Multicontrast MRI of remyelination in the central nervous system

NMR IN BIOMEDICINENMR Biomed. 2005;18:395–403Published online 8 August 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/nbm.972

Multicontrast MRI of remyelination inthe central nervous system

Doron Merkler,1y Susann Boretius,2*,y Christine Stadelmann,1 Tristan Ernsting,1 Thomas Michaelis,2

Jens Frahm2 and Wolfgang Bruck1

1Department of Neuropathology, Georg-August University Gottingen, Germany2Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut fur biophysikalische Chemie, Gottingen, Germany

Received 1 April 2005; Revised 7 June 2005; Accepted 10 June 2005

ABSTRACT: Although magnetic resonance imaging (MRI) represents the most sensitive tool for the detection of white

matter abnormalities in patients with multiple sclerosis (MS), the heterogeneity of MS placques severely hampers the

elucidation of specific pathophysiological processes. In order to identify putative MRI markers for de- and remyelination,

we employed the cuprizone mouse model which leads to a selective and reversible demyelination of the corpus callosum

with little or no axonal damage. Apart from histopathology, animals were studied with high-resolution three-dimensional

MRI in vivo using multiple contrasts. While individual MRI findings significantly correlated with electron microscopy, the

differentiation of regions with normal, demyelinated or remyelinated white matter by one contrast alone was less specific

than by histology or electron microscopy. However, an accurate MRI prediction of the in vivo myelin status was achieved by

a discriminant function analysis using a combination of T1, T2 and magnetization transfer contrast. With a correct

assignment of 95% of all animals examined, the procedure will allow for the survey of new therapeutic approaches aiming at

improved remyelination. Copyright # 2005 John Wiley & Sons, Ltd.

KEYWORDS: magnetic resonance imaging; remyelination; cuprizone

INTRODUCTION

Despite the fact that demyelination of white matter with arelative preservation of axons is the pathological hall-mark of multiple sclerosis (MS), recent studies reveal apronounced structural heterogeneity of MS plaques1,2

caused by multiple immunopathological mechanisms.3

This particularly applies to lesions with and withoutoligodendrocyte precursor recruitment and a correspond-ing presence or absence of remyelination.4 The hetero-geneous myelin pathology is further supported byquantitative histopathologic analyses of autopsies indi-cating that about 42% of MS lesions show some degree ofremyelination.5

While magnetic resonance imaging (MRI) has beendemonstrated to be the method of choice for the detectionof white matter abnormalities in patients with MS,6 theMRI equivalents of specific pathophysiologic processesunderlying MS lesions are largely unknown.7 For exam-ple, although demyelinated lesions in MS autopsies

differed significantly from remyelinated lesions withrespect to T1 contrast and magnetization transfer ratio(MTR), the groups showed considerable overlap.5 On theother hand, the time course of T1 hypointensities has beensuggested as an in vivo marker of remyelination,8 and in arat model of reversible demyelination, MTR decreasesduring demyelination were reported to recover almostcompletely upon remyelination.9

In order to evaluate the potential of different MRIcontrast parameters to identify reparative processes suchas remyelination and hence putative restoration of axonalconduction, we took advantage of the cuprizone mousemodel which allows for a consistent induction andreversal of demyelination of the corpus callosum,10,11

as also studied by T2-weighted MRI.12 Accordingly, theprimary aim was the elucidation of specific MRI markersfor white matter alterations that are directly related tomyelin loss and repair.

MATERIALS AND METHODS

Animals and cuprizone administration

All studies were performed in accordance with Germananimal protection laws and approved by the responsiblegovernmental authority (Bezirksregierung Braunsch-weig). Thirty-five male, 7–8-week-old C57/BL6 mice

Copyright # 2005 John Wiley & Sons, Ltd. NMR Biomed. 2005;18:395–403

*Correspondence to: S. Boretius, Biomedizinische NMR ForschungsGmbH am Max-Planck-Institut fur biophysikalische Chemie, AmFassberg 11, 37070 Gottingen, Germany.E-mail: [email protected] authors contributed equally to that work.

