Neuroprotective effects of cannabidiol following

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Neuroprotective effects of cannabidiol following concussion: A case study

A Thesis

Submitted to the Faculty of Graduate Studies

In Partial Fulfillment of the Requirements

For the Completion of

Master of Science

in

Kinesiology and Health Studies

University of Regina

By:

Jyotpal Singh

Regina, Saskatchewan

July 2019

Copyright 2019: J. Singh

UNIVERSITY OF REGINA

FACULTY OF GRADUATE STUDIES AND RESEARCH

SUPERVISORY AND EXAMINING COMMITTEE

Jyotpal Singh, candidate for the degree of Master of Science in Kinesiology & Health Studies, has presented a thesis titled, Neuroprotective Effects of Cannabidiol Following Concussion: A Case Study, in an oral examination held on July 22, 2019. The following committee members have found the thesis acceptable in form and content, and that the candidate demonstrated satisfactory knowledge of the subject material. External Examiner: Dr. Jane Alcorn, University of Saskatchewan

Supervisor: Dr. Patrick Neary, Faculty of Kinesiology & Health Studies

Committee Member: Dr. Darren Candow, Faculty of Kinesiology & Health Studies

Committee Member: Dr. Paul Bruno, Faculty of Kinesiology & Health Studies

Chair of Defense: Dr. Andrew Stevens, Faculty of Business Administration

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Abstract

Mild traumatic brain injury, such as concussion, can lead to physiological impairments.

Although these changes in physiology are not always characterized by symptoms, they

can be reflective of how the body is attempting to adjust and adapt to these

consequences. Cannabidiol can aid with some of the physiological and symptomatic

changes due to concussion, and the therapeutic properties of cannabidiol can be

enhanced when administered with other cannabinoids, specifically,

tetrahydrocannabinol. These cannabinoids can work together on the endogenous

cannabinoid system within an individual, thereby influencing their effects on

cerebrovascular and cardiovascular physiology. Specifically, the psychoactivity of the

cannabinoids can regulate cerebral blood flow parameters and heart rate metrics. The

purpose of this study was to observe the effects of different dosages of a 20:1

cannabidiol:tetrahydrocannabinol formulation on an individual suffering from post-

concussion like symptoms. Psychological assessments include the Patient Health

Questionnaire-9 item, Generalized Anxiety Disorder-7, 36-item Short Form Health

Survey, and the Positive and Negative Affect Schedule. Physiological assessment was

done using non-invasive near-infrared spectroscopy for cerebrovascular responses, and

electrocardiography (ECG) and finger plethysmography to record continuous

cardiovascular responses. End-tidal carbon dioxide was measured using a breath-by-

breath capnograph. The protocol consisted of: 5-minute sitting rest, 6 breaths·minute-1

paced breathing maneuver, a hypercapnic challenge (20sec breath-hold:40sec normal

breathing x 5 repeats), a 5-minute (20sec eyes closed:40sec eyes open x 5 repeats) object

identification protocol (“Where’s Waldo”), and a squat-stand baroreflex maneuver (0.05

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and 0.1 Hz frequency). Descriptive physiological results of the participant’s

cardiovascular and cerebrovascular parameters suggest the largest changes (322%

increase in deoxyhemoglobin and 53% increase in systolic pressure standard deviation

during 10 second squat stands; 130% increase in systolic pressure 10 seconds post 20

second breath holds) occurred when the participant consumed 2mL of the formulation

(40mgCBD:2mg THC) as compared to 1mL (20mgCBD:1mg THC) or 1.2mL

(24mgCBD:1.2mg THC) (a half dose at 9:30am and a half dose prior to sleep). These

results suggest that an optimal dosage is necessary for improved physiological

functioning post-concussion when using cannabis products. This research warrants

further investigation on cardiovascular and cerebrovascular metrics following

cannabinoid administration.

Keywords: Cannabinoid, Cannabidiol, Cerebrovascular, Cardiovascular,

Neuroprotection, Case Study

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Acknowledgements

I would like to thank my family and friends for all their support. I would also like to

thank Dr. Patrick Neary for his guidance, patience and encouragement throughout this

project.

Thank you to my supervisory committee, Dr. Darren Candow and Dr. Paul Bruno, for

their time and for providing further guidance with my project. I would also like to thank

Dr. Shanthi Johnson for her initial edits of my proposal and for helping me clarify some

of the details of the study. To the external examiner, Dr. Jane Alcorn, thank you for

agreeing to take on this role. Your questions and comments have helped to improvement

my thesis.

I would like to thank Dr. Gregory Kratzig for providing some of the equipment used in

this project.

Finally, I would like to thank the participant who self-administered the drug and came

back to the lab continuously for follow-up assessments to allow for the collection of such

valuable data.

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Table of Contents

List of Figures .............................................................................................................................. 10

List of Appendices ....................................................................................................................... 14

List of Abbreviations, Symbols, Nomenclature ........................................................................ 15

Introduction ................................................................................................................................. 17

1. Review of the Literature: Endocannabinoid Neurophysiology and Implication for

Concussion (A version of this review has been submitted for publication) ........................... 18

1.1 Introduction to Concussion .............................................................................................. 18

1.2 The Endocannabinoid System and Concussion Relationship ................................. 19

1.3 Cannabidiol Implications for Neuroprotection following Concussion ................... 24

1.3.1 Blood Brain Barrier ................................................................................................... 30

1.3.2 Brain Derived Neurotrophic Factors (BDNF) ......................................................... 32

1.3.3 Cognitive Capacity ..................................................................................................... 33

1.3.4 Cerebrovasculature .................................................................................................... 34

1.3.5 Cardiovascular Physiology in Relation to Concussion ........................................... 35

1.3.6 Neurogenesis ............................................................................................................... 36

1.3.7 Summary: Cannabidiol for Concussion ................................................................... 37

2.0 Study Objective, Rationale, and Hypotheses ...................................................................... 38

3.0 Methodology .......................................................................................................................... 41

3.1 Participant ......................................................................................................................... 41

3.2 Physiological Assessment Equipment .............................................................................. 43

3.3 Cerebrovascular Assessment Protocol ............................................................................ 46

3.5 Data Analysis ..................................................................................................................... 51

4.0 Results .................................................................................................................................... 52

4.1 Resting and 6 Breaths per minute (6-BPM) ................................................................... 52

4.2 Where’s Waldo and BH .................................................................................................... 59

4.3 10sec Squat Stand (10SS) and 5sec Squat Stand (5SS) .................................................. 67

4.4 Psychological Questionnaire Results ............................................................................... 75

5.0 Discussion ............................................................................................................................... 76

6.0 Conclusion and Limitations ................................................................................................. 85

7.0 References .............................................................................................................................. 88

8.0 Appendices ........................................................................................................................... 109

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8.1- Appendix A – Table 15. Breakdown of SCAT symptoms247 ...................................... 109

8.2- Appendix B – NIRS data at baseline for the full protocol ......................................... 110

8.3- Appendix C – Informed consent documentation and approval letter ...................... 111

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List of Tables

Table 1. Non CB1 and CB2 receptors stimulated by CBD and their relevance to

concussion pathophysiology. TRPV1=vanilloid receptor 1; A2a= adenosine receptor; 5-

HT1A = 5-hydroxytryptamine; PPARγ= Peroxisome proliferator-activated receptor

gamma; CBD= cannabidiol

Table 2. Initial demographics

Table 3. Participant characteristics at each visit

Table 4. NIRS parameters for resting state data. %TSI= Tissue Saturation Index; HbO2=

Oxyhemoglobin; HHb= Deoxyhemoglobin; tHb= Total Hemoglobin; HbDiff=

Hemoglobin difference; Day 0= 0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg THC;

Day 28= 40mg CBD:2mg THC; Day 42= 20mg CBD:1mg THC; Day 70= 24mg

CBD:1.2mg THC

Table 5. NIRS parameters for 6 breaths per minute. %∆ indicates the change from day 0.

%TSI= Tissue Saturation Index; HbO2= Oxyhemoglobin; HHb= Deoxyhemoglobin;

tHb= Total Hemoglobin; HbDiff= Hemoglobin difference; Day 0= 0mg CBD: 0mg

THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day 42= 20mg

CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

Table 6: Blood pressure metrics for rest and 6 breaths per minute (6-BPM) protocol. %∆

indicates the change from day 0. MAP=Mean arterial pressure; SV= Stroke volume; Q=

Cardiac output; SYS= Systolic pressure; DIAS= Diastolic pressure; Day 0= 0mg CBD:

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0mg THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day 42=

20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

Table 7. HRV parameters for 5-minute rest and 6 breaths per minute. HRV= Heart rate

variability; RR= R to R beats; SD= Standard Deviation; HR= Heart rate; Min=

Minimum; Max= Maximum; VLF=Very low frequency; LF= Low frequency; HF= High

frequency; SD1= Short term standard deviation; SD2= Long term standard deviation;

NNxx= Normal to normal intervals greater than 50ms; pNNxx= Proportion of the NNxx

in the total number of normal to normal intervals

Table 8. NIRS parameters for Where’s Waldo and breath hold protocols. %∆ indicates

the change from day 0. %TSI= Tissue Saturation Index; HbO2= Oxyhemoglobin; HHb=

Deoxyhemoglobin; tHb= Total Hemoglobin; HbDiff= Hemoglobin difference; Day 0=

0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC;

Day 42= 20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

Table 9. Blood pressure metrics for Where’s Waldo and breath hold protocols. %∆

indicates the change from day 0. MAP=Mean arterial pressure; SV= Stroke volume; Q=

Cardiac output; SYS= Systolic pressure; DIAS= Diastolic pressure; Day 0= 0mg CBD:



Table 10. HRV parameters for Where’s Waldo and breath holds. HRV= Heart rate




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Table 11. NIRS parameters for 10 sec and 5 sec squat stands. %∆ indicates the change

from day 0. %TSI= Tissue Saturation Index; HbO2= Oxyhemoglobin; HHb=

Deoxyhemoglobin; tHb= Total Hemoglobin; HbDiff= Hemoglobin difference; Day 0=



Table 12. Blood pressure metrics for 10 sec and 5 sec squat stands. %∆ indicates the

change from day 0. MAP=Mean arterial pressure; SV= Stroke volume; Q= Cardiac

output; SYS= Systolic pressure; DIAS= Diastolic pressure; Day 0= 0mg CBD: 0mg



Table 13. HRV parameters for 10 sec and 5 sec squat stands. HRV= Heart rate






Table 14. Psychological questionnaire results

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List of Figures

Figure 1. Overview of physiological consequences following concussion.

Figure 2. Typical cortical activation as revealed by near infrared spectroscopy. The red

line represents oxygenated hemoglobin and the blue line represents deoxygenated

hemoglobin. The green line represents the sum of the oxygenated and deoxygenated

hemoglobin, known as total hemoglobin. The shadow indicates the stimulus duration.

Adopted from Ferrari & Quaresima1. Hemoglobin difference is not shown here, but it is

the difference between oxygenated and deoxygenated hemoglobin (red line - blue line).

Figure 3. Overview of protocol to assess cardiovascular and cerebrovascular changes.

6BPM= 6 Breaths per minute; 5SS= 5 second squat; 10SS= 10 second squat; BH=

Breath hold; CVR= Cerebrovascular reactivity; NVC= Neurovascular coupling

Figure 4. Oxyhemoglobin (µM) changes from 5-minute rest (0 on y-axis) for 6 breaths

per minute protocol. Day 0= 0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg THC; Day

28= 40mg CBD:2mg THC; Day 42= 20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg

THC

Figure 5. Deoxyhemoglobin changes (µM) from 5-minute rest (0 on y-axis) for 6 breaths



THC

Figure 6. Total hemoglobin changes from 5-minute rest (0 on y-axis) for 6 breaths per

minute protocol. Day 0= 0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg THC; Day

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THC

Figure 7. Hemoglobin difference (Oxyhemoglobin - Deoxyhemoglobin) changes from 5-

minute rest (0 on y-axis) for 6 breaths per minute protocol. Day 0= 0mg CBD: 0mg



Figure 8. Oxyhemoglobin changes from 5-minute rest (0 on y-axis) for Where’s Waldo

and breath hold protocols. Day 0= 0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg


CBD:1.2mg THC

Figure 9. Deoxyhemoglobin changes from 5-minute rest (0 on y-axis) for Where’s

Waldo and breath hold protocols. Day 0= 0mg CBD: 0mg THC; Day 14= 40mg

CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day 42= 20mg CBD:1mg THC; Day

70= 24mg CBD:1.2mg THC

Figure 10. Total hemoglobin changes from 5-minute rest (0 on y-axis) for Where’s




Figure 11. Hemoglobin difference (oxyhemoglobin-deoxyhemoglobin) changes from 5-

minute rest (0 on y-axis) for Where’s Waldo and breath hold protocols. Day 0= 0mg

CBD: 0mg THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day

42= 20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

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Figure 12. Average of 10 seconds post breath hold minus the average of each of the 20

second breath holds. Units for MAP (mean arterial pressure): mmHg; SV (stroke

volume): mL; SYS (systolic): mmHg; DIAS (diastolic): mmHg. Day 0= 0mg CBD: 0mg


CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC.

Figure 13. Oxyhemoglobin changes from 5-minute rest (0 on y-axis) for 10 second squat

and 5 second squat protocols. Day 0= 0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg


CBD:1.2mg THC

Figure 14. Deoxyhemoglobin changes from 5-minute rest (0 on y-axis) for 10 second

squat and 5 second squat protocols. Day 0= 0mg CBD: 0mg THC; Day 14= 40mg



Figure 15. Total hemoglobin changes from 5-minute rest (0 on y-axis) for 10 second




Figure 16. Hemoglobin difference (oxyhemoglobin-deoxyhemoglobin) changes from 5-

minute rest (0 on y-axis) for 10 second squat and 5 second squat protocols. Day 0= 0mg

CBD: 0mg THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day

42= 20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

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Figure 17. Average of first 6 seconds of each squat minus the average of the last 4

seconds of each squat for the 10 second squat protocol. Units for MAP (mean arterial

pressure): mmHg; SYS (systolic): mmHg; DIAS (diastolic): mmHg. Day 0= 0mg CBD:


20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC.

Figure 18: The entire protocol for the participant’s baseline visit. The red line represents

oxyhemoglobin (HbO2), the blue line represents deoxyhemoglobin (HHb), the green line

represents the sum of HbO2 and HHb, known as total hemoglobin (tHb) and the yellow

line represents hemoglobin difference (HbDiff), the difference between HbO2 and HHb.

6-BPM: 6-Breaths per minute; BH: Breath Hold; 10SS: 10 Second Squats; 5SS: 5

Second Squats.

