INTRODUCTION

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ARTERIAL BARORECEPTORS ORIGINATING in the aortic arch and carotid sinus bifurcation contribute importantly to the short-term regulation of arterial blood pressure (ABP) by altering heart rate (HR) primarily through their influence on parasympathetic outflow to the sinoatrial (SA) node (22). Since the development of the variable-pressure neck chamber, carotid baroreflex (CBR) control of HR has been extensively investigated in humans (9, 18, 25). However, due primarily to limitations in techniques that can be employed, only a few studies have attempted to examine HR reflex regulation by the aortic baroreflex (ABR) in humans (11, 19, 27, 28, 33). This limited work in humans has utilized the approach of administering vasoactive drugs to alter systemic blood pressure in combination with the application of neck pressure or neck suction (NS) to negate the influence of the carotid baroreceptors. In general, these investigations have indicated that the ABR predominates in the baroreflex control of HR with the CBR only contributing ~30% (11, 27-29, 33).

Several points must be considered when interpreting findings from studies using pharmacological agents. Primarily, the systemic administration of vasoactive drugs to alter ABP may have directly affected the SA node (14, 34), modulated cardiac vagal motorneuron firing (4), or induced a direct central effect on baroreflex function (15). Moreover, steady-state infusions of phenylephrine and nitroprusside were often used in combination with the prolonged application of neck pressure and NS. Thus it is likely that both CBR and ABR adaptation and/or resetting may have occurred (6, 8). In addition, studies using steady-state infusions of vasoactive drugs elicit sustained alterations in ABP. The maintenance of a fixed pressure at the baroreceptors may not represent a true physiological stimulus to the ABR and CBR as that occurring during more dynamic changes in ABP, such as when one stands up. This is particularly true for the carotid baroreceptors, which respond more vigorously to rapid rather than slow changes in pressure (2). To alleviate such concerns, our laboratory (33) and others (11) have attempted to present a more dynamic alteration in ABP to the baroreceptors via bolus injections of vasoactive agents. However, this technique is subject to the previously mentioned limitations inherent to administration of pharmacological agents.

Another important consideration in the examination of cardiac baroreflex control is the rapidity with which the aortic and carotid baroreceptors alter HR in response to changes in ABP. Human baroreceptor-cardiac reflex latencies are reported to be in the range of 240-475 ms (7, 21). This short response latency is due to the dominance of cardiac vagal motorneurons in eliciting baroreflex-mediated changes in HR (22). Taking this into consideration, we designed the current investigation we designed to evaluate the HR response to acute systemic hypotension evoked by unilateral arterial thigh cuff inflation-deflation alone and in combination with the gradual application of NS at the immediate onset of and throughout the period of cuff release. The NS strategy was employed to closely mimic the decrease in mean arterial pressure (MAP) below baseline and to counteract the changes in carotid sinus pressure evoked by cuff release. We rationalized that the more dynamic and transient change in ABP induced by cuff release combined with the concurrent gradual application of NS would allow for a more complete analysis of the rapid baroreflex control of HR. We hypothesized that this methodology would elucidate a greater role for the CBR in the reflex regulation of HR than has been previously reported.

	METHODS

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Ten healthy men participated in this study. The group mean (±SE) age, height, and weight were 27 ± 1 yr, 184 ± 2 cm, and 75 ± 2 kg, respectively. Each subject was advised of the testing procedures and potential risks of participation in the study and provided written informed consent, which was approved by the Ethics Committee of Copenhagen (KF 01-008/98). All subjects were free of any known cardiovascular disease and were not taking any prescribed or over-the-counter medications. Strenuous physical activity and alcohol consumption was prohibited 24 h before the experimental day, and subjects were asked to abstain from caffeinated beverages and tobacco smoking 12 h before the testing.

