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Effects of lipoic acid on oxidative stress in rat striatum after pilocarpine-induced seizures
Neurochemistry International 56 (2010) 16–20
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Effects of lipoic acid on oxidative stress in rat striatum after pilocarpine-induced seizures Gardenia Carmen Gadelha Milita˜o, Paulo Michel Pinheiro Ferreira, Rivelilson Mendes de Freitas * Laboratory of Physiology and Pharmacology, Federal University of Piaui, Rua Cı´cero Eduardo, s/n, Junco, Picos, 64, 600-000, Piauı´, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 June 2009 Received in revised form 16 August 2009 Accepted 18 August 2009 Available online 26 August 2009
The relationship between free radical and scavenger enzymes has been found in the epilepsy and reactive oxygen species have been implicated in seizure-induced neurodegeneration. It has been suggested that pilocarpine-induced seizures is mediated by increases in oxidative stress. Current researches have suggested that antioxidant compounds may afford some level of neuroprotection against the neurotoxicity of seizures in cellular level. The objective of the present study was to evaluate the neuroprotective effects of lipoic acid (LA) in rats, against the observed oxidative stress during seizures induced by pilocarpine. Wistar rats were treated with 0.9% saline (i.p., control group), LA (20 mg/kg, i.p., LA group), pilocarpine (400 mg/kg, i.p., P400 group), and the association of LA (20 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before of administration of LA (LA plus P400 group). After the treatments all groups were observed for 1 h. The enzyme activities as well as the lipid peroxidation and nitrite concentrations were measured using spectrophotometric methods and the results compared to values obtained from saline and pilocarpine-treated animals. Protective effects of LA were also evaluated on the same parameters. In P400 group there was a significant increase in lipid peroxidation, nitrite level and glutathione peroxidase (GPx) activity. However, no alteration was observed in superoxide dismutase (SOD) and catalase activities. Antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content as well as increased the SOD, catalase and GPx activities in rat striatum after seizures. Our findings strongly support the hypothesis that oxidative stress in striatum occurs during seizures induced by pilocarpine, proving that brain damage induced by the oxidative process plays a crucial role in seizures pathogenic consequences, and also imply that strong protective effect could be achieved using LA. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Lipid peroxidation Nitrite Superoxide dismutase Catalase Glutathione peroxidase Striatum Seizures
1. Introduction Free radicals have been implicated in development of seizures and status epilepticus (SE) induced by pilocarpine (Castagne et al., 1999). The mechanism behind seizures-induced oxidative stress is not well understood, but several explanations have been proposed. These include excitotoxicity associated with excessive neurotransmitter release, oxidative stress leading to free radical damage (Andreoli and Mallett, 1997). Recently, several studies have examined the role of oxidative stress on pilocarpine-induced seizures, possibly via the formation of free radicals. Free radicals and reactive oxygen species (ROS) are generated during oxidative metabolism and can inflict damage on all classes of cellular macromolecules, eventually leading to cell death (McCord, 1989; Walz et al., 2000). Oxidative stress is attractive as a possible mechanism for the pilocarpine-induced seizures for
* Corresponding author. Tel.: +55 89 3422 4389; fax: +55 89 3422 4389. E-mail address: [email protected] (R.M. de Freitas). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.08.009
many reasons. The brain processes large amounts of O2 in relatively small mass, and has a high content of substrates available for oxidation in conjunction with low antioxidant activities, making it extremely susceptible to oxidative damage (Bergamini et al., 2004). In addition, certain regions of central nervous system (CNS), such as the striatum, may be particularly sensitive to oxidative stress because of their low endogenous levels of vitamin E, an important biochemical antioxidant, relatively to other brain regions. Such a depressed defense system may be adequate under normal circumstances. However, in pro-oxidative conditions, such as during seizures, these low antioxidant defenses can predispose the brain to oxidative stress. During acute phase of seizures have been observed an increase in nitrite formation (Freitas et al., 2005), a potent free radical known to be cytotoxic to neurons and glial cells. The role of oxidative stress in seizures induced by pilocarpine is also supported by studies showing beneficial effects of antioxidant compounds during seizures exposure (Barros et al., 2007; Xavier et al., 2007; Freitas, 2009). It has been reported the neuroprotective effects of ascorbic acid and lipoic acid against pilocarpine-induced
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seizures in adult rats. Lipoic acid (LA), an essential cofactor for mitochondrial enzymes and a natural antioxidant, has been explored for the treatment of neurodegenerative diseases (Bergamini et al., 2004). Neuropathogenic dysfunction due to cell death via apoptosis is one of the important consequences of oxidative stress that could be diminished using antioxidant such as LA. In present study we evaluated the lipoic acid effects on oxidative stress in the striatum of adult rats during acute phase of pilocarpine-induced seizures
inhibition of this reaction was measured to assess SOD activity. The standard assay substrate mixture contained 3.0 mL xanthine (500 mM), 7.44 mg cytochrome c, 3.0 mL KCN (200 mM), and 3.0 mL EDTA (1 mM) in 18.0 mL 0.05 M sodium phosphate buffer, pH 7.0. The sample aliquot (20 mL) was added to 975 mL of the substrate mixture plus 5 mL xanthine oxidase. After 1 min, the initial absorbance was recorded and the timer was started. The final absorbance after 6 min was recorded. The reaction was followed at 550 nm. Purified bovine erythrocyte SOD (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 SOD activity. SOD activity in the samples was determined from this curve, and the results expressed as U/mg of protein.
2. Materials and methods
2.5. Determination of catalase activity
2.1. Animal procedures Adult male Wistar rats (200–280 g) maintained in a temperature controlled room (26 2 8C) with a 12-h light/dark cycle with food and water ad libitum were used. All 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). The following substances were used: pilocarpine hydrochloride and alpha-lipoic acid (Sigma, Chemical USA). All doses are expressed in milligrams per kilogram and were administered in a volume of 10 ml/kg injected intraperitoneally (i.p.). In a set of experiments, the animals were divided in four groups and treated with lipoic acid (20 mg/kg, i.p., n = 36) or 0.9% saline (i.p., n = 36) and 30 min later, they received pilocarpine hydrochloride (400 mg/kg, i.p.), and in this 30-min interval rats were observed for the occurrence of any change in behavior. The treatments previously described represent the LA plus P400 and P400 groups, respectively. Other two groups received 0.9% saline (i.p., n = 36, control group) or lipoic acid alone (20 mg/kg, i.p., n = 24, LA group). After the treatments, the animals were placed in 30 cm 30 cm chambers to record: latency to first seizure (any one of the behavioral indices typically observed after pilocarpine administration: wild running, clonus, tonus, clonic–tonic seizures (Turski et al., 1983)), number of animals that died after pilocarpine administration. Previous work have shown that the numbers of convulsions and deaths occurring within 1 and 24 h post injection always follow the same pattern, so we decided to observe the animals for 1 h as pilocarpine-induced convulsions occur in 30–60 min and deaths within 1–24 h after pilocarpine injection. The survivors were killed by decapitation and their brains dissected on ice to remove striatum for determinations of lipid peroxidation levels, nitrite formation, superoxide dismutase, catalase and glutathione peroxidase activities. The pilocarpine group was constituted by those rats that presented seizures; SE for over 30 min and that did not died within 1 h. The drug dosages were determined from both dose–response studies, including pilocarpine (data not shown), and observations of the doses currently used in animals studies in the literature. The doses used are not equivalent to those used by humans because rats have different metabolic rates.
