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RESEARCHARTICLEOpenAccessComparisonofthevasodilatorresponsesofisolatedhumanandratmiddlemeningealarteriestomigrainerelatedcompoundsGustafGrnde1,SienekeLabruijere2,KristianAgmundHaanes3,AntoinetteMaassenVanDenBrink2andLarsEdvinsson1,3*AbstractBackground:Migraineattacksoccurspontaneouslyinthosewhosufferfromthecondition,butmigraine-likeattackscanalsobeinducedartificiallybyanumberofsubstances.
Previouslypublishedevidencemakesthemeningesalikelysourceofmigrainerelatedpain.
Thisarticleinvestigatestheeffectofseveralvasodilatorsonmeningealarteriesinordertofindaconnectionbetweentheeffectofasubstanceonameningealvesselanditsabilitytoartificiallyinducemigraine.
Methods:Amyographsetupwasusedtotestthevasodilatorpropertiesofthesubstancesacetylcholine(ACh),sodiumnitroprusside(SNP),sildenafil,prostaglandinE2(PGE2),pituitaryadenylatecyclaseactivatingpeptide-38(PACAP-38),calcitoningene-relatedpeptide(CGRP)andNaClbufferonmeningealarteriesfromhumanandrat.
Anunpairedt-testwasusedtostatisticallycomparethemeanEmax(%)atthehighestconcentrationofeachsubstancetotheEmax(%)ofNaClbuffer.
Results:Inthehumanexperiments,allsubstancesexceptPACAP-38hadanEmax(%)higherthantheNaClbuffer,butthedifferencewasonlysignificantforSNPandCGRP.
Forthehumansamples,clinicallytestedantimigrainecompounds(sumatriptan,telcagepant)wereappliedtotheisolatedarteries,andbothinducedasignificantdecreaseoftheeffectofexogenouslyadministratedCGRP.
Inexperimentsonratmiddlemeningealarteries,pre-contractedwithPGF2α,similartendencieswereseen.
Whenthepre-contractionwasswitchedtoK+inaseparateseriesofexperiments,CGRPandsildenafilsignificantlyrelaxedthearteries.
Conclusions:Stillnodefiniteanswercanbegivenastowhypainisexperiencedduringanattackofmigraine.
Noclearcorrelationwasfoundbetweentheefficacyofasubstanceasameningealarteryvasodilatorinhumanandtheabilitytoartificiallyinducemigraineorthemechanismofaction.
Vasodilatationcouldbeanessentialtrigger,butonlyinconjunctionwithotherunknownfactors.
Thevasculatureofthemeningeslikelycontributestothepropagationofthemigrainalcascadeofsymptoms,butmoreresearchisneededbeforeanyconclusionscanbedrawnaboutthenatureofthiscontribution.
Keywords:Vasodilation;Middlemeningealartery;Migraine;CGRP*Correspondence:Lars.
Edvinsson@med.
lu.
se1DepartmentofMedicine,InstituteofClinicalScience,LundUniversity,22184Lund,Sweden2DepartmentofInternalMedicine,DivisionofVascularMedicineandPharmacology,ErasmusMCRotterdam,POBox2040,3000CARotterdam,TheNetherlands3DepartmentofClinicalExperimentalResearch,CopenhagenUniversityHospital,Glostrup,Denmark2014Grndeetal.
;licenseeSpringer.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
Grndeetal.
TheJournalofHeadacheandPain2014,15:22http://www.
thejournalofheadacheandpain.
com/content/15/1/22BackgroundTheduramateranditsvasculature,themiddlemenin-gealartery(MMA)andthevenoussystemhaveforde-cadesbeencentraltomanyhypothesesaimedtoexplainmigrainepathophysiology[1,2].
RayandWolffshowedinthe1940sthatdirectstimulationoftheduramatermayresultinheadachewithmigraine-likequalities[3].
Furtherresearchduringthelasttwodecadeshasdemon-stratedthatsensitizationandlocalinflammationintheduramatercanbeelicitedbyvariousagentsandstimu-lationparadigms[4].
Thelocaladministrationofan"in-flammatorysoup"orthedirectstimulationatdifferentpartsoftheduramatermayresultnotonlyinvasoactiveresponsesbutsometimesalsoinmastcelldegranulationandinplasmaextravasation.
Histamine,amajoraminereleasedfrommastcells,isknowntoinduceamigraine-likepainuponinfusionintotestsubjectspreviouslydiagnosedwithmigraine[5].
Somemigraine-associatedsymptoms,includingthecharacteristicpain,couldthere-forestemfromtheactivationofmeningealmastcellsandasepticinflammationinitiatedbyhistaminerelease[6].
