Hereditary retinal degeneration and photoreceptor apoptosis

目的研究遗传性视网膜变性的发生机制。方法对生后不同鼠龄ＲＣＳ（ＲｏｙａｌＣｏｌｅｇｅｏｆＳｕｒｇｅｏｎｓ）和ＳＤ（Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ）大鼠的视网膜进行光镜观察、计算机自动图象分析和凋亡细胞的ＴＵＮＥＬ（ＴｄＴ－ｍｅｄｉａｔｅｄｂｉｏｔｉｎ－ｄＵＴＰｎｉｃｋ－ｅｎｄｌａｂｅｌ－ｉｎｇ）检测。结果与同龄ＳＤ大鼠相比，ＲＣＳ大鼠出生后１５ｄ，视细胞（ｐｈｏｔｏｒｅｃｅｐｔｏｒ，ＰＲＣ）外节膜盘堆积，以后相继出现内节排列紊乱、消失，细胞核固缩，从生后３０ｄ到６０ｄ，ＰＲＣ迅速消失。从出生后４０ｄ，视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞出现增殖和排列紊乱。１００ｄ时，ＲＰＥ层出现新生血管。ＴＵＮＥＬ检测，ＲＣＳ大鼠视网膜ＰＲＣ阳性数目从２５ｄ起开始逐渐增加，３５ｄ时达高峰。结论ＲＣＳ大鼠的视网膜变性以ＰＲＣ凋亡为主，ＲＰＥ紊乱和视网膜新生血管是视网膜变性的晚期病变。     

遗传性视网膜谱性视细胞凋亡与视网膜诱生型一氧化氮合酶活性的时程变化

目的 研究遗传性视网膜变性视细胞凋亡与诱生型一氧化氮合酶活性之间的关系。方法 对生后不同鼠龄RCS大鼠和Wistar大鼠的视网膜进行凋亡细胞的TUNEL检测、计算机自动图像分析及诱生型一氧化氮合酶活性的测定。结果 RCS大鼠视网膜视细胞自生后第25天出现凋亡,第30-35天达高峰;视细胞凋亡初期,即生后第25天,视网膜诱生型一氧化氮合酶活性达高峰。结论 在遗传性视网膜变性的视网膜。诱生型一氧化合酶激活可能在视细胞凋亡中起诱导作用。     

RCS大鼠视细胞超微结构观察

目的 观察遗传性视网膜变性大鼠视细胞超微结构改变。方法 电子显微镜观察出生后9－60天皇家外科学院（RCS）大鼠及正常SD大鼠视网视细胞超微结构。结果 与正常SD大鼠相比,RCS大鼠从出生后15天,视细胞相继出现视杆外节脱落的膜盘堆集,外节膜盘排列紊乱,视杆内节椭圆体线粒体空泡化,视肌样质结构消失,细胞核浓缩,浓缩的染色质沿核膜排列呈“新月形”,最终细胞体消失。结论 RCS大鼠遗传性视网膜变性中视细胞死亡具有细胞凋亡的形态特征。     

半胱氨酸天冬氨酸酶3在遗传性视网膜变性中的表达

目的 观察半胱氨酸天冬氨酸酶3（caspase 3)mRNA在RCS大鼠视网膜中的表达，分析caspase 3特异抑制剂Ac-DEVD-CHO对视细胞凋亡的影响。方法 应用逆转录－多聚酶链反应（RT－PCR）检测RCS大鼠出生后不同时间视网膜中caspase 3 mRNA的表达。应用TUNEL方法检测玻璃体内注射Ac-DEVD-CHO后视细胞凋亡的改变。结果 RCS大鼠出生后14天至60天视网膜caspase 3 mRNA均有表达，25天水平明显升高，达到高峰。SD大鼠视网膜15至60天的表达量无明显差异，与RCS大鼠14天水平接近。caspase 3特异抑制剂Ac-DEVD-CHO能显著抑制视细胞凋亡。结论 caspase 3可能在RCS大鼠视网膜变性视细胞凋亡中起关键作用。     

