MOTOR SYSTEM Dr. Zahoor Ali Shaikh. TRANSVERS SECTION OF SPINAL CORD.
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Transcript of Intrathecal interleukin-1β administration induces thermal hyperalgesia by activating inducible...
www.elsevier.com/locate/brainres
Brain Research 1015 (2004) 145–153
Research report
Intrathecal interleukin-1h administration induces thermal hyperalgesia
by activating inducible nitric oxide synthase expression
in the rat spinal cord
Chun-Sung Sunga,b, Zhi-Hong Wenc, Wen-Kuei Changb, Shung-Tai Hoc, Shen-Kou Tsaib,Yi-Chen Changc, Chih-Shung Wongc,*
aGraduate Institute of Medical Sciences, National Defense Medical Center, Taipei, TaiwanbDepartment of Anesthesiology, Veterans General Hospital-Taipei, Taipei, Taiwan
cDepartment of Anesthesiology, Tri-Service General Hospital and National Defense Medical Center,
#325, Section 2, Chenggung Road, Neihu 114, Taipei, Taiwan
Accepted 20 April 2004
Available online
Abstract
The effect of the pro-inflammatory cytokine interleukin-1h (IL-1h) on the inducible nitric oxide synthase-nitric oxide (iNOS-NO) cascadein nociceptive signal transduction was examined in the intact rat spinal cord. All rats were implanted with an intrathecal (i.t.) catheter; some
were also implanted with an i.t. microdialysis probe. The paw withdrawal latency to radiant heat was used to assess thermal hyperalgesia. The
iNOS protein expression in the spinal cord dorsal horn was examined by western blot analysis and NOS activity assay. NO production in the
CSF dialysate was also measured. IL-1h i.t. (100 ng) produced thermal hyperalgesia from 4 to 24 h after i.t. injection. The iNOS protein
expression was induced at 4 h after i.t. IL-1h injection, peaked at the 6th hour, and disappeared at 24 h. The iNOS activity showed a similar
time-dependent change as the iNOS protein expression. NO release increased by 1.1- to 1.9-fold between 4 and 12 h, also with a peak at the
6th hour, after i.t. IL-1h administration. Pretreatment with the iNOS inhibitor 1400W (10 Ag, i.t.) 1 h before i.t. IL-1h injection prevented all
the responses of IL-1h. Neither 1400W nor artificial CSF (aCSF) affected the thermal nociceptive threshold and NO production. These
results demonstrate that i.t. administration of IL-1h induced thermal hyperalgesia by activating the iNOS-NO cascade in the rat spinal cord.
On the basis of the present findings, we suggest that i.t. administration of iNOS inhibitors may have potential in the treatment of
inflammatory and neuropathic pain syndromes.
D 2004 Elsevier B.V. All rights reserved.
Theme: Sensory systems
Topic: Pain modulation: pharmacology; Pain: pathways
Keywords: Interleukin-1; Nitric oxide synthase; Nitric oxide; Hyperalgesia; Spinal cord
1. Introduction
Interleukin-1h (IL-1h), a pro-inflammatory cytokine, is
involved in the immune response and signal transduction
both in the periphery and the central nervous system (CNS)
[13,14,17,18,28,36]. It is released during inflammatory and
neuropathic pain conditions [8,34,39,43]. Samad et al. [34]
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.04.068
* Corresponding author. Tel.: +886-2-87927128; fax: +886-2-
87927127.
E-mail address: [email protected] (C.-S. Wong).
found that injection of complete Freund’s adjuvant (CFA)
into the rat’s hindpaw induced peripheral inflammation and
IL-1h upregulation (>10,000-fold) in the inflamed hindpaw
soon after CFA administration; this effect lasted for several
days and was correlated with the peripheral inflammation.
Moreover, in their study, the IL-1h concentration in the
CSF was increased 50- and 20-fold, at 2 and 4 h,
respectively, after CFA injection, and this was followed
by an increased cyclooxygenase-2 (COX-2) level in the
spinal cord. They also found that intrathecal (i.t.) injection
of IL-1h (50 ng) induced a 30-fold increase in COX-2
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153146
mRNA levels in the rat spinal cord. Administration of IL-
1h produces hyperalgesia; in contrast, blockade of IL-1hreduces pain [10,30,32,34,35,37]. It is apparent that IL-1his an important mediator in the cross-talk between the
nervous and immune systems, and also participates in
modulation of the nociceptive threshold.
