Evaluating astrocyte loss after traumatic brain injury in initiation of post-traumatic epilepsy

项目摘要 经过多年的假设，神经系统疾病是由直接损害神经元，我们现在知道， 受损的星形胶质细胞生理学和功能先于许多这些疾病的进展，并且对于这些疾病的进展至关重要。 疾病这一发现暗示了为什么专门针对神经元的抗癫痫药物 不能防止创伤性脑损伤（TBI）后癫痫的发展，这是最大的一组后天性癫痫。 癫痫十多年来，积累的数据显示星形胶质细胞变得反应性和失去活性， 它们的稳态功能是癫痫患者和动物正常神经元运作所必需的 模型然而，星形胶质细胞功能障碍和创伤后癫痫（PTE）之间的直接因果关系尚未得到证实。 脑外伤会引发星形胶质细胞增生这可能部分是由于TBI的复杂性， 诱发了许多并行的病理生物学机制星形胶质细胞增生主要在局灶性TBI中进行研究， 不同类型的反应性星形胶质细胞层围绕原发性脑损伤部位。然而，这种损伤类型 在不到10%的TBI患者中单独存在，并诱导了可能触发TBI的其他机制。 癫痫发作，限制了我们确定星形胶质细胞功能障碍与癫痫发作之间是否存在因果关系的能力。 存在PTE的发展。目前的PTE模型是由局灶性TBI引起的，但绝大多数人TBI 包括由脑组织的快速加速/减速引起的弥漫性或震荡性损伤。甚至 发生单一轻度弥漫性TBI的患者发生PTE的风险增加。一种新 PTE小鼠模型，重现弥漫性TBI无局灶性损伤。新的PTE模型 诱导自发性癫痫发作的发生率高于以前的PTE模型，但只有一个子集的细胞 和组织水平的变化，显著降低了基础病理学的复杂性。在此期间获得的数据 模型指出星形胶质细胞对弥漫性TBI的反应令人惊讶地不同，这表明星形胶质细胞的早期丧失可能是一个重要因素。 星形胶质细胞可能参与PTE的发生。然而，诱导星形胶质细胞的上游分子机制 必须确定神经元和邻近星形胶质细胞的损失和下游生理后果 最终找到阻断TBI向PTE进展的靶点。本建议旨在确定 星形胶质细胞损失的主要原因，使用改良的Folch提取和分级技术，以缩小 候选人名单。它进一步验证了星形胶质细胞的损失导致神经元和附近的星形胶质细胞 功能失调引发癫痫病灶的形成这个假设将使用一个 在PTE小鼠中或在细胞特异性消融后的成像、电生理和EEG记录的组合 玩家鉴于TBI的发病率在过去十年中有所增加，PTE作为TBI的终身并发症， TBI不仅使那些受影响的人衰弱，而且代表了世界上不断增加的社会和经济负担。 我们该提案将研究星形胶质细胞损失作为TBI后引发癫痫发生的根本原因，并将 为制定预防TBI向PTE进展的干预措施提供基础。

Project Summary After years of assuming that neurological diseases are caused by direct damage to neurons, we now know that impaired astrocyte physiology and function precedes and is essential for the progression of many of these diseases. This revelation hints toward the reason why anti-epileptic drugs that exclusively target neurons do not prevent the development of epilepsy after traumatic brain injury (TBI), the largest group of acquired epilepsies. For more than a decade, data have accumulated showing that astrocytes become reactive and lose their homeostatic functions indispensable for normal neuronal operation in epilepsy patients and animal models. Yet, a direct causal link between astrocyte dysfunction and post-traumatic epilepsy (PTE) has not been established beyond the fact TBI triggers astrogliosis. This may be in part due to the complexity of TBI, which induces many pathobiological mechanisms in parallel. Astrogliosis has mostly been studied in focal TBI, where layers of different types of reactive astrocytes surround a site of primary brain damage. Yet, this injury type presents in isolation in less than 10% of TBI patients and induces additional mechanisms that could trigger seizures, limiting our ability to determine if a causal relationship between astrocyte dysfunction and the development of PTE exists. Current PTE models are induced by focal TBI, but the vast majority of human TBIs include diffuse or concussive injury induced by rapid acceleration/deceleration of the brain tissue. Even patients who incur a single mild diffuse TBI are at increased risk for the development of PTE. Therefore, a new PTE mouse model that recapitulated diffuse TBI without focal injury was developed. This new PTE model induced spontaneous seizures at higher incidence than previous PTE models but with only a subset of cellular and tissue level changes, markedly reducing complexity of the underlying pathobiology. Data obtained in this model point to a surprisingly different response of astrocytes to diffuse TBI, suggesting that early loss of astrocytes may contribute to the development PTE. Yet, the upstream molecular mechanism inducing astrocyte loss and the downstream physiological consequences on neurons and neighboring astrocytes must be identified to ultimately find targets for interrupting the progression of TBI to PTE. This proposal aims to determine the primary cause for astrocyte loss using modified Folch extraction and fractionation techniques to narrow down the list of candidates. It further tests the hypothesis that astrocyte loss causes neurons and close-by astrocytes to become dysfunctional, initiating the formation of a seizure focus. This hypothesis will be tested using a combination of imaging, electrophysiology and EEG recordings in PTE mice or after specific ablation of cellular players. Given that the incidence of TBI has increased over the last decade, PTE as a lifelong complication of TBI is not only debilitating for those afflicted, but represents an ever-rising social and economic burden in the US. This proposal will examine astrocyte loss as a root cause initiating epileptogenesis after TBI, and will provide a basis for developing interventions that prevent the progression of TBI toward PTE.