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Diffuse Axonal and Tissue Injury in Patients With Multiple Sclerosis With Low Cerebral Lesion Load and No Disability
Nicola De Stefano, MD;
Sridar Narayanan, MSc;
Simon J. Francis, BSc;
Steve Smith, DPhil;
Marzia Mortilla, MD;
M. Carmela Tartaglia, BSc;
Maria L. Bartolozzi, MD;
Leonello Guidi, MD;
Antonio Federico, MD;
Douglas L. Arnold, MD
Arch Neurol. 2002;59:1565-1571.
ABSTRACT
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Background Although in situ pathological studies and in vivo magnetic resonance
(MR) investigations have shown that axonal injury can be significant in the
early stages of multiple sclerosis (MS), diffuse axonal injury is generally
considered a secondary event. Cerebral axonal damage can be specifically assessed
in vivo by measuring levels of brain N-acetylaspartate
(NAA, a specific index of axonal integrity detected by MR spectroscopy). Other
new MR measurements such as magnetization transfer ratio (MTr) or computed
estimation of brain volume can provide less specific indexes of tissue damage.
Objective To determine whether diffuse axonal and tissue injury is present in
patients with definite MS who do not show clinically significant disability.
Methods We measured brain NAA levels (normalized to creatine [Cr]), MTr values,
and cerebral volumes in patients with definite MS who had low T2-weighted
MR imaging lesion volumes and no clinical disability, and also in age-matched
healthy control subjects.
Results Values of central brain NAA/Cr and MTr in normal-appearing white matter
were significantly lower in the MS patients than in controls (P<.001). In contrast, total brain volumes were not significantly
different between these groups. Similar results were found for MS patients
with early disease (duration, <3 years) and with a particularly low cerebral
T2-weighted MR imaging lesion load ( 2 cm3).
Conclusions Cerebral NAA/Cr and MTr values are diffusely decreased in MS patients
with early disease, low demyelinating lesion load, and no significant disability.
This suggests that axonal and/or tissue injury begins very early in the course
of MS and might be at least partially independent of cerebral demyelination.
INTRODUCTION
MULTIPLE SCLEROSIS (MS) is an inflammatory demyelinating disease of
the central nervous system that causes severe clinical disability in young
adults. Traditionally, impairment of the central nervous system and the related
loss of function have been considered to be largely due to the demyelination
and consequent delay or block of electrical conduction by axons that are otherwise
substantially preserved. In the past decade, however, in vivo magnetic resonance
(MR) spectroscopy (MRS) studies of N-acetylaspartate
(NAA)1-5
and in situ postmortem studies6-8
have demonstrated that sparing of axons is only relative in MS and injured
or transected axons are a common finding in this disorder. This has led to
a reconsideration of the role of axonal injury in MS and, in particular, its
relevance to clinical disability.9-10
There is an increasing agreement that axonal loss plays a major role
in the pathology of MS11 and there exists both
in vivo and in situ evidence that axonal injury can be significant from the
early stages of the disease. However, the mechanisms by which axonal injury
occurs are not fully understood. For example, if axonal injury is simply a
bystander effect secondary to demyelination, then the degree of axonal injury
should be strongly related to the degree of demyelination. However, several
experimental studies suggest that axonal injury and dysfunction in MS may
be independent of the degree of demyelination12-16
and might begin to accumulate before clinical disability is evident.17
Axonal injury inside and beyond MS lesions can be evaluated with MRS
by measuring brain levels of NAA18 (an amino
acid localized almost exclusively in neurons and axons in the mature central
nervous system19-20) and, more
indirectly, by means of other MR indexes of tissue damage such as the magnetization
transfer (MT) ratio (MTr)21 or the computed
estimation of brain volume.22 In a number of
recent studies, these MR indexes have been demonstrated to be sensitive in
detecting early pathologic changes in brains of patients with MS.4, 23-26
Thus, our goal in the present study was to assess in vivo whether diffuse
cerebral axonal and tissue injury accrues in nondisabled MS patients who exhibit
little evidence of focal demyelination. To do this, we evaluated values of
NAA-creatine (Cr), MTr, and total cerebral volumes in the brains of a selected
group of patients with established MS who showed low volumes of cerebral T2-weighted
(T2-W) lesions on conventional MR imaging and absence of disability at clinical
examination.
