 |
 Previous Article | Next Article 
The Journal of Neuroscience, October 15, 1998, 18(20):8382-8393
Patterns of Status Epilepticus-Induced Neuronal Injury during
Development and Long-Term Consequences
Raman
Sankar1, 2, 3,
Don
H.
Shin1, 2, 3,
Hantao
Liu1, 3,
Andrey
Mazarati1, 3,
Anne
Pereira de
Vasconcelos4, and
Claude G.
Wasterlain1, 3
Departments of 1 Neurology and
2 Pediatrics, University of California Los Angeles School
of Medicine, Los Angeles, California 90095-1752, 3 Epilepsy
Research Laboratories, Veterans Affairs Medical Center, Sepulveda,
California 91343, and 4 Institut National de la Santé
et de la Recherche Médicale U 398, 67085 Strasbourg,
France
 |
ABSTRACT |
The lithium-pilocarpine model of status epilepticus (SE) was used
to study the type and distribution of seizure-induced neuronal injury
in the rat and its consequences during development. Cell death was
evaluated in hematoxylin- and eosin-stained sections and by electron
microscopy. Damage to the CA1 neurons was maximal in the 2- and
3-week-old pups and decreased as a function of age. On the other hand,
damage to the hilar and CA3 neurons was minimal in the 2-week-old rat
pups but reached an adult-like pattern in the 3-week-old animals, and
damage to amygdalar neurons increased progressively with age. The
3-week-old animals also demonstrated vulnerability of the dentate
granule cells. To evaluate neuronal apoptosis, we used terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling (TUNEL) stain, confocal fluorescence microscopy of ethidium
bromide-stained sections, electron microscopy, and DNA electrophoresis.
Neurons displaying all of those features of apoptotic death in response
to SE were seen in the CA1 region of the 2-week-old pups and in the
hilar border of the dentate granule cells of the 3-week-old animals.
Some (3/11) of the animals that underwent SE at 2 weeks of age and most
of the animals that underwent SE at 3 or 4 weeks of age (8/11 and 6/8,
respectively) developed spontaneous seizures later in life; the latter
showed SE-induced synaptic reorganization as demonstrated by Timm
methodology. These results provide strong evidence for the
vulnerability of the immature brain to seizure-induced damage, which
bears features of both necrotic and apoptotic death and contributes to
synaptic reorganization and the development of chronic epilepsy.
Key words:
status epilepticus; seizures; lithium; pilocarpine; hippocampus; apoptosis; necrosis; mossy fiber sprouting; epileptogenesis; rats; development
| |
INTRODUCTION |
In adult animals, seizures cause
selective neuronal death as a result of excessive neuronal activation,
even in the absence of systemic complications (Meldrum et al., 1973 ;
Nevander et al., 1985). Status epilepticus (SE) can produce neuronal
damage by excessive activation of both NMDA and non-NMDA ionotropic
glutamate receptors, and both NMDA and non-NMDA receptor antagonists
have demonstrated neuroprotective properties in various seizure
paradigms (Fujikawa, 1994, 1995; Penix and Wasterlain, 1994; Penix et
al., 1996). Different neuronal populations in the hippocampus
demonstrate selectively apoptotic or necrotic features of cell death in
response to perforant path stimulation, a model of focal SE (Sloviter
et al., 1996). Administration of kainic acid (KA) by intra-amygdaloid (Pollard et al., 1994), subcutaneous (Morrison et al., 1996), or
intraperitoneal (Filipkowski et al., 1994) route results in SE and
apoptosis of select populations of neurons. In the intra-amygdaloid model (Pollard et al., 1994), coadministration of diazepam prevented the seizures and apoptosis of neurons in the CA3 region, but not in the
amygdala, suggesting that the apoptotic hippocampal neurons reflect
seizure-induced excitotoxic damage.
In the developing brain, the effect of seizures on neuronal survival
and brain growth have been controversial (Camfield, 1997; Wasterlain,
1997). Excitotoxic neuronal death is influenced by brain maturation
(Portera-Cailliau et al., 1997a,b). Because apoptosis plays an
important role in early brain development it is possible that the
immature brain displays a special vulnerability to programmed cell
death in response to seizures. Animal models suggest the existence in
the young of both unique protective mechanisms as well as enhanced
vulnerability to specific types of experimental SE (Sankar et al.,
1995). Rat pups as young as 2 weeks demonstrate seizure-induced
elevation in serum neuron-specific enolase accompanied by histological
evidence of damage as a result of SE (Sankar et al., 1997b). Our
understanding of seizure-induced brain damage in immature animals
remains patchy because available reports are based on a limited
sampling of models and ages (Albala et al., 1984; Okada et al., 1984;
Sperber et al., 1991, 1992; Thompson and Wasterlain, 1997; Thompson et
al., 1998), or they have examined specific aspects such as EEGs (Hirsch
et al., 1992) or behavior (Liu et al., 1994) without a detailed study
of the histopathology.
The cholinergic agent pilocarpine (PC) produces limbic and generalized
SE in rodents accompanied by widespread brain damage in mature rats
(Turski et al., 1983). The use of PC-induced seizures as a model for
studying SE and SE-induced brain damage and epileptogenesis has been
well described (Cavalheiro, 1995; Cavalheiro et al., 1996).
Furthermore, the susceptibility of rats to PC-induced seizures appeared
to be age-dependent (Cavalheiro et al., 1987). Clifford and
collaborators (1987) have shown that pretreatment with lithium (Li)
potentiates the epileptogenic action of PC (Honchar et al., 1983; Jope
et al., 1986), reduces mortality, and avoids many of the peripheral
cholinomimetic side effects of PC.
We report here the pattern of apoptosis and necrosis resulting from SE
induced by the administration of lithium and pilocarpine in both
developing and adult rat brains. Both morphological and biochemical
methods have been used to detect the pattern of seizure-induced brain
damage at different developmental stages. We also report the
development of spontaneous seizures and seizure-associated synaptic
plasticity in the mossy fiber terminals as visualized by Timm
histochemistry.
Some of the results have appeared previously in abstract form (Sankar
et al., 1997a).
| |
MATERIALS AND METHODS |
Animals. Wistar rats (Simonsen Labs, Gilroy, CA) of
either sex were used in this study. The Committees on Animal Research at the University of California, Los Angeles, and the Sepulveda Veterans Affairs Medical Center approved all protocols. Rat pups of 2, 3, and 4 weeks of age and mature rats (12-16 weeks of age) were given
3 mEq/kg lithium chloride (Sigma, St. Louis, MO) intraperitoneally on
the day before the induction of SE. Seizures progressing to SE were
induced by subcutaneous injection of 60 mg/kg pilocarpine hydrochloride
(Sigma). Control animals were given an equal volume of saline
subcutaneously. Rats were observed for behavioral evidence of seizures.
