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수의학석사학위논문

PercutaneousUltrasound-guided

CholecystographywithUltrasound

ContrastAgentinDogs

개에서 초음파 조영제를 이용한

초음파 유도하의 경피적 담낭조영술

2015년 2월

서울대학교 대학원

수의학과 임상수의학(수의방사선과학)전공

지서연

수의학석사학위논문

PercutaneousUltrasound-guided

CholecystographywithUltrasound

ContrastAgentinDogs

개에서 초음파 조영제를 이용한

초음파 유도하의 경피적 담낭조영술

2015년 2월

서울대학교 대학원

수의학과 임상수의학(수의방사선과학)전공

지서연

Percutaneousultrasound-guided

cholecystographywithUltrasound

ContrastAgentindogs

Director:ProfessorMincheolchoi

SeoyeounJi

MajorinClinicalVeterinaryMedicine(VeterinaryRadiology)

DepartmentofVeterinaryMedicine

TheGraduateSchool

SeoulNationalUniversity

Abstract

Differentiating hepatocellular disease versus biliary obstruction can be

challengingindogspresentedforicterus.Thepurposeofthisprospectivestudy

was to determine the feasibility ofpercutaneous contrastultrasound-guided

cholecystographyindogs.Tennormaldogsweighing7.6-13.0kg(median9.8

kg)wererecruited.Alldogswereconsiderednormalbasedoncompleteblood

count, serum chemistry profile, ultrasound examination, and percutaneous

radiographic cholecystography. Percutaneous contrast ultrasound-guided

cholecystographywasperformedusing0.5mlofcommerciallyavailablecontrast

agentandtwoconventionalultrasoundmachinesforsimultaneousscanningat

twodifferentlocations.Twoobserversindependentlyevaluatedthetimetoinitial

detection ofcontrastin the proximalduodenum and duration ofcontrast

enhancement via visual monitoring. Dynamic contrast enhancement was

calculatedusingtime-intensitycurves.Mean(±SD)andmedian(range)oftime

toinitialdetectionwere8.60sec(±3.35)and8.0sec(2.0-11.0),respectively,

andmeanandmediandurationwere50.45sec(±23.24)and53.0sec(20.0-

70.0),respectively.Mean,medianandrangeofpeakintensitywere114.1mean

pixelvalue(MPV)(SD ±30.7),109.2MPV and79.7-166.7,respectively,and

mean,median,andrangeoftimetopeakintensitywere26.1sec(SD±7.1sec),

24.0secand19.0-41.0sec,respectively. Findingsindicatedthatpercutaneous

contrastultrasound-guidedcholecystographyisafeasibletechniquefordetecting

andquantifying patencyofthebileductinnormaldogs.Futurestudiesare

neededtoassessthediagnosticutilityofthistechniquefordogswithbiliary

obstruction.

Keywords: Percutaneous ultrasound-guided cholecystography, Ultrasound

contrastagent,SonoVue

Studentnumber:2011-21675

CONTENTS

INTRODUCTION......................................................................................1

MATERIALSAND METHODS.........................................................2

1.Experimentalanimals.......................................................................2

2.Percutaneousradiographiccholecystography..................................3

3.Percutaneousultrasound-guidedcholecystography........................4

4.Imageassessment............................................................................7

5.Statisticalanalysis............................................................................8

RESULTS.....................................................................................................8

DISCUSSION............................................................................................15

REFERENCES..........................................................................................17

ABSTRACT IN KOREAN..................................................................21

- 1 -

INTRODUCTION

Common causes oficterus in dogs include intravascular hemolysis,

hepatocellulardiseaseandbiliaryobstruction.Hemolysiscanbediagnosedby

evaluating completeblood counts foranemia and urinalysis forhemoglobin.

