NeuroImage 59 (2012) 2678–2688

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Assessment of lesion pathology in a new animal model of MS by multiparametricMRI and DTI

Susann Boretius a,c,⁎,1, Angelika Escher b,1, Tobias Dallenga b, Claudia Wrzos b, Roland Tammer a,c,Wolfgang Brück b,c, Stefan Nessler b, Jens Frahm a,c, Christine Stadelmann b,c

a Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut für biophysikalische Chemie, 37070 Göttingen, Germanyb Institute of Neuropathology, University Medical Center, Georg-August-University, 37099 Göttingen, Germanyc DFG Center of Molecular Physiology of the Brain, 37073 Göttingen, Germany

Abbreviations: AD, axial diffusivity; APP, amyloidbrain-barrier; Cup, Cuprizone; DTI, diffusion tensor imaimmune encephalomyelitis; FA, fractional anisotropy; Gdtriamine pentaacetic acid; GFAP, Glial fibrillary acidic promagnetic resonance imaging; MS, multiple sclerosis;glycoprotein;MTR,magnetization transfer ratio; RD, radiaROI, region of interest; PAS, periodic acid Schiff.⁎ Corresponding author at: Biomedizinische NMR Fors

Institut für biophysikalische Chemie, 37070 Göttingen, GeE-mail address: sboreti@gwdg.de (S. Boretius).

1 equal contribution.

1053-8119/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.neuroimage.2011.08.051

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 June 2011Revised 5 August 2011Accepted 17 August 2011Available online 2 September 2011

Keywords:CuprizoneEAEmagnetic resonance imaging (MRI)diffusion tensor imaging (DTI)multiple sclerosis (MS)

Magnetic resonance imaging (MRI) is the gold standard for the detection of multiple sclerosis (MS) lesions.However, current MRI techniques provide little information about the structural features of a brain lesionwith inflammatory cell infiltration, demyelination, gliosis, acute axonal damage and axonal loss. To identifymethods for a differentiation of demyelination, inflammation, and axonal damage we developed a novelmouse model combining cuprizone-induced demyelination and experimental autoimmune encephalomyeli-tis. MS-like brain lesions were assessed by T1-weighted, T2-weighted, and magnetization transfer MRI aswell as by diffusion tensor imaging (DTI). T2-weighted MRI differentiated control and diseased mice, whileT1-weighted MRI better reflected the extent of inflammation and axonal damage. In DTI, axonal damageand cellular infiltration led to a reduction of the axial diffusivity, whereas primary demyelination after cupri-zone treatment was reflected by changes in radial but not axial diffusivity. Importantly, alterations in radialdiffusivity were less pronounced in mice with demyelination, inflammation, and acute axonal damage, indi-cating that radial diffusivity may underestimate demyelination in acute MS lesions. In conclusion, the com-bined information from different DTI parameters allows for a more precise identification of solelydemyelinated lesions versus demyelinated and acutely inflamed lesions. These findings are of relevance foroffering individualized, stage-adapted therapies for MS patients.

precursor protein; BBB, bloodging; EAE, experimental auto--DTPA, gadolinium diethylenetein; LFB, Luxol fast blue; MRI,MOG, myelin oligodendrocytel diffusivity; RF, radiofrequency;

chungs GmbH am Max-Planck-rmany.

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© 2011 Elsevier Inc. All rights reserved.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory demyelinatingdisorder of the central nervous system. The histopathological hall-mark of this disease is the demyelinated plaque, which is associatedwith perivascular and parenchymal inflammatory cell infiltrates inthe acute disease stage. There is a variable extent of axonal injury be-tween patients, and several markers of tissue destruction have beenestablished in magnetic resonance imaging (MRI) studies. Newlyappearing, hyperintense areas on T2-weighted images, togetherwith contrast enhancement on T1-weighted images after Gd-DTPA

administration are commonly judged as active MS lesions (Brucket al., 1997) whereas chronic hypointensities on T1-weighted imagesare mainly associated with severe tissue damage (van Waesbergheet al., 1999). However, these techniques do not precisely characterizethe underlying lesion pathology.

