| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Department of Physiology, Institute of Fundamental and Clinical Movement Sciences, and 2Department of Pharmacology-Toxicology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
Submitted 8 March 2006 ; accepted in final form 5 July 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
|---|
endothelium-dependent dilation; cold pressor test; exercise
The sympathetic nervous system serves as a key modulator of cardiovascular and other physiological functions in humans (33). Interestingly, several conditions with impaired FMD responses are also characterized by an increased sympathetic activity (8, 11). Moreover, when acute stimulation of the sympathetic nervous system in healthy subjects is applied, an impaired FMD is observed in the brachial artery (9, 17, 23). However, these findings are not universal and seem to depend on the nature of the stress stimulus (9, 15).
Advancing age is associated with a chronic increased sympathetic activity (29), augmented vasoconstrictor responsiveness to acute sympathetic stimulation during exercise (19), and enhanced postexercise FMD in the brachial artery of older women (16). In addition, the age-related reductions in limb blood flow are reported to be mediated largely by the chronically elevated 
-adrenergic sympathetic tone (7). However, the effects of the age-related chronically elevated sympathetic tone on the impaired FMD responses of the superficial femoral artery are unknown. Therefore, the aim of the study is to assess the effects of reduction of the sympathetic responsiveness or stimulation of the sympathetic nervous system on the FMD of the superficial femoral artery in healthy older and young men. We hypothesize that reducing the sympathetic responsiveness of the chronically elevated sympathetic nerve activity in older men will restore flow-mediated vasodilation. This implies that local reduction of sympathetic activity enhances flow-mediated vasodilation.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ten young healthy men (22 ± 3 yr) and eight healthy older men (69 ± 2 yr) (Table 1) volunteered to participate in this study. All subjects were normotensive (blood pressure <160/90) and nonsmoking and were free of overt chronic cardiovascular disease as assessed by medical history and physical examination. None of the subjects used medication known to interfere with the cardiovascular system, and subjects were further evaluated by ECG at rest. The individuals with ankle-brachial pressure index <0.90, plaque formation, and/or clinical characteristics of arthrosclerosis were excluded. The study was approved by the hospital ethics committee. All subjects gave their written, informed consent before participation. All studies were performed according to the Declaration of Helsinki.
|
All subjects were tested on four separate days, all at the same time of the day. On day 1, baseline endothelium-dependent (FMD) and -independent (nitroglycerin; NTG) dilation of the superficial femoral artery was measured. Day 2 started with an exercise test, leading to local sympatholysis in the exercised limb. On the third day, the FMD and NTG responses were measured during sympathetic stimulation using the cold pressor test (CPT). The sequence of days 1–3 was randomized. During day 4, peripheral vascular changes were assessed during the CPT and the exercise test.
Protocol
Day 1: baseline. After a 12-h overnight fast, experiments were performed in the morning. Subjects refrained from caffeine, nicotine, chocolate, kiwi, vitamin C supplements, and alcohol for at least 18 h before the test. Room temperature was controlled at 23 ± 1°C. Subjects were positioned comfortably on a bed in the supine position. The lower legs rested on a 14-cm-high platform. A cuff (12 cm) was placed proximally around the upper leg and was connected to a rapid cuff inflator (Hokanson, Bellevue, WA). After an acclimatization period of 30 min, resting characteristics of the superficial femoral artery were measured (Fig. 1). Subsequently, the occlusion cuff was inflated to 220 mmHg suprasystolic pressure for 10 min to measure FMD. After a resting period of at least 30 min, a single spray of sublingual NTG (400 µg), an exogenous nitric oxide (NO) donor, was administered to determine the endothelium-independent vasodilation of the superficial femoral artery (4), which is indicative for smooth muscle function. During testing, arterial blood pressure was measured continuously using a portable blood pressure device (Finapres; TNO, Amsterdam, The Netherlands) that was connected to the middle phalanx of the index or middle finger of the left hand.
|
-adrenergic receptors (or its signaling).
