Inhibition of IL-6 signaling: A novel therapeutic approach to treating spinal cord injury pain

PAIN�

154 (2013) 1115–1128

w w w . e l s e v i e r . c o m / l o c a t e / p a i n

Inhibition of IL-6 signaling: A novel therapeutic approach to treating spinalcord injury pain

Jutatip Guptarak a, Sheshali Wanchoo a, Julieann Durham-Lee a, Yewen Wu a, Dragoslava Zivadinovic a,Adriana Paulucci-Holthauzen a,b, Olivera Nesic a,⇑a Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-1072, USAb Optical Microscopy Core, University of Texas Medical Branch, Galveston, TX 77555-1072, USA

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 December 2012Received in revised form 3 February 2013Accepted 15 March 2013

Keywords:Spinal cord injury painBody weightMechanical allodyniaInterleukin 6 receptor antibodyGabapentinAstrocytesgp130GLT-1

0304-3959/$36.00 Published by Elsevier B.V. on behahttp://dx.doi.org/10.1016/j.pain.2013.03.026

⇑ Corresponding author. Tel.: +1 409 772 3658.E-mail address: [email protected] (O. Nesic).

To characterize the contribution of interleukin-6 (IL-6) to spinal cord injury pain (SCIP), we employed aclinically relevant rat contusion model of SCIP. Using Western blots, we measured IL-6 levels in lumbarsegments (L1-L5), at the lesion site (T10), and in the corresponding lumbar and thoracic dorsal root gan-glia (DRG) in 2 groups of similarly injured rats: (a) SCI rats that developed hind-limb mechanical allo-dynia (SCIP), and (b) SCI rats that did not develop SCIP. Only in SCIP rats did we find significantlyincreased IL-6 levels. Immunocytochemistry showed elevated IL-6 predominantly in reactive astrocytes.Our data also showed that increased production of IL-6 in hyperreactive astrocytes in SCIP rats mayexplain still-poorly understood astrocytic contribution to SCIP. To test the hypothesis that IL-6 contrib-utes to mechanical allodynia, we treated SCIP rats with neutralizing IL-6 receptor antibody (IL-6-R Ab),and found that one systemic injection abolished allodynia and associated weight loss; in contrast to gaba-pentin, the analgesic effect lasted for at least 2 weeks after the injection, despite the shorter presence ofthe Ab in the circulation. We also showed that IL-6-R Ab partially reversed SCI-induced decreases in theprotein levels of the glutamate transporter GLT-1 12 hours and 8 days after Ab injection, which mayexplain the lasting analgesic effect of the Ab in SCIP rats. A link between reactive astrocytes IL-6-GLT-1has not been previously shown. Given that the humanized IL-6-R Ab tocilizumab is Food and DrugAdministration-approved for rheumatoid arthritis, we are proposing tocilizumab as a novel and poten-tially effective treatment for SCIP.

Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

1. Introduction

Chronic pain after traumatic spinal cord injury (SCI) is a devas-tating and debilitating condition that affects about 65% of SCI pa-tients [20]. Although several medications have been suggestedfor the treatment of SCI pain (SCIP) – including morphine, alfenta-nil, pregabalin, gabapentin, valproate, carbamazepine, and amitrip-tyline [4] – these medications may have unfavorable risk/benefitratios with only modest analgesic effects and potentially seriousside effects [13,29,98].

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in a widerange of biological processes, including neuropathologies [9,76,99].IL-6 acts by binding to the IL-6 receptor (IL-6-R), a process thattriggers the association of this complex with the glycoproteingp130. However, IL-6-R also exists in a soluble form, produced

lf of International Association for

either by shedding or by alternative splicing [68], that in complexwith IL-6 also activates gp130 [77,88], even in the absence of themembrane-bound IL-6-R.

Trauma to the central nervous system induces significantupregulation of IL-6 [1,41], including SCI in humans and in animalmodels [6,83,84]. Our earlier report demonstrates that IL-6 mRNAlevels are robustly increased as early as 6 hours after SCI [60]. Moststudies examining IL-6 in SCI have investigated acute IL-6 changes,spanning hours to days after trauma [32,83,104]. It appears thatearly upregulation of IL-6 after SCI is detrimental, as Okada et al.[64] and Mukaino et al. [53] have demonstrated that blockingacute IL-6 signaling with the neutralizing IL-6-R antibody (Ab) sig-nificantly improves motor recovery of SCI mice.

Although few, there are reports that demonstrate chronic in-creases in IL-6 after SCI. Interestingly, these reports also show thatpersistent IL-6 increases after SCI appear to correlate with thedevelopment of chronic pain both in SCI patients [19] and in ananimal model of SCI [25]. Therefore, an association between IL-6

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levels and established SCIP has been found, but the contribution ofIL-6 signaling to chronic pain development and/or maintenanceafter SCI has not been examined. In contrast, the contribution ofIL-6 to chronic pain in peripheral neuropathies is well established.In rats, a direct injection of IL-6 causes hypersensitivity to thermaland mechanical stimuli [11,22,63,69]. For example, intrathecal IL-6injection produces allodynia and hyperalgesia after peripheralnerve injury, while intrathecal anti-IL-6 Ab treatment decreasesallodynia [2].

Therefore, our goal in this study was to investigate the proteinlevels and cellular sources of IL-6 in chronically injured spinalcords relative to the development of SCIP (ie, hind-limb mechanicalallodynia) in a rat model of contusion SCI, to test the hypothesisthat inhibition of IL-6 signaling alleviates SCIP, and to shed lighton the mechanisms underlying the analgesic effects of IL-6inhibition.

2. Methods

2.1. Animals

2.1.1. Contusion injuryMale Sprague-Dawley rats from Harlan (Indianapolis, IN, USA)

weighing 225-250 g were anesthetized by intraperitoneal (i.p.)injection of pentobarbital (50 mg/kg). A detailed description ofthe procedure is given in our previous report [28]. Briefly, a lami-nectomy of the 10th thoracic (T10) vertebra was performed to ex-pose the spinal cord. Animals were contused at T10 with theInfinite Horizons Impactor (Precision Systems and Instrumenta-tion, LLC, Fairfax Station, VA, USA) using a force of 200 kdynes,which produces a motor impairment characterized by occasionalweight-supported steps with no fore limb-hind limb coordination.The surgical site was closed by suturing the muscle and staplingthe skin, followed by the superficial application of 0.3 mL of 1%lidocaine. Injured animals received the analgesic buprenorphine(0.1 mg/kg) subcutaneously twice a day for 3 days. Injured animalsalso received the antibiotic baytril (2.7 mg/kg) subcutaneouslytwice a day until bladder function returned. The bladders of SCIanimals were manually emptied twice daily until normal functionreturned. All procedures complied with the recommendations inthe Guide for the Care and Use of Laboratory Animals [57] and wereapproved by the University of Texas Medical Branch Animal Careand Use Committee. Control age-matched animals were not sub-jected to any part of the surgical or postsurgical care protocols.We have decided to use only naïve rats as controls, as describedin our previous report [28].

2.1.2. Number of ratsIn this study we used 15 control (uninjured rats) and 58 rats

that underwent contusion SCI. To equalize the injury levels inall SCI rats, only SCI rats whose score on the Basso, Beattie, andBresnahan Scale (BBB score) was 0 or 1 on day 1 post-SCI wereused for further analyses [7]. We further selected 51 SCI rats withsimilar BBB scores at day 21 post-SCI. Of 51 injured rats, 36 SCIrats showed consistent mechanical allodynia in both hind limbsat all time points (ie, 21, 28, 35, and 42 days after SCI). Theremaining 15 SCI rats manifested changes in mechanical thresh-olds that did not meet our criteria for SCI pain (as described inSection 2.5.2.) and thus were considered SCI rats without pain.SCI rats with allodynia (the ‘‘SCI pain’’ group) were divided into4 experimental groups: 1) SCI rats that received a single i.p. injec-tion of IL-6-R Ab 28 days after SCI (n = 12); 2) SCI rats that re-ceived one i.p. injection of vehicle (n = 10); 3) SCI rats thatreceived daily i.p. injections of gabapentin from 28 days until35 days after SCI (GBP; n = 7); and 4) SCI rats that received dailyi.p. injections of vehicle (n = 7).

2.2. IL-6 Receptor antibody treatment

We used neutralizing mouse IL-6-R Ab (#AF 1830; R&D Sys-tems, Minneapolis, MN, USA), since rat IL-6-R Ab was not commer-cially available. The rat and mouse IL-6-R share 90% homology,while human IL6-R shares only 50% homology with rat/mouseIL-6-R. This neutralizing Ab was raised against the extracellular re-gion of the IL-6-R Ab, and so should recognize both soluble IL-6-R(sIL-6-R) and membranous or full-length rat IL-6-R. Wu et al. [102]confirmed that this Ab effectively neutralized IL-6-R in mouse Tcells. Since systemic administration is more clinically relevant,we have chosen to inject IL-6-R Ab i.p. Intraperitoneal injectionsof neutralizing anti-mouse IL-6-R Ab have already been tested ina mouse model of SCI [53,64]. The authors have shown that onei.p. injection of IL-6-R Ab (100 mg/kg) given immediately afterSCI alters the inflammatory response, decreases astrocytic activa-tion, and improves motor recovery, but they did not measure SCIP.Given that commercially available IL-6-R Ab was diluted in phos-phate-buffered saline, we injected phosphate-buffered saline i.p.into vehicle-treated SCI rats.