Nitric oxide (NO), a short-lived diffusible molecule of
great biological importance, plays a key role in host defense,
signal transduction, and neurotransmission. There is increas-
ing evidence that NO is also involved in nociceptive
processing after inflammation and neuropathy [26]. Three
isoforms of nitric oxide synthase (NOS) are known; two are
constitutively expressed (nNOS and eNOS), and the third is
inducible NOS (iNOS). Both nNOS (neuronal) and eNOS
(endothelial) are activated in response to physical stimuli,
and rapidly produce low levels of NO. In contrast, iNOS is
activated by stimuli such as IL-1h, and produces a high
level of NO over a long period of time [6,23]. Upregulation
of NOS expression and subsequent NO production in the
spinal cord contributes to pain following nociceptive stim-
ulation in animals [20,26,33,41,46]. i.t. administration of
NOS inhibitors significantly reduces nociceptive behavior in
response to peripheral inflammation [31,33,46]. Moreover,
IL-1h was found to increase both iNOS expression and NO
production in cultured cells [12,15]. Although both IL-1hand the NOS-NO cascade have been suggested to modulate
nociception, there is little information concerning the inter-
action between IL-1h and the iNOS-NO cascade in the
spinal cord. We hypothesized that, i.t. IL-1h causes a
markedly increased NO production via induction of iNOS
expression in the rat spinal cord. If an IL-1h induced
elevation in NO can be confirmed, it might reveal a
significant pathophysiological pathway of intrathecal IL-
1h-induced thermal hyperalgesia. The present study was
designed to examine the role of the NOS-NO cascade, and
its correspondence to nociceptive behaviors, in the spinal
cord of i.t. IL-1h-treated rats.
2. Materials and methods
2.1. Construction and implantation of the intrathecal
catheter and microdialysis probe
The i.t. catheter was constructed using a 9-cm long
polyethylene (PE5) tube and a 3.5-cm long silastic tube.
The silastic tube was inserted into the PE5 tube and sealed
with epoxy resin and silicon rubber. In addition, the spinal
microdialysis probe construction technique was modified
and adapted in previous studies [21,45]. The microdialysis
probe was constructed using two 7 cm PE5 tubes (0.008
inch inner diameter, 0.014 inch outer diameter) and a 4-cm
cuprophan hollow fiber (300 Am outer diameter, 200 Aminner diameter, 50 kDa molecular weight cut-off; DM-22,
Eicom, Kyoto, Japan). To make the probe firm enough for
implantation, a Nichrome-Formavar wire (0.0026 in.; A-M
System, Everret, WA, USA) was passed through a polycar-
bonate tube (194 Am outer diameter, 102 Am inner diameter;
0.7 cm in length) and the cuprophan hollow fiber (active
dialysis region), and connected to a PE5 catheter with epoxy
glue. The fiber was then bent in the middle section of the
cuprophan hollow fiber, forming a ‘‘U’’ shaped loop. The
two ends of the dialysis fiber, consisting of silastic tubes,
were sealed with silicon sealant. The dead space of the
dialysis probe was 8 Al. During in vitro measurements, the
recovery rate of the dialysis probe was 40% at an infusion
rate of 5 Al/min. Using this technique, it was possible to
measure levels of CSF amino acids for up to 12 days after
implantation.
2.2. Animal preparation and intrathecal drug delivery
Male Wistar rats (National Laboratory Animal Breed-
ing and Research Center, Taipei, Taiwan), weighing 350–
400 g, were implanted with an i.t. catheter either with or
without an i.t. microdialysis loop probe under chloral
hydrate anesthesia (350 mg/kg i.p.). After implantation,
all rats were allowed a 5-day recovery before use. All
rats were housed individually in cages with ad libitum
food and water, and maintained under a standard 12:12
h light–dark cycle at room temperature. For the nocicep-
tive behavioral test, western blotting and NOS activity
assay experiments, the rats were implanted with an i.t.
catheter, while, for the microdialysis study, they were also
implanted with a microdialysis probe. Rats with any
neurological deficits were excluded from the study. The
treatment and use of the animals conformed to the
guidelines of the International Association for the Study
of Pain [47], and were approved by the Animal Care and
Use Committee of our institute.
