A demonstration of a switch of cell type in human fetal eye tissues in vitro: pigmented cells of the iris or the retina can transdifferentiate into lens

Pigmented cells of iris or retina from two human fetuses at 12 weeks after conception were cultured in vitro. In early stages of culturing (up to about 20 days), all cultured cells lost pigment granules. “Lentoid bodies” appeared at about 45 days in primary cultures of iris cells. Primary cultures of retinal pigment cells were repigmented by 30 days. When a small number of repigmented cells was transferred into secondary cultures, colonies with “lentoid bodies” were formed by 40 days. The lens nature of “lentoid bodies” was confirmed by immunofluorescence and immunoelectrophoresis using anti-rat lens serum which crossreacted with human crystallins.     

Mercapturic acid pathway enzymes in bovine ocular lens, cornea, retina and retinal pigmented epithelium

Analogous to the liver, ocular tissues contain large concentrations of glutathione and are exposed to potentially damaging chemical compounds. Since glutathione has been shown to have a detoxification function, via mercapturic acid production in the liver, we investigated whether glutathione has a similar function in ocular tissues. We have demonstrated the presence of all of the enzymes involved in the mercapturic acid pathway i.e. glutathione S-transferase, gamma-glutamyl transpeptidase, cysteinylglycinase, and N-acetyl transferase, in the ocular tissues of bovine lens, cornea, retina, and retinal pigmented epithelium. Therefore glutathione may have another function in ocular tissues, that of the detoxification of xenobiotics.     

Lens epithelial cells synthesize and secrete ceruloplasmin: effects of ceruloplasmin and transferrin on iron efflux and intracellular iron dynamics

Although an essential nutrient, iron can catalyze damaging free radical reactions. Therefore elaborate mechanisms have evolved to carefully regulate iron metabolism. Ceruloplasmin, a protein with ferroxidase activity, and transferrin, an iron binding protein have important roles in maintaining iron homeostasis in cells. Since oxidative damage is a hallmark of cataractogenesis, it is essential to determine iron's role in lenticular physiology and pathology. In the current study of lens epithelial cells, the effects of ceruloplasmin and transferrin on intracellular distribution and efflux of iron were determined. Both ceruloplasmin and transferrin increased iron efflux from these cells and their effects were additive. Ceruloplasmin had significant effects on extracellular iron distribution only in cases of iron overload. Surprisingly, both transferrin and ceruloplasmin had significant effects on intracellular iron distribution. Under physiological conditions, ceruloplasmin increased iron incorporation into the storage protein, ferritin. Under conditions of iron overload, it decreased iron incorporation into ferritin, which is consistent with increased efflux of iron. Measurements of an intracellular chelatable iron pool indicated that both transferrin and ceruloplasmin increased the size of this pool at 24 h, but these increases had different downstream effects. Finally, lens epithelial cells made and secreted transferrin and ceruloplasmin. These results indicate an important role for these proteins in iron metabolism in the lens.     

Imaging retinal mosaics in the living eye

Adaptive optics imaging of cone photoreceptors has provided unique insight into the structure and function of the human visual system and has become an important tool for both basic scientists and clinicians. Recent advances in adaptive optics retinal imaging instrumentation and methodology have allowed us to expand beyond cone imaging. Multi-wavelength and fluorescence imaging methods with adaptive optics have allowed multiple retinal cell types to be imaged simultaneously. These new methods have recently revealed rod photoreceptors, retinal pigment epithelium (RPE) cells, and the smallest retinal blood vessels. Fluorescence imaging coupled with adaptive optics has been used to examine ganglion cells in living primates. Two-photon imaging combined with adaptive optics can evaluate photoreceptor function non-invasively in the living primate retina.     

Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid

The discoveries of gene variants associated with macular diseases have provided valuable insight into their molecular mechanisms, but they have not clarified why the macula is particularly vulnerable to degenerative disease. Its predisposition may be attributable to specialized structural features and/or functional properties of the underlying macular RPE/choroid. To examine the molecular basis for the macula's disease susceptibility, we compared the gene expression profile of the human RPE/choroid in the macula with the profile in the extramacular region using DNA microarrays. Seventy-five candidate genes with differences in macular:extramacular expression levels were identified by microarray analysis, of which 29 were selected for further analysis. Quantitative PCR confirmed that 21 showed statistically significant differences in expression. Five genes were expressed at higher levels in the macula. Two showed significant changes in the macular:extramacular expression ratio; another two exhibited changes in absolute expression level, as a function of age or AMD. Several of the differentially expressed genes have potential relevance to AMD pathobiology. One is an RPE cell growth factor (TFPI2), five are extracellular matrix components (DCN, MYOC, OGN, SMOC2, TFPI2), and six are related to inflammation (CCL19, CCL26, CXCL14, SLIT2) and/or angiogenesis (CXCL14, SLIT2, TFPI2, WFDC1). The identification of regional differences in gene expression in the RPE/choroid is a first step in clarifying the macula's propensity for degeneration. These findings lay the groundwork for further studies into the roles of the corresponding gene products in the normal, aged, and diseased macula.     

New ultrastructure of rat RPE cells: basal intracytoplasmic tubules

Retinal pigment epithelial cells have prominent basal folds facing Bruch's membrane. In addition to folds I have observed intracytoplasmic tubules 60-90 nm in diameter in the basal cytoplasm of rat pigment epithelial cells. The tubules arise from the basal plasma membrane and open to the extracellular space. The tubules are most evident when intravenous horseradish peroxidase is used as a tracer. The tracer leaks out of the fenestrated choriocapillaris, into the extracellular space of the pigment epithelium and into the tubules. Electron microscopy at 1000 KV (High Voltage Electron Microscopy) confirms the tubular nature of these structures and their continuity with folds or the plasma membrane facing Bruch's membrane. The tubules are also observed in tissue not infiltrated with peroxidase. Morphometry shows that the tubules occupy about 21% of the surface area of the basal plasma membrane. Tubules appear plentiful where folds are reduced, and reduced where folds are plentiful; the tubules may be a different conformation of the normally slit-like fold extracellular space. The tubules are observed in all quadrants of the retina; centrally and peripherally; in young and adult rats and in pigmented and albino rats. The tubule's function may be linked to that of the folds, from which many of them arise.     

Light-induced Apoptosis: Differential Timing in the Retina and Pigment Epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

Selenium and non-selenium glutathione peroxidase activities in selected ocular and non-ocular rat tissues

Selenium (Se)-glutathione peroxidase activities were measured using H₂O₂ as a substrate in retina, retinal pigment epithelium (RPE), lens, brain, liver and testes from rats either deficient or supplemented with vitamin E and selenium for 20–30 weeks. In the supplemented rats, the RPE was found to be particularly rich in Se-glutathione peroxidase activity, being second only to liver in specific enzyme activity. Non-Se-glutathione peroxidase activities were also estimated in the same tissues utilizing an organic hydroperoxide substrate. Except for the retina and testes, only the Se-glutathione peroxidase activity could be detected to any significant extent in tissues from rats fed the supplemented diet. In rats fed the deficient diet there is a large decrease in the Se-enzyme activity for all tissues examined except brain. Except for brain and perhaps testes, tissues from deficient rats also show an increase in the fraction of non-Se-enzyme activity contributed to total glutathione peroxidase activity. For RPE and testes, the ratio of non-Se-enzyme activity to Se-enzyme activity is estimated to be about one-tenth the ratio found in other tissues examined in the deficient rats.In a previous publication (Katz, Stone and Dratz, 1978) we have shown that RPE and testes from vitamin E and Se deficient rats are particularly sensitive to the accumulation of a yellow autofluorescent pigment which is thought to be indicative of lipid peroxidation. Other retinal tissues, as well as brain and liver, accumulate much less of this pigment. The relatively low ratio of non-Se-enzyme to Se-enzyme activity observed in the RPE and testes of deficient rats may be related to the apparent susceptibility of these tissues to lipid peroxidation.     

