Carbon Dioxide (CO2): A Pilot Study of a Hypothesized Mechanism to Explain Cognitive Impairment
Phase: NA
Conditions: Healthy
Sponsor: Rutgers, The State University of New Jersey

Study Protocol 
 
Carbon Dioxide (CO2): A Pilot Study of a Hypothesized Mechanism to Explain Cognitive 
Impairment 
NCT 05292378 
November 18, 2024 

INTERVENTIONAL
RESEARCH PROTOCOL TEMPLATE
(HRP-503a)
STUDY INFORMATION

Title of Project:
Carbon Dioxide: A Pilot Study of a Hypothesized Mechanism to Explain Cognitive
Impairment

Principal Investigator Name
Howard M. Kipen, MD, MPH

Principal Investigator Div. & Dept.
EOHSI/Environmental and Occupational Health
Rutgers School of Public Health

Principal Investigator Contact Info:
Kipen@eohsi.rutgers.edu
EOHSI Room 208
170 Frelinghuysen Road
Piscataway, New Jersey 08854
848-445-6091

Protocol Version and Date:
V10, 09.30.2024
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Protocol Title: CO2 and Cognition
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Table of Contents
Skip To Section: Hold CTRL + Click (Below) To Follow Link in Blue
1.0
Research Design
1.1
Purpose/Specific Aims
1.2
Research Significance
1.3
Research Design and Methods
1.4
Preliminary Data
1.5
Sample Size Justification
1.6
Study Variables
1.7
Drugs/Devices/Biologics
1.8
Specimen Collection
1.9
Data Collection
1.10
Timetable/Schedule of Events
2.0
Project Management
2.1
Research Staff and Qualifications
2.2
Research Staff Training
2.3
Other Resources
2.4
Research Sites
3.0
Multi-Center Research
4.0
Subject Considerations
4.1
Subject Selection and Enrollment Considerations
4.2
Obtaining Identifiable Information About Non-Subjects
4.3
Number of Subjects
4.4
Consent Procedures
4.5
Special Consent Populations
4.6
Economic Burden and/or Compensation For Subjects
4.7
Risks of Harm/Potential for Benefits to Subjects
5.0
Special Considerations
5.1
Health Insurance Portability and Accountability Act (HIPAA)
5.2
Family Educational Rights and Privacy Act (FERPA)
5.3
Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations)
5.4
General Data Protection Regulation (GDPR)
5.5
 NJ Access to Medical Research Act (Surrogate Consent) 
6.0
Data Management Plan
6.1
Data Analysis
6.2
Data Security
6.3
Data Safety And Monitoring
6.4
Reporting Results
6.5
Secondary Use of Data
7.0
Research Repositories – Specimens and/or Data
8.0
Approvals/Authorizations
9.0
Bibliography
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1.0 Research Design
1.1 Purpose/Specific Aims
The purpose of the study is to provide a mechanistic basis for the experimental phenomenon of CO2-
induced cognitive impairments in humans. Our highly novel focus is on biochemical changes caused 
by CO2 through its interactions with reactive oxygen and nitrogen species (RONS). We speculate that 
elevated levels of CO2 alter the actions of RONS within cells and that this leads to changes in their 
function.
A.
Objectives 
a.
Use the Strategic Management Simulations (SMS) test, the Stroop Color-Word Test (SCWT), 
and the Wisconsin Card Sorting Test (WCST) to analyze the effects of CO2 on cognitive 
function in healthy volunteers.
b.
Determine whether CO2 inhalation results in nitrosative and nitrative modification of target 
biomolecules in polymorphonuclear leukocytes (PMNs) and whether this correlates with PMN 
activation and oxidative stress.
c.
Determine whether CO2 inhalation results in cerebral vascular leak and whether this correlates 
with PMN activation and oxidative stress. 
d.
In Phase 3 - determine whether CO2 inhalation results in cerebral vascular changes in vessel 
diameter and blood flow and whether this correlates with PMN activation and oxidative stress. 
In this pilot study our goal is to demonstrate the feasibility of using a novel noninvasive measure 
conjunctival these vascular changes to correlate with data we already collect in a, b, and c 
above.
B.  Hypotheses / Research Question(s)
We hypothesize that exposure of humans to inhaled CO2 at common indoor levels will lead to 
cognitive decrements due to CO2-mediated nitrosation and nitration of intracellular biomolecules in 
PMNs, which cause cellular activation and oxidative stress.
1.2 Research Significance
Carbon dioxide (CO2) is expired by people at a concentration of 40,000 ppm and is ubiquitous. Indoors, 
CO2 accumulates in proportion to the number of individuals in a room or enclosed space, making indoor 
levels substantially greater than outdoor levels. CO2 itself is generally considered non-toxic at levels up 
to 5,000 ppm, the federal standard for workplaces set by OSHA (U.S. Department of Labor). Based on 
this, guidelines for the International Space Station and U.S. submarines are to maintain CO2 
concentrations <5,000 ppm (National Research Council 2007). Levels up to 2,000 ppm are commonly 
measured in schools, businesses and homes, and levels up to 4,500 ppm are detectable in cars (Hudda 
and Fruin 2018) or inside the face masks of health care workers’ N95 respirators (Roberge, Coca et al. 
2010). CO2 levels below 1,000 ppm are widely regarded as acceptable (U.S. EPA , Roberge, Coca et 
al. 2010, Allen, MacNaughton et al. 2019) despite the fact that indoor values in excess of 1,000 ppm 
are strongly correlated with non-specific symptoms of sick building syndrome, as well as poor school 
performance (Seppanen, Fisk et al. 1999, Fisk 2017). High CO2 is commonly regarded only as an 
indicator of inadequate ventilation rather than a threat to health (National Research Council 2007). 
Since 2012 four groups have published ten papers using varying experimental designs to explore acute 
(~2 hr) effects of real-world CO2 concentrations (600-2,500 ppm) on cognitive performance of human 
subjects (Roberge, Coca et al. 2010, Kajtár and Herczeg 2012, Satish, Mendell et al. 2012, Allen, 
MacNaughton et al. 2016, Zhang, Wargocki et al. 2016, Liu, Zhong et al. 2017, Zhang, Wargocki et al. 
2017, Rodeheffer, Chabal et al. 2018, Allen, MacNaughton et al. 2019). Four of the studies reported an 
association between CO2 and decreased cognitive performance (Kajtár and Herczeg 2012, Satish, 
Mendell et al. 2012, Allen, MacNaughton et al. 2016, Allen, MacNaughton et al. 2019). All of these were 
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rigorous (within subject cross-over, double blind) studies and two demonstrated concentration-
dependent impairment using a highly sensitive, validated measure of cognitive function, the Strategic 
Management Simulations (SMS) (Satish, Mendell et al. 2012, Allen, MacNaughton et al. 2016, Azuma, 
Kagi et al. 2018). A third rigorous study was performed more realistically, and provocatively, with 
professional airline pilots in a flight simulator (Allen, MacNaughton et al. 2019). Conversely, the null or 
inconclusive studies used less demanding traditional neuropsychological tests, non-within subject 
designs, and/or impure CO2 (Roberge, Coca et al. 2010, Zhang, Wargocki et al. 2016, Liu, Zhong et al. 
2017, Zhang, Wargocki et al. 2017, Rodeheffer, Chabal et al. 2018).
Without a fundamental mechanism, one cannot properly assess the harm that may follow CO2 
exposure. Our proposed studies are focused on elucidating mechanisms at both the physiological and 
biochemical level that underlie cognitive impairments associated with CO2 exposure in humans. These 
mechanistic human studies are highly significant as there are major implications to systemically 
substandard performance at school, on aircraft, and in business. We intend to move the science beyond 
observation so that this important public health concern can be addressed both scientifically and at a 
regulatory level.
Chemically, CO2 lacks direct toxic reactivity. However, due to the dielectrics of the molecule, the central 
carbon is susceptible to nucleophiles such as superoxide anion, nitric oxide and peroxynitrite, 
generating caged radical intermediates. The reaction of CO2 with peroxynitrite generates a carbonate 
and nitrogen dioxide coordinated intermediate (Pryor, Lemercier et al. 1997, Zhang, Squadrito et al. 
1997) that favors nitrosation and nitration of targets over simple oxidation of targets such as glutathione 
(Zhang, Squadrito et al. 1997, Squadrito and Pryor 1998, Uppu, Lemercier et al. 1998). This can have 
a major impact on signal transduction pathways and cellular responses (Gow, Farkouh et al. 2004). 
This is supported by findings that treatment of isolated human PMNs with CO2, at realistic levels, 
enhances cellular activation as measured by an increased oxidative burst and the release of 
IL1βcontaining microparticles (Thom, Bhopale et al. 2017). Similar effects on PMNs were observed in 
rodents following CO2 inhalation (2,000-4,000 ppm), a response correlated with brain vascular leak 
(Thom, Bhopale et al. 2017). Importantly, vascular damage in the brain was abrogated by pre-treatment 
of mice with IL1β antibodies or an IL1β receptor antagonist. Moreover, when naïve mice were injected 
with IL1β containing microparticles derived from CO2 treated PMNs, vascular damage was 
recapitulated, while destruction of microparticles abrogated the vascular injury with no change in PMN 
activation. These data are in accord with reports that IL1β and other members of the IL1 family (e.g., 
IL18, IL33) induce vascular leak (Fahey and Doyle 2019). These findings suggest a causal link between 
PMN activation by CO2, microparticle/IL1β generation, changes in cerebral vessels and blood flow, and 
cognitive impairments, consistent with the relationship between inflammation and cognition (Shields, 
