Epileptogenic Changes in Local Network Structure Following Injury (Project 2)

严重创伤性脑损伤(TBI)导致创伤性癫痫(PTE)，伴随着NO潜伏期 在大约20%的平民人口中，有约20%的人被扣押。影响一个人能否发展的因素 颅脑损伤后的癫痫包括损伤的严重程度，患者的人口统计，以及大范围的特征不佳的 潜伏期内发生的细胞和分子过程。发展有效的干预措施 预防PTE的治疗将关键取决于识别最早的分子途径，这些分子途径是特定的 会导致癫痫的。在生物物理学相关模型中获取这样的数据在技术上是具有挑战性的。 啮齿动物脑损伤后的神经炎症过程已被广泛研究，但临床可转译 调查结果一直不佳。对人类和大型轮脑动物的研究主要局限于大型- 标尺脑电记录及体外生理和组织学分析。四个发现从以下几个方面突出 这些研究是癫痫脑的特征：1)损伤部位的胶质细胞增生，2)GABA介导的突触活动 这是兴奋性的，而不是抑制性的，3)非发作性超同步电活动的流行，以及4) 宏观(全脑)层面的功能网络连接中断。几行出版的和 初步数据表明，脑外伤导致的胶质组织产生一种细胞外基质，稳定地降低 胞外氯化物。这反过来又使GABA反转电位(EGABA)去极化。 我们假设，由此产生的去抑制将增加神经元活动的同步性，从 损伤部位，也就是形成胶质疤痕的地方。我们最近开发了一个新皮质后皮质的猪模型- 创伤性癫痫和成像工具(荧光团、大型动物双光子显微镜和辅助设备 技术)以单神经元精度和多模式(氯化物)纵向研究癫痫灶 和钙)荧光数据。在这个项目中，我们将同时对细胞内或细胞外的氯化物和 损伤前、潜伏期和自发性癫痫发作出现后的钙活性。我们 也会在化学上改变细胞外基质(进而改变细胞外的氯化物)来测试原因 胞外氯化物和功能连接之间的联系。我们将使用这些数据来评估 EGABA去极化改变导致功能性网络连通性癫痫改变的假说 这些变化与临床可测量的癫痫表型相关。 这一丰富的数据集将为以下抗PTE治疗的发展提供信息。如果细胞外液的减少 氯化物与增加神经元活动和网络连接有关，然后是改变 胶质细胞外基质(如基质金属蛋白酶)的形成可能会阻止神经元的同步化 潜伏期内，防止PTE本身。如果细胞内氯化物的增加与 增加神经元活性和功能连接，然后是细胞内蛋白酶抑制剂或转运 抑制剂可能更有效。如果神经元活动和网络连接与氯化物无关 变化，但预测PTE，那么可能有必要专注于开发直接 操纵受损神经元的神经元活动和功能网络连接。

Severe traumatic brain injury (TBI) results in post-traumatic epilepsy (PTE), following a latent period of no seizures, in approximately 20% of the civilian population. The factors that influence whether a person develops epilepsy following TBI include severity of injury, patient demographic, and a wide swath of poorly characterized cellular and molecular processes that take place during the latent period. Developing effective interventional therapies to prevent PTE will depend critically on identifying the earliest molecular pathways that are specifically epileptogenic. Acquiring such data in biophysically relevant models has been technically challenging. Neuroinflammatory processes following TBI in rodents have been widely studied, but clinical translatability of findings has been poor. Studies in humans and large gyrencephalic animals have been largely confined to large- scale electrographic recordings and ex vivo physiological and histological analysis. Four findings stand out from these studies as hallmarks of an epileptic brain: 1) gliosis at the site of injury, 2) GABA-mediated synaptic activity that is excitatory rather than inhibitory, 3) a prevalence of non-ictal hypersynchronous electrical activity, and 4) disruption of functional network connectivity at the macro (whole-brain) level. Several lines of published and preliminary data suggest that gliotic tissue resulting from TBI produces an extracellular matrix that stably lowers extracellular chloride. This, in turn, depolarizes the GABA reversal potential (EGABA). We hypothesize that the resulting disinhibition will increase the synchronicity of neuronal activity, beginning at the site of injury, where the gliotic scar forms. We have recently developed a porcine model of neocortical post- traumatic epilepsy and the imaging tools (fluorophores, large-animal 2-photon microscope, and supporting technologies) to longitudinally study the epileptic focus with single-neuron precision and multimodal (chloride and calcium) fluorescence data. In this project, we will simultaneously image intra or extracellular chloride and calcium activity before injury, during the latent period, and after the emergence of spontaneous seizures. We will also chemically alter the extracellular matrix (which in turn alters extracellular chloride) to test for a causal link between extracellular chloride and functional connectivity. We will use these data to evaluate the hypothesis that depolarizing changes in EGABA produces epileptogenic changes in functional network connectivity and that these changes correlate with clinically measurable epileptic phenotypes. This rich dataset will inform development of anti-PTE treatments as follows. If a decrease in extracellular chloride is associated with increased neuronal activity and network connectivity, then treatments that alter the formation of gliotic extracellular matrix (e.g. matrix metalloproteinases) may prevent neuronal synchronization during the latent period and prevent PTE itself. If an increase in intracellular chloride is associated with increased neuronal activity and functional connectivity, then intracellular protease inhibitors or transport inhibitors may be more effective. If neuronal activity and network connectivity are uncorrelated with chloride changes, but predictive of PTE, then it may be necessary to focus on developing treatments that directly manipulate neuronal activity and functional network connectivity in injured neurons.