Diaphragm Stimulation After Human Spinal Cord Injury: Effects on Respiratory Neural Drive and Function
Conditions: Cervical Spinal Cord Injury
Sponsor: University of Florida

 
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Protocol #:  201902285 
IRB version:  10/09/2019  
PI version:  2/3/2022 
 
Title: Diaphragm Pacing in Individuals with Spinal 
Cord Injuries 
 
NCT 04179799 
Study IRB-Approved Protocol 
 
2/3/22 
 
 

 
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1. Project Title:  
Diaphragm stimulation after human spinal cord injury: Effects on respiratory neural drive 
and function  
2. Investigators: 
PI: Emily J. Fox, PT, DPT, PhD 
UF Dept. of Physical Therapy & Brooks Rehabilitation 
Co-I: Alicia K. Vose, PhD, CCC-SLP 
 
 
University of Florida 
Co-I: Brian Yorkgitis, D.O.               
 
 
UF Health Shands Jacksonville 
Co-I: Kathryn Doughty, DPT 
 
 
 
Brooks Rehabilitation 
Co-I: Clayton Wauneka, PhD 
 
 
 
Brooks Rehabilitation 
Co-I: Amy Redding, M.Ed., CCC-SLP 
 
 
Brooks Rehabilitation 
Study Coordinator: Jennifer Mull, MSN, RN, CCRC  
UF Health Shands Jacksonville 
Study Coordinator: Yohan Diaz Zuniga, MD  
 
UF Health Shands Jacksonville 
Study Coordinator: Hannah Snyder, MS, ACSM-EP 
Brooks Rehabilitation 
3. Abstract: 
Respiratory dysfunction is the leading cause of death in individuals with spinal cord injuries (SCIs). 
Nearly one-quarter of all SCI cases involve injury to the upper spinal cord segments which impairs 
neural activation of the diaphragm muscle and compromises breathing.  Although mechanical 
ventilation can be life-saving after cervical SCI (C-SCI), it also triggers rapid and profound 
diaphragm muscle atrophy, thereby complicating (or even preventing) ventilator weaning. 
Intramuscular diaphragm stimulation, or diaphragm pacing, was developed to replace long-term 
ventilator support and is now used acutely post C-SCI (<4 months following injury) to promote 
ventilator weaning. Our group is part of a national team that initially reported 80% of C-SCI 
patients who underwent diaphragm pacing were successfully weaned from mechanical ventilation. 
Moreover, following electrode placement, gains in respiratory function were reported; remarkably, 
36% of patients who received diaphragm pacing recovered independent breathing, enabling 
removal of the pacing electrodes. More recently, our team assessed the effects of diaphragm 
stimulation (pacing) longitudinally, from days to weeks after onset of diaphragm pacing 
demonstrating improvements in respiratory outcomes and EMG diaphragm activation (details 
below). However, there are clear gaps in how such data can be interpreted to determine the 
unique effects of diaphragm pacing since many factors affected outcomes in our longitudinal 
assessments (i.e. patient comorbidities, secondary complications after SCI, variances in ICU care, 
etc). Thus, these newly proposed experiments will examine the effects of diaphragm stimulation 
within carefully controlled test sessions and examine effects immediately after a controlled 
period of stimulation. This will allow us to determine the unique effects of diaphragm stimulation. 
Specifically, we will measure respiratory function and diaphragm activation prior to and following 
two ~1-hour time blocks and a single ~12-hour time block of diaphragm stimulation in patients with 
traumatic SCI. Our team is experienced in working with these patients and the conduct of these 
measurements. Overall, the proposed experiments will allow us to assess the effects of 
diaphragm stimulation on respiratory neural drive and breathing function in individuals with acute, 
traumatic C-SCI.  
4.  Introduction & Specific Aim: 

 
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Respiratory dysfunction is a leading cause of death in individuals with spinal cord injuries; 
indeed, individuals with cervical lesions are at the greatest risk for respiratory complications 
leading to mortality.1-3 Cervical spinal cord injury (C-SCI) causes primary neurological 
impairment of the respiratory muscles. Since 40% of C-SCI cases involve C1 to C5 injury, 
phrenic nerve activation will be impaired, weakening diaphragm contractions. Although 
mechanical ventilation can be lifesaving after C-SCI, it triggers rapid and profound diaphragm 
muscle atrophy, thereby complicating or preventing ventilator weaning.4,5  Intramuscular 
diaphragm stimulation, or diaphragm pacing, was developed to replace ventilator support and is 
now used acutely to promote ventilator weaning in individuals with C-SCIs (< 4 months post C-
SCI).6  Our group is part of a national team that reported that the majority (80%) of patients with 
C-SCI who received diaphragm pacing were subsequently weaned from mechanical ventilation.  
Diaphragm pacing may improve respiratory function since 36% of patients receiving diaphragm 
pacing recovered independent breathing, enabling removal of the pacing electrodes.6   
 
