An anomalous vagorenal reflex pathway in the cat

EXPERIMENTAL

NEUROLOGY

An Anomalous F. R. CALARESU,

87,278-290

(1985)

Vagorenal

Reflex Pathway in the Cat

M. M. KNUEPFER,

J. CIRIELLO,

AND A. STELLA’

Department of Physiology, University of Western Ontario. London, Ontario N6A Kl,

Canada

Received June 27. 1984; revision received August 21. 1984 Although physiological investigations support the view that the innervation to the kidney is primarily sympathetic in origin, there is anatomic evidence suggesting direct vagal projections to the kidney. We examined electrophysiologically the possibility that neural connections exist between the cervical vagus and renal nerves. Electrical stimulation of the peripheral segment of the cut cervical vagus evoked electrical activity in the central segment of cut renal nerve of chloralose-anesthetized, paralyzed cats. The evoked potentials (vagorenal responses) displayed components with peak latencies of about 50, 120, and 500 ms. Another peak at about 175 ms was also seen in some cases. In addition, a period of postexcitatory depression occurred between approximately 180 and 400 ms after delivery of the stimulus. Evoked responses were recorded in the contralateral as well as the ipsilateral renal nerves. In contrast, stimulation of the central cut end of renal nerves did not elicit responses in the cervical vagus. Vagorenal responses were not altered by cutting the subdiaphragmatic vagus indicating that the abdominal vagus was not involved in this response. Electrical activity in renal nerves elicited by vagal stimulation could be eliminated by either ganglionic blockade or by cutting or cooling the splanchnic nerves. Finally, supraspinal ischemia abolished the vagorenal response. These data suggest that a vagorenal reflex pathway exists and that the potentials recorded in renal nerves are due to activation of aberrant sensory fibers traveling from the peripheral segment of the cut cervical vagus to the central nervous system, where they excite a sympathetic efferent pathway to the kidney. 0 1985 Academic PI=, IX.

INTRODUCTION It is well known that the majority of efferent fibers to the kidney originate in the sympathetic nervous system (3, 8, 22, 34). In addition, a direct vagal ’ This work was supported by the Medical Research Council of Canada and the Ontario Heart Foundation. Dr. Ciriello is a Canadian Heart Foundation Scholar. Dr. Knuepfer was supported by a postdoctoral fellowship from the U.S. National Institutes of Health (HL0633601); his present address is Dept. of Biomedical Engineering, Johns Hopkins Univ., 720 Rutland Ave., Baltimore, MD 21205. The critical comments by Dr. L. P. Schramm in the preparation of the manuscript are gratefully acknowledged. Dr. Stella was on leave from Clinica Medica IV, Universita di Milano, Italy. Please address correspondence to Dr. Calaresu. 278 0014-4886/85 $3.00 Copynghl 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved

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contribution to the renal nerve plexus or to the kidney has been described anatomically by many authors (8, 19, 22, 24, 34). Those studies were based primarily on the observation that vagal fibers appeared to make connections with renal nerves. In contrast, physiological experiments have produced equivocal results as they demonstrated that stimulation of the efferent vagus in the dog produced either no effect on renal blood flow (6, 18) or a decrease in renal blood flow (36). We tested the hypothesis that there are connections between the vagus and renal nerves in cats. The first series of experiments identified and characterized electrophysiologically the responses in renal nerves during stimulation of the peripheral segment of the cut cervical vagus; these experiments have been presented in a preliminary report (35). In the second series attempts were made to trace electrophysiologically the connection between the cervical vagus and renal nerves by examining the possible involvement of the subdiaphragmatic vagus, the splanchnic nerves, and the central nervous system in renal nerve responses to vagal stimulation. METHODS Nineteen cats (1.8 to 4.2 kg) were anesthetized with alpha-chloralose (60 mg/kg i.v. initially, supplemented by additional doses of 30 mg/kg at 6- to 8-h intervals) after induction with ethyl chloride and ether. The femoral artery and vein were cannulated for monitoring arterial pressure and drug administration, respectively. The arterial pressure was recorded on a Grass 7 polygraph and the pressure pulse was used to trigger a cardiotachometer for determination of heart rate. After insertion of a tracheal cannula and all surgical procedures, the animals were paralyzed with decamethonium bromide (Sigma, St. Louis, MO., 0.5 mg/kg, i.v. initially and additional doses when necessary) and artificially ventilated. The rectal temperature was maintained at 37 f 0.5”C by a heating pad connected to a Yellow Springs 73 temperature regulator. Both cervical vagi and nodose ganglia were dissected free of adjacent tissue and the vagi were cut just distal to the nodose ganglia. The peripheral segment of each cervical vagus was placed on bipolar stainless-steel stimulating electrodes in a pool of warm Dow Corning 360 medical fluid (Dow Corning, Midland, Mich.). A schematic diagram of the experimental preparation is shown in Fig. 1. The left kidney was exposed through a lateral incision in the abdominal wall. Branches of the renal nerve were dissected free from connective and fat tissues and, when possible, from small blood vessels, and were then placed on bipolar stainless-steel recording electrodes and covered with warm medical fluid. Electrical activity was recorded using a Grass P 15 preamplifier with a bandpass from l-10 Hz to 300-1000 Hz connected to a Tektronix

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FIG. I. Schematic diagram of experimental preparation and of sites of recording (voltmeter) and stimulation (coil) of neural pathways. A cooling loop is shown around the splanchnic nerves.

R5103N storage oscilloscope, from which Polaroid photographs could be obtained. Evoked potentials were averaged using a Neurolog 750 Signal Averager (Medical Systems, Great Neck, N.Y.), displayed on the oscilloscope and photographed. In some experiments responses were averaged and stored using a Neurograph STA-1 microprocessor (Medical Systems). In addition, some responses were examined using a Neurolog 200 spike discriminator and peristimulus time histograms were generated on the Neurograph STA1 microprocessor. All values of latencies of responses were expressed as mean + SE. To eliminate changes in cardiovascular variables during stimulation of the cervical vagi, atropine methylbromide (K & K Laboratories, Plainview, N.Y.; 2 mg/kg, i.v.) was administered, after determining the threshold currents to induce bradycardia (10-s train of rectangular pulses of 0.5 ms at 25 Hz). Ganglionic transmission was blocked by hexamethonium bromide (Sigma; 10 mg/kg, i.v.).

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Experiments designed to examine the route of conduction of evoked potentials required similar procedures with some modifications. First, atropine was not administered in order to verify adequate stimulation of the vagus throughout the experiment because these preparations were usually maintained for longer periods. Second, the greater and lesser splanchnic nerves were isolated and prepared for electrical stimulation according to a procedure described elsewhere (7). Third, the dorsal branch of the subdiaphragmatic vagus was dissected and prepared for either recording or stimulation. In some experiments, the effects of reversible blockade of the splanchnic nerves were examined by cooling the nerves. This was done using a hook of 15-gauge stainless-steel tubing entirely insulated with epoxy resin except on the concave portion of the hook where contact with the nerve was made. A precooled solution of 20% ethanol in water was pumped through the tubing to cool the nerve. The solution was maintained at 0 to 4°C before it entered the tubing and was measured at 5 to 8°C at the outflow. To investigate the role of supraspinal structures on the responses evoked in renal nerves by vagal stimulation, the effect of severe ischemia of the brain on the evoked responses was examined in three experiments. Central ischemia was induced by ligating the carotid arteries and occluding the vertebral arteries for 10 to 30 min. Cerebral ischemia was assumed to have occurred if the mean arterial pressure decreased to 60 to 70 mm Hg. At completion of experiments, the animals were perfused intracardially with 0.9% physiologic saline followed by 10% Formalin. In these fixed animals the renal and splanchnic nerves, celiac plexus, and the vagi were carefully dissected and examined to verify their anatomic identity. RESULTS Characterization of Evoked Potentials in Renal Nerves. In the initial experiments, single-pulse electrical stimulation (duration 0.5 to 2 ms) of the peripheral segment of the contralateral cervical vagus at three times the threshold current for bradycardia consistently elicited potentials in the renal nerves. To obtain a complete description of all potentials evoked in renal nerves it was necessary to record from more than one of the renal nerve branches. Therefore the components of the responses to vagal stimulation are a combination of the responses observed in more than one fiber bundle. In later experiments to examine the route of conduction, the most responsive renal nerve bundle was used to compare evoked potentials before and after an intervention. This procedure precluded obtaining a complete description of all peaks for each animal because this could usually be observed only by recording from more than one branch, as described above.

