Download - Imaging correlates of axonal swelling in chronic multiple sclerosis brains

Imaging Correlates of Axonal Swelling inChronic Multiple Sclerosis Brains

Elizabeth Fisher, PhD,1 Ansi Chang, MD,2 Robert J. Fox, MD,3 Jean A. Tkach, PhD,4 Therese Svarovsky, BS,2

Kunio Nakamura, BS,1 Richard A. Rudick, MD,3 and Bruce D. Trapp, PhD2

Objective: T2-weighted magnetic resonance imaging is a sensitive tool for monitoring progression of multiple sclerosis, but itdoes not provide information on the severity of the underlying tissue damage. Measurement of T1 hypointensities and magne-tization transfer ratio (MTR) can potentially distinguish lesions with more severe tissue damage. The objective of this study wasto use image-guided pathology to determine histological differences between lesions that are abnormal only on T2-weightedimages versus lesions that are abnormal on T2-weighted, T1-weighted, and MTR images.Methods: A total of 110 regions were selected from postmortem magnetic resonance images of 10 multiple sclerosis patients.Regions were classified into three magnetic resonance imaging–defined categories: normal-appearing white matter; abnormal onT2-weighted image only (T2-only); and abnormal on T2-weighted, T1-weighted, and MTR images (T2T1MTR). Myelin status,lesion activity, astrocytosis, serum protein distribution, axonal area, and axonal loss were evaluated histopathologically.Results: Comparisons between groups showed that T2T1MTR regions were more likely to be demyelinated (83% comparedwith 55% of T2-only regions) and more likely to be chronic inactive lesions (68% compared with 0% of demyelinated T2-onlyregions). There was no difference between T2-only and T2T1MTR regions in axonal area, but there was a significant differencein axonal count, indicating that axons in the T2T1MTR regions were enlarged relative to those in T2-only regions.Interpretation: Axonal swelling and axonal loss were major pathological features that distinguish T2T1MTR regions fromT2-only regions.

Ann Neurol 2007;62:219–228

Magnetic resonance imaging (MRI) is an objective andsensitive tool for diagnosing and monitoring multiplesclerosis (MS) patients.1–3 MRI has also been proposedas a tool for understanding MS pathogenesis in vivo.However, its usefulness in this regard is limited becauseconventional MRI is inherently nonspecific for distinctpathological processes. The nonspecific nature of MRIlesions is also one likely explanation for the weak cor-relations between lesion volumes and the progressionof clinical disability.4–8 MRI signal contrast arises fromdifferences in water and lipid content and the macro-molecular environment of adjacent tissues. In theory,all focal MS pathological processes, including blood–brain barrier (BBB) breakdown, inflammation, demy-elination, axonal loss, and gliosis, lead to an alterationof water and lipid content, and hence conspicuous le-sions on T2-weighted MRI (provided the affected re-gion is large enough to be detected). Additional MRsequences often are applied to distinguish underlying

pathology, or at least to determine lesion severity. Forexample, lesions that are hypointense on T1-weightedimages, or “black holes,” are generally considered tohave more severe tissue destruction than lesions thatare isointense on T1-weighted images, based on corre-lations with axonal loss,9–11 stronger correlations todisability,12,13 and association with chronic stage of le-sion development.14 T1 hypointense regions have alsobeen shown to have lower N-acetylaspartate comparedwith T1 isointense lesions, and normal white matterusing magnetic resonance spectroscopy.15,16 Similarly,decreased magnetization transfer ratio (MTR) in le-sions has been shown to be correlated to increased dis-ability,17,18 reduced N-acetylaspartate,19 demyelina-tion,20 and axonal loss.11

Previous postmortem and in vivo (biopsy) studieshave identified the histopathological characteristicsof different MRI features, such as T2 hypointen-sity,14,21 gadolinium enhancement,9,22,23 T1 hypoin-

From the Departments of 1Biomedical Engineering and 2Neuro-sciences, Lerner Research Institute; 3Mellen Center for MultipleSclerosis Treatment and Research, Cleveland Clinic; and 4Depart-ment of Radiology, Case Western Reserve University/UniversityHospitals Cleveland, Cleveland, OH.

Received Oct 10, 2006, and in revised form Jan 31, 2007. Acceptedfor publication Feb 2, 2007.

Current address for T. Svarovsky: Department of Pathology, Wis-consin National Primate Research Center, Madison, WI.

This article includes supplementary materials available via theInternet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat

Published online April 11, 2007 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.21113

Address correspondence to Dr Fisher, Department of BiomedicalEngineering ND20, Cleveland Clinic Foundation, 9500 EuclidAvenue, Cleveland, OH 44195. E-mail: [email protected]

ORIGINAL ARTICLES

© 2007 American Neurological Association 219Published by Wiley-Liss, Inc., through Wiley Subscription Services

tensity,9,11,14,24 and decreased MTR.11 Most of the ev-idence to date supports the idea that one canpathologically subclassify lesions in vivo using a singleMRI data set according to the following rules25: (1) T2hyperintensities are pathologically nonspecific, andtherefore represent MS lesions in all phases of patho-genesis; (2) gadolinium-enhancing lesions are regionswith breakdown of the BBB and possibly infiltration ofhematogenous leukocytes; (3) T1 hypointensities areMS lesions with axonal loss and severe tissue destruc-tion; and (4) lesions with decreased MTR representMS lesions with demyelination and axonal loss. In sup-port of this general concept, correlations between le-sion volumes and disability have been found to bestronger for T1 lesion volume than for the total T2lesion volume12; however, these correlations are stillquite modest in most studies, ranging from 0.22 to0.54.12,13 Notably, the lack of strong correlations isalso related to difficulties with measurement of clinicaldisease progression, in addition to limitations of cranialMRI. However, the correlations are not much strongerwhen an objective measure of disease severity, such asbrain atrophy, is used as the dependent variable. Cor-relations between T1 lesion volume and brain atrophyare about the same as or lower than correlations be-tween T2 lesion volume and atrophy, in the range of0.4 to 0.5, and T1 lesion volume is not an indepen-dent predictor of subsequent brain atrophy.26–28

