Comparingtheeffectsofdifferenttissuefixationprotocolsonpost-mortemmurinebrainMRIversushistology

Applicants:FredBloggs;BobJones;TomSmith

ScientificabstractMagneticresonanceimaging(MRI)hasestablisheditselfasapowerfultoolfordetectionanddiagnosticevaluationofneuropathologicalconditionsinthehumanbrain.However,thehighresolutionsrequiredtoprobepathologicalmiscrostructuraltissuechangesinvivoarechallengingbecauseoftheassociatedlongscantimes.Post-mortemMRIisanincreasinglypopularalternativeduetoitsunrestrictedtimelimitations,therebyallowinghighspatialresolutiontobeachieved.However,imagingbrainspost-mortemrequirestissuefixing,whichpresentsitsowntechnicalchallenges.IthasbeenwellestablishedthatthefixationprocessadverselyaffectsT1,T2,andtheapparentdiffusioncoefficient.Thesevaluescanbepartiallyrecoverablebysoakingfixedtissueinphosphatebufferedsolution,therebyreplacingfixativeinthetissuewithwater.However,thefactthatMRIpropertiesarenotfullyrecoveredtotheirnon-fixedvaluessuggeststhatinferencesfrompost-mortemMRIdatamaybeconfounded.Inthisworkweseektoinvestigatetheeffectsofdifferentfixationprotocolsonthetissuemicrostructure,viahistologicalsectioningandstaining,andviatheireffectsonrelaxometricanddiffusiontensormeasurements.Wewillinvestigatepost-mortemintervalandfixationdurationinthreecandidatefixatives(twoaldehydefixativesandonenon-aldehydefixative),andwillcorrelatetheresultingMRImeasurementswithhistology.Wehypothesizethatthenon-aldehydefixative(osmiumtetroxide),whichpreservesmyelin,willyieldimprovedMRImeasuresversusthestandardaldehydefixatives,thatareidealforgeneraltissuepreservationpurposesduetoproteincrosslinkingbutthatdonotpreservelipids.LaysummaryMagneticresonanceimaging(MRI)isabletoproduceexquisiteimagesofthehumanbodynon-invasively.However,toimprovetheefficacyofMRIasaclinicaltool,itisimportanttounderstandeveryaspectofthesignalthatisdetectedandtheunderlyingmicroscopicpropertiesofthetissuethataffectit.Inordertoprobetissueinthisdetail,itisnecessarytoprescribeverylongscantimes.Thesedurationsbecomeunrealisticforapersontoendureasthesizescaleoftheinformationweseektoextractfromthedatadecreases.Becauseofthisweproposetostudypost-mortembraintissue.Thisisaveryattractiveapproachsincescandurationconsiderationsbecomelessimportant.However,inordertostabilizetissueovertheselongscantimes,itisnecessarytotreatthetissuewithchemicalstopreventdegradation.Unfortunately,ittheprocessofchemicalstabilizationdeleteriouslyaltersthepropertiesthatproducetheMRIsignal.Inordertoallowresearchestomakeareliablelinkbetweeninvivodataandpost-mortemdata,themicrostructuralchangesthatoccurduetochemicalstabilizationneedstobemoreclearlyunderstood.Onlythencanpost-mortemfindingsspecifictodiseasesbecorrelatedwithdataacquiredintheclinic.Inthiswork,weseektoinvestigatetheeffectsthatvariouschemicalstabilizationprotocolshaveontissuemicrostructureandtheeffectthesechangeshaveontheMRIsignal.Thiswillprovideinformationthatoptimisesbrainstabilizationtechniquesandimprovecorrelationbetweenpost-mortemfindingsandclinicalMRImeasurements.