Abbreviations used: APP, amyloid precursor protein; BBB, blood–brain barrier; FLASH, fast low-angle shot; FSE, fast spin-echo; GFAP,glial fibrillary acidic protein; LFB, Luxol fast blue; MTR, magnetiza-tion transfer ratio; TE, echo time; TR, repetition time.

were purchased from Jackson Laboratories. Twenty ani-mals were fed 0.2% (wt/wt) cuprizone (Sigma, St Louis,MO, USA) in ground breeder chow ad libitum for6 weeks. At this time point (after 6 weeks of cuprizone)15 animals were examined by MRI and 10 of these weresacrificed for histology. The remaining 10 animals werekept on normal diet without cuprizone for further 6 weeksto study spontaneous remyelination. Fifteen age-matchedanimals served as controls and were maintained on anormal diet for the duration of the experiments. Micewere bred and maintained in a pathogen-free facility on aregular day/night cycle.

MRI

Mice were anesthetized by i.p. injection of medetomidine(1.5 mg/kg) and ketamine (150 mg/kg) for induction.Subsequently, animals were intubated with a polyethy-lene endotracheal tube and artificially ventilated. An-esthesia was maintained using 0.5–1.0% isoflurane inoxygen. Respiration was monitored by a signal derivedfrom a home-made pressure transducer fixed to theanimal’s chest. The animals were placed in a proneposition on a purpose-built plate holder with an adjus-table nose cone. The rectal temperature was held constantat 37� 1 �C by water blankets.

All MRI studies were conducted at 2.35 T using anMRBR 4.7/400 mm magnet (Magnex, Scientific, Abingdon,UK) equipped with a DBX spectroscopy and imagingsystem (Bruker Biospin, Ettlingen, Germany). For ex-citation a Helmholtz coil (100 mm) was used and com-bined with an elliptical surface coil (20� 12 mm) forsignal detection. At 6 and 12 weeks after starting thecuprizone treatment, three-dimensional T1-weighted (3DFLASH, TR/TE¼ 17/7.58 ms, �¼ 25�) and T2-weightedimages (3D FSE, TR/TE¼ 3000/98.25 ms, 16 echos,inter-echo-spacing¼ 12.5 ms) were obtained with an iso-tropic spatial resolution of 117mm.13 Maps of the mag-netization transfer ratio (MTR) were based on a spindensity-weighted sequence (3D FLASH, TR/TE¼30/7.60 ms, �¼ 5�) with and without off-resonance RFirradiation (Gaussian-shaped RF pulse, frequency offset5 kHz, mean amplitude 200 Hz corresponding to a flipangle of 1045�).14 The entire MRI session took about 5 h.After MRI, the animals were sacrificed and prepared forhistology. Five of the 20 cuprizone-treated animals un-derwent a follow-up study with identical MRI examina-tions before and after 6 weeks of cuprizone as well asafter an additional 6 weeks without cuprizone. Age-matched animals receiving normal diet showed negligiblevariance and were pooled into one control group.

Comparisons of MRI and histologic data were per-formed in a transverse section located about 0.7 mmanterior to the bregma. A quantitative region-of-interestanalysis was based on MRI signal intensities in the lateraland medial part of the corpus callosum normalized to the

intensity of the cerebrospinal fluid. MTR values weregiven in percentages and calculated as the quotient(M0�Msat)/M0 with M0 the signal of the unperturbedspin density-weighted MR images and Msat the signalafter off-resonance RF irradiation.

Tissue preparation and staining

Subsequent to their final MRI examination, animalsreceived an overdose of ketamine/medetomidine andwere perfused transcardially with 4% paraformaldehyde.Brains were removed and postfixed in 4% paraformalde-hyde overnight. Segments of brains were prepared forparaffin or electron microscopy sections as describedpreviously.15 Paraffin sections were stained convention-ally with Luxol fast blue (LFB), periodic acid Schiff andBielschowksy’s silver impregnation for detection ofmyelin and axonal structures, respectively. Immunohis-tochemistry was performed by biotin–avidin or alkalinephospatase/anti-alkaline phosphatase technique. We usedthe following primary antibodies: rabbit anti-glial fibril-lary acidic protein (GFAP, Dako, Germany) for judgmentof astrogliosis; rat anti-Mac3 (Pharmingen, USA) fordetection of activated microglia/macrophages; andmouse anti-human amyloid precursor protein (APP, clone22C11, Chemicon, USA) as a marker for acute axonaldamage. For each animal group electron microscopy wasperformed in a randomly selected subset of animals(controls, n¼ 7; 6 weeks on cuprizone, n¼ 6; and12 weeks, n¼ 7, after the start of cuprizone). Forthis purpose, sections from epon-embedded, glutaralde-hyde-fixed corpus callosum were cut and stained withtoluidine blue. The tissue was then trimmed and reor-iented so that ultrathin cross sections of midline corpuscallosum could be cut and treated with uranyl acetate andlead citrate.

