In vivo MR-angiography_JANSEN_Published5June2017_GOLD VoR

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Jansen, C. H. P., Reimann, C., Brangsch, J., Botnar, R. M., & Makowski, M. R. (2017). In vivo MR-angiography
for the a ssessment of aortic aneurysms in an experimental mouse model on a clinical MRI scanner: Comparison
with high-frequency ultrasound and histology. PLoS ONE, 12(6), e0178682.
https://doi.org/10.1371/journal.pone.0178682

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RESEARCHARTICLE
In vivo MR-angiography for the assessment of
aortic aneurysms in an experimental mouse
model on a clinical MRI scanner: Comparison
with high-frequency ultrasound and histology
ChristianH.P.Jansen1☯,CarolinReimann2☯*,JuliaBrangsch2,Rene´M.Botnar1,3,4,5,6,
MarcusR.Makowski1,2,3
a1111111111 1 King’sCollegeLondon,DivisionofImagingSciencesandBiomedicalEngineering,London,United
Kingdom,2 DepartmentofRadiology,Charite,Berlin,Germany,3 BHFCentreofExcellence,King’sCollege
a1111111111
London,London,UnitedKingdom,4 WellcomeTrustandEPSRCMedicalEngineeringCenter,King’s
a1111111111
CollegeLondon,London,UnitedKingdom,5 NIHRBiomedicalResearchCentre,King’sCollegeLondon,
a1111111111
London,UnitedKingdom,6 SchoolofEngineering,PontificiaUniversidadCatolicadeChile,Santiago,Chile
a1111111111
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Citation:JansenCHP,ReimannC,BrangschJ,
BotnarRM,MakowskiMR(2017)InvivoMR-
angiographyfortheassessmentofaortic Background
aneurysmsinanexperimentalmousemodelona
MR-angiographycurrentlyrepresentsoneoftheclinicalreference-standardsfortheassess-
clinicalMRIscanner:Comparisonwithhigh-
mentofaortic-dimensions.Forexperimentalresearchinmice,dedicatedpreclinicalhigh-
frequencyultrasoundandhistology.PLoSONE12
(6):e0178682.https://doi.org/10.1371/journal. fieldMRIscannersareusedinmoststudies.ThistypeofMRIscannerisnotavailablein
pone.0178682 mostinstitutions.TheaimofthisstudywastoevaluatethepotentialofMR-angiographyper-
Editor:DavidePacini,UniversitadegliStudidi formedonaclinicalMRscannerfortheassessmentofaorticaneurysmsinanexperimental
Bologna,ITALY mousemodel,comparedtoapreclinicalhigh-resolutionultrasoundimagingsystemand
Received:January20,2017 histopathology.
Accepted:May17,2017
Methods
Published:June5,2017
AllinvivoMRimagingwasperformedwithaclinical3TMRIsystem(PhilipsAchieva)
Copyright:©2017Jansenetal.Thisisanopen
equippedwithaclinicalgradientsystemincombinationwithasingle-loopsurface-coil(47
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which mm).AllMRsequenceswerebasedonclinicallyusedsequences.Forultrasound,adedi-
permitsunrestricteduse,distribution,and catedpreclinicalhigh-resolutionsystem(30MHzlineartransducer,Vevo770,VisualSonics)
reproductioninanymedium,providedtheoriginal
wasused.AllimagingwasperformedwithanApoEknockoutmouse-modelforaorticaneu-
authorandsourcearecredited.
rysms.Histopathologywasperformedasreference-standardatallstagesofaneurysm
DataAvailabilityStatement:Allrelevantdataare
development.
withinthepaper.
