Reoxygenation After Cardiac Arrest II (REOX II Study)
Phase: PHASE1
Conditions: Cardiac Arrest
Sponsor: The Cooper Health System

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STUDY PROTOCOL 
 
 
 
REOXYGENATION AFTER CARDIAC ARREST II (REOX II) STUDY 
 
 
 
SPONSOR: NATIONAL INSTITUTES OF HEALTH 
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE 
 
 
 
GRANT # R01 HL112815:  NEUROLOGICAL AND COGNITIVE EFFECTS OF  
HYPEROXIA AFTER CARDIAC ARREST 
 
 
 
VERSION 1, 12/3/2015 
 
 
 
 
 
 
 
 
Principal Investigator: 
Stephen Trzeciak, MD, MPH 
Professor and Head of Critical Care Medicine 
Cooper University Health Care 
Cooper Medical School of Rowan University 
Camden, New Jersey  08103 
Phone: 856-342-2633 
Email: trzeciak-stephen@cooperhealth.edu 
 
 
 

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A. SIGNIFICANCE 
Out-of-hospital cardiac arrest (OHCA) is common, lethal, and neurologically devastating for many 
survivors, but tremendous potential exists to improve outcomes with post-resuscitation interventions.  
 
A.1. Scope of the problem 
OHCA is common and lethal. Approximately 295,000 
Americans are treated by Emergency Medical Services for 
OHCA annually1, with an overall survival to hospital discharge of 
<10%, making OHCA a leading cause of death in the U.S.2 
Victims of OHCA are relatively young (median age 67 years), 
and women are affected as often as men. Often, OHCA is the 
first manifestation of cardiovascular disease, with two-thirds of 
arrests occurring without prior recognition of an underlying 
condition.3 Relative to other common emergency conditions, the 
number of deaths from OHCA is extremely high (Table 1).2,4,5 
 
Even if initial resuscitation from OHCA is successful, the rate of 
subsequent death and disability is staggering, primarily due to 
anoxic brain injury. Although cardiopulmonary resuscitation (CPR) 
and defibrillation may be successful in achieving return of 
spontaneous circulation (ROSC), the subsequent 
ischemia/reperfusion insult may have crippling neurological effects. 
Anoxic brain injury is the most common (~70%) cause of death 
among patients resuscitated from OHCA.6 In a large (n=8736) U.S. 
multi-center cohort study of patients who survived to intensive care 
unit (ICU) admission following successful resuscitation from cardiac 
arrest, our research team found that 50% of patients did not survive 
to hospital discharge.7 Of those who did survive, the majority of 
patients were functionally dependent, and more than half of these 
patients were discharged to a long-term care facility (Figure 1). 
Furthermore, survivors discharged with functional independence are considered to have “good outcomes”; 
however, substantial cognitive and psychomotor deficits are common among these survivors. Recently, our 
research team published the results of an NIH-funded study of neuropsychological deficits among OHCA 
survivors (see Preliminary Studies in section C.3.7).8 Among patients who recovered alertness but remained 
confused at 7 days, we performed neuropsychological testing at 90 days post-arrest. We found that while two-
thirds of patients manifested mild deficits in memory and psychomotor function with other cognitive domains 
preserved, one-third of patients manifested severe memory and psychomotor deficits and impairment in all 
cognitive domains. Overall, only ~25% of these patients were able to return to work. In summary, patients 
resuscitated from OHCA commonly suffer severe neurological injury leading to death or long-term 
disability. Research on new methods to improve outcomes from this devastating condition is a crucial 
and urgent priority. Given the magnitude of death and disability that OHCA imparts upon Americans, 
even modest improvements would provide a major public health benefit. 
 
A.2. Importance of post-resuscitation interventions. 
Until recently, neurological injury after resuscitation from cardiac arrest was largely thought to be untreatable. 
Now it is recognized that the trajectory of this disease can in fact be modulated.9 Although the initial ischemic 
insult has already occurred, therapeutic interventions applied after ROSC may mitigate the severity of 
reperfusion injury. Neuronal death after reperfusion is a dynamic process that does not occur immediately 
after ROSC.  Laboratory investigations demonstrate that histopathological changes in the brain commonly take 
several hours or days after ROSC to manifest.9  These data indicate that a window of opportunity exists after 
ROSC in which brain injury may be attenuated. Target temperature management (TTM), a strategy to control 
body temperature for 12-24 hours after ROSC, is the first proven therapy to reduce brain injury after 
resuscitation from cardiac arrest. Two landmark clinical trials found significantly improved neurological 
outcomes for patients treated with TTM after OHCA due to ventricular fibrillation (VF).10,11 These pivotal 
Figure 1: Outcomes for 8736 patients admitted to an 
intensive care unit after successful resuscitation from 
cardiac arrest (Trzeciak et al Crit Care Med 2009). 
Table 1: Annual U.S. deaths from out-of-hospital cardiac 
arrest (OHCA) compared to other emergency conditions. 
Disease 
Mortality (est.) 
OHCA 
265,000 
Severe sepsis 
215,000 
Acute myocardial 
infarction 
141,000 
Stroke 
137,000 
Trauma 
124.000 

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findings were the first evidence that brain injury after ROSC is in fact treatable. The American Heart 
Association (AHA) now recommends TTM for adults with OHCA due to VF who remain comatose after ROSC 
(Class I recommendation).12 Although TTM represents a truly groundbreaking discovery, the mortality and 
disability among OHCA victims treated with TTM remain unacceptably high.9 Finding additional post-
ROSC interventions to further attenuate reperfusion injury and improve outcomes is a crucial research priority 
for resuscitation science with potential impact for thousands of Americans annually.13 
 
A.3. Significance of the proposed research  
Another potentially modifiable factor in post-resuscitation care is the amount of oxygen delivered to the brain 
after reperfusion. Current resuscitation guidelines recommend a fraction of inspired oxygen (FiO2) of 100% 
during CPR, as this may help achieve ROSC; however, clinicians frequently maintain high FiO2 for prolonged 
and highly variable periods of time after circulation is restored to ensure adequate oxygenation.9 This strategy 
may not be innocuous, as there appears to be a paradox regarding oxygen delivery to the injured brain. 
Insufficient cerebral oxygen delivery can exacerbate cerebral anoxia, but hyperoxia (i.e. excessively high 
arterial oxygen partial pressure [PaO2] and saturation [SaO2] due to an excessively high FiO2) potentially 
accelerates reperfusion injury. Preclinical studies have found that hyperoxia after ROSC worsens brain 
damage, evidenced on functional neurological testing14-16 and histopathology,17,18 and worsens survival.19 The 
postulated mechanism is that hyperoxia drives an increase in formation of reactive oxygen species. This 
increase may cause oxidative impairment of mitochondrial respiration and cerebral energy metabolism 
(Figure 2). Oxidative modification to mitochondrial proteins may exacerbate the reperfusion-dependent 
inactivation of brain pyruvate dehydrogenase complex (PDHC), the only bridge between anaerobic and aerobic 
metabolism.20 Increased reactive oxygen species may also activate the mitochondrial permeability transition 
pore (PTP) to release NAD(H) into the cytosol, depleting a vital metabolic cofactor. These changes may impair 
oxidative phosphorylation and cerebral adenosine triphosphate (ATP) generation.20 Metabolic failure may 
ensue, resulting in 
decreased cerebral 
consumption of glucose 
and oxygen, increased 
production of lactate, and 
delayed neuronal cell 
death.18,21 In addition, 
increased reactive 
oxygen species may 
impair electron transport 
chain activity by forming 
mitochondrial membrane 
pores that release 
cytochrome c into the 
cytosol.20 Release of 
cytochrome c may trigger 
caspase-dependent 
apoptosis. Apoptosis 
may also be triggered as 
mitochondrial dysfunction 
disrupts cellular calcium 
ion homeostasis, resulting in an influx of intracellular calcium. Increased reactive oxygen species may also 
cause oxidation of brain lipids (i.e. lipid peroxidation), which may have physiologic effects (e.g. alteration of 
blood flow, neutrophil chemoattraction) as well as cellular toxic effects (e.g. excitotoxicity, 
neurodegeneration).15,19 Increased reactive oxygen species may also promote cellular inflammatory reactions, 
specifically the activation of microglia and astrocytes in the neuronal microenvironment leading to increased 
neuronal cell death.14 Lastly, hyperoxia may also have a direct vasoconstrictor effect, which may reduce 
cerebral blood flow after ROSC, exacerbating ischemic injury.22,23 
Figure 2: Mitochondrial effects of hyperoxia after ROSC. Hyperoxia increases reactive oxygen species, 
which may inhibit pyruvate dehydrogenase complex (PDHC), and open mitochondrial membrane 
permeability transition pore (PTP), depleting NAD(H). These effects inhibit adenosine triphosphate (ATP) 
generation, leading to metabolic failure. Increased reactive oxygen species may also, via Bcl-2-associated 
proteins (Bax/Bak), induce mitochondrial release of cytochrome C, triggering caspase-dependent apoptosis.  
[TCA = tricarboxylic acid; NAD = nicotinamide adenine dinucleotide; ETC = electron transport chain] 

