Effects of Levetiracetam in Lipid Peroxidation Level, Nitrite–Nitrate Formation and Antioxidant Enzymatic Activity in Mice Brain After Pilocarpine-Induced Seizures

Abstract

Oxidative stress has been implicated in a large number of human degenerative diseases, including epilepsy. Levetiracetam (LEV) is a new antiepileptic agent with broad-spectrum effects on seizures and animal models of epilepsy. Recently, it was demonstrated that the mechanism of LEV differs from that of conventional antiepileptic drugs. Objectifying to investigate if LEV mechanism of action involves antioxidant properties, lipid peroxidation levels, nitrite–nitrate formation, catalase activity, and glutathione (GSH) content were measured in adult mice brain. The neurochemical analyses were carried out in hippocampus of animals pretreated with LEV (200 mg/kg, i.p.) 60 min before pilocarpine-induced seizures (400 mg/kg, s.c.). The administration of alone pilocarpine, 400 mg/kg, s.c. (P400) produced a significant increase of lipid peroxidation level in hippocampus. LEV pretreatment was able to counteract this increase, preserving the lipid peroxidation level in normal value. P400 administration also produced increase in the nitrite–nitrate formation and catalase activity in hippocampus, beyond a decrease in GSH levels. LEV administration before P400 prevented the P400-induced alteration in nitrite–nitrate levels and preserved normal values of catalase activity in hippocampus. Moreover, LEV administration prevented the P400-induced loss of GSH in this cerebral area. The present data suggest that the protective effects of LEV against pilocarpine-induced seizures can be mediated, at least in part, by reduction of lipid peroxidation and hippocampal oxidative stress.

INTRODUCTION

The epilepsies are a group of clinical syndromes affecting more than 50 million people worldwide (Patel, 2004). The pilocarpine model is a useful animal model to investigate the development of neuropathology of seizures and temporal lobe epilepsy (Cavalheiro et al., 1994; Freitas et al., 2003). In this model, the initial precipitating injury is characterized by a prolonged status epilepticus (SE), which causes neuronal loss and mossy fiber sprouting in hippocampus and spontaneous recurrent seizures (Cavalheiro et al., 1991; Bonan et al., 2000).

Although the mechanism of pilocarpine-induced seizures and SE is not completely understood, it is known that it depends on muscarinic activation and also on enzymatic alterations in several systems (Naffah-Mazzacoratti et al., 2001; Liu et al., 2002). Following the toxicity induced by an initial cholinergic phase, a distinct noncholinergic phase occurs, in which excessive glutamate and aspartate release induces SE in this epilepsy model (McDonough and Shih, 1997).

Oxidative stress is classically defined as a redox unbalance with an excess of oxidants or a defect in antioxidants (Sies, 1985). It has been implicated in a large number of human degenerative diseases, affecting a wild variety of physiological functions such as inflammatory diseases, cancer, and neurological diseases, including epilepsy (Patel, 2004). Reactive oxygen species are normally produced in the brain, such as superoxide, hydroxyl radical, and the hydrogen peroxide (Dugan and Choi, 1999). When produced in excess, reactive oxygen species can cause tissue injury, including lipid peroxidation, DNA damage, and enzyme inactivation (Halliwell and Gutteridge, 1999).

Lipid peroxidation can be induced by many chemicals and in many tissue injuries, and has been suggested as a possible mechanism for the neurotoxic effects of convulsive process (Sawas and Gilbert, 1985; Waltz et al., 2000). Animal studies suggest that epileptic seizures result in free radical production and oxidative damage to cellular proteins, lipids, and DNA (Patel, 2004). Previous reports show that pilocarpine-induced seizures produce several changes in parameters related to generation and elimination of oxygen free radicals (Freitas et al., 2004a,b).

Another important point to discuss is that the role of nitric oxide (NO) in seizures remains controversial since both pro- and anticonvulsant properties are attributed to the molecule depending on the used experimental model (Kirkby et al., 1996). However, several lines of evidence suggest that NO produced by the activation of neuronal nitric oxide synthase (nNOS) triggers seizures (Murashima et al., 2002; Rajasekaran et al., 2003).