However,differentiatinghepatocellulardiseasefrom biliaryobstructionisusually

more difficult(1,2).Conventionalultrasonography has proven beneficialin

evaluatingbiliarydisease(3-5).However,thismodalityhasdiagnosticlimitsfor

biliaryobstructionbecauseductaldilationalonemaynotbeareliableindicator

ofexistingbiliaryobstruction(6-9),andobstructivediseasemayoccurwithout

ductaldilatation (10-12).Both radiographic cholecystography and endoscopic

retrogradecholangiography arefeasiblemethodsforopacifying bileductsand

evaluating biliary patency (13-15).However,radiographic cholecystography

requiresfluoroscopy forrealtimemonitoring and requiresradiation exposure.

Endoscopicretrogradecholangiographyalsorequiresaskilledoperator,especially

fortheratherdifficultcanulationoftheduodenalpapillainsmall-sizeddogs

(16-17).

Ultrasoundcontrastagentsconsistofmicrobubblesthatcontainagas

stabilizedbyashell.Theuseofsuchagentshasimprovedthesensitivityof

ultrasoundduetohighacousticimpedanceattheinterfacebetweenthebubble

- 2 -

andthesurroundingmedium.Inhumans,ultrasoundcontrastagentshavebeen

used for intravenous and intra-cavitary applications (18,19).Intra-biliary

contrast-enhanced ultrasound has been reported as highly effective in the

diagnosis ofobstructive biliary disease in humans (20,21).An important

limitationofcontrast-enhancedultrasonographyisthenecessityforspecialized

contrast-specificultrasoundtechniques,butsomeintra-biliaryprocedureshave

demonstrated that biliary leakage can be evaluated using conventional

ultrasonographywithalow-mechanicalindexmode(22,23).

The purpose of the current study was to determine whether

percutaneouscontrastultrasound-guidedcholecystographyisafeasibletechnique

fordetectingandquantifyingbiliarypatencyattheleveloftheduodenalpapilla

indogs.

MATERIALSAND METHODS

1.Experimentalanimals

AllproceduresinvolvingdogswereapprovedbytheGuidefortheCare

andUseofLaboratoryAnimalsofSeoulNationalUniversity,Korea.Tenadult

- 3 -

beaglesweighing7.6to13.0kg(median:9.8kg)andranginginagefrom 3to

7years(median:5.1years)wererecruited.Normalstatusofeachdog was

assessed based on history,physicalexamination,complete blood cellcount,

serum chemistryprofileandultrasoundexamination.

2.Percutaneousradiographiccholecystography

To prove biliary tract patency, all dogs underwent percutaneous

radiographic cholecystography based on a modified version ofa previously

publishedstudyprotocol(14).Foodwaswithheldfor12-18hoursbeforethe

investigation.Aftersedationwithacepromazineandatropine,generalanesthesia

wasinducedwithpropofolandwasmaintainedwithisoflurane/oxygeninhalation.

Alldogsreceivedbroad-spectrum antibioticsandcefazolinsodium (20mg/kg

bodyweight(BW),intravenous)tominimizesepticcomplications.Thehairover

theabdomenwasclipped,andtheskinsurfacewascleanedwith70% isopropyl

alcohol.Thegallbladderwasscannedbyconventionalultrasonography(Sonoace

9900®,Medison,Korea) usingalineartransducer(5-12MHz)witheachdogin

leftlateralrecumbency.Underultrasoundguidance,a23G chibaneedlewas

passedthroughtheventralabdominalwallslightlytotherightofthemidlineat

thecaudalborderofthexiphoid,andthegallbladderwaspunctured.Following

styletremoval,theneedlewasconnectedbymeansofanextensiontubetoa

- 4 -

three-way stopcock with an empty 10 mlsyringe and a second syringe

containing20mlofcontrastmedium mixedwitha1:1ratioof300mgI/ml

iohexol(Omnipaque300®,GEHealthcare,Co.Cork,Ireland)injectionandsterile

salinesolution.Fiveto10mlofthebilewasaspiratedalongthetube,and20

mlofthecontrastmedium wasinjectedslowlyunderfluoroscopy(DXG-525RF,

Dong-AX-rayco.Ltd.,Korea) monitoring.

