The Role of Hyperoxia in the Emergency Department Treatment of Acute Ischemic Stroke
Phase: NA
Conditions: Stroke, Acute
Sponsor: University of Colorado, Denver

The Role of Hyperoxia in the Emergency Department Treatment of Acute Ischemic Stroke
Principal Investigator – Layne Dylla
Study Protocol and Statistical Analysis Plan
NCT#03904017
Document Date 11/29/2021

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The Role of Hyperoxia in the Emergency Department Treatment of Acute Ischemic Stroke 
Principal Investigator – Layne Dylla 
 
 
1. PURPOSE OF STUDY  
Every year in the United States alone, approximately 795,000 people will experience a new 
or recurrent stroke, with the vast majority of these being an acute ischemic stroke (AIS)1. For 
every minute that a stroke goes untreated, 1.9 million neurons die, underscoring the 
importance of early identification and treatment2. Additionally, it is estimated that half of all 
stroke patients enter the healthcare system via Emergency Medical Services (EMS)3. As 
such, evaluating potential interventions in the prehospital and emergency department (ED) 
phases of care is a key, yet often ignored, component of stroke treatment. Administration of 
15L/min oxygen via a partial non-rebreather facemask to create systemic hyperoxia is a 
relatively low cost, potentially beneficial part of stroke care that could be implemented early 
in the prehospital and ED phases of care. However, this therapy has had conflicting results, 
with little data evaluating its potential benefits early after stroke onset, in the ED and 
prehospital phases of care, where most preclinical data suggests a potential benefit. The 
central hypothesis of this study is that hyperoxia during the acute ischemic period of a stroke 
prior to potential recanalization decreases infarct volume and improves patient outcomes. 
This clinical trial will evaluate the potential therapeutic role of hyperoxia when applied in the 
immediate ischemic period following a stroke in the controlled ED setting and will evaluate 
the effects of hyperoxia in stroke patients on the production of markers of free radical 
damage and inflammatory markers associated with hyperoxic lung injury.  
 
2. BACKGROUND AND RATIONALE  
Hyperoxia in stroke – therapeutic mechanism and biomarkers: Ischemic strokes are 
characterized by an infarct core comprised of cells having suffered irreversible damage in 
the setting of hypoxia that will progress to complete necrosis. This is surrounded by a 
“penumbra” of brain tissue, which is not yet irreversibly destined for cell death4. This 
penumbral zone contains cells whose metabolic and structural integrity have been 
compromised, but the damage is not irreversible should the cells be provided sufficient 
oxygen and metabolic substrates in a timely fashion. After the initial ischemic event, the 
penumbral tissue immediately surrounding the infarct core undergoes depolarization and 
subsequently increases its metabolic rate. Coupled with compromised cerebral blood flow 
and low oxygen supply, this creates a hypoxic environment that perpetuates tissue 
destruction and infarct size5,6. Tissue damage resulting from this hypoxia is mediated in part 
by a shift to anaerobic metabolism which in turn leads to production of excess amounts of 
oxygen and nitrogen free radicals and ultimately lipid peroxidation, protein denaturation, 
DNA damage and activation of apoptotic pathways resulting in cell death7,8.  
 
The utility of biomarkers to lend insight into the molecular pathophysiology of stroke and 
to act as potential biomarkers of diagnosis, hemorrhagic conversion, and ultimately stroke 
severity remains an area of active research. In stroke, it is difficult to directly measure the 
production of reactive oxygen species (ROS) given their short half-life within brain tissue 
itself, let alone in peripheral serum. Previous groups have shown that increased production 
of downstream products of free radical-induced lipid peroxidation leads to neurologic 
deterioration9. MMP-9 is one matrix metalloproteinase that is activated downstream of these 
oxidative stress pathways and contributes to destruction of the blood brain barrier and 
increased cerebral edema after stroke. MMP-9 also correlates with infarct volume, clinical 
stroke severity, and hemorrhagic transformation after thrombolysis10,11. IL-6 is an 
inflammatory cytokine that is increased during and after AIS in both astrocytes and microglia 

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and in peripheral leukocytes. Increased 
peripheral concentration of IL-6 in stroke 
correlates with stroke severity and the 
development of post-infarction infections12,13. 
One potential mechanism of hyperoxia therapy 
based on data in rodent models suggests that 
the provision of increased levels of PaO2 will 
raise the amount of oxygen available to the 
ischemic tissues and prevent further 
progression down this pathway toward cell 
death (Figure 1). 
 
Hyperoxia in preclinical models: In rodent models of middle cerebral artery occlusion 
(MCAO), hyperoxia supplied shortly after the initiation of ischemia results in return of 
penumbral brain tissue oxygenation levels to near pre-ischemic levels and increases 
cerebral blood flow to ischemic tissues14-19. Furthermore, there is no increase in production 
of markers of ROS. In other rodent models, hyperoxia even blunted the production of matrix 
metalloproteinase-9 (MMP-9) and subsequently increased levels of two tight junction 
proteins involved in maintaining the blood brain barrier20,21. Rodent models also correlated 
this decrease in ROS with decreases in markers of cell apoptosis14,22. However, in gerbil 
brain ischemia models, hyperoxia was shown to increase ROS and lipid peroxidation23. This 
potential mechanism for adverse effects of hyperoxia has not been evaluated in humans. 
We will examine the effects of hyperoxia in AIS on these biomarkers (IL-6 and MMP-9) and 
others. 
While the above beneficial effects of hyperoxia were observed in rodents using a 
transient MCAO model to carefully control for the potential negative effects of oxygen during 
reperfusion, using a permanent middle cerebral artery occlusion (pMCAO) model also saw 
beneficial effects of hyperoxia24. This model even showed an increase in systemic vascular 
resistance similar to prior observations in humans. However, despite the increase in 
systemic vascular resistance, hyperoxia still showed improved outcomes compared to 
controls and did not show a significant difference in cerebral blood flow to the contralateral 
hemisphere. 
Furthermore, the window in which hyperoxia is supplied is a key determinant of the 
potential therapeutic benefit. Mice treated with hyperoxia during the acute ischemic phase of 
MCAO showed improved outcomes including increased partial pressure of oxygen in the 
penumbral tissue, improved function on neurobehavioral testing, and lower levels of 
necrosis25,26. Importantly, these benefits were lost when hyperoxia was extended into the 
period of reperfusion where hyperoxia was associated with increased ischemic tissue 
damage and cerebral edema27.  
 
Hyperoxia in humans: A pilot study in which patients were treated with 45L/min humidified 
oxygen via facemask started within 12 hours of their last known well (LKW) and continued 
for 8 hours demonstrated reduced markers of anaerobic metabolism and increased markers 
of neuronal integrity and mitochondrial function in the hyperoxia treated group28. Hyperoxia 
was also associated with an initial improvement in neurological function (measured by the 
NIHSS) and a significant difference in penumbral salvage at 4 hours (~70% in controls vs 
120% in hyperoxia)29. However, the difference did not meet significance at 24 hours  When 
hyperoxia was applied for even longer periods of time there was also no significant 
neurological improvement30. Thus, like in the preclinical models, the duration of the 
hyperoxia may influence outcomes. Additionally, the dose of “hyperoxia” likely affects the 
potential therapeutic benefit. In patients treated with “hyperoxia” in the form of 3L nasal 
Cerebral blood flow
Cerebral O2
Depolarization
Metabolic Rate – shift to anaerobic metabolism 
Lipid peroxidation, DNA damage, denaturation proteins
Activation of cell death pathways
Disruption blood-brain barrier
MMP-9
Neuroinflammation
IL-6
Infarct size
Disability
Depolarization
Metabolic Rate – shift to anaerobic metabolism 
Lipid peroxidation, DNA damage, denaturation proteins
Activation cell death pathways
Disruption blood-brain barrier
MMP-9
Neuroinflammation
IL-6
Infarct size
Disability
A
B
Figure 1: Proposed mechanism of hypoxic injury in AIS (A) and the mitigation of these effects with 
hyperoxia (B).
Cerebral blood flow
Cerebral O2

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cannula for the first 24 hours after admission, there was no difference in neurological 
outcomes31,32.  
As a group, these human studies were relatively small studies with an overall positive 
effect of hyperoxia when given at high levels and started relatively early after AIS onset. 
However, when initiated at lower supplemental oxygen levels and/or treated further from the 
onset of ischemia, there was no significant improvement in patient outcomes nor evidence 
of harm. Hyperoxia therapy is most likely to benefit patients in the early stages after initial 
ischemia by limiting progression to final infarct volume while awaiting spontaneous 
recanalization. This is supported by the most promising studies which used 45L/min oxygen 
therapy for 8 hours in patients presenting within 12 hours from LKW. Thus, we have 
designed our study to focus on early administration, prior to potential reperfusion, of high 
levels of supplemental oxygen, targeting a PaO2 of 200-400mmHg as in the rodent models.  
 
