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Astrocytes are a specific immunological target in Rasmussen's encephalitis.

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Astrocytes Are a Specific Immunological
Target in Rasmussen’s Encephalitis
Jan Bauer, PhD,1 Christian E. Elger, MD,2 Volkmar H. Hans, MD,3 Johannes Schramm, MD,4
Horst Urbach, MD,5 Hans Lassmann, MD,1 and Christian G. Bien, MD2
Objective: The current histopathological criteria of Rasmussen’s encephalitis (RE) include the presence of T-cell–dominated
inflammation, microglial activation, neuronal loss, and astrocytic activation. An in vitro study, however, suggested glutamate
receptor 3 (GluR3) antibody–mediated astrocytic loss. Therefore, we investigated astrocytic apoptosis and loss in situ.
Methods: Histochemical, immunohistochemical, terminal deoxynucleotidyltransferase–mediated biotin-dUTP nick end labeling
and in situ hybridization techniques were applied to paraffin sections of 20 RE cases, 6 healthy control subjects, and 6 paraneoplastic encephalomyelitis, 10 Ammon’s horn sclerosis, and 5 focal cortical dysplasia cases.
Results: Astrocytic apoptosis and subsequent loss of these cells is a specific feature of RE. Such lesions are not found in the
control groups. In RE, astrocytic apoptosis and loss was present both in cortical and in white matter areas. Astrocytes in these
tissues showed major histocompatibility complex class I expression. Furthermore, granzyme-B⫹ lymphocytes were found in close
apposition to astrocytes bordering astrocyte-deficient lesions. Granzyme-B⫹ granules in these lymphocytes were polarized and
faced the astrocytic membranes. No evidence was found for an antibody-mediated destruction.
Interpretation: We suggest a specific attack by cytotoxic T lymphocytes as a possible mechanism responsible for astrocytic
degeneration in RE. The loss of astrocytes might play a role in neuronal dysfunction, seizure induction, and enhancement of
neuronal cell death.
Ann Neurol 2007;62:67– 80
Rasmussen’s encephalitis (RE) is an inflammatory, unihemispheric brain disorder that mainly affects children,1,2 although adult cases have been described.3–5
The clinical course of RE is characterized by intractable
focal onset seizures, namely, epilepsia partialis continua6 and progressive deterioration of functions associated with the affected hemisphere. The patients finally reach a residual stage with a decrease in seizure
frequency and a stable neurological deficit.7,8 Inflammation is one of the characteristic features that accompanies neuropathological changes such as microglia activation and the presence of microglial nodules,
neuronal loss, and astrogliosis.9
Since the first description of RE, several pathophysiological mechanisms have been suggested. Early studies described viral infections with enterovirus, Epstein–
Barr virus, cytomegalovirus, or herpes simplex
virus.10 –15 However, none of the studies could conclu-
sively link a specific virus to RE. Studies from McNamara’s group demonstrated that immunization of
rabbits with the glutamate receptor 3 (GluR3) produces a disease resembling RE and serum samples of
patients contain anti-GluR3 antibodies.16,17 Furthermore, these anti-GluR3 antibodies could activate the
glutamate receptor and might trigger the epileptic seizures.18 In addition, these antibodies can destroy neurons and astrocytes either directly, by excess stimulation of the receptor ion channel, or indirectly, by
complement-mediated cell death.19 –21 Recent studies,
however, demonstrated that anti-GluR3 antibodies are
not specific for RE, and numerous RE cases were
found to be GluR3 antibody–negative.22–24 Recently,
we provided evidence for another mechanism of cell
death in RE by showing that cytotoxic T cells may
destroy neurons by the release of granzyme-B (GrB).25
There are several reasons to investigate whether, besides
From the 1Division of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria; and Departments of 2Epileptology, 3Neuropathology, 4Neurosurgery, and 5Radiology/Neuroradiology, University of Bonn, Bonn, Germany.
Received May 20, 2006, and in revised form Mar 7, 2007. Accepted
for publication Mar 13, 2007.
This article includes supplementary materials available via the Internet at
Address correspondence to Dr Bauer, Division of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.
Published online May 14, 2007 in Wiley InterScience
( DOI: 10.1002/ana.21148
© 2007 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
neurons, astrocytes are damaged in RE. First, antiGluR3 antibodies destroyed astrocytes in vitro, suggesting that these cells might be affected in RE lesions as
well. In addition, astrocytic loss recently also has been
shown in animal models for epileptogenesis.26 –28 This
study demonstrates that astrocytic loss is a feature of
RE and suggests that its extent might affect physiological functions of neurons in the compromised areas.
Subjects and Methods
Brain Specimen Collection
The study was performed on resective epilepsy surgery tissue
or diagnostic brain biopsies from 20 RE patients studied at
the Epilepsy Center of the University of Bonn8 and, for
comparison, 6 control brains (autopsies from the Center of
Brain Research, Vienna, Austria), 10 noninflammatory epilepsy surgery cases of Ammon’s horn sclerosis in pharmacoresistent epilepsy patients (AHS, from Bonn, Germany), 5 epilepsy surgical cases of focal cortical dysplasia (FCD, from
Bonn), and 6 cases of paraneoplastic encephalitis (PE; autopsies, Bonn/Vienna). Diagnosis of RE was based on recently
published diagnostic criteria.29 See Table 1 for further demographic data.
