Transspinal direct current stimulation for multiple sessions alters neuronal excitability but not homosynaptic inhibition in people with and without Spinal Cord Injury

Transspinal direct current stimulation for multiple sessions alters neuronal excitability but not homosynaptic inhibition in people with and without Spinal Cord Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transspinal direct current stimulation for multiple sessions alters neuronal excitability but not homosynaptic inhibition in people with and without Spinal Cord Injury Maria Knikou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7365193/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Experimental Brain Research → Version 1 posted You are reading this latest preprint version Abstract Transspinal stimulation with direct current or at intensities and frequencies that produce intermittent depolarization of motoneurons can be an adjunct treatment strategy for spasticity and recovery of movement in persons with spinal cord injury (SCI). The main objective of this study was to assess neuroplasticity after multiple sessions of transspinal direct current stimulation (tsDCS) in people with and without SCI. Nine SCI and 10 noninjured subjects received daily cathodal tsDCS over Thoracic 10 while supine with an average stimulation intensity of 2.28 ± 0.02 mA. SCI and noninjured subjects received an average of 15 and 10 stimulation sessions, respectively. Before and 1–2 days post intervention, we assessed changes in soleus H-reflex recruitment input-output curves, homosynaptic depression and postactivation depression. tsDCS for approximately 1 hour did not alter the strength of homosynaptic depression in both subject groups, but reversed postactivation depression to facilitation in AIS C-D subjects. tsDCS resulted in depression of reflex excitability in both subject groups, but without significant changes in clinically assessed hyperreflexia. The results indicate decreased reflex hyperexcitability without recovery of spinal inhibitory control in the injured human spinal cord after tsDCS. More systematic investigations are needed to delineate the tsDCS-induced neuroplasticity of spinal neuronal networks in people with SCI and thus be able to develop effective treatments. Transspinal DC stimulation H-reflex Spinal Cord Injury Neuromodulation Neurorecovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction After spinal cord injury (SCI), recovery using neuromodulation strategies via stimulation that work synergistically with activity-dependent neuroplasticity is in great need. The impaired function of spinal circuitry and corticospinal drive and impaired processing of afferent input by the spinal circuits, and the decline in transmission of uninjured fibers lead to an altered excitability state, one of the most debilitating complications (Knikou 2007 ; Arvanian et al. 2009 ; Barthelemy et al. 2010; Tansey et al. 2012 ). Motor overactivity results in involuntary movements, co-contraction of antagonistic muscles, hyperreflexia, ankle clonus, and increased muscle tone, which all constitute clinical manifestations of spasticity. These clinical sequalae of pathological muscle tone cause pain and fatigue, disturb sleep, restrict daily activities like walking, sitting, and bathing, and can affect significantly rehabilitation efforts. The altered excitability state can be targeted via transspinal (or transcutaneous spinal cord) stimulation. Multiple sessions of transspinal stimulation with alternated current at low frequencies (0.2 Hz) decreases soleus H-reflex excitability, upregulates homosynaptic inhibition, decreases spasticity, and increases the net motor output of motoneurons over multiple segments in people with SCI (Knikou and Murray 2019 ; Murray and Knikou 2019 ). Further, transspinal stimulation at frequencies up to 15 Hz contributes to recovery of assisted standing, while frequencies ranging from 25 to 120 Hz are adopted for recovery of stepping after SCI (Minassian et al. 2016 ; Rejc et al. 2017 ; Sayenko et al. 2019 ; Zaaya et al. 2021 ), with significant neuromodulation effects on spinal locomotor pathways (Skiadopoulos and Knikou 2024 ) and cortical and corticospinal activity in healthy humans (Pulverenti et al. 2019 ; Murray et al. 2019 ), supporting for widespread neuronal excitability changes and development of neuroplasticity upon multiple sessions. Another form of transspinal stimulation that induces neuronal changes is direct current delivered at constant low intensity. In spinal cord preparations of spinalized mice, transspinal direct current stimulation (tsDCS) alters neuronal excitability that coincides with a significant increase of glutamate analog, D-2,3-(3)H-aspartate (D-Asp) (Ahmed and Wieraszko 2012 ). Moreover, tsDCS paired with sciatic nerve stimulation alters muscle tone and increases complex multi-joint movement amplitude in spinalized or anesthetized mice (Ahmed 2013 , 2014 ). In 17 healthy humans met allele carriers and 17 Val homozygotes anodal tsDCS induced a progressive leftward shift of recruitment curve of the H reflex during the stimulation that persisted for at least 15 min after current offset in Val/Val individuals (Lamy and Boakye 2013 ). Furthermore, 15-min of anodal tsDCS increased spinal reflex excitability during the stimulation, while both cathodal and sham tsDCS had no significant effects (Lamy et al. 2012 ). A single session of tsDCS has been linked to modulation of activity in lemniscal, spinothalamic, and segmental motor systems (Cogiamanian et al. 2011 , 2012 ), as well as to modulation of cortical mechanisms that persists for 30 minutes post stimulation (Murray et al. 2018 ). Reports on neuroplasticity and neurorecovery via tsDCS in people with SCI are scarce. Collectively, the main objective of this study was to assess the effects of multiple sessions of tsDCS on soleus H-reflex excitability, homosynaptic inhibition, and clinical measures of hyperreflexia in people with chronic SCI and compare the results to those observed in a group of noninjured subjects. To meet our main objective, before and after an average of 14.67 ± 0.47 sessions in 9 SCI and 10 sessions in 10 noninjured subjects, the soleus H-reflex recruitment curve, homosynaptic depression, postactivation depression and clinical measures of hyperreflexia were assessed. Methods Participants All experimental and intervention procedures were approved by the City University of New York’s Institutional Biomedical Committee (IRB Number 515055) and conducted in accordance with the standards of the Declaration of Helsinki. Eligible participants gave informed, written consent prior to enrollment. Inclusion was considered if: 1) ages were between 18–70 years; 2) persons were free of ferromagnetic material in the brain and/or spine; and 3) no contraindications to brain or spinal stimulation were present. Individuals with SCI were included if the injury was chronic (more than 6 months) and at or above Thoracic 12. Nine individuals with chronic SCI (Table 1 ) and 10 healthy volunteers (7 female; 27.2 ± 5 years, mean ± SD) free of musculoskeletal or neurological disorders completed the study. Five individuals with chronic SCI had a neurological deficit grade D on the American Spinal Injury Association Impairment Scale (AIS), 2 had AIS B, and 2 had AIS A, while the level of SCI ranged from Cervical 4 to Thoracic 11. Individuals with motor complete SCI were also included to assess changes in the presence of minimal descending and afferent input. Table 1 Demographics and injury characteristics of participants with chronic spinal cord injury (SCI). ID Gender Age (yrs) Post injury (yrs) Level of injury Motor score # of sessions attended (tsDCS) List of medication AIS Cause of injury LL RL R01 M 53 5 C7 B Ocean wave-related 0 0 17 Baclofen 20 mg 4xD † ; Cymbalta 60 mg 1xD; Oxybutynin 5 mg 3xD R06 M 36 4.5 T2 B MVC 0 0 13 None R09 F 20 7 T1 D SX 24 23 16 None R11 M 39 7 T9 D GSW 25 25 15 Gabapentin 800 mg 3xD; Baclofen 10 mg 3xD ‡ R14 M 46 1 T3 A Skiing 0 0 15 None R16 M 57 4 C4 D Fall & spinal stenosis 23 21 15 Gabapentin 400 mg 4xD; Baclofen 10 mg 5xD; Oxybutynin 10 3xD; Oxycontin 5 mg 2xD R17 M 31 4 C5 B Diving into shallows 0 0 14 None R18 M 61 17 C5-6 D MVC 25 25 15 Synthroid 225 mg 1Xd R19 M 60 2 C6 D Fall 18 17 15 Oxybutynin 10mg 2x/day; Pravachol 40mg 1x/day The level of injury corresponds to the vertebral level. Following the American Spinal Injury Association impairment scale (AIS) assessment, motor scores are indicated based on the manual muscle test of five key muscles and evaluated as 5 = normal muscle power, 4 = active movement against gravity with slight resistance, 3 = active movement against gravity, 2 = active movement with gravity eliminated, 1 = trace muscle contraction, 0 = no contraction. M: male; F: female; C: cervical; T: thoracic; MVC = Motor vehicle crash; SX = Surgery; GSW = Gunshot wound; xD = Times daily. † Participant delayed ingestion of baclofen until after each experiment. ‡ Participant ceased baclofen prior to the duration of the study. Ұ Participant ceased baclofen after the first day of experiments. [Insert Table 1 here] Surface electromyography (EMG) Surface EMG was recorded with single bipolar differential electrodes (MA300-28, Motion Lab Systems Inc., Baton Rouge, LA) placed on lightly shaved, abraded and cleansed skin over the left and or the right soleus muscle of individuals with SCI, and the right soleus muscle of individuals without SCI. Electrode placement was secured with transparent Tegaderm film (3M Healthcare, St Paul, Minnesota, USA) for the duration of the experiments. EMG signals were amplified, filtered (10–1000 Hz), sampled (2000 Hz) using a 1401-Plus system (Cambridge Electronics Design Ltd., Cambridge, UK) or LabVIEW scripts and stored for offline analysis. Intervention: Multiple sessions of tsDCS Cathodal tsDCS, known to have greater and/or more persistent neuromodulatory effects beyond that of anodal stimulation (Bindman et al. 1964 ; Bocci et al. 2014 ; Murray et al. 2018 ), was delivered using a direct current stimulator (neuroConn DC stimulator PLUS, neuroCare Group GmbH, München, Germany). The active square steel mesh housed in a rubber electrode (cathode; 3.2 cm x 3.2 cm; Amrex-Zetron Inc, California, USA) and covered by a sponge soaked in 0.9% saline solution was positioned centrally over vertebra Thoracic 10 to 12 corresponding to Lumbar 1–5 spinal segments. The position was determined via manual palpation of the spinal processes starting from Cervical 7, confirmed two vertebrae above the attachment point of the twelfth rib, and marked with a non-toxic surgical pen for constancy across stimulation sessions. A second saline-soaked square reference electrode (anode, same type as the cathode; 10.16 cm x 10.16 cm) was placed on the abdomen left of the umbilicus to avoid vital organs. This montage is known to produce maximal electric field potentials in a longitudinal direction along the spinal cord (Parazzini et al. 2014 ). For all subjects, tsDCS was delivered daily during weekdays, excluding holidays, whilst the subject was lying supine with knee and hip joints flexed at 30° and supported by pillows. Individuals with SCI received an average of 14.67 ± 0.47 stimulation sessions for 50.25 ± 2.25 min per session (Table 1 ). Healthy control subjects received 10 stimulation sessions for an average of 44.96 ± 0.27 min per session. Depending on the subjects’ availability, additional sessions were performed to ensure the neurophysiological recordings did not occur the day after a 2-day break (e.g. weekend). Stimulation intensity was delivered in blocks of 5–10 minutes, ramping up/down from the designated intensity over 20 s, until a total of 45 minutes of stimulation was given. To keep skin irritations minimal due to daily stimulation sessions, the intensity ranged between 1.25 and 3.0 mA, with an average stimulation intensity of 2.28 ± 0.02 mA. Both subject groups received similar intensities across the entire intervention despite the small increases and decreases. Current density and total charge never exceeded 0.26 mA/cm 2 and 878.91 mC/cm 2 in any stimulation session, respectively. A maximal average current density of 0.23 mA/cm 2 and a maximal average total charge of 630.43 mC/cm 2 was delivered across sessions for SCI subjects. Similar values were adopted for uninjured control subjects with a maximum average of 0.23 mA/cm 2 and 620 mC/cm 2 , respectively. These were within the safety limits of 2.3 mA/cm 3 for current density threshold during invasive spinal stimulation and 25 mA/cm 3 for pulse electrical stimulation known to cause tissue damage (McCreery et al. 1990 ; Wesselink et al. 1998 ; Cogiamanian et al. 2012 ). Due to the intensity of stimulation delivered, it was impossible to blind the participants to the sessions and therefore no sham group was conducted. Post-session tsDCS questionnaires were randomly completed throughout the intervention to establish presence of any adverse events. No significant changes were noted in the blood pressure of any participant during the stimulation sessions and/or experiments. The major complaint was skin redness or irritation (SCI: 69.2%; Controls: 37.5%) mainly due to the pressure of the electrode against the skin, subsiding within a few hours, but some had a slight skin rash from the salt water being applied daily. Mild reports of tingling (SCI: 38.5%; Controls: 44%), and burning or itchy sensations (SCI: 15.4%; Controls: 12.5%) during the ramp-up and down phase of stimulation were also reported. Lastly, mild back (SCI: 15.4%) and neck (Controls: 19%) pain possibly due to position requirements of the study, and a mild-moderate but transient headache (Controls: 19%) were also reported. Neurophysiological outcome measurements before and 1-day after multiple sessions of tsDCS The soleus H-reflex was evoked according to methods we have extensively used previously in our laboratory (Knikou 2008 ). With the subject seated, relaxed, and both feet supported by a footrest, a stainless-steel circular plate of 4 cm 2 in diameter (anode electrode) was secured proximally to the patella of the right leg. A rectangular