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Sample records for retinal cells adenosine

  1. Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina.

    Science.gov (United States)

    Reichenbach, A; Dettmer, D; Brückner, G; Neumann, M; Birkenmeyer, G

    1985-03-22

    Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.

  2. Advances in Retinal Stem Cell Biology

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    Andrea S Viczian

    2013-01-01

    Full Text Available Tremendous progress has been made in recent years to generate retinal cells from pluripotent cell sources. These advances provide hope for those suffering from blindness due to lost retinal cells. Understanding the intrinsic genetic network in model organisms, like fly and frog, has led to a better understanding of the extrinsic signaling pathways necessary for retinal progenitor cell formation in mouse and human cell cultures. This review focuses on the culture methods used by different groups, which has culminated in the generation of laminated retinal tissue from both embryonic and induced pluripotent cells. The review also briefly describes advances made in transplantation studies using donor retinal progenitor and cultured retinal cells.

  3. Extracellular adenosine controls NKT-cell-dependent hepatitis induction.

    Science.gov (United States)

    Subramanian, Meenakshi; Kini, Radhika; Madasu, Manasa; Ohta, Akiko; Nowak, Michael; Exley, Mark; Sitkovsky, Michail; Ohta, Akio

    2014-04-01

    Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls. Transfer of A2AR-deficient NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT-cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT-cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Retinal Cell Degeneration in Animal Models

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    Masayuki Niwa

    2016-01-01

    Full Text Available The aim of this review is to provide an overview of various retinal cell degeneration models in animal induced by chemicals (N-methyl-d-aspartate- and CoCl2-induced, autoimmune (experimental autoimmune encephalomyelitis, mechanical stress (optic nerve crush-induced, light-induced and ischemia (transient retinal ischemia-induced. The target regions, pathology and proposed mechanism of each model are described in a comparative fashion. Animal models of retinal cell degeneration provide insight into the underlying mechanisms of the disease, and will facilitate the development of novel effective therapeutic drugs to treat retinal cell damage.

  5. Detrimental effects of adenosine signaling in sickle cell disease

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    Zhang, Yujin; Dai, Yingbo; Wen, Jiaming; Zhang, Weiru; Grenz, Almut; Sun, Hong; Tao, Lijian; Lu, Guangxiu; Alexander, Danny C; Milburn, Michael V; Carter-Dawson, Louvenia; Lewis, Dorothy E; Zhang, Wenzheng; Eltzschig, Holger K; Kellems, Rodney E; Blackburn, Michael R; Juneja, Harinder S; Xia, Yang

    2016-01-01

    Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease. PMID:21170046

  6. Gene expression profiles in adenosine-treated human mast cells ...

    African Journals Online (AJOL)

    Gene expression profiles in adenosine-treated human mast cells. ... SW Kang, JE Jeong, CH Kim, SH Choi, SH Chae, SA Jun, HJ Cha, JH Kim, YM Lee, YS ... beta 4, ring finger protein, high-mobility group, calmodulin 2, RAN binding protein, ...

  7. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

  8. Regulation of Taurine transporter activity in cultured rat retinal ganglion cells and rat retinal Muller Cells

    International Nuclear Information System (INIS)

    Eissa, Laila A.; Smith, Sylvia B.; El-sherbeny, Amira A.

    2006-01-01

    Diabetic retinopathy is one of the most common complications of diabetes. The amino acid taurine is believed to play an antioxidant protective role in diabetic retinopathy through the scavenging of the reactive species. It is not well established whether taurine uptake is altered in retina cells during diabetic conditions. Thus, the present study was designed to investigate the changes in taurine transport in cultures of rat retinal Muller cells and rat retinal ganglion cells under conditions associated with diabetes. Taurine was abundantly taken up by retinal Muller cells and rat retinal ganglion cells under normal glycemic condition. Taurine was actively transported to rat Muller cells and rat retinal ganglion cells in a Na and Cl dependant manner. Taurine uptake further significantly elevated in both type of cells after the incubation with high glucose concentration. This effect could be attributed to the increase in osmolarity. Because Nitric Oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we also determined the activity of taurine transporter in cultured rat retinal Muller cells and rat retinal ganglion cells in the presence of the NO donors, SIN-1 and SNAP. Taurine uptake was elevated above control value after 24-h incubation with low concentration of NO donors. We finally investigated the ability of neurotoxic glutamate to change taurine transporter activity in both types of cells. Uptake of taurine was significantly increased in rat retinal ganglion cells when only incubated with high concentration of glutamate. Our data provide evidence that taurine transporter is present in cultured rat retinal ganglion and Muller cells and is regulated by hyperosmolarity. The data are relevant to disease such as diabetes and neuronal degeneration where retinal cell volume may dramatically change. (author)

  9. Stem Cell Therapies in Retinal Disorders

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    Aakriti Garg

    2017-02-01

    Full Text Available Stem cell therapy has long been considered a promising mode of treatment for retinal conditions. While human embryonic stem cells (ESCs have provided the precedent for regenerative medicine, the development of induced pluripotent stem cells (iPSCs revolutionized this field. iPSCs allow for the development of many types of retinal cells, including those of the retinal pigment epithelium, photoreceptors, and ganglion cells, and can model polygenic diseases such as age-related macular degeneration. Cellular programming and reprogramming technology is especially useful in retinal diseases, as it allows for the study of living cells that have genetic variants that are specific to patients’ diseases. Since iPSCs are a self-renewing resource, scientists can experiment with an unlimited number of pluripotent cells to perfect the process of targeted differentiation, transplantation, and more, for personalized medicine. Challenges in the use of stem cells are present from the scientific, ethical, and political realms. These include transplant complications leading to anatomically incorrect placement, concern for tumorigenesis, and incomplete targeting of differentiation leading to contamination by different types of cells. Despite these limitations, human ESCs and iPSCs specific to individual patients can revolutionize the study of retinal disease and may be effective therapies for conditions currently considered incurable.

  10. Retinal stem cells and potential cell transplantation treatments

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    Tai-Chi Lin

    2014-11-01

    Full Text Available The retina, histologically composed of ten delicate layers, is responsible for light perception and relaying electrochemical signals to the secondary neurons and visual cortex. Retinal disease is one of the leading clinical causes of severe vision loss, including age-related macular degeneration, Stargardt's disease, and retinitis pigmentosa. As a result of the discovery of various somatic stem cells, advances in exploring the identities of embryonic stem cells, and the development of induced pluripotent stem cells, cell transplantation treatment for retinal diseases is currently attracting much attention. The sources of stem cells for retinal regeneration include endogenous retinal stem cells (e.g., neuronal stem cells, Müller cells, and retinal stem cells from the ciliary marginal zone and exogenous stem cells (e.g., bone mesenchymal stem cells, adipose-derived stem cells, embryonic stem cells, and induced pluripotent stem cells. The success of cell transplantation treatment depends mainly on the cell source, the timing of cell harvesting, the protocol of cell induction/transplantation, and the microenvironment of the recipient's retina. This review summarizes the different sources of stem cells for regeneration treatment in retinal diseases and surveys the more recent achievements in animal studies and clinical trials. Future directions and challenges in stem cell transplantation are also discussed.

  11. Cell Therapy Applications for Retinal Vascular Diseases: Diabetic Retinopathy and Retinal Vein Occlusion.

    Science.gov (United States)

    Park, Susanna S

    2016-04-01

    Retinal vascular conditions, such as diabetic retinopathy and retinal vein occlusion, remain leading causes of vision loss. No therapy exists to restore vision loss resulting from retinal ischemia and associated retinal degeneration. Tissue regeneration is possible with cell therapy. The goal would be to restore or replace the damaged retinal vasculature and the retinal neurons that are damaged and/or degenerating from the hypoxic insult. Currently, various adult cell therapies have been explored as potential treatment. They include mesenchymal stem cells, vascular precursor cells (i.e., CD34+ cells, hematopoietic cells or endothelial progenitor cells), and adipose stromal cells. Preclinical studies show that all these cells have a paracrine trophic effect on damaged ischemic tissue, leading to tissue preservation. Endothelial progenitor cells and adipose stromal cells integrate into the damaged retinal vascular wall in preclinical models of diabetic retinopathy and ischemia-reperfusion injury. Mesenchymal stem cells do not integrate as readily but appear to have a primary paracrine trophic effect. Early phase clinical trials have been initiated and ongoing using mesenchymal stem cells or autologous bone marrow CD34+ cells injected intravitreally as potential therapy for diabetic retinopathy or retinal vein occlusion. Adipose stromal cells or pluripotent stem cells differentiated into endothelial colony-forming cells have been explored in preclinical studies and show promise as possible therapies for retinal vascular disorders. The relative safety or efficacy of these various cell therapies for treating retinal vascular disorders have yet to be determined.

  12. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid

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    Sanie-Jahromi Fatemeh

    2012-04-01

    Full Text Available Abstract Background Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers during treatment of human retinal pigment epithelium (RPE cells with amniotic fluid (AF, RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age. Results Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1 confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation. Conclusion Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

  13. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid.

    Science.gov (United States)

    Sanie-Jahromi, Fatemeh; Ahmadieh, Hamid; Soheili, Zahra-Soheila; Davari, Maliheh; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Deezagi, Abdolkhalegh; Pakravesh, Jalil; Bagheri, Abouzar

    2012-04-10

    Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers) during treatment of human retinal pigment epithelium (RPE) cells with amniotic fluid (AF), RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age. Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1) confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation. Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

  14. Melanopsin retinal ganglion cell loss in Alzheimer's disease

    DEFF Research Database (Denmark)

    La Morgia, Chiara; Ross-Cisneros, Fred N; Koronyo, Yosef

    2015-01-01

    OBJECTIVE: Melanopsin retinal ganglion cells (mRGCs) are photoreceptors driving circadian photoentrainment, and circadian dysfunction characterizes Alzheimer's disease (AD). We investigated mRGCs in AD, hypothesizing their contribution to circadian dysfunction. METHODS: We assessed retinal nerve...

  15. Stem cell therapy for retinal diseases

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    Garcia, José Mauricio; Mendonça, Luisa; Brant, Rodrigo; Abud, Murilo; Regatieri, Caio; Diniz, Bruno

    2015-01-01

    In this review, we discuss about current knowledge about stem cell (SC) therapy in the treatment of retinal degeneration. Both human embryonic stem cell and induced pluripotent stem cell has been growth in culture for a long time, and started to be explored in the treatment of blinding conditions. The Food and Drug Administration, recently, has granted clinical trials using SC retinal therapy to treat complex disorders, as Stargardt’s dystrophy, and patients with geographic atrophy, providing good outcomes. This study’s intent is to overview the critical regeneration of the subretinal anatomy through retinal pigment epithelium transplantation, with the goal of reestablish important pathways from the retina to the occipital cortex of the brain, as well as the differentiation from pluripotent quiescent SC to adult retina, and its relationship with a primary retinal injury, different techniques of transplantation, management of immune rejection and tumorigenicity, its potential application in improving patients’ vision, and, finally, approaching future directions and challenges for the treatment of several conditions. PMID:25621115

  16. Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

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    Dongchun Liang

    Full Text Available Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

  17. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    DEFF Research Database (Denmark)

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin

    2010-01-01

    and that the contraction induced secretion of VEGF is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent.......The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into the thigh muscle of seven male subjects and dialysate was collected at rest, during infusion of adenosine...... and contraction caused secretion of VEGF (pcontraction induced secretion of VEGF protein was abolished by the A(2B) antagonist enprofyllin and markedly reduced by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells...

  18. In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells

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    Gray, Daniel C.; Merigan, William; Wolfing, Jessica I.; Gee, Bernard P.; Porter, Jason; Dubra, Alfredo; Twietmeyer, Ted H.; Ahamd, Kamran; Tumbar, Remy; Reinholz, Fred; Williams, David R.

    2006-08-01

    The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

  19. Role of adenosine signalling and metabolism in β-cell regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, Olov, E-mail: olov.andersson@ki.se

    2014-02-01

    Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?.

  20. Role of adenosine signalling and metabolism in β-cell regeneration

    International Nuclear Information System (INIS)

    Andersson, Olov

    2014-01-01

    Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?

  1. Stem cells in retinal regeneration: past, present and future.

    Science.gov (United States)

    Ramsden, Conor M; Powner, Michael B; Carr, Amanda-Jayne F; Smart, Matthew J K; da Cruz, Lyndon; Coffey, Peter J

    2013-06-01

    Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field.

  2. Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

    Czech Academy of Sciences Publication Activity Database

    Žurovec, Michal; Doležal, Tomáš; Gaži, Michal; Pavlová, Eva; Bryant, P. J.

    2002-01-01

    Roč. 99, č. 7 (2002), s. 4403-4408 ISSN 0027-8424 R&D Projects: GA ČR GA204/01/1022; GA AV ČR IAA5007107 Institutional research plan: CEZ:AV0Z5007907 Keywords : adenosine daminase * minimal medium Subject RIV: CE - Biochemistry Impact factor: 10.701, year: 2002

  3. Adenosine: an activity-dependent axonal signal regulating MAP kinase and proliferation in developing Schwann cells.

    Science.gov (United States)

    Stevens, Beth; Ishibashi, Tomoko; Chen, Jiang-Fan; Fields, R Douglas

    2004-02-01

    Nonsynaptic release of ATP from electrically stimulated dorsal root gangion (DRG) axons inhibits Schwann cell (SC) proliferation and arrests SC development at the premyelinating stage, but the specific types of purinergic receptor(s) and intracellular signaling pathways involved in this form of neuron-glia communication are not known. Recent research shows that adenosine is a neuron-glial transmitter between axons and myelinating glia of the CNS. The present study investigates the possibility that adenosine might have a similar function in communicating between axons and premyelinating SCs. Using a combination of pharmacological and molecular approaches, we found that mouse SCs in culture express functional adenosine receptors and ATP receptors, a far more complex array of purinergic receptors than thought previously. Adenosine, but not ATP, activates ERK/MAPK through stimulation of cAMP-linked A2(A) adenosine receptors. Both ATP and adenosine inhibit proliferation of SCs induced by platelet-derived growth factor (PDGF), via mechanisms that are partly independent. In contrast to ATP, adenosine failed to inhibit the differentiation of SCs to the O4+ stage. This indicates that, in addition to ATP, adenosine is an activity-dependent signaling molecule between axons and premyelinating Schwann cells, but that electrical activity, acting through adenosine, has opposite effects on the differentiation of myelinating glia in the PNS and CNS.

  4. Single cell transcriptome profiling of developing chick retinal cells.

    Science.gov (United States)

    Laboissonniere, Lauren A; Martin, Gregory M; Goetz, Jillian J; Bi, Ran; Pope, Brock; Weinand, Kallie; Ellson, Laura; Fru, Diane; Lee, Miranda; Wester, Andrea K; Liu, Peng; Trimarchi, Jeffrey M

    2017-08-15

    The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types. © 2017 Wiley Periodicals, Inc.

  5. Dorsal raphe nucleus projecting retinal ganglion cells: Why Y cells?

    Science.gov (United States)

    Pickard, Gary E.; So, Kwok-Fai; Pu, Mingliang

    2015-01-01

    Retinal ganglion Y (alpha) cells are found in retinas ranging from frogs to mice to primates. The highly conserved nature of the large, fast conducting retinal Y cell is a testament to its fundamental task, although precisely what this task is remained ill-defined. The recent discovery that Y-alpha retinal ganglion cells send axon collaterals to the serotonergic dorsal raphe nucleus (DRN) in addition to the lateral geniculate nucleus (LGN), medial interlaminar nucleus (MIN), pretectum and the superior colliculus (SC) has offered new insights into the important survival tasks performed by these cells with highly branched axons. We propose that in addition to its role in visual perception, the Y-alpha retinal ganglion cell provides concurrent signals via axon collaterals to the DRN, the major source of serotonergic afferents to the forebrain, to dramatically inhibit 5-HT activity during orientation or alerting/escape responses, which dis-facilitates ongoing tonic motor activity while dis-inhibiting sensory information processing throughout the visual system. The new data provide a fresh view of these evolutionarily old retinal ganglion cells. PMID:26363667

  6. Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases.

    Science.gov (United States)

    Ding, Suet Lee Shirley; Kumar, Suresh; Mok, Pooi Ling

    2017-07-28

    The use of multipotent mesenchymal stem cells (MSCs) has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action.

  7. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment......ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment...

  8. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment......ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment...

  9. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    International Nuclear Information System (INIS)

    Schousboe, A.; Frandsen, A.; Drejer, J.

    1989-01-01

    Evoked release of [ 3 H]-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and [3H]-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP

  10. Retinal progenitor cell xenografts to the pig retina

    DEFF Research Database (Denmark)

    Warfvinge, Karin; Kiilgaard, Jens Folke; Lavik, Erin B

    2005-01-01

    To investigate the survival, integration, and differentiation of mouse retinal progenitor cells after transplantation to the subretinal space of adult pigs.......To investigate the survival, integration, and differentiation of mouse retinal progenitor cells after transplantation to the subretinal space of adult pigs....

  11. Human bone marrow mesenchymal stem cells for retinal vascular injury.

    Science.gov (United States)

    Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Jonas, Jost B; Xu, Liang; Zhang, Wei

    2017-09-01

    To examine the potential of intravitreally implanted human bone marrow-derived mesenchymal stem cells (BMSCs) to affect vascular repair and the blood-retina barrier in mice and rats with oxygen-induced retinopathy, diabetic retinopathy or retinal ischaemia-reperfusion damage. Three study groups (oxygen-induced retinopathy group: 18 C57BL/6J mice; diabetic retinopathy group: 15 rats; retinal ischaemia-reperfusion model: 18 rats) received BMSCs injected intravitreally. Control groups (oxygen-induced retinopathy group: 12 C57BL/6J mice; diabetic retinopathy group: 15 rats; retinal ischaemia-reperfusion model: 18 rats) received an intravitreal injection of phosphate-buffered saline. We applied immunohistological techniques to measure retinal vascularization, spectroscopic measurements of intraretinally extravasated fluorescein-conjugated dextran to quantify the blood-retina barrier breakdown, and histomorphometry to assess retinal thickness and retinal ganglion cell count. In the oxygen-induced retinopathy model, the study group with intravitreally injected BMSCs as compared with the control group showed a significantly (p = 0.001) smaller area of retinal neovascularization. In the diabetic retinopathy model, study group and control group did not differ significantly in the amount of intraretinally extravasated dextran. In the retinal ischaemia-reperfusion model, on the 7th day after retina injury, the retina was significantly thicker in the study group than in the control group (p = 0.02), with no significant difference in the retinal ganglion cell count (p = 0.36). Intravitreally implanted human BMSCs were associated with a reduced retinal neovascularization in the oxygen-induced retinopathy model and with a potentially cell preserving effect in the retinal ischaemia-reperfusion model. Intravitreal BMSCs may be of potential interest for the therapy of retinal vascular disorders. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley

  12. Adenosine signaling promotes regeneration of pancreatic β-cells in vivo

    Science.gov (United States)

    Andersson, Olov; Adams, Bruce A.; Yoo, Daniel; Ellis, Gregory C.; Gut, Philipp; Anderson, Ryan M.; German, Michael S.; Stainier, Didier Y. R.

    2012-01-01

    Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes. PMID:22608007

  13. Retinal Structure Measurements as Inclusion Criteria for Stem Cell-Based Therapies of Retinal Degenerations.

    Science.gov (United States)

    Jacobson, Samuel G; Matsui, Rodrigo; Sumaroka, Alexander; Cideciyan, Artur V

    2016-04-01

    We reviewed and illustrated the most optimal retinal structural measurements to make in stem cell clinical trials. Optical coherence tomography (OCT) and autofluorescence (AF) imaging were used to evaluate patients with severe visual loss from nonsyndromic and syndromic retinitis pigmentosa (RP), ABCA4-Stargardt disease, and nonneovascular age-related macular degeneration (AMD). Outer nuclear layer (ONL), rod outer segment (ROS) layer, inner retina, ganglion cell layer (GCL), and nerve fiber layer (NFL) thicknesses were quantified. All patients had severely reduced visual acuities. Retinitis pigmentosa patients had limited visual fields; maculopathy patients had central scotomas with retained peripheral function. For the forms of RP illustrated, there was detectable albeit severely reduced ONL across the scanned retina, and normal or hyperthick GCL and NFL. Maculopathy patients had no measurable ONL centrally; it became detectable with eccentricity. Some maculopathy patients showed unexpected GCL losses. Autofluorescence imaging illustrated central losses of RPE integrity. A hypothetical scheme to relate patient data with different phases of retinal remodeling in animal models of retinal degeneration was presented. Stem cell science is advancing, but it is not too early to open the discussion of criteria for patient selection and monitoring. Available clinical tools, such as OCT and AF imaging, can provide inclusion/exclusion criteria and robust objective outcomes. Accepting that early trials may not lead to miraculous cures, we should be prepared to know why-scientifically and clinically-so we can improve subsequent trials. We also must determine if retinal remodeling is an impediment to efficacy.

  14. Retinal stem cells and regeneration of vision system.

    Science.gov (United States)

    Yip, Henry K

    2014-01-01

    The vertebrate retina is a well-characterized model for studying neurogenesis. Retinal neurons and glia are generated in a conserved order from a pool of mutlipotent progenitor cells. During retinal development, retinal stem/progenitor cells (RPC) change their competency over time under the influence of intrinsic (such as transcriptional factors) and extrinsic factors (such as growth factors). In this review, we summarize the roles of these factors, together with the understanding of the signaling pathways that regulate eye development. The information about the interactions between intrinsic and extrinsic factors for retinal cell fate specification is useful to regenerate specific retinal neurons from RPCs. Recent studies have identified RPCs in the retina, which may have important implications in health and disease. Despite the recent advances in stem cell biology, our understanding of many aspects of RPCs in the eye remains limited. PRCs are present in the developing eye of all vertebrates and remain active in lower vertebrates throughout life. In mammals, however, PRCs are quiescent and exhibit very little activity and thus have low capacity for retinal regeneration. A number of different cellular sources of RPCs have been identified in the vertebrate retina. These include PRCs at the retinal margin, pigmented cells in the ciliary body, iris, and retinal pigment epithelium, and Müller cells within the retina. Because PRCs can be isolated and expanded from immature and mature eyes, it is possible now to study these cells in culture and after transplantation in the degenerated retinal tissue. We also examine current knowledge of intrinsic RPCs, and human embryonic stems and induced pluripotent stem cells as potential sources for cell transplant therapy to regenerate the diseased retina. Copyright © 2013 Wiley Periodicals, Inc.

  15. Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future

    Directory of Open Access Journals (Sweden)

    Mingyue Luo

    2018-01-01

    Full Text Available As a constituent of blood-retinal barrier and retinal outer segment (ROS scavenger, retinal pigmented epithelium (RPE is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

  16. Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

    Science.gov (United States)

    Luo, Mingyue; Chen, Youxin

    2018-01-01

    As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

  17. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Inagaki, A.; Novak, Ivana

    2016-01-01

    Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl......− channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (Vte......) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 μM, which corresponded to the value obtained on whole-cell Cl− currents in Capan-1 single cells. The effects of adenosine on Vte, an equivalent short-circuit current (Isc), and whole-cell Cl− currents were inhibited...

  18. Advances in Bone Marrow Stem Cell Therapy for Retinal Dysfunction

    Science.gov (United States)

    Park, Susanna S.; Moisseiev, Elad; Bauer, Gerhard; Anderson, Johnathon D.; Grant, Maria B.; Zam, Azhar; Zawadzki, Robert J.; Werner, John S.; Nolta, Jan A.

    2016-01-01

    The most common cause of untreatable vision loss is dysfunction of the retina. Conditions, such as age-related macular degeneration, diabetic retinopathy and glaucoma remain leading causes of untreatable blindness worldwide. Various stem cell approaches are being explored for treatment of retinal regeneration. The rationale for using bone marrow stem cells to treat retinal dysfunction is based on preclinical evidence showing that bone marrow stem cells can rescue degenerating and ischemic retina. These stem cells have primarily paracrine trophic effects although some cells can directly incorporate into damaged tissue. Since the paracrine trophic effects can have regenerative effects on multiple cells in the retina, the use of this cell therapy is not limited to a particular retinal condition. Autologous bone marrow-derived stem cells are being explored in early clinical trials as therapy for various retinal conditions. These bone marrow stem cells include mesenchymal stem cells, mononuclear cells and CD34+ cells. Autologous therapy requires no systemic immunosuppression or donor matching. Intravitreal delivery of CD34+ cells and mononuclear cells appears to be tolerated and is being explored since some of these cells can home into the damaged retina after intravitreal administration. The safety of intravitreal delivery of mesenchymal stem cells has not been well established. This review provides an update of the current evidence in support of the use of bone marrow stem cells as treatment for retinal dysfunction. The potential limitations and complications of using certain forms of bone marrow stem cells as therapy are discussed. Future directions of research include methods to optimize the therapeutic potential of these stem cells, non-cellular alternatives using extracellular vesicles, and in vivo high-resolution retinal imaging to detect cellular changes in the retina following cell therapy. PMID:27784628

  19. Troxler Fading, Eye Movements, and Retinal Ganglion Cell Properties

    Directory of Open Access Journals (Sweden)

    Romain Bachy

    2014-12-01

    Full Text Available We present four movies demonstrating the effect of flicker and blur on the magnitude and speed of adaptation for foveal and peripheral vision along the three color axes that isolate retinal ganglion cells projecting to magno, parvo, and konio layers of the LGN. The demonstrations support the eye movement hypothesis for Troxler fading for brightness and color, and demonstrate the effects of flicker and blur on adaptation of each class of retinal ganglion cells.

  20. Retinal pigment epithelium culture;a potential source of retinal stem cells.

    Science.gov (United States)

    Akrami, Hassan; Soheili, Zahra-Soheila; Khalooghi, Keynoush; Ahmadieh, Hamid; Rezaie-Kanavi, Mojgan; Samiei, Shahram; Davari, Malihe; Ghaderi, Shima; Sanie-Jahromi, Fatemeh

    2009-07-01

    To establish human retinal pigment epithelial (RPE) cell culture as a source for cell replacement therapy in ocular diseases. Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered.

  1. Efficacy and Safety of Human Retinal Progenitor Cells

    Science.gov (United States)

    Semo, Ma'ayan; Haamedi, Nasrin; Stevanato, Lara; Carter, David; Brooke, Gary; Young, Michael; Coffey, Peter; Sinden, John; Patel, Sara; Vugler, Anthony

    2016-01-01

    Purpose We assessed the long-term efficacy and safety of human retinal progenitor cells (hRPC) using established rodent models. Methods Efficacy of hRPC was tested initially in Royal College of Surgeons (RCS) dystrophic rats immunosuppressed with cyclosporine/dexamethasone. Due to adverse effects of dexamethasone, this drug was omitted from a subsequent dose-ranging study, where different hRPC doses were tested for their ability to preserve visual function (measured by optokinetic head tracking) and retinal structure in RCS rats at 3 to 6 months after grafting. Safety of hRPC was assessed by subretinal transplantation into wild type (WT) rats and NIH-III nude mice, with analysis at 3 to 6 and 9 months after grafting, respectively. Results The optimal dose of hRPC for preserving visual function/retinal structure in dystrophic rats was 50,000 to 100,000 cells. Human retinal progenitor cells integrated/survived in dystrophic and WT rat retina up to 6 months after grafting and expressed nestin, vimentin, GFAP, and βIII tubulin. Vision and retinal structure remained normal in WT rats injected with hRPC and there was no evidence of tumors. A comparison between dexamethasone-treated and untreated dystrophic rats at 3 months after grafting revealed an unexpected reduction in the baseline visual acuity of dexamethasone-treated animals. Conclusions Human retinal progenitor cells appear safe and efficacious in the preclinical models used here. Translational Relevance Human retinal progenitor cells could be deployed during early stages of retinal degeneration or in regions of intact retina, without adverse effects on visual function. The ability of dexamethasone to reduce baseline visual acuity in RCS dystrophic rats has important implications for the interpretation of preclinical and clinical cell transplant studies. PMID:27486556

  2. Live-cell imaging: new avenues to investigate retinal regeneration

    Directory of Open Access Journals (Sweden)

    Manuela Lahne

    2017-01-01

    Full Text Available Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish (Danio rerio possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

  3. Müller stem cell dependent retinal regeneration.

    Science.gov (United States)

    Chohan, Annu; Singh, Usha; Kumar, Atul; Kaur, Jasbir

    2017-01-01

    Müller Stem cells to treat ocular diseases has triggered enthusiasm across all medical and scientific communities. Recent development in the field of stem cells has widened the prospects of applying cell based therapies to regenerate ocular tissues that have been irreversibly damaged by disease or injury. Ocular tissues such as the lens and the retina are now known to possess cell having remarkable regenerative abilities. Recent studies have shown that the Müller glia, a cell found in all vertebrate retinas, is the primary source of new neurons, and therefore are considered as the cellular basis for retinal regeneration in mammalian retinas. Here, we review the current status of retinal regeneration of the human eye by Müller stem cells. This review elucidates the current status of retinal regeneration by Müller stem cells, along with major retinal degenerative diseases where these stem cells play regenerative role in retinal repair and replacement. Copyright © 2016. Published by Elsevier B.V.

