Download Evidence of sympathetic ®bers in the male rat pelvic nerve

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
1
Service 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
The distal colon and the lower urinary and genital
tracts receive neural supply from both sympathetic
and parasympathetic divisions of the autonomic
nervous system. In the rat parasympathetic preganglionic neurons going to the pelvis are located in the
parasympathetic nucleus of the sacral spinal cord
and their axons run in the pelvic nerve.1;2 Sympathetic preganglionic neurons to the pelvic organs
project from the T13-L2 spinal cord to the inferior
mesenteric ganglion and further travel distally in the
hypogastric nerves. Other sympathetic ®bers travel
caudally in the paravertebral sympathetic chain and
could reach the pelvic organs either through the
pudendal or pelvic nerves.3±6 In humans small
trunks issued from the sacral sympathetic chain
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 connective tissue.9,10 In human pelvic nerves, histochemical evidence of both cholinergic and
adrenergic neural ®bers has been provided.11,12
The composition of the pelvic nerves is of clinical
importance as the nerves may be lesioned during
Correspondence: Dr F Giuliano.
Received 5 February 1997; accepted 12 July 1997
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
applicable to all mammalian species investigated
thus far, including man.6 In the male rat, pelvic
neuroanatomy has been previously described
and appears simpler when compared to larger
species.13±16 In this species the pelvic and hypogastric nerves consist of ®ne bundles which are easily
identi®able and both project onto the pelvic plexus,
which ®gures as one large ganglion: the major pelvic
ganglion. Accordingly the rat appears as a suitable
model for anatomical and physiological studies on
pelvic neuroanatomy.17±19 The aim of the present
study was to demonstrate the mixed composition of
the pelvic nerve, both sympathetic and parasympathetic, and to specify the origin of sympathetic ®bers
in the pelvic nerve of the male rat.
Materials and methods
Seventeen adult male Sprague Dawley rats weighing
300±500 g were used in this study. Rats were
anesthetized with ketamine (150 mg/kg i.p., ImalgeÁne 500, RhoÃne-MeÂrieux, Lyon, France) and dissections were performed with the aid of a Zeiss
dissecting microscope equiped with a Zeiss camera.
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
(LSC) were studied in 10 rats via a medial
suprapubic incision and a transperitoneal approach.
Retrograde labeling studies were performed using
horseradish peroxidase in 2 rats (30% in sterile
water, HRP type VI, Sigma, Saint-Quentin-Fallavier,
France) and Fast Blue in 3 rats (2% in sterile water,
FB, Sigma). The left pelvic nerve was dissected free
and cut 3 mm proximal to the major pelvic ganglion.
Its central cut end was immersed in one or the other
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
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
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
®xative solution containing 1% paraformaldehyde,
1.25% glutaraldehyde in 0.1 M sodium phosphate
buffer (pH 7.4) at room temperature. After perfusion
the L1-S4 LSC and the L6-S1 spinal cord were
dissected and immersed in a solution of 30%
sucrose in phosphate buffer (pH 7.4, 4 C) for 12±
16 h. Twenty micrometers thick longitudinal sections of LSC and transverse sections
of the spinal cord were cut on the cryostat and
mounted on gelatin-coated slides. HRP activity was
revealed with tetramethylbenzidine as the chromogen.20 Following the reaction with hydrogen peroxide, the slices were washed in acetate buffer
(10 mM, pH 4.8), counterstained with neutral red,
dehydrated and mounted with permount. Sections
were examined with a light (HRP) or ¯uorescence
(FB) Leitz microscope equiped with an A1 ®lter
(Ploempack system). The long axis of densely
labeled neurons with clearly de®ned nuclei was
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 immediately incubated in 1075 M 3H-noradrenaline (Amersham, Les Ulis, France) for 20 min at 37 C 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 4 C 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 nuclear 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
coated glass slides, then contrasted with uranyl
acetate and lead citrate. A thin layer of carbon was
vaporized on the slides which were dipped into
Ilford L4 nuclear track emulsion diluted 1:4 in
water. After 1 month exposure, the autoradiographs
were developed in Microdol Kodak. CelloõÈdin
membranes were detached on water and mounted
on 22-P-mesh grids. After thinning of the celloidin
®lm during 2.5 min in isoamylacetate, the autoradiographs were examined with a Philips EM 300
electron microscope.
