Non-invasive Cervical Electrical Stimulation for SCI
Phase: NA
Conditions: Spinal Cord Injuries
Sponsor: Bronx VA Medical Center

Non-invasive Cervical Electrical Stimulation for SCI
NCT03414424 
PROTOCOL AND STATISTICAL ANALYSIS PLAN
Cover page created: 28 September 2023
Protocol: 9 December 2020

 
PART II 
GUIDE TO RESEARCH PROPOSAL 
 
1. SPECIFIC AIMS 
Aim 1: Map the cellular and synaptic targets of cervical electrical stimulation (CES). 
CES circuit interactions at both the spinal and supraspinal levels will be characterized using a common 
structure comprising an electrical or magnetic conditioning stimulus followed by a test stimulus delivered 
at a range of intensities, sites, and interstimulus intervals (ISI). This will shed insight into which circuits 
CES activates, and how CES circuits interact with circuits activated by other exogenous neural stimuli. 
Hypothesis 1.1: Conditioning CES will potentiate spinal motor neuron responses to test TMS pulses. 
Depending on the timing of the effect, this would support the mechanism of heterosynaptic summation 
between segmental Ia input and descending corticospinal input1, or sensory cortical facilitation of motor 
cortex excitability2–4, or both. 
Hypothesis 1.2: Conditioning CES will increase persistence of spinal motor neuron F-responses to 
retrograde stimulation over the median and ulnar nerves. This would further establish that CES could 
modulate motor neuron excitability at the segmental level. 
Hypothesis 1.3: Conditioning subthreshold CES will reduce spinal motor neuron H-responses to afferent 
stimulation over the median nerve. This would indicate the potential for CES to reduce hyperactive muscle 
stretch responses (spasticity) via homosynaptic interactions.  
Aim 2: Determine optimal CES parameters for acutely facilitating concurrent wrist and hand 
movements.  
Volitional limb movements depend on the same corticospinal and motor neuron circuits as those activated 
by TMS and F-waves. Therefore, if CES facilitates TMS and/or F-responses, CES may also be able to 
facilitate volitional limb movements. The experiments in Aim 2 will shed light on this clinically relevant 
question.   
Hypothesis 2.1: Subthreshold CES will facilitate concurrent wrist and hand muscle activation, supporting 
CES’s ability to positively modulate corticospinal and/or motor neuron excitability. This would present an 
opportunity to directly translate this paradigm for clinical benefit by combining repetitive subthreshold CES 
with repetitive task-oriented physical exercise training. 
Hypothesis 2.2: High-intensity suprathreshold CES will transiently inhibit concurrent wrist and hand muscle 
activation. If observed, this ‘spinal silent period’ would shed insight into mechanisms underlying the ‘cortical 
silent period’ noted when cortical TMS is delivered during volitional contraction5,6. 
 
2. BRIEF REVIEW OF RESULTS AND CURRENT STATE OF KNOWLEDGE 
Roughly 60% of spinal cord injuries occur at the cervical level7. Most injuries are anatomically incomplete. 
A large body of evidence in incomplete SCI as well as other forms of brain injury suggest that externally 
activating spared nerve circuits, whether by exercise, drugs, or electromagnetic stimulation, augments 
circuit and physiological function8–16. This proposal focuses on non-invasive electrical stimulation over the 
spinal cord. Below, we will detail the state of this field, highlighting the gaps in mechanistic understanding 
and therapeutic application toward SCI. We will thereby establish the rationale for testing and applying our 
novel technique of non-invasive cervical electrical stimulation. 
Non-invasive brain stimulation 
Transcranial magnetic stimulation (TMS) uses a transient focal magnetic field to induce action potentials 
in the underlying brain. TMS over the motor cortex transduces action potentials to descending corticospinal 
fibers17,18. Repetitive TMS (rTMS) at 1 Hz is generally inhibitory, whereas at 5 Hz or greater, rTMS is 
generally excitatory11. Higher-frequency (usually 50 Hz) ‘theta burst’ TMS may induce longer-lasting effects 
that are either inhibitory or excitatory based on burst patterns18,19. Seminal TMS mechanistic studies will 
serve as guides for some of the experiments in this proposal17–21. 
Direct current stimulation (DCS) delivers a low-power electric current that modulates neuronal excitability22. 
In distinction from TMS, DCS delivers exclusively tonic, subthreshold stimulation23. Various cranial and 

