Oxidative stress in rat brain during experimental status epilepticus: effect of antioxidants

Abstract

Antioxidants have been proposed as a treatment for diseases of the central nervous system. However, few studies actually studied their effects in the brain. To test central actions of antioxidants, we used the lithium-pilocarpine (Li-Pilo) model of status epilepticus (SE) in the rat in which seizures are accompanied by significant oxidative stress. We used in vivo microdialysis to determine isoprostane levels during SE in real time and brain homogenates for other measures of oxidative stress. Six different antioxidants were tested in acute and preventive experiments (vitamin C, vitamin E, ebselen, resveratrol, n-tert-butyl-α-phenylnitrone and coenzyme Q10). None of the antioxidants had an effect when given acutely during SE. In contrast, when antioxidants were given for 3 days prior to seizure induction, vitamins C and E reduced isoprostane formation by 58% and 65%, respectively. Pretreatment with the other antioxidants was ineffective. In brain homogenates prepared after 90 min of seizures, SE decreased the ratio of reduced vs. oxidized glutathione (GSH/GSSG ratio) from 60.8 to 7.50 and caused a twofold increase of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and protein carbonyls. Pretreatment with vitamin C or vitamin E mitigated these effects and increased the GSH/GSSG ratio to 23.9 and 28.3, respectively. Again, the other antioxidants were not effective. We conclude that preventive treatment with vitamin C or vitamin E ameliorates seizure-induced oxidative damage in the brain. Several well-studied antioxidants were inactive, possibly due to limited brain permeability or a lack of chain-breaking antioxidant activity in hydrophilic compounds.

Keywords: ascorbic acid; coenzyme Q10; ebselen; isoprostanes; resveratrol; α-tocopherol.

FIGURE 1

Flow diagram of the acute series of experiments. Diaz, diazepam; HPC, hippocampus; LiCl, lithium chloride; Pilo, pilocarpine; SE, status epilepticus.

FIGURE 2

Flow diagram of the preventive series of experiments. Diaz, diazepam; GSH/GSSG ratio, ratio of reduced vs. oxidized gluthatione; HPC, hippocampus; LiCl, lithium chloride; SE, status epilepticus; MDA, malondialdehyde; Pilo, pilocarpine; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

FIGURE 3

Extracellular isoprostane concentrations in the ventral hippocampus before SE (“Basal”), during SE (“Pilocarpine”, indicated as blue background) and after administration of diazepam (“Diazepam”) (acute series of experiments, cf. Figure 1). Antioxidant or saline were injected 90 min after pilocarpine administration. Data are presented as means ± SEM, given as absolute values. Treatments: (A) vitamin C (n = 9) and vitamin E (n = 6); (B) n-tert-butyl-α-phenylnitrone (PBN; n = 7) and coenzyme Q10 (Q10; n = 7); (C) ebselen (n = 6) and resveratrol (n = 7). Injections of saline (n = 8) served as controls. Statistics (two-way ANOVA with Bonferroni´s post-test): (A) F_2,20 = 0.27; p = 0.77. (B) F_2,20 = 0.14; p = 0.87. (C) F_2,18 = 0.07; p = 0.93.

FIGURE 4

Extracellular isoprostane concentrations in the ventral hippocampus before SE (“Basal”), during SE (“Pilocarpine”, indicated as blue background) and after administration of diazepam (“Diazepam”) (preventive series of experiments, cf. Figure 2). Rats received seven dosages of antioxidant or saline every 12 hours prior to seizure induction. Data are presented as means ± SEM, given as absolute values. Treatments: (A) vitamin C (n = 8) and vitamin E (n = 7); (B) n-tert-butyl-α-phenylnitrone (PBN; n = 6) and coenzyme Q10 (Q10; n = 8); (C) ebselen (n = 7) and resveratrol (n = 8). Pretreatment with saline (n = 7) served as control. Statistics (two-way ANOVA with Bonferroni’s post-test): **p < 0.01 versus saline. (A) F_2,19 = 2.23; p = 0.13. (B) F_2,18 = 0.19; p = 0.83. (C) F_2,19 = 0.58; p = 0.57.

FIGURE 5

Glutathione ratio and protein carbonyls measured in brain homogenates after 2 h of SE. Rats received seven dosages of antioxidant or saline every 12 hours prior to seizure induction (preventive series of experiments, cf. Figure 2). SE was not induced in control animals. Data are presented as means ± SEM. Abbreviations: PBN, n-tert-butyl-α-phenylnitrone; Q10, coenzyme Q10. (A) Ratio of reduced vs. oxidized glutathione (GSG/GSSG ratio). Number of experiments as indicated (n = 6–8). Statistics (one-way ANOVA with Dunnett’s multiple comparison test): F_7.49 = 28.66; p < 0.0001. ***, p < 0.001 vs. control. ###, p < 0.001 vs. saline. (B) Concentrations of protein carbonyls in brain homogenates. Data are presented as means ± SEM, normalised to protein. Number of experiments as indicated (n = 6–10). Statistics (one-way ANOVA with Dunnett’s multiple comparison test): F_7.48 = 4.557; p < 0.001.; *, p < 0.05; **, p < 0.01 vs. control.

FIGURE 6

8-hydroxy-2′-deoxyguanosine and malondialdehyde measured in brain homogenates after 2 h of SE. Rats received seven dosages of antioxidant or saline every 12 hours prior to seizure induction (preventive series of experiments, cf. Figure 2). SE was not induced in control animals. Abbreviations: PBN, n-tert-butyl-α-phenylnitrone (PBN); Q10, coenzyme Q10. (A) Concentrations of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Data are presented as means ± SEM, normalised to protein. Number of experiments as indicated (n = 6–10). Statistics (one-way ANOVA with Dunnett’s multiple comparison test): F_7.49 = 7.073; p < 0.0001. *p < 0.05; ***p < 0.001 vs. control. (B) Concentrations of malondialdehyde (MDA) in brain homogenates. Data are presented as means ± SEM. Number of experiments as indicated (n = 6, 7). Statistics (one-way ANOVA): F_7,43 = 1.175; p = 0.34.