Three-dimensional imaging and analysis of entire peripheral nerves after repair and reconstruction

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Article

Reference

Three-dimensional imaging and analysis of entire peripheral nerves after repair and reconstruction

BIKIS, Christos, et al.

Abstract

We wanted to achieve a three-dimensional (3D), non-destructive imaging and automatic post-analysis and evaluation of reconstructed peripheral nerves without involving cutting and staining processes.

BIKIS, Christos, et al . Three-dimensional imaging and analysis of entire peripheral nerves after repair and reconstruction. Journal of Neuroscience Methods , 2018, vol. 295, p. 37-44

PMID : 29179953

DOI : 10.1016/j.jneumeth.2017.11.015

Available at:

http://archive-ouverte.unige.ch/unige:154949

Disclaimer: layout of this document may differ from the published version.

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ContentslistsavailableatScienceDirect

Journal of Neuroscience Methods

j ou rn a l h om epa g e : w w w . e l s e v i e r . c o m / l o c a t e / j n e u m e t h

Three-dimensional imaging and analysis of entire peripheral nerves after repair and reconstruction

Christos Bikis

^a

, Lucas Degrugillier

^b

, Peter Thalmann

^a

, Georg Schulz

^a

, Bert Müller

^a,∗^∗^∗

, Simone E. Hieber

^a,∗

, Daniel F. Kalbermatten

^c,d

, Srinivas Madduri

^b,c,e,∗∗

aBiomaterialsScienceCenter,DepartmentofBiomedicalEngineering,UniversityofBasel,Switzerland

bCenterforBioengineeringandRegenerativeMedicine,DepartmentofBiomedicalEngineering,UniversityofBasel,Switzerland

cDepartmentofPlastic,Reconstructive,AestheticandHandSurgery,BaselUniversityHospital,Switzerland

dDepartmentofPathology,BaselUniversityHospital,Switzerland

eDepartmentofBiomedicine,UniversityofBasel,Switzerland

h i g h l i g h t s

•Micro-scaledetailsof3Dnerveﬁber and vessel reorganization upon repair.

•Distinct differentiation between nerve and connective tissue in a label-freemanner.

•Perineuriummembranes and small capillariesvisualizedwithisotropic 4␮mresolution.

•Quantitativemeasuresprovedsignif- icance for assessing repair quality, p<0.01.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Articlehistory:

Received26August2017 Receivedinrevisedform 23November2017 Accepted23November2017 Availableonline26November2017

Keywords:

␮CT

X-raytomography Axonalregeneration Anatomy

Morphology Micrometer Non-destructive Nerveconduits Nervereconstruction 3Dimaging

a b s t r a c t

Background:Wewantedtoachieveathree-dimensional(3D),non-destructiveimagingandautomatic post-analysisandevaluationofreconstructedperipheralnerveswithoutinvolvingcuttingandstaining processes.

Newmethod:Weusedalaboratory-basedmicrocomputedtomographysystemforimaging,aswellasa customanalysisprotocol.Thesamplepreparationwasalsoadaptedinordertoachieve3Dimageswith truemicrometerresolutionandsuitablecontrast.

Results:Analysisoftheacquiredtomogramsenabledthequantitativeassessmentof3Dtissuestructures, i.e.,surfacemorphology,nervefascicles,nervetissuevolume,geometry,andvascularregrowth.The resultingdatashowedsigniﬁcantdifferencesbetweenoperatedanimalsandnon-operatedcontrols.

Comparisonwithexistingmethods:Ourapproachavoidsthesamplingerrorassociatedwithconventional 2Dvisualizationapproachesandholdspromiseforautomationoftheanalysisoflargeseriesofdatasets.

Conclusions:Wehavepresentedapotentialwayfor3Dimagingandanalysisofentireregeneratednerves non-destructively,pavingthewayforhigh-throughputanalysisoftherapeuticconditionsoftreating adultnerveinjuries.

Abbreviations:NC,nerveconduit;␮CT,X-raymicrotomography;ROI,regionofinterest;SNR,signal-to-noise-ratio.

∗Correspondingauthorat:BiomaterialsScienceCenter,DepartmentofBiomedicalEngineering,UniversityofBasel,Gewerbestrasse14,4123Allschwil,Switzerland.

∗∗Correspondingauthorat:CenterforBioengineeringandRegenerativeMedicine,DepartmentofBiomedicalEngineering,UniversityHospitalBasel,Spitalstrasse21/Peters- graben4,4031Basel,Switzerland.