4 
 
These data from animal models support the hypothesis that hyperoxia after ROSC may worsen outcomes in 
post-resuscitation patients. However, clinical data are few. Three preliminary retrospective multi-center 
analyses have been published. Two of these were published by our research team.24,25 In a large (n=6326) 
cohort of patients admitted to one of 120 U.S. ICUs after cardiac arrest, we found that post-ROSC hyperoxia 
was common, an independent predictor of in-hospital death, and associated with lower functional 
independence among survivors.24 In a secondary analysis using the same ICU database, we found that a 
linear, “dose-dependent” relationship exists between supranormal PaO2 after ROSC and mortality25 
(see Preliminary Studies in sections C.3.1 and C.3.2). However, a recent study from the Australian and New 
Zealand Intensive Care Society (ANZICS) investigators reported somewhat different results.26 Using a 
retrospective methodology that replicated our initial study, they found that among 12,000 patients admitted to 
an ICU after cardiac arrest, patients with hyperoxia (defined by our previous methodology as a PaO2 >300 
mmHg) had significantly higher mortality (59% [95%CI 56-61%]) than patients with normoxia (47% [95% CI 45-
50%]), but not patients with hypoxia (60% [95% CI 59-61%]). In a multivariable model adjusting for illness 
severity, hyperoxia had an odds ratio for mortality of 1.2 (95% CI 1.1-1.6). However, when statistical models 
with a number of additional adjustments were applied, the association with increased mortality was lost. They 
concluded that hyperoxia has no consistently reproducible independent association with mortality.26 Therefore, 
whether hyperoxia is associated with poor outcome in post-resuscitation patients remains unclear. 
Given that post-ROSC hyperoxia exposure is extremely common25, this unanswered question represents a 
critical knowledge gap for resuscitation science. Accordingly, this project will generate new, critically 
important knowledge about a fundamental element of post-cardiac arrest care. 
 
B. INNOVATION 
Supplemental oxygen administration is considered a cornerstone of effective CPR, and the belief that 
increased oxygen delivery is beneficial is historically one of the most basic assumptions of resuscitation. In this 
application, we challenge the present paradigm regarding the role of supplemental oxygen in the treatment 
of cardiac arrest. Supplemental oxygen may be a drug with important toxicity after ROSC. Specifically, we 
propose an alternative hypothesis that hyperoxia after ROSC due to excessive supplemental oxygen is 
associated with harm. Hypotheses that challenge the prevailing thinking are the hypotheses with the greatest 
capacity to produce results that challenge conventional clinical practice. This project is further innovative in that 
we aim to identify a new potential therapeutic target in post-resuscitation care. Figure 3 below displays the 
classical Chain of Survival paradigm for the treatment of cardiac arrest victims, with a new final link 
emphasizing the importance of post-resuscitation care.27 Rather than studying a completely new treatment 
strategy (e.g. hypothermia), the proposed study will generate new knowledge about a standard component of 
supportive care (i.e. supplemental oxygen 
administration) that may be contributing to poor 
outcomes. This project is further innovative in that we 
will measure neuropsychological function among 
survivors at 180 days. Historically, cardiac arrest 
studies have used crude outcome measures.28 For 
example, the most often used outcome, Cerebral 
Performance Category (CPC), requires no cognitive 
testing. The CPC-1 is compatible with cognitive 
impairment, so the precise nature of residual deficits is 
largely unknown. To date, we are only aware of 5 high 
quality prospective studies of neuropsychological 
deficits after cardiac arrest published in the literature8,29-32 (including work from our research team8), and these 
studies only included 245 patients combined. Recently, a consensus conference on outcome measures in 
cardiac arrest research (led by the AHA and with participation of the NIH and FDA)33 stressed the importance 
of cognitive and functional outcomes, thus we are responsive to a recognized need for innovation in the field. 
This project is further innovative in that we will use the Emergency Department (ED) as our main clinical 
laboratory. In contrast to other clinical research networks in the U.S. that focus on pre-hospital elements of 
resuscitation or other neurological emergencies, the Emergency Medicine Shock Research Network 
(EMShockNet) (described in section C.2) is focused on the ED phase of therapy and continuum of care to the 
ICU. Because reduction of supplemental oxygen in OHCA patients typically begins in the ED and is continued 
Figure 3: The new Chain of Survival paradigm 
including postresuscitation care. 

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in the ICU, our network is an ideal environment to test this hypothesis. We are not aware of any U.S. hospital-
based cardiac arrest clinical research consortium, and thus EMShockNet is a unique resource. Lastly, this 
project is innovative in that it uses gas chromatography negative ion chemical ionization mass spectrometry 
(GC/NICI/MS) to identify novel biomarkers of post-resuscitation oxidative injury (i.e. plasma/urine 
isoprostanes and isofurans). Due to their GC/NICI/MS characteristics, isoprostane and isofuran assays are 
innovative methodologies that apply advanced technologies. 
34,35 
C. APPROACH 
C.1. The investigators 
  
 
 
 
The Principal Investigator, Stephen Trzeciak, MD, MPH, has extensive experience related to the proposed 
topic, established long-standing collaborative relationships with the research team proposed here, and is highly 
committed and uniquely suited to lead the proposed multi-center study. Our investigators have proven track 
records of NIH funding and successful publication in the domain of cardiac arrest and resuscitation. The 
proposed investigators have collaborated on and coauthored (i.e. two or more authors from our research team) 
30 peer-reviewed original science papers published in the biomedical literature in the 36 months preceding this 
application.  
 
 
 
Investigator 
 
 
Role 
 
 
Qualifications 
 
 
Responsibilities 
 
 
Stephen Trzeciak, MD, MPH 
 
 
PI 
 
 
Dr. Trzeciak is co-founder of EMShockNet. His research has been funded by NIH, 
Shock Society, Emergency Medicine Foundation, and American Heart Association. 
He has prior experience as PI directing a prospective multi-center (EMShockNet) 
study using high frequency oxygenation measurements and analyses of 
cumulative oxygenation exposure over time in the resuscitation phase of therapy.34 
 
 
Overall PI and 
study director 
overseeing all 
aspects of the 
project. 
 
Alan E. Jones, MD 
 
Co-I 
 
 
Dr. Jones is co-founder of EMShockNet. He is Vice Chair for Research, 
Department of Emergency Medicine, UMC. He has >60 papers related to the 
resuscitation of critically ill patients, and was PI on a pivotal NIH-funded 
(GM076652) EMShockNet trial recently published in JAMA35 (see D.2. below). 
 
 
Site PI  (UMC) 
 
 
Nathan I. Shapiro, MD, MPH 
 
 
Co-I 
 
 
Dr. Shapiro is co-founder of EMShockNet. He is Vice Chair for Research, 
Department of Emergency Medicine, BIDMC. He has extensive experience as PI 
on multi-center observational studies, including work funded by NIH (HL091757), 
and has >80 papers related to the resuscitation of critically ill patients. 
 
 
Site PI  (BIDMC) 
 
 
Jeffrey A. Kline, MD 
 
 
Co-I 
 
 
Dr. Kline is Vice Chair of Emergency Medicine at MH. He is a renowned leader in 
Emergency Medicine research.  He was awarded the 2009 Excellence in Research 
Award from the Society for Academic Emergency Medicine. He has a long track 
record of NIH funding and over 150 papers related to critically ill patients.  
 
 
Site PI (MH) 
 
 
Benjamin S. Abella, MD, MPhil 
 
 
Co-I 
 
 
Dr. Abella is Director of Clinical Research, Center for Resuscitation Science, HUP. 
He has >60 papers in the field of cardiac arrest including work funded by NIH 
(HL083082). He was a co-author on the 2010 CPR guidelines. He established and 
directs a highly successful training course for post-cardiac arrest care. 
 
 
Site PI  
(HUP and PRES) 
 
 
J. Hope Kilgannon, MD 
 
 
Co-I 
 
 
Dr. Kilgannon has extensive and unique experience related to the proposed 
research. She was first author (with Dr. Trzeciak as senior author) on both of our 
recent papers on hyperoxia after cardiac arrest24, 25 (see Preliminary Studies). 
 
 
Site PI  (CUH) 
 
 
Ramona Hopkins, PhD 
 
Co-I 
 
 
Dr. Hopkins is a Professor of Psychology and Neuroscience, Brigham Young 
University. She has conducted extensive prior research assessing long-term 
cognitive, psychiatric, physical, and quality of life outcomes in survivors of critical 
illness and anoxia/hypoxia, including NIH/NHLBI ARDS Network-2 studies. 
 
Oversee all 
aspects of 
neuropsychological 
testing. 
 
