Minocycline and Fluorocitrate Suppress SpinalNociceptive Signaling in Intrathecal IL-1b–InducedThermal Hyperalgesic Rats

CHUN-SUNG SUNG,1,2 CHEN-HWAN CHERNG,3 ZHI-HONG WEN,4 WEN-KUEI CHANG,1,2 SHI-YING HUANG,4

SHINN-LONG LIN,3 KWOK-HON CHAN,1,2AND CHIH-SHUNG WONG5*

1Department of Anesthesiology, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China2School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China3Department of Anesthesiology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan,Republic of China4Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center,National Sun Yat-Sen University, Kaohsiung, Taiwan, Republic of China5Department of Anesthesiology, Cathay General Hospital, Taipei, Taiwan, Republic of China

KEY WORDSinterleukin-1b; spinal cord; microglia; astrocyte; MAPK;iNOS

ABSTRACTWe previously demonstrated that intrathecal IL-1b causedthermal hyperalgesia in rats. This study was conducted toexamine the effects and cellular mechanisms of glial inhibi-tors on IL-1b–induced nociception in rats. The effects ofminocycline (20 lg), fluorocitrate (1 nmol), and SB203580 (5lg) on IL-1b (100 ng) treatment in rats were measured bynociceptive behaviors, western blotting of p38 mitogen-acti-vated protein kinase (MAPK) and inducible nitric oxidesynthase (iNOS) expression, cerebrospinal fluid nitric oxide(NO) levels, and immunohistochemical analyses. Theresults demonstrated that intrathecal IL-1b activatedmicroglia and astrocytes, but not neurons, in the dorsalhorn of the lumbar spinal cord, as evidenced by morphologi-cal changes and increased immunoreactivity, phosphoryl-ated p38 (P-p38) MAPK, and iNOS expression; the activa-tion of microglia and astrocytes peaked at 30 min andlasted for 6 h. The immunoreactivities of microglia andastrocytes were significantly increased at 30 min (6.6- and2.7-fold, respectively) and 6 h (3.3- and 4.0-fold, respec-tively) following IL-1b injection, as compared with salinecontrols at 30 min (all P < 0.01). IL-1b induced P-p38MAPK and iNOS expression predominantly in microgliaand less in astrocytes. Minocycline, fluorocitrate, orSB203580 pretreatment suppressed this IL-1b–upregulatedP-p38 MAPK mainly in microglia and iNOS mainly inastrocytes; minocycline exhibited the most potent effect.Minocycline and fluorocitrate pretreatment abrogated IL-1b–induced NO release and thermal hyperalgesia in rats.In conclusion, minocycline, fluorocitrate, and SB203580effectively suppressed the IL-1b–induced central sensitiza-tion and hyperalgesia in rats. VVC 2012 Wiley Periodicals, Inc.

INTRODUCTION

The proinflammatory cytokine interleukin-1b (IL-1b) ishighly inducible and known to mediate the pain response;elevated IL-1b concentrations in the cerebrospinal fluid(CSF) were observed in patients with complex regional

pain syndrome and rat models of inflammatory and neu-ropathic pain (Alexander et al., 2005; Samad et al., 2001;Whitehead et al., 2010). Previous studies have demon-strated that exogenous administration of IL-1b in thecentral nervous system (CNS) causes pain (Rady andFujimoto, 2001; Sung et al., 2004). In the CNS, astrocytesprovide trophic support, regulate synaptic transmission,maintain the homeostasis of the microenvironment, andmediate innate immunity, while microglia serve as resi-dent immune cells, producing and releasing proinflamma-tory cytokines such as IL-1b in response to stress,trauma, infection, inflammation, and degeneration(Newman, 2003; Srinivasan et al., 2004). In addition tothe well-known role of neurons in nociception (Vardehet al., 2009), astrocytes and microglia also play importantroles in nociceptive processing, particularly followinginflammation or nerve injury (Clark et al., 2007; Itoet al., 2009; Milligan et al., 2001; Raghavendra et al.,2003). Studies have demonstrated that glial inhibitorscan attenuate inflammatory or neuropathic pain (Clarket al., 2007; Hua et al., 2005; Milligan et al., 2001; Wat-kins et al., 1997). Upon inflammatory or neuropathicstimulation, p38 mitogen-activated protein kinase (p38MAPK) is phosphorylated in activated neurons and glialcells (Hua et al., 2005; Sorkin et al., 2009; Xu et al.,2007). IL-1b was shown to induce p38 MAPK phosphoryl-ation in neurons (Srinivasan et al., 2004), astrocytes (Wuet al., 2004), and mixed glial cells (Pinteaux et al., 2002).Similarly, we previously demonstrated that intrathecal(i.t.) IL-1b administration induces thermal hyperalgesiaand activates the p38 MAPK/inducible nitric oxide syn-thase (iNOS) signaling cascade in the rat spinal cord (SC)

Grant sponsor: National Science Council of Taiwan, Republic of China; Grantnumbers: NSC93-2314-B-075-089, NSC94-2314-B-075-031, and NSC95-2314-B-075-096-MY2; Grant sponsor: Taipei Veterans General Hospital, Taiwan, Republic ofChina; Grant numbers: VGH-94-342, V95C1-119, V96C1-116, and V97A-001.

*Correspondence to: Chih-Shung Wong, Department of Anesthesiology, CathayGeneral Hospital, #280, Sec. 4, Renai Rd., Taipei 10630, Taiwan, Republic ofChina. E-mail: w82556@gmail.com

Received 7 January 2012; Accepted 15 August 2012

DOI 10.1002/glia.22415

Published online in Wiley Online Library (wileyonlinelibrary. com).

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VVC 2012 Wiley Periodicals, Inc.

(Sung et al., 2004, 2005). However, it is not yet clearwhich types of cells are activated by IL-1b. Intrathecalinjection of fluorocitrate, p38 MAPK inhibitors, or IL-1receptor antagonists (IL-1ra) all effectively reversed theallodynia in zymosan-induced sciatic inflammatory painin rats; these data suggest that the expression of p38MAPK and IL-1b in spinal glia is important in the devel-opment and maintenance of neuroinflammatory pain(Milligan et al., 2003). Moreover, intrathecal injection offluorocitrate dose-dependently reduced mechanicalhypersensitivity in hind paw incision-induced postopera-tive pain in rats, suggesting that astrocytic activationcontributes to mechanical hypersensitivity (Obata et al.,2006). In a rat model of postoperative pain, spinal astro-cytes were activated within 24 h, and profound activationof microglia and astrocytes was observed at day 3 afterplantar incision (Wen et al., 2009). Moreover, microgliaare activated following L5 spinal nerve transection andhind paw incision in rats (Ito et al., 2009). These reportssupport the role of spinal astrocytes and microglia inpostoperative and neuropathic pain development.

In this study, we hypothesized that spinal glial cellsare activated in response to IL-1b administration andare responsible for i.t. IL-1b–induced nociceptive signal-ing. Accordingly, we examined the cell-type distributionof IL-1b–induced p38 MAPK/iNOS nociceptive signalingin the rat SC by pharmacological intervention. We foundthat i.t. IL-1b injection rapidly activated spinal astro-cytes and microglia and upregulated p38 MAPK/iNOSnociceptive signaling, subsequently resulting in thermalhyperalgesia in rats. Moreover, minocycline and fluoroci-trate effectively ameliorated the IL-1b–induced neuroin-flammatory pain in rats.

