自噬在青光眼发病机制中的研究进展

青光眼是一组以视网膜神经节细胞(RGCs)渐进性死亡及其轴突慢性退化为特征的神经退行性疾病,主要与病理性眼压升高有关。自噬,即细胞自我"消化",是一种细胞降解、回收机制。过度自噬或自噬受损均可能导致细胞功能障碍,甚至死亡。近年的研究表明,自噬与不同因素导致的青光眼小梁网细胞功能异常和RGCs凋亡及视神经退行性改变密切相...     

自噬与青光眼关系的研究进展

自噬是溶酶体降解或再循环利用细胞器、蛋白质等胞内物质成分的过程,在细胞内环境稳态中发挥重要作用.近年来的研究表明,自噬与众多眼病包括青光眼的发生和发展有着密切联系.自噬可能是导致小梁网细胞功能异常的重要因素之一,其对视网膜神经节细胞发挥保护作用还是促进其死亡,仍存在争议.目前与青光眼有关的自噬基因的研究主要是OPTN基因,其编码的蛋白质optineurin与正常眼压性青光眼的发生发展相关.通过调控自噬而保护视网膜神经节细胞免于损伤可能是青光眼视神经保护的一种新方法.     

Müller细胞对青光眼视网膜神经节细胞影响的研究进展

视网膜神经节细胞 (RGCs)的过度凋亡是青光眼病理改变的基础.Müller细胞作为视网膜的主要神经胶质细胞,对于维持神经元的完整性、代谢、内环境稳态以及信号转导等均具有重要的作用.随着对Müller细胞研究的逐渐深入,发现Müller细胞不仅参与了青光眼性RGCs的凋亡机制,而且还参与了RGCs的代偿性保护机制.那么Müller细胞是如何对RGCs起作用,它又是通过什么机制参与青光眼引起的RGCs凋亡以及代偿性保护作用呢?就这些问题的最新研究进展进行综述.     

线粒体动力学与视网膜神经节细胞

线粒体动力学是指细胞中的线粒体不断地分裂、融合、移动、运输和线粒体自噬等,这些动态的过程在调节线粒体的形态与功能中发挥关键作用,并对细胞的存活、代谢、功能等有重要影响.视网膜神经节细胞(RGCs)作为视网膜中一类特殊且重要的神经元,对线粒体的动力学改变特别敏感.有关常染色体显性遗传性视神经萎缩疾病的研究发现,控制线粒体融合的相关基因与RGCs功能密切相关.实验性青光眼模型提示,眼压升高引起RGCs的线粒体分裂增多,改变调节线粒体融合基因的表达,最终诱导RGCs的凋亡;线粒体在RGCs中的正常运输和分布对于RGCs轴突的功能至关重要.以上遗传性和实验性视神经病变的研究表明,线粒体动力学在调节RGCs的生存中发挥着核心作用,通过调控线粒体动力学来保护RGCs可能是一个非常有前景的治疗策略.本文将对线粒体动力学的主要内容和RGCs中的线粒体动力学进行阐述.     

自噬在青光眼和视网膜退行性病变中的研究进展

自噬是细胞的管家程序,是细胞维持内环境稳定的必需途径。主要通过移除错误折叠的蛋白和损伤的细胞器来实现目的。自噬途径涉及到人体多种疾病,包括肿瘤、神经退行性疾病以及传染性疾病。神经退行性疾病与许多眼部疾病的病理机制密切相关,比如青光眼以及年龄相关性黄斑病变。此外,眼内细胞持续暴露于各种应激因子中,可导致自噬的发生,自噬通过回收利用代谢前体细胞,或者促进受损细胞的死亡来提高整体存活率。自噬与凋亡也被证实在不同的实验环境中存在协同和拮抗作用,而这一切与许多疾病的病理机制息息相关。本文对自噬的机制及调控进行了回顾,并总结了较新的有关青光眼中视网膜神经元自噬的理论以及运用自噬的治疗手段。     

