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The potential for pathophysiologic changes in cardiovascular function due to an imbalance in myocardial innervation by the sympathetic and parasympathetic branches of the autonomic nervous system may be greater in immature animals in whom neural connections may not be fully established. This hypothesis is supported by many studies on the postnatal development of reflex control of the circulation(1) and by our findings that vagal parasympathetic neurons are relatively mature(2), whereas stellate ganglion neurons are relatively immature(3, 4) in neonatal swine. These findings led us to question whether experimentally produced dominance of cardiac parasympathetic innervation after stellate ganglionectomy would unmask a vagally mediated bradycardia and hypotension occurring either spontaneously or in response to afferent stimulation. In the latter case, stimulation of cardiopulmonary receptors at vagal afferent C fiber endings is known to affect cardiovascular parameters and is easily accomplished by right atrial or ventricular injections of a variety of chemical stimuli (e.g. serotonin, nicotine, acetylcholine, PDG, PBG, CO2) as well as by increased interstitial fluid volume due to pulmonary congestion(5–7). Stimulation of these receptors causes reflex bradycardia, apnea, and hypotension in the adult rat(6, 8), cat(7, 9), and rabbit(10, 11). We have reported previously that neonatal swine exhibited a similar rapid (latency 2-3 s) response pattern to chemical stimulation of cardiopulmonary receptors(12, 13).

In the present study, we have used PBG(10, 14) to stimulate cardiopulmonary receptors in the presence and absence of cardiac sympathetic innervation in neonatal and 2-mo-old swine. Unilateral and BSG were used to produce an imbalance in cardiac innervation such that vagal activity would predominate. The stellate ganglia were selected for ablation inasmuch as we have previously reported that these ganglia are the primary location of postganglionic cardiac sympathetic neurons in piglets(3, 4). Furthermore, the influence of respiratory effects of PBG(10, 11) on the cardiovascular responses were minimized by controlling pulmonary ventilation. Therefore we were able to determine: (1) whether the cardiovascular responses to PBG, particularly the previously observed AVB effects of PDG(12), are a function of postnatal age, and (2) whether a dominance of vagal tone would alter the pattern of cardiovascular responses to PBG in developing piglets.

METHODS

The study was approved by our Institutional Animal Use Committee. The experimental procedures complied with the “Guide for the Use and Care of Animals” approved by the council of American Physiologic Society, as well as with Federal and State regulations. Swine used in these studies were raised in the Division of Laboratory Animal Resources in our Institution.

We chose two different age groups of swine to assess the role of postnatal development: 1 wk of age, allowing comparison with our earlier study on neurally intact animals given PDG(12), and 8 wk of age because these animals exhibited the adult pattern of cardiopulmonary responses to veratrum alkaloids(13). Animals were initially anesthetized with the steroidal anesthetic Saffan (12 mg/kg, intramuscularly, Vet Drugs, Ltd.) which does not affect the cardiovascular system(15). An external jugular vein was cannulated for continuous infusion of Saffan at 6-12 mg/kg/h, during surgery; after the conclusion of surgical procedures the infusion rate was lowered to 6 mg/kg/h. To control the influence of the respiratory response on HR after right atrial injection of PBG, all animals were tracheotomized, paralyzed with decamethonium bromide (0.1 mg/kg, i.v.) and artifically ventilated with 100% O2. Needle electrodes were placed s.c. to monitor lead II of the ECG. A femoral artery was cannulated and the catheter advanced into the abdominal aorta for both the continuous monitoring of AoP and periodic (every 30 min) determinations of arterial blood gases and pH. A femoral vein was canulated for infusion of 5% dextrose solution. pH and Pco2 were maintained within physiologic ranges whereas Po2 was >13 kPa (100 mm Hg). If necessary, injections of 7.5% bicarbonate solution were given to correct pH and Pco2.

The chest wall was opened and retracted by methods previously reported(16). In brief, the mediastinum was separated from the chest wall by hand and the pericardium was left intact. After thoracotomy, ventilation was adjusted, as needed, to maintain adequate lung expansion. An end-expiratory load of 1-2 cm H2O was applied to minimize atelectasis.

Stellate ganglionectomy. The method used for stellate ganglionectomy has been described elsewhere by us(3, 16). Briefly, the stellate ganglia was exposed beneath the parietal pleura posteriorly between the first and second ribs. All nerve branches running into and out of the ganglia were isolated, ligated, and cut. Then ganglia were removed and stored in 10% formalin for later histologic verification(16).

Recordings and calculations. AoP and ECG were recorded on a Grass model 7C polygraph and on VCR tape for later statistical analyses. HR(beats/min) and the maximum R-R interval (seconds) were calculated on polygraph records taken at 25 mm/s over a period of 1 min during baseline conditions and after injection of test solutions. The occurrence of AVB during each period was determined by the appearance of P waves in the ECG without a subsequent QRS complex(12).

