Oxidative stress in the hippocampus after pilocarpine-
induced status epilepticus in Wistar rats
Rivelilson M. Freitas, Silva
ˆ
nia M. M. Vasconcelos, Francisca C. F. Souza, Glauce S. B. Viana
and Marta M. F. Fonteles
Department of Physiology and Pharmacology, Laboratory of Neuropharmacology, School of Medicine, Federal University of Ceara
´
, Fortaleza,
Brazil
Status epilepticus (SE) is a neurological emergency
with an associated mortality of 10–12% [1]. Pilocar-
pine-induced seizure models have provided information
on the behavioral and neurochemical characteristics
associated with seizure activity [2,3]. Other studies sug-
gest permanent changes in different biochemical sys-
tems during SE. An increase in lipid peroxidation, a
decrease in GSH content, and excessive free radical
formation may occur during SE induced by pilocarpine
[4,5].
This model can be used to investigate the develop-
ment of neuropathology in SE [6]. Despite numerous
studies clearly indicating the importance of enzyme
activity in the epileptic phenomenon, the mechanisms
by which these enzymes influence SE are not com-
pletely understood [7,8]. Therefore, we decided to
study enzymatic activity related to oxidative stress
mechanisms during SE [9].
Oxidative stress, which is defined as the over-produc-
tion of free radicals, can dramatically alter neuronal
function and has been related to SE [10,11]. It is partic-

,
subcutaneous). Both groups were killed 24 h after treatment. After the
induction of status epilepticus, there were significant increases (77% and
51%, respectively) in lipid peroxidation and nitrite concentration, but a
55% decrease in GSH content. Catalase activity was augmented 88%, but
superoxide dismutase activity remained unaltered. These results show evi-
dence of neuronal damage in the hippocampus due to a decrease in GSH
concentration and an increase in lipid peroxidation and nitrite content.
GSH and catalase activity are involved in mechanisms responsible for elim-
inating oxygen free radicals during the establishment of status epilepticus in
the hippocampus. In contrast, no correlations between superoxide dismutase
and catalase activities were observed. Our results suggest that GSH and
catalase activity play an antioxidant role in the hippocampus during status
epilepticus.
Abbreviations
ROS, reactive oxygen species; SE, status elipticus.
FEBS Journal 272 (2005) 1307–1312 ª 2005 FEBS 1307
inefficiency of the electron-carrying components of the
mitochondrial transport chain, monoamine degradation,
xanthine oxidase reaction, and metabolism of arachidon-
ic acid. However, the superoxide produced can be meta-
bolized by superoxide dismutase which is present in
both cytosol (copper–zinc-associated isoform) and mito-
chondria (manganese-associated isoform) [14,15].
Reactive oxygen species (ROS), such as superoxide,
hydroxyl radical, nitric oxide, nitrite, nitrate and H
2
O
2
,

sniffing, paw licking, rearing and wet dog shakes that
persist for 10–15 min), clonic movements of forelimbs,
head bobbing and tremors [21,22]. These behavioral
changes progress to motor limbic seizures as previously
described by Tursky et al. [23]. Limbic seizures persist
for 30–50 min, progressing to SE. In the latter experi-
ments, 63% of animals died during the 24 h observa-
tion period.
Lipid peroxidation and nitrite and GSH content
in the hippocampus of adult rats after
pilocarpine-induced SE
Lipid peroxidation and nitrite and GSH concentrations
are presented in Fig. 1. Lipid peroxidation was
markedly increased in this model compared with cor-
responding values for the control group. After pilocar-
pine-induced SE, there was a significant (77%) increase
in thiobarbituric-acid-reacting substances [T(14) ¼
18.282; P < 0.0001]. SE produced a significant increase
in hippocampal nitrite content of 51% [T(18) ¼ 25.959;
P < 0.0001] compared with the control group. On the
other hand, a 55% decrease in GSH concentration
[T(10) ¼ 27.452; P < 0.0001] compared with the con-
trol group was detected (Fig. 1).
Superoxide dismutase and catalase activities
in the hippocampus of adult rats after
pilocarpine-induced SE
Table 1 shows superoxide dismutase and catalase activ-
ities in the hippocampus after seizures and SE induced
by pilocarpine. Post hoc comparison of means indicated
similar superoxide dismutase activity [T(16) ¼ 0.5892;

In normal conditions, there is a steady-state balance
between the production of nitric oxide and metabolites
(nitrite and nitrate) and their destruction by antioxid-
ant systems. Our results show an increase in nitrite
formation after SE, suggesting that there is a possible
increase in concentrations of ROS, which are often
involved in neuronal damage [7,15]. Other studies have
shown that nitrite and nitrate concentrations are not
raised in epileptic patients [27]. Other mechanisms may
be associated with the increase in ROS levels in the
epilepsy model as well as in neurodegeneration
observed in epileptic humans [18,28].
During ROS scavenging, glutathione disulfide pro-
duction and GSH reduction occur. When the balance
between ROS formation and ROS elimination is func-
tionally normal, there is GSH recovery [29]. As men-
tioned above, we can conclude that during SE there is
over-formation of free radicals and ⁄ or a deficiency of
antioxidant systems, as evidenced by the augmented
nitrite content, the unaltered superoxide dismutase
activity, and the GSH consumption, all of which char-
acterize oxidative stress.
Our findings show that pilocarpine induces SE,
which can produce alterations in superoxide dismutase
and catalase activities in different areas, thereby pro-
tecting the brain from neuronal damage induced by
lipid peroxidation products [11]. However, we found
no changes in hippocampal superoxide dismutase
activity. It is unlikely that the unaltered superoxide
dismutase activity is related to the mechanisms

