sensors Article New Stimulation Device to Drive Multiple Transverse Intrafascicular Electrodes and Achieve Highly Selective and Rich Neural Responses Thomas Guiho 1,2, * , Victor Manuel López-Álvarez 3,4 , Paul Čvančara 5 , Arthur Hiairrassary 1,2 , David Andreu 1,2,6 , Thomas Stieglitz 5 , Xavier Navarro 3,4 and David Guiraud 1,2,6 1 Camin Team, National Institute for Research in Computer Science and Automation (Inria), 34095 Montpellier, France; arthur.hiairrassary@neurinnov.com (A.H.); david.andreu@neurinnov.com (D.A.); david.guiraud@inria.fr (D.G.) 2 Camin Team, Laboratoire d’Informatique, de Robotique et de Microélectronique de Montpellier, Université de Montpellier, 34095 Montpellier, France 3 Department of Cell Biology, Physiology and Immunology and Institute of Neurosciences, Universitat Autonoma de Barcelona (UAB), 08193 Bellaterra, Spain; vmlopal@gmail.com (V.M.L.-Á.); xavier.navarro@uab.cat (X.N.) 4 Centro de Investigacion Biomedica en Red en Enfermedades Neurodegenerativas (CIBERNED), 08193 Bellaterra, Spain 5 Laboratory for Biomedical Microtechnology, Department of Microsystems Engineering—IMTEK, University of Freiburg, 79110 Freiburg, Germany; paul.cvancara@imtek.uni-freiburg.de (P.Č.);   Thomas.Stieglitz@imtek.uni-freiburg.de (T.S.) 6 Neurinnov, 34095 Montpellier, France Citation: Guiho, T.; López-Álvarez, * Correspondence: thomas.guiho@inria.fr V.M.; Čvančara, P.; Hiairrassary, A.; Andreu, D.; Stieglitz, T.; Navarro, X.; Abstract: Peripheral Nerve Stimulation (PNS) is a promising approach in functional restoration Guiraud, D. New Stimulation Device following neural impairments. Although it proves to be advantageous in the number of implan- to Drive Multiple Transverse tation sites provided compared with intramuscular or epimysial stimulation and the fact that it Intrafascicular Electrodes and does not require daily placement, as is the case with surface electrodes, the further advancement Achieve Highly Selective and Rich Neural Responses. Sensors 2021, 21, of PNS paradigms is hampered by the limitation of spatial selectivity due to the current spread 7219. https://doi.org/10.3390/ and variations of nerve physiology. New electrode designs such as the Transverse Intrafascicular s21217219 Multichannel Electrode (TIME) were proposed to resolve this issue, but their use was limited by a lack of innovative multichannel stimulation devices. In this study, we introduce a new portable Academic Editors: Kei Masani, multichannel stimulator—called STIMEP—and implement different stimulation protocols in rats to Milos Popovic and Samuel C.K. Lee test its versatility and unveil the potential of its combined use with TIME electrodes in rehabilitation protocols. We developed and tested various stimulation paradigms in a single fascicle and thereafter Received: 17 September 2021 implanted two TIMEs. We also tested its stimulation using two different waveforms. The results Accepted: 26 October 2021 highlighted the versatility of this new stimulation device and advocated for the parameterizing Published: 29 October 2021 of a hyperpolarizing phase before depolarization as well as the use of small pulse widths when stimulating with multiple electrodes. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: stimulation selectivity; stimulation device; intrafascicular electrode; peripheral nerve; published maps and institutional affil- neuroprosthesis iations. 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Neuroprostheses are active medical devices aiming at restoring impaired physiological This article is an open access article functions via the injection of electrical charges close to excitable cells, especially neurons. distributed under the terms and Among the disorders that can benefit from these technologies is Spinal Cord Injury (SCI) [1]. conditions of the Creative Commons Characterized by a breakdown in supraspinal communication, SCI invariably results in Attribution (CC BY) license (https:// alterations of the sublesional functions and leads to visceral (sexual, urinary etc.), sensory creativecommons.org/licenses/by/ (anesthesia, pain) and motor (paraplegia, tetraplegia) deficits that have a dramatic impact 4.0/). on a patient0 s quality of life [2,3]. Sensors 2021, 21, 7219. https://doi.org/10.3390/s21217219 https://www.mdpi.com/journal/sensors Sensors 2021, 21, 7219 2 of 17 The preservation of excitatory properties of the sublesional tissue after SCI assists in configuring approaches to interface with the preserved cells so as to activate them vis electrical stimulation and to promote functional rehabilitation. Electrical stimulation thus implies a broad spectrum of clinical applications for functional restoration ranging from breathing facilitation by phrenic nerve stimulation [4] to bladder [5,6], bowel [7,8], sexual and motor [9–11] functions. Concerning the latter, several modalities of stimulation are currently investigated according to electrodes positioning—and design—distinguishing the spinal, intramuscular, epimysial, surface and Peripheral Nervous System (PNS) approaches. PNS is of particular interest as it overcomes most muscle stimulation constraints, i.e., the activation of many muscles from a single electrode (limiting surgical complexity and technical failures) and finer stimulation settings (reducing power consumption and opening new possibilities for hardly accessible muscles). Indeed, many muscles are controlled by the same nerve, and the application of targeted stimulations should, in theory, enable the selective activation of several muscles, since the nerve fibers controlling a specific muscle are grouped in the same fascicle [12,13]. Concerning afferent pathway stimulation, sensory feedback restoration is a potential application for such a technology as it requires selective stimulations to evoke specific sensations. Multicontact cuffs [14,15] and multicontact intrafascicular electrodes [16] already successfully elicited a large range of phantom hand sensations, providing richer and bidirectional hand prosthesis interfaces. However, despite these advantages, the potentially limiting factors of the electrical stimulation in comparison with physiological activation still need to be considered before PNS stimulation becomes a standard procedure [17]. Electrode design is of high relevance in PNS efficiency since it influences the paths of current dissipation, and the excitation threshold and spatial selectivity vary with the distance of the fibers to the stimulation sites. Nerve fibers that are far from the electrode are only slightly affected by stimulation while the nearest fibers are preferentially depolarized. Due to the peripheral nerve structure, two stimulation modalities were considered—i.e., stimulation using electrodes with contacts in close connection with the nerve surface [18] or via electrodes directly inserted through the nerve [19]. The first mentioned class belongs to the “cuff electrodes” family or to cuff electrode-related technologies (e.g., the nerve flattening FINE [20]) and preferentially activates fibers at the periphery while the second generation of the aforementioned electrodes belongs to the family of “intrafascicular elec- trodes” and targets the fibers within the nerve. The latter are either inserted longitudinally (Longitudinal IntraFascicular Electrode or LIFE [21]) or transversally (Transverse Intrafasci- cular Multichannel Electrode or TIME [22–24]) to specifically target fascicles in superficial and deeper nerve areas. Another limitation in PNS spread is linked to disparities in the activation thresholds of the different nerve fiber populations. Depending on their diameter and their conductive properties (presence/absence of myelin sheath), nerve fibers become more or less sensitive to stimulation. Thus, the large diameter-myelinated fibers are preferably activated at low currents. In order to manage this phenomenon, numerous simulation studies investigated the impact of stimulation parameters—i.e., the pulse width (PW), intensity (I), frequency (f)—on the relative recruitment of fiber populations using computational models [25–29]. Alongside these simulations, experimental techniques for obtaining a normal recruitment order have been described in the literature [30]. Among them, an anodal block is an extensively studied procedure that relies on the hyperpolarization of nerve fibers near an anode to selectively block evoked-action potentials from larger fibers [31]. In the same way, subthreshold depolarizing prepulses that are close to the excitation threshold exploit the accommodation of the fiber membrane and the inactivation of voltage-dependent sodium channels to influence the recruitment order [32]. The C-fibers’ selective activation was also investigated using slowly rising ramp pulses [33] while the high frequency block method uses rectangular or sinusoidal pulses of frequency 3–5 kHz to block activation of the nerve by preserving it in a refractory period [34]. Sensors 2021, 21, 7219 3 of 17 According to these guidelines, the optimization of PNS strategies requires the refining of stimulation parameters and the delivery of complex waveforms through multiple con- tacts dispatched among several multichannel electrodes. This implies a complex waveform design on the synchronous output and an increasing number of wires between the stimulus generator and the electrode contacts (poles). A programmable multidimensional stimulus waveform provides the opportunity to conduct research on artificial-to-natural interfaces in order to achieve an efficient and minimally aggressive activation [35–38]. In contrast, the vast majority of the solutions that have been tested on humans have been based on central- ized implants through which the wires output to monopolar or bipolar electrodes [39–41]. Two exceptions can be noted: BION technology, where bipolar stimulation is provided by injectable autonomous units [42], and the LARSI project, which is aimed at multipolar stimulation [43] localized to the sacral roots. In this study, we present a new portable multichannel stimulation device—called STIMEP—designed to automatically deliver sequences of complex stimulation patterns for up to four electrodes simultaneously. We tested this device in vivo by stimulating rats’ sciatic nerve with TIME electrodes along three modalities. Firstly, we finely tuned the stimulation parameters—intensity and pulse width—and investigated their impact on nerve fibers within a single fascicle; secondly, we implemented simultaneous stimulations of several TIMEs for the same nerve and studied spatial selectivity; and lastly, we tested two different stimulation patterns. We used these protocols to ensure stimulator performances and safety but also to gain further insights regarding the potential of TIME electrodes for optimized PNS strategies. 2. Materials and Methods 2.1. General Description of the Stimulation Device—STIMEP Platform STIMEP is a wearable neural stimulator designed and developed by the CAMIN team (former DEMAR team, INRIA/University of Montpellier) in association with the Axonic company (Sophia Antipolis, Vallauris, France). The hardware and embedded software architectures [44] were developed in compliance with European Union directives (90/385, 93/342 directives and EN 62304 before the application of the new EU regulation MDR2017/745) for active medical devices so as to permit its use for human clinical trials. STIMEP is piloted by means of the SYNERGY neurostimulation software (Camin team, INRIA, Montpellier, France). The STIMEP platform allows for the control of 64 contacts (56 active sites and 8 ground sites) of implanted electrodes, divided across 4 insulated ports of 14 capacitive coupled active outputs and 2 direct-coupling references. The stimulation device consists of a con- troller (SOC device) that drives and coordinates the 4 distributed stimulation units (DSU) able to drive up to 4 TIME devices simultaneously and independently (Table 1, Figure 1a,b). The controller embeds and executes a set of functions [45], for instance, those dedicated to “Contact Check” (qualitative impedance measurement), “Threshold Determination” (increasing stimulation to determine the sensation threshold), “Therapy” (for protocol- dependent multisite stimulation), etc., and controls the set of DSU depending on the given function. Each DSU is composed of an analog ASIC stimulation front-end (CAFE24 chip— designed by the company MXM under CAMIN licensing [WO2006/027473]) and a digital architecture based on FPGA that allows for communication functions, the sequencing of stimulations, the control of stimulation profiles and safety monitoring by means of reference models (ensuring contact and nerve integrity). The SYNERGY software was developed to configure and pilot the STIMEP controller, thus leading to the generation of complex stimulation patterns by adjusting both the stimulation parameters and waveforms on many independent channels (depending on the executed function). Sensors 2021, 21, 7219 4 of 17 Sensors 2021, 21, 7219 4 of 18 Table 1. Technical specifications of STIMEP stimulator. Table 1. Technical specifications of STIMEP stimulator. Parameter Value Parameter Value Type of stimulation Type of stimulation Constant current stimulation Constant current stimulation 14active 14 active sites sites (capacitively (capacitively coupled) coupled) Number Numberofof channels perper channels stimulation unitunit stimulation 22references references (non (non coupled) coupled) Number of distributed Number stimulation of distributed units (DSU) stimulation units 4 (56 active sites in total) 4 (56 active sites in total) (DSU) Weight 150 g (W × L × H) DimensionsWeight 81 × 130 × g21 mm3 150 Dimensions (W × L × H) 81 × 130 2–508 × 21 µs mm 3 Pulse width (2 µs2–508 µs resolution) Pulse width (2 µs resolution) 10–2540 µA Intensity (10 10–2540 µA µA resolution) Intensity (10 µA resolution) 3 ranges: low/mid/high 3 ranges: Low range: up to 8low/mid/high channels per electrode Low range: (DSU) up to 8->channels 4 to 58 Hzper electrode Frequency Mid range:(DSU) up to 4->channels per electrode 4 to 58 Hz Frequency Mid range:(DSU) up to -> 4 to 111 Hz 4 channels per electrode High range: up to 2 channels per electrode (DSU) -> 4 to 111 Hz (DSU) -> 4 to 200 Hz High range: up to 2 channels per electrode Passive discharge 150 µs minimum duration (DSU) -> 4 to 200 Hz ChannelPassive capacitive coupling discharge 330 nF duration 150 µs minimum Channel capacitive Output voltage coupling 330 19 VnF Output Poweringvoltage 19 V USB or external battery Powering USB or external battery Autonomy (external battery) 8 hours Autonomy (external battery) 8 hours Figure Figure1. STIMEP architecture 1. STIMEP and and architecture bench testing. bench (a) Diagram testing. of STIMEP (a) Diagram hardware of STIMEP and (b)and hardware picture of the corresponding device at the same scale. (c) Coordinated DSU stimulations with different fre- (b) picture of the corresponding device at the same scale. (c) Coordinated DSU stimulations quencies, intensities and pulse widths (oscilloscope screenshot). with different frequencies, intensities and pulse widths (oscilloscope screenshot). Sensors 2021, 21, 7219 5 of 17 Each DSU embeds a scheduler, thereby allowing for sequencing stimulations, in accordance with a given frequency (Figure 1c). The remote configuration and control of the (set of) DSU is ensured by the controller, depending on the executed function, for coordinating and modulating stimulations. The controller also generates synchronization signals to trigger the recording devices. 