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Intrathecal interleukin-1β administration induces thermal hyperalgesia by activating inducible nitric oxide synthase expression in the rat spinal cord
Brain Research 1015 (2004) 145 – 153 www.elsevier.com/locate/brainres
Research report
Intrathecal interleukin-1h administration induces thermal hyperalgesia by activating inducible nitric oxide synthase expression in the rat spinal cord Chun-Sung Sung a,b, Zhi-Hong Wen c, Wen-Kuei Chang b, Shung-Tai Ho c, Shen-Kou Tsai b, Yi-Chen Chang c, Chih-Shung Wong c,* a
Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan b Department of Anesthesiology, Veterans General Hospital-Taipei, Taipei, Taiwan c Department 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) cascade in 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] * Corresponding author. Tel.: +886-2-87927128; fax: +886-287927127. E-mail address: [email protected] (C.-S. Wong). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.04.068
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
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mRNA levels in the rat spinal cord. Administration of IL1h produces hyperalgesia; in contrast, blockade of IL-1h reduces pain [10,30,32,34,35,37]. It is apparent that IL-1h is 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 increasing 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 stimulation 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-1h and the NOS-NO cascade have been suggested to modulate nociception, there is little information concerning the interaction 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 IL1h-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 Am inner 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 polycarbonate 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 Breeding 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 nociceptive 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 spontaneous nociceptive behaviors (e.g., vocalizations and flaccidity) in three rats. The dose of 1400W was decreased to 10 Ag and 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,
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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 IL1h, 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.
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(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.
2.3. Behavioral assessment 2.5. NOS activity assay 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 17.6 F 0.4 s, and the cut-off time was set at 22 F 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-mercaptoethanol, 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% methanol) 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 Laboratories, 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
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 3 L-[ H]arginine with a final volume of 50 Al for 30 min at 37 jC, either with or without NOS inhibitor NG-nitro-Larginine 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]citrulline by centrifugation at 12,500 g for 30 s. The scintillation cocktail fluid was added to the eluate, containing L-[3H]citrulline, and then the radioactivity was quantitated 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)
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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 Instruments, 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 dihydrochloride] 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 (Buckinghamshire, UK). Reduced NADPH (h-nicotinamide adenine dinucleotide phosphate) was purchased from Sigma (USA). 2.8. Data analysis All data are presented as mean F 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-induced thermal hyperalgesia After i.t. injection of IL-1h (100 ng), thermal hyperalgesia was observed with the PWL reduced to 50 – 60% of baseline level between 4 and 24 h (Fig. 1). This hypersensitivity 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 hyperalgesia (Fig. 1).
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 Ag pretreatment 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 mean F 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.
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 (5530 F 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 significantly inhibited the IL-1h-induced iNOS activity to 1.8% (4th hour) and 0.5% (6th hour) of that induced by IL-1h injection (Fig. 3). Moreover, iNOS inhibitor 1400W pre-
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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-1h injection 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 mean F S.E.M. (n = 3) at each time-point in (B). *p < 0.05 indicated a significant difference from baseline value.
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.
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-
Fig. 3. The time course of iNOS activity changes in lumbar spinal cord after i.t. injection of IL-1h (100 ng) with or without i.t. 1400W (10 Ag) pretreatment. Rats were sacrificed at baseline (prior to drug injection) and at 2, 4, 6, 8, 10, 12 and 24 h, respectively after each drug injection and iNOS activity was determined by the stoichiometric conversion of L-[3H]arginine to L-[3H]citrulline by using the Cayman NOS assay kit. The data are expressed as count per minute per Ag of total protein (cpm/Ag protein). All the data are presented as mean F S.E.M for 4 – 6 rats. *p < 0.05 indicated a significant difference from baseline value in the IL group, p < 0.05 compared to the 1400W + IL group at the same time-point.
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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 mean F 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.
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 expression. The present results show that i.t. injection of the proinflammatory cytokine, IL-1h, induced hyperalgesia, which was produced by iNOS protein induction and activation, 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-1h injection 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 inflammation; IL-1h expression was increased at 2 and 4 h in the CSF after peripheral inflammation induction, and this was followed 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 upregulation 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 mechanical 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 injection (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
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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-bromo7-nitroindazole (selective nNOS inhibitor) or aminoguanidine (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 upregulated at 24 h (late phase) in wild type mice [42]. i.t. pretreatment 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. pretreatment 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. Maiho¨fner 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 zymosan, and they suggested that iNOS contributed to zymosaninduced thermal hyperalgesia. In addition, Wu et al. [46] demonstrated that intradermal injection of capsaicin produced 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 7NI blocked formalin-induced long-term thermal hyperalgesia in rats and suppressed formalin-induced soluble guanylate cyclase a1 subunit expression in the spinal cord as well. This study suggested that nNOS played a role in formalininduced long-term thermal hyperalgesia. IL-1h has been reported in inflammatory and neuropathic 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 inhibitor pretreatment significantly attenuated thermal hyper-
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algesia and NO release. iNOS is not constitutively expressed in vivo, and its expression is induced by lipopolysaccharide 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 immunopositive 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, Maiho¨fner 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 capsaicin, 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. administration of an iNOS inhibitor blocked the thermal hyperalgesia 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
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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]. Similarly 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 antinociceptive 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 pretreatment, 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 ameliorated 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-1h and 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 Defense (DOD-93-2-08) of the Republic of China.