Only rats displaying behavioral manifestations of seizures described
previously (Jope et al., 1986; Hirsch et al., 1992) were used. The
selected animals underwent blood gas monitoring 5 min before the
administration of PC, and 2, 4, and 6 hr after. We have previously
recorded electrographic seizures from both the hippocampus and parietal
cortex of selected animals of each age and have previously published
selected epochs (Kornblum et al., 1997). A detailed ontogenetic study
of the EEG during lithium-pilocarpine (LiPC)-induced SE has been
published by Hirsch et al. (1992).
Histology. The animals were subjected to methoxyflurane
(Mallinckrodt, Mundelein, IL) anesthesia 24 hr after the pilocarpine treatment. Subsequently, they underwent transcardiac perfusion-fixation with PBS followed by 4% phosphate-buffered paraformaldehyde.
Brains were left in situ for 2 hr and then put in 4%
paraformaldehyde overnight. Each brain was then dehydrated in graded
ethanol solutions, cleared with D-limonene (Fisher, Santa
Clara, CA), embedded in paraffin, sectioned to 8 µm thickness,
deparaffinized and hydrated, and stained with modified hematoxylin and
eosin. Slides were examined under fluorescent light so that cells that
sustained injury and became acidophilic (eosinophilic) could be
identified by their bright eosin fluorescence as described in detail
earlier (Sankar et al., 1997b). Bilateral sections of the dorsal
hippocampus approximating the region  3.8 mm posterior to bregma
(Paxinos and Watson, 1982) were used for cell counts of subfields CA1,
CA3, and hilus. A few animals were maintained for 6 months after SE and
then killed for examination of Nissl-stained sections. Tissue was cut
into 30 µm sections and stained with cresyl violet.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling staining. Paraffin-embedded sections were labeled with a commercially available in situ labeling kit
(Oncor, Gaithersburg, MD). Briefly, sections were deparaffinized in
D-limonene (Fisher), incubated with proteinase K (Oncor)
followed by terminal deoxynucleotidyl transferase and
biotinylated-16-dUTP. The reaction product was visualized with
diaminobenzidine (Sigma) and lightly counterstained with 1% methyl
green (Sigma).
Ethidium bromide staining. Some animals were perfused as
described above, and the brains were removed, cryoprotected in 30% sucrose, and then cut in 30 µm frozen sections. Sections were soaked
in D-limonene (Fisher), hydrated to water, stained in
ethidium bromide (EtBr) (0.1 µg/ml) for 3 min, rinsed in water, and
then dehydrated before mounting. Slides were viewed under fluorescent light using a rhodamine filter.
Optical sections were also obtained with an Olympus IX-70 microscope
equipped with an epifluorescence adapter and a 100× oil immersion objective lens. Images were recorded with the CELLscan System
(Scanalytics, Billerica, MA) equipped with a SenSys CCD camera system
(Photometrics, Tucson, AZ), a piezoelectric z-axis focus
device, and a computer-controlled excitation light shutter. The light
haze contributed by fluorescently labeled structures located above and
below the plane of optimal focus was mathematically reassigned to its
proper places of origin (Exhaustive Photon Reassignment software,
Scanalytics) after accurate characterization of the blurring function
of the optical system. The blurring function of the optical system (the
point-spread function) was characterized by imaging a through-focus
series of optical sections of a 0.2-µm-diameter fluorescent bead
(Molecular Probes, Eugene OR) using the same optical conditions as
those used to obtain the specimen image. The patented Exhaustive Photon
Reassignment (EPR) process successively iterates estimates of the true
signal and intensity distribution until such time as the estimate, when
blurred by the point-spread function, yields the image as detected by
the camera. This can be expressed as follows: I = O * PSF, where I = image of the
object as detected by the camera, O = the true object
as it exists on the microscope slide, * = convolution,
PSF = point-spread function image stack as measured
from the optical setup. During restoration, EPR uses the PSF image data
to refocus light and haze in the raw specimen image.
DNA extraction and agarose gel electrophoresis. Samples of
hippocampi (8-10) were dissected from fresh brains of rats (24 hr
after SE), immediately frozen in dry ice, and stored at 70°C until
use. DNA was purified using the technique of Iwasa et al. (1996).
Hippocampi were incubated overnight in digestion buffer [100
mM NaCl, 10 mM Tris-Cl, pH 8.0, 25 mM EDTA, pH 8.0, 0.5% SDS, 0.1 mg/ml proteinase K,
0.1 mg/ml RNase A (Sigma)]. DNA was extracted using equal proportions
of the lysate and phenol, chloroform, and isoamyl alcohol (Life
Technologies, Gaithersburg, MD). The aqueous layer was incubated at
37°C with additional RNase A (0.1 mg/ml) for 60-90 min. Phenol
extraction was repeated and DNA was precipitated with 2.5 vol of
ethanol and 1/10 vol of sodium acetate and placed in dry ice for 30 min. Precipitated DNA was spun at 11,000 rpm (14,500 × g) for 15 min and washed three times with 50 ml cold 70%
ethanol. The supernatant was then decanted, and the pellet was
air-dried for 15 min. Pellet was resuspended in 200-500 µl of 10 mM Tris-Cl, pH 8.5, in 55°C shaking water bath overnight.
Spectrophotometry revealed A260/280 ratios of
1.6-2.1, indicating relatively pure DNA in concentrations of ~1-4
µg/µl. DNA (20 µg per lane) was run on 2% agarose gels
containing 0.5% EtBr at 4 V/cm gel length. The gel was viewed under UV
transillumination (302 nm) and photographed with a Polaroid camera.
Electron microscopy. Some animals were perfused with PBS (20 ml for 2-week-old animals, 30 ml for 3-week-old animals) followed by
3% glutaraldehyde (50 ml for 2-week-old animals, 60 ml for 3-week-old
animals) (Electron Microscopy Sciences, Ft. Washington, PA) and 1%
paraformaldehyde (Fisher) in 0.1 M phosphate buffer (PB),
pH 7.4. The brain was removed and post-fixed for 1 d and then cut
on a vibratome into 100 µm sections. The sections were washed with
PB, stained for 1 hr in 2% OsO4 (Electron Microscopy Sciences), and stained en block in 2% aqueous uranyl
acetate (Mallinckrodt) for 30 min. The sections were then dehydrated in
ascending graded solutions of alcohol for 10 min each and then placed
in 1:1 100% EtOH/propylene oxide for 5 min followed by two incubations
in propylene oxide for 5 min. The sections were then incubated in 1:1
and 1:3 propylene oxide/Durcupan (Fluka, Buchs, Switzerland) for 1 hr
each followed by Durcupan overnight. The sections were placed between
strips of Aclar plastic and cured for 24 hr at 60°C, after which they
were cut on an ultramicrotome. They were placed on single hole grids,
covered by butvar, stained with 6% uranyl acetate and lead citrate,
and examined with a Philips Electron Microscope 201C.
Timm histochemistry. Rats that had been subjected to
LiPC-induced SE 4 months earlier and control rats were anesthetized
with methoxyflurane (Mallinckrodt) and perfused transcardially with an
aqueous solution of 250 ml of 0.1% (w/v) sodium sulfide, followed by
250 ml of 4% paraformaldehyde solution. The brains were removed and
left in a 30% (w/v) sucrose solution in the fixative overnight at
4°C. Coronal 30 µm frozen sections were developed in the dark for
30 min in a 6:3:1 mixture of gum arabic (20%, w/v), hydroquinone (5.6%, w/v), citric acid-sodium citrate buffer with 1.5 ml of a
silver nitrate solution (17%) (Danscher, 1981).