The findings for lesion MTR measurements are sim-ilar. MTR is decreased in MS brains and varies widelyin MS lesions, from extremely low to nearly normalvalues.29 MTR is reduced in gadolinium-enhancing le-sions because of acute inflammation, and it subse-quently recovers over time in a subset of lesions, butremains low in others.30 Variations in lesion MTR alsorelate to degree of demyelination,20 remyelination,31

and degree of axonal loss.11 Correlations between meanlesion MTR and disability vary widely between studies,from nonexistent19,32 to relatively strong (�0.4 to�0.7).17,18

In general, the expectations that measurements ofthe more severe lesions, such as T1 hypointensitieswith decreased MTR, would be strongly correlatedwith disease progression have not been met. Theseweaker than expected correlations led to persistentquestions on the ability to differentiate lesions basedon MRI. Such a capability would be highly beneficialin the evaluation of new neuroprotective therapies, as away to distinguish potentially reparable lesions from ir-reparable lesions. The purpose of this study was to useimage-guided pathology to directly determine the ma-jor histological differences between lesions that are ab-normal only on T2-weighted images versus lesions thatare abnormal on T2-weighted, T1-weighted, and MTRimages.

Subjects and MethodsOverviewMS brain tissue was acquired through the tissue procurementprogram established at the Cleveland Clinic Foundation. Pa-tients and their families signed an institutional review board–approved advance directive to donate brain and spinal cordtissue for research purposes. Demographic and disease-relatedinformation for each patient was entered into a database.The same protocol was followed for each patient: Within afew hours of death, the cadaver was brought to the imagingfacility for an MRI examination followed by rapid autopsy.The brain and spinal cord were removed, and one cerebralhemisphere was immediately fixed in 4% paraformaldehydefor at least 4 weeks. After fixation, the hemisphere was re-imaged in a custom-designed slicing box for coregistrationpurposes. The brain tissue was sliced coronally in the box,and each slice was numbered, stored, and subsequently pho-tographed. The cadaver images were registered to the post-fixation images so that the image planes corresponded withthe tissue slices. Lesions were segmented and classified in thecadaver images using automated image analysis programs.Using the registered MRIs, we generated region maps to in-dicate regions of interest (ROIs) for histological analysis.

Magnetic Resonance Imaging DetailsImaging was performed on a 1.5-Tesla MR scanner (VI-SION; Siemens, Erlangen, Germany). The MRI protocolconsisted of a three-dimensional T1-weighted magnetizationprepared rapid acquisition gradient-echo image, a T2-weighted fluid-attenuated inversion recovery image (FLAIR),a T2-weighted fast spin-echo image, a T1-weighted spin-echo image, and an MTR image calculated from proton den-sity–weighted three-dimensional gradient-echo images ac-quired with and without a magnetization transfer pulse. Themagnetization prepared rapid acquisition gradient-echo im-age was acquired with 1 � 1 � 1mm resolution, and all theother images had 0.9 � 0.9 � 3mm resolution. Detailedsequence parameters are provided in Supplementary Table 1(see supplementary online materials). For the postfixationimage, the fixed cerebral hemisphere was placed in a custom-designed slicing box before imaging to aid in image to tissuecolocalization. The precision-machined polycarbonate boxhas sliding walls that lock in place to securely hold the tissueduring imaging and slicing. Two walls of the box have ver-tical knife-guide slots positioned 10mm apart with 2mm gel-filled cylindrical wells above and below each slot. When fixedtissue is imaged in the box before slicing, the gel markers aredetectable on the MRI and indicate the orientation and po-sition of each slice plane.

Image analysis was performed using software developedin-house (BIP; Department of Biomedical Engineering,Cleveland Clinic Foundation, Cleveland, OH). The analysisfor the cadaver images consisted of brain segmentation, brainparenchymal fraction (BPF) calculation, lesion segmentation,and lesion classification. Brain segmentation and BPF calcu-lation were performed on the FLAIR image using a fully au-tomated program, as described previously.28,33 T2 lesionswere segmented in the brain-masked FLAIR image using amodified version of the iterated conditional modest (ICM)algorithm.34 The segmented T2 lesion regions were used to

220 Annals of Neurology Vol 62 No 3 September 2007

guide the automated segmentation of lesions in the T1 andMTR images. First, the T2 lesion regions were overlaid oncoregistered T1 and MTR images. For each image type, theintensity statistics of the normal-appearing brain tissue in theneighborhood surrounding each lesion were calculated, andthresholds for abnormal tissue within each lesion region weredetermined. Abnormal was defined as any voxel in the T1 orMTR image that was colocalized within a T2 lesion and hadan intensity less than the mean intensity minus one standarddeviation of the local nonlesion tissue. Therefore, classifica-tion of regions as abnormal on T1 and MTR images wasbased on low intensity relative to neighboring normal-appearing white matter (NAWM), not by global threshold-ing. Lesions were subsequently classified into two types: re-gions of T2 hyperintensity that were normal appearing onT1 and MTR (T2-only), and regions of T2 hyperintensitythat were hypointense on T1 and MTR (T2T1MTR). Thefirst type represented regions hypothesized to be of lesser se-verity, whereas the second type represented regions hypoth-esized to be of greater severity. For each brain, ROIs withineach type of lesion and within NAWM were delineated forthe generation of region maps (see description later in thisarticle). FLAIR, T1, and MTR contrast ratios were calcu-lated for each ROI as the mean intensity within the regiondivided by the mean intensity within all of the NAWMROIs for the same brain.