Funding:ThestudywasfundedbytheBritish
Results
HeartFoundationwww.bhf.org.uk(PG/09/061)and
theDeutscheForschungsgemeinschaft[http:// MR-angiographyonaclinical3Tsystemenabledtheclearvisualizationoftheaorticlumen
www.dfg.de/](MA5943/3-1/andMA5943/4-1/9-
andaneurysmaldilationatdifferentstagesofaneurysmdevelopment.Aclosecorrelation
1).Thefundershadnoroleinstudydesign,data
(R2=0.98;p<0.001)withhistologicalareameasurementswasfound.Additionally,agood
collectionandanalysis,decisiontopublish,or
preparationofthemanuscript.Otherwise,thereare agreementbetweenMRandultrasoundareameasurementsinsystole(R2=0.91;p<
PLOSONE|https://doi.org/10.1371/journal.pone.0178682 June5,2017 1/14
InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
nofinancialorotherrelationsthatcouldleadtoa 0.001)anddiastole(R2=0.94;p<0.001)weremeasured.Regardinginterobserverrepro-
conflictofinterest. ducibility,MRImeasurementsyieldedasmaller95%confidenceintervalandacloserinter-
Competinginterests:Theauthorshavedeclared readercorrelationcomparedtoultrasoundmeasurements(-0.37–0.46;R2=0.97vs.-0.78–
thatnocompetinginterestsexist. 0.88;R2=0.87).
Conclusion
ThisstudydemonstratesthatMR-angiography,performedonaclinical3TMRscanner,
enablesthereliabledetectionandquantificationoftheaorticdilatationatdifferentstagesof
aneurysmdevelopmentinanexperimentalmousemodel.
Introduction
Cardiovasculardiseases,includingaorticaneurysms,currentlyrepresentthemaincauseof
deathinWesternsocieties.Especiallytheincidenceofabdominalaorticaneurysms(AAAs)is
steadilyincreasing,especiallyinthelast20years[1,2].Oneofthemainfactorsforthisincrease
inincidenceistheprogressiveagingofthegeneralpopulation.Currentlytheincidenceof
abdominalaorticaneurysmsisestimatedtobearound5%inthegeneralpopulationolderthan
50years[3,4].Thedevelopmentofabdominalaorticaneurysmsisassociatedwithdifferent
causes,whichincludeaorticinfection,disordersofconnectivetissuesandtraumaticevents[5,
6].However,inmostcases,theexactinitiatingeventandpathophysiology,underlyingthe
developmentisnotfullyunderstoodyet[7].Inmostcases,abdominalaorticaneurysmsare
associatedwithaprogressivedilationoftheaorticlumen.Ifthisprocesscontinues,abdominal
aorticrupturewithfatalconsequencescanbetheresult[8].
Inclinicalpractice,thescreeningforandtheevaluationofabdominalaorticaneurysmscan
beperformedusingdifferentimagingmodalities[9].Theseimagingmodalitiesincludemagnetic
resonanceimaging(MRI),computedtomography(CT)andultrasound(US).Eachimaging
techniqueisassociatedwithspecificadvantagesanddisadvantages.MRIisuniqueinasensethat
itenablesthehigh-resolution3Dvisualizationoftheaortawithouttheneedforcontrastagentor
ionizingradiation.ThemaindisadvantageofMRIistherelativelylongscantime,comparedto
e.g.CT.ThemainadvantageofCTisthatimagingcanbeperformedwitharelativelyhighspatial
resolutioninashorttime,howeverCTangiographyisdependentontheuseofiodinatedcon-
trastagents.Ultrasoundhastheadvantage,thatitisawidelydistributedimagingtechniqueavail-
ableinmostclinicalcenters.Oneofitsmaindisadvantagesistheoperatordependence,whichis
especiallyrelevantinthecontextoffollowupexaminations.
Inmagneticresonanceimaging,differenttechniquescanbeusedforthevisualizationof
vessels[9].AsMRcontrastagentshavebeenrecentlylinkedtosideeffectssuchasnephrogenic
systemicfibrosis(NSF),non-contrastenhancedtechniquesaregaininginpopularity[10].In
anexperimentalsettingwithsmallanimals,suchasmice,non-contrastenhancedtechniques
haveseveraladvantages.Themainadvantageisthatimagingcanberepeatedlongitudinallyat
limitlesstimepointsinasingleanimalwithouttheneedforintervention.
