Effects of intrathecal administration of newer antidepressants on mechanical
allodynia in rat models of neuropathic pain
Tetsuya Ikedaa, Yasushi Ishidab,*, Rumi Naonoa, Ryuichiro Takedab, Hiroshi Abeb, Tadashi Nakamurac,
aDivision of Neurobiology, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
bDepartment of Psychiatry, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
cDepartment of Anesthesia, Junwakai Memorial Hospital, Miyazaki 880-2112, Japan
Neuropathic pain is defined as a chronic or persistent pain
resulting from an injury to the nervous system. The hallmarks of
this type pain include an enhanced response to noxious stimuli,
thermal and mechanical hyperalgesia and a pain response to
previously non-noxious stimuli, known as allodynia (Courteix
et al., 1993; Elliott, 1994; Woolf and Doubell, 1994; Calcutt et al.,
1996). Neuropathic pain may be due to a pathophysiological
alteration of the peripheral and/or central nervous system
including spinal dorsal horn neurons (Zimmermann, 2001;
Campbell and Meyer, 2006) and is resistant to opiate analgesics.
Antidepressants, especially tricyclic antidepressants (TCAs) are
widely used as the first-line drugs for the treatment of neuropathic
pain including diabetic neuropathy (Mico ´ et al., 2006; Wong et al.,
2007). The mechanisms underlying the antinociceptive effects of
TCAs are complicated and are associated with various substrates at
the supraspinal, spinal, or peripheral level when they are
administered systemically (Sawynok et al., 2001; Mico ´ et al.,
2006). Recently, newer antidepressants such as serotonin and
noradrenaline reuptake inhibitor (SNRI) and selective serotonin
reuptake inhibitor (SSRI) have been introduced and the function of
these antidepressants is better understood than TCAs. However, it
is unclear whether SNRIs such as milnacipran and SSRIs such as
paroxetine or fluvoxamine have similar antinociceptive effects.
The spinal cord is important as an active site of antidepressant-
mediated antinociception (Hwang and Wilcox, 1987). The inhibi-
tion of pain transmission by the descending systems has been
studied by focusing on the neuromodulatory functions of
noradrenaline (NA) and serotonin (5-HT) at the synapses between
dorsal horn neurons and primary afferents (Yoshimura and Furue,
2006). It is presumed that antidepressants alter nociceptive
thresholds and neuropathic pain is, at least in part, inhibited by
a blockade of NA and 5-HT reuptake and by holding them at a
certain level in the synaptic clefts of the spinal cord. Indeed, SSRIs
and SNRIs elicit an antinociceptive or antiallodynic effect on some
acute and chronic neuropathic pain in rodent models following
intrathecal administration (Obata et al., 2005; King et al., 2006) or
Neuroscience Research 63 (2009) 42–46
A R T I C L E I N F O
Received 6 August 2008
Received in revised form 1 October 2008
Accepted 3 October 2008
Available online 15 October 2008
Chronic constriction injury
von Frey test
A B S T R A C T
Antidepressants, especially tricyclic antidepressants (TCAs) are widely used for the treatment of various
types of chronic and neuropathic pain. The antinociceptive effects of TCAs are, however, complicated.
Therefore, two kinds of newer antidepressants whose functions have been more fully clarified were
selected, milnacipran, a serotonin and noradrenaline reuptake inhibitor (SNRI) and paroxetine and
fluvoxamine, which are selective serotonin reuptake inhibitors (SSRIs). The antiallodynic effects of
intrathecal administration of these newer antidepressants were examined in two rat models of
neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve and streptozotocin (STZ)-induced
diabetic neuropathy. The antiallodynic effect of these antidepressants was evaluated using the von Frey
test. The intrathecal administration of milnacipran had an antiallodynic effect in both CCI and STZ-
induced diabetic rats in a dose-dependent manner. On the other hand, the intrathecal administration of
either paroxetine or fluvoxamine elicited little antiallodynic effect in CCI rats, while both SSRIs had
antiallodynic effects in the STZ-induced diabetic rats in a dose-dependent manner. These results indicate
a considerable difference to exist in the development and/or maintenance between these two animal
models of neuropathic pain and suggest that each of these three antidepressants may be effective for the
treatment of diabetic neuropathic pain.