Image analysis for the postfixation images consisted of de-tection of the markers in the slicing box, localization of thecut planes, and registration with the cadaver images. Thresh-olding and connected-components labeling were used toidentify the cerebral hemisphere and the markers. The best-fit plane equations were determined from sets of markers us-ing a program encoded with the dimensions of the slicingbox. The postfixation image was registered to the cadaverimages using the Iterative Closest Point algorithm to deter-mine the optimal three-dimensional affine transform (threerotations � three translations � uniform scaling).35 Theequations of the cut planes were then used to extract theindividual planes from the analyzed cadaver image volumesthat corresponded to each tissue slice.

Lesion maps were generated for each tissue slice from thecorresponding MRI planes. A 10mm grid was overlaid onthe image planes to provide a frame of reference. The out-lines of ROIs that corresponded to each type of lesion (T2-only and T2T1MTR) and NAWM were transferred to theregion maps. These maps were then used to guide tissuesampling for the histological analysis.

Immunocytochemistry DetailsROIs were removed, cryoprotected, and sectioned (30�mthick) on a freezing-sliding microtome. The free-floating sec-tions (30�m thick) were microwaved in 10mM citric acidbuffer (pH 6.0) for 5 minutes, incubated in 3% hydrogenperoxide and 10% Triton X-100 (Sigma, St. Louis, MO) inphosphate-buffered saline for 30 minutes, and immuno-stained by the avidin-biotin complex procedure with diami-nobenzidine tetrahydrochloride as a chromagen, as describedpreviously.36 Sections stained for serum protein were not mi-crowaved. Sections stained for IgG were processed using theavidin-biotin complex procedure with diaminobenzidine tet-

rahydrochloride, whereas horseradish peroxidase–conjugatedfibrinogen and albumin were visualized with diaminobenzi-dine tetrahydrochloride. Sections were also double labeled foraxons and myelin utilizing immunofluorescent procedures, asdescribed previously.36 A list of antibodies is provided inSupplementary Table 2.

MRI-defined regions were histologically classified basedon the presence or absence of myelin staining proteolipidprotein (PLP), activity of the lesion, and the serum proteindetection. The activity of lesions were subdivided into active,chronic active, and chronic inactive. Active lesions were de-fined as hypercellular throughout the lesion area, chronic ac-tive lesions had a hypercellular border of major histocompat-ibility complex (MHC) class II–positive cells and ahypocellular center, and chronic inactive lesions were definedas hypocellular throughout.37 If myelin was present in theROI, the microglia were examined for activation markersand changes in shape. Microglia exhibiting both MHC classII expression and retracting and thickening of processes werenoted as “activated.” The presence of serum proteins in eachregion was categorized as either diffuse or cellular staining,specific to axons and glial cells. Sections that were positive inat least one serum protein stain were considered positive.These sections were compared with non-MS control tissue(to distinguish lesions and myelinated white matter, becausemany of the tissue samples contained gliosis). Sectionsstained for SMI 32 and 31 were examined for axonal mea-surements. Axonal area was estimated using a computer-aided technique, similar to that described previously.38 Mul-tiple confocal micrographs for each region were digitized(40� magnification), transferred to a workstation, automat-ically thresholded, and quantified. Axonal area was calculatedin red-channel images as the number of thresholded pixelsmultiplied by 100% and divided by the total number of pix-els in the fixed-size image (250 � 250�m). Results from sixnonoverlapping areas were averaged to give the final axonalarea for each region. The measurement error, as determinedby the mean standard deviation of repeated analyses on thesame area, was 1.7%. Axonal count was determined automat-ically in the same thresholded images as the number of ob-jects greater than four pixels in size. For each region, an ax-onal diameter index was calculated as the mean axonal areadivided by the axonal count.

Data AnalysisEach region was categorized based on MRI characteristics(NAWM, T2-only, or T2T1MTR) and on histopathologicalfeatures. For each MRI-based group, the number of regionswith each specific histopathological feature was determinedand compared between MRI groups to identify any majordifferences. The �2 test was used to test for differences be-tween MRI groups for lesion activity (active vs chronic activevs chronic inactive). Fisher’s exact test was used to test fordifferences between MRI groups for myelin (myelinated vsdemyelinated) and serum proteins (diffuse vs cellular).Kruskal–Wallis one-way analysis of variance with Dunn’s testfor multiple comparisons was applied to test for differencesin axonal measurements among the three MRI groups. Con-trast ratios and axonal measurements were compared betweenMRI and histopathology groups using the nonparametric

Fisher et al: MRI of Axonal Swelling in MS 221

Mann–Whitney U test. Spearman’s rank correlation coeffi-cients were calculated to determine the relation between T1and MTR contrast ratios and axonal measurements in allMRI lesions and in the T2-only and T2T1MTR lesiongroups separately.

ResultsTissue AcquisitionThis report includes data from 10 confirmed MS casesthat were imaged and processed through the brain do-nation protocol. Demographic and disease-related de-tails are provided in Supplementary Table 3. All 10patients (6 female and 4 male patients) had secondary-progressive MS. The mean (standard deviation) agewas 56 (9.7) years, disease duration was 31 (11) years,Extended Disability Status Scale score at the time ofdeath was 8.6 (1.0), BPF was 0.77 (0.06), and brainweight was 1,151.1 (145) gm. The mean time betweendeath and brain removal was 5.4 hours. Imaging wasperformed immediately before autopsy.

Comparison of the lesion maps and photographsconfirmed that good coregistration was achieved be-tween the MRIs and tissue slices for each case. TheSupplementary Figure shows an example lesion map/tissue slice pair and the MR image with the lesiontypes superimposed. A total of 110 MRI-defined re-gions, which were mapped onto coregistered tissue slice

images, were sectioned and stained for histologicalanalysis: 29 NAWM regions, 40 T2-only regions, and41 T2T1MTR regions. The MRI region types weredistributed across all 10 brains. All regions identifiedradiologically were evaluated histologically. The meancontrast ratios and histological characteristics for eachMRI group are summarized in the Table. Comparisonof image contrast ratios between the MRI groups con-firmed that each group was significantly different fromthe other two. Examples of each MRI region type andcorresponding histology are shown in Figures 1(NAWM), 2 (T2-only), and 3 (T2T1MTR).