3.Percutaneousultrasound-guidedcholecystography

Percutaneous ultrasound-guided cholecystography was performed on

average4.5days(4-5)afterradiographiccholecystographyforalldogs.This

procedurerequiredtwoultrasoundmachinesforsimultaneousscanningattwo

different locations. We additionally used a portable ultrasound machine

(Sonovet®,Medison,Korea).Tobereadyforuse,5mLof0.9% sodium chloride

solution was injected into a bottle ofultrasound contrastagent(SonoVue,

Bracco Imaging,Milan,Italy) for dispersion.Patient preparation,including

generalanesthesiaandtheapproachforthepercutaneouspunctureofthegall

bladder,werethesameasforthepercutaneousradiographiccholecystography.

Initially,thebilewasremovedfrom thegallbladderasfollows:a23G chiba

needlepuncturedthegallbladderundertheguidanceofaportableultrasound

withacurvedarraytransducer(4-9MHz).Followingstyletremoval,theneedle

- 5 -

wasconnectedbymeansofanextensiontubetoathree-waystopcockwithan

empty10mlsyringeandasecond10mlsyringecontaining10mlof0.9%

sodium chloridesolution.Tenmlofthebilewasaspiratedalongthetube,and

10mlof0.9% sodium chloridesolutionwasinjected(FIG.1A).Thisprocedure

wasrepeatedthreetimestocompletelywashthebileoutofthegallbladder.

Then,0.5mlofthecontrastagentwasmixedwith10mL of0.9% sodium

chloridesolutionina10mLinjectionsyringe.Beforeinjectionofthecontrast

agentintothegallbladder,ascanoftheproximalduodenum wasperformed

using conventional ultrasound with a 5-12 MHz linear transducer.The

transducerwasplacedattherightlateralninthtotwelfthintercostalspaces

neartheventralmidline.Theproximalduodenum wasscannedinalongitudinal

section,andthetransducerwasnotsubsequentlymoved.TheMI(Mechanical

Index)valuewasswitchedtolessthan0.2.Thegainsettingwasregulatedto

obtainananechoicbowellumenorthecentralhyperechoiclinearisingfrom the

bowellumen.Thecontrastagentdispersion wasslowly injected through an

extensiontubeforapproximately30secunderultrasoundguidance(FIG.1B).

Simultaneously,ascanoftheproximalduodenum wasperformedfor90sec.All

theimageswererecordedandsavedtotheharddisk forlaterreview and

documentation.Inalldogs,completebloodcellcounts,serum chemistryprofiles

and abdominal ultrasound examinations were performed 7 days after

- 6 -

percutaneous ultrasound-guided cholecystography to assess possible

complicationsofthestudy.

FIG.1.Biledrainagefrom gallbladderandinjectionofultrasoundcontrast

agentintothegallbladderinadog.(A)Identificationofthegallbladderand

insertion ofneedleareperformedunderultrasoundguidancetodrainagebile

from thegallbladder.(B)Theprobewaspositionedontheintercostralspace

and the proximalduodenum underwent carefulgrey-scale imaging.After

identification oftheproximalduodenum,Theultrasound contrastagentwas

slowlyinjectedthroughaextensiontube.