2.2. In Vivo Validation of the Stimulation Device and Selectivity Study 2.2.1. Experimental Set-Up Surgical procedures were performed in 2 female Sprague-Dawley rats (280–300 g) under pentobarbital anesthesia (40 mg/kg intraperitoneal i.p.) and in accordance with protocols approved by the Ethical Committee of the Universitat Autonoma de Barcelona. The right sciatic nerve was exposed at mid-thigh and gently dissected from the surrounding tissue between the sciatic notch and the knee. The TIMEs were implanted with the aid of a dissecting microscope to ensure the correct positioning of the device. The needle attached to the central axis of the TIME device was transversally inserted into the sciatic nerve proximal to its trifurcation at the knee before transferring the stimulation sites into the nerve. Since interfascicular selectivity—i.e., the selective activation of fibers laying in separate nerve fascicles—is relatively easy to assess by positioning different contact sites in the selected fascicles [22], we mainly focused our analysis on intrafascicular selectivity. As the TIMEs that were used were originally designed for human trials, only two contacts were positioned within the rat sciatic nerve concurrently (i.e., one contact in the left side and one in the right side). The required stimulation was provided by a STIMEP stimulator and configured using SYNERGY software. Different stimulation paradigms were tested while the elec- tromyographic signals were acquired. Three muscles that were innervated by the sciatic nerve were monitored throughout the procedure. The compound muscle action potentials (CMAPs) from the Plantar Interossei (PL), Gastrocnemius (GM) and Tibialis anterior (TA) muscles were recorded using pairs of EMG needle electrodes that were directly inserted in each muscle. 2.2.2. TIME Implants The TIME implants were developed and designed by the Laboratory for Biomedi- cal Microtechnology (Department of Microsystems Engineering—IMTEK, University of Freiburg, Freiburg im Breisgau, Germany) [23]. The implants consist of an L-shaped thin-film part that is connected, via the microflex interconnection technique (MFI) [46], to a screen-printed ceramic. Opposite to the MFI, a custom made 10 cm long cable was soldered. The cable consists of 16 helically wound MP35N wires which are insulated by polyesterimide (PEI). The wires are protected by a medical grade silicone rubber tubing (Helix Medical Europe GmbH, NuSIL MED-4750, Kaiserslautern, Germany), which is also contains silicone rubber. The cable terminates in a 16 channel nano neuroconnector by Omnetics (NCP-16-DD, Omnetics Connector Corporation, Minneapolis, MN, USA) for the electrical connection with the stimulation device. The exposed parts that will be implanted are additionally covered with medical grade silicone rubber (MED 1000, NuSIL Technology LLC, Carpintaria, CA, USA). The thin-film element uses polyimide as the substrate and insulation material. It incorporated 16 active sites, including 14 independent stimulation sites of 80 µm in diameter and 2 ground sites (GND) of 109 hexagonal arranged contacts (80 µm in diameter; total area of about 0.55 mm2 ) which are connected amongst themselves under the polyimide. The polyimide is fabricated through standard lithographic processes in a class 5 cleanroom. Platinum is used as the conducting material for the tracks. To ensure good adhesion to the polyimide, silicon carbide is used as an adhesion promoter. In order to increase the charge injection, a highly porous sputtered iridium oxide film is used on the active sites. The openings are obtained via reactive ion etching. Sensors 2021, 21, 7219 6 of 17 Sensors 2021, 21, 7219 6 of 18 Itsdesign Its design(version (versionTIME-4H) TIME-4H)isissymmetric, symmetric,so sothat thatafter afterthe thepeeling peelingof ofthe thethin-film thin-film electrodefrom electrode froma asilicon silicon wafer wafer (used (used forfor fabrication), fabrication), it canit can be folded be folded in theinassembly the assembly pro- procedure cedure fromfrom a U ashape U shape to antoLan L shape shape (similar (similar to previous to previous publications publications for TIME-3H for the the TIME- 3H [47]). [47]). ThisThis allows allows for the for the integration integration of aofsurgical a surgical needle needle witha asuture with suturesling sling(Prolene, (Prolene, EH7900G, Johnson & Johnson Medical Co., New Brunswick, EH7900G, Johnson & Johnson Medical Co., New Brunswick, NJ, USA), which facilitates NJ, USA), which facilitates thetransverse the transverseimplantation implantation through through thethe targeted targeted nerve. nerve. Once Once folded, folded, the contacts the contacts are are dis- distributed tributed across across twotwo compartments compartments positioned positioned onon each each side side ofofthe theelectrode electrode(7(7stimulation stimulation sitesand sites and11ground). ground).For Forthethe purpose purpose of of implantation, implantation, thethe stimulation stimulation sites—separated sites—separated by by a pitch of 0.75 mm—are inserted transversally within the nerve a pitch of 0.75 mm—are inserted transversally within the nerve while the grounds are while the grounds are disposed distally from the implantation site in parallel—but still in disposed distally from the implantation site in parallel—but still in close contact—to the close contact—to the nervesurface. nerve surface.The Thepitch pitchofofthe theleft leftand andright rightactive activesite siteisisdisplaced, displaced,relative relativetotoone oneanother, another, to half the pitch (0.375 mm) to facilitate multi-fascicles stimulation to half the pitch (0.375 mm) to facilitate multi-fascicles stimulation (Figure 2a). (Figure 2a). Figure 2. Transverse Intrafascicular Multichannel Electrode (TIME) and implantation procedure. (a) Photograph of the Figure TIME-4H2. Transverse Intrafascicular implant. Each Multichannel TIME consists Electrode of 14 independent (TIME) contacts stimulation and implantation procedure. and 2 ground sites. (b)(a) PhotographofofTIME Implantation the TIME-4H implant. Each TIME consists of 14 independent stimulation contacts and 2 ground devices into the sciatic nerve proximal to its trifurcation at the knee especially for protocol 2. sites. (b) Implantation of TIME devices into the sciatic nerve proximal to its trifurcation at the knee especially for protocol 2. 