Müller glial cells--the mediators of vascular disorders with vitreomacular interface pathology in diabetic maculopathy

The key to identifying the type of diabetic maculopathy is determining the status of posterior vitreous adhesion. In the pathological state, the breakdown of the internal and external blood-retina barrier is evident, however the mechanism is usually complex. The common denominator for these disorders are Müller glial cells, which mediate in maintaining the blood-retina barrier by linking the vessels, neurons and the vitreous in anatomical network and into functional dependence. The breakdown of the blood-retina barrier results in proliferation of Müller cells. Molecular changes in these cells increase endothelial barrier properties, but also induce pathological processes on the vitreo-retinal junction, resulting in increased adhesiveness of the collagen fibers of vitreous to retinal internal limiting membrane. The ability of Müller cells to reactive gliosis is influenced by the healthy functioning of the retinal pigment epithelium, which is a source of trophic factors necessary for appropriate Müller cells morphogenesis. Vitrectomy with the removal of ILM eliminates the vitreofoveal interface pathology, additionally provoking reactive gliosis within the macula. Intraoperative use of anti-VEGF supports short-term tightness of the blood-retina barrier in the perioperative neuralgic period. In the future, supplying astrocytes may be a strategy that will allow not only the inhibition of pathological neovascularization but also the restoration of the physiological network of capillaries in avascular retina areas. The delivery of recombinant PEDF allows for the recovery of Müller cells, and thus creates the conditions favourable for the survival of nerve cells in loss of retinal homeostasis.     

Leukocyte diapedesis in vivo induces transient loss of tight junction protein at the blood-retina barrier

PURPOSE: Although much is now understood about the molecular structure of tight junctions (TJs), little is known about the regulation of their function during neural inflammatory disease processes in vivo. The mechanisms by which leukocytes transmigrate the blood-retina barrier (BRB) without affecting endothelial permeability are controversial. METHODS: Confocal immunofluorescence microscopy of ex vivo retinal wholemounts was used to study BRB integrity during leukocyte adhesion and migration during experimental autoimmune uveoretinitis (EAU). Western blot analysis was used to measure levels of TJ proteins in EAU retina and RPE and in normal retina or RPE cultured with cytokines or chemokines. RESULTS: No evidence for discontinuity or other weakness of the endothelial or epithelial barrier at tricellular corners was observed, and maximum disruption of TJ protein expression was focused in retinal venules correlating with sites of leukocyte extravasation. Areas of maximum TJ protein loss in vivo also correlated with redistribution or loss of ensheathing astrocyte processes on venules but not adjacent capillaries or arterioles. Exposure of normal choroidal and retinal explants ex vivo to cytokines and chemokines alone did not downregulate total occludin-1 or claudin-3 TJ protein expression. CONCLUSIONS: The data presented herein support an active role for leukocytes in TJ disruption and blood-retina barrier (BRB) breakdown during retinal inflammation and further implicate venule microenvironment as a key factor in leukocyte recruitment to retinal tissue in vivo.     

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.     

Dysfunction and repair of the blood-retina barrier following white light exposure: a fluorophotometric and histologic study.

The purpose of this study was to pinpoint the site of blood-retina barrier disruption after white light exposure and determine the course of barrier repair. The retinas of 25 anaesthetized pigmented rabbits were exposed for 1 hr to the light of a xenon arc lamp filtered to eliminate ultraviolet and infrared light. The light intensities selected were near the threshold intensity causing visible retinal lesions in order to evaluate the function of the blood-retina barrier (BRB) in this range. Functional assessment of the BRB was made with vitreous fluorophotometry (VF), and electron microscopy (EM) after intra-arterial administration of horseradish peroxidase (HRP) as tracer. In 11 of the 14 rabbits exposed to threshold intensity (90-110 mW cm-2; retinal field of illumination, 0.64 cm2), a breakdown of the BRB was demonstrated by a 2-40-fold increase in the permeability of the BRB for fluorescein and by transcellular passage of HRP through the retina pigment epithelium (RPE). All 11 rabbits developed oedematous fundus lesions. Within a week, pigmentary alterations of the fundus were seen on ophthalmoscopy, while the BRB permeability for fluorescein and HRP had returned to normal. EM of the retina showed slight swelling of RPE during the period of increased permeability but no alterations of the neuroretina. After functional barrier repair, the RPE cells demonstrated irregularity of the melanin pigment alignment and some loss of the monocellular arrangement. In six rabbits exposed to subthreshold light intensity (65-89 mW cm-2) no fundus lesion developed and EM evaluation of the BRB was normal.2+ remains altered.     

