挫伤性视网膜病变中光感受器细胞凋亡与氧化损伤的实验研究

目的　探讨挫伤性视网膜病变中视网膜损伤的凋亡机制及诱因。方法　以 3J能量自由落体的方式制作兔眼挫伤性视网膜病变模型 ,通过光、电镜及TUNEL染色法观察视网膜病变及细胞凋亡情况 ,测定视网膜匀浆中MDA含量与SOD活性的变化 ,并行统计学分析。结果　以 3J能量挫伤后的视网膜病变中存在着光感受器细胞凋亡现象。随挫伤时间推移 ,视网膜MDA的含量逐渐升高 (P <0 .0 1) ,SOD活性在挫伤后早期反射性升高 (P <0 .0 1) ,3d时明显下降 (P <0 .0 1) ,且与凋亡细胞出现的时间一致。结论　细胞凋亡是挫伤性视网膜病变的一个重要机制。活性氧自由基是挫伤性视网膜病变中光感受器细胞凋亡的重要诱因。     

挫伤性视网膜病变中光感受器细胞凋亡与相关基因表达的研究

目的探讨挫伤性视网膜病变中Bax、Bcl-2表达与光感受器细胞凋亡的关系。方法自由落体法制作视网膜挫伤模型,行HE染色观察大鼠视网膜组织的病变情况;末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记(TUNEL)法检测感光细胞凋亡情况;PV免疫组织化学法检测视网膜组织中Bax、Bcl-2的表达。结果TUNEL染色显示挫伤后3d组视网膜内可见较多阳性表达的细胞,分布在外核层。余实验组未测到阳性着色细胞。Bax在挫伤后4h即有表达,随时间延长表达逐渐增加,至挫伤后3d达高峰,至7d、14d组表达阴性。Bcl-2的表达在各组均为阴性。结论细胞凋亡是挫伤性视网膜病变的重要机制之一,Bcl-2/Bax相对比值改变可能对细胞凋亡的发生起一定的诱导作用。     

挫伤性视网膜病变大鼠P53基因表达与光感受器细胞凋亡的关系

目的 观察鼠眼挫伤性视网膜病变后不同时间点视网膜感光细胞的改变及凋亡相关基因p53的表达.方法 自由落体法制作视网膜挫伤模型.49只SD大鼠随机分为正常对照组和视网膜挫伤组,后者又按照挫伤后时间的不同分为1 h、4 h、1 d、3 d、7 d、14 d组,每只大鼠左眼为实验眼,右眼为实验对照眼.行HE染色观察大鼠视网膜组织的病变情况;TUNEL法检测感光细胞凋亡情况;免疫组织化学法检测视网膜组织中p53的表达.结果 TUNEL染色显示正常对照组及实验对照组大鼠视网膜组织未见凋亡阳性细胞.视网膜挫伤后3 d视网膜内可见较多阳性表达的细胞,分布在外核层.挫伤后1h、4 h、1 d、7 d、14 d,大鼠视网膜组织均未见到凋亡细胞.p53在挫伤后4 h即有表达,分布在外核层,随时间延长表达逐渐增加,至挫伤后3 d达高峰,至挫伤后7 d、14 d未见阳性表达.挫伤后4 h、1 d和3 d,视网膜p53阳性细胞光密度值(OD值)差异比较有显著性意义(P＜0.01).正常对照组及实验对照组大鼠视网膜组织未见p53阳性表达.结论 大量感光细胞的凋亡是挫伤性视网膜病变的重要机制之一,p53基因的表达可能介导了光感受器细胞凋亡的过程.     

