The University of Western Australia



Cardiovascular Research Group

School of Exercise and Sport Sciences

The University of Western Australia

Lab Head: Winthrop Professor Daniel J Green

Experimental Procedures

The health of peripheral arteries is predictive of coronary vascular risk and can be improved through exercise and other therapies. Cerebrovascular health is associated with a lower incidence of cerebrovascular disease, and maintenance of cognitive function with aging.

Ours laboratory is an international leader in the development and optimisation of peripheral and cerebral vascular function assessment in humans. As well as focusing on the function and structure of large arteries (i.e. macrovasculature), we have developed techniques to assess microvascular function and structure in vivo.

Considering that vascular disease occurs in both macro and microvascular beds, and presents as both structural and functional abnormalities, the tests outlined below provide the most comprehensive non-invasive assessment of the vasculature in humans. With these tests, we aim to assess vascular health and disease status in individuals, to characterize vascular structure and function, to improve early diagnosis and prediction of vascular disease and to optimise and individualise the management of arterial health.

Peripheral Vasculature

Macrovascular tests: Function.

Flow Mediated Dilation

Macrovascular function will be assessed by measuring conduit artery responses to an imposed shear stress stimulus across the endothelial cell lining. We have developed world leading methods for the quantification of artery function in vivo, based on image analysis software used in combination with non-invasive high resolution Duplex ultrasound technology (Figures 1 and 2).(6, 15) Brachial and femoral FMD are typically assessed in view of the different propensity of the sites for disease. FMD, which is endothelium- and nitric oxide (NO)-mediated,(17) will be assessed along with vascular smooth muscle sensitivity to a NO donor (glyceryl trinitrate 400 micrograms, sub-lingual).Each of these tests can be performed following ischaemic handgrip or large muscle (leg exercise) to assess maximal or dilator capacity and, hence, structural adaptation of conduit and resistance arteries.

Endothelial dysfunction is a precursor to atherosclerotic disease. In 1992, Deanfield and Celermajer introduced a method to assess conduit artery endothelial function using high resolution ultrasound.(9) This technique, termed flow mediated dilation (FMD), has been technically enhanced[pic](13, 34) and formalised and in the past 2 decades.(30) FMD is largely mediated by endothelial vasodilators, particularly nitric oxide (NO),(17) which has myriad anti-atherogenic effects.(11) FMD in peripheral arteries is strongly correlated with coronary artery function(29) and independently predicts cardiac events in subjects with CV disease or risk factors and also in asymptomatic subjects.(18) A 1% decrease in FMD is associated with a 13% increase in CV event risk.(18) In addition, recent analysis of the FATE study suggested that small artery or microvascular function, assessed using hyperaemic flow during the FMD test, strongly predicts prognosis in healthy men.(2)

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Figure 1: Description of flow mediate dilation technique for in vivo assessment of conduit artery endothelium-mediated vasodilation

[pic]Figure 2. Edge detection and wall tracking software developed by Prof Green. Assessment of diameter, flow and shear across the cardiac cycle.(6, 15) Left: Diameter is calculated from >300 measures within the region of interest at 30Hz. Velocity is also calculated via waveform envelope algorithm. Right: output of software – continues diameter changes and shear rate stimulus.

Brachial Artery Endothelium Dependent Vasodilation- After 15-minutes of supine rest the participant’s arm is extended to ~80° from the torso and a rapid inflation/deflation pneumatic cuff is placed on the forearm distal to the olecranon process. Duplex ultrasound (B mode images and Doppler velocities; Terason 3200, USA) is then used to acquire an image of the brachial artery. Following a one-minute baseline, the forearm cuff is inflated to 220 mmHg for 5 minutes. Following deflation of the cuff, participants continue to rest supine for an additional 3 minutes. Duplex ultrasound images are recorded throughout this entire test. This is a safe protocol and has been approved by the UWA, RPH and FSH ethics committees on multiple occasions.

Femoral Artery Endothelium Dependent Vasodilation- After 15-minutes of supine rest the participant’s lower leg is slightly elevated by about 15cm with a foam pad and a rapid inflation/deflation pneumatic cuff is placed around the leg ~15cm below the inguinal ligament (slightly above the knee). Duplex ultrasound (B mode images and Doppler velocities; Terason 3200, USA) is then used to acquire an image of the superficial femoral artery. Following a one-minute baseline, the pneumatic cuff is inflated to 220 mmHg for 5 minutes. Following deflation of the cuff, participants continue to rest supine for an additional 5 minutes. Duplex ultrasound images are recorded throughout this entire test. This is a safe protocol and has been approved by the UWA, RPH and FSH ethics committees on multiple occasions.

