Elsevier

Neurobiology of Disease

Volume 45, Issue 2, February 2012, Pages 774-785
Neurobiology of Disease

Interictal spikes, seizures and ictal cell death are not necessary for post-traumatic epileptogenesis in vitro

https://doi.org/10.1016/j.nbd.2011.11.001Get rights and content

Abstract

Clinical studies indicate that phenytoin prevents acute post-traumatic seizures but not subsequent post-traumatic epilepsy. We explored this phenomenon using organotypic hippocampal slice cultures as a model of severe traumatic brain injury. Hippocampal slices were cultured for up to eight weeks, during which acute and chronic electrical recordings revealed a characteristic evolution of spontaneous epileptiform discharges, including interictal spikes, seizure activity and electrical status epilepticus. Cell death exhibited an early peak immediately following slicing, and a later secondary peak that coincided with the peak of seizure-like activity. The secondary peak in neuronal death was abolished by either blockade of glutamatergic transmission with kynurenic acid or by elimination of ictal activity and status epilepticus with phenytoin. Withdrawal of kynurenic acid or phenytoin was followed by a sharp increase in spontaneous seizure activity. Phenytoin's anticonvulsant and neuroprotective effects failed after four weeks of continuous administration. These data support the clinical findings that after brain injury, anticonvulsants prevent seizures but not epilepsy or the development of anticonvulsant resistance. We extend the clinical data by showing that secondary neuronal death is correlated with ictal but not interictal activity, and that blocking all three of these sequelae of brain injury does not prevent epileptogenesis in this in vitro model.

Highlights

► Organotypic hippocampal cultures are an in vitro model of epileptogenesis. ► Model enables longitudinal studies of seizures and cell death. ► Spontaneous seizures induce ongoing neuronal death. ► Preventing spontaneous seizures prevents ongoing neuronal death. ► Blocking seizure activity and neuronal death does not prevent epileptogenesis.

Introduction

Traumatic brain injury (TBI) is a major cause of acquired epilepsy (Pitkanen et al., 2011). Following a latent period of months to years, recurrent spontaneous seizures occur in up to 53% of veterans with severe military head injury (Raymont et al., 2010, Salazar et al., 1985), and 17% of civilian patients with severe head injury (Annegers et al., 1998). It has been hypothesized that neuronal death and axon damage resulting from trauma (Blumbergs et al., 1995, Graham et al., 2000) initiate the process of epileptogenesis, or development of epilepsy (Ben-Ari and Dudek, 2010, Staley et al., 2005). Consequences of neuron damage could include the loss of inhibition due to death of interneurons (Cossart et al., 2001, de Lanerolle et al., 1989, Kobayashi and Buckmaster, 2003, Sloviter, 1987), and axonal sprouting (Cronin and Dudek, 1988, Okazaki et al., 1995, Sutula et al., 1989) due to deafferentation (Laurberg and Zimmer, 1981, Steward and Vinsant, 1978, Sutula and Dudek, 2007) leading to hyperexcitability (Esclapez et al., 1999, Smith and Dudek, 2001) and spontaneous seizures (Jefferys, 2003). However, seizures can also cause neuronal death. It is widely accepted that prolonged seizures (status epilepticus) result in neuronal necrosis (Meldrum, 2002, Meldrum and Brierley, 1973, Meldrum et al., 1973). The possibility that even brief, spontaneous seizures can kill neurons has been raised by MRI studies demonstrating progressive atrophy in patients with intractable epilepsy (Bernhardt et al., 2009, Fuerst et al., 2003), evidence of apoptosis in brain tissue resected for seizure control (Henshall et al., 2004) and the correlation of cortical volume loss with seizure frequency in post-traumatic epilepsy (Raymont et al., 2010). It is difficult to establish causality from these observational studies. For example, cortical volume loss in patients with post-traumatic epilepsy may reflect a more severely epileptogenic initial injury, or more frequent post-traumatic seizures may lead to progressive volume loss. However, knowledge of causality is necessary to design rational antiepileptogenic therapies (Giblin and Blumenfeld, 2010, Pitkanen, 2010) and to know whether the benefits of seizure control include neuroprotection and/or suppression of epileptogenesis (Loscher and Brandt, 2010). The current paradigm for treatment of post-traumatic epilepsy includes treatment with anticonvulsants such as phenytoin (Chen et al., 2009, Temkin, 2009), although anticonvulsants tested to date have not demonstrated antiepileptogenic efficacy (Schierhout and Roberts, 2001, Temkin, 2001, Temkin, 2009). On the other hand, long-term monitoring studies indicate that experimental epilepsy continues to worsen after the first seizure (Williams et al., 2009), supporting the possibility that seizures themselves contribute to epileptogenesis. Open questions include whether recurrent spontaneous epileptiform activity induces neuronal death, whether such neuronal death worsens epilepsy, and whether effective anticonvulsant therapy alters the course of epilepsy.

Here, we use organotypic hippocampal slice cultures as a model of post-traumatic epileptogenesis to monitor the events following brain injury and the effects of treatment with phenytoin. We and others have previously reported that these cultures become spontaneously epileptic after a latent period in vitro (Dyhrfjeld-Johnsen et al., 2010, McBain et al., 1989). We follow both epileptogenesis and ongoing neuronal death in the same cultures using chronic electrical recordings and sequential measurement of cell death markers to test whether spontaneous ictal or interictal activity causes cell death, whether suppression of spikes or seizures is neuroprotective, and whether suppression of spikes, seizures, and ictal neuronal death has antiepileptogenic effects.