  4. Effect of adenosine on the growth of human T-lymphocyte leukemia cell line MOLT-4

    Czech Academy of Sciences Publication Activity Database

    Štreitová, Denisa; Weiterová, Lenka; Hofer, Michal; Holá, Jiřina; Horváth, Viktor; Kozubík, Alois; Znojil, V.

    2007-01-01

    Roč. 25, č. 6 (2007), s. 419-426 ISSN 0735-7907 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : MOLT-4 leukemia cell s * cell growth * adenosine Subject RIV: BO - Biophysics Impact factor: 2.106, year: 2007

  5. Hypoxia-ischemia and retinal ganglion cell damage

    Directory of Open Access Journals (Sweden)

    Charanjit Kaur

    2008-08-01

    Full Text Available Charanjit Kaur1, Wallace S Foulds2, Eng-Ang Ling11Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2Singapore Eye Research Institute, SingaporeAbstract: Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1α and its target genes such as vascular endothelial growth factor (VEGF and nitric oxide synthase (NOS. Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca2+ which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.Keywords: retinal hypoxia, retinal ganglion cells, glutamate receptors, neuronal injury, retina

  6. Stem cells in clinical trials for treatment of retinal degeneration.

    Science.gov (United States)

    Klassen, Henry

    2016-01-01

    After decades of basic science research involving the testing of regenerative strategies in animal models of retinal degenerative diseases, a number of clinical trials are now underway, with additional trials set to begin shortly. These efforts will evaluate the safety and preliminary efficacy of cell-based products in the eyes of patients with a number of retinal conditions, notably including age-related macular degeneration, retinitis pigmentosa and Stargardt's disease. This review considers the scientific work and early trials with fetal cells and tissues that set the stage for the current clinical investigatory work, as well the trials themselves, specifically those either now completed, underway or close to initiation. The cells of interest include retinal pigment epithelial cells derived from embryonic stem or induced pluripotent stem cells, undifferentiated neural or retinal progenitors or cells from the vascular/bone marrow compartment or umbilical cord tissue. Degenerative diseases of the retina represent a popular target for emerging cell-based therapeutics and initial data from early stage clinical trials suggest that short-term safety objectives can be met in at least some cases. The question of efficacy will require additional time and testing to be adequately resolved.

  7. Human amniotic fluid promotes retinal pigmented epithelial cells' trans-differentiation into rod photoreceptors and retinal ganglion cells.

    Science.gov (United States)

    Ghaderi, Shima; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Davari, Maliheh; Jahromi, Fatemeh Sanie; Samie, Shahram; Rezaie-Kanavi, Mozhgan; Pakravesh, Jalil; Deezagi, Abdolkhalegh

    2011-09-01

    To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

  8. Differential response of Drosophila cell lines to extracellular adenosine

    Czech Academy of Sciences Publication Activity Database

    Fleischmannová, J.; Kučerová, Lucie; Šandová, Kateřina; Steinbauerová, Veronika; Brož, Václav; Šimek, Petr; Žurovec, Michal

    2012-01-01

    Roč. 42, č. 5 (2012), s. 321-331 ISSN 0965-1748 R&D Projects: GA MŠk(CZ) LC06077 Grant - others:AV ČR(CZ) KJB501410801; European Community´s Seventh Framwork Programme (FP7/2007-2013)(CZ) 229518 Institutional research plan: CEZ:AV0Z50070508 Institutional support: RVO:60077344 Keywords : adenosine recycling * nucleoside transport * Mbn2 Subject RIV: CE - Biochemistry Impact factor: 3.234, year: 2012 http://www.sciencedirect.com/science/article/pii/S0965174812000033

  9. The circadian response of intrinsically photosensitive retinal ganglion cells.

    Directory of Open Access Journals (Sweden)

    Andrew J Zele

    Full Text Available Intrinsically photosensitive retinal ganglion cells (ipRGC signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central or intrinsic (retinal network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18-30 years with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC and outer retina (cone photoreceptors was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux. Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin retinal ganglion cells mediate this circadian variation.

  10. Timing the Generation of Distinct Retinal Cells by Homeobox Proteins

    Science.gov (United States)

    Decembrini, Sarah; Andreazzoli, Massimiliano; Vignali, Robert; Barsacchi, Giuseppina; Cremisi, Federico

    2006-01-01

    The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of “sensors” in which green fluorescent protein translation is under control of the 3′ untranslated region (UTR), we found that the 3′ UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities. PMID:16903786

  11. Timing the generation of distinct retinal cells by homeobox proteins.

    Directory of Open Access Journals (Sweden)

    Sarah Decembrini

    2006-09-01

    Full Text Available The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR, we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

  12. Roles of the adenosine receptor and CD73 in the regulatory effect of γδ T cells.

    Directory of Open Access Journals (Sweden)

    Dongchun Liang

    Full Text Available The adenosine A2A receptor (A2AR, the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses.

  13. THE MODULATORY ROLE OF TAURINE IN RETINAL GANGLION CELLS

    Science.gov (United States)

    Jiang, Zheng; Bulley, Simon; Guzzone, Joseph; Ripps, Harris; Shen, Wen

    2017-01-01

    Taurine (2-aminoethylsuphonic acid) is present in nearly all animal tissues, and is the most abundant free amino acid in muscle, heart, CNS and retina. Although it is known to be a major cytoprotectant and essential for normal retinal development, its role in retinal neurotransmission and modulation is not well understood. We investigated the response of taurine in retinal ganglion cells, and its effect on synaptic transmission between ganglion cells and their pre-synaptic neurons. We find that taurine-elicited currents in ganglion cells could be fully blocked by both strychnine and SR95531, glycine and GABAA receptor antagonists, respectively. This suggests that taurine-activated receptors might share the antagonists with GABA and glycine receptors. The effect of taurine at micromolar concentrations can effectively suppress spontaneous vesicle release from the pre-synaptic neurons, but had limited effects on light-evoked synaptic signals in ganglion cells. We also describe a metabotropic effect of taurine in the suppression of light-evoked response in ganglion cells. Clearly, taurine acts in multiple ways to modulate synaptic signals in retinal output neurons, ganglion cells. PMID:23392924

  14. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    Science.gov (United States)

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

  15. Retinal Biochemistry, Physiology and Cell Biology.

    Science.gov (United States)

    Smith, Ricardo Luiz; Sivaprasad, Sobha; Chong, Victor

    2016-01-01

    The vitreous, the vasculature of the retina, macular pigments, phototransduction, retinal pigment epithelium, Bruch's membrane and the extracellular matrix, all play an important role in the normal function of the retina as well as in diseases. Understanding the pathophysiology allows us to target treatment. As ocular angiogenesis, immunity and inflammation are covered elsewhere, those subjects will not be discussed in this chapter. © 2016 S. Karger AG, Basel.

  16. Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    Yi Zhao

    2017-04-01

    Full Text Available IntroductionMany antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells.MethodsCultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS in the presence of dead and dying cells, their supernatants (SNs, or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo.ResultsThe stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment.ConclusionInosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

  17. Transplanting Retinal Cells using Bucky Paper for Support

    Science.gov (United States)

    Loftus, David J.; Cinke, Martin; Meyyappan, Meyya; Fishman, Harvey; Leng, Ted; Huie, Philip; Bilbao, Kalayaan

    2004-01-01

    A novel treatment for retinal degenerative disorders involving transplantation of cells into the eye is currently under development at NASA Ames Research Center and Stanford University School of Medicine. The technique uses bucky paper as a support material for retinal pigment epithelial (RPE) cells, iris pigment epithelial (IPE) cells, and/or stem cells. This technology is envisioned as a treatment for age-related macular degeneration, which is the leading cause of blindness in persons over age 65 in Western nations. Additionally, patients with other retinal degenerative disorders, such as retinitis pigmentosa, may be treated by this strategy. Bucky paper is a mesh of carbon nanotubes (CNTs), as shown in Figure 1, that can be made from any of the commercial sources of CNTs. Bucky paper is biocompatible and capable of supporting the growth of biological cells. Because bucky paper is highly porous, nutrients, oxygen, carbon dioxide, and waste can readily diffuse through it. The thickness, density, and porosity of bucky paper can be tailored in manufacturing. For transplantation of cells into the retina, bucky paper serves simultaneously as a substrate for cell growth and as a barrier for new blood vessel formation, which can be a problem in the exudative type of macular degeneration. Bucky paper is easily handled during surgical implantation into the eye. Through appropriate choice of manufacturing processes, bucky paper can be made relatively rigid yet able to conform to the retina when the bucky paper is implanted. Bucky paper offers a distinct advantage over other materials that have been investigated for retinal cell transplantation - lens capsule and Descemet's membrane - which are difficult to handle during surgery because they are flimsy and do not stay flat.

  18. Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

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    Jeffery Glen

    2008-05-01

    Full Text Available Abstract Background The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs. At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+ showed that (1 the total number of RGC axons projected by the retina and (2 the proportions that are sorted into the ipsilateral and

  19. Differentiation of Pluripotent Stem Cells to Retinal Pigment Epithelial Cells: An Approach Toward Retinal Degenerative Diseases Treatment

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    Maryam Parvini

    2013-10-01

    Full Text Available Pluripotent stem cells as the cells with a capacity for self-renewal and differentiation into various specificcell types have been highly regarded in regenerative medicine studies. To repair the eye disease damages, thedifferentiation into retinal pigment epithelial cells of pluripotent stem cells has gained great importance inrecent decades because the inappropriate function of these cells is the main cause of degenerative diseases suchas the age-related macular degeneration. Millions of people in the world suffer this disease.To restore the damaged cells and, finally, to improve the vision, numerous studies have been conducted on usingpluripotent stem cells, their differentiation into retinal pigment epithelial cells, and finally, their applicationin cell therapy. Based on this, many researchers have attempted to produce highly efficient retinal pigmentepithelial cells, such that they show a proper function after transplant, along with the host cells. In this reviewarticle, the importance and the role of pigment epithelial cells, as well as, the studies on the in vitro productionof these cells were examined

  20. Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

    Science.gov (United States)

    Froger, Nicolas; Cadetti, Lucia; Lorach, Henri; Martins, Joao; Bemelmans, Alexis-Pierre; Dubus, Elisabeth; Degardin, Julie; Pain, Dorothée; Forster, Valérie; Chicaud, Laurent; Ivkovic, Ivana; Simonutti, Manuel; Fouquet, Stéphane; Jammoul, Firas; Léveillard, Thierry; Benosman, Ryad; Sahel, José-Alain; Picaud, Serge

    2012-01-01

    Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases. PMID:23115615

  1. Transplantation of rat embryonic stem cell-derived retinal progenitor cells preserves the retinal structure and function in rat retinal degeneration.

    Science.gov (United States)

    Qu, Zepeng; Guan, Yuan; Cui, Lu; Song, Jian; Gu, Junjie; Zhao, Hanzhi; Xu, Lei; Lu, Lixia; Jin, Ying; Xu, Guo-Tong

    2015-11-09

    Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. We have successfully differentiated

  2. Curcumin Attenuates Staurosporine-Mediated Death of Retinal Ganglion Cells

    OpenAIRE

    Burugula, Balabharathi; Ganesh, Bhagyalaxmi S.; Chintala, Shravan K.

    2011-01-01

    The functional effect of curcumin, a free radical scavenger and an herbal medicine from Indian yellow curry spice, Curcuma longa, on protease-mediated retinal ganglion cell death was investigated. These results show, for the first time, that curcumin indeed prevents the protease-mediated death of RGCs, both in vitro and in vivo.

  3. Retinal progenitor cell xenografts to the pig retina

    DEFF Research Database (Denmark)

    Warfvinge, Karin; Kiilgaard, Jens Folke; Klassen, Henry

    2006-01-01

    We evaluated the host response to murine retinal progenitor cells (RPCs) following transplantation to the subretinal space (SRS) of the pig. RPCs from GFP mice were transplanted subretinally in 18 nonimmunosuppressed normal or laser-treated pigs. Evaluation of the SRS was performed on hematoxylin-eosin...

  4. Processing of natural temporal stimuli by macaque retinal ganglion cells

    NARCIS (Netherlands)

    Hateren, J.H. van; Rüttiger, L.; Lee, B.B.

    2002-01-01

    This study quantifies the performance of primate retinal ganglion cells in response to natural stimuli. Stimuli were confined to the temporal and chromatic domains and were derived from two contrasting environments, one typically northern European and the other a flower show. The performance of the

  5. Astrocytes and Müller cells changes during retinal degeneration in a transgenic rat model of retinitis pigmentosa.

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    Laura eFernández-Sánchez

    2015-12-01

    Full Text Available Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer of P23H versus SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.

  6. Astrocytes and Müller Cell Alterations During Retinal Degeneration in a Transgenic Rat Model of Retinitis Pigmentosa

    Science.gov (United States)

    Fernández-Sánchez, Laura; Lax, Pedro; Campello, Laura; Pinilla, Isabel; Cuenca, Nicolás

    2015-01-01

    Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes, and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer (GCL) of P23H vs. SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina. PMID:26733810

  7. Chitosan Feasibility to Retain Retinal Stem Cell Phenotype and Slow Proliferation for Retinal Transplantation

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    Girish K. Srivastava

    2014-01-01

    Full Text Available Retinal stem cells (RSCs are promising in cell replacement strategies for retinal diseases. RSCs can migrate, differentiate, and integrate into retina. However, RSCs transplantation needs an adequate support; chitosan membrane (ChM could be one, which can carry RSCs with high feasibility to support their integration into retina. RSCs were isolated, evaluated for phenotype, and subsequently grown on sterilized ChM and polystyrene surface for 8 hours, 1, 4, and 11 days for analysing cell adhesion, proliferation, viability, and phenotype. Isolated RSCs expressed GFAP, PKC, isolectin, recoverin, RPE65, PAX-6, cytokeratin 8/18, and nestin proteins. They adhered (28 ± 16%, 8 hours and proliferated (40 ± 20 cells/field, day 1 and 244 ± 100 cells/field, day 4 significantly low (P95% and phenotype (cytokeratin 8/18, PAX6, and nestin proteins expression, day 11 on both surfaces (ChM and polystyrene. RSCs did not express alpha-SMA protein on both surfaces. RSCs express proteins belonging to epithelial, glial, and neural cells, confirming that they need further stimulus to reach a final destination of differentiation that could be provided in in vivo condition. ChM does not alternate RSCs behaviour and therefore can be used as a cell carrier so that slow proliferating RSCs can migrate and integrate into retina.

  8. Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

    Science.gov (United States)

    Eldred, Megan K; Charlton-Perkins, Mark; Muresan, Leila; Harris, William A

    2017-03-15

    To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process. © 2017. Published by The Company of Biologists Ltd.

  9. Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

    Science.gov (United States)

    Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

    2006-01-01

    An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

  10. Endogenous retinal neural stem cell reprogramming for neuronal regeneration

    Directory of Open Access Journals (Sweden)

    Romain Madelaine

    2017-01-01

    Full Text Available In humans, optic nerve injuries and associated neurodegenerative diseases are often followed by permanent vision loss. Consequently, an important challenge is to develop safe and effective methods to replace retinal neurons and thereby restore neuronal functions and vision. Identifying cellular and molecular mechanisms allowing to replace damaged neurons is a major goal for basic and translational research in regenerative medicine. Contrary to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system, including retina. This regenerative process depends on endogenous retinal neural stem cells, the Müller glial cells. Following injury, zebrafish Müller cells go back into cell cycle to proliferate and generate new neurons, while mammalian Müller cells undergo reactive gliosis. Recently, transcription factors and microRNAs have been identified to control the formation of new neurons derived from zebrafish and mammalian Müller cells, indicating that cellular reprogramming can be an efficient strategy to regenerate human retinal neurons. Here we discuss recent insights into the use of endogenous neural stem cell reprogramming for neuronal regeneration, differences between zebrafish and mammalian Müller cells, and the need to pursue the identification and characterization of new molecular factors with an instructive and potent function in order to develop theurapeutic strategies for eye diseases.

  11. Veratridine increases the survival of retinal ganglion cells in vitro

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    S.P.F. Pereira

    1997-12-01

    Full Text Available Neuronal cell death is an important phenomenon involving many biochemical pathways. This degenerative event has been studied to understand how the cells activate the mechanisms that lead to self-destruction. Target cells and afferent cells play a relevant role in the regulation of natural cell death. We studied the effect of veratridine (1.5, 3.0, 4.5 and 6.0 µM on the survival of neonatal rat retinal ganglion cells in vitro. Veratridine (3.0 µM, a well-known depolarizing agent that opens the Na+ channel, promoted a two-fold increase in the survival of retinal ganglion cells kept in culture for 48 h. This effect was dose-dependent and was blocked by 1.0 µM tetrodotoxin (a classical voltage-dependent Na+ channel blocker and 30.0 µM flunarizine (a Na+ and Ca2+ channel blocker. These results indicate that electrical activity is also important for the maintenance of retinal ganglion cell survival in vitro

  12. Real-Time Imaging of Retinal Ganglion Cell Apoptosis

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    Timothy E. Yap

    2018-06-01

    Full Text Available Monitoring real-time apoptosis in-vivo is an unmet need of neurodegeneration science, both in clinical and research settings. For patients, earlier diagnosis before the onset of symptoms provides a window of time in which to instigate treatment. For researchers, being able to objectively monitor the rates of underlying degenerative processes at a cellular level provides a biomarker with which to test novel therapeutics. The DARC (Detection of Apoptosing Retinal Cells project has developed a minimally invasive method using fluorescent annexin A5 to detect rates of apoptosis in retinal ganglion cells, the key pathological process in glaucoma. Numerous animal studies have used DARC to show efficacy of novel, pressure-independent treatment strategies in models of glaucoma and other conditions where retinal apoptosis is reported, including Alzheimer’s disease. This may forge exciting new links in the clinical science of treating both cognitive and visual decline. Human trials are now underway, successfully demonstrating the safety and efficacy of the technique to differentiate patients with progressive neurodegeneration from healthy individuals. We review the current perspectives on retinal ganglion cell apoptosis, the way in which this can be imaged, and the exciting advantages that these future methods hold in store.

  13. Versatile functional roles of horizontal cells in the retinal circuit.

    Science.gov (United States)

    Chaya, Taro; Matsumoto, Akihiro; Sugita, Yuko; Watanabe, Satoshi; Kuwahara, Ryusuke; Tachibana, Masao; Furukawa, Takahisa

    2017-07-17

    In the retinal circuit, environmental light signals are converted into electrical signals that can be decoded properly by the brain. At the first synapse of the visual system, information flow from photoreceptors to bipolar cells is modulated by horizontal cells (HCs), however, their functional contribution to retinal output and individual visual function is not fully understood. In the current study, we investigated functional roles for HCs in retinal ganglion cell (RGC) response properties and optokinetic responses by establishing a HC-depleted mouse line. We observed that HC depletion impairs the antagonistic center-surround receptive field formation of RGCs, supporting a previously reported HC function revealed by pharmacological approaches. In addition, we found that HC loss reduces both the ON and OFF response diversities of RGCs, impairs adjustment of the sensitivity to ambient light at the retinal output level, and alters spatial frequency tuning at an individual level. Taken together, our current study suggests multiple functional aspects of HCs crucial for visual processing.

  14. Effect of glial cell line-derived neurotrophic factor on retinal function after experimental branch retinal vein occlusion

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Dornonville de la Cour, Morten; Kyhn, Maria Voss

    2012-01-01

    The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs.......The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs....

  15. Adenosine: an activity-dependent axonal signal regulating MAP kinase and proliferation in developing Schwann cells

    OpenAIRE

    Stevens, Beth; Ishibashi, Tomoko; Chen, Jiang-Fan; Fields, R. Douglas

    2004-01-01

    Nonsynaptic release of ATP from electrically stimulated dorsal root gangion (DRG) axons inhibits Schwann cell (SC) proliferation and arrests SC development at the premyelinating stage, but the specific types of purinergic receptor(s) and intracellular signaling pathways involved in this form of neuron–glia communication are not known. Recent research shows that adenosine is a neuron–glial transmitter between axons and myelinating glia of the CNS. The present study investigates the possibility...

  16. Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

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    Kim Chan

    2007-10-01

    Full Text Available Abstract Background Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5. RGC-5 cells were cultured in a closed hypoxic chamber (5% O2 with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38 and nuclear factor-kappa B (NF-κB were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF, a well-known protective neurotrophin for retinal ganglion cells. Results After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-κB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-κB, while BDNF suppressed phosphorylation of ERK and p38. Conclusion Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-κB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia.

  17. Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

    Science.gov (United States)

    Hong, Samin; Lee, Jong Eun; Kim, Chan Yun; Seong, Gong Je

    2007-01-01

    Background Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5). RGC-5 cells were cultured in a closed hypoxic chamber (5% O2) with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH) assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38) and nuclear factor-kappa B (NF-κB) were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for retinal ganglion cells. Results After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-κB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-κB, while BDNF suppressed phosphorylation of ERK and p38. Conclusion Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-κB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia. PMID:17908330

  18. Transcriptome of Atoh7 retinal progenitor cells identifies new Atoh7-dependent regulatory genes for retinal ganglion cell formation.

    Science.gov (United States)

    Gao, Zhiguang; Mao, Chai-An; Pan, Ping; Mu, Xiuqian; Klein, William H

    2014-11-01

    The bHLH transcription factor ATOH7 (Math5) is essential for establishing retinal ganglion cell (RGC) fate. However, Atoh7-expressing retinal progenitor cells (RPCs) can give rise to all retinal cell types, suggesting that other factors are involved in specifying RGCs. The basis by which a subpopulation of Atoh7-expressing RPCs commits to an RGC fate remains uncertain but is of critical importance to retinal development since RGCs are the earliest cell type to differentiate. To better understand the regulatory mechanisms leading to cell-fate specification, a binary genetic system was generated to specifically label Atoh7-expressing cells with green fluorescent protein (GFP). Fluorescence-activated cell sorting (FACS)-purified GFP(+) and GFP(-) cells were profiled by RNA-seq. Here, we identify 1497 transcripts that were differentially expressed between the two RPC populations. Pathway analysis revealed diminished growth factor signaling in Atoh7-expressing RPCs, indicating that these cells had exited the cell cycle. In contrast, axon guidance signals were enriched, suggesting that axons of Atoh7-expressing RPCs were already making synaptic connections. Notably, many genes enriched in Atoh7-expressing RPCs encoded transcriptional regulators, and several were direct targets of ATOH7, including, and unexpectedly, Ebf3 and Eya2. We present evidence for a Pax6-Atoh7-Eya2 pathway that acts downstream of Atoh7 but upstream of differentiation factor Pou4f2. EYA2 is a protein phosphatase involved in protein-protein interactions and posttranslational regulation. These properties, along with Eya2 as an early target gene of ATOH7, suggest that EYA2 functions in RGC specification. Our results expand current knowledge of the regulatory networks operating in Atoh7-expressing RPCs and offer new directions for exploring the earliest aspects of retinogenesis. © 2014 Wiley Periodicals, Inc.

  19. Apelin is a novel angiogenic factor in retinal endothelial cells

    International Nuclear Information System (INIS)

    Kasai, Atsushi; Shintani, Norihito; Oda, Maki; Kakuda, Michiya; Hashimoto, Hitoshi; Matsuda, Toshio; Hinuma, Shuji; Baba, Akemichi

    2004-01-01

    There has been much focus recently on the possible functions of apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, in cardiovascular and central nervous systems. We report a new function of apelin as a novel angiogenic factor in retinal endothelial cells. The retinal endothelial cell line RF/6A highly expressed both apelin and APJ transcripts, while human umbilical venous endothelial cells (HUVECs) only expressed apelin mRNA. In accordance with these observations, apelin at concentrations of 1 pM-1 μM significantly enhanced migration, proliferation, and capillary-like tube formation of RF/6A cells, but not those of HUVECs, whereas VEGF stimulates those parameters of both cell types. In vivo Matrigel plug assay for angiogenesis, the inclusion of 1 nM apelin in the Matrigel resulted in clear capillary-like formations with an increase of hemoglobin content in the plug. This is the first report showing that apelin is an angiogenic factor in retinal endothelial cells

  20. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  1. Adenosine derived from Staphylococcus aureus-engulfed macrophages functions as a potent stimulant for the induction of inflammatory cytokines in mast cells

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Kim, Chan-Hee; Ryu, Kyoung-Hwa

    2011-01-01

    In this study, we attempted to isolate novel mast cell-stimulating molecules from Staphylococcus aureus. Water-soluble extract of S. aureus cell lysate strongly induced human interleukin- 8 in human mast cell line-1 and mouse interleukin-6 in mouse bone marrow-derived mast cells. The active...... adenosine receptor blocker, verified that purified adenosine can induce interleukin-8 production via adenosine receptors on mast cells. Moreover, adenosine was purified from S. aureusengulfed RAW264.7 cells, a murine macrophage cell line, used to induce phagocytosis of S. aureus. These results show a novel...

  2. Effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

    International Nuclear Information System (INIS)

    Barton, R.W.

    1985-01-01

    Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF

  3. Retinal Ganglion Cell Loss in Diabetes Associated with Elevated Homocysteine

    Directory of Open Access Journals (Sweden)

    Kenneth S. Shindler

    2009-11-01

    Full Text Available A number of studies have suggested that homocysteine may be a contributing factor to development of retinopathy in diabetic patients based on observed correlations between elevated homocysteine levels and the presence of retinopathy. The significance of such a correlation remains to be determined, and potential mechanisms by which homocysteine might induce retinopathy have not been well characterized. Ganapathy and colleagues1 used mutant mice that have endogenously elevated homocysteine levels due to heterozygous deletion of the cystathionine-β-synthase gene to examine changes in retinal pathology following induction of diabetes. Their finding that elevated homocysteine levels hastens loss of cells in the retinal ganglion cell layer suggests that toxicity to ganglion cells may warrant further investigation as a potential mechanism of homocysteine enhanced susceptibility to diabetic retinopathy.

  4. Radiochromatographic determination of activity of adenosine deaminase and purine nucleoside phosphorylase in blood cells

    International Nuclear Information System (INIS)

    Pechan, I.; Rendekova, V.; Pechanova, E.; Krizko, J.

    1982-01-01

    Expeditious and sensitive methods are described for determining the activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in human lymphocytes and erythrocytes. ADA and PNP activity is determined on the basis of the reaction of (U- 14 C)adenosine or (8- 14 C)inosine with the lysate of human blood cells. Reaction products are separated using paper chromatography. Following the measurement of the radioactivity of spots of adenosine, inosine and hypoxanthine, a calculation is made of ADA and PNP activity from the results of the said measurements. On a sample of 52 clinically healthy people average ADA and PNP activity in isolated lymphocytes was found to be (51.6+-18.8) and (185.6+-94.7) pcat/10 6 cells and in erythrocytes (9.8+-2.98) and (17.1+-3.19) pcat/mg of proteins, respectively. The advantage of the method is the small amount of sample needed (1 to 2 ml) which allows its application in pediatrics. (Ha)

  5. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

  6. Novel cellular bouton structure activated by ATP in the vascular wall of porcine retinal arterioles.

    Science.gov (United States)

    Misfeldt, Mikkel Wölck; Aalkjaer, Christian; Simonsen, Ulf; Bek, Toke

    2010-12-01

    The retinal blood flow is regulated by the tone of resistance arterioles, which is influenced by purinergic compounds such as adenosine and adenosine 5'-triphosphate (ATP) released from the retinal tissue. However, it is unknown what cellular elements in the perivascular retina are responsible for the effect of purines on the tone of retinal arterioles. Porcine retinal arterioles were loaded with the calcium-sensitive fluorophore Oregon green. The vessels were mounted in a confocal myograph for simultaneous recordings of tone and calcium activity in cells of the vascular wall during stimulation with ATP and adenosine, with and without modifiers of these compounds. Additionally, immunohistochemistry was used to localize elements with calcium activity in the vascular wall. Hyperfluorescence indicating calcium activity was recorded in a population of abundant round boutons interspersed in a network of vimentin-positive processes located immediately external to the smooth muscle cell layer but internal to the perivascular glial cells. These structures showed calcium activity when the vessel was relaxed with ATP but not when it was relaxed with adenosine. Ryanodine reduced calcium activity in the boutons, whereas the ATP antagonist adenosine-5'-O-(α, β- methylene diphosphate) reduced calcium activity in both the boutons and vascular tone. The vasodilating effect of purines in porcine retinal tissue involves ATP-dependent calcium activity in a layer of cellular boutons located external to the vascular smooth muscle cells and internal to the perivascular glial cells.

  7. Effect of Monocular Deprivation on Rabbit Neural Retinal Cell Densities

    OpenAIRE

    Mwachaka, Philip Maseghe; Saidi, Hassan; Odula, Paul Ochieng; Mandela, Pamela Idenya

    2015-01-01

    Purpose: To describe the effect of monocular deprivation on densities of neural retinal cells in rabbits. Methods: Thirty rabbits, comprised of 18 subject and 12 control animals, were included and monocular deprivation was achieved through unilateral lid suturing in all subject animals. The rabbits were observed for three weeks. At the end of each week, 6 experimental and 3 control animals were euthanized, their retinas was harvested and processed for light microscopy. Photomicrographs of ...

