Hello everybody, welcome to my iBiology presentation.
This is part 2 of a series on multiple sclerosis and remyelination.
The part 1 presentation is given by professor Mikael Simons from
the Technical University of Munich. I'm Christine Stadelmann from the
Institute of Neuropathology in Gottingen, and I'm now in the following
presentation going to talk about multiple sclerosis pathology and
also all the prospects that we see for remyelination.
MS is a disease that affects approximately 2.5 million people
worldwide. It is a disease that normally starts in young adulthood.
And that is quite the complex pathogenesis. Now it is believed
that there is a genetic predisposition for the disease, but that
it is complimented by environmental factors. These factors
together lead to an aberrant or destructive immune response
that targets the central nervous system. With regards to the
genetic predisposition, we know now that polymorphisms in
immune regulatory genes are relevant for the predisposition
to get the disease. On the other hand, environmental factors
such as EBV, vitamin D, and smoking affect the risk of attracting
-- or contracting the disease. With regard to the disease course, MS
is quite special. In that it basically changes its face. At the beginning,
MS normally presents as a relapsing remitting disease with almost
full recovery between the relapses. However, after several years
of disease, many patients, around 70%, convert into a secondary progressive
disease phase where this ability accumulation basically occurs
without superimposed relapses. So basically, without seemingly
peripheral immune activation. On the contrary, brain volume changes
occur basically right from the very start. And that's with reduction
in brain, and the decrease may be a real issue with regards to the
development of the secondary progressive disease phase.
What about the pathology of multiple sclerosis? Here what you see
is a fixed brain section with the very prominent periventricular
brain lesions that you see here. And these grayish areas
are very prominent, very clear, you see them on both sides
here. On both hemispheres of the brain. And these represent
fully demyelinated multiple sclerosis lesions. Apart from this
periventricular area, other predilection sites are the optic nerves,
the subpial cortical area that I will come to a bit later, the spinal
cord, the brainstem, and also the cerebellum.
What are now the key features of MS pathology?
One the one hand, clearly the focal demyelinated lesions
that we will discuss in depth in the next minute, but also the
diffuse brain pathology seems to be more and more important.
Especially with increased disease duration. And this includes
meningeal and parenchymal T cell infiltration, diffuse
microglia activation, neuroaxonal damage and loss, and also
then brain atrophy that is probably the cause of all those.
So in this presentation, I'll mainly focus on the focal lesion
pathology that is observed in MS. This is a typical chronic
demyelinated lesion, you see the myelin in blue, you see the
demyelinated lesion in rose, you also see that this lesion is
periventricular localized, so very characteristic for an established
MS lesion. You see a very sharp lesion edge. You see basically
that the lesion is hypocellular in the lesion center, and you don't
see accumulation of cells at the lesion edge. So this
seemingly is more or less a scar with very little or no
ongoing disease activity. Quite different from what I've shown you before,
is this chronic active or so called "smoldering" lesion. There  just
by almost bare eye in this slide here, you see that there is an
excentration of cells here at the lesion border around this
lesion area here. And if you do some immunohistochemistry,
and look at the slide in more detail, you see that there is
accumulation basically of myeloid cells. Of macrophages and
activated microglia. And what is also very important, nuclear
at the lesion center, there practically no microglia activation. Also no
phagocyte recruitment is found. This microglia activation, phagocyte
recruitment, myeloid cell activation, is very much accompanied by
acute axonal damage. And the image that you see here is right
from this lesion border. So from this smoldering lesion border,
with apparently some residual demyelinated activity.
And what you really look at in this APP immunohistochemistry
is disturbance of axonal transport, so you look at accumulated
APP here in the axons. And what we know from animal studies
and studies in brain trauma, that this APP protein stays there for
around three weeks. So basically, this means that these axons
have been damaged or demyelinated within the last three weeks.
Or it means that they are persistently functionally impaired.