Experimentalmousemodelsarethemostwidelyusedanimalmodelsforthepreclinical
investigationofdiseases[11].Thesemodelsenableresearcherstoinvestigatethedevelopment
ofdiseasesandtheinfluenceofgeneticmodulationorpharmacologicaltherapiesondisease
development.Additionally,mostnoveldrugsareinitiallytestedandvalidatedinanimalmod-
elspriortoclinicaltrials.Formostpreclinicalstudies,histologicalanalysisisperformedto
evaluateandquantifyinvivochanges.However,noninvasiveimagingtechniquesaregaining
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InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
inimportance.Themainadvantageofsuchanapproachisthatthenumberofrequiredani-
malsforastudyisdramaticallyreduced.
Inthiscontextmorphologicalaswellasmolecularimagingmethods,includingPET(posi-
tronemissiontomography),SPECT(single-photonemissioncomputedtomography),MRI,
CTandultrasoundarethemostfrequentlyusedtechniques.Inthisgroupofmodalities,MRI
hasseveraladvantagesincludingauniquesofttissuecontrastincombinationwiththe3D
acquisitionofmorphologyandfunction[12,13].ThemajorityofMRIstudiesinmiceareper-
formedwithdedicatedpreclinicalscannerswithanultra-highfieldstrength(4.7–16.4Tesla).
Manyinstitutionsdonothavethesekindofpreclinicalimagingsystemsavailableasitusually
requiresdedicatedpersonnel,includingMRphysicists,torunandmaintainthesesystems.
Advantagesinhardwaredevelopmentandsequencedesignhavemadeitpossibletoperform
smallanimalimaginginclinicalMRIscanners.
Inthisstudy,weevaluatedthepotentialofawidelyavailableclinical3TtheMRIsystemfor
theassessmentofaorticaneurysmdevelopmentinamousemodel.Weusedthemostfre-
quentlyinvestigatedandbestvalidatedmousemodel,whichisbasedontheApoE-/-mousein
combinationwithangiotensinIIinfusion[14–17].Suchamousemodelishighlyrelevantas
abdominalaorticaneurysmsrepresentacardiovasculardiseasewithseverecomplications.
Manyaspectsofthisexperimentalmodelarecomparabletohumandiseaseincludingan
increasedincidenceofhyperlipidemia[14].Besideshyperlipidemia,otherfactorssuchas
hypertensionandcysticnecrosisoftheaorticwallalsoplayanimportantroleduringthedevel-
opmentofaorticaneurysms.Especiallyinthecontextofthoracicaorticaneurysms,apotential
associationwithcysticmedialnecrosishasbeendescribedbypreviousstudies[18,19].
Theaimofthisstudywastotestthepotentialandreliabilityofaclinical3TMRIsystemfor
theperformanceofMR-angiographyinamousemodelofaorticaneurysm,comparedtoa
dedicatedpreclinicalhighresolutionultrasoundimagingsystem.Histologicalanalysiswas
usedasreferencestandard.