2.2.3. Stimulation Paradigms 2.2.3. Stimulation Paradigms Prior to the animal experiments, we designed three original protocols to ensure both Priorcompliance STIMEP to the animal experiments, with prerequisitewe designed technical three and original safety protocolsand specifications to ensure to assessbothad- STIMEP compliance with prerequisite technical and safety specifications vanced TIME-dedicated PNS paradigms. First, we performed finely tuned current intensity and to assess ad- vanced scanning TIME-dedicated PNS paradigms. with different pulse widths using First, we performed contacts in a singlefinely tuned fascicle. Wecurrent inten- automatically sity scanning with different pulse widths using contacts in a single generated the strength duration curve and used these data to identify the most energyfascicle. We automat- ically generated efficient the strength configurations duration around curve and the chronaxie. Inused these data the second step,towe identify implantedthe most en- two elec- ergy efficient configurations around the chronaxie. In the second step, we trodes within the same sciatic nerve along different axis and ensured the STIMEP ability to implanted two electrodes drive severalwithin DSUsthesimultaneously. same sciatic nerveWealong different further axis and capitalized ensured on this the STIMEP opportunity abil- to explore ity to drive several DSUs simultaneously. We further capitalized on the potential of a multi-TIME device approach and aimed to improve spatial selectivity this opportunity to explore the potential of a multi-TIME device approach and aimed to improve by targeting different fascicles and/or portions of the nerve. Finally, we tested two dif- spatial se- lectivity by targeting ferent stimulation different patterns by fascicles reversingand/or portions the polarity of of thethe nerve.symmetric original Finally, webiphasic tested two different pulse (bipolar stimulation patterns byofreversing configurations—one the two the polarityapplied references of the original symmetric in association withbi- an phasic pulse (bipolar configurations—one of the two references applied active contact to enable current flow)—i.e., the original stimulation with a depolarizing in association with anfollowed pulse, active contact by an to enable current interpulse of 100 µsflow)—i.e., [48] and athe original stimulation repolarizing phase, while withthea second depo- larizing pulse, followed by an interpulse of 100 µs [48] and a repolarizing pattern was constituted by a hyperpolarizing pulse followed by an interpulse (100 µs) and phase, while the second pattern was a depolarizing constituted by a hyperpolarizing pulse followed by an interpulse (100 phase. µs) and a depolarizing phase. • Protocol 1: Scanning with intensity for different pulse widths using a single TIME implant In the first rat, a single TIME was implanted in the sciatic nerve (TIME#1). The impact of the stimulation parameters on stimulation selectivity in one fascicle, i.e., the Sensors 2021, 21, 7219 7 of 18 • Protocol 1: Scanning with intensity for different pulse widths using a single TIME Sensors 2021, 21, 7219 7 of 17 implant In the first rat, a single TIME was implanted in the sciatic nerve (TIME#1). The impact of the stimulation parameters on stimulation selectivity in one fascicle, i.e., the tibial fas- tibial cicle, fascicle, was evaluated was evaluated byseveral by testing testingscans several scans of intensity of intensity which which ranged ranged from 5 to from 300 µA5 to 300 µA in full scale and the pulse widths, which were 20, 50, 100, 200 and in full scale and the pulse widths, which were 20, 50, 100, 200 and 400 µs. The scanning 400 µs. The thatscanning that was was conducted conducted using decreasingusing decreasing current current increments—from increments—from 20 down 20 down to 2.5 µA—were to 2.5 µA—were parameterized to ensure STIMEP accuracy and also to parameterized to ensure STIMEP accuracy and also to identify the best stimulation identify the best pa- stimulation parameters for both stimulation selectivity and power consumption rameters for both stimulation selectivity and power consumption (Figure 3). (Figure 3). Figure3.3.Algorithm Figure Algorithm illustrating illustrating parameterization parameterization ofof SYNERGY SYNERGY software software to to implement implement Protocol Protocol 1. 1. Protocol 1 was designed to perform scanning in intensities with different pulse widths using Protocol 1 was designed to perform scanning in intensities with different pulse widths using a a TIME implanted in a single fascicle (TIME#1). This experiment was implemented to ensure STIMEP ability TIME implanted in a single fascicle (TIME#1). This experiment was implemented to ensure STIMEP to automatically deliver finely tuned stimulations but also to assess the relative impact of both in- ability to automatically deliver finely tuned stimulations but also to assess the relative impact of both tensity and pulse width in intrafascicular stimulation selectivity. intensity and pulse width in intrafascicular stimulation selectivity. • Protocol 2: Stimulation using two DSU simultaneously to drive two TIME implants • Protocol 2: Stimulation using two DSU simultaneously to drive two TIME implants Two TIMEs were implanted in the sciatic nerve of the second rat. One implant Two TIMEs were implanted in the sciatic nerve of the second rat. One implant (TIME#2) was connected to STIMEP′s0 first Distributed Stimulation Unit (DSU #1) and dis- (TIME#2) was connected to STIMEP s first Distributed Stimulation Unit (DSU #1) and tally inserted while the second one (TIME#3) was implanted more proximally and was distally inserted while the second one (TIME#3) was implanted more proximally and was connected to the fourth DSU (DSU #4—Figure 2b). Two contacts per electrode (one on the connected to the fourth DSU (DSU #4—Figure 2b). Two contacts per electrode (one on the right and one on the left side) were placed in the nerve at the same time for a total of four implanted contacts. Sensors 2021, 21, 7219 8 of 17 Both the stimulator ability to drive multi TIME implants and the relative impact of multi-TIME stimulation approaches on selectivity were investigated by using appropriate parameters for the stimulation frequencies of the two DSU. An objective comparison of muscle activities when stimulating with a single or both TIMEs was made viable by doubling the frequency of one DSU relative to the other. This resulted in synchronized pulse delivery and alternations of stimulation pulses for either one or two of the TIME implants. Stimulations frequencies of 8 and 4 Hz were retained for multi-TIME DSU parameter- izing while scanning was performed with various intensities, from 60 to 300 µA (by steps of 60 µA) with 8 and 4 repetitions respectively, symmetrical biphasic pulses—to generate recruitment curves. Two sessions were performed successively to compare the results obtained with single a TIME stimulation—with TIME#2 or TIME#3 alternatively—to those achieved when using both the DSUs. Tests were first performed with the two contacts on the right side before switching to those on the left side (Supplementary Figure S1). • Protocol 3: Implementation of two stimulation waveforms The versatility of STIMEP was further investigated by assessing the impact of the stimulation waveform on selectivity. A new stimulation profile was programed using the SYNERGY platform and experiments performed in protocol 2 were reproduced using an inverted stimulation profile. In other words, the original symmetrical biphasic pattern was inverted to precede the second depolarizing phase by an initial hyperpolarizing phase (Supplementary Figure S2). 