Acute axonal damage in multiple sclerosis is most over time

Brain (2002), 125, 2202±2212
Acute axonal damage in multiple sclerosis is most
extensive in early disease stages and decreases
over time
Tanja Kuhlmann,1 Gueanelle Lingfeld,1 Andreas Bitsch,2 Jana Schuchardt1 and Wolfgang BruÈck1,*
of Neuropathology, ChariteÂ, HumboldtUniversitaÈt, Berlin and 2Department of Neurology,
Ruppiner Kliniken GmbH, Neuruppin, Germany
1Department
Correspondence to: Professor Dr med Wolfgang BruÈck,
Institut fuÈr Neuropathologie, ChariteÂ, Campus VirchowKlinikum, Augustenburger Platz 1, D-13353 Berlin,
Germany
E-mail: [email protected]
*Present address: InstituÈt fuÈr Neuropathologie,
Georg-August-UniversitaÈt, Robert-Koch-Str 40, 37075,
GoÈttingen, Germany
E-mail: [email protected]
Multiple sclerosis is characterized morphologically by
the key features demyelination, in¯ammation, gliosis
and axonal damage. In recent years, it has become
more evident that axonal damage is the major morphological substrate of permanent clinical disability. In our
study, we investigated the occurrence of acute axonal
damage determined by immunocytochemistry for amyloid precursor protein (APP) which is produced in neurones and accumulates at sites of recent axon
transection or damage. The numbers of APP-positive
axons in multiple sclerosis lesions were correlated with
the disease duration and course. Most APP-positive
axons were detected within the ®rst year after disease
onset, but acute axonal damage was also detected to a
minor degree in lesions of patients with a disease duration of 10 years and more. This effect was not due to
the lack of active demyelinating lesions in the chronic
disease stage. Late remyelinated lesions (so-called shadow plaques) did not show signs of axon destruction.
The number of in¯ammatory cells showed a decrease
over time similar to that of the number of APP-positive
axons. There was a signi®cant correlation between the
extent of axon damage and the numbers of CD8-positive
cytotoxic T cells and macrophages/microglia. Our
results indicate that a putative axon-protective treatment should start as early as possible and include strategies preventing T cell/macrophage-mediated axon
destruction and leading to remyelination of axons.
Keywords: multiple sclerosis; axon damage; APP
Abbreviations: APP = amyloid precursor protein; MBP = myelin basic protein; MOG = myelin oligodendrocyte
glycoprotein; MRS = magnetic resonance spectroscopy; NAA = N-acetylaspartate; NAWM = normal-appearing white
matter; PPMS = primary progressive multiple sclerosis; PPWM = periplaque white matter; RRMS = relapsing±remitting
multiple sclerosis; SPMS = secondary progressive multiple sclerosis
Introduction
Multiple sclerosis is a disease of the CNS characterized by
multifocal in¯ammation, demyelination, gliosis and axonal
loss. In recent years, the signi®cance of axonal degeneration
for permanent neurological de®cits in multiple sclerosis
patients has been emphasized. Magnetic resonance spectroscopy (MRS) of multiple sclerosis plaques revealed a
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correlation of disability and reduced levels of N-acetylaspartate (NAA) (Matthews et al., 1998), which is a biochemical marker found exclusively in neurones and their processes
(Birken and Oldendorf, 1989). Also the volume of hypointense lesions (`black holes') in T1-weighted MRI and the
extent of brain atrophy, both putative markers of axonal
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Summary
Acute axonal damage in multiple sclerosis
Material and methods
Patients
We retrospectively investigated brain tissue from 39 patients
(23 biopsies from 21 patients and 18 autopsies). Two patients
underwent two biopsies from different CNS sites at consecutive time points during progression of disease. Biopsies had
been performed for diagnostic reasons to exclude neoplastic
or infectious diseases. Informed consent had been obtained
from each patient. None of the study authors was involved in
decision making with respect to biopsy. When the biopsies
were taken, none of the patients ful®lled the criteria of
clinically de®nite multiple sclerosis according to the Poser
criteria (Poser et al., 1983). The study was approved by the
Ethics Committee of The University of GoÈttingen.