Diffusion-weighted MRI in the form of diffusion tensor imaging(DTI)may bemore suitable for addressing tissuemicrostructure and in-tegrity, because it allows for a quantitative determination of the orien-tational dependence of water diffusion. In myelinated fiber tracts thewater diffusivity has a strong directionality, reflected by high values ofthe fractional anisotropy (FA), which was found to be reduced in bothlesions (Filippi et al., 2001; Lowe et al., 2006; Roosendaal et al., 2009;Seewann et al., 2009) and normal-appearing white matter (Bammeret al., 2000; Ciccarelli et al., 2001; Giorgio et al., 2010; Werring et al.,1999) of MS patients. A reduction of the water diffusivity parallel tothe fiber tracts (λ1=axial diffusivity) has been observed after axonaldamage in different animal models such as pericontusional traumaticinjury (MacDonald et al., 2007) and experimental autoimmune enceph-alomyelitis (EAE) in subacute (DeBoy et al., 2007) and chronicstages of the disease (Sun et al., 2007; Wu et al., 2007). On the otherhand, an increase of the diffusivity perpendicular to the fiber tracts(λ2+3=radial diffusivity) has been demonstrated in a number of

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demyelinating and dysmyelinating animal models (Shamy et al., 2010;Song et al., 2002; Song et al., 2005). However it is a matter of debate,whether the radial diffusivity remains sensitive to demyelination inthe presence of significant inflammation and axonal damage (Buddeet al., 2008), in particular, as it requires a more complex interpretationof the DTI eigenvalues under pathological conditions (Alexander et al.,2007; Wheeler-Kingshott and Cercignani, 2009).

Each of the currently available animal models for MS reflects dif-ferent pathophysiological and neuropathological aspects of thehuman disease. EAE is caused by myelin-specific CD4 T cells, whichorchestrate a CNS inflammatory response composed of mononuclearcell infiltrates primarily in the spinal cord and optic nerve. In themost widely used EAE model induced by active immunization ofC57BL/6 mice with MOG-peptide35-55, massive inflammation is asso-ciated with very little primary demyelination but moderate to severeaxonal damage. However, inflammatory brain lesions are rare in thismodel.

In contrast, confluent demyelinated areas in the brain can begenerated in toxin-induced demyelination models, such as cuprizone(biscyclohexanone oxaldihydrazone), a copper-chelator admixed tothe chow (Matsushima and Morell, 2001), or by the local injection oflysolecithin (Hall, 1972). Both toxin-induced animal models, however,lack a substantial T cell mediated inflammation and, at least in thecase of cuprizone, fail to yield a prominent axonal pathology, thuslimiting the comparability to the human disease. Because all lesionconstituents are likely to contribute to theMRI contrasts,we establishedan animal model with MS-like lesions in the brain by immunizingcuprizone-fed mice with MOG35-55 (Cup-EAE). With this approach,considerable numbers of T cells were recruited into the demyelinatedcorpus callosum, significantly increasing the axonal damage in this area,while the extent of demyelination remained unaffected. We obtainedT1- and T2-weighted images, maps of the magnetization transfer ratio(MTR), and diffusion tensor maps in cuprizone-fed mice (Cup),Cup-EAE mice, and healthy age-matched controls.

A direct assessment of the relationship between these MRI-derivedparameters and the underlying pathology revealed the followingmain results: (i) Cup-EAE mice had reduced signal intensities onT1-weighted images compared to the Cup group; (ii) axial diffusivitywas significantly attenuated only in the Cup-EAE mice; (iii) comparedto controls radial diffusivity was increased only in the Cup but not inthe Cup-EAE group. Accordingly, the radial diffusivity seems to underes-timate the extent of demyelination, because its expected increase iscounterbalanced by inflammation and axonal damage in the acute dis-ease stage. We therefore conclude that the use of multiple parameterscan improve the specificity for a discrimination of respective tissuealterations.

Material and methods

Animals

A total of 31 female C57BL/6 mice aged between 6 and 8 weekswere included in this study (Table 1). Twenty-six mice were fedwith 0.25% (wt/wt) cuprizone (Sigma, St Louis, MO, USA) in groundbreeder chow ad libitum over 2 weeks followed by a 2 week courseof 0.15% cuprizone (Cup group). Thirteen of them were additionallyimmunized with myelin oligodendrocyte glycoprotein peptide

Table 1Numbers of animals per group analyzed by MRI and DTI.