that catalase would be one of the enzymes with aug-
mented activity, as this effect was not observed for the
superoxide dismutase.
Evidence for the role of free radicals in SE has been
found by using exogenously enzymatic and nonenzy-
matic antioxidant treatment for protection against
seizures and SE-induced neuronal damage [15,26]. A
steady-state level of superoxide and H
2
O
2
is always
present in cells as a result of normal metabolism.
Superoxide dismutase and catalase are responsible for
degradation of superoxide and H
2
O
2
, respectively, and
the balance between these antioxidant enzymes is rele-
vant for cell and neuronal functions [8,18]. The fact
that an increase in catalase activity may not result in
neurotoxic effects during SE indicates that basal ROS
production is damaging to the neurons and should be
controlled [9,28].
The biochemical alterations observed can produce
neuronal damage in the hippocampus. Our results indi-
cate that SE alters brain antioxidant defenses and that
there may be extensive participation of enzymes in sei-
zures. Further studies need to be carried out to ascer-

Male Wistar rats (250–280 g; 2 months old) were used. Ani-
mals were housed in cages with free access to food and
water and with a standard light ⁄ dark cycle (lights on at
07:00 h). The experiments were performed according to the
Guide for the Care and Use of Laboratory Animals of the
US Department of Health and Human Services, Washing-
ton, DC (1985). Control animals received 0.9% saline sub-
cutaneously (control group; n ¼ 48), and the pilocarpine
group were treated with a single dose of pilocarpine hydro-
chloride (400 mgÆkg
)1
; subcutaneous; n ¼ 43). Behavioral
changes were observed over 24 h. The variables assessed
were: number of peripheral cholinergic signs, tremors, ste-
reotyped movements, seizures, SE and mortality. SE was
defined as continuous seizures for a period longer than
30 min. SE was induced by method of Turski et al. [23].
For biochemical assays, both pilocarpine and control
groups were killed by decapitation 24 h after treatment.
Their brains were dissected on ice to remove the hippocam-
pus for determination of lipid peroxidation, nitrite content,
GSH concentration, and superoxide dismutase and catalase
activities. Detailed criteria for determining the periods after
pilocarpine administration have been reported by Cavalhe-
iro et al. [32]. The pilocarpine group consisted of rats that
had seizures, SE for a period longer than 30 min, and that
did not die within 24 h of observation.
Determination of lipid peroxidation and nitrite
content
For all of the experimental procedures, 10% (w ⁄ v) homo-

ured using a microplate reader. Nitrite concentration was
determined from a standard nitrite curve generated using
NaNO
2
.
Determination of GSH
GSH in the pilocarpine group (n ¼ 10) and control animals
(n ¼ 10) was analyzed. The hippocampus was homogenized
in 0.02 m EDTA. Immediately thereafter, 10% (w ⁄ v) homo-
genates were assayed for GSH as described by Sedlak &
Lindsay [34], and the results expressed in lgÆ(g tissue wet
weight)
)1
.
Determination of superoxide dismutase and
catalase activities
The hippocampus was ultrasonically homogenized in 1 mL
0.05 m sodium phosphate buffer, pH 7.0. Protein concen-
tration was measured by the method of Lowry et al. [35].
The 10% homogenates were centrifuged (800 g, 20 min),
and the supernatants used to assay superoxide dismutase
and catalase. Superoxide dismutase activity in the pilocar-
pine group (n ¼ 8) and control animals (n ¼ 10) was
assayed by using xanthine and xanthine oxidase to generate
superoxide radicals [24]. They react with 2,4-iodophenyl-
3,4-nitrophenol-5-phenyltetrazolium chloride to form a red
formazan dye. The degree of inhibition of this reaction was
measured to assess superoxide dismutase activity. The
standard assay substrate mixture contained 3 mL xanthine
(500 lm), 7.44 mg cytochrome c, 3.0 mL KCN (200 lm),

O
2
in 50 mL 0.05 m sodium
phosphate buffer, pH 7.0. The sample aliquot (20 lL) was
added to 980 lL substrate mixture. The initial absorbance
was recorded after 1 min, and the final absorbance after
6 min. The reaction was followed at 230 nm. A standard
curve was established using purified catalase (Sigma,
St Louis, MO, USA) under identical conditions. All samples
were diluted with 0.1 mmolÆL
)1
sodium phosphate buffer
Oxidative stress after status epilepticus in rats R. M. Freitas et al.
1310 FEBS Journal 272 (2005) 1307–1312 ª 2005 FEBS
(pH 7.0) to provoke a 50% inhibition of the diluent rate
(i.e. the uninhibited reaction). Results are expressed as
mmolÆmin
)1
Æ(lg protein)
)1
[36,37].
Statistical analysis
Results are expressed as means ± SEM for the number of
experiments, with all measurements performed in duplicate.
The Student–Newman–Keuls test was used for multiple
comparison of means of two groups of data. Differences
were considered significant at P < 0.05. Differences in
experimental groups were determined by two-tailed analysis
of variance.
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