MATERIALS AND METHODSAnimal Preparation and Intrathecal Drug

Delivery

All experiments were approved by the InstitutionalAnimal Care and Use Committee of our institution andconformed to the guidelines of the Committee forResearch and Ethical Issues of the International Associa-tion for the Study of Pain. Adult male pathogen-free Wis-tar rats (weight, 260–320 g) were purchased from theNational Laboratory Animal Center (Taipei, Taiwan).Construction of the i.t. catheter and microdialysis probeand animal preparation were conducted as previouslydescribed (Sung et al., 2004, 2005). In brief, rats wereanesthetized with pentobarbital (50 mg/kg, intraperito-neal), and an i.t. catheter was implanted with or withouta microdialysis probe. Postoperative care included subcu-taneous amoxicillin (150 mg/kg) and wound lidocaineinfiltration. Rats were allowed to recover for 4 daysbefore experimentation and were housed individuallywith ad libitum food and water on a standard 12:12-hlight/dark cycle at room temperature. Both i.t. catheterand microdialysis tubes were perfused with artificialCSF (aCSF) every other day to prevent obstruction. Ratswith neurological deficits were excluded from the study.

On day 5 after i.t. catheterization, various drugs wereadministered through the catheter, followed by 10 lLaCSF to flush the catheter. Rats were randomlyassigned to one of seven groups: (i) control rats wereinjected with saline at a volume of 5 lL; (ii) IL grouprats received IL-1b (100 ng, i.t.); (iii) SB 1 IL group ratswere injected with the p38 MAPK inhibitor SB203580(SB, 5 lg, i.t.) 1 h before IL-1b injection; (iv) Mino grouprats received the microglial inhibitor minocycline (Mino,20 lg, i.t.); (v) Mino 1 IL group rats received minocy-cline (20 lg, i.t.) given 1 h before IL-1b injection; (vi) FCgroup rats received the astrocyte metabolic inhibitor flu-orocitrate (FC, 1 nmol, i.t.); and (vii) FC 1 IL group ratsreceived fluorocitrate (1 nmol, i.t.), injected 1 h beforeIL-1b injection. Doses of IL-1b, SB203580, fluorocitrate,and minocycline were chosen according to previous stud-ies (Fonnum et al., 1997; Ledeboer et al., 2005; Sunget al., 2005).

Nociceptive Behavioral Assessment

Paw withdrawal latency (PWL) to radiant heat wasused to assess thermal hyperalgesia (UGO Basile Biolog-ical Instruments, Comerio, Italy). Rats were placed inplastic cages on a glass platform, and the heat sourcewas positioned directly beneath the right hind paw. Theheat intensity was adjusted to obtain an average PWLof 17.6 6 0.4 s, and the cut-off time was set at 22 6 0.4s to prevent tissue damage. PWL was assessed at base-line (before i.t. injection) and at 2, 4, 6, 8, 10, and 12 hafter drug injection.

Analysis of Nitric Oxide in Spinal CSF Dialysates

A CMA102 microinfusion pump with a gastight Ham-ilton syringe was used to continuously perfuse aCSFthrough one end of the microdialysis probe at a rate of5 lL/min, and the other end was used to collect CSFdialysates. Dialysates were collected in polypropylenetubes on ice prior to drug administration (baseline) andevery 30 min for 12 h after drug administration; sam-ples were then frozen at 280�C until use. Nitric oxide(NO) concentrations were measured by chemilumines-cence detection (NOA 280; Sievers Instruments, Boulder,CO) as described previously (Sung et al., 2004, 2005).

Spinal Cord Sample Preparation andWestern Blotting

Dorsal parts of the rat lumbar SC were collected 30min or 6 h after i.t. drug injection by exsanguinationunder isoflurane anesthesia, immediately frozen, andstored at 280�C until use. Samples were homogenizedin ice-cold lysis buffer, and tissue extracts were electro-phorezed and transferred to a polyvinylidene difluoride(PVDF) membrane as described previously (Sung et al.,2005). The membrane was blocked with 5% nonfat dry

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milk in Tris-buffered saline (TBS) for 30 min at roomtemperature, followed by incubation with primary anti-bodies overnight at 4�C. The primary antibodies used inthis study were mouse anti-iNOS monoclonal antibodies(1:1000; Transduction Laboratories, Lexington, KY) andrabbit polyclonal p38 or P-p38 MAPK antibodies(1:1,000; PhosphoPlus p38 MAPKinase Antibody Kit;Cell Signaling Technology, Beverly, MA). The membranewas washed, blocked with 5% nonfat dry milk in a mix-ture of TBS and Tween 20, and then incubated withappropriate horseradish peroxidase-conjugated second-ary antibodies for 1 h at room temperature. Bound anti-bodies were detected by chemiluminescence; the imageswere subsequently visualized, and the density of eachband was measured using a computer-assisted imageanalysis system (BioSpectrum 600; UVP, Upland, CA).PVDF membranes were stripped and reblotted with dif-ferent antibodies. Densitometry was used to evaluatethe density of the bands as relative densities comparedwith the background. SC b-actin was used as the inter-nal control for protein loading, and data were expressedas a ratio of the protein of interest to b-actin. In con-trast, the relative densities of P-p38 MAPK bands werecalculated and normalized relative to the total p38MAPK of each sample.

Immunohistochemical Assay

Rat SC samples were subjected to immunohistochemi-cal assay to detect the expression of P-p38 MAPK (10min, 30 min, and 6 h) and iNOS (30 min and 6 h) afterIL-1b injection. Cell markers for neurons (NeuN), astro-cytes (glial fibrillary acidic protein, GFAP), andmicroglia (CD11b/c) were used to identify the cell-typedistribution of P-p38 MAPK or iNOS protein in IL-1b–stimulated cells of the rat SC. The effects of glial inhibi-tors on IL-1b–induced phosphorylation of p38 MAPKand iNOS expression were assessed at 10 min (n 5 3 ineach group), 30 min (n 5 4 in each group), and 6 h (n 5

4 in each group) after IL-1b administration. Rats wereanesthetized with intraperitoneal pentobarbital (65 mg/kg) and transcardially perfused with 200 mL saline and4% paraformaldehyde in 0.1 M phosphate-buffered sa-line (PBS; pH 7.4). The lumbar SC enlargement wasrapidly removed and dissected, postfixed in 4% parafor-maldehyde in 0.1 M PBS for 2 h and washed in PBSthree times; subsequently, the tissues were transferredto 30% sucrose in PBS at 4�C until the tissue sank andsaturated and were then mounted in optimal cuttingtemperature compound and stored at 280�C until use.The SC was cut into sections (20-lm thick) on a cryostatset at 225�C, mounted serially onto microscope slides,and processed for immunofluorescence studies. Sectionswere air-dried on microscope slides for 30 min at roomtemperature, immersed in acetone/methanol (1:1) for 10min at 220�C, and washed three times in ice-cold 0.1 MPBS, then pre-incubated with 4% normal goat serumdiluted in 0.01% Triton X-100 in PBS for 1 h at roomtemperature. After washing in ice-cold PBS, sections