Section snippets

Materials and methods

All animal use protocols conformed to the guidelines of the National Institutes of Health and the Massachusetts General Hospital Center for Comparative Medicine on the use of laboratory animals.

Model of post-traumatic epileptogenesis in vitro

We recorded spontaneous activity from rat organotypic hippocampal cultures (n = 7) grown on MEAs for up to 37 DIV (Fig. 1B). All slice cultures developed epileptic activity. Raster plots were constructed from electrical data (Fig. 1C), with interictal and ictal activities pseudocolored based on spike frequency (Fig. 1D). Activity recordings reveal that organotypic cultures go through a latent period before developing epileptiform discharges, analogously to TBI patients (Annegers et al., 1998). In

Discussion

This study demonstrates clear relationships between ongoing seizure activity and accelerated neuronal death in vitro that was prevented by successful anticonvulsant therapy. However, prevention of ictal and interictal activity and ictal cell death did not prevent epileptogenesis.

The temporal definition of status epilepticus, a medical emergency comprised of unremitting seizures, remains controversial (Lowenstein et al., 1999, Shinnar and Hesdorffer, 2010) because the duration of seizure

Conclusions

Organotypic hippocampal cultures reproduce many of the salient features of severe post-traumatic epilepsy, and are therefore a good model to study epileptogenesis and effects of anticonvulsant and antiepileptogenic drugs in vitro. We found substantial evidence that spontaneous recurrent electrical seizures resulting from initial trauma cause secondary neuronal death. We also found that prevention of spontaneous electrical seizures has a neuroprotective, but not an antiepileptogenic effect.

Acknowledgments

This work was supported by National Institute of Neurological Disorders and Stroke (NINDS) and Epilepsy Foundation (EFA).

References (64)

  • O. Steward et al.

    Collateral projections of cells in the surviving entorhinal area which reinnervate the dentate gyrus of the rat following unilateral entorhinal lesions

    Brain Res.

    (1978)
  • T.P. Sutula et al.

    Unmasking recurrent excitation generated by mossy fiber sprouting in the epileptic dentate gyrus: an emergent property of a complex system

    Prog. Brain Res.

    (2007)
  • A.M. White et al.

    Efficient unsupervised algorithms for the detection of seizures in continuous EEG recordings from rats after brain injury

    J. Neurosci. Methods

    (2006)
  • J.F. Annegers et al.

    A population-based study of seizures after traumatic brain injuries

    N. Engl. J. Med.

    (1998)
  • Y. Ben-Ari et al.

    Primary and secondary mechanisms of epileptogenesis in the temporal lobe: there is a before and an after

    Epilepsy Curr.

    (2010)
  • A.T. Berg

    The natural history of mesial temporal lobe epilepsy

    Curr. Opin. Neurol.

    (2008)
  • B.C. Bernhardt et al.

    Longitudinal and cross-sectional analysis of atrophy in pharmacoresistant temporal lobe epilepsy

    Neurology

    (2009)
  • P.C. Blumbergs et al.

    Topography of axonal injury as defined by amyloid precursor protein and the sector scoring method in mild and severe closed head injury

    J. Neurotrauma

    (1995)
  • E. Bonfoco et al.

    Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
  • P.S. Buckmaster

    Prolonged infusion of tetrodotoxin does not block mossy fiber sprouting in pilocarpine-treated rats

    Epilepsia

    (2004)
  • G.E. Chatrian

    Report of the Committee on Terminology

  • G. Chatrian et al.

    Glossary of terms most commonly used by clinical electroencephalographers

    Electroencephalogr. Clin. Neurophysiol.

    (1974)
  • J.W. Chen et al.

    Posttraumatic epilepsy and treatment

    J. Rehabil. Res. Dev.

    (2009)
  • R. Cossart et al.

    Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy

    Nat. Neurosci.

    (2001)
  • J.S. Duncan

    Antiepileptic drugs and the electroencephalogram

    Epilepsia

    (1987)
  • J. Dyhrfjeld-Johnsen et al.

    Interictal spikes precede ictal discharges in an organotypic hippocampal slice culture model of epileptogenesis

    J. Clin. Neurophysiol.

    (2010)
  • M. Esclapez et al.

    Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy

    J. Comp. Neurol.

    (1999)
  • D. Fuerst et al.

    Hippocampal sclerosis is a progressive disorder: a longitudinal volumetric MRI study

    Ann. Neurol.

    (2003)
  • K.A. Giblin et al.

    Is epilepsy a preventable disorder? New evidence from animal models

    Neuroscientist

    (2010)
  • K.D. Graber et al.

    A critical period for prevention of posttraumatic neocortical hyperexcitability in rats

    Ann. Neurol.

    (2004)
  • D.I. Graham et al.

    Recent advances in neurotrauma

    J. Neuropathol. Exp. Neurol.

    (2000)
  • T.O. Grondahl et al.

    Epileptogenic effect of antibiotic drugs

    J. Neurosurg.

    (1993)
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