The participant was a 39-year old male who suffered a diffuse TBI in the frontal lobe region resulting from a motor vehicle accident. The accident occurred 7 years prior to start of the study. At the onset of the study, the participant was diagnosed by his medical team with symptoms of mild motor impairment affecting the right upper- and lower-limbs and higher brain dysfunction, which were the results of the TBI (see Supplementary Materials: Participant history). The participant was enrolled in the study aiming to improve upper-limb function using FEST. The participant was informed about the study objectives and signed a written informed consent in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the local institutional research ethics committee at the University of Tokyo.
Functional electrical stimulation was delivered using the Compex Motion system (Compex, Switzerland). Electrical stimulation was used to activate the muscles by applying a rectangular, biphasic, and asymmetric charge balanced stimulation pulses at a frequency of 40 Hz and 300 mus pulse width. Electrical stimulation was applied on the muscles using surface electrodes (5 x 5 cm square electrodes on larger muscles and 2 cm diameter circular electrodes on smaller muscles). During each FEST session, the therapist determined the stimulation levels for each muscle by gradually increasing the FES amplitude in 1 mA increments until they identified palpable contractions. The stimulation amplitude was then set to 150% of the amplitude that evoked palpable contractions, and adjusted if necessary, to produce smooth muscle contractions (for average amplitudes, see Supplementary Materials: FES).
The FEST training protocol is summarized in Figure 1. Training was delivered over the course of 3-months (12-weeks), with 3 sessions per week, each lasting 45-60 min (Figure 1A). Each FEST session consisted of three functional training protocols, consistent to previous FEST protocols ( and), which are illustrated in Figure 1B (see Supplementary Materials: FES). In each protocol, participant performed a specific functional task, including grasping a water bottle (palmar grasp), bringing an object to his mouth (hand-mouth), and pointing toward a target (pointing forward). For each trial, the participant was first asked to attempt to perform the task, while the therapist triggered a pre-programmed FES sequence to assist voluntary efforts.
Timeline of assessments is summarized in Figure 1A. Assessments were carried out to evaluate cortical and corticospinal circuits associated with upper-limbs as well as upper-limb functional performance and clinical scores. Short-term cortical changes were assessed once per week over the course of 12-weeks of training immediately before and after each FEST session using transcranial magnetic stimulation (TMS). Long-term assessments were carried out every 6-weeks over the course of the 12-weeks of FEST and during the 12-weeks follow-up after the intervention was complete. Specifically, long-term changes were assessed before the training at baseline (Pre), after 6-weeks of the training (During), and immediately after 12-weeks of FEST (Post0), as well as 6-weeks after FEST was completed (Post1), and 12-weeks after FEST was completed (Post2). Long-term cortical changes and corticospinal excitability were evaluated using TMS and functional magnetic resonance imaging (fMRI), while functional performance was assessed using an instrumented drawing test and clinical scores.
Transcranial magnetic stimulation sessions were carried out during both short-term and long-term assessments. During the assessments, participant remained seated comfortably on the chair with the right hand supported on the table. Electromyographic (EMG) activities were recorded using bipolar Ag/AgCl surface electrodes (Vitrode F-150S, Nihon Kohden, Tokyo, Japan) from the right (intervention) hand: (i) first dorsal interosseous (FDI) and (ii) abductor pollicis brevis (APB) muscles. A ground electrode was placed on the elbow of the right arm. It was ensured that the EMG electrodes were placed roughly on the same locations of the muscle between assessment days. EMG signals were band-pass filtered (15-1,000 Hz), amplified (1,000x; MEG-6108, Nihon Kohden, Tokyo, Japan) and sampled at 4,000 Hz using an analog-to-digital converter (Powerlab/16SP, AD Instruments, Castle Hill, Australia).
Using a mono-phasic magnetic stimulator (Magstim 200, Magstim Co., Whitland, United Kingdom) through a figure-of-eight coil, single-pulse TMS was delivered over the left primary motor cortex (M1) area that was optimal for inducing motor evoked potentials (MEPs) in the right FDI. The "hot spot" location was determined by detecting the point with the highest MEPs from the FDI (target) muscle and defined with respect to cranial landmarks as references during the baseline assessment (Pre). The same "hot spot" location was used to center the grid for all TMS map assessments (Pre, During, Post0, Post1, and Post2), while the exact location was confirmed on each day for single-location MEP assessments. The MEPs were always evoked with the participant keeping voluntary contraction at 10% maximal voluntary contraction (MVC) effort of the FDI muscle during the finger pinch task since there were no visible MEP responses at rest during baseline assessments (Pre). Contractions were maintained by holding a force sensor (OKLU-100K-S1-H18, Frontier Medic, Hokkaido, Japan) with his right thumb and index fingers, while the force level was shown on a visual display. The motor threshold (MT) for evoking MEPs was the minimum TMS intensity to elicit peak-to-peak amplitudes of at least 50 muV from the FDI muscle in five of ten consecutive trials. It was ensured that the MEPs of the APB muscle could also be evoked and recorded simultaneously.