Table 2: The team of investigators. [ARDS = acute respiratory distress syndrome; EMShockNet = Emergency Medicine Shock Research Network; 
CUH = Cooper University Hospital; UMC = University of Mississippi Medical Center; BIDMC = Beth Israel Deaconess Medical Center; MH = 
Methodist Hospital, Indianapolis, IN; HUP = Hospital of the University of Pennsylvania; PRES = Penn-Presbyterian Medical Center] 

6 
 
C.2. Our research network 
The Emergency Medicine Shock Research Network was founded in 2007. Its mission is to conduct 
innovative clinical investigation for the advancement of generalizeable knowledge about circulatory shock 
states in humans. Although the first EMShockNet studies were focused on septic shock, we have expanded 
our research portfolio to focus on the most severe manifestation of circulatory shock, namely cardiac arrest.  
Our network has already demonstrated the ability to successfully complete multicenter studies of the 
size and complexity proposed in this application. To date we have enrolled more than 800 patients in 
prospective research protocols in the Emergency Department (ED) including real-time data collection in the ED 
and through the ICU course. We performed the largest randomized trial published to date comparing different 
goal-directed resuscitation strategies in ED patients with septic shock (JAMA 2010; 303(8):739-46). Therefore 
we are experienced at enrolling patients in ED-based time-sensitive research protocols in the 
resuscitation phase of patient care. In addition to the original EMShockNet sites (CUH, BIDMC, CMC), four 
additional centers will participate in the proposed study. The University of Mississippi became an EMShockNet 
site in 2010 when Dr. Jones was recruited to UMC from CMC. MH became an EMShockNet site at the onset of 
REOX I. Both HUP (which has previously enrolled patients in EMShockNet research36) and Penn-Presbyterian 
will participate, with Dr. Abella as PI for both sites. Dr. Abella’s team has been enrolling OHCA patients in 
prospective research protocols at both sites since 2004.  
 
Our sites also have extensive experience implementing aggressive clinical treatment protocols for 
critically ill patients in the ED setting, and analyzing the effects using a before-and-after study design. 
Specifically, we have effectively implemented protocols for post-cardiac arrest care and hemodynamic 
optimization for sepsis.37-42 Importantly, we also have experience continuing these aggressive treatment 
protocols into the ICU. Compared to our previous treatment protocols, which required the introduction of 
invasive hemodynamic monitoring or other complex procedures in the ED setting, our protocol for rapid FiO2 
optimization after resuscitation from cardiac arrest is relatively straight-forward and quite feasible. Our sites’ 
clinical rationale for implementing the protocol in this application is that rapid FiO2 optimization is now 
recommended in the American Heart Association (AHA) Guidelines for Cardiopulmonary Resuscitation and 
Emergency Cardiovascular Care43 (and is now included in Advanced Cardiac Life Support [ACLS]) and a 
consensus statement on post-resuscitation care from the International Liaison Committee on Resuscitation 
(ILCOR).9 Therefore, the implementation of a rapid FiO2 optimization protocol after resuscitation from cardiac 
arrest will be consistent with current consensus treatment guidelines, but at the same time will generate the 
most valuable clinical data to date regarding this fundamental element of post-cardiac arrest care. 
 
C.3. Preliminary studies from the research team proposed in this application 
C.3.1. Arterial hyperoxia after resuscitation from cardiac arrest is associated with poor outcome. 
Kilgannon JH, Jones AE, Shapiro NI, Angelos MG, Milcarek B, Hunter K, Parrillo JE, Trzeciak S.  
Association between arterial hyperoxia following resuscitation from cardiac arrest and in-hospital mortality. 
JAMA 2010; 303(21):2165-71.  
     Using Project IMPACT, a database of ICU admissions from 120 U.S. 
hospitals, we tested the hypothesis that exposure to hyperoxia (defined as 
a PaO2 >300 mmHg on the first arterial blood gas recorded in the ICU) was 
associated with increased in-hospital death among 6326 patients admitted 
to an ICU after cardiac arrest. We found that the patients with hyperoxia 
had significantly higher in-hospital mortality (63% [95% CI 60%-66%]) 
compared to patients with normoxia (45% [95% CI 43%-48%]; proportion 
difference 18% [95% CI 14%-22%]), and also compared to patients with 
hypoxia (57% [95% CI 56%-59%]; proportion difference, 6% [95% CI, 3%-
9%]). Survival over time appears in Figure 4. In a model controlling for 
potential confounders (e.g., age, preadmission functional status, comorbid 
conditions, vital signs, and other physiological indices) including sensitivity 
analyses that adjusted for hospital factors and propensity of hyperoxia 
exposure, hyperoxia had an odds ratio for death of 1.8 (95% CI, 1.5-2.2). 
Among survivors, hyperoxia was associated with a lower proportion of functional independence at discharge 
compared to patients with normoxia (29% vs. 38%, respectively; proportion difference, 9% [95% CI, 3%-15%]. 
Figure 4: In-hospital death between 
hyperoxia and normoxia groups. 

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C.3.2. Dose-dependent relationship between supranormal PaO2 and risk of death after cardiac arrest.  
Kilgannon JH, Jones AE, Parrillo JE, Dellinger RP, Milcarek B, Hunter K, Shapiro NI, Trzeciak S.  
Relationship between supranormal oxygen tension and outcome after resuscitation from cardiac arrest.  
Circulation 2011; 123(23):2717-22.  
     Using the same ICU database, we tested the hypothesis that a linear, dose-dependent relationship exists 
between post-resuscitation supranormal oxygen tension (highest PaO2 over the first 24 hours in the ICU) and 
in-hospital mortality. Among 4459 patients, the median PaO2 was 231 (IQR 149-349) mmHg. Over ascending 
ranges of oxygen tension, we found significant linear trends of increasing in-hospital mortality and decreasing 
survival as functionally independent, p<0.0001 (Figure 5). On multivariable analysis, a 100 mmHg increase in 
PaO2 was associated with a 24% increase in mortality risk (odds ratio 1.24 [95% CI 1.18-1.31] (Figure 6).  
 
 
 
C.3.3. Development of a clinical protocol for rapid FiO2 optimization. 
The method of rapid FiO2 optimization in REOX II must be comprised of a detailed protocol (see Methods 
section below) rather than a simple directive to titrate FiO2 to keep SaO2 or PaO2 in a predetermined range, 
because rigorous uniformity is needed in the approach across all study sites, including uniformity in the 
increments and frequency of FiO2 reductions. The therapeutic goal of our protocol is a PaO2 of 60-99 mmHg, 
because this is the PaO2 range that was associated with the lowest risk of poor outcome in our previously 
published work.25 We also use PaO2 (measured by arterial blood gas [ABG] analysis) as the ultimate goal 
rather than SaO2 measured by pulse oximetry because an SaO2 value <100% on pulse oximetry monitoring 
does not always exclude supranormal PaO2. The protocol in this application begins with very rapid reduction of 
FiO2 as much as possible according to SaO2 values, and when FiO2 is maximally reduced by SaO2 an ABG is 
measured, followed by finer adjustment of FiO2 to achieve a PaO2 60-99 mmHg. The protocol must not only 
prescribe each downward titration of FiO2 but it must also include detailed limbs for upward titration of FiO2 to 
account for potential “overshoot” in FiO2 reduction. We acknowledge that the flowchart for FiO2 titration in the 
Methods section (Figure 9) may appear complex, but our experience is that this level of detail is needed in 
order to ensure that FiO2 optimization will not only be rapid and effective, but also safe. We believe using this 
protocol will allow for rapid achievement of the target PaO2 after ROSC and no significant hypoxic events 
related to overshoot.  
 
 
 
Figure 5 (left): Proportions of 
in-hospital mortality and 
independent functional status 
among survivors over 
increasing oxygen tensions. 
We found linear trends for 
increasing mortality and 
decreasing functional 
independence with increasing 
oxygen tension, p<0.0001. 
  
Figure 6 (right): Odds ratios 
(solid line) and 95% confidence 
intervals (dotted lines) for in-
hospital death with increasing 
increments of oxygen tension. 

8 
 
C.3.4. Optimal assessment of oxidative stress in vivo.  
Roberts LJ, and colleagues. Biomarkers of Oxidative Stress Study II:  Are oxidation products of lipids, proteins, 
and DNA markers of CCL4 poisoning? Free Radic Biol Med 2005;38(6):698-710. 
     Oxidative stress refers to the overproduction of oxygen free radicals and/or an insufficiency of 
antioxidant/radical-detoxifying systems resulting in deleterious modification of proteins, nucleic acids, and lipids 
(i.e. lipid peroxidation). The proposed study will assess the degree of oxidative stress in post-resuscitation 
patients, and therefore will require the most sensitive and specific biomarkers of oxidant injury in vivo. The 
optimal biomarkers to measure remained unclear until the Biomarkers of Oxidative Stress Study (BOSS) that 
was recently conducted by the National Institute of Environmental Health Sciences (NIEHS). Dr. Jack Roberts 
(Vanderbilt University), who is among the Key Personnel on this application, was one of the NIEHS-BOSS 
Principal Investigators. It was a comprehensive, multi-laboratory validation study directly comparing multiple 
different methods of assessing oxidative stress using identical samples from a rat model of massive oxidative 
injury (acute carbon tetrachloride poisoning). The purpose of the study was to identify the most reliable, 
sensitive and specific noninvasive biomarkers of oxidative damage. The study compared isoprostanes 
(prostaglandin F2-like compounds that are formed nonenzymatically by free radical-induced peroxidation of 
arachidonic acid), lipid hydroperoxides, thiobarbituric acid-reactive substances (TBARS), malondialdehyde 
(MDA), protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2V-deoxyguanosine, 
leukocyte DNA–MDA adducts, and DNA-strand breaks. Isoprostanes (IsoPs) measured in plasma or urine 
by gas chromatography negative ion chemical ionization mass spectrometry (GC/NICI/MS) were found 
to be the most reproducible, sensitive, and specific biomarkers of in vivo oxidative stress. None of the 
other biomarkers tested were found to be sufficiently reliable for oxidative stress assessment in vivo. Dr. 
Roberts and his laboratory have two decades of experience with these techniques for measuring IsoPs. 
 
