Glial cell and glaucomatous optic neuropathy

Glaucomatous optic neuropathy is a chronic disease accompanied by visual field loss, cupping of optic nerve head, and apoptosis of retinal ganglion cells (RGCs). The mechanism of glaucomatous optic neuropathy is unknown but glial cells play an important role in glaucomatous optic nerve damage and the repair process. We review the role of glial cells in the remodeling of optic nerve head, apoptosis of RGCs and immune reactions in glaucoma.     

The neurobiology of cell death in glaucoma

Glaucoma is an optic neuropathy in which the optic nerve axons are damaged, resulting in death of retinal ganglion cells (RGCs). The primary region of damage is thought to be the optic nerve head (ONH), with the lateral geniculate nucleus (LGN) and optic radiations to the visual cortex being secondarily affected. Neurotrophin deprivation resulting from optic nerve injury is thought to cause RGCs to die by apoptosis by inhibition of cell survival pathways. However, disruption of retrograde axonal transport is not the only mechanism associated with optic nerve damage and RGC death, and thus, an additional mechanism of injury is likely to be involved in glaucomatous optic neuropathy.     

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。     

胶质细胞活化相关炎症在青光眼视神经病变中的研究进展 △

青光眼是一种以视网膜神经节细胞(RGCs)及其轴突凋亡为特征的视神经退行性病变。越来越多的研究表明炎症和免疫反应在青光眼视神经病变中具有重要作用。在高眼压动物实验中,抑制早期胶质细胞活化及减少炎症因子释放,对RGCs和视神经具有保护作用。本文重点就星形胶质细胞和小胶质细胞活化及其产生的炎症因子,特别是视网膜及视盘部胶质细胞释放的炎症因子在青光眼视神经病变过程中的作用及相关机制进行综述,希望能给青光眼的研究和治疗带来新的启发。     

Primary retinal ganglion cells for neuron replacement therapy

Optic nerve damage as a result of trauma, ischemia, glaucoma or other forms of optic neuropathy disease, leads to disconnection between the eye and brain and death of retinal ganglion cells (RGCs), causing permanent loss of vision. Therapeutic options for treating optic neuropathy are limited and represent a signiifcant unmet medical need. Development of a regenerative strategy for replacement of lost RGCs lies at the core of the future cell-based therapy for these conditions. Successful long-term restoration of visual function depends on the type of cells for transplantation. Primary RGCs of neonatal mice are now reported to have the potential for serving such a purpose.     

Differentiation of retinal ganglion cells from induced pluripotent stem cells: a review

Glaucoma is a common optic neuropathy that is characterized by the progressive degeneration of axons and the loss of retinal ganglion cells (RGCs). Glaucoma is one of the leading causes of irreversible blindness worldwide. Current glaucoma treatments only slow the progression of RGCs loss. Induced pluripotent stem cells (iPSCs) are capable of differentiating into all three germ layer cell lineages. iPSCs can be patient-specific, making iPSC-derived RGCs a promising candidate for cell replacement. In this review, we focus on discussing the detailed approaches used to differentiate iPSCs into RGCs.     

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。     

Neuroprotective effects of BDNF and GDNF in intravitreally transplanted mesenchymal stem cells after optic nerve crush in mice

AIM: To assess the neuro-protective effect of bone marrow mesenchymal stem cells (BMSCs) on retinal ganglion cells (RGCs) following optic nerve crush in mice. METHODS: C56BL/6J mice were treated with intravitreal injection of PBS, BMSCs, BDNF-interference BMSCs (BIM), and GDNF-interference BMSCs (GIM) following optic nerve crush, respectively. The number of surviving RGCs was determined by whole-mount retinas and frozen sections, while certain mRNA or protein was detected by q-PCR or ELISA, respectively. RESULTS: The density (cell number/mm2) of RGCs was 410.77±56.70 in the retina 21d after optic nerve crush without any treatment, compared to 1351.39±195.97 in the normal control (P<0.05). RGCs in BMSCs treated eyes was 625.07±89.64/mm2, significantly higher than that of no or PBS treatment (P<0.05). While RGCs was even less in the retina with intravitreal injection of BIM (354.07+39.77) and GIM (326.67+33.37) than that without treatment (P<0.05). BMSCs injection improved the internal BDNF expression in retinas. CONCLUSION: Optic nerve crush caused rust loss of RGCs and intravitreally transplanted BMSCs at some extent protected RGCs from death. The effect of BMSCs and level of BDNF in retinas are both related to BDNF and GDNF expression in BMSCs.     