After measurement of the resting characteristics of the superficial femoral artery, subjects performed an incremental maximal cycling test. The exercise bout was performed on a leg cycling ergometer (Lode, Angio300, Groningen, The Netherlands), using a multistage protocol. Young men increased their workload by 20 W/min, starting at 20 W, until exhaustion, whereas older men used steps of 10 W/min. Oxygen consumption was measured continuously to determine physical fitness of both groups using a gas analyzer (Jaeger Benelux, Breda, The Netherlands). Peak oxygen consumption (
O2 peak) was analyzed as the mean of the last minute of the maximal exercise test. In addition, blood lactate levels (Roche Diagnostics, Mannheim, Germany) were measured, and heart rate was recorded continuously. Within 3 min after cessation of the exercise test, subjects were placed in the supine position, and an occlusion cuff was inflated to 220 mmHg for 5 min to assess the FMD during the period of local blunted sympathetic responsiveness (Fig. 1). To account for differences in oxygen consumption compared with the previous days, the time of occlusion was adapted to equalize the shear stress stimulus (16). After a resting period of 15 min, NTG was administered to assess the endothelium-independent dilation of the superficial femoral artery. This time window is associated with local postexercise sympatholysis (14).
Day 3: CPT. Experiments performed on day 1 were repeated with addition of the CPT. This is a widely accepted and well-validated model to stimulate the sympathetic nervous system. After 9 min of arterial occlusion, the CPT was started by immersion of the left hand into ice water (4°C) (10, 39) and was continued for 5 min (Fig. 1). Using this protocol, we ensured that maximal sympathetic stimulation, normally present during the initial minutes of the CPT (40), was present during measurement of the FMD. Leg vascular resistance was assessed before and during the CPT. The mean of the three highest values of leg vascular resistance during the CPT was used to calculate the percent change in vascular resistance compared with baseline (20). After a 30-min break, the CPT was also performed in combination with the endothelium-independent dilation. The CPT started 1 min after sublingual administration of NTG. Using this protocol, we ensured that maximal sympathetic stimulation (i.e., initial minutes of the CPT) (40) coincided with the maximal response of NTG (i.e., 3–6 min after administration) (data not shown).
Day 4: additional experiments. To assess the effect of the CPT and exercise bout on hemodynamics and sympathetic control, additional experiments were performed. Leg blood flow and vascular resistance, superficial femoral artery diameter, heart rate, and mean arterial pressure were measured before, during, and after a CPT. Leg blood flow was examined using venous occlusion plethysmography, while the diameter was simultaneously measured with an echo-Doppler machine. Subsequently, an incremental maximal test was performed to induce functional sympatholysis. To examine changes in the regulation of sympathetic control, a second CPT was performed within 3 min after cessation of the cycling exercise. Again, hemodynamics were recorded before, during, and after the postexercise CPT. These additional experiments were extended in a subgroup of healthy young men (23 ± 3 yr), who performed a CPT and an incremental maximal test as described above. To examine the duration of the local attenuation of the sympathetic system, leg blood flow (plethysmography) and vascular resistance were examined in two subjects until 20 min postexercise. To examine the localization of the attenuated sympathetic responsiveness, the two other subjects underwent a postexercise CPT, while we simultaneously examined leg and forearm blood flow (plethysmography) and vascular resistance. In this subgroup of the present subjects, the CPT-induced change in leg vascular resistance has an acceptable coefficient of variation (CV) of 13.7% (n = 4).
Measurements
Resting diameter, FMD, and NTG.