The striatum was ultrasonically homogenized in 1 mL 0.05 M sodium phosphate buffer, pH 7.0. Protein concentration was measured by the method of Lowry et al. (1951). The 10% homogenates were centrifuged (800 g, 20 min), and the supernatants used to assay catalase activity. Catalase activity was measured in the LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6) and control (n = 9) groups by the method that uses H2O2 to generate H2O and O2 (Chance and Maehly, 1955). Protein concentration was measured by the method of Lowry et al. (1951). The activity was measured by the degree of this reaction. The standard assay substrate mixture contained 0.30 mL H2O2 in 50 mL 0.05 M sodium phosphate buffer, pH 7.0. The sample aliquot (20 mL) was added to 980 mL of the 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 mmol/L sodium phosphate buffer (pH 7.0), to provoke a 50% inhibition of the diluent rate (i.e. the uninhibited reaction). Results are expressed as mmol/min/mg of protein (Chance and Maehly, 1955; Maehly and Chance, 1954). 2.6. Determination of glutathione peroxidase (GPx) activity GPx was measured in the LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6) and control (n = 9) groups by method described by Sinet et al. (1975) using t-butyl-HPx as substrate. The striatum was homogenized in PBS 0.05 M (pH 7.4) in buffer solution (1 mg/5 ml) and the protein concentration was measured according to the method described by Lowry et al. (1951). After homogenization, 30 mg of striatal protein was added to 500 ml of PBS pH 7.0 containing 10 3 M reduced glutathione, 2 units of yeast glutathione reductase (Sigma type III) and 2 10 4 NADPH. After 10 min at 37 8C, the reaction was initiated by the addition of t-butyl-HPx to a final concentration of 10 3 M, under constant agitation. The oxidation of NADPH was calculated using extinction coefficient for NADPH of 6.22 103 at 340 nm and the reaction was made for 5 min. Results are expressed as mU per mg of protein.
2.2. Determination of lipid peroxidation levels 2.7. Western blot analysis For all of the experimental procedures, 10% (w/v) homogenates of the area of the brain investigated were prepared for all groups. Lipid peroxidation levels in the LA plus P400 group (n = 6), P400 group (n = 6), LA group (n = 6) and control animal (n = 9) were analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley (1990). Briefly, the samples were mixed with 1 mL 10% trichloroacetic 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-acidreacting substances were determined from the absorbance at 535 nm. The results above were expressed as nmol of malondialdehyde (MDA)/g wet tissue. 2.3. Determination of nitrite content To determine nitrite contents of control rats (n = 9), LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6), the 10% (w/v) homogenates were centrifuged (800 g, 10 min). The supernatants were collected, and nitric oxide production was determined based on the Griess reaction (Green et al., 1981). Briefly, 100 mL supernatant was incubated with 100 mL of the Griess reagent at room temperature for 10 min. A550 was measured using a microplate reader. Nitrite concentration was determined from a standard nitrite curve generated using NaNO2. 2.4. Determination of superoxide dismutase activity The striatum was ultrasonically homogenized in 1 mL 0.05 M sodium phosphate buffer, pH 7.0. Protein concentration was measured by the method of Lowry et al. (1951). The 10% homogenates were centrifuged (800 g, 20 min), and the supernatants used to assay superoxide dismutase (SOD) activity. SOD activity in the LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6) and control animals (n = 9) was assayed by using xanthine and xanthine oxidase to generate superoxide radicals (Flohe and Otting, 1984). They react with 2,4-iodophenyl-3,4nitophenol-5-phenyltetrazolium chloride to form a red formazan dye. The degree of
For immunoblotting, striatum homogenates were mixed with protein loading buffer (roti-Load 1, Carl Roth GmbH, Karlsruhe, Germany) according to manufacturer’s procedure and placed in a heating bath (95 8C) for 5 min. Proteins were separated using SDS-PAGE (gradient gels from 5% to 25%). The protein amount loaded per lane was 10 Ag. After separation, the proteins were stained with Coomassie Brilliant Blue or transferred to nitrocellulose paper and unspecific protein binding sites were blocked with blocking buffer (Chemicon International, Hofheim, Germany). The blots were incubated overnight with the primary antibodies against (1) catalase (polyclonal, UBI, Lake Placid, NY, USA, 1:1500), (2) Mn-SOD (polyclonal, Assayama, Japan, 1:800) and (3) GPx (polyclonal, Assayama, 1:400), followed by incubation with horseradish peroxidase-conjugated secondary antibody (goat antirabbit IgG+ peroxidase, Boehringer Mannheim GmbH, Germany, 1:1000). Immunoreactivity was visualized using the ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). 2.8. Statistical analyses Results of latency to first seizure and neurochemical alterations were compared using ANOVA and the Student–Newman–Keuls test as post hoc test, because these results show a parametric distribution. The number of animals that seized and the number that survived were calculated as percentages (percentage seizures and percentage survival, respectively), and compared with a nonparametric test (x2). In all situations statistical significance was reached at p less-than-or-equals, slant 0.05. The statistical analyses were performed with the software GraphPad Prism, Version 3.00 for Windows, GraphPad Software (San Diego, CA, USA).
3. Results Pilocarpine induced the first seizure at 35.00 0.70 min. All the animals studied showed generalized tonic–clonic convulsions with
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Fig. 1. Effects of LA in status of lipid peroxidation level and nitrite content in rat striatum after seizures induced by pilocarpine. Male rats (250–280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, intraperitoneal, i.p., n = 6, P400 group), LA group with lipoic acid (20 mg/kg, i.p., n = 6, LA group) and the control animals with 0.9% saline (i.p., n = 9, control group). The LA plus pilocarpine group was treated with LA (20 mg/kg, i.p.) and 30 min before of administration received pilocarpine (400 mg/kg, i.p., n = 6, LA plus P400 group). Animals were observed for 1 h and then killed. Results are mean + S.E.M. for the number of animals shown inside in parentheses. The differences in experimental groups were determined by analysis of variance. ap < 0.05 as compared to control animals (tStudent–Neuman–Keuls test); bp < 0.05 as compared to P400 group (t-Student– Neuman–Keuls test).
