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Ganglionic distribution of afferent neurons innervating the canine heart and cardiopulmonary nerves
Journal of the Autonomic Nervous System, 26 (1989) 213-222
213
Elsevier JAN 00923
Ganglionic distribution of afferent neurons innervating the canine heart and cardiopulmonary nerves D a v i d A. H o p k i n s 1 a n d J. A n d r e w A r m o u r 2 Department of 1 Anatom)~ and 2 Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, NS (Canada) (Received 7 July 1988) (Revised version received 23 January 1989) (Accepted 26 January 1989)
Key words: Sensory; Dorsal root ganglion; Nodose ganglion; Horseradish peroxidase Abstract The ganglionic distribution of the perikarya of afferent axons in cardiopulmonary nerves or the heart was studied in 64 dogs by injecting horseradish peroxidase into physiologically identified cardiopulmonary nerves or different regions of the heart. In 6 additional dogs, horseradish peroxidase was injected into the aortic arch, pericardial sac, left ventricular cavity or the skin. After injections into cardiopulmonary nerves, retrogradely labeled perikarya were found in the ipsilateral nodose ganglion and the ipsilateral CT-T 7 dorsal root ganglia. After injections into different regions of the heart, retrogradely labeled neurons were found in the nodose ganglia bilaterally and in the C r - T 6 dorsal root ganglia bilaterally. Many more retrogradely labeled neurons were found in the nodose ganglia in comparison to the dorsal root ganglia. The largest numbers of retrogradely labeled perikarya in the dorsal root ganglia occurred in the "1"2_4 ganglia following nerve or heart injections. Following injections into specific regions of the heart or individual physiologically identified cardiopulmonary nerves, regional distributions of labeled neurons could not be identified within or among ganglia with respect to the structures injected. Perikarya in dorsal root ganglia which were labeled after heart injections ranged in area from 436-3280 /~m2 (X = 1279 + 51 S.E.M.) while after skin injections labeled perikarya ranged in area from 224-5701 /Lm2 (X = 1631 + 104 S.E.M.). The results show that the afferent innervation of the canine heart is provided by neurons located throughout the nodose ganglia and to a lesser degree in the C r - T 6 dorsal root ganglia bilaterally. The bilateral distribution of cardiac afferent neurons raises questions regarding mechanisms underlying unilateral symptoms frequently associated with heart disease.
Introduction Myelinated and unmyelinated afferent axons which arise from mechanoreceptors or chemoreceptors located in the heart, vena cavae, pulmonary arteries and aorta course centrally via cardiopulmonary nerves [1] to the vagosym-
Correspondence: D.A. Hopkins, Department of Anatomy, Faculty of Medicine, Dalhonsie University, Halifax, Nova Scotia, B3H 4H7, Canada.
pathetic complexes [8,11,30] or subclavian ansae and thoracic rami [7,9,17,25,27] to cell bodies in the nodose [14,21] or dorsal root ganglia [21,23]. The cell bodies of cardiac afferent dorsal root ganglion neurons have generally been reported to be located in dorsal root ganglia from the first to the 5th thoracic segments [20,23,29]. More recently, studies of the distribution of sensory perikarya innervating specific cardiopulmonary nerves or cardiac structures have been undertaken. It has been reported that afferent axons in the cat inferior cardiac nerve arise from cell bodies in the
0165-1838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
214
T1-7 [23] or the C s - T 9 [15] dorsal root ganglia and those in the region of the cat's left anterior descending coronary artery from cell bodies in the C s - T 6 dorsal root ganglia [28]. However, except for these few studies in which specific structures were investigated, the locations of the afferent cell bodies which project axons in various cardiopulmonary nerves or which innervate different regions of the heart remain unknown. In the present study, the dorsal root and nodose ganglia obtained from dogs in which physiologically identified cardiopulmonary nerves or specific regions of the heart were injected with horseradish peroxidase (HRP) have been studied in order to determine: (1) the ganglionic locations of cardiopulmonary afferent perikarya, (2) the relative contributions of the nodose and dorsal root ganglia to heart innervation and (3) the segmental organization of the afferent neurons which project axons into the cardiopulmonary nerves and the heart. In addition, comparisons were made between cardiac and skin afferents.
Materials and Methods
Experimental material was obtained from 70 mongrel dogs which had been used to study the locations of cardiac sympathetic efferent postganglionic neurons [3,4,13] and cardiac parasympathetic efferent preganglionic neurons [12,13]. Dogs of either sex (12-20 kg in weight) were sedated with ketamine hydrochloride (1 m g / k g i.m.) and anesthetized with sodium thiopental (25 m g / k g i.v.). Positive pressure respiration was initiated using a Bird Mark 7A respirator. A lead II E K G was recorded and a femoral artery was cannulated in order to record blood pressure. A unilateral thoracotomy was performed via the 3rd or 4th intercostal space in order to expose the thoracic autonomic nerves, heart and great thoracic vessels. In 40 dogs in which cardiopulmonary nerves were studied, Walton-Brodie strain gauge arches were sutured to the surfaces of an atrium a n d / o r a ventricle. Subsequently, cardiopulmonary nerves, the functional anatomy of which has been described previously [4,5], were identified, exposed
and stimulated for up to 10 s using a Grass SD9 square wave stimulator. The stimulus parameters (10 Hz, 5 ms, 4 V) were monitored on a Tektronix 510 3N oscilloscope and the cardiovascular responses were monitored on a Beckman Dynograph. When a cardiopulmonary nerve had been identified anatomically and the function of the efferent axons in that nerve demonstrated physiologically, 3-25 #l of aqueous 30% H R P (Boehringer) were injected into the nerve using a 5 0 / t l Hamilton syringe with a 22-gauge needle as described previously [3]. The procedure of identifying an intrathoracic structure as a cardiopulmonary nerve was necessary in order to confirm that such a structure was indeed a cardiopulmonary nerve and not a lymphatic vessel or fibrous tissue. Although this procedure does not identify afferent axons arising from the heart, it does establish that these structures contain cardiac efferent axons and since each efferent nerve also contains afferent axons [1,5], the presence of afferent axons in identified cardiopulmonary nerves is assured. In 24 dogs, 15-250/~l of 30% aqueous H R P or 5% wheat germ a g g l u t i n i n - H R P (Sigma) were injected into various regions of the heart (Fig. 1). In 3 dogs H R P was injected into the inner arch of the
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Fig. 1. Drawing of the ventral and dorsal surfaces of the heart and great thoracic vessels showing the locations of the injection sites (modified from Hopkins and Armour 3 and reproduced by permission from the Journal of Comparative Neurology). Injection sites in the atrioventricular nodal region (59, 74) and left ventricular ventral papillary muscle (64), marked by India ink and identified at autopsy, are shown by stippling.