Group T1w, T2w, MTR DTI Contrast-enhanced MRI

Cup-EAE 5 5 3Cup 5 5 3Control 5 -

(MOG35-55) at day 17 and boosted at day 24 after starting the cupri-zone feeding (Cup-EAE group). Pertussis toxin (PTX, List BiologicalLaboratories, London, UK) was injected on days 0, 2, and 9 after im-munization (300 ng i.p.). Five mice served as controls and were main-tained on a normal diet for the duration of the experiment (Fig. 1). Allexperiments were performed in compliance with relevant laws andinstitutional guidelines. Animal experiments were approved by localauthorities.

MRI

For MRI animals were initially anesthetized using a chamber per-vaded with 5% isoflurane in oxygen. Subsequently, the mice wereintubated and kept under anesthesia with 1–1.5% isoflurane in oxy-gen and ambient air (1: 1.5). Respiration was monitored by a signalderived from a pressure transducer fixed to the animal's chest. To en-sure comparable slice orientations, the mice were placed in a proneposition with the head fixed to a teeth and palate holder using theGöttingen animal bed (Boretius et al., 2009). The rectal temperaturewas held constant at 36±1 °C by water blankets.

One group of 15 animals consisting of 5 controls, 5 Cup and 5 Cup-EAE mice underwent MRI measurements at 2.35 T using an MRBR4.7/400 mm magnet (Magnex, Scientific, Abingdon, UK) equippedwith a DBX spectroscopy and imaging system (Bruker Biospin, Ettlingen,Germany). For excitation aHelmholtz coil (100 mm)was used and com-bined with an elliptical surface coil (20 mm×12 mm) for signal detec-tion. After 4 weeks of cuprizone treatment T1-weighted images (3DFLASH, TR/TE=17/7.6 ms, flip angle 25°) and T2-weighted images (3DFSE, TR/TE=3000/98 ms, 16 echoes, inter-echo-spacing=12.5 ms)were obtained with an isotropic spatial resolution of 117 μm. MTRmaps were based on a spin density-weighted sequence (3D FLASH, TR/TE=30/7.6 ms, flip angle 5°) with and without off-resonance radiofre-quency (RF) irradiation (Gaussian-shaped RF pulse, frequency offset5 kHz, mean amplitude 200 Hz corresponding to a flip angle of1045°). Percent MTR was calculated as MTR=(PD – MT) / PD×100with PD and MT the MRI signal intensities without and with RF irra-diation, respectively. The entire measurement time was about 5 hours.

To evaluate possible disruption of the blood-brain barrier (BBB),T1-weighted images (3D FLASH, TR/TE=17/4.2 ms, flip angle 25°,measurement time: 20 min) were obtained at 9.4 T (Bruker BiospinGmbH, Germany) before and 10 min after i.v. injection of 0.5 mmol/kggadolinium diethylene triamine pentaacetic acid (Gd-DTPA) in addi-tional 3 Cup and 3 Cup-EAE mice.

Fig. 1. Study design: 26 of a total of 31mice (Table 1)were fedwith a high dose (0.25%) ofcuprizone in ground breeder chow ad libitum for 2 weeks followed by a 2 week treatmentwith low dose (0.15%). Half of the cuprizone-treated mice where immunized with 300 μgmyelin oligodendrocyte glycoprotein (MOG)-peptide35-55 at days 17 and 24 of cuprizonefeeding. One group of animals was studied byMRI (5 Cup, 5 Cup-EAE) and another groupby DTI (5 Cup, 5 Cup-EAE). Five mice receiving a normal diet throughout the study servedas controls for each group. Three additional animals per treatment group underwentcontrast-enhanced MRI.

Fig. 2. Definition of axial section and region of interest. (Top) Mid-sagittal section of aT1-weighted MRI data set from a control mouse. The vertical line demonstrates thesection shownbelow. (Middle) Color-codedmap of themaindiffusion direction of a controlmousewith red=left-right, blue=rostral-caudal and green=anterior posterior. (Bottom)Outline of the region-of-interest used for DTI analyses of the corpus callosum. A similar ROIwas selected on T2-weighted images and transferred to the corresponding T1-weightedimages and MTR maps.