2.2.4. Signal Processing EMG signals were amplified (×1000 for PL, ×100 for GM and ×200 for TA—P511AC Amplifiers, Grass Instrument Co, West Warwick, RI, USA) and band-pass filtered (5 Hz to 20 kHz for high-pass and low-pass filtering respectively) before sampling—20 ksamples/s— and their acquisition using a PowerLab system (ADInstrument, Sydney, Australia). EMG signals were first synchronized to the STIMEP trigger output before the calculation of the Root Mean Square (RMS) for a 10 ms time limit beginning 1 ms after the stimulation using Labchart software (ADInstruments, Sydney, Australia). The RMS was defined for each acquisition channel—i.e., PL, GM and TA—and each stimulation pulse before averaging for each configuration using custom software written in Matlab (Mathworks, Natick, MA, USA). 3. Results 3.1. STIMEP Successfully Delivered Finely Tuned Stimulations That Potentiate Intrafascicular Stimulation Selectivity The first stimulation protocol (Figure 3) aimed both to ensure the ability of STIMEP to comply with a precise microstimulation and also to investigate the impact of both the stimulation intensity and the pulse width on selectivity when implanting one TIME in a single fascicle. Data obtained after scanning in intensities were processed to obtain the RMS values. These values were then combined to plot recruitment curves (Figure 4a) and chronaxie-rheobase-like curves (Figure 4b). We focused our analysis on intrafascicular selectivity and compared the recruitment profile of two muscles innervated by the tibial fascicle—i.e., the plantar interossei and the gastrocnemius muscles [24]. Shorter pulse widths of primarily 20 µs, but also of 50 and 100 µs, increased the range between the activation threshold and the maximal recruitment for both muscles, generating a larger range whereby the level of recruitment slowly increases as a function of stimulation intensity. The recruitment curves suggested a two stage recruitment profile for the plantar interossei muscle—especially for the left stimulation contacts and the short pulse widths (Figure 4a—PW = 20 and 50 µs)—as a plateau phase was apparent at the middle of the curve at 0.10 V RMS for a full scale activation of 0.34 V RMS. After comparing plantar interossei and gastrocnemius muscle recruitment plots for a 20 µs pulse width (Figure 4a—Left contact), an increase in the selectivity range was observed as the two-stage activation of the plantar interossei muscle potentiated the recruitment gap between both muscles (Plantar Sensors 2021, 21, 7219 9 of 17 interossei recruitment blocked at the plateau stage for intensities up to 300 µA—0.094 V RMS—while Gastrocnemius saturation was reached below 200 µA—0.87 V RMS). This was further confirmed by chronaxie curves presenting a very low activation of Plantar interossei (0.05 V RMS) and maximum recruitment of gastrocnemius muscle (0.8 V RMS) for medium intensities around 150 µA (Figure 4b). On the contrary, even small increments in intensity—down to 2.5 µA between two successive intensities—did not prove to lengthen the selectivity window between activation threshold and maximum recruitment when stimulating with larger pulse widths. This was highlighted by the relative superimposition Sensors 2021, 21, 7219 of recruitment curves for several intensity steps when stimulating with 50 and 100 9 ofµs18 (Figure 4a). Figure4.4.Recruitment Figure Recruitmentand andchronaxie chronaxiecurves curvesobtained obtainedafter afterperforming performingprotocol protocol1.1.(a) (a)Recruitment Recruitment curves of Plantar interossei and Gastrocnemius muscles after stimulating with either aacontact curves of Plantar interossei and Gastrocnemius muscles after stimulating with either contacton on the left or on the right side of the TIME#1 implant. Muscle activity is displayed as the RMS of the the left or on the right side of the TIME#1 implant. Muscle activity is displayed as the RMS of compound muscle action potential. (b) Chronaxie-rheobase-like curves plotted as a result of these the compound muscle action potential. (b) Chronaxie-rheobase-like curves plotted as a result of stimulations, presenting the relative recruitment of both Plantar Interossei and Gastrocnemius mus- these cles. stimulations, presenting the relative recruitment of both Plantar Interossei and Gastrocnemius muscles. Shorter pulse widths of primarily 20 µs, but also of 50 and 100 µs, increased the range between the activation threshold and the maximal recruitment for both muscles, generat- ing a larger range whereby the level of recruitment slowly increases as a function of stim- ulation intensity. The recruitment curves suggested a two stage recruitment profile for the plantar interossei muscle—especially for the left stimulation contacts and the short pulse widths (Figure 4a—PW = 20 and 50 µs)—as a plateau phase was apparent at the middle Sensors 2021, 21, 7219 10 of 17 3.2. STIMEP Successfully Drove Two TIME Implants Simultaneously and Increased Spatial Selectivity Stimulations tested after the implantation of two TIMEs displayed consistently dif- ferent responses, discarded the stimulation crosstalk between DSU and highlighted the Sensors 2021, 21, 7219 11 of 18 reliable functionality of the STIMEP device (Figure 5a,b). Figure 5. Raw signals recorded during implementation of the second stimulation protocol and corresponding recruitment Figure 5. Rawcurves. signals recorded during implementation of the second stimulation protocol and corresponding recruitment (a) Example of raw data obtained when stimulating with left contacts for a 300 µA intensity (i.e., DSU#1 = 4 Hz, curves. (a) Example DSU#4 =of rawI =data 8 Hz, 1, j = obtained when stimulating 2 and k = 4—configuration with based on left contacts Supplementary for S1). Figure a 300(b) µA intensity Close-up on the(i.e., three DSU #1 = 4 Hz, DSU muscles CMAPs #4 = 8 Hz, I = 1, obtained j = 2 and k = after stimulating with based 4—configuration either aon single (DSU#1) or two Figure Supplementary DSUs (DSU#1 andClose-up S1). (b) DSU#4)—These signals on the threecorre- muscles CMAPs spond to those marked with a star in (a). (c) Recruitment curves obtained for each muscle following the implementation obtained afterofstimulating the stimulationwith either protocol. Notea that single (DSU #1) RMS the “cumulated or two DSUs T#2T#3” (DSU curve #1 and to corresponds DSU #4)—These the calculation signals of the RMS correspond to those marked with a star in (a). (c) Recruitment curves obtained for each muscle following the implementation of the stimulation protocol. Note that the “cumulated RMS T#2T#3” curve corresponds to the calculation of the RMS theoretical value