视神经再生的研究进展

视神经属于中枢神经的一部分,损伤后难以再生。视神经损伤通常伴随视网膜神经节细胞(retinal ganglioncells,RGCs)的持续性凋亡及视神经变性坏死,引起视力损害甚至完全失明。目前针对视神经再生的基础研究主要集中于保护和维持视神经损伤后RGCs的存活、促进RGCs轴突再生及重建视神经功能。本文以RGCs保护、轴突再生及视神经功能重建等为关键词,查询国内外最新视神经再生研究类文献,并分析整理,从抗氧化应激、提供外源性细胞因子、炎症刺激、抗胶质瘢痕、基因调控等方面阐述近年的视神经再生研究进展,以期对后续的基础研究开展及临床转化有所帮助。     

视神经再生的实验研究进展

视神经是中枢神经的一部分，损伤后将无法再生，继而引起进一步视力损害。根据目前视神经损伤后视网膜神经节细胞（retinal ganglion cells，RGCs）轴突再生的基础研究，视神经损伤后必须采取以下有效措施:提高RGCs内在的再生潜力，改善生长抑制环境，优化RGCs神经再生，而诱导再生轴突靶向延伸是理想的促进视神经的再生与修复方式。本文查阅国内外最新实验性视神经再生研究类文献，从调控眼内炎症因子、提供合适外源性神秘生长因子、激活RGCs再生潜能、阻断抑制性轴突再生信号传导、给予适当的再生刺激信号、改善抑制性细胞外微环境等方面阐述促进视神经再生的研究现状，以期对早日实现基础研究成果尽快向临床应用转化有所帮助。     

大鼠视神经横断伤及夹挫伤后视网膜神经节细胞变化及与损伤时间关系

目的 观察大鼠视神经横断伤及夹挫伤后,视网膜神经节细胞(RGCs)形态学变化、区别及在不同时间的计数变化,探讨其与视神经损伤经过时间的关系,为视神经损伤的病理机制及损伤经过时间的推断提供一定的依据.方法 采用大鼠球后视神经横断伤/夹挫伤动物模型,在伤后不同时间处死动物并取材,HE 染色,光镜下观察RGCs的动态变化.结果 视神经损伤后RGCs数日均严重下降,2周内RGCs快速减少,3～7 d为RGCs快速减少期,2周以后缓慢减少;但横断伤组3 d以后各个时期RGCs计数下降幅度与夹挫伤组相比更明显.结论 视神经损伤导致了视网膜形态结构的变化,RGCs丢失的严重程度与损伤类型及时问呈相关性.     

大鼠外伤性视神经损伤模型的建立

目的建立大鼠定量视神经损伤模型,为研究视神经损伤的发病机制及治疗效果奠定基础。方法健康Sprague-Dawley大鼠27只,随机分为3组,分别为A组(损伤组)12只、B组(假损伤对照组)12只、C组(正常对照组)3只。A组暴露视神经,应用40g力的视神经夹在大鼠眼球后2mm处夹视神经30s,B组仅暴露视神经,C组不做任何处理。A、B组按损伤后不同时间分为3d组、7d组、14d组、28d组,采用双上丘注射50g.L-1荧光金标记双眼视网膜神经节细胞(retinal ganglion cells RGCs)。视网膜铺片荧光照相,计算RGCs计数,RGCs标识率及RGCs丧失率。结果 C组与B组RGCs计数及RGCs标识率比较,无明显差异;A组与B组各时间点RGCs计数及RGCs标识率比较,均有明显差异;A组视神经损伤后不同时间RCCs计数逐渐下降(3d:152.26±25.12,7d:111.19±20.32,14d:101.23±17.19,28d:94.86±18.26),14d组与28d组比较无统计学意义,其他时间点比较有统计学意义,视神经损伤后RGCs标识率随时间延长渐进性降低(3d:79.35%±8.29%,7d:59.76%±7.79%,14d:53.26%±7.26%,28d:51.29%±3.26%),而RGCs丧失率随时间延长渐进性增加(3d:20.65%±3.15%,7d:40.24%±5.63%,14d:46.74%±4.37%,28d:48.71%±5.12%)。结论应用40g力视神经夹在大鼠眼球后2mm处夹视神经30s能成功建立定量视神经损伤动物模型。     