视网膜挫伤兔模型的病理学观察

目的:建立视网膜挫伤的动物模型,观察视网膜损伤后的形态特点。方法:选取40只健康成年无眼疾青紫蓝兔,随机分为挫伤后1,3h;1,3,7,14,30d及正常对照共8组,每组5只,选取右眼为致伤眼,以改良Allen重击法制备兔单眼挫伤性视网膜病变模型,挫伤后获取兔眼标本,以光镜及电镜观察视网膜神经感觉层的病理变化,目镜测微尺(0.01mm)对视网膜神经纤维层(nerve fiber layer,NFL)厚度、内核层(inner nucler layer,INL)厚度进行测量,并对视网膜神经节细胞(ganglion cell,GC)计数。结果:挫伤后1,3h组和1d组NFL明显增厚(P<0.05),而7d组和14d组NFL明显变薄(P<0.05),3d组和30d组NFL厚度与正常对照组比较无显著性差异(P>0.05);各挫伤组GC计数明显少于正常对照组(P<0.05)。电镜显示挫伤后3h组;1,3d组视网膜神经感觉层均出现较多的,具有凋亡形态学与生化改变特征的凋亡细胞,其中在3d组视网膜神经感觉层凋亡细胞数量达到高峰,7d组显著下降。结论:视网膜水肿和神经感觉层细胞凋亡是挫伤性视网膜病变的一个重要机制。     

复方丹参注射液治疗兔视网膜挫伤的实验研究

目的:研究复方丹参注射液(Danshen injection)在实验性兔视网膜挫伤时对视网膜细胞凋亡的干预作用。方法:青紫兰兔44只分为实验对照组和复方丹参注射液治疗组。以改良Allen’s重击法制备兔右眼挫伤性视网膜病变模型,两组在建立模型后1,3h;1,3,7,14,28d分别取眼球。另设立正常对照组2只青紫兰兔,不作任何处理,在实验观察结束时取眼球。采用原位末端标记法(TUNEL)和透射电子显微镜观察视网膜细胞凋亡情况,并进行细胞计数。结果:视网膜挫伤后存在着神经感觉层细胞凋亡现象。在实验对照组和复方丹参注射液组两组中,正常对照组、1h,28d组几乎不见凋亡细胞,挫伤后3h;1,3d组在视网膜各细胞层均出现较多的、具有凋亡形态学与生化改变特征的凋亡细胞,其中在3d组视网膜感觉层细胞凋亡细胞数量达到高峰。复方丹参注射液治疗组的凋亡细胞数比实验对照组少,有显著性差异(P<0.01)。结论:实验性视网膜挫伤中,复方丹参注射液治疗能抑制视网膜细胞凋亡的发生。     

视网膜挫伤后神经感觉层细胞凋亡的实验研究

目的 了解实验性视网膜挫伤后神经感觉层细胞凋亡情况,并进一步探讨凋亡的发生机制,为临床治疗提供理论基础.方法 40只健康成年无眼疾青紫兰兔,随机数字表法分为挫伤后1 h、3 h、1 d、3 d、7 d、14 d、1个月及正常对照组8组,每组5只,选取右眼为致伤眼,以改良Allen重击法制备兔单眼挫伤性视网膜病变模型;分别于挫伤后1 h、3h、1 d、3 d、7 d、14 d、1个月获取兔眼标本,以电镜及TUNEL法观察视网膜神经感觉层细胞的凋亡情况.结果 视网膜挫伤后存在着神经感觉层细胞凋亡现象,正常对照组、14 d和1个月组几乎不见凋亡细胞,挫伤后3 h、1 d、3 d在视网膜各细胞层均出现较多的、具有凋亡形态学与生化改变特征的凋亡细胞,其中在3 d组视网膜感觉层凋亡细胞数量达到高峰.视网膜神经感觉层细胞数目在3 h、1 d、3 d组及7 d组之间两两比较均有显著统计学意义(P＜0.01);3 h、1 d、3 d组及7 d组与正常对照组、1 h、14 d、1个月组之间两两比较均有显著统计学意义(P＜0.01);正常对照组、1 h、14 d、1个月组之间两两比较无显著性意义(P＞0.05).结论 视网膜挫伤后,神经感觉层细胞凋亡的发生是视网膜功能恢复不良的重要原因.     