Comparingtheeffectsofdifferenttissuefixationprotocolsonpost-mortemmurinebrainMRIversushistologyBackground andMotivation:Magnetic resonance imaging (MRI) is an increasinglypopular technique forprobing themicroscopicarchitectureof thebrain.Higherspatial resolution ispreferable for itsability torevealbothstructuralandfunctionalinformationwithhigherfidelity.Thepredominantconfoundingfactorsaffectingthehigh-resolutionsrequiredforthisgoalfrominvivodataaremotion(e.g.respiratory,pulsatile,patient)andtheexperimentalduration.Thiscanleadtoanunfeasiblerequirementonthesubject/patienttoremainmotionless,andtounreasonablescandurations.Analternativetoinvivoimagingistheuseoffixedpost-mortembrains.Thishaslongbeenastrategyusedinpre-clinicalmodels, most notably inmurine brain1-2, achieving extraordinarily high resolutions (on the order of tens of microns).Interestintheuseofpost-mortemscanninginwholehumanbrainhasalsobeguntoattractattention3,particularlywiththeadventof‘brainbanks’.However,thereisconsiderablevariabilityinthefixationpreparationsusedacrosssites,andaknownimpactontheMRIparametersofinterest.Fractionalanisotropy(FA)andapparentdiffusioncoefficient(ADC)havebothbeenreportedtodecreasewithincreasedpostmorteminterval(PMI–thetimebetweendeathandfixation)4.TissuesamplesstoredinfixativepriortoimaginghaveshowndecreasedtissueADC,protondensity,andT2/T2*comparedwithinvivoexperiments4-8,andthesechangesarefixativedependent9.Someofthesepropertieshavebeenshowntobepartiallyrecoverable by soaking the tissue sample in phosphate buffer solution (PBS)10, a process likely associated with thereplacement of free water with that of fixative. However, the recoverability of the MRparametersisinconsistent.Thecurrentprevalentfixationpreparationformaintainingtissuemorphologyisachievedbythecrosslinkingofsolubleproteinswithstructuralproteinstoformgels,renderingtheminsoluble.Varying concentrations of aldehyde solutions are typically used – specifically formaldehyde,glutaraldehyde, or somemixture of these (e.g. Karnovsky'sMixture).Whilemaintenance of invivoanatomicalrelationshipisachieved,manymicrostructuralchangesoccurthatcouldfurtheraffectthehigh-resolutionMRsignal.Generalfixationartefactsincludetissuevolumechangesandtheslowdriftofunfixedmaterialfromtheirsourcelocation11.Ofparticularconcern,inthecaseof formaldehyde, is the fact that lipidsarenot fixed11. This leaves them free todissolve in thexylene stage of dehydration, a specific problem as it leaves myelin, a major target ofmicrostructural imaging, susceptible to degradation and/or elimination11 (Fig 1)12. In order torealise the potential of post-mortemMRI there is an urgent need to determine the optimumfixationpreparationanddefinetheeffectsontheMRIvariablesofinterest.Specific Aims and Hypotheses: In this work we propose to further the understanding offixation-relatedmicrostructuraltissuechangesandtheireffectsontheMRsignalbystudyingathigh resolution the post-mortem murine brain. Specifically, we propose to investigate the effects of PMI and fixationduration (FD – effectively the interval between fixation and scanning) on microstructural viability. Neuropathologichistology will be correlated with quantitative relaxometric and diffusion MRI. We hypothesize that the use of a newfixative,osmiumtetroxide,whichismorespecificallytailoredtopreservingultrastructure(i.e.myelin),willshowgreaterrobustness of MRI parameters to post-mortem fixation than the more conventional fixatives of formaldehyde andglutaraldehyde. The long-term aim of thisworkwould be the translational application for improved fixation of wholehumanbrain,optimisedforMRimaging.Thespecificaimsandhypothesesofthisgrantare:

1. TomeasurechangesinquantitativerelaxometricanddiffusionMRIparametersduetovaryingbothPMIandFD.2. PerformcomparativehistologytocorrelatethechangesinAim1withtheunderlyingmicrostructuralchanges.3. To test thehypothesis that osmium tetroxidewill exhibit improvedmyelin integrity compared to conventional