single 1 ms pulse was delivered by a custom-built constant current stimulator to the posterior tibial nerve at the popliteal fossa. The most optimal stimulation site was established via a handheld monopolar stainless steel head electrode used as a probe. An optimal stimulation site corresponded to the site that Ia afferents could be excited at lower stimulation intensities without the M-wave being present. The monopolar electrode was then replaced by a pregelled disposable electrode (SureTrace, Conmed, Utica, NY, USA) that was maintained under constant pressure throughout the experiment with an athletic wrap. Soleus H-reflex homosynaptic and postactivation depression The soleus M-wave and H-reflex were evoked using a constant current stimulator (DS7A, Digitimer Ltd., UK) with a 1.0 ms square pulse at 0.2 Hz. The H-reflex was evoked on the ascending part of the recruitment curve and was 25–40% of the maximal M-wave (Mmax). For homosynaptic depression, 15 H-reflexes were recorded at 0.1, 0.125, 0.2, 0.33, and 1.0 Hz. For postactivation depression, 20 paired stimuli were delivered randomly at the interstimulus intervals (ISIs) of 500, 300, 100, and 60 ms with a constant stimulation frequency of 0.2 Hz. Soleus H-reflex excitability and recruitment of alpha motoneurons Before and 1-day after tsDCS, the soleus H-reflex recruitment curve was assembled with subjects seated. The right posterior tibial nerve at the popliteal fossa was stimulated at 0.2 Hz and at least 120 responses were recorded via a customized stimulator randomly at increasing stimulation intensities. The LABVIEW software controlled and adjusted the stimulation intensity through a LabVIEW custom-made script and saved each value along with the triggering pulse and EMG response. This experimental approach ensured that the H reflex was continuously evoked at different intensities and not with uniformly increasing or decreasing consecutive values. Clinical outcome measurements before and 1-day after tsDCS : The self-reported Penn spasm frequency and Penn severity scales along with the modified Ashworth scale (Adams et al. 2007 ; Savic et al. 2007 ), were evaluated in individuals with SCI before and after tsDCS. Ankle clonus was scored as 3: sustained clonus, 2: prolonged but fatigable (20–100 beats per minute), 1: brief but fatigable (1–20 beats per minute), and 0: absent. Data analysis and statistics Offline analysis of the M-waves and H-reflexes for homosynaptic and postactivation depression was performed using Spike 2 software (Cambridge Electronics Design Ltd., Cambridge, UK). For homosynaptic depression, the area under the rectified H-reflex waveform was measured for each response evoked at 0.125, 0.2, 0.33, and 1.0 Hz, and normalized to the mean homonymous H reflex amplitude evoked at 0.1 Hz. For postactivation depression, the area under the rectified H-reflex waveform was measured for each response evoked by paired stimuli at 500, 300, 100 and 60 ms. The second H reflex (H2; H-reflex evoked by the 2nd pulse) was then normalized to the first H reflex (H1; H-reflex evoked by the 1st pulse) within each pair and averaged. For each subject, the peak-to-peak amplitude of the non-rectified soleus M wave and H reflex recorded at different stimulation intensities to assemble the recruitment curve, were measured offline with custom developed algorithms in LABVIEW (LabVIEW 8.2, National Instruments Ltd.,) and were normalized to the associated maximal M-wave. Then, the normalized soleus M-waves or H-reflexes were plotted against the actual stimulation intensities, and a Boltzmann sigmoid function (Eq. 1; SigmaPlot 11, Systat Software Inc., California, USA) was fitted to the data. From Eq. 1, the R 2 , slope of the function (m), and stimulus intensity corresponding to 50% of the maximal M-wave or maximal H-reflex (S50-Mmax, S50-Hmax) was established. For the M-waves, the stimulation intensities were normalized to the homonymous S50-Mmax before and after intervention. For the soleus H-reflex, the stimulation intensities were normalized to the homonymous S50-Mmax or to the S50-Hmax observed at baseline. For each and across subjects, the normalized soleus M-waves and H-reflexes were grouped based on increments of 0.05 multiples of S50-Mmax or S50-Hmax. \(\:\text{M}\left(\text{s}\right)=\frac{\text{M}\text{m}\text{a}\text{x}}{(1+\text{exp}\left(\text{m}\left(\text{S}50-\text{s}\right)\right))\:},\:\text{H}\left(\text{s}\right)=\frac{\text{H}\text{m}\text{a}\text{x}}{(1+\text{exp}\left(\text{m}\left(\text{S}50-\text{s}\right)\right))\:}\) [Eq. 1] Statistical analysis was performed using SigmaPlot (Systat Software Inc., California, USA). For each neurophysiological assessment, H-reflexes were grouped as AIS C-D, AIS A-B, and noninjured subjects before and after transspinal direct current stimulation. A repeated measures analysis of variance (rmANOVA) was conducted separately for normalized H reflexes recorded at different stimulation frequencies, upon nerve stimuli at different ISIs, and for M-wave and H-reflex recruitment curves grouped based on multiples of S50-Mmax or S50-Hmax. When a statistically significant main effect was found, post-hoc Bonferroni t -tests for multiple comparisons were used to test for significant interactions. Significant changes in clinical assessments were established with a paired t -test. For all statistical tests, significance was determined at p < 0.05. Results are presented as mean values and standard deviation (SD). Results Homosynaptic depression of the soleus H-reflex before and after multiple sessions of tsDCS in noninjured and SCI subjects Representative non-waveform averages recorded before and 1-day after tsDCS from one noninjured and one SCI subject are indicated in Fig. 1 A. While the soleus H-reflex did decrease in amplitude as stimulation to the posterior tibial nerve occurred at lower frequencies in both subjects, tsDCS appeared not to affect the strength of homosynaptic depression. [Insert Fig. 1 near here] The overall amplitude of the soleus H-reflex before and after multiple sessions of tsDCS recorded at different stimulation frequencies is indicated separately for AIS A-B, AIS C-D and noninjured subjects in Fig. 1 B. The soleus H-reflexes at 0.125 (8s), 0.2 (5s), 0.33 (3s), and 1.0 (1s) Hz were normalized to the homonymous mean reflex amplitude recorded at 0.1 (10s) Hz. In AIS A-B group, the soleus H-reflexes varied significantly across stimulation frequencies (F 3,40 = 19.15, p < 0.001) but there were no significant differences before and after tsDCS (F 1,40 = 0.02, p = 0.87). Similarly, in AIS C-D group the statistically significant differences of soleus H-reflexes across stimulation frequencies (F 3,56 = 8.31, p < 0.001) occurred with no significant differences before and after tsDCS (F 1,56 = 0.1, p = 0.75). Last, in noninjured subjects the soleus H-reflexes varied significantly across stimulation frequencies (F 3,72 = 75.3, p < 0.001; 2-way rmANOVA) but there were no significant differences before and after tsDCS (F 1,72 = 0.52, p = 0.47). These results support that some homosynaptic depression is present after SCI but of lesser strength compared to noninjured subjects, and that its strength remained unaltered by multiple sessions of tsDCS in both noninjured and SCI subjects. Postactivation depression of the soleus H-reflex before and after multiple sessions of tsDCS in noninjured and SCI subjects Representative examples of non-rectified soleus H-reflex waveform averages recorded upon paired tibial nerve stimuli at ISIs ranging from 500 to 60 ms from one noninjured and one SCI subject before and 1-day after tsDCS are indicated in Fig. 2 . It is evident that the soleus H reflex was significantly depressed upon paired tibial nerve stimuli and almost abolished at the ISI of 60 ms for both noninjured and SCI subjects. [Insert Fig. 2 near here] The overall amplitude of the soleus H-reflex before and 1-day after multiple sessions of tsDCS recorded at different ISIs of paired pulses is indicated separately for AIS A-B, AIS C-D and noninjured subjects in Fig. 3 . For all graphs, the H-reflex evoked by the second pulse (H-reflex2) is shown as a percentage of the H-reflex evoked by the first pulse (H-reflex 1) within the same sweep or paired pulses. In the AIS A-B group, the soleus H-reflexes varied significantly across ISIs (F 3,40 = 31.4, p < 0.001) but not as a function of time (F 1,40 = 0.39, p = 0.53; 2-way rmANOVA). In the AIS C-D group, the H-reflex 2 was statistically significant different from H-reflex 1 as a function of ISIs (F 3,56 = 64.45, p < 0.001) and time (F 1,56 = 8.62, p = 0.005). Holm-Sidak pairwise multiple comparisons showed that the H-reflex at the 300 ms was significantly different before and after tsDCS (t = 3.17, p = 0.003). Last, in noninjured subjects the soleus H-reflexes varied significantly across ISIs (F 3,72 = 23.27, p < 0.001) but not as a function of time (F 1,72 = 0.54, p = 0.46; 2-way rmANOVA). These results support for presence of postactivation depression after SCI, and that its strength was not affected by tsDCS in motor complete SCI and noninjured subjects but reversed to facilitation at 300 ms ISI in motor incomplete SCI. [Insert Fig. 3 near here] Recruitment of soleus motoneurons before and after multiple sessions of tsDCS in noninjured and SCI subjects To assess changes in the excitability state and recruitment order of alpha motoneurons by muscle spindle group Ia afferents, the soleus M-wave and H-reflex recruitment curves were assembled in noninjured and SCI subjects while seated before and 1-day after tsDCS. The soleus H-reflexes and M-waves from all noninjured subjects plotted against multiples of stimulation intensities along with the sigmoid fit are shown in Fig. 4 A-B. The soleus H-reflexes varied significantly across stimulation intensities (F 13,247 = 45.24, p < 0.001) but not based on the time of testing (F 1,247 = 0.34, p = 0.56; 2-way rmANOVA for H-reflexes grouped across subjects from 0.26 to 0.91 xS50-Mmax), while an interaction between normalized intensities and time was not present (F 13 = 0.228, p = 0.99). Non-significant differences in the M-wave recruitment curves (Fig. 4 B) as a function of time were present (F 25,411 = 9.75, p = 0.35; 2-way rmANOVA for M-waves grouped across subjects from 0.26 to 1.51 xS50-Mmax). However, we should note that when the normalized H-reflexes were grouped across subjects with stimulation intensities normalized to the predicted S50-Hmax at baseline from 0.46 to 1.41 xS50-Hmax (Fig. 4 C), 2-way rmANOVA showed significant differences across stimulation intensities (F 19,333 = 17.5, p < 0.001) and as a function of the time of testing (F 1,333 = 12.21, p < 0.001). [Insert Fig. 4 near here] The soleus H-reflexes and M-waves from all AIC C-D subjects along with the sigmoid fit are shown in Fig. 5 A-B. The soleus H-reflexes varied significantly across stimulation intensities (F 15,187 = 15.27, p < 0.001) and also based on the time of testing (F 1,187 = 16.09, p < 0.001; 2-way rmANOVA for H-reflexes grouped across subjects from 0.26 to 1.01 xS50-Mmax), but an interaction between normalized intensities and time was not present (F 15 = 0.34, p = 0.99). Non-significant differences in the M-wave recruitment curves (Fig. 5 B) as a function of time were present (F 25,258 = 22.17 p = 0.35; 2-way rmANOVA for M-waves grouped across subjects from 0.26 to 1.51 xS50-Mmax). In the case of AIS C-D subjects, when the normalized H-reflexes were grouped across subjects with stimulation intensities normalized to the predicted S50-Hmax at baseline from 0.26 to 1.31 xS50-Hmax (Fig. 5 C), 2-way rmANOVA showed significant differences across stimulation intensities (F 21,252 = 15.68, p < 0.001) and as a function of the time of testing (F 1,252 = 5.36, p = 0.021). This result suggests that the H-reflex depression after tsDCS was present regardless of the normalization factor used for stimulation intensities (S50Mmax or S50Hmax) although only in this subject group the peak slope, estimated as (m×Hmax)/4, was reduced after tsDCS (13.57 ± 9.71; p = 0.04, t-test) compared to before tsDCS (47.77 ± 51.75). [Insert Fig. 5 near here] The soleus H-reflexes and M-waves from all AIC A-B subjects along with the sigmoid fit are shown in Fig. 6 A-B. The soleus H-reflexes varied significantly across stimulation intensities (F 15,140 = 11.57, p < 0.001) but were not significantly different after tsDCS (F 1,140 = 0.87, p = 0.35; 2-way rmANOVA for H-reflexes grouped across subjects from 0.26 to 1.01 xS50-Mmax). Non-significant differences in the M-wave recruitment curves (Fig. 6 B) as a function of time were present (F 25,189 = 0.77, p = 0.38; 2-way rmANOVA for M-waves grouped across subjects from 0.26 to 1.51 xS50-Mmax). In the case of AIS A-B subjects, when the normalized H-reflexes were grouped across subjects with stimulation intensities normalized to the predicted S50-Hmax at baseline from 0.66 to 1.41 xS50-Hmax (Fig. 6 C), 2-way rmANOVA showed significant differences across stimulation intensities (F 15,148 = 9.25, p < 0.001) and as a function of the time of testing (F 1,148 = 72.25, p < 0.001). From the sigmoid fit to the H-reflex normalized to the homonymous Mmax and plotted against actual values of stimulation intensities, the m function of the slope was decreased in AIS A-B (from 1.61 ± 0.87 to 0.91 ± 0.28, p = 0.04), the peak slope was decreased in AIS C-D (from 47.77 ± 51.75 to 13.57 ± 9.71, p = 0.04), and the Hmax as a percentage of the Mmax was decreased in AIS C-D (from 81.91 ± 12.18 to 60.06 ± 14.13, p = 0.004). No changes were observed in any sigmoid parameters (data not shown) in noninjured subjects. [Insert Fig. 6 near here] Clinically evaluated hyperreflexia before and after tsDCS in individuals with SCI In Table 2 , the results of clinical assessments of hyperreflexia performed before and 1/2-days after cessation of tsDCS are indicated. The Penn spasm frequency, spasm severity and ankle clonus in both legs were not statistically significantly different before and after tsDCS ( p > .05 for all). Table 2 Clinical assessments of hyperreflexia before and after tsDCS in SCI. Penn Spasm Frequency Penn Spasm Severity Ankle Clonus (Left leg) Ankle Clonus (Right leg) Subject ID Before After Before After Before After Before After R01 a 3.0 2.0 3.0 2.0 3.0 2.0 1.0 0.0 R06 b 3.0 3.0 2.0 2.0 0.0 0.0 1.0 0.0 R09 2.0 1.0 2.0 2.0 1.0 0.0 1.0 1.0 R11 2.0 1.0 2.0 1.0 0.0 0.0 0.0 0.0 R14 c 1.0 2.0 2.0 1.0 0.0 0.0 0.0 0.0 R16 0.0 1.0 1.0 2.0 0.0 0.0 1.0 1.0 R17 2.0 1.0 1.0 2.0 2.0 3.0 2.0 3.0 R18 1.0 2.0 1.0 2.0 1.0 1.0 1.0 1.0 R19 3.0 3.0 3.0 3.0 2.0 1.0 2.0 1.0 p -value 0.4 0.5 0.33 0.29 For each subject, the self-reported Penn Spasm Frequency and Penn Spasm Severity are indicated before and after repeated cathodal transspinal stimulation. Ankle clonus for both left and right legs are indicated for before and after transspinal stimulation and evaluated as 0: absent clonus, 1: fatigable, 1–20 beats per minute, 2: fatigable, 20–100 beats per minute, 3: non-fatigable, continuous sustained clonus. P -values are indicated for before and after tsDCS comparisons based on a paired t-test. a : has cutaneous sensation but absent sensation of temperature. b Movement at the hips initiates spasms and ankle clonus. c flexor and extensor spasms were present in both legs. [Insert Table 2 near here] Discussion This is the first study on spinal neuroplasticity in both noninjured and SCI persons 1–2 days after multiple daily sessions of cathodal tsDCS. We used the soleus H-reflex, a neurophysiological probe for functional activity and recovery of spinal neuronal circuits and motoneurons as well as a biomarker of hyperreflexia and spasticity (Knikou 2008 , 2010). We found that daily sessions of tsDCS for approximately 1 hour did not alter the strength of homosynaptic depression in both subject groups, reversed postactivation depression to facilitation in AIS C-D, produced depression of reflex excitability in both subject groups that was revealed when intensities were normalized to the S50-Hmax at baseline, while clinically assessed hyperreflexia remained unaltered. While there are no systematic investigations on spinal inhibition after multiple sessions of tsDCS in SCI persons, a single 20-min session of anodal tsDCS did not affect homosynaptic depression of the flexor carpi radialis (FCR) H-reflex in noninjured subjects (Donges et al. 2017). In contrast, the soleus H-reflex homosynaptic depression in noninjured subjects was either decreased or increased after anodal and cathodal tsDCS, respectively (Winkler and Straube 2010). We have previously reported unaltered homosynaptic depression after a single or multiple sessions in healthy control subjects and increase of homosynaptic depression in SCI after multiple sessions of cathodal transspinal stimulation that produces intermittent depolarization of myelinated afferent fibers and motoneurons over multiple segments (Knikou et al. 2015 ; Knikou and Murray 2019 ). In this study we found that homosynaptic depression, although present but weaker in SCI compared to noninjured subjects, remained unaltered by tsDCS (Fig. 1 B). Homosynaptic depression is produced by repetitive discharges of Ia afferents and mediated by reduced transmitter release at the Ia-motoneuron synapse reducing the strength of transsynaptic depolarization of alpha motoneurons (Magladery and McDougal 1950). Because homosynaptic depression requires consecutive activation of the same group Ia afferents and occurs at the same Ia-motoneuron synapse without any contribution from motor axons, we conclude that tsDCS did not affect this circuit which is known to alter after SCI (Mailis and Ashby 1990 ). Our findings are consistent with the unaltered clinically evaluated ankle clonus (Table 2 ), and the upregulated excitability of myelinated nerve fibers and dorsal horn field potentials beyond the polarization period during cathodal direct current applied locally (Jankowska 2017 ; Jankowska et al. 2017 ). Although homosynaptic depression is not related to the classical presynaptic inhibition that requires activation of a diverse network of inhibitory interneurons and concomitant primary afferent depolarization, it does gate proprioceptive inputs in resting individuals. Homosynaptic depression is regularly used as an indicator of calcium release probability while the spasticity and spasms that partly result from malfunction of this neuronal mechanism have been partly associated with enhanced activation of calcium-mediated persistent inward currents (Norton et al. 2008 ; D’Amico et al. 2013; ElBasiouny et al. 2010 ). Therefore, we can conclude that tsDCS did not effectively affected persistent inward currents exerted at the somata and dendrites of motoneurons. Postactivation depression induced by paired stimuli to the tibial nerve was present at baseline in both AIS A-B and AIS C-D subjects but of lesser strength compared to noninjured subjects consistent to our previous reports (Knikou and Murray 2019 ) and reversed to postsynaptic facilitation after tsDCS in AIS C-D subjects at the ISI of 300 ms (Fig. 3 ). A conditioning H1 stimulus on a testing H2 stimulus results in depression of H2 responses that is maximal at 225 ms while recovery of reflex depression starts at 300 ms (Taborikova and Sax 1969). Because removal of inhibition occurred at long latencies, the effects have been attributed to long loop reflex circuits (Chofflon et al. 1982), suggesting that this type of postactivation depression is not mediated by similar mechanisms to that of homosynaptic depression. Removal of depression at the ISI of 300 ms can be attributed to potentiation of postsynaptic excitation or intercurrent facilitation (Taborikova and Sax 1969). Long-term potentiation (LTP) induced by tsDCS might have mediated this facilitation as LTP is a long-lasting increase in synaptic efficacy that persists for hours or days. LTP is primarily driven by an increase in volume and ionotropic glutamate receptor density in postsynaptic spines, while increased neurotransmitter release at presynaptic terminals is also involved (Bliss and Gardner-Medwin 1973 ; Hayashi 2022 ; Choi et al. 2018 ). We should be cautious with this assumption because LTP-mediated plasticity mechanisms in the spinal cord require high frequency stimulation (Bear and Malenka 1994 ). While the neuronal mechanisms are not clear, multiple sessions of tsDCS reversed postactivation depression to facilitation which may further contribute to spasticity and spasms in SCI persons. The maximal H-reflex amplitude was suppressed in AIS C-D subjects (Fig. 5 A). In all subject groups, when the H-reflexes were grouped with stimulation intensities being normalized to the predicted S50-Hmax at baseline a generalized depression of soleus H-reflex excitability was apparent (Figs. 4 - 6 C). This suggests that grouping of H-reflexes per multiples of stimulation intensities shifted to the right after tsDCS increasing the threshold of excitability and thus reducing the recruitment of motoneurons. Our findings are consistent with the suppressed soleus H-reflex recruitment input-output curve after a single session of cathodal tsDCS in healthy subjects (Murray et al. 2018 ), and multiple sessions of transspinal stimulation in SCI (Knikou and Murray 2019 ). However, facilitation of the long latency tibialis anterior stretch reflex has also been reported after cathodal tsDCS (Therkildsen et al. 2022 ). The changes in the H-reflex recruitment input-output curve suggest for altered responsiveness of motoneurons to proprioceptive afferent inputs in the injured human spinal cord after tsDCS. Limitations The research project that this study was a part of concentrated on the effects of transspinal stimulation with direct and alternated current in noninjured and SCI subjects. Due to the nature of the research project and to the fact that subjects can differentiate between active and sham conditions at these intensities (Murray and Knikou 2019 ), we did not test stimulation under sham conditions. Further, an investigation of spinal interneuronal circuits involving presynaptic and postsynaptic inhibitory mechanisms was not conducted. More systematic investigations are needed to delineate the tsDCS-induced neuroplasticity in people with and without SCI. Conclusion This study provides evidence on neurophysiological changes after multiple sessions of tsDCS in noninjured and SCI persons. tsDCS did not affect homosynaptic depression, reversed postactivation depression to facilitation, produced a generalized depression of soleus H-reflex excitability, but did not reduce clinically assessed hyperreflexia in individuals with motor incomplete and complete SCI. It remains to be shown if these effects are transferred to functional changes in movement. Declarations Acknowledgments The author gratefully thanks all research participants, their families and caregivers for their time and dedication to the study, and the Klab4Recovery staff for administration of all stimulation sessions and their help during data acquisition. Declaration of Conflicting Interests The author declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding This work was supported by the Craig H. Neilsen Foundation (Grant No. 339705) awarded to MK. References Adams MM, Ginis KA, Hicks AL (2007) The spinal cord injury spasticity evaluation tool: development and evaluation. Arch Phys Med Rehab 88:1185-1192. doi: 10.1016/j.apmr.2007.06.012 Ahmed Z, Wieraszko A (2012) Trans-spinal direct current enhances corticospinal output and stimulation-evoked release of glutamate analog, D-2,3-³H-aspartic acid. J Appl Physiol 112:1576-1592. doi: 10.1152/japplphysiol.00967.2011 Ahmed Z (2013) Effects of cathodal trans-spinal direct current stimulation on mouse spinal network and complex multijoint movements. J Neurosci 33:14949-14957. doi: 10.1523/JNEUROSCI.2793-13.2013 Ahmed Z (2014) Trans-spinal direct current stimulation alters muscle tone in mice with and without spinal cord injury with spasticity. J Neurosci 34:1701-1709. doi: 10.1523/JNEUROSCI.4445-13.2014 Arvanian VL, Schnell L, Lou L, Golshani R, Hunanyan A, Ghosh A, Pearse DD, Robinson JK, Schwab ME, Fawcett JW, Mendell LM (2009) Chronic spinal hemisection in rats induces a progressive decline in transmission in uninjured fibers to motoneurons. Exp Neurol 216:471-480. doi: 10.1016/j.expneurol.2009.01.004 Bączyk M, Jankowska E (2012) Long-term effects of direct current are reproduced by intermittent depolarization of myelinated nerve fibers. J Neurophysiol 120:1173-1185. doi: 10.1152/jn.00236.2018 Barthélemy D, Willerslev-Olsen M, Lundell H, Conway BA, Knudsen H, Biering-Sørensen F, Nielsen JB (2010) Impaired transmission in the corticospinal tract and gait disability in spinal cord injured persons. J Neurophysiol 104:1167-1176. doi: 10.1152/jn.00382.2010 Bear MF, Malenka RC (1994). Synaptic plasticity: LTP and LTD. Curr Opin Neurobiol 4:389-399. Bindman LJ, Lippold OC, Redfearn JW (1964) The action of brief polarizing currents on the cerebral cortex of the rat (1) during current flow and (2) in the production of long-lasting after-effects. J Physiol 172:369–382. Bliss TV, Gardner-Medwin AR (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the unanaestetized rabbit following stimulation of the perforant path. J Physiol 232:357-374. doi: 10.1113/jphysiol.1973.sp010274 Bocci T, Vannini B, Torzini A, Mazzatenta A, Vergari M, Cogia-manian F, Priori A, Sartucci F (2014) Cathodal transcutaneous spinal direct current stimulation (tsDCS) improves motor unit recruitment in healthy subjects. Neurosci Lett 578:75–79. doi: 10.1016/j.neulet.2014.06.037 Choi JH, Sim SE, Kim JI, Choi DI, Oh J, Ye S, Lee J, Kim T, Ko HG, Lim CS, Kaang BK (2018) Interregional synaptic maps among engram cells underlie memory formation. Science 360(6387):430-435. doi: 10.1126/science.aas9204 Cogiamanian F, Ardolino G, Vergari M, Ferrucci R, Ciocca M, Scelzo E, Barbieri S, Priori A (2012) Transcutaneous spinal direct current stimulation. Front Psychiatry 3:63. doi: 10.3389/fpsyt.2012.00063 Cogiamanian F, Vergari M, Schiaffi E, Marceglia S, Ardolino G, Barbieri S, Priori A (2011) Transcutaneous spinal cord direct current stimulation inhibits the lower limb nociceptive flexion reflex in human beings. Pain 152:370-375. doi: 10.1016/j.pain.2010.10.041 D'Amico JM, Murray KC, Li Y, Chan KM, Finlay MG, Bennett DJ, Gorassini MA (2013) Constitutively active 5-HT2/α1 receptors facilitate muscle spasms after human spinal cord injury. J Neurophysiol 109:1473-1484. doi: 10.1152/jn.00821.2012 ElBasiouny SM, Schuster JE, Heckman CJ (2010) Persistent inward currents in spinal motoneurons: important for normal function but potentially harmful after spinal cord injury and in amyotrophic lateral sclerosis. Clin Neurophysiol 121:1669-1679. doi: 10.1016/j.clinph.2009.12.041 Hayashi Y (2022) Molecular mechanism of hippocampal long-term potentiation - Towards multiscale understanding of learning and memory. Neurosci Res 175:3-15. doi: 10.1016/j.neures.2021.08.001 Jankowska E, Hammar I (2021) The plasticity of nerve fibers: the prolonged effects of polarization of afferent fibers. J Neurophysiol 126:1568-1591. doi: 10.1152/jn.00718.2020 Jankowska E, Kaczmarek D, Bolzoni F, Hammar I (2017) Long-lasting increase in axonal excitability after epidurally applied DC. J Neurophysiol 118:1210-1220. doi: 10.1152/jn.00148.2017 Jankowska E (2017) Spinal control of motor outputs by intrinsic and externally induced electric field potentials. J Neurophysiol 118:1221-1234. doi: 10.1152/jn.00169.2017 Knikou M, Dixon L, Santora D, Ibrahim MM (2015) Transspinal constant-current long-lasting stimulation: a new method to induce cortical and corticospinal plasticity. J Neurophysiol 114:1486-99. doi: 10.1152/jn.00449.2015 Knikou M, Murray LM (2019) Repeated transspinal stimulation decreases soleus H-reflex excitability and restores spinal inhibition in human spinal cord injury. PLoS One 14(9):e0223135. doi: 10.1371/journal.pone.0223135 Knikou M (2007) Plantar cutaneous input modulates differently spinal reflexes in subjects with intact and injured spinal cord. Spinal Cord 45:69-77. doi: 10.1038/sj.sc.3101917 Knikou M (2008) The H-reflex as a probe: pathways and pitfalls. J Neurosci Methods 171:1-12. doi: 10.1016/j.jneumeth.2008.02.012 Lamy JC, Boakye M (2013) BDNF Val66Met polymorphism alters spinal DC