青光眼疾病中视网膜胶质细胞的作用

青光眼是一种视网膜视神经的退行性病变,以视网膜神经节细胞(RGCs)的凋亡为基本病理基础,但其凋亡的具体机制尚未完全阐明.目前,大量在体及体外研究发现,视网膜胶质细胞,如Müller胶质细胞、星形胶质细胞和小胶质细胞与青光眼的发生和发展,特别是RGCs的损伤密切相关.胶质细胞上存在众多神经递质受体、离子通道、表面标志物及效应分子,同时也可释放活性因子,因此胶质细胞除了传统认为的具有对神经元的支持营养作用外,还积极参与神经元之间的信息传递过程.在青光眼条件下,胶质细胞可被激活并产生许多生理、生化及形态学改变,一方面可释放神经保护因子,启动神经保护程序;另一方面在胶质细胞过度激活时也会影响其正常生理功能或释放有害因子,加重视网膜神经元的损伤.这2种作用在青光眼中常常并存,但目前这些作用的潜在机制和相关信号通路尚不十分清楚,因而青光眼神经元损伤后胶质细胞是如何重建或进一步破坏神经元功能需要进行更多的研究.本文对视网膜Müller细胞、星形胶质细胞及小胶质细胞的基本生理特征,以及在青光眼病理过程中各自的功能变化进行综述.     

Muller细胞对青光眼视网膜神经节细胞影响的研究进展

视网膜神经节细胞（RGCs）的过度凋亡是青光眼病理改变的基础。Mller细胞作为视网膜的主要神经胶质细胞,对于维持神经元的完整性、代谢、内环境稳态以及信号转导等均具有重要的作用。随着对Mller细胞研究的逐渐深入,发现Mller细胞不仅参与了青光眼性RGCs的凋亡机制,而且还参与了RGCs的代偿性保护机制。那么Mller细胞是如何对RGCs起作用,它又是通过什么机制参与青光眼引起的RGCs凋亡以及代偿性保护作用呢？就这些问题的最新研究进展进行综述。     

急性视网膜缺血再灌注损伤后大鼠视网膜副凋亡和自噬的发生

目的　探讨大鼠急性视网膜缺血再灌注损伤（retinal ischemia-reperfusion injury,RIRI）后不同时间点视网膜中副凋亡及自噬的发生。方法　将30只健康成年SD雄性大鼠随机分为RIRI组及正常对照组。利用高眼压前房灌注法建立SD大鼠急性RIRI动物模型。正常对照组不做任何处理。急性RIRI后1 d、3 d、7 d、28 d各时间点取各组大鼠的视网膜组织,利用透射电子显微镜（transmission electron microscopy,TEM）检测视网膜神经节细胞（retinal ganglion cells,RGCs）副凋亡及自噬的发生;利用免疫荧光染色法检测视网膜微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3,LC3）的表达。结果　急性RIRI后1 d持续至28 d,TEM可见RGCs细胞质空泡数量较正常对照组增高。急性RIRI后1 d持续至28 d,TEM可见RGCs细胞质中自噬体数量增加。正常对照组RGCs的细胞质中自噬体每50 μm²平均为0.79个,急性RIRI后7 d自噬体的平均数量达到高峰,每50 μm²平均为2.29个,与正常对照组相比差异具有统计学意义（P<0.05）。免疫荧光法检测发现,与正常对照组相比,急性RIRI后1 d开始,RGCs的细胞质中LC3表达增加,并在整个实验期间持续高表达,正常对照组RGCs层的LC3阳性细胞百分比为15.90%,急性RIRI后1 d、28 d时LC3阳性细胞百分比分别为46.95%和52.30%,与正常对照组相比差异均具有统计学意义（均为P<0.05）。结论　急性RIRI后RGCs涉及副凋亡和自噬的激活,各种类型的程序性细胞死亡可作为单一细胞死亡的形式或多种细胞死亡形式共存,参与急性RIRI视网膜损伤。     

横向定量牵拉损伤对大鼠视网膜神经节细胞自噬水平的影响

目的：研究不同程度牵拉力对大鼠视网膜神经节细胞(RGCs)存活率和神经传导功能的影响,探讨RGCs自噬水平对上述指标的影响。

方法：选取健康雄性SD大鼠30只随机分为空白组、假手术组、0.15N组、0.3N组、0.6N组,每组各6只。模型组采用横向定量牵拉法制作视神经损伤大鼠模型。空白组大鼠不予处理。假手术组仅暴露视神经,不予牵拉。造模后第1、3d行闪光视觉诱发电位(f-VEP)检查,第3d取视网膜组织行Brn-3a免疫组织化学染色观察RGCs存活情况,透射电子显微镜观察自噬小体,蛋白质印迹法检测LC3BⅡ/Ⅰ蛋白表达水平。

