Oral gabapentin treatment accentuates nerve and responses following constriction in Wistar rats

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Neuroscience

Letters

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

Oral

gabapentin

treatment

accentuates

nerve

and

peripheral

inflammatory

responses

following

experimental

nerve

constriction

in

Wistar

rats

Carlos

C. Câmara

a,b

_,

_Heitor

_F.

_Ramos

a

_,

_Alan

_P.

_da

_Silva

a

_,

_Celina

_V.

_Araújo

c

_,

Antoniela

S. Gomes

e

_,

_Mariana

_L.

_Vale

e

_,

_André

_Luiz

_R.

_Barbosa

e

_,

_Ronaldo

_A.

_Ribeiro

e

_,

Gerly

A.C.

Brito

d,e

_,

_Carlos

_Maurício

_C.

_Costa

a

_,

_Reinaldo

_B.

_Oriá

c,d,∗

a_Laboratory_of_Experimental_{Neurophysiology,}_Department_of_Physiology_and_{Pharmacology,}_School_of_Medicine,_Federal_University_of_Ceará, Fortaleza,CE,Brazil

b_Laboratory_of_{Neurophysiology,}_School_of_Veterinary,_Federal_and_Rural_University_of_the_SemiArid,_Mossoro,_RN,_Brazil

c_Laboratory_of_Tissue_Healing,_Ontogeny_and_Nutrition,_Institute_of_Biomedicine_of_the_Brazilian_SemiArid,_Federal_University_of_Ceara,_Fortaleza,_CE,_Brazil d_Department_of_Morphology,_School_of_Medicine,_Federal_University_of_Ceara,_Fortaleza,_CE,_Brazil

e_Laboratory_of_Inflammation_and_Cancer,_Department_of_Physiology_and_{Pharmacology,}_School_of_Medicine,_Federal_University_of_Ceara,_Fortaleza,_CE,_Brazil

h

i

g

h

l

i

g

h

t

s

•Anervepro-inflammatoryeffectofgabapentintreatmentwasidentified.

•Gabapentinincreasedcarrageenan-inducedpawedemaandperitonealcellmigration. •Concernofgabapentinwidespreaduseinsystemicinflammatorydiseaseswasraised.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received8August2013 Receivedinrevisedform 30September2013 Accepted3October2013

Keywords: Gabapentin Sciaticnerve Neuropathicpain Cytokine Inflammation

a

b

s

t

r

a

c

t

Gabapentin(GBP)isananti-convulsivedrugoftenusedasanalgesictocontrolneuropathicpain.This studyaimedatevaluatingwhetheroralGBPtreatmentcouldimprovenerveinflammationresponseafter sciaticnerveconstrictioninassociationwithselectedpainandmotorspontaneousbehaviorassessments inWistarrats.Weevaluatednervemyeloperoxidase(MPO)andinflammatorycytokinesonthe5thday post-injury,timeinwhichnerveinflammationisongoing.Inaddition,theroleofGBPon carrageenan-inducedpawedemaandperitonealcellmigrationwasanalyzed.GBPwasgivenbygavageatdoses of30,60and120mg/kg,60minpriortochronicconstrictionofthesciaticnerve(CCSN)andduring5 dayspost-injury,12/12h.CCSNanimalstreatedwithsalinewereusedascontrolsandforbehavioral andinflammationassessmentsuntreatedsham-operatedratswerealsoused.Onthe5thday,GBP(60 and120mg/kg)alleviatedheat-inducedhyperalgesiaandsignificantlyincreaseddeltawalkingscoresin CCSNanimals,thelattersuggestingexcitatoryeffectsratherthansedation.GBP(60mg/kg)significantly increasednerveMPO,TNF-␣,andIL-1␤levels,comparingwiththesalinegroup.GBP(120mg/kg)reduced theanti-inflammatorycytokineIL-10nervelevelscomparedwiththeCCSNsalinegroup.Furthermore, GBP(60and120mg/kg)increasedcarrageenan-inducedpawedemaandperitonealmacrophage migra-tioncomparedwiththeCCSNsalinegroup.AltogetherourfindingssuggestthatGBPaccentuatesnerve andperipheralinflammatoryresponse,howeverconfirmeditsanalgesiceffectlikelyduetoan indepen-dentCNS-mediatedmechanism,andraisesomeconcernsaboutpotentialGBPinflammatorysideeffects inwidespreadclinicaluse.