stimulation-induced plasticity in humans. J Neurophysiol 110:109-116. doi: 10.1152/jn.00116.2013 Lamy JC, Ho C, Badel A, Arrigo RT, Boakye M (2012) Modulation of soleus H reflex by spinal DC stimulation in humans. J Neurophysiol 108:906-914. doi: 10.1152/jn.10898.2011 Li T, Chen J (2025) Spinal cord stimulation for functional restoration in spinal cord injury: A narrative review. Cureus 17(2):e78610. doi: 10.7759/cureus.78610 Mailis A, Ashby P (1990) Alterations in group Ia projections to motoneurons following spinal lesions in humans. J Neurophysiol 64:637-647. doi: 10.1152/jn.1990.64.2.637 McCreery DB, Agnew WF, Yuen TG, Bullara L (1990) Charge density and charge per phase as cofactors in neural injury induced by electrical stimulation. IEEE Trans Biomed Eng 37:996-1001. doi: 10.1109/10.102812 Minassian K, McKay WB, Binder H, Hofstoetter US (2016) Targeting lumbar spinal neural circuitry by epidural stimulation to restore motor function after spinal cord injury. Neurotherapeutics 13:284-294. doi: 10.1007/s13311-016-0421-y Murray LM, Islam MA, Knikou M (2019) Cortical and subcortical contributions to neuroplasticity after repetitive transspinal stimulation in humans. Neural Plast 2019:4750768. doi: 10.1155/2019/4750768 Murray LM, Knikou M (2019) Repeated cathodal transspinal pulse and direct current stimulation modulate cortical and corticospinal excitability differently in healthy humans. Exp Brain Res 237:1841-1852. doi: 10.1007/s00221-019-05559-2 Murray LM, Knikou M (2019) Transspinal stimulation increases motoneuron output of multiple segments in human spinal cord injury. PLoS One 14(3):e0213696. doi: 10.1371/journal.pone.0213696 Murray LM, Tahayori B, Knikou M (2018) Transspinal direct current stimulation produces persistent plasticity in human motor pathways. Sci Rep 8:717. doi: 10.1038/s4159 8-017-18872-z Norton JA, Bennett DJ, Knash ME, Murray KC, Gorassini MA (2008) Changes in sensory-evoked synaptic activation of motoneurons after spinal cord injury in man. Brain 131:1478-1491. doi: 10.1093/brain/awn050 Parazzini M, Fiocchi S, Liorni I, Rossi E, Cogiamanian F, Vergari M, Priori A, Ravazzani P (2014) Modeling the current density generated by transcutaneous spinal direct current stimulation (tsDCS). Clin Neurophysiol 125:2260–2270. doi: 10.1016/j.clinph.2014.02.027 Pulverenti TS, Islam MA, Alsalman O, Murray LM, Harel NY, Knikou M (2019) Transspinal stimulation decreases corticospinal excitability and alters the function of spinal locomotor networks. J Neurophysiol 122:2331-2343. doi: 10.1152/jn.00554.2019 Rejc E, Angeli CA, Atkinson D, Harkema SJ (2017) Motor recovery after activity-based training with spinal cord epidural stimulation in a chronic motor complete paraplegic. Sci Rep 7:13476. doi: 10.1038/s41598-017-14003-w Savic G, Bergstrom EMK, Frankel HL, Jamous MA, Jones PW (2007) Inter-rater reliability of motor and sensory examinations performed according to American Spinal Injury Association standards. Spinal Cord 45:444-451. doi: 10.1038/sj.sc.3102044 Sayenko DG, Rath M, Ferguson AR, Burdick JW, Havton LA, Edgerton VR, Gerasimenko YP (2019) Self-assisted standing enabled by non-invasive spinal stimulation after spinal cord injury. J Neurotrauma 36:1435-1450. doi: 10.1089/neu.2018.5956 Skiadopoulos A, Knikou M (2024) Tapping into the human spinal locomotor centres with transspinal stimulation. Sci Rep 14:5990. doi: 10.1038/s41598-024-56579-0 Tajali S, Balbinot G, Pakosh M, Sayenko DG, Zariffa J, Masani K (2024) Modulations in neural pathways excitability post transcutaneous spinal cord stimulation among individuals with spinal cord injury: a systematic review. Front Neurosci 18:1372222. doi: 10.3389/fnins.2024.1372222 Tansey KE, McKay WB, Kakulas BA (2012) Restorative neurology: consideration of the new anatomy and physiology of the injured nervous system. Clin Neurol Neurosurg 114:436-440. doi: 10.1016/j.clineuro.2012.01.010 Therkildsen ER, Nielsen JB, Beck MM, Yamaguchi T, Lorentzen J (2022) The effect of cathodal transspinal direct current stimulation on tibialis anterior stretch reflex components in humans. Exp Brain Res 240:159-171. doi: 10.1007/s00221-021-06243-0 Wesselink WA, Holsheimer J, Boom HBK (1998) Analysis of current density and related parameters in spinal cord stimulation. IEEE Trans Rehabil Eng 6:200–207. doi: 10.1109/86.681186 Zaaya M, Pulverenti TS, Knikou M (2021) Transspinal stimulation and step training alter function of spinal networks in complete spinal cord injury. Spinal Cord Ser Cases 7:55. doi: 10.1038/s41394-021-00421-6 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Experimental Brain Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7365193","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504707803,"identity":"ac4a2a39-9817-419d-85a9-dcd131313c25","order_by":0,"name":"Maria Knikou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie3PMUvDQBjG8ScG0uWtXStpv8NBIBIo9rOEwLlUF0E6lJApU3BWEPwKTs4XDs4l4ppVhE4d4pZB0CM10OXEbkXuT8K94e4HF8BmO8CYAPQj4ZH+agDqd5zMQKItUR1xbv9C5qJbqu6sSzs7RhI+v7yJFvXlMUnlz1bpBIMqfCfMpo/CQKpzVhZorrxhzv2FkgRanAYEHhiJ4BCEJs5HFPoXmZ7HeiDI2ETY6xrlJ+otibK0J19mUnPoy1RxPixC38ncnohfyBpywlSck0qiQv+LR/z65J4lwZ3xYtz92Cxl/FAkZd2u0vloIJ/Gm+XZ9MZAfuDO7On3iJjpqCmn3VfYbDbbf+4bDZpZYXvw+8IAAAAASUVORK5CYII=","orcid":"","institution":"The City University of New York","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"","lastName":"Knikou","suffix":""}],"badges":[],"createdAt":"2025-08-13 12:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7365193/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7365193/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00221-025-07164-y","type":"published","date":"2025-09-25T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89861031,"identity":"24668a5f-6906-404b-a214-e8af4452f904","added_by":"auto","created_at":"2025-08-25 20:55:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3834235,"visible":true,"origin":"","legend":"\u003cp\u003eSoleus H-reflex homosynaptic depression before and after tsDCS in individuals with and without SCI (A).\u003cstrong\u003e \u003c/strong\u003eNon-rectified waveform averages of soleus H reflexes recorded at different stimulation frequencies from a noninjured subject and from an individual with AIS B SCI. (\u003cstrong\u003eB).\u003c/strong\u003eSoleus H reflexes evoked at different stimulation frequencies before (grey) and 1-day after (orange) multiple sessions of cathodal tsDCS are indicated separately for AIS A-B, AIS C-D, and noninjured subjects. On the abscissa, the stimulation frequency is indicated. Ordinate indicates the soleus H reflexes normalized to the H-reflex evoked at 0.1 Hz. Error bars denote the SD.\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/e1501a9a6bbb5e545b06bf7f.png"},{"id":89861470,"identity":"a8106401-ddec-4855-b5fe-451cac13f49d","added_by":"auto","created_at":"2025-08-25 21:03:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2272405,"visible":true,"origin":"","legend":"\u003cp\u003eNon-rectified waveform averages of soleus H reflexes evoked upon paired stimuli delivered to the posterior tibial nerve at interstimulus intervals (ISIs) of 500, 300, 100, and 60 ms. H-reflexes are shown for an individual with AIS B SCI and a noninjured subject. In both subjects, the H-reflex at 60 ms is nearly abolished.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/2c7ccc2767d4417eb25c08ab.png"},{"id":89861471,"identity":"44427ad7-b084-4ea9-a182-6d5886df94b0","added_by":"auto","created_at":"2025-08-25 21:03:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1245957,"visible":true,"origin":"","legend":"\u003cp\u003eSoleus H-reflex postactivation depression before and after tsDCS in individuals with and without SCI.\u003cstrong\u003e \u003c/strong\u003eSoleus H reflexes evoked at different interstimulus intervals before (grey) and 1-day after (orange) multiple sessions of cathodal tsDCS are indicated separately for AIS A-B, AIS C-D, and noninjured subjects. On the abscissa, the interstimulus interval is indicated. Ordinate indicates the soleus H reflex 2 amplitudes expressed as a percentage of the homonymous H-reflex 1. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 for before and after intervention. Error bars denote the SD.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/7466ab35d976637e834d5583.png"},{"id":89861034,"identity":"056296d1-20e9-49c9-af13-2ed9bf671416","added_by":"auto","created_at":"2025-08-25 20:55:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3203792,"visible":true,"origin":"","legend":"\u003cp\u003eSoleus H-reflex and M-wave recruitment input/output curves before and after transspinal direct stimulation (tsDCS) in noninjured subjects. The soleus H-reflex \u003cstrong\u003e(A)\u003c/strong\u003e and M-wave \u003cstrong\u003e(B)\u003c/strong\u003e input/output curves from all AIS C-D subjects before (black circles) and after (orange circles) 1-2 days after an average of 15 sessions of tsDCS. The corresponding sigmoid function fitted to the responses are shown. Both the soleus H-reflexes and M-waves were normalized to the homonymous maximal M-wave and grouped in multiples of stimulation intensities normalized to the homonymous 50% of the maximal M-wave. \u003cstrong\u003e(C)\u003c/strong\u003e Soleus H-reflexes were normalized to the homonymous maximal M-wave and grouped in multiples of stimulation intensities normalized to the 50% of the maximal H-reflex observed at baseline.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/cee3937b244e5ea67c1f5445.png"},{"id":89861032,"identity":"4e0b31e5-85c4-4e09-a7eb-69e62d8728d6","added_by":"auto","created_at":"2025-08-25 20:55:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2432442,"visible":true,"origin":"","legend":"\u003cp\u003eSoleus H-reflex and M-wave recruitment input/output curves before and after transspinal direct stimulation (tsDCS) in AIS C-D subjects. The soleus H-reflex \u003cstrong\u003e(A)\u003c/strong\u003e and M-wave \u003cstrong\u003e(B)\u003c/strong\u003e input/output curves from all AIS C-D subjects before (black circles) and after (orange circles) 1-2 days after an average of 15 sessions of tsDCS. The corresponding sigmoid function fitted to the responses are shown. Both the soleus H-reflexes and M-waves were normalized to the homonymous maximal M-wave and grouped in multiples of stimulation intensities normalized to the homonymous 50% of the maximal M-wave. \u003cstrong\u003e(C)\u003c/strong\u003e Soleus H-reflexes were normalized to the homonymous maximal M-wave and grouped in multiples of stimulation intensities normalized to the 50% of the maximal H-reflex observed at baseline.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/0df766021f296fa27e00631e.png"},{"id":89861912,"identity":"25f23e56-28af-43dc-9897-b88c2666b924","added_by":"auto","created_at":"2025-08-25 21:11:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3172424,"visible":true,"origin":"","legend":"\u003cp\u003eSoleus H-reflex and M-wave recruitment input/output curves before and after transspinal direct stimulation (tsDCS) in AIS A-B subjects. The soleus H-reflex \u003cstrong\u003e(A)\u003c/strong\u003e and M-wave \u003cstrong\u003e(B)\u003c/strong\u003e input/output curves from all AIS A-B subjects before (black circles) and after (orange circles) 1-2 days after an average of 15 sessions of tsDCS. The corresponding sigmoid function fitted to the responses are shown. Both the soleus H-reflexes and M-waves were normalized to the homonymous maximal M-wave and grouped in multiples of stimulation intensities normalized to the homonymous 50% of the maximal M-wave. \u003cstrong\u003e(C)\u003c/strong\u003e Soleus H-reflexes were normalized to the homonymous maximal M-wave and grouped in multiples of stimulation intensities normalized to the 50% of the maximal H-reflex observed at baseline.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/7c161e3af6f929fc1b714482.png"},{"id":92431195,"identity":"3240aa98-5304-4049-871c-de96e5089d99","added_by":"auto","created_at":"2025-09-29 16:08:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17578781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7365193/v1/2657ae9a-fe85-4a79-bfea-79ba6765cc12.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transspinal direct current stimulation for multiple sessions alters neuronal excitability but not homosynaptic inhibition in people with and without Spinal Cord Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfter spinal cord injury (SCI), recovery using neuromodulation strategies via stimulation that work synergistically with activity-dependent neuroplasticity is in great need. The impaired function of spinal circuitry and corticospinal drive and impaired processing of afferent input by the spinal circuits, and the decline in transmission of uninjured fibers lead to an altered excitability state, one of the most debilitating complications (Knikou \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Arvanian et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Barthelemy et al. 2010; Tansey et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Motor overactivity results in involuntary movements, co-contraction of antagonistic muscles, hyperreflexia, ankle clonus, and increased muscle tone, which all constitute clinical manifestations of spasticity. These clinical sequalae of pathological muscle tone cause pain and fatigue, disturb sleep, restrict daily activities like walking, sitting, and bathing, and can affect significantly rehabilitation efforts.