Understanding relationships between skin microvascular and limb macrovascular structure and function assessed by optical coherence tomography and high-resolution ultrasound in response to physiological stimuli (flow-mediated dilation, local heating, carbon dioxide and glyceryl trinitrate) in healthy younger and older adults.

This is a multiple-visit, mechanistic laboratory study in healthy adults. After initial contact, potential participants will attend a screening visit at the Cardiovascular Research Group Laboratory, University of Western Australia, where the study will be explained, written informed consent obtained, and eligibility assessed using a health questionnaire, resting blood pressure, height, and weight. Eligible participants will then complete 3 formal assessment visits. These visits will be conducted on separate mornings between 08:00 and 12:00 in a thermoneutral room with controlled temperature and humidity. Before each formal visit, participants will fast for 8 hours and abstain from alcohol, caffeine, tea, chocolate, and strenuous exercise for at least 12 hours, while maintaining normal hydration. The order of Visits 1-3 will be randomly assigned using computer-generated randomisation. Visits will be scheduled at least 24 hours apart, with scheduling determined by participant availability and, for premenopausal women, menstrual-cycle phase requirements. Visit 1 will include body composition assessment using dual-energy X-ray absorptiometry (DXA), microvascular and macrovascular responses to carbon dioxide (CO2), and cardiorespiratory fitness testing on a cycle ergometer using a metabolic cart. The DXA scan will take less than 20 minutes. For the CO2 protocol, after 15 minutes of supine rest, participants will breathe through a mouthpiece connected to a three-way valve, beginning with a 2-minute room-air baseline. Internal carotid artery and forearm skin responses will then be recorded using duplex ultrasound, optical coherence tomography (OCT), and laser Doppler flowmetry (LDF) during inhalation of two mild hypercapnic gas mixtures (3% and 6% CO2, 21% O2, balance N2) for 4 minutes each, separated by a 7-minute room-air recovery period, followed by a further 5-minute room-air recovery. After completion of the CO2 vascular assessment, participants will undergo 15 minutes of passive recovery before the cardiorespiratory fitness test. The cardiorespiratory fitness test will be a ramp cycle ergometer protocol to volitional fatigue and will take less than 30 minutes. Visit 2 will include upper-limb vascular assessments. After 15 minutes of supine rest, local heating will be applied to the ventral forearm using a heating patch that increases from 33 degrees Celsius to 42 degrees Celsius at a rate of 1 degree Celsius every 10 seconds and is then maintained at 42 degrees Celsius for 30 minutes. OCT will be performed at baseline and after heating, and LDF will be recorded continuously throughout the procedure. After completion of local heating, participants will rest supine for 15 minutes before flow-mediated dilation (FMD) testing. FMD of the brachial artery and adjacent forearm skin will then be assessed using duplex ultrasound, OCT, and LDF during a 1-minute baseline, 5-minute forearm cuff inflation to 220 mmHg, and 3 minutes of recovery after cuff release. After FMD, participants will rest for 10 minutes before glyceryl trinitrate (GTN) testing. GTN responses of the brachial artery and forearm skin will then be assessed after a 1-minute baseline using a single sublingual spray of GTN (400 micrograms), with continuous duplex ultrasound, OCT, and LDF recording for 10 minutes after administration. Visit 3 will include lower-limb vascular assessments. After 15 minutes of supine rest, local heating will be applied to the dorsum of the foot using the same temperature protocol as Visit 2, with OCT collected at baseline and after heating and continuous LDF monitoring throughout the procedure. After completion of local heating, participants will rest supine for 15 minutes before flow-mediated dilation (FMD) testing. FMD of the superficial femoral artery and dorsum of the foot skin will then be assessed using duplex ultrasound, OCT, and LDF during a 1-minute baseline, 5-minute cuff inflation to 220 mmHg, and 5 minutes of recovery after cuff release. After FMD, participants will rest for 10 minutes before glyceryl trinitrate (GTN) testing. GTN responses of the femoral artery and dorsum of the foot skin will then be assessed after a 1-minute baseline using a single sublingual spray of GTN (400 micrograms), with continuous duplex ultrasound, OCT, and LDF recording for 10 minutes after administration. Male participants will attend the 3 formal assessment visits only, with a total time commitment of approximately 6 hours. Premenopausal women aged 18-40 years will attend 3 additional visits repeating the same formal vascular assessment protocols. Three visits will be conducted during the early follicular phase and three visits during the late follicular phase. Menstrual-cycle phase will be determined using menstrual-cycle history and a digital ovulation kit. Postmenopausal women aged 60-75 years will attend the first 3 formal assessment visits only. All procedures will be delivered face-to-face on an individual basis by trained cardiovascular physiology researchers using a standardised written protocol.