快速退变性视网膜变性感光细胞超微结构变化分析

目的 了解快速退变性遗传性视网膜变性的感光细胞在出生后早期发生的形态学变化,为临床治疗提供依据.方法 取出生后不同时间的rd小鼠及正常对照小鼠各6只的视网膜,经光镜、扫描电镜和透射电镜观察感光细胞的形态发生发育和结构变化过程,比较二者的动态变化和形态学差异.结果 rd鼠生后1周开始出现感光细胞节段变短,内节段内线粒体变性改变多见;偶见感光细胞核旁胞浆内出现肿胀变形的线粒体.2周时节段层变薄,内节段结构已不完整,外节膜盘少见,变形且排列不整齐;外核层细胞层数明显减少,可见核固缩及染色质凝聚,偶见外丛状层部分神经突起内出现变性的线粒体.3周时节段层近消失,外核层只剩一层细胞,胞浆内可见髓样结构;外丛状层变薄.4周时内节段高度变形,视网膜色素上皮层与外核层之间出现大量不成形结构.外核层仅残存少许胞体,胞浆内出现多量不成形结构.外丛状层极薄,部分区域已消失.结论 rd鼠在出生后早期就发生感光细胞的变性改变,细胞内线粒体改变明显.其感光细胞变性发生早,进展快,呈快速退变特点.     

小胶质细胞活化与rd小鼠遗传性视网膜变性

目的研究小胶质细胞活化与rd小鼠遗传性视网膜变性的关系。方法对出生后8、10、12、14、16及18d的rd小鼠及对照小鼠视网膜进行感光细胞凋亡TUNEL法检测及形态计量学分析。CD11b免疫组织化学染色标记视网膜小胶质细胞。结果rd小鼠出生后10d视网膜感光细胞层开始出现TUNEL染色阳性细胞,第16d达到高峰。视网膜小胶质细胞在rd小鼠出生后10d开始活化,第14d达到高峰。小胶质细胞向感光细胞层的迁移与感光细胞凋亡之间存在紧密的时间和空间关系。结论rd小鼠视网膜变性以感光细胞凋亡为主。小胶质细胞活化可能在视网膜变性过程中发挥重要作用。     

中药复方制剂对rds小鼠感光细胞凋亡的干预作用研究

目的观察中药复方制剂对先天性视网膜变性小鼠视网膜感光细胞凋亡的影响。方法以黄芪等药组成复方制剂1。对先天性视网膜变性动物模型rds小鼠,采用原位末端转移酶标记(TUNEL)方法及组织病理学技术,观察复方制剂1作用后,小鼠视网膜感光细胞数量及感光细胞凋亡的变化。结果仔鼠2周时,中药组小鼠视网膜感光细胞核数与对照组相比无明显差别,两组感光细胞凋亡率分别为1.6%及6.5%,组间差异有显著性(P<0.01);4周时中药组感光细胞核数目较对照组多,差异有显著统计学意义(P<0.01),两组感光细胞凋亡率分别为3.3%及8.5%,组间差异有显著统计学意义(P<0.01)。结论中药复方制剂1可以延缓rds小鼠视网膜色素变性过程中感光细胞凋亡的发展。     

MCP-1在rd小鼠视网膜变性过程中的表达

目的 探讨单核细胞趋化因子(MCP-1)在rd小鼠视网膜变性过程中的表达.方法 RT-PCR反应测定rd小鼠出生后8、10、12、14、16、18 d视网膜MCP-1 mRNA的表达.原位杂交及免疫组织化学检测高峰期MCP-1 mRNA及蛋白在视网膜的定位表达.结果 与正常对照鼠相比,MCP-1 mRNA在rd小鼠视网膜的表达水平于出生后第8 d开始升高,12 d达高峰.MCP-1 mRNA及蛋白的表达出现于视网膜内层,尤其在内核层细胞及节细胞核周围.结论 趋化因子MCP-1在rd小鼠视网膜变性过程中表达升高,提示MCP-1可能在rd小鼠感光细胞凋亡中发挥作用.     