215 aorta, the pericardial sac or the left ventricular cavity. In another group consisting of 3 dogs which were sedated with ketamine hydrochloride, the skin over the ulnar surface of the left forelimb, the skin adjacent to the left cranial thoracic nipple or the skin over the left mandibular angle were injected with 50-120 /al of HRP. Following the above procedures the thoracic cavity was closed and air was withdrawn for those animals which received thoracotomies, and antibiotics (Penlong 1 ml i.m.) and analgesics (Demerol 5-10 m g / k g i.m.) were administered. On the third postoperative day the animals were re-anesthetized with an overdose of sodium pentobarbital and the chest was rapidly opened. Following occlusion of the descending aorta at the level of the diaphragm, the animals were perfused via the ascending aorta with 2 - 4 litres of physiological saline (0.9%) followed by 6 litres of fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer (pH 7.4). Both nodose ganglia of all dogs and dorsal root ganglia bilaterally in all but 11 of the dogs were collected from the C 2 to the Ts levels and stored in 30% sucrose in phosphate buffer until processed. Serial 40-/~m-thick frozen sections were cut and processed for H R P histochemistry [19] and then counterstained with Neutral red and examined with bright- and dark-field microscopy. The distribution of labeled afferent neurons was assessed by counting the numbers of labeled neurons in each ganglion. In order to estimate the perikaryal dimensions of dorsal root ganglion afferent neurons associated with the heart, the maximum diameter, perimeter and area of perikarya labeled after heart injections were determined from camera lucida drawings with the aid of a Zeiss IBAS image analysis system. Perikaryal dimensions were also assessed for representative cases in which a cardiopulmonary nerve or the skin was injected. For each ganglion containing labeled perikarya at least 10 unlabeled perikarya with a visible nucleolus were also measured. Unlabeled cells were chosen by virtue of their proximity to the labeled cells and by selecting a range of cell sizes in the ganglia. Correction factors were not applied to measurements of cell sizes due to the fact that the relatively sparse
labeling in the ganglia made double counting unlikely. In addition, correction factors depend on being able to see the nucleolus which was often not visible because of the intensity of the labeling (Fig. 2).
Results
Retrogradely labeled neurons were observed in the nodose and dorsal root ganglia after injections of H R P into cardiopulmonary nerves and heart (Table I). In general, nerve injections resulted in larger numbers of labeled perikarya in nodose and dorsal root ganglia than did heart injections. In all of the dogs described in the present analysis, retrogradely labeled neurons have been previously identified in thoracic autonomic ganglia or the medulla oblongata [12,13].
Cardiopulmonary nerve injections Injections into certain nerves resulted in a greater degree of labeling in ganglia than other nerves. Injections into the right recurrent, right caudal vagal, left intermediate medial, and the left recurrent cardiopulmonary nerves resulted in the greatest number of labeled neurons in the nodose ganglia. Injections into the right recurrent and left caudal pole cardiopulmonary nerves resulted in the heaviest labeling of afferent neurons in the dorsal root ganglia. Nodose ganglia. Of the 40 dogs which had cardiopulmonary nerve injections, the ipsilateral nodose ganglia in 22 (55%) contained retrogradely labeled neurons. In this group of animals, all labeling was ipsilateral except for one contralateral nodose ganglion which contained two retrogradely labeled neurons. After nerve injections there was an average of 285 + 93 (range 2-1392) labeled neurons in the ipsilateral nodose ganglia in those animals which had labeled neurons. Following injections into right-sided nerves, there were 249 _ 133 (range 2-1392) labeled neurons in the right nodose ganglia and following left-sided nerve injections 333 + 140 (range 2-1101) in the left nodose area. Dorsal root ganglia. Of the 29 dogs from which dorsal root ganglia were collected following
216 TABLE I
Summary of experiments and percentages of labeled ganglia Injection sites
Left cardiopulmonary nerves Right cardiopulmonary nerves Total of nerves Heart Aorta Pericardial sac Left ventricular cavity Skin
Numbers of dogs studied
Numbers and percentages of dogs with labeled perikarya
N odose
DR G
N odose
DR G
21 19 40 24 1 1 1 3
21 8 29 24 1 1 1 3
11 (52%) * 11 (58%) * * 22 (55%) 22 (92%) 1 1 0 0
11 (52%) * 6 (75%) * 17 (59%) 12 (50%) 1 1 0 3 *
* Labeled neurons located ipsilaterally only. * * Labeled neurons located ipsilaterally with the exception of one case (a right caudal vagal cardiopulmonary nerve injection which had in addition two labeled perikarya in the contralateral nodose ganglion).
cardiopulmonary nerve injections, the ipsilateral dorsal root ganglia in 19 (66%) contained retrogradely labeled neurons. Retrogradely labeled perikarya (Figs. 2, 3) were observed in the ipsilateral dorsal root ganglia from the C T - T 7 levels (Fig. 4). The largest numbers of retrogradely labeled cells were present in the T 2_ 4 dorsal root ganglia after nerve injections (Fig. 4). Following cardiopulmonary nerve injections the maximum number of labeled cells found in one dorsal root ganglion was 260. This was the left T 2 dorsal root ganglion following a left caudal pole nerve injection of 6 ktl of H R P (Fig. 3). After cardiopulmonary nerve injections, an average of 4.2 __+10.4 (S.E.M.) dorsal root ganglia were labeled and these ganglia contained in total an average of 75.2 + 30 labeled neurons. In contrast, after heart injections an average of 2.3 + 0.6 ganglia were found to have a total of 13.5 ___4.4 labeled neurons. Labeled neurons in the dorsal root ganglia had a smaller mean diameter than unlabeled neurons or than neurons labeled after skin injections (Fig. 5; Table II).
Heart injections Nodose ganglia. In 24 dogs in which the heart was injected, in only one did an injection (one of the two atrio-ventricular nodal injections) fail to result in labeling of either the nodose or dorsal root ganglia. In the dogs which received heart injections, 22 (92%) had retrogradely labeled neu-
rons in the nodose ganglia and in all but 3 of these the nodose ganglia were labeled bilaterally. Following heart injections there was an average of 38 + 18 (range 1-411) labeled neurons in the right nodose ganglia and 25 + 10 (range 1-216) labeled neurons in the left nodose ganglia in those animals which had labeled neurons. Following injections of H R P into specific regions of the heart labeled neurons were located throughout the nodose ganglia bilaterally in almost every instance. Because labeled neurons were found to be scattered throughout the ganglia, there was no clear indication of a topographical organization within individual nodose or dorsal root ganglia which was also the case after nerve injections. Dorsal root ganglia. Twelve (50%) of the dogs with heart injections had retrograde labeling in the dorsal root ganglia. The numbers and locations of labeled neurons varied among animals, even when the injection sites in the heart were similar. After heart injections right dorsal root ganglia were labeled from C r - T 5, while on the left labeling extended from T I _ 6. Following cardiac injections the maximum number of labeled neurons found in a dorsal root ganglion was 43 (the left T 4 dorsal root ganglion following injection of 250 ~tl of H R P into the cranial epicardium of the right and left ventricles, dog 69). Overall, after heart injections an average of 2.3 + 0.6 ganglia were found to have a total of 13.5 ___4.4 labeled neurons. With
217
Fig. 2. Photomicrographs of retrogradely labeled perikarya in sensory ganglia. A: left T3 dorsal root ganglion of dog 33 (left caudal pole cardiopulmonary nerve, 10 pl). B: area enclosed by box in A. C: right nodose ganglion of dog 58 (left ventricular apical epicardium, 45 pl). D: right nodose ganglion of dog 36 (right recurrent cardiopulmonary nerve, 15 ~1). Calibration bars: A, D ffi 250 /~m; B = 50 pm; C = 40/~m.
