Optimizing Organotypic Slices to Study Epileptogenesis

描述（由申请人提供）：NINDS今年发布了U系列和R系列RFA，以发现预防脑损伤后癫痫发展的药物。对RFA反应的限速因素是缺乏脑损伤后癫痫的高度可扩展模型，以及量化癫痫发生的方法。海马切片是创伤性脑损伤的一个引人注目的模型，这些切片的器官型培养为严重剪切损伤后的恢复提供了一个独特的窗口。我们重新发现了器官型切片培养作为创伤后癫痫发生的加速模型的实用性，20年后首次描述了该制剂中的癫痫活性。癫痫研究界失去了20年来使用这个非常强大的模型，主要是因为不充分的标准化造成了不充分的可重复性。我们建议优化用于癫痫发生研究的器官型切片制备，重点关注3个关键问题以最大化其效用：首先，量化该制备中癫痫发生的最佳方法是什么？我们已经开发了连续电图记录和自动癫痫发作检测算法，使致癫痫切片培养物阵列的并行处理成为可能。我们将确定我们设计的用于定量体内癫痫发生的方法是否可以应用于这些培养阵列。第二，在切片培养中癫痫发生的最佳培养条件是什么？这种制剂从未被优化用于癫痫的研究，因此我们需要确定代谢底物和产物不会限制癫痫活性的量。第三，可以使用什么替代标记来加速使用该系统进行筛查？虽然从电描记记录癫痫发生的量化是金标准，我们的初步数据表明，有潜在的有前途的标志物，如细胞外乳酸水平和细胞内氯和钙水平，可用作荧光测定的基础，这将大大加快与此模型的吞吐量。总之，这些研究将使一个新的，快速的，高度可扩展的，易于转移的，可量化的创伤后癫痫发生的测定，将加速发病机制的基础研究和治疗策略的搜索。

DESCRIPTION (provided by applicant): The NINDS has issued U- and R-series RFAs this year to discover drugs to prevent the development of epilepsy after brain injury. The rate-limiting factors in the response to the RFAs are the lack of highly scalable models of epilepsy after brain injury, and the means to quantify epileptogenesis. The hippocampal slice is a compelling model of traumatic brain injury, and organotypic cultures of these slices provide a unique window into recovery after severe shear injury. We re-discovered the utility of the organotypic slice culture as an accelerated model of post traumatic epileptogenesis 20 years after epileptic activity in this preparation was first described. The epilepsy research community lost 20 years of use of this exceptionally powerful model largely because inadequate standardization created a perception of inadequate reproducibility. We propose to optimize the organotypic slice preparation for the study of epileptogenesis, focusing on 3 key questions to maximize its utility: First, what are the best means to quantify epileptogenesis in this preparation? We have developed methods for continuous electrographic recordings and automated seizure detection algorithms that make feasible the parallel processing of arrays of epileptogenic slice cultures. We will determine whether the methods that we have devised for quantification of in vivo epileptogenesis can be applied to these culture arrays. Second, what are the optimum culture conditions for epileptogenesis in slice cultures? This preparation was never optimized for the study of epilepsy, so we need to ascertain that metabolic substrates and products do not limit the quantity of epileptic activity. Third, what surrogate markers can be used to accelerate the use of this system for screening? Although quantification of epileptogenesis from electrographic recordings is the gold standard, our preliminary data suggest that there are potentially promising markers such as extracellular lactate levels and intracellular chloride and calcium levels that can be used as the basis of fluorescence assays that will substantially accelerate throughput with this model. Together, these studies will enable a new, rapid, highly scalable, readily transferrable, and quantifiable assay of post traumatic epileptogenesis that will accelerate both fundamental research into pathogenesis and the search for therapeutic strategies.