The Role of Gliotransmission in Cerebral Ischemia

描述(申请人提供)：作为一种主要的神经疾病，急性脑缺血约占人类所有中风的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是至关重要的。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，星形胶质细胞通过增强钙依赖的谷氨酸释放(神经胶质传递)来诱导神经元的兴奋性毒性反应，从而导致缺血诱导的神经元死亡和脑损伤。包括2-P显微镜、电生理学、病毒转导和转基因小鼠在内的各种最先进的技术将被用来检验这一假说。我们有三个具体的假设：1)局灶性脑缺血诱导缺血中心和半影区星形胶质细胞钙离子兴奋性增强，并介导这些胶质细胞释放谷氨酸。利用2-P活体钙离子成像，我们将研究缺血区星形胶质细胞钙信号的时空动态，并表征钙振荡的特性。利用药物干预和星形胶质细胞特有的分子遗传学方法，包括病毒转导和转基因小鼠，我们将确定光凝后星形胶质细胞钙离子兴奋性的分子基础和特性。2)星形胶质细胞在缺血后钙超兴奋期刺激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，我们将确定神经胶质传递对缺血后神经元兴奋的影响。具体地说，我们将确定星形胶质细胞是否在缺血后刺激含NR2B的NMDAR(NR2B-NMDAR)介导的神经元兴奋。3)星形胶质细胞通过钙依赖的胶质细胞传递加重缺血诱导的迟发性神经元死亡和脑损伤。利用免疫组织化学和神经元死亡分析，我们将确定神经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-NMDAR是否参与神经胶质传递介导的神经元死亡。虽然许多研究表明星形胶质细胞在脑缺血损伤后的脑损伤中可能起作用，但对这类胶质细胞生物学特性的缺乏以及体内星形胶质细胞特异性操作的缺乏阻碍了我们对其在发病机制中作用的理解。通过检验这一新的假说，即星形胶质细胞内和星形胶质细胞之间的钙信号变化通过胶质传递导致迟发性神经元死亡，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，并提供治疗途径和靶点，潜在地改善脑缺血后的神经元死亡和脑损伤。 公共卫生相关性：脑缺血是一种主要的神经疾病，对公共健康有重大影响。了解脑缺血导致脑损伤的细胞和分子机制对于提供潜在的治疗途径将损伤降至最低是至关重要的。这项拟议的项目将确定大脑中一种名为星形胶质细胞的非神经细胞在调节脑缺血后神经元死亡和脑损伤方面的新角色。该项目的结果将对缺血诱导的神经变性的原因得出全新的见解。

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. PUBLIC HEALTH RELEVANCE: Cerebral ischemia is a leading neurological disorder and has a major impact on public health. Understanding the cellular and molecular mechanism by which ischemia induces brain damage is essential for providing potential therapeutic avenues to minimize the damage. The proposed project will determine the novel role of a non-neuronal cell of the brain called astrocyte in regulating neuronal death and brain damage following ischemia. Results from this project will derive entirely new insights into the causes of ischemia-induced neurodegeneration.