| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
Circulatory Control Laboratory, Department of Physiology and Bioengineering Institute, University of Auckland, Auckland, New Zealand
Submitted 24 March 2005 ; accepted in final form 24 February 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
telemetry; central nervous system
Within the literature, the term baroreceptors has been loosely defined as receptors located within the periphery that sense pressure e.g., cardiopulmonary, renal, arterial, etc. With regard to the bursting pattern of SNA, it has been implied that it is the signal from arterial baroreceptors, i.e., carotid and aortic receptors, that provides the timing cues for when the bursts should occur. Timing in this context indicates the relation of an individual burst to the arterial pressure wave. Thus phasic input from the receptors in the carotid sinus and aortic arch provide information to the central nervous system that regulates two of the key features of SNA: the timing of bursts and, importantly, changes in the mean level of SNA in response to short-term changes in blood pressure. Therefore, the regulation over the average level of SNA and the timing of when bursts occur has been thought to be regulated by the same central nervous system cell groups and processes.
With the development of tools for telemetry-based long-term recordings of SNA (3), it has now become possible to explore the influence of the various reflex pathways in the control of SNA under "home cage" conditions. In this study, we examine the possibility that the mechanisms regulating the mean level of SNA in response to short-term changes in blood pressure may differ from the key cueing signal that regulates the timing of the bursts of SNA.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Experiments were conducted in 24 New Zealand White rabbits of either sex with initial weights of 2.4 to 3.5 kg and were approved by the University of Auckland Animal Ethics Committee. The rabbits were housed individually in cages (height = 45 cm, width = 65 cm, and depth = 65 cm). The rabbits were fed daily (100 g standard rabbit pellets, supplemented with hay, carrot, and apple) at 0900, and water was available ad libitum. The room was kept at a constant temperature (18°C) and dark-light cycle (lights on from 0600 to 1800).
Anesthesia was induced using intravenous administration of propofol (Diprivan; 10 mg/kg) followed by intubation and then maintenance with halothane. The rabbits were divided into two groups, arterial baroreceptor intact and denervated (SAD). All rabbits were instrumented with a radiotelemetry transmitter to record arterial pressure (model PA-D70; Data Sciences International, St Paul, MN). This was implanted via an abdominal incision, and the area around the iliac bifurcation was exposed. The cannula of the transmitter was inserted into a branch of the iliac artery (circumflexa ilium profunda) and advanced so that the tip of the catheter lay in the abdominal aorta, 
1 cm above the iliac bifurcation but well below the renal artery. The cannula was tied into position, the body of the transmitter was placed in the abdominal cavity, and the incision was closed. During the same procedure, the rabbits also underwent either SAD (n = 9) or a sham surgery (n = 10) where the baroreceptor nerves were exposed but not cut. The aortic depressor nerves were located in the cervical region between the vagus and the sympathetic trunk using a dissecting microscope; all nerves branching off either the vagus or the superior laryngeal nerve were separated free and sectioned near their junction. The left and right carotid sinuses were exposed and arterial baroreceptors denervated by cutting all the visible nerves between the internal and external carotid arteries and stripping these vessels.
Validation of SAD
Before implantation of the nerve recording amplifier, the efficacy of the SAD was confirmed under conscious conditions by examining the heart rate responses to a rapid infusion of phenylephrine (100 µl of 1 mg/ml iv) and sodium nitroprusside (100 µl of 1 mg/ml iv) over 30 s. Only animals with a heart rate change of no more than 10 beats/min in response to a 20-mmHg increase or decrease in blood pressure were subsequently considered to be SAD. In sham-operated (intact) rabbits, the change in heart rate was between 80 and 110 beats/min for a similar change in arterial pressure.
At least 2 wk after SAD surgery, a telemetry-based implantable nerve amplifier [model 2003/01; Telemetry Research, Auckland, New Zealand, (www.telemetryresearch.com)] was inserted via a flank incision with the electrodes coiled around the left renal nerve, and the electrode and nerve were coated in a Silicone elastomer (Kwik-sil; World Precision Instruments) (3). To avoid movement artifacts affecting the SNA signal, the implantable amplifier was placed as close to the nerve site as possible.
After each surgery, the rabbits were treated prophylactically with an antibiotic (enrofloxacin, Baytril; Bayer; 5 mg/kg sc daily for 5 days) and analgesic (ketoprofen, Ketofen; Rhone Merieux, Essex, UK; 2 mg/kg sc, daily for 3 days). As soon as the rabbits regained consciousness they were returned to their home cages. A heating pad was placed in the cage for 24 h after surgery.