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A second group of 15 mice comprising 5 controls (same mice as ingroup 1), 5 Cup, and 5 Cup-EAE underwent DTI (half Fourier diffusion-weighted single-shot STEAM, 125×125×500 μm3 resolution) at 9.4 T(Bruker Biospin GmbH, Germany) using a birdcage resonator (inner di-ameter 72 mm) for excitation and a saddle-shaped surface coil for signaldetection (Bruker Biospin, Ettlingen, Germany). Diffusion preparationinvolved two b values (0 and 1000 s mm−2) in 12 different directions(6 different gradients and their opposite values, measurement time: 3hours). The eigenvalues (λ1-3) of the diffusion tensorwere used to calcu-late parameter maps on a pixel by pixel basis for the axial diffusivity(AD) with AD=λ1 the largest eigenvector, the radial diffusivity (RD)with RD=(λ2+λ3) / 2, and the fractional anisotropy (FA) according to

FA ¼ffiffiffi32

r ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðλ1−ADC Þ2þðλ2−ADC Þ2 þ ðλ3−ADC Þ2

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiλ21 þ λ2

2 þ λ23

q :

Fig. 3. MS-like pathology in the corpus callosum of Cup-EAE mice in comparison to Cup micCup-EAE mice (scale bar 500 μm). Bielschowsky staining: no apparent axonal reduction (scCup-EAE mice (scale bar 50 μm). CD3: marked infiltration of T cells into the demyelinatedMac-3:. Mac-3: marked microglia activation in Cup mice, but no significant further increasCup mice, but, similarly, no further increase in Cup-EAE mice (scale bar 100 μm).

Quantitative analysis

Comparisons of MRI and histological data were performed in atransverse section located about 0.1 mm anterior to the bregma,where quantitative region-of-interest (ROI) analyses focused on thecorpus callosum as shown in Fig. 2. This region was mainly selectedbecause of the negligible number of crossing fibers that otherwisemight result in misleading FA values. ROIs were selected on color-coded maps of the main diffusion direction including only pixelswith a left-right orientation (marked red in Fig. 2). A similar ROIwas selected on T2-weighted images and transferred to the corre-sponding T1-weighted images and MTR maps. To allow for a compar-ison of T1- and T2-weighted images between animals the MRI signalintensity in the corpus callosum was normalized (T1c, T2c) by the in-tensity of the cerebrospinal fluid (Merkler et al., 2005).

Histopathology

After MRI, the animals received an overdose of ketamine/medetomidine and were perfused transcardially with 4% paraformalde-hyde in PBS. Brainswere removed andpostfixed in 4%paraformaldehydein PBS overnight. Brainswere dissected and embedded into paraffin. Par-affin sectionswere stainedwith hematoxylin and eosin (H&E), Luxol fastblue/periodic acid Schiff (LFB/PAS), and Bielschowsky's silver impregna-tion for detection of cellular structures, myelin and axons, respectively.Immunohistochemistry was performed by a biotin-avidin or alkalinephosphatase/anti-alkaline phosphatase technique. The following prima-ry antibodies were used: rabbit anti-glial fibrillary acidic protein(GFAP, Dako, Germany) for judgment of astrogliosis, rat anti-Mac3(Pharmingen, USA) for detection of activated microglia/macrophages,mouse anti-human amyloid precursor protein (APP, clone 22 C11,Chemicon, USA) as a marker for acute axonal damage, rat anti-CD3(Serotec, Oxford, UK) to detect infiltrating T cells, biotinylated horseanti immunoglobulin G (IgG; Vector Laboratories, Burlingame, USA),and rabbit anti fibrinogen (Dako, Germany) to assess the blood brainbarrier (BBB) permeability to large plasma proteins. For all primaryantibodies except IgG microwave pretreatment in 10 mM citric acidbuffer, pH 6.0 was performed.

Morphometry

5 Cupmice and 5 Cup-EAEmice underwent detailed histomorpho-logical analysis. Demyelination in the corpus callosum was scored inLFB/PAS stained sections using a semiquantitative scoring system(0=no demyelination; 1=1/3 of the corpus callosum demyelinated;2=between 1/3 and 2/3 of the corpus callosum demyelinated;3=more than 2/3 of the corpus callosum demyelinated (Matsushimaand Morell, 2001). The total number of Mac-3-positive microglia/macrophages, CD3-positive T cells and APP-positive axons in the cor-pus callosum was counted using an ocular morphometric grid at 400-fold magnification. Cell or axonal spheroid densities, respectively, aregiven as cells or axonal spheroids per mm2. We evaluated at least sixrandomly selected optical fields in the area of the corpus callosum peranimal.

