Vector analysis of porosity evidences bone loss at the epiphysis in the BTX rat model of disuse osteoporosis

Vector analysis of porosity evidences bone loss at the epiphysis in the BTX rat model of disuse

osteoporosis

D. Chappard * , H. Libouban

GEROMGroupeEtudesRemodelageOsseuxetbioMatériaux–LHEA,IRIS-IBSInstitutde Biologieen Santé,CHU d'Angers,49933AngersCedex,France

1. Introduction

Botulinumtoxin(BTX) injectionsare now routinelyusedin clinicalpracticetotemporarilyparalyzemuscleactivity.¹This leadstoatransientmuscleparalysiswhichisfullyreversible

in afew months.^2–4BTX typeA isproduced byClostridium botulinum that courses botulism, a food poisoning. BTX specifically inhibitsthe acetylcholine release at the neuro- muscularsynapse.BTXwasinitiallyproposedforitsabilityto reduce facial wrinkles.⁵ BTX injections have also been proposedtotreatmusclespasmsinvariouslocations.⁶ article info

Articlehistory:

Received4September2015 Accepted25April2016 Availableonlinexxx

Keywords:

BTX

Disuseosteoporosis Epiphysis

Vectoranalysis Porosity

abstract

Introduction: Botulinumtoxin(BTX)injectedinamusclecausesparalysiswithasubsequent bone loss. It represents a modelof disuse osteoporosis. Although boneloss has been regularlyevaluatedatthemetaphysisoflongbones,littleisknownconcerningthebone changesoccurringintheepiphysis.

Animalsandmethods: TenCopenhagenmaleratsreceivedasingleBTXinjectionintheMus quadricepsfemorisontherightsideandunilateralparalysisdevelopedinthefollowingdays.

Animalswereeuthanizedafter28days;femurandtibiawereharvestedandanalyzedby microCT.Vectoranalysisofporositywasappliedtothe2Dsectionsandproducedafrontal imagewithmappinginpseudo-colors.Thisallowsquantitativeanalysisattheepiphysisand metaphysis.‘‘Hotspot’’wereevidencedandindicatedboneloss.Quantitativeanalysisof theseimageswasdonebydecompositionoftheR,GandBplanesandderivingtheratioofR +Gpixelsonthewholepixelnumber.

Results:Atthemetaphysis,thisratiowascorrelatedwithmeasurementofthebonevolume obtainedbymicroCT.Attheepiphysis,whichhasacomplexshapein3D,themethodeasily identifiedtheboneloss.

Discussion: Paralysisofaunilateralquadricepsinducesbonelossatthemetaphysisofthe longbones.However,theepiphysis,havingareducedboneremodelingisalsoconcernedby disuse.MicroCTanalysisofthispartofthebonesisdifficultduetoitscomplexshapein3D.

Vectoranalysisisanewandrobustmethodtoquantifybonelossinsuchcomplexareas.

#2016AnatomicalSocietyofIndia.PublishedbyElsevier,adivisionofReedElsevier India,Pvt.Ltd.Allrightsreserved.

*Correspondingauthorat:GEROM–LHEA,IRIS-IBSInstitutdeBiologieenSanté,CHUd'Angers,UniversityofAngers,49933Angers Cedex,France.Tel.:+33244688349;fax:+33244688351.

E-mailaddress:[email protected](D.Chappard).

Availableonlineatwww.sciencedirect.com

ScienceDirect

journalhomepage:www.elsevier.com/locate/jasi

http://dx.doi.org/10.1016/j.jasi.2016.04.002

0003-2778/#2016AnatomicalSocietyofIndia.PublishedbyElsevier,adivisionofReedElsevierIndia,Pvt.Ltd.Allrightsreserved.