∗∗∗Correspondingauthorat:BiomaterialsScienceCenter,DepartmentofBiomedicalEngineering,UniversityofBasel,Gewerbestrasse14,4123Allschwil,Switzerland.

E-mailaddresses:[email protected](B.Müller),[email protected](S.E.Hieber),[email protected](S.Madduri).

https://doi.org/10.1016/j.jneumeth.2017.11.015

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38 C.Bikisetal./JournalofNeuroscienceMethods295(2018)37–44 1. Introduction

Peripheralnerveinjuriesconstituteamedicalproblemofcon- siderablesigniﬁcance, both intheclinic,aswellas inresearch.

Reportedcasesshowanincreasingprevalence,withseveralhun- dred thousand new patients affected annually (Kingham and Terenghi,2006).Furthermore, thecapacity of thenervous sys- temforself-healingisapriorilimitedcomparedtoothersystems (Hsuetal.,2013).Takentogether,thesetwofactorscontributetoa substantialsocio-economicalburden,associatedwithworkleave, healthcareexpensesandchronicdisability.

Asfarastheexistingtherapeuticinterventionsareconcerned, end-to-endsuturingandautologousnervegraftingarethecurrent choicesoftreatment(Haftek,1976;Stangetal.,2005).Neverthe- less,axonalregenerationveryoftenremainschallengingandthe functionaloutcomeisunsatisfactory.Indetail,end-to-endsutur- ingleadstoareducedstretchingcapacityinalmostoneoutoffour cases,duetomorphologicalandmicro-anatomicalalterationsat thesiteoftheintervention.Thesedrawbacksincludetheeffects of Wallerian degeneration,as well as ﬁbrosisand tissue adhe- sionsoccurringbotharoundandinsidethenerve(Kannanetal., 2005). When the direct end-to-end suturing is not an option, nervegraftingis needed,autologous graftsbeingthegoldstan- dard.Nevertheless,thisapproachisstill associatedwithseveral shortcomingssuchasscarformation,donorsitemorbidity,sizeand modalitymismatch(Johnsonetal.,2005;Weisetal.,2012).

Itisforalltheabovereasonsthatresearchonbiodegradable nerveconduits (NC)hasgained increasingimportanceover the last30years(MadduriandGander,2012).Avarietyofmaterials areusedwithorwithoutgrowthpromotingagents,allowingfor thenervetoregrowinsidetheNC.However,fullfunctionalrecov- eryitselfremainsanunmetchallenge(Yangetal.,2007;Moore etal.,2009).Furthermore,thereturningof functiondependsto someextentontheexact3Dmicroanatomyofthenerve,aswell asthemorphologyoftheconduit-nervecomplex.However,histol- ogy,whichisthegoldstandardofassessinganatomicalrecovery, is inherently2D. Therefore, it shouldideally be complemented bya compatible, 3D approach. Currently, ultrasonography(US) andmagneticresonanceimaging(MRI)arebeingusedinclinical practiceforthe3Dimagingofperipheralnerves,inparticularfor diagnosisandformonitoringperipheralneuropathies(Gargetal., 2017;Wu etal., 2017;Walker,2017;Willsey etal.,2017).The spatialresolution,however,islimitedtoafractionofamillime- ter(Willseyetal.,2017).The3Dimagingofperipheralnervesby USandMRIwithtruemicrometerresolution,however,remains elusive.

Towards this goal, recent developments include the visualization of rat sciatic nerves by a laboratory micro computed tomography (␮CT)system,using Lugol’siodine (Hopkins et al., 2015;Pixleyetal.,2016).Aimingtoeliminatetheneedforacontrast agent,wehaveillustratedtheuseofalaboratory␮CTsystemforthe visualizationandquantiﬁcationofunstained,parafﬁn-embedded reconstructednervesinsideacollagenNC(Bikisetal.,2016,2017).

Thevisualizationisperfectlycompatiblewithhistologicalstudies basedonparaffinembedding.Inparticular,thenon-destructive microcomputed tomographymeasurements canbecarried out directlyafterparaffinembeddingandpriortothehistologicaleval- uation,withtheonlyaddedprocessstepofmeltingthecylindrically shapedparaffin-embeddedtissuetoobtainthestandardhistolog- icalparaffinblockforthesubsequenthistologicalsectioning.

