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Cystatin C expression is associated with granule cell dispersion in epilepsy.

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Cystatin C Expression Is Associated with
Granule Cell Dispersion in Epilepsy
Terhi J. Pirttilä, MSc,1 Anni Manninen, BMed,1 Leena Jutila, MD,2 Jari Nissinen, MSc,1
Reetta Kälviäinen, MD, PhD,2 Matti Vapalahti, MD, PhD,3 Arto Immonen, MD,3 Leo Paljärvi, MD, PhD,4,5
Kari Karkola, MD, PhD,6 Irina Alafuzoff, MD, PhD,4 Esa Mervaala, MD, PhD,7
and Asla Pitkänen, MD, PhD1,2
Human temporal lobe epilepsy (TLE) is associated with cellular alterations (eg, hilar cell death, neurogenesis, and granule cell dispersion) in the dentate gyrus but their underlying molecular mechanism are not known. We previously
demonstrated increased expression of cystatin C, a protease inhibitor linked to both neurodegeneration and neurogenesis,
during epileptogenesis in the rat hippocampus. Here, we investigated cystatin C expression in the dentate gyrus in
chronic epilepsy and its association with neuronal loss and neurogenesis. In both rats with epilepsy and human patients
with TLE, cystatin C expression was increased in glial cells in the molecular layer of the dentate gyrus, being most
prominent in cases with granule cell dispersion. In patients with TLE, high cystatin C expression associated with greater
numbers of polysialylated neural cell adhesion molecule–positive newborn cells in the molecular layer, although the
overall number was decreased, indicating that the newborn cells migrate to abnormal locations in the epileptic dentate
gyrus. These data thus demonstrate that cystatin C expression is altered during the chronic phase of epilepsy and suggest
that cystatin C plays a role in network reorganization in the epileptic dentate gyrus, especially in granule cell dispersion
and guidance of migrating newborn granule cells.
Ann Neurol 2005;58:211–223
Temporal lobe epilepsy (TLE) is associated with hippocampal sclerosis, which includes selective neuronal
loss in the CA1, CA3, and hilar regions.1,2 A large subpopulation of patients with TLE also has granule cell
loss in the dentate gyrus.3 Furthermore, granule cells
often undergo several other neuroplastic changes in the
human epileptic brain, including mossy fiber sprouting,4,5 dendritic alterations,6 and dispersion.7,8 Dispersion of the granule cells into abnormal locations in the
molecular layer of the dentate gyrus occurs in about
40% of patients who underwent surgery for drugrefractory TLE, and it is associated with severe loss of
hilar cells.7,9,10
Neurogenesis (ie, proliferation, migration, and differentiation of neuronal progenitor cells) occurs
throughout life in the dentate gyrus in humans and
other mammals.11,12 According to the animal data, seizure activity is one of the most potent factors that increase neurogenesis.13 Therefore, one can assume that
in epilepsy the dentate gyrus must maintain molecular
machinery to orchestrate the genesis, migration, differentiation, and integration of newborn granule cells into
the neuronal network, particularly in patients with frequent seizures.
Cystatin C is a potent inhibitor of lysosomal proteinases, including cathepsins B, H, and L.14 In an experimental model of ischemia in rat, cystatin C is involved in neuronal death.15 Furthermore, a
glycosylated form of cystatin C, together with fibroblast growth factor–2, stimulates neurogenesis in rat
brain in vivo.16 Thus, cystatin C has multiple effects
on both neuronal birth and death. In humans, cystatin
C is linked to several neurodegenerative disorders, including hereditary hemorrhage with amyloidosis of Icelandic type where a variant of cystatin C protein is deposited as amyloid fibrils on the walls of cerebral
arteries17 and has susceptibility to late-onset Alzheimer’s disease.18,19
In rats undergoing status epilepticus (SE)–induced
epileptogenesis, the expression of both the cystatin C
From the 1A. I. Virtanen Institute for Molecular Sciences, University of Kuopio; Departments of 2Neurology, 3Neurosurgery, and
4
Pathology, Kuopio University Hospital, Kuopio; 5Department of
Pathology, Laboratory Centre, Tampere University Hospital, Tampere; and Departments of 6Pathology and Forensic Medicine and
7
Clinical Neurophysiology, Kuopio University Hospital, Kuopio,
Finland.
Published online Jul 27, 2005, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20545
Address correspondence to Dr Pitkänen, A. I. Virtanen Institute for
Molecular Sciences, University of Kuopio, P.O. Box 1627, FIN-70
211, Kuopio, Finland. E-mail: asla.pitkanen@uku.fi
Received Jan 6, 2005, and in revised form Apr 8. Accepted for publication May 17, 2005.
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
211
gene and protein are increased in the hippocampus
proper20 –23 and in the dentate gyrus.22 The time of
increased cystatin C expression parallels the time of
prominent neurodegeneration and neurogenesis after
SE.13,24 These observations led us to hypothesize that
cystatin C expression is associated with alterations in
the architecture of the dentate gyrus in the chronic
phase of epilepsy, that is, during spontaneous recurrent
seizures.
To examine this hypothesis, we used a rat model of
TLE and studied the animals after they developed
spontaneous recurrent seizures, that is, epilepsy. In addition, we studied hippocampal specimens from human patients who underwent surgery for drugrefractory TLE. We addressed the following three
questions: Is the expression of cystatin C altered during
chronic stages of epilepsy? Is the expression of cystatin
C related to neuronal death, granule cell dispersion, or
neurogenesis in the dentate gyrus? Are the data from
rats and humans comparable?
Materials and Methods
Animals and Induction of Temporal Lobe Epilepsy
Adult male Sprague–Dawley rats (Harlan, AD Horst, the
Netherlands) weighing 300 to 350gm were used. All animal
procedures were approved by the Animal Care and Use
Committee of the University of Kuopio, Kuopio, Finland,
and were conducted in accordance with the guidelines set by
the European Community Council Directives 86/609/EEC.