We reported that a single BTX injection in the mus quadricepscanleadtoarapidbonelossduetothe muscle paralysis.⁷Thebonelosswas evidencedattheuppertibia metaphysisandlowerfemoralmetaphysis.Thesedatawere confirmed inotherlaboratory animals(mice, rabbits,cats, etc.)and byusingvarious methods such ashistomorpho- metry and microcomputed tomography (microCT).^8–11 In mostofthesepapers, the metaphysisisanalyzedbecause itcontainsalargeamountoftrabecularboneinthesecondary spongiosa, immediately under the growth plate. However, bonelossalsoconcernscorticalbonetoalesserextend.^12–15It isnowclearthatmuscleatrophycausedbythetoxinleadsto disusewithasecondaryboneloss.¹⁶BTXisresponsiblefor muscle paralysis in the injected muscle but also in the neighboringmuscles.¹¹

Bonelossinducedbydisuseismoreintenseinboneswitha previoushighremodeling. Inthe rat,boneloss wasfound morepronouncedinthetibialmetaphysisthanatthefemur's one.⁷However,boneremodelingoftheepiphysealbonehas poorly been considered. Trabeculae of the epiphysis are thickerthaninthe metaphysisandtheremodelingrate is lower.^17,18

Thesecharacteristicshavebeenattributedtothecontinu- ousloadingoftheepiphyseswhichpreservesthethicknessof their trabeculaethroughout life.¹⁷ However,the epiphyseal bone is difficult to analyze in rodents due to the reduced volumeofthisarea.Histomorphometricanalysisisnoteasy andmicroCTisdifficultduetothecomplexshapeofthisbony subpart.Moreover,wedevelopedofanewanalysistechnique applied to microCT sections of objects witha complex 3D shape(vectoranalysisofporosity).¹⁹Inthepresentstudy,we conductedavectoranalysisofporosityoftheepiphysesand metaphysesof thefemursandtibaeinaseriesofratswith BTX-inducedparalysisoftheMusquadricepsfemoris.Thebones were analyzed by microCT and the sections were further treatedbyvectoranalysis.

2. Material and methods

TenCopenhagenmaleratsweighing48537gwereusedin this study. Generation of the modelhas been describedin detailelsewherebyourgroup.^7,20Briefly,animalsreceiveda single injection of BTX (Botox^® – Allergan France, 2IU dissolvedin0.4mLphysiologicalsaline)intheMusquadriceps femorisoftherighthindlimb(hereafterreferredastheBTX- injectedside).A0.4mLinjectionofsalinewasdoneintheleft hindlimb(non-injectedside).Paralysisoftherighthindlimb waseffectiveasearlyas2dayspost-injectionandlastupto2 months followed by progressive recovery. Animals were sacrificed28dayspost-injection.Thisprocedurewasapproved bythe Universityof Angersethicalcommittee(Agreements 49028 and01732.01) and performedin accordancewiththe European regulationfor the use of animal inexperimental procedures.

2.2. X-raymicrocomputedtomography

MicroCTanalysiswasperformedontibiaswithaSkyscan1172 microtomograph (Bruker-Skyscan, Kontich, Belgium) equipped with an X-ray tube workingat 70kV/100mA. An isotropicvoxelsizewasfixedat13.4mm,therotationstepat 0.258andexposurewasperformedwitha0.5mmaluminum filter.TheCTAnSoftware(Skyscan,release1.14.11)wasusedto measurethebonemassatthetibiauppermetaphysisandthe lower femur metaphysis after binarization with a global threshold.Thefirstimageselectedfor analysiswaslocated just under the primary spongiosa (characterized by thin trabeculae) and then extended on 300 sections toward the bone shaft. The volume of interest (VOI) was designed by interactivelydrawingapolygononeach2Dsection.Onlyafew

Fig.1–MicroCTimageofbonesofthesameratafterBTXinjectionontherightside.(A)Rightfemur;(B)leftfemur;(C)right tibia;(D)lefttibia.Notethereducedamountofthetrabecularvolumeparticularlyintherighttibiametaphysis.Alsonotethe largertrabeculaeintheepiphysesthaninthemetaphysis.

numberofpolygonsneedtobedrawn(e.g.onthefirstsection, severalatthemiddle,andonthefinalsection)sincearoutine facilitycalculatedalltheintermediarymasksbyinterpolation.

