Rev. bras. ortop. vol.50 número1

w w w . r b o . o r g . b r

Original

article

Plasma

cytokine

expression

after

lower-limb

compression

in

rats

夽

Mauricio

Wanderley

Moral

Sgarbi

,

Bomfim

Alves

Silva

Júnior

,

Carmem

Maldonado

Peres

_,

_Tatiana

_Carolina

_Alba

_Loureiro

_,

_Rui

_Curi

_,

Francisco

Garcia

Soriano

_,

_Daniel

_Araki

_Ribeiro

_,

_Irineu

_Tadeu

_Velasco

a_{MedicalSchool,UniversityofSãoPaulo(USP),SãoPaulo,SP,Brazil}

b_{DepartmentofBiosciences,FederalUniversityofSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil}

a

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t

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c

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Articlehistory:

Received11October2013

Accepted23January2014

Availableonline31December2014

Keywords:

Crushsyndrome

Animalmodels

Interleukins

Tumornecrosisfactor␣

a

b

s

t

r

a

c

t

Objectives: Muscleinjuryduetocrushing(musclecompressioninjury)isassociatedwith

systemic manifestationsknownas crushsyndrome. A systemicinflammatoryreaction

mayalsobetriggeredbyisolatedmuscleinjury.Theaimofthisstudywastoinvestigate

theplasmalevelsofinterleukins(IL)1,6and10andtumornecrosisfactoralpha

(TNF-␣),whicharemarkersforpossiblesystemicinflammatoryreactions,afterisolatedmuscle

injuryresultingfromlower-limbcompressioninrats.

Methods:MaleWistarratsweresubjectedto1hofcompressionoftheirlowerlimbsbymeans

ofarubberband.TheplasmalevelsofIL1,6and10andTNF-␣weremeasured1,2and4h

aftertheratswerereleasedfromcompression.

Results:TheplasmalevelsofIL10decreasedinrelationtothoseoftheothergroups,witha

statisticallysignificantdifference(p<0.05).Themethoduseddidnotdetectthepresenceof

IL1,IL6orTNF-␣.

Conclusion: OurresultsdemonstratedthatthechangesinplasmalevelsofIL10thatwere

foundmayhavebeenasignofthepresenceofcirculatinginterleukinsinthismodelof

lower-limbcompressioninrats.

©2014SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora

Ltda.Allrightsreserved.

Expressão

de

citoquinas

plasmáticas

após

compressão

de

membros

inferiores

de

ratos

Palavras-chave:

Síndromedeesmagamento

r

e

s

u

m

o

Objetivos:Alesãomuscularporesmagamento(lesãoporcompressãomuscular)está

asso-ciadaamanifestac¸õessistêmicasconhecidascomosíndromedoesmagamento.Areac¸ão

夽

WorkdevelopedintheMedicalSchooloftheUniversityofSãoPaulo(USP),SãoPaulo,SP,Brazil.

∗ _{Correspondingauthor.}

E-mail:[email protected](M.W.M.Sgarbi).

http://dx.doi.org/10.1016/j.rboe.2014.12.004

106

Modelosanimais

Interleucinas

Fatordenecrosetumoral␣

inflamatóriasistêmicapodetambémserdesencadeadapelalesãomuscularisolada.O

obje-tivodesteestudofoiinvestigarosníveisplasmáticosdeinterleucinas(IL)1,6,10eTNF-␣,

marcadoresdeumapossívelreac¸ãoinflamatóriasistêmica,apósalesãomuscularisolada

resultantedacompressãodemembrosinferioresderatos.

Métodos: RatosWistarmachosforamsubmetidosaumahoradecompressãodosmembros

inferioresporumafaixadeborracha.OsníveisplasmáticosdeIL1,6,10eTNF-␣foram

medidosuma,duasequatrohorasapósaliberac¸ãodacompressão.

Resultados: OsníveisplasmáticosdeIL10diminuíramquandocomparadoscomoutros

gru-poscomdiferenc¸aestatisticamentesignificante(p<0,05).Nãohouvedetecc¸ão,pelométodo,

dapresenc¸adeIL1,6eTNF-␣.

Conclusão: Nossosresultadosdemonstraramqueasalterac¸õesdosníveisplasmáticosdeIL

10encontradaspodemserumsinaldapresenc¸adeinterleucinascirculantesnessemodelo

decompressãodemembrosinferioresderatos.

©2014SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier

EditoraLtda.Todososdireitosreservados.