In this study,we have improvedboth thespatial resolution andcontrast,achievinghigh-resolution3Dimagingofregenerated nervesbyusingadvancedlaboratory-based␮CT,wherethespatial resolutionwasbetterthanthedistancebetweenneighboringslices fortypicalhistologicalsectioning.Forthis,wepreparedcollagenNC andimplantedtheminratstobridgea10mm-longsciaticnervegap

injury.Threemonthspost-operatively, regeneratednerve tissue wasexplantedandprocessedforimaging.Theincreaseinspatial resolutionwascoupledwithanincreaseinthecontrastdifference betweenanatomicalstructures,owingtoanimprovedscanning anddatatreatmentprocedure.Thus,theenhancedimagequality appearstobesufficienttoassesstheregeneratedperipheralnerves ina truly3Dway,downtothetruemicrometerlevel. Hopkins etal.(2015)havealreadydemonstratedaclearrelationbetween theanatomicalfeaturesofexaminednervesrecognizedinmicro computedtomographyandhistology.Asconsequence,theauthors havefocusedthestudyonthethree-dimensionalCTimages.For theinvestigatednerves,surfacelandmarksofthesamplecanalso beidentified,allowingfortheselectionoftheoptimalcuttingplane forthehistologicalslices(Stalderetal.,2014).Thisstudypavesthe wayforthestandardizationofthenon-destructivemonitoringof regeneratednervesandeventuallyforhighthroughputanalysisin thefieldofnerveregeneration.

2. Materialsandmethods 2.1. NCfabrication

Collagen NCs were produced using spinning mandrel tech- nology,asillustratedpreviously(Maddurietal.,2010).Insoluble collagen (2.5%, w/w) was swollen in 1M acetic acid and then homogenizedwithahigh-speedmixerat10,000rpm(Polytron^®, Kinematica, Lucerne, Switzerland) for a duration of 1min. The homogeneouscollagendispersionwasappliedviaasyringeonto aspinninggold-coatedmandrel(diameterof1.5mm),installedin asidewaysreciprocatingapparatus,andthesolventwasdriedoff underlaminarairﬂow.Subsequently,theresultingtubeswereneu- tralizedbyincubationin0.1Mdi-sodiumhydrogenphosphate(pH of7.4)for1h.Thetubeswereﬁnallycutinto14mmlongspec- imens,whichwerecross-linkedbysubjectingthecollagentubes toadehydro-thermaltreatment(DHT)atatemperatureof110^◦C anda pressure of20mbar for aperiod of5 days.Theresulting tubesexhibitedanouterandinnerdiameterof2.5mmand1.5mm, respectively.

2.2. Animalexperiments

Allanimalsweretreatedincompliancewiththeethicalpermis- sionapproved(25,212)fromtheVeterinaryOfﬁceoftheKanton Basel-Stadt(Basel,Switzerland).ThreefemaleSpragueDawleyrats weighing250–300g,abouteightweeksold,werehousedunder standardtemperatureandlightconditions.Leftsciaticnervewas surgicallyoperatedfor creating10mmgap.Theresultingnerve gapwasbridgedusing14mmcollagenNC(Maddurietal.,2010) andexplanted12weekspost-operativelyforoutcomeanalysis.The normalnerves werecollectedfromtheun-operated sideofthe sameanimals,thusrespectingthe3Rsoftheanimalexperimen- tation.

2.3. Standardhistologicalprocessing

Afterexcision,theperipheralnerveswereprocessedfollowinga standardhistologyprotocolforformalinfixation–paraffinembed- ding.Indetail,afterexcision,thenerveswerestraightenedbyfirmly tuggingthemfrombothendsbymeansofsurgicalforceps,fixed inhistology-gradeformalin,anddehydratedinascendingethanol solutions.Subsequently,theyweretransferredtoxylolandthen perfusedin aliquid paraffin–polymermixture(Leica Paraplast).

Thelonghealthynervewascutintwopartsandresultingsegments wereplacedlongitudinallyawayfromeachother,thuscreatingthe gapbetweenthetwonervesegmentsthatmayrepresenttheartiﬁ- cialnervegapcreatedinthepresentstudy.Unlessotherwisestated,

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suchgapwasonlyforexperimentalcharacterizationandexcluded fromthefurthermeasurementanalysis.

