Acute Intermittent Hypoxia to Enhance Motor Function After Spinal Cord Injury
Phase: NA
Conditions: Spinal Cord Injuries
Sponsor: University of Florida

Intermittent Hypoxia to Enhance Motor Function after Spinal Cord Injury 
NCT03071393 
University of Florida IRB protocol 2016-1680 
Study protocol 5/22/2019 
 
 
 

1. 
Title: Intermittent hypoxia to enhance motor function after spinal cord injury: a pilot 
study. 
 
2. 
Investigators: 
Principal Investigator: 
 Emily Fox, PT, DPT, PhD 
Co-Investigators and study staff: 
 Tommy Sutor, MS, CSCS 
 Kathryn Doughty, DPT, NCS 
 Alicia Vose, MS, CCC-SLP 
 Joseph Welch, PhD 
 Kelly Hawkins, DPT, NCS 
 Christy Conroy, MSPT 
 Arian Vistamehr, PhD 
 Paul Freeborn, BS (Study Coordinator) 
 
3. 
Abstract: Spinal cord injury (SCI) affects the trunk muscles that control respiration 
and posture. Decreased respiratory muscle function can lead to diseases of the 
respiratory system, which are the primary cause of death and significant cause of re-
hospitalization after SCI. Deficits in postural muscle function affect one’s ability to 
balance, safely maintain a seated position, or ambulation after SCI, severely impacting 
daily activities such as self-care and feeding skills.  Most people have incomplete SCI, 
and therefore have spared motor pathways. Thus, the majority of people with SCI would 
potentially benefit from treatments that strengthen spared motor pathways. One such 
treatment that strengthens spared motor pathways after spinal cord injury is acute 
intermittent hypoxia. 
 Acute intermittent hypoxia induces an episodic release of serotonin near motor 
neurons, beginning an intracellular cascade of events which results in an increased 
affinity of the motor neuron for excitatory neurotransmitters. Through this mechanism, 
spared motor pathways after SCI can potentially be strengthened by acute intermittent 
hypoxia. A number of studies have demonstrated improved function in individuals with 
SCI with daily (5-10 days) mild episodes of acute intermittent hypoxia.  Benefits have 
been demonstrated in ventilation, maximal ankle plantarflexion torque, walking speed and 
walking endurance. Based on the mechanisms of action and the demonstrated benefits 
of acute intermittent hypoxia, this intervention also may improve function of muscles that 
control respiration and posture.  
This study will test the hypothesis that a single session of acute intermittent 
hypoxia will increase strength and activation of the trunk muscles that control respiration 
and posture, leading to improved scores on functional assessments in individuals with 
SCI. Our long term goal is to better understand the therapeutic potential of acute 
intermittent hypoxia combined with physical rehabilitation for individuals with SCI. 
 
4. 
Background: 
      Most individuals, regardless of the severity of their SCI, maintain some intact spinal 
connections, creating an anatomical connection to the brain after injury (Flynn et al., 2011; 
Sherwood et al., 1992; McKay et al., 2004; Squair et al., 2016.) Different types of 

interventions, such as physical therapy, occupational therapy, electrical stimulation, or a 
combination thereof seek to take advantage of these intact connections, including spared 
motor pathways, to enhance function after SCI. The abundance of research on functions 
such as walking or grip strength has led to many intervention options for rehabilitating 
these abilities. However, research and interventions to enhance strength of trunk muscles 
which control respiration and posture is lacking, and therefore many people with SCI 
experience respiratory and postural impairments (Wang, 2016). For example, forceful 
respiratory maneuvers such as clearing the airway of secretions depend highly on 
abdominal and trunk motor function (De Troyer et al., 1986; Yokoba et al., 2003). Due to 
weak trunk muscles which contribute to these respiratory maneuvers, respiratory 
complications remain the second-leading cause of re-hospitalization after SCI (Kirshblum 
et al., 2004.) A majority of people with SCI would benefit from improved strength and 
activation of trunk muscles that control respiration and posture. Acute intermittent hypoxia 
may be a way to strengthen and activate those muscles through spared motor pathways. 
      The mechanisms by which acute intermittent hypoxia strengthens and increases 
activation of muscles after SCI has been established by extensive animal research. 
Briefly, acute intermittent hypoxia activates the carotid body chemoreceptors, triggering 
intermittent serotonin release in or near spinal motor nuclei. Subsequent serotonin 
receptor activation triggers new brain-derived neurotrophic factor synthesis, enhancing 
synaptic input, motor output, and the strength of muscle contractions (Gonzalez-Rothi et 
al., 2015; Baker-Herman et al., 2004; Fuller et al., 2003). Regarding functional recovery, 
rodent studies demonstrate acute intermittent hypoxia improves breathing and forelimb 
function (Lovett-Barr et al., 2012), and complex locomotor tasks (Prosser-Loose et al., 
2015) after SCI. In each of these aforementioned studies, no negative reactions to 
intermittent hypoxia were reported for any of the rodents. 
      Human clinical studies have yielded results similar to those of animal studies. Acute 
intermittent hypoxia can enhance ventilation (Tester et al., 2014; Sankari et al., 2015), 
ankle strength (Trumbower et al., 2012; Lynch et al., 2016), walking speed, and walking 
endurance (Hayes et al., 2014; Navarrete-Opazo et al., 2016b) after SCI. In these human 
studies, no adverse events were reported.  Subjects tolerated the intervention and were 
unable to distinguish between a sham and experimental condition. 
 
