Clinical trial NCT02323698
Conditions: Spinal Cord Injuries
Keywords: hypoxia spinal cord injury rehabilitation strength walk function low oxygen
Sponsor: Spaulding Rehabilitation Hospital
Start date: 2019-01-01; Completion date: 2022-02-19
Enrollment: 36 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Accumulating evidence suggests that repeatedly breathing low oxygen levels for brief periods (termed intermittent hypoxia) is a safe and effective treatment strategy to promote meaningful functional recovery in persons with chronic spinal cord injury (SCI). The goal of the study is to understand how caffeine may augment the effects of intermittent hypoxia on motor function and spinal plasticity (ability of the nervous system to strengthen neural pathways based on new experiences) following SCI.

Detailed description:
The investigators will examine the effects of acute intermittent hypoxia (AIH) as a possible therapeutic intervention to promote functionally useful motor recovery. In this sub-study, the investigators will assess changes in leg motor function in response to repetitive AIH with and without caffeine.

Participants will receive caffeine+AIH, placebo+AIH, caffeine+SHAM in a randomized order. Before each intervention round, subjects will be asked to avoid caffeine-containing substances for 48 hrs (\> 5\* half-life of \~7 hrs) prior to arrival to control for baseline plasma levels of caffeine. Subjects will then ingest capsules containing either placebo (dextrose) or caffeine (up to 6mg/kg). Capsules will be prepared by Johnson Compounding \& Wellness. Blood samples will be collected before and after the breathing intervention to assess caffeine concentrations within the body.

During and after each intervention, both the rate and extent of magnitude changes in voluntary and involuntary muscle response behaviors important for walking will be compared between interventions within participants. Repeated measurements will be collected on all subjects that participate in the multiple interventions.

Arms: arm 1: Subjects with chronic, motor-incomplete SCI receive Caffeine then AIH arm 2: Subjects with chronic, motor-incomplete SCI receive Placebo then AIH arm 3: Subjects with chronic, motor-incomplete SCI receive Caffeine then SHAM
Interventions: intervention 1: Caffeine intervention 2: AIH intervention 3: Placebo intervention 4: SHAM
Intervention descriptions: intervention 1: Subjects will ingest capsules containing caffeine (up to 6mg/kg). Experiments will begin 30min after consumption to approximately coincide with peak plasma concentrations.Throughout the 30min wait time and experimentation, blood pressure and heart rate will be monitored. intervention 2: Participants will breathe intermittent low oxygen via air generators. The generators will fill reservoir bags attached to a non-rebreathing face mask. Oxygen concentration will be continuously monitored to ensure delivery of FIO2 (fraction of inspired oxygen) = 0.10±0.02 (hypoxia). Throughout experimentation, blood pressure and heart rate will be monitored. intervention 3: This is a placebo counterpart to the caffeine drug. Subjects will ingest capsules containing dextrose. Experiments will begin 30min after consumption to mimic the caffeine drug protocol. Throughout experimentation, blood oxygenation, blood pressure and heart rate will be monitored. intervention 4: This is a placebo counterpart to breathing intermittent low oxygen. Participants will breathe intermittent room air via air generators. The generators will fill reservoir bags attached to a non-rebreathing face mask. Oxygen concentration will be continuously monitored to ensure delivery of FIO2 (fraction of inspired oxygen) = 0.21±0.02 (normoxia). Throughout experimentation, blood pressure and heart rate will be monitored.

Protocol documents:
Prot_SAP_000.pdf: 
1 
Table of Contents
I.
Checklist  ...................................................................................................................................... . 2
II.
Protocol Summary ........................................................................................................................  7 
III.
Statistical Analysis Plan ...............................................................................................................  31 
IV.
Flowchart ......................................................................................................................................  48 
V.
Enrollment Table ...........................................................................................................................  49 
VI.
Caffeine Dosing Table ...................................................................................................................  50 
Supplemental Material


1 
SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and 
related documents* 
Section/item 
Item # Description 
Page 
Administrative information 
Title 
1 
Descriptive title identifying the study design, population, 
interventions, and, if applicable, trial acronym 
M, 1 
Trial registration 
2a 
Trial identifier and registry name. If not yet registered, 
name of intended registry 
M, 3 
2b 
All items from the World Health Organization Trial 
Registration Data Set 
SP, 4 
Protocol version 
3 
Date and version identifier 
SP, 4 
Funding 
4 
Sources and types of financial, material, and other support SP, 4 
Roles and 
responsibilities 
5a 
Names, affiliations, and roles of protocol contributors 
SP, 5 
5b 
Name and contact information for the trial sponsor 
SP, 5 
5c 
Role of study sponsor and funders, if any, in study design; 
collection, management, analysis, and interpretation of 
data; writing of the report; and the decision to submit the 
report for publication, including whether they will have 
ultimate authority over any of these activities 
SP, 5 
5d 
Composition, roles, and responsibilities of the coordinating 
centre, steering committee, endpoint adjudication 
committee, data management team, and other individuals 
or groups overseeing the trial, if applicable (see Item 21a 
for data monitoring committee) 
SP, 5 
Introduction 
Background and 
rationale 
6a 
Description of research question and justification for 
undertaking the trial, including summary of relevant studies 
(published and unpublished) examining benefits and 
harms for each intervention 
M, 5 
6b 
Explanation for choice of comparators 
SP, 6 
Objectives 
7 
Specific objectives or hypotheses 
SP, 7 


 
2 
Trial design 
8 
Description of trial design including type of trial (eg, parallel 
group, crossover, factorial, single group), allocation ratio, 
and framework (eg, superiority, equivalence, noninferiority, 
exploratory) 
SP, 7 
Methods: Participants, interventions, and outcomes 
 
Study setting 
9 
Description of study settings (eg, community clinic, 
academic hospital) and list of countries where data will be 
collected. Reference to where list of study sites can be 
obtained 
SP, 7 
Eligibility criteria 
10 
Inclusion and exclusion criteria for participants. If 
applicable, eligibility criteria for study centres and 
individuals who will perform the interventions (eg, 
surgeons, psychotherapists) 
SP, 7 
Interventions 
11a 
Interventions for each group with sufficient detail to allow 
replication, including how and when they will be 
administered 
SP, 8 
11b 
Criteria for discontinuing or modifying allocated 
interventions for a given trial participant (eg, drug dose 
change in response to harms, participant request, or 
improving/worsening disease) 
SP, 10 
11c 
Strategies to improve adherence to intervention protocols, 
and any procedures for monitoring adherence (eg, drug 
tablet return, laboratory tests) 
SP, 10 
11d 
Relevant concomitant care and interventions that are 
permitted or prohibited during the trial 
SP, 10 
Outcomes 
12 
Primary, secondary, and other outcomes, including the 
specific measurement variable (eg, systolic blood 
pressure), analysis metric (eg, change from baseline, final 
value, time to event), method of aggregation (eg, median, 
proportion), and time point for each outcome. Explanation 
of the clinical relevance of chosen efficacy and harm 
outcomes is strongly recommended 
SP, 12 
Participant timeline 13 
Time schedule of enrolment, interventions (including any 
run-ins and washouts), assessments, and visits for 
participants. A schematic diagram is highly recommended 
(see Figure) 
SP, 12 
Sample size 
14 
Estimated number of participants needed to achieve 
study objectives and how it was determined, including 
clinical and statistical assumptions supporting any sample 
size calculations 
SP, 13 


 
3 
Recruitment 
15 
Strategies for achieving adequate participant enrolment to 
reach target sample size 
SP, 13 
Methods: Assignment of interventions (for controlled trials) 
 
Allocation: 
 
 
 
Sequence 
generation 
16a 
Method of generating the allocation sequence (eg, 
computer-generated random numbers), and list of any 
factors for stratification. To reduce predictability of a 
random sequence, details of any planned restriction (eg, 
blocking) should be provided in a separate document that 
is unavailable to those who enrol participants or assign 
interventions 
SP, 14 
Allocation 
concealment 
mechanism 
16b 
Mechanism of implementing the allocation sequence (eg, 
central telephone; sequentially numbered, opaque, sealed 
envelopes), describing any steps to conceal the sequence 
until interventions are assigned 
SP, 14 
Implementation 16c 
Who will generate the allocation sequence, who will enrol 
participants, and who will assign participants to 
interventions 
SP, 14 
Blinding (masking) 17a 
Who will be blinded after assignment to interventions (eg, 
trial participants, care providers, outcome assessors, data 
analysts), and how 
SP, 15 
 
17b 
If blinded, circumstances under which unblinding is 
permissible, and procedure for revealing a participant’s 
allocated intervention during the trial 
SP, 15 
Methods: Data collection, management, and analysis 
 
Data collection 
methods 
18a 
Plans for assessment and collection of outcome, baseline, 
and other trial data, including any related processes to 
promote data quality (eg, duplicate measurements, training 
of assessors) and a description of study instruments (eg, 
questionnaires, laboratory tests) along with their reliability 
and validity, if known. Reference to where data collection 
forms can be found, if not in the protocol 
SP, 15 
 
18b 
Plans to promote participant retention and complete follow-
up, including list of any outcome data to be collected for 
participants who discontinue or deviate from intervention 
protocols 
SP, 15 
Data management 19 
Plans for data entry, coding, security, and storage, 
including any related processes to promote data quality 
(eg, double data entry; range checks for data values). 
Reference to where details of data management 
procedures can be found, if not in the protocol 
SP, 16 


 
4 
Statistical methods 20a 
Statistical methods for analysing primary and secondary 
outcomes. Reference to where other details of the 
statistical analysis plan can be found, if not in the protocol 
SP, 17 
 
20b 
Methods for any additional analyses (eg, subgroup and 
adjusted analyses) 
SP, 17 
 
20c 
Definition of analysis population relating to protocol non-
adherence (eg, as randomised analysis), and any 
statistical methods to handle missing data (eg, multiple 
imputation) 
SP, 17 
Methods: Monitoring 
 
Data monitoring 
21a 
Composition of data monitoring committee (DMC); 
summary of its role and reporting structure; statement of 
whether it is independent from the sponsor and competing 
interests; and reference to where further details about its 
charter can be found, if not in the protocol. Alternatively, an 
explanation of why a DMC is not needed 
SP, 18 
 
21b 
Description of any interim analyses and stopping 
guidelines, including who will have access to these interim 
results and make the final decision to terminate the trial 
SP, 18 
Harms 
22 
Plans for collecting, assessing, reporting, and managing 
solicited and spontaneously reported adverse events and 
other unintended effects of trial interventions or trial 
conduct 
SP, 18 
Auditing 
23 
Frequency and procedures for auditing trial conduct, if any, 
and whether the process will be independent from 
investigators and the sponsor 
SP, 18 
Ethics and dissemination 
 
Research ethics 
approval 
24 
Plans for seeking research ethics committee/institutional 
review board (REC/IRB) approval 
SP, 18 
Protocol 
amendments 
25 
Plans for communicating important protocol modifications 
(eg, changes to eligibility criteria, outcomes, analyses) to 
relevant parties (eg, investigators, REC/IRBs, trial 
participants, trial registries, journals, regulators) 
SP, 18 
Consent or assent 26a 
Who will obtain informed consent or assent from potential 
trial participants or authorised surrogates, and how (see 
Item 32) 
SP, 19 
 
26b 
Additional consent provisions for collection and use of 
participant data and biological specimens in ancillary 
studies, if applicable 
SP, 19 


 
5 
Confidentiality 
27 
How personal information about potential and enrolled 
participants will be collected, shared, and maintained in 
order to protect confidentiality before, during, and after the 
trial 
SP, 19 
Declaration of 
interests 
28 
Financial and other competing interests for principal 
investigators for the overall trial and each study site 
SP, 19 
Access to data 
29 
Statement of who will have access to the final trial dataset, 
and disclosure of contractual agreements that limit such 
access for investigators 
SP, 19 
Ancillary and post-
trial care 
30 
Provisions, if any, for ancillary and post-trial care, and for 
compensation to those who suffer harm from trial 
participation 
SP, 19 
Dissemination 
policy 
31a 
Plans for investigators and sponsor to communicate trial 
results to participants, healthcare professionals, the public, 
and other relevant groups (eg, via publication, reporting in 
results databases, or other data sharing arrangements), 
including any publication restrictions 
SP, 19 
 
31b 
Authorship eligibility guidelines and any intended use of 
professional writers 
SP, 19 
 
31c 
Plans, if any, for granting public access to the full protocol, 
participant-level dataset, and statistical code 
SP, 19 
Appendices 
 
 
 
Informed consent 
materials 
32 
Model consent form and other related documentation given 
to participants and authorised surrogates 
SP, 20 
Biological 
specimens 
33 
Plans for collection, laboratory evaluation, and storage of 
biological specimens for genetic or molecular analysis in 
the current trial and for future use in ancillary studies, if 
applicable 
SP, 20 
Abbreviations: M, manuscript; SP, supplemental study protocol documentation. 
Chan A et al. SPIRIT 2013 Statement: Defining standard protocol items for clinical trials. Ann 
Intern Med 2013; 158:200-207. 
 


