Retinal ganglion cell death and optic nerve degeneration by genetic ablation in adult mice

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER™ transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.     

睫状神经节烧灼加视神经剪断术治疗绝对期青光眼

目的观察睫状神经节烧灼及视神经剪断术治疗伴有剧烈疼痛的绝对期青光眼的疗效。方法对15例(15眼)确诊为绝对期青光眼并伴有剧烈疼痛者,采用睫状神经节烧灼加视神经剪断术进行治疗。术中先行外直肌剪断。然后剪断视神经,翻转眼球暴露睫状神经节用烧灼器行睫状神经节烧灼。结果15例(15眼)术后1周内所有症状全部消失。出院后3a内定期追踪观察,无1例复发,保留眼球有效率为100％。结论睫状神经节烧灼加视神经剪断术方法简单,易于开展,在绝对期青光眼的治疗中有较好的疗效。     

氨基胍对视神经损伤后视网膜神经节细胞的保护性作用

视神经损伤是眼科常见疾病,多并发于颅脑外伤,预后不良,常致患者失明。由于视神经损伤的发病机制尚未完全明了,所以迄今为止其治疗仍是国内外眼科界的一大难题。现将视神经损伤后视网膜神经节细胞（retinal ganglion cells,RGCs）凋亡及氨基胍（Aminogunidine,AG）对其保护性作用做一综述。     

大剂量甲基泼尼松龙对大鼠视神经挤压伤后RGC凋亡的影响

目的　观察大剂量甲基泼尼松龙 (MP)对大鼠视神经挤压伤后视网膜神经节细胞 (RGC)凋亡的影响。方法　大鼠 12 6只分为正常对照组 18只 3 6眼、损伤组 54只 54眼和MP治疗组 54只 54眼。伤后 4、7和 14d ,通过视网膜铺片和切片相结合的技术 ,进行细胞原位凋亡检测以及Bcl 2和Bax阳性细胞计数 (免疫组化SP法 )。结果　正常对照组有少量凋亡细胞 ,Bcl 2和Bax阳性细胞数少 ;伤后 4、7、14d凋亡细胞大量增加 ,治疗组凋亡细胞各时间点均低于损伤组 ,差异有显著性 (P <0 0 5) ;治疗组Bcl 2阳性细胞数各时间点均高于损伤组 ,Bax阳性细胞数均低于损伤组 ,差异有显著性 (P <0 0 5)。结论　大剂量MP能抑制视神经挫伤后RGC的凋亡 ,上调bcl 2基因的表达和下调bax基因的表达可能是MP治疗作用机制之一     

兔视神经损伤后视网膜神经节细胞MDA/SOD的变化

目的:观察兔视神经损伤后视网膜神经节细胞(retinal ganglion cell,RGC)丙二醛(malondialdehyde,MDA)/超氧化物歧化酶(superoxide dismutase,SOD)的变化。方法:选用新西兰白兔32只,分为对照组和损伤组,损伤组再根据损伤后不同时间分为3,7,14d组,每组8只16眼。采用硫代戊巴比妥酸法和黄嘌呤氧化法分别测定视网膜MDA/SOD含量。结果:损伤实验组MDA含量分别为5.95±0.78,7.67±0.64和8.29±1.02μmol/g,均明显高于正常对照组(3.82±0.54μmol/g);实验各组SOD含量分别为510±44,415±29和398±36μkat/g,均明显低于正常对照组(727±52μkat/g)。结论:自由基代谢在兔视神经损伤后RGC凋亡中起着重要作用,MDA/SOD含量测定在RGC凋亡的研究中有一定的意义。     

