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ReviewAreviewofmethodsforassessmentofcoronarymicrovasculardiseaseinbothclinicalandexperimentalsettingsAxelR.Pries1,2,HelmutHabazettl1,2,GiuseppeAmbrosio Correspondingauthor.Tel/fax:39051347290.E-mailaddress:raffaele.bugiardini@unibo.it;rabugi@netscape.netPublishedonbehalfoftheEuropeanSocietyofCardiology.Allrightsreserved.TheAuthor2008.Forpermissionspleaseemail:journals.permissions@oxfordjournals.org.CardiovascularResearch(2008),165doi:10.1093/cvr/cvn136 CORE Metadata, citation and similar papers at core.ac.uk Provided by RERO DOC Digital Library maybeacauseofvasculardysfunction,themostfrequentcausebeingendothelialdysfunction.Anormallyfunctioningvascularendotheliumisrequiredforappropriatedilatationofarteries.Endothelialcellsproduceseveralmediatorswithvasorelaxing,anti-proliferative,anti-thrombotic,andanti-adherenteffects,suchasnitricoxide,prostacyclin,endothelium-derivedhyperpolarizingfactor,andC-typenatriureticpeptide.Thesefactorsarebalancedbythereleaseofsubstanceswithopposingeffects,suchasendothelin,thromboxaneA,prostaglandinH,andsuperoxideanion.Impairmentofendothelium-dependentdilatationshiftsanetdilatorresponsetoavarietyofstimulitoanetconstrictorresponse.3.CoronarymicrovasculardiseaseindifferentclinicalscenariosTherearethreebroadcategoriesrelatedtopossibleabnorm-alitiesofthecoronarymicrocirculation.Therstisreferredtoastheoccurrenceofischaemicheartdiseaseintheabsenceofangiographicallysignicantcoronaryatherosclerosisandcouldresultfrominammationand/orabnormalvasomotorregulationviaendothelial-dependentandindependentpath-However,itshouldbeconsideredthatanormalornear-normalangiographydoesnotnecessarilyruleoutthepresenceofalargehiddenatheroscleroticburden.Thesecondcategoryreferstoinadequatepost-PCIand/orpost-thrombolysiscoronaryreperfusionentailinganearlyphasereferredtoasmicrovascularobstructioninvolvingmicroembolicmechanisms,andalaterphaseofmicrovasculardysfunction,whichmayrepresentcorollariesofreperfusioninjury,includingtissueoedema,neutrophilaggregation,andfree-radicalrelease.Finally,thethirdcategoryismicrovasculardysfunctioninthecontextofepi-cardialvesseldisease.3.1AcutecoronarysyndromeintheabsenceofsignicantcoronaryatherosclerosisThisphenomenonhasbeenconsideredformanyyearsasanangiographiccuriosity.Newdatasuggestthatthisisnolongerappropriate.Patientswithchestpainandnormalornear-normalcoronaryangiogramsbelongtoagroupinwhichtheprognosisisnotnecessarilyasbenignaspreviouslyespeciallyifimpairedendothelialfunctionisdiag-Recentworkrevealedthatthe1yearmortalityrateofpatientswithacutecoronarysyndrome(ACS)butwithoutobstructivecoronaryarterydiseaseishigh(1.0%),butthesepatientshavearelativelylowerincidenceofdeathcomparedwithpatientswithobstructivecoronaryarterydiseaseinthesameclinicalsetting(3.9%).deathrateinpatientswithchronicstablesymptomsrangesfrom0.6%peryearintheabsenceofobstructivecor-onarylesionsto2.0%inthepresenceofsuchlesions.PatientswithACSare,therefore,usuallyathigherriskthanpatientswithstableeffort-relatedanginaevenintheabsenceofcoronaryobstructions.Thisdemonstratesthatweneedmoreinformationonmechanismsunderlyingmyo-cardialischaemiaandcardiacdamageinpatientspresentingwithanACSandnon-obstructiveepicardialcoronarydisease.Wealsoneedreliablemethodsofriskstraticationofpatientswithnon-obstructivecoronarydisease.3.2ChronicstableanginainpatientswithangiographicallysmoothnormalcoronaryarteriesRecurrentchronicstableanginainpatientswithangiogra-phicallysmoothcoronaryarteriesandanabnormalstresstestresponsehasbeenreferredtoasCardiacSyndrome1,21,22InasubsetofpatientswithSyndromeX,microvas-culardysfunctioncanbedemonstratedandthisentityiscommonlyreferredtoasmicrovascularangina.Microcirculatorydysfunctionhasrecentlybeenassociatedwithheartfailureand/ordiabetesandhypertension.4,5,24Early-stageatherosclerosisisalsoassociatedwithendo-thelialdysfunctionandmaycontributetomicrovasculardys-functioninlessdenedclinicalsubsetsofpatients.23,25,26Finally,microvascularvasomotordysfunctionmayoccurindependentlyofepicardialdiseaseinhearttransplantreci-pients,suggestingdifferententitiesofimmune-mediatedcardiacallograftvasculopathy,whichmayhaveprognosticimportanceforthedeteriorationofleftventricularfunc-Abetterunderstandingofthemechanismsofrecur-rentchronicanginaintheabsenceofcoronarydiseaseatangiographyshouldallowassigninganincreasingfractionofthesepatientstodistinctpathophysiologicalentities,which,inturn,maysupportthedevelopmentofmorespecictreatmentoptions.3.3Persistent/recurrentanginaaftersuccessfulrevascularizationInmanycases,percutaneousorsurgicalrevascularizationresultsinsuperiorsymptomaticreliefofanginaandimprovesexercisetolerancecomparedwithmedicaltherapy,butthebenetsareseldomcomplete.Itiscommonclinicalexperiencetoseethatasubstantialport
ionofpatientsstillcontinuestoexperiencesymptomsundervariouscircumstances.Thelong-termeffectsofPCIincomparisonwithanalternativepathofcontinuedmedicaltreatmenthavebeenreviewedrecently.6,29At5years,26%ofpatientsinthePCIgroupand28%ofthoseinthemedicaltherapygroupshowedpersistingangina.SimilarobservationswerereportedinamultinationalprospectivestudycomparingtherelativebenetsofCABGandPCIinpatientswithmultivesseldiseasepotentiallyamenabletostentimplantation.Althoughseveralmechanismsmaybeconsideredtoexplainthepersistenceofanginaafterarevascu-larizationprocedure(incompleterevascularization,graft/PTCAfailure,anddiseaseprogressioninnativecoronaryarteries),theunexpectedprevalenceofanginaaftersuccess-fulrevascularizationsupportsthehypothesisthatpersistingalterationsofmicrocirculationcontributetothepathogenesisofischaemia.31,323.4NoreowInpatientswithacutemyocardialinfarction,inadequatemyocardialperfusionaftersuccessfulcoronaryrecanaliza-tion(noreow)isassociatedwithadversecardiovascularNoreowmaybeassociatedwithlackofpatencyorwithlossofanatomicintegrityofmicrovessels.Theformerispotentiallyreversible,whereasthelatterisassociatedwithdenitivetissuedamage.Multiplemechanismsmayaccountforthisnding.Necropsystudieshavedemonstratedthepresenceofthrombiinthecoronarymicrovasculaturefrompatientswhodiedofacutemyocardialinfarction. A.R.Priesetal. mechanismsmayaccountforthisnding.Fibrinolysisgener-ateselevatedlevelsoffreethrombin.Thereleaseofvasoactivemediatorsfromactivatedthrombocytesmayimpairregionalow(microvascularspasm)andcontributetotheno-reowphenomenon.Oxygenfreeradicalsgen-eratedsoonafterthereleaseoftheepicardialobstructionmayinactivatenitricoxide,thusimpairingendothelium-mediatedvasodilatation,ordirectlycausemicrovascularFlowmayalsobeimpairedbyadhesionofleuko-cytestomicrovascularendothelium.Inadequatemyocar-dialperfusionaftersuccessfulcoronaryrecanalizationisassociatedwithadversecardiovascularevents.reowmaybeassociatedwithlackofpatencyorwithlossofanatomicintegrityofmicrovessels.Theformerispoten-tiallyreversible,whereasthelatterisassociatedwithdenitivetissuedamage.AlthoughGPIIb/IIIaglycoproteinreceptorinhibitiongenerallyamelioratesdecitsincoron-aryowreserveafteracutemyocardialinfarctiontreatedwithprimaryPCIandimprovesprognosisinthesepatients,apotenttherapeuticstrategytopreventorimprovetheno-reowphenomenonisstillmissing.3.5MicrovasculardysfunctioninthecontextofepicardialvesseldiseaseCoronaryarterystenosisdecreasesowreserveinthevascu-larbeddistallytoastenoticepicardialartery.Evenearlyatheroscleroticlesionsareassociatedwithimpairedendo-thelialcoronarybloodowregulation.Inadditiontotheeffectsofastenosisonowreservewithinitsownbed,cor-onarystenosisandocclusionalsodecreaseowreserveinadjacentnon-stenoticbedsinbothexperimentalanimalmodelsandclinicalpopulations.Importantly,globalcoron-aryowreservepredictscardiovasculareventsinpatientswithonlyminimalorintermediateepicardiallesions.alsopredictsdevelopmentofrestenosisaftercoronarystentimplantation.Bloodow-mediatedshearstressontheendotheliumofepicardialvesselscontrolsthereleaseofnitricoxideandotherfactors,regulatingthevascularmilieuandthereforemayinterferewithprogressionofatherosclerosisorrestenosisdevelopment.Theintegrityofbloodowregulationinthemicrocirculationmaythushavepotentialeffectsonthedevelopmentofepicardial4.ExperimentalassessmentofcoronaryInvestigatingalterationsofmicrocirculatoryfunctionunderavarietyofpathophysiologicalconditionsisaveryactiveareaofresearch,whichhasmuchcontributedtoourunder-standingofmicrocirculation.Manyexperimentalmodelsareavailabletoinvestigatethecoronarymicrocirculation;eachhasitsownpeculiarities,strengths,eldofapplication,andRats,pigs,anddogshavebeenusedmostfrequentlyasmodelsinthestudyofintramyocardialcirculationtovalidateimagingtechniques,toexaminepostmortempathology,toelucidatethepathophysiologyofmicrovasculartone,andtostudytheeffectivenessofpharmacologicalinterventionsinpromotingmicrovasculardilation.Experimentalmodelstostudymicrovasculardiametersand/orperfusionaswellascellularandmolecularmechanismsinclude(i)insitumeasurements;(ii)isolatedheartpreparations;(iii)isolatedvesselapproaches;(iv)culturedcoronarymicrovascularendothelialandsmoothmusclecells.Modelsforintravitalmicroscopyofthebeatingheartinopen-chestanimalshavebeenpublishedmorethan30yearsago.Later,microvas-culardiametermeasurementsbyintravitalmicroscopyincon-junctionwithluminalpressuredeterminationhavebeenperformedtoanalysetheresistancedistributionwithinthecoronarymicrovasculatureatrestandduringdipyridamole-inducedvasodilation.Also,differentlongitudinalgradientsintheresponsivenessofcoronaryarteriolestoadenosineandandinalpha-1and-2receptor-mediatedcor-onaryconstriction,havebeenidentied.Aneedle-probeallo
wedtoidentifydifferencesbetweenresponsesinepicardialvs.endocardialarterioles.InsitumeasurementsPerfusioninvivomaybemeasuredinvasivelywithintracor-onaryDopplerwiresorwithowsensorspositionedaroundthevesselinopen-chestpreparationornon-invasivelywithpositronemissiontomography(PET)andmagneticresonanceimaging(MRI).Manyofthesetechniquescanalsobeappliedtohumansubjectsincontrasttotheuseofradioactiveorcolour-labelledmicrospheres,whichremainsthegoldstan-dardofregionalmyocardialbloodowassessmentintheexperimentalsetting.Thesemethodologieshavebeenemployedtoinvestigateavarietyofissues,suchasthelossofendothelial-dependentrelaxationafterischaemia/reperfusionandtheroleofoxygenradicals,ofno-reowphenomenonandofmyocardialhiber-andtheimpactofcoronaryriskfactorsoncoronaryowreserve.4.2IsolatedheartpreparationsExvivoisolatedheartmeasurementsofmicrovascularper-fusion(rabbit,rat,guineapig,andmouse)allowtoaccu-ratelyinvestigateintrinsicregulationofcoronarybloodowintheabsenceofneuronalorhormonalinuenceswithouttheconfoundingeffectsofchangesinhaemody-namics.Typicaltopicsincludethenitricoxide-dependentregulationofcoronarybloodowandstudiesofthepathwaysofneutrophil/vesselwallandneutrophil/plate-let/vesselwallinteractionsduringpost-ischaemicreperfu-sion.Arrestingisolatedratheartswithtetrodotoxinalsoallowsformicroscopicinvestigationofmicrovasculardiam-eterresponsesatconstantmetabolicdemand.4.3IsolatedvesselapproachesandculturedcoronarymicrovascularcellsAssessmentofdiameter/toneresponsesinarteriolestypicallyisolatedbysurgicalexcisionofsubcutaneoustissueisaveryelegant,althoughtechnicallydemanding,method.Itcanbeemployedtotestthereactivityproleofthemicrocirculationwithrespecttoendothelin,arachidonicacidmetabolites,catecholamines,andothervasoactivesubstances.Thistech-niquehasalsobeenusedinhumanstoexaminetheendo-thelialfunction/dysfunctionoflargeandsmallarteriesessentialhypertension.Recently,coronaryvesselsobtainedfromhumanatrialappendagesremovedduringcar-diopulmonarybypasswerealsostudied.Onestepfurther,culturedcoronarymicrovascularendothelialandsmooth Assessingthecoronarymicrocirculation musclecellsallowtocharacterizevascularionchannels,whichmodulatevascularsmoothmuscleinmicrovessels,andtoelucidatebiochemicalpathwaysbywhichendogenousvasoactivefactorsexerttheirinuence.Regrettably,therearenoanimalmodelstrulymimickingnon-obstructiveanginaorCMD;infact,tosuccessfullydevelopsuchmodels,theunderlyingmechanismsneedtobebetterknown.Atthemoment,usefulinformationcanbegatheredbyinvestigatingclinicalconditionscharacter-izedbyalterationofcoronaryarteryreactivityintheabsenceofovertatherosclerothicstenosis.5.Measuringmyocardialmicrocirculatoryperfusioninhumans5.1Non-invasivescreeningforabnormalvascularfunctioninhumansCommonlyusedtestsforscreeningforabnormalvasomotorregulationaresinglephotonemissioncomputedtomography(SPECT)stressmyocardialperfusionimagingforpatientswhocanexerciseanddipyridamoleoradenosine-inducedvasodilationinconjunctionwithSPECTimagingorechocar-diographyforpatientswhocannotexercise.techniquessufferfromthelimitationthatonlyrelativecom-parisonofperfusioncanbemade,withnoabsoluteesti-matesofbloodow.Additionalmethodsarepromising,includingX-raycomputedtomography(CT),echoDoppler,quantitativecontrastechocardiography,andindicator-basedtechniquessuchasPETandMRITable1).Suchmeasure-mentoftissueperfusion,however,doesnotassessthemicrocirculationindependently,inthatthesemeasuresinterrogatetheowresistanceofboththeepicardialarteryandthemicrocirculation.Inthepresenceofxedordynamic(vasomotortone)epicardialstenoses,microvas-cularowresistancecannotbedirectlyestimated.However,someinsightscanbedrawn.Indeed,ifaninterventionlowerscomputedresistanceinastenoicvascularbedbuthasnoeffectinthesamepatientinanon-stenoticbed,itwouldbereasonabletoconcludethatthedeclineinresist-anceobservedinthestenoticbedreectsachangeinepi-cardialratherthanmicrovascularresistances.Conversely,ifthenon-stenoticvascularbed,butnotthestenosisregion,exhibitsadeclineinresistance,itwouldbelogicaltoconcludethatmicrovascularresistanceinthestenosticzonewasnearminimalandthatepicardialresistancewasunchangedbytheintervention.6.SurrogateindexesforcalculatingcoronarymicrovascularresistanceTherearesomeopportunitiestodevelopsurrogateindexesofmicrovascularowresistanceinthecoronaryarterialtreeonthebasisofinvasivelymeasuredcoronaryowpar-ameters.Theseindexesarebasedoncoronaryowpar-ametersandhamperedbythefactthatmyocardialowhastobemeasuredinvasively.6.1CoronaryandfractionalowreserveMeasuringtheowresponsetothedownstreammicrovascularbedduringtheinfusionofendothelium-dependentandendothelium-independentdilatorsintoacoronaryarteryhasbeenusedtodirectlyassesscoronarymicrovascularfunc-tion(Figure1Areducedcoronaryowreserveinthe