AfirstVOIcontainingonlytrabecularboneandmarrowcavity was drawn and a global threshold was used to selectthe trabeculae.²¹ The trabecular bone volume (BV/TV, in %) representingthepercentageofthecancellousspaceoccupied bytrabecular bonewasdeterminedaccordingtoguidelines andnomenclatureproposedbytheAmericanSocietyforBone andMineralResearch.²²

2.3. Analysisofmandibular3Dporositybyavector projectionalgorithm

Thestacksofbinarized2Dsectionsweretransferredtoalab- madesoftwarewritteninMatlab(MathWorks,Natick,MA) release7.10.Thealgorithmhasbeenextensivelydescribed elsewhere.¹⁹ Before analysis, the sesamoid bones were removedfromtheimagesbecausetheyproduceartifactual results.Onthebinarizedimages,porositywasvisibleinwhite andboneinblack.Foreach(x,y)binarizedimageofthestack, thepores(i.e.whitepixels)whichbelongtothesameimage columnxreceivedthesamepseudo-coloraccordingtoalook uptable(LUT).ALUTisatransferfunctionwhichdetermines whatscreenvaluescorrespondtoimagepixelintensityvalues atallx-ycoordinatesintheimage.Therepresentationofthe LUTappearsinFig.3.A3Dmodelwasreconstructedfromthe subsequent colorized images the ImageJ 3D plugin (NIH, http://rsb.info.nih.gov/ij/index.html). A frontal image was savedinthetifformatwiththecolorizedLUTandanalyzedby ImageJ.Withthismethod,thelargeporousareas(indicating thedisappearanceoftrabeculae)arein‘‘hotcolors’’(i.e.red, orangeandyellow)andareasrichintrabeculaeorcortical boneappearinblue(‘‘coldcolors’’).Themethodtoanalyze quantitativelyRGBimageswasproposedbyseveralauthors andisbased onthe 3histogramsofthe image.^23,24 Inthe additiveRGBcolormodel(Red-Green-Blue),eachimagecanbe decomposedin3planes(red,greenandblue).Thehistogram ofeachbitplanewasobtainedwithImageJandtransferredto an Excel spreadsheet (Microsoft Co). Then, only values comprisedbetween 20 and255 graylevelwereselectedto discardirrelevantdata.Theywereconvertedinpercentofthe wholenumberofpixelsandtheratioRofthe(red+green) pixels to the whole number of pixels was derived. This method allowsdeterminationofthe fractionofthe image containing‘‘hotcolors’’becauseyellowisobtainedbyadding redandgreenpixelsvaluesintheRGBmode.Rwasmeasured separatelyinthemetaphysis(anareacorrespondingtothe BV/TVmeasurementsdonein3D)andtheepiphysisofeach bone.

2.4. Statisticalanalysis

StatisticalanalysiswasperformedusingtheSystatstatistical softwarerelease13.0(SystatSoftwareInc.,SanJose,CA).All Table1–MorphometricanalysisofthetibiaeandfemursbymicroCTandvectoranalysis.

Femur Tibia

BTX-injected Non-injected BTX-injected Non-injected

BV/TV(%) 40.70.5 48.71.3^a 41.10.8 50.40.5^a

Rmetaphysis(%) 57.21.9 47.31.5^a 60.60.8 53.41.3^a

Repiphysis(%) 54.91.9 35.92.9^a 49.01.2 33.01.4^a

a Indicatesasignificantdifferencewiththeoppositeside.

Fig.2–MicroCTimagesofafemoralratepiphysis.The imagewasobtainedbyremovalimagebyimagethe primaryandsecondaryspongiosaonthewholestackof imagesusingPhotoshopCS5(Adobe).(A)Upperview showingthetwocondylesandtheintercondylarnotch.(B) Lowerviewshowingthefourexpansionsoftheprimary spongiosaandtheirimpingementdeepinsidethe epiphysis.(C)Lateralviewwiththepresenceofhornsin variousdirections.

datawereexpressedasmeanstandarderrorofthemean (SEM). Differences between the right and left bones were comparedusingtheMannandWhitney'sUtest.Differences wereconsideredsignificantwhenp<0.05.