结果：造模后第3d,与假手术组比较,模型组大鼠f-VEP P2潜伏期延长,振幅降低,视网膜组织中RGCs存活率降低,LC3BⅡ/Ⅰ蛋白表达水平降低,且各组大鼠视网膜组织中均可见自噬小体。

结论：视神经牵拉伤会降低大鼠早期视网膜自噬水平,导致RGCs死亡和相应的神经传导功能障碍,且不同牵拉力造成的损伤程度不同,RGCs存活情况可能与其自噬水平有关。     

自噬在年龄相关性黄斑变性和视网膜脱离中的研究进展

自噬是维持细胞正常功能和内环境稳定的一种关键的自我保护机制,其在生长发育、适应、肿瘤抑制、老化、先天性和获得性免疫中扮演着重要角色.近年来的研究表明,自噬与众多眼病,如角膜营养不良、白内障、青光眼和视网膜疾病等的发生和发展有着密切联系.在年龄相关性黄斑变性(AMD)中,自噬异常损害视网膜色素上皮(RPE)细胞的功能,促进脂褐质形成并且参与玻璃膜疣的积累.在视网膜脱离(RD)中,自噬既能保护光感受器细胞,也能促进光感受器细胞的死亡.本文就自噬的分子机制、其在AMD和RD中的作用以及自噬作用的转变和水平的变化与损伤的时间、强弱及性质的相关性进行综述.     

Autophagy in axonal degeneration in glaucomatous optic neuropathy

The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.     

The physiological and pathophysiological roles of the autophagy lysosomal system in the conventional aqueous humor outflow pathway: More than cellular clean up

During the last few years, the autophagy lysosomal system is emerging as a central cellular pathway with roles in survival, acting as a housekeeper and stress response mechanism. Studies by our and other labs suggest that autophagy might play an essential role in maintaining aqueous humor outflow homeostasis, and that malfunction of autophagy in outflow pathway cells might predispose to ocular hypertension and glaucoma pathogenesis. In this review, we will collect the current knowledge and discuss the molecular mechanisms by which autophagy does or might regulate normal outflow pathway tissue function, and its response to different types of stressors (oxidative stress and mechanical stress). We will also discuss novel roles of autophagy and lysosomal enzymes in modulation of TGFβ signaling and ECM remodeling, and the link between dysregulated autophagy and cellular senescence. We will examine what we have learnt, using pre-clinical animal models about how dysregulated autophagy can contribute to disease and apply that to the current status of autophagy in human glaucoma. Finally, we will consider and discuss the challenges and the potential of autophagy as a therapeutic target for the treatment of ocular hypertension and glaucoma.     

两种方法建立大鼠慢性青光眼模型的对比研究

目的评价2种大鼠慢性青光眼模型的效果。方法通过单纯烙闭3条大鼠巩膜上静脉（单纯手术组,n=19）和烙闭巩膜上静脉同时给予5-氟尿嘧啶（5-fluorouracil,5-Fu）（手术给药组,n=23）2种方法制作大鼠慢性高眼压模型,观察术前和术后1d、3d、5d、1周、2周、1个月和2个月早晚眼压变化,并于术后1周、2周、1个月和2个月摘除眼球,光镜观察大鼠视网膜各层组织厚度变化,荧光金逆行标记视网膜神经节细胞（retinal ganglion cells,RGCs）并计数,TUNEL染色,比较2组模型检测指标。结果2组模型大鼠术后眼压均明显升高,随着高眼压时间持续延长,RGCs逐渐减少及视网膜神经纤维层逐渐变薄,剩余RGCs数量逐渐减少,但2组间差异无统计学意义（P〉0.05）。结论持续高眼压导致大鼠RGCs的病理改变过程及其结果符合青光眼视神经损伤的病理特点。单纯烙闭大鼠巩膜上静脉即可获得稳定有效的慢性青光眼模型。     