  8. Intrinsically photosensitive retinal ganglion cell function in relation to age

    DEFF Research Database (Denmark)

    Herbst, Kristina; Sander, Birgit; Lund-Andersen, Henrik

    2012-01-01

    The activity of melanopsin containing intrinsically photosensitive ganglion retinal cells (ipRGC) can be assessed by a means of pupil responses to bright blue (appr.480 nm) light. Due to age related factors in the eye, particularly, structural changes of the lens, less light reaches retina. The aim...... of this study was to examine how age and in vivo measured lens transmission of blue light might affect pupil light responses, in particular, mediated by the ipRGC....

  9. Oxygen-induced retinopathy in mice with retinal photoreceptor cell degeneration.

    Science.gov (United States)

    Zhang, Qian; Zhang, Zuo-Ming

    2014-04-25

    It is reported that retinal neovascularization seems to rarely co-exist with retinitis pigmentosa in patients and in some mouse models; however, it is not widely acknowledged as a universal phenomenon in all strains of all animal species. We aimed to further explore this phenomenon with an oxygen-induced retinopathy model in mice with retinal photoreceptor cell degeneration. Oxygen-induced retinopathy of colored and albino mice with rapid retinal degeneration were compared to homologous wild-type mice. The retinas were analyzed using high-molecular-weight FITC-dextran stained flat-mount preparation, hematoxylin and eosin (H&E) stained cross-sections, an immunohistochemical test for vascular endothelial growth factor (VEGF) distribution and Western blotting for VEGF expression after exposure to hyperoxia between postnatal days 17 (P17) and 21. Leakage and areas of non-perfusion of the retinal blood vessels were alleviated in the retinal degeneration mice. The number of preretinal vascular endothelial cell nuclei in the retinal degeneration mice was smaller than that in the homologous wild-type mice after exposure to hyperoxia (Poxygen-induced retinopathy was positively correlated with the VEGF expression level. However, the VEGF expression level was lower in the retinal degeneration mice. Proliferative retinopathy occurred in mice with rapid retinal degeneration, but retinal photoreceptor cell degeneration could partially restrain the retinal neovascularization in this rapid retinal degeneration mouse model. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Retinal ganglion cell topography and spatial resolving power in penguins.

    Science.gov (United States)

    Coimbra, João Paulo; Nolan, Paul M; Collin, Shaun P; Hart, Nathan S

    2012-01-01

    Penguins are a group of flightless seabirds that exhibit numerous morphological, behavioral and ecological adaptations to their amphibious lifestyle, but little is known about the topographic organization of neurons in their retinas. In this study, we used retinal wholemounts and stereological methods to estimate the total number and topographic distribution of retinal ganglion cells in addition to an anatomical estimate of spatial resolving power in two species of penguins: the little penguin, Eudyptula minor, and the king penguin, Aptenodytes patagonicus. The total number of ganglion cells per retina was approximately 1,200,000 in the little penguin and 1,110,000 in the king penguin. The topographic distribution of retinal ganglion cells in both species revealed the presence of a prominent horizontal visual streak with steeper gradients in the little penguin. The little penguin retinas showed ganglion cell density peaks of 21,867 cells/mm², affording spatial resolution in water of 17.07-17.46 cycles/degree (12.81-13.09 cycles/degree in air). In contrast, the king penguin showed a relatively lower peak density of ganglion cells of 14,222 cells/mm², but--due to its larger eye--slightly higher spatial resolution in water of 20.40 cycles/degree (15.30 cycles/degree in air). In addition, we mapped the distribution of giant ganglion cells in both penguin species using Nissl-stained wholemounts. In both species, topographic mapping of this cell type revealed the presence of an area gigantocellularis with a concentric organization of isodensity contours showing a peak in the far temporal retina of approximately 70 cells/mm² in the little penguin and 39 cells/mm² in the king penguin. Giant ganglion cell densities gradually fall towards the outermost isodensity contours revealing the presence of a vertically organized streak. In the little penguin, we confirmed our cytological characterization of giant ganglion cells using immunohistochemistry for microtubule

  11. In vivo imaging of the retinal pigment epithelial cells

    Science.gov (United States)

    Morgan, Jessica Ijams Wolfing

    The retinal pigment epithelial (RPE) cells form an important layer of the retina because they are responsible for providing metabolic support to the photoreceptors. Techniques to image the RPE layer include autofluorescence imaging with a scanning laser ophthalmoscope (SLO). However, previous studies were unable to resolve single RPE cells in vivo. This thesis describes the technique of combining autofluorescence, SLO, adaptive optics (AO), and dual-wavelength simultaneous imaging and registration to visualize the individual cells in the RPE mosaic in human and primate retina for the first time in vivo. After imaging the RPE mosaic non-invasively, the cell layer's structure and regularity were characterized using quantitative metrics of cell density, spacing, and nearest neighbor distances. The RPE mosaic was compared to the cone mosaic, and RPE imaging methods were confirmed using histology. The ability to image the RPE mosaic led to the discovery of a novel retinal change following light exposure; 568 nm exposures caused an immediate reduction in autofluorescence followed by either full recovery or permanent damage in the RPE layer. A safety study was conducted to determine the range of exposure irradiances that caused permanent damage or transient autofluorescence reductions. Additionally, the threshold exposure causing autofluorescence reduction was determined and reciprocity of radiant exposure was confirmed. Light exposures delivered by the AOSLO were not significantly different than those delivered by a uniform source. As all exposures tested were near or below the permissible light levels of safety standards, this thesis provides evidence that the current light safety standards need to be revised. Finally, with the retinal damage and autofluorescence reduction thresholds identified, the methods of RPE imaging were modified to allow successful imaging of the individual cells in the RPE mosaic while still ensuring retinal safety. This thesis has provided a

  12. Stem Cell-Based Therapeutic Applications in Retinal Degenerative Diseases.

    OpenAIRE

    Huang Yiming; Enzmann Volker; Ildstad Suzanne T

    2011-01-01

    Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epithelium (RPE) affect millions of people worldwide. Retinal degeneration (RD) is found in many different forms of retinal diseases including retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma. Effective treatment for retinal degeneration has been widely investigated. Gene-replacement therapy has been shown to improve visual function in inheri...

  13. Retinal bipolar cells: elementary building blocks of vision.

    Science.gov (United States)

    Euler, Thomas; Haverkamp, Silke; Schubert, Timm; Baden, Tom

    2014-08-01

    Retinal bipolar cells are the first ‘projection neurons’ of the vertebrate visual system—all of the information needed for vision is relayed by this intraretinal connection. Each of the at least 13 distinct types of bipolar cells systematically transforms the photoreceptor input in a different way, thereby generating specific channels that encode stimulus properties, such as polarity, contrast, temporal profile and chromatic composition. As a result, bipolar cell output signals represent elementary ‘building blocks’ from which the microcircuits of the inner retina derive a feature-oriented description of the visual world.

  14. Investigating the role of retinal Müller cells with approaches in genetics and cell biology.

    Science.gov (United States)

    Fu, Suhua; Zhu, Meili; Ash, John D; Wang, Yunchang; Le, Yun-Zheng

    2014-01-01

    Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology.

  15. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    Directory of Open Access Journals (Sweden)

    Aaron H Fronk

    2016-07-01

    Full Text Available The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included.

  16. A3 Adenosine Receptors Modulate Hypoxia-inducible Factor-1a Expression in Human A375 Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Stefania Merighi

    2005-10-01

    Full Text Available Hypoxia-inducible factor-1 (HIF-1 is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% 02, adenosine upregulates HIF-1α protein expression in a dose-dependent and timedependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2 protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1a and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.

  17. Effect of Monocular Deprivation on Rabbit Neural Retinal Cell Densities.

    Science.gov (United States)

    Mwachaka, Philip Maseghe; Saidi, Hassan; Odula, Paul Ochieng; Mandela, Pamela Idenya

    2015-01-01

    To describe the effect of monocular deprivation on densities of neural retinal cells in rabbits. Thirty rabbits, comprised of 18 subject and 12 control animals, were included and monocular deprivation was achieved through unilateral lid suturing in all subject animals. The rabbits were observed for three weeks. At the end of each week, 6 experimental and 3 control animals were euthanized, their retinas was harvested and processed for light microscopy. Photomicrographs of the retina were taken and imported into FIJI software for analysis. Neural retinal cell densities of deprived eyes were reduced along with increasing period of deprivation. The percentage of reductions were 60.9% (P < 0.001), 41.6% (P = 0.003), and 18.9% (P = 0.326) for ganglion, inner nuclear, and outer nuclear cells, respectively. In non-deprived eyes, cell densities in contrast were increased by 116% (P < 0.001), 52% (P < 0.001) and 59.6% (P < 0.001) in ganglion, inner nuclear, and outer nuclear cells, respectively. In this rabbit model, monocular deprivation resulted in activity-dependent changes in cell densities of the neural retina in favour of the non-deprived eye along with reduced cell densities in the deprived eye.

  18. The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

    Science.gov (United States)

    Mølck, Christina; Ryall, James; Failla, Laura M; Coates, Janine L; Pascussi, Jean-Marc; Heath, Joan K; Stewart, Gregory; Hollande, Frédéric

    2016-12-01

    Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A 2b adenosine receptor (A 2b -AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A 2b -AR to the behavior of colorectal cancer cells. The A 2b -AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A 2b -AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Adenosine and preeclampsia.

    Science.gov (United States)

    Salsoso, Rocío; Farías, Marcelo; Gutiérrez, Jaime; Pardo, Fabián; Chiarello, Delia I; Toledo, Fernando; Leiva, Andrea; Mate, Alfonso; Vázquez, Carmen M; Sobrevia, Luis

    2017-06-01

    Adenosine is an endogenous nucleoside with pleiotropic effects in different physiological processes including circulation, renal blood flow, immune function, or glucose homeostasis. Changes in adenosine membrane transporters, adenosine receptors, and corresponding intracellular signalling network associate with development of pathologies of pregnancy, including preeclampsia. Preeclampsia is a cause of maternal and perinatal morbidity and mortality affecting 3-5% of pregnancies. Since the proposed mechanisms of preeclampsia development include adenosine-dependent biological effects, adenosine membrane transporters and receptors, and the associated signalling mechanisms might play a role in the pathophysiology of preeclampsia. Preeclampsia associates with increased adenosine concentration in the maternal blood and placental tissue, likely due to local hypoxia and ischemia (although not directly demonstrated), microthrombosis, increased catecholamine release, and platelet activation. In addition, abnormal expression and function of equilibrative nucleoside transporters is described in foetoplacental tissues from preeclampsia; however, the role of adenosine receptors in the aetiology of this disease is not well understood. Adenosine receptors activation may be related to abnormal trophoblast invasion, angiogenesis, and ischemia/reperfusion mechanisms in the placenta from preeclampsia. These mechanisms may explain only a low fraction of the associated abnormal transformation of spiral arteries in preeclampsia, triggering cellular stress and inflammatory mediators release from the placenta to the maternal circulation. Although increased adenosine concentration in preeclampsia may be a compensatory or adaptive mechanism favouring placental angiogenesis, a poor angiogenic state is found in preeclampsia. Thus, preeclampsia-associated complications might affect the cell response to adenosine due to altered expression and activity of adenosine receptors, membrane transporters

  20. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  1. Melanopsin-expressing retinal ganglion cells: implications for human diseases

    DEFF Research Database (Denmark)

    La Morgia, Chiara; Ross-Cisneros, Fred N; Hannibal, Jens

    2011-01-01

    In the last decade, there was the seminal discovery of melanopsin-expressing retinal ganglion cells (mRGCs) as a new class of photoreceptors that subserve the photoentrainment of circadian rhythms and other non-image forming functions of the eye. Since then, there has been a growing research...... interest on these cells, mainly focused on animal models. Only recently, a few studies have started to address the relevance of the mRGC system in humans and related diseases. We recently discovered that mRGCs resist neurodegeneration in two inherited mitochondrial disorders that cause blindness, i...

  2. Progranulin deficiency causes the retinal ganglion cell loss during development.

    Science.gov (United States)

    Kuse, Yoshiki; Tsuruma, Kazuhiro; Mizoguchi, Takahiro; Shimazawa, Masamitsu; Hara, Hideaki

    2017-05-10

    Astrocytes are glial cells that support and protect neurons in the central nervous systems including the retina. Retinal ganglion cells (RGCs) are in contact with the astrocytes and our earlier findings showed the reduction of the number of cells in the ganglion cell layer in adult progranulin deficient mice. In the present study, we focused on the time of activation of the astrocytes and the alterations in the number of RGCs in the retina and optic nerve in progranulin deficient mice. Our findings showed that the number of Brn3a-positive cells was reduced and the expression of glial fibrillary acidic protein (GFAP) was increased in progranulin deficient mice. The progranulin deficient mice had a high expression of GFAP on postnatal day 9 (P9) but not on postnatal day 1. These mice also had a decrease in the number of the Brn3a-positive cells on P9. Taken together, these findings indicate that the absence of progranulin can affect the survival of RGCs subsequent the activation of astrocytes during retinal development.

  3. Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shao-Yun, E-mail: jiangshaoyun@yahoo.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Tianjin 300070 (China); Wang, Jian-Tao, E-mail: wangjiantao65@hotmail.com [Eye Center, Tianjin Medical University, 64 Tongan Road, Tianjin 300070 (China); Dohney Eye Institute, Keck School of Medicine, University of Southern California, 1355 San Pablo Street, DOH 314, Los Angeles, CA 90033 (United States)

    2010-05-14

    Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.

  4. Melanopsin expressing human retinal ganglion cells

    DEFF Research Database (Denmark)

    Hannibal, Jens; Christiansen, Anders Tolstrup; Heegaard, Steffen

    2017-01-01

    microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1...

  5. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    International Nuclear Information System (INIS)

    Wilson, J.M.; Danos, O.; Grossman, M.; Raulet, D.H.; Mulligan, R.C.

    1990-01-01

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans

  6. Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely re...

  7. Structural analysis of retinal photoreceptor ellipsoid zone and postreceptor retinal layer associated with visual acuity in patients with retinitis pigmentosa by ganglion cell analysis combined with OCT imaging

    Science.gov (United States)

    Liu, Guodong; Li, Hui; Liu, Xiaoqiang; Xu, Ding; Wang, Fang

    2016-01-01

    Abstract The aim of this study was to examine changes in photoreceptor ellipsoid zone (EZ) and postreceptor retinal layer in retinitis pigmentosa (RP) patients by ganglion cell analysis (GCA) combined with optical coherence tomography (OCT) imaging to evaluate the structure–function relationships between retinal layer changes and best corrected visual acuity (BCVA). Sixty-eight eyes of 35 patients with RP and 65 eyes of 35 normal controls were analyzed in the study. The average length of EZ was 911.1 ± 208.8 μm in RP patients, which was shortened with the progression of the disease on the OCT images. The average ganglion cell–inner plexiform layer thickness (GCIPLT) was 54.7 ± 18.9 μm in RP patients, while in normal controls it was 85.6 ± 6.8 μm. The GCIPLT in all quarters became significantly thinner along with outer retinal thinning. There was a significantly positive correlation between BCVA and EZ (r = −0.7622, P retinal layer changes from a new perspective in RP patients, which suggests that EZ and GCIPLT obtained by GCA combined with OCT imaging are the direct and valid indicators to diagnosis and predict the pathological process of RP. PMID:28033301

  8. Perspectives of Stem Cell-Based Therapy for Age-Related Retinal Degenerative Diseases.

    Science.gov (United States)

    Holan, Vladimir; Hermankova, Barbora; Kossl, Jan

    2017-09-01

    Retinal degenerative diseases, which include age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and glaucoma, mostly affect the elderly population and are the most common cause of decreased quality of vision or even blindness. So far, there is no satisfactory treatment protocol to prevent, stop, or cure these disorders. A great hope and promise for patients suffering from retinal diseases is represented by stem cell-based therapy that could replace diseased or missing retinal cells and support regeneration. In this respect, mesenchymal stem cells (MSCs) that can be obtained from the particular patient and used as autologous cells have turned out to be a promising stem cell type for treatment. Here we show that MSCs can differentiate into cells expressing markers of retinal cells, inhibit production of pro-inflammatory cytokines by retinal tissue, and produce a number of growth and neuroprotective factors for retinal regeneration. All of these properties make MSCs a prospective cell type for cell-based therapy of age-related retinal degenerative diseases.

  9. Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

    Science.gov (United States)

    Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

    2017-08-01

    Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Activation of retinal stem cells in the proliferating marginal region of RCS rats during development of retinitis pigmentosa.

    Science.gov (United States)

    Jian, Qian; Xu, Haiwei; Xie, Hanping; Tian, Chunyu; Zhao, Tongtao; Yin, ZhengQin

    2009-11-06

    Retinal stem cells (RSCs) have been demonstrated at the proliferating marginal regions from the pars plana of ciliary body to the ciliary marginal zone (CMZ) in adult lower vertebrates and mammals. Investigations in the lower vertebrates have provided some evidence that RSCs can proliferate following retinal damage; however, the evidence that this occurs in mammals is not clear. In this study, we explored RSCs proliferation potential of adult mammalian in proliferating marginal regions of Royal College of Surgeons (RCS) rats, an animal model for retinitis pigmentosa (RP). The proliferation was evaluated using BrdU labeling, and Chx-10 as markers to discern progenitor cell of CMZ in Long-Evan's and RCS rats at different postnatal day (PND) after eye opening. We found that few Chx-10 and BrdU labeled cells in the proliferating marginal regions of Long-Evan's rats, which significantly increased in RCS rats at PND30 and PND60. Consistent with this, Chx-10/Vimentin double staining cells in the center retina of RCS rats increased significantly at PND30 after eye opening. In addition, mRNA expression of Shh, Ptch1 and Smo was up-regulated in RCS rats at PND60 compared to age-matched Long-Evan's rats, which revealed Shh/ptc pathway involving in the activation of RSCs. These results suggest that RSCs in the mammalian retinal proliferating marginal regions has the potential to regenerate following degeneration.

  11. Temporal Progression of Retinal Progenitor Cell Identity: Implications in Cell Replacement Therapies

    Directory of Open Access Journals (Sweden)

    Awais Javed

    2017-12-01

    Full Text Available Retinal degenerative diseases, which lead to the death of rod and cone photoreceptor cells, are the leading cause of inherited vision loss worldwide. Induced pluripotent or embryonic stem cells (iPSCs/ESCs have been proposed as a possible source of new photoreceptors to restore vision in these conditions. The proof of concept studies carried out in mouse models of retinal degeneration over the past decade have highlighted several limitations for cell replacement in the retina, such as the low efficiency of cone photoreceptor production from stem cell cultures and the poor integration of grafted cells in the host retina. Current protocols to generate photoreceptors from stem cells are largely based on the use of extracellular factors. Although these factors are essential to induce the retinal progenitor cell (RPC fate from iPSCs/ESCs, developmental studies have shown that RPCs alter fate output as a function of time (i.e., their temporal identity to generate the seven major classes of retinal cell types, rather than spatial position. Surprisingly, current stem cell differentiation protocols largely ignore the intrinsic temporal identity of dividing RPCs, which we argue likely explains the low efficiency of cone production in such cultures. In this article, we briefly review the mechanisms regulating temporal identity in RPCs and discuss how they could be exploited to improve cone photoreceptor production for cell replacement therapies.

  12. Progranulin increases phagocytosis by retinal pigment epithelial cells in culture.

    Science.gov (United States)

    Murase, Hiromi; Tsuruma, Kazuhiro; Kuse, Yoshiki; Shimazawa, Masamitsu; Hara, Hideaki

    2017-12-01

    Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

    Directory of Open Access Journals (Sweden)

    Thomas A Mendel

    Full Text Available Retinal vasculopathies, including diabetic retinopathy (DR, threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR, ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area. ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction. Treatment of ASCs with transforming growth factor beta (TGF-β1 enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection.ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated

  14. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    2017-01-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  15. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  16. Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

    Science.gov (United States)

    Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

    2002-01-01

    The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID

  17. Melanopsin-expressing retinal ganglion cells are resistant to cell injury, but not always

    DEFF Research Database (Denmark)

    Georg, Birgitte; Ghelli, Anna; Giordano, Carla

    2017-01-01

    Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs deputed to non-image forming functions of the eye such as synchronization of circadian rhythms to light-dark cycle. These cells are characterized by unique electrophysiological, anatomical and biochemical properties...

  18. Regenerating reptile retinas: a comparative approach to restoring retinal ganglion cell function.

    Science.gov (United States)

    Williams, D L

    2017-02-01

    Transection or damage to the mammalian optic nerve generally results in loss of retinal ganglion cells by apoptosis. This cell death is seen less in fish or amphibians where retinal ganglion cell survival and axon regeneration leads to recovery of sight. Reptiles lie somewhere in the middle of this spectrum of nerve regeneration, and different species have been reported to have a significant variation in their retinal ganglion cell regenerative capacity. The ornate dragon lizard Ctenophoris ornatus exhibits a profound capacity for regeneration, whereas the Tenerife wall lizard Gallotia galloti has a more variable response to optic nerve damage. Some individuals regain visual activity such as the pupillomotor responses, whereas in others axons fail to regenerate sufficiently. Even in Ctenophoris, although the retinal ganglion cell axons regenerate adequately enough to synapse in the tectum, they do not make long-term topographic connections allowing recovery of complex visually motivated behaviour. The question then centres on where these intraspecies differences originate. Is it variation in the innate ability of retinal ganglion cells from different species to regenerate with functional validity? Or is it variances between different species in the substrate within which the nerves regenerate, the extracellular environment of the damaged nerve or the supporting cells surrounding the regenerating axons? Investigations of retinal ganglion cell regeneration between different species of lower vertebrates in vivo may shed light on these questions. Or perhaps more interesting are in vitro studies comparing axon regeneration of retinal ganglion cells from various species placed on differing substrates.

  19. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures.

    Science.gov (United States)

    Vergara, M Natalia; Gutierrez, Christian; O'Brien, David R; Canto-Soler, M Valeria

    2013-04-01

    Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the

  1. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  2. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  3. Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

    Science.gov (United States)

    Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

    2014-03-01

    Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  4. Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies

    Science.gov (United States)

    Sadek, Jouliana; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J.; Sahai, Michelle A.; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H.; Elemento, Olivier; Kaye, Kenneth M.

    2017-01-01

    Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase–inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI–sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers. PMID:28504647

  5. Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

    Science.gov (United States)

    Nayar, Utthara; Sadek, Jouliana; Reichel, Jonathan; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J; Sahai, Michelle A; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Hassane, Duane; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H; Warren, J David; Elemento, Olivier; Kaye, Kenneth M; Cesarman, Ethel

    2017-06-01

    Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

  6. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS 2, 3 and 4 in Astrocyte-Like Cells.

    Directory of Open Access Journals (Sweden)

    Till Nicolas Eusemann

    Full Text Available The "regulators of g-protein signalling" (RGS comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

  7. Orexin-A potentiates L-type calcium/barium currents in rat retinal ganglion cells.

    Science.gov (United States)

    Liu, F; Weng, S-J; Yang, X-L; Zhong, Y-M

    2015-10-01

    Two neuropeptides, orexin-A and orexin-B (also called hypocretin-1 and -2), have been implicated in sleep/wake regulation, feeding behaviors via the activation of two subtypes of G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). While the expression of orexins and orexin receptors is immunohistochemically revealed in retinal neurons, the function of these peptides in the retina is largely unknown. Using whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that orexin-A increased L-type-like barium currents (IBa,L) in ganglion cells (GCs), and the effect was blocked by the selective OX1R antagonist SB334867, but not by the OX2R antagonist TCS OX2 29. The orexin-A effect was abolished by intracellular dialysis of GDP-β-S/GPAnt-2A, a Gq protein inhibitor, suggesting the mediation of Gq. Additionally, during internal dialysis of the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor U73122, orexin-A did not change the IBa,L of GCs, whereas the orexin-A effect persisted in the presence of the phosphatidylcholine (PC)-PLC inhibitor D609. The orexin-A-induced potentiation was not seen with internal infusion of Ca(2+)-free solution or when inositol 1,4,5-trisphosphate (IP3)-sensitive Ca(2+) release from intracellular stores was blocked by heparin/xestospongins-C. Moreover, the orexin-A effect was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, but was eliminated when PKC was inhibited by bisindolylmaleimide IV (Bis-IV)/Gö6976. Neither adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) nor guanosine 3',5'-cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway was likely involved, as orexin-A persisted to potentiate the IBa,L of GCs no matter these two pathways were activated or inhibited. These results suggest that, by activating OX1R, orexin-A potentiates the IBa,L of rat GCs through a distinct Gq/PI-PLC/IP3/Ca(2+)/PKC signaling pathway. Copyright

  8. Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa

    NARCIS (Netherlands)

    Alves, Celso Henrique; Pellissier, Lucie P; Vos, Rogier M; Garcia Garrido, Marina; Sothilingam, Vithiyanjali; Seide, Christina; Beck, Susanne C; Klooster, J.; Furukawa, Takahisa; Flannery, John G; Verhaagen, J.; Seeliger, Mathias W; Wijnholds, J.

    2014-01-01

    In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and

  9. Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

    Directory of Open Access Journals (Sweden)

    Torres P.M.M.

    1998-01-01

    Full Text Available Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC. These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

  10. Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

    Directory of Open Access Journals (Sweden)

    Boriana K Tchernookova

    Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

  11. Personalized Medicine: Cell and Gene Therapy Based on Patient-Specific iPSC-Derived Retinal Pigment Epithelium Cells.

    Science.gov (United States)

    Li, Yao; Chan, Lawrence; Nguyen, Huy V; Tsang, Stephen H

    2016-01-01

    Interest in generating human induced pluripotent stem (iPS) cells for stem cell modeling of diseases has overtaken that of patient-specific human embryonic stem cells due to the ethical, technical, and political concerns associated with the latter. In ophthalmology, researchers are currently using iPS cells to explore various applications, including: (1) modeling of retinal diseases using patient-specific iPS cells; (2) autologous transplantation of differentiated retinal cells that undergo gene correction at the iPS cell stage via gene editing tools (e.g., CRISPR/Cas9, TALENs and ZFNs); and (3) autologous transplantation of patient-specific iPS-derived retinal cells treated with gene therapy. In this review, we will discuss the uses of patient-specific iPS cells for differentiating into retinal pigment epithelium (RPE) cells, uncovering disease pathophysiology, and developing new treatments such as gene therapy and cell replacement therapy via autologous transplantation.

  12. Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy

    NARCIS (Netherlands)

    van der Lelij, A.; Rothova, A.; Stilma, J. S.; Hoekzema, R.; Kijlstra, A.

    1990-01-01

    Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from

  13. Caspases in retinal ganglion cell death and axon regeneration

    Science.gov (United States)

    Thomas, Chloe N; Berry, Martin; Logan, Ann; Blanch, Richard J; Ahmed, Zubair

    2017-01-01

    Retinal ganglion cells (RGC) are terminally differentiated CNS neurons that possess limited endogenous regenerative capacity after injury and thus RGC death causes permanent visual loss. RGC die by caspase-dependent mechanisms, including apoptosis, during development, after ocular injury and in progressive degenerative diseases of the eye and optic nerve, such as glaucoma, anterior ischemic optic neuropathy, diabetic retinopathy and multiple sclerosis. Inhibition of caspases through genetic or pharmacological approaches can arrest the apoptotic cascade and protect a proportion of RGC. Novel findings have also highlighted a pyroptotic role of inflammatory caspases in RGC death. In this review, we discuss the molecular signalling mechanisms of apoptotic and inflammatory caspase responses in RGC specifically, their involvement in RGC degeneration and explore their potential as therapeutic targets. PMID:29675270

  14. Induced pluripotent stem cells (iPSC)-derived retinal cells in disease modeling and regenerative medicine.

    Science.gov (United States)

    Rathod, Reena; Surendran, Harshini; Battu, Rajani; Desai, Jogin; Pal, Rajarshi

    2018-02-12

    Retinal degenerative disorders are a leading cause of the inherited, irreversible and incurable vision loss. While various rodent model systems have provided crucial information in this direction, lack of disease-relevant tissue availability and species-specific differences have proven to be a major roadblock. Human induced pluripotent stem cells (iPSC) have opened up a whole new avenue of possibilities not just in understanding the disease mechanism but also potential therapeutic approaches towards a cure. In this review, we have summarized recent advances in the methods of deriving retinal cell types from iPSCs which can serve as a renewable source of disease-relevant cell population for basic as well as translational studies. We also provide an overview of the ongoing efforts towards developing a suitable in vitro model for modeling retinal degenerative diseases. This basic understanding in turn has contributed to advances in translational goals such as drug screening and cell-replacement therapies. Furthermore we discuss gene editing approaches for autologous repair of genetic disorders and allogeneic transplantation of stem cell-based retinal derivatives for degenerative disorders with an ultimate goal to restore vision. It is pertinent to note however, that these exciting new developments throw up several challenges that need to be overcome before their full clinical potential can be realized. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Role of inflammatory cells and adenosine in lung ischemia reoxygenation injury using a model of lung donation after cardiac death.