More recently, our team assessed the effects of diaphragm stimulation (pacing) longitudinally, 
from days to weeks after onset of diaphragm pacing and showed improvements in respiratory 
outcomes including increased tidal volume, forced vital capacity, and maximum inspiratory 
pressures. Preliminary analyses also highlight increased EMG diaphragm activation evident after 
use of pacing. Thus, accumulating evidence suggests that diaphragm pacing exceeds the current 
standard of care (i.e. mechanical ventilation), and may represent a rehabilitation strategy to 
improve and even restore lost respiratory function. However, there are clear gaps in how such 
data can be interpreted to determine the unique effects of diaphragm pacing since many factors 
affected outcomes in our longitudinal assessments (i.e. patient comorbidities, secondary 
complications after SCI, variances in ICU care, etc). Thus, a primary goal in this protocol is to 
examine the effects of diaphragm stimulation within carefully controlled test sessions. Here, we 
propose to systematically evaluate the impact of diaphragm pacing on diaphragm neuromuscular 
activation (EMGs) and breathing capacity after two ~1-hour time blocks and a single ~12-hour 
time block of diaphragm stimulation in adults with acute, traumatic C-SCIs.  This work represents 
an essential step in determining the efficacy of intramuscular diaphragm stimulation and its effects 
on respiratory function and neural control of the diaphragm following C-SCI.  
Research AIM:  
The aim of this protocol is to test the hypothesis that diaphragm pacing enhances 
neuromuscular activation of the diaphragm in adults with C-SCIs and improves respiratory 
function. We will test the hypothesis by recording activity of the diaphragm muscle 
(electromyograms; EMGs) from intramuscular pacing electrodes. Recording from these surgically 
implanted electrodes allows comparisons of electromyogram recordings before and after short 
periods of diaphragm pacing to better isolate the unique contribution of diaphragm pacing on 
neuromuscular diaphragm activation.  This approach, in association with respiratory assessments, 
will enable us to better determine the unique impact of diaphragm pacing in patients with acute, 
traumatic C-SCIs. 
5. Background & Preliminary Data: 
SCI and respiratory dysfunction. One of the most serious and direct consequences of C-SCI 
is impaired breathing function.  Indeed, respiratory dysfunction is a leading cause of death after 
SCI, and individuals with cervical injuries have the greatest risk for respiratory complications.1-3  
C-SCI interrupts descending spinal pathways to respiratory motor neurons, thereby causing 
respiratory muscle paralysis or paresis.12  These primary respiratory control deficits are 

 
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exacerbated by inadequate respiratory defense and high rates of respiratory infection, 
atelectasis and altered breathing mechanics.13 In 40% of C-SCI cases, the upper cervical 
segments (C1-C5) are involved,3 causing severe respiratory impairment; these individuals often 
require mechanical ventilation.  More than two-thirds of acute C-SCI patients will require 
ventilatory support (usually mechanical ventilation), and 40% will require continued ventilatory 
support after discharge from acute care.14  
Mechanical ventilation. Mechanical ventilation can be lifesaving and is the current standard of 
care when C-SCI patients cannot sustain independent breathing.2,15  Unfortunately, mechanical 
ventilation causes rapid and profound diaphragm muscle atrophy, thereby complicating or even 
preventing ventilator weaning.4  Ventilators essentially assume the role of respiratory muscles, 
leading to diaphragm muscle inactivity and profound atrophy and weakness.  Numerous studies 
have demonstrated that mechanical ventilation induces diaphragm dysfunction,16 a syndrome 
known as ventilator-induced diaphragm dysfunction or VIDD.17,18  VIDD has been observed in 
animal models19 and humans18,20,21 with prolonged ventilatory support.  Even brief periods (18-
69 hours) of mechanical ventilation cause atrophy.20,22  SCI patients on mechanical ventilation 
experience greater rates of respiratory infections and complications, primary determinants in the 
duration of hospitalization.24  Moreover, ventilator-dependent individuals with SCI are 39 times 
more likely to die during the first year post-SCI, and 3.5 times more likely to die after the first 
year post-injury.25,26   
Intramuscular diaphragm stimulation (diaphragm pacing). Intramuscular stimulation of the 
diaphragm, or diaphragm pacing, was pioneered by Onders and colleagues to replace long term 
mechanical ventilation.11,31,32  The method is now used acutely post C-SCI to promote ventilator 
weaning and avoid ventilator-associated complications.6  Compared with mechanical ventilation, 
patients using diaphragm pacing experience fewer respiratory infections exhibit improved 
speech and olfaction and report overall better quality of life.10,33-35  Moreover, accumulating 
evidence, including both animal and humans reports, indicates that intramuscular diaphragm 
stimulation mitigates the negative impact of combined ventilatory support and SCI-induced 
inactivity.36,37  Dr. Martin and colleagues extended this work, demonstrating that even brief, 
intermittent stimulation (1 min/ per hour) improves muscle fiber mitochondrial respiration in the 
stimulated hemidiaphragm in humans undergoing mechanical ventilation during cardiothoracic 
surgery.23   
Recent Data  
Since this report, our group recently studied longitudinal outcomes in 11 adults with chronic  C-
SCI who received surgical placement of a diaphragm pacer due to failure to wean from 
mechanical ventilation. Of the 11 participants, all had a traumatic cervical SCI, C1-C4, ASIA 
impairment scale A or B, motor complete (except 1 individual whose injury was classified as AIS 
C). A brief summary of respiratory and EMG outcomes is below. 
Of the 11 adults who participated in this study, 9 of the 11 were successfully weaned from the 
ventilator and 6 out of those 9 were successfully weaned from the diaphragm pacer, meaning 
they resumed independent respiration and the diaphragm pacer was able to be stopped and in 
many cases, removed. Preliminary analyses of respiratory outcomes from these individuals 
have focused on comparing two early tests to determine the impact of diaphragm stimulation. 
Early tests were conducted ~1-7 days after pacer implant (Early 1) and then ~5-7 days later 
(Early 2). We also include data from the participant’s final test, though the number of test 
sessions varied since some participants transferred to inpatient rehabilitation where testing was 
ongoing and others transferred to other facilities or remained in acute care due to complicated 