SUBJECTS AND METHODS
STUDY POPULATION
Sixty patients (41 women and 19 men; age range, 18-54 years; mean, 35
years) with clinically definite MS27 but without
clinical disability (Expanded Disability Status Scale [EDSS],28
<2) were chosen from the population followed up at the MS clinics of the
Montreal Neurological Institute and Hospital, Montreal, Quebec (MNH, n = 26),
and of the Institute of Neurological Sciences of the University of Siena,
Siena, Italy (Siena; n = 34). Patients from both sites had a relatively short
disease duration (range, 0.4-13.0 years; median, 2.7 years) and were all classified
as having the relapsing-remitting form of the disease. All patients were relapse-
and drug treatment–free for at least 1 month before study entry. The
ethics committees of both institutions approved the study. Informed consent
was obtained from all participating subjects.
MR EXAMINATIONS
All patients were examined using the same MR protocol, which included
combined proton MR imaging and MRS imaging examinations of the brain. A transverse
dual-echo, turbo spin-echo sequence (repetition time, 2075 milliseconds; first
echo time, 30 milliseconds; second echo time, 90 milliseconds; 256 x
256 matrix; 1 signal average; and 250-mm field of view) yielding proton density
and T2-W images with 50 contiguous 3-mm slices was acquired parallel to the
line connecting the anterior and posterior commissures. Subsequently, an MT
sequence was performed to acquire 2 transverse T1-W, gradient echo images,
1 without (No Sat) and 1 with (Sat) MT saturation pulses (repetition time,
35 milliseconds; echo time, 10 milliseconds; 256 x 256 matrix; 1 signal
average; and 250-mm field of view). This sequence yielded image volumes of
50 slices, 3 mm thick, oriented to match the proton density–T2 acquisition
exactly. The MT pulse was a 1.2-millisecond on-resonance (radio-frequency
field strength, 20 µT), placed just before each slice-selective excitation.29
The MR images were used to select an intracranial volume of interest
(VOI) for spectroscopy measuring approximately 100 mm anteroposterior x
20 mm craniocaudal x 90 mm left to right. This was centered on the corpus
callosum to include mostly white matter of both hemispheres. Two-dimensional
spectroscopic images were obtained using a 90°-180°-180° pulse
sequence (repetition time, 2000 milliseconds; echo time, 272 milliseconds;
250-mm field of view; 32 x 32 phase-encoding steps; and 1 signal average
per step) as previously described.30 Water
suppression was achieved by placing frequency-selective excitation pulses
at the beginning of the MRS imaging sequence.31
Before the water-suppressed acquisition, another MRS image was acquired without
water suppression (repetition time, 850 milliseconds; echo time, 272 milliseconds;
250-mm field of view; and 16 x 16 phase-encoding steps) to allow for
B0 homogeneity correction.
MR DATA ANALYSIS
Lesion Volumes and MT
The T2-W lesions were classified by a single observer (S.J.F.) with
the use of a user-supervised thresholding technique. Lesion borders were determined
primarily on proton density–weighted images, but information from T2-W
and T1-W images were also considered. As the aim of the study was to study
a population with established MS and low disease burden, MS patients were
included only if they had a T2-W lesion volume of less than 20 cm3
(about 1.5% of the total brain tissue volume). This condition was met in 57
of the 60 MS patients; thus, data from 3 patients were excluded from the analysis.
Percentage difference MTr images were calculated on a voxel-by-voxel basis
according to the following equation:
(after thresholding above the noise background), as previously described.32 Once T2-W lesions and normal-appearing brain were
classified, the MTr values of these regions were calculated. Only values of
MTr in the normal-appearing white matter (NAWM) were considered here. These
were calculated by taking consistent samples of white matter from 5 regions
(corona radiata and centrum semiovale, frontal lobe, genu of corpus callosum,
splenium of corpus callosum, and occipital lobe) (Figure 1). The MTr for NAWM was then obtained by averaging the mean
MTr from each region. Values of MTr for NAWM in the control group were obtained
in the same way.
