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RESEARCHARTICLEOpenAccessExpressionof5-HT3receptorsandTTXresistantsodiumchannels(NaV1.
8)onmusclenervefibersinpain-freehumansandpatientswithchronicmyofascialtemporomandibulardisordersNikolaosChristidis1,2*,IsabellKang3,BrianECairns3,4,UjendraKumar3,XudongDong3,5,AnnikaRosén6,7,SigvardKopp1andMalinErnberg1,2AbstractBackground:Previousstudieshaveshownthat5-HT3-antagonistsreducemusclepain,buttherearenostudiesthathaveinvestigatedtheexpressionof5-HT3-receptorsinhumanmuscles.
Also,tetrodotoxinresistantvoltagegatedsodium-channels(NaV)areinvolvedinperipheralsensitizationandfoundintrigeminalganglionneuronsinnervatingtheratmassetermuscle.
Thisstudyaimedtoinvestigatethefrequencyofnervefibersthatexpress5-HT3A-receptorsaloneandincombinationwithNaV1.
8sodium-channelsinhumanmusclesandtocompareitbetweenhealthypain-freemenandwomen,thepain-freemasseterandtibialisanteriormuscles,andpatientswithmyofascialtemporomandibulardisorders(TMD)andpain-freecontrols.
Methods:Threemicrobiopsieswereobtainedfromthemostbulkypartofthetibialisandmassetermusclesofsevenandsixhealthymenandsevenandsixage-matchedhealthywomen,respectively,whiletraditionalopenbiopsieswereobtainedfromthemostpainfulspotofthemasseteroffivefemalepatientsandfromasimilarregionofthemassetermuscleoffivehealthy,age-matchedwomen.
Thebiopsieswereprocessedbyroutineimmunohistochemicalmethods.
ThebiopsysectionswereincubatedwithmonoclonalantibodiesagainstthespecificaxonalmarkerPGP9.
5,andpolyclonalantibodiesagainstthe5-HT3A-receptorsandNaV1.
8sodium-channels.
Results:Asimilarpercentageofnervefibersinthehealthymasseter(85.
2%)andtibialis(88.
7%)musclesexpressed5-HT3A-receptors.
TheexpressionofNaV1.
8by5-HT3Apositivenervefibersassociatedwithconnectivetissuewassignificantlyhigherthannervefibersassociatedwithmyocytes(P.
05,Studentst-test).
Eachbarrepresentstheaveragefromallthesectionsobtainedfromeachmuscletype.
Theerrorbarsdepictstandarderrorofthemean.
Table3Presentationofthemean(±SD)nervefiberdensityandtheirdistributionwithinthesectionsNervefiberdensityFiberspersection(n=)Location(%)ofpositivefibersMyocytesConnectivetissueMenWomenMenWomenMenWomenMicrobiopsiesMassetermuscleHealthy(n=12)6.
7±1.
85.
6±1.
576±15%78±20%45±20%40±24%TibialismuscleHealthy(n=14)6.
7±1.
98.
9±1.
995±2%77±13%26±17%38±17%Thetablepresentsthemeannervefiberdensity(i.
e.
fiberspersection),thepercentagedifferenceofpositivefiberswithinthemuscletissue,andthedistribution(eitherinassociationwithmyocytesorintheconnectivetissuesurroundingthemyocytes).
Therearemicrobiopsiesfromthemassetermusclefromsixmenandsixwomen,whiletherearemicrobiopsiesfromsevenmenandsevenwomenfromthetibialismuscle.
Therewerenosignificantdifferences.
Christidisetal.
TheJournalofHeadacheandPain2014,15:63Page7of12http://www.
thejournalofheadacheandpain.
com/content/15/1/63women(Table4),buttherewerenosignificantdiffer-encesinthereceptorexpressionbetweenthetwobiopsytechniques.
Inthemicrobiopsygroup,70.
5%ofthefibersassociatedwithmyocytesexpressed5-HT3Areceptorswhilethecorrespondingnumberwas62.
5%inthetrad-itionalbiopsygroup.
Further29.
