神经球蛋白对急性青光眼所致小鼠视网膜神经节细胞后续凋亡的作用研究

As one of the leading causes of blindness, glaucoma affects 70 million people worldwide.Lowering intraocular pressure(IOP) could partly slow down the progression of glaucomatous damage; however, clinical evidence suggests that glaucoma patients vary greatly in their susceptibility to elevated IOP：part of glaucoma patients' visual field get worse after the IOP has been controlled to normal or even lower by surguries or drugs. The underlying mechanism is still unclear. Neuroglobin(Ngb) is reported to colocalize to the mitochondria and to reversibly bind oxygen,likely to supply oxygen to neurons. In 2011, The applicant found,for the first time in the world, Ngb can ameliorate the damaging effects of continuous ocular hypertension to Retinal Ganglion Cell（RGC）through decreasing oxidative stress and preserving the function of mitochondria. However, it is so far not been reported that whether Ngb has protective effect on the RGC loss after transient high IOP which is common in clinic，because with the advances of modern medical technologies, most of the glaucoma patients's IOP level could be controlled to normal or even lower. Our former study indicates that the level of Reactive Oxidative Species(ROS) in mice retina still continued increasing with the amount of RGC continued decreasing after 8 weeks from the onset of acute high IOP in mice, even the IOP has been decreased to normal. Therefore, it is reasonalbe to speculate that Ngb has protective effect on the RGC apoptosis caused by acute high IOP. To prove this hypothesis, Our experiment plans to: (1)Establish a novel,stable acute glaucoma mouse model;(2)At different timepoints after acute high IOP, compare the Ngb expression profile and RGC survival rate between wide type(WT)and Ngb high expressed transgene (Ngb-Tg)mice, to investigate the function of Ngb after elevated IOP;(3)Through measure the ROS and ATP level in retina, try to figure out the mechanism of Ngb's effcet to RGC. We plan to investigate for the first time application of mouse transgenic technology to investigate functional significance of Ngb overexpression in RGC in acute glaucoma and explore the Ngb potential mechanisms regulating RGC survival after stressed conditions. It may offer novel protective strategies to treat or prevent the degeneration of neurons and optic nerves in glaucoma patients.

相当一部分青光眼患者在眼压控制后视网膜神经节细胞（RGC）仍持续丢失，其机制尚不清楚。神经球蛋白（Ngb）具有高亲氧性，能促进氧向线粒体内转运。申请人首次发现在持续高眼压状态下Ngb通过清除活性氧成分（ROS）等机制能对小鼠RGC有保护作用。但Ngb对临床上眼压已降至正常,而RGC仍持续凋亡的患者是否有保护作用，迄今国内外尚无相关研究或报道。本项目在前期研究中,发现小鼠急性高眼压模型在8周后眼压虽早已降至正常，但视网膜中ROS含量持续增高,且RGC进行性丢失。是否Ngb对急性高眼压所致RGC后续凋亡具有保护作用? 为回答这一问题，本研究拟(1)复制小鼠急性高眼压模型；（2）比较野生型和Ngb高表达小鼠不同时间点视网膜中Ngb的表达及RGC存活率；(3)进一步在体内实验层面和分子层面探讨急性高眼压所致RGC后续凋亡的原因以及Ngb对RGC的保护机制，为青光眼视神经保护治疗探索一个新方向。

青光眼是世界范围内第二大致盲性眼病，目前现代化的降眼压治疗手段即使能够将大部分青光眼患者眼压降到正常甚至更低，但相当一部分患者的视网膜神经节细胞(Retinal Ganglion Cell, RGC)仍持续凋亡、视野进行性丢失。因此，视神经保护的引入更加具有重要的临床意义。本项目拟依据前期研究结果（持续高眼压状态下神经球蛋白（Neuroglobin， Ngb）通过清除活性氧成分等机制能对小鼠RGC有保护作用）的基础上进一步验证: Ngb是否对急性青光眼发作后所致RGC持续凋亡仍具有保护作用，并探寻其机制及预测其在临床应用的可能性。主要研究内容及结果如下：.1. 已按计划成功构建小鼠急性高眼压模型.采用前房注入聚苯乙烯微球的方法，已成功构建急性高眼压小鼠模型；通过对微球直径及注射量等参数的调节，确定了诱导急性高眼压状态稳定的条件为前房注入15μm直径的微球2μL，这样可诱导小鼠眼压在术后第三天逐渐上升，最高峰值可达30mmHg，持续至2周可降至正常。.2. 已按计划验证 β-Ⅲ-tubulin(Tuj-1)免疫荧光标记物的准确性.采用Tuj-1免疫组化标染和经典的荧光金逆向标染法分别对对照组及高眼压组小鼠进行RGC标染，在视网膜铺片及切片上对标染阳性细胞计数并做统计分析，结果证实两种标染法间无统计学差异，因此Tuj-1可以作为一种可靠的标记物标染RGC。由于其较荧光金标染更为简便、经济，为以后的实验奠定了基础。.3. 已按计划验证Ngb对急性高眼压后RGC持续损伤的保护作用.小鼠在进行15μm微球或PBS注射后于不同时间点处死，测量急性高眼压所致Ngb-Tg及WT小鼠RGC持续丢失率，发现Ngb-Tg小鼠各时间点RGC丢失率小于WT小鼠，结果有统计学差异，这证实了Ngb对急性青光眼所致RGC的持续损伤仍具有保护作用。.4. 已按计划对Ngb神经保护作用的机制进行探讨.在诱导WT及Ngb-Tg小鼠急性高眼压后不同时间点处死以检测视网膜中过氧化物及ATP水平。结果发现急性高眼压后，WT小鼠组较Ngb-Tg小鼠组视网膜中过氧化物为高，而ATP水平为低，这提示Ngb的神经保护作用可能是通过清除过氧化物这一通道得以实现。.本项目的研究内容均顺利完成，下一步拟研究Ngb蛋白制剂能否直接注入玻璃体腔内、在眼内的药代动力学及靶器官毒性等，希望有机会推进其药物化可能。