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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: daipau@kma.lt
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.
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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.
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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-
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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,
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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).
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