In one study, Trumbower et al. (2012) reported a mean increase in voluntary ankle 
torque of 82% in individuals with incomplete spinal cord injury, following 15, 60-90 second 
exposures of 9% oxygen alternated with 15, 60-second exposures to room air. In another 
study, Navarrete-Opazo et al. (2016b) found significant improvements in 10-meter walk 
times and 6 minute walk test differences for individuals with spinal cord injury. These 
results were accomplished after four weeks of repeated walking training and repeated 
bouts of 15, 90-second exposures to 9% oxygen alternated with 15, 90-second exposures 
to room air. In both studies, results were significantly improved over control subjects who 
received sham treatment, and no adverse events were reported for any subject.   
Research examining the effects of acute intermittent hypoxia on trunk muscles that 
control respiration and posture has not been conducted. However, based on the 
mechanisms and prior studies demonstrating benefits of intermittent hypoxia, we expect 
acute intermittent hypoxia to improve the strength and activation of trunk muscles that 
control respiration and posture. The fundamental hypothesis guiding this proposal is a 

single session of acute intermittent hypoxia improves trunk muscle strength and 
activation, thereby improving respiratory strength and postural function after SCI. 
 
5. 
Specific Aims: 
Specific Aim 1: test the hypothesis that a single session of acute intermittent hypoxia 
improves respiratory strength in individuals with SCI compared to a sham treatment. 
Respiratory strength will be quantified using standardized measures of maximal 
inspiratory pressure, maximal expiratory pressure, forced vital capacity, and mouth 
occlusion pressure (P0.1). Muscle activation during the respiratory tests will be quantified 
using surface electromyography (EMG). 
 
Specific Aim 2: test the hypothesis that a single session of acute intermittent hypoxia will 
enhance postural strength and function in individuals with SCI compared to a sham 
treatment. Postural strength will be assessed in a seated position with standardized 
procedures, using a hand-held force dynamometer. Postural function will be quantified by 
assessing performance in 4 tests of sitting ability contained within the Neuromuscular 
Recovery Scale. Additionally, trunk and lower extremity muscle activation during postural 
tasks will be assessed using EMG. Additional non-invasive sensors may be worn to 
assess joint angles or forces applied during testing.  
 
Aim 2a: Postural control and trunk strength contributes significantly to the ability to 
stand and walk. For subjects who are able to safely stand or walk short distances, a 10-
meter walk test, 30-second chair stand test, or timed up-and-go test may be performed. 
Trunk and lower extremity muscle EMG will be recorded during testing. Sensors also may 
be worn to assess joint angles or forces generated during these tests. 
 
6. 
Research Plan: 
Inclusion criteria: 
1) Male or female, ages 18-65; 2) medically stable; 3) >6 months post-SCI; 4) SCI 
affecting segments between C4-T12; 5) no other known neurological disorders; 6) no 
severe musculoskeletal impairments, open wounds, or skin lesions that would limit 
participation in functional assessments; 7) able to provide informed consent 
 
Exclusion criteria: 
1) 
Not meeting any of the above inclusion criteria; 2) presence of a self-reported 
uncontrolled medical condition including, but not limited to: cardiovascular disease; sleep 
apnea; obstructive lung disease; severe neuropathic or chronic pain; severe recurrent 
autonomic dysreflexia; 3) severe, untreated bladder or urinary tract infection; 4) women 
who report being pregnant or test positive on a pregnancy test. 
 
We aim to screen 40 individuals with SCI and enroll 30 subjects in this study.  
 
Recruitment and enrollment 
The target population will be individuals with SCI. Study procedures will take place at 
Brooks Rehabilitation in Jacksonville, FL, and the University of Florida’s Health Science 
Center Department of Physical Therapy Research Laboratories.  Subjects will be 
recruited in four ways. 

1) 
The study team will notify Brooks clinicians about the study via e-mail. The study 
team may also notify other clinicians who work with individuals with SCI through e-mail. 
Those clinicians may then notify their patients about the study, who may then contact Dr. 
Emily Fox, the principal investigator, or other study staff for more information.  
2) 
Potential subjects also may be informed of the study via IRB approved flyers, which 
will be hung in the Brooks Health System, the Health Science Center at UF, and in the 
community. Contact information will be provided on the flyer so that interested potential 
subjects may contact Dr. Emily Fox for more information. 
3)  
Information regarding the study also may be provided through word of mouth or 
general presentations such as at support groups and community meetings (i.e. such as 
meetings for those with SCIs).  
4)  
All individuals admitted to Brooks Health System sign a Brooks Notice of Privacy 
Practices upon admission which indicates their information may be used for research. 
This is a standard practice for the Brooks Health System facilities. For individuals who 
sign this form, Brooks Research Recruitment personnel have permission to access their 
medical records for research-related purposes. Therefore, Brooks Research Recruitment 
personnel would be aware of our study (as it is being conducted at a Brooks facility) and 
would include this as one of several potential research study opportunities when 
contacting Brooks patients. This procedure is independent of our research study 
procedures and is carried out by Brooks Research Recruitment personnel; potential 
participants will only be contacted for screening for this specific study if they indicate to 
Brooks Research Recruitment personnel that they want to learn more about our study.  
 
