Gabapentin attenuates neuropathic pain and improves nerve myelination after chronic sciatic constriction in rats

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ContentslistsavailableatScienceDirect

Neuroscience

Letters

j ou rn a l h om epa 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

Research

paper

Gabapentin

attenuates

neuropathic

pain

and

improves

nerve

myelination

after

chronic

sciatic

constriction

in

rats

Carlos

C. Câmara

a,b

,

Celina

V. Araújo

b

,

Kalina

Kelma

Oliveira

de

Sousa

b

,

Gerly

A.C.

Brito

c

,

Mariana

L. Vale

c

_,

_Ramon

_da

_Silva

_Raposo

e

_,

_Fabiana

_Evaristo

_Mendonc¸

_a

d

_,

Bruno

S. Mietto

d

_,

_Ana

_Maria

_B.

_Martinez

d

_,

_Reinaldo

_B.

_Oriá

b,∗ a_Laboratory_of_{Neurophysiology,}_Federal_and_Rural_University_of_the_{SemiArid—UFERSA,}_Mossoro,_RN,_Brazil

b_Laboratory_of_Tissue_healing,_Ontogeny_and_Nutrition,_Department_of_Morphology,_Faculty_of_Medicine,_Federal_University_of_Ceara,_Fortaleza,_CE,_Brazil c_Laboratory_of_Inflammation_and_Cancer,_Department_of_Physiology_and_{Pharmacology,}_Faculty_of_Medicine,_Federal_University_of_Ceara,_Fortaleza,_CE, Brazil

d_Department_of_Pathology,_Faculty_of_Medicine,_HUCFF,_Health_Science_Center,_Federal_University_of_Rio_de_Janeiro,_Rio_de_Janeiro,_RJ,_Brazil e_Experimental_Biology_Core,_University_of_Fortaleza,_Fortaleza,_CE,_Brazil

h

i

g

h

l

i

g

h

t

s

•Gabapentinimprovesmyelinbasicproteinexpressionintheinjuredsciaticnerve.

•Gabapentinamelioratesneuropathicpainbehaviors.

•Gabapentinhasadualroleinimprovingneuropathicpainandnervemyelinationfollowingsciaticnerveinjury.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23February2015

Receivedinrevisedform11July2015

Accepted16September2015

Availableonline25September2015

Keywords:

Gabapentin

Sciaticnerve

Neuropathicpain

Nervemyelination

a

b

s

t

r

a

c

t

Gabapentin(GBP)isananti-convulsivedrugoftenusedasanalgesictocontrolneuropathicpain.This

studyaimedatevaluatingoralGBPtreatment(30,60,120mg/kg,60minpriortochronicconstriction

ofthesciaticnerve(CCSN)along15-daytreatmentpost-injury,12h/12h)bymonitoringspontaneous

andinduced-painbehaviorsinWistarratson5thand15thdayspost-injuryduringearlyneuropathic

events.CCSNanimalsreceivingsalinewereusedascontrols.Anotheraimofthisstudywastoevaluate

GBPeffectsonmyelinbasicprotein(MBP)onthe5thand15thdayspost-injuryandnerve

morphol-ogybytransmissionelectronmicroscopytoaddressnerveregeneration.Onthe5thand15thdays,GBP

(60mg/kg)reducedneuropathicpainbehaviors(scratchingandbiting)intheipsilateralpawand

allevi-atedmechanicalallodyniaincomparisonwiththeneuropathicsalinegroup.GBPsignificantlyincreased

climbingandrearingbehaviorsinCCSNandCCSN-freeanimalssuggestingincreasedmotoractivityrather

thansedation.Wefoundthree-foldsignificantincreaseinMBPexpressionbywesternblotsonthe15th

daywhencomparedtocontrols.Inaddition,GPB(60mg/kg)improvednerveaxonal,fiberandmyelinarea

15dayspost-surgery.Inconclusion,GBPalleviatedmechanicalandthermalallodyniaandspontaneous

pain-relatedbehaviorsandimprovedlaternervemorphology.OurfindingssuggestthatGBPimprove

nerveremyelinationafterchronicconstrictionofthesciaticnerve.

