 ± 1.15 Finterven. (2, 16) = 3.61 0.051 0.102 foll-pre (mV) 1.3 −0.20 ± 0.66 0.39 ± 0.65 −0.01 ± 1.03 Finterac. (12, 96) = 3.73 <0.001 0.109 1.4 0.13 ± 1.14 0.34 ± 0.72 1.24 ± 0.70 1.5 −0.47 ± 0.88 0.76 ± 0.63 1.05 ± 0.57 0.9 0.02 ± 0.07 −0.004 ± 0.01 0.01 ± 0.02 1 0.01 ± 0.09 −0.01 ± 0.02 0.02 ± 0.05 1.1 0.01 ± 0.04 −0.03 ± 0.11 0.001 ± 0.03 Fintensity (6, 48) = 0.96 0.460 0.023 C3-C4 recruitment: diff 1.2 0.01 ± 0.03 −0.04 ± 0.11 0.004 ± 0.02 Finterven. (2, 16) = 0.99 0.395 0.057 post-pre (mV) 1.3 −0.03 ± 0.09 −0.04 ± 0.11 −0.01 ± 0.03 Finterac. (12, 96) = 1.69 0.081 0.037 1.4 0.03 ± 0.09 −0.06 ± 0.11 0.01 ± 0.04 1.5 0.04 ± 0.10 −0.003 ± 0.09 −0.02 ± 0.04 0.9 0.03 ± 0.09 0.02 ± 0.05 0.01 ± 0.02 1 0.02 ± 0.09 −0.01 ± 0.03 0.04 ± 0.10 1.1 0.01 ± 0.05 −0.002 ± 0.11 0.01 ± 0.02 Fintensity (6, 48) = 0.31 0.929 0.008 C3-C4 recruitment: diff 1.2 0.02 ± 0.05 0.004 ± 0.02 0.02 ± 0.03 Finterven. (2, 16) = 0.78 0.474 0.036 foll-pre (mV) 1.3 0.03 ± 0.04 0.01 ± 0.02 0.001 ± 0.03 Finterac. (12, 96) = 1.10 0.373 0.035 1.4 0.04 ± 0.08 −0.01 ± 0.05 0.01 ± 0.03 1.5 0.06 ± 0.08 0.001 ± 0.04 0.01 ± 0.03 0.9 0.003 ± 0.03 −0.001 ± 0.03 0.01 ± 0.02 1 −0.001 ± 0.03 0.04 ± 0.10 0.01 ± 0.03 1.1 0.004 ± 0.02 −0.01 ± 0.05 0.01 ± 0.03 Fintensity (6, 48) = 2.14 0.066 0.041 C6-C7 recruitment: diff 1.2 −0.03 ± 0.09 −0.01 ± 0.06 −0.01 ± 0.03 Finterven. (2, 16) = 0.67 0.525 0.023 post-pre (mV) 1.3 −0.04 ± 0.11 −0.02 ± 0.04 0.001 ± 0.05 Finterac. (12, 96) = 0.51 0.906 0.023 1.4 −0.03 ± 0.08 −0.01 ± 0.05 −0.004 ± 0.05 1.5 −0.02 ± 0.12 0.01 ± 0.04 0.01 ± 0.06 0.9 0.01 ± 0.02 −0.004 ± 0.03 0.02 ± 0.02 1 −0.0004 ± 0.03 0.03 ± 0.09 0.02 ± 0.02 1.1 0.01 ± 0.02 −0.001 ± 0.04 0.02 ± 0.04 Fintensity (6, 48) = 0.40 0.878 0.010 C6-C7 recruitment: diff 1.2 −0.02 ± 0.10 0.01 ± 0.02 0.003 ± 0.03 Finterven. (2, 16) = 0.16 0.850 0.005 foll-pre (mV) 1.3 0.02 ± 0.04 0.01 ± 0.02 −0.02 ± 0.08 Finterac. (12, 96) = 0.72 0.725 0.039 1.4 0.01 ± 0.09 0.004 ± 0.03 0.004 ± 0.04 1.5 −0.01 ± 0.14 0.002 ± 0.02 0.03 ± 0.06 diff post-pre: differences between post-pre evaluation; diff foll-pre: differences between baseline-follow up evaluation. 0.9 0.01 ± 0.02 −0.004 ± 0.03 0.02 ± 0.02 1 −0.0004 ± 0.03 0.03 ± 0.09 0.02 ± 0.02 C6-C7 recruit- 1.1 0.01 ± 0.02 −0.001 ± 0.04 0.02 ± 0.04 Fintensity (6,48) = 0.40 0.878 0.010 ment: diff foll-pre 1.2 −0.02 ± 0.10 0.01 ± 0.02 0.003 ± 0.03 Finterven. (2.16) = 0.16 0.850 0.005 (mV) 1.3 0.02 ± 0.04 0.01 ± 0.02 −0.02 ± 0-08 Finterac. (12,96) = 0.72 0.725 0.039 1.4 0.01 ± 0.09 0.004 ± 0.03 0.004 ± 0.04 J. Clin. Med. 2021, 10, 3278 8 of 16 1.5 −0.01 ± 0.14 0.002 ± 0.02 0.03 ± 0.06 diff post-pre: differences between post-pre evaluation; diff foll-pre: differences between baseline-follow up evaluation. 3.1.1. Functional 3.1.1. Functional and Motor and Motor Strength Strength Assessment Assessment Box and Block test. There was a significant Box and Block test. There was a significant effect of of effect time timeand andinteraction interaction in in thethe rawraw data (Table 2) and an effect of time, intervention and interaction in the normalized data (Table 2) and an effect of time, intervention and interaction in the normalized data. data. The The percentage percentage change change of of number number of of boxes boxes during during follow-up follow-up was significantly was significantly higher higherforfor 90%90%and 110% and 110% intensity intensityof of eEmceEmcwith with respect respectto to 80% 80% (Figure (Figure 2A). 2A). Grip force Grip during force MVC. during MVC. RM-ANOVA RM-ANOVAshowed showedsignificant significanteffect effectof ofthe thetime timeand and inter- interac- action in the tion in the raw raw data; data;and andeffect effectofoftime, time,intervention intervention and and interaction interaction in in thethe normalized normalized data. data. Intervention simple effect in percentage changes of MVC showed Intervention simple effect in percentage changes of MVC showed higher maximum muscle higher maximum muscle grip strength grip strength withintensity with 90% 90% intensity of eEmcofthaneEmcwiththan 110%withand110% 80%and 80%intervention at post at post inter- and vention and follow-up follow-up time pointstime points (Figure (Figure 2B). 