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Autoradiographic study of the right stellate ganglionic fibers to the cat heart
Journal of the Autonomic Nervous System, 2 (1980) 105--I 15
105
O E~evier/North-Holland BiomedicalPren
AUTORADIOGRAFHIC STUDY OF THE RIGHT STELLATE GANGLIONIC FIBERS TO THE CAT HEART
MICHAEL L. NIEHOFF and JAMES M. SULLIVAN Deportment of Anatomy, St. Louis Uniue~ity School of Medicine, St. Louis, Mo 63t04 (U.S.A.)
(Received December 3rd, 1979) (Accepted March 25th, 1!)80) K e y w o r d s : autoradiography -- sympathetic
fiber -- postganglionic liber -steUate ganglion -- cardiac plexus -- autonomic nervous sy:~tem
ABSTRACT Autoradiographic techniques have been employed to demonstrate the distribution of the postganglionic sympathetic cardiac fibers originating from the right stellate ganglion in 3 cats. After exposing the ganglion through a right thoracotomy, a total of 500 #Ci of tritiated leucine was injected into the right stellateganglion of each cat. After 3 days the animals were sacrificed by vascular perfusion. The injected ganglia, the heart and great ~essels were processed for autoradiography. The majority of the right stellate ganglionic fibers travelled between the trachea and aortic arch, and entered the cardiac plexus. Continuing through the plexus, these fibers coursed between the ascending aorta and the pulmonary arterial trunk to form a periarterial plexus around the left coronary artery and its branches. Most of. the fibers followed the distribution of ~.he ventral descending branch of the left coronary artery; a few followed the circumflex artery. A conspicuous subepicardial plexus was derived frcm the periarterial plexus and was most prominent in the cranial half of the le't ventricle. Other fibers adding from the right stellate ganglion descended between the cranial vena ca~a and the right branch of the pulmonary artery. These ramified on the dorsolateral surface of the right atrium, and also formed a large bundle which passed caudally through the interatrial septum.
INTRODUCTION Studies have utilized various morphological techniques to investigate the sympathetic cardiac innervation. McKibben and Getty [9] investigated
106
the sympathetic ir~nervation of the cat heart employing microdissection techniques. Sympathetic fibers from the right stellate ganglion were observed passing through the cardiac plexus and continuing around the caudal surface of the ascending aorta to the left side of the heart. Nerve fibers were also observed travelling with the right and left coronary arteries to innervat~ ~,¢ coronary vasculature, right ventricle, half of the left ventricie, interventricular septum, and the cranial ventral walls of the left and right auricles. In this same study nerve fibers originating within the right stellate ganglion were seen passing between the right branch of the coronary artery and cranial vena cava to ramify on the dorsolateral surface of the right atrium. Silver impregnation studies [ 10,11,131 and fluorescence studies [ 1,2,3,7] have also contributed much information regarding intrinsic cardiac innervation. Generally, sympathetic fibers have been observed travelling either with the coronary vasculature as periarterial plexuses or with the myofibers in the myocardium. Krokhina [71 using fluorescent .nicroscopy observed that all preterminal nerve fibers first travel with the coronary arteries and their branch~.s. Nerve terminals were then observed diverging from the periarterial plextLses, passing into the myocardium, and were found associated with myofib4:rs. Although light microscopy and fluorescence techniques have contributed greatly to the characteristics of the extracardiac and intracardiac inne,-'vation, these techniques give no indication as to the specific origin of the nerve fibers. Autoradio~aphic procedures have only recently been employed to investigate the innervation of the heart [4,5,12,14] and to trace other components of the autonomic nervous system [6,15,16]. By utilizing the physiologic process of axoplasmic transport, autoradiographic techniques can isolate a specific porti(~n of the sympathetic cardiac distribution, allowing its investigation and an accurate interpretation of the origin and destination of nerve fibers. Thelefore, the intent of the present study was to demonstrate the cardiac sympathetic distribution specifically from the right stellate ganglion. MATERIALS A N D Mk'THODS Three adult mongrel cats weighing between 3.0 and 4.0 kg were used for this study. In preparation for surgery the animals were anesthetized with 33 mg/kg of ketamine hydrochloride (Ketaset) i.m. After intubation for positive pressure ventilation a thoracotomy was performed within the first intercostal space on the right side. The right steIIate ganglion, always found dorsal to the first intercostal space and lying on the ventrolateral surface of the right longus colli muscle, was isolated, and 0.1 cc of lidocaine hydrochloride (xylocaine hydrochloride) was used '~o block the postganglionic sympathetic fibers prcducing an antiarrhythmic effect. A total of ~00 /~Ci of tritiated leucine (L-[4,5-JH(N)], N e w England Nuclear) diluted with sterile distilled water to 10 ~l was injcct~Jd into the
107
