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253
Key anatomic data for the use of rat heart in electrophysiological
studies of the intracardiac nervous system
Darius Batulevičius1, 2, Neringa Paužienė2, Dainius H. Pauža2
1
Institute for Biomedical Research
2
Department of Human Anatomy, Kaunas University of Medicine, Lithuania
Key words: heart, cardiac innervation, anatomy, autonomic nervous system, acetylcholinesterase.
Summary. The aim of the present study was to elucidate both the topography and architecture
of the rat intrinsic cardiac nerve plexus, as this species becomes a frequent mammalian model
for electrophysiological investigations of the intracardiac nervous system. Fifteen adult rats
were examined employing histochemistry for acetylcholinesterase to visualize the intracardiac
nerve plexus in hearts. Extracardiac nerves entering the rat heart were found amid aorta and
pulmonary trunk as well as along both right and left cranial veins. The nerves from the arterial
part of the heart hilum extended directly to the ventricles but the nerves from the venous part of
the hilum interconnected among themselves forming a nerve plexus of the cardiac hilum on the
heart base. Within the rat epicardium, intrinsic nerves clustered into six routes by which they
selectively projected to different rat heart regions. Ventral wall of the ventricles was supplied by
three neural subplexuses, dorsal ventricular wall – by one subplexus; each atrium received nerves
from two distinct subplexuses. In conclusion, this morphological study demonstrates that rat
intracardiac nerve plexus compounds to anatomical scheme of the same plexus in human, therefore
rat is a usable model for electrophysiological experiments of the intracardiac nervous system.
Introduction
Intrinsic cardiac nervous system (ICNS) plays a
paramount role in regulating the heart rate, conduction, myocardial contractile force and coronary blood
flow (1). The structural organization of the ICNS has
been described for many experimental animals and
human (2–9). Recent studies of the architecture of
ICNP in both human and dog suggest that whole ICNP
may be subdivided into seven ganglionated subplexuses
(7, 8). Based on the architecture of the neural subplexuses, it was proposed that precise denervation of
the discrete heart regions and/or total denervation of
the heart is possible via the destruction of the selected
neural subplexuses (7, 8).
Although the rat has been widely used as a mammalian model to investigate the behavior of the ICNS
(10–13), the morphology of the rat ICNP is not well
understood. So far, very few studies have been aimed
to elucidate precisely the three-dimensional architecture of the rat ICNP employing distinct kinds of whole
mount preparations (3, 4, 14, 15). Nevertheless, there
are clear contradictions regarding 1) neural inputs to
the rat heart, and 2) concerning the architecture of the
neural routes inside the rat heart.
The aims of the present study were: 1) to investigate the morphology of the rat intracardiac nerve plexus
and 2) to determine whether the intracardiac nerve
plexus of this species compounds to anatomical scheme
of the same plexuses in dogs and humans reported previously (7, 8).
Material and methods
The study was performed on 15 adult (2–4 months
of age, 200–250 g in weight) Wistar rats of both sexes.
The rats were anesthetized by a lethal dose of sodium
thiopental (100 mg/kg i. p.) after an intraperitoneal
injection of 1000 units of heparin in accordance with
local and state guidelines for the care and use of laboratory animals (State permission number 0018).
Total heart preparations
The rats were perfused intracardially as was described previously by D. H. Pauza et al. (7, 8, 14).
Following perfusion, the atrial walls were distended
in situ by the aid of transmyocardial injection of warm
20% water solution of gelatin into atrial and ventricular
chambers. When the injected gelatin became stiff in
the cardiac chambers and sinuses of the vessels, the
heart was removed from the chest. Subsequently, the
Correspondence to D. H. Pauža, Laboratory for Neuromorphology, Department of Human Anatomy,
Kaunas University of Medicine, A. Mickevičiaus 9, 44307 Kaunas, Lithuania. E-mail: daipau@kmu.lt
254
Darius Batulevičius, Neringa Paužienė, Dainius H. Pauža
remnants of pericardium, pulmonary arteries and fat
pads were separated from the heart base using microsurgical instruments to reveal the neural plexuses
situated there. In this way prepared hearts were fixed
for 15 minutes in 4% paraformaldehyde solution in
0.1 M phosphate buffer.
Staining of the intracardiac nerve plexus
Histochemistry for acetylcholinesterase was used
to stain the intracardiac nerve plexus in total hearts.
