BRAIN DEVELOPMENT IN EXPERIMENTAL EPILEPSY

这个项目的长期目标是了解癫痫发作和大脑发育之间的关系。在这个项目的最后阶段，我们确定了某些类型的癫痫发作，在没有全身并发症的情况下，可以损害未成熟的大脑。我们也有初步证据表明，这种损害反过来又会导致癫痫。这项应用有两个目标：第一，确定癫痫诱导的未成熟脑神经元损伤是否会导致神经元凋亡，并了解其机制。第二，找出癫痫导致的未成熟大脑损伤是否会导致癫痫。我们将在未成熟大鼠癫痫持续状态模型中研究神经元死亡的机制。我们将仔细监测生理变量，如动脉血压或血气，以确定该模型中的神经元损伤是癫痫本身的结果。我们将确定神经元损伤的时间进程，用电子显微镜和共聚焦荧光显微镜描述其在CA1锥体、内齿状颗粒细胞和齿状脑门大神经元中的形态；我们将确定神经元的死亡是否依赖于蛋白质的合成；我们将通过TUNEL法寻找限制性内切酶早期破坏DNA的证据，以确定单个细胞中的双链DNA断裂；通过琼脂糖凝胶电泳的梯形条带；我们将通过免疫细胞化学和Western blots寻找caspase激活的证据，使用从肌动蛋白和聚ADP-核糖聚合酶中切割的蛋白分解片段的抗体，通过使用caspase的抑制剂，通过原位杂交和Northern blots检测单个caspase的表达增加。这一现象的时间进程将揭示导致特定细胞凋亡的生化级联。我们还将使用特定的抑制剂来研究大多数神经元的坏死性和凋亡性死亡依赖于M胆碱能受体、NMDA受体和一氧化氮合酶的活性。其次，我们将通过监测处于癫痫持续状态的慢性动物在P15、P21、P28或成年时的癫痫发作，来确定癫痫诱导的未成熟脑损伤是否致痫。我们将在癫痫持续状态结束后长期应用抗惊厥药物，并研究其对自发性癫痫发作发展的影响。

The long-term objective of this project is to understand the relationship between seizures and brain development. During the last period of this project, we established that some types of seizures, in the absence of systemic complications, can damage the immature brain. We also have preliminary evidence that this damage is, in turn, epileptogenic. This application has two goals: first, to determine whether seizure-induced neuronal injury in the immature brain leads to neuronal apoptosis, and to understand its mechanism. Second, to find out whether seizure-induced damage in the immature brain is epileptogenic. We will study the mechanism of neuronal death in the lithum-pilocarpine model of status epilepticus in the immature rat. We will carefully monitor physiological variables such as arterial blood pressure or blood gases, to ascertain that neuronal injury in the that model is the result of the seizures themselves. We will determine the time course of neuronal injury, describe its morphology by electron-microscopy and confocal fluorescesence microscopy in the CA1 pyramids, the inner dentate granule cells, and the large neurons of the dentate hilus; we will determine whether neuronal death is dependent upon protein synthesis; we will look for evidence of early DNA breakdown by restricted endonucleases by the TUNEL method for identify double- stranded DNA breaks in individual cells; and by laddering on agarose gel electrophoresis; we will look for evidence of caspase activation by immunocytochemistry and in Western blots using antibodies against proteolytic fragments cleaved from actin and from poly ADP-ribose polymerase, by using inhibitors of caspases, by using in situ hybridization and Northern blots to detect increased expression of individual caspases. The time course of this phenomena will reveal the biochemical cascade leading to apoptosis in specific cells. We will also use specific inhibitors to study the dependency of most necrotic and apoptotic death of neurons upon the activation of muscarinic cholinergic receptors, of NMDA receptors, and upon the activity of nitric oxide synthase. Second, we will determine whether seizure induced damage in the immature brain is epileptogenic by monitoring seizures in chronic animals subjected to status epilepticus at P15, P21, P28, or as adults. We will chronically administer anticonvulsants after the end of status epilepticus, and study their effects on the development of spontaneous seizures.