Measurements

Protocols

Cuff release. All testing was performed with the subjects in the supine position. Before the start of any protocol and actual data collection, each subject was familiarized with the equipment and procedures. The initial experimental protocol started with a 5-min baseline period followed by inflation of a unilateral arterial thigh cuff (300 Torr) for 9 min, after which the cuff was deflated and arterial baroreflex control of HR was assessed over the initial 20 s of cuff release. Subjects were instructed to maintain a normal breathing pattern throughout the cuff release period to minimize any confounding effects of respiration on the ensuing HR responses. We assumed that both the carotid and aortic baroreceptors were deactivated by the acute drop in ABP induced by cuff release and, therefore, the responses would accurately characterize the arterial baroreflex control of HR. A control cuff release trial always occurred first. This was necessary to examine the characteristic pattern of the ABP response (i.e., the rate and magnitude by which blood pressure fell after cuff release). After the initial control trial, another control cuff release trial and two suction cuff release trials were performed in a randomized order with ~25 min in between trials to allow all cardiovascular variables to return to basal levels.

Cuff release with NS. During the suction trials, the experimental protocol was repeated with the application of NS to the anterior two-thirds of the neck through a malleable lead collar during the initial 20 s of cuff release. The NS used was determined using a pressure transmission value of 68% (i.e., NS = control MAP/0.68). Transmission characteristics were derived taking into consideration previous (17) and more recent findings (24). Incomplete transmission of neck pressure and suction to the carotid sinus region has been well documented. Therefore, we felt that it was important to correct the NS if we were to successfully counteract the fall in pressure at the carotid sinus and minimize the contribution of the CBR to the overall arterial baroreflex-mediated HR response. Because of the rapidity with which the baroreflex-mediated HR responses occur, the application of NS was performed simultaneously with cuff release and graded to match the rate of decrease in MAP during the control trial. This was accomplished by observing the rate at which blood pressure fell after cuff release during the initial control trial as well as direct monitoring of the actual beat-to-beat changes in MAP induced by cuff release. In this way, we were able to more completely counteract the drop in carotid sinus transmural pressure and negate the CBR contribution to the HR response. Changes in estimated carotid sinus transmural pressure (i.e., MAP minus chamber pressure) from baseline were calculated over the initial 20 s after cuff release to assess the efficacy of the NS maneuver in limiting the fall in pressure at the carotid sinus. We assumed that during this phase of testing, changes in carotid sinus transmural pressure induced by cuff release would be minimized and, therefore, the responses would estimate the ABR control of HR.

NS alone. During this part of the experiment, carotid cardiac and MAP responses were examined independent of cuff release. Twenty seconds of NS at a level and pattern identical to that used to counteract the CBR during cuff release in the suction trials were applied while the subject rested quietly. Two trials were performed for each subject with a minimum of 3 min between trials. The amount of NS utilized in the current investigation ranged from 12 to 29 Torr. Subjects were again instructed to maintain a normal breathing pattern throughout the period of data collection.

Data Analyses

Statistical Analyses

	RESULTS

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Baseline and Cuff Inflation

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Changes in the content of oxygenated Hb and oxygenated myoglobin (HbO2 + MbO2) in the ischemic leg during cuff inflation and release with (suction) and without (control) neck suction (NS). Symbols denote group means ± SE and are presented as the change from the 30-s average recorded between 4.5 and 5 min of the baseline period. No differences occurred at any time point between the control and suction trials.

Cuff Release

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Changes in mean arterial pressure (MAP) and estimated carotid sinus transmural pressure (ECSP) during cuff release with and without NS. A: beat-to-beat responses from 1 subject; B: group means for all 10 subjects. ECSP was calculated as MAP minus chamber pressure. Symbols denote group means ± SE and are presented as the change from the 30-s average recorded between 8.5 and 9 min of the cuff inflation period. * Significant differences in MAP between control and suction trials (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Heart rate (HR) responses to cuff release with and without NS. A: beat-to-beat responses from the same subject presented in Fig. 2; B: group means for all 10 subjects. Symbols denote group means ± SE and are presented as the change from the 30-s average recorded between 8.5 and 9 min of the cuff inflation period. * Significant differences between control and suction trials (P < 0.05).