Individuals who are potentially interested in enrollment in the study will have ample 
opportunity to ask questions, talk with study staff and review the informed consent 
document.  If the individual indicates that he/she would like to determine if they qualify to 
participate in the study, they will be asked to complete a basic screening questionnaire to 
determine if they are eligible to participate. The screening may occur over the phone or 
in-person depending on the preference of the potential participant. The IRB-approved 
screening form, designed to avoid collection of protected health information, (see 
attached) will ask subjects general information about their eligibility. This information may 
include health status, physical functioning, history of neurologic impairments, the date of 
their SCI, and other information pertaining to inclusion/exclusion criteria. If an individual 
completes the screening process, is eligible to participate, and would like to enroll, a study 
staff member will meet with them to complete the informed consent process.  The 
informed consent form (ICF) will be used to guide the process.  The potential participant 
will have the opportunity to review the ICF ahead of time.  Potential participants will be 
informed there may be no direct benefit of participation. Study staff will be available to 
answer questions.  The informed consent process will occur prior to study procedures but 
may occur on the same visit as the anticipated study procedures.  The informed consent 
process also may be scheduled on a day prior to study procedures based on the 
preference of the potential participant.  We anticipate that some potential participants may 
want to meet with study staff ahead of time and others will prefer to complete the informed 
consent form and procedures on a single visit. 
 

Participants may be asked to participate in the following activities during an assessment. 
They will be informed that they may choose not to perform any aspect of the study. 
 
Overview 
This pilot study will utilize a single blind, placebo-controlled, randomized, repeated 
measures experimental design. Participants will be asked to come in for two visits at least 
7 days apart. If study procedures are not completed in the two sessions (such as due to 
equipment malfunction or unexpected time restrictions/events), an additional session may 
be required to complete procedures. This will be scheduled at the participant’s 
convenience. Participants will be asked to wear comfortable, casual clothing for 
participation ahead of time, during their phone screening. In the first visit, participants will 
be asked to complete a demographic health and function questionnaire. Questions may 
address their injury level, medical history, impact of their condition on physical functioning, 
breathing function, and their walking or other functional abilities. Participants may then be 
asked to undergo standardized assessments of muscle strength and sensory function. 
Following collection of this baseline information, the remainder of the first visit will be 
similar to all subsequent visits. Visits will include: 
 Pre-assessments of respiratory strength, postural strength and function, EMG of 
trunk muscles, and trunk positioning using non-invasive sensors. For subjects who 
can stand or walking with assistance, ambulation assessments may be conducted 
while also recording trunk and lower extremity EMGs, and joint angles or forces 
using non-invasive sensors. 
 15 short exposures to hypoxic air, or a sham exposure, in a randomized order 
(details below) 
 Post-assessments of respiratory strength, postural function, ambulation, EMG of 
trunk muscles, and trunk and joint positioning using non-invasive sensors 
 
Each session will last about 3 hours and no longer than 6 hours.  Rest breaks will be 
provided at regular intervals and at any time the participant requests.  A study clinician 
will oversee the study procedures and monitor the participants, including a baseline 
assessment of vital signs (blood pressure, heart rate, and blood oxygen saturation) for 
safety.  
 
For participants who provided consent for photography and/or video, recordings or 
photographs may be obtained during their session.  Recordings or photographs will only 
occur based on the consent provided by each participant.  The participant will be informed 
that they may refuse the use of photography or video recordings at any time during the 
procedures.   
 
Study Procedures 
Participants will be asked not to ingest any caffeine within four hours of each visit, as 
caffeine can affect the response to acute intermittent hypoxia. After consenting, female 
subjects of potential childbearing age will be asked to take a pregnancy test. A pregnancy 
test will not be required if a medical condition that prohibits pregnancy, such as 
hysterectomy, is reported. Participants will be able to sit or lie on a mat table comfortably 
during set-up. Instrumentation will generally include EMG sensors over the trunk, 

respiratory, and lower extremity muscles, which may be secured with hypoallergenic tape. 
Electrogoniometers to measure joint angles may be attached at the hips, knees, or along 
the vertebral column with double-sided medical tape. Pressure sensors also may be 
placed under the heel or ball of the subject’s feet, and worn in the shoes. All sensors are 
non-invasive and will be removed at the conclusion of testing. 
 
1) 
Baseline and demographic assessments:  
Participants may undergo the ISNCSCI (International Standards for the Neurological 
Classification of Spinal Cord Injury) examination. The ISNCSCI is a standardized 
assessment used to determine the motor and sensory impairment and severity of a spinal 
cord injury (Kirshblum et al., 2011; previously known as the ASIA examination based on 
the American Spinal Injury Association). A study clinician will complete this assessment 
using standardized procedures. Subjects may also be asked general demographic 
information via and IRB-approved demographics form about their injury, rehabilitation 
history, current health status, and current activity and exercise levels.  
 