Value of the Congenital Hypertrophy of the Retinal Pigment Epithelium in the Diagnosis of Familial Adenomatous Polyposis

BACKGROUND: Several kinds of congenital hypertrophy of the retinal pigment epithelium (CHRPE) have been described in patients with familial adenomatous polyposis (FAP). This study aims to assess which properties of CHRPE better predict FAP and investigate whether a relationship exists between specific CHRPE characteristics and FAP variants. METHODS: We examined 286 subjects, Group I--patients with FAP plus individuals "at risk"; n = 173; Group II--controls n = 113. Retinal lesions were classified in five types (A-E) and different characteristics (distribution, number, shape, size, pigmentation and site) were evaluated. RESULTS: The most common lesions in affected subjects were types A-D (83.4%) whilst in the "at risk" and control groups were type E. Greater numbers of lesions and bilateral distribution occurred more frequently among affected subjects than in other participants (p < 0.001). Large lesions with mixed pigmentation were associated with polyposis (p > 0.5). Controls had solitary CHRPE lesions (3.5%) and types C and E lesions (23%). The cumulative sensitivities and specificities of CHRPE were 42 and 97%, respectively. CHRPE was most common among those with classical FAP, but no specific characteristic was associated with any particular FAP variant. CONCLUSIONS: Pigmented fundal lesions are highly pleomorphic and represent the variable expression of a common genetic defect of growth regulation. No association was found between CHRPE characteristics and specific FAP variants.     

Management of crystalline lens dislocation into the anterior chamber in a victim of domestic violence

A 53-year-old woman presented with a right eye injury due to domestic violence. The crystalline lens in that eye was completely dislocated into the anterior chamber, fixated by the cornea and the iris. Fundoscopy showed a mild vitreous haemorrhage and a giant retinal tear. A modified lensectomy with a fragmatome was performed. During this procedure the capsule was preserved first to minimise the risk of corneal damage and second to reduce vitreous traction, which would repair the giant retinal tear.     

单眼斜视性弱视患者黄斑神经节细胞复合体厚度分析

目的：通过检测单眼斜视性弱视患者双眼黄斑区视网膜神经节细胞复合体厚度,探讨弱视的程度与视网膜神经节细胞厚度的相关性,以及斜视性弱视患者黄斑区神经节细胞复合体结构是否存在异常。
　　方法：选取临床单眼斜视性弱视患者26例52眼。采用美国傅立叶域光学相干断层扫描仪( fourier-domain optical coherence tomography,FD-OCT) iVue同时检测双眼黄斑区神经节细胞复合体厚度,并比较黄斑区神经节细胞复合体厚度与患者最佳矫正视力的相关性。
　　结果：黄斑区神经节细胞复合体厚度( macular ganglion cell complex, mGCC )分为三个区域测量：中心区、内圈(3mm )区、外圈(6mm )区。在斜视性弱视的眼(strabismic amblyopia eye,SAE)测量结果分别是50.74±21.51,101.4±8.51,114.2±9.455μm;在对侧对照眼(contralateral sound eyes,CSE)测量结果分别是43.79±11.92,92.47±25.01,113.3±12.88μm。斜视性弱视眼的黄斑区视神经节细胞复合体厚度与对侧眼相比无显著差异(P>0.05)。最佳矫正视力与黄斑区神经节细胞复合体厚度有相关性,视力矫正越好弱视程度越低的黄斑区神经节细胞复合体厚度相对增厚,黄斑区下方区域的神经节细胞复合体厚度与视力矫正的相关性优于上方。
　　结论：黄斑中心区神经节细胞复合体厚度与弱视矫正视力之间有相关性,虽然mGCC厚度在SAE和CSE无显著差异,但测量mGCC厚度对了解弱视程度有临床意义。     

An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice

PurposeThe purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light.MethodsScotopic and photopic electroretinogram (ERG) responses in young (3–6 months) and aged (23–26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-¹³C-glucose or U-¹³C-glutamine with gas chromatography-mass spectrometry (GC-MS), O₂ consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay.ResultsScotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable.ConclusionsOur examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.     

