Assessing seizure liability using multi-electrode arrays (MEA)
Introduction
It has been reported that 6.1% of new onset seizures are drug related and up to 9% of cases of status epileptics presenting to the emergency department may result from drug toxicity (Thundiyil et al., 2011). Not only drugs targeting CNS but those targeting infection, cardiovascular, respiratory, metabolic, gastrointestinal, oncologic and other therapeutic areas can cause seizures (Easter et al., 2009). Drug induced seizures are severe adverse events and can have profound negative impact on drug development. Unfortunately, seizure potential of drug candidates is not typically evaluated until late stage in preclinical discovery, during in vivo toxicology studies, and the timing of this assessment is such that positive findings of seizure liability could result in the need to identify alternate clinical candidates, significantly impacting timelines. Development of a reliable, standardized, in vitro seizurogenicity assay that could be implemented early in discovery would be a valuable tool for the industry.
Recently, several groups developed in vitro (Easter et al., 2007; Markgraf et al., 2014) or in vivo methods (Leiser et al., 2011; Metea et al., 2015) to evaluate the drug induced seizure liability at the early preclinical drug discovery stage. These in vitro systems use conventional electrophysiological recording techniques to measure electrically evoked synaptic activity from rat hippocampal slices. The assays are predictive for seizurogenic effects of a wide range of compounds associated with seizure induction in man. However, such techniques have limitations including being more technically difficult, requiring an experienced electrophysiologist and invasive microelectrodes to record spontaneous or evoked potentials. Also, measurements are highly localized to the regions where single recording electrodes are placed and this can result in misleading or false negative results.
Unlike conventional brain slice recording technique, which utilizes fine extracellular electrodes inserted into the vicinity of a cell or cells to measure field potentials within a tissue, multi-electrode array (MEA) is a technology that allows extracellular recording from many electrodes imbedded in the bottom of the MEA well without the need to penetrate into the tissues. The electrical activity across a brain slice can be measured at multiple points where the imbedded electrodes contact the tissue. Stimulation and recording electrodes can be selected from any of the electrodes based on the position of the brain slice. Therefore, the chance of producing false negatives may be lower than that of the conventional extracellular recording technique (Steidl et al., 2006). Additionally, in comparison with conventional microelectrode recording techniques, the MEA technique allows for long-term analysis of the spatiotemporal distribution of network-level electrical activity, as well as stable recording that is less sensitive to factors related to tissue impalement with microelectrodes such as mechanical vibration (Liu et al., 2012).
Another way to detect drug-induced seizure liability using MEA technology involves the measurement of spike activity in native neuronal cells that are dispersed and then cultured in the well of the MEA dish (van Vliet et al., 2007). The neurons grow and form synaptic networks during culture, and field potential recordings across the array of electrodes can be used to evaluate seizure liability during application of a test article. This is a particularly useful method of evaluating seizure liability of drugs that are poorly permeable and slow to reach the interior region of tissue preparations. In this study, the utility of MEA technology for assessing drug-induced seizure liability was evaluated in both hippocampal brain slices and in cultured primary hippocampal neurons.
Section snippets
Rat cultured hippocampal neuron assay
All animal and study related activities were conducted in accordance with applicable Standard Operating Procedures, and current guidelines for animal welfare (National Research Council for the Care and Use of Laboratory Animals, 2011, Animal Welfare Act (AWA), 1966, as amended in 1970, 1976, 1985, and 1990, and the AWA implementing regulations in Title 9, Code of Federal Regulations, Chapter 1, Subchapter A, Parts 1–3). Rat fetal (E18) hippocampi were removed by microdissection and collected in
Vehicle and time control
DMSO was used as vehicle in both rat cultured hippocampal neuron and hippocampal slice assays. In the neuron assay, the spike rates remained stable after cumulative additions of DMSO up to 0.3%, the maximal concentration used, and over a period of time up to 60 min (data not shown, n = 38 from 4 slices). Similarly, in the brain slice assay, DMSO, up to 0.3%, did not exhibit any effect on FP area, which remained stable across a period of time up to 75 min (data not shown, n = 15, from 3 wells).
Acetaminophen
Discussion
In the present study, 9 seizurogenic compounds and one non-seizurogenic compound were tested in vitro for seizurogenic activity using hippocampal primary neurons and brain slices and MEA. Both assays showed good correlation for most compounds, although the cultured neuron assay showed higher sensitivity to a few of compounds.
Standard extracellular microelectrode recording of electrical activity in brain slices has been used to understand seizure mechanisms and for risk assessment (Easter et
Limitations
These studies are limited by the relatively small number of compounds tested in these two assays. Further studies on additional seizurogenic and negative control compounds will enhance our understanding of how best to use hippocampal brain slice or cultured primary neurons in seizure risk assessment. Another limitation is that these assays focused on hippocampus and seizures may be elicited via a number of different brain regions (Foldvary-Schaefer and Unnwongse, 2011). It will be interesting
Conclusion
These findings demonstrate the utility of MEA technology for seizure liability assessment using either rat hippocampal brain slice or rat embryonic hippocampal neurons. The cultured embryonic hippocampal neurons were more sensitive to some known seizurogenic substances than brain slices, and further studies will investigate possible reasons.
Acknowledgments
The authors would like to thank Mrs. Lucy Sun, who passed away in 2012, for her significant contributions during the early stage of this study and Mr. Rong-An Zhang for his help in statistical analysis.
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