However,localadministrationofcalcitoningene-relatedpeptide(CGRP),whichisasensoryneuropeptideintheduramaterandaknownmigrainogenicsubstance[7],resultsonlyinvasodilatation[8].
DespitethefactthatmastcellshaveCGRPreceptors[9,10],noactivationorsensitizationofmeningealnociceptorscouldbede-tectedupontopicalorsystemicadministrationofthiscompoundinrats[8].
ApartfromCGRPandhistamine,othersubstancesthatuponintravenousinfusioncanprovokemigraine-likeattacksinhumanspreviouslydiagnosedwithmigraineareprostaglandinE2(PGE2),pituitaryadenylatecyclaseactivatingpeptide-38(PACAP-38),epoprostenol(prosta-cyclin,PGI2),sildenafilandnitricoxide(NO)[11-15].
Theonlycommonfactoramongthesesubstancesistheirvaso-dilatorproperties,butstillthereexistseveralothervasodi-latorswhichdonotinducemigraine-associatedsymptomsuponinfusion.
Vasoactiveintestinalpolypeptide(VIP)andcarbachol,bothassociatedwiththeparasympatheticsys-tem,arenotedexamples,althoughtheirabilitytodilatemeningealarteriesinvivoremainstobeverified[16,17].
Thisshowsthatfactorsapartfromthevasodilatationhavetobetakenintoconsideration.
Thereisstillcontroversyregardingwhethervasodilatationdirectlycontributestoanyofthemigraine-relatedsymptomsorwhetherthisisjustaside-phenomenon.
Severalhypothesesexistthatdownplaytheroleofvasodilatation,includingtheobser-vationofprodromalaurasymptomsprecedinganyactiv-ityinthevasculature[18],thatthevasoconstrictortriptandrugsarenotaneffectivetreatmentinallmigrainepatientsandthattheextravasculartissuesurroundingtheduralarteriescouldbetoorigidtoallowforanysignificantex-pansionofthevesseldiameter[19].
Wehaverecentlyinvestigatedthevasoactiveeffectsofseveralsubstancesoncerebralarteriesfromhumanandratinvitroinordertofindcommondenominatorsthatwouldallowustounderstandmoreaboutthepossiblefac-torsbehindthemigraine-associatedsymptoms[20].
How-ever,cerebralarteriesaredifferentfrommeningealarteriesregardingseveralmorphologicalaspects,includingrecep-torexpression,anatomicalorigin(internalcarotidartery(ICA)versusexternalcarotidartery(ECA))andthelackofablood–brainbarrier(BBB)inthemeningealarteries[21].
Nevertheless,bothvascularregionsreceivesensoryinputfromthefirstdivisionofthetrigeminalganglion[22].
Thereforeitispossiblethatthetwoarterytypescon-tributetomigrainedifferentlyifthereindeedisavascularcomponent.
Theaimofthepresentstudyistoevaluatewhetherthereisaconnectionbetweentheabilityofasubstancetodilatemeningealarteriesandthepreviouslyreportedmigrainogenicpropertiesofeachsubstance.
Itisusefultoverifywhethervasodilatationofthemeningealarteriesindeedisthetriggerforthemigraine-likepaintriggeredbythesesubstances.
SincethemeningealarterieslackBBBproperties,systemicdrugsarefreelydiffusibletoendothe-lialandsmoothmusclereceptorstoelicitavasomotorre-sponse.
Asecondarygoalofthisstudyistocomparethefunctionalaspectofthemiddlemeningealartery(MMA)betweenthespeciesbecauseratisoftenusedinmechanis-ticstudiesduetothedifficultyinobtaininghumantissuesamples.
MethodsTheexperimentalprotocol(M11104)wasapprovedbytheAnimalProtocolReviewcommitteeattheUniversityofLund.
Allhumanarteryprocedureswerecarriedoutstrictlyaccordingtonationallawsandguidelinesandap-provedbytheEthicalCommitteeattheUniversityofLund(LU-818-01)andthelocalEthicsCommitteeattheErasmusMC,Rotterdam.
ObtainingtheMMAA.
FreshsamplesofMMAwereobtainedfromthemen-ingesofmaleSprague–Dawleyratsbythefollowingproced-ure.
TheratswereanesthetizedusingCO2anddecapitated.
Thecraniumwasopenedfromthetopandthebrainre-moved,whichexposedtheduramater.
ThecraniumandduramaterweredividedsagitallyandeachhalfwasplacedinabuffersolutioncomposedofNaCl119mM,NaHCO315mM,KCl4.
