It is thought that the autonomic cardiac nerves distributing to the heart comprise the sympathetic and parasympathetic cardiac nerves, and that the parasympathetic cardiac nerve downregulates the heart rate, while the sympathetic cardiac nerve upregulates rate (Armour, 1996). Both are controlled by the cardiovascular center of the medulla oblongata and play a crucial role in cardiac function. Especially, the sympathetic cardiac nerve is indispensable for maintaining the blood pressure by controlling cardiovascular function. Thus, it is important to know the routes and distribution of the sympathetic cardiac nerves. A great deal of work has been carried out on heart innervation in mammals, including humans. Such studies on cardiac nerve supplies began with the macroscopic observations of His (1891) and were rapidly developed by Perman (1924) using developmental biological methods. Since then, regarding the autonomic cardiac innervations of humans, many detailed investigations have been reported by Licata (1954), White (1957), Randall and McNally (1960), Mizeres (1963), Navaratnam (1965), Fukuyama (1982), Janes et al. (1986), Kawashima (2005), Kawashima and Sasaki (2005), Kawashima et al. (2005), and Tanaka (2005).
In addition, many comparative anatomical studies have been carried out on autonomic cardiac innervation in various vertebrate species, such as the calf (Shaner, 1930), rat (Gómez, 1958; Burkholder et al., 1992; Batulevičius et al., 2004), chick (Kuratani and Tanaka, 1990; Verberne et al., 1998, 1999), musk shrew (Tanaka et al., 1998), guinea pig (Batulevičius et al., 2005), dog (Randall and Rohse, 1956; Itoh, 1960; Randall et al., 1963, 1968, 1972, 1989; Szentivanyi et al., 1967; Armour and Randall, 1975, 1985; Armour et al., 1975; Norris and Randall, 1977; Rinkema et al., 1982; Hopkins and Armour, 1984, 1989; Brandys et al., 1986; Ardell et al., 1988; Armour, 1988; Gagliardi et al., 1988; Brugnaro et al., 2003), cat (Phillips et al., 1986), and primates (Riegele, 1926; Randall et al., 1971; Billman et al., 1989; Kawashima et al., 2001, 2005). Especially, Armour and Randall (1975, 1985) contributed to our knowledge of cardiac function using methods such as retrograde tracing and electrophysiology in dogs. However, it is difficult to follow the route of nerves from their sympathetic origin to the target organ anatomically because the sympathetic postganglionic nerve fibers are long, complex, and thinner than the parasympathetic ones.
In this study, we examined the route of sympathetic cardiac nerves from their origin to the heart in the house musk shrew using a whole-mount immunostaining method. This animal is a member of the insectivores thought to be related to immediate ancestors of extant mammals (Sharma, 1958; Oda and Kondo, 1977; Romer and Parsons, 1977) and has been employed in various studies because of its primitive form compared with those of rodents generally used as laboratory animals (Kondo et al., 1985; Sakai et al., 2002; Yi et al., 2003, 2004). In this animal, symmetric large blood vessels, which have disappeared in most mammals during their evolutionary development, are preserved and optimal for providing clues about the arterial and venous portae, that is, the entrances to the heart of arteries and veins, because great vessels are considered to play a role as a guide for the peripheral sympathetic cardiac nerves from their origin to the heart. Whole-mount immunostaining is very useful to trace the three-dimensional structure of fine sympathetic cardiac nerves without making serial sections (Ishikawa et al., 1986; Kuratani et al., 1988a, 1988b). Previously, Tanaka et al. (1998) reported on the parasympathetic cardiac nerves in the house musk shrew, but no reports have been made on the sympathetic cardiac nerves in this species.
This study aimed to elucidate the routes of the sympathetic cardiac nerves from their origin to the heart using the house musk shrew, which has preserved primitive blood vessels, using a whole-mount immunostaining method.
MATERIALS AND METHODS
House musk shrews (Suncus murinus) were bred in an air-conditioned room and supplied water and laboratory chow ad libitum under light conditions of a 12-hr light and dark cycle. In this study, 10 adult (5 females and 5 males, weighing 40–50 g) house musk shrews were used. The experimental protocol and care of animals were in accordance with the Guidelines for the Care and Use of Laboratory Animals in Kanazawa University (Kanazawa, Japan).