Pathological Features of Different MagneticResonance Imaging Region TypesAll NAWM regions were myelinated on histological in-spection. There was no evidence of serum proteins inthe NAWM regions, but 90% of NAWM regions con-tained activated microglia. Axonal area was highly vari-able in NAWM, ranging from 7 to 29%. The mean(standard deviation) percentage of axonal area was17.3% (4.2%). Axonal count in NAWM ranged from3,751 to 8,056, with a mean of 6,119 (936).

The T2-only group was heterogenous, with only55% of these regions showing demyelination. Activatedmicroglia were associated with all of the myelinated

Table. Magnetic Resonance Imaging and Histological Characteristics for Each Magnetic Resonance Imaging RegionType

Characteristics NAWM(n � 29)

T2-only(n � 40)

T2T1MTR(n � 41)

Mean T2 contrast ratio (SD) 1.0 (0.11) 1.3 (0.21) 1.4 (0.27)

Mean T1 contrast ratio (SD) 1.0 (0.04) 0.96 (0.06) 0.80 (0.10)

Mean MTR contrast ratio (SD) 1.0 (0.03) 0.91 (0.06) 0.67 (0.13)

Myelinated 100% (29/29) 45% (18/40) 17% (7/41)

Activated microglia 90% (26/29) 100% (18/18) 100% (7/7)

Demyelinated 0 55% (22/40) 83% (34/41)

Active 27% (6/22) 6% (2/34)

Chronic active 73% (16/22) 26% (9/34)

Chronic inactive 0 68% (23/34)

Astrocytosis 48% (14/29) 93% (37/40) 98% (40/41)

Serum proteins 0 100% (40/40) 100% (41/41)

Diffuse 72% (29/40) 37% (15/41)

Cellular 28% (11/40) 63% (26/41)

Axons

Mean % area (SD) 17.3% (4.2) 12.0% (4.3) 11.9% (4.4)

Mean count (SD) 6,119 (936) 3,930 (902) 2,711 (815)

NAWM � normal-appearing white matter; T2T1MTR � T2-weighted, T1-weighted, and magnetization transfer ratio; SD � standarddeviation; MTR � magnetization transfer ratio.


T2-only regions. Nearly all of the T2-only regions,both myelinated and demyelinated, had astrocytosis(93%), and all had either diffuse (72%) or cellular(28%) serum proteins. When the demyelinated T2-only regions were subcategorized based on lesion activ-ity, 27% were active and 73% were chronic active, butnone was chronic inactive lesions. The mean (standarddeviation) percentage of axonal area for T2-only re-gions was 12.0% (4.3%), which was significantly less

than for NAWM regions (p � 0.001). Mean axonalcount was 3,930 (902), which was also significantly lessthan in NAWM (p � 0.001). Mean axonal diameterindex was not significantly different from NAWM (3.2compared with 2.8, p � 0.52)

In comparison with the T2-only regions, a signifi-cantly larger percentage (83%) of the T2T1MTR re-gions were demyelinated (Fisher’s exact test, p �0.008). All of the myelinated T2T1MTR regions had

Fig 2. Magnetic resonance images (MRIs) and histology for a typical T2-only region. (A) T2-weighted fluid-attenuated inversionrecovery MRI; arrow marks the region of interest used for the histology sections below. (B) T1-weighted MRI; (C) Magnetizationtransfer ratio image. (D) Corresponding tissue slice with region of interest outlined in yellow. (E) Myelin-stained section (PLP)showing loss of myelin. (F) Major histocompatibility complex class II staining for lesion activity showing staining at lesion edge (ie,a chronic active lesion). (G) Serum protein staining (IgG) showing diffuse staining throughout the lesion. Scale bars � 1mm(E–G). V � ventricle.

Fig 1. Magnetic resonance images (MRIs) and histology for a typical normal-appearing white matter (NAWM) region. (A) T2-weighted fluid-attenuated inversion recovery MRI; arrow marks the region of interest used for the histology sections in E, F, and G.(B) T1-weighted MRI. (C) Magnetization transfer ratio image. (D) Corresponding tissue slice with region of interest outlined inyellow. (E) Myelin-stained section (PLP) showing no loss of myelin. (F) Major histocompatibility complex class II staining for lesionactivity showing activated microglia. (G) Serum protein staining (IgG) showing no evidence of serum protein staining. Scale bars �1mm (E–G).


activated microglia. Demyelinated T2T1MTR regionswere repopulated by activated microglia, which hadshorter and fewer processes than those in NAWM re-gions. T2-only demyelinated regions contained phago-cytic macrophages if acute or activated microglia andoccasional macrophages if chronic active. As in the T2-only regions, nearly all of the T2T1MTR regions, bothmyelinated and demyelinated, had astrocytosis (98%),and all had either diffuse (37%) or cellular (63%) se-rum proteins. There was a significantly greater propor-tion of serum proteins found in the cellular compart-ment in the T2T1MTR regions compared with theT2-only regions (p � 0.002). When the demyelinatedT2T1MTR regions were subcategorized based on le-sion activity, only 6% were active, 26% were chronicactive, and 68% were chronic inactive lesions. This dis-tribution of lesion activity was significantly differentfrom that of the T2-only regions (�2 test � 24.8; p �0.0001). The mean percentage of axonal area for theT2T1MTR regions was 11.9% (4.4%), which was lessthan the NAWM regions (p � 0.001), but not signif-icantly different from the T2-only regions. However,mean axonal count was significantly less in theT2T1MTR regions compared with both the NAWMand T2-only groups (p � 0.001), with a mean of2,711 (815). Correspondingly, the mean axonal diam-eter index was significantly greater in T2T1MTR re-gions compared with the other two groups (p �0.001). Figure 4 is a graph of the axonal area, count,and diameter measurements relative to NAWM. Im-ages depicting axonal morphology in the three MRIgroups are provided in Figure 5.

