Matched increases in cerebral artery shear stress, irrespective of stimulus, induce similar changes in extra-cranial arterial

diameter in humans

*Kurt J Smith1 PhD, *Ryan L Hoiland2 MSc, Ryan Grove1BSc (Hon), Hamish McKirdy1 BSc (Hon), Louise Naylor 1 PhD, Philip N Ainslie2 PhD, Daniel J Green1,3 PhD

1Cardiovascular Research Group, School of Human Sciences (Exercise and Sport Science),

The University of Western Australia, Perth, Australia;2Centre for Hearth Lung and Vascular Health, School of Health and Exercise Sciences,

University of British Columbia,3Research Institute for Sport and Exercise Sciences, John Moores Liverpool University, UK

*Authors contributed equally to this manuscript

Corresponding Author:Kurt J. Smith

Kurt.smith@uwa.edu.au610431698502

School of Human Sciences M40835 Stirling Highway, Nedlands, 6000The University of Western Australia

Word Count: 4571Tables: 2Figures: 2

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Abstract

The mechanistic role of arterial shear stress in the regulation of cerebrovascular responses to

physiological stimuli (exercise and hypercapnia) is poorly understood. We hypothesized that,

if shear stress is a key regulator of arterial dilation, then matched increases in shear induced

by distinct physiological stimuli, would induce similar dilation of the large extra-cranial

arteries. Participants (n=10), participated in three 30-min experimental interventions, each

separated by ≥48hours: 1) mild hypercapnia (FICO2:~0.045); 2) submaximal cycling (EX;

60%HRreserve); or 3) resting (time-matched control, CTRL). Blood flow, diameter and shear

rate were assessed (via Duplex ultrasound) in the internal carotid and vertebral arteries (ICA,

VA) at baseline, during and following the interventions. Hypercapnia and EX produced

similar elevations in blood flow and shear rate through the ICA and VA (p<0.001), which

were both greater than CTRL. Elevations in ICA and VA diameter from baseline in response

to hypercapnia (5.3±0.8 and 4.4±2.0%) and EX (4.7±0.7 and 4.7±2.2%) were also similar,

and greater than CTRL (p<0.001). Our findings indicate that matched levels of shear,

irrespective of their driving stimulus, induce similar extra-cranial artery dilation. We

demonstrate, for the first time in humans, an important mechanistic role for the endothelium

in regulating cerebrovascular response to common physiological stimuli in vivo.

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Introduction

In conduit arteries such as the coronary,1 brachial,2 radial,3 and femoral,4,5 it is well

established that intra-arterial shear stress regulates endothelium-dependent vasoreactivity. It

is also well-known that episodic increases in shear can induce anti-atherogenic adaptation in

paracrine transduction pathways, including the nitric oxide (NO)-dilator system [reviewed in:

6]. Shear-mediated stimulation of the endothelium is a key mechanism responsible for

vasodilation through these conduit arteries in humans.6

Exercise increases blood flow to the brain in humans.7 Traditionally, the explanation for this

has been attributed to a combination of physiological stimuli, including changes in arterial

blood gases, blood pressure and metabolism. 8,9 Another possible mechanism, however,

involves changes in arterial shear stress and consequent endothelium-mediated dilation.10

Recently, we demonstrated vasodilation of extra-cranial feed arteries as a result of

hypercapnia induced increases in brain blood flow and shear stress in vivo.11,12 However, the

impact of shear stress on cerebrovascular responses during exercise has not previously been

investigated.

In the current study, we propose that shear stress is involved in regulating cerebrovascular

function during exercise in humans. We therefore examined the impact of matched elevations

in arterial shear stress, induced by distinct and common physiological mechanisms: Exercise

or hypercapnia. We examined the hypothesis that similar patterns of dilation of the large

extra-cranial arteries would be observed irrespective of the different stimuli induced by

exercise or hypercapnia.

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Methods

Participants

Ten healthy male participants (age, 25±6 years; weight, 74.14±7.03kg; height, 2.11±0.21m)

were recruited for the study. All participants were non-smokers, with a body mass index <30,

free of cardiovascular, respiratory, cerebrovascular, musculoskeletal and/or metabolic

diseases. The study was approved by the University of Western Australia’s Human Research

Ethics Committee. The participants were informed of all experimental procedures and

associated risk. Participants provided written informed consent before the commencement of

the study.

Experimental design

After inclusion, participants were tested at the Cardiovascular Research Laboratory on three

occasions separated by ≥48hours at the same time of day. Each visit consisted of one of three

randomly assigned interventional protocols (Control [CTRL], Hypercapnia [CO2], or

Exercise [EX]). Subjects arrived after fasting for a minimum of 6 hrs, with 24 hr abstinence

from alcohol, caffeine and vigorous exercise. Upon arrival, participants were instrumented

and underwent a 10 min rest period in a semi-recumbent position. Baseline (BL) recordings

of the primary outcome measures were then collected during a further 5-minute period of

quiet rest. Following baseline measures, subjects participated in one of three interventions: 1)

30-minutes of hypercapnia (see below); 2) 30-minutes of exercise at ~60% heart rate (HR)

reserve; or 3) 30-minutes of rest (time-control day). Cerebrovascular (transcranial Doppler

and duplex vascular ultrasound) and cardiorespiratory assessments were collected throughout

each condition.