\u003c/p\u003e\u003cp\u003eThe altered excitability state can be targeted via transspinal (or transcutaneous spinal cord) stimulation. Multiple sessions of transspinal stimulation with alternated current at low frequencies (0.2 Hz) decreases soleus H-reflex excitability, upregulates homosynaptic inhibition, decreases spasticity, and increases the net motor output of motoneurons over multiple segments in people with SCI (Knikou and Murray \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Murray and Knikou \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further, transspinal stimulation at frequencies up to 15 Hz contributes to recovery of assisted standing, while frequencies ranging from 25 to 120 Hz are adopted for recovery of stepping after SCI (Minassian et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rejc et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sayenko et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zaaya et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with significant neuromodulation effects on spinal locomotor pathways (Skiadopoulos and Knikou \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and cortical and corticospinal activity in healthy humans (Pulverenti et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Murray et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), supporting for widespread neuronal excitability changes and development of neuroplasticity upon multiple sessions.\u003c/p\u003e\u003cp\u003eAnother form of transspinal stimulation that induces neuronal changes is direct current delivered at constant low intensity. In spinal cord preparations of spinalized mice, transspinal direct current stimulation (tsDCS) alters neuronal excitability that coincides with a significant increase of glutamate analog, D-2,3-(3)H-aspartate (D-Asp) (Ahmed and Wieraszko \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Moreover, tsDCS paired with sciatic nerve stimulation alters muscle tone and increases complex multi-joint movement amplitude in spinalized or anesthetized mice (Ahmed \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In 17 healthy humans met allele carriers and 17 Val homozygotes anodal tsDCS induced a progressive leftward shift of recruitment curve of the H reflex during the stimulation that persisted for at least 15 min after current offset in Val/Val individuals (Lamy and Boakye \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, 15-min of anodal tsDCS increased spinal reflex excitability during the stimulation, while both cathodal and sham tsDCS had no significant effects (Lamy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A single session of tsDCS has been linked to modulation of activity in lemniscal, spinothalamic, and segmental motor systems (Cogiamanian et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), as well as to modulation of cortical mechanisms that persists for 30 minutes post stimulation (Murray et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eReports on neuroplasticity and neurorecovery via tsDCS in people with SCI are scarce. Collectively, the main objective of this study was to assess the effects of multiple sessions of tsDCS on soleus H-reflex excitability, homosynaptic inhibition, and clinical measures of hyperreflexia in people with chronic SCI and compare the results to those observed in a group of noninjured subjects. To meet our main objective, before and after an average of 14.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 sessions in 9 SCI and 10 sessions in 10 noninjured subjects, the soleus H-reflex recruitment curve, homosynaptic depression, postactivation depression and clinical measures of hyperreflexia were assessed.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003e All experimental and intervention procedures were approved by the City University of New York\u0026rsquo;s Institutional Biomedical Committee (IRB Number 515055) and conducted in accordance with the standards of the Declaration of Helsinki. Eligible participants gave informed, written consent prior to enrollment. Inclusion was considered if: 1) ages were between 18\u0026ndash;70 years; 2) persons were free of ferromagnetic material in the brain and/or spine; and 3) no contraindications to brain or spinal stimulation were present. Individuals with SCI were included if the injury was chronic (more than 6 months) and at or above Thoracic 12.\u003c/p\u003e\u003cp\u003eNine individuals with chronic SCI (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and 10 healthy volunteers (7 female; 27.2 \u0026plusmn; 5 years, mean \u0026plusmn; SD) free of musculoskeletal or neurological disorders completed the study. Five individuals with chronic SCI had a neurological deficit grade D on the American Spinal Injury Association Impairment Scale (AIS), 2 had AIS B, and 2 had AIS A, while the level of SCI ranged from Cervical 4 to Thoracic 11. Individuals with motor complete SCI were also included to assess changes in the presence of minimal descending and afferent input.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographics and injury characteristics of participants with chronic spinal cord injury (SCI).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAge (yrs)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePost injury (yrs)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLevel of injury\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eMotor score\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e# of sessions attended (tsDCS)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eList of medication\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eAIS\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eCause of injury\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOcean wave-related\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eBaclofen 20 mg 4xD\u003csup\u003e\u0026dagger;\u003c/sup\u003e; Cymbalta 60 mg 1xD; Oxybutynin 5 mg 3xD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMVC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eT9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGSW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eGabapentin 800 mg 3xD; Baclofen 10 mg 3xD\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSkiing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eFall \u0026amp; spinal stenosis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eGabapentin 400 mg 4xD; Baclofen 10 mg 5xD; Oxybutynin 10 3xD; Oxycontin 5 mg 2xD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDiving into shallows\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC5-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMVC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eSynthroid 225 mg 1Xd\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eFall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eOxybutynin 10mg 2x/day; Pravachol 40mg 1x/day\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003eThe level of injury corresponds to the vertebral level. Following the American Spinal Injury Association impairment scale (AIS) assessment, motor scores are indicated based on the manual muscle test of five key muscles and evaluated as 5\u0026thinsp;=\u0026thinsp;normal muscle power, 4\u0026thinsp;=\u0026thinsp;active movement against gravity with slight resistance, 3\u0026thinsp;=\u0026thinsp;active movement against gravity, 2\u0026thinsp;=\u0026thinsp;active movement with gravity eliminated, 1\u0026thinsp;=\u0026thinsp;trace muscle contraction, 0\u0026thinsp;=\u0026thinsp;no contraction. M: male; F: female; C: cervical; T: thoracic; MVC\u0026thinsp;=\u0026thinsp;Motor vehicle crash; SX\u0026thinsp;=\u0026thinsp;Surgery; GSW\u0026thinsp;=\u0026thinsp;Gunshot wound; xD\u0026thinsp;=\u0026thinsp;Times daily. \u003csup\u003e\u0026dagger;\u003c/sup\u003e Participant delayed ingestion of baclofen until after each experiment. \u003csup\u003e\u0026Dagger;\u003c/sup\u003e Participant ceased baclofen prior to the duration of the study. \u003csup\u003eҰ\u003c/sup\u003e Participant ceased baclofen after the first day of experiments.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e[Insert Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e here]\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSurface electromyography (EMG)\u003c/h3\u003e\n\u003cp\u003eSurface EMG was recorded with single bipolar differential electrodes (MA300-28, Motion Lab Systems Inc., Baton Rouge, LA) placed on lightly shaved, abraded and cleansed skin over the left and or the right soleus muscle of individuals with SCI, and the right soleus muscle of individuals without SCI. Electrode placement was secured with transparent Tegaderm film (3M Healthcare, St Paul, Minnesota, USA) for the duration of the experiments. EMG signals were amplified, filtered (10\u0026ndash;1000 Hz), sampled (2000 Hz) using a 1401-Plus system (Cambridge Electronics Design Ltd., Cambridge, UK) or LabVIEW scripts and stored for offline analysis.\u003c/p\u003e\n\u003ch3\u003eIntervention: Multiple sessions of tsDCS\u003c/h3\u003e\n\u003cp\u003eCathodal tsDCS, known to have greater and/or more persistent neuromodulatory effects beyond that of anodal stimulation (Bindman et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1964\u003c/span\u003e; Bocci et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Murray et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), was delivered using a direct current stimulator (neuroConn DC stimulator PLUS, neuroCare Group GmbH, M\u0026uuml;nchen, Germany). The active square steel mesh housed in a rubber electrode (cathode; 3.2 cm x 3.2 cm; Amrex-Zetron Inc, California, USA) and covered by a sponge soaked in 0.9% saline solution was positioned centrally over vertebra Thoracic 10 to 12 corresponding to Lumbar 1\u0026ndash;5 spinal segments. The position was determined via manual palpation of the spinal processes starting from Cervical 7, confirmed two vertebrae above the attachment point of the twelfth rib, and marked with a non-toxic surgical pen for constancy across stimulation sessions. A second saline-soaked square reference electrode (anode, same type as the cathode; 10.16 cm x 10.16 cm) was placed on the abdomen left of the umbilicus to avoid vital organs. This montage is known to produce maximal electric field potentials in a longitudinal direction along the spinal cord (Parazzini et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor all subjects, tsDCS was delivered daily during weekdays, excluding holidays, whilst the subject was lying supine with knee and hip joints flexed at 30\u0026deg; and supported by pillows. Individuals with SCI received an average of 14.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 stimulation sessions for 50.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 min per session (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Healthy control subjects received 10 stimulation sessions for an average of 44.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 min per session. Depending on the subjects\u0026rsquo; availability, additional sessions were performed to ensure the neurophysiological recordings did not occur the day after a 2-day break (e.g. weekend).\u003c/p\u003e\u003cp\u003eStimulation intensity was delivered in blocks of 5\u0026ndash;10 minutes, ramping up/down from the designated intensity over 20 s, until a total of 45 minutes of stimulation was given. To keep skin irritations minimal due to daily stimulation sessions, the intensity ranged between 1.25 and 3.0 mA, with an average stimulation intensity of 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mA. Both subject groups received similar intensities across the entire intervention despite the small increases and decreases. Current density and total charge never exceeded 0.26 mA/cm\u003csup\u003e2\u003c/sup\u003e and 878.91 mC/cm\u003csup\u003e2\u003c/sup\u003e in any stimulation session, respectively. A maximal average current density of 0.23 mA/cm\u003csup\u003e2\u003c/sup\u003e and a maximal average total charge of 630.43 mC/cm\u003csup\u003e2\u003c/sup\u003e was delivered across sessions for SCI subjects. Similar values were adopted for uninjured control subjects with a maximum average of 0.23 mA/cm\u003csup\u003e2\u003c/sup\u003e and 620 mC/cm\u003csup\u003e2\u003c/sup\u003e, respectively. These were within the safety limits of 2.3 mA/cm\u003csup\u003e3\u003c/sup\u003e for current density threshold during invasive spinal stimulation and 25 mA/cm\u003csup\u003e3\u003c/sup\u003e for pulse electrical stimulation known to cause tissue damage (McCreery et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Wesselink et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Cogiamanian et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Due to the intensity of stimulation delivered, it was impossible to blind the participants to the sessions and therefore no sham group was conducted.\u003c/p\u003e\u003cp\u003ePost-session tsDCS questionnaires were randomly completed throughout the intervention to establish presence of any adverse events. No significant changes were noted in the blood pressure of any participant during the stimulation sessions and/or experiments. The major complaint was skin redness or irritation (SCI: 69.2%; Controls: 37.5%) mainly due to the pressure of the electrode against the skin, subsiding within a few hours, but some had a slight skin rash from the salt water being applied daily. Mild reports of tingling (SCI: 38.5%; Controls: 44%), and burning or itchy sensations (SCI: 15.4%; Controls: 12.5%) during the ramp-up and down phase of stimulation were also reported. Lastly, mild back (SCI: 15.4%) and neck (Controls: 19%) pain possibly due to position requirements of the study, and a mild-moderate but transient headache (Controls: 19%) were also reported.\u003c/p\u003e\n\u003ch3\u003eNeurophysiological outcome measurements before and 1-day after multiple sessions of tsDCS\u003c/h3\u003e\n\u003cp\u003eThe soleus H-reflex was evoked according to methods we have extensively used previously in our laboratory (Knikou \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). With the subject seated, relaxed, and both feet supported by a footrest, a stainless-steel circular plate of 4 cm\u003csup\u003e2\u003c/sup\u003e in diameter (anode electrode) was secured proximally to the patella of the right leg. A rectangular single 1 ms pulse was delivered by a custom-built constant current stimulator to the posterior tibial nerve at the popliteal fossa. The most optimal stimulation site was established via a handheld monopolar stainless steel head electrode used as a probe. An optimal stimulation site corresponded to the site that Ia afferents could be excited at lower stimulation intensities without the M-wave being present. The monopolar electrode was then replaced by a pregelled disposable electrode (SureTrace, Conmed, Utica, NY, USA) that was maintained under constant pressure throughout the experiment with an athletic wrap.