And all this is of course really relevant for our understanding of
progressive disease. And this is just a schematic illustration
of this smoldering lesion activity. Basically, that is a hallmark
really of multiple sclerosis and is the most common lesion type
observed in patients with progressive disease. What about the
very early lesion? I want to say that one rarely has the chance
to even see early lesions because a regular typical MS
patient does not undergo brain biopsy, of course. So you
really see that only under the certain circumstances, especially when
the clinical presentation or the MRI presentation was not
entirely clear. What you see here is the same staining as before,
with PAS histochemistry, and again the myelin in blue. And again the
lesion is rose. You see a hypercellularity there, you see all this
hypercellularity is even increased at the lesion edge. And if you
do some immunohistochemistry and you look at the macrophage
and all the activated microglia, you again see clearly accentuated
lesion edge here. Again, speaking for the centrifugal lesion evolution
basically. But of course here in this quite early lesion, you see
also that the lesion is filled with macrophages in the lesion center.
With regards to astrocytes, you may see beginning astrocyte
reaction, reactive astrogliosis in the lesion itself, as well as at the
lesion edge. And this is quite important with regard to the animal
model lesion that I'm going to show you in a minute. With regards
to T cell infiltration, this may be very scarce in this early lesion
that I've just shown you. If you look here, this is CD8-positive
cells that we're looking at here. If you look at the CD3s, you might have
about twice the number that I'm showing here. So quite scarce.
But getting a bit more with lesion evolution. With regard to
our final goal in a lesion remyelination, of course the assessment of
mature oligodendrocytes, oligodendrocyte precursors, is very relevant
in the lesions. And this is for example here, a staining immunohistochemistry
for NogoA, for mature oligodendrocytes. And as it looks, the mature
oligodendrocytes do not seem to be very much impaired here by this
lesion formation. You see a bit of a reduction here in the active
lesion edge, but not very much so. It even seems that the protein
is upregulated in the lesion, compared to the clear white matter.
What we very much like to do in our research, but also in within clinical
practice, is to really stage our demyelinated lesions with regards
to demyelinated activity. To be able to compare lesions between patients
and also lesions between different diseases. To compare basically
equal stages of lesion formation. What we use for the purposes
in the one hand, the presence of myelin degradation products in
macrophages. We use major and minor myelin proteins and with the
concept that the major myelin proteins need longer to be
digested. So if you find still minor myelin proteins, such as MOGs,
cAMP, MAG, in the macrophages. This basically indicates
a more recent lesion. What also proves very, very useful
is some macrophage activation markers, such as MRP14.
That really highlight recently invaded monocytes from the blood
stream. So it's a very useful marker used for lesion age determination
in our setting. When do which lesions occur? When are they
most prominent? And there I think what is very important for you is
that on the one hand, the green lesions that are depicted here,
these are the actively demyelinating lesions. They occur in
monophasic disease, they occur in relapsing remitting disease,
they also occur still a bit in the more chronic disease forms, such
as a secondary progressive and primary progressive MS.
But in these later lesion stages, it's really the chronic
smoldering or the chronic active lesions that predominate
also of course the inactive, so called burned out lesions. And
also in part, the shadow plaques. And the shadow plaques are
here really indicated in rose. So just for comparison, my plan here is
to also show you a bit at least of NMO lesion pathology.
To have a comparison with MS and to look at the myelin
pathology that may be different in this clinical setting. So these
are really spinal cord sections from a neuromyelitis optica patient.
And just by looking here at the LFB/PAS and also the macrophage
immunohistochemistry, you see a huge lesion in the dorsal
funiculus and also of the ventrolateral spinal cord area here.
And just from looking at the macrophage staining, you might
say that from the density that you see, that the ventrolateral
lesion is much younger, much more recent. Importantly,
in 2004, Vanda Lennon has demonstrated that NMO,
neuromyelitis optica, is not a spectrum disease like multiple sclerosis
or a variant of multiple sclerosis, but really a completely different disease
entity. And that she demonstrated that anti-AQP4 antibodies
are really a clear serum signature of the disease. Neuromyelitis
optica is basically a disease characterized by spinal and optical
nerve involvement. And of course, not all patients with these
clinical signs and symptoms have anti-AQP4 antibodies.