ganglion. Two to three separate injections were made using a 50 #1 Hamilton syringe with a 30~auge, regular bevel, Yale hypodermic needle. The injections were made between the cranial and caudal poles of the g~mglion. Following a 3 day survival period, the animals were an~stiletlzed with 22 mg/kg of sodium pentobarbital and sacrificed by left transventricular perfusion. Perfusion consisted of two litersof normal saline followed by two litersof 1 0 % buffered neutral formalin. The right stellate ganglion and its branches, the heart and great vessels, and the trachea were removed for microscopic study. The tissues were further fixed in buffered neutral formalin for a m i n i m u m of 4 days, dehydrated in graded alcohols, cleared in xylol, infiltrated, and embedded in paraffin. The ganglia were cut in cross-sections at 15 # m and 1 of every 3--5 sections was mounted on a slide. Serial sections of the heart tissuewere cut in longitudinal or cross~sections at 15 ~ m and mounted on slides. After deparaffinizing, hydrating, and drying, the sections were processed for autoradiography as described by Foody et al. [15 ]. The ganglion tissue was exposed for 10--13 days and was processed using Kodak D-19 developer and Kodak Ektaflt,w ~ixer [5]. The slides were subsequently stained with a 1 % aqueous cresyl violet, dehydrated in alcohols, cleared in xylol, and ¢overslips applied using l'ermount. The exposure time for the heart tissue u ~ 4 or 6 weeks. All the uneven numbered slides containing heart tissue from cat 1 were exposed for 4 weeks and then developed as described above. The slides from cat 2 were exposed for 6 weeks and subsequently developed. After 4- and 6-week~xposure periods, alternate uneven numbered slides from animal 3 were developed. Therefore, slides 1,5,9, etc. were developed after 4 weeks exposure, and slides 3,7,11, etc. were developed after 6 weeks exposure. The developed heart tissue slides from all 3 animals were subsequently stained with Harris' hematoxyl~u and alcoholic eosin. The remaining sections of heart tissue not processed for autoradiography were stained with a modified Masson's trichrome stain to better visualize the cardiac nerve fibers and the conduction system, i.e. sinoatrial and atrioventricular nodes, bundle of His, bundle branches, and Purkinje system. These sections were post-fixed in Bouin's solution at 56°C for 60 rain, washed in running tap water, and stained in Weigert's iron hematoxylin, chromotrope 2R, and light green. The sections were examined under light-fieldand dark-field microscopy using a Leitz Wetzler Ortholux II microscope. RESULTS
Microscopic examination of the injected stellate ga,~giia revealed typical cytological features characteristic of normal neurons, indicating an absence of central chromatolysis. Therefore, the neurons or their axons were not damaged during the leucine injection. Dark.field microscopy of the ganglia revealed a moderate to heavy label throughout, and nerve bundles arising
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Fig. 1. Dark-field photomicrograph of the injected ganglion illustrating the heavily labeled neurons (arrows). 160x. from the ganglia also contained l~eavy concentrations o f label. Fig. 1 is a dark-field photomicrograph illustrating the labeling typical o f the neurons within the ganglia. Terminal portions of sympathetic postga~glionic axons have been shown by ultras~uctural studies [17] to consist of varicosities 0.5--0.2 jam ir diameter alternating with intervaricose segments that can be less than 0.2 jam in diameter. This places the intervaricose ~ g m e n t s beyond the resolution of the light microscope. Therefore, I he present study considered only those autoradiographic silver grains observed overlying histologically identifiable nerve.us tissue to be evidence of autoradiographically labeled axons. It was quite evident upon dark-field examination that the heart tissue of all 3 animals clearly demonstrated nerve bundles with concentrations of tritiated label. Heavily labeled nerve fibers were seen along the right lateral and ~,entral aspects of the trachea. As these fibers approached the heart they were located between the trachea and the arch of the aorta (Fig. 2). The majority of labeled cardiac nerve fibe'.'s, after coursing through the cardiac plexus, formed bundles of nerves which passed between the ascending aorta and the pulmonary arterial trunk to reach the left side of the heart (Fig. 3). These nerve bundles continued into the vicinity of the left coronary artery as it emerged from the left aortic sinus (Fig. 7). Almost immediately the left coronary artery divided into the circumflex artery and the ventral descending branch of the left coronary artery. The descending branch traveled ventrocaudally in the ventral interventricular sulcus where it gave off several branches which distributed to the ventricular myocardium and the interventricular septum. The large nerve bundles seen coursing with the left coronary artery also bifurcated and could be seen following the distribution of the descending branc.h of the left coronary artery and, to
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a lesser extent, the circumflex artery (Fig. 7). These nerves were seen following the arteries into the m y o c ~ r d i u m and the interventricular septum (Fig. 4), and appeared to be located within the adventitial layer of the vessels.
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Fig. 4. Dark-fiel.'t photomicrograph illustrating periarterial nerves (arrows) of an artery (A) located in the left ventrieular myocardium (M). 160x.