6mM,MgCl21.
2mM,NaH2PO41.
2mM,CaCl21.
5mMandglucose5.
5mM.
TheduramaterwascarefullyseparatedfromthecraniumandmovedtoaPetridish,whereitwassubmergedinbuffersolution,stretchedandsuspendedbyneedles.
TheMMAwasremovedfromtheduramaterbyadissectingmicroscope,cutintoseg-ments1–2mmlongthatwereplacedinparalleltissueGrndeetal.
TheJournalofHeadacheandPain2014,15:22Page2of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22bathsofice-coldbicarbonatebuffersolutionaeratedwithagascomposedof95%O2and5%CO2,witharesult-ingpHof7.
4[23].
B.
HumansamplesofintracranialMMAbrancheswereacquiredfrompatientsundergoingneurosurgeryattheUniversityHospitalofLundandtheErasmusUniversityMedicalCenter(Rotterdam,theNetherlands).
Thevesselsegmentsthemselveswerefromvisuallyhealthytissue.
ThearterysampleswereuponremovalimmediatelyplacedincoldDulbecco'smodifiedEagle'smedium(DMEM,Gibco,Invitrogen,Carlsbad,CA,USA)inLundandincoldmediumM199(Gibco,Invitrogen,Carlsbad,CA,USA)inRotterdamandimmediatelytransportedtothelaboratorywheretheywerepreparedinanidenticalwaytotheratMMA.
MyographyEachsegmentofMMAwasmountedonapairofthinmetalwires(human:40μm,rat:20/25μm)inanarterialmyograph.
Onewirewasconnectedtoamicrometerscrew,allowingforfineadjustmentofthevasculartonebyvaryingthedistancebetweenthewires.
Theotherwirewasconnectedtoaforcedisplacementtransducer,pairedwithananalogue-digitalconverter(ADInstruments,Oxford,UK).
DatawasrecordedonacomputerusingaPowerLabunit(ADInstruments).
Theaeratedbicarbonatebufferwasheatedto+37°CandusedtosubmergethewireswiththeMMA-segmentduringtheexperiment.
Thesegmentwasnormalized,attaining90%oftheinternalcircumferencethatafullyrelaxedvesselwouldhaveunderatransmuralpressureof100mmHgor50mmHg(seebelow).
AreferencevalueforthecontractilecapacityofthesegmentwasdeterminedbytemporarilyreplacingpartoftheNaClinthebuffersolutionwith60mMK+.
Theeffectsonthesegmentofthedifferentvaso-activesubstanceswastestedbyfirstrecordingthespon-taneousrestingdiameterofthearterysegmentanddefineitasthemaximumdilatationpossible.
Next,theseg-mentwaspre-contractedwith106MprostaglandinF2α(PGF2α)toachieveastabletensionforthedur-ationoftheexperiment.
Finally,concentrationsofthetestedsubstancesintherangeof1010-105M(theexactrangevariedbetweensubstances)wereappliedcumula-tivelyandthevesselresponsewasrecorded.
BecauseoftheunstableresponsetoPGF2αinthefirstratex-periments,wechangedtheprotocolandused30mMK+asamorestablecontraction.
Inaddition,thetrans-muralpressurewasloweredto90%of50mmHgintheseexperiments.
Arterysegmentswereomittedfromthestudyiftheyfailedtofulfilltheinclusioncriteria.
Segmentsincludedinthefinalcalculationswererequiredtorespondtoini-tialtestingwithK+andtothepre-contractionwithamaximumcontractilecapacityofatleast1mNforhu-manarteriesand0.
1mNforratarteries.
StudieswithclinicallytestedantimigrainecompoundsThevesselsegmentswereprecontractedwith30mMK+.
Afterprecontractionthevesselsegmentsweredilatedwith4nMCGRP.
Whenastabledilatationwasachieved,su-matriptanortelcagepantwasaddedtothearteriesincon-centrationscorrespondingtotheCmax(160nMand5μM,forthe100mgand300mgoraldoses,respectively)[24-26],aswellasinconcentrationscorrectedforplasmaproteinbinding.
TheCmaxforsumatriptanwascorrectedfor17.
5%plasmaproteinbinding[27],whiletheCmaxfortelcagepantwascalculatedusingaplasmaproteinbindingof96.
4%[28].
CalculationsThemaximumdilatoryresponse(Emax(%))foreachtestedsubstancewasobtainedforthevesselsegmentsasthepercentagetonusrelaxedbythemaximumconcentra-tionofsubstancefromthePGF2α-orK+-inducedpre-contractiontowardstherestingtonussampledbeforethelowestconcentrationofsubstancewasadded.
InthecaseofsignificantrelaxationthenegativelogarithmoftheconcentrationofsubstancerequiredfordilatationofthevesselsegmentstohalfoftheirEmax(%).
ThepEC50wasobtainedforthepurposeofcomparingthepotencyofthedifferentsubstances,calculatedusingGraphPadPrismsoftware(GraphPadSoftware,SanDiego,California,USA).
Ifatestsubjectcontributedwithmorethanonevesselsegmenttothetestingofasubstance,allresultsobtainedfromthatindividualregardingthatsubstancewerepooledtogetherintoanaveragebeforefurtherprocessingofdata.
ThehypothesiswasthateachsubstancewouldinducevasodilatationandtodeterminethesignificanceofthedilatationanunpairedttestwasperformedusingtheGraphPadPrismsoftware(GraphPadSoftware,SanDiego,California,USA.
)comparingtheaverageEmax(%)ofthehighestconcentrationofeachsubstancetotheaverageEmax(%)ofNaClbufferattheendofthesession.
FigureswerecompiledwhereitisshownhowthePGF2α/K+precontraction(100%)duringasessionisre-storedtowardstheoriginalrestingbaselineatdifferentconcentrationsofeachofthesubstancestestedandNaClbuffer(Figures1,2,3,4,and5).
Thecontractilere-sponsestotelcagepantandsumatriptanwereexpressedaspercentageofthepreviousrelaxantresponseto4nMCGRPinthesamesegment(Figure2).
Alldataarepresentedasmean±S.
E.
M.
StatisticalanalysisandpEC50calculationswereperformedusingGraphpadPrism5software.
StatisticalsignificancewasacceptedatP<0.
05.
Grndeetal.
TheJournalofHeadacheandPain2014,15:22Page3of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22ChemicalsThevasoactivesubstancesusedinthepresentstudywere:acetylcholinechloride(ACh),sodiumnitroprusside(SNP),sildenafil,PACAP-38(allpurchasedfromSigmaAldrich),PGE2(LarodanfineChemicalsAB).
Theinhibi-torsusedwerethefollowing:Telcagepant(MK-0974)(dissolvedinDMSOandfurtherdilutedinH2O)(MSD,WhitehouseStation,NJ,USA),sumatriptan(dissolvedinH2O)(SigmaAldrich).
ResultsFunctionalresultsfromtheMMAIntracranialMMAsegmentsfrom6humanswereusedtotestthevasoactivepropertiesofsixdifferentcompoundsincludingNaClbufferbutexcludingCGRP.
Thevesselsweredividedintoseveralsegmentsof1–2mminlengthandstudiedinparalleltissuebaths.
Noteverysegmentwasexposedtoallcompounds.
Vesselsfrom4additionalhumanpatientswereusedforthetestingofCGRP.
Figure1TheeffectofsubstancesonhumanmiddlemeningealarteriesprecontractedwithPGF2α.
TherelaxationrelativetoPGF2αinducedtoneinhumanmeningealarteriesbyincreasingconcentrationsofeachofthetestedsubstances(A-F)andNaClbuffer(G).
Valuesaregivenasmean±SEM(n=4–6),wherePGF2αprecontractionissettobe100%.
Grndeetal.
TheJournalofHeadacheandPain2014,15:22Page4of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22HumanMMA(100mmHg,pre-contractedwithPGF2α)Ofthecompoundstested,SNPandCGRPonhumanarteryshowedsignificantdilatationcomparedtoNaCl,withCGRPbeingthestrongestvasodilator(Figure1,Table1).
AChshowedanon-significanttendencyofrelaxation.
PGE2,PACAP-38andsildenafilshowednovasomotorac-tivitydistinctfromthegraduallossofpre-contractionseenwhenusingonlyNaClbuffer(Figure1,Table1).
Theveryhighconcentrationofsildenafil(10μM)wasnottestedonhumanvessels.
HumanMMAandCGRP(100mmHg,pre-contractedwith30mMK+)Forhumansamplestheclinicallytestedantimigrainecompoundswereappliedtotheisolatedarteriesthatthathadbeenprecontractedwith30mMK+.
CGRPin-ducedasignificantvasodilatationofmeningealarteries(84±10%ofprecontractionwith30mMK+).
Thecon-centrationscorrespondingtotheCmaxobtainedafteroraladministrationof100mgsumatriptan(160nM)or300mgtelcagepant(5μM)bothleadtoasignificantde-creaseoftheeffectoftheexogenousadministratedCGRP(8±18%and8±8%,respectively)(Figure2).