The dose-dependent effect of IL-1h, at various doses
(100, 200 and 500 ng, i.t.), on nociception and iNOS protein
expression in the rat spinal cord were examined. A reduction
of the PWL and an increase in iNOS expression in the rat
spinal cord were observed in a dose-dependent manner with
a similar time course between 4 to 24 h. Therefore, the
lowest dose of IL-1h, 100 ng, was used for the followed
experiments. The role of iNOS on IL-1h-induced thermal
hyperalgesia was examined by i.t. injection of 1400W (a
highly selective iNOS inhibitor); 50 Ag of 1400W was i.t.
injected in 10 rats, which unfortunately produced spontane-
ous nociceptive behaviors (e.g., vocalizations and flaccidity)
in three rats. The dose of 1400W was decreased to 10 Agand no spontaneous nociceptive behaviors were observed
with this dose in 10 rats; therefore, 10 Ag of 1400W was
chosen for the following experiments.
On the sixth day after i.t. catheter implantation, the rats
were assigned to one of four groups and various drugs
were injected i.t.: (1) the control aCSF group received 22
Al of artificial cerebrospinal fluid (aCSF; 151.1 mM Na+,
2.6 mM K+, 122.7 mM Cl�, 21.0 mM HCO3�, 0.9 mM
Mg2 +, 1.3 mM Ca2 +, 2.5 mM HPO42�, 3.5 mM dextrose,
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153 147
bubbled with 5% CO2 in 95% O2, adjusted to a pH of
7.3); (2) the 1400W group received 10 Ag (2 Al) of 1400W(an iNOS inhibitor), followed by 20 Al of aCSF to flush
the catheter; (3) the IL group received 100 ng (2 Al) of IL-1h, followed by 20 Al of aCSF to flush the catheter; (4)
the 1400W+ IL group received 10 Ag (2 Al) of 1400W,
which was flushed with 10 Al of aCSF 1 h before i.t.
injection of IL-1h (100 ng, 2 Al), which in turn was
flushed with 10 Al of aCSF.
2.3. Behavioral assessment
The paw withdrawal latency (PWL) to radiant heat was
used to assess thermal hyperalgesia by the plantar test
(Biological Research Apparatus Type 7370, Plantar Test;
UGO Basile, Comerio, Italy). Rats were placed in plastic
cages on a glass platform and the heat source was
positioned directly beneath the right hind paw. The heat
intensity was adjusted to obtain an average PWL of
17.6F 0.4 s, and the cut-off time was set at 22F 0.4 s
to prevent tissue damage. The PWL was assessed prior to
i.t. drug delivery (baseline) and at 1, 2, 3, 4, 5, 6, 8, 10,
12, and 24 h after drug delivery.
2.4. Western blot analysis
The rats were killed at 2, 4, 6, 8, 10, 12, or 24 h after
drug injection, and the spinal cords were rapidly removed.
The dorsal part of the lumbar spinal cord was dissected and
immediately frozen and stored at � 80 jC until use. The
spinal cord samples were homogenized by sonication in a
cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2%
Triton X-100, 0.1 mM EDTA, 0.1 mM EGTA, 100 Ag/ml of
phenylmethylsulfonyl fluoride, 1 Ag/ml of aprotinin), then
centrifuged at 100,000� g at 4 jC for 35 min. The protein
content in the supernatant was determined by the Lowry
method [19]. An equal volume of sample buffer (2% SDS,
10% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoe-
thanol, and 50 mM Tris–HCl, pH 7.2) was added into the
lysate samples (with equal protein content). The lysate
buffer, containing protein 24 Ag, was added and separated
on a 7.5% SDS polyacrylamide gel at 150 V for 75 min.
After transferring the proteins to a polyvinylidene difluoride
(PVDF) membrane (0.45 M pore size, Immobilonk-P,
Millipore, Bedford, MA. USA) in a transfer buffer (50
mM Tris–HCl, 380 mM glycine, 1% SDS and 20% meth-
anol) at 125 mA at 4 overnight, the membrane was blocked
with 5% non-fat dry milk in Tween-20 in Tris-buffered
saline (TTBS; 0.1% Tween-20, 20 mM Tris–HCl, 137 mM
NaCl, pH 7.4) for 30 min at room temperature. The
membranes were then incubated with mouse anti-iNOS
monoclonal antibodies (1:1,000 dilution; Transduction Lab-
oratories, Lexington, KY, USA) for 3 h at room temperature,
washed in TTBS for 10 min three times, blocked with 5%
non-fat dry milk in TTBS, and incubated with horseradish
peroxidase-conjugated secondary anti-mouse IgG antibody
(1:2,000 dilution; Transduction Laboratories, Lexington,
KY, USA) for another 1 h at room temperature. Finally,
bound antibody was detected by chemiluminescence (ECL;
Perkin-Elmer, Boston, MA, USA) using X-ray film
(KODAK X-OMAT LS, Kodak, Rochester, NY, USA).
