INTRODUCTION

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THE USE OF CHANGES in neck collar pressures to noninvasively model carotid baroreflex function in humans has been used in a variety of experimental paradigms. These include exercise (11, 12, 14), extended bed rest (3), and lower body positive (19) and negative pressures (18, 20). Along with an increased range of experimental investigations, the use of a wide diversity of neck chamber designs has been incorporated ranging from collars that enclose the entire head and neck (6) to small individual chambers that are capable of isolating unilateral carotid sinus stimulation (15). One major limitation or concern regarding this methodology is the possibility that the pressure transmitted to the carotid sinus and therefore the alteration of the carotid sinus pressure (CSP) may not be the same as the recorded external neck chamber pressure. Subsequently, calculated modeling parameters of the carotid baroreflex obtained from these data may not be representative of the true parameters of the carotid baroreflex. In 1976, Ludbrook et al. (9) examined the internal transmission characteristics of providing an external pressure stimulus to the neck in humans at rest using a 20-cm fluid-filled catheter advanced to the proximity of the carotid sinus. This classic experiment reported that the transmission of neck pressure/neck suction (NP/NS) stimuli was asymmetrical with transmissions of 64 and 86% for negative and positive pressures, respectively.

One important consideration to the chamber design is ensuring that the collar is capable of transmitting the external stimulus to the carotid sinus without obstruction. The study by Ludbrook et al. (9) did not measure the location of the carotid bifurcation and placed the catheter tip at the same anatomical location on each subject. Also, they recorded only transmission of NP/NS during resting conditions. The question of internal transmission of external NP/NS during exercise when increases in the activity of muscular tissue of the neck or elevated arterial pressure may alter carotid transmural pressure and thus transmission characteristics of external stimuli was not examined. Additionally, the variability of carotid sinus location and the surrounding anatomical structures within the sealed chamber must be considered. Furthermore, it is well known that there are specific challenges in using a fluid-filled catheter to measure tissue pressure. Guyton et al. (7) provides an extensive review and discussion of the challenges of measuring tissue pressure. Application of these limitations to the current methodology could include the possibility of the catheter tip becoming occluded by surrounding tissue or the environment of the fluid tip being altered by rapid and intense pressure changes induced by NP/NS. The data from Ludbrook et al. (9) was not confirmed by Thron et al. (24), who measured the transmission of external neck pressure to the esophagus in humans and reported nearly complete internal transmission from the external stimulus. Shubrooks (21) also found almost complete transmission to external neck pressure in dogs that were instrumented with a surgically placed balloon catheter at the carotid sinus. Recently, Smith et al. (22) utilized a balloon-tipped pressure transducer (Mikro-Tip, Millar Instruments; Houston, TX) placed into the rectus femoris muscle to examine changes in intramuscular pressure to external lower body positive pressure. They found 100% internal transmission of positive pressures of 45 Torr. These data indicate that external pressure upon the neck may be transmitted internally at a greater efficiency than previously accepted.

Since the description of a smaller and more versatile neck collar by Eckberg et al. (5), derivatives of this design have been utilized extensively and provide several advantages, including subject comfort and a better vacuum and pressure seal. There have been no studies to examine the transmission characteristics of this often-used NP/NS collar design. In the protocols that used prolonged neck pressure stimuli of greater than 5 s, confounding responses involving aortic baroreflex feedback responses to the carotid baroreflex induced changes in arterial pressure result in a decreased closed-loop estimate of maximal gain (17). Therefore, longer NP/NS perturbations likely underestimate the isolated inhibitory contribution of the carotid baroreflex in the regulation of arterial pressure.

The purpose of this investigation was to characterize the internal transmission of carotid sinus manipulation of the neck chamber adapted from Eckberg et al. (5). Two different catheters, which utilize different pressure transduction methodology, were used to record internal tissue pressure during the application of pulsatile neck pressures with two protocols that are commonly used in developing models of isolated carotid baroreflex function of humans. The NP/NS protocols were completed at rest and during exercise. In addition, we examined the variability of carotid sinus location in relation to the anatomical fit of the neck collar and any subsequent alterations in internal transmission of NP/NS stimuli.

	METHODS

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Subjects. Nineteen healthy male subjects volunteered to participate in this study. Each subject completed a written informed consent as approved by the Ethical Committee of the Fredricksberg Municipalities, Copenhagen, Denmark. The group means (±SE) for age, height, and weight were 24.6 ± 0.5 yr, 182.9 ± 2.9 cm, and 74.5 ± 2.4 kg, respectively.

Procedures. Each experiment consisted of repeated carotid sinus stimulation with the NP/NS technique. Subjects were instrumented and placed in a semirecumbent position. The NP/NS profiles were conducted both at rest, while the head was maintained in a position to allow relaxation of the neck musculature, during a Valsalva maneuver to investigate the effect of increased internal tissue pressure on transmission characteristic, and during low-intensity dynamic leg exercise. Dynamic exercise consisted of 7 min of cycle ergometry at 30% maximum oxygen uptake.

