Cervical and Lumbosacral Transspinal Stimulation to Reconnect the Injured Human Spinal Cord
Phase: NA
Conditions: Spinal Cord Injuries (Complete and Incomplete)
Sponsor: Maria Knikou, PT, MBA, PhD

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Official title: 
Cervical and Lumbosacral Transspinal Stimulation to Reconnect the Injured 
Human Spinal Cord 
 
 
NCT number: Not assigned yet 
 
 
Document Date: 9/10/2025 
 
 

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Study Protocol 
Determine if cervical and lumbosacral transspinal stimulation reconnects the injured human 
spinal cord, strengthens the weak remnant neuronal pathways, and augments the benefits of 
locomotor training. 
We will perform a pilot clinical trial on paired cervical and lumbar transspinal alone at rest or 
during locomotor training in people with SCI to establish safety based on adverse events, and 
efficacy based on clinical assessments and neurophysiological biomarkers. We hypothesize that 
cervical and lumbosacral transspinal stimulation administered alone at rest improves non-somatic 
functions and decreases spasticity, but interlimb coordination along with walking performance 
improves more in the group that receives cervical and lumbosacral transspinal stimulation during 
assisted stepping. 
Participants 
A total of 24 people with incomplete SCI (AIS B, C, D) will be enrolled to participate and will 
randomly be assigned to receive cervical and lumbosacral transspinal stimulation alone at rest or 
during assisted stepping. 
Intervention 
Cervical 
and 
lumbosacral 
transspinal 
tonic 
stimulation 
(Figure 1) will be delivered at a 
frequency of 30 Hz (charge-
balanced, symmetric, biphasic 
rectangular pulses of a 1-ms width 
per phase; DS8R, Digitimer Ltd., 
UK) at paresthesia levels or higher 
depending on each subject’s 
comfort while supine. This group 
will provide evidence on changes 
of neurophysiological and clinical 
outcomes attributed solely to 
cervical 
and 
lumbosacral 
transspinal stimulation. 
Another 
group 
will 
receive 
cervical 
and 
lumbosacral 
transspinal tonic stimulation at 30 
Hz during assisted stepping with 
the Lokomat 6 Pro ®.  In both 
groups, the soleus TEP threshold 
will be tracked over the course of 
treatment, as this relates to 
responsiveness 
of 
spinal 
motoneurons. 
Locomotor training will depend 
on the ability of each participant to 
Figure 1. Schematic illustration of the surface electrodes for cervical 
and thoracolumbar transspinal stimulation. Active electrodes are 
placed on the cervical and thoracolumbar spinal cord and reference 
electrodes on the clavicles and iliac crests. 

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step without foot dragging.  Over the training course, we will use the chart in Figure 2 to adjust 
body weight support, ankle straps position, and leg guidance force (Knikou 2013).  Specifically, 
the tension of the ankle straps will be adjusted based on the right and left tibialis anterior muscle 
strength evaluated every 2 weeks.  Body weight support and leg guidance force will be adjusted 
based on presence or absence of knee buckling during standing.  Body weight support, ankle straps 
position, and leg guidance force may affect both neurophysiological biomarkers and clinical 
outcomes, but they will be individualized as is the case during outpatient rehabilitation. 
 
Figure 2. Locomotor training progression protocol. The training protocol considers factors that are used to 
determine decreases in body weight support (BWS), increases in treadmill speed, and position of toe straps of the 
ankle braces over the course of the BWS robotic-assisted step training. 
 
Criteria for Selection of Therapeutic Intervention Parameters 
Transspinal stimulation will be delivered at 30 Hz because this frequency facilitates voluntary 
movement, modulates spinal circuits, and produces rhythmic motor activity in both legs 
(Gerasimenko et al. 2015; Danner et al. 2015).  Medium frequencies increase neural excitability 

