International Journal of Impotence Research (1997) 9, 179±185 ß 1997 Stockton Press All rights reserved 0955-9930/97 $12.00 Evidence of sympathetic ®bers in the male rat pelvic nerve by Gross anatomy, retrograde labeling and high resolution Autoradiographic study

F Giuliano1, P Facchinetti2, J BernabeÂ3, G Benoit1, A Calas2 and O Rampin3

1Service d'Urologie, HoÃpital de BiceÃtre, 78 du GeÂneÂral Leclerc, 94270 Le Kremlin BiceÃtre, France and 2Institut des Neurosciences, URA CNRS 1488, 7 quai Saint-Bernard, Universite Paris VI, 75252 Paris Cedex, France and 3Laboratoire de Neurobiologie des Fonctions VeÂgeÂtatives, I.N.R.A., 78352 Jouy-en-Josas Cedex, France

Several arguments exist in various animal species and man for the presence of a sympathetic component in the pelvic nerve, classically regarded as parasympathetic. We tested this hypothesis in the male rat. Nerve bundles issued from the sacral region of the paravertebral sympathetic chain and reaching the S1 spinal nerve were identi®ed. Neurons in the sacral parasympathetic nucleus of the L6-S1 spinal cord and in the L2-S1 paravertebral sympathetic chain were retrogradely labeled from the pelvic nerve. Radioautography evidenced labeling of unmyelinated ®bers in the pelvic nerve following in vitro incubation with 3H-noradrenaline. A population of sympathetic ®bers issued from the lumbosacral sympathetic chain exists in the pelvic nerve of the male rat. This qualitative study provides a morphological basis to uncover the role of the sympathetic out¯ow present in the pelvic nerve.

Keywords: sympathetic ®bers; pelvic nerve; paravertebral sympathetic chain; lumbosacral spinal cord; urogenital tract