Tests for malingering in ophthalmology

Simulation can be defined as malingering, or sometimes functional visual loss (FVL). It manifests as either simulating an ophthalmic disease (positive simulation), or denial of ophthalmic disease (negative simulation). Conscious behavior and compensation or indemnity claims are prominent features of simulation. Since some authors suggest that this is a manifestation of underlying psychopathology, even conversion is included in this context. In today’s world, every ophthalmologist can face with simulation of ophthalmic disease or disorder. In case of simulation suspect, the physician’s responsibility is to prove the simulation considering the disease/disorder first, and simulation as an exclusion. In simulation examinations, the physician should be firm and smart to select appropriate test(s) to convince not only the subject, but also the judge in case of indemnity or compensation trials. Almost all ophthalmic sensory and motor functions including visual acuity, visual field, color vision and night vision can be the subject of simulation. Examiner must be skillful in selecting the most appropriate test. Apart from those in the literature, we included all kinds of simulation in ophthalmology. In addition, simulation examination techniques, such as, use of optical coherence tomography, frequency doubling perimetry (FDP), and modified polarization tests were also included. In this review, we made a thorough literature search, and added our experiences to give the readers up-to-date information on malingering or simulation in ophthalmology.     

Endothelin-1 and endothelin receptors in light-induced retinal degeneration

We have studied the distribution of endothelinergic molecules: prepro-endothelin-1 (PPET-1), endothelin-1 (ET-1), and receptors A and B (ET-A) and (ET-B) in the retina of mice. The localization of these molecules in normal mice was compared to their localization in retinas from animals submitted to continuous illumination during 1, 6, 9 or 18 days. We also evaluated the distribution of smooth muscle actin (SMA) and glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthase (GS). PPET-1 immunoreactivity mainly appeared in retinal pigment epithelium (RPE) and cells of the ganglion cell layer (GCL), whereas ET-1 immunoreactivity was present in the RPE, outer plexiform layer (OPL) and astrocytes. Astrocytes exhibited the strongest immunostaining in the retina. ET-A immunoreactivity was observed in endothelium, RPE, OPL and cells of the GCL. By contrast, ET-B immunoreactivity could be detected in endothelial cells, horizontal cells and astrocytes. Astrocytes of the optic nerve also exhibited ET-1, ET-A, and ET-B immunoreactivities. After light-induced degeneration, there was an increase of RPE immunostaining. Degeneration of photoreceptors was accompanied by disappearance of immunoreactivity in the OPL. However, ET-A immunoreactivity appeared in the amacrine sublayer of the INL. There was an enormous increase in astrocytes and its cell processes. The increase of astrocytic immunoreactivities for ET-1 and ET-B was confirmed by quantitative image analysis. Growth of astrocytic cell processes was most marked around retinal blood vessels. Our findings indicate that there are at least three endothelinergic pathways in the normal retina: (1) between the RPE and choriocapillaris, (2) at the OPL, and (3) between blood vessels, astrocytes and cells of the GCL. After light-induced degeneration of photoreceptors, endothelinergic molecules were overexpressed at the RPE and astrocytes, but mostly disappeared from the OPL.     

Ultrastructural and immunocytochemical changes in retinal pigment epithelium, retinal glia, and fibroblasts in vitreous culture.

Retinal pigment epithelial (RPE) cells, retinal glial, and fibroblasts, three cell types believed to play a role in the pathogenesis of epiretinal membrane formation, were maintained in vitreous culture to determine the influence of vitreous on their ultrastructure and expression of cytokeratin, glial fibrillary acidic protein (GFAP), vimentin, and glutamine synthetase (GS). Using a highly sensitive, preembedding technique for the immunolocalization of these antigens at the ultrastructural level, most RPE cells were found to lose cytokeratin and vimentin within 1 day after seeding on irradiated vitreous. The percentage of keratin-positive cells then increased with time in culture. If the vitreous was placed on RPE cells cultured in monolayer instead of placing the cells on the vitreous, keratin and vimentin were expressed these intermediate filament proteins diminished with time. Glutamine synthetase was found in RPE cells grown in monolayer with or without a vitreous overlay, but not in RPE cells grown on the surface of vitreous. Retinal glial grown on vitreous showed a time-dependent decrease in the number of cells expressing GFAP and a corresponding increase in cells expressing vimentin or GS. Some fibroblasts in vitreous culture expressed vimentin but not the other antigens evaluated. A substantial number of cells in each culture did not stain positively for cytokeratin, GFAP, vimentin, or GS. All three cell types showed phenotypic diversity at the ultrastructural level with each cell type being capable of assuming the same morphologic appearance under certain conditions. These results demonstrate the phenotypic plasticity of RPE cells, retinal glia, and fibroblasts when grown in contact with vitreous and provide further evidence that neither ultrastructure, intermediate filament protein expression, nor the presence of GS is sufficient to determine the cell type of origin of cells in epiretinal membranes.     

