Brachial Artery Flow Mediated Dilation is Reduced by Facial Cooling
Matthew S. Roy, Jonathan W. Edwards, William C. Rose,
Raju Y. Prasad, David G. Edwards
Department of Health, Nutrition, and Exercise Sciences,
University of Delaware, Newark, DE 19716
Running Head: Facial cooling reduces flow mediated dilation
Address for correspondence:
David G. Edwards, PhD
Department of Health, Nutrition, and Exercise Sciences
541 South College Avenue
142 HPL
Newark, DE 19716
Phone: (302) 831-3363
Fax: (302) 831-3693
Email: dge@udel.edu

Abstract
A reduction in brachial artery flow-mediated dilation (FMD) has been observed during sympathetic activation via cold pressor testing. In the current study, we tested the hypothesis that local cooling of the face would result in a reduction FMD. Nine healthy men (22

1 years) underwent baseline assessment of brachial artery FMD using high resolution ultrasound and Doppler blood velocity during a standard 5 minute occlusion protocol. Following a 20 minute period of rest, facial cooling was induced by placing a gel pack (0

C) on the forehead and FMD was reassessed. Brachial artery diameters and blood velocity were used to calculate the shear rate area under the curve (AUC) from cuff deflation to maximum diameter in order to quantify the stimulus for dilation.
Systolic and diastolic pressures were increased during facial cooing (p<0.05). Brachial artery FMD was reduced during facial compared to baseline (8.5

2.1 vs. 5.9

1.7%; p<0.05). Brachial artery diameter prior to cuff inflation was unaltered by facial cooling
(3.8

0.3 vs. 3.8

0.4mm). Shear rate AUC from cuff deflation to peak diameter did not differ between baseline and facial cooling conditions. Additionally, FMD normalized to shear rate AUC from cuff release to peak diameter was reduced by facial cooling
(p<0.05). Thus, facial cooling resulted in a reduction in brachial artery FMD that appears to be due to impaired dilation in response to a given shear stimulus.
Key Words: cold, endothelial-dependent dilation

Introduction
Sympathetic nervous system activation via the cold pressor test (feet in ice water) has been demonstrated to impair brachial artery flow-mediated dilation (FMD)(5, 12). Facial cooling results in a robust increase in muscle sympathetic nerve activity (MSNA) (8) and blood pressure (1, 8) similar to placing the hand or foot in ice water and thus may also impair brachial artery FMD.
Placing the hand in ice water likely results in excitation of cold and pain receptors (11) and these receptors are also likely involved in facial cooling induced increases in MSNA
(8). A greater increase in MSNA and blood pressure occurs in response to ice placed on the forehead compared to the hand (8) suggesting that facial cooling results in greater sympathetic activation. It has been postulated that these differences are due differing distribution of cold and pain receptors as well as mechanoreceptors on the forehead compared to the hand (8). Thus the face, and in particular the forehead, may be an important region in mediating the cardiovascular response to cold exposure (8).
Facial cooling may impair FMD similarly to placing the feet in ice water. When individuals are fully clothed, cold exposure is often limited to the face, which may be accompanied by pain depending on temperature and wind (6). Facial cooling may impair brachial artery FMD by reducing reactive hyperemia and subsequently the shear stimulus for dilation, or by reducing the endothelial responsiveness to the shear stimulus for dilation. The purpose of this study was to determine the effect of acute sympathetic nervous system activation via facial cooling on brachial artery FMD and the shear

stimulus. We hypothesized that facial cooling would reduce FMD but not the shear stimulus for dilation.
Methods
Subjects
Nine apparently healthy men, as assessed by medical history questionnaire, participated in this study (age,
22 ± 1 years; mass, 75 ± 2 kg; height, 177 ± 3 cm).
Subjects were non-smokers, and were asked to refrain from caffeine, alcohol, and exercise for at least 24 hours prior to testing and reported to the lab following at least a
4 hour fast. All procedures were reviewed and approved by the Institutional Review
Board and all subjects gave written informed consent.
Instrumentation
Heart Rate and Blood Pressure.
A single lead ECG was recorded throughout the experiment and used to calculate heart rate (HR) (Dinamap Dash 2000, GE Medical
Systems, Milwaukee, WI). The analog output connection on the Dinamap Dash 2000 was used to transmit heart rate data to a National Instruments PCI-6221 8-channel DAQ board (National Instruments Corporation, Austin, Texas). The DAQ board was used to synchronously record all analog data at a sample rate of 1 KHz. Automated oscillometric arterial blood pressure (BP) was also determined with the Dinamap Dash
2000.