Axonal regeneration of retinal ganglion cells depending on the distance of axotomy in adult hamsters

PURPOSE: To examine the relationship between the distance of axotomy and axonal regeneration of injured retinal ganglion cells (RGCs) systematically and the effect of a predegenerated (pretransected or precrushed) peripheral nerve (PN) graft on axonal regeneration of RGCs axotomized at a definite distance (0.5 mm from the optic disc) in comparison with a normal PN graft. METHODS: The optic nerve (ON) was transected intraorbitally at 0.5, 1, 1.5, 2, or 3 mm or intracranially at 6 to 8 mm from the optic disc, and a PN graft was transplanted onto the ocular ON stump in adult hamsters. Four weeks after grafting, the number of RGCs regenerating their injured axons into the PN graft was investigated in all animals. RESULTS: The number of regenerating RGCs decreased significantly when the distance of axotomy increased from 0.5 to 7 mm. A precrushed PN graft was shown to enhance more injured RGCs to regenerate axons than a normal or pretransected PN graft. CONCLUSIONS: The distance of axotomy on the ON of adult hamsters is critical in determining the number of regenerating RGCs. Thus, experimental strategies to repair the damaged ON by PN transplantation is to attach a precrushed PN graft as close to the optic disc as possible to obtain optimal axonal regeneration of the axotomized RGCs.     

Optic Nerve Engraftment of Neural Stem Cells

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm² vs. 199 ± 58 RGCs/mm²; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.     

金丝桃素对视神经损伤大鼠视网膜节细胞的保护作用

目的观察金丝桃素对视神经损伤大鼠视网膜节细胞的保护作用。方法24只SD大鼠随机分为正常对照组、单纯夹伤组、生理盐水对照组、金丝桃素治疗组4组,每组6只（12眼）。对所有大鼠行双上丘注射2％荧光金逆行标记节细胞,7d后,对单纯夹伤组、生理盐水对照组、金丝桃素治疗组进行球后视神经钳夹．同时在生理盐水对照组、金丝桃素治疗组玻璃体内分别注入生理盐水和金丝桃素5ul,14d后进行视网膜节细胞的计数。采用SPSS13．0统计软件对所得数据进行t检验。结果视神经夹伤后14d,存活的视网膜节细胞显著减少。单纯夹伤组节细胞存活率为50％,生理盐水对照组节细胞存活率为52％,金丝桃素治疗组节细胞存活率为68％。金丝桃素治疗组相比单纯夹伤组和生理盐水对照组,存活的节细胞明显要多（P〈0．05）。结论玻璃体内注射金丝桃素能减少大鼠视神经损伤后视网膜神经节细胞的死亡率．对视网膜节细胞有保护作用。     

干细胞在视神经损伤修复中的应用

许多致盲性眼病是由视网膜神经节细胞（RGCs）损伤造成的,目前临床上缺乏有效的治疗方法。最近的研究显示干细胞可以替代或再生RGCs,从而有望修复受损的视功能。这些结果提供了新颖的视神经再生模式,但具体到临床应用,仍存在一系列问题需要解决。就应用干细胞再生视神经的研究进展进行综述。     