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Examples of responses evoked in renal nerves are shown in Fig. 2A, B, and C. During stimulation of the contralateral vagus three different components were observed: an early response with a mean peak latency of 52 + 5 ms (N = 8) a response with a mean peak latency of 116 + 10 ms (IV = 8), and a late response with a mean peak latency of 503 f 12 ms (N = 8). Electrical stimulation of the ipsilateral cervical vagus elicited similar responses with mean peak latencies of 54 f 5, 130 f 5, and 494 + 15 ms, respectively. An additional component with a mean peak latency of 174 f 5 ms for ipsilateral stimulation and 175 k 3 ms for contralateral stimulation was evoked in four of eight animals. No differences were observed between potentials evoked by single-pulse stimulation and those evoked by trains of five pulses at 200 Hz. Finally, a decrease in spontaneous nerve activity was observed in all animals after the activity evoked by vagal stimulation. This “silent period” lasted from 176 f 10 to 386 + 20 ms A

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FIG. 2. Electrical responses in renal nerves evoked by stimulation of peripheral segment of cut contralateral cervical vagus. Arrows indicate time of stimulation. A and B-top trace is 5 superimposed sweeps of renal nerve activity after vagal stimulation; bottom trace is average of 64 sweeps of activity shown in top trace. C-average of 64 sweeps of renal nerve activity after vagal stimulation showing a late component with a peak latency of approximately SO0 ms as well as a component at approximately 130 ms. D-record obtained from same nerve bundle shown in B after administration of hexamethonium (10 mg/kg, i.v.); top trace 5 superimposed sweeps, bottom trace average of 64 sweeps.

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from the time of delivery of the stimulus to the vagus nerve. The silent period was defined as a decrease in discriminated multiunit activity greater than 50% of the prestimulus spontaneous activity rate using a peristimulus time histogram (cf. Fig. 4A, C). The silent period was also readily apparent as a decrease in variability of the average response (Fig. 3A). After determining the responses to vagal stimulation, hexamethonium was administered to block ganglionic transmission. Ganglionic blockade prevented all responses in renal nerves to stimulation of the distal cut cervical vagus nerves. An example of the effects of hexamethonium is shown in Fig. 2D. Identijcation of a Vagorenal Reflex Pathway. Experiments were done to identify the pathway of the responses to vagal stimulation recorded in renal nerves. Because these animals did not receive atropine a slight decrease in heart rate (17 f 4 beats per minute) occurred during stimulation of the cervical vagus at 0.5 or 0.33 Hz. There was no significant change in mean arterial pressure during stimulation. Attempts were made in two cats to elicit an evoked potential in the distal segment of the cut cervical vagus after stimulation of the renal nerves. No responses were observed in either the ipsilateral or contralateral cervical vagus.

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3. A-example of an averaged renal nerve response to 50 stimuli at 0.33 Hz delivered to the contralateral cervical vagus. B-responses to the same stimulus (50 sweeps) after cutting the greater splanchnic nerve. FIG.