- 7 -

4.Imageassessment

Imagemediasoftware(Gomplayer2.2,gretechco.,Korea)wasusedto

view theimagesandtoexportselectedframesforanalysis.Twoexperienced

veterinaryradiologists(S.J.andB.K.)inourdepartmentsubjectivelyevaluated

contrastenhancementin the proximalduodenallumen.The two observers

independently measured the time to initialdetection following injection and

durationbyviewingtheimages.Thetimetoinitialdetectionfollowinginjection

wasdefinedasthetimewhencontrastenhancementintheproximalduodenum

wasfirstdetected.Thedurationwasdefinedastheperiodoftimeinwhich

contrastenhancementwas observed in the proximalduodenum.The visual

patternofcontrastenhancementwasalsodescribed.Forquantitativeanalysis,

individualultrasound images were acquired by a video frame grabberand

capturedatarateofoneframepersecfor90sec.Consecutivemeanpixel

value(MPV)measurementswereobtained using imagesoftware(NIH 1,43,

NationalInstitutesofHealth,USA)byasingleobserver(S.J.).A regionof

interest(ROI)wasdrawninthemoststronglyechogenicareaoftheduodenal

papillaafterinjectionofthecontrastagent.Standardizedtime-intensitycurves

depictingthesignalintensityplottedagainsttimewerecreatedbasedonselected

ROI,andpeakintensityandtimetopeakintensityafterinjectionwererecorded.

- 8 -

5.Statisticalanalysis

Statisticalanalysiswasperformedwithastandardcomputersoftware

program (SPSS 21.0.0.2,SPSS Inc.,Chicago,USA)by theobserver(S.J.).

Intra-observerrepeatabilitywasevaluatedbymeansofanintraclasscorrelation

coefficient(ICC).

RESULTS

Duringpercutaneousradiographiccholecystography,thecontrastmedium

wasobservedtoemptyintotheduodenum inthirtyminutesinalldogs(FIG2).

Beforetheinjection ofthecontrastagentinto thegallbladder,the

sonographic appearance ofthe proximalduodenum wasnormalin alldogs.

There were centralhyperechoic lines arising from the bowellumen and

intermittentsmallamountsofechogenic materialswithin the lumen due to

mucusorfoodremnants.

Duringtheinjectionofthecontrastagent,theechogenicsignalinthe

gallbladderwasimmediatelyvisible(FIG.3).Echogenicfluidbegantogradually

- 9 -

andnon-homogeneouslydispersewithinthelumenoftheproximalduodenum.

Then,echogenicfluid with athin and strong stream wasidentified overa

periodofsecondsatthelevelofthepresumedduodenalpapilla(FIG.4)and

wasfollowed by a progressively decreasing enhancement.Thispattern was

repeatableandirregular.

- 10 -

FIG.2.Radiographicimagesofthepercutaneousradiographiccholecystography.

During percutaneousradiographiccholecystography,thecontrastmedium was

observedtoemptyintotheduodenum intendogs.

- 11 -

FIG.3.Ultrasoundimagesofthegallbladderandcomparisonsoftheechogenic

signalbefore(A)andafter(B)percutaneousinjection ofultrasoundcontrast

agent.Afterinjectionofthecontrastagent,anechogenicsignalwasimmediately

visible.

FIG. 4.Longitudinalultrasound images of the proximalduodenum after

percutaneousinjectionofultrasoundcontrastagent.Markedechogenicfluidwith

athinandstrongstream (arrows)wasidentifiedatthelevelofthepresumed

duodenalpapilla.

- 12 -

Parameters Mean±SD ICC

TTI(s)

Observer1 8.60(±3.06)0.981

Observer2 8.60(±3.78)

8.60(±3.35)

Duration(s)

Observer1 54.10(±20.53)0.876

Observer2 46.80(±26.26)

50.45(±23.24)

The mean (±SD)and median (range)time to the initialdetection

followinginjectionwere8.60sec(±3.35)and8.0sec(2.0-11.0),respectively,

andthemeanandmediandurationwere50.45sec(±23.24)and53.0sec(20.0-

70.0), respectively. Intra-observer repeatability was excellent for both

measurements(intraclasscorrelationcoefficientfortimetoinitialdetection:0.981,

intraclasscorrelationcoefficientforduration:0.876)(Table1).

Table 1.Intraobserver Correlations for VisualMeasurements of Contrast

Enhancement of the Proximal Duodenal Lumen Following Percutaneous

Ultrasound-GuidedCholecystographyinTenHealthyDogs

TTI,timetoinitialdetectionafterinjection;ICC,intraclasscorrelationcoefficient

- 13 -

A regionofinterestwasdrawnintheregionofafocal,thin,highly

echogenicareaofthepresumedduodenalpapilla.Theareawas30mm2and

wasmaintainedin thesameposition.Time-intensity curvesoftheareaare

depicted in FIG.3.Therewereinconsistentincreasesand decreasesin the

intensity,andpatternsofirregularintensity fluctuation wereobservedin all

dogs.