1. Introduction

Peripheralnerveinjury(PNI)isanescalatingproblem

world-wide mostly due to a growing prevalenceof societalviolence,

∗Correspondingauthorat:InstituteofBiomedicineandDepartmentof

Morphol-ogy,SchoolofMedicine,FederalUniversityofCeara.RuaCoronelNunesdeMelo,

1315,CEP:60430-270Fortaleza,CE,Brazil.

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

occupational and traffic accidents, especially in more

densely-populated urban areas. PNI may lead to a chronic condition

associatedwithneuropathicpainandnervefunctionloss,witha

tremendousimpactinhospitalcostsandincapacity[1].

Although peripheral nerve regeneration is possible, current

treatmentisstillunsatisfactory,especiallyfortheelderly.Novel

therapies to accelerate nerve regeneration are needed in

asso-ciationwithbenefitsinreducing neuropathicpain.Mechanisms

underlyingpainreliefwiththeuseofanticonvulsantsarea

promis-http://dx.doi.org/10.1016/j.neulet.2015.09.021

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ingtargetfortherapyimprovements[2],howevertodatenostudies

haveaddressedtheirpotentialdualbenefitinimprovingpainand

nerveregeneration.

The anticonvulsivant gabapentin (GBP) (1-(aminomethyl)

cyclohexaneaceticacid),onestructuralderivativeofthe

gamma-aminobutyricacid(GABA),isnowfurtherconsideredasaneffective

therapyforsomeformsofneuropathicandpost-surgicalpain[3].

GBP-specificinhibitionofcalcium–voltagedependent␣2␦subunits

[4]oractivationofproteinkinaseG-K+channels[5]hasbeen

impli-catedinreducingallodyniabyalteringneurotransmitterrelease.

Ourpreviousstudyhasdocumentedthebenefiteffectoforal

GBP (60 and 120mg/kg) treatment in improving heat-induced

hyperalgesiawithnervepro-inflammatory effectsina modelof

sciaticnerveconstrictioninWistarrats[6].However,thepotential

nerveregenerativeeffectofGBPhasbeenpoorlyexplored.

InthisstudyweexploredwhetherprolongedGBP treatment

improvessciaticnervemyelinationbyassessingnervemyelinbasic

protein (MBP) (a constitutive myelin protein)[7], spontaneous

motor-relatedactivity,andfinenervemyelinmorphology,

follow-ingsciaticnerveconstrictioninWistarrats.Inaddition,wediscuss

whetherbetternerveremyelinationandreducedmyelinprotein

debriscouldimproveneuropathicpainfollowingGBPtreatment.

2. Materialsandmethods

ProtocolsfromthisstudywereinaccordancewiththeBrazilian

CollegeforAnimalExperimentation(COBEA)andtheInternational

AssociationfortheStudyofPain(IASP)andwereapprovedbythe

AnimalCareandUseCommitteeofDepartmentofPhysiologyand

Pharmacology,FederalUniversityofCeara.

2.1. Animalstudies

160maleWistarratsweighingbetween250and300gfromthe

DepartmentofPhysiologyandPharmacologyvivariumatthe

Fed-eralUniversityofCearawereusedinthisstudy.Ratswerehoused

inatemperature-controlledroom(26±2◦_C)_with_free_access_to

waterandchowdietina12h/12hlight/darkcycles.Allsurgical

procedureswereperformedinthelaboratoryofExperimental

Neu-rologyintheDepartmentofPhysiologyandPharmacologyatthe

FederalUniversityofCeara.

2.2. Sciaticnervechronicconstriction

Inordertoinducetheexperimentalneuropathy,weusedthe

chronicconstrictionofthesciaticnerve(CCSN)model,describedby

BennettandXie[8]andmodifiedbySommeretal.[9].Animalswere

anesthetized with intraperitoneal injection of tribromoethilene

(25mg/kg),following trichotomyand anti-sepsisof thesurgery

field.A15-mmlongitudinalincisionoftherightthigh,atthelevel

ofthefemoraltrocateroftheposteriorlimb,wasusedtoaccessand

exposethesciaticnerveaftergluteusandfemoralbicepsdissection.

Weusedthree4–0cat-gutlooseligaturesontherightpawsciatic

nerve,1-mmawayfromthesciatictrifurcationinducingaslight

nerveischemia.Intheleftthigh,thesciaticnervewasexposed,

butremaineduntouchedandsurgeryclosedafterwards.Skinand

muscularlayersweresuturedwitha5–0mononylonthread.