were incubated overnight at 4�C with appropriate pri-mary antibodies in 0.01% Triton X-100 and 2% normalgoat serum in PBS. To detect the topographic distribu-tion of proteins expressed in the dorsal horn of lumbarSCs (LSCDH), anti-iNOS monoclonal antibodies (1:50dilution; BD Pharmingen, San Diego, CA) and anti-P-p38 MAPK monoclonal antibodies (1:100 dilution; CellSignaling Technology, Beverly, MA) were used. Primarymonoclonal antibodies against cell markers were used tolabel neurons with Alexa Fluor 488-conjugated anti-mouse NeuN (1:100 dilution; Chemicon, Temecula, CA),astrocytes with Alexa Fluor 488-conjugated anti-mouseGFAP (1:200 dilution; Molecular Probes, Eugene, OR),and microglia with Alexa Fluor 488-conjugated anti-ratCD11b/c (1:200 dilution; Serotec, Oxford, UK). Single im-munofluorescence was used to examine the temporalpatterns of cell activation and induction of P-p38MAPK/iNOS after drug administration. After washing threetimes in PBS, sections were incubated with Alexa Fluor488-labeled goat anti-mouse IgG (green fluorescence,1:400 dilution; Jackson ImmunoResearch Laboratories,West Grove, PA) or DyLight 549-conjugated donkey anti-rabbit secondary antibodies (red fluorescence, 1:400 dilu-tion; Jackson ImmunoResearch Laboratories, WestGrove, PA) for 40 min at room temperature, followed bythree more washes in PBS. Double immunofluorescentstaining was performed to investigate the effects of vari-ous drugs on the cell-type distribution of P-p38 MAPKand iNOS. For colocalization studies, sections were incu-bated with a mixture of each of the primary cell markerantibodies (anti-GFAP, anti-CD11b/c, or anti-NeuN) andanti-P-p38 MAPK or anti-iNOS overnight at 4�C, fol-lowed by incubation with a mixture of Alexa Fluor 488-and DyLight 549-conjugated secondary antibodies for 40min at room temperature. Finally, the sections werewashed and mounted, and examined under a Leica DM-6000CS fluorescence microscope (Leica, Wetzlar, Ger-many) equipped with a charge-coupled device SpotXplorer integrating camera (Diagnostic Instruments,Sterling Heights, MI), and analyzed by SPOT software(Diagnostic Instruments). Confocal double-immunostain-ing images were captured with a Leica TCS SP5 II con-focal microscope (Leica, Wetzlar, Germany). For pixelmeasurement and analysis with density-slicing andquantitative colocalization, Image J software (NationalInstitutes of Health, Bethesda, MD) was used.

For quantification of GFAP and CD11b/c immunofluo-rescence and determination of the number of immunore-active cells colabeled with P-p38 MAPK or iNOS inLSCDH, four randomly selected SC sections were meas-ured and averaged for each rat. Then, the mean of fourrats in each group was calculated. Image measurementand quantization were obtained for the LSCDH under203 and 403 objective lenses, and confocal images wereanalyzed under a 633 oil lens. The sizes and conditionsof the image captures and views were kept constant atthe ipsilateral LSCDH. After setting the threshold toeliminate background noise for specific signals, immuno-fluorescence intensities above the threshold were calcu-lated. Immunoreactivity (IR) data are expressed as the

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fold change in immunofluorescence intensity for samplesfrom the experimental group versus samples from thecontrol animals (set as 100%).

Drug Preparation

Recombinant rat IL-1b (R&D Systems, Minneapolis,MN) was dissolved in physiological saline at a concen-tration of 50 ng/lL. SB203580 (Calbiochem, La Jolla,CA) was dissolved in physiological saline at a concentra-tion of 1 lg/lL. Fluorocitrate and minocycline were pur-chased from Sigma-Aldrich (St. Louis, MO) and dis-solved in sterile physiological saline at concentrations of1 nmol/lL and 2 lg/lL, respectively. All drugs werefreshly prepared on the morning of the experiment.

Statistical Analysis

Data are expressed as the mean 6 S.E.M. Changes inPWL and NO concentrations are expressed as percen-tages of baseline values. For western blot analysis, theintensity of each test band was expressed as the inte-grated optical density calculated with respect to the av-erage optical density of the corresponding control groupband. Statistical analysis of protein expression, immuno-fluorescent intensity, and ratio of cell colocalization wereused in either 1-way or 2-way analysis of variance(ANOVA), followed by the Student–Newman–Keuls posthoc test. In addition, 2-way repeated measures ANOVAfollowed by the Student-Newman-Keuls post hoc test,with time (before and after intrathecal injection) as thewithin-subjects variable and grouping as the between-subjects variable were used to analyze PWL and NOconcentrations. Mauchly’s sphericity test was used tocheck the equal variance assumption of the differencesbetween levels of the repeated measures factor. TheHuynh-Feldt correction was applied if the assumption ofsphericity was violated. A P-value <0.05 was consideredstatistically significant. All statistical analyses werecarried out with the Statistical Package for the SocialSciences (SPSS) version 17.0 for Windows (SPSS,Chicago, IL).

RESULTSIL-1b Induces p38 MAPK Phosphorylation andiNOS Expression in Astrocytes and Microglia in

LSCDH

Our previous study demonstrated that upregulatedP-p38 MAPK protein expression could be observed from30–240 min, while iNOS protein expression peaked at 6h following IL-1b injection (Sung et al., 2005). Therefore,we assessed P-p38 MAPK-IR at 30 min and iNOS-IR at6 h after IL-1b injection. Double-labeled immunostain-ing identified very low expression of P-p38 MAPK-IR inscattered CD11b/c-immunostained microglia and GFAP-positive astrocytes, but none in NeuN-positive neurons,

at 30 min after saline injection (Fig. 1). P-p38 MAPK-IRdid not change at 10 min after IL-1b injection (data notshown). However, IL-1b markedly increased P-p38MAPK-IR by 144.8-fold at 30 min, as compared with sa-line-treated rats (F[4,15] 5 14.051; P < 0.001; Fig. 7B),and P-p38 MAPK-immunopositive cells exhibited glia-like morphology in the superficial laminae I and II ofLSCDH (Fig. 1D,E). Double immunolabeling for P-p38MAPK and cell markers revealed that P-p38 MAPK wasexpressed in both GFAP-immunopositive astrocytes (Fig.1D) and CD11b/c-immunopositive microglia at 30 min af-ter IL-1b injection (Fig. 1E); no expression was detectedin NeuN-immunoreactive neurons (Fig. 1F). Confocalmicroscopy revealed colocalization of P-p38 MAPK-IRwith GFAP (Fig. 1G) and CD11b/c (Fig. 1H); however,the majority of P-p38–positive cells were microglia (Fig.7C). In contrast, no iNOS-IR was observed in LSCDH ofsaline-injected rats. Moreover, IL-1b markedly increasedthe number of iNOS-positive cells compared with saline-injected controls, and iNOS-positive cells exhibited aglia-like morphology (Fig. 2). Double immunostainingdemonstrated that iNOS-IR was colocalized with GFAP-IR in astrocytes (Fig. 2D) and CD11b/c-IR in microglia(Fig. 2E) at 6 h after IL-1b injection, but not in NeuN-positive neurons (Fig. 2F). Confocal microscopy alsoshowed colocalization of iNOS with GFAP (Fig. 2G) andCD11b/c (Fig. 2H), and the majority of iNOS-positivecells were microglia (Fig. 8C,D). Astrocytes expressingP-p38 MAPK and/or iNOS became hypertrophic andmore ramified (Figs. 1G and 2G), and microglia express-ing P-p38 MAPK and/or iNOS became hypertrophic andmore amoeboid after IL-1b administration (Figs. 1H and2H). These data suggest that i.t. IL-1b activated bothspinal astrocytes and microglia, causing increased p38MAPK phosphorylation, iNOS expression, and morpho-logical changes.