Alsotheconcentrationsofbothsumatriptanandtelcagepantcor-rectedforplasmaproteinbindingabolishedtheeffectofexogenousCGRP(8±36%forsumatriptanand4±7%fortelcagepant,respectively)(Figure2).
RatMMA,(100mmHg,pre-contractedwithPGF2α)SNPandsildenafilshowedatendencytorelaxthearter-iesbuttheobserveddilatationwasnotstatisticallysig-nificant(Figure3,Table2).
ACh,PGE2andPACAP-38didnotdisplayanydiscerniblevasodilatationcomparedtoNaCl(Figure3,Table2).
Theresultsfromtheseratexperimentsshowedalargerspreadcomparedtotheex-perimentsusinghumanarterysegmentsoradifferentprotocol,andthushadahigherratioofnotpassingtheinclusioncriteria.
Forthefirstexperimentsabove,thesameprotocolasforthehumanvessels(100mmHgandpre-contractedwithPGF2α)wasusedtotestallsubstancesincludingNaClonMMAsegmentsfrom5rats.
Thesevesselsshowedalargevariationandhadahighratioofnotpassingtheinclusioncriteriaof0.
1mN.
Therefore,theprotocolwaschangedbyloweringthetransmuralpressureto50mmHgandtheexperimentswererepeatedusinganadditional5rats.
Thismarkedlyimprovedthesuccessrate.
Inaddition,theMMAspre-contractedwith30mMK+,didnothaveanegativedriftovertime(Figure4A).
AsamplerelaxationtoacumulativedoseofSNPisalsoshown(Figure4B).
RatMMA,(50mmHg,pre-contractedwith30mMK+)Theresultsshowedsignificantrelaxationuponexpos-uretoSNP,CGRPandsildenafilcomparedtotheNaClcontrolgroup,SNPbeingthestrongestvasodilatator(Figure5,Table3).
PGE2,AChandPACAP-38showednosignificantresponsecomparedtothecontrolgroup(Figure5,Table3).
Theseresultscorrelatewiththeten-dencyseenfortheaboveMMA.
DiscussionTheadditionofsubstancesonhumanvesselswasstud-iedatatransmuralpressureof100mmHgandpre-contractedwithPGF2α.
TwodifferentapproachesfortheratMMAswereused,andinthisarticletheresultsfrombothmethodsarepresented.
Loweringofthetransmuralpressurefrom100mmHgto50mmHggreatlyimprovedthesuccessrate,andthemoreconstant30mMK+pre-contractionimprovedreproducibility.
ThegradualfailingofthePGF2αpre-contractionandincreaseoftheK+-inducedpre-contractionwasmitigatedbycomparingthevasomotoractivityofthesubstanceswiththespontaneousactivityobservedwhensubjectingthevesselsegmentstoonlyNaClbuffer.
Itisimportanttoinformthereader,thattheisolationandinsertionofwiresintotheratMMA,destroystheendothelialcelllayerbecauseofthesmalldiameteroftheratMMA(around100μmino.
d.
).
ThelackofendotheliumcanbeseeninFigure4AbecauseAChinducesacontraction,mostlikelyactingonAChre-ceptorsonthesmoothmusclecells.
Theendothelialfunc-tioninthehumanvesselsisnotsubstantialeither,asnoFigure2EffectoftelcagepantandsumatriptanonCGRPinducedrelaxationofthemiddlemeningealartery.
Arterieswereprecontractedwith30mMK+,dilatedwith4nMCGRPandsumatriptanortelcagepantwassubsequentlyadded(*=p<0.
05).
Valuesaregivenas%oftheCGRPrelaxation(mean±SEM,n=5).
Grndeetal.
TheJournalofHeadacheandPain2014,15:22Page5of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22significantrelaxationtoAChisobserved.
Thelackoffullendothelialfunctionandthelackofsympathetictoneinthevesselsinthisstudycouldaltersomeoftheeffectsofthesubstancesadded.
Forexample,sildenafilhasbothendothelial-dependentand-independenteffects[29],whichcouldbedifferentinhumanandrat.
Theresultsfrombothtypesofexperimentsonrattis-suewereconsistentandshowedthatSNP,CGRPandsildenafilinducedvasodilatation,whileACh,PGE2andPACAP-38hadlittleeffect.
Whenusedonhumanves-sels,thesamemyographymethodyieldedsignificantresultsforSNPandCGRP,whereastrongdilatationwasobserved.