Hyperoxia in other diseases – safety concerns: The role of hyperoxia in stroke remains 
surrounded by controversy based on data from other disease states. Initial studies on the 
role of hyperoxia in myocardial infarction (MI) showed a reduction in the degree of 
myocardial necrosis33-35. However, further studies demonstrated potential harm through 
decreases in coronary blood flow, myocardial oxygen consumption and cardiac output, as 
well as through increases in systemic vascular resistance and myocardial reperfusion 
injury36-39.  
 
Looking at a broader population of cardiac patients, a meta-analysis of patients with 
PaO2 ranging from 234-604mmHg who presented with heart failure, coronary artery disease 
(CAD), recent coronary artery bypass grafting (CABG) surgery, or sepsis found that 
compared to controls, hyperoxia overall resulted in decreased heart rate, decreased cardiac 
output and increased systemic vascular resistance40. However, these changes were not 
consistent amongst all disease entities. CAD and post-CABG patients showed decreased 
heart rates, but this was not seen in heart failure or in sepsis patients. Cardiac output was 
decreased in CAD and heart failure patients but not in post-CABG nor in septic patients. 
Thus, while there continue to be concerns regarding potential harmful hemodynamic and 
cardiovascular effects of hyperoxia, these risks appear somewhat disease-state specific. At 
best, in states with a baseline impaired cardiac function, hyperoxia has no overall benefit, 
with the possibility for negative hemodynamic consequences. 
Hyperoxia is associated with cerebral vasoconstriction in healthy subjects. Thus, it is 
also important to evaluate hyperoxia in the context of other brain injury states in which this 
potential vasoconstriction could be detrimental, such as traumatic brain injury (TBI). In TBI, 
hyperoxia provided in the first 48 hours increases the partial pressure of oxygen in brain 
tissue and reduces brain tissue lactate levels, corresponding to an improved prognosis41. In 
a clinical trial of severe TBI patients, hyperoxia resulted in: 1) improved cerebral metabolic 
rates of oxygen, 2) decreased lactate levels, 3) increased brain tissue oxygen levels, and 4) 
no increase in markers of hyperoxic lung injury (as measured by surrogate lung 
inflammatory biomarkers, IL-6 and IL-8)42. Despite these findings, safety concerns stemming 
largely from the potential for acute oxygen toxicity leading to the accumulation of ROS and 
free radicals leading to lung injury and lipid peroxidation of brain tissue limit the use of 
hyperoxia in TBI. While this remains the leading argument for not using hyperoxia, a 
recently published pilot study examining the use of hyperoxia in severe TBI, again showed 
no difference in the production of ROS43.   
The most commonly proposed mechanism of potential harm from hyperoxia therapy is 
the increased production of ROS that could not only contribute to reperfusion injury, but 
might also lead to hyperoxic lung injury. This issue has also been evaluated in rodent and 
human models of critically ill patients, through induced endotoxemia. Here, brief hyperoxia 
with a PaO2 of ~400mmHg for 3.5 hours in humans resulted in no change in the ability to 

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mount a cytokine and inflammatory response44. Both hyperoxic and control subjects had 
similar patterns of expression of tumor necrosis factor alpha (TNF-), interleukin-6 (IL-6), IL-
8, or IL-10 in response to endotoxemia. Additionally, hyperoxia treatment did not clinically 
alter the leukocyte population produced in response to endotoxemia. Hyperoxic lung injury 
has been demonstrated in patients exposed to hyperoxia for long periods of time but has not 
been evaluated in those with brief hyperoxia exposures45,46. Furthermore, rodent MCAO 
models suggest that this risk is low in stroke given there was no evidence of such effects 
when therapy was instituted immediately after the ischemic event14-19. Thus, by limiting the 
oxygen therapy in this clinical trial to no more than 4 hours, the study will minimize this 
potential risk.  
 
Potential risks of hyperoxia in stroke: Further safety concerns regarding possible increased 
mortality resulting from hyperoxia therapy were raised in a multicenter retrospective study 
that determined the 28-day mortality in mechanically ventilated intensive care unit (ICU) 
stroke patients (including ischemic stroke, subarachnoid hemorrhage (SAH), and 
intracerebral hemorrhage (ICH)). This study found an increased mortality in stroke patients 
found to be hyperoxic at 24 hours after admission47. These patients were exposed to longer 
durations of hyperoxia than proposed in this current study. Additionally, upon subgroup 
analysis, increased mortality was only seen in the hyperoxic SAH and ICH groups compared 
to normoxic controls, and there was no significant difference in mortality in the AIS patients 
exposed to hyperoxia47. This was further supported by a second study of ICU stroke patients 
being mechanically ventilated that failed to demonstrate an association between 
oxygenation level (PaO2) and mortality48. The safety of hyperoxia in AIS patients was also 
questioned in a recent unpublished clinical trial (NCT00414726) studying hyperoxia, that 
randomized patients to room air or 45L/min via facemask. The trial was terminated early, 
after enrolling only 85 participants, for safety concerns49. However, upon closer blinded 
review of the results, only one death in each group was determined to be possibly related to 
the therapy. The remainder (16/43 deaths in the hyperoxia group, 6/41 deaths in normoxia 
group) were determined to be unrelated to therapy. Taken together, these data suggest that 
the safety of hyperoxia therapy is disease-specific and short exposures to hyperoxia prior to 
recanalization in AIS patients are safe. However, the beneficial immediate and long-term 
effects of brief hyperoxia in AIS are not fully understood. 
The results of the proposed study have important implications for the acute treatment of 
stroke. Oxygen therapy is a simple, low cost treatment that could be instituted at the point of 
first contact with the healthcare system for many patients, in the prehospital and ED phases 
of stroke care. While the European PROOF trial of hyperoxia for acute stroke recently began 
enrollment, it randomizes patients to receive 40L/min supplemental oxygen, a level that is 
not currently feasible in the prehospital setting and that lacks data to suggest it is necessary 
for a beneficial effect. Additionally, this therapy is to begin within four hours of subject’s non-
contrast CT-head and extends further into the period of potential reperfusion. Thus, 
understanding the effects of hyperoxia provided at more feasible and tolerable levels and 
proximate to the onset of symptoms has the potential to impact many patients who are 
otherwise lacking potential therapeutic options, some of whom are already being exposed to 
oxygen therapy in the prehospital setting. The proposed project will inform the design of a 
future clinical trial of hyperoxia and will provide the needed experience and preliminary data 
to develop a career in clinical emergency medicine research emphasizing the treatment of 
acute stroke. 
 
 
3. ADMINISTRATIVE ORGANIZATION   
This study will be conducted at the University of Colorado Hospital (UCH) Anschutz Medical 

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Campus and the UCH-Memorial Central Hospital. Subjects will be recruited, enrolled, and 
therapy initiated upon presentation to the Emergency Department. Arterial Blood Gas (ABG) 
samples will be drawn and immediately processed per their standard protocol for routine 
ABG testing. Venous serum samples will also be collected and processed for storage in the 
Emergency Medicine Research Lab or the clinical labs of the UCH-Memorial and 
subsequently transferred to their research storage. All serum biomarker analyses will occur 
in aggregate at the end of subject recruitment.  
 
4. STUDY DESIGN 
 
This is a multicenter, prospective, randomized, controlled, feasibility study to evaluate the 
feasibility of early brief hyperoxia therapy for AIS in the ED and to gather preliminary data on 
the potential therapeutic and adverse effects of such intervention. It is hypothesized that 
global hyperoxia treatment in the acute ischemic period of a stroke prior to potential 
recanalization will decrease infarct volumes and improve neurologic outcomes in AIS 
patients without increasing the production of markers correlating with ischemic-reperfusion 
injury and the hyperoxic lung injury. To test this hypothesis, subjects will be randomized 
equally between the placebo (medical air) and hyperoxia (supplemental oxygen) treatment 
groups.  
 