Morphometry

Electron micrographs were analyzed for fiber diameter,axon diameter and myelin thickness using the analySISDocu1 System (Soft Imaging System GmbH, Germany).16

We distinguished remyelinated fibers from residual mye-linated fibers by their thinner myelin sheath in relation tothe axon diameter17 using at least 200 fibers from eachmouse (n¼ 6–7 animals per group). G-ratios were de-fined as diameter of the axon divided by fiber diameter(i.e. axon and myelin).18,19 We calculated a G-ratio foreach fiber and subsequently averaged all G-ratios fromone brain. In addition, the number of non-myelinatedfibers in relation to all fibers was counted and expressedas percentage non-myelination.

The total number of Mac-3þ microglia/macrophages inthe lateral and medial part of corpus callosum wascounted using 400-fold magnification, and cellularity

396 D. MERKLER ET AL.


was calculated as cells per mm2. The extent of astro-gliosis was scored by superimposing a 25-point stereo-logic eyepiece on GFAP-stained sections of the corpuscallosum. Using 400-fold magnification the number ofastrocytic processes in direct contact with the randomlydistributed crosses was counted and divided by thenumber of total crosses within the evaluated region. Weevaluated at least six randomly selected optical fields inthe area of corpus callosum per animal.

Statistical analysis

Statistical comparisons between the animal groups (con-trols, 6 weeks, 12 weeks) were performed by one-wayANOVA followed by multiple t-tests with Bonferoniadjustment for multiple comparison if the F-test ofANOVA indicated statistically significant differences.For comparison of histological and MRI findings, weperformed a bivariate correlation analysis and calculatedthe Pearson correlation coefficient.

To evaluate the potential of different MRI contrasts todiscriminate between groups of cuprizone-treated ani-mals (6 weeks, 12 weeks) and controls, we performed adiscriminant function analysis using SPSS 11.0 for win-dows1 (Chicago, IL, USA). A set of linear functions ofthe predictors (MRI contrasts) was constructed accordingto D¼ b1x1þ b2x2þ � � � þ bnxnþ c, with bs as discrimi-nant coefficients, xs as input variables or predictors and cas a constant. Wilks’s lambda (�) was used to evaluate thequality of group separation. This is the sum of squareswithin the groups divided by the overall sum of squares,and ranges from zero (best group separation) to one. Theattribution to the three different groups was based on themaximum posterior probability of group membership.20

RESULTS

Light microscopic analyses of brain sections (Fig. 1, toprow) revealed an almost complete demyelination ofthe mouse corpus callosum 6 weeks after continuous

Figure 1. Light microscopy and MRI of the corpus callosum ofmice treated with cuprizone. (LFB) Luxol fast blue staining formyelin, (T2w) T2-weighted images, (T1w) T1-weighted imagesand (MTR) magnetization transfer ratio of the brain of (left)controls, (middle) mice after 6 weeks of cuprizone treatmentand (right) after an additional 6 weeks on normal diet andwithdrawal of the toxin (12 weeks). Arrows head indicate corpuscallosum

MULTICONTRAST MRI OF REMYELINATION 397


administration of cuprizone. Subsequent discontinuationof cuprizone treatment resulted in substantial remyelina-tion after an additional 6 weeks on a normal diet. Usingconventional staining for myelin,15 the remyelinatedcorpus callosum was indistinguishable from that of un-treated animals.

High-resolution three-dimensional MRI (Fig. 1, bot-tom three rows) was performed for all animals of the age-matched control group, the demyelinated group (6 weeks)and the remyelinated group (12 weeks) using T2-weighted (T2w) and T1-weighted (T1w) images as wellas MTR maps.13,14 Qualitatively, all contrast mechanismsled to an inversion of the native white matter contrast(control) in response to demyelination (6 weeks). Forexample, T2-weighted images which present with hy-pointense white matter in the normally myelinated corpuscallosum relative to cortex revealed pronounced hyper-intensities in response to demyelination. While remyeli-nation (12 weeks) eliminated the inversion of T1, T2 andmagnetization transfer contrasts, T1- and T2-weightedMRI signal intensities yielded similar but not identicalvalues as for controls.

