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Imaging correlates of axonal swelling in chronic multiple sclerosis brains.

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ORIGINAL ARTICLES
Imaging Correlates of Axonal Swelling in
Chronic 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 it
does not provide information on the severity of the underlying tissue damage. Measurement of T1 hypointensities and magnetization transfer ratio (MTR) can potentially distinguish lesions with more severe tissue damage. The objective of this study was
to use image-guided pathology to determine histological differences between lesions that are abnormal only on T2-weighted
images 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 on
T2-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% compared
with 55% of T2-only regions) and more likely to be chronic inactive lesions (68% compared with 0% of demyelinated T2-only
regions). There was no difference between T2-only and T2T1MTR regions in axonal area, but there was a significant difference
in 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 from
T2-only regions.
Ann Neurol 2007;62:219 –228
Magnetic resonance imaging (MRI) is an objective and
sensitive tool for diagnosing and monitoring multiple
sclerosis (MS) patients.1–3 MRI has also been proposed
as a tool for understanding MS pathogenesis in vivo.
However, its usefulness in this regard is limited because
conventional MRI is inherently nonspecific for distinct
pathological processes. The nonspecific nature of MRI
lesions is also one likely explanation for the weak correlations between lesion volumes and the progression
of clinical disability.4 – 8 MRI signal contrast arises from
differences in water and lipid content and the macromolecular environment of adjacent tissues. In theory,
all focal MS pathological processes, including blood–
brain barrier (BBB) breakdown, inflammation, demyelination, axonal loss, and gliosis, lead to an alteration
of water and lipid content, and hence conspicuous lesions on T2-weighted MRI (provided the affected region is large enough to be detected). Additional MR
sequences often are applied to distinguish underlying
pathology, or at least to determine lesion severity. For
example, lesions that are hypointense on T1-weighted
images, or “black holes,” are generally considered to
have more severe tissue destruction than lesions that
are isointense on T1-weighted images, based on correlations with axonal loss,9 –11 stronger correlations to
disability,12,13 and association with chronic stage of lesion development.14 T1 hypointense regions have also
been shown to have lower N-acetylaspartate compared
with T1 isointense lesions, and normal white matter
using magnetic resonance spectroscopy.15,16 Similarly,
decreased magnetization transfer ratio (MTR) in lesions has been shown to be correlated to increased disability,17,18 reduced N-acetylaspartate,19 demyelination,20 and axonal loss.11
Previous postmortem and in vivo (biopsy) studies
have identified the histopathological characteristics
of different MRI features, such as T2 hypointensity,14,21 gadolinium enhancement,9,22,23 T1 hypoin-
From the Departments of 1Biomedical Engineering and 2Neurosciences, Lerner Research Institute; 3Mellen Center for Multiple
Sclerosis Treatment and Research, Cleveland Clinic; and 4Department of Radiology, Case Western Reserve University/University
Hospitals Cleveland, Cleveland, OH.
This article includes supplementary materials available via the
Internet at http://www.interscience.wiley.com/jpages/0364-5134/
suppmat
Received Oct 10, 2006, and in revised form Jan 31, 2007. Accepted
for publication Feb 2, 2007.
Current address for T. Svarovsky: Department of Pathology, Wisconsin National Primate Research Center, Madison, WI.