For the purpose of this study, we define normoxia as a pulse oximetry of 94% or greater, 
although per AHA guidelines, supplemental oxygen is recommended only in patients with a 
pulse oximetry of less than 94%50. Based on rodent models that targeted systemic hyperoxia 
with a PaO2 of 200-400mmHg, hyperoxia will be defined as a PaO2 of >200mmHg. 
Previous literature demonstrated that 10-15L/min facemask resulted in the PaO2 of 425 +/- 
43mmHg in patients undergoing cardiac catheterization found to have clean coronary 
arteries and 403 +/- 49mmHg in those with CAD51. Thus, treatment with 15L/min should be 
sufficient to achieve this level of hyperoxia in this study population, and will be confirmed 
through ABG sampling at 4 hours after initiation of treatment intervention.  
 
Patients who are found to require supplemental oxygen according to AHA guidelines will be 
provided such intervention with careful documentation of the timing, duration, and dose of 
supplemental oxygen provided. 
 
The design will proceed as follows: (see Figure 2) 
▪ Randomize subjects between placebo and supplemental oxygen via partial non-
rebreather facemask at 15 L/min. 
▪ Collect study measurements 
▪ Perform 3-month follow up for mRS 
▪ The study duration is estimated at 30 
months to allow sufficient time for 
subjects enrollment and completion of 
their 3 month follow-up 
 
In order to minimize bias, efforts will be made 
to blind the observers to the control versus 
treatment group. However, medical air and 
oxygen have different ED wall supply 
connectors that could be potentially identified 
by both the patient and the study personnel. Thus, full blinding cannot be guaranteed. To 
minimize bias, ED staff will initiate a given intervention and they will be instructed to not tell 
the patient which was started on the patient. To avoid bias with regards to infarct lesion size, 
Screen potential participants
Initial CT-perfusion
Initial vital signs (heart rate (HR), blood pressure (BP), respiratory rate (RR), pulse 
oximetry (SpO2))
Consent for enrollment
Perform baseline assessments (initial NIHSS, serum sample #1)
Hyperoxia
Serum Sample #2
ABG
NIHSS #2
Repeat vitals 
Serum Sample #3
NIHSS #3
Final Infarct volume (MRI or CT-perfusion)
Repeat vitals
Final Assesment: mRS
T1
Randomize
T2  (T1 + 4hours)
T3 (T1+24hours)
T4 (T1+ 3months)
Figure 2: Schematic of study design and outcome measurements
Medical Air

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the automated RAPID software will be used to perform lesion measurements and screening 
for enrollment based on target mismatch. The CT-Perfusion and automated CT-ASPECT 
(Alberta Stroke Program Early Computed Tomography Score) have been previously used to 
identify patients who are early in their acute stroke trajectory with a small core infarct volume 
and remaining area suggestive of salvageable tissue52. Both are standardized quantitative or 
semi-quantitative measurements that can be automatically computed by RAPID software, 
thereby eliminating the subject assessments of providers to identify patients with potentially 
salvageable penumbral tissue. The assessment of the NIHSS will be extracted from the 
initial stroke team consult note and subsequent assessments will be video-recorded by 
study personnel. Videos of the assessment will be stored using a unique file name and 
stored in the secure School of Medicine Server. They will be reviewed in groups in a blinded 
fashion for scoring. At the conclusion of the study and required time for maintaining study 
data, all videos will be destroyed. In the case where videotaping is not permitted by a 
performance site, NIHSS-trained personnel (RN, MD, or PA) who are caring for the patient, 
will be ask to complete and score the NIHSS. In the event that the clinical team is unable to 
complete this assessment, the research team, also NIHSS-trained, will complete this 
assessment. In all cases, who/how the NIHSS was completed will be documented and 
blinding to the study intervention will be attempted when possible. 
 
The primary study outcome is to determine the feasibility of enrolling and randomizing AIS 
patients to a therapeutic intervention within less than one hour of ED presentation. 
Secondary outcomes of potential therapeutic effect to inform future clinical trial(s) include 
penumbral salvage at repeat imaging, change in NIHSS over the first 24 hours, and 3-month 
mRS. To assess potential adverse effects on markers of worsening stroke severity and 
possible hyperoxic lung injury, levels of MMP-9, IL-6, and IL-8 will be measured at initiation, 
4 hours and 24 hours. Further analysis of additional biomarkers will be considered in the 
future. 
 
 
4.1. SUBJECT POPULATION 
Eligible subjects will include acute ischemic stroke (AIS) patients aged 18 years or 
older who present to the UCH ED. There will be no specific selection of subjects based 
on sex, race or ethnicity, age, or socioeconomic status. We anticipate the study 
population to be representative of that of the greater Colorado Springs and Aurora, CO 
area with the exception that stroke prevalence increases with age and this will likely be 
reflected in the final study population.  
 
Subjects with a suspected stroke who are normoxic at baseline (room air SpO2≥94%) 
and have either 1) a target mismatch lesion on CT-perfusion [defined by RAPID 
automated software demonstrating a ratio of PWITmax>6 lesion volume/Infarct volume 
(with cerebral blood flow <30% normal) greater than 1.2 or 2) a non-contrast CT-head 
RAPID ASPECT score of 10 (suggesting a patient has not completed their infarct), a 
NIHSS≥1, and a LKW ≤12 hours will be screened for eligibility. While some 
vulnerable populations (i.e. children and prisoners) will be excluded, AIS by the nature 
of the disease has the potential to impair a subject’s capability to give informed 
consent and places them in the category of a vulnerable population. All subjects will be 
evaluated for the ability to give informed consent through use of the University of 
California, San Diego, Brief Assessment of Capacity to Consent (UABCC) Form53. In 
cases where the subject is determined to lack the ability to provide consent, the 
subject’s legally authorized representative (LAR) will be approached. Consent forms 
describing in appropriate detail the study interventions, study procedures, and risks will 

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be given to the participant and/or their LAR. If and when the study subject regains 
capacity, they will be approached for continued participation. If the subject/LAR 
consents for participation he/she will sign the informed consent form prior to any study 
procedures being done.  
 
In terms of potential pregnant women, it is anticipated that most women will be older 
than likely childbearing age. While supplemental oxygen use in pregnant women 
experiencing fetal distress is routine, the effects of maternal hyperoxia on a developing 
fetus are unknown. Those of likely childbearing potential will be excluded. If a subject 
withdraws for any reason from the study, he/she will be replaced to meet the 
enrollment goal when possible. Our target enrollment will be 200 patients (~100 per 
arm). 
 
 
 
4.2. INCLUSION / EXCLUSION CRITERIA 
Inclusion Criteria: In order to be eligible to participate in this study, an individual must 
meet all of the following criteria: 
1. Provision of signed and dated informed consent form from patient or legal authorized 
representative (LAR) 
2. Subject or LAR stated willingness to comply with all study procedures and availability 
for the duration of the study  
3. Males and female (of unlikely childbearing capacity) aged over 18 years 
4. Exhibiting signs and physical examination findings suggestive of an acute ischemic 
infarction (with either): 
a. A target mismatch profile of CT-perfusion or MRI determined by RAPID 
automated software to have a ratio of PWITmax>6 lesion volume/DWI lesion 
volume >1.2 and NIHSS ≥154  
b. A RAPID automated software calculated non-contrast CT-head ASPECT score of 10 
and LKW ≤12 hours (in patients with symptoms discovered upon waking, the LKW is 
defined at the midpoint between going to sleep and awakening based on previous 
studies that suggest most strokes during sleep occur close to awakening)55 
5. Normoxic; a pulse oximetry of 94% or greater at time of screening without the use of 
supplemental oxygen 
 