Experimental protocol. Twenty-nine animals were used in the present study. Arterial blood gases and pH were maintained in the normal range throughout each protocol. After control conditions were established for at least 40 min after surgery, equivalent volumes of a saline control solution(0.4 mL/kg) or PBG (Aldrich, Milwaukee, WI), diluted in saline solution (0.4 mL/kg) were injected within 3 s via a catheter inserted into the right atrium. The location of the tip of the right atrial catheter was verified at the end of all experiments.

Dose-response animals. To establish the minimum dose that elicited the full cardiovascular response to PBG, four 1-wk-old animals were given different dosages (20, 60, and 80 μg/kg) of PBG as in adult rabbits(14). These data indicated that a dose of 80 μg/kg elicited the full cardiovascular response, as was the case for PDG on our earlier studies on developing swine(12).

Control for repeated PBG injections. To verify that no tachyphylaxis or cumulative drug effects occurred after repeated PBG injections, three 1-wk-old and five 8-wk-old piglets received three sequential injections of saline and three sequential injections of PBG (80 μL/kg). The interval between 0.4 mL/kg injections of the same substance was 30 min.

Stellate ganglionectomy group. Each piglet (1 and 8 wk old,n = 6 in each group) was studied under the following sequential conditions with a 30-min interval between test conditions: baseline; 15 min after RSG; 15 min after BSG. Each animal was used to determine the AoP and ECG responses to a single injection of saline and then PBG (80 μg/kg) under baseline, RSG and BSG conditions. The interval between PBG injections was at least 30 min.

Bilateral cervical vagotomy group. To demonstrate that the cardiovascular effects elicted with PBG were mediated by the vagus, three 1- and three 8-wk-old piglets were given PBG before and after bilateral vagotomy with stellate ganglia intact, according to the protocol described above for the BSG. Both cervical vagi were isolated from vagosympathetic trunks in the neck and then transected(16).

Statistics. Data are presented as means ± SEM of absolute values at peak response (Tables 1, 3, and 4) or PBG responses expressed as percentage of saline control response (Fig. 3). analysis of variance for repeated measures was used to verify the reproducibility of repeated PBG injections in the same animals. HR, maximum R-R interval, and blood pressure changes to saline or PBG before and after denervations in the two age groups were examined by analysis of variance. If significance was obtained, a post hoc Tukey test was used. To determine whether any of the cardiovascular variables exhibited age-related changes, they were compared using independent group t tests. Preplanned comparisons among denervation effects were made using the t test with Bonferroni correction (the significance was established at p < 0.05/6 = 0.0083) applied to the responses to saline in the two age groups. All statistical analyses were carried out using the SPSS computer program. Except for the Bonferroni correction noted above, the level of probability for statistical significance was established at p< 0.05 for all analyses.

Table 1 Effects of repeated injections of PBG (80μg/kg) in control experiments on neurally intact piglets*
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Table 3 Cardiovascular responses to PBG (80μg/kg) injections in 1-wk-old piglets: effects of RSG and BSG
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Table 4 Cardiovascular responses to PBG (80μg/kg) injections in 8-wk-old piglets: effects of RSG and BSG
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Figure 3
figure 3

Bar graphs present age-related effects of ganglionectomy on maximum R-R interval to PBG injection (PBG response). Data presented as percentage of saline control responses (100%). (Mean ± SEM, n = 6.) *p < 0.05 effect of PBG on the maximum R-R interval in 8 wk before denervation was significantly smaller than that in 1-wk-old piglets.

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RESULTS

In the eight animals used to determine whether there were differences in cardiovascular responses due to repeated injections of PBG, we observed no significant differences in HR, maximum R-R interval, AVB occurrence, or AoP responses over time (Table 1). In all cases, the onset of PBG-induced alterations in cardiovascular parameters occurred simultaneously within 3 s after right atrial injections. The duration of PBG effects after single injection was less than 15 min.

Effects of PBG in neurally intact and denervated swine. As shown in Table 2, control data in neurally intact animals exhibited the expected differences in several maturational variables(1), i.e. significant increases in body weight from 1 to 8 wk were accompanied by a significant increase in mean AoP and a significant decrease in resting HR.

Table 2 Control conditions at onset of test series of injections in piglets* used for stellate ganglionectomy experiments
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One-week-old swine. In the neurally intact animals, changes in measured cardiovascular parameters were not observed after saline injections. However, PBG injections elicited a very rapid (within 3 s) stereotypical response: sinus bradycardia, AVB (100% of the animals), and hypotension(cf. panels A and B in Fig. 1). This response pattern to PBG was unaffected by RSG and BSG(cf. panels B and C in Fig. 1). Comparisons of saline and PBG-evoked changes in cardiovascular parameters in Tables 1 and 3 showed that only PBG elicited significant decreases in mean AoP and HR as well as significant increases in the maximum R-R interval. The HR decreased after RSG and BSG. However, neither RSG nor BSG altered AoP significantly. Furthermore, the PBG-evoked changes in AoP and the maximum R-R interval were similar regardless of degree of denervation, RSG versus BSG (see Table 3). All effects of PBG were abolished after bilateral vagotomy in the three separate animals tested, as in our earlier study with PDG(12).

