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ARTICLE Acute psychological and physical stress transiently enhances brachial artery flow-mediated dilation stimulated by exerciseinduced increases in shear stress Ingrid C. Szijgyarto, Veronica J. Poitras, Brendon J. Gurd, and Kyra E. Pyke
Abstract: Exercise elevates conduit artery shear stress and stimulates flow-mediated dilation (FMD). However, little is known regarding the impact of acute psychological and physical stress on this response. The purpose of this study was to examine the impact of the Trier Social Stress Test (TSST (speech and arithmetic tasks)) and a cold pressor test (CPT) with and without social evaluation (SE) on exercise-induced brachial artery FMD (EX-FMD). A total of 59 healthy male subjects were randomly assigned to 1 of 3 conditions: TSST, CPT, or CPT with SE. During 6 min of handgrip exercise, brachial artery EX-FMD was assessed before and 15 and 35 min poststress with echo and Doppler ultrasound. Shear stress was estimated as shear rate, calculated as brachial artery mean blood velocity/brachial artery diameter. Results are means ± SD. All conditions elicited significant physiological stress responses. Salivary cortisol increased from 4.6 ± 2.4 nmol/L to 10.0 ± 5.0 nmol/L (p < 0.001; condition effect: p = 0.292). Mean arterial pressure increased from 98.6 ± 12.1 mm Hg to 131.9 ± 18.7 mm Hg (p < 0.001; condition effect: p = 0.664). Exercise shear rate did not differ between conditions (p = 0.592), although it was modestly lower poststress (prestress: 72.3 ± 4.5 s−1; 15 min poststress: 70.8 ± 5.4 s−1; 35 min poststress: 70.6 ± 6.1 s−1; trial effect: p = 0.011). EX-FMD increased from prestress to 15 min poststress in all conditions (prestress: 6.2% ± 2.8%; 15 min poststress: 7.9% ± 3.2%; 35 min poststress: 6.6% ± 2.9%; trial effect: p < 0.001; condition effect: p = 0.611). In conclusion, all conditions elicited similar stress responses that transiently enhanced EX-FMD. This response may help to support muscle perfusion during stress. Key words: endothelial function, cortisol, conduit artery, FMD, HPA axis, mental stress, cold pressor test. Résumé : L’exercice physique augmente la contrainte de cisaillement des artères et stimule la dilatation dépendante du flux sanguin (« FMD »). Cependant, il y a peu d’études au sujet de l’impact du stress psychologique et physique sur cette réponse. Cette étude se propose d’examiner l’impact du test de stress social de Trier (« TSST ») et de l’épreuve au froid (« CPT ») et CPT avec et sans évaluation sociale (CPT avec SE) sur l’artère brachiale induit par l’exercice de la FMD (« EX-FMD »). On répartit aléatoirement 59 hommes en bonne santé dans l’une des trois conditions : TSST, CPT ou CPT avec SE. Durant un exercice de préhension manuelle d’une durée de 6 min, on évalue l’EX-FMD de la artère brachiale par échographie et écho-Doppler, avant, a` la 15e minute et a` la 35e minute suivant le stress. On estime la contrainte de cisaillement d’après le taux de cisaillement (« SR ») calculé comme suit : SR = vélocité moyenne du sang dans la BA/diamètre de la BA. Les résultats sont exprimés par la moyenne ± écart-type. Toutes les conditions suscitent un stress physiologique significatif. La concentration salivaire de cortisol augmente de 4,6 ± 2,4 nmol/L a` 10,0 ± 5,0 nmol/L (p < 0,001; l’effet de la condition présentant une p = 0,292). La pression artérielle moyenne augmente de 98,6 ± 12,1 mm Hg a` 131,9 ± 18,7 mm Hg (p < 0,001; l’effet de la condition présentant une p = 0,664). Le SR a` l’effort ne varie pas d’une condition a` l’autre (p = 0,592) même s’il est légèrement plus faible après le stress (préstress: 72,3 ± 4,5 s−1; 15 min poststress: 70,8 ± 5,4 s−1; 35 min poststress: 70,6 ± 6,1 s−1; l’effet de l’essai présentant une p = 0,011). L’EX-FMD augmente du moment précédant le stress a` la 15e minute poststress dans toutes les conditions (préstress 6,2 ± 2,8 %; 15 min poststress: 7,9 ± 3,2 %; 35 min poststress: 6,6 ± 2,9 %; l’effet de l’essai présentant une p < 0,001; l’effet de la condition présentant une p = 0,611). En conclusion, toutes les conditions suscitent des réponses similaires de stress qui améliorent temporairement l’EX-FMD. Ces réponses pourraient plaider en faveur de la perfusion musculaire durant un stress. Mots-clés : fonction endothéliale, cortisol, conduit artériel, FMD, axe HHS, stress mental, épreuve au froid.
Introduction The proper function of the endothelium lining the arteries is important for the maintenance of vascular health and regulation of vascular tone (Landmesser et al. 2004) . Conduit artery endothelial function can be assessed noninvasively by measuring endothelialdependent vasodilation in response to increases in shear stress that occur with increased blood flow (flow-mediated dilation; FMD) (Celermajer et al. 1992). Exercise induces a sustained intensity-dependent increase in conduit artery shear stress (Pyke and Jazuli 2011; Wray et al. 2011;
Pyke et al. 2008b; Green et al. 1996). It has been demonstrated that brachial and radial artery dilation during a short bout (6 min) of moderate intensity handgrip exercise is stimulated by shear stress and is not the result of a vasodilatory signal conducted upwards from the exercising muscle (Jazuli and Pyke 2011; Pyke et al. 2008b). Therefore, brachial artery diameter changes during exerciseinduced increases in shear stress under these conditions reflect FMD specifically (EX-FMD). Importantly, FMD in daily living occurs during exercise and may play an important role in perfusion (i.e., impaired EX-FMD may reduce perfusion) (Vita and Hamburg 2010;
Received 13 August 2013. Accepted 10 March 2014. I.C. Szijgyarto, V.J. Poitras, B.J. Gurd, and K.E. Pyke. School of Kinesiology and Health Studies, Queen’s University, Kingston, ON K7L 3N6, Canada. Corresponding author: Kyra E. Pyke (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 927–936 (2014) dx.doi.org/10.1139/apnm-2013-0384
Published at www.nrcresearchpress.com/apnm on 13 March 2014.
