Faculty of Science
EFFECTS OF ACUTE SYMPATHETIC ACTIVATION ON EYE TEMPERATURE USING
INFRARED THERMOGRAPHY
2017 | JAY HUGGINS

B.Sc. Honours thesis - Biology

EFFECTS OF ACUTE SYMPATHETIC ACTIVATION ON EYE TEMPERATURE USING
INFRARED THERMOGRAPHY

By
JAY HUGGINS
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENTS OF BIOLOGICAL SCIENCES
AND PHYSICAL SCIENCES
(Ecology and Environmental Biology)

This thesis has been accepted as conforming to the required standards by:
Dr. Mark Rakobowchuk (Ph.D.), Thesis Supervisor, Dept. of Biological Sciences
Mr. Les Matthews (M.Sc.) Co-Supervisor, Dept. of Respiratory Therapy
Dr. Lyn Baldwin (Ph.D.), Examining Committee member, Dept. of Biological Sciences

Dated this 4th of May, 2017, in Kamloops, British Columbia, Canada
©Jay Huggins, 2017

ABSTRACT
Using physiological markers to measure sympathetic activation can be used to infer pain
and stress in humans. To date, the only methods in humans that reproducibly do this are invasive
and poses an undesired risk to participants. Previous work on cattle (Bos taurus) has used infrared
thermography to measure the temperature of the lacrimal caruncle region and provides a promise
of a novel method for measuring stress and pain non-invasively. The current study attempted to
determine if this method could be transferred for use in humans. Sixteen participants were recruited
for the study and underwent temporary painful stimuli using validated methods (that have
previously been shown to induce sympathetic activity known as the cold pressor test and the
muscle chemoreflex). During the trials, measurements included temperature of the lacrimal
caruncle, heart rate, mean arterial blood pressure and pulse transit time. Following each trial, blood
was drawn to measure concentrations of norepinephrine and epinephrine in the plasma. An
enzyme-linked immunosorbent assay was then performed to attempt to quantify the catecholamine
concentrations, however, the standards of the concentrations were not reliably determined so these
results were excluded from the study. A two-way repeated measures analysis of variance was
performed with the factors of condition and time. A condition x time interaction was observed in
heart rate (df= 8, F= 8.020, p<0.01), mean arterial pressure (df= 6 F= 12.6, p<0.01), and pulse
transit time (df= 8, F= 2.269, p= 0.03), but not temperature of the lacrimal caruncle. The results of
this study suggest that infrared thermography is not a reliable tool to measure sympathetic
activation in humans. This study also suggests that changes in blood flow at the lacrimal caruncle
region in response to sympathetic activation may be more complicated than previously proposed.
Thesis Supervisor: Associate Professor Dr. Mark Rakobowchuk

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ACKNOWLEDGEMENTS
I am in great appreciation for all those who contributed to this project but I would like to
give a special thank you to Dr. John Church for loaning me the equipment to carry out this
project. Without this generosity, this project would not have been possible. A thank you to Justin
Mufford for some helpful advice on using the infrared camera. Lastly, a huge thank you to Dr.
Mark Rakobowchuk for his grants, immense help with this thesis and the knowledge he so kindly
gave me.

DEDICATION
I would like to dedicate this thesis to my parents, Mel and Patricia Huggins, for their
endless support throughout the untraditional journey my undergraduate education has been, and
Brittany Garneau, who I might get a chance to see when this is finished.

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CONTENTS
ABSTRACT .................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
DEDICATION ............................................................................................................................... iii
LIST OF FIGURES ....................................................................................................................... iv
INTRODUCTION .......................................................................................................................... 1
Anatomy of the Nervous System ................................................................................................ 1
Physiology of the pain response ................................................................................................. 1
Measuring Sympathetic Activation............................................................................................. 2
Non-invasively measuring sympathetic activation in cattle ....................................................... 5
Inducing acute SNA .................................................................................................................... 6
Objectives ................................................................................................................................... 6
MATERIALS AND METHODS .................................................................................................... 7
Ethics statement and participant criteria ..................................................................................... 7
Infrared Thermography ............................................................................................................... 7
Traditional cardiovascular measures........................................................................................... 8
Blood plasma handling and sampling ......................................................................................... 9
Experimental Design ................................................................................................................. 10
Statistical analysis ..................................................................................................................... 10