Monitoring for spontaneous seizures. Animals that underwent
LiPC-induced SE were selected for monitoring when they began to demonstrate behavioral seizures 3 months or longer after the initial experiments. Animals were anesthetized with ketamine (60 mg/kg) and
xylazine (15 mg/kg). A bipolar stimulating electrode was implanted into
the angular bundle of the perforant path (4.5 mm left of and 0.5 mm
posterior to lambda), and a bipolar recording electrode was implanted
into the granule cell layer of the ipsilateral dentate gyrus (2.2 mm
left of and 3.5 mm anterior to lambda). The depth of both electrodes
was optimized by finding the population spike of maximal amplitude,
evoked from the dentate gyrus by stimulation of the perforant path.
After recovery from surgery for 1 week, the animals were monitored for
24 hr a day with a video camera and continuous EEG using the Monitor 81 program from Stellate Software. The software was configured to detect
and save seizures and spikes. The EEGs were analyzed off-line using the
same software.
| |
RESULTS |
Behavioral effects
The 60 mg/kg dose of pilocarpine used in this study produced
cortical and subcortical SE in all the ages studied (EEG data not
shown). The latency to SE was 10-20 min for all ages. The 2-week-old
pups developed head bobbing followed by intermittent forelimb clonus
and hyperextension of the tail (which persisted for most of the
duration of the SE) and of the hindlimbs intermittently. The 3- and
4-week-old rats showed forelimb clonus leading to frequent kindled-like
seizures with rearing and falling, and occasional running seizures with
vocalization, which sometimes culminated in tonic extension and death.
The adults exhibited forelimb clonus and kindled-like seizures with
frequent periods of behavioral arrest during which chewing movements
and twitching of the face and/or vibrissae were often observed. All the
2-week-old rat pups survived the lithium-pilocarpine SE, whereas
approximately one-third of the 3- and 4-week-old animals and 50% of
the adult animals died. Arterial blood gases were sampled in the
2-week-old group (data not shown), and none of the animals experienced
any hypoxemia during SE.
Histological changes
In all four groups, extensive hippocampal and extra-hippocampal
neuronal death followed SE. However, both the nature and the distribution of this neuronal death were highly age-specific.
Neuronal damage in the CA1 subfield of the hippocampus (Fig.
1A-D) as ascertained
by the presence of "ischemic cell change" (Brown and Brierley,
1968) with a pyknotic nucleus and an eosinophilic cytoplasm was maximal
in the 2- and 3-week-old rat pups (in which 36 ± 5% and 34 ± 10% of CA1 neurons, respectively, had this appearance) and
decreased progressively with age to 25 ± 8% in the 4-week-old animals and 9 ± 3% (p < 0.05) in the
adults (Table 1).
View larger version (145K):
[in this window]
[in a new window]
|
Figure 1.
Histological lesions in lithium-pretreated rats 24 hr after SE from pilocarpine administration. CA1 of a 2-week-old
(A), 3-week-old (B), and
4-week old pup (C) shows a large number of
eosinophilic cells that fluoresce brightly (hematoxylin and eosin),
whereas the CA1 of a mature rat (D) has scattered
damage. Scale bar, 100 µm.
|
|
The neuronal damage in the CA3 region of the 2-week-old rats was barely
discernible at 1%. The damage at 3, 4, and 9-12 weeks (20 ± 9, 18 ± 7, and 14 ± 3%, respectively) did not differ
significantly (Table 1). Two-week-old rat pups were resistant to
SE-induced hilar damage. The extent of damage in the 3-week-old rat
pups (31 ± 2%) was comparable to that in the 4-week-old (27 ± 2%) and adult rats (28 ± 2%) (Table 1, Fig.
2A-D).
Damage to the dentate granule cells is also shown in Figures
2A-D. The 3-week-old rat pups demonstrated a special
vulnerability to SE-induced damage in this cell population (33 ± 7%) that is different from that seen in younger or older animals
(5 ± 0.5, 7 ± 2, and 5 ± 1% in 2-, 4-, and
9-12-week-old animals, respectively) (Table 1, Fig. 2A-D). The cell injury demonstrated by eosin
fluorescence at 24 hr after SE was confirmed as permanent loss of cells
by cresyl violet-stained sections in selected animals 6 months after
the initial SE (Fig.
3A-F). We have
reported previously on the age-related SE-induced damage to
extrahippocampal structures (Sankar et al., 1997b). The neocortex,
caudate, and septum showed damage scores similar to adults by 3 weeks
of age, whereas the damage to amygdala showed a trend toward
progressively increasing damage with age. Damage to the thalamus was
maximal in the 2- and 3-week-old rats and decreased with age (Sankar et
al., 1997b).
View larger version (142K):
[in this window]
[in a new window]
|
Figure 2.
Dentate granule cells and hilar interneurons are
damaged after pilocarpine seizures in rats pretreated with lithium.
Scattered eosin fluorescence is seen in a 2-week-old pup 24 hr after SE
(A). A 3-week-old pup shows extensive damage to
the hilar and outer granule cells (B). Damaged
hilar cells are also visible in a 4-week-old (C)
and an adult rat (D). Scale bar, 100 µm.
|
|
View larger version (175K):
[in this window]
[in a new window]
|
Figure 3.
Permanent cell loss is seen several months after
SE as pups. Cresyl violet staining shows a reduction of cells in the
CA1 region of a rat subjected to LiPC at 2 weeks
(A) when compared with that of a control
(B) 6 months after treatment. The hilus of a rat
treated with LiPC at 3 weeks (C) lacks many of
the large cells seen within the tip of the hilus and in the region
between the superior blade of the dentate granule cell layer and the
CA3c neurons of a control (D). Cell loss is also
seen in the CA3a region of a rat given LiPC at 3 weeks of age
(E) as compared with a control
(F). Scale bar (shown in A):
A, B, 200 µm; C-F, 100 µm.
|
|
Type of neuronal death
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling (TUNEL) stain-positive neurons were seen in large
numbers only in the 2- and 3-week-old pups after SE. The distribution
of these neurons was age-dependent, with the 2-week-olds demonstrating
such injury predominantly in the CA1 region (Fig.
4A,C), the subiculum,
and the thalamus, with lesser labeling of the inner dentate granule
cells. In the 3-week-old animals, only the inner layer of the dentate
granule cells (Fig. 4B,D) and a few thalamic neurons
were TUNEL-positive. Fluorescence microscopy of EtBr-stained sections
(Fig. 5A,B) revealed
fragmented nuclei in the same areas that were TUNEL-stained. In the
3-week-old animals, the neuronal injury delineated by the TUNEL and
EtBr methods (Figs. 4B,D, 5B) seen in the
inner layer of the granule cells was different from that seen in the
outer granular layer neurons visualized by their eosinophilic cytoplasm
(Fig. 2B). Two types of damage also appear to coexist
in the CA1 subfield of 2-week-old pups subjected to SE.
