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Annals of Hepatology
j ou rn a l h o m e pa g e :w w w . e l s e v i e r . e s / a n n a l s o f h e p a t o l o g y
Concise reviews
Visualising and quantifying intestinal permeability -where do we stand
Monica Rusticeanu
a,∗, Vincent Zimmer
b,c, Frank Lammert
caDepartmentofMedicine,KrankenhausVilshofen,Krankenhausstrasse32,94474VislhofenanderDonau,Germany
bDepartmentofMedicine,MarienhausklinikSt.JosefKohlhof,Klinikweg1-5,66539Neunkirchen,Germany
cDepartmentofMedicineII,SaarlandUniversityMedicalCenter,SaarlandUniversity,Homburg,Germany
a rt i c l e i nf o
Articlehistory:
Received30August2020 Accepted20September2020 Availableonline10October2020
Keywords:
intestinalpermeability livercirrhosis intestinalimaging functionalimaging CLE
a b s t ra c t
Intestinalpermeabilityisgettingmoreandmoreattentioningastrointestinalresearch.Althoughwell recognized,itsexactroleinhealthanddiseaseisyettobedefined.Therearemanymethodsofquantifying intestinalpermeability,butmostofthemfailtodelivertangibleinformationaboutthemorphological integrityoftheintestinalbarrier.Inthisreviewweaimtodescribeimagingoptionsfortheassessment ofintestinalbarrierintegrityandtheirpotentialrelevanceforclinicalpractice.Ourfocusisonconfocal laserendomicroscopy,whichisatthistimetheonlymethodforvisualizingnotonlyfunctionalbutalso morphologicalaspectsofthegutbarrierinvivo.
©2020Fundaci ´onCl´ınicaM ´edicaSur,A.C.PublishedbyElsevierEspa ˜na,S.L.U.Thisisanopenaccess articleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Accordingtocurrentdata,theintestinalbarrier’sintegritymight playakeyroleinheathanddisease.Theassessmentoftheintesti- nalpermeabilityisinsufficientlystandardizedinclinicalpractice, anditsclinicalrelevanceforspecificdiseasestatesremainstobe delineatedfurther.Inthisreview,wesummarizecurrentknowl- edgeonfunctionalimagingtoolsfortheassessmentofintestinal permeabilityandbarrierintegrity.Thelattercontributenotonlyto theprotectionofthehumanbodyagainstinvasionofpathogenic microorganismsand toxins,butalsototheabsorptionofnutri- ents vital for life. Factors that influence intestinalpermeability and barrier integrity are intestinal perfusion, systemic and/or gastrointestinalinfections,diet,drugsaswellaslife-styleandenvi- ronmentalfactors[1].Certaindiseasestatesareassociatedwith increasedintestinalpermeabilityfavoringmucosalinflammation,
Abbreviations:IBD,inflammatoryboweldisease;IBS,irritablebowelsyndrome;
CLE,confocallaserendomicroscopy;MRI,magneticresonancetomography;PET, positronemissiontomography;DCE-MRI,dynamiccontrast-enhancedmagnetic resonanceimaging;TNBS,2,4,6-trinitrobenzenesulfonicacid;CD,Crohndisease;
CE-MRI,ContrastenhancedMRI;18F-FDG,18F-Fluordesoxyglukose;TJ,tightjunc- tion;ZO-1,Zonulaoccludens-1;PCR,polymerasechainreaction;CLE,Confocal endomicroscopy;pCLE,probebasedCLE;CLD,chronicliverdiseases;UC,ulcerative colitis;IgE,immunoglobulinE;SBP,spontaneousbacterialperitonitis.
∗ Correspondingauthor.
E-mailaddresses:monicarusticeanu@gmail.com(M.Rusticeanu), vincent.zimmer@gmx.de(V.Zimmer),Frank.Lammert@uks.eu(F.Lammert).
resulting in the translocation of intraluminal components. Not onlyprimarygastrointestinaldiseasessuchasinflammatorybowel diseases(IBD) and irritablebowel syndrome(IBS) arelinkedto impairedbarrierfunction,butalsoawidearrayofpathologiesorig- inatingfromotherorgans.Therearedatademonstratinganaxis betweenchronicliverdiseasesandthegutbarrier,thushighlight- ingthecriticalinterplaybetweensystemicandintestinaldisease [2].