FIG.3.Time-intensitycurvesofaregionofinterest(ROI)forthreedogs.The

ROIwasdrawninthemoststronglyechogenicareaofthepresumedduodenal

papillaafterinjectionof ultrasoundcontrastagent.Thecurvesshowedirregular

fluctuationsanddifferentpatternsforeachdog.

- 14 -

Parameters Mean±SD Range

PI(MPV) 114.1±30.7 79.7– 166.7

TTP 26.1±7.1 19.0– 41.0

Themean,medianandrangeofpeakintensitywere114.1MPV (SD ±

30.7),109.2MPVand79.7-166.7,respectively,andthemean,medianandrange

oftimetopeakintensitywere26.1sec(SD±7.1sec),24.0secand19.0-41.0

sec,respectively(Table2).

Complete blood cellcounts,serum chemistry profiles and abdominal

ultrasound examinations performed 7 days after the percutaneous

ultrasound-guidedcholecystographyrevealednosignificantabnormalitiesinany

dog.

Table2.QuantitativeContrastEnhancementoftheDuodenalPapillainTen

HealthyDogsFollowingPercutaneousUltrasoundGuidedCholecystography

PI,peakintensity;TTP,timetopeakfrom injection;MPV,meanpixelvalue

- 15 -

DISCUSSION

Inthisstudy,wewereabletoobservenotonlycontrastenhancementin

theduodenallumenbutalsothelocationofthepresumedduodenalpapillainall

dogs.Thesuccessinidentifyingechogenicflow withintheproximalduodenum

indicatesthatthetechniquecanbeusedfortheevaluationofbiliarypatency.

Unlikethehumanstudies,wechosetoevaluatecontrastenhancementinthe

proximalduodenum insteadofthebileductsbecauseultrasoundscansofbile

ducts in healthy dogs are limited by underlying gastricand duodenalgas,

respiratorymotionandthesmallsizeofthebiliarystructures.Weusedtwo

ultrasoundmachinesbecauseultrasound-guidedinjectionofthecontrastagent

intothegallbladderandultrasoundexaminationoftheproximalduodenum were

performedsimultaneously.Despiteartifactsduetomucus,foodremnantsandthe

echogeniccentrallinefrom theduodenum,highlyechogenicfluidwasclearly

identifiedwithin theduodenum inalldogsafterthecontrastagentinfusion.

Additionally,thelocationofthepresumedduodenalpapillawasidentifiedasthe

contrastagentpassedthroughtheduodenalpapillawithafocal,thin,andstrong

echogenic stream.The time-intensity curve ofthe duodenalpapilla showed

inconsistentfluctuations.Theintensity fluctuationsmostlikely resulted from

contractionandrelaxationofthesphincterofOddiinthegallbladdertocontrol

- 16 -

the excessive pressure induced by the injection ofcontrastagent.Authors

proposethattheinconsistencypatternineachdogmayhavebeenassociated

with pressure-related factors,such as variable volume ofthe gallbladder,

gallbladderdistensibility,intrahepaticreflux,resistanceofthesphincterofOddi

andvariabledistributionofcontrastagentinthefluid.

Onelimitationofthestudyreportedherewasthechoicetouseasingle

doseof0.5mlofcontrastagent.Thischoicewasmadebecause0.1-1mL of

contrastagentdilutedin0.9% salinehasbeenrecommendedforextravascular

contrast-enhancedultrasonographyinhumans(24).Theoptimalcontrastagent

doseand concentration wasnottested in thecurrentstudy and should be

optimizedinfuturestudies.Anotherlimitationwasthatonlynormaldogswere

evaluated.Itremainsunknownwhetherthetechniquewouldbefeasibleandsafe

indogswithbiliaryobstructivediseases.

