Electrocardiographic changes during postnatal development in conscious swine with cardiac autonomic imbalance.

Using a conscious swine model, we studied the effects of different patterns of cardiac autonomic denervation on alterations of R-R and Q T intervals for 8 postnatal weeks. Newborn pigs were assigned randomly to four different groups: sham-operated controls (C), stellate ganglion ablation (SGX), either left (LSGX) or right (RSGX), and the right cardiac vagus nerve (RCVX) transection. The ECGs were recorded by telemetry while animals rested quietly or were judged behaviorally to be asleep. Analyses of the ECG included measurements of R-R and Q-T intervals, as well as corrected Q-T intervals (QTc). Poincaré plots were used to display age-related differences in R-R and Q-T intervals. For stellectomized animals, significantly prolonged R-R intervals were first observed at post-surgical week 3 in the RSGX group and at week 5 in the LSGX group. Significantly prolonged QTc was found only in the RSGX group. In the RCVX group, shortened QTc and R-R intervals were noted at 6 and 7 weeks after denervation. Furthermore, three of six RSGX animals (50%) and one of four RCVX animals (25%) exhibited marked pauses in sinus rhythm that were unrelated to changes in heart rate or to sinus arrhythmia. These results in conscious animals support our hypothesis that abnormal autonomic innervation of the heart during maturation, e.g., withdrawal of vagal cardiac modulation or asymmetry of sympathetic innervation, impairs cardiac electrical stability.

Rhythmicities in sympathetic discharge: a signal of cardiorespiratory integration in developing animals.

We have been pursuing various avenues of investigation to elucidate the postnatal maturation of neural regulation of cardiovascular and respiratory integration. In this paper we present our results from a systematic analysis of age-related modulations of sympathetic (SYMP) activity with respect to experimental alterations in baroreceptor afferent inputs. The three age groups of piglets were chosen based on different responses to a complex stimulus, i.e., the Valsalva maneuver. Postnatal maturation of SYMP activity was examined by spectral analysis of SYMP discharge using cross-power, full and partial coherence. Three general oscillations were observed in spontaneous SYMP discharges in the 0-30 Hz range. We divided that range into five frequency bands (0-2, 2-6, 6-12, 12-20, 20-30 Hz), which included periodicities in phase with both central respiratory activity and the cardiac cycle. Spectral analyses of SYMP activity after either baroreceptor activation (phenylephrine) or deactivation (nitroprusside) revealed that respiratory modulation was age-related across all frequencies while baroreceptor modulation was usually age-related within three of the five frequency bands. These results lead to questions concerning the possible role of the autonomic nervous system and/or central interactions between the respiratory and SYMP rhythm generators in the etiology of sudden infant death syndrome (SIDS).

Changes in R-R and Q-T intervals following cardiac vagotomy in neonatal swine.

Asymmetric innervation of the myocardium, especially a predominance of sympathetic innervation, may establish conditions whereby electrical instability could result. Using a swine animal model, we studied the effect of right cardiac vagal denervation on the variability of R-R and Q-T intervals. Newborn pigs were assigned randomly to two groups: sham-operated controls (C), or denervation of the right cardiac vagus nerve (RCVX). EKGs were recorded weekly until the two groups exhibited significant heart rate differences. Analysis of the EKG included measurements of R-R and Q-T intervals and corrected Q-T intervals (QTc). Poincaré plots were used to display age-related differences in R-R and Q-T intervals. For RCVX animals, decreased QTc and R-R intervals were noted at 6 and 7 weeks after denervation, respectively. Unexpectedly, one RCVX animal exhibited marked sudden pauses in sinus rhythm. These data indicated that reduced vagal cardiac modulation during development might alter cardiac electrical stability in conscious swine.

CO(2)-induced expression of c-fos in the nucleus of the solitary tract and the area postrema of developing swine.