Themeasurableintestinalpermeabilityasfunctionofparacel- lularintestinaltransportcapacitiesreflectstheintestinalbarrier function,which,inturn,iscloselyrelatedtointestinalhealth.The architectureofthegutbarriercomprisesvariousphysicalandcel- lularcomponents(mucus,epithelialandvascularcells,stroma).In addition,chemicalaspectsofthebarrierfunctionlikewisehaveto betakenintoaccount,suchascellularproducts,antimicrobialpep- tides,andgastrointestinalsecretions.Lastbutnotleast,thecritical contributionofintestinalmicrobiota,thelocalandthesystemic immunesystemaswellasgastrointestinalmotilitytogutintegrity hastobetakenintoaccount[3].
Therearevarioustoolsandmethodstoassessintestinalper- meability, which measure distinctcomponents of this complex system.Mostofthecurrentinstrumentsfor (semi-)quantitative assessment, e.g. lactulose and mannitol permeability or blood zonulin measurements,provide information about the increase inpermeability and,potentially, thedegreeoftissue alteration.
However,theseparametersarenotcapableofdistinguishingfunc- tionalchangesfromstructuralintestinaldamage.Likewise,they
https://doi.org/10.1016/j.aohep.2020.09.010
1665-2681/©2020Fundaci ´onCl´ınicaM ´edicaSur,A.C.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
providelittleinformationabouttheexactmechanismand/orlocal- izationofdisruptedgutpermeability.Inthisreview,wehighlight lesswell-establishedimagingtoolsforintestinalbarrierfunction assessmentwithemphasisonconfocallaserendomicroscopy(CLE).
CLEisanoveltoolthatintegratesinvivomicroscopicassessment intostandardendoscopy[4,5].Furthermore,availableoptionsfor thequantitationofdisturbedpermeabilityaswellascausesand localizationofthesefunctionalchangesaredescribed.
2. ImagingTools
Thereisanincreasingnumberofstudieslookingatthefunc- tionalaspectofintestinalpermeability,bothinvitroandinvivo, inanimalmodelsorpatients.Aswewillpresentfurtheron,most studies visualizingintestinalpermeability in vivo are usingthe techniqueofconfocalendomicroscopy,withinflammatorybowel diseasebeingthemostinvestigatedpathology.Otherconditions influencingtheintestinalpermeabilitystudiedwithCLEtothisdate inhumansareirritablebowelsyndromeandallergies.Otherinvivo imagingmethodsrevealedbyourPubMed-researchweremagnetic resonancetomography(MRI)andpositronemissiontomography (PET)
2.1. Magneticresonanceimaging(MRI)
Inastudypublishedin2015,Towneretal.[6]demonstrated alteredpermeabilityintheurinebladderandbowelinananimal model by meansof dynamic contrast-enhanced magneticreso- nanceimaging (DCE-MRI).In theirpreliminarystudy,theyused aconcentrationofcontrastmediathatreadilyallowedfordirect and quantifiableobservationofleakage outofthebladderwall.
Theyalsoobservedasecondarycontrast-enhancementinthecolon, indicatingabreakdownofthecolonicpermeabilitybarrierinhis- tologicallyundamaged mucosaafterexposureofthebladderto protaminesulfate[7]Inasecondpublication[8],thesameresearch grouplookedatthecrosstalkbetweencolonandbladderin2,4,6- trinitrobenzenesulfonicacid(TNBS)-inducedcolitisasestablished methodofinducingtransmuralandsegmentalcolonicinflamma- tionsimilartoCrohndisease(CD).
Contrast enhancedMRI (CE-MRI)isinroutine clinicaluseto determine sites and degree of intestinal inflammation in IBD.
Assessing gastrointestinal, hepatobiliary or urinary tract with this methodmaybeusefulinprovidinginformation abouthow intestinalinflammationinfluencesthepermeabilityinhistological healthytissueofotherorgansinIBDpatients.
2.2. Positronemissiontomography(PET)
The authors of this study [9], published 2011, used PET- technology to assess intestinal permeability and absorption of orally administrated 18F-Fluordesoxyglukose (18F-FDG) in rats, concludingthatthiswouldbeapowerfultoolforinvivoinves- tigationofthegastrointestinaldrugabsorption.
2.3. Histology
Anothermethodofassessinggutbarrierintegrityis,amongoth- ers,observingthesheddingoftheintestinalepithelialcells.There arevarioushistologicalmethodsofdescribingadisruptedbarrier, likeincreasedintercellularspaceasareflectionofdamagedtight junctions,quantifyingtheextracellulartightjunction(TJ)protein occludincomparedtotheintracellularZonulaoccludens-1(ZO-1) proteinbypolymerasechainreaction(PCR),adecreasesuggesting adisturbanceinthecellularmake-upoftightjunctions[10].