fixatives,andwillexhibitimprovedmyelinwatermeasurementretentionaswellasdiffusiontensorestimates.ScientificPlanMethods:AllprocedureswillbeperformedonanimalsfollowingprotocolsapprovedbytheInstitutionalAnimalCareandUse Committee (application submitted). Animals will be prepared for one of two cohorts: perfusion fixation (PF) orimmersionfixation(IF).ToinvestigateFDdependentchangesintissue,PFadultnudemice(n=36,12perfixative)willbedeeplyanaesthetisedwith60mg/kgpentobarbitalandsacrificedbyintercardialperfusionwith0.01MPBS, followedbyone of three fixative solutions: 4% paraformaldehyde (pH 7.4)/PBS, 3% gluteraldehyde (pH 7.3)/PBS, or 1% osmiumtetroxideinbufferedacetate/veronalsolution(pH7.4).Afterfixation,brainswillbeimmersedinthefixativeforupto8weeks.Threerandombrainsfromeachfixativesolutionwillbeimagedweekly,withtwobeingreturnedtosolutionandonebeingsentforhistologicalanalysis.TheimagingandhistologyscheduleisdescribedinTable1a.To study theeffectsofPMIon tissue integrity, IF adultnudemice (n=81)will bedeeply anaesthetisedwith isoflurane,sacrificedviacervicaldislocation,andthebrainplacedinPBS.Ateachtimepoint,9brainswillberemovedfromPBSandplacedinoneofthethreefixativesolutions(3each).Brainswillbeallowedtofixfor12hours,thenimaged.Afterimaging,one brain from each fixative cohortwill be sent to neuropathology for sectioning and staining (see schedule details inTable1b).Forboth fixationproceduresonlyasinglebrainwillbesectionedateach timepoint todecreasecohortsizes,minimisecost,while stillproviding sufficiently representativequalitative results. In contrast, threebrainswillbe imagedateachtimepointwithMRtoimprovestatisticalpowerofthequantitativeresults.Aim1:MRIimagingwillbeperformedona7Tsmall-boreVarianInovascanner.ThreesampleswillbeplacedinasinglecryovialfilledwithFluorinertforsusceptibilitymatching.3Dhigh-resolution(150µmisotropic)protondensity(PD),T1,

Fig 1. Myelin stained (a)Formalin and (b) osmiumtetroxide fixed equinenervetrunk.