? 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
* Corresponding author. Tel.: +81 985 852969; fax: +81 985 855475.
E-mail address: email@example.com (Y. Ishida).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/neures
0168-0102/$ – see front matter ? 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
systemic administration (Yokogawa et al., 2002; Aubel et al., 2004;
Iyengar et al., 2004). However, the mechanisms of the analgesic
effect of these antidepressants are still unclear, particularly in the
diabetic neuropathic pain model.
term insulin-deficient diabetes in rodents (Junod et al., 1967) and
leads to the development of neuropathic pain characterized by
hyperalgesia and allodynia (Lee and McCarty, 1990; Courteix et al.,
elicit antiallodynic effects in rats with spinal nerve ligation (Iyengar
et al., 2004; Obata et al., 2005) and SSRIs such as paroxetine
produced antiallodynic effects in STZ-induced diabetic rats (Aubel
neuropathic model rats have not been extensively examined. The
effects of intrathecal administration of newer antidepressants that
antiallodynic effect of these drugs at the spinal level. Therefore, the
antiallodynic effects of three antidepressants, milnacipran, parox-
etine and fluvoxamine, were examined in STZ-induced diabetic rats
and in rats with chronic constrictioninjury (CCI) of the sciatic nerve
the different animal models of neuropathic pain.
2. Materials and methods
All animal protocols were approved by the ethical committee
for animal experimentation at University of Miyazaki and followed
the guidelines for treatment of animals of the International
Association for the Study of Pain (Zimmermann, 1983). The
experiments were performed on adult male Sprague–Dawley rats
(Charls River Laboratories Japan, Yokohama, Japan) weighing 200–
250 g on the day of catheterizing. The rats were housed 2 or 3
animals per cage with free access to food and water and exposed to
12-h cycles of light–dark.
2.2. Intrathecal catheterisation
The antidepressants were administered through a catheter into
the subarachnoid space of the rats. All rats were catheterized
intrathecally by modifying the procedure described by Yaksh and
Rudy (1976) one week after adaptation to standard housing
conditions in the Experimental Animal Center of University of
Miyazaki. The catheters were made from polyethylene tubing (PE-
10, Becton Dickinson, San Jose, CA) by stretching in a hot water
of the catheter was inserted caudally into the subarachnoid space
of rat through a small slit in the atlanto-occipital membrane to
extend 7.5 cm beyond the slit under anesthetic conditions with
sodium pentobarbital (20 mg/kg, i.p.) and ketamine hydrochloride
(100 mg/kg, i.p.). The rostral part of the catheter was sutured to the
occipital muscle to immobilize the catheter and the wound was
closed in two layers with 3-0 silk thread. The catheterized rats
were housed in individual cages with free access to food and water
before and during the experiments. The rats showing visible signs
of tissue inflammation, paralysis or other neurological deficits
following catheter implantation during a 1-week recovery period
were excluded from the study.
2.3. Induction of chronic constriction injury
The catheterized animals were prepared with a unilateral
sciatic nerve constriction injury, according to the previously
described procedure (Mosconi and Kruger, 1996). Under deep
anesthesia with sodium pentobarbital (50 mg/kg, i.p.), the sciatic
nerve was exposed by separating the left biceps femoris muscle
with blunt forceps and then freed from surrounding connective
tissue. Using a sterilized stainless probe, two cuffs consisting of a
2–4 mm section of split polyethylene tube (PE-90, Becton
Dickinson, San Jose, CA) were applied to the exposed nerve at
approximately 0.5 mm intervals. The muscle layer and skin layer
were closed using 3-0 silk thread. This experiment was performed
using ratsin whichthe withdrawal thresholdof the ipsilateral hind
paw came down 2 g or less at 2–3 weeks after cuff-implantation.