兔视神经损伤后视网膜神经节细胞线粒体跨膜电位的变化观察

目的观察兔视神经损伤后视网膜神经节细胞（retinal ganglion cell,RGC）线粒体跨膜电位（mitochondrial transmembrane potentials,△ψm）的变化。方法选用新西兰白兔32只,分为对照组和损伤组,损伤组再根据损伤后不同时间分为3d、7d、14d组,共4组,每组8只兔16眼。采用Rh123染色,流式细胞术测定RGC△ψm。结果正常新西兰白兔RGC△ψm为3836.67±21.32。在损伤后3d、7d、14d,视网膜细胞△ψm分别为3055.41±7.74、1822.07±18.66、2617.38±7.37。各时间点的△ψm均明显低于正常对照组,且各时间点间△ψm差异有统计学意义（P〈0.05）。结论△ψm在RGC凋亡早期的观测中有一定的意义,并且能进行定量分析研究。     

Mouse models of retinal ganglion cell death and glaucoma

Once considered too difficult to use for glaucoma studies, mice are now becoming a powerful tool in the research of the molecular and pathological events associated with this disease. Often adapting technologies first developed in rats, ganglion cell death in mice can be induced using acute models and chronic models of experimental glaucoma. Similarly, elevated IOP has been reported in transgenic animals carrying defects in targeted genes. Also, one group of mice, from the DBA/2 line of inbred animals, develops a spontaneous optic neuropathy with many features of human glaucoma that is associated with IOP elevation caused by an anterior chamber pigmentary disease. The advent of mice for glaucoma research is already having a significant impact on our understanding of this disease, principally because of the access to genetic manipulation technology and genetics already well established for these animals.     

地塞米松对兔视神经钳夹伤后视网膜神经节细胞存活及Nogo-A表达的影响

目的观察地塞米松对家兔视神经钳夹伤后视网膜神经节细胞(retinal ganglion cell,RGC)存活的影响及损伤视神经、视网膜Nogo-A表达的变化。方法采用兔球后视神经钳夹伤模型,健康成年家兔分为正常对照组、损伤组、治疗组。分别于损伤后3d、7d、14d处死动物,观察单位面积RGC存活数量及损伤后Nogo-A在视神经、视网膜的表达变化。结果视神经损伤后,治疗组RGC的存活数高于损伤组及对照组(P<0.01)。对照组、损伤组损伤后3d、7d、14d视神经、视网膜Nogo-A表达增强,至损伤后7d达高峰;治疗组视神经、视网膜表达亦增强,各时间点均弱于损伤组、对照组,差异有显著统计学意义(P<0·01)。结论视神经损伤后,地塞米松能够增加RGC的存活数量,能够下调No-go-A的表达,这可能是地塞米松治疗作用机制之一。     

Citicoline and lithium rescue retinal ganglion cells following partial optic nerve crush in the rat

Citicoline and lithium (Li(-)) have been shown to support retinal ganglion cell (RGC) survival and axon regeneration in vitro. Optic nerve crush (ONC) is a model of both brain axonal injury and certain aspects of the glaucomatous degeneration of RGC. We have used this model to quantify protection offered to RGC by these drugs and to determine whether their effects are mediated by enhanced expression of the antiapoptotic protein Bcl-2. Adult rats (6-12 per group) were subjected to ONC accompanied by a contralateral sham operation. Animals were treated intraperitoneally with either vehicle, citicoline sodium (1g/kg daily for up to 7 days and 300 mg/kg daily afterwards), lithium chloride (30 mg/kg daily), or both drugs combined. Fluorogold was injected bilaterally into superior colliculi 1, 5 or 19 days after ONC. Labeled cells were counted under a fluorescence microscope 2 days after tracer injection. In a separate set of experiments the effects of treatments on expression of Bcl-2 in retinas were evaluated by immunohistochemistry. In vehicle-treated animals there was a progressive decrease of RGC density after crush. This decrease was attenuated in citicoline-treated animals 1 week and 3 weeks after the crush. In the lithium-treated group protection was even more pronounced. In animals treated with both drugs RGC protection was similar to that achieved by lithium alone. Bcl-2 immunoreactivity was seen predominantly in retinal ganglion cells. Its increase was recorded in the lithium and citicoline group as well as in animals treated with the combination of both drugs. Both citicoline and lithium protect RGC and their axons in vivo against delayed degeneration triggered by the ONC. Retinoprotective action of both drugs may involve an increase in Bcl-2 expression.     

