Investigating Hypercapnia to Treat Neurogenic Orthostatic Hypotension
Phase: NA
Conditions: Orthostatic Hypotension, Neurogenic Orthostatic Hypotension
Sponsor: University of Calgary

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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Investigating Functional Optical Coherence Tomography and 
Hypercapnia to Diagnose and Treat Neurogenic Orthostatic Hypotension 
 
Principal Investigator: 
Satish R. Raj, MD MSCI FRCPC 
Professor of Cardiac Science 
Libin Cardiovascular Institute of Alberta 
Cummings School of Medicine 
Phone: 403-210-6152 Fax: 403-210-9444 
Email: satish.raj@ucalgary.ca 
 
Co-Principal Investigator: 
Richard Wilson PhD 
 
Co-Investigators: 
Robert S Sheldon MD PhD FRCPC 
Carlos Morillo MD FRCPC 
Jacquie Baker PhD 
 
Sponsor:  
Libin Cardiovascular Institute  
 
 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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1.0 BACKGROUND 
1.1 Neurogenic Orthostatic Hypotension 
Regulation of tissue blood supply to vital organs such as the brain is met in large part by 
local adjustment of the microvasculature (autoregulation) and autonomic nervous system control 
of the cardiovascular system. Neurogenic Orthostatic Hypotension (NOH) is a cardinal example 
of when these systems fail, specifically, the reflexive responses mediated by the sympathetic 
branch of the autonomic nervous system. NOH is a debilitating condition associated with 
reduced quality of life, impaired function and is also an independent predictor of mortality1,2. 
NOH is clinically defined as a sustained reduction in systolic blood pressure (SBP) ≥20 mmHg 
or diastolic blood pressure of ≥10 mmHg within 3 minutes of standing or head-up tilt performed 
at 60° without an appropriate compensatory postural tachycardia3.  
Significant and persistent orthostatic hypotension can lead to inadequate cerebral blood flow, 
reduced cerebral perfusion pressure and orthostatic symptoms including light-headedness, pre-
syncope, and in some cases, syncope4. As cerebral blood flow and perfusion pressure are crucial 
factors in normal brain health and function, failure to adequately control these variables may 
significantly impact the functionality of essential brain networks. 
 
1.2 Physiology: The Normal Orthostatic Response 
Upon assuming the upright posture, there is an instantaneous shift of 500 to 1000 mL of 
blood to the capacitance vessels in the lower extremities and splanchnic circulation5. Venous 
return to the heart is decreased6 and therefore stroke volume and arterial pressure decrease7. 
These changes trigger the baroreflex via unloading of high-pressure arterial baroreceptors in the 
carotid sinus and aortic arch. This causes a reflex increase in sympathetic efferent nerve activity 
that increases heart rate and systemic vascular resistance, and concomitantly suppress 
parasympathetic activity to the heart, which also causes an increase in heart rate7. Hormonal 
mechanisms (e.g. activation of renin-angiotensin and endothelin systems) and skeletal muscles 
are also engaged during prolonged standing to maintain blood pressure (BP)8. In a healthy 
individual, HR initially increases by 10-20 bpm, and the systolic BP decreases by approximately 
5 mmHg7. Any abnormality in these autonomic or neurohumoral reflex pathways can result in 
altered hemodynamic responses during standing.  
Baroreflex is a homeostatic mechanism responsible for short-term regulation of arterial 
blood pressure, but the chemoreflex is also an important modulator of sympathetic activity. It has 
been shown that the interplay between these two mechanisms is a complex issue, and baroreflex 
both influences9–11 and is influenced by chemoreflex activation12. It has been demonstrated that 
changes in baroreceptor activity modulate ventilatory responses to chemoreceptor 
stimulation13,14.  
 