Histochemistry and Immunohistochemistry
For basic classification of inflammation, demyelination, and
diffuse white matter injury, sections were stained with hematoxylin and eosin, Luxol fast blue myelin stain, and
Bielschowsky silver impregnation. Immunohistochemical
stainings (primary antibodies used are depicted in Table 2)
were performed on 3 to 5␮m paraffin sections. Before staining, endogenous peroxidase was blocked by 30-minute incubation in methanol with 0.02% H2O2. This was followed
with antigen retrieval by heating the sections for 90 minutes
in EDTA (0.05M) in tris(hydroxymethyl)aminomethane
(Tris) buffer (0.01M, pH 8.5) in a household food steamer
device (MultiGourmet FS 20; Braun, Kronberg/Taunus,
Germany). To detect IgG and complement C9, we incubated sections with 0.03% protease from Streptomyces griseus
(Sigma, St. Louis, MO) for 15 minutes at 37°C. Sections
were then incubated with 10% fetal calf serum (FCS) in
0.1M phosphate-buffered saline (FCS/PBS). Next, primary
antibodies were applied in FCS/PBS at 4°C overnight. After
washing with PBS, secondary antibodies in PBS/FCS with
3% normal human serum were applied for 1 hour at room
temperature. We used biotinylated secondary antibodies at a
concentration of 1:200 (donkey anti–rabbit, sheep anti–
mouse; Amersham Pharmacia Biotech, Uppsala, Sweden). As
a third step, avidin peroxidase (1:100; Sigma) was used. For
the CD3, CD8, major histocompatibility complex (MHC)
class I, and GrB stainings, biotinylated tyramine enhancement was used as described previously.25 Immunoglobulin
staining was done with biotinylated sheep anti–human antibody (Amersham). Labeling was visualized with 3,3⬘
diaminobenzidine-tetrahydrochloride (Sigma) or aminoethyl
carbazole (Sigma).
In case of double labeling for caspase-3 with glial fibrillary
acidic protein (GFAP), 2⬘3⬘cyclic nucleotide 3⬘ phosphohydrolase (CNPase), or microtubule-associated protein 2
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(MAP2) or neuronal nuclei (NeuN), both primary antibodies were diluted in FCS/PBS and sections were incubated at
4°C overnight. After washing in PBS, a mixture of alkaline
phosphatase–conjugated goat anti–mouse antibodies (Jackson
ImmunoResearch, West Grove, PA) and biotinylated donkey
anti–rabbit (1:100; Amersham) antibodies were applied in
FCS/PBS with 3% normal human serum for 1 hour at room
temperature. As a third step, avidin peroxidase (1:100;
Sigma) was applied for 1 hour at room temperature. Next,
the alkaline phosphatase label was visualized with fast blue B
base (Sigma) as substrate. This was followed by visualization
of peroxidase with diaminobenzidine-tetrahydrochloride or
aminoethyl carbazole. Finally, sections were counterstained
with hematoxylin.
In Situ Hybridization
Detection of messenger RNA was performed as described
previously.30 In brief, paraffin sections were dewaxed, pretreated with 10␮g proteinase K (Sigma) in Tris-buffered saline (pH 7.2) and incubated with digoxigenin-labeled probes
specific for GFAP (gift from Melitta Schachner, Hamburg,
Germany) or proteolipid protein (PLP).30 Sections then were
incubated with alkaline phosphatase–labeled anti-digoxigenin
antibody (Boehringer-Mannheim, Mannheim, Germany)
and developed using Niro blue tetrazolium chloride/5-Bromo4-chloro-3-indolyl phosphate (NBT/BCIP), (BoehringerMannheim) as substrate. Sections stained for GFAP or PLP
mRNA were double stained with anti-GFAP antibodies with
alkaline phosphatase–conjugated secondary antibodies using
fast red as substrate. As a result, mRNA appears black,
whereas GFAP protein is stained red. Sections were counterstained with hematoxylin.
Terminal Deoxynucleotidyltransferase–Mediated
Biotin-dUTP Nick End Labeling
To detect cells with DNA fragmentation, we performed terminal deoxynucleotidyltransferase–mediated biotin-dUTP
nick end labeling (TUNEL) with the In Situ Cell Death Detection Kit (Alkaline Phosphatase) from Roche (Mannheim,
Germany). Paraffin sections were deparaffinized, treated with
chloroform, and air-dried. This was followed by incubation
with labeled dUTP in the presence of terminal transferase
according to the manufacturer’s guidelines. Sections were developed with NBT/BCIP. Subsequently, the sections were
stained for GFAP as described earlier. As a result, DNA fragmentation in the nucleus appears black, whereas GFAP protein appears red.
Confocal Laser Fluorescence Microscopy
Fluorescence immunohistochemistry was performed on paraffin sections as described earlier with few modifications. For
confocal fluorescent double labeling with primary antibodies
from different species (eg, rabbit anti-GFAP and mouse
anti-GrB), antibodies were applied simultaneously at 4°C
overnight. After washing with PBS, secondary antibodies consisting of goat anti–mouse Cy3 (1:200; Jackson ImmunoResearch) and biotinylated anti–rabbit (1:200; Amersham) were
applied simultaneously for 1 hour at room temperature. The
staining was finished by application of streptavidin-Cy2 (1:75;
Jackson ImmunoResearch) for 1 hour at room temperature. In
Table 1. Patient Data and Semiquantitative Quantification of Astrocytic and Neuronal Degeneration and Loss
Patient No.
and Sex
Age at
Onset of
Stage (mo)
Site of Specimen
(n ⫽ 20)
1, M
2, F
Frontal neocortex
Superior temporal gyrus
Precentral gyrus
3, F
Temporal neocortex
4, F
Superior frontal gyrus
5, M
6, M
Superior frontal gyrus
7, M
Frontal neocortex
8, F
Temporal neocortex
and parahippocampal
9, M
Medial frontal gyrus
10, F
Temporal neocortex/
rolandic region
11, F
Temporal and parietal
neocortex parahippocampal gyrus
12, M
Frontal and temporal
13, F
Inferior temporal gyrus
14, F
Temporal neocortex
15, M
Temporal neocortex
16, M
Amygdala parahippocampal gyrus hippocampus temporal
17, F
Superior frontal gyrus
18, M
Superior frontal gyrus
19, F
Parietal neocortex
20, M
Hippocampus with
Control subjects
(n ⫽ 6; 3
F, 3 M)
67.0 ⫾ 4.9
horn sclerosis
(n ⫽ 10,
5 M, 5 F)
17.8 ⫾ 9.9
Focal cortical
(n ⫽ 5; 3
M, 2 F)
30.6 ⫾ 18.6
(n ⫽ 6, 3
F, 3 M)
65.8 ⫾ 13.6
4.6 ⫾ 3.6
1 ⫽ prodromal stage; 2 ⫽ acute stage; 3 ⫽ residual stage (see Bien and colleagues8).
Presence of caspase-3–positive astrocytes or neurons, respectively.
Semiquantitative assessment, categories 1–3; for details, see Subjects and Methods.
For Patients 2 and 10, specimens from two surgical procedures were evaluated.
