Rev. bras. ortop. vol.51 número6

SOCIEDADE BRASILEIRA DE ORTOPEDIA E TRAUMATOLOGIA

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

Original

Article

Morphologic

study

of

different

treatments

for

gastrocnemius

muscle

contusion

in

rats

夽

Ana

Carolina

Brandt

de

Macedo

,

Julye

Leiko

Ywazaki

,

Rafael

Michel

de

Macedo

,

Lucia

Noronha

_,

_Anna

_Raquel

_Silveira

_Gomes

a_{UniversidadeFederaldoParaná,Curitiba,PR,Brazil}

b_{Associac¸ãodosPaiseAmigosdosExcepcionais(APAE),Pinhais,PR,Brazil} c_{HospitalCardiológicoCostantini,Curitiba,PR,Brazil}

d_{PontifíciaUniversidadeCatólicadoParaná,LaboratóriodePatologiaExperimental,Curitiba,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20July2015 Accepted7January2016 Availableonline15October2016

Keywords:

Musculoskeletalsystem Woundsandinjuries Musclestretchingexercises Ultrasonictherapy

a

b

s

t

r

a

c

t

Objective:Evaluatetheeffectsofultrasoundandstretchinginmorphologyafterratmuscle contusion.

Methods:MaleWistarrats(n=35,8–9weeks,271±14g)weredividedintofivegroups:control group(CG=3);lesiongroup(LG=8);lesion+ultrasoundgroup(LUG=8);lesion+stretching group(LSG=8);lesion+ultrasound+stretchinggroup(LUSG=8).Theultrasoundwasapplied inLUGandLUSGfromthethirdtotheseventhday,thedoseusedwas50%pulsed,0.5W/cm2_,

5min.Fromthetenthuntilthetwentyfirstday,passivestretchingwasperformed,infour repetitionslasting30seachwith30sofrest.Initialandfinalbodyweight,muscleweightand length,numberandsarcomerelength,musclefibercross-sectionalarea,andpercentageof collagenwereevaluatedafter22days.

Results:Thefinalbodyweightwashigherthantheinitialinallgroups.Thenumberof sar-comereswasstatisticallyhigherinLSGthanLUGandhigherinLUSGthanLUSandCG;in sarcomerelengthwashigherinLUGwhencomparedwithLSG(p<0.05).Thecrosssectional areainLGwashigherthanLSG,andthepercentageofcollagenwashigherinLGwhen comparedwithLSGandCG;inLUGwhencomparedwithLSGandCG;andinLUSGwhen comparedwithCG.

Conclusion: Thepassivestretchingprotocolinducedsarcomerogenesisandantifibroticeffect overthemusclesubmittedtocontusion.Ultrasound,eveninassociationwithstretching, wasnotsufficienttopreventfibrosisintheinjuredmuscle.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeOrtopedia eTraumatologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

夽

StudyconductedattheCentroUniversitário(UniBrasil),PontifíciaUniversidadeCatólicadoParanáandUniversidadeFederaldoParaná, Curitiba,PR,Brazil.

∗ _{Correspondingauthor.}

E-mail:[email protected](A.C.Macedo).

http://dx.doi.org/10.1016/j.rboe.2016.10.001

Estudo

morfológico

entre

diferentes

tratamentos

da

contusão

muscular

de

gastrocnêmio

em

ratos

Palavras-chave:

Sistemamusculoesquelético Ferimentoselesões Exercíciosdealongamento muscular

Terapiaporultrassom

r

e

s

u

m

o

Objetivo: Avaliarosefeitosdoultrassomterapêuticoe/oualongamento,namorfologiaapós contusãomuscularemratos.

Métodos: RatosWistarmachos(n=35,8-9semanas,271±14g)foramdivididosemcinco grupos:GrupoControle(GC=3);GrupoLesão(GL=8);GrupoLesão+Ultrassom(GLUS=8); GrupoLesão+Alongamento(GLA=8);GrupoLesão+Ultrassom+Alongamento(GLUSA=8). Aaplicac¸ãodoultrassomnoGLUSeGLUSAfoifeitadoterceiroaosétimodia,pulsado50%, 0,5W/cm2_{,5min.Dodécimoaovigésimoprimeirodiafoifeitooalongamentopassivono}

GLAeGLUSA,emquatrorepetic¸õesde30s,com30sdeintervalo,cadarepetic¸ão.Após22 dias,osratosforampesadoseosmúsculosdeambasaspatasforamretiradosparaanálise dopesoecomprimentomuscular,númeroecomprimentodossarcômeros,áreadesecc¸ão transversaeporcentagemdecolágeno.