SE, which leads to epileptogenesis and epilepsy, was triggered by electrical stimulation of the amygdala as described
in detail previously.25 Control animals (n ⫽ 7) had implanted electrodes but were not stimulated. The rats were
allowed to recover from SE spontaneously (mean duration of
the SE, 714 ⫾ 307 minutes). The occurrence of spontaneous
recurrent seizures was monitored daily during Weeks 9 and
13 after the stimulation using continuous (24hr/day) videoelectroencephalography monitoring with the system that
Nissinen and colleagues25 described. Spontaneous seizures
were identified from the electroencephalography recordings,
and an electrographic seizure was defined as a high-frequency
(⬎5 Hz), high-amplitude (more than two times baseline)
discharge that lasted at least 5 seconds. If an electrographic
seizure was observed, behavioral severity was analyzed from
the corresponding video recording. Only the rats that had
spontaneous epileptic seizures were used for further experiments (n ⫽ 8). The mean seizure frequency was 23.5 ⫾
25.2 seizures per day during the first follow-up (Week 9) and
23.8 ⫾ 24.0 seizures per day during the second follow-up
(Week 13).
Human Subjects
Autopsy tissue for control specimens
was available from 11 cases (4 women and 7 men; mean age,
54 ⫾ 20 years; range, 23– 81 years). Causes of death included pancreatitis (n ⫽ 1), pulmonary embolism (n ⫽ 2),
peritonitis (n ⫽ 1), cardiomyopathy (n ⫽ 1), alcohol intoxication (n ⫽ 1), stabbing (n ⫽ 1), suicide by hanging (n ⫽
CONTROL SUBJECTS.
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3), and other causes (n ⫽ 1). The postmortem delay was
14 ⫾ 7 hours. Subjects had no clinical evidence of dementia
or history of brain disease. Tissue from each case was studied
by a neuropathologist to confirm the absence of neurological
disease.
Epileptic
hippocampal specimens were available from 61 patients (30
women and 31 men; mean age, 34 ⫾ 10 years; range, 18 –56
years) who underwent epilepsy surgery at the Kuopio University Hospital, Kuopio, Finland, for drug-refractory TLE.
The duration of epilepsy at the time of surgery was 20 ⫾ 13
years. The causes associated with epilepsy included hippocampal sclerosis (n ⫽ 26), ischemic insult (n ⫽ 5), tumor
(n ⫽ 7), cortical dysplasia (n ⫽ 5), central nervous system
infection (n ⫽ 5), and head trauma (n ⫽ 1). No clear causal
factors could be identified in 12 patients (cryptogenic epilepsy). All patients underwent a thorough clinical, electrophysiological, neuropsychological, and imaging evaluation
before epilepsy surgery. All human material was obtained
from the Kuopio University Hospital. Permission for the
study was obtained from the Ethics Committee of the Kuopio University Hospital and the National Authority for Medicolegal Affairs. Patient consent was obtained according to
the Declaration of Helsinki.
PATIENTS WITH TEMPORAL LOBE EPILEPSY.
Processing of Tissue for Histology
Fourteen weeks after SE, control and epileptic rats were transcardially perfused with Timm protocol, postfixed, and cryoprotected as Nissinen and colleagues25 described previously.
The brains were serially cut with a sliding microtome into
30␮m-thick coronal sections (one-in-five series). Tissue
available from autopsy and epilepsy surgery was processed as
Mikkonen and colleagues26 described previously. Adjacent
sections were used for Nissl staining, cystatin C, and polysialylated neural cell adhesion molecule (PSA-NCAM) immunohistochemistry.
Assessment of Neuronal Degeneration and Granule
Cell Dispersion
Cytoarchitectonic boundaries, neuronal damage, and granule
cell dispersion in the rat and human hippocampus were analyzed from thionin-stained sections. In rats, the analyses
were performed from the temporal (ventral) end of the dentate gyrus, which corresponds to the anterior dentate gyrus in
humans that was available from patients with TLE.
In rats, hilar cell death was scored from three sections
(150␮m apart) starting at ⫺5.30mm from bregma according
to Paxinos and Watson.27 In humans, three adjacent representative sections (400␮m apart) from the resected tissue
were analyzed from each case. Severity of damage was categorized into four classes: normal (score 0, no neuronal loss
detected), mild (score 1, ⬍20% of the neurons were lost in
the particular subfield), moderate (score 2, 20 –50% of the
neurons lost), or severe (score 3, ⬎50% of the neurons lost).
The mean score ⫾ standard deviation was calculated from
the scored sections. This method provides an accurate and
semiquantitative description of neuronal loss that can be
used to compare severity of neurodegeneration among the
subjects included in this study but does not refer to actual
neuronal numbers. The semiquantitative scoring using an ordinal scoring scale was chosen because it is not possible to do
unbiased stereology in human surgical tissue samples where
the entire region of interest is not present. The data were
further subjected to nonparametric statistics, which are
stricter than parametric ones, and a bigger difference between
parameters is needed to demonstrate a statistical significance
in results.
Granule cell dispersion in rats was classified as present or
not present, and the thickness of the granule cell layer was
measured from the midportion of the temporal granule cell
layer using Stereo Investigator software in a Neurolucida
morphometry system (MicroBrightField, Colchester, VT). In
humans, the appearance of the granule cell layer was divided
into five categories based on the morphology and organization of the granule cell layer as follows: control, granule cell
layer is formed from a dense band of granule cells that are
close to each other and displays regular inner and outer borders (see Fig 4A); epileptic with nondispersed appearance,
granule cell layer in the epileptic specimens that resembles
the organization in control specimen (see Fig 4C); dispersion, the granule cell bodies are dispersed into the molecular
layer forming an irregular outer border (see Fig 4E); double
layer, granule cell bodies are organized in two layers (see Fig
4G); and death, granule cell death prevails (see Fig 4I) (modified from Houser7).
