Inflammatory control of blood-brain barrier integrity and epileptogenesis after seizures

项目摘要 在动物模型中积累的证据表明，癫痫持续状态期间大脑中接踵而至的炎症 (Se)可能在持续的缉获及其长期有害后果中发挥决定性作用，独立 感染或自身免疫的原因。对多种动物模型的研究表明，SE导致快速的 前脑中强烈的炎性级联反应涉及神经元、反应性星形胶质细胞、 激活的小胶质细胞，血管内皮细胞，并最终渗透到中性粒细胞和单核细胞从 血。不同炎症分子之间的病理生理相互作用，以及事件的顺序 导致它们的归纳，还没有被解剖。SE后广泛的细胞因子爆发和胶质细胞增生症被钝化 在COX-2基因去除仅限于COX-2所在的主要前脑神经元的小鼠中 通常由SE诱导，表明COX-2通路在SE诱导的炎症中起作用，包括 血脑屏障(BBB)，足以引起癫痫。先前的研究表明，EP2受体 介导了COX-2的大部分效应。我们假设与SE相关的发病率很大程度上是由于 小胶质细胞EP2受体，它调节细胞因子的产生，从而降解血脑屏障。我们的具体目标是：1. 为了验证激活的小胶质细胞而不是炎性单核细胞对EP2负责的假设- 调节癫痫发作后血脑屏障的分解；2.检验IL-2分泌的假说 介体和EPAC是SE后降低血脑屏障完整性的主要EP2信号通路； 3.确定EP2的激活在癫痫的发生发展中是否起主导作用，还是在癫痫的并发症中起主导作用 在SE之后。为了达到这些目的，我们采用了血脑屏障的体外培养模型和体内SE模型。 EP2的特定条件基因敲除。免疫组织化学、免疫印迹、流式细胞仪、定量逆转录聚合酶链式反应、脑电和行为学 进行化验。

Project Summary Accumulating evidence in animal models highlights that inflammation ensuing in the brain during status epilepticus (SE) may play a determinant role in ongoing seizures and their long-term detrimental consequences, independent of an infection or auto-immune cause. Studies in a multitude of animal models demonstrate that SE causes a rapid and intense inflammatory cascade in the forebrain involving interactions among neurons, reactive astrocytes, activated microglia, vascular endothelial cells and, eventually, infiltrating neutrophils and monocytes from the blood. The pathophysiological interactions among the various inflammatory molecules, and the sequence of events leading to their induction, have not yet been dissected. The broad cytokine burst and gliosis following SE is blunted in mice that have genetic ablations of COX-2 restricted to those principal forebrain neurons in which COX-2 is normally induced by SE, pointing to a role for COX-2 pathways in SE-induced inflammation including breakdown of the blood-brain barrier (BBB), which is sufficient to produce epilepsy. Previous work showed that the EP2 receptor mediates much of the COX-2 effect. We hypothesize that SE-related morbidity is largely due to activation of microglial EP2 receptors, which modulates production of cytokines that degrade the BBB. Our specific aims are: 1. To test the hypothesis that activated microglia rather than inflammatory monocytes are responsible for EP2- regulated BBB breakdown after seizures; 2. To test the hypothesis that IL- -secreted mediator and Epac the major EP2 signaling pathway that degrades the integrity of the blood-brain barrier after SE; 3. To determine whether EP2 activation plays a dominant role in the development of epilepsy or its comorbidities after SE. To address these aims we employ in vitro culture models of the BBB and in vivo SE models with cell- specific conditional knockouts of EP2. Immunohistochemical, western blot, FACS, qRT-PCR, EEG and behavioral assays are performed.