    Science.gov (United States)

    Smail, Hassiba; Baste, Jean-Marc; Gay, Arnaud; Begueret, Hugues; Noël, Romain; Morin, Jean-Paul; Litzler, Pierre-Yves

    2016-04-01

    The objective of this study is to analyze the role of inflammation in the lung ischemia reperfusion (IR) injury and determine the protective role of adenosine in an in vitro lung transplantation model. We used a hybrid model of lung donor after cardiac death, with warm ischemia in corpo of varying duration (2 h, 4 h) followed by in vitro lung slices culture for reoxygenation (1 h, 4 h and 24 h), in the presence or not of lymphocytes and of adenosine. To quantify the inflammatory lesions, we performed TNFα, IL2 assays, and histological analysis. In this model of a nonblood perfused system, the addition of lymphocytes during reoxygenation lead to higher rates of TNFα and IL2 after 4 h than after 2 h of warm ischemia (P < .05). These levels increased with the duration of reoxygenation and were maximum at 24 h (P < .05). In the presence of adenosine TNFα and IL2 decreased. After 2 h of warm ischemia, we observed a significant inflammatory infiltration, alveolar thickening and a necrosis of the bronchiolar cells. After 4 h of warm ischemia, alveolar cells necrosis was associated. This model showed that lymphocytes increased the inflammatory response and the histological lesions after 4 h of warm ischemia and that adenosine could have an anti-inflammatory role with potential reconditioning action when used in the pneumoplegia solution.

  16. Role of hemolysis in red cell adenosine triphosphate release in simulated exercise conditions in vitro.

    Science.gov (United States)

    Mairbäurl, Heimo; Ruppe, Florian A; Bärtsch, Peter

    2013-10-01

    Specific adenosine triphosphate (ATP) release from red blood cells has been discussed as a possible mediator controlling microcirculation in states of decreased tissue oxygen. Because intravascular hemolysis might also contribute to plasma ATP, we tested in vitro which portion of ATP release is due to hemolysis in typical exercise-induced strains to the red blood cells (shear stress, deoxygenation, and lactic acidosis). Human erythrocytes were suspended in dextran-containing media (hematocrit 10%) and were exposed to shear stress in a rotating Couette viscometer at 37°C. Desaturation (oxygen saturation of hemoglobin ∼20%) was achieved by tonometry with N2 before shear stress exposure. Cells not exposed to shear stress were used as controls. Na lactate (15 mM), lactic acid (15 mM, pH 7.0), and HCl (pH 7.0) were added to simulate exercise-induced lactic acidosis. After incubation, extracellular hemoglobin was measured to quantify hemolysis. ATP was measured with the luciferase assay. Shear stress increased extracellular ATP in a stress-related and time-dependent manner. Hypoxia induced a ∼10-fold increase in extracellular ATP in nonsheared cells and shear stress-exposed cells. Lactic acid had no significant effect on ATP release and hemolysis. In normoxic cells, approximately 20%-50% of extracellular ATP was due to hemolysis. This proportion decreased to less than 10% in hypoxic cells. Our results indicate that when exposing red blood cells to typical strains they encounter when passing through capillaries of exercising skeletal muscle, ATP release from red blood cells is caused mainly by deoxygenation and shear stress, whereas lactic acidosis had only a minor effect. Hemolysis effects were decreased when hemoglobin was deoxygenated. Together, by specific release and hemolysis, extracellular ATP reaches values that have been shown to cause local vasodilatation.

  17. Adenosine, but not guanosine, protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity.

    Science.gov (United States)

    Menezes, Camila Braz; Frasson, Amanda Piccoli; Meirelles, Lucia Collares; Tasca, Tiana

    2017-02-01

    Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5'-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  18. A defect in KCa3.1 channel activity limits the ability of CD8+ T cells from cancer patients to infiltrate an adenosine-rich microenvironment.

    Science.gov (United States)

    Chimote, Ameet A; Balajthy, Andras; Arnold, Michael J; Newton, Hannah S; Hajdu, Peter; Qualtieri, Julianne; Wise-Draper, Trisha; Conforti, Laura

    2018-04-24

    The limited ability of cytotoxic T cells to infiltrate solid tumors hampers immune surveillance and the efficacy of immunotherapies in cancer. Adenosine accumulates in solid tumors and inhibits tumor-specific T cells. Adenosine inhibits T cell motility through the A 2A receptor (A 2A R) and suppression of KCa3.1 channels. We conducted three-dimensional chemotaxis experiments to elucidate the effect of adenosine on the migration of peripheral blood CD8 + T cells from head and neck squamous cell carcinoma (HNSCC) patients. The chemotaxis of HNSCC CD8 + T cells was reduced in the presence of adenosine, and the effect was greater on HNSCC CD8 + T cells than on healthy donor (HD) CD8 + T cells. This response correlated with the inability of CD8 + T cells to infiltrate tumors. The effect of adenosine was mimicked by an A 2A R agonist and prevented by an A 2A R antagonist. We found no differences in A 2A R expression, 3',5'-cyclic adenosine monophosphate abundance, or protein kinase A type 1 activity between HNSCC and HD CD8 + T cells. We instead detected a decrease in KCa3.1 channel activity, but not expression, in HNSCC CD8 + T cells. Activation of KCa3.1 channels by 1-EBIO restored the ability of HNSCC CD8 + T cells to chemotax in the presence of adenosine. Our data highlight the mechanism underlying the increased sensitivity of HNSCC CD8 + T cells to adenosine and the potential therapeutic benefit of KCa3.1 channel activators, which could increase infiltration of these T cells into tumors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  19. Layer-by-Layer Bioprinting of Stem Cells for Retinal Tissue Regeneration

    Science.gov (United States)

    2016-12-01

    Precision Tissue Models”, Distinguished Seminar, Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, University of...in vitro drug screening and potential in vivo retinal neuron repair. The expansion of ganglion cells is tightly related to the spatial arrangement of...AWARD NUMBER: W81XWH-14-1-0522 TITLE: Layer-by-Layer Bioprinting of Stem Cells for Retinal Tissue Regeneration PRINCIPAL INVESTIGATOR

  20. Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

    OpenAIRE

    Foltz, LP; Clegg, DO

    2017-01-01

    We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embry...

  1. Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

    Science.gov (United States)

    Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

    2016-04-01

    The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hi

  2. Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Xue [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province (China); Wang, Ke, E-mail: wangke@jsinm.org [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province (China); Zhang, Kai [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province (China); Zhou, Fanfan [Faculty of Pharmacy, University of Sydney, New South Wales 2006 (Australia); Zhu, Ling [Save Sight Institute, University of Sydney, New South Wales 2000 (Australia)

    2016-10-15

    Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells. - Highlights: • atRAL induces oxidative stress-mediated apoptosis in ARPE-19 cells. • atRAL induces oxidative stress-mediated inflammation in ARPE-19 cells. • NF-κB is involved in atRAL-induced oxidative and nitrosative stresses.

  3. Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal

    International Nuclear Information System (INIS)

    Zhu, Xue; Wang, Ke; Zhang, Kai; Zhou, Fanfan; Zhu, Ling

    2016-01-01

    Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells. - Highlights: • atRAL induces oxidative stress-mediated apoptosis in ARPE-19 cells. • atRAL induces oxidative stress-mediated inflammation in ARPE-19 cells. • NF-κB is involved in atRAL-induced oxidative and nitrosative stresses.

  4. Unfolding Role of a Danger Molecule Adenosine Signaling in Modulation of Microbial Infection and Host Cell Response

    Directory of Open Access Journals (Sweden)

    Jaden S. Lee

    2018-01-01

    Full Text Available Ectonucleotidases CD39 and CD73, specific nucleotide metabolizing enzymes located on the surface of the host, can convert a pro-inflammatory environment driven by a danger molecule extracellular-ATP to an adenosine-mediated anti-inflammatory milieu. Accordingly, CD39/CD73 signaling has been strongly implicated in modulating the intensity, duration, and composition of purinergic danger signals delivered to host. Recent studies have eluted potential roles for CD39 and CD73 in selective triggering of a variety of host immune cells and molecules in the presence of pathogenic microorganisms or microbial virulence molecules. Growing evidence also suggests that CD39 and CD73 present complimentary, but likely differential, actions against pathogens to shape the course and severity of microbial infection as well as the associated immune response. Similarly, adenosine receptors A2A and A2B have been proposed to be major immunomodulators of adenosine signaling during chronic inflammatory conditions induced by opportunistic pathogens, such as oral colonizer Porphyromonas gingivalis. Therefore, we here review the recent studies that demonstrate how complex network of molecules in the extracellular adenosine signaling machinery and their interactions can reshape immune responses and may also be targeted by opportunistic pathogens to establish successful colonization in human mucosal tissues and modulate the host immune response.

  5. Novel Strategies for the Improvement of Stem Cells' Transplantation in Degenerative Retinal Diseases

    Science.gov (United States)

    Nicoară, Simona Delia; Șușman, Sergiu; Tudoran, Oana; Bărbos, Otilia; Cherecheș, Gabriela; Aștilean, Simion; Potara, Monica; Sorițău, Olga

    2016-01-01

    Currently, there is no cure for the permanent vision loss caused by degenerative retinal diseases. One of the novel therapeutic strategies aims at the development of stem cells (SCs) based neuroprotective and regenerative medicine. The main sources of SCs for the treatment of retinal diseases are the embryo, the bone marrow, the region of neuronal genesis, and the eye. The success of transplantation depends on the origin of cells, the route of administration, the local microenvironment, and the proper combinative formula of growth factors. The feasibility of SCs based therapies for degenerative retinal diseases was proved in the preclinical setting. However, their translation into the clinical realm is limited by various factors: the immunogenicity of the cells, the stability of the cell phenotype, the predilection of SCs to form tumors in situ, the abnormality of the microenvironment, and the association of a synaptic rewiring. To improve SCs based therapies, nanotechnology offers a smart delivery system for biomolecules, such as growth factors for SCs implantation and differentiation into retinal progenitors. This review explores the main advances in the field of retinal transplantology and applications of nanotechnology in the treatment of retinal diseases, discusses the challenges, and suggests new therapeutic approaches in retinal transplantation. PMID:27293444

  6. Further studies on the effect of adenosine cyclic monophosphate derivatives on cell proliferation in the jejunal crypts of rat.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1982-01-01

    1. Cell proliferation in the jejunal crypt epithelium of rat was measured using a stathmokinetic technique. 2. Sodium butyrate was found to promote jejunal crypt cell proliferation. 3. N6, O2'-Dibutyryl cyclic adenosine monophosphate (cAMP), N6-monobutyryl-cAMP and N6-monobutyryl-8-bromo-cAMP were found to inhibit cell proliferation when compared to sodium butyrate treated tissues. 4. 8-Chlorophenylthio-cAMP was found to inhibit cell division when compared to untreated animals. 5. O2'-Monobutyryl cAMP and 8-bromo-cAMP were not found to inhibit cell proliferation.

  7. Adenosine Receptors and Wound Healing

    Directory of Open Access Journals (Sweden)

    Bruce N. Cronstein

    2004-01-01

    Full Text Available Recent studies have demonstrated that application of topical adenosine A2A receptor agonists promotes more rapid wound closure and clinical studies are currently underway to determine the utility of topical A2A adenosine receptor agonists in the therapy of diabetic foot ulcers. The effects of adenosine A2A receptors on the cells and tissues of healing wounds have only recently been explored. We review here the known effects of adenosine A2A receptor occupancy on the cells involved in wound healing.

  8. A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

    Directory of Open Access Journals (Sweden)

    Janosch Peter Heller

    2015-11-01

    Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

  9. The clinical correlation of regulatory T cells and cyclic adenosine monophosphate in enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Shih-Min Wang

    Full Text Available Brainstem encephalitis (BE and pulmonary edema (PE are notable complications of enterovirus 71 (EV71 infection.This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD or BE group, and the autonomic nervous system (ANS dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP levels, and the regulatory T cell (Tregs profiles of the patients were determined.Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4(+CD25(+Foxp3+ and CD4(+Foxp3(+ T cells compared with patients with HFMD or BE. The expression frequency of CD4-CD8- was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4+Foxp3+ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.

  10. Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficient immunodeficiency.

    Science.gov (United States)

    Shaw, Kit L; Garabedian, Elizabeth; Mishra, Suparna; Barman, Provaboti; Davila, Alejandra; Carbonaro, Denise; Shupien, Sally; Silvin, Christopher; Geiger, Sabine; Nowicki, Barbara; Smogorzewska, E Monika; Brown, Berkley; Wang, Xiaoyan; de Oliveira, Satiro; Choi, Yeong; Ikeda, Alan; Terrazas, Dayna; Fu, Pei-Yu; Yu, Allen; Fernandez, Beatriz Campo; Cooper, Aaron R; Engel, Barbara; Podsakoff, Greg; Balamurugan, Arumugam; Anderson, Stacie; Muul, Linda; Jagadeesh, G Jayashree; Kapoor, Neena; Tse, John; Moore, Theodore B; Purdy, Ken; Rishi, Radha; Mohan, Kathey; Skoda-Smith, Suzanne; Buchbinder, David; Abraham, Roshini S; Scharenberg, Andrew; Yang, Otto O; Cornetta, Kenneth; Gjertson, David; Hershfield, Michael; Sokolic, Rob; Candotti, Fabio; Kohn, Donald B

    2017-05-01

    Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. ClinicalTrials.gov NCT00794508. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA

  11. Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

    Science.gov (United States)

    Eiraku, Mototsugu; Sasai, Yoshiki

    2011-12-15

    Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

  12. Retinal Astrocytes and GABAergic Wide-Field Amacrine Cells Express PDGFRα: Connection to Retinal Ganglion Cell Neuroprotection by PDGF-AA.

    Science.gov (United States)

    Takahama, Shokichi; Adetunji, Modupe O; Zhao, Tantai; Chen, Shan; Li, Wei; Tomarev, Stanislav I

    2017-09-01

    Our previous experiments demonstrated that intravitreal injection of platelet-derived growth factor-AA (PDGF-AA) provides retinal ganglion cell (RGC) neuroprotection in a rodent model of glaucoma. Here we used PDGFRα-enhanced green fluorescent protein (EGFP) mice to identify retinal cells that may be essential for RGC protection by PDGF-AA. PDGFRα-EGFP mice expressing nuclear-targeted EGFP under the control of the PDGFRα promoter were used. Localization of PDGFRα in the neural retina was investigated by confocal imaging of EGFP fluorescence and immunofluorescent labeling with a panel of antibodies recognizing different retinal cell types. Primary cultures of mouse RGCs were produced by immunopanning. Neurobiotin injection of amacrine cells in a flat-mounted retina was used for the identification of EGFP-positive amacrine cells in the inner nuclear layer. In the mouse neural retina, PDGFRα was preferentially localized in the ganglion cell and inner nuclear layers. Immunostaining of the retina demonstrated that astrocytes in the ganglion cell layer and a subpopulation of amacrine cells in the inner nuclear layer express PDGFRα, whereas RGCs (in vivo or in vitro) did not. PDGFRα-positive amacrine cells are likely to be Type 45 gamma-aminobutyric acidergic (GABAergic) wide-field amacrine cells. These data indicate that the neuroprotective effect of PDGF-AA in a rodent model of glaucoma could be mediated by astrocytes and/or a subpopulation of amacrine cells. We suggest that after intravitreal injection of PDGF-AA, these cells secrete factors protecting RGCs.

  13. Occlusion of retinal capillaries caused by glial cell proliferation in chronic ocular inflammation.

    Science.gov (United States)

    Bianchi, E; Ripandelli, G; Feher, J; Plateroti, A M; Plateroti, R; Kovacs, I; Plateroti, P; Taurone, S; Artico, M

    2015-01-01

    The inner blood-retinal barrier is a gliovascular unit in which glial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. During chronic ocular inflammation, microvascular complications can give rise to vascular proliferative lesions, which compromise visual acuity. This pathologic remodelling caused by proliferating Müller cells determines occlusion of retinal capillaries. The aim of the present study was to identify qualitative and quantitative alterations in the retinal capillaries in patients with post-traumatic chronic ocular inflammation or post-thrombotic vascular glaucoma. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in retinal inflammation. Our electron microscopy findings demonstrated that during chronic ocular inflammation, thickening of the basement membrane, loss of pericytes and endothelial cells and proliferation of Müller cells occur with irreversible occlusion of retinal capillaries. Angiogenesis takes place as part of a regenerative reaction that results in fibrosis. We believe that VEGF and pro-inflammatory cytokines may be potential therapeutic targets in the treatment of this disease although further studies are required to confirm these findings.

  14. Bone marrow-derived cells are differentially involved in pathological and physiological retinal angiogenesis in mice

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    Zou, He [Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8507 (Japan); Otani, Atsushi, E-mail: otan@kuhp.kyoto-u.ac.jp [Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8507 (Japan); Oishi, Akio; Yodoi, Yuko; Kameda, Takanori; Kojima, Hiroshi; Yoshimura, Nagahisa [Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8507 (Japan)

    2010-01-08

    Purpose: Bone marrow-derived cells have been shown to play roles in angiogenesis. Although these cells have been shown to promote angiogenesis, it is not yet clear whether these cells affect all types of angiogenesis. This study investigated the involvement of bone marrow-derived cells in pathological and physiological angiogenesis in the murine retina. Materials and methods: The oxygen-induced retinopathy (OIR) model was used as a retinal angiogenesis model in newborn mice. To block the influence of bone marrow-derived cells, the mice were irradiated with a 4-Gy dose of radiation from a {sup 137}Cs source. Irradiation was performed in four different conditions with radio dense 2-cm thick lead disks; (1) H group, the head were covered with these discs to protect the eyes from radiation; (2) A group, all of the body was covered with these discs; (3) N group, mice were completely unshielded; (4) C group, mice were put in the irradiator but were not irradiated. On P17, the retinal areas showing pathological and physiological retinal angiogenesis were measured and compared to the retinas of nonirradiated mice. Results: Although irradiation induced leukocyte depletion, it did not affect the number of other cell types or body weight. Retinal nonperfusion areas were significantly larger in irradiated mice than in control mice (P < 0.05), indicating that physiological angiogenesis was impaired. However, the formation of tuft-like angiogenesis processes was more prominent in the irradiated mice (P < 0.05), indicating that pathological angiogenesis was intact. Conclusions: Bone marrow-derived cells seem to be differentially involved in the formation of physiological and pathological retinal vessels. Pathological angiogenesis in the murine retina does not require functional bone marrow-derived cells, but these cells are important for the formation of physiological vessels. Our results add a new insight into the pathology of retinal angiogenesis and bolster the hypothesis that

  15. Bone marrow-derived cells are differentially involved in pathological and physiological retinal angiogenesis in mice

    International Nuclear Information System (INIS)

    Zou, He; Otani, Atsushi; Oishi, Akio; Yodoi, Yuko; Kameda, Takanori; Kojima, Hiroshi; Yoshimura, Nagahisa

    2010-01-01

    Purpose: Bone marrow-derived cells have been shown to play roles in angiogenesis. Although these cells have been shown to promote angiogenesis, it is not yet clear whether these cells affect all types of angiogenesis. This study investigated the involvement of bone marrow-derived cells in pathological and physiological angiogenesis in the murine retina. Materials and methods: The oxygen-induced retinopathy (OIR) model was used as a retinal angiogenesis model in newborn mice. To block the influence of bone marrow-derived cells, the mice were irradiated with a 4-Gy dose of radiation from a 137 Cs source. Irradiation was performed in four different conditions with radio dense 2-cm thick lead disks; (1) H group, the head were covered with these discs to protect the eyes from radiation; (2) A group, all of the body was covered with these discs; (3) N group, mice were completely unshielded; (4) C group, mice were put in the irradiator but were not irradiated. On P17, the retinal areas showing pathological and physiological retinal angiogenesis were measured and compared to the retinas of nonirradiated mice. Results: Although irradiation induced leukocyte depletion, it did not affect the number of other cell types or body weight. Retinal nonperfusion areas were significantly larger in irradiated mice than in control mice (P < 0.05), indicating that physiological angiogenesis was impaired. However, the formation of tuft-like angiogenesis processes was more prominent in the irradiated mice (P < 0.05), indicating that pathological angiogenesis was intact. Conclusions: Bone marrow-derived cells seem to be differentially involved in the formation of physiological and pathological retinal vessels. Pathological angiogenesis in the murine retina does not require functional bone marrow-derived cells, but these cells are important for the formation of physiological vessels. Our results add a new insight into the pathology of retinal angiogenesis and bolster the hypothesis that bone

  16. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    Science.gov (United States)

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Molecular Responses of Human Retinal Cells to Infection with Dengue Virus.

    Science.gov (United States)

    Carr, Jillian M; Ashander, Liam M; Calvert, Julie K; Ma, Yuefang; Aloia, Amanda; Bracho, Gustavo G; Chee, Soon-Phaik; Appukuttan, Binoy; Smith, Justine R

    2017-01-01

    Recent clinical reports indicate that infection with dengue virus (DENV) commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin- β 1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

  18. Molecular Responses of Human Retinal Cells to Infection with Dengue Virus

    Directory of Open Access Journals (Sweden)

    Jillian M. Carr

    2017-01-01

    Full Text Available Recent clinical reports indicate that infection with dengue virus (DENV commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin-β1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

  19. Recovery of cat retinal ganglion cell sensitivity following pigment bleaching.

    Science.gov (United States)

    Bonds, A B; Enroth-Cugell, C

    1979-01-01

    1. The threshold illuminance for small spot stimulation of on-centre cat retinal ganglion cells was plotted vs. time after exposure to adapting light sufficiently strong to bleach significant amounts of rhodopsin. 2. When the entire receptive field of an X- or Y-type ganglion cell is bleached by at most 40%, recovery of the cell's rod-system proceeds in two phases: an early relatively fast one during which the response appears transient, and a late, slower one during which responses become more sustained. Log threshold during the later phase is well fit by an exponential in time (tau = 11.5-38 min). 3. After bleaches of 90% of the underlying pigment, threshold is cone-determined for as long as 40 min. Rod threshold continues to decrease for at least 85 min after the bleach. 4. The rate of recovery is slower after strong than after weak bleaches; 10 and 90% bleaches yield time constants for the later phase of 11.5 and 38 min, respectively. This contrasts with an approximate time constant of 11 min for rhodopsin regeneration following any bleach. 5. The relationship between the initial elevation of log rod threshold extrapolated from the fitted exponential curves and the initial amount of pigment bleached is monotonic, but nonlinear. 6. After a bleaching exposure, the maintained discharge is initially very regular. The firing rate first rises, then falls to the pre-bleach level, with more extended time courses of change in firing rate after stronger exposures. The discharge rate is restored before threshold has recovered fully. 7. The change in the response vs. log stimulus relationship after bleaching is described as a shift of the curve to the right, paired with a decrease in slope of the linear segment of the curve. PMID:521963

  20. Apoptosis of rabbit retinal cell after eyeball rupture.

    Science.gov (United States)

    Tian, Xue-Min; Zhu, Yu

    2013-04-13

    To establish rabbit eyeball rupture model by air gun in order to observe and analyze the early injury condition and reasons of retinal cell after eyeball rupture. Forty eight healthy rabbits were randomly divided into control group and 1, 3, 6, 12 and 24 h after injury groups. After anesthesia, the rabbit eyeball rupture model was established by air gun. Then the early pathological changes of rabbit retina were observed, and apoptotic index (AI), oncosis index (OI), the relationship between the expression amounts of apoptosis-related genes and AI were analyzed. Obvious pathological lesion appeared in retina 6 h after injury. Irreversible damage occurred 12-24 h after injury. The results of AI and OI indicated that the OI peak appeared 6 h after injury and then gradually declined, while the AI increased with the prolongation of time, and the AI was higher than OI in 12 h after injury. Immunohistochemical results indicated that there was no obvious bcl-2 protein expression change. Compared with the control group and the 3, 6, 12 and 24 h after the injury groups, the expressions of p53 and Caspase-3 were significantly improved and peaked at 12 h (Peyeball rupture. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  1. The molecular basis of retinal ganglion cell death in glaucoma.

    Science.gov (United States)

    Almasieh, Mohammadali; Wilson, Ariel M; Morquette, Barbara; Cueva Vargas, Jorge Luis; Di Polo, Adriana

    2012-03-01

    Glaucoma is a group of diseases characterized by progressive optic nerve degeneration that results in visual field loss and irreversible blindness. A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of CNS neurons with their soma in the inner retina and axons in the optic nerve. Strategies that delay or halt RGC loss have been recognized as potentially beneficial to preserve vision in glaucoma; however, the success of these approaches depends on an in-depth understanding of the mechanisms that lead to RGC dysfunction and death. In recent years, there has been an exponential increase in valuable information regarding the molecular basis of RGC death stemming from animal models of acute and chronic optic nerve injury as well as experimental glaucoma. The emerging landscape is complex and points at a variety of molecular signals - acting alone or in cooperation - to promote RGC death. These include: axonal transport failure, neurotrophic factor deprivation, toxic pro-neurotrophins, activation of intrinsic and extrinsic apoptotic signals, mitochondrial dysfunction, excitotoxic damage, oxidative stress, misbehaving reactive glia and loss of synaptic connectivity. Collectively, this body of work has considerably updated and expanded our view of how RGCs might die in glaucoma and has revealed novel, potential targets for neuroprotection. Copyright © 2011. Published by Elsevier Ltd.

  2. Spatial consequences of bleaching adaptation in cat retinal ganglion cells.

    Science.gov (United States)

    Bonds, A B; Enroth-Cugell, C

    1981-01-01

    1. Experiments were conducted to study the effects of localized bleaching on the centre responses of rod-driven cat retinal ganglion cells. 2. Stimulation as far as 2 degrees from the bleaching site yielded responses which were reduced nearly as much as those generated at the bleaching site. Bleaching in the receptive field middle reduced responsiveness at a site 1 degrees peripheral more than bleaching at that peripheral site itself. 3. The effectiveness of a bleach in reducing centre responsiveness is related to the sensitivity of the region in which the bleach is applied. 4. Response reduction after a 0.2 degree bleach followed the same temporal pattern for concentric test spots of from 0.2 to 1.8 degrees in diameter, implying a substantially uniform spread of adaptation within these bounds. 5. A linear trade-off between fraction of rhodopsin and area bleached over a range of 8:1 yields the same pattern of response reduction, implying that the non-linear nature of bleaching adaptation is a property of the adaptation pool rather than independent photoreceptors. PMID:7320894

  3. Retinal Vasculitis

    Science.gov (United States)

    Rosenbaum, James T.; Sibley, Cailin H.; Lin, Phoebe

    2016-01-01

    Purpose of review Ophthalmologists and rheumatologists frequently miscommunicate in consulting on patients with retinal vasculitis. This report seeks to establish a common understanding of the term, retinal vasculitis, and to review recent papers on this diagnosis. Recent findings 1) The genetic basis of some rare forms of retinal vascular disease have recently been described. Identified genes include CAPN5, TREX1, and TNFAIP3; 2) Behçet’s disease is a systemic illness that is very commonly associated with occlusive retinal vasculitis; 3) retinal imaging including fluorescein angiography and other newer imaging modalities has proven crucial to the identification and characterization of retinal vasculitis and its complications; 4) although monoclonal antibodies to IL-17A or IL-1 beta failed in trials for Behçet’s disease, antibodies to TNF alpha, either infliximab or adalimumab, have demonstrated consistent benefit in managing this disease. Interferon treatment and B cell depletion therapy via rituximab may be beneficial in certain types of retinal vasculitis. Summary Retinal vasculitis is an important entity for rheumatologists to understand. Retinal vasculitis associated with Behçet’s disease responds to monoclonal antibodies that neutralize TNF, but the many other forms of non-infectious retinal vasculitis may require alternate therapeutic management. PMID:26945335

  4. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

    Directory of Open Access Journals (Sweden)

    Budd A Tucker

    2011-04-01

    Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

  5. Relationship between macular ganglion cell complex thickness and macular outer retinal thickness: a spectral-domain optical coherence tomography study.

    Science.gov (United States)

    Kita, Yoshiyuki; Kita, Ritsuko; Takeyama, Asuka; Anraku, Ayako; Tomita, Goji; Goldberg, Ivan

    2013-01-01

    To assess the relationship between macular ganglion cell complex and macular outer retinal thicknesses. Case-control study. Forty-two normal eyes and 91 eyes with primary open-angle glaucoma were studied. Spectral-domain optical coherence tomography (RTVue-100) was used to measure the macular ganglion cell complex and macular outer retinal thickness. Ganglion cell complex to outer retinal thickness ratio was also calculated. The relationships between the ganglion cell complex and outer retinal thicknesses and between the ganglion cell complex to outer retinal thickness ratio and outer retinal thickness were evaluated. There was a positive correlation between ganglion cell complex and outer retinal thicknesses in the normal group and the glaucoma group (r = 0.53, P variation in the outer retinal thickness. Therefore, when determining the ganglion cell complex, it seems necessary to consider the outer retinal thickness as well. We propose the ratio as a suitable parameter to account for individual variations in outer retinal thickness. © 2013 The Authors. Clinical and Experimental Ophthalmology © 2013 Royal Australian and New Zealand College of Ophthalmologists.