1.3 Chemoreflex  
The respiratory chemoreflex is an important modulator of sympathetic and parasympathetic 
activity, and consists of two types of receptors: central and peripheral. Central chemoreceptors 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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are located primarily in the medulla, and respond primarily to increases in PaCO2 (hypercapnia) 
and concomitant acidosis. The peripheral chemoreceptors are located in the aortic and carotid 
bodies, and respond to arterial hypoxia and hypercapnia14,15, with synergistic effects. 
Respiratory chemoreceptors therefore modulate the autonomic nervous system via changes in 
blood O2 and CO2 concentration. The inhibition of K+ channels by hypercapnia, hypoxia, or 
acidosis, depolarizes chemosensitive neurons, and increases their firing rate16,17. The activation 
of either the hypoxic or hypercapnic chemoreflex elicits increased ventilation, parasympathetic 
and sympathetic activation.  
 
1.4 The interactions between the Chemoreflex and Baroreflex 
There is a wealth of research demonstrating interactions between the chemoreflex and 
baroreflex. Baroreceptors can modulate chemoreflex activity and ventilation. For example, 
ventilatory responses to chemoreceptor stimulation are augmented by arterial baroreceptor 
unloading (during hypotensive stimuli) and are depressed by baroreceptor loading (during 
hypertensive stimuli).  
The opposite relationship also exists, where chemoreflex activation can modulate the 
baroreflex-mediated cardiovascular responses to orthostasis. In 2001, Shoemaker et al.18 
observed that mild hypercapnia caused an attenuated sympathetic and vascular response to head-
up tilt in seven healthy individuals. However, BP was better maintained during hypercapnia than 
hypocapnia, via elevated cardiac output. Despite attenuated sympathetic activity, the authors 
proposed the improved BP response was attributed to enhanced venous return related to blood 
volume redistribution from splanchnic capacitance vessels. Furthermore, Howden et al.19, in 
2004, demonstrated that hypercapnic conditions in healthy subjects induced an increase in peak 
heart rate and time to peak heart rate, which combined to improve tolerance to orthostatic stress 
simulated by lower body negative pressure.  
Kazimierska et al.20 recently investigated baroreflex sensitivity during repeated transitions 
between squatting and standing in normocapnia compared to hypercapnia with hypoxia, in a 
group of 40 healthy young volunteers. Their results showed that hypercapnia with hypoxia was 
associated with slightly improved BP control during rapid transitions between body positions, 
which could indicate alterations in hemodynamic adaptation and autonomic control arising from 
chemoreflex activation. Therefore, chemoreflex activation might constitute an attractive, and 
novel, therapeutic option for NOH patients.  
 
1.6 Neurogenic Orthostatic Hypotension and CO2 
In healthy individuals, hypercapnia helps improve orthostatic BPs by augmenting stroke 
volume and improving cardiac output. Improved venous return may be facilitated by increased 
ventilation and changes in intrathoracic pressures, and/or the resulting improved central 
circulation. The latter mechanism is particularly important in autonomic failure as the 
splanchnic-mesenteric capacitance bed represents a large volume, low resistance reservoir that 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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constitutes 25-30% of total blood volume. Maintenance of postural normotension is clinically 
important and a common therapeutic target in orthostatic hypotension21.  
Although the literature surrounding the effects of CO2 in autonomic failure are sparse, Lipp 
et al.22 found the ventilatory responses to hypercapnia and hypoxia were preserved in patients 
with Multiple System Atrophy; a sporadic and progressive pre-ganglionic disorder characterized 
by loss of medullary sympathoexcitatory neurons. Patients demonstrated a SBP increase of 18 
mmHg in response to hypercapnia. 
In addition to improving orthostatic BP, hypercapnia may serve an equally important role in 
symptomatic management, cerebral autoregulation and maintenance of cerebral perfusion 
pressures in patients with NOH.  
 