Statistical analyses

Statistical analyses were performed using SPSS 16 for Windows(SPSS Inc., Chicago, USA). For all two-group comparisons, the non-parametric Mann-Whitney U test was used. For correlation analyses,

e and untreated controls. LFB/PAS staining: similar extent of demyelination in Cup andale bar 100 μm). APP: little acute axonal damage in Cup mice, but marked increase incorpus callosum of Cup-EAE mice. T cells are scarce in Cup mice (scale bar 100 μm).

e of monocytes in Cup-EAE mice (scale bar 100 μm). GFAP: pronounced astrogliosis in

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Fig. 4. Increased T cell infiltration and axonal damage in Cup-EAE mice. The densities of CD3-positive T cells and APP-positive acutely damaged axons were significantly increased inCup-EAE compared with Cup mice. However, the numbers of astrocytes and activated microglia/macrophages (Mac-3+) were comparable in Cup and Cup-EAE mice.

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the Spearman's correlation coefficient was used and in all cases whencorrelation coefficients were given, correlations were significant atpb0.05 unless noted otherwise. The p values are given without ad-justment for multiple testing, but the adjusted levels of significancefor two and three two-group comparisons (0.025 and 0.017) arementioned where relevant.

To evaluate the potential of different MRI contrasts/DTI parame-ters to discriminate between groups of Cup mice, Cup-EAE mice andcontrols, discriminant function analysis was used. A set of linear func-tions of the predictors (MRI contrasts, DTI parameters) was con-structed according to D=b1x1+b2x2+…+bnxn+c, with bi as thediscriminant coefficients, xi as input variables or predictors and c asa constant. The attribution to the three different groups was basedon themaximumposterior probability of groupmembership (Backhauset al., 2003).

Results

Histopathology

Cup-EAE mice showed significantly higher numbers of CD3 positiveT cells in the corpus callosum compared to Cup mice without subse-quent immunization (p=0.008). Similarly, the density of APP-positive,acutely damaged axons significantly increased with MOG35-55

immunization (p=0.008). However, neither Cup nor Cup-EAE miceexhibited marked axonal loss as shown by Bielschowsky staining.Moreover, the extent of demyelination as assessed by LFB/PAS stainingwas comparable between Cup and Cup-EAE mice (mean score=2.4±0.1 and 2.5±0.2, respectively). Both groups harbored comparablenumbers of Mac-3 positive macrophages/activated microglia cellsand GFAP-positive astrocytes (Figs. 3 and 4).

MRI

T1- and T2-weighted images andMTRmaps of a control, a Cup anda Cup-EAE mouse are shown in Fig. 5. Results for quantitative groupanalyses are given in Fig. 6. Cuprizone feeding for 4 weeks resultedin a decrease of the signal intensity of the corpus callosum on T1-weighted images compared to controls (p=0.032, level of signifi-cance 0.016). The T1 signal intensity further dropped significantly inCup-EAE mice compared to the Cup group (p=0.008). In addition,cuprizone feeding increased the signal intensities of the corpus callo-sum on T2-weighted images compared to controls (p=0.008). How-ever, no difference in T2 signal intensities between Cup and Cup-EAEmice was observed. The MTR of the corpus callosum was attenuatedin both cuprizone-fed groups. However, compared to untreated con-trols, only the MTR reduction in Cup-EAE mice reached statistical sig-nificance (p=0.008).

Fig. 5. (Top) Axial T1-weighted images, (middle) T2-weighted images, and (bottom) magnetization transfer ratio (MTR) maps of a control, a Cup, and a Cup-EAE mouse. Arrowsindicate MRI contrast alterations of the corpus callosum compared to control.

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Contrast-enhanced MRI demonstrated a focal disruption of theBBB in the corpus callosum of Cup-EAE mice, whereas no increasedpermeability of the BBB to Gd-DTPA was observed in Cup mice. Thisis in line with the exclusive detection of large plasma proteins suchas immunoglobulin G (120 kD) and fibrinogen (340 kD) in the formergroup (Fig. 7).