status epilepticus (SE), and 60% survived the seizures. All animals pretreated with the LA selected for this study were observed for 1 h before pilocarpine injection and its manifested alterations in behavior, such as peripheral cholinergic signs (100%), tremors (50%), staring spells, facial automatisms, wet dog shakes, rearing and motor seizures (75%), which develop progressively within 1–2 h into a long-lasting SE (75%) (Table 1). Result shows that when administered at the dose (20 mg/kg) before pilocarpine, LA reduced by 65% the percentage of animals that seized (p < 0.0001), increased (240%) latency to the first seizure (119.00 1.02 min) (p < 0.0001) and increased (30%) the survival percentage (p < 0.0001) as compared with the pilocarpine-treated group. None of the animal that received injections of isotonic saline (control) or LA alone showed seizure activity (Table 1). Effects of LA in lipid peroxidation and nitrite concentrations during seizures induced by pilocarpine are presented in Fig. 1. Lipid peroxidation was markedly increased in pilocarpine group compared with corresponding values for the control group. During acute phase of seizures induced by pilocarpine was observed a significant (89%) increase in thiobarbituric-acid-reacting substances (p < 0.0001), when compared with the control group. Seizures induced by pilocarpine produced a significant increase in striatal nitrite content of 94% (p < 0.0001), when compared with the control group (Fig. 1). Post hoc comparison of means indicated a significant decreases of 42% and 43% in striatum of rats pretreated with LA in lipid peroxidation level (p < 0.0001) and nitrite content (p < 0.0001), when compared with the P400 group, respectively (Fig. 1). No one animal that received injections of LA alone showed alterations in lipid peroxidation level (p = 0.8809)
Fig. 2. Effects of LA in superoxide dismutase, catalase and glutathione peroxidase activities in rat striatum after seizures induced by pilocarpine. Male rats (250– 280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, intraperitoneal, i.p., n = 6, P400 group), LA group with lipoic acid (20 mg/kg, i.p., n = 6, LA group) and the control animals with 0.9% saline (i.p., n = 9, control group). The LA plus pilocarpine group was treated with LA (20 mg/kg, i.p.) and 30 min before of administration received pilocarpine (400 mg/kg, i.p., n = 6, LA plus P400 group). Animals were observed for 1 h and then killed. Results are mean + S.E.M. for the number of animals shown inside in parentheses. The differences in experimental groups were determined by analysis of variance. ap < 0.05 as compared to control animals (t-Student–Neuman–Keuls test); bp < 0.05 as compared to P400 group (t-Student–Neuman–Keuls test).
and nitrite content (p = 0.6675), when compared with the control group (Fig. 1). Fig. 2 shows the lipoic acid effects in SOD, catalase and GPx activities in the striatum during acute phase of seizures induced by pilocarpine. SOD (p = 0.8473) and catalase (p = 0.8648) activities in the striatum during acute phase of seizures no was markedly altered in pilocarpine group, when compared with corresponding values for the control group. However, post hoc comparison of means indicated a significant (43%) increase in striatal GPx activity during acute phase of seizures (p < 0.0012) when compared with the control group (Fig. 2). Post hoc comparison of means indicated a significant (53%) increase in striatal SOD activity of rats pretreated with LA (p < 0.0006) when compared with the P400 group. The pretreatment with LA also produced a significant increases in striatal catalase and GPx activities of 88% (p < 0.0001) and 19% (p < 0.0001) when compared with the P400 group, respectively. In addition, the pretreatment with LA, 30 min before administration of pilocarpine also produced significant increases of 57, 90 and 69% in SOD (p < 0.0006), catalase (p < 0.0001) and GPx (p < 0.0408) activities, when compared with corresponding values for the control group, respectively (Fig. 2). However, none of the adult rats that received lipoic acid alone (LA group) showed
G.C.G. Milita˜o et al. / Neurochemistry International 56 (2010) 16–20 Table 1 Effect of pretreatment with lipoic acid on pilocarpine-induced seizures and lethality in adult rats. Groups
Latency to first seizures (min)
Percentage seizures
Percentage survival
Number of animals/group
P400 LA plus P400 LA
35.00 0.70 119.00 1.02a 00
75 10b 00b,c
60 90b 100b,c
36 36 36
Animals were pretreated acutely, intraperitoneally, with lipoic acid and 30 min afterwards received pilocarpine 400 mg/kg, i.p. Results for latency to first seizure are expressed as mean S.E.M. of the number of experiments shown in the table. Result for percentage seizures and percentage survival are expressed as percentages of the number of animals from each experimental group. a p < 0.0001 as compared with P400 group (x2-test). b p < 0.0001 as compared with LA plus P400 group (x2-test). c p < 0.0001 as compared with P400 group (ANOVA and Student–Newman–Keuls test).
Fig. 3. Western blot analysis of Mn-SOD, GPx, and catalase in striatum of adult rats during acute phase of seizures induced by pilocarpine. Male rats (250–280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., P400 group), LA group with lipoic acid (20 mg/kg, i.p., LA group) and the control animals with 0.9% saline (i.p., n = 9, control group). The LA plus pilocarpine group was treated with LA (20 mg/kg, i.p.) and 30 min before of administration received pilocarpine (400 mg/kg, i.p., LA plus P400 group). Animals were observed for 1 h and then killed. The protein amount per lane was 10 Ag. Shown is one representative experiment of n = 4.
alterations in SOD (p = 0.9203), catalase (p = 0.9266) and GPx (p = 0.8913) activities, when compared with the control group (Fig. 2). To clarify the mechanism of lipoic acid on oxidative stress for the development of anticonvulsant effects in pilocarpine model, the following results of Western blot analysis of Mn-SOD, GPx and catalase in rat striatum homogenates were obtained. After treatment with the single acute seizure-inducing dose of 400 mg/kg pilocarpine, the total Mn-SOD, GPx and catalase activities no were changed in comparison with saline controls. The results obtained by Mn-SOD, GPx and catalase activities measurements could be further supported by Western blot analysis. Likewise, LA plus pilocarpine and LA groups did not affect the level of the Mn-SOD, GPx and catalase mRNA or protein, as tested by immunoblot analyses of striatal homogenates (Fig. 3). 4. Discussion The nervous system contains some antioxidant enzymes, including SOD and GPx that are expressed in higher quantities than catalase (Shivakumar et al., 1991). This spectrum of enzymatic defense suggests that the brain may efficiently metabolize superoxide but may have difficulties in eliminating