Figure 1
figure 1

Original polygraph traces of ECG and AoP responses to PBG in a 1-wk-old piglet. (A) Saline injection; (B) PBG injection (80 μg/kg) before ganglionectomy; (C) PBG injection (80μg/kg) after BSG. Vertical arrows denote start and end of injection; diagonal arrows denote P waves without subsequent QRS complex.

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Eight-week-old swine. As observed in the newborn swine, 8-wk-old animals exhibited no changes in cardiovascular parameters after saline injections (Fig. 2, panel A). In neurally intact animals, PBG injections usually evoked sinus bradycardia without hypotension (see Tables 1 and 4). AVB was observed in only three of six (50%) neurally intact animals. The example in Figure 2 (panel B) demonstrates PBG-induced decrease in HR with no concomitant hypotension or AVB. However, after either RSG or BSG, PBG elicited decreases of HR and AoP as well as AVB (cf. panels B and C in Fig. 2) in all animals (Table 4). Furthermore, the HR decreased significantly after RSG or BSG alone (Table 4). All effects of PBG were abolished by bilateral vagotomy (see Fig. 4) in the three separate animals tested.

Figure 2
figure 2

Original polygraph traces of ECG and AoP responses to PBG in an 8-wk-old piglet. (A) Saline injection; (B) PBG injection (80 μg/kg) before ganglionectomy; (C) PBG injection (80μg/kg) after BSG. Vertical arrows denote start and end of injection; diagonal arrows denote P waves without subsequent QRS complex.

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Figure 4
figure 4

Original polygraph traces of ECG and AoP showing effect of bilateral cervical vagotomy on responses to PBG injection in a 8-wk-old piglet. (A) Saline injection; (B) PBG injection (80μg/kg) before denervation; (C) PBG injection (80 μg/kg) after bilateral cervical vagotomy. Vertical arrows denote start and end of injection.

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Age-related differences in PBG response patterns. Unlike the younger swine in which PBG evoked a stereotypical response pattern regardless of whether the heart was neurally intact or sympathectomized, 8-wk-old animals exhibited differences which were dependent upon extent of the denervation (Fig. 3). For example, examination of the group data in Table 4 revealed that both the decreased mean AoP and increased max R-R interval after BSG differed significantly from those values obtained after RSG.

Statistical comparisons also revealed that the PBG-induced increase of max R-R interval differed in the two age groups. In Figure 3, the maximum R-R interval after PBG injection is presented as a percentage of saline control. The PBG-mediated increase in the maximum R-R interval was greater (p < 0.05) in the 1-wk-old group than in the 8-wk-old group when the animals were neurally intact. The disappearance of this difference after BSG emphasizes that 8-wk-old animals then resembled the younger ones.

DISCUSSION

The results of our control experiments suggest that maturation of cardiovascular responses to cardiopulmonary receptor stimulation in piglets(Tables 3 and 4) extends beyond the previously reported 3 wk [Table 2 in Schleman et al.(12)] to at least 8 wk. AVB was always a prominent response to PBG in 1-wk-old piglets, in addition to sinus bradycardia and hypotension. We have defined this pattern of response as the neonatal response pattern, because the incidence of AVB and hypotension decreased between 1 and 8 wk of age.

Maturation of the responses would involve components of a reflex pathway(1, 5, 10, 11). Evidence has been obtained that pulmonary J receptors are involved in the respiratory effects, whereas cardiac receptors are involved in the cardiovascular effects. For example, vagal afferent C-fiber endings present in the atria and ventricles are activated by increases in intracardiac pressure(6), by chemical stimulation with atrial injections of PDG in cats(7) or PBG in rabbits(11), and by Veratrum veride alkaloids in swine(13). Right atrial injections elicited respiratory and cardiovascular responses in adult cats(7) and rabbits(11) as did pulmonary arterial injections(5), whereas left atrial injections in cats elicited only cardiovascular responses(7). Pulmonary vagal denervation prevented only the respiratory responses in rabbits(11), whereas myocardial denervation by intrapericardial procaine prevented only the cardiovascular responses in cats(7) and rabbits(11). Furthermore, injections into the proximal aorta in adult cats(7) and rabbits(10), and in piglets(12) elicited only long latency changes in blood pressure that varied in direction, suggesting but not proving that PBG had not stimulated intracoronary reflexes.

