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Outer Segments (outer + segment)
Kinds of Outer Segments Selected AbstractsThe translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeletonCYTOSKELETON, Issue 10 2008Boris Reidel Abstract In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells. Cell Motil. Cytoskeleton 65: 785,800, 2008. © 2008 Wiley-Liss, Inc. [source] Cones perform a non-linear transformation on natural stimuliTHE JOURNAL OF PHYSIOLOGY, Issue 3 2010D. Endeman Visual information in natural scenes is distributed over a broad range of intensities and contrasts. This distribution has to be compressed in the retina to match the dynamic range of retinal neurons. In this study we examined how cones perform this compression and investigated which physiological processes contribute to this operation. M- and L-cones of the goldfish were stimulated with a natural time series of intensities (NTSI) and their responses were recorded. The NTSI displays an intensity distribution which is skewed towards the lower intensities and has a long tail into the high intensity region. Cones transform this skewed distribution into a more symmetrical one. The voltage responses of the goldfish cones were compared to those of a linear filter and a non-linear biophysical model of the photoreceptor. The results show that the linear filter under-represents contrasts at low intensities compared to the actual cone whereas the non-linear biophysical model performs well over the whole intensity range used. Quantitative analysis of the two approaches indicates that the non-linear biophysical model can capture 91 ± 5% of the coherence rate (a biased measure of information rate) of the actual cone, where the linear filter only reaches 48 ± 8%. These results demonstrate that cone photoreceptors transform an NTSI in a non-linear fashion. The comparison between current clamp and voltage clamp recordings and analysis of the behaviour of the biophysical model indicates that both the calcium feedback loop in the outer segment and the hydrolysis of cGMP are the major components that introduce the specific non-linear response properties found in the goldfish cones. [source] Correlation of fundus autofluorescence with photoreceptor morphology and functional changes in eyes with retinitis pigmentosaACTA OPHTHALMOLOGICA, Issue 5 2010Taku Wakabayashi Abstract. Purpose:, To assess and correlate fundus autofluorescence (FAF) characteristics with photoreceptor morphology and functional features in eyes with retinitis pigmentosa (RP). Methods:, Thirty-four eyes of 17 patients with RP were examined. We compared FAF images obtained by confocal scanning laser ophthalmoscopy with Spectral-domain optical coherence tomography (SD-OCT) and retinal function assessed by microperimetry. Results:, Normal FAF surrounded by a ring of increased FAF at the macular area was detected in 32 (94%) eyes. The diameter of the normal FAF was correlated significantly with the preserved area of the photoreceptor inner segment and outer segment (IS/OS) junction on SD-OCT (R = 0.939, p < 0.001). The area outside the ring was associated with loss of IS/OS junction and external limiting membrane (ELM). The ring of increased FAF demarcated the border between the central retina with preservation of the IS/OS junction and ELM, and the adjacent eccentric retina with loss of these bands. In two eyes of one patient, there was no preservation of normal FAF at the macula and the photoreceptor IS/OS junction was not detected on SD-OCT. The mean retinal sensitivity derived from microperimetry was correlated significantly with the area of normal FAF (R = 0.929, p = 0.007) and the preserved area of the IS/OS junction (R = 0.851, p = 0.032). Ten eyes had progressive reduction in size of the normal FAF inside the ring accompanied by decreased area of preserved IS/OS during 3.1 years. Conclusion:, FAF appears to reflect the integrity of the photoreceptor layer. It may serve as a secondary outcome measure for novel therapeutic strategies for RP. [source] What drives cell morphogenesis: A look inside the vertebrate photoreceptorDEVELOPMENTAL DYNAMICS, Issue 9 2009Breandán Kennedy Abstract Vision mediating photoreceptor cells are specialized light-sensitive neurons in the outer layer of the vertebrate retina. The human retina contains approximately 130 million of such photoreceptors, which enable images of the external environment to be captured at high resolution and high sensitivity. Rod and cone photoreceptor subtypes are further specialized for sensing light in low and high illumination, respectively. To enable visual function, these photoreceptors have developed elaborate morphological domains for the detection of light (outer segments), for changing cell shape (inner segments), and for communication with neighboring retinal neurons (synaptic terminals). Furthermore, rod and cone subtypes feature unique