Exclusion Criteria: An individual who meets any of the following criteria will be 
excluded from participation in this study: 
1. Current use of supplemental oxygen  
2. Prisoner 
3. Pregnancy or those of likely childbearing potential (i.e. age <50, no documented 
negative pregnancy in last 24 hours, no documented history of hysterectomy) 
4. Documented blood glucose <70mg/dL 
5. Concurrent treatment with another investigational drug or other intervention 
6. Documented history of any of the following chronic respiratory illness that require 
pulmonary vasodilators or supplemental oxygen at baseline: Chronic Obstructive 
Pulmonary Disorder (COPD), Emphysema, Interstitial Lung Disease, Restrictive 
Lung Disease, Pulmonary Hypertension, Pulmonary Fibrosis 
7. Documented history of any of the following autoimmune diseases: systemic lupus 
erythematosus, rheumatoid arthritis, scleroderma, primary biliary cholangitis, multiple 
sclerosis, inflammatory bowel disease 

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8. Currently being treated for an acute myocardial infarction and/or decompensated 
heart failure at the onset of initial ED presentation as reported by the ED provider 
9. Plans for treatment with either IV tPA (alteplase) or endovascular therapy 
 
4.3. STUDY INTERVENTIONS 
Enrolled subjects will be randomized to receive either brief hyperoxia treatment (defined 
as 15L/min supplemental oxygen) or medical air (at 15L/min) via a partial non-rebreather 
mask for four hours from the time of study initiation (T1). Supplemental oxygen is 
routinely used to correct and/or prevent hypoxemia (low blood oxygen content, defined 
as an arterial oxygen content of less than 60-75mmHg). To avoid invasive monitoring of 
arterial oxygen content, pulse oximetry is routinely used to monitor for hypoxia with 
values less than 90-94% considered low in most clinical scenarios. Unfortunately, there 
are limitations with pulse oximetry and based on the oxyhemoglobin dissociation curve 
and a SpO2 of 97% could represent any PaO2 above ~90mmHg. Thus, pulse oximetry 
alone is not an ideal mechanism to determine hyperoxia. Based on mouse models, this 
study targets a PaO2 of 200-400mmHg in the hyperoxia treatment group. In a previous 
study of cardiac catheterization patients, delivery of 10-15L/Min facemask oxygen 
resulted in average PaO2 in the 400s.   
 
In this study, oxygen is being used as a drug. However, an IND exemption is requested. 
We feel that the use of oxygen in this scenario to prevent hypoxemia in the ischemic 
tissue by creating a global hyperoxia environment meets the criteria for an IND 
exemption. Specifically, this protocol will: be conducted in compliance with requirements 
for IRB review and the requirements concerning the promotion and sale of drugs; not be 
in support of a new indication for use or change in labeling or advertising of oxygen; not 
invoke exemption from informed consent; not involve a route of oxygen administration 
or dosage level that significantly increase the risks of the drug product. Additionally, 
there is no consistent predetermined dosage of oxygen that can be universally applied 
to all clinical contexts to determine who needs supplemental oxygen and at what dose. 
This decision is left to the clinical judgement of the practicing medical provider. For 
example, in patients undergoing procedural sedation in the ED, supplemental oxygen 
may be provided despite having a pulse oximetry reading greater than 92-94% to 
prevent desaturation. According to the AHA guidelines for stroke patients, hypoxia that 
they suggest warrants supplemental oxygen use is defined as a pulse oximetry of less 
than 94%, but the data behind this exact recommendation is also very weak50.  
 
Supplemental oxygen wall connections and/or oxygen tanks are standard in every 
hospital room and ambulance. Additional oxygen tanks are available for patients during 
transport as needed. Oxygen will be administered using standard oxygen tubing and a 
partial non-rebreather facemask. It is approved for the treatment and preventions of 
hypoxia and hypoxemia. Hypoxia is often measured non-invasively by a pulse oximetry 
and thresholds to begin supplemental oxygen therapy vary based on disease entity and 
provider discretion. Medical oxygen can be supplied in numerous methods, but for the 
purpose this study it will be supplied at flow rates of 15L/min via a non-rebreather 
facemask. This generally is considered to provide a fraction of inspired oxygen (FiO2) of 
0.45-0.9 in most patients and should result in systemic hyperoxia with a PaO2 
>200mmHg56.  
 
 
5. RECRUITMENT METHODS 

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The target sample population will include adults over the age of 18 years with an acute 
ischemic stroke. The demographics of the sample will reflect that of Denver and Arapahoe 
Counties, the major geographical recruitment area – approximately 49.8% and 50.4% 
female, 54.5% and 59.9% White, 29.7% and 19.5% Hispanic, respectively. There will be no 
specific recruitment strategies for specific demographic populations. However, given that 
older age is associated with increased stroke incidence, it is estimated that most subjects 
will be elderly.  
 
All suspected stroke patients undergoing a rapid stroke evaluation with a CT-perfusion or 
non-contrast CT-Head with RAPID ASPECT score and evaluation by the stroke team at the 
University of Colorado Hospital will be considered for enrollment. Patients will be screened 
upon arrival to the ED by a trained Emergency Department Research Assistant or other 
study personnel using a standard recruitment script. Consent, enrollment, and initiation of 
treatment will be performed by a study personnel. At UCH-Memorial, only the study research 
assistants who are trained in the how to conduct clinical research, including the more 
rigorous consent process associated with research compared to routine clinical care will 
screen, consent, and enroll subjects. The site-PI and site-sub-I will be available for clinical 
questions. These research team members will undergo yearly training provided by the study 
PI to ensure familiarity with the consent process and study activities. Additionally, the study 
PI will review consent documents, screening logs, and subject assessments on a monthly 
basis to ensure compliance and she will be available in real time, by video chat or phone for 
any questions. 
 
During the treatment, the study team will stress the importance of the 3-month follow-up 
phone interview and two potential phone contacts will be gathered if possible. The 
remainder of the study activities will occur while the patient remains in the ED or 
hospitalized. No specific recruitment strategies will be employed for historically 
underrepresented minorities.  
 
This study will not include vulnerable populations with the exception of potentially cognitively 
impaired subjects due the possibility of altered mental status secondary to the ischemic 
injury from their stroke and elderly patients with cognitive impairment from age-associated 
diseases such as dementia. However, in order to protect these subjects, they will only be 
included when a LAR is available for consent. Given the short duration of intervention while 
patients remain hospitalized or in the ED and follow up limited to 3 months, we anticipated 
only a few subjects deemed to have capacity at enrollment will become classified as 
vulnerable populations (i.e. develop cognitive impairment or become incarcerated) during 
the study. However, all subjects will be reevaluated at 4 hours by study personnel for 
possible changes in their capacity to consent.  
 
Given that subjects will be committing to a follow-up phone interview, subjects will be 
provided a $25 gift card for their participation at the T3 time point.  
 
 
6. CONSENT PROCESS 
Upon identification of a subject meeting all inclusion and no exclusion criteria, subjects will 
be approached by study research personnel (either the PI or a Research Assistant) to obtain 
informed consent prior to initiation of any study activities. This will occur while patients are in 
the ED and under the primary care of the ED physician. Given the goal of randomization 
within one-hour of presentation to the ED, care will be taken to not interfere with ongoing 
medical care while still ensuring timely approach for enrollment. Consent forms describing in 

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appropriate detail the study interventions, study procedures, and risks will be given to the 
participant and/or their LAR. Consent forms will be Institutional Review Board (IRB)-
approved and the subject/LAR will be asked to read and review the document. A verbal 
explanation will also be provided in terms suited to the subject’s comprehension of the 
purposes, procedures, and potential risks of the study and of their rights as research 
participants. Participants will have the opportunity to carefully review the written consent 
form and ask questions prior to signing. The participants will have the opportunity to discuss 
the study with their family or surrogates and think about it prior to agreeing to participate. 
The participant/LAR will sign the informed consent document prior to any study procedures 
being done. Subjects/LARs will be informed that participation is voluntary and that they may 
withdraw from the study at any time, without prejudice. A copy of the informed consent 
document will be given to the participants for their records. The informed consent process 
will be conducted and documented in the source document (including the date), and the 
form signed, before the participant undergoes any study-specific procedures. A witness 
signature will be not be included on the consent form. However, a signature of the study 
personnel completing the consent is included and required. The rights and welfare of the 
participants will be protected by emphasizing to them that the quality of their medical care 
will not be adversely affected if they decline to participate in this study. 
 