2.4. ParafﬁnsampleprocessingspeciﬁcforX-rayimaging

Whenliquidparafﬁnperfusionwascompleted,thenerveswere removedfromthehistologicaltissueprocessor,placedinametal containerandleftfor24hinsideanovenatatemperatureof60^◦C.

Thisstep is important in removingthe majority ofair bubbles trappedinside oraroundthespecimen,thatcancause artifacts duringtheX-rayimagingandmakedataanalysismorecomplex.

Afterwards,thespecimenswerethoroughlywashedunderflowing liquidparaffin,toremovehigh-absorbingparticlesordebrisonthe samplesurfacethatwouldaffectimagingquality.Finally,thenerve wasdippedinparaffinseveraltimeswhileholdingfromoneedge, untilauniformcylinder-likespecimenwasformedandthencooled downto4^◦Cduringaperiodof15min.

2.5. Mountingofsamplesonholderscompatiblewiththe laboratoryCTsystem

Intheend,theparafﬁnsamplesweregluedtospecializedmetal sampleholdersusingacyanoacrylateglue.Carewastakentoensure thatthegluewouldcoverasmallareaofthenervebottom.Handling ofthespecimenwasperformedexclusivelybyforcepstoavoid depositsonthesamplesurfacethatwouldcauseimagingartifacts.

Afterthegluesettled,anyexcesswasremovedcarefullybyascalpel andthespecimenswerereadyfortheX-rayimaging.

2.6. Tomographymeasurementsandreconstruction

FortheX-raytomographymeasurements,weusedthe␮CTlab- oratorysystemphoenixnanotom^®m(phoenix|X-ray,GESensing&

InspectionTechnologiesGmbH,Wunstorf,Germany).Anaccelera- tionvoltageof60kVandabeamcurrentof280mAwereselected fortheoperationoftheX-raybeam.Overanangularrangeof360^◦, 1800projectionswereacquired.Theeffectivepixelsizewas4␮m andscanningtimewas2.5hfora1cm-longnerve.Afterreconstruc- tion,the16-bitdatasetisapproximately2GBinsize.

2.7. Datahandlingandvisualization

Fortheorientationwithinthelargedatasetsacquired,acom- merciallyavailable software is needed. The VGStudio MAX 2.1

(Volume Graphics GmbH, Heidelberg,Germany) was thusused bothforthethree-dimensionalrenderingandthevisualizationof theacquiredtomograms,cp.imagesinFigs.1and5.

2.8. Dataanalysis

Theentireprocess oftheautomatedanalysisis explainedin detailinBikisetal.(2017).Shortly,thenervesweresegmentedfrom theirsurroundingsbymeansofanintensitythresholding/median filtering/connectedcomponentanalysis.Afternervesegmentation, extractionoftheinvestigatedparameterswasperformedinafully automatedway.Forthesetasks,Matlab(MATLAB2016a,TheMath- Works,Inc.,Natick,Massachusetts,UnitedStates)wasused.The diagramsinFig.3wherepreparedusingtheproFitsoftware(pro Fit6.2.16,QuantumSoft,UetikonamSee,Switzerland).Forthesta- tisticalanalysis,thedatawereexpressedasmeans±SE.Student’st testwasusedtodeterminestatisticalsignificanceofdifferences (p<0.05 was considered statisticallysignificant). The statistical analysiswasperformedinGraphPadPrism7.00forMac(GraphPad Software,LaJollaCaliforniaUSA).

3. Results

3.1. Surfacemorphology

Aninherentadvantageof tomographywithrespecttotradi- tional 2D imaging approaches is the isotropic voxel resolution provided,enabling accuratesurfacereconstruction,bymeansof 3Drendering,whichisexemplarilydemonstratedinthepresent study.InFig.1,ahealthyandaregeneratednervearerendered, withtheirproximalanddistalendslocatedat2mmfromtheright andleftbordersoftheimages,respectively.

Forthereconstructednerve,startingnearitsproximalend,the markingsuturesandthescarringthathasoccurredaroundthem arevisible(yellow-coloredarrow).Musclefibersattachedtothe nervesurface(white-coloredarrow)arealsovisibleclosetothe proximalnerveend.Themiddlesectionofthereconstructednerve showsareduceddiameterwithrespecttotheends.Alineplotof thecrosssectionalareaispresentedbelow.Thedistalnerveendis presentedinlargermagnificationinFig.1(b,bottom),whichshows howthefibrinoidtissueenvelopsthesutures.