This investigation was performed to determine whether hypercapnic exposure elicited expression of the c-fos protooncogene product, FOS, in nucleus of the solitary tract (NTS) and area postrema (AP) neurons of developing swine. Mean arterial blood pressure (MAP) and heart rate (HR) were also monitored to evaluate whether numbers of neurons containing FOS were related to changes of MAP and HR. In each experiment, two litter-matched piglets were prepared simultaneously, i.e., Saffan anesthesia, paralysis, and artificial ventilation (100% O(2)). One animal was exposed to hypercapnia (1 h of 10% CO(2), balance oxygen), while the other continued to breathe 100% O(2). Animals were studied at three different ages: 5-8 days, 13-15 days, and 26-34 days old. In the NTS, FOS expression was prominent in regions corresponding to the general visceral afferent subdivision; the AP showed no such topographic distribution. The number of NTS and AP neurons with FOS in hypercapnic-exposed animals was significantly greater than those of unexposed animals. However, an age-related increase of FOS was observed only for NTS neurons, with the greatest number observed in 13- to 15-day-old animals. Increases of MAP, not HR, were noted during the early part of hypercapnia in the 5- to 8-day-old group; older animals exhibited no change of MAP. Our findings demonstrated that prolonged hypercapnic stimulation elicited FOS expression in AP and NTS neurons of developing animals, and that such expression was non-uniform, depending upon the region studied.

The area postrema of newborn swine is activated by hypercapnia: relevance to sudden infant death syndrome?

This study was performed to investigate a role of the neonatal area postrema (AP) in the chemoreceptor response to hypercapnia which is defective in sudden infant death syndrome (SIDS). AP responses to CO2 inhalation were monitored in 1 to 5 week old piglets by mapping neurons that were induced to express the c-fos gene product, Fos--a marker of functional activation. Interpretive confounds were minimized by controlling for hypoxia, the effects of surgical procedures and ambient environmental stressors on neuronal activity (c-fos expression). The AP demonstrated a powerful and reproducible response in neonatal swine breathing 10% CO2 for 1 h. Intensely immunolabeled nuclei were detected throughout the longitudinal extent of the circumventricular organ, and were especially heavily concentrated at rostral levels proximal to obex. Quantitative analysis verified statistically significant increases in numbers of cells that were induced to express Fos-like immunoreactivity (FLI) in the AP of CO2- stimulated piglets as compared to control groups. No detectable age-related differences were observed in AP response patterns. Conclusions. The AP responds to hypercapnic stress in the newborn piglet. A mature circumventricular organ response in the neonate may be crucial in defending against common environmental stressors, such as nicotine exposure--an emetic agent acting via the AP and a major risk factor in SIDS. Hence, a defect of the AP or its network may underlie a loss of state-dependent controls over cardiopulmonary reflex function in SIDS.

Presence of a non-NMDA glutamate receptor subtype in the sympathetic nervous system of neonatal swine.

For the first time, the GluR-1 subtype of AMPA receptor was identified in the sympathetic nervous system of neonatal swine, an animal model of human development and heart disease. The rationale was to seek evidence of a role ascribed to glutamate in cardiorespiratory regulation in the laboratory rat. The receptor was demonstrated with the avidin-biotin immunoperoxidase technique by using an affinity-purified polyclonal antibody judged to be specific to Glu-R1 in several species. Glu-R1 immunoreactivity was regionally distributed in the thoracic spinal gray, and present intracellularly in neurons and within the surrounding neuropil. Sympathetic preganglionic neurons in the intermediolateral cell column of upper and lower thoracic spinal segments were intensely labeled and surrounded by labeled neuropil. High concentrations of Glu-R1 distinguished laminae II: substantia gelatinosa and the outer region of lamina III. Laminae I and V of the dorsal horn but not IV contained immunolabeled neurons. Arrays of moderately immunoreactive perikarya extended from an intermediate zone of laminae VII to the central gray. Glia and perivascular processes were not labeled, confirming previous observations [Tachibana, M., Wenthold, R.J., Morioka, H., Petralia, R.S., 1994. Light and electron microscopic immunocytochemical localization of AMPA-selective glutamate receptors in the rat spinal cord. J. Comp. Neurol. 344, 431-454]. Neuronal staining patterns corroborated evidence in rats indicating a postsynaptic localization of Glu-R1 associated with plasma membranes and cytoplasmic organelles [Martin, L.J., Blackstone, C.D., Levey, A.I., Huganir, R.L., Price, D.L., 1993. AMPA glutamate receptor subunits are differentially distributed in rat brain. Neuroscience 53, 327-358.; Rubio, M.E., Wenthold, R.J., 1997. Glutamate receptors are selectively targeted to postsynaptic sites in neurons. Neuron 18, 939-950]. Our data predict a role for L-glutamate in postnatal development of cardiorespiratory reflexes in swine.

