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The topograpy of demyelination and
neurodegeneration in the multiple sclerosis
brain
Lukas Haider,
1,2,
* Tobias Zrzavy,
1,
* Simon Hametner,
1
Romana Ho
¨ftberger,
3
Francesca Bagnato,
4
Gu
¨nther Grabner,
5
Siegfried Trattnig,
5
Sabine Pfeifenbring,
6
Wolfgang Bru
¨ck
6
and Hans Lassmann
1
*These authors contributed equally to this work.
Multiple sclerosis is a chronic inflammatory disease with primary demyelination and neurodegeneration in the central nervous
system. In our study we analysed demyelination and neurodegeneration in a large series of multiple sclerosis brains and provide a
map that displays the frequency of different brain areas to be affected by these processes. Demyelination in the cerebral cortex was
related to inflammatory infiltrates in the meninges, which was pronounced in invaginations of the brain surface (sulci) and possibly
promoted by low flow of the cerebrospinal fluid in these areas. Focal demyelinated lesions in the white matter occurred at sites
with high venous density and additionally accumulated in watershed areas of low arterial blood supply. Two different patterns of
neurodegeneration in the cortex were identified: oxidative injury of cortical neurons and retrograde neurodegeneration due to
axonal injury in the white matter. While oxidative injury was related to the inflammatory process in the meninges and pronounced
in actively demyelinating cortical lesions, retrograde degeneration was mainly related to demyelinated lesions and axonal loss in the
white matter. Our data show that accumulation of lesions and neurodegeneration in the multiple sclerosis brain does not affect all
brain regions equally and provides the pathological basis for the selection of brain areas for monitoring regional injury and
atrophy development in future magnetic resonance imaging studies.
1 Centre for Brain Research, Medical University of Vienna, Austria
2 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Austria
3 Institute of Neurology, Medical University of Vienna, Austria
4 Department of Neurology, Multiple Sclerosis Center, University of Vanderbilt, Nashville, TN, USA
5 High Field MR Centre, Medical University of Vienna, Austria
6 Department of Neuropathology, University Medical Centre Go
¨ttingen, Germany
Correspondence to: Prof. Dr Hans Lassmann
Centre for Brain Research,
Medical University of Vienna
Spitalgasse 4, A-1090 Wien,
Austria
E-mail: hans.lassmann@meduniwien.ac.at
Keywords: multiple sclerosis; demyelination; neurodegeneration; cerebral veins; cerebral arteries
doi:10.1093/brain/awv398 BRAIN 2016: Page 1 of 9 |1
Received September 1, 2015. Revised November 9, 2015. Accepted November 18, 2015.
ßThe Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
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Introduction
Multiple sclerosis is a chronic disease of the CNS, specifically
featured by inflammation, widespread primary demyelination
and progressive neurodegeneration. A widely held concept of
the pathogenesis of the disease is that tissue injury in the brain
and spinal cord is initiated by T cell-mediated inflammation
and that demyelination and neurodegeneration are driven by
heterogeneous mechanisms, involving both adaptive and
innate immune systems (Lassmann et al., 2007). We and
others recently proposed that microglia activation, production
of reactive oxygen species and oxidative damage are key
mechanisms driving demyelination and neurodegeneration,
particularly in the progressive disease stage (Haider et al.,
2011;Fischer et al., 2013). In addition, mitochondrial
injury (Mahad et al.,2008;Campbell et al., 2011)mayfur-
ther propagate oxygen radical production (Murphy, 2009)
and amplify demyelination and neurodegeneration by energy
deficiency through histotoxic hypoxia (Trapp and Stys, 2009;
Witte et al., 2010). With disease progression, the intensity of
the inflammatory response declines, but oxidative injury and
mitochondrial damage are aggravated by additional factors
relatedtoageingofthepatientsandtotheaccumulationof
disease and lesion burden (Mahad et al.,2015).
If this concept is valid, besides density of veins and men-
ingeal inflammatory infiltrates also arterial anatomy of the
brain should influence the topographical distribution of
demyelinated lesions and neurodegeneration within the
CNS due to varying basic levels of oxygen tension (Desai
et al., 2014). In our study we analysed predilection sites of
demyelination and neurodegeneration and how these relate
to arterial and venous anatomy and inflammation or de-
myelination in multiple sclerosis.