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Figure 1. Examples of magnetization transfer
ratio (MTr) percentage difference images of a healthy control subject, illustrating
(dark regions) the 5 white matter regions (corona radiata and centrum semiovale,
frontal lobe, genu of corpus callosum, splenium of corpus callosum, and occipital
lobe) used for the calculation of white matter MTr values. In each subject,
these regions of interest span 6 slices, but only 2 (A and B) are illustrated
for brevity.
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Proton-MRS Imaging
Postprocessing of the raw proton–MRS imaging data was performed
as previously described.26 Metabolite resonance
intensities of NAA were determined automatically from peak areas relative
to a spline-corrected baseline and expressed as ratios to Cr (Figure 2). The resonance intensity of intravoxel Cr has been widely
used as an internal standard in MRS studies in vivo, as it is relatively equally
present in all brain cells and tends to be stable in nonacute pathology.3 Changes in apparent brain Cr concentrations have been
reported in MS in recent MRS studies attempting absolute quantitation. However,
all current quantitative approaches have important limitations when applied
to clinical studies and, in MS patients, have shown discrepant results in
lesions and NAWM.4, 33-37
In vitro MRS, which does not suffer from the limitations of in vivo quantitation,
has demonstrated that Cr does not change in normal-appearing tissues of the
brains of MS patients.38 Thus, as lesions accounted
for only a minimal portion of the large central VOI (mean, 1.5% [range, 0%-3%];
data not shown) in this group of MS patients, changes in Cr, although possible,
are unlikely and results were expressed as the intravoxel ratio of NAA to
Cr. The relative NAA/Cr values of the whole brain region were obtained by
averaging the NAA/Cr for all the voxels in the spectroscopic VOI for each
subject. Spectra at the edges of the VOI affected by chemical shift artifacts
associated with selective excitation were deleted before averaging.
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Figure 2. Magnetic resonance images in sagittal
(A) and transverse (B) orientation of a patient with multiple sclerosis, illustrating
the volume of interest (VOI) used for spectroscopic imaging (see grid in B)
and an example of the resulting spectra (C) from deep white matter voxels
(filled squares in B). Voxels at the edge of the VOI were omitted from analysis,
since they can show artifactual relative amplitudes. The ratios of N-acetylaspartate (NAA) to intravoxel creatine (Cr) in the remaining
voxels were averaged to obtain 1 value for each examination.
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Total Brain Volumes
On T1-W MR images, normalized volumes of the whole of the brain parenchyma
were measured using a method for brain volume measurement (the cross-sectional
version of the SIENA software39 [SIENAX; available
at: http://www.fmrib.ox.ac.uk/analysis/research/siena/]) (Figure 3). SIENAX uses a method to extract
the brain and skull from the MR images, as previously described.40
A tissue segmentation program41 is then used
to segment the extracted brain image into brain tissue, cerebrospinal fluid,
and background, yielding an estimate of total brain tissue volume. The original
MR images are registered to a canonical image in a standardized space (using
the skull image to provide the scaling cue), a procedure that provides a spatial
normalization factor for each subject. The estimate of brain tissue volume
for a subject is then multiplied by the normalization factor to yield the
normalized brain volume (NBV).
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Figure 3. Typical transverse T1-weighted
magnetic resonance images of a healthy control subject (A and B) and an illustrative
example of the SIENAX output (the cross-sectional version of the SIENA software39) (C and D). The normalized brain volume includes
only brain parenchyma and discards cerebrospinal fluid and other nonbrain
tissues, yielding an estimate of total brain tissue volume (see the "Subjects
and Methods" section).