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Research report
Intrathecal interleukin-1h administration induces thermal hyperalgesia by activating inducible nitric oxide synthase expression in the rat spinal cord Chun-Sung Sung a,b, Zhi-Hong Wen c, Wen-Kuei Chang b, Shung-Tai Ho c, Shen-Kou Tsai b, Yi-Chen Chang c, Chih-Shung Wong c,* a
Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan b Department of Anesthesiology, Veterans General Hospital-Taipei, Taipei, Taiwan c Department 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) cascade in 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] * Corresponding author. Tel.: +886-2-87927128; fax: +886-287927127. E-mail address: [email protected] (C.-S. Wong). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.04.068
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
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mRNA levels in the rat spinal cord. Administration of IL1h produces hyperalgesia; in contrast, blockade of IL-1h reduces pain [10,30,32,34,35,37]. It is apparent that IL-1h is 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 increasing 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 stimulation 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-1h and the NOS-NO cascade have been suggested to modulate nociception, there is little information concerning the interaction 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 IL1h-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 Am inner 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 polycarbonate 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 Breeding 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 nociceptive 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 spontaneous nociceptive behaviors (e.g., vocalizations and flaccidity) in three rats. The dose of 1400W was decreased to 10 Ag and 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,
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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 IL1h, 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.
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(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.
2.3. Behavioral assessment 2.5. NOS activity assay 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 17.6 F 0.4 s, and the cut-off time was set at 22 F 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-mercaptoethanol, 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% methanol) 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 Laboratories, 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
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 3 L-[ H]arginine with a final volume of 50 Al for 30 min at 37 jC, either with or without NOS inhibitor NG-nitro-Larginine 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]citrulline by centrifugation at 12,500 g for 30 s. The scintillation cocktail fluid was added to the eluate, containing L-[3H]citrulline, and then the radioactivity was quantitated 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)
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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 Instruments, 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 dihydrochloride] 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 (Buckinghamshire, UK). Reduced NADPH (h-nicotinamide adenine dinucleotide phosphate) was purchased from Sigma (USA). 2.8. Data analysis All data are presented as mean F 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-induced thermal hyperalgesia After i.t. injection of IL-1h (100 ng), thermal hyperalgesia was observed with the PWL reduced to 50 – 60% of baseline level between 4 and 24 h (Fig. 1). This hypersensitivity 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 hyperalgesia (Fig. 1).
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 Ag pretreatment 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 mean F 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.
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 (5530 F 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 significantly inhibited the IL-1h-induced iNOS activity to 1.8% (4th hour) and 0.5% (6th hour) of that induced by IL-1h injection (Fig. 3). Moreover, iNOS inhibitor 1400W pre-
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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-1h injection 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 mean F S.E.M. (n = 3) at each time-point in (B). *p < 0.05 indicated a significant difference from baseline value.
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.
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-
Fig. 3. The time course of iNOS activity changes in lumbar spinal cord after i.t. injection of IL-1h (100 ng) with or without i.t. 1400W (10 Ag) pretreatment. Rats were sacrificed at baseline (prior to drug injection) and at 2, 4, 6, 8, 10, 12 and 24 h, respectively after each drug injection and iNOS activity was determined by the stoichiometric conversion of L-[3H]arginine to L-[3H]citrulline by using the Cayman NOS assay kit. The data are expressed as count per minute per Ag of total protein (cpm/Ag protein). All the data are presented as mean F S.E.M for 4 – 6 rats. *p < 0.05 indicated a significant difference from baseline value in the IL group, p < 0.05 compared to the 1400W + IL group at the same time-point.
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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 mean F 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.
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 expression. The present results show that i.t. injection of the proinflammatory cytokine, IL-1h, induced hyperalgesia, which was produced by iNOS protein induction and activation, 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-1h injection 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 inflammation; IL-1h expression was increased at 2 and 4 h in the CSF after peripheral inflammation induction, and this was followed 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 upregulation 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 mechanical 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 injection (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
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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-bromo7-nitroindazole (selective nNOS inhibitor) or aminoguanidine (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 upregulated at 24 h (late phase) in wild type mice [42]. i.t. pretreatment 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. pretreatment 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. Maiho¨fner 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 zymosan, and they suggested that iNOS contributed to zymosaninduced thermal hyperalgesia. In addition, Wu et al. [46] demonstrated that intradermal injection of capsaicin produced 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 7NI blocked formalin-induced long-term thermal hyperalgesia in rats and suppressed formalin-induced soluble guanylate cyclase a1 subunit expression in the spinal cord as well. This study suggested that nNOS played a role in formalininduced long-term thermal hyperalgesia. IL-1h has been reported in inflammatory and neuropathic 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 inhibitor pretreatment significantly attenuated thermal hyper-
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algesia and NO release. iNOS is not constitutively expressed in vivo, and its expression is induced by lipopolysaccharide 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 immunopositive 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, Maiho¨fner 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 capsaicin, 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. administration of an iNOS inhibitor blocked the thermal hyperalgesia 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
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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]. Similarly 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 antinociceptive 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 pretreatment, 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 ameliorated 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-1h and 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 Defense (DOD-93-2-08) of the Republic of China.
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