Regulatory Peptides 86 (2000) 89–94
www.elsevier.com / locate / regpep
An interaction of opioids and galanin in dorsal horn of the spinal cord in
mononeuropathic rats
a
a,
b
Yan-Ping Zhang , Long-Chuan Yu *, Thomas Lundeberg
a
Department of Physiology, College of Life Science, and National Laboratory of Biomembranes and Membrane Biotechnology, Peking University,
Beijing 100871, People’ s Republic of China
b
Department of Physiology and Pharmacology, and Department of Surgery and Rehabilitation, Karolinska Institutet, 171 77 Stockholm, Sweden
Received 29 May 1999; received in revised form 22 August 1999; accepted 1 September 1999
Abstract
The present study was performed in rats with experimentally induced mononeuropathy after common sciatic nerve ligation. The
hind-paw withdrawal latencies to thermal and mechanical stimulation were increased significantly after intrathecal injection of 3 nmol of
galanin. The increased hind-paw response latencies induced by galanin were attenuated by following intrathecal injection of 22 nmol, but
not 11 or 2.75 nmol of the opioid receptor antagonist naloxone. Further, the increased hind-paw response latencies induced by galanin
were prevented by following intrathecal injection of 10 nmol of mu-opioid receptor antagonist, b-funaltrexamine (b-FNA), but not by 10
nmol of delta-opioid receptor antagonist, natrindole or 10 nmol of kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI).
Intrathecal 10 nmol of b-FNA alone had no significant effects on the hind-paw withdrawal responses. These results demonstrate the
existence of a specific interaction between galanin and opioids in the transmission of presumed nociceptive information in the spinal cord
of mononeuropathic rats. This interaction involves the activation of mu-opioid receptor.  2000 Elsevier Science B.V. All rights
reserved.
Keywords: Intrathecal; Naloxone; b-Funaltrexamine; Natrindole; Nor-Binaltorphimine
1. Introduction
Painful syndromes following injury of the peripheral
nerve are usually characterized by spontaneous burning
pain combined with hyperalgesia and allodynia to thermal
and / or mechanical stimulation [1]. To investigate the
mechanisms of neurogenic pain Bennett and Xie set up an
experimental model of mononeuropathy, induced by four
loose ligatures placed around the common sciatic nerve, in
rats [2]. It has been suggested that this model is more
similar to those seen in patients than complete sciatic nerve
section [2,3].
Studies have demonstrated that the neuropeptide galanin
*Corresponding author. Tel.: 1 86-10-6275-1867; fax: 1 86-10-62751850.
E-mail address: [email protected] (L.-C. Yu)
may be involved in endogenous anti-nociception in the
spinal cord [4,5]. The mRNA of galanin is up-regulated in
dorsal root ganglion (DRG) cells and in the superfacial
laminae of the dorsal horn of the spinal cord following
peripheral nerve injury [6–8]. These regions of the spinal
cord are known to receive the terminations of primary
afferent neurons that respond to noxious stimuli. Also
spinal opioids are known to be associated with endogenous
anti-nociception. In a recent study we have demonstrated
that intrathecal administration of galanin (1 or 3 nmol)
resulted in an anti-nociceptive effect in mononeuropathic
rats [9]. Also the antinociceptive effects of morphine were
antagonized by the galanin antagonist, galantide (M-15)
[10]. These observations suggest that there is a close
interaction between galanin and opioids in the spinal cord
in mononeuropathic rats.
The aim of the present study was to further the
0167-0115 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0167-0115( 99 )00091-9
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Y.-P. Zhang et al. / Regulatory Peptides 86 (2000) 89 – 94
investigation on the interaction of opioids and galanin in
the presumed spinal anti-nociception in mononeuropathic
rats by using non-selective opioid receptor antagonist
naloxone, and the selective mu-opioid receptor antagonist
b-funaltrexamine (b-FNA), the delta-opioid receptor antagonist natrindole, or the kappa-opioid receptor antagonist
nor-binaltorphimine (nor-BNI).
2. Materials and methods
2.1. Animal preparation
Adult male Sprague–Dawley rats (Experimental Animal
Centre of Beijing Medical University, Beijing, China)
weighing 250–350 g were used. The animals were individually housed in cages with free access to water and
food ad libition, and maintained in a room temperature of
24628C with a normal day / night cycle. All rats were
accustomed to the testing conditions for five days before
starting the experiment in order to decrease the stress
induced by handling and measurements. All experiments
were conducted according to the guidelines of the animal
ethical committee of Karolinska Institutet and every effort
was made to minimize animal suffering.