Materials and methods
Patient cohort
We performed our study on autopsy material from 51 patients
with multiple sclerosis and 38 age-matched controls without
neurological disease or focal brain lesions. The whole sample
included two cohorts. In the first cohort we analysed the topo-
graphical distribution of demyelinated lesions and neurodegen-
eration. This cohort consisted of hemispheric or double
hemispheric paraffin-embedded mid-thalamic sections from 19
multiple sclerosis cases and 20 age-matched controls
(Supplementary Tables 1 and 2). The cohort included two
cases with relapsing remitting multiple sclerosis, two cases with
primary progressive and 15 cases with secondary progressive
multiple sclerosis. All of these cases had long-lasting disease
(median age 68, range 44–90 years); disease duration (median
32, range 7.25–51 years). In the second cohort we analysed the
relation between inflammation, demyelination and neurodegen-
eration in routine sized sections. This cohort consisted of 46
cases and included 13 cases with acute multiple sclerosis
(Marburg, 1906), one case with relapsing remitting multiple
sclerosis, 13 cases with secondary progressive multiple sclerosis,
eight cases with primary progressive multiple sclerosis and one
case with subclinical multiple sclerosis, diagnosed through neuro-
pathology as well as 18 age-matched controls (Supplementary
Tables 3 and 4). The respective tissue blocks were selected
from an archival collection to provide sections with all types of
cortical multiple sclerosis lesions and with normal-appearing grey
matter as well as with subcortical or deep white matter lesions.
Neuropathological techniques and
immunohistochemistry
All sections were screened for inflammation, demyelination
and axonal damage in sections stained with haematoxylin/
eosin, Luxol Fast blue and Bielschowsky silver impregnation.
Cortical demyelinated lesions were identified in sections
stained by immunohistochemistry for proteolipid protein.
Iron accumulation within the brain was visualized by the
DAB-enhanced Turnbull blue staining (Hametner et al.,
2013). We screened for the presence of white matter or cortical
demyelinated lesions. Detailed lesion staging and lesion maps
were made for each tissue block.
Immunohistochemistry was performed on paraffin sections
using the primary antibodies and antigen retrieval techniques
listed in Supplementary Table 5. For demonstration of retro-
grade neuronal degeneration we stained the sections with anti-
bodies against phosphorylated neurofilaments (Koliatsos et al.,
1989). Neurons with oxidative injury were identified by the
presence of oxidized phospholipids within the neuronal cell
body and dendrites (Fischer et al., 2013).
Quantification for topographical
probability maps in hemispheric
sections: Sample 1
Demyelination and leukoaraiosis
Each demyelinated lesion in the brain of individual patients was
manually drawn in the patient’s own brain section map. Lesions
were identified by focal areas of myelin loss or sharply demar-
cated shadow plaques. All lesions identified in individual pa-
tients were then superimposed onto a virtual brain section of
thesamearea(Supplementary Fig. 1). The probability of a
lesion to occur in a specific brain area is provided by a colour
code (white means that no lesions were present in these loca-
tions, while dark red colour means that a lesion was present in
all 19 patients in this particular location; Fig. 1). Areas of leu-
koaraiosis in controls were identified in sections stained with
Luxol Fast blue. In a normal brain of young individuals only
diffuse differences in myelin density are seen between different
white matter areas (Supplementary Fig. 2A)incomparisonwith
the corpus callosum. Areas of leukoaraiosis were defined as
those with at least 30% reduction of myelin density in compari-
son to the myelin density in the corpus callosum, measured by
densitometry (Supplementary Fig. 2B).