One possible cause of an artifact is, that under some conditions, the bursts of SNA were somehow due to movement of the nerve-electrode assembly and thus an artifact of the recording procedure. To test the background noise level in three separate conscious baroreceptor-intact and three separate SAD rabbits, at least 3 days after nerve electrode implantation surgery, all SNA discharges were removed with ganglionic blockade (5 mg/kg bolus of pentolinium). The blood pressure and SNA responses for the first 3 min after injection were recorded, and the minimum blood pressure and SNA recordings obtained during this period were measured.
Anesthetized Experiments
In addition to the conscious animal experiments, two further series of experiments were conducted in an effort to determine which nerves were involved in the regulation of the timing of the sympathetic bursts and to consider the possibility that anesthesia effects the entrainment of sympathetic bursts.
Series one. In seven of the rabbits that had already been used for the conscious experiment, and thus SNA electrodes had already been implanted (animals were baroreceptor intact), anesthesia was induced via pentobarbital sodium (90–150 mg; Virbac Laboratories) and maintained by pentobarbital sodium infusion [30–50 mg/h, as previously described (9, 10)]. Animals were artificially ventilated. After recording a period of control SNA, a midline incision in the neck region was made, and the vagus was identified running alongside the carotid artery. Using an operating microscope, bilateral section of the vagus, carotid sinus nerves, and aortic depressor nerves was performed. The order in which the nerves were cut was randomized between animals.
Series two. In six baroreceptor-intact rabbits in which SNA electrodes had been implanted 5 days previously (surgical procedure as above), anesthesia was induced using intravenous administration of propofol (10 mg/kg), followed by intubation and then maintenance with halothane. While SNA and blood pressure were continuously recorded, the nerves of the arterial baroreceptors were cut (see surgical procedure in Series one). The average change in heart rate to phenylephrine and nitroprusside was 8 ± 3 beats/min denervated vs. 215 ± 6 beats/min intact rabbits. Only animals with a heart rate change of no more than 10 beats/min in response to a 20-mmHg increase or decrease in blood pressure were subsequently considered to be SAD. Animals were then allowed to recover from the anesthesia, and SNA was recorded for 2 days. Arterial baroreflexes and the timing of sympathetic discharges were determined under the following conditions: 1) conscious arterial baroreceptors intact, 2) anesthetized baroreceptors intact, 3) anesthetized with arterial baroreceptors denervated, and 4) conscious arterial baroreceptors denervated. Baroreceptor denervation was confirmed (same procedure as above) under conscious conditions 2 days after the baroreceptor denervation surgery. The recording of SNA was maintained throughout all these procedures.
Data Collection
The SNA signals were amplified between 10 and 20,000 times and band pass filtered between 50 and 5,000 Hz. This signal was used to audibly check the quality of the recording. The amplified signal was also full-wave rectified and integrated with a time constant of 20 ms (2, 3). The use of the integrator, often described as a leaky integrator, produces a signal that displays the bursts as a series of peaks or waves (14). All subsequent analysis was performed on this integrated signal. Quantification of the individual bursts of SNA was made using an automated algorithm detailed previously (14) in which bursts were defined on the basis of their crossing of a threshold (set at 20% of the average maximum burst amplitude). Briefly, two moving average low-pass filters with short- and long-time constants were used to assist in defining what constituted a burst based on an approximate shape. A refractory period was also used to define a minimum interval between bursts (normally set at 50 ms). This method allows every sympathetic burst to be identified and then its amplitude and interval to the next burst calculated.
In the experiments on conscious rabbits, the recording of both arterial pressure and SNA via telemetry allowed monitoring to take place remotely with rabbits housed in their home cages. Data for analysis was obtained 3–14 days after implantation surgery. In two animals, data were collected while animals were receiving antibiotic treatment. All analysis was done on data obtained with animals in awake conditions but not moving. In each animal, data were collected over several time periods for between 5 and 15 min throughout the day. In all experiments, the baroreflexes were determined again as previously described to obtain SNA data. Data were sampled at 500 Hz using an analog-to-digital data acquisition card (cat. no. AT-MIO64E-3; National Instruments, Austin, TX). All subsequent data collection and analysis were performed using a data acquisition program (Universal Acquisition and Analysis version 11; Telemetry Research) as previously described.
