THE ANATOMICAL RECORD 255:271–294 (1999) Anatomical Study of the Neural Ganglionated Plexus in the Canine Right Atrium: Implications for Selective Denervation and Electrophysiology of the Sinoatrial Node in Dog DAINIUS H. PAUZA,* VALDAS SKRIPKA, NERINGA PAUZIENE, AND RIMVYDAS STROPUS Department of Human Anatomy, Kaunas Medical University, Kaunas LT-3000, Lithuania ABSTRACT The aim of the present study was to elucidate the topography and architecture of the intrinsic neural plexus (INP) in the canine right atrium because of its importance for selective denervation of the sinoatrial node (SAN). The morphology of the intrinsic INP was revealed by a histochemical method for acetylcholinesterase in whole hearts of 36 mongrel dogs and examined by stereoscopic, contact, and electron microscopes. At the hilum of the heart, nerves forming a right atrial INP were detected in five sites adjacent to the right superior pulmonary veins and superior vena cava (SVC). Nerves entered the epicardium and formed a INP, the ganglia of which, as a wide ganglionated field, were continuously distributed on the sides of the root of the SVC (RSVC). The epicardiac ganglia located on the RSVC were differentially involved in the innervation of the sinoatrial node, as revealed by epicardiac nerves emanating from its lower ganglia that proceed also into the atrial walls and right auricle. The INP on the RSVC (INP-RSVC) varied from animal to animal and in relation to the age of the animal. The INP-RSVC of juvenile dogs contained more small ganglia than that of adult animals. Generally, the canine INP-RSVC included 434 ⫾ 29 small, 17 ⫾ 4 medium-sized, and 3 ⫾ 1 large epicardiac ganglia that contained an estimated 44,700, 6,400, and 2,800 neurons, respectively. Therefore, the canine right atrium, including the SAN, may be innervated by more than 54,000 intracardiac neurons residing mostly in the INP-RSVC. In conclusion, the present study indicates that epicardiac ganglia that project to the SA-node are distributed more widely and are more abundant Abbreviations: AChE, acetylcholinesterase; Ad, adipocyte; Ao, aorta; ar, artery; BL, blood vessel; CA, conus arteriosus; Co, collagen fibers; CS, coronary sulcus; CT, crista terminalis; D, dendrite; De, dendrite-like process; DL, dorsal lower zone of the RSVC; DU, dorsal upper zone of the RSVC; ENP-RSVC, epicardiac neural plexus on the RSVC; En, endocardium; Ep, epicardium; F, fibroblast process; FE, free edge of the right auricle; FHH, fat in the heart hilum; FP, fat pad; INP, intrinsic neural plexus; ISRAu, inferior surface of the RAu; IVC, inferior vena cava; LA, left atrium; LP, lipofuscin granules; LU, lateral upper zone of the RSVC; My, myocardium; N, nucleus; Ne, nerve; Nu, nucleolus; OSVC, orifice of the SVC; PFP, pulmonary fat pad; RA, righta- r 1999 WILEY-LISS, INC. trium; RAu, right auricle; RIVC, root of the inferior vena cava; RSPV, right superior pulmonary veins; RSVC, root of the superior vena cava; RV, right ventricle; SAN, sinoatrial node zone; Sat, satellite cells; Sch, Schwann cells; SVC, superior vena cava; TS, terminal sulcus; V, axon varicosity; VA, vena azygos; VFP, ventral fat pad; VIAS, ventral interatrial sulcus; VL, ventral lower zone of the RSVC; VU, ventral upper zone of the RSVC. *Correspondence to: Dr. Dainius H. Pauza, Laboratory of Neuromorphology, Department of Human Anatomy, Kaunas Medical University, A. Mickeviciaus Street 9, Kaunas LT-3000, Lithuania. E-mail: firstname.lastname@example.org Received 11 July 1998; Accepted 2 March 1999 272 PAUZA ET AL. than was previously thought. Therefore, both selective and total denervation of the canine SAN should involve the whole region of the RSVC containing the INP-RSVC. Anat Rec 255:271–294, 1999. r 1999 Wiley-Liss, Inc. Key words: heart innervation; intrinsic neural plexus; autonomic ganglia; intracardiac neurons; sinoatrial node; right atrium; anatomy; electron microscopy; dog The dog is an important animal model in which the influence of the intrinsic cardiac nervous system on the electrophysiological and dynamic properties of the heart, as well as on the mechanisms of genesis of arrhythmias, has been studied (Priola et al., 1980; Janes et al., 1986; Inoue et al., 1987, 1988; Black et al., 1993; Amano et al., 1994; Noble et al., 1993; Opthof et al., 1993; Kingma et al., 1994; Murphy et al., 1994a,b; Page et al., 1995; Armour et al., 1995; Horackova and Armour, 1995; Korczyn et al., 1982; Stramba-Badiale et al., 1990; Aidonidis et al., 1993; Mcguire et al., 1994; Huang et al., 1994; etc.). Remarkable progress in understanding the topography of the canine intracardiac ganglia, including the location of neural pathways to select heart regions, has been made by a range of physiological and anatomical investigations for the last 30 years (Randall et al., 1968, 1986a,b, 1987, 1988; Randall et al., 1992; Kaye et al., 1970; Geis et al., 1973; Armour and Randall, 1975; Priola et al., 1977, 1980; Randall and Ardell, 1985; Ardell and Randall, 1986; Gagliardi et al., 1988; Bluemel et al., 1990; Page et al., 1995). Using electrical stimulation of distinct extracardiac nerves, selective surgical intracardiac neurotomy, heart activity control and neurohistological examination of the excised pieces from the different heart sites, the locations of the intracardiac ganglia and nerve pathways in epicardiac fat pads on the ventral, lateral, and dorsal surfaces of canine right atrium, on the dorsal and ventral surfaces of the left atrium, as well as in the junction of the inferior vena cava (IVC) and the dorsal inferior surface of the right atrium have been determined. In spite of this progress, the structural organization of the canine intrinsic neural plexus (INP) which resides in the right atrium, including region of the sinoatrial node (SAN), remains insufficiently clear (Yuan et al., 1994a). The intricacy of the INP in this region may be implied from the physiological studies that investigated the responses of the canine heart rate to the cervical vagal or stellate ganglia stimulation after selective SA-nodal parasympathectomy and total intrapericardiac denervation (Geis et al., 1973; Armour and Randall, 1975; Ardell and Randall, 1986; Randall et al., 1980; Randall DC et al., 1992). These reports have indicated that in canine hearts sympathetic innervation of the SAN is along the superior vena cava (SVC), the interatrial groove, and around great arteries, while SA-nodal parasympathetic pathways enter the heart along the SVC, superior left atrium, and interatrial groove. However, in some experiments the intrinsic ganglion cells mediating nerve control of the SAN were found being located in the fat pad between aorta and pulmonary artery (Yuan et al., 1994b). Moreover, an interanimal variation of the distribution of right and left vagal branches was revealed in canine hearts by ablation of the superior vena caval-right atrial junction, azygos, right pulmonary vein complex, and the dorsal surfaces of the common pulmonary vein complex (Randall and Ardell, 1985). The overlapping of the parasympathetic pathways from either right and left vagi projecting to both sinoatrial and atrioventricular nodes was physiologically demonstrated in the loci along and between the right pulmonary artery, and around the right pulmonary veins on the dorsal surface of canine right atrium (Ardell and Randall, 1986), as well as the intrinsic neurons modulating myocardial blood flow were found situated within the same neural plexus (Kingma et al., 1994). These findings suggest that: 1) apparently multiple structural and functional interconnections occur within the INP of the canine SAN region, 2) the structural organization of the INP in the region of the canine SAN may be more complex than is generally recognized from the earlier studies, and 3) more detailed information concerning anatomical routes of the distribution of vagal and sympathetic nerves to the conductive tissue of canine SAN is not yet fully elucidated. The present study was undertaken in an effort to understand better the topography and architecture of the canine INP in the SA-nodal region, including neural connectivities of the ganglia and character of their variability in the whole right atrium. This paper also provides an analysis of the morphology of canine epicardiac ganglia in relation to their distribution, the age of animals and the number of neurons residing within them. As well, we intended to bridge the gap between gross and microscopic anatomy that is important not only to a comprehension of the structural organization of the INP, but also in that way to prepare a morphological analysis on which