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RESULTS ..................................................................................................................................... 11
Heart Rate ................................................................................................................................. 11
Mean Arterial Pressure ............................................................................................................. 12
Pulse Transit Time .................................................................................................................... 13
IRT measurements .................................................................................................................... 14
DISCUSSION ............................................................................................................................... 15
Interpreting the CPT results ...................................................................................................... 15
Evidence of SNA during the MCR ........................................................................................... 16
Future Directions and Conclusions ........................................................................................... 18
REFERENCES ............................................................................................................................. 20
APPENDIX .................................................................................................................................... A
STATEMENTS OF ETHICS APPROVAL ................................................................................... C

LIST OF FIGURES
Figure 1 The human eye with a circle around the region of interest, the lacrimal caruncle. .......... 2
Figure 2 An example of a microneurograph during control and post exercies muscle ischemia
(PEMI) (from Ichinose et al. 2008)................................................................................................. 3
Figure 3 Average heart rate (±𝑆𝐸) of participants over time with (a) representing the CPT being
significantly different from control, (b) showing MCR different from control, while (*)
represents a difference from baseline in the same condition (n= 10). .......................................... 12
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Figure 4 Mean arterial pressure (±𝑆𝐸) before and during control, musclechemo reflex (MCR)
and cold pressor test (CPT) in young healthy participants(n= 10). Letters indicates a significant
difference from control at the same time point and the asterisk indicates a difference from
baseline (n=10). ............................................................................................................................ 13
Figure 5 Pulse transit time throughout before and during control, muscle chemo reflex (MCR)
and cold pressor test (CPT) in healthy participants (n= 10). The letter (a) indicates a significant
difference from the control at the same time point and the (*) indicates a change from baseline.
....................................................................................................................................................... 14
Figure 6 Mean eye temperature before and during control, musclechemo reflex (MCR) and cold
pressor test (CPT) in healthy participants (±𝑆𝐸) measured at the lacrimal caruncle region (n=
10). ................................................................................................................................................ 15

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INTRODUCTION
Anatomy of the Nervous System
One of the primary functions of the nervous system is to detect and respond to the
environment. Some of this is done consciously, while much of it is done unconsciously and is
under the control of the autonomic nervous system (ANS). The ANS can be further divided into
the sympathetic (SNS) and parasympathetic nervous systems. The parasympathetic nervous
system can be described as the “rest and digest” system and is predominant in situations of low
perceived stress. During high stress situations, the parasympathetic nervous system will often be
supressed and the SNS will become active and respond to keep the body in homeostasis. One such
stressor that will activate the SNS is pain (Widmaier et al. 2014).

Physiology of the pain response
In humans, pain is sensed by nociceptors which can be located in muscle, skin, glands and
the digestive tract. These nociceptors will detect a painful stimulus and relay this signal to the
central nervous system (Widmaier et al. 2014). The signal will then travel up the afferent nerves
of the spinal cord into the brain where it will eventually be processed by either the reticulating
activating system or the thalamus (Van de Kar and Blair 1999; Carrasco and Van de Kar 2003).The
stimulus is then relayed to either the hippocampus or the amygdala. Either of these structures can
trigger a sympathetic response either through the stimulation of adrenergic neurons, leading to the
release of norepineprhine (NorEpi) into synaptic clefts, or stimulating the release of epinephrine
(Epi) from the adrenal medulla (Carrasco and Van de Kar 2003). Release of these catecholamines
leads to classic physiological responses to stressors such as changes in pupil diameter, increased
cardiac output and redistribution of peripheral blood flow.

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The response an effector will have to a catecholamine is dependent on the organ receiving
them. For example, the release of NorEpi from adrenergic neurons can cause the constriction of
blood vessels. NorEpi will diffuse across the synaptic cleft reaching α-adrenergic receptors in the
membranes of smooth muscle cells surrounding the blood vessels leading to a sustained
contraction. Epi will cause a similar response through binding of α-adrenergic receptors, but the
mode of delivery for Epi to the receptors is through the plasma rather than neurons. Changes in
blood flow will cause a slight change in the wavelength emitted from the lacrimal caruncle region
(Figure 1) which the infrared camera will convert and record as a difference in surface temperature
(Meola and Carlomagno 2004). A decrease in blood flow is assumed to be a decrease in the surface
temperature while an increase in blood flow should show an increase in surface temperature.