利用荧光金逆行示踪技术评价视神经不完全损伤后视网膜神经节细胞的存活率

目的　利用荧光金逆行示踪技术评价正常及视神经不完全损伤后的视网膜神经节细胞 (retinalganglioncells,RGCs)的数目。 方法　正常成年LongEvans大鼠 2 0只 ,体重(2 70± 2 0 ) g ,雌雄不限。按对照组、损伤后 7d、14d、2 1d分组 ,每组 5只大鼠。在球后视神经钳夹伤前 7d行荧光金逆行标记。 7d后损伤组大鼠用反向血管夹于左眼球后 2mm处夹视神经 10s。对照组大鼠只暴露视神经不行钳夹 ,分别于各时间点将大鼠用 4 %多聚甲醛灌注固定后 ,行全视网膜铺片 ,3h内在荧光显微镜下观察。在每张视网膜的上、下、鼻、颞侧距视盘 1/ 6、1/ 2、5 / 6半径处共拍摄 12张荧光照片。对照片上标记的RGCs进行计数 ,求平均值 ,计算损伤后各时间点剩余的RGCs与正常视网膜中RGCs的百分比。结果　视网膜铺片的RGCs边界清晰 ,并可见明显的细胞突起 ,血管走行区未见节细胞分布。正常组每张铺片的平均节细胞数为 (2 0 31± 2 87)个·mm-2 ,损伤后 7d的RGCs存活率为 71% ,14d存活率为 5 1% ,2 1d存活率为 35 %。结论　荧光金逆行标记是评价视神经损伤后RGCs存活率可靠并且有效的方法。     

蛇毒神经生长因子对大鼠视神经夹伤保护的电镜观察

目的研究蛇毒神经生长因子在视神经损伤后对视网膜神经节细胞的保护作用。方法将Wistar大鼠40只随机分为实验对照组和实验治疗组。制作实验性视神经夹伤模型,用头部宽1mm的微型血管夹夹伤大鼠右眼视神经后,实验治疗组向伤眼玻璃体腔内注入蛇毒神经生长因子100BU(0.025mL)。实验对照组向伤眼玻璃体腔内注入0.025mL平衡盐液。于损伤后第3d、7d、14d、30d、60d取材,用透射电镜观察不同时间段各组视网膜形态学变化。结果电镜下大鼠视网膜改变:实验治疗组和对照组电镜下均可见坏死和凋亡。伤后14d,实验治疗组视网膜微管数目比实验对照组较多,排列比较整齐。结论在视神经损伤早期,蛇毒神经生长因子能减轻视神经夹伤后微管的损坏,提高视网膜神经节细胞的存活数量,对视网膜神经节细胞有明显的保护作用。     

青光眼视神经保护治疗的研究进展

青光眼是由于眼压升高引起视乳头损害和视野缺损的一种致盲性眼病,其病理基础是视网膜神经节细胞及其轴突的进行性丢失。过去大量的研究都集中在降低眼压方面,现在视神经保护治疗作为一种通过阻止神经元死亡治疗青光眼的新策略已被普遍接受。我们从NMDA受体拮抗剂、神经营养因子、热休克蛋白、免疫系统等方面,总结了目前青光眼视神经保护治疗的研究进展。     