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Schrage et al. 2007; Duffy et al. 1999; Nabel et al. 1990). Given its functional relevance, it is important that the factors influencing EX-FMD are well understood. The nature of the shear stress stimulus is a critical determinant of the FMD response characteristics (Pyke and Tschakovsky 2005; Mullen et al. 2001). Most of what we know about human conduit artery FMD comes from examining responses to transient reactive hyperemia (RH) shear stress profiles created with the release of temporary limb occlusion (RH-FMD). Exercise produces a very different shear stress stimulus profile and the resulting FMD response may demonstrate distinct characteristics (e.g., unique transduction pathways and interaction with other vasoactive signals). In support of this, it has been shown that radial artery FMD in response to hand warming, which produces a modest sustained shear stress stimulus, may be mediated by distinct vasodilators vs. RH-FMD (Mullen et al. 2001). Indeed, although some factors appear to have a similar deleterious impact on both RH and EX-FMD (e.g., a long history of smoking and aging) (Grzelak et al. 2010; Gaenzer et al. 2001), a distinction is supported by observations that other vascular insults (e.g., a high fat meal) have a disparate influence (Padilla et al. 2006). Thus, the effect of a given stimulus on RH-FMD cannot be generalized to EX-FMD, and the influence of various acute and chronic stimuli on EX-FMD is understudied. The acute stress response involves activation of the hypothalamic– pituitary–adrenal (HPA) axis (responsible for regulation of the stress hormone cortisol) and the sympathetic nervous system. Many studies have noted that RH-FMD is impaired following acute mental (Broadley et al. 2005; Spieker et al. 2002; Ghiadoni et al. 2000) and physical (painful) stress (Lemke et al. 2011; Jambrik et al. 2005). The impact of stress on EX-FMD has been incompletely characterized, although the prospect of stress-induced impairment in EX-FMD has immediate functional relevance because it could decrease perfusion and exacerbate any existing potential for ischemia (Sheps et al. 2002; Gottdiener et al. 1994; Rozanski et al. 1988). A previous study by our group identified that an acute mental stress task did not impair brachial artery EX-FMD (Szijgyarto et al. 2013). However, no other stress stimuli were tested (e.g., physical stress), and cortisol concentration, which has been shown to play a role in impairing RH-FMD poststress (Broadley et al. 2005), was not increased by the stress task. Therefore, it remains unknown whether physical stress (painful stimuli) or mental stress responses that are accompanied by increased cortisol concentration impair EX-FMD. The purpose of the current study was to examine the impact of acute mental, physical, and combined stress tasks on EX-FMD. The selected tasks have been shown to stimulate robust activation of the HPA axis (Smeets et al. 2012; Schwabe et al. 2008; Dixon et al. 2004; al’Absi et al. 2002; Kirschbaum et al. 1993). We hypothesized that elevated cortisol poststress would be accompanied by impaired poststress EX-FMD.
Materals and methods Subjects A total of 59 healthy, pain-free, nonsmoking male subjects from the student population of Queen’s University in Kingston, Ontario, Canada, participated in this study. A medical screening form was used to exclude subjects with known symptoms or diagnosis of cardiovascular disease, circulatory disorders, ongoing chronic pain problems, and chronic or acute health problems that affect the neuroendocrine system. Subjects engaging in more than 5 h of physical activity per week or who had previous experience with ice baths were also excluded. The Health Sciences Human Research Ethics Board at Queen’s University approved the study protocol and all subjects signed a consent form that was approved by the same board. Subjects were instructed to abstain from alcohol, caffeine, and exercise for 12 h prior to the study and to abstain from eating for 6 h prior (Thijssen et al. 2011). Each
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subject visited the laboratory on 2 occasions. During the first occasion (the screening visit), subjects became familiar with the protocol and filled out the medical screening form and a physical activity recall questionnaire (Jacobs et al. 1993). During the second visit, subjects performed the study after being randomized to 1 of 3 conditions: the Trier Social Stress Test (TSST (mental stress)), the cold pressor test (CPT (physical stress)), or the CPT with social evaluation (SE (combined mental and physical stress)). All test sessions took place in a quiet temperature-controlled (20 °C) room between 1200 and 1800 hours. As shown in Fig. 1, the protocol in each condition required approximately 2 h and 10 min to complete. Participants were booked into time slots at either 1200 or 1500; the proportion of sessions at 1200 vs. 1500 were similar across conditions (1200 slot: TSST 53%, CPT 50%, CPT with SE 55%). Subject monitoring Heart rate (HR) was monitored continuously throughout the experiment with a 3-lead electrocardiogram (ECG) and mean arterial blood pressure (MAP) was measured continuously using finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, the Netherlands). BeatScope Easy software (BeatScope Easy version 01, Finapres Medical Systems) was used to estimate stroke volume and cardiac output (CO) from the blood pressure signal. Total peripheral resistance (TPR) was calculated as: TPR = MAP/CO. Brachial artery blood velocity and diameter Brachial artery blood velocity was measured continuously throughout the experiment with Doppler ultrasound operating at 4 MHz (Vivid i2 GE Medical Systems, Milwaukee, Wis., USA). The Doppler shift frequency spectrum was analyzed via a Multigon 500P TCD spectral analyzer (Multigon Industries, Yonkers, N.Y., USA) and mean velocity was determined as a weighted mean of the spectrum of Doppler shift frequencies. Brachial artery images were obtained using echo ultrasound functioning at 12 MHz in B-mode (Vivid i2 GE Medical Systems) as described previously (Pyke et al. 2008a). Images acquired on the Vivid i2 were recorded as audio video interleave (AVI) files on an independent computer (Camtasia Studio, TechSmith) via a video graphics array (VGA) to universal serial bus (USB) frame grabber (Epiphan Systems Inc.). Salivary cortisol sampling Saliva was obtained with Salivette saliva collectors (SARSTEDT) (Poll et al. 2007). Samples were centrifuged for 2 min at 4 °C at a rate of 2500 r/min and 1070g (IEC Centra-MP4R; International Equipment Company). Once spun, the samples rested for 20 min in the centrifuge after which they were stored in a –80 °C freezer for future analysis (Garde and Hansen 2005). Subjective stress and pain ratings Subjects were asked to rate their level of stress and pain on a scale from 0–10 (where zero was the least stress/pain possible and 10 was the worst stress/pain imaginable).