Moons et al. 2017, Jacobson, Kler et al. 2019), which we propose to investigate in humans. Inclusion 
of functional magnetic resonance imaging (fMRI) will provide visualization and enhanced 
characterization of the cerebral vascular leakage predicted by our hypothesized mechanism of CO2-
mediated cognitive toxicity. In Phase 3, the addition of computer-assisted intravital microscopy (CAIM) 
will provide noninvasive visualization of a representative part of the cerebral vasculature, giving further 
insight into the hypothesized mechanism. Positive results from our high risk-high reward translational 
studies will provide important mechanistic data on CO2 and impaired cognitive function that may lead 
to consideration of revisions to current acceptable CO2 exposure levels. 
We contend that CO2 mediates changes in the reactivity of reactive nitrogen and oxygen species 
(RONS) and that this increases PMN activation. Therefore, baseline oxidative stress defenses in the 
subjects may affect the response to CO2. In this context, the status of glutathione S-transferase, 
(GSTM1), a phase II antioxidant enzyme, may be critical to responsiveness. The GSTM1 null genotype, 
lacking GSTM1 protein, is associated with increased risk of adverse health effects due to exposure to 
air pollutants as we and others have shown (Schwartz, Park et al. 2005, Lee, Kim et al. 2022). GSTM1 
has been reported to be null in approximately 50% of Caucasians, African American, and Asian 
populations, consistent with the diverse recruiting pool of subjects in New Jersey and providing greater 
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power for statistical analysis than other GST’s with lower null frequencies (Mo, Gao et al. 2009). Based 
on these frequencies in our recruitment pool, we believe, GSTM1 genotype represents the best variable 
to examine a hypothesis that antioxidant status and thus oxidative stress are mechanistically important 
for understanding how CO2 could affect cognition though inflammation.
1.3 Research Design and Methods
The study is a crossover trial. Subjects and outcome assessors will be blinded to the CO2 exposure 
level. The study will be done in two phases. The main endpoint for Phase 1 will be markers of PMN 
activation and oxidative stress in blood samples. In Phase 2, fMRI will be added to the study 
procedures, and fMRI evidence of cerebral vascular leak will be added as an endpoint. In Phase 3, we 
are no longer using fMRI, but we have added computer-assisted intravital microscopy (CAIM), and 
evidence of changes in cerebral vessel diameter and blood flow will be added as endpoints
Research Procedures – Phase 1, 2, and 3
Subjects will participate in two 2.5-hour exposure sessions in the Controlled Environment Facility 
(CEF) located in the Rutgers Environmental and Occupational Health Sciences Institute (EOHSI) 
building. Concentrations of CO2 during the exposure session will be either 600 ppm (control) or 
2500 ppm (exposure). Concentrations in the CEF will be continuously monitored during the 
exposure, and while keeping ventilation constant, the flow rate will be adjusted to maintain CO2 
levels. The order of the exposure sessions will be randomized and the exposure sessions will be 
at least one week apart. Up to four subjects will participate in each exposure session.
During the exposure session, each subject will have their own desk and laptop computer for the 
neurobehavioral testing and be visually and audibly (via headphones) isolated from the other 
subject. Since face masks have been shown to increase the concentration of carbon dioxide (Rhee, 
Lindquist et al. 2021), subjects will be asked to remove face masks while they are in the chamber. 
Subjects will be acclimatized in the CEF for 60 minutes with the selected CO2 exposure 
concentration already stabilized in the chamber atmosphere before they enter. During the last 30 
minutes of acclimatization, subjects will be instructed on the neurobehavioral Strategic 
Management Simulations (SMS) test. Otherwise, they may read/study quietly without music or 
phone calls. The neurobehavioral testing will consist of the SMS test (about 1.25 hours) and the 
SCWT (Phase 1 – about 5 minutes) or the WCST (Phase 2 – about 15 minutes).
Height and weight will be measured at the first study visit.
Subject symptoms and air quality perceptions will be assessed via questionnaires before entering 
the chamber and following each exposure session.
Venous blood will be collected from subjects at three time points at each visit: immediately before 
entering the chamber (30 mL), immediately after the exposure (10 mL), and approximately four 
hours after exposure ended (30 mL).
In Phase 2 only, after each exposure, subjects will undergo a 60-minute fMRI scan during which 
they will participate in Multi-Source Interference Task (MSIT).
A.
In Phase 3 only, after each exposure, subjects will have up to five minutes of video recorded of the 
white of the eye using computer-assisted intravital microscopy (CAIM) CAIM is a non-invasive 
technology that quantifies microcirculation abnormalities. Data Points
SMS, SCWT, and WCST scores will be collected at each exposure. Biomarkers of PMN activation 
and oxidative stress will be measured before and after each exposure. In Phase 2, the presence of 
cerebral vascular leak will be assessed by fMRI. In Phase 3, microcirculation abnormalities will be 
recorded using CAIM and later analyzed in the lab of Dr. Joshua Miller.
B.
Study Duration
The study is expected to take two years. Each subject will participate in 2 study visits for a total 
time of approximately 16 hours. In Phase 3, each subject will participate in 2 study visits, for a total 
time of approximately 8 hours.
C.
Endpoints - NA
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1.4 Preliminary Data
Indoor CO2 levels were recently measured in 29 homes in New Jersey (Pro2020001323; A Randomized 
Crossover Trial of Portable Air Cleaners to Reduce Coronavirus Exposures at Home). The average 
CO2 concentration was 825 ppm with three homes having average levels exceeding 1500 ppm. Two 
homes were observed to have sustained periods (>1 hour) of exposure to levels exceeding 2500 ppm.
1.5 Sample Size Justification
Twelve subjects will be enrolled in phases 1 and 2 of the study. The sample size was based on finding 
a difference in in PMN activation and oxidative stress. Using published data (Thom, Bhopale et al. 
2017) and assuming that ex vivo experiments are conducted on repeated samples from the same 
individuals and an intra-subject correlation of 0.7, a sample size of 6 has over 95% power to detect an 
effect. An additional up to ten subjects will be enrolled in phase 3 of the study. The purpose of this 
phase 3 portion of the study is to demonstrate the feasibility of using the CAIM technique as an outcome 
variable in our studies. Significant results of the cognitive testing and PMN activation are already 
reported (Lu et al., ink press).  We do not anticipate statistically significant changes in this pilot 
investigation
1.6 Study Variables
A.   Independent Variables, Interventions, or Predictor Variables 
The independent variable is the CO2 concentration. Two levels of CO2 will be used during the 
exposure sessions: 600 ppm (control) and 2500 ppm (exposure).
We will also assess the GSTM1 genotype (present/absent) of the subject. In Phase 3 we will not 
assess the GSTM1 genotype of the subject.
B.  Dependent Variables or Outcome Measures 
The primary outcome measures are cognitive function assessed by SMS, SCWT, and WCST, 
biological markers of PMN activation and oxidative stress, and cerebral vascular leak assessed by 
fMRI with a 3D diffusion-prepared arterial spin labeling (ASL) perfusion sequence.
In Phase 3 of the study changes assessed in the bulbar conjunctiva as measured by the CAIM 
technique will be added as a primary outcome measure.
SMS testing assesses up to nine primary factors of cognitive function: basic activity level, applied 
activity, focused activity, task orientation, initiative, information search, information usage, breadth 
of approach, and basic strategy.
SCWT responses will assess error rates and response times. WCST results will be scored by the 
number of errors, perseverative responses, and completed categories.
Blood samples will be examined for oxidative burst, mitochondrial energy metabolism, and NLRP3 
inflammasome activity as reflected by activated caspase-1 and expression of markers of 
inflammasome activation (e.g., IL1β, IL18, MYD88). We will also assess microparticles (e.g., 
extracellular vesicles) in peripheral blood samples, their origin (e.g., PMNs), and their vasoactive 
cargo (e.g., IL1β, IL18, IL33) (4), as well as serum levels of these cytokines. A key component of 
our studies will be to examine changes in nitrosation and nitration of potential proteins (e.g., 
cytochrome c oxidase, aconitase, GAPDH, NFκB, PPARγ, PKCs) and lipids (e.g., nitro-oleic and 
nitro-linoleic acids) within PMNs. In Phase 1, RNA sequencing will be done to assess additional 
markers of PMN activation (at the pre-exposure and 4-hour post-exposure time points). 
1.7 Drugs/Devices/Biologics - NA
1.8 Specimen Collection
A.
Primary Specimen Collection
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
Types of Specimens: Blood will be collected at 3 time points for each exposure session (70 
mL total/exposure): pre-exposure (30 mL), post-exposure (10 mL), and 4 hours post-exposure 
(30 mL).