218 DOG 48 T2 DORSAL ROOT GANGLION LATERAL
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Fig. 3. Drawing showingthe distribution of retrogradely labeled perikarya in the left T2 dorsal root ganglion of dog 48 (left caudal pole cardiopulmonary nerve, 6 ~1). The numbers on the left refer to serial section numbers. respect to the size of labeled neurons, those in the dorsal root ganglia had a smaller m e a n diameter t h a n unlabeled n e u r o n s or t h a n n e u r o n s labeled after skin injections (Fig. 5; T a b l e II). • •
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Fig. 4. Histogram showing the average number of labeled neurons per ganglion in the C6-T7 dorsal root ganglia following heart or cardiopulmonary nerve injections. Error bars = S.E.M.
2
0 100
2000 4000 AREA(~tm2)
6000
Fig. 5. Histograms showing the areas of labeled cells in dorsal root ganglia as a percentage of the total cells labeled following injections of the (A) heart and (B) skin of the left forelimb.
Aortic arch, pericardial sac, left ventricular cavity and skin injections Following injection of H R P in the a d v e n t i t i a of the arch of the aorta, labeled n e u r o n s were present only in the left nodose ganglion a n d the left dorsal root ganglia (T3_6). W h e n a n injection was m a d e into the pericardial sac, m a n y lightly labeled perikarya were f o u n d in the n o d o s e ganglia bilaterally a n d a few lightly labeled cells were f o u n d in the C 6 and C v dorsal root ganglia. W h e n H R P was injected i n t o the left ventricular cavity n o labeled n e u r o n s were detected in any of the ganglia investigated. The largest n u m b e r s of labeled n e u r o n s in dorsal root ganglia occurred following injections in the skin. W h e n the skin of the left forelimb or the left chest was injected, labeled n e u r o n s were located in ipsilateral dorsal root ganglia from
219 TABLE II Dimensions [mean + S.E.M. (Range)] of labeled and unlabeled perikarya in dorsal root ganglia Tissue injected
n
Diameter (#m)
Perimeter (l~m)
Area (~m)
Heart
Labeled 139 Unlabeled 151 Labeled 26 Unlabeled 20 Labded 258 Unlabeled 148 Labeled 135 Unlabeled 64
52 ± 1 (28- 87) 67 ± 1 (30-118) 56 + 3 (34- 86) 70 ± 5 (41-116) 49 ± 1 (22-100) 64 4- 2 (29-123) 57 + 2 (23-123) 67 + 3 (30-116)
135 ± 177 ± 147 ± 186 ± 128 + 171 + 147 ± 175 ±
127942350 ± 1550 ± 2615 ± 1176 ± 2248 ± 1631 ± 2320 ±
Aorta Nerve Skin
3 (83-226) 4 (81-302) 7 (93-226) 12 (109-288) 2 (57-250) 4 (77-286) 5 (57-286) 7 (81-309)
51 102 143 304 38 107 104 108
(436-3280) (404-6482) (642-3471) (824-5234) (225-4218) (413-5770) (224-5 701) (457-7054)
* Mean + S.E.M. (range).
the C8-T 6 levels depending on the segmental level of the injection. After injections into the skin (1) over the left ulnar tubercle there were 1662 labeled neurons in the left T 1 dorsal root ganglion, (2) adjacent to the left nipple there were 1933 labeled neurons in the left T 4 dorsal root ganglion and (3) over the left mandibular angle there were 4211 labeled neurons in the left C 3 dorsal root ganglion. Following injection into the skin over the left elbow, labeled neurons were located in the ipsilateral "1"2_4 dorsal root ganglia, following injection into the skin of the left nipple they were located in the C 8 - T 7 ganglia and following injection into the skin over the left mandibular angle they were located in the C 2_ 4 dorsal root ganglia.
Discussion The results of the present investigation in which H R P was injected into physiologically identified cardiopulmonary nerves or specific regions of the heart show that the perikarya of afferent neurons which innervate canine cardiopulmonary nerves or the myocardium are located in the nodose ganglia and the C6-T 7 dorsal root ganglia. More neurons were labeled in the nodose ganglia in comparison to the dorsal root ganglia when a cardiopulmonary nerve was injected. In all of the cases studied efferent preganglionic parasympathetic or postganglionic sympathetic neurons were also labeled [3,12,13] whether or not labeling occurred in afferent neurons. Thus, when no labeled neurons were
found in the afferent ganglia this could not simply be ascribed to a failure of the label to be picked up by the structures injected. The possibility remains that the extent of the dorsal root ganglion innervation was underestimated if dorsal root ganglion afferent axons do not take up or transport H R P as well as other axons. Afferent axons in cadiopulmonary nerves are connected primarily with perikarya in ipsilateral ganglia, whereas afferent axons from the heart arise from perikarya in the nodose and dorsal root ganglia bilaterally. In contrast, afferent neurons with axons in the arch of the aorta of one animal arose from perikarya in the left nodose and left dorsal root ganglia. Following cardiopulmonary nerve injections labeled neurons were located in the ipsilateral C 7 - T 7 dorsal root ganglia while following injections into the heart labeled neurons were located in the dorsal root ganglia from the C6-T 5 levels, the greatest numbers of labeled afferent neurons occurring in the T2_4 dorsal root ganglia (Fig. 4). More cells were labeled in dorsal root ganglia following nerve injections than heart injections. The difference in the degree of labeling in the two groups of experiments may be due in part to the fact that cardiopulmonary nerves contain afferent axons arising from receptors in noncardiac tissue, such as pulmonary tissue, in addition to the cardiac afferent axons which arise from receptors in different regions of the heart [1]. Dorsal root ganglion perikarya were labeled following injection into each major cardiopulmonary nerve. Injections into the right recurrent, left inter-
220
mediate medial and left caudal pole cardiopulmonary nerves resulted in the greatest number of labeled neurons in both the nodose and dorsal root ganglia. In keeping with this, physiological evidence has demonstrated that these are major afferent cardiopulmonary nerves [1,2]. Interganglionic cardiopulmonary nerve injections resulted in labeling of neurons in the ipsilateral dorsal root ganglia but not in the nodose ganglia. This nerve contains efferent axons arising from sympathetic but not parasympathetic neurons [3,12] and thus the axons in this nerve are primarily related to the spinal cord and not the brainstem. This is also in agreement with physiological evidence which shows that this nerve contains cardiac afferent axons [1] and cardiac sympathetic efferent axons [5]. Similarly, the left caudal pole cardiopulmonary nerve contained relatively large numbers of afferent axons related to the dorsal root ganglia in comparison to the nodose ganglia. This latter nerve is known from anatomical [3] and physiological [5] studies to be primarily a sympathetic efferent cardiac nerve which contains few, if any, parasympathetic efferent axons [12]. Therefore, there appears to be a relative dissociation of both afferent and efferent axons in these nerves with respect to the brainstem and spinal cord. This dissociation is however not absolute since a few labeled cells were found in the ipsilateral nodose ganglia following injections of H R P into the left caudal pole nerve, a nerve which has no efferent parasympathetic axons [12]. An injection in one epicardial area would potentially label m a n y afferent axons in that area as well as axons projecting to other areas. Mechanoreceptors located in the endocardium usually have relatively small receptor fields and those in the epicardium or myocardium may have relatively large receptor fields [1]. It is known that one afferent axon may arise from mechanoreceptors located in two relatively widely separated regions of the heart [1] or even different thoracic organs [2]. Also, perikarya of an afferent neuron may project axons into two or more different nerves [26]. In addition, axons passing through injection sites to other cardiac regions could also take up HRP. It appears that afferent cell bodies with