Some single scattered apoptotic astrocytes in one case, in the absence of large loss of astrocytes.
ND ⫽ not determined.
Bauer et al: Astrocytic Degeneration in RE
Table 2. Primary Antibodies
Sternberger Monoclonals,
Lutherville, MD
Dakopatts, Hamburg, Germany Labvision, Fremont,
Sigma, St. Louis, MO
Chemicon, Temecula, CA
Labvision, Fremont, CA
Boehringer-Mannheim, Germany
Gift from Dr C. Linington,
Aberdeen, United Kingdom53
MHC class
I (HC10)
1:2,000 (C)
1:250 (F)
␣-Chain MHC
class I
Gift from Dr Ploegh, Harvard
Medical School, Boston,
1:1000 (C)
1:50 (F)
T cells
Dakopatts, Hamburg, Germany
1:250 (C)
T cells
Labvision, Fremont, CA
Activated caspase-3
in apoptotic
Becton Dickinson, San Diego,
1:1,000 (C)
1:50 (F)
Labvision, Fremont, CA
Macrophages, microglial cells
Dakopatts, Hamburg, Germany
Lytic complement
S. Piddlesden, University of
Cardiff, United Kingdom
IgG, plasma cells
Amersham Pharmacia Biotech,
Uppsala, Sweden
CNPase ⫽ 2⬘3⬘cyclic nucleotide 3⬘ phosphohydrolase; mAb ⫽ monoclonal antibody; GFAP ⫽ glial fibrillary acidic protein; polyAb ⫽
polyclonal antibody; MAP2 ⫽ microtubule-associated protein 2; NeuN ⫽ neuronal nuclei; APP ⫽ amyloid precursor protein; MAG ⫽
myelin-associated glycoprotein; C ⫽ Catalysed System Amplification (see Subjects and Methods); F ⫽ fluorescence; MHC ⫽ major
histocompatibility complex.
case of triple labeling for mouse anti-GFAP/mouse anti-GrB/
rabbit anti-CD3, the three primary antibodies were applied
simultaneously and the staining was finished as described earlier. This results in the presence of red GrB⫹ granules inside
of a green CD3⫹ T lymphocyte and red GFAP⫹ astrocytes.
Fluorescent preparations were examined using a confocal laser
scanning microscope (LSM 410; Carl Zeiss, Jena, Germany).
Analysis of Astrocyte, Neuronal, and Oligodendrocyte
Degeneration and Loss
Sections double stained for light microscopy (combinations
of caspase-3 with GFAP, S100␤, NeuN, MAP2, or CNPase)
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were analyzed for the presence of apoptotic astrocytes, neurons, and oligodendrocytes, and for loss of these cells.
First, whole sections from RE cases and control subjects
(no neurological disease, AHS, FCD, PE) were semiquantitatively studied for loss of GFAP⫹ astrocytes and MAP2⫹
neurons by two investigators (J.B., C.G.B.). Loss was scored
by division in three categories: (1) loss of some cells (mostly
around blood vessels), often found together with the presence of caspase-3⫹ astrocytes or neurons; (2) small- to
middle-sized areas (lesions) with loss of cells; and (3) large
(cortical or white matter) areas with loss of cells.
Second, we determined the magnitude of astrocytic and
neuronal loss. We assessed the lesional size, that is, area without GFAP or combined NeuN/MAP2 signal within the cortex
of each sample. As a first step, we analyzed the size of gray and
white matter of each sample as follows: The largest available
Luxol fast blue–stained section of each patient containing cortex and white matter was scanned at 1,000dpi. In these images, the cortical gray matter and underlying white matter
were outlined and measured using Scion Image (freeware,, an image processing and analysis
program based on National Institutes of Health Image. Next,
at 10⫻ objective magnification, a 100-point morphometric
ocular grid was sequentially superimposed over the whole section area. The size of GFAP- or NeuN/MAP2-deficient areas
was assessed by counting the number of grid points (intersections), which were not on all sides surrounded by GFAP⫹ or
NeuN/MAP2⫹ cells. The total GFAP- or NeuN/MAP2deficient area, indicated by the total number of “lesional” grid
points, was divided by the total cortical area of the specimen
and the result given as percentage of area devoid of GFAP⫹ or
NeuN/MAP2⫹ cells. In addition, this procedure was performed for GFAP⫹ cells in white matter.
Third, we analyzed loss of GFAP⫹ or S100⫹ astrocytes
and NeuN⫹ neurons in specific RE areas and compared the
numbers of cells with the numbers of astrocytes and neurons
in healthy control subjects. Areas in RE were divided into
three categories: (I) “RE GFAP normal,” no apparent loss of
GFAP reactivity; (II) “RE GFAP loss,” areas with lack of
GFAP (absence of GFAP-reactive cells); and (III) “RE GFAP
gliosis,” areas with dense fibrillary gliotic scar tissue. The
numbers of GFAP⫹, S100␤⫹, and MAP⫹ cells were measured in control and RE brain by superimposing a morphometric grid at 20⫻ objective magnification. Cell counting
started at the molecular layer (cortical layer 1) and was continued perpendicular to the meningeal lining until the white
matter was reached. Cortical areas were taken in the middle
of sulcal banks to standardize the procedure as much as possible, thereby avoiding the thinner sulcal bases and thicker
gyral crowns. The assessed areas are 0.5mm broad strips of
cortex of variable length (normally about 4mm) perpendicular to the cortical surface. The results of this quantification
procedure are given in cells per square millimeter of cortex.
Statistical Analysis
Two-sided Pearson’s or two-sided t tests were performed, as
appropriate; p ⬍ 0.05 was considered significant.
Qualitative Assessment of Apoptotic Astrocytic and
Neuronal Loss in Active Rasmussen’s Encephalitis
Neuronal destruction and loss is regarded as the hallmark of lesional pathology in RE.9 A further characteristic feature of RE is the presence of astrogliosis, that
is, the presence of hypertrophic, strongly stained,
GFAP⫹ reactive astrocytes. Here, however, we investigated astrocytic loss in RE.