Emergence of lung-inflation-related sympathetic nerve activity in spinal cord transected neonatal swine.

Sympathetic (SYMP) nerve activity in spinal intact neonatal swine is comprised of prominent bursts reflecting modulation by supraspinal structures involved in shaping central respiratory and baroreceptor activity. After spinal cord transection (SCT), we found no evidence of such modulation. SYMP activity was now related to the ventilatory cycle, exhibiting bursts only during lung inflation. Such activity suggests the emergence of latent spinal circuits which may have the capacity to regulate cardiovascular activity.

Induction of c-fos gene expression by spinal cord transection in the rat.

Sympathetic nerve activity is maintained after high spinal injury through circuits that remain in question. We evaluated patterns of c-fos gene induction as a monitor of spinal neurons responding to high spinal cord transection in the rat. Rats were anesthetized with isofluorane. Lower cervical or upper thoracic spinal segments were exposed, immersed in warm mineral oil and transected. Spinal cords were exposed but not transected in anesthetized controls. After 2.5 h, spinalized and control rats were perfused for immunocytochemistry. Cervical and thoracolumbar spinal segments and dorsal root ganglia were sectioned coronally. Tissues were incubated in primary, polyclonal antisera raised in rabbit or sheep against a peptide sequence unique to the N-terminal domain of Fos, and processed immunocytochemically. Neurons were induced to express Fos-like immunoreactivity (FLI), bilaterally, in the spinal gray, but not in primary sensory ganglia. Spinal cord transection induced neurons to express FLI in thoracic laminae I, IIo (outer substantia gelatinosa), Vre (lateral reticulated division), VII (lamina intermedia) and X, and the intermediolateral cell column. Lamina VIII was also labeled in spinal-injured but not in control animals. Immunolabeled nuclei were prominent in lumbar segments and were concentrated in the medial third of laminae I and IIo, and in laminae VII and X. Few cells were labeled in upper cervical or sacral segments. FLI was sparse in the spinal gray of controls and expressed mainly within the dorsal root entry zone of upper thoracic segments. Patterns of c-fos gene expression were site-specific and correlated with laminae that respond predominantly to noxious stimulation and that contain sympathetic interneurons. Laminae that are responsive to non-noxious stimuli and activated by walking, IIi, nucleus proprius, medial V and layer VI were not induced to express FLI. We conclude that neurons in specific spinal laminae that process high threshold afferents and that harbor neurons with sympathetic nerve-related activity are activated selectively by spinal cord transections. We hypothesize that peripheral afferents processed by spinal-sympathetic circuit neurons may regulate sympathetic discharge in the absence of supraspinal drive.

Preliminary evidence for the evolution in complexity of heart rate dynamics during autonomic maturation in neonatal swine.

Previous studies suggest that the autonomic nervous system plays an important role in the generation of complex heart rate dynamics. Therefore, we hypothesized that the complexity (irregularity) of cardiac interbeat intervals would evolve with the maturation of autonomic innervation to the heart. Twelve healthy newborn piglets were implanted with ECG transmitters and studied at one or more different ages up to 33 days of age, the period during which pigs develop functional sympathetic innervation of the heart from the stellate ganglia. Three animals underwent right stellate ganglionectomy, two a left stellate ganglionectomy, two a right cardiac vagotomy and five a sham procedure. The statistic, approximate entropy (ApEn), was used to quantify the regularity of interbeat interval fluctuations. Sham-operated animals showed an increase in the standard deviation (SD) and irregularity (ApEn) of cardiac interval fluctuations with increasing age. Right stellate ganglionectomized piglets had lower interbeat interval ApEn values, but similar SD's by 26-27 days of age compared to sham-operated animals. Left stellate ganglionectomy, which affects cardiac inotropy rather than chronotropy, had no effect on cardiac interval irregularity, while vagotomy had an indeterminant effect. The increasing irregularity of interbeat interval dynamics during autonomic maturation and the apparent attenuation of heartbeat irregularity when right stellate ganglion innervation is interrupted, provides empirical support for the notion that complex heartbeat dynamics in the mature animal are the result of a network of autonomic neural pathways that enables an organism to adapt to stress.