2.2.1. DoseWe have chosen to administer 1.5 mg/kg IL-6-R Ab, as a dose

that is higher than 0.75 mg/kg and lower than 4 mg/kg. Wu et al.[102] showed that i.p. injections of �0.75 mg/kg of the same neu-tralizing IL-6-R Ab used in our experiments effectively neutralizedIL-6-R in dermal lymphocytes. Because in our experiments theneutralizing Ab had to cross the blood–spinal barrier to affect painprocesses in injured spinal cords, we increased the Ab dose to1.5 mg/kg. However, we chose not to increase the dose to morethan 1.5 mg/kg, given that a 4-mg/kg dose of humanized IL-6-RAb showed adverse effects in rheumatoid arthritis (RA) patients.In 2010 the US Food and Drug Administration (FDA) approvedthe humanized monoclonal Ab against IL-6 receptor (tocilizumab,sold under the trade name ACTEMRA [Genentech USA Inc, SanFrancisco, CA, USA]) for the treatment of RA. The recommendedtocilizumab dose for the treatment of RA is 4 mg/kg given onceevery 4 weeks as a single intravenous infusion, followed by an in-crease to 8 mg/kg based on clinical response. Although the safetyand tolerability of tocilizumab has been confirmed in clinical trials,prolonged administration of therapeutic doses showed adverse ef-fects such as an increased risk of infections (although opportunisticinfections were rare), transaminitis, and neutropenia. No increasedrisk of malignancy, tuberculosis, or clinical hepatitis was noted.Although rare, a unique adverse event seen in clinical trials of toc-ilizumab has been gastrointestinal (GI) perforation (2.8 per 1000patients).

2.3. Assessing possible side effects of IL-6-R Ab

The most comprehensive description of tocilizumab safety andits side effects, described in Koike et al. [40] and Kaneko [37], isbased on data collected from 3881 RA patients. The most com-monly reported adverse events are abnormal laboratory values,such as rises in the hepatic function markers and rises in bloodcholesterol levels. However, there is no evidence that elevationsin cholesterol increase the risk of cardiovascular events. Infectionsoccurred at a frequency of 30.8/100 patient years, with seriousinfections at 9.1/100 patient years. It is generally accepted that toc-ilizumab therapy is well tolerated among RA patients; that is, theretention rate after 24 weeks of daily therapy is found to be79.5% [103]. However, the most serious adverse effect of toc-ilizumab was gut perforation, which occurred as 7 events per100 patient years [40], or as 3 events per 2188 patient years [61].

Therefore, in our study we assessed the effects of IL-6 R Ab onthe gross histological appearance of the intestines and liver,

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including the liver weight, in collaboration with the institutionalveterinarian, Jason S. Villano, DVM, MSc, MS, DACLAM. The grossmorphological examination did not reveal any effect of IL-6-R Abon the intestines (not shown), while the average liver weight inthe vehicle-treated SCI rats was 12.96 ± 2.04, and in IL-6-R Ab-trea-ted SCI rats, was 13.17 ± 1.25. Thus, we did not find any adverse ef-fects of a single i.p. injection of 1.5 mg/kg IL-6-R Ab on the GI tractof SCI rats.

2.4. Locomotor assessment

Hind-limb movement was assessed using the BBB scale [7]. BBBscores were collected daily on the first 14 days after injury, andonce weekly thereafter.

2.5. Mechanical allodynia

Mechanical allodynia was measured in a blinded fashion; theperson assessing behavior was not involved in animal surgeriesor drug injections. Rats were placed in plastic bottomless restrain-ers, on a mesh stand. The animals were allowed to acclimatize tothe restrainers for 30 minutes before the testing session, so thatexploratory and grooming activities stopped. A series of von Freyfilaments – 0.4-60 g – were used to stimulate the glabrous surfacesof the paw (avoiding the pads). The von Frey filament touched per-pendicularly with a force enough to bend the filament slightly. Apositive response was noted if the animal lifted the paw immedi-ately, flinched, or licked the paw [16,17]. Ambulation was classifiedas an ambiguous response, and the stimulus was repeated [16]. If anegative response was noted, the next von Frey filament (of a high-er strength) was tested. Similarly, for a positive response, the nextweaker von Frey filament was used [16]. Based on the report byDetloff et al. [24], 30 seconds were allowed to pass between 2 con-secutive von Frey filament applications. Our preliminary datashowed that mechanical thresholds were significantly higher withthe 30-second waiting time: without the waiting time, the thresh-old in uninjured rats was 10.75 ± 3 g, while the threshold in unin-jured rats with the waiting time was 31.2 ± 10.4 g, implying thatwithout the waiting time, artificially low mechanical thresholdsor threshold decay [15] occurred. Although detailed mechanismsunderlying threshold decay are not yet known, Detloff et al. [24]have shown that supraspinal processing protects against thresholddecay, and suggested that the anticipatory state in which thethreat of pain activates fear and emotional centers in the brain,can enhance pain [12]. Therefore, it is likely that allowing a suffi-ciently long time (30 seconds) in between von Frey applicationsdiminishes or eliminates anticipatory reactions and thus preventsan error of measuring artificially low thresholds.

2.5.1. Mechanical threshold valuesMechanical threshold values were calculated by using the for-

mula: 50% withdrawal value = 10(P

Xi)/N + d/N(A + C), where Xi arethe test levels (log values of von Frey filaments tested), N = numberof trials (10 in our calculations), and d = the interval between doselevels (the average of the differences between stimuli in log units).A and C were calculated based on the table presented in Dixon [26]and chosen using no (negative responses) and nx (positive re-sponses) during the trial. The average of the 2 50% withdrawal val-ues was calculated for each day tested, and used for the finalanalysis.

2.5.2. Characterization of mechanical allodynia in SCI ratsCommon signs of neuropathic pain in SCI patients include sev-

eral types of sensory abnormalities including mechanical allodyniathat may be present at or below the level of injury [97]. We there-fore assessed hind-limb thresholds to mechanical stimuli (gram

force) before and after SCI; for example, we measured thresholds3, 2, and 1 day before SCI (pre-SCI) and then once weekly from 2to 6 weeks after SCI. As shown in Fig. 1A, in the majority of SCI rats,SCI elicited hind-limb mechanical allodynia, that is, decreases inmechanical post-SCI thresholds by �50%-60% compared to pre-SCI levels, as early as 21 days after SCI (n = 31); thresholds at14 days after SCI were not significantly different from pre-SCI lev-els (not shown).

We used a cutoff criterion for identifying SCI rats that developpain based on our analysis of mechanical threshold variations innaïve rats (Fig. 1B; n = 15). As shown in this graph, mechanicalthresholds in naïve rats vary over time by ±23%, so we assumedthat only decreases in mechanical thresholds >23% can be treatedas SCI-induced mechanical allodynia. Furthermore, we also ana-lyzed whether this decrease lasted for at least 4 consecutive mea-surements over 4 weeks post-SCI (at 21, 28, 35, and 42 days).According to these criteria, we found that 68% of SCI rats developchronic allodynia (n = 36), consistent with clinical data showingthat not all SCI patients develop pain [21]. Fig. 1C presents the per-cent change in rats’ mechanical thresholds compared to their pre-SCI levels for both groups of rats: ‘‘SCI pain’’ (n = 36) and ‘‘SCI nopain’’ (n = 15). Negative percent values represent decreases inthresholds in the ‘‘SCI pain’’ group (averaging around 50% over4 weeks after SCI).

We also found that ‘‘SCI pain’’ rats showed 5% lower bodyweight than the ‘‘SCI no pain’’ group (Fig. 1D), a novel finding.The difference in body weight became significant 28 days afterSCI and continued to 42 days post-SCI, consistent with decreasedfood intake by rats experiencing chronic pain [30,44,45]. Althoughfeeding in male rats appears to be a protected behavior, Foo et al.[31] have shown that hypophagia occurs in rats when severe painis induced, or when rats are exposed to prolonged pain, as shownin Malick et al. [47]. Although the central mechanism underlyinghypophagia in pain states is still poorly understood, Malick et al.[47] have shown that hypothalamic neurons that normally inhibitappetite can also mediate the suppression of food intake by painsignals. Therefore, it is likely that the lower body weight in ourSCI rats with pain (Fig. 1D) resulted from decreased food intake,but that remains to be determined.