Systolic and diastolic vessel diameters of the superficial femoral artery were measured with an echo-Doppler device (Megas; ESAOTE, Firenze, Italy) with a 5- to 7.5-MHz broadband linear array transducer. Diameter images were made 3 cm distal to the bifurcation of the femoral artery. To measure resting diameter, two consecutive images in the longitudinal view were frozen at the peak systolic and end-diastolic phase. The mean diameter (D) was calculated by use of the following formula: 1/3·systolic diameter + 2/3·diastolic diameter. Before measurement of diameter images, from each artery, four images with a total of 12 velocity profiles were obtained and manually traced afterward by a single investigator. The average of these waveforms was used to calculate mean and peak blood flow (5). The angle of insonation for the velocity measurements was consistently at 60°, and the vessel area was adjusted parallel to the transducer. Mean blood flow (in ml/min) was calculated as 1/4·
(D)2·Vmean(cm/s)·60, where Vmean is mean blood cell velocity. Peak blood flow (in ml/min) was calculated as 1/4·(Ds)2·Vpeak(cm/s)·60, where Ds is systolic diameter. Mean wall shear rate (MWSR) (in s) was calculated as 4·Vmean/D, where Vpeak is peak blood cell velocity. Postocclusion diameters were obtained at 50, 60, 70, 90, 120, 180, and 240 s. Measurement of the post-NTG diameters started 2 min after administration of sublingual NTG, and the diameter was measured every 30 s for 6 min. FMD and NTG responses were expressed as the maximal relative diameter change in end-diastolic diameter from the baseline resting end-diastolic diameter. On day 2, 15 min of postexercise rest were not sufficient for the diameter to return to baseline level. Therefore, we used the baseline diameter of the FMD response. The ratio between the relative FMD response (%FMD) and the primary stimulus for vessel dilation (MWSR) was calculated. 
MWSR was defined as the difference between rest and peak response and was used to calculate the amount of vasodilation per stimulus during the FMD (21). This hyperemic velocity was recorded on videotape for the first 25 s after cuff release. The velocity profiles between 10 and 15 s after cuff release were analyzed by a single investigator. The average of these profiles was used to calculate the MWSR.
Leg blood flow and vascular resistance. To measure leg blood flow and calculate vascular resistance during the CPT on day 4, a 12-cm-width cuff was placed proximally around the upper leg. The strain gauge was placed at midthigh, at least 10 cm above the patella (37). The occlusion cuff was inflated, ECG triggered and within one heartbeat, to a cuff pressure of 50 mmHg (13). This pressure was sustained for nine heartbeats after which the cuff was instantaneously deflated (for 10 heartbeats). In our lab, this method to measure baseline leg blood flow (plethysmography) or vascular resistance is demonstrated to have a CV of 5.9 and 8.3%, respectively (37). Data were digitalized with a sample frequency of 100 Hz [Michigan Digital Automatic Computer (MIDAC), Instrumentation Department, Radboud University Nijmegen Medical Center] and analyzed by a customized computer program (Matlab, Mathworks). Blood flow (in ml·min–1·dl–1) was calculated as the slope of the volume change over a 4-s interval, starting directly after the cuff artifact (2 s). Mean arterial pressure (MAP) data of the Portapress were used to calculate vascular resistance [MAP/blood flow, in mmHg·ml–1·min–1·dl–1; arbitrary units (AU)].
Statistical Analysis
The main goal of this study was to compare the FMD responses between the different conditions. With a SD of 45% (16, 17), a relevant effect of the intervention of 50%, and an of 0.05, we calculated that at least eight subjects per group would be needed to achieve a power of 90%. Statistical analyses were performed using SPSS 11.0 computer software (SPSS, Chicago, Illinois). The effect of the three different sympathetic conditions on the FMD (dependent variable) in both groups (independent variable) was evaluated by a two-way ANOVA for repeated measurements (RM-ANOVA). Differences were subsequently analyzed with the least squares difference post hoc test. A Student's t-test for independent groups was used to examine differences in physical characteristics between the young and older men. Data are presented as means ± SD. The level of statistical significance was set at = 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Table 1 shows the physical characteristics of both study populations. Systolic and diastolic blood pressure, body mass, and body fat were significantly higher in older men. Maximum oxygen uptake in older men was significantly lower than in young men.