2.4. Induction of diabetic neuropathy
Diabetic rats were produced by a single injection of STZ (Sigma,
St. Louis, MO; 50 mg/kg, i.v.) prepared in 0.1 M sodium citrate
buffer (pH 4.4) into the tail vein of the catheterized animals.
Intravenous injection of STZ is known to induce insulin-deficient
diabetes by ablating pancreatic b cells (Arison et al., 1967; Junod
confirmed by measuring plasma glucose concentrations in blood
samples obtained from the tail vein using a glucose oxidase
impregnated strip and reflectance meter (GLUTEST ACE, Arkray
Factory, Shiga, Japan) and rats with blood glucose concentrations
greater than 350 mg/dl were considered to be diabetic. The plasma
glucose concentrations of STZ-treated rats were measured at four
time points, 1 day, 3 days, 1 week and 2 weeks following STZ
administration and mechanical allodynia was simultaneously
assessed at the same time points. The catheterized rats showing
diabetes and tactile allodynia 2–4 weeks after STZ injection were
used in this experiment.
2.5. Measurement of hind paw withdrawal threshold
In order to assess mechanical allodynia, the withdrawal thresh-
old of hind paws to mechanical stimulation was determinedusinga
Coast Medical Inc., Morgan Hill, CA) and was expressed in grams.
Eighteen filaments ranging from 0.008 to 100 g were used. The von
Frey filaments were applied using a platform constructed specifi-
mesh floor (Dynamic Planter Aesthesiometer, UGO BASILE, Italy).
The rats were allowed to acclimate to the box for 30 min after
hind paws was touched with different von Frey filaments through
mesh floor and the trial was repeated five times per filament at an
interval of a few seconds. The withdrawal threshold of each hind
paw was determined by increasing the stimulus strength from the
2 g filament until paw withdrawal occurred. A descending series of
the filaments were used when rats responded to the starting
filament. The lowest filament in grams that evoked withdrawal
responses at least two times out of five applications was considered
as the withdrawal threshold (Tal and Bennett, 1994). This test is
designed to measure the extent of mechanical allodynia seen in
neuropathic pain, such as CCI or diabetic model animals. The paw
withdrawal threshold in diabetic model rats was determined by an
average of the withdrawal thresholds of both hind paws, while that
of CCI rats was determined by an average of the withdrawal
thresholds ipsilateral to the injured hind paw.
Milnacipran hydrochloride, paroxetine hydrochloride or flu-
voxamine maleate was diluted to various concentrations in 10 ml
saline and administered intrathecally over a period of 30 s through
the catheter, and followed by 10 ml saline at the same rate to flush
the catheter. Paw withdrawal thresholds were determined 10 min
before and 5 and 30 min and 1, 2, 6 and 24 h after the injection of
T. Ikeda et al./Neuroscience Research 63 (2009) 42–46
2.6. Statistical analysis
The hind paw withdrawal thresholds at all time points were
expressed as the mean ? S.E.M. and analyzed using a two-way
Turkey’s post hoc honestly significant difference (HSD) test. The level
of significance was set at P < 0.05.
3.1. Effects of antidepressants on CCI-induced neuropathic pain
Eighteen out of 23 animals with implanted polyethylene cuffs
showed mechanical allodynia on the ipsilateral (left) hind paws.
The mean threshold was reduced from 23.2 ? 6.4 to 1.2 ? 0.3 g in
rats showing mechanical allodynia 2 weeks after surgery, while
the threshold in the contralateral hind paw (right) 2 weeks after
surgery (25.7 ? 4.8 g) showed little change from that before surgery
(24.9 ? 5.6 g).