A very pr~)rninent subepic~u'dial nerve plexus was seen over m u c h o f t h e left ventricle (Fig. 5a, b). 'I'ho nerve fibers c o n s t i t u t i n g this subepicardial plexus appeared to have their origin in t h e larger nerve b u n d l e s o f t h e periarterial plexus of the left c o r o n a r y artery and its branches. T h e subepicardial plexus was f o u n d i m m e d i a t e l y b e n e a t h t h e e p i c a r d i u m a n d was m~st pr~,valent in t h e cranial t w o - t h i r d s o f the left ventricle. This was espe('ially true on its ventral and lateral aspects where t h e m a j o r i t y of t h e large blood vessels ,,e~e iocated (Figs. t , 8 ) . As these subepicardial nerves
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a p p r o a c h e d the apex of the h e a r t t h e y b e c a m e smaller, less p u m e r o u s , and m o r e difficult to identify as labeled nerves. O t h e r nerve bundles with large c o n c e n t r a t i o n s of tritiated label over t h e m , a p p r o a c h e d the heart from the right stellate ganglion. They passed o n t o the dorsolateral surface of the right atrium and into the interatrial s e p t u m . In longitudinal ~ecl.ions these nerve bundles were followed along the dorsal surface of the cranial vena cava until they reached the right atrium (Fig. 8). Cross sections d e m o n s t r a t e d t h a t m o r e cranially the nerves were located b e t w e e n the cranial veY,a cava and the right branch of the p u l m o n a r y artery. More caudally, the nerve fibers were seen a p p r o a c h i n g and dispersing along the dorsolateral surface of the right atrium forming a subepicardial plexus. A major group of fibers were also seen passing caudally in the dorsal portion of the interatrial s e p t u m . This large bundle d e s c e n d e d to the m o s t caudal portion of the interatria" s e p t u m where it then turned ventrocranially (Fig. 6). The nerves then ascended cranially in the ventral aspect of the septum for a short distance and ramified a m o n g the septal myocardial cells. The sinoatrial and atrioventricular nodes, b u n d l e of His, and Purkinje system were also investigated. The sinoatrial n o d e was not positively identified with t h e histological m e t h o d s utilized. However, the other c o m p o n e n t s of the c o n d u c t i o n system were observed, and were found not to be innervated by labeled nerve fibers from the right stellate ganglion. Also~ few intrinsic cardiac ganglia were observed on the dorsal surface of the right atrium. A l t h o u g h labeled nerve fibers were passing in their vicinity, silver grains were n o t observed in direct appositian to the neurons ~vithin these ganglia. Figs. 7 and 8 are diagrams summarizing ~he observed cardiac distribution of the right stellate ganglion. Only arteries were observed to have peri-
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Fig. 7. Illustration of the left ventrolateral aspect of the cat heart demonstrating cardiac innervation. Solid lines, arteries: broken lines, periarterial plexuses; and dotted lines, subepicardia] plexuses.
vascular nerve bundles within their adventitia. This perial'terial plexus also gave rise to the subepicardial plexus formed over much of the left ventricle. DISCUSSION
Cardiovascular postganglionic pathways originating from the right stellate galiglion have been found coursing primarily to the left side of the heart to form periarterial and subepicardial plexuses. In the present study only the AORTIC ARCH_
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113
cardiac projections originating in the right stellate ganglion were investigated. Other ipsilateral or contralateral ganglia, i.e. the cranial cervical, vertebral, or intermediate ganglia, and their interganglionic connections with the right stellate were n o t examined. Instead, this study concentrated on the intracardiac fibers originating only in the right stellate ganglion. The present autoradiographic investigation confirms the predominantly left-sided perivascular distribution of the right stellate ganglion as observed by other investigators [9]. Nerve fibers were seen traveling with the left coronary artery and its branches to innervate most of the anterior and lateral cranial two-thirds of the left ventricle and the interventricular septum. However, contrary to the reports of McKibben and Getty [9], labeled nerve fibers were not observed to travel with the right coronary artery to innervate the right ventricle. McKibben and Getty [9] also reported nerve fibers from the right stellate ganglion passing between the right branch of the pulmonary artery and the cranial vena cava to ramify on the dorsolateral surface of the right atrium. A right atrial subepicardial plexus was also seen in the present investigation. Autoradiographically labeled nerves were observed descending from a position between the cranial vena cava and the right branch of the pulmonary artery. Some of these fibers ramified in the subepicardium of the dorsoiateral right atrium. However, a large bundle of these fibers passed medially to reach the interatrial septum. The majority of the nerves penetrated the interatrial septum where they traveled caudally in its dorsal aspect. This bundle descended to the most caudal portion of the septum where it then turned vel~trocranially to ascend a short distance, and then