2B). Figure 2. Functional and motor outcomes assessed by the Box and Block test and MVC. (A) percentage Figure 2. Functional and motor outcomes assessed by the Box and Block test and MVC. (A) percent- agechanges changes in in thethe number number of blocks moved of blocks in the moved inBox the and BoxBlock test with and Block testrespect to baseline. with respect Intervention to baseline. In- simple effect shows significant differencesdifferences at follow ofat90% ** ( p of= 0.004) **** p 0.05) neither in the raw nor in the normalized data. For the TMS-induced cortical MEPs at 120% of RMT, RM-ANOVA showed significant effect of intervention and interaction in the raw data. However, there was significant effect of time, intervention and interaction in the normalized data. According to intervention simple effect, the percentage change of MEP amplitude was significantly higher at 110% of eEmc with respect to 80% and to 90% at follow-up (Figure 4A). J. Clin. Med. 2021, 10, x FOR PEER REVIEW 9 of 16 tion. It was significantly higher with 90% of eEmc in comparison with 80% at post inter- J. Clin. Med. 2021, 10, 3278 9 of 16 vention; and with 90% and 110% in comparison with 80% of eEmc at follow-up evaluation (Figure 3B). Figure 3. F wave persistency and F/M wave ratio. (A) percentage changes in F wave persistency Figure with 3. F wave respect persistency to baseline. and F/M wave Intervention simpleratio. effect(A) percentage showed changes significant in F wave differences persistency of 90% eEmc with with respect to 80% ( p = 0.0079) and 110% eEmc ( p = 0.0005) at post and at follow eEmc respect to baseline. ** Intervention simple effect showed *** significant differences of 90% (* p = with 0.0304; respect ** p =to 0.0020); p =percentage 80% (**(B) 0.0079) and 110% eEmc changes in F/M( ratio *** p = 0.0005) at post and at follow (* p = 0.0304; ** p = with respect to baseline. Intervention simple effect 0.0020); (B) percentage changes in F/M ratio with respect to baseline. Intervention simple effect showed significant differences at post of 90% and 80% eEmc** (** p = 0.0032); and at follow of 80% showed significant* differences at post of 90% and 80% eEmc ( p = 0.0032); and at follow of 80% eEmc and 90% ( p = 0.0491) and 110% eEmc (** p = 0.0051). eEmc and 90% (* p = 0.0491) and 110% eEmc (** p = 0.0051). For recruitment curves of cortical MEP, there was a significant effect of intensity and Spinal MEPs recruitment curve. RM-ANOVA showed no significant differences in the interaction for difference between pre and post, and an effect of interaction for difference raw or the normalized data regarding results of the recruitment curve of spinal MEPs at between pre and follow-up. We found a significantly higher MEP amplitude with 90% and C3-C4 or at C6-C7 level. 110% of eEmc compared with 80% at post intervention (Figure 4B). The MEP amplitude wasCortical J. Clin. Med. 2021, 10, x FOR PEER REVIEW RMT, TMS-induced significantly corticaltesting higher at follow-up MEPs in and SICI. 90% andCortical 110% ofRMT eEmcand withSICI didto respect not 1080% of 16 change for 1.4×and 1.5 × RMT (Figure 4C), and in 110% with respect to 80% and 90% of eEmcinfor significantly at any eEmc stimulation intensity (p > 0.05) neither in the raw nor the1.1 normalized data. 4C). × RMT (Figure For the TMS-induced cortical MEPs at 120% of RMT, RM-ANOVA showed signifi- cant effect of intervention and interaction in the raw data. However, there was significant effect of time, intervention and interaction in the normalized data. According to interven- tion simple effect, the percentage change of MEP amplitude was significantly higher at 110% of eEmc with respect to 80% and to 90% at follow-up (Figure 4A). For recruitment curves of cortical MEP, there was a significant effect of intensity and interaction for difference between pre and post, and an effect of interaction for difference between pre and follow-up. We found a significantly higher MEP amplitude with 90% and 110% of eEmc compared with 80% at post intervention (Figure 4B). The MEP ampli- tude was significantly higher at follow-up testing in 90% and 110% of eEmc with respect to 80% for 1.4x and 1.5xRMT (Figure 4C), and in 110% with respect to 80% and 90% of eEmc for 1.1xRMT (Figure 4C). Figure 4. Cortical excitability outcomes. (A) percentage changes in cortical MEP