During clinical follow-up, 19 of the 21 biopsied patients
ful®lled the Poser criteria for clinically de®nite multiple
Table 1 Characteristics of patients
Female n = 24, male n = 15
Age 49 years (median), 15±71 (range)
23 biopsies and 18 autopsies
Clinical course
Relapsing±remitting
Secondary progressive
Primary progressive
Disease duration of autopsy patients 108
months (median), 30±336 months (range)
Disease duration in biopsied patients* 10
months (median), 0.5±168 months (range)
n = 23 (5 autopsies,
18 biopsies)
n = 5 (5 autopsies)
n = 11
(8 autopsies, 3 biopsies)
*Time from ®rst symptoms to biopsy.
sclerosis (two PPMS, 17 RRMS; for details see Table 1). One
patient had two relapses with similar symptoms, thus
ful®lling the criteria for clinically probable multiple sclerosis
according to Poser. Another patient showed all characteristics
of PPMS (Thompson et al., 2000). However, with the help of
longitudinal MRI data in some cases, all 21 patients ful®lled
the new diagnostic criteria for multiple sclerosis (McDonald
et al., 2001). Three patients entered a secondary progressive
disease course years after biopsy, but had a relapsing±
remitting course at biopsy. Therefore, these patients were
classi®ed as RRMS patients in the study. Biopsies were
performed between 0.5 and 168 months (median: 10 months)
after occurrence of the ®rst symptoms. The biopsied patients
included in the present study were selected from a larger
group of patients in whom an in¯ammatory demyelinating
CNS process was diagnosed at biopsy. The 21 biopsied
patients included in the present study had been the subject of
an earlier investigation in which the extent of acute axonal
damage was correlated with the stage of demyelinating
activity within the lesions (Bitsch et al., 2000).
Among the 18 autopsy cases, eight had PPMS, ®ve had a
relapsing±remitting disease course and ®ve had SPMS (for
details see Table 1). The patients who were autopsied had a
disease duration between 30 and 336 months (median: 108
months). No patients with a fulminant variant of multiple
sclerosis (Type Marburg) leading to early death were
represented in either the biopsy or autopsy group in the
study. The patient's clinical characteristics are summarized in
Table 1.
Histopathology
Biopsies were performed in different centres all over
Germany and sent to the Department of Neuropathology in
Berlin after completion of routine analyses. Autopsies were
also collected in Berlin's Department of Neuropathology.
Specimens were ®xed in 4% paraformaldehyde and embedded in paraf®n. Slices 4 mm thick were stained with
haematoxylin and eosin (HE), Luxol-fast blue (LFB), peri-
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damage, correlated with the degree of disability (Grimaud
et al., 1999; Fisher et al., 2000; Paolillo et al., 2000; Pelletier
et al., 2001).
Axonal damage can be determined histopathologically by
the extent of axonal loss relative to the normal-appearing
white matter (NAWM). In addition, acute axonal damage can
be detected by immunohistochemistry for the amyloid
precursor protein (APP) (Li et al., 1995; Bramlett et al.,
1997; Yam et al., 1997; Pesini et al., 1999). APP is found in
neurones and undergoes anterograde axonal transport. In the
case of an axonal transection, the transport is interrupted and
APP accumulates in the proximal axonal ends. As a
consequence, so-called APP-positive spheroids are formed
that persist for <30 days. A variety of different histopathological studies exist that investigated acute axonal damage in
multiple sclerosis (Ferguson et al., 1997; Trapp et al., 1998;
Bitsch et al., 2000; Kornek et al., 2000). However, to the best
of our knowledge, so far no report has been published that
correlates axonal damage and disease duration as carried out
in our study. We focused on acute axonal damage within the
lesions and the periplaque white matter (PPWM) and
correlated it with disease duration, in¯ammation and clinical
course.
