An increase in lipid peroxidation, a decrease in GSH content, and excessive free radical formation may occur during SE induced by pilocarpine [4,5].. Superoxide, a free radical, can be g
Trang 1induced status epilepticus in Wistar rats
Rivelilson M Freitas, Silvaˆnia 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-ularly facilitated in the brain, as the brain contains large quantities of oxidizable lipids and metals, and, moreover, has fewer antioxidant mechanisms than other tissues [8]
Free radicals are chemical entities characterized by an orbital containing an unpaired electron [12] This elec-tron confers on these molecules a selec-trong propensity to react with target molecules by giving or withdrawing one electron from the target molecules to complete their own orbital [13] Superoxide, a free radical, can be gen-erated in the brain by several mechanisms such as
Keywords
hippocampus; oxidative stress; pilocarpine;
seizures; status epilepticus
Correspondence
R M Freitas, Rua Frederico Severo 201,
Ap 103, Bl 07, Messejana, Fortaleza,
60830-310, Brazil
Tel ⁄ Fax: +55 85 3274 6091
E-mail: rivmendes@bol.com.br
(Received 23 October 2004, revised 28
November 2004, accepted 20 December
2004)
doi:10.1111/j.1742-4658.2004.04537.x
The role of oxidative stress in pilocarpine-induced status epilepticus was investigated by measuring lipid peroxidation level, nitrite content, GSH con-centration, and superoxide dismutase and catalase activities in the hippo-campus of Wistar rats The control group was subcutaneously injected with 0.9% saline The experimental group received pilocarpine (400 mgÆkg)1, 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.
Trang 2inefficiency 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 H2O2,
are normally produced in the brain H2O2 is converted
into water by catalase and glutathione peroxidase,
which involves GSH, a cofactor of this enzyme [5,8]
GSH is one of the most important agents of the cellular
antioxidant defense system [16] The resulting hydroxyl
radical reacts with nonradical molecules, transforming
them into secondary free radicals This reaction takes
place during lipid peroxidation and produces
hydroper-oxides [7,11] In the nervous system, the phenomenon
known as excitotoxicity has been related to
over-pro-duction of free radicals [17] Neuronal hyperactivity
and⁄ or excitotoxicity may induce an increase in free
rad-ical concentrations during pilocarpine-induced SE [18]
This work was performed to determine lipid
peroxida-tion, nitrite content, GSH concentraperoxida-tion, and
super-oxide dismutase and catalase activities in the hippocampus
of adult rats after SE induced by pilocarpine
Results
Behavioral alterations after treatment with
pilocarpine
According to previous studies [2,19,20], immediately
after pilocarpine administration, animals persistently
show behavioral changes, including initial akinesia,
ataxic lurching, peripheral cholinergic signs (miosis,
piloerection, chromodacriorrhea, diarrhea and
mastica-tory automatisms), stereotyped movements (continuous
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;
P ¼ N.S.] However, hippocampal catalase activity showed a marked (88%) increase [T(10)¼ 10.722;
P < 0.0001] compared with the control group (Table 1)
Discussion
SE and oxidative stress are thought to be closely inter-related Our findings show that GSH was reduced whereas lipid peroxidation and nitrite content were increased after SE Lipid peroxidation in the brain can
Fig 1 Biochemical alterations in the hippocampus of adult rats after pilocarpine-induced SE Male rats (250–280 g, 2 months old) were treated with a single dose of pilocarpine (400 mgÆkg)1, subcu-taneously) The control group was treated with 0.9% saline Animals were observed for 24 h and then killed Results are mean ± SEM for the number of animals shown inside the bars.
a P < 0.05 compared with control animals (Student-Newman-Keuls test) The differences in the experimental groups were determined
by analysis of variance.