But those who have, are designated then anti-AQP4 autoimmunity
and if you perform an indirect immunofluorescence assay in
these patients. For example on their cerebellar section, you would
see exactly what I show you here on this side. That is, you would
see a clear and very nice and fine delineation of the pial surface
on the one hand, and of the capillaries, as seen here on this right
brain section. Indicating that with the anti-APQ4 antibodies, you basically
label the astrocyte food processors that are in the brain capillaries.
Now knowing that basically an anti-astrocyte immune response is
causative of NMO and looking at the lesion morphology that you see
in the brain, you would probably expect a completely different
type of pathology. However, looking at the lesion here on the LFB
PAS histochemistry, you might really get the diagnosis wrong
and diagnose an MS lesion. If you don't look carefully enough.
However, going a bit deeper and looking at astrocytes, for example,
using GFAP immunohistochemistry. You see that astrocytes are not
usually destroyed in the lesion center, towards the lower right.
They are preserved at the upper left border here, where the
peri plaque white matter is situated. Going then further and really
staining for AQP4 itself, you see that this immunoreactivity is even
further decreased, compared to the structure protein GFAP.
Again, as an evidence that AQP4 antibodies here play a role in the
disease. If you then look at oligodendrocytes or mature and
oligodendrocyte precursor cells in the lesions using NogoA and
Olig2 as markers, as we did here. You see that oligodendrocytes
are largely absent from these acute lesions, completely destroyed
due to early lesion forming process in anti-APQ4 positive
neuromyelitis optica spectrum disorder. Looking then at the myelin
protein pathology a bit more closely, you see that major myelin
proteins, such as MBP and also PAP, are largely preserved
in these early lesions. And you would not be easily diagnosed
by demyelination here. Whereas if you look at MAG or CMP,
you'll find a total decrease or loss of these myelin proteins in the lesion.
So very indicative here of distinct pathological demyelinated process
going on. And what one might conclude from that, that there are at least
two principle mechanisms of demyelination. On the one hand,
a mechanism whereby the oligodendrocyte suffers from a primary
damage from oligodendroglial cell death. And the myelin sheath then
degenerates secondarily. And on the other hand, disease
settings of pathological circumstances, whether primary
target of the immune response is the myelin sheath. And
either is then removed by the macrophages concomitantly with the
oligodendrocyte. Or even the oligodendrocytes appear preserved
to a certain extent. In the next part, I would very much like
to come a bit back to what Mika has already discussed,
namely disease progression in multiple sclerosis.
And certainly, this is one of the characteristic features of the
disease. And again, as I said already, very special and so far,
also unresolved puzzle. And for decades now, neuropathologists
have tried to really establish the correlation of progressive disease
in their materials. What is very prominent in late stage MS patients is
certainly cortical pathology. And there especially, subpial cortical
demyelination, as delineated here with the arrows. Very characteristic
for chronic disease, very widespread in chronic patients. And
of course, as you may know, not detected by MR imaging.
So really, escaping our clinical pathological correlations
normally. Importantly, however, what should be mentioned is that
cortical demyelination is not only a feature of late stage
disease, but also occurs early in disease and may even
be present really right at the first disease bout, basically.
What may really contribute to the importance in chronic disease is
however, that ongoing remyelination that is very efficient in the
cortex may decrease with disease duration. And thus, leave
completely demyelinated subpial cortical areas that are of course
then very easy to visualize as seen here in this image.
This slide really serves two purposes. So the right hand scheme on
the one side, shows you the discrepancy between white matter
demyelination in green, and here mainly periventricular, as shown
here. And cortical demyelination shown in orange here, that really
outnumbers the volume of white matter demyelination here
by far. On the other hand, what is also easily visible on this
frontal brain section, and mainly seen on the left hand scheme here, is the
important brain atrophy that is found in quite a proportion of chronic
MS patients that also already starts early in the disease. And
maybe, a very first sign even. There I'm thinking mainly about
the MRI data on cortical atrophy, as in early sign of patients
that really, really convert into secondary progressive disease.
