Extensive studies in the adult have demonstrated that the sympathetic nervous system plays a central role in cardiovascular control. The maturation of the sympathetic nervous system before birth is poorly understood. In the present study, we directly recorded renal sympathetic nerve activity (renal SNA) in five preterm fetal sheep (99 ± 1 days gestation; term is 147 days). Recordings were performed in utero using a telemetry-based technique to alleviate movement artifact without anesthesia or paralysis. The preterm fetuses exhibited a coordinated discharge pattern in renal SNA, indicating many individual neurons active at approximately the same time. This is consistent with that observed previously in adult animals, although the frequency of the bursts was relatively low (0.5 ± 0.1 Hz). The discharges in renal SNA were entrained to the cardiac cycle (average delay between diastolic pressure and maximum renal SNA 319 ± 1 ms). The entrainment of the sympathetic discharges to the cardiac cycle indicates phasic baroreceptor input and that the underlying circuits controlling SNA within the central nervous system are active in premature fetuses.
- renal sympathetic nerve activity
although the sympathetic nervous system significantly contributes to cardiovascular control in the adult sheep, its role in fetal life is not well understood. Indirect pharmacological studies suggest that there may be basal sympathetic tone from early in gestation (2, 24). However, the only previous direct recordings of sympathetic nerve activity (SNA) in the fetus were performed near term (16, 27, 30). Further, other than the recent report from Lumbers et al. (16), these studies were performed under general anesthesia and fetal paralysis, which are not practical for significant periods of recording and are likely to substantially alter central nervous system (CNS) control of cardiovascular function (9). Thus it is important to develop ways to study SNA in fetal life without anesthesia or paralysis.
Ongoing SNA recorded in both humans and animals is classically described as displaying a coordinated bursting pattern, resulting from synchronized firing of many individual neurons at approximately the same time (17). This pattern is tightly regulated by specific cell groups within the CNS, which are believed to receive continual pulsatile input from baroreceptors, entraining the subsequent bursts of SNA to occur at a certain period of the cardiac cycle (1, 4, 8, 10, 18, 31). Over the past 15 years, a number of CNS regions have been identified as important to the regulation of the mean level and pattern of SNA in response to changes in blood pressure (11, 12, 21, 23, 28). Although the precise control mechanisms remain elusive, the timing and recruitment of sympathetic neurons are indicative of a highly regulated and complex system. An important unanswered question is whether this patterning and control are evident during fetal development before term?
If the fetus were shown to produce SNA with characteristics similar to that seen in the adult, it would be indicative of the development of the regulatory processes within the CNS and cardiovascular control in general. It would also provide an important framework for understanding how the fetus adapts to challenges in utero such as hypoxia and hemorrhage. In the present study, renal SNA was directly measured in chronically instrumented 0.7 gestation fetal sheep. Brain development at this age is equivalent to the preterm human fetus (28–32 wk gestation) (22). SNA recordings were performed using a telemetry-based technique. By placing the amplifier close to the nerve and removing the connection between the amplifier and the external lead, artifact was minimized, allowing continuous recordings to be performed in utero without anesthesia or paralysis. Two main questions were addressed by this study: 1) Does the preterm fetus have synchronized discharges of renal SNA? 2) If present, are discharges of renal SNA entrained to the cardiac cycle?
All procedures were approved by the Animal Ethics Committee of the University of Auckland. Five times-mated singleton Romney/Suffolk fetal sheep were instrumented at 99 ± 1 days gestation (term = 147 days). Food, but not water, was withdrawn 18 h before surgery. Ewes were given 5 ml of streptocin [procaine penicillin (250,000 IU/ml) and dihydrostreptomycin (250 mg/ml); Stockguard Labs, Hamilton, New Zealand] intramuscularly for prophylaxis 30 min before the start of surgery. Anesthesia was induced by intravenous injection of Alfaxan (alphaxalone, 3 mg/kg, Jurox, Rutherford, Australia), and general anesthesia was maintained using 2–3% halothane in O2. Ewes were allowed to breathe spontaneously, and the depth of anesthesia, maternal heart rate, and respiration were constantly monitored by trained anesthesiological staff. Under anesthesia, a 20-gauge catheter was placed in a maternal front leg vein for the duration of surgery, and the ewes were placed on a constant infusion isotonic saline drip (at an infusion rate of ∼250 ml/h) to maintain maternal fluid balance.