2) 
Standardized assessments of respiratory function 
These clinical tests of respiratory function will be administered according to standardized 
guidelines (American Thoracic Society, 2002; Miller et al., 2005). Clinical outcomes from 
these assessments will allow us to assess the level of respiratory function of subjects vs. 
predicted values derived from able-bodied individuals based on the subject’s age. 
Participants will complete up to, but no more than 10 trials of each test in order to ensure 
a maximal effort was obtained. Rest breaks will be allowed at the subject’s request as 
often and as long as they are needed. Oxygen saturation and breathlessness will be 
monitored for safety. The assessments may include: 
a) 
Maximum inspiratory pressure – an evaluation of inspiratory respiratory muscle 
strength, assessed using a digital respiratory pressure meter.   
b) 
Maximum expiratory pressure – an evaluation of expiratory respiratory muscle 
strength, assessed using a digital respiratory pressure meter. 
c) 
Forced vital capacity  – The forced vital capacity is an evaluation of how much air 
a person can forcefully exhale after a maximal inspiratory effort. Tests are conducted 
using a digital spirometer or respiratory monitor.  
d) 
Mouth occlusion pressure (P0.1) – P0.1 is an index of the neuromuscular drive to 
breathe, independent of lung mechanics. Tests are performed by having participants 
breathe naturally through a mouthpiece with their eyes closed. Upon completion of one 
breath, at random, the tester manually occludes the mouthpiece for less than half of one 
second. The pressure generated in the first 0.1 seconds of the participant’s initiation of 
inhalation will be recorded as the participant’s P0.1. 
 
 
 
3) 
Standardized assessments of functional ability 
3a)  
Functional ability of the trunk muscles 
The degree to which subjects can independently perform certain tasks that require trunk 
muscles will be assessed with the Neuromuscular Recovery Scale. Four components of 
this standard, reliable and responsive clinical test of functional ability for people with SCI 

will be used (Tester et al., 2016). Standardized instructions will be provided to the subjects 
for each component. A study staff member trained in administering the Neuromuscular 
Recovery Scale will supervise testing to ensure proper execution and subject safety. 
These components include: 
a) 
Sit – testing the subject’s ability to sit tall with proper posture when the feet 
are flat on the floor. 
b) 
Reverse Sit-up – subjects will be asked to lower themselves from sitting up 
to lying supine on a mat as slowly and controlled as possible. 
c) 
Sit-up – the subject’s ability to sit up from lying supine on a table while the 
feet are on the ground will be assessed. 
d) 
Trunk extension in sitting – while seated on a table with feet on the floor, 
subjects will be asked to lean forward with their arms hanging down, and then return to a 
seated position without using their hands. 
 
3b) 
Assessment of postural muscle strength in sitting 
A standardized and reliable protocol using a hand-held force dynamometer will be used 
to assess postural muscle strength for maintaining upright sitting (Larson et al., 2010). A 
study staff member will apply force to the participant’s chest (anterior strength), upper 
back (posterior strength), or either shoulder (lateral strength) while the participant is asked 
to resist the force to remain seated, unsupported, on a mat table. The force applied at the 
point that the participant loses balance or can no longer sit unsupported will be recorded 
as strength in that direction. This test of seated strength will be carried out by a study staff 
member experienced in administering the test, and the participant will also be spotted by 
an additional therapist or research assistant in case the participant is unable to balance 
themselves during the test. 
 
3c) Assessments of ambulation 
These are standard, clinical tests of functional ability that may be used to assess mobility 
in subjects who can safely attempt these tests. Outcomes from these assessments will 
allow us to quantify the level of functional mobility of our subjects, and if acute intermittent 
hypoxia affects levels of functional mobility. These assessments may include: 
a) Timed Up and Go Test – Assessment of walking balance, and fall risk based on 
the participant’s ability to stand up form a seated position, walking 10 meters, 
turn around and return to the chair and sit down. 
b) 10 meter walk test – Assessment of walking speed over a distance of 10 meters 
c) 30 second chair stand test – Assessment of lower body strength and power in 
mobility impaired populations, by assessing the maximum amount of times that a 
participant can safely stand up from a seated position in 30 seconds. 
 
4) 
EMG activation and activation patterns of respiratory, postural, and lower extremity 
muscles 
Muscle activity will be collected and measured by surface EMG. By analyzing the 
electrical activity generated during a muscle contraction, EMG analyses allow us to 
understand timing and amplitude of muscle activation as well as the frequency content of 
the EMG signal. Thus, we will be able to assess how the nervous system’s control of 

muscles is disrupted following SCI, and whether this control will be affected by acute 
intermittent hypoxia. 
 
Using standard surface preparation (cleaning the skin with water or alcohol pad, shaving 
of excess hair) and electrode placement, EMGs will be collected from postural muscles 
of the trunk, accessory respiratory muscles, and lower extremity muscles using surface 
EMG electrodes. EMG sensors will be attached to the skin with hypoallergenic medical 
tape. Before data collection, tests of each EMG channel may be performed to ensure 
clear signals and to check for crosstalk. EMG information will be used to assess when 
different postural, respiratory, or lower extremity muscles fire during the assessments, as 
well as when certain muscles fire in relation to one another. 
 
 
5) Trunk positioning and function using non-invasive sensors. 
Electrogoniometers may be placed on the hips, knees, or spinous process on the back of 
subjects to allow for quantification of the angle of the spine or hips achieved during each 
task. These sensors will be attached to the skin using double-sided, medical grade, 
hypoallergenic tape. Information gained from these movement sensors will allow for 
quantification of how a subject performs respiratory tests and postural muscle functional 
tasks. 
 
