Abstract
Understanding the mechanisms of epileptogenesis is essential to develop novel drugs that could prevent or modify the disease. Neuroinflammation has been proposed as a promising target for therapeutic interventions to inhibit the epileptogenic process that evolves from traumatic brain injury. However, it remains unclear whether cytokine-related pathways, particularly TNFα signaling, have a critical role in the development of epilepsy. In this study, we investigated the role of innate inflammation in an in vitro model of post-traumatic epileptogenesis. We combined organotypic hippocampal slice cultures, representing an in vitro model of post-traumatic epilepsy, with multi-electrode array recordings to directly monitor the development of epileptiform activity and to examine the concomitant changes in cytokine release, cell death, and glial cell activation. We report that synchronized ictal- and interictal-like activities spontaneously evolve in this culture. Dynamic changes in the release of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 were observed throughout the culture period (3 to 21 days in vitro) with persistent activation of microglia and astrocytes. We found that neutralizing TNFα with a polyclonal antibody significantly reduced ictal discharges, and this effect lasted for 1 week after antibody washout. Neither phenytoin nor an anti-IL-6 polyclonal antibody was efficacious in inhibiting the development of epileptiform activity. Our data show a sustained effect of the anti-TNFα antibody on the ictal progression in organotypic hippocampal slice cultures supporting the critical role of inflammatory mediators in epilepsy and establishing a proof-of-principle evidence for the utility of this preparation to test the therapeutic effects of anti-inflammatory treatments.
Highlights
Epilepsy is a chronic disease with recurrent and unprovoked seizures that seriously impacts on the quality of life of Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.astrocytes and the concomitant increased expression of proinflammatory cytokines such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNFα), and interleukin-6 (IL-6) following brain insults contribute to hyperexcitability and neuronal damage [13,14,15,16,17,18,19,20]
We performed power spectrum analysis in 0 to 30 Hz frequency range and found that the group of slices at the later stages (14-21 DIV) showed significantly higher power than the group at 3 to 7 DIV in the frequency range between 5 and 30 Hz (Fig. 1G). These results indicate a progression of epileptiform activity in Organotypic hippocampal slice cultures (OHSCs) which paralleled the age of the culture
The main findings of this study indicate that both age-dependent ictal progression and inflammation driven by activated microglia and astrocytes are present in the OHSCs
Summary
Murashima et al [21] reported a progressive increase of IL-1α, IL-1β, and TNFα expression during the development of epilepsy in epileptic mutant (EL) mice with secondarily generalized seizures. TNFR1 signaling increases glutamatergic neurotransmission by upregulating calcium-permeable AMPA and NMDA receptors and by inhibiting glutamate re-uptake by astrocytes [14, 25,26,27] which may promote seizure generation. It remains unclear whether TNFα signaling has a critical role in epileptogenesis and whether blocking TNFα activity with specific antibodies inhibits this pathologic process
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