Introduction surgery of the rectum or the bladder. The anatomical arrangement of the neural supply of the pelvic organs and distal colon seems to be generally The distal colon and the lower urinary and genital applicable to all mammalian species investigated tracts receive neural supply from both sympathetic thus far, including man.6 In the male rat, pelvic and parasympathetic divisions of the autonomic neuroanatomy has been previously described nervous system. In the rat parasympathetic pregan- and appears simpler when compared to larger glionic neurons going to the pelvis are located in the species.13±16 In this species the pelvic and hypogas- parasympathetic nucleus of the sacral spinal cord tric nerves consist of ®ne bundles which are easily and their axons run in the pelvic nerve.1;2 Sympa- identi®able and both project onto the pelvic plexus, thetic preganglionic neurons to the pelvic organs which ®gures as one large ganglion: the major pelvic project from the T13-L2 spinal cord to the inferior ganglion. Accordingly the rat appears as a suitable mesenteric ganglion and further travel distally in the model for anatomical and physiological studies on hypogastric nerves. Other sympathetic ®bers travel pelvic neuroanatomy.17±19 The aim of the present caudally in the paravertebral sympathetic chain and study was to demonstrate the mixed composition of could reach the pelvic organs either through the the pelvic nerve, both sympathetic and parasympa- 3±6 pudendal or pelvic nerves. In humans small thetic, and to specify the origin of sympathetic ®bers trunks issued from the sacral sympathetic chain in the pelvic nerve of the male rat. joining the pelvic nerves have been evidenced.7,8 Human pelvic nerves are very thin and ensheathed within a dense connective tissue. Their dissection is rather dif®cult and requires removal of the con- Materials and methods nective tissue.9,10 In human pelvic nerves, histo- chemical evidence of both cholinergic and Seventeen adult male Sprague Dawley rats weighing adrenergic neural ®bers has been provided.11,12 300±500 g were used in this study. Rats were The composition of the pelvic nerves is of clinical anesthetized with ketamine (150 mg/kg i.p., Imal- importance as the nerves may be lesioned during geÁne 500, RhoÃne-MeÂrieux, Lyon, France) and dis- sections were performed with the aid of a Zeiss Correspondence: Dr F Giuliano. dissecting microscope equiped with a Zeiss camera. Received 5 February 1997; accepted 12 July 1997 Topography of the pelvic nerve and its spinal origin Evidence of sympathetic ®bers in the male rat pelvic nerve F Giuliano et al 180 and dissection of the lumbosacral sympathetic chain acetate and lead citrate. A thin layer of carbon was (LSC) were studied in 10 rats via a medial vaporized on the slides which were dipped into suprapubic incision and a transperitoneal approach. Ilford L4 nuclear track emulsion diluted 1:4 in Retrograde labeling studies were performed using water. After 1 month exposure, the autoradiographs horseradish peroxidase in 2 rats (30% in sterile were developed in Microdol Kodak. CelloõÈdin water, HRP type VI, Sigma, Saint-Quentin-Fallavier, membranes were detached on water and mounted France) and Fast Blue in 3 rats (2% in sterile water, on 22-P-mesh grids. After thinning of the celloidin FB, Sigma). The left pelvic nerve was dissected free ®lm during 2.5 min in isoamylacetate, the autora- and cut 3 mm proximal to the major pelvic ganglion. diographs were examined with a Philips EM 300 Its central cut end was immersed in one or the other electron microscope. neuroanatomical tracer for 30 min, then the pelvic cavity was washed with saline. Abdominal muscles and skin were stitched in separate layers. In 2 rats Results the pelvic nerve was immersed in HRP after a ligation of the nerve was performed centrally to the application site. Three days following HRP injection Gross anatomy and 10 days after FB injection the rats were deeply anesthetized and intracardially perfused with 250 ml of a 0.9% saline solution followed by 1 l of The pelvic nerve originated from two joined bundles ®xative solution containing 1% paraformaldehyde, arising at the fusion of the L6-S1 spinal nerves 1.25% glutaraldehyde in 0.1 M sodium phosphate proximal to the sacral plexus. The pelvic nerve buffer (pH 7.4) at room temperature. After perfusion travelled with a branch of the inferior vesical artery the L1-S4 LSC and the L6-S1 spinal cord were and ran dorsally to the internal iliac vein. The major dissected and immersed in a solution of 30% pelvic ganglion was closely apposed by the pelvic sucrose in phosphate buffer (pH 7.4, 4C) for 12± fascia on the lateral side of the dorsal prostatic lobe 16 h. Twenty micrometers thick longitudinal sec- anterior to the lateral wall of the rectum (Figure 1) tions of LSC and transverse sections and was naturally masked to the ventral approach of the spinal cord were cut on the cryostat and by the distal vas deferens. The ganglion had an mounted on gelatin-coated slides. HRP activity was estimated surface of 3 mm2. revealed with tetramethylbenzidine as the chromo- The LSC laid on the anterior surface of the gen.20 Following the reaction with hydrogen per- vertebral column, embedded in the psoas muscle, oxide, the slices were washed in acetate buffer in the retroperitoneal space dorsal to the abdominal (10 mM, pH 4.8), counterstained with neutral red, aorta and to the caudal vena cava. The LSC dehydrated and mounted with permount. Sections consisted in two parallel trunks, sometimes fu- were examined with a light (HRP) or ¯uorescence sioned, along which several buldges occurred. Thin (FB) Leitz microscope equiped with an A1 ®lter rami connected the trunks to the vertebrae. Further (Ploempack system). The long axis of densely experiments con®rmed that buldges contained neu- labeled neurons with clearly de®ned nuclei was ronal cell bodies and therefore represented LSC measured. Labeled neurons were counted and correction for double counting was made according to Abercrombie.21 In one rat a 3 mm segment of the pelvic nerve was removed before its entry into the major pelvic ganglion. The fragment was immedi- ately incubated in 1075 M 3H-noradrenaline (Amer- sham, Les Ulis, France) for 20 min at 37C under constant stirring and oxygenation by air bubbling. The piece was then rinsed in saline for 5 min, ®xed with 4% glutaraldehyde in cacodylate buffer (0.1 M pH 7.4) for 1 h at 4C and immersed in 2% osmium tetroxide in the same buffer, dehydrated in ethanol and embedded in Epon resin. Semi-thin sections (2 mm) were radioautographied with Ilford KI nucle- ar emulsion diluted 1:1 in water, exposed 15 days and developed in Microdol Kodak. After staining with toluidine blue the sections were mounted with permount and observed with light microscopy. Ultrathin transversal sections (50 nm) were cut from this block and mounted on celloidin Figure 1 Schematic representation of a lateral view of the major coated glass slides, then contrasted with uranyl pelvic ganglion of the male rat. Evidence of sympathetic ®bers in the male rat pelvic nerve F Giuliano et al 181 ganglia. LSC gave off lumbar splanchnic nerves was 19 Æ 2 mm. The L2-S1 LSC ganglia contained which joined the intermesenteric nerve lying on the 182 Æ 21 neurons retrogradely labeled with FB ventral surface of the aorta. Interindividual variation (Figures 4 and 5a) and 106 Æ 7 neurons retrogradely was present in the number and location of fusions labeled with HRP (Figure 5b). The majority of between the two sympathetic trunks between L3 and labeled neurons was present in the L3-L5 LSC L6 LSC ganglia. Caudally to the aortic bifurcation, ganglia (Figure 6). Retrogradely labeled neurons the LSC was deeply located and twisted around the were oval-shaped, had a mean diameter of middle caudal artery, rendering dif®cult the identi- 12 Æ 2 mm and were homogeneously distributed in ®cation of sacral sympathetic ganglia in gross a ganglion. Following ligation of the pelvic nerve dissection. In 3 rats the LSC was carefully separated central to the site where HRP was applied respec- from the middle caudal artery. Two of these rats tively 12 and 15 neurons were labeled in the L2-S1 displayed a ®ne nerve bundle passing through psoas LSC. Semi-thin transversal sections of the pelvic major and iliacus muscles. This bundle was given nerve evidenced presence of several fascicles, some off by the cephalad part of the sacral segment of the of which displayed silver grain accumulations. The sympathetic chain and joined the S1 spinal nerve majority of axons was unmyelinated, contained (Figure 2). mitochondria, neurotubules and pro®les of endo- FB applied to the proximal cut end of the left plasmic reticulum, and was ensheathed with col- pelvic nerve retrogradely labeled 124 Æ 5 neurons in lagen ®bers. These axons appeared to be in close the L6 segment and 85 Æ 28 neurons in the S1 contact with myelinated ones. Clear vesicles were segment of the spinal cord homolaterally to the sparse and no varicosities or terminals were identi- injected nerve. In the transverse plane they formed a ®ed on examined grids. Positive radioautographic dense collection of closely packed cells in the lateral reactions were noticed upon unmyelinated ®bers, part of lamina VII (Figure 3). Labeled neurons were closely apposed with unmarked ones (Figure 7a triangular or oval-shaped and their mean diameter and b).

