The impact of a cold pressor test on brachial artery handgrip exercise-induced flow-mediated dilation

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1 586473VMJ / X Vsculr MedicineStuckless nd Pyke reserch-rticle2015 Originl Article The impct of cold pressor test on brchil rtery hndgrip exercise-induced flow-medited diltion Vsculr Medicine 2015, Vol. 20(5) The Author(s) 2015 Reprints nd permissions: sgepub.co.uk/journlspermissions.nv DOI: / X vmj.sgepub.com Troy JR Stuckless nd Kyr E Pyke Abstrct It is unknown how endothelil-dependent flow-medited diltion (FMD) stimulted by sustined, exercise-induced increse in sher stress (EX-FMD) is ffected by simultneous sympthoexcittory pinful stimulus. The purpose of this study ws to exmine the impct of cold pressor test (CPT) on brchil rtery EX-FMD elicited by hndgrip exerciseinduced increse in sher stress. Prticipnts were helthy mles (ge 21±2 yers) (n=28; 16 Experimentl group, 12 Control). Brchil rtery dimeter nd blood velocity were mesured using echo nd Doppler ultrsound, respectively. Sher stress ws estimted by sher rte (sher rte = blood velocity / dimeter) nd trgeted to rech 75 s 1 in ech of two EX-FMD trils in ll subjects. In the Experimentl group, the second EX-FMD tril ws ccompnied by simultneous foot immersion in ice wter (simultneous CPT). The sher rte stimulus did not differ between groups (p=0.823) or trils (p=0.726) (group tril interction: p=0.646) (verge exercise sher rte (men ± SD): 67.6±6.2 s 1 ). The CPT (experienced during EX-FMD tril 2 in the Experimentl group) incresed men rteril pressure (p<0.001) nd hert rte (p=0.002) reltive to the Control group. %EX-FMD ws not different between groups (p=0.508) or trils (p=0.592) (group tril interction: p=0.879) (EX-FMD: Experimentl group tril 1: 5.4±3.4%, tril 2: 5.6±2.6%; Control group tril 1: 6.0±3.7%, tril 2: 6.4±2.2%). In conclusion, the CPT did not impct concurrent EX-FMD, nd this indictes tht n cute pinful stimulus does not interfere with conduit rtery FMD responses during exercise in young helthy men. Keywords Blood vessels, brchil rtery, endothelil function, FMD, sher stress, sympthetic ctivtion, vsculr endothelium, vsodiltion Introduction The proper function of the endothelil cells lining the rteries is criticl for mintining vsculr helth. 1 In ddition to exerting n ntitherogenic, vsoprotective influence, helthy endothelil cells secrete vsodiltors nd influence perfusion. 2,3 A key stimulus for endothelil vsodiltor relese is blood flow-ssocited sher stress. Indeed, endothelil function is typiclly ssessed in humns by quntifying the mgnitude of diltion in response to n experimenter-imposed increse in sher stress (flowmedited vsodiltion, FMD). The stndrd non-invsive FMD ssessment protocol cretes trnsient sher stress stimulus vi the relese of temporry limb occlusion (rective hyperemi, RH). 4,5 Acute stimuli (e.g. single mel or stressful event) cn trnsiently impir RH-FMD in helthy individuls 6,7 nd this my hve importnt implictions if it occurs frequently nd endothelilly medited vsoprotection is similrly ttenuted. The nture of the sher stress stimulus profile is key determinnt of the FMD response chrcteristics. 8,9 FMD stimulted by RH versus more prolonged increses in sher stress my be mechnisticlly distinct. In contrst with the brief RH stimulus profile, exercise induces sustined, intensity-dependent increse in conduit rtery sher stress Importntly, FMD in dily living occurs during exercise (EX-FMD) nd my ply n importnt role in muscle perfusion (i.e. perfusion my be compromised if EX-FMD is impired). 2,13,14 Existing evidence indictes tht RH-FMD nd EX-FMD my hve differing vulnerbilities to impirment, lthough similr vulnerbility hs lso been found. 19,20 As whole, findings suggest tht the impct of stimulus or condition on RH-FMD cnnot be School of Kinesiology nd Helth Studies, Queen s University, Kingston, Ontrio, Cnd Corresponding uthor: Kyr E Pyke, Crdiovsculr Stress Response Lbortory, School of Kinesiology nd Helth Studies, Queen s University, Kingston, ON K7L 3N6, Cnd. Emil: pykek@queensu.c