Glyceryl Trinitrate [GTN] Mediated Endothelium Independent Vasodilation – Following a 1 minute baseline collection of brachial and femoral artery duplex ultrasound (Terason 3200, USA), a single sublingual spray of glyceryl trinitrate (GTN, 400 μg), a nitric oxide donor, is administered. Ten minutes of continuous ultrasound recording of diameter and velocity ensues. This is a safe protocol and has been approved by the UWA, RPH and FSH ethics committees on multiple occasions. GTN mediated dilation provides information regardi9ng the sensitivity of vascular smooth muscle cells to donated or exogenous nitric oxide. When considered in the context of FMD assessments, an index of endothelium-dependent and –independent dilator function can be determined.

Assessment of Arterial Stiffness

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Figure 3. Diagram illustrating the calculations used to quantify pulse wave velocity (PWV), where D is distance between the location of tonometry application at the carotid and femoral artery, and t is the time taken for the pressure wave from a single cardiac cycle to travel to the tonometry location.

Another biophysical property of large arteries, stiffness, can be reliably and non-invasively measured by determining arterial pulse wave velocity (PWV), which strongly relates to atherosclerotic disease.(31) Whilst assessing arterial properties distinct from NO-mediated function, PWV compliments FMD in that it also integrates the impact of multiple risk factors and genetic predisposition to CV disease. Ben-Shlomo et al. recently meta-analyzed individual patient data from 16 studies (n=17 635) and, after adjusting for age, sex and traditional CV risk factors, found that the increase in risk for CV events and mortality was 30% and 28%, respectively, for every 1 standard deviation change in PWV.[pic](4) This suggest that, for a 60-year old non-smoking, non-diabetic, normotensive and normolipidaelic male, a 1 m/sec increase in PWV leads to a 7% increase of the hazard for CV events.(31) The addition of PWV improved 10-year risk classification for intermediate risk subjects by 13%, indicating that it may enable better identification of high-risk populations who could benefit from aggressive CV preventative strategies. Collectively, these studies indicate that direct measures of large and small artery function provide independent prognostic information which equals or exceeds that from traditional risk factors.

Pulse Wave Velocity A measure of arterial stiffness, pulse wave velocity is assessed following 15-minutes of supine rest using the Sphygmocor Xcel (ATcor Medical Pty). A blood pressure cuff is placed on the left leg, approximately 200mm inferior from the inguinal ligament. The blood pressure cuff is inflated throughout the entire pulse wave assessment (< 3 minutes). A small tonometer is placed against the skin to measure the velocity in the carotid artery, approximately 30-50 mm above the sternal notch. The pulse wave velocity is then calculated from the offset of the femoral and carotid pulse-waves and the distance between the carotid tonometer site and the femoral artery cuff. This is a safe non-invasive protocol and has been approved by the UWA, RPH, SXCGH and FSH committee’s on multiple occasions.

Pulse Wave Analysis Pulse wave analysis is assessed following 15-minutes of supine rest using the Sphygmocor Xcel (ATcor Medical Pty). A blood pressure cuff is placed on the right arm, directly over the biceps brachii. The cuff is inflated for approximately 3 minutes, and measures systolic and diastolic brachial blood pressure, aortic systolic pressure, central pulse pressure and augmentation index. This is a safe non-invasive protocol and has been approved by the UWA, RPH, SXCGH and FSH committee’s on multiple occasions.

Macrovascular tests: Structure.

Intima Media Thickness/Conduit Artery Wall Thickness

The ARIC (Atherosclerosis Risk in Communities)(10) and Rotterdam studies(19) indicated that carotid IMT is associated with increased risk for adverse cerebral events (e.g. stroke), independent of other risk factors. Carotid IMT may possess superior predictive capacity for stroke than other measures of atherosclerotic risk, such as the presence of carotid plaques and the ankle–arm index.(19) Increased carotid IMT is also associated with increased coronary(19, 21) and peripheral vascular risk.(32) The annual change in carotid IMT is a surrogate for systemic atherosclerosis and provides prognostic information: a 0.1 mm increase in carotid artery IMT is associated with an increase in age- and sex-adjusted relative risk of 18% for stroke and 15% for myocardial infarction.(23)