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List of Appendices

Appendix A – Table 15. Breakdown of SCAT symptoms

Appendix B – NIRS data at baseline for the full protocol

Appendix C – Informed consent documentation and approval letter

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List of Abbreviations, Symbols, Nomenclature

Δ9-THC: Delta 9 tetrahydrocannabinol

2-AG: 2-arachidonoyl glycerol

5-HT1A: 5-hydroxytryptamine

5SS: 5 second squat stands

10SS: 10 second squat stands

AEA: Anandamide

BDNF: Brain derived neurotrophic factor

BH: Breath Holds

BL: Baseline visit (Day 0)

BP: Blood pressure

BPM: Breaths per minute

CBD: Cannabidiol

CB1 and CB2: Cannabinoid receptor 1 and 2

CO2: Carbon dioxide

DIAS: Diastolic

ECG: Electrocardiogram

fMRI: Functional magnetic resonance imaging

GABA: Gamma-aminobutyric acid

GAD-7: Generalized anxiety disorder scale 7 item

HbDiff: Hemoglobin difference

HbO2: Oxyhemoglobin

HF: High frequency power

HHb: Deoxyhemoglobin

HR: Heart rate

HRV: Heart Rate Variability

LF: Low frequency power

MAP: Mean arterial pressure

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MAPK: Mitogen activated protein kinase

NIRS: Near infrared spectroscopy

NMDA: N-methyl-d-aspartate

PANAS: Positive and negative anxiety scale

PHQ-9- Patient health questionnaire 9 item

PPAR: Peroxisome proliferator-activated receptors

Q: Cardiac output

SD: Standard deviation

SF-36: Short form 36 item survey

SV: Stroke volume

SYS: Systolic

TBI: Traumatic brain injury

tHb: Total hemoglobin

THC: Tetrahydrocannabinol

TRPV1: Vanilloid receptor 1

TSI: Tissue saturation index (%)

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Introduction

Over 10 million injuries in the world annually occur due to traumatic brain injuries

(TBI)2, meaning that there is a need for an efficient treatment to recover from these

injuries. TBIs can cause difficulty in day-to-day life and even return to normal

physiology does not assure that symptoms will clear3. Interestingly, the majority of head

injuries, such as concussions, are considered mild (mTBIs) and rarely receive full

medical treatment4. Alongside TBIs, there has been a huge increase in research being

done in the area of endogenous cannabinoids and on the cannabis plant itself5. Research

into the cannabinoids and the endocannabinoid system has recently hinted at potential

therapeutic targets, including the use of cannabinoids for TBIs2,6.

Cannabidiol (CBD) is a major non-intoxicating compound in both Cannabis Sativa

and Indica, and in the hemp plant, and shows therapeutic potential7,8. Interestingly, it is

thought that when CBD is administered alongside other cannabinoids, such as

tetrahydrocannabinol (THC or Δ9-THC), there is an entourage effect which elevates the

therapeutic properties of the cannabinoids9–11. For assistance across the blood brain

barrier, ATP-binding cassette transporters have been shown to aid in THC efflux12, and

with CBD’s ability to inhibit these transporters13, it’s possible that THC may exert it’s

therapeutic influence at the brain for longer periods14. Human research in this realm in

relation to concussion is very limited, likely due to ethical issues surrounding

administration of intoxicating and often illegal compounds such as THC, even if CBD

does have the potential to counteract THC psychosis15,16. However, numerous rat studies

have supported this idea of an entourage effect, having suggested changes in behavioural

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effects17 and THC metabolism18 when THC is co-administered with CBD. CBD is also

thought to enhance THC’s antinociceptive and hypolocomotive effects14.

To compliment some of this research and add to the existing body of literature, this

case study focused on the physiological effects of cannabinoids, specifically a

combination of CBD and THC, on an individual suffering from long-term (2 years)

mTBI like symptoms (often referred to as post-concussion symptoms). Considering the

prevalence of the entourage effect, it will also be important to discuss what the research

suggests regarding the effects of both endogenous (endo) and phyto (plant-based)

cannabinoids, specifically for concussion. While an animal model has suggested a role

for CBD to regulate glutamate and gamma-aminobutyric acid (GABA)19 responses

following mTBI, the fundamental underlying mechanisms are still not clear, especially

when considering human studies and their physiological role.

1. Review of the Literature: Endocannabinoid Neurophysiology and Implication for

Concussion (A version of this review has been submitted for publication)

1.1 Introduction to Concussion

Concussion (mTBI) related injuries have been a growing issue, both in adults and

youths20–23. While return to baseline after 3 weeks is common in ages 5 to 1422, post-

concussion syndrome (symptoms lasting over 3 months following mTBI) has been

shown to persist for over 6 months in 40% of mTBI patients24. Post-concussion

syndrome can have negative effects on the individuals day to day lives, as shown

through its negative associations with health related quality of life, assessed by the 36-

item Short-Form Health Survey and Perceived Quality of Life Scale24. Interestingly,

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persistence of one symptom was much more common than multiple symptoms (with

headaches, fatigue, forgetfulness and poor concentration among the most common)25,26,

and being female was a significant predictor post 12 months26.

With respect to the pathophysiology of concussion, following impact there is a large

disruption of the cellular membranes, which is thought to initiate the cascade of ionic

inflow which appears to sustain over time27,28. Excess glutamate release binding to N-

methyl-d-aspartate (NMDA) further accelerates this ionic inflow and intracellular

calcium (Ca2+) accumulation, possibly inducing mitochondria-centred cell damage27–29.

Much of the damage caused by concussion is thought to impair vital mechanisms in the

body, including heart rate variability biofeedback30,31, cerebral autoregulation32,

cerebrovascular reactivity33,34 and neurovascular coupling35 (Figure 1). Imaging

techniques, including functional near infrared spectroscopy and functional magnetic

resonance imaging have shown impairments in region specific functionality in patients

with persistent post-concussion symptoms (most notably in the dorsolateral prefrontal

cortex)36, and independent component analysis of fMRI data has further added evidence

suggesting altered functional connectivity between brain regions in mTBI. Taken

together, it is clear that concussion is a huge burden and requires a new treatment

modalities.

1.2 The Endocannabinoid System and Concussion Relationship

After the identification and synthesis of THC37, the scientific world expanded much

of their knowledge of the cannabis plant constituents. This led to the understanding of

the endocannabinoid system, one which is thought to have a regulatory role in pain

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processing and perception38, maintaining immune system homeostasis39, and influencing

cardiovascular functioning40. The endocannabinoid system consists of endogenous

cannabinoids N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoyl

glycerol (2-AG), enzymes for cannabinoid degradation, and at least 2 receptors coupled

through G-protein inhibition: cannabinoid receptor 1 (CB1) and cannabinoid receptor 2

(CB2)41–43. Other receptors sensitive to cannabinoids include vanilloid receptor 1

(TRPV1)44,45, adenosine receptors46,47, 5-hydroxytryptamine (5-HT1A)48,49, and G-

protein coupled receptors50.

The CB1 receptors are most abundant within the central nervous system, primarily

located on the axon and synaptic terminals of the neurons51, while the CB2 receptors are

much more concentrated within the peripheral nervous system and the immune

system51,52. Interestingly, CB2 receptors are thought to suffer from an “identity crisis”

due to their presence also being at the brain microglia53–55. Discussed in more detail in

the following section, activation of the CB1 receptor is thought to be responsible for the

common psychosis associated with THC43, while activation of the CB2 receptor can

attenuate inflammation and accelerate regeneration in many disease states, including

liver regeneration in an animal model of acute hepatitis56 and attenuation of

inflammation and tissue injury in an animal model of spinal cord injury54,57.

Furthermore, because microglia activation can result in release of proinflammatory

cytokines and reactive oxygen intermediates58, CB2’s presence at the microglia suggests

a protective purpose as CB2 receptor stimulation in CB1 receptor knockout mice

produced antinociceptive effects in response to inflammatory pain59. Therefore, the

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receptors are present in neural and peripheral tissue, suggesting involvement in many

physiological processes.

As mentioned above, concussion is thought to initiate a neurometabolic cascade28,

one which can lead to an energy crisis and disrupt autonomic functioning32,35, among

other physiological consequences (Figure 1)28–30,32,34,60–65. mTBIs generally occur due to

rotational or twisting forces (accelerations or decelerations)66,67 of the brain within the

skull, thereby resulting in a disruption in homeostasis. In comparison, the

endocannabinoid system is thought to have neuroprotective properties68,69, which could

therefore ameliorate these disturbances in physiological homeostasis. This may partly be

due to the fact the endocannabinoids are retrograde messengers with the ability to cause

depolarization-induced suppression of inhibition70. This mechanism suggests that once

neurotransmitters are released from the presynaptic neuron, they activate the

endocannabinoids of the postsynaptic neurons. These endocannabinoids are thought to

be produced in vivo where increased intracellular [Ca2+] is the major trigger for

synthesis71. The endocannabinoid ligands AEA and 2-AG42 are released from cells in a

stimulus-dependent manner by cleavage of membrane lipid precursors72–74. These

endocannabinoids travel back to the presynaptic neuron and bind to the CB1 receptors

which causes a decrease in neurotransmission across the synaptic cleft. This is done by

inhibition of adenylate cyclase (leading to a decrease in Na+ inflow) and calcium

channels, along with activation of the potassium channels70, thus potentially leading to

hyperpolarization as depolarization-induced suppression of inhibition decreases the

frequency of miniature inhibitory postsynaptic currents, thereby increasing the

percentage of synaptic failures70.

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The endocannabinoids are synthesized as needed by the body from arachidonic acid

of membrane phospholipids75. This “need” is generally in response to pain,

inflammation, anxiety, emotional and physical stressors, and pathological conditions.

Completion of the endocannabinoid signaling requires cellular re-uptake through a

carrier-mediated transport process followed by enzymatic degradation by enzyme fatty

acid amide hydrolase primarily for AEA to arachidonic acid and ethanolamine 76

monoacylglycerol lipase for 2-AG to glycerol and arachidonic acid75,77. The calcium-

dependent synthesis and release of endocannabinoids for its retrograde mechanism could

have a significant role in protecting the body by limiting overstimulation, such as

protection against the neurometabolic cascade following a mTBI28.

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Figure 1: Overview of physiological consequences following concussion.

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1.3 Cannabidiol Implications for Neuroprotection following Concussion

CBD is a cannabinoid found in the cannabis sativa plant that is well known for its

therapeutic potential but devoid of psychosis78,79. Cannabinoids, including CBD, are also

known to act on other receptors aside from the CB1 and CB2 receptors in the body (Table

1). Interestingly, CBD can inhibit fatty acid amide hydrolase, thereby inhibiting the

enzymatic hydrolysis and uptake of AEA80 from the synapse. This means that CBD can

therefore indirectly influence the effects of AEA in the endocannabinoid system, thereby

possibly allowing for sustained neuroprotective effects of AEA81,82. CBD has been

shown to be a negative allosteric modulator at the CB1 receptor as it reduced the efficacy

and potency of 2‐AG and (THC) on cells expressing CB1 receptors83. Because the CB2

receptor is thought to be involved in aiding with anti-inflammatory responses52, it

follows that CBD can act as a partial agonist at this receptor84 to further aid in

inflammation regulation.

TRPV1 is a ligand-gated ion channel and acts on afferent neurons where it is

expressed both pre-synaptically, and post-synaptically. This receptor is involved in the

sensation of pain and thermal hyperalgesia85. CBD can essentially function as a weak

TRPV-1 agonist, which may help explain its therapeutic ability in neuropathic pain80,86,

as 10mg/kg of CBD administered to rats with acute inflammation showed an

antihyperalgesic response86. Research by Benyo et al87 highlights that the activation of

TRPV1 is known to control vascular responses, thus hinting at the idea of CBD having

different action within the cerebral vasculature. Furthermore, the authors suggest that

cannabinoids have the ability to directly influence cerebrovascular resistance and blood

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perfusion of the brain87 and that all cerebrovascular control pathways contain cells

capable of being modulated by cannabinoids. Considering that concussion can impair

cerebrovascular reactivity33,34,88, it follows that CBD’s influence on TRPV1 can protect

against these changes.

Another group of receptors upon which CBD is known to act are adenosine

receptors, such as A2a receptors. These receptors can down-regulate the release of other

neurotransmitters such as dopamine and glutamate, and CBD is thought to increase brain

adenosine levels by reducing adenosine reuptake7. Furthermore, CBD-mediated

activation of A2a receptors may also allow for increased anti-inflammatory effects,

thereby further increasing CBD’s potential for enhancing neuroprotection89. Indeed,

CBD was shown to downregulate inflammatory biomarkers and attenuate microglia

activation in a viral model of multiple sclerosis by interacting with A2a receptors90.

Finally, it was recently suggested that CBD can blunt the THC-induced cognitive

impairment in an A2a receptor dependent manner in a mouse model91.

Another receptor with which CBD is known to directly act upon is the 5-HT1A

receptor, which is found in large concentrations in the brain, with large densities at the

prefrontal cortex, hippocampus and amygdala92,93. CBD has been shown to exhibit a high

potency for this receptor and serve as an agonist, to further stimulate the receptor’s

properties of decreasing anxiety, pain, and headaches. 5-HT1A is a G-protein coupled

serotonin receptor, which further suggests the potential of CBD to provide therapeutic

benefits48, similar to those of serotonin itself. For example, it was found that CBD can

enhance both serotonergic and glutamate cortical signalling, possibly allowing for rapid-

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acting antidepressant-like effects94. Furthermore, CBD was also shown to be

neuroprotective by inhibiting reuptake of 5HT48,95, resulting in decreased excitotoxicity,

inflammation and oxidative stress95.

Peroxisome proliferator- activated receptors (PPAR) gamma (γ) receptor is also

thought to be influenced by cannabinoids producing therapeutic effects96. PPARs are

nuclear receptor proteins that function as transcription factors and are essential in the

body’s ability to regulate energy homeostasis and metabolic function97. As such, their

involvement becomes increasingly important in stabilizing cell homeostasis. For

example, it was shown that CBD suppresses the increased permeability of the blood

brain barrier associated with oxygen–glucose deprivation by PPARγ and 5-HT1A

activation96,98. In relation to other neurodegenerative disorders, CBD reduces β-amyloid

expression96,99, and can possibly induce apoptosis in some forms of cancer cells96,100 via

PPAR activation. PPARγ is also thought to inhibit the expression of inflammatory

cytokines97, thereby suggesting possible CBD and PPARγ synergy to further regulate

inflammation. This was further shown by CBD’s inhibition of necrosis factor kappa b,

nitric oxide, tumor necrosis factor alpha and interleukin 1 beta, along with promoting

hippocampal neurogenesis. Furthermore, CBD’s neuroprotective effects were completely

abolished after introduction of a PPARγ antagonist101.

Finally, a receptor initially known as the endothelial cannabinoid receptor, now

thought to be G-protein-coupled receptor 18102,103, also serves as a receptor at which

abnormal CBD (ABN-CBD), a synthetic CBD product was shown to react102–104. This

receptor is believed to be in existence as administration of CBD to mice lacking the

27

cannabinoid receptors still resulted in hypotension and endothelium dependent

mesenteric vasodilation, yet administration of a CB1 antagonist was shown to block the

CBD effect87. It is therefore possible that CBD can help to regulate cerebral blood flow

due to its influence on vasomotor control, meaning that these receptors mediate

neuroprotective effects and regulate cell migration.

Concussion can result in a cascade of pro- and anti-inflammatory cytokines105, ionic

imbalance28, increased lactate accumulation due to increased glycolysis and oxidative

stress106,107, and impaired cerebral blood flow34. Through its influences on the

cerebrovasculature, its anti-inflammatory properties and its neuroprotective properties,

CBD can potentially help to reduce impairment following concussion. Furthermore, to

control for the calcium sequestration and ionic inflow61, it has been suggested that CBD

can regulate Ca2+ homeostasis against mitochondrial dysfunction and toxins, and Ca2+

dysregulation108.