 
spinal DCS configurations have shown promising effects in neurological and psychiatric disorders, as well 
as enhancement of normal function24–35. However, there is no technique to directly map how the low-energy 
current distributes within the body, or to determine how individual variations in injury characteristics affect 
that distribution36. Furthermore, the continuous nature of DCS makes it difficult if not impossible to elucidate 
timing-dependent synaptic changes. Therefore, although DCS has therapeutic potential, its underlying 
mechanisms are quite likely to remain a black box.  
Invasive spinal stimulation 
Epidural electric stimulation through implanted lumbar electrodes delivers tonic, motor-subthreshold 
stimulation, usually targeted toward the locomotor central pattern generator. In rodent SCI models, epidural 
stimulation combined with physical training and monoaminergic drug stimulation has led to recovered 
ability to walk on previously paralyzed hindlimbs37–39. This work has been translated into humans with 
chronic motor-complete spinal cord injury as high as the C7 level, resulting in regaining some volitional leg 
movement while stimulation is turned on8,13. These incredibly encouraging results support the utility of 
subthreshold spinal stimulation to improve neural circuit function. However, stimulator implantation carries 
the risks of invasive surgery – these risks are significantly higher in the cervical than lumbar spine.  
Non-invasive spinal stimulation  
Multiple transcutaneous spinal stimulation approaches have been developed, largely targeted at 
thoracolumbar locomotor circuits. Tonic stimulation over the T11 level at 3 Hz induces coordinated walking 
movements in uninjured volunteers40. Adding simultaneous stimulation at the C5 and L1 levels increases 
motion coordination and range41. High-intensity phasic stimulation over the C7-T1 or T10-L1 levels 
activates efferent fibers in ventral motor roots to elicit action potentials in arm or leg muscles, 
respectively42,43. 
Transcutaneous biphasic stimulation can also elicit action potentials through afferent fiber pathways44. 
Subthreshold transcutaneous stimulation over T11-T12 for 30 minutes at 50 Hz led to reduced leg 
spasticity and evidence for improved motor function in three subjects with motor-incomplete SCI45. This 
effect was most likely mediated by activation of afferent spinal roots and their segmental interneuronal 
connections. We plan to exploit subthreshold cervical transcutaneous stimulation targeted at similar 
afferent pathways. 
Temporally linked (paired) neuronal firing can lead to lasting effects on synaptic and neuronal excitability 
through Hebbian-like mechanisms such as long term potentiation and synaptic summation46–49. These 
paired stimulation techniques include paired associative stimulation (PAS), transspinal-transcortical 
stimulation, spinal associative stimulation, spike timing-dependent plasticity, and others4,50–57. In a 
demonstration of spike timing-dependent plasticity in the cervical cord, TMS over the hand motor cortex 
was paired with high-intensity electrical stimulation over the ulnar nerve at the wrist50. When repetitive (0.1 
Hz, 100 repetitions) paired pulses were timed so the anterograde TMS signal arrived at cervical motor 
neurons 1-2 ms before the retrograde ulnar nerve signal, both able-bodied volunteers and subjects with 
SCI demonstrated increased TMS-evoked potentials in hand muscles and improved hand dexterity for at 
least 30 minutes after the end of paired stimulation50. This suggested that synapses between upper and 
lower motor neurons underwent Hebbian-like strengthening. Other studies have found more evidence for 
the highly timing-dependent nature of synaptic effects, both at cervical and lumbar levels54,56,57.  
The advantages of non-invasive cervical electrical stimulation (CES) 
Our group recently developed a novel configuration of transcutaneous CES. Our technique draws on 
lessons learned from other forms of non-invasive spinal stimulation referenced above. We have already 
applied for and received FDA designation as non-significant risk, and we are conducting an IRB-approved 
human pilot study to establish basic CES mechanisms and safety. As presented in the preliminary data, 
CES (Figure 1) comfortably elicits action potentials over multiple spinal cord segments simultaneously in 
both arms. CES activates spinal motor neurons indirectly via nerve roots, targeting either afferent or 
efferent fibers depending on stimulus intensity. This afferent or efferent root-stimulation approach provides 
a flexible portal to access synapses between upper and lower motor neurons, even in contexts of damaged 
motor circuitry58.  

 
While we are excited to test CES paired with either peripheral nerve stimulation or TMS in our ongoing 
pilot study (clinicaltrials.gov NCT02469675), TMS requires bulky, expensive equipment that makes it highly 
impractical to use in conjunction with concurrent physical exercise. In this proposal, we will take a more in-
depth mechanistic approach to the CES technique itself, with the goal of establishing a single-modality 
stimulation paradigm that mediates beneficial synaptic plasticity in conjunction with physical exercise.  
We emphasize that CES is inherently different 
from popularly used types of non-invasive 
electrical stimulation such as direct current 
stimulation (DCS, described above) and functional 
electrical 
stimulation 
(FES). 
FES 
targets 
peripheral nerves with suprathreshold pulses 
designed to directly stimulate motor units within 
one or more large muscles at a time. Separate 
FES electrodes are required for each targeted 
nerve/muscle. Critically, FES engages motor units 
in non-physiological order, from largest to smallest 
– this causes excessive muscle fatigue59. In 
contrast, CES targets multiple root levels and both 
sides simultaneously, with the goal of using 
subthreshold intensity to amplify endogenous 
volitional neural circuit signaling. 
Gaps in mechanistic understanding of spinal stimulation 
As opposed to stimulation configuration and pairing with other modalities, the effect of timing between 
successive spinal cord stimuli has received less attention in comparison to similar experiments in the field 
of TMS. Extensive studies have defined rTMS effects over a range of pulse frequencies and patterns, in a 
broad variety of injury and disease contexts. A similarly rigorous definition of spinal stimulation timing 
effects has not yet been achieved. One reproducible finding has shown that at low suprathreshold 
intensities, a conditioning spinal pulse inhibits a second spinal pulse given 20-50 ms later1,44,45,60. These 
findings, which we have confirmed in our own studies, indicate that at these intensities, spinal stimulation 
triggers afferent sensory fibers that activate motor neurons via synapses that are subject to homosynaptic 
depression. However, these studies need to be supplemented with experiments that more deeply 
investigate basic mechanisms of circuit interactions. In the TMS field for example, conditioning pulses 
inhibit test pulses given either 1-3 or 100-200 ms later, and facilitate test pulses given 8-15 ms later. The 
effects of these time intervals have revealed important brain circuit interactions termed short-interval 
cortical inhibition (SICI), long-interval cortical inhibition (LICI), and intracortical facilitation (ICF), 
respectively18,21. Using conditioning and test stimuli over a broad range of time intervals, site, and modality 
combinations, we now expect to define similar types of circuit interactions mediating the response to CES. 
Our team is optimally positioned to address these gaps – we already conduct studies involving electrical 
and magnetic stimulation in a well-run clinical research center; and we have already obtained FDA and 
IRB clearance to conduct human studies using our novel CES technique. 
PRELIMINARY DATA 
Non-invasive, non-noxious cervical electrical stimulation (CES) 
One 5x10 cm electrode is placed longitudinally over the midline with the top edge 2 cm caudal to the C7 
spinous process (~T1-T3 vertebral levels). Another 5x10 cm electrode is placed horizontally centered over 
the thyroid cartilage, corresponding to the C4-C5 levels anteriorly (Figure 1). Two 5x10 cm common 
 
 
Figure 1. CES schematic. Biphasic electrical pulses are 
delivered noninvasively over the cervical spine. The 
cathode is placed posteriorly over T1-T3 levels, and the 
anode is placed anteriorly over C4-C5 levels. Ground 
electrodes are placed over the distal clavicles. Depicted 
schematically on left, photographically on right. 

 
ground electrodes are placed over the distal 
clavicles. Preliminary testing has shown that 
cathode-posterior biphasic (1 ms each phase) 
stimulation results in the best combination of 
subject tolerability, lowest motor threshold, and 
largest muscle response. We will use this 
configuration for all studies within the current 
proposal.  
In preliminary studies, 20 subjects (6 with SCI) 
have undergone over 120 sessions of CES 
without procedure-related significant adverse 
events to date. Each session involves between 
150-300 pulses of CES. All subjects have had 
easily obtainable CES responses. Across our 
studies involving TMS over hand motor cortex, 8 
out of 9 subjects with cervical SCI have had 
readily elicited TMS responses in hand muscles 
(Table 1). This supports the physiological 
feasibility and safety of performing CES in 
individuals with and without SCI.  
 