青光眼小鼠基因模型的研究进展

青光眼作为目前全球首位不可逆性致盲眼病，是具有遗传倾向的多因素复杂疾病，病理性眼压升高是其危险因素。关于青光眼的发病机制尚不完全清楚，现有研究多建立在动物模型的基础上，以小鼠为主要研究对象，通过实验诱导手段或转基因操作复建青光眼病理损伤过程，进一步研究相关的发病机制和病理变化。实验诱导构建青光眼小鼠模型技术经过诸多学者的研究，逐渐趋于成熟。而随着分子生物学和遗传学的研究深入，越来越多的研究集中在青光眼的相关疾病基因上，转基因小鼠模型成为近年的热点，与实验操作控制单一因素相比，基因编辑更能模拟出疾病发病的复杂过程。本文主要通过阐述相关青光眼小鼠转基因模型的研究进展，为今后研究中模型的选择提供更完整的方向与策略。     

米诺环素对视网膜神经节细胞保护作用的研究进展

视网膜神经节细胞作为中枢神经系统的一部分,是青光眼和视网膜疾病的主要受损细胞。米诺环素是一种半合成的四环素类衍生物,除广谱抗菌功效外,还具有抗氧化、抗凋亡和抑制小胶质细胞活化的作用,对神经元具有一定的保护作用。研究证明米诺环素对视网膜神经节细胞也具有保护作用,在视神经外伤、青光眼和多种视网膜疾病的研究中均显示了不同程度的效果。本文就米诺环素对视网膜神经节细胞的神经保护作用及其作用机制作一综述。     

Neuroprotection by sodium channel blockade with phenytoin in an experimental model of glaucoma

PURPOSE: Sustained influx of intracellular sodium through voltage-gated sodium channels is an important event in the cascade leading to degeneration of axons. This study tested the hypothesis that sodium channel blockade with phenytoin would result in neuroprotection of retinal ganglion cells (RGCs) and optic nerve axons in an experimental model of glaucoma. METHODS: Chronic elevation of rat intraocular pressure (IOP) leading to optic nerve damage was induced using the episcleral vein occlusion model. Before induction of glaucoma, a subset of animals was placed on phenytoin-containing chow; this treatment continued for 8 weeks. Quantitative counts of backfilled RGCs and optic nerve axons was performed to examine the effects of phenytoin on glaucoma-induced adverse neurodegeneration. RESULTS: Elevated IOP resulted in a significant decrease in density of RGCs, as well as dropout of axons within the optic nerve at 8 weeks after induction. In phenytoin-treated animals, however, the loss of RGCs was significantly reduced compared to vehicle-treated glaucomatous animals. Axon loss in the optic nerve was also reduced in phenytoin-treated animals, compared to controls. CONCLUSIONS: Orally delivered phenytoin was effective in protecting neurons in an animal model of glaucoma, and merits further examination as a potential therapeutic strategy.     

雷公藤甲素在慢性青光眼大鼠模型中对视网膜神经节细胞的保护

背景青光眼是一种以视神经损害为病理特点的常见致盲性眼病。目前,通过延缓或阻止病程的进展而保护视神经是青光眼研究的热点。目的研究中药雷公藤的有效提取成分雷公藤甲素对慢性青光眼大鼠模型视网膜神经节细胞（RGCs）的保护作用。方法选用清洁级Wistar雌性大鼠80只,采用房水释放联合激光房角光凝法建立慢性青光眼大鼠模型。右眼为激光眼,左眼为对照眼。激光光凝前3d起每日雷公藤甲素组大鼠腹腔给予雷公藤甲素5μg／kg直至处死,生理盐水组以同样的方式给予等量生理盐水。于术前,术后1、3、5d,1周及此后每周用Tono—PenXL眼压计监测眼压。激光光凝术后1、2、4、8周制作大鼠眼球视网膜铺片并行Nissl染色,对RGCs进行定量检测。结果激光眼术后1d眼压较术前增高,术后1周达高峰,持续约3周,4周时眼压恢复正常;对照眼各时间点眼压值与术前相比差异均无统计学意义（P〉0．05）。术后各时间点雷公藤甲素组激光眼与生理盐水组激光眼间眼压的差异无统计学意义（P〉0．05）。生理盐水组激光眼术后1周RGCs数量开始减少,术后4～8周RGCs存活数量明显下降;与生理盐水组激光眼相比,各时间点雷公藤甲素组激光眼RGCs数量明显增多,差异均有统计学意义（P〈0．05）。雷公藤甲素组激光眼与其对照眼相比,各时间点RGCs数目的差异均无统计学意义（P〉0．05）。结论雷公藤甲素能防止慢性青光眼大鼠模型的RGCs损伤,对RGCs具有保护作用,该作用并不依赖于眼压的下降,这可能为青光眼的治疗提出新思路。     