Published online April 11, 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21113
Address correspondence to Dr Fisher, Department of Biomedical
Engineering ND20, Cleveland Clinic Foundation, 9500 Euclid
Avenue, Cleveland, OH 44195. E-mail: fishere@ccf.org
© 2007 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
219
tensity,9,11,14,24 and decreased MTR.11 Most of the evidence to date supports the idea that one can
pathologically subclassify lesions in vivo using a single
MRI data set according to the following rules25: (1) T2
hyperintensities are pathologically nonspecific, and
therefore represent MS lesions in all phases of pathogenesis; (2) gadolinium-enhancing lesions are regions
with breakdown of the BBB and possibly infiltration of
hematogenous leukocytes; (3) T1 hypointensities are
MS lesions with axonal loss and severe tissue destruction; and (4) lesions with decreased MTR represent
MS lesions with demyelination and axonal loss. In support of this general concept, correlations between lesion volumes and disability have been found to be
stronger for T1 lesion volume than for the total T2
lesion volume12; however, these correlations are still
quite modest in most studies, ranging from 0.22 to
0.54.12,13 Notably, the lack of strong correlations is
also related to difficulties with measurement of clinical
disease progression, in addition to limitations of cranial
MRI. However, the correlations are not much stronger
when an objective measure of disease severity, such as
brain atrophy, is used as the dependent variable. Correlations between T1 lesion volume and brain atrophy
are about the same as or lower than correlations between T2 lesion volume and atrophy, in the range of
0.4 to 0.5, and T1 lesion volume is not an independent predictor of subsequent brain atrophy.26 –28
The findings for lesion MTR measurements are similar. MTR is decreased in MS brains and varies widely
in MS lesions, from extremely low to nearly normal
values.29 MTR is reduced in gadolinium-enhancing lesions because of acute inflammation, and it subsequently recovers over time in a subset of lesions, but
remains low in others.30 Variations in lesion MTR also
relate to degree of demyelination,20 remyelination,31
and degree of axonal loss.11 Correlations between mean
lesion 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 of
the more severe lesions, such as T1 hypointensities
with decreased MTR, would be strongly correlated
with disease progression have not been met. These
weaker than expected correlations led to persistent
questions on the ability to differentiate lesions based
on MRI. Such a capability would be highly beneficial
in the evaluation of new neuroprotective therapies, as a
way to distinguish potentially reparable lesions from irreparable lesions. The purpose of this study was to use
image-guided pathology to directly determine the major histological differences between lesions that are abnormal only on T2-weighted images versus lesions that
are abnormal on T2-weighted, T1-weighted, and MTR
images.
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Subjects and Methods
Overview
MS brain tissue was acquired through the tissue procurement
program established at the Cleveland Clinic Foundation. Patients and their families signed an institutional review board–
approved advance directive to donate brain and spinal cord
tissue for research purposes. Demographic and disease-related
information for each patient was entered into a database.
The same protocol was followed for each patient: Within a
few hours of death, the cadaver was brought to the imaging
facility for an MRI examination followed by rapid autopsy.
The brain and spinal cord were removed, and one cerebral
hemisphere was immediately fixed in 4% paraformaldehyde
for at least 4 weeks. After fixation, the hemisphere was reimaged in a custom-designed slicing box for coregistration
purposes. The brain tissue was sliced coronally in the box,
and each slice was numbered, stored, and subsequently photographed. The cadaver images were registered to the postfixation images so that the image planes corresponded with
the tissue slices. Lesions were segmented and classified in the
cadaver images using automated image analysis programs.
Using the registered MRIs, we generated region maps to indicate regions of interest (ROIs) for histological analysis.
Magnetic Resonance Imaging Details
Imaging was performed on a 1.5-Tesla MR scanner (VISION; Siemens, Erlangen, Germany). The MRI protocol
consisted of a three-dimensional T1-weighted magnetization
prepared rapid acquisition gradient-echo image, a T2weighted fluid-attenuated inversion recovery image (FLAIR),
a T2-weighted fast spin-echo image, a T1-weighted spinecho image, and an MTR image calculated from proton density–weighted three-dimensional gradient-echo images acquired with and without a magnetization transfer pulse. The
magnetization prepared rapid acquisition gradient-echo image was acquired with 1 ⫻ 1 ⫻ 1mm resolution, and all the
other images had 0.9 ⫻ 0.9 ⫻ 3mm resolution. Detailed
sequence parameters are provided in Supplementary Table 1
(see supplementary online materials). For the postfixation
image, the fixed cerebral hemisphere was placed in a customdesigned slicing box before imaging to aid in image to tissue
colocalization. The precision-machined polycarbonate box
has sliding walls that lock in place to securely hold the tissue
during imaging and slicing. Two walls of the box have vertical knife-guide slots positioned 10mm apart with 2mm gelfilled cylindrical wells above and below each slot. When fixed
tissue is imaged in the box before slicing, the gel markers are
detectable on the MRI and indicate the orientation and position of each slice plane.