In all RE patients, large parts of RE cortex showed
GFAP⫹ astrocytes with normal or activated morphology. Many patients, however, also showed some loss of
GFAP⫹ astrocytes around blood vessels or loss of astrocytes close to the meningeal lining (Figs 1B and
4Q). In about one third of the patients, loss of GFAP⫹
astrocytes was even more pronounced. Here, multiple
small- to middle-sized lesions with loss of GFAP reactivity were found (see Figs 1G and 4M). Finally, in
some patients, GFAP reactivity was absent in large
parts of the cortex. Often these lesions were bordered
by hypertrophic activated astrocytes strongly stained for
GFAP (see Figs 1I, 2D, and 4A). Because these lesions
were not completely devoid of nuclei, we also examined these lesions for the presence of other cells. Stainings for neurons (MAP2 or combination of NeuN with
MAP2) showed that cell bodies were largely missing
(see Figs 1F, H). Bielschowsky staining, in addition,
showed that, in cortical areas, the axonal density often
was largely reduced (not shown), whereas in white
matter lesions, GFAP loss was not always combined
with axonal injury (see underneath). Staining for oligodendrocytes cell bodies by CNPase and microglial cells
by CD68, however, showed that the oligodendrocytes
and microglial cells were present in normal numbers.
The range of astrocytic loss in all sections from all RE
patients was determined semiquantitatively and presented in Table 1.
Lack of staining for GFAP does not necessarily mean
that astrocytes are in fact lost. To confirm that there was
actual astrocytic loss, we performed additional immunohistochemical stainings for another marker for astrocytes
(always S100␤), as well as in situ hybridization (ISH) for
GFAP mRNA (on selected cases that were graded with
severe loss in Table 3). In areas with cortical loss of
GFAP⫹ astrocytes, we also found a loss of S100␤⫹ cells
(see Fig 1J) and of GFAP mRNA⫹ cells (see Fig 1L). In
contrast, ISH for PLP mRNA demonstrated numbers of
oligodendrocytes comparable with those in areas without
loss of astrocytes (see Fig 1M). This indicates that loss of
GFAP mRNA is not due to a general downregulation of
mRNA, but rather to a selective astrocytic loss. Taken
together, these findings confirm that loss of GFAP⫹ cells
does not merely reflect a loss of GFAP immunoreactivity
but is a real loss of astrocytes.
Staining for activated caspase-3 demonstrated the
presence of single caspase-3⫹ cells within otherwise
normal-appearing cortices (see Fig 1A). Double-labeling
studies for caspase-3 and GFAP demonstrated that these
caspase-3⫹ cells exhibited astrocytic morphology and
were either (weakly) GFAP⫹ (see Figs 1B, 1C, 4C, and
4M), had GFAP reactivity only in part of the cytoplasm
(see Figs 4E, F), or were GFAP⫺ (see Figs 1A, 1B, 4D,
and 4M). Besides the presence of activated caspase-3 immunoreactivity, these caspase-3⫹ or GFAP⫹ astrocytes
showed nuclear condensation (see Figs 1D and 4C) or
nuclear fragmentation (see Figs 1E and 4D). Moreover,
although in much lower numbers than caspase-3⫹ astrocytes, TUNEL staining in combination with GFAP
Bauer et al: Astrocytic Degeneration in RE
Fig 1. Astrocytic pathology in Rasmussen’s encephalitis (RE). (A–C) Immunohistochemistry (IHC) for glial fibrillary acidic protein
(GFAP) (blue) and caspase-3 (brown). (A) Two cells with morphology of astrocytes. Whereas the lower cell (arrowhead) is double
stained, the other one shows staining only for caspase-3 (brown), indicating loss of only GFAP reactivity in this stage. Original
magnification ⫻388. (B) An area without GFAP reactivity around a small blood vessel with caspase-3⫹ astrocyte-like cells in the
center, whereas on the edge a double-stained astrocyte (arrowhead) is shown. Original magnification ⫻308. (C) A greater magnification of the latter cell shows condensation of the nucleus (arrowhead), another criterion for apoptosis. Original magnification
⫻792. (D, E) Two examples of GFAP-positive astrocytes with condensed (D, arrowhead) or fragmented (E, arrowhead) nuclei
indicative of apoptosis. Original magnification ⫻990 (D, E). (F) Staining for microtubule-associated protein 2 (MAP2) shows a
cortical lesion with loss of MAP2⫹ neurons. Original magnification ⫻40. (G) The same area as in (F) stained for GFAP (blue)
and caspase-3 (brown). Original magnification ⫻40. The border of this lesion is graphically delineated and was projected in (F).
Inside the delineation this lesion is almost devoid of GFAP⫹ astrocytes. (H) Greater magnification of the rectangle in (F) showing
that left, above, and on the right side of the lesion MAP2-positive neuronal cell bodies (arrowheads) are still present. Original
magnification ⫻60. (I) Enlargement from the edge of the lesion (rectangle) from (G) showing the presence of caspase-3–stained
(brown, arrowheads) apoptotic glial cells. Original magnification ⫻161. (J) Double staining for S100␤ (blue) and caspase-3
(brown) in the middle of a cortical area shows some double-stained S100␤⫹ astrocytes (arrowheads). Original magnification
⫻308. (K) Double staining for CD68 (blue, microglial cells) and caspase-3 (red) shows that microglial cells are not apoptotic.
Original magnification ⫻244. (L) ISH for GFAP messenger RNA (mRNA; black) in combination with IHC for GFAP protein
(red) shows a small white matter area in which both GFAP mRNA and GFAP protein are lost. Original magnification ⫻96. (M)
ISH for proteolipid protein (PLP) mRNA in combination with GFAP IHC (red) does not show any downregulation of oligodendrocyte mRNA (black dots). Thus, no loss of oligodendrocytes is found. Original magnification ⫻96.
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Fig 2. Neurons and astrocytes in controls and various epileptic diseases. Staining for neuronal nuclei (NeuN) (A, C, E, G; original
magnification ⫻80) and glial fibrillary acidic protein (GFAP) (B, D, F, H; original magnification ⫻80) in the cortex (layers I
[left] to VI [right]) of control subjects (con; A, B), Rasmussen’s encephalitis patients (RE; C, D), cortical dysplasia patients (FCD;
E, F), and in the CA1 hippocampal region of Ammon’s horn sclerosis (AHS; G, H) patients. In RE, both neuronal loss and astrocyte loss is seen. In FCD, the NeuN staining shows a lower density of neurons and the presence of balloon cells (arrowheads).