Measuring Sympathetic Activation
Increased sympathetic outflow has been linked in the pathogenesis of many diseases with
some examples being left ventricular hypertrophy and type II diabetes (Esler 2000; Fisher et al.
2009). Chronic hypertension will lead to a stiffening of the arteries and subsequent hemodynamic
changes. Long term hemodynamic changes can lead to
increased left ventricular afterload which reduces
contractility and function of the heart (Lovic et al. 2017).
In regards to the pathogenesis of type II diabetes, acute
sympathetic nervous system activation (SNA) has been
shown to cause an increase in plasma insulin
Figure 1 The human eye with a circle around the
region of interest, the lacrimal caruncle.

concentrations which could potentially lead to the insulin

resistance observed in type II diabetes (Jamerson et al. 1993; Fisher et al. 2009). Another theory

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is that chronic vasoconstriction reduces blood flow to muscle causing slower glucose clearance
and increased insulin release following meals (Fisher et al. 2009).
The two mechanisms mentioned above are examples of the plethora of diseases related to
hyperactive SNA. For this reason, the ability to measure acute sympathetic nervous system
activation (SNA) is of value in a variety of fields for determining said mechanisms. Currently,
commonly used estimates of SNA include infusing catecholamines into the arteries of subjects and
recording the concentrations of venous return (known as catecholamine spillover), quantifying
blood plasma catecholamine concentrations and microneurography (Esler 2010).
Microneurography involves the insertion of a tungsten needle directly in to a nerve of
interest. This method is useful in gauging autonomic dysfunction by directly measuring the firing
rate of efferent nerve fibres (Mathias 2003). The number of impulses passing through this
individual nerve fibre can then be visualized by the number of voltage spikes detected by the
electrode with more bursts associating with greater
activity (Figure 2).
Although this method can provide great
detail about one particular nerve, differential firing
rates in the region of measurement can be incorrectly
interpreted as the firing rate for the entire body.
Furthermore,

although

microneurography

has

associations with catecholamine release, it does not
take into account neurotransmitter release from
Figure 2 An example of a microneurograph during
control and post exercies muscle ischemia (PEMI) (from
Ichinose et al. 2008)

adjacent nerves or modulation that could occur from
hormonal mechanisms (Vallbo et al. 2004). Insertion
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of the needle into the nerve is also a skill that can take multiple attempts, causing pain for the
participant. Increasing the pain of procedures can make participant recruitment more difficult.
Catecholamine measurements also represent another way to measure SNA and are
considered the gold standard for measuring the acute stress response. Norepinephrine and
epinephrine are responsible for the sympathetic response and quantifying their concentrations can
provide useful regional and whole body information, but this too has limitations. The half life of
catecholamines in the blood is 1-2 minutes so blood must be drawn quickly following interventions
(Hjemdahl 1993). The relative concentration of catecholamines that spillover into the blood is also
low, and it is estimated that between 5-10 % of norepinephrine actually reaches the blood plasma
(Sinski et al. 2006; Zygmunt and Stanczyk 2010).
Measuring catecholamine concentrations and microneurography are also invasive
procedures creating a risk of infection for participants. With the rise of multi-drug resistant
microbials, this may become an increasing concern for researchers. The development of a noninvasive method to measure SNA would avoid these risks, providing greater safety for participants
and easier recruitment for researchers.
In the past, a commonly used non-invasive method for measuring SNA was heart rate
variability (HRV). However, there are multiple lines of evidence indicating that HRV is not an
actual measure of sympathetic tone. Instead, it is believed that HRV is a measure of baroreflex
function (reviewed by Goldstein et al. 2011). There has yet to be a well validated non-invasive
measure to take the place of HRV.