5%ofthefibersassoci-atedwithconnectivetissuefibersexpressed5-HT3Areceptorsinthemicrobiopsygroup,whereasthecorre-spondingnumberwas37.
5%inthetraditionalbiopsygroup(Table2).
DiscussionMainfindingsThemainfindingsofthisstudywerethatthe5-HT3Are-ceptorishighlyexpressedinhumanmasseterandtibialismuscletissue,butthattherewerenodifferencesinex-pressionduetosex.
Further,thereweremoreimmuno-reactivenervefibersexpressing5-HT3receptorsandNaV1.
8sodiumchannelsinthemassetermuscleoffe-malepatientswithmyofascialTMDcomparedtohealthyfemalecontrols.
Thesefindingsindicatethat5-HT3re-ceptorsmightbeup-regulatedinmyofascialTMDandmaythereforeplayaroleinpaintransmission,henceprovidingthe5-HT3-receptorasatargetforfuturether-apeuticsorasapotentialbiomarkerofchronicmusclepain.
Secondarily,theresultsfromthisstudyindicatethatthemicrobiopsytechniqueprovidedsufficientmuscletissue,lessdiscomfortandpost-surgicalpainthanthetraditionalbiopsytechnique.
Therefore,themicrobiopsytechniquecanberegardedasavalidbiopsymethodforimmunohistochemicalanalysesofmuscletissue,andmay,thus,bepreferredtotraditionalopenbiopsiesinfuturestudiesorevenasadiagnostictool.
ReceptorexpressionTheimmunohistochemicalanalysisofthemusclesectionsindicatesthatthe5-HT3Areceptorishighlyexpressedbyhumanmasseterandtibialisanteriormusclenervefibers.
Toourknowledgetherearenootherstudiesthathavestudiedtheexpressionofthe5-HT3receptorinhumanmuscletissues.
ArecentstudydidshowthattheNMDAreceptorisalsoexpressedbymassetermusclenervefibers(45-50%)inhumansandrats[22].
However,resultsfromanimalstudiesregardingtheexpressionofthe5-HT3re-ceptorintrigeminalanddorsalrootganglionneurons[7,38]areconsistentwiththepresentstudy.
ItiswellestablishedthattheNaV1.
8sodiumchannelsplayanim-portantroleinnociceptionandchronicpainwhenfoundonsmallunmyelinatedandthinlymyelinatedsensoryneu-rons[24,25,29,30,39]sincetheycontributetoactionpo-tentialsinsensoryneurons[28,39,40]andareinvolvedinperipheralsensitization[23].
Theyhavefurtheralsobeenfoundinlargediametersensoryneuronsinhumanpainstates[41],aswellaslargediameterfibersinmouse[42]andrat[26,43]muscleafferentneurons.
Hence,thisTTXresistantsodiumchannelisnotjustlimitedtosmalldiam-eternervefibers[44].
InflammationhasbeenshowntoenhancenociceptivesignalingbytheNaV1.
8sodiumchan-nels[31,44-46],inpart,throughreleaseofinflammatorymediators,suchas5-HTandprostaglandinE2,whichdirectlyincreaseNaV1.
8mediatedcurrents[31,47].
Table4ComparisonofthetwotechniquesusedforobtainingmasseterbiopsiesfromhealthyvolunteersMicrobiopsies(n=18)Traditionalbiopsies(n=10)Meanweightofeachmusclesection19.
8(±2.
5)mg***58.
4(±9.
6)mgMeanvolumeofeachmusclesection13.
0(±1.
6)mm3***64.
4(±9.
9)mm3SurroundingtissuesNoYesTypeoftissueParotidgland(20%)Fatcells(30%)Adequatemusclesection100%100%NumberofPGP9.
5positivefiberspersection8.
2±1.
2*2.
7±0.
2Areaofwound1(±0.
04)mm2***10(±0.
7)mm2Post-surgicaldiscomfort3(±1)days8(±2)daysTypeofdiscomfortPalpatorytendernessoverthebellyofthemasseter(100%)Palpatorytendernessoverthebellyofthemassetermuscle(100%)Bleeding(6%)Limitedjawfunction(10%)Chewingdifficulties(50%)Discomfortfromsutures(100%)Extravisittoremovesutures(100%)Differencesarepresentedasmean(±SD)values.