The hearts were incubated for 1–3 hours at 4oC in the
medium described by M. J. Karnovsky and L. A. Roots
(16). Since the fat pads of the heart base were almost
non-permeable for the incubation media, an additional
precise dissection of those fat pads was usually
performed to expose the neural structures located there.
Following staining, the preparations were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer. The
preparations have been stored in the same paraformaldehyde without noticeable alterations for years.
Microscopy of the intracardiac nerve plexus
Total heart preparations were placed in distilled
water and examined in a transient light from a fiber
optic illuminator using a stereomicroscope (MBS-10,
Russia) at magnification 16-56X. Stereomicroscopically visible intrinsic nerves and ganglia were photographed using the digital camera (C-2500L, Olympus).
Results
Intracardiac inputs of extrinsic nerves
Extrinsic nerves entered the rat heart both in arterial and venous part of the heart hilum. In the arterial
part of the heart hilum (i. e. around the ascending aorta
and pulmonary trunk), the accessing nerves were distributed: 1) in the fat between aorta and pulmonary
trunk (89% of hearts examined), 2) on the dorsal (20%)
and 3) ventral (29%) sides of the pulmonary trunk and
4) on the lateral sides of aorta (7%) (Fig. 3).
In the venous part of the heart hilum (i. e. around
the cranial, caudal and pulmonary veins), the accessing
nerves were regularly found at the following locations:
1) on the medial side of the right cranial vein (RCV)
(100%), 2) on the medial side of the left cranial vein
(LCV) (100%), 3) on the dorsal side of the LCV (86%),
4) in the fat pad situated at the bifurcation of the
pulmonary trunk (75%), 5) on the dorsal wall of the
middle pulmonary sinus (MPS) (36%) (Figs. 1–3).
Occasionally, the accessing nerves were identified on
the dorsal (7%) wall of the left pulmonary sinus (LPS).
Morphology of the intrinsic cardiac nerve plexus
Extrinsic nerves accessing the heart from the arterial part of the cardiac hilum extended directly into the
epicardium of the ventricles (Fig. 3). In contrast, the
nerves entering the heart through the venous part of
the hilum extended to both neuronal clusters located
therein, forming in this way a particular nerve plexus
of the cardiac hilum (Fig. 1).
Nerve plexus of the cardiac hilum (NPCH)
The nerves accessing the heart via the medial side
of sinus of the RCV prolonged up to the right neuronal
cluster, whereas the nerves accessing the heart through
the sinuses of the LCV and pulmonary veins as well as
at the bifurcation of the pulmonary trunk penetrated
into the left neuronal cluster (Fig. 1). The abundant
commissural nerves interconnecting both neuronal clusters were identified in all hearts examined (Fig. 1).
The nerves extending forward from the neuronal clusters spread epicardially to the atria and ventricles and
endocardially into the interatrial septum.
The nerves originating from the right neuronal cluster coursed in the following directions: 1) to the ventral interatrial groove (100% of the preparations examined), 2) to the dorsal and lateral sides of the right
atrium and RCV sinus (95%), and 3) between the right
pulmonary sinus (RPS) and caudal vein (100%) (Fig.
1). The nerves deriving from the left neuronal cluster
extended by two paths: 1) by the ventral (87%) and 2)
dorsal (100%) sides of the left atrium (Fig. 1). On the
dorsal left atrial route, the nerves passed either between the RPS and MPS (100% of the preparations),
between the MPS and LPS (80%) or between the LPS
and LCV (93%) (Fig. 1).
Epicardiac neural subplexuses
Within the rat epicardium, intrinsic nerves are clustered into evident routes, by which they selectively
project to different atrial and/or ventricular regions.
Therefore, the morphology of the epicardial extensions
of the whole intracardiac neural plexus in the rat was
analyzed and described in accordance with these routes.
Due to comparison and coherency with earlier anatomical studies of the intrinsic cardiac neural plexuses
in human and dog (7, 8), the clustered into routes epicardial extensions of the intrinsic nerves were termed
as epicardiac neural subplexuses.
Right (RCS) and left (LCS) coronary subplexuses
The nerves of these two subplexuses distributed
exceptionally in the ventricles. The nerves of the RCS
entered the epicardium on the ventral and lateral walls
of both the pulmonary trunk and ascending aorta (Fig.