Resultados: Opesocorporalfinalfoimaiordoqueoinicialemtodososgrupos.Onúmerode sarcômerosfoimaiornoGLAemrelac¸ãoaoGLUSenoGLUSAemrelac¸ãoaoGLUSeaoGC; ocomprimentodossarcômerosfoimaiornoGLUScomparadocomoGLA(p<0,05).Aárea desecc¸ãotransversanoGLfoimaiordoquenoGLAeaporcentagemdecolágenofoimaior noGLcomparadocomoGLAeoGC;noGLUScomoGLAeoGC;noGLUSAcomoGC.

Conclusão: Oprotocolodealongamentopassivoinduziuasarcomerogêneseeapresentou efeitoantifibróticoemmúsculossubmetidosacontusão.Oultrassom,independentemente daassociac¸ãocomoalongamento,nãofoisuficienteparaimpedirafibrosenosmúsculos lesados.

©2016PublicadoporElsevierEditoraLtda.emnomedeSociedadeBrasileirade OrtopediaeTraumatologia.Este ´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Muscleinjuryaccountsfor60%ofsportsinjuries.1_Despiteits

highprevalence,therearefewstudiesonitstreatment,which hindersthestandardizationofphysicaltherapy.2_Amongthe

different forms of treatment, the most commonly recom-mended are:rest; cryotherapy/compression/elevation;early mobilization;laser;ultrasound;andexercisesofactive mobi-lization, passive stretching, and concentric and eccentric strengthening.2–5

Althoughitsbiologicaleffectsinacuteandchronic inflam-mationarenotwellunderstood,2,6–8 _{therapeuticultrasound}

(TUS)hasbeenrecommended. Piedadeetal.7 _assessedthe

effectsofpulsedapplicationonlaceratedgastrocnemius mus-cleofrats,andobservedasignificantincreaseinthenumber ofmyotubesintheregenerationzone14daysaftertreatment. However,despitesuchresults,theappropriatemodulationof the TUSparameters (frequency,intensity, mode,treatment time)isstillcontroversial.

In addition to treatment with electrothermal and pho-totherapeutic resources, exercise is recommended for the treatmentofmuscleinjury.Themostcommonlyprescribed exercise is muscle stretching.1,5 _{As with TUS, its}

bene-fits on the injured fiber are unclear and it still has alow level of evidence. Nevertheless, Hwang et al.,9 _in

experi-mental studies (rats), observed that the passive stretching doneduringtheinflammatory,proliferative,andregenerative phases reduced the muscle fibrosis area by 50%, increas-ingstrength and thenumber ofmyofiberswhen compared withthecontrolgroup,demonstratingabenefitformuscle

regeneration.Thebestresultsobtainedinthestudyoccurred when the stretching was initiated on the 14th day after injury.

Itisimportanttoemphasizethat bothTUSand muscle stretchingareindicatedforthetreatmentofmuscleinjury,2,5–9

butthereisstillnoconsensusonwhenandhowtoprescribe them.Moreover,itisunclearwhethertheassociationbetween thesetherapeuticmodalitiescan benefitthe muscle regen-erationmechanism.Thus,this studyaimedtoevaluatethe effectsofTUSand/orstretchingonmorphologyaftermuscle contusioninrats.

Methods

Sample

This was an experimental, prospective, randomized study. After approval by the Ethics Committee for Ani-mal Experimentation10 _{(EAEC) of the UFPR (Certificate No.}

491/2010), 35 young Wistar rats were selected (8–9 weeks, 271±14g).Theanimalswere grouped infourper standard plastic cage, in an environment of controlled light (12h light/darkcycle)andtemperature,withfreeaccesstowater andpelletedfeed.

Inclusioncriteria

Sampledistribution

The animals included (n=35) were randomized into five groups: control group (CG=3); lesion group (LG=8); lesion+ultrasound group(LUG=8);lesion+stretchinggroup (LSG=8);lesion+ultrasound+stretchinggroup(LUSG=8).