2.3. Drugsandtreatmentregimens

Gabapentin (GBP) (1-(aminomethyl) cyclohexaneacetic acid,

C9H17NO2)(Neurontin®

,Pfizer)capsulesweredissolvedin0.9%

salinesolutionandthengivenorallybygavageevery12hduring

eithera5or 15-daytreatmentcourse.GBPdosesof30,60 and

120mg/kgwereusedbasedonpreviousstudieswithgoodclinical

response[10].AsthemaximumGBPeffectoccurs60minafteroral

administration[11],thefirstdosewasgiven60minpriortothe

nervesurgeryandthelastdosewasgiven60minbeforebehavior

tests.

2.4. Neuropathicpainassessment

InordertoassesswhetherGBPtreatmentcouldameliorate

neu-ropathicpainfollowingsciaticnerveinjury,cohortanimalswere

evaluatedinspontaneousandinducedbehaviors.

2.4.1. Spontaneouspainbehaviors

Ratswerekeptinawoodencage(100×50×50cm)fora5-min

acclimationtimeandtesting.Theobservationswereconductedas

describedelsewhere[6].Eachexperimentaland controlratwas

observedduringatimeof30min.Positionedinfrontofthecage,

theobservercouldidentifyeachbehavioralcomponentandrecord

itusingacomputersoftware(Comporta®

)designedbyProf.

Mar-cusVale(FederalUniversityofCeara,Brazil).Experimenterswere

unawareoftheidentityoftheexperimentalgroups.

Thefirstobservationwasperformedbeforethesurgery

(base-line)and then onthe5th and 15thpost-surgery.Adeltamean

behaviorvaluewasderivedfrombaseline.Formeasurements(in

seconds),weconsideredthefollowingpain-relatedspontaneous

behaviors:(1)scratching:timespentraisingthehindleftorright

pawtoscratchpartsofthebodywithrapidmovementsofthepaw

andclaws;(2)biting:timespentpiercingtheskinwiththemouth

andteethontheleftorrightsideofthebody.

2.4.2. Inducedpainbehaviors

2.4.2.1. Mechanicalallodynia. InordertoassesstheGBP chronic

effectonCCSNmechanicalallodynia,thevonFreyassessmentwas

usedaccordingtoAzevedoandcolleagues[12].Briefly,the

test-ingconsistedofpokingthehindpawtoprovokeaflexionreflex

followed by a clearflinch response afterpawwithdrawal. Paw

stimulationwasrepeateduntiltheanimalpresentedthreesimilar

measurements(thedifferencebetweenthehighestandthe

low-estmeasurementshouldbelessthan10g).Animalsweretested

onthe5thand15thdaypost-CCSN.Theresultsarereportedasthe

withdrawalthreshold(g).

2.4.2.2. Coldacetoneallodynia.InordertoassesstheGBPchronic

treatment effect on CCSN-induced cold allodynia, the method

describedbyBennettandFlatters[13]wasused.Briefly,ratswere

placedinacryliccageswithawiregridfloor15–30minbeforethe

beginningofthetestsinaquietroom.Adrop(0.05mL)of

ace-tonewasplacedagainstthecenterofhindpawventralsideanda

stopwatchwasstarted.Responsestoacetoneweregradedtothe

following4-pointscale:0,noresponse;1,quickwithdrawal,flick

orstampofthepaw;2,prolongedwithdrawalorrepeatedflicking

ofthepaw;3,repeatedflickingofthepawwithlickingdirectedat

theventralsideofthepaw.Acetonewasappliedalternatelythree

timestoeachpawandtheresponsesscoredcategorically.Animals

weretestedonthe5thand15thdaypost-CCSN.

2.5. Spontaneousmotor-relatedbehaviors

InordertodifferentiateGBPtreatmentanalgesiceffectsfrom

sedation, we observed the control and experimental rats, as

describedabove(Section2.4.1),measuringtherearing(timespent

suspendingtheforefeetandsupportingthebodyonthehindpaws

andclimbingbehaviors(timespentclimbinga2-cmhighthreestep

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2.6. MyelinnervemorphologyandMBPexpression

In order to investigate nerve remyelination following CCSN

injury,weassessultra-structuralmyelinfeaturesandnervefine

histologyandMBPexpressionbywesternblottingand

immuno-histochemistryonday15thpost-challenge.