增生性玻璃体视网膜病变玻璃体切割物的细胞凋亡观察

目的观察增生性玻璃体视网膜病变玻璃体切割物的细胞凋亡情况 。方法收集60例不同分级的增生性玻璃体视网膜病变患者的玻璃体切割物,核苷酸末端转移酶介导dUTP缺口翻译法(TdT-mediated dUTP nick end label ling method,TUNEL法)标记凋亡细胞,光镜观察。结果60例患者的玻璃体切割物均有细胞凋亡的特征性改变,随着病变程度的加重,非色素细胞凋亡总数逐渐减少,并出现色素细胞凋亡。结论增生性玻璃体视网膜病变存在不同类型细胞的凋亡,细胞凋亡是调控其病变程度的重要机制之一。(中华眼底病杂志,1999,15:81-83)     

N-乙酰半胱胺酸对大鼠挫伤性视网膜病变的保护作用

目的:研究凋亡相关基因Bcl-2/Bax在大鼠挫伤性视网膜病变中的表达,以及N-乙酰半胱氨酸(N-acetyl-cysteine,NAC)对其表达的影响。方法:自由落体法制作视网膜挫伤模型。将SD大鼠随机分为正常组6只、模型组24只及治疗组24只。每组按挫伤后不同时间段分为1,3,7,14d,每个观察时段6只大鼠。HE染色光镜观察视网膜组织学变化,应用SABC免疫组织化学法检测大鼠视网膜挫伤后视网膜组织Bcl-2/Bax蛋白表达的变化。结果:挫伤性视网膜病变的损害主要集中在神经纤维层。挫伤后视网膜组织水肿,细胞紊乱,胞浆空泡样变,视网膜组织变薄,细胞丢失。NAC治疗组视网膜水肿程度有所改善,细胞紊乱,胞浆空泡样变,细胞丢失有所恢复。正常组及NAC治疗组视网膜组织Bax均未见表达,Bcl-2低表达。视网膜挫伤后1d时Bax表达开始增多,3d强阳性表达,7d表达有所减少,14d时表达进一步减少。Bcl-2低表达,未见明显变化。NAC治疗组各观察指标变化趋势基本与视网膜挫伤组相似,但表达明显减弱,两组相比较,于挫伤后1,3d和7d时差异有统计学意义(P<0.05)。Bcl-2在各时段的表达均较模型组有所增强,两组相比较,于挫伤后1,3d和7d时差异有统计学意义(P<0.01)。结论:在大鼠挫伤性视网膜病变中,NAC能够改善视网膜组织病理学损害并通过调节Bcl-2/Bax蛋白表达而抑制细胞凋亡,对视网膜挫伤具有保护作用。     

兔视网膜挫伤后Müller细胞Vimentin表达的变化

目的:探讨兔视网膜挫伤后Müller细胞波形蛋白(Vimentin)表达的变化方法:通过3J能量自由落体方式制作兔眼挫伤性视网膜病变模型,于伤后1/8,1,3,7,14d时处死动物取材,免疫组化染色和计算机图像分析仪检测视网膜挫伤后Müller细胞Vimentin的表达和分布.结果:视网膜挫伤后1d Vimentin开始阳性表达增强,7d达到高峰,14d略有下降.随着视网膜挫伤时间的延长,Vimentin的免疫染色范围也逐渐向外扩展,3d时免疫染色达外界膜,7d时视网膜全层都有表达,二组比较,各时段差别均P＜0.01,存在统计学差异.结论:视网膜挫伤后Müller细胞Vimentin反应动态增强.     

兔视网膜挫伤后Müller细胞Vimentin表达的变化

目的：探讨兔视网膜挫伤后Müller细胞波形蛋白（Vimentin）表达的变化 方法：通过3J能量自由落体方式制作兔眼挫伤性视网膜病变模型,于伤后1/8,1,3,7,14d时处死动物取材,免疫组化染色和计算机图像分析仪检测视网膜挫伤后Müller细胞Vimentin的表达和分布。 结果：视网膜挫伤后1d Vimentin开始阳性表达增强,7d达到高峰,14d略有下降。随着视网膜挫伤时间的延长,Vimentin的免疫染色范围也逐渐向外扩展,3d时免疫染色达外界膜,7d时视网膜全层都有表达,二组比较,各时段差别均P〈0.01,存在统计学差异。 结论：视网膜挫伤后Müller细胞Vimentin反应动态增强。     