重组人促红细胞生成素对小鼠视网膜光损伤的防护作用

目的　探讨重组人促红细胞生成素(rHEPO)通过小鼠血视网膜屏障的情况及其对视网膜光损伤的保护作用。方法　24只BALB/c小鼠腹腔注射rHEPO,采用酶联免疫吸附测定(ELISA)法检测小鼠视网膜中促红细胞生成素(EPO)的含量; 24只BALB/c小鼠建立光损伤动物模型,通过光镜和核苷酸末端转移酶介导的dUTP缺口标记(TUNEL)法观察实验组小鼠(12只,腹腔注射rHEPO)和对照组小鼠( 12只,腹腔注射生理盐水)视网膜细胞的变化情况。结果　腹腔注射rHEPO后2、4、6及8h小鼠100μg视网膜蛋白中EPO的含量分别为(0 .68±0 .24)、(1 .87±0 .37)、(0. 96±0 .24)及(0. 47±0. 13)mU,差异有统计学意义(F=2 .113,P<0 .05),在腹腔注射药物后4h视网膜中EPO的浓度达到高峰。随光照时间延长,光镜下可见对照组视网膜杆细胞的内、外节破坏明显加重,外核层逐渐变薄,出现核固缩,甚至核碎裂;实验组各观察时间点损伤变化均较轻,仅见感光细胞内、外节排列紊乱,形成空泡,外核层细胞排列稍紊乱,厚度无明显变化。荧光显微镜下观察,对照组视网膜外核层的凋亡细胞随光照时间延长不断增多,至光照后7d凋亡细胞数量减少;实验组视网膜外核层仅见少量凋亡细胞,光照后72h凋亡细胞的数量明显减少,至光照后7d几乎无凋亡细胞。实验组视网膜外核层凋亡细胞的数量     

Muller cell expression of glutamate cycle related proteins and anti-apoptotic proteins in early human retinal development

AIMS: The distribution of glutamate cycle related proteins (glutamine synthetase (GS) and GLAST) and anti-apoptotic proteins (Bcl-2 and Bcl-X) was investigated in Müller cells during early human retinal development, relative to the onset of expression of synaptophysin, a presynaptic vesicle protein. METHODS: Using frozen sections of human fetal eyes (13-22 weeks gestation) (n = 10), Bcl-2, Bcl-X, GS, GLAST, and synaptophysin immunoreactivities (IR) were imaged using fluorescence microscopy and plotted as a function of eccentricity from the incipient fovea. Frozen sections of adult human retina (n = 4) were immunolabelled with antibodies to Bcl-2 and Bcl-X. RESULTS: Müller cell immunoreactivity for GS, GLAST, and Bcl-2 was initially detected in the incipient fovea, and then at more peripheral locations with increasing age. Synaptophysin-IR appeared earlier than all other target proteins. Within the synaptophysin-IR region, mature (differentiated) Müller cells expressed both Bcl-2 and Bcl-X-IR from 13 weeks gestation, ahead of GS-IR and GLAST-IR that were first seen at 14 weeks gestation. Additionally, from as early as 13 weeks gestation, ganglion cells and immature neuronal progenitor cells across the entire retina expressed Bcl-2-IR and Bcl-X-IR, respectively. In adult retina, ganglion cells and some bipolar cells expressed Bcl-X but not Bcl-2. CONCLUSION: Müller cells express Bcl-2 and Bcl-X after synaptogenesis has commenced, but before the onset of GS and GLAST expression, suggesting a protective role for these proteins in Müller cells during the onset of glutamatergic transmission in early human retinal development.     