 
CES activates multiple arm muscles simultaneously 
Surface recording electrodes were placed in a non-disabled volunteer over the biceps (C5-C6), flexor carpi 
radialis (FCR, C6-C7), extensor carpi radialis (ECR, C6-C7), triceps (C7-C8), and abductor pollicis brevis 
(APB, C8-T1). At intensity levels sufficient to stimulate both proximal and distal arm muscles, relative 
latencies corresponded to the distance of each recording electrode from the cathode (Fig 2A). The hand 
myotomes more directly underlying the cathode responded at lower thresholds than the arm myotomes 
(Fig 2B). Additionally, muscles in both arms simultaneously and symmetrically respond to CES in non-
disabled subjects (Fig 2C).  
CES targets different circuits at different intensities 
At peri-threshold intensity, latencies to the APB muscle are up to ~3-5 ms longer than the peripheral motor 
conduction time (not shown). At higher stimulation intensities, latency is equal to or shorter than the 
peripheral motor conduction time. Similar observations have been made in studies of other transcutaneous 
spinal stimulation paradigms43,61,62. These findings are consistent with lower intensity stimuli acting via 
A  
 
 
 
 
 
 
 
 
B  
 
 
    
 
 
       C 
 
 
Figure 2. CES activates multiple muscles simultaneously. A) CES waveforms in one non-disabled volunteer 
depicted in raster format. CES stimulus at ~200% threshold. Note the proximal to distal gradient of latencies and 
amplitudes. B) Intensity-response curves of five different arm muscles, showing distal muscle selectivity. C) CES 
symmetrically activates homologous muscles on both sides 10 non-disabled subjects. FCR, flexor carpi radialis; 
ECR, extensor carpi radialis; ADM, abductor digiti minimi; APB, abductor pollicis brevis. 
Age 
Level 
ASIA 
Grade 
Duration 
MEP 
Threshold 
(% MSO) 
CES 
Threshold 
(mA) 
64 
C4 
D 
14yr 
60 
10 
54 
C4 
D 
4yr 
neg 
NT 
57 
C4 
D 
12yr 
57 
42 
40 
C4 
D 
15yr 
65 
24.5 
36 
C5 
D 
7yr 
69 
NT 
42 
C5 
D 
14yr 
58 
29 
40 
C5 
D 
5yr 
67 
NT 
29 
C8 
C 
3yr 
47 
32 
51 
C8 
D 
17yr 
34 
36 
Table 1 – Demographics and average TMS and CES 
resting motor thresholds to hand muscles in subjects with 
SCI. MEP, motor-evoked potential; MSO, maximum 
stimulator output; mA, milliampere; neg, no response; 
NT, CES not tested in that subject. 

 
afferent sensory fibers that synapse onto lower motor neurons, and higher intensity stimuli acting via 
efferent motor fibers ~2-4 cm distal to the cell bodies. The longer route traversed by lower-intensity stimuli 
explains the longer latency.  
These afferent versus efferent root transmission pathways are further demonstrated by the response to 
two sequential CES pulses given 40 ms apart (Figure 3). At low intensity, the amplitude of the second 
pulse is strongly reduced, suggesting post-activation synaptic depression as seen with H-reflexes and 
posterior root-muscle reflexes1,44,45,60. At high intensity, there is little to no decrement, suggesting direct 
efferent fiber axonal activation, which is not susceptible to post-activation depression.   
 
Subthreshold CES acutely facilitates muscle responses to cortical stimulation 
To test whether subthreshold CES (80-90% of motor 
threshold) could facilitate response to suprathreshold 
TMS (120% of motor threshold), CES was delivered 
either alone or paired such that the CES pulse arrived 
at cervical motor neurons 10 ms prior to TMS pulse 
arrival or 1.5-5.0 ms after TMS pulse arrival. 
Preliminary results show that subthreshold CES 
facilitates the response to TMS when the TMS pulse 
arrives first (Fig 4), with similar trends among non-
disabled and SCI subjects. Since TMS travels via the 
same corticospinal pathways that mediate volitional 
movement, the fact that subthreshold CES facilitates 
TMS suggests that it may also facilitate volitional 
movement. This is a finding with potential for direct 
clinical 
translation. 
Note, 
only 
three 
different 
interstimulus intervals (ISI) were tested in this 
experiment. A much wider range of intervals needs to 
be tested to better understand and optimize this 
phenomenon. 
 
Rationale:  
This proposal includes experiments designed to achieve both mechanistic insight and demonstration of 
therapeutic principle. CES is innovative: the delivery patterns and configurations proposed here are novel. 
CES is practical: non-invasive stimulation carries significantly lower cost, lower risk, and greater ability to 
widely implement than surgically implanted stimulation, especially at the cervical level. Unlike TMS, surface 
electrical stimulation may be easily combined with simultaneous physical exercise. This approach is 
 
Figure 4. Subthreshold CES acutely facilitates 
TMS-evoked potentials. Suprathreshold (120%) 
TMS and subthreshold (80-90%) CES were given 
alone or in combination. Response amplitudes were 
normalized to the response to TMS alone. 
Subthreshold CES facilitated the response to TMS 
alone when the TMS pulse arrived prior to CES, but 
not when the timing was reversed. Amp, amplitude. 
 A  
 
 
 
                 B 
 
  
 
 
             C  
 
 
 
 
 
 
D 
 
Figure 3. The CES circuit pathway is adjustable via stimulus intensity. APB recordings of double CES stimuli at 
40 ms intervals. A, At high intensity (200% of resting motor threshold), repeat CES stimulus (red lines) shows no 
decrement. B, At peri-threshold intensity, repeat CES stimulus results in significant amplitude decrement. C, Second 
stimulus response increases as stimulus intensity increases, indicating direct axonal activation, not synaptic 
transmission. Average ± SEM in 8 non-disabled subjects. D, CES activates sensory afferent roots (red) at lower 
intensity, and motor efferent roots (green) at higher intensity. Red 20 ms-scale bar same for A and B.  

 
broadly applicable: it could be used for individuals with other neurological injuries such as stroke, 
amyotrophic lateral sclerosis, multiple sclerosis, or traumatic brain injury. Finally, CES is compatible: it 
could be combined with drug or cell-based treatments to hopefully produce synergistic functional benefits 
in the future. 
 