DTI

Maps of FA, AD, and RD of a control, a Cup and a Cup-EAE mouseare presented in Fig. 8. The results of the ROI analyses of the corpuscallosum in Fig. 9 revealed comparable reductions of fractional

Fig. 6. Group analysis of T1-weighted and T2-weighted MRI signal intensities (normalized to tcontrol, Cup and Cup-EAEmice (n=5 each).While bold horizontal bars indicatemedian valuesof measurements, respectively. The difference in T1 between control and Cup group is statistic

anisotropy (FA) in both the Cup and the Cup-EAE group. However,Cup and Cup-EAE mice had distinct patterns of axial and radial diffu-sivity. Relative to controls, AD was significantly reduced in Cup-EAEmice (p=0.008), but not in Cup mice. In contrast, Cup mice showedsignificantly increased RD in the corpus callosum (p=0.008), whilesuch values were comparable in Cup-EAE and control mice (Fig. 9).

Discriminant Function Analysis

Combining the normalized signal intensities on T1- and T2-weighted images and MTR by a discriminant function analysis,86.7% (13/15) of all mice could be correctly assigned to their

he cerebrospinal fluid) and magnetization transfer ratios (MTR) in the corpus callosum of, boxes and bars represent the interquartile ranges (25th to 75th percentiles) and the rangeally not significant when adjusting for multiple comparisons (3 tests).

Fig. 7. (Top) Contrast-enhanced MRI of the blood-brain barrier in (middle, right) a Cupmouse and Cup-EAEmouse (second row) relative to (left) a precontrast image. (Lower rows)Histologically, the lesion shows marked infiltration of T cells (CD3) as well as tissue deposition of IgG (IgG) and fibrinogen (Fibrinogen). Right column: close-up of the area depictedin the middle column. Scale bar left and middle column: 500 μm, right column: 200 μm.

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respective treatment group. In particular, all control animals werecorrectly assigned, whereas 40% (2/5) of the treated mice were sortedinto the wrong cuprizone-fed group (Fig. 10, left). In more detail,functions 1 and 2 were 2.286 x T1c+36.489 x T2c – 0.211 x MTR –

32.587 and 8.943 x T1c+13.442 x T2c+0.466 x MTR – 46.888, re-spectively. The functional characteristics of the group centroids

(Fig. 10, black quadrates) were D1/D2=−6.092/-0.91 for controls,2.65/1.1 for Cup mice and 3.442/-1.009 for Cup-EAE mice.

Similarly, also FA, AD and RD valueswere subjected to a discriminantfunction analysis and resulted in an 86.7% (13/15) correct assignment ofall mice to their respective treatment group. However, 20% (1/5) of thecontrols were assigned to the Cup-EAE group and 20% (1/5) of the Cup

Fig. 8. Axial maps of fractional anisotropy (FA), axial diffusivity (AD), and radial diffusivity (RD) of a control, a Cup, and a Cup-EAE mouse. Arrows indicate parameter changes of thecorpus callosum compared to control.

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group to the controls. No false assignment was found between the twocuprizone-fed groups (Fig. 10, right). The corresponding functions1 and 2 were 54.421 x FA - 59.822 x AD+40.279 x RD+14.898and 2.065 x FA - 11.304 x AD+27.547 x RD - 6.676. The functionalcharacteristics of the group centroids (Fig. 10, black quadrates)were D1/D2=−0.852/-0.882 for controls, -1.237/0.78 for Cup and2.089/0.102 for Cup-EAE mice.

Correlation of DTI parameters with histopathology

In the two cuprizone-fed groups, we obtained strong correlationsbetween AD values and the number of APP-positive axonal spheroids(R=−0.87), the number of CD3-positive cells (T-cells) (R=−0.79),

Fig. 9. Group analysis of fractional anisotropy (FA), axial diffusivity (AD), and radial diffusivbold horizontal bars indicate median values, boxes and bars represent the interquartile ran

and the number of cell nuclei in general (R=−0.84). The RD valuescorrelated with the number of T cells (R=−0.87) and slightly lesswith the number of APP-positive axonal spheroids (R=−0.70). Nocorrelation was found between FA values and any of the quantifiedhistological parameters. Furthermore, no correlations were found be-tween any DTI parameter and the LFB score (extent of demyelin-ation), the number of GFAP-positive astrocytes, and the number ofMac-3-positive activated microglia/macrophages.