axons in a specific cardiac region or a specific cardiopulmonary nerve are not restricted to a specific ganglion or ganglionic region. For example, injections of H R P into the left ventricular apex or the left ventricular ventral papillary muscle would presumably label only the distal cardiac axons as few axons in these regions project to other regions of the heart. In these cases the distribution of afferent neuron labeling did not differ from that observed after injections in more cranial cardiac regions such as following injections into the base of the ventricles or in the atria. Thus, a topographical anatomical arrangement with respect to the distribution of afferent cardiac neurons could not be identified. Similarly, efferent postganglionic sympathetic neurons which innervate the heart [13] or cardiopulmonary nerves [3] also do not have a topographical anatomical arrangement. When injections of H R P were placed adjacent to the major coronary vessel it was likely that axons which accompanied that vessel were able to pick up the label. Axons which accompany a coronary vessel innervate distal regions of the heart perfused by that vessel, regions which can be relatively extensive [6]. Thus, labeling of neurons following such an injection would reflect the distribution of nerves over a relatively large region of the heart and not necessarily the innervation of the coronary arteries alone. The present study and a previous one [28] indicate that the nerves which accompany major coronary vessels contain sympathetic postganglionic axons [13] which innervate the distal myocardium [6] and afferent axons which arise primarily from the ganglia bilaterally. When H R P was injected into the left ventricular cavity no labeled neurons were identified. However, when H R P was injected into the pericardial cavity afferent neurons were lightly labeled. In this control experiment m a n y cells were labeled in the nodose ganglia bilaterally while only a few cells were labeled in the dorsal root ganglia. Presumably, such an injection would result in labeling of both myocardial and pericardial afferent axons. Thus, leakage of H R P from the injection site, particularly into the pericardial or intrathoracic spaces, could give rise to retrograde labeling in the
221
ganglia as it is possible that cardiac contraction could express H R P out of a needle hole and thus result in some spurious labeling. Injections of H R P into the skin were made into regions considered to be comparable to skin regions to which cardiac pain is referred in humans. Following injections into the skin of the left elbow, left thorax or left mandibular angle, only ipsilateral dorsal root ganglia contained labeled neurons, none being found in the nodose ganglia. There was a striking difference between the numbers of cells labeled in dorsal root ganglia after skin injections as compared to heart injections despite the fact that the range of amounts of neuroanatomicai tracer injected and injection sizes were similar. This suggests that the afferent innervation of the skin is denser than that of the heart. On average, perikarya labeled after heart injections were smaller and had a more restricted size range than after skin injections (Table II). Other investigations have reported that cardiac afferent perikarya are smaller than unlabeled or randomly selected dorsal root ganglion perikarya labeled after injections of H R P into the stellate ganglion or heart [23,28]. There are different types of cardiac receptors which have afferent axons with differing conduction velocities [1,2] and fiber diameter difference could conceivably affect transport of HRP. However, since skin injections resuited in labeling of perikarya of all sizes and heart injections resulted in labeling of smaller cells on average, as has been reported in other studies [28], it seems unlikely that there was differential uptake and transport of H R P by cardiac afferent neurons. The present results demonstrate that the origin of the afferent irmervation of any specific region of the canine heart is widespread with respect to the distribution within ganglia. Secondly, the segmental distribution of cardiac afferent neurons in the dorsal root ganglia is more widespread than is commonly thought which is in agreement with recent reports [14,23,28] and is more widespread than has been reported for the origins of efferent sympathetic preganglionic axons which traverse the stellate ganglion [16,24] or which innervate the heart [22]. Thirdly, the largest numbers of cardiac afferent neurons were located in the nodose
ganglia. The presence of cardiac afferent neurons in the nodose ganglia may account in part for the fact that cardiac pain may be referred to the neck or ear. It has been reported that stimulation of the stellate or second thoracic ganglia in man produces pain in the upper left thoracic region which radiates down the left arm, whereas stimulation of the left thoracic vagosympathetic complex produces no pain [29]. In addition, it has been reported that sectioning the left vagosympathetic complex does not alter the symptoms of angina pectoris [29]. White [29] concluded that anginal pain referred to the region of the angle of the jaw was not related to afferent axons in the vagosympathetic complex but rather to afferent axons in the spinal nerves. With reference to possible mechanisms of referred pain, it has been demonstrated that the perikaryon of one afferent neuron in the dorsal root ganglion can innervate the heart and an ulnar nerve [18]. As the greatest numbers of afferent neurons which innervate the heart are located in the nodose ganglia and afferent neurons which innervate the heart are relatively equally distributed bilaterally, whether comparing nodose or dorsal root ganglia, it is difficult to account for the distributions of thoracic pain related to heart disease on the basis of the locations of cardiac afferent neurons alone [10].
Acknowledgements The authors thank K.M. McCleave and R. Livingston for technical assistance. This work was supported by the Nova Scotia Heart Foundation and the Medical Research Council of Canada (Grant MA 10122).
References 1 Armour, J.A., Physiological behavior of thoracic cardiovascular receptors, Am. J. Physiol., 225 (1973) 177-185. 2 Armour, J.A., Thoracic and cardiovascular afferent nerves. In W.C. Randall (Ed.), Neural Regulation of the Heart, Oxford University Press, New York, 1977, pp. 13-42. 3 Armour, J.A. and Hopkins, D.A., Localization of sympathetic postganglionic neurons of physiologically identified cardiac nerves in the dog, J. Comp. Neurol., 202 (1981) 169-184.
222 4 Armour, J.A. and Hopkins, D.A., Anatomy of the extrinsic efferent autonomic nerves and ganglia innervating the mammalian heart. In W.C. Randall (Ed.), Neural Regulation of the Heart, Oxford University Press, New York, 1984, pp. 20-45. 5 Armour, J.A. and Randall, W.C., Functional anatomy of canine cardiac nerves, Acta. Anat. (Basel), 91 (1975) 510-528. 6 Brandys, J.C., Randall, W.C. and Armour, J.A., Functional anatomy of the canine mediastinal cardiac nerves located at the base of the heart, Can. J. Physiol. Pharmacol., 64 (1986) 152-162. 7 Casati, R., Lombardi, F. and Malliani, A., Afferent sympathetic unmyelinated fibers with left ventricular endings in cats, J. Physiol. (Lond.), 292 (1979) 135-148. 8 Coleridge, J.C.G., Hemingway, A., Holmes, R.L. and Linden, R.J., The location of atrial receptors in the dog: a physiological and histological study, J. Physiol. (Lond.), 136 (1957) 174-197. 9 Foreman, R.D., Viscerosomatic convergence onto spinal neurons responding to afferent fibers located in the inferior cardiac nerve, Brain Res., 137 (1977) 164-168. 10 Foreman, R.D., Blair, R.W. and Ammons, W.S., Neural mechanisms of cardiac pain. In F. Cervero and J.F.B. Morrison (Eds.), Visceral Sensation, Progress in Brain Res., Vol. 67, Elsevier Science Publishers, Amsterdam, 1986, pp. 227-243. 11 Hageman, G.R., Urthaler, F. and James, T.N., Neural pathways of a cardiogenic hypertensive reflex, Am. J. Physiol., 235 (1978) H345-H349. 12 Hopkins, D.A. and Armour, J.A., Medullary cells of origin of physiologically identified cardiac nerves in the dog, Brain Res. Bull., 8 (1982) 359-365. 13 Hopkins, D.A. and Armour, J.A., Localization of sympathetic postganghonic and parasympathetic preganglionic neurons which innervate different regions of the dog heart, J. Comp. Neurol., 229 (1984) 186-198. 14 Kalia, M. and Mesulam, M.-M., Brainstem projections of sensory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches, J. Comp. Neurol., 193 (1980) 467-508. 15 Kuo, D.C., Oravitz, J.J. and De Groat, W.C., Tracing of afferent and efferent pathways in the left inferior cardiac nerve of the cat using retrograde and transganglionic transport of horseradish peroxidase, Brain Res., 321 (1984) 111-118. 16 Lichtman, J.W., Purves, D. and Yip, J.W., Innervation of sympathetic neurons in the guinea-pig thoracic chain, J. Physiol. (Lond.), 298 (1980) 285-299.