Image analysis was performed using software developed
in-house (BIP; Department of Biomedical Engineering,
Cleveland Clinic Foundation, Cleveland, OH). The analysis
for the cadaver images consisted of brain segmentation, brain
parenchymal fraction (BPF) calculation, lesion segmentation,
and lesion classification. Brain segmentation and BPF calculation were performed on the FLAIR image using a fully automated program, as described previously.28,33 T2 lesions
were segmented in the brain-masked FLAIR image using a
modified version of the iterated conditional modest (ICM)
algorithm.34 The segmented T2 lesion regions were used to
guide the automated segmentation of lesions in the T1 and
MTR images. First, the T2 lesion regions were overlaid on
coregistered T1 and MTR images. For each image type, the
intensity statistics of the normal-appearing brain tissue in the
neighborhood surrounding each lesion were calculated, and
thresholds for abnormal tissue within each lesion region were
determined. Abnormal was defined as any voxel in the T1 or
MTR image that was colocalized within a T2 lesion and had
an intensity less than the mean intensity minus one standard
deviation of the local nonlesion tissue. Therefore, classification of regions as abnormal on T1 and MTR images was
based on low intensity relative to neighboring normalappearing white matter (NAWM), not by global thresholding. Lesions were subsequently classified into two types: regions of T2 hyperintensity that were normal appearing on
T1 and MTR (T2-only), and regions of T2 hyperintensity
that were hypointense on T1 and MTR (T2T1MTR). The
first type represented regions hypothesized to be of lesser severity, whereas the second type represented regions hypothesized to be of greater severity. For each brain, ROIs within
each type of lesion and within NAWM were delineated for
the generation of region maps (see description later in this
article). FLAIR, T1, and MTR contrast ratios were calculated for each ROI as the mean intensity within the region
divided by the mean intensity within all of the NAWM
ROIs for the same brain.
Image analysis for the postfixation images consisted of detection of the markers in the slicing box, localization of the
cut planes, and registration with the cadaver images. Thresholding and connected-components labeling were used to
identify the cerebral hemisphere and the markers. The bestfit plane equations were determined from sets of markers using a program encoded with the dimensions of the slicing
box. The postfixation image was registered to the cadaver
images using the Iterative Closest Point algorithm to determine the optimal three-dimensional affine transform (three
rotations ⫹ three translations ⫹ uniform scaling).35 The
equations of the cut planes were then used to extract the
individual planes from the analyzed cadaver image volumes
that corresponded to each tissue slice.
Lesion maps were generated for each tissue slice from the
corresponding MRI planes. A 10mm grid was overlaid on
the image planes to provide a frame of reference. The outlines of ROIs that corresponded to each type of lesion (T2only and T2T1MTR) and NAWM were transferred to the
region maps. These maps were then used to guide tissue
sampling for the histological analysis.
Immunocytochemistry Details
ROIs were removed, cryoprotected, and sectioned (30␮m
thick) on a freezing-sliding microtome. The free-floating sections (30␮m thick) were microwaved in 10mM citric acid
buffer (pH 6.0) for 5 minutes, incubated in 3% hydrogen
peroxide and 10% Triton X-100 (Sigma, St. Louis, MO) in
phosphate-buffered saline for 30 minutes, and immunostained by the avidin-biotin complex procedure with diaminobenzidine tetrahydrochloride as a chromagen, as described
previously.36 Sections stained for serum protein were not microwaved. Sections stained for IgG were processed using the
avidin-biotin complex procedure with diaminobenzidine tet-
rahydrochloride, whereas horseradish peroxidase–conjugated
fibrinogen and albumin were visualized with diaminobenzidine tetrahydrochloride. Sections were also double labeled for
axons and myelin utilizing immunofluorescent procedures, as
described previously.36 A list of antibodies is provided in
Supplementary Table 2.
MRI-defined regions were histologically classified based
on the presence or absence of myelin staining proteolipid
protein (PLP), activity of the lesion, and the serum protein
detection. The activity of lesions were subdivided into active,
chronic active, and chronic inactive. Active lesions were defined as hypercellular throughout the lesion area, chronic active lesions had a hypercellular border of major histocompatibility complex (MHC) class II–positive cells and a
hypocellular center, and chronic inactive lesions were defined
as hypocellular throughout.37 If myelin was present in the
ROI, the microglia were examined for activation markers
and changes in shape. Microglia exhibiting both MHC class
II expression and retracting and thickening of processes were
noted as “activated.” The presence of serum proteins in each
region was categorized as either diffuse or cellular staining,
specific to axons and glial cells. Sections that were positive in
at least one serum protein stain were considered positive.