Methods
Setupofanimalexperiments
ThisstudywascarriedoutinstrictaccordancewiththerecommendationsintheGuideforthe
CareandUseofLaboratoryAnimalsoftheUnitedKingdomHomeOfficeandisregulated
undertheAnimalsScientificProceduresAct1986(ASPA).ASPAhasrecentlybeenrevisedto
transposetotheEuropeanDirective2010/63/EUontheprotectionofanimalsusedforscien-
tificpurposes.TheprotocolwasapprovedbytheCommitteeontheEthicsofAnimalExperi-
mentsoftheKing’sCollegeLondon.Allanimalexperimentsinthisstudywereperformedin
accordancewiththeseinternationalregulations.Allinterventionwasperformedwithacombi-
nationanesthesia(Medetomidin,Midazolam,Fentanyl),andalleffortsweremadetominimize
suffering.Osmoticminipumps(Alzetmodel2004,DurectCorporation,Cupertino,CA,USA)
wereimplantedintoeightweeksoldmice.Forthisstudy,homozygouseightweeksoldC57BL/
6JApoE-knockoutmice(male)fromtheCharlesRiversLaboratorieswereused.Theanimals
werefedwithastandardlabdietandhousedinacleanbarrier.Theminipumpswereloaded
(loadingwasperformedassuggestedbythemanufacturer)exvivowithAngII(Sigma-Aldrich,
SaintLouis,MO,USA,),implantedsubcutaneouslyinthedorsalregionunderacombination
anesthesia(500μg/kgMedetomidin,50μg/kgFentanyl,5mg/kgMidazolam)andinfuseda
continuouslydoseof1microgramkg-1min-1intothemice[15,20].Atweekone,two,three
andfourafterAngIIinfusionMRIimagingwasperformedandvesselswereharvestedforhis-
tologicalanalysiseachweek(n=8pergroup).Asham-operatedgroup(controlgroup,n=6)
werealsoimplantedminipumpswhichinfusedsalineforfourweeks.Eightmicewerescanned
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InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
byMRIateachtimepoint.allanimalsweresacrificedforfurtherhistopathologicalanalysis
afterthefinalimagingsessions.Fortheimagingsession,micewereanesthetizedwithanintra-
muscularapplication[21,22]ofthesamecombinationofMedetomidin,Fentanyl,Midazolam
asmentionedabove.Inallmicewithabdominalaorticaneurysm,anexsanguinationinante-
riorperfusionwithphosphatebufferedsaline(100mmHg)wasperformedfollowingtheMR
imagingsession.Thiswasfollowedbyaperfusionwith10%formalinifvesselsampleswere
usedforhistology.Aorta,rightrenalarteryandthelastpairofintercostalarterieswasexcised
toallowanatomicalmatchingduringhistopathologicalprocessingofthesamples.
Animalhandlingandinvivomagneticresonanceimaging
Eachanimalwasinanesthesia(asdescribedearlier)andplacedinapronepositiononasurface
microscopycoil(Philipshealthcare,Best,theNetherlands).Allimagingwasperformedusinga
clinical3TAchievaMRsystem(PhilipsHealthcare,Best,TheNetherlands).Agradientsystem
withagradientstrengthof30mT/mandaslewrateof200T/m/swasused.Forthesequence
acquisition,adedicatedsoftwarepackageforcardiacimagingwasavailable.Thesignalwas
gainedusingamicroscopysingleloopcoilwithaninnerdiameterof47mm.Thecoilwas
placedinthemagneticcenteroftheboreoftheMRI.AnMRcompatiblebodytemperature
monitoringandheatingsystemwasusedtomaintainthetemperature(37ºdegreesCelsius)of
allanimalsduringtheentireacquisitionoftheMRdatasets(Model1025,SAInstruments
Inc.,StonyBrook,NY).AllMRimagingsequenceswerebasedonclinicalMRsequences.The
imagingprotocolincludedthefollowingsequences.AtthebeginningofaMRimagingproto-
colalow-resolutionscoutsequence(three-dimensionalgradientechosequence)wasusedto
forananatomicaloverviewandlocalizationoftheabdominalaorta.Thescoutscanwasper-
formedinthecoronalandtransverseorientationusingthefollowingparameters:field-of-view
(FOV)=200mm,matrix=320,slicethickness=2mm,TR/TE=20/5.8ms,flipangle=30˚
andslices=9.Followingatransverseorientationofatwo-dimensionaltime-of-flightangiog-
raphy(2DTOF)wasexecutedforaprecisevisualizationoftheabdominalaorta.The2Dtime-
of-flightsequencewasplannedtoincludetherenalarteriesasanatomicallandmarksinall
scans.Theimageparametersincluded:Slicethickness=0.5mm,inplanespatialresolution=0.3
x0.3mm(reconstructed0.13x0.13mm),imagingmatrix=160x160,fieldofview=20x20x
10mm,flipangle=60˚,echotime(TE)7.7msandrepetitiontime(TR)sequence=37ms.
Fold-oversuppressionwasactivated.Fold-overdirectionwasrighttoleft.Acartesianacquisi-
tionmodewasused.Amaximumintensityprojection(MIP)ina360-degreereconstruction
wasautomaticallyreconstructedbasedonthetime-of-flightangiography(Fig1).