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the hydrogen peroxide produced by this reaction (Castagne et al., 1999). In the present study we have examined whether the treatment with LA can reverse the alterations in the lipid peroxidation level, nitrite content, SOD, catalase and GPx activities in striatum observed during the acute phase of seizures induced by pilocarpine in adult rats. Generation of reactive oxygen species (ROS) is currently viewed as one of the process through which epileptic activity exert their deleterious effects on brain (Castagne et al., 1999). These ROS in the absence of an efficient defence mechanism cause lipid peroxidation (Halliwell and Gutteridge, 1999). Brain is particularly susceptible to peroxidation due to simultaneous presence of high levels of polyunsaturated fatty acids and iron (Halliwell and Gutteridge, 1989), which is the target of free radical. We have recorded the rise in lipid peroxidation level in rat striatum of rats. This is reflected by rise in TBARS level which may be related to its intermediate free radicals formed during seizures induced by pilocarpine. Fig. 1 summarizes the mean value of nitrite content in striatum of rats in P400 and LA plus P400 groups. It has been demonstrated that seizures induced by pilocarpine produces changes in nitric oxide metabolism and interacts with glutamatergic receptors to produced part of its stimulatory action on the CNS (Dymond and Crandall, 1976; Tran et al., 2005). The fall in nitrite content, after pretreatment with LA, is most readily explained as a consequence of inhibiting formation of radicals and by reduction of lipid peroxidation (Maczurek et al., 2008). Increased nitrite levels may cause lipid peroxidation in rat striatum during seizures (Freitas et al., 2005; Tejada et al., 2006). No alteration in striatal SOD and catalase activities was observed during acute phase of seizures, suggesting that these enzymes cannot be activated during this phase. Furthermore, other antioxidant systems such as GPx can be responsible by inhibition of neurotoxicity induced by seizures. Our results showed that LA during acute phase of seizures produces increase in SOD, catalase activities and GPx in rat striatum. The increase in these enzymes activities, after pretreatment with LA, is most readily explained as a necessary consequence of inhibiting formation of free radicals during seizures (Kudin et al., 2004; Maczurek et al., 2008; Michiels et al., 1994; Bellissimo et al., 2001). The GPx activities, quantified in the striatal formation of LA plus P400 and P400 groups showed an increase in values to that found for controls animals, except in those animals of LA group. This data suggests that H2O2, which is generated during superoxide dismutation, could be sufficiently removed from the striatum by GPx after pretreatment with LA. An elevation in free radical formation can be accompanied by an immediate compensatory increase in the activities of the free radical scavenging enzymes (Barros et al., 2007). Previous studies showed an increased in striatal catalase activity during 24 h of acute phase of seizures (Freitas et al., 2005; Santos et al., 2008). Other study showed an increase in striatal GPx activity after 1 h of seizures (Bellissimo et al., 2001). In our studies, the total activities of SOD, GPx and catalase no were changed during acute phase of seizures. The data of Western blot analysis now demonstrated evidence for the upregulation of SOD, GPx and catalase after pilocarpine-induced seizures. In addition, the data obtained with Western blot analysis confirmed our hypothesis that occurred only an increase in the enzymatic activities studied, since there was no change in protein contents of Mn-SOD, GPx and catalase in striatum. In addition, during the convulsive process, neuronal activities changes are accompanied by alterations in the cerebral metabolic rate (Dymond and Crandall, 1976). Our results have not shown any alterations in SOD and catalase activities within 1 h of acute phase of seizures. Considering that an increased metabolic demand can be observed during the epileptic activity we can suggest that these
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enzymes activities are not modified during this period. These findings might suggest that the antioxidant metabolism in this structure remains unaltered during first hour of seizures and that pretreatment with LA can produce an increase in these enzymes activity. Its compensatory mechanisms against oxidative stress observed during seizures can explain the anticonvulsant their actions verified during behavioral studies. The seizures induced by pilocarpine are prevented by LA, suggesting a role of free radical in controlling seizures installation and propagation. In fact, we found that pretreatment with LA is able to inhibit pilocarpine-induced seizures, SE and mortality of adult rats. In addition, the present data suggest evidence that free radical formation have a relevant role in the propagation and/or maintenance of convulsive activity. An increase in these enzymes activities, while free radical formation reduces, produces a significant decrease in the susceptibility to seizures, the results suggest that LA can present anticonvulsant effects. Therefore, LA represents a possible neuroprotective agent against risk factors of acute phase of seizures induced by pilocarpine. The results suggest the involvement of lipid peroxidation, nitrite and changes in antioxidant enzymatic systems in the mechanism of seizures induced by pilocarpine in rats. LA will provide further insights for neuroprotection and may lead to the development of effective therapeutic strategies against it, through modulation of brain antioxidant enzyme activities. References Andreoli, S.P., Mallett, C.P., 1997. Disassociation of oxidant-induced ATP depletion and DNA damage from early cytotoxicity in LLC-PK1 cells. Am. J. Physiol. 272, F729–F735. Barros, D.O., Xavier, S.M., Barbosa, C.O., Silva, R.F., Maia, F.D., Oliveira, A.A., Freitas, R.M., 2007. Effects of the vitamin E in catalase activities in hippocampus after status epilepticus induced by pilocarpine in Wistar rats. Neurosci. Lett. 41, 227– 230. Bellissimo, M.I., Amado, D., Abdalla, D.S.P., Ferreira, E., Cavalheiro, E.A., NaffahMazzacoratti, M.G., 2001. Superoxide dismutase, glutathione peroxidase activities and the hydroperoxide concentration are modified in the hippocampus of epileptic rats. Epilep. Res. 46, 121–128. Bergamini, C.M., Gambetti, S., Dondi, A., Cervellati, C., 2004. Oxygen, reactive oxygen species and tissue damage. Curr. Pharm. Des. 10, 111–112. Castagne, V., Gastschi, M., Lefevre, K., Posada, A., Clarke, P.G.H., 1999. Relationship between neuronal death and cellular redox status, focus on the developing nervous system. Prog. Neurophysiol. 59, 397–423. Chance, B., Maehly, A.C., 1955. Assay of catalases and peroxidases. Methods Enzymol. 2, 74–78. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as an index of lipid peroxidation. Methods Enzymol. 18, 421–431.