自噬在视网膜和眼部疾病中的研究进展

自噬维持细胞内成分降解和再循环的稳态,是一种关键的细胞质量控制机制。在应激反应中,自噬促进细胞成分的降解,以提供细胞代谢所需的营养物质和能量。视网膜是眼睛中转导和处理视觉信息的光敏组织,对物质和能量需求极高,基础水平的自噬对维持视网膜细胞的稳态和视觉系统的正常功能至关重要。本文总结了自噬途径参与青光眼、年龄相关性黄斑变性、糖尿病视网膜病变、视网膜营养不良和视网膜脱离等眼科疾病的最新研究,为未来通过调控自噬治疗眼部疾病提供理论依据。     

The bionic eye: a review

Visual prostheses including artificial retinal devices are a novel and revolutionary approach to the treatment of profound visual loss. The development of the field of visual prosthesis began with cortical prosthetic devices but since then, a variety of devices which target different sites along the visual pathway have been developed with the retinal prosthesis being the most advanced. We present a review of the history of these devices, an update on the current state of play and future prospects of this field.     

Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

The proteasome plays a fundamental role in processes essential for cell viability. A loss in proteasome function has been associated with aging, as well as a number of age-related diseases. Defining the mechanism(s) behind this loss in function will add important information regarding the molecular basis for aging. In the current study, we performed an age-based comparison of proteasome function and composition of subunits and regulatory proteins in the neural retina and retinal pigment epithelium (RPE) in Fischer 344 rats. In the RPE, there was no age-dependent difference in activity, subunit composition, or content of proteasome regulators, PA28 and PA700. In contrast, the aged neural retina demonstrated a significant reduction in the chymotrypsin-like activity and decreased degradation of both casein and casein modified by 4-hydroxynonenal. This loss in function could not be explained by differences in subunit composition, content of PA28 and PA700, or reversible modification of cysteine residues. To begin investigating the molecular basis for the age-associated decrement in proteasome function, we modified the cysteine residues in proteasome from young rats with the sulfhydryl-reactive chemical N-ethylmaleimide. We observed inhibition of the chymotrypsin-like activity and decreased degradation of casein that was comparable to that seen in aged retinas. Thus, chemical modification of cysteine provides an in vitro method that partially recapitulates aging proteasome. Further studies are required to confirm irreversible modification of functionally significant cysteine as a potential mechanism behind the age-related loss in proteasome function.     

CD81在正常大鼠视网膜色素上皮层的表达

目的:观察CD81在正常大鼠视网膜色素上皮层(retinal pigment epithelium,RPE)的表达。方法:用抗CD81抗体EAT2对正常大鼠RPE组织切片及体外培养RPE进行免疫组织化学染色,H121对RPE组织进行CD81蛋白的Western blot分析。结果:正常大鼠视网膜的色素上皮细胞膜表面呈CD81的阳性染色,体外培养的大鼠RPE也呈CD81阳性表达;Western blot分析RPE层蛋白出现CD81阳性条带。结论:正常大鼠的RPE层可以表达CD81。     

A novel cone visual cycle in the cone-dominated retina

The visual processing of humans is primarily reliant upon the sensitivity of cone photoreceptors to light during daylight conditions. This underscores the importance of understanding how cone photoreceptors maintain the ability to detect light. The vertebrate retina consists of a combination of both rod and cone photoreceptors. Subsequent to light exposure, both rod and cone photoreceptors are dependent upon the recycling of vitamin A to regenerate photopigments, the proteins responsible for detecting light. Metabolic processing of vitamin A in support of rod photopigment renewal, the so-called "rod visual cycle", is well established. However, the metabolic processing of vitamin A in support of cone photopigment renewal remains a challenge for characterization in the recently discovered "cone visual cycle". In this review we summarize the research that has defined the rod visual cycle and our current concept of the novel cone visual cycle. Here, we highlight the research that supports the existence of a functional cone-specific visual cycle: the identification of novel enzymatic activities that contribute to retinoid recycling, the observation of vitamin A recycling in cone-dominated retinas, and the localization of some of these activities to the Müller cell. In the opinions of the authors, additional research on the possible interactions between these two visual cycles in the duplex retina is needed to understand visual detection in the human retina.