1.5 Cerebral Autoregulation  
Regulation of tissue blood supply to vital organs, such as the brain, is met in large part by 
local adjustments of the microvasculature (autoregulation). In the brain, cerebral blood flow 
(CBF) is maintained over a wide range of systemic blood pressures via multiple overlapping 
autoregulatory processes, collectively known as cerebral autoregulation. Cerebral autoregulation 
is commonly modeled as static and dynamic, which describes the response of the brain to long-
term gradual and rapid alterations in BP, respectively23,24. 
In response to variations in perfusion pressure (mean arterial pressure minus intracranial 
pressure), an adaptation in cerebrovascular resistance will cause CBF to return to its baseline24. 
For instance, postural changes challenge cerebral autoregulation by reducing arterial pressure, 
cardiac output, and cerebral perfusion pressure. In response to reduced systemic pressures, 
cerebral vessels will dilate to maintain a relatively constant CBF. Standard orthostatic challenges 
are a common method to challenge cerebral autoregulation and better understand its role. 
Similarly, alterations in arterial blood gas partial pressures also influence the cerebral circulation. 
CO2 is a very potent cerebral vasodilator, and hypercapnia causes a 3% to 5% increase 
in CBF per mmHg rise in end-tidal CO2 (ETCO2). Hypocapnia induces vasoconstriction and can 
reduce CBF by 2% to 3% per mmHg reduction in ETCO2 25.  
Understanding the role of cerebral autoregulation in autonomic failure remains elusive. For 
example, patients with NOH experience orthostatic light-headedness, nausea, pre-syncope and 
even syncope, suggesting impaired cerebrovascular autoregulation and hypoperfusion. Some 
studies have reported impaired cerebral autoregulation in autonomic failure26, while other have 
found preserved autoregulation27–31 and both preserved and impaired autoregulation, depending 
on the underlying diagnosis32–34. The likely reason for these disparate findings is, in part, due to 
the considerable variation in techniques to quantify sympathetic activity and cerebral 
autoregulation. Techniques to measure cerebral autoregulation vary considerably, including the 
use of different collection tools, quantification methods and various algorithms to interpret and 
analyze different variables. Similarly, routine measurements of the microvasculature and 
sympathetic activity are lacking, or avoided as current methods are inaccurate (heart rate 
variability), lack temporal resolution (plasma norepinephrine), or are finicky and invasive 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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(microneurography)35. Microvascular techniques are equally impoverished. Functional MRI 
devices are costly for widespread use and have relatively poor temporal and spatial resolutions, 
and are often insufficient to directly resolve the microvasculature36. Laser Doppler Flowmetry 
provides non-invasive assessment of tissue microvascular hemoconcentration; however, the 
amount and depth of tissue penetration is not adequate for cerebral assessments. Therefore, 
calculation of total tissue flow is not possible37. Overall, the development of a clinically relevant 
method to assess cerebral autoregulation, sympathetic nerve activity and microvascular 
activity/reactivity is needed.  
 
1.6 Functional Optical Coherence Tomography 
To better understand the mechanistic underpinnings of NOH, and to explore the use of 
elevated Fractional Inspired CO2 (FiCO2) to treat it, we need a better way to monitor sympathetic 
activity and cerebrovascular perfusion. The imaging of cerebral tissue allows clinical 
investigation into a tissue bed, which is exquisitely sensitive to various vaso-active stimuli38. The 
eye is an extension of the brain with two distinct vascular beds: 1) The epiretinal vascular arcade 
supplies the superficial retinal layers resembling the circulation of the brain39,40. 2) The choroid, 
on the other hand is largely sympathetically regulated via α- and β-adrenergic receptors41,42, 
resembling peripheral vasculature.  
The imaging of vascular beds with such diverse regulation enables the assessment of 
microvascular autoregulatory and sympathetic mediated regulation, enabling a readout of 
microvascular and sympathetic activity. Optical coherence tomography (OCT) is an advanced 
imaging technology using near-infrared laser light providing images with a spatial resolution of 
4μm and scanning depth of 500μm43,44 within 1.5 seconds43,45,46. The fine resolution of OCT 
imaging parameters enables a resolution necessary to detect sensitive microvascular changes in 
response to sympathetic provocations. While OCT provides useful structural characteristics, 
Functional Optical Coherence Tomography (fOCT) provides information about tissue dynamics 
such as blood flow, oxygenation and vascular perfusion, expanding OCT’s clinical relevance. 
fOCT of the retinal and choroid vascular beds of the eye is being explored in Calgary to allow 
physiological monitoring of these essential variables. 
 