*P<.
05,unpairedt-test,***P<.
001,unpairedt-test.
Christidisetal.
TheJournalofHeadacheandPain2014,15:63Page8of12http://www.
thejournalofheadacheandpain.
com/content/15/1/63Also,5-HTisknowntoparticipateinpainmediationattheperipherallevel,actingonthe5-HT3receptors,whichmainlyappearonafferentnociceptivesensory,autonomicandentericneurons[16].
Giventhis,onecanassumethattheNaV1.
8positivefibersaresensoryneu-ronswithapotentialnociceptivefunction,consequentlyimplyingthatthemusclenervefibersthatco-expressthe5-HT3AreceptorsandNaV1.
8sodiumchannelsaremostprobablynociceptors.
Noteworthyisthesignificantlyhigheraverageoffiberspersectioninthemassetermuscleofpatientswithmyo-fascialTMD,whichcouldindicateaproliferationofnervefibersinthepainfulmuscletissue.
Thereissomeevidencethathyper-innervationofthetendonwithputa-tivenociceptivefibersoccursinAchillestendinosis[48,49].
ThelargernumberoffibersintenderregionsofthemassetermuscleofTMDpatientsfoundinthisstudycouldindicateanup-regulationof5-HT3recep-tors.
Thisfindingmayexplainpreviousfindingsthatalocalinjectionofthe5-HT3receptorantagonistgranise-tronwaseffectiveindecreasingmusclesensitivityinpa-tientswithlocalizedmyalgia[21]andfibromyalgia[50].
Themajorityofthe5-HT3AandNaV1.
8positivefibersinthemassetermusclewerefoundintheconnectivetis-sue,whichsuggeststhatmasticatorymusclepainmayemanateprimarilyfromtheconnectivetissue.
Thisthe-oryisbasedonthefindingsfromthemusclesectionswhereboththepatientsandhealthysubjectshadsignifi-cantlymoreNaV1.
8positivenervefibersinconnectivetissuecomparedtofibersassociatedwithmyocytes.
TheelevatedlevelsofserotonininthemassetermuscleinpatientswithmyofascialTMD[3]andtheveryhighex-pressionof5-HT3AandNaV1.
8positivefibersonsen-sorynervesassociatedwithconnectivetissue,showninthisstudy,supportthetheorythatthemusclenocicep-torsinpainfulmusclescouldbesensitizedbyserotoninandhencethatserotoninmayhaveanimportantroleinmyofascialTMD.
Apartfrompainmediation,the5-HT3receptorsareinvolvedinmanyeventsinthehumanbody.
Centrallytheyplayanimportantroleinpsychosis,anxiety,cogni-tionandeatingdisorders[51],butalsoinmotorsystemfunction.
Arecentstudyshowedthatactivationofthe5-HT3receptorinducedrhythmicmovementsofthehin-dlimbsinmice[52].
Intheperipherytheyhaveawell-knownroleinemeticpathways[53]andtheyhavearoleintheintestinaltone,activatingcolonicmigratingmotorcomplex,i.
e.
muscularmotoractivity[54].
PGP9.
5labelsbothsmallandlargediametermyelinatedfibers[55]andNaV1.
8isamarkerofsmalldiameterthinlymyelinatednociceptivefibers[44].
Withthisinmind,asthemajorityofthe5-HT3Apositivemusclenervefiberswerelocatednearmyocytesanddidnotco-expressNaV1.
8,ourfindingsimplythatthe5-HT3Apositivefibersinassociationwiththemyocytesarelikelyeitherproprio-ceptivemusclespindleafferentfibersormotoraxons[56].
ComparisonofthebiopsytechniquesTheresultsofthisstudyindicatethatthemicrobiopsytechniqueprovidedsufficientmuscletissueforimmuno-histochemistryandledtolessdiscomfortandpost-surgicalpainaswellasfewervisitsthanthetraditionalbiopsytechnique.