Using electron microscopy in a subset of animals(Fig. 2) we could demonstrate a high percentage ofcallosal axons to be non-myelinated or demyelinated

(57.1� 17.3%, n¼ 6) after 6 weeks of cuprizone relativeto controls (6.4� 4.7%, n¼ 7). After an additional6 weeks on a normal diet, the number of non-myelinatedor demyelinated fibers was again reduced (18.0� 7.9%,n¼ 7) but still above the level of controls [Fig. 2(D)].Correspondingly, the mean ratio of the axon thicknessto total fiber diameter including myelin [G-ratio,Fig. 2(E)] was significantly increased after 6 weeks ofcuprizone (0.894� 0.049, n¼ 6) compared with controls(0.729� 0.031, n¼ 7). After an additional 6 weeks with-out cuprizone, the G-ratio recovered to an intermediatevalue (0.796� 0.025, n¼ 7). These findings providefurther evidence that a large proportion of demyelinatedaxons is indeed capable of remyelinating after acutecuprizone intoxication. The results imply that electronmicroscopy permits a discrimination of normal myelina-tion, demyelination and remyelination.

Immunohistochemical staining against the amyloidprecursor protein (APP), a marker for acute axonaldamage,21 resulted in only a small number of damagedaxons in cuprizone-treated animals (Fig. 3). Accordingly,staining with Bielschowsky’s silver impregnation did notreveal an apparent reduction of axonal structures withinthe corpus callosum during and subsequent to cuprizonetreatment. The number of hypertrophic astrocytes with

Figure 2. Electron microscopy of the corpus callosum of mice treated with cuprizone.(A) Controls, (B) mice after 6 weeks of cuprizone treatment, and (C) after additional6 weeks of remyelination. (D) Percentage of non-myelinated fibers in controls (n¼7)and (E) ratio of the diameter of the axon to the axon plus myelin (G-ratio) after 6 weeksof cuprizone treatment (n¼6), and after an additional 6 weeks of remyelination(n¼ 7). While bold horizontal bars indicate median values, boxes and bars extendingoutside the boxes represent the interquartile ranges (25th to 75th percentiles) and therange of measurements excluding outliers (open circles), respectively. Asterisks andvertical bars indicate significant difference between time points (**p<0.001,*p< 0.005, Bonferroni corrected t-test)



stellate shape and thick processes was markedly in-creased after cuprizone and concurrent with demyelina-tion. A large portion of these ‘reactive’ astrocytes wasstill present 6 weeks after cuprizone discontinuation.Whereas microglia/macrophages were hardly detectablein the corpus callosum of controls (1.37� 1.76 cells/mm2), their number was significantly increased(442.6� 177.8 cells/mm2) after 6 weeks of cuprizonetreatment. Again, 6 weeks afterwards, the number ofmicroglia/macrophages was almost restored to controllevels (26.3� 24.8 cells/mm2). Together, histopathologyconfirmed that acute intoxication with cuprizone resultsin consistent and reversible demyelination of the corpuscallosum with concomitant astrocytic activation andtransient accumulation of microglia/macrophages. Thedegree of irreversible axonal damage, in terms of trans-

ected axons and subsequent axonal loss, was only mar-ginal.

To quantify the MRI results, we normalized the signalintensities from regions-of-interest in the lateral andmedial part of corpus callosum by the intensity of thecerebrospinal fluid (T1c, T2c). Because there was nosignificant difference between the results obtained forthe median and lateral part of the corpus callosum, thesubsequent analyses were performed with the combineddata set. Relative to controls, the corpus callosum ofdemyelinated animals (6 weeks) showed a significantincrease of the median T2c value, a corresponding de-crease in T1c, and a reduction of the MTR ( p¼ 7� 10�29,1� 10�17 and 1� 10�14, respectively, Bonferronicorrected t-test) as demonstrated by the results of a groupanalysis [Fig. 4(A)]. In remyelinated animals (6 further

Figure 3. Histopathology of the corpus callosum of mice treated with cuprizone.(Left) controls, (middle) mice after demyelination (6 weeks) and (right) mice afterremyelination (12 weeks). (APP) amyloid precursor protein: only very few damagedaxons were detected after 6 weeks of cuprizone treatment (arrow heads in insetshowing APP-positive axons). APP-positive axons were again almost absent after 12weeks (6 weeks after cuprizone withdrawal). (Biel) Bielshowsky’s silver impregna-tion: light microscopy did not reveal an apparent loss of axons during demyelination(6 weeks) or remyelination (12 weeks). (GFAP) anti-glial fibrillary acidic protein: thenumber of hypertrophic GFAP-positive cells was markedly increased after 6 weeks ofcuprizone. A considerable proportion of these ‘activated’ astrocytes was stilldetectable after remyelination (12 weeks). (Mac3) anti-Mac3: an increase in thenumber of activated microglia/macrophages was detectable after demyelination butvanished after remyelination



weeks on normal diet after having been 6 weeks oncuprizone), MTR was not significantly different fromthe control group (p¼ 0.055, Bonferroni corrected t-test).In contrast, and despite a trend towards normalization,namely a decrease in T2c and an increase in T1c duringremyelination, the T2c and T1c values remained signifi-cantly ( p¼ 5� 10�11 and 6� 10�6, respectively, Bon-ferroni corrected t-test) different from those of controls.

