Co-registration of DTI and MTR MRI Data to Quantitative Immunohistochemistry in a Novel Mouse Model of Inflammatory Cerebral Demyelination Simone P. Zehntner1, Alex P. Zijdenbos1, Barry J. Bedell1,2, Diego Cadavid3 1Biospective Introduction Results Focal Brain Lesion in C57Bl/6 MOG/EAE IgG The corpus callosum ROI was sub-parcellated into demyelinated (DeMyCC = blue) and myelinated (MyCC = pink) regions by thresholding the 3D MBP IHC data. The IgG ROI was defined by the IgG staining (IgG ROI = turquoise), while the contralateral ROI was defined as the mirror of the IgG staining region (contralateral mirror = gray). Representative IHC images illustrate pathological change at the site of the brain lesion (circles): loss of myelin integrity (MBP), blood-brain barrier permeability covering both white and grey matter (IgG), and microgliaactivation/macrophage-infiltration (IBA-1). Arrows indicate areas of inflammation in the right hemisphere. qIHC Demyelinated CC Co-Registered 3D MRI and 3D PERMITSTM MBP 0.205 ± 0.089 30 0.686 ± 0.101 IgG 0.053 ± 0.079 2,650 0.002 ± 0.003 qIHC IgG Lesion % Difference Mirror MBP 0.083 ± 0.043 76 0.109 ± 0.051 IgG 0.138 ± 0.076 3,450 0.004 ± 0.004 MTR MRI Map DTI FA Map DTI RD Map 2D Myelin IHC 3D MTR 3D DTI MRI Template Registration Linear & Non-Linear Transformations Linear & Non-Linear Transformations MRI Demyelinated CC % Difference Myelinated CC IgG Lesion % Difference Mirror 45.762 ± 3.59 87 52.598 ± 1.443 39.692 ± 2.574 88 44.140 ± 2.776 DTI-FA 0.31 ± 0.043 86 0.36 ± 0.033 0.272 ± 0.04 97 0.279 ± 0.033 DTI-MD 0.834 ± 0.097 109 0.763 ± 0.046 0.850 ± 0.092 112 0.759 ± 0.082 DTI-AD 1.099 ± 0.105 104 1.053 ± 0.051 1.078 ± 0.096 111 0.973 ± 0.092 DTI-RD 0.701 ± 0.097 113 0.617 ± 0.048 0.737 ± 0.092 113 0.652 ± 0.079 Summary MRI measures within the corpus callosum and IgG ROIs (mean ± s.d.; % difference) IgG qIHC Lesion Based Correlation Analysis IgG qIHC (superimposed on anatomical MRI) Histological Volume in MRI space Slice-to-Slice Registration MBP Individual qIHC measures within the corpus callosum and IgG ROIs MTR MTR Native Histological Volume MBP qIHC (superimposed on anatomical MRI) Reconstructed Histological Volume bregma +0.5mm 3D PERMITSTM Stacked 2D Coronal Sections MBP qIHC 7-9 Days Myelinated CC DTI-FA 7 Days DeMyCC MyCC IgG Lesion Mirror Summary qIHC measured as staining density (mean ± s.d. & % difference) DTI MD Map Focal Brain Lesion in C57Bl/6 MOG/EAE and MRI Acquisition 1 L intracranial cytokine (0.1 g mrTNF- & 0.1 g mrIFN-) % Difference MTR Anatomical (MTR Labeled) MRI DTI AD Map Methods Lesion-Based Analysis IBA1 IgG MBP DTI-FA The Theobjective objectiveof ofthis thisstudy studywas wasto todetermine determinethe theneuropathological neuropathologicalcorrelates correlatesof ofininvivo vivo magnetization magnetizationtransfer transferimaging imaging(MTI) (MTI)and anddiffusion diffusiontensor tensorimaging imaging(DTI) (DTI)MRI MRImeasures measures using a novel mouse model of inflammatory cerebral demyelination. Conventional MRI using a novel mouse model of inflammatory cerebral demyelination. Conventional MRI isisroutinely routinelyutilized utilizedto toevaluate evaluatethe theefficacy efficacyof oftherapeutic therapeuticagents agentsininmultiple multiplesclerosis sclerosis (MS) (MS)clinical clinicalstudies. studies. However, However,the therelationship relationshipbetween betweennon-conventional, non-conventional,ininvivo vivo imaging imagingmeasures measuresand andthe theunderlying underlyingpathophysiological pathophysiologicalprocesses processesremains remainspoorly poorly understood. understood. In Inorder orderto tomaximize maximizethe theinformation informationgleaned gleanedfrom