∗Correspondingauthorat:InstituteofBiomedicineoftheBrazilianSemiarid, SchoolofMedicine,FederalUniversityofCeara,RuaCoronelNunesdeMelo,1315, CEP:60430-270Fortaleza,CE,Brazil.Tel.:+5508533668239.

E-mailaddress:rbo5u@virginia.edu(R.B.Oriá).

1. Introduction

Neuropathicpainisadirectconsequenceofinjuryordisease causingdysfunctionoftheperipheralandcentralnervoussystem atdifferentlevels,mostly affectingthemiddle-agedand elderly withanescalatingglobalburden.

Treatmentforneuropathicpainisstillnotsatisfactoryformost of the patients especially those with a more severe condition,

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onestructuralderivativeofthegamma-aminobutyricacid(GABA), inadditiontobeingananti-convulsivedrug,isnowfurther con-sidered as an effective therapeutical drug for some forms of neuropathic and post-surgical pain [26]. Gabapentin effects of reducing allodynia may be associated with specific binding to calcium-voltagedependent␣2␦ subunits, reducing calciumcell influx withchanges in neurotransmitter release [13] or activa-tionofproteinkinase-G-K+channelpathways[24].Severalstudies have showngabapentin-related analgesiceffects in experimen-tallyinducedneuropathicpain[7,15].However,studiesassociating GBPwiththeinflammatoryresponsefollowingsciaticnerve con-strictionare still scarce. Thisis an importantissue since nerve inflammationmayinfluencedrugefficacyduringneuropathicpain treatment[30].

Hurleyetal.aftergivingasingledoseofGBP(300mg/kg)via gavage,2.5hfollowingcarrageenan-inducedintra-plantaredema inWistarrats,didnotfindsignificantacuteedemachange[16].

In this current study we explored whether prolonged GBP

treatment modulated inflammatory responses following sciatic nerveconstrictioninratsbyassessingtheinvolvementofnerve inflammatorycytokines,associatingtheseoutcomeswithselected pain-relatedandmotorbehaviors.Inaddition,theroleofGBPon carrageenan-inducedpawedemaandperitonealcellmigrationwas assessedtoevaluateaperipheralinflammatoryresponse.

2. Materialsandmethods

ProtocolsfromthisstudywereapprovedbytheAnimalCare andUseCommitteeoftheFederalUniversityofCearaandwerein accordancewiththeBrazilianCollegeforAnimalExperimentation (COBEA)andtheInternationalAssociationfortheStudyofPain (IASP)guidelines.

2.1. Animals

160maleWistarratsweighingbetween250and300gfromthe DepartmentofPhysiologyandPharmacologyvivariumatthe Fed-eralUniversityofCearáwereusedinthisstudy.Ratswerehoused inatemperature-controlledroom(26±2◦_C)_with_free_access_to water and chowdiet in a 12h/12hlight/darkcycles. All surgi-calprocedureswereperformedinthelaboratoryofExperimental NeurologyinthePhysiologyandPharmacologydepartmentatthe FederalUniversityofCeara.

2.2. Sciaticnervechronicconstriction

Weusedthechronicconstriction ofthesciaticnerve(CCSN) modeltoinducetheexperimentalneuropathy,describedby Ben-nettandXie,1988[2]andmodifiedbySommerandMyers[32]. Animalswereanesthetizedwithintraperitonealinjectionof tribro-moethylene(25mg/kg),followingtrichotomyandanti-sepsisofthe surgeryfield.A15-mmlongitudinalincisionoftherightthigh,at thelevelofthefemoraltrocateroftheposteriorlimb,wasusedto accessandexposethesciaticnerveaftergluteusandfemoralbiceps dissection.Weusedthree4-0cat-gutlooseligaturesontheright pawsciaticnerve,distancedapproximately1mmbetweenthe lig-aturesandproximal tothesciatictrifurcationinducinga slight nerveischemia.Intheleftthigh,thesciaticnervewasexposed,

usedbasedonapreviousstudyshowingitssafetyuseand anal-gesiceffectinamodelofneuropathicpain[18].Asthemaximum GBPeffectoccurs60minafteroral administration[17],thefirst dosewasgiven60minpriortothenervesurgeryand60minbefore eachbehaviortest.Followingthelastbehaviortestonthe5thday post-injurytheanimalswereeuthanizedandthesciaticnervewas removedtoconducthistologicalandmolecularbiologystudies.