Discussion

We have established a mouse model with brain lesions very similarto those found in the human disease MS and performed a detailed MRI

ity (RD) in the corpus callosum of control, Cup and Cup-EAE mice (n=5 each). Whileges (25th to 75th percentiles) and the range of measurements, respectively.

Fig. 10. Discriminant function analysis of (left) multi-contrast MRI and (right) multi-parametric DTI in the corpus callosum of control, Cup, and Cup-EAE mice. In both analysesabout 87% of all mice were assigned to the correct treatment group. MRI revealed the best prediction of control and affected mice, whereas DTI parameters allowed for the bestseparation between Cup and Cup-EAE mice. For details see text.

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characterization. The lesions were predominantly localized in the cor-pus callosum and characterized by a confluent demyelinated area,moderate T cell infiltration, and acute axonal damage, thus compris-ing all basic pathologic aspects of an acute MS lesion. Compared tothe Cup treatment, the extent of demyelination in the corpus callo-sum in mice receiving an additional immunization with MOG35-55

was similar, whereas T cells and early axonal pathology were signifi-cantly increased. These differences were reflected by different MRIand DTI patterns. The best separation between controls and affectedmice was obtained by combining T1, T2 and MTR contrasts, whereasthe combination of AD, RD, and FA values resulted in the best predic-tion of the type of pathological alteration. Although sample sizes wereat the lower limit for a discriminant function analysis, its power tocombine the strengths of different techniques is clearly illustrated inFig. 10.

Relative to controls, T2-weighted signal intensities of the corpus cal-losum increased to a similar extent in both Cup and Cup-EAE groups(Fig. 6). The observation that T2-weighted MRI is a sensitive but rathernon-specific indicator of tissue injury is in line with many previous re-ports (Barkhof, 2002; Boretius et al., 2008; Bruck et al., 1997; Werringet al., 2000). The severity of the diseasewasmuchbetter reflected by re-duced signal intensities on T1-weighted images (Fig. 6). Whilecuprizone-induced demyelination led to low T1-weighted signal inten-sities in the corpus callosum, significantly stronger reductions in theCup-EAE group are ascribed to the additional presence of T cell infil-trates and early axonal pathology. This is in agreement with resultsobtained in optic neuritismodels and clonal adoptive transfer EAE (Bor-etius et al., 2008; Nessler et al., 2007), where T1-weighted intensity re-ductions correlated with the density of cellular infiltrates.

We observed focal Gd-DTPA enhancement around the corpus cal-losum and ventricles of Cup-EAE but not Cup mice. In fact, the latterhave also previously been shown to present with an intact BBB forlarge solutes (Bakker and Ludwin, 1987; Kondo et al., 1987; Merkleret al., 2005). Gd-DTPA enhancement of Cup-EAE mice correspondedwith tissue deposition of IgG and fibrinogen in histology. Thus, Cup-EAE mice turned out to be a more appropriate model for acutehuman MS than pure cuprizone treatment or previous EAE models,where an inflammatory involvement of the cerebrum is only incon-sistently observed (Rausch et al., 2003).

Magnetization transfer techniques indirectly assess the amount ofwater protons associated with macromolecules such as myelin. In

previous experimental studies, cuprizone-induced demyelination wasassociated with MTR reductions (Merkler et al., 2005), which normal-ized after remyelination. However, in a guinea pig EAE model, acuteEAE lesions, although not demyelinated, also showed a significantMTR reduction (Dousset et al., 1992). In postmortem studies ofhuman MS lesions, MTR was correlated with the degree of myelinationand the number of axons (Schmierer et al., 2004). In our study, bothgroups of cuprizone-fed mice had attenuated MTR values compared tocontrols, but these reductions reached significance only for Cup-EAEmice (Fig. 6). Since the two groups exhibited a comparable degree of de-myelination and a negligible axonal loss, the stronger MTR reduction inthe Cup-EAE groupmay be explained by an additional loss ofmyelin perimage voxel due to axonal swelling, cellular infiltration, and edema.