Immunohistochemical Analysis of
Cystatin C Expression
To study cystatin C expression in chronic epileptic rat (sections sampled at 300␮m intervals starting at ⫺2.30mm from
bregma according the atlas of Paxinos and Watson27) and
human hippocampus (three sections sampled at 400␮m intervals), we immunohistochemically stained hippocampal
sections with a polyclonal rabbit anti–human cystatin C antibody (#A0451; DAKO, Glostrup, Denmark). Briefly, the
free-floating sections were treated with 1% H2O2 (rat) or 3%
H2O2 in 10% methanol (human) to remove the endogenous
peroxidase activity, after which human sections were treated
with 1% sodium borohydride for 15 minutes. Sections were
blocked with 10% normal goat serum (NGS) and 0.5% Triton X-100 (Fluka Chemie GmbH, Buchs, Germany) in
0.05M Tris-buffered saline (TBS, pH 7.4) before incubation
with a cystatin C antibody (1:40,000; DAKO) for 2 days at
⫹4°C. Sections were then washed (2% NGS in TBS) and
incubated with a biotinylated goat anti–rabbit IgG (1:200;
#BA-1000; Vector Laboratories, Burlingame, CA) diluted in
1% NGS, 0.3% Triton X-100 in TBS. After washing (2%
NGS in TBS), the sections were incubated with avidinbiotin-peroxidase complex according to the manufacturer’s
instructions (Vectastain ABC Standard, #PK-4000; Vector
Laboratories). The peroxidase activity was visualized in the
rat in 0.05% 3,3⬘-diaminobenzidine (DAB; Pierce Chemical
Company, Rockford, IL) and 0.04% H2O2 in TBS and in
humans in nickel-3-3⬘-DAB solution (0.5% nickel ammonium sulfate, 0.5mg/ml DAB, 10% ␤-D(⫹)- glucose, 0.2%
ammonium chloride, and 1␮l/ml glucose oxidase [#G-6891;
Sigma-Aldrich Chemical, St. Louis, MO]). Rat sections were
intensified with osmium tetroxide and thiocarbohydrazine
according to method of Lewis and colleagues28 before cover-
slipping with Depex (BDH Chemical, Poole, United Kingdom). Sections from control subjects and patients with epilepsy were processed simultaneously.
The specificity of the antibody was tested either by omitting the primary antibody or by preincubating the primary
antibody overnight with excess of human cystatin C protein
(0.5–10␮g/ml; Calbiochem, La Jolla, CA) both in rat and
human tissue. These resulted in the disappearance of all specific immunostaining in a concentration-dependent manner
(data not shown).
Cystatin C immunoreactivity (ir) was analyzed in the rat
in CA1 and CA3 subfields of the hippocampus and in the
dentate gyrus (molecular layer, granule cell layer, and hilus of
the dentate gyrus) from septal (five sections) and temporal
parts (two sections) of the hippocampus. In the human sections, cystatin C-ir was scored in CA1, CA2, and CA3 subfields of the hippocampus and in the dentate gyrus. Because
unbiased stereology is not possible in human surgical tissue
samples, we scored cellular cystatin C-ir semiquantitatively as
follows: score 0, no ir cells present; score 1, a few ir cells
present (Figs 1B and 2B; see also Fig 4B); score 2, a moderate number of ir cells (see Fig 4D); and score 3, a high
number of cystatin C-ir cells present (see Figs 1D, 2D, and
4F). According to the cellular morphology, neurons and glial
cells were scored separately in all areas. Mean value ⫾ standard deviation was calculated from the scored sections for
each area separately. The data were analyzed by using nonparametric statistics.
To analyze the cellular location of cystatin C in the human tissue, we double labeled control and epileptic sections
with cystatin C and an astrocytic marker GFAP (anti–glial
fibrillary acidic protein, Boehringer Mannheim, Germany),
or neuronal marker NeuN (antineuronal nuclei protein,
Chemicon International, Temecula, CA). The protocol was
essentially the same as discussed earlier but with minor modifications. The mixture of secondary antibodies, biotinylated
goat anti–rabbit IgG (Vector Laboratories) and Alexa 488
conjugated goat anti–mouse IgG (1:500; #A-11001; Molecular Probes, Leiden, the Netherlands), was applied overnight
followed by washing and 3 hours in Cy5-conjugated streptavidin (2␮g/ml; Jackson Immunoresearch, West Grove, PA).
Images were captured with a Nikon laser-scanning confocal
microscope (Nikon GmbH, Düsseldorf, Germany) equipped
with Ultra View LCI confocal imaging system (Perkin
Elmer, Fremont, CA). The excitation/emission wavelengths
of 488/525 (Alexa 488) and 647/700nm (Cy5) were used.
Analysis of Neurogenesis Using Polysialylated Neural
Cell Adhesion Molecule
To visualize the newborn immature neurons in the dentate
gyrus of human specimens, we stained 3 human sections (7
control subjects and 39 patients with epilepsy) with a mouse
monoclonal anti–PSA-NCAM antibody (12E3 IgM; 1:800; a
generous gift from Dr Tatsunori Seki, Tokyo, Japan29). The
specificity of the antibody in human tissue has been described earlier, and there is no cross-reactivity with other
proteins.26 The protocol was essentially the same as for cystatin C staining in human tissue with minor modifications.
The secondary antibody was a biotinylated goat anti–mouse
IgM (1:300; #BA-2020; Vector Laboratories), and the stain-
Pirttilä et al: Cystatin C in Chronic Epilepsy
213
Fig 1. Representative digital photomicrographs demonstrating cystatin C expression in the temporal dentate gyrus of a control and
an epileptic rat. (A, C) Thionin-stained sections; (B, D) the adjacent sections stained for cystatin C. In the control rat (B), light
cystatin C immunoreactivity (ir) was present in the molecular layer of the temporal dentate gyrus (arrows indicate cystatin C-ir
cells). In the chronic epileptic rat (D), there was an increase in cystatin C expression (arrows indicate cystatin C-ir cells). Note that
the morphology of the cells resembled that of glial cells (insets in B and D). Furthermore, in the chronic epileptic rat with increased cystatin C -ir, there was a substantial dispersion of the granule cell layer in the temporal dentate gyrus (C, two-headed arrow indicates the dispersed granule cell layer). g ⫽ granule cell layer; m ⫽ molecular layer of dentate gyrus. Scale bar ⫽ 50␮m
(A–D).
ing was visualized in nickel-3-3⬘-DAB. PSA-NCAM–immunopositive cells in the dentate gyrus were plotted and the
boundaries of the dentate granule cell layer were drawn with
a computer-aided digitizing system (Minnesota Datametrics,
St. Paul, MN). Then the length of the granule cell layer was
measured from the line drawings using a digitizing table
(Summasketch II Professional; Summagraphics Corp., CT)
and Sigma-Scan V3.92 MS-DOS software (Jandel Scientific,
San Raphael, CA). The number of PSA-NCAM–positive
cells in the subgranule zone, granule cell layer, and molecular
layer of the dentate gyrus were calculated. The results are
presented as the mean value (PSA-NCAM cells/mm) ⫾ standard deviation of three consecutive sections.