 
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discharge planning or medical needs. Figure 1 highlights the improvement in respiratory 
outcomes (tidal volume, forced vital capacity, and max inspiratory pressures) over the course of 
these time points. Also highlighted are pilot results from 3 individuals showing increased 
diaphragm activation indicated by greater EMG activity from Early 1 to Early 2 time points 
(Figure 2). 
 
 
Figure 1 
 
 
Figure 2 
Summary: Overall, these data combined with previous evidence 
suggests the possibility that diaphragm pacing is a potentially 
legitimate rehabilitation strategy to improve and even restore 
respiratory function.6-8,23  The goal of this current protocol is to 
further examine the unique effects of diaphragm pacing in 
carefully controlled sessions shortly after implantation of 
the pacer. This is an essential and critical next step in 
determining the efficacy of intramuscular diaphragm stimulation 
and its effects on respiratory neural drive and function after SCI.   
 
 
6. Research Plan: 
Inclusion and exclusion criteria.   
Ten adults will be recruited who are ≥18 years old with acute, traumatic cervical spinal cord 
injuries (C-SCIs), classified according to the American Spinal Injury Association (ASIA) 
Impairment Scale (AIS) as A-C (complete SCI (A); motor complete SCI (B); motor incomplete 
with minimal motor function (C)),58 affecting C1-C6 spinal cord segments, and who have been 
scheduled to undergo implantation of a diaphragm pacer, or who have recently received (in past 
5-days) implantation of intramuscular diaphragm pacing electrodes due to severe respiratory 
impairments and dependence on mechanical ventilation. The inclusion criteria are based on our 
prior work with SCI patients and the injury characteristics of SCI patients with intramuscular 
pacing electrodes per clinical indications.6,7   
 
Tidal Volume (ml)
↑39%
↑41%
0
200
400
600
Early 1
Early 2
Final
*
*
0
20
40
60
Early 1
Early 2
Final
Max Inspiratory Pressure (-cm H2O)
↑24%
↑48%
0
0.04
0.08
0.12
Early 1
Early 2
Amplitude (mV)
↑46%
-0.4
-0.2
0
0.2
0.4
-0.4
-0.2
0
0.2
0.4
EMG Amplitude during Max 
Inspiratory Pressure (n=3)

