锂对视网膜色素变性小鼠光感受器细胞凋亡的保护作用

目的 研究锂在视网膜色素变性小鼠模型上对光感受器细胞凋亡的神经保护作用.方法 实验研究.FVB/NJ视网膜色素变性小鼠出生后立刻用含锂的食物喂养,7和14 d时摘除眼球做视网膜冰冻切片,同时取血测血清锂浓度.HE、TUNEL和视杆细胞免疫荧光染色进行视网膜组织形态、光感受器细胞凋亡分析.结果 光镜下可见,对照组外核层可见TUNEL阳性细胞,出生后14 d外核层厚度和细胞层数(3或4层)明显低于7 d时的厚度(9或10层),而锂喂养组出生后14 d外核层的厚度和细胞层数明显高于对照组,与出生7 d时的外核层厚度及细胞层数无明显差异.结论 锂能够保护视网膜色素变性小鼠光感受器细胞免于凋亡.(中华眼科杂志,2008,44:248-252)     

小鼠实验性视网膜脱离后光感受器细胞的凋亡

目的 研究小鼠实验性视网膜脱离后光感受器细胞的凋亡情况。 方法 将成年C57Bl/6J小鼠36只分为2组：实验组小鼠18只左眼视网膜下注射1.4%透明质酸钠造成视网膜脱离，对照组小鼠18只左眼仅作巩膜穿刺。分别于手术后1、3、7和28 d摘除眼球，视网膜切片进行组织化学、免疫荧光染色，共聚焦显微镜检查。抗视锥和抗视杆细胞的抗体分别标记视锥和视杆细胞，dUTP缺口末段标记法(TUNEL)标记凋亡细胞。通过计数存活和凋亡的视锥和视杆细胞来定量光感受器细胞的凋亡和细胞丢失。 结果 凋亡细胞只存在于脱离部分视网膜的外核层，凋亡细胞在视网膜脱离后1 d即可检测得到，3 d时达到高峰，7 d后陡然减少。视网膜脱离后视杆和视锥细胞的死亡呈现同样的时程。 结论 凋亡是视网膜脱离后光感受器细胞死亡的主要病理改变。 (中华眼底病杂志, 2006, 22: 124-127)     

米诺环素对视网膜色素变性小鼠光感受器细胞的保护作用

目的 观察米诺环素对视网膜色素变性（RP）的rd小鼠［C3H/HeN (Pde6brd-/rd-)］RP过程的影响。方法 40只新生rd小鼠随机分为10组，5组为实验组，5组为对照组，每组4只小鼠。实验组，出生后每日腹腔注射米诺环素22.5 mg/kg；对照组，出生后每日腹腔注射生理盐水10 ml/kg。在出生后1、7、14、21、28 d各处死一组实验组和对照组小鼠，取眼球做组织学观察并行凋亡细胞检测，并对两组视网膜光感受器细胞数、外核层厚度以及凋亡细胞数目进行统计分析。结果 （1）rd小鼠出生后7 d光感受器细胞开始凋亡，14 d达高峰，28 d光感受器细胞完全消失；（2）出生后7 d实验组光感受器细胞数目和外核层厚度与对照组比较差异无统计学意义；（3）出生后14、21 d实验组光感受器细胞数目和外核层厚度多于对照组相应时间点，差异有统计学意义（14 d：t=-3.03、P=0.016，t=-4.469，P=0.004；21d: t=-8.782、P＜0.001，t=-3.497、P=0.004）；（4）出生后7、14 d实验组外核层凋亡细胞数目少于对照组相应时间点，差异有统计学意义（t=-3.497、P=0.004，t=-8.782、P＜0.0001）。结论 米诺环素在rd小鼠RP早期可以延缓光感受器细胞丢失，但不能完全阻止RP的发生。     

Selective photoreceptor cell loss in hypoxic ischemia

We studied the histopathologic changes in the brain and eye of a 5 1/2-month-old infant who suffered a profound hypotensive episode three days antemortem. Nuclear pyknosis and other histologic changes characteristic of ischemic cell necrosis were found throughout the brain and in the retina's photoreceptor-cell layer. These findings suggest that hypoxic ischemia may produce preferential damage to the photoreceptor-cell layer.     