2.6.1. Finenervehistologyandmorphometry

Anesthetizedratswereperfusedintracardiallywithasolution

containing4%paraformaldehydeand2%glutaraldehydein0.1M

phosphatebuffer(pH 7.4).After,a 5mm segment(5mmdistal

fromtheinjurysite)ofthe15thday-post-injurysciaticnervewas

removedandfixedwith2.5%glutaraldehydein0.1Mcacodilate

buffer(pH 7.4)for 2h and processedfor transmission electron

microscopy.Afterthat,segmentswerepost-fixedin1%osmium

tetroxideplus 0.8% potassium ferrocyanide and 5mM CaCl2 in

0.1Mcacodilatebuffer(pH7.4)during1h.Next,segmentswere

dehydrated in increasing concentrations of acetoneand finally

embeddedin Embed-812 resin (Electron Microscopy Sciences).

Semithin(1␮m)andultrathin(60␩m)sectionswereobtainedina

RMCMT-6000ultramicrotome.Thesemithinsectionswerestained

with1%toluidinebluesolutionandobservedandphotographed

underaZeissAxioskop2Pluslightmicroscopewith20×and40×

objectives.Theultrathinsectionswerecollectedoncoopergrids

andstained30mininuranylacetatefollowedby10mininlead

citrate.ElectronmicrographswereacquiredusingZeiss900

trans-missionelectronmicroscopeoperatedat120kV.

2.6.2. Nervemorphometry

Inordertoidentifymyelinatedfibers,fiveareaswere

systemati-callyselectedfromthesemi-thincrosssectionsofthenerveofeach

animalandwereimage-capturedinhighmagnificationby

Axiovi-sionRel.4.5(CarlZeissMicroimaging,Thornwood,NY,USA).The

totalof30areasfromeachgroupwasanalyzed.Thisquantification

wasperformedusingImageJavasoftware(Bethesda,MD,USA).

2.6.3. MBPimmunohistochemistry

Onthe15thdaypost-CCSN and underdeepanesthesia, rats

weretranscardiallyperfusedwithsalineandthenthedistalsciatic

nervesegmentwasharvested5mm-distaltotheligatures,

imme-diatelyimmersedinbufferedformaline(pH7.4)for24handsentto

thehistologycoreforimmunohistochemistryprocessing.

Immuno-histochemistryforMBP(anti-MBPdiluted1:200,SantaCruz,CA)

wasperformedusingthestreptavidin-biotin-peroxidasemethod

informalin-fixed,paraffin-embeddedtissuesections(4␮mthick)

aftercitratebufferantigenretrieval,accordingtomanufacturer’s

instructions.Immunolabelingwasvisualizedwiththechromogen

3,3-diaminobenzidine(DAB).Negativecontrolsectionswere

pro-cessedsimultaneously,asdescribedabovebutwiththeprimary

antibodybeingreplacedby5%PBS-BSA.Noneofthenegative

con-trolsshowedimmunoreactivity.Slideswerecounterstainedwith

Harry’shaematoxylin.

2.6.4. MBPimmunoblotting

A2.5cm-longnervetissueoftheproximalsciaticnervestump

washarvestedtoassesstheMPBonthe15thdayspost-surgery

andimmediatelysnap-frozeninliquidnitrogenandprocessedfor

westernblotting,asdescribedelsewhere[14].MBPhasbeenshown

tobehighlyexpressedintheproximalstumpthanthedistalone

14days-post-CCSN[15].

2.7. Statisticalanalyses

NormaldistributionwasassessedusingKolmogorov–Smirnov’s

test. Effects amongst groups were assembled using

one-way ANOVA with post-hoc Tukey for parametric data and

Kruskal–Walliswithpost-hocDunn’sfornon-parametricdata.In

nerve morphometry, we used Mann–Whitney test. Values are

shownasmean±SEM.