Statistical Analysis
Researchers were not blinded to the surgical status of the animals. For calculation of the overall mean levels of blood pressure and SNA, the 500-Hz sampled signal was binned every 2 s and saved to disk. The analysis of SNA derived from triggering off the blood pressure signals (see RESULTS) was conducted using data sampled at 500 Hz. All data was analyzed using a Student's unpaired t-test. The tests were considered significant if P < 0.05. Data are shown as the means ± SE.
Spectral Analysis
In seven conscious arterial baroreceptor-intact and seven conscious SAD rabbits, power spectral density was calculated for both blood pressure and SNA signals using MatLab (The Mathworks, Natick, MA). Five-minute segments of stationary data sampled at 500 Hz were split into four segments overlapping by 50% (60,000 data points/segment). A Hanning window was applied to each segment, and the average power spectral density plot was found from the four segments. In each animal, a distinct peak was seen at the frequency of the heart rate. The frequency span of this peak was found from the blood pressure spectrum. The power spectral density of SNA was then integrated over this frequency range and is presented as a percentage of the total power in the SNA signal. Transfer function analysis was used to calculate the gain between blood pressure and SNA (8) using the same data segments as above. Briefly, the cross spectrum of blood pressure and SNA was divided by the autospectrum of blood pressure, again using Welch's method and MatLab. The average gain was calculated across the heart rate frequency span found earlier. To give an indication of how well the blood pressure and SNA signals were coupled, coherence was also calculated (8). This is presented as the maximum coherence at the heart rate frequency.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Over a 24-h period of recording, arterial pressure demonstrated characteristically greater variability in the SAD rabbits compared with intact rabbits, as evidenced by the standard deviation of the 2-s averages of arterial pressure (9.4 ± 0.4 mmHg SAD vs. 5.8 ± 0.9 mmHg intact, P < 0.05). Baseline mean arterial pressure was not significantly different between the intact and SAD rabbits (83 ± 3 mmHg intact vs. 92 ± 4 mmHg SAD, 24-h means). Heart rate was significantly higher in SAD rabbits (256 ± 7 vs. 235 ± 7 beats/min).
Although one must take care in comparing SNA levels between animals, because differences in the contact between the nerve and recording electrode has the potential to alter the amplitude of the signal recorded, no significant differences in resting SNA levels between intact and SAD rabbits were apparent, either when measured as direct voltage levels or normalized to the maximum SNA achieved during a nasopharyngeal stimulation [50 ml of cigarette smoke puffed into the face of the rabbit over 2 s, (3)] (30 ± 6 units or 16 ± 3% in the intact rabbits vs. 35 ± 8 units or 21 ± 2% in the SAD rabbits). As described in MATERIALS AND METHODS, we were careful to confirm that in each SAD animal there was <10 beats/min change in heart rate in response to changes in the mean level of blood pressure, i.e., animals were sinoaortically denervated. This was confirmed again with regard to SNA once the amplifier had been implanted (Fig. 1). In intact rabbits, in response to full baroreflex determination [phenylephrine and sodium nitroprusside as previously described (3, 13)], the average range of SNA and heart rate was 36 ± 6 normalized units and 215 ± 10 beats/min, respectively.
|
Pattern of Sympathetic Bursts in Arterial Baroreceptor-Denervated Rabbits
Examination of the ongoing pattern of bursts of SNA indicated no differences between SAD and intact rabbits. Initial visualization was unable to identify which animals were SAD or arterial baroreceptor intact. When the individual bursts of SNA were detected using an algorithm based on the crossing of slow and fast filters (14), it was apparent that the variation in the burst amplitude was similar between SAD and intact rabbits, i.e., amplitude and width of the SNA bursts were similar for SAD and intact animals (11) (Fig. 2). The width of the bursts remained proportional to the amplitude of the bursts and was not altered in SAD rabbits.