Visual deterioration in patients with photoreceptor loss after retinal reattachment surgery

Graefe's Archive for Clinical and Experimental Ophthalmology - Assess the relationship between photoreceptor degeneration and visual function after retinal reattachment surgery (RRS) in a...     

NADPH氧化酶抑制剂对遗传性视网膜色素变性感光细胞凋亡的抑制作用

背景 我们先前的研究表明,rd小鼠遗传性视网膜色素变性（RP）过程中小胶质细胞活化与感光细胞的凋亡密切相关.研究显示,小胶质细胞中烟酰胺二磷酸腺苷（NADPH）氧化酶的活化在小胶质细胞活化及神经元损伤中发挥重要作用,但NADPH氧化酶在RP过程中作用机制及其抑制剂的作用有待探讨.目的 探讨rd小鼠发生RP过程中NADPH氧化酶产生活性氧簇（ROS）的活化反应及其抑制剂对感光细胞的保护作用. 方法 按抛掷硬币法将60只SPF级rd小鼠随机分为香荚兰乙酮注射组和PBS对照组,香荚兰乙酮注射组小鼠于出生后9d（P9）腹腔内注射NADPH氧化酶抑制剂香荚兰乙酮10 mg/kg（0.01 ml/kg）,每日1次,连续5d（至P13）;PBS对照组rd小鼠以同样方式注射等容量的PBS;C57 BL/6N小鼠10只不注射任何药物作为rd小鼠的野生对照鼠.各组小鼠于出生后14 d（P14）处死并制备视网膜冰冻切片,采用二氢乙锭（DHE）荧光染色法检测3个组小鼠视网膜中ROS的表达;采用实时定量PCR（real-time PCR）法测定2个组rd小鼠视网膜感光细胞中视紫红质mRNA的定量表达;采用苏木精-伊红染色法检查2个组rd小鼠视网膜外核层厚度.结果 DHE荧光染色表明,小鼠视网膜中ROS表达呈红色荧光,注药组小鼠视网膜外核层中ROS的红色荧光明显强于C57BL/6N野生鼠,但明显弱于PBS对照组.Real-time PCR检测表明,香荚兰乙酮注射组小鼠感光细胞中视紫红质mRNA相对表达量为4.21 ±0.33,明显低于PBS对照组的0.93±0.24,差异有统计学意义（t=2.360,P=0.000）;香荚兰乙酮注射组小鼠视网膜外核层厚度为（35.95±1.63） μm,明显厚于PBS对照组的（23.17±1.38） μm,差异有统计学意义（t=3.850,P=0.016）.结论 在rd小鼠视网膜感光细胞变性过程中,NADPH氧化酶生成ROS的活化反应明显增强;香荚兰乙酮能够延缓rd小鼠感光细胞的凋亡过程.     

大鼠实验性视网膜光损伤中的视细胞凋亡

目的 进一步探讨视网膜光损伤的发病机制。 方法 20只Wistar大鼠分为实验组、对照组,分别在光照后12,24,36小时摘除眼球,视网膜组织行HE染色和核苷酸末端转移酶介导的DUTP缺口翻译法(TdT-mediated dUTP nick end labelling method,TUNEL)标记凋亡细胞。 结果 光照后12小时,视杆细胞外节出现少量空泡变性;24小时后,外核层出现明显的细胞核破碎、浓染和DNA裂解;36小时后,视杆细胞内、外节溶解,外核层大量细胞核丢失。 结论 视细胞凋亡是大鼠实验性视网膜光损伤的重要机制之一。 (中华眼底病杂志, 1999, 15: 167-169)     

The zinc-binding domain of Nna1 is required to prevent retinal photoreceptor loss and cerebellar ataxia in Purkinje cell degeneration (pcd) mice

The Purkinje cell degeneration (pcd) mouse undergoes retinal photoreceptor degeneration and Purkinje cell loss. Nna1 is postulated to be the causal gene for pcd. We show that a BAC containing the Nna1 gene rescues retinal photoreceptor loss and Purkinje cell degeneration, confirming that Nna1 loss-of-function is responsible for these phenotypes. Mutation of the zinc-binding domain within the transgene destroyed its ability to rescue neuronal loss in pcd(5J) homozygous mice. In conclusion, Nna1 is required for survival of retinal photoreceptors and other neuron populations that degenerate in pcd mice. A functional zinc-binding domain is crucial for Nna1 to support neuron survival.     