Adult house musk shrews were first anesthetized with ether, then given an intraperitoneal injection of pentobarbital (0.5 mg/10 g weight). After the animal was completely anesthetized, the peritoneal cavity was opened and a catheter was inserted retrogradely into the abdominal aorta at a level immediately above the bifurcation of this artery into the common iliac arteries. Perfusion was commenced with 0.01 M phosphate buffer (PBS; pH 7.2), thereafter with 0.01 M PBS (pH 7.2) containing 4% paraformaldehyde (PFA). Following perfusion, blood vessels were labeled by the injection of neoprene latex (Showa DDE) through the same part. They were fixed overnight at 4°C in 0.1 M PBS containing 4% PFA, and the heart tissues, including their surroundings, were excised from the carotid sinus to the diaphragm. The specimens were then postfixed with the same solution overnight at 4°C.
The staining procedure was developed and verified in previous studies (Ishikawa et al., 1986; Kuratani et al., 1988a, 1988b) and used with some minor modifications as follows.
After the samples were washed with distilled water and rinsed in PBS, they were treated with hydrogen peroxidase to block the intrinsic peroxidase reaction. Then, they were incubated in freshly prepared 0.5% papain in 0.025 M Tris-HCl buffer (pH 7.6) for 1 hr. In whole-mount immunostaining, since the antigen is not exposed in contrast to serial sections, we augmented the antigen-antibody reaction by exposing more antigens using protease repeatedly. Then the samples were treated with 2.5%, 5.0%, and 10.0% sucrose in PBS for 30 min. The samples were frozen and thawed three times for the same reason as described above and incubated with primary antibody (monoclonal mouse antihuman neurofilament protein, or NFP; Dako, Denmark) in PBS containing 0.2% bovine serum albumin (BSA), 0.3% Triton X-100, and 0.1% sodium azide for 3–4 days at 4°C. After a thorough wash in PBS, they were incubated with a secondary antibody (HRP-conjugated affinity-purified sheep antimouse IgG; MBL, Japan) containing 0.2% BSA and 0.3% Triton X-100 for 3 days at 4°C. After the samples were washed thoroughly in PBS, they were treated with 0.05 M Tris-HCl buffer containing 0.002% 3,3′-diaminobenzidine tetrahydrochloride (DAB) and 0.01% (v/v) H2O2 for 1–3 days at 4°C to visualize the immunoreactivity. The stained samples were then stored in ethylene glycol to achieve transparency and observed with a stereoscopic microscope (Nikon SMZ-U).
Identification of Sympathetic Ganglia
In the cervical region, the cervical sympathetic ganglia and trunks were located on the bilateral sides of the spine and gave the gray rami communicantes to C1–C8, but did not accept the white rami communicantes. In the thoracic region, the thoracic sympathetic ganglia and trunks were located on the bilateral sides of the spine and on the ventral side of the head of the rib, gave the gray rami communicantes to the spinal cord of the thoracic region, and accepted the white rami communicantes. Also, the sympathetic trunks were made up of the sympathetic ganglia, which were longitudinally connected to each other by the interganglionic rami (Berry et al., 1995). In this study, the inferior cervical ganglia were not separately observed, but were fused with the most cranial thoracic sympathetic ganglia at the level of C7-T1. Therefore, we termed this larger one the stellate (cervicothoracic) ganglion.
Overview of Vascular and Autonomic Nervous Systems in House Musk Shrew
Arterial system of cervical and thoracic regions
In this species, the intercostal arteries were different from those of other mammals, including humans (Fig. 1a). The first to fifth intercostal arteries divided from the descending vertebral artery of Nishida (1985) instead of the subclavian artery, and those caudal to the sixth arose directly from the thoracic aorta. Significant variation of the arterial system was not observed among individuals in the 10 house musk shrews examined.
Venous system of cervical and thoracic regions
The left azygos vein accepted the left and right intercostal veins lower than the fifth level and then returned venous blood to the right atrium through the coronary sinus. The right azygos vein remained similar to that on the left side as a common duct of the second to fifth intercostal vein, then joined to the right anterior cardinal vein. The left anterior cardinal vein was maintained without any reduction to accept veins of the head and forelimbs and the left intercostal vein until T4, and allowed backflowing to the right atrium via the coronary sinus (Fig. 1b). Hence, the coronary sinus of this animal was more developed compared with that of humans, because it plays a crucial role in backflow to the right atrium. Significant variation of the venous system was not observed among individuals in the 10 house musk shrews examined.