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSoleus H-reflex homosynaptic and postactivation depression\u003c/strong\u003e\u003cp\u003eThe soleus M-wave and H-reflex were evoked using a constant current stimulator (DS7A, Digitimer Ltd., UK) with a 1.0 ms square pulse at 0.2 Hz. The H-reflex was evoked on the ascending part of the recruitment curve and was 25\u0026ndash;40% of the maximal M-wave (Mmax). For homosynaptic depression, 15 H-reflexes were recorded at 0.1, 0.125, 0.2, 0.33, and 1.0 Hz. For postactivation depression, 20 paired stimuli were delivered randomly at the interstimulus intervals (ISIs) of 500, 300, 100, and 60 ms with a constant stimulation frequency of 0.2 Hz.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSoleus H-reflex excitability and recruitment of alpha motoneurons\u003c/strong\u003e\u003cp\u003eBefore and 1-day after tsDCS, the soleus H-reflex recruitment curve was assembled with subjects seated. The right posterior tibial nerve at the popliteal fossa was stimulated at 0.2 Hz and at least 120 responses were recorded via a customized stimulator randomly at increasing stimulation intensities. The LABVIEW software controlled and adjusted the stimulation intensity through a LabVIEW custom-made script and saved each value along with the triggering pulse and EMG response. This experimental approach ensured that the H reflex was continuously evoked at different intensities and not with uniformly increasing or decreasing consecutive values.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eClinical outcome measurements before and 1-day after tsDCS\u003c/em\u003e: The self-reported Penn spasm frequency and Penn severity scales along with the modified Ashworth scale (Adams et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Savic et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), were evaluated in individuals with SCI before and after tsDCS. Ankle clonus was scored as 3: sustained clonus, 2: prolonged but fatigable (20\u0026ndash;100 beats per minute), 1: brief but fatigable (1\u0026ndash;20 beats per minute), and 0: absent.\u003c/p\u003e\n\u003ch3\u003eData analysis and statistics\u003c/h3\u003e\n\u003cp\u003eOffline analysis of the M-waves and H-reflexes for homosynaptic and postactivation depression was performed using Spike 2 software (Cambridge Electronics Design Ltd., Cambridge, UK). For homosynaptic depression, the area under the rectified H-reflex waveform was measured for each response evoked at 0.125, 0.2, 0.33, and 1.0 Hz, and normalized to the mean homonymous H reflex amplitude evoked at 0.1 Hz. For postactivation depression, the area under the rectified H-reflex waveform was measured for each response evoked by paired stimuli at 500, 300, 100 and 60 ms. The second H reflex (H2; H-reflex evoked by the 2nd pulse) was then normalized to the first H reflex (H1; H-reflex evoked by the 1st pulse) within each pair and averaged.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor each subject, the peak-to-peak amplitude of the non-rectified soleus M wave and H reflex recorded at different stimulation intensities to assemble the recruitment curve, were measured offline with custom developed algorithms in LABVIEW (LabVIEW 8.2, National Instruments Ltd.,) and were normalized to the associated maximal M-wave. Then, the normalized soleus M-waves or H-reflexes were plotted against the actual stimulation intensities, and a Boltzmann sigmoid function (Eq.\u0026nbsp;1; SigmaPlot 11, Systat Software Inc., California, USA) was fitted to the data. From Eq.\u0026nbsp;1, the R\u003csup\u003e2\u003c/sup\u003e, slope of the function (m), and stimulus intensity corresponding to 50% of the maximal M-wave or maximal H-reflex (S50-Mmax, S50-Hmax) was established. For the M-waves, the stimulation intensities were normalized to the homonymous S50-Mmax before and after intervention. For the soleus H-reflex, the stimulation intensities were normalized to the homonymous S50-Mmax or to the S50-Hmax observed at baseline. For each and across subjects, the normalized soleus M-waves and H-reflexes were grouped based on increments of 0.05 multiples of S50-Mmax or S50-Hmax.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{M}\\left(\\text{s}\\right)=\\frac{\\text{M}\\text{m}\\text{a}\\text{x}}{(1+\\text{exp}\\left(\\text{m}\\left(\\text{S}50-\\text{s}\\right)\\right))\\:},\\:\\text{H}\\left(\\text{s}\\right)=\\frac{\\text{H}\\text{m}\\text{a}\\text{x}}{(1+\\text{exp}\\left(\\text{m}\\left(\\text{S}50-\\text{s}\\right)\\right))\\:}\\)\u003c/span\u003e\u003c/span\u003e [Eq.\u0026nbsp;1]\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eStatistical analysis was performed using SigmaPlot (Systat Software Inc., California, USA). For each neurophysiological assessment, H-reflexes were grouped as AIS C-D, AIS A-B, and noninjured subjects before and after transspinal direct current stimulation. A repeated measures analysis of variance (rmANOVA) was conducted separately for normalized H reflexes recorded at different stimulation frequencies, upon nerve stimuli at different ISIs, and for M-wave and H-reflex recruitment curves grouped based on multiples of S50-Mmax or S50-Hmax. When a statistically significant main effect was found, post-hoc Bonferroni \u003cem\u003et\u003c/em\u003e-tests for multiple comparisons were used to test for significant interactions. Significant changes in clinical assessments were established with a paired \u003cem\u003et\u003c/em\u003e-test. For all statistical tests, significance was determined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Results are presented as mean values and standard deviation (SD).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eHomosynaptic depression of the soleus H-reflex before and after multiple sessions of tsDCS in noninjured and SCI subjects\u003c/em\u003e\u003c/p\u003e\u003cp\u003eRepresentative non-waveform averages recorded before and 1-day after tsDCS from one noninjured and one SCI subject are indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. While the soleus H-reflex did decrease in amplitude as stimulation to the posterior tibial nerve occurred at lower frequencies in both subjects, tsDCS appeared not to affect the strength of homosynaptic depression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe overall amplitude of the soleus H-reflex before and after multiple sessions of tsDCS recorded at different stimulation frequencies is indicated separately for AIS A-B, AIS C-D and noninjured subjects in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. The soleus H-reflexes at 0.125 (8s), 0.2 (5s), 0.33 (3s), and 1.0 (1s) Hz were normalized to the homonymous mean reflex amplitude recorded at 0.1 (10s) Hz. In AIS A-B group, the soleus H-reflexes varied significantly across stimulation frequencies (F\u003csub\u003e3,40\u003c/sub\u003e = 19.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but there were no significant differences before and after tsDCS (F\u003csub\u003e1,40\u003c/sub\u003e = 0.02, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.87). Similarly, in AIS C-D group the statistically significant differences of soleus H-reflexes across stimulation frequencies (F\u003csub\u003e3,56\u003c/sub\u003e = 8.31, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) occurred with no significant differences before and after tsDCS (F\u003csub\u003e1,56\u003c/sub\u003e = 0.1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.75). Last, in noninjured subjects the soleus H-reflexes varied significantly across stimulation frequencies (F\u003csub\u003e3,72\u003c/sub\u003e = 75.3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 2-way rmANOVA) but there were no significant differences before and after tsDCS (F\u003csub\u003e1,72\u003c/sub\u003e = 0.52, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.47). These results support that some homosynaptic depression is present after SCI but of lesser strength compared to noninjured subjects, and that its strength remained unaltered by multiple sessions of tsDCS in both noninjured and SCI subjects.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePostactivation depression of the soleus H-reflex before and after multiple sessions of tsDCS in noninjured and SCI subjects\u003c/em\u003e\u003c/p\u003e\u003cp\u003eRepresentative examples of non-rectified soleus H-reflex waveform averages recorded upon paired tibial nerve stimuli at ISIs ranging from 500 to 60 ms from one noninjured and one SCI subject before and 1-day after tsDCS are indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. It is evident that the soleus H reflex was significantly depressed upon paired tibial nerve stimuli and almost abolished at the ISI of 60 ms for both noninjured and SCI subjects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe overall amplitude of the soleus H-reflex before and 1-day after multiple sessions of tsDCS recorded at different ISIs of paired pulses is indicated separately for AIS A-B, AIS C-D and noninjured subjects in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. For all graphs, the H-reflex evoked by the second pulse (H-reflex2) is shown as a percentage of the H-reflex evoked by the first pulse (H-reflex 1) within the same sweep or paired pulses. In the AIS A-B group, the soleus H-reflexes varied significantly across ISIs (F\u003csub\u003e3,40\u003c/sub\u003e = 31.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not as a function of time (F\u003csub\u003e1,40\u003c/sub\u003e = 0.39, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.53; 2-way rmANOVA). In the AIS C-D group, the H-reflex 2 was statistically significant different from H-reflex 1 as a function of ISIs (F\u003csub\u003e3,56\u003c/sub\u003e = 64.45, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and time (F\u003csub\u003e1,56\u003c/sub\u003e = 8.62, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005). Holm-Sidak pairwise multiple comparisons showed that the H-reflex at the 300 ms was significantly different before and after tsDCS (t\u0026thinsp;=\u0026thinsp;3.17, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). Last, in noninjured subjects the soleus H-reflexes varied significantly across ISIs (F\u003csub\u003e3,72\u003c/sub\u003e = 23.27, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not as a function of time (F\u003csub\u003e1,72\u003c/sub\u003e = 0.54, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.46; 2-way rmANOVA). These results support for presence of postactivation depression after SCI, and that its strength was not affected by tsDCS in motor complete SCI and noninjured subjects but reversed to facilitation at 300 ms ISI in motor incomplete SCI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003e\u003cem\u003eRecruitment of soleus motoneurons before and after multiple sessions of tsDCS in noninjured and SCI subjects\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo assess changes in the excitability state and recruitment order of alpha motoneurons by muscle spindle group Ia afferents, the soleus M-wave and H-reflex recruitment curves were assembled in noninjured and SCI subjects while seated before and 1-day after tsDCS. The soleus H-reflexes and M-waves from all noninjured subjects plotted against multiples of stimulation intensities along with the sigmoid fit are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B. The soleus H-reflexes varied significantly across stimulation intensities (F\u003csub\u003e13,247\u003c/sub\u003e = 45.24, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not based on the time of testing (F\u003csub\u003e1,247\u003c/sub\u003e = 0.34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.56; 2-way rmANOVA for H-reflexes grouped across subjects from 0.26 to 0.91 xS50-Mmax), while an interaction between normalized intensities and time was not present (F\u003csub\u003e13\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.228, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.99). Non-significant differences in the M-wave recruitment curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) as a function of time were present (F\u003csub\u003e25,411\u003c/sub\u003e = 9.75, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35; 2-way rmANOVA for M-waves grouped across subjects from 0.26 to 1.51 xS50-Mmax). However, we should note that when the normalized H-reflexes were grouped across subjects with stimulation intensities normalized to the predicted S50-Hmax at baseline from 0.46 to 1.41 xS50-Hmax (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), 2-way rmANOVA showed significant differences across stimulation intensities (F\u003csub\u003e19,333\u003c/sub\u003e = 17.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and as a function of the time of testing (F\u003csub\u003e1,333\u003c/sub\u003e = 12.21, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe soleus H-reflexes and M-waves from all AIC C-D subjects along with the sigmoid fit are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B. The soleus H-reflexes varied significantly across stimulation intensities (F\u003csub\u003e15,187\u003c/sub\u003e = 15.27, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and also based on the time of testing (F\u003csub\u003e1,187\u003c/sub\u003e = 16.