Robert D. Wurster

Topography of cardiac ganglia in the adult human heart

Published descriptions of the topography of cardiac ganglia in the human heart are limited and present conflicting results. This study was carried out to determine the distribution of cardiac ganglia in adult human hearts and to address these conflicts. Hearts obtained from autopsies and heart transplant procedures were sectioned, stained, and examined. Results indicate that the largest populations of cardiac ganglia are near the sinoatrial and atrioventricular nodes. Smaller collections of ganglia exist on the superior left atrial surface, the interatrial septum, and the atrial appendage-atrial junctions. Ganglia also exist at the base of the great vessels and the base of the ventricles. The right atrial free wall, atrial appendages, trunk of the great vessels, and most of the ventricular myocardium are devoid of cardiac ganglia. These findings suggest modifications to surgical procedures involving incisions through regions concentrated with ganglia to minimize arrhythmias and related complications. Repairs of septal defects, valvular procedures, and congenital reconstructions, such as the Senning and Fontan operations, involve incisions through areas densely populated with cardiac ganglia. The current standard procedure for orthotopic heart transplantation severs cardiac ganglia and their projections to nodal and muscular tissue. One modification of the current heart transplantation procedure, involving bicaval anastomosis, preserves atrial anatomy and the cardiac ganglia. Preservation of cardiac ganglia within the donor heart may provide additional neuronal substrate for intracardiac processing and targets for regenerating nerve fibers to the donor heart.

Vagal postganglionic innervation of the canine sinoatrial node.

Differential, selective distribution of parasympathetic, postganglionic innervation to the atrioventricular nodal (AVN) region of the canine heart was recently described. Ablation of parasympathetic pathways to the AVN by disruption of the epicardial fat pad at the junction of the inferior vena cava and inferior left atrium did not interfere with normal vagal control of the sinoatrial node (SAN) function. In sharp contrast, surgical dissection of the fat pad overlying the right pulmonary vein-left atrial junction interrupted the major right and left vagal inputs to the SAN region. The pulmonary vein fat pad (PVFP) in the dog heart is triangular in shape with roughly equilateral dimensions of approximately 1 cm, its base extending from superior to inferior veins, and its apex extending nearly to the sinus nodal artery as it courses rostrally in the sulcus terminalis. Careful dissection of smaller fat pads around the circumference of the pulmonary veins and particularly over the rostral-dorsal surfaces of the right superior pulmonary vein and adjacent right atrium, completed SAN parasympathetic denervation. Care in making these dissections left the vagal supply to the AVN region essentially intact, and preserved the sympathetic supplies to both SAN and AVN regions. Autonomic ganglia, varying in size from 1 or 2 cells to 80-100 cells, were found scattered throughout the ventral PVFP (overlying and surrounding the right pulmonary vein-left atrial junction). The ganglia were generally imbedded in fatty connective tissue, although they commonly rested very close to, or were loosely surrounded by epicardial muscle. Ganglia were also found in smaller fat pads on the dorsal surfaces of the atrium between the azygos and the right superior pulmonary vein.

Monoamine- and Histamine-Synthesizing Enzymes and Neurotransmitters Within Neurons of Adult Human Cardiac Ganglia

BACKGROUND Cardiac ganglia were originally thought to contain only cholinergic neurons relaying parasympathetic information from preganglionic brain stem neurons to the heart. Accumulating evidence, however, suggests that cardiac ganglia contain a heterogeneous population of neurons that synthesize or respond to several different neurotransmitters and neuropeptides. Reports regarding monoamine and histamine synthesis and neurotransmission within cardiac ganglia, however, present conflicting information or are limited in number. Furthermore, very few studies have examined the neurochemistry of adult human cardiac ganglia. The purpose of this study was, therefore, to determine whether monoamine- and histamine-synthesizing enzymes and neurotransmitters exist within neurons of adult human cardiac ganglia. METHODS AND RESULTS Human heart tissue containing cardiac ganglia was obtained during autopsies of patients without cardiovascular pathology. Avidin-biotin complex immunohistochemistry was used to demonstrate tyrosine hydroxylase, L-dopa decarboxylase, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, tryptophan hydroxylase, and histidine decarboxylase immunoreactivity within neurons of cardiac ganglia. Dopamine, norepinephrine, serotonin, and histamine immunoreactivity was also found in ganglionic neurons. Omission or preadsorption of primary antibodies from the antisera and subsequent incubation with cardiac ganglia abolished specific staining in all cases examined. CONCLUSIONS Our results suggest that neurons within cardiac ganglia contain enzymes involved in the synthesis of monoamines and histamine and that they contain dopamine, norepinephrine, serotonin, and histamine immunoreactivity. Our findings suggest a putative role for monoamine and histamine neurotransmission within adult human cardiac ganglia. Additional, functional evidence will be necessary to evaluate what the physiological role of monoamines and histamine may be in neural control of the adult human heart.