2 410 Vsculr Medicine 20(5) Tble 1. Subject chrcteristics. Prmeter Experimentl group Control group p-vlue Age (yers) 20 ± 2 21 ± Weight (kg) 76 ± 9 77 ± Height (cm) 179 ± ± TRG (mg/dl) 70 ± ± HDL (mg/dl) 49 ± ± LDL (mg/dl) 98 ± ± GLU (mg/dl) 81 ± 6 81 ± Vlues re ± stndrd devition. TRG, triglycerides; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; GLU, glucose. n=8, Experimentl group; n=11, Control group. generlized to EX-FMD. Although connections between EX-FMD nd future crdiovsculr disese hve not been estblished, due to its physiologicl relevnce nd potentil functionl involvement in perfusion during exercise, it is importnt tht the fctors influencing EX-FMD re fully understood. Understnding the interction of multiple vsoctive stimuli is key to understnding in vivo vsculr function. Submersion of limb in n ice-wter slurry (cold pressor test, CPT) cutely increses sympthetic nervous ctivity, is perceived s pinful nd results in robust increse in men rteril pressure. 21,22 Superimposition of CPT with RH hs consistently been shown to impir brchil rtery RH-FMD; 23,24 however, the impct of CPT on FMD performed in conjunction with sustined increse in sher stress is unknown. The purpose of this study ws to determine whether brchil rtery EX-FMD is impired when CPT is performed during the period of exercise-induced sher stress elevtion. We hypothesized tht the CPT would result in n ttenuted EX-FMD response. Methods Prticipnts Twenty-eight young, helthy, mle non-smokers from the Queen s University (Kingston, Ontrio) community prticipted in the study. Subject chrcteristics re summrized in Tble 1. Sixteen prticipnts were recruited for the Experimentl group nd 12 prticipnts were recruited for the Control group. The study protocol ws pproved by the Helth Sciences Reserch Ethics Bord t Queen s University nd ll subjects signed consent form pproved by the sme bord. A medicl screening questionnire ws used to exclude subjects with known symptoms of or dignosis of crdiovsculr disese. All subjects fsted for 4 hours nd bstined from cffeine, lcohol nd exercise for 12 hours prior to the study. Following fmiliriztion visit, during which informed consent ws provided, subjects visited the lbortory on one occsion in the fternoon for prticiption in the experiment. All experiments were performed in quiet nd temperture-controlled room (~20 23 C). Subject monitoring Hert rte ws mesured using three-led electrocrdiogrm. Blood pressure ws mesured continuously with finger photoplethysmogrphy (Finometer; Finpres Medicl Systems, Amsterdm, The Netherlnds). BetScope Esy softwre v.01 (Finpres Medicl Systems) ws used to record n estimte of stroke volume derived from the finger blood pressure wve. Crdic output ws clculted s: crdic output = hert rte * stroke volume. Totl peripherl resistnce ws clculted s: totl peripherl resistnce = men rteril pressure / crdic output. Due to technicl difficulties we were unble to obtin BetScope recordings in four subjects; therefore, for totl peripherl resistnce, n=13 in the Experimentl group nd n=11 in the Control group. Brchil rtery blood velocity nd dimeter Blood velocity in the brchil rtery ws mesured continuously using Doppler ultrsound operting t 4 MHz (Vivid i2; GE Medicl Systems, London, ON, Cnd). The probe ws plced pproximtely 5 cm proximl to the ntecubitl foss, nd guide ws dhered to the skin to ensure the sme probe plcement between trils. Doppler ultrsound mesurements were tken t 68 insontion ngle for resons previously described. 