It has been reported that PBG stimulates cardiac and pulmonary unmyelinated vagal afferent C-fiber endings by exciting 5-hydroxytryptamine receptor subtype-3(10). 5-Hydroxytryptamine subtype-3 receptors are cation channels; activation of these receptors results in depolarization of vagal afferents by an action similar to the nicotinic effects of acetylcholine(17). The vagal afferents excited by PBG have been found to project to the nucleus tractus solitarius in rats(6, 8, 18) and in rabbits, to both nucleus tractus solitarius and the medullary depressor area i.e., caudal ventrolateral medulla(18). The present results confirm previous studies which demonstrated that the bradycardia and hypotension responses disappeared after bilateral cervical vagotomy(7, 10–12, 14). Thus the cardiovascular responses to PBG are mediated by the vagi and reflect both the vagal afferent pathway and the interactions between efferent cardiac sympathetic and parasympathetic innervation. The hypotensive effect of PBG may be attributable to the stimulation of medullary depressor areas(18) and to sympathetic withdrawal, because a PBG-mediated decrease in renal sympathetic nerve activity has been observed in adult rabbits(11). It should be noted that sympathetic control of the renal circulation is active in neonatal swine(1).

Another component in the reflex effects of PBG would be the well known limited responsiveness of immature ventricular myocardium to stretch imposed by increased diastolic filling during bradycardia. Examination of the prestellectomy data in Tables 3 and 4 reveals that PBG lowered blood pressure to about 61% of saline control in 1-wk-old animals and only to about 96% of control in 8-wk-old animals, whereas HR decreased to about 75% of control values at both ages. The failure of the immature heart to increase enough stroke volume to maintain blood pressure could account for this difference. On the other hand, our observation that, after BSG (see Tables 3 and 4), PBG injections lowered blood pressure to nearly the same level (to about 68% of control values) and decreased HR to about 84% of control values in both age groups suggests that maturation of the myocardial response to stretch may not play a major role in the hypotension.

In the present study, HR in 1- and 8-wk-old piglets decreased after RSG compared with the neurally intact state with no further decrease after BSG, revealing that the right stellate ganglion plays a major role in regulating HR, as expected(3). The disappearance of responses to PBG after bilateral cervical vagotomy in 1-wk-old neurally intact piglets confirmed that the cardiac vagi were functional at this age(1, 2, 12). The absence of different response patterns to PBG before and after stellate ganglionectomy in the 1-wk-old group indicates that vagal activity predominated even before ganglionectomy. This conclusion is further supported by our earlier findings suggesting that cardiac sympathetic innervation is not fully functional during the first postnatal month in swine(1, 3, 4).

In addition, PBG elicited a more pronounced hypotension after BSG than occurred in the baseline state or after RSG in 8-wk-old piglets. These results indicate that the left stellate ganglion plays a greater role in regulating cardiac ventricular function in 8-wk-old piglets compared with 1-wk-old piglets, as expected(3).

The lower incidence of AVB, absence of a hypotensive response, as well as a smaller increase in the maximum R-R interval to PBG in the neurally intact 8-wk-old piglets indicated that reflex vagal effects on cardiac conduction and blood pressure may be modulated by the maturing sympathetic innervation to the heart. Once the sympathetic system became adequately functional, the possibility of prolonged atrioventricular conduction induced by excitation of the cardiopulmonary receptors was significantly diminished. When the parasympathetic system was dominant after BSG in older swine, the neonatal response pattern to PBG injection appeared. This finding suggests that, if functional cardiac sympathetic innervation is delayed or not fully functional, the neonate might be vulnerable to the effects of activation of cardiopulmonary receptors by either internal or environmental stimuli. Such a delay in sympathetic innervation might reflect central (brain stem-spinal cord) immaturity and/or ganglionic or neuroeffector immaturity(1). This conclusion does not conflict with the present study, because what may be important is the presence of both sympathetic and vagal tone in an appropriate balance. When either sympathetic or parasympathetic outflow is compromised, an imbalance occurs, resulting in cardiac electrical instability.

There is considerable interest in the hypothesis of abnormal development of autonomic innervation as a possible mechanism in the etiology of SIDS(19). Our speculation about autonomic imbalance is reinforced by the report by Meny et al.(20), who used memory equipped cardiorespiratory monitors to describe intractable bradycardia in three infants latter diagnosed as SIDS cases, as well as in two other infants with bronchopulmonary dysplasia and severe bronchopneumonia. Because pulmonary interstitial edema is a pathophysiologic stimulus of cardiopulmonary receptors(5, 7), the infants studied by Meny et al.(20) might have succumbed as a result of cardiac and respiratory changes consequent to these receptors. We consider that one possible mechanism leading to SIDS may be a dominance of vagal tone in the presence of substances capable of exciting vagal afferent C-fiber discharge, such as nicotine(5, 21, 22).