  6. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    Directory of Open Access Journals (Sweden)

    Lorena Olivares-González

    Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  7. Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

    Science.gov (United States)

    Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

    2008-02-01

    Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

  8. NUTRITION AND VASCULAR SUPPLY OF RETINAL GANGLION CELLS DURING HUMAN DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Paul eRutkowski

    2016-04-01

    Full Text Available Purpose. To review the roles of the different vascular beds nourishing the inner retina (retinal ganglion cells during normal development of the human eye and using our own tissue specimens to support our conclusions.Methods. An extensive search of the appropriate literature included PubMed, Google scholar, and numerous available textbooks. In addition, choroidal and retinal NADPH-diaphorase stained whole mount preparations were investigated.Results. The first critical interaction between vascular bed and retinal ganglion cell (RGC formation occurs in the 6th-8th month of gestation leading to a massive reduction of RGCs mainly in the peripheral retina. The first three years of age are characterized by an intense growth of the eyeball to near adult size. In the adult eye, the influence of the choroid on inner retinal nutrition was determined by examining the peripheral retinal watershed zones in more detail.Conclusion. This delicately balanced situation of retinal ganglion cell nutrition is described in the different regions of the eye, and a new graphic presentation is introduced to combine morphological measurements and clinical visual field data.

  9. Density, proportion, and dendritic coverage of retinal ganglion cells of the common marmoset (Callithrix jacchus jacchus

    Directory of Open Access Journals (Sweden)

    F.L. Gomes

    2005-06-01

    Full Text Available We performed a quantitative analysis of M and P cell mosaics of the common-marmoset retina. Ganglion cells were labeled retrogradely from optic nerve deposits of Biocytin. The labeling was visualized using horseradish peroxidase (HRP histochemistry and 3-3'diaminobenzidine as chromogen. M and P cells were morphologically similar to those found in Old- and New-World primates. Measurements were performed on well-stained cells from 4 retinas of different animals. We analyzed separate mosaics for inner and outer M and P cells at increasing distances from the fovea (2.5-9 mm of eccentricity to estimate cell density, proportion, and dendritic coverage. M cell density decreased towards the retinal periphery in all quadrants. M cell density was higher in the nasal quadrant than in other retinal regions at similar eccentricities, reaching about 740 cells/mm² at 2.5 mm of temporal eccentricity, and representing 8-14% of all ganglion cells. P cell density increased from peripheral to more central regions, reaching about 5540 cells/mm² at 2.5 mm of temporal eccentricity. P cells represented a smaller proportion of all ganglion cells in the nasal quadrant than in other quadrants, and their numbers increased towards central retinal regions. The M cell coverage factor ranged from 5 to 12 and the P cell coverage factor ranged from 1 to 3 in the nasal quadrant and from 5 to 12 in the other quadrants. These results show that central and peripheral retinal regions differ in terms of cell class proportions and dendritic coverage, and their properties do not result from simply scaling down cell density. Therefore, differences in functional properties between central and peripheral vision should take these distinct regional retinal characteristics into account.

  10. Clinical efficacy of gene-modified stem cells in adenosine deaminase–deficient immunodeficiency

    Science.gov (United States)

    Shaw, Kit L.; Garabedian, Elizabeth; Mishra, Suparna; Barman, Provaboti; Davila, Alejandra; Carbonaro, Denise; Shupien, Sally; Silvin, Christopher; Geiger, Sabine; Nowicki, Barbara; Smogorzewska, E. Monika; Brown, Berkley; Wang, Xiaoyan; de Oliveira, Satiro; Choi, Yeong; Ikeda, Alan; Terrazas, Dayna; Fu, Pei-Yu; Yu, Allen; Fernandez, Beatriz Campo; Cooper, Aaron R.; Engel, Barbara; Podsakoff, Greg; Balamurugan, Arumugam; Anderson, Stacie; Muul, Linda; Jagadeesh, G. Jayashree; Kapoor, Neena; Tse, John; Moore, Theodore B.; Purdy, Ken; Rishi, Radha; Mohan, Kathey; Skoda-Smith, Suzanne; Buchbinder, David; Abraham, Roshini S.; Scharenberg, Andrew; Yang, Otto O.; Cornetta, Kenneth; Gjertson, David; Hershfield, Michael; Sokolic, Rob; Candotti, Fabio

    2017-01-01

    BACKGROUND. Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase–deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1–2.6) and granulocytes (VCN = 0.01–0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION. ClinicalTrials.gov NCT00794508. FUNDING. Food and Drug Administration Office of Orphan Product

  11. Progress of stem/progenitor cell-based therapy for retinal degeneration.

    Science.gov (United States)

    Tang, Zhimin; Zhang, Yi; Wang, Yuyao; Zhang, Dandan; Shen, Bingqiao; Luo, Min; Gu, Ping

    2017-05-10

    Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future.

  12. Adenosine A₂A receptors inhibit delayed rectifier potassium currents and cell differentiation in primary purified oligodendrocyte cultures.

    Science.gov (United States)

    Coppi, Elisabetta; Cellai, Lucrezia; Maraula, Giovanna; Pugliese, Anna Maria; Pedata, Felicita

    2013-10-01

    Oligodendrocyte progenitor cells (OPCs) are a population of cycling cells which persist in the adult central nervous system (CNS) where, under opportune stimuli, they differentiate into mature myelinating oligodendrocytes. Adenosine A(2A) receptors are Gs-coupled P1 purinergic receptors which are widely distributed throughout the CNS. It has been demonstrated that OPCs express A(2A) receptors, but their functional role in these cells remains elusive. Oligodendrocytes express distinct voltage-gated ion channels depending on their maturation. Here, by electrophysiological recordings coupled with immunocytochemical labeling, we studied the effects of adenosine A(2A) receptors on membrane currents and differentiation of purified primary OPCs isolated from the rat cortex. We found that the selective A(2A) agonist, CGS21680, inhibits sustained, delayed rectifier, K(+) currents (I(K)) without modifying transient (I(A)) conductances. The effect was observed in all cells tested, independently from time in culture. CGS21680 inhibition of I(K) current was concentration-dependent (10-200 nM) and blocked in the presence of the selective A(2A) antagonist SCH58261 (100 nM). It is known that I(K) currents play an important role during OPC development since their block decreases cell proliferation and differentiation. In light of these data, our further aim was to investigate whether A(2A) receptors modulate these processes. CGS21680, applied at 100 nM in the culture medium of oligodendrocyte cultures, inhibits OPC differentiation (an effect prevented by SCH58261) without affecting cell proliferation. Data demonstrate that cultured OPCs express functional A(2A) receptors whose activation negatively modulate I(K) currents. We propose that, by this mechanism, A(2A) adenosine receptors inhibit OPC differentiation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Retinal Oxygen Delivery and Metabolism in Healthy and Sickle Cell Retinopathy Subjects.

    Science.gov (United States)

    Shahidi, Mahnaz; Felder, Anthony E; Tan, Ou; Blair, Norman P; Huang, David

    2018-04-01

    Reduction in inner retinal oxygen delivery (DO2) can cause retinal hypoxia and impair inner retinal oxygen metabolism (MO2), leading to vision loss. The purpose of the current study was to establish measurements of DO2 and MO2 in healthy subjects and test the hypothesis that DO2 and MO2 are reduced in sickle cell retinopathy (SCR) subjects. Dual wavelength retinal oximetry and Doppler optical coherence tomography were performed in 12 healthy control and 12 SCR subjects. Images were analyzed to measure retinal arterial and venous oxygen content (O2A and O2V), venous diameter (DV), and total retinal blood flow (TRBF). Retinal arteriovenous oxygen content difference (O2AV), DO2, MO2, and oxygen extraction fraction (OEF) were calculated according to the following equations: O2AV = O2A - O2V; DO2 = TRBF * O2A; MO2 = TRBF * O2AV; OEF = MO2/DO2. Retinal DV and TRBF were higher in the SCR group as compared to the control group, whereas, O2A, O2V, and O2AV were lower in SCR group as compared to the control group. DO2, MO2, and OEF were not significantly different between control and SCR groups. MO2 and DO2 were linearly related, such that higher MO2 was associated with higher DO2. There was an inverse relationship between TRBF and OEF, such that lower TRBF was associated with higher OEF. Increased blood flow compensated for decreased oxygen content, thereby maintaining DO2, MO2, and OEF at predominately lower stages of SCR. Quantitative assessment of these parameters has the potential to advance knowledge and improve diagnostic evaluation of retinal ischemic conditions.

  14. Retinal Ganglion Cell Diversity and Subtype Specification from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Kirstin B. Langer

    2018-04-01

    Full Text Available Summary: Retinal ganglion cells (RGCs are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. : In this article, Langer and colleagues present extensive characterization of RGC subtypes derived from human pluripotent stem cells, with multiple subtypes identified by subtype-specific molecular markers. Their results present a more detailed analysis of RGC diversity in human cells and yield the use of different markers to identify RGC subtypes. Keywords: iPSC, retina, retinal ganglion cell, RGC subtype, stem cell, ipRGC, alpha RGC, direction selective RGC, RNA-seq

  15. Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases

    Science.gov (United States)

    Jones, Melissa K.; Lu, Bin; Girman, Sergey; Wang, Shaomei

    2017-01-01

    Cell-based therapeutics offer diverse options for treating retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). AMD is characterized by both genetic and environmental risks factors, whereas RP is mainly a monogenic disorder. Though treatments exist for some patients with neovascular AMD, a majority of retinal degenerative patients have no effective therapeutics, thus indicating a need for universal therapies to target diverse patient populations. Two main cell-based mechanistic approaches are being tested in clinical trials. Replacement therapies utilize cell-derived retinal pigment epithelial (RPE) cells to supplant lost or defective host RPE cells. These cells are similar in morphology and function to native RPE cells and can potentially supplant the responsibilities of RPE in vivo. Preservation therapies utilize supportive cells to aid in visual function and photoreceptor preservation partially by neurotrophic mechanisms. The goal of preservation strategies is to halt or slow the progression of disease and maintain remaining visual function. A number of clinical trials are testing the safety of replacement and preservation cell therapies in patients; however, measures of efficacy will need to be further evaluated. In addition, a number of prevailing concerns with regards to the immune-related response, longevity, and functionality of the grafted cells will need to be addressed in future trials. This review will summarize the current status of cell-based preclinical and clinical studies with a focus on replacement and preservation strategies and the obstacles that remain regarding these types of treatments. PMID:28111323

  16. Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype.

    Science.gov (United States)

    Huang, Chen; Zhang, Jing; Ao, Mingxin; Li, Ying; Zhang, Chun; Xu, Yonggen; Li, Xuemin; Wang, Wei

    2012-02-01

    Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction. Copyright © 2011 Wiley Periodicals, Inc.

  17. A case of red-cell adenosine deaminase overproduction associated with hereditary hemolytic anemia found in Japan.

    Science.gov (United States)

    Miwa, S; Fujii, H; Matsumoto, N; Nakatsuji, T; Oda, S; Asano, H; Asano, S

    1978-01-01

    A case of red cell adenosine deaminase (ADA) overproduction associated with hereditary hemolytic anemia is reported here. This appears to be the second report. Proband is a 38-year-old Japanese male who had hemoglobin, 15.8 g/100 ml; reticulocyte count, 4.5%; serum indirect bilirubin, 4.9 mg/100 ml; 51Cr-labeled red cell half-life, 12 days; red cells showed moderate stomatocytosis. His red cell ADA activity showed 40-fold increase while that of the mother showed 4-fold increase. The mother was hematologically normal. The father had a normal enzyme activity. The proband and the mother showed slightly high serum uric acid levels. The proband's red cell showed: ATP, 628 nmoles/ml (normal, 1,010--1,550); adenine nucleotide pool, 46% of the normal mean; 2,3-diphosphoglycerate content, 3,782 nmoles/ml (normal 4,170--5,300); increased oxygen affinity of hemoglobin, P50 of intact erythrocytes being 21.8 mmHg (normal, 24.1--26.1). Red cell glycolytic intermediates in the proband were low in general, and the rate of lactate production was low. Kinetic studies using crude hemolysate revealed a normal Km for adenosine, normal electrophoretic mobility but slightly abnormal pH curve and slightly low utilization of 2-deoxyadenosine. The ADA activity of lymphocytes was nearly normal.

  18. Coenzyme Q10 protects retinal cells from apoptosis induced by radiation in vitro and in vivo

    International Nuclear Information System (INIS)

    Lulli, M.; Witort, E.; Papucci, L.; Torre, E.; Schiavone, N.; Capaccioli, S.; Dal Monte, M.

    2012-01-01

    The key pathogenetic event of many retinopathies is apoptosis of retinal cells. Our previous studies have demonstrated that Coenzyme Q10 (CoQ10) prevents apoptosis of corneal keratocytes both in vitro and in vivo, by virtue of its ability to inhibit mitochondrial depolarization, independently of its free radical scavenger role. The aim of this study was to evaluate whether CoQ10 can protect cultured retinal cells and the retinas of rats from radiation-induced apoptosis, if instilled as eye drops in the cornea. In vitro experiments were carried out on cultured ARPE-19 or retinal ganglion cells (RGC)-5 cells pretreated with CoQ10 before eliciting apoptosis by ultraviolet (UV)- and γ-radiation, chemical hypoxia (Antimycin A) and serum starvation. Cell viability was evaluated by light microscopy and fluorescence activated cell sorting analysis. Apoptotic events were scored by time-lapse videomicroscopy. Mitochondrial permeability transition was evaluated by JC-1. The anti-apoptotic effectiveness of CoQ10 in retina was also evaluated by an in situ end-labeling assay in Wistar albino rats treated with CoQ10 eye drops prior to UV irradiation of the eye. CoQ10 substantially increased cell viability and lowered retinal cell apoptosis in response both to UV- and γ-radiation and to chemical hypoxia or serum starvation by inhibiting mitochondrion depolarization. In the rat, CoQ10, even when applied as eye drops on the cornea, protected all retina layers from ultraviolet radiation (UVR)-induced apoptosis. The ability of CoQ10 to protect retinal cells from radiation-induced apoptosis following its instillation on the cornea suggests the possibility for CoQ10 eye drops to become a future therapeutic countermeasure for radiation-induced retinal lesions. (author)

  19. Melanopsin retinal ganglion cells are resistant to neurodegeneration in mitochondrial optic neuropathies

    DEFF Research Database (Denmark)

    La Morgia, C; Ross-Cisneros, F.N.; Sadun, A.A.

    2010-01-01

    Mitochondrial optic neuropathies, that is, Leber hereditary optic neuropathy and dominant optic atrophy, selectively affect retinal ganglion cells, causing visual loss with relatively preserved pupillary light reflex. The mammalian eye contains a light detection system based on a subset of retinal...... ganglion cells containing the photopigment melanopsin. These cells give origin to the retinohypothalamic tract and support the non-image-forming visual functions of the eye, which include the photoentrainment of circadian rhythms, light-induced suppression of melatonin secretion and pupillary light reflex...... subjects as in controls, indicating that the retinohypothalamic tract is sufficiently preserved to drive light information detected by melanopsin retinal ganglion cells. We then investigated the histology of post-mortem eyes from two patients with Leber hereditary optic neuropathy and one case...

  20. Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

    Science.gov (United States)

    Davari, Maliheh; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

    2013-01-01

    Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

  1. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19...

  2. Potential of Gene Editing and Induced Pluripotent Stem Cells (iPSCs) in Treatment of Retinal Diseases.

    Science.gov (United States)

    Chuang, Katherine; Fields, Mark A; Del Priore, Lucian V

    2017-12-01

    The advent of gene editing has introduced the ability to make changes to the genome of cells, thus allowing for correction of genetic mutations in patients with monogenic diseases. Retinal diseases are particularly suitable for the application of this new technology because many retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA), are monogenic. Moreover, gene delivery techniques such as the use of adeno-associated virus (AAV) vectors have been optimized for intraocular use, and phase III trials are well underway to treat LCA, a severe form of inherited retinal degeneration, with gene therapy. This review focuses on the use of gene editing techniques and another relatively recent advent, induced pluripotent stem cells (iPSCs), and their potential for the study and treatment of retinal disease. Investment in these technologies, including overcoming challenges such as off-target mutations and low transplanted cell integration, may allow for future treatment of many debilitating inherited retinal diseases.

  3. Melanopsin-expressing retinal ganglion cells are resistant to cell injury, but not always.

    Science.gov (United States)

    Georg, Birgitte; Ghelli, Anna; Giordano, Carla; Ross-Cisneros, Fred N; Sadun, Alfredo A; Carelli, Valerio; Hannibal, Jens; La Morgia, Chiara

    2017-09-01

    Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs deputed to non-image forming functions of the eye such as synchronization of circadian rhythms to light-dark cycle. These cells are characterized by unique electrophysiological, anatomical and biochemical properties and are usually more resistant than conventional RGCs to different insults, such as axotomy and different paradigms of stress. We also demonstrated that these cells are relatively spared compared to conventional RGCs in mitochondrial optic neuropathies (Leber's hereditary optic neuropathy and Dominant Optic Atrophy). However, these cells are affected in other neurodegenerative conditions, such as glaucoma and Alzheimer's disease. We here review the current evidences that may underlie this dichotomy. We also present our unpublished data on cell experiments demonstrating that melanopsin itself does not explain the robustness of these cells and some preliminary data on immunohistochemical assessment of mitochondria in mRGCs. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  4. Induced Retro-Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA.

    Science.gov (United States)

    Nazemroaya, Fatemeh; Soheili, Zahra-Soheila; Samiei, Shahram; Deezagi, Abdolkhalegh; Ahmadieh, Hamid; Davari, Malihe; Heidari, Razeih; Bagheri, Abouzar; Darvishalipour-Astaneh, Shamila

    2017-10-01

    Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells.

    Directory of Open Access Journals (Sweden)

    Alba Galan

    Full Text Available Retinal ganglion cells (RGCs are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.

  6. Retinal Ganglion Cell Distribution and Spatial Resolving Power in Deep-Sea Lanternfishes (Myctophidae)

    KAUST Repository

    De Busserolles, Fanny; Marshall, N. Justin; Collin, Shaun P.

    2014-01-01

    Topographic analyses of retinal ganglion cell density are very useful in providing information about the visual ecology of a species by identifying areas of acute vision within the visual field (i.e. areas of high cell density). In this study, we

  7. Elucidating the role of AII amacrine cells in glutamatergic retinal waves.

    Science.gov (United States)

    Firl, Alana; Ke, Jiang-Bin; Zhang, Lei; Fuerst, Peter G; Singer, Joshua H; Feller, Marla B

    2015-01-28

    Spontaneous retinal activity mediated by glutamatergic neurotransmission-so-called "Stage 3" retinal waves-drives anti-correlated spiking in ON and OFF RGCs during the second week of postnatal development of the mouse. In the mature retina, the activity of a retinal interneuron called the AII amacrine cell is responsible for anti-correlated spiking in ON and OFF α-RGCs. In mature AIIs, membrane hyperpolarization elicits bursting behavior. Here, we postulated that bursting in AIIs underlies the initiation of glutamatergic retinal waves. We tested this hypothesis by using two-photon calcium imaging of spontaneous activity in populations of retinal neurons and by making whole-cell recordings from individual AIIs and α-RGCs in in vitro preparations of mouse retina. We found that AIIs participated in retinal waves, and that their activity was correlated with that of ON α-RGCs and anti-correlated with that of OFF α-RGCs. Though immature AIIs lacked the complement of membrane conductances necessary to generate bursting, pharmacological activation of the M-current, a conductance that modulates bursting in mature AIIs, blocked retinal wave generation. Interestingly, blockade of the pacemaker conductance Ih, a conductance absent in AIIs but present in both ON and OFF cone bipolar cells, caused a dramatic loss of spatial coherence of spontaneous activity. We conclude that during glutamatergic waves, AIIs act to coordinate and propagate activity generated by BCs rather than to initiate spontaneous activity. Copyright © 2015 the authors 0270-6474/15/351675-12$15.00/0.

  8. Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast...... cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p......-trifluorometroxyphenylnydrazone), which are known to block the energy-dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187-induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5...

  9. Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

    Science.gov (United States)

    Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Zhang, Wei; Lanza, Robert; Lu, Shi-Jiang; Jonas, Jost B; Xu, Liang

    2017-09-01

    To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 μl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 μl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. By integrating into

  10. SirT1—A Sensor for Monitoring Self-Renewal and Aging Process in Retinal Stem Cells

    Directory of Open Access Journals (Sweden)

    Chi-Hsien Peng

    2010-06-01

    Full Text Available Retinal stem cells bear potency of proliferation, self-renewal, and differentiation into many retinal cells. Utilizing appropriate sensors one can effectively detect the self-renewal and aging process abilities. Silencing information regulator (SirT1, a member of the sirtuin family, is a NAD-dependent histone deacetylase and an essential mediator for longevity in normal cells by calorie restriction. We firstly investigate the SirT1 mRNA expression in retinal stem cells from rats and 19 human eyes of different ages. Results revealed that SirT1 expression was significantly decreased in in vivo aged eyes, associated with poor self-renewal abilities. Additionally, SirT1 mRNA levels were dose-dependently increased in resveratrol- treated retinal stem cells. The expression of SirT1 on oxidative stress-induced damage was significantly decreased, negatively correlated with the level of intracellular reactive oxygen species production. Treatment with resveratrol could effectively further reduce oxidative stress induced by H2O2 treatment in retinal stem cells. Importantly, the anti-oxidant effects of resveratrol in H2O2-treated retinal stem cells were significantly abolished by knockdown of SirT1 expression (sh-SirT1. SirT1 expression provides a feasible sensor in assessing self-renewal and aging process in retinal stem cells. Resveratrol can prevent reactive oxygen species-induced damages via increased retinal SirT1 expression.

  11. Retinal vessel diameters decrease with macular ganglion cell layer thickness in autosomal dominant optic atrophy and in healthy subjects

    DEFF Research Database (Denmark)

    Rönnbäck, Cecilia; Grønskov, Karen; Larsen, Michael

    2014-01-01

    diameters (central retinal artery equivalent, CRAE, and central retinal vein equivalent, CRVE). Statistical analysis was corrected for age, gender, spherical equivalent refraction, axial length and mean arterial blood pressure (MABP) in a mixed model analysis. RESULTS: Retinal arteries and veins were...... ganglion cell-inner plexiform layer (GC-IPL) thickness (p = 0.0017 and p = 0.0057, respectively). CONCLUSION: Narrow retinal arteries and veins were associated not only with the severity of ADOA but with ganglion cell volume in patients with ADOA and in healthy subjects. This suggests that narrow vessels...

  12. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

    Science.gov (United States)

    Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

    2011-05-01

    Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

  13. Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation.

    Science.gov (United States)

    Vugler, Anthony; Carr, Amanda-Jayne; Lawrence, Jean; Chen, Li Li; Burrell, Kelly; Wright, Andrew; Lundh, Peter; Semo, Ma'ayan; Ahmado, Ahmad; Gias, Carlos; da Cruz, Lyndon; Moore, Harry; Andrews, Peter; Walsh, James; Coffey, Peter

    2008-12-01

    Healthy Retinal Pigment Epithelium (RPE) cells are required for proper visual function and the phenomenon of RPE derivation from Human Embryonic Stem Cells (HESC) holds great potential for the treatment of retinal diseases. However, little is known about formation, expansion and expression profile of RPE-like cells derived from HESC (HESC-RPE). By studying the genesis of pigmented foci we identified OTX1/2-positive cell types as potential HESC-RPE precursors. When pigmented foci were excised from culture, HESC-RPE expanded to form extensive monolayers, with pigmented cells at the leading edge assuming a precursor role: de-pigmenting, proliferating, expressing keratin 8 and subsequently re-differentiating. As they expanded and differentiated in vitro, HESC-RPE expressed markers of both developing and mature RPE cells which included OTX1/2, Pax6, PMEL17 and at low levels, RPE65. In vitro, without signals from a developing retinal environment, HESC-RPE could produce regular, polarised monolayers with developmentally important apical and basal features. Following transplantation of HESC-RPE into the degenerating retinal environment of Royal College of Surgeons (RCS) dystrophic rats, the cells survived in the subretinal space, where they maintained low levels of RPE65 expression and remained out of the cell cycle. The HESC-RPE cells responded to the in vivo environment by downregulating Pax6, while maintaining expression of other markers. The presence of rhodopsin-positive material within grafted HESC-RPE indicates that in the future, homogenous transplants of this cell type may be capable of supporting visual function following retinal dystrophy.

  14. Adenosine as a signaling molecule in the retina: biochemical and developmental aspects

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    ROBERTO PAES-DE-CARVALHO

    2002-09-01

    Full Text Available The nucleoside adenosine plays an important role as a neurotransmitter or neuromodulator in the central nervous system, including the retina. In the present paper we review compelling evidence showing that adenosine is a signaling molecule in the developing retina. In the chick retina, adenosine transporters are present since early stages of development before the appearance of adenosine A1 receptors modulating dopamine-dependent adenylate cyclase activity or A2 receptors that directly activate the enzyme. Experiments using retinal cell cultures revealed that adenosine is taken up by specific cell populations that when stimulated by depolarization or neurotransmitters such as dopamine or glutamate, release the nucleoside through calcium-dependent transporter-mediated mechanisms. The presence of adenosine in the extracellular medium and the long-term activation of adenosine receptors is able to regulate the survival of retinal neurons and blocks glutamate excitoxicity. Thus, adenosine besides working as a neurotransmitter or neuromodulator in the mature retina, is considered as an important signaling molecule during retinal development having important functions such as regulation of neuronal survival and differentiation.O nucleosídeo adenosina apresenta um importante papel como neurotransmissor ou neuromodulador no sistema nervoso central, inclusive na retina. Neste artigo apresentamos uma revisão das evidências que mostram que a adenosina é uma molécula sinalizadora na retina em desenvolvimento. Na retina de pinto, transportadores de adenosina estão presentes desde estágios precoces do desenvolvimento, antes do aparecimento dos receptores A1 que modulam a atividade adenilato ciclase dependente de dopamina ou dos receptores A2 que ativam diretamente a enzima. Experimentos usando culturas de células de retina revelaram que a adenosina é captada por populações celulares específicas que, quando estimuladas por despolarização ou por

  15. Adenosine A2B receptor: from cell biology to human diseases

    Science.gov (United States)

    Sun, Ying; Huang, Pingbo

    2016-08-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR’s functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases.

  16. Method to investigate temporal dynamics of ganglion and other retinal cells in the living human eye

    Science.gov (United States)

    Kurokawa, Kazuhiro; Liu, Zhuolin; Crowell, James; Zhang, Furu; Miller, Donald T.

    2018-02-01

    The inner retina is critical for visual processing, but much remains unknown about its neural circuitry and vulnerability to disease. A major bottleneck has been our inability to observe the structure and function of the cells composing these retinal layers in the living human eye. Here, we present a noninvasive method to observe both structural and functional information. Adaptive optics optical coherence tomography (AO-OCT) is used to resolve the inner retinal cells in all three dimensions and novel post processing algorithms are applied to extract structure and physiology down to the cellular level. AO-OCT captured the 3D mosaic of individual ganglion cell somas, retinal nerve fiber bundles of micron caliber, and microglial cells, all in exquisite detail. Time correlation analysis of the AO-OCT videos revealed notable temporal differences between the principal layers of the inner retina. The GC layer was more dynamic than the nerve fiber and inner plexiform layers. At the cellular level, we applied a customized correlation method to individual GCL somas, and found a mean time constant of activity of 0.57 s and spread of +/-0.1 s suggesting a range of physiological dynamics even in the same cell type. Extending our method to slower dynamics (from minutes to one year), time-lapse imaging and temporal speckle contrast revealed appendage and soma motion of resting microglial cells at the retinal surface.

  17. Development and degeneration of cone bipolar cells are independent of cone photoreceptors in a mouse model of retinitis pigmentosa.

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    Miao Chen

    Full Text Available Retinal photoreceptors die during retinal synaptogenesis in a portion of retinal degeneration. Whether cone bipolar cells establish regular retinal mosaics and mature morphologies, and resist degeneration are not completely understood. To explore these issues, we backcrossed a transgenic mouse expressing enhanced green fluorescent protein (EGFP in one subset of cone bipolar cells (type 7 into rd1 mice, a classic mouse model of retinal degeneration, to examine the development and survival of cone bipolar cells in a background of retinal degeneration. Our data revealed that both the development and degeneration of cone bipolar cells are independent of the normal activity of cone photoreceptors. We found that type 7 cone bipolar cells achieved a uniform tiling of the retinal surface and developed normal dendritic and axonal arbors without the influence of cone photoreceptor innervation. On the other hand, degeneration of type 7 cone bipolar cells, contrary to our belief of central-to-peripheral progression, was spatially uniform across the retina independent of the spatiotemporal pattern of cone degeneration. The results have important implications for the design of more effective therapies to restore vision in retinal degeneration.

  18. Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

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    Kati Juuti-Uusitalo

    Full Text Available Retinal pigment epithelial (RPE cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP, the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC and RPE derived from the hESC (hESC-RPE. Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

  19. Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration.

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    S N Leow

    Full Text Available To investigate the safety and efficacy of subretinal injection of human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs on retinal structure and function in Royal College of Surgeons (RCS rats.RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8 and placebo control group (n = 8. In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT. Retinal function was assessed by electroretinography (ERG 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies.No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells.Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.