Instrumentation. For each subject the location of the carotid sinus bifurcation was determined using Doppler ultrasound (Ultrasound scanner type 2000, B&K Medical; Gentofte, Denmark). The measurement of the carotid sinus location was referenced as the distance (cm) below the gonion of the mandible (the angular point at which the mandible changes from an anterior-posterior direction to a superior-inferior direction). This reference to the mandibular ridge was utilized to examine the carotid sinus location in relationship to the anatomical fit of the NP/NS collar, which was sealed tightly along the mandibular ridge. Local anesthesia (2 ml of lidocaine) was unilaterally administered percutaneously before either one of the two types of catheters were used. The location of the anesthesia administration was determined by measuring the length of the catheter introducer from the location of the carotid bifurcation. Because of the distance from the insertion point to the location of the carotid sinus bifurcation (4.5 cm), limited quantity of anesthesia, and superficial administration above the thick fascia covering the carotid sinus region, there was minimal risk of any anesthetic effect on the free nerve endings located at the carotid sinus. In 12 subjects, internal tissue pressure (TP) at the carotid sinus was measured by placing a 2-in., 19-gauge Teflon catheter (Ohmeda) percutaneously under the fascia so that the tip of the catheter was at a depth of 1.0-1.5 cm at the level of the pulse-Doppler echoed identification of the carotid sinus bifurcation. This catheter was chosen to examine the repeatability of previous research findings of internal tissue pressure recordings from an open-ended, fluid-filled catheter. The TP measured by the catheter was transduced utilizing a sterile disposable pressure transducer (PX-260, Baxter) and a pressure monitoring system (Dialogue 2000). The location of the catheter tip was verified by transcutaneous two-dimensional and Doppler ultrasound techniques. The transducer was zeroed to the level of the carotid sinus, and the catheter was kept patent by a continuous drip of saline solution at 3 ml/h. To eliminate potential limitations of recording tissue pressure with a fluid-filled system, a balloon-tipped pressure transducer (Mikro-Tip, Millar Instruments; Houston, TX) was utilized to record TP in seven additional subjects. With the balloon-tipped catheter, two anatomical locations were utilized. First, the catheter tip was advanced to a position just inferior to the mandibular ridge. The purpose of this location was to examine the transmission characteristics of NP/NS at the border of the rim of the NP/NS collar. This position would also represent the internal transmission to the carotid sinus region in an individual with an anatomically high carotid sinus bifurcation in reference to the mandibular ridge. The second catheter location was placed at the location of the carotid bifurcation, which in most subjects occurred in the central portion of the open aperture formed by the NP/NS collar. Heart rate (HR) was measured using a three-lead configuration and processed by an electrocardiogram (ECG) data computer (Dialogue 2000).

Carotid baroreceptor function. Neck pressure stimuli (NP/NS) were applied through a cushioned malleable lead collar that was placed around the anterior two-thirds of the neck. The collar creates a sealed open chamber with the superior border on the lower mandibular ridge and the inferior border along the clavicle thus encompassing the carotid sinus location and allowing for a wide variety of anatomical variability. Graded levels of pressure between +40 and 80 Torr were generated by a variable pressure vacuum source and controlled by two-way solenoid valves (model 8215B, Asco; Florham Park, NJ). For each pulse, the pressure within the neck collar was controlled manually and the external neck chamber pressure was measured by a pressure transducer (model DP45, Validyne Engineering; Northridge, CA). To minimize the respiratory-related modulation of HR and mean arterital pressure (MAP), all carotid baroreflex perturbations were conducted during a held breath at end expiration.

Data and statistical analysis. Sections of NP/NS data collection were separated from the raw data file and analyzed for internal transmission of the externally applied stimulus. The transmission percentage was the recorded internal TP divided by the recorded external chamber pressure multiplied by 100% [% transmitted = (TP/chamber pressure) × 100]. In addition, the latency between the external application and internal transmission of pressure stimuli was analyzed. Statistical analysis of the effect of applying correction factors for the fluid-filled catheter, balloon-tipped catheter, or no correction factor (uncorrected) was completed using one-way analysis of variance and Student-Newman-Keuls post hoc analysis. All variables are represented as means ± SE. Significance was accepted at P < 0.05.

