The Role of Gliotransmission in Cerebral Ischemia

描述（由申请人提供）：作为一种主要的神经系统疾病，急性脑缺血约占所有人类卒中的80%，对公众健康有重大影响。了解病理生理学对于开发治疗途径以最大限度地减少脑损伤至关重要。因此，该项目的目标是确定星形胶质细胞在缺血诱导的神经元死亡和脑损伤的小鼠模型中的新作用。我们的中心假设是，星形胶质细胞诱导神经元兴奋性毒性反应，通过增强钙依赖性谷氨酸释放（gliotransmission），从而有助于缺血诱导的神经元死亡和脑损伤。各种国家的最先进的技术，包括2-P显微镜，电生理学，病毒转导和转基因小鼠将被用来测试这一假设。我们有三个具体的假设：1）局灶性缺血诱导增强的钙离子兴奋性的星形胶质细胞在缺血的核心，以及在半影区和介导谷氨酸从这些胶质细胞的释放。使用2-P在体内Ca 2+成像，我们将研究缺血区域星形胶质细胞Ca 2+信号传导的时空动力学，并表征Ca 2+振荡的性质。使用药理学干预以及星形胶质细胞特异性分子遗传学方法，包括病毒转导和转基因小鼠，我们将确定分子基础和星形胶质细胞Ca 2+兴奋性的性质，以下光血栓形成。2)星形胶质细胞刺激N-甲基-D-天冬氨酸受体（NMDARs）介导的神经元兴奋期间，其Ca 2+的超兴奋性缺血。利用2-P显微镜和电生理学，我们将确定胶质传递对缺血后神经元兴奋的影响。具体而言，我们将确定星形胶质细胞是否刺激缺血后含NR 2B的NMDAR（NR 2B-NMDAR）介导的神经元兴奋。3)星形胶质细胞通过Ca 2+依赖性胶质传递加重缺血诱导的迟发性神经元死亡和脑损伤利用免疫组化和神经元死亡检测，我们将确定胶质传递在介导神经元死亡和脑损伤中的作用。此外，我们将测试NR 2B-NMDAR是否参与胶质传递介导的神经元死亡。虽然有许多研究表明，星形胶质细胞在脑损伤后缺血性损伤的潜在作用，这种类型的神经胶质细胞的生物学特性的知识缺乏，在体内的星形胶质细胞特异性的操作虚拟的情况下，阻碍了我们的进展，在了解其在发病机制中的作用。通过研究星形胶质细胞内和星形胶质细胞之间Ca 2+信号转导的改变通过胶质传递诱导迟发性神经元死亡的新假设，我们的研究将为星形胶质细胞在调节神经元兴奋性和兴奋性毒性中的生理和病理作用提供全新的见解。该项目的结果将推动神经胶质生物学领域的发展，并提供可能改善缺血后神经元死亡和脑损伤的治疗途径和靶点。

DESCRIPTION (provided by applicant): As a leading neurological disorder, acute cerebral ischemia accounts for approximately 80% of all human strokes and has a major impact on public health. Understanding the pathophysiology is essential to develop therapeutic avenues to minimize brain damage. Thus, the project goal is to determine the novel role of astrocytes in a mouse model of ischemia-induced neuronal death and brain damage. Our central hypothesis is that astrocytes induce neuronal excitotoxic responses through enhanced Ca2+-dependent glutamate release (gliotransmission) and consequently contribute to ischemia-induced neuronal death and brain damage. A variety of state-of-the-art technologies including 2-P microscopy, electrophysiology, viral transduction and transgenic mice will be used to test this hypothesis. We have three SPECIFIC hypotheses: 1) Focal ischemia induces enhanced Ca2+ excitability in astrocytes in the ischemic core as well as in the penumbra and mediates glutamate release from these glial cells. Using 2-P in vivo Ca2+ imaging we will study the spatial and temporal dynamics of astrocytic Ca2+ signaling in the ischemic region and characterize the properties of Ca2+ oscillations. Using pharmacological interventions as well as astrocyte-specific molecular genetic approaches including viral transduction and transgenic mice, we will identify the molecular basis and the properties of astrocytic Ca2+ excitability that follows photothrombosis. 2) Astrocytes stimulate N-methyl-D-aspartate receptors (NMDARs)-mediated neuronal excitation during the period of their Ca2+ hyperexcitability following ischemia. Using 2-P microscopy and electrophysiology, we will determine the effects of gliotransmission on neuronal excitation following ischemia. Specifically, we will determine whether astrocytes stimulate the NR2B- containing NMDAR (NR2B-NMDAR)-mediated neuronal excitation after ischemia. 3) Astrocytes exacerbate ischemia-induced delayed neuronal death and brain damage through Ca2+-dependent gliotransmission. Using immunohistochemistry and a neuronal death assay, we will determine the role of gliotransmission in mediating neuronal death and brain damage. Furthermore we will test whether NR2B-NMDARs are involved in gliotransmission-mediated neuronal death. Although there are many studies suggesting the potential role of astrocytes in brain damage following ischemic injury, the lack of knowledge of biological properties of this type of glial cell together with the virtual absence of in vivo astrocyte-specific manipulations has hampered our progress in understanding their role in pathogenesis. By examining the novel hypothesis that alterations in Ca2+ signaling within and among astrocytes induce delayed neuronal death through gliotransmission, our study will provide entirely new insights into the physiological and pathological role of astrocytes in regulating neuronal excitability and excitotoxicity. Results from this project will advance the field of glial biology and provide therapeutic avenues and targets that could potentially ameliorate neuronal death and brain damage following ischemia.