Please note that this IRB application involves similar neural stimulation procedures as already 
reviewed and approved in study HAR-15-001 and HAR-16-042. The HAR-16-042 protocol, “Acute 
interactions between electromagnetic stimulation and physical exercise”, was originally designed to 
obtain preliminary data to support several ongoing grant applications. Since that time, we have obtained 
funding for two new grants. Therefore, this and a concurrent new protocol being submitted by Dr. Yu-
Kuang Wu are now more closely tailored to the respective grants we are being awarded. 
 
 

 
3. PROCEDURES, METHODS AND EXPERIMENTAL DESIGN. 
Participants: Ages between 18 and 75. n=15 subjects with SCI. n=15 able-bodied volunteers. 
Inclusion criteria 
Able-bodied participants 
1. Age between 18 and 75 years; n=15 for both Aims; 
2. No known central or peripheral neurological disease or injury. 
SCI participants 
1. Age between 18 and 75 years; n=15 for Aims 1 and 2; 
2. Chronic (> 12 months) motor-incomplete SCI between neurological levels {C1-C8}; 
3. Score of 2, 3, or 4 (out of 5) on manual muscle testing of wrist extension, wrist flexion, finger 
extension, finger flexion, or finger abduction in left or right hand; 
4. Detectable F-wave responses of the left or right APB to median nerve stimulation and/or FDI to 
ulnar nerve stimulation; 
Exclusion criteria  
1. Multiple spinal cord lesions; 
2. History of seizures; 
3. Ventilator dependence or patent tracheostomy site; 
4. Use of medications that significantly lower seizure threshold, such as tricyclic antidepressants, 
amphetamines, neuroleptics, dalfampridine, and bupropion; 
5. History of stroke, brain tumor, brain abscess, or multiple sclerosis; 
6. History of moderate or severe head trauma (loss of consciousness for greater than one hour or 
evidence of brain contusion or hemorrhage or depressed skull fracture on prior imaging); 
7. History of implanted brain/spine/nerve stimulators, aneurysm clips, ferromagnetic metallic 
implants, or cardiac pacemaker/defibrillator; 
8. Significant coronary artery or cardiac conduction disease; 
9. Recent history (within past 6 months) of recurrent autonomic dysreflexia, defined as a syndrome 
of sudden rise in systolic pressure greater than 20 mm Hg or diastolic pressure greater than 10 
mm Hg, without rise in heart rate, accompanied by symptoms such as headache, facial flushing, 
sweating, nasal congestion, and blurry vision (this will be closely monitored during all screening 
and testing procedures); 
10. History of bipolar disorder; 
11. History of suicide attempt; 
12. Active psychosis; 
13. Heavy alcohol consumption (greater than equivalent of 5 oz of liquor) within previous 48 hours; 
14. Open skin lesions over the face, neck, shoulders, or arms; 
15. Pregnancy 
16. Unsuitable for study participation as determined by study physician. 
 
Recruitment: 
Recruitment will be accomplished by pre-existing relationships, physician referrals, and IRB-approved 
advertisements. Veterans with SCI who have an ongoing relationship with personnel of the Center of 
Excellence for the Medical Consequences of SCI, such as those who attend the pulmonary, endocrine, 
cardiovascular, gastroenterology, and SCI clinics, will be informed about the study. Physicians at the 
James J. Peters VA Medical Center will be informed of the goals and aims and the inclusion/exclusion 
criteria for this study. Interested non-veterans who contact us through word of mouth, public online sites 
such as clinicaltrials.gov, or through participation in other ongoing studies at the Center of Excellence will 
also be informed about the study. We will not contact any patients unless referring physicians provide us 
with assurance that the patient would be interested in receiving further information about this study.  
Eligibility Screening: 

 
Persons interested in participating will be assigned a number beginning at 1. To determine eligibility, 
interested participants will be asked the “yes” or “no” questions listed below. Persons who answer “no” to 
any of the following questions will not be eligible for the study: 
 
▪ 
Do you have a spinal cord injury, or are you an able-bodied volunteer? 
▪ 
If you have spinal cord injury, did the injury occur greater than 12 months ago? 
▪ 
Are you between the ages of 18 to 75 years? 
 
If the potential participant answers “yes” to all questions, then the informed consent process will continue 
by inviting the participant for an in-person interview.  
 
Enrollment: 
At the in-person interview, the study will be explained by one of the study team members. All study team 
members will be trained to obtain consent. The study will be explained in its entirety. Along with explaining 
each of the testing and evaluation procedures, the study team member will explain every possible risk that 
the participant may encounter. The potential participant will be told that there is a possibility that he or she 
will not be eligible if any of the exclusion criteria are found to be true (i.e. a screening failure). This is an 
investigational, observational study. As such, no direct permanent benefits will be expected. 
Potential participants will be encouraged to ask questions throughout the process. Potential participants 
will be informed of their right to withdraw at any time and that choosing to not participate will not infringe 
on any of their regular VA benefits or medical care. Once he/she has no further questions and the study 
team is confident that the potential participant fully understands the protocol and its risks, then the 
participant will be asked whether he or she is willing to sign the ICF.  
 