The intrathecal administration of milnacipran produced a dose-
dependent increase in the paw withdrawal threshold (Fig. 1A). The
paw withdrawal thresholds following intrathecal administration
of milnacipran at 8 concentrations, 10?1, 10?2, 10?3, 10?4, 10?5,
10?6, 10?7and 10?8M (treatment) 10 min before and 5, 30 min, 1,
2, 6 and 24 h after the administration (time-course) were
evaluated by a two-way ANOVA with repeated measures (treat-
ment ? time). Fig. 1A showed that there were significant effects of
the treatments [F(8,43) = 12.5, P < 0.0001] and time-course
[F(6,258) = 46.2, P < 0.0001] and significant treatment ? time
interactions [F(48,258) = 6.3, P < 0.0001]. Tukey’s post hoc HSD
tests showed that intrathecal administrations of 10?1, 10?2, 10?3,
10?4, and 10?5M milnacipran significantly increased the paw
withdrawal thresholds 1 h after administration, in comparison to
saline administration. At 30 min after administration, only 10?1M
of milnacipran produced a significantly larger paw withdrawal
threshold in comparison to that of saline.
On the other hand, intrathecal administration of paroxetine
(from 10?3to 10?6M) or fluvoxamine (from 10?2to 10?6M)
elicited little effect in the paw withdrawal threshold at any of the
concentrations and time points (Fig. 1B and C).
3.2. Effects of antidepressants on diabetic neuropathic pain
A single injection of STZ (50 mg/kg, i.v.) induced hypergly-
cemia and tactile allodynia in a time-dependent manner (Fig. 2).
The blood glucose level of 22 animals injected intravenously
with STZ was higher than 200 mg/dl at 2 weeks after STZ
injection. Furthermore, the blood glucose level of 17 animals
among them was over 400 mg/dl. Nineteen out of 22 hypergly-
cemic rats displayed tactile allodynia, since the threshold of
hind paw withdrawal response was below 1 g in the von Frey
test. The mean blood glucose level in 19 rats showing
mechanical allodynia was 438.5 ? 22.2 mg/dl at 2 weeks after
STZ injection and the mean values of left and right hind paw
withdrawal thresholds decreased from 14.8 ? 2.5 g to 0.7 ? 0.1 g at
2 weeks after STZ injection.
The intrathecal administration of milnacipran to diabetic
neuropathic rats increased the paw withdrawal threshold in a
dose-dependent manner (Fig. 3A). The paw withdrawal threshold
10?2, 10?3, 10?4, 10?5, 10?6and 10?7M (treatment) at 7 time
points (time-course) and evaluated by a two-way ANOVA with
repeated measures (treatment ? time). Fig. 3A demonstrates the
significant effects of the treatment [F(7,40) = 22.0, P < 0.0001] and
time-course [F(6,240) = 57.75, P < 0.0001] and the significant
Fig. 1. Time course of the effect of intrathecal administration of milnacipran (A),
paroxetine (B) or fluvoxamine (C) on the withdrawal threshold of the left hind paws
in CCI-induced neuropathic rats (n = 6 each antidepressant). The horizontal axis
represents the time before (?10 min) and after (5 and 30 min and 1, 2, 6 and 24 h)
injection of each antidepressant or saline and the vertical axis represents the mean
(?S.E.M.) paw withdrawal threshold expressed in grams.*P < 0.05, in comparison to
each corresponding value in saline treatment.
Fig. 2. Changes in the blood glucose levels and withdrawal thresholds of the hind
paws in streptozotocin (STZ)-induced diabetic rats. The bars demonstrate the
plasma glucose concentrations in mg/dl (left vertical axis) after intravenous
injection of STZ. The inserted sequential line chart shows a time course of the
average values of the withdrawal thresholds between the left and right hind paw,
expressed in grams (right vertical axis), at the each time point. All values represent
the mean ? S.E.M. for (n = 19).*P < 0.05, in comparison to the value of the withdrawal
thresholds at pretreatment of streptozotocin.