Theanimalswererandomizedbydrawing.TheCGGroup wasplacedthreetimesandtheLUG,LSG,LG,andLUSGwere placedeighttimesinto aplasticbagfordrawing.Afterthe animalswereweighed,thedrawingwasconducted,andthe animalswereassignedintoeachgroupandseparatedintothe cages(Fig.1).

Protocolforpromotingmusclecontusion

The animals were anesthetized with ketamine (95mg/kg) andxylazine(12mg/kg)intraperitoneallyandkeptinaprone

positionwith theright paw manuallyimmobilizedin knee extensionand90◦ _{dorsiflexionoftheanklejoint.Thelesion} wasproducedintherightgastrocnemiusmuscle(RGM)using adeviceconsistingofawoodenplatformwithahollow alu-minumtube,graduatedat5cm,placedperpendicularlytothe platform,aspreviouslydescribedbyMinamotoetal.11_(Fig.2).

Afterthelesion,theabsenceoftibialfracturewasassessedby meansofpalpationandhandling.1

Ultrasoundprotocol

For the application of TUS, a researcher immobilized the mousewhileanotherappliedthedevice.ToapplyTUS (cal-ibrated),theposteriorregionoftherightpawwasshavedand positionedinkneeextensionand90◦_{dorsiflexionoftheankle} joint,andTUSwasappliedonthemedialbellyoftheRGM.Gel wasthecontactmedium.Theeffectiveradiationareawas1

Adult male Winstar rats selected for the study

(n=35)

Outcomes: Body weight Muscle weight Muscle length

Number and length of sarcomeres Cross-section area of muscle fiber

Percentage of collagen Sample randomized

into 5 groups

Control group (CG=3)

Lesion group (LG=8)

Day 0 – lesion Day 0 – lesion

Day 3-7 – ultrasound

Day 22 – euthanasia

Day 22 – euthanasia

Day 22 – euthanasia

Day 22 – euthanasia

Day 22 – euthanasia

Day 3-7 – ultrasound Day 0 – lesion

Day 10-21 – stretching

Day 10-21 – stretching

Day 0 – lesion Lesion +

ultrasound group (LUG=8)

Lesion + stretching group

(LSG=8)

Lesion + ultrasound + stretching group

(LUSG=8) Included animals (n=35) which

did not present fracture associated with the muscle lesion and weighed between

250-300 g

Fig.2–Apparatusforperformingmusclelesion.

A,apparatusforinductionofmusclelesion,whichindicatesthemassofthe200-gprojectile(arrow),whichisslidfroma heightof37cm;B,arrowindicatestheprojectileontheventralregionofthegastrocnemiusmuscleduringthelesion procedure.

cm2_{.TheapparatususedwastheSonopulseespecial(Ibramed),} 1MHz, pulsedat100Hz,50%cycle, intensity7 _of0.5W/cm2_, andapplicationtimeof5min.TUSwasstarted72hafterthe lesion.

Protocolforstretchingthegastrocnemiusmuscle

Animalswereplacedinthesupinepositionwiththefrontlegs immobilizedbyaresearcher,sothatanotherresearchercould performpassivestretching.Tothisend,themaximumdorsal flexionoftheanklejointwasdonemanuallywiththeknee extended,asdescribedbyMattiello-Sverzutetal.12_The

pro-tocolwasmaintainedfor30s13_{duringeachrepetition,with}

anintervalof30s,fourrepetitions,14_{fivetimesaweek(from}

MondaytoFriday).Stretchingwasinitiatedonthe10thday afterthelesion.

Chronologicalpresentationoftheinterventionsinthe groups

Table1showsthesequenceofinterventionsineach exper-imentalgroup.Aspulsedultrasoundhasanti-inflammatory effects(amongothers),itwasdecidedtousethisfeature dur-ingtheinflammatoryphaseofthelesion.Inturn,stretching wasstartedinthefibroblastproliferationphase.9

Euthanasiaandmusclecollection

Theanimalsofallgroupswereanesthetizedwithketamine (95mg/kg) and xylazine (12mg/kg) intraperitoneally for removalofthe right and leftgastrocnemiusmuscles using scissorsandtweezers.Subsequently,theanimalswere eutha-nizedwithananestheticoverdose(ketamineandxylazineon the22thdayofthestudy).