Results
Gross anatomy
The pelvic nerve originated from two joined bundles
arising at the fusion of the L6-S1 spinal nerves
proximal to the sacral plexus. The pelvic nerve
travelled with a branch of the inferior vesical artery
and ran dorsally to the internal iliac vein. The major
pelvic ganglion was closely apposed by the pelvic
fascia on the lateral side of the dorsal prostatic lobe
anterior to the lateral wall of the rectum (Figure 1)
and was naturally masked to the ventral approach
by the distal vas deferens. The ganglion had an
estimated surface of 3 mm2.
The LSC laid on the anterior surface of the
vertebral column, embedded in the psoas muscle,
in the retroperitoneal space dorsal to the abdominal
aorta and to the caudal vena cava. The LSC
consisted in two parallel trunks, sometimes fusioned, along which several buldges occurred. Thin
rami connected the trunks to the vertebrae. Further
experiments con®rmed that buldges contained neuronal cell bodies and therefore represented LSC
Figure 1 Schematic representation of a lateral view of the major
pelvic ganglion of the male rat.
Evidence of sympathetic ®bers in the male rat pelvic nerve
F Giuliano et al
ganglia. LSC gave off lumbar splanchnic nerves
which joined the intermesenteric nerve lying on the
ventral surface of the aorta. Interindividual variation
was present in the number and location of fusions
between the two sympathetic trunks between L3 and
L6 LSC ganglia. Caudally to the aortic bifurcation,
the LSC was deeply located and twisted around the
middle caudal artery, rendering dif®cult the identi®cation of sacral sympathetic ganglia in gross
dissection. In 3 rats the LSC was carefully separated
from the middle caudal artery. Two of these rats
displayed a ®ne nerve bundle passing through psoas
major and iliacus muscles. This bundle was given
off by the cephalad part of the sacral segment of the
sympathetic chain and joined the S1 spinal nerve
(Figure 2).
FB applied to the proximal cut end of the left
pelvic nerve retrogradely labeled 124 5 neurons in
the L6 segment and 85 28 neurons in the S1
segment of the spinal cord homolaterally to the
injected nerve. In the transverse plane they formed a
dense collection of closely packed cells in the lateral
part of lamina VII (Figure 3). Labeled neurons were
triangular or oval-shaped and their mean diameter
was 19 2 mm. The L2-S1 LSC ganglia contained
182 21 neurons retrogradely labeled with FB
(Figures 4 and 5a) and 106 7 neurons retrogradely
labeled with HRP (Figure 5b). The majority of
labeled neurons was present in the L3-L5 LSC
ganglia (Figure 6). Retrogradely labeled neurons
were oval-shaped, had a mean diameter of
12 2 mm and were homogeneously distributed in
a ganglion. Following ligation of the pelvic nerve
central to the site where HRP was applied respectively 12 and 15 neurons were labeled in the L2-S1
LSC. Semi-thin transversal sections of the pelvic
nerve evidenced presence of several fascicles, some
of which displayed silver grain accumulations. The
majority of axons was unmyelinated, contained
mitochondria, neurotubules and pro®les of endoplasmic reticulum, and was ensheathed with collagen ®bers. These axons appeared to be in close
contact with myelinated ones. Clear vesicles were
sparse and no varicosities or terminals were identi®ed on examined grids. Positive radioautographic
reactions were noticed upon unmyelinated ®bers,
closely apposed with unmarked ones (Figure 7a
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.
181
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 sem (n ˆ 3).
Figure 3 (a) Photomicrograph of a transverse section of the
spinal cord at the level of S1 (20 mm thick, magni®cation: 6160).
Neurons were retrogradely labeled with Fast Blue. Cell bodies are
located in the sacral parasympathetic nucleus corresponding to
the intermediolateral part of the gray matter (IML). Calibration:
50 mm. (b) At a higher magni®cation ( 6680) labeled neurons
appeared triangular or oval-shaped. Calibration: 10 mm.
Discussion
The present study provides evidence that the pelvic
nerve of the rat contains sympathetic postganglionic
and parasympathetic preganglionic ®bers. We found
that the pelvic nerve was issued from L6 and S1
spinal nerves, in agreement with previous studies
conducted in male and female rats.2,19,22,23 Labeled
neurons present in the intermediolateral column of
the L6-S1 spinal cord con®rm that the cell bodies of
parasympathetic neurons are located in the sacral
parasympathetic nucleus.1,2 In humans, parasympathetic ®bers in the pelvic nerve arise in the S2-S4
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 L6S1 levels of the rat spinal cord correspond to the S2S4 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 sympathetic chain of the rat.25 The sacral part of the
sympathetic chain was dif®cult to individualize due
to its close apposition to the median caudal artery.