Very thin, light pressure sensors made of flexible plastic may be worn in the subjects’ 
shoes, under their heels and/or the ball of their feet. As the subjects will be seated with 
their feet on the ground during the tests of postural muscle function, the non-invasive 
pressure sensors may help give an idea of how much force the subject is imparting on 
the ground during different tasks. Since the seated tasks of postural muscle function are 
dynamic in nature, the amount of pressure subjects will have to apply to the ground to 
accomplish the tasks must change. Quantifying the amount of force the subject applies 
to the ground may better inform us how well they are doing a particular task. 
 
Delivering an acute intermittent hypoxia or a sham procedure 
Acute intermittent hypoxia is a safe, non-invasive technique to improve muscle strength 
and activation after SCI. All sessions will be monitored by a  study clinician with 
experience in both delivering acute intermittent hypoxia, and treating individuals with SCI. 
Hypoxic intervals will be delivered with a commercially available sports performance 
hypoxicator. A unit capable of delivering a fraction of inspired oxygen ranging from 21%-
9% will be used. Air will be delivered into a mask that will be secured comfortably over 
the subject’s nose and mouth. An SpO2 sensor that monitors oxygen saturation of the 
blood will be used to monitor the subject’s response to hypoxic intervals; if the user’s 
SpO2 falls below 80% during a round of hypoxia, the fraction of inspired oxygen delivered 
by the hypoxicator will be rapidly increased in order to bring the user’s SpO2 back over 
the safety threshold. 80% SpO2 is adequate to induce the effects of hypoxia without 
causing adverse safety events (Trumbower et al., 2012; Hayes et al., 2014; Lynch et al., 
2016; Navarrete-Opazo et al., 2016a, 2016b).  
 

Acute intermittent hypoxia will be provided to the subject by delivering 15 brief exposures 
(60 to 120 seconds) of hypoxic air alternated with 15 brief exposures (60 to 120 seconds) 
of room air. The amount of oxygen delivered during hypoxic exposures may range from 
15%-9% fraction of inspired oxygen, compared to 21% oxygen in normal atmospheric air. 
Various protocols have previously used intervals and oxygen concentrations within this 
range (Trumbower et al., 2012; Tester et al., 2014; Navarrete-Opazo et al., 2016b). To 
switch from hypoxic to room air, a four-way valve will be attached to the subject’s mask 
which can allow air from the hypoxicator, or ambient air from the room to flow into the 
mask. The amount of oxygen delivered and the length of the hypoxic and room air 
exposures will be adjusted during the protocol to maintain safety, tolerability, and a 
consistent hypoxic response of the subject. The total time of all episodes of alternating 
room and hypoxic air will be between 30-45 minutes. Alternatively, a sham protocol will 
be administered in which 21% fraction of inspired oxygen will be delivered by the 
hypoxicator during hypoxic intervals, and room air will be delivered through the four-way 
valve during room air intervals. 
Subjects will rest and be asked not to ingest any food or drink other than water for 30 
minutes after the delivery of acute intermittent hypoxia, because fullness of the stomach 
may impede full depression of the diaphragm and compromise maximal respiratory 
measures. Following this, maximal inspiratory pressure, maximal expiratory pressure, 
forced vital capacity, mouth occlusion pressure, postural strength assessments, 
Neuromuscular Recovery Scale components, and assessments of ambulation will be re-
assessed as outlined above. EMG electrodes, electrogoniometers, and pressure sensors 
will be worn during the acute intermittent hypoxia and rest period, and will be removed 
following the conclusion of all testing. 
 
Following the procedures for each visit, a $75.00 gift card or check will be provided to 
participants. Thus, upon full completion of two visits, subjects will have been provided 
with $150.00 following their participation in the study. If participants are asked to come in 
for a third session, an additional $75.00 will be provided. 
 
No standard therapies for people with SCI currently include acute intermittent hypoxia. 
Thus, this research does not intend to directly deliver therapeutic benefits to subjects. 
Rather, this research seeks to determine the therapeutic potential of acute intermittent 
hypoxia on respiratory strength and postural muscle function for people with SCI. As an 
alternative to participating in this study, subjects may not enroll or choose to withdraw at 
any time. 
 
Statistical Plan 
Percent changes in maximal inspiratory pressure, maximal expiratory pressure, forced 
vital capacity, mouth occlusion pressure, EMG parameters, spine and hip angles, postural 
strength measures, absolute Neuromuscular Recovery Scale scores, and assessments 
of ambulation will be compared pre- and post-intervention for both experimental 
conditions. Changes for each subject will be averaged, and the group mean change ± 
standard deviation will be reported and compared across experimental conditions. 
Statistical differences between the group means for each variable in each experimental 

condition will be compared using t-tests. Multiple comparisons will be accounted for. 
Statistical analyses will be carried out by the study investigators.  
 
