Interleukin-1β biosynthesis inhibition reduces acute seizures and drug resistant chronic epileptic activity in mice.
ABSTRACT Experimental evidence and clinical observations indicate that brain inflammation is an important factor in epilepsy. In particular, induction of interleukin-converting enzyme (ICE)/caspase-1 and activation of interleukin (IL)-1β/IL-1 receptor type 1 axis both occur in human epilepsy, and contribute to experimentally induced acute seizures. In this study, the anticonvulsant activity of VX-765 (a selective ICE/caspase-1 inhibitor) was examined in a mouse model of chronic epilepsy with spontaneous recurrent epileptic activity refractory to some common anticonvulsant drugs. Moreover, the effects of this drug were studied in one acute model of seizures in mice, previously shown to involve activation of ICE/caspase-1. Quantitative analysis of electroencephalogram activity was done in mice exposed to acute seizures or those developing chronic epileptic activity after status epilepticus to assess the anticonvulsant effects of systemic administration of VX-765. Histological and immunohistochemical analysis of brain tissue was carried out at the end of pharmacological experiments in epileptic mice to evaluate neuropathology, glia activation and IL-1β expression, and the effect of treatment. Repeated systemic administration of VX-765 significantly reduced chronic epileptic activity in mice in a dose-dependent fashion (12.5-200 mg/kg). This effect was observed at doses ≥ 50 mg/kg, and was reversible with discontinuation of the drug. Maximal drug effect was associated with inhibition of IL-1β synthesis in activated astrocytes. The same dose regimen of VX-765 also reduced acute seizures in mice and delayed their onset time. These results support a new target system for anticonvulsant pharmacological intervention to control epileptic activity that does not respond to some common anticonvulsant drugs.
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ABSTRACT: Neuropeptide Y has been shown to inhibit the immunological activity of reactive microglia in the rat cerebral cortex, to reduce N-methyl-D-aspartate current (I NMDA) in cortical neurons, and protect neurons. In this study, after primary cultured microglia from the cerebral cortex of rats were treated with lipopolysaccharide, interleukin-1β and tumor necrosis factor-α levels in the cell culture medium increased, and mRNA expression of these cytokines also increased. After primary cultured cortical neurons were incubated with the lipopolysaccharide-treated microglial conditioned medium, peak I NMDA in neurons increased. These effects of lipopolysaccharide were suppressed by neuropeptide Y. After addition of the neuropeptide Y Y1 receptor antagonist BIBP3226, the effects of neuropeptide Y completely disappeared. These results suggest that neuropeptide Y prevents excessive production of interleukin-1β and tumor necrosis factor-α by inhibiting microglial reactivity. This reduces I NMDA in rat cortical neurons, preventing excitotoxicity, thereby protecting neurons.Neural Regeneration Research 05/2014; 9(9):959-67. · 0.14 Impact Factor
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ABSTRACT: Epilepsy is one of the most common chronic brain disorders worldwide, affecting 1% of people across different ages and backgrounds. Epilepsy is defined as the sporadic occurrence of spontaneous recurrent seizures. Accumulating preclinical and clinical evidence suggest that there is a positive feedback cycle between epileptogenesis and brain inflammation. Epileptic seizures increase key inflammatory mediators, which in turn cause secondary damage to the brain and increase the likelihood of recurrent seizures. Cytokines and prostaglandins are well-known inflammatory mediators in the brain, and their biosynthesis is enhanced following seizures. Such inflammatory mediators could be therapeutic targets for the development of new antiepileptic drugs. In this review, we discuss the roles of inflammatory mediators in epileptogenesis.Mediators of Inflammation 01/2014; 2014:901902. · 3.88 Impact Factor
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ABSTRACT: The time course of cytokine dynamics after seizure remains controversial. Here we evaluated the changes in the levels and sites of interleukin (IL)-1β expression over time in the hippocampus after seizure.Japanese clinical medicine. 01/2014; 5:25-32.
Interleukin-1β Biosynthesis Inhibition Reduces Acute Seizures and
Drug Resistant Chronic Epileptic Activity in Mice
Mattia Maroso,1Silvia Balosso,1Teresa Ravizza,1Valentina Iori,1Christopher Ian Wright,2
Jacqueline French,3and Annamaria Vezzani1
1Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milano, 20156 Italy;2Vertex Pharmaceuticals
Inc., Cambridge, Massachusetts 02139; and3New York University Comprehensive Epilepsy Center, New York, New York 10016
Summary: Experimental evidence and clinical observations
indicate that brain inflammation is an important factor in
epilepsy. In particular, induction of interleukin-converting
enzyme (ICE)/caspase-1 and activation of interleukin (IL)-1β/
IL-1 receptor type 1 axis both occur in human epilepsy, and
contribute to experimentally induced acute seizures. In this
study, the anticonvulsant activity of VX-765 (a selective ICE/
caspase-1 inhibitor) was examined in a mouse model of chronic
epilepsy with spontaneous recurrent epileptic activity refractory
to some common anticonvulsant drugs. Moreover, the effects of
this drug were studied in one acute model of seizures in mice,
previously shown to involve activation of ICE/caspase-1.
Quantitative analysis of electroencephalogram activity was
done in mice exposed to acute seizures or those developing
chronic epileptic activity after status epilepticus to assess the
anticonvulsant effects of systemic administration of VX-765.
Histological and immunohistochemical analysis of brain tissue
was carried out at the end of pharmacological experiments in
epileptic mice to evaluate neuropathology, glia activation and
IL-1β expression, and the effect of treatment. Repeated
systemic administration of VX-765 significantly reduced
chronic epileptic activity in mice in a dose-dependent fashion
(12.5-200 mg/kg). This effect was observed at doses ≥50 mg/
kg, and was reversible with discontinuation of the drug.
Maximal drug effect was associated with inhibition of IL-1β
synthesis in activated astrocytes. The same dose regimen of
VX-765 also reduced acute seizures in mice and delayed their
onset time. These results support a new target system for
anticonvulsant pharmacological intervention to control epileptic
activity that does not respond to some common anticonvulsant
drugs. Key Words: Astrocytes, anticonvulsant drug,
inflammation, IL-1b, temporal lobe epilepsy.