Statistics
All statistical analyses were performed with SPSS for Windows (version 11.5; SPSS, Chicago, IL). Differences between
two groups were assessed using a nonparametric Mann–
Whitney U test. The statistical significances of multiple
groups were calculated using a nonparametric Kruskall–Wallis test followed by a post hoc test for multiple compari-
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sons.30 Correlations were assessed with a two-tailed Spearman’s correlation coefficient. A p value less than 0.05 was
considered significant.
Results
Rat Temporal Lobe Epilepsy
CYSTATIN C EXPRESSION IN THE CONTROL AND EPILEPTIC DENTATE GYRUS.
First, we immunohistochemi-
cally examined cystatin C expression in the septal and
temporal dentate gyrus of chronic epileptic rats. The
summary of the results is shown in Table 1. In control
animals, weak cystatin C-ir was present in all subregions (molecular layer, granule cell layer, and hilus)
that were analyzed (see Fig 1B). Cystatin C–positive
cells had both glial-like (see inset in Fig 1B) and
neuronal-like morphologies under light microscopy. In
epileptic animals, the density of cystatin C–expressing
cells with a glial morphology was increased in the septal hilus as compared with the control rats ( p ⬍ 0.01),
Fig 2. Representative digital photomicrographs demonstrating cystatin C expression and its cellular localization in the human dentate gyrus. (A, C) Thionin-stained sections; (B, D) the adjacent sections stained for cystatin C. In the control hippocampus (B), only
weak cystatin C-ir was present in the molecular layer and hilus of dentate gyrus. A robust increase in the number of cystatin C-ir
cells was observed in the epileptic hippocampus, especially in the molecular layer of the dentate gyrus (D). Note that the morphology
of the cystatin C-ir cells resembled that of glia (arrows in B and D), but there were also cells with neuronal-like morphology, particularly in the hilus of control hippocampus (arrowhead in B). (E, G) Morphology of cystatin C–immunoreactive cells with greater
magnification. Cystatin C colocalized with an astrocytic marker, glial fibrillary acidic protein (GFAP; F), and neuronal marker,
neuronal nuclei protein (NeuN; H), thus confirming the astrocytic and neuronal localization of cystatin C expression. cystC ⫽ cystatin C; g ⫽ granule cell layer; h ⫽ hilus; m ⫽ molecular layer of dentate gyros. Scale bar ⫽ 100␮m (A–D).
whereas the number of cystatin C–expressing cells with
a neuronal morphology was decreased ( p ⬍ 0.01). At
the temporal end, the most prominent increase in cystatin C-ir occurred in the molecular layer of the dentate gyrus ( p ⬍ 0.05; see Fig 1D) in cells with a glial
morphology (see insert in Fig 1D). There was, however, no change in neuronal cystatin C staining at the
temporal end compared with control subjects.
ASSOCIATION OF CYSTATIN C EXPRESSION WITH NEURONAL DAMAGE AND GRANULE CELL DISPERSION IN THE
RAT DENTATE GYRUS.
Previous studies suggest that
cystatin C has a role in neuronal cell death.15,31 In
the amygdala stimulation model of TLE, there is a
prominent loss of hilar cells25; thus, we investigated
the association between increased cystatin C expression and hilar cell death. In the analysis we focused
on the temporal end of the rat dentate gyrus because
it corresponds to the human anterior dentate gyrus,
which typically is removed during epilepsy surgery.
All epileptic animals had bilateral hilar cell death
(mean score, 2.4 ⫾ 0.8 on the side of seizure focus) compared with control animals ( p ⬍ 0.01).
A high number of spontaneous seizures was associated with more severe hilar cell damage (r ⫽ 0.853,
p ⬍ 0.05) and with increased cystatin C expression
Pirttilä et al: Cystatin C in Chronic Epilepsy
215
Table 1. Cystatin C Immunoreactivity in Different Regions of the Rat and Human Dentate Gyrus (mean ⫾ SD)
Hilus
Rat
Control (7)
Septal
Temporal
Epileptic (8)
Septal
Temporal
Human
Control (11)
Epileptic (61)
Symptomatic (49)
HC sclerosis (26)
Ischemia (5)
Tumor (7)
Dysplasia (5)
Infection (5)
Trauma (1)
Cryptogenic (12)
Granule Cell Layer
Molecular Layer
Neurons
Glia
Neurons
Glia
Neurons
Glia
1.6 ⫾ 0.3
1.9 ⫾ 0.8
1.3 ⫾ 0.3
2.1 ⫾ 0.5
1.8 ⫾ 0.6
1.9 ⫾ 0.7
1.1 ⫾ 0.2
1.1 ⫾ 0.4
0
0
1.8 ⫾ 0.5
1.7 ⫾ 0.5
1.2 ⫾ 0.3a
1.4 ⫾ 0.7
2.2 ⫾ 0.5b
2.6 ⫾ 0.5
1.3 ⫾ 0.4
1.4 ⫾ 0.4
1.3 ⫾ 0.2
1.3 ⫾ 0.4
0
0
2.1 ⫾ 0.6
2.6 ⫾ 0.5a
1.5 ⫾ 0.9
1.0 ⫾ 0.5a
1.0 ⫾ 0.4
1.1 ⫾ 0.6
1.0 ⫾ 0.0
1.0 ⫾ 0.2
1.0 ⫾ 0.0
0.9 ⫾ 0.4
1.0 ⫾ 0.0
0.9 ⫾ 0.4
1.3 ⫾ 0.9
1.1 ⫾ 0.5
1.1 ⫾ 0.6
1.1 ⫾ 0.6
1.0 ⫾ 0.0
1.1 ⫾ 0.3
1.4 ⫾ 1.1
1.0 ⫾ 0.0
2.0 ⫾ 0.0
1.2 ⫾ 0.4
1.5 ⫾ 0.9
1.1 ⫾ 0.5
1.2 ⫾ 0.5
1.2 ⫾ 0.6
1.1 ⫾ 0.3
1.0 ⫾ 0.1
1.3 ⫾ 0.7
1.0 ⫾ 0.0
1.0 ⫾ 0.0
1.0 ⫾ 0.0
0.3 ⫾ 0.5
0.5 ⫾ 0.5
0.5 ⫾ 0.5
0.5 ⫾ 0.6
0.2 ⫾ 0.4
0.4 ⫾ 0.5
0.6 ⫾ 0.5
0.6 ⫾ 0.5
1.0 ⫾ 0.0
0.6 ⫾ 0.5
0
0
0
0
0
0
0
0
0
0
0.6 ⫾ 0.5
1.4 ⫾ 0.6c
1.4 ⫾ 0.7b
1.6 ⫾ 0.7b
1.3 ⫾ 0.7
1.0 ⫾ 0.3
1.1 ⫾ 0.6
1.5 ⫾ 0.5
2.3 ⫾ 0.0
1.0 ⫾ 0.1
Rat data are from the left hemisphere, which was also the side of the seizure focus. There were no differences between the hemispheres
(Wilcoxon rank test).