fromMRI MRIdata dataand and appropriately appropriatelysteer steerclinical clinicaldevelopment developmentof ofnovel noveltherapeutics, therapeutics,we wehave haveperformed performedaa rigorous rigorouscorrelation correlationanalysis analysisbetween betweenininvivo vivo3D 3DMRI MRImeasures measuresand andgold-standard, gold-standard, post-mortem, post-mortem,quantitative quantitativeimmunohistochemistry immunohistochemistry(qIHC) (qIHC)measures measuresininmice micewith withfocal focal TM inflammatory/demyelinating inflammatory/demyelinatingcerebral cerebrallesions lesionsusing using3D 3DPERMITS PERMITSTM.. MOG35-55/CFA (s.c) Pertussis toxin (i.p.) Abstract # 1984 Poster # P06.124 Inc., Montreal, QC, Canada, 2McConnell Brain Imaging Centre, Montreal Neurological Institute, Montreal, QC, Canada, 3Biogen Idec Inc., Cambridge, MA, USA Post-mortem 3D Histology In vivo MRI Segmented 3D qIHC TM PERMITS PERMITSTMuses usesmulti-step, multi-step,image imageregistration registrationtotogenerate generate3D 3DqIHC qIHCvolumes volumes(Grand’Maison, (Grand’Maison,2013): 2013): •• Mouse Mousebrains brainstissue tissuewere werefixed fixedand andembedded embeddedininparaffin, paraffin,then thensectioned sectionedinto into55μm μmsections sectionswith with ~120 ~120levels levelscovering coveringthe theentire entirebrain brain •• Tissue sections were stained for Myelin Basic Protein (MBP), Immunoglobulin-G (IgG) or the Tissue sections were stained for Myelin Basic Protein (MBP), Immunoglobulin-G (IgG) or the microglial/macrophage microglial/macrophagemarker markerIBA-1 IBA-1and andcounterstained counterstainedwith withAcid AcidBlue Blue129 129(Zehntner, (Zehntner,2008) 2008) •• IHC IHCsections sectionswere weredigitized digitizedusing usingZeiss ZeissMIRAX MIRAXScan150 Scan150whole wholeslide slidescanner scanner •• Image Imageregistration registration employed employedbetween-section between-sectionalignment alignmentininaacoarse-to-fine coarse-to-finefashion, fashion,proceeding proceeding from froman aninitial initialcenter-of-mass center-of-massalignment, alignment,through throughaffine affinealignment, alignment,and andthen thenseveral severalpasses passesofofnonnonlinear linearbetween-section between-sectionalignment alignment •• Registration of the resulting 3D volume to an anatomical MRI template using a coarse-to-fine, Registration of the resulting 3D volume to an anatomical MRI template using a coarse-to-fine,multimultiresolution, resolution,nonlinear nonlinearregistration registrationprocess process •• Two-dimensional Two-dimensional(2D) (2D)qIHC qIHCmaps mapswere weregenerated generatedfor foreach eachsection sectionusing usingan anartificial artificialneural neuralnetwork network (ANN) (ANN)classifier classifier(Zijdenbos, (Zijdenbos,2002),in 2002),inaafully-automated fully-automatedmanner, manner,totogenerate generateunbiased, unbiased,binarized binarized (chromogen vs. non-chromogen) images (chromogen vs. non-chromogen) images •• The Theconcatenated concatenatedtransformations transformationsderived derivedfrom fromthe the3D 3Dreconstruction reconstructionprocess processwere werethen thenapplied appliedtoto the the2D 2DqIHC qIHCmaps mapstotogenerate generate3D 3DqIHC qIHCvolumes volumesofofMBP MBPand andIgG IgG Representative views of the co-registered MRI and 3D PERMITSTM data from an individual mouse brain. The anatomical volume was derived from the MT-labeled images. The MTR MRI parametric data is illustrated using a spectral color scale (low MTR signal in the colder colors and high MTR signal in the warmer colors). The DTI MRI maps (FA, MD, AD, and RD) are illustrated using grayscale (low signal in black and high signal in white). The cytokine-induced lesion is readily apparent in the left hemisphere in all MR images. The reconstructed histological volume utilized in 3D PERMITSTM was derived from the IHC tissue stain. The MBP and IgG qIHC maps represent the quantitative MBP and IgG load, respectively. References Grand’Masion, Grand’Masion,M., M.,Zehntner, Zehntner,S.P., S.P.,Ho, Ho,M.