Experimental seizures in rodents induce inflammatory
processes in brain regions in which epileptic activity
originates and spreads [1, 2]; in this respect, the
activation of the interleukin (IL)-1β/IL-1 receptor type
1 (R1) signaling in glia and neurons is a key event
contributing to intrinsic brain inflammation [3–12].
Paracrine and autocrine activation of this signaling by
the brain application of IL-1β exacerbates kainic acid- or
bicuculline-induced seizures in rats and mice [5, 11, 13],
and lowers the seizure threshold in febrile seizure models
[7, 8]. Conversely, IL-1 receptor antagonist (the naturally
occurring competitive antagonist of IL-1R1) mediates
powerful anticonvulsant effects in rodents [6, 13–15] and
mice over-expressing IL-1 receptor antagonist in astro-
cytes, or lacking IL-1R1, are intrinsically less suscep-
tible to seizures [7, 13]. These data indicate the
important involvement of elevated brain IL-1β levels
and the activation of IL-1R1 signaling in experimental
Our earlier studies explored whether seizures induced
acutely in naïve rats were affected by the blockade of IL-
1β biosynthesis. Using either pralnacasan or VX-765
(two selective inhibitors of interleukin-converting
enzyme (ICE)/caspase-1 , the key enzyme specifi-
cally involved in the production of the releasable and
biologically active form of IL-1β ), we found a
significant decrease in acute seizure activity induced by
intracerebral injection of kainic acid , and the arrest of
seizure generalization in the kindling model of epilepto-
There is evidence of increased levels of IL-1β and IL-
1R1, and ICE/caspase-1 activation  in surgically
Address correspondence and reprint requests to: Annamaria Vezzani,
Phd., Department of Neuroscience, Mario Negri Institute for Pharma-
cological Research, Milano, 20156 Italy. E-mail: vezzani@marionegri.
Mattia Maroso and Silvia Balosso contributed equally to this study.
Electronic supplementary material The online version of this article
(doi:10.1007/s13311-011-0039-z) contains supplementary material,
which is available to authorized users.
Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics
Vol. 8, 304Y315, April 2011* The American Society for Experimental NeuroTherapeutics, Inc.
resected epileptogenic tissue from pharmaco resistant
patients with temporal lobe epilepsy (TLE) or malforma-
tions of cortical development [10, 19–22],suggesting that
the IL-1β/IL-1R1 axis is activated in human epilepsy as
in experimental models.
The objective of this study was to establish whether
ICE/caspase-1 inhibition by VX-765 could suppress
spontaneous recurrent epileptic activity by using a mouse
model, which recapitulates some of the salient neuro-
pathological sequelae of TLE, and in which epileptic
activity does not respond to some commonly used anti-
epileptic drugs (AEDs) [23, 24]. Moreover, because
VX-765 effect on acute seizures was previously studied
only in rats, we extended this investigation to mice
injected with kainic acid, because this model involves the
activation of ICE/caspase-1 . We found that the
systemic administration of VX-765 significantly reduces
both acute seizures and spontaneous epileptic activity.
This anticonvulsant effect was associated with inhibition
of astrocytic IL-1β expression in the hippocampus,
whereas glial cell activation was not significantly
modified. The use of this anti-inflammatory strategy may
be envisaged for treating established pharmacoresistant
MATERIALS AND METHODS
Male C57BL6 mice (60 days old, 25 g; Charles River,
Calco, Italy) were housed at a constant temperature (23°C)
and relative humidity (60%) with free access to food and
animals and their care were conducted in accordance with
the ethically approved institutional guidelines that are in
compliance with national and international laws and policies
(EEC Council Directive 86/609, OJ L 358, 1, Dec.12, 1987;
Guide for the Care and Use of Laboratory Animals, United
States National Research Council, 1996).
Mouse model of acute seizures
Mice were surgically implanted with an injection guide
cannula and recording electrodes under deep Equithesin
anesthesia and stereotaxic guidance [11, 25]. Two
Nichrome-insulated bipolar depth electrodes (60 μm
OD; MOR Electric Heating Assoc. Inc, Comstock Park,
MI) were implanted bilaterally into the dorsal hippo-
campus (from bregma [mm]: nose bar 0; anteroposterior,
–1.8; lateral, ±1.5; and 2.0, below dura mater). A 23-
gauge cannula was unilaterally positioned on top of the
dura mater and glued to one of the depth electrodes for
the intrahippocampal infusion of kainic acid (“for more
detail see as follows”). The electrodes were connected to
a multipin socket, and together with the cannula of
injection they were secured to the skull by acrylic dental
cement. The correct position of the electrodes and
injection needle was evaluated by histological analysis
of brain tissue at the end of the experiments (“for more
detail see as follows”).
Intrahippocampal injection of kainic acid in freely
moving mice was done 7 days after surgery as
previously described [11, 25]. Kainic acid (7 ng in
0.5 μl; Sigma, Saint Louis, MO) was dissolved in
0.1 M phosphate-buffered solution (PBS, pH 7.4) and
injected unilaterally in the dorsal hippocampus by using
a needle protruding 2.0 mm from the bottom of the
guide cannula. This dose of kainic acid was proven to
induce electroencephalographic (EEG) ictal episodes in
the hippocampus in 100% of mice without mortality
[11, 25]. Selective cell loss in CA3 region of the
injected hippocampus is observed 3 to 7 days after
kainate injection [9, 11, 26].