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STATISTICAL ANALYSIS
At each site, MR data of MS patients were compared with those of an
age-matched healthy control (HC) group: 21 subjects at MNH (13 women and 8
men; age range, 22-57 years [mean, 35 years]) and 21 at Siena (12 women and
9 men; age range, 21-52 years [mean, 35 years]). Comparisons were made between
corresponding HC populations of each site (ie, Siena-HC vs MNH-HC) and between
the whole group of MS patients and HC subjects of both sites. In the latter
case, MR data were standardized at each site using a z
score transformation relative to the corresponding HC group. This allowed
avoiding potentially spurious results due to machine-related differences between
sites. The nonparametric Kruskal-Wallis 1-way analysis of variance on ranks
was used for the statistical analysis, and values were considered significant
at the .05 level. SYSTAT software (Version 9 for Windows; SPSS Inc, Chicago,
Ill) was used to perform statistical calculations.
RESULTS
The comparison of MR measurements from each center showed that NAA/Cr
and NBV values of the HC groups were not different between the 2 sites (NAA/Cr
in Siena-HC, 3.06 ± 0.2 and in MNH-HC, 3.14 ± 0.16; NBV in Siena-HC,
1467 ± 41 cm3 and in MNH-HC, 1476 ± 68 cm3; P>.10 for both). However, white matter MTr values were
significantly lower in the Siena-HC group than in the MNH-HC group (MTr in
Siena-HC, 35.3 ± 0.9 and in MNH-HC, 36.4 ± 0.4; P<.001). This was probably due to the sensitivity of MTr measurements
to subtle differences in hardware between MR scanners. However, as mentioned
before, all MR data measurements were standardized at each site using a z score transformation to correct for differences in HC
subjects at different sites and to allow comparisons between the whole groups
of HC subjects and MS patients.
In the whole group of MS patients without clinical disability, the standardized
levels of central brain NAA/Cr were significantly lower than those of HC subjects
(P<.001, Figure
4). Similarly, standardized MTr values were lower in the NAWM of
MS patients than in the white matter of HC subjects (P<.001, Figure 4). However, while the fully automated
estimation of standardized NBV showed a trend toward decreased values in the
MS group with respect to the age-matched HC group, this did not reach statistical
significance (P = .07, Figure 4).
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Figure 4. Box plots comparing the standardized
magnetic resonance measurements (ratio of N-acetylaspartate
to creatine [NAA/Cr], magnetization transfer ratio [MTr], and normalized brain
volume [NBV]) in the whole group of patients with multiple sclerosis (MS)
with a group of age-matched healthy control subjects (HC).
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When similar analyses were performed in MS patients grouped according
to duration of disease, 36 patients with early disease duration (<3 years)
still showed significantly lower brain NAA/Cr and MTr values than did HC subjects
(P = .001 and P = .004,
respectively; Figure 5). Furthermore,
a subgroup of 26 patients with minimal lesion volume (2 cm3
of T2-W MR imaging lesions, about 0.15% of the total brain volume) also showed
significantly low NAA/Cr and MTr (P<.05 for both; Figure 6). In both subgroups, patient values
of NBV were not different from those of HC subjects (P>.50
in both subgroups).
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Figure 5. Box plots of the standardized
magnetic resonance measurements (ratio of N-acetylaspartate
to creatine [NAA/Cr], magnetization transfer ratio [MTr], and normalized brain
volume [NBV]) in a selected subgroup of patients with multiple sclerosis (MS)
(n = 36) with short disease duration (<3 years) with respect to healthy
control subjects (HC).
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Figure 6. Box plots of the standardized
magnetic resonance (MR) measurements (ratio of N-acetylaspartate
to creatine [NAA/Cr], magnetization transfer ratio [MTr], and normalized brain
volume [NBV]) in a selected subgroup of patients with multiple sclerosis (MS)
(n = 26) with very low volume (2 cm3) of cerebral T2-weighted
MR imaging lesions with respect to healthy control subjects (HC).