SMI 31 positive neurons
Hemispheric sections were stained by immunocytochemistry for
phosphorylated neurofilaments. Neurons with signs of retro-
grade neurodegeneration were identified by their cytoplasmic
accumulation of phosphorylated neurofilaments, by their
enlarged cell body and the eccentric location of their nucleus
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Figure 1 Probability maps of multiple sclerosis patients. Probability maps of multiple sclerosis patients (A,Cand E) and age-matched
controls (B,Dand F). The colour codes indicate the probability of finding lesions (in % of cases Aand B), the cumulative frequencies of neurons
affected by retrograde neurodegeneration (C/D) and the cumulative frequencies of meningeal inflammatory cells (E/F) in a specific location of this
virtual brain slice. (A) The probability of demyelinating lesions in the white and grey matter of multiple sclerosis patients. (B) The probability of
leukoaraiosis in control subjects. (C) The cumulative number of neurons with retrograde degeneration in multiple sclerosis patients. (E) The
accumulated number of inflammatory cells in the leptomeninges of multiple sclerosis patients. The highest incidence of demyelination (A) in the
white matter is seen in the so-called watershed areas, which are located at the borders of the supply territories of the major cerebral arteries. In
contrast, cortical lesions are mainly concentrated in invaginations of the cortical surface, such as the cortical sulci, in regions with high incidence of
meningeal inflammatory infiltrates (E). Retrograde neurodegeneration is mainly seen in the deep cortical layers and the deep grey matter and in
part follows the putative fibre projection from white matter lesions into the cortex (C). In age-matched controls no plaques of primary
demyelination were present, but there were areas of diffuse white matter alterations (leukoaraiosis) in 43% of the cases investigated (blue areas in
B). Cortical neurodegeneration was much less pronounced compared to that seen in multiple sclerosis (D), but showed a topographically similar
distribution compared to that seen in multiple sclerosis patients. Inflammatory infiltrates were also seen in low numbers and incidence in the
meninges of the age-matched controls (F). Definitions of regions of interest: (G) venous density atlas from Grabner et al. (2014) depicts the
density of veins (red) in different brain areas. Brains were scanned in a 7 T MRI and a venous map of the normal human brain was created. (H)
Turnbull staining showing the iron distribution throughout the brain. (I) Our regions of interest in one hemisphere of the virtual brain map. Areas
of low CSF flow are indicated by blue, watershed area in purple and the basal ganglia in pink.
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(Fig. 2). The entire grey matter of the individual section was
screened by superimposing a grid of 5 mm
2
. Each cortical area
represented in the outlines of the grid was given four different
scores: Score 0: no positive neurons; score 1: 1–10 positive
neurons; score 2: 11–40 positive neurons and score 3: 440
positive neurons. The respective scores were inserted into the
map of the representative section in their exact location in the
grey matter. Having determined these values for each individual
patient, the results for all patients were superimposed into the
respective areas of a single virtual brain section. We then deter-
mined the average number of positive neurons for the individual
scores and multiplied the incidence of the scores in each given
brain area with this average number. This provided a summary
score of immune-reactive neurons for each cortical region,
derived from all patients included in this investigation. The sum-
mary scores were then graphically transferred into different col-
ours from light yellow to dark red, as depicted in Fig. 1.The
scales in the figure show the summative number of neurons with
SMI31 reactivity in a given cortical region (from 0 to 450).
Note that the numbers provided here are closely similar to the
numbers given from the small tissue block analysis in Fig. 2 (e.g.
average number of positive cells in highly affected regions of
4.7), considering the larger size of the grid (5 mm
2
)andthe
summation of 19 patients (4.7 519 = 446).
Meningeal inflammation
To determine meningeal inflammation in essence we used the
method described by Magliozzi et al. (2007) and Howell et al.
(2011). The presence of leucocytes was analysed in haematoxylin
and eosin stained sections in the microscope at an objective mag-
nification of 20, resulting in the determination of leucocyte
infiltrates in meningeal stretches of 0
.
23 mm per microscope
field. Inflammation was defined by the following scores: score
0: 55 leucocytes; score 1: 5–50 leucocytes and score 3: 450
leucocytes. Transformation of the data from individual patients
into the virtual lesion map followed the same principles as
described above for SMI31 positive neurons. The colour code
represents the summation of the numbers of leucocytes/0
.
23
mm
2
seen in the entire sample of 19 patients. When calculated
on the basis of single patients, the values described in our present
study are similar to those reported by Howell and co-authors
(2011) and in our previous study based on quantification of T
cells, B cells and plasma cells by immunocytochemistry (Frischer
et al., 2009). A much lower extent of meningeal inflammation
was reported previously in the study by Kutzelnigg et al. (2005).