Arteries of the lower limbs are also subject to the development of atherosclerosis and wall thickening is also found in the upper limbs of older subjects, in which plaque formation is not typically observed.(5) The presence of arterial wall thickening in atherosclerosis-prone and -resistant conduit arteries supports the idea that wall thickening occurs systemically, although it is not currently know whether this reflects systemic atherosclerosis or benign thickening. Increased femoral artery IMT correlates with traditional risk factors, such as waist circumference, blood pressure, cholesterol, insulin and smoking status(26, 27) and with measures of peripheral atherosclerotic disease, such as the ankle–brachial index,(28) suggesting that femoral IMT may be of prognostic relevance. Peripheral vascular disease is particularly prevalent in diabetes and 30% of the Raine cohort had, by 8 years of age, shown increased risk of diabetes with a cluster of metabolic abnormalities associated with overweight and obesity.[pic](20) Other studies have reported associations between femoral IMT and restenosis after percutaneous coronary intervention and with the severity and extent of coronary artery disease.(22) Increased brachial IMT also correlates, independent of other risk factors, with the presence of coronary disease and predicts future cardiovascular events in subjects undergoing coronary angiography.(12) These findings suggest that measures of IMT provide prognostically relevant information pertaining to the presence of atherosclerosis in vivo.

B-Mode vascular ultrasound (Terason 3200, USA) is used to record the intraluminal diameter and wall thickness (intima thickness) of the large conduit arteries (i.e., common carotid, brachial, femoral, popliteal arteries). We have developed customized edge-detection and wall tracking software to provides an investigator-independent assessment of arterial diameter and intima wall thickness across the cardiac cycle. We assess IMT in carotid, femoral and brachial arteries due to the complementary information which these measures may provide regarding the specific or generalised presence of atherosclerotic disease in the cerebral, coronary and peripheral circulations.

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Figure 4 Specialised edge-detection and wall-tracking software. Assessment of diameter, flow and shear across the cardiac cycle. Left: Diameter is calculated from ~400 measures within the region of interest at 30Hz. Velocity is also calculated via waveform envelope algorithm. Right panel: Assessment of wall thickness using validated automated algorithms. 

Cerebral Vascular Function

Cerebral blood flow velocity (CBFv) can be assessed by combining bilateral measures of middle (MCA) and posterior cerebral artery (PCA) flow velocities, and extra-cranial cerebral blood flow in the internal carotid and vertebral arteries (QICA & VA), using state-of-the-art transcranial Doppler (TCD) techniques, pioneered by Ainslie and Green (Fig 5). Cerebrovascular reactivity is assessed by constructing a dose-response curve to different partial pressures of arterial CO2. Impairment in cerebrovascular CO2 reactivity is an independent predictor of stroke risk and cognitive impairment.(24, 25) Dynamic cerebral perfusion pressure regulation (DCPPR) involves CBFv responses to blood pressure manipulation and is also strongly linked to adverse clinical outcomes.(3, 35) Finally, neurovascular coupling assesses responses to increased neural activity induced by repeated trials of voluntary eye movement (tracking) and cognitive tasks. Response times to these tests and the magnitude of change in brain blood flows provide indices of cerebral neurovascular function which provide a prognostic index of cerebrovascular health.(7) Direct measures of cerebrovascular function are novel and are linked to meaningful clinical outcomes. However, all studies to date have been undertaken in small samples and there are no data on antecedent developmental factors which contribute to cerebrovascular dysfunction and risk in humans.

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Figure 5. Schematic summary of the assessment battery of cerebrovascular measures of function and health developed by Ainslie and Green. Transcranial Doppler (TCD) measures are complemented by contemporaneous assessment of whole brain blood flow, derived from simultaneous high resolution ultrasound via insonation of the internal carotid and vertebral arteries (see Fig 5).

CO2 reactivity: Cerebral blood flow velocity (CBFv) and QICA; VA is measured in response to different "doses" of blood carbon dioxide (CO2), achieved by switching from room air to CO2 mixtures.

Dynamic Cerebral Perfusion Pressure Regulation (DCPPR): CBFv and QICA;VA can be assessed in response to different "doses" of BP manipulation. A lower body negative pressure (LBNP) chamber is used to precisely manipulate BP, or CBFv can be assessed during a squat-to-stand technique to stimulate orthostatic (baroreflex) and cardiovascular (exercise pressor and metabolic) reflexes.

Neurovascular coupling (NVC): CBFv assessed in response to neural activity induced by repeated voluntary eye movement. Response times provide prognostic indices of cerebrovascular health.

The reproducibility of MCAv and PCAv has a CV ................
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