Acute seizures assessment and quantification
EEG seizures induced by intrahippocampal injection
of kainic acid in mice have been extensively described
before [11, 25, 26]. Briefly, a 30-minute recording was
done before kainic acid injection to assess the basal EEG
pattern (FIG. 1a), and for 180 minutes after kainate
injection. Kainate was injected for 1 minute CMA/100
pump (CMA microdialysis AB, Solma, Sweden) using a
30-gauge injection needle connected to a 10.0 μl
Hamilton microsyringe via PE20 tubing; the needle was
left in place for 1 additional minute to avoid backflow
through the cannula. Ictal episodes (FIG. 1b, d) are
characterized by high frequency (7–10 Hz) and/or multi-
spike complexes, and/or high-voltage (700 μV-1.0 mV)
synchronized spikes simultaneously occurring in the
injected and contralateral hippocampi. The EEG record-
ing of each animal was analyzed visually to detect any
activity different from baseline. Seizure activity was
FIG. 1. Representative electroencephalographic (EEG) tracings
of acute seizures induced by intra-hippocampal kainate in vehi-
cle- or VX-765-treated mice. Tracings in (a) and (c) depict base-
line EEG activity recorded in the left hippocampi (LHP) and right
hippocampi (RHP) before kainate injection in the LHP (7 ng in
0.5 μl). Tracings in (b) and (d) depict kainate-induced seizure a-
ctivity (delimited by arrowheads) in mice pretreated with vehicle
or 200 mg/kg i.p. VX-765, respectively. VX-765 reduced seizure
number and duration as reported in Table 2.
305ICE/CASPASE-1 ROLE IN EPILEPTIC ACTIVITY
Neurotherapeutics, Vol. 8, No. 2, 2011
quantified by reckoning the time elapsed from kainic acid
injection to the occurrence of the first EEG seizure
(onset) and the total number and total duration of
seizures (reckoned by summing up the duration of every
ictal episode during the EEG recording period). Seizures
occurred with an average latency of ~10 minutes from
kainic acid injection, and then recurred for ~90 minutes
from their onset, which were associated with motor arrest
of the mice.
Chronic epileptic mice
We used a chronic model of spontaneous epileptic
activity that has been extensively described before
[23–25, 27–29], which develops in mice following non-
convulsive status epilepticus (SE) lasting for as long as
10 h, induced by unilateral intrahippocampal application of
200 ng in 50 nl kainic acid. Spontaneous epileptic activity
reproducibly occurs after 3 to 7 days on average from SE
induction (“for more detailsee as follows”) anditrecurs for
several months. Once established, this activity is not
associated with motor convulsions, it is stable, and it is
refractory to phenytoin, carbamazepine, and valproate ,
thus providing a model for focal epilepsy not responding to
some commonly used AEDs [24, 25, 27–29]. Histopatho-
logical changes are restricted to the hippocampus injected
with kainate and are similar to those described in human
mesial TLE including neuronal loss, granule cell disper-
sion, and sprouting [23–25, 27–29].
Status epilepticus induction
Mice were deeply anesthetized with Equithesin and
stereotaxically injected with kainic acid (200 ng in
50 nl phosphate-buffered saline; PBS, pH 7.4; Sigma-
Aldrich) unilaterally into the left dorsal hippocampus at
the following coordinates from bregma: mm, nose bar,
0; anteroposterior, –1.8; lateral, +1.7; and 1.9, below
dura . Kainate was injected over 1 min (CMA/100
pump) using a 30-gauge injection needle connected to a
0.5 μl Hamilton microsyringe via PE20 tubing. At the
end of the injection, the needle was left in situ for an
additional 2 minutes to limit backflow along the
injection track. After kainate injection, mice were
implanted with two nichrome-insulated bipolar depth
electrodes (60 μm OD) bilaterally into the dorsal
hippocampus (from bregma [mm]: nose bar 0; ante-
roposterior −1.8, lateral ±1.7 and 1.9 below dura
mater). The electrodes were connected to a multipin
socket and secured to the skull by acrylic dental
cement. After the mice awoke from anesthesia (i.e., 60
minutes on average), they underwent continuous EEG
analysis for 24 h to determine the occurrence of SE, as
defined by high amplitude, uninterrupted spiking activ-
ity with an average frequency of 12 to 18 Hz lasting for
at least 3 h. Then the mice were returned to their cages
for 6 weeks until EEG recording of spontaneous
epileptic activity was initiated. One additional group of
the same conditions, and used for immunohistochemical
analysis (“for more detail see as follows”).
Spontaneous epileptic activity
Six weeks after intrahippocampal kainate injection,
mice were EEG monitored to establish their baseline
hippocampal activity. EEG was monitored for 3 consec-
utive days from 9:00AM to 11:00AM and from 4:00PM to
6:00PM. These recordings provided a daily average
sampling of epileptic events and their duration in each
mouse, within the day–time interval of our pharmaco-
logical experiments. This protocol allowed us to select 21
of 23 mice with stable spontaneous epileptic activity
before starting the treatment (Table 1). Chronic epileptic
activity (FIG. 2) was defined by the appearance and
recurrence of subclinical paroxystic events such as: high-
voltage sharp waves (HVSW) (1–4 mV; 3–8 Hz; average
duration, 20 seconds; FIG. 2 panels a, a1), which are
most commonly observed in groups, and they are usually
unilateral, but they may also occur bilaterally; and
hippocampal paroxysmal discharges (HPD) that typically
start with large amplitude sharp waves (1–3 mV; 1–3 Hz;
FIG. 2 panels b, b1) followed by a train of spikes of
increasing frequency (0.5-1.0 mV; 10–20 Hz, panel b,b2)
and terminating with a deflection in the EEG (FIG. 2
panels b, b3). HPDs last 30 to 60 seconds on average,
occuring unilaterally (i.e., in the hippocampus originally
injected with kainate), and are typically followed by
isolated spikes; HPDs represent ~35% of chronic parox-
ysmal activity. Sporadic bilateral hippocampal paroxysmal
discharges may also occur (FIG. 2 panel d), but these are
rare events that were not considered in evaluating drug
effects. Spontaneous epileptic activity was quantified by
(HVSW and HPD) during a 2-h EEG recording period in
the morning (9:00–11:00AM) and afternoon (4:00–6:00PM)
sessions. HVSW and HPD did not occur during SE and
epileptogenesis, although they were always observed in the
chronic phase and were stable and reproducible in each
Isolated spikes or spike trains with a frequency of 1 to
3 Hz, and/or duration less than 20 seconds (FIG. 2 panel c)
were not considered in the quantitative analysis of epileptic
EEG activity was monitored using the GRASS 79D
EEG recording system (Astro-Med Industrial Park, West
Warwick, RI), the signal was digitalized with a PowerLab
16/S data acquisition system (ADInstrument Pty. Ltd., Bella
Vista, NSW, Australia) and analyzed with LabChart 7
software (ADInstrument Pty. LTD, Bella Vista NSW,
EEG analysis in acute and chronic models was done
by two independent investigators blinded to the treat-
306MAROSO ET AL.