大鼠视神经压榨伤模型的建立

目的建立大鼠标定性视神经损伤模型.方法健康SD大鼠28只,7只为正常对照组,只进行双上丘注射3%快蓝逆行标记视网膜神经节细胞(retinal ganglion cells,RGCs),另21只为标定性视神经损伤组,依损伤后存活时间的不同再分为A组(4d组)、B组(14d组)及C组(21d组),每组7只.21只大鼠以夹持力为40 g的特制视神经夹,在大鼠眼球后2 mm处夹持视神经4s,制成大鼠标定性视神经压榨伤模型,于处死前3d采用双上丘直接注射3%快蓝(fast blue)法标记双眼RGCs,将全视网膜铺片置于荧光显微镜下,在距视乳头1 mm处的颞上、颞下、鼻下、鼻上4处作荧光摄影(400 ×),并输入计算机经图像分析仪计数RGCs,按RGCs标识率进行统计学比较.RGCs标识率=损伤眼(右眼)RGCs数/未损伤眼(左眼)RGCs数×100%.结果正常大鼠的RGCs标识率右眼RGCs数/左眼RGCs数为99.79%±13.05%,左眼RGCs数/右眼RGCs数为101.86%±13.91%,无论是用左眼的RGCs数比右眼的RGCs数,或用右眼的RGCs数比左眼的RGCs数,其结果无显著性差异(P＞0.5).视神经损伤组的RGCs标识率A组(4d组)RGCs标识率为77.79%±7.11%;B组(14d组)RGCs标识率为63.76%±3.79%;C组(21d组)RGCs标识率为54.66%±4.75%.以上显示,损伤各组的RGCs标识率明显低于正常对照组(P＜0.05),且随着时间的推移,损伤A、B、C组的RGCs标识率渐进性降低.结论用特制的夹持力为40 g的视神经夹,夹持正常大鼠视神经4s,可造成部分性RGCs丧失,随大鼠存活时间的推移,RGCs呈渐进性丧失.眼科学报2001;1799～102.     

黄芪注射液对体外培养视网膜神经节细胞的保护作用

目的 视神经由视网膜神经节细胞(retinal ganglion cells,RGCs)轴突构成,一旦损伤难以再生,视神经保护是治疗视神经疾病的关键,本研究拟评价黄芪注射液的视神经保护作用.方法 体外培养RGCs,采用谷氨酸作为细胞凋亡诱导剂建立RGCs凋亡模型,并用不同浓度黄芪注射液加入细胞培养基进行干预,分别用MTT和流式细胞检测的方法进行细胞存活率及细胞凋亡相关检测.结果 黄芪注射液加入DMEM-F12培养基共培养RGCs 24 h后,MTT检测结果示终浓度为(0.10～0.40)×103g·L-1时黄芪注射液均可促进RGCs存活,其中0.20×103g·L-1、0.30×103g·L-1浓度促生长作用最好(P=0.040、0.005,n=10),谷氨酸浓度为3.2 mmol·L-1时可造成理想的RGCs凋亡模型,0.20×103g·L-1黄芪注射液可促进谷氨酸损伤的RGCs存活(P=0.08,n=10)并减少其细胞凋亡率(P=0.36,n=4).结论 黄芪注射液作为视神经保护中药制剂对RGCs具有一定保护作用,黄芪对RGCs的保护作用很可能跟抑制谷氨酸兴奋性毒性作用有关.     