GFAP reactivity is seen in hypertrophic astrocytes. In AHS (G, H; rectangle in the inset shows localization of G), the right side of
the CA1 region shows loss of neurons, whereas in the same area with GFAP, a dense gliosis is observed.
demonstrated the presence of astrocytes with DNA fragmentation (see Fig 4G). These three combined characteristics (activated caspase-3 reactivity, nuclear condensation, and DNA fragmentation) imply that astrocytes die
by apoptosis. The presence of caspase-3⫹/GFAP⫺ cells
raises the question whether some of these cells could be
microglial cells. Double staining for caspase-3 and
CD68, however, showed no CD68/caspase-3 coexpression (see Fig 1K), thus suggesting that these caspase-3⫹
cells are indeed astrocytes. Astrocytic loss and presence
of caspase-3⫹ astrocytes in the different sections from
the various RE patients are depicted in Table 1. Although, in most cases, the caspase-3⫹ cells had astrocytic
morphology, some of these cells were much larger and
had only one or two short processes, suggesting that they
were apoptotic neurons. However, these caspase-3⫹ neurons were found only rarely.
Two of 20 patients showed astrocytic apoptosis
without apparent loss of astrocytes (see Table 1; Patients 3 and 19). Caspase-3⫹ cells with astrocytic morphology were found in and on the borders of all lesions
with astrocytic loss (see Figs 1A, B).
Bauer et al: Astrocytic Degeneration in RE
Table 3. Areas with Astrocytic and Neuronal Loss
Loss ⴞ SEM
Mean Area
Loss ⴞ SEM
Loss ⴞ SEM
144.3 ⫾ 38.5
0.2 ⫾ 0.4
0.0 ⫾ 0.1
0.1 ⫾ 0.1
75.7 ⫾ 51.7
7.6 ⫾ 9.8
6.0 ⫾ 6.5
3.4 ⫾ 4.9
31.6 ⫾ 12.3
21.3 ⫾ 13.5
0.0 ⫾ 0.0
0.0 ⫾ 0.0
140.8 ⫾ 66.3
8.6 ⫾ 3.7
0.0 ⫾ 0.0
0.0 ⫾ 0.0
213.9 ⫾ 73.1
0.0 ⫾ 0.0
0.0 ⫾ 0.0
0.0 ⫾ 0.0
In control subjects (CON), Rasmussen’s encephalitis (RE) patients, and focal cortical dysplasia (FCD) patients, neocortical areas were
quantified. In Ammon’s horn sclerosis (AHS) patients, quantification was done only in the pyramidal layer of the cornu ammonis and
the granular layer of the dentate gyrus. In paraneoplastic encephalomyelitis (PE) patients, quantification was performed in inflammatory
SEM ⫽ standard error of the mean; NeuN ⫽ neuronal nuclei; MAP2 ⫽ microtubule-associated protein 2; GFAP ⫽ glial fibrillary
acidic protein.
The extent of loss of GFAP-reactive astrocytes and
of apoptotic (caspase-3⫹) astrocytes not only varied between patients but also between different biopsy regions (sections) from one patient and even within one
section. These differences in the degree of apoptosis
and loss of GFAP⫹ cells between the various sections,
which are summarized in Table 1, probably reflect different stages of astrocytic loss.
Astrocytic Destruction and Loss Is a Specific Feature
of Rasmussen’s Encephalitis Pathology
To investigate the specificity of astrocytic loss, we
quantified the degree of astrocytic and neuronal loss
in the cortex of RE patients and compared these with
healthy control subjects, brains of PE patients, and
brains of epilepsy patients (AHS and FCD). The degree of astrocytic loss (GFAP or S100␤) or neuronal
loss (NeuN/MAP2) in these control subjects and various patient groups is presented in Table 3. In FCD
and AHS cases, a considerable degree of neuronal loss
was found. In FCD, astrocytes in these areas with reduced neuronal density showed a hypertrophic morphology. In AHS, neuronal loss was colocalized with
fibrillary gliosis. Areas with loss of astrocytes, as seen
in RE, were found neither in FCD nor in AHS patients. Representative pictures from stainings for
GFAP and NeuN from control subjects and RE,
FCD, and AHS cases are depicted in Figure 2. Evidence for apoptosis of astrocytes in these brains (see
Table 1), except for some single apoptotic cells with
astrocytic morphology in one PE case, was absent.
Taken together, these results suggest that astrocytic
apoptosis and loss are specific features of RE.
Rasmussen’s Encephalitis Cortex with Astrocytic
Fibrillary Gliosis
Besides normal-appearing areas and areas with (combined) astrocytic and neuronal loss in RE, we also
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found areas with profound fibrillary astrocytic scar formation. These areas were present in a small number of
patients (N ⫽ 4; Patients 2, 9, 11, and 12 in Table 1).
All of these patients were in the residual clinical stage
(stage 3). NeuN or MAP2 immunohistochemistry, as
well as Bielschowsky’s silver impregnation in these lesions, demonstrated severe cortical atrophy with almost
complete loss of neurons and axons. A combined
GFAP/caspase-3 staining did not show apoptotic astrocytes in these areas.
Relation of Astrocytic and Neuronal Loss
Figure 3 shows the correlation of cortical areas devoid
of neurons versus the cortical areas devoid of astrocytes. In 13 of 20 RE patients, the area of astrocytic
and neuronal loss was less than 10% of the total cortex
investigated. The other seven patients showed more extensive areas of astrocytic (up to 26%) and neuronal
loss (up to 36%). Astrocytic and neuronal loss were
significantly correlated ( p ⫽ 0.01; see Fig 3), although
the extent of neuronal loss appears larger than that of
astrocytic loss. A representative pair of figures illustrates
a similar degree of loss of GFAP⫹ astrocytes and
MAP2⫹ or NeuN⫹ neurons (see Figs 1F, 1G, 2C, and
2D) in a cortical area.