|
Using the systolic pressure as a trigger, averaged recordings of SNA were obtained for seven SAD and seven intact rabbits. In each case, a distinct phase relationship between blood pressure and SNA was present in both SAD and intact rabbits (Fig. 3). The time between the diastolic pressure and the next maximum SNA voltage averaged 107 ± 12 vs. 105 ± 7 ms (SAD vs. intact, respectively), and the time between the systolic pressure and the next maximum SNA voltage averaged 144 ± 7 vs. 161 ± 13 ms (SAD vs. intact, respectively, not significant) (note that because of the variation in the heart rates between animals, it is not possible to produce an average figure across the group of animals for the timing relationship between blood pressure and SNA). In the triggered, averaged recordings of SNA from individual animals (Fig. 3), it was apparent in all animals that the fluctuation of the mean level of SNA with the cardiac cycle accounted for a significant percentage of the overall mean level of SNA. For example, in rabbit 1 (Fig. 3, top, left) the average range of SNA over the cardiac cycle is from 20 to 50 units. Even taking into account variations in the noise level between animals (generally, <10 units) this indicates that the overall mean level of SNA is predominantly composed of bursts of SNA that occur at a certain phase of the cardiac cycle and that the probability of bursts outside this phase relationship is low. This was not different between intact and SAD rabbits.
|
Short (5-min) segments of stationary SNA and blood pressure data underwent spectral analysis to determine the spectral power associated with the heart rate. In each animal, a distinct peak associated with the heart rate was evident in the power spectral density plot (Fig. 4), and the power associated with that peak was determined by calculating the area under that portion of the spectrum (12). In intact animals, 19 ± 4% of spectral power was around the heartbeat frequency. This was not significantly different from that seen in SAD animals (16 ± 3%). Transfer function analysis between blood pressure and SNA was used to determine the gain between the signals. If sympathetic discharges in SAD animals had lost their timing relative to the cardiac cycle, the transfer function gain would be expected to be reduced. However, average gain was 10.4 ± 4.3 in intact animals vs. 8.0 ± 1.7 in SAD animals (gain was calculated as the integration under the curve). A high coherence between blood pressure and SNA at the heart rate frequency in SAD animals also indicated that strong coupling remained between blood pressure waveform and the timing of SNA discharges (the mean of the maximum coherence was 0.67 ± 0.05 and 0.74 ± 0.08 in intact and SAD groups, respectively). The average coherence over the frequency window assigned for heart rate was 0.27 ± 0.03 (intact) vs. 0.40 ± 0.07 (SAD) animals.
|
One possibility is that the cardiac-related nature of the bursts of SNA was somehow due to movement of the nerve-electrode assembly and thus, an artifact of the recording procedure. This is unlikely because the complete loss of all sympathetic bursts was observed with ganglionic blockade (pentolinium 5 mg/kg bolus) in both intact and SAD rabbits (Fig. 5). Blood pressure was reduced in both groups between 20–30 mmHg (average reduction in blood pressure 24 ± 5 mmHg).
|
Series one: serial denervation. Initially under baroreflex intact anesthetized conditions (pentobarbital sodium anesthesia) the timing of SNA discharges was not different from the conscious condition as above (diastolic pressure to next maximum SNA voltage 107 ± 12 ms). Removal of either the carotid sinus nerve, the aortic depressor nerve, the vagus alone or the vagus and aortic depressor nerve together did not affect the timing of the SNA discharges and had only a minimal effect on the size of the bursts. Only when the carotid sinus nerves and aortic depressor nerves had both been cut, regardless of whether the vagotomy had been performed, was the entrainment between the SNA bursts and cardiac cycle eliminated (Fig. 6). Animals were maintained under anesthetic for a further 1 to 3 h but the timing relationship between SNA and blood pressure did not return.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Ongoing SNA has been classically described as having several characteristic features. First, that the activity of the postganglionic nerve fibers forms bursts where many of the individual axons fire at approximately the same time. Furthermore, although these bursts vary greatly in their amplitude, they occur at a regular phase of the pulse wave cycle, i.e., bursts are entrained. The experiments of Barman and Gebber (1) showed that after removal of baroreceptors, sympathetic discharges, while still occurring in bursts, were no longer entrained to the arterial pulse wave cycle. Such experiments confirmed that the central nervous system was inherently capable of generating bursts of SNA. The final classical feature is that the mean level of SNA is highly responsive to changes in arterial pressure. Thus an understanding has emerged that phasic input from arterial baroreceptors provides information to the central nervous system that regulates two of the key features of SNA: 1) the timing of bursts and 2) the mean level of SNA in response to short-term changes in blood pressure. Therefore, the regulation over the average level of SNA and the timing of when bursts occur has been thought to be regulated by the same central nervous system cell groups and processes. However, our results partly challenge these assertions because we observed in conscious animals that even when the mean level of SNA was unresponsive to changes in blood pressure the bursts were still being entrained by the cardiac cycle. Subsequent experiments in anesthetized animals indicated that immediately after the SAD procedure, the entrainment of sympathetic bursts was lost under anesthesia, but this returned quickly as the animals recovered from the anesthesia.