17 Malliani, A., Cardiovascular sympathetic afferent fibers, Reo. Physiol. Biochem. Pharmacol., 94 (1982)11-74. 18 McNeil, D.L. and Burden, H.W., Convergence of sensory processes from the heart and the left ulnar nerve onto a single afferent perikaryon: a neuroanatomical study in the rat employing fluorescent tracers, Anat. Rec., 214 (1986) 441-444. 19 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 20 Mitchell, G.A.G., Cardiovascular lnneroation, Livingstone, Edinburgh, London, 1956. 21 Nettleship, W.A., Experimental studies on the afferent innervation of the cat's heart, J. Comp. Neurol., 64 (1936) 115-133. 22 Norris, J.E., Foreman, R.D. and Wurster, R.D., Responses of the canine heart to stimulations of the first five ventral thoracic roots, Am. J. Physiol., 227 (1974) 9-12. 23 Oldfield, J. and McLachlan, E.M., Localization of sensory neurons traversing the stellate ganglion, J. Comp. Neurol., 182 (1978) 915-922. 24 Oldfield, J. and McLachian, E.M., An analysis of the sympathetic preganghonic neurons projecting from the upper thoracic spinal roots of the cat, J. Comp. Neurol., 196 (1981) 329-345. 25 Peters, S.R., Kostreva, D.R., Armour, J.A., Zuperku, E.J., Igler, F.O., Coon, R.L. and Kampine, J.P., Cardiac, aortic, pericardial and pulmonary vascular receptors in the dog, Cardiology, 65 (1980) 85-100. 26 Taylor, D.C.M. and Pierau, F.-K., Double fluorescence labelhng supports etectrophysiological evidence for dichotomizing peripheral sensory nerve fibers in rats, Neurosci. Lett., 33 (1982) 1-6. 27 Ueda, H., Uchida, Y. and Kamisaka, K., Distribution and responses of the cardiac sympathetic receptors to mechanically induced circulatory changes, Jpn. Heart J., 10 (1969) 70-81. 28 Vance, W.H. and Bowker, R.C., Spinal origins of cardiac afferents from the region of the left anterior descending artery, Brain Res., 258 (1983) 96-100. 29 White, J.C., Cardiac pain: anatomic pathways and physiologic mechanisms, Circulation, 16 (1957) 644-655. 30 Whitteridge, D., Afferent nerve fibres from the heart and lungs in the cervical vagus, J. Physiol. (Lond.), 107 (1948) 496-512.
213
Elsevier JAN 00923
Ganglionic distribution of afferent neurons innervating the canine heart and cardiopulmonary nerves D a v i d A. H o p k i n s 1 a n d J. A n d r e w A r m o u r 2 Department of 1 Anatom)~ and 2 Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, NS (Canada) (Received 7 July 1988) (Revised version received 23 January 1989) (Accepted 26 January 1989)
Key words: Sensory; Dorsal root ganglion; Nodose ganglion; Horseradish peroxidase Abstract The ganglionic distribution of the perikarya of afferent axons in cardiopulmonary nerves or the heart was studied in 64 dogs by injecting horseradish peroxidase into physiologically identified cardiopulmonary nerves or different regions of the heart. In 6 additional dogs, horseradish peroxidase was injected into the aortic arch, pericardial sac, left ventricular cavity or the skin. After injections into cardiopulmonary nerves, retrogradely labeled perikarya were found in the ipsilateral nodose ganglion and the ipsilateral CT-T 7 dorsal root ganglia. After injections into different regions of the heart, retrogradely labeled neurons were found in the nodose ganglia bilaterally and in the C r - T 6 dorsal root ganglia bilaterally. Many more retrogradely labeled neurons were found in the nodose ganglia in comparison to the dorsal root ganglia. The largest numbers of retrogradely labeled perikarya in the dorsal root ganglia occurred in the "1"2_4 ganglia following nerve or heart injections. Following injections into specific regions of the heart or individual physiologically identified cardiopulmonary nerves, regional distributions of labeled neurons could not be identified within or among ganglia with respect to the structures injected. Perikarya in dorsal root ganglia which were labeled after heart injections ranged in area from 436-3280 /~m2 (X = 1279 + 51 S.E.M.) while after skin injections labeled perikarya ranged in area from 224-5701 /Lm2 (X = 1631 + 104 S.E.M.). The results show that the afferent innervation of the canine heart is provided by neurons located throughout the nodose ganglia and to a lesser degree in the C r - T 6 dorsal root ganglia bilaterally. The bilateral distribution of cardiac afferent neurons raises questions regarding mechanisms underlying unilateral symptoms frequently associated with heart disease.
Introduction Myelinated and unmyelinated afferent axons which arise from mechanoreceptors or chemoreceptors located in the heart, vena cavae, pulmonary arteries and aorta course centrally via cardiopulmonary nerves [1] to the vagosym-
Correspondence: D.A. Hopkins, Department of Anatomy, Faculty of Medicine, Dalhonsie University, Halifax, Nova Scotia, B3H 4H7, Canada.
pathetic complexes [8,11,30] or subclavian ansae and thoracic rami [7,9,17,25,27] to cell bodies in the nodose [14,21] or dorsal root ganglia [21,23]. The cell bodies of cardiac afferent dorsal root ganglion neurons have generally been reported to be located in dorsal root ganglia from the first to the 5th thoracic segments [20,23,29]. More recently, studies of the distribution of sensory perikarya innervating specific cardiopulmonary nerves or cardiac structures have been undertaken. It has been reported that afferent axons in the cat inferior cardiac nerve arise from cell bodies in the
0165-1838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
214
T1-7 [23] or the C s - T 9 [15] dorsal root ganglia and those in the region of the cat's left anterior descending coronary artery from cell bodies in the C s - T 6 dorsal root ganglia [28]. However, except for these few studies in which specific structures were investigated, the locations of the afferent cell bodies which project axons in various cardiopulmonary nerves or which innervate different regions of the heart remain unknown. In the present study, the dorsal root and nodose ganglia obtained from dogs in which physiologically identified cardiopulmonary nerves or specific regions of the heart were injected with horseradish peroxidase (HRP) have been studied in order to determine: (1) the ganglionic locations of cardiopulmonary afferent perikarya, (2) the relative contributions of the nodose and dorsal root ganglia to heart innervation and (3) the segmental organization of the afferent neurons which project axons into the cardiopulmonary nerves and the heart. In addition, comparisons were made between cardiac and skin afferents.