Dymond, A.M., Crandall, P.H., 1976. Oxygen availability and blood flow in the temporal lobes during spontaneous epileptic seizures in men. Brain Res. 102, 191–196. Flohe, L., Otting, F., 1984. Superoxide dismutase assays. Methods Enzymol. 105, 93– 104. Freitas, R.M., Souza, F.C.F., Vasconcelos, S.M.M., Viana, G.S.B., Fonteles, M.M.F., 2005. Oxidative stress in the hippocampus after status epilepticus in rats. FEBS J. 272, 1307–1312. Freitas, R.M., 2009. The evaluation of effects of lipoic acid on the lipid peroxidation, nitrite formation and antioxidant enzymes in the hippocampus of rats after pilocarpine-induced seizures. Neurosci. Lett. 455, 140–144. Green, L.C., Tannenbaum, S.R., Goldman, P., 1981. Nitrate synthesis in the germfree and conventional rat. Science 212, 56–58. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford Science Publications, London. Halliwell, B., Gutteridge, J.M.C., 1989. Lipid Peroxidation: A Radical Chain Reaction. Free Rad. Biol. Med., Clarendon Press, Oxford. Kudin, A.P., Bimpong-Buta, N.Y., Vielhaber, S., Elger, C.E., Kunz, W.S., 2004. Characterization of superoxide-producing sites in isolated brain mitochondria. J. Biol. Chem. 279, 4127–4135. Lowry, H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 25–275. Maehly, A.C., Chance, B., 1954. The assay of catalases and peroxidases. Methods Biochem. Anal. 1, 357–359. Maczurek, A., Hager, J., Kenklies, M., Sharman, M., Martins, R., Engel, J., Carlson, D.A., Munch, G., 2008. Lipoic acid as an anti-inflammatory and neuroprotective treatment for Alzheimer’s disease. Adv. Drug Deliv. Rev. 60, 143–1470. Michiels, C., Raes, M., Toussaint, O., Remacle, J., 1994. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic. Biol. Med. 17, 235–248. McCord, J.M., 1989. Superoxide radical: controversies, contradiction and paradoxes. Proc. Soc. Exp. Biol. Med. 209, 112–117. Santos, L.F., Freitas, R.L., Xavier, S.M., Saldanha, G.B., Freitas, R.M., 2008. Neuroprotective actions of vitamin C related to decreased lipid peroxidation and increased catalase activity in adult rats after pilocarpine-induced seizures. Pharmacol. Biochem. Behav. 89, 1–5. Shivakumar, B.R., Ananndatheerthavarada, H.K., Ravindranath, V., 1991. Free radical scavenging system in developing rat brain. Int. J. Dev. Neurosci. 9, 181–185. Sinet, P.M., Michelson, A.M., Bazin, A., Lejeune, J., Jeerome, H., 1975. Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 subjects. Biochem. Biophys. Res. Commun. 67, 910–915. Tejada, S., Roca, C., Sureda, A., Rial, R.V., Gamundı´, A., Esteban, S., 2006. Antioxidant response analysis in the brain after pilocarpine treatments. Brain Res. Bull. 9, 587–592. Tran, T.D., Jackson, H.D., Horn, K.H., Goodlett, C.R., 2005. Vitamin E does not protect against neonatal ethanol-induced cerebellar damage or deficits in eye blink classical conditioning in rats. Alcohol Clin. Exp. Res. 29, 117–129. Turski, W.A., Cavalheiro, E.A., Schwartz, M., Czuczwar, S.J., Kleinrok, Z., Turski, L., 1983. Limbic seizures produced by pilocarpine in rats: a behavioural, electroencephalographic and neuropathological study. Behav. Brain Res. 9, 315–335. Xavier, S.M.L., Barbosa, C.O., Barros, D.O., Silva, R.F., Oliveira, A.A., Freitas, R.M., 2007. Vitamin C antioxidant in hippocampus of adult Wistar rats after seizures and status epilepticus induced by pilocarpine. Neurosci. Lett. 420, 7–79. Walz, R., Moreira, J.C.F., Benfato, M.S., Quevedo, J., Schorer, N., Vianna, M.M.R., Klamt, F., Dal-Pizzol, F., 2000. Lipid peroxidation in hippocampus early and late after status epilepticus induced by pilocarpine of kainic acid in Wistar rats. Neurosci. Lett. 291, 179–182.
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Effects of lipoic acid on oxidative stress in rat striatum after pilocarpine-induced seizures Gardenia Carmen Gadelha Milita˜o, Paulo Michel Pinheiro Ferreira, Rivelilson Mendes de Freitas * Laboratory of Physiology and Pharmacology, Federal University of Piaui, Rua Cı´cero Eduardo, s/n, Junco, Picos, 64, 600-000, Piauı´, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 June 2009 Received in revised form 16 August 2009 Accepted 18 August 2009 Available online 26 August 2009
The relationship between free radical and scavenger enzymes has been found in the epilepsy and reactive oxygen species have been implicated in seizure-induced neurodegeneration. It has been suggested that pilocarpine-induced seizures is mediated by increases in oxidative stress. Current researches have suggested that antioxidant compounds may afford some level of neuroprotection against the neurotoxicity of seizures in cellular level. The objective of the present study was to evaluate the neuroprotective effects of lipoic acid (LA) in rats, against the observed oxidative stress during seizures induced by pilocarpine. Wistar rats were treated with 0.9% saline (i.p., control group), LA (20 mg/kg, i.p., LA group), pilocarpine (400 mg/kg, i.p., P400 group), and the association of LA (20 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before of administration of LA (LA plus P400 group). After the treatments all groups were observed for 1 h. The enzyme activities as well as the lipid peroxidation and nitrite concentrations were measured using spectrophotometric methods and the results compared to values obtained from saline and pilocarpine-treated animals. Protective effects of LA were also evaluated on the same parameters. In P400 group there was a significant increase in lipid peroxidation, nitrite level and glutathione peroxidase (GPx) activity. However, no alteration was observed in superoxide dismutase (SOD) and catalase activities. Antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content as well as increased the SOD, catalase and GPx activities in rat striatum after seizures. Our findings strongly support the hypothesis that oxidative stress in striatum occurs during seizures induced by pilocarpine, proving that brain damage induced by the oxidative process plays a crucial role in seizures pathogenic consequences, and also imply that strong protective effect could be achieved using LA. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Lipid peroxidation Nitrite Superoxide dismutase Catalase Glutathione peroxidase Striatum Seizures
1. Introduction Free radicals have been implicated in development of seizures and status epilepticus (SE) induced by pilocarpine (Castagne et al., 1999). The mechanism behind seizures-induced oxidative stress is not well understood, but several explanations have been proposed. These include excitotoxicity associated with excessive neurotransmitter release, oxidative stress leading to free radical damage (Andreoli and Mallett, 1997). Recently, several studies have examined the role of oxidative stress on pilocarpine-induced seizures, possibly via the formation of free radicals. Free radicals and reactive oxygen species (ROS) are generated during oxidative metabolism and can inflict damage on all classes of cellular macromolecules, eventually leading to cell death (McCord, 1989; Walz et al., 2000). Oxidative stress is attractive as a possible mechanism for the pilocarpine-induced seizures for
* Corresponding author. Tel.: +55 89 3422 4389; fax: +55 89 3422 4389. E-mail address: [email protected] (R.M. de Freitas). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.08.009
many reasons. The brain processes large amounts of O2 in relatively small mass, and has a high content of substrates available for oxidation in conjunction with low antioxidant activities, making it extremely susceptible to oxidative damage (Bergamini et al., 2004). In addition, certain regions of central nervous system (CNS), such as the striatum, may be particularly sensitive to oxidative stress because of their low endogenous levels of vitamin E, an important biochemical antioxidant, relatively to other brain regions. Such a depressed defense system may be adequate under normal circumstances. However, in pro-oxidative conditions, such as during seizures, these low antioxidant defenses can predispose the brain to oxidative stress. During acute phase of seizures have been observed an increase in nitrite formation (Freitas et al., 2005), a potent free radical known to be cytotoxic to neurons and glial cells. The role of oxidative stress in seizures induced by pilocarpine is also supported by studies showing beneficial effects of antioxidant compounds during seizures exposure (Barros et al., 2007; Xavier et al., 2007; Freitas, 2009). It has been reported the neuroprotective effects of ascorbic acid and lipoic acid against pilocarpine-induced
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seizures in adult rats. Lipoic acid (LA), an essential cofactor for mitochondrial enzymes and a natural antioxidant, has been explored for the treatment of neurodegenerative diseases (Bergamini et al., 2004). Neuropathogenic dysfunction due to cell death via apoptosis is one of the important consequences of oxidative stress that could be diminished using antioxidant such as LA. In present study we evaluated the lipoic acid effects on oxidative stress in the striatum of adult rats during acute phase of pilocarpine-induced seizures
inhibition of this reaction was measured to assess SOD activity. The standard assay substrate mixture contained 3.0 mL xanthine (500 mM), 7.44 mg cytochrome c, 3.0 mL KCN (200 mM), and 3.0 mL EDTA (1 mM) in 18.0 mL 0.05 M sodium phosphate buffer, pH 7.0. The sample aliquot (20 mL) was added to 975 mL of the substrate mixture plus 5 mL xanthine oxidase. After 1 min, the initial absorbance was recorded and the timer was started. The final absorbance after 6 min was recorded. The reaction was followed at 550 nm. Purified bovine erythrocyte SOD (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 SOD activity. SOD activity in the samples was determined from this curve, and the results expressed as U/mg of protein.