When the patient’s capacity for informed consent is questioned due to the nature of the 
stroke itself or other mental impairment, capacity will be determined using the UABCC Form. 
This method of determining capacity consists of a 10-item questionnaire that can be 
delivered in 5-10minutes and has been found to have high sensitivity and specificity and 
good interrater reliability53. If a subject is determined to not have capacity for consent, the 
LAR will be identified and approached for consent. If and when the study subject regains 
capacity, they will be approached for continued participation. If the subject/LAR consents for 
participation he/she will sign the informed consent document prior to any procedures being 
done specifically for this study.  
 
After a patient consents for participation in the study, he/she will be randomized to either 
hyperoxia or medical air treatment. Randomization will occur in blocks of 20 using excel and 
subject treatment assignments will be determined prior to initiation of the study and 
enrollment of subjects.  
 
In addition to the traditional paper consent process, the ongoing COVID-19 pandemic has 
necessitated the use of an optional electronic consent (eConsent) process. Bringing a paper 
consent form and pen to the bedside of a patient with known or suspected COVID-19 and 
then taking these out of the room would violate infection control principles and policies. 
Additionally, the current pandemic presents challenges to obtaining consent from the LAR 
who may not be physically present in the ED at the time of consent due to a variety of 
reasons including limited visitation policies and/or the need to self-quarantine. Therefore, an 
eConsent process will also be used. In these cases, if a subject has decisional capacity 
and/or the LAR is physically present in the ED, but there is concern for COVID-19 infection, 
the research team will call the subject on their hospital room phone or personal phone and 
request to email the subject/LAR a copy of the eConsent form. The same process for 
informed consent, including explanation of the consent process and study, and opportunity 
for questions, will occur via telephone. At the end of the conversation, the subject/LAR will 
be asked to complete the eConsent Survey via REDCap. The research team and witness 
signatures will also be documented via REDCap. The same process will be used if the 
subject lacks decisional capacity and the LAR is not physically present in the ED for 
consent.  

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As blood will be collected as part of the study protocol for analysis of serum biomarkers of 
stroke, subjects will also be consented to have any remaining serum maintained in CU 
repository in the Emergency Medicine Research Lab. Blood sample collection for research 
purposes will not be optional for subjects participating in the study, as reflected in the 
consent form. However, use of the blood sample for future research is optional. Blood 
samples will be obtained from consenting subjects and immediately processed for storage in 
freezers with unique sample identifiers. Subjects will not have access to any individual 
results from this study or such future analysis. Future analysis of blood samples markers will 
be subject to COMIRB requirements, and approval for the initiation and continuance of such 
activities. Upon completion of this study, blood values and correlating clinical information will 
be linked and all information will be de-identified. All blood analyses will be completed in 
aggregates, not at the individual subject level.  
 
 
7. STUDY PROCEDURES 
As part of the screening and enrollment process, patients with suspected AIS will have 
either a non-contrast CT head with RAPID calculation of ASPECT score or a CT-perfusion 
head. Using standard stroke alert treatment protocols at this institution, the RAPID software 
is used in most potential stroke patients at our institution and will be used to calculate infarct 
and penumbral volume and targeted diffusion/perfusion mismatch for this study. As part of 
the screening process, medical charts and EMS reports will be accessed by study personnel 
as needed to ensure potential subjects meet all inclusion/exclusion criteria. The screening 
and enrollment procedures along with collection of initial vitals will occur almost 
simultaneously given the very acute nature of the presentation and desired short interval 
from ED presentation to randomization. No data will be collected during the screening 
process that is not part of routine patient care prior to subject enrollment. Routine patient 
demographics, past medical history, medications, and prehospital assessments will be 
obtained from chart review. No research data that is not already part of routine patient care 
will be entered into the subject medical records. Randomization will occur in a 1:1 fashion 
between the two treatment groups. The estimated study duration is 30 months to allow for 
full enrollment of subjects and the 3-month mRS assessment. Each subject will be followed 
for a total of 90 days from enrollment to final assessment. No results from this study will be 
shared with subjects.  
 
Once consented and enrolled in the study, subjects will have the following procedures: (See 
Table 1 for Schedule of Activities). 
1. Infarct progression: 
a. Initial CT-perfusion head or non-contrast CT-Head with RAPID ASPECT score – 
multimodal CT-head imaging will be obtained upon ED presentation according to 
local stroke protocols. Targeted mismatch criteria will be based on previous 
standards identified in the DEFUSE 3 trial, using RAPID generated CT maps 
(RAPID, iSchema View, Menlo Park, CA)57. This study measurement will be 
completed as part of the screening process to ensure eligibility with regards to a 
target mismatch lesion. However, upon completion of the CT-perfusion or non-
contrast CT-Head, the total volume of at-risk tissue (both penumbral and 
infarcted tissue) will also be determined and recorded. This is part of the routine 
medical treatment for acute stroke patients. 
b. Follow-up MRI-head, or a repeat CT-head in patients unable to obtain an MRI, 
will be obtained at roughly T3 according to standard stroke imaging protocols. 
The total volume of infarcted tissue will be determined and recorded. Repeat 

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imaging usually between 24-72 hours is part of the standard of care for most 
stroke patients to ensure there has not been hemorrhagic conversion. Only these 
repeat imaging studies that were routinely obtained for standard patient care will 
be used for further determination of infarct progression. Specific study-dedicated 
imaging will not be obtained.   
c. The ratio of the volume of at-risk tissue (T0 – penumbral and infarcted tissue) to 
the volume of final infarct at subsequent reimaging will be compared between the 
placebo and hyperoxia groups. Volume assessments will be generated from both 
the CT-perfusion or non-contrast CT-Head at screening and the subsequent MRI 
using RAPID (iSchema View). These image files contain PHI, but are de-
identified prior to processing by RAPID under the below process and PHI 
remains within the UCHealth firewall. 
 
2. Analysis of serum markers of free radical ischemic-reperfusion injury and hyperoxic lung 
injury: 
a. Whole blood and serum samples will be collected using routine phlebotomy and 
venipuncture procedures. Serum will be collected in standard blood collection 
tubes, subsequently isolated from whole blood and stored in small aliquots for 
further analysis. All samples will be labeled with unique study identifiers to 
ensure blinded analysis but still allowing for correlation with specific patient 
outcomes. To decrease variability between analysis of serum markers, serum will 
be collected and stored in the ED cold storage facility until samples can be run in 
batches.  
b. Analysis of specific markers (MMP-9, IL-6 and IL-8): Serum levels will be 
determined using the Millipore custom-designed Milliplex multiplex assays in 
accordance with the manufacturer recommended protocol. This assay uses 
bead-based immunodetection of multiple biomarkers simultaneously from 25uL 
of serum in 96-well plates. The technology also contains internal controls to help 
ensure accurate determination of serum levels of biomarkers. All sample analysis 
will be performed on a blinded basis in batches, using a single Luminex xMap 