Overview of autonomic nerves of cervical and thoracic regions
The sympathetic cardiac nerves and parasympathetic cardiac branches of the vagal nerve were clearly distinguishable from surrounding tissues using antineurofilament protein (NFP) antibodies (Fig. 1c). The cervical sympathetic chain descended from the superior cervical ganglion along the dorsal medial side of the common carotid artery, turned to the lateral side at the border of the cervix and thorax, and shifted to the thoracic sympathetic trunk. The superior cervical ganglion was observed at the site of bifurcation of the common carotid artery into the internal and external carotid arteries. The middle cervical ganglion was smaller than the superior cervical ganglions and was located at the level of C2–C3. The inferior cervical ganglion was not observed independently, but was instead fused with the first thoracic sympathetic ganglion to form the stellate ganglion at the border of the neck and thorax. The thoracic sympathetic trunk was made up of 11 pairs of sympathetic trunk ganglia and was located on the bilateral sides of the spine and on the ventral side of the head of each rib.
The main trunk of the vagus nerve, on the other hand, ran along the lateral side of the common carotid artery, descended between the aortic arch and the dorsal side of the heart on the right side, and on the left side passed around the ventral side of the left subclavian artery dorsal to the heart. On both sides of the neck, the trunks of this nerve fused to form the esophageal plexuses along the esophagus, after sending smaller branches to the lung.
Origin and Route of Sympathetic Cardiac Nerves
The sympathetic nerve branches originating from the superior cervical ganglion on the left side descended toward the aortic arch, forming nerve plexuses along the common carotid artery, accepting nerve fibers of the middle cervical ganglion and vagus nerves along the way (Fig. 2). At the level of C7, the cervical sympathetic trunk sprouted two branches ventral to the subclavian artery, one of which accepted the parasympathetic cardiac branch of the vagus nerve at the level of T2, and descended along the aortic arch with the main trunk of the vagus nerve to form the nerve plexuses. The other ran together with the parasympathetic cardiac branch and descended ventral to the aortic arch along the common carotid and subclavian arteries. After reaching the arterial porta, they formed nerve plexuses and supplied nerves to the ventral wall of the ventricle (Fig. 3a and b).
Three nerve branches originated from the left stellate ganglion. The first ran together with the subclavian artery to contribute to the plexuses of the arterial porta. The second descended to reach the left anterior cardinal vein via the intercostal vein, accepting nerve fibers from the vagus nerve to form a nerve plexus. The third ran along the descending vertebral artery with the nerve fibers of the thoracic sympathetic ganglia from T2 to T3 and joined with nerve fibers derived from T4. These ramified fibers ran along the lateral and medial sides of the left azygos vein (Figs. 2 and 3c). The former then divided into a nerve fiber taking a caudal course along the azygos vein, and the other entered the coronary sinus. The latter went to the arterial and venous portae along the aortic arch and coronary sinus, respectively (Fig. 2, dorsal view). The nerve branches originating from the thoracic sympathetic ganglia at T5 and T6 reached the left azygos vein via the intercostal artery or vein to extend into the space between the left azygos vein and the thoracic aorta (Figs. 2 and 3d). These branches joined added the parasympathetic cardiac branches at the entrance of the coronary sinus to enter the sinus venarum cavarum corresponding to the dorsal wall of the right atrium and also descended caudally with the nerve branch derived from T4 along the left azygos vein (Fig. 2, ventral view). Some nerve branches ran parallel to the coronary sinus and sent many fine nerves toward the dorsal wall of the ventricle (Fig. 3e). Furthermore, one of the nerve branches reaching the coronary sinus contributed to the plexus of the pulmonary venous sinus, while the other deflected to the lateral side of the left ventricle and descended along the left ventricle marginal vein toward the apex of the heart (Fig. 3f).
The nerve branches originating from the cervical sympathetic ganglia or trunk on the right side reached the arterial porta along the aortic arch similar to those on the left side, then extended to the ventral wall of the ventricle along the coronary arteries (Figs. 2 and 3a and b).
The nerve branches originating from the right stellate and thoracic sympathetic ganglia or trunk until T5 on the right side supplied the same arterial porta as those of the left side (Fig. 2).