morphological variations of these specialized characteristics. Here, we review the major aspects of vertebrate photoreceptor morphology and key genetic mechanisms that drive their formation. These mechanisms are necessary for cell differentiation as well as function. Their defects lead to cell death. Developmental Dynamics 238:2115,2138, 2009. © 2009 Wiley-Liss, Inc. [source] Retina expresses a novel variant of the ryanodine receptorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007Varda Shoshan-Barmatz Abstract Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+ -independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with ~ 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel. [source] Evidence for RPE65-independent vision in the cone-dominated zebrafish retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Helia B. Schonthaler Abstract An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11- cis -retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an all- trans to 11- cis -retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all- trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11- cis -retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11- cis -retinal for cone vision. [source] Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004Afrim H. Bytyqi Abstract Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE,/, retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE,/, mouse their processes become structured within two ,novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE,/, retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE,/, mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration. [source] Pigment epithelium-derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epitheliumGLIA, Issue 1 2001Monica M. Jablonski Abstract In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments. GLIA 35:14,25, 2001. © 2001 Wiley-Liss, Inc. [source] Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: Interaction-restricted diffusionIUBMB LIFE, Issue 1 2008Vladlen Z. Slepak Abstract Many signaling proteins change their location within cells in response to external stimuli. In photoreceptors, this phenomenon is remarkably robust. The G protein of rod photoreceptors and rod transducin concentrates in the outer segments (OS) of these neurons in darkness. Within ,30 minutes after illumination, rod transducin redistributes throughout all of the outer and inner compartments of the cell. Visual arrestin concurrently relocalises from the inner compartments to become sequestered primarily within the OS. In the past several years, the question of whether these proteins are actively moved by molecular motors or whether they are redistributed by simple diffusion has been extensively debated. This review focuses on the most essential works in the area and concludes that the basic principle driving this protein movement is diffusion. The directionality and light dependence of this movement is achieved by the interactions of arrestin and transducin with their spatially restricted binding partners. © 2007 IUBMB IUBMB Life, 60(1): 2,9, 2008 [source] Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Tuomas Ryhänen Abstract The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells. [source] GRK1 and GRK7: Unique cellular distribution and widely different activities of opsin phosphorylation in the zebrafish rods and conesJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Yasutaka Wada Abstract Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7,1 and 7,2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the Vmax value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency. [source] Differential distribution of voltage-gated potassium channels Kv 1.1,Kv1.6 in the rat retina during developmentJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2007M. Höltje Abstract The discharge behavior of neurons depends on a variable expression and sorting pattern of voltage-dependent potassium (Kv) channels that changes during development. The rodent retina represents a neuronal network whose main functions develop after birth. To obtain information about neuronal maturation we analyzed the expression of subunits of the Kv1 subfamily in the rat retina during postnatal development using immunocytochemistry and immunoelectron microscopy. At postnatal day 5 (P5) all the ,-subunits of Kv1.1,Kv1.6 channels were found to be expressed in the ganglion cell layer (GCL), most of them already at P1 or P3. Their expression upregulates postnatally and the pattern and distribution change in an isoform-specific manner. Additionally Kv1 channels are found in the outer and inner plexiform layer (OPL, IPL) and in the inner nuclear layer (INL) at different postnatal stages. In adult retina the Kv 1.3 channel localizes to the inner and outer segments of cones. In contrast, Kv1.4 is highly expressed in the outer retina at P8. In adult retina