Our data demonstrate that acute axonal damage occurs
early during disease and lesion formation. APP-positive
axons can be detected in all stages of demyelinating activity
and also in the PPWM. Acute axonal damage is most
prominent within the ®rst year after disease onset. In
relapsing±remitting (RRMS) and secondary progressive
multiple sclerosis (SPMS), acute axonal damage is signi®cantly higher in early stages of the disease than after a disease
duration of 10 years or more. In contrast, in primary
progressive multiple sclerosis (PPMS) there are no signi®cant
differences over time. Our data indicate that a putative
neuroprotective treatment could be most effective if started at
the beginning of the disease during the relapsing±remitting
stage.
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T. Kuhlmann et al.
odic acid±Schiff (PAS) and Bielschowsky's silver impregnation. Immunohistochemical staining was performed with a
biotin±avidin or an alkaline phosphatase/anti-alkaline phosphatase technique. The primary antibodies were anti-myelin
basic protein (anti-MBP, Boehringer Mannheim, Mannheim,
Germany), anti-proteo-lipid protein (anti-PLP), anti-myelin
oligodendrocyte glycoprotein (anti-MOG, Dr Chris
Linington, Max-Planck-Institut fuÈr Neurobiologie, Munich,
Germany), anti-KiM1P (macrophages, Dr Radzun,
University of GoÈttingen, Germany), anti-27E10 and antiMRP 14 (activated macrophages, BMA Biomedicals, Augst,
Switzerland), anti-CD3 (T cells, Dako, Denmark) anti-CD8
(cytotoxic T cells, Dako, Denmark), anti-IgG (Plasma cells,
Dako, Denmark) and anti-APP (Boehringer Mannheim,
Germany).
Classi®cation of multiple sclerosis lesions
Morphometry
The number of APP-positive axons as well as macrophages
and T cells stained with the corresponding antibodies was
determined in at least 10 standarized microscopic ®elds of
10 000 mm each de®ned by an ocular morphometric grid. In
the text and ®gures, the mean number of cells/mm2 is given.
Statistics
For statistical analysis, non-parametric tests were performed
(Mann±Whitney test, Spearman rank correlation, Kruskal±
Wallis test). In cases of multiple comparisons, Dunn's
multiple comparison test was performed. All tests were
classi®ed as signi®cant if the P value was <0.05. The
Results
Acute axonal damage, in¯ammation and disease
duration
Patients were divided into four groups according to disease
duration (time from ®rst symptoms to biopsy/autopsy): group
I, 0±1 year (12 biopsies); group II, 1±5 years (four autopsies,
®ve biopsies), group III, 5±10 years (seven autopsies, three
biopsies); and group IV, >10 years (seven autopsies, three
biopsies).
Acute axonal damage in multiple sclerosis lesions was
determined in sections stained for APP (Fig. 1). The highest
number of APP-positive axons was found in group I (Fig. 2A).
Signi®cantly more axons stained for APP in group I than in
group II (P < 0.001), group III (P < 0.01) and group IV
(P < 0.001). There were also signi®cantly more APP-positive
axons in group II compared with group III (P < 0.05).
From earlier studies, it is known that there are more APPpositive axons in active lesions than in demyelinated or
remyelinating plaques. To test whether the signi®cant higher
accumulation of APP-positive axons in group I is the result of
an unbalanced distribution of active lesions, we compared
active, demyelinated and remyelinating lesions of the groups
I±IV. As shown in Fig. 3A, active lesions of group I revealed
the highest number of APP-positive axons, indicating that the
high level of acute axonal damage in group I is, in fact, caused
by an increased number of transected axons rather than an
unbalanced distribution of active demyelinating lesions
(Fig. 1A±C). Numbers of APP-positive axons were signi®cantly higher in group I than in group III (P < 0.05) and in
group IV (P < 0.01). In demyelinated and remyelinating
lesions, the number of APP-positive axons was relatively low
in all groups, and no signi®cant differences between the
groups were observed (Fig. 3B and C). In late remyelinating
lesions, with few exceptions, only little APP staining was
seen (Figs 1D±F and 3D). These results demonstrate that
acute axonal damage is (i) an early event during multiple
sclerosis; (ii) highest at the beginning of the disease; and (iii)
low in remyelinated lesions.