钳夹法造成大鼠视网膜神经节细胞过量丢失

目的评估钳夹视神经对视网膜神经节细胞(retinal ganglion cells,RGCs)的损伤程度。方法取38只雄性Wistar大鼠,夹持组(n=30)按夹持时间12s、9s、6s、3s、1s分为A-E组,F为反身夹持组,每组各5只大鼠。沿大鼠眼球颞侧暴露视神经,于球后2mm处用90g微型视神经夹夹持视神经,另有40g反向镊在球后2mm处夹持视神经,每只大鼠均左眼手术,右眼正常对照,假手术组(n=8)大鼠左眼仅手术暴露视神经球后段,不予夹持。采用荧光金逆行标记RGCs,视网膜铺片计数,计算RGCs数量及存活率。结果假手术组左右眼RGCs密度相比无显著性差异,细胞密度分别为(2679±67)mm-2、(2689±53)mm-2(P=0.896);夹持组RGCs细胞密度明显下降,分别为(220±167)mm-2、(265±232)mm-2、(298±239)mm-2、(478±682)mm-2、(769±615)mm-2、(974±476)mm-2,夹持冲量(夹持力与时间的乘积)和RGCs存活率呈负相关。结论钳夹法可造成明确、定量的视神经损伤,但损伤量过大、稳定性较差,与临床外伤性视神经病变的发病实际尚有一定差距。     

横向定量牵拉法制作大鼠视神经精确损伤模型

目的:采用横向定量牵拉法制作大鼠视神经损伤模型,并利用荧光金逆行标记评价视神经牵拉伤后视网膜节细胞(retinal ganglion cells,RGCs)的存活率.方法:将25只雄性Wistar大鼠随机均分为5组,即假手术组和视神经牵拉伤后1、3、7、14d组.模型组用横向张力计牵拉左眼视神经,假手术组仅暴露左眼视神经但不予牵拉,各组以右眼为正常对照.用荧光金逆行标记,并观察假手术组及视神经牵拉伤后1、3、7、14d组RGCs的密度.结果:正常对照组RGCs形态多呈圆形或椭圆形,边界清楚,细胞外无明显荧光染料渗漏,部分可见明显细胞突起;而视神经牵拉伤后RGCs随时间延长而不断减少,细胞分布不均匀,并可见大量荧光渗漏及小胶质细胞.与正常对照组相比,假手术组RGCs形态和密度无明显差异(P＞0.05);视神经牵拉伤后第1、3、7、14d的RGCs数量进行性减少,且其密度均明显低于正常对照组(P＜0.01);视神经牵拉伤后第1、3、7、14d的RGCs存活率分另别为78.94％±0.92％、60.07％±0.90％、38.92％±1.42％和17.31％±0.97％.结论:横向定量牵拉法可以建立易于量化的视神经损伤模型,为进一步研究视神经损伤后的治疗方法提供有力工具.     

视神经损伤的基因治疗

视神经损伤因缺乏有效治疗手段,预后不良,有关其治疗方法的研究成为眼科学界的热点之一.目前研究认为原发性损伤后,继发的视网膜神经节细胞(RGCs)凋亡是造成视神经损伤的重要机制,及时有效地减少RGCs凋亡对视神经的保护具有重要作用.随着分子生物学技术的发展,视神经损伤的基因治疗备受关注,并有望从实验室走向临床,成为有效治疗手段.本文就视神经损伤基因治疗的现状及前景做一综述.     

视网膜神经节细胞钾离子通道的表达与功能

视网膜神经节细胞（retinal ganglion cells,RGCs）不可逆性凋亡是视神经损害的主要原因.在许多视神经相关疾病中如青光眼、年龄相关性黄斑变性、糖尿病视网膜病变、葡萄膜炎和玻璃体视网膜病变等,RGCs凋亡是视觉障碍的一个主要原因.RGCs的电性能主要取决于离子通道的存在.各种离子通道中,钾离子通道起到了非常关键的作用.本文就钾离子通道在RGCs上的表达及其功能进行综述.     