ramified among the septal myocardial cells. Throughout their course these septal nerve fibers traveled without accompanying blood vessels. It might be expected that these nerve bundles may have some terminations on the atrioventricular nodal tissue or other portions of the conduction system. However, there were no labeled nerve fibers observed i,l relation to the specialize~; ticsues of the heart. Using fluorescent microscopy, Ellison [3] also described nerve bundles within the interatrial septum. He reported these interatrial septal fibers as being large bundles coursing independent of the :oronary arterial distribution and dispersing within the septum. A pronounced left ventricular subepicardial plexus was also observed in the cranial portion of the left ventricle. The subepicardial nerves arose from the large nerve bundles that were seen traveling with the blanches of the left coronary artery as they penetrated through the myocardium. Using fluorescence microscopy, Ellison [3] stated that there are few nerves in the subepicardium of the ventricles. Other investigators [2,3,10], however, have reported a subepicardial plexus distributed in the stature of loose connective tissue directly beneath the epicardium in most areas of the heart. Mention should also be made of the postoperative survival time of 3 days utilized in this study. The estimated distance of axoplasmic transport from the right stellate ganglicn to the apex of the heart was 70--90 ram, relative to the overall size of the animal. Cat sympathetic ganglion cells have a fast
114
cc,mponent transport velocity of approximately 120 mm/day [8]. Therefore, it is be Jieved that a survival period of 3 days was adequate time to allow complete uptake and transport of the tritiated precursor by the neurons. This period of time, however, does not allow sufficient transport time for the slow velocity component of 1 mm/day [3] which would have required an impractical survival period. In conclusion, the autoradiographic technique utilized in this study employed the physiologic process of orthograde axopiasmic transport to demonstrate the cardiac distribution of the right stellate postganglionic fibers. Thi.q ganglion was found to have a predominantly left4ided distribution, forming periarterial and subepicardial plexuses over much of the left ventricle. A right atrial subepicardial plexus and interatrial septal fibers were also ob~rved. ACKNOWLEDGEMENTS
The authors wish to thank Ms. Ann Smith and Ms. Jill Warhoover for typing this manuscript. This study was supported in part by U S P H S Grant F R 05388. REFERENCES 1 Dahlstrom, A., Fuxe, K., Mya-Tc, M. and Zetterstrom, B.E.M., Observations on adrenergic innervation of the dog heart, Amer. J. Physiol., 209 (1965) 689--692. 2 Dolezel, S., Gerova, M., Gero, J., Sladek, T. and Vaska, J., Adrenergic innervation of the coronary arteries and the myocardium, Acts Anat. (Basel), 100 (1978) 306--316. 3 Elliaon, J.P., The adrenergic cardiac nerves of the cat, Amer. J. Anat., 139 (1974) 209--226. 4 Food,y, L.T., D'Agrosa, L.S. and Connors, N.A., Autoradiography of sympathetic pogtgangiionic fibers to the canine heart, Physiologist, (abstr.), 21 (1978) 38. 5 Foody, L.T., Niehoff, M.N., D'Agrosa, L.S., Connors, N.A. and Sullivan, J.M., The orthograde labeling of postgangiionic sympathetic cardiac nerves in the dog and cat as demonstrated by autoradiography, J. Auton. Nerv. Syst., 1 (1979) 77--91. 6 Kelts, K.A., Whitlock, D.G., Ledbury, P.A. and Reese, B.A., Poltganglionic connections between sympathetic ganglia in the solar plexus of the cat demonstrated autoradio~'aphicaIly, Exp. NeuroI., 63 (1979) 120--134. 7 Krokhina, E.M., The adrenergic component of the effector heart innervation. Histochemical study by the method of fluorescent microscopy, Acts Anat. (Basel), 74 (1969) 214--227. 8 Livett, B.G., Geffen, L.B. and Austin, L., Proximo-distal transport of | 14C] noradrenaline and protein in sympathetic nerves, J. Neurochem., 15 (1968) 931--939. 9 McKibben, J.S. and Getty, R., A comparative morphologic ,tudy of the cardiac inncrvstion in domestic animals. II. The feline, Amer. J. Anat., 122 (1968) 545---554. 10 Mikhail, Y., Intrinsic nerve supply of the ventricles of the heart, Acts Anat. (Basel), 76 (1970) 2 8 9 - 2 9 8 . 11 Mikhail, Y. and Kamel, I., Nerve endings in the atrial muscle of the heart, Acta Anat. (Basel), 82 (1972) 138--144. 12 Niehoff, M.L. ~nd Sullivan, J.M., Efferent cardiac projections of the right stellate ganglion in t~.e cat, Neurosci. Abstr., 4 (1978) 23.
115 13 Qayyum, M.A., Innervation of the heart of the Indian cat Felil caius, Acta Anat. (Bs~I), 85 (1973) 395--406. 14 Sullivan, J.M. and Connors, N.A., An autoradiographic study of vagal preganglionic fibers to rat bronchi, heart, and lower esophagus, Neuroeei. Abstr., 4 (1978) 25. 15 Whitlock, D.G., Gilmartin, P. and Ledbury, P.A., Computer and autoradiographic analysiJs of nerve fibers in the mammalian thoracoabdominal visceral nervous system, J . Anat. (Basel), (abstr.), 128 (1978) 651--662. 16 Whitlock, D.G., Ledbury, P.A., Land, L.J. and Lehman, R.A., Innervation of some deep ~issues in the thoracolumbar region of mammals, Neurosci. Abstr., 3 (1977) 464. 17 Yamauchi, A., Ultras'ructure of the mammalian heart. In C.E. Challice and S. Viragh (Eds.), Ultrastructure of the Mammalian Heart. Academic Press, New York, 1973, pp. 127--178.