Procedures: 
General: All procedures are performed in a seated position with elbows at roughly 90 degrees and hands 
resting on a pillow in pronated position. Electrophysiological measurements are made using the dominant 
or stronger affected arm. Safety (blood pressure, heart rate, pulse oxygenation, peak expiratory flow rate, 
and structured symptom questionnaire) and tolerability are closely monitored throughout all experiments 
(see Human Subjects section).  
Electromyography (EMG): EMG is recorded using surface sensors with 300x preamplification, 15-2,000 
Hz bandwidth, and internal grounding (Motion Lab Systems). EMG input is collected at a sample rate of 
5,000 Hz via digital acquisition board and customized LabVIEW software (National Instruments). Muscles 
recorded may include abductor pollicis brevis (APB), abductor digiti minimi (ADM), first dorsal interosseous 
(FDI), flexor carpi radialis (FCR), extensor carpi radialis (ECR), and/or biceps brachii. 
TMS: TMS is performed using a MagPro X100 stimulator (MagVenture). The hand motor cortex ‘hotspot’ 
is found while monitoring EMG motor response. Location of the hotspot and all subsequent TMS stimuli 
are tracked using a neural navigation system (Brainsight). Resting motor threshold is determined by 
delivering pulses with increasing intensity until motor responses of ≥ 50 V are observed in the APB or FDI 
muscle in at least 5 out of 10 stimuli.  
CES: CES is performed using two DS7A nerve stimulators or a dual DS8R stimulator (Digitimer) linked to 
deliver biphasic pulses (1 ms each phase; cathodal first). The cathode is a 5x10 cm surface electrode 
(Natus) placed longitudinally with the top edge ~2.5 cm caudal to the C7 spinous process (~T1-T3 vertebral 
levels posteriorly). The 5 x 10 cm anode is placed horizontally over the thyroid cartilage, corresponding to 
the C4-C5 levels anteriorly. Two 5x10 cm common ground electrodes are placed over the distal clavicles. 
Stimulus intensity ranges from 0-80 mA. Motor threshold is determined analogously to the method used in 
TMS. Blood pressure, heart rate, pulse oximetry, and peak expiratory flow rate are monitored every three 
minutes. 
Peripheral nerve stimulation: Stimulation is delivered using a DS7A or a DS8R nerve stimulator and dual 
surface electrodes (20 mm apart). M-wave and F-reflex responses are triggered over the median and ulnar 

 
nerves at the wrist, recording over the APB and FDI muscles, respectively. F-wave pulse intensity is ~110-
120% of the intensity that results in maximal compound motor action potential (CMAP). H-reflex responses 
are triggered over the median nerve at the elbow, recording over the FCR muscle. H-reflex pulse intensity 
will be calibrated to result in H-reflex amplitude ~20-25% of maximal CMAP63. Pulse width is 0.2 ms for 
M/F wave stimulation and 1.0 ms for H-reflex stimulation. 
Timing: Peripheral motor conduction time is calculated using F-wave and M-wave latencies using the 
formula (LatencyM+LatencyF – 1) ÷ 264. Central motor conduction time is calculated as the TMS-evoked 
potential latency minus the peripheral conduction time. These values are used to precisely synchronize 
arriving TMS, CES, and peripheral pulses at cervical motor neurons in the relevant experiments. 
Replication: For Aim 1, testing will occur over three sessions. For Aim 2, testing will occur over two 
sessions. This will allow confirmation of key intensity and timing parameters defined for each subject over 
different sessions, improving reliability of the findings. 
Autonomic monitoring: In addition to standard vital sign monitoring of blood pressure, heart rate, pulse 
oxygenation, and forced vital capacity, real-time changes in cardiovascular and respiratory function will be 
monitored: A three lead electrocardiogram (ECG) (UFI; Morro Bay, CA. #Resp1EKG) will be used to 
determine HR and heart rate variability (HRV); recording electrode will be in the V6 position. These data 
will be intermittently monitored during study visits. The ECG data will be viewed in real-time and stored on 
a secured desktop computer for future analysis using LabVIEW graphical software for instrumentation 
(National Instruments, Austin, TX, USA). Beat-to-beat finger BP will be monitored using photo-
plethysmography for assessment of BP variability and baroreceptor reflex activity (CNSystems 
Medizintechnik; Graz, Austria; # CNAP Monitor 500). Impedance plethysmography will be used to monitor 
respiration rate (Biopac Systems, Inc.; Goleta, CA.; # RSP100C). These data will be viewed in real time 
and stored on a secured desktop computer for future analysis using LabVIEW.  
 
Aim 1: Map the cellular and synaptic targets of cervical electrical stimulation (CES). 
This Aim investigates fundamental CES mechanisms. The experiments share a common structure 
comprising an electrical conditioning stimulus delivered within 0-300 ms of a test stimulus delivered at a 
range of intensities and sites. This will shed insight into which circuits CES activates, and how CES circuits 
interact with circuits activated by other exogenous neural stimuli. 
Aim 1 Outcomes: 
• 
Amplitudes: Peak-to-peak amplitudes from an average of 8-10 responses per condition.  
• 
Persistence (F-waves): Persistence is defined as the percent of positive F-wave responses out of 25 
pulses. 
CES-TMS interactions:  
• 
Goal: Measure interactions between afferent cervical nerve roots and descending corticospinal inputs 
into the cervical cord.  
• 
Methods: Test TMS pulse intensity will be 120% of motor threshold. Conditioning CES pulse intensity 
will be between 30-95% of motor threshold, delivered within 300 ms of the test pulse. For conditioning 
CES, either single or “theta bursts” (3 pulses at 50 Hz)19 will be delivered. All pulse combinations will 
be delivered at a rate of 0.1 Hz, in pseudorandom parameter order, until 8-10 responses per parameter 
have been recorded. 
• 
Hypothesis 1.1: Conditioning subthreshold CES will potentiate motor neuron response to test TMS 
pulses. 
• 
Interpretation: If subthreshold CES facilitation occurs at ISI between 2-20 ms, this would support the 
mechanism of heterosynaptic summation between segmental Ia input and descending corticospinal 
input1. If facilitation occurs at ISI between 20-60 ms, this would support the mechanism of sensory 
cortical facilitation of motor cortex excitability (based on analogous findings using peripheral nerve 
stimulation4,2,3). Because TMS travels along the same corticospinal and subcortical circuits used during 
volitional movement, this would also set the basis for using subthreshold CES to facilitate motor 