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Experimental procedures

Cerebrovascular assessments

Non-invasive insonation via 2MHz transcranial Doppler ultrasound (TCD; Spencer

Technologies, Seattle, WA) was used to assess blood velocity in the middle (MCAv) and

posterior (PCAv) cerebral arteries. The cerebral arteries were identified and optimised

according to their signal depth, waveform and velocities, according to previously published

guidelines.13 The MCAv and PCAv were continuously recorded at baseline, throughout the

30-minutes of each intervention, and during post-intervention assessments.

Blood velocity and diameter of the internal carotid artery (ICA) and vertebral (VA) arteries

(VA) were measured using a 10-15MHz multi-frequency linear array vascular ultrasound

(Terason T3200, Teratech, Burlington, MA).14 Whilst ICA recordings were captured at 10,

20 and 30 minutes of the intervention, the VA recording were captured at 15 and 25 minutes

of the intervention. Ultrasound assessments were performed by one scanner and required

approximately 5-minutes to achieve a single scan. Thus, a standardized timing of ICA and

VA collection was applied to ensure similar time-points were collected throughout each

experimental condition. Baseline and post-intervention recordings (5–10 minutes post) were

collected for both the ICA and VA. All of the ICA and VA recordings were screen captured

and stored as video files for offline analysis.15

Cardiorespiratory measurements

All cardiorespiratory variables were sampled continuously throughout the protocol at 1000Hz

via an analogue-to-digital converter (Powerlab, 16/30; ADInstruments, Colorado Springs,

CO). Partial pressure of oxygen (PETO2) and carbon dioxide (PETCO2) were assessed using a

gas analyser (ADInstruments, Colorado Springs, CO), heart rate (HR) was measured by a 3-

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lead electrocardiogram (ECG; ADI bioamp ML132), and beat-to-beat blood pressure by

finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam,

Netherlands).

Experimental interventions

Hypercapnia (CO2) condition

Hypercapnia was used as a stimulus to elevate cerebral blood flow (CBF) and shear stress for

30 minutes via breathing an air mixture (4.5% CO2, 21% O2, and balanced N2) through a

spirometer connected to a Douglas bag. The participants were seated throughout in a semi-

recumbent position. Simultaneous assessment of intracranial velocity (measured by

transcranial Doppler of the left MCA and right PCA) and beat-by-beat extracranial blood

flow (measurement by Duplex ultrasonography of the ICA and VA) was assessed along with

beat-to-beat arterial pressure (Finometer Pro, Amsterdam, Netherlands). Continuous

monitoring of MCAv was used as an index of real-time change in cerebrovascular shear

stress. If necessary, the concentration of inspired CO2 was altered (range 3-6%) to achieve the

desired increase in arterial shear stress (~30% above baseline). The decision to use a 30%

increase in shear stress was chosen based off of pilot data indicating this to be the most

reliable increase achievable in both CO2 and EX protocols. This allowed CO2 and EX shear to

be matched irrespective of study order.

Exercise condition

Participants cycled at submaximal intensity [~60% heart rate (HR) reserve = 100-120bpm]

for 30-minutes to maintain a steady increase in CBF measures throughout the exercise

condition. As with the hypercapnia intervention, continuous intracranial blood velocity and

extra-cranial blood flow were measured at the same time points during the intervention. The

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MCAv during the 30-minute intervention was closely monitored to ensure that an elevated

CBF and shear stress was achieved (~30% above baseline, to match the CO2 condition).

Time-control condition

Participants sat in a semi-recumbent position for 30-minutes. Continuous intracranial blood

velocity and extra-cranial blood flow were measured at the same time points as the

interventions above.

Statistics

Statistical and graphing analysis was performed using GraphPad PRISM 6.01 software

(GraphPad Software, LaJolla, CA, USA). All parameters were compared within-subjects

using 2-way ANOVA repeated-measures. Bonferroni-correction for multiple comparisons

were used for all post-hoc analysis. Statistical significance was assumed at p<0.05. All data

are reported as mean ± SD unless otherwise specified.

Results

All cardiorespiratory and cerebrovascular values, for the anterior (ICA, MCA) and posterior

(VA, PCA) cerebral arteries, are provided in tables 1 and 2.

Cardiorespiratory measures

No changes were observed for any of the cardiorespiratory measures assessed during the 30-

minute CTRL condition (Table 1 and 2).