High Resolution Ultrasound and Doppler Blood Velocity. Longitudinal images of the left brachial artery were obtained using a 10 MHz linear phased array ultrasound transducer
(SonoSite TITAN, Bothell, WA). Once the location and image of the brachial artery was determined the transducer was clamped into place (Flexbar, Islandia, NY) to prevent movement. A 4 MHz continuous wave Doppler probe (Neurovision 500P, Multigon
Industries, Inc., Yonkers, NY) was positioned distal to the ultrasound transducer and taped in place over the brachial artery. The probe insonation angle was 45° relative to the skin. The peak Doppler blood velocity signal was transmitted to the DAQ board at 1
KHz. Brachial artery images were transmitted from the SonoSite TITAN to a National
Instruments IMAQ PCI-1411 image acquisition board by way of an S-Video connection.
Images were captured by the IMAQ board and saved at a frequency of 20 frames per second. The 20 Hz image acquisition frame rate was determined by image buffering capability and Sonosite output frequency.
Experimental Protocol
Trials involved noninvasive assessment of brachial artery FMD (control) followed by repeat assessment of FMD during facial cooling. Subjects were studied supine with the left arm supported at heart level. A blood pressure cuff was placed on the proximal forearm just below the antecubital crease. Control measurements were performed following 15 minutes of supine rest. After baseline images and blood velocity were obtained the cuff was rapidly inflated (AG101 rapid cuff inflator, Hokanson, Bellevue,
WA) to 200 mmHg for 5 minutes. Brachial artery images were recorded during the last
15 seconds of occlusion and continued for 2 minutes following cuff release for

determination of peak diameter change and the shear rate stimulus. Twenty minutes of quiet rest separated the control trial from the facial cooling trial. Facial cooling was performed by applying a cold gel pack (0ºC) to the forehead. A damp paper towel formed a barrier between the gel pack and forehead. The gel pack was secured in place using an ACE bandage. Gel packs were kept in a thermostat controlled standard freezer. Facial cooling was performed for a total of 9 minutes. Following 2 minutes of facial cooling the occluding cuff was inflated. Two minutes was chosen because peak sympathetic nervous system activity during ice on forehead facial cooling has been observed at 90 seconds and peak systolic and diastolic pressure by 2 minutes (8). The cold stimulus was maintained for an additional 7 minutes: 5 minutes of cuff occlusion followed by 2 minutes post cuff release for determination of peak diameter. In order to maintain the cold stimulus for the entire 9 minute protocol the gel pack was replaced with a new gel pack at 4 minutes. Blood pressure was assessed before, at 2 minutes
(cuff inflation), and at 7 minutes (cuff deflation) during each trial.
Data Analysis
Brachial artery diameter was determined using custom designed automated edge detection software in National Instruments LabVIEW 8.0. The software allowed for identification of a vessel region of interest (ROI) and defining an intensity threshold which was used to identify the vessel walls within the selected ROI. Edge detection settings were saved and used for all ultrasound images from a given subject. Peak diameter was determined after applying a 3-second-wide median filter to each data point. FMD was expressed as percent change from baseline (15 second average).