obtained by linear summation of the individual responses. Similarly, the presence of two curves corresponding to the stimulation with two DSU is linked to the protocol repetition after the reversal of the stimulation frequencies on both DSUs. Sensors 2021, 21, 7219 11 of 17 Relative positioning of the stimulation contact within the TIME implant proved crucial as the amplitude of the evoked CMAPs varied when stimulating with contacts located on the left or on the right side of the TIME implants—maximum responses over all muscles were consistently observed for the stimulation of the left contacts (Figure 5c). The raw EMG signals, evoked after the synchronous stimulation with two DSUs, revealed the relative cumulative effects as compared with the EMGs produced when stimulating with a single DSU (Figure 5a,b). The RMS values of the evoked EMGs were treated separately depending on their origin, i.e., a stimulation with one or two TIMEs and whether the stimulation was conducted using either the left or the right side contacts. For each muscle, these data were normalized by the maximum RMS value across all stimulation modalities. Recruitment curves were then plotted to characterize the effect of these stimulation modalities on muscle recruitment (Figure 5c). Using the RMS responses to single DSU stimulation (from both TIME#2 and TIME#3), a theoretical ceiling RMS value was calculated assuming the complete independence of the responses obtained following stimulation with these two TIME implants. This value was obtained by a linear summation of both TIME individual responses, by assuming the independence of the unitary responses (orange curve in Figure 5c, the Hypothesis of independence between responses evoked by stimulating with different single DSU). The use of two contacts from different TIME implants activated all the monitored muscles while only two muscles were activated by each implant independently—TIME#2 (DSU #1) recruited the Gatrocnemius and Tibialis anterior muscle while TIME#3 activated the Gastrocnemius and the Plantar interossei. This suggests the activation of different fiber populations and an increase in the options potential movements. The recruitment profiles obtained when stimulating with both TIMEs synchronously confirm the visual impressions from the raw EMGs by presenting the cumulative effects of the individual responses (Figure 5c). Indeed, two tailed paired t-tests across all investigated intensities failed to display any significant differences between ceiling RMS values calculated after the linear summation of individual responses and the actual data for all the muscles and every investigated contact (t4 = −1.88, P = 0.13; t4 = 0.69, P = 0.53; t4 = 1.22, P = 0.29 when stimulating with left contact and t4 = 0.68, P = 0.53; t4 = 0.16, P = 0.88; t4 = 1.99, P = 0.11 when stimulating with right contact for Plantar interossei, Gastrocnemius and Tibialis anterior muscles respectively). 3.3. STIMEP Generated Complex Waveforms and Underlines the Relative Impact of Polarity on Stimulation Selectivity Using TIME Implants The versatility of the STIMEP device was further investigated by implementing an- other complex stimulation waveform. This inverted biphasic profile—i.e., depolarizing pulse preceded by a hyperpolarizing phase (protocol 3—inverted polarity relative to the original biphasic stimulation profile) was also used to investigate the relative influence of the stimulation pattern on the recruitment of the monitored muscles. None of the remaining parameters (frequency, intensity and pulse width) were altered from those of the previous protocol (protocol 2). RMS values were normalized using the same maximum RMS value predefined in subsection b., and, for each muscle, recruitment curves comparing results of both protocols are compiled in Figure 6. Recruitment curves showed a saturation for the higher RMS values when stimulating with a hyperpolarizing pre-pulse preceding the depolarizing pulse. The increase is due to inverted polarity when stimulating with TIME#2 (DSU #1) implant, and proved to be of an even higher magnitude than when stimulating with two TIMEs simultaneously with the original pattern for the Gastrocnemius and Tibialis anterior muscles (one tailed paired t-test across all intensities, t4 = 2.74, P = 0.03 and t4 = 3.92, P = 0.01 respectively for left contact and t4 = 2.26, P = 0.04; t4 = 2.175, P = 0.049 for right contact). In the same way, the recruitments of the Plantar interossei and the Gastrocnemius proved significantly higher when stimulating with DSU #4 using the right contact of TIME#3 (paired t-test across all intensities, t4 = 3.34, P = 0.014 and t4 = 2.6, P = 0.03 respectively) although the paired t-test failed to reveal any statistical potentiation when stimulating with the left contact 3.3. STIMEP Generated Complex Waveforms and Underlines the Relative Impact of Polarity on Stimulation Selectivity Using TIME Implants The versatility of the STIMEP device was further investigated by implementing an- Sensors 2021, 21, 7219 other complex stimulation waveform. This inverted biphasic profile—i.e., depolarizing 12 of 17 pulse preceded by a hyperpolarizing phase (protocol 3—inverted polarity relative to the original biphasic stimulation profile) was also used to investigate the relative influence of the stimulation pattern on the recruitment of the monitored muscles. None of the remain- (t4 =parameters ing 1.45, P = 0.11 (frequency, −1.15, Pand and t4 =intensity = 0.84 when pulse comparing width) with from were altered stimulation those ofwith two the pre- TIMEs and t = 1.47, P = 0.11 and t = − 0.55, P = 0.7 respectively after comparison vious protocol (protocol 2). RMS values were normalized using the same maximum RMS 4 4 with the original pattern delivered by DSU #4 alone). This further highlighted more value predefined in subsection b., and, for each muscle, recruitment curves comparing pronounced improvements results after stimulating of both protocols with contacts are compiled in Figurelocated 6. on the right side of the electrode. Figure 6. Figure 6. Recruitment Recruitmentcurves obtained curves after obtained stimulating after withwith stimulating two distinct stimulation two distinct patterns. stimulation In this figure, patterns. In thisboth “Pattern figure, both 2” curves were obtained after performing the third protocol. Note that the little horizontal bold line indicates the maximum “Pattern 2” curves were obtained after performing the third protocol. Note that the little horizontal bold line indicates the RMS value used to normalize the data (corresponds to y = 1). maximum RMS value used to normalize the data (corresponds to y = 1). Recruitment curves showed a saturation for the higher RMS values when stimulating 4. Discussion with a hyperpolarizing pre-pulse preceding the depolarizing pulse. The increase is due to The implemented protocols included the fine modulation of pulse width and intensity inverted polarity when stimulating with TIME#2 (DSU#1) implant, and proved to be of an starting at 20 µs or 5 µA which gradual decreased to 5 µs and 2.5 µA respectively, the even higher magnitude than when stimulating with two TIMEs simultaneously with the synchronized steering of two TIMEs implants and the inversion of biphasic