In a second series of analyses, we investigated the
composition of the in¯ammatory in®ltrates in multiple
sclerosis plaques in correlation to disease duration. Lesions
were in®ltrated mainly by macrophages and CD3-positive T
cells (Fig. 2B±D). Signi®cantly more CD3 cells were seen in
group I compared with all other groups (Fig. 2B; group I
versus II, P < 0.05; I versus III, P < 0.01; I versus III, P < 0.01).
Most CD8-positive lymphocytes were found in group I
(group I versus II, P < 0.05; I versus IV, P < 0.05). The
highest number of macrophages was observed in group I,
whereas no signi®cant differences were found between
groups II, III and IV (Fig. 2D; group I versus III, P < 0.05).
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All lesions ful®lled the generally accepted criteria for the
diagnosis of multiple sclerosis (Prineas, 1985; Allen, 1991;
Lassmann, 1998). Lesional activity was classi®ed as
described in detail earlier (BruÈck et al., 1995). Brie¯y, early
active lesions (n = 24) were located at the plaque border,
partially demyelinated and in®ltrated by numerous MBP- and
MOG-positive macrophages. In late active lesion areas
(n = 22), demyelination was more advanced and macrophages
contained MBP- but no MOG-positive myelin debris.
Demyelinated lesions (n = 57) were completely demyelinated
and full of macrophages/microglia that contained PASpositive material but no MOG- or MBP-positive myelin
degradation products. In early remyelinating plaques (n = 15),
thin, irregularly formed myelin sheaths were seen as well as a
massive in®ltration by macrophages/microglia and T cells. In
late remyelinating lesions (n = 18), remyelination is more
advanced (shadow plaques) and only a few in¯ammatory
cells can be found. The PPWM (n = 81) showed no signs of
demyelination.
GraphPad PRISMÔ software was used (Graph Pad Software,
Inc., San Diego, CA, USA).
Acute axonal damage in multiple sclerosis
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Fig. 1 APP-positive axons in active demyelinating (A±C) and remyelinating (D±E) lesions. (A) Demyelinated lesion with an active
demyelinating border of a patient with a disease duration longer than 10 years (group IV). The area of active demyelination is marked by
arrows. (B) Numerous LFB-positive macrophages are seen (LFB staining, see arrows). (C) In the area of active demyelination, many
APP-positive axons were found (immunohistochemistry for APP, see arrows). (D) Late remyelinating lesion (shadow plaque) of a patient
with a disease duration of 5±10 years (group III). (E) Thin, irregularly formed myelin sheaths that are characteristic for remyelinating
lesions are shown, see arrows (immunohistochemistry for MBP). (F) In these lesions, only a few APP-positive axons were found, see
arrow (immunohistochemistry for APP).
Interestingly, only few plasma cells were seen, and the
number of plasma cells was similar in all groups (Fig. 2E).
Acute axonal damage and in¯ammation within
the periplaque white matter
Numerous APP-positive axons were detected within the
PPWM of multiple sclerosis lesions. We studied the number
of APP-positive axons in correlation to disease duration.
Highest numbers of APP-positive axonal spheroids were
identi®ed in the PPWM of patients who were biopsied within
1 year after onset of ®rst symptoms (group I) (Fig. 2F; group I
versus IV, P < 0.05). In the other groups, only few APPpositive axons were observed in the PPWM. In¯ammatory
in®ltrates were seen perivascularly and diffusely distributed
through the otherwise normal appearing PPWM. They
consisted mainly of macrophages and T cells (Fig. 2G±I).
The number of macrophages and CD3-positive lymphocytes
within the PPWM was similar in all groups (Fig. 2G and H),
whereas most CD8-positive T cells were found in group I
patients (Fig. 2H; group I versus II, P < 0.05; I versus IV,
P < 0.05).
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T. Kuhlmann et al.
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Fig. 2 Acute axonal damage and in¯ammation in lesions and periplaque white matter. Number of (A and
F) APP-positive axons, (B and G) CD3- and (C and H) CD8-positive T cells, (D and I) macrophages/
microglia and (E) plasma cells in lesions and in periplaque white matter of patients with different disease
duration (I, 0±1 year; II, 1±5 years; III, 5±10 years; IV, >10 years). Values are given as means 6 SD.