Wistar鼠和RCS鼠视网膜内一氧化氮合酶的分布

目的 观察正常Ｗｉｓｔａｒ大鼠和患遗传性视网膜病变的ＲＣＳ鼠视网膜内一氧化氮合酶（ＮＯＳ）的分布。方法 ７４天Ｗｉｓｔａｒ鼠和７４天ＲＣＳ鼠各６眼分为２组,冰冻切片,用ＮＡＤＰＨ黄递酶组织染色法（ＮＤＰ）,光镜下观察。结果 一氧化氮合酶主要存在于Ｗｉｓｔａｒ鼠,ＲＣＳ鼠的无长突细胞内,在双侧锯齿缘、赤道部和后极部内丛层均有分布,二者间的数量有差异。结论 一氧化氮合酶分布在Ｗｉｓｔａｒ鼠,ＲＣＳ鼠的无长突细胞,但视细胞萎缩对含一氧化氮合酶的阳性无长突细胞数量有影响。     

鼠视网膜感光细胞移植术后超微结构观察

目的 以遗传性视细胞变性动物ＲＣＳ鼠为受体,Ｗｉｓｔａｒ鼠为供体,观察纯视锥、杆细胞移植术后ＲＣＳ鼠视网膜外层超微结构的变化。方法 应用视网膜板层切削技术或准分子激光技术制备供体层状视细胞;视网膜板层外路移植技术,获得视网膜移植动物模型。术后２周及４周时处死动物,眼球常规切片后于光镜及透射电镜下观察受体视网膜。结果 移植的视细胞大多成层排列于受体鼠的视网膜色素上皮层及内颗粒层之间。重新出现的外网状     

眼眶横纹肌肉瘤N—ras癌基因点突变及rasp21,p53蛋白的 …

研究眼眶横纹肌肉瘤（ｒｈａｂｄｏｍｙｏｓａｒｃｏｍａ，ＲＭＳ）Ｎ－ｒａｓ癌基因突变ｒａｓｐ２１、ｐ５３蛋白的异常表达，探讨基因突变在眼眶ＲＭＳ发病中的作用。方法应用特异性高突变区位不同的突变型寡核苷酸探针及突型变型ｒａｓｐ２１、ｐ５３单克隆抗体，对２１眼眶ＲＭＳ组织进行ＤＮＡ斑点杂交和免疫组织化学分析。结果４例眼眶ＲＭＳ细胞发生Ｎ－ｒａｓ癌基因１２密码子第２个碱基突变，５例眼眶ＲＭＳ组织中发生６１     

不同放射剂量下大鼠视网膜组织形态与氧化应激因子的变化

目的　通过直线加速器照射大鼠眼部制作放射性视网膜病变大鼠模型,观察大鼠视网膜组织形态与氧化应激因子的变化。方法　选取４５只２个月龄ＳＤ大鼠,随机分为正常对照组、１０Ｇｙ组、３０Ｇｙ组,每组１５只。直线加速器照射眼部,单次剂量１０Ｇｙ。１０Ｇｙ组放射１次。３０Ｇｙ组每周放射１次,连续３周,合计剂量３０Ｇｙ。放射后正常饲养３个月,处死各组大鼠并取眼球。采用ＨＥ染色观察视网膜组织形态,黄嘌呤法测视网膜超氧化物歧化酶（ｓｕｐｅｒｏｘｉｄｅｄｉｓｍｕｔａｓｅ,ＳＯＤ）活性、丙二醛（ｍａｌｏｎａｌｄｅｈｙｄｅ,ＭＤＡ）含量以及ＥＬＩＳＡ法测定活性氧自由基（ｒｅａｃｔｉｖｅｏｘｙｇｅｎｓｐｅｃｉｅｓ,ＲＯＳ）含量。结果　造模后３个月,３０Ｇｙ组视网膜各层组织结构松散,神经节细胞层及内核层水肿,１０Ｇｙ组模型大鼠视网膜仅神经节细胞层轻微水肿。与正常对照组相比,１０Ｇｙ组ＳＯＤ活性、ＭＤＡ及ＲＯＳ含量分别为（８８．８０±１６．４４）Ｕ·ｍｇｐｒｏｔ－１（Ｐ＜０．０５）、（１１．０２±４．０２）ｎｍｏｌ·ｍｇｐｒｏｔ－１（Ｐ＜０．０１）、（１６．８９±６．０２）Ｕ·ｍＬ－１（Ｐ＜０．０５）,差异均有统计学意义;３０Ｇｙ组ＳＯＤ活性、ＭＤＡ及ＲＯＳ含量分别为（７８．５０±１８．０９）Ｕ·ｍｇｐｒｏｔ－１、（１５．２６±５．３４）ｎｍｏｌ·ｍｇｐｒｏｔ－１、（２８．６６±８．８２）Ｕ·ｍＬ－１,三者差异均有显著统计学意义（均为Ｐ＜０．０１）。与１０Ｇｙ组比较,３０Ｇｙ组ＭＤＡ含量和ＲＯＳ含量均升高（均为Ｐ＜０．０５）。结论　直线加速器放射造模时,３０Ｇｙ为最佳放射造模剂量,造模后大鼠视网膜ＳＯＤ活性降低,ＭＡＤ及ＲＯＳ含量均升高,可能是放射性视网膜病变的发病基础。     