pulses polarity original pattern for the Gastrocnemius and Tibialis anterior muscles (one tailed paired t- revealing a fine gradual modulation of both the selectivity and the muscle recruitment with test across all intensities, t4 = 2.74, P = 0.03 and t4 = 3.92, P = 0.01 respectively for left contact intrafascicular TIME implants. and tThe 4 = 2.26, P = 0.04; t4 = 2.175, P = 0.049 for right contact). In the same way, the impact of the stimulation parameters was evaluated by performing multiple intensity series for several pulse widths in a single fascicle. Interestingly, the factor that mostly impacted selectivity was the reduction of the pulse length. For a pulse of 20 µs, a downturn of the recruitment curve was observed, enabling the finer modulation of muscular activation. In our experiments, the use of a short pulse widths efficiently widens the activation gap between plantar interossei and gastrocnemius muscles by slowing down the recruitment of the former. Although the stimulation intensity did not seem to significantly influence stimulation selectivity alone, the combination of short pulses with a low increment seems the best approach to optimize selectivity. The implantation of two TIME devices enabled the individualization of two distinct patterns of response with one mobilizing both the plantar interossei and gastrocnemius Sensors 2021, 21, 7219 13 of 17 muscles while the other recruited the tibialis anterior and the gastrocnemius. A further comparison of experimental data derived from the synchronized use of the two TIME im- plants with the linear summation of their unitary activities revealed no marked differences, which led to the omittance of the occurrence of nonlinear phenomenon. We thus produced an increase in the stimulation selectivity by successfully activating several independent pools of nerve fibers located in different areas of the sciatic nerve. These results proved reliable after stimulating with both the left and the right side of the TIMEs, although a clear reduction in amplitude was observed when stimulating with the right contacts. To summarise, the implantation of several TIMEs allowed for an increase in the spatial selectivity of motor fibers recruitment. Lastly, the use of inverted polarity, including a hyperpolarizing phase before the depolarizing pulse, resulted in a potentiation of both plantar interossei and tibialis anterior recruitment without a modification of the selectivity profile—which was evident for the case the left side contacts with an activation of Pl and GM muscles for TIME#2 and of the TA and the GM for TIME#3. To provide an overview, both the added hyperpolarizing pre- pulse and the use of small pulse widths enhanced the selectivity of nerve fibers recruitment when stimulating with the TIME implants. As previously mentioned, these results further highlight the necessity for power- ful stimulation tools that allow for the implementation of complex stimulation patterns as well as an appropriate adjustment of the stimulation parameters on multiple chan- nels/electrodes. The STIMEP platform presented in this paper meets these requirements and should ultimately allow for further refined parameters for selective stimulation. In many ways, the results presented in this paper are consistent with previous studies modeling the peripheral nerve responses to stimulation. The effects of an initial hyper- polarizing phase and of small pulse widths had already been assessed using extraneu- ral cuffs [14,15] and longitudinal intraneural electrodes (only two pulse widths were investigated—i.e., 20 and 50 µs [19]) but without the inclusion of transfascicular implants. Extrapolating from our results, we suggest that similar stimulation properties should be used for both intraneural (longitudinal and transversal) and extraneural electrodes. In the context of selective peripheral nerve stimulations, the results suggest the use of biphasic pulses— first charge balanced with a hyperpolarizing phase—with small pulse widths (about 20 µs) and an interphase delay of 100 µs between the two active phases [49]. Our selectivity study constitutes only a fragmented approach that is insufficient to investigate the peak potential of TIME electrodes for the selective stimulation of the periph- eral nerve—approaches such as “depolarizing prepulses” [32] and “rising ramps” [33] have yet to be tested. Further, the STIMEP limitation in frequency prevents the implementation of “high frequency block” protocols [34] (Table 1) while an “anodal block” procedure is not appropriate with TIME implants as both anodes and cathodes are required to be located along the nerve fibers [31]. The implementation of symmetrical biphasic stimulation patterns may have limited data interpretation as either phase could have been responsible for muscle recruitment. The use of a small interpulse delay—100 µs as advised by Mortimer 2000 [48] and further recommended by Maciejasz et al. [49]—combined with a 20 kHz sampling frequency further precluded the analyses of the latency that could have assisted in identifying the role of hyperpolarizing prepulses as implemented in pattern#2. However, the use of symmetric charge-balanced pulses was considered to be reasonable so as to avoid nerve damage during peripheral nerve stimulation and to also maximize the range of frequency that could be delivered using STIMEP. Only two contacts for each TIME were implanted within the rat sciatic nerve at the same time, underlining the need for further experiments in a larger animal model or for the resizing of the implant. Indeed, an increase in the number of contacts implanted transversely within the nerve favor an increase in selectivity, as previously shown in Badia et al. [24]. Furthermore, the lack of a histological study prevents the bridging of electrophysiology and anatomy. Sensors 2021, 21, 7219 14 of 17 Nevertheless, the rat model proved adequate to obtain a proof of concept and safety assessment but also to ensure the fulfillment of the specifications that supported the validity of the hypothesis that was tested in the aforementioned protocols; especially since the use of the final design of both the STIMEP and TIME implants allowed for the validation of the stimulation set-up prior to the implementation of a clinical trial in amputees [50–55]. During this trial, stimulating with TIME electrodes improved the richness and accuracy of the evoked sensations compared to those elicited via multicontact cuff electrodes [14,15] in upper limb amputees. Patients were thus able to “feel” the object and greatly enhance their manipulation abilities (Figure 7a) [50–53]. In the same way, the overall system—STIMEP and TIME implants—was successfully used in real-time to modulate neural stimulation in lower limb amputees when walking. In practice, these patients were able to “feel” both the ground and the knee angle of their prosthetic device which greatly improved their walking performances (Figure 7b) [54,55]. Moreover, in both cases, phantom pain significantly Sensors 2021, 21, 7219 decreased throughout the trial. Nevertheless, these studies failed to fully investigate 15 of 18 the potential of STIMEP for functional recovery. Figure7.7. Clinical Figure Clinicaltrials trialsperformed performedusing