Kv1.4 occurs in rod inner segments (RIS) near the connecting cilium where it colocalizes with synapse associated protein SAP 97. By using confocal laser scanning microscopy we showed a differential localization of Kv1.1-1.6 to cholinergic amacrine and rod bipolar cells of the INL of the adult retina. © 2006 Wiley-Liss, Inc. [source] Lipofuscin and Macular DegenerationNUTRITION REVIEWS, Issue 10 2003George Wolf DPhil The accumulation of the autofluorescent pigment lipofuscin in the retina that occurs with aging has been explained as a side effect of the visual cycle. It occurs when two molecules of all- trans -retinal condense with one molecule of phosphatidylethanolamine in the discs of the rod outer segments, and is followed by uptake into retinal pigment epithelium (RPE) and conversion to the stable A2E, a pyridinium bisretinoid that is toxic to RPE cells. The accumulation of A2E, the major component of lipofuscin causes RPE cell apoptosis, thereby explaining age-related macular degeneration and macular degeneration characteristic of Stargardt disease. The drug isotretinoin (13- cis - retinoic acid) prevents accumulation of A2E in mice by slowing down the visual cycle and might therefore be used to prevent macular degeneration. [source] Salamander Blue-sensitive Cones Lost During Metamorphosis,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Ying Chen The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors,two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase. [source] Effect of Digitonin on the Rhodopsin Meta I-Meta II Equilibrium,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2005Istvan Szundi ABSTRACT Absorbance difference spectra were recorded from 10 ,s to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20°C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin. [source] Photoreceptor and ganglion cell topographies correlate with information convergence and high acuity regions in the adult pigeon (Columba livia) retinaTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2009Angeliza Querubin Abstract The fovea and area dorsalis are high acuity vision regions in the pigeon retina. However, the degree of neural convergence (an important determinant of acuity) has not been quantified consistently in this bird. The purpose of the study was to determine the topographic density changes and degree of photoreceptor to ganglion cell convergence in the fovea and the area dorsalis. Total photoreceptor and ganglion cell densities were calculated on the horizontal and vertical meridia. In four eyes, retinal topography was mapped for photoreceptors and ganglion cells. Rod density was quantified by counting anti-rod opsin-stained outer segments across the retina. The ratio of cone photoreceptors to ganglion cells, a rough measure of information convergence, was calculated. The fovea and the red field contained significantly higher mean cone and ganglion cell densities compared with the yellow field. Rods were missing from the fovea. Outside the fovea, rods comprised 20% of the photoreceptor population, with no significant density changes across the retina. The ratio of photoreceptors to ganglion cells was highest in the yellow field, suggesting a high degree of information convergence and low acuity. Our data indicate that convergence of cones onto ganglion cells in the red field is similar to that observed in the fovea. Convergence ratios in both the fovea and red field suggest greater visual acuity compared to that of the surrounding yellow field, which is consistent with the higher visual acuities that have been reported in these regions. J. Comp. Neurol. 517:711,722, 2009. © 2009 Wiley-Liss, Inc. [source] Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrityTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007Rafal Farjo Abstract We examined the molecular, structural, and functional consequences on cone photoreceptors of the neural retinal leucine zipper knockout (Nrl,/,) mice when only one allele of retinal degeneration slow (Rds) is present (Rds+/,/Nrl,/,). Quantitative RT-PCR and immunoblot analysis were used to assess the expression levels of several phototransduction genes; electroretinography was used to assess quantitatively the retinal responsiveness to light; and immunohistochemistry and ultrastructural analysis were used to examine retinal protein distribution and morphology, respectively. In Rds/Nrl double-null mice, S-cones form dysmorphic outer segments that lack lamellae and fail to associate properly with the cone matrix sheath and the outer limiting membrane. In Rds+/,/Nrl,/, mice, cones form oversized and disorganized outer segment lamellae; although outer limiting membrane associations are maintained, normal interactions with cone matrix sheaths are not, and photoreceptor rosette formation is observed. These retinas produce significantly higher photopic a-wave and b-wave amplitudes than do those of Rds,/,/Nrl,/, mice, and the levels of several cone phototransduction genes are significantly increased coincidently with the presence of Rds and partial lamellae formation. Thus, as in rod photoreceptors, expression of only one Rds allele is unable to support normal outer segment morphogenesis in cones. However, cone lamellae assembly, albeit disorganized, concomitantly permits outer limiting membrane association, and this appears to be linked to photoreceptor rosette formation in the rodless (cone-only) Nrl,/, retina. In addition, photoreceptor gene expression alterations occur in parallel with changes in Rds levels. J. Comp. Neurol. 