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compared to low frequencies that produce the opposite effects.  Recent studies have shown that 
transspinal stimulation improves trunk control, likely because of the bilateral leg extension 
produced at higher intensities (Sayenko et al. 2018 and personal observations).  This motivated us 
to use 30 Hz for transspinal stimulation.  There is a possibility that transspinal stimulation delivered 
during assisted stepping blocks walking-related proprioceptive signals from reaching the spinal 
cord (Formento et al. 2018; Knikou and Murray 2018).  However, only when cervical and 
lumbosacral transspinal stimulation is delivered during stepping we will be able to establish if this 
stimulation targets neurons connecting the enlargements of the spinal cord.  A consensus regarding 
the ‘dosage’ of locomotor training (intensity, volume, duration) has not been reached, with 20 - 
120 sessions being reported (Knikou 2013; Morrison et al. 2018).   Each SCI participant enrolled 
in the pilot clinical trial will receive 5 sessions/week because it is superior to 2 or 3 sessions/week 
(Deng et al. 2019; Zhao et al. 2022). 
Neurophysiological Biomarkers Before and After Intervention 
For both groups we will perform state-of-the-art experiments to establish changes in 
neurophysiological biomarkers. 
General Experimental Procedures 
Electromyographic (EMG) recordings: Following standard skin preparation, single differential 
bipolar surface EMG electrodes (Motion Lab Systems Inc., Baton Rouge, LA, USA) will be placed 
bilaterally on the flexor carpi radialis (FCR), extensor carpi radials (ECR), biceps brachii (BIC), 
triceps brachii (TRIC), soleus (SOL), tibialis anterior (TA), rectus femoris (RF), vastus lateralis 
(VL), and semitendinosus (ST) muscles. EMG electrodes will be secured with 3M Tegaderm 
transparent film (3M, St. Paul, MN, USA). All EMG signals will be filtered with a cut-off 
frequency of 20 - 1000 Hz (1401 plus running Spike 2; Cambridge Electronic Design, Cambridge, 
UK) and sampled at 2000 Hz. 
Transspinal stimulation: We will use our standard methods for transspinal stimulation (Einhorn et 
al. 2013; Knikou 2014; Murray and Knikou 2017, 2019; Knikou and Murray 2019; Pulverenti et 
al. 2022a, 2022b; Skiadopoulos et al. 2022). For cervical transspinal stimulation, a self-adhesive 
electrode (Uni-PatchTM, 10.2 x 5.1 cm2, Wabasha, MA, USA), will be placed over Cervical 3 - 
Thoracic 1 and two electrodes (anode; same type and size as the cathode) connected to act as one 
will be placed on each clavicle.  For lumbosacral transspinal stimulation, a self-adhesive electrode 
(Uni-PatchTM, 10.2 x 5.1 cm2, Wabasha, MA, USA), will be placed over Thoracic 10 to Lumbar 
1/2 and two electrodes connected to act as one (anode) will be placed on each iliac crest. For 
cervical or lumbosacral single pulse transspinal stimulation we will use a DS7A or DS7AH 
stimulator (Digitimer, Hertfordshire, UK). 
Posterior tibial nerve stimulation for soleus H-reflex: With subjects seated and both feet supported 
by a footrest, a stainless-steel plate of 4 cm2 in diameter (anode electrode) will be secured 
approximately 1 cm proximally to the patella.  The optimal stimulation site of the right posterior 
tibial nerve will be probed with a 1-ms monophasic square-wave pulse via a hand-held monopolar 
stainless-steel head electrode (Knikou 2008) and will correspond to the site that the soleus H-reflex 
is elicited without a preceding soleus M-wave at low stimulation intensities and when stimulation 
intensity is increasing the soleus M-wave has a similar shape to that of the soleus H-reflex.  When 
the optimal site is identified, the monopolar electrode will be replaced by a pre-gelled disposable 
electrode (SureTrace, Conmed, NY, USA) that will be maintained under constant pressure 
throughout the experiment with athletic foam pre-wrap. 