evoked by TMS 120% of RMT with Figure 4. respect toCortical excitability baseline. Interventionoutcomes. simple(A) percentage effect showedchanges in cortical significant MEP evoked differences by of at follow TMS 120% 110% eEmcof RMT withwith respect respect to 80% (* p = 0.0126) and 90% eEmc (*** p = 0.0005); (B) difference in cortical MEP amplitude between post and pre time= to baseline. Intervention simple effect showed significant differences at follow of 110% eEmc with respect to 80% (* p 0.0126) and 90% eEmc (*** p = 0.0005); (B) difference in cortical MEP amplitude between post and pre time points. Intensity points. Intensity simple effects showed significant differences of 80% eEmc with respect 90% and 110% of eEmc at 1.4 simple effects showed significant differences of+++ 80% eEmc with respect 90% and 110% of eEmc at 1.4 (* p = 0.0390, + p = (* p = 0.0390, + p = 0.0133) and 1.5 (** p = 0.0042, p = 0.0008) RMT cortical MEP; (C) difference in cortical MEP amplitude .0133) and 1.5 (** p = 0.0042, +++p = 0.0008) RMT cortical MEP; (C) difference in cortical MEP amplitude between follow and between follow and pre time points. Intensity simple pre time points. Intensity simple effects showed significant effectsdifferences showed significant differences of 80% and 110% eEmcof 80% and at 1.2 (+ p110% eEmc1.4 = 0.0162), at (1.2 ++++ (p+

0.05). Cortical RMT, TMS-induced cortical MEPs and SICI. RM-ANOVA showed significant effect of time in the raw and in the normalized data for cortical RMT, but not for SICI. On Figure 5. Changes in MVC, F wave persistency and F/M wave ratio. (A) percentage changes in grip force during MVC according to baseline. Intervention simple effect showed differences at post (* p = 0.0144) and follow (** p = 0.0011); (B) percentage changes in F wave persistency according to baseline. Intervention simple effects showed significant differences J. Clin. Med. 2021, 10, 3278 12 of 16 at post (** p = 0.0073) and follow (* p = 0.0191); (C) percentage changes in F/M ratio according to baseline. Intervention simple effect showed significant differences at post (** p = 0.0016). Spinal SpinalMEPs MEPsrecruitment recruitment curve. curve. RM-ANOVA RM-ANOVA did didnot notshow showany anysignificant significantdifferences differencesin inraw rawand andnormalized normalized data of the recruitment curve of spinal MEP evoked with data of the recruitment curve of spinal MEP evoked with stimulation stimula- tion either either at C3-C4 at C3-C4 or C6-C7 or C6-C7 (p >(p > 0.05). 0.05). Cortical Cortical RMT, TMS-inducedcortical RMT, TMS-induced corticalMEPs MEPsand andSICI. SICI.RM-ANOVA RM-ANOVAshowed showedsignificant significant effect effect of time in the raw and in the normalized data for cortical RMT, butnot of time in the raw and in the normalized data for cortical RMT, but notfor forSICI. SICI.On On the the other hand, TMS-induced cortical MEPs at 120% RMT showed significant effectof other hand, TMS-induced cortical MEPs at 120% RMT showed significant effect of interaction interactionininthe theraw rawand andininthe thenormalized normalizeddata. data.The Thecortical corticalMEP MEPamplitude amplitudewas washigher higher inin50% 50%than thaninin100% 100%handgrip handgripstrength strengthintervention interventionatatfollow-up follow-up(Figure (Figure6A). 6A). Figure Figure6.6.Cortical Corticalexcitability excitabilityoutcomes. outcomes.(A) (A)percentage percentagechanges changesinincortical corticalMEP MEPfrom from120% 120%ofofRMT RMTaccording accordingtotobaseline. baseline. Intervention Interventionsimple simpleeffect effectshowed showed significant significant differences differences at follow follow;(*** (B) pdifference = 0.0017);amplitude (B) difference amplitude between between post and post pre cortical and pre cortical MEP recruitment. Intensity simple effects showed significant differences at 1.5 RMT multiple MEP recruitment. Intensity simple effects showed significant differences at 1.5 RMT multiple (** p = 0.0059); (C) difference (** p= 0.0059); (C) difference amplitude between follow and pre MEP recruitment. Intensity simple effects showed amplitude between follow and pre MEP recruitment. Intensity simple effects showed significant differences at 1.5 RMT significant differences at 1.5 RMT multiple (** p = 0.0063). multiple (** p = 0.0063). In Inthe therecruitment recruitmentcurve curveof ofcortical corticalMEPs, MEPs,RM-ANOVA RM-ANOVAshowed showedsignificant significanteffect effectof of intensity intensity and interaction between pre and post, and effect of interaction between preand and interaction between pre and post, and effect of interaction between pre and follow-up. Additionally, 100% of handgrip strength intervention significantly increased the cortical MEP amplitude at 1.5 × RMT with respect to 50% of MVC at post intervention and at follow-up (Figure 6B,C). 