a
p ⬍ 0.05 as compared with the control; bp ⬍ 0.01 as compared with the control; cp ⬍ 0.001 as compared with the control.
SD ⫽ standard deviation.
in the molecular layer of the dentate gyrus (r ⫽
0.737, p ⬍ 0.05), but there was no clear correlation
between increased cystatin C expression and hilar cell
death.
Based on our earlier observations, we estimated that
the rats had been epileptic for about 10 weeks (median
time to epilepsy is about 4 weeks; see Nissinen and
colleagues25). Of eight epileptic animals, six (75%) had
granule cell dispersion at the temporal end of the dentate gyrus, whereas there was no dispersion at the septal
end. In control animals, the thickness of the granule
cell layer at the temporal end was 51 to 77␮m, whereas
in chronic epileptic rats with dispersion, it was 119 to
165␮m ( p ⬍ 0.05). In epileptic rats, neither doublelayer formation nor granule cell loss was detected. At
the temporal end, the hilar cell damage was more severe in animals with granule cell dispersion than in animals with no dispersion (mean damage score, 2.6 ⫾
0.6 vs 0.1 ⫾ 0.4; p ⬍ 0.01). Furthermore, the animals
with granule cell dispersion had more intense cystatin
C-ir in the molecular layer compared with the animals
with no dispersion (mean cystatin C-ir score, 2.7 ⫾
0.5 vs 1.8 ⫾ 0.5; p ⬍ 0.05).
Human Temporal Lobe Epilepsy
CYSTATIN C EXPRESSION IN THE DENTATE GYRUS IN CONTROL SUBJECTS AND PATIENTS WITH TEMPORAL LOBE EPILEPSY. In control specimens, there was weak cystatin
C-ir in all areas of the dentate gyrus (hilus, granule cell
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layer, and molecular layer; see Table 1 and Fig 2B).
The morphology of the cystatin C–positive cells resembled that of glia (see Fig 2E), but neuronal profiles
were also observed, especially in the hilus (see Figs 2B,
G), but also in CA1, CA2, and CA3 regions (data not
shown). In the epileptic specimens, however, there was
a robust increase in cystatin C-ir cells with glial morphology in the molecular layer of the dentate gyrus as
compared with control specimens ( p ⬍ 0.001; see Fig
2D). In contrast, there was a reduction in cystatin
C–labeled neurons in the hilus of the dentate gyrus in
the epileptic specimens in comparison with control
subjects ( p ⬍ 0.05). In addition, cystatin C-ir in gliallike cells was also increased in the CA1 ( p ⬍ 0.05),
CA2 ( p ⬍ 0.001), and CA3 ( p ⬍ 0.01) subfields of
the hippocampus proper in epileptic specimens (data
not shown). Double labeling and confocal microscopy
further confirmed that cystatin C was expressed in astrocytes and neurons in the brains of control subjects
and human patients with epilepsy (see Figs 2F, H).
Next, we studied whether increased cystatin C expression in the molecular layer of the dentate gyrus was
related to a specific etiology. The results are summarized in Table 1. Cystatin C expression in the molecular layer was particularly increased in patients with
symptomatic TLE ( p ⬍ 0.01). Within this category, a
subgroup of patients with hippocampal sclerosis exhibited the most pronounced cystatin C expression compared with control subjects ( p ⬍ 0.01; see later).
ASSOCIATION OF CYSTATIN C EXPRESSION WITH NEURONAL DAMAGE AND GRANULE CELL DISPERSION IN THE
HUMAN DENTATE GYRUS.
Cystatin C-ir in the molec-
ular layer of the dentate gyrus was associated with hilar
cell damage, that is, more pronounced cystatin C expression in the molecular layer was observed in subjects
with more severe cell death in the hilus (r ⫽ 0.356,
p ⬍ 0.01). There was, however, no correlation between
cystatin C-ir and the severity of epilepsy for duration
of epilepsy or number of seizures during the year before surgery, although the longer duration of epilepsy
typically was associated with more pronounced hilar
cell death (r ⫽ 0.329, p ⬍ 0.05).
There was no association between cystatin C-ir in
the molecular layer and granule cell death as assessed
by scoring the loss of neurons in the granule cell layer.
There was, however, a difference in the degree of cystatin C-ir between different categories of granule cell
dispersion including the control group ( p ⬍ 0.01; Fig
3). Representative samples of cystatin C-ir in different
types of granule cell dispersion are shown in Figure 4.
In control specimens, there was only weak cystatin C-ir
in the molecular layer (see Figs 3B and 4B). Patients
with TLE and nondispersed granule cell layer had
slightly enhanced cystatin C-ir in the molecular layer
compared with that in control specimens (see Figs 3
and 4D). When granule cells were dispersed to the molecular layer, there was, however, a robust increase in
cystatin C-ir in the molecular layer compared with
control subjects ( p ⬍ 0.01; see Figs 3 and 4F). Also,
when the dispersion of granule cells was more pronounced, that is, granule cells formed a double-layer–
like structure, cystatin C-ir tended to be greater than in
control subjects (see Fig 3).