Acute axonal damage in multiple sclerosis
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Correlation of acute axonal damage and
in¯ammation
The extent of acute axonal damage in the entire group of
lesions correlated signi®cantly with the number of CD8positive T cells (P < 0.0005) and macrophages/microglia
(P < 0.0001). In the PPWM, a signi®cant correlation was only
found for CD8-positive T cells (P < 0.0001). However,
differences were observed when grouping the lesions according to the disease duration. In lesions obtained early in the
disease (groups I and II, disease duration <5 years), a positive
correlation was found between the number of APP-positive
axons and macrophages/microglia (P = 0.005), but not for
CD8-positive T cells. The opposite was found in the PPWM
at this disease stage. Acute axonal damage signi®cantly
correlated with the number of CD8-positive T cells
(P = 0.0001, r = 0.79). In the late disease stages (groups III
and IV, disease duration >5 years), in both the lesions and the
PPWM, a signi®cant correlation was found between acute
axonal damage and numbers of CD8-positive T cells
(P < 0.01) and macrophages/microglia (P < 0.005). These
data suggest that different components of the in¯ammatory
in®ltrate may contribute differentially to axon damage in the
demyelinating lesions and the PPWM.
Acute axonal damage and disease course
We also studied the number of APP-positive axons in
patients of different clinical courses and disease durations.
In patients with a relapsing±remitting disease course,
there were signi®cantly more APP-positive axons at the
beginning of the disease than after 10 years (Fig. 4A;
group I versus IV, P < 0.001). In autopsies from patients
with SPMS, all lesions were older than 1 year. Again,
APP expression was more pronounced in early lesions
(Fig. 4B; group III versus IV, P < 0.001). In PPMS, there
were no signi®cant differences between different disease
durations after correction for multiple comparisons
(Fig. 4C). We also investigated whether there was a
difference between male and female patients with respect
to acute axonal damage, but no signi®cant variations
between the sexes were detected.
Discussion
Our present data show that acute axonal damage is an early
event during the development of multiple sclerosis lesions.
We observed the highest number of APP-positive axons, a
marker of acute axonal damage, in lesions of patients with a
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Fig. 3 Disease duration and acute axonal damage in different stages of demyelinating activity. APP-positive axons in (A) active
demyelinating, (B) remyelinating, (C) inactive remyelinated and (D) late remyelinating lesions in patients with different disease duration
(I, 0±1 year; II, 1±5 years; III, 5±10 years; IV, >10 years). Horizontal bars indicate the median.
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T. Kuhlmann et al.
disease duration of <1 year. Our data also suggest that axonal
damage is a continuous process that can be found in patients
who suffer from multiple sclerosis for >10 years independently of the clinical course. The number of in¯ammatory cells
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Fig. 4 Acute axonal damage in different disease courses. Acute
axonal damage in patients with (A) relapsing±remitting; (B)
secondary progressive; and (C) primary progressive disease course
and different disease durations (I, 0±1 year; II, 1±5 years; III, 5±10
years; IV, >10 years). Bars indicate the median.
runs through a decrease over time similar to the number of
APP-positive axons. In addition, acute axonal damage can
also be seen in the PPWM with a similar time course to that
inside the lesions. The extent of axon damage correlates with
in¯ammation. However, especially in the early disease
evolution, different components of the immune system
(CD8-positive T cells and macrophages/microglia) seem to
be involved differentially in this process in the lesions and the
NAWM.
Axonal loss can be detected indirectly by MRS (Arnold
et al., 1990; Matthews et al., 1998) and MRI techniques
(BruÈck et al., 1997; van Walderveen et al., 1998) in vivo. In
MRS, axonal dysfunction or loss is associated with reduced
levels of NAA (De Stefano et al., 1995) that is located mainly
in neurones and axons (Birken and Oldendorf, 1989). It is
therefore a suitable marker of axonal loss (Simmons et al.,
1991). Hypointense T1 lesions and a decreased magnetization
transfer ratio (MTR) are also associated with tissue destruction and axonal loss (BruÈck et al., 1997; van Walderveen
et al., 1998; van Waesberghe et al., 1999). T1 lesion
hypointensity (Grimaud et al., 1999; Paolillo et al., 2000),
decreased MTR and reduced NAA levels all correlate with an
increased disability (Gass et al., 1994; De Stefano et al.,
1995, 1998; Davie et al., 1995, 1999). Some studies show
abnormal low NAA/Cr (creatine) values in early stages of
multiple sclerosis and a reduction of the NAA/Cr level that
even precedes signi®cant disability (De Stefano et al., 2001).