Neurotherapeutics, Vol. 8, No. 2, 2011
ment, who visually reviewed all the EEG tracings.
Deviation of ≤5% from concordance was considered
acceptable; otherwise, EEG tracing was additionally
analyzed by a third person.
Mouse model of acute seizures.
46 mice were used in these experiments. VX-765 (Vertex
Pharmaceuticals Inc.), which dissolved in 0.1% Tween 80 in
0.5% hydroxy-ethyl-cellulose in distilled water was injected
at 12.5, 50, 100, and 200 mg/kg i.p. in different groups of
mice daily (i.e., at 9:00AM) for 3 consecutive days, and on
day 4 a bolus injection was done 45 minutes before kainic
acid application. This repeated treatment regimen using
200 mg/kg VX-765 was previously shown to provide brain
765) able to block enzyme activity (70–140 nM, measured
2 h after the last drug administration), and showed
anticonvulsant effects on acute seizures induced in rats by
kainic acid  and in rapid kindling. An EEG recording
was made for 3 h after kainic acid administration (“for more
details see previous information”); a 30-minute EEG
recording similar to baseline was required in each mouse
before ending the experiment.
A total number of
Chronic epileptic mice.
21 epileptic mice and randomly divided them into 3
experimental groups to be treated with 12.5 mg/kg i.p.
(n=6), 50 mg/kg i.p. (n=6), or 200 mg/kg i.p. (n=9) VX-
765. We chose this study design to be able to test the
anticonvulsant effect of the different drug doses on
chronic epileptic activity at approximately the same
time (i.e., ~6 weeks) after SE induction in mice. The
larger number of mice in the group treated with
200 mg/kg VX-765 dose was required by the post-hoc
immunohistochemical analysis (“for details see as
Amongthe 9 epileptic mice to be treated with200 mg/kg
VX-765, we randomly chose 5 mice (Table 1, numbers
5–9) to confirm previous literature evidence that chronic
epileptic activity in this model was unaffected by some
conventional AEDs . This group of 5 epileptic mice
was treated with a single dose of 50 mg/kg phenytoin i.p.,
7 days before starting 200 mg/kg VX-765 treatment
(protocol is shown in FIG. 3 panel a). This dose of
phenytoin was previously shown to reduce acute or
spontaneous seizures in rodents within 2 h after a single
drug administration [31, 32].
For each VX-765 dose regimen, the following treat-
ment protocol was adopted in each epileptic mouse
We used a total number of
Table 1. Baseline Spontaneous Epileptiform Activity Assessed by EEG Analysis in Chronic Epileptic Mice used for Pharm-
Mouse I.D.Time in Epileptic Activity (min)
Mice were used as follows: numbers 1 to 9 were injected with 200 mg/kg VX-765; numbers 10 to 15 were injected with 50 mg/kg VX-765;
numbers 16 to 21 were injected with 12.5 mg/kg VX-765. Mice numbers 5 to 9 were also treated with 50 mg/kg phenytoin before VX-765;
mice numbers 10 to 15 were also treated with 3 mg/kg dexamethasone after treatment with 50 mg/kg VX-765. (“For details on treatment
protocols see Methods and FIG. 3.”).
EEG=electroencephalographic; I.D. = identification.
307ICE/CASPASE-1 ROLE IN EPILEPTIC ACTIVITY
Neurotherapeutics, Vol. 8, No. 2, 2011
(FIG. 3 panel b): after 3 days of EEG baseline recording,
on day 4 of the vehicle of VX-765 was intraperitoneally
injected twice (9:00AM and 4:00PM), and no changes in
baseline were observed (not shown). Then epileptic mice
received VX-765 twice a day (9:00AM and 4:00PM) for 4
consecutive days, and EEG recording was done for 2 h
after each drug administration since this drug was proven
to be effective for at least 2 h in the acute seizure model
. The last drug administration was followed by a 3-day
wash-out period during which EEG was recorded daily
from 9:00 am to 11:00 am; the time required by each
epileptic mouse to recover its pre-injection baseline
activity after drug withdrawal (after day 4 of treatment,
i.e., the eighth drug administration) was used to estimate
the duration of VX-765 anticonvulsant action.
At the end of the pharmacological experiments with
50 mg/kg VX-765 (including the 3 day wash-out period),
the epileptic mice (Table 1, numbers 10–15) were
recorded continuously between 9:00AM and 5:00PM for
FIG. 2. Representative electroencephalographic (EEG) tracings of
chronic epileptic activity induced by intra-hippocampal kainate in
mice. Chronic epileptic activity develops in mice 3 to 7 days after
unilateral intra-hippocampal injection (left hippocampus [LHP]) of
kainate (200 ng in 50 nl) causing SE. Tracings in (a) (boxed area is
enlarged in a1) and (b) (boxed areas are enlarged in b1, b2, b3) de-
duration, 20 seconds) and unilateral hippocampal paroxysmal dis-
charges (HPDs) that typically start (arrowhead in panel b) with large
amplitude sharp waves (1–3 mV; 1–3 Hz; panel b1) followed by a
train of spikes of increasing frequency (0.5–1.0 mV; 10–20 Hz, pa-
in panel b3). HVSW and HPDs were inhibited by VX-765 admin-
istration (see FIG. 4b–d). Panel (c) depicts interictal activity consist-
ing of isolated spikes or spike trains (1–3 Hz; duration, <20
seconds); panel (d) shows bilateral hippocampal paroxysmal dis-
(d) were not included in the quantification of chronic epileptic acti-
vity. RHP=right hippocampus.