Introduction

Ithasbeenwellestablishedthatmusculoskeletaltraumais

associatedwithasignificantinflammatoryresponsemediated

byacomplexbiologicalsystemofchemicalmediators.1_These

mechanismsarepresent inmuscleinjuries.2 _Inadditionto

thelocalinflammatoryresponse,distantorgansaresubjectto

theeffectsofthesemediators.3 _{Thesystemicinflammatory}

responsesyndrome(SIRS)isaseverecomplicationfromthis

process.1,4

Thecrushsyndromeisasystemicrepercussionfrom

mus-cleinjuries,especiallywhenthelowerlimbsareaffected.5_The

typeofmuscleinjuryassociatedwithcrushsyndromeoccurs

throughcompression,suchasinthesituationofvictimsunder

debris.Classically,thereisextravasationofmyoglobininthe

circulation,withrenalrepercussionsandinmanycases,

kid-neyfailure.6 _{Othermechanisms relatedtocrushsyndrome}

havebeenstudied,withspecialinterestintheinflammatory

response.7,8

Cytokinesarechemicalmediatorsthatarestudiesin

rela-tiontoinflammationandSIRS.Theyactaschemotacticfactors

for inflammatory cells such as neutrophils and are

capa-ble of promotingsystemic clinical responses suchas pain

andfever.9_{Productionofanti-inflammatorycytokinesoccurs}

simultaneously during the process,and this diminishes or

eveneliminatestheinflammation.10_{Today,severaldifferent}

cytokinesareknown.Amongtheonesthathavebeenmost

studied, interleukin (IL) 1, IL6 and TNF-␣ (tumor necrosis

factoralpha)arecharacterizedbybeingstronglyassociated

withtheinflammatoryprocess.IL10ischaracterizedbyits

antagonistfunctionintherelationtothefirstthree,i.e.its

anti-inflammatoryaction.Theproportionsbetweenproductionof

proandanti-inflammatorycytokinesseemtodeterminethe

severityoftheinflammatoryresponse.3

Werecentlydevelopedanexperimentalmodelfor

com-pressionofthelowerlimbsofratswiththecharacteristicsof

amuscleinjuryduetocrushing.8_Withtheaimof

contribut-ing towards understanding the physiopathology ofmuscle

compressionatthecellularandmolecularlevels,we

inves-tigatedinthepresent paper,whetherplasmacytokinesare

presentinratessubjectedtolower-limbmusclecompression,

inanexperimentalmodelthatsimulatesmuscleinjurydueto

crushing.

Materials

and

methods

Afterobtainingapprovalfromtheethicscommitteeforanimal

research, adultWistarrats weighing250–300g(n=24) were

deprivedoffoodbutcontinuedtohavefreeaccesstowater

overa12hperiodbeforetheexperiments.Theanimalswere

thensubjectedtogeneralanesthesia(pentobarbital

intraperi-toneally,30mg/kg;and2%xylazineintramuscularly,5mg/kg)

whilespontaneousrespirationwasmaintainedforthestartof

theexperiments.Afterthese24animals’clinicalconditionhad

beenstabilized,theywererandomizedintofourgroups(n=6):

threegroupsaccordingtotheirlengthsofsurvivalafterrelease

fromcompression(1h,6hand4h)andacontrolgroup(n=6).

Astripofelasticrubber(Esmarchbandage)of10cminwidth

and80cminlengthwasfirmlyappliedaroundbothhindlimbs

simultaneously, by the same researcher (Fig. 1). The

pres-sureproducedbythebandagewasmeasuredas300mmHg,

using avariationofthe Whitesidesmethod,with the

ban-dagestillapplied(Fig.2).11_{After1h,thebandagewasremoved.}

Theanesthesiawastoppedupwhennecessary.Inaccordance

withthepreviousrandomization,theratsweresacrificed1,

2or4hafterremovalofthebandage.Thecontrolswerekept

undergeneralanesthesiafor4h,withoutcompressionorany

other procedure.Theratswere sacrificedusing

pentobarbi-talintraperitoneally,atadoseof80mg/kgandbloodsamples

(2mL)werecollectedbymeansofpuncturingtheheartusing

aneedleandsyringe.Thebloodsamplesthatweretakenwere

centrifuged immediately (4◦C_{) and the plasma was frozen}

(−80◦C)forsubsequentanalysisonthecytokines.The

exper-imentaldesignofthismodelwaspreviouslyestablishedand

usedbyourresearchgroup.8

Expression

of

interleukins

1,

6

and

10

and

TNF-

␣

Blood samples were collected (2mL) by means of cardiac

Fig.1–Esmarchbandageappliedtothehindlimbs, wrappedaroundthem10times.