  20. Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration.

    Science.gov (United States)

    Leow, S N; Luu, Chi D; Hairul Nizam, M H; Mok, P L; Ruhaslizan, R; Wong, H S; Wan Abdul Halim, Wan Haslina; Ng, M H; Ruszymah, B H I; Chowdhury, S R; Bastion, M L C; Then, K Y

    2015-01-01

    To investigate the safety and efficacy of subretinal injection of human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats. RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies. No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells. Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.

  1. Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

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    Griffiths T Daniel

    2009-05-01

    Full Text Available Abstract Background The Retinal Pigmented Epithelium (RPE is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD, Retinitis Pigmentosa (RP, Fundus Flavimaculatus (FFM, Best's disease and Age-related Macular Degeneration (AMD. We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. Results The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. Conclusion The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal

  2. Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

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    Álcio Coutinho de Paula

    2015-04-01

    Full Text Available Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR was used to evaluate expression of apoptosis-inducing caspases (8 and 3. Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10% or necrotic (<1% activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

  3. Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells.

    Science.gov (United States)

    Yuan, Jing; Yu, Jian-Xiong

    2016-05-01

    Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

  4. Cell-type specific roles for PTEN in establishing a functional retinal architecture.

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    Robert Cantrup

    Full Text Available The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and

  5. A quest for the best retinal pigment epithelium (stem) cell replacement therapy

    NARCIS (Netherlands)

    Bennis, A.

    2017-01-01

    In this thesis the focus of study lies on the retinal pigment epithelium (RPE), a monolayer of pigmented cells that lie underneath the photoreceptors (PR). The PR are specialized type of neurons that are capable of converting the incoming light into electric and neurochemical signals to the brain.

  6. Loss of Melanopsin-Expressing Retinal Ganglion Cells in Patients With Diabetic Retinopathy

    DEFF Research Database (Denmark)

    Obara, Elisabeth Anne; Hannibal, Jens; Heegaard, Steffen

    2017-01-01

    Purpose: Photo-entrainment of the circadian clock is mediated by melanopsin-expressing retinal ganglion cells (mRGCs) located in the retina. Patients suffering from diabetic retinopathy (DR) show impairment of light regulated circadian activity such as sleep disorders, altered blood pressure...

  7. An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

    Science.gov (United States)

    Tang, Zhongshu; Zhang, Shuihua; Lee, Chunsik; Kumar, Anil; Arjunan, Pachiappan; Li, Yang; Zhang, Fan; Li, Xuri

    2011-01-01

    Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness. The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy. Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result. PMID:21540827

  8. Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

    Science.gov (United States)

    Ströh, Sebastian; Puller, Christian; Swirski, Sebastian; Hölzel, Maj-Britt; van der Linde, Lea I S; Segelken, Jasmin; Schultz, Konrad; Block, Christoph; Monyer, Hannah; Willecke, Klaus; Weiler, Reto; Greschner, Martin; Janssen-Bienhold, Ulrike; Dedek, Karin

    2018-02-21

    In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light

  9. Spontaneous oscillatory rhythms in the degenerating mouse retina modulate retinal ganglion cell responses to electrical stimulation

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    Yong Sook eGoo

    2016-01-01

    Full Text Available Characterization of the electrical activity of the retina in the animal models of retinal degeneration has been carried out in part to understand the progression of retinal degenerative diseases like age-related macular degeneration (AMD and retinitis pigmentosa (RP, but also to determine optimum stimulus paradigms for use with retinal prosthetic devices. The models most studied in this regard have been the two lines of mice deficient in the β-subunit of phosphodiesterase (rd1 and rd10 mice, where the degenerating retinas exhibit characteristic spontaneous hyperactivity and oscillatory local field potentials (LFPs. Additionally, there is a robust ~10 Hz rhythmic burst of retinal ganglion cell (RGC spikes on the trough of the oscillatory LFP. In rd1 mice, the rhythmic burst of RGC spikes is always phase-locked with the oscillatory LFP and this phase-locking property is preserved regardless of postnatal ages. However, in rd10 mice, the frequency of the oscillatory rhythm changes according to postnatal age, suggesting that this rhythm might be a marker of the stage of degeneration. Furthermore when a biphasic current stimulus is applied to rd10 mice degenerate retina, distinct RGC response patterns that correlate with the stage of degeneration emerge. This review also considers the significance of these response properties.

  10. Protein kinase A mediates adenosine A2a receptor modulation of neurotransmitter release via synapsin I phosphorylation in cultured cells from medulla oblongata.

    Science.gov (United States)

    Matsumoto, Joao Paulo Pontes; Almeida, Marina Gomes; Castilho-Martins, Emerson Augusto; Costa, Maisa Aparecida; Fior-Chadi, Debora Rejane

    2014-08-01

    Synaptic transmission is an essential process for neuron physiology. Such process is enabled in part due to modulation of neurotransmitter release. Adenosine is a synaptic modulator of neurotransmitter release in the Central Nervous System, including neurons of medulla oblongata, where several nuclei are involved with neurovegetative reflexes. Adenosine modulates different neurotransmitter systems in medulla oblongata, specially glutamate and noradrenaline in the nucleus tractussolitarii, which are involved in hypotensive responses. However, the intracellular mechanisms involved in this modulation remain unknown. The adenosine A2a receptor modulates neurotransmitter release by activating two cAMP protein effectors, the protein kinase A and the exchange protein activated by cAMP. Therefore, an in vitro approach (cultured cells) was carried out to evaluate modulation of neurotransmission by adenosine A2a receptor and the signaling intracellular pathway involved. Results show that the adenosine A2a receptor agonist, CGS 21680, increases neurotransmitter release, in particular, glutamate and noradrenaline and such response is mediated by protein kinase A activation, which in turn increased synapsin I phosphorylation. This suggests a mechanism of A2aR modulation of neurotransmitter release in cultured cells from medulla oblongata of Wistar rats and suggest that protein kinase A mediates this modulation of neurotransmitter release via synapsin I phosphorylation. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  11. Retinal dendritic cell recruitment, but not function, was inhibited in MyD88 and TRIF deficient mice

    OpenAIRE

    Heuss, Neal D; Pierson, Mark J; Montaniel, Kim Ramil C; McPherson, Scott W; Lehmann, Ute; Hussong, Stacy A; Ferrington, Deborah A; Low, Walter C; Gregerson, Dale S

    2014-01-01

    Background Immune system cells are known to affect loss of neurons due to injury or disease. Recruitment of immune cells following retinal/CNS injury has been shown to affect the health and survival of neurons in several models. We detected close, physical contact between dendritic cells and retinal ganglion cells following an optic nerve crush, and sought to understand the underlying mechanisms. Methods CD11c-DTR/GFP mice producing a chimeric protein of diphtheria toxin receptor (DTR) and GF...

  12. Exposure of Human Lung Cancer Cells to 8-Chloro-Adenosine Induces G2/M Arrest and Mitotic Catastrophe

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    Hong-Yu Zhang

    2004-11-01

    Full Text Available 8-Chloro-adenosine (8-CI-Ado is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt and H1299 (p53-depleted to 8-CI-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-CI-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.

  13. Rapid and coordinated processing of global motion images by local clusters of retinal ganglion cells.

    Science.gov (United States)

    Matsumoto, Akihiro; Tachibana, Masao

    2017-01-01

    Even when the body is stationary, the whole retinal image is always in motion by fixational eye movements and saccades that move the eye between fixation points. Accumulating evidence indicates that the brain is equipped with specific mechanisms for compensating for the global motion induced by these eye movements. However, it is not yet fully understood how the retina processes global motion images during eye movements. Here we show that global motion images evoke novel coordinated firing in retinal ganglion cells (GCs). We simultaneously recorded the firing of GCs in the goldfish isolated retina using a multi-electrode array, and classified each GC based on the temporal profile of its receptive field (RF). A moving target that accompanied the global motion (simulating a saccade following a period of fixational eye movements) modulated the RF properties and evoked synchronized and correlated firing among local clusters of the specific GCs. Our findings provide a novel concept for retinal information processing during eye movements.

  14. The stimulatory adenosine receptor ADORA2B regulates serotonin (5-HT synthesis and release in oxygen-depleted EC cells in inflammatory bowel disease.

    Directory of Open Access Journals (Sweden)

    Rikard Dammen

    Full Text Available We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion.The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2 was compared to NECA (adenosine agonist, MRS1754 (ADORA2B receptor antagonist and SCH442146 (ADORA2A antagonist on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo.HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (~90% of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5'-ASA treatment was confirmed in a TNBS-model.Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

  15. Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

    Directory of Open Access Journals (Sweden)

    Jasmin Balmer

    2015-07-01

    Full Text Available Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3 both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg. Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE cells, primary retinal cells, and the cone photoreceptor (PRC cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1 was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

  16. Translating induced pluripotent stem cells from bench to bedside: application to retinal diseases.

    Science.gov (United States)

    Cramer, Alona O; MacLaren, Robert E

    2013-04-01

    Induced pluripotent stem cells (iPSc) are a scientific and medical frontier. Application of reprogrammed somatic cells for clinical trials is in its dawn period; advances in research with animal and human iPSc are paving the way for retinal therapies with the ongoing development of safe animal cell transplantation studies and characterization of patient- specific and disease-specific human iPSc. The retina is an optimal model for investigation of neural regeneration; amongst other advantageous attributes, it is the most accessible part of the CNS for surgery and outcome monitoring. A recent clinical trial showing a degree of visual restoration via a subretinal electronic prosthesis implies that even a severely degenerate retina may have the capacity for repair after cell replacement through potential plasticity of the visual system. Successful differentiation of neural retina from iPSc and the recent generation of an optic cup from human ESc invitro increase the feasibility of generating an expandable and clinically suitable source of cells for human clinical trials. In this review we shall present recent studies that have propelled the field forward and discuss challenges in utilizing iPS cell derived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment for patients suffering from genetic retinal disease.

  17. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    Science.gov (United States)

    2016-03-01

    diabetic retinopathy . Life Sci. 2013 Jul 30;93(2-3):78-88. doi: 10.1016/j.lfs.2013.05.024. Epub 2013 Jun 12.PMID:23770229 7 AIMS: This study was...undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling...reported recently that adenosine kinase upregulated in retinal tissue of diabetic retinopathy (Elsherbiny et al., 2013). Adenosine kinase (ADK) converts

  18. Pten Regulates Retinal Amacrine Cell Number by Modulating Akt, Tgfβ, and Erk Signaling.

    Science.gov (United States)

    Tachibana, Nobuhiko; Cantrup, Robert; Dixit, Rajiv; Touahri, Yacine; Kaushik, Gaurav; Zinyk, Dawn; Daftarian, Narsis; Biernaskie, Jeff; McFarlane, Sarah; Schuurmans, Carol

    2016-09-07

    All tissues are genetically programmed to acquire an optimal size that is defined by total cell number and individual cellular dimensions. The retina contains stereotyped proportions of one glial and six neuronal cell types that are generated in overlapping waves. How multipotent retinal progenitors know when to switch from making one cell type to the next so that appropriate numbers of each cell type are generated is poorly understood. Pten is a phosphatase that controls progenitor cell proliferation and differentiation in several lineages. Here, using a conditional loss-of-function strategy, we found that Pten regulates retinal cell division and is required to produce the full complement of rod photoreceptors and amacrine cells in mouse. We focused on amacrine cell number control, identifying three downstream Pten effector pathways. First, phosphoinositide 3-kinase/Akt signaling is hyperactivated in Pten conditional knock-out (cKO) retinas, and misexpression of constitutively active Akt (Akt-CA) in retinal explants phenocopies the reduction in amacrine cell production observed in Pten cKOs. Second, Akt-CA activates Tgfβ signaling in retinal explants, which is a negative feedback pathway for amacrine cell production. Accordingly, Tgfβ signaling is elevated in Pten cKO retinas, and epistatic analyses placed Pten downstream of TgfβRII in amacrine cell number control. Finally, Pten regulates Raf/Mek/Erk signaling levels to promote the differentiation of all amacrine cell subtypes, which are each reduced in number in Pten cKOs. Pten is thus a positive regulator of amacrine cell production, acting via multiple downstream pathways, highlighting its diverse actions as a mediator of cell number control. Despite the importance of size for optimal organ function, how individual cell types are generated in correct proportions is poorly understood. There are several ways to control cell number, including readouts of organ function (e.g., secreted hormones reach functional

  19. Adenosine 5′-Triphosphate Metabolism in Red Blood Cells as a Potential Biomarker for Post-Exercise Hypotension and a Drug Target for Cardiovascular Protection

    Directory of Open Access Journals (Sweden)

    Pollen K. Yeung

    2018-05-01

    Full Text Available The importance of adenosine and ATP in regulating many biological functions has long been recognized, especially for their effects on the cardiovascular system, which may be used for management of hypertension and cardiometabolic diseases. In response to ischemia and cardiovascular injury, ATP is broken down to release adenosine. The effect of adenosine is very short lived because it is rapidly taken up by erythrocytes (RBCs, myocardial and endothelial cells, and also rapidly catabolized to oxypurine metabolites. Intracellular adenosine is phosphorylated back to adenine nucleotides via a salvage pathway. Extracellular and intracellular ATP is broken down rapidly to ADP and AMP, and finally to adenosine by 5′-nucleotidase. These metabolic events are known to occur in the myocardium, endothelium as well as in RBCs. Exercise has been shown to increase metabolism of ATP in RBCs, which may be an important mechanism for post-exercise hypotension and cardiovascular protection. The post-exercise effect was greater in hypertensive than in normotensive rats. The review summarizes current evidence in support of ATP metabolism in the RBC as a potential surrogate biomarker for cardiovascular protection and toxicities. It also discusses the opportunities, challenges, and obstacles of exploiting ATP metabolism in RBCs as a target for drug development and precision medicine.

  20. The Retinal Pigment Epithelium: a Convenient Source of New Photoreceptor cells?

    Directory of Open Access Journals (Sweden)

    Shu-Zhen Wang

    2014-01-01

    Full Text Available Recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. Current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (iPS cells to generate transplantable cells. Nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. Here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (RPE, to produce photoreceptor cells in situ in the eye. Unlike the neural retina, the RPE in adult mammals maintains cell proliferation capability. Furthermore, progeny cells from RPE proliferation may differentiate into cells other than RPE. The combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro-photoreceptor activity towards photoreceptor production. Studies using embryonic chick and transgenic mouse showed that indeed photoreceptor-like cells were produced in culture and in vivo in the eye using genedirected reprogramming of RPE cells, supporting the feasibility of using the RPE as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.

  1. Perspectives of Stem Cell-Based Therapy for Age-Related Retinal Degenerative Diseases

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Heřmánková, Barbora; Kössl, Jan

    2017-01-01

    Roč. 26, č. 9 (2017), s. 1538-1541 ISSN 0963-6897 R&D Projects: GA ČR(CZ) GA17-04800S; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : age-related retinal degenerative diseases * mesenchymal stem cells * stem cell therapy Subject RIV: FF - HEENT, Dentistry OBOR OECD: Ophthalmology Impact factor: 3.006, year: 2016

  2. Retinal input to efferent target amacrine cells in the avian retina

    Science.gov (United States)

    Lindstrom, Sarah H.; Azizi, Nason; Weller, Cynthia; Wilson, Martin

    2012-01-01

    The bird visual system includes a substantial projection, of unknown function, from a midbrain nucleus to the contralateral retina. Every centrifugal, or efferent, neuron originating in the midbrain nucleus makes synaptic contact with the soma of a single, unique amacrine cell, the target cell (TC). By labeling efferent neurons in the midbrain we have been able to identify their terminals in retinal slices and make patch clamp recordings from TCs. TCs generate Na+ based action potentials triggered by spontaneous EPSPs originating from multiple classes of presynaptic neurons. Exogenously applied glutamate elicited inward currents having the mixed pharmacology of NMDA, kainate and inward rectifying AMPA receptors. Exogenously applied GABA elicited currents entirely suppressed by GABAzine, and therefore mediated by GABAA receptors. Immunohistochemistry showed the vesicular glutamate transporter, vGluT2, to be present in the characteristic synaptic boutons of efferent terminals, whereas the GABA synthetic enzyme, GAD, was present in much smaller processes of intrinsic retinal neurons. Extracellular recording showed that exogenously applied GABA was directly excitatory to TCs and, consistent with this, NKCC, the Cl− transporter often associated with excitatory GABAergic synapses, was identified in TCs by antibody staining. The presence of excitatory retinal input to TCs implies that TCs are not merely slaves to their midbrain input; instead, their output reflects local retinal activity and descending input from the midbrain. PMID:20650017

  3. The ciliary margin zone of the mammalian retina generates retinal ganglion cells

    Science.gov (United States)

    Marcucci, Florencia; Murcia-Belmonte, Veronica; Coca, Yaiza; Ferreiro-Galve, Susana; Wang, Qing; Kuwajima, Takaaki; Khalid, Sania; Ross, M. Elizabeth; Herrera, Eloisa; Mason, Carol

    2016-01-01

    Summary The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live-imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. As Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2−/− mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity. PMID:28009286

  4. Diabetes Accelerates Retinal neuronal cell Death in A Mouse Model of endogenous Hyperhomocysteinemia

    Directory of Open Access Journals (Sweden)

    Preethi S. Ganapathy

    2009-01-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase ( cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs +/– mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs +/– and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs +/– non-DB cbs +/– DB cbs +/+ ; non-DB cbs +/+ . One group of diabetic cbs +/– mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs +/– had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs +/– and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 μm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  5. Diabetes Accelerates Retinal Neuronal Cell Death In A Mouse Model of Endogenous Hyperhomocysteinemia

    Directory of Open Access Journals (Sweden)

    Preethi S. Ganapathy

    2009-07-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase (cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs+/- mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs+/- and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs+/-; non-DB cbs+/-; DB cbs+/+; non-DB cbs+/+. One group of diabetic cbs+/- mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs+/- had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs+/- and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 µm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  6. Crocin prevents retinal ischaemia/reperfusion injury-induced apoptosis in retinal ganglion cells through the PI3K/AKT signalling pathway.

    Science.gov (United States)

    Qi, Yun; Chen, Li; Zhang, Lei; Liu, Wen-Bo; Chen, Xiao-Yan; Yang, Xin-Guang

    2013-02-01

    Crocin is a pharmacologically active component of Crocus sativus L. (saffron) and has been reported to be useful in the treatment of neuronal damage. In the present study, we investigated the neuroprotective effect of crocin on retinal ganglion cells (RGCs) after retinal ischaemia/reperfusion (IR) injury, and our results show that crocin acts through the PI3K/AKT signalling pathway. Retinal IR injury was induced by raising the intraocular pressure of Sprague-Dawley rats to 110 mmHg for 60 min. The neuroprotective effect of crocin was determined by quantifying the surviving RGCs and apoptotic RGCs following IR injury by means of retrograde labelling and TUNEL staining, respectively. The phosphorylated AKT protein level was determined by western blot and immunohistochemical analysis. To determine the extent to which the PI3K/AKT pathway contributes to the neuroprotective effect of crocin, experiments were also performed using the PI3K inhibitor LY294002. Compared with the IR + vehicle group, crocin (50 mg/kg) treatment enhanced RGC survival by approximately 36% and decreased RGC apoptosis by 44% after retinal IR injury. Western blot and immunohistochemical analysis demonstrated that the PI3K/AKT pathway was activated by crocin in the ganglion cell layer after retinal IR injury. Intravitreal injection of LY294002 blocked the neuroprotective effect of crocin on IR-induced RGC death. In conclusion, crocin prevents retinal IR-induced apoptosis of RGCs by activating the PI3K/AKT signalling pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Retinal ganglion cells in the eastern newt Notophthalmus viridescens: topography, morphology, and diversity.

    Science.gov (United States)

    Pushchin, Igor I; Karetin, Yuriy A

    2009-10-20

    The topography and morphology of retinal ganglion cells (RGCs) in the eastern newt were studied. Cells were retrogradely labeled with tetramethylrhodamine-conjugated dextran amines or horseradish peroxidase and examined in retinal wholemounts. Their total number was 18,025 +/- 3,602 (mean +/- SEM). The spatial density of RGCs varied from 2,100 cells/mm(2) in the retinal periphery to 4,500 cells/mm(2) in the dorsotemporal retina. No prominent retinal specializations were found. The spatial resolution estimated from the spatial density of RGCs varied from 1.4 cycles per degree in the periphery to 1.95 cycles per degree in the region of the peak RGC density. A sample of 68 cells was camera lucida drawn and subjected to quantitative analysis. A total of 21 parameters related to RGC morphology and stratification in the retina were estimated. Partitionings obtained by using different clustering algorithms combined with automatic variable weighting and dimensionality reduction techniques were compared, and an effective solution was found by using silhouette analysis. A total of seven clusters were identified and associated with potential cell types. Kruskal-Wallis ANOVA-on-Ranks with post hoc Mann-Whitney U tests showed significant pairwise between-cluster differences in one or more of the clustering variables. The average silhouette values of the clusters were reasonably high, ranging from 0.52 to 0.79. Cells assigned to the same cluster displayed similar morphology and stratification in the retina. The advantages and limitations of the methodology adopted are discussed. The present classification is compared with known morphological and physiological RGC classifications in other salamanders.

  8. Comparison of neurosphere-like cell clusters derived from dental follicle precursor cells and retinal Müller cells

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Petersen, Jørgen; Felthaus, Oliver

    2011-01-01

    Unrelated cells such as dental follicle precursor cells (DFPCs) and retinal Müller cells (MCs) make spheres after cultivation in serum-replacement medium (SRM). Until today, the relation and molecular processes of sphere formation from different cell types remain undescribed. Thus in this study we...... compared proteomes of spheres derived from MCs and DFPCs. 73% of 676 identified proteins were similar expressed in both cell types and many of them are expressed in the brain (55%). Moreover proteins are overrepresented that are associated with pathways for neural diseases such as Huntington disease...... or Alzheimer disease. Interestingly up-regulated proteins in DFPCs are involved in the biosynthesis of glycosphingolipids. These lipids are components of gangliosides such as GD3, which is a novel neural stem cell marker. In conclusion spheres from different types of cells have highly similar proteomes...

  9. Potentiating action of propofol at GABAA receptors of retinal bipolar cells

    DEFF Research Database (Denmark)

    Yue, Lan; Xie, An; Bruzik, Karol S

    2011-01-01

    Purpose. Propofol (2,6-diisopropyl phenol), a widely used systemic anesthetic, is known to potentiate GABA(A) receptor activity in a number of CNS neurons and to produce changes in electroretinographically recorded responses of the retina. However, little is known about propofol's effects...... on specific retinal neurons. The authors investigated the action of propofol on GABA-elicited membrane current responses of retinal bipolar cells, which have both GABA(A) and GABA(C) receptors. Methods. Single, enzymatically dissociated bipolar cells obtained from rat retina were treated with propofol...... + propofol) led to a progressive increase in peak response amplitude and, at higher propofol concentrations, additional changes that included a prolonged time course of response recovery. Pre-exposure of the cell to perfusing propofol typically enhanced the rate of development of potentiation produced...

  10. Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

    Science.gov (United States)

    Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

    2018-02-28

    Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

  11. Retina tissue engineering by conjunctiva mesenchymal stem cells encapsulated in fibrin gel: Hypotheses on novel approach to retinal diseases treatment.

    Science.gov (United States)

    Soleimannejad, Mostafa; Ebrahimi-Barough, Somayeh; Nadri, Samad; Riazi-Esfahani, Mohammad; Soleimani, Masoud; Tavangar, Seyed Mohammad; Ai, Jafar

    2017-04-01

    Retinitis pigmentosa (RP) and age related macular degeneration (AMD) are two retinal diseases that progress by photoreceptor cells death. In retinal transplantation studies, stem and progenitor cells inject into the sub retinal space or vitreous and then these cells can be migrate to the site of retinal degeneration and locate in the host retina and restitute vision. Our hypothesis suggests that using human conjunctiva stem cells (as the source for increasing the number of human stem cells progenitor cells in retina dysfunction diseases) with fibrin gel and also assessing its relating in vitro (cellular and molecular processes) and in vivo (vision tests and pathology) could be a promising strategy for treatment of AMD and RP disorders. In this idea, we describe a novel approach for retina tissue engineering with differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells in fibrin gel with induction medium contain taurine. For assessment of differentiation, immunocytochemistry and real time PCR are used for the expression of Rhodopsin, RPE65, Nestin as differentiated photoreceptor cell markers in 2D and 3D culture. The results show that fibrin gel will offer a proper 3D scaffold for CJMSCs derived photoreceptor cell-like cells. Application of immune-privileged, readily available sources of adult stem cells like human conjunctiva stem cells with fibrin gel would be a promising strategy to increase the number of photoreceptor progenitor cells and promote involuntary angiogenesis needed in retina layer repair and regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Retinal Ganglion Cell Distribution and Spatial Resolving Power in Deep-Sea Lanternfishes (Myctophidae)

    KAUST Repository

    De Busserolles, Fanny

    2014-01-01

    Topographic analyses of retinal ganglion cell density are very useful in providing information about the visual ecology of a species by identifying areas of acute vision within the visual field (i.e. areas of high cell density). In this study, we investigated the neural cell distribution in the ganglion cell layer of a range of lanternfish species belonging to 10 genera. Analyses were performed on wholemounted retinas using stereology. Topographic maps were constructed of the distribution of all neurons and both ganglion and amacrine cell populations in 5 different species from Nissl-stained retinas using cytological criteria. Amacrine cell distribution was also examined immunohistochemically in 2 of the 5 species using anti-parvalbumin antibody. The distributions of both the total neuron and the amacrine cell populations were aligned in all of the species examined, showing a general increase in cell density toward the retinal periphery. However, when the ganglion cell population was topographically isolated from the amacrine cell population, which comprised up to 80% of the total neurons within the ganglion cell layer, a different distribution was revealed. Topographic maps of the true ganglion cell distribution in 18 species of lanternfishes revealed well-defined specializations in different regions of the retina. Different species possessed distinct areas of high ganglion cell density with respect to both peak density and the location and/or shape of the specialized acute zone (i.e. elongated areae ventro-temporales, areae temporales and large areae centrales). The spatial resolving power was calculated to be relatively low (varying from 1.6 to 4.4 cycles per degree), indicating that myctophids may constitute one of the less visually acute groups of deep-sea teleosts. The diversity in retinal specializations and spatial resolving power within the family is assessed in terms of possible ecological functions and evolutionary history.

  13. Effects of the Macular Carotenoid Lutein in Human Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Xiaoming Gong

    2017-12-01

    Full Text Available Retinal pigment epithelial (RPE cells are central to retinal health and homoeostasis. Oxidative stress-induced damage to the RPE occurs as part of the pathogenesis of age-related macular degeneration and neovascular retinopathies (e.g., retinopathy of prematurity, diabetic retinopathy. The xanthophyll carotenoids, lutein and zeaxanthin, are selectively taken up by the RPE, preferentially accumulated in the human macula, and transferred to photoreceptors. These macular xanthophylls protect the macula (and the broader retina via their antioxidant and photo-protective activities. This study was designed to investigate effects of various carotenoids (β-carotene, lycopene, and lutein on RPE cells subjected to either hypoxia or oxidative stress, in order to determine if there is effect specificity for macular pigment carotenoids. Using human RPE-derived ARPE-19 cells as an in vitro model, we exposed RPE cells to various concentrations of the specific carotenoids, followed by either graded hypoxia or oxidative stress using tert-butyl hydroperoxide (tBHP. The results indicate that lutein and lycopene, but not β-carotene, inhibit cell growth in undifferentiated ARPE-19 cells. Moreover, cell viability was decreased under hypoxic conditions. Pre-incubation of ARPE-19 cells with lutein or lycopene protected against tBHP-induced cell loss and cell co-exposure of lutein or lycopene with tBHP essentially neutralized tBHP-dependent cell death at tBHP concentrations up to 500 μM. Our findings indicate that lutein and lycopene inhibit the growth of human RPE cells and protect the RPE against oxidative stress-induced cell loss. These findings contribute to the understanding of the protective mechanisms attributable to retinal xanthophylls in eye health and retinopathies.

  14. Spatially and Temporally Regulated NRF2 Gene Therapy Using Mcp-1 Promoter in Retinal Ganglion Cell Injury

    Directory of Open Access Journals (Sweden)

    Kosuke Fujita

    2017-06-01

    Full Text Available Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter-driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

  15. Therapeutic Effect of Bone Marrow Mesenchymal Stem Cells on Laser-Induced Retinal Injury in Mice

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    Yuanfeng Jiang

    2014-05-01

    Full Text Available Stem cell therapy has shown encouraging results for neurodegenerative diseases. The retina provides a convenient locus to investigate stem cell functions and distribution in the nervous system. In the current study, we investigated the therapeutic potential of bone marrow mesenchymal stem cells (MSCs by systemic transplantation in a laser-induced retinal injury model. MSCs from C57BL/6 mice labeled with green fluorescent protein (GFP were injected via the tail vein into mice after laser photocoagulation. We found that the average diameters of laser spots and retinal cell apoptosis were decreased in the MSC-treated group. Interestingly, GFP-MSCs did not migrate to the injured retina. Further examination revealed that the mRNA expression levels of glial fibrillary acidic protein and matrix metalloproteinase-2 were lower in the injured eyes after MSC transplantation. Our results suggest that intravenously injected MSCs have the ability to inhibit retinal cell apoptosis, reduce the inflammatory response and limit the spreading of damage in the laser-injured retina of mice. Systemic MSC therapy might play a role in neuroprotection, mainly by regulation of the intraocular microenvironment.