脑苷肌肽对视网膜变性小鼠发育过程的影响

目的:观察脑苷肌肽(CEGI)对视网膜变性模型(rds小鼠)发育过程的影响,旨在探求CEGI治疗视网膜变性类疾病的治疗效果,为临床用药提供客观依据和指导。方法:采用组织病理学技术(光、电镜)和原位末端转移酶标记(TUNEL)方法观察脑苷肌肽腹腔给药干预后视网膜组织形态、超微结构、感光细胞凋亡的变化。结果:生后14d(P14)是rds鼠视网膜发育最完善阶段,随后28～56d,视网膜外核层及内核层细胞层数逐渐减少,感光细胞凋亡细胞数逐渐增多,电镜观察有凋亡核变化以及内节和纤毛崩解。给予CEGI治疗后28d和56d,视网膜细胞层数较同日龄对照组增厚,凋亡细胞数减少,有统计学差异(P<0.05)。神经生长因子(NGF)与CEGI作用相似。结论:脑苷肌肽有促进视网膜细胞生长发育作用,对rds小鼠视网膜变性过程有一定的缓解作用。     

细胞因子Ⅱ在遗传性视网膜色素变性的转基因小鼠视网膜的表达研究

目的 探讨细胞因子Ⅱ(ealpain Ⅱ)在视网膜色素变性过程中表达的变化。以此窥视遗传性视网膜色素变性的转基因小鼠(rds小鼠)发病的某些可能因素。方法 以C3B小鼠为对照，选择不同生长时期的rds小鼠的视网膜组织，用免疫组化方法检测CalpainlI在不同生长阶段的小鼠模型视网膜中的是否表达及表达情况。结果 1周即可见CalpainlI在rds小鼠视网膜神经节细胞层的表达，2周左右内核层也见表达，4周后外核层也开始出现由强至弱的ealpainⅡ强阳性表达，最终至全部视网膜剩余组织中均可见ealpainⅡ的表达。结论 只有高钙才能激活ealpainⅡ的表达，研究中ealpainⅡ参与了rds小鼠视网膜细胞的变性死亡。因此，高钙是rds小鼠视网膜变性的一个可能因素。     

β趋化因子受体CCR1在遗传性视网膜变性小鼠视网膜中的表达

目的探讨B趋化因子主要的受体之一β趋化因子受体1（CCR1）在视网膜变性模型小鼠（rd小鼠）的视网膜变性过程中的表达及其在感光细胞凋亡中的病理作用。设计实验研究。研究对象出生后8、10、12、14、16及18天的rd小鼠各10只（共60只）及同龄C57BL／6N对照小鼠各10只（共60只）。方法小鼠断颈处死,取双眼眼球制作冰冻切片或分离新鲜视网膜组织。逆转录多聚酶链反应（RT．PCR）测定各鼠龄rd小鼠及对照鼠视网膜CCR1mRNA的表达水平。免疫组织化学法观察CCR1蛋白在各鼠龄rd小鼠视网膜的定位表达。CCR1在感光细胞及凋亡细胞中的表达由免疫荧光双标法确定。主要指标视网膜CCR1mRNA及蛋白的表达、CCR1的细胞定位及与凋亡细胞的关系。结果CCR1mRNA在对照组及各鼠龄rd小鼠视网膜中均有表达,但在出生后12、14天rd小鼠视网膜中表达明显升高（光密度值分别为0．986±0．17和1．152＋0．22,P=0．010和0．008）。CCR1阳性染色细胞开始出现于出生后8天的rd视网膜外核层,并于12及14天达到高峰。对照组视网膜外核层无CCRl阳性染色。CCR1与视紫红质、CD11b或TUNEL染色免疫荧光双标结果显示,CCR1表达于感光细胞而非小胶质细胞中,部分CCR1表达于凋亡的感光细胞中。结论在rd小鼠视网膜中CCR1表达于感光细胞并随其变性程度加重表达升高。CCR1的活化可能在rd小鼠感光细胞凋亡中发挥作用。     