Magneticresonanceimageanalysis
Signaltonoisemeasurements(SNR)wereperformeddirectlyproximaltotherightrenalartery
inthecontrolgroup(shamgroup,Fig1).TherightrenalarterywasclearlyvisibleinallMR
scans.Ifananeurysmwaspresent,signaltonoisemeasurements(SNR)wereperformedatthe
locationofthemaximalareasize(Fig2).MRimageanalysisofDICOMimageswasperformed
usingtheopensourceversionofOsiriX(version7.1,OsiriXfoundation).Timeofflight(TOF)
imageswereusedtolocalizeaorticaneurysms.Regionofinterests(ROIs)weremeasuredas
areasofsignalenhancementonTOFimagesfortheevaluationofsignalintensity.Regionof
interestsweredrawntodelineatethecompletevascularlumenforareproduciblemeasure-
ment.FortheROIssignaltonoiseratio(SNR)wascalculatedwithfollowingformula:Signal
tonoiseratioMR-angiography(MRA)=((aneurysmal)aorticlumensignal)/(standarddevia-
tionlumensignal).Suchanapproachwaschosentoallowforcomparabilitybetweentheanal-
ysismethodsforMRandultrasound.
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InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
Fig1.VisualizationoftheabdominalaortainacontrolmousebyMR-angiographyonaclinicalMR
systemincomparisontohigh-frequencyultrasound.Imagesdemonstratethevisualizationofthe
abdominalaortainasham-operatedApoE-/-mousebyinvivoMR-angiographyonaclinicalMRsystem(A)
anddedicatedhigh-frequencyultrasound(C)incomparisontohistology(B).ThereconstructedTOF
angiogram(A1,maximumintensityprojection(MIP))ofthesuprarenalpartofthenondilatedabdominalaorta
isshown.RedlinesindicatetheorientationofsubsequentlyperformedtransverseMRIsequences(A2,A3,
A4).Correspondingexvivohistologicalsections(B1-B6),ElasticavanGieson(EvG)stain(B1,B2,B3),
hematoxylineosin(HE)stain(B4,B5,B6)demonstrateanondilatedabdominalaortaatdifferentlevels(red
lines).High-frequencyultrasoundimages(C)ofanabdominalaortausingadedicatedimagingsystem(Vevo
770).Correspondinglongitudinal(C4)andtransversalimagingplanes(C1,C2,C3)areshown.
https://doi.org/10.1371/journal.pone.0178682.g001
Invivohigh-frequencyultrasoundimaging
Highresolutionultrasoundimagingwasperformedwithanimalsplacedonaautomatically
heatedtable(37˚)insupineposition.Priortoultrasoundimaging,allanimalsweredepilated
withhairremovalcream.Forultrasoundmeasurements,aVevo770ultrasoundimagingsys-
tem(VisualSonics,Toronto,Canada)witha30MHzlinearsignaltransducerwasused.For
Fig2.EvaluationoftheabdominalaortainanApoE-/-mousebyMR-angiographyonaclinicalMR
systemandhigh-frequencyultrasound.EvaluationofanabdominalaorticaneurysminanApoE-/-mouse
byinvivoMRIandultrasound4weekaftercontinuousinfusionofangiotensinII(4-weekgroup).Themaximum
intensityprojection(MIP)ofthetime-of-flight(TOF)angiogram(A1)demonstratesasignificantlydilatedaortic
lumen.Thelocationoftransverseslices(A2,A3)aredepictedbytheredlinesinA1.Correspondingexvivo
histologicalsections(ElasticavanGieson(EvG)stain(B1,B2),hematoxylineosin(HE)stain(B3,B4)confirm
thedilationoftheaorticlumen.MagnificationsofB2andB4highlightthesiteofruptureoftheelasticlaminaein
thetunicamediaoftheaortainEvGstainandHEstain.Correspondingultrasoundimagesofabdominalaorta
usingthededicatedhigh-frequencyUSimagingsystem(Vevo770)insagittal(C3)andtransversalorientation
(C1,C2).