POLYSIALYLATED NEURAL CELL ADHESION MOLECULE IMMUNOREACTIVITY IN THE HUMAN EPILEPTIC HIPPOCAMPUS. The hippocampus produces new neurons in the
adult mammalian brain,12 and seizures in experimental
animal models dramatically increase neurogenesis.13
Whether the seizure activity in human chronic epilepsy
leads to increased neurogenesis has, however, remained
under dispute. Because cystatin C expression is increased after seizures and previous studies have related
cystatin C to neurogenesis,16 we investigated the degree
of neurogenesis in the human epileptic tissue by using
an immature neuronal marker PSA-NCAM.
To estimate the effects of chronic seizures on the
genesis of newborn neurons in the dentate gyrus, we
analyzed the total number of PSA-NCAM–positive
cells. The results are shown in Figure 5A. In the human control brain, there were numerous PSA-NCAM–
positive cells present along the granule cell layer (8.2 ⫾
1.5 cells/mm). The density of PSA-NCAM–positive
cells in the TLE specimens was, however, greatly reduced (4.7 ⫾ 3.2 cells/mm; p ⬍ 0.01; see Fig 5A),
Fig 3. Graph showing cystatin C–immunoreactivity (ir) in
control subjects and patients with temporal lobe epilepsy (TLE)
and in subgroups of patients with TLE divided based on the
granule cell layer organization. Data are presented as mean ⫾
standard error of the mean. Note that the cystatin C-ir was
increased in the patients with TLE compared with control
subjects (***p ⬍ 0.001, Mann–Whitney U test). Within TLE
specimens, a subgroup of patients in which granule cells were
dispersed to the molecular layer had the most pronounced cystatin C expression (***p ⬍ 0.01 compared with the control,
Kruskall-Wallis followed by a post hoc test for multiple comparisons).
especially in the patients with symptomatic TLE
(4.5 ⫾ 3.3 cells/mm; p ⬍ 0.01; see Fig 5A). In this
subgroup of patients, hippocampal sclerosis (2.8 ⫾ 1.7
cells/mm) as the underlying etiology was particularly
associated with decreased levels of PSA-NCAM–positive cells in the dentate gyrus ( p ⬍ 0.01; see Fig 5A).
MIGRATION OF POLYSIALYLATED NEURAL CELL ADHESION
MOLECULE–POSITIVE CELLS IN THE DENTATE GYRUS AND
ITS ASSOCIATION WITH GRANULE CELL DISORGANIZATION AND CYSTATIN C EXPRESSION. Next, to estimate
the effects of chronic seizures on the migration of newborn neurons in the dentate gyrus, we analyzed the
number of PSA-NCAM–positive cells in the subgranule zone consisting of a two-cell diameter zone below
the granule cell layer, in the granule cell layer, and in
the molecular layer of the dentate gyrus. The results are
summarized in Table 2. In control specimens, the majority of PSA-NCAM–positive cells were located in the
subgranule zone (47%) and the granule cell layer
(49%). Few cells (3%) were in the molecular layer. In
the dentate gyrus of patients with TLE, 38% of all
PSA-NCAM–positive cells were located in the granule
cell layer compared with 49% in control subjects ( p ⬍
0.05). Also, the percentage of PSA-NCAM–positive
cells in the molecular layer was increased in epileptic
specimens (12%) compared with control samples (3%;
p ⬍ 0.05); that is, the surviving newborn granule cells
migrated to an abnormal location in the molecular
layer.
Pirttilä et al: Cystatin C in Chronic Epilepsy
217
Figure 4
218
Annals of Neurology
Vol 58
No 2
August 2005
When the granule cells were dispersed, the percentage of PSA-NCAM–positive cells in the molecular
layer tended to be greater than in control samples. Because of the limited number of patients in this category, the difference was not significant, although two
of four patients had a clearly increased number of PSANCAM–positive cells in the molecular layer compared
with control subjects (21% and 32% vs 3% in the control group). In cases with depleted granule cells, the
number of PSA-NCAM–positive cells in the molecular
layer was greater than in control subjects ( p ⬍ 0.05)
and comprised 28% of all PSA-NCAM–positive cells
compared with 3% in control subjects ( p ⬍ 0.01). A
greater percentage of PSA-NCAM–positive cells in the
molecular layer was also associated with greater density
of cystatin C-ir cells in the molecular layer (r ⫽ 0.371,
p ⬍ 0.05).
Discussion
These experiments were designed to elucidate whether
cystatin C expression is altered during the chronic
phase of epilepsy, and whether its expression is related
to typical TLE-associated neuropathological findings in
the dentate gyrus, such as hilar cell death, granule cell
dispersion, and neurogenesis. For this study, we used
hippocampal tissue samples from epileptic rats in
which the epilepsy was triggered by electrically induced
SE and from human patients with drug-refractory epilepsy who were treated with surgical resection of the
hippocampus.
Cystatin C Expression Is Upregulated in the
Molecular Layer of the Dentate Gyrus in Chronic
Temporal Lobe Epilepsy
To extend our previous study, which indicated an
acute increase in cystatin C expression after SE,23 we
studied whether cystatin C upregulation persists in epileptic rats. Our data demonstrated that in rats with
chronic recurrent seizures for about 2.5 months, cystatin C expression was robustly increased in the hilus of
the septal hippocampus and in the molecular layer of
the dentate gyrus in the temporal hippocampus compared with control subjects. Most of the immunopositive cells resembled glia, which is consistent with
our previous study that demonstrated glial localization of cystatin C by double labeling and confocal
Š
Fig 5. (A) Graph demonstrating the total number of polysialylated neural cell adhesion molecule (PSA-NCAM)–positive cells
in the dentate gyrus in the control subjects and patients with
temporal lobe epilepsy (TLE) and in subgroups of patients with
TLE divided based on the underlying etiology. The data are
presented as mean ⫾ standard error of the mean. Note that the
number of PSA-NCAM–positive cells was decreased in patients
with TLE compared with control subjects (**p ⬍ 0.01, Mann–
Whitney U test). In subgroups of TLE specimens, the decrease
in PSA-NCAM–positive cells was clearly evident in the group of
patients with symptomatic epilepsy (symptomatic, **p ⬍ 0.01,
Kruskall–Wallis followed by a post hoc test for multiple comparisons) and especially with hippocampal sclerosis (HC sclerosis,
**p ⬍ 0.01) as the underlying etiology. (B, C) Representative
digital photomicrographs demonstrating PSA-NCAM immunoreactivity in the dentate gyrus of a control subject (B) and a patient with symptomatic TLE (C). Arrows point to the PSANCAM–positive neuronal bodies in the subgranule zone (sgz).
