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- 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 strong 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. 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 , are normally produced in the brain. H 2 O 2 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 concentration, 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. Oxidative stress after status epilepticus in rats R. M. Freitas et al. 1308 FEBS Journal 272 (2005) 1307–1312 ª 2005 FEBS be 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 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 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 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- tain whether ROS are involved in the pathogenesis of temporal lobe epilepsy. Table 1. Superoxide dismutase [UÆ(mg protein) )1 ] and catalase [mmolÆmin )1 Æ(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. Group Superoxide dismutase Catalase 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). R. M. Freitas et al. Oxidative stress after status epilepticus in rats FEBS Journal 272 (2005) 1307–1312 ª 2005 FEBS 1309 Experimental 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; 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- genates of the area of the brain investigated were prepared for both groups. Lipid peroxidation in the pilocarpine group (n ¼ 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 (n ¼ 10) and pilocarpine group (n ¼ 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% H 3 PO 4 ⁄ 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride ⁄ 1% H 3 PO 4 ⁄ distilled water, 1 : 1 : 1 : 1, v ⁄ v ⁄ v ⁄ v) at room temperature for 10 min. A 550 was meas- 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), 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 (n ¼ 9) and pilocarpine (n ¼ 8) groups by the method that uses H 2 O 2 to generate H 2 O and O 2 [36]. The activity was measured by the degree of this reaction. The standard assay substrate mix- ture contained 0.30 mL H 2 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. 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. 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