All surgical procedures were performed using sterile techniques (6, 25, 26). The fetal head and upper chest were exposed through a midline cesarean incision and a small incision in the uterus. Polyvinyl catheters were placed in the fetal right brachial artery and the amniotic sac. A Teflon-coated, stainless-steel electrode (Cooner Wire, Chatsworth, CA) was sewn in the nuchal muscle to record electromyographic activity (EMG) as a measure of fetal movement, and a reference electrode was sewn over the occiput. ECG electrodes (Cooner Wire) were sewn across the fetal chest to record the fetal heart rate (FHR). The uterus was then closed in layers, and a second incision was made to expose the fetal hindlimbs and abdomen. The left femoral artery was isolated and catheterized to measure mean arterial blood pressure (MAP). The left kidney was exposed via a retroperitoneal incision, the renal sympathetic nerve was visualized with a surgical microscope (OPMI 1FC, Zeiss, Oberkochen, Germany), and the electrode coils of a telemetry-based implantable nerve amplifier (Telemetry Research Limited, www.telemetryresearch.com, Auckland, New Zealand) were coiled around the nerve. The electrode and nerve were insulated from the surrounding tissues with a coat of silicone elastomer (Kwik-sil, World Precision Instruments, FL) (5). The implantable amplifier was secured on the fetal back. In the first preparation an external aerial was used to receive the transmitted signal. However, because of intermittent signal loss in this preparation, subsequent experiments were performed with the aerial secured on the back of the fetus.
The uterus was then closed in layers. Antibiotics [80 mg gentamicin sulfate (80 mg/2 ml); Pharmacia and Upjohn, Rydalmere, NSW, Australia] were administered into the amniotic sac. All fetal catheters and leads were exteriorized through the maternal flank. The maternal long saphenous vein was catheterized to provide access for postoperative maternal care and euthanasia. The maternal skin incision was infiltrated with a long-acting local anesthetic, Marcain (bupivacaine hydrochloride 0.25% with adrenaline 1:400,000; Astra Zeneca, North Ryde, NSW, Australia).
Following surgery, sheep were housed in separate metabolic cages with access to water and food ad libitum. They were kept in a temperature-controlled room (16 ± 1°C, humidity 50 ± 10%), in a 12:12-h light-dark cycle. After surgery, the exteriorized catheters and leads were kept in an enclosed Perspex box suspended from the side of the ewes’ metabolic cage. Catheters were maintained patent by continuous infusion of heparinized isotonic saline (20 U/ml, 0.2 ml/h). Fetal brachial arterial blood was taken at the end of the study for blood gas analysis for the assessment of fetal health.
Recordings were started 2 h after the end of surgery, in the afternoon, and were continued for 16 h. Fetal MAP, corrected for maternal movement by subtraction of intra-amniotic pressure, FHR, derived from the ECG, fetal EMG, and renal SNA were recorded and saved continuously to disk for off-line analysis using custom data acquisition programs (LabView for Windows, National Instruments, Austin, TX). At the end of the protocol, the ewe and fetus were killed with an overdose of sodium pentobarbitol (30 ml of 300 mg/ml iv, Pentobarb 300, Chemstock International, Christchurch, New Zealand).
Renal SNA signals were amplified 50,000 times, filtered between 50 and 2,000 Hz, full-wave rectified, and integrated using a low-pass filter with a time constant of 20 ms (5). The level of activity between bursts of renal SNA was automatically set as background noise by the acquisition program. The analog signals were then digitized and continuously displayed and recorded at 500 Hz.