3). Proceeding along the right coronary groove, the
latter nerves branched out in the lateral and ventral
sides of the right ventricle (Fig. 3). The nerves supplyMEDICINA (2004) 40 tomas, Nr. 3
Animal model for electrophysiology of the intracardiac nervous system
255
Fig. 1. Schematic drawing of the rat heart base stained for acetylcholinesterase demonstrating
the structure of the nerve plexus of the cardiac hilum
Right neuronal cluster is located in the interatrial groove while the left neuronal cluster is located on the left atrium.
Note the accesses of the extracardiac nerves proceeding to both neuronal clusters located on the medial sides of both
right and left cranial veins as well as at the bifurcation of pulmonary trunk. Interrupted line delineates the area of the
heart hilum.
ing the LCS penetrated into epicardium between the
ascending aorta and pulmonary trunk, extended by the
left coronary groove and expanded on the lateral and
ventral sides of the left ventricle following the branches
of the left coronary artery (Fig. 3). As a rule, the LCS
contained more epicardial nerves than the RCS. It is
noteworthy that the LCS was well developed in all
hearts examined whereas the RCS was identified only
in 27% of the investigated hearts.
Ventral right atrial subplexus (VRAS)
The nerves of this subplexus derived from the venMEDICINA (2004) 40 tomas, Nr. 3
tral side of the right neuronal cluster of the nerve plexus
of the cardiac hilum and entered the right atrial epicardium in the superior ventral interatrial groove (Fig.
3). Fine branches of the ventral right atrial subplexus
reached the right auricle, left side of aorta root, and
even the superior ventral and lateral walls of the right
ventricle (Fig. 3).
Ventral left atrial subplexus (VLAS)
The sparse nerves of this subplexus were identified
in 87% of the hearts examined. This subplexus was
supplied by the nerves originated from the ventral side
256
Darius Batulevičius, Neringa Paužienė, Dainius H. Pauža
Fig. 2. Macrophotograph of the dorsal wall of the
rat heart stained for acetylcholinesterase demonstrating the structure of left dorsal neural subplexus
Arrow points to the nerves accessing the heart via the dorsal
side of the left cranial vein (LCV). Arrowheads indicate
the nerves extending to the dorsal wall of the ventricles
through the coronary groove. Note the prominent neural
network on the sinus of the LCV. LA, left atrium; LAu,
left auricle; LPS, left pulmonary sinus; LV, left ventricle;
MCV, middle cardiac vein; PVLV, posterior vein of the
left ventricle.
a
of the left neuronal cluster of the NPCH. The VLAsubplexal nerves extended mainly to the ventral side
of the left atrium (Fig. 3). Some thin nerves of the
VLAS overlapped with the nerves from the VRAS
during their course to ventral coronary groove.
Dorsal right atrial subplexus (DRAS)
The nerves of the DRAS originated from the dorsal and lateral sides of the right neuronal cluster of the
NPCH (Fig. 3). Between the sinuses of the RCV and
RPS the DRA-subplexal nerves coursed to the left side
of the caudal vein and branched out in the dorsal and
lateral sides of the right atrium sending thin branches
to the right auricle and sinus node (Fig. 3). In addition, the sparse nerves extending from the lateral side
of the right neuronal cluster to the region of the rat
sinus node along the dorsal wall of the RCV were identified in 43% of the hearts examined.
Left dorsal subplexus (LDS)
Compared with previous subplexuses, the LDS was
structurally the most complicated. The exceptional feature of this subplexus was the large epicardial ganglia
located persistently along or between the nerves on the
dorsal side of the left atrium (Fig. 3). The LD-subplexal
nerves were derived from: 1) the dorsal side of the right
neuronal cluster, 2) the dorsal side of the left neuronal
cluster, 3) the dorsal side of the LCV sinus, and 4) the
dorsal side of pulmonary sinuses (Fig. 2 and 3). From
the epicardial ganglia scattered on the dorsal side of
left atrium the nerves extended towards the dorsal coronary groove (Fig. 3). On their route to the sinus of the
b
Fig. 3. Scheme of six neural subplexuses in the rat heart viewed ventrally (a) and dorsally (b)
Arrows indicate the location, course and target regions of the neural subplexuses. Interrupted line delineates the limit of
the heart hilum. Dotted areas mark the locations of intrinsic cardiac neurons. Ao, aorta; CV, caudal vein; DRA, dorsal
right atrial subplexus; LC, left coronary subplexus; LD, left dorsal subplexus; LPS, left pulmonary sinus; MPS, middle
pulmonary sinus; PT, pulmonary trunk; RC, right coronary subplexus; RPS, right pulmonary sinus; VLA, ventral left
atrial subplexus; VRA, ventral right atrial subplexus.