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Non-invasively measuring sympathetic activation in cattle
In the field of animal physiology, researchers have suggested a novel non-invasive way to
measure SNA in cattle (Bos taurus) using infrared thermography (IRT). Specifically, researchers
have shown that cattle exhibit alterations in the temperatures of the lacrimal caruncle region of the
eye in response to stressful or painful interventions such as prodding, epinephrine infusion, and
castration (Stewart et al. 2008; Stewart, Verkerk, et al. 2010; Stewart, Webster, et al. 2010).
Previous work in cattle shows temperature in the lacrimal caruncle region decreases in response
to increased SNA (Stewart, Webster, et al. 2010), suggesting vasoconstriction and decreased blood
flow. As α-adrenergic receptors are the dominant receptor in the smooth muscle cells of the
conjunctival bed (the vessel system controlling blood flow to the lacrimal caruncle region of the
eye), SNA should cause a decrease in blood flow to the region. Further evidence of α-adrenergic
predominance in the conjunctiva may be supported by the decreased eye temperature in cattle
following an epinephrine infusion (Stewart, Webster, et al. 2010). However, some studies have
shown an increase in temperature at the lacrimal caruncle region (Stewart, Verkerk, et al. 2010).
It is assumed that an increase in blood flow is occurring at the lacrimal caruncle in these cases.
These experiments may activate another pain induced pathway involving a deep visceral pain such
as castration. The mechanisms for this increase in blood flow are discussed below.
With many of the physiological processes in mammals being conserved, IRT represents a
potential solution to measuring SNA non-invasively in humans. A recent study of humans
involving visceral pain measured temperature in the lacrimal caruncle during tooth extraction
(under local anaesthetia) also found an increase compared to pre-tooth extraction temperatures
(Kolosovas-Machuca et al. 2016). This increase in eye temperature may be due to a release of the
vasodilator nitric oxide (NO) from the endothelial cells (Stewart, Verkerk, et al. 2010) or the initial
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drop in eye temperature may have gone undetected (Stewart, Schaefer, et al. 2008). It is also
feasible that subjects experienced some minor autonomic failure via the mechanisms that occur
during neurally mediated syncope (mechanisms discussed in Kaufmann 1997).

Inducing acute SNA
Two commonly used methods to induce SNA are the cold pressor test (CPT) and the
muscle chemoreflex (MCR). The use of the CPT dates backs to the 1980’s and involves
participants placing their feet in an ice-water slurry activating a participant’s thermoreceptors
(Smirnova et al. 2013; Mizeva et al. 2015). This cold stimulus is enough to activate the SNS which
is evidenced by increases in plasma catecholamine concentrations, heart rate (HR), aortic blood
pressure and vasoconstriction of peripheral blood vessels (Victor et al. 1987; Smirnova et al. 2013;
Kalfon et al. 2015; Mizeva et al. 2015). This vasoconstriction decreases arterial compliance and
has been shown to increase pulse wave velocity (Nichols 2005; Kalfon et al. 2015) which can be
measured by the time it takes the pressure wave to leave the left ventricle to the time it reaches a
point on the radial artery.
The muscle chemoreflex has also been shown to induce SNA. The MCR has shown
increases in both norepinephrine and epinephrine plasma concentrations (Bouloux et al. 1985;
Dyson et al. 2006; Kaur et al. 2015). Typical of other SNA interventions, the MCR will also cause
an increase in blood pressure and pulse wave velocity (Scherrer et al. 1990; Figueroa et al. 2010).
Both the CPT and MCR have been widely used to induce SNA and cause changes in vascular tone.

Objectives
The aims of this study are to determine: if IRT is a viable method to noninvasively measure
SNA in humans; to validate whether IRT is measuring SNA in humans using other indicators of

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SNA; and to better understand the vascular conductance observed in the conjunctiva bed during
SNA. Since the receptors of the conjunctiva bed are primarily α-adrenergic, it is expected that
vasoconstriction will occur and a decrease in eye temperature will be observed during this study
via the physiological mechanisms discussed above.

MATERIALS AND METHODS
Ethics statement and participant criteria
The procedures of this study were approved by the Human Ethics board of Thompson
Rivers University. Sixteen participants between the ages of 18 and 35 were recruited for the study.
Participant exclusion criteria included; regular tobacco users, medication use, or known
cardiovascular disease. On the day of testing participants were also asked to abstain from
stimulants (caffeine) and depressants (alcohol). During testing participants were also asked to
remove contact lenses and corrective eyewear which could potentially impact temperature
measurements.