504:619,630, 2007. © 2007 Wiley-Liss, Inc. [source] Fine Structure of the Retinal Pigment Epithelium, Bruch's Membrane and Choriocapillaris in the HorseANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2000H. Altunay Summary The fine structure of the retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaris was investigated by light and transmission electron microscopy in both the tapetal and non-tapetal fundus of the horse eye. In all locations, the RPE consisted of a single layer of low cuboidal cells. The epithelial cells were joined laterally by apically located tight junctions. These cells displayed numerous basal infoldings and abundant thin apical processes which enclosed the rod outer segments. The epithelial cell nuclei were large and located basally. Within the epithelial cells, smooth endoplasmic reticulum was very abundant, while rough endoplasmic reticulum was scarce. polysomes and mitochondria, which often display a ring-shaped struccture, were abundant. Melanosomes were abundant in the non-tapetal area but absent in the tapetal area. Bruch's membrane was pentalaminate throughout the retina. The endothelium of the choriocapillaris was heavily fenestrated. [source] 2131: Human pluripotent stem cells provide excellent source of functional pigment epithelial cellsACTA OPHTHALMOLOGICA, Issue 2010H SKOTTMAN Purpose Defined differentiation of functional RPE cells from human embryonic (hESC) or induced pluripotent stem cells (iPSC) is a prerequisite for their use in individualised disease modelling, drug discovery and transplantation for retinal diseases. In this study we report differentiation of RPE cells from hESC and iPSC in condition enabling easy translation to clinical quality cell production. Methods Pluripotent stem cells were produced on human fibroblast feeder cells in serum-free medium. The differentiation of the cells was induced using bFGF and feeder cell removal approach under serum-free conditions. The pigmentation and RPE morphology of the cells were analysed and the expression of genes and proteins characteristics for RPE cells were studied. The in vitro functionality of the cells was analysed using ELISA measurements and phagocytosis of photoreceptor outer segments. The integrity of the generated RPE layer was analysed using transepithelial electric resistance (TEER) measurements. Results With our differentiation method, we were able to generate RPE cells with satisfying efficiency. The typical pigmented cobblestone-like morphology and expression of RPE specific markers were confirmed at gene and protein level. The differentiated cells were able to phagocytose and secreted growth factors typical for RPE cells. In addition, cells formed a well polarized epithelium with high integrity, exhibiting good TEER values. Conclusion We have developed progressive differentiation protocol for production of functional RPE cells from hESC and iPSC. The developed production method is currently translated to defined and animal component free conditions enabling clinical grade cell production. [source] Choroidal neovascularization associated with cancer-associated retinopathyACTA OPHTHALMOLOGICA, Issue 5 2010Giuseppe Querques Abstract. Purpose:, To report an unusual association between cancer-associated retinopathy (CAR) associated with invasive thymoma and choriodal neovascularization (CNV), treated by photodynamic theraphy (PDT). Methods:, A 39-year-old man affected with thymoma and paraneoplastic syndrome (myasthenia gravis and diarrhoea) was observed between October 1997 and September 2007. The patient developed progressive visual dysfunction including bilateral visual acuity loss and concentric constriction of visual fields. Ophthalmological, immunological and systemic examinations were performed. Immunological evaluations included an assessment of antibody activity by indirect immunohistochemistry on sectioned rhesus monkey eye, and Western blot reactions upon an extract of pig retina. Results:, Fundus ophthalmoscopy and fluorescein angiography revealed retinal vessel attenuation and retinal pigment epithelium degeneration. Electroretinogram suggested both rod and cone dysfunction. Indirect immunohistochemistry identified antibody activity within the photoreceptor outer segments. Western