In this study, however, meningeal inflammation was assessed by
the presence of large inflammatory infiltrates, representing men-
ingeal follicle-like aggregates as defined by Magliozzi et al.
(2007). Examples of meningeal inflammatory infiltration, as
determined in this study, are provided in Supplementary Fig. 3.
Collation of the lesion map
Manually quantified counts from the microscope were plotted
into patients’ individual scans with Adobe Photoshop
TM
.Inthe
next step these results were manually transposed from the indi-
vidual scans to an average brain (Supplementary Fig. 1). These
average brain data were then quantified with ImageJ at a reso-
lution of 10000 pixels/image and imported into IBM SPSS
TM
.
The raw results were then corrected for the area that the regions
of interest covered on each individual patient’s Klu
¨ver-Pas scan,
to control for variation of section level within the mid-thalamus
and interindividual variation. Venous density was determined in
a 7 T MRI venous atlas of the normal human brain (Fig. 1G)
(Grabner et al., 2014), based on venous-sensitive susceptibility
weighted imaging. A corridor of 1 cm adjacent to border
zones of the anterior-, medial-, posterior- cerebral and anterior
choroidal arterial territory was considered as watershed area
(Fig. 1I) (van der Zwan and Hillen, 1991). Iron content in dif-
ferent brain regions was depicted by Turnbull staining (Fig. 1H)
(Hametner et al.,2013). Structures in the fissura longitudinalis
cerebri, in the sulcus lateralis and individual sulci were con-
sidered as areas with low CSF flow (Fig. 1I).
Quantification of retrograde neuro-
degeneration and oxidative injury in
standard tissue sections: Sample 2
To determine the relation between oxidative neuronal injury or
retrograde neuronal degeneration with demyelination and menin-
geal inflammation, we analysed sections from small tissue blocks,
counted the number of neurons with immunoreactivity for oxi-
dized phospholipids (E06) or phosphorylated neurofilament. The
immunostained sections were overlaid by a morphometric grid (a
´
0
.
0576 mm
2
). Regions of interest were: normal-appearing grey
matter (in controls/multiple sclerosis), leucocortical lesions (ex-
tending over the cortex-white matter border), subpial lesions
and normal-appearing cortex adjacent to large subcortical
white matter lesions. The activity of cortical lesions was charac-
terized by the presence of PLP-reactive myelin degradation prod-
ucts in macrophages or microglia (Fischer et al., 2013). Neuronal
cells were defined by nuclear morphology in the haematoxylin
counterstaining. Neurons with phosphorylated neurofilament-re-
active somata were manually counted in 10 fields per region of
interest. Neurons with signs of E06 reactivity were divided into
three groups according to Fischer et al. (2013) (Fig. 2). Average
counts per square millimetre were calculated for each region of
interest per case and compared by statistical analysis.
Statistical analysis
Statistical analysis was performed with IBM SPSS
TM
. Due to
uneven distribution of our data, non-parametric tests were
applied. Descriptive statistics included median value and
range. Differences between two groups were assessed with
Wilcoxon Mann Whitney U-tests. In case of multiple testing
(comparison of more than two groups), significant values were
corrected with the Bonferroni procedure. Interdependence of
variables was evaluated by the Spearman non-parametric cor-
relation test. The reported P-values are results of two-sided
tests. A P-value 40
.
05 was considered statistically significant.