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InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
imagingandanatomicalcolocalizationthesuprarenalabdominalaortamorethan20dynamic
2D-transverseandsagittalimageswereacquiredinallanimals.Cinetransversalandsagittal
imageswerereconstructedbytheECG-basedkilohertzvisualization(EKV)technique.The
resultingimageswereanalyzedduringend-diastoleandduringend-systole.
Analysisofinvivohigh-frequencyultrasoundimaging
Signaltonoisemeasurements(SNR)wereperformedinthesameanimalatthesamelocationas
MR-angiographymeasurements.Ifananeurysmwaspresent,measurementswereperformedat
thelocationofthemaximalareasize.Ifnoaneurysmwaspresent,measurementswereper-
formeddirectlyproximaltotherightrenalartery.Toobtaincomparablemeasurementsbe-
tweenMRIandultrasounddatasetscomparabletechniquesfortheassessmentoftheSNRwere
applied.Regionofinterests(ROIs)weremeasuredasareasofsignalenhancementofluminal
aorticsignal.Theregionofinterestweredrawntodelineatethecompletevessel.FortheROIs
signaltonoiseratio(SNR)wascalculatedwithfollowingformula:Signaltonoiseratioultra-
sound(US)=((aneurysmal)aorticlumensignal)/(standarddeviationlumensignal).
Aorticaneurysmmorphometry
Aorta,rightrenalarteryandthelastpairofintercostalarterieswasexcisedtoallowprecise
anatomicalmatchingbetweenMRI,ultrasoundandhistopathology.Theleftrenalarteryand
thelastpairofintercostalarterywerethemainlandmarksfortheco-registration.Themor-
phometricalanalysiswasperformedusingelastin-stainedsections(Miller’sElasticavanGieson
stain)andImageProPlussoftware(ImageProPlus,MediaCybernetics).
Histologicalanalysisofaorticaneurysms
Histologicalanalysiswasperformedinthesameregionofaortathatwasimagedwithmagnetic
resonanceimagingandultrasound.Invivoandexvivomorphometricdatacouldthereforebe
directlycompared.Surgicallyremovedaorticaneurysmswereprocessedovernightforfurther
histologicalprocessing.Segmentedaorticaneurysmswereembeddedinparaffinandwerecut
fromtheproximalendoftheaneurysmevery40μminto6μmthickserialsections.After
dewaxingandrehydration,thesectionswerestainedusingMiller’sElasticavanGiesonstain
(EvG)andhematoxylinandeosin(HE).
Interobserveragreementsmagneticresonanceangiographyand
ultrasoundmeasurements
Fortheassessmentoftheinterreadervariabilitytwoinvestigatorsperformedtheaorticmea-
surements.Allimageswereanalyzedindependentlyinarandomizedorderandblindedtothe
accordingotherimagingmodalities.Areasizeswererecordedforeachmeasurement.
Statisticalanalysis
Dataareexpressedasmean±standarddeviation.AStudent’sttest(two–tailed,unpaired)was
usedtocomparecontinuousvariablesandverifythestatisticalsignificancebetweensham
(control)andtreatedaortas.Ifmorethantwogroupswereinvestigated,avarianceanalysis
(ANOVA)andBonferronicorrectionwasperformedforstatisticalcomparison.Interobserver
agreementsforexvivoandinvivomeasurementswereassessedusingBland-Altmanplots,
whichweregeneratedfortherawvolumedatatodisplaythespreadofdataandthelimitsof
agreement.Linearregressionwasappliedtodeterminetherelationshipbetweenaremeasure-
mentsonMRI,ultrasoundandhistology.