胚胎发育中神经视网膜对血视网膜外屏障结构形成的作用

目的 研究胚胎发育中神经视网膜对血视网膜外屏障结构形成的作用。 方法 将孵化7、10、14 d的鸡胚眼各150、120和90只，分批分别剥离视网膜神经上皮层（RNE）和色素上皮层（RPE）。RNE用来制备不同条件的培养液（7drcS F3、10drcSF3和14drcSF3），将7 d和14 d的RPE细胞分别培养到12 mmTranswell滤膜上,其顶房分别用SF3或上述条件培养液，底房均用SF2培养液。形成单层RPE细胞后，测定此上皮层的电阻(TER)。最后固定细胞，用细胞免疫组织化学方法和透射电子显微镜检测RPE细胞间紧密连接形成的情况。 结果 不同培养液(SF3/S F2, 7drcSF3/SF2,10drcSF3/SF2, 14drcSF3/SF2)培养7d和14d鸡胚眼的RPE细胞TER之间的差异有非常显著性的意义(P＜0.01)。电子显微镜结果显示神经视网膜条件培养液可以使RPE细胞产生细胞极性，有效地促使紧密连接条带形成连续的网状结构。 结论 神经视网膜对血视网膜外屏障结构的形成起积极的促进作用。 （中华眼底病杂志，2004，20：237-240）     

脑源性神经营养因子治疗视网膜色素变性的研究进展

脑源性神经营养因子(BDNF)是一种由脑组织合成并广泛分布于中枢神经系统的小分子碱性蛋白.研究发现,BDNF对视网膜神经节细胞(RGCs)、视网膜感光细胞(RPCs)和视网膜色素上皮(RPE)细胞均有保护、营养及抗凋亡作用.视网膜色素变性(RP)是由于RPCs及RPE细胞凋亡引起的视网膜退行性疾病.RP动物模型证实了BDNF的长期给药对于RP的治疗价值.然而BDNF在体内的半衰期较短,且无法跨越血-视网膜屏障由循环系统输送到视网膜,这给BDNF用于RP的治疗带来了挑战.为了使BDNF在眼内可以稳定持续地释放,多种新型给药方式已被尝试,包括基因工程技术、细胞移植技术、高分子材料缓释系统及滴眼液等.本文就BDNF对RP治疗的研究现状及BDNF的新型给药方式做一综述.     

Polyamine-dependent migration of retinal pigment epithelial cells

PURPOSE: Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration. METHODS: Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of ((14)CO(2))(.) Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody. RESULTS: The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm. CONCLUSIONS: Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.     

AII amacrine cells express the MT1 melatonin receptor in human and macaque retina

AII amacrine cells are critical interneurons in the rod pathway of mammalian retina, active primarily in dim lighting conditions. Melatonin, a neuromodulator produced at night in the retina, is believed to induce retinal adaptation to dim lighting conditions in most vertebrate species examined to date, including humans. We hypothesized that melatonin may influence retinal light adaptation by acting on AII cells directly and thus investigated whether melatonin receptors were expressed in AII neurons. Postmortem nonpathological eyes from four human donors as well as two eyes from two Macaque Fasicularis monkeys were analyzed. Double immunocytochemistry was performed using an anti-MT(1) antibody and an antibody to calretinin, an AII marker. Analysis utilized confocal microscopy. A polyclonal anti-calretinin antibody labelled amacrine cells exhibiting the distinct AII morphology, in both human and macaque retina. MT(1) immunoreactivity in macaque retina was similar to human staining, in that horizontal, amacrine and ganglion cell bodies were stained, as were inner segments of photoreceptors. In human retina 86% of calretinin positive cells expressed the MT(1) receptor peripherally, whereas centrally, 78% colocalization was observed. In the macaque retina, 100% of AII amacrine cells expressed MT(1) immunoreactivity both centrally and peripherally. That virtually all AII neurons express the MT(1) receptor in both human and macaque retina, may provide the first evidence demonstrating a role for melatonin in AII regulation, furthering the hypothesis of melatonin function in retinal light adaptation.     

