Oxidative stress in the hippocampus during experimental seizures can be ameliorated with the antioxidant ascorbic acid

Abstract

Ascorbic acid has many nonenzymatic actions and is a powerful water-soluble antioxidant. It protects low density lipoproteins from oxidation and reduces harmful oxidants in the central nervous system. Pilocarpine-induced seizures have been suggested to be mediated by increases in oxidative stress. Current studies have suggested that antioxidant compounds may afford some level of neuroprotection against the neurotoxicity of seizures. The objective of the present study was to evaluate the neuroprotective effects of ascorbic acid (AA) in rats, against the observed oxidative stress during seizures induced by pilocarpine. Wistar rats were treated with 0.9% saline (i.p., control group), ascorbic acid (500 mg/kg, i.p., AA group), pilocarpine (400 mg/kg, i.p., pilocarpine group), and the association of ascorbic acid (500 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before of administration of ascorbic acid (AA plus pilocarpine group). After the treatments all groups were observed for 6h. 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 ascorbic acid were also evaluated on the same parameters. In pilocarpine group there was a significant increase in lipid peroxidation and nitrite level. However, no alteration was observed in superoxide dismutase and catalase activities. Antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content as well as increased the superoxide dismutase and catalase activities in hippocampus of adult rats after seizures induced by pilocarpine. Our findings strongly support the hypothesis that oxidative stress in hippocampus 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 a strong protective effect could be achieved using ascorbic acid.

Figure 1

Effects of ascorbic acid (AA) in status of lipid peroxidation level in hippocampus of adult rats 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), AA group with ascorbic acid (500 mg/kg, i.p., n = 6, VIT C) and the control animals with 0.9% saline (i.p., n = 9, Control). The AA plus pilocarpine group was treated with ascorbic acid (500 mg/kg, i.p.) 30 min after pilocarpine injection (400 mg/kg, i.p., n = 6, AA plus P400). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 6 h. For neurochemical determinations of lipid peroxidation level, all groups were killed by decapitation 6 h after the treatment and their brains were dissected on ice to remove the hippocampus. The cerebral area studied was ultrasonically homogenized in 1 ml of 0.05 M phosphate buffer, pH 7.0, and the homogenates 10% were centrifuged (800 xg/20 min) and the supernatant of the homogenates was used for lipid peroxidation level investigation. The lipid peroxidation level in the hippocampus during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley. Results are expressed as means + S.E.M. for the number of animals shown inside in parenthesis. Results were expressed as nmol of malondialdehyde (MDA)/g wet tissue. The Student-Newman-Keuls test was used for multiple comparisons of means of two groups of data. Differences in experimental groups were determined by two-tailed analysis of variance. Differences were considered significant at p < 0.05. ^ap < 0.05 as compared to control animals (t-Student-Neuman-Keuls test); ^bp < 0.05 as compared to pilocarpine group (t-Student-Neuman-Keuls test).

Figure 2

Effects of ascorbic acid (AA) in status of nitrite content in hippocampus of adult rats 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), AA group with ascorbic acid (500 mg/kg, i.p., n = 6, AA) and the control animals with 0.9% saline (i.p., n = 9, Control). The AA plus pilocarpine group was treated with ascorbic acid (500 mg/kg, i.p.) 30 min after pilocarpine injection (400 mg/kg, i.p., n = 6, AA plus P400). The cerebral area studied was ultrasonically homogenized in 1 ml of 0.05 M phosphate buffer, pH 7.0, and the homogenates 10% were centrifuged (800 g/20 min) and the supernatant of the homogenates was used for nitrite level investigation. The nitrite content in the hippocampus during seizure activity was analyzed by measuring the nitrite and nitrate in homogenates, as previously described by Green et al. Results are expressed as means + S.E.M. for the number of animals shown inside in parenthesis. Results were expressed as nM. The Student-Newman-Keuls test was used for multiple comparisons of means of two groups of data. Differences in experimental groups were determined by two-tailed analysis of variance. Differences were considered significant at p < 0.05. ^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).

Figure 3

Effects ascorbic acid (AA) in superoxide dismutase activities in hippocampus of adult rats 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), AA group with ascorbic acid (500 mg/kg, i.p., n = 6, AA) and the control animals with 0.9% saline (i.p., n = 9, Control). The AA plus pilocarpine group was treated with ascorbic acid (500 mg/kg, i.p.) 30 min after pilocarpine injection (400 mg/kg, i.p., n = 6, AA plus P400). The cerebral area studied was ultrasonically homogenized in 1 ml of 0.05 M phosphate buffer, pH 7.0, and the homogenates 10% were centrifuged (800 xg/20 min) and the supernatant of the homogenates was used for superoxide dismutase activity investigation. The superoxide dismutase activity in the hippocampus during seizure activity was analyzed by measuring the superoxide dismutase activity in homogenates, as previously described by Flohe and Otting, 1989. Results are expressed as means + S.E.M. for the number of animals shown inside in parenthesis. Results were expressed as U/mg of protein. The Student-Newman-Keuls test was used for multiple comparisons of means of two groups of data. Differences in experimental groups were determined by two-tailed analysis of variance. Differences were considered significant at p < 0.05. ^ap < 0.05 as compared to control animals (t-Student-Neuman-Keuls test); ^bp < 0.05 as compared to pilocarpine group (t-Student-Neuman-Keuls test).

Figure 4

Effects of ascorbic acid (AA) in catalase activities in hippocampus of adult rats 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), AA group with ascorbic acid (500 mg/kg, i.p., n = 6, VIT C) and the control animals with 0.9% saline (i.p., n = 9, Control). The AA plus pilocarpine group was treated with ascorbic acid (500 mg/kg, i.p.) 30 min after pilocarpine injection (400 mg/kg, i.p., n = 6, AA plus P400). The cerebral area studied was ultrasonically homogenized in 1 ml of 0.05 M phosphate buffer, pH 7.0, and the homogenates 10% were centrifuged (800 xg/20 min) and the supernatant of the homogenates was used for catalase activity investigation. The catalase activity in the hippocampus during seizure activity was analyzed by measuring the catalase activity in homogenates, as previously described by Chance and Maehly, 1955. Results are expressed as means ± S.E.M. for the number of animals shown inside in parenthesis. Results were expressed as mmol/min/mg of protein. The Student-Newman-Keuls test was used for multiple comparisons of means of two groups of data. Differences in experimental groups were determined by two-tailed analysis of variance. Differences were considered significant at p < 0.05. ^ap < 0.05 as compared to control animals (t-Student-Neuman-Keuls test); ^bp < 0.05 as compared to pilocarpine group (t-Student-Neuman-Keuls test).