DETAILED STUDY PROTOCOL 
BWH/MGH Human Subjects Research Application 
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TABLE OF CONTENTS 
I.
LIST OF ABBREVIATIONS .......................................................................................................................................... 2 
II.
ADMINISTRATION INFORMATION ......................................................................................................................... 4 
1.
TITLE .............................................................................................................................................................................. 4 
2.
TRIAL REGISTRATION ...................................................................................................................................................... 4 
3.
PROTOCOL VERSION ........................................................................................................................................................ 4 
4.
FUNDING ......................................................................................................................................................................... 4 
5.
ROLES AND RESPONSIBILITIES (DELEGATION OF AUTHORITY) ........................................................................................ 5 
III.
INTRODUCTION ............................................................................................................................................................ 5 
6.
BACKGROUND AND RATIONALE ..................................................................................................................................... 5 
7.
OBJECTIVES .................................................................................................................................................................... 6 
8.
TRIAL DESIGN ................................................................................................................................................................. 7 
IV.
METHODS: PARTICIPANTS, INTERVENTIONS, AND OUTCOMES ................................................................. 7 
9.
STUDY SETTING .............................................................................................................................................................. 7 
10.
ELIGIBILITY CRITERIA ................................................................................................................................................ 7 
11.
INTERVENTIONS ......................................................................................................................................................... 8 
12.
OUTCOMES ............................................................................................................................................................... 10 
13.
PARTICIPANT TIMELINE ............................................................................................................................................ 11 
14.
SAMPLE SIZE ............................................................................................................................................................ 13 
15.
RECRUITMENT .......................................................................................................................................................... 13 
16.
ASSIGNMENT OF INTERVENTIONS (FOR CONTROLLED TRIALS) ................................................................................. 14 
17.
BLINDING (MASKING) ............................................................................................................................................... 15 
18.
DATA COLLECTION, MANAGEMENT, AND ANALYSIS ................................................................................................ 15 
19.
DATA MANAGEMENT ................................................................................................................................................ 17 
20.
STATISTICAL METHODS ............................................................................................................................................ 17 
V.
MONITORING ............................................................................................................................................................... 18 
21.
DATA MONITORING .................................................................................................................................................. 18 
22.
HARMS ..................................................................................................................................................................... 18 
23.
AUDITING ................................................................................................................................................................. 18 
VI.
ETHICS AND DISSEMINATION ................................................................................................................................ 18 
24.
RESEARCH ETHICS APPROVAL .................................................................................................................................. 18 
25.
PROTOCOL AMENDMENTS ........................................................................................................................................ 18 
26.
CONSENT OR ASSENT ............................................................................................................................................... 19 
27.
CONFIDENTIALITY .................................................................................................................................................... 19 
28.
DECLARATION OF INTERESTS ................................................................................................................................... 19 
29.
ACCESS TO DATA ..................................................................................................................................................... 19 
30.
ANCILLARY AND POST-TRIAL CARE ......................................................................................................................... 19 
31.
DISSEMINATION POLICY ........................................................................................................................................... 19 
VII. APPENDICES ................................................................................................................................................................. 20 
32.
INFORMED CONSENT MATERIALS ............................................................................................................................. 20 
33.
BIOLOGICAL SPECIMEN PROTOCOLS ......................................................................................................................... 20 


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I. 
List of Abbreviations 
AIH: acute intermittent hypoxia 
AIS: American Spinal Injury Association Impairment Scale 
α: alpha level, established Type I error rate for power analyses 
ANOVA: analysis of variance 
ANCOVA: analysis of covariance 
BP: blood pressure 
BL: baseline 
BDNF: brain-derived neurotrophic factor 
C2: spinal cord cervical level 2 
C: caffeine 
CI: confidence interval 
CRP: c-reactive protein 
Di: ith day of intervention 
ERK MAP: extracellular signal-regulated kinases; mitogen-activated protein kinases 
ECG: electrocardiography 
5-HT2: receptor that binds endogenous serotonin neurotransmitter 
FiO2: fraction of inspired oxygen 
F: follow-up 
Gq: guanosine nucleotide-binding protein 
HIPAA: Health Insurance Portability and Accountability Act 
HR: heart rate 
iSCI: incomplete spinal cord injury 
LEMS: lower extremity motor score 
L5: spinal cord lumbar level 5 
MAP: mitogen-activated protein kinases 
m: meter 
min: minute 
mmHg: millimeter of mercury 
P: placebo 
RR: respiratory rate 
SCI: spinal cord injury 
s: second 


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6MWT: 6-minute walk test 
SBP: systolic blood pressure 
SCATS: spinal cord assessment tool for spastic reflexes 
SCI-FAI: spinal cord injury functional ambulatory index 
SDB: sleep-disordered breathing 
SpO2: blood oxygen saturation level 
10MWT: 10-meter walk test 
TrkB: tyrosine kinase B receptor 
TUG: timed up-and-go test  
WISCI: walking index for spinal cord injury 
 
 
 


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II. Administration Information 
1. Title 
Mechanisms of intermittent hypoxia-induced motor recovery in persons with SCI 
2. Trial registration 
ClinicalTrials.gov Identifier: NCT02323698 
Registry name: Effects of caffeine and intermittent hypoxia on leg function in human spinal cord 
injury 
3. Protocol version 
Detailed Protocol Summary, Last updated: September 27, 2021 
4. Funding 
National Institutes of Health, HD081274 
Wings for Life Foundation WFL US-026/14 
 
 


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5. Roles and responsibilities (delegation of authority) 
  
Study Title: Mechanisms of intermittent hypoxia-induced motor recovery in persons with SCI  
IRB #: 2017P001940 
 
 
 
 
      
Principal Investigator: Randy Trumbower, PT, PhD 
Sponsors: Spaulding, NIH-NICHD, Wings for Life International  
 
 
 
Study Site: Spaulding Rehabilitation Hospital    
 
List delegated study related tasks and dates of involvement for each staff member in accordance with institutional guidelines 
and Good Clinical Practice (GCP). All IRB approved study staff should sign and initial this log.  The PI should acknowledge 
delegation by signing his/her initials after each entry and at study ‘close out’ to attest to the fact that the list is complete, 
accurate and that all staff are accounted for.  We will update this log in a timely manner as new personnel are added and/or 
study roles change. 
 
First Name 
Last Name 
Title 
Start Date 
End Date 
Activities* 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
  
 
 
 
 
  
 
 
 
 
  
 
 
 
 
  
 
 
 
 
 
  
 
 
 
 
  
 
 
Activity codes: 
1 
Obtain Informed 
Consent 
2 
Assess Eligibility 
Criteria 
3 
Maintain IRB & 
Regulatory 
Documents 
4 
Essential 
Documentation 
5 
Obtain Medical 
History 
6 
Concomitant 
Medication Review 
7 
Dispense Study 
Drug/Device 
8 
Perform Protocol 
Tasks with Study 
Subjects 
9 
Review Lab and 
Procedure Results 
 
10 
Safety Monitoring 
 
11 Data Monitoring 
 
12 
Collection, Handling, 
and Shipment of 
Samples 
 
13 
AE Report to 
Sponsor and/or 
IRB 
 
14 
CRF Queries 
 
15 
Perform Physical 
Exam 
 
 16 
  AE Grading and 
Attribution 
 
 
III. Introduction 
 
6. Background and Rationale 
Spinal cord injury (SCI) disrupts connections between the brain and spinal cord, causing life-long 
paralysis and reduced mobility. As such, restoring walking ability remains a highly valued goal 
for persons with SCI.1 Even small improvements in walking skills that enable a person with SCI 
to stand, walk within the home, or negotiate spaces not accessible to wheelchairs can translate into 
significant empowerment, health benefits, and improvement in quality of life. Because most spinal 


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injuries are incomplete, there are at least some spared neural pathways to the motor neurons that 
can initiate and coordinate movements necessary for walking recovery. Although injury-induced 
plasticity in spared spinal pathways enables partial spontaneous recovery of respiratory and 
somatic motor function,2, 3 the extent of this recovery is slow, variable and frustratingly limited.4 
Thus, there is a critical need for new strategies that augment spinal plasticity and subsequently 
improve walking ability in persons with SCI. 
Acute intermittent hypoxia (AIH) induces spinal plasticity, strengthening connections to motor 
neurons.5-9 Considerable progress has been made towards an understanding of cellular mechanisms 
giving rise to AIH-induced respiratory plasticity.7 Repetitive exposure to AIH enhances the 
expression of plasticity-promoting proteins in respiratory motor nuclei9-11 and elicits profound 
recovery of breathing capacity in spinally injured rats.12 Key steps in AIH-induced plasticity 
include episodic release of serotonin near respiratory motor neurons, initiating new synthesis of 
brain derived neurotrophic factor (BDNF) and subsequently activating its high affinity receptor, 
TrkB 5. “Downstream” events include the activation of extracellular signal-regulated kinases 
(ERK); mitogen-activated protein (MAP) kinases and postulated insertion of glutamate receptors 
at the synapse between respiratory pre-motor and motor neurons. Excitingly, these changes extend 
beyond the respiratory system. Colleagues also observed that AIH enhances expression of these 
same proteins in non-respiratory motor neurons.8  
Although dAIH may be a viable method to enhance walking after iSCI, the mechanisms underlying 
dAIH-induced motor recovery may be constrained by competing neural mechanisms of motor 
facilitation. While dAIH induces facilitation predominantly via serotonin-dependent mechanisms 
13, even mild hypoxia causes ATP release from glia and other sources in the nervous system, 
leading to an accumulation of extracellular adenosine. Subsequent activation of A2A receptors 
elicits a distinct form of motor facilitation 14 that is known to compete with the serotonin-dependent 
pathway via cross-talk inhibition 15, 16. Consequently, systemic administration of a selective A2A 
receptor antagonist, MSX-3, reduced cross-talk inhibition and increased AIH-induced long-term 
motor facilitation in the phrenic and hypoglossal nerves by nearly 50% and 20%, respectively, in 
rats 16. The impact of adenosine A2A receptor antagonists on AIH-induced motor recovery have 
not yet been studied in persons with incomplete SCI (iSCI).  
Our working hypothesis is that reducing crosstalk inhibition during dAIH with a well-tolerated 
A2A antagonist (caffeine) will enhance AIH-induced improvement of walking function in persons 
with chronic iSCI.  
7. Objectives 
The main objective of this clinical trial is to evaluate the effects of a common, safe adenosine A2A 
antagonist, caffeine, on recovery of walking following dAIH in persons with chronic iSCI. While 
highly specific A2A receptor antagonists, such as MXS-3 and ZM2412385, block cross-talk in 
rats,16 their safety in SCI is not known. On the other hand, caffeine offers the ideal blend of known 
safety, metabolism, and efficacious A2A receptor antagonism.17 Even at low plasma concentrations 
(1-10μM) after one cup of coffee, caffeine antagonizes A2A receptors,17 and the efficacy of 
moderate caffeine in blocking neural A2A receptors is illustrated by the neuroprotective effects of 
regular caffeine consumption in dopaminergic transmission in Parkinson’s Disease.18 Although 
caffeine has many neurobiological actions, caffeine’s actions at physiologically relevant doses (1-
100 μM), including the proposed dose of up to 6 mg/kg body weight, are mediated by adenosine 
receptor antagonism versus phosphodiesterase inhibition, release of intracellular calcium stores, 


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or other site.19 Our study will quantify the daily (5 consecutive days) effects of caffeine (e.g., 
4 mg/kg) as an adjunctive therapy to dAIH for restoring walking function in persons with chronic 
iSCI. This dose has known safety in iSCI and substantial antagonism to A2A receptors.17, 20, 21  
8. Trial Design 
We will conduct a placebo-controlled, cross-over, block-randomized intervention study to assess 
the efficacy of daily caffeine prior to AIH on improving walking function in persons with chronic, 
motor-incomplete SCI.  
IV. Methods: Participants, interventions, and outcomes 
9. Study setting 
The following laboratory will be used as the primary site of this study: 
INSPIRE Lab at Spaulding Hospital 
1575 Cambridge Street 
Cambridge MA 02138 USA 
 
10. Eligibility criteria 
Potential subjects will be informed about the objectives of the study and must meet all inclusion 
and exclusion criteria (see Appendix 1 for sample Informed Consent Form). They will be required 
to read and sign approved consent and HIPAA forms prior to participation to allow access to 
medical information from their physicians.  
Inclusion criteria: Participation requirements for SCI participants include: 1) 18 to 75 years old; 2) 
medically stable with medical clearance from physician to participate; 3) SCI at or below C2 and 
at or above L5 with at least some lower extremity motor function preserved below the neurologic 
level; 4) non-progressive etiology of spinal injury; 4) AIS A-D at initial screen; 5) at least 6 months 
post-injury (chronic). We plan to choose subjects greater that 6 months post-injury to ensure 
minimal confounding effects of spontaneous neurological recovery during the experiments. This 
will mean that changes in sensorimotor performance are more likely due to the interventions 
associated with the research study.  
Exclusion criteria: Individuals will be excluded due to: 1) severe concurrent illness or pain, 
including unhealed decubiti, severe neuropathic or chronic pain syndrome, infection (e.g. bladder), 
hypertension, cardiovascular disease, pulmonary disease, severe osteoporosis (history of 
fractures), active heterotopic ossification in the lower extremities, or history of peripheral nerve 
injury in the legs; 2) < 24 on Mini-Mental Exam 22; 3) severe autonomic dysreflexia; 4) history of 
cardiovascular/pulmonary complications; 5) pregnancy because of unknown effects of AIH on a 
fetus, although women of childbearing potential will not otherwise be excluded; 6) undergoing 
concurrent physical therapy; 7) history of diabetes. Caffeine can potentially alter sugar metabolism 
and, thus, persons diagnosed with diabetes will be excluded from participating in the caffeine 
portion of this study; 8) history of cirrhosis. Cirrhosis is known to increase the elimination time of 
caffeine and, thus, persons diagnosed with cirrhosis will be excluded from participating in the 
caffeine portion of this study; and 9) history of caffeine allergies or intolerances. Participants with 
a history of caffeine allergies or intolerances will be excluded from participating in the caffeine 
study.  
Inclusion of women and minorities: We will recruit persons with SCI to include women and 
minorities. To protect against possible unknown side effects of dAIH, women who are pregnant 


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may not take part in this study as the effects of dAIH on the developing fetus have not been studied. 
If a woman is of childbearing ability, birth control will be recommended for use throughout the 
study.  
Our study coordinator, study physicians, physical therapists, PI (Trumbower), and research 
collaborators recruited subjects using a variety of strategies shown effective in the past. First, the 
team identified potential subjects using Institutional Review Board (IRB) approved resources to 
inform persons with SCI that are currently admitted to inpatient rehabilitation/outpatient units of 
Spaulding Rehabilitation Hospital, Boston University Medical Center, Massachusetts General 
Hospital, Gaylord hospital, Albany Medical Center, and Maine Medical Center. Subjects also will 
be recruited by word of mouth and flyers at these facilities. Eligible subjects screened at INSPIRE 
Laboratory, Spaulding Rehabilitation Hospital Cambridge, MA. At the screening visit, subjects 
informed of this project, their potential involvement, the possible benefits, and risks, and their right 
to terminate participation at any time without penalty. Qualifying subjects in agreement with the 
study details asked to sign the informed consent and HIPAA form approved by Partners IRB. All 
potential subjects entered the study according to the inclusion/exclusion criteria above and 
following medical clearance. Experiments will be conducted at the Spaulding Rehabilitation 
Hospitals. 
11. Interventions 
a. Intervention Procedures: This placebo-controlled study will examine the combined effects of 
caffeine (vs. placebo) and AIH (vs. SHAM) on walking performance in persons with chronic, 
iSCI. Participants will receive three combinatorial interventions in random order: 
placebo+AIH, caffeine+AIH, caffeine+SHAM. The order of interventions will be block 
randomized. The initial screening and evaluation procedures are similar for all participants. 
For each intervention, participants will complete up to 8 visits/intervention (1 
baseline/intervention + 5 treatment days/intervention + 2 follow-up days/intervention). 
Participants will complete a total of 24 visits (8 visits/intervention x 3 interventions) after the 
initial screen. Participant enrollment and allocation will be completed during the screening 
visit. Baseline will be recorded up to 3 days prior to the first intervention.  
Caffeine intervention (caffeine or placebo): Before each intervention round, subjects will be 
asked to avoid caffeine-containing substances for 48 hrs. (half-life ~7 hrs.)21, 23 prior to arrival 
to control for baseline plasma levels of caffeine.20 Subjects will then ingest capsules containing 
either placebo (sucrose) or caffeine (up to 400 mg). USP and FCC grade caffeine and placebo 
capsules will be sourced to a private independent compounding pharmacy (Johnson 
Compounding Pharmacy, Waltham MA, USA). The study PI (Trumbower) will hold a Class 
VI Drug License and the study physician (Slocum) will write the prescription for caffeine or 
placebo and have the prescription sent to the compounding pharmacy. The compounding 
pharmacy will then dispense the caffeine or placebo to the subject for use in the study. 
Administration will also be overseen by a physician member of the study team.  
Persons experiencing caffeine-induced adverse side effects (e.g., dizziness) will be excluded. 
Since caffeine is a diuretic, we will encourage subjects to empty bladders prior to AIH (or 
SHAM). To monitor caffeine levels and identify subject-to-subject differences in absorption, 
blood samples will be collected before capsule consumption and post-AIH or SHAM 
intervention on days 1 and 5. 17, 24, 25 
 