IL-1b Increases GFAP and CD11b/c,but not NeuN, IR

In the saline-control group, GFAP-immunopositiveastrocytes had a less ramified morphology in LSCDH,and the intensity of GFAP immunofluorescence washigher at 30 min than at 6 h (100% vs. 36.1%; F[1,30] 59.258; P 5 0.005; Fig. 3A,B). In contrast, i.t. IL-1binduced time-dependent astrocyte activation manifestedby a strong GFAP-IR, increased cell number, and hyper-trophic morphology, from 30 min to 6 h (Fig. 3A). Two-way ANOVA followed by Student-Newman-Keuls posthoc test showed a significant effect for grouping(F[4,30]5 11.352; P < 0.001) and time (F[1,30]5 9.258;P 5 0.005). IL-1b significantly increased GFAP-IR at 30min (2.7-fold), as compared with 6 h (1.4-fold), whennormalizing GFAP-IR for each group at 30 min and 6 hto that of the control group at 30 min (P 5 0.005;Fig. 3B). However, the magnitude of GFAP-IR increasewas higher at 6 h than at 30 min after IL-1b injection(4.0- vs. 2.7-fold, respectively) when GFAP-IR of eachgroup was normalized to that of the control group at

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Fig. 1. Cellular localization of P-p38 MAPK in astrocytes and micro-glia in the dorsal horn of rat lumbar spinal cords (LSCDH) at 30 minafter i.t. IL-1b injection. A–F: (Double-labeled immunofluorescencemerged images) Depict cells labeled with cell-specific green marker(GFAP, CD11b/c, or NeuN) and P-p38 MAPK (red staining) in spinalcord (SC) sections (20-lm thick) from saline-injected (A, B, and C) andIL-1b–injected (D, E, and F) rats. P-p38 MAPK immunoreactivity (IR)was detected in both GFAP-positive astrocytes (yellow signal, arrows in

D) and CD11b/c-positive microglia (yellow signal, arrows in E) after IL-1b injection. Confocal microscopy demonstrated colocalization of P-p38MAPK IR with GFAP in astrocytes (arrows in G) and CD11b/c in micro-glia (arrows in H). Most of the P-p38 MAPK-positive cells are microglia.Magnification: 4003 (A–F) and 6303 (G and H). Scale bars 5 100 lm(A–F) and 10 lm (G and H). [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

Fig. 2. Cellular localization of iNOS in astrocytes and microglia inLSCDH at 6 h after i.t. IL-1b injection. Immunofluorescence micro-graphs show double labeling of iNOS (red) and GFAP for astrocytes(green in A and D), CD11b/c for microglia (green in B and E), andNeuN for neurons (green in C and F) in SC sections (20 lm thick) fromsaline-injected controls rats (A, B, and C) and IL-1b–injected rats (D,E, and F). IR of iNOS was observed in both GFAP-positive astrocytes

(yellow signal, arrows in D) and CD11b/c-positive microglia (yellow sig-nal, arrows in E) at 6 h after IL-1b injection, as shown by the mergedimages. Confocal microscopy showed the colocalization of iNOS-IR withGFAP in astrocytes (arrows in G) and CD11b/c in microglia (arrows inH). Magnification: 4003 (A–F) and 6303 (G and H). Scale bars 5 100lm (A–F) and 10 lm (G and H). [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]

each indicated time point (Fig. 3C). CD11b/c-immuno-positive microglia with a resting ramified morphology inLSCDH and similar CD11b/c-IR at 30 min and 6 h wereobserved after saline injection in the control group(100 vs. 82.1%; F[1,30] 5 9.205; P 5 0.005; Fig. 4A,B).In contrast, IL-1b induced microglial activation, mani-fested by increased CD11b/c-IR, a higher cell density,and hypertrophic and amoeboid morphology, at 30 minand 6 h (Fig. 4A). IL-1b resulted in a significantlygreater increase in CD11b/c-IR at 30 min (5.8-foldincrease) than at 6 h (2.8-fold increase) when normaliz-ing the CD11b/c-IR data of each group at 30 min and 6h to that of the control group at 30 min (P 5 0.005; Fig.4B). The magnitude of IL-1b–induced microglia activa-tion was greater than that of astrocyte activation at 30min (5.8- vs. 2.7-fold, respectively), but was similar at 6h (1.4- vs. 2.8-fold, respectively) when normalizing thedata from GFAP-IR or CD11b/c-IR of each group to thatof the control group at each indicated time (Figs. 3C and4C). In contrast, IL-1b did not alter NeuN-IR at either30 min or 6 h (data not shown). These results suggestedthat i.t. IL-1b injection activated both microglia andastrocytes in LSCDH, with a stronger activation at 30min than at 6 h, and was more prominent in microgliathan in astrocytes.

SB203580, Minocycline, and Fluorocitrate InhibitIL-1b–activated Astrocytes and Microglia

Pretreatment with SB203580, minocycline, or fluoroci-trate markedly inhibited the IL-1b–induced astrocyteactivation at both 30 min and 6 h (Fig. 3B,C). Further-more, pretreatment with SB203580, minocycline, and flu-orocitrate exerted a similar effect on the attenuation of IL-1b–increased GFAP-IR in the rat LSCDH both at 30 min(80.2, 79.1, and 75.2% reductions, respectively; F[4,15] 536.978; all P < 0.001) and 6 h (82.9, 83.5, and 80.7% reduc-tions, respectively; F[4,15] 5 34.383; all P < 0.001). How-ever, there were no differences in CD11b/c-IR detectedamong SB 1 IL, Mino 1 IL, and FC 1 IL groups (P > 0.05;Fig. 3C). Two-way ANOVA followed by Student-Newman-Keuls post hoc test showed a significant difference both ingrouping (F[4,30] 5 11.352; P < 0.001) and time (F[1,30]5 9.258; P 5 0.005) on GFAP-IR. Moreover, i.t. pretreat-ment with SB203580, minocycline, or fluorocitrate alsosignificantly inhibited IL-1b–induced microglial activa-tion at 30 min and 6 h (Fig. 4B,C). A similar time-depend-ent downregulation of CD11b/c-IR from 30 min to 6 h wasobserved in IL, SB 1 IL, Mino 1 IL, and FC 1 IL groups(Fig. 4B). Two-way ANOVA followed by Student-Newman-Keuls post hoc test showed a significant effect both for

Fig. 3. Temporal patterns of the astrocytic response to various drugsin LSCDH. Representative immunofluorescent images of astrocytesstained with GFAP (green) in the LSCDH sections (20 lm thick) areshown, depicting the density and morphology of the control (saline), IL-1b (IL), SB 1 IL-1b (SB 1 IL), minocycline 1 IL-1b (Mino 1 IL), andfluorocitrate 1 IL-1b (FC 1 IL) groups at 30 min and 6 h (panel A,magnification: 1003). Insets depict GFAP-immunopositive astrocytes(A). Quantification of GFAP-IR after normalization of GFAP-IR data for

each group at 30 min and 6 h to that of the control group at 30 min(B). Quantification of GFAP-IR after normalization of GFAP-IR datafrom each group to that of the control group at each indicated time (C).n 5 4 rats per group. *, P < 0.05 compared with the saline treated con-trol at the indicated time points. #, P < 0.05 compared with IL-1b–injected rats at the indicated time points. Scale bars 5 200 lm in A; 50lm in insets. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

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grouping (F[4,30] 5 13.425; P < 0.001) and time (F[1,30]5 9.258; P 5 0.005) on CD11b/c-IR. In addition, pretreat-ment with SB203580, minocycline, and fluorocitratemarkedly attenuated the IL-1b–increased CD11b/c-IR at

both 30 min (82.1, 71.8, and 71.6% reductions, respec-tively; F[4,15] 5 11.582; all P � 0.002) and 6 h (75.9, 88.3,and 58.3% reductions, respectively; F[4,15] 5 19.758; allP � 0.001; Fig. 4C). Similarly, no significant differences in