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InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
Results
Assessmentofdifferentstagesofabdominalaorticaneurysmsby
magneticresonanceangiographyonaclinicalMRsystem
ThecontinuousangiotensinIIinfusioninApoE-/-miceresultedinthedevelopmentandpro-
gressionofabdominalaorticaneurysms.Intheshamgroupthedevelopmentofabdominalaor-
ticaneurysmswasnotobserved.Usingtheclinical3TMRIsystemtheaorticdilationat
differentstagesofaneurysmdevelopmentcouldbeclearlyvisualized.Intheshamgroupan
averageaorticareaof1.16±0.12mm2wasmeasured(Fig3).AfteroneweekofangiotensinII
infusionanaverageareaof2.3±0.7mm2wasmeasured.Afurtherdilationwasobservedafter
threeandfourweekswithanaverageaorticareaof2.94±0.8mm2and3.79±1.12mm2(Fig3).
Comparisonofsignaltonoisemeasurementsofmagneticresonance
angiographywithpreclinicalhigh-frequencyultrasoundsystem
Inclinicalpractice,theluminalarea/diameterisassessedbasedonaTOFangiogramoracon-
trastenhancedangiograminMRI.Inultrasounddiametermeasurementsareusuallyper-
formedintheB-mode.Therefore,weusedtheaccordingimagesinourstudyforsignal
measurements.Signaltonoisemeasurements(SNRs)wereperformedinthesameanimalat
thesamelocationinboththeMRangiogramandultrasoundimages(Fig4).Signaltonoise
measurementsyieldasignificantly(p<0.001)highervaluesforMR-angiographycompared
toultrasoundimages(7.6±2.4versus2.1±0.9).
Comparisonoflumenmeasurementsofmagneticresonance
angiographyandpreclinicalhigh-frequencyultrasoundsystemwith
histopathology
BoththeMR-angiographyacquiredonaclinicalMRIsystemandthededicatedpreclinical
high-frequencyultrasoundsystemenabledareliabledifferentiationofthedifferentstagesof
aorticaneurysms.Allinvivomeasurementswerecorrelatedwithmeasurementsonexvivo
Fig3.TimecourseofaorticdilatationassessedbyMR-angiographyonaclinicalMRsystem.Invivo
assessmentofthedimensionsofabdominalaorticaneurysmmeasuredintheTOFangiographyinanApoE-/-
mousemodelofaorticaneurysms.Theluminalaorticareasweremeasuredinvivoafterone,two,threeandfour
weeksofangiotensinIIinfusion.Inthecontrolgroup(shamgroup)anaverageaorticareaof1.16±0.12mm2
wasmeasured.AfteroneweekofangiotensinIIinfusionanaverageareaof2.3±0.7mm2wasmeasured.A
furtherdilationwasobservedafterthreeandfourweekswithanaverageaorticareaof2.94±0.8mm2and
3.79±1.12mm2.ToF:Timeofflight.
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InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
Fig4.SignaltonoisemeasurementsoftheaorticlumenonaclinicalMRsystemandonadedicated
high-frequencyultrasoundsystem.Thisfigureshowsthatmagneticresonanceangiography(MRA,black
bar)demonstratedasignificantly(p<0.001)highersignaltonoiseratio(SNR)comparedtoultrasound(US,
greybar).MRIandultrasoundmeasurementswereperformedatcomparablelocationsoftheaorta.Thetime-
of-flighttechniqueinMRandtheB-modeinultrasoundaretechniqueswhicharealsofrequentlyusedina
clinicalsetting.
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histology(Elastica-van-Giessonstain,referencestandard).AreameasurementsoninvivoMR
angiogramsshowedtheclosestcorrelationwithexvivomeasurements(R2=0.98;p<0.001,
Table1),whileinvivomeasurementsslightlyandsystemicallyoverestimatedthesizeofthe
aneurysmalarea(Fig5).Thiscanbeexplainedbytheshrinkageofthehistologicalspecimens
followingtheprocessingofthetissuesamples.Inhigh-frequencyultrasound,systolicand
diastolicareameasurementsshowedastrong,howeverslightlylower,correlationwithexvivo
histologycomparedtotheMR-angiography.Thecorrelationcoefficientforsystolicareamea-
surementswasR2=0.91(p<0.001)andfordiastolicareameasurementsR2=0.93(p<
0.001).ComparabletoMRmeasurements,ultrasoundmeasurementsinbothcardiacphases
resultedinaslightsystemicoverestimationoftheareaoftheaorticaneurysm.Thiscanbe
explainedbytheshrinkageofthehistologicalspecimensfollowingtissueprocessing.