What is really underlying features that contribute to this atrophy
of the MS brain? And mainly to the cortical atrophy. And there
during the last years, really the notion appeared in neuropathology
that it's not really that neuropathological damage or damage to
neurons and axons. It's not only related to focal lesions. And does not
even correlate well with focal lesions, but rather is a diffuse
phenomenon. And there is work by Doron Merkler that very much
contributes. Where they show a spine loss in multiple sclerosis
cortex, independent of focal demyelination. What could now
remyelination add to our picture of multiple sclerosis?
Remyelination in MS has been discussed for decades.
And especially prominent was John Prineas delineating
the morphology of remyelination in the MS brain, as shown here
in this Annuals of Neurology paper, which very nicely showed
the axons with a thin myelin sheath that indicate remyelination
here in this MS plaque. To the left hand side, what you really see
is a fully remyelinated shadow plaque. And I think this is really
the goal, this is where we would like to go. This is what we
would like to achieve for all the lesions, for all of our patients.
So have them fully remyelinated after a certain time.
Is that goal that is useful in any way? And Mikael has already
discussed that remyelination very much seems to be the most
or is maybe the most axonal protective therapy that we may
have. However, I think some further work needs to be done
to really formally demonstrate that, especially in vivo in the patient.
We did a bit of work in that direction, and showing here, for example,
that the axonal density is higher in remyelinated compared to
demyelinated lesion areas. However, of course, one has to be
conscious here, what is the hen and what is the egg.
It may also be that remyelination just occurred much more
easily in lesion areas that were in much less damaged and
probably at a higher axonal and probably also higher OPC
density here. But still, remyelinated areas in general look much
better than demyelinated areas. What about oligodendroglia
that we certainly need if we want to induce and stimulate
remyelination? Are they there in chronic lesions? When are they
lost? Part of these questions are still open. And we tried to
get closer to some of these aspects in recent work, where we looked at
oligodendroglial density in cortical demyelinated lesions. And where we
saw that really in the chronic cortical demyelination setting, the
NogoA positive cells, as well as the Olig2 positive cells, went
very much down. However, in earlier lesions, oligodendroglia
densities were much better, and even better than normal.
So even a stimulation of oligodendroglia densities. However,
I think we're still lacking the knowledge about the modes and also the
timing of cell death. And probably our therapies should not
wait too long, but rather target earlier lesions if possible.
A huge step forward with regard to our aim of stimulating
pro-remyelinative therapies is certainly detection of remyelination
of myelin in vivo. And there, I think in the last years, the important
advances have been made with PET imaging. And this is a study hereby
Bruno Stankoff that I'm showing here from Paris, who follows up
the patients and then identifies individual lesions and identifies
basically myelin repair, remyelination in these patients. And I think this will
be very useful for the future. On the one hand, to establish
the neuron and axon protective effect of remyelination, and
on the other hand, as an in vivo readout for our pro-remyelinative
therapies. So to sum up what I've said, MS is pathologically very
characteristic. It shows a characteristic centrifugal lesion. We still have to
identify what really underlies this specific lesion,  or pattern of lesion
formation. In addition, however, they are substantially diffuse and non-focal
pathology. And also inflammatory and noninflammatory correlates
of progressive disease, and they are not yet fully understood.
And we really need to know more about that to fully be able
to treat this aspect of disease in our patients. Also the pathomechanisms
of myelin damage and oligodendrocyte death are not yet fully understood.
And, as Mika also mentioned, remyelination is efficacious in only
a small proportion, approximately 20%, of patients.
So, I would clearly identify as research goals to further improve
our understanding of MS pathogenesis and lesion evolution.
To also formally demonstrate the neuroprotective effect of
remyelination, ideally in vivo using the new imaging technologies.
And then also from the cell biological side, to identify means to
protect and stimulate OPCs in evolving and established MS
lesions. With that of course, I'd like to thank all the people who contributed
this work, and you for listening. Thanks a lot.