25 A Multigon 500P TCD spectrl nlyzer (Multigon Industries, Yonkers, NY, USA) ws used to nlyze the Doppler shift frequency spectrum, where men velocity ws determined s weighted men of the spectrum of Doppler-shift frequencies. The voltge output ws smpled continuously (PowerLb; ADInstruments, Colordo Springs, CO, USA) nd stored for lter nlysis (LbChrt; ADInstruments). Brchil rtery imges were obtined with the sme probe using echo ultrsound t 12 MHz in B-mode (Vivid i2). Prmeters on the ultrsound mchine were set to optimize longitudinl B-mode imges of the lumen rteril wll interfce. The imge dt were recorded s.vi files on n independent computer (Cmtsi Studio; TechSmith Corp., Okemos, MI, USA) with VGA to USB frme grbber (Epiphn Systems Inc., Ottw, ON, Cnd). Experimentl procedure Prticipnts ly supine, nd their left rm ws extended to the side for ultrsound mesurements. Blood pressure mesurements were mde on the right rm. After pproximtely 30 minutes of supine rest, prticipnts performed

3 Stuckless nd Pyke 411 Figure 1. The experimentl protocol. A minimum of 10 minutes, or the time until the brchil rtery dimeter returned to bseline, ws llowed between EX-FMD trils. The CPT involved plcing the right foot in n ice bth for 3 minutes, followed by removl of the right foot nd simultneous submersion of the left foot for 3 minutes. (EX-FMD: hndgrip exercise-induced flowmedited vsodiltion; EX-FMD+CPT: EX-FMD with CPT performed during the hndgrip exercise bout.) the first EX-FMD tril. Following minimum 10-minute rest (witing until the brchil rtery dimeter hd returned to bseline), second EX-FMD tril ws performed. This ws identicl to the first tril in the Control group. In the Experimentl group, the second tril ws performed with submersion of one foot in ice wter (CPT) for the durtion of the exercise period (Figure 1). EX-FMD. In ech EX-FMD tril, brchil rtery blood velocity nd dimeter were recorded for 1 minute t bseline nd then during 6 minutes of hndgrip exercise. During the hndgrip exercise, isometric hndgrip force feedbck (percent mximl voluntry contrction) ws displyed continuously (PowerLb). Subjects chieved the required force nd durtion for ech contrction by displcing the force redout line to the desired level in time with 1-second contrction / 5-second relxtion duty cycle metronome. 26 In both trils the gol ws to rech nd mintin trget brchil rtery sher rte of 75 s 1 during exercise. Sher rte (n estimte of sher stress without blood viscosity) ws clculted s: sher rte = men blood velocity / vessel dimeter. This trget ws selected becuse it hs previously been shown to result in robust FMD response nd becuse it requires n exercise intensity tht cn be comfortbly mintined for 6 minutes. 15 The blood velocity required to chieve the trget sher rte ws clculted for ech subject s follows: required blood velocity = 75 s 1 * brchil rtery dimeter. To estimte the exercise intensity necessry to chieve the required blood velocity, subjects performed series of contrctions t vrious % mximl voluntry contrctions during the fmiliriztion visit. During the EX-FMD tril the intensity ws djusted by n experimenter who coched the subject to mintin the desired blood velocity. CPT. The CPT ws performed concurrently with the hndgrip exercise during the second EX-FMD tril in the Experimentl group. After the 1-minute bseline period, prticipnts begn the hndgrip exercise. At this point, lb personnel immersed one of the prticipnt s feet in the icewter bth, which ws mintined t constnt temperture (4.5 ± 0.5 C). This foot ws kept in the bth for 3 minutes nd then ws removed s the opposite foot ws submerged for 3 minutes. The CPT therefore totled 6 minutes in durtion, ligned with the 6 minutes of hndgrip exercise. All prticipnts completed the full 6 minute CPT. Blood lipids nd glucose. At the end of the ultrsound dt collection, smll smple of blood ws tken by finger prick for nlysis of blood lipid nd glucose levels using Cholestech LDX system (Alere Inc., Ottw, ON, Cnd). Due to technicl difficulties, this smpling nd nlysis ws only completed in eight subjects in the Experimentl group nd 11 subjects in the Control group. Dt nlysis Hemodynmic vribles. Hert rte, men rteril pressure nd totl peripherl resistnce dt were nlyzed offline nd compiled into 3-second verge time bins (LbChrt). For ech vrible, 1-minute verge bseline vlue nd pek 1-minute verge during exercise were reported. Brchil rtery dimeter. Brchil rtery imge files were nlyzed using utomted edge detection softwre (FMD/Blood Flow Acquisition nd Anlysis; Reed Electronics, Perth, WA, Austrli) s previously described. 