2.0 SIGNIFICANCE 
The effects of hypercapnia on CBF and the cardiovascular system, under orthostatic stress 
conditions, have been tested in a limited number of healthy subjects, with the net effect of 
improving orthostatic tolerance. Therefore, a novel solution to counter the acute effects of NOH 
is to transiently increase sympathetic activity by stimulating the peripheral and central 
respiratory chemoreceptors with elevated FiCO247,48.   
The results of this study will: 1) improve our understanding of the effects of hypercapnia in 
patients with NOH, and 2) provide robust evidence to support or reject the use of gas trapping 
devices in the management of NOH patients, which could translate to clinical settings adding 
options to manage symptoms and improve quality of life for patients. 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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Additionally, to better understand the cerebral autoregulatory underpinnings of NOH, and to 
explore the use of elevated FiCO2 to treat it, we need a better way to monitor sympathetic 
activity and cerebrovascular perfusion. Application of the recently developed fOCT techniques 
provides a completely novel method to measure sympathetic activity in this unique cohort, gain 
valuable insight into cerebral autoregulation and potentially provide diagnostic value. 
 
3.0 OUTCOMES, OBJECTIVES and HYPOTHESES 
This is a proof-of-concept study to evaluate hypercapnia as a novel therapeutic intervention 
to improve blood pressure and orthostatic tolerance in patients with NOH. In addition, we will 
aim to evaluate functional OCT as an advance, non-invasive tool to measure sympathetic and 
metabolic cerebrovascular control.  
The specific objectives of the current proposal are to apply hypercapnia during fOCT 
monitoring in patients with NOH and healthy controls to: (a) evaluate and compare the effects of 
hypercapnia on cardiovascular and cerebrovascular responses to better understand basic 
chemoreflex and baroreflex physiology in  patients with NOH, (b) determine if a device that 
transiently increases FiCO2 in response to postural changes will have efficacy as a non-drug 
therapeutic and (c) evaluate fOCT as a novel advanced tool to measure sympathetic and 
metabolic components of cerebral autoregulation in patients with autonomic failure. 
We hypothesize that: a) increasing FiCO2 will improve the cerebrovascular and 
cardiovascular response to standing in patients with NOH, b) we will be able to translate the 
results of the current study to a clinical setting and c) fOCT will reliably distinguish between 
patients with NOH and healthy controls adding value as a novel tool to measure sympathetic 
control.  
 
 
 
 
4.0 INCLUSION/EXCLUSION CRITERIA:  
Inclusion Criteria (for all participants):  
• Age ≥18 years  
• Male and Female  
• Non - smokers. 
• Able and willing to provide informed consent. 
• Ability to travel to Libin Cardiovascular Institute Autonomic Testing Lab at the University of 
Calgary, Calgary, AB. 
 
Inclusion Criteria for Patients 
• Physician diagnosis of Neurogenic Orthostatic Hypotension  
 
Exclusion Criteria (for all participants):  
• Medical therapies or medications which could interfere with testing of autonomic function 
• Participants with somatization or severe anxiety symptoms will be excluded  
• Pregnant or breast-feeding females 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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• Inability to tolerate mask for the duration of the study  
• Subjects who require portable oxygen at rest or with exercise 
• Subjects with chronic heart failure or severe pulmonary disease who are unable to climb one 
flight of stairs due to shortness of breath.  
• Presence of failure of other organ systems or systemic illness that can affect autonomic function 
or the participant’s ability to cooperate. These include: dementia, alcohol and/or drug abuse, 
cerebrovascular disease, kidney or liver disease, surgical procedures where the nerves of the 
sympathetic nervous system have been cut. 
• Other factors which in the investigator’s opinion would prevent the participant from completing 
the protocol, including poor compliance during previous studies. 
 
Additional Exclusion Criteria for Healthy Controls 
• The presence of any dysfunction relating to the autonomic nervous system.  
In addition, in accordance with the University of Calgary requirements for in-person activities, 
all students, faculty, staff and visitors to campus, including research participants, are required to 
show proof of full vaccination status against COVID-19.  
 