Fig. 4. Temporal patterns of the microglial response in LSCDH. Rep-resentative immunofluorescent micrographs of microglia stained withCD11b/c (green) in LSCDH sections (20 lm thick) show the microgliadensity and morphology of the control (saline), IL-1b (IL), SB 1 IL-1b(SB 1 IL), minocycline 1 IL-1b (Mino 1 IL), and fluorocitrate 1 IL-1b(FC 1 IL) groups at 30 min and 6 h (panel A, magnification: 1003).Insets depict CD11b/c-immunopositive microglia (A). Quantification ofCD11b/c-IR after normalization of the CD11b/c-IR data of each group at

30 min and 6 h to that of the control group at 30 min (B). Quantificationof CD11b/c-IR after normalization of the data for either GFAP-IR orCD11b/c-IR to that of the control group at each indicated time (C). n 5 4rats per group. *, P < 0.05, compared with saline-treated controls at theindicated time points. #, P < 0.05, compared with IL-1b–injected rats atthe indicated time points. Scale bars 5 200 lm in A; 50 lm in insets.[Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

Fig. 5. Effects of minocycline and fluorocitrate on IL-1b–inducedthermal hyperalgesia in rats. PWL for (A) minocycline (Mino) and (B)fluorocitrate (FC) were plotted against time in rats intrathecallytreated with saline (control, n 5 6), IL-1b (n 5 6), minocycline alone(Mino; n 5 6) or pretreatment 1 h before IL-1b treatment (Mino 1 IL,

n 5 8), and fluorocitrate alone (FC; n 5 6) or pretreatment 1 h beforeIL-1b treatment (FC 1 IL; n 5 8). Values were normalized to the base-line and expressed as the mean 6 SEM. *, P < 0.05 compared with sa-line treated controls. #, P < 0.05 compared with IL-1b–injected rats. (,P < 0.05 versus baseline data.

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CD11b/c-IR were present among SB 1 IL, Mino 1 IL, andFC 1 IL groups (P > 0.05).

Pretreatment with Minocycline or FluorocitratePrevents Intrathecal IL-1b–induced Thermal

Hyperalgesia in Rats

IL-1b induced significant thermal hyperalgesia in rats ina time-dependent manner (26.8–48%; P < 0.001), with anadir at 6 h, of the PWL from 4–12 h, as compared with thebaseline (Fig. 5). Saline, minocycline, and fluorocitrate allexerted no effect on the PWL in na€ıve rats (Fig. 5A,B). Incontrast, pretreatment with minocycline and fluorocitratesignificantly abolished the IL-1b–induced reduction ofPWL from 4–12 h, as compared with the IL-1b group (P <0.001 and P< 0.005, respectively; Fig. 5A,B). No significantdifferences were observed between Mino 1 IL and FC 1 ILgroups (P > 0.05). Two-way repeated measures ANOVA,followed by Student-Newman-Keuls post hoc test, showeda significant effect for grouping (F[5,34] 5 28.558; P <0.001) and time (F 5 30.206; df5 3.581; P < 0.001) and forthe interaction between grouping and time (F5 3.459; df517.907; P < 0.001). The results demonstrated that i.t. pre-treatment with astrocyte or microglia inhibitors could sup-press IL-1b–induced thermal hyperalgesia in rats.

Glial or p38 MAPK Inhibitors AttenuateIL-1b–induced p38 MAPK Phosphorylation

and iNOS Expression

Western blot analysis confirmed that IL-1b increasedP-p38 MAPK expression by 2.3-fold (F[6,35] 5 7.024; P

< 0.001; Fig. 6A) and induced iNOS protein expressionby 77.6-fold (F[6,35] 5 190.519; P < 0.001; Fig. 6B), ascompared with the saline-treated controls. Pretreatmentwith SB203580 significantly attenuated IL-1b–inducedp38 MAPK phosphorylation (44.6% reduction; P 5

0.003) and iNOS expression (92.3% reduction; P <0.001; Fig. 6). Intrathecal treatment with either minocy-cline or fluorocitrate alone did not affect the expressionof P-p38 MAPK (P > 0.05; Fig. 6A) or iNOS (P > 0.05;Fig. 6B). In contrast, minocycline pretreatment signifi-cantly attenuated IL-1b–induced upregulation of P-p38MAPK (42.4% reduction; P 5 0.005) and iNOS protein(94.5% reduction; P < 0.001), as compared with IL-1b–injected rats (Fig. 6). Similarly, fluorocitrate pretreat-ment attenuated IL-1b–induced thermal hyperalgesia(Fig. 5B) and suppressed P-p38 MAPK (44.5% reduction;P 5 0.003) and iNOS protein expression (93.2% reduc-tion; P < 0.001; Fig. 6). IL-1b also markedly increasedthe P-p38 MAPK-IR (144.8-fold) at 30 min comparedwith that of the saline-injected control (F[4,15] 5

14.051; P < 0.001; Fig. 7B); P-p38 MAPK-IR wasobserved in both GFAP-positive astrocytes (5.8 6 1.6%vs. 0.2 6 0.1%; F[4,15] 5 7.269; P 5 0.003) and CD11b/c-positive microglia (32.1 6 3.9% vs. 0.63 6 0.11%;F[4,15] 5 37.421; P < 0.001), as compared with the sa-line control at 30 min (Fig. 7C). In addition, the extentof IL-1b–induced P-p38 MAPK-IR expression wasgreater in microglia than in astrocytes (51.5- vs. 37.7-fold; P < 0.05). At 30 min after IL-1b injection, P-p38MAPK-IR colocalized primarily in CD11b/c-IR microgliaand, to a lesser extent, in GFAP-IR astrocytes in LSCDH(Fig. 7A). IL-1b–induced P-p38 MAPK-IR was signifi-cantly attenuated by pretreatment with minocycline

Fig. 6. Effects of various treatments on IL-1b–induced p38 MAPK phos-phorylation and iNOS protein expression in rat SCs. P-p38 MAPK and p38MAPK levels were examined 30 min after drug treatment by western blot-ting. Semi-quantitative analysis was used to determine P-p38 MAPK/p38MAPK density ratios (A). The expression of iNOS and b-actin proteins inthe dorsal part of the lumbar SC 6 h after drug treatment was also deter-

mined and expressed as iNOS/b-actin density ratios (B). The intensity ofeach test band was expressed as the integrated optical density (IOD) calcu-lated with respect to the average optical density of the corresponding controlgroup band. Control values are defined as 100%, and values are presentedas the mean 6 SEM (n 5 6 for each group). *, P < 0.05, compared with thesaline-injected controls. #, P< 0.05, compared with IL-1b–injected rats.

