1,765.28 SUB TOTAL:

Haemodynamic function can be evaluated using the cold pressor test (CPT), which measures blood

pressure and HR in response to an external cold stimulus, typically a cold water ice slurry, and is a

useful predictor of future hypertension (Godden et al., 1955; Victor et al., 1987; Kasagi et al., 1995;

Zhao et al., 2012). In healthy individuals, the cold stress typically induces vascular sympathetic

activation leading to vasoconstriction and a concomitant sustained increase in blood pressure,

although the HR response demonstrates high inter-individual variability (Victor et al., 1987; Mourot et

al., 2009). Blood pressure reactivity is calculated as the difference between the baseline and peak blood pressure during the cold stimulus, with greater or “hyper” reactivity corresponding with a

greater risk of developing hypertension and CVD (Zhao et al., 2012). However, no best practice

guidelines exist concerning what constitutes a “normal” or “hyper” response to the external cold

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An index of coronary artery disease risk and cranial conduit artery endothelial function can be

calculated through assessment of carotid artery reactivity (CAR%; carotid artery diameter), via an

ultrasound scan of the carotid artery performed during the CPT (Rubenfire et al., 2000). During

exposure to the cold stimulus, healthy individuals typically demonstrate vasodilation of the carotid

artery, which is independent of carotid intima-media thickness (cIMT) (Rubenfire et al., 2000). Recent

data demonstrates a good correlation between the response of the coronary arteries and CAR%, and

that the presence of traditional cardiovascular risk factors, in addition to older age, is associated with

lower CAR% (van Mil et al., 2017). Assessing CAR% via the CPT, therefore, represents a valuable

method of assessing cardiovascular risk and the potential changes in CAR% following periods of altered

lifestyle behaviours.

Following a 20-minute supine rest, participants were comfortably positioned to enable them to easily

move their left hand off the bed without excessive bodily movement. Participants were asked to

slightly extend their neck to allow simultaneous ultrasound imaging of the left common carotid artery

for the duration of the CPT. A 10 MHz multifrequency linear array probe, attached to a high-resolution

2D duplex ultrasound machine (Terason u-Smart 3300, Teratech, Burlington, MA, USA) was used to

assess the left common carotid artery reactivity (diameter and blood flow velocity). Following a 1-

minute baseline period, participants were instructed to immerse their left hand (up to the wrist) in

iced slush (1-5°C) for 3-minutes. Participants were instructed to breathe normally throughout the CPT

and to avoid breath holding/hyperventilation, whilst remaining as still as possible. The reproducibility

of the CPT is generally considered to be stable following both short-term (Durel et al., 1993; Saab et

al., 1993; Fasano et al., 1996) and long-term (Fahrenberg et al., 1987; Sherwood et al., 1997; Hassellund et al., 2010; Zhao et al., 2012) studies of <2-weeks and between 1 to 18-years, respectively.

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Beat-to-beat arterial blood pressure and 5-lead ECG were recorded online throughout the CPT

(LabChart 8.0, AD Instruments, Dunedin, New Zealand), from which SBP and DBP were exported in 10-

second intervals. BP reactivity was calculated as the difference between baseline BP and the peak BP

recorded during the 3-minute hand immersion. Custom-designed edge detection software (described

earlier in section 3.3.2.1) was used to analyse CAR%. Baseline diameter and blood flow were taken in

10-second intervals for 1-minute prior to hand immersion. Post-immersion data was analysed in 10-

second intervals, from which peak diameter change (maximum dilation/constriction) and area-under-

the-curve (AUC) for the diameter change during the CPT (CARAUC) were calculated. Peak diameter

change relates to the dilation or constriction of the carotid artery, with the directional change being

determined by a positive or negative CARAUC, representing dilation or constriction, respectively.

Typically, greater CAR% is observed in younger versus older individuals, and in healthy individuals

compared to those exhibiting greater cardiovascular risk profiles (van Mil et al., 2017).

3.5 Cerebrovascular Function

Transcranial Doppler ultrasound (TCD) is a useful, non-invasive tool that provides high temporal

resolution for measuring blood velocity in cerebral vessels, measured in centimetres per second (cm∙s-

1_{) (Willie et al., 2011). As vessel diameter is unknown, TCD is not a measure of blood flow per se, rather}

it measures blood velocity of an insonated vessel and gives a reliable index of blood flow, providing

the angle of the Doppler probes remain constant (Giller et al., 1998; Schreiber et al., 2000; Ainslie &

Duffin, 2009). Compared to other, often limited in availability and more expensive, methods of

assessing cerebrovascular function, such as functional magnetic resonance imaging (fMRI), positron

emission tomography (PET) and dynamic perfusion computer tomography (PCT), TCD is advantageous

in being portable, allowing repeated measures and continuous monitoring of cerebral blood flow

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particularly in assessing integrative cerebrovascular function through cerebral reactivity,

autoregulation and neurovascular coupling (van Beek et al., 2008; Willie et al., 2011).