Experimental procedure All subjects arrived at the laboratory at either 1200 or 1500 h. Saliva samples and stress/pain ratings were taken at specific time points in the protocol as described in Fig. 1. Subjects were instrumented and then performed 2 maximal voluntary contractions (MVCs). Following this, subjects acclimatized to the laboratory environment for 1 h before commencing the study. During this period, a clear brachial artery ultrasound image and blood velocity signal were acquired and the position of the ultrasound probe was marked on the subjects’ arm. Subjects also performed a brief series of contractions to identify the contraction intensity (%MVC) that elicited the blood velocity required to achieve a shear rate of 75 s−1. The blood velocity required to achieve the target shear rate Published by NRC Research Press
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Fig. 1. Protocol timeline. Each subject was randomly assigned to one of the following conditions: (A) Trier Social Stress Test, (B) the cold pressor test (CPT), or (C) CPT with social evaluation. Black arrows indicate time points where salivary cortisol samples and subjective stress/ pain ratings were taken. EX-FMD, exercise-induced brachial artery flow-mediated dilation; MAT, mental arithmetic test.
was calculated for each subject as required velocity = 75 s−1 × brachial artery diameter. To perform the calculation, the brachial artery diameter for each subject was estimated by manual caliper placement on the ultrasound image before the trials started. The 75 s−1 target shear rate was selected because pilot work indicated that it results in a substantial EX-FMD response. At the end of the acclimatization period, the first EX-FMD trial (prestress FMD) was performed. Subjects were then given instructions regarding the stress task (i.e., the condition) to which they had been randomly assigned (TSST, CPT, or CPT with SE). An additional EX-FMD trial was performed 15 min (15 min poststress EX-FMD) and 35 min (35 min poststress EX-FMD) following the stress task. The timing of the stress task to FMD assessment impacts mechanisms of stress–endothelial function interaction (Poitras and Pyke 2013), and these times were chosen to correspond with the peak elevation in cortisol (Schommer et al. 2003). RH-FMD has also been shown to be impaired during this poststress period (Ghiadoni et al. 2000). Following the last EX-FMD trial, a fingerprick blood test was performed to measure blood lipids and glucose (Cholestech, LDX; Alere Inc.). EX-FMD test Baseline brachial artery blood velocity and diameter were recorded for 1 min, followed by 6 min of handgrip exercise, and 3 min of recovery. During the handgrip exercise, subjects were instructed to contract at the intensity that was chosen during the acclimatization period. Isometric handgrip force feedback was displayed continuously for the subjects on a computer dataacquisition system (Powerlab; ADInstruments). Subjects achieved the target force and duration for each contraction by displacing the force readout line to the desired level in time with a 2 s contraction and 3 s relaxation duty cycle metronome. Brachial artery blood velocity was monitored throughout the trial, and an experimenter coached the subjects through minor increases and decreases in force production to maintain the desired blood velocity target. This protocol is similar to previous reports (Jazuli and Pyke 2011; Pyke and Jazuli 2011).
Stress tasks TSST During the instructions for the TSST, a panel of 3 unfamiliar female peers entered the room to serve as an audience (the panel). The investigator then asked the subject to take on the role of a job applicant who had been invited for a personal interview with the company’s staff managers (i.e., the panel). Subjects were told that after a preparation period (10 min), they should deliver a 5 min speech designed to convince the panel of their high degree of suitability for a vacant position. Further, it was announced that a voice frequency analysis would be performed on their taperecorded speech and a video analysis of their performance would also be conducted. Following the speech, the panel then asked the subject to serially subtract the number 13 from 1022 as fast and as accurately as possible for 5 min (mental arithmetic test). In a standard manner, subjects were occasionally told they were incorrect even when they responded correctly (Fig. 1A presents the timeline of this protocol). At the end of the visit, the investigator debriefed the subject regarding the goal of the study and informed them that neither a voice frequency nor a video analysis would be performed (Kirschbaum et al. 1993). CPT Following a set of instructions and preparation period (10 min), subjects in the CPT condition were asked to place their right foot in an ice water slurry maintained at a temperature of 4 ± 0.2 °C for 2.5 min or until the task was intolerable (Fig. 1B). Following 2.5 min of foot immersion, subjects were instructed to remove their right foot while simultaneously placing their left foot in the same ice water slurry for another 2.5 min. CPT with SE Subjects in the CPT with SE condition performed the same protocol as described for the CPT with the addition of a female peer and a video recorder present in the room (Schwabe et al. 2008) Published by NRC Research Press
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(Fig. 1C). Subjects were told that the female peer was taking notes on their facial expressions and overall reaction to the task. Of the subjects that performed a CPT, 3 removed their feet from the bath prior to the 2.5 min time target (minimum single foot submersion time in these subjects was 30 s). These subjects had similar hemodynamic responses to the rest of the group and were included in the analysis. Data analysis Brachial artery blood velocity Blood velocity was analyzed offline in 3 s average time bins using the data-acquisition software program LabChart (AD Instruments) as described previously (Pyke and Jazuli 2011; Thijssen et al. 2011). Brachial artery diameter Blood vessel diameter was analyzed with an updated version of the automated edge-detection software package described in Woodman et al. (2001) (Encoder FMD & Bloodflow v3.0.3, Reed Electronics) as described previously (Szijgyarto et al. 2013). The experimenter was blinded to the EX-FMD trial (prestress, 15 