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(97.1% reduction; P < 0.001), SB203580 (82.3% reduc-tion; P 5 0.001), and fluorocitrate (75.9% reduction; P 5

0.002; Fig. 7B). Similarly, the percentage of GFAP-immunopositive astrocytes with concomitant P-p38MAPK immunofluorescence staining following IL-1badministration was significantly reduced from 5.8 6

1.6% to 0.3 6 0.1% by minocycline pretreatment (P 5

0.004), 1.7 6 0.7% by SB203580 pretreatment (P <0.05), and 2.2 6 0.6% by fluorocitrate pretreatment,compared with that of IL-1b–injected rats at 30 min (P< 0.05; Fig. 7C). Moreover, the percentage of P-p38MAPK immunofluorescence colocalized with CD11b/c-immunopositive microglia was dramatically reducedfrom 32.1 6 3.9% (IL-1b) to 0.9 6 0.4% by minocyclinepretreatment, 5.8 6 2.1% by SB203580 pretreatment,and 7.7 6 1.7% by fluorocitrate pretreatment (all P <0.001; Fig. 7C). Pretreatment with minocycline exertedsimilar effects on the reduction of IL-1b–induced p38MAPK phosphorylation in both microglia and astrocytesin LSCDH (97.1 and 94.8%, respectively). However, pre-treatment with SB203580 and fluorocitrate inhibited IL-1b–induced P-p38 MAPK expression in microglia (81.8and 75.9% reductions, respectively) and in astrocytes(71.6 and 61.7% reductions, respectively). Together, theincreased GFAP and CD11b/c expression at 30 min byi.t. IL-1b administration revealed the following changes:

(1) IL-1b–induced P-p38 MAPK upregulation was moreprominent in microglia than in astrocytes; (2) the inhibi-tory effects of minocycline, fluorocitrate, and SB203580pretreatment on the IL-1b–induced increase in P-p38MAPK-IR was also more pronounced in microglia thanin astrocytes (Figs. 3 and 4). Similarly, IL-1b markedlyincreased the intensity of iNOS-IR (1859-fold) at 6 hcompared with that of the saline control in the ratLSCDH (F[4,15] 5 362.488; P < 0.001; Fig. 8B). Quanti-fication of IL-1b–induced iNOS-immunopositive glialcells revealed that iNOS was primarily localized inCD11b/c-IR microglia (64.2%) and, to a lesser extent, inGFAP-IR astrocytes (21.8%) at 6 h after IL-1b adminis-tration (Fig. 8A,C). GFAP-positive astrocytes with iNOS-IR expression (19.6 6 4.7% vs. 0%; F[4,15] 5 17.424; P< 0.001) and CD11b/c-positive microglia with iNOS-IRexpression (50.2 6 7.2% vs. 0%; F[4,15] 5 14.01; P <0.001) were significantly increased at 6 h after IL-1badministration, as compared with that of saline-injectedcontrol rats (Fig. 8D). At 6 h, the IL-1b–induced iNOS-IR expression was significantly attenuated by pretreat-ment with minocycline (97.1% reduction; P < 0.001),SB203580 (82.3% reduction; P < 0.001), and fluoroci-trate (75.9% reduction; P < 0.001; Fig. 8B). The distribu-tion of iNOS-immunopositive cells was significantlyaltered by SB203580, minocycline, and fluorocitrate

Fig. 7. Effects of IL-1b, SB203580, minocycline, and fluorocitrate onthe detection of P-p38 MAPK-IR. Representative images of double-la-beled immunofluorescent staining show colocalization (yellow) of P-p38MAPK (red) and either GFAP (green) or CD11b/c (green) in LSCDH af-ter various treatments (A). Histograms depict P-p38 MAPK immunoflu-orescent intensity after various treatments (B). Histograms depict thepercentage of GFAP-positive astrocytes and CD11b/c-positive microglia

colocalized with P-p38 MAPK-IR after various treatments (C). n 5 4rats per group. Scale bars 5 10 lm. For the quantification in B and C,data were normalized to the saline control group and expressed as thefold change (mean 6 SEM; 4-section measurement per rat, n 5 4 rats ineach group) compared with control SCs. *, P < 0.05 compared with con-trols. #, P < 0.05 compared with the IL group. [Color figure can beviewed in the online issue, which is available at wileyonlinelibrary.com.]

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pretreatment, mainly in CD11b/c-stained microglia (94.3,86.8, and 89.3%, respectively) and, to a lesser extent, inGFAP-stained astrocytes (1.6, 7.7, and 6.1%, respectively;Fig. 8C). The percentage of GFAP-IR astrocytes withiNOS expression at 6 h after IL-1b administration wasdramatically reduced from 19.6 6 4.7% (in IL-1b–injectedrats) to 0.05 6 0.02% by minocycline pretreatment, 0.016 0.00% by SB203580 pretreatment, and 0.23 6 0.14%by fluorocitrate pretreatment (all P < 0.001; Fig. 8D).Similarly, the percentage of CD11b/c-IR microglia withiNOS expression at 6 h after IL-1b administration wasalso significantly reduced from 50.2 6 7.2% (IL-1b) to13.8 6 2.6% by minocycline pretreatment (P 5 0.001),24.1 6 7.5% by SB203580 pretreatment (P 5 0.021), and16.0 6 2.9% by fluorocitrate pretreatment (P 5 0.002;Fig. 8D). The reduction in IL-1b–induced iNOS-IR bypretreatment with SB203580, minocycline, and fluoroci-trate was greater in astrocytes (99.9, 99.8, and 98.8%reductions, respectively) than in microglia (52.0, 72.6,and 68.1% reductions, respectively). IL-1b–inducediNOS-IR, apparent 6 h after IL-1b administration, wasmainly found in microglia and, to a lesser extent, inastrocytes, and the effects of minocycline, fluorocitrate,and SB203580 pretreatment on attenuation of IL-1b–

induced iNOS-IR were more obvious in astrocytes than inmicroglia (Figs. 3 and 4).

IL-1b–induced NO Release by Activated SpinalAstrocytes and Microglia

Two-way repeated measures ANOVA, followed by Stu-dent-Newman-Keuls post hoc test, showed a significanteffect for time (F 5 3.921; df 5 2.594; P 5 0.016) andfor the interaction between grouping and time (F 5

16.77; df 5 7.783; P < 0.001) on NO contents in CSFdialysates. Intrathecal IL-1b injection resulted in amarked increase in NO concentration in CSF dialysatesat 4 h (105% increase), peaking at 6–6.5 h (190%increase), and lasting for 12 h (107% increase), as com-pared with the baseline (all P < 0.001; Fig. 9). Minocy-cline pretreatment decreased NO concentrations in CSFdialysates from 11–12 h, as compared with the baselinelevel, and decreased from 11.5 to 12 h, as compared withsaline controls (all P < 0.05; Fig. 9A). IL-1b–induced NOrelease was markedly inhibited, even below baseline, byminocycline pretreatment from 4–12 h, as comparedwith the IL-1b–injected group (all P < 0.001; Fig. 9A).

Fig. 8. Effects of IL-1b, SB203580, minocycline, and fluorocitrate oniNOS IR in spinal glial cells. Representative double-labeled immunoflu-orescent staining shows colocalization (yellow) of iNOS (red) withGFAP (green) or CD11b/c (green) in LSCDH after various treatments(A). Histograms depict the iNOS immunofluorescent intensities aftervarious treatments (B). Histograms depicting the cell-type distributionof iNOS-immunopositive cells revealed the percentage of iNOS-positivecells co-expressing either GFAP-IR or CD11b/c-IR after various treat-

ments (C). Histograms depicting the percentage of glial cells coexpress-ing iNOS-IR after various treatments (D). For the quantification in (B),data were normalized to the saline-injected control group and expressedas the fold change (mean 6 SEM; 4-section measurement per rat, n 54 rats in each group). Scale bars 5 10 lm. *, P < 0.05 compared withcontrols. #, P < 0.05 compared with the IL group. [Color figure can beviewed in the online issue, which is available at wileyonlinelibrary.com.]

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Fluorocitrate pretreatment decreased NO concentrationin CSF dialysates at 3, 4.5, and 5.5 to 12 h when com-pared with the baseline level, and from 11.5 to 12 hwhen compared with the saline control (P < 0.05; Fig.9B). Similarly, IL-1b–induced NO release was markedlyinhibited by fluorocitrate pretreatment from 4–12 h, ascompared with the IL-1b–injected group (all P < 0.001;Fig. 9B). Together, the attenuation of GFAP-, CD11b/c-,and iNOS-IRs by glial inhibitors indicated that the re-versal of IL-1b–induced NO release by pretreatmentwith minocycline or fluorocitrate was due to inhibition ofspinal astrocyte and microglia activity.

