Characterising sex and ethnicity-based differences in cerebrovascular dynamics using a CO2 stimulus.

Participant Experience Summary (3 interactions): Interaction 1 – Recruitment and Consent: The participant pool will be recruited from posters advertised around the city (Auckland and Gisborne) and campuses (University of Auckland and Matai Medical Research Institute). Upon expressed interest to the recruiter, the recruiter will explain the research either over the phone, by email, or in person. Should the potential participant wish to join the study, a meeting with a researcher and the potential participant will be booked to complete the informed consent process over the phone or video call. The recruiter will then pass on the participants contact information to imaging centre administration to schedule Interactions 2 and 3. Interaction 2 – Visit 1: Demographics, BP measurement, and Mock Scan: Timing: Approximately 1-2 weeks after interaction 1. The participant will be booked for a one-hour visit to the scanning centre. 24 hours before the visit, the participant will be asked to abstain from alcohol, exercise, and be encouraged to have good sleep. Participants will be asked to not eat 2 hours before the visit to minimise the effects of digestion in the measurements. The participant will receive a reminder email the day before for visit for pre-scan preparation and the researcher will verbally confirm adherence on the day. Provided the participant has followed the pre scan preparation: During the visit: • A member of the research team will first complete the demographic questionnaire form with the participant. At this stage, and prior to measurement, the option to have a female or male chaperone will be offered to the participant during the time the researcher is collecting measurements. The main role of the chaperone is to be physically present during the consultation and to directly observe all contact between the researcher and participant. The chaperone will ensure the protocol is followed and correctly documented, and intervene if the researcher acts inappropriately. The chaperone will adhere to the Medical Council of New Zealand Information Sheet and Policy 2020 standards. [https://www.mcnz.org.nz/support/support-for-patients/chaperones/]. • The participant will then be asked to test several gas masks for an ideal fit and to lie down. A finger heart rate monitor will be attached, the mask will be placed on the participant, and headphones will be provided playing MRI scanner sounds. This will simulate the MRI experience to prepare the participant before the scan. This will last for 10 minutes. Five minutes into rest, 3 brachial blood pressure cuff measurements will be taken for consistent measurement followed by arterial tonometry (a pencil based pressure reader) to measure pulse wave velocity (PWV) by reading the blood signal at the neck and femoral artery. A measure of distance between both measurements will also be collected using a tape measure. • The participant will then practice breathing a carbon dioxide enriched gas blend (gas blend (5% CO2, 21% O2, and 74% Nitrogen) for 10 minutes to make sure they are comfortable with the sensation, which may provoke anxiety. 5% CO2 is completely safe for the human body and little to no sensation is expected. It can comfortably be inhaled and has been used in MRI scanners before at higher percentages for 15 minutes at a time in a young and older cohort [Miller:10.3389/fphys.2018.01096]. Their blood pressure will be taken thrice again at 5 minutes with CO2, followed again by the arterial tonometry. Interaction 3 – Visit 2: MRI, Ultrasound (US), and Blood Sample: Timing: As soon as possible after interaction 2. Estimated 1 week. The participant will be booked by imaging centre admin for a two-hour visit. Availability for this visit will be confirmed during the scheduling of visit 1. 24 hours before the visit, the participant will be asked to abstain from alcohol, exercise, and be encouraged to have good sleep. Participants will be asked to not eat 2 hours before the scan to minimise the effects of digestion. The participant will receive a reminder email the day before for visit for pre-scan preparation and the researcher will verbally confirm adherence on the day. Provided the participant has followed the pre scan preparation: • The participant and researcher will move into the MRI scanner module and similar equipment to the mock scan will be placed on the participant. • The participant will then be imaged using the MR protocol detailed in the next section. The scan is non-invasive and does not involve any harmful substances or needles. Midway through (~12 minutes into scan), the CO2 enriched gas blend will be delivered and the participant will breath the gas for ~10-12 minutes (dependent on participant heart rate) to image changes in vascular flow. We are aware of the time commitment and comfort of participants, and as such have developed the entire MRI scanning protocol to be approximately 30 minutes. • After the MRI scan, the participant will be immediately transported with the research team to the ultrasound scan room where they will receive a cardiac echo ultrasound scan which will take approximately 30 minutes. 