 
responses during physical training programs (see Aim 2). Subjects with incomplete cervical SCI are 
expected to have reduced CES-TMS facilitation compared to uninjured subjects (see Figure 4). 
CES-peripheral nerve interactions:  
• 
Goal: Measure interactions between afferent and efferent cervical nerve roots, segmental interneuronal 
circuits, orthodromically conducting peripheral sensory fibers, and antidromically conducting peripheral 
motor fibers.  
• 
Methods: F-wave pulse intensity will be ~110-120% of the intensity that results in maximal compound 
motor action potential (CMAP). H-reflex pulse intensity will be calibrated to result in H-reflex amplitude 
~20-25% of maximal CMAP63. Conditioning CES pulse intensity will be set between 30-175% of motor 
threshold, delivered within 300 ms of test pulses. For subthreshold conditioning CES, either single or 
“theta bursts” (3 pulses at 50 Hz)19 will be delivered. All pulse combinations will be delivered at a rate 
of 0.1 Hz, in pseudorandom parameter order, until 8 (CES-H combinations) or 25 (CES-F 
combinations) responses per parameter have been recorded. 
• 
Hypothesis 1.2: Conditioning CES will increase persistence (number of responses per 25 stimuli) of 
median (APB) and ulnar (FDI) F-responses elicited up to 50 ms later. 
• 
Hypothesis 1.3: Conditioning CES will reduce amplitude of median (FCR) H-responses elicited up to 
50 ms later. 
• 
Speculative hypothesis 1.2: Conditioning suprathreshold CES will block spinal motor neuron F-
responses delivered up to 10 ms later.  
• 
Interpretation: These experiments will lead to multiple insights. Through segmental interactions, 
confirmation of Hypothesis 1.2 would further demonstrate the ability of CES to positively modulate 
motor neuron excitability, and confirmation of hypothesis 1.3 would indicate the potential for CES to 
reduce spasticity45. Confirmation of speculative hypothesis 1.2 would demonstrate that at high 
intensity, CES pulses travel via efferent nerve roots and collide with retrogradely traveling F-waves.  
Aim 1 data analysis: Peak-to-peak amplitude of all conditioned pulses will be normalized to the amplitude 
of unconditioned pulses. For each interaction paradigm, analysis of variance (ANOVA) with factors of 
subject group, stimulus intensity, interstimulus interval, and muscle will be performed. Post hoc pairwise 
comparisons will be made using Tukey’s method. Power: Using a conservative predicted effect size of 0.4 
for each interaction paradigm, >80% power will be achieved with a sample size of 10 for each analysis 
(repeated measure ANOVA) with four factors). 15 subjects in each group (SCI, able-bodied) will undergo 
testing. 
Aim 1 Timeline (Table 2): 
Each subject will undergo three testing sessions on separate days, with a minimum of 24 hours and a 
maximum of two weeks between visits. Per subject, Aim 1 participation would therefore take 3 to 28 days. 
30 subjects will be enrolled, resulting in 90 total testing sessions. A conservative projection of completing 
two testing sessions per week would result in a timeline of 45 weeks or 11-12 months for completing Aim 
1. 
Aim 2: Determine optimal CES parameters for acutely facilitating concurrent wrist and hand 
movements. 
Volitional limb movements depend on the same corticospinal and spinal motor circuits as those activated 
by TMS and F-waves. Therefore, since preliminary data shows that subthreshold CES facilitates TMS 
responses, CES may also be able to facilitate volitional limb movements. The experiments in Aim 2 will 
shed light on this clinically applicable question. 
Volitional motor tasks: Maximal voluntary contraction (MVC) will be defined as the largest response from 
3 attempts at maximal effort prior to testing. Effort will be measured using a customized dynamometer or 
the root mean square (RMS) of electromyographic activity in the target muscles. The tasks are: opposition 
between the tips of the thumb and third finger (C8-T1 levels), or wrist extension laterally against a mounted 
load cell (C6 level). A combined finger opposition/wrist extension task will also be tested. Subjects will be 
instructed to perform tasks at target effort levels based on real-time display of ongoing EMG or force output. 

 
CES-motor task interaction: Subjects will be given a synchronized audio and visual cue to perform volitional 
motor tasks at 100%, 50%, or 10-20% of MVC. Volitional tasks will be performed for a maximum of 4 
continuous seconds. At least 10 seconds will elapse between each CES/motor task combination. 
Conditioning CES pulses (or sham pulses at 0% intensity) will be delivered at intensity ranging between 
30%-175% of CES resting motor threshold. For subthreshold conditioning CES, either single or “theta 
bursts” (3 pulses at 50 Hz)19 will be delivered. Motor task, effort level, and CES parameters will be varied 
in pseudorandom order, until 8-10 responses per parameter have been recorded. 
Aim 2 Outcomes: 
• 
Facilitation: At each muscle, the effect of CES pulses on RMS electromyographic activity over the 
subsequent 200 ms (in 25 ms bins) will be measured and normalized to the baseline RMS from the 
preceding 100 ms. 
• 
Silent period: At each muscle, the duration of electromyographic silence is measured from the end of 
the CES-evoked potential until the resumption of volitional muscle activity (defined as mean rectified 
EMG amplitude less than or greater than mean baseline activity + 2 standard deviations68, 
respectively). 
Hypothesis 2.1: Subthreshold CES will facilitate concurrent wrist and hand muscle activation. 
Hypothesis 2.2: High-intensity CES will transiently inhibit concurrent wrist and hand muscle activation. 
Interpretation: Confirmation of Hypothesis 2.1 would substantiate the predicted findings of Hypotheses 1.1 
and 1.2 and our early preliminary data that indicate CES’s ability to positively modulate corticospinal and/or 
motor neuron excitability. We expect optimal facilitation when subthreshold CES is delivered at closer to 
motor threshold intensity. We do not know whether the effects will differ depending on intensity of volitional 
effort, but if so, further experiments could investigate the roles played by peripheral, spinal, and supraspinal 
components of effort intensity69. Furthermore, these experiments would shed light on whether CES 
modulates ongoing motor neuron activity differently at different spinal levels during level-specific motor 
tasks (thumb-finger opposition C8-T1, wrist extension C6). Most importantly, confirmation of Hypothesis 
2.1 would represent an opportunity to directly translate and test this paradigm for clinical benefit by 
combining repetitive subthreshold CES with repetitive task-oriented physical exercise training. 
If high-intensity CES inhibits concurrent volitional activity as predicted by Hypothesis 2.2, this would be 
analogous to the ‘cortical silent period’ (CSP) noted when cortical TMS is delivered during volitional 
contraction5,6. The early phase of CSP (the first 50-75 ms after onset of the cortical motor-evoked potential) 
is mediated by poorly understood spinal mechanisms5. Our observations of the onset and duration of a 
‘spinal silent period’ (SSP) in response to CES would shed mechanistic light on this phenomenon. 
Aim 2 Data analysis: Rather than peak-to-peak amplitude, the effects of CES on concurrent volitional 
muscle contraction will be measured using RMS as described above. Repeated-measure ANOVA with 
factors of subject group, volitional effort intensity, CES intensity, post-stimulus timebin, and muscle will be 
performed. Post hoc pairwise comparisons will be made using Tukey’s method. Although we can only 
speculate on the predicted effect size, with use of a conservative effect size of 0.4 for each interaction 
paradigm, >80% power will be achieved with a sample size of 10 for each analysis (repeated measure 
ANOVA with five factors). 15 subjects in each group (SCI, able-bodied) will undergo testing. 
Aim 2 Timeline: 
Each subject will undergo two testing sessions on separate days, with a minimum of 48 hours and a 
maximum of three weeks between visits. Per subject, Aim 2 participation would therefore take 3 to 21 days. 
30 subjects will be enrolled, resulting in 60 total testing sessions. A very conservative projection of 
completing 1.5 testing sessions per week would result in a timeline of 40 weeks or 10 months for 
completing Aim 2. 
Overall timeline: 