Eachmusclewasseparatelyweighedonananalytical bal-anceandtheirlengthwasmeasuredbycaliper.Themuscles werelongitudinallydividedinhalfusingascalpelblade.The lateralportionwasdiscardedandthemedialportionwas lon-gitudinallydivided inhalf.Halfofthesamples(lateral)had theirdistalendsfixedintherestingpositionwithultrafine needlesandweremaintainedfor3hinglutaraldehyde(2.5%), then nitric acid (30%)for 48h, and subsequently storedin glycerol(50%).Thelateralhalfwassubjectedtoroutine pro-ceduresforassessingthenumberofsarcomeresinseries,as describedbyWilliamsandGoldspink.15_{Themedialhalfofthe}

RGM was fixedin10% formalin formorphologicalanalysis ofcross-sectional area ofmuscle fibers (CSAMF) and colla-gen percentage.Leftgastrocnemiusmuscle(LGM)wasused ascontrol.

Thenumberofsarcomereswasassessedtoverifywhether the combination of ultrasound and/or stretching could

Table1–Sequenceofinterventionsoftheexperiment.

Groupintervention LG LUG LSG LUSG CG

Contusion Day0 Day0 Day0 Day0

Ultrasound 3rdto7thday 3rdto7thday

Stretching 10thto21stday 10thto21stday

Euthanasia 22ndday 22ndday 22ndday 22ndday 22ndday

Fig.3–Photomicrographofanisolatedmusclefiber(100× objective).Arrow:eachstripinthearrowheadrepresentsa sarcomerethatwascountedalong100␮m.

potentiatetheincreaseinthenumberofsarcomeresinseries afterlesion. In turn,the cross sectional area and the per-centage of collagen were assessed to verify whether the combination ofultrasoundand/or stretching could prevent muscleatrophyandreducethepercentageofconnective tis-sue after muscle lesion.All of these mechanisms may be affectedaftermusclelesion.

Identifyingthenumberofsarcomeresinseries

For preparation of histological slides, five fibers were iso-lated(usingultrafinetipforceps)ofeachmuscle.Thefibers werephotographedunderalightmicroscope(100×objective, immersion).Oneachfiber,thenumberofsarcomeresinseries wasidentifiedin100␮m;threefieldsofdifferentdifferent

pho-tomicrographswere used along each muscle fiber, totaling 300␮m.

Aftercountingthenumber ofsarcomeresinthe 300␮m

area,thetotalnumberofsarcomereswasestimatedand cor-relatedwiththetotallengthofthemuscle,measured with calipersatthedayofeuthanasia(Fig.3).Thus,thefollowing equationwasused:numberofsarcomeresin300␮m,

multi-pliedbymusclelength,dividedby300.

Therefore,thetotalsarcomerenumberandlengthineach isolated fiber were estimated by correlation between the numberofidentifiedsarcomeresandthetotallengthofthe muscle.15–18

CSAMFandpercentageoftypeI_andIIIcollagen

For analysis of the CSAMF,the material was stained with hematoxylinand eosin. Thephotomicrographs were made usingalightmicroscope at20× magnification. Tomeasure CSAMF, 100 muscle fibers were randomly selected in the central histologicalsection,using cross-sections of 8␮m.19

CSAMF was measured with the image program Pro Plus 4.0,and the unitofmeasurement adoptedwas the square micrometer(␮m2).Thefinalarithmeticmeanregardingthe

100measuredfiberswascalculated(Fig.4).

For analysis of collagen percentage, the material was stained withsirius red. Photomicrographs were made in a lightmicroscopeat20xmagnificationforhistologicalslides, usingcross-sectionsof8␮m.Theimageswereanalyzedusing

thesoftwareImageProPlus4.0.Aftercalibration,apreviously polarizedimagewaschosenbytheprogramtoestablisha pat-tern ofred and green,which was quantifiedinall images. Thisimagewasconsidered asa“mask”,which was super-imposeduponanyotherimagesforidentificationofcertain

Fig.4–Photomicrographofahistologicalcross-sectionof thegastrocnemiusmuscletomeasureCSAMFusingthe

ImageProPlus4.0software.Outlineinwhite:outlineto measurethecross-sectionalareaofmusclefiber(CSAMF).

colors.Afterthe“mask”wasoverlaidoneachimage,the pro-gramcalculatedthepercentageofredandgreencolors,which correspondtothemature(typeI_{)andimmature(type}III₎ col-lagenoftheendomysiumandperimysial,respectively.20_The

resultswereexpressedaspercentageofmaturecollagen(I_, red)andimmaturecollagen(III,green),andthesumofbothis equalto100%(Fig.5).