The spinal cord h-actin protein was used as the control.
Densitometry was used to express the density of the band as
a relative density compared to the background. The relative
densities of the bands were calculated and standardized
using the density of the h-actin band.
2.5. NOS activity assay
NOS activity was determined by the stoichiometric
conversion of L-[3H]arginine to L-[3H]citrulline by NOS
assay kit (Cayman, Ann Arbor, MI, USA). The spinal cord
tissues were homogenized with a Dounce homogenizer
(Tenbroeck Tissue Grinder) in an ice-cold homogenization
buffer (25 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM
EGTA, 1 Ag/ml aprotinin, 100 Ag/ml phenylmethylsulfonyl
fluoride). The homogenates were centrifuged at 10,000� g
for 15 min at 4 jC, and the supernatants were extracted and
kept on ice. The protein concentration of the supernatant
was measured by the Lowry method with bovine serum
albumin as standard. Then, 10 Al of spinal cord extract
supernatant, corresponding to 15 Ag of total protein, was
incubated with reaction buffer provided in the kit and
supplemented with freshly prepared reduced NADPH and
L-[3H]arginine with a final volume of 50 Al for 30 min at
37 jC, either with or without NOS inhibitor NG-nitro-L-
arginine methyl ester hydrochloride (L-NAME HCl, 1
mM). The reaction was terminated by adding 400 Al of
the stop buffer (HEPES 50 mM, pH 5.5, and EDTA 5
mM), followed by 100 Al of the equilibrated resin to the
reaction mixture to bind the residual L-[3H]arginine. Then,
the mixtures were transferred to the spin cups in cup
holders, and L-[3H]arginine was separated from L-[3H]cit-
rulline by centrifugation at 12,500� g for 30 s. The
scintillation cocktail fluid was added to the eluate, contain-
ing L-[3H]citrulline, and then the radioactivity was quanti-
tated in a liquid scintillation counter (Beckman LS6500;
USA). iNOS activity was calculated as the difference in
activity between samples in the presence of L-NAME
(1 mM) or not. EGTA/EDTA (1 mM) was added to all
samples to chelate CaCl2 and calmodulin in the reaction
mixture. NOS activity was expressed as counts per minute
per Ag of total protein (cpm/Ag protein).
2.6. CSF sampling and NO analysis
One end of the microdialysis tube was connected to a
micro-syringe pump (CMA102, Acton, MA, USA) for
continuous aCSF infusion, and the other end was used to
collect the CSF dialysate. The microdialysis tube was
perfused with aCSF at a flow rate of 5 Al/min, and dialysates
were collected prior to each drug administration (baseline)
Fig. 1. Effects of various i.t. drugs treatments on the thermal nociceptive
threshold in rats. Values are normalized to the baseline (before i.t. drug
delivery) of each group. aCSF, artificial CSF (n= 6); IL, IL-1h 100 ng
(n = 8); 1400W, 1400W 10 Ag (n = 8); 1400W+ IL, 1400W 10 Agpretreatment 1 h prior to IL-1h 100 ng (n= 8). The PWL was obtained
by the plantar test. The maximal cut-off time was set at 22 s. The results are
illustrated as meanF S.E.M. *p< 0.05 indicated a significant difference
from baseline value in the same group, p< 0.05 compared to the control
group (aCSF i.t. injection) at the same time-point.
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153148
and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 10, 12, and 24
h after i.t. administration of drugs. The dialysate samples
were collected in polypropylene tubes and frozen at � 80
jC until used for NO analysis.
The total amount of NOx (NO +NO2�+NO3
�) in the
samples was used to determine the NO concentration in
the CSF. NOx in the dialysates was analyzed by using a
chemiluminescence detector (NOA 280, Sievers Instru-
ments, Boulder, CO, USA). The method was modified from
those described in previous studies [2,3]. The standard curve
was generated by using sodium nitrate and 0.1 M vanadium
(III) chloride, heated to 90 jC in a water-jacketed purge
vessel constantly bubbled with oxygen-free nitrogen. The
NOx signals from the chemiluminescence detector were
displayed and analyzed by using a computer-based data
recording and analyzing system. Background concentrations
of NOx with different agents in aCSF were measured and
subtracted. All standards and samples were analyzed in
duplicate.