After release of the thigh cuff, the content of HbO₂ and MbO₂ increased ~50 µM to 16 ± 2 and 15 ± 2 µM above baseline values during control and suction, respectively (Fig. 1), indicating a similar ischemic stimulus and hyperemic response was elicited during both experimental paradigms. Likewise, changes in pulse pressure during cuff release were similar between control and suction trials. The range of changes in pulse pressure from baseline were from 0 ± 1 to 6 ± 1 mmHg during control and 1 ± 1 to 7 ± 1 mmHg during suction.

Estimated cardiac baroreflex responsiveness assessed over the initial 10 s of cuff release indicated that the maintenance of carotid sinus pressure during cuff release (suction) caused an ~50% reduction in baroreflex responsiveness (Table 2 and Fig. 4). The HR/MAP response was 0.85 ± 0.14 beats · min¹ · mmHg¹ during control and 0.40 ± 0.13 beats · min¹ · mmHg¹ during suction (P < 0.05). Similarly, the R-R interval/MAP response was 14.1 ± 2.6 ms/mmHg under control conditions and was reduced to 7.7 ± 2.8 ms/mmHg during suction (P < 0.05). These responses were consistent with results obtained from linear regression analysis of HR and MAP, which indicated a reduction of baroreflex gain in all subjects during the suction trial (Table 2). A significant relationship between HR and MAP was found during both control and suction in 8 of 10 subjects with mean r values of 0.89 ± 0.03 and 0.79 ± 0.03, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Comparison of cardiac baroreflex responsiveness between control and suction cuff release trials. Estimated cardiac baroreflex responsiveness was assessed as the change in HR or R-R interval per change in MAP, and comparisons were made at 0-5, 6-10, and 1-10 s of cuff release with and without NS. * Significant differences between control and suction trials (P < 0.05).

NS Alone

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View larger version (16K): [in this window] [in a new window]   Fig. 5.   HR, ECSP, and MAP responses to 20 s of NS alone at a level identical to that used to counteract the carotid baroreflex during the suction cuff release trials. Symbols denote group means ± SE and are presented as changes from the mean 30-s HR and MAP just before the application of NS.

	DISCUSSION

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With the use of a nonpharmacological method of unilateral arterial cuff inflation-deflation with and without the gradual application of NS, we were able to assess the relative contributions of the carotid and aortic baroreceptors to the reflex-mediated changes in HR during a dynamic and transient decrease in systemic blood pressure. Results suggest that the carotid baroreceptors play a more important role in the reflex regulation of HR during acute systemic hypotension than previously reported. In contrast to earlier studies in which ~30% of the baroreflex-mediated changes in HR were attributed to the CBR (11, 27-29, 33), we identified a nearly equivalent role for the CBR and ABR in the reflex control of HR. These findings open the possibility that alterations in arterial baroreflex function noted with aging and certain disease states such as hypertension may be mediated by the CBR to a greater extent than would be expected (16, 20). This may be particularly prominent with acute and transient decreases in blood pressure as occur during or- thostatic challenges (30). Findings in patients with surgical denervation of the carotid sinus baroreceptors support such speculation, because these individuals have severe impairment in blood pressure control during orthostasis (31). In fact, recent data indicate that bilateral carotid sinus denervation results in a chronic impairment in the rapid reflex control of blood pressure that remains up until 10 yr postsurgery in patients (32). Collectively, these findings identify the critical role that carotid baroreceptors play in controlling blood pressure in humans.

In the present investigation, the application of NS to counteract the decrease in pressure at the carotid sinus during cuff release caused a greater decrease in MAP, signifying the importance of the CBR in the maintenance of ABP. This larger drop in MAP corresponded to an attenuated increase in HR compared with control conditions when both the CBR and ABR were deactivated. Thus minimizing the contribution of the CBR to the arterial baroreflex-mediated increase in HR caused a 44% reduction in the HR response to acute hypotension (Fig. 3). These findings are in agreement with previous data indicating the importance of the CBR in the reflex tachycardic response to systemic hypotension induced by lower body negative pressure (1). Moreover, unlike the application of lower body negative pressure, unilateral cuff release does not alter central venous pressure (10), and thus aortic and carotid baroreflex responses are not confounded by the unloading of the cardiopulmonary baroreceptors. Likewise, given that central venous pressure remains unchanged, alterations in stroke volume should have been kept to a minimum and, therefore, should not have effected pulsatile vascular distention after cuff release.