For theoverview image of the healthy nerve (Fig. 1a, top), presentedforcomparison,themostprominentdifferencetothe reconstructednerveisthemajorsurfacegroove(redarrow)caused

Fig.1. Threedimensionalvisualizationoftomographydatafromahealthy(a)andareconstructednerve(b),withalengthofapproximately8and15mm,respectively.The surroundingparafﬁnhasbeenmadedigitallytransparentpriortothis3Drendering.Thered-coloredarrowindicatesthesurfacegroovebetweenthemajornervefascicles andthewhite-coloredarrowsshowmuscleﬁbersonthesurfaceofthenerves.Theyellow-coloredarrowsindicatethetwoendsofthereconstructednerve.

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40 C.Bikisetal./JournalofNeuroscienceMethods295(2018)37–44

Fig.2. Crosssectionalandlongitudinalviewsofhealthy(a)andregenerated(b)ratsciaticnervesrespectively,togetherwithselectedlineplotsandhistogramsofselected regionsofinterest(c).Thegreen,redandbluedashedlinesandsubfigureframesshowtherelativepositionofthedepictedcutsthroughthe3Ddatasets.Theincludedline plotsaretakenalongtheorange-coloreddashedlines.Inthelongitudinalviews,theproximalanddistalendsofthenervesarelocatedat2mmfromtherightandleftborders oftheimages,respectively.Intheseviews,onerecognizesthatthehealthynerveiscomprisedoftwoparts,whichshowonlyasmallgapandareembeddedintoparaffin together.TheblackdotsinthehistogramindicatethenumberofpixelscorrespondingtoaspecificattenuationvalueandthecurvesareGaussiansfittedtotheexperimental data,correspondingtoair(red),paraffin(green)andnerve(blue).Theattenuationvaluesarerelatedtothecurrentsetupanddonotrepresentthephysicalquantityusedfor monochromaticradiation.Thescalebarscorrespondtoalengthof1mm.

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bytheexistenceoftwomainnervefascicles.Moremuscleﬁbers areseenattachedtothenervesurface(white-coloredarrow),com- paredtotheregeneratednerve.Themeandiameterofthesemuscle ﬁbersisaround50␮m,inreasonableagreementwiththeliterature values(AlnaqeebandGoldspink,1987).Azoom-inontothesurface ofthehealthynerve(Fig.1a,bottom)betterrevealstheirrod-like structureandparallelarrangement.

3.2. Structuralanalysisofreconstructednerve

Anotherdistinctive advantageof3Dtomographicdataisthe abilitytoselectanyvirtualcuttingplane,asillustratedinFig.2, wherethreeorthogonalcuttingplanesatonceareusedtobetter revealtheinternalstructuresandthedegreeoforganizationofthe nervesinvestigated.Thewhitecolorstructuresappearinginlongi- tudinalandcross-sectionalimagesofbothnervesamplesindicate theaxonsandassociatedmyelintissue(Hopkinsetal.,2015;Pixley etal.,2016).Fission-like-structuresexistinginthecrosssections showtheorganizationofthenervefasciclesinallthetissuesam- ples.Theepineuriumandperineuriummembranesaswellasthe vesselsaredepictedwhite,duetoahigherlocalX-rayabsorption, whilethesurroundingmedium(air)isshownasblack,duetomin- imalX-rayabsorption.Parafﬁnsurroundingthenervesisshownin gray,accordingtothelocalX-rayabsorption.

For the reconstructed nerve data, the cross-sectional view obtainedfromitsproximalend,revealstheexistenceoftwomain nervefascicles,dividedbyoneprominentperineuriummembrane.

Nomajorvesselcanbedetectedinthiscross-sectionalview,aseasy asinthehealthynerve(Fig.2).Inaddition,theconnectivetissue surroundingthefasciclesisenvelopingthemmorecloselythanfor thecaseofthehealthynerve,whereboththeconnectivetissueand theepineuriummembraneareseparatedfromthefourmainnerve fasciclesvisibleandatplacesshowdifferingX-rayabsorption.

Thelongitudinalviewsclearlyrevealedtheinternalstructures suchasthefascicleorientationand3Dorganization.Intherecon- structednerves,thefasciclesarenotaseasilydistinguishable,asitis thecasefortheirhealthycounterpart.Moreover,thereconstructed nerveshowsa randomarrangementoffascicles, that,especially arounditsproximalanddistalendbecomessocomplicatedthat someofthemareperpendiculartothemajornerveaxis.Bothnerve thicknessanddegreeoffascicleanisotropyaremaximizedatthe proximalanddistalnerve ends,comparedtoitsthinnermiddle section.