C.3.5. Assessment of oxidative stress with exposure to hyperoxia.  
Roberts LJ, et al. Discovery of lipid peroxidation products formed in vivo 
with a substituted tetrahydrofuran ring (isofurans) that are favored by 
increased oxygen tension. Proc Natl Acad Sci 2002;99(26):16713-18.  
     Dr. Roberts and his laboratory were the investigators who discovered 
isofurans (IsoFs), which are isomeric products of lipid peroxidation 
characterized by a substituted tetrahydrofuran ring. Isofuran assays are 
essential for the proposed study. Under conditions of increasing ambient 
oxygen concentrations (e.g. high FiO2 producing supranormal PaO2) an 
oxygen molecule is added to the carbon-centered radical to yield a 
peroxyl radical, and this favors the production of IsoFs instead of IsoPs 
(Figure 7). These reactions are competing, mutually exclusive, and 
differentially regulated by the ambient oxygen concentration. Although IsoPs are an excellent assessment 
of oxidative stress in general, under hyperoxic conditions measurement of IsoPs alone would underestimate 
the extent of lipid peroxidation. A complete picture across all FiO2 concentrations is only possible with 
measurement of both IsoPs and IsoFs. Given that IsoFs are a remarkably sensitive indicator of hyperoxia-
induced oxidative injury, and the proposed study is focused on hyperoxia exposure, measuring IsoFs in 
addition to IsoPs in this study is vital. Importantly, and pertinent to the feasibility of the proposed study, IsoF 
and IsoP assays are remarkably stable and therefore are applicable to stored specimens. In addition, IsoFs 
and IsoPs are quantified in the same GC/NICI/MS assay using a single purification protocol, allowing for 
maximum analytical simplicity. We will measure plasma IsoPs and IsoFs in this study. 
 
C.3.6. Association between isofuran levels after resuscitation and clinical outcome.  
Roberts LJ, et al. Preterm resuscitation with low oxygen causes less oxidative stress, inflammation, and 
chronic lung disease. Pediatrics 2009;124(3):e439-49.  
     In this clinical trial comparing resuscitation with 90% FiO2 versus 30% FiO2 at the time of delivery among 
extremely low gestational age neonates, Dr. Roberts and colleagues assessed in vivo oxidative stress by 
measuring urine isofurans by GC/NICI/MS. They found that neonates resuscitated with 30% FiO2 had 
significantly lower isofuran levels over the first day after resuscitation compared to those resuscitated with 90% 
FiO2, and with this reduction in isofurans, neonates in the 30% FiO2 arm also had improved clinical outcomes 
(reduction in acute lung injury and bronchopulmonary dysplasia). 
Figure 7: Differential effects on isofuran and 
isoprostane formation by ambient oxygen. 
Excessive ambient oxygen favors isofurans. 

9 
 
 
C.3.7. Neuropsychological deficits among survivors of out-of-hospital cardiac arrest.  
Alexander MP, Lafleche G, Schnyer D, Lim C, Verfaellie M. Cognitive and functional outcome after out of 
hospital cardiac arrest. J Int Neuropsychol Soc 2011; 17(2):364-8.  
     This NIH-funded (R01HD046442) 
prospective study determined the nature 
and severity of residual cognitive deficits 
for survivors of OHCA. Thirty OHCA 
patients who recovered alertness within 
1-3 days of ROSC but remained 
confused at one week, and 30 non-
OHCA coronary care admissions 
(matched controls) were administered 
neuropsychological testing at 90 days in 
five cognitive domains: memory, 
executive function, lexical-semantic, 
visuoperceptual, and psychomotor 
(Figure 8). Subjects fell into two deficit 
profiles. Two-thirds had only mild 
memory deficits and borderline 
psychomotor deficits compared to 
controls; 40% in this mild group had 
returned to work. The other one-third of 
patients had severe impairments in all 
domains. In the severe group, only as 
impairment in memory or psychomotor 
domains increased did impairments in other domains appear. Follow-up evaluation at one year revealed 
discouraging little further cognitive improvement over the levels reached at 90 days. Only one additional 
subject returned to work. In addition, we assessed health-related quality of life (QOL) at one year using the 
Sickness Impact Profile-68 (SIP-68)44, a measure of health-related functional status in rehabilitation medicine, 
and found that the OHCA patients reported more adverse effects on health than the cardiac control patients 
due to poorer psychological health and social participation, not due to effects on physical health.  
The proposed study will use a similar methodology for neuropsychological testing and functional 
assessment at 180 days after resuscitation among survivors.  
Figure 8: Composite z-scores – mean and standard deviation – for neuropsychological 
function in five cognitive domains among cardiac controls (CC), all out-of-hospital cardiac 
arrests (OHCA), and mild and severely impaired OHCA patients separately. Deficits after 
OHCA were most common and most severe in memory and psychomotor domains. 

10 
 
D.1. RESEARCH DESIGN AND METHODS 
D.1.1 Specific Aims 
 
D.1.2. Overview 
Design: Prospective protocol implementation study with an observational comparison group [REOX II 
(intervention component) versus REOX I (observational component)] 
Participants: Adult patients resuscitated from cardiac arrest 
Enrollment location: Emergency Departments of 6 urban academic medical centers 
Intervention: Implementation of a clinical protocol for rapid FiO2 optimization after ROSC from cardiac arrest. 
Primary outcome: Good neurological outcome (defined as Modified Rankin Scale <3)45 at hospital discharge. 
Secondary outcomes: Neuropsychological tests and ability to return to work among survivors at 180 days. 
Mechanistic outcomes: Oxidative stress markers (IsoPs/IsoFs) in plasma 0-6 hours after ROSC. 
Estimated sample size and study duration: 280 patients (205 from REOX I and 75 from REOX II). 
 
D.2. REOX II Study Protocol 
Study setting and population: This study is the second phase in a two-phase study. The initial phase, REOX 
I, was an observational-only study. As in REOX I, REOX II will identify subjects with return of spontaneous 
circulation (ROSC) after cardiac arrest in 6 hospitals (Cooper University Hospital, Camden, NJ; Hospital of the 
University of Pennsylvania and Penn-Presbyterian Medical Center, both in Philadelphia, PA; University of 
Mississippi Medical Center, Jackson, MS; Methodist Hospital, Indianapolis, IN; and Beth Israel Deaconess 
Medical Center, Boston, MA). The inclusion criteria are: age > 17 years; cardiac arrest; ROSC achieved; 
comatose (Glasgow Coma Scale motor score <6) after ROSC; and clinician intent to treat with target 
temperature management. The exclusion criteria are: presumed 
etiology of arrest is trauma, hemorrhage or sepsis; resident of a 
nursing home or other long-term care facility; known or clinically 
apparent pregnancy; known lack of commitment to aggressive 
support by next of kin; and any other condition, that, in the opinion 
of the investigator, would preclude the subject from being a 
suitable candidate (e.g. end stage chronic illness with no 
reasonable expectation of survival to hospital discharge). 
 
Patient identification and recruitment: Each of the participating 
centers currently has a mechanism in place for real-time 
notification of study personnel when an OHCA subject arrives in 
the emergency department (ED) or when a cardiac arrest occurs 
in-hospital. Each center has previously validated its notification 
mechanism to ensure that subjects will not be missed and the 
population of potentially eligible subjects will be consecutive 
cardiac arrest subjects. Study personnel will assess patient 
eligibility and will immediately enroll subjects meeting all inclusion 
and no exclusion criteria. 
 
Informed consent: In line with REOX I (observational-only 
component), in REOX II we will begin initial collection of cardiac 
arrest and post-resuscitation data immediately after ROSC. As per 
the inclusion criteria all subjects will be comatose during the initial 
post-ROSC period and therefore will not be able to consent 
themselves for the study intervention (i.e. protocol for rapid FiO2 
optimization after cardiac arrest). Once a subject is identified to 
have all inclusion and no exclusion criteria, we will approach 
surrogates (i.e. next of kin or legally authorized representative) 
Screen patient
Consent can be 
obtained within 4 hours 
of ROSC
Consent can be 
obtained after 4 hours 
of ROSC
Enroll in REOX II 
(interventional)
Enroll in REOX I 
(observational)
Patient excluded 
for inability to 
obtain consent
Patient meets all 
inclusion and no 
exclusion criteria
Yes
Yes
No
No
Figure 9 Flow diagram for enrollment  
We will test if a protocol for rapid FiO2 optimization following ROSC from cardiac arrest is associated with: 
• 
Reduced in vivo oxidative stress during the post-resuscitation phase of therapy (Aim 1) 
• 
Reduced neurological disability at hospital discharge (Aim 2) 
• 
Reduced neuropsychological and functional deficits among survivors at 180 days (Aim 3) 

11 
 
within the first four hours after ROSC for written informed consent and 
HIPAA authorization prior to implementation of the study intervention. If 
communication with a surrogate within the first four hours after ROSC is 
not achieved the subject will not be eligible for the study intervention. 
However, for subjects who are excluded from the study intervention due 
to lack of informed consent within the first four hours after ROSC, we will 
approach subjects or surrogates at a later time for written informed 
consent and HIPAA authorization for participation in the observational 
only (REOX I) component of the study including (a) use of plasma 
samples for research purposes (both stored samples obtained at the time 
of initial resuscitation and new samples); (b) determination of Modified 
Rankin Scale at hospital discharge; and (c) follow-up visits for 
neuropsychological testing at 180 days (Figure 9).  
 