2. Materials and methods
2.5. Determination of catalase activity
2.1. Animal procedures Adult male Wistar rats (200–280 g) maintained in a temperature controlled room (26 2 8C) with a 12-h light/dark cycle with food and water ad libitum were used. All 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). The following substances were used: pilocarpine hydrochloride and alpha-lipoic acid (Sigma, Chemical USA). All doses are expressed in milligrams per kilogram and were administered in a volume of 10 ml/kg injected intraperitoneally (i.p.). In a set of experiments, the animals were divided in four groups and treated with lipoic acid (20 mg/kg, i.p., n = 36) or 0.9% saline (i.p., n = 36) and 30 min later, they received pilocarpine hydrochloride (400 mg/kg, i.p.), and in this 30-min interval rats were observed for the occurrence of any change in behavior. The treatments previously described represent the LA plus P400 and P400 groups, respectively. Other two groups received 0.9% saline (i.p., n = 36, control group) or lipoic acid alone (20 mg/kg, i.p., n = 24, LA group). After the treatments, the animals were placed in 30 cm 30 cm chambers to record: latency to first seizure (any one of the behavioral indices typically observed after pilocarpine administration: wild running, clonus, tonus, clonic–tonic seizures (Turski et al., 1983)), number of animals that died after pilocarpine administration. Previous work have shown that the numbers of convulsions and deaths occurring within 1 and 24 h post injection always follow the same pattern, so we decided to observe the animals for 1 h as pilocarpine-induced convulsions occur in 30–60 min and deaths within 1–24 h after pilocarpine injection. The survivors were killed by decapitation and their brains dissected on ice to remove striatum for determinations of lipid peroxidation levels, nitrite formation, superoxide dismutase, catalase and glutathione peroxidase activities. The pilocarpine group was constituted by those rats that presented seizures; SE for over 30 min and that did not died within 1 h. The drug dosages were determined from both dose–response studies, including pilocarpine (data not shown), and observations of the doses currently used in animals studies in the literature. The doses used are not equivalent to those used by humans because rats have different metabolic rates.
The striatum was ultrasonically homogenized in 1 mL 0.05 M sodium phosphate buffer, pH 7.0. Protein concentration was measured by the method of Lowry et al. (1951). The 10% homogenates were centrifuged (800 g, 20 min), and the supernatants used to assay catalase activity. Catalase activity was measured in the LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6) and control (n = 9) groups by the method that uses H2O2 to generate H2O and O2 (Chance and Maehly, 1955). Protein concentration was measured by the method of Lowry et al. (1951). The activity was measured by the degree of this reaction. The standard assay substrate mixture contained 0.30 mL H2O2 in 50 mL 0.05 M sodium phosphate buffer, pH 7.0. The sample aliquot (20 mL) was added to 980 mL of the 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 mmol/L sodium phosphate buffer (pH 7.0), to provoke a 50% inhibition of the diluent rate (i.e. the uninhibited reaction). Results are expressed as mmol/min/mg of protein (Chance and Maehly, 1955; Maehly and Chance, 1954). 2.6. Determination of glutathione peroxidase (GPx) activity GPx was measured in the LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6) and control (n = 9) groups by method described by Sinet et al. (1975) using t-butyl-HPx as substrate. The striatum was homogenized in PBS 0.05 M (pH 7.4) in buffer solution (1 mg/5 ml) and the protein concentration was measured according to the method described by Lowry et al. (1951). After homogenization, 30 mg of striatal protein was added to 500 ml of PBS pH 7.0 containing 10 3 M reduced glutathione, 2 units of yeast glutathione reductase (Sigma type III) and 2 10 4 NADPH. After 10 min at 37 8C, the reaction was initiated by the addition of t-butyl-HPx to a final concentration of 10 3 M, under constant agitation. The oxidation of NADPH was calculated using extinction coefficient for NADPH of 6.22 103 at 340 nm and the reaction was made for 5 min. Results are expressed as mU per mg of protein.
2.2. Determination of lipid peroxidation levels 2.7. Western blot analysis For all of the experimental procedures, 10% (w/v) homogenates of the area of the brain investigated were prepared for all groups. Lipid peroxidation levels in the LA plus P400 group (n = 6), P400 group (n = 6), LA group (n = 6) and control animal (n = 9) were analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley (1990). Briefly, the samples were mixed with 1 mL 10% trichloroacetic 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-acidreacting substances were determined from the absorbance at 535 nm. The results above were expressed as nmol of malondialdehyde (MDA)/g wet tissue. 2.3. Determination of nitrite content To determine nitrite contents of control rats (n = 9), LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6), the 10% (w/v) homogenates were centrifuged (800 g, 10 min). The supernatants were collected, and nitric oxide production was determined based on the Griess reaction (Green et al., 1981). Briefly, 100 mL supernatant was incubated with 100 mL of the Griess reagent at room temperature for 10 min. A550 was measured using a microplate reader. Nitrite concentration was determined from a standard nitrite curve generated using NaNO2. 2.4. Determination of superoxide dismutase activity The striatum was ultrasonically homogenized in 1 mL 0.05 M sodium phosphate buffer, pH 7.0. Protein concentration was measured by the method of Lowry et al. (1951). The 10% homogenates were centrifuged (800 g, 20 min), and the supernatants used to assay superoxide dismutase (SOD) activity. SOD activity in the LA plus P400 group (n = 6), P400 group (n = 6) and LA group (n = 6) and control animals (n = 9) was assayed by using xanthine and xanthine oxidase to generate superoxide radicals (Flohe and Otting, 1984). They react with 2,4-iodophenyl-3,4nitophenol-5-phenyltetrazolium chloride to form a red formazan dye. The degree of
For immunoblotting, striatum homogenates were mixed with protein loading buffer (roti-Load 1, Carl Roth GmbH, Karlsruhe, Germany) according to manufacturer’s procedure and placed in a heating bath (95 8C) for 5 min. Proteins were separated using SDS-PAGE (gradient gels from 5% to 25%). The protein amount loaded per lane was 10 Ag. After separation, the proteins were stained with Coomassie Brilliant Blue or transferred to nitrocellulose paper and unspecific protein binding sites were blocked with blocking buffer (Chemicon International, Hofheim, Germany). The blots were incubated overnight with the primary antibodies against (1) catalase (polyclonal, UBI, Lake Placid, NY, USA, 1:1500), (2) Mn-SOD (polyclonal, Assayama, Japan, 1:800) and (3) GPx (polyclonal, Assayama, 1:400), followed by incubation with horseradish peroxidase-conjugated secondary antibody (goat antirabbit IgG+ peroxidase, Boehringer Mannheim GmbH, Germany, 1:1000). Immunoreactivity was visualized using the ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). 2.8. Statistical analyses Results of latency to first seizure and neurochemical alterations were compared using ANOVA and the Student–Newman–Keuls test as post hoc test, because these results show a parametric distribution. The number of animals that seized and the number that survived were calculated as percentages (percentage seizures and percentage survival, respectively), and compared with a nonparametric test (x2). In all situations statistical significance was reached at p less-than-or-equals, slant 0.05. The statistical analyses were performed with the software GraphPad Prism, Version 3.00 for Windows, GraphPad Software (San Diego, CA, USA).