sampleswerecentrifugedat1000g(4◦_{C)for10minandthe}

supernatant (approximately 1mL of plasma) was placed

inEppendorf plastictubes and stored at−80◦C for

subse-quentanalysis.Theinterleukins1,6and10andTNF-␣were

expressedinaccordancewiththemanufacturer’sinstructions

(PharMingen– OptEia®_{, BDBiosciences, FranklinLakes,NJ,}

USA).Firstly,thespecificantibodieswereaddedtothewells

ofthe readingplate:biotinylatedanti-rat IL1,anti-rat IL6,

anti-ratIL10orTNF-␣monoclonalantibodies(PharMingen–

OptEia®_{).Theplateswereincubatedfor12h(4}◦_C).Thewells

were washed five times with saline solutionin phosphate

buffer(PBS)with0.05%Tween-20(polyoxyethylenesorbitan

monooleate,Sigma®_{,USA)(standardsolution).Followingthis,}

200␮Lofthesalinesolutioninphosphatebuffer(pH=7.0)with

10%fetalbovineserumwasaddedtotheplates,whichwere

incubated at4◦_{C for30min. Theplates werethen washed}

using the standard solution. The first columnof the plate

(eightwells)received100␮Loftheantigensolution(IL1,IL6,

IL10orTNF-␣)atincreasingdilutions(PharMingen–OptEia®_).

Followingthis,100␮Lofeach ofthe sampleswasadded to

thecellsofthesecondcolumn(thefirstcolumn,asalready

described,wasreservedforconstructionofthestandardcurve

ofeachoftheinterleukinsstudied).Eachsamplewasstudied

intwocontiguouscellssothatwewouldbeabletousethe

meanfromthevaluesobtained.Theincubationperiodforthis

phasewas2handthiswasagainfollowedbywashingthecells

fivetimeswiththestandardsolution.Monoclonalantibodies

bonded with biotin were added to the monoclonal plates

(Biotinylatedanti-rat; PharMingen–OptEia®_).After

incuba-tionfor1h,thewellswereagainwashedusingthestandard

solution.Atthisstage,100␮Lofhorseradishperoxidase(HRP)

conjugatedwithavidinwasincorporatedintoeachwell.The

plates were incubated for 30min and were washed seven

timeswiththe standardsolution. Inthe final stage,100␮L

Fig.2–ModifiedWhitesidesmethod:15mLsyringefilled withair,withembolusundertraction,connectedtoa three-waytapthatledtooneplastictubecontainingair andanothertubehalf-filledwithphysiologicalserum.One tubeconnectedtoamercurysphygmomanometerandthe othertubetoaneedlebetweentheskinandtheEsmarch bandage.WiththestandardizedapplicationoftheEsmarch bandage(wrapped10timesaroundthelegs),thepressure neededtomovetheliquidmeniscuswas300mmHg. Diagramoftheexperiment(thescalesdonotcorrespondto realityinordermakethemethodeasiertounderstand).

oftetramethylbenzidineandhydrogenperoxidewereadded.

Theplateswereincubatedfor30min.Thefinalresultswere

obtainedusingaspectrophotometer (wavelengthof570nm

forIL1,IL6andIL10;andwavelengthof450nmforTNF-␣).

Statisticalanalysis

ThestatisticswereanalyzedusingtheSigmaStat® _software

(SigmaStatforWindows,version1.0,copyright1992–1994,

Jan-delCorporation).ThenonparametricKruskal–Wallistestwas

used (ANOVA ranking test)and the resultswere described

in terms of the medianand interquartile range. Post-tests

werethenusedtocomparepairsinthegroups.Forthis,the

Student–Newman–Keuls t-test was used. To compare pairs

aftertheKruskal–Wallistest,theDunntestwasused.

Results

The animals’ plasma was investigated for the presenceof

pro-inflammatoryinterleukins(IL1,IL6andTNF-␣)and

anti-inflammatory interleukins (IL 10). Withinthe limits of the

methodused,nopresenceofpro-inflammatoryinterleukins

108

Control

Interleukin 10 (pg/ml)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

1 hour 2 hours 4 hours

Fig.3–Variationsinplasmalevelsofinterleukin10found duringtheexperiment.*Significantwhenthegroups evaluated1and4hafterreleaseofthecompressionwere compared(p<0.05).

animals studiedand, afteran initialincrease, it presented

aprogressive declineinplasma concentration (Fig.1). The

meansobtainedwere0.09pg/mL(range:0.073–0.17pg/mL)for

the control; 0.142pg/mL (range: 0.085–0.259pg/mL) for 1h;

0.09pg/mL(range:0.061–0.121pg/mL)for2h;and0.03pg/mL

(range:0.020–0.047pg/mL)for4h(Kruskal–WallisAnalysisof

VarianceonRanks).Atthelaststudytime(4hafterrelease

ofthemusclecompression),thisdecreaseacquiredstatistical

significanceinrelationtothe1hgroup(Dunntest;p<0.05)

(Fig.3).