Our initial interpretation from the conscious animal studies was that the arterial baroreceptors do not regulate the timing of the sympathetic discharges but rather regulation arises from nonarterial baroreceptor inputs, such as cardiopulmonary regions. However, our experiments in anesthetized animals, in which baroreceptor denervation removes the entrainment, seem to complicate this assertion. Under anaesthetized conditions, clearly, arterial baroreceptors do regulate the timing of the sympathetic discharges; however, it would appear that there is some redundancy in the system, such that under conscious conditions, afferent information from other sources is sufficient to allow the SNA to remain entrained to the cardiac cycle. Possible sources of afferent information allowing the entrainment of the SNA bursts to the cardiac cycle are either the cardiopulmonary or aberrant arterial baroreceptor afferents traveling in the vagus. A limitation of the present experiments is that we were not able to precisely determine the source of the afferent information. Although we assume that the remaining afferents are carried in the vagus, no experiments in conscious animals with vagal denervation were carried out because of the adverse effect such a denervation has on the welfare of the animals. Given that the vagus remained intact in all of our conscious rabbit experiments, we conclude that, although the timing input is initially disturbed after the SAD procedure, the baroreceptor afferents within the vagus are sufficient to entrain the sympathetic bursts to the arterial pressure waveform, even if those afferents are not capable of regulating the mean level of SNA.
One important finding obtained from the halothane anesthetized experiments was that the entrained time interval between the diastolic pressure and the maximum SNA does not appear to be fixed. Animals under anesthesia had a considerably longer time interval (Fig. 7). In addition, there was considerable variation between animals in the time interval. The focus of our study is on the nature of the timing relationship rather than a detailed quantitative analysis of how the timing may be altered by different stimuli. Clearly, this could be a fruitful area for further investigation. Similarly, it should be noted that these data were obtained in rabbits and translation to other species would need further studies.
Close examination of the original neurogram from a single SAD rabbit in Fig. 2 shows a high level of ongoing SNA bursts where there are often more than one burst per cardiac cycle. On first glance, this may appear to negate our proposal that bursts are entrained to occur at a particular period in the cardiac cycle; however, the averaged analysis shown in subsequent figures (e.g., Fig. 3) reflects the probability when a burst occurs, i.e., a high probability at a certain phase and lower probability outside this period. Therefore, bursts can and do occur at different periods of the cardiac cycle, but their probability is highest 100–120 ms after the diastolic period. Importantly, this patterning was not altered by baroreceptor denervation. The phenomenon of why more than one burst of SNA occurs per cardiac cycle cannot be explained on the basis of the data obtained in the present study, but it is an important area for future research.
Arterial baroreceptor denervation is a common approach used for exploring neural control of the cardiovascular system (17–20). However, it is also likely that section of the aortic and carotid sinus nerves does not remove 100% of arterial baroreceptor fibers (5). The classical approach to assessing whether animals are completely baroreceptor denervated or not is the absence of a heart rate of SNA response to a brief pressor or depressor agent (22). Although cutting the aortic depressor and carotid sinus nerves may eliminate the baroreflex response to changes in blood pressure, our results indicate that this does not necessarily mean that the central nervous system is not receiving any information on blood pressure. It would seem that the central nervous system responds to the remaining afferent nerve fibers, such as those traveling in the vagus in a "sensitive trigger-like" fashion, i.e., it is possible that a very small input is sufficient to entrain the total SNA output to a certain phase of the cardiac cycle. These observations suggest that the experimental approach of arterial baroreceptor denervation requires care when interpreting the resulting data.
In conclusion, we propose that the central mechanisms regulating changes in the mean level of SNA in response to changes in blood pressure appear to be different from those mechanisms regulating the timing (entrainment) of the sympathetic bursts.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
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 |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S.-J. Guild, C. J. Barrett, F. D. McBryde, B. N. Van Vliet, and S. C. Malpas Sampling of cardiovascular data; how often and how much? Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R510 - R515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Booth, L. Bennet, C. J. Barrett, S.-J. Guild, G. Wassink, A. J. Gunn, and S. C. Malpas Cardiac-related rhythms in sympathetic nerve activity in preterm fetal sheep Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R185 - R190. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