Due to its neuroprotective capabilities, CBD has been shown to have some potential

in helping individuals with TBIs. Mice induced with bilateral common carotid artery

occlusion were administered 10mg/kg CBD which attenuated hippocampal

neurodegeneration and white matter injury, increased hippocampal brain derived

neurotrophic factor protein levels, stimulated neurogenesis and promoted dendritic

restructuring109. Furthermore, CBD administration in mice both before and after middle

cerebral artery occlusion showed that CBD suppressed the impairment in cerebral blood

flow by the failure of cerebral microcirculation after reperfusion, providing further

evidence of CBD’s neuroprotective properties110. Adding to this evidence of

28

neuroprotective potential, CBD administration to piglets exposed to acute hypoxia-

ischemia increased the brain activity post-ischemia back to normal as shown by brain

electrical activity, cerebral tissue oxygenation, and neurobehavioral responses, such as

motor performance111.

29

Table 1: Non CB1 and CB2 receptors stimulated by CBD and their relevance to

concussion pathophysiology. TRPV1=vanilloid receptor 1; A2a= adenosine

receptor; 5-HT1A = 5-hydroxytryptamine; PPARγ= Peroxisome proliferator-

activated receptor gamma; CBD= cannabidiol

Receptor Implications for Concussion Dose and Model Reference

TRPV1 Antihyperalgesic effect in

response to inflammation

Male Wistar rats with acute

inflammation; CBD 10mg/kg

Costa et al.

200486

5-HT1A Decrease in excitotoxicity,

inflammation and oxidative

stress

Increase in cerebral blood flow

1-2-day old male piglets; CBD

1mg/kg

Male middle cerebral artery occluded

mice; CBD 0.1 to 10mg/kg

Pazos et al.

201395

Mishima et al.

2005112

PPARγ Suppress the increase in the

blood brain barrier permeability

(also an effect of 5-HT1A)

Promotion of hippocampal

neurogenesis and reduction in

inflammatory biomarkers

Human brain microvascular

endothelial cells and human

astrocytes; CBD 100nM, 1μM and

10μM

Adult male Sprague-Dawley rats;

CBD 10mg/kg

Hind et al.

201698

Esposito et al.

2011101

A2a Downregulate inflammatory

biomarkers and attenuate

microglia activation

Reduce inflammatory

biomarkers and glutamate in

hypoxic ischemia

Female mice; CBD 5mg/kg

Forebrain of 7-to-10-day old C57BL6

mice; CBD 0.1 to 1000μM

Mecha et al.

201390

Castillo et al.

201089

30

With respect to THC, it is known to act upon the CB1 receptor7,74,113 and it can

allow for depolarization-induced suppression of inhibition to occur. Research has

suggested that THC can exert different pharmacological responses depending on

location. For example, it is thought to be a full agonist at the CB1 receptor when

considering the GABA receptors at the hippocampus114, whereas other research has

shown that THC exerts both agonist and antagonist effects following acute

administration in a mouse model of hypothermia115. Because of the potential of THC to

act agonistically on the CB1 receptor, this can lead to the decrease of cyclic adenosine

monophosphate by inhibition of the enzyme adenylate cyclase116, induce potassium

efflux by stimulating A-type and G-protein coupled inward rectifying potassium

channels and decrease calcium influx by inhibiting voltage dependent N and P/Q-type

calcium channels117. This can explain why THC administration can help with reducing

and managing pain. The direct influence of THC on the cannabinoid receptor also

suggests that CB1 activation can cause psychotic effects. Thus, an ensemble of high CBD

levels and low THC levels can be the most beneficial for medicinal purposes, as CBD

administration can help to attenuate these increases in psychosis9,118. Currently, there is

also ongoing research to observe the effects of a 20:1 CBD:THC composition on

children with refractory epileptic encephalopathy119, thereby further suggesting

neurophysiological benefits following administration.

1.3.1 Blood Brain Barrier

Concussions can further alter cerebrovascular actions by disrupting the blood

brain barrier integrity120. Cannabinoids, specifically 2-AG and dexanabinol, a synthetic

31

cannabinoid also known as HU-211, have been shown to decrease pro-inflammatory

cytokines such as tumor necrosis factor-alpha121 and necrosis factor kappa b122.

Cytokines have been shown to overexpress following concussion105, which can lead to a

heightened sensitivity for disease, such as multiple sclerosis or Alzheimer’s, at the blood

brain barrier123. Furthermore, head injuries tend to enhance the activity of water-soluble

antioxidants in the brain124, along with an increase in 2-AG, which can further increase

the levels of these antioxidants125, suggesting a regulator ability for the endocannabinoid

in neuroprotection with the blood brain barrier126. The blood brain barrier is important as

it controls the amount of material transported into the brain, and in combination with CB

receptors can limit and protect the brain against the influx of neurotoxins, immune cells

and macromolecules127, thus allowing maintenance of an optimal extracellular

environment in the brain87. This allows for prevention of apoptosis, glia cell activation

and scar tissue formation, among other symptoms caused by these substances. Although

both CB1 and CB2 receptors are involved in the blood brain barrier, 2-AG is largely

effective as a full CB2 receptor agonist128, which may help explain its anti-inflammatory

properties52,89. Due to CBD’s lipophilic properties, it can easily pass the blood brain

barrier7. While research pertaining to CBD’s influence at the blood brain barrier in

humans is limited, it has been shown that CBD can preserve the barrier’s integrity

(permeability), possibly by protecting against the loss of tight junction proteins by acting

on PPARγ and 5‐HT1A receptors98,129.

Adding to this idea of neuroprotection, oxidative stress is greatly modulated by

CBD as well130–133. Following a TBI, it is evident that there is an increase in free radical

production, such as reactive oxygen species134,135. Under oxidative stress, the protective

32

effect of CBD is thought to be mediated by a decrease in reactive oxygen species

production131. This can be due to the molecular structure of CBD, potentially allowing it

to act as an antioxidant136,137. Post TBI, NMDA-receptor binding by glutamate allows for

intracellular accumulation of Ca2+, causing cellular damage via proteases, reactive

oxygen species, and mitochondrial impairment29,61. NMDA-induced retinal neurotoxicity

was also shown to be treated by intravenous injection of CBD to the eye at 2mg/kg,

which further shows the antioxidative properties of CBD, as it reduced both oxidative

and nitrative stress113.

1.3.2 Brain Derived Neurotrophic Factors (BDNF)

BDNF is a protein which promotes the survival of nerve cells. It is known to

increase the frequency of miniature excitatory postsynaptic currents, possibly

strengthening excitatory, glutamatergic synapses and weakening inhibitory, GABAergic

synapses138,139. In cases involving encephalopathy, administration of 5mg/kg CBD was

shown to help restore BDNF levels in mice through the activation of the 5-HT1A

receptor140. Furthermore, withdrawal from amphetamines can also diminish BDNF levels

due to oxidative stress141, which have been shown to be blocked by the administration of

60mg/kg dosages of CBD in an animal model142,143, suggesting CBD may have a great

potential for aiding in addiction recovery. Finally, 30mg/kg dosages of CBD have been

shown to upregulate BDNF following cerebral malaria through the nitric synthase

pathway144. This ability of CBD to increase BDNF expression can suggest an important

role in neuroprotection, leading to decreased neuronal damage142. Furthermore, BDNF is

thought to enhance neurogenesis138, further suggesting CBD’s restorative potential. At

33

the prefrontal cortex and hippocampus, CBD was shown to elevate BDNF levels,

resulting in sustained antidepressant-like effects145. Considering that BDNF levels can be

impaired following TBI146,147, and there is a correlation between BDNF serum levels and

cognitive impairments148, it follows that CBD administration post-concussion can further

aid in the recovery process.

1.3.3 Cognitive Capacity

Cognitive capacity is the total amount of information the brain can retain at any

moment. fMRI is a neuroimaging method used to measure brain activity by observing

correlations relative to blood flow, thus allowing measurements of neural mechanisms of

cognitive capacities149. Using this fMRI technique for example, it was found that THC

and CBD have distinct effects on regional brain activation150, sometimes even

completely opposite effects150,151 when 10mg THC, 600mg CBD, or placebo capsules

were administered to 14 healthy volunteers. Furthermore, using fMRI, the study found

that THC decreased activation of bilateral temporal cortices during auditory processing,

and was associated with increases in anxiety, intoxication, and positive psychotic

symptoms. It also influenced visual processing. This was completely different to the

CBD administration, which never had any reductive effects on areas such as visual

processing, but also showed no significant symptomatic effects such as psychotic

symptoms and increases in anxiety150,151, suggesting that CBD alone can provide more

therapeutic benefits than THC.

Interestingly, THC and CBD have also shown opposite effects on cognition-

related brain activation. It has been suggested that the harmful effect of cannabis might

34

be driven by high THC/low CBD compositions152, as shown by poor performances on

verbal memory task153, reduced emotional processing accuracy154, and poorer verbal

memory performance118. Combination of CBD and THC allows a reduction in the

psychotic effects of THC, and thus CBD is able to partially prevent the detrimental

effects of THC on working memory118,152. Finally, higher THC negatively impacts on

memory and psychological well-being, while lower psychosis-like symptoms were found

in those whose hair samples had CBD155, further showing the greater therapeutic

potential of CBD as compared to THC.

1.3.4 Cerebrovasculature

Human studies have demonstrated a reduced mortality and morbidity following

TBI owing to the ability of CBD to significantly reduce inflammation via the reduction

in microglia activation and pro-inflammatory cytokines75,125,142. As discussed earlier,

during brain trauma the endocannabinoid system is activated by the rise in intracellular

calcium. Activation of the endocannabinoid system suggests that it is part of the

compensatory repair mechanism for the brain156. Since cerebral blood flow is tightly

regulated by myogenic, endothelial, metabolic and neural mechanisms, all major cell

types involved in cerebrovascular control pathways are capable of synthesizing

endocannabinoids87. Finally, CBD administration has been shown to increase cerebral

blood flow following middle cerebral artery occlusion by action on 5-HT1A

receptors112,157. As such, it is logical to hypothesize that the endocannabinoid system

modulates the regulation of cerebral circulation158 under both physiological and

pathophysiological conditions87.

35

Regulation of blood flow following a traumatic brain injury can be beneficial in

assuring adequate nutrient supply to areas of altered metabolic activity. This

compensatory phenomena in healthy individuals is internally regulated through a

mechanism known as neurovascular coupling. While no direct studies have shown

CBD’s influence on this mechanism, the role of the endocannabinoid system in cerebral

circulation87 and the presence of TRPV1 at the sensory vagal afferent neurons and the

tunica of blood vessels40 does give CBD potential. Concussion may lead to changes in

the neurovascular coupling response35,65,159, and CBD’s ability to regulate blood flow160

and influence pial vessel responses by regulating vascular effects in combination with

protecting the blood brain barrier161. Keeping in mind that the cannabinoid receptors and

TRPV1 are located at the cerebrovasculature87, CBD’s ability to sustain AEA by

inhibiting fatty acid amide hydrolase162 suggests an indirect effect on CB receptors.

Further stimulation of CB2 receptors by AEA can allow for regulation of nitric oxide,

thereby potentially suppressing neuroinflammatory reactions163, which suggests great

potential in combination with the CB1 receptor stimulation to promote neuroprotection81.

1.3.5 Cardiovascular Physiology in Relation to Concussion

CBD has also been shown to exert its protective influences over the

cardiovascular system. For example, diabetes is known to cause dysfunction and cardiac

autonomic abnormalities164 by mechanisms such as hyperglycemia-induced

overproduction of reactive oxygen species and impaired antioxidant enzyme activities.

Administration of ABN-CBD has been shown to attenuate all these effects in rats, and

even promote vagal responses (such as decreased heart rate and mean arterial pressure),

36

supported by a high frequency increase in the ECG R-R index spectral analysis165. It is

important to note that heart rate variability impairments have been noted following

concussion30,166,167, although the specific physiological phenomena is still unknown. As

concussion can occur due to any transient neurologic dysfunction resulting from a

biomechanical force28, it seems logical that an injury leading to autonomic dysfunction

may further lead to changes in heart rate variability. Furthermore, decreased global heart

rate variability is also thought to be related to arrhythmic mortality31,168. CBD’s ability to

reduce arrhythmic events169 suggests further potential to treat cardiovascular

complications arising from concussion. It has been shown that there are some changes in

heart rate variability when getting the participant to exercise32,166, but the data is still

unclear as to what the changes imply. Regardless, it is clear that dysfunction occurs

following concussion, and considering the effect of ABN-CBD to influence endothelial

cannabinoid receptors, it follows that cannabinoids can help regulate the cardiovascular

system following dysfunction.

1.3.6 Neurogenesis

Neuronal cell death is usually a result of increased intracellular [Ca2+], as this

increases glutamate release across the synapse, activation of NMDA receptors and

thereby increasing [Ca2+]. This can further lead to an increase in enzyme activity,

specifically enzymes which can induce apoptosis. The ability of cannabinoids to inhibit

N- and P/Q-type calcium channels170, and the ability of CBD to induce a hyperpolarizing

shift in the steady state inactivation potentials of the T-type calcium channels171 suggests

37

that there is potential for CBD in neurogenesis and protection against neurodegenerative

processes.

Endocannabinoids are known to be produced by neural progenitor cells which

can stimulate neural progenitor cell proliferation at the hippocampal and sub-ventricular

zones via CB1 receptors, as documented by neurosphere generation172. CBD has also

been suggested to have a restorative effect on regions of the hippocampus which may be

damaged due to prolonged cannabis use173. Furthermore, mice with knocked-out fatty

acid amide hydrolase enzyme express a higher concentration of 2-AG, which has been

shown to induce astrogliogenesis174. The mechanism behind the cannabinoids’ ability to

regulate neurogenesis seems to involve the mitogen activated protein kinase (MAPK)

pathway, activation of which allows neural progenitor cell proliferation, whereas the CB1

-mediated inhibition of the proliferation is due to the attenuation of sustained MAPK

activity 172,175. These results suggest that CBD and the endocannabinoids themselves can

be key compounds in neurogenesis.

1.3.7 Summary: Cannabidiol for Concussion

Overall, CBD seems to have an untapped potential of medicinal properties. In

combination with THC, the effects of CBD are not fully understood in regard to its

physiology. As stated earlier, cannabinoids generally tend to stimulate cannabinoid

receptors and work to decrease neurotransmission while also exhibiting psychotic effects

42. CBD can stimulate other receptors (see Table 1) and thus has the ability to utilize the

medicinal properties similar to cannabinoids such as THC, but without the psychotic

effects. An ensemble including both THC and CBD has been suggested to allow for the

38

most beneficial results 176 as CBD has the potential to block the THC intoxicating

effects, but retain the neuroprotective effects such as protection against free oxygen

species 136 and restoring BDNF levels 177.