Optic nerve protection, regeneration, and repair in the 21st century: LVIII Edward Jackson Memorial lecture

PURPOSE: To present the current status and clinical implications of optic nerve protection, repair, and regeneration after experimental injury in mammals, including nonhuman primates. DESIGN: Optic nerve and neuro-ophthalmology experimental study review. METHOD: Synthesis of experimental data regarding experimental studies of optic nerve protection, repair, and regeneration. RESULTS: Under certain conditions, mammalian retinal ganglion cells can be prevented from dying despite injury to the cell bodies or their axons, injured mammalian retinal ganglion cells whose axons have degenerated can be induced to extend new axons, and regenerating axons can reach their correct targets in the central nervous system. In addition, stem cells can be induced to become retinal ganglion cells. CONCLUSIONS: It may soon be possible to preserve and restore vision in persons whose sight is threatened or has been lost from disease or damage to the optic nerve.     

视网膜神经节细胞的保护和修复

视网膜神经节细胞(RGCs)的进行性死亡是许多视网膜和视神经疾病发展到最后的必经之路。长期以来一直认为，由于抑制性环境的存在，视神经损伤后不能再生和修复，现在研究证实，在特定的条件下，尽管RGCs的胞体或轴突受损，仍能免于死亡，而且变性的轴突能再生，并能与靶组织建立突触联系。对RGCs的保护和修复的研究进展作一综述。     

Müller细胞与视网膜神经再生研究进展

视网膜退行性疾病是以视网膜神经元凋亡为主要病理过程的一类致盲性眼病,视网膜神经元受损后再生困难,Müller细胞是参与视网膜发育、受损及再生过程的重要胶质细胞。近年研究证明,Müller细胞是视网膜神经元的内源性替代来源,为视网膜神经再生的优秀靶标。本文根据视网膜神经再生过程、Müller细胞与视网膜神经再生相关因素进行综述,为神经再生研究提供新方向。     

促红细胞生成素及其受体与视神经视网膜疾病

促红细胞生成素（EPO）具有促进红系祖细胞增生和分化的作用,其在光诱导性视网膜变性、视网膜缺血一再灌注损伤、急慢性高眼压、视神经损伤、多发性硬化性视神经炎、糖尿病视网膜病变（DR）、早产儿视网膜病变（ROP）等视神经视网膜疾病模型中的神经保护作用也受到了关注。就EPO及其受体在视网膜的分布、其在视神经视网膜病变模型中的表达情况及其对视网膜神经元的保护作用和机制方面的研究进展进行综述。     

Clinical electrophysiology of the optic nerve and retinal ganglion cells

Clinical electrophysiological assessment of optic nerve and retinal ganglion cell function can be performed using the Pattern Electroretinogram (PERG), Visual Evoked Potential (VEP) and the Photopic Negative Response (PhNR) amongst other more specialised techniques. In this review, we describe these electrophysiological techniques and their application in diseases affecting the optic nerve and retinal ganglion cells with the exception of glaucoma. The disease groups discussed include hereditary, compressive, toxic/nutritional, traumatic, vascular, inflammatory and intracranial causes for optic nerve or retinal ganglion cell dysfunction. The benefits of objective, electrophysiological measurement of the retinal ganglion cells and optic nerve are discussed, as are their applications in clinical diagnosis of disease, determining prognosis, monitoring progression and response to novel therapies.Subject terms: Optic nerve diseases, Retina     

Mesenchymal stem cells for repairing glaucomatous optic nerve

Glaucoma is a common and complex neurodegenerative disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Currently, there is no effective method to address the cause of RGCs degeneration. However, studies on neuroprotective strategies for optic neuropathy have increased in recent years. Cell replacement and neuroprotection are major strategies for treating glaucoma and optic neuropathy. Regenerative medicine research into the repair of optic nerve damage using stem cells has received considerable attention. Stem cells possess the potential for multidirectional differentiation abilities and are capable of producing RGC-friendly microenvironments through paracrine effects. This article reviews a thorough researches of recent advances and approaches in stem cell repair of optic nerve injury, raising the controversies and unresolved issues surrounding the future of stem cells.     