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core instrument. Further undetermined biomarkers may also be analyzed 
separately and/or in conjunction with these selected biomarkers. This technology 
has the ability to analyze related biological pathways that may be related to 
underlying pathophysiology that is being assessed through the analysis of MMP-
9, IL-6 and IL-8. 
3. Functional Outcomes: 
a. NIHSS score: The NIH Stroke Scale (NIHSS) is a standardized tool to measure 
stroke severity. Study observers will be required to complete the National Stroke 
Association NIHSS certification online course. As part of the training, interrater 
reliability between study observers will be determined. This assessment is part of 
all initial stroke evaluations by ED and/or neurology physicians. Subsequent 
NIHSS assessments at T1 and T2 will be video-recorded by trained study 
personnel and/or EDRAs. However, in the case the videotaping is not permitted 
by a performance site, NIHSS-trained personnel (RN, MD, or PA) who are caring 
for the patient, will be asked to complete and score the NIHSS. In the event that 
the clinical team is unable to complete this assessment, the research team, also 
NIHSS-trained, will complete this assessment. In all cases, who/how the NIHSS 
was completed will be documented and blinding to the study intervention will be 
attempted when possible. The preference will be to have the score determined in 
a blinded manner upon review of the assessments at a subsequent time point.  
b. mRS scoring: The modified Rankin Scale, mRS, is another functional scale that 
assesses for neurologic disability on a scale of 0-6 (0-asymptomatic; 6-death). A 
historical mRS will be extracted from the patient chart and/or calculated at patient 
presentation. Patients and/or their caregivers will be interviewed by phone at 
3months using a series of standardized questions regarding their neurologic 
disability to determine this score. 
4. Arterial puncture and ABG – Hyperoxia (PaO2 >200mmHg) will be confirmed by an ABG 
sample from subjects. These will be obtained by licensed medical personnel trained to 
perform arterial punctures (i.e. nurses, physicians, respiratory therapists). Standard 
procedures for obtaining an arterial blood sample will be employed to reduce the risk of 
complications. To decrease variability between ABG analysis, ABGs will be performed using 
standard operating procedures. The site of the arterial puncture and correlating extremity 
will be evaluated for potential complications (increased pain, discoloration, erythema, 
hematoma formation, bleeding). If there are any concerning findings on these exams, the 
study PI will be notified and the subject will be monitored again at T2 and/or until the event is 
felt to resolve. 
5. Vital signs - The determination of need for additional supplemental oxygen in a patient 
that becomes hypoxic (pulse oximetry<94%) will be determined by the subject’s treating 
physician irrespective of study interventions. Should a patient be found to require additional 
supplemental oxygen while enrolled in the study, this will be noted and the patient will 
continue through the final study assessments. The patient’s treating physician will also be 
responsible for routine monitoring and interpretation of other vital signs such as blood 
pressure and heart rate. If at study assessment time points the study personnel note a 
subject to have significant vital sign abnormalities (SpO2<94%, SBP>180mmHg, 
DBP>110mmHg, SBP<90mmHg, HR>120, HR<50), the study PI and patient’s treating 
physician will be notified to ensure proper documentation. Interventions will be left to the 
discretion of the patient’s treating physician. 
 
 
 
 
 

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Table 1: Schedule of Activities: 
Procedures 
Baseline/Enrollment 
ED presentation -T0 
(ED arrival +/-60min) 
T1 (T0+4hours  
+/- 20min) 
T2 (T0+24hours 
+/- 4hours) 
 T3 - Final 
Assessment 
(T0+3mo 
+/- 2weeks) 
Informed consent 
X 
 
 
 
Demographics 
X 
 
 
 
Medical history 
X 
 
 
 
Randomization 
X 
 
 
 
Administer study intervention 
 
X 
 
 
Vital signs: HR, BP, RR, SpO2 
X 
X 
X 
 
ABG 
 
X 
 
 
CT-perfusion or CT-head with ASPECTS 
X 
 
 
 
Repeat MRI-head (CT-head if unable to get 
MRI)a 
 
 
X 
 
Serum sampleb 
X 
X 
X 
 
NIHSS 
X 
X 
X 
 
mRS 
X 
 
 
X 
Adverse event review and evaluation 
 
X 
X 
 
a only routine re-imaging will be obtained 
b tested for selected biomarkers (IL-6, IL-8, and MMP-9) 
 
Assessment of study intervention adherence: 
1. Compliance with study interventions will be directly observed at the given time points. If 
at any observation period a subject is found not to be compliant with the study intervention, 
medical records will be reviewed to determine the timing of termination of the supplemental 
oxygen/medical air.  
2. All cases of non-compliance with study interventions will be recorded. 
 
 
8. RISKS TO SUBJECTS 
Hyperoxia has been explored in various disease states. While meta-analysis in myocardial 
infarction did not show an increase in mortality, hyperoxia has been shown to have negative 
cardiovascular and hemodynamic effects in cardiac patients. In some, but not all cardiac 
disease states, there were decreases in heart rate and cardiac output and increases in 
systemic vascular resistance40. These potential decreases in cardiac output and increases in 

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systemic vascular resistance could negatively impact stroke patients, especially those with 
perfusion dependent lesions.  
 
The most commonly proposed potential harm from hyperoxia therapy is the increased 
production of reactive oxygen and nitrogen species that not only may contribute to 
reperfusion injury, but may also lead to hyperoxic lung injury. Rodent MCAO models 
suggest that this risk is low given there was no evidence of such effects when therapy was 
instituted immediately after the ischemic event14-19. This issue has also been evaluated in 
rodent models and in human models in critically ill patients. In an endotoxemia model 
(induced by the administration of lipopolysaccharide derived from E.coli), brief hyperoxia 
with a PaO2 of ~400mmHg for 3.5 hours in humans resulted in no change in the ability to 
mount a cytokine and inflammatory response44. Both hyperoxic and control subjects had 
similar patterns of expression of tumor necrosis factor alpha (TNF-), interleukin-6 (IL-6), IL-
8, or IL-10 in response to endotoxemia. Additionally, hyperoxia treatment did not clinically 
alter the leukocyte population produced in response to endotoxemia, nor the ability for 
neutrophils to undergo phagocytosis. Hyperoxic lung injury has been demonstrated in 
patients exposed to hyperoxia for long periods of time but has not been evaluated in those 
with brief hyperoxia exposures45,46. Thus, by limiting the oxygen therapy in this clinical trial to 
no more than 4 hours, the study will minimize this potential risk.  
 
Further safety concerns regarding possible increased mortality resulting from hyperoxia 
therapy were also raised with a multicenter retrospective study that determined the 28-day 
mortality in all mechanically ventilated intensive care unit (ICU) stroke patients (including 
ischemic stroke, subarachnoid hemorrhage (SAH), and intracerebral hemorrhage (ICH)). 
This study found an increased mortality in all stroke patients found to be hyperoxic (defined 
as a 24-hour post-admission PaO2 >300mmHg)47. However, upon subgroup analysis, 
increased mortality was only seen in the hyperoxic SAH and ICH groups compared to 
normoxic controls, and there was no significant difference in mortality in the AIS patients 
exposed to hyperoxia (43% in-hospital death at 28 day in normoxic patients vs 57% in 
hyperoxic patients, p=0.13)47. This was further supported by a second study of ICU stroke 
patients being mechanically ventilated that failed to demonstrate an association between 
oxygenation level (PaO2) and mortality48. The safety of hyperoxia in AIS patient was also 
questioned in a recent unpublished clinical trial (NCT00414726) studying hyperoxia, that 
randomized patients to room air or 45L/min via facemask. The trial was terminated early, 
after enrolling only 85 participants, for safety concerns49. However, upon closer blinded 
review of the results, only one death in each group was determined to be possibly related to 
the therapy. The remainder (16/43 deaths in the hyperoxia group, 6/41 deaths in normoxia 
group) were determined to be unrelated to therapy. However, the exact cause of death was 
not reported. Additionally, there similar rates of cardiac disorders between the hyperoxia vs 
normoxia groups (4/43 vs 5/41) and pulmonary related adverse events (3/43 vs 2/41). As 
such, a similarly designed clinical study in Europe began recently. Taken together, the data 
suggests that the safety of hyperoxia therapy is disease-specific and short exposures to 
hyperoxia in AIS patients is safe. However, the exact immediate and long-term effects of 
brief hyperoxia in AIS are not fully understood. 
 
In this study, the patients will have both a venipuncture for serum samples and an arterial 
blood gas (ABG) analysis performed. While venipuncture for various laboratory analyses is 
part of the standard of care for stroke patients, only a select number of patients would 
normally be exposed to this procedure – usually limited to patients with questionable 
respiratory status. ABGs carry the risk of pain associated with the needle puncture and 
increased risk of bleeding and/or hematoma formation, arterial vasospasm causing distal 

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ischemia that affects the distal circulation in patients without good collateral blood flow, and 
infection. These are significant risks compared to venipuncture. However, these 
complications from an ABG are still rare when performed by trained personnel. Additionally, 
while there are no alternatives to verify arterial oxygen content, the number of ABGs 
performed is limited to a single draw after the institution of hyperoxia treatment to verify 
hyperoxia. 
 
 
10. POTENTIAL BENEFITS TO SUBJECTS 
Preclinical and limited human data suggests that this treatment may improve neurologic 
outcomes in a disease that is a leading cause of world-wide morbidity and mortality. The 
study intervention has the potential to increase cerebral blood flow to the penumbral tissue 
at risk for cell death, decrease the production of free radicals leading to reperfusion injury, 
help maintain the blood-brain barrier, and thereby decrease the final infarction volume and 
improve neurologic outcomes in stroke patients14-17,20-22,26,28,58. Additionally, in a study that 
provided supplemental oxygen via a 40% venturi mask compared to nasal cannula in 
severe stroke patients, there was also a non-significant trend toward lower rates of in-
hospital mortality and co-morbidities such as fever, pneumonia, and respiratory failure58.  
 