Electrical stimulation facilitates rat facial nerve recovery from a crush injury

Objective To study the effect of electrical stimulation on accelerating facial nerve functional recovery from a crush injury in the rat model. Study Design Experimental. Method The main trunk of the right facial nerve was crushed just distal to the stylomastoid foramen, causing right-sided facial paralysis in 17 Sprague-Dawley rats. An electrode apparatus was implanted in all rats. Nine rats underwent electrical stimulation and eight were sham stimulated until complete facial nerve recovery. Facial nerve function was assessed daily by grading eyeblink reflex, vibrissae orientation, and vibrissae movement. Results An electrical stimulation model of the rat facial nerve following axotomy was established. The semi-eyeblink returned significantly earlier (3.71 + 0.97 vs 9.57 + 1.86 days post axotomy) in stimulated rats (P = 0.008). Stimulated rats also recovered all functions earlier, and showed less variability in recovery time. Conclusion Electrical stimulation initiates and accelerates facial nerve recovery in the rat model as it significantly reduces recovery time for the semi-eyeblink reflex, a marker of early recovery. It also hastens recovery of other functions.

In vitro growth inhibition of growth factor-stimulated meningioma cells by calcium channel antagonists

Studies have shown that a majority of meningiomas contain receptors for platelet-derived growth factor and epidermal growth factor and that these growth factors promote the proliferation of meningioma cells in culture. Although the mechanism of action has not been elucidated, intracellular calcium appears to be part of the signal transduction mechanism. Because alterations in intracellular calcium could interrupt this pathway and decrease cellular proliferation, we investigated the effects of calcium channel-blocking agents on the growth of meningioma cells in vitro. Primary meningioma cell cultures were established, and the cells were characterized by light and electron microscopy and by immunohistochemical studies. Then, the cultures were given growth factors and/or various calcium channel antagonists, and growth rates were measured. A dose-response decrease in cell growth was seen when verapamil, nifedipine, or diltiazem (voltage-dependent calcium channel-blocking agents) was added to serum-containing media. Also, these drugs blocked the growth stimulation of epidermal growth factor and platelet-derived growth factor in a similar fashion. Dantrolene, which inhibits the release of sequestered intracellular calcium, was also an effective blocker of the mitogenic stimulation of these growth factors.

Accelerating functional recovery after rat facial nerve injury: Effects of gonadal steroids and electrical stimulation

Objective We investigated the combined effects of electrical stimulation and testosterone propionate on overall recovery time in rats with extracranial crush injuries to the facial nerve. Study Design Male rats underwent castration 3 to 5 days prior to right facial nerve crush injury and electrode implantation. Rats were randomly assigned to two groups: crush injury + testosterone or crush injury with electrical stimulation + testosterone. Recovery was assessed by daily subjective examination documenting vibrissae orientation/movement, semi-eye blink, and full eye blink. Results Milestones of early recovery were noted to be significantly earlier in the groups with electrical stimulation, with/without testosterone. The addition of testosterone to electrical stimulation showed significant earlier return of late recovery parameters and complete overall recovery. Conclusion Electrical stimulation may decrease cell death or promote sprouting to accelerate early recovery. Testosterone may affect the actual rate of axonal regeneration and produce acceleration in functional recovery. By targeting different stages of neural regeneration, the synergy of electrical stimulation and testosterone appears to have promise as a neurotherapeutic strategy for facial nerve injury.

Inhibition of cell growth and intracellular Ca2+ mobilization in human brain tumor cells by Ca2+ channel antagonists.

The effects of various Ca2+ channel agonists and antagonists on tumor cell growth were investigated using U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines. Classical Ca2+ channel antagonists, verapamil, nifedipine, and diltiazem, and inorganic Ca2+ channel antagonists, Ni2+ and Co2+, inhibited growth of these tumor cells in a dose-dependent manner. Except Ni2+, these Ca2+ channel antagonists did not induce a significant cytotoxicity, suggesting that the growth-inhibitory effects of these drugs may be the result of the influence on the proliferative signaling mechanisms of these tumor cells. In contrast, Bay K-8644, a Ca2+ channel agonist, neither enhanced the growth of tumor cells nor increased intracellular Ca2+ concentration, indicating that voltage-sensitive Ca2+ channels may not be involved in tumor cell proliferation. Moreover, growth-inhibitory concentrations of Ca2+ channel antagonists significantly blocked agonist (carbachol or serum)-induced intracellular Ca2+ mobilization, which was monitored using Fura-2 fluorescence technique. These results suggest that the inhibition of the growth of human brain tumor cells induced by Ca2+ channel antagonists may not be the result of interaction with Ca2+ channels, but may be the result of the interference with agonist-induced intracellular Ca2+ mobilization, which is an important proliferative signaling mechanism.