Induction of c-fos gene expression by spinal cord transection in Sus scrofa.

Functional responses of primary sensory afferents and spinal cord were monitored in swine subjected to a high cervical (C1) spinal transection. Two and a half hours after transection, dorsal root ganglia and cervical and thoracolumbar spinal segments were processed immunocytochemically for the c-fos gene product, Fos and related antigens. In spinal-transected animals, Fos-like immunoreactivity (FLI) was induced in spinal laminae I, V, VII and X and the intermediolateral cell column but not in sensory ganglia as compared to controls: spinal-intact age-matched littermates. Spinal laminae expressing FLI harbor sympathetic and somatic interneurons and may aid in maintaining sympathetic outflow.

Anatomy of medullary and peripheral autonomic neurons innervating the neonatal porcine heart.

Gross and microscopic anatomical investigations were carried out in 14 piglets aged from 4 to 66 days. True Blue (7-50 microliters) and Diamidino Yellow (7-50 microliters) were injected individually into 2 different cardiac sites (the right atrial ganglionated plexus, the inferior vena cava, inferior atrial ganglionated plexus, the right atrium or the right ventricle). Gross anatomy: Globular superior cervical and nodose ganglia, elongated stellate ganglia, multiple small middle cervical ganglia and multiple small mediastinal ganglia along the course of cardiopulmonary nerves were identified. Microscopic anatomy: Neurons innervating specific cardiac regions or intrinsic cardiac ganglionated plexuses were distributed relatively evenly among stellate (primarily in their cranial poles) and middle cervical ganglia bilaterally, fewer labeled neurons being located in the superior cervical and mediastinal ganglia bilaterally. Parasympathetic efferent preganglionic neurons associated with either intrinsic cardiac ganglionated plexus studied were identified primarily throughout the ventrolateral region (the external formation) of the nucleus ambiguus bilaterally. Labeled neurons were also identified throughout the right and left nodose ganglia. Individual neurons did not project axons to different cardiac regions, as no double-labeled neurons were identified. No correlation between age and the numbers and locations of labeled neurons was apparent. Thus, porcine sympathetic efferent neurons which innervate individual cardiac regions, including intrinsic cardiac ganglionated plexuses, lie scattered primarily throughout the right and left mediastinal and middle cervical ganglia as well as the cranial poles of stellate ganglia at birth, apparently changing little during the first 2 months of age. Porcine cardiac parasympathetic efferent preganglionic neurons are located primarily in the external formation of the nucleus ambiguus bilaterally at birth. The numbers of afferent cardiac neurons distributed throughout the nodose ganglia bilaterally also change little during that time. It is concluded that most of the autonomic neurons which innervate the heart are in place at birth.

Effects of cardiac autonomic imbalance on postnatal changes in the electrocardiographic Q-T interval in conscious swine.

The effects of partial autonomic denervation of the heart rate and Q--T interval were examined during maturation in swine. Four groups of newborns were prepared: right stellate ganglionectomy (RSG), left stellate ganglionectomy (LSG), right cardiac vagotomy (RCV) and sham-operated. Swine were studied postsurgically for eight weeks. Unexpected deaths of unknown cause occurred in five of the 20 denervated swine but in none of the 10 sham-operated controls. Prolongation of the Q--T interval was greatest in RSG animals and least in RCV as compared to controls. The results indicate that we have successfully developed an animal model of a long Q--T syndrome in swine.

Age-related effects of cardiac sympathetic denervation on the responses to cardiopulmonary receptor stimulation in piglets.