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During the CO2 and EX, MAP increased (p<0.01), but returned toward baseline values post-

intervention (Tables 1 and 2). The magnitude of increase in MAP was similar between the

CO2 and EX conditions, except for the post-intervention time point (EX was lower;

p<0.001).,\ Both CO2 and EX MAP data were significantly higher throughout the intervention

period compared to the CTRL trial (p<0.001).

The CO2 and CTRL conditions did not elicit any changes in HR. In contrast, the EX

condition was associated with an elevated HR compared to baseline values and by

comparison to the other conditions during the intervention period. Both PETO2 and PETCO2

were elevated throughout the CO2 intervention (p<0.001), compared to baseline values and

also when compared to the CTRL condition. EX had a small increase on PETCO2 compared to

baseline values, but not to the same extent as the CO2 condition.

Cerebrovascular measures

No changes in either MCAv or PCAv were observed during CTRL. When all time points

were considered, similar mean increases, from BL, were observed in response to CO2

(28.1±3.4 and 23.3±2.8%) and EX (27.7±3.8 and 19.6 ± .3.0%) in the in MCAv and PCAv,

respectively (p<0.001). All MCAv and PCAv values were greater during the EX and CO2

conditions relative to the CTRL data (Figure 1; p<0.001). Both the EX and CO2 conditions

stimulated similar magnitudes of increase in blood flow, blood velocity and shear rate (p <

0.001) in the ICA and VA, relative to baseline value. Furthermore, blood flow, velocity and

shear rate data collected under the CO2 and EX conditions were elevated compared to CTRL

throughout the intervention period (Figure 1; p<0.001).

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No changes ICA or VA diameter were observed during CTRL. In contrast, ICA and VA

diameters increased from baseline values during both CO2 and EX (Table 1; p<0.001). The

increases in diameter, in both arteries, were higher than the changes observed in the CTRL

condition at all time-points, except for the VA diameters 10 minutes post intervention (Figure

2). There were no differences in the changes in diameter observed when the CO2 and EX

conditions were compared.

Linear Regression

Linear regression analysis between the peak changes in diameter and shear stress indicated

significant correlation coefficient during the CO2 (R2 = 0.47; p< 0.01) and EX (R2 = 0.31;

p<0.01) conditions (see Figure 4). Multiple regression analysis using changes in blood

pressure as a covariate to predict the relationship between shear stress and ICA dilation

during the CO2 condition revealed an enhanced correlation coefficient (R2 = 0.61; p< 0.01);

however, no significant improvement was observed using changes in CO2 as a covariate. In

contrast, multiple regression analysis using changes in CO2 as a covariate during the EX

condition, revealed an enhanced correlation coefficient between the peak shear and dilation

(R2 = 0.53; p< 0.01), but not for changes in BP. No significant change in the improvement in

the covariate linear regression was observed between shear stress and diameter using changes

in both CO2 and blood pressure during either the CO2 or EX conditions.

Discussion

Our findings indicate that matched increases in cerebrovascular shear stress, irrespective of

the eliciting stimulus (CO2 or EX), induce similar increases in extra-cranial artery diameter in

humans. This study therefore provides novel evidence that arterial shear stress, alongside

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changes in blood gases and pressure, is an important stimulus regulating cerebral conduit

artery dilation in vivo. Given that repetitive episodic increases in shear stress beneficially

impact arterial function and structure,6 our novel observation provides a mechanistic basis for

linking exercise, physical activity and cerebrovascular health in humans.

It is well established that endothelium-dependent mechanisms play a key role in shear stress

mediated vasodilation of coronary and peripheral arteries in humans.1,3-5,16 These studies have

established a role for paracrine hormones such as NO and prostacyclin in vasomotor

regulation, for example, in response to exercise. In animals, classic studies have established

that in vivo increases in flow and shear stress through cerebral conduit arteries (e.g. basilar

artery) trigger endothelial-mediated vasodilation.17 Recently, in a well-controlled mouse

model, Raignault et al.18 illustrated that the cerebrovascular endothelium optimally couples

shear stress to eNOS-mediated dilation under physiological pulse pressures, in contrast to

static flow conditions. These findings strongly infer that changes in the diameter of cerebral

arteries are responsive to a pulsatile environment and that shear stress sensitivity and

consequent production of NO are optimised under in vivo conditions.18 It is also well

established that carotid artery remodelling as a result of chronic changes in flow is dependent

upon the presence of a functional endothelial layer,19 implicating shear- and NO-mediated

mechanisms in arterial structural adaptation. Collectively these findings, in animals, support a

key role for shear-mediated endothelial-dependent dilation of extra- and intra-cranial cerebral

conduit arteries, although it is also true that some studies indicate a role for non-shear

mediated NO-dependent (ie. eNOS) pathways in cerebral microvascular dilation 20-22. Few

studies, however, have addressed the impact of changes in arterial shear on human cerebral

vasodilator function in vivo.