Correlations between MRI contrast ratios (FLAIR,T1, or MTR) and percentage axonal area ranged fromweak for T1 and MTR (Spearman’s rank correlationcoefficient [SRCC] � 0.25 and 0.27; p � 0.01) tomoderate for T2 (SRCC � �0.39; p � 0.0001). Incontrast, correlations between MRI contrast ratios andaxonal count were strong for all three parameters, par-ticularly for MTR (SRCC � �0.52, 0.71, and 0.79for FLAIR, T1, and MTR, respectively; p � 0.0001for all). Correlations between MRI contrast ratios and

Fig 4. Plot of percentage axonal area, axonal count, andswelling index in each magnetic resonance imaging group(gray bars denote T2-weighted imaging only; black bars de-note T2-weighted, T1-weighted, and magnetization transferratio abnormal [T2T1MTR]) relative to the means fornormal-appearing white matter (NAWM; hatched bars) re-gions. sd � standard deviation.

Fig 3. Magnetic resonance images (MRIs) and histology for a typical T2-weighted, T1-weighted, and magnetization transfer ratioabnormal (T2T1MTR) region. (A) T2-weighted fluid-attenuated inversion recovery MRI; arrow marks the region of interest usedfor the histology sections below. (B) T1-weighted MRI. (C) MTR image. (D) Corresponding tissue slice with region of interest out-lined in yellow. (E) Myelin-stained section (PLP) showing loss of myelin. (F) Major histocompatibility complex class II staining forlesion activity showing hypointense staining throughout the lesion (ie, a chronic inactive lesion). (G) Serum protein staining (IgG)showing cellular staining throughout the lesion. Scale bars � 1mm (E–G). V � ventricle.


axonal measurements were also determined in the de-myelinated regions separately (n � 56) to identify thepotential contribution of axonal swelling or loss toMRI characteristics independent of myelin. In the de-myelinated subset, which included 22 T2-only and 34T2T1MTR regions, there were no correlations at allbetween MRI contrast ratios and percentage of axonalarea. There was also no correlation between FLAIRcontrast ratio and axonal count. However, the correla-tions between T1 and MTR contrast ratios and axonalcount were still significant (T2: SRCC � 0.44, p �0.0009; MTR: SRCC � 0.41, p � 0.002). MRI con-trast ratios and axonal measurements were not corre-lated to postmortem time.

When the regions were subdivided based on the his-topathological features, FLAIR, T1, and MTR contrastratios were all significantly different in the myelinatedregions as compared with the demyelinated regions(1.1 vs 1.3 for median FLAIR contrast ratio; 0.97 vs0.86 for median T1 contrast ratio; and 0.97 vs 0.77 formedian MTR contrast ratio; p � 0.001 for all com-parisons). The demyelinated regions were then subdi-vided by lesion activity. Median FLAIR contrast ratiosfor active, chronic active, and chronic inactive lesionswere 1.26, 1.27, and 1.33, respectively; median T1contrast ratios were 0.96, 0.93, and 0.76, respectively;and median MTR contrast ratios in these three groupswere 0.90, 0.86, and 0.59, respectively. The chronicinactive regions had significantly lower T1 contrast ra-tio and MTR contrast ratios compared with the activeand chronic active groups, but there were no other sig-nificant differences between activity-based groups.

Other ObservationsThis study was primarily focused on pathologicalchanges that were most likely to contribute to the vari-able MRI characteristics in the three types of regions.However, we also examined several other cellular com-ponents that showed little correlation in distinguishingbetween T2-only and T2T1MTR lesions. For example,general T-cell density as identified by CD3 stainingmade up less than 0.5% of the total parenchymal areain both T2-only and T2T1MTR regions. Perivascularcuffs represented less than 1% of T2-only orT2T1MTR lesion areas. Other cellular componentssuch as phagocytic and foamy macrophages were abun-dant only in active lesions, which only made up 7.3%of the total regions in this study, and thus could notcontribute to either T1 or MTR differences observedoverall. Activated microglia were present in varying de-grees, but were most abundant in T2-only regions andleast abundant in T2T1MTR regions.

DiscussionTo investigate the validity of MRI-based lesion differ-entiation, we imaged postmortem MS brains in situand applied a new image-to-tissue coregistrationmethod for localization of tissue samples for his-topathological analyses based on MRI characteristics.Although no single histological features clearly distin-guished T2-only regions from T2T1MTR regions, sev-eral important differences were observed. Comparedwith the T2-only regions, the T2T1MTR regions weremore likely to be demyelinated (83% of theT2T1MTR group compared with 55% of the T2-only

Fig 5. Confocal images of sections stained with polyclonal myelin basic protein antibody (green) and axonal markers SMI 31 and32 (red). (A) Normal-appearing white matter (NAWM) region showing normal myelination. (B) Demyelinated T2-only region. (C)Demyelinated T2-weighted, T1-weighted, and magnetization transfer ratio abnormal (T2T1MTR) region. (D) Red-channel imageof NAWM region in (A) with percentage axonal area � 18.0% and axonal count � 6,420, or 105% of the mean for allNAWM regions. (E) Red-channel image of T2-only region in (B) with percentage axonal area � 13.8% and axonal count �3,665, or 60% of the mean for NAWM. (F) Red-channel image of T2T1MTR region with percentage axonal area � 13.0% andaxonal count � 1,748, or 29% of mean for NAWM. Scale bar � 20�m (A–F).


group). Demyelinated regions from both MRI groupshad significantly greater FLAIR contrast ratio andlower T1 and MTR contrast ratios than myelinated re-gions. Demyelinated T2T1MTR regions were muchmore likely to be chronic inactive lesions, whereas twothirds of the regions in this group were chronic inac-tive; none of the T2-only lesions was classified as such.T2T1MTR regions were also more likely to have se-rum proteins located intracellularly rather than dif-fusely, as in the majority of T2-only regions. Acute orrecent breakdown of the BBB, therefore, is a consistentfeature of T2-only regions. Demyelination does not ap-pear to be a consistent consequence of BBB breakdownbecause only 55% of the T2-only regions were demy-elinated. Surprisingly, there was no difference in thearea occupied by axons between the two groups, eventhough there were significantly fewer axons inT2T1MTR regions. In addition to demyelination andaxonal loss, axonal swelling is a significant determinantof T1 hypointensity and MTR abnormality.