These sections were compared with non-MS control tissue
(to distinguish lesions and myelinated white matter, because
many of the tissue samples contained gliosis). Sections
stained for SMI 32 and 31 were examined for axonal measurements. Axonal area was estimated using a computeraided technique, similar to that described previously.38 Multiple confocal micrographs for each region were digitized
(40⫻ magnification), transferred to a workstation, automatically thresholded, and quantified. Axonal area was calculated
in red-channel images as the number of thresholded pixels
multiplied by 100% and divided by the total number of pixels in the fixed-size image (250 ⫻ 250␮m). Results from six
nonoverlapping areas were averaged to give the final axonal
area for each region. The measurement error, as determined
by the mean standard deviation of repeated analyses on the
same area, was 1.7%. Axonal count was determined automatically in the same thresholded images as the number of objects greater than four pixels in size. For each region, an axonal diameter index was calculated as the mean axonal area
divided by the axonal count.
Data Analysis
Each region was categorized based on MRI characteristics
(NAWM, T2-only, or T2T1MTR) and on histopathological
features. For each MRI-based group, the number of regions
with each specific histopathological feature was determined
and compared between MRI groups to identify any major
differences. The ␹2 test was used to test for differences between MRI groups for lesion activity (active vs chronic active
vs chronic inactive). Fisher’s exact test was used to test for
differences between MRI groups for myelin (myelinated vs
demyelinated) and serum proteins (diffuse vs cellular).
Kruskal–Wallis one-way analysis of variance with Dunn’s test
for multiple comparisons was applied to test for differences
in axonal measurements among the three MRI groups. Contrast ratios and axonal measurements were compared between
MRI and histopathology groups using the nonparametric
Fisher et al: MRI of Axonal Swelling in MS
221
Mann–Whitney U test. Spearman’s rank correlation coefficients were calculated to determine the relation between T1
and MTR contrast ratios and axonal measurements in all
MRI lesions and in the T2-only and T2T1MTR lesion
groups separately.
Results
Tissue Acquisition
This report includes data from 10 confirmed MS cases
that were imaged and processed through the brain donation protocol. Demographic and disease-related details are provided in Supplementary Table 3. All 10
patients (6 female and 4 male patients) had secondaryprogressive MS. The mean (standard deviation) age
was 56 (9.7) years, disease duration was 31 (11) years,
Extended Disability Status Scale score at the time of
death was 8.6 (1.0), BPF was 0.77 (0.06), and brain
weight was 1,151.1 (145) gm. The mean time between
death and brain removal was 5.4 hours. Imaging was
performed immediately before autopsy.
Comparison of the lesion maps and photographs
confirmed that good coregistration was achieved between the MRIs and tissue slices for each case. The
Supplementary Figure shows an example lesion map/
tissue slice pair and the MR image with the lesion
types superimposed. A total of 110 MRI-defined regions, which were mapped onto coregistered tissue slice
images, were sectioned and stained for histological
analysis: 29 NAWM regions, 40 T2-only regions, and
41 T2T1MTR regions. The MRI region types were
distributed across all 10 brains. All regions identified
radiologically were evaluated histologically. The mean
contrast ratios and histological characteristics for each
MRI group are summarized in the Table. Comparison
of image contrast ratios between the MRI groups confirmed that each group was significantly different from
the other two. Examples of each MRI region type and
corresponding histology are shown in Figures 1
(NAWM), 2 (T2-only), and 3 (T2T1MTR).
Pathological Features of Different Magnetic
Resonance Imaging Region Types
All NAWM regions were myelinated on histological inspection. There was no evidence of serum proteins in
the NAWM regions, but 90% of NAWM regions contained activated microglia. Axonal area was highly variable in NAWM, ranging from 7 to 29%. The mean
(standard deviation) percentage of axonal area was
17.3% (4.2%). Axonal count in NAWM ranged from
3,751 to 8,056, with a mean of 6,119 (936).