晶状体损伤对视神经损伤后RGCs的保护作用及其机制

背景大鼠Miiller细胞提取液能够促进离体培养的视网膜神经节细胞（RGCs）存活及轴突的再生,伴晶状体损伤的视神经外伤眼RGCs存活率提高,但Milller细胞和晶状体损伤在促进RGCs存活方面的关系鲜见报道。目的探讨伴晶状体损伤的视神经外伤眼Mtiller细胞对RGCs存活的促进作用及其机制。方法清洁级成年Wistar大鼠48只按随机数字表法随机分为伪手术组、视神经损伤组、晶状体联合视神经损伤组。伪手术组大鼠手术中暴露但不损伤视神经,视神经损伤组大鼠行视神经横断伤,晶状体联合视神经损伤组行视神经横断伤联合晶状体针刺伤,并导致晶状体混浊。术后7d及14d各组分别取8只大鼠处死后制备视网膜标本。采用苏木精一伊红染色观察各组大鼠视网膜和RGCs的形态学改变,采用免疫组织化学法检测各组大鼠视网膜内核层胶质纤维酸性蛋白（GFAP）标记的Muller细胞,光学显微镜下计数各组大鼠RGCs数量及GFAP阳性标记的Muller细胞数量。结果术后7d及14d,伪手术组大鼠RGCs的数量分别为（52．98±1．90）个／高倍视野和（51．81±3．09）个／高倍视野,差异无统计学意义（t=0．910,P=0．378）;术后14d视神经损伤组大鼠RGCs数量为（22．67±1．94）个／高倍视野,明显少于术后7d的（36．61±1．69）个／高倍视野,差异有统计学意义（t=15．312,P=0．000）;术后14d晶状体联合视神经损伤组RGCs数量为（35．69±1．80）个／高倍视野,明显少于术后7d的（50．76±2．77）个／高倍视野,差异有统计学意义（t=12．920,P=0．000）。术后7d及14d,晶状体联合视神经损伤组存活的RGCs数量均多于视神经损伤组,差异均有统计学意义（7d：t=102．840,P=0．000;14d：t=164．020,P=0．000）;术后14d晶状体联合视神经损伤组存活的RGCs：牧量少于伪手术组,差异有统计学意义（t=187．04,P=0．034）。术后7d及14d,伪手术组大鼠视网膜内核层均未见GFAP阳性标记的Muller细胞;视神经损伤组大鼠内核层GFAP阳性标记Muller细胞数量分别为（29．38＋2．04）个／高倍视野和（19．07±2．14）个／高倍视野,差异有统计学意义（t=-9．868,P=0．000）。晶状体联合视神经损伤组大鼠内核层GFAP阳性标记的Muller细胞数量分别为（48．96±2．80）个／高倍视野和（46．73±1．50）个／高倍视野,差异无统计学意义（t=1．987,P=0．067）。术后7d及14d,晶状体联合视神经损伤组大鼠内核层GFAP阳性Muller细胞数量均较视神经损伤组增多,差异均有统计学意义（7d：t=-15．997,P=0．000;14d：t=-29．938,P=0．000）。结论在视神经损伤合并晶状体刺伤时,晶状体损伤可诱导Muller细胞活化,进而促进视神经损伤后RGCs的存活。     

人脐血干细胞对大鼠部分性视神经损伤保护作用机制的研究

背景视神经损伤后将导致视网膜神经节细胞（RGCs）的凋亡,而凋亡的重要机制是内质网应激（ERS）,减弱ERS可能对RGCs起到保护作用。目的探讨ERS在大鼠视神经损伤中的机制及人脐血干细胞对大鼠部分性视神经损伤后RGCs的保护作用。方法采用40g自制夹钳夹持102只SD大鼠的左眼视神经制作部分性视神经钳夹伤动物模型,用随机数字表法将动物分为模型损伤组和人脐血干细胞组,每组51只,均取左眼为视神经损伤眼,右眼为正常对照眼。人脐血于细胞组大鼠左眼造模后立即将10川人脐血干细胞注入玻璃体腔。分别在造模后3、7、14、21、28d各处死3只大鼠,苏木精一伊红染色后光学显微镜下观察大鼠RGCs形态学的改变,并对存活的RGCs进行计数。在造模后3、12、24、48、72h及1周各处死6只大鼠,分别进行TUNEL法检测2组大鼠RGCs的凋亡率及逆转录聚合酶链反应（RT—PCR）法检测上述时间点GRP78 mRNA和CHOP mRNA在2组大鼠视网膜中的表达。结果模型损伤组和人脐血干细胞组随造模时间的延长,RGCs存活的数量明显下降,差异均有统计学意义（F目目=20．100,P=0．007）,与模型损伤组相比,人脐血干细胞组RGCs存活的数量下降缓慢。各时间点间模型损伤组及人脐血干细胞组RGCs存活的数量明显低于正常对照组,差异均有统计学意义（P〈0．01）,而人脐血干细胞组RGCs的数量明显高于正常对照组,差异有统计学意义（P〈0．01）。TUNEL检测表明,人脐血干细胞组在造模后24h内未见RGCs凋亡,而模型损伤组可见大量TUNEL阳性细胞出现,造模后48h～1周人脐血干细胞组RGCs凋亡率明显低于模型损伤组,差异有统计学意义（P〈0．01）。人脐血干细胞组视网膜GRP78 mRNA表达较强,CHOP mRNA表达微弱,与模型损伤组比较差异均有统计学意义（P〈0．01）。结论ERS参与了大鼠部分性视神经损伤后RGCs的凋亡机制,人脐血干细胞对大鼠部分性视神经损伤后RGCs起保护作用。     