Analternativemethodthathasbeenusedinrodentswithsomesuccessisthecranialwin-dowmethodwheresubstancescanbeappliedtopic-allythroughathinnedcalvarium[30],butwiththismethodquantificationisdifficult,astheconcentrationofactivesubstancethatreachesthevesselisdifficulttocontrol,hencepEC50andEmaxcannotreliablybecalcu-lated.
Further,therearemorefactors(e.
g.
,bloodpressure)thatcaninfluencetheresultsthanwhenusingisolatedvesselsinvitro.
Fortheexperimentsonhumanvessels,thegreatestdifferenceinresponsebetweenMMAandthepreviouslypublisheddataforMCA[20]wasseenwhensubjectingthevesselstosildenafilandPACAP-38,whichinbothcasesconstitutedastrongerdilatationintheMCA.
ThiswassurprisinginthecaseofsildenafilsincetherearepreviousreportsofratMMAcontaininghigherlevelsofphosphodieterasetype5(PDE5)thanreportedforratMCA[31].
Ifthesameconditionswerevalidforhumanvessels,thehigherconcentrationofthetargetenzymeintheMMAmeansthatsildenafilshouldhavehadmoreeffectthere.
However,theeffectisalsodependentonbasalNOandcGMPlevels,andwecannotruleoutthattherecouldbeaninfluenceoftheageofthehumancomparedtotheratvessels.
Inaddition,thesympathetictoneinvivocouldbecounteractedbyincreasedNOproduction,whichwouldnotoccurinoursetup.
There-fore,sildenafilcouldbeeffectiveinvivo,butnotinthemyographs.
Itishardtodrawconclusionsaboutthedif-ferenceinEmax(%)ofPACAP-38duetopreviouscon-flictingobservationsoftheactivityofPACAP-38inrat.
PreviouslypublishedinvitroexperimentswherehumanFigure3TheeffectofsubstancesonratmiddlemeningealarteriesprecontractedwithPGF2α.
Therelaxationinducedinratmeningealarteriespre-contractedwithPGF2αatincreasingconcentrationsofeachofthetestedsubstances(A-E)andNaClbuffer(F).
Valuesaregivenasmean±SEM(n=3–5),wherePGF2αprecontractionissettobe100%.
Grndeetal.
TheJournalofHeadacheandPain2014,15:22Page6of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22intracranialMMAwassubjectedtothesubstancesug-geststhatPACAP-38couldindeeddilatehumanMMA(Emax(%)=34±12;pEC50=6.
9±0.
1)[32]andthattheabsenceofvasodilatationobservedintheratexperi-mentsinthepresentstudyiseitherduetolackofreceptorsortechnicalissues.
However,thereisnodatasuggestinganendothelialresponsetoPACAP-38.
ThissuggeststhattheratisnotagoodmodelforstudiesofthePACAP-38mechanisms.
Previousinvivostudiesexistwheremagneticreson-anceangiography(MRA)wasusedtomeasurethediam-eteroftheextracranialpartoftheMMAoflivehumantestsubjectsbefore,duringandafterspontaneousandartificiallyinducedepisodesofmigraine.
Intravenousin-fusionofCGRP[33],PACAP-38[34]andnitroglycerine[35]inhealthynon-migraineurswereshowntoincreasethediameteroftheextracranialMMA.
Thesesubstancesareknowntoinduceaheadachelackingthemigraine-relatedcharacteristicsinnon-migraineursandthistypeofpaincoincidedwiththeobservedvasculardilatation.
WhenCGRPwasinfusedtomigraineurs,theresultswerereproducedandanadditional,migraine-likeheadachewithdelayedonsetoccurred[36].
Ontheotherhand,spontaneousmigraineattacksreportedinpatientswereassociatedwithamodestincreaseindiameter(amagni-tudeof11.
4-13.
0%)oftheMCAandtheintracranialparts(cavernousandcerebral)oftheICA[37].
Theintracranialbasilarartery(BA)andtheextracranialvesselsofECA,superficialtemporalartery(STA),thecervicalpartoftheICAandtheextracranialpartoftheMMAwerenotsig-nificantlydilated.
Migraine-abortingtriptandrugshadaconstrictingef-fectonarteriesoutsideoftheBBB,liketheMMAandtheextracranialpartoftheICA,butnotoncerebralar-teriesincludingtheintracranialpartoftheICA.
Regard-lessofwhetheranepisodeofmigrainewastriggerednaturallyorbyaninfusedsubstance,thepainwasmiti-gatedandtheextracranialMMAcontractedfromtheuseoftriptandrugs.