By comparing electron microscopy and MRI findingswe found a correlation of the G-ratio with T2c (r¼ 0.74,p< 0.001), T1c (r¼�0.62, p< 0.01) and MTR(r¼�0.48, p< 0.05). Similar highly significant resultswere obtained for a correlation of the percentages of non-myelinated axons with T2c (r¼ 0.76, p< 0.0001), T1c(r¼�0.62, p< 0.001), and MTR (r¼�0.60, p< 0.01).Although these findings confirm a direct pathophysiolo-gical relevance of the observed MRI signal alterations,the group analysis of all three MRI contrasts showedconsiderable overlap between demyelinated and remye-linated animals as well as controls when considering T2c,T1c and MTR alone [Fig. 4(A)]. Because this observationmay have been caused by interindividual differences, wecarried out intraindividual follow-up studies in an addi-tional cohort of five different animals. The resultingtemporal evolution of T2c, T1c, and MTR [Fig. 4(B)]

before and after feeding of cuprizone (6 weeks), as wellas after withdrawal (12 weeks), confirmed the findings ofthe group analysis. Despite quantitative differences, allanimals responded with an MRI contrast inversion dueto demyelination which at least partly recovered afterremyelination.

To more objectively assess the MRI ability to differ-entiate between normal myelination, demyelination andremyelination, we used discriminant function analysis.Whereas MTR resulted in a correct prediction of only40%, T2c or T1c contrast alone correctly assigned 80% ofthe regions/lesions in the remyelinated group (12 weeks).This outcome was improved by a combination of twoMRI contrasts. Table 1 summarizes respective predictionstrengths expressed as Wilks’s lambda (�) and the per-centage of correct classifications. The combination of T2cand T1c resulted in a correct classification of 93.8% ofall animals examined with �¼ 0.127, that is 12.7% ofscattering is not explainable by the group difference.However, the most accurate prediction of myelinstatus was obtained by using all three contrasts, yieldinga correct assignment of 95.0% of all animals with�¼ 0.089 (Table 1). The separation of groups isvisualized in a three-dimensional scatter plot of MTR,T2c and T1c together with the results of the discriminant

Figure 4. MRI of the corpus callosum of mice treated with cuprizone. MRI signal intensities of T2- and T1-weighted images normalized to the intensity of the cerebrospinal fluid (T2c, T1c) and the magnetization transferratio were determined in the lateral and medial part of corpus callosum. (A) Group analysis of controls (n¼15),demyelinated animals (6 weeks, n¼15) and remyelinated animals (12 weeks, n¼ 10). While bold horizontalbars indicate median values, boxes and bars extending outside the boxes represent the interquartile ranges (25thto 75th percentiles) and the range of measurements excluding outliers (open circles), respectively. Asterisks andvertical bars indicate significant difference between time points (**p<0.001, Bonferroni corrected t-test). (B)Intraindividual time courses of five different animals before (0 weeks) and after cuprizone (6 weeks) as well asfurther 6 weeks after the treatment period (12 weeks)



analysis (Fig. 5). These parameters were obtained bycombining the data from the median and lateral part ofthe corpus callosum as essentially the same parameterswere obtained in separate analyses.