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Median nerve stimulation for FCR H-reflex: The FCR H-reflex will be evoked by percutaneous 
stimulation of the right median nerve with rectangular shocks of 1-ms duration every 5 s delivered 
by a constant current stimulator (DS7A, Digitimer, Hertfordshire, UK).  A hand-held bipolar 
stainless-steel electrode will be placed medial to the brachial artery on the cubital fossa and used 
as a probe to determine the most optimal stimulation site (Knikou 2008).  This site will correspond 
to the one during which at low stimulation intensities Ia afferents could selectively be excited with 
absent activation of motor axons (M-wave), and the shape of the FCR M-wave is similar to that of 
the H-reflex at both low and high stimulation intensities. When the optimal stimulation site is 
identified, the bipolar electrode will be maintained in place via an athletic wrap. 
Before and one/two days after completion of the 20 sessions, we will assemble the 1) soleus and 
FCR H-reflex and arm-leg TEP recruitment input-output curves with subjects seated or supine 
while at rest, 2) the FCR and soleus H-reflex rate-dependent depression, and the soleus H-reflex 
and TEPs phase-dependent modulation during assisted stepping. 
Recruitment input-output curves: For the recruitment curves, we will deliver single pulses of 1 ms 
duration at increasing intensities to the posterior or median nerves to evoke the soleus and FCR H-
reflexes. Similarly, single 1 ms pulses at increasing intensities will be delivered to the cervical or 
low thoracic levels to assemble the arm and leg TEPs. We will analyze recruitment curves by 
fitting a sigmoid function and estimate predicted parameters of physiological importance like the 
threshold excitability and slope or gain of the system according to methods we have extensively 
utilized on the lab (Skiadopoulos et al. 2022). 
Soleus/FCR H-reflex rate-dependent depression: To examine restoration of homosynaptic 
depression exerted at the Ia/motoneuron synapse, soleus H-reflexes following tibial nerve 
stimulation with a 1-ms pulse will be recorded from seated subjects seated at inter-pulse intervals 
of 1, 3, 5, 8, and 10 s.  The same method will be used for FCR H-reflex following stimulation of 
the median nerve with a bipolar electrode. Homosynaptic depression is greatest at 1 s and fully 
recovers at inter-pulse intervals of 8 or 10 s (Crone and Nielsen 1989).  The main outcome measure 
will be the H-reflex 1/10 s ratio (Aymard et al. 2000).  Using a linear mixed model, we expect a 
30% decrease in the 1/10s ratio of post-intervention compared to baseline in both groups, as 
indicated by a return of rate-dependent reflex depression. 
Soleus H-reflex and TEPs phase-dependent modulation during stepping: Each participant will 
stand while wearing an upper body harness connected to overhead pulleys. While standing with 
BWS as necessary, the soleus H-reflex and M-wave recruitment curves will be assembled. Then, 
each participant will step with the assistance of the Lokomat, and we will record soleus H-reflexes 
randomly across 16 equal time bins into which each step cycle is divided. The tibial nerve 
stimulation intensity will be adjusted in real-time online to evoke H-reflexes such that their 
corresponding M-waves are 2-8 % of the Mmax evoked 80 ms after the H-reflex at each bin. Soleus 
H-reflexes during stepping will be collected before and after the intervention at Lokomat settings 
matched to before and the last training session. This will establish changes to matched settings and 
reflex adaptation at more demanding settings.  During stepping, H-reflexes will be measured as 
peak-to-peak amplitude, accepted for M-waves ranging from 2-8 % of the Mmax, normalized to the 
Mmax, and averaged for each bin of the step cycle. The main outcome will be the control and 
conditioned soleus H-reflex amplitude normalized to the Mmax at mid-stance and mid-swing 
phases. We will record the leg TEPs during assisted stepping at 1.2 right soleus TEP threshold. 
Stimulations will be triggered based on the foot switch signal. 

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Clinical Measures Before and After Intervention 
For each subject, at baseline and 1/2 days after completion of 20 intervention sessions, we will 
assess via questionnaires improvements in bladder and bowel function via the University of 
Michigan Spinal Cord Injury Model Systems Spinal Cord Injury Bowel and Bladder Treatment 
Index Short Form (SCI-BBTI-SF), sexual function via the PROMIS® V2.0 Brief Profile Sexual 
Function and Satisfaction (for male and female). Additionally, we will clinically evaluate spasticity 
via the Modified Ashworth Score and walking ability via the 2-minute and 10-meter walking tests. 
Data Reduction and Statistical Analysis 
All responses will be measured as peak-to-peak amplitude. H-reflexes recorded at different 
stimulation intensities (recruitment curve) will be normalized to the soleus maximal M-wave 
(Mmax) evoked by tibial nerve stimulation. This will counteract differences in muscle fiber 
composition across subjects. Stimulation intensities will be normalized to the intensity 
corresponding to the threshold of the H-reflex or TEP, as estimated based on the sigmoid function 
fit to the data. Normalized H-reflexes will be plotted against the normalized intensities and grouped 
based on time of testing. Similar analysis will be performed for the arm/leg TEPs with the only 
difference that they will be normalized to the homonymous TEP maximal amplitude. 
For each dependent variable, we will perform descriptive statistics including frequencies and 
percentages for categorical variables and means and percentile distributions for continuous 
variables to examine the distribution of outcomes and describe the study population, followed by 
Shapiro-Wilk’s test for normal distribution and Mauchly’s test of sphericity for homogeneity of 
variances.  If sphericity is not established, the Greenhouse-Geisser correction statistic will be used.  
We will group each dependent variable based on time of testing (before and after the intervention), 
and subject group (2 groups; 1st group cervical-lumbosacral transspinal stimulation at rest, 2nd 
group cervical-lumbosacral transspinal stimulation during stepping).  A repeated measures 
analysis of covariance (ANCOVA) one-way ANOVA along with Bonferroni post-hoc t-tests ( = 
0.05) at 2 x 2 levels (2: time, 2: groups) to test the main and interaction effects among subject 
groups will be performed separately for each outcome.  Further, repeated measures ANCOVA will 
be performed for each outcome separately with gender, age, level of SCI and clinically evaluated 
baseline walking function as separate covariates. 
 
 
 

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