The insufficient efficacy of modern anticonvulsive drugs used in clinical practice, as well as wide global distribution of epilepsy, makes the design of novel compounds and study of its mechanisms of action very important (Brunbech and Sabers, 2002). Levetiracetam (Keppra^®, LEV), the S-enantiomer of a-ethyl-2-oxo-1-pyrrolidine acetamide, is a new antiepileptic agent that has broad-spectrum effects on partial and generalized seizures in several animal models of epilepsy (Löscher and Hönack, 1993; Gower et al., 1995; Klitgaard et al., 1998). Currently, many investigations have been carried out in order to clarify the mechanism of action of this drug.

The clinical effectiveness of LEV has been reported in patients with partial refractory epilepsy (Cereghino et al., 2000; Dooley and Plosker, 2000). Although its mechanism of action remains to be further characterized, it seems unrelated to any modulation of neuronal voltage-gated Na⁺ or low-voltage-activated Ca²⁺ (T-type) channels (Zona et al., 2001). However, experimental evidence, both in vitro and in vivo, suggests a role of both calcium channels N-type (Lukyanetz et al., 2002) and GABAergic activity in LEV effects (Poulain and Margineanu, 2002; Rigo et al., 2002). Other investigations show that LEV delays rectifier K⁺ currents in hippocampal neurons (Madeja et al., 2003) and also it is able to bind to SV2A protein, a synaptic vesicle protein that is an essential protein implicated in the control of exocytosis (Lynch et al., 2004). Klitgaard et al. (2003) demonstrated that LEV is able to counteract seizure-induced alterations in amino acid concentrations and to suppress the synchronization of neuronal spike and burst firing in hippocampus of rats without affecting normal neuronal excitability. Our group has postulated a modulation of the muscarinic cholinergic system by LEV in mice hippocampus on pilocarpine-induced model (Oliveira et al., 2005). These results support a novel mechanism of action for LEV, encouraging study of the drug to try to characterize its antiepileptic properties further.

The present work proposes to investigate whether LEV may alter pilocarpine-induced changes in antioxidant enzymes (catalase and reduced glutathione), in lipid peroxidation level, and nitrite–nitrate formation in mice hippocampus.

MATERIALS AND METHODS

Animals and Experimental Protocol

Experimentally naive, male Swiss mice, from the Animal House of the Federal University of Ceará, weighing 25–30 g, housed in home cages in a light/dark cycle and with water and food ad libitum, were used. The experiments were performed according to the Guide for the care and use of laboratory, the US Department of Health and Human Services, Washington, DC (1985).

Experiments were conducted between 8:00 and 10:00 a.m. Animals were acutely injected with LEV 200 mg/kg, i.p., or 0.9% NaCl, the vehicle of dissolution of all used drugs, and, 60 min later, they received pilocarpine hydrochloride, 400 mg/kg, s.c. (P400). Other two groups received only LEV (LEV200, 200 mg/kg, i.p.) or 0.9% NaCl (Control group).

Animals were closely observed for behavioral changes (appearance of peripheral cholinergic signs, such as miosis, piloerection, chromodacryorrhea, diarrhea, masticatory and stereotyped movements), latency to the appearance of the first seizure, SE, and death, for 1 h after the pilocarpine injection. In P400 group, brains were taken just after death; in LEV200 + P400 group, the survivors were killed by decapitation and brains were immediately removed. In control and LEV200 groups, animals were killed 1 h after injections and brains were immediately removed. The brains of all tested groups were dissected on ice to remove hippocampus for neurochemistry assays.

Our results of the observed behavioral alterations were previously shown in Oliveira et al. (2005), when we described that all animals treated with P400 presented peripheral cholinergic signs (miosis, piloerection, chromodacriorrhea, diarrhea, masticatory) and stereotyped movements (continuous sniffing, paw licking, and rearing), followed by motor limbic seizures in 100% (40/40) of the tested animals. The convulsive process persisted and built up to a SE in 90% (36/40) of these mice, leading to death of all animals. LEV200 administration, 60 min before P400, increased the latency to the onset of the first seizure, protected 53% (20/38) of the animals from seizures; reduced the occurrence of SE in 58% (22/38); increased the death's latency by 116%, and protected 61% (23/38) from death as compared to P400. No peripheral cholinergic signs and stereotyped movements were observed as compared to P400. Animals that received injections of 0.9% saline (Control) or LEV200 alone showed no seizure activity.

Enzymatic Assays

Immediately after decapitation, hippocampus was dissected for preparation of homogenates 10% (w/v) in 0.05 M phosphate buffer, pH 7.4 or EDTA 0.02M (GSH measurements) for the enzymatic assays. The protein concentration was measured according to the method described by Lowry et al. (1951).