Table1. Summaryofresultsfrominvivomagneticresonanceimaging,ultrasoundandexvivohistology.
Mean SD 95%CI R2 pvalue
MRIvsUSsystole -0.26 0.34 -0.94 to 0.42 0.91 <0.001
MRIvsUSdiastole 0.03 0.26 -0.49 to 0.54 0.94 <0.001
MRIvsHistology -0.50 0.26 -1.01 to 0.01 0.98 <0.001
USsystolevsHistology -0.25 0.31 -0.86 to 0.37 0.91 <0.001
USdiastolevsHistology -0.53 0.33 -1.20 to 0.14 0.93 <0.001
InterobserverMRI 0.04 0.21 -0.37 to 0.46 0.96 <0.001
InterobserverUS 0.05 0.42 -0.78 to 0.88 0.87 <0.001
https://doi.org/10.1371/journal.pone.0178682.t001
PLOSONE|https://doi.org/10.1371/journal.pone.0178682 June5,2017 8/14
InvivoMRangiographyonaclinicalMRscannerfortheassessmentofaorticaneurysms
Fig5.InvivoareameasurementsontheTOFMR-angiographyanddedicatedhigh-frequencyultrasound
comparedtoexvivohistopathology.Theclosestcorrelation(A)betweeninvivomeasurementsandexvivo
histologywasfoundfortheTOFangiogram(R2=0.98;p<0.001).Thiscanbeexplainedbythehighersignalto
noiseratiosofMRAcomparedtoultrasound.Ahigh,howeverslightlylowerhoweversignificantcorrelation(B,C)
wasmeasuredforultrasoundinsystole(R2=0.92;p<0.001)anddiastole(R2=0.93;p<0.001).Aslight
overestimationofluminalareaswasmeasuredforbothMR-angiographyandultrasound.Thiscanbeexplained
withtheshrinkageofthehistologicalspecimensfollowingtheprocessingofthetissuesamples.MRA:Magnetic
resonanceangiography.
https://doi.org/10.1371/journal.pone.0178682.g005
Thistablesummarizestheresultsfromthedifferentinvivoimagingmodalities(MRI,ultra-
soundinsystoleanddiastole,includinginterobservervariability)andexvivohistology.
Regardinginvivomeasurements,theclosestcorrelationwasfoundbetweenMRIandultra-
soundmeasurementsindiastole.MRIshowedtheclosestcorrelationwithareameasurements
onexvivohistology.InterobservervariationwassmallerforMRIcomparedtoultrasound.
95%CI:95%confidenceinterval.
Comparisonoflumenmeasurementsofmagneticresonance
angiographywithhigh-frequencyultrasoundsystem
LuminalareameasurementsonTOFMR-angiographyshowedaclosecorrelationwithmeasure-
mentsderivedfromthededicatedpreclinicalhigh-frequencyultrasoundsysteminsystoleand
diastole(Fig6).However,thecorrelationwasslightlyhigherindiastole(R2=0.94;p<0.001)
comparedtothesystole(R2=0.91;p<0.001).AstheTOFangiographycontinuouslyacquires
imagesthroughoutsystoleanddiastole,theresultingimagereflectsthelargerdiameteracquired
indiastole,assummationeffectsoccur.Therefore,aslightlybettercorrelationwasfoundbetween
TOFangiographyandultrasoundimagesacquiredindiastole.
Interobserveragreementsmagneticresonanceangiographyand
ultrasoundmeasurements
InterobservercorrelationforareameasurementsinMR-angiographyshowedaclosecorrela-
tionbetweenbothimagereaders(R2=0.96;p<0.001)(Fig7).Theassociated95%confidence
interval(CI)forthecorrelationrangewas-0.73to0.46.Interobservercorrelationforareamea-
surementsforhigh-frequencyultrasoundalsoshowedastrongcorrelationbetweenboth
PLOSONE|https://doi.org/10.1371/journal.pone.0178682 June5,2017 9/14