Discussion

Thereareseveralexperimentalmodelsforstudyinginjuries

duetomusclecompressionandcrushsyndrome.7,8,12–15_From

ourmodel,theresultsdemonstratedsignificantalterationsin

IL10levels,4hafterreleaseofthecompression.

Althoughthemostreproducibleresultshavebeenfound

throughexperimentalmodels,clinicaltrialshavecorrelated

highplasmacytokinelevelsandsevereconditionspresented

bypatientswithmultipletraumaticinjuries.16_Recently,

mod-elsforcrushsyndromehavefocusednotonlyontheclassical

findingsofcrushsyndrome,butalsoonsystemicalterations

relatedtotheinflammatoryresponse.7,8_{However,amongthe}

studiesintheliteraturethatwereviewed,noneofthem

corre-latedcrushsyndromewiththeproductionofinterleukins1,6

and10andTNF-␣intheplasma,asseeninthepresentstudy.

Interleukinsare polypeptidesproduced byinflammatory

cellsandtheyactatsitesclosetowheretheyareproduced.17

Damagedmusclefibersmayproducecytokinessuchas

TNF-␣, IL 1 ␤ and IL 6 (2). When there is high production of

interleukins,extravasationofthesesubstancestotheplasma

occurs. Under these biological conditions, some of these

cytokines serve as triggers for a systemic inflammatory

reaction.17

SomeinterestingresultshaveindicatedthatIL10hasan

importantregulatoryroleinimmunologicalandinflammatory

responsesbecauseofitscapacitytoinhibittheproductionof

pro-inflammatorycytokinesbymonocytes.18_{IL10reducesthe}

levelsofinflammationinducedbyTNF-␣inendothelialcells,

such asthe productionofreactive oxygen speciesand the

adherence ofleukocytes tothe endothelium.19_Itisunclear

whether IL10isconnectedwithmuscleinjury,but thereis

someevidencethatmoderatetohighlevelsofIL10inhibitthe

productionofIL1andIL6undersuchconditions.20_Our

find-ings(increasedIL10andnon-identificationofinflammatory

cytokines)maybeexplainedbytheseinteractionsbetweenIL

10andinflammatorycytokines.

Inthismodel,pro-inflammatorycytokineswerenotfound

intheplasmaaftertheinjurycausedbytheisolated

compres-sionofthehindlimbsoftherats.Inreviewingtheliterature,we

founddatathatmayhelpindiscussingwhythesesubstances

werenotdetected.ThereissomeevidencethatIL6mayonly

beproducedinmusclesthatarecontractedatthetimeofthe

trauma.2_{Becauseoftheeffectoftheanesthesia,themuscles}

inthemodelproposedherewouldhavebeenrelaxedatthe

timeofapplyingthecompression,andthismaycorroborate

thenegativeresultsfoundregardinginflammatorycytokine

production.Furthermore,itispossiblethatsubtlevariations

intheconcentrationsofIL1,IL6andTNF-␣maynothavebeen

detectedinthismodelduetothelimitsofthemethodology

usedorevenbecauseofthetimesthatwechosefordetection

ofcytokines(1,2and4h),sinceitisknownthatinterleukins

may haveahalf-life ofonlyafew minutes.Itisimportant

toemphasizethatIL10was present1hafterreleasing the

compressionandthattherewasaprogressivedecreaseinits

plasmaconcentrationsoverthecourseoftheexperiment.As

statedearlier,studieshavesuggestedthatthefinal

inflamma-toryreactiondependsontheresultantbetweenthepro-and

anti-inflammatorymediators.20_{Indirectly,thedecreaseinthe}

plasmaconcentrationofIL10mayberelatedtothepresenceof

circulatingIL1,IL6andTNF-␣.Inconclusion,althoughIL10is

ananti-inflammatorycytokine,thelevelsencounteredmaybe

anindirectsignofthepresenceofinflammatoryinterleukins

inthismodelofmusclecompressioninrats.

Conclusions

Ourexperimentalmodelformuscleinjuryduetocompression

(muscleinjuryduetocrushing)demonstratedthepresence

and variationinlevels ofIL10inthe plasma, withapeak

1hafterthecompressionwasreleasedandadecreaseinthe

valuesfound,4hafterthisrelease.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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