FIG. 3. Schematic representation of the treatment protocols in
chronic epileptic mice. Panel (a) depicts the treatment protocol
used in 5 randomly chosen epileptic mice: after establishing the
baseline of spontaneous epileptic activity, the mice were treated
intraperitoneally with a single dose of 50 mg/kg phenytoin. Then
they underwent a 3-day wash-out period before entering proto-
col (b) (200 mg/kg VX-765). Panel (b) depicts the treatment pro-
tocol of 21 epileptic mice with VX-765: 3 days of
electroencephalographic (EEG) baseline recording was done, a-
nd on day 4, the vehicle of VX-765 was injected intraperitoneally
twice (at 9:00AM and 4:00PM), and on the following day, the ep-
ileptic mice received VX-765 twice a day (at 9:00AM and 4:00PM)
for 4 consecutive days, and the EEG recording was done for 2 h
after each drug administration. The last drug administration was
followed by a 3-day wash-out period, and each day EEG was
recorded (at 9:00AM to 11:00AM) to evaluate the time required by
each epileptic mouse to recover its pre-injection baseline activity
after drug withdrawal. Mice injected with VX-765 were killed for
histological analysis (i.e., 5 mice at the time of maximal drug e-
ffect and 4 mice after drug wash-out). Panel (c) depicts the tre-
atment protocol with dexamethasone: 6 epileptic mice at the end
of treatment with 50 mg/kg VX-765, according to protocol (b),
including the 3-day wash-out period were recorded by EEG co-
ntinuously (between 9:00AM and 5:00PM) for 3 additional days
(days 1–3) to monitor if the baseline epileptic activity had resu-
med and was stable. Then the vehicle was subcutaneously inj-
ected on day 4 (at 9:00AM and 4:00PM); on day 5, the baseline
was again recorded (between 9:00AM and 11:00AM), followed by
a subcutaneous injection of dexamethasone in alcohol formula-
tion (3 mg/kg). An EEG recording was continued for an additional
6 h (i.e., until 5:00PM). This procedure was repeated for 3 cons-
ecutive days. A 3-day wash-out period followed the last dex-
amethasone dose. d=day; DEX=dexamethasone.
308MAROSO ET AL.
Neurotherapeutics, Vol. 8, No. 2, 2011
3 additional days (days 1–3) to monitor if baseline EEG
epileptic activity was resumed and stable. Then the
vehicle of dexamethasone (DEX) was injected subcuta-
neously on day 4 (9:00AM and 4:00PM), and again no
changes in baseline were observed (not shown). Baseline
was again recorded in each mouse on day 5 between 9:00
AM and 11:00AM, followed by a subcutaneous injection of
3 mg/kg DEX in alcohol formulation  and an EEG
recording that was continued for an additional 6 h (i.e.,
until 5:00PM). This procedure was repeated for 3
consecutive days (see the protocol in FIG. 3 panel c).
We studied IL-1β expression, glia activation, and
leukocytes (granulocytes, T-lymphocytes, and macro-
phages) extravasation in the hippocampus of epileptic
mice treated with 200 mg/kg VX-765 and killed
randomly, either at the end of drug wash-out (when
spontaneous epileptic activity had resumed, i.e., day 10 in
FIG. 4c; n=4) or at the time of maximal anticonvulsant
effect (day 7 in FIG. 4c; n=5).
Mice were deeply anesthetized using Equithesin and
perfused via ascending aorta with 50 mM cold PBS (pH
7.4), followed by chilled 4% paraformaldehyde in 0.1 M
PBS. The brains were post-fixed for 90 minutes at 4°C,
and were then transferred to 20% sucrose in PBS for 24 h
at 4°C. The brains were rapidly frozen in −50°C
isopentane for 3 minutes and stored at −80°C until
assayed. Serial cryostat coronal sections (40 μm) were
cut from all brains throughout the septotemporal exten-
sion of the hippocampus  and were collected in
0.1 M PBS. Four series of 9 adjacent slices were used,
with 1 slice for each marker: first, second, third, seventh,
eighth, and ninth slices, respectively, were used for IL-
1β, glial-fibrillary acidic protein (GFAP), CD11b, gran-
ulocyte (Gr-1), macrophages (CD68), and T-lymphocytes
(CD3); the fourth and fifth slices were used for double
immunostaining; and the sixth slice was used for Nissl
staining. Primary and secondary antibodies and exper-
imental procedures were those previously shown to
determine a specific immunohistochemical signal of the
protein of interest in rodent brain slices [10, 25, 34]. For
IL-1β or glia markers (GFAP, CD11b), slices were
incubated at 4°C for 10 minutes in 70% methanol and 2%
H2O2in Tris HCl-buffered saline (TBS, pH 7.4), followed
by 30-minute incubation in 10% in fetal calf serum in 1%
(for IL-1β) or 0.1% (for glia markers) Triton X-100 in TBS.
Then slices were incubated overnight with the primary
antibody to IL-1β (1:200; Santa Cruz Biotechnology), to
mouse GFAP (1:2500; Chemicon), a selective marker of
marker of monocyte/microgliacells,at 4°C in10%fetal calf
serum in 1% Triton X-100 in TBS. For macrophages,
granulocytes, and T-lymphocytes, slices were incubated at
room temperature for 10 minutes in 1% H2O2in TBS,
followed by 1 h incubation in 10% normal goat serum in
0.3% Triton X-100 in TBS. Then slices were incubated
Serotec), a selective marker of macrophages, human CD3
(1:150; Dako), a selective marker of T-lymphocytes, or
Pharmingen), a marker of granulocytes, at 4°C in 4%
normal goat serum in 0.1% Triton X-100 in TBS.
Immunoreactivity was tested by the avidin-biotin-perox-
idase technique (Vector Labs, INC, Burlingame, CA); the
sections were reacted using diaminobenzidine, and the signal
was amplified by nickel ammonium only for IL-1β. No
immunostaining was observed by incubating the slices with
the primary antibodies pre-absorbed with the corresponding
peptides, or without the primary antibodies [10, 25, 34].