By participating in this study, patients will be part of research that furthers the 
understanding of the potential therapeutic role of hyperoxia. Additionally, to date, there are 
limited potential therapeutic interventions for stroke, with few patients being eligible for tPA 
or presenting with a lesion and timeframe that is amenable to endovascular therapy. This 
study will shed light on these potentials for this novel therapy for stroke.  
 
 
11. COSTS FOR PARTICIPATION 
There will be no additional costs to the subject resulting from study activities.  
 
 
12. PAYMENT FOR PARTICIPATION  
While participation in the study is voluntary, subjects will be asked to agree to a follow-up 
telephone interview 3-months after their enrollment. To compensate them for their time, 
subjects completing all study interventions including this 3-month interview will receive a $25 
gift card or check. This will be disbursed after the completion of the interview by mail. If a 
subject is unable to complete the study, they will not be paid for their participation. There will 
be no other forms of compensation and/or reimbursement. 
 
 
13. SUBJECT WITHDRAWALS 
Due to the brief nature of the intervention limited to only 4 hours of therapy, it is not 
anticipated that many (if any) subjects will be withdrawn by the investigator from the 
research without their consent.  An investigator may discontinue the study intervention for 
the following reasons: significant study intervention non-compliance (more that 20 minutes 
of non-compliance with study intervention); if any clinical adverse event (AE), laboratory 
abnormality, or other medical condition or situation occurs such that continued participation 
in the study would not be in the best interest of the participant; If the participant meets an 
exclusion criterion (either newly developed or not previously recognized) that precludes 
further study participation. 
 

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Discontinuation of hyperoxia treatment arm does not mean discontinuation from the study, 
and remaining study procedures should be completed as indicated by the study protocol. If a 
clinically significant finding is identified (including, but not limited to: an alternative diagnosis 
besides stroke, evidence of hypoxia prior to enrollment, baseline requirement of 
supplemental oxygen use) after enrollment, the investigator or qualified designee will 
determine if any change in participant management is needed. Any new clinically relevant 
finding will be reported as an adverse event (AE). 
 
The data to be collected at the time of study intervention discontinuation will include the 
following: reason for discontinuation; time of discontinuation; medically necessitated 
interventions as a result of discontinuation. 
 
Participants are free to withdraw from participation in the study at any time upon request. 
The reason for participant discontinuation or withdrawal from the study will be recorded on 
the Case Report Form (CRF). Subjects who sign the informed consent form, are randomized 
and receive the study intervention, and subsequently withdraw, are withdrawn or discontinue 
from the study, will be not be replaced. 
 
 
14. PRIVACY AND CONFIDENTIALITY OF SUBJECTS AND RESEARCH DATA 
Participant confidentiality and privacy will be ensured through careful data handling 
procedures. All paper forms for study documentation will be stored in locked cabinets, and 
only research staff will have access to the documents. Serum samples will be immediately 
coded into a secure, HIPAA-compliant database (either REDCap or a password-protect 
secure server at the University of Colorado). All study data will be de-identified after chart 
extraction and stored in the database to maximize the security of the electronic data. All 
data will be secured in locked offices and all electronic data will be stored on encrypted 
servers, as per institutional policy. All computer systems at the University of Colorado 
Anschutz Medical Campus have the level and scope of security exceeding that established 
by the HIPAA Security Rules. Imaging data is processed using RAPID AI. In cases where 
images are not automatically processed by RAPID, i.e. subsequent MRI, these raw DICOM 
files containing PHI will be processed with RAPID technology within the hospital firewall 
(see figure above regarding RAPID DUA and de-identification overview). No data will be 
shared with anyone who is not directly involved with the study, and findings will only be 
reported in aggregate.   
 
In order to obtain required clinical information, study personnel will require access to the 
subject’s medical charts through eRecord. Only the predetermined information will be 
reviewed and collected. Subjects will not be re-contacted outside of the scheduled study 
activities if they have chosen to opt-out of future contact. Data and unused samples will be 
stored for a minimum of 6 years after the completion of the study.  
 
 
15. DATA / SAMPLE STORAGE FOR FUTURE USE 
The field of biomarkers in stroke research is used to help predict outcomes and better 
understand the pathophysiology behind stroke is rapidly changing. To ensure simplicity of 
study design and to enhance the feasibility of this study, only three biomarkers, MMP-9, IL-6 
and IL-8 have been chosen upon initial review to be included in biomarker analysis. 
However, additional biomarkers may also be chosen for further analysis and serum will be 
stored within the ED Cold Storage space for future analysis that could improve the 
understanding of the biological mechanism underpinning the effects of hyperoxia in AIS. 

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These samples will not be analyzed on an individual basis, but rather analyzed and reported 
in aggregates. Thus, it represents minimal additional risk to study subjects to store the 
serum for future biomarker analysis. The physical samples will be de-identified and coded 
such that they may be linked to the clinical information for the patient, which will also be de-
identified after all data collection for the study has been completed. All samples will be 
stored in a HIPAA-compliant, restricted access ED Cold Storages space. Only study 
personnel will have access to the samples. Samples will aliquoted into smaller vials to help 
limit degradation of samples through repeated freeze-thaw cycles for up to 6 years. A 
password protected document on a UC secure server will be used to track the usage of 
individual aliquots of samples. 
 
During the conduct of this study, an individual participant can choose to withdraw consent to 
have biological specimens stored for future research. However, withdrawal of consent with 
regard to biosample storage may not be possible after the conclusion of this study once 
samples have been de-identified. Samples will only be released for future analysis after a 
formal request process. The process will include a request for a COMIRB approved-protocol 
and/or protocol amendment from an individual or team of individuals associated with this 
original study. The released sample and accompanying clinical information will be de-
identified. 
 
 
16. DATA AND SAFETY MONITORING PLAN 
Only trained research assistants at UCH-Memorial will be responsible for consent of 
subjects, with support of the site-PI, site-sub-I and/or PI as needed (see consent section for 
details). The research assistants at UCH-Memorial will be responsible for completion of 
patient assessments and reporting of potential safety issues and/or protocol violations to 
the site-PI, site-sub-I and PI. In addition, the PI will review data assessments uploaded to 
REDCap monthly for completion. Site screening/enrollment logs will be reviewed monthly. 
Study personnel at both sites will undergo an initial training by the PI at onset of study 
which will include review of the study protocol, inclusion/exclusion criteria, the consent 
process, methods of sample collection and processing, patient assessments, and 
standardized chart review methods. The trained research assistants at both sites will have 
complete required human subjects research trainings and specific training to ensure 
accuracy of the NIHSS scoring prior to completing these assessments on any subjects. A 
standard operating procedures (SOP) manual will also be provided for references. Training 
will be refreshed annually or sooner if there are protocol changes and/or any compliance 
concerns. 
 
Safety oversight will be under the direction of a Safety Monitoring Committee (SMC), 
composed of individuals with the appropriate expertise, including stroke, neurocritical care, 
and emergency medicine. Members of the SMC will include members of the investigative 
team. However, measures will be in place to minimize perceived conflict of interest.  The 
SMC will meet at least semiannually to assess safety and efficacy data on each arm of the 
study.  
 