However,itshouldbenotedthatthesestudiesdidnotincludetheintracranialbranchesoftheMMA,whichisthesourceofthevesselsegmentsusedpresentlyintheinvitrostudies,anditisstillun-knownhowthispartofthearterywouldbeaffectedinvivobyaspontaneousepisodeofmigraineorathera-peuticdoseofsumatriptan.
Thequestionisrelevant,be-causeallarteriesshowntosignificantlydilateduringanepisodeofmigraineareallintracranial.
IntracranialmeningealarterieswouldbeuniqueinthattheyarebothFigure4Comparisonof30mMK+andPGF2αonratmiddlemeningealarteries,andsamplerelaxation.
A)Samplecontractioninducedbyof30mMK+orPGF2αinratmeningealartery.
Onecanobservetheslightpositivedriftfor30mMK+andslightthenegativedriftforPGF2α.
B)Samplerelaxationinanarteryprecontractedwith30mMK+andthecumulativerelaxationofaddedsodiumnitroprusside(SNP).
Smallplateauswereallowedbeforenextconcentrationwasadded.
Table1HumanMMA,100mmHg,pre-contractedwithPGF2αCompoundEmax(%)pEC50(M)p-valueACh(n=6)17±6N.
A.
0.
44SNP(n=6)32±6~3.
50.
034Sildenafil(n=6)11±2N.
A.
0.
91PGE2(n=6)8±1N.
A.
0.
62PACAP-38(n=5)9±3N.
A.
0.
89CGRP(n=4)62±88.
1±0.
190.
0015NaCl(n=4)10±4N.
A.
p-valueforcomparingagonistversustheNaCleffect.
N.
A.
=NotApplicable.
Valuesare±SEM,ngiveninparenthesis.
Table2RatMMA,100mmHg,pre-contractedwithPGF2αCompoundEmax(%)pEC50(M)p-valueACh(n=3)33±22N.
A.
0.
68SNP(n=3)70±188.
3±0.
70.
066Sildenafil(n=3)52±20~70.
23PGE2(n=3)23±12N.
A.
0.
82PACAP-38(n=4)22±14N.
A.
0.
93NaCl(n=3)23±4N.
A.
p-valueforcomparingagonistversustheNaCleffect.
N.
A.
=NotApplicable.
Valuesare±SEM,ngiveninparenthesis.
Grndeetal.
TheJournalofHeadacheandPain2014,15:22Page7of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22intracranialandwithoutBBBproperties.
Theyaretherebyatypeofarterythatcouldpossiblybebothdilatedduringanepisodeofmigraineandcontractedbydrugs.
Intheinvitrostudypresentedhere,theeffectoftwowell-knownclinicallytestedantimigrainecompoundswereappliedtotheisolatedhumanMMAs.
Bothsumatriptan(5-HTre-ceptoragonist)andtelcagepant(aCGRPreceptorantagon-ist)significantlyreducedtheCGRPinducedvasodilation.
ThisshowsthatthevasoactiveclinicallytestedcompoundscanexerttheireffectdirectlyontheMMA.
Invivoexperimentsinrat,usingthecranialwindowmethodtodirectlyviewandapplysubstancestotheFigure5TheeffectofsubstancesonratmiddlemeningealarteriesprecontractedwithK+.
Therelaxationinducedinratmeningealarteriespre-contractedwithK+atincreasingconcentrationsofeachofthetestedsubstances(A-F)andNaClbuffer(G).
Valuesaregivenasmean±SEM(n=5),where30mMK+precontractionissettobe100%).
Table3RatMMA,50mmHg,pre-contractedwith30mMK+CompoundEmax(%)pEC50(M)p-valueACh(n=5)26±13N.
A.
0.
85SNP(n=5)67±22;6.
1±1.
30.
015Sildenafil(n=5)40±175.
7±3.
80.
0069PGE2(n=5)31±5N.
A.
0.
311PACAP-38(n=5)27±8N.
A.
0.
94CGRP(n=5)1±6~80.
0015NaCl(n=5)24±5N.
A.
p-valueforcomparingagonistversustheNaCleffect.
N.
A.
=NotApplicable.
Valuesare±SEM,ngiveninparenthesis.
Grndeetal.
TheJournalofHeadacheandPain2014,15:22Page8of10http://www.
thejournalofheadacheandpain.
com/content/15/1/22intracranialportionoftheMMA,haveyieldedsimilarresultstotheinvivoobservationsinhumanregardingtheextracranialpartofthevessel.
TheMMAofratswassignificantlydilatedbyα-andβCGRP[38,39],sildenafil[31],VIP,PACAP-38,PACAP-27[40],histamine[41]andACh[42].