to base further anatomical and physiological investigations of the canine intrinsic cardiac nervous system. MATERIALS AND METHODS Hearts of 36 mongrel dogs of both sexes (18 male, 18 female) and various age (22 adults, 14 juveniles) were used in this study. Juveniles were mostly suckling pups (2–30 days old) and weighed 0.3–2.0 kg, while dogs considered to be adult weighed 9–20 kg. The animals were anesthetized by a lethal dose of sodium thiopental (100 mg/kg i.p.) after an intraperitoneal injection of 1,000 units of heparin in accordance with local and state guidelines for the care and utilization of laboratory animals. Following thoracotomy, the heart was stopped in diastole with injection of warm (37°C) 20 ml saline with 1 ml of 3M KCl into the inferior vena cava. A metal catheter was inserted transmyocardially into the left ventricle chamber and the heart was perfused by saturated with oxygen and carbon dioxide (95% of O2 and 5% of CO2) 35°C saline. Chemical composition of the saline (pH 7.3) was (in mM): NaCl, 170; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 2.5; glucose, 11.5. To examine the INP stereomicroscopically a special technique CANINE RIGHT ATRIAL NEURAL PLEXUS 273 developed in this study was used (see below). This technique allowed us to investigate the INP on the total (non-sectioned) and pressure-distended canine hearts. Preparations Following washing out of blood from the coronary vessels and cardiac chambers, the canine hearts were prepared as follows. Since the walls of both atria were flabby after perfusion, special balloon-tip catheters inserted through the SVC and left pulmonary veins into the both atrial chambers were utilized to distend the atria via an injection of the saline into the balloon-tips of the catheters. The pressure-bloated hearts were removed from the chest and placed into a chamber with room temperature saline where the remainders of pericardium, pulmonary arteries and fat pads were carefully separated from the heart base by using microsurgical instruments in order to uncover the neural plexuses situated there. The prepared hearts were fixed by immersion for 2 hr at 4°C in 4% paraformaldehyde solution in 0.1 M phosphate buffer. In order to stain intrinsic neural elements from the endocardiac side, the pressure-distended hearts were kept in the same paraformaldehyde solution for up to 4 hr so that the walls of atria and great vessels became stiff and did not collapse when the balloon-tip catheters were removed. Through open orifices of the veins a staining solution could easily enter the atrial and ventricular chambers and stain the nerves laying both beneath and within the endocardium. Following fixation, the hearts were washed at 4°C in saline solution containing hyaluronidase (0.5 mg/100 ml, SERVA) and tetraisopropylphospharamide (iso-OMPA, 0.5 mM, SIGMA), which acts as an inhibitor of pseudocholinesterase. The duration of incubation in saline was dependent on the heart size: small hearts were incubated for shorter periods (up to 5 hr), while large ones were incubated for up to 24 hr. Staining of Intrinsic Nerves and Ganglia A histochemical method for acetylcholinesterase (AChE) was used to stain the INP in whole hearts. The hyaluronidase-treated hearts were incubated for periods 4–24 hr, depending on the organ size, on a rotator at 4°C in the medium described by Karnovsky and Roots (1964). Chemical composition of the incubation medium (pH 5.6) was (in mM): Na acetate buffer, 60; acetyltiocholine iodide (SERVA), 2; Na citrate, 15; CuSO4, 3; K3Fe(CN) 6, 0.5; iso-OMPA, 0.5. Additionally, the incubation medium was supplemented by Triton-X 100 up to 1% and hyaluronidase (0.5 mg/100 ml) to enhance the permeability of the organ to reagents. From time to time during the incubation the staining of the epicardiac neural structures was monitored by a contact microscope LUMAM K-1 (LOMO, Leningrad), special objectives of which contained protruding frontal lenses that flattens an examining surface and keeps it in focus (for details see Barskii et al., 1976). The incubation medium was renewed every 5–12 hr depending on the color of the medium. Turbid incubation medium was immediately refreshed to minimize precipitation of the reaction products in nonspecific sites on the heart surfaces. The deepness of the permeability of the Karnovsky-Roots medium through the epicardium into heart wall was clearly dependent on the duration of incubation as well as on the structure of the epicardium. Fatty, thick and compact Fig. 1. Photomicrographs of cross-sections through a right atrial wall to demonstrate the greater permeability of the Karnovsky-Roots incubation medium into the myocardium (My) and endocardium (En) through the epicardium (Ep) in adult (A) and juvenile (B) dogs during the same incubation time. Dark zones on photographs are myocardium stained for acetylcholinesterase. White arrow in A indicates the cross-section of an AChE-positive epicardiac nerve. Scale bar ⫽ 100 µm for A, B. epicardiac regions of the adult dogs were less permeable to reagents of incubation media than those of the juvenile animals (Fig. 1). Following staining, preparations were fixed in neutral 4% formalin in 0.1 M phosphate buffer. These have been stored in the same formalin without noticeable alterations for 10 years. The Mapping of the Canine INP Stereomicroscopic examinations of the preparations were performed at magnification of 15–20⫻ in distilled water. Most features of the INP were readily visible with a transmitted tungsten light from a fiber optic illuminator introduced into the atrial chambers through the orifices of pulmonary veins and SVC. However, where necessary, additional observations were performed by using a contact microscope LUMAM K-1 to specify the particularities of the INP or improve their delineation. All stereomicroscopically visible intrinsic nerves and ganglia on each preparation were photographed at 4⫻ magnification using an MIKROPLANAR objective (F ⫽ 65 mm; LOMO, Leningrad) and Kodak Academic (black/white) films. The contours of atria, auricles, sinuses of the great vessels, nerves, ganglia and fat pads were outlined on the transparent paper from the photographs or their collages at final 20⫻ enlargement. Though the contours drawn for the INP did not reflect some details of the heart surface, it was considered that the contours reliably rendered the morphology of the INP. The location of the canine SAN was topographically determined according to the descriptions of Palate et al. (1995) and by the density of the fine network of AChE-positive nerve fibers (Fig. 2) which in mammals is considered to be higher within the SAN in 274 PAUZA ET AL. Fig. 2. Lateral view of the epicardiac neural plexus on the canine right atrium that has been visualized histochemically for acetylcholinesterase. Note the more intensively stained SA-nodal region above the terminal sulcus (TS) and the numerous epicardiac ganglia of various size on the sinus of the superior vena cava (SVC). Scale bar ⫽ 1 mm. comparison with its surroundings (Hayashi et al., 1970; Bojsen-Moller and Tranum-Jensen, 1971; Kent et al., 1974; Roberts et al., 1989; Crick et al., 1997). Fine details of the INP, including nerve cells situated solely in the SAN region, were additionally identified by contact, routine brightfield, and electron microscopic examinations as follows. Quantitative Analysis To evaluate the epicardiac neural plexus located around the canine sinus of the superior vena cava quantitatively, intramural ganglia from 22 adult and 14 juvenile dogs were counted at magnifications of 8 or 16⫻ by using a stereoscopic microscope MBS-10 (LOMO, Leningrad) with an eyepiece containing a grid of 225 squares. Each of its squares was 1 mm2. In the present work ganglia occupying more than one square were considered to be large, while ganglia less were considered to be medium-sized. Ganglia classified as small ranged from a few neurons to as large as a quarter of a square (Fig. 3A). Since the density of the ganglia within the INP was variable, the dorsal, lateral, and ventral regions around the SVC were conventionally divided into their upper and lower zones to evaluate quantitatively the regional distribution of those ganglia (Fig. 4A). However, it should be pointed out that in adult dogs accurate counting of ganglia was hindered by fatty tissue that accumulates as fat pads in the epicardium after birth. Because of the fat pads, some epicardiac ganglia could be obscured from view (Fig. 3B, C). Therefore, the number of ganglia in the adult right atrial areas is presumably higher than has been calculated in the present work, although