 
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Exclusion criteria include: progressive neuromuscular diseases such as multiple sclerosis and 
myasthenia gravis, history of neurologic injuries such as stroke or prior SCI, chest wall injuries 
or deformities likely to influence breathing, pregnancy or cognitive impairments limiting study 
participation. 
Subject recruitment. Patients with acute, traumatic C-SCIs, scheduled to receive intramuscular 
diaphragm pacing OR who have recently received a diaphragm pacer (within past 5-days) will 
be recruited from UF Health Shands Jacksonville, a Level-1 trauma hospital serving North 
Florida. Potential study participants will be identified and informed of our study by Dr. Brian 
Yorkgitis (study Co-I and expert in surgical diaphragm pacer implantation). UF Health Shands 
provides care to more than 500 acute, traumatic SCI patients annually, including 100 C-SCI 
patients.  From this population, more than 50 patients are estimated to have C-SCIs categorized 
as AISA-C and are potential candidates for intramuscular diaphragm pacing.  We aim to 
complete successful tests in 5 individuals. To complete these tests, we anticipate enrollment of 
10 individuals who meet study inclusion criteria. Study procedures will be conducted at UF 
Health Jacksonville. Dr. Yorkgitis routinely provides medical and surgical care to this study 
population. 
Of note, we will aim to recruit and enroll participants once they are scheduled to undergo 
diaphragm pacing (i.e. before pacer placement) so that the baseline tests can be performed 
prior to implantation and post-implant assessments can be performed shortly after implantation 
of the diaphragm pacer.  We recognize that this process may not always be feasible due to 
complexities in the patient’s medical condition or status. In some cases a patient may undergo 
implantation of the diaphragm pacer and the device may not be turned on for days. We, 
therefore, may enroll participants up to 5-days following pacer implantation. This strategy will 
help ensure that we meet enrollment goals and include those who would like to participate but 
had delayed use of the pacer. 
Dr. Yorkgitis  will inform potential participants about the study and study-related procedures.  
Because Dr.  Yorkgitis  provides medical care and/or performs surgical diaphragm pacer 
implantation to this C-SCI population, he will identify potential participants who meet the 
inclusion/exclusion criteria.  The specified criteria (determination of SCI injury level and status, 
respiratory function, medical history) are routinely part of this patient population’s medical care 
and medical history. If a potential participant would like to learn more about the study, detailed 
information will be provided to the patient and their caregivers/family.  The informed consent 
document will be used to guide this process. Dr. Yorkgitis  also will notify the PI, Dr. Fox or a 
study Co-I that a patient would like to learn about the study and may be potentially interested in 
enrolling.  Study coordinators, Jennifer Mull or Yohan Diaz Zuniga, who work closely with the 
study physicians, also may notify Dr. Fox and study Co-I. Based on this, Dr. Fox and/or a study 
Co-I will discuss the study with the patient and caregivers/family.  
Study design & overview.  
A single-factor quasi-experimental time-series study design is proposed.  We will systematically 
evaluate diaphragm activation from EMGs recorded from intramuscular pacing electrodes and 
test inspiratory muscle strength and respiratory function in a small group of patients with acute, 
C-SCIs who have undergone implantation of diaphragm pacing electrodes.   
 
An overview of the assessments is below, with details outlined in the subsequent 
sections. 

 
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Assessment Overview: We will study a small cohort of patients with acute C-SCIs following 
implantation of intramuscular diaphragm pacing wires. All individuals will meet inclusion criteria 
and will complete the informed consent process. Experiments are designed to determine if 
diaphragm stimulation (pacing) can produce neuroplastic changes in respiratory neural drive to 
the diaphragm and increase respiratory function. Neural drive to the diaphragm will be assessed 
by recording electromyograms (EMGs) from the intramuscular pacing wires following each of 3 
blocks of diaphragm stimulation (Figure 3). Concurrent with these recordings from the 
diaphragm, respiratory function will be assessed using standardized clinical respiratory 
measures.  
Diaphragm EMGs will be recorded from the intramuscular diaphragm pacing electrodes and 
respiratory function will be assessed during non-stimulated respiration (diaphragm pacing unit 
turned off).   Diaphragm EMGs will be recorded from the intramuscular pacing electrodes with a 
custom connector that attaches to the external pacing electrode wires and interfaces with an 
EMG acquisition system. Concurrent with EMG recordings, each assessment will include tests 
of maximal inspiratory pressures to assess diaphragm muscle strength and maximal activation.  
Respiratory function also will be assessed by measurement of standard spirometry (tidal 
breathing, forced vital capacity or FVC).  
To classify the level and severity of each subject’s C-SCI, a clinical ASIA neurologic 
examination will be performed in accordance with the International Standards for Neurological 
Classification of Spinal Cord Injury. This standardized clinical examination will allow us to 
characterize segmental sensory and motor function.   
Baseline Assessments 
After obtaining informed consent for participation in the study, Dr. Fox or designated members 
of the study team (Alicia K. Vose, PhD, Kathryn Doughty (Cavka), DPT) will review the 
participant’s medical records to obtain study-related medical and demographic data. Information 
obtained from the medical record will include information pertaining to health and medical 
history, details pertaining to the SCI, reports of medications and test results that could influence 
the individual’s participation in the research procedures..  Additionally, clinical tests of sensory 
and motor function will be conducted to classify the level and severity of the individual’s SCI.  
The tests of sensory and motor function are part of the American Spinal Injury Association 
(ASIA) Impairment Scale and the International Standards for Neurological Classification of 
Spinal Cord Injury.  These clinical assessments are part of standard clinical care and include 
tests of sensation (dermatomes) and muscle strength via manual muscle testing (myotomes). 
These routine, standardized tests are done to clinically determine which segmental levels of the 
spinal cord demonstrate normal or impaired function.  These tests also aid in determining if the 
SCI is clinically complete or incomplete.  Determination of complete vs. incomplete is based on 
the sensory and motor function in the lowest sacral segments which control sensation and 
motor function at the anal sphincter.  These standardized clinical tests are part of routine 
examination following spinal cord injury.    
Baseline respiratory testing will be initiated once per day, up to 3 times on the days prior to 
diaphragm pacer implantation (Figure 3). Baseline respiratory tests include measurements of 
standard spirometry (tidal breathing, forced vital capacity or FVC) and maximum inspiratory 
pressures. We recognize that enrolling patients prior to diaphragm pacer placement may not 
always be feasible and we, therefore, may enroll participants up to 5-days following pacer 
implantation and will not obtain baseline (pre-implant) respiratory measures in these individuals.  
Pre-stimulation Assessment (post-diaphragm pacer placement)* 