Retinal degeneration,apoptosis and the c-fos gene

In the past few years, the interest in the research field of apoptosis in the retina has been growing rapidly. We will give a short overview of apoptosis in the context of retinal degeneration and summarize recent data obtained in our laboratory. Based on our findings, we will also discuss possible future strategies to influence apoptotic cell death in the retina and to modulate the time course of retinal dystrophies. Apoptosis is the final common pathway of photoreceptor cell death in several retinal dystrophies as well as in light-induced photoreceptor degeneration. We investigated potential signal transducers for apoptosis in our laboratory and found an essential role of the immediate-early gene product c-Fos in light-induced photoreceptor degeneration. This is of particular interest in light of the finding that c-fos is continuously upregulated concomitant with apoptotic photoreceptor death in animal models of the retinal dystrophy retinitis pigmentosa. Interference with c-fos expression or function might therefore represent a novel means to influence the time course of retinal dystrophies, which are at present incurable diseases.     

Ceramide is a mediator of apoptosis in retina photoreceptors

PURPOSE: The precise mechanisms involved in photoreceptor apoptosis are still unclear. In the present study, the role of ceramide, a sphingolipid precursor that induces apoptosis on cellular stress, was investigated in relation to the activation of cell death in photoreceptors. METHODS: Rat retina neuronal cultures, with or without docosahexaenoic acid (DHA), were treated with the ceramide analogue acetylsphingosine (C2-ceramide), and with a glucosylceramide synthase inhibitor. Ceramide synthesis in cultures treated with the oxidant paraquat was evaluated with [3H]palmitate. The effect of inhibitors of ceramide de novo synthesis, fumonisin B1 and cycloserine, on photoreceptor apoptosis was investigated. Apoptosis, mitochondrial membrane potential, and Bcl-2 expression were determined. RESULTS: Addition of C2-ceramide induced photoreceptor apoptosis. Paraquat increased formation of [3H]ceramide in photoreceptors, compared with the control, whereas inhibition of ceramide synthesis, immediately before paraquat treatment, prevented paraquat-induced photoreceptor apoptosis. Fumonisin also reduced photoreceptor apoptosis during early development in vitro. DHA, the retina major polyunsaturated fatty acid, which protects photoreceptors from oxidative stress-induced apoptosis, completely blocked C2-ceramide-induced photoreceptor death, simultaneously increasing Bcl-2 expression. Inhibiting glucosylceramide synthase, which catalyzes ceramide glucosylation, before ceramide or paraquat treatment blocked DHA's protective effect. CONCLUSIONS: The results suggest that oxidative stress stimulated an increase in ceramide levels that induced photoreceptor apoptosis. DHA prevented oxidative stress and ceramide damage by upregulating Bcl-2 expression and glucosylating ceramide, thus decreasing its intracellular concentration. This shows for the first time that ceramide is a critical mediator for triggering photoreceptor apoptosis in mammalian retina and suggests that modulating ceramide levels may provide a therapeutic tool for preventing photoreceptor death in neurodegenerative diseases.     

Preventing retinal detachment-associated photoreceptor cell loss in Bax-deficient mice

PURPOSE: To characterize photoreceptor cell apoptosis and cell loss in a mouse model of experimental retinal detachment (RD), and to use the technology of mouse genetics to study the molecular mechanisms underlying RD-associated photoreceptor degeneration. METHODS: Retinal detachments were created in adult wild-type and Bax-deficient mice by subretinal injection of 1.4% sodium hyaluronate. At 1, 3, 7, and 28 days after injection, animals were killed, eyes enucleated, and retinal sections studied by histochemistry, immunofluorescence labeling, and confocal microscopy. Rods and cones were labeled, and apoptotic cells were identified with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Photoreceptor cell apoptosis and cell loss were assessed quantitatively by counting both surviving and TUNEL-positive rods and cones. RESULTS: TUNEL-positive cells were found within the outer nuclear layer (ONL) of the detached portions of the retina. They were detected in the detached retina on day 1, peaked on day 3, and dropped precipitously after day 7 after RD. Photoreceptor cell loss of both rods and cones followed a similar time course after RD. Moreover, deletion of the proapoptotic gene Bax in a knockout mouse model abolished the RD-associated photoreceptor cell degeneration. CONCLUSIONS: Apoptosis is a major mechanism leading to photoreceptor cell death after RD. Blockage of the activity of the proapoptotic molecule Bax in a knockout mouse model prevents photoreceptor cell apoptosis and cell loss. These data suggest that the Bax-mediated apoptotic signaling pathway plays a critical role in RD-associated photoreceptor cell death.     