During short-term and long-term assessments, the input-output relationship between TMS stimulation intensity and MEP responses amplitude was obtained by applying TMS at 60, 70, 80, 90, and 100% of the TMS stimulator intensity. The exact "hot spot" location was confirmed on each assessment day with the starting point as the location defined during the baseline (Pre) assessment. Three trials were performed at each TMS intensity and the responses obtained for each muscle (FDI and APB) at each intensity. Since MEPs were recorded during active contractions at 10% MVC, it was also possible to record the cortical silent period (CSP) of the MEPs from the same trials. Three CSP trials were also calculated from the responses evoked at 70% of the stimulator output. Post processing evaluation revealed that it was not possible to elicit clear APB (non-target muscle) CSP response during the Pre assessment, resulting in removal of data from long-term assessment analysis. Moreover, APB response during the short-term assessment day 11 were unclear, also resulting in removal of CSP and input-output data for that assessment day.
During long-term assessments, MEP maps of corticospinal responses of each muscle were recorded by applying TMS at 70% of the stimulation output, which was determined to be the 120% MT stimulation intensity during the baseline (Pre) assessment and remained unchanged. During each assessment, the participant was asked to keep voluntary contractions at 10% of MVC of the FDI muscle. The MEP map was centered at the FDI "hot spot" location, which was defined with respect to cranial landmark during the baseline (Pre) assessment and remained unchanged. The MEP map was then expanded to the surrounding points on the 10 x 10 cm grid with a 1 cm resolution (100 cm2 area) around the "hot spot" location using pre-determined markings on a tight-fitting cap. Three stimuli were delivered at each location in a semi-randomized order at a rate of approximately every 6 s and averaged to obtain a peak-to-peak amplitude response for each location.
During fMRI sessions, which were carried out during long-term assessments, the participant remained in the supine position in an MRI scanner (MAGNETOM Prisma, Siemens, Germany) and was asked to perform: (i) hand grip and (ii) finger pinch force matching tasks with the right (intervention) hand, while holding a force sensor (OKLU-100K-S1-H18, Frontier Medic, Hokkaido, Japan). The force matching tasks was a trapezoidal pursuit consisting of four phases: rest, ascending, keep, and descending, each lasting 10 s. The target force level (keep phase) was set to 20% of the MVC effort, while the ascending and descending phase linearly increased and decreased to the target force over the course of 10 s. The participant could see the target force on the visual display, which they attempted to match during the experimental trials. A total of four force matching tasks were repeated within each session with a rest period of 20 s between tasks. One hand grip task session and one finger pinch task session were performed on each assessment day, which were conducted in separate scans. The MVC levels were determined prior to the experiment for the hand grip and finger pinch tasks. During fMRI assessments, the participant was asked to follow the target force trajectories as precisely as possible. All MRI images were acquired using a 3T MRI scanner (MAGNETOM Prisma, Siemens, Germany). Functional T2*-weighted echo-planar images that reflect blood oxygenation level-dependent (BOLD) responses as well as high-resolution T1-weighted structural images were collected (see Supplementary Materials: fMRI data acquisition).
To evaluate upper-limb fine motor function, which was carried out during long-term assessments, the participant was asked to perform: (i) tracing and (ii) target tracking tasks of a sine wave (wavelength: 50 mm, amplitude: 25 mm, and distance: 150 mm) using an instrumented tablet system (TraceCoder  Version 1.0.8, Surface Pro4, SystemNetwork, Osaka, Japan). During the assessments, the participant was comfortably seated in a chair with his elbow on the table and flexed at 90 . During the tracing task, the participant was instructed to follow the outline of a sine wave at his preferred speed without a moving target, while during the target tracking task, the participant was instructed to follow the moving target on the tablet screen which moved on a sine wave at 12 mm/s. For both tasks, the participant was asked to draw as precisely as possible. Two trials, each consisting of three sine waves, were recorded for each of the tracing and tacking tasks. Before each assessment day, a practice period of approximately 1 min was allowed to prevent any learning effects and to allow the participant to assume a comfortable position for the assessments.
Clinical scores, which were evaluated during long-term assessments, included functional independence measure (FIM), Fugl-Meyer assessment (FMA), and Motor Activity Log (MAL). All tests were performed by the same trained therapist.
All MEP analysis was performed using a custom program written in MATLAB (The MathWorks Inc., United States). To evaluate the input-output curve relationship between the TMS stimulation intensity and the MEP responses for the FDI and APB muscles, MEP peak-to-peak amplitudes of each muscle for each of the three repeated trials at each stimulation intensity (60, 70, 80, 90, and 100% of the TMS stimulator output) were first calculated. The MEP amplitudes were plotted relative to the TMS stimulation intensity and a linear fit line was obtained using simple linear regression. The slope of the linear regression line was used to define the three repeated trial gain parameters of the input-output relationship curve.
The CSP duration was defined for each muscle for three repeated trials as the time between the end of the MEP (i.e., where EMG activity was below 3SD of mean pre-stimulus activity) and the time at which the post-stimulus EMG returned to the pre-stimulus EMG activity (i.e., where EMG activity exceeded 3SD of the mean pre-stimulus activity).