View larger version (144K):
[in this window]
[in a new window]
|
Figure 4.
Comparison of the distribution of a TUNEL-stained
section in 2-week-old and 3-week-old rat. Note that TUNEL label in the
CA1 of the 2-week-old pup (A, C) overlaps
with the hematoxylin- and eosin-labeled cells (Fig.
1A), whereas the TUNEL label in the inner granule
cell layer in a 3-week-old pup (B,
D) is distinct from the fluorescent cells visualized
under UV light (Figs. 1B,
2B). Scale bar (shown in A):
A, B, 200 µm: C, D, 45 µm.
|
|
View larger version (83K):
[in this window]
[in a new window]
|
Figure 5.
Ethidium bromide staining in the CA1 of a
2-week-old rat (A) and the inner granule cell
layer of a 3-week-old rat (B).
Arrowheads indicate fragmented nuclei. Scale bar, 18 µm. Inset in B shows an exhaustive photon reassignment
view of chromatin concretions in an apoptotic nucleus (6 µm
diameter).
|
|
Electron micrographs of CA1 in 2-week-old pups (Fig.
6A) and of the inner
granule cell region and hilus (Fig. 6B) of the
3-week-olds clearly demonstrated the presence of distinctive nuclear
and cytoplasmic changes suggestive of necrosis in some neurons and of
apoptosis in others. Twenty-four hours after SE, many CA1 neurons
displayed chromatin condensation coexisting with intact cytoplasmic and nuclear membranes and with relatively intact cytoplasmic organelles. In
that same CA1 region, however, swollen neurons with ruptured membranes,
extensive cytoplasmic damage, and all the hallmarks of necrosis were
also seen. In the 3-week-old dentate gyrus, inner granule cells with
chromatin margination and nearly intact cytoplasms were seen in close
proximity to clearly necrotic hilar neurons.
View larger version (191K):
[in this window]
[in a new window]
|
Figure 6.
Electron micrographs in rat pups. The CA1 of a 2 week-old rat (A) shows an apoptotic cell with
condensed cytoplasm (arrow) and several necrotic cells
with shrunken nuclei and vacuolated cytoplasm
(arrowheads). EM of the granule cells near the hilar
border of a 3 week-old rat (B) shows several
apoptotic neurons. CA1 cells from a control 2-week-old pup and dentate
granule cells from a control 3-week-old pup are shown in
C and D, respectively. Scale bar, 4 µm.
|
|
The DNA extracted from the hippocampi of rats of various ages subjected
to LiPC SE and of their age-matched controls was studied by agarose gel
electrophoresis (Fig. 7). Laddering was
not evident in the lanes in which DNA from control rats was placed.
Discrete bands corresponding to fragments of DNA with a periodicity
approximating 180 base pairs, suggesting oligonucleosomal breaks, were
seen best in the hippocampal DNA from the 2- and 3-week-old rats that underwent SE. A less prominent degree of laddering was also seen in the
lanes containing DNA from the hippocampi and neocortices (the latter
not shown) of 4-week-old and adult rats that underwent SE, although we
did not see a significant number of TUNEL-positive neurons, or
apoptotic bodies, at the two time points (24 and 72 hr) studied in
those animals.
View larger version (111K):
[in this window]
[in a new window]
|
Figure 7.
Paired agarose gel electrophoresis of hippocampal
DNA from LiPC-treated (left lanes) and age-matched
control rats (right lanes). Laddering pattern is visible
in rats undergoing SE at all ages. Lanes in
A-D correspond to 2-, 3-, and 4-week-old and adult
rats, respectively. Shown at left is 100 base pair
standard.
|
|
Epileptogenesis
Three of the 11 rats subjected to SE at 2 weeks of age that were
observed for at least 4 months developed spontaneous seizures that
resembled Racine's (1972) stages 3-5 in kindled animals and seizures
triggered by handling. The latter were characterized by a few myoclonic
jerks sometimes leading to a stage 3-5 seizure. Of the 11 rats
subjected to SE at 3 weeks of age, eight (and six out of eight that
experienced SE at 4 weeks) displayed spontaneous seizures after a
latent period of <3 months, shorter than that seen in the animals
subjected to SE at 2 weeks of age. Recordings of spontaneous seizures
from a rat that underwent LiPC SE at 2 weeks is shown in Figure
8A, whereas Figure
8B shows multiple spontaneous seizures recorded from
a rat that experienced LiPC SE at 3 weeks of age.
View larger version (108K):
[in this window]
[in a new window]
|
Figure 8.
Spontaneous seizures and sprouting in adult rats
after treatment with LiPC as pups. A, A hippocampal EEG
recording accompanied by a stage 5 behavioral convulsion 6 months after
SE at 2 weeks of age. B, Continuous monitoring of a rat
3 months after LiPC treatment at 3 weeks of age shows several
spontaneous seizures during a 24 hr period. Each line represents 2 hr.
Black bar shows an electrographic seizure captured by
the software, accompanied by stage 4 behavioral convulsions.
Electrographic seizure is outlined by the dashed line.
Mossy fiber sprouting is not seen in control rats treated at 2 weeks
(C) and 3 weeks of age (D).
E, Timm staining of the mature rat in A
that was given LiPC at 2 weeks of age is not discernible from an
age-matched control (C), whereas dense Timm
staining of the inner molecular layer is seen in a pup given LiPC at 3 weeks of age (F). Scale bar, 500 µm.
|
|
Mossy fiber synaptic reorganization
The degree of staining seen in the rats that developed spontaneous
seizures after undergoing SE at 2 weeks of age (Fig.
8E) was not distinguished from that of controls (Fig.
8C). The pattern of Timm staining of the inner molecular
layer seen in a rat with spontaneous seizures that had undergone SE at
3 (Fig. 8F) and 4 weeks of age is similar to that
seen in adults.
| |
DISCUSSION |
We have used different methods known to be sensitive to
distinctive mechanisms that may lead to neuronal damage as a result of
the recurrent excitation caused by SE during development. Previous studies by others (Albala et al., 1984; Sperber et al., 1991, 1992)
have focused on specific aspects of hippocampal response to seizures
such as hilar cell loss and mossy fiber sprouting and have remarked on
the relative resiliency of the immature brain to seizures. Our results
show enhanced vulnerability to CA1 neurons during early development at
a stage when hilar injury and mossy fiber synaptic reorganization are
not prominent. The damage seen at this age appears to involve both
necrosis, as evidenced by the eosinophilic cells, and apoptosis as
demonstrated by various techniques that included TUNEL staining,
visualization of fragmented nuclei under fluorescence microscopy of
EtBr-stained sections, and electron microscopy. We have also presented
DNA electrophoresis data compatible with the process of apoptosis.