Older Men (Days 1–3)
Resting characteristics of the superficial femoral artery before the FMD were not different among the three conditions (Table 2). The uncorrected and shear rate-corrected FMD and NTG response in older men was significantly lower compared with the young men (t-test; P = 0.001, P = 0.04, and P = 0.02, respectively).
|
MWSR) showed similar results (see 
Fig. 3). The NTG response was not different among the three conditions (Table 3).
|
|
|
In young men, the resting diameter of the superficial femoral artery before the exercise bout was significantly higher compared with the resting diameters before measurement of the baseline (P = 0.01) and CPT FMD (P = 0.002). Resting blood flow, heart rate, and MAP were not different among the 3 days (Table 2).
The baseline FMD of the superficial femoral artery in young men was not different from the FMD during the CPT or from the postexercise FMD (Table 4). Also, after correction for the eliciting stimulus (MWSR), the FMD was not different among baseline condition, during the CPT, and postexercise (see Fig. 3). Due to nausea, postexercise NTG response in one subject was not measured. The NTG response was not different among the three conditions (Table 4).
|
After cessation of the exercise bout, arterial pressure in young men was significantly decreased, whereas older men showed no change in arterial pressure compared with preexercise values (Fig. 2). The postexercise CPT showed no change in vascular resistance, heart rate, and MAP in both groups (Fig. 2). This suggests a postexercise local attenuation of the sympathetic responsiveness in the leg.
CPT (Day 4)
Because of technical problems, one subject from the older group was not included in the analysis. Leg vascular resistance increased significantly during the CPT in young (P < 0.001) as well as in older men (P = 0.01, Fig. 2A). Heart rate did not change, whereas MAP significantly increased, suggesting sympathetic stimulation in both groups (Fig. 2, B and C). Superficial femoral arterial diameter did not change during the CPT in young or in older men (ANOVA: P = 0.38 and 0.27, respectively). Also, in the subgroup of two healthy young men, the CPT-induced increase in leg vascular resistance was attenuated after exercise (preexercise, 130 ± 63%; postexercise, 11 ± 1%). Interestingly, these subjects demonstrate a similar relative change in forearm vascular resistance between pre- and postexercise CPT (preexercise, 211 ± 82%; postexercise, 261 ± 63%). In addition, the other two subjects showed a markedly lower leg vascular resistance during the FMD time window (8–6 AU) compared with baseline (16 ± 4 AU). This suggests that the exercise bout attenuates the constrictor response in the leg but not in the (nonactive) forearm during the time window in which we examine the FMD response.
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Unique to this study is the application of the CPT and the maximal exercise test, which stimulates or reduces, respectively, the responsiveness of the sympathetic nervous system to examine the effect on the FMD. The noninvasive nature of these interventions is an important advantage. Because these interventions have not been used before in this setting, some aspects of our approach should be discussed. First, plasma norepinephrine or muscle sympathetic nerve activity was not assessed in the current study. Yet, we are confident that the CPT effectively increased sympathetic activity, since this has been described in previous studies (22, 24) and because the CPT markedly elevated arterial blood pressure in both groups, comparable with previous studies (9, 23). This pressor effect is primarily induced by a sympathetic vasoconstriction response as demonstrated by the elevated leg vascular tone.
Second, to assess whether the maximal exercise bout successfully reduced local sympathetic responsiveness in the legs during assessment of the FMD, we quantified the postexercise CPT response in the time window of the FMD. In contrast to the marked increase in leg peripheral resistance during the preexercise CPT, no change in leg vascular resistance was observed after maximal exercise. The lack of changes in leg vascular tone suggests successful attenuation of the sympathetic responsiveness in the legs after maximal cycling exercise. Regarding the physiological underlying mechanism, one may hypothesize that a part of this mechanism is in accordance with the mechanism suggested for exercise-induced hypotension. Halliwill et al. (14) demonstrated that postexercise hypotension is partly mediated through a vascular component (less vasoconstriction with any increase in sympathetic activity).