Fig. 2 also shows two line plots through the selected cross sectional cutsof the 3Ddataset, indicating a spatial resolution close to the effective pixel size for both measurements. His- tograms of thelocal X-rayabsorption fromselected regions of the two datasets are also presented. They contain three Gaus- sianpeakscorrespondingtoair(red),paraffin(green)andnerve tissue (blue), which can be identified by intensity threshold- ing (Müller et al., 2002). The area under the histogram curve includes all voxels of the selected region of interest (ROI), and the area under each peak provides the volume of its related specimen component. For our measurements, the fact that the Gaussians fit the data very well indicates the mean- ingful applicability of photon statistics. For each Gaussian, the expectation value gives the peak position corresponding to a specific component and the standard deviation character- izes howbroad the distributionis. Based onthese parameters, thesignal-to-noise ratio(SNR)of each specific componentwas calculatedas:SNR(component)=|component−air|/air.TheSNR forbothparaffinandnervewasalmostequalforbothmeasure- ments (SNR_healthy(paraffin)=34.31±0.02; SNRregenerated (paraffin)=37.12±0.02, SNR_healthy(nerve)=47.99±0.03; SNRregenerated

(nerve)=44.74±0.03).

Fig.3. Crosssectionalareaalongtworepresentativenerves.Thehealthynerve showsanalmostconstantvalue,whereastheregeneratednerveexhibitscharac- teristicmodulations.

Fig.4. Comparisonofgeometricalparametersforthreehealthyandthreeregen- eratednerves,calculatedautomaticallyafternervesegmentation.Singleasterisk indicatesap-valuesmallerthan0.05anddoubleasteriskindicatesap-valuesmaller than0.01.

3.3. Nervethicknessproﬁle

Afterthenervehasbeensegmentedfromitssurroundingparaf- ﬁn,plottingthecross-sectionalareaoverthenervelengthprovides usefulinformationonthenerveregenerationprocess,asseenin Fig.3,where onerepresentative healthyand onereconstructed nerve arepresentedfromeachgroup.Therearetwodistinctive peaksinthecurveofthereconstructednerve,indicatingtheprox- imal(left) anddistal(right)end.Theregionbetweenthesetwo peakscorrespondstothereconstructednervesegment.Forthecase ofahealthynerve,itsthicknessproﬁlerevealedbymeasurement ofcross-sectionalarearemainsrelativelyconstantalongitsentire length.

3.4. Nervegeometry

We have alsocalculated several geometrical parameters for thenerves investigated, in anautomated process. Fig.4 shows theresultsofthetwogroupsofhealthyandreconstructednerves (n=3),aswellastheircomparison.Theparameterschosenforthe investigationwerethemedianand maximumcrosssection,the averagecrosssections(definedasvolume/length),thehalfmid- rangecross-section(definedasthedistanceofmaximumtothe median)andthecrosssectionabsmid-range(definedasthesum of thedistanceof each cross sectionalong the nervelength to

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Fig.5. Intensity-thresholdedvesselsofahealthy(a)andareconstructed(b)nerve,withalengthofapproximately3and7mm,respectively.Thesurroundingparafﬁnhas beenmadedigitallytransparentandthenervevolumehasbeenmadesemi-transparenttoallowforabettervisualizationofthevesselstructure.Thediameterofthevessels indicatedbythewhite-coloredarrowsisapproximately0.04and0.02mmforthehealthyandthereconstructednerve,respectively.

themedian).Indetail,astronglystatisticallysignificantdifference wasobservedbetweenthereconstructedandthehealthygroup forthemedian cross section(p=0.0026). Therewasalsoa statisticallysignificant differencebetweenthe two groups for the averagecrosssection(definedasvolume/length)andthecrosssec- tionabs(absolute)mid-range(definedasthesumofthedistance ofthecrosssectionateachpositionalongthenervetothemedian crosssection),withp=0.0178andp=0.0419,respectively.Finally, atendencyforastatisticallysignificantdifferencebetweenthetwo groupswasfoundforthemaximumcrosssection(p=0.0890).