5.0 INTERVENTION  
The RespirAct™ system (Thornhill Research Inc., Toronto, Canada) is a computer-controlled 
gas blender providing CO2, O2 and nitrogen for a subject to inhale while breathing for the 
purpose of controlling the concentrations of the respective blood gases. The RespirAct™ records 
inspired and exhaled gas concentrations, which can be recalled, analyzed and graphed. Data is 
collected breath-by-breath including end tidal O2 and CO2, the length of inspiration and 
expiration for the breath, the respiration rate and the tidal volume per breath. The RespirAct™ 
system is an investigational use device, distributed by Thornhill Research to collaborators for use 
in IRB/REB approved research studies, and has been utilized in several publications in healthy 
individuals and clinical populations49,50. Subjects will be fitted with a facemask, which will be 
connected to a tube supplied with gas from RespirAct™ system. 
 
6.0 ENROLLMENT, RANDOMIZATION and BLINDING  
6.1 Recruitment  
Patients will be recruited from the Calgary Autonomic Investigation and Management Clinic, 
and the practices of Drs. Satish R. Raj, Carlos A Morillo, and Robert S. Sheldon, including past 
research study participants. We will also recruit participants from the community who contact us 
directly about their interest in our studies. 
 
6.2 Randomization:  
Each study participant will complete 5 Active Stand Tests on a single day. Subjects will be 
fitted with a facemask connected to a tube supplied with gas from the RespirAct™ system: 
a) Free breathing: Normal (not coached) breathing (no ETCO2 clamp)  

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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b) Baseline: Normal not coached breathing with ETCO2 clamped at baseline levels 
c) Low Hypercapnia: Normal (not coached) breathing with ETCO2 clamped at +5 mmHg 
above eucapnia 
d) High Hypercapnia: Normal (not coached) breathing with ETCO2 clamped at +10 mmHg 
above eucapnia 
e) Hypercapnia + Hypoxia: Normal (not coached) breathing with ETCO2 clamped at +10 
mmHg above eucapnia and ETO2 clamped at 50 mmHg.   
 
The study will involve a partial randomization. The order of interventions b-d will be 
randomized to account for any possible effects associated with repeated Active Stand Tests. 
Randomization will be performed using a pre-prepared randomization table. Protocol A will 
always be performed first to determine individual eucapnic levels. Protocol E will always be 
performed last to due to the long lasting hemodynamic and sympathetic changes associated with 
hypoxia. 
 
7.0 STUDY PROCEDURE  
7.1 Informed Consent 
The informed consent will be sent by email to the patients in advance. We will give each 
patient a chance to ask questions about the protocol and have these questions answered to their 
satisfaction. Written informed consent will be documented prior to engaging in study-related 
procedures. 
 
7.2 Holding Pre-Existing Medications  
Participants will be asked to hold their clinical medications if possible, but this will not be 
mandatory. We will make note of the medications used by each participant at the time of the 
study.  
7.3 Study Day 
7.3.1 Instrumentation  
 Patient will be instrumented at least 2h post-prandial or fasting on an empty bladder. 
Participants will be asked to abstain from alcohol, caffeine and exercise for a period of 12 
hours prior to testing. Participants will be allowed to drink water on the morning of study.  
 Skin electrodes will be applied for continuous ECG and HR monitoring.  
 BP will be monitored continuously using a finger volume clamp method (using beat-to-beat 
blood pressure monitoring) and calibrated with intermittent brachial cuff measurements.  
 From the continuous BP waveform, we can get an estimate of stroke volume, cardiac output, 
and systemic vascular resistance (Modelflow, FMS, Amsterdam, Netherlands).  
 Middle cerebral artery blood flow velocity (CBFv) will be assessed using a digital 
transcranial Doppler system (PMD150 Unilateral digital transcranial Doppler, License 
number 73603). 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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 Subjects will be fitted with a facemask. The facemask will be connected to a tube supplied 
with gas from a programmable gas mixing system (RespirAct™; Thornhill Research Inc., 
Toronto, Canada). 
 Oxygen saturation and End-tidal CO2 will be monitored by the RespirAct™ system. 
 Data will be digitally sampled and collected digitally for offline analysis 
 
7.3.2 Study Design  
 Review procedure and reconfirm consent. 
 The participant will be asked about co-morbid disorders, and current pharmacological and 
non-pharmacological treatments for NOH.  
 The participant will be instrumented as discussed above 
 Participant will be seated comfortably  
 The participant will rest comfortably in a seated position for 10 minutes prior for baseline 
data collection  
 Each study participant will complete all 5 Active Stand Test in a single day. 
 