Analysisofresults

Toassessnormalityandhomoscedasticity,theShapiro–Wilk and Levene tests were performed, respectively. Inter- and intra-group comparisons were made by one-way ANOVA

posthocTukey unequalHSDforparametric values;for non-parametricvalues,theKruskal–Wallistestwasused.Values were considered significant when p≤0.05.Statistica 7 was usedforthestatisticalanalyses.

Results

Bodyweight

Asignificantincreasewasobservedbetweentheinitialand finalweightinallgroups.Regardingabsoluteweight,thefinal bodyweightintheLGwashigherthanintheLUSG;thevaluein theLUGwashigherthanintheLUSG;andthevalueintheCG washigherthanthatintheLSGandintheLUSG.Inrelative difference,asignificantincreasewasnotedbetweenLGand LSG;LGandLUSG;LUGandLSG;andLUGandLUSG(Table2).

Weightandlengthofthegastrocnemiusmuscle

Therewasnostatisticallysignificantdifferenceinthe com-parisonofweightandmusclelengthbetweentheRGM and theLGMandamongthegroups(Fig.6AandB).

Estimatednumberofsarcomeresinseries(ENSS)

Fig.5–Photomicrographsofbladeswithtransversehistologicalsectionsofthegastrocnemiusmusclestainedwithsirius red.

A,imageshowsthecrosssectionofthegastrocnemiusmusclestainedwithsiriusred;whitearrowshowstheareastained inred,identifyingtypeIcollagen(mature)andtheasterisktheareaingreencorrespondstotypeIIIcollagen(immature);B, imageshowsaphotomicrographofthecross-sectionofthegastrocnemiusmuscleoverlappedbythe“mask”.Thenumbers representthevaluesoftheareasmarkedinredandgreencolors,identifiedbythesoftware.

intergroupcomparison,asignificantincreaseoftheENSSin theRGMwasobservedbetweentheLSGandLUG;LUSGand LUG;andLUSGandCG(Fig.6C).

SarcomerelengthandCSAMF

Inthecomparisonoftheright andleft sidesintheLSG, it wasfoundthatsarcomerelengthwasgreaterintheLGMthan intheRGM.Inthecomparisonbetweengroups,anincreased lengthofthesarcomeresoftheRGMwasobservedbetween LUGandLSG(Fig.6D).CSAMFoftheRGMwasgreaterinthe LGwhencomparedwithLSG(Fig.7A).

AnalysisoftypeI_(mature)andIII_{(immature)collagens}

Therewasnostatisticallysignificantdifferenceinthe com-parisonbetweenRGMandLGMineachexperimentalgroup (p>0.05). In the intergroup analysis of RGM, a significant increase(p<0.05)wasfoundinthepercentagesofmature col-lagen(typeI_{)inLG andLSG(40}±12%vs.24±14%);LGand CG(40±12%vs.21±9%);LUGandLSG(37±10%vs.24±14%);

LUGandCG(38±10%vs.21%);andLUSGandCG(36±12%vs.

21±9%)(Fig.7B).

Discussion

Passivemanualstretchingincreasedthenumberof sarcom-eresinseriesandpreventedtheincreaseintypeI_collagenin injuredmuscles.However,theultrasoundprotocol,regardless ofassociationwithstretching,showednoantifibroticeffectin thesemuscles.

Inrelationtobodyweight,allgroupsshowedanincrease whencomparingtheinitialwiththefinalweight.The abso-lutefinalbodyweightwashigherintheCGthanintheLSG and inthe LUSG;theLG andLUG had greaterweight than theLUSG.Therefore,thegroupsthatunderwentthehighest amountofstimulation(contusion,ultrasound,andstretching) mayhavepresentedanegativeinterferenceinabsolutebody weightgain,sincetheCGhadthehighestfinalbodyweight.