Retinal ganglion cell death is not prevented by application of tetrodotoxin during optic nerve regeneration in the frog Hyla moorei

Following extracranial optic nerve crush in the adult frog Hyla moorei, regeneration takes place to restore topographically organised visual projections, despite partial depletion of the retinal ganglion cell population. In the present study, the right optic nerve was crushed and tetrodotoxin (TTX) repeatedly injected into the right eye to abolish electrical activity mediated by sodium channels in ganglion cell axons. At 70-78 days post-crush, the number and distribution of live cells in the ganglion cell layer were assessed from cresyl violet-stained wholemounts. After regeneration, cell numbers in TTX-treated animals fell by a mean of 32.6% in comparison with their unoperated partner retinae. This value was very similar to the 32.4% mean fall found after regeneration for animals receiving no injections of TTX. Furthermore, distributions of surviving cells were comparable for the two groups. We conclude that sodium-mediated electrical activity within retinal ganglion cells does not control the extent or pattern of their death during optic nerve regeneration.     

Influence of interleukin-1 beta induction and mitogen-activated protein kinase phosphorylation on optic nerve ligation-induced matrix metalloproteinase-9 activation in the retina

Ischemic damage to the retina is a multifaceted process that results in irreversible loss of ganglion cells and blinding disease. Although the mechanisms underlying ischemia-induced ganglion cell death in the retina are not clearly understood, we have recently reported that retinal damage induced by ligation of the optic nerve results in increased matrix metalloproteinase-9 (MMP-9) synthesis and promotes ganglion cell loss. In this study, we have investigated the roles of IL-1beta and mitogen activated protein kinases in MMP-9 induction in the retina. Optic nerve ligation led to a transient increase in IL-1beta and MMP-9 levels and phosphorylation of p42/p44 mitogen activated protein kinases (extracellular signal-regulated kinases, ERK1 and ERK2) in the retina. We found no significant increase in phosphorylation of p38 MAP kinase or c-jun N-terminal kinases indicating that ERK1/2 plays a major role in MMP-9 induction. Intravitreal injection of IL-1 receptor antagonist (IL-1Ra) or MAP kinase inhibitor U0126 significantly decreased both ERK1/2 phosphorylation and MMP-9 induction suggesting that interruption of this cascade might attenuate retinal damage. In support of this, intravitreal injection of IL-1Ra and U0126 offered significant protection against optic nerve-induced retinal damage. These results suggest that optic nerve ligation-induced IL-1beta promotes retinal damage by increasing MMP-9 synthesis in the retina.     

Expression of myocilin/TIGR in normal and glaucomatous primate optic nerves

Myocilin/TIGR was the first molecule discovered to be linked with primary open angle glaucoma (POAG), a blinding disease characterized by progressive loss of retinal ganglion cells. Mutations in myocilin/TIGR have been associated with age of disease onset and severity. The function of myocilin/TIGR and its role in glaucoma is unknown. Myocilin/TIGR has been studied in the trabecular meshwork to determine a role in regulation of intraocular pressure. The site of damage to the axons of the retinal ganglion cells is the optic nerve head (ONH). The myocilin/TIGR expression was examined in fetal through adult human optic nerve as well as in POAG. Myocilin/TIGR was expressed in the myelinated optic nerve of children and normal adults but not in the fetal optic nerve before myelination. Also examined was the expression in monkeys with experimental glaucoma. The results demonstrate that optic nerve head astrocytes constitutively express myocilin/TIGR in vivo in primates. Nevertheless, myocilin/TIGR is apparently reduced in glaucomatous ONH. The colocalization of myocilin/TIGR to the myelin suggests a role of myocilin/TIGR in the myelinated optic nerve.