in juvenile animals epicardiac ganglia were not obscured and therefore represented more accurate counts. To assess a heterogeneity of the ganglia, the number of neurons within ganglia was estimated in the 4–6-µm-thick serial sections by counting the stained nucleoli of the nerve cells (Fig. 3D). Moreover, in order to estimate a relationship between the number of neurons residing within ganglion and its appearance, 56 ganglia from three canine hearts stained for AChE were photographed via a contact microscope LUMAM K-1 (LOMO, Leningrad) at a magnification of 10⫻ before their cutting into serial sections (Fig. 3C, D). Photographed ganglia were extirpated from the right atrial wall by the aid of a diamond microscalpel, postfixed en block in 1% osmium tetroxide, rinsed in 0.1 M phosphate buffer, dehydrated in a graded ethanol series, and embedded in a mixture of Epon 812 and Araldite. In capsule embedding molds the ganglia were orientated on the same plane as they were observed by a contact microscope. Serial plane sections of the ganglia were cut using an ultramicrotome TESLA BS490 A (Czechoslova- CANINE RIGHT ATRIAL NEURAL PLEXUS 275 Fig. 3. A: Contact micrograph illustrating the comparative size of the large (black arrow), medium-sized (white arrow) and small (arrowheads) epicardiac ganglia that have been stained for acetylcholinesterase. Squares represent 1 mm2. B: Contact micrograph demonstrating the ganglionated epicardiac neural plexus from the right atrium of adult dog. Note a few small ganglia (white arrowheads) that are situated above fatty epicardium and seen better than those (black arrowheads) that are located more deeply in the epicardiac fat, beneath the superficial ones and are very weakly noticeable. C: Contact micrograph to illustrate an epicardiac ganglion (arrow) which appears medium-sized by a contact microscope. Following cutting into serial sections, it was identified as a large ganglion containing 985 neurons. Arrowheads indicate four small superficial ganglia that were entirely stained for acetylcholinesterase. D: Section through the medium-sized epicardiac ganglion shown in panel C. Some nucleoli are indicated by the arrows. Scale bar ⫽ 0.5 mm for B,C; 65 µm for D. kia) and stained with methylene blue as described above. Sections were examined with a light microscope MEOPTA (Czechoslovakia) at a magnification of 100⫻ or 450⫻, depending on a ganglion size. However, some sections have been inevitably lost during a cutting and/or staining procedures. Therefore, the estimated number of neurons in some ganglia, especially in those that were scattered throughout 30–40 sections, could be underestimated. tures, usually lasting 30–40 min at room temperature, was monitored by a contact microscope. Following visualization of nerve cells and fibers, the methylene blue solution was replaced with the saline and respective tissue samples of about 1 ⫻ 1 mm were excised from SAN region by using microsurgical instruments. Subsequently, the extirpated tissue samples were immersed into a fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cocadylate buffer (pH 7.4) for at least 4 hr at room temperature or for overnight at 4°C. Afterwards, the samples were postfixed for 2 hr with 1% osmium tetraoxide solution in 0.1 M cacodylate buffer (pH 7.4), dehydrated through a graded ethanol series and embedded in a mixture of Epon 812 and Araldite. Semithin sections of 1 µm were stained with methylene blue according to Ridgway (1986) and examined with a Jeneval (Carl Zeiss, Germany) microscope. Photographs were taken with the Electron Microscopic Investigations The ultrastructural study of the INP was performed on 19 samples from three adult mongrel dogs of either sexes. Following heart perfusion with saline in a chamber, the INP was visualized by the supravital staining with methylene blue. The dyeing 0.002% solution of methylene blue (MERCK, Darmstadt) had been prepared in the aforementioned saline (pH 7.3). The staining of the nerve struc- 276 PAUZA ET AL. Fig. 4. A: Drawing of a cranial view of the canine heart to illustrate the approximate limits (dashed lines) of the zones that define the regional distribution of the epicardiac ganglia on the root of the superior vena cava. Dotted line indicates the limits of the heart hilum. B: Drawing of a cranial view of the canine heart indicating the percent of the intracardiac nerves that innervate right atrium and presumably are involved into the innervation of the canine SA-node via an epicardiac neural plexus on the root of the superior vena cava. Dotted lines and shading indicate the limits of the heart hilum. Dotted areas (I–III) show the locations of the supplementary epicardiac ganglia of the right atrium that were not part of the innervation of the canine SA-node. CANINE RIGHT ATRIAL NEURAL PLEXUS same microscope using Kodak TMX 100 (black and white) films. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with an electron microscope Tesla BS 500 (Czechoslovakia) using Svema (Ukraine) electron image films. Statistical Analysis All results shown in the tables are expressed as mean ⫾ standard error. n represents the number of samples from which quantitative data were obtained. The statistical significance of the difference between the means was performed with Student’s unpaired test. Significance was accepted at P ⬍ 0.05. RESULTS Entries of the Mediastinal Nerves Extending Into the Right Atrium As shown previously, in dogs as well as in other mammals, extracardiac nerves enter the heart through a specific site termed by us the hilum of the heart (Pauza et al., 1997a). Although in the present work we did not analyze how locations of the nerves, proceeding from the heart hilum to the SAN region via a neural plexus, were interdependent, within the heart hilum these nerves were detected in the sites indicated in Figure 4B. In all heart preparations, the right atrial nerves were revealed amid the right superior pulmonary veins opposite a massive fat pad, which was termed the pulmonary vein fat pad (Randall et al., 1987). In 63% of the preparations, these nerves were identified between the right superior pulmonary veins and superior vena cava. The latter nerve groups proceeded to the SA-nodal region by the dorsal surface of the right atrium. In 66% of the hearts, the epicardiac nerves extended to the SAN from the ventral interatrial sulcus, where in adult dogs a fat pad was regularly located. In the literature, this fat pad is named either the ventral (Randall et al., 1987) or the medial superior vena caval (Chiou et al., 1997). On the ventral surface of the SVC the second ventral nerve pathway to the canine SAN was present in 88% of the examined preparations. Finally, nerves passing to the canine SAN were also detected on the lateral surface of the SVC in 34% of the preparations. The number of the nerves in each entry as well as a thickness of the nerves varied from animal to animal, but commonly two to four nerves could be resolved in every nerve entry. It should be pointed out that these nerves were more easily visualized in juvenile than in adult dogs. In adults, the origins of the epicardiac nerves were commonly overlaid by fat pads within the heart hilum and in their epicardia. As mentioned, epicardiac fat pads on the right atrium of the adult dogs were consistently located in two sites: at the right superior pulmonary veins (pulmonary fat pad) and in the superior portion of the ventral interatrial sulcus beyond the ascending aorta (ventral fat pad). These pads are the expansions of fat from the heart hilum into the epicardium of the right atrium. Moreover, a third fat pad laying at the origin of the superior free edge of the right auricle was repeatedly located in adult dogs (Fig. 5). Architecture of the INP on the Root of the Superior Vena Cava (RSVC) The right atrial epicardiac nerves formed a complex neural plexus, which ganglia as a wide ganglionated field 277 were continuously distributed on every side of the RSVC (Fig. 5). The upper limits of this ganglionated field were mostly confined by the reflection of the parietal pericardium into the epicardium, while lower ones reached the root of the IVC, dorsally; the terminal sulcus, laterally; and the middle of the anterior wall of the right atrium, ventrally. Supplementary small epicardiac ganglia were occasionally distributed beyond the terminal sulcus on the right atrium, as well as within the boundaries of heart hilum on the medial surface of the SVC (Fig. 4B). In adult dogs, the majority of the nerves extending from the heart hilum to the ganglionated field, ramified therein and their thin branches composed a many