4. Discussion In a previous study we reported that eEmc combined with hand training improves hand grip force and increases spinal and corticospinal excitability in comparison to each intervention tested alone [8]. In the present study, we further investigated if the levels of both eEmc intensity and muscle strength during training could influence the results for neuromodulation at the cervical spinal cord. The results of this study show that eEmc at 90% intensity of spinal RMT of the APB muscle combined with maximal handgrip strength induced better hand function and muscle strength than the other conditions assayed. The results of F wave persistency and F/M ratio and of cortical MEP recruitment curves reflected stronger plastic changes in the spinal and cortical pathways of eEmc at 90% intensity compared to eEmc at intensities of 80% or 110%. Most of these effects were maintained at least for one hour following the intervention. On the other hand, eEmc at 110% intensity induced better hand function and higher F/M ratio and cortical plasticity changes with respect to eEmc at 80%. In contrast, eEmc at 80% of intensity did not show any noticeable effect on hand function, spinal and cortical excitability. In the second part of the study, eEmc at 90% intensity was combined with training at the maximal (100%) or at half (50%) MVC in hand grip strength. With 100% MVC, there was higher muscle strength and more plastic changes at spinal cord, measured by F wave persistency and F/M ratio, and at cortical level, measured by cortical MEP recruitment curve, than with J. Clin. Med. 2021, 10, 3278 13 of 16 50% MVC. Most of these effects were also maintained at least one hour during follow-up testing. In contrast, with the 50% MVC of grip strength there was only a significant increase in cortical excitability measured by cortical MEP. 4.1. Effects of eEmc Intensity There is no consensus in the literature about which intensity of electrical stimulation may modulate more effectively the functional outputs of the cervical spinal cord. Thus, previous studies have used different methods to select the eEmc intensity. In our previous study in healthy individuals, we empirically used a stimulation intensity at 90% of spinal RMT of the APB muscle [10], whereas Benavides et al. [5] had used 100% of RMT of the Biceps brachii muscle in SCI and healthy individuals. Gad et al. [6] adjusted the eEmc to enable maximal grip strength or submaximal isometric hand movement without causing discomfort in SCI subjects. Freverty et al. [7] used varying combinations of eEmc parameters to obtain optimal facilitation of voluntary hand grip by identification of the relative activation levels of the motor pools, and combined eEmc with medication (Buspirone) without hand training. Studying cervical SCI subjects, other authors used different strategies, such as beginning eEmc at 50 mA and adjusting the intensity based on the functional task performance and subjective feedback during the intervention [9], increasing stimulus intensity in 10 mA intervals from 10 to 120 mA [8], or from 5 to 68 mA [19]. Despite the different stimulus intensity used for eEmc, the most likely direct mecha- nism of eEmc occurs via transcutaneous tonic spinal activation by elevating spinal networks excitability [12] and may activate interneuronal pathways that generate action potentials on motoneurons within a motor pool in a more normal stochastic time frame [13]. As such, eEmc is hypothesized to potentiate the generation of postsynaptic excitatory potentials and thus shift the spinal motor network excitability closer to the excitation threshold. The con- stantly changing postsynaptic potentials