	RESULTS

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Anatomical location of carotid sinus. The Doppler-measured anatomical locations of the carotid sinus bifurcation in the initial 12 subjects indicated wide variability from 0 to 7 cm below the gonion. To determine whether this variability was repeatable and descriptive of the population or only a rare occurrence, Doppler ultrasound was conducted on 83 additional randomly selected hospital employees (n = 95). Figure 1 illustrates the frequency distribution of the location of the right and left carotid sinus bifurcation referenced as the distance below the gonion of the mandible. The location of the carotid sinus bifurcation by Doppler ultrasound in the total sample population measured 3.2 ± 0.1 cm (median 3.0 cm, range 0-6 cm) on the right side and 3.6 ± 0.2 cm (median 3.5 cm, range of 0-7.5 cm) on the left side.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Frequency distribution of right and left carotid sinus bifurcation. Location of right and left carotid sinus bifurcation was measured from the gonion of the mandible (n = 95). Bifurcation location determined by Doppler ultrasound. Location of the right bifurcation was 3.2 ± 0.1 cm (median 3.0 cm, range 0-6.5 cm) and the left bifurcation was 3.6 ± 0.2 cm (median 3.5 cm, range 0-7.5 cm) below the gonion (angle of the mandible). Values reported are means ± SE.

Transmission characteristics of NP/NS. The details of the transmission characteristics across the dynamic range of NP/NS stimuli for both protocols are shown in Table 1. The results from both positive and negative pressure transmitted through the open fluid-filled catheter were not significantly different (P > 0.05) to those previously reported by Ludbrook et al. (9) with 84.7 ± 1.3 and 64.2 ± 1.7% transmitted internally for positive and negative pressures, respectively. The transmission characteristics of external NP/NS were significantly higher (P < 0.05) than previously reported fluid-filled catheter data when measured by a balloon-tipped catheter. Positive pressure was transmitted at 89.1 ± 1.4%, whereas negative pressure was 82.4 ± 1.9% when recorded at the average anatomical location of the carotid sinus within the open aperture of the sealed collar. The internal transmission at the higher catheter location near the rim of the sealed collar was significantly reduced (P < 0.05) to 62.0 ± 3.0 and 44.0 ± 3.7% for positive and negative pressures, respectively, compared with the lower recording site at the carotid sinus bifurcation.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Rapid pulse train carotid baroreflex stimulus and cardiovascular reflex responses. Schematic illustration represents one rapid pulse carotid baroreflex train (+40 to 80 Torr) and the resulting reflex responses in heart rate (A) and blood pressure (B). C: pressure recorded within the external neck chamber. D: pressure recorded at the carotid sinus with the fluid-filled catheter. E: expanded overlap view of the external chamber pressure and internal neck pressure recorded by the balloon-tipped catheter on a separate rapid pulse train illustrating the temporal congruency and improved internal transmission.

	DISCUSSION

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The major findings in this investigation were that 1) the transmission of external neck pressure to the carotid sinus tissue was higher than had been previously reported (9); 2) the transmission profiles were unchanged from rest to exercise; 3) an increase in local tissue pressure from the Valsalva technique did not affect the transmission profiles; and 4) correction of internal transmission resulted in minimal and nonsignificant changes in calculated carotid baroreflex parameters. The data collected utilizing the open fluid-filled catheter for 5-s pulses resulted in transmission recordings very similar to those reported by Ludbrook et al. (9). However, the pitfalls that may occur with the measurement of tissue pressure by a transmission methodology designed for a fluid-fluid interface resulted in some major inaccuracies. Tissue occlusion of the catheter tip or alterations in the fluid-tissue environment, as could occur with the rapid, high-intensity pressure changes of NP/NS, may have accounted for at least part of the incomplete transmission recorded with the open fluid-filled catheters. The balloon-tipped catheter does not rely on a fluid interface. Therefore, the transmission of external pressure recorded with this method does not have the same limitations in recording tissue pressure and would reflect tissue pressures more faithfully. Whereas in some subjects, the transmission recordings in the fluid-filled catheter did not reflect the square-wave generation recorded by the neck chamber pressure transducer and resulted in a curvilinear pressure profile, the balloon catheter demonstrated insignificant delay in transmission of external pressure and mirrored the external neck chamber pressure square-wave pressure change. Additionally, the reduced pressure transmission recorded with the balloon catheter at the superior position of the rim of the collar indicated that internal transmission of NP/NS was not uniform within the aperture of this collar design. This discovery has critical relevance in those cases where the anatomical location of the carotid sinus lies in a superior/inferior location approximating the rim of the sealed neck chamber and to those investigators using the single-sided chamber design (15).