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Recently we characterised the time-course and response of ICA dilation to changes in shear

using duplex ultrasound with high temporal resolution edge-detection and wall-tracking in

healthy subjects.11 We concluded that, in response to a sustained hypercapnic stimulus (5-

mins), dilation of the ICA occurs subsequent to marked increases in intra-arterial blood flow

and shear stress. Carter et al.,10 observed a significant relationship (R2 = 0.46) between the

peak shear response and the magnitude of ICA dilation during hypercapnic stimulus. In a

subsequent follow-up experiment,12 a more transient increase in PETCO2 (30 secs) triggered

similar ICA vasodilation to those observed during a sustained 4-min stimulus. Hoiland et

al.,11 also observed a significant relationship (R2 = 0.44) between the shear stress area under

the curve and the magnitude of ICA dilation following the transient hypercapnic bolus. Since

the transient changes in PETCO2 elicited vasodilation following the hypercapnic stimulus, the

notion of shear-mediated dilation of the ICA was further confirmed. The current study

advances these findings by illustrating that similar changes in shear stress during moderate

intensity exercise - and hypercapnia - elicit a similar increases in extra-cranial artery dilation

of both the ICA and VA in humans. This is further corroborated by the observed relationship

(Fig 4) between the peak changes in shear stress and the maximal dilation of the ICA during

exercise (R2 = 0.34) and CO2 (R2 = 0.47).

This study is one of the few to have assessed extra-cranial artery responses to exercise in

humans. Whilst some previous studies have assessed blood flow responses in the ICA during

exercise,7,23,24 these have not specifically focused on changes in arterial diameter or shear

stress. Furthermore, to the best of our knowledge, no study has reported changes in ICA and

VA diameter or shear during and following a commonly undertaken bout of exercise. We

observed significant ICA and VA dilation during exercise in all subjects. Our findings

therefore raise the possibility that exercise-mediated and shear-driven increases in the

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bioavailability of endothelium-derived dilators such as NO may counteract other vasoactive

pathways, such as the increase in sympathetic drive associated with some forms of exercise.

Indeed, functional sympatholysis is a well-established phenomenon in skeletal muscle

arterioles during exercise.25,26 However, the role of sympatholysis in the brain is unclear, and

mechanism(s) of vasomotion and adrenoceptor distribution differ from the large to smaller

cerebral arteries.27

This is the first study that has assessed VA diameter and shear relationships during EX, CO2

and CTRL conditions in humans. Our findings indicate that the vertebral arteries exhibit

dilator responses which are similar to those observed in the ICA. Only two previous studies,

to our knowledge, have recorded VA blood flows during exercise in humans, but these did

not address changes observed in either diameter or shear.7,24 When considered in concert with

our contemporaneous MCA and PCA observations, our ICA and VA data therefore indicate

that cerebral shear and artery dilation occur in both the anterior and posterior cerebral

circulation in response to both EX and CO2 in humans.

An interesting secondary finding of the present study relates to change in shear and artery

diameter following the cessation of exercise. We continued data collection in the post-

exercise period, at different time-points in the ICA (5 minutes post) and VA (10 minutes

post). Dilation remained elevated 5 minutes post-exercise, whereas it had returned to near

resting baseline levels 10-min post-exertion. These findings provide some insight into the

time-course of change in extra-cranial artery diameter following a physiological relevant

exercise duration and intensity.

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There are several limitations of the current experiment. We recruited and studied young male

subjects. The impact of cyclical changes in sex hormones on shear-mediated cerebrovascular

function and health would be an important follow up study in women (as would aging and

menopause). Although our findings suggest that exercise or hypercapnia elicited shear-

mediated endothelial dependent dilation of the ICA and VA, we did not utilize a NO

antagonist to address specific endothelial pathways. However, a previous study following a

sublingual administration of an NO donor (nitroglycerin spray) observed vasodilation in the

common carotid artery28 that was of similar magnitudes to the dilation that we observed in the

ICA. Future studies, involving more invasive approaches, for example endothelial denudation

and/or pharmacological blockade, would advance our understanding of the endothelial

dependency of extra-cranial dilation. Similarly, experiments involving deliberate

manipulation of the magnitude of shear (e.g., different levels of exercise or PETCO2) and

assessment of consequential diameter change in extra-cranial arteries are germane. It is

unlikely that our findings are explained solely as a result of the increases in blood pressure,

per se. Increases in intra-arterial pressure, and associated transmural pressure, are typically

associated with myogenic mediated vasoconstriction,29 however, it is unknown how this

directly influences shear mediated vasodilation in extracranial arteries. Our multiple linear

regression calculations suggest that changes in BP during sustained hypercapnia positively

reinforce shear mediated dilation. Also, PETCO2 was somewhat elevated above baseline in

each condition, and linear regression analysis between ICA dilation and shear stress during

exercise is enhanced when changes in PETCO2 was used as a covariate. We are therefore

unable to exclude the possibility that CO2 directly induces extra-cranial dilation. However,

we have recently provided evidence10,11 that part of the influence of CO2 on extra-cranial

vasodilation is likely due, in part, to shear stress which is supported by our findings of an

enhanced linear regression analysis when CO2 is used as a covariate (Fig 4). Indeed, when a