Each nerve branch arising from the sympathetic ganglia at the level of T2–T5 ran along the intercostal vein and reached the right anterior cardinal vein via the right azygos vein. These branches joined fibers from the vagus nerve, then descended along the right anterior cardinal vein toward the sinus venarum cavarum of the right atrium and joined to form plexuses with the nerve branch running along the coronary sinus from the left side. One sympathetic nerve contributed to the plexus of the pulmonary venous sinus and to the middle cardiac vein toward the dorsal wall of the right ventricle (Fig. 3e), while the other extended to the crux of the heart near the sinoatrial node (Fig. 3g). The nerve branch originating from the thoracic sympathetic ganglion at T6 reached the thoracic aorta separately without passing along the intercostal artery and joined the corresponding sympathetic nerve arising at the same level from the left side. One of the nerves originating from the thoracic sympathetic ganglia or trunk at T4–T6 on the left side coursed caudally along the ventral side of the left azygos vein (Fig. 2) and was divided at the midpoint between T8 and T9. One of which ran toward the esophagus along the esophageal artery or vein between the mediastinal pleura and joined with the vagal nerve on the esophageal plexuses (Figs. 4a and b). The other descended caudally along the left azygos vein, joined a nerve branch from the thoracic sympathetic trunk at the midpoint between T9 and T10, and reached the esophageal plexuses from the midpoint between T10 and T11.
Of the two nerve branches originating at the level of T7 on the right side (Fig. 4c–e), the first reached the thoracic aorta independently to join a branch arising from T6 at the midpoint between T6 and T7, then running toward the esophagus along the esophageal artery within mediastinal pleura at the same level. The second T7 branch reached the thoracic aorta in conjunction with the nerve branch deriving from T8 via the seventh intercostal artery, joined the nerve branch descending along the aorta at the level of T7, and ran toward the esophageal artery within the mediastinal pleura at the same level. The sympathetic nerve fibers from T8 and T9 had a route similar to that described for fibers arising from T7.
Moreover, these nerve fibers reaching the esophageal plexuses were macroscopically traced to the lung from the esophageal plexuses via the bronchial artery or vein within the mediastinal pleura (Fig. 4f) and were composed of the nerve branches from the aortic arch plexus and those deriving from the stellate ganglion and the thoracic sympathetic trunk until T6 at the arterial and venous portae.
In this study of the house musk shrew, the sympathetic cardiac nerves were macroscopically categorized into the following three categories on the basis of their origin, nerve routes, and terminal portions: innervation of the cervical sympathetic ganglia and trunk mainly to the arterial porta of the heart; innervation of the stellate and thoracic sympathetic ganglia at the level of T2–T5 or T6 to both the arterial and venous portae of the heart by diverging along the pathway; and innervation of the thoracic sympathetic ganglia at the level of T4–T9 to the esophagus, lung, and heart via the blood vessels within the mediastinal pleura.
The origins of these nerves were approximately bilaterally symmetrical, but some differences were observed in their pathways. This might have resulted from bilateral differences of the blood vessels that carried nerve fibers to the target organ. In other words, the differences in the left and right blood vessels may lead to prominent differences in the nerve courses of the sympathetic cardiac nerves. The existence of the superior cardiac nerve originating from the superior cervical ganglion has been controversial. The location of this ganglion and the course of the nerve vary greatly among species, and both may be absent in other species. Perman (1924) asserted that the sympathetic cardiac nerves in vertebrates originate only from the middle cervical sympathetic and stellate ganglia. Janes et al. (1986) also reported that all the sympathetic cardiopulmonary nerves originates from the stellate ganglia and the caudal halves of the cervical sympathetic trunks below the level of the cricoid cartilage; these authors reported that none originated from the superior cervical ganglia or directly from the cranial cervical sympathetic trunks in humans. However, Mitchell (1953) and Ellison and Williams (1969) also indicated that there are multiple anastomoses among the superior sympathetic, middle, and inferior cardiac nerves and the parasympathetic cardiac branches of the vagus nerve in human adults and embryos. In the present study, the nerve originating from the superior cervical ganglion descended to reach the aortic arch along the common carotid artery, adding nerve branches from the middle cervical sympathetic ganglion and the vagus nerve along the way, but no independent nerve originating from the middle cervical sympathetic ganglion was observed to reach the heart. The data in the present study accord with those reported by Mitchell (1953) and Ellison and Williams (1969). Recently, Kawashima et al. (2005) reported in humans and monkeys that the superior sympathetic cardiac nerves running toward the heart after accepting the nerves from the middle cervical sympathetic ganglion and the parasympathetic vagus nerve. Therefore, the nerve branch from the superior cervical ganglion observed in the house musk shrew may correspond to the superior sympathetic cardiac nerve in humans and monkeys and supply the aortic arch in the arterial porta.