min poststress, and 35 min poststress) while performing the analysis. The diameter data were compiled into 3 s time bins allowing alignment with the 3 s average velocity data. EX-FMD is reported as the percent change in diameter from baseline to the average diameter recorded during each minute of handgrip exercise (%EX-FMD). The absolute change in diameter is the average diameter during handgrip exercise minus the baseline diameter. Where only a single EX-FMD value is reported, it describes the response in the last minute of exercise. Shear rate was calculated using the 3 s average velocity and diameter data (shear rate = mean blood velocity/brachial artery diameter). The mean shear rate from the last 5 min of the handgrip exercise protocol (the steady state period) was reported as the shear rate stimulus during the handgrip exercise. Hemodynamic variables MAP, HR, and TPR were analyzed offline and compiled into 3 s average time bins. For the EX-FMD trials, a 1 min baseline was calculated. Average hemodynamic values include all subjects in the study (N = 59) with the exception of TPR (n = 55; TSST: n = 17, CPT: n = 19, CPT: n = 19) because the data files were lost. To assess hemodynamic reactivity (HR, MAP, and TPR) the peak (1 min average) response during the stress tasks is reported. Salivary cortisol Cortisol was measured using high sensitivity salivary cortisol enzyme immunoassay kits (cat. no. 1-3002 Salimetrics, State College, Pa., USA). Cortisol samples were thawed on the day of the assay and spun at 1500 r/min and 380g for 15 min. Saliva samples were assayed in duplicate according to the manufacturer’s instructions. Samples that had a >15% coefficient of variation were repeated on another plate, with the rerun value used to reject one of the original replicates (Harkness et al. 2011). The high control, measured at 1.0 g/dL, had an intra-assay coefficient of variation of 3.25% and an interassay coefficient of variation of 3.65%. The low control, measured at 0.10 g/dL, had an intra-assay coefficient of variation of 5.06% and an interassay coefficient of variation of 6.93%. Salivary cortisol agrees well with plasma cortisol concentrations (Dorn et al. 2007). Changes in cortisol with stress were calculated by subtracting the prestress sample (taken immediately after the prestress FMD test) from the peak cortisol response. Cortisol responders were identified as having an increase of at least 2 nmol/L (Schwabe et al. 2008). Blood lipids Total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and glucose were as-
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Table 1. Subject characteristics. Condition, mean±SD
Age (y) BMI (kg/m2) TC (mmol/L)a TRG (mmol/L)b HDL (mmol/L)c LDL (mmol/L)d Glucose (mmol/L)e
TSST (n = 19)
CPT (n = 20)
CPT+SE (n = 20)
p
19.2±1.5 23.2±2.8 4.04±0.61 0.98±0.45 1.38±0.37 2.3±0.59 4.69±0.45
19.8±2.5 24.3±2.9 4.27±0.68 0.90±0.30 1.31±0.37 2.6±0.67 4.92±0.46
21.3±2.7 24.8±3.6 4.24±0.68 1.06±0.47 1.39±0.40 2.4±0.66 4.63±4.8
0.020* 0.234 0.270 0.561 0.785 0.433 0.161
Note: BMI, body mass index; CPT, cold pressor test; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SE, social evaluation; TC, total cholesterol; TRG, triglyceride; TSST, Trier Social Stress Test. aTSST: n = 18; CPT: n = 16; CPT+SE: n = 17. bTSST: n = 14; CPT: n = 16; CPT+SE: n = 17. cTSST: n = 17; CPT: n = 17; CPT+SE: n = 19. dTSST: n = 13; CPT: n = 16; CPT+SE: n = 15. eTSST: n = 18; CPT: n = 17. *p < 0.05; subject age was significantly higher in the CPT+SE vs. the TSST condition.
sessed from capillary tube blood samples using a Cholestech LDX system (Alere Inc.). In some cases, concentrations were outside the measurement range set by the Cholestech LDX machine. For some samples, this may have been because of sample dilution with interstitial fluid due to excess finger pressure application when puncture site blood flow was low. The number of subjects included in the reported values for each parameter appears in the footnote of Table 1. Statistical analyses One-way ANOVA was used to detect a main effect of condition (TSST, CPT, CPT with SE; between subjects) for subject characteristics. Two-way ANOVAs were performed to examine the effects of condition (between subjects) and trial (prestress, 15 min poststress, 35 min poststress; within subjects) on diameter and baseline hemodynamic variables. Two-way ANOVAs were also performed to examine the effects of condition and time (prestress vs. peak) on cortisol, hemodynamic reactivity data, and subjective stress/pain ratings. A three-way ANOVA with the factors condition, trial, and time (each minute of the handgrip exercise test; within subjects) was used to assess EX-FMD over the course of the exercise bout. Significant main effects and interactions were examined using Bonferroni post hoc tests. To examine the impact of cortisol responses on EX-FMD, responses were also compared pooled across condition and split according to cortisol response (top quarter vs. bottom quarter). The relationship between change in EX-FMD vs. change in cortisol was examined with linear regression and outliers were identified via Cook’s distance test and removed. Significance was set at p < 0.05. All results are means ± SD.
Results Subject characteristics Average age, body mass index (BMI), and blood glucose and lipid profiles across conditions are shown in Table 1. Age was modestly higher in the CPT with SE condition compared to the TSST condition (p = 0.021). No effect of condition was detected for any other variable. Hemodynamic reactivity and subjective stress/pain ratings associated with the stress tasks The hemodynamic responses to each condition are shown in Table 2. There were significant increases in MAP, HR, and TPR (p < 0.001) and this was irrespective of condition for MAP (p = 0.664) and TPR (p = 0.841). However, peak HR was higher in the TSST condition compared to the CPT (p < 0.001) and the CPT with SE (p < 0.001) conditions. Subjective stress and pain ratings are shown in Table 2. Published by NRC Research Press
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Table 2. Stress response characteristics. Time
MAP (mm Hg) TSST CPT CPT+SE HR (bpm) TSST CPT CPT+SE TPR (mm Hg/(L·min)) TSST CPT CPT+SE Salivary cortisol (nmol/L) TSST CPT CPT+SE Salivary cortisol (responders only) (nmol/L) TSST CPT CPT+SE Stress rating (0–10 scale) TSST CPT CPT+SE Pain rating (0–10 scale) TSST CPT CPT+SE