Annotation: Blood samples will be annotated with the study ID, the exposure session number, 
and the time point (pre and post). No identifiers will be used on the specimen containers.

Transport: Specimens collected pre-exposure and 4 hours post-exposure will be carried from 
EOHSI to Dr. Gow’s lab in the Ernest Mario School of Pharmacy by a research assistant. In 
Phase 1 only, after processing, an aliquot of RNA isolated from the PMNs will be shipped to 
Baylor College of Medicine for RNA sequencing on a fee-for-service basis. Shipping will be 
done by a laboratory assistant with current IATA training.

Processing: Laboratory assistants in Dr. Gow’s lab will process the blood collected pre-
exposure and 4 hours post-exposure. Specimens collected immediately post-exposure will be 
processed by research assistants at EOHSI, Room 145.

Storage: Processed specimens will be stored in -80oC freezers in EOHSI Room 232 until 
analysis in Dr. Gow’s lab or, in Phase 1 only, shipping to Baylor College of Medicine. Access 
to the room is controlled by key card.

Disposition: Specimens sent to Baylor University will be destroyed after all data have been 
reviewed and approved. Remaining specimens in Dr. Gow’s lab will be stored indefinitely for 
use only by the study investigators. The link between the study ID and the subject identity will 
be destroyed when the study is closed.
B.
Secondary Specimen Collection - NA
1.9 Data Collection
A.
Primary Data Collection

Location: The SMS, SCWT, and WCST data collection will take place in the controlled 
exposure facility (CEF) of Rutgers Environmental and Occupational Health Sciences Institute 
(EOHSI). In Phase 2, the fMRI will be done at the Center for Advanced Human Brain Imaging, 
119 Staged Research Building, Piscataway, NJ. In Phase 3, the SMS data collection will take 
place in the CEF at EOHSI, and digitized conjunctival images taken using the CAIM technique 
will take place in the EOHSI Clinical Center subject room (room 145).