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bolus of CO2 is administered and arterial diameter is measured following restoration of

PETCO2 and BP to normal values, vasodilation occurs. This vasodilation is approximately

50% in magnitude of the vasodilation observed during a steady state, or persistent CO2

stimulus. Therefore, while data to support direct vasodilatory mechanisms on dilation in the

ICA or VA do not currently exist in humans, a decrease in small intracranial arteriole

resistance and consequent increase in extracranial shear stress clearly possesses a significant

role in cerebrovascular regulation. Stimulation of the peripheral chemoreceptors is associated

with sympathoexcitation, and usually associated with systemic arterial vasoconstriction rather

than the dilation we observed. Finally, we acknowledge that the limitations of TCD impair

our ability to extrapolate our extracranial diameter responses to changes in shear stress, to

intracranial changes that may have simultaneously occurred in the MCA and PCA. However,

the intention of our study was to focus on the ICA and VA responses, with intracranial

measures collected primarily in order to confirm that matching of shear in each intervention

was successful.

Conclusion

We have demonstrated, for the first time in humans, that matched increases in shear stress

through the extra-cranial cerebral conduit arteries in response to distinct stimuli induce

similar levels of vasodilation. Alongside previous evidence, largely derived from animal and

in situ preparations 30,31, our findings regarding vasodilator changes in both the anterior and

posterior extra-cranial conduit arteries suggest an important mechanistic role for the

endothelium in regulating cerebrovascular function in humans in response to both 30 minutes

of moderate hypercapnia and exercise. Since previous studies indicate that some clinical

population’s (stroke, Alzheimer’s, etc)32,33 exhibit impaired carotid and MCA responses to

physiological challenges , our cerebral flow mediated dilation approach may prove useful in

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the characterisation of cerebrovascular health in humans. Interventions that enhance

endothelial function may mitigate age-related decline in cerebrovascular vasodilator function

in humans.

Disclosures: None

Acknowledgements

Kurt J Smith, is supported by a Natural Sciences and Engineering Council of Canada Post-

Doctoral Fellowship. Ryan L. Hoiland was supported by a UBC School of Medicine

Friedman’s Scholarship. Prof’ Ainslie is a Canadian Research Chair in Cerebrovascular

Physiologist. Winthrop Professor Green is a National Health and Medical Research Council

Principal Research Fellow (1080914). This work was supported by NHMRC Project grant

(APP1045204)

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References

1. Berdeaux A, Ghaleh B, Dubois-Randé JL, Vigué B, La Rochelle CD, Hittinger L, Giudicelli JF. Role of vascular endothelium in exercise-induced dilation of large epicardial coronary arteries in conscious dogs. Circulation. 1994;89:2799–2808.

2. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992;340:1111–1115.

3. Rathore S. Impact of catheter insertion using the radial approach on vasodilatation in humans. Clinical Science. 2010;118:633.

4. Kooijman M, Thijssen DHJ, De Groot PCE, Bleeker MWP, Van Kuppevelt HJM, Green DJ, Rongen GA, Smits P, Hopman MTE. Flow-mediated dilatation in the superficial femoral artery is nitric oxide mediated in humans. The Journal of Physiology. 2007;586:1137–1145.

5. Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension. 1986;8:37.

6. Green DJ, Hopman MTE, Padilla J, Laughlin MH, Thijssen DHJ. Vascular Adaptation to Exercise in Humans: Role of Hemodynamic Stimuli. Physiological Reviews. 2017;97:495–528.

7. Smith KJ, Wildfong KW, Hoiland RL, Harper M, Lewis NCS, Pool A, Smith SL, Kuca T, Foster GE, Ainslie PN. Role of CO2 in the cerebral hyperemic response to incremental normoxic and hyperoxic exercise. Journal of Applied Physiology. 2016;120:jap.00490.2015–854.

8. Smith KJ, Ainslie PN. Regulation of cerebral blood flow and metabolism during exercise. Experimental Physiology. 2017; https://doi.org/10.1113/EP086249.

9. Ogoh S, Ainslie PN. Cerebral blood flow during exercise: mechanisms of regulation. J Appl Physiol. 2009;107:1370–1380.

10. Bolduc V, Thorin-Trescases N, Thorin E. Endothelium-dependent control of cerebrovascular functions through age: exercise for healthy cerebrovascular aging. AJP: Heart and Circulatory Physiology. 2013;305:H620–33.

