Early hippocampal oxidative stress is a direct consequence of seizures in the rapid electrical amygdala kindling model

Summary

Epilepsy is characterised by recurrent seizures, which are manifestations of aberrant cortical neuronal firing. It is unclear whether oxidative stress is a cause or consequence of seizure-related hippocampal neuronal loss or whether it occurs concomitantly with the initiation of cell death pathways. We utilised the rapid electrical amygdala kindling (REAK) model which does not induce cell death to examine early seizure-induced oxidative stress in wildtype and superoxide dismutase 2 (Sod2) +/− mice, which lack 50% of Sod2 activity and are therefore known to be more susceptible to mitochondrial oxidative stress. A significant increase in lipid peroxidation and superoxide production was noted in the hippocampi of wildtype mice and a more delayed response observed in Sod2 +/− mice at early time-points post-seizures, but protein carbonylation levels appeared unchanged. A 10-fold increase in superoxide production was seen in the Sod2 +/− CA2 neurons, indicating that Sod2 plays an important role in protecting the CA2 region of the hippocampus from seizure-induced free radical damage. Early hippocampal cell death was undetectable in wildtype or Sod2 +/− mice post-seizures. We were able to demonstrate that hippocampal oxidative stress occurred as a direct consequence of seizures rather than downstream of activation of cell death pathways. We were also able to show that this increase in oxidative stress was not sufficient to cause cell death within the time window investigated. Our data indicates that a possible upregulation of endogenous antioxidant activity might exist within selective hippocampal sectors in the Sod2 +/− mice that are as yet unknown.

Introduction

Mesial temporal lobe epilepsy (MTLE) is a neurological disorder characterised by recurrent seizures and hippocampal sclerosis, which involves a selective loss of neurons in hippocampal fields CA1, CA3 and CA4 (Hauser et al., 1993). During epileptic seizures, increased glutamate levels and the activation of NMDA receptors causes a dysregulation of neuronal calcium homeostasis and the activation of cell death pathways (Manev et al., 1989, Michaels and Rothman, 1990). The rise in intracellular Ca²⁺ stimulates the generation of reactive oxygen species (ROS) which enhances the opening of the mitochondrial permeability transition pore and can also have an impact on cell viability (Duchen, 2000). Thus, glutamate excitotoxicity following seizures can cause mitochondrial oxidative stress which can then lead to cell death. However, there may also be a feedback system of cell death leading to increased excitability, seizures, and subsequently, further cell death (Malis and Bonventre, 1986, Dykens et al., 1987). It remains to be clarified whether overproduction of free radicals in epilepsy occurs downstream of cellular apoptotic and necrotic processes, or whether it is a direct consequence of seizures. An understanding of the biochemical causes and consequences of epileptic seizures is necessary to develop targeted therapeutics for epilepsy (Waldbaum and Patel, 2010).

Superoxide dismutase 2 or Sod2 is an antioxidant enzyme that is located on the matrix side of the inner mitochondrial membrane (Weisiger and Fridovich, 1973). Sod2 −/− mice are neonatal lethal and survive for approximately 5 and 21 days when on a CD1 and C57Bl6 background, respectively, confirming the deleterious effects of unquenched mitochondrial ROS production (Li et al., 1995, Lebovitz et al., 1996). Kainate-induced seizures result in an increase in mitochondrial superoxide production, and hippocampal neuronal loss is reduced in transgenic mice over-expressing MnSOD (Liang et al., 2000). Sod2 +/− mice appear normal but exhibit increased sensitivity to oxidative stress and increased oxidative damage (Williams et al., 1998, Van Remmen et al., 2003), and are therefore regarded as a model of accelerated mitochondrial oxidative stress (Lebovitz et al., 1996, Kokoszka et al., 2001). In humans, Sod2 activity is significantly lower in the epileptic cerebral cortex compared to controls, strongly implicating the overproduction of mitochondrial superoxide in the pathogenesis of epilepsy (Eun et al., 2004).

Here, we hypothesised that oxidative stress is a direct effect of seizures rather than a consequence of seizure-induced cell death, which we investigated in an animal model of MTLE known as the rapid electrical amygdala kindling (REAK) model. Early seizure-induced cell death was not expected in these mice and therefore we were able to isolate the effects of seizures alone in the hippocampus independently of the activation of cell death pathways (Smith et al., 2005). We also hypothesised that if enhanced oxidative stress was a cause of hippocampal cell death, then the Sod2 +/− mice would be more susceptible to hippocampal cell death in the acute phase following REAK-evoked seizures.