This result strongly suggests that increased sensitivity of thehind-limb flexion (Fig. 1C) is the manifestation of allodynic behav-ior rather than SCI-induced hyperreflexia [74]. Although rats in the‘‘SCI pain’’ group had slightly lower BBB scores (Fig. 1E; the maxi-mal difference was 1 BBB score unit at 21 days after SCI), the differ-ence was not statistically significant. We also did not findsignificant correlation between BBB scores and allodynia at 28 daysafter SCI (Pearson correlation r = 0.28; n = 40).

To further confirm that decreases in mechanical thresholds ade-quately model SCIP and that paw withdrawal is unlikely to behyperreflexia, we treated 7 SCIP rats with gabapentin (GBP) and7 SCIP rats with vehicle. Similar to clinical settings, we tested theeffect of GBP on already-established allodynia, starting 28 daysafter SCI. As presented in Fig. 1F, we found a significant analgesiceffect of acutely administered GBP (50 mg/kg i.p., equivalent tothe dose used in patients), although GBP was not effective in 2SCI rats, which agrees with the clinical findings that not all peoplewith chronic pain respond to GBP treatment [51]. In Fig. 1F wepresent threshold changes 2 hours after GBP injections 35 and42 days after SCI. However, GBP was not effective if the measure-ments were delayed by more than 6 hours after GBP injection, de-spite daily injections of this medication, as has already beenreported by Densmore et al. [23]. In agreement with the transientpain-relieving effect of GBP, we found that SCI rats treated withGBP had the same body weight changes as did SCI rats treated withvehicle (Fig. 1G). Although GBP treatment did not significantly af-fect locomotor recovery of SCI rats (Fig. 1H), GBP-treated rats

Fig. 1. Characterization of mechanical allodynia in spinal cord injury (SCI) rats. (A) Mechanical stimuli (gram force) applied with von Frey filaments to rats’ hind limbs thatelicited paw withdrawal. The thresholds for paw withdrawals were measured in both hind limbs (and then averaged): before SCI (the first bar represents an average value formechanical thresholds obtained during 3 consecutive days before contusion SCI) and then once weekly after SCI (up to 42 days post-SCI). (B) Mechanical thresholds were alsomeasured in uninjured (naïve) rats once weekly to assess normal variations in withdrawal thresholds of uninjured rats over time. The maximal variation was 23% changefrom the baseline value (dashed line). (C) In a group of similarly injured rats, we usually find that �30% of SCI rats do not develop allodynia. On the Y-axis is the percentchange in mechanical thresholds at various times after SCI, compared to pre-SCI values (=100%). Positive numbers indicate an increase in mechanical threshold values (graybars = ‘‘SCI rats without pain’’), while negative numbers indicate decreases in mechanical thresholds. Shown are % changes in left (L) and right (R) hind limbs. We did not findsignificant differences in thresholds between the left and right hind limbs. However, significant decreases in mechanical thresholds were found in a subpopulation of SCI ratsat all time points, in both hind limbs (black bars = P < 0.05); those rats were considered rats that developed allodynia, in contrast to other SCI rats whose thresholds mostly didnot change from the baseline value, or even increased 42 days after SCI (gray bars). (D) Body weight (grams) of SCI rats decreased in the first 7 days post-SCI, and thenincreased progressively over time. However, SCI rats that developed allodynia had significantly lower body weight than SCI rats that did not develop allodynia, starting at28 days after SCI (P < 0.05). (E) Locomotor recovery scores (Basso, Beattie, and Bresnahan Scale [BBB]) were not statistically different between SCI rats that developed vs thosethat did not develop allodynia. (F) The effect of gabapentin (GBP) injections (intraperitoneal [i.p.]) on mechanical thresholds of SCI rats that developed allodynia (theirmechanical thresholds were significantly reduced, by �50%, at 28 days after SCI, before GBP injections). Mechanical thresholds of SCI rats that received GBP, if measuredwithin 2 hours post injection, were markedly increased (and not different from baseline, pre-SCI values). (G) SCI rats received one i.p. GBP injection daily from 28 to 42 daysafter SCI (represented in the graph as a dashed line). However, even daily administration of GBP did not affect body weight of GBP-treated SCI rats; their body weight wasindistinguishable from vehicle-treated SCI rats, and both were significantly lower than body weights of SCI rats that did not develop allodynia (P < 0.05). (H) Locomotorrecovery (BBB scores) was not statistically different in GBP-treated vs vehicle-treated SCI rats.


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ceased to improve from 28 to 42 days after SCI, in contrast to vehi-cle-treated SCI rats, whose BBB scores continued to increase up to42 days after SCI.

In sum, our characterization of hind-limb allodynia in a rat mod-el of SCI used in this study mimics some of the basic clinical featuresof SCI-related neuropathic pain: it occurs below the level of injury;it develops weeks after SCI and may last indefinitely; it does not oc-cur in all SCI rats (<30%); it is effectively, but transiently, reduced byGBP; and it is not affected by GBP in �30% of SCI rats.

2.6. Blood collection

Before tissue collection, rats were deeply anesthetized, theirchests opened to expose the heart, and 1 mL of blood drawn from

the left ventricle and transferred to 1.5 mL heparin-coated centri-fuge tubes. The plasma was separated from the blood cells by cen-trifugation at 4000 rpm, 4�C, for 20 minutes, then collected andstored at �80�C until later use for Western blot analyses.

2.7. Protein extraction

Animals were anesthetized by i.p. injection of pentobarbital(150 mg/kg) and sacrificed by transcardial perfusion with 0.9% sal-ine containing 1 unit of heparin per mL. T10 (lesion epicenter) andlumbar (L1-L5) spinal cord segments and the corresponding thoracic(T9-T11) and L1-L5 dorsal root ganglia (DRG) were removed andstored at �80�C. For protein extraction, 200 lL of homogenizationbuffer [10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic


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acid (HEPES), 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid(EDTA), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM DTT,0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 lg/mL antipain,2 lg/mL chymostatin, 2 lg/mL pepstatin, 2 lg/mL leupeptin], with1� phosphatase inhibitors (78426; Thermo Scientific, Waltham,MA, USA), was added to each individual spinal cord segment (or col-lection of DRG) for homogenization on ice with a pestle (05-559-27;Fisher Scientific, Pittsburgh, PA, USA). After homogenization, thesamples were vortexed for up to 1 minute and centrifuged at 5900rcf for 8 minutes at 4�C. The resulting supernatant, which containedthe crude cytosolic/whole-cell extract, was separated from the pel-leted nuclei, cellular debris, and intact cells. Nuclear proteins wereextracted from the nuclear pellet by resuspension in extraction buf-fer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMDTT, 1 mM PMSF, 2 ug/mL antipain, 2 ug/mL chymostatin, 2 ug/mLpepstatin, 2 ug/mL leupeptin), with 1� phosphatase inhibitors.The pellets from individual T10 segments were resuspended in 70-100 lL of buffer, with uninjured animals requiring the larger buffervolumes. Resuspension was followed by 20 minutes of vortexing at1400 rpm at 4�C, and subsequent centrifugation at 16,000 rcf for10 min at 4�C. The supernatant contained the nuclear protein, andall protein concentrations were determined using the BioRad Pro-tein Assay (500-0006; BioRad, Hercules, CA, USA).

2.8. Electrophoresis and Western blotting

Samples containing 40 lg of protein (from spinal cord, DRG, orplasma) were mixed with an appropriate volume of 6� super-denaturing sample buffer (350 mM Tris-HCl, pH 6.8, 1 M urea,6.0% 2-mercaptoethanol, 9.3% DTT, 18.0% SDS, 0.06% bromophenolblue, 30.0% glycerol), and equal amounts of protein were loadedonto a sodium dodecyl sulfate-polyacrylamide gel; the sampleswere not boiled to avoid aggregation of membranous proteins[82]. The samples were separated by electrophoresis at 150 V for4.5 hours, and the proteins transferred overnight to an Immobi-lon-P (Millipore Corporation, Billerica, MA, USA) polyvinylidenedifluoride membrane at 4�C and 25 V.

The membranes were reversibly stained with Ponceau S (0.5%w/v Ponceau S, 1.0% acetic acid) to confirm the transfer of proteins,and destained in water, then incubated for 1 hour at room temper-ature (RT; 23�C) in blocking solution (5.0% nonfat dry milk in Tris-buffered saline with 0.2% Tween-20 [TBS-T]), and then incubatedwith the appropriate primary Ab diluted in blocking solution for1 hour at RT, or overnight at 4�C. After washing in TBS-T, the mem-branes were incubated for 1 hour at RT with a horseradish perox-idase-conjugated secondary Ab diluted in blocking solution, thenagain washed in TBS-T (3� 20 minutes). Peroxidase activity wasdetected using the Amersham enhanced chemiluminescence light-ing system (ECL, RPN2106, Amersham Biosciences, Piscataway, NJ,USA) and exposure to X-ray film. The films were scanned andquantified by spot densitometry using the AlphaEase program, ver-sion 5.5 (Genetic Technologies, Miami, FL, USA).