Materials and Methods
Experimental material was obtained from 70 mongrel dogs which had been used to study the locations of cardiac sympathetic efferent postganglionic neurons [3,4,13] and cardiac parasympathetic efferent preganglionic neurons [12,13]. Dogs of either sex (12-20 kg in weight) were sedated with ketamine hydrochloride (1 m g / k g i.m.) and anesthetized with sodium thiopental (25 m g / k g i.v.). Positive pressure respiration was initiated using a Bird Mark 7A respirator. A lead II E K G was recorded and a femoral artery was cannulated in order to record blood pressure. A unilateral thoracotomy was performed via the 3rd or 4th intercostal space in order to expose the thoracic autonomic nerves, heart and great thoracic vessels. In 40 dogs in which cardiopulmonary nerves were studied, Walton-Brodie strain gauge arches were sutured to the surfaces of an atrium a n d / o r a ventricle. Subsequently, cardiopulmonary nerves, the functional anatomy of which has been described previously [4,5], were identified, exposed
and stimulated for up to 10 s using a Grass SD9 square wave stimulator. The stimulus parameters (10 Hz, 5 ms, 4 V) were monitored on a Tektronix 510 3N oscilloscope and the cardiovascular responses were monitored on a Beckman Dynograph. When a cardiopulmonary nerve had been identified anatomically and the function of the efferent axons in that nerve demonstrated physiologically, 3-25 #l of aqueous 30% H R P (Boehringer) were injected into the nerve using a 5 0 / t l Hamilton syringe with a 22-gauge needle as described previously [3]. The procedure of identifying an intrathoracic structure as a cardiopulmonary nerve was necessary in order to confirm that such a structure was indeed a cardiopulmonary nerve and not a lymphatic vessel or fibrous tissue. Although this procedure does not identify afferent axons arising from the heart, it does establish that these structures contain cardiac efferent axons and since each efferent nerve also contains afferent axons [1,5], the presence of afferent axons in identified cardiopulmonary nerves is assured. In 24 dogs, 15-250/~l of 30% aqueous H R P or 5% wheat germ a g g l u t i n i n - H R P (Sigma) were injected into various regions of the heart (Fig. 1). In 3 dogs H R P was injected into the inner arch of the
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215 aorta, the pericardial sac or the left ventricular cavity. In another group consisting of 3 dogs which were sedated with ketamine hydrochloride, the skin over the ulnar surface of the left forelimb, the skin adjacent to the left cranial thoracic nipple or the skin over the left mandibular angle were injected with 50-120 /al of HRP. Following the above procedures the thoracic cavity was closed and air was withdrawn for those animals which received thoracotomies, and antibiotics (Penlong 1 ml i.m.) and analgesics (Demerol 5-10 m g / k g i.m.) were administered. On the third postoperative day the animals were re-anesthetized with an overdose of sodium pentobarbital and the chest was rapidly opened. Following occlusion of the descending aorta at the level of the diaphragm, the animals were perfused via the ascending aorta with 2 - 4 litres of physiological saline (0.9%) followed by 6 litres of fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer (pH 7.4). Both nodose ganglia of all dogs and dorsal root ganglia bilaterally in all but 11 of the dogs were collected from the C 2 to the Ts levels and stored in 30% sucrose in phosphate buffer until processed. Serial 40-/~m-thick frozen sections were cut and processed for H R P histochemistry [19] and then counterstained with Neutral red and examined with bright- and dark-field microscopy. The distribution of labeled afferent neurons was assessed by counting the numbers of labeled neurons in each ganglion. In order to estimate the perikaryal dimensions of dorsal root ganglion afferent neurons associated with the heart, the maximum diameter, perimeter and area of perikarya labeled after heart injections were determined from camera lucida drawings with the aid of a Zeiss IBAS image analysis system. Perikaryal dimensions were also assessed for representative cases in which a cardiopulmonary nerve or the skin was injected. For each ganglion containing labeled perikarya at least 10 unlabeled perikarya with a visible nucleolus were also measured. Unlabeled cells were chosen by virtue of their proximity to the labeled cells and by selecting a range of cell sizes in the ganglia. Correction factors were not applied to measurements of cell sizes due to the fact that the relatively sparse
labeling in the ganglia made double counting unlikely. In addition, correction factors depend on being able to see the nucleolus which was often not visible because of the intensity of the labeling (Fig. 2).
Results
Retrogradely labeled neurons were observed in the nodose and dorsal root ganglia after injections of H R P into cardiopulmonary nerves and heart (Table I). In general, nerve injections resulted in larger numbers of labeled perikarya in nodose and dorsal root ganglia than did heart injections. In all of the dogs described in the present analysis, retrogradely labeled neurons have been previously identified in thoracic autonomic ganglia or the medulla oblongata [12,13].
Cardiopulmonary nerve injections Injections into certain nerves resulted in a greater degree of labeling in ganglia than other nerves. Injections into the right recurrent, right caudal vagal, left intermediate medial, and the left recurrent cardiopulmonary nerves resulted in the greatest number of labeled neurons in the nodose ganglia. Injections into the right recurrent and left caudal pole cardiopulmonary nerves resulted in the heaviest labeling of afferent neurons in the dorsal root ganglia. Nodose ganglia. Of the 40 dogs which had cardiopulmonary nerve injections, the ipsilateral nodose ganglia in 22 (55%) contained retrogradely labeled neurons. In this group of animals, all labeling was ipsilateral except for one contralateral nodose ganglion which contained two retrogradely labeled neurons. After nerve injections there was an average of 285 + 93 (range 2-1392) labeled neurons in the ipsilateral nodose ganglia in those animals which had labeled neurons. Following injections into right-sided nerves, there were 249 _ 133 (range 2-1392) labeled neurons in the right nodose ganglia and following left-sided nerve injections 333 + 140 (range 2-1101) in the left nodose area. Dorsal root ganglia. Of the 29 dogs from which dorsal root ganglia were collected following
216 TABLE I
Summary of experiments and percentages of labeled ganglia Injection sites
Left cardiopulmonary nerves Right cardiopulmonary nerves Total of nerves Heart Aorta Pericardial sac Left ventricular cavity Skin
Numbers of dogs studied
Numbers and percentages of dogs with labeled perikarya
N odose
DR G
N odose
DR G
21 19 40 24 1 1 1 3
21 8 29 24 1 1 1 3
11 (52%) * 11 (58%) * * 22 (55%) 22 (92%) 1 1 0 0
11 (52%) * 6 (75%) * 17 (59%) 12 (50%) 1 1 0 3 *
* Labeled neurons located ipsilaterally only. * * Labeled neurons located ipsilaterally with the exception of one case (a right caudal vagal cardiopulmonary nerve injection which had in addition two labeled perikarya in the contralateral nodose ganglion).
cardiopulmonary nerve injections, the ipsilateral dorsal root ganglia in 19 (66%) contained retrogradely labeled neurons. Retrogradely labeled perikarya (Figs. 2, 3) were observed in the ipsilateral dorsal root ganglia from the C T - T 7 levels (Fig. 4). The largest numbers of retrogradely labeled cells were present in the T 2_ 4 dorsal root ganglia after nerve injections (Fig. 4). Following cardiopulmonary nerve injections the maximum number of labeled cells found in one dorsal root ganglion was 260. This was the left T 2 dorsal root ganglion following a left caudal pole nerve injection of 6 ktl of H R P (Fig. 3). After cardiopulmonary nerve injections, an average of 4.2 __+10.4 (S.E.M.) dorsal root ganglia were labeled and these ganglia contained in total an average of 75.2 + 30 labeled neurons. In contrast, after heart injections an average of 2.3 + 0.6 ganglia were found to have a total of 13.5 ___4.4 labeled neurons. Labeled neurons in the dorsal root ganglia had a smaller mean diameter than unlabeled neurons or than neurons labeled after skin injections (Fig. 5; Table II).