2.7. Chemicals
1400W [N-(3-(aminomethyl)benzyl)acetamidine dihy-
drochloride] was purchased from Calbiochem (La Jolla,
CA, USA) and dissolved at 5 Ag/Al concentration in
aCSF. Rat recombinant IL-1h was purchased from R&D
Systems (Minneapolis, MN, USA) and dissolved at 50
ng/Al concentration in aCSF. L-[3H]arginine (1 ACi/Al)was purchased from Amersham Biosciences (Bucking-
hamshire, UK). Reduced NADPH (h-nicotinamide ade-
nine dinucleotide phosphate) was purchased from Sigma
(USA).
2.8. Data analysis
All data are presented as meanF S.E.M. Data were
analyzed by using one-way analysis of variance (ANOVA),
and followed by the post-hoc Tukey test. A p value < 0.05
was considered statistically significant.
3. Results
3.1. Effect of iNOS inhibitor 1400W on IL-1b-inducedthermal hyperalgesia
After i.t. injection of IL-1h (100 ng), thermal hyper-
algesia was observed with the PWL reduced to 50–60% of
baseline level between 4 and 24 h (Fig. 1). This hyper-
sensitivity to the heat was significantly increased at 4 h,
peaked at 6 h, and lasted for 24 h after IL-1h i.t.
administration. Either aCSF or 1400W (10 Ag) alone did
not affect the PWL to the heat, whereas i.t. pretreatment
with 1400W (10 Ag) 1 h prior to IL-1h (100 ng) injection
significantly attenuated the IL-1h-induced thermal hyper-
algesia (Fig. 1).
3.2. Effects of i.t. injection of IL-1b on iNOS protein
expression and NO production in the spinal cord
As shown in Fig. 2, i.t. injection of IL-1h (100 ng)
induced iNOS protein expression in the dorsal horn of the
rat lumbar spinal cords. The expression was first observed at
4 h, peaked at 6 h, and disappeared at 24 h after injection.
The time course of iNOS activity change was measured in
lumbar spinal cord after IL-1h 100 ng i.t. injection. As
shown in Fig. 3, there was no apparent iNOS activity in the
i.t. aCSF injected rats, while the appearance and increase of
iNOS activity were found after IL-1h i.t. injection in the rat
spinal cord. After 2 h, a marked time-dependent increase of
iNOS activity was observed between 4 and 12 h with a peak
at 6 h (5530F 856 cpm/Ag protein). The iNOS activity
returned to the baseline at 24 h after IL-1h injection. As
shown in Fig. 4, i.t. injection of IL-1h (100 ng) caused a
marked increase in NO concentration in the CSF. The NO
concentrations were increased by 1.1- to 1.9-fold of baseline
between 4 and 12 h, peaked at 6 h, then gradually fell to the
basal level 24 h after IL-1h injection.
3.3. Effect of iNOS inhibitor 1400W on iNOS protein
expression and NO production in the spinal cord
Neither aCSF nor the selective iNOS inhibitor 1400W i.t.
injection induced iNOS protein expression and activity in
the rat spinal cord (Figs. 2 and 3); however, i.t. pretreatment
with 1400W (10 Ag) 1 h before IL-1h i.t. injection signif-
icantly inhibited the IL-1h-induced iNOS activity to 1.8%
(4th hour) and 0.5% (6th hour) of that induced by IL-1hinjection (Fig. 3). Moreover, iNOS inhibitor 1400W pre-
Fig. 2. Intrathecal injection of IL-1h (100 ng) induced iNOS protein expression in the rat spinal cord. Time course of iNOS protein upregulation after i.t. IL-1hinjection was shown after western blot analysis (A) and densitometry of iNOS protein bands (B). Naive meant that rats had not received any surgical or
pharmacologic manipulation, whereas aCSF meant that rats were intrathecally injected with aCSF and were sacrificed 6 h after injection. Values of optical
density were expressed as meanF S.E.M. (n= 3) at each time-point in (B). *p< 0.05 indicated a significant difference from baseline value.
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153 149
treatment also significantly inhibited IL-1h-induced NO
production, whereas i.t. injection of 1400W alone did not
affect NO release (Fig. 4). This inhibitory effect of the
1400W pretreatment indicates that iNOS is involved in the
signaling of spinal IL-1h-induced NO release.
Fig. 3. The time course of iNOS activity changes in lumbar spinal cord after i.t. in
Rats were sacrificed at baseline (prior to drug injection) and at 2, 4, 6, 8, 10, 1
determined by the stoichiometric conversion of L-[3H]arginine to L-[3H]citrulline
minute per Ag of total protein (cpm/Ag protein). All the data are presented as me
baseline value in the IL group, p< 0.05 compared to the 1400W+ IL group at th
4. Discussion
In the present study, thermal hyperalgesia was observed 4
h after IL-1h injection and lasted for 24 h. The iNOS
selective inhibitor 1400W prevented this thermal hyper-
jection of IL-1h (100 ng) with or without i.t. 1400W (10 Ag) pretreatment.