人黑色素瘤细胞侵袭转移恶性表型获得的分子学基础

目的 探讨具有不同潜在转移能力的黑色素瘤细胞恶性表型的演进及其侵袭转移的机制。方法 采用细胞培养,ｐ５３蛋白免疫组化染色,ＰＣＲ－ＳＳＣＰ检测,流式细胞术（ＰＣＭ）,蛋白酶活性分析（Ｚｙｍｏｇｒａｐｈｙ）及裸鼠体内异种接种实验。结果 人黑色素瘤细胞中存在有ｐ５３基因的突变瘤细胞表面６７ＫＤ ＬＮ－Ｒ的荧光阳性率和全部细胞的平均荧光强度大小顺序为ＷＭ４５１〉ＷＭ９８３Ａ〉ＷＭ１３４１Ｂ〉ＷＭ３５。金     

RDS／peripherin表达载体的构建和在COS—1细胞的表达

目的 构建大鼠ＲＤＳ／ｐｅｒｉｐｈｅｒｉｎ真核表达载体ｐＡＬＴＥＲ－ＲＤＳ,并观察在ＣＯＳ－１细胞中的表达,方法 将全长度的ＲＤＳ／ｐｅｒｉｐｈｅｒｉｎｃＤＮＡ（１．５ｋｂ）插入到真核表达载体ｐＡＬＴＥＲ－ＭＡＸ中,构建了ＲＤＳ真核表达载体ｐＡＬＴＥＲ－ＲＤＳ,采用电穿孔技术将其转染ＣＯＳ－１细胞,转染４８ｈ后,采用ＲＴ－ＰＣＲ检测ＲＤＳ／ｐｅｒｉｈｐｅｒｉｎｃＤＮＡ的表达,结果 采用ＲＴ－ＰＣＲ     

Light treatment enhances photoreceptor survival in dystrophic retinas of Royal College of Surgeons rats.

PURPOSE: To determine whether treatment with bright light elicits a protective response that enhances photoreceptor survival in Royal College of Surgeons (RCS) rats with inherited retinal degeneration. METHODS: RCS rats were illuminated for 10 to 12 hours with 130 foot-candles (fc) of white or green light. Untreated littermates that were kept under low cyclic light levels were used as control subjects. Photoreceptor survival was determined by quantitative analysis of photoreceptor nuclei and ultrastructural assessment of cellular organization. Basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) gene expression were determined at the mRNA and protein levels. RESULTS: Treatments of RCS rats with a single dose of bright light on postnatal day 23 (P23) greatly enhanced photoreceptor survival. Ultrasturctural analysis revealed intact inner segments in light-treated retinas, whereas in untreated retinas only remnants of inner segments were observed. By P42, numerous viable nuclei were counted in the posterior retina of light-treated rats, whereas most of the remaining nuclei in untreated RCS rat retinas were highly pyknotic. At 2.5 days after treatment with a single dose of bright light, bFGF gene expression was significantly higher than in untreated RCS rat retinas. By P42, bFGF protein levels were still significantly higher in the treated retinas. CONCLUSIONS: Exogenous bFGF has been shown to promote photoreceptor survival in the RCS rat retina. Thus, the increased bFGF expression that was measured in the light-treated RCS rat retinas may be a protective response to light stress, which supports the observed rescue of photoreceptors in light-treated RCS rat retinas.     

Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration

The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon’s (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic μ-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic μ-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new pharmaceutical treatments for retinitis pigmentosa.     