Franck and Schwartzkroin (1984) had found preferential loss of CA1
neurons in immature rabbits subjected to SE induced by systemic KA and
had observed the similarity of this lesion to that induced by ischemia
and/or hypoxia. It was subsequently shown in hippocampal slices
obtained from immature rabbits that the rise in extracellular potassium
after repetitive stimulation was regulated less effectively in CA1
compared with CA3 (Haglund and Schwartzkroin, 1990). More recently,
such vulnerability of the CA1 neurons to LiPC SE has been demonstrated
in immature rabbits in the absence of hypoxia (Thompson and Wasterlain,
1997). In the rat, Hamon and Heinemann (1988) had shown that during the second postnatal week, the apical dendrites of pyramidal neurons in
area CA1 become more sensitive to NMDA, which is expressed by big
influxes of calcium at this level. Harris and Teyler (1984) showed that
long-term potentiation in area CA1 of the rat was maximal at 15 d.
Excitability comparable to that seen in fully mature animals could be
demonstrated in the CA1 region after postnatal day 14, whereas the
inhibitory processes did not reach an adult stage of maturation until
several weeks later (Michelson and Lothman, 1989). Our observation of
preferential CA1 damage at 2 weeks of age is consistent with the
observations of the development of synaptic inhibition lagging in CA1
compared with area CA3 (Swann et al., 1989) or the dentate gyrus
(Bekenstein and Lothman, 1991). Target kainate receptor development at
the mossy fiber terminals and amygdala reached maturation (Berger et
al., 1984) only toward the end of the third week such that the hilus
and CA3 are resistant to SE-induced damage in the 2-week-old rat.
In the CA1 region of the 2-week-old rat, both acidophilic neurons and
TUNEL-positive neurons are seen in close proximity to each other. Our
electron micrographs confirm the presence of nuclear profiles
suggesting necrosis in the same region as those suggesting apoptosis.
It has been shown that both apoptosis and necrosis can result from
qualitatively similar types of excitotoxic stimulus, with the intensity
of NMDA receptor activation deciding the process (Bonfoco et al.,
1995). The neuronal damage seen maximally in the CA1 of our 2 week-old
rats is attributable to the seizures, because hypoxia was not
encountered in these animals. Furthermore, hypoxia at
PaO2 of 20 mmHg for 20 min without ischemia did
not result in cell loss or even morphological evidence of injury as evidenced by the lack of expression of heat-shock protein in mature rats (Pearigen et al., 1996).
Damage to the hilus and CA3 in this model of SE appears to be at least
as severe in the 3-week-old rat pups as in mature rats, but 2-week-old
pups show little or no damage. The development of vulnerability to
seizure-induced damage in the hilus and CA3 may be attributed to the
maturation of the mossy fiber terminals. This was suggested by Nitecka
et al. (1984) in a detailed study of SE-induced damage in the KA model
that documented progressive damage to limbic structures from postnatal
day 18. Previous studies demonstrating resistance to SE-induced hilar
and CA3 damage in the developing brain have compared mature rats only
to 2-week-old pups (Albala et al., 1984; Sperber et al., 1991, 1992) in
the kainic acid model or have used groupings such as 15- to 21-d-old rats in the PC model of SE (Cavalheiro et al., 1987).
Recent studies in immature animals have suggested that certain methods
such as acid fuchsin staining (Chang and Baram, 1994) and silver
degeneration methods (Toth et al., 1998) may delineate reversible
neuronal injury rather than serve as markers of dying neurons. We have
demonstrated that the pattern of neuronal damage discernible by eosin
fluorescence at 24 hr is reflected as permanent cell drop-out in cresyl
violet-stained sections obtained from surviving rats several months
after SE.
We saw evidence of damage to the granule cells of the dentate gyrus
mainly in the 21-d-old rat pups. It is striking that strongly eosinophilic neurons are seen mainly in the outermost layers of the
granule cells, whereas scattered TUNEL-positive neurons are seen along
the hilar border. The distribution of TUNEL-positive neurons in our
experiments is reminiscent of the results reported by Goodman et al.
(1993) in ischemic rat pups, and by Bengzon et al. (1997) after
kindling or KA administration in adult rats. Apoptosis of granule cells
has also been shown after repetitive perforant path stimulation
(Sloviter et al., 1996) and after adrenalectomy (Sloviter et al., 1993;
Hu et al., 1997), in mature rats. Under these circumstances, apoptosis
does not appear to be restricted to the granule cells along the hilar
border. In hypoxic-ischemic rat pups, the distribution of apoptosis
was limited to the inner granule cells that did not express the
calcium-binding protein calbindin D28k, suggesting a mechanism for this
age-dependent vulnerability, which might also apply to our pups.
Weiss et al. (1996) reported a significantly different pattern of DNA
fragmentation in the hippocampus of rats subjected to KA-induced SE. It
is likely that the pattern of DNA fragmentation detected by a
particular technique is influenced by the timing after the excitotoxic
insult as well as the variations in the technique. Didier et al. (1996)
have suggested that the in situ nick translation method
using DNA polymerase 1 is very sensitive and labels single-strand
breaks and that such damage may be reversible, whereas the nick end
labeling with terminal deoxynucleotidyl transferase identifies
double-strand breaks.
Charriaut-Marlangue and Ben-Ari (1995) cautioned on the use of the
TUNEL stain as the sole criterion for the demonstration of apoptosis
and suggested that methods such as Hoechst 33258 staining be used to
supplement the data. We have used EtBr staining in such a manner. It is
of interest that significant TUNEL labeling was not seen in our
experiments in regions where extensive eosinophilic neurons are
encountered in animals older than the 3 week-old pups. This is
consistent with the results of Fujikawa et al. (1997) who did not see
significant evidence for apoptosis in mature rats subjected to LiPC SE.
On the other hand, "DNA laddering" on agarose gel is widely
presented as evidence of apoptosis based on the suggestion that
necrosis would lead to a "smear" DNA pattern (Walker et al., 1994).
Our results show that laddering may be seen when there is extensive
necrosis, although cells matching the morphological criteria for
apoptosis may be extremely rare. Similar observations have been made by
Fujikawa et al. (1997). These results are consistent with recent
reports that examined the consequences of excitotoxic exposure of
cultured murine cortical neurons (Gwag et al., 1997; Sohn et al.,
1998). In these studies, early cell swelling and early damage to the
plasma membrane were observed when the integrity of the nuclear
membrane was preserved along with DNA ladders. Hence, we would like to
sound a cautionary note on the use of agarose gel electrophoresis of
extracted DNA as a sole or major criterion for apoptosis.
Rat pups that were subjected to SE at 2 weeks of age developed
spontaneous seizures after a longer latency than those that underwent
SE at P21, and in much smaller (3/11 vs 8/11, respectively) numbers.
Even the rats that developed spontaneous seizures after SE at 2 weeks
of age did not show evidence of sprouting as seen by Timm stain. A
recent report had suggested (Longo and Mello, 1997) that spontaneous
seizures could develop after SE in the absence of mossy fiber
sprouting. This conclusion was based on experiments in which sprouting
had been blocked by the administration of cycloheximide, a protein
synthesis inhibitor, without abolishing the development of spontaneous
seizures. Our results do not distinguish whether sprouting and synaptic
reorganization of the mossy fiber pathway is a cause or result of
repeated seizures.
