Alpha7-烟碱型胆碱能受体（α7-nAChRs）在青光眼视网膜神经保护中的作用及机制研究

The neuroprotection mechanism of a7-nicotinic acetylcholine receptors (a7-nAChRs) in glaucoma retina has received increasing attention. Recent studies have determined that the activation of a7-nAChRs could protect cultured RGCs from glutamate-induced excitotoxicity by stimulating anti-apoptotic pathways. However, the role and synaptic mechanism linking nAChRs to neuroprotection in glaucoma retina is unknown. Our previous work showed that activating a7-nAChRs could increase the release of inhibitory GABA and glycine in RGCs, but have no effect in releasing the glutamate. Base on the previous finding, this study will examine the changes in α7-nAChRs protein expression in chronic ocular hypertension rats model and investigate the regulating mechanism of activating and inhibiting α7-nAChRs in the synaptic transmistter of the inner retina as well as RGCs’ biology membrane characteristics, K+ currents and the reactivity of NMDA/AMPA receptors in both normal and glaucoma rats, using whole-cell patch clamp electrophysiology, pharmacological methods, immunohistochemistry and western blot. Through in vivo and ex vivo experiments, this research has great significance in demonstrating the retinal neuroprotection mechanism of a7-nAChRs at the synaptic level, providing theoretical foundation for the clinical experiments of the optic nerve protectant.

Alpha7-烟碱型胆碱能受体（α7-nAChRs）对青光眼视网膜的神经保护作用受到关注。资料表明：在培养的视网膜神经节细胞(RGCs)，α7-nAChRs通过激活抗凋亡通路对抗谷氨酸兴奋毒性。但α7-nAChRs在青光眼视网膜中的神经保护作用及突触调节机制不明。我们前期工作证明激活RGCs中α7-nAChRs，可增加自发性GABA和甘氨酸的释放，对谷氨酸的释放无效。在此基础上，本项目拟采用全细胞膜片钳电生理、药理学、免疫组织化学和western blot等方法，研究慢性高眼压大鼠视网膜中α7-nAChRs表达量的变化；研究激活和抑制α7-nAChRs对正常和病变大鼠内层视网膜突触传递以及RGCs上的生物膜特性、K+电流和NMDA/AMPA受体反应性的调控机制。通过在体和离体实验，本项目对阐明α7-nAChRs在突触水平的视网膜神经保护机制有重要意义，为视神经保护剂的临床实验提供理论依据。

过度激活谷氨酸受体诱发的兴奋毒性会导致青光眼疾病的发生。细胞的死亡/存活依赖于兴奋性和抑制性通路的平衡。Alpha7烟碱受体（a7-nAChR）激动剂能够阻断培养的视网膜节细胞（RGC）中的谷氨酸诱发的兴奋毒性。然而，在大鼠慢性青光眼模型中a7-nAChR激动剂PNU-282987的神经保护作用和突触机制不明。我们发现高眼压（IOP）可以下调视网膜a7-nAChR的表达。IOP后，视网膜电图在体评估明视负波反应PhNR的幅度下降与RGCs的丢失呈正相关。PNU-282987有效增加RGCs存活和功能，减少RGCs中TUNEL阳性细胞数目。在大鼠视网膜切片的RGCs中用全细胞膜片钳记录到微小型GABA能抑制性突触后电流（mIPSCs），mIPSCs的基础频率和延时时间在对照和病理的RGCs中有显著差异。用Western blotting和免疫荧光法发现GABA合成酶（谷氨酸脱羧酶65/67，GAD65/67）和GABA在青光眼视网膜中表达缺失，这种缺失可以被PNU-282987逆转。PNU-282987增加mIPSCs的频率和幅度。在突触后水平，PNU-282987增强GABA诱发的受体电流的幅度。然而，微小型谷氨酸能兴奋性突触后电流（mEPSCs）的频率和幅度在对照和病理RGCs中无差异。PNU-282987不影响mEPSCs的频率和幅度。但PNU-282987可减少突触后NMDA诱发的受体电流的幅度。此外高眼压可以导致GABAA受体的a1亚基表达下调，NMDA受体的NR2B亚基表达上调，这些变化均可被PNU-282987逆转。PNU-282987所介导的保护作用可以被玻璃体腔注射GABAA受体拮抗剂阻断。在慢性高眼压模型中，激活a7-nAChR可以正向调控GABA能，负向调节谷氨酸能系统。从而使PNU-282987导致视网膜节细胞环路发生去兴奋作用，减少其兴奋毒性。

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