10 The dimeter dt were compiled into 3-second time bins for lignment with blood velocity nd clcultion of sher rte (see below). EX-FMD ws clculted s the percent chnge in dimeter from bseline to the pek 1-minute verge during exercise. Brchil rtery blood velocity. Blood velocity ws nlyzed offline in 3-second verge time bins (LbChrt). The blood velocity dt were ssembled into 1-minute verges, nd the blood velocity for ech tril is reported s n verge of the stedy-stte period (lst 5 minutes of exercise). Sher rte. The following eqution ws used to clculte sher rte: sher rte = men blood velocity / vessel dimeter. This ws chieved using ligned 3-second verge dimeter nd blood velocity time bins. The sher rte dt were ssembled into 1-minute verges nd the sher rte for ech tril is reported s n verge of the stedy-stte period (lst 5 minutes of exercise). Sttisticl nlysis The dt re reported s mens ± stndrd devition. To compre subject chrcteristics between groups, t-tests were used. ANOVA nlysis (fctors group (between-subjects; Experimentl nd Control) nd tril (within-subjects; trils

4 412 Vsculr Medicine 20(5) Tble 2. Hemodynmic, rteril nd force prmeters. Prmeter Experimentl Control ANOVA result Tril 1 Tril 2 Tril 1 Tril 2 Group Tril Group Tril Bseline HR (bpm) 59 ± 9 58 ± 7 51 ± 6 52 ± MAP (mmhg) 91 ± ± ± 7 91 ± < TPR (mmhg/l/min) 17.7 ± ± ± ± Dimeter (cm) 0.35 ± ± ± ± Velocity (cm/s) 6 ± 2 6 ± 2 6 ± 2 6 ± Sher (s 1 ) 16 ± 5 17 ± 5 14 ± 5 15 ± Exercise HR (bpm) 62 ± 8 70 ± 8 b 59 ± 8 59 ± 9 c MAP (mmhg) 98 ± ± 10 b 96 ± ± 11 c <0.001 <0.001 TPR (mmhg/l/min) 18.5 ± ± ± ± Velocity (cm/s) 24 ± 3 25 ± 3 28 ± 5 28 ± %MVC 32 ± 12% 33 ± 14% 43 ± 18% 43 ± 17% Vlues re ± stndrd devition. HR, hert rte; MAP, men rteril pressure; TPR, totl peripherl resistnce; MVC, mximl voluntry contrction. n=13, Experimentl group; n=11, Control group. b Significnt difference from tril 1 within group. c Significnt difference from tril 2 in Experimentl group. 1 nd 2)) ws used to compre ll other vribles. Post hoc nlysis to explore significnt interctions ws done vi pirwise comprisons (t-tests nd pired t-tests s pproprite). The level of significnce ws set t p < 0.05 for ll tests. All sttistics were clculted using SPSS (SPSS Inc., Chicgo, IL, USA) or Sigm Plot (Systt Softwre Inc., Sn Jose, CA, USA). Results Subject chrcteristics The verge ge, height, weight, nd blood lipid profiles of the prticipnts re shown in Tble 1. Group differences pproched significnce for ge (higher in the Control group; p=0.054) nd LDL (lower in the Control group; p=0.054) only. Hemodynmic vribles The verge hert rte, men rteril pressure nd totl peripherl resistnce t bseline nd during hndgrip exercise re shown in Tble 2. Hert rte. Bseline hert rte did not differ between trils (min effect of tril p=0.928); however, hert rte ws higher in the Experimentl group versus the Control group (min effect of group p=0.012) (group tril interction p=0.487). During hndgrip exercise, there ws significnt min effect of group (p=0.023) nd tril (p=0.001), nd significnt interction between group nd tril (p=0.001). Post hoc nlysis reveled tht hert rte ws significntly greter in tril 2 versus tril 1 in the Experimentl group (p<0.001) but not in the Control group (p=0.868). In ddition, hert rte ws not significntly different between groups in tril 1 (p=0.262), but it ws significntly greter in the Experimentl group in tril 2 (p=0.002). Men rteril pressure. Bseline men rteril pressure did not differ between groups (min effect of group p=0.211); however, it ws significntly greter in tril 2 versus tril 1 (min effect of tril p<0.001) (group tril interction p=0.174). During exercise, there ws min effect of group (p=0.014) nd tril (p<0.001), nd significnt interction between group nd