11. Carter HH, Atkinson CL, Haynes A, Robey E, Smith KJ, Ainslie PN, Hoiland RL, Green DJ. Evidence for Shear Stress-Mediated Dilation of the Internal Carotid Artery in Humans. Hypertension. 2016;68:1217–1224.

12. Hoiland RL, Smith KJ, Carter HH, Lewis NCS, Tymko MM, Wildfong KW, Bain AR, Green DJ, Ainslie PN. Shear-mediated dilation of the internal carotid artery occurs independent of hypercapnia. Am J Physiol Heart Circ Physiol. 2017;:ajpheart.00119.2017.

13. Willie CK, Colino FL, Bailey DM, Tzeng YC, Binsted G, Jones LW, Haykowsky MJ, Bellapart J, Ogoh S, Smith KJ, Smirl JD, Day TA, Lucas SJ, Eller LK, Ainslie PN. Utility of transcranial Doppler ultrasound for the integrative assessment of cerebrovascular function. Journal of Neuroscience Methods. 2011;196:221–237.

15

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368369370

371372

373374375

376377

378379

380381382

383384

385386

387388389

390391392

393394395

396397398399

14. Thomas KN, Lewis NCS, Hill BG, Ainslie PN. Technical recommendations for the use of carotid duplex ultrasound for the assessment of extracranial blood flow. AJP: Regulatory, Integrative and Comparative Physiology. 2015;309:R707–20.

15. Woodman RJ, Playford DA, Watts GF, Cheetham C, Reed C, Taylor RR, Puddey IB, Beilin LJ, Burke V, Mori TA, Green D. Improved analysis of brachial artery ultrasound using a novel edge-detection software system. J Appl Physiol. 2001;91:929–937.

16. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol. 1986;250:H1145–9.

17. Fujii K, Heistad DD, Faraci FM. Flow-mediated dilatation of the basilar artery in vivo. Circ Res. 1991;69:697–705.

18. Raignault A, Bolduc V, Lesage F, Thorin E. Pulse pressure-dependent cerebrovascular eNOS regulation in mice. J Cereb Blood Flow Metab. 2016;37:413–424.

19. Langille BL, O'Donnell F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. J Vasc Surg. 1986;231:405–407.

20. Bertalanffy H, Kawase T, Toya S. In vivo effect of visible light on feline cortical microcirculation. Acta Neurochir (Wien). 1993;121:174–180.

21. Wang Q, Pelligrino DA, Koenig HM, Albrecht RF. The role of endothelium and nitric oxide in rat pial arteriolar dilatory responses to CO2 in vivo. J Cereb Blood Flow Metab. 1994;14:944–951.

22. Wang Q, Pelligrino DA, Baughman VL, Koenig HM, Albrecht RF. The role of neuronal nitric oxide synthase in regulation of cerebral blood flow in normocapnia and hypercapnia in rats. J Cereb Blood Flow Metab. 1995;15:774–778.

23. Trangmar SJ, Chiesa ST, Stock CG, Kalsi KK, Secher NH, Gonzalez-Alonso J. Dehydration affects cerebral blood flow but not its metabolic rate for oxygen during maximal exercise in trained humans. The Journal of Physiology. 2014;592:3143–3160.

24. Sato K, Ogoh S, Hirasawa A, Oue A, Sadamoto T. The distribution of blood flow in the carotid and vertebral arteries during dynamic exercise in humans. The Journal of Physiology. 2011;589:2847–2856.

25. Remensnyder JP, Mitchell JH, Sarnoff SJ. Functional sympatholysis during muscular activity. Observations on influence of carotid sinus on oxygen uptake. Circ Res. 1962;11:370–380.

26. Hearon CM, Dinenno FA. Regulation of skeletal muscle blood flow during exercise in ageing humans. The Journal of Physiology. 2015;594:2261–2273.

27. Brassard P, Tymko MM, Ainslie PN. Sympathetic control of the brain circulation: Appreciating the complexities to better understand the controversy. Auton Neurosci. 2017; http://dx.doi.org/10.1016/j.autneu.2017.05.003

28. Naqvi TZ, Hyuhn HK. Cerebrovascular mental stress reactivity is impaired in hypertension. Cardiovasc Ultrasound. 2009;7:32.

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421422423

424425426

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429430

431432433

434435

29. Atkinson CL, Lewis NCS, Carter HH, Thijssen DHJ, Ainslie PN, Green DJ. Impact of sympathetic nervous system activity on post-exercise flow-mediated dilatation in humans. The Journal of Physiology. 2015;593:5145–5156.

30. Hoi Y, Gao L, Tremmel M, Paluch RA. In vivo assessment of rapid cerebrovascular morphological adaptation following acute blood flow increase: Laboratory investigation. J Neurosurg 2008; 109(6): 1141-1147.