2.4. Spontaneousmotorbehaviors

TofindoutwhetherGBPcouldamelioratemotorbehaviorsafter sciaticnerveinjuryandrule-outapossiblesedationeffect,cohort animalswerekeptinawoodencage(100cm×50cm×50cm)for a5-minacclimationtime.Theobservingcage(withPlexiglas)with thefloorcoveredwithwoodshavingswasplacedinaslightly illu-minatedandsilentroomforbehavioraltesting.Positionedinfront ofthecage,theobservercouldidentifyeachbehavioralcomponent andrecord itusing acomputersoftware (Comporta®₎_designed byProf.MarcusVale(FederalUniversityofCeará,Fortaleza, CE, Brazil).Eachanimalwasobservedforresting-sleepingandwalking behaviorand observedduring a30min-time.Thefirst observa-tionwas60mbeforethesurgery(baseline)andthenonthe5th daypost-surgeryand60mafterthelasttreatment.Adeltamean behaviorvaluewasderivedfrombaseline(priortosurgery)and5th daypost-surgerytimepoints.Cumulativeresting-sleeping behav-iorwasmeasured(inseconds) iftheanimalswere foundlying immobileonthebedding.Cumulativewalkingbehaviorwasalso measured(inseconds).Experimenterswereunawareofthe iden-tityoftheexperimentalgroups.

2.5. Thermaltest

InordertoconfirmtheGBPanalgesiceffectontheCCSN,we evaluatedpain-inducedresponsestothermalstimuli.Testsusing noxious(46◦_C)_stimulation_were_performed_involving_immersion oftherat’shindpawina bathuntilthewithdrawalorstruggle wasobserved.After15sofexposure(cut-off),thethermal stimu-luswasremovedandthewithdrawallatencywasdetermined.The testswereperformedondayzero(preoperativeday)andonthe 5thpostoperativeday60minafterGBP-treatment.Adeltascore wascalculatedforfurtheranalyses.

2.6. Nerveinflammationmarkers

In order to evaluate GBP effect on nerve inflammation, we obtained snap-frozen sciatic nerves following CCSN for nerve

cytokine measurements by immunoenzymatic assays and by

immunohistochemistry.

2.6.1. ELISAassaysfornervecytokines

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Fig.1.Gabapentin(GBP)treatment(30,60and120mg/kg)effectonthespontaneousbehaviorsresting-sleeping(A)andwalking(B)andonthethermaltest(C).Atleast n=10animals/group.Data(mean±SEM)areexpressedasdeltascores.Forthethermaltestcut-off:15s.*p<0.05vs.salinegroupbyANOVA.

2.6.2. Nervemyeloperoxidase(MPO)assay

TheMPOenzymeisfoundprimarilyinazurophilicgranulesfrom inflammatorycellsandhasbeenextensivelyusedasabiochemical markerforinflammatorycellinfiltrationintovarioustissues.After 5daysofGBP(60and120mg/kg)treatment(12/12h),thesciatic nerveMPOactivityassaywasmeasuredasdescribedelsewhere [20].ResultswerereportedasMPOunits/mgoftissue.CCSNand shamoperatedratsreceivingsalinewereusedascontrols.

2.6.3. Immunohistochemistryofnervecytokines

Under deep anesthesia, rats were transcardially perfused withsalineand then thedistal sciaticnerve segment was har-vested5mm-distaltotheligatures,and immediatelyimmersed inbufferedformaline(pH7.4)for24handsenttothehistology

corefor immunohistochemistryprocessing.

Immunohistochem-istry for TNF-␣, IL-1␤, and the macrophage marker-Iba-1was performed using the streptavidin–biotin–peroxidase method in formalin-fixed, paraffin-embedded tissue sections(4␮m thick), usingpolyclonalrabbitanti-mouseantibodies(SantaCruz Biotech-nology, SantaCruz, CA), as described elsewhere [20]. Negative controlsectionswereprocessedsimultaneouslywiththeprimary antibody being replaced by PBS-BSA 5%. None of the negative controls showed immunoreactivity. Slides were counterstained withHarry’shaematoxylin,dehydratedinagradedalcoholseries, clearedinxylene,andcoverslipped.