Process of Data Collection: An online SMS test will be administered; a research assistant, 
trained in test administration, will provide instruction and be available to answer any questions 
during testing. Online versions of the SCWT and WCST will also be administered. The fMRI will 
follow a standard sequence (see below) to assess vascular leak.

Timing and Frequency: The tests will be administered once during each exposure session. 
The SMS test will take 75 minutes each time, and the SCWT will take 5 minutes. In Phase 2, 
the WCST will take 15 minutes. The fMRI will take approximately 1 hour.

Procedures for Audio/Visual Recording: NA

Study Instruments: The neurobehavioral SMS test is a highly sensitive measurement of higher 
order decision-making that has been used successfully to assess cognitive effects of allergy 
medication and alcohol, as well as exposure to CO2 (Streufert, DePadova et al. 1988, Streufert, 
Pogash et al. 1994, Breuer and Satish 2003, Satish, Streufert et al. 2004, Satish, Mendell et al. 
2012, Azuma, Kagi et al. 2018, Allen, MacNaughton et al. 2019).  Another advantage of SMS is 
that parallel forms are available which can be used to control for practice effects from repeated 
administrations. Importantly, numerous publications support SMS test retest reliability (r = 0.72 
to 0.94) for parallel forms of the test (Streufert, DePadova et al. 1988, Streufert, Pogash et al. 
1994, Breuer and Streufert 1995, Satish, Streufert et al. 2004, Satish, Mendell et al. 2012, Allen, 
MacNaughton et al. 2016), as well as predictive validity of SMS for real world situations such as 
surgical residents’ decision making (Satish, Streufert et al. 2001). Once the task commences, 
messages appear on the computer with instructions and participants are asked to respond with 
a drop-down menu of possible decisions. Although the same amount of information is presented 
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at fixed time points to each participant during the session, they are free to make decisions at 
any time, similar to real world situations. Scores are generated by the computer, based on 
previous factor analysis to reflect the participant’s performance on the following measures: 
Activity – number of actions taken; Speed – speed of response to information; Responsiveness: 
orientation to task; Initiative: actions with creativity; Information: activities to seek information; 
Emergency: actions in response to emergency events; Breadth: use of multiple approaches for 
problem solving; Planning: plans for future action; Strategy: multiple interactive activities toward 
complex goals (Streufert, DePadova et al. 1988, Satish, Mendell et al. 2012).
The SCWT is a commonly used measure of executive function (Scarpina and Tagini 2017). The 
test consists of three conditions (naming the color of dots, of neutral words, and of the color of 
words printed in incongruent colors). Each condition consists of 24 items. Error rates and 
response times are evaluated.
The WCST is another commonly used measure of executive function and cognitive flexibility 
(Miles, Howlett et al. 2021). We will use the computerized 64-item version.
For fMRI, a 3D diffusion-prepared pseudo-continuous arterial spin labeling (ASL) perfusion 
sequence allows mapping of water exchange across the blood-brain barrier (BBB) without 
contrast, facilitating identification of vascular leak (Shao et al., 2018;). After removal of any 
metal, each participant will be positioned in a Siemens 3T Siemens Prisma with a 64-channel 
head coil. After a 1-minute scout navigator scan is collected for positioning of scans, a 5-minute 
structural T1-weighted scan will be collected. This will be followed by an approximately 10-
minute scan to determine BBB diffusion and blood oxygenation level dependent (BOLD) 
imaging of the Multi-Source Interference Task (MSIT). The MSIT is a validated fMRI task that 
reliably and robustly activates a subject’s cingulo-frontal-parietal cognitive/attention network 
(CFP network) to further assess cognition (Bush and Shin, 2006). An additional 14 minutes of 
resting-state fMRI to measure functional connectivity will be collected at the end of the scan. 
During all scans, a trained MRI technician will observe the participant through a large window 
and communicate by two-way microphone.

