Key anatomic data for the use of rat heart in electrophysiological studies of the intracardiac nervous system

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1 253 Key anatomic data for the use of rat heart in electrophysiological studies of the intracardiac nervous system Darius Batulevičius 1, 2, Neringa Paužienė 2, Dainius H. Pauža 2 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, 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, Kaunas, Lithuania. daipau@kmu.lt

2 254 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 4 o C 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 supply-

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 ventral 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

4 256 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. 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 a 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.

5 Animal model for electrophysiology of the intracardiac nervous system 257 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 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).

6 258 Darius Batulevičius, Neringa Paužienė, Dainius H. Pauža 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 Batulevičius 1, 2, Neringa Paužienė 2, Dainius H. Pauža 2 Kauno medicinos universiteto 1 Biomedicininių tyrimų institutas, 2 Žmogaus 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, Kaunas. El. paštas: daipau@kmu.lt References 1. Ardell JL. Structure and function of mammalian intrinsic cardiac neurons. In: Armour JA, Ardell JL, editors. Neurocardiology. New York, Oxford: Oxford University Press; p Pardini BJ, Patel KP, Schmid PG, Lund DB. Location, distribution and projections of intracardiac ganglion cells in the rat. J Auton Nerv Syst 1987;20: Burkholder T, Chambers M, Hotmire K, Wurster RD, Moody S, Randall WC. Gross and microscopic anatomy of the vagal innervation of the rat heart. Anat Rec 1992;232: de Souza RR, Gama EF, de Carvalho CA, Liberti EA. Quantitative study and architecture of nerves and ganglia of the rat heart. Acta Anat 1996;156: Cheng Z, Powley TL, Schwaber JS, Doyle FJ 3rd. Projections of the dorsal motor nucleus of the vagus to cardiac ganglia of rat atria: an anterograde tracing study. J Comp Neurol 1999;410: Leger J, Croll RP, Smith FM. Regional distribution and extrinsic innervation of intrinsic cardiac neurons in the guinea pig. J Comp Neurol 1999;407: Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000; 259: Pauza DH, Skripka V, Pauziene N. Morphology of the intrinsic cardiac nervous system in the dog: a whole-mount study employing histochemical staining with acetylcholinesterase. Cells Tissues Organs 2002;172: Arora RC, Waldmann M, Hopkins DA, Armour JA. Porcine intrinsic cardiac ganglia. Anat Rec 2003;271A: Maccarrone C, Jarrott B. Differential effects of surgical sympathectomy on rat heart concentrations of neuropeptide Y-immunoreactivity and noradrenaline. J Auton Nerv Syst 1987;21: Sun LS, Legato MJ, Rosen TS, Steinberg SF, Rosen MR. Sympathetic innervation modulates ventricular impulse propagation and repolarisation in the immature rat heart. Cardiovasc Res 1993;27: Yaoita H, Sato E, Kawaguchi M, Saito T, Maehara K, Maruyama Y. Nonadrenergic noncholinergic nerves regulate basal coronary flow via release of capsaicin-sensitive neuropeptides in the rat heart. Circ Res 1994;75: Igawa A, Nozawa T, Yoshida N, Fujii N, Inoue M, Tazawa S, et al. Heterogeneous cardiac sympathetic innervation in heart failure after myocardial infarction of rats. Am J Physiol Heart Circ Physiol 2000;278H: Pauza DH, Skripkiene G, Skripka V, Pauziene N, Stropus R. Morphological study of neurons in the nerve plexus on heart base of rats and guinea pigs. J Auton Nerv Syst 1997;62:1-

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