Taken together, we were able to separate cortical areas into three categories: (I) areas without apparent astrocytic or neuronal loss, but in some cases, in the
presence of single apoptotic cells (underneath referred
to as RE “GFAP normal”); (II) areas with loss of
GFAP-reactive astrocytes and neurons in the presence
of apoptotic astrocytes (RE “GFAP loss”); and (III) areas with dense GFAP⫹ fibrillary gliotic scar formation
and severe loss of neurons, in the absence of apoptotic
astrocytes (RE “GFAP gliosis”).
We quantified the densities of neurons and astrocytes in the three cortical areas (areas without GFAP
loss [I], with GFAP loss [II], and with GFAP gliosis
reactive hypertrophic astrocytes surrounded the larger
lesions. Immunohistochemical staining for CNPase (see
Fig 4B) and ISH for PLP (see Fig 1L) demonstrated
that oligodendrocytes and myelin in these white matter
areas were unaffected. In addition, Bielschowsky silver
stain for axons (see Fig 4I) and staining for ␤-amyloid
precursor protein (see Fig 4J) showed acute axonal injury in some areas. However, astrocytic loss was not
coupled to axonal injury of traversing fiber tracts. The
percentage of areas with loss of astrocytes in subcortical
white matter ranged from 0 to 22.0% (average ⫾ standard error: 2.0 ⫾ 1.8%).
Possible Mechanisms Responsible for Astrocytic Loss
Fig 3. Quantification of astrocytic and neuronal loss in cortical areas from Rasmussen’s encephalitis (RE) patients. In 20
cortical sections from 20 RE cases, the areas (average area ⫾
standard error ⫽ 75.7 ⫾ 4.5; minimal area ⫽ 20.2mm2;
maximal area ⫽ 223.3mm2) with loss of glial fibrillary acidic
protein (GFAP; astrocytes) or microtubule-associated protein
2/neuronal nuclei (MAP2/NeuN; neurons) was quantified.
Areas with loss of astrocytes ranged from 0 to 37%, whereas
areas with loss of neurons ranged from 0 to 26%.The correlation between astrocyte and neuronal loss was significant (p ⫽
[III]) and compared those with the respective cells in
control cortices. These results are shown in Figure 5.
Densities of GFAP⫹ and S100␤⫹ cells in “GFAP normal” areas did not differ from those in control brain.
However, the numbers of neurons in these areas were
decreased as compared with the cortex in control subjects. In areas with GFAP loss, the numbers of all cell
types were significantly diminished. In gliotic areas (RE
“GFAP gliosis”), the numbers of GFAP⫹ and S100␤⫹
cells were increased in comparison with the control
subjects, as well as with the other RE specimens. Staining for NeuN, however, demonstrated a loss of neurons as compared with the control subjects and the
other RE subgroups.
astrocyte death has been noted after treatment with
anti-GluR3 antibodies,21 we investigated whether immunoglobulin or complement deposition on astrocytes
was present in our material. In most cases, a diffuse
staining for immunoglobulin and complement C9neo
was observed around blood vessels (see Figs 4K, L). In
areas with astrocytic loss, IgG and C9neo deposition
on astrocytes, however, was absent. This suggests that a
complement-mediated killing of these cells can be
ruled out. Furthermore, 4 of our 20 patients have been
tested for serum anti-GluR3 antibodies. None of them
was found to be positive.25
Severe brain inflammation may result in hypoxia-like
tissue injury that shows specific loss of myelinassociated glycoprotein immunoreactivity31 and could
be responsible for damage to both neurons and astrocytes. To check whether the loss of GFAP immunoreactivity could also be the result of ischemia, we stained
for PLP mRNA and myelin-associated glycoprotein
immunoreactivity in areas showing loss of GFAP⫹
cells. ISH demonstrated a loss of GFAP mRNA⫹ cells
(see Fig 1L), but no loss of PLP mRNA (see Fig 1M)
or myelin-associated glycoprotein (not shown) in the
same areas. These results suggest that loss of astrocytes
or neurons does not result from hypoxia-like tissue injury.
Astrocytic Apoptosis and Loss Also Affect the
Subcortical White Matter
Astrocytic apoptosis and loss in RE cases are not restricted to cortical areas. To our surprise, we discovered
small and large areas devoid of GFAP⫹ astrocytes
deeply in the subcortical white matter (see Fig 4A).
Many of these white matter lesions showed caspase-3⫹
or TUNEL⫹ astrocytes within as well as on the edge of
these lesions (see Figs 4C–H). Similar to cortical areas,
Previously, we showed that cytotoxic T lymphocytes
interacting with neurons are a considerable component
of the infiltrating inflammatory cells in RE.25 To investigate whether astrocytic cell death could be mediated by cytotoxic T lymphocytes, we performed double
(GFAP and GrB) and triple (GFAP, CD3, and GrB)
stainings. Cytotoxic T cells were present around blood
vessels and on the border of lesions in which astrocytes
were dying or already lost (see Figs 4N, Q). Often,
Bauer et al: Astrocytic Degeneration in RE
Figure 4
single or multiple cytotoxic T cells were seen in close
apposition to astrocytes. GrB⫹ granules in several lymphocytes were polarized and facing the astrocyte surface
(see Figs 4N–P) similar to that described in in vitro
GrB studies.32 Because MHC class I expression is a
prerequisite for antigen-specific cytotoxicity mediated
by CD8⫹ T cells, we performed stainings with for
MHC class I. Although weaker than on endothelial
cells, lymphocytes, neurons, and microglial cells, astrocytes indeed were MHC class I⫹ (see Fig 4R). The
online supplementary data show to what extent loss of
astrocytes, neurons, and gliosis may be reflected in
MRI recordings.
The current histopathological criteria for the diagnosis
of RE include the presence of T-cell–dominated inflammation, microglial activation, and microglial nod-
Annals of Neurology
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July 2007
ules, as well as neuronal loss and astrocytic activation.
This study suggests that astrocytic apoptosis and loss,
most probably induced by a cytotoxic T-cell response
toward astrocytes, is a common finding in RE.
The major arguments for the presence of astrocytedeficient lesions are the observations of areas that specifically lack GFAP or S100␤ protein, GFAP mRNA,
as well as the presence of apoptotic astrocytes at the
borders of those regions. Oligodendrocytes, stained for
PLP mRNA or CNPase, and CD68⫹ microglial cells
were present in normal numbers, indicating that these
lesions are not necrotic or hypoxic in nature.