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FIG. 4. Peristimulus time histograms of discriminated multifiber activity in a renal nerve bundle in response to stimulation of the ipsilateral vagus (50 stimuli at 0.33 Hz). A-control response; B-response after cooling the greater and lesser splanchnic nerves; C-response to the same stimulus 60 min after rewarming the splanchnic nerves; D-response obtained after supraspinal &hernia.

Role of Dorsal Subdiaphragmatic Vagus. Single-pulse stimulation of the dorsal subdiaphragmatic vagus in seven animals did not elicit responses in renal nerves. Conversely, in three animals stimulation of the renal nerves did not evoke a compound action potential in the dorsal subdiaphragmatic vagus. Finally, in four animals after cutting the dorsal subdiaphragmatic vagus, stimulation of the distal segment of the cut cervical vagus still elicited responses in renal nerves.

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Role of the Splanchnic Nerves in Vagorenal Responses.In four animals the dependence of vagorenal responses on the splanchnic nerves was examined by cutting or cooling the greater and lesser splanchnic nerves. The evoked potentials elicited by stimulation of the cervical vagus were attenuated by cutting the greater splanchnic nerve. Responses could be eliminated by cooling both the greater and lesser splanchnic nerves (Fig. 4B), and they could be elicited again within 30 to 60 min after removing the cold block (Fig. 4C). These findings clearly demonstrated the dependence of the vagorenal responses on the splanchnic nerves. Role of the Central Nervous System in Vagorenal Responses.Ischemia of the supraspinal central nervous system was used in three cats to determine whether or not supraspinal structures were necessary for vagorenal responses. The vagorenal responses were abolished after central ischemia (Fig. 4D). Anatomical Dissection. The left renal, left splanchnic, and vagus nerves were dissected to determine the connections between these nerves. In all cases, one or more branches of the dorsal subdiaphragmatic vagus projected to the dorsal aspect of the stomach and to the celiac plexus composed of the celiac and superior mesenteric ganglia. The left greater and lesser splanchnic nerves were also seen projecting to the celiac plexus entering the ganglia very close to one another and near the entry of the vagal branches. Finally the left renal nerves were seen to make connections with the caudal portion of the superior mesenteric ganglion. Similar connections have been described by others (16, 23). DISCUSSION The anatomic route taken by vagal fibers to the abdomen is through the dorsal and ventral subdiaphragmatic vagi. The ventral branch (also referred to as the anterior gastric plexus) innervates the pyloric region of the stomach and the dorsal branch (posterior gastric plexus) projects to the lesser curvature of the stomach and to the celiac ganglia (23, 29, 31). Though many authors have described direct connections between the abdominal vagus and the renal nerve plexus in humans (10, 19, 22, 24), in cats (8, 3 l), and in dogs (21, 34), Christensen and co-workers (8) and Shvalev (34) differed from the others because they concluded that vagal fibers did not enter the kidney. An examination of the possible contribution of the parasympathetic division of the autonomic nervous system to the innervation of the kidney has also failed to provide histologic evidence for vagal projections to the kidney (28, 37). Despite these few exceptions, anatomical texts unequivocably state that direct projections from the vagus to the kidney exist based on anatomic evidence obtained by dissection of abdominal