3. Results

3.1. GBPeffectonneuropathicpainbehaviors

After5-days of GBP treatment, all tested doses (30, 60 and

120mg/kg)causedsignificantreductionsinthescratching

behav-ioroftherightpaw(93,86.7,94.4,and83.6%,respectively)when

comparedtoneuropathicsalinegroup(p<0.01).GBP(60mg)lowed

thebitingbehavior(131.8%),asopposedtotheneuropathicsaline

group(p<0.01).InCCSN-freerats,thosebehaviorswerebarelyseen

(N=12).Onthe15thdaypost-surgery,allneuropathicpain-related

behaviors(scratchingandbiting)wereworse(∼2and10-foldmore,

respectively).GBP(60mg)markedlyimprovedthescratchingand

bitingbehaviors (76 and 73%,respectively) compared withthe

untreatedchallenged-group(Fig.1A–D).

3.1.1. GBPeffectonvonFrey’sandcoldacetoneallodynia

Onthe5thdaypost-constriction,allGBPdosescaused

signifi-cantincreaseintherightpawwithdrawalthresholdinrelationto

theneuropathicsalinegroup.GBPatdosesof60and120mgwere

responsibleforthemaximum effect,providingthreshold

catch-upof45.9%and57.7%,respectively,inrelationtotheneuropathic

salinegroup,Fig.1E(p<0.00001).Onthe5thdaypost-constriction,

thescoreforcoldallodyniawasreduced(42.2%)by120mgofGBP

incomparisonwiththeneuropathicsalinegroup.The30and60mg

dosesleadtoreductiontrendsbutneitherreachedastatistical

dif-ference(Fig.1G).Onthe15thday,GBPtreatment(60mg)improved

mechanicalandthermalallodyniacomparedwiththeuntreated

CCSN-group(p<0.01)(Fig.1FandH).GBPtreatmentdidnotinduce

significantdifferencesinthecontra-lateralCCSN-freepaw(datanot

shown).

3.2. GBPeffectonspontaneousmotor-relatedbehaviors

GBP(60mg)raisedthedeltascoresofrearingcomparedwith

the untreated CCSN-rats on day 5 post-nerve constriction. No

differences were found for the rearing behavior on the 15th

post-challenge.60and120mgofGBP-treatedCCSNratsshowed

significantincreaseinclimbingwhencomparedwiththe

neuro-pathicsalinegroup,p<0.05 ontheday5 post-surgery.AllGBP

testeddosesimprovedclimbing15dayspost-surgerycompared

withtheuntreatedchallenged-group(SupplementaryFig.1).

Supplementarymaterial related tothis article found, in the

onlineversion,athttp://dx.doi.org/10.1016/j.neulet.2015.09.021.

3.3. Nervemorphology

Onthe15thday,qualitativeobservationofsemi-thincross

sec-tionsofthesciaticnerve showedhighernumber ofmyelinated

fiberswithinthechallengedandGBP-treatedgroupasopposedto

thenervesoftheneuropathicsalinegroup.Ultra-thinnerve

cross-sectionsrevealedthicker and higherdensity ofmyelinsheaths.

Inaddition, thenerves oftheneuropathic salinegroupshowed

nervefiberswithsignsofdegeneration,includingseveralmyelin

ovoids,amongpreservedand/orregeneratedfibersandsomefibers

withthinmyelinlamellae.InjuredsciaticnervetreatedwithGBP

showed many preserved and/or regenerated fibers among few

myelinovoids(Supplementary Figs.2 and3).Furthermore,oral

GBP(60mg/kg)improvedby63.3,32.8and28.2%insciaticnerve

axonal and fiber areas and myelin area, respectively, in

CCSN-challenged rats when compared with untreated nerve-injured

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Scratching (right paw)

Sham

Sal

30

60

12

0

10

20

30

40

50 GBP

*

_*

#

A

5° d

ay

D

e

lta

tim

e

(s

.)

Biting

(righ

t paw)

Sham

Sal

30

60

12

0 -5

0

5

10 C

#

*

GBP

5°day

De

lt

a t

im

e

(

s.

)

Sham

Sa

l

30

60

12

0 -50

0

50

100

*

GBP

15° day

D

*

De

lt

a T

ime

(s

.)

Sham

Sal

30

60

120

0

20

40

60

80 _#

*

B

GBP

15° day

De

lt

a T

ime

(s

.)