31. Paravicini TM, Miller AA, Drummond GR, Sobey CG. Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase. J Cereb Blood Flow Metab. 2005;26:836–845.

32. Glodzik L, Randall C, Rusinek H, de Leon MJ. Cerebrovascular reactivity to carbon dioxide in Alzheimer's disease. J Alzheimers Dis. 2013;35:427–440.

33. Gupta A, Chazern JL, Hartman M. Cerebrovascular Reserve and Stroke Risk in Patients With Carotid Stenosis or Occlusion: A Systematic Review and Meta-Analysis. J Vasc Surg 2013;57:1720.

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List of Tables

Table 1. Anterior cerebral blood flow and cardiorespiratory measurements during control

(CTRL), carbon dioxide (CO2), and exercise (EX) interventions (n=10).

Table 2. Posterior cerebral blood flow measurements during control (CTRL), carbon dioxide

(CO2), and exercise (EX) interventions (n=8)

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Table 1. Anterior cerebral blood flow and cardiorespiratory measurements during control (CTRL), carbon dioxide (CO2), and exercise (EX) interventions (n=10).

Time (min)Measure Condition BL 10 20 30 Post INT

Mean SD Mean SD Mean SD Mean SD Mean SD

MAP (mmHg)CTRL 88 11 90 8 90 8 93 8 91 9

**CO2 87 7.6 100*φ 10 104*φ 14 102*φ 14 96 11EX 82φ 11 102*φ 10 102*φ 9.5 100*φ 7.7 86*ѱ 15.8

HR (bpm)CTRL 64 7 64 9 67 7 67 7 63 7

**CO2 66 8 70 9 74 9 70 7 63 7EX φѱ 70 8 115φѱ 11 115φѱ 7 118φѱ 7 80φѱ 11

PetO2 (mmHg)CTRL 95.2 6.8 97.7 3.2 98.5 5 100.4 6.9 96 6.2

**CO2 φ 97.4 5 128*φ 2.9 130*φ 1.9 130 *φ 2.3 96 7.8EX ѱ 95.2 6.5 96.6ѱ 7.8 95.8ѱ 7.6 96.0ѱ 7.8 96.8 4.1

PetCO2 (mmHg)CTRL 42.7 4 42.4 2.7 41.9 3.6 41.3 3.6 41.6 3.4

**CO2 φ 42.3 3 46.9*φ 2.4 46.3*φ 2.3 46.2*φ 2.2 41.3 2.5EX 41.6 3.1 44.7* 4.9 44.7* 4.5 44.4φ 5.2 40.8 2.4

QICA (ml.min-1)CTRL 297 24.8 289 20.8 278 21.4 278 24.4 264 24.1

**CO2 262 18.8 368*φ 23.8 380*φ 30.5 354*φ 25.1 256 16.1EX 274 16.9 361*φ 19.7 368*φ 29.6 394*φ 26 282 24.1

ICA Diam (mm)CTL 5.2 0.2 5.2 0.2 5.2 0.2 5.2 0.2 5.2 0.2

**CO2 5.0 φ 0.1 5.3* 0.1 5.3* 0.1 5.4* φ 0.1 5.2 0.1EX 5.1 0.2 5.2 0.2 5.4* 0.2 5.4* φ 0.2 5.3 0.2

ICA Vel (cm.s-1)CTRL 41.1 1.5 40.8 1.2 39.7 1.7 39.6 1.6 38.3 2.2

**CO2 39 2.1 49.5*φ 2.8 49.7*φ 2.7 49.3*φ 2.5 36.9 1.4EX 38 1.6 47.5*φ 1.5 47.4*φ 2.3 49.8*φ 2.5 39.7 1.9

ICA shearCTRL 316 11.3 314 12.1 305 14.2 304 12.9 294 16.6

**CO2 309 15.5 372* 18.0 369*φ 17.4 362*φ 14.2 286 12.2EX 297 12.4 366* 14.6 354*φ 14.1 371*φ 18.0 298 17.0

MCAv (cm.s-1)CTRL 66 3 66 3 65 3 63 3 63 3

**CO2 64 6 83*φ 7 83*φ 7 82*φ 7 68 5EX 58 3 75*φ 4 75*φ 5 74*φ 5 60 3

CVC(ml.min-1/mmHg)

CTRL3.4 0.3 3.2 0.3 3.1 0.3 3.0 0.3 2.9 0.3

NSCO23.0 0.2 3.7 0.2 3.7 0.2 3.5 0.3 2.7 0.1

EX3.5 0.3 3.5 0.2 3.6 0.3 3.9 0.2 3.5 0.4

Time: * = different from BL; p < 0.05, COND: φ = different from CTRL; ѱ = different from CO2; p < 0.01, INT: ** = significant COND x TIME interaction; φ = different from CTRL; ѱ = different from CO2; p < 0.001