The effects of right stellate ganglionectomy (RSG) and bilateral stellate ganglionectomy (BSG) on cardiovascular responses to phenyl biguanide (PBG, 80 micrograms/kg) were studied in 1- and 8-wk-old piglets. Animals were anesthetized with Saffan, paralyzed, thoractomized, and ventilated with 100% O2. Recordings of the ECG (lead II) and aortic pressure (AoP) were used to compute the maximum R-R interval, heart rate (HR), and mean AoP, and to determine the occurrence of atrioventricular conduction block (AVB). Right atrial injections of PBG in 1-wk-old piglets elicited AVB as well as decreases in AoP and HR in all animals; this response pattern was not altered by either RSG or BSG. The PBG response of neurally intact 8-wk-old animals was comprised of a decrease of HR without change in AoP; AVB occurred in three of six animals. After RSG or BSG, AoP decreased along with decreased HR, and now AVB occurred in all animals; changes of AoP and maximum R-R interval were greater after BSG than after RSG. These results suggest that the stellate ganglia exert a neuroprotective influence on cardiovascular function, requiring some degree of maturation for expression. Our findings support the hypothesis that an imbalance of cardiac autonomic innervation favoring parasympathetic activity may produce immature responses to cardiopulmonary afferent stimulation in older maturing animals.

Hypotension-induced expression of the c-fos gene in the medulla oblongata of piglets.

Neural networks that mediate the reflex response to baroreceptor withdrawal were explored in Sus scrofa. Induction of c-fos was used as a monitor of synaptic activity in response to hypotension sustained by systemic administration of a peripheral vasodilator, sodium nitroprusside. Patterns of c-fos gene expression were compared between Saffan-anesthetized experimental animals and age-matched normotensive controls administered vehicle. Effects of other variables were controlled including 1 h preoperative accommodation to the novel environment, anesthesia, blood gases and pH. Identical post-stimulus survival periods were allowed for accumulation of transcript. The c-fos protein, Fos, was identified immunocytochemically with two rabbit antisera raised against amino acids 1-131 of Fos or residues 4-17 of synthetic human transcript. Fos was identified in catecholaminergic neurons labeled with an antiserum to tyrosine hydroxylase (TH). Fos was induced in the nucleus tractus solitarii (NTS) of hypotensive piglets. Neurons encoding Fos matched projection patterns of first order visceral afferents. Induction was prominent in the dorsolateral nucleus coinciding with the baroreceptor field. Indices of increased neuronal activity were evident in other baroreceptor terminal sites, e.g., medial subnucleus, the medial commissural field, the intermediate subnucleus and a ventral A2 noradrenergic area. In reticular formation c-fos protein was induced in circumscribed columns in the lateral tegmental field (LTF) extending from facial nucleus to calamus scriptorius. Catecholaminergic (TH-positive) neurons expressed Fos in the porcine C1 and A1 areas of ventrolateral medulla. Fos was also induced in a dorsal intermediate reticular zone of LTF. Minor or inconsistent differences between experimental and control were observed in nucleus raphe pallidus, rostral paramedian reticular formation, upper thoracic intermediolateral cell column, and stellate ganglia. In conclusion, baroreceptor withdrawal in young animals induced patterns of neuronal response along established cardiovascular reflex pathways.

Postganglionic sympathetic discharge in neonatal swine.

We hypothesized that cardiac and respiratory modulation of postganglionic peroneal activity appeared in an age-related manner. In anesthetized, paralyzed and artificially ventilated piglets, simultaneous recordings of efferent phrenic and peroneal discharges were obtained during hyperoxia (fraction of inspired oxygen, FiO2 = 1.0) and hypoxia (FiO2 = 0.1). Spectral analyses of peroneal and aortic blood pressure signals revealed peaks at the cardiac frequency (3.25-5.0 Hz). Coherence analysis showed that these two signals were highly correlated at those frequencies, providing evidence for baroreceptor entrainment. Statistically significant (p < 0.05) increases of coherence values were observed during hypoxic stimulation. Such results were observed in most animals despite age, and provided evidence of a potent mechanism for insuring vasomotor tone even in newborn animals. In contrast, spontaneous respiration-related peroneal discharges were observed only in animals > or = 20 d old. In animals < 20 d old, hypoxic stimulation elicited respiration-related discharges in peroneal activity. In many cases, peroneal hypoxic discharges exhibited an immature biphasic response pattern despite the presence of a mature response pattern of phrenic activity. Such findings suggest a developmental lag in the linkages of respiratory and sympathetic controlling networks.