However, MR techniques are not able to differentiate
between acute and long-standing axonal damage. MRS,
MRI and MTR can only re¯ect the accumulation of axonal
damage/dysfunction. In contrast, histopathological studies
are able to distinguish between acute axonal damage and
permanent loss of axons. The latter can be measured by the
number of axons counted after Bielschowsky's stain or
immunohistochemical neuro®lament stainings. In contrast,
APP is a marker of acute axonal damage. It persists in
transected axons for a period of <30 days (Li et al., 1995;
Pierce et al., 1996; Bramlett et al., 1997; Yam et al., 1997)
In histopathological studies, a signi®cant reduction of axon
density was found in multiple sclerosis lesions (Lovas et al.,
2000). An average axonal loss of 59±82% was observed in
demyelinated and remyelinated multiple sclerosis plaques
compared with the PPWM (Mews et al., 1998). The acute
axonal damage determined by APP staining or axonal
spheroids was investigated in acute (Ferguson et al., 1997;
Kornek et al., 2000), early (Bitsch et al., 2000) and late
chronic (Kornek et al., 2000) multiple sclerosis cases. From
these studies, it is well known that axonal damage is an early
event during development of multiple sclerosis plaques and
that the highest degree of acute axonal damage is associated
with active demyelination (Ferguson et al., 1997; Bitsch et al.,
2000; Kornek et al., 2000)
Our data con®rm these investigations. We see the highest
numbers of APP-positive axons in active demyelinating
plaques. However, APP-positive axons are also present in
early remyelinating and to a much less degree in demyeli-
Acute axonal damage in multiple sclerosis
progressive clinical disability in PPMS. However, more
studies comparing axon density and loss in different clinical
courses are required to con®rm this conclusion.
We also investigated the PPWM with regard to axonal
damage and in¯ammation. Correlating disease duration and
the number of APP-positive axons, most APP-positive axons
were observed within the ®rst year after disease onset. This
was associated with an increased in®ltration of the PPWM
with CD8-positive T lymphocytes, which may indicate a
substantial role for these cells in axonal destruction. We
found APP-positive axons in the PPWM without any signs of
demyelination. These ®ndings indicate that axonal damage
occurs without visible demyelination or even precedes
demyelination. These results are in line with earlier studies
that investigated acute axonal damage in PPWM of experimental allergic encephalomyelitis (EAE) animals and multiple sclerosis patients (Bitsch et al., 2000; Kornek et al.,
2000). Changes in the white matter can be found not only in
the PPWM but also in white matter far away from lesions, the
so-called NAWM. So far, only few histological studies have
investigated the morphological changes that occur in the
NAWM. Allen and McKeown (1979) reported on an
increased gliosis, small round cell in®ltrates and an increased
number of macrophages. A recent detailed analysis of the
NAWM of a patient with acute multiple sclerosis described
axonal changes (Bjartmar et al., 2001). Abnormalities of the
NAWM can also be detected with magnetic resonance
techniques (for a review see Filippi et al., 1999b).