Renal SNA was analyzed in three ways. First, hourly averages of renal SNA, MAP, and FHR were analyzed over the first 16 h after recovery from surgery. As the magnitude of the renal SNA signal is dependent on contact area of the electrodes and the nerve, integrated renal SNA was normalized by expressing the signal as a percentage of the average activity over 16 h for each fetus.
Second, to investigate the coordination between the bursting pattern in sympathetic activity and the cardiac cycle, averages of 1-s intervals of blood pressure and SNA were obtained using the systolic pressure as a trigger, for 100–200 epochs, at 0, 4, 8, 12, and 16 h after recovery from surgery (18). The times from the nadir of diastolic pressure or the peak of systolic pressure and the following peak of the mean renal SNA burst were calculated manually. The peaks in SNA occurred close to the diastolic nadir; therefore, the burst in SNA that followed systole was used for the calculations. Third, renal SNA burst frequency (bursts per second) and burst occurrence (bursts per 100 heartbeats) were calculated from the last 5 min of the study.
Statistical analysis was performed using SPSS (SPSS, Chicago, IL). Changes in cardiovascular variables over time were tested using repeated-measures ANOVA. Statistical significance was accepted when P < 0.05. Data are expressed as the means ± SE. Where a significant effect of time was found, post hoc comparisons were made using the least significant difference test.
Biochemistry and cardiovascular measurements.
Values for fetal biochemical measurements during the recording period were within our laboratory standards for this stage of gestation (26): fetal blood pH, 7.37 ± 0.01; PaCO2, 39.2 ± 3.5 mmHg; PaO2, 22.3 ± 1.4 mmHg; lactate, 0.8 ± 0.1 mmol/l; and glucose 0.9 ± 0.1 mmol/l. There was a significant effect of time on integrated renal SNA (ANOVA, P < 0.05). Integrated renal SNA was greatest in the 2nd h after surgery (121 ± 14%) and lowest at 15 h (86 ± 4%; Figure 1); however, on post hoc analysis, there were no significant differences between individual data points. MAP did not change significantly over the recording period. FHR fell significantly from a maximum of 212 ± 8 bpm in the 2nd h of recording to a minimum of 189 ± 8 bpm (ANOVA, P < 0.001).
Relationship between renal SNA discharges and the cardiac cycle.
A phasic relationship between blood pressure and integrated nerve activity was found in all fetuses. Typical examples from two separate fetuses are shown in Fig. 2. The mean interval between diastolic nadir and peak renal SNA was 319 ± 1 ms (range 308 to 325 ms). The mean interval between peak systolic pressure and peak renal SNA was 513 ± 5 ms (range 488 to 546 ms). There was no significant change in either interval over the recording period.
Overall level of sympathetic nerve activity.
Examples of renal SNA recordings from an adult and a fetal sheep are shown in Fig. 3, A and B. In the adult sheep, bursts of sympathetic activity occurred with nearly every cardiac cycle (C. May, Howard Florey Institute, Melbourne, Australia, unpublished data). In contrast, in the fetus, many cardiac cycles had no corresponding burst in sympathetic activity. At the end of the recording period, the average burst frequency in the preterm fetuses was 0.5 ± 0.1 Hz, and the burst occurrence was 16.4 ± 4.8 bursts per 100 heart beats.
Renal SNA signal verification.
ECG interference, characterized by sharp deflections synchronous with pulses in arterial blood pressure, was not seen in any recordings in the present study. An example of ECG interference, from a fetus excluded from the study, is shown in Fig. 4. In all fetuses, bursts of renal SNA occurred independently of fetal nuchal activity (an example is shown in Fig. 5).
This is the first study to directly record sympathetic activity and describe the characteristics of its discharge pattern in fetuses at 0.7 of gestation. By using an adapted telemetry technique similar to that previously reported in the adult rabbit (5), we were able to place the nerve amplifier close to the nerve and separate the amplifier from the external lead. This reduced movement artifacts and allowed recordings to be performed without the confounding effects of paralysis or anesthesia (Fig. 5).