blots on the retina revealed that most of the patient's antibody activity was focused upon a retinal protein antigen approximating 145 kD. These findings share the commonalities of size and retinal distribution of the interphotoreceptor retinoid-binding protein (IRBP), a recognized autoantigen. The surgically resected mediastinal tumour was diagnosed as invasive thymoma. No other malignancy has since been found throughout nearly 10 years of follow-up. In March 2006, the patient developed a subfoveal CNV in his left eye, which was treated by PDT. Conclusion:, We describe the third case of paraneoplastic retinopathy associated with invasive thymoma. This is the first example of CAR involving autoantibodies reactive with a retinal protein having the characteristics of the IRBP, and is also the first complicated by CNV treated by PDT. [source] Value of internal limiting membrane peeling in surgery for idiopathic macular hole and the correlation between function and retinal morphologyACTA OPHTHALMOLOGICA, Issue thesis2 2009Ulrik Correll Christensen MD Abstract. Idiopathic macular hole is characterized by a full thickness anatomic defect in the foveal retina leading to loss of central vision, metamorphopsia and a central scotoma. Classic macular hole surgery consists of vitrectomy, posterior vitreous cortex separation and intraocular gas tamponade, but during the past decade focus has especially been on internal limiting membrane (ILM) peeling as adjuvant therapy for increasing closure rates. With increasing use of ILM peeling and indocyanine green (ICG) staining, which is used for specific visualization of the ILM, concerns about the safety of the procedure have arisen. At present, it is not known whether ICG-assisted ILM peeling potentially reduces the functional outcome after macular hole surgery. The purpose of the present PhD thesis was to examine whether ICG-assisted ILM peeling offers surgical and functional benefit in macular hole surgery. We conducted a randomized clinical trial including 78 pseudophakic patients with idiopathic macular hole stages 2 and 3. Patients were randomly assigned to macular hole surgery consisting of (i) vitrectomy alone without instrumental retinal surface contact (non-peeling), (ii) vitrectomy plus 0.05% isotonic ICG-assisted ILM peeling or (iii) vitrectomy plus 0.15% trypan blue (TB)-assisted ILM peeling. Morphologic and functional outcomes were assessed 3, 6 and 12 months after surgery. The results show that surgery with ILM peeling, for both stages 2 and 3 macular holes, is associated with a significantly higher closure rate than surgery without ILM peeling (95% versus 45%). The overall functional results confirm that surgery for macular hole generally leads to favourable visual results, with two-thirds of eyes regaining reading vision (,20/40). Macular hole surgery can be considered a safe procedure with a low incidence of sight-threatening adverse events; the retinal detachment rate was 2.2%. Visual outcomes in eyes with primary hole closure were not significantly different between the intervention groups; however, for the stage 2 subgroup with primary macular hole closure, there was a trend towards a better mean visual acuity in the non-peeling group (78.2 letters) compared to the ICG-peeling group (70.9 letters), p = 0.06. Performing repeated macular hole surgery was associated with a significant reduction in functional outcome indicating that primary focus should be on closing the macular hole in one procedure. Morphological studies of closed macular holes with contrast-enhanced optical coherence tomography (OCT) found thinning and discontinuity of the central photoreceptor layer matrix that were highly specific for predicting the likelihood of an eye having regained reading vision 12 months after macular hole surgery. Additionally, healing after macular hole surgery appeared to begin with the contraction of the inner aspect of the retina, forming a roof over a subfoveal fluid-filled cavity, and to end with a gradual restoration of the anatomy in the outer layers of the retina at the junction of the photoreceptor inner and outer segments. We found the more intact this structure was on contrast-enhanced OCT 3 months after macular hole surgery, the better the visual acuity after 12 months, whereas late rather than early resolution of subfoveal fluid had no impact on final visual outcome. The use ILM peeling and intraoperative dyes did not have any functionally important effects on postoperative macular structure. Based on the above findings, we conclude that ILM peeling should be performed in all cases of full thickness macular hole surgery. The use of 0.05% intraoperative isotonic ICG with short exposure time appears to be a safe alternative in stage 3 macular hole surgery, whereas a slight reduction in functional potential not can be excluded when performing 0.05% isotonic ICG-assisted ILM peeling in