-K., M.-K.,Hébert, Hébert,F., F.,Wood, Wood,A., A.,Carbonell, Carbonell,F., F.,Zijdenbos, Zijdenbos,A.P., A.P.,Hamel, Hamel, E., E.,Bedell, Bedell,B..J. B..J. Early Earlycortical corticalthickness thicknesschanges changespredict predictβ-amyloid β-amyloiddeposition depositionininaamouse mousemodel modelofof Alzheimer's Alzheimer'sdisease. disease. Neurobiol. Neurobiol.Dis., Dis.,2013, 2013,doi: doi:10.1016/j.nbd.2013.02.005 10.1016/j.nbd.2013.02.005 Zehntner, Zehntner,S.P., S.P.,Chakravarty, Chakravarty,M.M., M.M.,Bolovan, Bolovan,R.J., R.J.,Chan, Chan,C., C.,Bedell, Bedell,B.J. B.J. Synergistic Synergistictissue tissuecounterstaining counterstaining and andimage imagesegmentation segmentationtechniques techniquesfor foraccurate, accurate,quantitative quantitativeimmunohistochemistry. immunohistochemistry. J.J.Histochem. Histochem. Cytochem., Cytochem.,56: 56:873-880, 873-880,2008. 2008. Zijdenbos, Zijdenbos,A.P., A.P.,Forghani, Forghani,R., R.,Evans, Evans,A.C. A.C. Automatic Automatic“pipeline” “pipeline”analysis analysisofof3-D 3-DMRI MRIdata datafor forclinical clinicaltrials: trials: application applicationtotomultiple multiplesclerosis. sclerosis. IEEE IEEETrans. Trans.Med. Med.Imaging, Imaging,21: 21:1280-1291, 1280-1291,2002. 2002. Acknowledgements & Disclosure This Thisstudy studywas wassupported supportedby byBiogen BiogenIdec IdecInc. Inc. MBP MBP IgG IgG rDeMyCC = 0.755 rMyCC = 0.502 rDeMyCC = 0.749 rMyCC = 0.677 rIgG Lesion = 0.117 rMirror = -0.231 rIgG Lesion = -0.616 rMirror = -0.101 Correlation analysis of the MTR and DTI-FA MRI measures within the corpus callosum and IgG ROIs Conclusions •• The Thefocal focallesions lesionswere wereclearly clearlyvisible visibleon onthe theMR MRimages, images,and andco-registration co-registrationbetween betweenMRI MRIand and TM qIHC qIHCdata datawas wasachieved achievedusing usingBiospective’s Biospective’sPERMITS PERMITSTMtechnology. technology. •• The Thelesions lesionsdemonstrated demonstratedvariable variabledegrees degreesofofdemyelination demyelinationand andIgG IgGstaining stainingacross acrossanimals. animals. •• Strong Strongcorrelations correlationswere wereobserved observedbetween betweendemyelination demyelinationand andboth bothMTR MTR&&DTI-FA DTI-FAininthe the demyelinated demyelinatedcorpus corpuscallosum callosumROI. ROI. •• ItItwill be important to examine these correlations in the context of remyelination. will be important to examine these correlations in the context of remyelination. •• AAstrong strongcorrelation correlationwas wasalso alsoobserved observedbetween betweenIgG IgGand andDTI-FA DTI-FAininthe thecortical corticalROI ROI •• The The combination combinationofofininvivo vivoMRI MRIand andpost-mortem post-mortem3D 3DqIHC qIHCstudies studiesisisaavaluable valuablestrategy strategyfor for interrogating interrogatingthe therelationship relationshipbetween betweenquantitative quantitativeneuroimaging neuroimagingand andgold-standard gold-standard neuropathology neuropathologymeasures. measures. •• This Thisunique uniqueapproach approachisisexpected expectedto toprovide provideimproved improvedinterpretation interpretationofofMRI MRIdata datafrom fromprepreclinical and clinical clinical and clinicaltherapeutic therapeuticefficacy efficacystudies. studies.
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