2.0 Study Objective, Rationale, and Hypotheses

The objective of this project was to investigate the effect of different dosages of a

high CBD and low THC cannabis formulation on cerebrovascular and cardiovascular

physiology in a case study participant suffering long-term symptoms following a

concussion. It was hypothesised that consumption of a high CBD/low Δ9-THC ensemble

will not adversely affect cerebrovascular and cardiac physiology in this participant but

will rather improve overall physiological function. As mentioned before, CBD has been

shown to influence cerebrovascular and cardiovascular physiology, by regulating blood

flow and reducing neuro-inflammation101,160,178. The mechanisms, however, are still not

completely understood, but research has confirmed that the human endocannabinoid

system helps to regulate cerebral circulation to provide these beneficial effects179. Since

cerebral blood flow is tightly regulated by myogenic, endothelial, metabolic and neural

mechanisms, and because all major cell types involved in cerebrovascular control

pathways, such as smooth muscle, neurons, and astrocytes are capable of synthesizing

endocannabinoids87, it is logical to hypothesize that the endocannabinoid system

modulates the regulation of cerebral circulation under both physiological and

pathophysiological conditions. Thus, if CBD and THC are capable of producing

therapeutic effects, what is the physiological response of CBD and THC administration

during concussion recovery relative to the cardiovascular and cerebrovascular indices?

39

Limited research is available to demonstrate a safe and effective dosage level of

high CBD. It has been reported that high levels of CBD are well tolerated in humans180

and do not cause THC like psychotic symptoms. Literature values report that beneficial

effects in some pathological conditions can be attained with levels from 300-600mg

(Huntington’s disease) to 1500mg per day for 4-weeks (schizophrenia)181. Furthermore,

even 6000mg of CBD has been shown to be well tolerated when administered on a daily

basis182. There is less available information on the effects of high CBD/low THC on

cerebrovascular and cardiovascular physiology in concussed individuals. One study

however, using normal healthy male volunteers were administered an acute dosage of

400mg on two separate occasion’s 1-week apart to investigate whether CBD can alter

resting regional cerebral blood flow160. They showed there was a significant decrease in

subjective anxiety and increased mental sedation, and the SPECT imaging results

showed that CBD significantly modulated the resting activity in the limbic and

paralimbic cortical areas. Although this study provides valuable information regarding

the benefit of CBD as an anxiolytic agent, a dose-response relationship to changes in

cerebral blood flow was not possible to confirm as blood samples were not taken. It is

difficult to correlate these results to concussed individuals, but the underlying potential

remains. Recent research documents the importance of knowing the plasma

concentration of CBD to understand the pharmacokinetic relationship79. Furthermore, the

chronic effects of daily CBD administration are less clear, and what is the relationship

between sublingual administration and blood toxicity levels still requires clarity. This

information is important so that proper guidelines can be established for both healthy and

pathological conditions when individuals are seeking to use medical cannabis on a daily

40

basis. Thus, if there are adverse effects on brain physiological mechanisms, such as

neurovascular coupling and cerebrovascular reactivity, it is important to understand what

the potential complication is.

What is not known is: 1) does a high CBD/low THC ensemble modulate the

cerebrovascular response by altering neurovascular coupling and cerebrovascular

reactivity, 2) are all dosages safe and tolerable to demonstrate improved cerebrovascular

and cardiovascular function, and 3) does a daily high CBD/low THC ensemble

administration help improve overall quality of life?

Research Outcomes and Hypotheses

The proposed study was innovative as it is assessing the cerebrovascular and

cardiovascular physiological effects of different dosages of high CBD/low THC

administration in a concussed individual. In doing so, a greater understanding of the

medicinal, and by extension, the physiological and pharmacological properties of a high

CBD/low THC ensemble was demonstrated. In using this specific dosage, this project

seeked to address the following outcomes and hypothesis:

1) To investigate whether a high CBD/low THC ensemble will alter

physiological function as it is hypothesised that cerebrovascular and

cardiovascular function in a concussed individual will not be adversely

affected.

2) To subjectively assess the overall improvement in the participant’s quality of

life, and thus to hypothesize that there will be an improved quality of life.

41

3.0 Methodology

3.1 Participant

The purpose of this case study research project was to investigate the responses

of a high CBD/low THC ensemble ((20mg/mL:1mg/mL CBD:THC; CanniMed Oil 1:20

(THC:CBD)) on cerebrovascular and cardiovascular physiology when administered post-

concussion30,32,35. The participant (female, 57 years, post-menopausal) was medically

confirmed as having post-concussion syndrome approximately 2 years prior to

assessment by the family physician. She presented with numerous symptoms (See

Appendix B) at baseline. The participant was assessed at baseline (Day 0) prior to

cannabis consumption, 2 weeks (Day 14) following 2 mL (40mg CBD:2mg THC) daily

intake of the formulation, 4 weeks (Day 28) following 2 mL (40mg CBD:2mg THC)

daily intake of the same formulation, 6 weeks (Day 42) following a reduction to 1 mL

(20mg CBD:1mg THC) daily intake of the same formulation, and 10 weeks (Day 70)

following 1.2 mL (24mg CBD:1.2mg THC) daily intake of the same formulation. The

participant took a half dose at 9:30am and a half dose prior to sleep, while the

assessment was conducted at 11am. Because the participant was self-administering the

study product on her own (sublingually), the researcher had no control of this regimen.

The participant was asked to list of all medications and supplements prior to

commencing this study. The participant maintained a logbook to record daily physical

activity and symptoms, and the participant did not consume any other cannabis or

tobacco products throughout the duration of the study.

A Short form-36 Health Survey Questionnaire183 was completed prior to each

assessment to measure subjective states of mood and general health related to emotional

42

and physical health and attitudes before and following each dosage administration. The

Positive and Negative Affect Schedule (PANAS)184 was also completed prior to each

assessment to measure positive and negative affect levels in this study185. Finally, the

Patient Health Questionnaire-9 (PHQ-9)186 and Generalized Anxiety Disorder-7 (GAD-

7)187 questionnaires were completed at each visit. Prior to starting this study ethical

approval was granted by the University of Regina Research Ethics Board (REB 2018-

223).

Table 2: Initial demographics of participant

Age (years) Height (cm) Reasons for Physician Care Prescription Medications

57 165 Depression and Concussion Ranitidine 75mg/day

Citalopram 30mg/day

Amitriptyline 10mg/day

43

Table 3: Participant characteristics at each visit

Hours since meal

Time of Half Doses

Alcohol (last 24 hours)

Caffeine (last 24 hours)

Sleep (hours)

Exercise (last 24 hours)

SCAT Symptom Score*

Body Mass (kg)

Day 0 2.5 None

No Yes (2.5 hrs) 8 No 8 60.8

Day 14 (40mg CBD: 2mg THC) 2.5

1.5 hours prior to testing and before sleeping No

Yes (2.5 hrs) 8 No 12 60.8

Day 28 (40mg CBD: 2mg THC) 1.5

1.5 hours prior to testing and before sleeping

Yes (16 hrs)

Yes (2 hrs) 8 No 3 60.8

Day 42 (20mg CBD: 1mg THC) 2

1.5 hours prior to testing and before sleeping

Yes (16 hrs)

Yes (2 hrs) 9 No 0 60.5

Day 70 (24mg CBD: 1.2mg THC) 2

1.5 hours prior to testing and before sleeping No Yes (2hrs) 9.5 No 2 60.0

*=See Table 15 in Appendix B for list and severity of symptoms

3.2 Physiological Assessment Equipment

All equipment used to assess cerebrovascular and cardiovascular physiology was

non-invasive and operational in our fully equipped and functioning research laboratory,

including:

Near infrared spectroscopy (NIRS)

Cerebral oxygenation was monitored in the right prefrontal cortex using a single

channel Portalite system (Artinis Medical, The Netherlands), with data collected at 10

Hz for higher temporal resolution188.

44

As depicted by Figure 2, NIRS allows for relative measures of hemodynamic

responses, being applied either at the muscle189 or cerebral landmarks1,88,190. NIRS

devices are valuable due to their portability and non-invasive methodology. The probe

placed over the right prefrontal cortex returns wavelengths indicative of hemoglobin

activity. Light is transparent in the NIR region of the electromagnetic spectrum (approx.

650– 1000 nm)1, thereby easily bypassing biological tissue. Using a reflectance mode

technique191, the absorbance spectra of hemoglobin can allow for measurement of

hemodynamic activity in the cerebrovasculature. Deoxyhemoglobin, (HHb; measured

within 650nm-1000nm) and oxyhemoglobin (HbO2; measured as a broad peak within

700-1150nm)191,192 can lead to calculation of total hemoglobin (tHb=HbO2+HHb), which

has been correlated to cerebral blood volume193,194, and hemoglobin difference

(HbDiff=HbO2-HHb) measures, which is thought to reflect a change in oxygenation195.

It’s important to note that an increase in HbO2 in combination with a decrease in HHb

reflects an increase in local arteriolar vasodilatation, increasing local cerebral blood flow

and cerebral blood volume1. Absorbance is measured through the use of the modified

Beer–Lambert law, an equation which relates the pathlength of NIR light to the

concentration and absorption spectra of tissue chromophores191, allowing for

measurement of tissue oxygen saturation191.

45

Figure 2: Typical cerebral cortical activation as revealed by near infrared spectroscopy.

The red line represents oxygenated hemoglobin and the blue line represents

deoxygenated hemoglobin. The green line represents the sum of the oxygenated and

deoxygenated hemoglobin, known as total hemoglobin. The shadow indicates the

stimulus duration. Adopted from Ferrari & Quaresima1. Hemoglobin difference is not

shown here, but it is the difference between oxygenated and deoxygenated hemoglobin

(red line - blue line).

The prefrontal cortex is vital for executive function196. For this study, important

major functions of the prefrontal cortex197 include empathy198, fear modulation199, self-

reflection and insight200, emotional responses201, autonomic nervous system

regulation202, regulation of cognitive flexibility203, intuition204,205 and sympathy and

attuned communication197,206. These responses and communication between the

46

prefrontal cortex and cognitive control207, influences on memory, attention and stress

signaling pathways207–210 , suggest that the prefrontal cortex can be a valuable area to

measure hemodynamic responses following injury such as concussion. With respect to

the equipment, placing the NIRS probe between the ventromedial and ventrolateral

prefrontal cortex allows avoidance of the sinus, makes the recording easily reproducible,

and represent a location of poor skin blood flow to ensure neural activity leads to the

measured cortical responses88.

Finapres Nova

Cardiovascular indices were recorded by monitoring continuous blood pressure

using finger plethysmography32. The NOVA (Finapres Medical Systems, The

Netherlands) comes complete with 3-lead electrocardiogram (ECG) to record heart rate

variability (HRV) indices and heart rate. Software is included to examine hemodynamic

variables (stroke volume, cardiac output, systolic and diastolic volume index).

Gas Analysis

End-tidal carbon dioxide and oxygen was monitored to assess cerebrovascular

reactivity 211,212. This was recorded into a data acquisition software (PowerLab 8/32

Amplifier, LabChart Software; ADInstruments, Colorado Springs, CO) and analyzed as

a change against atmospheric pressure. The gas analyzers were calibrated using primary

standard gases (16.0% O2, 4.0% CO2, balance N2) before all assessments.

3.3 Cerebrovascular Assessment Protocol

Prior to testing, participant demographic information was completed to inform

the researcher of caffeine, alcohol, and medication intake, as well as exercise and sleep

47

activity from the prior night. Finally, the participant also completed the Sport

Concussion Assessment Tool (SCAT) symptom scale to record changes in concussion-

like symptoms. The participant was encouraged to eat a small meal at least 2 hrs before

testing and asked to void their bladder within 30 minutes of arrival. Upon arrival to the

laboratory on the first occasion, the participant completed the necessary paperwork

(informed consent, medical forms including any prescription drugs currently taking,

SF36 Health Survey Questionnaire, PANAS, PHQ-9, and GAD-7). The participant had a

NIRS probe (Portalite) placed on the right prefrontal cortex to allow for cerebral

hemodynamic measurements. A 3-lead ECG (Finapres) was attached to the participant,

with the ground ECG electrode placed on the left anterior superior iliac spine and the

two main leads under the middle portion of each clavicle, known as a Lead I

configuration. The plethysmograph cuff was placed on the left middle finger, zeroed

according to guidelines, and the height corrector was placed at the level of the heart

(NOVA, Finapres). Finally, the capnograph was calibrated using atmospheric pressure

and primary standard gases (16.0% O2, 4.0% CO2, balance N2). The participant was re-

assessed at the same time of the day to avoid circadian changes. The testing protocol

(Figure 3) included:

(1) 5-minute sitting rest to establish spontaneous physiological baseline values,

(2) 6 breaths/minute paced breathing maneuver repeated for 5-minutes 213,

(3) 2-minute sitting rest to re-establish baseline physiology

48

(4) a hypercapnic breath hold (BH) challenge (20sec breath hold, followed by

40sec of normal breathing and repeated five consecutive times) to document

vascular responses for a total of 5 minutes158,214.

(4) 2-minute washout period to re-establish baseline physiology,

(5) 5-minute (20sec eyes closed:40sec eyes open x 5 repeats) object identification

protocol (“Where’s Waldo”) to act as a complicated visual search paradigm

that involves searching on a computer screen for an object character of a

specific color and shape (“Waldo”) that is hidden in a field of distracters of

similar colors and shapes to assess neurovascular coupling 212

(6) 2-minute washout period to re-establish baseline physiology,

(7) a squat-stand baroreflex maneuver (squat down to 90 degrees, stand-up;

performed at 10sec:10sec (0.05Hz) and 5sec:5sec (0.01Hz), repeated for 5

minutes) to assess flow-pressure reactivity and dynamic autoregulatory

control 32,215. A 3-minute wash-out period (2-minute seated; 1-minute

standing) was incorporated between the two maneuvers.

49

Figure 3: Overview of protocol to assess cardiovascular and cerebrovascular changes.

6BPM= 6 Breaths per minute; 5SS= 5 second squat; 10SS= 10 second squat; BH=

Breath hold; CVR= Cerebrovascular reactivity; NVC= Neurovascular coupling

All data was downloaded and stored following each test session, and then

analysed off-line in the days immediately following testing to keep the results up-to-date.

The 6 breaths/min maneuver is included to assess for heart rate variability changes

during the 5 minute baseline period which may be due to random breathing patterns, as

increased respiratory rates have been shown to cause a significant reduction of the

balance between long- and short-term heart rate variability213.

50

The hypercapnic test allows for monitoring of serial changes in cerebrovascular

reactivity. As partial pressure of CO2 increases, CBF also increases and washes out CO2

from brain tissue, reducing the central chemoreceptor stimulus. Thus, any reduction in

the sensitivity of CBF to CO2 increases the overall sensitivity of the central chemoreflex

response to CO2 changes, which will consequently lead to dysfunction of cerebrovascular

reactivity 216. Continued physiological assessment for 5 minutes while the participant

was guided through the BH protocol provided a better insight regarding cerebrovascular

reactivity 217.

The object identification protocol can elicit an elevation of cerebral blood

velocity within both the posterior and middle cerebral artery supplied regions of the

brain212. With the posterior cerebral artery being the primary source of blood supply to

the visual processing areas of the cerebral cortex, measurement using NIRS can show the

direct metabolic changes involved with the neurovascular coupling effect along with

how cannabidiol can affect this phenomena.