contextofanormalintravascularultrasoundornormalfrac-tionalowreserveindicatesmicrocirculatorydysfunction.Currentdualsensorpressureandowwiresallowrelativelysimplemeasurementsofcoronaryowreserve.techniquemayprovidefurtherinsightintocoronaryperfusionphysiologyviaassessmentoftheowpattern(e.g.diastolicdecelerationtime,systolicowreversal)andallowtocalculateindexesofabsolutecoronarybloodowandmicrocirculatoryresistance(takingintoaccountsystemichaemodynamicsandvesselsize,derivedbyquantitativecor-onaryangiographyorintracoronaryultrasound).6.2IndexofmicrovascularresistanceDistalcoronarypressuremultipliedbythehyperaemicmeantransittime(whichisinverselycorrelatedtoabsoluteow,asmeasuredsimultaneouslywiththecoronarypressurewire)maybeusedtoroughlyestimatemicrovascularresist-ance(indexofmicrovascularresistance).Importantly,thepressuredropisafunctionofthesquareofowacrossacoronarylesionandthusisgeometricallynotproportion-allyrelated.Changesinowresistanceofthevascularbeddistaltothetipofthecathetercanbedetected.However,allresistancecalculationsbasedonthistechniqueignoreveryimportantphysiologicalissuessuchasthetruebackpressureinthecoronarycirculationandcoronarycapacitanceandsomaynotalwaysprovideanaccurateesti-mateoftheparameterofinterest,namelymicrovasculartone.Inaddition,preliminarydatashowthatbloodowvaluescannotbereadilycomparedamongdifferentpatients Table1TechniquesforcardiacassessmentofmicrovascularfunctionMethodQuanticationTracerSpatialRecordingNon-invasiveSPECTNoneRadioisotopesVerylowLongPETPerfusion(mL/min/g)goldRadioisotopes(cyclotron-generated)LowLongCTPerfusion(mL/min/g)ContrastagentVeryhighLowMRIPerfusion(mL/min/g)ContrastagentModerateModerateUltrasoundPerfusion(mL/min/g)MicrobubblesHighRealtimeInvasiveDopplerwireFlowvelocity(mm/s)NoneSelectiveassessmentintargetvesselterritoryThermo-dilutionBloodow(mL/min)Saline(bodytemperature)CTFCNoneContrastagent A.R.Priesetal. becausetheabsoluteresistancevariesdependinguponthetissuevolumesuppliedbytherespectiveartery.6.3ThrombolysisinMyocardialInfarctionFrameCountTheCorrectedThrombolysisInMyocardialInfarction(TIMI)FrameCount(CTFC)providesasimpleindexofcoronaryowandmyocardialperfusion.CTFCisanestablishedmethodtoprovideasemiquantitativecategorizationofepicardialbloodow,withtheimplicitassumptionthatslowowintheabsenceofsignicantstenosiswouldreectimpairmentofthemyocardialmicrocirculatoryperfusion.CTFChasbeenusedasmeasureofmicrovascularintegrityandseemstopredictoutcome.ChangesinCTFCnotexplainedbyconcur-rentchangesinepicardialminimallumendiameter(MLD)maybeattributedtotheresistancetoowatthelevelofcor-onarymicrocirculation.TheconceptoftheCTFC/MLDratiomaythusbeintroducedasacompositemeasureofmicrovascu-larstructureandfunction.Yetthevalueofthistechnique,too,needstobefullyelucidated.MeasurementsofbloodowusingtheTIMIframecountmethodologyrequireserialreproductioninpatientsnotundergoingPCIorthrombolysisasacontrolgroup.Thesedataareatpresentnotavailable.7.PeripheralindexesOnealternativeapproachtoinvasivecardiacproceduresenablingtheassessmentofalterationsinmicrovascularfunctionandstructureisbasedontheassumptionthatmicrovascularalterationsinperipheraltissuesmightreectcorrespondingalterationsintheheart.Thishasbeenconrmedforendothelialdysfunctioninpatientswithsystemicriskfactorsforatherosclerosissuchasdia-betes,hypercholesterolaemia,orhypertensionandforpatientswithgeneticalterationsofmicrovascularrespon-However,onlyfewstudieshavesystematicallyassessedadirectcorrelationbetweenperipheralandcoron-aryvasodilatorcapacity,mostofthemlimitedbycomparingdifferententitiesintheperipheralandcoronaryvasculaturewithrespecttovesselsizeandagonisttested.Itwasshownthattheperipheralperfusionresponsetotransientforearmischaemia(seealsowhatfollowsfordetails)doesnotcorre-latewithdipyridamole-inducedmyocardialhyperaemia.Thismightindicatedifferentmechanismsofmicrovascularactivationorregulationandadvocateadequatecareinrespectiveextrapolations.However,peripheraltechniquesassummarizedinwhatfollows(Table2)arelessinvasiveand/orlessexpensiveandtimeconsumingcomparedwithcoronarycatheterizationornon-invasivescreeningstrat-egiesandmayallowamoredirectassessmentofinvolvedmicrovascularmechanisms.7.1Brachialarterypost-ischaemicreowBrachialarterydilationinresponsetodifferentstimulicanbemeasuredwithhigh-resolutionultrasoundscanners.Totestendothelium-dependentdilatation,owchangesareinducedbyforearmischaemiafollowedbypost-ischaemichyperaemicreow.Insubjectswithintactendothelialfunc-tion,thistypicallyresultsinabrachialarterydiameterincreaseof10%,anditsexactquanticationrequirescon-siderabletechnicalskillandexperience.Dataobtainedarethencomparedwiththediameterresponseaftersublin-gualapplicationofglyceryltrinitrate,anendothelium-inde-pende
ntdilator.Thistechniqueisnon-invasive,butitassessesendothelialfunctioninalargeconduitarteryandnotinmicrovessels.Nevertheless,theresultsseemtocorre-latewithresponsesinskinmicrocirculation.7.2VenousocclusionplethysmographyInordertoassessmicrovascularendothelialfunctionbyvenousocclusionplethysmography,thebrachialarteryis Figure1Set-upofintracoronaryDopplermeasurementsofthecoronarymicrocirculation.Bloodowvelocity(APV,averagepeakvelocity)aswellasowpat-terns(e.g.DT,diastolicdecelerationtimeorsystolicowreversal)maybeassessed.Volumentricbloodowmaybecalculatedwithadditionalassessmentofcoronarylumenarea,approximatedfromquantitativecoronaryangiography.Vasoreactivityofthecoronarymicrocirculationcanbeexpressedaschangeofcoronarybloodowinresponsetodifferentstimuli(e.g.acetylcholineforendothelialstimulationoradenosineformaximalvasodilatorcapacity).Assumingconstantepicardialvesselsize,coronaryowreservemaybeexpressedastheratiobetweenmaximal(adenosine-induced)andbasalAPV(modied,witpermissionfromUrban&Vogel,Munich,fromSchachingerV,ZeheirAM.Coronarymicrocirculation.Pathophysiology,clinicalrelevance,andimportanceforregenerativetherapyaftermyocardialinfarction. Assessingthecoronarymicrocirculation cannulatedfortheinfusionofendothelium-dependent(e.g.acetylcholine)and-independent(e.g.Na-nitroprusside)vasodilators,renderingthistechniquemoderatelyinvasive.Ithasbeensuccessfullyusedtodemonstratethebenecialeffectsofstatinsorathirdgenerationonendo-thelialfunctioninpatientswithhypercholesterolaemiaandhypertension,respectively.Moreover,vasodilatorcapacityoftheperipheralmicrocirculationassessedbyvenousocclu-sionplethysmographypredictscardiovasculareventsinpatientswithstablecoronaryarterydiseaseorheartaswellasinACS,withthosepatientsathighestlong-termriskinwhomACS-associatedimpairmentofthemicrocir-culationdoesnotrecoverwithinthefollowingweeks.Recently,ngerplethysmographyhasbeenintroduced,whichprovidesfurtherinformationonpulsatileowpatternsandallowstheassessmentofnon-invasivestimuli(reactive7.3LaserDoppleruxmetryLaserDoppleruxmetry(LDF)isbecomingincreasinglypopularfortheestimationofskinmicrovascularbloodow.Thesurfaceinvestigatedisilluminatedwithalaserspot.Thefrequencyofthelaserlightisalteredbymultiplereectionsatowingredbloodcells.Theresultingfre-quencyshiftinlightreectedfromthetissueisproportionaltotheproductofredcellnumberandowvelocity(equaltoaux)inthesampleregion,whichisusuallyahalfspherewithadiameterof1mm.Sincethelocalperfusionintheskinisveryheterogeneous,itishelpfultoassesslargerareasofthetissuesurfacebytwo-dimensionalscan-ningLDFimaging.AdditionalinformationonmicrovascularfunctionmaybeobtainedbyspectralanalysisofthelaserDoppleruxtimeseriesusingwavelettransforms.Oscil-lationsinskinperfusionwithfrequenciesaround0.1and0.01Hzareconsideredtoreectmyogenicandendothelialactivities,respectively.Incombinationwithlocaldrugapplicationbyiontophoresis,theLDFtechniqueallowsfornon-invasivestudyofmicrovas-cularresponsestoavarietyofpharmacologicalstimuli.Ion-tophoresisreliesondrugmoleculescarryingapositiveornegativechargewhichmigrateacrosstheskinwhenarespectivepotentialisapplied,indirectproportiontothecurrentapplied.Thusminutequantitiesofavasoactivedrugcanbeadministeredtothemicrovasculatureunderinvesti-gationinacontrolledfashion,withoutcausingsystemiceffects.AcetylcholineandNa-nitroprussidearetypicalsub-stancestoassessendothelium-dependentand-independentvasodilatorresponses.Incontrasttothetechniquesdis-cussedearlier,thismethodalsoallowsfortheapplicationofvasoconstrictors.Alpha-adrenergicvasoregulationhasbeenanalysedusingiontophoreticdeliveryoftyramine,phentola-mine,bretylium,andnorepinephrine.Interestingly,ionto-phoreticallydeliveredurotensinIImayinducevasodilationintheskinmicrovasculatureofhealthycontrolsubjectsbutcon-strictioninpatientswithchronicheartfailure.Thus,iontophoresisinconjunctionwithlaserDopplerowmetryorimagingseemsmostattractivefordetailedstudyofperipheralmicrovascularfunctionbecauseitisnotonlycompletelynon-invasivebutalsoallowsfortheassessmentofendothelium-dependentand-independentvasodilatorreserveandofvasoconstrictorsensitivity. Table2TechniquesforperipheralassessmentofmicrovascularfunctionandstructureMethodTargetvesselsStimulusCommentsNon-invasiveBrachialarterypost-ischaemicConduitarteryShearstressValidatedtopredictclinicalLaserDopplerux(imaging)/iontophoresisCutaneousmicrovesselsVasoactivedrugsIontophoresis:localapplicationofabroadselectionofdrugs;currentmayhavedirecteffectonlocalmicrovasculartoneClinicalintravitalmicroscopyCutaneous,mucosal,orretinalmicrovesselsNoneInformationonmicrovascularstructureandfunction;limitedrangeoftissuesandparameterstobeanalysedInvasiveVenousocclusionplethysmography(forearmbloodow)AllforearmmicrovesselsV
asoactivedrugsValidatedtopredictclinicaloutcome;problem:systemicrecirculationofdrugsBiopsySubcutaneous/muscularmicrovesselsNoneAnalysisofmicrovascularstructure;nofunctionalinformation Figure2LaserDoppleruxtracingsofskinperfusioninapatientwithPar-vovirusB19-inducedcardiomyopathy(B19)andahealthysubject(Control)during5minofrestand10minofiontophoreticapplicationofacetylcholine(Ach)andNa-nitroprusside(NP)at0.02mA. A.R.Priesetal. Figure2showsanexampleoflaserDoppleruxtracingsofskinperfusioninapatientwithParvovirusB19-inducedcardiomyopathy.7.4ClinicalintravitalmicroscopyInadditiontofunctionaldecits,apparentmicrovasculardysfunctionmayalsobeduetostructuralalterationsofthemicrovasculaturesuchasrarefactionand/orremodel-Evaluationofmicrovasculardensityrequiresdirectvisualizationofmicrovascularnetworksbyclinicalintravitalmicroscopy,whichispossibleonlyinselecttissues.Capillaroscopyofthenailfoldororaltissues(gingi-val,sublingual,orbuccalmucosa)allowstostudycapillarymorphology,capillarydensity,andcapillarybloodvel-ocity.Theintroductionofnewclinicalintravitalmicroscopes(e.g.orthogonalpolarizationspectralimaging)hasimprovedimagequalityandeaseofapplicationandrendersadditionaltissuessuchassublingualorbuccalmucosaaccessible.Specicimageanalysisapproachesadditionallyallowthemeasurementofarteriolarorvenularbloodowvelocityandpulsatility.Arteriolarmorphologycanalsobeanalysedbyretino-graphy.Focalorgeneralizedluminalnarrowing,aswellasarteriovenousnicking,i.e.theindentationanddisplace-mentofvenulesbycrossingarteriolesowingtostructuralchangesinthearteriolarwall,hasbeenassociatedwithmetabolicsyndrome,coronaryheartandstroke.Themajordisadvantageofusingretinographyfortheanalysisofarteriolarremodellingisthequalitativenatureoftheresults.Acommonproblemofallintravitalmicroscopyapproachesliesinthedifcultyofextractingquantitativedatafromtheimages,whichusuallyrequirestimeconsumingoff-lineanalysisofdigitizedvideosequences.7.5SubcutaneousbiopsyInordertoobtainquantitativedataonarteriolarwallthick-nesstolumenratio,histologicalanalysisoftissuefrombiopsiesisindispensable.Incontrasttocardiactissue,sub-cutaneousfatorglutealmusclespecimenscanbecollectedinsufcientquantitieswithlittlediscomforttotheTosupporttheassumptionthatmicrovascularalterationsoccurnotonlyintheheartorthebrainbutinalltissuesthroughoutthebody,resultsobtainedinthesespecimenscanbecomparedwithretinographyndingsandwithfunctionaldataobtainedinthecoronaryandinperiph-eralcirculations.Thesamespecimensmayalsobeusedtoinvestigatethemechanismsresponsiblefortheobservedmorphologicalalterations.8.PerspectivesAlthoughmanystudieshavesupportedtheroleofCMDinexplainingchestpainsymptomsinavarietyofclinicalsettings,convincingevidenceofacausalrelationshipbetweenmyocardialischaemiaandCMDandofitspathophy-siologicalmechanismsissparse.Wethereforestronglyemphasizethatinvestigationsdesignedtoelucidatetheregulationofthecoronarymicrocirculationinhealthanddiseaseshouldincorporatethebesttechnologicaladvancesinmolecularbiology,biochemistry,imaging,andphysiologi-calmeasurements.Futureresearchneedstoencompassbothbasicandclini-calinvestigationsandtocompareperipheralinvestigationsofmicrocirculationwithparallelmeasurementsintheheart.Developmentandtestingofnewandimproveddiag-nosticproceduresforCMDareneededaswell.Currently,thecoronarymicrocirculationmovesintothefocusofnewtherapeuticstrategiesofcardiacdiseasedrivenbyrecentinsightsinto,e.g.endothelialeffectsofdrugssuchasstatinsorACE-inhibitors,ortherestorativefunctionofendothelialprogenitorcellsandhaematopoieticstem/progenitorcells.Establishedandexperimentalthera-piesforthepreventionandtreatmentofmicrovasculardys-functionshouldbetestedinanimalandcell-basedmodels.Inaddition,clinicallyorientedinvestigationswillbeindis-pensabletounderstandtheroleofthecoronarymicrocircu-lationincardiacdiseaseandtogainmorepreciseinsightsintotherelationshipsbetweencoronarymicrovasculardys-function,induciblemyocardialischaemia,andadverseoutcome.Areassuchasgeneticpredispositiontomicrovas-culardiseaseinresponsetohypertensionand/orothercar-diovascularriskfactorsalsodeserveemphasis.9.ConclusionsTheauthorsperceivethatthereisanimpressivelackofclinicalandpathophysiologicalinformationontheroleofCMDincardiacdisease.Thisisinpartduetothegapbetweencurrentexperimentalstudiesofthecoronarymicrocirculationandclinicalinvestigationsinthisarea.Increasedcollaborationbetweenbasicscienceandclinical-orientedresearchersshouldhelpbridgingthisgapandhelpdevelopimprovedexperimentalandclinicalapproachestoassessandtreatCMD.Conictofinterest:nonedeclared.Funding1.KaskiJC.Pathophysiologyandmanagementofpatientswithchestpainandnormalcoronaryarteriograms(cardiacsyndromeX).Circulation2.BugiardiniR,BaireyMerzCN.Anginawithnormalcoronaryarteries:achang
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