2.2. Surgical procedures and intrathecal catheter
implantation
Rats were anesthetized with intra-peritoneal sodium
pentobarbital (45 mg / kg). In order to create a mononeuropathy model, nerve ligation was performed according to
the method of Bennett and Xie [2]. Briefly, rat’s left sciatic
nerve was exposed for 8–10 mm at the level of the mid
thigh. Four loose ligatures (4.0 chronic gut) were made
around the dissected nerve with a 1.0–1.5 mm interval
between each of them. The ligation was carefully manipulated so that the nerve was barely constricted. The skin
incision was closed with 4-0 silk sutures.
An intrathecal catheter was implanted in each rat
immediately following nerve ligation. A sterilized salineflushed polyethylene (PE-10) tube was inserted into the
subarachnoid space through a slit in the atlanto-occipital
membrane. The caudal end of the catheter was gently
threaded into the lumbar enlargement. The catheter was
fixed and the skin wound was closed with 4-0 silk sutures.
All rats received an intra-muscular injection of potassium
penicillin (30,000 IU / rat) after operation in order to
prevent infection. Approximately 10–20% of the operated
rats exhibited post-surgical motor deficient (e.g. limb
paralysis), and these rats were excluded from the experiment.
2.3. Nociceptive tests
Intrathecal administrations were performed during 5–12
days after left sciatic nerve ligation as during these days
the abnormal presumed ‘‘pain’’ behaviour is at a relatively
stable level [2,3]. Also, 5–12 days after left sciatic nerve
ligation a dramatic increase in galanin mRNA is detected
[11–14]. The hind-paw withdrawal latency (HWL) in
seconds to thermal stimulation and the hind-paw withdrawal threshold (HWT) in grams to mechanical stimulation
were measured. The thermal response was assessed using
the hot-plate test. To ensure that the entire ventral surface
of the hind-paw was in contact with the hot-plate the knee
joint was kept extended manually during the test. The rats
left or right hind-paw was separately placed manually on
the hot-plate which was maintained at a temperature of
528C (51.8–52.48C) [9,15–17]. The response to mechanical stimulus was determined using the Randall Selitto Test
(Ugo Basile, Type 7200, Italy). Mechanical stimulation
was applied to the dorsal surface of the hind-paw by a
wedge-shaped pusher at a loading rate of 30 g / s and the
threshold required to initiate the struggle response was
measured. The values obtained before intrathecal injection
were regarded as the basal HWL or HWT. The HWL and
HWT recorded during subsequent experiments were expressed as percentage change of the mean basal level for
each rat (% change of HWL or HWT). Each rat was tested
with both nociceptive tests.
2.4. Chemicals
Solutions for intrathecal administration were prepared
with sterilized saline, each with a volume of 10 ml: (1) 3
nmol of galanin (Galanin, Bachem, Feinchemikalien AG,
Switzerland); (2) 2.75 (0.5 mg), 11 (4 mg) or 22 (8 mg)
nmol of naloxone (naloxone hydrochloride, Sigma Chemical Company, St. Louis, MO) respectively; (3) 10 nmol of
b-funaltrexamine (b-FNA hydrochloride; Research Biochemicals, MA), natrindole (naltrindole hydrochloride;
Research Biochemicals), or nor-binaltorphimine (nor-BNI
hydrochloride; Research Biochemicals). Control groups
were given 10 ml of 0.9% saline.
2.5. Statistical analysis
Data from nociceptive tests were presented as
mean6SEM. The difference between groups was determined by two-way analysis of variance (ANOVA) for
repeated measures or Student’s t-test where applicable, and
*P , 0.05, **P , 0.01 and ***P , 0.001 were considered
as significant differences. The notation of Fleft / left , Fleft / right
or Fright / right expresses the F value in ANOVA, and left or
right means left HWL (or HWT) or right HWL (or HWT).
Y.-P. Zhang et al. / Regulatory Peptides 86 (2000) 89 – 94
3. Results
3.1. Effects of intrathecal administration of naloxone on
the galanin-induced increase in HWL and HWT in
mononeuropathic rats
Thirty-two rats received intrathecal injection of 3 nmol
of galanin followed, 10 min later, by either 2.75 (n 5 8),
11 (n 5 8) or 22 nmol (n 5 8) of naloxone, or 10 ml of
0.9% saline as a control (n 5 8).