Transient protective effect of caspase inhibitors in RCS rat

In most retinal degenerations in humans and in animal models, photoreceptor cells die by apoptosis. Although the biochemical features are similar in all apoptotic cells, different molecular events lead the cell to death. In the present study we used a rat model of inherited retinal degeneration, the RCS rats, to investigate the involvement of the proteases, caspases and/or calpains, in photoreceptor apoptosis. In the first experiments, rats were untreated or injected intravitreally at post natal day 27 (P27) with the large broad spectrum caspase inhibitor, ZVAD, the calpain inhibitor, MuhPhe, or with the vehicle, DMSO. Retinal status was evaluated at P35 and P42 by electroretinography, morphometry and apoptotic nuclei detection. DMSO and MuhPhe had no effect on RCS retinas as evidenced by equivalent loss of function and equivalent number of apoptotic cells than in untreated group. ZVAD transiently reduced apoptotic cells and preserved photoreceptor function at P35 but not at P42. These results suggest that caspases but not calpains are involved in retinal degeneration in the RCS. In the second experiments, RCS rats were injected twice at P27 and P35 with ZVAD or DMSO. Although ZVAD-treated retinas were preserved at P35 compared to the DMSO controls, the second injection of ZVAD did not extend the preserving effect to P42. Moreover, a single injection of ZVAD at P35 had no preserving effect at P42. All these data taken together suggest that caspases do not play a pivotal role after P35. In a fourth set of experiments, we used specific caspase inhibitors to elucidate which caspase was activated. The caspase-1/4 inhibitor (YVAD) or the caspase-3/7 inhibitor (DEVD) were injected intravitreally at P27 and retinal status was evaluated at P35 and P42. Electroretinograms and apoptotic nuclei detection demonstrated that YVAD and DEVD preserved photoreceptors at P35 but not at P42. These results suggest that both caspase-1/4 and caspase-3/7 play a major role in the apoptotic pathway between P27 and P35 in retinal degeneration of RCS rats. In this study, we show that 1/ the photoreceptor apoptotic process in the RCS rat involves caspases but not calpains, and 2/ the retinal degeneration seems to be composed of different phases involving different molecular players. Indeed, we have demonstrated that caspases are playing a major role at P35, but not at P42.     

Immunohistochemical localization of basic fibroblast growth factor in mature and developing retinas of normal and RCS rats.

Basic fibroblast growth factor (bFGF) delays photoreceptor degeneration when injected intraocularly in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy. In the present study, we have determined the localization of endogenous bFGF in retinas of normal and RCS rats during the normal developmental period (postnatal days 0-20) and the period of photoreceptor degeneration in RCS rats (days 20-90). bFGF was localized immunohistochemically by indirect immunoperoxidase using two different polyclonal antibodies and one monoclonal antibody against bFGF. bFGF was present in retinas as early as birth, and remained through adult age. Controls using either PBS, non-immune IgG or antibody preabsorbed with bFGF peptide were devoid of label. In normal rats between the ages of birth and postnatal day (P) 4, bFGF was found in developing ganglion cells, superficial blood vessels, some of the innermost cells in the neuroblastic layer, developing horizontal cells, and retinal pigment epithelial (RPE) cells. Between P0 and P4, the intensity of staining increased significantly in horizontal cells. From P6-P10, some cells in the inner nuclear layer remained positive, but horizontal cell staining became less intense in the central retina. The superficial vessels, ganglion cells and RPE cells also remained positive for bFGF. At P20-25, when the retina was essentially mature, bFGF was found in RPE cells, most cells of the ganglion cell layer, and many cells of the inner nuclear layer, but horizontal cells and blood vessels showed a lower concentration of bFGF than they did at younger ages. At P45 and older, blood vessels no longer showed bFGF immunoreactivity. The staining pattern in RCS rats was indistinguishable from that for normal rats at all ages examined. These results show that bFGF is present in the developing and adult rat retina in some neural cells, in addition to vessels and RPE cells. The transient elevated expression of bFGF immunoreactivity in developing horizontal cells and blood vessels suggests a possible role for this growth factor in retinal development. In addition, if RCS retinas possess any difference in bFGF localization or concentration compared to normal retinas, it must be too small to detect by immunohistochemical means, or at least with the reagents used.     