 


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Safety considerations for caffeine: Caffeine is commonly used on a daily basis, with an average 
adult consumption of up to ~6 mg of caffeine per kg of body weight and heavy consumers 
consuming ≥ 9 mg/kg,19, 26 including in persons with SCI 27. High doses of > 6 mg/kg caffeine 
have been tested in humans for athletic performance enhancement without reported adverse 
effects.20, 28, 29 In persons with SCI, 6 mg/kg has been shown enhance athletic performance 
without reported adverse effects 20, 27 and no significant or unsafe changes in blood pressure, 
heart rate, or respiration were seen.27 Thus, up to 6 mg/kg is within a reasonable and safe range, 
and that caffeine was safe and well-tolerated by persons with SCI. 
Breathing intervention (AIH or SHAM): Breathing interventions will begin 30 min after 
caffeine or placebo to approximately coincide with peak plasma caffeine concentrations.30 
Participants will don a latex-free, full non-rebreather mask with a custom neoprene head strap 
while they sit comfortably in a semi-reclined chair. Intermittent gas mixtures will be delivered 
via automatic adjustment of one-way valves attached to a hypoxia generator [HYP123, 
Hypoxico Inc., USA].31 We will deliver preset air mixtures of FIO2=0.09±0.02 (hypoxia) or 
FIO2=0.21±0.02 (normoxia) for 1.5min at 1 min intervals of room air breathing. The generator 
will fill reservoir bags attached to a non-rebreathing facemask worn by subjects. Oxygen 
concentration will be continuously monitored [OM-25RME; Maxtec Inc, USA]. We will take 
precautions to ensure safety during interventions by monitoring 5-lead electrocardiography 
(ECG) and recording oxyhemoglobin saturation via pulse oximetry (SpO2) and heart rate (HR) 
at 2-second intervals. Respiratory rate (RR) and blood pressure (BP) will be recorded every 5th 
breathing interval [Dash 4000 Patient Monitor, GE]. We also will record SpO2, HR, RR, and 
BP at the start and end of walking practice and again at the end of each treatment session. 
Safety considerations for AIH: The physiological effects of AIH in humans are well studied 
and the methods proposed here deemed safe. Daily AIH-induced enhancements in breathing 
capacity, limb strength, and walking function occurred without significant adverse events in 
humans with chronic SCI.32-34 Although prolonged chronic exposure to more severe and longer 
duration hypoxia causes serious morbidity,35 considerable data show that repetitive exposure 
to brief mild AIH elicits meaningful functional benefits without pathology.8, 36, 37 With mild 
AIH, the severity of hypoxemia, number of episodes, and exposure duration are low (15 
episodes per day, 5 days) versus the chronic hypoxia experienced in obstructive sleep apnea 
(80-400 episodes per day for years 35, 38). Daily AIH (10 episodes/day, 7 days) showed no 
evidence for hypertension or weight loss 9 or hippocampal gliosis or cell death in rats with 
SCI.8 Prior study in persons with SCI found no adverse events associated with mild exposures 
of single-day and dAIH (5 days), and all subjects tolerated exposures well.32, 33 No changes in 
mean arterial blood pressure or heart rate were found before and after each treatment or 
between baseline and the end of the exposure week. No maladaptive changes in cognitive or 
spasticity were found either, as measured by the Mini-Mental State Examination and Spinal 
Cord Assessment Tool for Spasticity. 
We have taken great care and consideration in establishing a safe AIH protocol for persons 
with SCI. Rodent studies were developed using modest protocols of dAIH (10, 5 min episodes; 
7 days 8, 9). In humans, we selected even more modest dosing (15, 1.5 min episodes; up to 5 
days).32, 33 Our mild AIH dose of 1.5-min duration is based on human studies that delivered 
short doses of hypoxia (SaO2=80-85%) without report of adverse events.32, 33, 39, 40 We also 
based our AIH protocol on the pattern-sensitive effects of dAIH on respiratory motor control 
that were more dependent on the intermittency and repetition than the duration of the 


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episodes.5, 7, 41-43 Such pattern-sensitivity is common to many models of neuroplasticity 44 
including serotonin-dependent plasticity.45 Daily exposures in rats (7 days) allowed for further 
amplification of the gains without eliciting maladaptive changes, such as cell death, 
hypertension, or weight loss.8, 9 
b. Criteria for discontinuing or modifying allocated interventions: The interventions will be 
stopped, and immediate care provided if any significant adverse effects, such as rapid heart 
rate, dizziness, nausea, or a significant change in blood pressure, are detected or self-reported 
by the study participant. Participants with diabetes, cirrhosis, a history of autonomic 
dysreflexia, caffeine allergies or sensitivities, or females who are pregnant will be excluded 
from this portion of the study. Due to the diuretic effects of caffeine, subjects will be asked to 
empty their bladders prior to consumption. Throughout the 30 min wait time and 
experimentation, blood pressure and heart rate will be monitored. Any adverse effects will be 
reported and assessed. Our study team will be prepared to discontinue treatment and offer 
immediate care if any significant adverse effects, such as rapid heart rate, systemic 
hypertensive event, dizziness, or nausea are detected or self-reported by the subject. We will 
define a systemic hypertensive event as a systolic pressure exceeding 140 mmHg and/or 
diastolic pressure exceeding 90 mmHg.46, 47 
c. Strategies to improve adherence to intervention protocols: To improve adherence to 
intervention protocols, participants will complete these protocols within the INSPIRE 
Laboratory. We will provide participants with a stipend and reimburse them for travel and 
lodging costs.  
d. Relevant concomitant care and interventions permitted or prohibited during the trial: Patient 
Monitoring Equipment. Patient Monitor Systems will be used to monitor hemodynamic 
changes during and following intermittent hypoxia [Smith Medical Systems, USA; Nonin, 
USA; Welsh-Allyn, USA; Biopac Systems Inc, USA and Masimo; Irvine, CA, USA]. These 
devices provide estimates of blood pressure, heart rate, oxygen saturation, and pulse rate 
variability. These measures will be monitored continuously during breathing protocols to 
ensure subject safety. The blood pressure cuff transducer will be secured around the upper arm 
during pressure measurements.  
We realize concomitant medication use (e.g., bowel/bladder dysfunction, systemic 
hypertension, pain, spasticity, anti-depressant medications) may confound the effects of our 
interventions. Due to participant safety and comfort, we will not limit prescribed medication 
use. However, we will require those receiving medication to maintain dose 1 week prior to and 
during our interventions as we did in our previous study.33, 48 We will work closely with 
physicians to ensure medication dosing is stable before enrollment and during interventions. 
We also will log daily medication intake for each participant throughout study and assess for 
possible drug-AIH interactions and changes in prescribed drug dosages at follow-ups. If a 
patient must change medication for safety, they can readily withdraw from the study at any 
time. 
12. Outcomes 
The primary outcome measure for this clinical trial is walking speed. We will measure walking 
speed using the 10-meter walk test (10MWT). The 10MWT is considered the best available 
measure to assess walking function in persons with iSCI.49 Speed also is the gold-standard measure 
of improved walking function in other clinical populations and use of this measure will allow us 


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to assess outcomes relative to other published studies. At the end of each training session, we will 
assess the 10MWT speed relative to baseline to determine if the participants achieved a minimum 
clinically important difference (MCID) of 0.06 m/s change on the 10MWT.50 We will also use 
standardized functional assessments to evaluate functional walking capacity via the 6MWT, and 
functional mobility via the TUG test.51 The TUG and 10MWT have also been used to distinguish 
functional ambulation status and progression from non-ambulatory to ambulatory in the acute 
setting.52 Our team has experience with all of these measures and testing and analysis will be 
performed according to our previously published reports.33, 48 
13. Participant timeline 
Participants will be randomized to a sequence of three interventions (Table 1). Participants will 
participate in a single-day screening and 24 visits (1 baseline/intervention + 5 treatment 
days/intervention + 2 follow-up days/intervention). Participant enrollment and allocation will be 
completed during the screening visit. Baseline visits will occur up to 3 days prior to the first 
treatment. A two-week washout will follow the last treatment day. This washout duration is more 
conservative than washout durations reported in previous animal and human intermittent hypoxia 
studies.53, 
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Table 1. Timing Schedule for enrollment, interventions, and assessments 
  
Enrollment 
Baseline 
Intervention 
Follow-ups/Washout 
Timepoints (days) 
-5 
0 
1 
2 
3 
4 
5 
8 
14 
Screening and Allocation Timing 
Eligibility screen 
x 
 
 
 
 
 
 
 
 
Informed consent 
x 
 
 
 
 
 
 
 
 
Medical clearance 
x 
 
 
 
 
 
 
 
 
Allocation 
 
x 
 
 
 
 
 
 
 
Drug script 
 
x 
 
 
 
 
 
 
 
Intervention Timing  
Placebo+AIH 
 
 
x 
x 
x 
x 
x 
 
 
Caffeine+AIH 
 
 
x 
x 
x 
x 
x 
 
 
Caffeine+SHAM 
 
 
x 
x 
x 
x 
x 
 
 
Assessment Timing 
Primary outcome 
 
 
 
 
 
 
 
 
 
• 
Walking speed 
(10MWT, s) 
 
x 
 
 
 
 
x 
x 
x 
Other outcomes 
 
 
 
 
 
 
 
 
 
• 
Voluntary leg strength  
 
x 
 
 
 
 
x 
x 
x 
• 
Spasticity 
 
x 
 
 
 
 
x 
x 
x 
• 
Spasm frequency  
 
x 
 
 
 
 
x 
x 
x 
• 
Walking function 
 
x 
 
 
 
 
x 
x 
x 
• 
Walking distance 
(6MWT, m) 
 
x 
 
 
 
 
x 
x 
x 
• 
Walking Balance (TUG, 
s) 
 
x 
 
 
 
 
x 
x 
x 
• 
Blinding Questionnaire 
 
 
x 
x 
x 
x 
x 
 
 
• 
Weight (kg) 
 
x 
 
 
 
 
 
x 
x 
Safety Outcomes 
 
 
 
 
 
 
 
 
 
• 
Pain (0-10) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Blood pressure (mmHg) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Heart rate (BPM) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Oxygen saturation (%) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Memory (0-30) 
 
x 
x 
x 
x 
x 
x 
x 
x 
Biomarkers 
 
 
 
 
 
 
 
 
 
• 
CYP1A2 Polymorphism  
 
x 
 
 
 
 
 
 
 
• 
Caffeine serum level 
(mg/L) 
 
 
x 
 
 
 
x 
 
 
• 
Inflammation (CRP) 
 
 
x 
 
 
 
x 
 
 
 
 
 


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14. Sample size 
Twenty individuals with chronic (> 6 months post injury), motor-incomplete SCI (i.e., American 
Spinal Injury Association Impairment Scale (AIS) C or D) will be recruited for the study. Prior to 
the start of this clinical trial, we estimated the number of participants needed to achieve study 
objectives. In particular, our sample size computation focused on treatments: caffeine+dAIH 
versus placebo+dAIH alone. Our preliminary study on the effects of caffeine prior to single AIH 
sequence (vs placebo+SHAM) on 10MWT showed a decrease of 2.4s±1.2s. Under the hypothesis 
of an additive effect of caffeine+dAIH (vs. placebo+dAIH), we anticipate the difference in walking 
speed (10MWT time) change between treatments would be approximately 0.06m/s. With N=20 
cross-over participants (includes 17% attrition rate), a repeated measures ANOVA comparing 4 
treatments across 4 time points, using pooled SD of 1.3s across days, will be sensitive to detect a 
difference at power of 1 – β = 0.80 (f=0.7, F3,12=3.5; ρ=0.4, α=.05).  
15. Recruitment 
Our study coordinator (Barth), study physicians (Slocum, Zafonte), physical therapists, PI 
(Trumbower), and research collaborators will recruit subjects using a variety of strategies shown 
effective in the past. First, the team will identify potential subjects using Institutional Review 
Board approved resources to inform persons with SCI that are currently admitted to inpatient and 
outpatient settings at Spaulding Rehabilitation Hospital, Boston University Medical Center, 
Massachusetts General Hospital, Gaylord Hospital, Albany Medical Center, and Maine Medical 
Center. Subjects will be recruited by word of mouth and flyers at these facilities. This project also 
will have access to clinical resources at Spaulding Rehabilitation Network. 
Prospective participants will be contacted initially by phone or in-person and informed of the 
methods, inclusion/exclusion criteria, and purpose of the study. We also will provide a description 
of this study on ClinicalTrials.gov (NCT02323698), as required by U.S. law. In all cases, 
recruitment will not be coercive, will not involve undue inducements, and will accurately reflect 
the study.  
Potential subjects will be identified by the following sources: 
• Attending physicians or therapists may refer their subjects to the study. We will provide 
physicians, therapists, and clinics with study information sheets, letters, and flyers. 
Prospective subjects will be encouraged to contact the study co-investigators. 
• Flyers posted in public areas across the Boston-land region, in the outpatient specialist 
clinics, or other private locations with given permission. 
• Internet, e-mail, digital signage, and newspaper advertisements. 
• Advertisings posted in Boston public transportation (The T). 
• Patients who have received care within the Mass General Brigham Healthcare system will 
be recruited Patient Gateway. We will use the Research Patient Data Registry (RPDR) to 
identify potential participants using strict search criteria which will include spinal cord 
injury diagnosis, age (18 to 75), and date of injury; we will exclude individuals who have 
“opted out” of receiving research announcements. 
• Possible participants may also be identified through their medical records and their 
physicians might be asked to inform the subjects about the study. 