Finally, the squat-stand baroreflex maneuvers assessed dynamic autoregulatory

control. The squat-stand protocol has been shown to stimulate the baroreflex and it has

been suggested autonomic function is dysregulated following concussion, which was

shown using this maneuver 32,215. Thus, heart rate and blood pressure standard deviations

were analyzed for changes following drug administration. Because biological variance

can be impaired when one part of a system no longer functions within a healthy

range32,88,218,219, standard deviation measures were also analyzed.

51

3.4 Statistics

Because this was a case study, the statistical analysis was completed as

descriptive statistics. This included percent changes in cardiovascular and

cerebrovascular responses following each protocol.

3.5 Data Analysis

The NIRS software package provided tools for marking events and biasing the

chromophore concentration during testing. After zeroing all traces at the start of the rest

period, all data were exported (Microsoft Excel; Microsoft Office Home, Redmond, WA,

USA) at 10 Hz, as relative changes in HbO2, HHb, tHb and HbDiff concentrations, and

an absolute change in %TSI. The NIRS quality control factor (QCF) was then inspected

to ensure the quality of the signal (99.87-99.98%) was not below 98% which would

indicate that extraneous light had contaminated the recording88. The 5 minutes of resting

data was averaged out to provide a reference value. Each following data point was

subtracted from the previous 5 minutes of seated resting value (thereby setting each

resting period as “zero”) to provide a relative change from baseline response. The

average change of the 6 BPM, Where’s Waldo, BH, 10SS, and 5SS protocols were then

graphed (Figures 4-11; 13-16).

The respiratory and cardiovascular indices were recorded at 400 Hz into a data

acquisition software (PowerLab 8/32 Amplifier, Labchart Software; ADInstruments,

Colorado Springs, CO) to allow for time alignment. To analyze changes in HRV30, the

ECG signal was exported at 250Hz and analyzed using an in-house software for removal

of any ectopic beats and artifacts to ensure that all heartbeats were of sinus origin. After

52

acquiring the filtered R-R intervals, they were uploaded to Kubios HRV (Version 3.0.1

HRV Biosignal Analysis and Medical Imaging Group, Finland) to analyze linear, non-

linear and Poincare plot parameters. Blood pressure metrics (cardiac output (Q), stroke

volume (SV), mean arterial pressure (MAP), systolic pressure (SYS), and diastolic

pressure (DIAS) were exported at 2.5Hz. To observe the effects of medullary

chemoreceptor stimulation due to increases in the arterial carbon dioxide (CO2) pressure

(PaCO2) during the 20s breath hold, the 20 seconds of each breath hold was averaged

and subtracted from the average of the following 10 seconds after each breath hold

(Figure 12). To assess for changes in pressure alleviation during the 10SS, the first 6

seconds of each squat was averaged and then subtracted from the average of the last 4

seconds of each squat to provide an indication of the baroreflex for all the blood pressure

indices (Figure 17).

4.0 Results

4.1 Resting and 6 Breaths per minute (6-BPM)

Near Infrared Spectroscopy

All resting and 6BPM NIRS data are presented in Table 4. Because the data were

zeroed at the start of the 5-minute resting period, all resting data presented (besides

%TSI which is an absolute measure), showed the average relative change from the same

zero starting point. Day 14 (40mg CBD:2mg THC) and 28 (40mg CBD:2mg THC)

showed the largest change as there was an overall negative change from the zero point

during the 5 minutes of rest for HbO2, tHb, and HbDiff. HbO2 and HbDiff also had a

more negative change at day 70 (-135.2%). HHb was more positive relative to the zeroed

53

point at all days compared to baseline. Finally, %TSI over the resting period was much

greater at days 14 (40mg CBD:2mg THC), 28 (40mg CBD:2mg THC) and 42 compared

to both baseline and day 70. During the 6-BPM protocol (Table 5; Figures 4-7), with

respect to the change from the 5-minute resting data, there was a decrease at all days for

%TSI. HbO2 and HbDiff decreased at day 14 (40mg CBD:2mg THC) and 28 (40mg

CBD:2mg THC) relative to baseline, and they both increased at day 70. HHb increased

relative to baseline at days 14 (40mg CBD:2mg THC), 28 (40mg CBD:2mg THC) and

42. tHb changes were less consistent, with a large decrease at day 28 (40mg CBD:2mg

THC), and an increase at all other visits relative to baseline. These data further suggests

greater oxygen utilization at days 14 (40mg CBD:2mg THC) and 28 (40mg CBD:2mg

THC) in comparison to baseline, day 42 (20mg CBD:1mg THC) or day 70 (24mg

CBD:1.2mg THC).

Table 4: NIRS parameters for resting state data. %∆ indicates the change from Day 0.

Rest

%TSI %∆ HbO2 %∆ HHb %∆ tHb %∆ HbDiff %∆

DAY 0 70.0±0.7 0.3±0.6 -0.3±0.3 0.0±0.3 0.5±0.8

DAY 14 79.2±0.3 13.1 -1.1±0.3 -500.1 0.0±0.1 104.8 -1.1±0.2 -21731.0 -1.1±0.4 -304.2

DAY 28 77.2±0.2 10.2 -1.4±0.5 -612.7 0.1±0.1 141.4 -1.3±0.5 -25917.0 -1.5±0.5 -379.2

DAY 42 73.6±0.7 5.1 0.3±0.6 21.0 0.1±0.2 126.2 0.4±0.5 7923.8 0.3±0.7 -51.9

DAY 70 70.3±0.3 0.33 -0.1±0.4 -132.8 0.1±0.1 137.7 0.0±0.3 130.9 -0.2±0.5 -135.2





54

Table 5: NIRS parameters for 6 breaths per minute. %∆ indicates the change from day 0.

6 Breaths per Minute


DAY 0 2.4±0.5 1.2±0.5 -1.3±0.2 0.2±0.4 2.8±0.6

DAY 14 1.2±0.5 -48.4 1.0±0.6 -32.2 -0.7±0.2 43.6 0.3±0.6 41.4 1.8±0.7 -37.5

DAY 28 -0.7±0.3 -129.6 -0.8±0.4 -152.2 -0.3±0.1 81.2 -1.0±0.4 -609.4 -0.5±0.4 -119.3

DAY 42 1.9±0.3 -19.0 2.2±0.5 45.8 -0.6±0.1 54.9 1.6±0.5 693.5 2.8±0.5 -0.9

DAY 70 1.9±0.3 -22.5 1.8±0.3 19.7 -1.3±0.1 -1.6 0.5±0.3 136.0 3.1±0.3 11.3





Figure 4: Oxyhemoglobin (µM) changes from 5-minute rest (0 on y-axis) for 6 breaths



THC

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

Day 0 Day 14 Day 28 Day 42 Day 70

µM

HbO2 6BPM

55

Figure 5: Deoxyhemoglobin changes (µM) from 5-minute rest (0 on y-axis) for 6 breaths



THC

Figure 6: Total hemoglobin changes (µM) from 5-minute rest (0 on y-axis) for 6 breaths



THC

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0


µM

HHb 6BPM

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5


µM

tHb 6BPM

56

Figure 7: Hemoglobin difference (oxyhemoglobin - deoxyhemoglobin) (µM) changes

from 5-minute rest (0 on y-axis) for 6 breaths per minute protocol. Day 0= 0mg CBD:



Blood Pressure Metrics

All absolute blood pressure metrics are presented in Table 6. Resting state blood

pressure showed large variations at each visit. Relative to baseline, there was an increase

in SYS and MAP at each visit except day 28 (40mg CBD:2mg THC), while DIAS also

increased at day 14 (40mg CBD:2mg THC), 42 and 70. SV decreased at each visit and Q

did as well, except at day 28 (40mg CBD:2mg THC). Interestingly, the largest change

for each variable occurred at day 42 (20mg CBD:1mg THC) for the rest period. During

6-BPM, there was a decrease in SYS, DIAS and MAP at days 14 (40mg CBD:2mg

THC) and 28 (40mg CBD:2mg THC). There was a decrease in SV and Q at all days

relative to baseline. The largest change during the 6BPM occurred at day 70. It is also

very important to note that the standard deviation (SD) of MAP, SYS, and DIAS was

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4


µM

HbDiff 6BPM

57

always lowest at the baseline visit. During the 6-BPM protocol, this SD was highest at

day 70 (24mg CBD:1.2mg THC).

Table 6: Blood pressure metrics for rest and 6 breaths per minute (6-BPM) protocol. %∆

indicates the change from day 0.

MAP (mmHg) %∆

SV (mL) %∆

Q (L/min) %∆

SYS (mmHg) %∆

DIAS (mmHg) %∆

REST

Day 0 85.8±2.3

88.8±6.1 5.5±0.3 128.8±5.6

64.3±1.4

Day 14 90.9±3.7 6.0

71.8±7.8

-19.1 4.7±0.4 -15.2 133.0±8.8 3.2

69.9±2.0 8.7

Day 28 76.6±5.2

-10.7

83.2±10.3 -6.2 5.9±0.6 7.5 112.9±10.7

-12.4

58.5±3.0 -9.1

Day 42 101.8±4.4 18.7

58.7±5.8

-33.9 3.9±0.3 -28.6 146.8±9.4 14.0

79.4±2.6 23.5

Day 70 99.9±4.0 16.4

62.7±6.2

-29.4 4.1±0.3 -26.1 144.8±9.2 12.4

77.4±2.2 20.4

6-BPM

Day 0 77.4±5.1

90.7±13.6 5.6±0.7 115.8±11.6

58.3±2.9

Day 14 74.9±6.1 -3.3

76.2±13.1

-16.0 5.0±0.7 -11.2 108.5±13.1 -6.3

58.0±3.1 -0.4

Day 28 74.7±5.6 -3.5

82.3±14.4 -9.3 5.5±0.8 -2.0 109.6±12.6 -5.3

57.2±3.2 -1.8

Day 42 90.2±6.0 16.4

64.7±9.8

-28.7 4.3±0.5 -24.7 129.7±13.5 12.0

70.4±2.9 20.9

Day 70 91.4±7.6 18.0

64.6±10.4

-28.8 4.2±0.6 -25.2 131.8±16.4 13.8

71.2±3.7 22.2

MAP=Mean arterial pressure; SV= Stroke volume; Q= Cardiac output; SYS= Systolic

pressure; DIAS= Diastolic pressure; Day 0= 0mg CBD: 0mg THC; Day 14= 40mg



All HRV metrics are presented in Table 7. It is important to note that for the

resting data, due to the volume of artifact and excessive noise, the baseline data were not

included. However, the SD of R-R intervals was very consistent at both rest and 6BPM.

However, SD HR was also consistent for 6BPM with each visit showing a greater

increase in SD HR relative to baseline. There was also an increase in low frequency (LF,

ms2) and a decrease in high frequency (HF, ms2) at each visit in comparison to baseline.

58

%LF increased and %HF decreased at each day relative to baseline for 6BPM. Total

power (ms2) also increased at each visit during 6-BPM, and the LF/HF ratio did as well.

There was also a small decrease in SD1 at each visit and a small increase at SD2 (except

Day 42 (20mg CBD:1mg THC) is approximately the same as baseline) (Table 7).

Finally, SD2/SD1 increased at each visit during 6-BPM and sample entropy decreased at

each visit. Due to influences of respiratory sinus arrhythmia, resting HRV parameters are

difficult to assess. However, the data presented does suggest minimal changes in resting

HRV at each visit.

Table 7: HRV parameters for 5-minute rest and 6 breaths per minute

REST 6BPM

HRV Day

0 Day

14 Day

28 Day

42 Day

70 Day 0 Day

14 Day

28 Day

42 Day

70

Mean RR (ms) 923.1 844.3 896.7 924.6 958.6 906.7 886.1 909.3 915.3

SD RR (ms) 23.0 25.4 24.0 23.7 54.8 54.5 55.9 54.2 56.6

Mean HR (beats/min) 65.0 71.1 66.9 64.9 62.6 66.2 67.7 66.0 65.6

SD HR (beats/min) 1.6 2.1 1.8 1.7 3.6 4.1 4.3 4.0 4.1

Min HR (beats/min) 63.1 66.9 62.7 62.2 55.3 59.3 58.6 58.1 57.7

Max HR (beats/min) 66.8 76.8 70.7 67.2 68.2 74.7 75.5 72.7 72.0

NNxx (beats) 3.0 6.0 0.0 1.0 51.0 31.0 32.0 31.0 31.0

pNNxx (%) 0.9 1.7 0.0 0.3 16.5 9.4 9.5 9.5 9.5

VLF (ms^2) 117.8 178.3 264.1 185.5 498.7 434.8 263.0 317.7 323.3

LF (ms^2) 32.2 73.3 46.0 34.6 1586 1919 2089 2044 2162

HF (ms^2) 351.6 225.2 233.2 289.9 259.6 205.9 170.0 147.3 158.6

LF (%) 6.4 15.4 8.5 6.8 67.6 74.9 82.8 81.4 81.8

HF (%) 70.1 47.2 42.9 56.8 11.1 8.0 6.7 5.9 6.0

Total power (ms^2) 501.9 476.9 543.3 510.2 2347 2560 2523 2509 2645

LF/HF ratio 0.1 0.3 0.2 0.1 6.1 9.3 12.3 13.9 13.6

SD1 (ms) 17.5 16.6 14.8 15.0 24.0 21.4 21.7 21.4 21.3

SD2 (ms) 27.5 31.7 30.6 29.9 73.7 74.2 76.0 73.7 77.2

SD2/SD1 ratio 1.6 1.9 2.1 2.0 3.1 3.5 3.5 3.4 3.6

Approximate entropy 1.0 1.2 1.0 1.1 0.9 0.7 0.9 0.7 0.7

Sample entropy 1.7 1.8 1.5 1.7 1.2 0.8 1.1 0.8 0.9

HRV= Heart rate variability; RR= R to R beats; SD= Standard Deviation; HR= Heart

rate; Min= Minimum; Max= Maximum; VLF=Very low frequency; LF= Low frequency;

59

HF= High frequency; SD1= Short term standard deviation; SD2= Long term standard

deviation; NNxx= Normal to normal intervals greater than 50ms; pNNxx= Proportion of

the NNxx in the total number of normal to normal intervals

4.2 Where’s Waldo and BH


NIRS data are presented in Table 8. For the Where’s Waldo protocol, there was a

decrease in %TSI at each visit compared to baseline. HbO2 and HbDiff decreased at each

visit except day 70 relative to baseline. tHb decreased at each day except day 42 (20mg

CBD:1mg THC), while HHb increased at each day except day 70 (24mg CBD:1.2mg

THC). Furthermore, for the Where’s Waldo protocol there was an increase in variability

relative to baseline at day 42 (20mg CBD:1mg THC) for %TSI and an increase at each

day except day 42 (20mg CBD:1mg THC) for HHb. There was a slight increase in

variability at day 42 (20mg CBD:1mg THC) for HbDiff during the Waldo protocol. For

the BH protocol, there was a decrease in all NIRS parameters compared to rest, with the

exceptions being HHb ((increase at days 28 (40mg CBD:2mg THC), 42 (20mg

CBD:1mg THC) and 70 (24mg CBD:1.2mg THC)) and tHb ((increase at day 42 (20mg

CBD:1mg THC)). In regard to the variability of the signal, consistent patterns included a

decrease at each testing day for each NIRS parameter during the BH. These results are

also graphically represented in Figures 8-11.