intrinsic to spinal networks [20] can contribute to a constantly changing population of interneurons being asynchronously activated in a ran- dom pattern [21]. According to our results, it can be interpreted that eEmc, at higher levels when delivered at 90% stimulus intensity, brings interneurons and motoneurons closer to motor threshold and, therefore, more likely to respond to descending drive, and elevates the excitability of spinal cord networks and possibly also of motor cortex. In addition, sensory afferents at the level of dorsal roots and/or the dorsal column spinal pathways may be affected by the tSCS and thus contribute differentially to elevating or suppressing spinal and/or supraspinal neural excitability [14,15,21]. Despite we cannot preclude this mechanism, considering that the stimulation was applied at C3 level, far cranial from the APB muscle root innervation segment (C8-T1), we hypothesize that the effect on muscle response is more likely due to stimulation of descending pathways and premotoneuronal network than to the stimulation of proprioceptive fibers within posterior roots. From our results, it appears that there is a window of intensity for the neuromodula- tory effects of eEmc. The effects of an eEmc intensity of 90% of spinal RMT of the target muscle exceeded the effects of lower (80%) and higher (110%) stimulus intensities. It is unclear, however, why only the 90% level of intensity enabled higher voluntarily controlled drive to produce stronger contractions of hand training, which subsequently strengthens the neuromuscular network. It is, noteworthy that eEmc at 110% intensity augmented hand muscle strength less than 90% intensity but induced more plastic changes at cortical level than 90% and 80% intensities. Perhaps the higher eEmc intensity can elicit impulses that are conducted antidromically to the cortex or induce a greater facilitatory effect among brain networks, inducing more effectively plastic changes at the cortical than the spinal level. Thus, our data suggest that eEmc at suprathreshold intensity modulates more effectively the cortical excitability than at subthreshold intensities, but hand function, muscle strength and spinal cord excitability were modulated more effectively by subthreshold intensity of 90% spinal RMT. J. Clin. Med. 2021, 10, 3278 14 of 16 4.2. Effects of Handgrip Strength during Training Our results indicate that the combination of maximal (100% of MVC) handgrip strength with eEmc further increases the hand muscle strength and induces more ef- fectively plastic changes at spinal cord and cortical levels in comparison to lower (50%) handgrip strength. There is no consensus either about what kind of hand training could help to modulate, more effectively, spinal cord motor function in healthy individuals or following SCI. Indeed, only a few studies on eEmc at cervical cord level combined tSCS with hand training. In the study by Gad et al. [6] with cervical SCI subjects, two tasks were performed: isometric maximum handgrip contraction or voluntary rhythmic efforts of submaximal contraction (opening and closing the hand). In another study on a single SCI subject, a functional task combined with eEmc aimed to address hand grasp and pinch bilaterally [9]. In healthy individuals, we previously used maximal handgrip force for hand training combined with eEmc [10]. Several observations made after a single training or stimulation session suggest that short-term plastic changes occur [5,10,22]. Performing the maximum handgrip strength combined with eEmc at 90% of stimulus intensity modulates more effectively hand motor function and induces more effectively neurophysiological changes at spinal cord and cortical levels than when performing lower hand grip strength. According to our results, activity-based plasticity depends on the level of hand grip strength for hand training and the eEmc intensity for neuromodulation. Activity-based neural plasticity mechanisms involve both functional modifications of existing synapses and structural changes that alter the synaptic connectivity of neurons (formation, removal and remodeling of synaptic buttons and even dendrites) [22]. A single exposure to activity- dependent plasticity by physical training combined with electrical stimulation may induce these short-term changes that in turn could promote occurrence of long-term plastic- ity under repeated exposure that involves mechanisms such as long-term potentiation and depression, morphological changes of dendrites, synaptogenesis and axonal branch- ing/regeneration [23]. 