3. Results Pilocarpine induced the first seizure at 35.00 0.70 min. All the animals studied showed generalized tonic–clonic convulsions with
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Fig. 1. Effects of LA in status of lipid peroxidation level and nitrite content in rat striatum after seizures induced by pilocarpine. Male rats (250–280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, intraperitoneal, i.p., n = 6, P400 group), LA group with lipoic acid (20 mg/kg, i.p., n = 6, LA group) and the control animals with 0.9% saline (i.p., n = 9, control group). The LA plus pilocarpine group was treated with LA (20 mg/kg, i.p.) and 30 min before of administration received pilocarpine (400 mg/kg, i.p., n = 6, LA plus P400 group). Animals were observed for 1 h and then killed. Results are mean + S.E.M. for the number of animals shown inside in parentheses. The differences in experimental groups were determined by analysis of variance. ap < 0.05 as compared to control animals (tStudent–Neuman–Keuls test); bp < 0.05 as compared to P400 group (t-Student– Neuman–Keuls test).
status epilepticus (SE), and 60% survived the seizures. All animals pretreated with the LA selected for this study were observed for 1 h before pilocarpine injection and its manifested alterations in behavior, such as peripheral cholinergic signs (100%), tremors (50%), staring spells, facial automatisms, wet dog shakes, rearing and motor seizures (75%), which develop progressively within 1–2 h into a long-lasting SE (75%) (Table 1). Result shows that when administered at the dose (20 mg/kg) before pilocarpine, LA reduced by 65% the percentage of animals that seized (p < 0.0001), increased (240%) latency to the first seizure (119.00 1.02 min) (p < 0.0001) and increased (30%) the survival percentage (p < 0.0001) as compared with the pilocarpine-treated group. None of the animal that received injections of isotonic saline (control) or LA alone showed seizure activity (Table 1). Effects of LA in lipid peroxidation and nitrite concentrations during seizures induced by pilocarpine are presented in Fig. 1. Lipid peroxidation was markedly increased in pilocarpine group compared with corresponding values for the control group. During acute phase of seizures induced by pilocarpine was observed a significant (89%) increase in thiobarbituric-acid-reacting substances (p < 0.0001), when compared with the control group. Seizures induced by pilocarpine produced a significant increase in striatal nitrite content of 94% (p < 0.0001), when compared with the control group (Fig. 1). Post hoc comparison of means indicated a significant decreases of 42% and 43% in striatum of rats pretreated with LA in lipid peroxidation level (p < 0.0001) and nitrite content (p < 0.0001), when compared with the P400 group, respectively (Fig. 1). No one animal that received injections of LA alone showed alterations in lipid peroxidation level (p = 0.8809)
Fig. 2. Effects of LA in superoxide dismutase, catalase and glutathione peroxidase activities in rat striatum after seizures induced by pilocarpine. Male rats (250– 280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, intraperitoneal, i.p., n = 6, P400 group), LA group with lipoic acid (20 mg/kg, i.p., n = 6, LA group) and the control animals with 0.9% saline (i.p., n = 9, control group). The LA plus pilocarpine group was treated with LA (20 mg/kg, i.p.) and 30 min before of administration received pilocarpine (400 mg/kg, i.p., n = 6, LA plus P400 group). Animals were observed for 1 h and then killed. Results are mean + S.E.M. for the number of animals shown inside in parentheses. The differences in experimental groups were determined by analysis of variance. ap < 0.05 as compared to control animals (t-Student–Neuman–Keuls test); bp < 0.05 as compared to P400 group (t-Student–Neuman–Keuls test).
and nitrite content (p = 0.6675), when compared with the control group (Fig. 1). Fig. 2 shows the lipoic acid effects in SOD, catalase and GPx activities in the striatum during acute phase of seizures induced by pilocarpine. SOD (p = 0.8473) and catalase (p = 0.8648) activities in the striatum during acute phase of seizures no was markedly altered in pilocarpine group, when compared with corresponding values for the control group. However, post hoc comparison of means indicated a significant (43%) increase in striatal GPx activity during acute phase of seizures (p < 0.0012) when compared with the control group (Fig. 2). Post hoc comparison of means indicated a significant (53%) increase in striatal SOD activity of rats pretreated with LA (p < 0.0006) when compared with the P400 group. The pretreatment with LA also produced a significant increases in striatal catalase and GPx activities of 88% (p < 0.0001) and 19% (p < 0.0001) when compared with the P400 group, respectively. In addition, the pretreatment with LA, 30 min before administration of pilocarpine also produced significant increases of 57, 90 and 69% in SOD (p < 0.0006), catalase (p < 0.0001) and GPx (p < 0.0408) activities, when compared with corresponding values for the control group, respectively (Fig. 2). However, none of the adult rats that received lipoic acid alone (LA group) showed
G.C.G. Milita˜o et al. / Neurochemistry International 56 (2010) 16–20 Table 1 Effect of pretreatment with lipoic acid on pilocarpine-induced seizures and lethality in adult rats. Groups
Latency to first seizures (min)
Percentage seizures
Percentage survival
Number of animals/group
P400 LA plus P400 LA
35.00 0.70 119.00 1.02a 00
75 10b 00b,c
60 90b 100b,c
36 36 36
Animals were pretreated acutely, intraperitoneally, with lipoic acid and 30 min afterwards received pilocarpine 400 mg/kg, i.p. Results for latency to first seizure are expressed as mean S.E.M. of the number of experiments shown in the table. Result for percentage seizures and percentage survival are expressed as percentages of the number of animals from each experimental group. a p < 0.0001 as compared with P400 group (x2-test). b p < 0.0001 as compared with LA plus P400 group (x2-test). c p < 0.0001 as compared with P400 group (ANOVA and Student–Newman–Keuls test).