去小胶质细胞化对糖尿病小鼠视网膜光感受器细胞的影响

目的观察去小胶质细胞化对早期糖尿病小鼠视网膜光感受器细胞的影响。方法选取6～8周龄的SPF级雄性C57BL/6J小鼠作为实验动物,未经处理的5只作为空白对照组(A组),10只采用链脲佐菌素(STZ)腹腔注射法成功诱导出糖尿病后的小鼠随机等分为B、C两组。A、B组继续喂养标准实验饲料4周,C组在喂养1周标准实验饲料后添加含290 mg·kg^-1 PLX3397(集落刺激因子1受体拮抗剂)的AIN-76A(标准饮食配制的啮齿类实验动物纯化饲料)3周以去除小胶质细胞。4周后于同一时间点处死各组动物,眼球标本均于处死后即刻获取并固定。制备视网膜石蜡切片,HE染色后光学显微镜下观察视网膜结构;小胶质细胞特异性抗体P2ry12免疫荧光化学法检测小胶质细胞在视网膜上的分布,并测算平均吸光度(D)值以间接代表小胶质细胞的数量;TUNEL法测定光感受器细胞的凋亡情况;将上述观察指标在三组间进行比较。结果光镜观察视网膜结构显示,与A组相比,B组神经纤维层变水肿,内丛状层、内核层及外核层排列变疏松;C组也出现上述改变,但程度较轻。小胶质细胞的免疫荧光化学检测结果显示:A组可在视网膜内层...     

A pathologic study of photoreceptor cell death in retinal photic injury

In the studies of retinal photic injury in the rat model, about 14-47% of the photoreceptor cell loss occurs in the first 24 hours. To understand the mechanism of this massive cell death and subsequent dissolution, we studied the early morphologic changes and examined the effect of cycloheximide, a protein synthesis inhibitor, on photic injury in rats. Groups of 2 dark-adapted albino Lewis rats were sacrificed immediately after 8, 16 or 24 hr of continuous exposure to green fluorescent light (intensity, 160-180 foot-candles; wavelength, 490-560 nm). An additional 2 rats were sacrificed 8 hr after a 24 hr light exposure, and 2 animals served as unexposed controls. The morphologic findings of the degenerating photoreceptor cells were assessed by light and electron microscopy. The integrity of the nuclear chromatin was investigated using a monoclonal anti-DNA antibody. Most of the photoreceptor cell loss was observed between 16 and 24 hr of exposure. No inflammatory or macrophagic cells were seen. Different stages of nuclear condensation and chromatin margination could be defined. The chromatin showed a progressive decrease in DNA labelling density. Scattered photoreceptor cells showed early cytoplasmic densification. To study the effect of cycloheximide, 4 rats were treated with 5 mg/kg subcutaneously at the start of a 24 hr exposure period and were sacrificed 6 hr after the exposure. Four untreated animals served as exposed controls for morphometric comparison of the outer nuclear layer (ONL). The control rats showed a 24% decrease in the thickness of the ONL when compared to cycloheximide-treated rats (p less than 0.001). The observations of mitochondrial changes and early DNA digestion were consistent with necrosis as the mechanism of cell death. However, in scattered photoreceptor cells, cytoplasmic densification, margination of nuclear chromatin, the lack of associated inflammation and the protective effect of cycloheximide were suggestive of apoptosis as another mechanism of cell death.     