tril (p<0.001). Post hoc nlysis reveled tht men rteril pressure during exercise ws significntly greter during tril 2 versus tril 1 in the Experimentl group (p<0.001) but not in the Control group (p=0.095). In ddition, exercise men rteril pressure ws not different between groups in tril 1 (p=0.480), but it ws significntly greter in the Experimentl group versus the Control group in tril 2 (p<0.001). Totl peripherl resistnce. Bseline totl peripherl resistnce ws not significntly different between groups (min effect of group p=0.866); however, tril 2 totl peripherl resistnce ws significntly greter thn tril 1 totl peripherl resistnce (min effect of tril p=0.032) (group tril interction p=0.599). Similrly, during exercise totl peripherl resistnce ws not significntly different between groups (min effect of group p=0.444); however, totl peripherl resistnce in tril 2 ws significntly greter thn totl peripherl resistnce in tril 1 (min effect of tril p=0.005) (group tril interction p=0.127). Sher rte Bseline sher rte ws not different between groups (min effect of group p=0.325) or trils (min effect of tril p=0.461) (group tril interction p=0.671). As expected with trgeting of sher rte, there ws no effect of group (min effect of group p=0.823) or tril (min effect of tril p=0.726) (group tril interction p=0.646) on the sher rte stimulus during exercise (Figure 2). The velocity reched to chieve the sher rte ws slightly higher in the Control group (p=0.039) (Tble 2).

5 Stuckless nd Pyke 413 Figure 2. The verge sher rte stimulus during the lst 5 minutes of the hndgrip exercise (stedy-stte period). (Error brs re ± stndrd devition.) Figure 3. Percent exercise-induced flow-medited diltion (%EX-FMD). (Error brs re ± stndrd devition.) Bseline dimeter nd %EX-FMD There ws no significnt effect of group (min effect of group p=0.093) or tril (min effect of tril p=0.605) (group tril interction p=0.133) on bseline dimeter (Tble 2). There ws lso no significnt effect of group (min effect of group p=0.508) or tril (min effect of tril p=0.592) (group tril interction p=0.879) on %EX-FMD (Figure 3). Hndgrip exercise intensity The verge % mximl voluntry contrction throughout the 6 minutes of exercise ws not significntly different between groups (min effect of group p=0.075) or trils (min effect of tril p=0.607) (group tril interction p=0.760) (Tble 2). Discussion The purpose of this study ws to perform the first exmintion of the impct of CPT on concurrent hndgrip EX-FMD. A robust response to the CPT ws indicted by increses in hert rte nd men rteril pressure; however, brchil rtery EX-FMD mesured simultneously ws not different from the Control condition. These results indicte tht the physiologicl response evoked by the CPT did not impct the bility of the brchil rtery endothelium to respond to exercise-induced increses in sher stress nd suggests tht in this popultion, n cute, sympthoexcittory stimulus does not interfere with conduit rtery FMD responses during exercise. This is in contrst to previous observtions of impired FMD responses to brief sher stress stimulus during CPT, 23,24 nd suggests tht the impct of CPT-stimulted signls my be sher stress

6 414 Vsculr Medicine 20(5) profile-specific. An intct response to n exercise-induced sher stress stimulus would tend to support ctive muscle perfusion in the presence of pinful/stressful stimuli. CPT FMD interction Severl studies hve exmined the impct of CPT nd other cutely stressful nd pinful stimuli on endothelil function. 23,24,27 Sympthetic nervous ctivity, epinephrine, nd hemodynmic responses increse shortly fter the onset of stressor 28 nd begin to return to bseline immeditely post-stressor, while other responses re significntly delyed (e.g. pek cortisol concentrtions occur minutes following stressor onset 28 ). Therefore, the stressssocited physiologicl signls present to interct with FMD differ depending on the timing of FMD evlution to stressor ppliction. 