Third, the dramatically elevated postexercise oxygen demand of the leg muscles could alter postocclusive shear rate stimulus, which is the primary trigger for the FMD response (21). As such, we adjusted the duration of the postexercise leg arterial occlusion (using a 5-min instead of 10-min occlusion) and achieved a similar shear rate stimulus after exercise compared with baseline FMD or CPT FMD in both groups (Tables 3 and 4). Nevertheless, FMD responses were also analyzed after correction for the main vasodilating stimulus, i.e., MWSR (21).
Finally, changes in responsiveness or bioavailability of NO may lead to changes in FMD responses during acute stimulation or reduction. The response to exogenous NO of the superficial femoral artery in young and older men does not differ among baseline, postexercise, or CPT conditions. Although postexercise timing of the FMD and NTG responses was not similar, our results suggest that the response of the superficial femoral artery to exogenous NO is independent of sympathetic nerve activity, which is in agreement with a previous study by Hijmering et al. (17) reporting no effect of sympathetic stimulation on brachial dilation to exogenous NO.
FMD After Local Attenuation of Sympathetic Responsiveness
Our results demonstrate a significantly lower FMD of the superficial femoral artery in older men compared with healthy young men. This difference between young and older men may be explained by an attenuated NO bioavailability with aging or by age-related changes in the regulation of the sympathetic nervous system in the legs. Dinneno et al. (7) indicated in an elegant study that the reduction in basal limb blood flow with human aging is mediated largely by an augmented sympathetic -adrenergic vasoconstriction. The age-related increase in sympathetic nerve activity (29) may also attenuate the FMD response in older men. Parallel to our hypothesis, local blunting of the sympathetic responsiveness through exercise in older men increased the corrected FMD response toward normal values in healthy controls (%FMD/MWSR) (Fig. 3). Moreover, a significant negative correlation is reported among the three different conditions of sympathetic nerve activity (exercise, baseline, and CPT) and the FMD response in older men (Fig. 4). These results suggest that the impaired FMD of the superficial femoral artery in older men may be explained partly by age-related elevation of sympathetic nerve activity. Our findings are in agreement with those of Harvey et al. (16). They reported a significant increase in postexercise FMD of the brachial artery in postmenopausal women, while young premenopausal women showed no change after exercise.
|
Whereas local sympatholysis in older men restored the FMD response, young men reported no change. One may speculate about a possible "ceiling effect" of the FMD in healthy young men; the higher FMD response would be difficult to increase any further. However, we compared a subpopulation of the older men with the highest FMD values (n = 4) with a subset of the young men (n = 4) with matched FMD values. The older men showed an increase in FMD response during the postexercise FMD. In contrast, two young men demonstrated a decreased FMD response, while the other two showed an increased FMD response. As such, this argues against a possible ceiling effect in young men in our study.
FMD After Sympathetic Stimulation
In contrast with previous studies in the brachial artery (9, 15, 17, 23), we reported that acute stimulation of the sympathetic nervous system, using the CPT, did not alter the FMD response of the superficial femoral artery in the young as well as older men. To date, evidence is increasing that forearm and leg vasculature show different responses to similar stimuli (27, 28, 30, 31). Whether a limb difference may explain our findings can be questioned. Therefore, in a subpopulation of the young men from our study (n = 6), we measured the FMD of the brachial artery with and without the CPT. Similar to previous studies in the brachial artery (9, 17, 23), we found that the brachial artery FMD decreased significantly during sympathetic stimulation (FMD/MWSR 0.024 ± 0.003, CPT FMD/MWSR 0.014 ± 0.003; t-test, P = 0.03). These results suggest that limb differences (either in the response to the CPT or in the sensitivity of the FMD for sympathetic stimulation) may account for different FMD responses of the brachial and femoral artery during acute sympathetic stimulation in the subgroup of young men. This interesting finding could be extended by future research.