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COMMENT
Because NAA is localized to neurons and axons in adult human brain19, 42 and correlates strongly with axonal
density,20, 43 levels of brain
NAA detected by proton-MRS imaging can be interpreted, with some rare exceptions,44-45 as a surrogate of neuronal and axonal
integrity. Large decreases of NAA have been observed in numerous spectroscopic
studies inside and beyond MS lesions1-2,4-5,18
and have been demonstrated to occur, to a lesser degree, also in the NAWM
of MS patients from the early disease stages.26
Results of the present study extend these previous observations26
by showing that significant decreases of NAA can be detected in the NAWM of
MS patients with very low disease duration, in the absence of substantial
focal brain demyelination and before permanent clinical disability becomes
evident. Given that NAA decreases were confined to demyelinating lesions in
brains of patients with clinically isolated syndromes who subsequently developed
MS,46 our observations also suggest that diffuse
cerebral axonal injury rapidly accumulates in the early stages of the disease.
In addition to decreases of NAA/Cr, we also found decreases of MTr in
the NAWM in this group of nondisabled MS patients. Magnetization transfer
imaging of the brain is based on the interactions between the free water protons
and protons attached to macromolecules, and a low MTr indirectly reflects
tissue (matrix) damage.47-48 Several
studies have demonstrated marked MTr reductions in lesions and NAWM of patients
with MS.49-50 As recent studies
have shown that focal MTr decreases in NAWM can occur before lesion appearance
on conventional MR imaging,51-53
a low MTr in the NAWM may reflect subtle, microscopic, or molecular pathology
of myelin in macroscopically normal white matter. Edema, astrocytic proliferation,
perivascular inflammation, and demyelination may all contribute to a decreased
amount of water bound to macromolecules in the NAWM and, as a consequence,
reduced MTr.47 These pathologic features, however,
are not prominent in the NAWM in early MS. A potential mechanism for subtle
molecular alteration of myelin remains to be determined, but its presence
is suggested by the fact that MTr values are decreased in the white matter
of patients with MS who have completely normal results of conventional MR
imaging of their brain.23 In addition, since
MTr decreases in postmortem brain of MS patients also correlate with axonal
loss,21 it may be possible that membrane alterations
associated with axonal injury also contribute to the decreases in MTr.
In this cross-sectional study, we did not find significant differences
in NBV between MS patients and age-matched HC subjects, although there was
a trend suggesting the presence of modest brain atrophy in the patient group
as a whole. Significant atrophy has been recently reported in the brains of
MS patients in both cross-sectional and longitudinal studies that used automated
or semiautomated measures of brain volume.22, 25, 54-56
In particular, significant losses of brain volume have been found in MS patients
with mild disability,22 and significantly increased
rates of ventricular enlargement have been reported in early-stage MS patients57 and in patients with clinically isolated syndromes
who later developed MS.24 Differences between
these published data and those presented herein could be the result of the
lower sensitivity of cross-sectional measurements of total brain volume with
respect to measurements of atrophy rates and the particularly mild clinical
condition of the patients included in our study. Indeed, by showing a trend
toward NBV decreases in MS patients with a low lesion load and the absence
of disability, we suspect that the present results are not really in conflict
with previous findings. Notably, the differences in the relative magnitude
of decreases in NAA/Cr and NBV in our study suggest that decreases in NAA
(which can result from axonal dysfunction, decreases in axonal density, and
axonal loss), and decreases in NBV (which reflect a less pathologically specific
tissue loss) do not always occur in parallel. Brain atrophy should be considered
a later event that is not necessarily proportional to axonal injury. It follows
that in vivo measures of total axonal injury and loss should be based on measurements
of both decreases in brain volume and decreases of NAA density in remaining
brain tissue.
By restricting our analysis to MS patients with a low volume of T2-W
lesions, we sought evidence that the abnormalities in NAWM could occur independently
of focal demyelinating lesions. As reported in a number of previous studies,4, 32, 49, 52, 58
decreases of NAA and MTr are more pronounced inside demyelinating lesions
than in the NAWM of MS patients. However, the presence of significant decreases
of NAA/Cr and MTr in our subgroup of patients in whom lesions occupied less
than 0.2% of the total brain tissue (the patient subgroup with 2 cm3 of T2-W MR imaging lesions) suggests that axonal and tissue injury
in MS might accrue, at least in part, independently of focal cerebral demyelination.