4.3. Limitations of the Study It has to be noted that the sample size in this study was relatively small, limiting statistical power and interpretation of the results. Four days of experiments and the long duration of each experiment may result in fatigue factors, limiting the number of replicates that could be recorded for neurophysiological parameters, resulting in more variability. The only follow-up assessment period was for one hour after the end of session, thus providing no comparison for other longer timeframes. Nevertheless, the obtained results open a work frame for further studies refining the conditions of combinatory neuromodulation for SCI patients. 5. Conclusions Taken together, this study demonstrates an approach to assess two modulatory in- terventions, eEmc and activity-dependent intervention at the supraspinal, and spinal and peripheral levels. It also provides basic physiological responses in healthy subjects, which will help the design of future studies involving other dysfunctional conditions. The findings of the study indicate that the stimulus intensity for eEmc is relevant for improving hand function and/or muscle strength, as well as hand muscle strength level and recruitment of motor units within a motor pool during training in conjunction with electrical neuromodulation. The data also demonstrate both the immediacy and short-term persistence of at least one hour after intervention. Interestingly, our results also allow us to hypothesize that spinal and cortical excitability may be modulated differently depending on the eEmc intensity. Therefore, both the electrical stimulus intensity of eEmc and the intensity of the voluntary effort during training should be considered when optimizing rehabilitation protocols for restoring motor performance. J. Clin. Med. 2021, 10, 3278 15 of 16 Author Contributions: Conceptualization, H.K., M.R.-C., Á.F., G.G.-A. and J.V.; methodology, H.K., M.R.-C. and J.V.; software, M.R.-C.; formal analysis, H.K. and M.R.-C.; validation, H.K., M.R.-C., G.G.-A. and J.V.; investigation, H.K., M.R.-C., Á.F., L.G., I.S., G.G.-A. and J.V.; resources, H.K., M.R.-C., E.O., G.G.-A. and J.V.; data curation, H.K., M.R.-C. and L.G.; writing—original draft preparation, H.K., M.R.-C. and X.N.; writing—review and editing, H.K., M.R.-C., V.R.E., L.G., E.O., I.S., J.V., Y.G., Á.F., G.G.-A. and X.N.; visualization, H.K. and M.R.-C.; supervision, H.K. and G.G.-A.; project administration, H.K., G.G.-A. and J.V.; funding acquisition, H.K., G.G.-A. and J.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by H2020-ERA-NET Neuron (AC16/00034) to G.G.-A. and J.V.; AES 2019 ISCIII (PI19/01680) to J.V.; Fundació La Marató de TV3 2017 (201713.31) to G.G.-A.; Premi Beca “Mike Lane” 2019-Castellers de la Vila de Gràcia to H.K.; and National Institutes of Health Grant 1R01 NS102920-01A1 to Y.G. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Institut Guttmann (protocol code 2017255 and date of approval: 29 April 2017). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions. Conflicts of Interest: V.R.E. and Y.G. hold shareholder interest in NeuroRecovery Technologies and hold inventorship rights on intellectual property licensed by The Regents of the University of California to NeuroRecovery Technologies and its subsidiaries. Y.G. also holds shareholder interest in Cosyma Inc., St. Petersburg, Russia. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. References 1. Gerasimenko, Y.P.; Lu, D.C.; Modaber, M.; Zdunowski, S.; Gad, P.; Sayenko, D.G.; Morikawa, E.; Haakana, P.; Ferguson, A.R.; Roy, R.R.; et al. Noninvasive Reactivation of Motor Descending Control after Paralysis. J. Neurotrauma 2015, 32, 1968–1980. [CrossRef] [PubMed] 2. Megía García, A.; Serrano-Muñoz, D.; Taylor, J.; Avendaño-Coy, J.; Gómez-Soriano, J. Transcutaneous Spinal Cord Stimulation and Motor Rehabilitation in Spinal Cord Injury: A Systematic Review. 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