105
O E~evier/North-Holland BiomedicalPren
AUTORADIOGRAFHIC STUDY OF THE RIGHT STELLATE GANGLIONIC FIBERS TO THE CAT HEART
MICHAEL L. NIEHOFF and JAMES M. SULLIVAN Deportment of Anatomy, St. Louis Uniue~ity School of Medicine, St. Louis, Mo 63t04 (U.S.A.)
(Received December 3rd, 1979) (Accepted March 25th, 1!)80) K e y w o r d s : autoradiography -- sympathetic
fiber -- postganglionic liber -steUate ganglion -- cardiac plexus -- autonomic nervous sy:~tem
ABSTRACT Autoradiographic techniques have been employed to demonstrate the distribution of the postganglionic sympathetic cardiac fibers originating from the right stellate ganglion in 3 cats. After exposing the ganglion through a right thoracotomy, a total of 500 #Ci of tritiated leucine was injected into the right stellateganglion of each cat. After 3 days the animals were sacrificed by vascular perfusion. The injected ganglia, the heart and great ~essels were processed for autoradiography. The majority of the right stellate ganglionic fibers travelled between the trachea and aortic arch, and entered the cardiac plexus. Continuing through the plexus, these fibers coursed between the ascending aorta and the pulmonary arterial trunk to form a periarterial plexus around the left coronary artery and its branches. Most of. the fibers followed the distribution of ~.he ventral descending branch of the left coronary artery; a few followed the circumflex artery. A conspicuous subepicardial plexus was derived frcm the periarterial plexus and was most prominent in the cranial half of the le't ventricle. Other fibers adding from the right stellate ganglion descended between the cranial vena ca~a and the right branch of the pulmonary artery. These ramified on the dorsolateral surface of the right atrium, and also formed a large bundle which passed caudally through the interatrial septum.
INTRODUCTION Studies have utilized various morphological techniques to investigate the sympathetic cardiac innervation. McKibben and Getty [9] investigated
106
the sympathetic ir~nervation of the cat heart employing microdissection techniques. Sympathetic fibers from the right stellate ganglion were observed passing through the cardiac plexus and continuing around the caudal surface of the ascending aorta to the left side of the heart. Nerve fibers were also observed travelling with the right and left coronary arteries to innervat~ ~,¢ coronary vasculature, right ventricle, half of the left ventricie, interventricular septum, and the cranial ventral walls of the left and right auricles. In this same study nerve fibers originating within the right stellate ganglion were seen passing between the right branch of the coronary artery and cranial vena cava to ramify on the dorsolateral surface of the right atrium. Silver impregnation studies [ 10,11,131 and fluorescence studies [ 1,2,3,7] have also contributed much information regarding intrinsic cardiac innervation. Generally, sympathetic fibers have been observed travelling either with the coronary vasculature as periarterial plexuses or with the myofibers in the myocardium. Krokhina [71 using fluorescent .nicroscopy observed that all preterminal nerve fibers first travel with the coronary arteries and their branch~.s. Nerve terminals were then observed diverging from the periarterial plextLses, passing into the myocardium, and were found associated with myofib4:rs. Although light microscopy and fluorescence techniques have contributed greatly to the characteristics of the extracardiac and intracardiac inne,-'vation, these techniques give no indication as to the specific origin of the nerve fibers. Autoradio~aphic procedures have only recently been employed to investigate the innervation of the heart [4,5,12,14] and to trace other components of the autonomic nervous system [6,15,16]. By utilizing the physiologic process of axoplasmic transport, autoradiographic techniques can isolate a specific porti(~n of the sympathetic cardiac distribution, allowing its investigation and an accurate interpretation of the origin and destination of nerve fibers. Thelefore, the intent of the present study was to demonstrate the cardiac sympathetic distribution specifically from the right stellate ganglion. MATERIALS A N D Mk'THODS Three adult mongrel cats weighing between 3.0 and 4.0 kg were used for this study. In preparation for surgery the animals were anesthetized with 33 mg/kg of ketamine hydrochloride (Ketaset) i.m. After intubation for positive pressure ventilation a thoracotomy was performed within the first intercostal space on the right side. The right steIIate ganglion, always found dorsal to the first intercostal space and lying on the ventrolateral surface of the right longus colli muscle, was isolated, and 0.1 cc of lidocaine hydrochloride (xylocaine hydrochloride) was used '~o block the postganglionic sympathetic fibers prcducing an antiarrhythmic effect. A total of ~00 /~Ci of tritiated leucine (L-[4,5-JH(N)], N e w England Nuclear) diluted with sterile distilled water to 10 ~l was injcct~Jd into the
107