胚胎干细胞源性视网膜前体细胞在rd鼠视网膜下腔移植后的分化

目的 研究绿色荧光蛋白标记的胚胎干细胞(green fluorescent protein-embryonic stem cells,GFP-ESC)源性视网膜前体细胞在rd小鼠视网膜下腔移植后的存活分化情况.方法 将GFP-ESC 源性视网膜前体细胞移植到伴有视网膜感光细胞变性和继发性视网膜神经节细胞变性的rd 小鼠视网膜下腔,移植后1周、4周、8周取材行免疫荧光细胞化学检测,观察移植细胞存活、分化情况.对移植细胞进行核型分析,并接种裸鼠眼内观察是否成瘤,评价其安全性.结果 GFP-ESC 源性视网膜前体细胞移植眼内可存活、整合到宿主变性视网膜层间.1周时 GFP 阳性细胞移行于外层视网膜并呈视杆细胞标志抗原rhodop-sin阳性:4周时整合于内层视网膜,共表达成熟神经元标志抗原神经丝蛋白-200、微管相关蛋白-2,突触标志抗原synaptophysin检测阳性;8周时移至邻近玻璃体腔形成节细胞样结构,表达节细胞标志抗原Thy1.1;移植细胞维持正常二倍体核型,术后8周未见排斥反应及瘤性增殖.结论 GFP-ESC源性视网膜前体细胞在变性视网膜中可存活、整合,并有优先向变性的感光细胞及.神经节细胞类型分化的倾向,有望为青光眼等视神经视网膜病变的细胞移植治疗提供安全有效的种子细胞.     

p53和MDM2在3种RP小鼠视网膜中的表达

目的 了解凋亡相关基因p53和MDM2与视网膜色素变性（retinitis pigmentosa,RP)发病的关系。方法 以RP模式动物rds小鼠，rd小鼠，C3H小鼠及正常对照C3B小鼠为材料，提取不同发病时间（出生后7，12，17，23，29，37和50d)视网膜的总RNA，以β-actin和L19核糖体蛋白基因为内对照，RT－PCR分析小鼠发病过程中视网膜p53和MDM2的表达。结果 在C3B小鼠视网膜中，p53和MDM2的表达完全同步。出生后7d，高表达；7－12d期间，表达量迅速下降；12－50d期间，表达水平低，但基本稳定；p53和MDM2在rds小鼠的表达水平低，但基本上是同步，稳定表达。在rd小鼠中，p53的表达变化不大，但MDM2的表达则与p53不同步，类似于C3B小鼠MDM2的表达。在C3H小鼠的视网膜，p53和MDM2的表达也不同步。7－23d期间，p53的表达基本稳定；自23d后逐步下降，其中23－29d期间，p53的表达下降最快。而MDM2的表达总体变化不大。结论：在这3种RP模式动物中，虽然凋亡相关基因和MDM2存在着表达差异，但其发病过程中视网膜细胞的变性死亡可能是通过p53非依赖型途径进行的。     

Comparison of the lens crystallin proteins from normal, rd, and rds mutant mice utilizing specific monoclonal antibodies

The lens proteins from three lines of congenic mice which are homozygous for the gene retinal degeneration (rd) or retinal degeneration slow (rds) or carrying the normal alleles (normal) were analyzed by SDS-PAGE and by immunological reactivity to specific monoclonal antibodies. The lens proteins of normal, rd, and rds mice showed a similar developmental pattern between postnatal day 0 and postnatal day 30. The expression of the 25000 molecular weight (MW) beta-crystallin polypeptide which appears postnatally in the normal lens was not affected by retinal abnormalities in the mutant mice. It is concluded that the regulation of the 25000 MW beta-crystallin polypeptide is not dependent upon differentiation or maintenance of the photoreceptor outer segments or continued presence of the photoreceptor cells.     

Localization of an endogenous substrate for cyclic AMP-stimulated protein phosphorylation in retina

Incubation of mouse or rabbit whole retina homogenates in the presence of [gamma 32P]-ATP and Mg2+ leads to the phosphorylation of various proteins, as demonstrated using SDS-polyacrylamide gel electrophoresis and autoradiography. The phosphorylation of one protein (ca. approximately equal to 31000 mol. wt) was increased by cyclic AMP in both species, with half-maximal stimulation at 5 x 10(-7)M. Cyclic GMP was also active, but much less potent. Protein phosphorylation patterns were compared in retina homogenates from normal mice (C57BL/6J), from adult C57BL/6J mice homozygous for the retinal degeneration gene (rd/rd) in which rod photoreceptor cells are absent, and from 21-day-old 020/Cpb mice homozygous for the retinal degeneration slow gene (rds/rds) in which only the outer segments of the rod photoreceptors are missing. The 31 K protein was only present in normal and in 21-day-old rds/rds mice. When rabbit retina was microdissected into outer segment, inner segment plus outer nuclear, and inner retina layers, cyclic AMP-stimulated phosphorylation of the 31 K protein was evident only in the inner segment plus outer nuclear layer. These data indicated the presence of a specific, endogenous substrate for a cAMP-dependent protein kinase which is found in the inner portions of rod photoreceptor cells.     