Figure 2 Gross anatomical preparation of sacral sympathetic trunks as viewed from an anterior approach in the male rat. The rectum has been removed. In A: photograph of dissected sacral sympathetic chain and ®rst sacral spinal nerve is displayed. B: Diagrammatic representation of nerves identi®ed on photograph in A. Scale bar: 1 mm. Evidence of sympathetic ®bers in the male rat pelvic nerve F Giuliano et al 182

Figure 4 Segmental distribution of left sympathetic trunk postganglionic neurons projecting in the left pelvic nerve. These neurons were retrogradely labeled with Fast Blue. Data are expressed as mean Æ sem (n ˆ 3).

sacral spinal nerves.7,8 In the cat, parasympathetic neurons projecting to the pelvic nerves are located in the S1-S3 spinal cord.24 Thus, it is likely that L6- S1 levels of the rat spinal cord correspond to the S2- S4 spinal levels in humans and to the S1-S3 levels in the cat. Individual variations in the arrangement of paravertebral sympathetic ganglia were found at the lumbar level in the rat. Fusion of both lumbar paravertebral sympathetic trunks often occurred but was not constant and locations of cross-connections differed, con®rming a previous description of anatomical organization of the paravertebral sympa- thetic chain of the rat.25 The sacral part of the Figure 3 (a) Photomicrograph of a transverse section of the sympathetic chain was dif®cult to individualize due spinal cord at the level of S1 (20 mm thick, magni®cation: 6160). to its close apposition to the median caudal artery. Neurons were retrogradely labeled with Fast Blue. Cell bodies are located in the sacral parasympathetic nucleus corresponding to Small nerve ®laments joining the sacral sympathetic the intermediolateral part of the gray matter (IML). Calibration: chain and the S1 spinal nerve may represent a 50 mm. (b) At a higher magni®cation ( 6680) labeled neurons contribution of the LSC either to the pelvic or to the appeared triangular or oval-shaped. Calibration: 10 mm. pudendal nerve. The latter takes its origin in the sacral plexus formed by the L6-S1 spinal nerve trunk and the lumbosacral trunk composed of the 26 Discussion L3-L5 spinal nerves. In cats and dogs connections between LSC and the pelvic nerve have previously been evidenced.27,28 In humans nerve bundles The present study provides evidence that the pelvic mainly issued from the fourth sacral sympathetic nerve of the rat contains sympathetic postganglionic ganglion and joining the pelvic plexus have also and parasympathetic preganglionic ®bers. We found been described.8,9 In human embryos the pelvic that the pelvic nerve was issued from L6 and S1 plexus receives neural ®bers from the sacral sympa- spinal nerves, in agreement with previous studies thetic trunk and its ganglia by rami connecting the conducted in male and female rats.2,19,22,23 Labeled sympathetic chain and the sacral nerves.29 Thus in neurons present in the intermediolateral column of various species including humans anatomical path- the L6-S1 spinal cord con®rm that the cell bodies of ways joining the sacral sympathetic chain and the parasympathetic neurons are located in the sacral pelvic nerves or the pelvic plexus exist. parasympathetic nucleus.1,2 In humans, parasympa- LSC neurons retrogradely labeled from the pelvic thetic ®bers in the pelvic nerve arise in the S2-S4 nerve con®rm these anatomical ®ndings. The differ- Evidence of sympathetic ®bers in the male rat pelvic nerve F Giuliano et al 183