29 We hve previously observed tht the EX-FMD response is modestly enhnced 15 but not 35 minutes following CPT in young helthy men. 22 To our knowledge, this is the only previous study exmining EX-FMD in reltion to CPT. In the present study, EX-FMD ws mesured concurrently with the CPT experience, nd we found no significnt difference from Control conditions (Figure 3). Tken together, these dt suggest tht EX-FMD is unffected by the signls present during CPT, nd is modestly enhnced by the signls tht shortly follow CPT cesstion. This is in contrst to observtions tht CPT performed concurrently with RH-induced increses in sher stress impirs brchil rtery RH-FMD by ~35 70%. 23,24 These studies lso used foot immersion, with 5-minute 23 or 10-minute 24 durtion. Rective hyperemi nd hndgrip exercise elicit very distinct sher stress profiles in the brchil rtery, which likely influence the trnsduction pthwys, nd potentilly the vsodiltors, tht re involved in the respective FMD responses. 8,12,30 33 Differing vulnerbilities of RH-FMD nd EX-FMD to cute stimuli hve previously been reported by our group 16 nd others. 17 Given the contrst of our findings with the previous work indicting impired RH-FMD during CPT, our findings suggest tht the impct of CPT signls on FMD my be non-uniform nd specific to the nture of the sher stress stimulus. These dt dd to the evidence indicting tht n observed impct of given stimulus on RH-FMD should not be generlized to EX-FMD. Although not mesured directly here, the CPT hs been demonstrted to rise sympthetic nervous ctivity, indicted by n increse in the rte of peronel nerve bursts 21 nd n increse in circulting norepinephrine. 24 In ddition, cold exposure stimultes nociceptors 34 nd is perceived s both pinful nd stressful. 22 Interventions tht cutely elevte sympthetic nervous ctivity hve been shown to impir RH-FMD in some, 35 but not ll, 24 cses, nd sympthetic nervous ctivity my hve been involved in impiring endothelil function in previous CPT studies. 23,24 It is thought tht impirment could be due to blunting of diltion due to superimposed vsoconstrictor signl or neurl interctions with endothelil function. 36 The hemodynmic responses to the CPT re secondry to sympthetic ctivtion, nd the mgnitude of the increse in men rteril pressure (~18 mmhg) observed in the present study is similr to previous reports in which RH-FMD ws observed to be impired. 23,24 However, we did not detect significntly higher totl peripherl resistnce during tril 2 in the Experimentl group (EX-FMD test + CPT) versus tril 2 in the Control group (EX-FMD test lone) (no interction between group nd tril, p=0.127) (Tble 2). This suggests lck of pronounced peripherl vsoconstriction response to the CPT. It is therefore possible tht in individuls with more profound constrictor response EX-FMD my be dversely ffected by the CPT. However, explortory nlysis indicted no reltionship between the chnge in totl peripherl resistnce with the CPT nd the chnge in EX-FMD in the Experimentl group (dt not shown). Significnce of EX-FMD responses The use of exercise-induced increses in sher stress for investigting conduit rtery FMD hs been vlidted previously. Specificlly, it ws demonstrted tht brchil nd rdil rtery diltion during brief (6-minute) bout of hndgrip exercise is due to increses in sher stress (i.e. it is FMD) nd not vsodiltory signl tht is conducted upwrds from the ctive muscle vsculr bed. 10,11 EX-FMD is understudied; however, smll body of evidence indictes tht it provides informtion tht is relevnt to rteril helth. First, FMD in response to exercise or other sustined, modest increses in sher stress hs been shown to be impired in some popultions t incresed risk for crdiovsculr disese (ptients with type 1 dibetes 18 nd smokers 15,19 ). However, this hs not lwys been found (ptients with hypercholesterolemi 8 ). Future reserch is required to determine if, like RH-FMD, 37 EX-FMD hs some disese prediction vlue. EX-FMD cn ply role in tissue perfusion, nd if this diltory contribution were compromised in vulnerble popultions nd vsculr beds (e.g. in the hert in coronry rtery disese popultion or in the leg in peripherl rtery disese popultion), it might contribute to cliniclly importnt ischemi. 