Soluble retinal proteins associated with photoreceptor cell death in the rd mouse

In the mouse, the homozygous presence of the rd gene results in the genetically programmed death of the photoreceptor cells of the retina. Using congenic strains of mice and a novel, sensitive, immunological approach for visualizing unique retinal proteins, we identified four bands of protein whose concentrations are regulated by the homozygous presence of the gene for retinal degeneration. Since these proteins (with apparent molecular weights of 23, 33, 55, and 69 kD) are present in normal adult mouse retinas and absent from rodless retinas, and from other mouse non-retinal tissues including brain, heart, kidney and liver, the data support the identification of these proteins as being retina specific. These proteins are not peculiar to the normal mouse retina; but rather, all four (23, 33, 55 and 69 kD) are common to rat retina; three (23, 33, and 55 kD) are common to bovine retina; and presently at least two, 23 and 69 kD, are clearly detectable in normal, adult human retina. The temporal appearance and disappearance of the four retinal specific protein bands coincide with the morphological maturation and degeneration of the photoreceptor cell population. Collectively, the present data suggest that one or more may be photoreceptor specific. These observations present the first step in the identification and characterization of specific soluble proteins correlated with the biochemical phenotype of the rd gene and the death of photoreceptor cells of the retina.     

视网膜脱离后光感受器细胞死亡机制及潜在治疗策略

视网膜脱离(RD)是临床上常见的致盲眼病之一,患者术后视功能恢复不理想.近年来研究表明,光感受器细胞死亡是导致RD后视功能损伤的主要原因,其发生机制复杂,涉及半胱氨酸蛋白酶依赖性的经典凋亡途径、内质网应激等非半胱氨酸蛋白酶依赖性凋亡途径以及坏死性凋亡、自噬等多种信号通路及其相互作用.阐明RD光感受器细胞的死亡机制,并在此基础上采取联合抑制多条死亡通路、干预上游靶点和加强内源性保护等策略有助于保护光感受器细胞,最终改善RD患者的视功能.本文综述了近年来RD光感受器细胞死亡机制的研究进展,探讨了可能存在的干预靶点以及未来潜在的治疗策略.     

Elevated retinal zeaxanthin and prevention of light-induced photoreceptor cell death in quail

PURPOSE: Inferential evidence indicates that macular pigments (lutein and zeaxanthin) protect photoreceptors and/or retard age-related macular degeneration. These experiments tested the hypothesis that retinal zeaxanthin prevents light-induced photoreceptor cell death. METHODS: Retinal damage was assessed in quail fed a carotenoid-deficient (C-) diet for 6 months. Groups of 16 birds (8 male, 8 female) were fed a C- diet supplemented with 35 mg 3R,3'R-zeaxanthin for 1, 3, or 7 days; one group was continued on C- diets. Half of each group was exposed to intermittent 3200-lux white light (10 1-hour intervals separated by 2 hours in dark). After 14 additional hours in the dark, one retina of each quail was collected for HPLC analysis, and the contralateral retina was embedded in paraffin for counts of apoptotic nuclei. RESULTS: After 7 days' supplementation, concentrations of zeaxanthin in serum, liver, and fat had increased by factors of 50.8, 43.2, and 6.5, respectively (all P < 0.001). In contrast, retinal zeaxanthin fluctuated significantly upward on day 3, but there was no net change on day 7. The number of apoptotic rods and cones in light-damaged eyes correlated significantly and inversely with zeaxanthin concentration in the contralateral retina (r = -0.61; P < 0.0001 and r = -0.54; P < 0.002), but not with serum zeaxanthin. Similar correlations were observed with retinal lutein, which correlated strongly with retinal zeaxanthin (r = 0.95; P < 0.0001). CONCLUSIONS: Retinal zeaxanthin dose dependently reduced light-induced photoreceptor apoptosis; elevated serum levels did not. These data provide the first experimental evidence that xanthophyll carotenoids protect photoreceptors in vivo.     

