Interictal spikes, seizures and ictal cell death are not necessary for post-traumatic epileptogenesis in vitro
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)
- et al.
Microfluidics and multielectrode array—compatible organotypic slice culture method
J. Neurosci. Methods
(2009) - et al.
Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats
Brain Res.
(1988) - et al.
Hippocampal interneuron loss and plasticity in human temporal lobe epilepsy
Brain Res.
(1989) - et al.
A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity
J. Immunol. Methods
(1988) - et al.
Organotypic slice cultures: a technique has come of age
Trends Neurosci.
(1997) - et al.
Epilepsy after head injury. Residual risk after varying fit-free intervals since injury
Lancet
(1973) - et al.
An enzyme-release assay for natural cytotoxicity
J. Immunol. Methods
(1983) - et al.
Rat hippocampal slices ‘in vitro’ display spontaneous epileptiform activity following long-term organotypic culture
J. Neurosci. Methods
(1989) Concept of activity-induced cell death in epilepsy: historical and contemporary perspectives
Prog. Brain Res.
(2002)- et al.
Anti-epileptogenesis in rodent post-traumatic epilepsy models
Neurosci. Lett.
(2011)
Collateral projections of cells in the surviving entorhinal area which reinnervate the dentate gyrus of the rat following unilateral entorhinal lesions
Brain Res.
Unmasking recurrent excitation generated by mossy fiber sprouting in the epileptic dentate gyrus: an emergent property of a complex system
Prog. Brain Res.
Efficient unsupervised algorithms for the detection of seizures in continuous EEG recordings from rats after brain injury
J. Neurosci. Methods
A population-based study of seizures after traumatic brain injuries
N. Engl. J. Med.
Primary and secondary mechanisms of epileptogenesis in the temporal lobe: there is a before and an after
Epilepsy Curr.
The natural history of mesial temporal lobe epilepsy
Curr. Opin. Neurol.
Longitudinal and cross-sectional analysis of atrophy in pharmacoresistant temporal lobe epilepsy
Neurology
Topography of axonal injury as defined by amyloid precursor protein and the sector scoring method in mild and severe closed head injury
J. Neurotrauma
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.
Prolonged infusion of tetrodotoxin does not block mossy fiber sprouting in pilocarpine-treated rats
Epilepsia
Report of the Committee on Terminology
Glossary of terms most commonly used by clinical electroencephalographers
Electroencephalogr. Clin. Neurophysiol.
Posttraumatic epilepsy and treatment
J. Rehabil. Res. Dev.
Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy
Nat. Neurosci.
Antiepileptic drugs and the electroencephalogram
Epilepsia
Interictal spikes precede ictal discharges in an organotypic hippocampal slice culture model of epileptogenesis
J. Clin. Neurophysiol.
Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy
J. Comp. Neurol.
Hippocampal sclerosis is a progressive disorder: a longitudinal volumetric MRI study
Ann. Neurol.
Is epilepsy a preventable disorder? New evidence from animal models
Neuroscientist
A critical period for prevention of posttraumatic neocortical hyperexcitability in rats
Ann. Neurol.
Recent advances in neurotrauma
J. Neuropathol. Exp. Neurol.
Epileptogenic effect of antibiotic drugs
J. Neurosurg.
Cited by (52)
Variability of seizure-like activity in an in vitro model of epilepsy depends on the electrical recording method
2020, HeliyonCitation Excerpt :These changes in the network may result in generation of seizures with different patterns. There may also be ongoing cell death in slices due to seizures (Berdichevsky and Dzhala, 2012), which in turn may cause compensatory changes in the network. Machine learning has been used to estimate the phase of oscillations in the brain (McIntosh and Sajda 2020).
GABA <inf>A</inf> receptor-mediated networks during focal seizure onset and progression in vitro
2019, Neurobiology of DiseaseCitation Excerpt :A relevant issue to consider in the context of in vitro experiments is the notion that seizure-like events (SLEs) do not occur spontaneously in vitro (see Dulla et al., 2018). With the exception of long-term organotypic hippocampal slice cultures maintained in vitro for at least 1 week (Berdichevsky et al., 2012), in vitro studies of epileptic seizure-genesis are based on SLEs induced by a precipitating condition. This applies also to studies performed on brain slices obtained from chronically epileptic animals and in cortical and hippocampal slices obtained from human tissue from therapeutic surgical resections performed in patients with focal epilepsy resistant to pharmacological treatment (for review see Jones et al., 2016; Raimondo et al., 2017; Dulla et al., 2018).
Organotypic Hippocampal Slice Cultures as a Model of Posttraumatic Epileptogenesis
2017, Models of Seizures and Epilepsy: Second Edition
- 1
These authors contributed equally to this work.