2.9. Antibodies

Rabbit anti-IL-6 (Abcam # Ab 6672); dilution 1: 500. SecondaryAbs: Goat anti-rabbit IgG-HRP (Southern Biotech [Birmingham, AL,USA] # 4052-05); dilution 1:5000. Goat anti-mouse IL-6-R (R&DSystems #AF 1830); dilution: 1 lg/mL. Secondary: donkey anti-goat horseradish peroxidase-conjugated immunoglobulin G (IgG-HRP; R&D Systems # HAF 109); dilution: 1:3000. Mouse anti-glialfibrillary acidic protein (GFAP; Millipore # MAB360); dilution: 1:8000. Secondary: goat anti-mouse IgG-HRP (Southern Biotech #1034-05); dilution: 1:5000. Rabbit anti-gp130 (Santa Cruz Biotech-nology [Dallas, TX, USA] # SC-655); dilution: 1:500. Secondary:Goat anti-rabbit IgG-HRP (Southern Biotech # 4052-05); dilution:

1:5000. Goat anti-myelin oligodendrocyte glycoprotein (Abcam[Cambridge, MA, USA] # Ab 28766); dilution: 1:1000. Secondary:donkey anti-goat IgG-HRP (R&D Systems # HAF 109); dilution:1:3000. Mouse anti-neuronal nuclei (NeuN; Millipore # MAB377).Secondary: goat anti-mouse IgG-HRP (Southern Biotech # 1034-05); dilution: 1:5000. Mouse anti-phosphorylated neurofilament(pNF; Covance [Princeton, NJ, USA] # SMI-31R); dilution: 1:1000.Goat anti-mouse IgG-HRP (Southern Biotech # 1034-05); dilution:1:5000. Guinea pig anti-GLT-1 (Chemicon [Temecula, CA, USA] #1783); dilution: 1:1200. Secondary: goat anti-guinea pig IgG-HRP(Chemicon # AP108P); dilution: 1:3000. Rabbit anti-Iba-1 (WakoUSA, New York, NY, USA# 016-20001 [WB]); dilution: 1:500(1 lg/mL). Secondary: goat anti-rabbit IgG-HRP (Southern Biotech#4052-05); dilution: 1:5000. Mouse anti-b-actin (Sigma [St. Louis,MO, USA] # A5441); dilution: 1:80,000. Secondary: goat anti-mouse IgG-HRP (Southern Biotech # 1034-05); dilution: 1:5000.

2.10. Immunofluorescence

Animals were anesthetized with pentobarbital i.p. as above, andsacrificed by transcardial perfusion with 0.9% saline, followed by4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer. Spinalcords were removed, postfixed overnight at 4�C in 4% paraformal-dehyde, and cryoprotected in 30% sucrose in phosphate buffer for3 days. T10 and L4 spinal cord segments were then embedded inOCT (optimal cutting temperature) compound (Fisher 14-373-65)and cut into 30-lm transverse sections on a sliding microtome.Three 10-minute shaking washes of the floating frozen sectionswere conducted in Petri dishes containing cold 0.05-M tris-buf-fered saline (TBS) before incubation in blocking solution (0.05 MTBS, 0.3% Triton-X, 5% normal goat serum [NGS], 0.3% bovine serumalbumin [BSA]) for 45 minutes at RT, with orbital shaking, in achamber for free-floating tissue sections. The sections were thenincubated with primary Abs overnight at RT with shaking in a solu-tion of 0.05 M TBS, 0.3% Triton-X, 0.3% BSA, and 1% NGS. Sectionswere rinsed with 0.05 M TBS 3 times, 10 minutes each wash, atRT with shaking. The appropriate secondary Abs were diluted in0.05 M TBS, 0.3% Triton-X, 0.3% BSA, and 1% NGS for incubationwith the sections for 2 hours at RT with shaking; the chamberswere covered for protection from light.

The sections were washed in 0.05 M TBS (4� at RT, 10 minuteseach wash, with shaking), mounted on gelatin-coated glass slides,and dried before the addition of mounting medium with the nuclearstain DAPI (H-1200; Vector Laboratories; Burlingame, CA, USA), andapplication of a coverslip. The slides were kept at RT for at least20 minutes, then at 4�C for at least 24 hours before storage at�20�C until viewing. Primary Abs used include: rabbit anti-IL-6 (Ab-cam # Ab 6672); dilution 1:100; mouse anti-NeuN (Millipore#MAB377); dilution: 1:5000; NeuN recognizes a predominantly nu-clear protein specifically expressed in mature neurons, identified byKim et al. (2009) [38] as the RNA-binding protein FOX-3. Mouse anti-TUJ-1 (Covance #MMS-435P). TUJ-1 recognizes Class III b-tubulinspecifically expressed in neurons; dilution: 1:10,000. Mouse anti-GFAP (Millipore #MAB360); dilution: 1:1000; Mouse anti-OX-42(#MCA275G; SERATEC, Goettingen, Germany); dilution: 1:200. Thisantibody recognizes the rat equivalent of human CD11b, the recep-tor for the iC3b component of complement. The antigen is expressedon macrophages (resident and activated) and in microglial cells (res-ident and activated); mouse anti-CC1 (Abcam # ab16794); dilution:1:100. The mature oligodendrocyte marker APC (or CC1) is an antag-onist of b-catenin and results in a reduction in Wnt signaling [66].Mouse anti-RECA-1, rat endothelial cell antigen (AbD SEROTECMCA970R); dilution: 1:200. Guinea pig anti-GLT-1 (Chemicon#1783); dilution: 1:2000; rabbit anti-gp130 (Santa Cruz Biotechnol-ogy # SC-655 812); dilution: 1:600. Secondary antibodies used in-clude anti-mouse IgG AlexaFluor 488/green (#A11001, 1:2000;


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Invitrogen, Life Technologies, Grand Island, NY, USA), and anti-rab-bit IgG AlexaFluor 568/red (Invitrogen #A11011, 1:2,000). We alsoincluded a ‘‘no-primary Ab’’ control, in which all other steps wereperformed except addition of the primary Ab. However, with allAbs used in the study, this experiment gave no staining.

2.11. Image acquisition and analysis

Confocal images were acquired with a Zeiss LSM-510 Meta con-focal microscope (Carl Zeiss, Oberkochen, Germany) with a 20�,0.75 NA, 40�, 0.95 NA, and a 63� oil immersion objective (1.4NA). Multi-track sequential acquisition was done with excitationlines at 488 nm for Alexa 488 and 543 nm for Alexa 594. Respectiveemissions were collected with a 505-530 nm and a 560-615 nm fil-ter. Frame size was 1024 � 1024, and final image was a collectionof an 8-frame Kalman-averaging. Pinhole was properly adjusted togive the best confocal resolution according to the objective numer-ical aperture and utilized wavelengths. Optical slices were constantin all channels (488 and 543 nm), being 0.8 lM for 60� oil objec-tive and 1 lM for 40� objective. Image processing was done withMetaMorph 7.1.3.0 (Molecular Devices, Sunnyvale, CA, USA) bysubtraction of background from each frame on each independentchannel, and for some of the images a low pass filter of 2 by 2was applied to improve signal-to-noise ratio. A relative fluores-cence intensity of IL-6 immunolabeling was analyzed using Meta-Morph software. Background fluorescence was discounted from allimages. Neuronal cell bodies were traced by drawing a region ofinterest delineated by NeuN co-staining. An average intensity ofIL-6 immunolabeling within region of interest was calculated in5 sections per segment (T10 or L4), per rat. Three rats per experi-mental group were used in the analysis.

2.12. Statistical analysis

All statistical tests were evaluated at the a level of 0.05, with 2-tailed t-test, using the SPSS program (IBM, Armonk, NY, USA). Formultiple-group comparisons, data are analyzed using analysis ofvariance. The Least Significant Difference multiple-comparisonspost hoc test is used to determine P values (<0.05). In all ourgraphs, the symbols are used to denote the following:

#Significant difference (P < 0.05) between uninjured rats, andSCI rats that do not develop pain (‘‘SCI no pain’’), or betweenuninjured rats and SCI rats that develop pain (‘‘SCI pain’’);⁄Significant difference (P < 0.05) between SCI rats that do notdevelop pain (‘‘SCI no pain’’) and SCI rats that develop SCIP(‘‘SCI pain’’ group).