大鼠视神经夹伤模型中莫尼定的视网膜节细胞保护作用

目的 :研究莫尼定对大鼠视神经夹伤模型视网膜神经节细胞的保护作用。方法 :实验用SD大鼠 2 0只随机分为用药组 8只和对照组 12只。所有大鼠右眼用 40 g微型视神经夹紧贴球后夹持视神经 60秒 ,左眼未做夹持。用药组于夹伤前1小时及夹伤后每日腹腔注射莫尼定 1mg/kg ,阴性对照组于夹伤前 1小时及夹伤后每日腹腔注射生理盐水 5ml/kg ,实验观察2 8天。实验结束前 4天双上丘注射 3 %荧光金逆行标记视网膜神经节细胞。做视网膜铺片 ,距离视乳头中心上下左右各2mm拍摄照片 ,使用CPAS图像分析软件做节细胞定量分析 ,节细胞存活率 =右眼节细胞密度 /左眼节细胞密度× 10 0。结果 :用药组、对照组节细胞存活率分别为 61 0 1%和 53 48% ,两者之间存在显著性差异 (P =0 .0 3 5)。结论 :在大鼠视神经夹伤模型中 ,莫尼定具有明显的视网膜节细胞保护作用     

Leber遗传性视神经萎缩病外显率的影响因素研究进展

Leber遗传性视神经萎缩病(Leber's Hereditary Optic Neuropathy, LHON)的发生是由于视网膜神经节细胞死亡进而导致视神经萎缩,表现为急性或亚急性双眼视力下降,是目前研究最为广泛的由线粒体基因突变引起的母系遗传病之一。超过90%的LHON是由线粒体基因组的3个原发致病突变之一所致,即G11778A、T14484C和G3460A。LHON临床表型不同,外显率的不同以及发病率的性别差异都提示可能存在其他相关基因(核基因和线粒体基因组)在其发挥着重要作用。本文主要对近二十年来该病的分子遗传学,特别是可能的外显率影响因素的研究进展作一阐述,为该病预防与临床治疗提供参考。     

