Optimizing Organotypic Slices to Study Epileptogenesis

描述(申请人提供)：NINDS今年已经发布了U系列和R系列RFA，以发现防止脑损伤后癫痫发展的药物。对RFA反应的速度限制因素是缺乏高度可扩展的脑损伤后癫痫模型，以及量化癫痫发生的手段。海马片是一个引人注目的创伤性脑损伤模型，这些脑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。在该制剂的癫痫活性首次被描述20年后，我们重新发现器官型切片培养作为创伤后癫痫发生的加速模型的用途。癫痫研究界失去了20年来使用这种特别强大的模型的机会，这主要是因为标准化不充分造成了人们对重复性不足的看法。我们建议优化用于癫痫发生研究的器官型切片制备方法，重点解决三个关键问题，以最大限度地发挥其作用：第一，在这种制备方法中，量化癫痫发生的最佳方法是什么？我们已经开发了连续脑电记录和自动癫痫发作检测算法的方法，使致痫切片培养阵列的并行处理成为可能。我们将确定我们设计的体内癫痫发生的量化方法是否可以应用于这些培养阵列。第二，薄片培养致痫的最适培养条件是什么？这种制剂从来没有在癫痫的研究中得到优化，所以我们需要确定代谢底物和产品不会限制癫痫活动的数量。第三，可以使用哪些替代标记来加速使用该系统进行筛查？虽然根据脑电记录量化癫痫的发生是金标准，但我们的初步数据表明，有潜在的有希望的标志物，如细胞外乳酸水平和细胞内氯和钙水平，可以用作荧光分析的基础，从而大大加快该模型的吞吐量。总之，这些研究将使创伤后癫痫发生的新的、快速的、高度可扩展的、容易转移的和可量化的分析成为可能，这将加速对发病机制的基础研究和治疗策略的探索。

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.