The presence of coherence in sympathetic and phrenic activities in a developing mammal.

To determine whether are development changes in the baroreceptor and central respiratory modulation of sympathetic activity, we used ordinary and partial coherence spectral analyses on cervical and splanchnic sympathetic activity in swine 1-36 days old. Removal of baroreceptor influences from cervical sympathetic and splanchnic spectra using partialization shows that 3-6 Hz peaks are due to baroreceptors since coherence decreased in > 19 days old while remaining unchanged in < 2 weeks old piglets. The 8-12 Hz band (present in normal coherence after 21 days) was revealed in piglets < 14 days old after removal of respiratory modulation by partialization; similarly increased coherence was also observed in the 16-18 Hz band through 3 weeks. Thus, use of partial power and coherency is a useful tool for unmasking the complex relationship found in developing SYMP outflows. These results suggest that there is a period of reorganization within the SYMP rhythm generating circuits, which may be essential for normal development.

Development of a swine animal model for the study of sudden infant death syndrome.

An animal model with selective cardiac autonomic denervation was developed in neonatal swine for the purpose of future studies concerning the possible role of abnormal cardiac innervation in the etiology of sudden infant death syndrome. Twenty-six 4- to 8-day old piglets were randomly assigned to one of the following treatment groups: right stellate ganglionectomy (RSG), left stellate ganglionectomy (LSG), right cardiac vagotomy (RCV), and sham-operated control group. Piglets were anesthetized with Saffan and mechanically ventilated on room air. The thoracic wall was opened, and the sternum was divided, permitting exposure of pleura, heart, and lungs. After completion of one of these operative procedures, the thoracic wall was closed in layers. On successful recovery from anesthesia and extubation, piglets were returned to their sows. Postoperative administration of antibiotics was performed during the first week. Electrocardiograms were recorded once a week over an 8-week period. The sham-operated control group had a postnatal increase in QTc and decrease in heart rate. The RSG group had significantly longer QTc than the sham-operated control group QTc, whereas the RCV group had significantly shorter QTc interval than sham-operated controls. Surgical complications caused the death of two piglets (hemorrhage in one and aspiration in the other), infection caused the death of one piglet 2 weeks after surgery, and three died of unknown causes (one from the RSG group and two from the RCV group). The cause of death for these three animals could not be determined (necropsy was not revealing) because they were not monitored at the time of their demise.(ABSTRACT TRUNCATED AT 250 WORDS)

NADPH-diaphorase activity in piglet intestinal mucosa.

The distribution of the enzyme synthesizing nitric oxide (NO) has been characterized in several mammalian enteric nervous systems. Two methods, immunohistochemical staining, employing anti-nitric oxide synthase antibodies, and histochemical localization of NADPH-diaphorase (NADPH-D), have given the same results. On the other hand, few studies have investigated nitric oxide synthase (NOS) in the gastrointestinal mucosa. Our study demonstrated the presence and distribution of the enzyme, NADPH-D, throughout all layers of the neonatal piglet intestinal tract. In the neonatal piglet, NADPH-D activity was found in nerve fibers parallel to the circular and to the longitudinal muscles and in the ganglion cells of Auerbach's plexus. However, the majority of NADPH-D activity was localized to the mucosa. Furthermore, the most intense activity in the mucosa was observed in villous epithelial cells. Other mucosal cells which were NADPH-D positive included the glandular epithelium and crypt cells. In addition, glandular epithelium in the deeper submucosa had very strong NADPH-D activity. Our results support the hypothesis that locally produced NO mediates physiological functions in the intestinal mucosa and submucosa.

Neuronal activity of the stellate ganglia in neonatal swine.

In anesthetized, paralyzed, and artificially ventilated neonatal pigs, neuronal activity was recorded extracellularly from the intact right stellate ganglion. Of 301 investigated neurons, only a few had spontaneous activity: 27 neurons generated activity either during lung inflation or deflation, and 1 neuron generated activity related to the cardiac cycle. The remaining 273 neurons were activated by stroking of body hair or by light pressure applied to various somatic sites. These data suggest that much of the spontaneous activity in the stellate ganglia appear later during development.