Ethnographic Studies, Interviews, Or Observation: NA

Subject Identifiers: Tests will be coded by subject ID and exposure session number.
B.
Secondary Data Collection - NA
1.10 Timetable/Schedule of Events - NA
2.0 Project Management
2.1 Research Staff and Qualifications
Howard Kipen, MD, MPH, Professor, Environmental and Occupational Health, Rutgers School of Public 
Health. Dr. Kipen specializes in controlled exposure-based experimental studies of individuals’ 
responses to changes in air pollutants, such as we propose here. He has led multiple federally funded 
field studies such as those proposed here including a recently completed study with portable air 
cleaners.
Andrew Gow is a Professor of Pharmacology at Rutgers University. He has 30 years of experience in 
academic research and has held positions at Princeton University, Temple University, University of 
Pennsylvania, and Duke University. Dr. Gow’s primary research focus is nitric oxide chemistry and he 
has published extensively on the mechanisms of cardiac hypertrophy, environmental effects on nitric 
oxide signaling, pulmonary function and mechanisms of disease, and the role of nitric oxide in 
controlling oxygen delivery and inflammation. Dr. Gow’s work has led to the understanding of S-
nitrosothiols as a major factor in controlling nitric oxide function and has yielded technologies related 
to a variety of pathologies including persistent pulmonary hypertension of the newborn, the 
regeneration of stored blood, cystic fibrosis, sickle cell anemia, and asthma. Dr. Gow holds a BSc 
(Biochemistry) from the University of Edinburgh, a MSc in Physical Education, and a PhD in Exercise 
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Physiology from Temple University. He has published over 120 papers and has been cited over 7,500 
times with an h-index of 41. He has given invited lectures across the world, including Japan, Germany, 
Russia, and the UK. Dr. Gow is a chartered chemist and a member of the American Physiological 
Society, the American College of Sports Medicine, AAAS, ASPET, SOT, and ATS; as well as being a 
past president of the International Nitric Oxide Society. He is a former personal trainer and rugby player.
Nancy Fiedler, Professor, Department of Environmental and Occupational Health, Rutgers School of 
Public Health and Deputy Director of the Environmental and Occupational Health Sciences Institute.  
Dr. Fiedler is a clinical psychologist and routinely performs neuropsychological evaluations of workers 
and community members who report symptoms related to neurotoxicant exposures.  
David Zald, PhD, Henry Rutgers Professor of Psychiatry and Director of the Center for Advanced 
Human Brain Imaging Research (CAHBIR). He received training as a neuropsychologist and 
neuroimaging researcher. He has been conducting functional neuroimaging research for over a quarter 
of a century. 
Joshua Miller, PhD, Professor and Chair in the Department of Nutritional Sciences in the School of 
Environmental and Biological Sciences. His recent research primarily focuses on the relationships 
among vitamins (B12, folate, B6, and D), homocysteine, and neurodegenerative diseases, age-related 
cognitive decline, and cancer. He has extensive experience in the measurement of various biomarkers 
of B vitamin status in large-scale epidemiological studies. He is currently subcontracted co-PI on an 
NIH-funded study of B12 status, cognitive decline, and incident dementia (AG059011 – PF Jacques, 
lead PI) and a subcontracted co-investigator on an NIH-funded intervention trial of vitamin D 
supplements to slow cognitive decline in older adults (AG051618 – J Olichney, PI). With respect to the 
present proposal, he has co-authored papers utilizing computer-assisted intravital microscopy (CAIM) 
to assess the peripheral microvasculature in sickle cell disease patients, and now endeavors to expand 
this novel technology to the study of early nutritional and vascular biomarkers of brain injury and age-
related cognitive decline. His lab has purchased and operates a CAIM which will be transported to 
EOHSI for this pilot study.
Gina Roslan, BS, Graduate student in the Department of Nutritional Sciences. She has worked with 
computer-assisted intravital microscopy (CAIM) in Dr. Miller’s laboratory on a previous project and is 
trained in its use. Along with Dr. Miller, she will assist in the training of research staff on the operation 
of the CAIM.
Kathy Black, PhD, MPH, Senior Research Associate, Rutgers Environmental and Occupational Health 
Sciences Institute. Dr. Black will act as the study coordinator and will be responsible for the training 
and supervision of the study research assistants. Dr. Black has over 20 years of experience 
coordinating environmental health studies, including Dr. Kipen’s recent study using air cleaners in 
homes.
2.2 Research Staff Training
Dr. Kipen and Dr. Black will review all study procedures with the personnel prior to any enrollment. Dr. 
Fiedler will train the study staff to administer both the SMS and VST. Dr. Black will directly supervise 
the study research assistants and review the study records to ensure that consent has been properly 
documented and all data recorded. Dr. Kipen will meet weekly with the study team to discuss study 
progress. In Phase 2, Dr. Zald, director of the CAHBIR, will oversee the MRI protocol. The person 
running the MRI scanner must have received certified training to run the MRI scanner and must have 
Level 3 safety training consistent with the operating procedures of the CAHBIR. Any assistants on the 
project must have at least Level 2 safety training. The person reviewing the screening form must have 
at least Level 2 training and is required to notify the person running the scanner that the review sheet 
had no items of note or that the sheet requires secondary review if any questions on the review sheet 
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were scored in the affirmative. Dr. Zald will oversee the overall study, whereas the person operating 
the scanner directly oversees the MRI data collection during the session itself. In Phase 3, Dr. Miller 
and Gina Roslan will train study staff in the procedures for CAIM.
2.3 Other Resources 
Laboratory: Dr. Gow occupies approximately 1500 sq. ft. of newly renovated laboratory space in the 
Ernest Mario School of Pharmacy building (room 009) on the Busch Campus of Rutgers University; this 
laboratory, along with 2000 sq ft of adjacent laboratories (room 001) are available to perform laboratory 
studies outlined in this application. The School of Pharmacy is connected by an enclosed walkway to 
the Rutgers Environmental and Occupational Health Sciences Institute (EOHSI), which houses the 
laboratories, equipment and facilities for performing human exposure studies (see Clinical) below). Dr. 
Gow’s laboratories are fully equipped with all of the basic and specialized equipment needed for the 
proposed, including analytical balances, pH meters, refrigerators, freezers, low and high speed 
centrifuges, water baths, gel electrophoresis equipment and power supplies, perfusion pumps, 
histology stations, a thermocycler, a hybridization oven, spectrophotometer, fluorescence plate reader, 
Shandon cytocentrifuge, microcentrifuge, and microwave, and an NOA-280i nitric oxide analyzer. Dr. 
Gow’s laboratories have dedicated for tissue culture facilities which include laminar flow hoods (4), 
incubators (4), cell culture and fluorescent microscopes with digital imaging. Also available for use in 
the laboratories are desk top computers and printers for data analysis and processing. Major shared 
equipment in the School of Pharmacy and the EOHSI building available for this project include 
ultracentrifuges, freezers, scintillation and gamma counters and gel scanners. Common cold rooms 
and dark rooms are also available for use.
Controlled Environmental Facility (CEF): Human CO2exposures will be conducted in the EOHSI 
CEF under the direction of Dr. Laurent.  The CEF consists of three major components: 1) the exposure 
chamber; 2) the air pollutant generation system; and 3) the control systems. The CEF is a large 
stainless steel room (7.3 ft. high x 13.5 ft. wide x 9 ft. deep; volume=887 cubic feet) in which the air 
flow, temperature and humidity can be varied and well controlled.  The airflow rate through the CEF 
can vary from 100 to 700 ft3/min. The CEF operating temperature range is from 55° to 80° ± 1° F.  The 
relative humidity range is 10% to 80% ± 2%.  A divider can separate the chamber into two work station 
compartments, so that two subjects can be exposed simultaneously.  For the 600 ppm exposure, 
ambient air that supplies the exposure chamber will be treated using a series of processes, which 
include cooling/heating, humidification/dehumidification, and filtration through a carbon filter, a KMnO4 
purifier, and HEPA filters. The clean air enters the CEF through two diffusers located in the ceiling and 
exits through the perforated stainless steel floor without recirculation. Eight small brushless fans (to 
prevent unwanted particle generation from brush degradation) are used to ensure that the air is well 
mixed at the edges and throughout the stainless steel chamber. The temperature and relative humidity 
will be maintained at 72±1ºF and 45±2%, respectively, during each exposure session.  While the CEF 
can be operated under either a positive or negative pressure of 0.1 inches of water, a negative pressure 
will be used to avoid CO2 releases from the CEF to the surrounding rooms. A high purity (>99.999) CO2 
gas cylinder will be used to generate the desired CO2 level within the CEF. The tank will be directly 
connected to a port leading to the inlet air for the CEF prior to the baffle system that is designed to mix 
the target agent(s) with the intake air before entering the CEF. The CO2 concentration will be 
continuously monitored by the CEF operator to be within 10% of the target value and the tank flow rate 
will be adjusted using a fine needle valve to maintain that level throughout the exposure scenario. Since 
people are a source of CO2, adjustments to the gas flow will be made as needed across the exposure 
session based on the number of people in the CEF and their breathing levels. In addition, there is a 
one-way mirror and a microphone so the participants can be monitored to verify that they are not feeling 
any distress. If the CO2 level is not maintained or distress is indicated, then the experiment is stopped 
with the subjects brought out of the exposure session and evaluated by a physician from EOHSI clinic 
in the same building. Subjects enter the chamber through an airlock with two doors, one opening to the 
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outer room and the other opening into the chamber. Each door has a sensor which, when the door is 
opened, activates a sliding bolt to the other door, preventing it from being opened.  When the door is 
closed, the sensor deactivates the bolt and either door can then be opened. This prevents both doors 
from being opened simultaneously and thus prevents air exchange between the outer room and the 
chamber. There is a 4-foot wide emergency exit in the front of the chamber leading directly to the outer 
room. The latches on the doors are designed to prevent anyone from being locked in the room. Other 
safety features include a smoke detector, a sprinkler system and battery operated emergency lighting. 
Researchers can view subjects participating in human exposure studies through a 4 x 6 ft. one-way 
mirror in the front of the CEF. There is a system for continuous electrocardiograph recording and 
monitoring of subjects during exposure. There is a lavatory in the chamber for extended exposure 
studies. 
The Clinical Center for Environmental and Occupational Health: The EOHSI Clinical Center is the 
leading facility in NJ for workplace health. Directed by Dr. Kipen, there are 8 nurses and 4 FTE 
physicians in the EOHSI Clinical Center. The Clinical Center provides a wide variety of services 
including diagnosis, treatment and rehabilitation related to workplace exposures and injuries, clinical 
toxicology consultation, management of medical surveillance programs, biomonitoring for toxins, and 
evaluation of indoor air quality. The EOHSI Clinical Center also provides medical monitoring and 
treatment for ~2,500 WTC rescue, recovery and clean-up workers as part of a consortium of eight 
NIOSH designated Centers of Excellence. The clinical staff occupies space two floors below the CEF 
in the EOHSI building. Clinical Center staff will be on-call during exposures.
The Center for Advanced Human Brain Imaging: CAHBIR is equipped with a 3T Siemens Prisma 
with a 64-channel head coil. The center is equipped with a crash cart if a subject needs immediate 
intervention while at the scanner, and the MR technologist is trained in its use.
2.4 Research Sites
The study will take place at Rutgers-EOHSI (170 Frelinghuysen Road, Piscataway, NJ 08854). All 
data and specimen collection will take place at EOHSI including recruitment, CO2 exposure, 
neurobehavioral testing, and blood collection. 
Specimens will be analyzed at Rutgers-EMSOP (160 Frelinghuysen Road, Piscataway, NJ 08854). 
The fMRI will take place at the CAHBIR, 119 Staged Research Building, Piscataway, NJ.
3.0 Multi-Center Research - NA
4.0 Subject Considerations
4.1 Subject Selection and Enrollment Considerations 
A.
Method to Identify Potential Subjects
Subjects will be broadly recruited from the University and local communities. A screening 
questionnaire will be administered to determine eligibility.
B.
Recruitment Details
Approved flyers will be posted around the University and local community.  Study information will 
also be posted on the EOHSI website.  
If interested, subjects will call or email the study coordinator or research assistants. A screening 
questionnaire will be administered to determine eligibility. The screening questionnaire will be 
reviewed by the study physician for approval prior to scheduling a study visit. In addition to the 
screening questionnaire, a pregnancy test will be given to all female participants prior to each 
exposure session. The short-term CO2 exposures used in this study are seen in everyday life, 
including schools and workplaces, and are not expected to cause clinically significant or persistent 
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health effects. Phase 2 participants will be given an additional screening questionnaire for fMRI 
compatibility to be completed before the MRI scan. No additional physical screening will be used. 
Pregnancy and current asthma have been included in the exclusion list out of an abundance of 
caution. 
C.
Subject Screening