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 2-way rmANOVA for H-reflexes grouped across subjects from 0.26 to 1.01 xS50-Mmax), but an interaction between normalized intensities and time was not present (F\u003csub\u003e15\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.99). Non-significant differences in the M-wave recruitment curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) as a function of time were present (F\u003csub\u003e25,258\u003c/sub\u003e = 22.17 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35; 2-way rmANOVA for M-waves grouped across subjects from 0.26 to 1.51 xS50-Mmax). In the case of AIS C-D subjects, when the normalized H-reflexes were grouped across subjects with stimulation intensities normalized to the predicted S50-Hmax at baseline from 0.26 to 1.31 xS50-Hmax (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), 2-way rmANOVA showed significant differences across stimulation intensities (F\u003csub\u003e21,252\u003c/sub\u003e = 15.68, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and as a function of the time of testing (F\u003csub\u003e1,252\u003c/sub\u003e = 5.36, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021). This result suggests that the H-reflex depression after tsDCS was present regardless of the normalization factor used for stimulation intensities (S50Mmax or S50Hmax) although only in this subject group the peak slope, estimated as (m\u0026times;Hmax)/4, was reduced after tsDCS (13.57\u0026thinsp;\u0026plusmn;\u0026thinsp;9.71; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04, t-test) compared to before tsDCS (47.77\u0026thinsp;\u0026plusmn;\u0026thinsp;51.75).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe soleus H-reflexes and M-waves from all AIC A-B subjects along with the sigmoid fit are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B. The soleus H-reflexes varied significantly across stimulation intensities (F\u003csub\u003e15,140\u003c/sub\u003e = 11.57, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but were not significantly different after tsDCS (F\u003csub\u003e1,140\u003c/sub\u003e = 0.87, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35; 2-way rmANOVA for H-reflexes grouped across subjects from 0.26 to 1.01 xS50-Mmax). Non-significant differences in the M-wave recruitment curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) as a function of time were present (F\u003csub\u003e25,189\u003c/sub\u003e = 0.77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.38; 2-way rmANOVA for M-waves grouped across subjects from 0.26 to 1.51 xS50-Mmax). In the case of AIS A-B subjects, when the normalized H-reflexes were grouped across subjects with stimulation intensities normalized to the predicted S50-Hmax at baseline from 0.66 to 1.41 xS50-Hmax (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), 2-way rmANOVA showed significant differences across stimulation intensities (F\u003csub\u003e15,148\u003c/sub\u003e = 9.25, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and as a function of the time of testing (F\u003csub\u003e1,148\u003c/sub\u003e = 72.25, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFrom the sigmoid fit to the H-reflex normalized to the homonymous Mmax and plotted against actual values of stimulation intensities, the m function of the slope was decreased in AIS A-B (from 1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 to 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04), the peak slope was decreased in AIS C-D (from 47.77\u0026thinsp;\u0026plusmn;\u0026thinsp;51.75 to 13.57\u0026thinsp;\u0026plusmn;\u0026thinsp;9.71, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04), and the Hmax as a percentage of the Mmax was decreased in AIS C-D (from 81.91\u0026thinsp;\u0026plusmn;\u0026thinsp;12.18 to 60.06\u0026thinsp;\u0026plusmn;\u0026thinsp;14.13, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). No changes were observed in any sigmoid parameters (data not shown) in noninjured subjects.\u003c/p\u003e\u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e near here]\u003c/p\u003e\n\u003ch3\u003eClinically evaluated hyperreflexia before and after tsDCS in individuals with SCI\u003c/h3\u003e\n\u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the results of clinical assessments of hyperreflexia performed before and 1/2-days after cessation of tsDCS are indicated. The Penn spasm frequency, spasm severity and ankle clonus in both legs were not statistically significantly different before and after tsDCS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;.05 for all).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical assessments of hyperreflexia before and after tsDCS in SCI.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003ePenn Spasm Frequency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003ePenn Spasm Severity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eAnkle Clonus\u003c/p\u003e\u003cp\u003e(Left leg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eAnkle Clonus\u003c/p\u003e\u003cp\u003e(Right leg)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubject ID\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eFor each subject, the self-reported Penn Spasm Frequency and Penn Spasm Severity are indicated before and after repeated cathodal transspinal stimulation. Ankle clonus for both left and right legs are indicated for before and after transspinal stimulation and evaluated as 0: absent clonus, 1: fatigable, 1\u0026ndash;20 beats per minute, 2: fatigable, 20\u0026ndash;100 beats per minute, 3: non-fatigable, continuous sustained clonus. \u003cem\u003eP\u003c/em\u003e-values are indicated for before and after tsDCS comparisons based on a paired t-test. \u003csup\u003ea\u003c/sup\u003e: has cutaneous sensation but absent sensation of temperature. \u003csup\u003eb\u003c/sup\u003e Movement at the hips initiates spasms and ankle clonus. \u003csup\u003ec\u003c/sup\u003e flexor and extensor spasms were present in both legs.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e[Insert Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e near here]\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first study on spinal neuroplasticity in both noninjured and SCI persons 1\u0026ndash;2 days after multiple daily sessions of cathodal tsDCS. We used the soleus H-reflex, a neurophysiological probe for functional activity and recovery of spinal neuronal circuits and motoneurons as well as a biomarker of hyperreflexia and spasticity (Knikou \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, 2010). We found that daily sessions of tsDCS for approximately 1 hour did not alter the strength of homosynaptic depression in both subject groups, reversed postactivation depression to facilitation in AIS C-D, produced depression of reflex excitability in both subject groups that was revealed when intensities were normalized to the S50-Hmax at baseline, while clinically assessed hyperreflexia remained unaltered.\u003c/p\u003e\u003cp\u003eWhile there are no systematic investigations on spinal inhibition after multiple sessions of tsDCS in SCI persons, a single 20-min session of anodal tsDCS did not affect homosynaptic depression of the flexor carpi radialis (FCR) H-reflex in noninjured subjects (Donges et al. 2017). In contrast, the soleus H-reflex homosynaptic depression in noninjured subjects was either decreased or increased after anodal and cathodal tsDCS, respectively (Winkler and Straube 2010). We have previously reported unaltered homosynaptic depression after a single or multiple sessions in healthy control subjects and increase of homosynaptic depression in SCI after multiple sessions of cathodal transspinal stimulation that produces intermittent depolarization of myelinated afferent fibers and motoneurons over multiple segments (Knikou et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Knikou and Murray \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study we found that homosynaptic depression, although present but weaker in SCI compared to noninjured subjects, remained unaltered by tsDCS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Homosynaptic depression is produced by repetitive discharges of Ia afferents and mediated by reduced transmitter release at the Ia-motoneuron synapse reducing the strength of transsynaptic depolarization of alpha motoneurons (Magladery and McDougal 1950). Because homosynaptic depression requires consecutive activation of the same group Ia afferents and occurs at the same Ia-motoneuron synapse without any contribution from motor axons, we conclude that tsDCS did not affect this circuit which is known to alter after SCI (Mailis and Ashby \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Our findings are consistent with the unaltered clinically evaluated ankle clonus (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and the upregulated excitability of myelinated nerve fibers and dorsal horn field potentials beyond the polarization period during cathodal direct current applied locally (Jankowska \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jankowska et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Although homosynaptic depression is not related to the classical presynaptic inhibition that requires activation of a diverse network of inhibitory interneurons and concomitant primary afferent depolarization, it does gate proprioceptive inputs in resting individuals. Homosynaptic depression is regularly used as an indicator of calcium release probability while the spasticity and spasms that partly result from malfunction of this neuronal mechanism have been partly associated with enhanced activation of calcium-mediated persistent inward currents (Norton et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; D\u0026rsquo;Amico et al. 2013; ElBasiouny et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, we can conclude that tsDCS did not effectively affected persistent inward currents exerted at the somata and dendrites of motoneurons.\u003c/p\u003e\u003cp\u003ePostactivation depression induced by paired stimuli to the tibial nerve was present at baseline in both AIS A-B and AIS C-D subjects but of lesser strength compared to noninjured subjects consistent to our previous reports (Knikou and Murray \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and reversed to postsynaptic facilitation after tsDCS in AIS C-D subjects at the ISI of 300 ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A conditioning H1 stimulus on a testing H2 stimulus results in depression of H2 responses that is maximal at 225 ms while recovery of reflex depression starts at 300 ms (Taborikova and Sax 1969). Because removal of inhibition occurred at long latencies, the effects have been attributed to long loop reflex circuits (Chofflon et al. 1982), suggesting that this type of postactivation depression is not mediated by similar mechanisms to that of homosynaptic depression. Removal of depression at the ISI of 300 ms can be attributed to potentiation of postsynaptic excitation or intercurrent facilitation (Taborikova and Sax 1969). Long-term potentiation (LTP) induced by tsDCS might have mediated this facilitation as LTP is a long-lasting increase in synaptic efficacy that persists for hours or days. LTP is primarily driven by an increase in volume and ionotropic glutamate receptor density in postsynaptic spines, while increased neurotransmitter release at presynaptic terminals is also involved (Bliss and Gardner-Medwin \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Hayashi \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Choi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We should be cautious with this assumption because LTP-mediated plasticity mechanisms in the spinal cord require high frequency stimulation (Bear and Malenka \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). While the neuronal mechanisms are not clear, multiple sessions of tsDCS reversed postactivation depression to facilitation which may further contribute to spasticity and spasms in SCI persons.\u003c/p\u003e\u003cp\u003eThe maximal H-reflex amplitude was suppressed in AIS C-D subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In all subject groups, when the H-reflexes were grouped with stimulation intensities being normalized to the predicted S50-Hmax at baseline a generalized depression of soleus H-reflex excitability was apparent (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This suggests that grouping of H-reflexes per multiples of stimulation intensities shifted to the right after tsDCS increasing the threshold of excitability and thus reducing the recruitment of motoneurons. Our findings are consistent with the suppressed soleus H-reflex recruitment input-output curve after a single session of cathodal tsDCS in healthy subjects (Murray et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and multiple sessions of transspinal stimulation in SCI (Knikou and Murray \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, facilitation of the long latency tibialis anterior stretch reflex has also been reported after cathodal tsDCS (Therkildsen et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The changes in the H-reflex recruitment input-output curve suggest for altered responsiveness of motoneurons to proprioceptive afferent inputs in the injured human spinal cord after tsDCS.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eThe research project that this study was a part of concentrated on the effects of transspinal stimulation with direct and alternated current in noninjured and SCI subjects. Due to the nature of the research project and to the fact that subjects can differentiate between active and sham conditions at these intensities (Murray and Knikou \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), we did not test stimulation under sham conditions. Further, an investigation of spinal interneuronal circuits involving presynaptic and postsynaptic inhibitory mechanisms was not conducted. More systematic investigations are needed to delineate the tsDCS-induced neuroplasticity in people with and without SCI.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides evidence on neurophysiological changes after multiple sessions of tsDCS in noninjured and SCI persons. tsDCS did not affect homosynaptic depression, reversed postactivation depression to facilitation, produced a generalized depression of soleus H-reflex excitability, but did not reduce clinically assessed hyperreflexia in individuals with motor incomplete and complete SCI. It remains to be shown if these effects are transferred to functional changes in movement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author gratefully thanks all research participants, their families and caregivers for their time and dedication to the study, and the Klab4Recovery staff for administration of all stimulation sessions and their help during data acquisition.