Infrared Thermography
To determine lacrimal caruncle region temperature, a FLIR E-60 Infrared Thermography
Camera (FLIR Systems Inc., Burlington, ON) was used. The infrared (IR) camera was calibrated
before measurements on each day of testing. Participants were seated 1 m away from the camera
and instructed to stare at the lens during the trials. Emissivity was set at 0.98.
Eye temperature measurements were analyzed using the FLIR Tools + software (FLIR
Systems Inc., Burlington, ON). Mean temperature was taken every ten seconds in the lacrimal
caruncle region of the right eye. In circumstances where the eye was partially or fully closed at the

7

ten second mark, the temperature was recorded from the next available frame that the eye was fully
open.

Traditional cardiovascular measures
Throughout each trial, beat to beat blood pressure was recorded using an applanation
tonometer (Millar Inc., Houston, TX), and heart rate was determined by 3 lead ECG in the V5
configuration (AD Instruments, Colorado Springs, CO). Beat to beat blood pressure and heart rate
were analyzed using LabChart software (AD Instruments, Colorado Springs, CO) and used to
calculate an indirect measure of arterial compliance known as pulse transit time (PTT). Ten
measurements of PTT were taken approximately every 50 seconds during the 5-minute
intervention trials and the control trial and averaged to give a value minute-by-minute. During the
60 second pre-intervention time, five measurements of PTT were taken and averaged for
comparison with the post intervention times. PTT was measured using PowerLab Software (AD
Instruments, Colorado Springs, CO) and was measured using the double derivative method. The
calculation is commonly measured as the time from the peak of the QRS complex to the start of
the pressure wave recorded at the radial artery (Murray 2001) (Figure 3).

8

Figure 3 A sample recording of PTT. In the top panel, a pulse pressure wave recorded at the radial artery. The bottom panel
shows a standard electrocardiogram. The space between the two arrows represents one measurement of PTT.

Traditional blood pressure measurements were also taken throughout the trials using the
OMRON 3 Series automated blood pressure cuff (OMRON Healthcare, Hoofddorp, Netherlands)
to record mean arterial blood pressure (MAP).

Blood plasma handling and sampling
Blood was drawn at the end of each trials to measure plasma epinephrine and
norepinephrine levels. Two mL of blood was sampled from participants into EDTA blood tubes.
The sample was then centrifuged at 500 g and the 150-300 μL of plasma was extracted and stored
at -70° C until analysis. Concentrations were determined using the Abnova enzyme-linked
immunosorbent assay (ELISA) kit (Abnova, Taipei City, Taiwan). The protocol laid out by the
Abnova user kit was followed with the exception of insufficient plasma. In these cases, the amount
of plasma extracted was noted and multiplied to determine final concentrations.

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Experimental Design
The study took place in the Ken Lepin Science Building at Thompson Rivers University.
Participants performed three separate trials: a control trial; a cold pressor test (CPT); and a muscle
chemoreflex test (MCR). For the control trial, participants were asked to gaze straight ahead whilst
in the seated position five minutes while blood pressure and heart rate were recorded. During the
CPT, an ice-water slurry was created that was approximately 5°C. For the MCR trial, blood
pressure cuffs were secured directly below the patella; then inflated and maintained above
200mmHg for the intervention. The order of the trials was randomized to mitigate the effects of
white coat syndrome. To allow the participant to return to baseline physiological norms after the
interventions and blood draws, a ten-minute rest period was given between each trial and if their
blood pressure had not returned to baseline additional time was added. The order participants
completed the trials was randomized. For both treatment trials, eye temperature was recorded for
one minute prior to the intervention and continued throughout the trial. After one minute, the
intervention began. The treatment continued for four minutes, or for as long as the participants
could tolerate the discomfort.

Statistical analysis
Statistical calculations were performed on the software program SPSS version 22.0 (IBM,
Armonk, NY). A two-way repeated measure analysis of variance (ANOVA) was performed with
the factors of condition (control, MCR and CPT) and time (Baseline, 1min, 2min, 3min, and 4min).
A post hoc analysis was done using Sidak correction. Variables determined using this method
included PTT, HR, and eye temperature. Due to variations in sampling time of blood pressure,
only four time points could be reliably recorded for each participant and this was reflected in the
statistical calculation.
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In situations where participants could not complete an intervention, or in instances where
artefact measurements temporarily obstructed the ability to record reliable datum points, the
average increase from baseline levels was used to fill omitted data points. This was done by
averaging recorded values at each time point, converting intervention averages to a percentage
relative to baseline, and then adding the appropriate increase or decrease relative to that
participants baseline value. Filling these omissions was necessary to allow for a two-way repeated
measures ANOVA to be performed. This was done to have minimal impact on the statistical
analysis.
Two participants were unable to complete the full length of the CPT intervention due to
cold sensitivity. The first 6 participants had data recorded in an incorrect file format and were
excluded from the data set for that reason. Due to an inability to accurately determine standardized
controls through ELISA, the results of the assay can only be reported as a non-result.