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nerves. As mentioned by von Niederhlusem (37), the belief that the sympathetic system must be juxtaposed with the parasympathetic system in each organ has probably led to the presumption that the vagus innervates the kidney. Our observations demonstrated electrophysiologically that there are no direct vagal projections to the kidney. Instead, we have demonstrated the existence of a reflex pathway between the cut peripheral segment of the cervical vagus and renal nerves. This demonstration is based on the following findings. First, the dorsal subdiaphragmatic vagus does not appear to be a part of the reflex pathway because cutting this nerve does not affect the vagorenal reflex. Second, evoked potentials in the renal nerve were not observed after stimulation of the dorsal subdiaphragmatic vagus. Third, the reflex was shown to be dependent on ganglionic transmission and on conduction through the splanchnic nerves, indicating a sympathetic efferent limb of the pathway. Finally, it was shown that the reflex is dependent on supraspinal structures but is not dependent on activation of vagal fibers projecting to the medulla. Although it may be argued that the disappearance of the vagorenal reflex after cerebral &hernia may have been related to &hernia of the spinal cord secondary to the hypotension (mean arterial pressure of 60 to 70 mm Hg) present in our animals, this is an unlikely explanation, as it has been shown elsewhere that blood flow in the spinal cord remains constant for a range of mean arterial pressures of 50 to 135 mm Hg ( 14). We therefore interpret our results to indicate that the vagorenal reflex depends on the integrity of supraspinal structures. These data are consistent with previous observations showing that electrical stimulation of the abdominal or thoracic vagus had no effect on renal blood flow in dogs (6, 18). Our data also support the observation that electrical stimulation of renal nerves did not elicit evoked activity in the cervical vagus in two cats tested (5). It could, however, be suggested that the very small number of cholinergic fibers described by Khamitov and Shvalev (13) in the renal nerves of the dog, if they were vagal in origin, might be difficult to identify in whole-nerve preparations. It is more likely that those previously described choline& fibers are efferent sympathetic preganglionic fibers described by other authors in the renal nerves (2, 4, 16, 22, 34). The presence of an afferent pathway through the celiac plexus to the abdominal vagus was recently demonstrated to exist in the rat by recording evoked potentials in the celiac branch of the abdominal vagus after stimulation of the hepatic branch of the splanchnic nerve, suggesting that some fibers within the celiac branch of the dorsal subdiaphragmatic vagus may carry afferent fibers from abdominal organs (27). Because we were unable to obtain an evoked response in the abdominal or cervical vagus

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during stimulation of the renal nerves, it is not likely that afferent renal projections in the cat follow a similar route as afferent hepatic fibers in the rat. After eliminating the dorsal subdiaphragmatic vagus from consideration as the pathway mediating renal nerve responses to vagal stimulation, the nature of the potentials elicited in renal nerves was examined in detail. The conduction latencies of primary peaks at 130 ms and 450 to 500 ms coincide with evoked responses in renal nerves after stimulation of a variety of other nerves including the saphenous, intercostal, radial, inferior cardiac, and renal nerves (7, 9, 33, 38). Similar renal nerve responses evoked by ipsilateral or contralateral renal nerve stimulation (7) were interpreted to be due to excitation of sensory A fibers (early component) and of C fibers (late component). In fact, the potentials described in the present study are characteristic of somatosympathetic and viscerosympathetic reflexes ( 15) although the latency of the evoked responses is about 30% longer than that observed in most other reflex responses. As suggested elsewhere (33), the latency of sympathetic reflex responses is dependent on the proximity of afferent and efferent nerves to the central nervous system. Since the fibers mediating the vagorenal reflex probably join the sympathetic nerves and project to the spinal cord, the latency of the A fiber component would be expected to be somewhat longer than that observed after stimulation of shorter reflex pathways such as the cardiorenal reflex response seen approximately 95 ms after stimulation of the stellate ganglion (38). A postexcitatory depression of sympathetic nerve activity was also observed. This silent period has been described by many investigators as a typical response to reflex activation of sympathetic pathways [see (15)]. The evidence obtained in our study suggests strongly the existence of a vagorenal reflex arc as depicted in Fig. 5 with characteristics similar to other somatosympathetic and viscerosympathetic reflexes. An early response (approximately 50 ms) in the renal nerve was observed after vagal stimulation. The latency of this response was slightly longer than that of spinal reflexes identified by other investigators (9, 32). Because the other responses were also delayed about 30% compared with those described by other investigators, we suggest that a longer afferent limb may also exist for this early response. As this response was eliminated by ganglionic blockade, we conclude that it was mediated through the preganglionic sympathetic nerves to the kidney. Because this early response was not examined in detail after supraspinal ischemia it is not known whether or not this is a spinal reflex, although its latency supports this possibility. The afferent limb of the vagorenal reflex described in these experiments is relayed through an anomalous route (see Fig. 5) i.e., a pathway in the