layered network that encompassed each epicardiac ganglion and was almost devoid of thicker nerves. Orientations of the slender nerves within the ganglionated field were irregular and the thin nerves, entering and leaving the ganglia, were morphologically similar. However, the lower parts of the ganglionated field included thicker nerves. Since these nerves originated within or beyond the epicardiac ganglionated field, they were called postganglionated nerves.1 The postganglionated nerves extended to their target regions, in which they vanished from sight in two ways: either via penetration into the myocardium or by becoming gradually thinner in the epicardium (Figs. 6, 7A, B, 9, and 12). Epicardiac ganglia of the right atrium within the ganglionated field appeared to be differently involved in the innervation of the canine SAN. In the heart preparations it is evident that postganglionated nerves which originated from ganglia in the upper zones of the ganglionated field extend into the sinus of the SVC, including SA-nodal region, while postganglionated nerves from the lower zones also innervated additional regions of the right and even left atria (Figs. 6, 7A, B). For instance, the epicardiac nerves proceeding from the ganglia adjacent to the pulmonary fat pad extended widely into the right atrium and dorsal surface of the right auricle, while the inferior surface of the right auricle was consistently innervated by the postganglionated nerves from the ventral lower zone of the ganglionated field (Figs. 5, 7A, B). Occasionally, both nerve groups joined supplementary ganglionated fields situated at the right lateral and right anterior coronary sulcus, respectively. In comparison with the ganglionated field on the RSVC, these fields were small and contained no more than 30 small ganglia (Figs. 6C, D, 7A). Because these ganglia were interconnected with the epicardiac nerves accompanying the principal right coronary artery along its course from the arterial part of the heart hilum, their occurrence in the overlapping of the separate neural plexuses may be related to an associative role of those ganglia. An analysis of the serial sections of epicardiac fat pads from the adult dogs revealed that ganglia were scattered throughout whole their depth (Fig. 8). However, the ganglia found beneath or within epicardiac fat pads were very sparse in comparison with the ganglia or single neurons distributed superficially in the epicardium of the canine RSVC (Fig. 13A). 1The term ‘‘postganglionated nerves’’ must be clearly differentiated from the ‘‘axons of postganglionic neurons’’ because epicardiac postganglionated nerves presumably contain both efferent (sympathetic and parasympathetic postganglionic) and afferent (sensory) nerve fibers. 278 PAUZA ET AL. Fig. 5. Drawing of a reconstruction of the distribution of the AChE-positive epicardiac neural ganglionated plexus of the superior vena caval root on the straightened right atrium in an adult dog, illustrating the complexity of this plexus. The boxed areas (A–C) have been photographed by a contact microscope at magnification 10⫻ and three microphotographs of those areas (A,B,C) demonstrate those areas in the reconstruction. Scale bar ⫽ 0.5 mm for A–C. Fig. 6. Photomacrographs illustrating a variability of the epicardiac neural plexus on the root of the superior vena cava in juvenile dogs. A,B: Dorsal view. C,D: Ventral view of the right atria. White arrows indicate the supplementary epicardiac ganglia on the inferior surface of the right auricle. Black arrowheads, the thicker epicardiac nerves that course towards or into innervation targets. White arrowheads, nerves in the heart hilum that supply the ganglionated neural plexus on the RSVC. Scale bar ⫽ 1 mm for A–D. 280 PAUZA ET AL. Fig. 7. A,B: Innervation pattern of the right atrium in juvenile dogs. A: The third supplementary ganglionated field adjacent to the coronary sulcus (CS) on the lateral surface of the right atrium. Black arrowheads indicate the epicardiac nerves that proceed from the heart hilum along principal coronary artery; white arrowheads indicate the thin postganglionated nerves that course from the epicardiac ganglia located in the vicinity of pulmonary fat pad. B: Postganglionated epicardiac nerves (white arrowheads) that proceed from the pulmonary fat pad (PFP) and form a perivascular nerve network on a superficial blood vessel (BL). Black arrowheads indicate the sites at which epicardiac nerves penetrate into myocardium. C–E: Contact micrographs illustrating both the small ganglia and scattered AChE-positive neurons on the canine sinoatrial node (C), as well as the neuron cluster (D) and capsulated ganglion (E) on the RSVC of adult dogs. Scale bar ⫽ 1 mm for A, B; 150 µm for C; 450 µm for D; 630 µm for E. In contrast to adult dogs, in juvenile animals the epicardiac neural plexus on the RSVC (ENP-RSVC) within the ganglionated field contained a number of nerves that were thicker than others and congregated with the thin nerves passing from or through epicardiac ganglia (Figs. 6B–D, 9). Moreover, in the ENP-RSVC of juvenile dogs, nerves that proceeded from the heart hilum to the right atrial regions overleaping in full or in part the epicardiac ganglia were identified (Fig. 9). As a rule, in long-term incubated heart preparations the latter nerves were slightly positive CANINE RIGHT ATRIAL NEURAL PLEXUS 281 Fig. 8. Cross section of a right atrial wall at a pulmonary fat pad to show the distribution of the intrinsic ganglia (black arrowheads) and nerves (white arrowheads) therein. The boxed areas (A,B) are depicted in upper right hand (A) and lower left hand (B) insets at higher magnification. Scale bar ⫽ 200 µm for main micrograph, 55 µm for both insets. for AChE, while a standard incubation in Karnovsky-Root medium was not capable of revealing them at all. Usually, these nerves were located on the lateral or ventral surfaces of the SVC and proceeded towards the right auricle and SAN. The postganglionated nerves coursing into innervation targets from the ganglia either penetrated deeply into the myocardium or encompassed the epicardiac coronary blood vessels (Fig. 7B). Although the postganglionated nerves were invisible in the deeper layers of the myocardium, it might be supposed that they range between the myocardiac muscle bundles because in the pre-endocardiac layer comparatively thick nerves passing on amid the atrial myocytes were readily observable (Fig. 10 A). It seems that in the myocardiac-endocardiac junctional layer those nerves composed a well-developed nerve network, from which thinner nerves ramified to the atrial myocytes, blood vessels or endocardium (Fig. 10A, B, E). In the right atrium this endocardiac nerve network was principally irregular (Fig. 10F), but in the vicinity of the terminal crista its thin nerves were directed to the canine SAN (Fig. 10G). In the right atrial endocardium the fine AChEpositive nerve bundles were arranged in a few layers that formed a sparse meshwork (Fig. 10C) containing two types of nerve endings. Most numerous were the free nerve endings (Fig. 10D) that were very similar to loose ‘‘endnets’’ demonstrated by the aid of a confocal microscopy (Cheng et al., 1997a). AChE-positive nerve endings of the second type were more compact (Fig. 11) and resembled the ‘‘flower-spray’’ endings examined by Cheng et al. (1997a,b). The flower-spray nerve endings were scarcer than loose ‘‘end-nets’’ and distributed predominantly in the endocardium of the root of the SVC. However, a few ‘‘flower-sprays’’ were also detected in the myocardium adjacent to the canine SAN (Fig. 11B). Unlike the epicardiac, the endocardiac nerve meshwork was mostly aganglionated, except a few preparations, in which three to six Fig. 9. Ventral view of the epicardiac neural plexus on the RSVC in a juvenile dog stained histochemically for acetylcholinesterase. White arrows indicate the intrinsic nerves that are less AChE-positive and proceed from the heart hilum into innervation target overleaping in full or in part the epicardiac ganglia; black arrows indicate the nerves in the heart hilum that supply the ganglionated neural plexus on the RSVC; small black arrowheads indicate small ganglia; large black arrowheads indicate medium-sized ganglia; white arrowheads indicate large ganglia. Note the more intensively stained SA-nodal region above the terminal sulcus (TS) and numerous epicardiac ganglia of various size on the RSVC. Scale bar ⫽ 1 mm. Fig. 10. Morphological patterns of the endocardiac nerve network in the canine right atrium that have been stained for acetylcholinesterase. A–D: Serial light micrographs, taken