Rep roducibility in our lab for this technique is 1.3±1.1% and 1.9±1.6% (coefficient of variation) for baseline and peak brachial diameters, respectively.
Doppler data was averaged and down sampled to 20 Hz to match the ultrasound frame rate and used to calculate flow, conductance, and shear rate as:
Flow (ml · min -1 ) =
 (1/2 · vessel diameter) 2 · (1/2 · V peak
)
Conductance (ml · min -1 · mmHg -1 ) = flow · mean arterial pressure -1
Shear rate (s -1 ) = 4 · V peak
· vessel diameter -1 where V peak
= centerline velocity.
It has been shown that the shear rate area under the curve (AUC) from cuff release to peak diameter best represents the reactive hyperemia shear stimulus for FMD (16).
Therefore, FMD was normalized to the shear rate AUC from cuff release to peak diameter.
Statistics
Unpaired t-tests were performed to compare differences between trials. A 2 X 3
(condition x time) analysis of variance with repeated measures was used to compare differences in blood pressure and heart rate. Bonferroni-adjusted post-hoc analyses were performed to determine differences within and between conditions. Statistical analysis was performed using the SPSS statistical program version 13 (SPSS, Inc.). An alpha level of 0.05 was required for statistical significance.

Results
Heart rate and blood pressure data are presented in Table 1. There were no differences in baseline measurements between facial cooling and control conditions.
Heart rate remained unchanged during control trials and facial cooling trials. Systolic and diastolic blood pressures did not change during control trials but were increased
(p<0.05) at 2 and 7 minutes during the facial cooling trial.
Flow, conductance, and shear rate data from cuff release to 1 minute post are shown in
Figure 1. Data were averaged over 3 seconds for display purposes. The flow AUC from cuff release to peak diameter or from cuff release to 1 minute post did not differ between control and facial cooling trials (Figure 1A). However, conductance AUC from cuff release to peak diameter trended toward being lower in the facial cooling trial
(p=0.06) and conductance AUC from cuff release to 1 minute post was lower in the facial cooling trial (p<0.05; Figure 1B). Shear rate AUC from cuff release to peak diameter or to 1 minute post did not differ between control and facial cooling trials
(Figure 1C).
Brachial artery diameter prior to cuff inflation was unaltered by facial cooling (3.8

0.3 vs. 3.8

0.4mm). Facial cooling resulted in a reduction in brachial artery FMD (8.5

2.1 vs. 5.9

1.7%; p<0.05) (Figure 2A). This difference persisted when FMD was corrected for the shear rate AUC from cuff release to peak diameter (Figure 2B) or shear rate AUC from cuff release to 1 minute (data not shown).

Discussion
The purpose of this study was to examine the effect of acute facial cooling on brachial artery FMD. The primary findings of this study were: 1) brachial artery FMD was reduced by acute facial cooling; and 2) the reduction in brachial artery FMD was not due to a change in the stimulus for FMD, as estimated by shear rate AUC. These findings suggest that the dilation in response to a given shear stimulus is impaired during acute facial cooling.
Facial cooling results in a robust increase in MSNA (8) and blood pressure (1, 8, 9).
Consistent with previous studies (1, 8, 9), facial cooling increased blood pressure in the present study. The increase in blood pressure occurred by 2 minutes and was maintained for 7 minutes following application of cold indicating elevated sympathetic activation was maintained through release of the occlusion cuff. Previous studies utilizing a cold pressor test (feet in ice water) to increase sympathetic nervous system activation have observed impaired brachial artery FMD (5, 12).
We utilized facial cooling instead of placing the foot in ice water. It has been proposed that the face, and in particular the forehead, may be an important region in mediating the cardiovascular response to cold exposure (8). Thus, our finding that facial cooling impaired brachial artery FMD may have important implications for understanding the increased occurrence of cardiovascular events in the cold. We used a standard 5 minute occlusion period to assess FMD similar to Lind et al. (12) but quantified the shear stimulus for dilation by calculating the shear rate AUC. Facial cooling resulted in vasoconstriction, as evidenced by a lower conductance AUC, however, shear rate AUC