In conclusion, we have shown that the pattern of seizure-induced cell
death is related to development. Apoptosis and necrosis can result from
seizures in an age-dependent distribution, and even young animals can
develop spontaneous seizures as a consequence of early seizures. The
demonstration of seizure-related apoptosis in the immature brain may
have implications for the development of neuroprotective agents
tailored for use in the young.
| |
FOOTNOTES |
Received May 27, 1998; revised Aug. 3, 1998; accepted Aug. 7, 1998.
R.S. was supported by Clinical Investigator Development Award NS01792
from the National Institute of Neurological Disorders and Stroke
(NINDS), National Institutes of Health. C.G.W. was supported by Grant
NS13515 from NINDS, National Institutes of Health, and by the Research
Service of the Veterans Health Administration. We thank Roger Baldwin,
Rosie Lezama, Steve Shinmei, and Don Santos for technical
assistance.
Correspondence should be addressed to Dr. Raman Sankar, Pediatric
Neurology (22-474 Marion Davies Children's Center), Box 951752, UCLA School of Medicine, Los Angeles, CA
90095-1752.
| |
REFERENCES |
-
Albala BJ,
Moshé SL,
Okada R
(1984)
Kainic-acid-induced seizures: a developmental study.
Brain Res
315:139-148[Medline].
-
Bekenstein JW,
Lothman EW
(1991)
A comparison of the ontogeny of excitatory and inhibitory neurotransmission in the CA1 region and dentate gyrus of the rat hippocampal formation.
Dev Brain Res
63:237-243[Medline].
-
Bengzon J,
Kokaia Z,
Elmer E,
Nanobashvili A,
Kokaia M,
Lindvall O
(1997)
Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures.
Proc Natl Acad Sci USA
94:10432-10437[Abstract/Free Full Text].
-
Berger ML,
Tremblay E,
Nitecka L,
Ben-Ari Y
(1984)
Maturation of kainic acid seizure-brain damage syndrome in the rat. III. Postnatal development of kainic acid binding sites in the limbic system.
Neuroscience
13:1095-1104[Web of Science][Medline].
-
Bonfoco E,
Krainc D,
Ankarcrona M,
Nicotera P,
Lipton SA
(1995)
Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.
Proc Natl Acad Sci USA
92:7162-7166[Abstract/Free Full Text].
-
Brown AW,
Brierley JB
(1968)
The nature, distribution and earliest changes of anoxic-ischemic nerve cell damage in the rat brain as defined by the optical microscope.
Br J Exp Pathol
49:87-106[Web of Science][Medline].
-
Camfield PR
(1997)
Recurrent seizures in the developing brain are not harmful.
Epilepsia
38:735-737[Web of Science][Medline].
-
Cavalheiro EA
(1995)
The pilocarpine model of epilepsy.
Ital J Neurol Sci
16:33-37[Web of Science][Medline].
-
Cavalheiro EA,
Silva DF,
Turski WA,
Calderazzo FL,
Bortolotto ZA,
Turski L
(1987)
The susceptibility of rats to pilocarpine-induced seizures is age-dependent.
Brain Res
465:43-58[Medline].
-
Cavalheiro EA,
Santos NF,
Priel MR
(1996)
The pilocarpine model of epilepsy in mice.
Epilepsia
37:1015-1019[Web of Science][Medline].
-
Chang D,
Baram TZ
(1994)
Status epilepticus results in reversible neuronal injury in infant rat hippocampus: novel use of a marker.
Dev Brain Res
77:133-136[Medline].
-
Charriaut-Marlangue C,
Ben-Ari Y
(1995)
A cautionary note on the use of the TUNEL stain to determine apoptosis.
NeuroReport
7:61-64[Web of Science][Medline].
-
Clifford DB,
Olney JW,
Maniotis A,
Collins RC,
Zorumski CF
(1987)
The functional anatomy and pathology of lithium-pilocarpine and high-dose pilocarpine seizures.
Neuroscience
23:953-968[Web of Science][Medline].
-
Danscher G
(1981)
Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electronmicroscopy.
Histochemistry
71:1-16[Web of Science][Medline].
-
Didier M,
Bursztajn S,
Adamec E,
Passani L,
Nixon RA,
Coyle JT,
Wei JY,
Berman SA
(1996)
DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures.
J Neurosci
16:2238-2250[Abstract/Free Full Text].
-
Filipkowski RK,
Hetman M,
Kaminska B,
Kaczmarek L
(1994)
DNA fragmentation in rat brain after intraperitoneal administration of kainate.
NeuroReport
5:1538-1540[Web of Science][Medline].
-
Franck JE,
Schwartzkroin PA
(1984)
Immature rabbit hippocampus is damaged by systemic but not intraventricular kainic acid.
Brain Res
315:219-227[Medline].
-
Fujikawa DG
(1995)
Neuroprotective effect of ketamine administered after status epilepticus onset.
Epilepsia
36:186-195[Web of Science][Medline].
-
Fujikawa DG,
Daniels AH,
Kim JS
(1994)
The competitive NMDA receptor antagonist CGP 40116 protects against status epilepticus-induced neuronal damage.
Epilepsy Res
17:207-219[Web of Science][Medline].
-
Fujikawa DG,
Cai BB,
Shinmei SS,
Allen SG
(1997)
Neuronal death induced by lithium-pilocarpine-induced seizures is necrotic, not apoptotic.
Soc Neurosci Abstr
23:1112.
-
Goodman JH,
Wasterlain CG,
Massarweh WF,
Dean E,
Sollas AL,
Sloviter RS
(1993)
Calbindin-D28k immunoreactivity and selective vulnerability to ischemia in the dentate gyrus of the developing rat.
Brain Res
606:309-314[Web of Science][Medline].
-
Gwag BJ,
Koh JY,
DeMaro JA,
Ying HS,
Jacquin M,
Choi DW
(1997)
Slowly triggered excitotoxicity occurs by necrosis in cortical cultures.
Neuroscience
77:393-401[Web of Science][Medline].
-
Haglund MM,
Schwartzkroin PA
(1990)
Role of Na-K pump potassium regulation and IPSPs in seizures and spreading depression in immature rabbit hippocampal slices.
J Neurophysiol
63:225-239[Abstract/Free Full Text].
-
Hamon B,
Heinemann U
(1988)
Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus.
Brain Res
466:286-290[Medline].
-
Harris KM,
Teyler TJ
(1984)
Developmental onset of long-term potentiation in area CA1 of the rat hippocampus.
J Physiol (Lond)
346:27-48[Abstract/Free Full Text].
-
Hirsch E,
Baram TZ,
Snead III OC
(1992)
Ontogenic study of lithium-pilocarpine-induced status epilepticus in rats.
Brain Res
583:120-126[Web of Science][Medline].
-
Honchar MP,
Olney JW,
Sherman WR
(1983)
Systemic cholinergic agents induce seizures and brain damage in lithium-treated rats.
Science
220:323-325[Abstract/Free Full Text].