RESULTS
Heart Rate
There was a condition x time interaction for HR (df= 8, F= 8.020, p<0.01), MAP (df= 6
F= 12.6, p<0.01), and PTT (df= 8, F= 2.269, p= 0.03). Post hoc comparisons showed a significant
difference in HR during the MCR trial during the third (95± 3 (SE) bpm) and fourth (99± 3 bpm)
minutes compared to the control at the same time points and baseline (84± 4 bpm, p= 0.05).
During the CPT, HR increased from 88±4 bpm at rest to 105±4 bpm after one minute submersion
of the feet and remained elevated until three minutes after which heart rate returned to values not
different from baseline (Figure 3).

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Figure 3 Average heart rate (±𝑆𝐸) of participants over time with (a) representing the CPT being significantly different from control,
(b) showing MCR different from control, while (*) represents a difference from baseline in the same condition (n= 10).

Mean Arterial Pressure
During the CPT, MAP increased from baseline (94± 2 mmHg) immediately upon
immersion of the feet to 100± 2 mmHg and remained elevated for the remainder of the trial
(p<0.05). In contrast to CPT, MAP did not increase significantly until the 2nd minute of the MCR
trial going from 93± 2 mmHg to 98± 2 mmHg and remained elevated throughout the remainder
of the trial relative to baseline (p< 0.05, Figure 4). MAP was also significantly different from the
control (91±1 mmHg) in the second minute.

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Figure 4 Mean arterial pressure (±𝑆𝐸) before and during control, muscle chemoreflex (MCR) and cold pressor test (CPT) in young
healthy participants(n= 10). Letters indicates a significant difference from control at the same time point and the asterisk indicates
a difference from baseline (n=10).

Pulse Transit Time
PTT was 0.185± 0.09 s at baseline and decreased during the first minute of submersion to
0.174± 0.09 s during the CPT (Figure 5). The PTT remained lower relative to baseline for the
remainder of the trial. Compared to the control (0.186±0.09 s), PTT was also significantly lower
during the first minute of the intervention (p<0.05) but not during the second, third and fourth
minutes. MCR (0.184±0.09 s) was not significantly different than the control. During the MCR,
PTT steadily decreased following the inflation until the fourth minute 0.179± 0.01 s at which point
a significant reduction was apparent from baseline, but not from the control (0.191± 0.09 s)
(p<0.05).

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Figure 5 Pulse transit time throughout before and during control, muscle chemo reflex (MCR) and cold pressor test (CPT) in
healthy participants (n= 10). The letter (a) indicates a significant difference from the control at the same time point and the (*)
indicates a change from baseline.

IRT measurements
Eye temperature did not display any discernable response under any perturbation. At
baseline, means were within a narrow range and averaged 35.2±0.2°C, only deviating slightly
from this mean indicating no specific trend (Figure 6).

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Figure 6 Mean eye temperature before and during control, musclechemo reflex (MCR) and cold pressor test (CPT) in healthy
participants (±𝑆𝐸) measured at the lacrimal caruncle region (n= 10).

DISCUSSION
Interpreting the CPT results
The clear increases of HR and MAP from control and baseline levels show the expected
and classic response to acute SNA achieved through both the MCR and CPT (Mourot et al. 2009;
Kalfon et al. 2015). Although HR and MAP increases are typical of SNA, their measurements
should be interpreted with caution as heart rate can increase due to parasympathetic withdrawal as
opposed to sympathetic activation. This parasympathetic withdrawal will cause an increase in
cardiac output (𝑄̇ ) which can increase blood pressure in turn as seen from the equation (MAP=
total peripheral resistance x 𝑄̇ ). A potentially more useful estimate of SNA for this study may be
PTT. A change in PTT implies that a change in arterial stiffness occurred via vasoconstriction in
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peripheral blood vessels as constricted blood vessels stiffen and propagate pule waves with greater
velocity (Kalfon et al. 2015). The current study saw PTT decrease in a way that closely mirrors
the expected SNA. Also, it is likely the closest non-invasive measure mechanistically since it is
influenced most by changes in vascular tone and the circulatory system lacks parasympathetic
innervation. No trend was observed by the IRT in the lacrimal caruncle region. This may be
because changes in lacrimal caruncle temperature are more complex than previously proposed.

