Macrovascular Function – Flow-Mediated Dilation

Conduit artery endothelium-dependent function was measured using the FMD technique, which

provides an assessment of peripheral conduit artery diameter following a brief period of distal limb

ischaemia (Thijssen et al., 2011). Shear stress is the key physiological stimulus evoking endothelium-

mediated vasodilation during the FMD response (Melkumyants et al., 1989; Thijssen et al., 2011) and

is associated with dose-dependent increases in artery diameter (Betik et al., 2004; Padilla et al., 2009).

Increased shear stress is detected by cell membrane mechanoreceptors, following which a signalling

cascade is activated and subsequently stimulates the production and release of vasoactive substances

that diffuse across the endothelial cell membrane into the smooth muscle cell (Thijssen et al., 2011).

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subsequently, vasorelaxation. Biological variability in between-subject FMD responses largely arises

from differences in the transduction of the vasodilatory responses to the smooth muscle cells, in

addition to the structural characteristics of the vessel wall, such as the wall-to-lumen ratio, potentially

influencing the resultant diameter change (Thijssen et al., 2011). FMD assessment, therefore, provides

a means assessing endothelial function via interrogation of such biological differences and allows the

macrovascular impact of lifestyle factors, disease status, exercise training status and various

interventions to be examined.

Simultaneous assessment of the brachial artery, at the distal third of the non-dominant upper arm,

and the femoral artery of the right leg was performed (Figure 3.5). The non-dominant arm was

extended and positioned at an angle of ~80° from the torso, whilst the right leg was extended in a

comfortable position. Rapid inflation and deflation pneumatic cuffs (D.E. Hokanson, Bellevue, WA,

USA), connected to a rapid inflator (D.E. Hokanson, Bellevue, WA, USA), were positioned on the

forearm, immediately distal to the olecranon process, and around the right thigh, proximal to the

patella, to provide the stimulus for ischaemia (Thijssen et al., 2011). With a stable image obtained, the

ultrasound parameters were set to optimise the longitudinal B-mode image of the lumen-arterial wall

interface. Continuous Doppler velocity assessment was collected using the lowest insonation angle

(<60°), which was standardised across all measures. Baseline images for the assessment of resting

vessel diameter, shear rate and flow were recorded for 1-minute, following which the occlusion cuffs

were inflated (>220 mmHg) for 5-minutes to completely block the arterial inflow. Diameter and

velocity recordings resumed 30-seconds prior to cuff deflation and continued for 3-minutes

thereafter, according to methodological guidelines (Woodman et al., 2001; Thijssen et al., 2011;

Greyling et al., 2016). Peak brachial artery diameter, peak blood flow velocity and the time taken to

reach these peaks post cuff release were recorded. Ultrasound images were recorded using

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dependent upon the adherence to current methodological guidelines (Thijssen et al., 2011), which

were followed closely for all assessments performed during the current research programme.

Figure 3.5 Simultaneous assessment of brachial and femoral flow-mediated dilation (FMD).

Figure 3.6 Schematic representation of diameter and shear-stress responses following cuff deflation, in response to a 5-minute ischaemic stimulus during FMD assessment. The grey area represents the shear rate area-under-the-curve (SRAUC), which is considered to be the main stimulus for peak diameter. Taken from (Thijssen et al., 2011).

58 3.3.2.1 Artery diameter and blood flow analysis

Post-test analysis of brachial and femoral artery diameter and velocity was performed using custom-

designed edge-detection and wall-tracking software (Dicom Encoder, V.3.0.5 LabVIEW V.7.0, National

Instruments Corporation), which is largely independent of investigator bias and provides continuous

measurements of arterial diameter and blood flow velocity. The software is written in icon-based

graphical programming language (LabVIEW V.7.0, National Instruments) and uses an IMAQ vision

toolkit for image handling. Arterial diameter on the B-mode image and velocity on the Doppler strip

were calibrated for each individual assessment. An optimal region of interest (ROI) was identified for

analysis on the B-mode image, chosen according to the image quality depicting a clear distinction

between the vessel walls and lumen (Figure 3.7). Within the selected ROI, a pixel-density algorithm

measures the mean diameter changes according to changes in pixel density from the far- and near-

wall lumen-intima interface via a Rake routine, which measures 30 points per second throughout the

analysis. The edge-detection algorithm assessed the peak velocity envelope from the Doppler gate

which was placed in the centre of the artery. Mean diameter derived from the B-mode ROI was

subsequently synchronised with the velocity derived from the Doppler ROI at 30 Hz.

From the synchronised diameter and velocity data, blood flow (the product of cross-sectional area

and Doppler velocity) and shear rate (four times the velocity divided by the diameter) were calculated

at 30 Hz (Black et al., 2008a). Peak blood flow was taken over the initial 10-second post cuff deflation,

with peak artery FMD defined as the percentage change of the post cuff deflation peak diameter from

baseline diameter. Total shear rate (SRAUC) was calculated, which is considered to be the main

stimulus of the FMD response, rather than peak shear rate. All data were written to file and retrieved

for analysis in the custom-designed analysis software package. The edge-detection and wall-tracking

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measurements (coefficient of variation = 6.7%) compared to manual methods, whilst simultaneously

reducing observer error (Woodman et al., 2001).

A B

Figure 3.7 Ultrasound image of the brachial artery and blood flow velocity trace during A. baseline, and B. post cuff deflation during flow-mediated dilation (FMD).