3. Results

3.1. AssessmentofbonemassonmicroCTimagesofthe metaphysis

Fig.1illustratesthebonelossinthesetwobonesfromthe same animal. The trabecular bone volume was markedly decreased at the metaphysis of thetwo boneson the BTX injectedside.A 16.4%decreasewasobservedatthefemur anda 18.4%atthetibia(Table1).Fig.2illustratesthecomplex shapeofafemoralepiphysisin3Dafterinteractiveelimination oftheprimaryandthesecondaryspongiosafromthestackof 2Dimagescontaining.Eachimagewasprocessedseparately.It islikelythatitisimpossibletoselectthetrabecularboneinthis area because softwares cannot adapt to such complicated boundaries.

3.2. Assessmentofboneporosityonimageswithvector analysis

Fig.3illustratestheaspectofthefemursandtibiaofthesame rat imaged after vector analysis of porosity. The highest porous zones (corresponding to areas with an increased trabecularresorption)appearin‘‘hotcolors’’i.e.redtoyellow.

Thebonelossiseasilydemonstratedinthemetaphysesof bothbones.Intheepiphyses,thepresenceof‘‘hotspots’’is alsoclearlyevidencedandcorrespondstoaboneloss.Inallthe ratsoftheseries,themaximumareaswithincreasedporosity wereobservedattheinnerfemoralcondyle.Inthetibia,the

‘‘hottestareas’’werealsoobservedattheinnerplateau.

ThedifferenceinR reached20.9% at the femoral meta- physisand13.5%atthetibialmetaphysis.Intheepiphyses,the differencewas52.9%atthefemurand48.4%atthetibia.

The histograms of the Red, Green and Blue planes of metaphysealROIsintheinjectedandnon-injectedsideofa femurappearinFig.4.Onthenon-injectedside,thehistogram frequencyexhibitsalargepeakforthebluecomponentwhile Fig.3–AnalysisofthesamebonesthaninFig.1,byvectoranalysis.Thepresenceof‘‘redspots’’indicatesareaswhere porosityisincreased(A)rightfemur;(B)leftfemur;(C)righttibia;(D)lefttibia.TheLUTappearsontherightsideofthefigure.

1 2 3

Percent

20 100 180 255

0 1 2 3

Uninjected side

BTX-injected side

A

B

Fig.4–TheRed,GreenandBluehistogramsofthe metaphysisofthefemurin(A)theun-injectedside(left limb)and(B)theinjectedside(rightlimb).Notetheincrease numberofredpixelsinthehistogramandthereductionin thepercentofbluepixelsafterBTXinjection.Each histogramisfiguredwithitsrepresentativecolor.

thegreenandredhistogramsaremoreflattened.OntheBTX- injected side (Fig. 4B), the peak of the blue histogram is markedlyreducedandtheredhistogramexhibitsalargepeak.

TheRratioisillustratedinFig.5andTable1inthedifferent bones and their metaphyseal and epiphyseal subparts.

Noteworthy,BV/TVmeasuredbymicroCTand R,estimated at the metaphysis were well linearly correlated (r= 0.65;

p=0.001).

4. Discussion

Striated muscles are under the control of the voluntary nervous system which controls body movements. The motoneuronsofthespinalcordtransmitinformationtothe muscles viatheir axon whichends atthe neuromuscular synapse.Acetylcholineistheneurotransmitterreleasedby the axon and BTX blocks its release by destroying the intracellular SNARE system involved in the fusion of the synapticvesicleswiththe cellmembrane.^2–4Theparalysis occurs within 2 days after injection ofthe toxin and the musclefunction recovery is observed within two months.