60

Table 8: NIRS parameters for Where’s Waldo and breath hold protocols. %∆ indicates

the change from day 0.


Waldo

Day 0

1.4± 0.3 1.4±0.5 -0.4±0.1 1.0±0.6

1.7± 0.5

Day 14

-0.0± 0.2 -100.1 -0.2±0.2 -112.8 -0.0±0.1 99.4 -0.2±0.3 -118.0

-0.2± 0.2 -109.8

Day 28

-1.1± 0.3 -177.3 -0.9±0.4 -166.9 0.5±0.1 221.9 -0.4±0.5 -145.5

-1.4± 0.4 -179.0

Day 42

0.4± 0.4 -70.0 1.1±0.5 -21.8 0.3±0.1 180.7 1.4±0.6 40.3

0.8± 0.5 -56.6

Day 70

0.9± 0.2 -34.9 1.4±0.3 4.2 -0.5±0.2 -32.2 0.9±0.5 -6.7

1.9± 0.2 10.3

BH

Day 0

1.8± 0.9 2.3±0.6 -0.5±0.3 1.8±0.3

2.8± 0.9

Day 14

1.3± 0.5 -29.6 0.7±0.3 -70.7 -0.5±0.2 -2.7 0.2±0.2 -89.8

1.2± 0.5 -58.1

Day 28

-0.2± 0.3 -113.3 -0.1±0.3 -104.5 0.2±0.1 136.9 0.1±0.3 -96.0

-0.3± 0.4 -110.0

Day 42

0.9± 0.5 -53.3 1.9±0.4 -18.0 0.4±0.2 182.9 2.3±0.3 24.9

1.5± 0.6 -46.2

Day 70

0.8± 0.5 -56.1 2.0±0.5 -13.8 -0.3±0.3 48.4 1.8±0.2 -4.8

2.3± 0.7 -19.8





Figure 8: Oxyhemoglobin changes (µM) from 5-minute rest (0 on y-axis) for Where’s



70= 24mg CBD:1.2mg THC.

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

WALDO BH

µM

HbO2 Change from Rest


61

Figure 9: Deoxyhemoglobin changes (µM) from 5-minute rest (0 on y-axis) for Where’s




Figure 10: Total hemoglobin changes (µM) from 5-minute rest (0 on y-axis) for Where’s



70= 24mg CBD:1.2mg THC.

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

WALDO BH

µM

HHb Change from Rest


-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

WALDO BH

µM

tHb Change from Rest


62

Figure 11: Hemoglobin difference (oxyhemoglobin-deoxyhemoglobin) (µM) changes

from 5-minute rest (0 on y-axis) for Where’s Waldo and breath hold protocols. Day 0=



Blood Pressure Metrics: Where’s Waldo and BH

All absolute blood pressure metric data, including percent changes from day 0,

are presented in Table 9. There was an increase in MAP, SYS, and DIAS at both the

Where’s Waldo and the BH protocols at each day relative to baseline. There was a

decrease in stroke volume and cardiac output each day relative to baseline for both the

Where’s Waldo and BH protocols. The largest change for all blood pressure metrics

occurred at day 42 (20mg CBD:1mg THC) for the Where’s Waldo protocol and at day

70 (24mg CBD:1.2mg THC) for all the BH blood pressure metrics. However, the

greatest percent change for SV was at day 42 (20mg CBD:1mg THC) compared to day

70 (24mg CBD:1.2mg THC). As mentioned before, to observe the effects of medullary

chemoreceptor stimulation due to increases in the arterial carbon dioxide (CO2) pressure

(PaCO2) during the 20s BH, the 20 seconds of each breath hold was averaged and

-3

-2

-1

0

1

2

3

4

5

WALDO BH

µM

HbDiff Change from Rest


63

subtracted from the average of the following 10 seconds after each BH. MAP, SYS and

DIAS all increased at day 14 (40mg CBD:2mg THC) and 28 (40mg CBD:2mg THC)

compared to baseline and decreased at day 42 (20mg CBD:1mg THC) before increasing

again at day 70 (24mg CBD:1.2mg THC). Stroke volume had the opposite response as it

decreased at days 14 (40mg CBD:2mg THC) and 28 (40mg CBD:2mg THC), increased

at day 42 (20mg CBD:1mg THC) and then decreased again at day 70 (24mg CBD:1.2mg

THC), all relative to baseline (Figure 12). With respect to the SD of the blood pressure

signal, during the BH protocol, there was an increase in this variability for both the SYS

and DIAS measures at each visit following baseline, while the MAP signal increased at

both day 14 (40mg CBD:2mg THC) and day 28 (40mg CBD:2mg THC). There was a

decrease in SD for both SV and Q during the BH protocol at each visit.

64

Table 9: Blood pressure metrics for Where’s Waldo and breath hold protocols. %∆

indicates the change from day 0.

MAP (mmHg) %∆

SV (mL) %∆

Q (L/min) %∆

SYS (mmHg) %∆

DIAS (mmHg) %∆

Waldo

Day 0

76.6±4.0

88.7±7.9 5.3±0.4

113.5±7.5

58.1± 2.8

Day 14

90.1±4.0 17.6

70.8±6.4 -20.2 4.6±0.4 -13.2

130.9±8.5 15.4

69.6± 2.2 19.8

Day 28

87.9±3.1 14.9

71.0±5.8 -19.9 4.7±0.3 -10.4

126.7±6.9 11.7

68.5± 1.8 18.0

Day 42

97.3±3.6 27.0

54.9±5.3 -38.1 3.5±0.3 -33.5

136.9±8.0 20.6

77.4± 2.1 33.3

Day 70

96.8±4.8 26.4

55.5±6.5 -37.4 3.6±0.4 -32.5

136.3±10.4 20.1

77.0± 2.5 32.5

BH

Day 0

86.9±6.0

89.9±9.2 5.4±0.5

131.3±9.3

64.7± 5.0

Day 14

97.0±6.5 11.7

72.6±8.1 -19.3 4.7±0.4 -13.2

143.9±11.1 9.6

73.6± 5.0 13.8

Day 28

91.1±7.9 4.9

74.5±10.7 -17.2 5.1±0.6 -7.1

133.6±11.9 1.7

69.9± 6.6 8.0

Day 42

105.3±4.9 21.2

57.6±5.7 -36.0 3.8±0.3 -30.7

151.9±8.9 15.7

82.0± 3.8 26.7

Day 70

108.7±5.6 25.0

58.7±5.9 -34.7 3.8±0.3 -30.3

158.2±9.9 20.5

83.9± 4.4 30.0





65

Figure 12: Average of 10 seconds post breath hold minus the average of each of the 20

second breath holds. Units for MAP (mean arterial pressure): mmHg; SV (stroke

volume): mL; SYS (systolic): mmHg; DIAS (diastolic): mmHg. Day 0= 0mg CBD: 0mg



All HRV metrics are presented in Table 10. Most notably, for Where’s Waldo

there was a decrease in SD RR each day relative to the baseline measure. For BH, SD

RR increased each day relative to baseline. Also, SD of HR also increased during BH

relative to baseline. Minimum and maximum heart rate also increased relative to baseline

at each day for both the Where’s Waldo and the BH protocols. Fast Fourier

transformation spectral changes were also of interest, with %HF decreasing at each day

and %LF increasing at each day post baseline during Where’s Waldo, with a similar

pattern during BH, although an increase in %LF at day 70 (24mg CBD:1.2mg THC)

during BH relative to baseline. The LF/HF ratio also increased each day post baseline for

Where’s Waldo and only increased at day 28 (40mg CBD:2mg THC) and 70 (24mg

CBD:1.2mg THC) for BH.

-20

-15

-10

-5

0

5

10

15

20

MAP SV SYS DIAS

10 Seconds post Breath Hold

DAY 0 DAY 14 DAY 28 DAY 42 DAY 70

66

The non-linear parameter included Poincare plots to observe changes in SD1 and

SD2. SD1 decreased each day relative to baseline for both protocols, while SD2

decreased for Where’s Waldo and increased during BH. These results led to an increase

in the SD1/SD2 ratio at each day for both protocols. Finally, both sample entropy and

approximate entropy were also parameters of interest, and while approximate entropy

decreased each day relative to baseline for both protocols, sample entropy increased

during the Where’s Waldo protocol and decreased during the BH protocol. In regard to

end-tidal carbon dioxide (PETCO2), the highest value during exhalation following the 20

seconds of breath hold was consistent at 37.8 mmHg being the lowest (day 70; 24mg

CBD:1.2mg THC) and 39.2 mmHg being the highest (day 28; 40mg CBD:2mg THC).

67

Table 10: Heart rate variability parameters for Where’s Waldo and breath holds

Waldo BH

HRV Day 0 Day

14 Day

28 Day

42 Day

70 Day

0 Day

14 Day

28 Day

42 Day

70

Mean RR (ms) 1004.

0 923.3 898.0 935.0 931.3 991.

5 920.2 880.7 914.4 927.0

SD RR (ms) 25.2 21.3 22.9 20.0 19.2 40.2 40.2 48.4 43.8 44.8

Mean HR (beats/min) 59.8 65.0 66.8 64.2 64.4 60.5 65.2 68.1 65.6 64.7

SD HR (beats/min) 1.5 1.5 1.7 1.4 1.3 2.5 2.9 3.7 3.1 3.1

Min HR (beats/min) 57.7 62.2 63.6 62.0 62.5 56.6 60.3 61.9 60.4 58.5

Max HR (beats/min) 63.7 68.2 70.1 67.1 67.4 65.8 69.9 73.7 70.3 69.8

NNxx (beats) 20.0 2.0 4.0 1.0 1.0 29.0 9.0 11.0 7.0 9.0

pNNxx (%) 6.9 0.6 1.2 0.3 0.3 9.7 2.8 3.2 2.1 2.8

VLF (ms^2) 163.8 172.2 147.0 288.6 132.3 1167 1251 1835 1535 1222

LF (ms^2) 45.9 121.2 70.3 55.6 76.1 130.

7 114.5 138.8 126.6 159.4

HF (ms^2) 161.6 151.2 142.7 75.3 104.9 313.

8 285.4 225.7 256.7 260.6

LF (%) 12.3 27.3 19.5 13.3 24.3 8.1 6.9 6.3 6.6 9.7

HF (%) 43.5 34.0 39.6 18.0 33.5 19.5 17.3 10.3 13.4 15.9

Total power (ms^2) 371.9 444.6 360.0 419.6 313.4 1612 1651 2200 1918 1642

LF/HF ratio 0.3 0.8 0.5 0.7 0.7 0.4 0.4 0.6 0.5 0.6

SD1 (ms) 19.4 14.3 13.9 10.5 10.0 19.9 16.4 16.3 15.2 15.4

SD2 (ms) 29.9 26.5 29.2 26.3 25.2 53.3 54.5 66.6 60.1 61.6

SD2/SD1 ratio 1.5 1.9 2.1 2.5 2.5 2.7 3.3 4.1 4.0 4.0

Approximate entropy 1.2 1.1 1.2 1.1 1.1 1.1 0.9 1.1 0.9 0.9

Sample entropy 1.5 1.7 1.9 1.7 1.9 1.4 0.9 1.2 0.8 1.0

PETCO2 (mmHg)* 38.0 38.2 39.2 38.9 37.8






*Average 10 seconds of PETCO2 post exhalation following the 20s BH

4.3 10sec Squat Stand (10SS) and 5sec Squat Stand (5SS)


All NIRS data are presented in Table 11. Interestingly, there was a decrease in

%TSI, HbO2, and HbDiff at each day during both 10SS and 5SS. tHb decreased at each

day during the 10SS, but it increased 33.6% at day 42 (20mg CBD:1mg THC) during the

68

5SS. HHb on the other hand increased each day during both the 10SS and the 5SS. For

all NIRS parameters, the largest changes happened at day 28 (40mg CBD:2mg THC).

Variability, as measured by standard deviation was inconsistent from day-to-day relative

to baseline. For 10SS, variability was at its highest at day 42 (20mg CBD:1mg THC) for

%TSI, day 28 (40mg CBD:2mg THC) for HbO2, day 42 (20mg CBD:1mg THC) for

HHb, day 28 (40mg CBD:2mg THC) for tHb, and day 28 (40mg CBD:2mg THC) for

HbDiff. For 5SS, variability for HbDiff was highest at baseline; tHb and HHb were

highest at day 42 (20mg CBD:1mg THC); HbO2 was highest at day 14 (40mg CBD:2mg

THC) and %TSI was highest at day 42 (20mg CBD:1mg THC).

Table 11: NIRS parameters for 10 sec and 5 sec squat stands. %∆ indicates the change

from day 0.


10SS

Day 0 2.9±0.6 4.1±1.1

-0.6±0.3 3.5±1.0 4.7±1.2

Day 14

-0.8±0.3

-126.9 0.6±0.9 -84.8 0.2±0.2

130.7 0.8±1.1 -76.6 0.4±0.8 -90.8

Day 28

-2.2±0.7

-175.8

-1.3±1.4

-132.8 1.4±0.4

322.7 0.0±1.4 -98.9

-2.7±1.5

-157.8

Day 42

-1.2±0.8

-141.5 2.1±1.0 -49.5 1.0±0.5

264.9 3.1±1.0 -11.0 1.0±1.3 -77.8

Day 70

-0.7±0.3

-123.0 2.3±1.0 -44.1 0.3±0.4

144.7 2.6±1.0 -26.2 2.0±1.1 -57.3

5SS

Day 0 2.9±0.4 3.7±0.6

-0.4±0.2 3.2±0.7 4.1±0.7

Day 14 0.3±0.3 -88.3 1.0±0.8 -73.2 0.5±0.2

220.6 1.5±0.9 -53.9 0.5±0.7 -88.5

Day 28

-2.8±0.3

-195.8

-2.3±0.8

-163.7 1.9±0.2

554.7

-0.4±0.9

-112.5

-4.3±0.6

-204.3

Day 42 0.1±0.5 -97.6 2.8±0.7 -22.8 1.5±0.3

453.2 4.3±1.0 33.6 1.3±0.6 -67.4

Day 70 1.0±0.3 -67.5 2.3±0.6 -38.5 0.4±0.2

194.1 2.7±0.7 -18.1 1.9±0.5 -54.6





69

Figure 13: Oxyhemoglobin changes (µM) from 5-minute rest (0 on y-axis) for 10 second




Figure 14: Deoxyhemoglobin changes (µM) from 5-minute rest (0 on y-axis) for 10

second squat and 5 second squat protocols. Day 0= 0mg CBD: 0mg THC; Day 14=

40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day 42= 20mg CBD:1mg THC;

Day 70= 24mg CBD:1.2mg THC

-4

-3

-2

-1

0

1

2

3

4

5

6

10SS 5SS

µM

HbO2 Change from Rest


-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

10SS 5SS

µM

HHb Change from Rest


70

Figure 15: Total hemoglobin changes (µM) from 5-minute rest (0 on y-axis) for 10

second squat and 5 second squat protocols. Day 0= 0mg CBD: 0mg THC; Day 14=

40mg CBD:2mg THC; Day 28= 40mg CBD:2mg THC; Day 42= 20mg CBD:1mg THC;

Day 70= 24mg CBD:1.2mg THC

Figure 16: Hemoglobin difference (oxyhemoglobin-deoxyhemoglobin) (µM) changes

from 5-minute rest (0 on y-axis) for 10 second squat and 5 second squat protocols. Day

0= 0mg CBD: 0mg THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg

THC; Day 42= 20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

-2

-1

0

1

2

3

4

5

6

10SS 5SS

µM

tHb Change from Rest


-6

-4

-2

0

2

4

6

8

10SS 5SS

µM

HbDiff Change from Rest


71

Blood Pressure Metrics

All absolute blood pressure metric data are presented in Table 12. There is an

increase in MAP, SYS, and DIAS at both the 10SS and the 5SS at each day relative to

baseline. There is a decrease in stroke volume and cardiac output each day relative to

baseline at both the 5SS and the 10SS. The largest change for all blood pressure metrics

occurred at day 70. This pattern was also consistent for all blood pressure metrics. To

observe the pressure alleviation effects of the baroreflex during the 10SS32, the first 6

seconds of each squat was averaged and subtracted from the average of the last 4

seconds on each squat (Figure 17). Interestingly, pressure alleviation decreased from

baseline at both day 14 (40mg CBD:2mg THC) and day 28 (40mg CBD:2mg THC) for

MAP, SYS and DIAS. However, pressure alleviation increased greater than baseline at

day 70 (24mg CBD:1.2mg THC) compared to baseline, and at day 42 (20mg CBD:1mg

THC) for DIAS as well.