急性眼压升高所致视网膜、视神经及视交叉胶质细胞的早期反应

背景 中枢神经系统以及视网膜中的胶质细胞与神经元关系十分紧密,胶质细胞在神经元损伤和修复过程中发挥着重要作用.急性眼压升高引起的视网膜、视神经及视交叉各部位胶质细胞的早期反应特点以及其与视神经损伤的关系目前尚不清楚. 目的 探讨大鼠视网膜、视神经及视交叉的胶质细胞对急性高眼压的早期反应,同时观察神经前体细胞标志物巢蛋白( nestin)在反应性胶质细胞中的表达. 方法 成年雌性Wistar大鼠9只,分为正常对照组3只和急性高眼压组6只,急性高眼压组大鼠采用右眼前房灌注生理盐水的方法升高大鼠眼压至110 mmHg,持续60 min.于术后第3天和第7天用过量麻醉法处死各组动物各3只,摘出眼球分离视神经和大脑标本,并制作冰冻切片.利用Nissl染色的方法测量高眼压眼视网膜内层厚度,观察视网膜和视交叉的大体形态.用βⅢ-tubulin免疫荧光染色法标记视神经内的视网膜神经节细胞(RGCs)轴突,用胶质纤维酸性蛋白(GFAP)和nestin双重标记显示视网膜、视神经及视交叉的胶质细胞反应,并对两组结果进行比较.结果 正常大鼠的视网膜、视神经以及视交叉内均可见到一定量的GFAP阳性胶质细胞,但nestin的表达量很低.急性眼压升高后的第3天,视网膜内丛状层厚度明显变薄,RGCs数目较损伤前减少约46％.视网膜内胶质细胞GFAP的表达显著增加,细胞突起由神经纤维层伸展至整个视网膜,增生的胶质细胞内可见nestin的明显表达.视神经内RGCs轴突发生变性样改变,GFAP阳性胶质细胞内nestin的表达较眼压升高前明显增加.同损伤眼相对应的一侧视交叉的横断面积减小,出现大量星状GFAP和nestin共表达的胶质细胞.以上改变在眼压升高后第7天更趋明显.结论 急性眼压升高早期即可引起RGCs的丢失及轴突的变性,视觉神经元改变的同时伴随胶质细胞的反应,增生的胶质细胞表达神经前体细胞的标志物.视网膜与视神经和视交叉的改变在时间上具有一定的同步性.     

Histopathological Changes of Inner Retina,Optic Disc,and Optic Nerve in Rabbit with Advanced Retinitis Pigmentosa

We observed the histopathological changes of retinal ganglion cells (RGCs), optic disc, and optic nerve in rabbit with advanced retinitis pigmentosa (RP). Wild-type (WT) and rhodopsin transgenic (Tg) of RP rabbits were used at age 24 months. Light and electron microscopy were used to observe the retina, optic disc, and optic nerve. RGCs were also confirmed by immunofluorescent staining with a TUJ-1 monoclonal antibody. In addition to the rod and cone degeneration, we observed the astrocyte infiltration of the optic disc due to the damage of small RGCs and nerve fibres and atrophy of small optic nerve fibres. They subsequently lead to the optic disc excavation and atrophy of the optic nerve. Consequently, our histopathological study clarified that not only the outer retina but also the inner retina, the optic disc, and the optic nerve were also affected in the late stages of RP rabbit.     

睫状神经营养因子对大鼠急性高眼压眼视网膜神经节细胞的保护作用

背景 青光眼可以引起视网膜神经节细胞(RGCs)凋亡,据报道睫状神经营养因子(CNTF)对外伤性视神经损伤有修复作用,其是否对青光眼视神经病变有保护作用尚少见报道. 目的 观察CNTF对大鼠急性高眼压眼RGCs的保护作用.方法 24只Wistar大鼠双眼采用眼前房平衡盐液加压灌注法建立大鼠急性高眼压模型,造模前2d左眼玻璃体内注入0.5μg CNTF 5μl,右眼以同样的方法注射磷酸钠溶液5μl,另取3只正常大鼠作为正常对照.造模后1、3、7、14 d过量麻醉法处死动物并摘除眼球,制备视网膜组织学切片后采用苏木精-伊红染色法进行形态学观察,光学显微镜下计数RGCs数目;采用免疫组织化学染色法观察RGCs层谷氨酸的表达情况.结果 正常对照组大鼠视网膜各层排列整齐,细胞边界清晰;模型对照组大鼠RGCs细胞膜、细胞核均发现异常改变,有细胞空泡样变;CNTF治疗组大鼠造模后变性的RGCs数量少.与模型对照组比较,造模后3、7、14 d CNTF治疗组RGCs数目明显增加,差异均有统计学意义(均P=0.000).免疫组织化学染色表明,造模后3～7d,CNTF治疗组RGCs层谷氨酸阳性细胞数分别为(5.50±1.04)个／3个高倍视野和(6.00±1.41) 个/3个高倍视野,明显低于模型对照组的(9.00±2.91)个／3个高倍视野和(10.83±1.94)个／3个高倍视野,差异均有统计学意义(均P=0.000),而造模后1d和14 d两组间谷氨酸阳性细胞数的差异均无统计学意义(P=0.578、0.180).结论 CNTF能够下调急性高眼压眼谷氨酸在RGCs中的表达,从而对RGCs提供保护作用.