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FIG. 5. A schematicrepresentationof the proposedafferent(on the left) and efferent(on the right) limbs of a vagorenal reflex. Dashed lines are suggestedpathwaysof the reflex. The number of synapsesin the pathwaysis unknown. cervical vagus not projecting directly to the medulla. The vagus nerve has been shown to contain a few cell bodies together with a large number of parasympathetic motor and sensory fibers from thoracic and abdominal viscera ( 1, 11, 20, 29). Neural connections between the nodose and the superior cervical ganglion and between the thoracic vagus and sympathetic nerves have long been known to exist (12, 30). Using more recent histochemical techniques, sympathetic fibers have been verified to exist in the cervical vagus originating from the superior cervical ganglia (25, 26) and in the abdominal vagus originating from the stellate ganglia (17). As nerves designated as parasympathetic and sympathetic have been shown to contain both afferent and efferent fibers, it is likely that the atferent limb of the vagorenal reflex passes through cervical or thoracic sympathetic nerves into the spinal cord. The function of the afferent fibers in the vagus examined in this study is, as yet, unknown. ‘The data suggest that these fibers originate in thoracic or cervical regions since stimulation of the abdominal vagus does not elicit vagorenal reflexes. It is possible that these projections represent an alternative pathway for cardiopulmonary afferent fibers. Although further experiments will be necessary to elucidate the physiologic role of vagorenal reflexes, it is interesting to note that previous experimental results showing that stimulation of the distal cut end of the cervical vagus elicited renal vasoconstriction

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(36) can be accounted for by the existence of the vagorenal reflex pathways demonstrated in our experiments. REFERENCES I. AGOSTONI, E., J. E. CHINNOCK, M. DEBURGH DALY, AND J. G. MURRAY. 1957. Functional and histological studies of the vagus nerve and its branches to the heart, lungs and abdominal viscera in the cat. J. Physiol. (London) 135: 182-205. 2. BARAJAS,L., AND P. WANG. 1975. Demonstration of acetylcholinesterase in the adrenergic nerves of the renal glomerular arterioles. J. Uhrustruc. Rex 53: 244-253. 3. BARAJAS,L., AND P. WANG. 1978. Myelinated nerves of the rat kidney: a light and electron microscopic autoradiographic study. J. Ulfrasfruc. Rex 65: 148-162. 4. BARAJAS, L., P. WANG, AND S. DE SANTIS. 1976. Light and electron microscopic localization of acetylcholinesterase activity in the rat renal nerves. Am J. Annf. 147: 219-234. 5. BEACHAM, W. S., AND D. L. KUNZE. 1969. Renal receptors evoking a spinal vasomotor reflex. J. Physiol. (London) 201: 73-85. 6. BRADFORD, J. R. 1889. The innervation of the renal blood vessels.J. Physiol. (London) lo: 358-407.

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35. STELLA, A., J. CIRIELLO, AND F. R. CALARESU. 1982. Electrical activity in renal nerves evoked by stimulation of cervical vagus in the cat. Sot. Neurosci. Abstr. 8: 722. 36. TAKEUCHI, J., E. UCHIDA, S. NAKAYMA, T. TAKEDA, S. YAGI, G. INOUE, ANDH. UEDA. 1962. Experimental studies on the nervous control of the renal circulation-effect of the electrical stimulation of vagal and other somatic nerves and of the carotid sinus reflex on the renal circulation (III). Jpn. Heart J. 3: 259-268. 37. VON NIEDERH~~USERN,W. 1953. La question du parasympathique &al: recherches sur la limite inf&ieure du domaine du nerf vague. J. Ural. (Paris) 59: 565-577. 38. WEAVER, L. C. 1977. Cardiopulmonary sympathetic afferent influences on renal nerve activity. Am. J. Physiol. 233: H592-H599.