In the present study, nerve branches originating from the stellate and the thoracic sympathetic ganglia or trunk from T2–T6 mainly innervated arterial and venous portae of the heart. Some previous studies indicated that the nerve deriving from the stellate ganglion reaches the arterial porta. For instance, Perman (1924) reported that the inferior cardiac nerves deriving from the stellate ganglion innervated the plexus of the coronary artery of the heart in mammals. Mitchell (1953) as well as Fukuyama (1982) also suggested that the nerve descending along the dorsal side of the aortic arch reaches the cardiac plexus in humans. However, Janes et al. (1986) reported that the sympathetic cardiac nerves originating from the right stellate ganglion reach the right atrium via the superior vena cava in humans. In addition, it has been asserted that the nerve branches deriving from the stellate ganglion innervate the venous porta in Japanese monkeys and humans (Kawashima et al., 2001; Kawashima, 2005; Kawashima and Sasaki, 2005). In the present study, the large blood vessels connecting to the heart were bilaterally symmetrical, but those in other mammals, including humans, were asymmetrical, so there are thus fundamental vascular differences between the house musk shrew and other mammals, including humans. Asymmetrical arrangement of the blood vessels may have caused the asymmetrical nerve course constitution deriving from the stellate ganglion and thoracic sympathetic ganglia or trunk until T6 in primates, in contrast with the symmetrical arrangement in the house shrew. To resolve this issue, it will be necessary to perform comparative studies in mammals with different arrangements of blood vessels in the trunk.
On the other hand, in the present study, the nerve branches originating from the thoracic sympathetic ganglia at T4–T9 innervated both the arterial and venous portae and then the heart through the esophagus and lung via blood vessels within the mediastinal pleura. The origin of mammalian sympathetic cardiac nerves in the thoracic region has been controversial. Perman (1924) concluded that no cardiac nerves arise from the sympathetic trunk in man below the second thoracic segment, and Licata (1954) questioned whether the nerve branches deriving from the thoracic sympathetic trunk reach the heart. Janes et al. (1986) also added that the sympathetic cardiac nerves arising from the thoracic sympathetic ganglia are not observed in humans. However, Perman (1924) traced nerves arising from T3–T6 into the cardiac plexus in certain Artiodactyla, and Kuntz and Morehouse (1930) traced nerves arising from T2–T5 into the cardiac plexus in humans. Mizeres (1963) found that the cardiac nerve in the thoracic region arising from T1–T4 or T5 and also their nerve branches are related to nerve plexuses of the lung, heart, and aortic arch in humans. Ellison and Williams (1969) reported the sympathetic cardiac nerves arise from T2–T6 in the human embryo. Moreover, Fukuyama (1982) mentioned that the nerve branches deriving from the thoracic sympathetic ganglia or trunk at T2–T4 or T5 innervate the heart and lung (cardiopulmonary nerve), and it has been reported that sympathetic cardiac nerves originating from T4–T5 descend obliquely along the intercostal vein, after being diverted at T7 or T8, to ascend toward the heart along the aorta in adult humans and Japanese monkeys (Kawashima et al., 2001; Kawashima, 2005). Tanaka (2005) recently reported that the sympathetic nerve branches originating from T5–T8 reach the oblique sinus of the pericardium via the bifurcation of the trachea in human cadavers. In a physiological study, Randall and McNally (1960) suggested that nerve fibers originating from the upper four or five thoracic segments contribute to cardiac acceleration in humans. In contrast to the findings of these authors, Itoh (1960), using a pathological method in dogs, indicated that the nerve center of algesia of the heart is pervasive in the range from C1 to T10. In the present study, nerve branches originating from the thoracic sympathetic ganglia at T4–T9 reached the thoracic aorta along the intercostal artery, then ran toward the esophagus along with the esophageal artery within the mediastinal pleura to reach the lung and the arterial and venous portae of the heart, in addition to nerve fibers from sympathetic thoracic ganglia from T2–T5 or T6. There have been no previous reports on the nerve courses of sympathetic cardiac nerves arising from below T6 and running to the heart along the blood vessels within the mediastinal pleura like those in this study. Therefore, the present study suggests that the caudalmost limit of theorigin of sympathetic cardiac nerves deriving from the thoracic sympathetic ganglia or trunk and running tothe heart along the blood vessels within the mediastinal pleura in mammals, including humans, is likely to be located further caudally than presently thought.
The authors thank Mr. Tsuneo Nakamura for his continuous efforts in breeding the house musk shrews, Mr. Yoshitake Shiraishi for technical assistance with the photographs, and Mrs. Ikuko Koizumi for help in preparing the manuscript.