-
Hu Z,
Yuri K,
Ozawa H,
Lu H,
Kawata M
(1997)
The in vivo time course for elimination of adrenalectomy-induced apoptotic profiles from the granule cell layer of the rat hippocampus.
J Neurosci
17:3981-3989[Abstract/Free Full Text].
-
Iwasa M,
Maeno Y,
Inoue H,
Koyama H,
Matoba R
(1996)
Induction of apoptotic cell death in rat thymus and spleen after a bolus injection of methamphetamine.
Int J Legal Med
109:23-28[Web of Science][Medline].
-
Jope RS,
Morrisett RA,
Snead OC
(1986)
Characterization of lithium potentiation of pilocarpine-induced status epilepticus in rats.
Exp Neurol
91:471-480[Web of Science][Medline].
-
Kornblum HI,
Sankar R,
Shin DH,
Wasterlain CG,
Gall CM
(1997)
Induction of brain derived neurotrophic factor mRNA by seizures in neonatal and juvenile rat brain.
Mol Brain Res
44:219-228[Medline].
-
Liu Z,
Gatt A,
Werner SJ,
Mikati MA,
Holmes GL
(1994)
Long-term behavioral deficits following pilocarpine seizures in immature rats.
Epilepsy Res
19:191-204[Web of Science][Medline].
-
Longo BM,
Mello LE
(1997)
Blockade of pilocarpine- or kainate-induced mossy fiber sprouting by cycloheximide does not prevent subsequent epileptogenesis in rats.
Neurosci Lett
226:163-166[Web of Science][Medline].
-
Meldrum BS,
Vigouroux RA,
Brierley JB
(1973)
Systemic factors and epileptic brain damage. Prolonged seizures in paralyzed, artificially ventilated baboons.
Arch Neurol
29:82-87[Abstract/Free Full Text].
-
Michelson HB,
Lothman EW
(1989)
An in vivo electrophysiological study of the ontogeny of excitatory and inhibitory processes in the rat hippocampus.
Dev Brain Res
47:113-122[Medline].
-
Morrison RS,
Wenzel HJ,
Kinoshita Y,
Robbins CA,
Donehower LA,
Schwartzkroin PA
(1996)
Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death.
J Neurosci
16:1337-1345[Abstract/Free Full Text].
-
Nevander G,
Ingvar M,
Auer R,
Siesjo BK
(1985)
Status epilepticus in well-oxygenated rats causes neuronal necrosis.
Ann Neurol
18:281-290[Web of Science][Medline].
-
Nitecka L,
Tremblay E,
Charton G,
Bouillot JP,
Berger ML,
Ben-Ari Y
(1984)
Maturation of kainic acid seizure-brain damage syndrome in the rat. II. Histopathological sequelae.
Neuroscience
13:1073-1094[Web of Science][Medline].
-
Okada R,
Moshé SL,
Albala BJ
(1984)
Infantile status epilepticus and future seizure susceptibility in the rat.
Brain Res
317:177-183[Medline].
-
Paxinos G,
Watson C
(1982)
In: The rat brain in stereotaxic coordinates. Sydney: Academic.
-
Pearigen P,
Gwinn R,
Simon RP
(1996)
The effects in vivo of hypoxia on brain injury.
Brain Res
725:184-191[Web of Science][Medline].
-
Penix LP,
Wasterlain CG
(1994)
Selective protection of neuropeptide containing dentate hilar interneurons by non-NMDA receptor blockade in an animal model of status epilepticus.
Brain Res
644:19-24[Web of Science][Medline].
-
Penix LP,
Thompson KW,
Wasterlain CG
(1996)
Selective vulnerability to perforant path stimulation: role of NMDA and non-NMDA receptors.
Epilepsy Res [Suppl]
12:63-73[Medline].
-
Pollard H,
Charriaut-Marlangue C,
Cantagrel S,
Represa A,
Robain O,
Moreau J,
Ben-Ari Y
(1994)
Kainate-induced apoptotic cell death in hippocampal neurons.
Neuroscience
63:7-18[Web of Science][Medline].
-
Portera-Cailliau C,
Price DL,
Martin LJ
(1997a)
Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum.
J Comp Neurol
378:70-87[Web of Science][Medline].
-
Portera-Cailliau C,
Price DL,
Martin LJ
(1997b)
Non-NMDA and NMDA receptor-mediated excitotoxic neuronal deaths in adult brain are morphologically distinct: further evidence for an apoptosis-necrosis continuum.
J Comp Neurol
378:88-104[Web of Science][Medline].
-
Racine RJ
(1972)
Modification of seizure activity by electrical stimulation. II. Motor seizure.
Electroencephalogr Clin Neurophysiol
32:281-294[Web of Science][Medline].
-
Sankar R,
Wasterlain CG,
Sperber EF
(1995)
Seizure-induced changes in the immature brain.
In: Brain development and epilepsy (Schwartzkroin PA,
Moshé SL,
Swann JW,
Noebels JL,
eds), pp 268-288. New York: Oxford UP.
-
Sankar R,
Shin DH,
Lezama R,
Liu H,
Santos D,
Pulera M,
Allen S,
Wasterlain CG
(1997a)
Seizure-induced apoptosis and necrosis in the developing brain is age-dependent.
Soc Neurosci Abstr
23:807.
-
Sankar R,
Shin DH,
Wasterlain CG
(1997b)
Serum neuron-specific enolase is a marker for neuronal damage following status epilepticus in the rat.
Epilepsy Res
28:129-136[Web of Science][Medline].
-
Sloviter RS,
Dean E,
Neubort S
(1993)
Electron microscopic analysis of adrenalectomy-induced hippocampal granule cell degeneration in the rat: apoptosis in the adult central nervous system.
J Comp Neurol
330:337-351[Web of Science][Medline].
-
Sloviter RS,
Dean E,
Sollas AL,
Goodman JH
(1996)
Apoptosis and necrosis induced in different hippocampal neuron populations by repetitive perforant path stimulation in the rat.
J Comp Neurol
366:516-533[Web of Science][Medline].
-
Sohn S,
Kim EY,
Gwag BJ
(1998)
Glutamate neurotoxicity in mouse cortical neurons: atypical necrosis with DNA ladders and chromatin condensation.
Neurosci Lett
240:147-150[Web of Science][Medline].
-
Sperber EF,
Haas KZ,
Stanton PK,
Moshé SL
(1991)
Resistance of the immature hippocampus to seizure-induced synaptic reorganization.
Dev Brain Res
60:88-93[Medline].
-
Sperber EF,
Haas KZ,
Moshé SL
(1992)
Developmental aspects of status epilepticus.
Int Pediatr
7:213-222.
-
Swann JW,
Brady RJ,
Martin DL
(1989)
Postnatal development of GABA-mediated synaptic inhibition in rat hippocampus.
Neuroscience
28:551-561[Web of Science][Medline].
-
Thompson K,
Wasterlain C
(1997)
Lithium-pilocarpine status epilepticus in the immature rabbit.
Dev Brain Res
100:1-4.