Therewerenosignificantdifferencesinmuscleweightand length,perhapsduetotheshortinterventionperiodandthe

Table2–Effectsofultrasoundand/orstretchingonthebodyweightofmice.

Groups Initialweight(g) Finalweight(g) Relativedifference(%) pANOVA

LG 267± 11 350± 18a,b _31±7e _0.02

LUG 266±10 352±19a,c _32±10f _0.01

LSG 271±11 323±18a _{19±8 0.00}

LUSG 270±14 318±16a _{17±3 0.00}

CG 299±16 383±24a,d _{28±6 0.00}

LG,lesiongroup;LUG,lesion+ultrasoundgroup;LSG,lesion+stretchinggroup;LUSG,lesion+ultrasound+stretchinggroup;CG,controlgroup.

a _{Comparedwiththeinitialbodyweight.} b _{ComparedwithLUSG(p=0.02).} c _{ComparedwithLUSG(p=0.01).}

d_{ComparedwithLSG(p=0.00)andLUSG(0.001).} e _{ComparedwithLSG(p=0.00)andLUSG.} f _{(p=0.000)comparedwithLSGandLUSG.}

0 0.5 1 1.5 2 2.5

CG LUSG LSG LUG LG

Muscle weight (g)

0 5 10 15 20 25 30

CG LUSG LSG LUG LG

Muscle length (mm)

0 2000 4000 6000 8000 10 000 12 000

CG LUSG LSG LUG

a _{Compared with the right gastrocnemius muscle of the LUG (P=.001).} b

Compared with the right gastrocnemius muscle of the CG (P=.04).

c _{Compared with the left gastrocnemius muscle (P=.001).}

d _{Compared with the right gastrocnemius muscle of the LSG (P=.001).} e

Compared with the left gastrocnemius muscle of the LSG (P=.001). Mean ± standard deviation.

LG

ENSS

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

CG LUSG LSG LUG LG

Length of the sarcomeres (

µ

m)

Right Left B

a a, b, c

a

d

C

D

A

B

Fig.6–Effectsofultrasoundand/orstretchinginweightandmusclelength,number,andlengthofsarcomeres.A,muscle weight;B,musclelength;C,thenumberofsarcomeresinseries;D,lengthofthesarcomeres.

ENSS,estimateofthenumberofsarcomeresinseries;LG,lesiongroup;LUG,lesion+ultrasoundgroup;LSG, lesion+stretchinggroup;LUSG,lesion+ultrasound+stretchinggroup;CG,controlgroup.

volumeofthe stretchingprotocol,that is,onlytwoweeks, fortwominuteseachsession.Coutinhoetal.19_observedan

increasemusclelengthafter40minstretchingforthreeweeks. Therefore,theinterventionperiodandthevolumeof stretch-ingcontributetolongitudinalmusclegrowth.However,the measurementofthecross-sectionalareaofmusclefibersand thenumberofsarcomeresinseriescountarethemost accu-ratemethodsforassessingmuscletrophisminparallelandin series,respectively.21

Regarding the number of sarcomeres in series, the LSG presented a higher number, demonstrating the effect of stretchingonthesarcomerogenesisofinjuredmuscles.This differencemayalsobeduetothevolumeofstimulus,asthe LUGreceivedonlyfiveTUSapplicationsandtheLSGreceived ten stretching sessions.19 _{The combination of TUS with}

stretching(LUSG)contributed totheincreaseinsarcomeres inserieswhencomparedwiththeLUG,demonstratingonce

againthestretching-stimulatedsarcomerogenesis,whichwas alsocrucialtothegrowthofsarcomeresininjuredmuscles. Furthermore,thisoutcomewasconfirmedthroughthe obser-vationthatthenumberofsarcomeresinseriesintheLUSG washigherthan intheCG.Thus,thisisthefirst confirma-tionofanincreaseinthenumberofsarcomeresinseriesin previously injured muscles. Thissuggests that the adapta-tionresultingfromtheassociationofTUSwithstretchingwas notonlyachangeinthepassivetensionofthelengthofthe muscle-tendonunit,beingsufficienttoinducemorphological adaptationofthelongitudinalmuscle.22