Estimations of cardiac baroreflex responsiveness in the present investigation indicated that the CBR contributed importantly to the reflex control of HR during dynamic and transient decreases in MAP (Table 2 and Fig. 4). Although previous investigations have noted that both carotid and aortic baroreceptors contribute to the reflex control of HR, the overall findings have indicated that the ABR dominates arterial baroreflex control of HR with the CBR only contributing minimally (11, 19, 27, 28, 33). The discrepancy between our findings and those previously reported was most likely due to differences in experimental paradigms. Several of these investigations used steady-state infusions of phenylephrine and nitroprusside along with prolonged application of neck pressure and NS to assess baroreflex function (11, 19, 27, 28). Thus it is likely that baroreflex resetting may have occurred because estimates of aortic baroreceptor resetting have been reported in the range of 20-30 s (6). In addition, adaptations between carotid sinus transmural pressure and afferent nerve firing may occur with the prolonged application of neck pressure and NS (9). Although our laboratory (33) and others (11) have attempted to deliver more dynamic alterations in pressure by using bolus injections of nitroprusside and phenylephrine, the conclusions of these investigations also indicated an apparent dominance of the ABR in the reflex control of HR. However, it should be noted that findings from these studies might have been limited by the use of vasoactive substances. These drugs may have direct effects on sinus nodal function (14, 34), cardiac vagal activity (4) and/or central baroreceptor processing (15).

In the present investigation, we used a nonpharmacological technique of cuff release with NS (suction) and without NS (control) and discerned a significantly greater role for the CBR in the baroreflex control of HR than has previously been reported. That is, counteraction of the decrease in pressure at the carotid sinus with NS during cuff release caused cardiac baroreflex responsiveness to be decreased by ~50% (Table 2 and Fig. 4). This finding was true whether expressed as changes in the R-R interval or HR. To avoid possible baroreflex adaptation or resetting and to more completely assess baroreflex control of cardiac vagal motorneurons, estimations of cardiac baroreflex responsiveness were only assessed over the initial 10 s of cuff release. This was the period of time when MAP was decreasing rapidly and HR was continually increasing (Figs. 2 and 3). Thus neither baroreflex adaptation nor resetting should have occurred. Moreover, baroreflex responsiveness was not different between the time periods of 0-5 and 6-10 s, as indicated in Fig. 4.

Interestingly, the application of NS alone at a peak level identical to that used to counteract the CBR during cuff release indicated that CBR-mediated changes are well preserved over 20 s (Table 3). Although HR responses have been shown to be well sustained during 2 min of neck pressure and over the first 15 s of NS (19), other investigations have suggested that changes in HR or the R-R interval may be more transient when using NS (7, 8). The reason for these differences is unclear. We suggest that the application of NS during held expiration, as is commonly practiced, limits the time period that can be taken to assess CBR-mediated decreases in HR. The greatest beat-to-beat change in HR occurs early in response to NS even before it is terminated, as noted in this study (Fig. 5) and by others (7, 8). However, assessment of CBR responses over longer periods, as was permitted in the current investigation, indicates that the average change in HR, although slightly reduced, may be well preserved throughout 20 s (3).

The application of the unilateral arterial thigh cuff (300 Torr) for 9 min under resting conditions caused slight but significant increases in MAP during both the control and suction trials (Table 1). Previous investigations have indicated that under resting conditions, cuff occlusion-induced ischemia does not elicit a muscle metaboreflex-mediated increase in blood pressure (26). Thus the mechanism causing this elevation in MAP is unclear. It is plausible that the slight increases observed were the result of nocioreceptor activation; however, the subjects did not report any pain during the occlusion period. Nevertheless, metaboreceptor and/or nocioreceptor activation cannot be completely excluded. However, we suggest that the interpretation of our results would not be affected, because presumably both baroreceptor populations would be equally influenced due to common mechanisms of central neural processing (23). In addition, these stimuli were present under both control and suction conditions.