Brn3b基因对视神经损伤条件下视网膜神经节细胞的保护作用

目的　探讨Brn3b过表达对视神经损伤条件下视网膜神经节细胞（retinal ganglion cells,RGCs）的保护作用。方法　选取雄性健康成年BALB/c小鼠60只,用微型视神经夹将小鼠右眼球后视神经夹持损伤,按视神经损伤后的天数依次分为第1、3、5、7、14天组,小鼠左侧正常眼作为空白组,明确视神经损伤天数条件。选取雄性健康成年BALB/c小鼠40只,用微量进样器以小鼠右眼玻璃体内注射的方法将腺相关病毒载体转染小鼠视网膜,建立Brn3b过表达模型并分组:Brn3b过表达组［转染Brn3b过表达腺相关病毒载体（Brn3b overexpressed adeno-associated viral vector,AAV-CMV-Brn3b）］和阴性对照组［转染空白腺相关病毒载体（blank adeno-associated viral vector,AAV-CMV-GFP）阴性对照物］,每组20只;之后,取Brn3b过表达组和阴性对照组各10只,用微型视神经夹将小鼠右眼球后视神经夹持损伤,构建模拟小鼠视神经损伤（controlled optic nerve crush,CONC）模型并分组:CONC-Brn3b过表达组和CONC-阴性对照组。利用视网膜铺片和切片免疫荧光标记相关蛋白表达量,检测Brn3b过表达对视神经损伤条件下RGCs、Brn3b和Caspase3蛋白表达的影响,并对其共定位情况做出统计分析。结果　与阴性对照组相比,Brn3b过表达组小鼠视网膜Brn3b的表达水平明显增加。在小鼠CONC模型制作后的第7天RGCs的总凋亡数量达到65%,第14天RGCs的凋亡数量未见进一步改变。免疫荧光标记的蛋白表达量及其共定位分析显示,在视神经损伤条件下,与CONC-阴性对照组相比,CONC-Brn3b过表达组小鼠RGCs的凋亡量以及凋亡因子Caspase3的表达量均明显减少,差异均有统计学意义（均为P＜0.001）。结论　Brn3b基因对视神经损伤条件下RGC具有明确的保护作用,Brn3b基因对凋亡因子Caspase3的表达可能具有一定的抑制作用。     

Optical coherence tomography evaluation of the optic nerve head neuro‐retinal rim in glaucoma

Clinical examination of the optic disc is a fundamental component of any ophthalmic evaluation, but it is especially important for diagnosis and management of glaucoma. The purpose of this article is to: (1) review the limitations inherent to clinical examination; (2) outline the rationale for adopting into clinical practice quantitative measures of the optic nerve head neuro‐retinal rim tissue integrity derived from current optical coherence tomography imaging approaches; (3) describe recent developments in this area; and (4) highlight a few avenues of active research that hold promise for future translation to clinical practice.     

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     

去乙酰化酶Sirt1在视神经视网膜疾病中的神经保护作用

作为一类广泛参与细胞能量代谢、周期控制和免疫应答等生命过程的重要蛋白,去乙酰化酶Sirt1还具有明显的神经元保护作用。同时,Sirt1在许多视网膜疾病动物模型的干预中展示了不可忽视的作用。本文对这一领域的研究进展进行了综述,主要包括：在糖尿病视网膜病变中,Sirt1能够抑制视网膜的慢性炎症反应、减少视网膜新生血管生成并清除视网膜细胞的高血糖记忆;在视神经炎和青光眼模型中,Sirt1能保护视网膜神经节细胞,减少其凋亡;而在年龄相关性黄斑变性模型中,Sirt1能保护视网膜色素上皮细胞的活力和功能,并降低补体因子引起的视网膜自身免疫反应。在脉络膜新生血管生成过程中,Sirt1可能具有促血管增生作用,但尚需进一步研究。由于Sirt1活性对于减轻多种视网膜疾病的组织损伤均具有重要作用,因此Sirt1是治疗视网膜病变很有前景的靶点。     