Therapeutic interventions: Table 3 displays the standard elements of 
post-resuscitation care across all centers. All sites are experienced in 
target temperature management after ROSC and target 32-36ºC for 24 
hours using external cooling devices (Arctic Sun, Medivance, USA, or 
Gaymar III, Gaymar Industries, USA). Using a standardized order set for 
target temperature management induction, we demonstrated successful 
achievement of target temperature in 96% of patients and a median time 
from ROSC to target temperature of four hours (i.e. faster than the 
landmark randomized trial) in our routine clinical care.40 All standard elements in Table 3 reflect current AHA 
recommendations.12 All subjects in this study will receive standard post-resuscitation care. Since none of the 
sites previously had a protocol in place for FiO2 titration after ROSC and our standardized order sets for post-
resuscitation care do not have orders for FiO2 titration, subjects in REOX I (observational only component) had 
clinician-directed FiO2 titration after ROSC.  
 
For the interventional component of this study we 
will implement the protocol for rapid FiO2 
optimization after cardiac arrest (Figure 10) at all 
sites. Once a subject is identified and written 
informed consent is obtained from a surrogate, 
the study intervention (i.e. rapid FiO2 optimization 
protocol) will be activated by the investigator who 
receives notification in real-time. (see “Patient 
identification and recruitment” above). The on 
call investigator will communicate directly with 
the clinical care team at the bedside and will 
ensure activation and adherence to the clinical 
protocol. Subjects enrolled in the study 
intervention will receive standard post-
resuscitation care in Table 3 plus rapid FiO2 
optimization according to Figure 10. Our sites 
have an outstanding track record of sustained 
clinical adherence to new resuscitation protocols 
for multi-year time periods.37-42 Subjects who are 
excluded from the study intervention due to lack 
of informed consent within the first four hours 
after ROSC, will still be eligible for the 
observational only aspect of this study (REOX I). 
These subjects will receive standard post-
resuscitation care outlined in Table 3, but will not 
have activation of the rapid FiO2 optimization.  
Figure 10: Protocol for rapid FiO2 optimization after ROSC 
Table 3: Standard elements of post-cardiac 
arrest care across all centers. (TH = 
therapeutic hypothermia; GCS = Glasgow 
Coma Scale; ROSC = return of spontaneous 
circulation) 
 
Standard care elements 
  
 
24/7 capability for interventional cardiac 
catheterization (if needed) 
 
 
Standardized patient selection for TH  
(i.e. GCS motor <6 after ROSC) 
 
 
Target temperature goal for TH of 32-36ºC 
 
 
Maintain TH target temperature 24 hours 
 
 
Controlled rewarming to avoid 
hyperpyrexia after TH completion 
 
 
24/7 capability for goal-directed 
hemodynamic support interventions 
 
 
24/7 capability for continuous 
electroencephalographic monitoring 
 
 
Evidence-based approach to neurological 
prognostication (e.g. wait >72 hours after 
ROSC before support limitations for poor 
neurological prognosis) 
 
FiO2, fraction of inspired oxygen; PaO2, partial pressure of arterial oxygen;  
SaO2, arterial oxygen saturation 
Yes to 
ALL? 
Cancel Protocol 
NO 
Eligibility Criteria: 
Cardiac arrest with return of spontaneous circulation (ROSC)? 
Comatose after ROSC? 
Mechanically ventilated? 
Intent to initiate target temperature management protocol? 
YES  
Maintain FiO2 1.0 
SaO2 
>93%? 
YES  
NO 
Not eligible for further 
FiO2 de-escalation.  
PaO2 
60-99mmHg? 
NO 
> 99mmHg 
Decrease FiO2 by 0.1 (do 
not titrate FiO2 <0.21) 
Recheck PaO2 in 15 
minutes 
Increase FiO2 by 0.1 
Recheck PaO2 in 15 
minutes 
YES  
< 60mmHg 
NO 
GOALS ACHIEVED 
Use FiO2 titration increments of 0.1 (as above) to maintain SaO2 93-96% 
Check PaO2 as clinically indicated 
Check PaO2 
(1) Decrease FiO2 by 0.1  
(do not titrate FiO2 <0.21) 
(2) Monitor SaO2 for 2 minutes 
NO 
SaO2 
>90? 
FiO2 
0.21? 
NO 
YES  
NO 
Eligible for further 
FiO2 de-escalation.  
FiO2 
0.21? 
YES  
(1) Increase FiO2 by 0.1  
(2) Monitor SaO2 for 2 minutes 
FiO2 
1.0? 
Cancel Protocol 
NO 
YES  
YES  

12 
 
Instead these patients will have clinician-directed FiO2 titration after ROSC. Data will be prospectively collected 
on these patients (see “Data Collection” below) and we will approach subjects or surrogates at a later time for 
written informed consent and HIPAA authorization for participation in the observational component of the 
study. 
 
Data collection: We will collect baseline data (e.g. demographics, comorbidities, etc.) and intra-arrest data 
(e.g. location, initial rhythm, length of downtime, etc.) using the Utstein template for cardiac arrest research46 
including the recommended variables for post-resuscitation research.47 We will capture multiple indicators of 
post-resuscitation care quality in accordance with the 2010 AHA guidelines.12 As mandated by the protocol 
(Figure 10) above, our standardized order set for post-resuscitation care will include multiple arterial blood gas 
(ABG) analyses for partial pressure of arterial oxygen (PaO2) measurement after ROSC. As part of standard 
care each institution already obtains ABG analyses at 0- and 6-hours after ROSC. Any additional ABG 
analyses required to achieve the PaO2 goal described in Figure 10 will be considered for research purposes 
and will be funded by the study. We will record all ABG results over the initial 36 hours after ROSC. We will 
capture SaO2 continuously for the initial 36 hours after ROSC. We will also capture every adjustment in FiO2 
from respiratory therapy records and the precise time of the adjustment. We will obtain plasma samples after 
ROSC at 0- and 6-hours after ROSC and ship the samples to the 
laboratory of our collaborator Jack Roberts (Vanderbilt) for measurement 
of isoprostanes (IsoPs) and isofurans (IsoFs) by gas chromatography 
negative ion chemical ionization mass spectrometry (GC/NICI/MS). IsoP 
and IsoF assays are remarkably stable and therefore are applicable to 
stored specimens. We can measure IsoPs and IsoFs on clinical plasma 
samples obtained immediately after ROSC because (after informed 
consent is secured) we can use samples left over in the clinical laboratory 
that are routinely discarded. 
 
Outcome measures: The primary outcome will be neurological disability 
at hospital discharge assessed with the Modified Rankin Scale (mRS),48,49 
a well-validated and widely used outcome measures in stroke clinical 
trials. The scale runs from 0-6, from perfect health without symptoms to 
death (Table 4). All raters will be trained and certified in mRS 
assessment50 and will use a structured questionnaire and interview, which 
have been shown to produce very good interobserver reliability.51,52 We 
will measure the Full Outline of UnResponsiveness (FOUR) Score53,54 at 
72 hours post-ROSC (Table 5). The FOUR Score consists of four 
components with a maximum score of four for 
each component and allows for assessing 
intubated patients and brainstem reflexes. We 
will also perform neuropsychological testing at 
180 days after resuscitation for all patients who 
survived to hospital discharge.  We will use an 
NHLBI-sponsored methodology for performing 
centralized, telephone-based cognitive testing, 
which has been previously validated for use in 
multi-center clinical studies.55-60 
Neuropsychological testing will use validated 
instruments across five cognitive domains: (1) 
attention, Wechsler Adult Intelligence Scale-IV-
digit span; (2) reasoning, Wechsler Adult 
Intelligence Scale-IV-similarities; (3) immediate 
and delayed memory, Wechsler Memory Scale-
III-logical memory I and II; (4) verbal fluency, 
Controlled Oral Word Association Test; and (5) 
executive functioning, Hayling Sentence 
Completion Test. We will also assess 
Table 4: Modified Rankin Scale  
 
Scale 
 
 
Description 
 
 
0 
 
 
No symptoms. 
 
 
1 
 
No significant disability. Able to 
carry out all usual activities, 
despite some symptoms. 
 
 
2 
 
Slight disability. Able to look 
after own affairs unassisted, but 
unable to carry out all previous 
activities. 
 
 
3 
 
Moderate disability. Requires 
some help, but able to walk 
unassisted. 
 
 
4 
 
Moderately severe disability. 
Unable to walk and attend to 
own bodily needs unassisted. 
 
 
5 
 
Severe disability. Requires 
constant nursing care and 
attention, bedridden, 
incontinent. 
 