3. Results

3.1. Increased IL-6 levels in chronically injured spinal cords areassociated with hind-limb allodynia

Using Western blots, we analyzed IL-6 protein levels in 2 groupsof SCI rats, with and without SCIP (Fig. 2). IL-6 is a glycoproteinwith a variable molecular mass due to different glycosylation pat-terns, ranging from 19-21 kDa (nonglycosylated form) to �30 kDa(fully glycosylated form). It has been shown that the 23- to 25-kDaIL-6 forms are O-glycosylated, while the 28- to 30-kDa species areboth O- and N-glycosylated [46]. We compared IL-6 bands in unin-jured spinal cords with the unmodified recombinant rat IL-6 pro-tein (rrIL-6; R&D Systems #506-RL/CF; Fig. 2A) of expected22 kDa molecular weight (MW), and found that the lowest MWband in spinal cord samples clearly corresponded to the unmodi-fied rrIL-6 protein. As shown in Fig. 2A and B (marked with ar-rows), IL-6 Western blots of uninjured and injured spinal cord

samples also showed multiple bands whose higher MWs likely cor-respond to IL-6 forms that are either O-glycosylated (�25 kD) orboth O- and N-glycosylated (�30 kD). Given that the 22-kDa bandcorresponds to the unmodified rrIL-6 protein, we quantified onlythat band (presented in all bar graphs in Fig. 2D). However, we alsofound that altered intensities of IL-6 bands with higher molecularweights (25 or 30 kDa) closely paralleled changes in the 22-kDband (Table 1), that is, the intensity of those bands was signifi-cantly higher only in ‘‘SCI pain’’ rats, suggesting that the observedIL-6 level changes in SCI rats that developed pain did not dependon the altered extent of posttranslational modifications, but ratheron increased production of IL-6.

Using Western blots, we assessed changes in IL-6 levels at thelesion site (T10) and in lumbar segments (pooled L1-L5) 42 daysafter SCI, as SCI-induced changes in all those regions may influencethe processing of sensory information originating in the hindlimbs. Given that DRG play a key role in neuropathic sensitivity[100,101,107], we also assessed changes in thoracic and lumbarDRG.

We performed Western blots for 3 rats in an ‘‘SCI no pain’’ groupand 4 rats in an ‘‘SCI pain’’ group, whose mechanical thresholdchanges after SCI are presented in Figure 2C1. All rats in the ‘‘SCIpain’’ group showed consistently decreased mechanical thresholdsat 28, 35, and 42 days after SCI compared to their pre-SCI levels, incontrast to the ‘‘SCI no pain’’ rats, whose mechanical thresholdswere not significantly different from their pre-SCI levels. However,the BBB scores in both groups were similar (8 ± 1). We also mea-sured levels of the myelin oligodendrocyte glycoprotein (MOG) atthe lesion site (T10); its loss at the lesion site reflects the severityof injury, as we showed in Durham-Lee et al. [27]. MOG levels inboth the ‘‘pain’’ and ‘‘non-pain’’ groups of SCI rats were indistin-guishable (Fig. 2C2), suggesting that the initial damage (ie, MOGlevels) and the extent of recovery at 42 days post-SCI (ie, BBBscores) were approximately the same in both groups of SCI rats,and that allodynia did not develop as a result of increased severityof injury in SCIP rats.

IL-6 levels in thoracic or lumbar DRG were not affected byeither SCI or the development of allodynia (Fig. 2D); however, IL-6 levels in lumbar segments of the ‘‘SCI pain’’ group were signifi-cantly higher than the IL-6 levels in uninjured rats or the ‘‘SCI nopain’’ group (Fig. 2B and D; [2.7 fold, P = 0.013]). In contrast, IL-6levels were significantly reduced (by �25%) at the lesion site(T10; Fig. 2D) in both SCI groups (#P < 0.01), reflecting the loss ofIL-6-expressing cells (neurons).

3.2. Cellular localization of IL-6

Using double immunofluorescence staining for IL-6 (red; Fig. 3)and various cell markers (green; Fig. 3), we analyzed the cellularlocalization of IL-6 in uninjured (Fig. 3A) and injured spinal cords(Fig. 3B) in lumbar segments (L4) and at T10 isolated from SCI ratsthat developed hind-limb mechanical allodynia 42 days after SCI(n = 3/cell marker/group). We found IL-6 protein expressed in neu-rons, axons, and astrocytes in both uninjured and injured spinalcords, but not in microglia, oligodendrocytes, or parenchymalblood vessels.

IL-6 staining in large ventral horn neurons in L4 (labeled withNeuN, a neuronal marker; Fig. 3A and B) was confirmed in ahigh-magnification image (Fig. 3B1): cytoplasmic IL-6 was easilyidentified, but was not found in neuronal nuclei intensely labeledwith NeuN. We performed quantitative analysis of IL-6 immunola-beling in cell bodies of large-diameter ventral horn neurons inuninjured (n = 3) and SCI pain rats (n = 3), in both T10 and L4 seg-ments (5 sections/segment) co-labeled with neuronal markerNeuN. An average intensity of IL-6 labeling per cell differed signif-icantly between 2 experimental groups (Table 2; SD).

Fig. 2. Association between hind-limb allodynia and interleukin (IL)-6. (A) The IL-6 antibody (Ab) used in Western blots in our study generated a band of the expectedmolecular weight (MW; 22 kD) in uninjured spinal cord samples (lumbar segments L1-L5 were pooled). The same Ab recognized recombinant rat IL-6, which gave a band ofthe same MW. This band was subsequently quantified in all our experiments. (B) A representative IL-6 Western blot in lumbar (L1-L5 segments) isolated from 3 uninjured, 3spinal cord injury (SCI) rats that did not develop allodynia, and 4 SCI rats that developed allodynia (their threshold changes are presented in 2-C1). Black arrows indicate otherpossible IL-6 bands of higher MWs, representing posttranslational modification of IL-6. Beta-actin was used as a control for loading in all our experiments. The unmodified IL-6 band (22 kDa) had the same intensity in uninjured and injured lumbar segments in rats that did not develop allodynia, but was markedly higher in SCI rats that developallodynia. (C1) The percent change in mechanical thresholds for both groups of SCI rats. (C2) A representative Western blot of myelin oligodendrocyte glycoprotein (MOG;25 kDa) shows that SCI rats that developed allodynia had similar levels of MOG change as SCI rats that did not develop allodynia. Average values of quantified MOG bands forboth groups of SCI rats are written below the blots. (D) Bar graphs representing quantified IL-6 bands in 3 groups of rats (uninjured, SCI no pain, and SCI pain) in variousregions: lumbar segments (pooled L1-L5), lumbar dorsal root ganglia (DRGs; pooled L1-L5 DRGs), lesion site (T10), and lesion site DRGs (pooled T9, T10, and T11 DRGs). Theintensities of the IL-6 bands were first normalized to beta-actin, and then to values obtained for uninjured rats. Therefore, the Y-axis represents the fold change in IL-6 levelscompared to uninjured rats (=1). A significant change (P < 0.05) between uninjured and either SCI group is indicated by ‘‘#,’’ while a significant difference between 2 SCIgroups is indicated by ‘‘⁄,’’ here and throughout this paper. (E) A representative Western blot depicting dramatic changes in the neuronal nuclei (NeuN) band (expressed onlyin neuronal somata; only nuclear proteins were used for this analysis) and in the phosphorylated neurofilaments (PNF, expressed in axons) at the lesion site 42 days after SCI.

Table 1Western blot analysis of IL-6 bands.

22 kDa 25 kDa 30 kDa

Fold change SD Fold change SD Fold change SD

Uninjured 1 0.02 1 0.09 1 0.05SCI no pain 1.12 0.38 1.13 0.01 1.14 0.26SCI pain 2.65* 0.67 1.31** 0.07 1.79*** 0.37

SCI, spinal cord injury.* P = 0.013.** P = 0.015.*** P = 0.039.


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A marked increase in neuronal IL-6 labeling was found at the le-sion site (by 5.3-fold), and a more modest 1.9-fold increase in L4segments of SCI rats that developed pain. However, whether therewas a difference in neuronal IL-6 labeling between SCI rats that de-velop and SCI rats that do not develop pain remains to bedetermined.

IL-6 expression was also confirmed in axons (Fig. 3C): TUJ-1-la-beled axons (green) in the L4 segment of a ‘‘SCI pain’’ rat (42 daysafter SCI) were co-labeled with IL-6, especially intensely in the graymatter. Figure 3C1 presents a high-magnification image of TUJ-1-labeled IL-6-expressing axons in the dorsal column (marked witha yellow arrow in Fig. 3C).