Regarding blood pressure variability, for MAP, SYS, and DIAS, there was an

increase in SD at each visit relative to baseline. For MAP and SYS, SD increased the

largest SD occurred at day 28 (40mg CBD:2mg THC), whereas the largest SD for DIAS

occurred at day 70 (24mg CBD:1.2mg THC). Finally, for both Q and SV, SD decreased

at each visit relative to baseline.

72

Table 12: Blood pressure metrics for 10 sec and 5 sec squat stands. %∆ indicates the

change from day 0.

MAP

(mmHg) %∆ SV (mL) %∆ Q

(L/min) %∆ SYS

(mmHg) %∆ DIAS

(mmHg) %∆

10SS

Day 0 103.8±17.2 91.01±12

.6 6.9±1.4 160.7±27.

0 75.4±12.8

Day 14 108.4±22.3 4.5

73.9±12.5

-18.9 5.8±1.2

-16.9

165.9±36.5 3.2 79.7±16.3 5.8

Day 28 107.4±24.4 3.5

69.6±10.3

-23.6 5.7±1.1

-18.1

162.7±41.4 1.2 79.8±16.8 5.9

Day 42 114.7±23.1

10.5

66.3±10.0

-27.3 5.3±1.1

-24.1

172.7±38.0 7.5 85.6±16.6

13.6

Day 70 121.3±23.7

16.9

65.1±10.2

-28.5 5.2±1.1

-25.1

183.1±38.1

13.9 90.4±17.4

20.1

5SS

Day 0 96.4±16.4 97.5±10.

8 7.8±1.1 148.7±24.

4 70.2±13.1

Day 14 112.8±20.2

17.1 72.2±9.3

-26.0 5.9±0.8

-23.7

170.8±30.5

14.9 83.9±16.0

19.4

Day 28 114.0±22.3

18.3 68.8±9.6

-29.4 5.9±0.9

-23.8

171.6±34.5

15.4 85.2±17.2

21.3

Day 42 116.0±21.9

20.4 67.5±9.1

-30.8 5.7±0.9

-27.0

173.7±32.4

16.9 87.2±17.5

24.2

Day 70 120.2±21.8

24.7 63.1±9.5

-35.3 5.3±0.9

-32.0

178.8±32.1

20.3 90.9±17.6

29.4





73

Figure 17: Average of first 6 seconds of each squat minus the average of the last 4

seconds of each squat for the 10 second squat protocol. Units for MAP (mean arterial

pressure): mmHg; SYS (systolic): mmHg; DIAS (diastolic): mmHg. Day 0= 0mg CBD:


20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC.

HRV Metrics

All HRV metrics are presented in Table 13. Most notably, for 10SS, SD RR

increased each day relative to the baseline measurement. Consequently, HR SD

increased each day relative to baseline as well. Furthermore, SD2 from the Poincare

plot213 also increased each day relative to baseline in contrast to SD1, which decreased

each day relative to baseline. Fast Fourier transformation spectral changes were

inconsistent, with %High frequency (%HF) increasing at day 14 (40mg CBD:2mg THC)

post baseline but decreased at all other days. Finally both sample entropy and

approximate entropy increased at day 14 (40mg CBD:2mg THC), but decreased at days

28 (40mg CBD:2mg THC), 42 (20mg CBD:1mg THC) and 70 (24mg CBD:1.2mg

THC). These changes were not consistent with the 5SS, as shown by decreases in SD of

-20

-15

-10

-5

0

5

10

15

20

25

30

35

MAP SYS DIAS

Blood Pressure Alleviation during 10SS

DAY 0 DAY 14 DAY 28 DAY 42 DAY 70

74

RR intervals and inconsistent changes in the majority of HRV metrics relative to

baseline. However, there was an increase in %LF and %HF during 5SS each day relative

to the baseline measure. Finally, both SD1 and SD2 were decreased each day relative to

baseline which was consistent with the 10SS; there was an increase in the SD2/SD1 ratio

each day relative to baseline.

Table 13: HRV parameters for 10 sec and 5 sec squat stands.

10SS 5SS

HRV Day 0 Day 14

Day 28

Day 42

Day 70 Day 0

Day 14

Day 28

Day 42

Day 70

Mean RR (ms) 795.2 774.0 732.4 758.4 754.2 751.8 727.3 694.5 710.0 714.3

SD RR (ms) 70.6 71.4 80.6 80.9 80.4 82.9 70.7 65.7 76.8 75.0

Mean HR (beats/min) 75.5 77.5 81.9 79.1 79.6 79.8 82.5 86.4 84.5 84.0

SD HR (beats/min) 6.6 7.1 8.8 8.2 8.1 8.0 7.4 7.4 8.6 8.0

Min HR (beats/min) 63.9 65.0 66.8 63.9 63.9 62.1 65.6 65.7 64.8 64.0

Max HR (beats/min) 85.5 89.7 96.7 94.1 91.6 90.9 91.8 96.5 97.5 94.3

NNxx (beats) 41.0 27.0 34.0 35.0 37.0 25.0 10.0 17.0 10.0 14.0

pNNxx (%) 11.1 7.2 8.4 9.0 9.5 6.6 2.5 4.0 2.4 3.4

VLF (ms^2) 539.0 774.4 721.6 1310.

1 1233.

8 2044.

4 1694.

1 1226.

9 1847.

0 1740.

5

LF (ms^2) 3033.

6 3124.

0 5380.

7 4707.

6 4383.

8 158.4 239.6 198.9 285.5 115.9

HF (ms^2) 245.4 369.0 212.7 226.3 326.1 90.6 153.2 87.1 97.8 130.9

LF (%) 79.4 73.2 85.2 75.4 73.8 6.9 11.48 13.15 12.8 5.8

HF (%) 6.4 8.6 3.4 3.6 5.5 4.0 7.3 5.8 4.4 6.6

Total power (ms^2) 3820.

9 4269.

1 6315.

4 6244.

1 5944.

2 2294.

0 2087.

0 1513.

0 2230.

4 1987.

8

LF/HF ratio 12.4 8.5 25.3 20.8 13.4 1.8 1.6 2.3 2.9 0.9

SD1 (ms) 30.2 21.5 20.4 21.2 24.2 23.4 15.7 16.7 14.3 18.7

SD2 (ms) 95.0 98.8 112.2 112.5 111.1 114.6 98.6 91.2 107.6 104.3

SD2/SD1 ratio 3.2 4.6 5.5 5.3 4.6 4.9 6.3 5.5 7.5 5.6

Approximate entropy 1.0 1.1 0.8 0.9 0.9 0.8 0.9 0.8 0.8 0.7

Sample entropy 1.1 1.2 0.7 0.8 0.7 0.7 0.9 0.7 0.7 0.6






75

4.4 Psychological Questionnaire Results

The questionnaires were intended to assess the participant’s mood, mental health,

state of possible depression and anxiety. Overall, there was very little change in the

absolute score of any of the questionnaires. It is important to note that there was an

increase in difficulty according to the participant in her ability to work, take care of

things at home or get along with other people as assessed by the PHQ-9 at days 42

(20mg CBD:1mg THC) and 70 (24mg CBD:1.2mg THC), when the participant dropped

her dose from days 14 and 28 (40mg CBD:2mg THC).

Clinical questionnaires are often assessed by their minimal clinically important

difference (MCID). While this was not a clinical case study, it is worth mentioning that

an increase of 5 in the PHQ-9 is considered significant220, and this change was not seen

from any day. A change of 4 points is considered as the MCID for the GAD-7 which was

also not seen in the participant221. The PANAS positive (30.6±7.9) and negative

(16.7±6.4) values all fell within the normal, non-clinical range for females184, with the

negative values being on the lower end. Finally, the SF-36 data was also within normal

ranges222.

Table 14: Psychological questionnaire results


PHQ-9 3 (ND) 2 (ND) 2 (ND) 4 (SD) 3 (SD)

GAD-7 0 (ND) 2 (ND) 0 (ND) 1 (ND) 0 (ND)

PANAS-P 32 35 36 30 32

PANAS-N 11 14 10 12 11

SF-36 Sum 2380 2690 2655 2375 2395

PHQ-9= Patient Health Questionnaire; GAD-7=General Anxiety Disorder-7 PANAS-

P/N=The Positive and Negative Affect Schedule Positive/Negative; SF-36=Short Form

Health Survey 36-item

76

5.0 Discussion

This case study examined the effects of a high CBD/low THC on cerebrovascular

and cardiovascular physiology. To the authors knowledge this the first such study to

report prefrontal cortex oxygenation, blood pressure and heart rate metric changes in a

participant self-administering medical cannabis while experiencing post-concussion

symptoms. Overall, these results suggest that for this participant, there was a beneficial

(positive) effect with the greatest physiological responses occurring at day 28 when the

participant was consuming 2mL of the high CBD/low THC study drug daily for 28

consecutive days. Much of the data, especially blood pressure variability, suggest that

there is a sustained beneficial physiological effect even when lower dosages (1mL and

1.2mL) of the study drug was continued to be consumed up to 70 days post-

administration when compared to baseline before starting the drug administration (day

0).

Rest

The 5-minute resting period established a daily baseline to compare the results of

the other maneuvers within the protocol. At rest, %TSI remained elevated during days 14

(40mg CBD:2mg THC) and 28 (40mg CBD:2mg THC) when the participant was taking

2mL per day of the study drug. This suggests an oxygen-rich right prefrontal cortex,

thereby implying that the extraction rate of oxygen from hemoglobin is not a limitation;

i.e., on day 14 (40mg CBD:2mg THC) and 28 (40mg CBD:2mg THC) vs baseline (day

0) pre-administration, cellular metabolism is elevated even under resting conditions.

This implies the administration of high CBD/low THC simulated the endocannabinoid

77

system to enhance functional adaptations in cellular metabolism that were otherwise

potentially downregulated in this participant. Because the changes in NIRS are all

relative, the high %TSI and the continuous increase in HHb and decrease in HbO2 at day

28 (40mg CBD:2mg THC) does suggest availability of oxygen at the brain, however,

there is a faster rate of extraction than delivery (as shown by the 379% decrease in

HbDiff on Table 4). Research by Ceprián et al223 supports this contention, where it was

shown when rats were administered single doses of 5mg/kg CBD there was a reduction

in metabolic derangement and damage due to middle cerebral artery occlusion, reduction

in excitotoxicity and neuro-inflammation, and this was also supported by Maroon &

Bost7, who stated that CBD’s ability to reduce cerebral hemodynamic impairment may

be due to CBD’s ability to reduce fatty acid amide hydrolase, thereby increasing AEA

levels6,7. Furthermore, with CBD’s potential to inhibit fatty acid amide hydrolase162,

there may also be an increase to CB receptor stimulation not induced by THC, but rather

AEA. Taken together, these results suggest more energy availability at rest at days 14

(40mg CBD:2mg THC), 28 (40mg CBD:2mg THC) and 42 (20mg CBD:1mg THC) as

compared to day 0.

6-Breaths per Minute

The 6-BPM maneuver was designed to assess for physiological changes while

taking respiratory sinus arrythmia (RSA) into consideration224 which functions to

minimize the mechanical work done by the heart. This ensures an optimal environment

for gas exchange during breathing by matching perfusion to heart rate224. 6-BPM has

been shown to be an efficient method to assess accurate HRV measures213 and these

78

measures can provide more insight into the overall physiological changes. The decrease

in HbO2 and the increase in HHb from the 5-minute resting baseline at days 14 (40mg

CBD:2mg THC) and 28 (40mg CBD:2mg THC) might suggest an increase in metabolic

activity and oxygen extraction, as confirmed by the decrease in HbDiff, as more negative

values for HbDiff likely represent higher oxygen consumption225,226. It therefore follows

that HbDiff can act as a surrogate measure to reflect oxygen extraction in the tissue.

Furthermore, the increase in the SD of MAP, SYS and DIAS all further suggest

that a 2mL intake of the drug ensemble induces a larger physiological response as

compared to the 1mL and 1.2mL intake (Table 6). Finally, the HRV parameters are also

of great interest during this maneuver35,213. Results showed an increase in the LF (0.04-

0.15 Hz; %, ms2) and a decrease in HF (0.15-0.40 Hz; %, ms2). The LF power is

generally interpreted to reflect sympathetic, parasympathetic and baroreceptor activity

during resting conditions, while the HF power is thought to be indicative of vagal or

parasympathetic activity227. Therefore, these results may be indicative of an increase in

the baroreflex response to accommodate for the large fluctuations occurring in heart rate.

Where’s Waldo

Where’s Waldo has been shown to induce large increases in cerebral artery blood

flow velocity212, thereby showing great neurovascular coupling potential. With an

increase in neural activation during Where’s Waldo, there must be an increase in nutrient

demand to accommodate for the increase in cerebral metabolism. Thus, the measurement

of prefrontal cortex oxygenation using NIRS can provide valuable insight to the

metabolic activity. The decrease in HbO2 and increase in HHb at days 14 (40mg

79

CBD:2mg THC) and 28 (40mg CBD:2mg THC) with respect to the 5 minutes of rest

suggests that the Where’s Waldo maneuver elicited a greater rate of oxygen extraction in

comparison to the resting period on day 0. Both the HbO2 and HHb responses did elevate

back to the baseline level at days 42 (20mg CBD:1mg THC) and 70 (24mg CBD:1.2mg

THC), thereby suggesting a decreased rate of extraction when 1 and 1.2 mL per day of

the drug were consumed in comparison to the 2mL per day intake. Because glutamate

binding to NMDA receptors can influence neuronal release of Ca2+ activated nitric oxide

and thereby inducing vasodilation228, CBD’s ability to regulate intracellular Ca2+ build

up108 following traumatic brain injury28 can limit the nitric oxide production and

therefore limit vasodilation. This can interrupt the large energy crisis response following

concussion which tends to be due to increases in extracellular Ca2+, and therefore help

aid against the increased metabolism (accelerated glycolysis) common in post concussive

pathophysiology28,229.