实验性兔视网膜挫伤后不同时段的视网膜厚度研究

目的探讨实验性兔视网膜挫伤后不同时段视网膜厚度的变化。方法40只健康成年无眼疾青紫兰兔，随机数字表法分为挫伤后1、3h组，1、3、7、14d组，1个月组及正常对照组共8组，每组5只，选取右眼为致伤眼，以改良Allen’s重击法，致伤能量约为2．87J（E=mgh），制备兔眼挫伤性视网膜病变模型，分别于挫伤后1、3h，1、3、7、14d，1个月处死兔子，摘除眼球并取材，通过光镜观察视网膜的情况，目镜测微尺（0．010mm）对视网膜神经纤维层（NFL）厚度、内核层（INL）厚度进行测量，并对视网膜神经节细胞（GC）计数。结果挫伤后1、3h组和1d组NFL明显增厚（P〈0．05），而7d组和14d组NFL明显变薄（P〈0．05），3d组和1月组NFL厚度与正常对照组比较差异无显著性意义（P〉0．05）；挫伤后1、3h组、1d组INL明显增厚（P〈0．05），而7、14d组明显变薄（P〈0．05），3d组和1个月组INL厚度与正常对照组比较差异无显著性意义（P〉0．05）；各挫伤组GC计数明显少于正常对照组（P〈0．05）。结论视网膜厚度的改变是挫伤性视网膜病变的一个重要病理现象。     

Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors

PURPOSE. Simple sphingolipids control crucial cellular processes in several cell types. Previous work demonstrated that sphingolipids, such as ceramide, sphingosine, and sphingosine-1-phosphate, are key mediators in the regulation of survival, differentiation, and proliferation of retina photoreceptors. Ceramide-1-phosphate (C1P) regulates growth and survival in several cell types; however, little is known concerning its functions in the retina. Whether C1P also participates in controlling photoreceptor development was also explored. METHODS. Rat retina neuronal cultures were supplemented with 1 to 10 μM C1P. Proliferation was determined by evaluating 5-bromo-2-deoxyuridine (BrdU) uptake and the number of mitotic figures and differentiation by evaluating opsin and peripherin expression by immunocytochemistry and Western blot. Apoptosis was inhibited with the pan caspase inhibitor ZVADFMK and evaluated by TUNEL assay, propidium iodide/annexin V, and DAPI labeling. Preservation of mitochondrial membrane potential was evaluated. RESULTS. C1P enhanced BrdU uptake and increased mitosis in retinal progenitors. C1P addition advanced photoreceptor differentiation, enhancing opsin and peripherin expression and stimulating development of the apical processes in which these proteins were concentrated. In the absence of these trophic factors, photoreceptors degenerated after 4 days in vitro, and at day 6, almost 50% of photoreceptors were apoptotic. C1P decreased photoreceptor apoptosis, reducing this percentage by half. Inhibiting caspase activity reduced photoreceptor apoptosis in the controls, but did not increase opsin expression, implying that C1P has separate effects on differentiation and survival. CONCLUSIONS. These results suggest for the first time that C1P is a novel mediator that has multiple functions in photoreceptors, initially regulating their proliferation and then promoting their survival and differentiation.     