usingthe theSTIMEP STIMEPNeurostimulator. Neurostimulator. (a)(a) Implantation Implantation of of TIME TIME electrodes electrodes transversally transversally in in upper limb nerves and stimulation with STIMEP device to restore rich sensations in amputees [50–53]. (b) Combined upper limb nerves and stimulation with STIMEP device to restore rich sensations in amputees [50–53]. (b) Combined use of use of TIME implants and STIMEP to optimize walking performances in lower-limb amputees [54,55]. (c) Restoration of TIME implants and STIMEP to optimize walking performances in lower-limb amputees [54,55]. (c) Restoration of upper- upper-limb movements in individuals with spinal cord injury using multicontact cuff electrodes and STIMEP neurostim- limb movements in individuals with spinal cord injury using multicontact cuff electrodes and STIMEP neurostimulator ulator (ongoing clinical trial—manuscript in preparation). (ongoing clinical trial—manuscript in preparation). The same core stimulation system was also used with complex multicontact cuff elec- The same core stimulation system was also used with complex multicontact cuff trodes [56,57] that allow for the selective stimulation of the upper limb muscles responsi- electrodes [56,57] that allow for the selective stimulation of the upper limb muscles re- ble for hand and wrist movements through radial and median nerve stimulations in indi- sponsible for hand and wrist movements through radial and median nerve stimulations in viduals with a spinal cord injury. STIMEP is currently being used in a second clinical trial individuals with a spinal cord injury. STIMEP is currently being used in a second clinical that is ongoing (clinical trial identifier: NCT04306328—manuscript in preparation, Figure trial that is ongoing (clinical trial identifier: NCT04306328—manuscript in preparation, 7c), following Figure these two 7c), following preliminary these experiments. two preliminary experiments. The results of this study support The results of this study support the the versatility versatility of of this this new new stimulation stimulation platform. platform. STIMEP and its associated software, SYNERGY, enabled the automated STIMEP and its associated software, SYNERGY, enabled the automated delivery of complex delivery of com- plex sequences sequences of stimulation, of stimulation, the tuning the fine fine tuning of parameters of parameters (i.e., intensity, (i.e., intensity, pulsepulse widthwidth and frequency), the steering of up to four stimulation units independently—for aatotal and frequency), the steering of up to four stimulation units independently—for totalof of 56 stimulation channels—and the programming of multiple pulse 56 stimulation channels—and the programming of multiple pulse waveforms. Alongside waveforms. Alongside these intrinsic these intrinsicmechanisms, mechanisms,the thedimensions dimensionsofofSTIMEP STIMEPmakemake it it anan ideal ideal portable portable stimula- stimulator tor for both clinical trials and ambulatory for both clinical trials and ambulatory protocols. protocols. 5. Conclusions 5. In this paper, In paper, we weintroduce introducenewnewstimulation stimulationtools and tools present and several present strategies several de- strategies signed to optimize designed the the to optimize use use of TIME implants of TIME in neural implants stimulation. in neural By implanting stimulation. several By implanting several electrodes electrodes in thenerve in the same sameand nerve and tuning tuning the stimulation the stimulation parameters, parameters, we wereweable weretoable val- idate the overall STIMEP platform and to also identify several axes of improvement and to confirm predictions from the computational studies. Among these results, the use of short pulses and the introduction of a hyperpolarizing pre-pulse seem to be of particular interest for selectivity optimization. In the same way, the implantation of several elec- trodes increases the size of the stimulated area and greatly enhances the overall potential Sensors 2021, 21, 7219 15 of 17 to validate the overall STIMEP platform and to also identify several axes of improvement and to confirm predictions from the computational studies. Among these results, the use of short pulses and the introduction of a hyperpolarizing pre-pulse seem to be of particular interest for selectivity optimization. In the same way, the implantation of several electrodes increases the size of the stimulated area and greatly enhances the overall potential of such an approach. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/s21217219/s21217219/s1, Figure S1: Algorithm illustrating parameterization of SYNERGY software to implement Protocol 2. Protocol 2 was designed to perform stimulation using two DSU simultaneously (DSU #1 and DSU #4) in order to drive two TIME implanted in different fascicles (TIME#2 and TIME#3). 5 stimulation intensities were tested—from 60 to 300 µA—with constant pulse width (20 µs) and multiple repetitions (either 4 or 8 repetitions for the 4 Hz or the 8 Hz stimulation). This experimental session aimed to assess STIMEP ability to deliver synchronized stimulation on different DSU but also to highlight the potential of a multi-TIME approach for rehabilitation purposes. Figure S2: Algorithm corresponding to Protocol 3. 15 stimulation intensities were tested—from 20 to 300 µA—with constant pulse width (20 µs) and frequency (5 Hz). This third protocol was designed to investigate the impact of stimulation pattern on selectivity by reversing pulses polarity—experiments were performed using the same experimental set-up as protocol 2—i.e., using two TIMEs (TIME#2 and TIME#30 connected to DSU #1 and DSU #4 respectively. Author Contributions: Conceptualization, T.G., A.H., D.A., T.S., X.N. and D.G.; methodology, T.G., V.M.L.-Á., P.Č., A.H. and D.G.; software, T.G., A.H. and D.A.; validation, T.S., X.N. and D.G.; formal analysis, T.G. and D.G.; investigation, T.G., V.M.L.-Á., A.H. and X.N.; resources, D.A., T.S., X.N. and D.G.; data curation, T.G. and D.G.; writing—original draft preparation, T.G.; writing—review and editing, T.S., X.N. and D.G.; visualization, T.G.; supervision, D.A., T.S., X.N. and D.G.; project administration, D.A., T.S., X.N. and D.G.; funding acquisition, T.S., X.N. and D.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the EPIONE grant. EU Grant FP7-HEALTH EPIONE (Natural sensory feedback for phantom limb pain modulation and therapy) # 602547. Institutional Review Board Statement: All experimental procedures performed were approved by the Ethical Committee of the Universitat Autonoma de Barcelona in accordance with the European Communities Council Directive 2010/63/EU, and in compliance with the NIH Guide for Care and Use of Laboratory Animals. Informed Consent Statement: This study is not involving humans. The last figure is only illustrating the potential use of STIMEP stimulator. Data Availability Statement: Data sharing is not applicable to this article. Conflicts of Interest: D.A. and D.G. are shareholders of NEURINNOV, but the company wasn0 t yet created at the time of the study thus “The authors declare no conflict of interest”. References 1. Wyndaele, M.; Wyndaele, J.J. 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