The pattern electroretinogram in retinal and optic nerve disease

A retrospective analysis was performed on the transient and steady-state pattern electroretinograms recorded from 42 patients with glaucoma, 13 patients with senile dementia of the Alzheimer's type, 58 patients with diabetes mellitus, and 92 control subjects to evaluate the pattern of electroretinographic changes associated with retinal and optic nerve disease. The amplitudes of both the initial positive component (N1 to P1) and the subsequent negative component (P1 to N2) of the transient (4 rps) responses were measured. From these measurements the (P1 to N2)/(N1 to P1) was derived. The N1 to P1 amplitude of the steady-state pattern electroretinogram also was measured. In the glaucoma patients all three amplitude measures, as well as the amplitude ratio of the components of the transient response, were reduced significantly compared with age-matched controls (p < 0.05). A similar pattern was detected in the patients with Alzheimer's disease, but in this case the only statistically significant amplitude reduction was in the steady-state pattern electroretinogram. A different pattern was observed among the diabetic patients (both with and without retinopathy). Only minor reductions in the amplitude of the transient pattern electroretinogram, which were not statistically significant, were noted. In addition, the ratio of the amplitudes of the components of the transient response did not differ from age-matched controls. The amplitude of the steady-state pattern electroretinogram was reduced in diabetics, but this was significant only for those patients with retinopathy (p < 0.01). These findings support the suggestion that an analysis of both the positive and negative components of the pattern electroretinogram may be useful for differentiating the contributions of retinal and optic nerve dysfunction to visual impairment. The results also indicate that in both retinal and optic nerve disease the steady-state pattern electroretinogram can be an earlier sign of dysfunction than the transient pattern electroretinogram.     

溴莫尼定治疗大鼠视神经夹挫伤机制探讨

目的观察0.2%酒石酸溴莫尼定对视神经夹挫伤大鼠视网膜形态及bcl-2/bax表达的影响,探讨其作用机制。方法雌性SD大鼠90只随机分为正常组、模型组、治疗组,每组30只。60只大鼠用视神经钳夹法制作SD大鼠视神经夹挫伤模型,治疗组30只鼠给予0.2%酒石酸溴莫尼定点眼,于实验的l、3、5、7、14、21d处死大鼠,用苏木精-伊红染色计数各组鼠视网膜神经节细胞（RGCs）的数量,用透射电镜观察和比较各组鼠视网膜的超微结构改变,免疫组织化学染色检测鼠视网膜中bcl-2及bax的表达。结果正常组视网膜结构正常,治疗组较模型组视网膜形态损伤减轻。造模后3～21d,模型组和治疗组较正常对照组RGCs数量明显减少（P〈0.05）,但治疗组较模型组RGCs数量明显增加（P〈0.01）。造模后5～7d,治疗组bcl-2在鼠视网膜中的表达量较模型组明显增加（P〈0.01）,但bax表达量明显减少（P〈0.01）。模型组和治疗组的bcl-2在鼠视网膜中的表达量较正常组增加但bax表达量明显减少（P〈0.05）。结论0.2%酒石酸溴莫尼定对大鼠视神经夹挫伤有一定的治疗作用,其机制与抑制凋亡有关。     

经瞳孔温热疗法对大鼠视神经钳夹视网膜神经节细胞的保护作用

目的探讨经瞳孔温热疗法（TTT）阈下反应对BN大鼠视神经钳夹后视网膜神经节细胞（RGCs）的保护作用。方法采用阈下TTT对BN大鼠视网膜进行照射后3d,通过逆行标记RGCs的方法,对TTT＋视神经钳夹组（A组）、TTT＋假手术组（B组）、单纯视神经钳夹组（C组）和空白对照组（D组）在视神经钳夹后1、2、4周进行RGCs计数并比较;检测视网膜TTT阈下反应的热休克蛋白70（HSP70）表达;观察TTT阈下反应对视网膜的影响。结果视神经钳夹后4周,A组RGCs数显著高于C组（P=0.006）,而1周和2周时2组之间差异无统计学意义（P〉0.05）;各时间点B组和D组的RGCs数差异无统计学意义（P〉0.05）。视网膜经阈下TTT干预后,HSP70表达高于对照眼。阈下TTT照射能引起视网膜组织形态上的改变。结论阈下TTT可显著提高视神经钳夹4周后RGCs的存活数量;其保护机制可能与诱导视网膜内源性HSP70表达、启动内源性保护机制有关。