2.7. Peripheralinflammationmodels

InordertoevaluateapossibleGBPeffectonotherperipheral inflammatory conditions, we subjected CCSN-free rats to well-knownmodelsofinflammation.

2.7.1. Carrageenan-inducedpawedema

In order to study peripheral GBP inflammatory effects in rats without sciatic nerve injury, paw edema was induced by intra-plantar injection of carrageenan (100␮g/paw, 0.1ml) in

experimental rats receiving a 5-day GBP treatment (60 and

120mg/kg,12/12h).Ratsreceivingsalinewereusedascontrols. Carrageenanwasinjectedintheipsilateralhindpaw60minafter

lastdoseofGBP.Pawvolumewasmeasuredby

plesthysmome-try(Ugo-Basile7140Plesthysmometer)immediatelybefore(basal volume)andat1,2,3,4and5haftercarrageenanadministration. Resultswereexpressedaspawvolumevariation(ml),calculated bysubtractingpawbasalvolumefromthespecificvolumeattimes 1,2,3or4h.

Furthermore,pawskinsampleswereremovedinasubsetof experimental rats to measure skin MPO activity 5h after car-rageenaninjection.

2.7.2. Inflammatorycellmigrationintoperitonealcavity

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Fig.2. Gabapentin(GBP)treatment(60and120mg/kg)effectonTNF-␣(A),IL-1␤(B),andIL-10(C)levelsintheinjuredsciaticnervedetectedbyELISA.N=atleast5 forallgroups.Data(mean±SEM)areexpressedinpicograms/ml.*p<0.05vs.salinegroup(Sal),byANOVA.(D)Oralgabapentintreatment(60and120mg/kg)increases myeloperoxidase(MPO)activityintheinjuredsciaticnerve.N=6(allgroups).Data(mean±SEM)areexpressedasunits/milligramofnervetissue.*p<0.05vs.salinegroup (Sal)byANOVA.

intraperitoneallyinjectedcarrageenan(300␮g/1.0ml)60minafter thelastdoseofGBPin experimentalratsreceivinga5-dayGBP treatment(60and120mg/kg,12/12h).Ratsreceivingsalinewere usedascontrols.Threehoursaftercarrageenaninjection,animals wereeuthanatized andperitonealfluidwascollected. Totaland differentialcellcountswereperformedasdescribedbySouzaand Ferreira(1985)[6].

2.8. Statistics

NormaldistributionwasassessedusingKolmogorov–Smirnov’s

test. Effects amongst groups were assembled using one-way

ANOVAforparametricdata.Incaseofp<0.05,Tukey’stestwas appliedtodrawmultiplecomparisonsamongstgroups.Insome behavioral tests, independent Student’s T test was used when appropriate.Valuesareshownasmean±SEM.

3. Results

3.1. GBPeffectonspontaneousandinducedbehaviors

3.1.1. Locomotionbehavior

After5-daysof GBPtreatment, oralGBP (60and 120mg/kg) caused significant reductions in the resting-sleeping behavior (delta=−72.95±49.6; delta=−49.53±43.74s,

respec-tively) in comparison with the neuropathic saline group

(delta=150±38.58s) (p<0.01),Fig.1A.On theotherhand,GBP

(60mg/kg)increasedthewalkingbehavior(delta=42.50±9.82s.), asopposedtotheneuropathicsalinegroup(delta=−16.60±9.55s, p<0.05)(Fig. 1B), therefore showingthat GBP was not ableto inducesedationbutratherinducedanexcitatoryeffect.

3.2. Thermaltest

In the thermal test performed to experimental rats on

the 5th day post-injury, GBP (60 and 120mg/kg)

signifi-cantly increased the ipsilateral hind limb (operated)’s with-drawallatencytime(delta=4.04±1.01s,p<0.001and5.64±0.57s, p<0.01,respectively),comparedwiththeneuropathicsalinegroup (−1.06±0.65s)(Fig.1C).