Inclusion Criteria
Inclusion criteria include:

Age between 18 and 30 years (inclusive)

History of COVID-19 vaccination

Weigh at least 110 pounds

Exclusion Criteria
Exclusion criteria include:

Colorblindness

Inability to hear verbal instructions

Cardiovascular disease, including a history of stroke

Diabetes requiring the use of insulin

Pregnancy

Current asthma (an asthma attack within the past five years)

History of a severe anxiety or panic disorder

Medications for anxiety disorder

Ever experienced a panic attack

Medications which may affect cognition such as beta-blockers and CNS depressants

Respiratory symptoms in the previous 4 weeks

Use of sedating cold/allergy medications in the previous week

Use of marijuana in the previous week

Consumption of alcohol in the previous 24 hours
Additional exclusion criteria for Phase 2 participants are:
•
History of head trauma or neurosurgery or neurological disorder
•
Ferrous material implanted in or on the body, including surgical clips, bullets, electrical devices 
such as a pacemaker, or nonremovable ferrous jewelry (fillings in teeth and permanent retainers 
are permitted). 
•
Individuals with surgical pins or plates above the neck are excluded. Surgical pins or plates below 
the neck are exclusions, except when the material is fixed to bone, and considered acceptable by 
the Reference Manual for Magnetic Resonance Safety. Implants and Devices, 2020 Edition. Almost 
all recent orthopedic implants are made of materials that are not ferromagnetic and therefore are 
safe for scanning, and even though some screws are still made of ferromagnetic materials these 
are firmly screwed into bone. In cases where the material is unknown or deemed unsafe for 
scanning by the Reference Manual for Magnetic Resonance Safety. Implants and Devices, 2020 
Edition, the participant will be excluded.
•
History of eye injury involving metallic materials, shavings in eyes, or welding without a face mask
•
Lead/iron tattoos
•
Claustrophobia (history of significant anxiety in closed places). 
•
Back problem that would prevent the subject from laying still comfortably for up to 90 minutes. 
•
Deafness
Additional exclusion criteria for Phase 3 participants only:
•
Contra-indications for CAIM
o
Inability to keep head still and eyes open during CAIM recordings  
o
Current or recent contact lens use 
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D.
Privacy Protections
Prospective subjects must respond to posted study information by contacting investigators. 
Individuals will not be approached for participation. Contact information collected during recruitment 
will be stored in the REDCap database, accessible only to the study team.
4.2 Obtaining Identifiable Information About Non-Subjects - NA
4.3 Number of Subjects
A. Total Number of Subjects
34 subjects will complete the study. To compensate for voluntary withdrawals, up to 44 subjects may 
be enrolled.
B. Total Number of Subjects If Multicenter Study - NA
C. Feasibility
Given the size of the University community, broad inclusion criteria, and the small number of 
subjects, we expect to be able to complete testing in a timely manner.
4.4 Consent Procedures
A. Consent Process

Location of Consent Process
Screening will take place over the phone. If the subject is on any medications, the list of 
medications will be reviewed by the study physician (Dr. Kipen) to determine eligibility. If 
eligible, an appointment will be made for a study visit. Subjects will be consented in the EOHSI 
Clinical Center (Room 145). The consent will be given to the study subject and the subject will 
be asked to review the consent prior to the study visit. At the start of the first exposure visit, the 
consent will be reviewed with the subject and signed by both the study participant and the 
investigator. After consent, the subject’s height and weight will be collected and, if female, a 
pregnancy test will be administered.

