Combination antioxidant therapy prevents epileptogenesis and modifies chronic epilepsy

Abstract

Many epilepsies are acquired conditions following an insult to the brain such as a prolonged seizure, traumatic brain injury or stroke. The generation of reactive oxygen species (ROS) and induction of oxidative stress are common sequelae of such brain insults and have been shown to contribute to neuronal death and the development of epilepsy. Here, we show that combination therapy targeting the generation of ROS through NADPH oxidase inhibition and the endogenous antioxidant system through nuclear factor erythroid 2-related factor 2 (Nrf2) activation prevents excessive ROS accumulation, mitochondrial depolarisation and neuronal death during in vitro seizure-like activity. Moreover, this combination therapy prevented the development of spontaneous seizures in 40% of animals following status epilepticus (70% of animals were seizure free after 8 weeks) and modified the severity of epilepsy when given to chronic epileptic animals.

Keywords: Epileptogenesis; Keap1-Nrf2 pathway; NADPH oxidase; Oxidative stress; Spontaneous seizures.

Fig. 1

Combination of antioxidant therapies is neuroprotective in vitro. (A) Representative image of synchronous oscillatory Ca²⁺ signal in neurons indicating seizure like activity induced by replacement of artificial CSF (aCSF) with low Mg²⁺ aCSF. (B) Normalised Rhodamine123 fluorescence of neurons 10, 20 and 30 min treated with aCSF (n = 6 experiments (exp.)], low Mg²⁺ (n = 6 exp.), and treated acutely with AEBSF either alone (50 μM; n = 6 exp.) or with RTA (RTA 408, 200 nM, preincubation for 24 h; n = 6 exp., F(3,20) = 30.201, P < 0.001). (C) Normalized rates of ROS generation at different time points in neurons exposed to aCSF (n = 5 exp.), low Mg²⁺ (n = 5 exp.) or neurons treated acutely with AEBSF either alone (50 μM; n = 6 exp.) or with RTA 408 (200 nM, preincubation for 24 h; n = 6 exp., F(3,18) = 61.299, P < 0.001). Note that the combination therapy has a greater effect at later time point (15 min) than AEBSF only (P = 0.015). (D) The percentage of neuronal death in cultures following 2 h exposure to aCSF (n = 7), low Mg²⁺ (n = 7), and treatment with AEBSF either alone (50 μM; n = 7 exp.) or with RTA 408 (200 nM, preincubation for 24 h; n = 7 exp., F(3,24) = 19.699, P < 0.001]. Combination of both treatments has a greater effect versus single treatment (P = 0.029). Data (mean ± s.e.m.) were analysed by either repeated measures one-way ANOVA (B and C) or one-way ANOVA (D) followed by Tukey's post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus low Mg²⁺ condition.

Fig. 2

Combination antioxidant therapy increases total antioxidant capacity following status epilepticus. Total antioxidant capacity normalized to Trolox standard in plasma (A), cortex (B) and hippocampus (C) of rats 24 h following KA induced SE (2 h). A-C: n = 6, data are expressed as mean ± s.e.m, (A: F(4,25) = 16.625, P < 0.001; B: F(4,25) = 2.389, P = 0.078; C: F(4,25) = 22.594, P < 0.001), one way-ANOVA followed by Tukey’s post hoc. *P < 0.05, **P < 0.01, ***P < 0.001 vs. KA + Vehicle group. n.s. P > 0.05.

Fig. 3

Combination of NOX inhibition and Nrf2 activation suppresses the development of epilepsy following status epilepticus in rats. (A) Bar charts of seizure frequency (seizures\week; mean ± s.e.m) of animals following KA induced status epilepticus (2 h), treated immediately after termination of SE (Diazepam 5 mg/kg) with single administration of either vehicle (10% DMSO/saline; n = 10), RTA 408 25 mg/kg (n = 7), AEBSF 50 mg/kg (n = 7) or RTA 408 25 mg/kg and AEBAF 50 mg/kg (n = 10). F(3,300) = 8.005, P < 0.001 by generalized log-linear mixed model followed by sequential Bonferroni post hoc test. *P < 0.05, vs. Vehicle; #P < 0.05, RTA 408 + AEBSF vs. RTA 408; ^P < 0.05, RTA 408 + AEBSF vs. AEBSF. (B) Cumulative number of seizures of animals in A. Data are plotted on a logarithmic scale after incrementing each total seizure count by 1 to avoid zero values. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test (C) Combination therapy increases the latent period, F(3,24) = 11.197, P < 0.001, by one-way ANOVA followed by Tukey’s post hoc, ***P < 0.001 vs. KA + Vehicle group. (D) The pie charts illustrate percentage of animals seizure free for the whole study and for the last 5 weeks following KA induced status epilepticus treated with either vehicle (left) or with combination of RTA 408 (25 mg/kg) and AEBSF (50 mg/kg) (right). (E) Normalized seizure frequency (seizures\week; mean±s.e.m) of animals following KA induced SE (2 h) treated with either vehicle (n = 6; red) or with combination of RTA 408 (25 mg/kg) and AEBSF (50 mg/kg) (n = 6; blue) for 3 days, 12 weeks after SE. F(1,100) = 6.737, P = 0.011 by generalized log-linear mixed model on weeks 1–10, followed by sequential Bonferroni post hoc test). *P < 0.05, **P < 0.01 (F) Cumulative number of post-treatment seizures (mean ± s.e.m) for the two groups in E. P = 0.022 Student’s t-test.