ganglion. Two to three separate injections were made using a 50 #1 Hamilton syringe with a 30~auge, regular bevel, Yale hypodermic needle. The injections were made between the cranial and caudal poles of the g~mglion. Following a 3 day survival period, the animals were an~stiletlzed with 22 mg/kg of sodium pentobarbital and sacrificed by left transventricular perfusion. Perfusion consisted of two litersof normal saline followed by two litersof 1 0 % buffered neutral formalin. The right stellate ganglion and its branches, the heart and great vessels, and the trachea were removed for microscopic study. The tissues were further fixed in buffered neutral formalin for a m i n i m u m of 4 days, dehydrated in graded alcohols, cleared in xylol, infiltrated, and embedded in paraffin. The ganglia were cut in cross-sections at 15 # m and 1 of every 3--5 sections was mounted on a slide. Serial sections of the heart tissuewere cut in longitudinal or cross~sections at 15 ~ m and mounted on slides. After deparaffinizing, hydrating, and drying, the sections were processed for autoradiography as described by Foody et al. [15 ]. The ganglion tissue was exposed for 10--13 days and was processed using Kodak D-19 developer and Kodak Ektaflt,w ~ixer [5]. The slides were subsequently stained with a 1 % aqueous cresyl violet, dehydrated in alcohols, cleared in xylol, and ¢overslips applied using l'ermount. The exposure time for the heart tissue u ~ 4 or 6 weeks. All the uneven numbered slides containing heart tissue from cat 1 were exposed for 4 weeks and then developed as described above. The slides from cat 2 were exposed for 6 weeks and subsequently developed. After 4- and 6-week~xposure periods, alternate uneven numbered slides from animal 3 were developed. Therefore, slides 1,5,9, etc. were developed after 4 weeks exposure, and slides 3,7,11, etc. were developed after 6 weeks exposure. The developed heart tissue slides from all 3 animals were subsequently stained with Harris' hematoxyl~u and alcoholic eosin. The remaining sections of heart tissue not processed for autoradiography were stained with a modified Masson's trichrome stain to better visualize the cardiac nerve fibers and the conduction system, i.e. sinoatrial and atrioventricular nodes, bundle of His, bundle branches, and Purkinje system. These sections were post-fixed in Bouin's solution at 56°C for 60 rain, washed in running tap water, and stained in Weigert's iron hematoxylin, chromotrope 2R, and light green. The sections were examined under light-fieldand dark-field microscopy using a Leitz Wetzler Ortholux II microscope. RESULTS
Microscopic examination of the injected stellate ga,~giia revealed typical cytological features characteristic of normal neurons, indicating an absence of central chromatolysis. Therefore, the neurons or their axons were not damaged during the leucine injection. Dark.field microscopy of the ganglia revealed a moderate to heavy label throughout, and nerve bundles arising
108
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Fig. 1. Dark-field photomicrograph of the injected ganglion illustrating the heavily labeled neurons (arrows). 160x. from the ganglia also contained l~eavy concentrations o f label. Fig. 1 is a dark-field photomicrograph illustrating the labeling typical o f the neurons within the ganglia. Terminal portions of sympathetic postga~glionic axons have been shown by ultras~uctural studies [17] to consist of varicosities 0.5--0.2 jam ir diameter alternating with intervaricose segments that can be less than 0.2 jam in diameter. This places the intervaricose ~ g m e n t s beyond the resolution of the light microscope. Therefore, I he present study considered only those autoradiographic silver grains observed overlying histologically identifiable nerve.us tissue to be evidence of autoradiographically labeled axons. It was quite evident upon dark-field examination that the heart tissue of all 3 animals clearly demonstrated nerve bundles with concentrations of tritiated label. Heavily labeled nerve fibers were seen along the right lateral and ~,entral aspects of the trachea. As these fibers approached the heart they were located between the trachea and the arch of the aorta (Fig. 2). The majority of labeled cardiac nerve fibe'.'s, after coursing through the cardiac plexus, formed bundles of nerves which passed between the ascending aorta and the pulmonary arterial trunk to reach the left side of the heart (Fig. 3). These nerve bundles continued into the vicinity of the left coronary artery as it emerged from the left aortic sinus (Fig. 7). Almost immediately the left coronary artery divided into the circumflex artery and the ventral descending branch of the left coronary artery. The descending branch traveled ventrocaudally in the ventral interventricular sulcus where it gave off several branches which distributed to the ventricular myocardium and the interventricular septum. The large nerve bundles seen coursing with the left coronary artery also bifurcated and could be seen following the distribution of the descending branc.h of the left coronary artery and, to
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a lesser extent, the circumflex artery (Fig. 7). These nerves were seen following the arteries into the m y o c ~ r d i u m and the interventricular septum (Fig. 4), and appeared to be located within the adventitial layer of the vessels.
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Fig. 3. Dark-field photomicrograph of a large well labeled nerve bundle (arrows) located between the a~ending aorta (not shown) and the pulmonary arterial trunk (P). 63~.
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Fig. 4. Dark-fiel.'t photomicrograph illustrating periarterial nerves (arrows) of an artery (A) located in the left ventrieular myocardium (M). 160x.