VIPisinterestinginthatitisnotamigrainogenicsubstance[16].
IthasnotbeenestablishedwhateffectVIPcouldhaveonhumanisolatedMMAinvivo,butsinceVIPisknowntodilatehumanMMAbranchesinvitro[32]onecanassumethattheinvivoeffectwouldmirrorthatfoundintherat.
Vasodilatationwithoutmigraine-relatedpainspeaksagainstthedilationoftheMMAasadirectcauseofthepain.
Aswiththeeffectofothersubstancesonmeningealarteries,thereareconflictingreportsregardingVIP.
Nosignificantdila-tationwasobservedduringinvitroexperimentsusingthemyographymethodontheMMA[43].
UsingthepressurizedarteriographymethodcerebralandMMAwerefoundtorelaxtotheadditionofVIPandPACAP[44,45].
PACAPwasmorepotentthanVIP[44]whilethereversewasseeninthecerebralartery[45].
Whenthevasomotorresponsesfromourexperimentsarecomparedwiththemigrainogenicpropertiesofthetestedsubstances,thereseemstobelittlecorrelationbe-tweentheabilityofasubstancetoinducemigraine-likeattacksandtheirvasoactivepropertiesinhumanmenin-gealarteries.
Sildenafil,PGE2andPACAP-38showednotendencyofdilatationofthearteriesinthecurrentstudy,despitehavingknownmigrainogenicproperties.
Onemaywonderwhyandhowthemigrainogenicsub-stancesinduceheadacheifvasodilatationofmeningealorcerebralarteriesisnotthemechanism.
Endothelialnitricoxide(NO)isnotthekeymoleculesincetheintra-cellularpathwaysactivatedbythemigrainogenicsub-stancesCGRPandPACAP-38arenotdependentofNOinthecranialvessels[20].
Theremainingfactorsaretheperi-vascularandduralnervefibers.
Ithasbeenshownbeforethattheduramatercontainsarichsupplyofsensorysub-stancePandCGRPfibers,andofsympatheticnoradren-alineandneuropeptideY,butonlyaminoramountofparasympatheticVIPandPACAPcontainingfibers[46].
Itistentativetosuggestthatlocalreleaseorsystemicadmin-istrationofthevarioussubstancesmaymodifytheactivityoftheduralnervefibersviaspecificreceptorsormecha-nisms.
Thisclearlydeservesfutureattention.
ConclusionThelackofcorrelationbetweenthevasoactiveandmigrainogenicpropertiesofthetestedsubstancesleadsustotheconclusionthatdirectvasodilatationofintra-cranialmeningealarteriesismostlikelynotthesoletrig-geroftheartificialmigraine-likepainexperiencedbymigrainesufferersuponinfusionofthesesubstancesintothebloodstream.
Thisopinionisbasedonthefactthatseveralmigrainogenicsubstancesinducednosignificantvasodilatationwhenappliedinvitrotohumanmeningealarteries.
Wecanhowevernotruleoutthatthecranialpainandmigrainogenicpropertiesassociatedwiththeinfusionofthesesubstancescouldhavearelationtocra-nialvasodilationinvivo.
Theinvivovasodilatoryresponsestothesubstancescouldbeaffectedbythevasculartone,endothelialinterplayandtheactivationofperivascularnervefibersaroundtheduralarteriesorinthetrigeminalganglion.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsConceivedanddesignedtheexperiments:GG,SL,KAH,AMvdB,LE.
Performedtheexperiments:GG,SL,KAH.
Analyzedthedata:GG,SL,KAH,AMvdB.
Contributedreagents/materials/analysistools:AMvdB,LE.
Wrotethepaper:GG,SL,KAH,AMvdB,LE.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementThetechnicalassistancefromElisabethNilssonisgreatlyappreciated.
FundingDr.
AntoinetteMaassenVanDenBrinkwassupportedbytheNetherlandsOrganizationforScientificResearch(Vidigrant917.
11.
349).
Prof.
LarsEdvinssonwassupportedbytheSwedishReseachCouncil(grantno5958)andaCentreofExcellencegrantfromtheLundbeckfoundation,Denmark.
Authordetails1DepartmentofMedicine,InstituteofClinicalScience,LundUniversity,22184Lund,Sweden.
2DepartmentofInternalMedicine,DivisionofVascularMedicineandPharmacology,ErasmusMCRotterdam,POBox2040,3000CARotterdam,TheNetherlands.
3DepartmentofClinicalExperimentalResearch,CopenhagenUniversityHospital,Glostrup,Denmark.
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