Building on this vasoconstrictive mechanism, it follows that there was a large

overall increase in MAP, SYS and DIAS, with a decrease in Q and SV, possibly

implying a greater vasoconstrictive effect when searching for Waldo following the

ensemble administration. This implication is further supported by the large decrease in

both Q and SV. Interestingly these patterns were consistent even when the participant

reduced the intake to 1mL and 1.2mL per day, as blood pressure continued to increase

and both Q and SV continued to decrease. Rather than a dose-dependent response, this

suggests that there may be a pooled effect on blood pressure when taking the

cannabinoids. It’s possible that the ensemble induced upregulation of the receptors

(TRPV1, CB1, CB2, 5-HT1A) most likely due to CBD’s influence on AEA2,230, in

80

combination with decreasing excitatory post synaptic potentials and excitatory

neurotransmitter release2,6,95, which can be responsible for vasoconstrictive responses.

Finally, regarding HRV results, most notably there was an increase in LF (%, ms2) and

decrease in HF (%, ms2), along with an increase in SD2/SD1 and LF/HF ratios. These

results may be due to the metabolically demanding Where’s Waldo task, suggesting a

greater rate of energy utilization. Because there is a likely decrease in vasodilation

mediators such as nitric oxide following CBD administration231, it follows that the

increase in blood pressure in response to a metabolically demanding task shifts

autonomic function to a more sympathetic state. As such, %LF, LF/HF and SD2/SD1 are

all considered to be expressive of sympathetic dominance227, and following mTBI, it is

possible that this sympathetic state (decrease HF and increase LF) does represent a

recovery period as it can be indicative of decreases in Ca2+ accumulation232.

Breath Hold

Changes in cerebrovascular reactivity have been documented before following a

hypocapnic challenge when utilizing 20 second breath holds in a concussed population

88,214. As breath holding results in a linear increase in PaCO2, changes in

cerebrovasculature vasodilation are expected233. It’s been suggested that a possible

mismatch may occur between the metabolic demands of a concussed brain and the

delivery of blood to the area234,235. Interestingly, this case study suggests an overall

decrease in HbO2 in relation to 20 seconds of breath holds and 40 seconds of breathing

normally at 2mL and 1mL per day administrations, with little to no change when taking

1.2mL. It is also important to note that this same pattern was consistent with all NIRS

81

parameters when observing the change at 20 seconds only. Dysregulation of neuronal

metabolism following brain injury can be induced due to increases in calcium-

sequestration, possibly resulting in microvascular changes, thereby altering cerebral

oxygenation and blood volume. CBD’s ability to regulate internal calcium changes108

suggests a possible interaction to reduce any further damage. The variability of the signal

is in contrast to results from a previous study by Bishop and Neary88, who showed that

the variability of acutely concussed athletes tends to decrease in comparison to controls.

The participant in this case study had a decrease in variability compared to their baseline

assessments during each of the visits, however, it is important to note that she was not

acutely concussed. Furthermore, the overall decrease in HbO2 is consistent with changes

noted by Bishop and Neary88, as they found participants assessed at days 7 to 14 post

mTBI had decreased HbO2 levels in comparison to controls. The consistent decrease in

SV and Q in relation to the increase in MAP, SYS and DIAS suggest an increased

vasoconstrictive response, which is contrary to previous research236 which showed that

single oral doses of CBD decrease blood pressure. Much of this may be due to the nature

of this participant having post-concussion like symptoms, and therefore the complex

pathophysiology does suggest that the decrease in vasodilation mediators perhaps

rebalance the blood flow and nutrient demand mismatch, and this may contribute to these

results.

The specific ratio of CBD to THC or whether THC is included in the ensemble is

also influential. CBD is thought to cause an ‘accommodating’ effect in the presence of

THC, and can therefore induce a large decrease in blood pressure237. Combined with the

increase in LF, and MAP, SYS and DIAS variability, this suggests an increase in the

82

baroreflex response during BH, thereby possibly allowing a more sensitive response by

the chemoreceptors to the increase in PaCO2. This response can be seen at day 28 (40mg

CBD:2mg THC; Figure 12), as the first 10 seconds of normal breathing following the 20

second breath hold elicits the largest increase in MAP, SYS and DIAS, and the largest

decrease in SV. Taken together, suggests an increase in vasoconstriction.

This sustained beneficial increase in blood pressure is likely due to the presence

of both THC and CBD in the composition. Because CBD (and THC) stimulation of

TRPV140 can potentially lead to an increase in endothelial nitric oxide synthase, this

should theoretically result in vasodilation. As mentioned earlier it’s possible that the

pathological state of the participant may have induced a greater intracellular Ca2+

response. Therefore, by reducing the excitatory neurotransmitter release, a reduction in

vasodilation is very plausible. A combination of both CBD and THC may have also

resulted in a combination of stimulating both the CB and non-CB receptors sensitive to

THC and CBD, thereby possibly furthering the vasoconstrictive response. Changes

regarding a decrease in blood pressure and an increase in heart rate has been noted in

healthy volunteers236, but the complexity of a concussion or traumatic brain injury in

combination with modulation of the endocannabinoid system following injury makes it

very difficult to assume the same response would be shown in a pathological case.

Squat Stand Maneuvers

The 5 (5SS) and 10 (10SS) second squat stands were designed to initiate a

baroreflex response and to assess changes in pressure alleviation. The degree of pressure

alleviation reflects dynamic cerebral autoregulation32,238. The 5SS was designed to assess

83

overall HRV and blood pressure variability utilizing a similar stance to the 10SS but

done at a higher frequency (0.05 Hz for 5SS compared to 0.10 Hz for 10SS). As per

unpublished data in the exercise physiology laboratory35, the NIRS data showed an

increase in variability for the 10SS for all variables when consuming the ensemble

formulation, with day 28 (40mg CBD:2mg THC) having the greatest increase in HbO2,

tHb, and HHb at 2mL administration. This does suggest that the brain is less resistant to

changes induced by the squat stand maneuver.

For 5SS, the increase in MAP, SYS and DIAS in combination with a decrease in

SV and Q does further suggest a more baroreflexive response over the duration of the

study, especially when considering the increase in SD RR and SD HR when taking the

study drug as compared to baseline. This increase in HRV is consistent with the increase

in blood pressure variability. The 5SS induced a physiological response similar to the

10SS, with an increase in blood pressure variability when having administered the

ensemble. Previous research by Wright et al239 used a 5SS challenge in an athletic

population that experienced mild traumatic brain injury, and showed that injury can

result in delayed changes in vascular resistance.

As per Figure 17, pressure alleviation remained low during days 14 (40mg

CBD:2mg THC) and 28 (40mg CBD:2mg THC), suggesting a change in dynamic

cerebral autoregulation. Interestingly, this change is similar to mTBI patients in the study

by Bishop et al32. This further supports greater induced vasoconstriction following

ensemble administration, thereby delaying vasodilation following a large increase in

84

blood pressure, as the participant in this case study was suffering from post-concussive

issues rather than acute concussion changes.

In Relation to Symptoms

While changes in the mood, depression and anxiety questionnaires were largely

negligible, the changes associated with the participant’s day-to-day activities as recorded

in her logbook were substantially different. The participant did state “jittery” feelings

and feeling a lot more “alert” when returning on day 28 (40mg CBD:2mg THC) for

assessment. Because the participant was self-administering her dosage, and because she

was feeling much better physically, she decided to try to reduce her dosage (to 1mL).

However, it is important to note that the participant did report feeling mild headaches

and troubles sleeping when she lowered her dose. This further suggests a “pooled”

accumulative effect of the drug, as the brain may have still been at an alerted state due to

possible upregulation of TRPV1, CB, 5-HT1A, A2a and other receptors involved in

endocannabinoid signaling. Furthermore, the participant also stated being a lot more

active at days 14 (40mg CBD:2mg THC) and 28 (40mg CBD:2mg THC) as compared to

her normal day-to-day activities. In response to these results, it is possible that the body

was extracting oxygen at a greater rate than normal and inducing vasoconstriction as a

protective mechanism to counterbalance all the substantial changes in the quality of life

on a day-to-day basis, as noted in her logbook. The participant recorded her daily quality

of life on a 0-10 scale. At day 0 she had a rating of 5, which improved to an average of

6.6 by day 14 (40mg CBD:2mg THC), 6.9 by day 28 (40mg CBD:2mg THC), dropped

to 6.2 by day 42 (20mg CBD:1mg THC) and went back up to 6.7 by day 70. These self-

85

assessments therefore do suggest that the participant did believe that administration of

the ensemble had a positive influence on her quality of life.

6.0 Conclusion and Limitations

Overall these results suggest that a high CBD and low THC combination have a

complex physiological response in an individual still experiencing concussion-like

symptoms. While the participant did note subjective changes and improvements in her

quality of life, the measured parameters indicate that the participant’s physiology varied

greatly depending on the dose of the daily administered drug. This is an important

finding and suggests that an optimal dosage is necessary for improved physiological

functioning post-concussion. Taking 2mL of the study drug per day for the first 2 weeks

showed an overall improvement in physiology for prefrontal cortex oxygenation, blood

pressure, and HRV. This continued administration of 2mL for another 2 weeks (28 days),

seemed to provide a “pooled” effect as suggested by the trends in the hemoglobin

parameters as measured by NIRS, the increases in SD of the blood pressure parameters

that suggest a more responsive and alert cardiovascular system. An increase in cerebral

metabolism (as indicated by an increase in HHb and decrease in HbO2 with a higher

absolute %TSI) may also suggest that the body is responding most strongly in

accordance with the 2mL administration per day and therefore allowing a greater rate of

oxygen extraction. Overall, in combination with the subject’s subjective self-assessment,

the objective physiological results reported here also suggest benefits to the participant’s

health and well-being when administered the high CBD and low THC study drug.

86

The largest limitation to the study would be its case study approach. While

assessing these changes in a single individual does provide valuable insight to the

pathophysiology, a larger scale study utilizing similar dosages can provide a stronger

statistical case for the use of a drug such as the one used in this study. Thus, a double

blinded, dose-escalation, randomized controlled trials to show the differences in

physiological responses following different CBD dose administration is needed. This

would show whether or not there is an increase in tolerance and dependence on CBD,

and clinical trials for post-concussion syndrome and acute concussion are needed to

better understand the therapeutic effects of CBD in these pathologies.

In regard to the assessment, the participant was also on medications (listed in

Table 2) which may have a possible metabolic interaction with CBD and/or THC, as

CBD is an inhibitor of cytochrome P450 enzymes, and thus, it is possible that CBD can

interfere with the metabolism of THC and medicinal products240. These medications can

also have an influence on the measured physiological parameters as well, such as blood

flow and cardiovascular metrics241,242. The participant also consumed alcohol 16 hours

prior (1 glass; 5% alcohol content) to the visits on days 28 (40mg CBD:2mg THC) and

42 (20mg CBD: 2mg THC). Alcohol consumption can increase antidiuretic hormone and

potentially lead to dehydration243. This can affect cardiovascular parameters such as

cardiac output and mean arterial pressure244, and thereby may have influenced some of

the results attained during the protocol. However, it is important to note that the alcohol

intake was not excessive and as such, 16 hours would provide a large enough time period

to presume that there is likely no influence on the individual’s physiology. Finally, the

participant did consume caffeine (1 cup tea) approximately 2 to 2.5 hours prior to each

87

assessment. While this was consistent at each visit, caffeine can antagonize adenosine

receptors245, and exert changes in cardiovascular and cerebrovascular responses.

Furthermore, because CBD can increase adenosine levels, there may be an interaction

between CBD and caffeine in response to either antagonizing or agonizing the adenosine

receptors, specifically at A2a receptors246.

The Portalite NIRS device used in this study only provides information from the

right prefrontal cortex, meaning there may be potential changes occurring on other

regions of the brain which may have been able to provide more insight to the cerebral

hemodynamic responses. A more robust multi-channel fNIRS in combination with a

transcranial Doppler can potentially provide more insight to functional changes between

the different regions, as well as changes in flow pressure reactivity and cerebral artery

blood flow velocity.

88

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8.0 Appendices

8.1- Appendix A – Table 15. Breakdown of SCAT symptoms247


Headache 0 0 0 0 1

"Pressure in head" 0 1 0 0 0

Neck pain 0 0 0 0 0

Balance problems or dizzy 0 1 0 0 0

Nausea or vomiting 0 0 0 0 0

vision problems 0 0 0 0 0

hearing problems/ringing 0 0 0 0 0

"Don't feel right" 1 1 0 0 0

Feeling "dinged" or "dazed" 1 1 0 0 0

Confusion 0 0 0 0 0

Feeling slowed down 1 1 0 0 1

Feeling like "in a fog" 1 1 0 0 0

Drowsiness 1 0 0 0 0

Fatigue or low energy 1 1 0 0 0

More emotional than usual 0 0 0 0 0

Irritability 0 0 0 0 0

Difficulty concentrating 1 1 0 0 0

Difficulty remembering 1 1 0 0 0

Sadness 0 0 0 0 0

Nervous or anxious 0 0 0 0 0

Trouble falling asleep 0 3 3 0 0

Sleeping more than usual 0 0 0 0 0

Sensitivity to light 0 0 0 0 0

Sensitivity to noise 0 0 0 0 0

TOTAL 8 12 3 0 2

Day 0= 0mg CBD:0mg THC; Day 14= 40mg CBD:2mg THC; Day 28= 40mg CBD:2mg

THC; Day 42= 20mg CBD:1mg THC; Day 70= 24mg CBD:1.2mg THC

110

8.2- Appendix B – NIRS data at baseline for the full protocol

Figure 18: The entire protocol for the participant’s baseline visit. The red line represents

oxyhemoglobin (HbO2), the blue line represents deoxyhemoglobin (HHb), the green line

represents the sum of HbO2 and HHb, known as total hemoglobin (tHb) and the yellow

line represents hemoglobin difference (HbDiff), the difference between HbO2 and HHb.

6-BPM: 6-Breaths per minute; BH: Breath Hold; 10SS: 10 second Squats; 5SS: 5 second

Squats

-4

-2

0

2

4

6

8

10

1

69

8

13

95

20

92

27

89

34

86

41

83

48

80

55

77

62

74

69

71

76

68

83

65

90

62

97

59

10

45

6

11

15

3

11

85

0

12

54

7

13

24

4

13

94

11

46

38

15

33

5

16

03

2

16

72

9

17

42

6

18

12

3

18

82

0

19

51

7

20

21

4

20

91

12

16

08

22

30

5

23

00

2

23

69

9

Example of NIRS Data Baseline Visit

HbO2 HHb tHb HbDiff

REST

REST REST REST REST

6-BPM WALDO BH 10SS 5SS

111

8.3- Appendix C – Informed consent documentation and approval letter

See attached form.

112

113

114

115

116

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