 
6 
 
Dead 
 
Table 5: Full Outline of UnResponsiveness Score 
Component 
Scoring 
Eye Response 
4 = eyelids open or opened, tracking, or blinking to command 
3 = eyelids open but not tracking 
2 = eyelids closed but open to loud voice 
1 = eyelids closed but open to pain 
0 = eyelids remain closed with pain 
Motor 
Response 
4 = thumbs-up, fist, or peace sign 
3 = localizing to pain 
2 = flexion response to pain 
1 = extension response to pain 
0 = no response to pain or generalized myoclonus status 
Brainstem 
Reflexes 
4 = pupil and corneal reflexes present 
3 = one pupil wide and fixed 
2 = pupil or corneal reflexes absent 
1 = pupil and corneal reflexes absent 
0 = absent pupil, corneal, and cough reflex 
Respiration 
4 = not intubated, regular breathing pattern 
3 = not intubated, Cheyne-Stokes breathing pattern 
2 = not intubated, irregular breathing 
1 = breathes above ventilator rate 
0 = breathes at ventilator rate or apnea 
 

13 
 
orientation and judgment using the COGNISTAT- Neurobehavioral Cognitive Status Exam (NCSE). Functional 
outcome assessment will use validated measures of ability to return to work. The telephone-based cognitive 
battery allows for uniformity in cognitive assessment for patients enrolled across all study sites. Subjects who 
complete the telephone-based cognitive battery will be compensated $100 for their time. Other measures: 
Post-ROSC hyperoxia may also have extracerebral effects. We will calculate the extracerebral Sequential 
Organ Failure Assessment (SOFA) score,61 which quantifies respiratory, cardiovascular, renal, hepatic, and 
coagulation system failure for 0-6 hours and 6-36 hours. We have experience using SOFA as an outcome 
measure to quantify organ failure.61-64 These data will permit secondary analyses of the association between 
hyperoxia exposure and non-neurologic organ failure (i.e. total SOFA minus the neurological component61).  
 
Analysis 
We will begin the analysis with descriptive statistics for the entire cohort. We will report categorical data as 
proportions with 95% confidence intervals and continuous data as means with standard deviations or medians 
with interquartile ranges as appropriate. Using the data collected for PaO2 and SaO2 over time as described 
above, we will compute the time-weighted average (TWA) PaO2 and SaO2 over the first 24 hours after ROSC. 
Computing TWA is a common method for quantifying exposures over time in epidemiological research,65-67 
including studies of exposures and subsequent cognitive function.68 The equations are: 
 
 
 
We will build a multivariable regression model to test the association between TWA-PaO2 and mRS at hospital 
discharge, adjusting for covariates previously demonstrated to be associated with poor neurological outcome 
after cardiac arrest. Covariates to be considered include: (1) pre-arrest comorbidities (Charlson index69), (2) 
prolonged downtime (downtime > 20 minutes) (yes/no), (3) initial arrest rhythm (pulseless electrical 
activity/asystole vs. ventricular tachycardia/ventricular fibrillation), and (4) arrest location (in-hospital vs. out-of-
hospital).70-75 In addition, in order to account for patients with lung injury (i.e. inability to maintain adequate 
oxygenation) we will add PaO2/FiO2 < 100mmHg (yes/no) to the regression model. We will build separate 
models to test the association between TWA-PaO2 (adjusting for the five covariates above) and each of the 
five cognitive domains in our neuropsychological testing. We will then repeat these analyses using TWA-SaO2 
as the exposure variable of interest. This approach will not only allow us to test for an independent association 
between the exposure and neurological disability and neuropsychological deficits, but will also allow us to 
pinpoint one or more types of cognitive deficit (e.g. attention, executive function) most closely associated with 
the exposure. Given outcome measures that are quantitative (i.e. z-score for each cognitive domain) or semi-
quantitative (i.e. mRS at hospital discharge), we will treat these outcomes as continuous dependent variables 
for modeling. Using continuous measures of the exposure and outcomes (rather than collapsing the data into 
categorical variables) will permit detection of subtle effects that could be missed if only categorical analyses 
were employed. 
 
Secondary analyses: We will compare proportions of good neurological outcome [defined as a mRS ≤ 3 (based 
on the preliminary data of our collaborator, Dr. Clifton Callaway, University of Pittsburgh)],45 between subjects 
who receive the study intervention (i.e. rapid FiO2 optimization) vs. those who do not receive the study 
intervention, using binomial test. Among survivors, we will compare the 180-day neuropsychological measures 
(composite z-scores for each cognitive domain) between the same two groups using t-test or Mann-Whitney U 
as appropriate with corrections for multiple comparisons. We will compare the proportions of patients able to 
return to work between the two groups using binomial test. We will compare plasma IsoPs/IsoFs at each time 
point between the two groups using t-test or Mann-Whitney U as appropriate with corrections for multiple 
comparisons. 
 
Sample Size:  The sample size calculation in the initial study proposal was based on the primary outcome, 
mRS at hospital discharge, and we planned to enroll a total of 266 subjects. To ensure adequate power to test 
the relationships between the above independent variables and cognitive domains, we assumed a medium 
relationship strength between the six independent variables and each of the five cognitive domains (R2 = 0.15).  
In order to achieve 80% power and an a = 0.05 we would require a minimal of 85 subjects to undergo 
neuropsychological testing at 180 days. This sample size will be among the largest prospective studies of 
TWA-PaO2 = [(PaO2a * Time a) + (PaO2b * Time b) + … (PaO2z * Time z)] / (Time a + Time b + … Time z) 
 
TWA-SaO2 = [(SaO2a * Time a) + (SaO2b * Time b) + … (SaO2z * Time z)] / (Time a + Time b + … Time z) 

14 
 
neuropsychological outcomes that we are aware of in the literature.8,29-32 Assuming a 15% drop out rate for lost 
to follow up for neuropsychological testing at 180 days we would require 98 subjects to be discharged with a 
mRS ≤ 4. Based on preliminary data from REOX I we expect 35% of patients will be discharged with a mRS ≤ 
4 and undergo cognitive testing at 180 days. We would therefore require a minimal total sample size of 280 
subjects between REOX I and II combined for the multivariable testing. We have enrolled 200 subjects in 
REOX I to date. We anticipate additional subjects to be enrolled in REOX I and therefore plan for 75 subjects 
to be enrolled in REOX II to ensure a total sample size of at least 280 subjects. 
 
Overview of the safety monitoring plan and serious adverse event tracking:  In order to help ensure 
adequate protection of human subjects enrolled in this clinical study, we established an independent three-
member Data Safety Monitoring Board (DSMB) (see DSMB Charter). The Chair of the DSMB will be Dr. 
Christopher W. Jones. Dr. Jones is an established investigator and is currently the Director of Clinical 
Research, Department of Emergency Medicine, Cooper University Hospital, Camden, NJ. The DSMB will be 
charged with reviewing all serious adverse event reports and will also perform interim analysis of the data after 
one-third (n=25) and two-thirds (n=50) of the patients have been enrolled in REOX II. Given neither REOX I 
nor REOX II are blinded, all data reviewed by the DSMB will be unmasked. At both interim analyses the 
committee will be asked to render a decision as to whether the trial should be continued, modified, or stopped 
based on the safety profile of the interim data. We have defined the following a priori as a reportable serious 
adverse event (SAE): (1) any events resulting in death or repeat cardiac arrest; (2) sustained hypoxia (defined 
as SaO2 < 88% for more than three consecutive minutes); (3) heart rate < 50 for greater than one minute; (4) 
documented cyanosis at any time; or (5) withdrawal of a subject from the trial for any reason. We will define 
excessive rates of any single SAE a priori as a >15% difference in the prevalence (% of total number in each 
group) between the two groups [i.e. subjects who receive the study intervention (i.e. rapid FiO2 optimization) 
vs. those who do not receive the study intervention] at either interim analysis. A clinician can withdraw a 
subject from the study for worsening clinical status including worsening respiratory status (based on clinical 
judgment, arterial blood gas analysis, or increasing FiO2 requirement). The site PIs at each collaborating 
institution will be responsible for reporting all predefined SAEs, as well as any other adverse events felt to be 
related to the study protocol, to the coordinating site, Cooper University Hospital. In addition to reporting all 
adverse events to the DSMB, all adverse events will be submitted to each institution’s IRB for review. 
 