Proapoptotic bcl-2 family members,Bax and Bak,are essential for developmental photoreceptor apoptosis

PURPOSE: Apoptosis has been implicated in retinal development and degeneration, but the specific apoptotic pathways used are incompletely understood. The purpose of this study was to characterize the roles in retinal development of the proapoptotic Bcl-2 family members Bax and Bak. METHODS: Eyes from mice at postnatal day (P)7, during the peak of developmental apoptosis in the retina, were processed for TdT-dUTP terminal nick-end labeling (TUNEL) to determine whether Bax knockout or double Bax/Bak knockout causes a defect in developmental apoptosis. Adult (>2-month-old) eyes from wild-type, Bak(-/-), Bax(-/-), and Bax(-/-)Bak(-/-) mice were analyzed by histology and immunocytochemistry to identify persistent retinal cells. RESULTS: Adult Bax(-/-)Bak(-/-) eyes showed significant increases in the number of inner retinal cells, with an almost complete absence of TUNEL-positive cell death at P7. Some of these persistent cells in the inner retina notably included rod photoreceptors that normally undergo apoptosis after failure to migrate to the outer retina. These inner nuclear layer (INL) rods contained markers of early rod differentiation: rod opsin, arrestin, and recoverin. However, they did not form ectopic outer segments or contain the associated markers ROM-1, peripherin-2, and RP1. CONCLUSIONS: Bax and Bak are important for retinal development and are the first apoptotic factors identified as essential for developmental photoreceptor apoptosis. Future studies will investigate the potential role of Bax and Bak in mediating pathologic photoreceptor death.     

Multiple, parallel cellular suicide mechanisms participate in photoreceptor cell death

Photoreceptor degeneration in human photoreceptor dystrophies and in the relevant animal models has been thought to be executed by one common mechanism -- caspase-mediated apoptosis. However, recent experiments have challenged this concept. In previous experiments, analyzing gene expression in the degenerating rd/rd mouse retina, we have suggested that the gene defect leads to oxidative stress and altered metabolism, which may induce caspase-dependent and caspase-independent cell death mechanisms such as the activation of cystein-proteases, lysosomal proteases, autophagy and complement-mediated lysis. In this study we asked two questions. First, whether a temporal analysis of these different mechanisms during the course of degeneration would enable us to establish a causal relationship between these events; and second, whether photoreceptor degeneration in different models of photoreceptor dystrophies occurs by activating the same mechanisms. Three models of photoreceptor degeneration were chosen in which photoreceptor degeneration is caused by different events: the rd/rd mouse (calcium overload); the rds/rds mouse (structural defect); and light-damage (LD; oxidative stress). Marker genes were selected for the identified processes. PCR-analysis on laser capture microdissection samples was used to verify the expression of these genes in the rod photoreceptor layer. A temporal relationship between the processes was established at the mRNA level, using quantitative RT-PCR. The time course of gene expression was compared to that of cell loss (loss of rows of photoreceptor nuclei) and apoptosis (TUNEL labeling). Apoptosis and autophagy was analyzed using enzymatic assays. The time course of apoptosis and TUNEL labeling coincide in all three models. Complement-activated lysis was found to either parallel (rd/rd and rds/rds) or precede (LD) the development of TUNEL-positive cells. Autophagy was determined to parallel (rd/rd and LD) or lag (rds/rds) behind the development of TUNEL-positive cells. In all three models, glucose metabolism was found to be increased significantly prior to the onset of cell death, but then dropped in parallel with the loss of cells. The presence of the marker genes was verified by laser capture microdissection, and apoptosis (caspase activity) and autophagy (lysozyme and cathepsin activity) were verified in retina extracts. These results provide evidence that irrespective of whether photoreceptor degeneration is triggered by gene defects (lack of beta-PDE or rds/peripherin) or environmental stress (light-damage), a number of pro-apoptotic mechanisms are triggered leading to the degeneration of the photoreceptor cells. The temporal pattern of the different pathways suggests that the non-caspase-dependent mechanisms may actively participate in the demise of the photoreceptors, rather than represent a passive response of the retina to the presence of dying cells. Thus, unless the common upstream initiator for a given photoreceptor dystrophy is found, multiple rescue paradigms need to be used to target all active pathways.