After incubation with the primary IL-1β antibody, slices
were incubated in biotinylated secondary anti-goat anti-
peroxidase (HRP) and the signal was revealed with
tyramide conjugated to fluorescein using the TSA ampli-
fication kit (NEN Life Science Products, Boston, MA).
Sections were subsequently incubated with the following
primary antibodies: mouse anti-GFAP (1:2500) or rat anti-
mouse CD11b (1:1000). Fluorescence was detected using
anti-mouse or anti-rat secondary antibody conjugated with
Alexa546 (Molecular Probes, Leiden, The Netherlands).
Slide-mounted sections were examined with an Olympus
BX61 and confocal system FV500; Hamburg, Germany)
using dual excitation of 488 nm (Laser Ar) and 546 nm
(Laser He–Ne green) for fluorescein and Alexa546,
respectively. The emission of fluorescent probes was
collected on separate detectors. To eliminate the possibility
of bleed through between channels, the sections were
scanned in a sequential mode.
Statistical analysis of data
Data are the mean±standard error of the mean (n=
number of individual samples). The effects of treatments
Inc., La Jolla, CA) using absolute values. The effects of
by repeated measures of analysis of variance followed by
Tukey’s test using raw data. Differences due to the
treatments were considered significant with a P<0.05.
Anticonvulsant activity of VX-765
Table 2 shows the dose-response effect of VX-765 in
the mouse model of acute seizures induced by intra-
hippocampal injection of kainic acid. FIG. 1 depicts
309ICE/CASPASE-1 ROLE IN EPILEPTIC ACTIVITY
Neurotherapeutics, Vol. 8, No. 2, 2011
representative EEG tracings from kainate-injected mice
treated with vehicle (FIG. 1a, b) or 200 mg/kg VX-765
injected mice (FIG. 1c, d). An average 50% and 64%
decrease in the number of seizures and their total duration
was observed with 50 to 200 mg/kg VX-765 (Table 2). The
timetoonset of the first seizurewas significantly delayedat
doses of 100 and 200 mg/kg. No effects of 12.5 mg/kg on
seizure parameters were observed (Table 2).
Table 1 reports the quantification of time spent in
spontaneous epileptic activity in each mouse used for
pharmacological evaluation of VX-765 effect. In the 2-h
EEG recording during the morning and afternoon
FIG. 4. Panels (a–c): Anticonvulsant effects of VX-765 and dexamethasone in chronic epileptic mice. Electroencephalographic (EEG)
activity was recorded and quantified for 3 days (days 1–3) in C57BL/6 epileptic mice, 6 weeks after kainate-induced status epilepticus
(200 ng in 50 nl). Mice (n=21) with a stable baseline of spontaneous epileptic activity (days 1–3; “see Table 1 for row values”) were
injected daily intraperitoneally with 12.5 (n=6), 50 (n=6), or 200 mg/kg VX-765 (n=9) (twice a day at 9:00AM and at 4:00PM) from day 4 to
day 7 (“see Methods for details”) (panels a–c). EEG activity was measured continuously after each drug injection for 2 h, then disconti-
nued until the subsequent injection. In each experimental group, VX-765 treatment was withdrawn at day 7 (after the 4:00PM injection),
and EEG recordings were made for the subsequent 3 days (days 8–10) to monitor when pre-injection baseline was resumed after drug
wash-out. Panel (d) depicts the maximal reduction in epileptic activity, as assessed for each dose of VX-765 at day 7 (after the 4:00PM
injection). Panel (e) shows data from a subgroup of epileptic mice (n=6) treated with dexamethasone (3 mg/kg s.c.) for 3 days (“see
Methods and FIG. 3c”). Data from day 1 of treatment only are shown because the same effect was observed in the subsequent 2 days.
Data in panels (a–e) are presented as mean±standard error of the mean of epileptic activity, expressed as a percentage of the corres-
ponding pre-injection baseline (baseline raw data are shown in Table 1). *p<0.05; **p<0.01 versus baseline by repeated measures
analysis of variance followed by Dunnett’s test. DEX=dexamethasone.
310 MAROSO ET AL.
Neurotherapeutics, Vol. 8, No. 2, 2011
sessions, daily spontaneous epileptic activity (“see
representative tracings in FIG. 2 panels a and b”)
was stable and reproducible in each epileptic mouse
(i.e., ~6 weeks after status epilepticus). FIG. 4a–d
shows the dose-dependent anticonvulsant effect of
VX-765 in the 2-h EEG recordings (i.e., from 9:00AM
to 11:00AM, and from 4:00PM to 6:00PM) after each
daily injection. The systemic administration of VX-
765 in epileptic mice dose- and time-dependently
reduced the time spent in epileptic activity during the
4 days of treatment (FIG. 4a–d): a maximal 50 to
75% reduction was achieved using 50 and 200 mg/kg,
respectively (FIG. 4d) (days 6 and 7 in FIG. 4b, c), as
compared to respective pre-injection baselines (days 1–3
in FIG. 4b, c); a dose of 12.5 mg/kg was ineffective
(FIG.4a, d).Theanticonvulsant effect ofVX-765beganon
day 2 of treatment (i.e., day 5 in FIG. 4b, c; corresponding
to the third and fourth injections). After reaching the
maximal anticonvulsant effect (50 and 200 mg/kg at day 7,
the 4:00PM injection), VX-765 administration was inter-
rupted: seizure activity was still significantly reduced for
15 h after drug withdrawal (i.e., day 8), and then it
recovered to pre-injection baseline within the next 24 h
(day 9 in FIG. 4b, c).
To assess the effect of an anti-inflammatory steroid drug,
epileptic mice (Table 1, numbers 10–15) were injected
daily with 3 mg/kg s.c. DEX after 2-h baseline recording;
this treatment was repeated for 3 days (“see Methods and
FIG.3c for details”).DEX treatment reduced spontanenous
epileptic activity by 30% on average (p<0.01) between the
second and fourth hour after each daily injection. Then
mice invariably recovered their pre-injection baseline
epileptic activity within the fourth and sixth hour after
drug injection (FIG. 4e).
