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中风的80%，并对公众健康产生重大影响。了解病理生理学是必要的发展治疗途径，以尽量减少脑损伤。因此，该项目的目标是确定星形胶质细胞在小鼠缺血诱导的神经元死亡和脑损伤模型中的新作用。我们的中心假设是星形胶质细胞通过增强Ca2+依赖性谷氨酸释放（胶质传递）诱导神经元兴奋毒性反应，从而导致缺血诱导的神经元死亡和脑损伤。各种最先进的技术，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验证这一假设。我们有三个特定的假设：1)局灶性缺血诱导缺血核心和半暗带星形胶质细胞Ca2+兴奋性增强，并介导这些胶质细胞的谷氨酸释放。利用2-P体内Ca2+成像，我们将研究缺血性区域星形细胞Ca2+信号的时空动态，并表征Ca2+振荡的特性。利用药物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，我们将确定光血栓形成后星形胶质细胞Ca2+兴奋性的分子基础和特性。2)星形胶质细胞在缺血后的Ca2+高兴奋性期间刺激n -甲基- d -天冬氨酸受体（NMDARs）介导的神经元兴奋。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。具体来说，我们将确定星形胶质细胞在缺血后是否刺激含有NR2B-的NMDAR （NR2B-NMDAR）介导的神经元兴奋。3)星形胶质细胞通过Ca2+依赖性胶质传递加剧缺血诱导的延迟性神经元死亡和脑损伤。利用免疫组织化学和神经元死亡实验，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NR2B-NMDARs是否参与胶质传递介导的神经元死亡。尽管有许多研究表明星形胶质细胞在缺血性脑损伤中的潜在作用，但由于缺乏对这类胶质细胞生物学特性的了解，以及体内星形胶质细胞特异性操作的缺乏，阻碍了我们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Ca2+信号的改变通过胶质传递诱导延迟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.