Thisisinagreementwithapreviousstudycomparingthemicrobiopsytechniquewiththetraditionalopenbiopsytechniqueinthevastuslateralisofthequadricepsfemorismuscle[57].
Further,themicrobiopsytechniqueseemstobemoreprecise,sincebyusingtheBardTruGuidecoaxialneedle,thesurgeonismorelikelytoremovetissuefromtheexactregionofinterestwithoutdamagingthesurroundingtissue[35].
Itwasalsoshownthatthemusclesectionsfromthemicro-biopsiescontainedlessofthesurroundingtissues,suchaspartsofthesalivaryglandsthantraditionalopenbiopsies(Table4).
Takentogetherthisshowsthatthemicrobiopsytechniqueisavalidbiopsymethodforimmunohistochem-icalanalysesofmuscletissueandhence,maybepreferredtotraditionalopenbiopsiesinfuturestudiesorevenasadiagnostictool.
DifferencesduetosexoranatomicallocalizationIncontrasttoapreviousstudy[7]indicatingthattheex-pressionof5-HT3receptorsbytrigeminalganglionneu-ronsinnervatingthemassetermusclewashigherinfemalerats,thisstudyshowednosignificantdifferenceintheexpressionof5-HT3Areceptorsinthemasseterortibialismuscles,whenmenandwomenwerecompared.
Inpreviousstudies,womenreportedmorepainthanmenwhenserotoninwasinjectedintothemassetermuscle[13],andalargerpainareawhenhypertonicsa-linewasinjected[10].
Basedonthosestudiesonecouldhaveexpectedasignificantsexdifferencewithalargeramountofnervefibersco-expressing5-HT3AreceptorsandNaV1.
8sodiumchannelsinwomen.
Further,aprevi-oushumanexperimentalstudyshowedthatsystemicad-ministrationofthe5-HT3receptorantagonistgranisetronincreasedthePPToverthetibialisanteriormuscleinmenbutnotinwomen[11].
Asapossibleexplanationforthisdifferencecouldhavebeenthatthenumberofreceptorsdiffersbetweenmenandwomen,however,thefindingsofthepresentstudydonotsupportthisexplanation.
Itshouldbenotedthatalimitationofthisstudyisthesmallsamplesize(6–7individualsofeachsex),whichmayhavebeeninsufficienttopermitidentificationofsex-relateddif-ferencesin5-HT3Areceptorexpression.
Therewasadifferencebetweenthetwoskeletalmus-clessinceamajorityofthefibersco-expressing5-HT3AreceptorsandNaV1.
8sodiumchannelswerefoundintheconnectivetissueinthemassetermusclewhileinChristidisetal.
TheJournalofHeadacheandPain2014,15:63Page9of12http://www.
thejournalofheadacheandpain.
com/content/15/1/63thetibialismusclethesefiberswerefoundtobeassoci-atedwithmyocytes.
Thetibialismuscleisrarelyexposedtochronicpainincontrasttothemassetermuscleandisfurtherinnervatedbythedeepperonealnervewhichmajorroleistocontractthemusclefibersinordertodorsiflexandinvertthefoot[58].
Incontrasttothetibi-alisanteriormuscle,themassetermuscleexpressedalargeramountofputativelynociceptivenervefibersintheconnectivetissue.
Thisisaninterestingdifferencebetweenmusclesthatcouldbeaconsequenceofthedif-ferentinnervationwitheithertrigeminalbranches(mas-setermuscle)orspinalbranches(tibialismuscle)andfunctionalityofthenervesregardingmotorandsensoryfunctions[58].
Therefore,thelargeramountofputa-tivelynociceptivenervefibersintheconnectivetissueofthemassetermuscleandthedifferenceininnervationmightpartlyexplainthemorefrequentpresenceofchronicmyalgiainthemassetermuscle.
StudylimitationsOnelimitationofthestudyis,asmentioned,thesmallsamplesizewhichmighthaveaffectedtheoutcomere-gardingsex-relateddifferences.
Anotherpossiblelimita-tionofthestudyistheexclusioncriterion"useofanalgesicoranti-inflammatorymedicationduringthe24hoursprecedingthebiopsy".