In accordance with Riban et al. , 50 mg/kg
phenytoin (Table 1, tested in mice numbers 5–9 ) did
not affect spontaneous EEG epileptic activity in the 2-h
post-injection period (percentage changes of time in
epileptic activity vs respective pre-injection baseline was
As we previously described in Maroso et al. ,
Nissl-stained sections of the hippocampi of epileptic
mice showed unilateral neuronal cell loss reminescent of
hippocampal sclerosis, involving pyramidal cell layers
and the hilus of the dentate gyrus, and dispersion of
granule cells (FIG. 5a–c). In accordance with Riban et al.
, this pattern was restricted to the KA-injected (kainic
acid) side (FIG. 5b) because the contralateral non-
injected side (FIG. 5c) had normal histology similar to
naïve mice (FIG. 5a) (“for details also see Bouilleret et
al. , Riban et al. , Maroso et al. , and
Antonucci et al. ”).
Immunohistochemical analysis of hippocampal sec-
tions from epileptic mice after wash-out of VX-765
(FIG. 5e, h) showed activation of GFAP-positive astro-
cytes (FIG. 5e vs d) and CD11b-positive microglia
(FIG. 5h vs g) in kainate injected hippocampi. IL-1β
expression was not observed in control mice (FIG. 5j);
whereas it was increased in GFAP-positive astrocytes in
epileptic mice (FIG. 5k vs j; co-localization in panels
FIG. 5k1–k3). No expression of IL-1β in CD11b-
positive microglia was found (FIG. 5k inset). A similar
glia activation and IL-1β expression pattern were
observed in the hippocampus contralateral to kainate
injection (not shown).
of its maximal anticonvulsant effect, showed no IL-1β
expression in the hippocampus (FIG. 5l vs k). Although
astrocytes did not express IL-1β during VX-765 treatment
(FIG. 5l1–l3), the astrocytes still showed activated pheno-
type (FIG. 5f vs e and d). Similarly, VX-765 did not alter
microglia activation (FIG. 5i vs h and g).
Scattered perivascular CD68-immunoreactive macro-
phage-like cells were found near blood vessels in the
hippocampi of epileptic mice (FIG. 5h inset), similarly to
what was observed in VX-765 treated mice (not shown);
these cells were absent in control mice (not shown).
Granulocytes and T cells were not detected in brain
parenchyma in all experimental groups (not shown).
Table 2. Dose-Response Effect of VX-765 on Acute Seizures
Onset time (minutes)
Number of seizures
Time in seizures (minutes)
VX-765 (12.5, 50, 100, 200 mg/kg) was injected intraperitoneally once a day for 3 consecutive days; on day 4, the drug was injected 45 minutes
before kainic acid. Kainate (7 ng in 0.5 μl) was injected unilaterally in the left hippocampus in freely moving C57BL6 mice (n=7–-12).
*p<0.05;†p<0.01 versus vehicle-injected mice by one-way analysis of variance followed by Tukey’s post hoc test.
311ICE/CASPASE-1 ROLE IN EPILEPTIC ACTIVITY
Neurotherapeutics, Vol. 8, No. 2, 2011
FIG. 5. Histopathology, interleukin (IL)-1β expression, and glia activation in the hippocampus of epileptic mice, and effect of VX-765.
Nissl-stained sections of the hippocampi of representative (a) vehicle-injected and (b, c) epileptic mice. (b) The hippocampus injected
with kainate shows histopathology reminescent of hippocampal sclerosis, involving cell loss in pyramidal cell layers and degeneration of
hilar interneurons, and dispersion of granule cells. (c) The contralateral non-injected side has normal histology similar to (a) vehicle-
injected mice. (d–l) These panels show representative micrographs of CA3 hippocampal sections depicting (d–f) GFAP-positive astro-
cytes, (g–i) CD-11b-positive microglia, and (j–l) IL-1β immunostaining in (d, g, and j) vehicle-injected mice, and (e, f, h, i, k, and l) epileptic
mice. (e, h, and k) Show the hippocampus of an epileptic mouse at the end of 200 mg/kg VX-765 wash-out period (i.e., at day 10 in
FIG. 4c); (f, i, and l) show the hippocampus of a representative mouse killed at the time of maximal anticonvulsant drug effect (i.e., at day
7 in FIG. 4c). (d–i, insets) show 4-fold magnification of glial cells to show their phenotypic activation in (e, f, h, and i) epileptic tissue vs (d,
g) control tissue. (h, inset) shows a 2-fold magnification of perivascular CD68-positive macrophages. Co-localization panels (k1–l3) show
IL-1β expression in activated astrocytes in the hippocampus of epileptic mice; note the inhibition of IL-1β expression in VX-765 treated
mice (l1–l3). IL-1β signal did not co-localize with CD11b signal denoting lack of IL-1β localization in microglia (k, inset). Scale bar in (a–c)
250 μm; (d–l) 100 μm. CA1=Cornus Ammoni’s 1; CA3=Cornus Ammoni’s 1; h=hilus.
312MAROSO ET AL.