Fig. 3. Cellular localization of interleukin (IL)-6. (A) IL-6 immunolabeling (red) in uninjured lumbar segment L4. IL-6 was co-labeled with the neuronal marker neuronalnuclei (NeuN; green). Calibration lines: 500 lm. (B) IL-6 and NeuN co-labeling in injured lumbar L4, isolated 42 days after spinal cord injury (SCI) from a rat thatdemonstrated significant mechanical allodynia (>50%) from 21 to 42 days after SCI. Calibration lines: 500 lm. (B1) High-magnification image of ventral horn neurons (markedwith white arrow in B) co-labeled with IL-6 and NeuN in injured lumbar L4, isolated 42 days after SCI from a rat that demonstrated significant mechanical allodynia (�50%)from 21 to 42 days after SCI. Calibration line: 50 lm. (C) IL-6 was also co-labeled with the axonal marker TUJ-1 (green). Calibration line: 500 lm. (C1) High-magnificationimage of dorsal column axons (labeled with yellow arrow in C). Calibration line: 50 lm. (D) A comparison of IL-6 + glial fibrillary acidic protein (GFAP) double labeling in asection of uninjured T10 with the corresponding double labeling in a section of an age-matched lesion site (T10; 42 days) showed significantly increased IL-6 in activatedastrocytes in injured spinal cords. Calibration line: 200 lm. (D1) High-magnification image of white matter astrocytes identified with GFAP (green) showing IL-6 expression inastrocytic processes and in cell bodies. Calibration line: 20 lm. (E) Astrocytic expression of IL-6 (red) was also confirmed in injured lumbar L4, isolated 42 days after SCI froma rat that showed significant mechanical allodynia (�50%) from 21 to 42 days after SCI. A yellow arrowhead in ‘‘merged’’ image marks IL-6 labeling in dorsal roots. Whitearrows mark astrocytes in ventrolateral white matter. The white star marks extraparenchymal blood vessel expressing IL-6. Calibration lines: 500 lm. (E1) High-magnification image of IL-6-expressing astrocytes in ventrolateral white matter (marked with white arrows in E). Calibration line: 20 lm. (E2) A high-magnification image ofIL-6 labeling in dorsal roots indicating IL-6 expression in sensory axons and likely in satellite cells. Calibration line: 20 lm. (F) Double labeling of IL-6 and the microglial/macrophage marker OX-42 in injured lumbar L4, isolated 42 days after SCI from a rat that showed significant mechanical allodynia (�50%) from 21 to 42 days after SCI.Calibration line: 50 lm.

Table 2Intensity of IL-6 labeling

T10 L4

Fold change SD Fold change SD

Uninjured 1 0.112 1 0.05SCI pain 5.32* 0.056 1.9** 0.30

T10 = 10th thoracic segment; L4 = 4th lumbar segment; SCI, spinal cord injury.* P = 0.00008.** P = 0.047.


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We found markedly increased IL-6 expression in astrocytesboth at T10 (Fig. 3D) and L4 (Fig. 3E) that closely paralleled astrog-lial activation (ie, increased GFAP labeling, green), consistent withWestern blot data (Fig. 4B). A high-magnification image of astro-cytes in injured white matter (Fig. 3D1) showed IL-6 not only inGFAP-labeled astrocytic processes, but also in astrocytic cellbodies. Although quantitative analysis of astrocytic IL-6 immu-nolabeling at T10 of ‘‘SCI no pain’’ vs astrocytic IL-6 in ‘‘SCI pain’’rats was not performed, a hind-limb allodynia-associated differ-ence in astrocytic IL-6 at T10 was unlikely, since GFAP levels atT10 were indistinguishable between ‘‘SCI no pain’’ and ‘‘SCI painrats’’ (Fig. 4B). However, GFAP levels in lumbar segments of SCI rats


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with allodynia closely paralleled IL-6 increases associated withallodynia (Fig. 4), even with the same-fold increase (2.7-fold) forboth IL-6 and GFAP (Figs. 2D and 4B), suggesting that activatedastrocytes are an important source of increased IL-6 in SCI rats thatdevelop allodynia.

Given that microglia upregulate IL-6 if exposed to inflammatorysignals [79], we also analyzed whether IL-6 was expressed inmicroglia/macrophages labeled with anti-OX-42 antibody in in-jured spinal cords. However, we did not find co-labeling of IL-6with a microglial/macrophage marker (OX-42, green; Fig. 3F).The IL-6 labeling (red) in Fig. 3F is likely astrocytic, based on theshape of those IL-6-expressing cells in the proximity of microglia,and immunolabeling shown in Fig. 3D and E. We also did not findIL-6 co-labeling with the oligodendrocyte marker CC1 (not shown)or with the endothelial cell marker RECA-1 (not shown). However,large extraparenchymal blood vessels were labeled with IL-6(Fig. 3E; marked with asterisk).

The apparent contradiction between observed IL-6 decreases atthe lesion site detected by Western blot analyses and increasedIL-6 expression in reactive astrocytes, documented in immunocy-tochemical analyses, likely resulted from the massive loss of IL-6-expressing neurons and axons after SCI (Fig. 2E). Lost neuronsand axons at the lesion site were in part replaced with IL-6-non-expressing cells (such as microglia/macrophages), so neuronaland axonal proteins in the whole-cell extracts used in Westernblots were replaced with proteins expressed in IL-6 non-expressingcells, and likely by other abundantly expressed proteins at thelesion epicenter such as extracellular matrix proteins. Noticeably

Fig. 4. Astrocytic activation parallels interleukin (IL)-6 changes associated with allodyniasegments (L1-L5, pooled) used in Fig. 2. (B) Quantitative analysis of GFAP levels in the saganglia.

increased IL-6 immunolabeling in reactive astrocytes at T10 (�2fold) can also partially explain why the overall decreases in IL-6levels at the lesion site detected in Western blots were markedlylower (30%) than the overall loss of IL-6-expressing neurons/axons(�90%; Fig. 2E). As shown in representative Western blots inFig. 2E, there was a �90% decrease in the intensity of bands specificfor NeuN and for phosphorylated neurofilament (pNF; axonal mar-ker). Although decreases in NeuN and pNF also reflect the dysfunc-tion of surviving neurons in chronically injured spinal cords[35,81,92], the decreases predominantly represented the actualloss of neurons and axons at the lesion site, consistent with ourprevious report [58].

IL-6 immunolabeling was not performed in DRG, although wefound IL-6 expression in dorsal roots (Fig. 3E merged, marked withyellow arrow), in both axons and satellite cells that are visible inFigure 2E2, consistent with the report by Vazquez et al. [93]. How-ever, our IL-6 Western blot analysis did not indicate any differencebetween ‘‘SCI no pain’’ and ‘‘SCI pain’’ rats in DRG (Fig. 2D), so wedid not further pursue IL-6 labeling in DRG.

3.3. IL-6-R Ab exerts robust and lasting analgesic effects

Our data presented in Figs. 2 and 3 suggested that increased IL-6 levels may significantly contribute to hind-limb mechanical allo-dynia in SCI rats. To test this hypothesis, we examined the effect ofneutralizing IL-6-R Ab on mechanical allodynia in SCI rats. Wechose to use IL-6-R Ab in part because the FDA has approvedhumanized IL-6-R Ab (tocilizumab, ACTEMRA) for the treatment

. (A) A representative glial fibrillary acidic protein (GFAP) Western blot in the lumbarme samples that were described in Fig. 2D. SCI, spinal cord injury; DRG, dorsal root

Fig. 5. Interleukin (IL)-6R antibody (Ab) abolishes mechanical allodynia. (A)Western blot performed with the IL-6-R Ab used for intraperitoneal (i.p.) injectionsshowed that this Ab recognizes mouse recombinant IL-6-R (R&D Systems Cat#506-RL/CF). Two Western blot lanes were loaded with 50 and 100 ng of rIL-6-R. Thestrongest intensity band had the expected molecular weight (MW) of 55 kDa. Therewas only one band of the same MW detected in uninjured and injured spinal cordssamples. Here are presented 2 lumbar (L1-L5) spinal cord samples isolated 42 daysafter spinal cord injury (SCI). (B) The percentage changes in hind-limb mechanicalthresholds were measured in 22 SCI rats; they all showed an �70% decrease inmechanical thresholds in both the left and right hind limbs (LH and RH,respectively) 28 days after SCI. After threshold measurements, 12 SCI rats wereinjected i.p. with 1.5 mg/kg IL-6-R Ab (marked with black arrow), and those ratsshowed significantly smaller% decrease in mechanical thresholds 35 and 42 daysafter SCI (gray bars) compared to vehicle-injected SCI rats (black bars). (C) Bloodwas collected 12 hours after IL-6-R Ab injection from 2 vehicle- and 2 Ab-injectedrats, and also 8 days after injections from 3 vehicle- and 3 Ab-injected SCI rats. Theblood levels of IL-6-R Ab (gray arrow) were assessed by Western blots, as presentedhere (vertical arrows mark blood samples isolated from SCI rats that received IL-6-RAb injections). The last lane was loaded with IL-6-R Ab, here presented with 2exposure times (vertical gray arrows). The lower exposure time (that yielded nosignal in blood samples) shows the position of the Ab band that was expected anddetected in blood samples. Although present 12 hours after injection, the amount ofIL6R Ab in the blood was markedly lower than the injected dose. A white arrowmarks an unidentified blood protein that reflected equal loading of all samples. (D)SCI rats treated with IL-6-R Ab (gray bar) gained �5% more body weight from 28 to42 days after SCI than did vehicle-treated SCI rats (black bar). (E) Locomotorrecovery (Basso, Beattie, and Bresnahan [BBB] scores) of SCI rats treated with IL-6-RAb (gray line; n = 12) was the same as the locomotor recovery of vehicle-treated SCIrats (black line; n = 10), before and after drug or vehicle injections (marked witharrow). Inserted bar graph: Similar levels of injury in the 2 groups were alsoreflected by the recovery of bladder functions that occurred 7 days after SCI in bothgroups (7.29 ± 1.22 days for vehicle-treated and 7.33 ± 1.66 days for IL-6-R Ab-treated SCI rats).