2 and 24 h, respectively after each drug injection and iNOS activity was
by using the Cayman NOS assay kit. The data are expressed as count per
anF S.E.M for 4–6 rats. *p< 0.05 indicated a significant difference from
e same time-point.
Fig. 4. Time-course of NO release into the CSF after i.t. various drugs treatment. i.t. administration of IL-1h 100 ng caused a marked increase in NO release. i.t.
injection of 1400W 10 Ag 1 h before IL-1h treatment dramatically decreases NO release. The NO concentration in the CSF dialysate was analyzed by using a
chemiluminescence detector. Values are normalized to the baseline (before i.t. drug delivery) for each group. Data are presented as meanF S.E.M for 6 rats.
*p< 0.05 compared to baseline in the same group, p< 0.05 compared to the 1400W group at the same time-point.
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153150
algesia and the corresponding biochemical changes. I.t.
administration of IL-1h induced iNOS protein expression
and the subsequent NO release, in a time-dependent manner,
in the rat spinal cord. iNOS protein induction and activation
were observed at 4 h after IL-1h injection, peaked at 6 h,
then declined gradually, and disappeared at 24 h. This
increase in NO level in the CSF after IL-1h injection was
accompanied by an upregulation of iNOS protein expres-
sion. The present results show that i.t. injection of the
proinflammatory cytokine, IL-1h, induced hyperalgesia,
which was produced by iNOS protein induction and acti-
vation, and the subsequent NO production. i.t. pretreatment
with 1400W blocked the synthesis of NO and reduced the
thermal hyperalgesia which were induced by the i.t. IL-1hinjection in rats.
IL-1h was found to increase in the spinal cord in animal
models of neuropathy and peripheral inflammation
[8,34,39,40,43]. Samad et al. [34] found that injection of
CFA into the hindpaw of rats induced peripheral inflamma-
tion; IL-1h expression was increased at 2 and 4 h in the CSF
after peripheral inflammation induction, and this was fol-
lowed by an increasing level of COX-2 mRNA in the spinal
cord. i.t. injection of IL-1h (50 ng) induced an increase in
the COX-2 mRNA level in the rat spinal cord, which was
similar to the central effect of CFA-induced COX-2 upre-
gulation in the spinal cord. They also found that i.t.
administration of IL-1h receptor antagonist (IL-1ra, 6 Ag)or IL-1h-converting enzyme (ICE) inhibitor YVAD (0.5 Ag)blocked the induction of spinal COX-2 mRNA and the
central prostanoid production, and attenuated the mechani-
cal hypersensitivity produced by peripheral inflammation
[34]. They concluded that IL-1h was a major contributor in
CFA-induced inflammatory pain. Peripheral inflammation
upregulated central IL-1h expression [39]; in addition, i.t.
administration of IL-1ra and soluble tumor necrosis factor
receptor attenuated the mechanical allodynia following
peripheral neuropathy [40]. These results suggest that spinal
IL-1h may contribute to the development of nociceptor
hypersensitivity to the peripheral inflammation.
Peripheral IL-1h administration produces hyperalgesia in
rats [11,44], yet the nociceptive and anti-nociceptive effect
of central IL-1h are still debated. In the rats, researchers
have found that intracerebroventricular (i.c.v.) injection of
IL-1h exerts biphasic responses on thermal and mechanical
nociceptive thresholds depending upon the dosage. i.c.v.
injection of IL-1h causes hyperalgesia at lower doses, but in
contrast, causes analgesia at higher doses [5,24,25,29].
Different nociceptive behavioral responses to IL-1h i.t.
administration have also been reported. Falchi et al. [10]
reported thermal hyperalgesia in rats after i.t. IL-1h injec-
tion (at the dose of 50 or 500 pg) by the hot-plate test. In the
present study, we found that i.t. injection of recombinant rat
IL-1h (100 ng) produced thermal hyperalgesia in rats 4
h after injection and lasted for 24 h. However, Watkins et al.
[44] reported that i.t. injection of human recombinant IL-1h(50 ng) did not produce any effect on the nociceptive
threshold assessed by a modified tail-flick test in rats.