Acute Intermittent Hypoxia Safety Profile 
Acute intermittent hypoxia is a safe, non-invasive, relatively inexpensive way to 
strengthen spared motor pathways after SCI. With the mild to moderate acute intermittent 
hypoxia protocols used in neurorehabilitation research (Trumbower et al., 2012; Hayes et 
al., 2014; Navarrete-Opazo, 2016b), the severity of hypoxia, number of episodes, and 
exposure duration are low (e.g. 10-15, 1-2 minute episodes per day). The acute 
intermittent hypoxia protocols for this study are not similar to these more severe hypoxia 
exposures, as may be experienced during sleep apnea (Gozal & Kheirandish-Gozal, 
2008).Chronic intermittent hypoxia experienced in obstructive sleep apnea is severe and 
can consist of 80-400 episodes per night for many years, and is known to have detrimental 
systemic effects. While too high a dosage of intermittent hypoxia can be dangerous, lower 
doses, such as those previously published in acute intermittent hypoxia studies, can be 
both safe and beneficial after SCI (Navarrete-Opazo & Mitchell, 2014). All studies to date 
have demonstrated acute intermittent hypoxia-induced functional benefits in humans with 
SCI without adverse events or unwanted side effects (Trumbower et al., 2012; Hayes et 
al., 2014; Tester et al., 2014; Sankari et al., 2015; Lynch et al., 2016; Navarrete-Opazo 
et al., 2016a, 2016b). In these studies, changes in SpO2, blood pressure and heart rate 
were closely monitored, and no adverse events were reported. All study participants 
tolerated procedures without discomfort, and most were unable to distinguish acute 
intermittent hypoxia versus sham normoxia. In rodent models, repetitive acute intermittent 
hypoxia does not cause hypertension or weight loss (Wilkerson et al., 2009), hippocampal 
gliosis or neuronal cell death (Lovett-Barr et al., 2012; Satriotomo et al., 2012). 
Accumulating data indicate that acute intermittent hypoxia is safe and will induce 
meaningful gains in respiratory function in adults with chronic incomplete SCI. 
 
Safety monitoring 
The testing procedures are not particularly strenuous, but could be tiring for some 
individuals.  All participants will be given rest breaks and informed that they may rest or 
terminate their participation at any time. During brief hypoxic episodes, subjects may 
become sleepy. If subjects appear to be falling asleep they will be asked to stay awake 
and report any concerns or feeling of discomfort. The criteria for termination include 
subject complaints of confusion, dyspnea, onset of angina, and/or atypical bradycardia 
(drop in heart rate greater than 10 beats per minute). In addition, should the subject’s 
heart rate exceed 70% of their age-predicted maximum (220 – age) then the assessment 
will halt. In this case, the subject will be asked to rest while blood pressure and heart rate 
are monitored and will resume only when they return to acceptable values. If any of these 
conditions persist after rest, the individual’s primary physician may be called and referred 
for evaluation. If the individual complains of angina at rest, loss of consciousness occurs, 
or cardiac arrest, emergency medical services through 911 will be called immediately.  
 
SpO2 will be continuously monitored throughout the study. If SpO2 drops below a safety 
threshold of 80%, the fraction of inspired oxygen delivered by the hypoxicator will be 
rapidly increased until the subject is back over 80% SpO2. This threshold is chosen based 

on 80% being the average number the SpO2 has dropped to in previous studies without 
any adverse events occurring (Trumbower et al., 2012; Hayes et al., 2014; Lynch et al., 
2016; Navarrete-Opazo et al., 2016a). If the subject’s SpO2 remains under 80% or 
continues to decline after the percent oxygen delivered has been increased, the mask will 
be removed and the procedure will be aborted. In the event that a subject reports 
discomfort and does not wish to continue, the mask will be removed and the procedure 
will be aborted.  None of these adverse events are anticipated, however if they occur, 
each will be handled using the aforementioned methods. 
 
Confidentiality and protecting subject privacy 
The risks related to collecting and sharing protected health information and study-related 
data will be addressed in several ways.  First, upon enrollment in the study, a study ID 
will be assigned to the participant so that all study-related information pertaining to the 
subject uses the study ID. The document that links study-IDs to participant information 
will be stored in a separate location, not with the study-related data.  The study 
procedures will be conducted in private locations at Brooks Rehabilitation or University of 
Florida Department of Physical Therapy laboratories, so that participant privacy may be 
maintained when conducting procedures such as clinical testing, administering 
questionnaires, or answering individual questions.  All study procedures will be conducted 
in limited access areas with minimal traffic and personnel.   
Protected health information collected as part of this study will be limited to the minimal 
necessary information to carry out procedures in this approved protocol. All hard-copy 
study related documents will be stored in locked file cabinets in locked offices.   All 
electronic study related information and data will be stored on password protected 
computers and on password-protected institutional servers or encrypted electronic 
storage devices.  Only approved study personnel will have access to research records.    
 