Fig.1.Effectofchronicgabapentin(GBP)treatmentonscratching(A,B),biting(C,D)behaviorsandonvonFrey’spressurestimulus(E,F),andcoldacetone(G,H)testing

oftheipsilateralhindpawinCCSN-ratsoneitherthe5◦_or_15th_day._At_least_n₌₈_animals_per_group_were_used._GBP_30,_60,₁₂₀_mg/g_was_given_by_daily_gavage_(12/12_h).

Behavioraldata(mean±SEM)areexpressedindeltavalues.Inpressurestimulus,dataareexpressedinthreshold(g).Incoldstimulustest,dataareexpressedinscores.

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Pressur

e stimulus

Sham

Sa

l

30

60

12

0

20

40

60

80 #

*

GBP

E

5°day

Th

re

sh

ol

d

(g

.)

15° day

Sham

Sa

l

30

60

12

0

20

40

60

80 GBP

*

#

F

Th

re

sh

ol

d (

g

.)

Cold stimulus (10°C)

Sham

Sal

30

60

120

0

2

4

6

8

10 GBP

*

#

15° day

H

Sc

or

e

Sham Sa

l

30

60

12

0

2

4

6

8 #

*

GBP

G

5° day

Sc

o

re

(6)

CCSN-challengedratswereworsecomparedwiththeshamgroup

(SupplementaryFig.4).TheGratiowasnotfounddifferentbetween

theGBP-treatedinjurednervesasopposed tothecontrols,

sug-gestingboththemyelinandtheaxonproportionallyimprovedin

diameter.

3.4. GBPeffectonthesciaticnerveMBPexpression

Atthetimepointofthe15thdaypost-injury,GBP(60mg/kg)

significantlyincreased(overthreefold,p<0.05)MBPnerve

expres-sion,ascomparedwiththeneuropathicsalinegroup.Inaddition,

theMBPimmunostainingwasmarkedlyseenintheGBP(60mg/kg)

treatedratsafter15daysoftreatmentfollowingsciaticnerve

con-striction(SupplementaryFig.5).Atthetimepointofthe5thday

post-injuryanincreasedinMBPnerveexpressionwasseeninthe

GBP(60mg)treatedgroupalbeitnotreachingstatistically

signifi-cance(datanotshown).

4. Discussion

Inthis study,weaddressedGBP effects onneuropathic pain

behavior (5th and 15thdaypost-sciatic ligature)and on nerve

myelinationparametersfollowingchronicsciaticnerve

constric-tioninrats.

Ourdatahaveshownreducedbitingandscratchingbehaviors

inchallengedrats,followingGBPtreatmentonthe5thand15th

daypost-nerveconstriction.Earlystudieshavedemonstratedthat

ipsilateralscratchingtotheinjuredhindlimbisarecognized

hall-markfeatureofneuropathicpain[16]withapeakonthe14thday

post-nerveconstriction[17].Bitinglikewisehasbeenimplicated

inneuropathicdistressbutwithadistinctevolutioninmodelsof

chronicarthritis-associatedneuropathy[18].

OralGBPtreatment(12/12h)atdosesof30, 60,and120mg

inducedsignificantreductionsinthescratchingtime(intheright

ipsilateralpaw)spentbytheneuropathicratsincomparisonwith

theuntreatedgroup.However,thisfindingwasnotseenina

dose-dependentmanner,in factthebesteffectwasseen witha GBP

doseof 30mg/kg.Interestingly,Kayser andChristensenfounda

benefitwiththesameGBPdose(30mg/kgi.p.)onthevocalization

thresholds(asupraspinal-derivedbehavior)followingsciaticnerve

constrictioninrats[11].Ontheotherhand,ourdatashowthatGBP

(60mg/kg)wasmoreeffectiveinreducingthebitingbehavior.

Bagriyaniketal.havefounddeficitsinmotor-relatedbehaviors

(openfieldandrotarodtest)14dayspost-nerveconstrictioninrats

[19].DivergingfromGustafssonetal.findingsthatshowdecreased

locomotionandrearingbehaviorsafteracuteandcumulativedoses

ofGBP(200␮mol/kg,∼40mg/kg)[20],wefoundthatprolonged

GBPtreatmentincreasedmotor-relatedbehaviordeltascores

(rear-ingandclimbing),ascomparedtocontrolsthereforesuggesting

thatoralGBPdidnotinducesedativeeffectsintheseanimals.