Whereas local sympatholysis in older men restored the FMD response, stimulation did not significantly decrease the FMD. However, a negative correlation is present between the level of sympathetic nerve stimulation and FMD response. In addition, subanalysis of the older men with the highest baseline FMD showed a decrease of the FMD response during the CPT. Apparently, the already impaired FMD, which makes a further decrease more difficult, may explain why the decrease in FMD after CPT in older men did not reach statistical significance. Our data may suggest that baseline FMD is a determinant of the increase in postexercise FMD. However, when we corrected for age effects, we did not find a significant correlation (R = 0.40, P = 0.11).
Limitations
The timing of the CPT related to the FMD and NTG may be an important determinant of the overall effect on the FMD. In previous studies, the CPT started 7 min (9) or 4 min (23) before cessation of the arterial occlusion cuff. Because maximal sympathetic activity is present during the first minutes of the CPT (40), we started the test 1 min before cuff release. This ensured that the peak sympathetic activity coincided with the maximal postocclusive diameter.
A second possible limitation of the study is the difference in resting diameters in the young men. Variation of this diameter, while the maximal diameter is unaltered, leads to different FMD values. Before the exercise FMD, we found a predilated diameter of the superficial femoral artery in young men compared with the resting diameters for baseline and CPT FMD. Although the absolute and relative diameter increase during the postexercise FMD in young men was lower than for baseline and CPT FMD, it was not statistically significant. Moreover, the maximal absolute diameters during FMD were not different among the 3 days (Table 3). Therefore, the larger resting diameter in young men before the exercise test did not alter the major outcomes of our study.
A CPT normally provides a strong general sympathetic stimulus leading to a generalized increase in vascular tone. Our additional experiments showed that a postexercise CPT leads to a local attenuated vascular response in the thigh but not the forearm in young men. We were not able to prove that these responses were sympathetically mediated and/or age dependent, although there is no compelling reason to suspect otherwise. In addition, one of our subjects may be diagnosed as borderline hypertensive (resting systolic blood pressure varied between 140 and 145 mmHg). This may have some implications for our findings, since we were interested in physiological aging. On the basis of the minimal increase in systolic blood pressure, we argue that this has not importantly influenced our results.
Clinical Relevance
Of interest, in a recent point-counterpoint, it was debated whether the brachial artery FMD represents NO-mediated endothelial function (12) or whether the sympathetic nervous system also influences the FMD response (38). The results of our study indicate that decreased FMD response of the superficial femoral artery in older men does not necessarily reflect an impaired NO bioavailability but might in part be explained by an increase in sympathetic nerve activity. This indicates that the sympathetic nervous system also influences the superficial femoral artery FMD, at least in older men. Parallel to our findings, a recent paper by Harvey et al. (16) demonstrated an increased postexercise FMD in the brachial artery in older women. These findings should be kept in mind when interpreting the FMD, especially in subjects with an elevated activity of the sympathetic nervous system.
In conclusion, we have demonstrated that older men had an impaired FMD response of the superficial femoral artery, which increased by local blunting of the sympathetic responsiveness but remained unaltered during sympathetic activation. These data suggest that the lower FMD of the superficial femoral artery in older men can, at least in part, be explained by elevated sympathetic nerve activity. In addition, in healthy young men, sympathetic nerve activity does not alter the FMD of the superficial femoral artery, whereas sympathetic activation using the CPT decreases the FMD of the brachial artery. Limb differences in vascular control may underlie these findings.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Dhindsa, S. M. Sommerlad, A. E. DeVan, J. N. Barnes, J. Sugawara, O. Ley, and H. Tanaka Interrelationships among noninvasive measures of postischemic macro- and microvascular reactivity J Appl Physiol, August 1, 2008; 105(2): 427 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. J. Thijssen, M. Kooijman, P. C. E. de Groot, M. W. P. Bleeker, P. Smits, D. J. Green, and M. T. E. Hopman Endothelium-dependent and -independent vasodilation of the superficial femoral artery in spinal cord-injured subjects J Appl Physiol, May 1, 2008; 104(5): 1387 - 1393. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P < 0.001 between pre- and post-CPT. 
P < 0.05 between pretraining and postexercise.