Heart injections Nodose ganglia. In 24 dogs in which the heart was injected, in only one did an injection (one of the two atrio-ventricular nodal injections) fail to result in labeling of either the nodose or dorsal root ganglia. In the dogs which received heart injections, 22 (92%) had retrogradely labeled neu-
rons in the nodose ganglia and in all but 3 of these the nodose ganglia were labeled bilaterally. Following heart injections there was an average of 38 + 18 (range 1-411) labeled neurons in the right nodose ganglia and 25 + 10 (range 1-216) labeled neurons in the left nodose ganglia in those animals which had labeled neurons. Following injections of H R P into specific regions of the heart labeled neurons were located throughout the nodose ganglia bilaterally in almost every instance. Because labeled neurons were found to be scattered throughout the ganglia, there was no clear indication of a topographical organization within individual nodose or dorsal root ganglia which was also the case after nerve injections. Dorsal root ganglia. Twelve (50%) of the dogs with heart injections had retrograde labeling in the dorsal root ganglia. The numbers and locations of labeled neurons varied among animals, even when the injection sites in the heart were similar. After heart injections right dorsal root ganglia were labeled from C r - T 5, while on the left labeling extended from T I _ 6. Following cardiac injections the maximum number of labeled neurons found in a dorsal root ganglion was 43 (the left T 4 dorsal root ganglion following injection of 250 ~tl of H R P into the cranial epicardium of the right and left ventricles, dog 69). Overall, after heart injections an average of 2.3 + 0.6 ganglia were found to have a total of 13.5 ___4.4 labeled neurons. With
217
Fig. 2. Photomicrographs of retrogradely labeled perikarya in sensory ganglia. A: left T3 dorsal root ganglion of dog 33 (left caudal pole cardiopulmonary nerve, 10 pl). B: area enclosed by box in A. C: right nodose ganglion of dog 58 (left ventricular apical epicardium, 45 pl). D: right nodose ganglion of dog 36 (right recurrent cardiopulmonary nerve, 15 ~1). Calibration bars: A, D ffi 250 /~m; B = 50 pm; C = 40/~m.
218 DOG 48 T2 DORSAL ROOT GANGLION LATERAL
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Fig. 3. Drawing showingthe distribution of retrogradely labeled perikarya in the left T2 dorsal root ganglion of dog 48 (left caudal pole cardiopulmonary nerve, 6 ~1). The numbers on the left refer to serial section numbers. respect to the size of labeled neurons, those in the dorsal root ganglia had a smaller m e a n diameter t h a n unlabeled n e u r o n s or t h a n n e u r o n s labeled after skin injections (Fig. 5; T a b l e II). • •
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Fig. 4. Histogram showing the average number of labeled neurons per ganglion in the C6-T7 dorsal root ganglia following heart or cardiopulmonary nerve injections. Error bars = S.E.M.
2
0 100
2000 4000 AREA(~tm2)
6000
Fig. 5. Histograms showing the areas of labeled cells in dorsal root ganglia as a percentage of the total cells labeled following injections of the (A) heart and (B) skin of the left forelimb.
Aortic arch, pericardial sac, left ventricular cavity and skin injections Following injection of H R P in the a d v e n t i t i a of the arch of the aorta, labeled n e u r o n s were present only in the left nodose ganglion a n d the left dorsal root ganglia (T3_6). W h e n a n injection was m a d e into the pericardial sac, m a n y lightly labeled perikarya were f o u n d in the n o d o s e ganglia bilaterally a n d a few lightly labeled cells were f o u n d in the C 6 and C v dorsal root ganglia. W h e n H R P was injected i n t o the left ventricular cavity n o labeled n e u r o n s were detected in any of the ganglia investigated. The largest n u m b e r s of labeled n e u r o n s in dorsal root ganglia occurred following injections in the skin. W h e n the skin of the left forelimb or the left chest was injected, labeled n e u r o n s were located in ipsilateral dorsal root ganglia from
219 TABLE II Dimensions [mean + S.E.M. (Range)] of labeled and unlabeled perikarya in dorsal root ganglia Tissue injected
n
Diameter (#m)
Perimeter (l~m)
Area (~m)
Heart
Labeled 139 Unlabeled 151 Labeled 26 Unlabeled 20 Labded 258 Unlabeled 148 Labeled 135 Unlabeled 64
52 ± 1 (28- 87) 67 ± 1 (30-118) 56 + 3 (34- 86) 70 ± 5 (41-116) 49 ± 1 (22-100) 64 4- 2 (29-123) 57 + 2 (23-123) 67 + 3 (30-116)
135 ± 177 ± 147 ± 186 ± 128 + 171 + 147 ± 175 ±
127942350 ± 1550 ± 2615 ± 1176 ± 2248 ± 1631 ± 2320 ±
Aorta Nerve Skin
3 (83-226) 4 (81-302) 7 (93-226) 12 (109-288) 2 (57-250) 4 (77-286) 5 (57-286) 7 (81-309)
51 102 143 304 38 107 104 108
(436-3280) (404-6482) (642-3471) (824-5234) (225-4218) (413-5770) (224-5 701) (457-7054)
* Mean + S.E.M. (range).
the C8-T 6 levels depending on the segmental level of the injection. After injections into the skin (1) over the left ulnar tubercle there were 1662 labeled neurons in the left T 1 dorsal root ganglion, (2) adjacent to the left nipple there were 1933 labeled neurons in the left T 4 dorsal root ganglion and (3) over the left mandibular angle there were 4211 labeled neurons in the left C 3 dorsal root ganglion. Following injection into the skin over the left elbow, labeled neurons were located in the ipsilateral "1"2_4 dorsal root ganglia, following injection into the skin of the left nipple they were located in the C 8 - T 7 ganglia and following injection into the skin over the left mandibular angle they were located in the C 2_ 4 dorsal root ganglia.