RCS大鼠视网膜内核层变性的超微结构研究

目的　证实RCS大鼠视网膜内核层存在神经原变性。方法　利用光学显微镜和透射电子显微镜观察出生后第 18、2 0、2 8、3 5、42、45、5 6、60、70和 10 0d的RCS大鼠视网膜和正常SD大鼠视网膜组织结构的变化。并用TUNEL方法证实视网膜神经细胞存在凋亡。结果　和正常SD大鼠视网膜比较 ,RCS大鼠 18～ 2 0d开始视网膜变性 ,光感受器细胞死亡 ,内核层细胞也有不同程度的变性。RCS大鼠在出生后 2 5d ,视网膜外核层细胞核TUNEL呈阳性 ,出生后 3 5～ 45d呈强阳性 ,视网膜内核层细胞核TUNEL标记呈阳性。结论　RCS大鼠视网膜内核层细胞存在神经原变性 ,视网膜内核层细胞死亡存在凋亡这一方式。跨神经原变性很可能是这些细胞变性的机制。RCS大鼠视网膜外核层细胞存在神经原变性 ,其死亡以凋亡为主     

Accumulation of neurocan,a brain chondroitin sulfate proteoglycan,in association with the retinal vasculature in RCS rats

PURPOSE: To examine whether and how the retinal distribution of the chondroitin sulfate proteoglycan neurocan is affected after photoreceptor cell loss and whether it correlates with the multiple secondary cellular changes that accompany the photoreceptor degeneration. METHODS: Retinas from normal rats (Sprague-Dawley; postnatal days [P]0-P70), RCS rats with dystrophic retinas (P0-P300), RCS-rdy(+) congenic rats with nondystrophic retinas (P0-202), and rhodopsin mutant rats, P23H (P0-P257) and S334ter (P0-P220), were processed for immunohistochemistry using a polyclonal antibody to rat neurocan. RESULTS: The overall distribution of neurocan was similar in all retinas examined. Neurocan immunostaining was detected over the nerve fiber layer, the plexiform layers, the photoreceptor outer segments region, and the ciliary epithelium. With age, labeling throughout the plexiform layers decreased continuously. In RCS rats however, conspicuous labeling was also seen in association with retinal vessels, from P15 onward. CONCLUSIONS: Accumulation of neurocan in association with the retinal vasculature does not correlate with photoreceptor cell loss, because it was not observed in the rhodopsin mutant rats. During the earliest stages of the disease, accumulation of debris in the subretinal space in RCS rats may be sufficient per se to initiate a cascade of metabolic changes that result in accumulation of neurocan. With time, the neurocan accumulated perivascularly may, by interaction with other matrix molecules, modulate at least some of the vascular alterations observed in this animal model.     

Visual response properties of retinal ganglion cells in the royal college of surgeons dystrophic rat

PURPOSE: Alterations in retinal ganglion cell response patterns were profiled in dystrophic Royal College of Surgeons (hereafter RCS) rats over the first 100 postnatal days as a baseline for retinal rescue and vision restoration strategies. This method enabled the evaluation of the extent to which postreceptoral neuronal attributes in degenerating retinas mirror inferred declines in photoreceptor function. METHODS: Single-unit responses from large retinal ganglion cells were recorded from age-matched dystrophic RCS (RCS-rdy(-)) and congenic RCS-p(+) (hereafter wild-type or wt) rats were recorded in vitro under visual control. Cells were profiled with conventional spatial and flux stimulus modulations. RESULTS: Ganglion cell single unit and population attributes alter slowly over the course of photoreceptor degeneration in dystrophic RCS rats, with significant decreases in apparent receptive field size, contrast sensitivity, and threshold sensitivity detected by the first month of life. Spatial frequency tuning and contrast responses were extremely weak by postnatal day (P)76, paralleled by a progressive decline in signal-to-noise (S-N) ratio to roughly unity by postnatal day (P)107. This decline was only a simple loss of responsivity, as background firing rates increased substantially over time. Whereas wt retinas were dominated by ON-center cells (15/23 cells), dystrophic animals were dominated by OFF-center cells by P47 (24/27 cells). CONCLUSIONS: The first definitive signs of degeneration in dystrophic RCS rats are parallel decreases in ganglion cell threshold sensitivity and receptive field size, followed by deterioration in spatial summation. Arguably, these changes can be qualitatively explained as photoreceptor signaling losses. However, the apparent shift in population profile from ON- to OFF-center ganglion cells long before loss of the b-wave at P90 implies that a reactive mechanism such as bipolar cell rewiring and/or transformation of neuronal phenotypes occur during the early phase of photoreceptor stress, before rod and cone death.