 
Registration:  This study will be registered on clinicaltrials.gov 
 
 
 
 
 
 
 

15 
 
References 
 
1. 
Nichol G, Thomas E, Callaway CW, et al. Regional variation in out-of-hospital cardiac arrest incidence 
and outcome. Jama. Sep 24 2008;300(12):1423-1431. 
2. 
Association AH. Heart disease and stroke statistics: 2010 update. Dallas, TX2010. 
3. 
Zheng ZJ, Croft JB, Giles WH, Mensah GA. Sudden cardiac death in the United States, 1989 to 1998. 
Circulation. Oct 30 2001;104(18):2158-2163. 
4. 
Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe 
sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Critical care 
medicine. Jul 2001;29(7):1303-1310. 
5. 
Surgeons ACo. National Trauma Data Bank Report 2006. Chicago, IL2006. 
6. 
Laver S, Farrow C, Turner D, Nolan J. Mode of death after admission to an intensive care unit following 
cardiac arrest. Intensive care medicine. Nov 2004;30(11):2126-2128. 
7. 
Trzeciak S, Jones AE, Kilgannon JH, et al. Significance of arterial hypotension after resuscitation from 
cardiac arrest. Critical care medicine. Nov 2009;37(11):2895-2903; quiz 2904. 
8. 
Alexander MP, Lafleche G, Schnyer D, Lim C, Verfaellie M. Cognitive and functional outcome after out 
of hospital cardiac arrest. J Int Neuropsychol Soc. Mar 2011;17(2):364-368. 
9. 
Neumar RW, Nolan JP, Adrie C, et al. Post-cardiac arrest syndrome: epidemiology, pathophysiology, 
treatment, and prognostication. A consensus statement from the International Liaison Committee on 
Resuscitation (American Heart Association, Australian and New Zealand Council on Resuscitation, 
European Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart 
Foundation, Resuscitation Council of Asia, and the Resuscitation Council of Southern Africa); the 
American Heart Association Emergency Cardiovascular Care Committee; the Council on 
Cardiovascular Surgery and Anesthesia; the Council on Cardiopulmonary, Perioperative, and Critical 
Care; the Council on Clinical Cardiology; and the Stroke Council. Circulation. Dec 2 
2008;118(23):2452-2483. 
10. 
Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. The New England 
journal of medicine. Feb 21 2002;346(8):549-556. 
11. 
Bernard SA, Gray TW, Buist MD, et al. Treatment of comatose survivors of out-of-hospital cardiac 
arrest with induced hypothermia. The New England journal of medicine. Feb 21 2002;346(8):557-563. 
12. 
Peberdy MA, Callaway CW, Neumar RW, et al. Part 9: post-cardiac arrest care: 2010 American Heart 
Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. 
Circulation. Nov 2;122(18 Suppl 3):S768-786. 
13. 
Gazmuri RJ, Nolan JP, Nadkarni VM, et al. Scientific knowledge gaps and clinical research priorities for 
cardiopulmonary resuscitation and emergency cardiovascular care identified during the 2005 
International Consensus Conference on ECC and CPR Science with Treatment Recommendations. A 
consensus statement from the International Liaison Committee on Resuscitation; the American Heart 
Association Emergency Cardiovascular Care Committee; the Stroke Council; and the Cardiovascular 
Nursing Council. Resuscitation. Dec 2007;75(3):400-411. 
14. 
Hazelton JL, Balan I, Elmer GI, et al. Hyperoxic reperfusion after global cerebral ischemia promotes 
inflammation and long-term hippocampal neuronal death. Journal of neurotrauma. Apr 2010;27(4):753-
762. 
15. 
Liu Y, Rosenthal RE, Haywood Y, Miljkovic-Lolic M, Vanderhoek JY, Fiskum G. Normoxic ventilation 
after cardiac arrest reduces oxidation of brain lipids and improves neurological outcome. Stroke; a 
journal of cerebral circulation. Aug 1998;29(8):1679-1686. 
16. 
Zwemer CF, Whitesall SE, D'Alecy LG. Cardiopulmonary-cerebral resuscitation with 100% oxygen 
exacerbates neurological dysfunction following nine minutes of normothermic cardiac arrest in dogs. 
Resuscitation. Mar 1994;27(2):159-170. 
17. 
Danilov CA, Fiskum G. Hyperoxia promotes astrocyte cell death after oxygen and glucose deprivation. 
Glia. May 2008;56(7):801-808. 
18. 
Vereczki V, Martin E, Rosenthal RE, Hof PR, Hoffman GE, Fiskum G. Normoxic resuscitation after 
cardiac arrest protects against hippocampal oxidative stress, metabolic dysfunction, and neuronal 
death. J Cereb Blood Flow Metab. Jun 2006;26(6):821-835. 
19. 
Mickel HS, Vaishnav YN, Kempski O, von Lubitz D, Weiss JF, Feuerstein G. Breathing 100% oxygen 

16 
 
after global brain ischemia in Mongolian Gerbils results in increased lipid peroxidation and increased 
mortality. Stroke; a journal of cerebral circulation. Mar-Apr 1987;18(2):426-430. 
20. 
Fiskum G, Danilov CA, Mehrabian Z, et al. Postischemic oxidative stress promotes mitochondrial 
metabolic failure in neurons and astrocytes. Annals of the New York Academy of Sciences. Dec 
2008;1147:129-138. 
21. 
Richards EM, Fiskum G, Rosenthal RE, Hopkins I, McKenna MC. Hyperoxic reperfusion after global 
ischemia decreases hippocampal energy metabolism. Stroke; a journal of cerebral circulation. May 
2007;38(5):1578-1584. 
22. 
Dyson A, Stidwill R, Taylor V, Singer M. The impact of inspired oxygen concentration on tissue 
oxygenation during progressive haemorrhage. Intensive care medicine. Oct 2009;35(10):1783-1791. 
23. 
Lu J, Dai G, Egi Y, et al. Characterization of cerebrovascular responses to hyperoxia and hypercapnia 
using MRI in rat. Neuroimage. May 1 2009;45(4):1126-1134. 
24. 
Kilgannon JH, Jones AE, Shapiro NI, et al. Association between arterial hyperoxia following 
resuscitation from cardiac arrest and in-hospital mortality. Jama. Jun 2 2010;303(21):2165-2171. 
25. 
Kilgannon JH, Jones AE, Parrillo JE, et al. Relationship between supranormal oxygen tension and 
outcome after resuscitation from cardiac arrest. Circulation. 2011;(in press). 
26. 
Bellomo R, Bailey M, Eastwood GM, et al. Arterial hyperoxia and in-hospital mortality after resuscitation 
from cardiac arrest. Crit Care. Mar 8 2011;15(2):R90. 
27. 
Nolan JP, Soar J, Zideman DA, et al. European Resuscitation Council Guidelines for Resuscitation 
2010 Section 1. Executive summary. Resuscitation. Oct 2010;81(10):1219-1276. 
28. 
Trzeciak S, Jones AE, Kilgannon JH, et al. Outcome measures utilized in clinical trials of interventions 
for post-cardiac arrest syndrome: a systematic review. Resuscitation. Jun 2009;80(6):617-623. 
29. 
Roine RO, Kajaste S, Kaste M. Neuropsychological sequelae of cardiac arrest. Jama. Jan 13 
1993;269(2):237-242. 
30. 
Sauve MJ, Doolittle N, Walker JA, Paul SM, Scheinman MM. Factors associated with cognitive 
recovery after cardiopulmonary resuscitation. Am J Crit Care. Mar 1996;5(2):127-139. 
31. 
Tiainen M, Poutiainen E, Kovala T, Takkunen O, Happola O, Roine RO. Cognitive and 
neurophysiological outcome of cardiac arrest survivors treated with therapeutic hypothermia. Stroke; a 
journal of cerebral circulation. Aug 2007;38(8):2303-2308. 
32. 
van Alem AP, de Vos R, Schmand B, Koster RW. Cognitive impairment in survivors of out-of-hospital 
cardiac arrest. American heart journal. Sep 2004;148(3):416-421. 
33. 
Becker LB, Aufderheide TP, Geocadin RG, et al. Primary outcomes for resuscitation science studies: a 
consensus statement from the American Heart Association. Circulation. Nov 8 2011;124(19):2158-
2177. 
34. 
Trzeciak S, Jones AE, Shapiro NI, et al. A prospective multicenter cohort study of the association 
between global tissue hypoxia and coagulation abnormalities during early sepsis resuscitation. Critical 
care medicine. Apr 2010;38(4):1092-1100. 
35. 
Jones AE, Shapiro NI, Trzeciak S, Arnold RC, Claremont HA, Kline JA. Lactate clearance vs central 
venous oxygen saturation as goals of early sepsis therapy: a randomized clinical trial. Jama. Feb 24 
2010;303(8):739-746. 
36. 
Pope JV, Jones AE, Gaieski DF, Arnold RC, Trzeciak S, Shapiro NI. Multicenter study of central 
venous oxygen saturation (ScvO(2)) as a predictor of mortality in patients with sepsis. Ann Emerg Med. 
Jan 2010;55(1):40-46 e41. 
37. 
Gaieski DF, Band RA, Abella BS, et al. Early goal-directed hemodynamic optimization combined with 
therapeutic hypothermia in comatose survivors of out-of-hospital cardiac arrest. Resuscitation. Apr 
2009;80(4):418-424. 
38. 
Gaieski DF, Fuchs B, Carr BG, et al. Practical implementation of therapeutic hypothermia after cardiac 
arrest. Hosp Pract (Minneap). Dec 2009;37(1):71-83. 
39. 
Jones AE, Focht A, Horton JM, Kline JA. Prospective external validation of the clinical effectiveness of 
an emergency department-based early goal-directed therapy protocol for severe sepsis and septic 
shock. Chest. Aug 2007;132(2):425-432. 
40. 
Kilgannon JH, Roberts BW, Stauss M, et al. Use of a standardized order set for achieving target 
temperature in the implementation of therapeutic hypothermia after cardiac arrest: a feasibility study. 
Acad Emerg Med. Jun 2008;15(6):499-505. 