Astrocyte death has been shown in several studies of
animal models for epilepsy.26 –28,33,34 Therefore, the
possibility exists that astrocytic loss is not specific for
RE but is a consequence of epileptic seizures per se.
Alternatively, astrocytes may die from an unspecific reaction to inflammation. Astrocytic loss, however, was
not present in noninflammatory focal epilepsy controls
(AHS and FCD) or in PE cases (ie, inflammatory control). In AHS cases, however, severe gliosis was seen in
the hippocampal CA1 region where maximal neuronal
loss is observed. This scarlike gliosis is comparable with
gliotic areas in the severely affected RE specimens. It
could still be possible that, during the early stages of
AHS, hippocampal astrocytes are lost. On the other
hand, epileptic seizures in these AHS cases were
present at the time of epilepsy surgery, and astrocyte
death, if generated by the seizures, could be expected.
Therefore, astrocytic loss does not appear to be a
consequence of epileptic seizures or an unspecific inflammatory reaction and should be considered specific
for RE.
Double labeling of GFAP with caspase-3 showed
that only a fraction of the caspase-3⫹ cells contain
GFAP reactivity in parts of the cytoplasm, whereas
others, although showing an astrocytic morphology,
fail to express GFAP completely. Furthermore, we
found much lower numbers of GFAP⫹/TUNEL⫹ cells
than cells double labeled for GFAP and caspase-3.
These findings suggest that, in astrocytes, upregulation
of caspase-3 is followed by a degradation of GFAP,
suggesting that GFAP itself is a substrate for caspase-3.
Comparable results were found in neurons in glutamate excitotoxity studies in vitro.35
An important question is the reason for the specific
astrocyte death described here. A previous study21 provided evidence that treatment of mixed neural cell cultures with anti-GluR3 antibodies induces astrocyte cell
death in these cultures. However, although we cannot
completely rule out a role of anti-GluR3 or other antibodies,36 it is unlikely that antibodies are largely involved in astrocytic damage in vivo. Our results show
that none of our patients, despite a disturbed blood–
brain barrier and severe inflammatory response, shows
IgG or complement C9neo deposition on astrocytes.
Moreover, all 4 of the 20 patients tested previously for
the presence of serum GluR3 antibodies were negative
for these antibodies25 but showed unequivocal astrocytic loss. This is corroborated by publications24,37 that
question the role of anti-GluR3 antibodies in RE because these antibodies were detected in only some of
the RE cases and have been found at an equal frequency in noninflammatory epileptic disorders.
Another possible explanation for astrocytic damage
could be an indirect phenomenon, namely, the lack of
production of “astrotrophic” factors by neurons. We
have tried to find evidence for this by correlating neu-
Fig 4. Astrocytic degeneration and loss in white matter. (A) Double staining for glial fibrillary acidic protein (GFAP; blue) and
caspase-3 (brown) showing an area with loss of GFAP-reactive astrocytes (arrowhead indicates small vessel). Original magnification
⫻75. (B) Same area as in (A) stained for CNPase, showing only a mildly reduced staining intensity, indicative of well-preserved
oligodendrocytes and myelin. Original magnification ⫻75. (C–F) Double staining for caspase-3 (brown/red) and GFAP (blue).
Two examples of caspase-3⫹ cells in and on the border of the lesion as seen in (A). (C) This caspase-3–positive cell still shows some
GFAP immunoreactivity in the cytoplasm (arrowhead). Nuclear counterstain (hematoxylin) shows a condensed nucleus (arrow), another indication for apoptosis. Original magnification ⫻990. (D) In this caspase-3–positive cell, GFAP reactivity is absent. Nuclear
counterstain (arrow) shows fragmentation of the nucleus. Original magnification ⫻990. (E, F). Two examples of caspase-3 and
GFAP double-labeled apoptotic astrocytes. GFAP reactivity (arrowhead, blue) is seen only in part of the cytoplasm of the astrocytes.
Original magnification ⫻792 (E), ⫻673 (F). (G) Terminal deoxynucleotidyltransferase–mediated biotin-dUTP nick end labeling
(TUNEL) staining (black) in combination with GFAP (red) shows two GFAP-positive astrocytes with DNA fragmentation in the
nucleus (arrows), another indication of apoptotic cell death. Original magnification ⫻330 (G). (H–L) Various stainings in the
white matter of a lesion with GFAP loss. (H) Double staining for GFAP (blue) and caspase-3 (brown) shows the presence of multiple apoptotic astrocytes (arrowheads) in this white matter lesion. Original magnification ⫻890. (I) Bielschowsky stain for axons
shows the absence of acute axonal damage. Original magnification ⫻200. (J) Double labeling for GFAP (brown, arrows point out
astrocytic processes) and amyloid precursor protein (blue) shows the loss of astrocytic cell bodies. Except for a single axonal spheroid
(arrowhead), no axonal pathology is evident. Original magnification ⫻277. (K) Staining for immunoglobulins shows some leakage
around blood vessels without binding to astrocytes. Original magnification ⫻ 185. (L) Staining for C9neo shows a diffuse reactivity
around the same blood vessel as in (K), but no deposition on astrocytes. Original magnification ⫻185. (M–R) Inflammation and
astrocyte damage. (M) Staining for GFAP (red) and caspase-3 (green). An astrocyte in the center shows staining for caspase-3 in the
absence of GFAP. Two other GFAP⫹ astrocytes show some caspase-3 reactivity in the cytoplasm. Original magnification ⫻675.
(N–P) Triple staining for GFAP (red), CD3 (green), and granzyme-B (GrB; red granules inside of lymphocytes). (N) A lesion with
two astrocytes (arrowheads) showing swollen cell bodies and degeneration of processes. Original magnification ⫻720. In the surrounding area, several lymphocytes are seen in close contact with astrocyte processes. A lymphocyte in apposition to an astrocyte (arrow), which is further enlarged in (O) (original magnification ⫻3,600), showing GrB⫹-granules polarized toward the astrocyte
process. (P) Another example of a lymphocyte with polarized GrB⫹ granules facing an astrocyte process. Original magnification
⫻900. (Q) Multiple lymphocytes (green) are attached to a degenerating perivascular astrocyte (red). In the top left corner a normal
astrocyte is seen. Original magnification ⫻720. (R) Double staining for major histocompatibility complex (MHC) class I (green)
and GFAP (red) shows a neuron (left from the astrocyte) and blood vessels strongly stained for MHC class I. Astrocytic cell bodies
and processes (arrowheads) also stain positive for MHC class I, although less strongly than neurons and endothelial cells. Original
magnification ⫻1,350.