The T2-only group was heterogenous, with only
55% of these regions showing demyelination. Activated
microglia were associated with all of the myelinated
Table. Magnetic Resonance Imaging and Histological Characteristics for Each Magnetic Resonance Imaging Region
Type
Characteristics
NAWM
(n ⴝ 29)
T2-only
(n ⴝ 40)
T2T1MTR
(n ⴝ 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
Activated microglia
Demyelinated
100% (29/29)
45% (18/40)
90% (26/29)
100% (18/18)
0
17% (7/41)
100% (7/7)
55% (22/40)
83% (34/41)
Active
27% (6/22)
6% (2/34)
Chronic active
73% (16/22)
Chronic inactive
0
Astrocytosis
48% (14/29)
Serum proteins
0
26% (9/34)
68% (23/34)
93% (37/40)
98% (40/41)
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 ⫽ standard
deviation; MTR ⫽ magnetization transfer ratio.
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Fig 1. Magnetic resonance images (MRIs) and histology for a typical normal-appearing white matter (NAWM) region. (A) T2weighted 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 in
yellow. (E) Myelin-stained section (PLP) showing no loss of myelin. (F) Major histocompatibility complex class II staining for lesion
activity showing activated microglia. (G) Serum protein staining (IgG) showing no evidence of serum protein staining. Scale bars ⫽
1mm (E–G).
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 T2only regions were subcategorized based on lesion activity, 27% were active and 73% were chronic active, but
none was chronic inactive lesions. The mean (standard
deviation) percentage of axonal area for T2-only regions was 12.0% (4.3%), which was significantly less
than for NAWM regions ( p ⬍ 0.001). Mean axonal
count was 3,930 (902), which was also significantly less
than in NAWM ( p ⬍ 0.001). Mean axonal diameter
index was not significantly different from NAWM (3.2
compared with 2.8, p ⫽ 0.52)
In comparison with the T2-only regions, a significantly larger percentage (83%) of the T2T1MTR regions 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 inversion
recovery MRI; arrow marks the region of interest used for the histology sections below. (B) T1-weighted MRI; (C) Magnetization
transfer 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.
Fisher et al: MRI of Axonal Swelling in MS
223
Fig 3. Magnetic resonance images (MRIs) and histology for a typical T2-weighted, T1-weighted, and magnetization transfer ratio
abnormal (T2T1MTR) region. (A) T2-weighted fluid-attenuated inversion recovery MRI; arrow marks the region of interest used
for the histology sections below. (B) T1-weighted MRI. (C) MTR 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 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.
activated microglia. Demyelinated T2T1MTR regions
were repopulated by activated microglia, which had
shorter and fewer processes than those in NAWM regions. T2-only demyelinated regions contained phagocytic macrophages if acute or activated microglia and
occasional macrophages if chronic active. As in the T2only regions, nearly all of the T2T1MTR regions, both
myelinated and demyelinated, had astrocytosis (98%),
and all had either diffuse (37%) or cellular (63%) serum proteins. There was a significantly greater proportion of serum proteins found in the cellular compartment in the T2T1MTR regions compared with the
T2-only regions ( p ⫽ 0.002). When the demyelinated
T2T1MTR regions were subcategorized based on lesion activity, only 6% were active, 26% were chronic
active, and 68% were chronic inactive lesions. This distribution of lesion activity was significantly different
from that of the T2-only regions (␹2 test ⫽ 24.8; p ⬍
0.0001). The mean percentage of axonal area for the
T2T1MTR regions was 11.9% (4.4%), which was less
than the NAWM regions ( p ⬍ 0.001), but not significantly different from the T2-only regions. However,
mean axonal count was significantly less in the
T2T1MTR regions compared with both the NAWM
and T2-only groups ( p ⬍ 0.001), with a mean of
2,711 (815). Correspondingly, the mean axonal diameter index was significantly greater in T2T1MTR regions 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. Images depicting axonal morphology in the three MRI
groups are provided in Figure 5.
