代谢型谷氨酸受体5对大鼠脑缺血损伤后神经突起生长的作用和机制研究

Metabotropic glutamate receptors (mGluRs) are broadly distributed in brain and play some important roles. However, little is known about their role in regulating neuroplasticity, such as the growth neurite and the formation of synapse. Based on our previous work that showed mGluR 5 played crucial roles in neural regenreation after ischemic brain injury via stimulating prolifeartion and differentiation of neural stem cells, here we are aiming to investigate the effect and the mechanism of mGluR 5 in reestablishment of neural structure via enhancing the growth of neurite and the formation of synapse. Hypoxia cell model and animal model (middle cerebral artery occlusion) are going to be used in the present study. Expression and function of mGluR 5 are altered either by retroviral-mediated gene transfer or by using specific agonists/antagonists of mGluR 5. Then, following observations will be undertook: 1) In vivo observation of funcitonal recovery from ismechmia by using behavioral observation and functional magnetic resonance imaging (fMRI); 2) In vivo and in vitro observation of neuronal structure (including axons and dendrites) and function by dendritic analysis and axonal tracing to analyze the effect of mGluR 5 in structural reestablishment of brian after ischemia; 3) In vivo and in vitro observation of memebrane ion channel, particularly calcium channel, and sigmaling pathways, including MAPK/PI3K, CREB and NF-κB,to explore the mechanism of mGluR 5. This work will provide some new evidences to show that mGluR 5 is also critically needed for the structural and functional recoverry after brain injury. It also suggests that mGluRs promote neural recovery via enhancing both neuronal regeneration and structural/functional reestablishment. The greater understanding of the effect of mGluRs on neuroplasticity may lead to devising better therapeutic strategies for the treatment of ischemic brain injury.

代谢型谷氨酸受体广泛存在于脑内并发挥多种神经功能，但其对神经元结构的调节作用鲜有研究。本项目拟在前期工作基础上对代谢型谷氨酸受体5（mGluR5）促进脑缺血损伤后神经突起生长作用和机制进行研究。分别在神经细胞缺氧和大鼠MCAO模型上，利用基因调控技术或受体激动剂/拮抗剂改变mGluR5的表达或效应，进而采用形态学、分子生物学、神经示踪、动物行为学及功能影像学等方法，观察mGluR5对脑缺血损伤后神经功能恢复的影响；离体和在体条件下观察神经元轴、树突形态及功能变化，分析 mGluR5对脑缺血损伤后脑结构重塑的影响；观察神经元胞体以及突起部位细胞膜钙离子通道、胞内MAPK/PI3K信号及核内CREB、NF-κB信号变化，探索mGluR5调节脑缺血后神经突起生长机制；阐明代谢型谷氨酸受体对中枢神经系统突触重建、神经结构重塑的调节作用，为脑损伤后内源性修复治疗研究提供重要理论和实验依据。

代谢型谷氨酸受体5亚型（mGluR5）广泛参与神经元的存活、分化、突起生长，突触可塑性和神经回路的形成。然而，有关它在神经元极化和轴突生长中的作用知之甚少。在这项研究中，我们应用mGluR5选择性激动剂CHPG和拮抗剂MTEP处理培养的海马神经元，观察神经元的极化和轴突生长，并进一步探索可能的细胞内信号转导通路。结果表明，通过smi312（轴突标记物）和Tuj-1表达（所有突起的标记物）免疫荧光双标记方法，发现MTEP处理能显著减少极化神经元的比例和长度。Western blot分析显示MTEP也可抑制Akt的活化和核转录因子（NF-κB），以及p65的磷酸化水平降低。此外，Akt抑制剂LY294002处理导致神经元极化延迟和轴突生长迟缓，同时抑制磷酸化p65的核转位。我们的结论是，mGluR5可能在大鼠神经元的发育形态分化过程中调节神经元和轴突生长的极性，其机制可能与细胞内信号转导通路Akt–NF-κB有关。