Astheyareheldtogetherbytightjunctions,anydisruptionof theequilibriumbetweenepithelialcellsheddingatthevillustip
andgenerationofnewcellsinthecryptcanaffectthehomeostasis andpermeabilityofthegut.It canbeassumedthatsheddingof intestinalcellsfromtheepithelialmonolayer,whichcanbeseen underthemicroscope,causestransientgapsinthebarrierwitha correspondingimpactonintestinalpermeability[11].
Themechanismunderlyingthisphenomenonof pathological cellshedding, observed in animal models of inflammation and humanintestinalconditionslikeIBDremainspoorlyunderstood, butthereisasoundrationaletobelievethatitmightstemfrom systemicandintestinalinflammatorydiseaseandadysfunctional gutbarrier[12].
Studiesinvestigatingthelightmicroscopicaspectoftheintesti- nalmucosainlivercirrhosishaveshownsignsofphysicalinjury likeepithelialcellshortening,nucleardisarrayanddegenerative changesof thecytoplasmpredominantly in cells locatedatthe tipofthevilli,aswellaspronouncedextrusionofdeadcells.Fur- thermore,atasubmicroscopiclevellivercirrhosiswaslinkedtoa widenedtightjunctioncomplexaswellasdisruptionoftheactin cytoskeleton,mitochondrialdysfunctionwithconsecutiveswelling andchangesinpermeability[13,14]aswellaschangesinsugar compositionatthebrushborder[15].Attheimmunohistochemi- callevel,theexpressionofTJproteinsoccludinandclaudin-1was repressedincirrhosis.Ofnote,thesefindingswereevenmorepro- nouncedinpatientswithdecompensatedcirrhosis[16].
2.4. Confocalendomicroscopy(CLE)
Thisimagingmethodisbasedontissueilluminationaftersys- temicapplicationoffluorescein,whichhighlightstheextracellular matrix[17].ThisallowsCLEtoassesstheinternalmicrostructure oftissueswithinanatomicaltracts,i.e.gastrointestinal(butalso urinaryorrespiratory),throughanendoscope.Currently,toour knowledge,theonlydeviceonthemarketprovidingthistechnol- ogyhasthetradenameCellvizioisafiberedconfocalmicroscopic system.Itusestheso-calledprobebasedCLE(pCLE)whereaprobe ispassedthroughtheworkingchannelofastandardgastroscope orcolonoscope[18].
A488-nmwavelengthlasersystemisused.Therearedifferent probesfordifferentindicationsavailableandtheconfocalimages aresteamedat aframerateof 12framespersecond,obtaining real-timevideosofthemucosa[19].
Confocalminiprobesaremadeofadistaltip,afiberwithapro- tectivesheathandaconnector.WhenconnectedtotheCellvizio systemthroughtheirconnector,confocalminiprobestransportthe scannedlaserbeamthroughthesheathedfibertothesiteofobser- vationincontactwiththedistal tipandcapturethefluorescent lightemittedbackfromthetissue,inordertoprovidein-vivoflu- orescenceimagingoftissues.Thepartsoftheconfocalminiprobes thatgetincontactwiththepatientconsistsofbiocompatibleglass, plasticandmetalderivatives.
HeresomeexamplesofintestinalimagesobtainedwiththeCel- lviziopCLEsystem:
Fig.1
The method permitsin vivo microscopy of the humangas- trointestinalmucosaduringendoscopy,providing optical ¨virtual biopsies¨.Endomicroscopyhasbeenappliedforthestudyofseveral diseases,withmoststudiesfocusingoninflammationandneopla- sia,suchasBarrettesophagus,gastriccancer,celiacdisease,IBD orcolorectalneoplasia.CLEfacilitatesthestudyofpathophysio- logicaleventsintheirnaturalenvironment(functionalimaging) [20–24].Toourknowledge,theroleofendomicroscopyinpatients withhepatobiliarydiseaseshasbeenconfinedtoimagingthebil- iarytree[25]buthasnotbeenusedsofartoevaluatetheintestinal mucosainpatientswithchronicliverdiseases(CLD).Ofnote,sugar absorptiontests(lactuloseandmannitol)havebeenusedtoassess intestinalpermeabilityand mucosalbarrierfunctioninCLD,but
Fig.1. (A):intestinalvilliwithintraepithelialgap(blackarrow)andincreasedluminalfluorescence(whitearrow)asasignofincreasedpermeability.(B):intestinalvilli withoutgaps,intravascularfluorescence(blackarrow),noincreasedintraluminalfluorescence.
therearenodataatthispointthatdemonstratesignsofintestinal barrierdysfunctioninvivoinhumanliverdiseasebyCLE.