3.3. GBPeffectonsciaticnerveinflammation

3.3.1. ELISAassay

Sciaticnerveconstrictiontountreatedratsinducedasignificant increaseinthenerveTNF-␣levels(p<0.05).BothGBPdoses(60 and 120mg) significantly increased (120.9% and 92%, respec-tively)sciaticnerveTNF-␣level,ascomparedtotheneuropathic salinegroup (Fig. 2A). In addition,sciatic nerve constriction to untreatedratsinducedasignificantrise(almost3-foldincrease) inthenerveIL-1␤levels(p<0.05)comparedtothecontrolsham group.Nevertheless,GBP(60mg)wasabletoincreasenerveIL-1␤

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nervelevelsascomparedtotheneuropathicsalinegroupandas comparedwithGBPatdoseof60mg(Fig.2C).

Furthermore, GBP (60mg) markedly increased (982.16%,

p<0.001)nerveMPOactivityasopposedtotheneuropathicsaline group.However,ahigherdose(120mg)didnotshowdifferences fromthecontrol(Fig.2D).

3.3.2. IL-1ˇ,TNF-˛,andIba-1immunohistochemistry

Cross-sections from the sciatic nerve showed more diffuse immunostainingforpro-inflammatorycytokinesIL-1␤and

TNF-␣,followingchronicnerveconstrictiononthe5thdaypost-injury, asopposedtothecontrolgroup(supplementaryFig.1).In addi-tion,increasedIba-1immunolabelingwasfoundespeciallywithin endoneuralSchwanncells.OralGBP(60mg/kg)markedlyincreased nerve expressionof IL-1␤,TNF-␣, andthemacrophage marker,

Iba-1. The immunolabeling was found mostly in Schwann and

macrophage-typecells(supplementaryFig.1).

Supplementary material related to this article can be

found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2013.10.010.

3.4. GBPeffectonperipheralinflammation

3.4.1. Carrageenan-inducedpawedema

OralGBP at doses of60 and 120mgincreased

carrageenan-induced paw edema three hours after carrageenan

intra-plantar injection (delta=0.46±0.04 and 0.56±0.06ml, i.e. 53%

and 87%, respectively), as compared to the saline group

(delta=0.21±0.04). GBP (120mg/kg) also significantly elevated carrageenan-inducedpawedemaonthe2thand4thhour (sup-plementaryFig.2A).Furthermore,GBP(120mg)increasedby96% (33.98±6.94MPOunits/mgoftissue)MPOactivityinthepawskin when comparedtosalinegroup (17.33±2.34MPOunits/mg tis-sue),datanotshown.

Supplementary material related to this article can be

found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2013.10.010.

3.4.2. Carrageenan-inducedcellmigrationtotheperitoneal cavity

OralGBP(60mg)significantlyincreased(262.19%)peritoneal totalcellmigration(10.25±1.63×10−3_cells),_as_compared_to_the challengedsalinegroup(2.83±0.68×10−3_cells)._Albeit_not reach-ingasignificantlevel,therewasatrendofincreasecellmigrationby the120mgdose(5.93±1.7×10−3_cells,_109.54%)_when_compared tosalinegroup(supplementaryFig.2B).Regardingdifferentialcell count,wefoundthatGBP(60mg/kg)causeda29.6-foldsignificant increaseinmacrophagemigration,ascomparedtotheneuropathic salinegroup,howeverGBPtreatmentatthedoseof120mg/kgdid notcauseanysignificanteffect.Likewise,GBPdo notcauseany significanteffectonneutrophilmigrationtotheperitonealcavity (datanotshow).

4. Discussion

Our studiesconfirmed theanalgesiceffect of GBP usingthe thermaltestfollowingsciaticnerveinjury,furthersupportingthe beneficialGBProleonneuropathicpain.DEVRYetal.usingthe samemodelalsodemonstrateantihyperalgesicandanti-allodynic effectsofGBPonthedoseof50mg/kg,i.p.(27thdaypost-injury) [14].

In contrast to Gustafsson et al. who have shown reduced

locomotion and rearing behaviors after acute and cumulative doses of GBP (350␮mol/kg, ∼60mg/kg) [12], we found that prolonged GBP treatment increasedwalking and reduced rest-sleepingdeltascoresascomparedtocontrols.Wespeculatethat

this effect may be associated with increased serotonin levels, sincebloodserotoninwasfoundincreasedinhealthyyoungmen afterchronicGBP treatment[29] and sincehyperactive starved

animals show increased serotonin central neurotransmission

[27].