stage 2 macular hole surgery. [source] New perspectives of optical coherrence tomography in diagnosis and follow-up of macular holesACTA OPHTHALMOLOGICA, Issue 2009SA KABANAROU Purpose To compare Time Domain (TD) with Spectral Domain (SD) OCT for imaging macular holes, identify retinal pathology and correlate anatomical morphology after surgical intervention for hole closure with visual outcome. Methods 34 eyes of 34 patients with idiopathic macular holes stage II- IV were included in this study. Comparative studies were performed with both SD OCT (Heidelberg, Germany) and TD OCT (Stratus) using standard scanning protocols of 6 radial 6-mm scans through the fovea. All patients underwent a standard three port- pars plana vitrectomy. Postoperatively, all patients were evaluated using both OCTs. ETDRS visual acuities were recorded pre- and post-operatively. Results In general TD and SD OCTs showed comparable images of macular holes. However, the boundary line between the inner and outer segments of the photoreceptors was better imaged with the SD OCT preoperatively and postoperatively. Poor visual acuity postoperatively was measured mainly in cases with morphological disruption in this boundary line despite hole closure. Conclusion SD OCT imaging enhances the visualization of retinal anatomy in macular holes relative to TD OCT. [source] Complement factor H and factor B expression in RPE cellsACTA OPHTHALMOLOGICA, Issue 2008Purpose Age-related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface. Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT-PCR. Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch's membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF-alpha and IL-6, whereas the expression of CFB was positively regulated by TNF-alpha and IFN-gamma. Short-term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long-term incubation of RPE cells with POS significantly down-regulated CFH expression but up-regulated CFB expression. Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS. [source] Dissection and cotransplantation of large pieces of RPE and neural retina; effect of protease K on the developmentACTA OPHTHALMOLOGICA, Issue 1 2000Rajesh Kumar Sharma ABSTRACT. Purpose: This study attempts to cotransplant large pieces of the RPE and neural retina in the subretinal space of rabbits by using protease K for dissection of the donor tissue, and to investigate the effect of dissection technique on the development of the grafts. Methods: Eyes from 15-day-old pigmented rabbit embryos were partly digested by protease K to assist dissection of sclera and the choroid from RPE and neural retina. Large pieces of RPE and the neural retina thus obtained were cotransplanted into the eyes of adult albino rabbits who were allowed to survive for up to 63 days. The transplants were examined under light microscope. Results: It was possible to transplant large sheets of RPE and neural retina together. Both the RPE and the neural retina survived after cotransplantation. Retinal pigment epithelium survived in layers, but at places formed clusters. In cotransplants neural retina formed rosettes, developed gliosis, and photoreceptors failed to develop outer segments, possibly due to the action of protease K. Conclusion: Proteases seem to be injurious for the development of the neural retina. [source] Pore-Forming Properties of Alamethicin F50/5 Inserted in a Biological MembraneCHEMISTRY & BIODIVERSITY, Issue 6 2007Natascia Vedovato Abstract The pore-forming properties of native and synthetic alamethicins were investigated in photoreceptor rod outer segments (OS) isolated from frog retina, and recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS within less than 50,ms by means of a computer-controlled micro-perfusion system. Once blocked with light, the main OS endogenous conductance, the OS membrane resistance was >1,G,, allowing low-noise and high-resolution recordings. Currents of ca. 700,pA were recorded in symmetric K+ (100,mM) and Ca2+ (1,mM), upon applying 1,,M of alamethicin F50/5 or its [L -Glu(OMe)7,18,19] analogue to the OS membrane (clamped at ,20,mV). In the latter peptide, the Gln residues at positions 7, 18, and 19 were substituted with side-chain esterified Glu residues. For both peptides, the current activated exponentially, with a delay from peptide application, and exponentially returned to zero without any delay, upon removing the peptide from the external solution. The delay as well as the activation (,a) and deactivation (,d) time constants of the current produced by the modified alamethicin were much slower, and the current noise was much larger, with respect to the corresponding values for alamethicin F50/5. Therefore, the above three Gln residues are not a key factor for pore formation, but the [L -Glu(OMe)7,18,19] analogue produces larger pores with a lower probability of formation. [source] | ||||||||||||||||||||||||||||