at several depths of focus and demonstrating a ‘‘shifting’’ of AChE-positive nerve network from the myocardium (A) through the myocardiac-endocardiac junction (B) up to the deep (C) and superficial (D) layers of the endocardium. Note the comparatively thick nerves (N) in the myocardium and very few free nerve endings in the endocardium (arrows). E–G: Contact micrographs illustrating a morphology of the AChE-positive neural plexus that has been surveyed from the endocardiac side on the anterior wall of the right atrium (E), in the vicinity of the crista terminalis (F) and sinoatrial node (G). H: Macrograph demonstrating the location of a few endocardiac ganglia (arrows) that were observed through the orifice of the superior vena cava. Scale bar ⫽ 50 µm for A–D; 460 µm for E–G; 1.23 mm for G. 284 PAUZA ET AL. Fig. 11. Examples of nerve endings (arrows) in the canine right atrial endocardium (A) and myocardium (B) that have been stained for acetylcholinesterase. Scale bar ⫽ 50 µm for A, B. TABLE 1. Distribution of small, medium-sized and large epicardiac ganglia in various zones of the RSVC in juvenile and adult dogs (according to Figure 4A) Zones of RSVC Juvenile n Adult n Juvenile ⫹ adult Min–max Small ganglia UD LD UL Sinoatrial UV LV Average in RSVC 54 ⫾ 9 99 ⫾ 9 70 ⫾ 6 102 ⫾ 6 79 ⫾ 11 101 ⫾ 6 470 ⫾ 26 8 8 7 7 9 9 9 49 ⫾ 7 110 ⫾ 24 64 ⫾ 9 107 ⫾ 11 70 ⫾ 6 86 ⫾ 4 401 ⫾ 49 7 7 7 7 8 8 10 52 ⫾ 6 104 ⫾ 12 67 ⫾ 5 105 ⫾ 6 74 ⫾ 6 94 ⫾ 4 434 ⫾ 29 23–110 37–224 37–99 75–141 33–124 63–141 166–606 Medium-sized ganglia UD LD UL Sinoatrial UV LV Average in RSVC 4⫾1 1 ⫾ 0.5 1 ⫾ 0.4 1 ⫾ 0.6 1 ⫾ 0.5 5⫾2 12 ⫾ 3 8 8 7 7 9 9 9 3⫾1 3⫾1 3⫾2 n/f 6⫾3 6⫾3 22 ⫾ 6 7 7 7 7 8 8 10 3⫾1 2⫾1 2⫾1 0.4 ⫾ 0.3 6⫾2 6⫾2 17 ⫾ 4 0–11 0–8 0–11 0–4 0–8 0–24 0–55 Large ganglia UD LD UL Sinoatrial UV LV Average in RSVC 0.6 ⫾ 0.4 n/f 0.1 ⫾ 0.1 n/f n/f 0.1 ⫾ 0.1 0.7 ⫾ 0.3 7 7 7 7 9 9 9 0.1 ⫾ 0.1 n/f n/f n/f 0.3 ⫾ 0.2 2 ⫾ 0.7 4.7 ⫾ 2 7 7 7 7 8 8 10 0.4 ⫾ 0.2 n/f 0.1 ⫾ 0.1 n/f 0.1 ⫾ 0.1 1 ⫾ 0.3 3⫾1 0–3 n/f 0–1 n/f 0–1 0–4 0–19 Ganglia of all types Average in RSVC 486 ⫾ 26 9 428 ⫾ 46 10 456 ⫾ 28 183–621 n/f ⫽ not found, i.e. no ganglia were detected in that zone. small endocardiac ganglia were identified in the vicinity of the SAN (Fig. 10H). Variations in Morphology of the ENP-RSVC A major finding of the present study is that the canine SAN and other right atrial regions were innervated by a rich neural plexus, the nerve entries of which in the heart hilum as well as the ganglionated field on the RSVC had characteristic locations. However, the structural organization of the ENP-RSVC did vary from animal to animal and in relation to animal age. In order to study the variability of the ENP-RSVC, the plexus was analyzed according to regional distribution of the different sizes of epicardiac ganglia (Table 1). It should be, however, kept in mind that numerous small epicardiac ganglia were also revealed: 1) on the inferior surface of the right auricle, 2) beyond and close to the terminal sulcus, and 3) near the coronary sulcus on the lateral surface of the right atrium, as well as a few endocardiac ganglia in front of the sinoatrial node (Figs. 4B; 10H). Since these ganglia, except for the endocar- Fig. 12. Epicardiac neural plexus on the RSVC in an adult dog, stained histochemically for acetylcholinesterase. Black arrows indicate the remainders of the pericardium on the superior vena cava; white arrows indicate the postganglionated nerves that innervate the ventral right atrial wall and inferior surface of the right auricle; small black arrowheads indicate the small ganglia; large black arrowheads indicate medium-sized ganglia; white arrowheads indicate the large ganglia. Scale bar ⫽ 1 mm. 286 PAUZA ET AL. TABLE 2. Correlation coefficients between the numbers of small and other epicardiac ganglia of the RSVC in juvenile and adult dogs Type of ganglia Medium-sized Large Medium-sized ⫹ Large Juvenile n Adult n Juvenile ⫹ adult ⫺0.03 0.39 0.01 7 7 7 ⫺0.44 ⫺0.52 ⫺0.49 10 10 10 ⫺0.42 ⫺0.48 ⫺0.46 diac ones, were inconstant and not clearly related to the nerve supply of the canine SAN, their number was not included into Table 1. In both juvenile and adult dogs, the small ganglia were less abundant in the upper than in the lower parts of the RSVC, including SA-nodal region (Table 1). In comparison with adult animals, the ENP-RSVC of juvenile dogs contained significantly more small ganglia. Since the hearts of juvenile animals were markedly smaller than those of adults, the ENP-RSVC of the latter was obviously more sparse (Fig. 12). Generally, the ENP-RSVC included approximately 434 small epicardiac ganglia, but a few examined canine hearts had up to 600 small ganglia in the ENP-RSVC (Table 1). Both medium-sized and large epicardiac ganglia were more plentiful in adult than in juvenile animals (Table 2). They were mostly located on the ventral side of the RSVC, but medium-sized ganglia were also common on the dorsal side, where six ganglia of such a type were distributed, at the average. SA-nodal zone had no medium-sized and large ganglia in adult dogs, as well as the large ganglia were not determined in the lower dorsal and the upper ventral zones of the RSVC in both juvenile and adult animals (Table 1). On the other hand, the lower ventral zone in adult dogs as well as the upper dorsal zone in juvenile dogs were the most numerous in large epicardiac ganglia. Nevertheless, in both age groups of animals either medium-sized or large epicardiac ganglia within the examined neural plexus were rarer than the small ganglia. Therefore, it is evident that small epicardiac ganglia accumulated the biggest quantity of intracardiac nerve cells on the canine RSVC. Based on our data (Tables 1 and 3), it may be concluded that in general the small, mediumsized, and large epicardiac ganglia of the ENP-RSVC, containing 103, 377, and 945 neurons per ganglion, totaled 44,700, 6,400, and 2,800 nerve cells in those ganglia, respectively. This means that at least 54,000 intracardiac nerve cells were presumably involved into the innervation of the canine right atrium, including the SAN. In addition, our findings did not show a correlation between the numbers of small and other ganglia of the ENP-RSVC in juvenile dogs (Table 2). Nevertheless, a trend towards negative correlation between numbers of small and large ganglia was present in adult animals (Table 2), but this did not reach statistical significance. Morphology of the Epicardiac Ganglia and Nerve Cells Located on the Canine RSVC Although classifying of all identified ganglia as small, medium-sized, or large ones was at first based exclusively on their relative two-dimensional size (see Materials and Methods), the quantitative analysis of the neuronal nucleoli within ganglia substantiated that differences in the number of the nerve cells in small, medium-sized, and large epicardiac ganglia were statistically significant. Moreover, the number of neurons was also significantly different in all types of ganglia from juvenile and adult animals, except the medium-sized ganglia of adult dogs. As seen in Table 3, small and medium-sized ganglia from juvenile dogs contained more nerve cells than the same sized ganglia from adult animals. However, differences between neuron numbers of large ganglia from juvenile and adult animals, as well as the neuron numbers in their epicardiac ganglia in general, were statistically insignificant. Light microscopic examinations of the serial sections revealed the basis of the quantitative differences in numbers of neurons within epicardiac ganglia in the juvenile and adult animals. At the light microscopic level, it was confirmed that neurons within a ganglion may be distributed compactly and/or loosely (Figs. 3D; 13D). Small compact ganglia sometimes consisted of the same number of nerve cells as the large ones in area, but loose in structure. These observations illustrate that the 2D size of the epicardiac ganglion was frequently not related to the number of neurons residing inside the epicardiac ganglia (Fig. 14). Nevertheless, as previously mentioned, the mean number of neurons in all types of ganglia differed in general and, therefore, a partition of the epicardiac ganglia into three types according to their 2D sizes should be applicable in further studies. With the aid of light and electron microscopy two types of epicardiac ganglia were identified in the canine ENPRSVC. Nerve cells and fibers in the ganglia of the first type were surrounded by a capsule of perineurial cells, whereas the ganglia of the second type were devoid of such perineurial capsules and their ganglion cells epicardiac were distributed