7. 
Possible Discomforts and Risks: 
 
Discomforts and risks from acute intermittent hypoxia are minimal. Many studies 
in animals (Gonzalez-Rothi, 2015; Baker-Herman, 2004; Fuller, 2003; Lovett-Barr, 2012; 
Prosser-Loose, 2015; Satriotomo, 2016) and humans (Trumbower et al., 2012; Tester et 
al., 2014; Hayes et al., 2014; Sankari et al., 2015; Navarrete-Opazo, 2016a, 2016b; Lynch 
et al., 2016) have established that acute intermittent hypoxia can be delivered safely with 
minimal risk of adverse events. In a human study with a sham control, subjects were not 
able to distinguish whether they were receiving acute intermittent hypoxia or the sham 
procedure (Hayes et al., 2014). All sessions will be monitored by a study clinician who 
has experience working with individuals with SCI and in delivering acute intermittent 
hypoxia.  
Some participants may find the mask used during acute intermittent hypoxia 
uncomfortable, so the mask will be adjusted as necessary to ensure optimal comfort of 
the subject. Subjects may also feel sleepy during the brief episodes of hypoxia. However, 
this feeling typically lasts only for the duration of the hypoxic episode, as the episodes of 
hypoxia are alternated with breathing room air. Additionally, the percent of oxygen 
delivered will be increased, if necessary, to keep subjects above the safety threshold of 
80% SpO2. All subjects will undergo the acute intermittent hypoxia protocol in a 

comfortable chair with armrests and a headrest to ensure both comfort and safety if the 
subject does become sleepy. 
 
Discomfort and risks of injury from functional assessments are minimal.  All 
procedures are non-invasive and will be supervised by a study clinician with experience 
in testing breathing, postural muscle strength and function, and ambulation with 
individuals with SCI.  Participants may rest at any time and may opt not to perform any of 
the study procedures.  Participant responses will be closely monitored.  During seated 
tasks of postural muscle function or ambulation, subjects may be assisted as necessary 
to safely complete the tasks. 
The non-invasive sensors affixed with tape or wrapping may cause some mild 
discomfort or redness.  Removal of the tape may be uncomfortable for some participants 
and participants will be made aware prior to application and removal.  Some participants 
may not be accustomed to the study activities and may become fatigued during the 
testing.  Rest breaks will be provided as needed. 
 
8.        Possible Benefits: 
Participants may experience short-term improvements in total volume of air breathed in 
the hours following the procedure. Participants may also experience increased strength 
in postural muscles in the hours following the procedure. Any responses are not expected 
to last more than four hours. As these effects have not been tested formally in humans 
with SCI, responses will likely be variable across participants. 
Future populations may benefit from knowledge of how acute intermittent hypoxia affects 
the outcomes of rehabilitation for people with SCI. Since all risks to the subjects are 
minimal, the risks are reasonable in relation to the knowledge that could potentially be 
gained from this pilot study. 
 
9. 
Conflict of Interest: 
There are no conflicts of interests relating to any of the investigators and this protocol 
beyond the professional benefit from academic publication or presentation of the results. 
 
 

References Cited 
American Thoracic Society/European Respiratory S (2002) ATS/ERS Statement on 
respiratory muscle testing. American journal of respiratory and critical care medicine 
166:518-624. 
 
Baker-Herman TL, Fuller DD, Bavis RW, Zabka AG, Golder FJ, Doperalski NJ, Johnson 
RA, Watters JJ, Mitchell GS (2004) BDNF is necessary and sufficient for spinal 
respiratory plasticity following intermittent hypoxia. Nature neuroscience 7:48-55. 
 
Cardenas DD, Hoffman JM, Kirshblum S, McKinley W (2004) Etiology and incidence of 
rehospitalization after traumatic spinal cord injury: a multicenter analysis. Archives of 
physical medicine and rehabilitation 85:1757-1763. 
 
De Troyer A, Estenne M, Heilporn A (1986) Mechanism of active expiration in 
tetraplegic subjects. The New England journal of medicine 314:740-744. 
 
Flynn JR, Graham BA, Galea MP, Callister RJ (2011) The role of propriospinal 
interneurons in recovery from spinal cord injury. Neuropharmacology 60:809-822. 
 
Fuller DD, Johnson SM, Olson EB, Jr., Mitchell GS (2003) Synaptic pathways to phrenic 
motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord 
injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 
23:2993-3000. 
 
Gonzalez-Rothi EJ, Lee KZ, Dale EA, Reier PJ, Mitchell GS, Fuller DD (2015) 
Intermittent hypoxia and neurorehabilitation. Journal of applied physiology (Bethesda, 
Md : 1985) 119:1455-1465. 
 
Gozal D, Kheirandish-Gozal L (2008) Cardiovascular morbidity in obstructive sleep 
apnea: oxidative stress, inflammation, and much more. American journal of respiratory 
and critical care medicine 177:369-375. 
 
Hayes HB, Jayaraman A, Herrmann M, Mitchell GS, Rymer WZ, Trumbower RD (2014) 
Daily intermittent hypoxia enhances walking after chronic spinal cord injury: a 
randomized trial. Neurology 82:104-113. 
 
Kirshblum SC, Burns SP, Biering-Sorensen F, Donovan W, Graves DE, Jha A, 
Johansen M, Jones L, Krassioukov A, Mulcahey MJ, Schmidt-Read M, Waring W 
(2011) International standards for neurological classification of spinal cord injury 
(revised 2011). The journal of spinal cord medicine 34:535-546. 
 
Larson CA, Tezak WD, Malley MS, Thornton W (2010) Assessment of postural muscle 
strength in sitting: reliability of measures obtained with hand-held dynamometry in 
individuals with spinal cord injury. Journal of neurologic physical therapy : JNPT 34:24-
31. 
 

Lovett-Barr MR, Satriotomo I, Muir GD, Wilkerson JE, Hoffman MS, Vinit S, Mitchell GS 
(2012) Repetitive intermittent hypoxia induces respiratory and somatic motor recovery 
after chronic cervical spinal injury. The Journal of neuroscience : the official journal of 
the Society for Neuroscience 32:3591-3600. 
 