17 
 
41. 
Shapiro NI, Howell MD, Talmor D, et al. Implementation and outcomes of the Multiple Urgent Sepsis 
Therapies (MUST) protocol. Critical care medicine. Apr 2006;34(4):1025-1032. 
42. 
Trzeciak S, Dellinger RP, Abate NL, et al. Translating research to clinical practice: a 1-year experience 
with implementing early goal-directed therapy for septic shock in the emergency department. Chest. 
Feb 2006;129(2):225-232. 
43. 
Peberdy MA, Callaway CW, Neumar RW, et al. Part 9: post-cardiac arrest care: 2010 American Heart 
Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. 
Circulation. Nov 2 2010;122(18 Suppl 3):S768-786. 
44. 
Post MW, de Bruin A, de Witte L, Schrijvers A. The SIP68: a measure of health-related functional status 
in rehabilitation medicine. Arch Phys Med Rehabil. May 1996;77(5):440-445. 
45. 
Rittenberger JC, Raina K, Holm MB, Kim YJ, Callaway CW. Association between Cerebral 
Performance Category, Modified Rankin Scale, and discharge disposition after cardiac arrest. 
Resuscitation. Aug 2011;82(8):1036-1040. 
46. 
Jacobs I, Nadkarni V, Bahr J, et al. Cardiac arrest and cardiopulmonary resuscitation outcome reports: 
update and simplification of the Utstein templates for resuscitation registries: a statement for healthcare 
professionals from a task force of the International Liaison Committee on Resuscitation (American 
Heart Association, European Resuscitation Council, Australian Resuscitation Council, New Zealand 
Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart Foundation, 
Resuscitation Councils of Southern Africa). Circulation. Nov 23 2004;110(21):3385-3397. 
47. 
Langhelle A, Nolan J, Herlitz J, et al. Recommended guidelines for reviewing, reporting, and conducting 
research on post-resuscitation care: the Utstein style. Resuscitation. Sep 2005;66(3):271-283. 
48. 
Farrell B, Godwin J, Richards S, Warlow C. The United Kingdom transient ischaemic attack (UK-TIA) 
aspirin trial: final results. J Neurol Neurosurg Psychiatry. Dec 1991;54(12):1044-1054. 
49. 
van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J. Interobserver agreement for the 
assessment of handicap in stroke patients. Stroke; a journal of cerebral circulation. May 
1988;19(5):604-607. 
50. 
Quinn TJ, Lees KR, Hardemark HG, Dawson J, Walters MR. Initial experience of a digital training 
resource for modified Rankin scale assessment in clinical trials. Stroke; a journal of cerebral circulation. 
Aug 2007;38(8):2257-2261. 
51. 
Wilson JT, Hareendran A, Grant M, et al. Improving the assessment of outcomes in stroke: use of a 
structured interview to assign grades on the modified Rankin Scale. Stroke; a journal of cerebral 
circulation. Sep 2002;33(9):2243-2246. 
52. 
Wilson JT, Hareendran A, Hendry A, Potter J, Bone I, Muir KW. Reliability of the modified Rankin Scale 
across multiple raters: benefits of a structured interview. Stroke; a journal of cerebral circulation. Apr 
2005;36(4):777-781. 
53. 
Stead LG, Wijdicks EF, Bhagra A, et al. Validation of a new coma scale, the FOUR score, in the 
emergency department. Neurocritical care. 2009;10(1):50-54. 
54. 
Wijdicks EF, Bamlet WR, Maramattom BV, Manno EM, McClelland RL. Validation of a new coma scale: 
The FOUR score. Annals of neurology. Oct 2005;58(4):585-593. 
55. 
Christie JD, Biester RC, Taichman DB, et al. Formation and validation of a telephone battery to assess 
cognitive function in acute respiratory distress syndrome survivors. Journal of critical care. Jun 
2006;21(2):125-132. 
56. 
Mikkelsen ME, Christie JD, Lanken PN, et al. The adult respiratory distress syndrome cognitive 
outcomes study: long-term neuropsychological function in survivors of acute lung injury. Am J Respir 
Crit Care Med. Jun 15 2012;185(12):1307-1315. 
57. 
Mikkelsen ME, Shull WH, Biester RC, et al. Cognitive, mood and quality of life impairments in a select 
population of ARDS survivors. Respirology. Jan 2009;14(1):76-82. 
58. 
Needham DM, Dinglas VD, Bienvenu OJ, et al. One year outcomes in patients with acute lung injury 
randomised to initial trophic or full enteral feeding: prospective follow-up of EDEN randomised trial. 
Bmj. 2013;346:f1532. 
59. 
Needham DM, Dinglas VD, Morris PE, et al. Physical and cognitive performance of patients with acute 
lung injury 1 year after initial trophic versus full enteral feeding. EDEN trial follow-up. Am J Respir Crit 
Care Med. Sep 1 2013;188(5):567-576. 
60. 
Taichman DB, Christie J, Biester R, et al. Validation of a brief telephone battery for neurocognitive 

18 
 
assessment of patients with pulmonary arterial hypertension. Respiratory research. 2005;6:39. 
61. 
Roberts BW, Kilgannon JH, Chansky ME, et al. Multiple organ dysfunction after return of spontaneous 
circulation in postcardiac arrest syndrome. Crit Care Med. Jun 2013;41(6):1492-1501. 
62. 
Jones AE, Trzeciak S, Kline JA. The Sequential Organ Failure Assessment score for predicting 
outcome in patients with severe sepsis and evidence of hypoperfusion at the time of emergency 
department presentation. Critical care medicine. May 2009;37(5):1649-1654. 
63. 
Trzeciak S, McCoy JV, Phillip Dellinger R, et al. Early increases in microcirculatory perfusion during 
protocol-directed resuscitation are associated with reduced multi-organ failure at 24 h in patients with 
sepsis. Intensive care medicine. Dec 2008;34(12):2210-2217. 
64. 
Sciences NIoGM. Randomized trial of inhaled nitric oxide to augment tissue perfusion in sepsis 
(NCT00608322).  clinicaltrials.gov. Accessed May 1, 2011. 
65. 
Doria A, Wojcik J, Xu R, et al. Interaction between poor glycemic control and 9p21 locus on risk of 
coronary artery disease in type 2 diabetes. JAMA : the journal of the American Medical Association. 
Nov 26 2008;300(20):2389-2397. 
66. 
Egi M, Bellomo R, Stachowski E, et al. The interaction of chronic and acute glycemia with mortality in 
critically ill patients with diabetes. Critical care medicine. Jan 2011;39(1):105-111. 
67. 
Finney SJ, Zekveld C, Elia A, Evans TW. Glucose control and mortality in critically ill patients. JAMA : 
the journal of the American Medical Association. Oct 15 2003;290(15):2041-2047. 
68. 
Jacobson AM, Musen G, Ryan CM, et al. Long-term effect of diabetes and its treatment on cognitive 
function. The New England journal of medicine. May 3 2007;356(18):1842-1852. 
69. 
Murray SB, Bates DW, Ngo L, Ufberg JW, Shapiro NI. Charlson Index is associated with one-year 
mortality in emergency department patients with suspected infection. Acad Emerg Med. May 
2006;13(5):530-536. 
70. 
Gaul GB, Gruska M, Titscher G, et al. Prediction of survival after out-of-hospital cardiac arrest: results 
of a community-based study in Vienna. Resuscitation. Oct 1996;32(3):169-176. 
71. 
Hajbaghery MA, Mousavi G, Akbari H. Factors influencing survival after in-hospital cardiopulmonary 
resuscitation. Resuscitation. Sep 2005;66(3):317-321. 
72. 
Langhelle A, Tyvold SS, Lexow K, Hapnes SA, Sunde K, Steen PA. In-hospital factors associated with 
improved outcome after out-of-hospital cardiac arrest. A comparison between four regions in Norway. 
Resuscitation. Mar 2003;56(3):247-263. 
73. 
Nadkarni VM, Larkin GL, Peberdy MA, et al. First documented rhythm and clinical outcome from in-
hospital cardiac arrest among children and adults. Jama. Jan 4 2006;295(1):50-57. 
74. 
Peberdy MA, Kaye W, Ornato JP, et al. Cardiopulmonary resuscitation of adults in the hospital: a report 
of 14720 cardiac arrests from the National Registry of Cardiopulmonary Resuscitation. Resuscitation. 
Sep 2003;58(3):297-308. 
75. 
Tok D, Keles GT, Toprak V, Topcu I. Assessment of in-hospital cardiopulmonary resuscitation using 
Utstein template in a university hospital. Tohoku J Exp Med. Apr 2004;202(4):265-273. 
76. 
Davis DP, Idris AH, Sise MJ, et al. Early ventilation and outcome in patients with moderate to severe 
traumatic brain injury. Crit Care Med. Apr 2006;34(4):1202-1208. 
77. 
Del Castillo J, Lopez-Herce J, Matamoros M, et al. Hyperoxia, hypocapnia and hypercapnia as 
outcome factors after cardiac arrest in children. Resuscitation. Dec 2012;83(12):1456-1461. 
78. 
Roberts BW, Kilgannon JH, Chansky ME, Mittal N, Wooden J, Trzeciak S. Association between 
postresuscitation partial pressure of arterial carbon dioxide and neurological outcome in patients with 
post-cardiac arrest syndrome. Circulation. May 28 2013;127(21):2107-2113. 
79. 
Roberts BW, Kilgannon JH, Chansky ME, Mittal N, Wooden J, Trzeciak S. Response to letter regarding 
article, "association between postresuscitation partial pressure of arterial carbon dioxide and 
neurological outcome in patients with post-cardiac arrest syndrome". Circulation. Jan 7 
2014;129(1):e10.