Ongoing Consent
Study activities will be discussed with the subject at the start of each exposure visit. All 
questions will be answered.

Individual Roles for Researchers Involved in Consent
Screening and consenting may be done by the coordinator or a research assistant. 

Consent Discussion Duration
The consent process will begin during screening when a brief explanation of the study is given 
to the subject. After screening, the consent form will be given to the subject to review prior to 
the exposure study visit. At a minimum, the subject will be given at least overnight to review 
the consent, but most appointments will be scheduled at least several days in advance. At the 
first exposure visit, the consent form will be reviewed with the subject and all questions will be 
answered before signing; this discussion is expected to take approximately 30 minutes.

Coercion or Undue Influence
Subjects are not approached individually, rather the study will be discussed only with subjects 
responding to a study flyer.

Subject Understanding
The key study elements will be specifically mentioned during the consent discussion and all 
questions will be answered.

Protecting Privacy
Consent discussions will take place in a private office prior to participation. No additional 
information about the subject will be collected until after consent is obtained.
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B.
Waiver or Alteration of Consent Process - NA
C.
Documentation of Consent

Documenting Consent
The consent form will be stored in REDCap. Both the participant and study investigator will 
sign the consent form. A copy of the signed consent form will be emailed to the participant.

Waiver of Documentation of Consent (i.e., will not obtain subject’s signature) - NA
4.5 Special Consent Populations
A.
Enrolling Minors-Subjects Who Are Not Yet Adults - NA
B.
Enrolling Wards of the State - NA
C.
Enrolling Non-English-Speaking Subjects - NA
D.
Enrolling Adults Lacking Decision-Making Capacity (Surrogate Consent) - NA
E.
Special Consent Considerations - NA
4.6 Economic Burden and/or Compensation for Subjects
A. Expenses - NA
B.
Compensation/Incentives
Subjects will be paid $100 for each exposure visit (up to $200 total).
C Compensation Documentation
Compensation will be documented in the REDCap database. Additionally, subjects will be asked 
to sign a voucher. Copies of the voucher will be stored in a locked cabinet in a private office 
(Room 147) at EOHSI.
. 
4.7 Risks of Harm/Potential for Benefits to Subjects
A.
Description of Risks of Harm to Subjects

Reasonably Foreseeable Risks of Harm
Carbon dioxide exposure: CO2 itself is generally considered non-toxic at levels up to 5,000 
ppm, the federal standard for workplaces set by OSHA ((U.S. Department of Labor). The CO2 
levels used in the exposures will not exceed those often indoors in conference rooms and 
schools; these levels have been used previously to assess changes in cognitive function with 
no adverse events. There are no known persistent sequelae to acute exposures in healthy 
individuals. Subjects may increase their respiratory rate during the high CO2 exposure, but this 
is transient. Inhalation of CO2 at approximately 35,000 ppm will reliably induce panic attack 
symptoms in those with a history of such attacks, but this is not reported at the order-of-
magnitude lower concentrations that we will use.
Venipuncture: Slight pain, some bleeding, or bruising may occur when blood is drawn. A total 
of 100 ml of blood (50 ml per study visit) will be required for the planned assays.  
Phase 3 only:
MRI Risks: There are no known health risks associated with the magnetic field produced by 
the 3.0T scanner in healthy adults. The FDA has indicated that they consider MR imaging on 
machines up to 4.0T to pose no risk. Exposure to high magnetic fields is associated with 
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primary or secondary risks in certain patient populations (e.g., patients with pacemakers), but 
all such patient populations are excluded. Subjects with aneurysm clips, neural stimulators, 
possible metal fragments in the eyes, cochlear implants, artificial cardiac valves, iron based 
facial tattoos, and body piercings that are not removable are excluded from participation. The 
only other risks associated with scanning are claustrophobia while in the magnet, physical 
discomfort from lying still in the magnet, and the loud sound of the magnet.

Incidental findings: The MRI exam could reveal a neural abnormality. Learning of such an 
abnormality could cause psychological distress. 

Risk of Harm from an Intervention on a Subject with an Existing Condition - NA
The study will not enroll subjects with existing conditions.

Other Foreseeable Risks of Harm
There is a risk of possible loss of confidentiality, but no sensitive information is collected in 
the study.

Observation and Sensitive Information - NA
B.
Procedures which Risk Harm to Embryo, Fetus, and/or Pregnant Subjects - NA
C.
Risks of Harm to Non-Subjects - NA
D.
Assessment of Social Behavior Considerations - NA
E.
Minimizing Risks of Harm

All subjects will be screened prior to scheduling, and the responses will be reviewed by the 
study physician PI prior to enrollment. Subjects with a diagnosis of anxiety disorder, history of 
panic attacks, or taking medication for anxiety disorder are excluded from the study, again out 
of an abundance of caution, as our concentrations are not known to be associated with this 
outcome. All female subjects will receive a urine pregnancy test prior to exposure. Subjects will 
be informed in the consent document that they will be given the results of the pregnancy test. 
Although the CO2 exposure levels used in the study are common in schools, many indoor 
workplaces, and submarines and spacecraft, subjects will be informed that these levels have 
been associated with temporary declines in cognitive function. During the 2-hour exposure, 
subjects will be visually monitored and can speak with the technicians at will.  Physician 
consultation is available if needed. In Phase 2, subjects will be asked to complete a standard 
online screening form at the Brain Health Institute to confirm eligibility for the fMRI. An MRI 
screening questionnaire is used to rule out the presence of any medical problems that would 
cause a risk in the magnet. The participants’ answers to the screening questionnaire are 
reviewed by the scan operator who must have completed Level 2 Safety Training in accordance 
with CAHBIR Standard Operating Procedures. Claustrophobia in the magnet is limited by 
excluding subjects with a history of claustrophobia. Subjects are excluded who do not feel they 
could comfortably lie still in the scanner, or comfortably wear the passive shim mouthpiece. 
Subjects wear headphones to reduce the sound of the magnet. Minor physical comforts are 
reduced by use of comfortable pillows for neck and leg support while in the scanner. 
Communication is maintained with the subject through use of a microphone and headphones. 
The subject is also given a button to press should they decide to stop the experiment or capture 
the experimenter’s attention.  All scans at the Center for Advanced Human Brain Imaging 
Research (CAHBIR) are screened by the center’s MRI technologist. If there is any sign of an 
abnormality (liberal criteria), the scan data is reviewed by a certified neuroradiologist through 
a contract with University Radiology Group (URG). 
If the URG neuroradiologist reports that an abnormality may be clinically significant and 
recommends referral or further evaluation, the study PI will communicate this finding to the 
participant if it is deemed clinically significant. Note: participants are not shown or told about 
any potential abnormality until after the neuroradiologist’s evaluation to avoid any unnecessary 
distress given that the majority of observations in a nonclinical young adult population are 
clinically nonsignificant. 
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In phase 3 we introduce computer assisted intravital microscopy (CAIM). There are no known 
risks associated with CAIM, which is very similar to a slit-lamp exam done at routine optometry 
exams.