DISCUSSION

The present findings demonstrate that the combination ofmultiple MRI contrasts dramatically improves the pre-diction of the myelin status of a lesion, and in particular,

allows for the in vivo characterization of remyelinatedwhite matter. Whereas the limitations of single-contrastMRI are likely to at least partially account for the poorcorrelation of morphologic involvement and clinicalfindings in MS,22 the reliable discrimination of remyeli-nated lesions from normal myelination and demyelina-tion by a combination of three readily available MRIcontrasts promises new insights into the evolution ofMS lesions. It certainly represents a prerequisite for thein vivo monitoring and evaluation of therapeutic ap-proaches devised to promote remyelination. Pertinent

Table 1. Discriminant function analysis using a combination of two or three MRI contrasts: T1c, T2c and MTR

Prediction strength (%) Total predictionstrength (%)

Control 6 weeks 12 weeks

T1c and T2c �¼ 0.127 Control group 93.3 0.0 6.76 weeks (demyelinated) 0.0 93.3 6.7 93.812 weeks (remyelinated) 0.0 5.0 95.0

T1c and MTR �¼ 0.178 Control group 76.7 0.0 23.36 weeks (demyelinated) 0.0 93.3 6.7 82.512 weeks (remyelinated) 20.0 5.0 75.0

T2c and MTR �¼ 0.152 Control group 90.0 0.0 10.06 weeks (demyelinated) 0.0 86.7 13.3 86.312 weeks (remyelinated) 15.0 5.0 80.0

T1c and T2c and MTR �¼ 0.089 Control group 93.3 0.0 6.76 weeks (demyelinated) 0.0 93.3 6.7 95.012 weeks (remyelinated) 0.0 0.0 100.0

Wilks’s lambda (�) ranges from zero (best group separation) to one. The prediction strength is expressed as the percentage of all data points (lateraland medial part of the corpus callosum) from either the controls (n¼ 30) or demyelinated (6 weeks, n¼ 30) and remyelinated animals (12 weeks,n¼ 20). Total prediction strengths refer to the total number of correctly assigned animals.

Figure 5. Discriminant function analysis of multicontrast MRI. Normalized signal intensities ofT1- and T2-weighted images (T1c, T2c) and the magnetization transfer ratio were obtained inthe lateral and medial part of corpus callosum of myelinated (control), demyelinated (6 weeks)and remyelinated (12 weeks) mice. (A) Scatter plot and (B) discriminant function analysis. D1

and D2 represent the two discriminant functions. The functional characteristics of the groupcentroids were D1/D2¼3.45/�0.13 for controls, as well as�3.63/�0.11 and 0.27/0.36 for thedemyelinated and remyelinated group, respectively. Deviations between real and predictedgroup membership are indicated by white and black circles



strategies include the application of growth factors,23 thetransplantation of myelin forming stem cells24,25 or theuse of intravenous immunoglobulin therapies.26,27

Our accomplishment is primarily based on the MRIappearance of remyelinated lesions, which differ fromthat of normal myelination. This observation is in linewith data derived from human autopsies5 and animalstudies such as a primate model of MS in commonmarmosets28 and a rat model of reversible demyelina-tion,9 which also demonstrates that remyelinated lesionsmay lead to an abnormal MRI signal. In general, how-ever, linking a particular MRI feature to a specificpathology has been hampered by the simultaneous in-volvement of myelin and axonal structures and thepresence of inflammatory cell infiltrates29 as well as afocal and temporary breakdown of the blood–brain bar-rier (BBB). Animal studies showed that enhancing MRIlesions reflect a breakdown of the BBB30,31 and areassociated with macrophage activation.32 In contrast, inthe cuprizone model the observed MRI signal changesduring cuprizone treatment most likely reflect an increasein the extracellular space due to depletion of myelin. It isfollowed by a tendency towards normalization after theend of cuprizone treatment, which is in line with theformation of new myelin. It is noteworthy that the MRIfindings in the cuprizone model may not be ascribed to theoccurrence of axonal loss and inflammation as illustratedby the histology (Fig. 3). In more detail, the cuprizonetreated animals reveal only a very moderate degree ofinflammation in comparison with experimental autoim-mune encephalomyelitis. In agreement with previousstudies,33,34 we did not find a distortion of the BBB asevidenced by the lack of gadolinium enhancement in thedemyelinated corpus callosum (data not shown).

In conclusion, multicontrast MRI may address onefeature of the structural heterogenity of MS lesions,namely the characterization of demyelination and remye-lination. Although, in general, additional factors such asinflammation, axon loss or gliosis contribute to the MRIappearance of MS lesions, such questions may be ad-dressed using the cuprizone treatment for geneticallymodulated mice or animals with experimental autoim-mune encephalomyelitis.

With respect to remyelination, the present findingsprovide a significant step toward the MRI elucidation ofthe pathophysiological processes involved in MS. It maycontribute to the management of individual patients by adiscrimination of subjects with and without spontaneousremyelination and a subsequent assessment of myelin-targeted therapies in suitable subgroups.

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Multicontrast MRI of remyelination in the central nervous system

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