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Correlations between MRI contrast ratios (FLAIR,
T1, or MTR) and percentage axonal area ranged from
weak for T1 and MTR (Spearman’s rank correlation
coefficient [SRCC] ⫽ 0.25 and 0.27; p ⬍ 0.01) to
moderate for T2 (SRCC ⫽ ⫺0.39; p ⬍ 0.0001). In
contrast, correlations between MRI contrast ratios and
axonal count were strong for all three parameters, particularly for MTR (SRCC ⫽ ⫺0.52, 0.71, and 0.79
for FLAIR, T1, and MTR, respectively; p ⬍ 0.0001
for all). Correlations between MRI contrast ratios and
Fig 4. Plot of percentage axonal area, axonal count, and
swelling index in each magnetic resonance imaging group
(gray bars denote T2-weighted imaging only; black bars denote T2-weighted, T1-weighted, and magnetization transfer
ratio abnormal [T2T1MTR]) relative to the means for
normal-appearing white matter (NAWM; hatched bars) regions. sd ⫽ standard deviation.
Fig 5. Confocal images of sections stained with polyclonal myelin basic protein antibody (green) and axonal markers SMI 31 and
32 (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 image
of NAWM region in (A) with percentage axonal area ⫽ 18.0% and axonal count ⫽ 6,420, or 105% of the mean for all
NAWM 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% and
axonal count ⫽ 1,748, or 29% of mean for NAWM. Scale bar ⫽ 20␮m (A–F).
axonal measurements were also determined in the demyelinated regions separately (n ⫽ 56) to identify the
potential contribution of axonal swelling or loss to
MRI characteristics independent of myelin. In the demyelinated subset, which included 22 T2-only and 34
T2T1MTR regions, there were no correlations at all
between MRI contrast ratios and percentage of axonal
area. There was also no correlation between FLAIR
contrast ratio and axonal count. However, the correlations between T1 and MTR contrast ratios and axonal
count were still significant (T2: SRCC ⫽ 0.44, p ⫽
0.0009; MTR: SRCC ⫽ 0.41, p ⫽ 0.002). MRI contrast ratios and axonal measurements were not correlated to postmortem time.
When the regions were subdivided based on the histopathological features, FLAIR, T1, and MTR contrast
ratios were all significantly different in the myelinated
regions as compared with the demyelinated regions
(1.1 vs 1.3 for median FLAIR contrast ratio; 0.97 vs
0.86 for median T1 contrast ratio; and 0.97 vs 0.77 for
median MTR contrast ratio; p ⬍ 0.001 for all comparisons). The demyelinated regions were then subdivided by lesion activity. Median FLAIR contrast ratios
for active, chronic active, and chronic inactive lesions
were 1.26, 1.27, and 1.33, respectively; median T1
contrast ratios were 0.96, 0.93, and 0.76, respectively;
and median MTR contrast ratios in these three groups
were 0.90, 0.86, and 0.59, respectively. The chronic
inactive regions had significantly lower T1 contrast ratio and MTR contrast ratios compared with the active
and chronic active groups, but there were no other significant differences between activity-based groups.
Other Observations
This study was primarily focused on pathological
changes that were most likely to contribute to the variable MRI characteristics in the three types of regions.
However, we also examined several other cellular components that showed little correlation in distinguishing
between T2-only and T2T1MTR lesions. For example,
general T-cell density as identified by CD3 staining
made up less than 0.5% of the total parenchymal area
in both T2-only and T2T1MTR regions. Perivascular
cuffs represented less than 1% of T2-only or
T2T1MTR lesion areas. Other cellular components
such as phagocytic and foamy macrophages were abundant only in active lesions, which only made up 7.3%
of the total regions in this study, and thus could not
contribute to either T1 or MTR differences observed
overall. Activated microglia were present in varying degrees, but were most abundant in T2-only regions and
least abundant in T2T1MTR regions.
Discussion
To investigate the validity of MRI-based lesion differentiation, we imaged postmortem MS brains in situ
and applied a new image-to-tissue coregistration
method for localization of tissue samples for histopathological analyses based on MRI characteristics.