Measurements of Lipid Peroxidation

Lipid peroxides formation was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in the homogenates, as previously described by Huong et al. (1998). The samples were briefly mixed with 50 mM potassium phosphate monobasic buffer pH 7.4 and catalytic system of formation of free radicals (FeSO₄ 0.01 mM and ascorbic acid 0.1 mM), and then incubated at 37°C for 30 min. The reaction was stopped with 0.5 mL of trichloroacetic acid 10%, then the samples were centrifuged (3000 rpm/15 min), the supernatants were retrieved and mixed with 0.5 mL of thiobarbituric acid 0.8%, then heated in a boiling water bath for 15 min and after this period, immediately kept cold in a bath of ice. Lipid peroxidation was determined by the absorbance at 532 nm and was expressed as μmol of malondialdehyde (MDA)/mg of protein.

Nitrite–Nitrate Determination

In order to assess the effects of treatments with respective drugs on nitric oxide (NO) production, nitrite–nitrate levels were determined in the mouse brain homogenates immediately after decapitation in all groups. After centrifugation (800 × g/10 min), the supernatant of homogenate was collected and the production of NO was determined based on Griess reaction (Green et al., 1981; Radenovic and Selakovic, 2005). Briefly, 100 μL of supernatant was incubated with 100 μL of Griess reagent (sulfanilamine in 1% H₃PO₄/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/1% H₃PO₄/distilled water, 1:1:1:1) at room temperature for 10 min. The absorbance was measured at 550 nm via a microplate reader. The standard curve was prepared with several concentrations of NaNO₂ (ranging from 0.75 to 100 μM) and was expressed as μM.

Evaluation of Catalase Activity

Catalase activity was measured by the method that employs hydrogen peroxide to generate H₂O and O₂ (Maehly and Chance, 1954). The activity was measured by degree of this reaction. The standard assay substrate mixture contained 0.30 mL of hydrogen peroxide in 50 mL of 0.05 M phosphate buffer, pH 7.0. The sample aliquot (20 μL) was added to 980 μL of substrate mixture. After 1 min, initial absorbance was recorded and final absorbance was read after 6 min. The reaction was followed at 230 nm. A standard curve was established using purified catalase (Sigma, MO, USA) under identical conditions. All samples were diluted with 0.1 mmol/ L phosphate buffer (pH 7.0) to provoke an inhibition 50% of diluent rate (i.e., the uninhibited reaction) and results were expressed as μM/min/μg protein.

Measurements of Glutathione (GSH) Levels

GSH levels were evaluated to estimate endogenous defenses against oxidative stress. The method was based on Ellman's reagent (DTNB) reaction with free thiol groups. Hippocampus homogenates 10% (w/v) in EDTA 0.02 M were added to a 50% trichloroacetic acid solution. After centrifugation (3000 rpm/15 min), the supernatant of homogenate was collected and the production levels of GSH were determined as described by Sedlak and Lindsay (1998). Briefly, the samples were mixed with 0.4 M Tris-HCl buffer, pH 8.9 and 0.01 M DTNB. GSH level was determined by the absorbance at 412 nm and was expressed as ng of GSH/g wet tissue.

Drugs

Levetiracetam (Keppra^®) was obtained from UCB Pharmaceutical Sector (Chemin du Foriest, Belgium). Pilocarpine HCl was purchased from ICN (CA, USA). All other chemicals were of analytical grade.

Statistical Analysis

Data were expressed as mean ± SEM. ANOVA was used for the statistical analysis followed by Student–Newman–Keuls test to identify differences between experimental groups. Values of p < 0.05 were considered significant.

RESULTS

The treatment of the animals only with P400 resulted in high levels of MDA in hippocampus when compared with the control group (only saline 0.9%). However, the treatment with LEV before P400 induced a decrease in the levels of lipid peroxidation as compared to the P400 group. The animals that had received only LEV had not shown alterations in the MDA levels when compared with the controls (Control: 6.79 ± 0.22; P400: 8.41 ± 0.24; LEV200: 6.90 ± 0.34; LEV200 + P400: 5.89 ± 0.54—results were expressed in μmol of malondialdehyde (MDA)/mg of protein) (Fig. 1).

Fig. 1.