 
 
 
4. Possible RISKS and protective actions 
For subjects with SCI, there is a risk of falling during transfers between different testing positions. To 
minimize this risk, a clinician will be at the participant’s side at all times to assess comfort and provide 
manual assistance/stabilization as necessary.  
The surface electromyography recording and stimulating electrodes and tape have adhesive backing. 
Therefore, minimal risks such as transient skin irritation at the sites of surface electrode application may 
occur. Areas with excessive hair will be shaved prior to adhesive application. 
Electrical stimulation involves currents up to 80 milliamperes. Electrical pulses may be transiently irritating 
or painful. Stimulation intensity will be reduced, or testing halted, if a subject is too uncomfortable. Electrical 
stimulation of the upper spinal cord may theoretically alter activity in vagal or other autonomic circuits. The 
most likely adverse risks of autonomic activation would be nausea, light-headedness, diaphoresis, or 
syncope. There is no risk of current crossing over cardiac muscle with the electrode configurations used 
in this protocol. Nevertheless, to provide further caution against cardiac damage or arrhythmia, subjects 
who 
have 
significant 
coronary 
artery 
disease, 
cardiac 
conduction 
disease, 
implanted 
pacemaker/defibrillators, or history of recurrent autonomic dysreflexia (defined in Exclusion Criteria) will 
be excluded from participation.  
In subjects with SCI above the T6 vertebral level, there is a risk of autonomic dysreflexia – this is a 
potentially dangerous increase in blood pressure with simultaneous decrease in heart rate, usually 
accompanied by symptoms such as headache, facial flushing, sweating, nasal congestion, and blurry 
vision. There is a theoretical risk that it could occur in response to magnetic or electrical stimulation. 
Potential subjects who have a history of recurrent autonomic dysreflexia within the past 6 months will be 
excluded. If autonomic dysreflexia is suspected during a procedure, the procedure will be halted 
immediately. Head position, bowel, bladder, and other triggers will be addressed according to a standard 
algorithm70. Subjects who experience autonomic dysreflexia during any session will be withdrawn from 
further participation in the protocol. 
To provide further caution against any adverse cardiac or autonomic event, the procedure will be closely 
monitored for cardiac or dysautonomic side effects with pulse oximetry, blood pressure, and spirometric 
measurements. Additionally, subjects will be monitored in real time for changes in cardiovascular and 
respiratory function using a three-lead electrocardiogram, photo-plethysmography, and impedance-
plethysmography. Any change in mean arterial pressure or pulse oximetry of greater than 15% from 
baseline, accompanied by symptoms such as sudden shortness of breath, chest pain, significant 
headache, or diaphoresis, will lead to immediate cessation of the protocol and further medical evaluation. 
Furthermore, if cardiac or dysautonomic side effects occur during the screening visit, the subject will be 
ineligible for further participation in the study. A medical doctor will be on the premises at all times during 
stimulation protocols. This technique has received formal designation by the FDA as Non-significant Risk 
(Q150053). 
Transcranial magnetic stimulation (TMS) carries several potential risks. Most of these risks are much 
greater during application of repetitive TMS (defined as pulses given at a frequency of 1 per second or 
Stage 
AB 
SCI 
Visits 
Duration (wks) 
Months 
1-6 
7-12 
13-18 
19-24 
Startup 
 
 
 
 
X 
 
 
 
Aim 1 
15 
15 
3 
1-4 
   XXXX XXXXX 
 
 
Aim 2 
15 
15 
2 
1-3 
 
 
XXXXX XXX 
Analysis/ 
Dissemination 
 
 
 
 
 
 
 
    XX 
Table 2 – Study timeline. Extra time is allotted for study startup, data analysis, and 
dissemination of results. See also Timeline descriptions in proposal text. 