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Table 2. Posterior cerebral blood flow and cardiorespiratory measurements during Control (CTRL), carbon dioxide (CO2), and Exercise (EX)(n=8)

Time (min)Measure Condition BL 15 25 Post Int

Mean SD Mean SD Mean SD Mean SD

MAP (mmHg)

CTRL 92 3 93 3 90 4 93 4**CO2 94 3 102 5 106*φ 6 98 5

EX 87 4 102*φ 3 101*φ 3 88 4

HR (BPM)

CTRL 63 2 65 3 65 3 64 3**CO2 64 2 69 3 72*φ 3 65 3

EX ѱ 68 2 114*φѱ 2 118*φѱ 2 80*φѱ 3

PetO2(mmHg)

CTRL 97.5 2.4 104.7 5.3 100 1.4 99.3 1.3**CO2 φ 97.5 2.4 129.9*φ 0.8 130.3*φ 1.3 100.1 0.9

EX φ ѱ 95.9 1.7 94.1 φѱ 3.0 96.0ѱ 2.2 97.9 1.6

PetCO2(mmHg)

CTRL 40.2 2.3 40.4 4.5 40.2 2.2 41 0.9**CO2 φ 41.3 1.3 46.4*φ 0.8 46.1*φ 1 41 0.9

EXφ 41.4 1.1 45.7φ 1.9 44.5 1.5 40.1 1.1

QVA

(ml.min-1)

CTRL 113 20.6 111 21.6 103 19.7 103 17.9**CO2 100 13.1 141* 16.1 142* 20 95 12.9

EX 90 16.1 140* 26.2 143* 26.1 87 12.3

VA Diam(cm)

CTL 3.9 0.1 3.9 0.2 3.9 0.2 3.9 0.1CO2 3.9φ 0.2 4.2φ 0.2 4.2*φ 0.2 4.0φ 0.1 **EX 3.7φ ѱ 0.2 3.9*ѱ 0.2 4.1*ѱ 0.2 3.7φѱ 0.1

VA Vel(cm.s-1)

CTRL 27.7 3.3 27.3 3.3 25.5 2.9 24.4* 3**CO2 24.7 φ 1.9 31.6* φ 2.2 31.2* 2.2 23.4 2

EX 24.7 φ 2.0 31.1* φ 2.6 31.5* 2.5 24.1 1.7

VA shearCTRL 282 28 279 27 261 25 249* 26

**CO2 251 φ 14 305*φ 19 297*φ 16 237 17EX 268 16 322*φ 20 316*φ 20 261 16

PCAv(cm.s-1)

CTRL 49 3 47 3 47 3 46 3**CO2 φ 51 4 63*φ 5 63*φ 4 52 4

EX 48 4 58*φѱ 5 56*φѱ 4 45*ѱ 3

CVC(ml.min-1/mmHg)

CTRL 1.2 0.2 1.2 0.3 1.1 0.2 0.9 0.1NSCO2 1.1 0.1 1.4 0.2 1.4 0.2 1.0 0.1

EX 1.1 0.2 1.4 0.3 1.5 0.3 1.0 0.2Time: * = different from BL; p < 0.05, COND: φ = different from CTRL; ѱ = different from CO2; p < 0.01, INT: ** = significant COND x TIME interaction; φ = different from CTRL; ѱ = different from CO2; p < 0.001

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Figures

Figure 1. Middle (MCAv) and posterior (PCAv) cerebral blood flow velocity (Panels A &

C), internal carotid (ICA) and vertebral (VA) artery Shear (Panels B and D), pre

(BL) during (10,15, 25, 30 minutes, respectively) and POST (5 and 10 minutes

respectively) of the control (CTRL), carbon dioxide (CO2) and exercise (EX)

interventions. φ indicate differences between CO2 and CTRL (p<0.001); τ indicates

differences between EX and CTRL (p<0.001).

Figure 2. Changes in diameter in the internal carotid (ICA), and vertebral (VA) arteries

during (10, 15, 20, 25 and 30 mins, respectively) as well as POST (5 and 10 min,

respectively) control (CTRL) carbon dioxide (CO2) and exercise (EX)

interventions. Straight line connectors indicate significance between experimental

and control conditions (p<0.001).

Figure 3. Arterial blood pressure [MAP]) and end-tidal carbon dioxide [PETCO2] at baseline

(BL), during (10, 15, 20, 25, 30 mins) as well as post exercise during the control

(CTRL), carbon dioxide (CO2) and exercise (EX) conditions. φ indicate differences

between CO2 and CTRL (p<0.001); τ indicates differences between EX and CTRL

(p<0.001).

Figure 4. Relationship between changes in arterial shear stress and dilation of the internal

carotid artery during the carbon dioxide (CO2; r = 0.69) and exercise (EX; r = 0.58)

conditions. Control data is included in both the CO2 and EX figures (p<0.01).

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