Oxidative stress in murine Theiler's virus-induced temporal lobe epilepsy
Graphical abstract
Introduction
Temporal lobe epilepsy or TLE, the most common form of acquired epilepsy is initiated by a variety of insults including traumatic brain injury, stroke, status epilepticus and infections, which can cause early seizures, and following a latent period, lead to the development of spontaneous seizures or epilepsy. The cascade of biochemical, molecular and structural alterations following a precipitating injury and culminating in the development of epilepsy, i.e., epileptogenesis, is thought to involve processes such as neuronal loss, gliosis, axonal sprouting, neurogenesis and inflammation (Dudek and Staley, 2011, Sharma et al., 2007). A recent study indicates that patients that exhibit seizures during viral encephalitis are 22 times more likely to develop epilepsy than the control population (Misra et al., 2008). Thus patients with encephalitis are at high risk for developing epilepsy. A novel mouse model of infection-induced TLE which recapitulates clinical observations has been recently developed which offers a unique opportunity to study the molecular mechanism(s) underlying epileptogenesis and to identify novel therapeutic strategies (Libbey et al., 2008). Theiler's murine encephalomyelitis virus (TMEV) infected C57BL/6J mice show acute behavioral seizures between 3 and 7 days post-infection (dpi), exhibit neurodegeneration and glial activation in the hippocampus. A significant proportion of mice surviving the infection develop epilepsy after a latent period (Kirkman et al., 2010, Stewart et al., 2010a, Stewart et al., 2010b). In addition, the brains of TMEV infected mice show increased expression of mRNA for proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), during the acute seizure period (Kirkman et al., 2010). Given that TNF-α receptor 1 and IL-6 knockout mice have a significantly reduced incidence of seizures during the acute TMEV infection period, inflammation seems to play an important role in the induction of seizures in this animal model (Kirkman et al., 2010).
Oxidative stress is an important mechanism known to occur following brain injuries, sufficient to cause epilepsy (Liang et al., 2000). Endogenous antioxidants can overcome normal production of reactive oxygen and nitrogen species (ROS and RNS). However, their excessive production can overwhelm the natural antioxidant defenses and shift the redox state to a more oxidized environment which can lead to oxidative damage of various cellular targets. In fact, both mitochondrial and extracellular ROS play a role in mediating seizure-induced neuronal death (Liang et al., 2000, Patel et al., 2005). Additionally, oxidative stress has been shown to occur throughout epilepsy development in chemoconvulsant models of TLE (Waldbaum and Patel, 2010, Patel, 2004, Liang and Patel, 2006). Whether oxidative stress is a common mechanism underlying diverse epileptogenic injuries is unclear. We hypothesized that oxidative stress occurs in the Theiler's virus infection model of TLE for the following reasons. (1) Viral infections often cause increased formation of ROS and RNS either due to direct effects of the virus on the cells or as a consequence of host inflammatory responses to the infections (Schwarz, 1996, Valyi-Nagy et al., 2000). In response to viral infections, increased levels of cytokines, chemokines and other inflammatory mediators can directly damage mitochondria, resulting in oxidative stress. Herpes simplex virus (HSV) and Japanese encephalitis virus (JEV) are among the most common viruses which cause encephalitis and are both associated with acute seizures in patients (Theodore, 2014). Acute and chronic HSV-1 infection in mice results in inflammation and oxidative damage to the neurons and non-neuronal cells in the brain (Valyi-Nagy and Dermody, 2005). JEV infection has also been shown to stimulate the formation of oxidative stress in rat cultured cortical glial cells and in an acute JEV rat model (Liao et al., 2002, Srivastava et al., 2009). (2) Oxidative stress and mitochondrial dysfunction have the potential to lower seizure threshold by a variety of mechanisms including impaired ATP production (Jamme et al., 1995) and altered expression of transporters and enzymes crucial in the homeostasis of synaptic levels of neurotransmitter and intracellular calcium levels, thus tilting the balance of synaptic neurotransmission towards hyperexcitation (Waldbaum and Patel, 2010). Oxidative stress can further damage neurons by directly inducing apoptosis or necrosis and such aberrant neuronal loss can facilitate seizure generation (Kannan and Jain, 2000). Therefore, both inflammation and oxidative stress following viral infection may contribute to the development of acute seizures in the TMEV model.
While inflammation has been well documented in the TMEV model of infection-induced epilepsy, it is currently unknown if oxidative stress is observed during the acute seizure stage in TMEV infected mice. Therefore, the goal of the present study was to investigate the time course of oxidative stress in the TMEV-infection mouse model of TLE. We report here that TMEV-infected animals have a significant depletion of reduced glutathione (GSH), increase in oxidized glutathione (GSSG) levels, as well as an increase in 3-nitrotyrosine/tyrosine (3NT/Tyr) ratio. This data suggests that oxidative stress occurs in the TMEV model of CNS infection-induced epilepsy coincident with inflammation and acute seizure activity.
Section snippets
Animals
Male C57BL/6 mice aged between 4 and 5 weeks old were purchased from Jackson Laboratory (Bar Harbor, ME, USA). After arrival, mice were allowed to acclimatize for 3 days prior to the experiment. Mice were provided food and water ad libitum and kept in a facility providing 12 h of light and lark cycle starting at 6 am. All the procedures performed were in accordance with the guidelines provided and approved by the Institutional Animal Care and Use Committee of the University of Utah.
Treatment of mice and seizure monitoring
Mice were
Acute behavioral seizures in TMEV-infected mice
Behavioral seizures occur between 3 and 7 dpi in the TMEV model (Stewart et al., 2010a). Accordingly, seizures were not observed in animals sacrificed prior to day 3. As it was not possible to predict which animals would have gone on to develop seizures, the number of animals was increased (n = 8 per group) for the TMEV samples that were obtained at 8 hpi and at 1 and 2 dpi. The remaining animals (n = 26) were then assessed for seizure activity and 81% of those animals were observed to have had at
Discussion
This study demonstrates for the first time, that acute seizures resulting from TMEV infection in mice leads to the occurrence of oxidative and nitrosative stress which along with inflammation, might be contributing to the development of epilepsy in this model. We have shown that (1) the glutathione redox status is significantly impaired and (2) 3-nitrotyrosine levels are significantly increased following acute seizures in the TMEV model of infection-induced epilepsy. These results are
Acknowledgments
This work was funded by the Skaggs Scholar Award (KSW and MP), R01 NS065434 (KSW) and 1R01NS086423 (M.P.)
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