p
Prestress
Peaka
98.8±11.2 97.7±11.8 99.4±13.6
136.3±21.7 129.5±14.5 130.2±19.5
63.4±7.1 62.0±11.9 62.8±6.0
95.1±15.3* 79.0±11.9 78.0±7.7
19.4±6.8 18.8±5.2 17.9±6.5
26.8±13.3 25.2±6.2 25.6±9.8
4.7±2.8 4.8±2.9 4.4±1.3
11.3±5.5 10.1±5.5 8.5±3.6
4.5±2.8 4.8±3.4 3.9±1.0
12.5±5.2 12.1±5.3 10.7±2.9
3.4±2.3 2.5±1.4 2.4±1.6
5.4±2.4 5.1±2.1 4.8±2.0
1.3±1.8 1.2±1.6 0.9±0.8
1.2±1.7* 6.6±1.4 6.4±1.7
Timeb
Conditionc
Time×condition
ARTICLE Acute psychological and physical stress transiently enhances brachial artery flow-mediated dilation stimulated by exerciseinduced increases in shear stress Ingrid C. Szijgyarto, Veronica J. Poitras, Brendon J. Gurd, and Kyra E. Pyke
Abstract: Exercise elevates conduit artery shear stress and stimulates flow-mediated dilation (FMD). However, little is known regarding the impact of acute psychological and physical stress on this response. The purpose of this study was to examine the impact of the Trier Social Stress Test (TSST (speech and arithmetic tasks)) and a cold pressor test (CPT) with and without social evaluation (SE) on exercise-induced brachial artery FMD (EX-FMD). A total of 59 healthy male subjects were randomly assigned to 1 of 3 conditions: TSST, CPT, or CPT with SE. During 6 min of handgrip exercise, brachial artery EX-FMD was assessed before and 15 and 35 min poststress with echo and Doppler ultrasound. Shear stress was estimated as shear rate, calculated as brachial artery mean blood velocity/brachial artery diameter. Results are means ± SD. All conditions elicited significant physiological stress responses. Salivary cortisol increased from 4.6 ± 2.4 nmol/L to 10.0 ± 5.0 nmol/L (p < 0.001; condition effect: p = 0.292). Mean arterial pressure increased from 98.6 ± 12.1 mm Hg to 131.9 ± 18.7 mm Hg (p < 0.001; condition effect: p = 0.664). Exercise shear rate did not differ between conditions (p = 0.592), although it was modestly lower poststress (prestress: 72.3 ± 4.5 s−1; 15 min poststress: 70.8 ± 5.4 s−1; 35 min poststress: 70.6 ± 6.1 s−1; trial effect: p = 0.011). EX-FMD increased from prestress to 15 min poststress in all conditions (prestress: 6.2% ± 2.8%; 15 min poststress: 7.9% ± 3.2%; 35 min poststress: 6.6% ± 2.9%; trial effect: p < 0.001; condition effect: p = 0.611). In conclusion, all conditions elicited similar stress responses that transiently enhanced EX-FMD. This response may help to support muscle perfusion during stress. Key words: endothelial function, cortisol, conduit artery, FMD, HPA axis, mental stress, cold pressor test. Résumé : L’exercice physique augmente la contrainte de cisaillement des artères et stimule la dilatation dépendante du flux sanguin (« FMD »). Cependant, il y a peu d’études au sujet de l’impact du stress psychologique et physique sur cette réponse. Cette étude se propose d’examiner l’impact du test de stress social de Trier (« TSST ») et de l’épreuve au froid (« CPT ») et CPT avec et sans évaluation sociale (CPT avec SE) sur l’artère brachiale induit par l’exercice de la FMD (« EX-FMD »). On répartit aléatoirement 59 hommes en bonne santé dans l’une des trois conditions : TSST, CPT ou CPT avec SE. Durant un exercice de préhension manuelle d’une durée de 6 min, on évalue l’EX-FMD de la artère brachiale par échographie et écho-Doppler, avant, a` la 15e minute et a` la 35e minute suivant le stress. On estime la contrainte de cisaillement d’après le taux de cisaillement (« SR ») calculé comme suit : SR = vélocité moyenne du sang dans la BA/diamètre de la BA. Les résultats sont exprimés par la moyenne ± écart-type. Toutes les conditions suscitent un stress physiologique significatif. La concentration salivaire de cortisol augmente de 4,6 ± 2,4 nmol/L a` 10,0 ± 5,0 nmol/L (p < 0,001; l’effet de la condition présentant une p = 0,292). La pression artérielle moyenne augmente de 98,6 ± 12,1 mm Hg a` 131,9 ± 18,7 mm Hg (p < 0,001; l’effet de la condition présentant une p = 0,664). Le SR a` l’effort ne varie pas d’une condition a` l’autre (p = 0,592) même s’il est légèrement plus faible après le stress (préstress: 72,3 ± 4,5 s−1; 15 min poststress: 70,8 ± 5,4 s−1; 35 min poststress: 70,6 ± 6,1 s−1; l’effet de l’essai présentant une p = 0,011). L’EX-FMD augmente du moment précédant le stress a` la 15e minute poststress dans toutes les conditions (préstress 6,2 ± 2,8 %; 15 min poststress: 7,9 ± 3,2 %; 35 min poststress: 6,6 ± 2,9 %; l’effet de l’essai présentant une p < 0,001; l’effet de la condition présentant une p = 0,611). En conclusion, toutes les conditions suscitent des réponses similaires de stress qui améliorent temporairement l’EX-FMD. Ces réponses pourraient plaider en faveur de la perfusion musculaire durant un stress. Mots-clés : fonction endothéliale, cortisol, conduit artériel, FMD, axe HHS, stress mental, épreuve au froid.
Introduction The proper function of the endothelium lining the arteries is important for the maintenance of vascular health and regulation of vascular tone (Landmesser et al. 2004) . Conduit artery endothelial function can be assessed noninvasively by measuring endothelialdependent vasodilation in response to increases in shear stress that occur with increased blood flow (flow-mediated dilation; FMD) (Celermajer et al. 1992). Exercise induces a sustained intensity-dependent increase in conduit artery shear stress (Pyke and Jazuli 2011; Wray et al. 2011;
Pyke et al. 2008b; Green et al. 1996). It has been demonstrated that brachial and radial artery dilation during a short bout (6 min) of moderate intensity handgrip exercise is stimulated by shear stress and is not the result of a vasodilatory signal conducted upwards from the exercising muscle (Jazuli and Pyke 2011; Pyke et al. 2008b). Therefore, brachial artery diameter changes during exerciseinduced increases in shear stress under these conditions reflect FMD specifically (EX-FMD). Importantly, FMD in daily living occurs during exercise and may play an important role in perfusion (i.e., impaired EX-FMD may reduce perfusion) (Vita and Hamburg 2010;
Received 13 August 2013. Accepted 10 March 2014. I.C. Szijgyarto, V.J. Poitras, B.J. Gurd, and K.E. Pyke. School of Kinesiology and Health Studies, Queen’s University, Kingston, ON K7L 3N6, Canada. Corresponding author: Kyra E. Pyke (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 927–936 (2014) dx.doi.org/10.1139/apnm-2013-0384
Published at www.nrcresearchpress.com/apnm on 13 March 2014.