Note that the number of PSA-NCAM–positive cells is decreased
in the TLE specimen (C) compared with the control sample
(B). g ⫽ granule cell layer; m ⫽ molecular layer of dentate
gyrus; sgz ⫽ subgranule zone; TLE ⫽ temporal lobe epilepsy.
Scale bar ⫽ 50␮m (B, C).
Fig 4. Representative digital photomicrographs demonstrating the association between the cystatin C immunoreactivity (ir) and granule
cell dispersion in the human dentate gyrus. (A, C, E, G, I) Cytoarchitectonic organization of the granule cell layer in thionin-stained
preparations. (B, D, F, H, J) Adjacent sections stained for cystatin C. (A, B) In control specimens, there was weak cystatin C-ir present
in the molecular layer of the dentate gyrus (B). (C, D) A slight increase in cystatin C-ir was evident in epileptic dentate gyrus with
nondispersed granule cell layer. (E, F) When granule cells were dispersed to the molecular layer, there was robust cystatin C expression
in the molecular layer (F). (G, H) Also, in the dentate gyrus with a bilaminated granule cell layer (double layer), there was increased
cystatin C expression (H). (I, J) When granule cells had died, cystatin C expression was still above the control levels (J). The arrows in
B, D, F, H indicate cystatin C-ir cells. g ⫽ granule cell layer; m ⫽ molecular layer of dentate gyrus. Scale bar ⫽ 100␮m (A–J).
Pirttilä et al: Cystatin C in Chronic Epilepsy
219
Table 2. Distribution of PSA-NCAM–Immunopositive Cells in Different Phases of Granule Cell Dispersion in the Human Dentate
Gyrus (mean ⫾ SD)
Subgranule Zone
Control (7)
Epileptic (39)
Nondispersed (21)
Dispersion (5)
Double layer (6)
Death (7)
PSANCAM
(cells/mm)
3.9 ⫾ 1.6
2.6 ⫾ 2.2a
3.6 ⫾ 2.4
1.3 ⫾ 1.3a
1.6 ⫾ 1.0
1.3 ⫾ 0.8a
Granule Cell Layer
Percentage
of total
PSANCAM
(cells/mm)
47 ⫾ 14
49 ⫾ 16
53 ⫾ 15
46 ⫾ 7
54 ⫾ 17
33 ⫾ 14
4.0 ⫾ 0.9
1.8 ⫾ 1.2b
2.2 ⫾ 1.1a
1.5 ⫾ 2.0c
1.1 ⫾ 0.9c
1.3 ⫾ 0.5c
Molecular Layer
Percentage
of total
PSANCAM
(cells/mm)
Percentage
of total
Total,
PSANCAM
(cells/mm)
49 ⫾ 9
38 ⫾ 14a
38 ⫾ 12a
42 ⫾ 19
37 ⫾ 17
38 ⫾ 15
0.2 ⫾ 0.2
0.5 ⫾ 0.6
0.4 ⫾ 0.4
0.3 ⫾ 0.2
0.2 ⫾ 0.2
1.1 ⫾ 1.0a
3⫾3
12 ⫾ 12a
8⫾7
13 ⫾ 13a
7⫾8
28 ⫾ 15a
8.2 ⫾ 1.5
4.7 ⫾ 3.2c
6.3 ⫾ 3.4
1.6 ⫾ 0.5c
2.9 ⫾ 1.5c
3.9 ⫾ 1.5
p ⬍ 0.05; bp ⬍ 0.001;
p ⬍ 0.01 as compared with the control.
a
c
PSA-NCAM ⫽ polysialylated neural cell adhesion molecule; SD ⫽ standard deviation.
microscopy.23 Based on light microscopic analysis, the
increase in cystatin C was caused by an increased number of cystatin C–expressing cells in the dentate gyrus,
as well as denser immunostaining of the cells, which
suggests an increased amount of cystatin C protein in
the cells. These data thus support the report by
Aronica and colleagues,22 which shows persistent upregulation of cystatin C three months after SE induced
by electrical stimulation of the hippocampus in rat.
To further elucidate whether a similar increase in
cystatin C expression occurs in human epilepsy, we
studied the hippocampal specimens from patients with
drug-refractory TLE. As a control, we used autopsied
human hippocampal specimens that showed light cystatin C expression in all hippocampal subfields studied.
In the epileptic hippocampus, however, there was a robust upregulation of cystatin C expression, particularly
in the molecular layer of the dentate gyrus. Also, other
subregions of the hippocampus, the CA1, CA2, and
CA3 regions, had increased cystatin C-ir (data not
shown). To our knowledge, this is the first report demonstrating increased cystatin C expression in human
epilepsy. Increased levels of cystatin C in the human
brain have been described previously in degenerating
neurons of patients with Alzheimer’s disease.32 Based
on the morphology of the immunoreactive cells in the
light microscopic analysis, cystatin C localized to glial
cells in the molecular layer of the dentate gyrus. Neurons are rarely seen in this layer, and no cystatin C-ir
cells resembling neurons were seen in the molecular
layer. In other hippocampal regions (hilus, CA1, CA2,
and CA3 regions), the majority of cystatin C-ir cells
were glial-like, but there were also cells with a
neuronal-like morphology (data not shown). The astrocytic and neuronal localization of cystatin C was confirmed by double labeling and confocal microscopy.
Our data thus are consistent with previous observations
220
Annals of Neurology
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No 2
August 2005
that cystatin C in the human brain is expressed by astrocytes and neurons.32–34
Taken together, cystatin C expression is increased in
the molecular layer of the dentate gyrus both in rats
with frequent spontaneous recurrent seizures and in patients who underwent surgery for drug-refractory epilepsy. Because the dentate gyrus undergoes a variety of
neuropathological changes during epilepsy, cystatin C
might have a regulatory role in these alterations.