T. Ikeda et al./Neuroscience Research 63 (2009) 42–46
treatment ? time interactions [F(42,240) = 11.8, P < 0.0001].
Tukey’s post hoc HSD tests showed the intrathecal administration
of 10?1, 10?2and 10?3M milnacipran to significantly increase the
paw withdrawal thresholds 1 h after administration, in compar-
ison to that of saline.
The intrathecal administration of paroxetine or fluvoxamine
also increased the paw withdrawal threshold (Fig. 3B and C,
respectively). There were significant effects of the treatment
[F(5,30) = 13.9; F(6,35) = 12.0, P < 0.0001 in both cases] and time-
course [F(6,180) = 30.8; F(6,180) = 87.0; P < 0.0001 in both cases]
and showed significant treatment ? time interactions [F(30,180) =
10.4; F(36,210) = 12.7; P < 0.0001 in both cases]. Tukey’s post hoc
HSD tests showed that intrathecal administration of 10?3M
paroxetine and 10?2, 10?3, 10?4
and 10?5M fluvoxamine
significantly increased the paw withdrawal threshold 1 h after
administration, in comparison to that of saline (Fig. 3B and C).
In the present study, the antiallodynic effects of three
antidepressants (milnacipran, paroxetine and fluvoxamine) were
evaluated in two different rat models of neuropathic pain. This
study demonstrated for the first time that the intrathecal
administration of milnacipran, paroxetine or fluvoxamine elicited
rats. Interestingly, intrathecal administration of paroxetine or
fluvoxamine attenuated tactile allodynia in STZ-induced diabetic
rats, but not in CCI rats although the intrathecal administration of
milnacipran produced antiallodynic effects in both rat models of
neuropathic pain. This suggests that there may be a considerable
difference in the development and/or maintenance of these two
animal models of neuropathic pain.
Previous studies showed the increased numbers of serotonin 5-
HT1Aand 5-HT2receptors in the brain stem and cortex in STZ-
induced diabetic rats (Sandrini et al., 1997; Sumiyoshi et al., 1997).
This finding suggest that the 5-HT receptor plasticity is induced in
related to the antiallodynic effects of the SSRIs (paroxetine and
fluvoxamine) in the present study.
A single systemic injection of STZ (i.v.) increased the blood
glucose levelto over 400 mg/dl at2 weeks after STZinjection inthe
present study. Furthermore, in association with the increased
blood glucose levels, the paw withdrawal thresholds were
markedly reduced. As reported previously (Lee and McCarty,
1990; Courteix et al., 1993; Calcutt et al., 1996), it seems likely that
the development of tactile allodynia is positively correlated with
progress of hyperglycemia.
The threshold of the paw withdrawal response was markedly
reduced in the side ipsilateral to CCI at 2 weeks after cuff
implantation. The decreased paw withdrawal threshold was dose-
dependently reversed by intrathecal administration of milnaci-
of SNRIs including milnacipran increased the paw withdrawal
threshold in the spinal nerve ligation (SNL) model that is another
neuropathic pain model.
both the NA and 5-HT reuptake systems (Mochizucki, 2004).
However, the antiallodynic effect of milnacipran in CCI rats might
the serotonergic system at the spinal level, since the intrathecal
administration of SSRIs, paroxetine and fluvoxamine, showed little
antiallodynic effect. This speculation is also supported by the
findings that the upregulation of NA synthesis is observed in the
locus coeruleus and the density of noradrenergic innervation
increased in the spinal dorsal horn of CCI mice (Ma and Eisenach,
2003). In addition, the synergistic interaction between the nora-
drenergic and serotonergic systems may contribute to the anti-
allodynic effects of milnacipran because of the evidence that
intrathecal co-administration of methysergide, an antagonist of 5-
SNL rats as well as co-administration of yohimbine, an a2
adrenoceptor antagonist (Obata etal.,2005). The failureofthe SSRIs
in the present study to cause antiallodynic effect in CCI rats would
appear to corroborate the above explanation about the dominant
contribution of NA neurons and/or the synergistic interaction
between the NA and 5-HT systems. To elucidate how the
noradrenergic and serotonergic systems are involved in the
mechanisms of antiallodynic effect of milnacipran in CCI and STZ-
Fig. 3. Time course of the effect of intrathecal administration of milnacipran (A),
paroxetine (B) or fluvoxamine (C) on the withdrawal threshold of the left and right
hindpawsinSTZ-induceddiabeticneuropathicrats(n = 6eachantidepressant).The
horizontal axis represents the time before (?10 min) and after (5 and 30 min and 1,
2, 6 and 24 h) injection of each antidepressant or saline and the vertical axis
represents the mean (?S.E.M.) paw withdrawal threshold expressed in grams.