Trang 3be induced by many chemical compounds and brain
injury such as epilepsy [24,25] The brain is more
vul-nerable to injury by lipid peroxidation products than
other tissues [8] Moreover, lipid peroxidation is an
index of irreversible neuronal damage of cell
mem-brane phospholipid and has been suggested as a
poss-ible mechanism of epileptic activity [11,18,26]
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 repro-duction 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
involved in the initiation and⁄ or propagation of
seizures induced by pilocarpine Our results are in agreement with another study showing unaltered superoxide dismutase activity after 24 h, suggesting that superoxide dismutase activity only changes during the initiation of seizures [14] When studying this epi-lepsy model, we found increased catalase activity in the hippocampus, indicating that this enzyme, in association with GSH, provides neuroprotection against the increase in lipid peroxidation and nitrite content These data suggest that the hippocampus does not use superoxide dismutase as the major free-radical-scavenging system [9,30] It probably uses other scav-enging systems (catalase and GSH)
Pilocarpine-induced SE produces several changes in variables related to the generation and elimination of oxygen free radicals in adult rats [18,30] An increase
in free radical formation is accompanied by an imme-diate compensatory increase in catalase activity, which may be a long-term compensatory mechanism inclu-ding activity modulation of enzymes [31] In addition,
in the normal physiological state, changes in neuronal activity are accompanied by alterations in the meta-bolic rate (oxygen and energy metabolism) [1,8], which induce modifications in cerebral blood flow [10] In pathological states, blood flow may not occur in the same way There is clinical and experimental evidence
of alterations in oxygen levels because of reduced oxygen availability after SE [10] Considering that increased metabolic demand was observed, we suggest 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 H2O2 is always present in cells as a result of normal metabolism Superoxide dismutase and catalase are responsible for degradation of superoxide and H2O2, 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-tain whether ROS are involved in the pathogenesis of temporal lobe epilepsy
Table 1 Superoxide dismutase [UÆ(mg protein))1] and catalase
[mmolÆmin)1Æ(lg protein))1] activities in the hippocampus of adult
rats after pilocarpine-induced SE Male rats (250–280 g, 2 months
old) were treated with a single dose of pilocarpine (400 mgÆkg)1,
subcutaneously) The control group was treated with 0.9% saline.
Animals were observed for 24 h and then killed Results are
mean ± SEM for the number of animals shown in parentheses.
The differences in experimental groups were determined by
ana-lysis of variance.
Control 2.35 ± 0.14 (10) 14.50 ± 0.65 (9)
Pilocarpine 2.45 ± 0.10 (8) 27.25 ± 1.03 (8) a
a P < 0.05 compared with control animals (Student–Newman–Keuls
test).
Trang 4Experimental procedures
Treatment of animals and preparation of samples
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; n¼ 48), and the pilocarpine
group were treated with a single dose of pilocarpine
hydro-chloride (400 mgÆkg)1; subcutaneous; n¼ 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-genates of the area of the brain investigated were prepared
for both groups Lipid peroxidation in the pilocarpine
group (n¼ 7) and control animals (n ¼ 9) was analyzed by
measuring thiobarbituric-acid-reacting substances in
homo-genates, as previously described by Draper & Hadley [33]
Briefly, the samples were mixed with 1 mL 10%
trichloro-acetic acid and 1 mL 0.67% thiobarbituric acid They were
then heated in a boiling water bath for 15 min, and butanol
(2 : 1, v⁄ v) was added to the solution After centrifugation
(800 g, 5 min), thiobarbituric-acid-reacting substances were
determined from the absorbance at 535 nm
To determine nitrite content of the control rats (n¼ 10)
and pilocarpine group (n¼ 10), the 10% (w ⁄ v) homogenates
were centrifuged (800 g, 10 min) The supernatants were
col-lected, and nitric oxide production was determined based on
the Griess reaction [25] Briefly, 100 lL supernatant was
incubated with 100 lL of the Griess reagent [1%
sulfanila-mide in 1% H3PO4⁄ 0.1% N-(1-naphthyl)ethylenediamine
dihydrochloride⁄ 1% H3PO4⁄ distilled water, 1 : 1 : 1 : 1,
v⁄ v ⁄ v ⁄ v) at room temperature for 10 min A550was
meas-ured using a microplate reader Nitrite concentration was
determined from a standard nitrite curve generated using NaNO2
Determination of GSH
GSH in the pilocarpine group (n¼ 10) and control animals (n¼ 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 (n¼ 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), and 3.0 mL EDTA (1 mm) in 18.0 mL 0.05 m sodium phos-phate buffer, pH 7.0 The sample aliquot (20 lL) was added to 975 lL of the substrate mixture plus 5 lL xan-thine oxidase After 1 min, the initial absorbance was recor-ded and the timer was started The final absorbance after
6 min was recorded The reaction was followed at 550 nm Purified bovine erythrocyte superoxide dismutase (Randox Laboratories, Belfast, Northern Ireland, UK) was used under identical conditions to obtain a calibration curve showing the correlation of the inhibition percentage of formazan dye formation and superoxide dismutase activity Superoxide dismutase activity in the samples was deter-mined from this curve, and the results expressed as UÆ(mg protein))1
Catalase activity was measured in the control (n¼ 9) and pilocarpine (n¼ 8) groups by the method that uses H2O2to generate H2O and O2[36] The activity was measured by the degree of this reaction The standard assay substrate mix-ture contained 0.30 mL H2O2 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
Trang 5(pH 7.0) to provoke a 50% inhibition of the diluent rate
(i.e the uninhibited reaction) Results are expressed as
mmolÆmin)1Æ(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
Acknowledgements
This work was supported by a research grant from the
Brazilian National Research Council (CNPq) R.M.F
is a fellow of the CNPq The technical assistance of
Maria Vilani Rodrigues Bastos and Steˆnio Gardel
Maia are gratefully acknowledged
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