Results
Topographical distribution of
demyelinated lesions in the multiple
sclerosis brain
Demyelinated lesions in the cerebral cortex
As reported previously, cortical demyelination was domi-
nated by widespread subpial lesions (Bo et al., 2003;
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Figure 2 Neurodegeneration in the multiple sclerosis cortex. Oxidative injury in cortical neurons is reflected by two different neuronal
changes. (A–D) The first is the intense and diffuse immunoreactivity for oxidized phospholipids (E06 immunoreactivity) in neurons (B), associated
with beading and fragmentation of cell processes (indicated by the arrowhead) and neuronal apoptosis (Fischer et al., 2013). This is mainly seen in
active cortical lesions, but in lower frequency also in inactive lesions and the normal-appearing multiple sclerosis cortex (grey bars in A). The
second is reflected by a granular, lipofuscin-like immunoreactivity in neurons (C). This is significantly more frequent (white bars in A) in the
multiple sclerosis cortex compared to controls, but within multiple sclerosis its incidence is not significantly different between active and inactive
lesions. (D) A normal neuron without immunoreactivity for oxidized phospholipids in the multiple sclerosis cortex. (E–H) Retrograde neuro-
degeneration is reflected by the cytoplasmic accumulation of phosphorylated neurofilaments (F). This is frequently associated with ballooning of
neurons and an eccentric location of the nerve cell nucleus (Gand H). Retrograde neurodegeneration is most prominently seen in the cortex
adjacent to demyelinated lesions with axonal transection in the white matter (E). *P50.05; **P50.01; ***P50.001.
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Kutzelnigg et al., 2005), while intracortical and cortico-sub-
cortical lesions were less frequent (Fig. 1A). Demyelinated
lesions were seen along the entire cortical ribbon, but their
incidence and size were significantly larger in cortical sulci,
and in deep invaginations of the brain surface, such as
the cingulate gyrus and in the insular cortex (Fig. 1A and
Table 1). The topographical distribution of meningeal in-
flammatory infiltrates, which were increased in cerebral
sulci and in deep invaginations of the brain surface
(Supplementary Fig. 3), was significantly associated with
subpial cortical demyelination (R = 0.646; P= 0.003) (Fig.
1A and E).
Demyelinated lesions in the white matter
The highest frequency of lesions was seen in the periven-
tricular white matter, but in addition the lesions accumu-
lated in a fan-shaped pattern expanding from the
periventricular region towards the deep and juxta-cortical
white matter (Fig. 1A). As it can be seen from the venous
density map of the brain (Fig. 1G), areas with a high
venous density are likely to harbour demyelinated white
matter lesions, but not all brain areas with high venous
density are equally affected. However, precipitation of
focal white matter lesions occurs in the border areas of
the territories of blood supply from the major cerebral
arteries (the so-called watershed areas) (Fig. 1A and I;
Table 1).
No focal demyelinated lesions were observed in the white
or grey matter of control patients. However, 43% of con-
trol patients showed areas of variable size with diffuse re-
duction of myelin, representing leukoaraiosis or small
vessel disease of the white matter, which is typically present
in a subset of aged control patients (Fig. 1B and
Supplementary Fig. 2).
Oxidative injury in cortical neurons
In our study we distinguished between two different pat-
terns of neurodegeneration. The first was oxidative injury,
reflected by the cytoplasmic accumulation of oxidized
phospholipids (Fig. 2A–D). They were either present in
granular form, resembling lipofuscin granules (Fig. 2C),
or in a diffuse distribution within the entire cytoplasm of
neurons and dendrites. The latter was associated with den-
dritic beading and fragmentation, suggesting active neuro-
degeneration in the course of oxidative stress (Fig. 2B). The
highest incidence of neurons with diffuse cytoplasmic accu-
mulation of oxidized phospholipids was seen in actively
demyelinating cortical lesions (Fig. 2A), characterized by
the presence of abundant meningeal inflammation, micro-
glia activation at the lesion edges and the presence of
macrophages with early myelin degradation products at
sites of active demyelination in the cortex (Fischer et al.,
2013). In addition, we found neurons with diffuse or
granular accumulation of oxidized phospholipids also in
inactive cortical lesions, irrespective of their type and inde-
pendent from the presence of subcortical demyelinated le-
sions (Fig. 2A). Neurons with accumulation of oxidized
phospholipids were also present in the normal appearing
grey matter of multiple sclerosis patients (Fig. 2A). The
incidence of neurons with oxidative injury was significantly
higher in the multiple sclerosis cortex in comparison to
controls and correlated with inflammation in the meninges,
covering the respective cortical areas (R = 0.469;
P= 0.008).