DISCUSSION

In this study, we demonstrated that i.t. IL-1b adminis-tration induced thermal hyperalgesia, which was associ-

ated with early reactive astrocytosis and microgliosis inthe rat LSCDH. Both p38 MAPK phosphorylation andiNOS protein expression were identified predominantlyin activated microglia and, to a lesser extent, in acti-vated astrocytes in response to IL-1b administration.Minocycline, fluorocitrate, or SB203580 pretreatmentattenuated IL-1b–induced thermal hyperalgesia andassociated astrocytic and microglial activation, p38MAPK phosphorylation and iNOS protein expression inthe SC, and NO release in CSF dialysates. Based onthese findings, we concluded that i.t. IL-1b activatedmicroglia and astrocytes in LSCDH; these activated cellswere responsible for the induction of p38 MAPK/iNOSnociceptive signaling in the SC. Minocycline, fluoroci-trate, and SB203580 pretreatment markedly attenuatedIL-1b–induced reactive astrocytosis and microgliosis,subsequently inhibiting downstream signaling andreducing thermal hyperalgesia in rats.

Upregulated IL-1b expression was observed in manyanimal models of pain. Intrathecal IL-1b promoted theC-fiber–induced nociceptive reflex in both normal andCFA-induced monoarthritic rat SC (Constandil et al.,2009; Samad et al., 2001; Whitehead et al., 2010). Bothperipheral and central administration of IL-1b inducedhyperalgesia in rats (Falchi et al., 2001; Oka et al.,1993), whereas i.t. administration of IL-1ra blockedinflammatory and neuropathic pain (Samad et al., 2001;Watkins et al., 1997; Zhang et al., 2008). Our previousstudy also demonstrated that i.t. IL-1b (100, 200, and500 ng) produced a time- and dose-dependent reductionin PWL to heat stimuli, and induced iNOS proteinexpression in SC and NO release in CSF (Sung et al.,2004). Intrathecal pretreatment with the iNOS inhibitor1400W effectively blocked IL-1b–induced thermal hyper-algesia, iNOS protein induction, and NO release in rats(Sung et al., 2004). Moreover, we observed a time-de-pendent upregulation of P-p38 MAPK protein expressionin the rat SC at 30–240 min following IL-1b administra-tion, which was earlier than iNOS protein expressionand NO production (Sung et al., 2005). In the samestudy, neither the extracellular signal-regulated kinasenor c-Jun NH2-terminal kinase was activated. Further-more, i.t. pretreatment with SB203580 significantlyinhibited IL-1b–upregulated P-p38 MAPK and almostcompletely blocked subsequent iNOS expression, NOrelease, and thermal hyperalgesia. Furthermore, i.t. pre-treatment with IL-1ra effectively abrogated IL-1b–induced P-p38 MAPK upregulation, iNOS/NO produc-tion, and thermal hyperalgesia (Sung et al., 2005). Ourpresent results further demonstrated that IL-1bincreased microglia and astrocyte IR and stimulated P-p38 MAPK and iNOS colocalization with microglia andastrocytes in LSCDH. This study provides further evi-dence of IL-1b–induced glial activation underlying spi-nal nociceptive sensitization.

Glial cells are activated in response to insults, includ-ing trauma, ischemia, and pathogens. Activation ofastrocytes and microglia is important to the pathophysi-ology of the CNS (Hains and Waxman, 2006; Ji andSuter, 2007; Ledeboer et al., 2005; Tsuda et al., 2004;

Fig. 9. Time course of NO concentration changes in spinal CSF dial-ysates after various treatments. Intrathecal saline injection alone didnot affect NO levels in rat spinal CSF dialysates (n 5 5). IL-1bincreased NO release from 4–12 h after injection (n 5 6). Intrathecalpretreatment with minocycline or fluorocitrate 1 h prior to IL-1b injec-tion completely blocked IL-1b–induced NO release (n 5 8 for eachgroup). Values were normalized to the baseline (before i.t. drug deliv-ery) and are expressed as the mean 6 SEM. *, P < 0.05 versus saline-injected controls. #, P < 0.05 versus IL-1b–injected rats. (, P < 0.05 ver-sus baseline.

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Wen et al., 2009). Accumulating evidence suggests thatIL-1b is a key activator of astrocytes and microglia inthe CNS; activated microglia and astrocytes produce IL-1b in response to pathological insults (Guo et al., 2007;Ledeboer et al., 2005). IL-1b induces and maintainsastrocytic and microglial activation through type 1 IL-1receptors (IL-1R1) (Liberto et al., 2004; Pinteaux et al.,2002). Brain injury-induced gliosis and IL-1b productionwere absent in IL-1R1-knockout mice (Basu et al., 2002;Lin et al., 2006). Studies further demonstrated that IL-1ra attenuated this reactive microgliosis and astrocyto-sis, as well as nerve injury, inflammation, and pain(Fiorentino et al., 2008; Vogt et al., 2008). In this study,activation of spinal astrocytes and microglia by IL-1bwas evident by morphological changes and increases inthe number and intensity of GFAP- and CD11b/c-IR.Moreover, increased p38 MAPK phosphorylation andiNOS expression were also observed in activated astro-cytes and microglia. Double immunostaining revealed amarked increase in P-p38 MAPK- and iNOS-immuno-stained cells in LSCDH after i.t. IL-1b injection. Theseproteins localized predominantly in microglia and, to alesser extent, astrocytes. IL-1b–induced astrocytic andmicroglial activation was stronger at 30 min than at 6h, and this effect was more pronounced in microgliathan in astrocytes. The above results further confirmedthat spinal astrocytes and microglia are the targets ofIL-1b signaling.

Microglia have been shown to interact with astrocytes,contributing to downstream biochemical and behavioralresponses to noxious stimuli (Raghavendra et al., 2004;Watkins et al., 1997). Additionally, activated microgliarelease mediators that promote astrocyte activation inLSCDH in response to SC injuries, inflammation, andspinal nerve ligation (SNL), and blocking these micro-glia-released mediators attenuated astrocyte activationand pain in rodents (Gu et al., 2011; Miyoshi et al.,2008; Raghavendra et al., 2004; Watkins et al., 1997).Furthermore, activated astrocytes have also been shownto release mediators, which, in turn, activate spinalmicroglia in SNL or focal brain-injured rodents (Davaloset al., 2005; Lindia et al., 2005). These findings suggestastrocyte-microglia interactions, which would facilitatenociceptive responses. In this study, IL-1b activatedboth spinal microglia and astrocytes, causing morpholog-ical changes, elevated GFAP- and CD11b/c-IR, and up-regulated P-p38 MAPK and iNOS protein expression at30 min and 6 h after IL-1b injection; these effects werefollowed by thermal hyperalgesia (from 4–12 h) in rats.Moreover, the magnitude of microglial activation wasgreater than that of astrocytes in LSCDH. Therefore, wesuggest that activation of microglia and astrocytes ispivotal in the induction and maintenance of IL-1b–induced thermal hyperalgesia. Fluorocitrate, an aconi-tase inhibitor, can suppress the tricarboxylic acid cycle,cause citrate accumulation, reduce glutamine produc-tion, and deprive the cell of energy (Fonnum et al.,1997; Hassel et al., 1992). Experiments conducted inastrocyte-brain endothelial cell cocultures and brain tis-sue culture has indicated that the primary effect of fluo-