Bauer et al: Astrocytic Degeneration in RE
Fig 5. Numbers of glial fibrillary acidic protein–positive (GFAP⫹; hatched bars), S100␤⫹ (black bars), and neuronal nuclei–
positive (NeuN⫹; white bars) cells in defined cortical areas. GFAP⫹, S100␤⫹, and NeuN⫹ cells were counted in the cortex of
healthy control subjects and in the three different regions (I: normal numbers of GFAP⫹ cells; II: areas with loss of GFAP⫹ cells;
III: areas with GFAP⫹ gliotic cells) found in Rasmussen’s encephalitis (RE) patients. In the RE “GFAP normal” group, the numbers of GFAP⫹ and S100␤⫹ cells do not differ from the GFAP⫹ and S100␤⫹ cells in control subjects. The number of NeuN⫹
neurons, however, is significantly lower (*1p ⫽ 0.004). In the RE “GFAP loss” group, all three parameters are lower than in the
control group (*2p ⫽ 0.01; *3p ⫽ 0.037; *4p ⫽ 0.004). The number of neurons do not differ from those in the RE “GFAP normal” group. In the RE “GFAP gliosis” group, the numbers of GFAP⫹ (*5p ⫽ 0.011) and S100␤⫹ (*6p ⫽ 0.011) cells are significantly greater than in control subjects, whereas the NeuN⫹ cells are significantly lower than in control subjects (*7p ⫽ 0.011),
lower than in the RE “GFAP normal” group (p ⫽ 0.006), and lower than in the RE “GFAP loss” group (p ⫽ 0.036). The pictures on top show representative GFAP stainings for these different groups (from left to right: control subjects, RE “GFAP normal,”
RE “GFAP loss,” and RE “GFAP gliosis”).
ronal and astrocytic loss in cortical areas. Indeed, our
results show that astrocytic loss and neuronal loss can
be found in identical areas and to a similar degree. In
addition, however, there are areas with sole loss of neurons and, more important, also areas of astrocytic apoptosis and loss in white matter. Axonal stainings indicated that in the white matter astrocytic lesions,
axonal damage was absent, and thus loss of “astrotrophic” support by neurons may not be responsible for
astrocytic apoptosis and loss.
Another option for astrocytic loss can be specific
damage by cytotoxic T lymphocytes. Recent findings
by us25 show that cytotoxic T lymphocytes filled with
GrB⫹-positive granules are found in close apposition
to MHC class I⫹ neurons. Here we show that in areas with degradation of astrocytes, such interactions
between T lymphocytes and astrocytes are readily
found. As in the interaction of lymphocytes with neurons and as shown in in vitro studies,38 we found
polarization of cytotoxic granules toward the astrocytic surface. In our samples, MHC class I molecules
were expressed in and on astrocytes. Furthermore,
MHC class I localization on astrocytes in RE has
Annals of Neurology
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July 2007
been described previously by immunohistochemical
staining for the associated membrane protein ␤2microglobulin.39 As Christinck and colleagues40 have
shown, only minimal expression of MHC class I antigens is needed for an interaction between a cytotoxic T cell and its target cell. Thus, induction of
apoptosis in astrocytes probably is generated by a
GrB-mediated, MHC class I–restricted T-cell response. A further argument for a role of cytotoxic T
cells in the pathogenesis of RE comes from our tacrolimus (a T-cell–specific immunosuppressant) studies.41 Eight patients included in this study have received long-term treatment with tacrolimus. Six of
eight patients have not or only mildly progressed by
motor deterioration and hemispheric cerebral tissue
loss. The other two patients progressed and finally
underwent hemispherectomy. Five of these eight patients have been presented previously with a shorter
follow-up.41 The underlying reason for T-cell cytotoxicity toward astrocytes and neurons, however, remains to be elucidated. Although it may be an autoimmune condition, cytotoxicity against a slowly
spreading local viral infection might even better ex-
plain the unilateral, centrifugal degeneration present
in RE.42 Many different viruses can cause alterations
of cytoskeletal structures.43 Moreover, in vitro infection of astrocytoma cell lines with measles virus specifically disrupts the GFAP cytoskeleton.44 Therefore,
it is not inconceivable that an as yet unknown virus
may contribute to the loss of GFAP in RE. Destruction could be explained as an effect of the infection
itself, as well as by a cytotoxic T-cell response to the
infected cells (ie, astrocytes and neurons). Similar
conclusions were drawn in an earlier immunohistochemical study by Farrell and colleagues,39 who also
mentioned loss of GFAP immunoreactivity in cortical
areas of RE but did not further pursue this issue.
Astrocytes conduct a large number of functions.45– 48 Pathophysiologically important in RE may
be the roles of astrocytes in energy metabolism, in
maintaining potassium homeostasis, and in the catabolism of GABA and glutamate, neurotransmitters critically involved in epileptic processes. Hansson and coworkers’ studies,49 for instance, suggest that glial
glutamate transporters are essential for maintaining
low extracellular glutamate levels, as well as for preventing chronic glutamate neurotoxicity. In some
forms of human epilepsy, impaired potassium buffering by astrocytes may contribute to seizure generation
or perpetuation.50 Finally, several reports indicate
that hypertrophic astrocytes can aggravate or induce
seizures by glutamate release51 or by upregulation of
adenosine kinase, a key regulator of the anticonvulsant adenosine.52 Because both dying and activated
hypertrophic astrocytes are present in RE, it is impossible to define in which state these astrocytes influence neuronal activity most. Either way, astrocytic pathology in RE may be considered to play a role in
neuronal dysfunction and may contribute both to the
induction of seizures and to the progressive deterioration of functions associated with the affected hemisphere.
This study was supported by the Austrian “Fonds zur Förderung der
wissenschaftlichen Forschung” (P16063-B02).
We thank H. Breitschopf, U. Köck, A. Kury, and M. Leisser for
expert technical assistance.
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