between a connective tissue of epicardium (Fig. 13B, C). The latter ensembles of nerve cells in the canine epicardium, including single nerve cells situated closely on the SAN, resembled neuron clusters more than typical intrinsic autonomic ganglia. Both types of ganglia were recognizable with the contact microscope in the total heart preparations stained for AChE (Fig. 7C–E). With the contact microscope, it was apparent that the epicardiac ganglia of the juvenile dogs on the RSVC were obviously unequal according to stainability for AChE. Repeatedly, ganglia located side by side were positive or negative for AChE (Fig. 14B). The same was characteristic for the medium-sized and large ganglia, certain parts of which were stained both AChE-positively and AChEnegatively. The nuclei of single and ganglion nerve cells from the ENP-RSVC were usually located eccentrically and their cytoplasm contained typical neuronal organelles, including lipofuscin granules (Figs. 13 and 15). All neurons examined were enfolded by satellite cell sheaths (frequently with a few layers) that had numerous interruptions containing the fine dendrite-like processes derived from neuron somata. The satellite cell sheaths of epicardiac neurons were completely invested by capsules of the collagen fibers that resembled the fine collagen PlenkLendlaw network of the nerve fibers in the endoneurial space of peripheral nerves described by Ushiki and Ide (1990) and Pannese (1994) (Fig. 15A). The fibroblast processes usually separated single neurons or neuron clusters from the neighboring adipocytes and cardiomyocytes. However, the layer of fibroblasts was frequently interrupted or even absent and, thereby, intrinsic neurons were separated from other tissues by satellite cells and 287 CANINE RIGHT ATRIAL NEURAL PLEXUS TABLE 3. Numbers of neurons in small, medium-sized, and large epicardiac ganglia from the RSVC in juvenile and adult dogs Type of ganglia Small Medium-sized Large At the average per all ganglia Juvenile n Adult n Juvenile ⫹ adult Min–max 159 ⫾ 30* 561 ⫾ 173* 602 ⫾ 258* 360 ⫾ 80 13 7 5 25 42 ⫾ 9* 284 ⫾ 61* 1296 ⫾ 478* 353 ⫾ 109 12 14 5 31 103 ⫾ 20* 377 ⫾ 74* 945 ⫾ 218* 356 ⫾ 69 3–345 66–985 113–2860 3–2860 *The differences are statistically significant at the level 0.05. sheaths of fine collagen fibers. The immediate surroundings of the single neurons from the SA-nodal epicardium were not occupied by nerve fibers and our observations did not reveal any axosomatic synapses on them. No synapses were identified on fine dendrite-like processes that were a usual feature of the single nerve cells. In contrast, neurons in epicardiac ganglia were enmeshed in nerve bundles and fibers that were mainly unmyelinated. The axon terminals within unmyelinated nerve fibers had abundant varicosities containing numerous round, small, clear, and a few dense-cored vesicles (Fig. 15B). Although in the ganglion neuropil most nerve terminals were wrapped into Schwann cells, the adherent axon varicosities within nerve fibers as well as presumptive axodendritic synapses adjacent to the spine-like processes of dendrites were frequent as well (Fig. 15B). DISCUSSION This is the first anatomical demonstration of the routes of the intracardiac nerves proceeding from the heart hilum into the right atrium on whole canine hearts. In this study the richly developed epicardiac neural plexus with a number of ganglia of different in sizes around the RSVC was demonstrated to be more extensive and numerous than was considered previously. According to the earlier neuroanatomical investigation of Yuan et al. (1994a), the intrinsic ganglia nearest to the SAN were located on: 1) ventral, lateral and dorsal surfaces of the right atrium, 2) mid-dorsal surface of the two atria, and 3) dorsal caudal surface of the right and left atria adjacent to the origin of the canine IVC. In this work a blunt dissection and 1% solution of methylene blue were used to stain the intrinsic nerves and ganglia in small extirpated and fixed in 10% formalin blocks. This particular method did not allow the authors to determine which intrinsic ganglia were interconnected with the canine SA-nodal region, as well as how those ganglia were distributed therein. Our findings display that all ganglia located around the RSVC are presumably associated with the canine SAN via epicardiac plexus. Furthermore, in the study of Yuan and associates (1994a) 154 intrinsic ganglia of different size per dog were found in both atria, on average. The majority of those ganglia were distributed in the inferior atrial plexus at the origin of the IVC on the dorsal inferior plexus of the two atria and in the right plexus on the ventral, lateral and dorsal surfaces of the right atrium. In accordance with our results, the ENP-RSVC was more ganglionated and contained 456 ⫾ 28 epicardiac ganglia per animal at the average (Table 1). Some of the ganglia were small and composed of 103 neurons at the average, while others were larger and complicated in structure (Table 3, Fig. 14). For example, on the ventral surface of the RSVC we frequently detected ganglia that contained more than 2,000 nerve cells per ganglion. This study assessed the variety of epicardiac ganglia distributed in the ENP-RSVC. As seen from Table 3, small and medium-sized ganglia of adult dogs contain fewer of nerve cells than the same ganglia in juvenile animals. In contrast to small and medium-sized ganglia, large ganglia of adult dogs include two times more neurons per ganglion than large ganglia in juveniles. However, small, mediumsized, and large ganglia taken together contain on average almost equal numbers of neurons in both juvenile and adult animals. Based on this, it can be hypothesized that remodeling of epicardiac ganglia in the ENP-RSVC takes place for a period postnatally. Our data demonstrate that majority of intracardiac neurons of juvenile dogs are assembled into small ganglia, while following birth some small ganglia may fuse and form large or medium-sized ganglia. Since small and medium-sized ganglia of adult animals contain fewer nerve cells than ones of juveniles (Table 3), probably a decomposition of primary small ganglia occurs in adult dogs after birth too. As it was previously established, the postnatal addition of satellite cells to postganglionic parasympathetic neurons and neuronal enlargement occur during the first 8 weeks after birth (Pomeroy et al., 1996). Therefore, both the postnatal addition of satellite cells and fusion of primary small ganglia may explain the equal sizes and different numbers of neurons in small ganglia of juvenile and adult animals. In addition, the quantitative results of distribution of epicardiac ganglia in ENP-RSVC confirm the remodeling of ganglia following birth (Table 1). The largest number of small ganglia in juveniles as well as the larger number of large and medium-sized ganglia in adults together with the fewer total number of all ganglia per ENP-RSVC in adult animals support a hypothesis of possible union or fusion of some epicardiac ganglia in adult dogs. Electron microscopic investigation revealed that majority of large ganglia in the canine ENP-RSVC have a sheath of perineurial cells that surround ganglion cells at periphery. In contrast, smaller ganglia and single neurons distributed on the RSVC are deprived of these sheaths and only capsules of the satellite cells envelop neuron somata. Apparently, in epicardiac connective tissue a role of perineurial cells becomes insignificant and, thereby, intrinsic neurons organized as their clusters on the canine RSVC differ from extrinsic autonomic ganglia. Our ultrastructural examination revealed only axodendritic synapses in epicardiac ganglia of the RSVC. These findings confirm the recordings of Yuan et al. (1994a) that canine intracardiac neurons form predominantly axodendritic synapses in the ganglion neuropil. Interestingly, a similar predominance of axodendritic synapses and paucity of axosomatic syn- 288 PAUZA ET AL. Figure 13. CANINE RIGHT ATRIAL NEURAL PLEXUS apses has been identified in humans (Armour et al., 1997), whereas intrinsic cardiac ganglia of cats, rats, guinea pigs, and rabbits are reported to have axosomatic synapses as well (Ellison and Hibbs, 1976). With respect to the neuroanatomical methods of this study, it is not guaranteed that all neural components of the INP within thick and fatty canine epicardium were exposed and examined in the obtained preparations. Since previous studies usually dealt with a sectioned material, a question remains whether the incubation medium of Karnovsky-Roots is capable of penetrating fully into the deeper layers of the