-
Thompson K,
Holm AM,
Schousboe A,
Popper P,
Micevych P,
Wasterlain C
(1998)
Hippocampal stimulation produces neuronal death in the immature brain.
Neuroscience
82:337-348[Web of Science][Medline].
-
Toth Z,
Yan XX,
Haftoglou S,
Ribak CE,
Baram TZ
(1998)
Seizure-induced neuronal injury: vulnerability to febrile seizures in an immature rat model.
J Neurosci
18:4285-4294[Abstract/Free Full Text].
-
Turski WA,
Cavalheiro EA,
Schwarz M,
Czuczwar S-LJ,
Kleinrok Z,
Turski L
(1983)
Limbic seizures produced by pilocarpine in rats: behavioural, electroencephalographic and neuropathological study.
Behav Brain Res
9:315-335[Web of Science][Medline].
-
Walker PR,
Weaver VM,
Lach B,
LeBlanc J,
Sikorska M
(1994)
Endonuclease activities associated with high molecular weight and internucleosomal DNA fragmentation in apoptosis.
Exp Cell Res
213:100-106[Web of Science][Medline].
-
Wasterlain CG
(1997)
Recurrent seizures in the developing brain are harmful.
Epilepsia
38:728-734[Web of Science][Medline].
-
Weiss S,
Cataltepe O,
Cole AJ
(1996)
Anatomical studies of DNA fragmentation in rat brain after systemic kainate administration.
Neuroscience
74:541-551[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18208382-12$05.00/0
This article has been cited by other articles:
|
|
|
|
 
L.-H. Zeng, N. R. Rensing, and M. Wong
The Mammalian Target of Rapamycin Signaling Pathway Mediates Epileptogenesis in a Model of Temporal Lobe Epilepsy
J. Neurosci.,
May 27, 2009;
29(21):
6964 - 6972.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. C. Glass and E. Wirrell
Controversies in Neonatal Seizure Management
J Child Neurol,
May 1, 2009;
24(5):
591 - 599.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. W. Loepke and S. G. Soriano
An Assessment of the Effects of General Anesthetics on Developing Brain Structure and Neurocognitive Function
Anesth. Analg.,
June 1, 2008;
106(6):
1681 - 1707.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Pisani, C. Cerminara, C. Fusco, and L. Sisti
Neonatal status epilepticus vs recurrent neonatal seizures: Clinical findings and outcome
Neurology,
December 4, 2007;
69(23):
2177 - 2185.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Walter, B. L. Murphy, R. Y. K. Pun, A. L. Spieles-Engemann, and S. C. Danzer
Pilocarpine-Induced Seizures Cause Selective Time-Dependent Changes to Adult-Generated Hippocampal Dentate Granule Cells
J. Neurosci.,
July 11, 2007;
27(28):
7541 - 7552.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Sankar and J. M. Rho
Do Seizures Affect the Developing Brain? Lessons From the Laboratory
J Child Neurol,
May 1, 2007;
22(5_suppl):
21S - 29S.
[Abstract]
[PDF]
|
|
|
|
|
|
|
L. K. Friedman
CALCIUM: A Role for Neuroprotection and Sustained Adaptation
Mol. Interv.,
December 1, 2006;
6(6):
315 - 329.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Dube, C. Richichi, R. A. Bender, G. Chung, B. Litt, and T. Z. Baram
Temporal lobe epilepsy after experimental prolonged febrile seizures: prospective analysis
Brain,
April 1, 2006;
129(4):
911 - 922.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Parfenova, P. Carratu, D. Tcheranova, A. Fedinec, M. Pourcyrous, and C. W. Leffler
Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression
Am J Physiol Heart Circ Physiol,
June 1, 2005;
288(6):
H2843 - H2850.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Sankar and G. L. Holmes
Mechanisms of Action for the Commonly Used Antiepileptic Drugs: Relevance to Antiepileptic Drug-Associated Neurobehavioral Adverse Effects
J Child Neurol,
August 1, 2004;
19(1_suppl):
S6 - S14.
[Abstract]
[PDF]
|
|
|
|
|
|
|
R. Sankar and G. L. Holmes
Mechanisms of Action for the Commonly Used Antiepileptic Drugs: Relevance to Antiepileptic Drug--Associated Neurobehavioral Adverse Effects
J Child Neurol,
January 1, 2004;
19(1_suppl):
S6 - S14.
[Abstract]
[PDF]
|
|
|
|
|
|
|
G. L. Holmes
Seizure-induced neuronal injury: Animal data
Neurology,
November 12, 2002;
59(90095):
S3 - 6.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. H. Mohajeri, K. Saini, J. G. Schultz, M. A. Wollmer, C. Hock, and R. M. Nitsch
Passive Immunization against beta -Amyloid Peptide Protects Central Nervous System (CNS) Neurons from Increased Vulnerability Associated with an Alzheimer's Disease-causing Mutation
J. Biol. Chem.,
August 30, 2002;
277(36):
33012 - 33017.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. P. Miller, J. Weiss, A. Barnwell, D. M. Ferriero, B. Latal-Hajnal, A. Ferrer-Rogers, N. Newton, J. C. Partridge, D. V. Glidden, D. B. Vigneron, et al.
Seizure-associated brain injury in term newborns with perinatal asphyxia
Neurology,
February 26, 2002;
58(4):
542 - 548.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Kubova, R. Druga, K. Lukasiuk, L. Suchomelova, R. Haugvicova, I. Jirmanova, and A. Pitkanen
Status Epilepticus Causes Necrotic Damage in the Mediodorsal Nucleus of the Thalamus in Immature Rats
J. Neurosci.,
May 15, 2001;
21(10):
3593 - 3599.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. K. McCabe, D. C. Silveira, M. R. Cilio, B. H. Cha, X. Liu, Y. Sogawa, and G. L. Holmes
Reduced Neurogenesis after Neonatal Seizures
J. Neurosci.,
March 15, 2001;
21(6):
2094 - 2103.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Schmid, P. Tandon, C. E. Stafstrom, and G. L. Holmes
Effects of neonatal seizures on subsequent seizure-induced brain injury
Neurology,
November 1, 1999;
53(8):
1754 - 1754.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Liu, Y. Cao, A. I. Basbaum, A. M. Mazarati, R. Sankar, and C. G. Wasterlain
Resistance to excitotoxin-induced seizures and neuronal death in mice lacking the preprotachykinin A gene
PNAS,
October 12, 1999;
96(21):
12096 - 12101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Theodore and C. G. Wasterlain
Do early seizures beget epilepsy?
Neurology,
September 1, 1999;
53(5):
898 - 898.
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Koh, T. W. Storey, T. C. Santos, A. Y. Mian, and A. J. Cole
Early-life seizures in rats increase susceptibility to seizure-induced brain injury in adulthood
Neurology,
September 1, 1999;
53(5):
915 - 915.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. P. Roux, M. A. Colicos, P. A. Barker, and T. E. Kennedy
p75 Neurotrophin Receptor Expression Is Induced in Apoptotic Neurons After Seizure
J. Neurosci.,
August 15, 1999;
19(16):
6887 - 6896.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
Compound via MeSH