Moreover, Souter et al. [38] reported that i.t. administration
of murine recombinant IL-1h did not affect the nociceptive
threshold in rats, at dose of 100 ng in a 3-h observation. In
contrast, Ji et al. showed an anti-nociceptive effect after rat
recombinant IL-1h (10 and 100 ng) i.t. injection in rats
assessed by the plantar test, which appeared from 5 to 30
min after injection [16]. Our results were similar to some
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153 151
studies but different from others. These controversial results
might be due to differences in experimental conditions, such
as the source of IL-1h, the methods used for thermal
threshold evaluation, the time point of observation on
thermal threshold, and the dose in i.t. delivery.
To date, evidence shows that different isoforms of NOS
may contribute to peripheral inflammatory pain. Osborne
and Coderre found that i.t. administration of either 3-bromo-
7-nitroindazole (selective nNOS inhibitor) or aminoguani-
dine (AG, selective iNOS inhibitor) effectively attenuated
carrageenan-produced thermal hyperalgesia in rats and that
iNOS contributed to the thermal hyperalgesia in the late
stage of carrageenan-induced inflammatory pain, while
nNOS played a role throughout the whole course [27].
Tao et al. found that intraplantar (ipl.) carrageenan injection
produced thermal hyperalgesia, which peaked at 4 h and
persisted for 24 h after carrageenan injection, in both wild
type and iNOS knockout mice, and the iNOS mRNA
expression in the lumbar spinal cord was markedly upregu-
lated at 24 h (late phase) in wild type mice [42]. i.t. pre-
treatment with selective iNOS inhibitor L-N-(1-iminoethyl)-
lysine had no effect in the early phase (2–6 h) but
significantly attenuated the carrageenan-produced reduction
of PWL in wild type mice in the late phase (24 h), and the
nNOS protein expression was significantly up-regulated at
24 h after carrageenan (ipl.) injection in the iNOS knockout
mice when compared to that in wild type mice [42]. i.t. pre-
treatment with the selective nNOS inhibitor, 7-nitroindazole
(7-NI), not only delayed the onset of thermal hyperalgesia in
the early phase but also significantly attenuated the thermal
hyperalgesia in the late phase after carrageenan treatment in
iNOS knockout mice. Therefore, Tao et al. [42] suggested
that iNOS might be sufficient, but not essential, for the late
phase of the carrageenan-induced thermal hyperalgesia.
Maihofner et al. [20] also found that both iNOS and nNOS
proteins were upregulated in the mouse spinal cord 168
h after injection (ipl.) of zymosan. Meller et al. [22] found
that i.t. injection of AG dose-dependently reduced the
thermal hyperalgesia produced by injection (ipl.) of zymo-
san, and they suggested that iNOS contributed to zymosan-
induced thermal hyperalgesia. In addition, Wu et al. [46]
demonstrated that intradermal injection of capsaicin pro-
duced mechanical allodynia in rats, and upregulated both
iNOS and nNOS protein expression in the rat spinal cord
from 20 to 150 min after capsaicin injection. In contrast, Tao
and Johns [41] found that intraperitoneal pretreatment of 7-
NI blocked formalin-induced long-term thermal hyperalge-
sia in rats and suppressed formalin-induced soluble guany-
late cyclase a1 subunit expression in the spinal cord as well.
This study suggested that nNOS played a role in formalin-
induced long-term thermal hyperalgesia.
IL-1h has been reported in inflammatory and neuro-
pathic pain conditions [8,34,40,43]. Our present study was
targeted at i.t. IL-1h-produced spinal iNOS activation and
nociceptive responses, and we found that i.t. iNOS inhib-
itor pretreatment significantly attenuated thermal hyper-
algesia and NO release. iNOS is not constitutively
expressed in vivo, and its expression is induced by lipo-
polysaccharide or cytokines. In contrast to nNOS and
eNOS, iNOS generates high concentrations of NO (n rather
than pmolar) and this level of synthesis is sustained for
hours, days, or even longer [6,23]. There is a lag phase of
several hours between cell activation and NO production,
reflecting the time taken for mRNA and protein synthesis
[6]. Casamenti et al. directly injected IL-1h into the right
nucleus basalis of rats and found that iNOS immunopos-
itive microglia surrounded the injection site at 7 days after
IL-1h injection; meanwhile, a transient increase in reactive
nitrogen intermediates (NO2�) was present 7 days after
injection [4]. Moreover, Maihofner et al. demonstrated an
increase of iNOS protein expression in the spinal cord at 7
days after injection (ipl.) of zymosan [20]. The latency and
time course of the induced iNOS expression in the spinal
cord may be related to the characteristics of the insults.