IntheLUG,thelengthofthesarcomeresintheRGMwas lowerthanintheLGM;comparingRGMs,thelengthof sarcom-ereswaslowerintheLUGthanintheLUSG.Theseoutcomes confirm the sarcomerogenesis observed in the LSG, since, accordingtoKoh,23_{skeletalmusclecanincreaseordecrease}

0 2000 4000 6000 8000 10 000 12 000 14 000 16 000 18 000

A

B

Cross-sectional area (

µ

m²

)

LUG LG a

LSG LUSG CG

Right Left

0 10 20 30 40 50 60 70 80 90 100

LG

Percentage of the area of collagen

a

LUG b

LUSG LSG

c

CG

% Red D % Red E % Green D % Green E

a _{Compared with the right gastrocnemius muscle of the LUG (P=.01).} b _{Compared with the LSG (p =.00) and CG (P=.00).}

c _{Compared with the LSG (p = 0,00) and CG (P=.00).}

Mean ± standard deviation

Fig.7–Ultrasoundand/orstretchingeffectsonCSAMF(A)andonthepercentageoftheareaoftypeIcollagen(mature,red) andtypeIIIcollagen(immature,green)(B).

R,right;L,left;LG,lesiongroup;LUG,lesion+ultrasoundgroup;LSG,lesion+stretchinggroup;LUSG,

lesion+ultrasound+stretchinggroup;CG,controlgroup;RedR(darkgraycolumn),typeIcollagenoftheRgastrocnemius; RedL(graycolumn),typeIcollagenoftheLgastrocnemius;GreenR(whitecolumn),typeIIIcollagenoftheR

gastrocnemius;GreenL(blackcolumn),typeIIIcollagenoftheLgastrocnemius.

sarcomereinwhichmaximummusclestrengthwillbe pro-ducedduringcontraction.Thus,thismechanismmighthave occurredintheintragroupLSGandinthecomparisonwith theLUG,since,asincreasednumbersofsarcomeresinseries wereobservedintheLSG,therewasadecreaseofthe sarcom-erelengthinthisgroup;thereversepatternwasidentifiedin theLUG.

Regarding CSAMF, it was observed that this value was higher in the LG than in the LSG; however, no difference wasobservedwhencomparingwiththeCG.Thebodyweight ofthe LGincreasedby31%,vs. 19%inthe LSG,whichmay haveinfluencedtheCSAMF,despitethefactthatnoincrease inmuscleweightwasobserved.Inthepresentstudy, ultra-soundandstretchingdidnotinfluenceCSAMF.Marketetal.6

assessedtheeffectsofTUSandlow-intensitywalking exer-cisefor 20min on the treadmill (speed 14m/min)for four days,initiated24haftermechanicallesionofthe gastrocne-mius,andalsodidnotobserveanincreaseinCSAMF.Coutinho etal.19_{foundthat40-minstretchingincreasedCSAMFinthe}

immobilizedandstretchedgroupwhencomparedwiththe groupthatwas onlyimmobilized; thoseauthors concluded thatstretchingcanpreventmuscleatrophy,andits applica-tionisrelevantinimmobilizedmuscles.AstheLGpresenteda higherCSAMFthantheLSG,stretchingmayhaveinterferedin reducingedemaandregeneratingthesarcolemma,butitwas notsufficienttostimulateradial(transverse)musclegrowth, asreported inother studies.24,25 _{However,tofurther}

eluci-datethemechanismsinvolvedinchangesinmuscletrophism, studies analyzingthe muscle ultrastructureand regulatory genesaresuggested.

Theinjuredmusclesandthosethatdidnotundergo inter-vention(ultrasoundand/orstretching)showedanincreased

percentageoftypeI_{collagen,demonstratingthefibroticeffect} ofthelesion.Thiseffectwasalsoobservedwhenultrasound wasappliedinisolationandinassociationwithstretching. Conversely,whenstretchingwasperformedalone,the prolif-erationofcollagenwaslowerthanthatobservedinmuscles thatunderwentTUSalone.Therefore,itcanbeconcludedthat stretchingshowedanantifibroticeffectwhencomparedwith isolatedorcombinedultrasound.