As shown in Fig. 1, after injection of 3 nmol of galanin,
both the HWL and HWT to thermal and mechanical
stimulation increased in the four groups. In the control
group, after administration of 10 ml of 0.9% saline, the
galanin-induced increases in HWL and HWT lasted for
more than 40 min. After intrathecal injection of 22 nmol of
naloxone, the increased HWL and HWT was attenuated
significantly (in the thermal test: Fleft / left 5 7.44, P , 0.01;
Fright / right 5 11.20, P , 0.01; and in the mechanical test:
Fleft / left 5 12.72, P , 0.001) compared to the control
group, except right hind-paw in the mechanical test
(Fright / right 5 2.19, P 5 0.15). In the groups of receiving
91
intrathecally 2.75 (Thermal test: Fleft / left 5 0.26, P 5 0.62;
F right / right 5 1.02, P 5 0.32; mechanical test: Fleft / left 5
0.37, P 5 0.54; Fright / right 5 0.04, P 5 0.84) and 11 nmol
of naloxone (Thermal test: Fleft / left 5 0.24, P 5 0.62;
Fright / right 5 0.01, P 5 0.92; mechanical test: Fleft / left 5
0.25, P 5 0.62; F right / right 5 0.03, P 5 0.86), there were no
significant changes in HWL and HWT in comparison with
the control group.
3.2. Effects of intrathecal administration of b -FNA,
natrindole and nor-BNI on galanin-induced increase in
HWL and HWT
In order to investigate which type of opioid receptors
may be involved in the modulation of galanin-induced
increases in HWL and HWT, three types of selective
opioid antagonists were used. Twenty-eight rats received
intrathecal injections of 3 nmol of galanin followed, 10
min later, by either 10 nmol of b-FNA (mu-opioid
antagonist; n 5 7), 10 nmol of natrindole (delta-opioid
antagonist; n 5 7), 10 nmol of nor-BNI (kappa-opioid
Fig. 1. Effects of intrathecal injection of naloxone on the galanin-induced increase in HWL and HWT. Time 5 0 min: 3 nmol of galanin was injected
intrathcally; Time 5 10 min: intrathecal administration of 22 nmol (n 5 8), 11 nmol (n 5 8) or 2.75 nmol (n 5 8) of naloxone or 10 ml of 0.9% saline as a
control (n 5 6). HWL: hind-paw withdrawal latency to thermal test, HWT: hind-paw withdrawal threshold to mechanical test, vertical bars indicate SEM,
two-way ANOVA, **P , 0.01, ***P , 0.001 compared with control group.
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Y.-P. Zhang et al. / Regulatory Peptides 86 (2000) 89 – 94
antagonist; n 5 7), or 10 ml of 0.9% saline (n 5 7) as
control. The results are shown in Fig. 2.
After intrathecal injection of galanin the HWL to
thermal and HWT to mechanical stimulation increased
significantly in the all four groups. In the control group,
after administration of 10 ml of saline, the increase in
HWL and HWT to both stimuli lasted for about 40 min. 20
min after intrathecal injection of 10 nmol of b-FNA, the
increased HWL and HWT were prevented significantly in
the thermal test (Fleft / left 5 22.53, P , 0.001; Fright / right 5
16.79, P , 0.001) and in the mechanical test (Fleft / left 5
11.33, P , 0.01; Fright / right 5 10.63, P , 0.01) compared to
the control group. However, in the groups of receiving 10
nmol of natrindole (Thermal test: Fleft / left 5 1.67, P 5
0.21; Fright / right 5 0.45, P 5 0.51; mechanical test:
Fleft / left 5 0.09, P 5 0.76; Fright / right 5 2.26, P 5 0.14), and
of receiving 10 nmol of nor-BNI (Thermal test: Fleft / left 5
1.41, P 5 0.25; Fright / right 5 0.26, P 5 0.61; mechanical
test: Fleft / left 5 0.13, P 5 0.72; Fright / right 5 2.44, P 5 0.13),
there were no significant changes in HWL and HWT in
comparison with control group.
Rats with left sciatic nerve ligation received intrathecal
administration of 10 nmol of b-FNA (n 5 5). The results
are shown in Fig. 3. Comparing with pre-injection, the
injection of b-FNA alone showed no significant influence
on HWL and HWT.
4. Discussion
Recent work in our laboratory has shown that intrathecal
administration of galanin induce a significant increase in
hind-paw withdrawal latency in intact and ligated rats
[9,10]. Furthermore, the selective galanin receptor antagonist galantide attenuated the anti-nociceptive effect of
morphine. These results indicate that galanin and opioids,
in some extent, are involved in the transmission of
presumed nociceptive information in the spinal cord of
rats. The results of present study confirmed the existence
of specific interaction between galanin and opioids in the
spinal cord of neuropathic rats, show that this interaction is
related to the activation of mu-opioid receptors.