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of RA, which supports the clinical relevance of our experiments.We confirmed that the IL-6-R Ab used in our experiment recog-nized recombinant mouse sIL-6-R in Western blot assays(�55 kDa band; Fig. 5A), consistent with other reports[15,72,102]. Although we used a polyclonal anti-mouse IL-6-R Ab(a monoclonal anti-rat IL-6-R Ab was not commercially available),it recognized only one band (�55 kDa) in rat spinal cords, whichcorresponded in size to the recombinant sIL-6-R, thus indicatingthat there was minimal likelihood of this Ab neutralizing proteinsother than sIL-6-R. Whether this Ab also neutralizes membranousIL-6-R Ab remains to be determined.

IL-6-R Ab was injected 28 days post-SCI, after 2 measurementsof hind-limb allodynia (21 and 28 days) confirmed significantly re-duced thresholds in both hind limbs at both time points in a groupof SCI rats (‘‘SCI pain’’ group). As shown in Fig. 5B, one injection ofIL-6-R Ab significantly decreased hind-limb allodynia 35 days and42 days after SCI – or 1 and 2 weeks after the Ab injection(n = 12). Since the mechanical thresholds measured 42 days afterSCI in SCI rats treated with IL-6-R Ab did not differ from theirpre-SCI levels, we concluded that IL-6-R Ab abolished SCIP 2 weeksafter one i.p. administration. Previous studies have shown that i.p.-injected IL-6-R Ab can persist in the circulation for 3 days [53,64],so we tested if IL-6-R Ab was present in the serum of IL-6-R Ab-treated SCI rats 12 hours or 8 days after injection. As shown inFig. 5C, Western blot analyses of the blood immunoglobulins re-vealed that goat IgG (the neutralizing IL-6-R Ab was raised ingoats) was detectable 12 hours after injection, but not 8 days afterinjection (although various conditions were tested, ie, increasedantibody concentrations or exposure times), so the analgesic effectof the antibody 2 weeks after the drug injection likely resultedfrom long-term IL-6-R Ab-induced changes in nociceptive path-ways in SCIP rats.

We also found that the body weight of IL-6-R Ab-treated SCIrats increased by 6.23% from 28 to 42 days after SCI, while the in-crease in body weight of an SCI group treated with vehicle wasonly 1.92% (⁄P = 0.02). Furthermore, the increase in the bodyweight of IL-6-R Ab-treated SCI rats was similar to the weight gainrecorded in SCI rats that did not develop SCIP (Fig. 1D), suggestingthat IL-6-R Ab-treated SCI rats did not experience chronic pain.

BBB scores in IL-6-R Ab-treated and vehicle-treated SCI rats(Fig. 5E) were indistinguishable before and after Ab injection; from1 to 42 days post-SCI, so the analgesic effect of IL-6-R Ab or weightgain did not result from improved motor recovery of SCI rats.

3.4. IL-6-R Ab restores GLT-1 expression in chronically injured spinalcords

In this study we have not characterized the mechanisms under-lying the contributions of IL-6 to SCIP (this will be reportedelsewhere). However, we found that IL-6-R Ab treatment signifi-cantly affected SCI-induced changes in protein levels of the gluta-mate transporter GLT-1. This result: (a) confirmed thatsystemically administered IL-6-R Ab exerted a central effect onthe spinal cord cells; and (b) offered a possible explanation forthe mechanism underlying the analgesic effect of IL-6-R Ab in SCIPrats. In agreement with previous reports [39,65], we found mark-edly decreased protein levels of GLT-1 not only at the lesion site(T10; Fig. 6A), but also in lumbar segments of chronically injuredspinal cords (Fig. 6B; #P < 0.01). Our GLT-1 immunolabeling studiesalso confirmed distinctly decreased GLT-1 levels at the lesion site inchronically injured spinal cords (42 days post-SCI; Fig. 6C), in bothwhite and gray matter astrocytes, which was particularly visible inthe superficial layers of the dorsal horn (marked with white arrow).

One i.p. injection of IL-6-R Ab partially (�30%) but significantly(⁄P < 0.05) reversed SCI-induced decreases in GLT-1 levels 12 hoursafter injection at the lesion site (Fig. 6A). Interestingly, the same

effect was found even 8 days after IL-6-R Ab injection in lumbarsegments (Fig. 6B), despite the fact that IL-6-R Ab was not presentin the circulation of SCI rats 8 days after the injection (Fig. 5B). Co-immunolabeling of GLT-1 and gp130 (red; Fig. 6D) confirmed thatastrocytes in injured spinal cords co-expressed GLT-1 and gp130,thus confirming that gp130 activation in astrocytes by elevatedIL-6 in SCIP rats may affect GLT-1 levels, or that blocking of IL-6signaling (ie, prevention of activation of astrocytic gp130) with

Fig. 6. Interleukin (IL)-6R antibody (Ab) restores glutamate transporter GLT-1. (A) A representative Western blot of GLT-1 (�75 kDa band) in 2 age-matched uninjuredsamples (T10), 2 injured T10 segments (lesion site) 29 days after spinal cord injury (SCI) or 12 hours after vehicle injection, and 2 injured T10 samples 12 hours after IL-6-R Abinjection. The bar graph below shows a significant decrease in GLT-1 levels 29 days after SCI at the lesion site (black bar; #P < 0.001)). However, 12 hours after IL-6-R Abinjection, GLT-1 levels were significantly elevated (gray bar; ⁄P < 0.05). In the same samples, we also assessed changes in glial fibrillary acidic protein (GFAP) and themicroglia/macrophage marker Iba-1 (as reported in our previous report, Carlton et al. [14]). Although both GFAP and Iba-1 were markedly increased after SCI, they were notaffected by the injection of IL-6-R Ab. Beta-actin was used as a control for loading. (B) A representative Western blot of GLT-1 (�75 kDa band) in 3 age-matched uninjuredsamples (pooled L1-L5), 3 injured lumbar segments (pooled L1-L5) 35 days after SCI or 8 days after vehicle injection, and 3 injured lumbar samples (pooled L1-L5) 8 days afterIL-6-R Ab injection (gray bar). The bar graph below shows a significant decrease in GLT-1 levels 35 days after SCI even away from the lesion site, in lumbar segments (blackbar; #P < 0.001). However, 8 days after IL-6-R Ab injection, GLT-1 levels were significantly elevated (gray bar; ⁄P < 0.05). GFAP and Iba-1 levels in the same samples weresignificantly increased 35 days after SCI, but were not affected by IL-6-R Ab treatment. (C) GLT-1 immunolabeling (green) in T10 of an uninjured age- matched rat and at thelesion site (T10) of a SCI rat 42 days after SCI. White arrows point to markedly reduced GLT-1 in the dorsal horns of chronically injured spinal cords, indicating the significanceof GLT-1 decreases in pain processing in injured spinal cords. Substantial cavitations were present in injured spinal cords. Calibration line: 200 lm. (D) GLT-1-expressingastrocytes were co-labeled with gp130 (red). High-magnification images of double labeled (GLT-1 + gp130) white matter astrocytes, and also astrocytes in the vicinity of thecavity. Calibration lines: 50 lm.


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IL-6-R Ab can restore GLT-1 levels. We also showed that IL-6-R Abdid not affect GLT-1 by reducing overall astrocytic activation (in-creases in GFAP) at the lesion site (Fig. 6A) or in lumbar segments(Fig. 6B).

Given that microglial activation has been shown to play a keyrole in SCI pain [36], and that IL-6-R Ab treatment has demon-strated an effect on both microglia and macrophages in a mousemodel of SCI [33], we also assessed levels of microglia/macrophagemarker, Iba-1. However, no changes in Iba-1 levels were noted inIL-6-R Ab-treated SCI rats (Fig. 6A and B).

4. Discussion

4.1. Increased IL-6 and SCI pain

IL-6 induction after SCI has 2 phases, an acute phase, followedby a less studied, secondary or chronic phase. Acute IL-6 levelsmeasured in the cerebrospinal fluid of the most severely injuredSCI patients are significantly correlated with the severity of the in-jury [42], in agreement with data in animal models [105]. How-ever, the exceedingly high IL-6 levels detected in the first hours/days after SCI decline to the baseline level by 2-3 days after SCI[56,60], and are not correlated with the development of pain inSCI patients [42]. Although the time window for onset of this sec-ond wave of IL-6 increases after SCI has not been identified, it isbecoming increasingly clear that this delayed IL-6 upregulation is

associated with SCIP, in agreement with our findings presentedhere. For example, Detloff et al. [25] found IL-6 chronically upreg-ulated after SCI, but more so in SCIP rats, while Davies et al. [19]showed that IL-6 levels not only increase in the serum of chronicSCI patients, but are even higher in patients who develop pain.