 
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Following diaphragm pacer placement, the pre-stimulation assessment (prior to turning on the 
diaphragm stimulation) will be initiated approximately 18-24 hours after pacer implantation, once 
the patient is medically stable and able to participate. Waiting 18-24 hours after pacer 
implantation is standard of practice based on the procedures used by the diaphragm pacing 
surgical team (Dr. Yorkgitis). For individuals enrolled within 5-days after pacer implantation, the 
pre-stimulation assessment will occur as soon as possible after enrollment. The pre-stimulation 
assessment  will include clinical respiratory tests and recording of diaphragm EMGs which are 
detailed in sections below. Pre-stimulation assessment will take approximately 2 hours to 
complete. To obtain a clear pre-stimulation assessment, two sets of tests will be conducted, 
about 1 hour apart.  Ample time for rest breaks will be provided. The participant will be closely 
monitored and testing will be adjusted for each participant’s comfort and medical needs. 
Post-tests (following periods of diaphragm stimulation)* 
Post-testing will occur after 3 different blocks of diaphragm stimulation, and will occur with the 
stimulator turned off (Figure 3). These blocks are contiguous, meaning block 2 will begin 
immediately after post-testing in block 1, and block 3 will begin immediately after post-testing in 
block 2. See Figure below. Of note, turning the stimulator off and on is a typical occurrence in 
the medical setting. This allows patients to get used to the stimulation and also is necessary to 
change batteries in the control box. This externalized control box is easily accessible and has 
simple controls for turning the unit on and off. Post-tests will include the same battery of clinical 
respiratory assessments and diaphragm EMGs as pre-testing and will take approximately 1 
hour to complete. The duration of stimulation varies for each block:  
 
Block 1: ~1 hour of stimulation (no longer than 2 hours*) followed by post-test 1 
Block 2: ~ 1 hour of stimulation (no longer than 2 hours*) followed by post-test 2 
Block 3: ~ 12 hours of stimulation (no longer than 24 hours*) followed by post-test 3 
 
Figure 3 
 
Dr. Yorkgitis will provide medical oversight of procedures.  Assessments will be conducted by the 
PI, Dr. Fox and/or Alicia K. Vose, PhD and Kathryn Doughty (Cavka), DPT. A study research 
assistant may assist with the assessments.  The study team has extensive experience with these 
assessments. Assessments are conducted in the intensive care unit, with the patient’s care team 
easily accessible.  