7.3.3 For each Active Stand Test (AST) 
 Physiological signals will be digitally sampled throughout the study  
 Up to 10-minute baseline data collection (seated position) 
 Subjects will be introduced to each gas condition while seated 1 min prior to the AST. 
 Participants will actively stand and remain upright for up to 5 minutes  
 Standing will be terminated early if systolic blood pressure falls below 70 mmHg, SpO2 
levels drop below 80%, or the patient requests the test be stopped. 
 At least 5-minute recovery data collection after returning to the seated position, or until 
normalization of ETCO2 to baseline rest level. 
 We will document hemodynamic parameters (BP, cardiac output, HR and CBFv) and 
respiratory parameters (tidal volume, respiratory rate, ETO2, and ETCO2) during seated 
position and each Active Stand Test. 
 We will assess acute orthostatic tolerance using the Vanderbilt Orthostatic Symptoms Score 
(VOSS) at the end of each Active Stand Test. 
 
 
7.3.4 ETCO2 Baseline Levels  

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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The baseline ETCO2 levels will be defined during the first 10 min baseline, and will be 
calculated from the mean ETCO2 over the last 5 min of first baseline phase.  
 
7.3.5 Specific Active Stand Test Protocols (AST):                                  
1. Free breathe: AST with Normal Breathing (no ETCO2 clamp): 
 ETCO2 will not be clamped (i.e. participants will breathe freely); they will stand for up to 
5 minutes                             
2. Baseline: AST with Normal Breathing (ETCO2 clamped at baseline):  
 The ETCO2 will be clamped at baseline levels. Participants will then be asked to stand for 
up to 5 min 
3. Hypercapnia: AST with Normal (Not Coached) Breathing and Low Hypercapnia 
 1 min before Active Stand Test, the ETCO2 will be ramped up to 5mmHg above eucapnic 
levels, the participant will then stand for up to 5 min 
4. Hypercapnia: AST with Normal (Not Coached) Breathing and High Hypercapnia  
 1 min before Active Stand Test, the ETCO2 will be ramped 10mmHg above eucapnic 
levels, the participant will then stand for up to 5 min                                                                                     
5. Hypoxia and Hypercapnia 
 1 min before Active Stand Test, the ETCO2 will be ramped up to 10mmHg above 
eucapnic levels and ETO2 will be ramped down to 50mmHg. Participant will then stand 
up for up to 5 min 
 
The study will be conducted in a human physiology research and procedure room. The study will 
take approximately 2.5 hours. 
 
8.0 STUDY OUTCOME MEASURES  
The primary outcome measures will be:  
1) The magnitude of the hemodynamic response (ΔHR/ΔBP difference between HR/BP from 
seated to peak HR/BP during standing) for each intervention.  
2) Choroid and retinal (surrogates for peripheral sympathetic activity and cerebral 
autoregulation, respectively) perfusion density and reactivity 
3) Comparison of NOH with control to determine the potential of fOCT to distinguish NOH 
patients from controls 
 
Secondary outcome measures will be the ΔCBFv upon standing and the VOSS symptom score 
for each intervention. Additional outcome measures will include ventilatory rate and tidal 
volume at different levels of ETCO2, and the changes when moving from seated to standing. 
- 
Seated parameters will be calculated as the mean value (HR, ETCO2, CBFv) between the 
8th and 9th minute of seated baseline. 
- 
Peak parameters during standing will be defined as the mean value during the first min 
and between the 2rd and 5th min (mean at each 30 seconds)  
- 
VOSS Symptom Rating at the end of each 5 min Active Stand Test 
  

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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9.0 STATISTICAL CONSIDERATIONS 
9.1 Primary Analysis  
The effects of ETCO2 level and posture on all measured variables will be assessed using a 
repeated measures two-way analysis of variance (ANOVA) procedure and post-hoc analysis.  
To determine diagnostic potential of fOCT, OCT scans (choroid and retinal perfusion 
densities) will be compared between NOH patient and healthy controls using an independent t-
test. 
 