Discussion The results of the present investigation in which H R P was injected into physiologically identified cardiopulmonary nerves or specific regions of the heart show that the perikarya of afferent neurons which innervate canine cardiopulmonary nerves or the myocardium are located in the nodose ganglia and the C6-T 7 dorsal root ganglia. More neurons were labeled in the nodose ganglia in comparison to the dorsal root ganglia when a cardiopulmonary nerve was injected. In all of the cases studied efferent preganglionic parasympathetic or postganglionic sympathetic neurons were also labeled [3,12,13] whether or not labeling occurred in afferent neurons. Thus, when no labeled neurons were
found in the afferent ganglia this could not simply be ascribed to a failure of the label to be picked up by the structures injected. The possibility remains that the extent of the dorsal root ganglion innervation was underestimated if dorsal root ganglion afferent axons do not take up or transport H R P as well as other axons. Afferent axons in cadiopulmonary nerves are connected primarily with perikarya in ipsilateral ganglia, whereas afferent axons from the heart arise from perikarya in the nodose and dorsal root ganglia bilaterally. In contrast, afferent neurons with axons in the arch of the aorta of one animal arose from perikarya in the left nodose and left dorsal root ganglia. Following cardiopulmonary nerve injections labeled neurons were located in the ipsilateral C 7 - T 7 dorsal root ganglia while following injections into the heart labeled neurons were located in the dorsal root ganglia from the C6-T 5 levels, the greatest numbers of labeled afferent neurons occurring in the T2_4 dorsal root ganglia (Fig. 4). More cells were labeled in dorsal root ganglia following nerve injections than heart injections. The difference in the degree of labeling in the two groups of experiments may be due in part to the fact that cardiopulmonary nerves contain afferent axons arising from receptors in noncardiac tissue, such as pulmonary tissue, in addition to the cardiac afferent axons which arise from receptors in different regions of the heart [1]. Dorsal root ganglion perikarya were labeled following injection into each major cardiopulmonary nerve. Injections into the right recurrent, left inter-
220
mediate medial and left caudal pole cardiopulmonary nerves resulted in the greatest number of labeled neurons in both the nodose and dorsal root ganglia. In keeping with this, physiological evidence has demonstrated that these are major afferent cardiopulmonary nerves [1,2]. Interganglionic cardiopulmonary nerve injections resulted in labeling of neurons in the ipsilateral dorsal root ganglia but not in the nodose ganglia. This nerve contains efferent axons arising from sympathetic but not parasympathetic neurons [3,12] and thus the axons in this nerve are primarily related to the spinal cord and not the brainstem. This is also in agreement with physiological evidence which shows that this nerve contains cardiac afferent axons [1] and cardiac sympathetic efferent axons [5]. Similarly, the left caudal pole cardiopulmonary nerve contained relatively large numbers of afferent axons related to the dorsal root ganglia in comparison to the nodose ganglia. This latter nerve is known from anatomical [3] and physiological [5] studies to be primarily a sympathetic efferent cardiac nerve which contains few, if any, parasympathetic efferent axons [12]. Therefore, there appears to be a relative dissociation of both afferent and efferent axons in these nerves with respect to the brainstem and spinal cord. This dissociation is however not absolute since a few labeled cells were found in the ipsilateral nodose ganglia following injections of H R P into the left caudal pole nerve, a nerve which has no efferent parasympathetic axons [12]. An injection in one epicardial area would potentially label m a n y afferent axons in that area as well as axons projecting to other areas. Mechanoreceptors located in the endocardium usually have relatively small receptor fields and those in the epicardium or myocardium may have relatively large receptor fields [1]. It is known that one afferent axon may arise from mechanoreceptors located in two relatively widely separated regions of the heart [1] or even different thoracic organs [2]. Also, perikarya of an afferent neuron may project axons into two or more different nerves [26]. In addition, axons passing through injection sites to other cardiac regions could also take up HRP. It appears that afferent cell bodies with
axons in a specific cardiac region or a specific cardiopulmonary nerve are not restricted to a specific ganglion or ganglionic region. For example, injections of H R P into the left ventricular apex or the left ventricular ventral papillary muscle would presumably label only the distal cardiac axons as few axons in these regions project to other regions of the heart. In these cases the distribution of afferent neuron labeling did not differ from that observed after injections in more cranial cardiac regions such as following injections into the base of the ventricles or in the atria. Thus, a topographical anatomical arrangement with respect to the distribution of afferent cardiac neurons could not be identified. Similarly, efferent postganglionic sympathetic neurons which innervate the heart [13] or cardiopulmonary nerves [3] also do not have a topographical anatomical arrangement. When injections of H R P were placed adjacent to the major coronary vessel it was likely that axons which accompanied that vessel were able to pick up the label. Axons which accompany a coronary vessel innervate distal regions of the heart perfused by that vessel, regions which can be relatively extensive [6]. Thus, labeling of neurons following such an injection would reflect the distribution of nerves over a relatively large region of the heart and not necessarily the innervation of the coronary arteries alone. The present study and a previous one [28] indicate that the nerves which accompany major coronary vessels contain sympathetic postganglionic axons [13] which innervate the distal myocardium [6] and afferent axons which arise primarily from the ganglia bilaterally. When H R P was injected into the left ventricular cavity no labeled neurons were identified. However, when H R P was injected into the pericardial cavity afferent neurons were lightly labeled. In this control experiment m a n y cells were labeled in the nodose ganglia bilaterally while only a few cells were labeled in the dorsal root ganglia. Presumably, such an injection would result in labeling of both myocardial and pericardial afferent axons. Thus, leakage of H R P from the injection site, particularly into the pericardial or intrathoracic spaces, could give rise to retrograde labeling in the
221
ganglia as it is possible that cardiac contraction could express H R P out of a needle hole and thus result in some spurious labeling. Injections of H R P into the skin were made into regions considered to be comparable to skin regions to which cardiac pain is referred in humans. Following injections into the skin of the left elbow, left thorax or left mandibular angle, only ipsilateral dorsal root ganglia contained labeled neurons, none being found in the nodose ganglia. There was a striking difference between the numbers of cells labeled in dorsal root ganglia after skin injections as compared to heart injections despite the fact that the range of amounts of neuroanatomicai tracer injected and injection sizes were similar. This suggests that the afferent innervation of the skin is denser than that of the heart. On average, perikarya labeled after heart injections were smaller and had a more restricted size range than after skin injections (Table II). Other investigations have reported that cardiac afferent perikarya are smaller than unlabeled or randomly selected dorsal root ganglion perikarya labeled after injections of H R P into the stellate ganglion or heart [23,28]. There are different types of cardiac receptors which have afferent axons with differing conduction velocities [1,2] and fiber diameter difference could conceivably affect transport of HRP. However, since skin injections resuited in labeling of perikarya of all sizes and heart injections resulted in labeling of smaller cells on average, as has been reported in other studies [28], it seems unlikely that there was differential uptake and transport of H R P by cardiac afferent neurons. The present results demonstrate that the origin of the afferent irmervation of any specific region of the canine heart is widespread with respect to the distribution within ganglia. Secondly, the segmental distribution of cardiac afferent neurons in the dorsal root ganglia is more widespread than is commonly thought which is in agreement with recent reports [14,23,28] and is more widespread than has been reported for the origins of efferent sympathetic preganglionic axons which traverse the stellate ganglion [16,24] or which innervate the heart [22]. Thirdly, the largest numbers of cardiac afferent neurons were located in the nodose
ganglia. The presence of cardiac afferent neurons in the nodose ganglia may account in part for the fact that cardiac pain may be referred to the neck or ear. It has been reported that stimulation of the stellate or second thoracic ganglia in man produces pain in the upper left thoracic region which radiates down the left arm, whereas stimulation of the left thoracic vagosympathetic complex produces no pain [29]. In addition, it has been reported that sectioning the left vagosympathetic complex does not alter the symptoms of angina pectoris [29]. White [29] concluded that anginal pain referred to the region of the angle of the jaw was not related to afferent axons in the vagosympathetic complex but rather to afferent axons in the spinal nerves. With reference to possible mechanisms of referred pain, it has been demonstrated that the perikaryon of one afferent neuron in the dorsal root ganglion can innervate the heart and an ulnar nerve [18]. As the greatest numbers of afferent neurons which innervate the heart are located in the nodose ganglia and afferent neurons which innervate the heart are relatively equally distributed bilaterally, whether comparing nodose or dorsal root ganglia, it is difficult to account for the distributions of thoracic pain related to heart disease on the basis of the locations of cardiac afferent neurons alone [10].
Acknowledgements The authors thank K.M. McCleave and R. Livingston for technical assistance. This work was supported by the Nova Scotia Heart Foundation and the Medical Research Council of Canada (Grant MA 10122).
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