 
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*Given the medical condition of these participants, we will adjust timing or omit a test if 
necessary, as to not interfere with the participant’s medical care.  Furthermore, the timing of 
tests and duration of stimulation may vary slightly given the constraints of patients being in the 
intensive care unit (ICU) and coordination with nurses, physicians, and ICU scheduling. 
Additionally, these parameters may vary based on the safety and medical monitoring of each 
subject as directed by Dr. Yorkgitis.* 
During the assessments, photographs and/or video recordings may be obtained.  These 
recordings will be done with the participant’s knowledge and consent for the type of recording 
being obtained.  Photographs and video recordings will be used to communicate study 
procedures with the study team and to monitor the assessment process.  Video and 
photographic data will be used to assess how individuals with SCIs perform and respond to the 
testing and to communicate the study procedures.  Video and photographic information will be 
used in a manner based on the level of consent obtained from each participant.    
Diaphragm Stimulation: This protocol will not alter the implantation of the diaphragm pacer. This 
study will assess those who have received or who are planned to receive the device based on 
their medical needs and prior medical assessments (not associated with this study). Diaphragm 
stimulation settings are determined intraoperatively or immediately postoperatively by the 
surgeon (Dr. Yorkgitis) to maximize stimulation effectiveness and also to ensure patient comfort.  
Parameters such as stimulation amplitude, frequency, and breaths per minute are adjusted 
within a pre-set range based on the device usage guidelines.  Typical values are 15-25mA at a 
frequency of 30 or 50Hz, and at a rate of 12-18 breaths per minute;39,40 we expect stimulation 
parameters to be in this range.   
Assessment of neuromuscular activation of the diaphragm. Neuromuscular activation of the 
diaphragm will be assessed by recording diaphragm EMGs from the surgically-implanted 
intramuscular stimulating electrodes.11,75  This approach will allow for comparisons of EMG 
recordings across time and reduces the methodological limitations associated with surface or 
percutaneous EMG approaches.9  EMGs will be recorded during non-stimulated respiration 
(diaphragm pacer turned off) and simultaneously with assessments of respiratory function.  
These assessments may include maximal inspiratory pressures and assessments of resting 
tidal breathing and measurement of flow volumes using standard spirometry.76,77  
Primary outcomes to characterize neuromuscular activation of the diaphragm and assess 
changes in diaphragm activation associated with intramuscular diaphragm stimulation will 
include: amplitude and timing characteristics (raw, filtered EMGs and normalized);76,77 neural 
respiratory drive to evaluate diaphragm muscle activation during tidal breathing relative to 
activation capacity during maximal inspiratory maneuvers;77,78 and time-frequency 
characteristics of the diaphragm EMGs (wavelet analysis).75,79-81  Methods pertaining to 
diaphragm EMG signal acquisition and processing are explained below.   
Diaphragm EMG acquisition and signal processing. The PI has extensive experience with EMG 
acquisition and analyses in humans.69,70  EMGs will be recorded from the externalized pacing 
wires which connected to each of four intramuscular electrodes (2 in each hemidiaphragm).  
The implanted, remote sub-cutaneous electrode will serve as the ground.11  A custom-connector 
(Synapse Biomedical, Oberlin, OH) will be used to link the exposed pacing wires to our EMG 
acquisition system.  EMG signals will be amplified and band pass filtered (10 Hz to 1000 Hz), 
acquired at a sampling frequency of 2 kHz, and saved to a computer for off-line analysis.   
To acquire the diaphragm EMG signals, the pacer will be shut off to allow connection to the 
EMG acquisition system.  It is feasible to shut off the diaphragm pacer for several reasons.  

 
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First, most of the individuals with SCI have residual respiratory control and are able to maintain 
sufficient respiration for a short time period.  It is part of standard procedures to turn off the 
pacer on a regular basis to change the pacer batteries.  Consistent with this process (turning off 
the pacer when changing the batteries), supplementary respiratory support will be provided as 
needed (i.e. mechanical ventilation) and oxygen saturation will be monitored at all times to 
ensure adequate respiration (>90% O2 saturation) during the assessments.  Second, all 
assessments will be overseen by the participant’s clinical medical team (ICU) with oversight 
from the participant’s physician.  All assessments will include cardiorespiratory monitoring.  
Finally, an assessment will be halted at any time the patient indicates distress or indicates that 
he or she would like to stop the assessment.  In this case, the pacer will be re-connected and 
turned on and respiratory support will continue until the patient is comfortable.    
Neuromuscular activation outcomes. Raw, filtered EMGs, as well as normalized signals will be 
assessed for timing and amplitude characteristics.  Normalization will be based on maximal 
amplitude of the signals recorded during tests of maximal inspiratory pressure and sniff nasal 
inspiratory pressure.76,78,89  Signals will be full-wave rectification and amplitudes calculated 
based on the root mean square or integral of the signal.  Comparisons of normalized signal 
amplitudes, burst timing, and burst durations will provide evidence of changes in neuromuscular 
activation of the diaphragm in association with intramuscular stimulation of the diaphragm and 
following C-CSI.  Increased EMG amplitudes are indicative of greater motor unit recruitment and 
suggest gains in muscle force generation.65,75,90  
To determine the relationship between diaphragm muscle activation during resting tidal 
breathing and diaphragm activation capacity during maximal inspiratory maneuvers, the relative 
level of “neural respiratory drive” will be calculated from the normalized diaphragm EMGs.  
Neural respiratory drive is a measurement approach used to quantify the respiratory 
load/capacity relationship and is a marker of respiratory disease severity.78,89,91,92  Changes in 
neural respiratory drive are associated with changes in respiratory function and breathlessness 
during exercise in patient populations.78,92  Neural respiratory drive will be calculated based on 
the root mean square of the diaphragm EMG signals during resting tidal breathing normalized to 
the maximal EMG values obtained during the maximal inspiratory maneuvers.89  
Finally, time-frequency characteristics of diaphragm EMGs will be assessed using wavelet 
analysis.75,80,93  Continuous wavelet analysis using a Morlet wavelet will be used to decompose 
the diaphragm EMG signals into individual frequency components as a function of time. The 
Morlet wavelet will be used because it provides a reasonable balance between time and 
frequency localization.94  Wavelet transforms of the EMG signals will be calculated using a base 
algorithm in Matlab (The Mathworks, Natick MA) developed by Torrence and Compo and 
available at URL: http://paos.colorado.edu/research/wavelets.95  Comparisons of the frequency 
characteristics of the EMGs recorded during different respiratory maneuvers and longitudinally 
will provide novel insight regarding motor unit recruitment, firing frequencies, muscle fiber 
changes, as well as central activation of phrenic motor neurons.75,80,81 
Higher frequencies of the EMG suggest greater recruitment of larger muscle fibers and faster 
conduction velocities.  For instance, a shift toward higher frequencies during more forceful 
maneuvers or over-time would suggest recruitment of larger, more forceful motor units with 
higher recruitment thresholds.96,97  Such changes would support our previous observations8,23 
and others11,36  that intramuscular stimulation promotes neural and muscular adaptations.    
Assessments of diaphragm muscle strength and respiratory function.  