Evidence of SNA during the MCR
Relative to CPT, the results of the MCR test are slightly ambiguous. As expected increases
in HR and MAP were observed. In the final minute of measurement, PTT showed a reduction from
baseline, but never differed from control. It is possible that participants may not have been
sufficiently stimulated for sympathetic activation to occur but this is unlikely as there was no
significant difference from the control during the strongest measure of SNA recorded in this test,
the PTT. However, it is important to note that there was an obvious trend with PTT decreasing as
time passed. A longer time under local ischemia, or an increased sample size would likely produce
the expected drop in PTT in future studies.
Results from the CPT test are more pronounced and conclusive. PTT abruptly decreased
during the CPT in the minute following immersion of the feet. Collectively, HR, MAP and PTT
measurements convincingly show acute SNA was achieved during the CPT. However, the failure
for IRT to detect any changes during this perturbation suggests that the IRT camera is not sensitive
enough to detect changes in vascular conductance in humans, or the physiological response to
SNA is different, or less pronounced at the conjunctiva bed relative to cattle. Another explanation
is that increased perfusion occurs from the increase in HR which is nullified by vasoconstriction

16

in the conjunctiva bed. These two explanations are not satisfactorily answered from the results of
this study so little more can be inferred regarding it.
Similar results to this study have been reported before. Young and healthy subjects
underwent the Von Frey monofilament test, where an electrical stimulation of 1 Hz was
superficially applied for 30 s at two separate body locations. No change in temperature at the
lacrimal caruncle was observed, however, a subjective, researcher scored pain measurement,
suggested that the stimulus may have been insufficient to cause pain in young and healthy subjects
(Barney et al. 2015). Interestingly, the same study found patients with a neurodegenerative
disorder classified as neural ceroid lipofuscinosises (NCL) did receive sufficient pain which
suggests they may have increased pain sensitization. The IRT of the lacrimal caruncle in this group
displayed an increase in eye temperature (Barney et al. 2015). Assessing pain in children with
NCL is difficult as language is often compromised in this population so evidence is often based
on observation (Barney et al. 2015). However, from parental surveys and subjective pain scoring,
there is reason to believe pain is a symptom regularly experienced by these individuals (Santavuori
et al. 1993; Mannerkoski et al. 2001; Barney et al. 2015). As this disorder causes a deterioration
of the central nervous system (Mannerkoski et al. 2001), the sympathetic response becomes
difficult to predict and these results provide inadequate information to the general population.
A study by Kolosovas-Machuca et al. (2016) also found a significant increase in eye
temperature during tooth extraction. The physiological response to tooth extraction may differ in
that the response to a deep visceral pain may cause a release of NO (Stewart, Verkerk, et al. 2010).
This study also saw an increase in HR during the tooth extraction so the increase in 𝑄̇ may have
caused an increase in perfusion pressure to the conjunctiva bed. This tooth extraction study also
lacked a control group and patients were placed under a regional anaesthetic which blocks
17

peripheral nerves from transmitting the pain signal and may act as a local vasodilator (Woodward
2008). The vasodilation could explain the increased temperature to the conjunctiva bed, or there
was a regional withdrawal of sympathetic activity.

Future Directions and Conclusions
Plasma catecholamine levels could not be reliably determined in the current study. This is
unfortunate as significant increases in these levels would provide a robust confirmation that SNA
was achieved. Future studies should include measures such as plasma catecholamine levels,
catecholamine spillover or microneurography. As previously mentioned, these methods are well
accepted measures of SNA and would provide another line of evidence regarding the capabilities
of IRT. A study measuring temperatures of the lacrimal caruncle during infusions of
catecholamines would be of value as well. Epinephrine infusion was shown to decrease
temperature in the lacrimal caruncle of cattle (Stewart, Webster, et al. 2010), so a different
response in humans would suggest different mechanisms predominate or that IRT cannot measure
SNA in human subjects.
To ensure blood flow changes are significant enough to be detectable in humans, measuring
blood flow in the lacrimal caruncle during the CPT and MCR using a technique such as Laser
Doppler Flowmetry would provide valuable information. Additionally, ultrasound imaging during
the interventions performed on cattle would provide valuable insight into the mechanisms
controlling blood flow to the conjunctiva bed and could help other researchers attempting to use
this tool in other mammalian taxa.
Although there is some evidence that IRT is an effective measure of SNA in cattle, studies
in humans have yet to provide convincing evidence that this tool is transferable. In cattle,