Fig. 3. Western blot analysis of Mn-SOD, GPx, and catalase in striatum of adult rats during acute phase of seizures induced by pilocarpine. Male rats (250–280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., P400 group), LA group with lipoic acid (20 mg/kg, i.p., LA group) and the control animals with 0.9% saline (i.p., n = 9, control group). The LA plus pilocarpine group was treated with LA (20 mg/kg, i.p.) and 30 min before of administration received pilocarpine (400 mg/kg, i.p., LA plus P400 group). Animals were observed for 1 h and then killed. The protein amount per lane was 10 Ag. Shown is one representative experiment of n = 4.
alterations in SOD (p = 0.9203), catalase (p = 0.9266) and GPx (p = 0.8913) activities, when compared with the control group (Fig. 2). To clarify the mechanism of lipoic acid on oxidative stress for the development of anticonvulsant effects in pilocarpine model, the following results of Western blot analysis of Mn-SOD, GPx and catalase in rat striatum homogenates were obtained. After treatment with the single acute seizure-inducing dose of 400 mg/kg pilocarpine, the total Mn-SOD, GPx and catalase activities no were changed in comparison with saline controls. The results obtained by Mn-SOD, GPx and catalase activities measurements could be further supported by Western blot analysis. Likewise, LA plus pilocarpine and LA groups did not affect the level of the Mn-SOD, GPx and catalase mRNA or protein, as tested by immunoblot analyses of striatal homogenates (Fig. 3). 4. Discussion The nervous system contains some antioxidant enzymes, including SOD and GPx that are expressed in higher quantities than catalase (Shivakumar et al., 1991). This spectrum of enzymatic defense suggests that the brain may efficiently metabolize superoxide but may have difficulties in eliminating
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the hydrogen peroxide produced by this reaction (Castagne et al., 1999). In the present study we have examined whether the treatment with LA can reverse the alterations in the lipid peroxidation level, nitrite content, SOD, catalase and GPx activities in striatum observed during the acute phase of seizures induced by pilocarpine in adult rats. Generation of reactive oxygen species (ROS) is currently viewed as one of the process through which epileptic activity exert their deleterious effects on brain (Castagne et al., 1999). These ROS in the absence of an efficient defence mechanism cause lipid peroxidation (Halliwell and Gutteridge, 1999). Brain is particularly susceptible to peroxidation due to simultaneous presence of high levels of polyunsaturated fatty acids and iron (Halliwell and Gutteridge, 1989), which is the target of free radical. We have recorded the rise in lipid peroxidation level in rat striatum of rats. This is reflected by rise in TBARS level which may be related to its intermediate free radicals formed during seizures induced by pilocarpine. Fig. 1 summarizes the mean value of nitrite content in striatum of rats in P400 and LA plus P400 groups. It has been demonstrated that seizures induced by pilocarpine produces changes in nitric oxide metabolism and interacts with glutamatergic receptors to produced part of its stimulatory action on the CNS (Dymond and Crandall, 1976; Tran et al., 2005). The fall in nitrite content, after pretreatment with LA, is most readily explained as a consequence of inhibiting formation of radicals and by reduction of lipid peroxidation (Maczurek et al., 2008). Increased nitrite levels may cause lipid peroxidation in rat striatum during seizures (Freitas et al., 2005; Tejada et al., 2006). No alteration in striatal SOD and catalase activities was observed during acute phase of seizures, suggesting that these enzymes cannot be activated during this phase. Furthermore, other antioxidant systems such as GPx can be responsible by inhibition of neurotoxicity induced by seizures. Our results showed that LA during acute phase of seizures produces increase in SOD, catalase activities and GPx in rat striatum. The increase in these enzymes activities, after pretreatment with LA, is most readily explained as a necessary consequence of inhibiting formation of free radicals during seizures (Kudin et al., 2004; Maczurek et al., 2008; Michiels et al., 1994; Bellissimo et al., 2001). The GPx activities, quantified in the striatal formation of LA plus P400 and P400 groups showed an increase in values to that found for controls animals, except in those animals of LA group. This data suggests that H2O2, which is generated during superoxide dismutation, could be sufficiently removed from the striatum by GPx after pretreatment with LA. An elevation in free radical formation can be accompanied by an immediate compensatory increase in the activities of the free radical scavenging enzymes (Barros et al., 2007). Previous studies showed an increased in striatal catalase activity during 24 h of acute phase of seizures (Freitas et al., 2005; Santos et al., 2008). Other study showed an increase in striatal GPx activity after 1 h of seizures (Bellissimo et al., 2001). In our studies, the total activities of SOD, GPx and catalase no were changed during acute phase of seizures. The data of Western blot analysis now demonstrated evidence for the upregulation of SOD, GPx and catalase after pilocarpine-induced seizures. In addition, the data obtained with Western blot analysis confirmed our hypothesis that occurred only an increase in the enzymatic activities studied, since there was no change in protein contents of Mn-SOD, GPx and catalase in striatum. In addition, during the convulsive process, neuronal activities changes are accompanied by alterations in the cerebral metabolic rate (Dymond and Crandall, 1976). Our results have not shown any alterations in SOD and catalase activities within 1 h of acute phase of seizures. Considering that an increased metabolic demand can be observed during the epileptic activity we can suggest that these
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enzymes activities are not modified during this period. These findings might suggest that the antioxidant metabolism in this structure remains unaltered during first hour of seizures and that pretreatment with LA can produce an increase in these enzymes activity. Its compensatory mechanisms against oxidative stress observed during seizures can explain the anticonvulsant their actions verified during behavioral studies. The seizures induced by pilocarpine are prevented by LA, suggesting a role of free radical in controlling seizures installation and propagation. In fact, we found that pretreatment with LA is able to inhibit pilocarpine-induced seizures, SE and mortality of adult rats. In addition, the present data suggest evidence that free radical formation have a relevant role in the propagation and/or maintenance of convulsive activity. An increase in these enzymes activities, while free radical formation reduces, produces a significant decrease in the susceptibility to seizures, the results suggest that LA can present anticonvulsant effects. Therefore, LA represents a possible neuroprotective agent against risk factors of acute phase of seizures induced by pilocarpine. The results suggest the involvement of lipid peroxidation, nitrite and changes in antioxidant enzymatic systems in the mechanism of seizures induced by pilocarpine in rats. LA will provide further insights for neuroprotection and may lead to the development of effective therapeutic strategies against it, through modulation of brain antioxidant enzyme activities. References Andreoli, S.P., Mallett, C.P., 1997. Disassociation of oxidant-induced ATP depletion and DNA damage from early cytotoxicity in LLC-PK1 cells. Am. J. Physiol. 272, F729–F735. Barros, D.O., Xavier, S.M., Barbosa, C.O., Silva, R.F., Maia, F.D., Oliveira, A.A., Freitas, R.M., 2007. Effects of the vitamin E in catalase activities in hippocampus after status epilepticus induced by pilocarpine in Wistar rats. Neurosci. Lett. 41, 227– 230. Bellissimo, M.I., Amado, D., Abdalla, D.S.P., Ferreira, E., Cavalheiro, E.A., NaffahMazzacoratti, M.G., 2001. Superoxide dismutase, glutathione peroxidase activities and the hydroperoxide concentration are modified in the hippocampus of epileptic rats. Epilep. Res. 46, 121–128. Bergamini, C.M., Gambetti, S., Dondi, A., Cervellati, C., 2004. Oxygen, reactive oxygen species and tissue damage. Curr. Pharm. Des. 10, 111–112. Castagne, V., Gastschi, M., Lefevre, K., Posada, A., Clarke, P.G.H., 1999. Relationship between neuronal death and cellular redox status, focus on the developing nervous system. Prog. Neurophysiol. 59, 397–423. Chance, B., Maehly, A.C., 1955. Assay of catalases and peroxidases. Methods Enzymol. 2, 74–78. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as an index of lipid peroxidation. Methods Enzymol. 18, 421–431.
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