In conclusion, findings from the current study indicated that

percutaneousultrasound-guidedcholecystography using anultrasoundcontrast

agentisfeasibleandsafeforevaluationofbiliarypatencyandlocalizationofthe

duodenalpapillainnormaldogs.Futurestudiesareneededtodeterminethe

utilityofthistechniquefordiagnosingobstructivebiliarydiseaseindogs.

- 17 -

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국문초록

개에서 초음파 조영제를 이용한

초음파 유도하의 경피적 담낭조영술

서울대학교 대학원

수의학과 임상수의학 (수의방사선과학)전공

지서연

황달이 있는 개에서 간세포성 질환과 담도폐색을 구분하여 진단하는 것은

어려운 문제이다.본 연구의 목적은 담도폐색을 평가하기 위하여 지금까지 소동물

에서 적용해본 적이 없는 초음파 조영제를 이용한 초음파 유도하의 경피적 담낭조

영술을 실시하고 그 유용성을 확인하고자 하는 것이다.

본 연구에는 체중이 7.6-13.0kg(중간값 9.8kg)이며 건강에 문제가 없

는 열 마리의 개가 사용되었다.모든 개들은 연구에 앞서 건강상태를 확인하기 위

하여 전혈검사,혈청검사,흉복부 방사선 및 복부초음파 검사를 실시하였고 담도개

- 22 -

통상태를 평가하기 위하여 기존의 담도폐색 유무를 확인하기 위한 일반적인 진단방

법인 방사선 조영제를 이용한 경피적 담낭조영술을 실시하였다.건강 및 담도개통

상태에 이상이 없음을 확인한 개들에 대해 0.5ml의 초음파 조영제를 이용하여 초

음파 유도하의 경피적 담낭조영술을 실시하였다.초음파상에서 근위 십이지장에서

의 초음파 조영제에 의한 조영증강 과정을 기록하고 이를 두명의 관찰자가 각각 조

영제 주입후 조영증강이 발생하는 시간과 조영증강이 유지되는 기간을 측정하였다.

또한 근위 십이지장내 조영증강이 발생하는 관심영역에 대하여 시간-강도 곡선을

그렸다.

본 실험 결과,초음파 조영제 주입후 조영증강이 발생하는 시간의 평균,중

간값,범위는 각각 8.60sec(±3.35)과 8.0sec,2.0-11.0sec였고 조영증강이 유

지되는 기간의 평균,중간값,기간은 각각 50.45sec(±23.24),53.0sec,20.0-70.0

sec였으며 두 관찰자간의 유의적인 차이는 확인되지 않았다.또한 근위 십이지장

내 조영증강이 발생하는 관심영역의 최대 신호강도의 평균,중간값,범위는 각각

114.1MPV (SD±30.7),109.2MPV,79.7-166.7MPV 였고 최대 신호강도가 확

인되는 시간의 평균,중간값,범위는 각각 26.1sec(SD±7.1sec),24.0sec,19.0-

41.0sec였다.

본 실험을 통해 초음파 조영제를 이용한 초음파 유도하의 경피적 담낭조영

술이 정상개의 담도개통성을 보여줄 수 있음을 확인하였고 추후 폐쇄성 담도계 질

환에 대한 진단적 도구를 연구하는데 이용될 수 있을 것으로 사료된다.

INTRODUCTIONMATERIALS AND METHODS1.Experimental animals2.Percutaneous radiographic cholecystography3. Percutaneous ultrasound-guided cholecystography4. Image assessment5. Statistical analysis

RESULTSDISCUSSIONREFERENCESABSTRACT IN KOREAN

9INTRODUCTION 1MATERIALS AND METHODS 2 1.Experimental animals 2 2.Percutaneous radiographic cholecystography 3 3. Percutaneous ultrasound-guided cholecystography 4 4. Image assessment 7 5. Statistical analysis 8RESULTS 8DISCUSSION 15REFERENCES 17ABSTRACT IN KOREAN 21