  16. Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

    International Nuclear Information System (INIS)

    Chung, Hyewon; Yoon, Young Hee; Hwang, Jung Jin; Cho, Kyung Sook; Koh, Jae Young; Kim, June-Gone

    2009-01-01

    Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death

  17. The trophic effect of ouabain on retinal ganglion cells is mediated by IL-1β and TNF-α

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    Salles von-Held-Ventura, Juliana; Mázala-de-Oliveira, Thalita; Cândida da Rocha Oliveira, Amanda; Granja, Marcelo Gomes; Gonçalves-de-Albuquerque, Cassiano Felippe; Castro-Faria-Neto, Hugo Caire; Giestal-de-Araujo, Elizabeth

    2016-01-01

    Ouabain is a steroid hormone that binds to the enzyme Na + , K + – ATPase and stimulates different intracellular pathways controlling growth, proliferation and cell survival. IL-1β and TNF-α are pleiotropic molecules, conventionally regarded as pro-inflammatory cytokines with well-known effects in the immune system. In addition, IL-1β and TNF-α also play important roles in the nervous system including neuroprotective effects. Previous data from our group showed that ouabain treatment is able to induce an increase in retinal ganglion cell survival kept in mixed retinal cell cultures. The aim of this work was to investigate if IL-1β and TNF-α could be mediating the trophic effect of ouabain on retinal ganglion cells. Our results show that the trophic effect of ouabain on retinal ganglion cell was inhibited by either anti-IL-1β or anti-TNF-α antibodies. In agreement, IL-1β or TNF-α increased the retinal ganglion cells survival in a dose-dependent manner. Accordingly, ouabain treatment induces a temporal release of TNF-α and IL-1β from retinal cell cultures. Interestingly, TNF-α and IL-1β regulate each other intracellular levels. Our results suggest that ouabain treatment triggers the activation of TNF-α and IL-1β signaling pathways leading to an increase in retinal ganglion cell survival. - Highlights: • Pro-inflammatory cytokines regulates the ouabain effect on RGC survival. • Ouabain treatment modulates the intracellular levels of TNF-α and IL-1β. • Ouabain induces the release of TNF-α and IL-1β in retinal cell cultures.

  18. The trophic effect of ouabain on retinal ganglion cells is mediated by IL-1β and TNF-α

    Energy Technology Data Exchange (ETDEWEB)

    Salles von-Held-Ventura, Juliana; Mázala-de-Oliveira, Thalita; Cândida da Rocha Oliveira, Amanda; Granja, Marcelo Gomes [Departamento de Neurobiologia, Programa de Neurociências, Outeiro de São João Batista s/n CEP: 24020-150, Universidade Federal Fluminense, Niterói, RJ (Brazil); Gonçalves-de-Albuquerque, Cassiano Felippe; Castro-Faria-Neto, Hugo Caire [Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Departamento de Fisiologia e Farmacodinâmica, Av., no 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ (Brazil); Giestal-de-Araujo, Elizabeth, E-mail: egiestal@vm.uff.br [Departamento de Neurobiologia, Programa de Neurociências, Outeiro de São João Batista s/n CEP: 24020-150, Universidade Federal Fluminense, Niterói, RJ (Brazil)

    2016-09-09

    Ouabain is a steroid hormone that binds to the enzyme Na{sup +}, K{sup +} – ATPase and stimulates different intracellular pathways controlling growth, proliferation and cell survival. IL-1β and TNF-α are pleiotropic molecules, conventionally regarded as pro-inflammatory cytokines with well-known effects in the immune system. In addition, IL-1β and TNF-α also play important roles in the nervous system including neuroprotective effects. Previous data from our group showed that ouabain treatment is able to induce an increase in retinal ganglion cell survival kept in mixed retinal cell cultures. The aim of this work was to investigate if IL-1β and TNF-α could be mediating the trophic effect of ouabain on retinal ganglion cells. Our results show that the trophic effect of ouabain on retinal ganglion cell was inhibited by either anti-IL-1β or anti-TNF-α antibodies. In agreement, IL-1β or TNF-α increased the retinal ganglion cells survival in a dose-dependent manner. Accordingly, ouabain treatment induces a temporal release of TNF-α and IL-1β from retinal cell cultures. Interestingly, TNF-α and IL-1β regulate each other intracellular levels. Our results suggest that ouabain treatment triggers the activation of TNF-α and IL-1β signaling pathways leading to an increase in retinal ganglion cell survival. - Highlights: • Pro-inflammatory cytokines regulates the ouabain effect on RGC survival. • Ouabain treatment modulates the intracellular levels of TNF-α and IL-1β. • Ouabain induces the release of TNF-α and IL-1β in retinal cell cultures.

  19. Inhibition of A2A Adenosine Receptor Signaling in Cancer Cells Proliferation by the Novel Antagonist TP455

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    Stefania Gessi

    2017-12-01

    Full Text Available Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A1, A2A, A2B, and A3 receptor (AR subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A2AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A2AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl-N5-(2-methoxybenzyl[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455. Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The A2AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of A2AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective A2AAR agonist 2-p-(2-carboxyethylphenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680, concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the A2AAR antagonist TP455, as well as by the reference A2AAR blocker 4-(2-[7-amino-2-(2-furyl[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethylphenol (ZM241385. As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of A2AAR stimulation was induced through phospholipase C (PLC and protein kinase C-delta (PKC-δ. In addition, we evaluated, through the AlphaScreen SureFire phospho(p protein assay, the kinases enrolled by A2AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT, extracellular regulated kinases (ERK1/2, and c-Jun N-terminal kinases (JNKs. Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was

  20. Bilateral orbital infarction and retinal detachment in a previously undiagnosed sickle cell hemoglobinopathy African child

    Science.gov (United States)

    Helen, Onakpoya Oluwatoyin; Ajite, K. O.; Oyelami, O. A.; Asaleye, C. M.; Adeoye, A. O.

    2013-01-01

    Bone infarction involving the orbit in sickle cell disease is not common. Bilateral orbital infarction in a previously undiagnosed sickle cell hemoglobinopathy has not been previously reported. In this report, we present a case of an 11-year-old previously undiagnosed sickle cell disease Nigerian girl with severe acute bilateral orbital infarction and retinal detachment to highlight that hemoglobinopathy induced orbital infarction should be considered in African children with acute onset proptosis with or without previous history of sickle cell hemoglobinopathy. PMID:23901183

  1. The long noncoding RNA RNCR2 directs mouse retinal cell specification

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    Blackshaw Seth

    2010-05-01

    Full Text Available Abstract Background Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation. Results We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation. Conclusion These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.

  2. Adenosine receptors and caffeine in retinopathy of prematurity.

    Science.gov (United States)

    Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

    2017-06-01

    Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A 1 R, A 2A R, A 2B R) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays.

    Science.gov (United States)

    Johnen, Sandra; Meißner, Frank; Krug, Mario; Baltz, Thomas; Endler, Ingolf; Mokwa, Wilfried; Walter, Peter

    2016-01-01

    Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue.

  4. Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays

    Directory of Open Access Journals (Sweden)

    Sandra Johnen

    2016-01-01

    Full Text Available Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT, used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe or mixtures of iron-platinum (Fe-Pt and iron-titanium (Fe-Ti acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28 cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue.

  5. Possible mechanisms of retinal function recovery with the use of cell therapy with bone marrow-derived stem cells

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    Rubens Camargo Siqueira

    2010-10-01

    Full Text Available Bone marrow has been proposed as a potential source of stem cells for regenerative medicine. In the eye, degeneration of neural cells in the retina is a hallmark of such widespread ocular diseases as age-related macular degeneration (AMD and retinitis pigmentosa. Bone marrow is an ideal tissue for studying stem cells mainly because of its accessibility. Furthermore, there are a number of well-defined mouse models and cell surface markers that allow effective study of hematopoiesis in healthy and injured mice. Because of these characteristics and the experience of bone marrow transplantation in the treatment of hematological disease such as leukemia, bone marrow-derived stem cells have also become a major tool in regenerative medicine. Those cells may be able to restore the retina function through different mechanisms: A cellular differentiation, B paracrine effect, and C retinal pigment epithelium repair. In this review, we described these possible mechanisms of recovery of retinal function with the use of cell therapy with bone marrow-derived stem cells.

  6. New medium used in the differentiation of human pluripotent stem cells to retinal cells is comparable to fetal human eye tissue.

    Science.gov (United States)

    Wang, Xiaobing; Xiong, Kai; Lin, Cong; Lv, Lei; Chen, Jing; Xu, Chongchong; Wang, Songtao; Gu, Dandan; Zheng, Hua; Yu, Hurong; Li, Yan; Xiao, Honglei; Zhou, Guomin

    2015-06-01

    Human pluripotent stem cells (hPSCs) have the potential to differentiate along the retinal lineage. However, most induction systems are dependent on multiple small molecular compounds such as Dkk-1, Lefty-A, and retinoic acid. In the present study, we efficiently differentiated hPSCs into retinal cells using a retinal differentiation medium (RDM) without the use of small molecular compounds. This novel differentiation system recapitulates retinal morphogenesis in humans, i.e. hPSCs gradually differentiate into optic vesicle-shaped spheres, followed by optic cup-shaped spheres and, lastly, retinal progenitor cells. Furthermore, at different stages, hPSC-derived retinal cells mirror the transcription factor expression profiles seen in their counterparts during human embryogenesis. Most importantly, hinge epithelium was found between the hPSC-derived neural retina (NR) and retinal pigment epithelium (RPE). These data suggest that our culture system provides a new method for generating hPSC-derived retinal cells that, for the first time, might be used in human transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. In vitro activation of retinal cells: estimating location of stimulated cell by using a mathematical model

    Science.gov (United States)

    Ziv, Ofer R.; Rizzo, Joseph F., III; Jensen, Ralph J.

    2005-03-01

    Activation of neurons at different depths within the retina and at various eccentricities from the stimulating electrode will presumably influence the visual percepts created by a retinal prosthesis. With an electrical prosthesis, neurons will be activated in relation to the stimulating charge that impacts their cell membranes. The common model used to predict charge density is Coulomb's law, also known as the square law. We propose a modified model that can be used to predict neuronal depth that takes into account: (1) finite dimensions related to the position and size of the stimulating and return electrodes and (2) two-dimensional displacements of neurons with respect to the electrodes, two factors that are not considered in the square law model. We tested our model by using in vitro physiological threshold data that we had obtained previously for eight OFF-center brisk-transient rabbit retinal ganglion cells. For our most spatially dense threshold data (25 µm increments up to 100 µm from the cell body), our model estimated the depth of one RGC to be 76 ± 76 µm versus 87 ± 62 µm (median: SD) for the square law model, respectively. This difference was not statistically significant. For the seven other RGCs for which we had obtained threshold data up to 800 µm from the cell body, the estimate of the RGC depth (using data obtained along the X axis) was 96 ± 74 versus 20 ± 20 µm for the square law and our modified model, respectively. Although this difference was not statistically significant (Student t-test: p = 0.12), our model provided median values much closer to the estimated depth of these RGCs (Gt25 µm). This more realistic estimate of cell depth predicted by our model is not unexpected in this latter data set because of the more spatially distributed threshold data points that were evaluated. Our model has theoretical advantages over the traditional square law model under certain conditions, especially when considering neurons that are

  8. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  9. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells

    International Nuclear Information System (INIS)

    Lim, B.; Apperley, J.F.; Orkin, S.H.; Williams, D.A.

    1989-01-01

    Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. The authors have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. They report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy

  10. Effect of duration and severity of migraine on retinal nerve fiber layer, ganglion cell layer, and choroidal thickness.

    Science.gov (United States)

    Abdellatif, Mona K; Fouad, Mohamed M

    2018-03-01

    To investigate the factors in migraine that have the highest significance on retinal and choroidal layers' thickness. Ninety patients with migraine and 40 age-matched healthy participants were enrolled in this observational, cross-sectional study. After full ophthalmological examination, spectral domain-optical coherence tomography was done for all patients measuring the thickness of ganglion cell layer and retinal nerve fiber layer. Enhanced depth imaging technique was used to measure the choroidal thickness. There was significant thinning in the superior and inferior ganglion cell layers, all retinal nerve fiber layer quadrants, and all choroidal quadrants (except for the central subfield) in migraineurs compared to controls. The duration of migraine was significantly correlated with ganglion cell layer, retinal nerve fiber layer, and all choroidal quadrants, while the severity of migraine was significantly correlated with ganglion cell layer and retinal nerve fiber layer only. Multiregression analysis showed that the duration of migraine is the most important determinant factor of the superior retinal nerve fiber layer quadrant (β = -0.375, p = 0.001) and in all the choroidal quadrants (β = -0.531, -0.692, -0.503, -0.461, -0.564, respectively, p  layer quadrants (β = -0.256, -0.335, -0.308; p  = 0.036, 0.005, 0.009, respectively) and the inferior ganglion cell layer hemisphere (β = -0.377 and p = 0.001). Ganglion cell layer, retinal nerve fiber layer, and choroidal thickness are significantly thinner in patients with migraine. The severity of migraine has more significant influence in the thinning of ganglion cell layer and retinal nerve fiber layer, while the duration of the disease affected the choroidal thickness more.

  11. Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

    Science.gov (United States)

    Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

    2016-04-01

    We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

  12. Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology.

    Directory of Open Access Journals (Sweden)

    Shaomei Wang

    Full Text Available BACKGROUND: Retinitis pigmentosa (RP is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS rat, a well-established animal model for RP. METHODOLOGY/PRINCIPAL FINDINGS: Animals received syngeneic MSCs (1x10(6 cells by tail vein at an age before major photoreceptor loss. PRINCIPAL RESULTS: both rod and cone photoreceptors were preserved (5-6 cells thick at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. CONCLUSIONS/SIGNIFICANCE: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort

  13. Epiretinal transplantation of human bone marrow mesenchymal stem cells rescues retinal and vision function in a rat model of retinal degeneration.

    Science.gov (United States)

    Tzameret, Adi; Sher, Ifat; Belkin, Michael; Treves, Avraham J; Meir, Amilia; Nagler, Arnon; Levkovitch-Verbin, Hani; Rotenstreich, Ygal; Solomon, Arieh S

    2015-09-01

    Vision incapacitation and blindness associated with incurable retinal degeneration affect millions of people worldwide. In this study, 0.25×10(6) human bone marrow stem cells (hBM-MSCs) were transplanted epiretinally in the right eye of Royal College Surgeons (RCS) rats at the age of 28 days. Epiretinally transplanted cells were identified as a thin layer of cells along vitreous cavity, in close proximity to the retina or attached to the lens capsule, up to 6 weeks following transplantation. Epiretinal transplantation delayed photoreceptor degeneration and rescued retinal function up to 20 weeks following cell transplantation. Visual functions remained close to normal levels in epiretinal transplantation rats. No inflammation or any other adverse effects were observed in transplanted eyes. Our findings suggest that transplantation of hBM-MSCs as a thin epiretinal layer is effective for treatment of retinal degeneration in RCS rats, and that transplanting the cells in close proximity to the retina enhances hBM-MSC therapeutic effect compared with intravitreal injection. Copyright © 2015. Published by Elsevier B.V.

  14. NGF protects corneal, retinal, and cutaneous tissues/cells from phototoxic effect of UV exposure.

    Science.gov (United States)

    Rocco, Maria Luisa; Balzamino, Bijorn Omar; Aloe, Luigi; Micera, Alessandra

    2018-04-01

    Based on evidence that nerve growth factor (NGF) exerts healing action on damaged corneal, retinal, and cutaneous tissues, the present study sought to assess whether topical NGF application can prevent and/or protect epithelial cells from deleterious effects of ultraviolet (UV) radiation. Eyes from 40 young-adult Sprague Dawley rats and cutaneous tissues from 36 adult nude mice were exposed to UVA/B lamp for 60 min, either alone or in the presence of murine NGF. Corneal, retinal, and cutaneous tissues were sampled/processed for morphological, immunohistochemical, and biomolecular analysis, and results were compared statistically. UV exposure affected both biochemical and molecular expression of NGF and trkA NGFR in corneal, retinal, and cutaneous tissues while UV exposure coupled to NGF treatment enhanced NGF and trkA NGFR expression as well as reduced cell death. Overall, the findings of this in vivo/ex vivo study show the NGF ability to reduce the potential UV damage. Although the mechanism underneath this effect needs further investigation, these observations prospect the development of a pharmacological NGF-based therapy devoted to maintain cell function when exposed to phototoxic UV radiation.

  15. Effect of monocular deprivation on rabbit neural retinal cell densities

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    Philip Maseghe Mwachaka

    2015-01-01

    Conclusion: In this rabbit model, monocular deprivation resulted in activity-dependent changes in cell densities of the neural retina in favour of the non-deprived eye along with reduced cell densities in the deprived eye.

  16. Induced pluripotent stem cells for retinal degenerative diseases: a ...

    Indian Academy of Sciences (India)

    2009-12-31

    Dec 31, 2009 ... anisms of these diseases is still very limited and no radical drugs are available. Induced .... Induced pluripotent stem cells are ES-like pluripotent cells capable of .... lation to test whether immunorejection with the latter is in-.

  17. Intraocular Injection of ES Cell-Derived Neural Progenitors Improve Visual Function in Retinal Ganglion Cell-Depleted Mouse Models

    Directory of Open Access Journals (Sweden)

    Mundackal S. Divya

    2017-09-01

    Full Text Available Retinal ganglion cell (RGC transplantation is a promising strategy to restore visual function resulting from irreversible RGC degeneration occurring in glaucoma or inherited optic neuropathies. We previously demonstrated FGF2 induced differentiation of mouse embryonic stem cells (ESC to RGC lineage, capable of retinal ganglion cell layer (GCL integration upon transplantation. Here, we evaluated possible improvement of visual function by transplantation of ES cell derived neural progenitors in RGC depleted glaucoma mice models. ESC derived neural progenitors (ES-NP were transplanted into N-Methyl-D-Aspartate (NMDA injected, RGC-ablated mouse models and a pre-clinical glaucoma mouse model (DBA/2J having sustained higher intra ocular pressure (IOP. Visual acuity and functional integration was evaluated by behavioral experiments and immunohistochemistry, respectively. GFP-expressing ES-NPs transplanted in NMDA-injected RGC-depleted mice differentiated into RGC lineage and possibly integrating into GCL. An improvement in visual acuity was observed after 2 months of transplantation, when compared to the pre-transplantation values. Expression of c-Fos in the transplanted cells, upon light induction, further suggests functional integration into the host retinal circuitry. However, the transplanted cells did not send axonal projections into optic nerve. Transplantation experiments in DBA/2J mouse showed no significant improvement in visual functions, possibly due to both host and transplanted retinal cell death which could be due to an inherent high IOP. We showed that, ES NPs transplanted into the retina of RGC-ablated mouse models could survive, differentiate to RGC lineage, and possibly integrate into GCL to improve visual function. However, for the survival of transplanted cells in glaucoma, strategies to control the IOP are warranted.

  18. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice.

    Science.gov (United States)

    Fox, Donald A; Hamilton, W Ryan; Johnson, Jerry E; Xiao, Weimin; Chaney, Shawntay; Mukherjee, Shradha; Miller, Diane B; O'Callaghan, James P

    2011-11-01

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ~25 and ~40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    Science.gov (United States)

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  20. Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions

    Directory of Open Access Journals (Sweden)

    María de Lourdes Mora-García

    2016-10-01

    Full Text Available Abstract Background In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs from bone marrow and other “classic” sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs on effector T lymphocytes through the purinergic pathway. Methods We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs. We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+ lymphocyte function through the generation of adenosine (Ado. Results We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. Conclusions This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

  1. Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

    Science.gov (United States)

    de Lourdes Mora-García, María; García-Rocha, Rosario; Morales-Ramírez, Omar; Montesinos, Juan José; Weiss-Steider, Benny; Hernández-Montes, Jorge; Ávila-Ibarra, Luis Roberto; Don-López, Christian Azucena; Velasco-Velázquez, Marco Antonio; Gutiérrez-Serrano, Vianey; Monroy-García, Alberto

    2016-10-26

    In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

  2. Collectin-11 Is an Important Modulator of Retinal Pigment Epithelial Cell Phagocytosis and Cytokine Production.

    Science.gov (United States)

    Dong, Xia; Wu, Weiju; Ma, Liang; Liu, Chengfei; Bhuckory, Mohajeet B; Wang, Liping; Nandrot, Emeline F; Xu, Heping; Li, Ke; Liu, Yizhi; Zhou, Wuding

    2017-01-01

    In this paper, we report previously unknown roles for collectin-11 (CL-11, a soluble C-type lectin) in modulating the retinal pigment epithelial (RPE) cell functions of phagocytosis and cytokine production. We found that CL-11 and its carbohydrate ligand are expressed in both the murine and human neural retina; these resemble each other in terms of RPE and photoreceptor cells. Functional analysis of murine RPE cells showed that CL-11 facilitates the opsonophagocytosis of photoreceptor outer segments and apoptotic cells, and also upregulates IL-10 production. Mechanistic analysis revealed that calreticulin on the RPE cells is required for CL-11-mediated opsonophagocytosis whereas signal-regulatory protein α and mannosyl residues on the cells are involved in the CL-11-mediated upregulation of IL-10 production. This study is the first to demonstrate the role of CL-11 and the molecular mechanisms involved in modulating RPE cell phagocytosis and cytokine production. It provides a new insight into retinal health and disease and has implications for other phagocytic cells. © 2017 S. Karger AG, Basel.

  3. Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID

    Science.gov (United States)

    Sauer, Aisha V.; Brigida, Immacolata; Carriglio, Nicola; Jofra Hernandez, Raisa; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L.; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna

    2012-01-01

    Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)–mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA–treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA−/− Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA–treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA–treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID. Trials were registered at www.clinicaltrials.gov as NCT00598481/NCT00599781. PMID:22184407

  4. Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

    Science.gov (United States)

    Sauer, Aisha V; Brigida, Immacolata; Carriglio, Nicola; Hernandez, Raisa Jofra; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna; Aiuti, Alessandro

    2012-02-09

    Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

  5. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Donald A., E-mail: dafox@uh.edu [College of Optometry, University of Houston, Houston, TX (United States); Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Department of Pharmacology and Pharmaceutical Sciences, University of Houston, Houston, TX (United States); Hamilton, W. Ryan [Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Johnson, Jerry E. [Department of Natural Sciences, University of Houston-Downtown, Houston, TX (United States); Xiao, Weimin [College of Optometry, University of Houston, Houston, TX (United States); Chaney, Shawntay; Mukherjee, Shradha [Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Miller, Diane B.; O' Callaghan, James P. [Toxicology and Molecular Biology Branch, Health Effects Research Laboratory, Centers for Disease Control and Prevention-NIOSH, Morgantown, WV USA (United States)

    2011-11-15

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was {<=} 1, {<=} 10, {approx} 25 and {approx} 40 {mu}g/dL, respectively, on PN10 and by PN30 all were {<=} 1 {mu}g/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: Black-Right-Pointing-Pointer Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: {<=} 1, {<=} 10, 25 and 40 {mu}g/dL Black

  6. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    International Nuclear Information System (INIS)

    Fox, Donald A.; Hamilton, W. Ryan; Johnson, Jerry E.; Xiao, Weimin; Chaney, Shawntay; Mukherjee, Shradha; Miller, Diane B.; O'Callaghan, James P.

    2011-01-01

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ∼ 25 and ∼ 40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: ► Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: ≤ 1, ≤ 10, 25 and 40 μg/dL ► Gestational lead exposure dose-dependently decreased the number of TH

  7. Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

    Directory of Open Access Journals (Sweden)

    József Jászai

    2011-03-01

    Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

  8. ROCK-1 mediates diabetes-induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy.

    Science.gov (United States)

    Rothschild, Pierre-Raphaël; Salah, Sawsen; Berdugo, Marianne; Gélizé, Emmanuelle; Delaunay, Kimberley; Naud, Marie-Christine; Klein, Christophe; Moulin, Alexandre; Savoldelli, Michèle; Bergin, Ciara; Jeanny, Jean-Claude; Jonet, Laurent; Arsenijevic, Yvan; Behar-Cohen, Francine; Crisanti, Patricia

    2017-08-18

    In diabetic retinopathy, the exact mechanisms leading to retinal capillary closure and to retinal barriers breakdown remain imperfectly understood. Rho-associated kinase (ROCK), an effector of the small GTPase Rho, involved in cytoskeleton dynamic regulation and cell polarity is activated by hyperglycemia. In one year-old Goto Kakizaki (GK) type 2 diabetic rats retina, ROCK-1 activation was assessed by its cellular distribution and by phosphorylation of its substrates, MYPT1 and MLC. In both GK rat and in human type 2 diabetic retinas, ROCK-1 is activated and associated with non-apoptotic membrane blebbing in retinal vessels and in retinal pigment epithelium (RPE) that respectively form the inner and the outer barriers. Activation of ROCK-1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. In RPE cells, actin cytoskeleton remodeling and membrane blebs in RPE cells contributes to outer barrier breakdown. Intraocular injection of fasudil, significantly reduces both retinal hypoxia and RPE barrier breakdown. Diabetes-induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.

  9. Effect of eye NGF administration on two animal models of retinal ganglion cells degeneration

    Directory of Open Access Journals (Sweden)

    Valeria Colafrancesco

    2011-01-01

    Full Text Available The aim of this study was to investigate the effect of nerve growth factor (NGF administration on retinal ganglion cells (RGCs in experimentally induced glaucoma (GL and diabetic retinopathy (DR. GL was induced in adult rats by injection of hypertonic saline into the episcleral vein of the eye and diabetes (DT was induced by administration of streptozoticin. Control and experimental rats were treated daily with either ocular application of NGF or vehicle solution. We found that both animal models present a progressive degeneration of RGCs and changing NGF and VEGF levels in the retina and optic nerve. We then proved that NGF eye drop administration exerts a protective effect on these models of retinal degeneration. In brief, our findings indicate that NGF can play a protective role against RGC degeneration occurring in GL and DR and suggest that ocular NGF administration might be an effective pharmacological approach.

  10. Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

    Directory of Open Access Journals (Sweden)

    Michele Bertacchi

    2015-10-01

    Full Text Available Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

  11. Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata.

    Science.gov (United States)

    Filgueira, Daza de Moraes Vaz Batista; Guterres, Laís Pereira; Votto, Ana Paula de Souza; Vargas, Marcelo Alves; Boyle, Robert Tew; Trindade, Gilma Santos; Nery, Luiz Eduardo Maia

    2010-01-01

    The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm(-2) UVA, 0.07 and 0.9 J cm(-2) UVB, 20 nmβ-PDH (pigment dispersing hormone) or 10 μm SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo. Cultured cells were exposed to 250 μm L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo. SIN-1 did not induce pigment dispersion in the cell cultures. L-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent. © 2010 The Authors. Journal Compilation. The American Society of Photobiology.

  12. Lack of FasL expression in cultured human retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Kaestel, C G; Madsen, H O; Prause, J U

    2001-01-01

    Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune...... privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western...... blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from...

  13. Regulation of CD8+ T cell responses to retinal antigen by local FoxP3+ regulatory T cells

    Directory of Open Access Journals (Sweden)

    Scott W McPherson

    2012-06-01

    Full Text Available While pathogenic CD4 T cells are well known mediators of autoimmune uveoretinitis, CD8 T cells can also be uveitogenic. Since preliminary studies indicated that C57BL/6 mice were minimally susceptible to autoimmune uveoretinitis induction by CD8 T cells, the basis of the retinal disease resistance was sought. Mice that express β-galactosidase (βgal on a retina-specific promoter (arrβgal mice were backcrossed to mice expressing green fluorescent protein and diphtheria toxin receptor under control of the Foxp3 promoter (Foxp3-DTR/GFP mice, and to T cell receptor transgenic mice that produce βgal specific CD8 T cells (BG1 mice. These mice were used to explore the role of regulatory T cells in the resistance to retinal autoimmune disease. Experiments with T cells from double transgenic BG1 x Foxp3-DTR/GFP mice transferred into Foxp3-DTR/GFP x arrβgal mice confirmed that the retina was well protected from attempts to induce disease by adoptive transfer of activated BG1 T cells. The successful induction of retinal disease following unilateral intraocular administration of diphtheria toxin to deplete regulatory T cells showed that the protective activity was dependent on local, toxin-sensitive regulatory T cells; the opposite, untreated eye remained disease-free. Although there were very few Foxp3+ regulatory T cells in the parenchyma of quiescent retina, and they did not accumulate in retina, their depletion by local toxin administration led to disease susceptibility. We propose that these regulatory T cells modulate the pathogenic activity of βgal-specific CD8 T cells in the retinas of arrβgal mice on a local basis, allowing immunoregulation to be responsive to local conditions.