One of the intriguing questions about these secondary IL-6changes after SCI is what processes determine individual variabil-ity in the amplitude of IL-6 changes that are associated with SCIPdevelopment. Interindividual variability in IL-6 production hasbeen documented in genetically distinct strains of rodents. Forexample, injecting the same amount of bacterial lipopolysaccha-ride into B6 and A/J mice induced markedly different circulatinglevels of various inflammatory mediators [20]; B6 mice showedhigher IL-6 levels than A/J mice. Interestingly, it has also beenshown that the genotype of B6 mice determines their markedly en-hanced sensitivity to nociceptive stimuli [50]. Given that ourexperiments used outbred Sprague-Dawley rats, it is possible thattheir genetic variability predetermined their proinflammatory re-sponse to SCI and production of IL-6 in chronically injured spinalcords, and thus the development of SCIP.

4.2. Astrocytic induction of IL-6

Our immunocytochemical analyses showed that the predomi-nant source of elevated IL-6 in SCIP rats was activated astrocytes.Although the contribution of neuronal IL-6 to overall IL-6 increases


154 (2013) 1115–1128

in SCIP rats was not analyzed, it nevertheless cannot be ruled out.However, the investigation of the neuronal/axonal IL-6 in SCIP wasbeyond the scope of this study.

Similar to our findings, IL-6 was found in neurons and astrocytesafter global brain ischemia [55], but in neurons and microglia afterfocal cerebral ischemia [86]. Therefore, it appears that the glialsource of IL-6 induction depends on the extent of the injury andaccompanying inflammatory processes. For example, proinflamma-tory tumor necrosis factor alpha and IL-1 both induce IL-6 synthesisin astrocytes, but not in microglia. In contrast, granulocyte/macro-phage colony-stimulating factor triggers IL-6 production in microg-lia, but not in astrocytes [87], while lipopolysaccharide triggers IL-6production in both cell types in vitro [79]. However, inflammatorysignaling upstream of IL-6 induction in astrocytes in chronically in-jured spinal cords remains to be determined. Our analyses also re-vealed that elevated IL-6 levels in lumbar astrocytes reflectedastrocytic activation selectively increased in SCIP rats (as describedin Nesic et al. [58]), rather than IL-6-mediated activation of astro-cytes. Although IL-6 can activate astrocytes in pain conditions[90], our data showed that IL-6-R Ab does not affect GFAP levelsin SCIP rats (Fig. 6A and B). Therefore, increased production of IL-6 in hyperreactive astrocytes in SCIP rats may explain still-poorlyunderstood astrocytic contribution to SCIP, but elevated IL-6 cannotexplain increased astrocytic activation in SCIP rats.

Since we found IL-6 significantly induced in reactive astrocytesin lumbar and thoracic segments of SCIP rats, it is likely that in-creased IL-6 can be found throughout injured spinal cords, as weand others have shown persistently activated astrocytes from lum-bar to cervical segments [14,43,59]. Although we showed that onlyIL-6 increases in lumbar segments are directly correlated with thedevelopment of hind-limb allodynia, it is nevertheless probablethat IL-6 upregulation elsewhere (eg, at T10 or possibly at otherspinal cord, and even brain regions) can also affect nociception inhind limbs. For example, pathological changes in nociceptive path-ways might be initiated by IL-6 in lumbar segments of a specificsubpopulation of SCI rats (ie, SCIP rats), but could be further signif-icantly amplified by elevated IL-6 at the lesion site or in other re-gions, all along the ascending pain pathways. Although we didnot examine it in this study, it is possible that astrocyte inductionof IL-6 at T10 would correlate with the allodynia in the thoracic re-gions (ie, girdling).

4.3. IL-6-R Ab-induced analgesia and restoration of GLT-1

In 2010, the FDA approved the humanized monoclonal antibodyagainst IL-6 receptor (tocilizumab, sold under the trade name ACT-EMRA) for the treatment of RA, and a year later for the treatment ofjuvenile idiopathic arthritis in children. Although IL-6-R Ab has notbeen studied in SCI pain, tocilizumab has recently been tested insciatica patients [62]. This study showed that tocilizumab allevi-ates chronic pain in sciatic patients more effectively than dexa-methasone, the first-line treatment for that painful condition.

Humanized monoclonal Abs have been used as therapeuticagents in various diseases, including such neuropathological condi-tions as Alzheimer disease [91], glioblastoma [10], and multiplesclerosis [70]. The use of systemically administered humanizedmonoclonal Abs to treat neuropathological conditions depends onthe Abs ability to penetrate the blood–brain barrier. A growing bodyof evidence indicates that Abs can cross this barrier in animals andhumans [5,34,49,52,67,78,80], although only a fraction of an Ab canpenetrate from serum into the central nervous system parenchymaunder normal conditions [52,78]. However, therapeutic Abs modi-fied to be bispecific (for the main target, eg, IL-6-R, and for thetransferrin receptor) can penetrate the intact blood–brain barriermuch more readily [3,106]. Alternatively, therapeutic Abs can bealso markedly more penetrating if the barrier is compromised

[75]. Since the blood–spinal cord barrier remains chronically com-promised after SCI [18,59], it is likely that a portion of the IL-6-R Abinjected systemically in our experiments crossed this compromisedbarrier, and so could affect pain processing, including impairedfunction of the astrocytic glutamate transporter GLT-1 in injuredspinal cords.

GLT-1 is abundantly expressed in astrocyte membranes that arein close association with synapses [94], so any decreases in GLT-1levels will significantly impair glutamate uptake, and thus can am-plify glutamatergic signaling in nociceptive pathways. Althoughdecreased expression of GLT-1 and decreased glutamate uptakehave been shown to contribute to the development of neuropathicpain in various pathological conditions [8,85,95], our results arethe first to indicate that decreased GLT-1 levels also have an impor-tant role in SCIP. Since the restoration of decreased GLT-1 levels invarious models of neuropathic pain by ceftriaxone, amitriptyline,or propentofylline causes significant analgesia [48,73,89], it islikely that restoration of GLT-1 levels in IL-6-R Ab-treated SCI ratscan explain the pain-relieving effect of this Ab. Moreover, the last-ing and widespread effect of IL-6-R Ab on GLT-1 levels (8 days afterinjection) that coincide with the enduring analgesic effects of IL-6-R Ab (8 days after injection) further support a link between theanalgesia and restoration of GLT-1 levels in injured spinal cords.

Since decreased GLT-1 levels are typically found in activatedastrocytes [54], we also tested whether IL-6-R Ab restored GLT-1by inhibiting overall astrocytic activation. However, we did notfind a significant effect of IL-6-R Ab on GFAP levels. Therefore,our results suggest that significant analgesia in SCI rats can beachieved by selective targeting of GLT-1, and does not require non-specific and broad inhibition of astrocytic activation, which can bedetrimental in SCI [96]. However, whether GLT-1 restoration is theonly nociceptive (or astrocytic) process affected by IL-6-R Ab re-mains to be determined.

4.4. IL-6-R Ab vs gabapentin (GBP)

GBP is most often recommended as a first-line treatment forSCIP, although it provides only transient relief, so its use requiresrepeated daily dosing over an extended period. Unfortunately, pro-tracted use of GBP aggravates its serious side effects, which includedizziness, confusion, somnolence, and edema, so patients often re-fuse to continue the treatment. We found a similarly transient anal-gesic effect of GBP in SCIP rats, consistent with the findings ofDensmore et al. [23]. In contrast, the analgesic effect of IL-6-R Ablasted for at least 2 weeks after a single systemic injection, thussuggesting that the pain-relieving effect of IL-6-R Ab is long-lastingand may even be permanent, which would be a considerable clin-ical advantage over GBP. Furthermore, we also found that IL-6-R Absignificantly reduced allodynia in all SCI rats that developed pain,as opposed to GBP (ineffective in �30% of SCI rats), similar to theclinical findings where Putzke et al. [71] showed that �33% ofSCI patients who were able to tolerate the side effects of GBP didnot respond to that pain treatment.

Therefore, our study is the first to indicate that humanized IL-6-RAb should be considered as a novel and promising treatment for SCIpain, and warrants further preclinical and clinical investigations.

Conflict of interest statement

The authors declare that no conflicts of interest exist.

Acknowledgements

This work was funded by the Mission Connect, a project of TIRRFoundation, Moody Center for Brain and Spinal Cord Injury,


154 (2013) 1115–1128 1127

University of Texas Medical Branch at Galveston. We thank Dr. Ja-son Velano for the gross morphological and histological assess-ments of the liver and GI tract in our experimental animals. Wealso thank Dr. David Konkel for critically editing the manuscript.

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Inhibition of IL-6 signaling: A novel therapeutic approach to treating spinal cord injury pain

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