Since the first direct sympathetic nerve recordings were carried out in the 1930s, two main properties of efferent nerve activity have been described (1, 8). The first is that sympathetic nerve activity occurs in bursts, which consist of the synchronized activation of many nerve fibers at approximately the same time. The second is that the discharges are entrained to the cardiac cycle. Both of these features were strongly evident in the renal SNA recorded in the preterm fetus. Previous studies in adult animals have shown that the bursting pattern is an intrinsic property of the cells within the CNS, such as those within the paraventricular nucleus and rostral ventrolateral medulla (11, 12, 21, 23, 28). The present study therefore suggests that the circuitry within the CNS is developed and active at this maturational stage.
The renal SNA recorded from all fetuses showed strong coordination with the cardiac cycle in a pattern similar to that seen in adult animals. The entrainment of sympathetic discharges to the cardiac cycle is widely believed to be dependent on input from baroreceptors to a network of cell groups within the CNS (as recently reviewed in Ref. 13). An interesting observation was that the delay between the nadir of diastole and peak renal SNA in the fetus was approximately twice that reported in adult animals (14, 18). Although not specifically investigated, the increased delay could be due to a number of factors, such as the speed of propagation along nerve fibers or delayed transmission within the CNS related to incomplete myelination (3).
A recent study by Malpas et al. (18) indicated that the baroreflex control of the mean level of sympathetic activity and the timing of the discharges with the cardiac cycle are independently controlled. The former is dependent on either aortic or carotid baroreceptor innervation and the latter hypothesized to only require limited input from afferent baroreceptor fibers that may travel within the vagus. The present investigations indicate that the circuitry involved in coordinating sympathetic discharges with the cardiac cycle is also present in the preterm fetus. An important area for future studies is to determine whether baroreflex control of the mean level of activity is also present at this stage of gestation. Limited previous studies have demonstrated that baroreflex-mediated control of heart rate exists in the preterm fetus (7, 29); however, it is unknown to what extent baroreflex-mediated control of the sympathetic nervous system determines vascular tone at this age.
The overall frequency of SNA discharges (both per 100 heartbeats and per second) was relatively low in the fetus compared with that in adult animals (15, 19). It is generally observed that bursts of activity in the adult sheep occur with every second or third cardiac cycle (19), whereas in the fetus bursts occurred on average with every sixth cardiac cycle (Fig. 3). A ‘low’ resting level of SNA is consistent with previous studies that used pharmacological intervention to determine the contribution of the sympathetic nervous system to basal blood pressure and heart rate (2, 24). This low background level of SNA, however, does not necessarily mean that the sympathetic nervous system at this stage of gestation cannot increase. For example, previous studies in the preterm fetus have reported rapid peripheral vasoconstriction in response to umbilical cord occlusion (26), suggesting that SNA can increase in utero during homeostatic challenges. The technique described in this paper provides a useful methodology to allow future studies to dissect the contribution of sympathetic activity to normal fetal cardiovascular regulation.
A potential limitation of this study is that ganglionic blockade was not performed, leaving the possibility that the SNA signal might include movement artifacts from surrounding vessels or ECG interference. Such artifacts induced by movement or ECG are highly coordinated, occurring with every pulse wave in the case of ECG or pulse wave movement or synchronous with the fetal movement and so readily detected, as shown in Fig. 4.
The primary objective of this study was to record renal SNA in the preterm fetus without anesthesia or paralysis. The occurrence of bursts in sympathetic nerve activity and entrainment of discharges with the cardiac cycle are key findings that help to establish that the intricate circuitry controlling SNA in the adult is functional in the preterm fetus. Although further studies are required to test the functionality of preterm fetal sympathetic activity, this study provides an important tool for future investigations.
This study was funded by grants from the Health Research Council of New Zealand, the Auckland Medical Research Foundation and Lottery Health New Zealand. L. C. Booth was supported by a University of Auckland Doctoral Scholarship.
The authors gratefully acknowledge Professor Clive May at the Howard Florey Institute, Melbourne, Australia for advice and the adult sheep sympathetic nerve recording.
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- Copyright © 2007 the American Physiological Society