Williams et al.26 _{stated that dailystretching ofmuscles}

shortenedduetoimmobilizationpreventsthedepositionof connectivetissueinthemuscletissue.ThestudybyHwang etal.9 _{corroboratedtheseresults.Theyassessedtheeffects}

ofstretchingoninjuredmusclesandobserveditsantifibrotic effect. These authors indicated that stretching should be startedonthe14thdayafterlesion.However,inthepresent study stretching wasinitiatedonthe 10thday; despitethe earlyinitiation,thefibrinolyticeffectwasstillobserved. Jarvi-nenetal.27_{havesuggestedthattheincreaseintramuscular}

connectivetissuecontributestofunctionaldeficits,producing tensilestrength,compliance,andmusclestiffness.Stretching programsmaycontributetoincreasemuscleextensibility,due tothereorganizationofintramuscularconnectivetissue.28

The LUG had a higher percentage of mature collagen in relation to the LSG and the CG. The increased density ofconnective tissue,associatedwith adecreased numbers ofsarcomeres, whichwas foundin LUG relativetoLSG, is characteristic of shortened skeletal muscle under fibrosis process.9,19,21,26_{Thisdemonstratesthatultrasoundusedalone}

foronlyfivedayshasnofibrinolyticeffect.Rantanenetal.29

lesion.Theauthors foundanincreasedproductionof fibro-blastsafter musclecontusion inrats from the groupsthat hadundergoneatleastfivesessionsofpulsedTUS.Piedade etal.,7_{starteddailyapplicationsof1MHzpulsedTUSat50%,}

intensityof0.57W/cm2_{,for5min,throughthesliding} tech-nique,andobservedtheearlyonsetoftypeI_{collagenfibers} fourdaysaftergastrocnemiusmusclelesioninrats;abetter structuralarrangementandbetteralignmentofmyotubesin formationwasobservedinthegrouptreatedwithTUS.These authorssuggestedthatTUScanstimulateearlyaggregationof thistypeofcollagen.

AlthoughnoincreaseinthepercentageoftypeI_collagen (mature)wasobservedinthepresentstudy,theauthorsdid notassessthearrangement,thatis,theorientationofthe col-lagenfibers.Thus,itisnotpossibletopredictwhetherthis increaseintypeI_{collagenmaygeneratefunctionaldeficits.} Therefore,futurestudiesshouldusepolarizingmicroscopyfor analysisofbirefringenceofintramuscularconnectivetissue28

andrangeofmotion.

Theoutcomesofthisstudyindicateimportantclinical per-spectives,demonstrating thatapassive stretching protocol performedtendaysaftermusclelesioninduced sarcomeroge-nesisandpresentedanantifibroticeffect,indicatingapossible improvementofskeletalmuscleextensibility.Thus, respec-ting the limitations regarding direct extrapolations of the presentresultshumans,itcanbehypothesizedthat stretch-ingexercisescouldbeprescribedforthetreatmentofmuscles injuredbycontusion.

Study

limitations

Thisstudywaslimitedbytheuseofananimalmodel,which producesalowerlevelofscientificevidence.Therefore, ran-domized controlled clinical trials should be performed to assessthe effectsofultrasound,whether ornotassociated withstretching,ininjured skeletal muscleofhumans.The authorsrecommendtheuseofelectronmicroscopytoassess sarcomere length in future studies. Furthermore, analyses withmolecularbiologytechniquesshouldbeusedto investi-gatethegenesinvolvedintheprocessesofrepairandtropism ofskeletalmuscle,especiallyresultingfromtheapplication ofthetwotherapeuticmodalities,ultrasoundandstretching. Biomechanicalassaysforanalysisofmuscle-tendonunit ten-sionarealsoimportanttoelucidatetheviscoelasticproperties inresponsetoultrasoundand/orstretching.

Conclusion

Thepassivestretchingprotocolinducedsarcomerogenesisin injuredmuscles.Furthermore,stretchingalonehadan antifi-broticeffect,whileultrasound,regardlessofassociationthe stretching,wasnotsufficienttopreventtheincreaseof colla-genIininjuredmuscles.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors would like to thankthe FederalUniversity of Paraná;theNationalCouncilforScientificandTechnological Development(CNPq, ProcessNo.474303/2011-0andProcess No. 308696/2012-3); the Pontifícia Universidade Católica do Paraná(PUC/PR);andUnibrasil.

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