Importance of carotid sinus location and collar position. The wide variation in the location of the carotid sinus had not been reported with regard to potential methodological problems with the NP/NS technique. To illustrate the diversity that may occur in the anatomical location of the carotid sinus, magnetic resonance images (MRI) of the carotid bifurcation were obtained and matched to the cervical vertebra images of two individuals (see Fig. 3). The Doppler ultrasound measurement of the carotid sinus for the individual in Fig. 3A was measured at the point of the gonion (0 cm) of the mandible. The MRI image placement of the carotid sinus in Fig. 3A was at the C2-C3 vertebral level. This vertebral locus was higher than the commonly used thyroid cartilage anatomical landmark, which occurs at the level of C3-C4. The Doppler ultrasound measurement of the carotid sinus for the individual in Fig. 3B was measured at a point 7 cm below the gonion of the mandible. The MRI image placement of the carotid sinus in Fig. 3B was at the C7 vertebra. The sealed neck collar was placed along the lower mandible with the rim of the collar in contact with the gonion. This was the purpose of utilizing the gonion of the mandible as an external landmark rather than the thyroid cartilage. Although the average carotid sinus location was ~3 cm below the gonion of the mandible, the observed variability of the anatomical location coupled with the decreased internal transmission characteristics recorded just below the rim of the sealed collar does bring into question the possibility of poor transmission of the applied stimulus in individuals with a carotid sinus anatomical location near the gonion of the mandible or the clavicle.

View larger version (86K): [in this window] [in a new window]   Fig. 3.   Magnetic resonance images of variation in carotid bifurcation location. Images of two individual subjects that represent the range of the anatomical location of the carotid sinus bifurcation. Arrow, location of the carotid bifurcation. Dotted line, location of the carotid bifurcation to the corresponding level of cervical vertebrae. The carotid bifurcation for subject A measured externally 0.5 cm below the gonion (angle of the mandible) by Doppler ultrasound and corresponded to C3 level. The carotid bifurcation for subject B measured externally 7.0 cm below the gonion, corresponding to the C7 level. Anatomical variation in carotid sinus location may be of concern with specific neck collar designs and the neck pressure/neck suction technique.

Transmission of external pressure stimuli during exercise. One concern of utilizing NP/NS with exercise was that increases in tension of the musculature of the neck would further attenuate the transmission of external pressure or during the sustained 5-s NP/NS protocol the internal pressure would diminish due to tissue adaptation. In reference to increased muscular contraction attenuating an external pressure stimulus, a recent study by Smith and colleagues (22) found that the intramuscular pressure transmission of a sustained external positive pressure (lower body positive pressure of +45 Torr) was maintained during rest and throughout maximal muscular contraction. Also, Aratow and colleagues (1) reported further uninhibited transmission of lower body negative pressure to the muscle of the thigh during contraction. Therefore, it was unlikely that the transmission characteristics of NP/NS would be diminished or altered even with mild to moderate tension increases in the neck musculature due to the fact that transmission of positive pressure and the concurrent changes in tissue was uniform even at elevated external pressures (80 Torr). The findings of the present investigation indicated that the transmission percentages of NP/NS measured by the balloon-tipped catheter were consistent between rest and exercise conditions and even with increased muscular and tissue pressure due to a Valsalva technique. Therefore, calculations based on estimated CSP would include the same error across experimental conditions, and any reflex response changes measured would then reflect alterations in the integration of afferent information from the altered CSP and the reflex peripheral responses rather than alterations in pressure transmission characteristics.

Effect of correcting CSP on carotid baroreflex parameters. One area that would benefit from correcting the estimated CSP and calculated carotid baroreflex for accurate transmission percentages is the comparison between animal and human studies on carotid baroreceptor function. Animal studies often have the advantage of direct measurement of CSP (4, 10, 23, 25, 26). The measured variables of reflex function in these studies should reflect very accurate physiological values and are often different from the reported variables in human studies (2, 11, 13, 14, 16) where estimations of CSP alteration are mathematically derived. Additionally, animal studies often use methodological approaches that allow cardiac output and peripheral resistance to reach more of a steady-state environment to evaluate carotid baroreflex function. This steady-state evaluation is difficult to reproduce in human investigations using NP/NS. The short stimulus duration in rapid pulse and 5-s protocols reflect both parasympathetic and sympathetic components, although there is inadequate time for maximal sympathetic responses to occur. The consistently higher calculated gains reported in animal studies may in part be attributed to technical differences in experimental design. However, the correction of the external chamber pressure for transmission percentage with the neck pressure technique in human studies may bring the calculated reflex gains between animal and human studies closer together.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of correcting carotid sinus pressure stimulus on the logistic curve fit and calculated parameters. The correction of the measured neck chamber pressure stimulus for the internal transmission characteristics recorded with a fluid-filled catheter result in an increased maximal gain, and threshold pressure and a reduced saturation pressure compared with values obtained with transmission correction from a balloon-tipped catheter or without a correction factor. Curves are representative of carotid baroreflex data from 10 subjects/20 rapid pulse trains. Group data for maximal gain, threshold and saturation are means ± SE. *Significant difference between parameters calculated with fluid-filled catheter correction and uncorrected calculated carotid baroreflex parameters (P < 0.05). There was no significant difference in calculated carotid baroreflex parameters between the balloon-tipped correction factor and uncorrected calculations (P > 0.05).