Table 1 Quantitative evaluation of demyelination and neurodegeneration in multiple sclerosis and control brains in
relation to watershed areas and cortical invaginations
Watershed Non-watershed Invaginations Surface
MS cortex DM 15.5 8.2 17.3 5.5
(3.4–71.3) (2–30) (4.9–49.5) (2.7–27.3)
MS cortex ND 43 820.3 2.3
(16.3–51.1) (4.7–12.1) (12.4–33.4) (1.4–3.6)
MS WM DM 31.3 12.1 n.a. n.a.
(18.4–62) (4.9–22.6)
MS DGM DM 25 11.7 n.a. n.a.
(11.6–55.8) (7.4–16.5)
MS DGM ND 83.7 11 n.a. n.a.
(28.9–115.8) (4.3–24.3)
CO cortex ND 0.42 0.14 0.7 0
(0–3.1) (0–1.6) (0–3.4) (0–0.3)
CO DGM ND 0 0 n.a. n.a.
(0–1.6) (0–0.4)
Demyelination and neurodegeneration are significantly more pronounced (bold numbers in the table) in watershed areas and within the cortex in invaginations of the brain surface, as
defined in Fig. 1I.
Demyelination is expressed as the number of pixels with demyelination in a region of interest, divided by the total area of this region of interest of the same patient.
Neurodegeneration is expressed as the number of pixels with retrograde neurodegeneration (correcting for the levels low, intermediate and severe), divided by the total grey matter
area of the same patient.
All P-values result of a two-sided Man-Whitney U-test and were subjected to Bonferroni correction for multiple testing.
MS = multiple sclerosis; CO = controls; WM = white matter; DGM = deep grey matter (basal ganglia, thalamus, hypothalamus); DM = demyelination; ND = retrograde
neurodegeneration.
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Retrograde neurodegeneration in
cortex and deep grey matter nuclei
The second pattern of neuronal pathology represents retro-
grade neurodegeneration (Fig. 2E–H). When axons are
transected, their associated neuronal cell bodies react with
a morphological change called central chromatolysis. This
process is characterized by the accumulation of phosphory-
lated neurofilament protein within the perinuclear and den-
dritic neuronal cytoplasm, an increase in cytoplasmic
volume and an eccentric dislocation of the neuronal nucleus
(Fig. 2F–H) (Koliatsos et al., 1989;Martin et al., 1999).
Overall, we found an increased number of neurons with
retrograde degeneration in the multiple sclerosis cortex in
comparison to the cortex of age-matched controls (Fig. 2E).
The highest incidence of neurons with cytoplasmic accumu-
lation of phosphorylated neurofilaments was present within
cortico-subcortical lesions and in normal-appearing cortex
adjacent to subcortical white matter plaques. In contrast,
only very few neurons with central chromatolysis were seen
in subpial lesions (Fig. 2E).
We then used a similar approach in entire double hemi-
spheric brain sections to evaluate the global incidence of
retrograde degeneration in the brain (Fig. 1C). The highest
incidence of neurons with cytoplasmic accumulation of
phosphorylated neurofilaments was present in deep grey
matter nuclei (mainly in the thalamus and the globus pal-
lidus) and in the depth of cortical sulci, mainly affecting
neurons in the fifth and sixth cortical layer. Neurons with
signs of retrograde degeneration were topographically
related to the location of demyelinated lesions in the
white matter (Fig. 1A and C) and, thus, also accumulated
in watershed areas (Table 1). In addition, we found a much
higher incidence of neurons with retrograde neurodegenera-
tion in cortical sulci compared to cortical gyri (Fig. 1C and
Table 1) and a substantial number of these neurons also
contained oxidized phospholipids in their cytoplasm
(Fischer et al., 2013).
In comparison to age-matched controls, we found a sig-
nificantly increased incidence of neurons with retrograde
degeneration in multiple sclerosis patients, but the topo-
graphical distribution of affected neurons was similar in
controls compared to that seen in multiple sclerosis patients
(Fig. 1C and D). In controls, retrograde neurodegeneration
was topographically related to diffuse periventricular white
matter abnormalities (Fig. 1B and D).
Discussion
Our data on lesion topography in the multiple sclerosis
brain suggest that several different factors contribute to
their formation (Mahad et al., 2015). In line with current
concepts, inflammation, microglia activation, oxidative
injury and energy deficiency due to mitochondrial damage
may be key factors, but their relative contribution may
differ depending upon type and location of the lesions.