The FA value has been shown to be sensitive to changes of myeli-nation and axonal integrity (Beaulieu, 2002; Beaulieu and Allen,1994) and its reduction has been observed in a broad spectrum ofbrain disorders (Cheng et al., 2010; Kubicki et al., 2007; Pierpaoli etal., 2001; Riley et al., 2010; Stebbins and Murphy, 2009). In ourstudy, FA of the corpus callosum was significantly reduced in bothcuprizone-treated groups, but could not differentiate demyelinationfrom demyelination with inflammation and acute axonal damage.This may be explained by the fact that the FA reflects all three eigen-values of the diffusion tensor, so that the increased mean of λ2 andλ3 in the Cup group and the reduced λ1 in the Cup-EAE group maylead to a similar FA reduction.

In contrast to the FA findings, the separate consideration of theeigenvalues substantially improved the tissue characterization. Inparticular, the AD was significantly reduced only in Cup-EAE mice andshowed a strong negative correlation with APP deposits. This is in linewith DTI studies of mice and rats with different degrees of axonaldamage (Budde et al., 2008; MacDonald et al., 2007; Sun et al., 2006;Wu et al., 2007). The AD of Cupmice did not differ from those of controlmice. This is in accordance with a study of shiverer mice having intactaxons (Song et al., 2003), but contrary to demyelinating modelswith lysolecithin (DeBoy et al., 2007), and probably reflects model-dependent differences in the extent of axonal pathology. Moreover,AD was significantly correlated with the number of CD3 positiveT cells, which may hinder and reduce the diffusivity by infiltratingthe aligned axon bundles. This aspect could also be of relevance fora number of DTI studies which reported normal and even increasedAD values in MS lesions (Lowe et al., 2006; Roosendaal et al., 2009).

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Besides differences in the numbers of inflammatory cells which mayinfluence the AD, putative diffusion restrictions inside damaged butpersisting axons might be counterbalanced by an overall increase indiffusivity due to tissue loss. Of note, Cup and Cup-EAE mice didnot exhibit marked axonal loss as shown by neurofilament (datanot shown) and Bielschowsky stainings.

The RD has been proposed to be an in vivo marker for the degreeof myelination in animal models (Shamy et al., 2010; Song et al.,2002; Song et al., 2005) and human brain (Giorgio et al., 2010;Lowe et al., 2006; Pierpaoli et al., 2001). However, RD of the spinalcord in mice with EAE did not significantly correlate with demyelin-ation (Budde et al., 2008). In our model, the extent of demyelinationwas comparable in Cup and Cup-EAE mice, but RD was significantlyincreased only in the group with toxic, non-inflammatory demyelin-ation. Increases in the number of inflammatory cells and the extentof early axonal damage seem to counterbalance the effect of demye-lination on RD. One may speculate that the accumulation of APP de-posits reduces the diffusivity inside the axons in all directions, whilemicroglia and T cells similarly reduce the diffusivity by filling theinteraxonal space which is enlarged as a result of demyelination.Therefore, the use of RD values alone may underestimate the degreeof demyelination in diseases with accompanying inflammation andacute axonal damage such as MS.

A higher diagnostic certainty could be achieved by combiningdifferent MRI results with the use of a discriminate function analy-sis. While a best separation between affected and unaffected micewas realized by combining T1, T2 and MTR contrasts, the best pre-diction with respect to the pathologic Cup and Cup-EAE groupswas achieved by combining AD, RD, and FA values as rather directparameters of tissue microstructure. These results clearly indicatethat a more extended discriminant function analysis of both MRIand DTI parameters promises even further advances in the future.At this stage, a combined analysis was precluded by the extensivemeasuring times for all acquisitions and the impaired generalhealth of cuprizone-treated mice which required the use of twogroups of animals for separate studies of MRI and DTI.

In summary, using a novel mouse model reflecting both extensivedemyelination and inflammation with acute axonal damage in thecorpus callosum, the present study demonstrates that a more precisecharacterization of the underlying pathological substrate is feasibleusing MRI-based diffusion parameters. In addition, our study indicatesthat care has to be taken when assigning a certain DTI quantity suchas the RD value to a specific pathological substrate such as the degreeof myelination. However, the combination of information obtainedfrom the different DTI parameters substantially increases the accuracyof the prediction of the lesion microstructure.

Acknowledgement

The authors would like to thank Sina Bode for excellent assistancewith animal handling and quantitative data analysis as well as UtaScheidt, Angela Dettmar and Katja Schulz for excellent support forhistology and immunohistochemistry.

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