Downregulation of Thy1 in retinal ganglion cells in experimental glaucoma

PURPOSE: Thy1 is a surface glycoprotein uniquely expressed in retinal ganglion cells (RGCs) in retina. The aim of this study was to investigate the expression change of Thy1 in a model of experimental glaucoma. METHODS: The change of protein and mRNA levels of Thy1 in the retina were studied using stereological counts of back-labeled RGCs, Western blot analysis, immunohistochemistry, and laser capture microdissection (LCM) of RGCs with quantitative PCR analysis of mRNA in a model of experimental glaucoma. LCM after optic nerve crush was also performed to evaluate Thy1 mRNA levels after a different injury. RESULTS: After 10 days of elevated IOP, there was a 34% loss of RGC number, Thy1 protein decreased 60% in eyes with elevated intraocular pressure (IOP), and Thy1 mRNA levels decreased 51% in RGCs. Both protein and mRNA level change of Thy1 is to a much greater extent than RGC number loss. CONCLUSIONS: The current results confirm that Thy 1 mRNA levels do not reflect the number of RGCs present and extend this to include a parallel decrease in Thy1 protein levels. These results suggest that Thy1 serves as an early marker of RGC stress, but not a marker of RGC loss, in models of retinal damage.     

Neuroprotection in glaucoma - Is there a future role?

In glaucoma, the major cause of global irreversible blindness, there is an urgent need for treatment modalities that directly target the RGCs. The discovery of an alternative therapeutic approach, independent of IOP reduction, is highly sought after, due to the indirect nature and limited effectiveness of IOP lowering therapy in preventing RGC loss. Several mechanisms have been implicated in initiating the apoptotic cascade in glaucomatous retinopathy and numerous drugs have been shown to be neuroprotective in animal models of glaucoma. These mechanisms and their potential treatment include excitotoxicity, protein misfolding, mitochondrial dysfunction, oxidative stress, inflammation and neurotrophin deprivation. All of these mechanisms ultimately lead to programmed cell death with loss of RGCs. In this article we summarize the mechanisms involved in glaucomatous disease, highlight the rationale for neuroprotection in glaucoma management and review current potential neuroprotective strategies targeting RGCs from the laboratory to the clinic.     

Advances in retinal ganglion cell imaging

Glaucoma is one of the leading causes of blindness worldwide and will affect 79.6 million people worldwide by 2020. It is caused by the progressive loss of retinal ganglion cells (RGCs), predominantly via apoptosis, within the retinal nerve fibre layer and the corresponding loss of axons of the optic nerve head. One of its most devastating features is its late diagnosis and the resulting irreversible visual loss that is often predictable. Current diagnostic tools require significant RGC or functional visual field loss before the threshold for detection of glaucoma may be reached. To propel the efficacy of therapeutics in glaucoma, an earlier diagnostic tool is required. Recent advances in retinal imaging, including optical coherence tomography, confocal scanning laser ophthalmoscopy, and adaptive optics, have propelled both glaucoma research and clinical diagnostics and therapeutics. However, an ideal imaging technique to diagnose and monitor glaucoma would image RGCs non-invasively with high specificity and sensitivity in vivo. It may confirm the presence of healthy RGCs, such as in transgenic models or retrograde labelling, or detect subtle changes in the number of unhealthy or apoptotic RGCs, such as detection of apoptosing retinal cells (DARC). Although many of these advances have not yet been introduced to the clinical arena, their successes in animal studies are enthralling. This review will illustrate the challenges of imaging RGCs, the main retinal imaging modalities, the in vivo techniques to augment these as specific RGC-imaging tools and their potential for translation to the glaucoma clinic.