 
more frequently), which will not be conducted in this study. We will be using a MagPro X100 device 
(MagVenture). This device has FDA 510(k) clearance (approval #K091940) for peripheral nerve 
stimulation, and has been cleared as a non-significant risk device by this and numerous other IRBs for 
research and clinical use. The most serious risk of TMS is induction of seizures. TMS-induced seizures 
are usually focal, but in some cases can become generalized. To minimize this risk, participants with 
underlying brain injury that increases the risk of TMS-induced seizures will be excluded from participation 
– this includes moderate or severe traumatic brain injury, stroke, tumor, multiple sclerosis, or abscess (see 
full list under Exclusion Criteria). Additionally, participants taking medications that significantly lower 
seizure thresholds, such as anti-psychotics, amphetamines, tricyclic antidepressants, bupropion, and 
dalfampridine will be excluded. Furthermore, the applied stimulus intensity will be kept below 200% of the 
motor threshold for each muscle. This intensity and single-pulse frequency fall far below the recommended 
safe guidelines delineated by an international workshop on TMS safety (Rossi et al. 2009). Furthermore, 
participants with implanted devices with electromagnetic properties, such as spine stimulators, deep brain 
stimulators, vagal nerve stimulators, cardiac pacemakers, cochlear implants, or aneurysm clips, will be 
excluded. The investigator who will be performing the TMS protocol (Noam Harel) is a neurologist 
experienced in treating seizures. There is no risk of seizure from electrical stimulation below the brain, as 
performed in this protocol. 
There is a theoretical risk of repetitive TMS causing acute psychotic or manic symptoms in patients with 
depression. This risk is not clearly above the risk for sham-TMS, and it is not clearly above the natural rate 
of psychotic or manic symptoms that may arise in subjects with depression. Regardless, this protocol does 
not meet the definition of repetitive TMS, and any subject with history of bipolar disease, active psychotic 
symptoms, or history of suicide attempt will be excluded 
TMS pulses generate loud auditory clicks. Hence, all participants will wear earplugs during the procedure. 
TMS may also cause scalp tingling sensations or pain that is almost always mild and transient. This occurs 
approximately half as frequently in participants exposed to sham-stimulation. This is much less common 
using single-pulse TMS than repetitive TMS. Our protocol will use only single-pulse TMS.  
The research team has delivered TMS pulses at 5 to 10-second intervals extensively in prior and ongoing 
IRB-approved clinical studies. A standardized form to assess TMS side effects is used at the end of each 
TMS session. To repeat, the use of only single pulse TMS, as well as all the other precautions and 
exclusion criteria we will follow in our TMS protocol, far exceed the recommended guidelines established 
by an international group of TMS experts 71. 
The Brainsight optical TMS tracking system (Rogue Research) uses passive reflectors placed on the 
subject’s head and the TMS magnet to track the magnet’s position and orientation relative to the subject’s 
brain in real time. This is a passive detection device, with no penetrating electromagnetic stimulation. The 
infrared optical tracking portion of the system meets all applicable conformity standards (ANSI/AAMI 
ES60601-1:2005 +C1:2009 +A2:2010; see appendices), The infrared optical tracking portion of this system 
has been incorporated into numerous surgical tracking systems that have obtained FDA 510(k) approval 
in the setting of invasive brain and organ surgery. However, when used in combination with non-invasive 
TMS for research, it is not considered a medical device that requires FDA approval. There is no risk to the 
subject from using this tracking system. 
Study coordinators and research assistants will be trained to conduct all procedures in a safe and effective 
manner that produces reproducible results. Study personnel with experience working with SCI participants 
will supervise all sessions. All staff will have undergone the appropriate training to use the equipment 
properly and safely. All subjects will be thoroughly questioned after each procedure to ensure that all 
possible adverse events, expected and unexpected, are ascertained. Study personnel will refer 
participants to the proper medical or psychological resources for any identified conditions or problems as 
a consequence of the research. 
In the event of a serious adverse event, it will be reported to the IRB within 24 hours and study interventions 
will be discontinued until the study physician states that it is safe to resume the study. Adverse events will 

 
be recorded in a data sheet and reported annually to the IRB. When appropriate, necessary medical or 
professional intervention will be provided for any serious or regular adverse event warranting treatment. 
Any unexpected complications that may occur will be discussed with the study physician and/or the 
participant’s SCI physician. Dr. Miroslav Radulovic, a board-certified internist, will serve as the study 
physician.  
 
Protection against Risk 
Most of the information that participants provide will not be identifiable. Participant data results will be 
stored on the VA network in a password-protected file. No identifiable information will be linked to this file. 
The study team members will have a separate file of participant contact information, also stored on the VA 
network. Participants will be assured that any “hard copies” of their contact information or data will be kept 
in a securely locked cabinet in a locked private office. This data will not be destroyed. There is minimal risk 
of a breach of confidentiality or data security.  
Study coordinators and research assistants will be trained to conduct all procedures in a safe and effective 
manner that produces reproducible results. Study personnel with experience working with SCI participants 
will supervise all sessions. All staff will have undergone the appropriate training to use the equipment 
properly and safely. All subjects will be thoroughly questioned after each procedure to ensure that all 
possible adverse events, expected and unexpected, are ascertained. Study personnel will refer 
participants to the proper medical or psychological resources for any identified conditions or problems as 
a consequence of the research. 
In the event of a serious adverse event, it will be reported to the IRB within 24 hours and study interventions 
will be discontinued until the study physician states that it is safe to resume the study. Adverse events will 
be recorded in a data sheet and reported annually to the IRB. When appropriate, necessary medical or 
professional intervention will be provided for any serious or regular adverse event warranting treatment. 
A formal Data Safety Monitoring Board will not be needed for this study. 
Provisions for keeping data confidential are established. All electronic data will be kept on the secure VA 
network. All intake forms are de-identified according to HIPAA regulations. Consent forms, along with any 
other forms containing identifiable information, are kept in a locked filing cabinet. Only members of the 
investigative team will be able to use the information. 
Any unexpected complications that may occur will be discussed with the study physician and/or the 
participant’s SCI physician. Dr. Miroslav Radulovic, a board-certified internist, will serve as the study 
physician.  
5. SIGNIFICANCE OF THIS RESEARCH. 
The information obtained in this study may be useful scientifically to the subjects taking part in the study 
and to other researchers and patients. This could provide new information that leads to improved non-
invasive techniques for strengthening nerve transmission after central nervous system injury.  
Participants may achieve transient neurophysiological benefits. However, the study is not designed to provide long-
lasting direct benefit. Rather, study participation may lead to new knowledge that could be applied toward the SCI 
population as a whole. 
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