9.2 Secondary Analysis  
The secondary analysis will compare the effects of ETCO2 level and posture on CBFv using 
a repeated measures two-way analysis of variance (ANOVA) procedure and post-hoc analysis. 
VOSS Symptom Rating will be completed at end of each 5 min Active Stand Test protocol and 
compared between ETCO2 levels in a similar manner. 
Other secondary analyses will include the magnitude of ΔETCO2 from seated to standing, 
and peak standing ETCO2 in the no gas intervention (Protocol D), during first min and between 
the 2rd and 5th min (mean at each 30 seconds) 
 
9.3 Sample Size Calculation  
There are limited existing data regarding the effect of hypercapnia in NOH patients. 
Therefore, we can only estimate the sample size for the current pilot study. Based on the limited 
number of previous studies in similar patient populations, whose sample sizes ranged from 7 to 
16, we aim to recruit 40 patients with NOH, and 40 healthy controls. This sample size will allow 
for additional sex-based comparisons 
 
10.0 RISKS and INCONVENIENCES 
BP cuff: Some may find the arm or finger cuffs uncomfortable, or to keep their arm in a 
relatively fixed position. 
 
Electrodes: Sticky electrodes will be put on the chest and the limbs to record electrical activity 
from your heart. This can occasionally cause a rash.  
 
Active Stand Test: There might be light-headedness, tremor, headache, nausea or feelings of 
faintness during active standing. These symptoms usually resolve rapidly upon return to the 
seated position. To ensure patient safety, hemodynamic (BP, HR, stroke volume) will be 
continuously monitored throughout the entire study, and standing will be terminated early if 
systolic blood pressure falls below 70 mmHg, SpO2 levels drop below 80%, or the patient 
requests the test be stopped. Expert personnel will be present at all times.  
 
Transcranial Doppler (TCD): Ultrasound gel will be applied which may cause a rash. The 
Doppler probe will be in direct contact with the head, which may be uncomfortable. The 
headband/apparatus will be snug, but there will be opportunities to loosen it off during the 
recovery periods, and the headband is adjustable.  

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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RespirAct: Subjects will be fitted with a facemask, which might be uncomfortable to the 
participant. The mask will be connected to a tube supplied with gas from an automated, non-
invasive programmable gas mixing system (RespirAct™ Thornhill Medical). Briefly, the 
RespirAct employs an automated gas blender to adjust the composition and flow of CO2 and O2 
to a sequential gas-delivery mask and breathing circuit. The RespirAct™ system is an 
investigational use device, distributed by Thornhill Research to collaborators for use in IRB/REB 
approved research studies, and has been utilized in several publications in healthy individuals 
and clinical populations. 
 
Breathing Protocol: The subjects will breathe three different inspiratory gas mixtures, which are 
not typically encountered in day-to-day life. Hypercapnia is similar to re-breathing air but not to 
a point where the oxygen is exhausted. The levels we will use are the same as in expired air 
during moderate exercise. Rebreathing will be a combination of both hypoxia (low oxygen) and 
hypercapnia as mentioned above. Hypoxia is similar to high altitude and affects cardiovascular 
and cardiorespiratory indices similarly, including increased heart rate and breathing. Some 
participants have described the sensation of a mild headache; however, this is diminished quickly 
upon cessation of the breathing exercise. Syncope is rare and uncommon in response to the 
composition of gases employed within this study. Hyperventilation may induce a light-headed 
sensation. This dissipates quickly upon return to normal ventilatory rate. To ensure patient 
safety, hemodynamic (BP, HR, stroke volume) will be continuously monitored throughout the 
entire study, and we will have expert personnel present at all times. 
 
Function Optical Coherence Tomography (fOCT): We will use a state-of-the-art commercially 
available OCT machine (Zeiss Cirrus 5000) that is used by ophthalmologists around the world. 
The OCT uses a weak and harmless near infra-red laser beam to illuminate the retina and 
choroid. Participants need to rest their chin and forehead on support bars which can be 
uncomfortable. However, we have added padding to support the chin and reduce discomfort. 
During scans, participants may also experience mild pain or eye fatigue equivalent to looking 
into a bright light. These sources of discomfort will be mitigated by positioning participants 
appropriately using back supports, placing the machine at the appropriate height using a table 
with fine height adjustment and limiting each scan to ~3-5 seconds. 
 