Several potential limitations in the design and interpretation of the present investigation should be considered. First, conclusions regarding the relative roles of the CBR and ABR should be interpreted cautiously due to the uncertainty of the interactive relationship between these two baroreceptor populations. Animal studies have indicated that whichever of the arterial baroreceptor populations is denervated last is the more powerful of the two; thereby, suggesting that when either baroreflex is eliminated the response of the other reflex is enhanced (12). However, the degree to which one baroreceptor population compensates for the other in humans remains unknown and will most likely never be defined because this would require denervation of one of the baroreceptor populations. Nevertheless, in the current study, even if the ABR was enhanced when NS was applied to counteract pressure changes at the carotid sinus, the HR response was still significantly attenuated. Therefore, if anything, the importance of the CBR may be even greater than suggested by the current findings. Second, despite our attempts to correct for incomplete transmission to the carotid sinus during the suction trial, some deactivation or activation of the CBR cannot be discounted because it is likely that transmission of the stimulus varies between subjects and neck collars used (17, 24). The precise effectiveness with which NS counteracted carotid baroreceptor deactivation during suction is unknown, because this would require carotid catheterization. However, on the basis of estimated carotid sinus transmural pressure, we believe that this methodology effectively minimized input to the CBR during cuff release. Third, we cannot exclude possible influences of pulse pressure on the baroreflex-mediated changes in HR (5). However, only slight and insignificant changes in pulse pressure were noted between the control and suction trials. Therefore, given that pulse pressure was not different and that the hyperemic response appeared equivalent between the control and suction trials (Fig. 1), we rationalized that the stimulus to the aortic baroreceptors was similar during all trials and that the only difference during the suction trials was the maintenance of carotid sinus transmural pressure. Fourth, we cannot fully discount possible influences on the baroreceptors evoked by cuff release itself that were not accounted for (e.g., flow changes). However, these influences would presumably be identical under both conditions of cuff release. Finally, the nonpharmacological technique of cuff release only provides a means for studying arterial baroreflex control of HR during hypotension and, therefore, care should be taken in extrapolating these data to cardiac baroreflex control during hypertension. Nevertheless, we submit that utilization of the cuff inflation-deflation technique provides an advantageous method to examine arterial baroreflex control of HR.

Because of the short response latency of the baroreceptor-cardiac reflex, we suggest that the dynamic and transient decrease in MAP induced by cuff release allowed a more complete analysis of cardiac vagal control by the arterial baroreceptors. Moreover, using this technique, it was important to counteract changes in carotid sinus transmural pressure with the simultaneous and gradual application of NS. In a previous investigation with the use of examining arterial baroreflex control of muscle sympathetic nerve activity, NS was applied when the MAP response reached its nadir after cuff release. Use of the technique in this manner elicited only small differences between control and suction HR responses (10). We reasoned that by waiting to apply NS the CBR may have contributed via cardiac vagal motorneurons to the HR response during the suction trial and, therefore, significant differences between the ABR and CBR control of HR were minimized. Findings from the present investigation suggest this was likely the case. Another distinct advantage of cuff inflation-deflation is that, unlike vasoactive drug administration, this methodology provides a means to evaluate reflex changes in ABP. Furthermore, this represents a more physiological stimulus to the ABR and CBR because blood pressure is not artificially maintained at a particular level.

In summary, we demonstrated the importance of the CBR in the maintenance of ABP as the gradual application of NS to counteract decreases in pressure at the carotid sinus during cuff release caused a greater decrease in MAP. Furthermore, the attenuated HR response elicited during cuff release with NS combined with the profound effect of CBR stimulation with NS alone on HR and MAP indicated that the CBR plays a major role in the control of ABP and reflex regulation of HR. Collectively, these data suggest that the carotid baroreceptors are pivotal to the reflex regulation of HR and maintenance of ABP during dynamic and transient decreases in systemic blood pressure. As such, changes in the sensitivity of the carotid baroreflex may significantly contribute to the alterations in autonomic control that manifest with aging or after the development of cardiovascular disease.