Since wallerian degeneration of axons traversing lesions is unlikely to account
for much of the NAA decrease seen in these patients with very low lesion loads,
the diffuse axonal abnormality must result from nonlesional abnormalities
associated either with subtle myelin abnormality that is not visible on conventional
MR imaging or with subtle axonal abnormality, possibly due to the indirect
effects of inflammation.59 That diffuse decreases
of NAA are to some extent reversible with immunomodulatory therapy60 is consistent with a role for inflammation in these
decreases.
How can widespread axonal injury occur in the brains of MS patients
without clinical evidence of disability? Experimental and functional MR imaging
data may provide an answer. Neurons can function as dynamic electrogenic machines
with electroresponsive properties that change in response to pathologic insults.61 However, in the initial disease stages, both adaptive
cortical reorganization (by the "unmasking" of latent pathways)62
and ion channel redistribution63-64
can achieve complete functional recovery and cause neuronal degeneration to
remain subclinical. Development of permanent disability occurs later, when
a threshold of axonal loss is reached and compensatory resources of the central
nervous system are exhausted.65-67
Thus, axonal injury does occur in MS, even in the absence of clinical disability,
and can be detected and monitored by pathologically specific MR measures.
In conclusion, results of our study indicate that cerebral NAA/Cr and
MTr values are diffusely decreased in brains of patients with early MS, minimal
focal T2-W lesion volume, and no clinical evidence of disability. This finding
suggests that axonal and/or tissue injury might be to some extent independent
of focal cerebral demyelination and is probably initially well compensated
for by brain plasticity. Decreases of total brain volume do not necessarily
occur in parallel with decreases of NAA and, therefore, both NAA and brain
atrophy should be assessed to determine the true total extent of axonal injury
and loss.
AUTHOR INFORMATION
Accepted for publication May 29, 2002.
Author contributions: Study concept and design (Drs De Stefano and Arnold, Mr Narayanan, and Ms Tartaglia);
acquisition of data (Drs De Stefano, Mortilla, Bartolozzi,
Guidi, and Arnold; Messrs Narayanan and Francis; and Ms Tartaglia);
analysis and interpretation of data (Drs De Stefano, Smith,
Federico, and Arnold and Messrs Narayanan and Francis); drafting of
the manuscript (Drs De Stefano, Smith, and Federico and
Messrs Narayanan and Francis); critical revision of the manuscript
for important intellectual content (Mr Narayanan and Drs
Mortilla, Bartolozzi, Guidi, and Arnold); statistical expertise (Drs De Stefano, Smith, and Bartolozzi and Mr Narayanan);
administrative, technical, and material support (Messrs
Narayanan and Francis and Drs Mortilla, Bartolozzi, and Federico);
and study supervision (Dr Arnold).
This study was supported by grants from the Multiple Sclerosis Society
of Canada (Toronto, Ontario) and the Medical Research Council of Canada (Ottawa,
Ontario). Dr De Stefano was supported by a PAR (Progetto Ateneo di Ricerca)
grant of the University of Siena. Dr Federico was supported by a grant from
MURST (Ministero dell'Università e della Ricerca Scientifica e Tecnologica,
Rome, Italy). Dr Arnold was supported by grants from the Multiple Sclerosis
Society of Canada and the Medical Research Council of Canada.
Corresponding author and reprints: Nicola De Stefano, MD, Institute
of Neurological Sciences, Viale Bracci 2, 53100, Siena, Italy (e-mail: destefano{at}unisi.it).
From the Institute of Neurological Sciences and NMR Centre, University
of Siena, Siena, Italy (Drs De Stefano, Mortilla, and Federico); Department
of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital,
Montreal, Quebec (Messrs Narayanan and Francis, Ms Tartaglia, and Dr Arnold);
Centre for Functional Magnetic Resonance Imaging of the Brain, Department
of Clinical Neurology, University of Oxford, Oxford, England (Dr Smith); and
Neurology Unit, Hospital of Empoli, Empoli, Italy (Drs Bartolozzi and Guidi).
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