Asstatedabove,intestinalepithelialcellsandtightjunctions playacrucialroleinmaintainingtheintactgutbarrier.Ontheone hand,theyassuretheabsorptivecharacteroftheintestine,onthe othertheyarenecessarytopreventpotentiallyharmfulcompo- nentsfromenteringthesystemiccirculation[26,27].Representing a highcellularturnovertissue, theintestinalepitheliumiscon- stantlyregeneratedfromstemcellsoriginatingatthecryptbase.
Theymigratetothetopofthevilliastheymatureandfromwhere theyare shedintime [27]. Thecellshedding isresponsiblefor epithelialgaps,whichcanbedirectlyobservedinreal-timebyCLE inhumans[28,29].Severalstudiesconfirmedthatincreasedintesti- nalpermeabilitycanbevisualizedbyCLE,especiallyinpatients withIBD,butalsoinIBSandfoodallergy.
TheepithelialgapdensityvisualizedbyCLEhasbeensuggested asquantitativeparameterforintestinalpermeability.Itwasshown that the epithelial gapdensity of theterminal ileum is signifi- cantly higherthatthat ofcontrolswithoutIBD,inboth CDand ulcerativecolitis(UC)patients.Interestingly,gapdensitydidnot
correlatewithdiseaseactivity,hencefurtherstudiesareneededto betterunderstandthepotentialsignificanceoftheseobervations inIBD[30].Sincecellsheddingisaphysiologicalregenerativephe- nomenon,thequalitativeobservationofepithelialgapsmaynotbe sufficienttodistinguishphysiologicalversuspathologicalgutper- meability.In2011Liuetal.[31]describedamethodofdefininggap densitybycountingtheepithelialgapsper1,000cellsinadequately imagedvilliintheterminalileumofIBDpatients.Thisdefinition wasalsousedbyTurcotteetal.[32]whodescribedincreasedgap densityinIBSpatientscomparedwithcontrolsinasmallsample ofsixversus32individuals.Thereby,theywereabletoachieve adiagnosticaccuracyforthediagnosisofIBSwith62%sensitiv- ity,89%specificity,83%positivepredictivevalue,and73%negative predictivevalue.
Fritscher-Ravensetal.[33]usedCLEtechnologyinIBSpatients toassesstheirresponsetospecificfoodcomponentsbyanalyz- ingintraepithelial lymphocytesand fluid extravasationthrough epithelial leaks. Using this method, theyestablished a diagno- sisof(immune-mediated)foodallergyinpatientswithnegative immunoglobulinE(IgE)andskin-pricktests.Inthisstudy,several
CLEcriteriaforthedetectionofbarrierdisruptionweresuggested:
densityofintraepitheliallymphocytes,epithelialbreaks,luminal fluoresceinleakage,presenceoffluoresceinbetweenenterocytes andwideningoftheintervillousspace.
3. Discussion
Althoughanemergingfield,imagingmethodsforintestinalper- meabilityassessmentremainimmatureandinevolution.Radiology methodsmightprovideinvivoinformationaboutthefunctional integrityoftheintestinalbarrierbutfailtocharacterizemorpho- logicalchangesthatleadtopermeabilitychange.Asofnow,CLE representstheonlyimagingmethodthatmayprovidedetailedcel- lularandanatomicinformationaswellasfunctionalalterationsof theintestinalbarrierinrealtime.
Thishasbeenpioneeredinapotentialclinicalapplicationby Fritscher-Ravensfordiagnosisandmonitoringofdistincttypesof foodallergies[33].Furtherpotentialclinicalutilityofthisinnova- tivetechnologymaylieinprognosticationoftreatmentresponses anditsfine-tuningintheIBDfield[34].
Functional imaging with the CLE method also allows for testing of emerging therapies in IBS contributing to better understandingthepathophysiologicmechanismsunderlyingthis condition.
Extraintestinalconditionscanbeinfluencedbytheintestinal permeabilityandintegrityofthegutbarrier.Itiswellknownthat in individuals withlivercirrhosis and ascites,disturbedintesti- nalpermeabilityfavoursthedevelopmentofspontaneousbacterial peritonitis (SBP) as a potentially deleterious clinical complica- tion. Anearlydiagnosisand risk stratificationby quantification ofimpairedgutbarriermayallowfortargetedinterventionand preventive measures, such as pre-emptive antibiotic prophy- laxis.
Severalstudiesconfirmedincreasedintestinalpermeabilityin liver cirrhosis, however, these disturbances have not yet been addressedthoroughlybyinvivofunctionalimagingsuchasCLE.
Amongothers,increasedgutpermeabilityisconsideredanessen- tialandearlyfactorinthepathophysiologyofSBPaswellasother infectiouscirrhosiscomplicationsrelatedtobacterialtranslocation [35,36].
Allthesechangescouldnot,toourknowledge,bevisualizedin realtimewithinvivoimagingmethods.
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