How-ever,wecannotruleoutanexcitatoryeffectofGBPenhancingthese

behaviors.Morestudiesareneededtodissectbetterthesefindings.

In accordance withour data, LaBuda and Little [10] used a

spinalnerve(L5)ligaturemodelandfoundincreasedpaw

with-drawn threshold(tactile von Frey) withGBP treatment(30, 60

and120mg/kgi.p.)ascomparedwiththesalinecontrolafter1h

post-surgery.Theanti-allodynicGBPeffectcouldbeexplainedby

blockageofthe␣2␦intheCav2.1calciumchannelsubunits[21].In

addition,GBPcansuppressinvivoandinvitroectopicdischargesin

thesciaticnerveafferentfibersfromneuropathicanimalsbutnot

fromnormalanimals[22].

MBPandPLPconstitute85%oftheproteinfoundinthemyelin

sheathandhelptostabilizethemyelinstructurescaffoldingthe

lipid component [23]. Setton-Avruj et al. studied the temporal

course of MBP nerve expression (3, 7 and 14 days), after

sci-aticnerveconstrictionin adultrats,distallytotheligature,and

foundthat therewas amaxiumpeak of MBPexpression seven

dayspost-constriction,reducingthereafter.Intheproximalnerve

stump, a maximum peak of MBP was found on the 14th day

post-constriction[24].SimilarlytoSetton-Avruj findings,aMBP

redistribution was seen in the distal stump of injured nerves,

suggesting an increased cellularphagocytosis and scatteringof

MBP debris. MBP redistribution maybe ongoing during

simul-taneous processes of distal Wallerian degeneration and nerve

regeneration.Noteworthy,axon-releasedMBPmaybedigestedby

metaloproteinase-9togenerateMBP84-104andMBP68-86

frag-mentpeptidesthatarestronglyimmunogenic,activatingT-cells

andcausingT-cell-mediatedmechanicalallodynia[25].Our

pre-vious[6]andcurrentfindings suggestthatGBP-induced myelin

debrisremovalfromtheinjurednervesiteandbetterregenerating

axonscouldbebeneficialtoimproveneuropathicpainbyreducing

MBPdigestedfragmentsofthenervemillieu.

MacrophagesandSchwanncellsbothcooperateduringmyelin

degeneration,removingmyelinandaxondebristhatmayfacilitate

nerveregeneration[26].Inaddition,accordingtoHall(1978),rapid

Schwanncellproliferation(3–4dayspost-nerveinjury)iskey

fac-tortopromoteaxonalregeneration.Removalofmyelindebrisby

macrophage-likeactivatedcellsisanimportantfactor

beneficiat-ingnerverecoveryaftersciaticnerveconstriction,asmyelindebris

couldbeinhibitorytonerveregeneration, sinceseveral

myelin-associatedinhibitorsofaxonregenerationhavebeenfoundinthe

peripheralmyelin[27,28].

Onestudyhasfoundnonerveregenerativeeffectwithchronic

injectedpregabalin,aGBPanalogous,(10mg/kgbydaily

subcuta-neousinjection)atthe21thdayfollowingsciaticnervecrush[29],

thismaybeduetoadifferentdoseused.Inlightofourfindings,

Machadoetal.havedocumentedbenefitsofa highdoseoforal

GBP(300mg/kg)inimprovingtheareaanddensityofmyelinated

fibers30daysfollowingstretch-inducednerveinjuryinWistarrats

[30].

Morestudiesarewarrantedtoappreciate inmoredetail the

mechanisms,time-courseandfinehistologyofmyelinremovaland

remodelingduringGBPpro-myelinationeffectsintheirassociation

withtheameliorationofneuropathicpain.

5. Conclusion

In summary, altogether our findings reinforce the analgesic

effectsofGBPandsuggestabeneficialroleofGBPonnerve

mor-phologyfollowingsciaticnerveinjury,throughmodulationofMBP

expressionandmyelinremodeling.

Funding

ThisstudywassupportedbyFUNCAPandCNPq.

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