3.5. Vascularregrowth

Vesselsshow a higher intensity valuethan parafﬁn or ner- voustissue,namelyaround44,000forthehistogramspresented inFig.2.Duetoalownumberofvesselvoxelsthehistogramdoes notreveal themseparately. Thevesselnetwork is visualizedin Fig.5usingthresholding.Intensitythresholdingrevealedinterest- ingobservationsassociatedwithvascularregrowth.Theresultsof thissegmentationprocessareseeninFig.5,revealingthevascular networkofa healthy(Fig.5a)andreconstructed(Fig.5b)nerve, respectively.In detail, forthe caseof thereconstructed nerves, themeandiameterofthedetectedvesselsissmaller, compared tohealthy nerves. In addition, a quick overview of the recon- structednerve (Fig.5b)revealedthe existenceof severalsmall signalscharacterizedbyhighX-rayabsorptionattheproximaland distal nerveends (green-coloredarrows). These structuresmay indicatehaematomas.

Insidethereconstructednerve,themorphologyandorganiza- tionofvesselsappearedtobeheterogeneous.Thevisualinspection ofthehigh-resolutionCTdataindicatesthatthenerveendscontain alessorganizedvesselstructureandhaverandomorientation.Itis difﬁculttoprovethisobservation,sincethenumberofsegmented vesselsisrelativelylow.Vesseldensityisalsohighernearthetwo endsofthenerve.Incontrast,theregionbetweentheproximaland distalpartsofthereconstructednerveexhibitsamoreorganized

vesselstructure;vesselsaregenerallybiggerindiametercompared totheonesatthenerveendsandarelessfrequentlybranched.They aremostlyparallelonetoanother,followingthenerve axisand residingveryclosetoitssurface(magenta-coloredarrow).

4. Discussion

4.1. Spatialresolutionandimagecontrast

ThelineplotspresentedinFig.2runalongtheparaffin/airedge andwereusedtodeterminethespatialresolutionclosetothepixel size,asdescribed earlier(Thurner etal.,2004).Fromthemean andstandarddeviationvaluesoftheGaussiansfittedtothehis- togramspresentedinFig.2,theSNRforthemicrostructureswas calculated(Schulzetal.,2012).TheSNRwasalmostidenticalfor thecaseofparaffin(consideredtobeahomogeneousstructure)in bothmeasurements.Togetherwiththefactthatspatialresolution wasalsofoundsimilarforbothmeasurements,scanningquality isconsideredconsistent.ComparedtoUSandMRI,whicharethe modalitiesof choiceforperipheralnerve3Dimaging inclinical practice(Willseyetal.,2017), thespatialresolutionachievedin thisstudyisatleasttwoordersofmagnitudebetterineachofthe threeorthogonaldirections.Comparedtorecentsimilar␮CTstud- ies,e.g.Hopkinsetal.(2015),Pixleyetal.(2016),spatialresolution isatleastanorderofmagnitudebetterineachorthogonaldirection.

Althoughourapproachneedsnostainingagentatall,theobtained tomogramcontrastisalsogreatlyincreased,allowingforthedis- crimination between nerve fascicles and connective tissue, an unsolvedproblemforsimilarstudiesupuntilnow(Hopkinsetal., 2015).Thesuperiorimagequalitymakesevenanautomatednerve segmentationandextractionofquantitativeparameterspossible.

In this study, the mostnotable difference betweenthe two measurementsisthebetterseparationoftheparafﬁnandnerve peak of thehistogramsfor the case ofthe healthy nerve com- paredtotheregeneratedone,asseenintheimagesofFig.2.We have alreadysome indicationsthat duringthe ﬁrstdays ofthe

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samplebeingembeddedinparafﬁn,contrastdifferencebetween structuresincreasesasthetimepasses,mostprobablyduetoan ongoingperfusionprogress.Itisalsopossible,thatacombination ofanexistingdifferencebetweengroupsandapreparationeffect couldhavetakenplace; onehypothesisis,thattheregenerating nerve,duetolesscompactbundlepackingormyelinmaturity,is physicallylessstableandthereforegetsmoreaffectedduringthe dehydrationandsubsequentparafﬁnperfusion.

4.2. Thicknessproﬁlealongthenerves

DiagramsasshowninFig.3canbeusedtoassessthelengthof thezonesintheregeneratingnerves,aswellasquicklyrevealpos- siblesegmentationerrors.Forexample,thenumeroussmallspikes ofthecross-sectionalareaalongtheendofthehealthynervewere causedbysuboptimalsegmentation,duetoconebeamartifacts.