Small nerve ®laments joining the sacral sympathetic
chain and the S1 spinal nerve may represent a
contribution of the LSC either to the pelvic or to the
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
L3-L5 spinal nerves.26 In cats and dogs connections
between LSC and the pelvic nerve have previously
been evidenced.27,28 In humans nerve bundles
mainly issued from the fourth sacral sympathetic
ganglion and joining the pelvic plexus have also
been described.8,9 In human embryos the pelvic
plexus receives neural ®bers from the sacral sympathetic trunk and its ganglia by rami connecting the
sympathetic chain and the sacral nerves.29 Thus in
various species including humans anatomical pathways joining the sacral sympathetic chain and the
pelvic nerves or the pelvic plexus exist.
LSC neurons retrogradely labeled from the pelvic
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 distribution 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 horseradish peroxidase (arrows) applied to the pelvic nerve. Calibration: 10 mm.
ence in the nature of the retrograde tracers used,
HRP and FB respectively, may explain the differences 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 hypogastric 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
widely distributed. It may be partly explained by
the fact that the pelvic nerve contains neurons
destined to different pelvic viscera. Bilateral location of retrogradely labeled neurons occurred in the
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
3
H-noradrenaline. Magni®cation: 69000; calibration: 1 mm. (b)
Detail from a: positive radioautographic reaction upon one
fascicle of unmyelinated axons. Magni®cation: 630000; calibration 300 nm.
Evidence of sympathetic ®bers in the male rat pelvic nerve
F Giuliano et al
184
LSC. It is therefore possible that postganglionic
axons in the LSC cross over to the opposite side. In
our study the number of retrogradely labeled
neurons in the sacral parasympathetic nucleus and
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 .
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.
However retrograde labelling from the pelvic
nerve in this species suggests that the lumbosacral
spinal cord contribute a contingent of ®bers in the
pelvic nerve. It is also unclear whether preganglionic parasympathetic neurons may give off several
axons. This fact would lead to an overestimation of
the contribution of sacral preganglionic neurons
following lesion of the lumbosacral spinal cord. The
cat's pelvic nerve contains twice as many postganglionic sympathetic ®bers as preganglionic parasympathetic ®bers.24 In the rhesus monkey 40% of
the pelvic nerve's efferent ®bers originate in the LSC
ganglia.32 The ultrastructural study of the rat's
pelvic nerve revealed the presence of several
fascicles with few myelinated and many unmyelinated axons, con®rming a report of 80% unmyelinated axons in the rat pelvic nerve.31 The
noradrenaline uptake ability of some unmyelinated
axons supports the view that the pelvic nerve
contains catecholaminergic ®bers which are very
likely postganglionic sympathetic axons.33 In human pelvic nerves, catecholaminergic ®bers have
been identi®ed using formaldehyde ¯uorescence
technique.11 Since the pelvic plexus provides
innervation to the pelvic organs, neurons arising in
the paravertebral sympathetic trunks and conveyed
by the pelvic nerve may be destined to various
pelvic viscera. The cat urinary bladder receives
sympathetic ®bers issued from the LSC.34 In the
female rat, sympathetic innervation to the bladder
also arises from the pelvic nerves.30 Nevertheless
the physiological role of the sympathetic ®bers in
the pelvic nerves remains unknown. In dogs, cats,
rabbits and rats electrical stimulation of the lower
part of the sympathetic chain elicits subsidence of
erection.34ÿ39 Sympathetic ®bers issued from the
lumbar sympathetic chain may reach their targets
either through the pelvic or the pudendal nerves. In
the rat electrical stimulation of the L4-L5 LSC elicits
a response on the dorsal nerve of the penis.40 There
is no evidence for a role of sympathetic ®bers in the
pelvic nerve in the control of erection. These ®bers
may contribute to the control of micturition or
secretion and motility of sex accessory glands.34
Recent data suggest that sympathetic ®bers in the
pelvic nerve and sympathetic chain provide input
into the testes.41 Furthermore some labelling of
neurons in the lumbosacral sympathetic chain from
the penis has been demonstrated.42 In conclusion in
the rat the pelvic ganglion receives sympathetic
inputs from both the hypogastric nerve and the
paravertebral sympathetic chain. Fibers issued from
the latter run in the pelvic nerve.
Acknowledgements
Pr Jacques Taxi and Mrs Sylvette Gougis are gratefully acknowledged for their help in the present
experiment.
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