Assessment of safety measures and adverse events: 
Study personnel and the subject’s treating physician will regularly monitor subjects for safety 
measures and adverse events. This includes practices that are associated with routine 
patient care (i.e. vital sign assessments, assessment of neurologic deterioration, 
development of respiratory compromise). Specific monitoring includes the following: 

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1. Vital signs - The determination of need for additional supplemental oxygen in a patient 
that becomes hypoxic (pulse oximetry < 94%) will be determined by the subject’s 
treating physician irrespective of study interventions. Should a patient be found to 
require additional supplemental oxygen while enrolled in the study, this will be noted and 
the patient will continue through the final study assessments. The patient’s treating 
physician will also be responsible for routine monitoring and interpretation of other vital 
signs such has blood pressure and heart rate. If at study assessment time points the 
study personnel note a subject to have significant vital sign abnormalities (SpO2<94%, 
SBP>200mmHg, DBP>110mmHg, SBP<90mmHg, HR>120, HR<50), the study PI and 
patient’s treating physician will be notified to ensure proper documentation. Interventions 
will be left to the discretion of the patient’s treating physician. 
2. Arterial puncture and ABG – Hyperoxia (PaO2>200mmHg) will be confirmed by 
obtaining an ABG in subjects. Personnel trained in obtaining arterial punctures (i.e. 
nurse, physician or respiratory therapist) will be notified and available to obtain the ABG 
four hours after initiation of study intervention. Standard procedures for obtaining an 
arterial blood sample will be employed to reduce the risk of complications. To decrease 
variability between ABG analysis, ABGs will be performed using standard operating 
procedures. The site of the arterial puncture and correlating extremity will be evaluated 
for potential complications (increased pain, discoloration, erythema, hematoma 
formation, bleeding) immediately after obtaining the ABG and at T2. 
3. Hemorrhagic conversion – as part of the standard neurological care, patients are 
monitored for neurological deterioration. Patients exhibiting neurological decline will 
receive a CT-head and/or further imaging for evaluation of potential hemorrhagic 
conversion at the discretion of the treating physician. Based on previous studies in AIS, 
it is expected that between 13-43% of subjects will undergo spontaneous hemorrhagic 
conversion on imaging59. Any such cases will be reported as an adverse event. 
4. Development of respiratory compromise – While patients are continuously assessed by 
their treating medical team, study personnel will specifically assess study subjects at the 
T0+4hour and T0+24hour evaluations for worsening respiratory status and the need for 
additional supplemental oxygen. At the final 3-month assessment, patients’ medical 
records will also be reviewed for evidence of respiratory deterioration while the patient 
was hospitalized and up to this final assessment including: need for additional 
supplemental oxygen, need for non-invasive positive pressure ventilation, mechanical 
ventilation, development of pulmonary edema, development of pneumonia. 
5. Development of post-stroke infections – Neuroinflammation after a stroke also results in 
immunosuppression. As such, this places patients are increased risk of post-stroke 
infections, the two most common being pneumonia and urinary tract infections which 
may account for up to 30% of deaths after an AIS60. Additionally, there may be sex-
based differences in post-stroke infections that could be related to underlying hormone 
status61. As such, patients will be screened at the 3-month assessment to determine if 
they experienced any post-stroke hospitalizations and/or treatment for pneumonia, 
urinary tract infections, sepsis, or other infections. In addition to asking subjects directly 
at this assessment, their medical record will also be reviewed for re-admissions while it 
is being reviewed for the above respiratory compromise.  
 
 
Definition and Classification of Adverse Events: An adverse event means any untoward 
medical occurrence, unintended sign, symptom, or disease temporarily associated with the 
use of an intervention in humans, whether or not considered intervention-related. All AEs will 
be documented and analyzed further. Subjects will be monitored for signs of an AE at 
T0+4hr and T0+24hours.  

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An adverse event (AE) or suspected adverse reaction is considered "serious" if it results in 
any of the following outcomes: death, a life-threatening adverse event, prolongation of 
existing hospitalization, a persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions. Important medical events that may not result in 
death, be life-threatening, or require hospitalization may be considered serious when, based 
upon appropriate medical judgment, they may jeopardize the participant and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition.  
 
For adverse events (AEs), the following guidelines will be used to describe severity.  
 
• 
Mild – Events require minimal or no treatment and do not interfere with the 
participant’s daily activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with functioning 
and/or the need for further supplemental oxygen or antibiotic treatment. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events may include need for mechanical 
ventilation, vasoactive infusion, are usually considered potentially life-threatening or 
incapacitating.   
 
The PI will be responsible for determining whether an adverse event (AE) is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of 
the event is not consistent with the risk information previously described for the study 
intervention. 
 
The following AEs are considered to be expected:  
1. 
Bleeding and/or hematoma formation at sight of arterial puncture 
2. 
Pain at the sight of arterial puncture lasting after collection of ABG 
3. 
Hemorrhagic conversion in up to ~40% of study subjects 
 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the 
attention of study personnel during study observation time points or upon review by a study 
monitor. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be 
captured on the appropriate case report form (CRF). Information to be collected includes 
event description, time of onset, clinician’s assessment of severity, relationship to study 
intervention (assessed only by those with the training and authority to make a diagnosis), 
and time of resolution/stabilization of the event. All AEs occurring while on study must be 
documented appropriately regardless of relationship. All AEs will be followed to adequate 
resolution. 
 
Any medical condition that is present at the time that the participant is screened will be 
considered as medical history and not reported as an AE. However, if the study participant’s 
condition deteriorates at any time during the study, it will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the duration 
of the event at each level of severity to be performed. AEs characterized as intermittent 
require documentation of onset and duration of each episode. 
 

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The study PI will record all reportable events into a database with start dates occurring any 
time after randomization until completion of the study. At T0+24hours, study personnel will 
inquire about the occurrence of AE/SAEs since the last observation point. Solicited and 
unsolicited events will be captured. Unsolicited AEs will be identified by asking participants if 
they “have noticed anything different since beginning the treatment after the onset of your 
stroke.” Solicited AEs will include: 1) any trouble breathing, 2) development of pneumonia, 
3) need for additional oxygen therapy, 4) hemorrhagic conversion of the stroke. Events will 
be followed for outcome information until resolution or study completion. 
 
 
17. DATA ANALYSIS PLAN 
The primary endpoint of this study is feasibility. We hypothesize that the challenges of 
enrolling and randomizing AIS patients to a therapeutic intervention within less than one 
hour of ED presentation can be addressed. The number of potential subjects meeting the 
inclusion criteria and the relative rates of enrollment over time will be tracked. Additionally, 
the timing to enrollment and initiation of treatment intervention will be monitored. The 
primary endpoint of this study is feasibility as measured by the mean time from presentation 
to the ED to randomization and initiation of intervention. We hypothesize that we will be able 
to enroll, randomize and initiate treatment intervention in less than one hour for 80% of the 
enrolled subjects.  
 
This study is not powered to detect significant differences in secondary or exploratory 
outcomes. However, analysis of secondary outcomes will occur both on an intention-to-treat 
and a per-protocol basis. Penumbral salvage will be reported as an average and as a 
standard deviation of the mean. We plan to enroll a total of 200 patients. Based on previous 
data that showed a decrease in the relative infarct volume growth (infarct volume at 
presentation/infarct volume at 4 hours) in patients treated with hyperoxia (87.8 +/-22%) 
compared to controls (149.1 +/- 41%)29, 5 patients per arm would be needed to detect a 
similar difference with a power of 90% and type I error rate () of 0.0562. However, this effect 
was seen in a small study with less than 10 patients per treatment arm. Thus, such dramatic 
changes in infarct volume may not be expected, but even a smaller reduction in infarct 
volume will be clinically important as small reductions in infarct volume can have profound 
clinical effects if it is in a key cerebral territory. If you assume 20% of patients for some 
reason do not receive standard follow-up imaging to allow for calculation of infarct 
progression, a standard deviation of 41%, 80% power and  of 0.05, the proposed sample 
size will be able to detect an absolute difference in infarct volume growth of 18% between 
the control and hyperoxia. 
 
In terms of effects on clinical patient outcomes, both the effect on 3-month mRS and NIHSS 
will be determined. The DEFUSE-3 study found the median NIHSS score in AIS patients 
evaluated for possible extended-window thrombectomy was 16 with an interquartile range of 
10-2063. Based on these results and our proposed sample size for feasibility and a 29% loss 
to follow-up, we will be able to detect a two-point difference in NIHSS between the hyperoxia 
and control groups at 24hours with 80% power and a type I error () of 0.05. Given the large 
potential number of confounding events between the time of study intervention and a 3-
month mRS, this will be analyzed on an exploratory basis. The average 3-month mRS in 
both the hyperoxia and control groups will be compared. Finally, an exploratory analysis of 
the effects of hyperoxia on biomarkers of stroke severity and hyperoxic lung injury will be 
evaluated over time. The change in level of biomarkers (MMP-9, IL-6, and IL-8) over time 
will be analyzed and reported using the mean and standard deviation and will be depicted 
graphically. 

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