Certificate of Confidentiality - NA

Provisions to Protect the Privacy Interests of Subjects
All data and specimen will be coded by a Study ID. All identifiable information will be stored in 
REDCap and accessible only to the study team.
F.
Potential Direct Benefits to Subjects
There is no direct benefit to study subjects.
5.0 Special Considerations
5.1 Health Insurance Portability and Accountability Act (HIPAA) – NA
5.2 Family Educational Rights and Privacy Act (FERPA) - NA
5.3 Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations) - NA
5.4 General Data Protection Regulation (GDPR) - NA
5.5 NJ Access to Medical Research Act (Surrogate Consent) - NA
6.0 Data Management Plan
6.1 Data Analysis
Means, standard deviations, percentiles and histograms will summarize the distributions of outcome 
variables. For skewed distributions, transformation such as the logarithmic distribution will be taken to 
stabilize variance. Initially, multivariate analysis of variance (MANOVA) will be used to examine 
differences between exposures in the behavioral outcomes measured during exposure and transposed, 
if necessary, with a random effect to control for correlation between the same outcome across exposure 
sessions. If a significant effect of exposure is detected, post-hoc analyses will include separate 
ANOVAs for each outcome again focusing on the effect of the exposure on the outcomes measured at 
the end of each exposure session. Sensitivity analyses will control for any baseline characteristics in 
these analyses. Other biomarkers will also be studied using analysis of variance. 
6.2 Data Security
All study data will be stored in REDCap is a secure, password-protected web-based application 
designed to support data capture for research studies. A unique study participant ID will be assigned 
in REDCap at the time of screening. Specimens, for both Dr. Gow’s lab and Baylor University, and SMS 
data will be coded only with the study ID and Exposure visit number. All data used for analysis will be 
fully de-identified upon data export from REDCap and stored on an encrypted, password-protected 
computer network to which only study investigators and staff have access. De-identification of data will 
consist of removal of all personal identifiers from analytic files. Investigators will keep the data, including 
links to personal identifiers, until study closure.  Collaborators at Baylor University will not have access 
to identifiers.
In Phase 2, the MRI form is kept in a locked file cabinet and is only accessible by the staff of the Center 
for Advanced Human Brain Imaging Research and individuals related to Rutgers University who are 
involved in research safety oversight. CAHBIR has contracted with Flywheel and Google Cloud 
Services to provide an encrypted cloud-based data transfer, preprocessing, and archiving environment 
for MRI data, which allows access to numerous automated data quality control and processing steps, 
with 24/7 support. All neuroimaging data will be archived on Google Cloud via the contract with 
Flywheel. 
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In Phase 3, the data collected using CAIM will be stored in university secure restricted Box folder and 
will only be accessible to authorized research personnel.
6.3 Data and Safety Monitoring - NA
6.4 Reporting Results
A.
Individual Subjects’ Results 
Individual results will not be given to the subject because no clinically relevant results will be 
obtained during the study. In Phase 2, T1-weighted scan pictures of the brain are shared with the 
participants who complete the MRI. 
All scans at the Center for Advanced Human Brain Imaging Research (CAHBIR) are screened by 
the center’s MRI technologist. If there is any sign of an abnormality (liberal criteria), the scan data 
is reviewed by a certified neuroradiologist through a contract with University Radiology Group 
(URG). If the URG neuroradiologist reports that an abnormality may be clinically significant and 
recommends referral or further evaluation, the study PI will communicate this finding to the 
participant if it is deemed clinically significant. Note: participants are not shown or told about any 
potential abnormality until after the neuroradiologist’s evaluation to avoid any unnecessary distress 
given that the majority of observations in a nonclinical young adult population are clinically 
nonsignificant. Incidental findings are shared with the subject only if the neuroradiologist 
determines that they warrant further evaluation or treatment.
B.
Aggregate Results
Research results will be posted on the EOHSI website.
C.
Professional Reporting
Results will be presented at scientific conferences and published in peer-reviewed literature.
D.
Clinical Trials Registration, Results Reporting and Consent Posting
The study is a Basic Experimental Study Involving Humans (BESH) and will be registered on 
clinicaltrials.gov. As required, the consent form and results will be posted. 
6.5 Secondary Use of the Data - NA
7.0 Research Repositories – Specimens and/or Data
Remaining specimens at Rutgers University will be stored for future research only by the study 
investigators; they will not be distributed to others. The specimens will be stored in -80oC freezers at EOHSI 
Room 232. Freezer temperatures are monitored remotely, and notifications are sent in cases of malfunction. 
Access to the freezer room is controlled by key card. 
8.0 Approvals/Authorizations - NA
9.0 Bibliography
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Donnelly-McLay and J. D. Spengler (2019). "Airplane pilot flight performance on 21 maneuvers in a flight 
simulator under varying carbon dioxide concentrations." J Expo Sci Environ Epidemiol 29(4): 457-468.
Allen, J. G., P. MacNaughton, U. Satish, S. Santanam, J. Vallarino and J. D. Spengler (2016). "Associations 
of Cognitive Function Scores with Carbon Dioxide, Ventilation, and Volatile Organic Compound 
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Breuer, K. and S. Streufert (1995). The strategic management simulation (SMS): a case comparison 
analysis of the German SMS version. Corporate Training for Effective Performance. M. Mulder and R. O. 
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Rodeheffer, C. D., S. Chabal, J. M. Clarke and D. M. Fothergill (2018). "Acute Exposure to Low-to-
Moderate Carbon Dioxide Levels and Submariner Decision Making." Aerosp Med Hum Perform 89(6): 
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bioeffluents on perceived air quality, self-assessed acute health symptoms, and cognitive performance." 
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