Corticospinal representation MEP maps were calculated from the MEP peak-to-peak amplitudes of each point on the 100 cm2 area (10 x 10 cm map with 1 cm resolution). The three repeated trials for each point were first averaged and normalized with the peak MEP amplitude on the map for each assessment day. The MEP map was then constructed from the average MEP amplitudes from each point on 10 x 10 cm grid using MATLAB's "gridfit" function to define 2,500 partitions within 100 cm2 area. Finally, activated area on the 100 cm2 map was calculated by taking the ratio of the number of partitions where the approximated MEP exceeded 50% of maximum MEP (aMEP50%) relative to all partitions (Ntotal = 2,500): , where areamap is 100 cm2.
All fMRI data analysis was performed using Statistical Parametric Mapping (SPM12, Wellcome Trust Center for Neuroimaging, London, United Kingdom) software implemented in MATLAB (The MathWorks Inc., United States). First, data preprocessing procedures were applied (see Supplementary Materials: fMRI data processing). If the head motion remained over 2 mm, the scans would be considered for removal from subsequent analysis. However, the participant's head motion always remained within 2 mm during all scan, thus no trials were removed. After the preprocessing, the general linear model regression to the time course data was obtained to estimate the amount of neural activation. Whole brain analysis was performed to depict the general features of brain activations during the hand grip and finger pinch tasks. First, the brain regions where the BOLD signals increased during the hand grip and finger pinch were depicted by evaluating the T-values obtained from each session to contrast a task specific voxel by voxel activation map. The threshold was set at voxel level p < 0.001 (uncorrected) and cluster level p < 0.050 family-wise error correction (FWE).
Next, the region of interest (ROI) was set in six anatomical hand areas defined bilaterally: primary motor cortex (M1: x = +- 37, y = -21, and z = 58), sensory cortex (S1: x = +- 40, y = -24, and z = 50), secondary somatosensory cortex (S2: x = +- 58, y = -27, and z = 30), parietal rostroventral area (PR: x = +- 54, y = -13, and z = 19), supplementary motor area (SMA: x = +- 20, y = -8, and z = 64), premotor cortex (PM: x = +- 8, y = -6, and z = 64). These ROI regions were chosen based on the previous studies that investigated cortical effects of FES and implemented as 10 mm diameter spheres centered around each defined coordinate. In addition, the most activated voxel (peak voxel) in the contralateral M1 region was calculated to define the most active ROI location. A control region was defined as the hippocampus gyrus (HC: x = -22, y = -34, and z = -8 for contralateral and x = 32, y = -30, and z = -8 for ipsilateral), which was not associated with hand movements. Significant activation maps during both finger pinch and hand grip tasks for all assessment points were also computed to compare the ROI results (see Supplementary Table 1). The BOLD signal time-series data from all ROIs was extracted and calculated as the percent signal change for each force matching phase volume (ascending, keep, and descending) relative to the mean BOLD signal in the rest phase volume. The task was repeated four times, resulting in 12 measurements for each assessment point.
Tracing and target tracking tasks were evaluated using the following parameters to assess performance: (i) sum of error - difference between the target coordinates of the sine wave and participant's pen in the x direction (medio-lateral), y direction (antero-posterior), and xy direction (sum of squared error); (ii) velocity - mean velocity during the tasks; (iii) acceleration - mean acceleration during the tasks; and (iv) pressure - mean pressure exerted during the tasks. The parameters were calculated for each full sine wave and the task was repeated two times, resulting in six measurements for the tracing and sine wave tracking tasks for each assessment point. All parameters were calculated using a custom program written in MATLAB (The MathWorks Inc., United States).
Clinical scores for the FIM, FMA, and MAL tests were tabulated and evaluated by a trained occupational therapist and compared between different assessment points.
Short-term TMS assessments were analyzed using paired samples t-test to compare the input-output curve slope and CSP between assessment points (before and after). Long-term TMS assessments were analyzed using the one-way repeated measures analysis of variance (ANOVA) to compare the input-output curve slope and CSP between assessment points (Pre, During, Post0, Post1, and Post2). Same statistical procedures were applied to compare long-term fMRI cortical activations during hand grip and finger pinch tasks in the peak activated voxel in M1 as well as in the contralateral and ipsilateral hemisphere in each ROI (M1, S1, S2, PR, SMA, PM, and HC), as well as drawing task error (x, y, and xy directions), velocity, acceleration, and pressure between assessment points. For long-term assessments, when significant results were found on the ANOVA, post hoc multiple comparisons with Holm adjustment to correct for comparison between assessment time points were conducted to compare Pre to other assessment points. Parametric tests were chosen since the Shapiro-Wilk test was used to confirm that most data were normally distributed. Short-term assessments were performed before and after each FEST session over the 12-weeks, while long-term assessments were performed on repeated trials on each assessment point. Statistical comparisons were performed using SPSS Statistics (IBM Corp., Armonk, NY, United States). Significance level for all tests was set to p < 0.050.