18

deviations in temperature averaged 0.14°C making it plausible that this method can detect changes
in SNA via the mechanisms discussed, but only at levels of pain not generally desired or ethically
acceptable for most human research situations. If IRT is to be used as a tool in human pain research,
it is imperative that future studies provide validation of this technique through stronger measures
such as plasma catecholamine concentrations and microneurography.
Manual selection of the region of interest for IRT measurements is also a severe limitation
in the use of IRT. Differences in inter and intra-examiner scoring has been previously recorded
which suggests there is a current lack of reproducibility for this method. Standardized protocols
and automated software that can objectively calculate the region of interest need to be developed
and validated if the use of IRT is to persist in humans. These steps will greatly increase the
reliability and speed of analysis for this method. In the interim, averaging the scores of multiple
examiners may help mitigate differences in scoring (Fernández-Cuevas et al. 2015).
The findings of this study suggest that IRT is not a useful measurement in determining
SNA in humans. Changes in HR, MAP and PTT during the interventions provide convincing
evidence that SNA was achieved and IRT measurements did not mirror these results. Going
forward, PTT appears to be the most reliable measure of SNA in humans as it involves changes in
vascular tone which is controlled by the SNS.

19

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22

APPENDIX
Table 1 Means, standard error and 95 % confidence interval of temperature in the lacrimal caruncle region during each testing
condition.

95% Confidence Interval

Condition

Mean

Std. Error

Lower Bound

Upper Bound

MCR

35.168

.265

34.569

35.767

CPT

35.306

.162

34.939

35.672

Control

35.017

.231

34.495

35.539

Table 2 Mean, standard error and 95 % confidence interval of heart rate for the duration of each condition.

95% Confidence Interval

Condition

Mean

Std. Error

Lower Bound

Upper Bound

MCR

92.1

2.4

86.5

97.8

CPT

96.9

3.8

88.3

105.4

Control

85.6

3.1

78.6

92.7

A

Table 3 Mean, standard error and 95 % confidence intervals of mean arterial pressure through each condition.

95% Confidence Interval

Condition

Mean

Std. Error

Lower Bound

Upper Bound

MCR

97.3

1.9

93.2

101.4

CPT

99.6

1.3

96.8

102.4

Control

91.3

1.3

88.4

94.3

Table 4 Means, standard error and 95 % confidence interval of PTT during each condition.

95% Confidence Interval

Condition

Mean

Std. Error

Lower Bound

Upper Bound

MCR

.184

.009

.163

.205

CPT

.178

.008

.159

.197

Control

.190

.009

.170

.211

B

STATEMENTS OF ETHICS APPROVAL

December 07, 2016
Mr. Jay Huggins
Faculty of Science
Thompson Rivers University
File Number: 101341
Approval Date: December 07, 2016
Expiry Date: December 06, 2017
Dear Mr. Jay Huggins,
The Research Ethics Board has reviewed your application titled 'The Effects of Sympathetic
Nervous System Activation on Eye Temperature Using Infrared Thermography.'. Your
application has been approved. You may begin the proposed research. This REB approval, dated
December 7, 2016, is valid for one year less a day: December 06, 2017.
Throughout the duration of this REB approval, all requests for modifications, renewals and
serious adverse event reports are submitted via the Research Portal. To continue your proposed
research beyond December 06, 2017, you must submit a Renewal Form before December 06,
2017. If your research ends before December 06, 2017, please submit a Final Report Form to
close out REB approval monitoring efforts.
If you have any questions about the REB review & approval process, please contact the Research
Ethics Office via 250.852.7122. If you encounter any issues when working in the Research
Portal, please contact the Research Office at 250.371.5586.
Sincerely,

C

Andrew Fergus
Chair, Research Ethics Board

D