Upregulation of iNOS mRNA was detected in the spinal
cord 24 h after carrageenan injection (ipl.) in mice [42],
and iNOS protein upregulation in the spinal cord was
increased 20 min after intradermal injection of capsaicin
in rats [46]. Taken together, these results suggest that it
takes several hours to days for the induction of iNOS
mRNA and protein expression after stimulation. In the
present study, we intrathecally injected IL-1h in rats and
found an upregulation of iNOS protein in the rat spinal
cord. Compared with capsaicin-and carrageenan-induced
peripheral inflammatory pain models, upregulation of
iNOS protein in the rat spinal cord was detected 4 h after
i.t. administration of IL-1h in our present study, which was
later than that produced by intradermal injection of capsa-
icin, but earlier than that produced by carrageenan injection
(ipl.) [42]. The latency of iNOS protein expression in the
spinal cord induced by IL-1h injection (i.t.) might be due
to the time lag of intracellular signalling transduction,
activation and translocation of nuclear transcription factors,
gene transcription and translation, and spinal sensitization.
It is worth further investigation on the intracellular events
after IL-1h administration.
In in vivo experiments, i.t. IL-1h (10 ng) injection
upregulated the expression of iNOS mRNA in rat spinal
cords [22]; in addition, IL-1h (10 U) injected into the
nucleus basalis activated iNOS-immunopositive microglias
with the production of NO in the rats [4]. Similarly,
upregulation of iNOS protein expression and increase of
NO release from the spinal cord were also observed in
peripheral inflammatory pain models [20,46]. i.t. adminis-
tration of an iNOS inhibitor blocked the thermal hyper-
algesia and mechanical allodynia induced by peripheral
inflammation [22,27,46]. These reports suggest that the
central iNOS-NO cascade played a role in nociceptive
processing and that IL-1h activates this iNOS-NO signal
transduction pathway. In our present study, except for
thermal hyperalgesia, i.t. administered IL-1h, at doses of
100, 200 and 500 ng, did not induce any neurological deficit
C.-S. Sung et al. / Brain Research 1015 (2004) 145–153152
or abnormal behavioral changes in rats. In the present study,
by Western blot analysis and NOS enzyme activity assay,
IL-1h induced a significant expression of iNOS protein in
the spinal cord as well as NO release in the CSF. From these
results, we suggest that, in a similar temporal domain, i.t.
injection of IL-1h activated the spinal iNOS-NO cascade,
which induced the thermal hyperalgesia.
In a rat zymosan intra-articular injection-induced arthritis
pain model, pretreatment with 1400W (a highly selective
iNOS inhibitor) inhibited the zymosan-induced articular
incapacitation and nociception [7]. Beauregard et al. [1]
demonstrated a time-dependent expression of iNOS protein
and NO production in cultured rabbit lacrimal gland acinar
cells, which were significantly antagonized by co-incubation
with 1400W. Furthermore, 1400W was found to inhibit the
IL-1h-induced iNOS mRNA and protein expression, and
nitrite release in human bronchial epithelial cells [9]. Simi-
larly to our present study, i.t. injection of IL-1h activated the
iNOS-NO cascade and induced thermal hyperalgesia, and i.t.
pretreatment of 1400W 10 Ag (at this dose, no antinocicep-
tive effect was observed by itself) significantly inhibited the
IL-1h-mediated iNOS protein expression and NO release in
the rat spinal cord. The IL-1h-induced iNOS protein activity
was almost completely eliminated by the 1400W pretreat-
ment, and the IL-1h-induced thermal hyperalgesia was also
reversed.
In conclusion, we found that i.t. injection of IL-1h results
in activation of the iNOS-NO signal transduction cascade in
the rat spinal cord, which leads to thermal hyperalgesia.
Inhibition of iNOS by i.t. 1400W pretreatment significantly
reduced the IL-1h-induced iNOS-NO activation and ame-
liorated i.t. IL-1h-induced thermal hyperalgesia. On the
basis of the present study, we suggest that administration
of an i.t. iNOS inhibitor is a potential treatment for the
central iNOS-NO-mediated inflammatory and neuropathic
pain syndromes.
Acknowledgements
The authors wish to thank Dr. Yoshito Takano for his
helpful communications on the algesic effect of spinal IL-1hand Mr. Yao-Chang Chen for the statistical analysis. This
work was supported by grants from the National Science
Council (NSC 91-2314-B-075-070) and Ministry of De-
fense (DOD-93-2-08) of the Republic of China.
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