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• Presentations at local, state hospitals, and rehabilitation facilities that include public and 
VA medical centers in the U.S. Northeast (such as to rehabilitation staff and support groups 
at Spaulding and Partners Network, Boston University Medical Center, Gaylord Hospital, 
Albany Medical Center, and Maine Medical Center). 
• Attending public forums, conferences, or events at which the co-investigator will distribute 
IRB approved recruitment materials. 
• Social Media: Google Ads, Facebook, and Twitter – all advertisements will be submitted 
to (the way they appear on any social media site) and approved by the IRB prior to posting. 
• Partners clinicaltrials.partners.org site. 
• We will contact patients who have consented to be added to the Spaulding Rehabilitation 
Hospital’s Spinal Cord Injury Model Systems (SCIMS) Database who have identified that 
they wished to be contacted for additional research studies. Research staff recruiting from 
the SCIMS database are also added as study staff on the SCIMS protocol. 
Eligible participants will be screened at the designated site facilities (i.e., INSPIRE Laboratory, 
Spaulding Rehabilitation Hospital at Boston and at Cambridge sites). At the screening visit, 
subjects will be informed of this project, their potential involvement, the possible benefits and 
risks, and their right to terminate participation at any time without penalty. Qualifying subjects in 
agreement with the study details will be asked to sign the informed consent and HIPAA form 
approved by Spaulding Rehabilitation Hospital’s IRB. All potential subjects will enter the study 
according to the inclusion/exclusion criteria above and following medical clearance. Experiments 
will be conducted at the Spaulding Hospital in Cambridge.  
Prospective subjects will be contacted by telephone, mail or email and briefly informed of the 
methods, inclusion criteria, and purpose of the study. We also will provide a description of this 
study on http://www.ClinicalTrials.gov, as required by U.S. law. In all cases, recruitment will not 
be coercive, will not involve undue inducements, and will accurately reflect the study. 
We will recruit individuals who reflect the population of persons with SCI in the Northeast region 
of the United States, which is 19% women, 81% men, 67% Caucasian, 24% African American, 
8% Hispanic, 2% Asian, and 1% Native American. We do not yet know of any differences in 
responsiveness to AIH or caffeine according to the gender or race of persons with SCI. 
Children will not be included in this study. The proposed research is aimed at understanding the 
plasticity-inducing mechanisms of caffeine and repetitive acute intermittent hypoxia exposure in 
adults with chronic, incomplete spinal cord injury. A separate study would be required to address 
the confounding effects of childhood growth and development after spinal injury on the proposed 
intervention study – a topic best suited for future research once the underlying mechanisms of the 
intervention are better characterized. 
16. Assignment of interventions (for controlled trials) 
Assignment of interventions for study participants will be done using allocation sequence 
generation, concealment, and double-blinded implementation. 
• Allocation sequence generation – This study will consist of three blocks: caffeine+AIH, 
caffeine+SHAM, and placebo+AIH. We will generate the blocked randomization for the study 
participants using a random number list randomizer (Random.org).55 Participants will 


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randomly receive each intervention block with a minimum of 2-week wash-out between 
blocks. We will analyze post hoc differences in age, impairment score, assistive device used, 
baseline lower extremity motor scores, and male-female ratio using independent t-tests. 
• Concealment mechanism – We will conceal the intervention allocation from the evaluators 
performing the assessments, treatment administrators, and the study participant. 
• Implementation – The PI and biostatistician will generate the allocation sequences for the 
cross-over study. The trial coordinator will enroll participants and study staff who are not 
training or assessing the participants will assign the block randomized interventions. 
17. Blinding (masking) 
The PI, participants, care provider, and clinical evaluators will be made aware of possible air 
mixtures and caffeine interventions, but not made aware of the combinatorial intervention 
delivered. Based upon reports from participants (N=6) blinded during our prior study,33 4 of 6 
guessed incorrectly or were uncertain of the intervention received, suggesting our prior blinding 
were effective. 
18. Data collection, management, and analysis 
Data collection methods 
• Assessment and collection plans: Physical evaluations will be completed by the study’s 
licensed physical therapists or occupational therapists and/or trained research study staff. 
Functional ability of subjects will be evaluated using standard clinical assessments with high 
inter-rater reliability: American Spinal Injury Association Impairment Scale (AIS) lower 
extremity motor scores (LEMS) to quantify strength,56 and Spinal Cord Assessment Tool for 
Spastic Reflexes (SCATS), and the Penn Spasm Frequency Scale (PSFS) which is a self-report 
assessment of the frequency and severity of muscle spasms57 and modified Ashworth Scale to 
quantify spasticity.58 We also will record pain severity using the well-established Wong-Baker 
FACES scale, which shows high test-retest reliability and interpretability as well as good 
content and construct validity.59, 60 Clinical ratings of walking ability, including Walking Index 
for Spinal Cord Injury (WISCI) II 61 and/or SCI Functional Ambulation Index (SCI-FAI),62 
will be assessed based on video recordings of the 10MWT. 
We will quantify overground walking ability using the 10MWT.49 Subjects will perform 2 
trials each of the 10MWT at their fastest but safe speed with a minimum of 1-minute rest 
between trials. Average speed across the two 10MWT trials will be used for analyses. The 
validated assessment tool will provide an indication of return of our subject population to pre-
morbid activities following the initial neurological insult. The 10MWT has high inter-rater 
reliability, and the rating procedure can be accomplished in approximately 30 minutes. 
The screening and physical assessment procedures will last approximately 1.5 hours. 
To ensure safety, we also will assess pain, spasticity, systemic hypertension, and autonomic 
dysreflexia prior to and following caffeine and AIH interventions. We routinely measure pain, 
spasticity, and incidence of hypertension and autonomic dysreflexia as part of our study 
protocols in persons with chronic SCI.32, 33 We will make measurements at baseline, after 
session 5, and one-week follow-up, and again at two-weeks follow-ups.  
Quantify pain: We will assess pain severity using the Wong-Baker FACES scale of 0 (no pain) 
to 5 (extreme pain).59, 60  


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Quantify spasticity: We will assess spasticity using the Spinal Cord Assessment Tool for 
Spastic Reflexes (SCATS).58 We will quantify total lower extremity spasticity score using the 
cumulative sum of 3 SCATS subscales: clonus (0=no spasticity; 3=severe), flexor (0=no 
spasticity; 3=severe), and extensor (0=no spasticity; 3=severe).  
Quantify systemic hypertension incidence rate: In addition to monitoring during the 
intervention, we will measure systolic and diastolic BP at baseline, after sessions 5, and again 
at follow-ups. We will define a systemic hypertensive event as a systolic pressure exceeding 
140 mmHg and/or diastolic pressure exceeding 90 mmHg.46, 47  
We will compute hypertension incident rate for each intervention group as the number of 
hypertensive events divided by the total person-time. We define person-time in units of person-
measures (the sum of the total number of BP measurements) taken for each person. Person-
measures accounts for the total number of chances for detecting a hypertensive event and 
accounts for measurements not made due to drop-out or a disqualifying adverse event 63. 
Finally, we will compute Relative Risk for each Aim as the incidence rate in the dAIH groups 
over the incidence rate in the dSHAM groups.64 
Quantify autonomic dysreflexia incidence rate: We will assess the occurrence of autonomic 
dysreflexia in all intervention groups. Systolic blood pressure (SBP) will be measured as 
detailed in common methods. We will assess SBP at baseline, and before, during and 
immediately after breathing interventions, and again at follow-ups. Additional SBP 
measurements will be made if participants first present with symptoms characteristic of 
autonomic dysreflexia (e.g., headache, diaphoresis, blurred vision). An autonomic dysreflexia 
event will constitute a participant having a SBP increase from baseline of 20 mmHg or SBP 
greater than 150 mmHg with complaints of headache, diaphoresis, and/or blurred vision and 
will be diagnosed by our study team clinicians (physicians and physical therapists). An 
autonomic dysreflexia event will result in termination of study participation and require prompt 
evaluation by the team physicians and emergency medical team.  
We will compute autonomic dysreflexia incident rate for each group as the number of 
autonomic dysreflexia events divided by the total person-time. We define person-time in units 
of person-days (the number of days a person remains in the study). Person-days accounts for 
the total number of chances for detecting autonomic dysreflexia and accounts for days on 
which measurements were not made due to drop-out or a disqualifying adverse event.63 Finally, 
we will compute Relative Risk as the ratio of incidence rates between groups.64 
We previously demonstrated that daily exposures of AIH (up to 5 consecutive days) do not 
elicit adverse clinical events in persons with chronic SCI.32, 33 Similarly, rodents exposed to 
daily exposure for 7 consecutive days did not induce hippocampal cell death, changes in body 
mass, or hypertension,8, 9 including in animals early after injury.8 Determining whether dAIH 
or in combination protocols lead to the onset of significant pathology (pain, spasms, 
hypertension, autonomic dysreflexia) in persons with SCI is essential for establishing safe 
delivery of caffeine combined with AIH in this earlier stage of injury. 
Quantify inflammation: We will quantify inflammation using measures of C-reactive protein 
concentrations in the blood. 
• Participant retention plans: Whether or not you complete the whole study, we will pay you $25 
for each visit you do complete. We will also reimburse for up to $100 per visit in travel 


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expenses. Receipts for travel expenses are required. If eligible, we will reimburse for up to 
$150 per night for up to 12 nights in lodging expenses. Receipts for lodging expenses are 
required. Eligibility depends on travel distance of more than 60 miles to Spaulding Hospital. 
19. Data management 
Research materials will consist of data from human subjects including: (1) recordings of subject 
muscle activity, kinematics, and kinetics during overground walking (2) subject anthropometric 
measurements; (2) video images of the experiments; (3) biological (i.e., blood, saliva) samples; 
and (4) personal health information collected during screening. Assessment scores will be obtained 
during an initial screening conducted by the team physician (Slocum) and physical therapist.  
The clinical and physiological data will be obtained only for research purposes. Upon enrollment, 
study subjects will be assigned an ID number. The ID number will identify data in reports, public 
or private, and personal information kept confidential. Data will not be labeled with subject names; 
no code sheets or linkages between subject name and code number will be made on data collection 
sheets. Biological samples will be collected, de-identified, and immediately stored according to 
biohazard safety protocols. Testing results will not be provided to the subjects.  
Consent forms and HIPAA waivers that contain subject names will be stored separate from data 
sheets in a locked file cabinet in the PI’s laboratory. Coded data, which include computer files and 
computer video records, will be kept on a password protected local network in a secured lab area. 
Any forms/computer files containing the subject’s personal information and their ID number will 
be housed in a locked office/laboratory space and accessed only by the investigators or other study 
personnel unless required by law. Study records can be opened by court order or produced in 
response to a subpoena or a request for production of documents unless a Certificate of 
Confidentiality is in place for this study. 
20. Statistical methods 
All data will be managed using REDCap65, MS 365 Excel 16.54 (Microsoft, USA), and MATLAB 
R2020a (Mathworks Inc, USA) and will be analyzed using SPSS® Statistics 26 (64-bit edition, 
IBM, USA). Descriptive statistics, including means, standard deviations and 95% confidence 
intervals of the within-participant pre-post change for each intervention will calculated for all 
measures. The baseline characteristics of the participants will be compared using parametric and 
non-parametric inferencing. 
Hypothesis testing: To evaluate our main hypothesis that caffeine prior to AIH enhances walking 
recovery, we will perform a linear mixed model with fixed effects at a significance at the p=0.05 
level.66 Intervention (caffeine+dAIH, caffeine+SHAM, placebo+dAIH) and time (day) will be the 
fixed main effects, with subject as random effect and walking speed (10MWT) as repeat measures. 
Differences from BL will be compared between and within interventions at D5, F1, and F2. 
Bonferroni corrections will be made for multiple comparisons. If distributions are not normal, we 
will perform comparable nonparametric tests. If baseline measures are significantly different 
between sessions (interventions), an analysis of co-variance (ANCOVA) will be used to analyze 
the data. We predict walking speed (10MWT) will be greater after daily caffeine+AIH as compared 
to after daily placebo+AIH and daily caffeine+SHAM. 
Blinding integrity analysis: we plan to rigorously quantify blinding integrity here. We will ask 
participants and raters to guess the intervention received at the end of each intervention and 
indicate guess confidence using a Likert scale.67, 68 We will use a contingency table and Fischer’s 


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Exact Test to determine if the probability of correct guessing is different from chance. Using 
multivariate logistic regressions, we also will assess factors that may influence guessing (i.e., 
adverse events, perceived effects, and sensorimotor changes). In the event of a medical emergency, 
we will unblind the PI, study participants, and care provider. We also will notify IRB of treatment 
allocation during adverse event reporting as necessary. 
V. 
Monitoring 
21. Data monitoring 
While this study is not under FDA review, all investigators have completed/will complete training 
requirements at Spaulding Rehabilitation Hospital. Staff training will include Protection of Human 
Subjects that will familiarize our team with updates to Good Clinical Practices (i.e., CITI 
certification), and other principles related to the ethical and appropriate conduct of clinical 
research. The Principal Investigator will regularly review study procedures with the Medical 
Monitor (monthly) and study staff (bi-weekly), and annual reviews will be conducted by the sites’ 
Office of Clinical Research to ensure compliance with applicable regulations. The monitoring plan 
will include review of elements required by U.S. 21 CFR 50 and ICH E6, Section 4.8. Onsite 
monitoring visits will be conducted annually. Adverse event reporting and corrective actions will 
be reported to the Institutional Review Boards and DOD Sponsor as soon as possible to ensure 
participant safety. 
The PI (Trumbower) will be responsible for monitoring the completeness of all data and source 
documents. The Principal Investigator will monitor the informed consent procedures in accordance 
with the Informed Consent Compliance Checklist of Partners HealthCare Systems. Subject 
data/protocol adherence will be monitored by the research coordinator at each step in the study 
including.  
22. Harms 
Checklists and note pages are used to note any deviations or omissions from the protocols. Any 
clinical/health related issues would be immediately presented to the subject to determine 
appropriate notification (i.e., current physician or appropriate specialist). Based on the seriousness 
of the situation Drs. Slocum or Zafonte may be contacted to provide clinical guidance on the 
appropriate course of action as per our Laboratory Medical Emergency Safety Plan. 
23. Auditing 
The MGB performs routine internal audits of laboratories on an annual basis. The audits will be 
performed independent from the investigators and sponsors.  
VI. 
Ethics and Dissemination 
24. Research ethics approval 
The study will be conducted in accordance with the principles of GCP. A favorable ethical opinion 
will be obtained from the appropriate REC and local R&D approval will be obtained prior to 
starting the study. The study will not start until a Clinical Trial Authorization is obtained from the 
appropriate Regulatory Authority.  
25. Protocol amendments 
Procedures defined in the study protocol will be carefully reviewed by the PI and research staff 
prior to the time of study initiation. Any changes to the protocol in the form of an amendment must 
be submitted to the IRB. We also plan to communicate important protocol modifications such as 