Earliest studies on multiple sclerosis pathology have al-
ready established that white matter lesions are formed
around inflamed veins (Rindfleisch, 1863) and this is con-
firmed by recent studies using high field MRI (Tallantyre
et al., 2008). Similarly, cortical lesions are associated with
perivenous inflammation in early and aggressive multiple
sclerosis cases (Lucchinetti et al., 2011) and active subpial
demyelination was found to be related to inflammation in
meninges (Kutzelnigg et al., 2005;Howell et al., 2011;
Choi et al., 2012) and were most frequent in invaginations
of the brain surface (Kutzelnigg and Lassmann, 2006).
Their location, shape and patterns of active demyelination
are compatible with the view that they are driven by a
soluble factor, produced in the inflammatory infiltrates of
the meninges, which diffuses into the cortex and triggers
demyelination either directly or indirectly through micro-
glia activation. Demyelinating and/or cytotoxic activity
has been observed in the CSF of multiple sclerosis patients
and has been ascribed to specific autoantibodies or other
cytotoxic molecules (Lisak et al., 2012;Vidaurre et al.,
2014). In addition, essentially similar cortical lesions have
been induced in experimental models in rats and primates,
associated with meningeal inflammation and the presence
of demyelinating antibodies directed against myelin oligo-
dendrocyte glycoprotein (Pomeroy et al., 2005;Storch
et al., 2006). There is a flow of CSF within the arachnoid
compartment of the meninges at the outer surface of the
brain hemispheres (Abbott, 2004), which is likely to be
dynamically restricted in the cerebral sulci and deep inva-
ginations of the brain surface in the insular and cingulate
cortex. This may explain the preferential accumulation of
inflammation and subpial cortical demyelination at these
sites.
Recent data suggest that oxidative injury at least in part
mediates demyelination and neurodegeneration in the mul-
tiple sclerosis brain. Oxidative injury can trigger mitochon-
drial dysfunction and subsequent energy failure, a process
termed histotoxic or ‘virtual’ hypoxia (Aboul-Enein et al.,
2003;Trapp and Stys, 2009). This process may be ampli-
fied by genuine hypoxia through increased energy con-
sumption around inflamed vessels, and the consequence
of energy failure is amplified in areas with low arterial
perfusion and oxygen supply (Davies et al., 2013). This
may explain the accumulation of lesions in the multiple
sclerosis brain in the so-called watershed areas (Brownell
and Hughes, 1962). Our findings are in parallel with pre-
vious in vivo imaging studies showing that in patients with
SPMS, lesions tend to accumulate in areas of low perfusion,
while this is not seen in early disease stages (Holland et al.,
2012). These data indicate that in early multiple sclerosis
lesions are formed at any site of the brain, but that some of
these early lesions disappear due to resolution of oedema
and remyelination, while those located in areas of low
blood perfusion persist due to the higher degree of tissue
damage (Holland et al., 2012). In addition, oxidative injury
and mitochondrial damage is amplified in the progressive
stages of the disease by factors unrelated to the
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inflammatory process, such as the age and disease burden-
related microglia activation and mitochondrial gene dele-
tions or accumulation of iron in the ageing human brain
(Mahad et al., 2015). Thus, low blood perfusion and
oxygen tension may amplify tissue damage more severely
in lesions formed in the progressive stage than in those
arising at earlier phases of the disease. However, different
patterns of tissue injury have been seen in patients with
early (acute) multiple sclerosis (Lucchinetti et al., 2000)
and one of those is associated with profound oxidative
and mitochondrial injury (Aboul-Enein et al., 2003;
Mahad et al., 2008). It is likely that in such cases a similar
relation between brain blood perfusion and lesion topog-
raphy is present, but so far the low number of autopsy
cases available for pathological analysis precluded a sys-
tematic comparison of lesion location between these cases
and those following different patterns of demyelination.