heart wall to stain the neural elements located therein. According Baluk and Gabella (1989), hyaluronidase and Triton-X 100, as well as a prolonged duration and a lowered temperature of incubation, improve very much the permeability of the tissues to reagents from incubation medium and develop entirely the intrinsic neural plexus in wholemount preparation of the guinea pig trachea. However, connective tissue and fat as the barriers for incubation medium had a negative influence on AChE staining. Therefore, better results were obtained from the juvenile and newborn guinea pigs. This was confirmed in our study too, because intrinsic ganglia and nerves were stained weakly and pale in the fatty canine epicardia. Nevertheless, the INP of juvenile and gaunt adult dogs stained for AChE by our protocol was perfectly observable with the aid of dissecting or contact microscopes. On the other hand, the Karnovsky-Roots histochemical method was mainly developed to identify the cholinergic nerves. In spite of this, it has been established that non-cholinergic nerves and ganglia, including the adrenergic and sensory, contain detectable amounts of AChE as well (Giacobini, 1967). Therefore, it is likely that in this study all intrinsic nerve fibers were visualized following a prolonged period of incubation in KarnovskyRoots solution. Concerning our technique to map the intracardiac nerves and ganglia, it should be noted that a lack of optimal technique for this purpose has hindered neuromorphological investigations of the heart for a long time and to date very few anatomical methods have been applied to study the INP in mammalian hearts. The method of micromacrodissection developed by Worobiew (1925) has produced very interesting and, at the same time, limited results due to its applicability mainly for large hearts of such species as cattle. Although the micro-macrodissection was not enough sensitive to examine the canine hearts, Worobiew (1958) has, nevertheless, successfully determined the topography of the INP in human heart. The later investigators who utilized either technique of the wholemount preparation or traditional histological meth- Fig. 13. Micrographs of semithin sections of epicardiac ganglia (A–D) and solitary nerve cells (E) from the canine RSVC. A: Cross section of a right atrial wall at the SA-nodal artery (ar) to illustrate the locations of the ganglia (black arrowheads) and nerves (white arrowheads) in an epicardium (Ep). B,C: Clustered (B) and capsulated (C) types of epicardiac ganglia. Note the capsule of the perineurial cells (arrowheads) sheathing the ganglion cells and nerve fibers in panel C, and solely situated neuron (arrow) in panel B. D: Plane section of an epicardiac ganglion from the RSVC of juvenile dog. Note the scarceness of the neurons (arrowheads) disposed therein, in comparison with the neuron distribution on Figure 3D. E: Single epicardiac neuron situated between cardiomyocytes (My) and adipocytes (Ad) at the canine sinoatrial node. Scale bar ⫽ 100 µm for A, 44 µm for B; 38 µm for C; 84 µm for D; 18 µm for E. 289 ods, especially in combination with the methylene blue or cholinesterase staining, have ascertained additional data on the nerve networks in the mammalian epicardium, myocardium, endocardium, conduction system, and coronary vessels (Davies et al., 1952; King and Coakley, 1958; Smith, 1969; Bojsen-Moller and Tranum-Jensen, 1971; Roberts et al., 1989; Roberts, 1991; Yuan et al., 1994a). However, using these methods, mammalian hearts were, as a rule, cut or sectioned into separate pieces or serial sections in order to stain intrinsic nerves and ganglia located therein. Following staining, approximate locations of the ganglia in the heart were determined with respect to blood vessels or to reconstruct a whole INP from the separate pieces and sections. As a result, those reconstructions were very schematic drawings, in which a continuous view of the total INP was absent and neural connections between separate intrinsic ganglia and target regions were only speculatively demonstrated. The technique of the present study seemingly over came short comings of earlier techniques and introduced a possibility both to map simply and to evaluate three-dimensionally the total INP in non-sectioned hearts. Thus, we conclude that the histochemical method for AChE described above, gives beneficial results and is promising for further neuroanatomical investigations in the whole hearts. Findings of the present study are only in part consistent with the data that have been previously obtained in the canine heart examinations. Randall et al. (1986b, 1987) reported that the majority of parasympathetic pathways from right and left vagi to the SAN region in canine heart are distributed in the fat pad overlying the right pulmonary vein-left atrial junction. According to these authors, 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 also contain autonomic ganglia that influence the activity of the canine SAN. Our results demonstrate that in dogs numerous intrinsic ganglia, from or through which nerves course forward into the SAN region via neural plexus, are distributed more widely on the RSVC in epicardiac fat pads or outside them. Usually, the epicardiac ganglionated field on the RSVC occupies continuously the region from the right pulmonary veins on the dorsal surface of right up to the interatrial groove on the ventral surface of the right atrium. Intrinsic ganglia of this plexus, including numerous isolated nerve cells that overlay continuously the whole sinus of the SVC, extend to the terminal sulcus and mid-part of the ventral wall of right atrium. Anatomical data obtained during physiological experiments of Randall et al. (1985, 1986b, 1987) did not reveal many topographical and structural characteristics of the complex intrinsic neural plexus on the canine RSVC. In the work of Randall and associates cited above, the location of the nerve pathways to the SAN is related to epicardiac fat pads. The same observation concerning the distribution of the intracardiac neural elements can be noticed in the study of Billman and his coworkers (1989) where it was reported that although the triangular shaped pulmonary vein fat pad over-lying the canine atrium was absent in the monkey heart, a careful exploration of the epicardium over and around the pulmonary veins revealed many well defined but smaller fat pads in monkeys. Yuan et al. (1994a) also concluded that most intracardiac neurons were identified in ganglia embedded in the fat on the surface of the canine heart or in the interatrial septum, 290 PAUZA ET AL. Fig. 14. Contact micrographs of epicardiac ganglia from the ENPRSVC of juvenile (A–C) and adult (D–F) dogs that have been stained histochemically for AChE and subsequently examined in serial sections. Numbers indicate the number of nerve cells counted within them. Note the epicardiac ganglia (arrowheads) in panel B that were more AChE-positive than neighboring ones, as well as the epicardiac ganglia in panels C and D that were AChE-positive in part. Scale bar ⫽ 500 µm for A–E. Fig. 15. Electron micrographs of solitary (A) and ganglion (B) nerve cells that have been sampled in the epicardiac neural plexus at the canine sinoatrial node. A: Micrograph showing features of the solitary epicardiac neuron. B: Micrograph illustrating spines (S) of dendrite (D) of the ganglion nerve cell and unmyelinated nerve fibers that are situated in the vicinity of the dendrite and contain axon varicosities (V1–V5) with round, small, and clear vesicles. Arrow indicates the axodendritic synapse. Scale bar ⫽ 1.45 µm for A; 1 µm for B. 292 PAUZA ET AL. and very few ganglia were found located between atrial or ventricular muscle fascicles. According to our data, the pulmonary vein fat pad in canine hearts varies considerably in the shape and the size, frequently extends to the terminal sulcus or joins a fat pad overlaying on the anterior surface of the right atrium. In obese dogs the pulmonary vein fat pad is considerably increased in size, non-triangular in shape, and its location, therefore, should not be considered as a key site whence a majority of nerves course to the SAN. Moreover, in juvenile dogs it was noticed that the epicardiac fat pads occur very rarely on the heart surfaces, although the epicardiac neural plexus of the right atrium is well developed at that time. We suppose that accumulation of the fat pads in the epicardium alters from dog to dog, depends on the obesity of animal, and is only in part related to the location of epicardiac nerves and ganglia. With respect to the canine heart, we suggest that the