Acute seizure activity promotes lipid peroxidation, increased nitrite levels and adaptive pathways against oxidative stress in the frontal cortex and striatum

Abstract

Previous experiments have shown that the generation of free radicals in rat brain homogenates is increased following pilocarpine-induced seizures and status epilepticus (SE). This study was aimed at investigating the changes in neurochemical mechanisms such as lipid peroxidation levels, nitrite content, glutathione reduced (GSH) concentration, superoxide dismutase and catalase activities in the frontal cortex and the striatum of Wistar adult rats after seizures and SE induced by pilocarpine. The control group was treated with 0.9% saline and another group of rats received pilocarpine (400 mg/kg, i.p.). Both groups were sacrificed 24 h after the treatments. Lipid peroxidation level, nitrite content, GSH concentration and enzymatic activities were measured by using spectrophotometric methods. Our findings showed that pilocarpine administration and its resulting seizures and SE produced a significant increase of lipid peroxidation level in the striatum (47%) and frontal cortex (59%). Nitrite contents increased 49% and 73% in striatum and frontal cortex in pilocarpine group, respectively. In GSH concentrations were decreases of 54% and 58% in the striatum and frontal cortex in pilocarpine group, respectively. The catalase activity increased 39% and 49% in the striatum and frontal cortex, respectively. The superoxide dismutase activity was not altered in the striatum, but it was present at a 24% increase in frontal cortex. These results suggest that there is a direct relationship between the lipid peroxidation and nitrite contents during epileptic activity that can be responsible for the superoxide dismutase and catalase enzymatic activity changes observed during the establishment of seizures and SE induced by pilocarpine.

Keywords: catalase; frontal cortex; glutathione reduced; lipid peroxidation; nitrite content; striatum; superoxide dismutase.

Figure 1

Lipid peroxidation level is increased in the striatum during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 2

Lipid peroxidation level is increased in the frontal cortex during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 3

Nitrite content is increased in the striatum during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 4

Nitrite content is increased in the frontal cortex during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 5

Glutathione reduced concentration is decreased in the striatum during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 6

Glutathione reduced concentration is decreased in the frontal cortex during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 7

Superoxide dismutase activity is increased in the frontal cortex during seizure activity. Male rats (250–280 g, 2-months-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).

Figure 8

Catalase activity is increased in the striatum and frontal cortex during seizure activity. Male rats (250–280 g, 2-month-old) were treated with a single dose of pilocarpine (400 mg/kg, i.p., n = 7, pilocarpine group) and the control group with 0.9% saline (n = 9). The pilocarpine group was constituted by those rats that presented seizures, SE for over 30 min and that did not died within 24 h. For neurochemical determinations of lipid peroxidation level, the rats (pilocarpine and control groups) were killed by decapitation 24 h after the treatment and their brains were dissected on ice to remove the striatum. 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 lipid peroxidation level investigation. The lipid peroxidation level in the striatum during seizure activity was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in homogenates, as previously described by Draper and Hadley, 1990. 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 with control group (analysis of variance and Student-Newman-Keuls test).