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Craig H. Neilsen Foundation (Grant No. 339705) awarded to MK.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams MM, Ginis KA, Hicks AL (2007) The spinal cord injury spasticity evaluation tool: development and evaluation. Arch Phys Med Rehab 88:1185-1192. doi: 10.1016/j.apmr.2007.06.012\u003c/li\u003e\n\u003cli\u003eAhmed Z, Wieraszko A (2012) Trans-spinal direct current enhances corticospinal output and stimulation-evoked release of glutamate analog, D-2,3-\u0026sup3;H-aspartic acid. J Appl Physiol 112:1576-1592. doi: 10.1152/japplphysiol.00967.2011\u003c/li\u003e\n\u003cli\u003eAhmed Z (2013) Effects of cathodal trans-spinal direct current stimulation on mouse spinal network and complex multijoint movements. J Neurosci 33:14949-14957. doi: 10.1523/JNEUROSCI.2793-13.2013\u003c/li\u003e\n\u003cli\u003eAhmed Z (2014) Trans-spinal direct current stimulation alters muscle tone in mice with and without spinal cord injury with spasticity. J Neurosci 34:1701-1709. doi: 10.1523/JNEUROSCI.4445-13.2014\u003c/li\u003e\n\u003cli\u003eArvanian VL, Schnell L, Lou L, Golshani R, Hunanyan A, Ghosh A, Pearse DD, Robinson JK, Schwab ME, Fawcett JW, Mendell LM (2009) Chronic spinal hemisection in rats induces a progressive decline in transmission in uninjured fibers to motoneurons. Exp Neurol 216:471-480. doi: 10.1016/j.expneurol.2009.01.004\u003c/li\u003e\n\u003cli\u003eBączyk M, Jankowska E (2012) Long-term effects of direct current are reproduced by intermittent depolarization of myelinated nerve fibers. J Neurophysiol 120:1173-1185. doi: 10.1152/jn.00236.2018\u003c/li\u003e\n\u003cli\u003eBarth\u0026eacute;lemy D, Willerslev-Olsen M, Lundell H, Conway BA, Knudsen H, Biering-S\u0026oslash;rensen F, Nielsen JB (2010) Impaired transmission in the corticospinal tract and gait disability in spinal cord injured persons. J Neurophysiol 104:1167-1176. doi: 10.1152/jn.00382.2010\u003c/li\u003e\n\u003cli\u003eBear MF, Malenka RC (1994). Synaptic plasticity: LTP and LTD. Curr Opin Neurobiol 4:389-399.\u003c/li\u003e\n\u003cli\u003eBindman LJ, Lippold OC, Redfearn JW (1964) The action of brief polarizing currents on the cerebral cortex of the rat (1) during current flow and (2) in the production of long-lasting after-effects. J Physiol 172:369\u0026ndash;382.\u003c/li\u003e\n\u003cli\u003eBliss TV, Gardner-Medwin AR (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the unanaestetized rabbit following stimulation of the perforant path. J Physiol 232:357-374. doi: 10.1113/jphysiol.1973.sp010274\u003c/li\u003e\n\u003cli\u003eBocci T, Vannini B, Torzini A, Mazzatenta A, Vergari M, Cogia-manian F, Priori A, Sartucci F (2014) Cathodal transcutaneous spinal direct current stimulation (tsDCS) improves motor unit recruitment in healthy subjects. Neurosci Lett 578:75\u0026ndash;79. doi: 10.1016/j.neulet.2014.06.037\u003c/li\u003e\n\u003cli\u003eChoi JH, Sim SE, Kim JI, Choi DI, Oh J, Ye S, Lee J, Kim T, Ko HG, Lim CS, Kaang BK (2018) Interregional synaptic maps among engram cells underlie memory formation. Science 360(6387):430-435. doi: 10.1126/science.aas9204\u003c/li\u003e\n\u003cli\u003eCogiamanian F, Ardolino G, Vergari M, Ferrucci R, Ciocca M, Scelzo E, Barbieri S, Priori A (2012) Transcutaneous spinal direct current stimulation. Front Psychiatry 3:63. doi: 10.3389/fpsyt.2012.00063\u003c/li\u003e\n\u003cli\u003eCogiamanian F, Vergari M, Schiaffi E, Marceglia S, Ardolino G, Barbieri S, Priori A (2011) Transcutaneous spinal cord direct current stimulation inhibits the lower limb nociceptive flexion reflex in human beings. Pain 152:370-375. doi: 10.1016/j.pain.2010.10.041\u003c/li\u003e\n\u003cli\u003eD\u0026apos;Amico JM, Murray KC, Li Y, Chan KM, Finlay MG, Bennett DJ, Gorassini MA (2013) Constitutively active 5-HT2/\u0026alpha;1 receptors facilitate muscle spasms after human spinal cord injury. J Neurophysiol 109:1473-1484. doi: 10.1152/jn.00821.2012\u003c/li\u003e\n\u003cli\u003eElBasiouny SM, Schuster JE, Heckman CJ (2010) Persistent inward currents in spinal motoneurons: important for normal function but potentially harmful after spinal cord injury and in amyotrophic lateral sclerosis. Clin Neurophysiol 121:1669-1679. doi: 10.1016/j.clinph.2009.12.041\u003c/li\u003e\n\u003cli\u003eHayashi Y (2022) Molecular mechanism of hippocampal long-term potentiation - Towards multiscale understanding of learning and memory. Neurosci Res 175:3-15. doi: 10.1016/j.neures.2021.08.001\u003c/li\u003e\n\u003cli\u003eJankowska E, Hammar I (2021) The plasticity of nerve fibers: the prolonged effects of polarization of afferent fibers. J Neurophysiol 126:1568-1591. doi: 10.1152/jn.00718.2020\u003c/li\u003e\n\u003cli\u003eJankowska E, Kaczmarek D, Bolzoni F, Hammar I (2017) Long-lasting increase in axonal excitability after epidurally applied DC. J Neurophysiol 118:1210-1220. doi: 10.1152/jn.00148.2017\u003c/li\u003e\n\u003cli\u003eJankowska E (2017) Spinal control of motor outputs by intrinsic and externally induced electric field potentials. J Neurophysiol 118:1221-1234. doi: 10.1152/jn.00169.2017\u003c/li\u003e\n\u003cli\u003eKnikou M, Dixon L, Santora D, Ibrahim MM (2015) Transspinal constant-current long-lasting stimulation: a new method to induce cortical and corticospinal plasticity. J Neurophysiol 114:1486-99. doi: 10.1152/jn.00449.2015\u003c/li\u003e\n\u003cli\u003eKnikou M, Murray LM (2019) Repeated transspinal stimulation decreases soleus H-reflex excitability and restores spinal inhibition in human spinal cord injury. PLoS One 14(9):e0223135. doi: 10.1371/journal.pone.0223135\u003c/li\u003e\n\u003cli\u003eKnikou M (2007) Plantar cutaneous input modulates differently spinal reflexes in subjects with intact and injured spinal cord. Spinal Cord 45:69-77. doi: 10.1038/sj.sc.3101917\u003c/li\u003e\n\u003cli\u003eKnikou M (2008) The H-reflex as a probe: pathways and pitfalls. J Neurosci Methods 171:1-12. doi: 10.1016/j.jneumeth.2008.02.012\u003c/li\u003e\n\u003cli\u003eLamy JC, Boakye M (2013) BDNF Val66Met polymorphism alters spinal DC stimulation-induced plasticity in humans. J Neurophysiol 110:109-116. doi: 10.1152/jn.00116.2013\u003c/li\u003e\n\u003cli\u003eLamy JC, Ho C, Badel A, Arrigo RT, Boakye M (2012) Modulation of soleus H reflex by spinal DC stimulation in humans. J Neurophysiol 108:906-914. doi: 10.1152/jn.10898.2011\u003c/li\u003e\n\u003cli\u003eLi T, Chen J (2025) Spinal cord stimulation for functional restoration in spinal cord injury: A narrative review. Cureus 17(2):e78610. doi: 10.7759/cureus.78610\u003c/li\u003e\n\u003cli\u003eMailis A, Ashby P (1990) Alterations in group Ia projections to motoneurons following spinal lesions in humans. J Neurophysiol 64:637-647. doi: 10.1152/jn.1990.64.2.637\u003c/li\u003e\n\u003cli\u003eMcCreery DB, Agnew WF, Yuen TG, Bullara L (1990) Charge density and charge per phase as cofactors in neural injury induced by electrical stimulation. IEEE Trans Biomed Eng 37:996-1001. doi: 10.1109/10.102812\u003c/li\u003e\n\u003cli\u003eMinassian K, McKay WB, Binder H, Hofstoetter US (2016) Targeting lumbar spinal neural circuitry by epidural stimulation to restore motor function after spinal cord injury. Neurotherapeutics 13:284-294. doi: 10.1007/s13311-016-0421-y\u003c/li\u003e\n\u003cli\u003eMurray LM, Islam MA, Knikou M (2019) Cortical and subcortical contributions to neuroplasticity after repetitive transspinal stimulation in humans. Neural Plast 2019:4750768. doi: 10.1155/2019/4750768\u003c/li\u003e\n\u003cli\u003eMurray LM, Knikou M (2019) Repeated cathodal transspinal pulse and direct current stimulation modulate cortical and corticospinal excitability differently in healthy humans. Exp Brain Res 237:1841-1852. doi: 10.1007/s00221-019-05559-2\u003c/li\u003e\n\u003cli\u003eMurray LM, Knikou M (2019) Transspinal stimulation increases motoneuron output of multiple segments in human spinal cord injury. PLoS One 14(3):e0213696. doi: 10.1371/journal.pone.0213696\u003c/li\u003e\n\u003cli\u003eMurray LM, Tahayori B, Knikou M (2018) Transspinal direct current stimulation produces persistent plasticity in human motor pathways. Sci Rep 8:717. doi: 10.1038/s4159 8-017-18872-z\u003c/li\u003e\n\u003cli\u003eNorton JA, Bennett DJ, Knash ME, Murray KC, Gorassini MA (2008) Changes in sensory-evoked synaptic activation of motoneurons after spinal cord injury in man. Brain 131:1478-1491. doi: 10.1093/brain/awn050\u003c/li\u003e\n\u003cli\u003eParazzini M, Fiocchi S, Liorni I, Rossi E, Cogiamanian F, Vergari M, Priori A, Ravazzani P (2014) Modeling the current density generated by transcutaneous spinal direct current stimulation (tsDCS). Clin Neurophysiol 125:2260\u0026ndash;2270. doi: 10.1016/j.clinph.2014.02.027\u003c/li\u003e\n\u003cli\u003ePulverenti TS, Islam MA, Alsalman O, Murray LM, Harel NY, Knikou M (2019) Transspinal stimulation decreases corticospinal excitability and alters the function of spinal locomotor networks. J Neurophysiol 122:2331-2343. doi: 10.1152/jn.00554.2019\u003c/li\u003e\n\u003cli\u003eRejc E, Angeli CA, Atkinson D, Harkema SJ (2017) Motor recovery after activity-based training with spinal cord epidural stimulation in a chronic motor complete paraplegic. Sci Rep 7:13476. doi: 10.1038/s41598-017-14003-w\u003c/li\u003e\n\u003cli\u003eSavic G, Bergstrom EMK, Frankel HL, Jamous MA, Jones PW (2007) Inter-rater reliability of motor and sensory examinations performed according to American Spinal Injury Association standards. Spinal Cord 45:444-451. doi: 10.1038/sj.sc.3102044\u003c/li\u003e\n\u003cli\u003eSayenko DG, Rath M, Ferguson AR, Burdick JW, Havton LA, Edgerton VR, Gerasimenko YP (2019) Self-assisted standing enabled by non-invasive spinal stimulation after spinal cord injury. J Neurotrauma 36:1435-1450. doi: 10.1089/neu.2018.5956\u003c/li\u003e\n\u003cli\u003eSkiadopoulos A, Knikou M (2024) Tapping into the human spinal locomotor centres with transspinal stimulation. Sci Rep 14:5990. doi: 10.1038/s41598-024-56579-0\u003c/li\u003e\n\u003cli\u003eTajali S, Balbinot G, Pakosh M, Sayenko DG, Zariffa J, Masani K (2024) Modulations in neural pathways excitability post transcutaneous spinal cord stimulation among individuals with spinal cord injury: a systematic review. Front Neurosci 18:1372222. doi: 10.3389/fnins.2024.1372222\u003c/li\u003e\n\u003cli\u003eTansey KE, McKay WB, Kakulas BA (2012) Restorative neurology: consideration of the new anatomy and physiology of the injured nervous system. Clin Neurol Neurosurg 114:436-440. doi: 10.1016/j.clineuro.2012.01.010\u003c/li\u003e\n\u003cli\u003eTherkildsen ER, Nielsen JB, Beck MM, Yamaguchi T, Lorentzen J (2022) The effect of cathodal transspinal direct current stimulation on tibialis anterior stretch reflex components in humans. Exp Brain Res 240:159-171. doi: 10.1007/s00221-021-06243-0\u003c/li\u003e\n\u003cli\u003eWesselink WA, Holsheimer J, Boom HBK (1998) Analysis of current density and related parameters in spinal cord stimulation. IEEE Trans Rehabil Eng 6:200\u0026ndash;207. doi: 10.1109/86.681186\u003c/li\u003e\n\u003cli\u003eZaaya M, Pulverenti TS, Knikou M (2021) Transspinal stimulation and step training alter function of spinal networks in complete spinal cord injury. Spinal Cord Ser Cases 7:55. doi: 10.1038/s41394-021-00421-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Transspinal DC stimulation, H-reflex, Spinal Cord Injury, Neuromodulation, Neurorecovery","lastPublishedDoi":"10.21203/rs.3.rs-7365193/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7365193/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTransspinal stimulation with direct current or at intensities and frequencies that produce intermittent depolarization of motoneurons can be an adjunct treatment strategy for spasticity and recovery of movement in persons with spinal cord injury (SCI). The main objective of this study was to assess neuroplasticity after multiple sessions of transspinal direct current stimulation (tsDCS) in people with and without SCI. Nine SCI and 10 noninjured subjects received daily cathodal tsDCS over Thoracic 10 while supine with an average stimulation intensity of 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mA. SCI and noninjured subjects received an average of 15 and 10 stimulation sessions, respectively. Before and 1\u0026ndash;2 days post intervention, we assessed changes in soleus H-reflex recruitment input-output curves, homosynaptic depression and postactivation depression. tsDCS for approximately 1 hour did not alter the strength of homosynaptic depression in both subject groups, but reversed postactivation depression to facilitation in AIS C-D subjects. tsDCS resulted in depression of reflex excitability in both subject groups, but without significant changes in clinically assessed hyperreflexia. The results indicate decreased reflex hyperexcitability without recovery of spinal inhibitory control in the injured human spinal cord after tsDCS. More systematic investigations are needed to delineate the tsDCS-induced neuroplasticity of spinal neuronal networks in people with SCI and thus be able to develop effective treatments.\u003c/p\u003e","manuscriptTitle":"Transspinal direct current stimulation for multiple sessions alters neuronal excitability but not homosynaptic inhibition in people with and without Spinal Cord Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 20:55:40","doi":"10.21203/rs.3.rs-7365193/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3eb619ed-54c9-4c60-b0ed-0d7856508283","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:07:33+00:00","versionOfRecord":{"articleIdentity":"rs-7365193","link":"https://doi.org/10.1007/s00221-025-07164-y","journal":{"identity":"experimental-brain-research","isVorOnly":false,"title":"Experimental Brain Research"},"publishedOn":"2025-09-25 15:58:19","publishedOnDateReadable":"September 25th, 2025"},"versionCreatedAt":"2025-08-25 20:55:40","video":"","vorDoi":"10.1007/s00221-025-07164-y","vorDoiUrl":"https://doi.org/10.1007/s00221-025-07164-y","workflowStages":[]},"version":"v1","identity":"rs-7365193","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7365193","identity":"rs-7365193","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}