Paralysiscausesdisuseoftheinjectedlimbleadingtoarapid boneloss.⁷TheeffectofBTXonbonelossiscumulativewith otherfactors knownto reduce bonemass such ascastra- tion.^25,26orhindlimbunloading.²⁷Atthebonecelllevel,bone loss is due to a sudden increase in osteoclast number associated with a progressive decline of the osteoblastic formationofnewbone.²⁸Sincetheoriginaldescriptionofthe model, at least 40 publications have been presented and pharmacological or physical counter measures have been evaluated.²⁹However,uptonow,littleconsiderationhasbeen paidtotheepiphysesbecausethisbonesubpartisdifficultto analyze.Becauseoftheheterogeneityintrabecularreparti- tionandtheverycomplexshapeofthisbone(seeFig.2),avery limitednumberofstudieshavebeendoneonbonelossinthis areainanimalmodelsofosteoporosis.Inthepresentstudy, weperformeda‘‘virtualdissection’’ofafemoralepiphysisto showthe3Dcomplexityofafemoralepiphysisandtoexplain whyitisimpossibletoselectthewholetrabecularboneinside

the cortical shell of the epiphysis.Although somestudies havepresenteddataonboneremodelingattheepiphysisin laboratoryanimals,theyaremostoftenconcernedwith2D microCT images,³⁰ histological sections,^31,32 or scanning electronmicroscopicimages.³¹Toourknowledge,onlytwo papershavepresentedaquantitativeanalysisoftrabecular bonebymicroCTattheepiphysisbutdeterminationofthe VOIwaspoorlydescribedandno3Dmodelwaspresented.^33,34 Here,thebonelossinducedbytheBTX-relateddisusewas confirmed at the metaphysis of mature rat bones by a quantitativeanalysisusingthededicatedsoftwareavailable formicroCT.Vectoranalysisofporositywasinitiallydevel- opedbyourgrouptoanalyzethecomplexityoftheporesin various biomaterials presenting a complex shape.¹⁹ The method was also successfully used to characterize the alveolarbonelossatthemandibleinratswithparalysisof masticatory muscles.³⁵ A very similar approach was pre- sentedpreviouslytoappreciatethethinningofcorticalbone atthehiponhumanCTscanswithmappingpseudo-colorsat thesurfaceofthe3Dmodel.^36,37Here,wefoundthatvector analysis couldbeappliedtoevaluate thebone lossin the metaphysisandepiphysisofratsintheBTXmodel.MicroCT valuesandRwerewellcorrelatedinthemetaphysis,making themethodvalidtoextrapolatetheresultstotheepiphysis.

However, the variations inR arenot comparable between differenttypesofbonesbecausethebonewidthinfluences the parameter.So,Rcanonlybeusedtocomparechanges occurringinthesamebone.Inaddition,themethodallowsan immediatevisualperceptionoftheboneloss inthese two skeletalpieces.

5. Conclusion

BTXinducesbonelossafterparalysis ofasinglemuscle.It occursbothatthefemurandtibiaonwhichthedifferentheads of the muscle are inserted. Disusecauses boneloss at the trabecularenvelopeofthemetaphysisandepiphysis.Vector analysisofporositycanbequantifiedbyanalysisoftheRGB histogramsoftheimages.

Conflicts of interest

Theauthorshavenonetodeclare.

Acknowledgments

ThisworkwasmadepossiblebygrantsfromNationalministry of Research. Many thanks to Mrs. Lechat for secretarial assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,intheonlineversion,atdoi:10.1016/j.jasi.2016.04.002.

metaphysis

FEMUR

0.00 0.14 0.28 0.42 0.56 0.70

Ratio red+green/total (in %)

epiphysis

TIBIA

metaphysis epiphysis p < 0.03

p < 0.01 p < 0.01

p < 0.001

Fig.5–Quantitativeanalysisoftheratio(red+green)pixels onthetotalnumberofpixelsinthemetaphysisand epiphysisofthefemurandtibiaofratshavingreceiveda BTXinjectionintheMusquadricepsfemoris.BTX-injected sideinred(right);non-injectedsideinblue(left).

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