Lynch M, Duffell L, Sandhu M, Srivatsan S, Deatsch K, Kessler A, Mitchell GS, 
Jayaraman A, Rymer WZ (2016) Effect of acute intermittent hypoxia on motor function 
in individuals with chronic spinal cord injury following ibuprofen pretreatment: A pilot 
study. The journal of spinal cord medicine:1-9. 
 
McKay WB, Lim HK, Priebe MM, Stokic DS, Sherwood AM (2004) Clinical 
neurophysiological assessment of residual motor control in post-spinal cord injury 
paralysis. Neurorehabilitation and neural repair 18:144-153. 
 
Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R, Enright 
P, van der Grinten CP, Gustafsson P, Jensen R, Johnson DC, MacIntyre N, McKay R, 
Navajas D, Pedersen OF, Pellegrino R, Viegi G, Wanger J, Force AET (2005) 
Standardisation of spirometry. The European respiratory journal 26:319-338. 
 
Navarrete-Opazo A, Mitchell GS (2014) Therapeutic potential of intermittent hypoxia: a 
matter of dose. American journal of physiology Regulatory, integrative and comparative 
physiology 307:R1181-1197. 
 
Navarrete-Opazo A, Alcayaga J, Testa D, Quinteros AL (2016a) Intermittent Hypoxia 
Does not Elicit Memory Impairment in Spinal Cord Injury Patients. Archives of clinical 
neuropsychology : the official journal of the National Academy of Neuropsychologists 
31:332-342. 
 
Navarrete-Opazo A, Alcayaga J, Sepulveda O, Rojas E, Astudillo C (2016b) Repetitive 
intermittent hypoxia and locomotor training enhances walking function in incomplete 
spinal cord injury subjects: A randomized, triple-blind, placebo-controlled clinical trial. 
Journal of neurotrauma. 
 
Prosser-Loose EJ, Hassan A, Mitchell GS, Muir GD (2015) Delayed Intervention with 
Intermittent Hypoxia and Task Training Improves Forelimb Function in a Rat Model of 
Cervical Spinal Injury. Journal of neurotrauma 32:1403-1412. 
 
Sankari A, Bascom AT, Riehani A, Badr MS (2015) Tetraplegia is associated with 
enhanced peripheral chemoreflex sensitivity and ventilatory long-term facilitation. 
Journal of applied physiology (Bethesda, Md : 1985) 119:1183-1193. 
 
Satriotomo I, Dale EA, Dahlberg JM, Mitchell GS (2012) Repetitive acute intermittent 
hypoxia increases expression of proteins associated with plasticity in the phrenic motor 
nucleus. Experimental neurology 237:103-115. 
 

Satriotomo I, Nichols NL, Dale EA, Emery AT, Dahlberg JM, Mitchell GS (2016) 
Repetitive acute intermittent hypoxia increases growth/neurotrophic factor expression in 
non-respiratory motor neurons. Neuroscience 322:479-488. 
 
Sherwood AM, Dimitrijevic MR, McKay WB (1992) Evidence of subclinical brain 
influence in clinically complete spinal cord injury: discomplete SCI. Journal of the 
neurological sciences 110:90-98. 
 
Squair JW, Bjerkefors A, Inglis JT, Lam T, Carpenter MG (2016) Cortical and vestibular 
stimulation reveal preserved descending motor pathways in individuals with motor-
complete spinal cord injury. Journal of rehabilitation medicine 48:589-596. 
 
Tester NJ, Fuller DD, Fromm JS, Spiess MR, Behrman AL, Mateika JH (2014) Long-
term facilitation of ventilation in humans with chronic spinal cord injury. American journal 
of respiratory and critical care medicine 189:57-65. 
 
Tester NJ, Lorenz DJ, Suter SP, Buehner JJ, Falanga D, Watson E, Velozo CA, 
Behrman AL, Michele Basso D (2016) Responsiveness of the Neuromuscular Recovery 
Scale During Outpatient Activity-Dependent Rehabilitation for Spinal Cord Injury. 
Neurorehabilitation and neural repair 30:528-538. 
 
Trumbower RD, Jayaraman A, Mitchell GS, Rymer WZ (2012) Exposure to acute 
intermittent hypoxia augments somatic motor function in humans with incomplete spinal 
cord injury. Neurorehabilitation and neural repair 26:163-172. 
 
Wang YJ, Li JJ, Zhou HJ, Liu GL, Zheng Y, Wei B, Zhang Y, Hao CX, Kang HQ, Yuan 
Y, Gao LJ (2016) Surface electromyography as a measure of trunk muscle activity in 
patients with spinal cord injury: a meta-analytic review. The journal of spinal cord 
medicine 39:15-23. 
 
Wilkerson JE, Mitchell GS (2009) Daily intermittent hypoxia augments spinal BDNF 
levels, ERK phosphorylation and respiratory long-term facilitation. Experimental 
neurology 217:116-123. 
 
Yokoba M, Abe T, Katagiri M, Tomita T, Easton PA (2003) Respiratory muscle 
electromyogram and mouth pressure during isometric contraction. Respiratory 
physiology & neurobiology 137:51-60.