ATTENUATION OF HEART RATE CONTROL AND NEURAL DEGENERATION IN NUCLEUS AMBIGUUS FOLLOWING CHRONIC INTERMITTENT HYPOXIA IN YOUNG ADULT FISCHER 344 RATS

Chronic intermittent hypoxia (CIH) attenuates baroreflex control of heart rate (HR). In this study, we assessed whether CIH exposure reduced nucleus ambiguus (NA) control of HR and induced neural degeneration in the NA. Fischer 344 (age: 3-4 months) rats were exposed to either room air (RA: normoxia) or intermittent hypoxia for 35-50 days. At the end of these exposures, animals were anesthetized with pentobarbital. HR responses to arterial blood pressure (AP) changes induced by phenylephrine (PE) and sodium nitroprusside (SNP) were measured. In another set of rats, HR and AP responses to L-glutamate (L-Glu) microinjections (10 mM, 20 nl) into the left NA and electrical stimulation of the left cervical vagus nerve at 1-30 Hz (0.5 mA, 1 ms) for 20 s were measured. Brainstem slices at the level of -800, -400, 0, +400, +800 microm relative to the obex were processed in additional rats using Nissl staining. The NA was identified by retrogradely labeling vagal motoneurons using the tracer tetramethylrhodamine dextran (TMR-D) which was injected into the ipsilateral nodose ganglion. We found that CIH significantly 1) reduced the baroreflex control of HR (slope RA: -1.2+/-0.2 bpm/mmHg; CIH -0.5+/-0.1 bpm/mmHg; P<0.05); 2) attenuated the HR responses to l-Glu injections into the NA [HR: -280+/-15 (RA) vs. -235+/-16 (CIH) beats/min; P<0.05]; 3) augmented the HR responses to electrical stimulation of the vagus (P<0.05); 4) induced a significant cellular loss in the NA region (P<0.05). Thus, CIH induces a cell loss in the NA region which may contribute to attenuation of baroreflex sensitivity and NA control of HR following CIH.

Determination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG

Accurate estimates of cytosolic free Ca2+ with fluorescence indicators are dependent on the determination of the in situ dissociation constants (kd) of the intracellular dyes and the correction for background fluorescence. The in situ dissociation constant for Ca2+ and indicator dye Fura-2/AM varies significantly from the in vitro published values due to differences in ionic strength, pH, viscosity and Ca2+ buffering by intracellular lipids and proteins. During the course of a measurement, background fluorescence changes may occur as the result of endogenous fluorescent compounds and compartmentalized Fura-2 indicator. The in situ dissociation constant value was determined for human astrocyte cell line U373-MG by creating several known intracellular Ca2+ concentrations while measuring total fluorescence and background fluorescence values for each. The background fluorescence was not constant, rather it demonstrated a linear relationship with the free cytosolic Ca2+ concentrations and total fluorescence intensities. The Ca2+ dependent and total fluorescence dependent background was expressed as a linear equation and subtracted appropriately from the total intensity measurements. The in situ dissociation constant was determined to be 3-fold greater than in vitro measurements after the background was corrected. The experimentally determined standard linear equations for background quantitation and the in situ dissociation constant for this line produce accurate cytosolic free Ca2+ estimates.

Identification of the sympathetic preganglionic pathway to the cat stellate ganglion

Both intraspinal and segmental preganglionic pathways to sympathetic ganglia have been proposed based on different methods of horseradish peroxidase application. The present study mapped the projections of preganglionic neurons to the stellate ganglion in the cat by horseradish peroxidase injection into ganglia with various white rami intact. Central and peripheral cut ends of the severed white rami were tied to eliminate leakage of peroxidase from the ganglia. In control cats with all white rami intact, an average of 9186 neurons were labeled in the spinal cord ipsilateral to the injection from C8 to T9; the highest density of labeling occurred in the first and second thoracic segments. In cats with single white rami intact (T1, T2, or T3), neurons were labeled only in the spinal cord segment that corresponded to the intact ramus and one segment rostral to it. In additional experiments, the spinal cord was hemisected a few segments below, and ipsilateral to the injected ganglion with all rami intact. In these cats neurons were labeled with a similar distribution to control cats, indicating that the preganglionic pathway was extraspinal. Finally, experiments were performed on cats with a single ramus intact, but without ligatures around the cut rami. The distribution of neuronal labeling was similar to control cats (labeled cells from C8 to T9). These results demonstrate that peroxidase leakage from the stellate ganglion can result in an artifactual labeling which would indicate an intraspinal preganglionic pathway. Thus, the present experiments support a segmental distribution of upper thoracic sympathetic preganglionic neurons to the stellate ganglion in the cat, and do not provide evidence for long intraspinal pathways of preganglionic neurons before exit from the cord.