did not differ between conditions. Our findings that the shear rate AUC did not differ between facial cooling and cold conditions and that FMD normalized to shear rate AUC was lower than control suggests that facial cooling results in an impaired dilation in response to a given shear stimulus. This is consistent with the results from Dyson et al.
(5) who demonstrated that FMD was impaired during cold pressor testing but did not alter the shear rate profile. Interestingly these authors demonstrated that sympathetic activation via cold pressor testing was the only stimulus resulting in impaired FMD, in contrast to lower body suction, mental stress, and muscle metaboreflex activation (5); however this is not a universal finding (7, 10, 12, 17). Our data in combination with previous studies (5, 12) suggests that local cooling of the skin impairs FMD, however the cold pressor effect on FMD may be limb or vessel specific as it has been shown to impair popliteal (15) but not superficial femoral artery FMD (19).
Brachial artery FMD has been shown to be closely related to coronary endothelial function (2) and is reduced in various conditions including aging, hypertension, and coronary artery disease (3, 14, 21). Normal coronary arteries dilate in response to cold stimulus using cold pressor testing (13), aiding coronary blood supply. However, atherosclerotic coronary arteries constrict in response to sympathetic activation induced by cold pressor testing (13). Thus, facial cooling in an older diseased population may increase the likelihood of myocardial ischemia as a result of impaired perfusion.
It should be noted that our study involved direct application of cold to the forehead. A comparison of full face cooling to forehead only cooling demonstrated similar increases in systolic pressure with both techniques (1), suggesting that forehead cooling alone as

used in the present study elicits an equivalent pressure response to full facial cooling.
Direct application of cold to the forehead may not be the best representation of environmental exposure to cold. Blowing cold air on the face results in increased blood pressure that varies depending on the temperature and wind speed utilized (4, 6, 18,
20). However, the increased pressure observed in the present study is within the range of observed values found in those studies. The effects of facial exposure to cold air on
FMD should be investigated in the future.
In summary, brachial artery FMD is reduced by acute facial cooling and the reduction in brachial artery FMD was not due to a change in the stimulus for FMD, as estimated by shear rate. These findings suggest that dilation in response to a given shear stimulus is impaired in the brachial artery during facial cooling.

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Figure Legends
Figure 1.
Flow (A), conductance (B), and shear rate (C) following cuff release. Data were averaged over 3 seconds for display purposes. Flow AUC from cuff release to peak diameter or from cuff release to 1 minute post did not differ between control and facial cooling trials. Conductance AUC from cuff release to peak diameter trended toward being lower in the facial cooling trial (p=0.06) and conductance AUC from cuff release to 1 minute post was lower in the facial cooling trial. Shear rate AUC from cuff release to peak diameter or to 1 minute post did not differ between control and facial cooling trials.
Figure 2.
Brachial artery FMD (A) and FMD normalized for shear rate AUC from cuff release to peak diameter (B). * p <0.05 versus control.

Table 1.
Blood Pressure and Heart Rate Data
Control trial Facial Cooling trial
Heart Rate, beats · min -1
Baseline
51 ± 3
2 min 7 min
52 ± 3 51 ± 3
Baseline
53 ± 2
115 ± 4 119 ± 5 117 ± 5
2 min
54 ± 4
7 min
53 ± 3
116 ± 4 138 ± 6* † 139 ± 6* †
Systolic Pressure, mmHg
Diastolic Pressure, mmHg 59 ± 2 58 ± 2 56 ± 2 59 ± 2 76 ± 3* † 75 ± 3* †
Values are means ±SE. Heart rate and blood pressure data were collected before facial cooling or control, following 2 minutes of facial cooling or control which corresponded with cuff inflation, and following 7 minutes of facial cooling or control which corresponded with cuff release.
* p < 0.05 vs. b aseline; † p < 0.05 vs. control.

Figure 1
A
B
200
150
100
50
0
0
Facial Cooling
Control
10 20 30
Time (s)
40 50 60
3
Facial Cooling
Control
2
1
C
0
0 10 20 30
Time (s)
40 50 60
750
Facial Cooling
Control
500
250
0
0 10 20 30
Time (s)
40 50 60

Figure 2
A
10.0
8.0
6.0
4.0
2.0
*
0.0
B
0.0010
Control Facial Cooling
0.0008
0.0006
0.0004
*
0.0002
0.0000
Control Facial Cooling