Integration of tight junctions and claudins with the barrier functions of the retinal pigment epithelium

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier by regulating the movement of solutes between the fenestrated capillaries of the choroid and the photoreceptor layer of the retina. Blood-tissue barriers use various mechanisms to accomplish their tasks including membrane pumps, transporters, and channels, transcytosis, metabolic alteration of solutes in transit, and passive but selective diffusion. The last category includes tight junctions, which regulate transepithelial diffusion through the spaces between neighboring cells of the monolayer. Tight junctions are extraordinarily complex structures that are dynamically regulated. Claudins are a family of tight junctional proteins that lend tissue specificity and selectivity to tight junctions. This review discusses how the claudins and tight junctions of the RPE differ from other epithelia and how its functions are modulated by the neural retina. Studies of RPE-retinal interactions during development lend insight into this modulation. Notably, the characteristics of RPE junctions, such as claudin composition, vary among species, which suggests the physiology of the outer retina may also vary. Comparative studies of barrier functions among species should deepen our understanding of how homeostasis is maintained in the outer retina. Stem cells provide a way to extend these studies of RPE-retinal interactions to human RPE.     

Evaluation of indocyanine green toxicity to rat retinas

PURPOSE: To investigate the toxicity of indocyanine green (ICG) on retinal cells using cultured retinal pigment epithelium (RPE) cells and the effects of intravitreous injection of ICG into rat eyes. METHODS: Cultured RPE cells were exposed to various concentrations of ICG for 2 min, a viability assay was performed 1 day after exposure. For an in vivo study, 5 microl of ICG (5 or 25 mg/ml) were injected into the vitreous cavity of rat eyes, which were examined 1, 3 and 7 days after the injection by histological and glutamine synthetase (GS) immunohistological evaluation. RESULTS: Viabilities of RPE cells were decreased dependent on the ICG dose. In the histological evaluation, we observed differences of effects of ICG between the central retinal area and the peripheral area. ICG injection caused degeneration of all retinal layers in the central retinal area. GS immunoreactivities decreased by ICG injection, which corresponded to an area of severe destruction. CONCLUSION: A high concentration of ICG may cause toxic effects on retinal cells. Mueller cell dysfunction may play some role in the retinal toxicity caused by ICG.     

Induced expression of hematopoietic- and neurologic-expressed sequence 1 in retinal pigment epithelial cells during newt retina regeneration

Newts can regenerate their organs even as adults. For instance, when their neural retinas are completely removed by operation, the remaining retinal pigment epithelial (RPE) cells dedifferentiate to reconstruct neural retinas. To elucidate the molecular mechanisms of newt retina regeneration, we investigated genes upregulated in dedifferentiating RPE cells using differential display methods. We observed that a cDNA fragment of hematopoietic- and neurologic-expressed sequence 1 (Hn1) appeared to be induced within a few days of surgical removal of newt neural retina. Using an anti-HN1 antiserum against the recombinant HN1 protein, we carried out immunohistochemical analyses. The anti-HN1 antiserum recognized the plexiform layers and ganglion cell layer (GCL) but not the RPE cell layer in unoperated (normal) newt retinas. Using a glial fibrillary acidic protein antibody, Hn1 was shown to be possibly expressed in glial cells in normal neural retina. During retina regeneration, immunoreactivity for HN1 appeared in dedifferentiating RPE cells 10 days post-operation, and in retinal progenitor cells 18 days post-operation. Twenty seven days post-operation, HN1 immunoreactivity was localized in the plexiform layers and GCL as in the normal retina. Therefore, HN1 possibly plays an undefined role in dedifferentiating RPE cells and retinal progenitor cells during newt retina regeneration.     

Mechanisms of fluid accumulation in retinal edema

This paper reviews the anatomic and physiologic conditions which predispose to fluid accumulation within the retina. Retinal edema has its inception in disease that causes a breakdown of the blood-retinal barrier in retinal capillaries and/or the retinal pigment epithelium (RPE). Edema develops not only because protein and fluid enter the extracellular space, but because the external limiting membrane and the convoluted extracellular pathway within the retina limit the clearance of albumin and other large osmotically-active molecules. These molecules bind water to cause edema. Recognition of edema clinically is complicated by the facts that angiographic markers (fluorescein and ICG) do not match albumin in size, and that clinical leakage does not always correlate closely with tissue swelling or functional loss. Active water transport across the RPE is efficient at removing subretinal water, but the flow resistance of the retina limits RPE access to the water of retinal edema. Consideration of the pathophysiology of retinal edema may aid in the development of better strategies for managing retinal edema. This revised version was published online in July 2006 with corrections to the Cover Date.