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changes to eligibility criteria, outcomes, and analyses to trial participants, trial registry 
(clinicaltrials.gov), and journals.  
26. Consent or assent 
The research physician or physical therapist may be responsible for identifying suitable patients 
and obtaining consent the patient. However, the PI must confirm that the patient fulfills the eligibility 
criteria. The PI is responsible for ensuring informed consent is obtained before any protocol specific 
procedures are carried out. 
27. Confidentiality 
Assessment forms, reports, and other medical records must be identified in a manner that ensures 
participant confidentiality. All records will be kept in a secure storage area with limited access. 
The PI and study site staff involved with this study may not use for any purpose other than 
performance of the study, any data, confidential information disclosed to those individuals for the 
purpose of the study. Only authorized personnel will have access to these confidential files. 
Authorized FDA personnel have the right to inspect and copy all records pertinent to this trial. Use 
of study data when reporting results will be without identifiable reference to the participants. 
28. Declaration of interests 
There are no financial or competing interests for the PI at the start of this clinical trial. 
29. Access to data 
The study staff and PI will comply with the requirements of the Data Protection Act 1998 with 
regard to the collection, storage, processing, and disclosure of personal information and will 
uphold the Act’s core principles. Published results will not contain any personal data that could 
allow identification of individual participants.  
30. Ancillary and post-trial care 
We will offer the care needed to treat any injury that directly results from taking part in this 
research study. We reserve the right to bill the participant’s insurance company or other third 
parties, if appropriate, for the care they receive for the injury. We will try to have these costs paid 
for, but the participant may be responsible for some of them. For example, if the care is billed to 
your insurer, the participant will be responsible for payment of any deductibles and co-payments 
required by their insurer. 
31. Dissemination policy 
The clinical trial report will be used for dissemination within clinicaltrials.gov, scientific journals, 
and meetings. The PI has the right to publish orally or in writing the results of the study. On 
completion of the trial, a clinical study report will be prepared in accordance with GCP guidelines. 
The success of this trial depends entirely on the collaboration of a large number of doctors, 
therapists, neuroscientists, engineers, and other healthcare professionals. Those included in the 
Delegation Log will be included in any listing of collaborators. The primary trial publication will 
be drafted by a writing committee who will approve submission for publication.  
 
 


DETAILED STUDY PROTOCOL 
 
BWH/MGH Human Subjects Research Application  
 
 
20 
VII. 
Appendices 
32. Informed consent materials 
Participants will receive a copy of their signed informed consent forms. 
33. Biological specimen protocols 
We will collect blood samples from eligible participants by venipuncture for the purpose of 
accurately assessing caffeine dose, as well as C-Reactive Protein (CRP) levels. A licensed 
phlebotomist will draw blood using venipuncture per WHO guidelines (WHO, 2010) before and 
after combinatorial treatment on day one, and once after combinatorial treatment day five, totaling 
no more than 3 total venipuncture blood draws or 2 tablespoons of blood per time point. Collection 
of blood samples by venipuncture poses minimal risk to subjects. To minimize the number of 
venipunctures and total volume of blood drawn, we will only perform venipuncture at critical time 
points during the study. All samples to assess caffeine and CRP levels will be de-identified then 
allowed to coagulate and spun via centrifuge if needed. Serum samples will be transported to Quest 
Diagnostics, Inc. for analysis. Biohazard safety protocols will be strictly followed. Samples drawn 
to assess for additional markers will be de-identified, spun via centrifuge to extract and aliquot 
serum, frozen and securely stored at -80 °C until future analyses. 
Blood collection protocols: To monitor caffeine plasma concentration and levels of inflammation, 
we collected blood samples at the beginning and end of the study visit intervention, with a total of 
fewer than 50mL of blood taken during a single visit day. 
Genetic biomarker protocol: Subjects will participate in identifying genetic markers that may 
contribute to intervention responsiveness and motor recovery after SCI. Saliva samples will be 
collected after subjects are screened and deemed eligible to participate as defined by our inclusion 
and exclusion criteria. Samples will be collected using a standard passive drool protocol. The 
subject will be asked to collect saliva in their mouth and drool into a vial (DNA genotek OGR-500 
Vials, distributed by DNA Genotek Inc, USA) via a saliva collection aide (DNA Genotek Inc, 
USA). The vial will be sealed tightly and stored at ambient temperature, until transported, plated, 
and stored at the Partners Biobank until further analysis. BDNF, ApoE, and other genotypes will 
be determined. The results of genotyping will be evaluated in relation to performance levels 
achieved following treatments. All samples will be de-identified and stored and transported 
according to biohazard safety protocols. Genetic testing results will not be provided to the subjects.  
 


DETAILED STUDY PROTOCOL 
 
BWH/MGH Human Subjects Research Application  
 
 
21 
VIII. REFERENCES 
 
1. 
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16. Hoffman MS, Golder FJ, Mahamed S, Mitchell GS. Spinal adenosine A2A receptor inhibition 
enhances phrenic long term facilitation following acute intermittent hypoxia. The Journal of 
Physiology. 2010;588(1):255-266. doi:10.1113/jphysiol.2009.180075 


DETAILED STUDY PROTOCOL 
 
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22 
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25. Brice C, Smith A. Caffeine levels in saliva: associations with psychosocial factors and behavioural 
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27. Van Soeren M, Mohr T, Kjaer M, Graham TE. Acute effects of caffeine ingestion at rest in humans 
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28. Graham TE, Spriet LL. Metabolic, catecholamine, and exercise performance responses to various 
doses of caffeine. J Appl Physiol. 1995;78(3):867-874.  
29. Mohr M, Nielsen JJ, Bangsbo J. Caffeine intake improves intense intermittent exercise performance 
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1, 2011 2011;111(5):1372-1379. doi:10.1152/japplphysiol.01028.2010 
30. Newton R, Broughton LJ, Lind MJ, Morrison PJ, Rogers HJ, Bradbrook ID. Plasma and salivary 
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31. Tan AQ, Papadopoulos JM, Corsten AN, Trumbower RD. An automated pressure-swing absorption 
system to administer low oxygen therapy for persons with spinal cord injury. Exp Neurol. Jul 17 
2020;333:113408. doi:10.1016/j.expneurol.2020.113408 
32. Trumbower RD, Jayaraman A, Mitchell GS, Rymer WZ. Exposure to Acute Intermittent Hypoxia 
Augments Somatic Motor Function in Humans With Incomplete Spinal Cord Injury. Neurorehabil 
and Neural Repair. February 1, 2012 2012;26(2):163-172. doi:10.1177/1545968311412055 
33. Hayes HB, Jayaraman A, Herrmann M, Mitchell GS, Rymer WZ, Trumbower RD. Daily intermittent 
hypoxia enhances walking after chronic spinal cord injury: a randomized trial. Neurology. Jan 14 
2014;82(2):104-13. doi:10.1212/01.WNL.0000437416.34298.43 
34. Tester NJ, Fuller DD, Fromm JS, Spiess MR, Behrman AL, Mateika JH. Long-term facilitation of 
ventilation in humans with chronic spinal cord injury. American journal of respiratory and critical 
care medicine. Jan 1 2014;189(1):57-65. doi:10.1164/rccm.201305-0848OC 


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23 
35. Gozal D, Kheirandish-Gozal L. Cardiovascular morbidity in obstructive sleep apnea: oxidative 
stress, inflammation, and much more. American journal of respiratory and critical care medicine. 
Feb 15 2008;177(4):369-75. doi:10.1164/rccm.200608-1190PP 
36. Serebrovskaya TV, Manukhina EB, Smith ML, Downey HF, Mallet RT. Intermittent hypoxia: cause 
of or therapy for systemic hypertension? Exp Biol Med (Maywood). Jun 2008;233(6):627-50. 
doi:10.3181/0710-MR-267 
37. Burtscher M, Haider T, Domej W, et al. Intermittent hypoxia increases exercise tolerance in patients 
at risk for or with mild COPD. Respir Physiol Neurobiol. Jan 1 2009;165(1):97-103. 
doi:10.1016/j.resp.2008.10.012 
38. Prabhakar NR, Kumar GK. Oxidative stress in the systemic and cellular responses to intermittent 
hypoxia. Biol Chem. Mar-Apr 2004;385(3-4):217-21. doi:10.1515/BC.2004.015 
39. Aboubakr SE, Taylor A, Ford R, Siddiqi S, Badr MS. Long-term facilitation in obstructive sleep 
apnea patients during NREM sleep. J Appl Physiol (1985). Dec 2001;91(6):2751-7.  
40. Cutler MJ, Swift NM, Keller DM, Wasmund WL, Smith ML. Hypoxia-mediated prolonged 
elevation of sympathetic nerve activity after periods of intermittent hypoxic apnea. J Appl Physiol 
(1985). Feb 2004;96(2):754-61. doi:10.1152/japplphysiol.00506.2003 
41. McGuire M, Zhang Y, White DP, Ling L. Effect of hypoxic episode number and severity on 
ventilatory long-term facilitation in awake rats. J Appl Physiol. Dec 2002;93(6):2155-61. 
doi:10.1152/japplphysiol.00405.2002 
42. Baker TL, Mitchell GS. Episodic but not continuous hypoxia elicits long-term facilitation of phrenic 
motor output in rats. J Physiol. Nov 15 2000;529 Pt 1:215-9. doi:10.1111/j.1469-7793.2000.00215.x 
43. Mitchell GS, Baker TL, Nanda SA, et al. Invited review: Intermittent hypoxia and respiratory 
plasticity. J Appl Physiol. Jun 2001;90(6):2466-75.  
44. Mitchell GS, Johnson SM. Neuroplasticity in respiratory motor control. J Appl Physiol. Jan 
2003;94(1):358-74. doi:10.1152/japplphysiol.00523.2002 
45. Mauelshagen J, Sherff CM, Carew TJ. Differential induction of long-term synaptic facilitation by 
spaced and massed applications of serotonin at sensory neuron synapses of Aplysia californica. 
Learn Mem. Jul-Aug 1998;5(3):246-56.  
46. Franco V, Oparil S, Carretero OA. Hypertensive therapy: Part II. Circulation. Jun 29 
2004;109(25):3081-8. doi:10.1161/01.CIR.0000132615.44103.B1 
47. Franco V, Oparil S, Carretero OA. Hypertensive therapy: Part I. Circulation. Jun 22 
2004;109(24):2953-8. doi:10.1161/01.CIR.0000132614.41493.B5 
48. Tan AQ, Sohn WJ, Naidu A, Trumbower RD. Daily acute intermittent hypoxia combined with 
walking practice enhances walking performance but not intralimb motor coordination in persons 
with 
chronic 
incomplete 
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injury. 
Exp 
Neurol. 
Feb 
26 
2021:113669. 
doi:10.1016/j.expneurol.2021.113669 
49. van Hedel HJ, Wirz M, Dietz V. Assessing walking ability in subjects with spinal cord injury: 
validity and reliability of 3 walking tests. Research Support, Non-U.S. Gov't. Archives of Physical 
Medicine and Rehabilitation. Feb 2005;86(2):190-6. doi:10.1016/j.apmr.2004.02.010 
50. Mehrholz J, Kugler J, Pohl M. Locomotor training for walking after spinal cord injury. Cochrane 
Database 
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Nov 
14 
2012;11(11):CD006676. 
doi:10.1002/14651858.CD006676.pub3 
51. Podsiadlo D, Richardson S. The Timed “Up & Go”: A Test of Basic Functional Mobility for Frail 
Elderly Persons. Journal of the American Geriatrics Society (JAGS). 1991;39(2):142-148. 
doi:10.1111/j.1532-5415.1991.tb01616.x 
52. van Middendorp JJ, Hosman AJ, Pouw MH, Group E-SS, Van de Meent H. ASIA impairment scale 
conversion in traumatic SCI: is it related with the ability to walk? A descriptive comparison with 