Levels of TBARS (lipid peroxidation level) in hippocampus of adult mice treated or not with levetiracetam (200 mg/kg, i.p.) and administered or not with P400 (pilocarpine, 400 mg/kg, s.c.). Each bar represents the mean ± SEM, n = 7–10 mice/group. ^* p < 0.01 as compared to the other groups by ANOVA followed by Student–Newman–Keuls test. Abbreviation: TBARS, tiobarbituric-acid-reacting substances.

The administration of P400 alone resulted in high levels of nitrite–nitrate in hippocampus when compared to controls. However, when the mice had been managed with LEV before P400, a reduction in the nitrite–nitrate formation was observed when compared to the P400 group. The animals that had received only LEV did not have alteration in the hipocampal levels of nitrite–nitrate when compared with the control group (Control: 49.73 ± 1.04; P400: 59.18 ± 3.42; LEV200: 48.88 ± 1.41; LEV200 + P400: 45.53 ± 4.48—results were expressed in μM) (Fig. 2).

Fig. 2.

Content of nitrite–nitrate in hippocampus of adult mice treated or not with levetiracetam (200 mg/kg, i.p.) and administered or not with P400 (pilocarpine, 400 mg/kg, s.c.). Each bar represents the mean ± SEM, n = 5–6 mice/group. ^* p < 0.05 as compared to the other groups by ANOVA followed by Student–Newman–Keuls test.

In the P400 group, a significant increase of the activity of catalase in hippocampus was observed as compared to the control group. The values of treated animals only with LEV were similar to that observed in the control. The group treated with LEV before P400 had the catalase activity reduced in relation to the alone P400 group, showing that LEV pretreatment was capable to keep catalase activity to normal levels (Control: 37.56±3.26; P400: 59.62±5.46; LEV200: 37.10±3.05; LEV200+P400: 37.88±3.63—results were expressed in μM/min/μg protein) (Fig. 3).

Fig. 3.

Catalase activity in hippocampus of adult mice treated or not with levetiracetam (200 mg/kg, i.p.) and administered or not with P400 (pilocarpine, 400 mg/kg, s.c.). Each bar represents the mean ± SEM, n = 17–19 mice/group. ^* p < 0.01 as compared to the other groups by ANOVA followed by Student–Newman–Keuls test.

The treatment of the mice with P400 resulted in decreased levels of GSH in hippocampus when compared to control groups. LEV administration before P400 prevented the loss of GSH in this cerebral area. Animals treated with LEV alone did not show altered GSH levels when compared to control groups (Control: 1104.00 ± 81.42; P400: 850.80 ± 34.26; LEV200: 1063.01 ± 56.65; LEV200 + P400: 1020.00 ± 57.86—results were expressed in ng/g wet tissue) (Fig. 4).

Fig. 4.

Glutathione (GSH) levels in hippocampus of adult mice treated or not with levetiracetam (200 mg/kg, i.p.) and administered or not with P400 (pilocarpine, 400 mg/kg, s.c.). Each bar represents the mean ± SEM, n = 7–10 mice/group. ^* p < 0.05 as compared to the other groups by ANOVA followed by Student–Newman–Keuls test.

DISCUSSION

Membrane lipid derangements, including lipid peroxidation, have been reported to contribute significantly to paroxysmal membrane malfunction during epileptogenesis. Data in the literature have demonstrated that the enhanced free radical production and oxidative lipid damage occur during seizures and seizure-mediated neuronal injury (Frantseva et al., 2000b; Patel et al., 2001). Although the exact pathophysiological mechanism still needs to be clarified, the whole process can be related to the elevated intracellular concentration of Ca²⁺. A variety of biochemical processes, including the activation of membrane phospholipases, proteases, and nucleases that cause degradation of membrane phospholipids, proteolysis of cytoskeletone proteins, and protein phosphorylation (Dugan and Choi, 1994) are triggered during seizures. It also is now well established that SE is associated with neuronal damage, particularly in the hippocampus (Costa et al., 2004).

The results of the present study show that P400-induced (pilocarpine, 400 mg/kg, i.p) seizures lead to the increase of the lipid peroxidation in hippocampus of mice. Tejada et al. (2006) showed induced lipid peroxidation by pilocarpine in cortex and hippocampus of rats. In accordance with Freitas et al. (2004b), lipid peroxidation levels and nitrite content are increased during the acute period of P400-induced seizures in adult rats' hippocampus, striatum, and frontal cortex. In the current study, a rise in MDA levels was observed indicating the existence of lipid injury in the hippocampal region, in agreement with previous works (Dal-Pizzol et al., 2000; Frantseva et al., 2000a). These results show a direct evidence of lipid peroxidation during seizure activity that could be responsible for neuronal damage in the hippocampus of rats, during the establishment of P400 model of seizures.