Although no single histological features clearly distinguished T2-only regions from T2T1MTR regions, several important differences were observed. Compared
with the T2-only regions, the T2T1MTR regions were
more likely to be demyelinated (83% of the
T2T1MTR group compared with 55% of the T2-only
Fisher et al: MRI of Axonal Swelling in MS
225
group). Demyelinated regions from both MRI groups
had significantly greater FLAIR contrast ratio and
lower T1 and MTR contrast ratios than myelinated regions. Demyelinated T2T1MTR regions were much
more likely to be chronic inactive lesions, whereas two
thirds of the regions in this group were chronic inactive; none of the T2-only lesions was classified as such.
T2T1MTR regions were also more likely to have serum proteins located intracellularly rather than diffusely, as in the majority of T2-only regions. Acute or
recent breakdown of the BBB, therefore, is a consistent
feature of T2-only regions. Demyelination does not appear to be a consistent consequence of BBB breakdown
because only 55% of the T2-only regions were demyelinated. Surprisingly, there was no difference in the
area occupied by axons between the two groups, even
though there were significantly fewer axons in
T2T1MTR regions. In addition to demyelination and
axonal loss, axonal swelling is a significant determinant
of T1 hypointensity and MTR abnormality.
An important observation of this report is the identification of swollen axons as a marked feature in the
T2T1MTR regions (see Fig 5F). Based on visual inspection of axonal diameters in T2T1MTR lesions and
adjacent NAWM, the enlarged axonal diameters did
not result from selective loss of smaller diameter axons.
Rather, axons were abnormally swollen and much
larger than axons in NAWM. In a previous postmortem study of MS spinal cords, swollen axons were
found within demyelinated lesions in patients with
long-standing MS.39 These lesions had high signal intensity on T2-weighted MRIs, but T1-weighted and
MTR images were not acquired in that study. An important consequence of axonal swelling is that the
space occupied by axons is maintained despite the destruction of substantial numbers of axons, potentially
confounding measurements of brain atrophy. Thus, axonal swelling could partially explain why correlations
between T2T1MTR lesion volumes and BPF are
weaker than expected.
From this postmortem study, it is not possible to
determine whether all the axonal swelling occurred in
vivo. If postmortem changes contribute to axonal
swelling, these changes operate in demyelinated axons
and in more chronic lesions identified histologically
and by T2T1MTR changes. Axonal diameters were not
increased in demyelinated T2-only lesions. Furthermore, because there were no correlations between postmortem time and any of the MRI contrast ratios or
axonal measurements, it is likely that the axonal swelling occurred in vivo and is indicative of a slowly progressing necrotic death that eventually affects most
chronically demyelinated axons. Current concepts,40
supported by experimental animal models and molecular and morphological analysis of chronic MS brains,
implicate malfunction of ion channels and pumps that
226
Annals of Neurology
Vol 62
No 3
September 2007
maintain axoplasmic Na/K gradients during nerve conduction.41,42 Specifically, an imbalance of Na/K exchange will eventually lead to swelling and death of
chronically demyelinated axons. Increased axoplasmic
Ca and reduced adenosine triphosphate production appear to be essential contributors to the process of axonal swelling and axonal death.42
When all of the histological differences between
MRI groups are considered, it is clear that there is a
greater likelihood for a T2T1MTR region to have
more severe tissue damage than a T2-only region; however, the T2T1MTR regions exhibited a high degree of
pathological heterogeneity. The observation that there
is considerable overlap in the histopathological features
underlying different MRI lesion types confirms previous reports.9 –11,14,20,23,31,43 It is also consistent with
the weak correlations reported in most MRI-clinical
correlation studies. In an analysis of MRI characteristics of MS biopsy specimens, Brück and colleagues9
found that MRI features were not specific for distinct
pathology, but that T1 hypointensity was related to
several different factors, including extracellular edema,
axonal loss, and degree of demyelination. A more recent report noted that T1 hypointensity at the time of
biopsy did not correlate with axonal loss or demyelinating activity in a group of relapsing-remitting and
primary progressive patients.24 However, these findings
may not be representative of MS lesions in general because of the atypical MRI appearance of biopsied
MS lesions. Postmortem MRI-histopathological studies
have demonstrated strong correlations between T1
contrast ratio and residual axonal density,10,11 MTR
and axonal density,14 and a significant association between myelin status and MRI characteristics on T1 and
MTR images.31 Schmierer and colleagues20 report a
strong correlation between MTR and quantitative measurements of myelination and a moderate correlation
between T1 relaxation time and axonal count, but no
significant difference in axonal counts between lesions
that were hypointense versus those that were isointense
on T1-weighted MRI. As in van Waesberghe and colleagues’ study,11 we found that both MTR and T1
contrast ratios were highly correlated to axonal loss, as
was FLR contrast ratio, to a lesser extent. There was
also a significant correlation between MTR and axonal
area, but only in the T2T1MTR regions and not in
the set of all lesions combined. This finding suggests
that variance in MTR in the older, more chronic lesions may be determined mainly by axonal water, but
that variance in MTR in the less severe lesions may be
dominated by myelin content or extracellular water.