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24 
functional ambulation outcome measures in 273 patients. Spinal Cord. Jul 2009;47(7):555-60. 
doi:10.1038/sc.2008.162 
53. Rodway GW, Sethi JM, Hoffman LA, et al. Hemodynamic and molecular response to intermittent 
hypoxia (IH) versus continuous hypoxia (CH) in normal humans. Transl Res. Feb 2007;149(2):76-
84. doi:10.1016/j.trsl.2006.09.005 
54. Schlenker EH, Shi Y, Wipf J, Martin DS, Kost CK, Jr. Fructose feeding and intermittent hypoxia 
affect ventilatory responsiveness to hypoxia and hypercapnia in rats. J Appl Physiol. Oct 
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57. Adams MM, Ginis KAM, Hicks AL. The spinal cord injury spasticity evaluation tool: development 
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Statistical Analysis Plan 
9 January 2022 
1 
TABLE OF CONTENTS 
I.
ADMINISTRATION INFORMATION ......................................................................................................................... 2 
1.
TITLE AND TRIAL REGISTRATION .................................................................................................................................... 2 
2.
STATISTICAL ANALYSIS PLAN (SAP) VERSION 4.0 ......................................................................................................... 2 
3.
STUDY PROTOCOL VERSION 3.0 ..................................................................................................................................... 2 
4.
STATISTICAL ANALYSIS PLAN REVISIONS ........................................................................................................................ 3 
5.
ROLES AND RESPONSIBILITY ........................................................................................................................................... 3 
6.
STATISTICAL ANALYSIS PLAN APPROVAL ........................................................................................................................ 4 
II.
INTRODUCTION ............................................................................................................................................................ 5 
7.
BACKGROUND AND RATIONALE ..................................................................................................................................... 5 
8.
OBJECTIVES .................................................................................................................................................................... 5 
III.
METHODS: PARTICIPANTS, INTERVENTIONS, AND OUTCOMES ................................................................. 6 
9.
TRIAL DESIGN ................................................................................................................................................................. 6 
10.
RANDOMIZATION ....................................................................................................................................................... 6 
11.
SAMPLE SIZE .............................................................................................................................................................. 6 
12.
FRAMEWORK .............................................................................................................................................................. 7 
13.
STATISTICAL INTERIM ANALYSES AND STOPPING GUIDANCE ..................................................................................... 7 
14.
TIMING OF ANALYSIS ................................................................................................................................................. 7 
15.
TIMING OF OUTCOME ASSESSMENTS .......................................................................................................................... 8 
IV.
STATISTICAL PRINCIPLES ........................................................................................................................................ 9 
16.
LEVEL OF SIGNIFICANCE ............................................................................................................................................ 9 
17.
ADJUSTMENT FOR MULTIPLICITY ............................................................................................................................... 9 
18.
CONFIDENCE INTERVALS ............................................................................................................................................ 9 
19.
ADHERENCE AND PROTOCOL DEVIATIONS ................................................................................................................. 9 
20.
ANALYSIS POPULATIONS ............................................................................................................................................ 9 
V.
TRIAL POPULATION .................................................................................................................................................. 10 
21.
SCREENING DATA ..................................................................................................................................................... 10 
22.
ELIGIBILITY .............................................................................................................................................................. 10 
23.
RECRUITMENT .......................................................................................................................................................... 11 
24.
WITHDRAWAL/FOLLOW-UP ...................................................................................................................................... 12 
25.
BASELINE PATIENT CHARACTERISTICS ..................................................................................................................... 12 
VI.
ANALYSIS ...................................................................................................................................................................... 12 
26.
OUTCOME DEFINITIONS ............................................................................................................................................ 12 
27.
ANALYSIS METHODS ................................................................................................................................................ 13 
28.
MISSING DATA ......................................................................................................................................................... 14 
29.
ADDITIONAL ANALYSES ........................................................................................................................................... 14 
30.
HARMS ..................................................................................................................................................................... 14 
31.
STATISTICAL SOFTWARE .......................................................................................................................................... 14 
32.
LINKS TO ADDITIONAL SUPPORTING DOCUMENTATION ............................................................................................ 14 
33.
REFERENCES ............................................................................................................................................................ 15 


Statistical Analysis Plan 
 
9 January 2022 
 
 
2 
I. 
Administration Information 
1. Title and trial registration 
Title: Caffeine combined with low oxygen therapy boosts walking recovery in people with spinal 
cord injury 
Trial registration: ClinicalTrials.gov Identifier: NCT02323698, registry name: Effects of caffeine 
and intermittent hypoxia on leg function in human spinal cord injury 
2. Statistical analysis plan (SAP) Version 4.0 
Recruitment to the study ended on 19 March 2021 
Follow-up for the main results ended on 22 September 2021 
3. Study Protocol Version 3.0 
Last updated: September 27, 2021 
 
 


Statistical Analysis Plan 
 
9 January 2022 
 
 
3 
4. Statistical analysis plan revisions 
Revision history and justification 
Revision  Date 
Modification to SAP 
Reason 
1.0 
11/29/2017 
Change in primary study site 
PI changed institutions 
2.0 
11/29/2017 
Change in administration 
Change in roles and responsibilities 
of initial study staff due to relocation 
of study site. 
3.0 
11/21/2019 
Change in trial population 
Extended age and level of injury 
inclusion criterion due to new 
evidence of intervention benefits.1  
4.0 
05/11/2020 
Change in study methods 
Modify SAP to accommodate 
COVID-19 pandemic safety 
mandates 
 
5. Roles and responsibility  
Names, affiliations, and roles of SAP contributors 
Name 
Affiliation 
Role of SAP Contributor 
Randy Trumbower 
Harvard Medical School & 
Spaulding Rehabilitation Hospital 
Principal Investigator, wrote all 
version of SAP 
Stella Barth 
Spaulding Rehabilitation Hospital 
Lab Manager, contributed to final 
version of SAP 
Heather Hayes 
Emory University 
Co-Investigator, contributed to the 
first version of SAP  
Michael Epstein 
Emory University 
Statistician, contributed to the first 
version of study methods, statistical 
principles, and analyses 
Chris Tuthill  
Harvard Medical School & 
Spaulding Rehabilitation Hospital 
Postdoctoral Fellow, contributed to 
the final version of SAP 
Gordon Mitchell 
University of Florida 
Co-Investigator, contributed to the 
initial version of introduction and 
study methods 
 
 
 


Statistical Analysis Plan 
 
9 January 2022 
 
 
4 
6. Statistical analysis plan approval 
 
 
 
 
 
 
 
Final SAP Approval 
 
 
Version 4.0 
 
 
Date Finalized: 3 November 2021 
 
 
PI Name: Randy D Trumbower 
 
PI Email Address: randy.trumbower@mgh.harvard.edu 
 
 
 
 
Randy D Trumbower, Principal Investigator 
 
3 November 2021 
Print first and last name 
 
Date 
 
 
 
 
 
3 November 2021 
Signature 
 
Date 


Statistical Analysis Plan 
 
9 January 2022 
 
 
5 
II. Introduction 
 
7. Background and Rationale 
Spinal cord injury (SCI) disrupts connections between the brain and spinal cord, causing life-long 
paralysis and reduced mobility. As such, restoring walking ability remains a highly valued goal 
for persons with SCI.2 Even small improvements in walking skills that enable a person with SCI 
to stand, walk within the home, or negotiate spaces not accessible to wheelchairs can translate into 
significant empowerment, health benefits, and improvement in quality of life. Because most spinal 
injuries are incomplete, there are at least some spared neural pathways to the motor neurons that 
can initiate and coordinate movements necessary for walking recovery. Although injury-induced 
plasticity in spared spinal pathways enables partial spontaneous recovery of respiratory and 
somatic motor function,3, 4 the extent of this recovery is slow, variable and frustratingly limited.5 
Thus, there is a critical need for new strategies that augment spinal plasticity and subsequently 
improve walking ability in persons with SCI. 
Acute intermittent hypoxia (AIH) induces spinal plasticity, strengthening connections to motor 
neurons.6-10 Considerable progress has been made towards an understanding of cellular 
mechanisms giving rise to AIH-induced respiratory plasticity.8 Repetitive exposure to AIH 
enhances the expression of plasticity-promoting proteins in respiratory motor nuclei10-12 and elicits 
profound recovery of breathing capacity in spinally injured rats.13 Key steps in AIH-induced 
plasticity include episodic release of serotonin near respiratory motor neurons, initiating new 
synthesis of brain derived neurotrophic factor (BDNF) and subsequently activating its high affinity 
receptor, TrkB 6. “Downstream” events include the activation of extracellular signal-regulated 
kinases (ERK); mitogen-activated protein (MAP) kinases and postulated insertion of glutamate 
receptors at the synapse between respiratory pre-motor and motor neurons. Excitingly, these 
changes extend beyond the respiratory system. Colleagues also observed that AIH enhances 
expression of these same proteins in non-respiratory motor neurons.9  
Although dAIH may be a viable method to enhance walking after iSCI, the mechanisms underlying 
dAIH-induced motor recovery may be constrained by competing neural mechanisms of motor 
facilitation. While dAIH induces facilitation predominantly via serotonin-dependent mechanisms 
14, even mild hypoxia causes ATP release from glia and other sources in the nervous system, 
leading to an accumulation of extracellular adenosine. Subsequent activation of A2A receptors 
elicits a distinct form of motor facilitation 15 that is known to compete with the serotonin-dependent 
pathway via cross-talk inhibition 16, 17. Consequently, systemic administration of a selective A2A 
receptor antagonist, MSX-3, reduced cross-talk inhibition and increased AIH-induced long-term 
motor facilitation in the phrenic and hypoglossal nerves by nearly 50% and 20%, respectively, in 
rats 17. The impact of adenosine A2A receptor antagonists on AIH-induced motor recovery have 
not yet been studied in persons with incomplete SCI (iSCI).  
Our working hypothesis is that reducing crosstalk inhibition during dAIH with a well-tolerated 
A2A antagonist (caffeine) will enhance AIH-induced improvement of walking function in persons 
with chronic iSCI.  
8. Objectives 
The main objective of this clinical trial is to evaluate the effects of a common, safe adenosine A2A 
antagonist, caffeine, on recovery of walking following dAIH in persons with chronic iSCI. While 
highly specific A2A receptor antagonists, such as MXS-3 and ZM2412385, block cross-talk in 
rats,17 their safety in SCI is not known. On the other hand, caffeine offers the ideal blend of known 


Statistical Analysis Plan 
 
9 January 2022 
 
 
6 
safety, metabolism, and efficacious A2A receptor antagonism.18 Even at low plasma concentrations 
(1-10μM) after one cup of coffee, caffeine antagonizes A2A receptors,18 and the efficacy of 
moderate caffeine in blocking neural A2A receptors is illustrated by the neuroprotective effects of 
regular caffeine consumption in dopaminergic transmission in Parkinson’s Disease.19 Although 
caffeine has many neurobiological actions, caffeine’s actions at physiologically relevant doses (1-
100 μM), including the proposed dose of up to 6 mg/kg body weight, are mediated by adenosine 
receptor antagonism versus phosphodiesterase inhibition, release of intracellular calcium stores, 
or other site.20 Our study will quantify the daily (5 consecutive days) effects of caffeine (e.g., 
4 mg/kg) as an adjunctive therapy to dAIH for restoring walking function in persons with chronic 
iSCI. This dose has known safety in iSCI and substantial antagonism to A2A receptors.18, 21, 22  
III. Methods: Participants, interventions, and outcomes 
9. Trial Design 
We will conduct a placebo-controlled, cross-over, block-randomized intervention study to assess 
the efficacy of daily caffeine prior to AIH on improving walking function in persons with chronic, 
motor-incomplete SCI.  
10. Randomization 
Assignment of interventions for study participants will be done using allocation sequence 
generation, concealment, and double-blinded implementation. 
• Allocation sequence generation – This study will consist of three blocks: caffeine+AIH, 
caffeine+SHAM, and placebo+AIH. We will generate the blocked randomization for the study 
participants using a random number list randomizer (Random.org).23 Participants will 
randomly receive each intervention block with a minimum of 2-week wash-out between 
blocks. We will analyze post hoc differences in age, impairment score, assistive device used, 
baseline lower extremity motor scores, and male-female ratio using independent t-tests. 
• Concealment mechanism – We will conceal the intervention allocation from the evaluators 
performing the assessments, treatment administrators, and the study participant. 
• Implementation – The PI and biostatistician will generate the allocation sequences for the 
cross-over study. The trial coordinator will enroll participants and study staff who are not 
training or assessing the participants will assign the block randomized interventions. 
11. Sample size 
Twenty individuals with chronic (> 6 months post injury), motor-incomplete SCI (i.e., American 
Spinal Injury Association Impairment Scale (AIS) C or D) will be recruited for the study. Prior to 
the start of this clinical trial, we estimated the number of participants needed to achieve study 
objectives. In particular, our sample size computation focused on treatments: caffeine+dAIH 
versus placebo+dAIH alone. Our preliminary study on the effects of caffeine prior to single AIH 
sequence (vs placebo+SHAM) on 10MWT showed a decrease of 2.4s±1.2s. Under the hypothesis 
of an additive effect of caffeine+dAIH (vs. placebo+dAIH), we anticipate the difference in walking 
speed (10MWT time) change between treatments would be approximately 0.06m/s. With N=20 
cross-over participants (includes 17% attrition rate), a repeated measures ANOVA comparing 4 
treatments across 4 time points, using pooled SD of 1.3s across days, will be sensitive to detect a 
difference at power of 1 – β = 0.80 (f=0.7, F3,12=3.5; ρ=0.4, α=.05).  


Statistical Analysis Plan 
 
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7 
12. Framework 
This placebo-controlled study will examine the combined effects of caffeine and AIH results in 
greater walking performance than either intervention alone. However, this trial will not evaluate 
the potential superiority or equivalence of caffeine+AIH to standard of care treatments for persons 
with chronic iSCI.  
13. Statistical interim analyses and stopping guidance 
We do not plan to conduct an interim analysis. However, we will impose criteria for discontinuing 
or modifying allocated interventions. The interventions will be stopped, and immediate care 
provided if any significant adverse effects, such as rapid heart rate, dizziness, nausea, or a 
significant change in blood pressure, are detected or self-reported by the study participant. 
Participants with diabetes, cirrhosis, a history of autonomic dysreflexia, caffeine allergies or 
sensitivities, or females who are pregnant will be excluded from this portion of the study. Due to 
the diuretic effects of caffeine, subjects will be asked to empty their bladders prior to consumption. 
During and between interventions, blood pressure and heart rate will be monitored. Any adverse 
effects will be reported and assessed. Our study team will be prepared to discontinue treatment 
and offer immediate care if any significant adverse effects, such as rapid heart rate, systemic 
hypertensive event, dizziness, or nausea are detected or self-reported by the subject. We will define 
a systemic hypertensive event as a systolic pressure exceeding 140 mmHg and/or diastolic pressure 
exceeding 90 mmHg.24, 25 We will stop this trial if serious adverse events are directly attributed to 
the interventions under study. 
14. Timing of analysis 
All outcomes will be analyzed collectively. We will maintain concealment of the intervention 
allocation until the completion of data collection.  
 