The relationship between SE and oxygen reactive species is well known as the epileptiform activity causing excessive free radical production of reactive oxygen species, a factor believed to be involved in the mechanism leading to cell death and neurodegeneration (Frantseva et al., 2000b; Bellissimo et al., 2001; Freitas et al., 2004a). Our work shows a significant increase in nitrite–nitrate levels in hippocampus after P400 administration. The interaction between NO and free radicals produced during seizures could have potential consequences on the outcome of seizure-induced neuronal injury, since NO may cause neuronal damage in cooperation with other reactive oxygen species (Sah et al., 2002; Rajasekaran, 2005).

Seizures followed by a substantial increase in lipid peroxidation in brain tissue might be diminished by substances with antioxidant properties (Yamamoto and Tang, 1996; Gupta et al., 2001). Previous studies revealed that LEV has a novel spectrum of activity in experimental seizures models (Löscher and Hönack, 1993; Gower et al., 1995; Sharief et al., 1996). The mechanisms underlying the neuroprotective effect of LEV are not well understood. Considering its ability to block Ca²⁺ influx into the cells (Lukyanetz et al., 2002), it is possible to speculate an activity against the oxidative stress.

Marini et al. (2004) showed that LEV pretreatment was able to blunt brain MDA both in cortex and diencephalon of rats on kainic acid-induced seizures model. Our findings showed that LEV pretreatment before P400 administration was able to reduce lipid peroxidation and nitrite–nitrate levels in hippocampus, which could indicate antioxidant effects of LEV against pilocarpine-induced oxidative stress.

The conversion of hydrogen peroxide (H₂O₂) to H₂O can be made by catalase and glutathione peroxidase (that involves the reduced glutathione, a cofactor of this enzyme) (Simonié et al., 2000). An elevation in free radical formation can be accompanied by an immediate compensatory increase in the activities of the free radical scavenging enzyme and such increase can be related to compensatory mechanisms. The SE activates the oxygen reactive species scavenging enzymes, such as superoxide dismutase and catalase, indicating a cellular response to increased oxygen reactive species formation (Ferrer et al., 2000). GSH is an essential tripeptide, and endogenous antioxidant found in all animal cells. It reacts with the free radicals and can protect from singlet oxygen, hydroxide radicals, and superoxide radical damage (Shi et al., 1994).

Our data show enhanced catalase activity and reduced levels of reduced GSH in hippocampus after P400-induced seizures, demonstrating an alteration in antioxidative brain defenses. Freitas et al. (2004a) demonstrated that the P400-induced SE increases catalase activity in the frontal cortex, striatum, and hippocampus of rat's brain. Nascimento et al. (2005) showed an increase in activity of catalase in striatum of young rats, 1 h after P400-induced seizures. Similarly, Tejada et al. (2006) demonstrated an increase in the activity of catalase and other antioxidant enzymes involved in free radical scavenging in rat brain 2 h after pilocarpine treatment. Recently, Gibbs et al. (2006) reported, in an electrical stimulation model of SE, a specific pattern of biochemical change after SE. These data are consistent with the occurrence of oxidative stress and free radical production, including reductions in cellular levels of GSH and reduced activities of enzymes susceptible to oxidative stress within the tricarboxylic acid cycle.

Interestingly, LEV pretreatment preserved catalase activity in normal levels and prevented the loss of reduced GSH in hippocampus. Marini et al. (2004) showed that LEV pretreatment was able to prevent the loss of reduced GSH both in cortex and diencephalon of rats on kainic acid-induced seizures model. These results suggest that LEV could help the brain cells to counteract the pilocarpine-induced reactive oxygen species overproduction and the oxidative damage.

Thus, the results of our present study suggest that lipid peroxidation, nitrite–nitrate formation, and changes in antioxidant brain enzymes are involved in the pathophysiology of pilocarpine-induced seizures and SE. Our data showed that LEV displays activity against pilocarpine-induced oxidative stress in hippocampus, which might contribute to the drug ability to behave as a possible neuroprotective agent. However, LEV could prevent pilocarpine epilepsy by different mechanisms, even if it does not act directly on oxidative stress, since the protection against seizures per se is related to a reasonable decrease of oxidative stress. Further studies may be done to clarify the possible direct antioxidant effects of LEV.