2,13,14,38 If EX-FMD were vulnerble to impirment by elevted sympthetic nervous ctivity (elevted in the present study with the CPT, but lso elevted by mentl stress nd certin disese sttes), this might result in compromised perfusion, nd this could contribute to the reltionship between sympthetic ctivity nd mortlity. 39 While the present results do not support n impct of cute sympthetic ctivtion on EX-FMD, this my differ in other popultions. Further reserch is required to estblish the precise circumstnces nd popultions in which EX-FMD impirment is present nd cliniclly/functionlly relevnt. Limittions This study ws performed in young, helthy mles, nd the results cnnot be extended to other popultions. Also, muscle sympthetic nervous ctivity, epinephrine nd norepinephrine were not ssessed, nd chnges in these vribles might hve provided further insight regrding the response to the CPT nd its potentil to influence EX-FMD responses.

7 Stuckless nd Pyke 415 However, the observed hemodynmic response to the CPT ws similr to tht of other studies in which the CPT impired RH-FMD. This suggests tht our prticipnts responded typiclly nd to degree tht could influence some spects of endothelil function. Given the potentil for lsting impct of CPT on EX-FMD, 22 we could not counterblnce the order of the presenttion of the CPT in the Experimentl group (the CPT ws lwys experienced in tril 2). It is possible tht the impct of the CPT on EX-FMD might hve been different if it hd been experienced during the first EX-FMD tril; however, this seems unlikely given tht multiple, closely spced hndgrip exercise trils result in stble EX-FMD response (demonstrted in the Control group nd in Pyke nd Jzuli 40 ). Although it did not rech significnce (group tril interction p=0.174), the rise in bseline men rteril pressure from tril 1 to tril 2 ws lrger in the Experimentl group (6 mmhg vs 3 mmhg; Tble 2). It is possible tht this indictes some nticiption of the impending CPT in the Experimentl group, but this is unlikely to hve impcted the results given tht the much more robust elevtion in blood pressure during the CPT did not significntly impir the EX-FMD response. Finlly, we did not perform CPT to chrcterize the hemodynmic rectivity without simultneous hndgrip exercise, lthough previous work suggests tht hndgrip exercise does not lter hert rte nd blood pressure responses to CPT. 41 If the exercise did modulte rectivity to the CPT, this would not lter our primry conclusion tht the simultneous CPT did not hve deleterious impct on the EX-FMD response. Conclusions In popultion of young, helthy mles we found tht, when performed concomitntly with hndgrip exercise, CPT chieved with foot immersion in ice wter did not result in n impired brchil rtery EX-FMD response. This suggests tht, in this popultion, n cute pinful stimulus ssocited with n increse in sympthetic nervous ctivity does not interfere with peripherl conduit rtery endothelil responses to exercise. Future reserch is required to exmine responses in rnge of popultions nd to identify the impct of chroniclly elevted sympthetic nervous ctivity on EX-FMD. Declrtion of conflicting interest The uthors declre tht there is no conflict of interest. Funding This study ws funded by Nturl Sciences nd Engineering Reserch Council of Cnd (NSERC) Discovery Grnt nd by Cnd Foundtion for Innovtion nd Ontrio Ministry of Reserch nd Innovtion Leders Opportunity Funding to KE Pyke. TJR Stuckless ws supported by Ministry of Reserch Innovtion Erly Resercher Awrd to KE Pyke. References 1. Lndmesser U, Hornig B, Drexler H. Endothelil function: A criticl determinnt in therosclerosis? Circultion 2004; 109: II27 II Duffy SJ, Cstle SF, Hrper RW, Meredith IT. 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