Optimizing Organotypic Slices to Study Epileptogenesis

描述（由申请人提供）：国家药物研究中心今年已发出U和r系列射频识别，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。海马切片是创伤性脑损伤的一个令人信服的模型，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效用，在这种制备中癫痫活动首次被描述20年后。癫痫研究界浪费了20年的时间来使用这个异常强大的模型，主要是因为标准化不足造成了可重复性不足的感觉。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，以最大限度地发挥其效用：第一，在这种制备中，量化癫痫发生的最佳手段是什么？我们已经开发了连续电记录和自动癫痫检测算法的方法，使并行处理阵列的癫痫切片培养成为可能。我们将确定我们设计的用于体内癫痫发生定量的方法是否可以应用于这些培养阵列。其次，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动的数量。第三，可以使用哪些替代标记物来加速该系统的筛选使用？虽然从电记录中定量癫痫发生是金标准，但我们的初步数据表明，有潜在的有前途的标记，如细胞外乳酸水平和细胞内氯化物和钙水平，可以用作荧光分析的基础，这将大大加快该模型的吞吐量。总之，这些研究将使一种新的、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分析成为可能，这将加速对发病机制的基础研究和对治疗策略的探索。

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. PUBLIC HEALTH RELEVANCE: Organotypic brain slice cultures are a potentially powerful model of post traumatic epilepsy, but this preparation was never optimized for the study of epilepsy. We have the experience with both the culture and seizure quantification necessary to develop this preparation to the point that it can become a rapid, highly scalable, reproducible and transferrable, and quantifiable model of post traumatic epilepsy. Such a model will profoundly increase our capacity to seek new cures for epilepsy after brain injury.