A low-frequency (≤2 MHz) pulsed Doppler ultrasonic beam is emitted from the Doppler probe(s) and

crosses the intact skull to insonate the basal cerebral arteries through relatively thin bone ‘windows’

(Moppett & Mahajan, 2004; D’Andrea et al., 2016). Reflection of the ultrasonic beam off moving red

blood cells within the field of transmission is detected by the transducer within the Doppler probe(s)

(Figure 3.9), with the resultant Doppler shift being proportional to red blood cell velocity (DeWitt &

Wechsler, 1988; Willie et al., 2011). Assessment of cerebral perfusion is often difficult due to the

natural skull barrier and transmission of the ultrasonic beam can be influenced by the structural

characteristics of the cranial bones, with approximately 10-20% of individuals having inadequate

transtemporal acoustic windows, often related to age, gender and other factors (Marinoni et al., 1997;

D’Andrea et al., 2016).

Figure 3.10 Schematic depicting the components of the Doppler ultrasound probe, consisting of two piezoelectric elements; an ultrasound transmitter and a receiver for the returning echoes of detected moving red blood cells.

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The intracranial arteries of most notable clinical interest are the internal carotid artery (ICA), middle

cerebral artery (MCA), anterior cerebral artery (ACA) and posterior cerebral artery (PCA) (Figures 3.11

and 3.12). The MCA is the most frequently insonated artery due to the ease of access and signal quality

obtained through the temporal window. The MCA originates from the ICA and supplies blood to many

parts of the lateral cerebral cortex, carrying 50-60% of the ipsilateral ICA blood flow (Moppett &

Mahajan, 2004). Furthermore, the MCA receives ~80% of the blood volume delivered to the Circle of

Willis (Lindegaard et al., 1987) and is generally considered to represent blood flow to the brain

(Moppett & Mahajan, 2004). MCA blood velocity is typically detected at a depth of 45-60 mm, with

directional blood flow towards the Doppler probe(s) (Willie et al., 2011; Bouzat et al., 2014). Detailed

descriptions of the techniques used for insonating the MCA have previously been documented

elsewhere and are outlined in Table 3.1 (Moppett & Mahajan, 2004; Willie et al., 2011; D’Andrea et

al., 2016). The angle of insonation is important in ensuring accurate measurement of MCAv and it should remain constant, as the observed velocity is inversely proportional to the cosine angle of

incidence between the vessel and ultrasound beam (Moppett & Mahajan, 2004). The MCA provides

relatively easy signal acquisition with a small insonation angle and an acceptable error of ~15% has

been associated with insonation angles of up to 30° (Aaslid et al., 1982).

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Figure 3.12 Overview of the Circle of Willis that is responsible for delivering cerebral blood flow.

Table 3.1 Typical patterns for identifying cerebral arteries with ‘normal’ Circle of Willis anatomy and without intracranial or vascular pathology. Adapted from Moppett and Mahajan (2004).

Vessel Probe direction Depth (mm) Flow direction Ipsilateral carotid compression

Contralateral carotid compression

ACA Anterior 60-75 Away Flow reversal Increased

velocity

MCA Perpendicular 35-60 Toward Reduced velocity No change

PCA Posterior 55-70 Toward No change or

increased velocity

No change

Abbreviations; ACA, anterior cerebral artery; MCA, middle cerebral artery; PCA, posterior cerebral artery.

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In this thesis, TCD ultrasonography was used to continuously measure changes in MCAv both at rest

(steady-state) and in response to physical manoeuvres (repetitive sit-to-stand movements) that

induce fluctuations in BP and alterations in arterial carbon dioxide tension (cerebrovascular reactivity).

With the participant resting in a supine position, middle cerebral artery blood velocity (MCAv, 1 cm

distal to the MCA-ACA bifurcation) was continuously measured through the temporal window

bilaterally via TCD ultrasonography (Doppler-BoxX, DWL Compumedics, Germany). The temporal

window was located in the region immediately superior to the zygomatic arch, anterior to the tragus,

at which site acoustic gel was applied and a 2 MHz Doppler probe (DiaMon, DWL Compumedics,

Germany) was positioned on each side and adjusted until optimal signals were identified and

remained consistent, according to previous descriptions (Willie et al., 2011). An adjustable headband

(DiaMon, DWL Compumedics, Germany) was used to securely position the Doppler probes to ensure

that the same angle of insonation for continuous flow velocity measurement could be maintained and

any subtle movements prevented for the duration of the assessment (Figure 3.13).

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Specific criteria were used to insonate the correct vessel (MCA), with an insonation depth initially

being set at 50 mm and the Doppler probe being slowly manipulated perpendicular to the assumed

vessel direction. Upon identifying a strong signal of blood flow towards the transducer, the signal was

optimised through minimal corrective movements of the probe and adjustments to the signal gain. A

Doppler spectral waveform demonstrated the blood flow velocity profile on the M-mode screen,

which assisted in confirming identification of the MCA through mean and peak blood flow velocity

values, reading >50 cm∙s-1 _{and ~80 cm∙s}-1_{, respectively (Willie et al., 2011). However, the large}

variability in MCA peak blood velocity should be noted when identifying the MCA, as large variability

is often inherent in specific patient groups and in ageing populations (Moppett & Mahajan, 2004).

Standardisation of within-participant measures during repeated tests was achieved through recording

the vessel depth, optimisation settings, mean and peak MCAv values, in addition to using the MCA-

ACA bifurcation as an anatomical landmark. The same researcher positioned the Doppler probes for

repeat laboratory visits to ensure consistency of placement. Real-time MCAv was displayed and

recorded online at 1 kHz (LabChart version 8.0, AD Instruments, Dunedin, New Zealand).