Some cytological and initial biochemical observations on photoreceptors in retinas of rds mice

As a control for biochemical studies in progress, an ultrastructural study has been carried out on the deteriorating, 21-day photoreceptors of the 020/Cpb strain of mice, homozygous for retinal degeneration, slow (rds). At 21 postnatal days, outer segments were essentially lacking, but cilia erupting from the inner segments were common. A low percentage of cilia bore small cytoplasmic masses containing a few layered membranes, and rare inner segments possessed spherical aggregations of multilayered membranes. Pigment epithelial cells also possessed membranous aggregations in presumed phagosomes. While other parts of photoreceptors possessed the usual organelles of normal rods, inner segments were reduced in volume, and the layer of photoreceptor synaptic terminals was thinner. Mutant 21-day retinas possessed about two-thirds of the protein of normal 21-day retinas but 50% more protein than "rodless" (rd/rd) 21-day retinas. Surprisingly, while dark-adapted rds retinas possessed markedly lower levels of cyclic GMP as compared to controls, light-adaptation significantly reduced cyclic GMP and cyclic AMP levels, and biochemical data point to persistent light-modulated cyclic nucleotide levels in the photoreceptors.     

Accumulation of glial fibrillary acidic protein in Müller radial glia during retinal degeneration

Müller radial glia accumulate glial fibrillary acid protein (GFAP) in response to retinal injuries. We have studied the changes in cellular localization of GFAP in genetically caused retinal dystrophy in strains of cat and mouse: Abyssinian cats with progressive retinal dystrophy, and mice homo- and heterozygous for the retinal degeneration (rd) and retinal degeneration slow (rds) genes. The following observations were made: (1) Glial fibrillary acid protein-immunoreactive (GFAP-IR) radial Müller glia are present in normal cat and mouse retinae. (2) There is a general increase with age in numbers of GFAP-IR radial Müller glia, and other GFAP-IR elements in the retina of cat and mice with hereditary retinal dystrophy. (3) The increase in GFAP-accumulating Müller cells seems to proceed from peripheral to central retina in both cats and mice. This might reflect the direction of photoreceptor degeneration, which proceeds in the same direction in the retinal dystrophic cat and in mice bearing the rds gene. However, in the rd mouse photoreceptor degeneration proceeds from central to peripheral retina, indicating that GFAP accumulation is not a local direct effect of photoreceptor damage. (4) Comparing the different mutant mouse strains, there seem to be qualitative differences in the distribution of GFAP-IR elements. The numbers of tangential elements and fibrillary tangles in the inner plexiform and nuclear layers are highest in the +/+ rds/rds retina, followed by the +/+ rds/+, whereas such elements appear to be more scarce in rd/rd rds/rds, followed by the rd/rd +/+ and +/+ +/+ retinae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatio—temporate Expression of P53,Bcl—2 and Fas in the Retina of RD,a

Purpose: To investigate the expression status of p53, Fas and bcl-2 in the developmentof retinal degeneration in C3H, rd and rds mice.Methods: Expression of p53, Fas and bcl-2 in the retina of rd,rds and C3H mice as wellas in normal C3B mice in different periods was examined by immunohistochemicaltechnique.Results: The expression of Fas and p53 was not detected in normal C3B mice. Fas andp53 were expressed in the ganglionic layer in the early stage and then in the innernuclear layer, while the scale and intense increased in the ganglionic layer. There is nodifference of bcl-2 expression between the normal mice and mice with retinaldegeneration.Conclusion: Fas and p53 may be involved in the retinal cell death in rd, rds, and C3B,but bcl-2 may not. It is unknown why p53 and Fas appeared first in ganglion layer butnot in the outer nuclear layer where retinal cells death was noticed in early stage. EyeScience 2000; 16: 158 ~ 162.     