 
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Diaphragm muscle strength. PI, Dr. Fox, has experience with diaphragm and respiratory 
assessments. Maximal inspiratory maneuvers are typically used as an index of diaphragm 
muscle strength.98 Measures will be obtained in accordance with the American Thoracic Society 
testing guidelines100 and adjusted for SCI patients.101  Inspiratory pressures will be acquired 
using a digital respiratory pressure meter or a clinical respiratory monitor connected to a laptop 
computer.  Subjects will exhale to residual volume and then attempt a maximal inspiration for at 
least 2s in duration.  Subjects requiring mechanical ventilation will use a manometer connected 
to the subject’s endotracheal tube or tracheostomy and attached to a one-way valve, allowing 
only expiration.102 All maneuvers will be performed ~3-5 times with a minimum of 60s intervals 
between tests or rest breaks as needed by the participant. 
Respiratory function. In addition to the above assessments, respiratory function will be 
assessed by measurement of standard spirometry to measure forced vital capacity (FVC) and 
tidal breathing. A digital spirometer or clinical respiratory monitor connected to a laptop 
computer will be used to obtain forced flow volume curves/loops and to assess tidal breathing. 
Testing will be performed in accordance with the American Thoracic Society guidelines and 
adjusted for SCI patients.100,101  Percent predicted values will be calculated based on uninjured 
adults of the same age, gender and height.103   
Overall, a battery of clinical respiratory assessments will be used to measure inspiratory or 
diaphragm muscle strength, inspiratory pressures, and respiratory function.  Testing procedures 
will be standardized (consistent test positions, standardized procedures, randomized testing 
order) and conducted in accordance with established guidelines to allow for appropriate 
comparisons.100,101  Results from this battery of respiratory assessments will allow us to 
characterize changes in inspiratory strength and respiratory function across time and in 
association with diaphragm pacing, and to determine the association of such changes with 
diaphragm activation.  
Safety monitoring. Our protocol will not alter medical care.  Oversight of study procedures will 
be provided by Dr. Yorkgitis, and study collaborators and trauma surgeons who provide acute 
SCI care, including diaphragm pacer implantation.  The PI, Dr. Fox, will oversee all assessment 
procedures and will maintain regular communication with the study physicians and study team.   
Medical monitoring during assessments in the ICU hospital setting will include bedside 
monitoring of vital signs including heart rate, blood pressure, oxygen saturation, and may 
include electrocardiogram.  If the patient is receiving ventilator support, ventilator settings and 
pulmonary mechanics also will be monitored.  Additionally, subjects will be monitored for signs 
of autonomic dysreflexia which include complaints of headache, sweating above the lesion 
level, elevated blood pressure and bradycardia.  Standard guidelines when autonomic 
dysreflexia is suspected will be followed104 and appropriate medical care and monitoring will be 
provided.   
Statistical analysis. As mentioned above, we are conducting an initial proof of concept 
experiment in a small (n=10) sub population of individuals with C-SCI.  Summary statistics will 
be provided for demographic and clinical characteristics.  We do not expect the data to have a 
normal distribution and therefore plan to use non-parametric statistical approaches. A Mann-
Whitney U will be used to test for differences in the EMG and respiratory outcomes pre and post 
stimulator activation. 
Interpretation and possible outcomes  If study outcomes are consistent with our preliminary 
data6-8,23 and a published report,11 we expect to demonstrate increased neuromuscular 
activation of the diaphragm and improved respiratory function associated with intramuscular 