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Schrage et al. 2007; Duffy et al. 1999; Nabel et al. 1990). Given its functional relevance, it is important that the factors influencing EX-FMD are well understood. The nature of the shear stress stimulus is a critical determinant of the FMD response characteristics (Pyke and Tschakovsky 2005; Mullen et al. 2001). Most of what we know about human conduit artery FMD comes from examining responses to transient reactive hyperemia (RH) shear stress profiles created with the release of temporary limb occlusion (RH-FMD). Exercise produces a very different shear stress stimulus profile and the resulting FMD response may demonstrate distinct characteristics (e.g., unique transduction pathways and interaction with other vasoactive signals). In support of this, it has been shown that radial artery FMD in response to hand warming, which produces a modest sustained shear stress stimulus, may be mediated by distinct vasodilators vs. RH-FMD (Mullen et al. 2001). Indeed, although some factors appear to have a similar deleterious impact on both RH and EX-FMD (e.g., a long history of smoking and aging) (Grzelak et al. 2010; Gaenzer et al. 2001), a distinction is supported by observations that other vascular insults (e.g., a high fat meal) have a disparate influence (Padilla et al. 2006). Thus, the effect of a given stimulus on RH-FMD cannot be generalized to EX-FMD, and the influence of various acute and chronic stimuli on EX-FMD is understudied. The acute stress response involves activation of the hypothalamic– pituitary–adrenal (HPA) axis (responsible for regulation of the stress hormone cortisol) and the sympathetic nervous system. Many studies have noted that RH-FMD is impaired following acute mental (Broadley et al. 2005; Spieker et al. 2002; Ghiadoni et al. 2000) and physical (painful) stress (Lemke et al. 2011; Jambrik et al. 2005). The impact of stress on EX-FMD has been incompletely characterized, although the prospect of stress-induced impairment in EX-FMD has immediate functional relevance because it could decrease perfusion and exacerbate any existing potential for ischemia (Sheps et al. 2002; Gottdiener et al. 1994; Rozanski et al. 1988). A previous study by our group identified that an acute mental stress task did not impair brachial artery EX-FMD (Szijgyarto et al. 2013). However, no other stress stimuli were tested (e.g., physical stress), and cortisol concentration, which has been shown to play a role in impairing RH-FMD poststress (Broadley et al. 2005), was not increased by the stress task. Therefore, it remains unknown whether physical stress (painful stimuli) or mental stress responses that are accompanied by increased cortisol concentration impair EX-FMD. The purpose of the current study was to examine the impact of acute mental, physical, and combined stress tasks on EX-FMD. The selected tasks have been shown to stimulate robust activation of the HPA axis (Smeets et al. 2012; Schwabe et al. 2008; Dixon et al. 2004; al’Absi et al. 2002; Kirschbaum et al. 1993). We hypothesized that elevated cortisol poststress would be accompanied by impaired poststress EX-FMD.
Materals and methods Subjects A total of 59 healthy, pain-free, nonsmoking male subjects from the student population of Queen’s University in Kingston, Ontario, Canada, participated in this study. A medical screening form was used to exclude subjects with known symptoms or diagnosis of cardiovascular disease, circulatory disorders, ongoing chronic pain problems, and chronic or acute health problems that affect the neuroendocrine system. Subjects engaging in more than 5 h of physical activity per week or who had previous experience with ice baths were also excluded. The Health Sciences Human Research Ethics Board at Queen’s University approved the study protocol and all subjects signed a consent form that was approved by the same board. Subjects were instructed to abstain from alcohol, caffeine, and exercise for 12 h prior to the study and to abstain from eating for 6 h prior (Thijssen et al. 2011). Each
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subject visited the laboratory on 2 occasions. During the first occasion (the screening visit), subjects became familiar with the protocol and filled out the medical screening form and a physical activity recall questionnaire (Jacobs et al. 1993). During the second visit, subjects performed the study after being randomized to 1 of 3 conditions: the Trier Social Stress Test (TSST (mental stress)), the cold pressor test (CPT (physical stress)), or the CPT with social evaluation (SE (combined mental and physical stress)). All test sessions took place in a quiet temperature-controlled (20 °C) room between 1200 and 1800 hours. As shown in Fig. 1, the protocol in each condition required approximately 2 h and 10 min to complete. Participants were booked into time slots at either 1200 or 1500; the proportion of sessions at 1200 vs. 1500 were similar across conditions (1200 slot: TSST 53%, CPT 50%, CPT with SE 55%). Subject monitoring Heart rate (HR) was monitored continuously throughout the experiment with a 3-lead electrocardiogram (ECG) and mean arterial blood pressure (MAP) was measured continuously using finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, the Netherlands). BeatScope Easy software (BeatScope Easy version 01, Finapres Medical Systems) was used to estimate stroke volume and cardiac output (CO) from the blood pressure signal. Total peripheral resistance (TPR) was calculated as: TPR = MAP/CO. Brachial artery blood velocity and diameter Brachial artery blood velocity was measured continuously throughout the experiment with Doppler ultrasound operating at 4 MHz (Vivid i2 GE Medical Systems, Milwaukee, Wis., USA). The Doppler shift frequency spectrum was analyzed via a Multigon 500P TCD spectral analyzer (Multigon Industries, Yonkers, N.Y., USA) and mean velocity was determined as a weighted mean of the spectrum of Doppler shift frequencies. Brachial artery images were obtained using echo ultrasound functioning at 12 MHz in B-mode (Vivid i2 GE Medical Systems) as described previously (Pyke et al. 2008a). Images acquired on the Vivid i2 were recorded as audio video interleave (AVI) files on an independent computer (Camtasia Studio, TechSmith) via a video graphics array (VGA) to universal serial bus (USB) frame grabber (Epiphan Systems Inc.). Salivary cortisol sampling Saliva was obtained with Salivette saliva collectors (SARSTEDT) (Poll et al. 2007). Samples were centrifuged for 2 min at 4 °C at a rate of 2500 r/min and 1070g (IEC Centra-MP4R; International Equipment Company). Once spun, the samples rested for 20 min in the centrifuge after which they were stored in a –80 °C freezer for future analysis (Garde and Hansen 2005). Subjective stress and pain ratings Subjects were asked to rate their level of stress and pain on a scale from 0–10 (where zero was the least stress/pain possible and 10 was the worst stress/pain imaginable).