T2,T2*,andDTIdatawillbeacquiredovera24hourscantime.Allrelaxometrydatawillbeacquiredusinggoldstandardacquisitiontechniques,exploitingthelimitedtimerestrictionsrequired for post-mortem imaging (PD will use a conventional proton density weightedgradient echo sequence (GRE); T1, an inversion recovery spin-echo protocol with 6inversiontimes;T2,aCPMGacquisitionwithaminimumof6echoes;andT2*,amulti-GREacquisition).DTIdatawillbeacquiredusingdiffusion-weightedsteadystatefreeprecession(DW-SSFP).Thishasbeendemonstratedtoperformbetterthanitsspinechocounterpartat3T13andwithhigherSNRefficiencyat7T14.QuantitativeDTIresultswillbeproducedusingmodifiedversionsofDTIFITandBEDPOST(FSL)toaccountfortheDW-SSFPsignalmodel.Aim 2: Comparisons of quantitative results outlined in Aim 1 will be made betweenperfusion fixation duration time points, PMI time points, and PF vs. IF time points bothwithin a fixative solution cohort as well as across fixative solution cohorts. Statisticalanalyses ofmeanvalueswithinwhitemattermaskeddata andmajorwhitematter tractswill bemade across the same comparisons to evaluate the variability of the quantitativeresultsacrossthevaryingphysicalchanges.Onepointofparticularinterestistheeffectsofchangesinmyelinationduetothedifferingfixatives.T115andT216distributionsaswellasT2*fittingofthefreeinductiondecay17havealldemonstratedmulti-compartmentbehaviorduetomicrostructurallydependentvariations.Specifically,alloftheseexponential fitting-based results reflect the presence of a myelin water compartment where trapped waterbetweenmyelin layersexhibitsspecificT1,T2,andT2*values.Destructivechangestothelipidbilayerinmyelinduetofixationcouldbedetectedbyanyofthesemethodsbyseriallytrackingthechangeinthemyelinwater-specificcomponentofeach.CorrelatingchangesinthesemeasurementswithincreasedPMIandFDwouldsuggestchangesinthelipidbilayerof myelin. Histological staining for myelin will identify the extent to which changes in myelin microstructure haveoccurredandtowhatextentPMI,FD,andfixativecontribute.Aim3:Majortracts, includingthecorpuscallosumandcorticospinaltract,willbeidentifiedviatractographyinDTIdataandinhistologicalslides.ComparisonsofaveragequantitativerelaxometryandDTIresultsoverthesetractswillbemadebetween the differing fixation techniques, PMIs, FDs, and fixatives. Quantitative estimates of myelination will beperformed and correlated with relaxometry results and fractional anisotropy, the standard deviation of the fractionalanisotropy estimate, radial and axial diffusivity, and the accuracy of the primary diffusion direction estimate. OurhypothesissuggestsanimprovementinmyelinwaterT1,T2,andT2*retentionovertimeinosmiumtetroxidefixedbrainsduetolipidpreservationfromlipid/proteincrosslinking,andaloweruncertaintyestimateinthefiberdirectionswithinthesemajorwhitemattertracts.Impact: With increasing interest and implementation of post-mortem human brain imaging for improving non-destructiveanatomicalvisualizationofhumanbrainatthemicro-structural level,amorecompleteunderstandingoftheeffectsthatfixationhasonbraintissueandsubsequentMRImeasurementsisimperative.Apotentialapplicationforpost-mortem human brain imaging is aiding in detection of disease biomarkers. With improved understanding of thedifferences between post-mortem and in vivo tissue due to both natural tissue degradation and fixative-relateddestruction, and how those translate into changes in MR measurements, the identification of these biomarkers post-mortemcouldbemore readily translated tohuman invivomeasurements, therebypotentiallyaiding inearlydetectionand/ordiagnosisofneurologicaldisease.Environment:TheproposedresearchwillberealizedbythecooperationofthreeOxfordresearchdepartments/centres:Oxford Centre for Functional MRI of the Brain (FMRIB), the Nuffield Department of Clinical Neurology, and the GrayInstituteforRadiationOncologyandBiology(ROB).FMRIBisaworldrecognizedmultidisciplinarycentreattheforefrontofdevelopingtechniquesandmethodsforMRimaging,imageanalysis,basicscienceandtranslationalresearch.Bobjonesisanexpertatpost-mortemhumanbrain imagingandhasanestablishedbackground inboth invivoandpostmortempre-clinical animal experiments, experience that isdirectly applicable to the specific aimsof this study.TomSmithhasmorethan25yearsofresearchexperiencewithMRI,spanningboth diffusion and relaxometric imaging techniques. FredBloggs has expertise on diffusion imaging and sequenceprogramming. Collaborator Jim Johnson, the leadNeuropathologist from the Department of Clinical Neurologywillhelpwithsectionandstaining.Allscanningwill takeplaceona7TVariansmall-borescannerattheROB.

References:1Johnson(2002)Radiology,789-932Henkelmann(2006)MagnResonMed,1371-43McNabb(2009)NeuroImage,775-854D’Arceuil(2007)NeuroImage,64-85Pfefferbaum(2004)NeuroImage,6Sun(2003)MagnResonMed,743-87Sun(2005)MagnResonMed,1447-518Dawe(2009)MagnResonMed,810-89Shepherd(2009)NeuroImage,820-610Shepherd(2009)MagnResonMed,

26-3411Tofts(2002)TheoryandPracticeofHistologicalTechniques,Ch512http://www.upei.ca/~morph/webct/Modules/Nerve13Miller(2011)NeuroImage,167-8114Foxley(2014)NeuroImage,Inpress15Labadie(2014)MagnResonMed,375-8716MacKay(1994)MagnResonMed,673-717Sati (2013) NeuroImage, 268

BudgetJustification:1.Personnel:TBA,postdoctoralMRPhysicist(effort=24calendarmonths)withannualsalary(Grade07S3)of£400002.Equipment:Scanning£39000(39days,£1000/weekendday),Computer/monitor/harddrives£3000,softwarelicensesandclusteraccess£60003.Material:Adultnudemice£3000(120mice,£25each)4.Travel:Professionalconferencesandworkshops(e.g.,ISMRM,HBM)£5000(£1250each)5.Manuscriptpublishingcost:£4000(Colourprinting,etc.)

2yeartotal:£140000

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