 
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stimulation of the diaphragm.  This outcome would suggest a rehabilitative effect and that 
diaphragm pacing is a therapeutic strategy to promote respiratory function after C-SCI.  Further, 
changes in neuromuscular activation would provide evidence that diaphragm pacing activates 
myogenic pathways and could be a trigger for spinal respiratory plasticity.  These outcomes 
would justify and inform a larger diaphragm pacing trial and identify candidate mechanisms 
underlying restoration of function.       
It is possible (and perhaps likely) that some individuals will demonstrate gains in respiratory 
function and others will not. This outcome may be associated with variations in SCI lesion 
characteristics and spared spinal pathways mediating changes in respiratory control. This result 
would afford the chance to begin looking for determinant factors associated with ‘responders’ to 
diaphragm pacing and ‘non-responders’, i.e. individuals who benefit from pacing by weaning 
from mechanical ventilation, but who do not demonstrate gains in respiratory function or 
diaphragm activation.  Some individuals also may exhibit changes in neuromuscular activation, 
but no apparent gains in respiratory function.  This outcome also could suggest that changes in 
neuromuscular activation occur before an overt change in respiratory control is detectable.   
Participant compensation To compensate participants for their time and involvement in the 
assessment procedures, study participants will be paid fifty ($50.00) for each assessment. 
Payments will be made through the U of Florida Human Subject Payment system using 
payment cards. Since up to 8 assessments may be conducted (7 EMG/respiratory assessments 
and the ASIA sensory/motor exam), each participant may receive up to $400.00 for completion 
of study procedures.  
7. Possible Discomforts and Risks: 
There is a low risk of harm associated with the assessments in this protocol.  All procedures will 
be closely monitored and fully explained to the participants (using lay-language, understandable 
terms) so that they understand what to expect and the reasoning for the procedures.  They will be 
informed that they may refuse testing or discontinue study participation at any time.  For 
participants who have difficulty or who are unable to communicate verbally, a communication 
system will be implemented.  For instance, the study team will monitor facial expressions and use 
eye blinking by the participant to respond yes/no or signal discomfort.  Participants may be at risk 
for autonomic dysreflexia, although the incidence is low in patients with acute injuries.  
Participants will be informed to alert the study team if they notice symptoms consistent with 
autonomic dysreflexia (head ache, sweating above the lesion, elevated blood pressure).  The 
study team will closely monitor the participants and is trained to assess and respond to signs of 
dysreflexia.  All procedures will occur in the ICU setting with the participant’s medical team 
immediately available.  
The clinical ASIA snesory and motor examination of neurologic function is part of standard clinical 
assessments after SCI and is non-invasive.  There is no specific risk of this assessment which 
includes standard tests of sensation and motor function (strength).  Participants may feel 
frustrated or uncomfortable with the testing because they are unable to feel sensations and move 
their limbs as they did prior to SCI.  This may upset some individuals.  It may be uncomfortable for 
some participants to undergo testing in the peri-anal region.  This testing is part of the standard 
care after SCI.  Some participants may feel pain or uncomfortable sensations during the testing.  
The procedures will be adjusted to be as comfortable as possible and the participants will be 
closely monitored. 

 
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The respiratory assessments also are part of standard clinical care after SCI and in individuals 
with respiratory impairments.  These assessments may cause anxiety and may challenge an 
individual’s breathing and cause participants to feel out of breath.  These discomforts will be 
explained.  Participants will be closely monitored as previously described.  At any time the 
assessments may be discontinued and breathing support provided as indicated (i.e. re-connect to 
the ventilator) or turn back on the diaphragm stimulator or supplemental oxygen support. 
Recording of diaphragm EMGs from the external pacing wires is not part of clinical care.  This 
procedure does not pose direct risk or likelihood of harm.  It is a non-invasive procedure since the 
pacing wires are already implanted and externalized.  The participant may feel breathless or 
uncomfortable with the device turned off.  Some participants may feel nervous about turning the 
pacer off.  However, as previously described, this is part of standard procedures during regular 
battery changes and cleaning of the connectors.  Our team is experienced in working with patients 
and helping them feel ‘OK’ and safe with the pacer turned off.  The participant will be closely 
monitored and supplemental respiratory support will be readily available.  At any time the 
assessments may be discontinued and breathing support provided as indicated (i.e. re-connect to 
the ventilator or turn back on the diaphragm stimulator or supplemental oxygen support).    
8. Possible Benefits: 
There is no direct benefit to participants enrolled in this study.  This study will involve assessments 
to determine the effects of diaphragm pacing in adults with C-SCIs.  The implementation of this 
intervention (diaphragm pacing) will not be altered by this study.  Our goal is to monitor and study 
the effects of this intervention after short duration, carefully controlled sessions of stimulation.  The 
outcomes from this study may benefit future individuals with C-SCIs or other clinical populations 
with respiratory impairments.   
The risks associated with this study are reasonable because overall the risks are minimal and 
there is potential benefit to future individuals with C-SCIs, which is a severe catastrophic 
condition.  This study is an essential step in determining the efficacy of intramuscular diaphragm 
stimulation and its effects on respiratory function after SCI.  Outcomes from this study may alter 
and/or improve respiratory care post-C-SCI.  Currently, respiratory illness and complications are 
the leading cause of illness and death after SCI.  Advancements in respiratory care and long-term 
management are needed to improve outcomes post-SCI and improve health and quality of life 
after SCI.   
9. Conflict of Interest: 
The investigators associated with this research project have no known conflicts of interest.   
 
 
 
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