Neurotherapeutics, Vol. 8, No. 2, 2011
These results show a powerful anticonvulsant effect of
VX-765 (a specific inhibitor of ICE/caspase-1) after its
systemic administration in a mouse model of acute seizures
and in chronic epileptic mice with neuropathological
features mimicking TLE with hippocampal sclerosis [23–
25, 27–29]. As in human TLE, spontaneous epileptiform
activity in this mouse model is resistant to some common
AEDs  (as in the present study). We used spontaneous
epileptic activity to assess impact of drugs, rather than
spontaneous seizures. This activity is more consistent than
spontaneous seizures, which can be erratic, and this
approach does not require continuous video EEG monitor-
ing, which would substantially reduce the extent of experi-
ments wecould perform.We selected epileptic activity (“for
more detail see FIG. 2”) that is more consistent with the
subclinical seizures seen in humans during intracranial
Unlike other caspases, ICE/caspase-1 is specifically
required for processing the inactive precursor pro-IL-1β
to biologically active IL-1β , and for its subsequent
secretion from the cell. VX-765, which represents a new
class of specific ICE/caspase-1 protease inhibitors, is a pro-
drug with improved oral bioavailability that has been under
clinical development for the treatment of inflammatory and
autoimmune conditions [16, 37]. VX-765 has been shown
to block the release of IL-1β induced by ICE/caspase-1
activation by proinflammatory stimuli in organotypic
hippocampal slices [9, 38]; moreover, VX-765 reduced
acute seizures in kainate-treated rats with a concomitant
50% decrease in hippocampal levels of IL-1β . VX-765
was also able to block generalization of seizures during
rapid electrical kindling development in rats, and this effect
was associated with prevention of after discharge-induced
IL-1β expression in forebrain astrocytes . The anti-
convulsant effects of this class of drugs (pralnacasan, VX-
765) are specifically mediated by ICE/caspase-1 inhibition
because these effects were precluded in mice defective for
ICE/caspase-1 gene . These findings, together with the
evidence that ICE/caspase-1 knock-out mice are intrinsi-
cally less susceptible to kainate seizures , strongly
supports a key role of ICE/caspase-1 activation in the
mechanisms underlying seizure occurrence.
The activation of the IL-1β system in neurons and glia
during experimental seizures is corroborated by clinical
observations showing overexpression of IL-1β and IL-
1R1 and induction of ICE/caspase-1 in surgically
resected epileptic tissue from individuals with drug-
resistant seizures [10, 18, 21, 22]. Notably, the expres-
sion of IL-1β measured in human epileptogenic tissue
positively correlates with the frequency of seizures in the
same patients before surgery, highlighting a possible link
between the brain level of this cytokine and seizure
A mechanistic link between IL-1β and N-Methyl-D-
aspartate (NMDA) receptors has been recently demon-
strated to underlie the pro-convulsant action of this
cytokine . This mechanism consists of the IL-1R1-
dependent activation of neuronal sphingomyelinase and
Src kinases, resulting in phosphorylation of the NR2B
subunit of the NMDA receptors [11, 39] and enhanced
NMDA-mediated neuronal Ca2+
Pharmacological interference with this mechanism in
vivo prevents the pro-convulsant activity of IL-1β .
NR2B-expressing NMDA receptors are considered the
main targets of glutamate released by activated
astrocytes, leading to neuronal induction of slow inward
currents. These slow inward currents have a role in
neuronal synchronization and can trigger action potentials
in neurons; these currents are increased in models of
seizures and their pharmacological inhibition significantly
attenuates ictal events [40, 41]; therefore, the currents may
represent the crucial molecular event by which IL-1β
worsens seizures. IL-1β also inhibits the astrocytic
reuptake of glutamate  and may increase glutamate
release from glia via tumor necrosis factor-α production
, thus resulting in elevated extracellular glutamate
levels that promote hyperexcitability. The inhibition of IL-
1β production by VX-765 would prevent the increased
neuronal excitability mediated by the previously described
events, thus reducing epileptic activity. Notably, an anti-
inflammatory steroid drug, such as dexamethasone ,
which is used for medical treatment of pharmacoresistant
also effective in this model for partly reducing epileptic
activity. The effect of dexamethasone was transient lapsing
after 4 h of treatment. This relatively short-lasting effect as
compared to longer biological activity  and terminal
half-life  reported in rats might be due to species
differences in dexamethasone pharmacokinetics .
Besides its established anti-inflammatory properties,
dexamethasone anticonvulsant effect may be mediated by
its ability to reverse the increased serum corticosterone
concentrations, which are elicited by elevated IL-1β brain
levels via activation of the hypothalamic-pituitary adrenal
axis (HPA) axis [47, 48]. Thus, chronic high levels of
glucorticoids can produce proinflammatory effects in the
brain  and increase neuronal vulnerability to injury [50,
51]. This mechanism, although still speculative, may also
play a role in the anticonvulsant activity of VX-765.
Given the unmet needs in treatment-resistant epilepsy,
and the potential role of ICE/IL-1β in seizures, the
results from the previous and current pre-clinical studies
support testing the hypothesis that inhibition of ICE/
caspase-1 may be useful to treat intractable epilepsy in
humans. In a phase 2 clinical study of psoriasis, VX-765
was safe and well tolerated [52–54]. A phase 2 clinical
trial is currently underway to assess whether VX-765 can
be administered safely and reduces seizures in patients
313ICE/CASPASE-1 ROLE IN EPILEPTIC ACTIVITY
Neurotherapeutics, Vol. 8, No. 2, 2011
with treatment-resistant partial epilepsy when given for a
6-week duration . Pending the results of this initial
epilepsy study, it will be important to further understand
the safety and efficacy of longer duration treatment with
VX-765. Chronic administration is likely to be required,
but the potential effects of long-term inhibition of ICE/
caspase-1 are unknown.
In summary, the powerful anticonvulsant effect of VX-
765 on acute seizures and in chronic epileptic mice not
responsive to some conventional AEDs may open new
perspectives for a clinical use of selective ICE/caspase-1
inhibitors for the treatment of seizures in pharmacor-
esistant epileptic disorders. Moreover, because these
acute and chronic models of seizures are sensitive to
anti-inflammatory drugs, including antagonists of toll-
like receptor 4 , they may be useful to develop
pharmacological attempts to block brain inflammation for
attaining seizure control.
Acknowledgments: This study was supported by EPICURE
(LSH-CT-2006-037315) (A.V.) and Fondazione Monzino (A.
V.). Mattia Maroso received a fellowship from NeuroGlia (EU-
FP7-project 202167). The authors are grateful to Vertex
Pharmaceuticals Inc. for supplying VX-765 and to Irina
Kadiyala (Vertex Pharmaceuticals Inc.) for her support with
the drug formulation. Dr. C. I. Wright is employed at Vertex
Pharmaceuticals Inc. We also thank Dr. F. Noé and Dr. M. de
Curtis for their valuable contribution in EEG tracing analysis.
Full conflict of interest disclosure is available in the electronic
supplementary material for this article.
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