Increased Cystatin C Expression Is Associated with
Granule Cell Dispersion
In epileptic rats, the increased cystatin C expression
in the molecular layer of the temporal dentate gyrus
was most prominent when granule cells were dispersed. Dispersion was accompanied with severe hilar
cell loss, which, however, did not correlate with the
degree of cystatin C-ir in the molecular layer. Granule
cell dispersion in experimental TLE models has been
demonstrated previously in the septal hippocampus
of pilocarpine-treated rats and after intrahippocampal
injection of kainate in mice.35–37 Our report further
demonstrates that granule cell dispersion occurs in
the rat temporal hippocampus after electrically induced SE in the presence of increased cystatin C expression.
Also, in human patients with epilepsy, the cystatin
C-ir in the molecular layer of the dentate gyrus was
most strikingly increased when granule cells were dispersed to the molecular layer. In humans, granule cell
dispersion occurs particularly in patients with hippocampal sclerosis.7,9,38 Interestingly, in our series of
patients, the upregulation of cystatin C occurred particularly in patients with symptomatic epilepsy with
hippocampal sclerosis.
Because granule cell dispersion and the accompanied
hilar cell death were associated with increased cystatin
C expression in the molecular layer of the dentate gyrus, it raises a question whether the increase is primarily caused by hilar damage or dispersion, or whether
cystatin C increase precedes these phenomena. However, this is difficult to address when using human epileptic tissue where hilar cell death is almost always accompanied with granule cell dispersion. In our rat
model, however, there was hilar cell death in the septal
dentate gyrus, but no dispersion or increased cystatin C
in the molecular layer. Also, the notion that cystatin C
was not increased in the hilus, although robust cell
death and gliosis were in this area in patients with epilepsy, indicates that increased cystatin C expression in
the molecular layer of the dentate gyrus is related to
the granule cell dispersion rather than to cell death and
gliosis.
Reduced Number and Aberrant Migration of
Newborn Polysialylated Neural Cell Adhesion
Molecule–Positive Cells in the Dentate Gyrus of
Patients with Temporal Lobe Epilepsy
Neurogenesis that occurs in the dentate gyrus of adult
mammals is greatly increased by the seizure activity as
demonstrated by animal studies.13 Because the increased cystatin C expression after seizure activity also
occurs in the dentate gyrus, it is reasonable to speculate that it might play a role in seizure-induced neurogenesis. Because the data about the effects of
chronic epilepsy on neurogenesis in humans has been
rather controversial, we studied the neurogenesis in
our set of patients using PSA-NCAM as a marker for
newborn neurons. Consistent with previous observations by Mikkonen and colleagues,26 there were a
number of PSA-NCAM–positive neurons in the dentate gyrus of control specimens. In the group of patients with chronic TLE with frequent seizures, however, the degree of neurogenesis was reduced in
comparison with control brains, especially if the overall neuronal loss in the dentate gyrus was severe. This
supports the findings by Mikkonen and colleagues,26
who demonstrated decreased numbers of PSANCAM–positive cells in adult patients with TLE with
severe hippocampal damage. Decreased numbers of
PSA-NCAM–positive cells after seizures also have
been reported in pediatric patients with TLE.39 These
observations are further supported by recent data
from a kainate model of TLE demonstrating severely
declined neurogenesis in chronic epileptic animals
at 5 months after kainate.40 In conclusion, despite
the increased expression of cystatin C in chronic epilepsy, the process of neurogenesis is markedly impaired in the hippocampus of patients with chronic
TLE, especially in the presence of severe neuronal
damage.
This study also demonstrates that the molecular
layer of the dentate gyrus contained a greater propor-
tion of PSA-NCAM–positive cells in epileptic specimens compared with control samples, thus suggesting
that the newborn cells migrate to abnormal locations
in chronic epileptic tissue. These data further support
the idea that granule cell dispersion could be caused
by abnormal migration of newborn neurons to the
molecular layer,41 although we could not demonstrate
a significant increase in PSA-NCAM–positive cells in
the molecular layer in patients with granule cell dispersion due to limited number of patients in this category. These results thus demonstrate that the migration of newborn granule cells also is affected by
prolonged seizure activity.
Cystatin C Expression, Migration of Polysialylated
Neural Cell Adhesion Molecule–Positive Cells, and
Granule Cell Dispersion
It has been suggested that cystatin C has a role in
neurogenesis because it is required for the activity of a
major neurogenic protein, fibroblast growth factor2.16 Previous studies also have indicated that glial
cells might secrete factors that induce proliferation,
migration, and differentiation of neural progenitors.42,43 Thus, we hypothesize that glial cells expressing cystatin C in the molecular layer of the dentate
gyrus might provide cues for the migrating PSANCAM–positive cells, thereby contributing to the
formation of a dispersed granule cell layer. Our recent
studies further support this by showing that migration of newborn PSA-NCAM– or TUC-4 –positive
cells is reduced in cystatin C knock-out mice.44 The
exact mechanism by which cystatin C confers its
growth-promoting function on the cellular level requires further study.
Conclusions
This study investigated the expression of cystatin C in
chronic epilepsy and its association with common
neuropathological features of TLE. The data demonstrated that cystatin C expression was upregulated in
the molecular layer of the dentate gyrus during the
chronic stages of epilepsy both in the rat model of
TLE and in human patients with TLE. The expression was localized particularly to glial cells, and it was
associated with the granule cell dispersion rather than
with cell loss. Furthermore, the neurogenesis was decreased in patients with TLE, and the surviving newborn granule cells migrated to aberrant locations in
the molecular layer where increased cystatin C expression was present. These data suggest that cystatin C
plays a role in ongoing network reorganization in the
epileptic dentate gyrus, especially in granule cell dispersion and guidance of migrating newborn granule
cells.
Pirttilä et al: Cystatin C in Chronic Epilepsy
221
The study was supported by the Academy of Finland (A.P.), the
Research and Science Foundation of Farmos (T.J.P.), the Finnish
Medical Foundation (T.J.P.), the Finnish Medical Society Duodecim (T.J.P.), the Jenny and Antti Wihuri Foundation (T.J.P.), the
Lilly Foundation (T.J.P.), the North-Savo Regional Fund of the
Finnish Cultural Foundation (T.J.P.), the Sigrid Juselius Foundation (A.P.), and the Vaajasalo Foundation (A.P.).
We thank M. Lukkari and M. Tikkanen for their excellent technical
assistance.
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