Tape allergy reaction: A tape will be applied to fix the mask to the participant’s face, which may 
cause a rash or mild discomfort when removing. 
 
Privacy Risks: Patient identifiers will be used in this study. The investigators will comply with 
the patient privacy guidelines of the University of Calgary and applicable provincial and federal 
rules. 
 
COVID-19: This study involves in-person interactions, and therefore will require increased time 
within a health care facility and increased exposure to other people  
  
We cannot foresee any other risks, but there may be previously unknown or unforeseen risks. 
 
 

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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11.0 DATA and SAFETY PLAN   
11.1 Adverse Event (or Unanticipated Problem) Reporting  
Any adverse events of a serious nature will be reviewed immediately with the principal 
investigators. Serious adverse events will be reported in writing to the CHREB within 10 days of 
the PI’s notification of the event. All study adverse events will be summarized once a year, 
during the annual review reporting, for the CHREB. The research coordinator will be responsible 
for tracking adverse events in this study.  
The adverse event will be described with the following information: description of the event, 
outcome of the event, how long it lasted, whether the event required treatment or intervention, 
and the outcome.  
The definition of events is as follows:  
Mild – transient and mild in nature, with no treatment necessary.  
Moderate – some intervention and treatment necessary, but participant completely recovers.  
Severe – an event that results in hospitalization, disability, death or is life threatening.  
 
11.2 Data & Safety Monitor  
There will be no external Data & Safety monitor for this study.  
 
12.0 STUDY WITHDRAWAL or DISCONTINUATION  
12.1 Principal Investigator Initiated Withdrawal 
The principal investigator reserves the right to withdraw the participant from the study after 
they have provided informed consent, but before study completion. This could occur for one of 
many reasons, which include, but are not limited to: non-compliance with the protocol, a concern 
for participant safety, the availability of new knowledge that might affect continued participation 
in the study, or study termination.  
 
12.2. Study Participant Initiated Withdrawal  
Participants are free to withdraw from this study at any time. Withdrawal of consent or 
refusal to participate will not prejudice their health care.  
 
12.3 Data Post-Withdrawal  
If the participant is taken out of the study, or if the participant chooses to no longer be in the 
study, then we will stop collecting any information on the participant. With the participant’s 
permission we will keep the data already collected, and this will still be used for analysis.  
 
13.0 COMPENSATION  
We will cover the parking costs near TRW for participants during their study day. 
Participants will not be financially compensated for their time involved in this study. We are 
doing assessments that might generate knowledge about their medical condition and may 
indirectly be of benefit to them.  

 
 
 
PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
Page 14 of 18 
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14.0 PRIVACY and CONFIDENTIALITY ISSUES  
Protected Health Information will be used in this study. The investigators will comply with 
the patient privacy guidelines of the University of Calgary and applicable provincial and federal 
rules.  
The research team is comprised of experienced research nurses and research assistants who 
are aware of the importance of confidentiality of health information. Paper research records will 
be stored in a locked office. Digital records will be stored on password-protected University of 
Calgary computers/servers and in the University of Calgary Clinical Research Unit REDCap 
Database.  
Every effort will be made to publish and present the data from this study. At no time will any 
participant be identified in any such publication.  
 
15.0 FOLLOW-UP AND RECORD RETENTION 
 15.1 Follow Up  
There is no follow-up in this study. Once the physiological study day has been completed, 
the participant will be finished in this protocol. We may approach participants who participate in 
this study for further studies related to NOH. 
 
16.0 STUDY SPONSOR 
Departmental Funds and the start-up package of Dr. Satish R Raj will support this study.  
 
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Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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PI: Satish R. Raj MD MSCI FRCPC 
Co-PI: Richard Wilson PhD 
Post-Doctoral Fellow: Jacquie Baker PhD 
 
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