Inaddition,wefoundincreasedMPOlevelsintheinjuredsciatic nervefollowingGBPtreatment.MPOisfoundintheazurophilic granulesofmonocyte–macrophageandneutrophilinflammatory cells.Furthermore,followingGBPtreatment,increased immuno-labelingofmacrophage-typecells(includingSchwanncells)was seeninthesciaticnerve stroma(Iba-1immunolabeling).TNF-␣

up-regulationseeninthenervemilieucouldincreaseextra-cellular matrixmetalloproteases[31],ultimatelybreakingtheblood–nerve barrier and driving more migrating inflammatory cells to the endoneuriumstroma [22].AlthoughTNF-␣-related hyperalgesia involvingCfibershasbeendemonstratedfollowingsciaticnerve injury[35],theprimaryblockingeffectofGBPoncalciumchannels mayovercomethiseffect.

GBP-inducednerveexpressionofpro-inflammatorycytokines would be beneficial during sciatic nerve re-myelinization after injury. Accordingto Georgeet al. data[8], TNF-␣ nerve mRNA was foundelevatedon thefirst 12th hourand remains signif-icantly elevated on the 5th day post crush injury, a period in which myelindebrisremoval isconspicuousand thatcoincides withthepeak of macrophageinfluxto thenerve endoneurium (6day-post nerve constrictionin therat)[3,4].TNF-␣couldbe releasedbyactivatedSchwanncellswithautocrineandparacrine effects[28]andcouldrecruitadditionalextra-nervemacrophages [19],requiredtodegrademyelin.Activatedmacrophagesbymyelin fragmentscouldreleaseIL-12andmoreTNF-␣[5].Inourstudy, IL-10nervelevelswerefoundsimilartotheshamgrouponthe 5thdaypost-constriction,levelsthatweredecreasedbyGBP treat-ment (120mg/kg). George et al. found that endoneurial IL-10 immuoreactivityisacutelydeprived24h-followingsciaticnerve injury that is latelyrecoveredaround 5–7days post-injury[9]. IL-10isanti-inflammatorycytokinethatcounterbalancesTNF-␣

andIL-1␤increasedlevelsduringWalleriandegeneration[9].GBP mayhavearoletosustaintheinflammatory-drivenmyelin clear-ance.

IL-1␤can promoteaxonal outgrowthfollowing sciaticnerve injury,whichmaybemediatedbyNGF[33].IL-1␤producedby macrophagescanactivatenerveSchwanncellsandfibroblaststo release NGF [21]. In contrast, IL-1␤ anti-antibodies can impair neuralregeneration [10]. IL-1␤treated rats(100ng/ml), after2 weeks-followingsciaticnerveinjury,couldhastensensitivenerve fiberregenerationand promoteSchwann cellproliferation[33]. Furthermore, pro-inflammatory cytokines, such as IL-1␤, could driveimmune-mediatedphagocytosiswithlymphocyte involve-ment[34].

Our data supporttheconcept that fine-regulated inflamma-toryresponsesarebeneficialtonerveregeneration,likelydueto removalofmyelindebrisbymacrophage-likeactivatedcells,as myelindebriscouldbeinhibitorytonerveregeneration[34].The phagocytosisofmyelindebrisbySchwanncellsandmacrophages couldthereforefavoraxonalsprouting[33].

GBPtreatmentincreasedperitonealmacrophagemigrationand pawedemainducedbycarrageenan,suggestingthatGBPperseis apro-inflammatoryfactor,thatunexpectedeffecthighlightsthe needofcautionfor long-termGBPprescriptioninconditionsof systemicinflammatorydiseases.

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themechanisms,time course and finehistology nerve changes involvedintheGBPinductionofpro-inflammatorycytokinesand theireffectsonneuropathicpain.

5. Conclusion

Altogetherour findings suggest that GBP accentuates nerve andperipheralinflammatoryresponse,andconfirmeditsanalgesic effect,thatcouldbemainlyduetoanindependentCNS-mediated mechanism,andraisesomeconcernsofpotentialGBP inflamma-torysideeffectsinwidespreadclinicaluse.

Funding

ThisstudywassupportedbyBrazilianfundingagenciesFUNCAP andCNPq.

Conflictofinterest

Noconflictofinterestexistsforthisstudy.

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