An important observation of this report is the iden-tification of swollen axons as a marked feature in theT2T1MTR regions (see Fig 5F). Based on visual in-spection of axonal diameters in T2T1MTR lesions andadjacent NAWM, the enlarged axonal diameters didnot result from selective loss of smaller diameter axons.Rather, axons were abnormally swollen and muchlarger than axons in NAWM. In a previous postmor-tem study of MS spinal cords, swollen axons werefound within demyelinated lesions in patients withlong-standing MS.39 These lesions had high signal in-tensity on T2-weighted MRIs, but T1-weighted andMTR images were not acquired in that study. An im-portant consequence of axonal swelling is that thespace occupied by axons is maintained despite the de-struction of substantial numbers of axons, potentiallyconfounding measurements of brain atrophy. Thus, ax-onal swelling could partially explain why correlationsbetween T2T1MTR lesion volumes and BPF areweaker than expected.

From this postmortem study, it is not possible todetermine whether all the axonal swelling occurred invivo. If postmortem changes contribute to axonalswelling, these changes operate in demyelinated axonsand in more chronic lesions identified histologicallyand by T2T1MTR changes. Axonal diameters were notincreased in demyelinated T2-only lesions. Further-more, because there were no correlations between post-mortem time and any of the MRI contrast ratios oraxonal measurements, it is likely that the axonal swell-ing occurred in vivo and is indicative of a slowly pro-gressing necrotic death that eventually affects mostchronically demyelinated axons. Current concepts,40

supported by experimental animal models and molec-ular and morphological analysis of chronic MS brains,implicate malfunction of ion channels and pumps that

maintain axoplasmic Na/K gradients during nerve con-duction.41,42 Specifically, an imbalance of Na/K ex-change will eventually lead to swelling and death ofchronically demyelinated axons. Increased axoplasmicCa and reduced adenosine triphosphate production ap-pear to be essential contributors to the process of ax-onal swelling and axonal death.42

When all of the histological differences betweenMRI groups are considered, it is clear that there is agreater likelihood for a T2T1MTR region to havemore severe tissue damage than a T2-only region; how-ever, the T2T1MTR regions exhibited a high degree ofpathological heterogeneity. The observation that thereis considerable overlap in the histopathological featuresunderlying different MRI lesion types confirms previ-ous reports.9–11,14,20,23,31,43 It is also consistent withthe weak correlations reported in most MRI-clinicalcorrelation studies. In an analysis of MRI characteris-tics of MS biopsy specimens, Bruck and colleagues9

found that MRI features were not specific for distinctpathology, but that T1 hypointensity was related toseveral different factors, including extracellular edema,axonal loss, and degree of demyelination. A more re-cent report noted that T1 hypointensity at the time ofbiopsy did not correlate with axonal loss or demyeli-nating activity in a group of relapsing-remitting andprimary progressive patients.24 However, these findingsmay not be representative of MS lesions in general be-cause of the atypical MRI appearance of biopsiedMS lesions. Postmortem MRI-histopathological studieshave demonstrated strong correlations between T1contrast ratio and residual axonal density,10,11 MTRand axonal density,14 and a significant association be-tween myelin status and MRI characteristics on T1 andMTR images.31 Schmierer and colleagues20 report astrong correlation between MTR and quantitative mea-surements of myelination and a moderate correlationbetween T1 relaxation time and axonal count, but nosignificant difference in axonal counts between lesionsthat were hypointense versus those that were isointenseon T1-weighted MRI. As in van Waesberghe and col-leagues’ study,11 we found that both MTR and T1contrast ratios were highly correlated to axonal loss, aswas FLR contrast ratio, to a lesser extent. There wasalso a significant correlation between MTR and axonalarea, but only in the T2T1MTR regions and not inthe set of all lesions combined. This finding suggeststhat variance in MTR in the older, more chronic le-sions may be determined mainly by axonal water, butthat variance in MTR in the less severe lesions may bedominated by myelin content or extracellular water.Evidence of more extracellular water in the T2-only le-sions as compared with the T2T1MTR lesions (as de-termined by the presence of diffuse serum proteins)supports this explanation.

Another interesting observation in this study is that


45% of T2-only regions and 17% of T2T1MTR re-gions were histologically classified as myelinated. Mye-lin sheath thickness was appropriate for axonal diame-ters in these regions. Therefore, they were classified asnormally myelinated. None of the regions containedshadow plaques, although remyelination was oftenfound at the edge of some chronic active and chronicinactive lesions. The finding that such a high propor-tion of MRI-detected lesions are actually myelinatedappears counterintuitive, particularly about theT2T1MTR group, but not surprising because it is con-sistent with previous studies.11,14,20 De Groot and co-workers14 sampled postmortem brains based on MRIabnormalities and found that 48% of T2 lesions hadno apparent loss of myelin. They also found that 30%of the lesions that were classified as either mildly orseverely hypointense on T1 MRI had no apparent lossof myelin, similar to this study. In Schmierer and col-leagues’ study,20 44 of 98 ROIs selected by T2 hyper-intensity were discarded, mainly because of technicalproblems, but six regions were not studied becausethey were found to be normally myelinated. We ana-lyzed such regions to determine the potential source ofMRI signal change. Unlike NAWM, all of the myelin-ated T2-only regions had serum proteins and manyhad significantly reduced axonal area and axonal count,indicating MS pathology in the absence of colocalizeddemyelination.