A very pr~)rninent subepic~u'dial nerve plexus was seen over m u c h o f t h e left ventricle (Fig. 5a, b). 'I'ho nerve fibers c o n s t i t u t i n g this subepicardial plexus appeared to have their origin in t h e larger nerve b u n d l e s o f t h e periarterial plexus of the left c o r o n a r y artery and its branches. T h e subepicardial plexus was f o u n d i m m e d i a t e l y b e n e a t h t h e e p i c a r d i u m a n d was m~st pr~,valent in t h e cranial t w o - t h i r d s o f the left ventricle. This was espe('ially true on its ventral and lateral aspects where t h e m a j o r i t y of t h e large blood vessels ,,e~e iocated (Figs. t , 8 ) . As these subepicardial nerves
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a p p r o a c h e d the apex of the h e a r t t h e y b e c a m e smaller, less p u m e r o u s , and m o r e difficult to identify as labeled nerves. O t h e r nerve bundles with large c o n c e n t r a t i o n s of tritiated label over t h e m , a p p r o a c h e d the heart from the right stellate ganglion. They passed o n t o the dorsolateral surface of the right atrium and into the interatrial s e p t u m . In longitudinal ~ecl.ions these nerve bundles were followed along the dorsal surface of the cranial vena cava until they reached the right atrium (Fig. 8). Cross sections d e m o n s t r a t e d t h a t m o r e cranially the nerves were located b e t w e e n the cranial veY,a cava and the right branch of the p u l m o n a r y artery. More caudally, the nerve fibers were seen a p p r o a c h i n g and dispersing along the dorsolateral surface of the right atrium forming a subepicardial plexus. A major group of fibers were also seen passing caudally in the dorsal portion of the interatrial s e p t u m . This large bundle d e s c e n d e d to the m o s t caudal portion of the interatria" s e p t u m where it then turned ventrocranially (Fig. 6). The nerves then ascended cranially in the ventral aspect of the septum for a short distance and ramified a m o n g the septal myocardial cells. The sinoatrial and atrioventricular nodes, b u n d l e of His, and Purkinje system were also investigated. The sinoatrial n o d e was not positively identified with t h e histological m e t h o d s utilized. However, the other c o m p o n e n t s of the c o n d u c t i o n system were observed, and were found not to be innervated by labeled nerve fibers from the right stellate ganglion. Also~ few intrinsic cardiac ganglia were observed on the dorsal surface of the right atrium. A l t h o u g h labeled nerve fibers were passing in their vicinity, silver grains were n o t observed in direct appositian to the neurons ~vithin these ganglia. Figs. 7 and 8 are diagrams summarizing ~he observed cardiac distribution of the right stellate ganglion. Only arteries were observed to have peri-
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Fig. 7. Illustration of the left ventrolateral aspect of the cat heart demonstrating cardiac innervation. Solid lines, arteries: broken lines, periarterial plexuses; and dotted lines, subepicardia] plexuses.
vascular nerve bundles within their adventitia. This perial'terial plexus also gave rise to the subepicardial plexus formed over much of the left ventricle. DISCUSSION
Cardiovascular postganglionic pathways originating from the right stellate galiglion have been found coursing primarily to the left side of the heart to form periarterial and subepicardial plexuses. In the present study only the AORTIC ARCH_
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cardiac projections originating in the right stellate ganglion were investigated. Other ipsilateral or contralateral ganglia, i.e. the cranial cervical, vertebral, or intermediate ganglia, and their interganglionic connections with the right stellate were n o t examined. Instead, this study concentrated on the intracardiac fibers originating only in the right stellate ganglion. The present autoradiographic investigation confirms the predominantly left-sided perivascular distribution of the right stellate ganglion as observed by other investigators [9]. Nerve fibers were seen traveling with the left coronary artery and its branches to innervate most of the anterior and lateral cranial two-thirds of the left ventricle and the interventricular septum. However, contrary to the reports of McKibben and Getty [9], labeled nerve fibers were not observed to travel with the right coronary artery to innervate the right ventricle. McKibben and Getty [9] also reported nerve fibers from the right stellate ganglion passing between the right branch of the pulmonary artery and the cranial vena cava to ramify on the dorsolateral surface of the right atrium. A right atrial subepicardial plexus was also seen in the present investigation. Autoradiographically labeled nerves were observed descending from a position between the cranial vena cava and the right branch of the pulmonary artery. Some of these fibers ramified in the subepicardium of the dorsoiateral right atrium. However, a large bundle of these fibers passed medially to reach the interatrial septum. The majority of the nerves penetrated the interatrial septum where they traveled caudally in its dorsal aspect. This bundle descended to the most caudal portion of the septum where it then turned vel~trocranially to ascend a short distance, and then