Experimental procedure All subjects arrived at the laboratory at either 1200 or 1500 h. Saliva samples and stress/pain ratings were taken at specific time points in the protocol as described in Fig. 1. Subjects were instrumented and then performed 2 maximal voluntary contractions (MVCs). Following this, subjects acclimatized to the laboratory environment for 1 h before commencing the study. During this period, a clear brachial artery ultrasound image and blood velocity signal were acquired and the position of the ultrasound probe was marked on the subjects’ arm. Subjects also performed a brief series of contractions to identify the contraction intensity (%MVC) that elicited the blood velocity required to achieve a shear rate of 75 s−1. The blood velocity required to achieve the target shear rate Published by NRC Research Press
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Fig. 1. Protocol timeline. Each subject was randomly assigned to one of the following conditions: (A) Trier Social Stress Test, (B) the cold pressor test (CPT), or (C) CPT with social evaluation. Black arrows indicate time points where salivary cortisol samples and subjective stress/ pain ratings were taken. EX-FMD, exercise-induced brachial artery flow-mediated dilation; MAT, mental arithmetic test.
was calculated for each subject as required velocity = 75 s−1 × brachial artery diameter. To perform the calculation, the brachial artery diameter for each subject was estimated by manual caliper placement on the ultrasound image before the trials started. The 75 s−1 target shear rate was selected because pilot work indicated that it results in a substantial EX-FMD response. At the end of the acclimatization period, the first EX-FMD trial (prestress FMD) was performed. Subjects were then given instructions regarding the stress task (i.e., the condition) to which they had been randomly assigned (TSST, CPT, or CPT with SE). An additional EX-FMD trial was performed 15 min (15 min poststress EX-FMD) and 35 min (35 min poststress EX-FMD) following the stress task. The timing of the stress task to FMD assessment impacts mechanisms of stress–endothelial function interaction (Poitras and Pyke 2013), and these times were chosen to correspond with the peak elevation in cortisol (Schommer et al. 2003). RH-FMD has also been shown to be impaired during this poststress period (Ghiadoni et al. 2000). Following the last EX-FMD trial, a fingerprick blood test was performed to measure blood lipids and glucose (Cholestech, LDX; Alere Inc.). EX-FMD test Baseline brachial artery blood velocity and diameter were recorded for 1 min, followed by 6 min of handgrip exercise, and 3 min of recovery. During the handgrip exercise, subjects were instructed to contract at the intensity that was chosen during the acclimatization period. Isometric handgrip force feedback was displayed continuously for the subjects on a computer dataacquisition system (Powerlab; ADInstruments). Subjects achieved the target force and duration for each contraction by displacing the force readout line to the desired level in time with a 2 s contraction and 3 s relaxation duty cycle metronome. Brachial artery blood velocity was monitored throughout the trial, and an experimenter coached the subjects through minor increases and decreases in force production to maintain the desired blood velocity target. This protocol is similar to previous reports (Jazuli and Pyke 2011; Pyke and Jazuli 2011).
Stress tasks TSST During the instructions for the TSST, a panel of 3 unfamiliar female peers entered the room to serve as an audience (the panel). The investigator then asked the subject to take on the role of a job applicant who had been invited for a personal interview with the company’s staff managers (i.e., the panel). Subjects were told that after a preparation period (10 min), they should deliver a 5 min speech designed to convince the panel of their high degree of suitability for a vacant position. Further, it was announced that a voice frequency analysis would be performed on their taperecorded speech and a video analysis of their performance would also be conducted. Following the speech, the panel then asked the subject to serially subtract the number 13 from 1022 as fast and as accurately as possible for 5 min (mental arithmetic test). In a standard manner, subjects were occasionally told they were incorrect even when they responded correctly (Fig. 1A presents the timeline of this protocol). At the end of the visit, the investigator debriefed the subject regarding the goal of the study and informed them that neither a voice frequency nor a video analysis would be performed (Kirschbaum et al. 1993). CPT Following a set of instructions and preparation period (10 min), subjects in the CPT condition were asked to place their right foot in an ice water slurry maintained at a temperature of 4 ± 0.2 °C for 2.5 min or until the task was intolerable (Fig. 1B). Following 2.5 min of foot immersion, subjects were instructed to remove their right foot while simultaneously placing their left foot in the same ice water slurry for another 2.5 min. CPT with SE Subjects in the CPT with SE condition performed the same protocol as described for the CPT with the addition of a female peer and a video recorder present in the room (Schwabe et al. 2008) Published by NRC Research Press
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(Fig. 1C). Subjects were told that the female peer was taking notes on their facial expressions and overall reaction to the task. Of the subjects that performed a CPT, 3 removed their feet from the bath prior to the 2.5 min time target (minimum single foot submersion time in these subjects was 30 s). These subjects had similar hemodynamic responses to the rest of the group and were included in the analysis. Data analysis Brachial artery blood velocity Blood velocity was analyzed offline in 3 s average time bins using the data-acquisition software program LabChart (AD Instruments) as described previously (Pyke and Jazuli 2011; Thijssen et al. 2011). Brachial artery diameter Blood vessel diameter was analyzed with an updated version of the automated edge-detection software package described in Woodman et al. (2001) (Encoder FMD & Bloodflow v3.0.3, Reed Electronics) as described previously (Szijgyarto et al. 2013). The experimenter was blinded to the EX-FMD trial (prestress, 15 min poststress, and 35 min poststress) while performing the analysis. The diameter data were compiled into 3 s time bins allowing alignment with the 3 s average velocity data. EX-FMD is reported as the percent change in