Differences in the findings reported here and in pre-vious MRI-pathology correlation studies are likely dueto differences in techniques. In contrast with mostother studies, we performed postmortem imaging insitu before autopsy and used different MRI lesion de-tection, image-to-tissue coregistration, and histologymethods than those described for other studies.Schmierer and colleagues20 applied a surgical stereo-taxic frame system after brain removal on individualtissue slices to accurately mark ROIs based on MRIcoordinates for subsequent histological sampling.44 vanWaesberghe and colleagues11 also imaged single brainslices immediately after autopsy. To report axon den-sity, they used a visual ranking system from 0 to 100%based on Bodian staining, whereas we used acomputer-aided analysis method to determine the totalpercentage area occupied by axons based on SMI 32and 31 staining. We did not use Bodian staining inthis study because it binds to all filaments, includingthose in astrocytes, which creates a problem in the au-tomated thresholding step used for quantification.

ConclusionCurrently, T1 hypointensity and reduced MTR areconsidered the most practical way to distinguish MSlesions with more severe tissue destruction in vivo.This postmortem study of patients with long-standingMS confirms that although not all T1 hypointensities

with low MTR represent areas of severe tissue destruc-tion, there is a greater likelihood that they do corre-spond to demyelinated, chronic inactive lesions withsignificantly fewer and more swollen axons, consistentwith irreversible damage. Conversely, MRI-defined re-gions that are abnormal only on T2-weighted imagesare more likely to correspond to regions with less se-vere tissue damage, breakdown of the BBB, and rela-tively less axonal loss. Axonal swelling, a major distin-guishing feature between T2-only and T2T1MTRregions, is an indication of the axonal changes that re-sult from chronic demyelination and precede axonaldegeneration. Refinement of widely available MRI ac-quisition sequences, like the conventional T1-weightedand MTR images used for this study, may provide ameans of distinguishing demyelination, axonal loss,and axonal pathology in MS brains.

This study was supported by the NIH (National Institute of Neu-rological Disorders and Stroke, PO1 NS38667, E.F., A.C., R.J.F.,J.A.T., R.A.R., B.D.T.) and the National Center for Research Re-sources (Public Health Service, M01-RR018390, E.F., R.A.R.,B.D.T.).

We thank R. Klinkosz for technical assistance.

References1. McDonald WI, Compston A, Edan G, et al. Recommended

diagnostic criteria for multiple sclerosis: guidelines from the In-ternational Panel on the diagnosis of multiple sclerosis. AnnNeurol 2001;50:121–127.

2. Filippi M, Dousset V, McFarland HF, et al. Role of magneticresonance imaging in the diagnosis and monitoring of multiplesclerosis: consensus report of the White Matter Study Group. JMag Reson Imaging 2002;15:499–504.

3. McFarland HF, Barkhof F, Antel J, Miller DH. The role ofMRI as a surrogate outcome measure in multiple sclerosis. MultScler 2002;8:40–51.

4. Miller DH. Magnetic resonance in monitoring the treatment ofmultiple sclerosis. Ann Neurol 1994;36(suppl):S91–S94.

5. Filippi M, Paty DW, Kappos L, et al. Correlations betweenchanges in disability and T2-weighted brain MRI activity inmultiple sclerosis: a follow-up study. Neurology 1995;45:255–260.

6. Mammi S, Filippi M, Martinelli V, et al. Correlation betweenbrain MRI lesion volume and disability in patients with multi-ple sclerosis. Acta Neurol Scand 1996;94:93–96.

7. Weiner HL, Guttmann CR, Khoury SJ, et al. Serial magneticresonance imaging in multiple sclerosis: correlation with at-tacks, disability, and disease stage. J Neuroimmunol 2000;104:164–173.

8. Rovaris M, Comi G, Ladkani D, et al. European/CanadianGlatiramer Acetate Study Group. Short-term correlations be-tween clinical and MR imaging findings in relapsing-remittingmultiple sclerosis. AJNR Am J Neuroradiol 2003;24:75–81.

9. Bruck W, Bitsch A, Kolenda H, et al. Inflammatory centralnervous system demyelination: correlation of magnetic reso-nance imaging findings with lesion pathology. Ann Neurol1997;42:783–793.

10. van Walderveen MA, Kamphorst W, Scheltens P, et al. His-topathologic correlate of hypointense lesions on T1-weightedspin-echo MRI in multiple sclerosis. Neurology 1998;50:1282–1288.


11. van Waesberghe JH, Kamphorst W, De Groot CJ, et al. Axonalloss in multiple sclerosis lesions: magnetic resonance imaginginsights into substrates of disability. Ann Neurol 1999;46:747–754.

12. Truyen L, van Waesberghe JH, van Walderveen MA, et al. Ac-cumulation of hypointense lesions (“black holes”) on T1 spin-echo MRI correlates with disease progression in multiple scle-rosis. Neurology 1996;47:1469–1476.

13. Simon JH, Lull J, Jacobs LD, et al. A longitudinal study of T1hypointense lesions in relapsing MS: MSCRG trial of interferonbeta-1a. Neurology 2000;55:185–192.

14. De Groot CJ, Bergers E, Kamphorst W, et al. Post-mortemMRI-guided sampling of multiple sclerosis brain lesions: in-creased yield of active demyelinating and (p)reactive lesions.Brain 2001;124(pt 8):1635–1645.

15. van Walderveen MA, Barkhof F, Pouwels PJ, et al. Neuronaldamage in T1-hypointense multiple sclerosis lesions demon-strated in vivo using proton magnetic resonance spectroscopy.Ann Neurol 1999;46:79–87.