While the fast ﬂuctuations in the amplitude are caused by improper segmentation, the high and slow modulation of the regeneratingnerve crosssection comparedtoits healthycoun- terpartcanbeexplainedbythemechanismofnervedamageand regeneration itself.Anincreasedcross-sectional areais seen,as expected,in the proximaland distal endsof the reconstructed nerve,whichmayindicateoccurrenceofhemorrhage,inﬂamma- tionandscarring(Fig.3).Alsoasexpected,theregeneratedareais foundtoberelativelythinner,asseenforthecaseofthesampledis- playedinFig.1b.Thetwobundlesobservedinthecross-sectional viewarenotwellseparatedandareultimatelyjoinedfurtheralong thenerve,sothatatthethinnestpartoftheregeneratedregion,all fasciclesareenclosedbyonesingleperineuriummembrane.

4.3. Geometryofreconstructednervetissue

Itshouldbenotedthatthecalculatednervecross-sectionalarea wastakenwithrespecttotheplaneperpendiculartotherotation axisofthesampleandnottothenerve centerline.Forourcase this onlyhas aminor effecton theresults, becausethenerves werestraightenedas muchas possibleprior toembeddingand thenmountedwiththemajornerveaxisparalleltotherotation axis.Eventhoughthemediancrosssectionhasthestrongestdif- ferencebetweenthetwogroupsintermsofstatisticalsigniﬁcance, theaveragecrosssection(volume/length)cansometimesbeamore preferableparameterforcomparison,sinceitismorerobustwith respecttohowthesampleisembeddedormounted.

Crosssectionabsmid-rangeisamorerobustparametercom- paredtothehalfmid-rangecrosssectionbecauseit ignoresthe intra-groupdifferences in nervesize andis directlyaffectedby theexistenceoftransitionzonesintheregeneratednerves.This isbecausetheformertakesthedifferenceofthecrosssectionat everypointalongthenervecomparedtoitsmediancrosssection, whilethelaterismerelythedifferenceofonlytwovalues,namely maximumandmediancrosssectionofthenerve.

Tobeabletocomparegeometricalparameters,allmuscleor connectivetissuearoundthenervehastobeclearedasmuchas possibleduringextraction.Theregeneratednervesmustalsobe cutwithsimilarendpoints,e.g.usingthestitchesasguides.

4.4. Interpretationandchallengesofvesselanalysis

ThevisualinspectionoftheCTdataindicatesthatvesselssmaller thanthevoxelsizecanbedetectedduetothepartialvolumeeffect.

Theirhighintensityismainlyaresultoftheremainingdehydrated bloodinthevessellumen.Anelaborate,automatedsegmentation oftheentirevesseltreeremainsanopenchallenge.Giventhatno contrastagentwasused,thenon-connectedvesselnetworkvisual- izedcanbecausedbyinhomogeneousvesselperfusionbyparafﬁn, probablyduetotrappedairinsidethevessels.Nevertheless,there

isacleartendencyformore,shortervesselfragmentsintherecon- structednerveendsthatcanbeexplainedbytheangiogenesisat thissitesasaresponsetotraumaandasabasis forsubsequent regeneration.Inadditiontothat,itisveryprobablethatthehighly X-rayabsorbingfeaturesﬁlling largeareasatthereconstructed nerveendsarecausedbyhaemosidirindepositsduetohemorrhage attheseareas.

5. Conclusions

We have achieved a 3D visualization of regenerated nerves withtrue micrometerresolution usinga procedurethat allows subsequentcuttingandstaining.Finedetailsofmorphologyand microanatomyofnervetissuewerevisualized,owingtoimproved imagingprocedureandanupdateddataprocessing.Themethod hasbeenvalidatedwithasufficientnumberofsamplestoprovide findingswithstatisticalsignificance.Thequantitativeparameters ofregeneratednervesdetailedinthisstudyprovideasuitablemea- sureforassessingthequalityofnerveregenerationwithafocuson high-throughputanalysis.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictofinterest.

Acknowledgments

TheauthorsthankStephanFrankandJürgenHenchoftheNeu- ropathologyDepartment,BaselUniversityHospitalforallowingthe useofhistologicalequipment,aswellasSaschaMartinandStefan GentschoftheMechanicsShop,PhysicsDepartment,Universityof Basel,forfabricatingthecustomizedsampleholders.Theproject wassupportedbySNSFprojects144535and133802.

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