Figure 6 Diagrammatic representation of the segmental distribu- tion of postganglionic neurons in the lumbosacral sympathetic ganglia which were labeled with horseradish peroxidase applied to the left pelvic nerve in two rats (A and B).

Figure 5 (a) Photomicrograph of neurons in the sympathetic ganglion L5 which were labeled with Fast Blue applied to the pelvic nerve. Calibration: 10 mm. (b) Photomicrograph of neurons in the sympathetic ganglion L5 which were labeled with horse- radish peroxidase (arrows) applied to the pelvic nerve. Calibra- tion: 10 mm. ence in the nature of the retrograde tracers used, HRP and FB respectively, may explain the differ- ences between the number of labeled neurons in the LSC in each experience. Postganglionic sympathetic cell bodies which axons travel in the pelvic nerve are widely distributed in the L2-S1 sympathetic ganglia and are mainly located in the L3-L5 ganglia. In the female rat the sympathetic innervation of the bladder is conveyed in equal parts by the hypogas- tric and pelvic nerves.30 Postganglionic neurons destined to the bladder are numerous in the L6 LSC ganglion and sparse in the L1-L5 ganglia. In the male rat, retrograde tracing from the cavernous nerve labeled about 100 neurons in the paravertebral sympathetic chain mainly at the L5-L6 levels.31 In the present study LSC neurons which send axons in the pelvic nerve are more numerous and more Figure 7 (a) Radioautography of a cross section of the pelvic nerve in an adult male rat after the nerve was incubated in vitro in widely distributed. It may be partly explained by 3 the fact that the pelvic nerve contains neurons H-noradrenaline. Magni®cation: 69000; calibration: 1 mm. (b) Detail from a: positive radioautographic reaction upon one destined to different pelvic viscera. Bilateral loca- fascicle of unmyelinated axons. Magni®cation: 630000; calibra- tion of retrogradely labeled neurons occurred in the tion 300 nm. Evidence of sympathetic ®bers in the male rat pelvic nerve F Giuliano et al 184 LSC. It is therefore possible that postganglionic the rat the pelvic ganglion receives sympathetic axons in the LSC cross over to the opposite side. In inputs from both the hypogastric nerve and the our study the number of retrogradely labeled paravertebral sympathetic chain. Fibers issued from neurons in the sacral parasympathetic nucleus and the latter run in the pelvic nerve. in the LSC was comparable. Extended lesion of the LSC or ventral rhizotomy (T12-S2) decreased by 25% the number of ®bers in the rat pelvic nerve31. Acknowledgements To our knowledge there is no clear demonstration of a loss of neural ®bers in the pelvic nerve following lesion of the lumbosacral spinal cord in the rat. Pr Jacques Taxi and Mrs Sylvette Gougis are grate- However retrograde labelling from the pelvic fully acknowledged for their help in the present nerve in this species suggests that the lumbosacral experiment. spinal cord contribute a contingent of ®bers in the pelvic nerve. It is also unclear whether preganglio- nic parasympathetic neurons may give off several References axons. This fact would lead to an overestimation of the contribution of sacral preganglionic neurons 1 Hancock MB, Peveto CA. Preganglionic neurons in the sacral following lesion of the lumbosacral spinal cord. The spinal cord of the rat: an HRP study. Neurosci Lett 1979; 11: 1±5. cat's pelvic nerve contains twice as many postgan- 2 Nadelhaft I, Booth AM. The location and morphology of glionic sympathetic ®bers as preganglionic para- preganglionic neurons and the distribution of visceral affer- sympathetic ®bers.24 In the rhesus monkey 40% of ents from the rat pelvic nerve: a horseradish peroxidase study, J Comp Neurol 1984; 226: 238±245. the pelvic nerve's efferent ®bers originate in the LSC 3 Hancock MB, Peveto CA. A preganglionic autonomic nucleus 32 ganglia. 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