*P < 0.05, in comparison to each corresponding value in saline treatment.
T. Ikeda et al./Neuroscience Research 63 (2009) 42–46
NA and 5-HT receptor subtypes to the antiallodynic effect of Download full-text
milnacipran and the interactions between various NA and 5-HT
The administration of fluvoxamine and paroxetine, as well as
milnacipran, also significantly increased the paw withdrawal
threshold in diabetic rats at 1 h after injection. The antiallodynic
effect of paroxetine seems to be inconsistent with the results in the
previous study since acute intraperitoneal administration of
paroxetine had little influence on the paw withdrawal threshold
of mechanical hyperalgesia in diabetic rats (Aubel et al., 2004). This
might not sufficiently reverse the hyperalgesia by acute intraper-
itoneal administration. Alternatively,thisdiscrepancy might be due
to a difference in the route of paroxetine administration, namely,
either intrathecal or intraperitoneal administration.
In STZ-induced diabetic rats, 5-HT might be associated with the
reduction of tactile allodynia, at least in part, since fluvoxamine
and paroxetine had antiallodynic effects. Paroxetine and fluvox-
amine are SSRIs and have little effect on the NA reuptake system
(Claassen et al., 1977; Thomas et al., 1987). Furthermore, the
antiallodynic effect of fluvoxamine may be mediated by 5-HT2A/
2C receptors in the partial sciatic nerve ligation mouse model
(Honda et al., 2006). In addition, the serotonergic system might
play an important role in the reversion of allodynia at the spinal
level of STZ-injected rats. An electrophysiological study using rat
spinal cord slices revealed that 5-HT had inhibitory effects on Ad
and C fiber input to all types of superficial dorsal horn (SDH)
neurons, while NA inhibited C fiber input to transient central
neurons of SDH (Lu and Perl, 2007). Therefore, the reuptake
inhibition of 5-HT by SSRIs might have an inhibitory effect on the
Ad and C fiber input to SDH neurons and this may elicit the
antiallodynic effects by SSRIs in STZ-induced diabetic rats.
Newer antidepressants such as SNRIs and SSRIs, as well as TCAs,
inhibitory amino acids, adenosine, ion channels at the central and
peripheral levels (Sawynok et al., 2001; Mico ´ et al., 2006). These
might be related to the antiallodynic effects of milnacipran,
paroxetine and fluvoxamine. Further pharmacological and mor-
of antiallodynic action for antidepressants at the spinal or
supraspinal level in each model of chronic neuropathic pain.
Although there are still many questions to be clarified, the
present findings suggested that each of the three antidepressants,
milnacipran, paroxetine and fluvoxamine, may have utility in the
treatment of diabetic neuropathic pain.
We thank Ms. Fumiko Tsuda for excellent technical assistance.
This research was supported by Grants-in Aid for Scientific
Research from the Japan Society for the Promotion of Science.
Milnacipran, paroxetine and fluvoxamine were generous gift from
Asahi Kasei Pharma (Tokyo, Japan), GlaxoSmithKline (Brentford,
UK) and Solvay Pharmaceutical Co. (Tokyo, Japan), respectively.
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