fine atrial folds are more important than epicardiac fat pads. Unfortunately, to date these folds, with the exception a few (Worobiew, 1928), have not been described and named. Randall and associates (1983), as well as Randall and Ardell (1985) reported that precise course of the sympathetic pathways to the SAN in canine hearts remains to be demonstrated, but it is assumed that they follow a course along the dorsal wall of the common pulmonary artery into the plexus supplying the main left coronary artery and are distributed to the myocardium, presumably along coronary arterial pathway, from that point. Moreover, the works of Martins and Zipes (1980a,b) and Takahashi and Zipes (1983) suggest a differential distribution of sympathetics via epicardiac and parasympathetics via endocardiac routes to the ventricular myocardium, with vagal passage across atrioventricular groove, as determined physiologically. Results of the present study do not support this conclusion, because in canine endocardium we have identified only a fine nerve network that is presumably sensory in function, because many of these endocardiac AChE-positive nerve fibers had endings that resembled sensory receptors described by Cheng et al. (1997a,b) as vagal afferents in the atrial endocardium and wall of the aortic arch in rats. Similar structures as receptors in the canine atrial endocardium were stained with methylene blue and demonstrated by Holmes (1957). Concerning the routes of sympathetic nerves to SAN region, the present study suggests that in dogs these nerves proceed via the epicardiac neural plexus which presumably is mixed and consists of parasympathetic, sympathetic and sensory nerve fibers. Additionally, in canine heart preparations produced in this study it is seen that some epicardiac nerves are evidently less AChEpositive and spread from the heart hilum to the SAN region without ramifications at the intrinsic ganglia. Therefore, further immunohistochemical studies of the precise samples from the ENP-RSVC will allow us to confirm the possible functional nature of these nerves. To date it remains undetermined what the anatomical basis could be for the chronotropic responses of the SAN to stimulation of either vagi or ansae subclavia following dissection and painting with phenol the dorsal surface of the atria between the right and left pulmonary veins (Randall and Ardell, 1986). Anatomical findings of this study indicate that there are no epicardiac or endocardiac nerves interconnecting the dorsal surface of the left atrium and SAN region. However, in our preparations it was evident that ENP occurs between the canine SAN and 1) superior vena cava, 2) azygos vein, and 3) right pulmonary veins, i.e. between the locations that were oblated in the surgical experiments of Randall and Ardell (1985). Comparing the INP of the dogs to other mammals, it should be pointed out that there is scanty information on the architecture of the INP in the right atrium. BojsenMoller and Tranum-Jensen (1971) reported that in juvenile pigs extracardiac nerves enter the heart superiorly at the junction of the right and left atrium, forming a dorsal plexus with numerous ganglia therein. According to these authors, intrinsic nerves proceed towards the porcine SAN by two routes: 1) many of the nerves pass on the left of the SVC at it entrance in the atrium and, en route, run into a sinoatrial ganglion whence they continue to the SAN in the terminal sulcus. The sinoatrial ganglion of the pigs is situated in the antero-medial wall of the SVC immediately before it branches into the atrium; 2) other nerves cross the SVC in its anterior wall above the SAN to curve down the right lateral wall of the atrium, including the SAN. In canine heart we have described similar topography of the SA-nodal nerves, but the distribution of the canine right atrial ganglia is rather different in comparison with the porcine ones. In the human heart, intrinsic ganglia that were located close to the SAN region and could be thereby involved into a neural regulation of the SAN have been identified on: 1) the posterior superior surface of the right atrium adjacent to the junction of the superior vena cava and right atrium, and 2) the posterior surface of the right atrium adjacent to the interatrial groove (Armour et al., 1997). Nevertheless, to date no neural connections between these atrial ganglionated plexuses and human SAN region have been anatomically demonstrated. Using a wholemount preparation technique, Roberts and associates (1989) revealed in the rabbit intercaval region: 1) an inferior ganglionic complex located inferior to the SAN at varying distances from the terminal crista; 2) two or three moderately large nerves transversing the sinoatrial node parallel to the terminal crista; and finally 3) nerves entering the SAN region from the atrial septum, the SVC, IVC, and joining the inferior ganglionic complex. In rats ganglion cells innervating SAN region were found in a neural plexus of the heart hilum on the base of left atrium (Pauza et al., 1997b). In contrast, in guinea pigs the topography of ganglia supplying intrinsic nerves to the SAN is rather distinct from those in the rat because in guinea pigs these ganglia are situated in both the neural plexus of the heart hilum on the left atrium and the right atrium on the surfaces of the RSVC very close to SAN (Pauza et al., 1997b). Thus, we conclude that in mammals the intracardiac ganglion cells, innervating the SAN region, may be located moderately far from their target tissues. On the other hand, some intrinsic ganglia, distributed comparatively close to the SAN, may be not connected with the region where they are located, as has been shown in this study. In view of this, a second conclusion could be derived concerning a research methodology in heart neuroanatomy. It seems that a traditional histological technique, even with the sequential reconstructions from the serial sections, is less encompassing in revealing aspects of heart neuroanatomy than methods which utilize wholemount or total heart preparations. Although the occurrence of the intrinsic cardiac afferent and efferent (parasympathetic and sympathetic) neurons CANINE RIGHT ATRIAL NEURAL PLEXUS was recently ascertained in the canine heart (Murphy et al., 1994; Ter Horst et al., 1996), the functional and cytochemical properties of the neurons located in the vicinity of the canine SAN remain unknown, because to date no special studies devoted to studies of those neurons have been carried out. However, morphological features of the intracardiac neurons from the canine SAN region demonstrate that nerve cells with different functions are distributed in the ENP-RSVC (Xi et al., 1991). The occurrence of sensory, sympathetic and parasympathetic nerve cells within the SAN region supports the possibility that neurons of the ENP-RSVC may be involved in short local feedback loops necessary for a modulation of cardiac excitability and, therefore, in canine model either the selective SA-nodal parasympathectomy or its total surgical denervation by the extirpation of a few fat pads from the right atrium at the pulmonary veins should be more complicated than it was earlier thought. In summary, the findings of the present study demonstrate that canine right atrium is supplied by abundant nerves from the rich epicardiac plexus on the RSVC. This plexus contains on average of 456 ganglia of different sizes, in which on average of 54,000 nerve cells reside. Extracardiac nerves supplying this plexus enter the heart hilum via five sites and proceed into the right atrium both by the dorsal, ventral, and lateral surfaces of the RSVC. Taken together, our results indicate that intrinsic neural plexus innervating canine SAN is more complex and widely distributed than was considered previously. Therefore, the surgical ablation of the neural components during electrophysiological experiments in order to denervate the canine SAN should include a wide region around the root of the SVC. ACKNOWLEDGMENTS The technical assistance of Mrs. Gertruda Skripkiene throughout the study is gratefully acknowledged. We thank Dr. Gintautas Saulis for helpful comments on earlier draft of the manuscript. We are also grateful to Prof. David A. Hopkins from Dalhousie University in Halifax for his careful reading of the manuscript, constructive criticisms and friendly editorial assistance. Grant in partial support of this work was from Lithuanian State Science and Studies Foundation (No. 364). LITERATURE CITED Aidonidis I, Metz J, Gerstheimer F, Kubler W, Brachmann J. 1993. 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