diameter from baseline to the average diameter recorded during each minute of handgrip exercise (%EX-FMD). The absolute change in diameter is the average diameter during handgrip exercise minus the baseline diameter. Where only a single EX-FMD value is reported, it describes the response in the last minute of exercise. Shear rate was calculated using the 3 s average velocity and diameter data (shear rate = mean blood velocity/brachial artery diameter). The mean shear rate from the last 5 min of the handgrip exercise protocol (the steady state period) was reported as the shear rate stimulus during the handgrip exercise. Hemodynamic variables MAP, HR, and TPR were analyzed offline and compiled into 3 s average time bins. For the EX-FMD trials, a 1 min baseline was calculated. Average hemodynamic values include all subjects in the study (N = 59) with the exception of TPR (n = 55; TSST: n = 17, CPT: n = 19, CPT: n = 19) because the data files were lost. To assess hemodynamic reactivity (HR, MAP, and TPR) the peak (1 min average) response during the stress tasks is reported. Salivary cortisol Cortisol was measured using high sensitivity salivary cortisol enzyme immunoassay kits (cat. no. 1-3002 Salimetrics, State College, Pa., USA). Cortisol samples were thawed on the day of the assay and spun at 1500 r/min and 380g for 15 min. Saliva samples were assayed in duplicate according to the manufacturer’s instructions. Samples that had a >15% coefficient of variation were repeated on another plate, with the rerun value used to reject one of the original replicates (Harkness et al. 2011). The high control, measured at 1.0 g/dL, had an intra-assay coefficient of variation of 3.25% and an interassay coefficient of variation of 3.65%. The low control, measured at 0.10 g/dL, had an intra-assay coefficient of variation of 5.06% and an interassay coefficient of variation of 6.93%. Salivary cortisol agrees well with plasma cortisol concentrations (Dorn et al. 2007). Changes in cortisol with stress were calculated by subtracting the prestress sample (taken immediately after the prestress FMD test) from the peak cortisol response. Cortisol responders were identified as having an increase of at least 2 nmol/L (Schwabe et al. 2008). Blood lipids Total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and glucose were as-
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Table 1. Subject characteristics. Condition, mean±SD
Age (y) BMI (kg/m2) TC (mmol/L)a TRG (mmol/L)b HDL (mmol/L)c LDL (mmol/L)d Glucose (mmol/L)e
TSST (n = 19)
CPT (n = 20)
CPT+SE (n = 20)
p
19.2±1.5 23.2±2.8 4.04±0.61 0.98±0.45 1.38±0.37 2.3±0.59 4.69±0.45
19.8±2.5 24.3±2.9 4.27±0.68 0.90±0.30 1.31±0.37 2.6±0.67 4.92±0.46
21.3±2.7 24.8±3.6 4.24±0.68 1.06±0.47 1.39±0.40 2.4±0.66 4.63±4.8
0.020* 0.234 0.270 0.561 0.785 0.433 0.161
Note: BMI, body mass index; CPT, cold pressor test; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SE, social evaluation; TC, total cholesterol; TRG, triglyceride; TSST, Trier Social Stress Test. aTSST: n = 18; CPT: n = 16; CPT+SE: n = 17. bTSST: n = 14; CPT: n = 16; CPT+SE: n = 17. cTSST: n = 17; CPT: n = 17; CPT+SE: n = 19. dTSST: n = 13; CPT: n = 16; CPT+SE: n = 15. eTSST: n = 18; CPT: n = 17. *p < 0.05; subject age was significantly higher in the CPT+SE vs. the TSST condition.
sessed from capillary tube blood samples using a Cholestech LDX system (Alere Inc.). In some cases, concentrations were outside the measurement range set by the Cholestech LDX machine. For some samples, this may have been because of sample dilution with interstitial fluid due to excess finger pressure application when puncture site blood flow was low. The number of subjects included in the reported values for each parameter appears in the footnote of Table 1. Statistical analyses One-way ANOVA was used to detect a main effect of condition (TSST, CPT, CPT with SE; between subjects) for subject characteristics. Two-way ANOVAs were performed to examine the effects of condition (between subjects) and trial (prestress, 15 min poststress, 35 min poststress; within subjects) on diameter and baseline hemodynamic variables. Two-way ANOVAs were also performed to examine the effects of condition and time (prestress vs. peak) on cortisol, hemodynamic reactivity data, and subjective stress/pain ratings. A three-way ANOVA with the factors condition, trial, and time (each minute of the handgrip exercise test; within subjects) was used to assess EX-FMD over the course of the exercise bout. Significant main effects and interactions were examined using Bonferroni post hoc tests. To examine the impact of cortisol responses on EX-FMD, responses were also compared pooled across condition and split according to cortisol response (top quarter vs. bottom quarter). The relationship between change in EX-FMD vs. change in cortisol was examined with linear regression and outliers were identified via Cook’s distance test and removed. Significance was set at p < 0.05. All results are means ± SD.
Results Subject characteristics Average age, body mass index (BMI), and blood glucose and lipid profiles across conditions are shown in Table 1. Age was modestly higher in the CPT with SE condition compared to the TSST condition (p = 0.021). No effect of condition was detected for any other variable. Hemodynamic reactivity and subjective stress/pain ratings associated with the stress tasks The hemodynamic responses to each condition are shown in Table 2. There were significant increases in MAP, HR, and TPR (p < 0.001) and this was irrespective of condition for MAP (p = 0.664) and TPR (p = 0.841). However, peak HR was higher in the TSST condition compared to the CPT (p < 0.001) and the CPT with SE (p < 0.001) conditions. Subjective stress and pain ratings are shown in Table 2. Published by NRC Research Press
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Table 2. Stress response characteristics. Time
MAP (mm Hg) TSST CPT CPT+SE HR (bpm) TSST CPT CPT+SE TPR (mm Hg/(L·min)) TSST CPT CPT+SE Salivary cortisol (nmol/L) TSST CPT CPT+SE Salivary cortisol (responders only) (nmol/L) TSST CPT CPT+SE Stress rating (0–10 scale) TSST CPT CPT+SE Pain rating (0–10 scale) TSST CPT CPT+SE
p
Prestress
Peaka
98.8±11.2 97.7±11.8 99.4±13.6
136.3±21.7 129.5±14.5 130.2±19.5
63.4±7.1 62.0±11.9 62.8±6.0
95.1±15.3* 79.0±11.9 78.0±7.7
19.4±6.8 18.8±5.2 17.9±6.5
26.8±13.3 25.2±6.2 25.6±9.8
4.7±2.8 4.8±2.9 4.4±1.3
11.3±5.5 10.1±5.5 8.5±3.6
4.5±2.8 4.8±3.4 3.9±1.0
12.5±5.2 12.1±5.3 10.7±2.9
3.4±2.3 2.5±1.4 2.4±1.6
5.4±2.4 5.1±2.1 4.8±2.0
1.3±1.8 1.2±1.6 0.9±0.8
1.2±1.7* 6.6±1.4 6.4±1.7
Timeb
Conditionc
Time×condition