Evidence of more extracellular water in the T2-only lesions as compared with the T2T1MTR lesions (as determined 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 regions were histologically classified as myelinated. Myelin sheath thickness was appropriate for axonal diameters in these regions. Therefore, they were classified as
normally myelinated. None of the regions contained
shadow plaques, although remyelination was often
found at the edge of some chronic active and chronic
inactive lesions. The finding that such a high proportion of MRI-detected lesions are actually myelinated
appears counterintuitive, particularly about the
T2T1MTR group, but not surprising because it is consistent with previous studies.11,14,20 De Groot and coworkers14 sampled postmortem brains based on MRI
abnormalities and found that 48% of T2 lesions had
no apparent loss of myelin. They also found that 30%
of the lesions that were classified as either mildly or
severely hypointense on T1 MRI had no apparent loss
of myelin, similar to this study. In Schmierer and colleagues’ study,20 44 of 98 ROIs selected by T2 hyperintensity were discarded, mainly because of technical
problems, but six regions were not studied because
they were found to be normally myelinated. We analyzed such regions to determine the potential source of
MRI signal change. Unlike NAWM, all of the myelinated T2-only regions had serum proteins and many
had significantly reduced axonal area and axonal count,
indicating MS pathology in the absence of colocalized
demyelination.
Differences in the findings reported here and in previous MRI-pathology correlation studies are likely due
to differences in techniques. In contrast with most
other studies, we performed postmortem imaging in
situ before autopsy and used different MRI lesion detection, image-to-tissue coregistration, and histology
methods than those described for other studies.
Schmierer and colleagues20 applied a surgical stereotaxic frame system after brain removal on individual
tissue slices to accurately mark ROIs based on MRI
coordinates for subsequent histological sampling.44 van
Waesberghe and colleagues11 also imaged single brain
slices immediately after autopsy. To report axon density, they used a visual ranking system from 0 to 100%
based on Bodian staining, whereas we used a
computer-aided analysis method to determine the total
percentage area occupied by axons based on SMI 32
and 31 staining. We did not use Bodian staining in
this study because it binds to all filaments, including
those in astrocytes, which creates a problem in the automated thresholding step used for quantification.
Conclusion
Currently, T1 hypointensity and reduced MTR are
considered the most practical way to distinguish MS
lesions with more severe tissue destruction in vivo.
This postmortem study of patients with long-standing
MS confirms that although not all T1 hypointensities
with low MTR represent areas of severe tissue destruction, there is a greater likelihood that they do correspond to demyelinated, chronic inactive lesions with
significantly fewer and more swollen axons, consistent
with irreversible damage. Conversely, MRI-defined regions that are abnormal only on T2-weighted images
are more likely to correspond to regions with less severe tissue damage, breakdown of the BBB, and relatively less axonal loss. Axonal swelling, a major distinguishing feature between T2-only and T2T1MTR
regions, is an indication of the axonal changes that result from chronic demyelination and precede axonal
degeneration. Refinement of widely available MRI acquisition sequences, like the conventional T1-weighted
and MTR images used for this study, may provide a
means of distinguishing demyelination, axonal loss,
and axonal pathology in MS brains.
This study was supported by the NIH (National Institute of Neurological 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 Resources (Public Health Service, M01-RR018390, E.F., R.A.R.,
B.D.T.).
We thank R. Klinkosz for technical assistance.
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