Thiscriterionwasprimar-ilyinordertoavoidanyriskofincludingapatientwithmyofascialTMDamongthehealthyparticipants.
How-ever,24hoursisashortwash-outperiodforNSAIDswhichcouldhaveaffectedthebiopsyprocedurenegatively,forexamplethroughincreasedbleeding,sincetheuseofNSAIDsmightimpairthethrombocyteaggregationforupto8–10days.
UseofNSAIDmightalsohavesuppressedreceptorexpression.
Ifso,itislikelythatpatientswithmyfascialTMDwouldhavebeenaffectedmoreandinthatcasethedifferenceinfrequencywouldbeevengreaterthanfoundinthecurrentstudy.
Thestorageofthebiopsiesinparaffincouldbeanotherpossiblelimita-tionsincethistechniquemightblunttheantigenicityofthesamples,whichmaymakeitmoredifficulttodetectthereceptors.
Infuturestudiesthemusclebiopsiesaresuggestedtobefrozenimmediatelyin80°C.
ConclusionsThisstudyshowedthatthe5-HT3Areceptorishighlyexpressedbyhumanmasseterandtibialismusclenervefibers.
Further,agreaternumberofputativenociceptorswerefoundtoexpressthe5-HT3inthemassetermuscleoffemalepatientswithmyofascialTMDcomparedtohealthyfemalecontrols,whichsuggeststhat5-HT3re-ceptorsmightbeup-regulatedinmyofascialTMD.
Thisfindingcouldsupporttheuseof5-HT3receptorexpres-sioninmusclebiopsiesasabiomarkerofchronicmasti-catorymusclepain.
AbbreviationsTMD:Temporomandibulardisorders;5-HT:5-hydroxytryptamineserotonin;TTX:Tetrodotoxin;NaVs:Sodiumchannel;RDC/TMD:Researchdiagnosticcriteriafortemporomandibulardisorders;PBS:Phosphate-bufferedsaline.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsNC,contributedinthedesignofthestudy,bycollectingthematerial(microbiopsies),inthestatisticalanalysis,andasthemainauthorofthemanuscript.
IKcontributedintheanalysisofthematerialandintheresultsectionofthemanuscript.
BEC,contributedinthedesignofthestudy,intheanalysisofthematerial,inthestatisticalanalysis,andinthemanuscriptingeneral.
UK,contributedintheanalysisofthematerialandinthediscussionsectionofthemanuscript.
XD,contributedintheanalysisofthematerialandinthediscussionsectionofthemanuscript.
AR,contributedbycollectingthematerial(traditionalbiopsies)andinthediscussionsectionofthemanuscript.
SK,contributedinthedesignofthestudyandintheresultanddiscussionsectionofthemanuscript.
ME,contributedinthedesignofthestudy,inthestatisticalanalysis,andinthemanuscriptingeneral.
Allauthorsreadandapprovedthefinalmanuscript.
Authordetails1OrofacialPainandJawFunction,DepartmentofDentalMedicine,KarolinskaInstitutet,Huddinge,Sweden.
2ScandinavianCenterforOrofacialNeuroscience(SCON),Stockholm,Sweden.
3FacultyofPharmaceuticalSciences,UniversityofBritishColumbia,Vancouver,Canada.
4CenterforSensory-MotorInteraction,AalborgUniversity,Aalborg,Denmark.
5CollegeofStomatology,TianjinMedicalUniversity,Tianjin300070,P.
R.
China.
6OralandMaxillofacialSurgery,DepartmentofDentalMedicine,KarolinskaInstitutet,Huddinge,Sweden.
7OralandMaxillofacialSurgery,DepartmentofClinicalDentistry,UniversityinBergen,Bergen,Norway.
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Submityourmanuscripttoajournalandbenetfrom:7Convenientonlinesubmission7Rigorouspeerreview7Immediatepublicationonacceptance7Openaccess:articlesfreelyavailableonline7Highvisibilitywithintheeld7RetainingthecopyrighttoyourarticleSubmityournextmanuscriptat7springeropen.
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