MEDICINA (2004) 40 tomas, Nr. 3
Animal model for electrophysiology of the intracardiac nervous system
LCV, the nerves interconnected among themselves,
forming a prominent neural network accompanied
sometimes by small ganglia formed at intersections of
these nerves (Fig. 2). From the neural network on the
dorsal wall of left atrium and the LCV sinus the nerves
extended in the following ways: 1) the vast majority of
the nerves crossed the dorsal coronary groove and
spread on the dorsal and lateral surfaces of both ventricles, 2) a small group of the nerves branched out on
the lateral sides of the left atrium and left auricle, 3) a
few thin nerves coursed to the dorsal side of the right
atrium bypassing from underneath the root of the caudal vein, 4) one or several thick nerves coursed to the
dorsal interatrial groove where they penetrated first
into myocardium and then to endocardium of the interatrial septum (Fig. 2 and 3).
Discussion
Intracardiac access of extrinsic nerves
Our neurotopographical examinations demonstrate
that the extrinsic nerves access the rat heart both in the
arterial and venous part of the heart hilum. From the
arterial part of the heart hilum the nerves proceed
directly to the ventricles, while from the venous part
of the hilum the nerves form a particular nerve plexus
of the cardiac hilum located on the heart base. The
epicardial extensions of the rat intrinsic cardiac neural
plexus project to distinct atrial and/or ventricular
regions by six routes named by us as epicardial neural
subplexuses. It has been known from the earlier
investigations that the extrinsic nerves enter the rat heart
along the RCV (3, 4, 14, 15), along the LCV (3, 14,
15), through the bifurcation of pulmonary trunk (14)
as well as through the pulmonary veins (4). The present
observations not only specified the above-mentioned
accessing sites but also identified the new ones along
great heart arteries, i. e. aorta and the pulmonary trunk.
Architecture of the neural routes in the rat heart
The neural projections to certain regions of the rat
heart have been previously reported by several authors
(4, 14, 15). According to both D. H. Pauza et al (14)
and D. Batulevicius et al (15), the architecture of the
intrinsic cardiac nerve plexus of the rat may be
characterized by (1) NPCH located on the heart base,
(2) the nerves extending from this plexus epicardially
to both the anterior surface of the left atrium and the
dorsal surface of the right atrium and (3) the nerves
extending from this plexus endocardially towards the
dorsal part of the interatrial septum. The examination
carried out by D. H. Pauza et al (14) was restricted to
the atrial projections of the nerves from venous part of
the NPCH and, therefore, both the neural projections
MEDICINA (2004) 40 tomas, Nr. 3
257
from the arterial part of the heart hilum and the neural
projections in the ventricles as well as in the cardiac
septa were not identified.
The detailed scheme of the rat intrinsic cardiac nerve
plexus proposed by R. R. de Sousa et al (4) suggested
the following distinctive features of the architecture of
the nerves in the rat heart: 1) ganglia forming a complicated plexus around the pulmonary veins, 2) nerves
extending from the cranial part of this plexus to the
ventral wall of the ventricles, 3) nerves extending from
the dorsal part of this plexus to the dorsal wall of the
ventricles and, finally, 4) nerves extending from ganglia located in the interatrial groove to the right atrial
wall. It seems likely that the “complicated plexus
around the pulmonary veins” determined by R. R. de
Sousa et al (4) or at least a cranial part of this “complicated plexus” is entirely consistent with the nerve
plexus of the cardiac hilum identified by both D. H.