rocitrate is on astrocytes (Gesuete et al., 2011; S�a Santoset al., 2011). Fluorocitrate (100 fmol) injected into therostral ventromedial medulla selectively inhibited astro-cytic activation, but not microglial activation, in chronicinfraorbital nerve constriction injury rats (Wei et al.,2008). However, in SNL-induced neuropathic painmouse models, chronic i.t. injection of fluorocitrate (0.5nmol/day) did not inhibit microglial activation or painoccurring during the early induction phase (<7 day afterSNL), but significantly suppressed astrocyte and micro-glial activation and pain in the late maintenance phase(>7 day after SNL) in mice (Shibata et al., 2011). In con-trast, i.t. fluorocitrate (1 nmol) was shown to attenuateastrocytosis and microgliosis in rat models of peripheralinflammatory and neuropathic pain (Clark et al., 2007).As expected, our present results showed that fluoroci-trate pretreatment inhibited IL-1b–induced thermalhyperalgesia, upregulated P-p38 MAPK and iNOS pro-tein expression, and promoted NO release. However, wefound that fluorocitrate inhibited not only astrocyte acti-vation, but also microglial activation at both 30 min and6 h. Therefore, we hypothesized that the effect of fluoro-citrate on attenuating IL-1b–induced microglial activa-tion may be secondary to direct inhibition of astrocytes,in association with reduced production and release ofastrocytic mediators, which, in turn, suppressed theastrocyte-microglia interaction.

In our present study, an early and extensive activationof microglia and astrocytes with an increased numberand intensity of GFAP- and CD11b/c-IR cells and colocal-ization of P-p38 MAPK-IR and iNOS-IR were observedin the superficial laminae of LSCDH at 30 min and 6 hafter IL-1b injection. In addition, our data do not sup-port a direct effect of IL-1b on LSCDH neurons due tolack of P-p38 MAPK/iNOS immunofluorescence in neu-rons, at up to 6 h after i.t. IL-1b injection. However, wecannot exclude the possibility that IL-1b affects neu-rons. Neurons are known to be involved in the centralsensitization and maintenance of pain, and IL-1R1 hasbeen found in specific neuronal populations, includingneurons in the SC dorsal horn (Samad et al., 2001;Weyerbacher et al., 2010). IL-1b has been shown to acti-vate neurons via upregulation of downstream signalingcomponents, such as PKCg, Src tyrosine kinase, andcAMP response element-binding protein (Kawasakiet al., 2004; Srinivasan et al., 2004; Weyerbacher et al.,2010). Additionally, IL-1b can activate neuronalN-methyl-D-aspartate (NMDA) receptors to mediate painperception (Zhang et al., 2008). The antinociceptiveeffect of minocycline on inflammatory and neuropathicpain has been shown to depend on inhibition of spinalmicroglia activation (Hua et al., 2005; Ledeboer et al.,2005; Piao et al., 2006; Raghavendra et al., 2003), butnot on astrocytes or neurons (Clark et al., 2007; Hainsand Waxman, 2006; Raghavendra et al., 2003; Tikkaet al., 2001). However, minocycline has been shown toinhibit spinal astrocyte activation and mechanical allo-dynia in partial sciatic nerve-ligated rats (Nie et al.,2010). Minocycline can attenuate apoptosis, inflamma-tion, and glial activation; suppress reactive oxygen

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species production and glutamate release; and promotethe survival of lesioned neurons (Boyette-Davis andDougherty, 2011). In a whole-cell patch clamp study,minocycline directly inhibited both tetrodotoxin-sensitiveand tetrodotoxin-resistant Na1-channels in rat dorsalroot ganglion neurons, a process that has been sug-gested to produce peripheral analgesia (Kim et al.,2011). Furthermore, minocycline markedly reduces for-malin-induced c-Fos IR in spinal dorsal horn neuronsand inhibits synaptic transmission in the dorsal hornsubstantia gelatinosa neurons in a rat model of forma-lin-induced inflammatory pain (Cho et al., 2006). There-fore, the analgesic effects of minocycline pretreatmentmay not solely rely on inhibited glial activation. Takentogether, our data suggest that the activation of spinalastrocytes and microglia is necessary and sufficient fornociceptive processing and is correlated with the devel-opment of IL-1b–induced thermal hyperalgesia.

The upregulation of P-p38 MAPK in spinal microglia(Crown et al., 2008; Hains and Waxman, 2006; Huaet al., 2005; Ito et al., 2009; Piao et al., 2006; Sorkinet al., 2009; Tsuda et al., 2004; Wen et al., 2009) andastrocytes (Crown et al., 2008; Xu et al., 2007) has beenimplicated in spinal nociceptive processing in neuro-pathic and inflammatory pain in animals. Inhibition ofp38 MAPK attenuates neuropathy-induced astrocytosis,microgliosis, and allodynia in rodents (Crown et al.,2008; Ito et al., 2009; Xu et al., 2007). Minocycline hasbeen reported to inhibit p38 MAPK activation in spinalmicroglia, thus preventing thermal and mechanicalhyperalgesia following inferior alveolar and mental nerveinjuries or carrageenan injection in rat hind paws (Huaet al., 2005; Xu et al., 2007). Our previous study showedthat IL-1b–induced p38 MAPK phosphorylation wasprominent at 30 min, while iNOS expression was maxi-mal by 6 h after IL-1b treatment (Sung et al., 2004). Inthis study, immunohistochemical analysis demonstratedthat the intensity of P-p38 MAPK-IR in LSCDH was lowat 10 min after IL-1b injection; then, as expected, P-p38MAPK expression was significantly increased at 30 minand 6 h in both astrocytes and microglia, and this wasaccompanied by thermal hyperalgesia. Pretreatmentwith minocycline, fluorocitrate, and SB203580 markedlyattenuated IL-1b–induced P-p38 MAPK and iNOS immu-nofluorescence in spinal astrocytes and microglia; how-ever, the magnitude of inhibition of glial activation bythese inhibitors varied. All of these inhibitors suppressedIL-1b–induced p38 MAPK phosphorylation mainly inmicroglia, while suppression of iNOS expression wasobserved predominantly in astrocytes; minocycline wasthe most efficacious among these inhibitors. Theseresults suggest that spinal astrocytes and microglia arethe targets responsible for IL-1b–activated nociceptivesignaling, thus allowing the development of thermalhyperalgesia. Minocycline, fluorocitrate, and SB203580effectively inhibited IL-1b–induced reactive astrocytosis,microgliosis, and thermal hyperalgesia. These resultssuggest that glial and p38 MAPK inhibitors have thera-peutic potential in the treatment of IL-1b–mediatedcentral sensitization and inflammatory pain.

In summary, in this study, we found that intrathecalIL-1b administration activated astrocytes and microglia,thus leading to the activation of p38 MAPK/iNOS noci-ceptive signaling in SC and resulting in thermal hyperal-gesia in rats. This neuroinflammatory process can beblocked by i.t. pretreatment with glial cell inhibitors andp38 MAPK inhibitors. Our results indicate that spinalastrocytes and microglia are the targets of the proinflam-matory cytokine IL-1b in the induction of thermal hyper-algesia. This direct causal relationship between i.t. IL-1badministration and activation of spinal astrocytes andmicroglia suggests that modulation of spinal glial cell ac-tivity provides potential therapeutic strategy for treatinginflammatory and neuropathic pain in clinical practice;glial cell and p38 MAPK inhibitors may emerge as valua-ble therapeutic drugs for treating inflammatory pain.

ACKNOWLEDGMENTS

The authors thank Dr. Yu-Hsueh Wang for technicalassistance, Dr. Kuang-Yi Chang for statistical assis-tance, and the Clinical Research Core Laboratory of Tai-pei Veterans General Hospital, Taipei, Taiwan, Republicof China, for experimental support.

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