16. Li BS, Wang H, Gonen O. Brain metabolite profiles of T1-hypointense lesions in relapsing-remitting multiple sclerosis.AJNR Am J Neuroradiol 2003;24:68–74.

17. Gass A, Barker GJ, Kidd D, et al. Correlation of magnetizationtransfer ratio with clinical disability in multiple sclerosis. AnnNeurol 1994;36:62–67.

18. Grimaud J, Barker GJ, Wang L, et al. Correlation of magneticresonance imaging parameters with clinical disability in multi-ple sclerosis: a preliminary study. J Neurol 1999;246:961–967.

19. Pike GB, de Stefano N, Narayanan S, et al. Combined magne-tization transfer and proton spectroscopic imaging in the assess-ment of pathologic brain lesions in multiple sclerosis. AJNRAm J Neuroradiol 1999;20:829–837.

20. Schmierer K, Scaravilli F, Altmann DR, et al. Magnetizationtransfer ratio and myelin in postmortem multiple sclerosisbrain. Ann Neurol 2004;56:407–415.

21. Stewart WA, Hall LD, Berry K, Paty DW. Correlation betweenNMR scan and brain slice data in multiple sclerosis. Lancet1984;2:412.

22. Katz D, Taubenberger J, Raine C, et al. Gadolinium-enhancinglesions on magnetic resonance imaging: neuropathological find-ings. Ann Neurol 1990;28:243 (Abstract).

23. Nesbit GM, Forbes GS, Scheithauer BW, et al. Multiplesclerosis: histopathologic and MR and/or CT correlation in 37cases at biopsy and three cases at autopsy. Radiology 1991;180:467–474.

24. Bitsch A, Kuhlmann T, Stadelmann C, et al. A longitudinalMRI study of histopathologically defined hypointense multiplesclerosis lesions. Ann Neurol 2001;49:793–796.

25. Wayne Moore GR. MRI-clinical correlations: more than in-flammation alone—what can MRI contribute to improve theunderstanding of pathological processes in MS? J Neurol Sci2003;206:175–179.

26. Kalkers NF, Bergers E, Castelijns JA, et al. Optimizing the as-sociation between disability and biological markers in MS.Neurology 2001;57:1253–1258.

27. Fisher E, Rudick RA, Simon JH, et al. Eight-year follow-upstudy of brain atrophy in patients with MS. Neurology 2002;59:1412–1420.

28. Rudick RA, Fisher E, Lee JC, et al., Multiple Sclerosis Collab-orative Research Group. Use of the brain parenchymal fractionto measure whole brain atrophy in relapsing-remitting MS.Neurology 1999;53:1698–1704.

29. Dousset V, Grossman RI, Ramer KN, et al. Experimental al-lergic encephalomyelitis and multiple sclerosis: lesion character-ization with magnetization transfer imaging. Radiology 1992;182:483–491.

30. Richert ND, Ostuni JL, Bash CN, et al. Interferon beta-1b andintravenous methylprednisolone promote lesion recovery inmultiple sclerosis. Mult Scler 2001;7:49–58.

31. Barkhof F, Bruck W, De Groot CJ, et al. Remyelinated lesionsin multiple sclerosis: magnetic resonance image appearance.Arch Neurol 2003;60:1073–1081.

32. Rovaris M, Bozzali M, Rodegher M, et al. Brain MRI correlatesof magnetization transfer imaging metrics in patients with mul-tiple sclerosis. J Neurol Sci 1999;166:58–63.

33. Fisher E, Cothren RM, Tkach JA, et al. Knowledge-based 3Dsegmentation of MR images for quantitative MS lesion track-ing. SPIE Med Imaging 1997;3034:19–25.

34. Besag J. On the statistical analysis of dirty pictures. J R Stat Soc1986;48:259–302.

35. Besl PJ, McKay ND. Method for registration of 3-D shapes.IEEE Trans Pattern Anal Mach Intell 1992;14:239–255.

36. Trapp BD, Peterson J, Ransohoff RM, et al. Axonal transectionin the lesions of multiple sclerosis. N Engl J Med 1998;338:278–285.

37. Bo L, Mork S, Kong PA, et al. Detection of MHC class II-antigens on macrophages and microglia, but not on astrocytesand endothelia in active multiple sclerosis lesions. J Neuroim-munol 1994;51:135–146.

38. Bjartmar C, Kidd G, Mork S, et al. Neurological disability cor-relates with spinal cord axonal loss and reduced N-acetyl aspar-tate in chronic multiple sclerosis patients. Ann Neurol 2000;48:893–901.

39. Bergers E, Bot JC, De Groot CJ, et al. Axonal damage in thespinal cord of MS patients occurs largely independent of T2MRI lesions. Neurology 2002;59:1766–1771.

40. Stys PK. White matter injury mechanisms. Curr Mol Med2004;4:113–130.

41. Craner MJ, Newcombe J, Black JA, et al. Molecular changes inneurons in multiple sclerosis: altered axonal expression ofNav1.2 and Nav1.6 sodium channels and Na�/Ca2� ex-changer. Proc Natl Acad Sci USA 2004;101:8168–8173.

42. Dutta R, McDonough J, Yin X, et al. Mitochondrial dysfunc-tion as a cause of axonal degeneration in multiple sclerosis pa-tients. Ann Neurol 2006;59:478–489.

43. Barnes D, Munro PM, Youl BD, et al. The longstanding MSlesion. A quantitative MRI and electron microscopic study.Brain 1991;114(pt 3):1271–1280.

44. Schmierer K, Scaravilli F, Barker GJ, et al. Stereotactic co-registration of magnetic resonance imaging and histopathologyin post-mortem multiple sclerosis brain. Neuropathol ApplNeurobiol 2003;29:596–601.