ramified among the septal myocardial cells. Throughout their course these septal nerve fibers traveled without accompanying blood vessels. It might be expected that these nerve bundles may have some terminations on the atrioventricular nodal tissue or other portions of the conduction system. However, there were no labeled nerve fibers observed i,l relation to the specialize~; ticsues of the heart. Using fluorescent microscopy, Ellison [3] also described nerve bundles within the interatrial septum. He reported these interatrial septal fibers as being large bundles coursing independent of the :oronary arterial distribution and dispersing within the septum. A pronounced left ventricular subepicardial plexus was also observed in the cranial portion of the left ventricle. The subepicardial nerves arose from the large nerve bundles that were seen traveling with the blanches of the left coronary artery as they penetrated through the myocardium. Using fluorescence microscopy, Ellison [3] stated that there are few nerves in the subepicardium of the ventricles. Other investigators [2,3,10], however, have reported a subepicardial plexus distributed in the stature of loose connective tissue directly beneath the epicardium in most areas of the heart. Mention should also be made of the postoperative survival time of 3 days utilized in this study. The estimated distance of axoplasmic transport from the right stellate ganglicn to the apex of the heart was 70--90 ram, relative to the overall size of the animal. Cat sympathetic ganglion cells have a fast
114
cc,mponent transport velocity of approximately 120 mm/day [8]. Therefore, it is be Jieved that a survival period of 3 days was adequate time to allow complete uptake and transport of the tritiated precursor by the neurons. This period of time, however, does not allow sufficient transport time for the slow velocity component of 1 mm/day [3] which would have required an impractical survival period. In conclusion, the autoradiographic technique utilized in this study employed the physiologic process of orthograde axopiasmic transport to demonstrate the cardiac distribution of the right stellate postganglionic fibers. Thi.q ganglion was found to have a predominantly left4ided distribution, forming periarterial and subepicardial plexuses over much of the left ventricle. A right atrial subepicardial plexus and interatrial septal fibers were also ob~rved. ACKNOWLEDGEMENTS
The authors wish to thank Ms. Ann Smith and Ms. Jill Warhoover for typing this manuscript. This study was supported in part by U S P H S Grant F R 05388. REFERENCES 1 Dahlstrom, A., Fuxe, K., Mya-Tc, M. and Zetterstrom, B.E.M., Observations on adrenergic innervation of the dog heart, Amer. J. Physiol., 209 (1965) 689--692. 2 Dolezel, S., Gerova, M., Gero, J., Sladek, T. and Vaska, J., Adrenergic innervation of the coronary arteries and the myocardium, Acts Anat. (Basel), 100 (1978) 306--316. 3 Elliaon, J.P., The adrenergic cardiac nerves of the cat, Amer. J. Anat., 139 (1974) 209--226. 4 Food,y, L.T., D'Agrosa, L.S. and Connors, N.A., Autoradiography of sympathetic pogtgangiionic fibers to the canine heart, Physiologist, (abstr.), 21 (1978) 38. 5 Foody, L.T., Niehoff, M.N., D'Agrosa, L.S., Connors, N.A. and Sullivan, J.M., The orthograde labeling of postgangiionic sympathetic cardiac nerves in the dog and cat as demonstrated by autoradiography, J. Auton. Nerv. Syst., 1 (1979) 77--91. 6 Kelts, K.A., Whitlock, D.G., Ledbury, P.A. and Reese, B.A., Poltganglionic connections between sympathetic ganglia in the solar plexus of the cat demonstrated autoradio~'aphicaIly, Exp. NeuroI., 63 (1979) 120--134. 7 Krokhina, E.M., The adrenergic component of the effector heart innervation. Histochemical study by the method of fluorescent microscopy, Acts Anat. (Basel), 74 (1969) 214--227. 8 Livett, B.G., Geffen, L.B. and Austin, L., Proximo-distal transport of | 14C] noradrenaline and protein in sympathetic nerves, J. Neurochem., 15 (1968) 931--939. 9 McKibben, J.S. and Getty, R., A comparative morphologic ,tudy of the cardiac inncrvstion in domestic animals. II. The feline, Amer. J. Anat., 122 (1968) 545---554. 10 Mikhail, Y., Intrinsic nerve supply of the ventricles of the heart, Acts Anat. (Basel), 76 (1970) 2 8 9 - 2 9 8 . 11 Mikhail, Y. and Kamel, I., Nerve endings in the atrial muscle of the heart, Acta Anat. (Basel), 82 (1972) 138--144. 12 Niehoff, M.L. ~nd Sullivan, J.M., Efferent cardiac projections of the right stellate ganglion in t~.e cat, Neurosci. Abstr., 4 (1978) 23.
115 13 Qayyum, M.A., Innervation of the heart of the Indian cat Felil caius, Acta Anat. (Bs~I), 85 (1973) 395--406. 14 Sullivan, J.M. and Connors, N.A., An autoradiographic study of vagal preganglionic fibers to rat bronchi, heart, and lower esophagus, Neuroeei. Abstr., 4 (1978) 25. 15 Whitlock, D.G., Gilmartin, P. and Ledbury, P.A., Computer and autoradiographic analysiJs of nerve fibers in the mammalian thoracoabdominal visceral nervous system, J . Anat. (Basel), (abstr.), 128 (1978) 651--662. 16 Whitlock, D.G., Ledbury, P.A., Land, L.J. and Lehman, R.A., Innervation of some deep ~issues in the thoracolumbar region of mammals, Neurosci. Abstr., 3 (1977) 464. 17 Yamauchi, A., Ultras'ructure of the mammalian heart. In C.E. Challice and S. Viragh (Eds.), Ultrastructure of the Mammalian Heart. Academic Press, New York, 1973, pp. 127--178.