Statistical Analysis Plan 
 
9 January 2022 
 
 
8 
15. Timing of outcome assessments 
Timing of outcome assessments will occur during specified visits. The timepoints of these 
assessments along with the timepoints for enrollment and interventions are detailed in the timing 
schedule (Table 1).  
Table 1. Timing Schedule for enrollment, interventions, and assessments 
  
Enrollment 
Baseline 
Intervention 
Follow-ups/Washout 
Timepoints (days) 
-5 
0 
1 
2 
3 
4 
5 
8 
14 
Screening and Allocation Timing 
Eligibility screen 
x 
 
 
 
 
 
 
 
 
Informed consent 
x 
 
 
 
 
 
 
 
 
Medical clearance 
x 
 
 
 
 
 
 
 
 
Allocation 
 
x 
 
 
 
 
 
 
 
Drug script 
 
x 
 
 
 
 
 
 
 
Intervention Timing  
Placebo+AIH 
 
 
x 
x 
x 
x 
x 
 
 
Caffeine+AIH 
 
 
x 
x 
x 
x 
x 
 
 
Caffeine+SHAM 
 
 
x 
x 
x 
x 
x 
 
 
Assessment Timing 
Primary outcome 
 
 
 
 
 
 
 
 
 
• 
Walking speed 
(10MWT, s) 
 
x 
 
 
 
 
x 
x 
x 
Other outcomes 
 
 
 
 
 
 
 
 
 
• 
Voluntary leg strength  
 
x 
 
 
 
 
x 
x 
x 
• 
Spasticity 
 
x 
 
 
 
 
x 
x 
x 
• 
Spasm frequency  
 
x 
 
 
 
 
x 
x 
x 
• 
Walking function 
 
x 
 
 
 
 
x 
x 
x 
• 
Walking distance 
(6MWT, m) 
 
x 
 
 
 
 
x 
x 
x 
• 
Walking Balance (TUG, 
s) 
 
x 
 
 
 
 
x 
x 
x 
• 
Blinding Questionnaire 
 
 
x 
x 
x 
x 
x 
 
 
• 
Weight (kg) 
 
x 
 
 
 
 
 
x 
x 
Safety Outcomes 
 
 
 
 
 
 
 
 
 
• 
Pain (0-10) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Blood pressure (mmHg) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Heart rate (BPM) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Oxygen saturation (%) 
 
x 
x 
x 
x 
x 
x 
x 
x 
• 
Memory (0-30) 
 
x 
x 
x 
x 
x 
x 
x 
x 
Biomarkers 
 
 
 
 
 
 
 
 
 
• 
CYP1A2 Polymorphism  
 
x 
 
 
 
 
 
 
 
• 
Caffeine serum level 
(mg/L) 
 
 
x 
 
 
 
x 
 
 
• 
Inflammation (CRP) 
 
 
x 
 
 
 
x 
 
 
 


Statistical Analysis Plan 
 
9 January 2022 
 
 
9 
IV. Statistical Principles 
We plan to use a parametric linear mixed model since this method is sensitive to within subject 
changes and robust against variability, small sample sizes, and missing/unbalanced data sets as 
compared to other statistical models.26 If the results do not meet these model assumptions, we will 
apply alternative non-parametric tests. 
16. Level of significance  
All applicable statistical tests will be 2-sided and will be performed using a 5% significance level.  
17. Adjustment for multiplicity 
we do not plan to adjust for multiplicity. 
18. Confidence intervals 
Statistical comparisons will include 95% (2-sided) confidence intervals (CIs). 
19. Adherence and protocol deviations 
We will record a deviation (if the even does not significantly affect a participant’s rights, safety, 
or well-being, or trial outcomes), or a violation (if the deviation may potentially significantly 
impact the completeness, accuracy, and/or reliability of the trial data or that may significantly 
affect a patient’s rights, safety, or well-being). All protocol deviations/violations will be logged, 
but no formal statistical testing will be performed. 
Adverse events will be reported on an IRB-approved electronic Case Report Form (eCRF). Details 
will include the type of event, date of onset, duration, intensity, causality relationship to the study 
drug(s), and outcome. If an adverse event should occur, every attempt will be made to obtain as 
much information as possible about event evaluation and outcome. If a serious adverse event 
occurs, the treatment will be interrupted or discontinued at the physician investigator's discretion. 
All protocol deviations will be reported to the PI (Trumbower) and the IRB.  
Stella Barth will fulfill the responsibilities identified in the study’s delegation log. These 
responsibilities include collecting and tracking data forms and instituting quality control measures 
for data entry verification and study compliance. She will request further documentation such as 
physician and/or procedure notes in the event of observed or reported adverse events or deviations. 
They also will be responsible for auditing the database and confirming the overall integrity of the 
data. The PI (Trumbower) will ensure that all information pertaining to significant new 
developments and unanticipated adverse events are provided to the appropriate regulatory 
authorities, the Investigators, and to the IRB. Quality control measures for this study will be 
conducted at regular intervals ensure the highest standard of clinical research conduct. These 
inspections are conducted in order to verify adherence to the protocol and the completeness and 
accuracy of the data. 
20. Analysis populations 
In this cross-over trial, participants will receive the primary intervention (caffeine+AIH) as well 
as the control conditions (placebo+AIH, caffeine+SHAM). We will perform safety-related 
assessments on each study participant. We anticipate all participants will participate in the 3 
interventions as prescribed, will attend all test visits, and will not have any serious protocol 
violations.  
 


Statistical Analysis Plan 
 
9 January 2022 
 
 
10 
V. Trial Population 
21. Screening data 
There will be no statistical testing completed for clinical characteristics. We will summarize the 
sex, duration of SCI, level and severity of SCI, age, weight, as well as biomarker measures (see 
Table below). The categorical data will be presented using counts and percentages. The continuous 
variables (e.g., outcome measures) will be presented using the mean, median, standard deviation 
(SD), minimum, maximum, and number of participants with an observation (n). 
22. Eligibility 
Potential subjects will be informed about the objectives of the study and must meet all inclusion 
and exclusion criteria (see Appendix 1 for sample Informed Consent Form). They will be required 
to read and sign approved consent and HIPAA forms prior to participation to allow access to 
medical information from their physicians.  
Inclusion criteria: Participation requirements for SCI participants include: 1) 18 to 75 years old; 2) 
medically stable with medical clearance from physician to participate; 3) SCI at or below C2 and 
at or above L5 with at least some lower extremity motor function preserved below the neurologic 
level; 4) non-progressive etiology of spinal injury; 4) AIS A-D at initial screen; 5) at least 6 months 
post-injury (chronic). We plan to choose subjects greater that 6 months post-injury to ensure 
minimal confounding effects of spontaneous neurological recovery during the experiments. This 
will mean that changes in sensorimotor performance are more likely due to the interventions 
associated with the research study.  
Exclusion criteria: Individuals will be excluded due to: 1) severe concurrent illness or pain, 
including unhealed decubiti, severe neuropathic or chronic pain syndrome, infection (e.g. bladder), 
hypertension, cardiovascular disease, pulmonary disease, severe osteoporosis (history of 
fractures), active heterotopic ossification in the lower extremities, or history of peripheral nerve 
injury in the legs; 2) < 24 on Mini-Mental Exam 27; 3) severe autonomic dysreflexia; 4) history of 
cardiovascular/pulmonary complications; 5) pregnancy because of unknown effects of AIH on a 
fetus, although women of childbearing potential will not otherwise be excluded; 6) undergoing 
concurrent physical therapy; 7) history of diabetes. Caffeine can potentially alter sugar metabolism 
and, thus, persons diagnosed with diabetes will be excluded from participating in the caffeine 
portion of this study; 8) history of cirrhosis. Cirrhosis is known to increase the elimination time of 
caffeine and, thus, persons diagnosed with cirrhosis will be excluded from participating in the 
caffeine portion of this study; and 9) history of caffeine allergies or intolerances. Participants with 
a history of caffeine allergies or intolerances will be excluded from participating in the caffeine 
study.  
Inclusion of women and minorities: We will recruit persons with SCI to include women and 
minorities. To protect against possible unknown side effects of dAIH, women who are pregnant 
may not take part in this study as the effects of dAIH on the developing fetus have not been studied. 
If a woman is of childbearing ability, birth control will be recommended for use throughout the 
study.  
Our study coordinator, study physicians, physical therapists, PI (Trumbower), and research 
collaborators recruited subjects using a variety of strategies shown effective in the past. First, the 
team identified potential subjects using Institutional Review Board (IRB) approved resources to 
inform persons with SCI that are currently admitted to inpatient rehabilitation/outpatient units of 
Spaulding Rehabilitation Hospital, Boston University Medical Center, Massachusetts General 


Statistical Analysis Plan 
 
9 January 2022 
 
 
11 
Hospital, Gaylord hospital, Albany Medical Center, and Maine Medical Center. Subjects also will 
be recruited by word of mouth and flyers at these facilities. Eligible subjects screened at INSPIRE 
Laboratory, Spaulding Rehabilitation Hospital Cambridge, MA. At the screening visit, subjects 
informed of this project, their potential involvement, the possible benefits, and risks, and their right 
to terminate participation at any time without penalty. Qualifying subjects in agreement with the 
study details asked to sign the informed consent and HIPAA form approved by Partners IRB. All 
potential subjects entered the study according to the inclusion/exclusion criteria above and 
following medical clearance. Experiments will be conducted at the Spaulding Rehabilitation 
Hospitals. 
23. Recruitment 
Our study coordinator (Barth), study physicians (Slocum, Zafonte), physical therapists, PI 
(Trumbower), and research collaborators will recruit subjects using a variety of strategies shown 
effective in the past. First, the team will identify potential subjects using Institutional Review 
Board approved resources to inform persons with SCI that are currently admitted to inpatient and 
outpatient settings at Spaulding Rehabilitation Hospital, Boston University Medical Center, 
Massachusetts General Hospital, Gaylord Hospital, Albany Medical Center, and Maine Medical 
Center. Subjects will be recruited by word of mouth and flyers at these facilities. This project also 
will have access to clinical resources at Spaulding Rehabilitation Network. 
Prospective participants will be contacted initially by phone or in-person and informed of the 
methods, inclusion/exclusion criteria, and purpose of the study. We also will provide a description 
of this study on ClinicalTrials.gov (NCT02323698), as required by U.S. law. In all cases, 
recruitment will not be coercive, will not involve undue inducements, and will accurately reflect 
the study.  
Potential subjects will be identified by the following sources: 
• Attending physicians or therapists may refer their subjects to the study. We will provide 
physicians, therapists, and clinics with study information sheets, letters, and flyers. 
Prospective subjects will be encouraged to contact the study co-investigators. 
• Flyers posted in public areas across the Boston-land region, in the outpatient specialist 
clinics, or other private locations with given permission. 
• Internet, e-mail, digital signage, and newspaper advertisements. 
• Advertisings posted in Boston public transportation (The T). 
• Patients who have received care within the Mass General Brigham Healthcare system will 
be recruited Patient Gateway. We will use the Research Patient Data Registry (RPDR) to 
identify potential participants using strict search criteria which will include spinal cord 
injury diagnosis, age (18 to 75), and date of injury; we will exclude individuals who have 
“opted out” of receiving research announcements. 
• Possible participants may also be identified through their medical records and their 
physicians might be asked to inform the subjects about the study. 
• Presentations at local, state hospitals, and rehabilitation facilities that include public and 
VA medical centers in the U.S. Northeast (such as to rehabilitation staff and support groups 
at Spaulding and Partners Network, Boston University Medical Center, Gaylord Hospital, 
Albany Medical Center, and Maine Medical Center). 


Statistical Analysis Plan 
 
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12 
• Attending public forums, conferences, or events at which the co-investigator will distribute 
IRB approved recruitment materials. 
• Social Media: Google Ads, Facebook, and Twitter – all advertisements will be submitted 
to (the way they appear on any social media site) and approved by the IRB prior to posting. 
• Partners clinicaltrials.partners.org site. 
• We will contact patients who have consented to be added to the Spaulding Rehabilitation 
Hospital’s Spinal Cord Injury Model Systems (SCIMS) Database who have identified that 
they wished to be contacted for additional research studies. Research staff recruiting from 
the SCIMS database are also added as study staff on the SCIMS protocol. 
Eligible participants will be screened at the designated site facilities (i.e., INSPIRE Laboratory, 
Spaulding Rehabilitation Hospital at Boston and at Cambridge sites). At the screening visit, 
subjects will be informed of this project, their potential involvement, the possible benefits and 
risks, and their right to terminate participation at any time without penalty. Qualifying subjects in 
agreement with the study details will be asked to sign the informed consent and HIPAA form 
approved by Spaulding Rehabilitation Hospital’s IRB. All potential subjects will enter the study 
according to the inclusion/exclusion criteria above and following medical clearance. Experiments 
will be conducted at the Spaulding Hospital in Cambridge.  
Prospective subjects will be contacted by telephone, mail or email and briefly informed of the 
methods, inclusion criteria, and purpose of the study. We also will provide a description of this 
study on http://www.ClinicalTrials.gov, as required by U.S. law. In all cases, recruitment will not 
be coercive, will not involve undue inducements, and will accurately reflect the study. 
We will recruit individuals who reflect the population of persons with SCI in the Northeast region 
of the United States, which is 19% women, 81% men, 67% Caucasian, 24% African American, 
8% Hispanic, 2% Asian, and 1% Native American. We do not yet know of any differences in 
responsiveness to AIH or caffeine according to the gender or race of persons with SCI. 
Children will not be included in this study. The proposed research is aimed at understanding the 
plasticity-inducing mechanisms of caffeine and repetitive acute intermittent hypoxia exposure in 
adults with chronic, incomplete spinal cord injury. A separate study would be required to address 
the confounding effects of childhood growth and development after spinal injury on the proposed 
intervention study – a topic best suited for future research once the underlying mechanisms of the 
intervention are better characterized. 
24. Withdrawal/follow-up 
We will compute the completeness of follow-up as a ratio depicting the proportion of participants 
with a complete follow-up 10MWT time after randomization relative to the number of participants 
enrolled. There will be no statistical testing completed for assessing the adherence to allocated 
treatments. Reasons for lost data will be documented and detailed in the study’s CONSORT flow 
diagram.  
25. Baseline patient characteristics 
VI. Analysis 
26. Outcome definitions 
The primary outcome measure is change in walking speed (10MWT time, s) relative to baseline 
(BL). For all analyses, we will use the average of two 10MWT times at BL, D5, F1, and F2. Change 


Statistical Analysis Plan 
 
9 January 2022 
 
 
13 
in walking speed will correspond to the difference between post-intervention 10MWT and BL 
times. Please review Table 1 for timing of outcome assessments.  
All the summaries in this section will be equivalent to the main and the final results reports. There 
will be statistical testing completed for baseline testing of the primary outcome measure 
(10MWT). Since we plan to use a cross-over study design, we will compare the 10MWT baseline 
measurements between the interventions to evaluate the effects of the washout period.  
Assessment and collection plans: Physical evaluations will be completed by the study’s licensed 
physical therapists or occupational therapists and/or trained research study staff. Functional ability 
of subjects will be evaluated using standard clinical assessments with high inter-rater reliability: 
American Spinal Injury Association Impairment Scale (AIS) lower extremity motor scores 
(LEMS) to quantify strength,28 and Spinal Cord Assessment Tool for Spastic Reflexes (SCATS), 
and the Penn Spasm Frequency Scale (PSFS) which is a self-report assessment of the frequency 
and severity of muscle spasms29 and modified Ashworth Scale to quantify spasticity.30 We also 
will record pain severity using the well-established Wong-Baker FACES scale, which shows high 
test-retest reliability and interpretability as well as good content and construct validity.31, 32 Clinical 
ratings of walking ability, including Walking Index for Spinal Cord Injury (WISCI) II 33 and/or 
SCI Functional Ambulation Index (SCI-FAI),34 will be assessed based on video recordings of the 
10MWT.  
We will quantify secondary measures of walking function using the timed up-and-go test (TUG, 
walking initiation and balance), six-minute walk test (6MWT, walking endurance). These tests 
capture functional ambulation as well as the ability to initiate walking, exhibit high reliability and 
construct validity.35 Participants will perform 2 trials each of the TUG at their fastest but 
comfortable and safe speed with a minimum of 1-minute rest between trials. Average speed across 
the 2 TUG trials will be used for analyses. Subjects will perform the 6MWT once at their fastest, 
comfortable walking speed sustainable for 6 minutes. Distances will be recorded at 2 and 6 
minutes. Participants will use their least restrictive assistive device during these walking 
assessments. 
27. Analysis methods 
We plan to analyze within-participant pre-post changes in walking speed (10MWT) for each 
intervention. We plan to use a parametric linear mixed model for comparing our outcome measure 
since this method is sensitive to within subject changes and robust against variability, small sample 
sizes, and missing/unbalanced data sets as compared to other statistical models.26 If the results do 
not meet these model assumptions, we will apply alternative non-parametric tests.  
All data will be managed using REDCap36, MS 365 Excel 16.54 (Microsoft, USA), and MATLAB 
R2020a (Mathworks Inc, USA) and will be analyzed using SPSS® Statistics 26 (64-bit edition, 
IBM, USA). Descriptive statistics, including means, standard deviations and 95% confidence 
intervals of the within-participant pre-post change for each intervention will calculated for all 
measures. The baseline characteristics of the participants will be compared using parametric and 
non-parametric inferencing. 
Hypothesis testing: To evaluate our main hypothesis that caffeine prior to AIH enhances walking 
recovery, we will perform a linear mixed model with fixed effects at a significance at the p=0.05 
level.37 Intervention (caffeine+dAIH, caffeine+SHAM, placebo+dAIH) and time (day) will be the 
fixed main effects, with subject as random effect and walking speed (10MWT) as repeat measures. 
Differences from BL will be compared between and within interventions at D5, F1, and F2. 