Chronic constriction injury produced by loosely tying
four ligatures around the sciatic nerve is commonly used as
an animal model of neuropathic pain [2]. Large myelinated
fibres degenerate after constriction injury, but thinly myeli-
Fig. 2. Effects of intrathecal administration of b-FNA, natrindole and nor-BNI on the galanin-induced increase in HWL and HWT. Time 5 0 min: 3 nmol
of galanin was injected intrathecally; Time 5 10 min: intrathecal administration of 10 nmol of b-FNA (n 5 7), 10 nmol of natrindole (n 5 7), and 10 nmol
of nor-BNI (n 5 7). HWL: hind-paw withdrawal latency to thermal test, HWT: hind-paw withdrawal threshold to mechanical test, vertical bars indicate
SEM, two-way ANOVA, **P , 0.01, ***P , 0.001 compared with control group.
Y.-P. Zhang et al. / Regulatory Peptides 86 (2000) 89 – 94
Fig. 3. Effects of intrathecal injection of 10 nmol of b-FNA on HWL to
thermal (A) and HWT to mechanical (B) stimulation in mononeuropathic
rats. Number of rats in each group: n 5 5, data measured at pre-injection,
5 min and 10 min after the injection. Mean. SEM, vertical bars indicate
SEM. Student’s t-test (two tails). Left: ligated side, right: contralateral
side. HWL: hind-paw withdrawal latency; HWT: hind-paw withdrawal
threshold.
nated and unmyelinated fibres are less severely damaged
[18]. In rats with this injury, the decreases of HWL and
HWT in the affected hind limb are also observed but to a
lesser degree on the intact side, an effect also seen in our
previous studies [3,16,18].
Peripheral nerve injury induces significant synthesis and
up-regulation of galanin and galanin mRNA in the dorsal
root ganglion cells and dorsal horn neurons of the spinal
cord [6–8,11,19], which contrasts with the down-regulation of substance P [20]. Ma and Bisby [21] have reported
that after sciatic nerve section galanin immuno-reactive
neurons were increased in the ipsilateral dorsal root
ganglia, and that galanin immuno-reactive axonal fibres
were increased in the superficial laminae of the dorsal
horn. Furthermore, they found that partial sciatic nerve
injuries induced greater galanin up-regulation in mediumand large-sized dorsal root ganglion neurons than that seen
93
following a complete sciatic nerve section. The inhibitory
action of galanin on transmission of nociceptive information is supported by several studies [4,5,9,10]. It is
therefore tempting to suggest that the up-regulation of
galanin induced by peripheral nerve lesion is a compensatory response.
Data from the study of Przewlocka et al. [22] have
shown that the anti-nociceptive effects derived from opioid
agonist morphine can be enhanced by co-administration or
following injection of galanin or N-terminal galanin fragment. Also, Selve and Reimann [23,24] have reported that
the galanin receptor antagonist galantide and M35 attenuate spinal anti-nociceptive effects of opioid agonist
DAMGO, or morphine. All of these observations and
together with our recent and present results suggest that
galanin and opioids in the dorsal root ganglion and / or
spinal cord may serve as some intrinsic anti-nociceptive
factors that attenuate the transmission of afferent nociceptive information.
The inhibitory effect of opioids on the spinal nociceptive
transmission has been well documented, and both pre-and
post-synaptic mechanisms have been suggested to play a
role [1,15]. It is likely that the effect of opioids is mediated
at the dorsal horn level, as an increase in mu-opioid
receptors in the superficial dorsal horn has seen in mononeuropathic rats [25,26]. Also, using immunocytochemistry, Goff and collaborators [12] reported that muopioid receptor immunodensities in the superficial dorsal
horn was found to be increased from 4 to 28 days after
sciatic nerve constriction. The increases of mu-opioid
receptors observed after chronic constriction injury might
explain why an increased efficacy of opiates has been
reported in this model [27]. Also, the attenuated effects of
anti-nociception of galanin by mu-opioid receptor antagonist b-FNA in present experiment may relate to the
increase of mu-opioid receptors induced by peripheral
lesion.
Acknowledgements
This study was supported by funds from the Karolinska
Institutet Foundation, Foundation of Acupuncture and
Alternative Methods, the National Natural Science
Foundation of China (NSFC) and the Natural Science
Pre-Research Foundation of Peking University.
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