Development and degeneration of retina in rds mutant mice: light and electron microscopic observations in experimental chimaeras

Mice, homozygous for the rds gene, fail to develop the receptor outer segments and show a slow reduction of the outer nuclear layer. A series of 13 chimaeric mice was produced by combining morulae from albino rds/rds and pigmented normal (+/+) mice. At 3-4 weeks, variable stretches of visual cells without outer segments were observed together with stretches of visual cells with normal outer segments. The location of these areas was unrelated to the genotype of the overlying pigment epithelium. Phagosomes containing outer segment debris were present in albino pigment epithelial cells, located over normal outer segments, indicating normal functional properties of rds/rds pigment epithelial cells. At 9 months, regions with visual cell loss were observed underlying both types of pigment epithelial cells. Regions showing normal and intermediate thicknesses of the outer nuclear layer were seen more often than regions showing rds/rds type distribution. In another series of eight chimaeras, consisting of albino rds/rds and pigmented rd/rd genotypes, the eyes examined at 22 days showed more pronounced visual cell loss than in the rds----normal retinas at 9 months. Regions of the outer nuclear layer, containing a single row of cone perikarya, were similar to the rd/rd phenotype and differed from the phenotype of the double homozygous rd/rd rds/rds retina, which has a slower rate of degeneration than in rd/rd mice. Visual cell loss in these chimaeras at 9 months was similar to that in the rds/rds retina of the same age. The findings show that the expression of the rds gene, resulting in failure of outer segment development and eventual death of visual cells is unrelated to the genotype of the overlying pigment epithelial cells and suggest that the gene acts within the neural retina and possibly intracellularly in the visual cells.     

Retardation of photoreceptor degeneration in the detached retina of rd1 mouse

PURPOSE: To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice. METHODS: RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30. RESULTS: The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD. CONCLUSIONS: RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.     

A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae

Interphotoreceptor retinoid-binding protein (IRBP) was studied using immunochemical and immunocytochemical techniques in retinae of mice with allelic combinations at the rd and rds loci at different stages of development and degeneration. Until postnatal day 7 (P7), IRBP is located intracellularly in developing retinae of the different genotypes. Thereafter, IRBP is present mainly in the interphotoreceptor matrix. As previously noted, cell death is slowest in the heterozygous +/+,rds/+ mutant with loss increasing in order in +/+,rds/rds, rd/rd, rds/rds and rd/rd,+/+ animals. The IRBP content of the total retina also approximates this pattern, with lowest amounts by far in rd/rd, rds/rds and rd/rd,+/+ mutants (after P14). Interestingly though, IRBP loss significantly precedes visual cell loss in the rd/rd,rds/rds retina. In all the mutants, the remaining rod cells in the outer nuclear layer exhibit synthesis of intracellularly located IRBP at late stages of degeneration. In the single homozygous rd/rd,+/+ and the double homozygous rd/rd,rds/rds mutants, IRBP is present intracellularly during the entire degenerative process with somewhat less intracellular IRBP in the rd/rd,rds/rds mutant. Retinae of homozygous +/+,rds/rds and heterozygous +/+,rds/+ animals exhibit a normal distribution pattern of IRBP immunoreactivity until loss of photoreceptor cells becomes pronounced at later stages of the disease. Many of the remaining cells at this time are probably cone elements although they are structurally changed. Double labeling with IRBP and S-antigen demonstrates, in many but not all, the presence of both proteins in the same cell body. Immunocytochemistry clearly demonstrated the presence of IRBP in remaining photoreceptor cells at late stages of the disease. Thus, the biochemically measured loss of IRBP appears to be a complex process neither directly dependent on the loss of photoreceptor outer segments and reduced interphotoreceptor matrix space (e.g. there is a sustained IRBP level in rodless rds mutants) nor simply due to cell death (e.g. in the rd/rd,rds/rds mutant, IRBP loss significantly precedes cell loss). That this IRBP is mainly intracellular, however, may indicate an abnormality in secretion which, combined with other factors, induces a degenerated and less differentiated phenotype.