Statistical Analysis Plan 
 
9 January 2022 
 
 
14 
Bonferroni corrections will be made for multiple comparisons.  
Alternative statistical methods: If distributions are not normal, we will perform comparable 
nonparametric tests. If baseline measures are significantly different between sessions 
(interventions), an analysis of co-variance (ANCOVA) will be used to analyze the data.  
Blinding integrity analysis: we plan to rigorously quantify blinding integrity here. We will ask 
participants and raters to guess the intervention received at the end of each intervention and 
indicate guess confidence using a Likert scale.38, 39 We will use a contingency table and Fischer’s 
Exact Test to determine if the probability of correct guessing is different from chance. Using 
multivariate logistic regressions, we also will assess factors that may influence guessing (i.e., 
adverse events, perceived effects, and sensorimotor changes). In the event of a medical emergency, 
we will unblind the PI, study participants, and care provider. We also will notify IRB of treatment 
allocation during adverse event reporting as necessary. 
CYP1A2 analysis: we will perform subgroup analysis on our study cohort to determine the extent 
to which differences in caffeine metabolism due to CYP1A2 polymorphisms may affect 
responsiveness to the combinatorial treatment of caffeine+AIH.  
28. Missing data 
Rigorous efforts will be made to ensure complete outcome data. However, we anticipate that 
participants in this study cohort may be unable to complete the study requirements. Missing data 
will be reported and documented as we did in our previous clinical trial.40 We will document 
reasons for missing data and our approach to handle missing data during statistical analysis. We 
will account for potential outliers in our results using a conservative outlier labeling technique set 
forth by Hoaglin and Tukey.41  
29. Additional analyses 
We do not anticipate any additional statistical analyses methods.  
30. Harms 
Checklists and note pages are used to log any deviations or omissions from the protocols. Any 
clinical/health related issues would be immediately presented to the subject to determine 
appropriate notification (i.e., current physician or appropriate specialist). Based on the seriousness 
of the situation Drs. Slocum or Zafonte may be contacted to provide clinical guidance on the 
appropriate course of action as per our Laboratory Medical Emergency Safety Plan. 
31. Statistical software 
All data will be managed using REDCap36, MS 365 Excel 16.54 (Microsoft, USA), and MATLAB 
R2020a (Mathworks Inc, USA) and will be analyzed using SPSS® Statistics 26 (64-bit edition, 
IBM, USA). 
32. Links to additional supporting documentation 
Link to Data Management Plan 
Link to the Trial Master File and Statistical Master File 
Link to other standard operating procedures  
 


Statistical Analysis Plan 
 
9 January 2022 
 
 
15 
33. References 
 
1. 
Navarrete-Opazo A, Alcayaga J, Sepulveda O, Rojas E, Astudillo C. Repetitive Intermittent 
Hypoxia and Locomotor Training Enhances Walking Function in Incomplete Spinal Cord Injury Subjects: 
A Randomized, Triple-Blind, Placebo-Controlled Clinical Trial. J Neurotrauma. May 1 2017;34(9):1803-
1812. doi:10.1089/neu.2016.4478 
2. 
Ditunno PL, Patrick M, Stineman M, Ditunno JF. Who wants to walk? Preferences for recovery 
after SCI: a longitudinal and cross-sectional study. Spinal Cord. Jul 2008;46(7):500-6. 
doi:10.1038/sj.sc.3102172 
3. 
Goshgarian HG. Plasticity in respiratory motor control - Invited review: The crossed phrenic 
phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury. Journal of 
Applied Physiology. Feb 2003;94(2):795-810.  
4. 
Kaegi S, Schwab ME, Dietz V, Fouad K. Electromyographic activity associated with spontaneous 
functional recovery after spinal cord injury in rats. Research Support, Non-U.S. Gov't. The European 
journal of neuroscience. Jul 2002;16(2):249-58.  
5. 
Raineteau O, Schwab ME. Plasticity of motor systems after incomplete spinal cord injury. Nature 
Reviews Neuroscience. Apr 2001;2(4):263-273. doi:10.1038/35067570 
6. 
Baker-Herman TL, Fuller DD, Bavis RW, et al. BDNF is necessary and sufficient for spinal 
respiratory 
plasticity 
following 
intermittent 
hypoxia. 
Nat 
Neurosci. 
Jan 
2004;7(1):48-55. 
doi:10.1038/nn1166 
7. 
Fuller DD, Johnson SM, Olson EB, Jr., Mitchell GS. Synaptic pathways to phrenic motoneurons 
are enhanced by chronic intermittent hypoxia after cervical spinal cord injury. J Neurosci. Apr 1 
2003;23(7):2993-3000.  
8. 
Mahamed S, Mitchell GS. Is there a link between intermittent hypoxia-induced respiratory 
plasticity 
and 
obstructive 
sleep 
apnoea? 
Exp 
Physiol. 
Jan 
2007;92(1):27-37. 
doi:10.1113/expphysiol.2006.033720 
9. 
Lovett-Barr MR, Satriotomo I, Muir GD, et al. Repetitive intermittent hypoxia induces respiratory 
and somatic motor recovery after chronic cervical spinal injury. J Neurosci. Mar 14 2012;32(11):3591-
600. doi:10.1523/JNEUROSCI.2908-11.2012 
10. 
Wilkerson JE, Mitchell GS. Daily intermittent hypoxia augments spinal BDNF levels, ERK 
phosphorylation and respiratory long-term facilitation. Exp Neurol. May 2009;217(1):116-23. 
doi:10.1016/j.expneurol.2009.01.017 
11. 
Satriotomo I, Date E, Mitchel G. Thrice weekly intermittent hypoxia increases expression of key 
proteins necessary for phrenic long-term facilitation: a possible mechanism of respiratory metaplasticity? 
FASEB Journal. 2007; 
12. 
Satriotomo I, Vinit S, Flom A, Mitchel G. Repetitive Acute Intermittent Hypoxia Increases BDNF 
and TrkB Expression in Respiratory Motor Neurons: Dose Effects. FASEB Journal. 2011;(In Press) 
13. 
Barr M, Sibigtroth C, Mitchel G. Daily acute intermittent hypoxia improves respiratory function 
in rats with chronic cervical spinal hemisection. Faseb Journal. 2007; 
14. 
Dale-Nagle EA, Hoffman MS, MacFarlane PM, Mitchell GS. Multiple pathways to long-lasting 
phrenic motor facilitation. Adv Exp Med Biol. 2010;669:225-230.  
15. 
Golder FJ, Mitchell GS. Spinal Synaptic Enhancement with Acute Intermittent Hypoxia Improves 
Respiratory Function after Chronic Cervical Spinal Cord Injury. The Journal of Neuroscience. March 16, 
2005 2005;25(11):2925-2932. doi:10.1523/jneurosci.0148-05.2005 
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N=193 assessed for eligibility
January 2019 to December 2020
N=4
assigned to 
Placebo+tAIH
N=4
assigned to 
Caffeine+tAIH 
N=4
assigned to 
Caffeine+SHAM
N=34 Participants consented 
and block randomized
N=3
assigned to 
Caffeine+tAIH
N=4
assigned to 
Placebo+tAIH 
N=4
assigned to 
Caffeine+SHAM 
N=4
assigned to 
Caffeine+tAIH 
N=2
assigned to 
Caffeine+SHAM 
N=4
assigned to 
Placebo+tAIH 
Round 1
Round 2
Round 3
N=1 lost after Round 1 (S09)
• Due to change in availability (N=0)
• Due to COVID-19 pandemic (N=1)
N=22 lost during allocation
• No longer eligible due to change 
in medical status (N=8)
• Declined due to relocation (N=1) 
• Due to COVID-19 pandemic and 
commitment challenges (N=13)
Analysis
3 follow-ups (D5, D12, D19)
Enrollment
3 follow-ups (D5, D12, D19)
3 follow-ups (D5, D12, D19)
Allocation
N=169 lost during enrollment
• Due to unmet inclusion or exclusion criteria 
(N=169)
Supplemental Figure. Flowchart of prospective participant enrollment
N=1 lost after Round 2 (S10)
• Due to change in availability (N=1)
• Due to COVID-19 pandemic (N=0)
N=10 completed study
No serious adverse events
N=2 rounds lost during analysis 
S08, Round 2 removed from analysis due to 
baseline outlier
S09, Round 1 removed from analysis due to 
participant missing Rounds 2 and 3
N=5 follow-up days missing prior to analysis
S03, missing D19 during Round 2
S10, missing D5 during Round 2 
S12, missing D19 during Round 1, missing D5 
during Round 2, missed D12 during Round 3 
N=0 lost after Round 3
• Due to change in availability (N=0)
• Due to COVID-19 pandemic (N=0)


Table 1. Timing Schedule for enrollment, interventions, and assessments 
Enrollment 
Baseline 
Intervention 
Follow-ups/Washout 
Timepoints (days) 
-5
0 
1 
2 
3 
4 
5 
8 
14 
Screening and Allocation Timing 
Eligibility screen 
x 
Informed consent 
x 
Medical clearance 
x 
Allocation 
x 
Drug script 
x 
Intervention Timing 
Placebo+AIH 
x 
x 
x 
x 
x 
Caffeine+AIH 
x 
x 
x 
x 
x 
Caffeine+SHAM 
x 
x 
x 
x 
x 
Assessment Timing 
Primary outcome 
•
Walking speed
(10MWT, s)
x 
x 
x 
x 
Other outcomes 
•
Voluntary leg strength
x 
x 
x 
x 
•
Spasticity
x 
x 
x 
x 
•
Spasm frequency
x 
x 
x 
x 
•
Walking function
x 
x 
x 
x 
•
Walking distance
(6MWT, m)
x 
x 
x 
x 
•
Walking Balance
(TUG, s)
x 
x 
x 
x 
•
Blinding Questionnaire
x 
x 
x 
x 
x 
•
Weight (kg)
x 
x 
x 
Safety Outcomes 
•
Pain (0-10)
x 
x 
x 
x 
x 
x 
x 
x 
•
Blood pressure
(mmHg)
x 
x 
x 
x 
x 
x 
x 
x 
•
Heart rate (BPM)
x 
x 
x 
x 
x 
x 
x 
x 
•
Oxygen saturation (%)
x 
x 
x 
x 
x 
x 
x 
x 
•
Memory (0-30)
x 
x 
x 
x 
x 
x 
x 
x 
Biomarkers 
•
CYP1A2
Polymorphism
x 
•
Caffeine serum level
(mg/L)
x 
x 
•
Inflammation (CRP)
x 
x 


Supplemental Table. Physiological markers of caffeine dosage, absorption, and metabolism during first treatment day 
ID 
Body 
Weight (kg) 
Caffeine 
Dosage (mg) 
Placebo+AIH 
[Caffeine, mg/L] 
 Pre 
 Post 
Caffeine+AIH 
[Caffeine, mg/L] 
 Pre 
 Post 
Caffeine+SHAM 
[Caffeine, mg/L] 
 Pre 
 Post 
CYP1A2 
Genotype 
1 
81 
300 
1.4 
1.1 
2.8 
8.3 
2.5 
6.8 
CA 
2 
77 
300 
0.0 
0.0 
2.5 
9.8 
2.8 
8.5 
CA 
3 
45 
200 
3.6 
2.9 
1.0 
9.6 
5.3 
12.0 
AA 
4 
83 
300 
-
- 
0.0
6.4 
0.0 
7.6 
CA 
5 
58 
200 
-
- 
0.0
5.2 
0.0 
7.9 
CA 
6 
102 
400 
1.0 
3.4 
2.8 
12.6 
2.8 
7.0 
CA 
7 
90 
300 
0.0 
0.0 
0.0 
6.0 
0.0 
5.7 
AA 
8 
93 
400 
0.0 
0.0 
0.0 
7.0 
1.2 
6.0 
CA 
9 
86 
- 
- 
- 
- 
- 
- 
- 
- 
10 
68 
- 
- 
- 
- 
- 
- 
- 
- 
11 
73 
300 
0.0 
0.0 
0.0 
5.7 
0.0 
4.7 
CA 
12 
74 
300 
0.0 
0.0 
0.0 
5.6 
0.0 
5.8 
AA 
Mean±SD 
0.7±12 
0.8±1.4 
0.9±1.3 
7.6±2.4* 
1.4±1.7* 
7.1±2.0 
CYP1A2: cytochrome P450 family 1, subfamily A, member 2; SNP: Single nucleotide polymorphism; CA: CYP1A2 
heterozygous, CA alleles carrier; AA: homozygous, CYP1A2 AA alleles carrier. *Significant difference in serum caffeine 
concentration between post-treatment of Caffeine and Placebo at the Bonferroni-corrected, p=0.05 level.