15 minutes breathing regular air, and ~15 minutes breathing the gas blend. • Finally, the research team will accompany the participant to the blood clinic, where blood samples will be collected. MRI Protocol: Scan Time 30 minutes. Below describes the MR sequence and analysis plan/use. 1 - Structural T1-weighted: Measure total intracranial volume, grey matter, white matter, cerebrospinal 2 - Angiogram (Time of Flight): Localise the brain vessels to plan the 4D flow 3 - 4D Flow (3D phase contrast (0.75mm isotropic), Velocity encoding=90cm/s): Measure cardiac gated cerebral blood flow in the brain (focused on the circle of Willis) 4 - Functional fMRI: During initial CO2 inhalation, measure the initial response to CO2 bolus as a form of tissue reactivity Ultrasound Protocol: Scan Time 30 minutes Immediately following the MRI, an echocardiogram will be performed. This will be undertaken by an experienced echocardiographer using a cardiac ultrasound system. The echocardiogram will be undertaken in the MRI recovery area next to the MRI. At this stage, and prior to measurement, the option to have a female or male chaperone will be offered to the participant during the time the researcher is collecting measurements. Two-dimensional (2D), pulsed-Doppler, and colour tissue Doppler imaging will be performed from standard parasternal and apical transducer positions. All indices will be measured according to the recommendation of the American Society of Echocardiography: 1) Standard 2D left ventricular measurements (apical 4 and 2 chamber volumes) and left atrial size will be recorded. Left ventricular ejection fraction will be quantified using Simpson’s biplane method, left atrial volume will be obtained from Simpson’s biplane volume assessment or area length volume assessment in the apical views. 3D left ventricular volumes will be assessed. 2) Left ventricular diastolic parameters will include the combined mitral inflow and annular tissue Doppler velocities (with sample volume placed at the mitral annulus and the average value of the medial and lateral velocities will be used to derive the E/e’). 3) Left ventricular global longitudinal strain (GLS) will be measured using speckle tracking echocardiography (STE) in the three standard apical views and the average value recorded. If regional tracking is suboptimal in more than two myocardial segments in a single view, the calculation of GLS will be recorded as unobtainable. 4) RV systolic function will be assessed by tricuspid annular plane systolic excursion [TAPSE]. TAPSE is easily obtainable and has been shown to be an accurate reflection of RV global systolic function. Two 3D+t volumetric scans of one breath-hold each will be acquired to reconstruct 3D cardiac geometry and function within one cardiac cycle. Using echo analysis software, 3D measurements of left ventricular geometry and function such as: left ventricular mass, left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular ejection fraction, as well as 3D myocardial strain, will be derived from the 3D volumetric data. Finally, the raw imaging data will be converted to DICOM images, which will then be analysed using a model-based image processing software tool to construct a 3D computer model of the left ventricle for biomechanical analysis. Two 3D-doppler images will be taken in the left ventricle and aorta to allow for assessment of quantification of pressure via a non-invasive means. A Pulse Cor R6.5 Cardiovascular monitor will be used to measure aortic pressure and arterial stiffness. This is a standard automated blood pressure cuff and the measurements are no different to having a standard blood pressure measurement performed. The ultrasound imaging will be repeated breathing the CO2 enriched gas blend for reactivity measurements after free end tidal CO2 has normalised on the gas monitor. Each imaging round with and without gas will last about 15 minutes. Blood Collection: The participant and accompanying researcher will travel by foot or cab to the designated blood collection sites. The private third-party company will draw, store, process, and destroy the blood tissue (unless return is requested, an option by the companies named below). Blood Tests: Oestradiol (Estrogen) from plasma Progesterone from plasma. Haematocrit (red blood cell count, platelet count) from whole blood sample. Testosterone (Total) from plasma