Pauza et al (14) and D. Batulevicius et al (15). In addition, R. R. de Sousa et al (4) reported that 1) the
nerves innervating the ventral wall of the ventricles
originated from ganglia located cranial to pulmonary
veins, 2) the nerves for the dorsal wall of the ventricles
originated caudal to the pulmonary veins, 3) the nerves
for the right atrial wall originated both to the left of
RCV and in the interatrial groove and 4) the nerves
for the left atrial wall originated from ganglia located
to the left of pulmonary veins. Based on the results of
the present study it might be supposed that the neural
routes within the rat heart epicardium are more complicated than previously described by R. R. de Sousa
(4). In fact, our observations performed on the heart
preparations evidently suggest that 1) the ventral wall
of the ventricles is supplied by nerves from three
subplexuses (RC, LC and VRA), 2) the dorsal ventricular wall – from the LD subplexus only, 3) the right
atrium receives the nerves both from the VRA and DRA
subplexuses, and 4) the nerves for the left atrium proceed by VLA and LD subplexuses.
In the present study a staining for acetylcholinesterase was utilized to visualize the rat intracardiac
nerve plexus. Although it is possible that this histochemical technique did not allow to notice some noncholinergic nerve fibers proceeding within rat heart,
we imply that both the cholinergic and non-cholinergic
intracardiac nerve pathways have been labeled in the
present study as it has been previously shown that 1) a
predominant proportion of the intracardiac nerves
displayed acetylcholinesterase activity (17, 18) and 2)
absolute majority of the intracardiac nerves presumably
contained both cholinergic and non-cholinergic nerve
fibers (6).
Darius Batulevičius, Neringa Paužienė, Dainius H. Pauža
258
Conclusions
The epicardial part of the rat intrinsic cardiac nerve
plexus may be subdivided into six distinct neural subplexuses. Ventral wall of the rat ventricles is supplied
by three neural subplexuses, dorsal ventricular wall –
by one subplexus, each atrium is innervated by two
subplexuses. The anatomical scheme of the rat intrinsic
cardiac nerve plexus compounds to the description of
the same plexus in human, therefore rat is a usable
model for electrophysiological experiments of the
intracardiac nervous system.
Intrakardinės nervų sistemos elektrofiziologinių tyrimų modelio (žiurkės) širdies
nervinio rezginio anatominiai pagrindai
Darius Batulevičius1, 2, Neringa Paužienė2, Dainius H. Pauža2
Kauno medicinos universiteto 1Biomedicininių tyrimų institutas, 2Žmogaus anatomijos katedra
Raktažodžiai: širdis, širdies inervacija, anatomija, autonominė nervų sistema, acetilcholinesterazė.
Santrauka. Žmogaus širdies nervinis rezginys yra sudarytas iš atskirų nervinių subrezginių, kurie pasižymi
specifine topografija ir skirtingomis inervacijos zonomis. Darbo tikslas. Ištirti žiurkės, kaip populiaraus
neurokardiologinių eksperimentų modelio, širdies nervinio rezginio morfologiją ir nustatyti, ar žiurkės širdies
nerviniam rezginiui taip pat būdinga subrezgininė organizacija. Tyrime panaudotos 15 suaugusių žiurkių širdys.
Širdies nervinio rezginio struktūros išryškintos širdžių preparatuose histocheminiu acetilcholinesterazės metodu.
Nustatyta, kad ekstrakardiniai nervai žiurkės širdyje išsidėstę tiek arterinėje, tiek ir veninėje širdies vartų dalyse.
Iš širdies vartų arterinės dalies žiurkės nervai driekiasi į skilvelius, o į veninę vartų dalį einantys nervai suformuoja
širdies vartų nervinį rezginį. Iš širdies vartų nervinio rezginio ir širdies vartų arterinės dalies į epikardą einantys
nervai driekiasi į prieširdžius ir skilvelius šešiais nerviniais keliais. Šio tyrimo duomenimis, žiurkės kaip ir
žmogaus širdies nervinį rezginį sudaro specifiniai subrezginiai, todėl žiurkė yra tinkamas modelis elektrofiziologiniams intrakardinių nervų tyrinėjimams.
Adresas susirašinėjimui: D. H. Pauža, KMU Žmogaus anatomijos katedros Neuromorfologijos laboratorija,
A. Mickevičiaus 9, 44307 Kaunas. El. paštas: daipau@kmu.lt
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Received 17 November 2003, accepted 16 January 2004
Straipsnis gautas 2003 11 17, priimtas 2004 01 16
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