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

项目摘要 越来越多的动物模型证据表明，癫痫持续状态期间大脑中的炎症反应 (SE)可能在持续的癫痫发作及其长期有害后果中发挥决定性作用， 感染或自身免疫的原因在大量动物模型中的研究表明，SE引起快速的 以及前脑中涉及神经元，反应性星形胶质细胞， 活化的小胶质细胞、血管内皮细胞，以及最终从血管壁浸润的中性粒细胞和单核细胞。 血各种炎症分子之间的病理生理学相互作用，以及事件的顺序 导致他们的感应，还没有被解剖。SE后广泛的细胞因子爆发和神经胶质增生减弱 在对考克斯-2进行基因消融的小鼠中，局限于其中考克斯-2被 这表明考克斯-2通路在SE诱导的炎症中的作用，包括 血脑屏障（BBB），这足以产生癫痫。先前的研究表明，EP 2受体 介导了大部分的考克斯-2效应。我们假设SE相关的发病率很大程度上是由于 小胶质细胞EP 2受体，其调节降解BBB的细胞因子的产生。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负责EP 2 - 1的假设， 癫痫发作后调节的BBB破坏; 2.为了验证白细胞介素分泌的假设， 介质和Epac是SE后降低血脑屏障完整性的主要EP 2信号通路; 3.确定EP 2激活是否在癫痫或其合并症的发展中起主导作用 在SE之后为了实现这些目标，我们采用了BBB的体外培养模型和具有细胞的体内SE模型， EP 2的特异性条件性敲除。免疫组织化学、western blot、FACS、qRT-PCR、EEG和行为分析 进行测定。

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.