Topographic and morphologic analyses of retinal ganglion cell loss in old DBA/2NNia mice

PURPOSE: To evaluate the relationship between retinal ganglion cell (RGC) size, density distribution, and survival in senescent DBA2/NNia mice that develop pigmentary glaucoma. To evaluate the validity of nearest neighbor distance (NND), a measure of focal density for surviving RGCs in the retina, as a method to quantify RGC loss in mice. METHODS: Fifteen-month-old DBA2/NNia mice were labeled retrogradely with fluorogold. Retinas were flat mounted and imaged in their entirety using an epifluorescence microscope with a motorized stage. Digital maps of the retinal wholemounts were constructed to automatically count and establish spatial coordinates for RGCs over the entire retina. RGC size and NND were determined from these maps. RESULTS: RGC counts in the group of 15-month-old DBA/2NNia animals ranged from 22,330 to 92,157 cells per retina. Mean RGC cell size per retina ranged from 22.35 to 35.64 microm2 and correlated linearly with total RGC counts. NND distribution histograms were compared for retinas with variable degrees of RGC loss. The distribution of NNDs in each retina was skewed toward larger distance values in more affected retinas. In partially damaged retinas, areas with severe pathology coincided with areas of maximal loss of large RGCs, and areas of preserved RGCs correlated with larger cell sizes. CONCLUSIONS: Damaged retinas have a smaller mean cell size, indicating preferential loss of larger RGCs or size reduction of surviving cells. NND analysis of the RGC population in a retina is a useful measure of glaucomatous RGC loss. The skewed NND distribution of surviving RGCs and the finding that RGC loss correlates with a shift/amplitude change in the mode of the histogram and its tail suggests two different patterns of RGC loss possibly attributable to different pathologic processes in glaucomatous DBA/2 mice.     

Predisposing factors to light-induced photoreceptor cell damage: retinal changes in maturing rats

Retinal changes occurring during the period of growth and maturation of Long Evans pigmented rats were examined to obtain a better understanding of the basis for the age-dependency of light-induced photoreceptor cell damage. Susceptibility to light damage increased markedly between 30 and 60 days of age and to a lesser extent between 60 and 90 days. Although the retinal antioxidant vitamins E and C, and taurine showed a significant increase during the age-period studied, retinal lipid phosphorus and total protein increased by similar amounts indicating that the concentration of these nutrients was not changing. In contrast, rhodopsin content of the retina increased progressively by 44% between 30 and 90 days of age. While ROS length showed no appreciable change with age, rhodopsin per ROS length increased by 31% between 30 and 60 days of age and by 48% between 30 and 90 days. Determinations of ROS phospholipid to rhodopsin ratio and disks per ROS length indicated that rhodopsin did not become more concentrated in photoreceptor cells between 30 and 90 days. However, the 12% increase in ROS diameter between 30 and 90 days of age may partially account for the rhodopsin difference. These findings demonstrate an age-dependent association between greater rhodopsin per ROS length and increased susceptibility to retinal light damage. An increased metabolic demand on photoreceptor cells with greater rhodopsin may be an important factor influencing their destruction by light.     

Effects of macrophage and retinal pigment epithelial cell transplants on photoreceptor cell rescue in RCS rats

The effects of macrophage transplants on photoreceptor cell survival in retinas of Royal College of Surgeons (RCS) dystrophic rats were contrasted with RPE-cell transplants, sham-injection and surgical controls. The effects of these different treatments on the thickness and total area of the outer nuclear layer (ONL) were evaluated by light and electron microscopy at 1, 2 and 5 months after transplantation or surgical manipulations. Macrophage transplants into dystrophic retinas, although significantly reducing the debris zone thickness (p less than 0.01), had little effect on photoreceptor cell survival (2-3 cells thick ONL) after two months. In contrast, two months after RPE-cell transplantation, retinas exhibited an 8-10 cell thick ONL. Also, inner and outer segments of rescued photoreceptor cells were present, especially in areas directly beneath RPE-cell transplants. At the same time period, retinas injected with saline had a 2-3 cell thick ONL with no organized inner or outer segments. Furthermore, the affected ONL area in macrophage-transplanted or saline-injected retinas was significantly smaller than that seen in RPE-cell transplanted retinas (p less than 0.0001). Surviving photoreceptor cells were found only in the RPE-cell transplanted retinas five months after treatment. No effect on photoreceptor cell survival was seen in saline-injected, needle-inserted or incision-only retinas. Thus, transplantation of healthy RPE cells is an effective long-term therapeutic approach to correct the genetic defect in retinas of RCS dystrophic rats.