Regarding neurodegeneration, we identified two different
patterns in the multiple sclerosis brain. Acute nerve cell
injury, characterized by dendritic and axonal fragmentation
and cell changes of apoptosis or necrosis, was associated
with accumulation of oxidized lipids and DNA in affected
cells (Fischer et al., 2013). In our study this was mainly
seen in actively demyelinating lesions, less frequently in in-
active cortical lesions and rarely in the normal appearing
cortex. In addition, we found profound accumulation of
lipofuscin-like granules, which mainly contain oxidized
lipids and proteins (Ho
¨hn and Grune, 2013), within neu-
rons and glia in the entire multiple sclerosis cortex. The
significant correlation between the number of cortical neu-
rons with accumulation of oxidized lipids and meningeal
inflammation indicates that this type of neurodegeneration
may be in part driven by oxidative stress mediated by acti-
vated macrophages and microglia.
The second process of neurodegeneration in the multiple
sclerosis brain revealed cellular changes which are typical
for neurons after axonal transection (Koliatsos et al., 1989;
Martin et al., 1999). They were mainly present in the
cortex overlying subcortical demyelinated lesions, while
they were rare within intracortical subpial lesions.
Furthermore, their global distribution in the brain suggests
that neurons with signs of retrograde degeneration were
most frequent in cortical areas, which are topographically
related to areas with a high probability to harbour white
matter lesions (Kolasinski et al., 2012). The low incidence
of such changes in subpial cortical lesions can be explained
by the low incidence of axonal transection in comparison
to that in white matter lesions (Frischer et al., 2009) and by
the parallel loss of neurons and axons within these lesions
(Schmierer et al., 2010;Klaver et al., 2015). Interestingly, a
similar topographical distribution of neurons with retro-
grade degeneration, although much less numerous, was pre-
sent in the cortex of controls. Aged controls often show
diffuse periventricular white matter damage (leukoaraiosis),
which is associated with axonal transection (Brown and
Thore, 2011). Thus, periventricular white matter damage
in patients with progressive multiple sclerosis is not only
due to axonal transection in plaques, but also to age-
related injury. It is currently not possible to differentiate
the contribution of each of these mechanisms to the
global extent of damage in the periventricular normal-ap-
pearing white matter. It has, however, to be noted that the
white matter injury in leukoarayosis also occurs at sites of
low physiological blood perfusion in the brain and appears
to be driven by age-related pathological changes in the long
penetrating arteries providing the blood supply in these
areas (Brown and Thore, 2011).
Neurons with retrograde degeneration tended to be
located in the deeper cortical layers (layers 5 and 6), and
they were mainly present in cortical sulci. We have shown
previously that neurons with signs of retrograde degener-
ation frequently contained high amounts of oxidized
phospholipids (Fischer et al., 2013) and neurons with mito-
chondrial gene deletions are also predominantly seen in the
same cortical layers (Campbell et al., 2011). Retrograde
neurodegeneration is accomplished by apoptosis and asso-
ciated with oxidative damage of neuronal proteins and nu-
cleic acids (Martin et al., 1999). In addition, it has been
suggested that oxidative injury in white matter plaques
leads to mitochondrial gene deletions and that the defective
mitochondria are transported in a retrograde manner into
the cell body of cortical neurons. This process may further
amplify oxidative injury by electron leakage from defective
mitochondria (Mahad et al., 2015).
Our results may have consequences for MRI studies
focusing on brain atrophy. Brain atrophy is thought to be
a useful paraclinical outcome measure for the evaluation of
the effect of neuroprotective treatments (Filippi et al.,
2014). Careful analysis of regional atrophy in the brain
may further improve the diagnostic and predictive value
of MRI. Our current study may provide the neuropatho-
logical basis for regional stratification of atrophy analysis.
Acknowledgements
We thank Marianne Leiszer and Ulrike Ko
¨ck for expert
technical assistance.
Tissue samples and associated clinical and neuropatho-
logical data were supplied by the Multiple Sclerosis
Society Tissue Bank.
Funding
The study was funded by the Austrian Science Fund
(Project: P 24254 B19 and I 2114-B27 MELTRA-BBB).
Funded by the Multiple Sclerosis Society of Great Britain
and Northern Ireland, registered charity 207495.
Supplementary material
Supplementary material is available at Brain online.
8|BRAIN 2016: Page 8 of 9 L. Haider et al.
by guest on February 10, 2016http://brain.oxfordjournals.org/Downloaded from
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