Numerous studies detected decreased NAA levels within
NAWM and therefore indicated an axonal loss or dysfunction
(Matthews et al., 1996; Davie et al., 1997; Schiepers et al.,
1997; Fu et al., 1998). This axonal loss seems to be caused by
Wallerian degeneration of axons transected in multiple
sclerosis lesions (Evangelou et al., 2000)
The pathogenesis of axonal damage is still a matter of
debate. MRS studies demonstrate a temporary decrease of
NAA level (Davie et al., 1994). This is either a consequence
of phenomena that are reversible in principle, such as
oedema, or indicates that axonal damage or dysfunction
measured by magnetic resonance techniques is at least partly
reversible. Whether the axonal damage that is observed by
APP staining is permanent or transient is still unclear
(Ferguson et al., 1997). There is a strong interaction between
axons and myelin sheaths. Myelin sheaths are essential for
maintaining the axonal cytoskeleton. Therefore, it is conceivable that demyelination makes axons vulnerable to
axonal damage. However, the occurrence of acute axonal
damage in the PPWM makes it unlikely that this pathogenetic
pathway is the only one. Other mechanisms such as
in¯ammatory mediators, cellular or antibody-mediated damage may be also responsible for axonal damage. A variety of
different in¯ammatory mediators can be found in multiple
sclerosis lesions and in PPWM. The most important are
tumour necrosis factor-a (TNF-a), interferon-g and nitric
oxide (NO) (Bitsch et al., 1998; Koeberle and Ball, 1999;
Silber and Sharief, 1999). Both TNF-a and interferon-g
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nated lesions. In our material, the extent of acute axonal
damage depends on disease duration and clinical course. The
highest number of APP-positive axons was detected in active
lesions of patients with a disease duration <1 year. To exclude
that the signi®cant higher accumulation of APP in group I is
the result of an unbalanced distribution of active lesions, we
compared the number of APP-positive axons in active lesions
of groups I±IV. Active lesions are de®ned by the presence of
macrophages with myelin debris within their cytoplasm. In
early active lesions, macrophages contain MOG- and MBPpositive debris, and in late active lesions only MBP. The
digestion of the minor myelin protein MOG lasts about a
week, whereas MBP is removed completely after 2±3 weeks
(Lassmann, 1983; BruÈck et al., 1995), indicating that the age
of active lesions is similar in all patients. In active lesions of
group I, a signi®cantly higher number of APP-positive axons
is found compared with groups II±IV, suggesting that acute
axonal damage is highest at the beginning of the disease and
does not depend on an unbalanced distribution of active
lesions or different lesion ages. One could argue that there
may be a selection bias of cases included in our study, since
brain biopsies form a major part of our study and these
patients might suffer from an atypical clinical course at the
beginning of the disease. However, in our case collection,
there are no patients with a very fulminant disease course
leading to early death (Type Marburg), and all biopsied
patients developed a classical chronic multiple sclerosis
during follow-up. Additionally, biopsies were included in all
groups (I±IV), thus largely excluding a selection bias. Our
®ndings suggest that an axon-protective treatment, if available, should start immediately after disease onset and should
be continued during the following years, especially in patients
with a relapsing±remitting disease course. This is in agreement with clinical studies that showed a bene®t of
immunomodulatory treatment with b-interferon starting
early after disease onset (Jacobs et al., 2000; Comi et al.,
2001). Treatment in later disease stages resulted only in a
limited bene®t [Secondary Progressive Ef®cacy Clinical Trial
of Recombinant Interferon-beta-1a in MS (SPECTRIMS)
Study Group, 2001]. According to our data, acute axonal
damage becomes less prominent in the chronic disease stage.
As acute axonal damage and in¯ammatory in®ltrates are
associated with each other, the reduced number of APPpositive axons may be due to the mild in¯ammatory reaction
in completely demyelinated or late remyelinating lesions.
In particular, patients with a relapsing±remitting disease
course demonstrate prominent acute axonal damage in early
disease stages, whereas the amount of acute axonal damage in
patients with PPMS is generally lower and does not change
signi®cantly over time. This is in line with earlier studies
from our group in which the lowest number of APP-positive
axons was also found in PPMS (Bitsch et al., 2000).
Additionally, in MRI, the hypointense T1 lesion load is
lowest in patients with PPMS (Filippi et al., 1999a; van
Walderveen et al., 2001). These results indicate that acute
axonal damage in the lesions may not be the only cause of
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T. Kuhlmann et al.
Acknowledgement
The present study was supported by a grant from the
GemeinnuÈtzige Hertie-Stiftung.
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stimulate the release of NO by astrocytes and macrophages.
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Received January 28, 2002. Revised March 11, 2002.
Accepted April 25, 2002
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