  14. A comparison of spatial analysis methods for the construction of topographic maps of retinal cell density.

    Directory of Open Access Journals (Sweden)

    Eduardo Garza-Gisholt

    Full Text Available Topographic maps that illustrate variations in the density of different neuronal sub-types across the retina are valuable tools for understanding the adaptive significance of retinal specialisations in different species of vertebrates. To date, such maps have been created from raw count data that have been subjected to only limited analysis (linear interpolation and, in many cases, have been presented as iso-density contour maps with contour lines that have been smoothed 'by eye'. With the use of stereological approach to count neuronal distribution, a more rigorous approach to analysing the count data is warranted and potentially provides a more accurate representation of the neuron distribution pattern. Moreover, a formal spatial analysis of retinal topography permits a more robust comparison of topographic maps within and between species. In this paper, we present a new R-script for analysing the topography of retinal neurons and compare methods of interpolating and smoothing count data for the construction of topographic maps. We compare four methods for spatial analysis of cell count data: Akima interpolation, thin plate spline interpolation, thin plate spline smoothing and Gaussian kernel smoothing. The use of interpolation 'respects' the observed data and simply calculates the intermediate values required to create iso-density contour maps. Interpolation preserves more of the data but, consequently includes outliers, sampling errors and/or other experimental artefacts. In contrast, smoothing the data reduces the 'noise' caused by artefacts and permits a clearer representation of the dominant, 'real' distribution. This is particularly useful where cell density gradients are shallow and small variations in local density may dramatically influence the perceived spatial pattern of neuronal topography. The thin plate spline and the Gaussian kernel methods both produce similar retinal topography maps but the smoothing parameters used may affect

  15. A comparison of spatial analysis methods for the construction of topographic maps of retinal cell density.

    Science.gov (United States)

    Garza-Gisholt, Eduardo; Hemmi, Jan M; Hart, Nathan S; Collin, Shaun P

    2014-01-01

    Topographic maps that illustrate variations in the density of different neuronal sub-types across the retina are valuable tools for understanding the adaptive significance of retinal specialisations in different species of vertebrates. To date, such maps have been created from raw count data that have been subjected to only limited analysis (linear interpolation) and, in many cases, have been presented as iso-density contour maps with contour lines that have been smoothed 'by eye'. With the use of stereological approach to count neuronal distribution, a more rigorous approach to analysing the count data is warranted and potentially provides a more accurate representation of the neuron distribution pattern. Moreover, a formal spatial analysis of retinal topography permits a more robust comparison of topographic maps within and between species. In this paper, we present a new R-script for analysing the topography of retinal neurons and compare methods of interpolating and smoothing count data for the construction of topographic maps. We compare four methods for spatial analysis of cell count data: Akima interpolation, thin plate spline interpolation, thin plate spline smoothing and Gaussian kernel smoothing. The use of interpolation 'respects' the observed data and simply calculates the intermediate values required to create iso-density contour maps. Interpolation preserves more of the data but, consequently includes outliers, sampling errors and/or other experimental artefacts. In contrast, smoothing the data reduces the 'noise' caused by artefacts and permits a clearer representation of the dominant, 'real' distribution. This is particularly useful where cell density gradients are shallow and small variations in local density may dramatically influence the perceived spatial pattern of neuronal topography. The thin plate spline and the Gaussian kernel methods both produce similar retinal topography maps but the smoothing parameters used may affect the outcome.

  16. Effects of the neuroprotective drugs somatostatin and brimonidine on retinal cell models of diabetic retinopathy.

    Science.gov (United States)

    Beltramo, Elena; Lopatina, Tatiana; Mazzeo, Aurora; Arroba, Ana I; Valverde, Angela M; Hernández, Cristina; Simó, Rafael; Porta, Massimo

    2016-12-01

    Diabetic retinopathy is considered a microvascular disease, but recent evidence has underlined early involvement of the neuroretina with interactions between microvascular and neural alterations. Topical administration of somatostatin (SST), a neuroprotective molecule with antiangiogenic properties, prevents diabetes-induced retinal neurodegeneration in animals. The α 2 -adrenergic receptor agonist brimonidine (BRM) decreases vitreoretinal vascular endothelial growth factor and inhibits blood-retinal barrier breakdown in diabetic rats. However, SST and BRM effects on microvascular cells have not yet been studied. We investigated the behaviour of these drugs on the crosstalk between microvasculature and neuroretina. Expression of SST receptors 1-5 in human retinal pericytes (HRP) was checked. We subsequently evaluated the effects of diabetic-like conditions (high glucose and/or hypoxia) with/without SST/BRM on HRP survival. Endothelial cells (EC) and photoreceptors were maintained in the above conditions and their conditioned media (CM) used to culture HRP. Vice versa, HRP-CM was used on EC and photoreceptors. Survival parameters were assessed. HRP express the SST receptor 1 (SSTR1). Glucose fluctuations mimicking those occurring in diabetic subjects are more damaging for pericytes and photoreceptors than stable high glucose and hypoxic conditions. SST/BRM added to HRP in diabetic-like conditions decrease EC apoptosis. However, neither SST nor BRM changed the response of pericytes and neuroretina-vascular crosstalk under diabetic-like conditions. Retinal pericytes express SSTR1, indicating that they can be a target for SST. Exposure to SST/BRM had no adverse effects, direct or mediated by the neuroretina, suggesting that these molecules could be safely evaluated for the treatment of ocular diseases.

  17. Neuroprotective effect of He-Ying-Qing-Re formula on retinal ganglion cell in diabetic retinopathy.

    Science.gov (United States)

    Zhang, Cheng; Xu, Yu; Tan, Hor-Yue; Li, Sha; Wang, Ning; Zhang, Yinjian; Feng, Yibin

    2018-03-25

    He-Ying-Qing-Re Formula (HF) was empirically modified from Si-Miao-Yong-An Decoction (SD), which was recorded in the literature of Divine Doctor's Secret Transmission, and has been utilized for centuries to treat vasculopathy through clearing heat and accelerating bloodstream. HF has been used as an effective holistic treatment of diabetic retinopathy (DR) for decades and experimentally reported to ameliorate retinal condition in diabetic mice. Our study aims to investigate the effect of HF in preventing sustained hyperglycemia and hyperlipidemia-associated retinal ganglion cell (RGC) cell death and its possible mechanism. Chromatographic fingerprint of HF was obtained upon the UPLC-based analytic system; Diabetic retinopathy was established in streptozotocin (STZ) injection-induced hyperglycemic mice; Alterations of retinal structure was assayed by H&E staining. Expression of PSD-95 and CHOP in retinae was assessed by immunofluorescence; RGC cell line (mRGC) was used for in vitro study. Cell death was analyzed by flow cytometry; Intracellular reactive oxygen species (ROS) was measured by 2',7'-dichlorofluorescin diacetate (DCFDA); Apoptosis-related proteins and signaling were monitored with immunoblotting and colorimetric assay. Chlorogenic acid, ferulic acid, and rutin were identified in HF. HF attenuates the loss of RGCs, thinning of inner retinal layers in diabetic mice. Furthermore, expressions of Brn3a and PSD-95 were restored while CHOP level was downregulated upon HF treatment. In vitro study, HF alleviates H 2 O 2 -induced apoptosis of mRGCs and loss of postsynaptic protein via scavenging ROS and suppressing ATF4/CHOP-mediated endoplasmic reticulum stress and mitochondria-related pro-apoptotic factors, probably as cleaved-caspase-3, and phospho-p38 mitogen-activated protein kinase (MARK). Meanwhile, both pro-survival protein levels like Bcl-2/Bcl-xL and postsynaptic protein of PSD-95 were upregulated upon HF treatment. HF administration was a valid

  18. Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

    International Nuclear Information System (INIS)

    Wielgus, Albert R.; Zhao, Baozhong; Chignell, Colin F.; Hu, Dan-Ning; Roberts, Joan E.

    2010-01-01

    The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C 60 (OH) 22-26 ] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C 60 (OH) 22-26 in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 μM. Exposure to an 8.5 J.cm -2 dose of visible light in the presence of > 5 μM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 μM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of Φ = 0.05 in D 2 O and with a range of 0.002-0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage.

  19. Rescue of compromised lysosomes enhances degradation of photoreceptor outer segments and reduces lipofuscin-like autofluorescence in retinal pigmented epithelial cells.

    Science.gov (United States)

    Guha, Sonia; Liu, Ji; Baltazar, Gabe; Laties, Alan M; Mitchell, Claire H

    2014-01-01

    Healthful cell maintenance requires the efficient degradative processing and removal of waste material. Retinal pigmented epithelial (RPE) cells have the onerous task of degrading both internal cellular debris generated through autophagy as well as phagocytosed photoreceptor outer segments. We propose that the inadequate processing material with the resulting accumulation of cellular waste contributes to the downstream pathologies characterized as age-related macular degeneration (AMD). The lysosomal enzymes responsible for clearance function optimally over a narrow range of acidic pH values; elevation of lysosomal pH by compounds like chloroquine or A2E can impair degradative enzyme activity and lead to a lipofuscin-like autofluorescence. Restoring acidity to the lysosomes of RPE cells can enhance activity of multiple degradative enzymes and is therefore a logical target in early AMD. We have identified several approaches to reacidify lysosomes of compromised RPE cells; stimulation of beta-adrenergic, A2A adenosine and D5 dopamine receptors each lowers lysosomal pH and improves degradation of outer segments. Activation of the CFTR chloride channel also reacidifies lysosomes and increases degradation. These approaches also restore the lysosomal pH of RPE cells from aged ABCA4(-/-) mice with chronically high levels of A2E, suggesting that functional signaling pathways to reacidify lysosomes are retained in aged cells like those in patients with AMD. Acidic nanoparticles transported to RPE lysosomes also lower pH and improve degradation of outer segments. In summary, the ability of diverse approaches to lower lysosomal pH and enhance outer segment degradation support the proposal that lysosomal acidification can prevent the accumulation of lipofuscin-like material in RPE cells.

  20. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    OpenAIRE

    Jiang, M; Pandey, S; Tran, V T; Fong, H K

    1991-01-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

  1. Visual Field Defects and Retinal Ganglion Cell Losses in Human Glaucoma Patients

    Science.gov (United States)

    Harwerth, Ronald S.; Quigley, Harry A.

    2007-01-01

    Objective The depth of visual field defects are correlated with retinal ganglion cell densities in experimental glaucoma. This study was to determine whether a similar structure-function relationship holds for human glaucoma. Methods The study was based on retinal ganglion cell densities and visual thresholds of patients with documented glaucoma (Kerrigan-Baumrind, et al.) The data were analyzed by a model that predicted ganglion cell densities from standard clinical perimetry, which were then compared to histologic cell counts. Results The model, without free parameters, produced accurate and relatively precise quantification of ganglion cell densities associated with visual field defects. For 437 sets of data, the unity correlation for predicted vs. measured cell densities had a coefficient of determination of 0.39. The mean absolute deviation of the predicted vs. measured values was 2.59 dB, the mean and SD of the distribution of residual errors of prediction was -0.26 ± 3.22 dB. Conclusions Visual field defects by standard clinical perimetry are proportional to neural losses caused by glaucoma. Clinical Relevance The evidence for quantitative structure-function relationships provides a scientific basis of interpreting glaucomatous neuropathy from visual thresholds and supports the application of standard perimetry to establish the stage of the disease. PMID:16769839

  2. Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

    Science.gov (United States)

    Brew, Helen; Attwell, David

    1987-06-01

    Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

  3. Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

    International Nuclear Information System (INIS)

    Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

    2016-01-01

    Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

  4. Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

    Energy Technology Data Exchange (ETDEWEB)

    Ranaei Pirmardan, Ehsan [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Soheili, Zahra-Soheila [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Samiei, Shahram [Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran (Iran, Islamic Republic of); Ahmadieh, Hamid [Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Mowla, Seyed Javad [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ezzati, Razie [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Naseri, Marzieh [Department of Molecular Medicine, Faculty of Advanced Technology, Iran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2016-10-01

    Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

  5. Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells

    Science.gov (United States)

    Han, Meng; Blindewald-Wittich, Almut; Holz, Frank G.; Giese, Günter; Niemz, Markolf H.; Snyder, Sarah; Sun, Hui; Yu, Jiayi; Agopov, Michael; La Schiazza, Olivier; Bille, Josef F.

    2006-01-01

    Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

  6. Effects of elevated glucose concentration on cultured bovine retinal endothelial (BRE) cells

    International Nuclear Information System (INIS)

    Capetandes, A.; Gerritsen, M.E.

    1986-01-01

    Salient clinical features of diabetic retinopathy include capillary microaneurysm and neovascularization, which progress with the severity of the disease. It has been suggested that exposure of the retinal vascular cells to high glucose concentrations may play a causative role in the retinopathy. In the present study, the effects of variant media glucose concentrations on BRE cell growth were determined. Normal growth curves were obtained with glucose concentrations of 100, 450 and 600 mg%, but the replication rate was decreased with 600 mg%. To determine if elevated glucose concentrations also altered DNA synthesis, BRE cells cultivated with 100 and 600 mg% glucose demonstrated increased thymidine uptake and total DNA content compared to the 100 mg% group. Furthermore, vacuolation and increased cell diameter occurred in BRE cells cultivated 600 mg% compared to 100 mg% glucose. In conclusion, increases in media glucose concentrations result in a decreased cellular replication rate, increased DNA synthesis and increased cell diameter during the log phase of growth

  7. Honeybee retinal glial cells transform glucose and supply the neurons with metabolic substrate

    International Nuclear Information System (INIS)

    Tsacopoulos, M.; Evequoz-Mercier, V.; Perrottet, P.; Buchner, E.

    1988-01-01

    The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor cells (sensory neurons) constitute two distinct metabolic compartments. Retinal slices incubated with 2-deoxy[ 3 H]glucose convert this glucose analogue to 2-deoxy[ 3 H]glucose 6-phosphate, but this conversion is made only in the glial cells. Hence, glycolysis occurs only in glial cells. In contrast, the neurons consume O 2 and this consumption is sustained by the hydrolysis of glycogen, which is contained in large amounts in the glia. During photostimulation the increased oxidative metabolism of the neurons is sustained by a higher supply of carbohydrates from the glia. This clear case of metabolic interaction between neurons and glial cells supports Golgi's original hypothesis, proposed nearly 100 years ago, about the nutritive function of glial cells in the nervous system

  8. Honeybee Retinal Glial Cells Transform Glucose and Supply the Neurons with Metabolic Substrate

    Science.gov (United States)

    Tsacopoulos, M.; Evequoz-Mercier, V.; Perrottet, P.; Buchner, E.

    1988-11-01

    The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor cells (sensory neurons) constitute two distinct metabolic compartments. Retinal slices incubated with 2-deoxy[3H]glucose convert this glucose analogue to 2-deoxy[3H]glucose 6-phosphate, but this conversion is made only in the glial cells. Hence, glycolysis occurs only in glial cells. In contrast, the neurons consume O2 and this consumption is sustained by the hydrolysis of glycogen, which is contained in large amounts in the glia. During photostimulation the increased oxidative metabolism of the neurons is sustained by a higher supply of carbohydrates from the glia. This clear case of metabolic interaction between neurons and glial cells supports Golgi's original hypothesis, proposed nearly 100 years ago, about the nutritive function of glial cells in the nervous system.

  9. Downregulation of Lysyl Oxidase Protects Retinal Endothelial Cells From High Glucose-Induced Apoptosis.

    Science.gov (United States)

    Kim, Dongjoon; Mecham, Robert P; Trackman, Philip C; Roy, Sayon

    2017-05-01

    To investigate the effect of reducing high glucose (HG)-induced lysyl oxidase (LOX) overexpression and increased activity on retinal endothelial cell apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N) or HG (30 mM glucose) medium for 7 days. In parallel, RRECs were grown in HG medium and transfected with LOX small interfering RNA (siRNA), scrambled siRNA as control, or exposed to β-aminopropionitrile (BAPN), a LOX inhibitor. LOX expression, AKT activation, and caspase-3 activity were determined by Western blot (WB) analysis and apoptosis by differential dye staining assay. Moreover, to determine whether diabetes-induced LOX overexpression alters AKT activation and promotes apoptosis, changes in LOX expression, AKT phosphorylation, caspase-3 activation, and Bax expression were assessed in retinas of streptozotocin (STZ)-induced diabetic mice and LOX heterozygous knockout (LOX+/-) mice. WB analysis indicated significant LOX overexpression and reduced AKT activation under HG condition in RRECs. Interestingly, when cells grown in HG were transfected with LOX siRNA or exposed to BAPN, the number of apoptotic cells was significantly decreased concomitant with increased AKT phosphorylation. Diabetic mouse retinas exhibited LOX overexpression, decreased AKT phosphorylation, and increased Bax and caspase-3 activation compared to values in nondiabetic mice. In LOX+/- mice, reduced LOX levels were observed with increased AKT activity, and reduced Bax and caspase-3 activity. Furthermore, decreased levels of LOX in the LOX+/- mice was protective against diabetes-induced apoptosis. Findings from this study indicate that preventing LOX overexpression may be protective against HG-induced apoptosis in retinal vascular cells associated with diabetic retinopathy.

  10. Correspondence between visual and electrical input filters of ON and OFF mouse retinal ganglion cells

    Science.gov (United States)

    Sekhar, S.; Jalligampala, A.; Zrenner, E.; Rathbun, D. L.

    2017-08-01

    Objective. Over the past two decades retinal prostheses have made major strides in restoring functional vision to patients blinded by diseases such as retinitis pigmentosa. Presently, implants use single pulses to activate the retina. Though this stimulation paradigm has proved beneficial to patients, an unresolved problem is the inability to selectively stimulate the on and off visual pathways. To this end our goal was to test, using white noise, voltage-controlled, cathodic, monophasic pulse stimulation, whether different retinal ganglion cell (RGC) types in the wild type retina have different electrical input filters. This is an important precursor to addressing pathway-selective stimulation. Approach. Using full-field visual flash and electrical and visual Gaussian noise stimulation, combined with the technique of spike-triggered averaging (STA), we calculate the electrical and visual input filters for different types of RGCs (classified as on, off or on-off based on their response to the flash stimuli). Main results. Examining the STAs, we found that the spiking activity of on cells during electrical stimulation correlates with a decrease in the voltage magnitude preceding a spike, while the spiking activity of off cells correlates with an increase in the voltage preceding a spike. No electrical preference was found for on-off cells. Comparing STAs of wild type and rd10 mice revealed narrower electrical STA deflections with shorter latencies in rd10. Significance. This study is the first comparison of visual cell types and their corresponding temporal electrical input filters in the retina. The altered input filters in degenerated rd10 retinas are consistent with photoreceptor stimulation underlying visual type-specific electrical STA shapes in wild type retina. It is therefore conceivable that existing implants could target partially degenerated photoreceptors that have only lost their outer segments, but not somas, to selectively activate the on and off

  11. Traumatic central retinal artery occlusion with sickle cell trait.

    Science.gov (United States)

    Sorr, E M; Goldberg, R E

    1975-10-01

    An 8-year-old black boy with sickle cell trait struck his left brow and globe on the edge of a table and had immediate blurring of vision. Six days later visual acuity was light projection, and traumatic iritis with secondary glaucoma and perimacular edema were present. Fluorescein angiography indicated obstructed peripheral and perimacular arterioles and dye leakage from the disk.

  12. Estimation of adenosine triphosphate utilization of rat mast cells during and after anaphylactic histamine secretion

    DEFF Research Database (Denmark)

    Johansen, Torben

    1990-01-01

    during the time period of histamine secretion and immediately after its completion. During secretion the additional ATP-utilization above the basal level of ATP-synthesis was 0.51 pmol/10(3) cells. 2.5 min after cell activation, the rate of additional ATP-utilization was 0.30 pmol/10(3) cells...

  13. The effects of platelet gel on cultured human retinal pigment epithelial (hRPE cells

    Directory of Open Access Journals (Sweden)

    Sahar Balagholi

    2017-11-01

    Full Text Available The positive role of platelet gel (PG in tissue regeneration is well known, however, other characteristics of PG still remain to be determined. We investigated cellular and molecular changes in cultured human retinal pigment epithelial (hRPE cells when treated with different concentrations of PG named PG1, PG2, and PG3. hRPE cells were isolated from donor eyes of two newborn children, within 24 hours after their death. The cells were treated with three concentrations of PG for 7 days: 3 × 104/ml (PG1, 6 × 104/ml (PG2, and 9 × 104/ml (PG3. Fetal bovine serum was used as a control. Immunocytochemistry was performed with anti-RPE65 (H-85, anti-Cytokeratin 8/18 (NCL-5D3, and anti-PAX6 antibody. We used MTT assay to determine cell viability. Gene expressions of PAX6, MMP2, RPE65, ACTA2, MKI67, MMP9, and KDR were analyzed using real-time PCR. A significant increase in viability was observed for PG3-treated cells compared to control (p = 0.044 and compared to PG1 group (p = 0.027, on day 7. Cellular elongation together with dendritiform extensions were observed in PG-treated cells on days 1 and 3, while epithelioid morphology was observed on day 7. All cells were immunoreactive for RPE65, cytokeratin 8/18, and PAX6. No significant change was observed in the expression of MKI67 and PAX6, but the expressions of MMP2, MMP9, ACTA2, and KDR were significantly higher in PG2-treated cells compared to controls (p < 0.05. Our results indicate that increased concentration of PG and extended exposure time have positive effects on viability of hRPE cells. PG may be useful for hRPE cell encapsulation in retinal cell replacement therapy.

  14. PlGF gene knockdown in human retinal pigment epithelial cells.

    Science.gov (United States)

    Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Ahmadieh, Hamid; Rezaeikanavi, Mozhgan; Samiei, Shahram; Khalooghi, Keynoush

    2011-04-01

    To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

  15. Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Gnana-Prakasam, Jaya P.; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Reddy, Sushma K.; Martin, Pamela M.; Thangaraju, Muthusamy; Smith, Sylvia B.; Ganapathy, Vadivel

    2013-01-01

    Purpose. Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated. Methods. Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4. Results. Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells. Conclusions. RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells. PMID:23169885

  16. Modulation of gene expression of adenosine and metabotropic glutamate receptors in rat's neuronal cells exposed to L-glutamate and [60]fullerene.

    Science.gov (United States)

    Giust, Davide; Da Ros, Tatiana; Martín, Mairena; Albasanz, José Luis

    2014-08-01

    L-Glutamate (L-Glu) has been often associated not only to fundamental physiological roles, as learning and memory, but also to neuronal cell death and the genesis and development of important neurodegenerative diseases. Herein we studied the variation in the adenosine and metabotropic glutamate receptors expression induced by L-Glu treatment in rat's cortical neurons. The possibility to have structural alteration of the cells induced by L-Glu (100 nM, 1 and 10 microM) has been addressed, studying the modulation of microtubule associated protein-2 (MAP-2) and neurofilament heavy polypeptide (NEFH), natively associated proteins to the dendritic shape maintenance. Results showed that the proposed treatments were not destabilizing the cells, so the L-Glu concentrations were acceptable to investigate fluctuation in receptors expression, which were studied by RT-PCR. Interestingly, C60 fullerene derivative t3ss elicited a protective effect against glutamate toxicity, as demonstrated by MTT assay. In addition, t3ss compound exerted a different effect on the adenosine and metabotropic glutamate receptors analyzed. Interestingly, A(2A) and mGlu1 mRNAs were significantly decreased in conditions were t3ss neuroprotected cortical neurons from L-Glu toxicity. In summary, t3ss protects neurons from glutamate toxicity in a process that appears to be associated with the modulation of the gene expression of adenosine and metabotropic glutamate receptors.

  17. Ezh2 does not mediate retinal ganglion cell homeostasis or their susceptibility to injury.

    Directory of Open Access Journals (Sweden)

    Lin Cheng

    Full Text Available Epigenetic predisposition is thought to critically contribute to adult-onset disorders, such as retinal neurodegeneration. The histone methyltransferase, enhancer of zeste homolog 2 (Ezh2, is transiently expressed in the perinatal retina, particularly enriched in retinal ganglion cells (RGCs. We previously showed that embryonic deletion of Ezh2 from retinal progenitors led to progressive photoreceptor degeneration throughout life, demonstrating a role for embryonic predisposition of Ezh2-mediated repressive mark in maintaining the survival and function of photoreceptors in the adult. Enrichment of Ezh2 in RGCs leads to the question if Ezh2 also mediates gene expression and function in postnatal RGCs, and if its deficiency changes RGC susceptibility to cell death under injury or disease in the adult. To test this, we generated mice carrying targeted deletion of Ezh2 from RGC progenitors driven by Math5-Cre (mKO. mKO mice showed no detectable defect in RGC development, survival, or cell homeostasis as determined by physiological analysis, live imaging, histology, and immunohistochemistry. Moreover, RGCs of Ezh2 deficient mice revealed similar susceptibility against glaucomatous and acute optic nerve trauma-induced neurodegeneration compared to littermate floxed or wild-type control mice. In agreement with the above findings, analysis of RNA sequencing of RGCs purified from Ezh2 deficient mice revealed few gene changes that were related to RGC development, survival and function. These results, together with our previous report, support a cell lineage-specific mechanism of Ezh2-mediated gene repression, especially those critically involved in cellular function and homeostasis.

  18. Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells

    International Nuclear Information System (INIS)

    Choudhary, S.; Xiao, T.; Srivastava, S.; Zhang, W.; Chan, L.L.; Vergara, L.A.; Van Kuijk, F.J.G.M.; Ansari, N.H.

    2005-01-01

    Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H 2 O 2 , 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H 2 O 2 -, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H 2 O 2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3 H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST) (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD

  19. Leukaemic infiltration and cytomegalovirus retinitis in a patient with acute T-cell lymphoblastic leukaemia in complete remission.

    Science.gov (United States)

    Saldaña Garrido, J D; Martínez Rubio, M; Carrión Campo, R; Moya Moya, M A; Rico Sergado, L

    2017-03-01

    A 43-year-old woman in remission from T- cell acute lymphoblastic leukaemia was referred to our hospital with suspected leukaemic retinitis. The funduscopic examination of her left eye revealed multifocal yellow-white peripheral retinitis and retinal haemorrhage. The patient was treated for cytomegalovirus retinitis after an extended haematological investigation showed no abnormalities. Initial improvement was followed by papillitis in the left eye and motility restriction in the right eye. Magnetic resonance and lumbar puncture confirmed leukaemia relapse. Specific treatment was initiated with complete resolution. Ocular involvement may precede haematological leukaemia relapse. Physicians should be alerted when ocular symptoms appear in these cases. Copyright © 2016 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

  20. The immune privilege of the eye: human retinal pigment epithelial cells selectively modulate T-cell activation in vitro

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Lovato, Paola; Ødum, Niels

    2005-01-01

    PURPOSE: To examine the effect of human retinal pigment epithelial (RPE) cells on phytohemagglutinin (PHA) activation of T cells. METHODS: Resting peripheral blood lymphocytes (PBLs) were stimulated with PHA with or without the presence of gamma-irradiated RPE cells. Proliferation and the cell...... in cell culture supernatant was measured by ELISA. RESULTS: Human RPE cells were found to suppress PHA-induced proliferation, cyclin A, IL-2R-alpha and -gamma, and CD71 expression and decrease the production of IL-2; but RPE cells do not inhibit the PHA-induced expression of early activation markers CD69......, MHC class I and II, and of cyclin D of the PBLs. CONCLUSIONS: These results are the first to indicate that RPE cells impede generation of activated T cells by interfering with the induction of high-affinity IL-2R-alphabetagamma, IL-2 production, and the expression of CD71 and cyclin A....

  1. SPECIFIC ROLE OF LYMPHATIC MARKER PODOPLANIN IN RETINAL PIGMENT EPITHELIAL CELLS

    Science.gov (United States)

    Grimaldo, S.; Garcia, M.; Zhang, H.; Chen, L.

    2015-01-01

    Podoplanin is a small transmembrane glycoprotein widely known to be a marker for lymphatic endothelial cells. In this study, we identify a novel localization of podoplanin in the retinal pigment epithelium (RPE), a cellular monolayer critically involved in the visual process. Using a small interfering RNA (siRNA)-mediated gene silencing approach, we have also demonstrated, for the first time, that podoplanin depletion in human RPE cells leads to a marked reduction of cell aggregates and tight junctions. Additionally, the podoplanin-depleted cells also exhibit a significantly lower rate of proliferation. These data together indicate that podoplanin plays a crucial role in RPE cell functions. Further investigation on this factor may reveal novel mechanisms and therapeutic strategies for RPE-related eye diseases, such as proliferative retinopathy and age-related macular degeneration. PMID:21226415

  2. Adenosine A2A Receptor and IL-10 in Peripheral Blood Mononuclear Cells of Patients with Mild Cognitive Impairment

    Directory of Open Access Journals (Sweden)

    Beatrice Arosio

    2011-01-01

    Full Text Available Adenosine suppresses immune responses through the A2A receptor (A2AR. This study investigated the interleukin 10 (IL-10 genetic profile and the expression of A2AR in peripheral blood mononuclear cells (PBMCs of patients with mild cognitive impairment (MCI, Alzheimer disease (AD, and age-matched controls to verify, if they may help distinguish different forms of cognitive decline. We analyzed the IL-10 genotype and the expression of A2AR in 41 subjects with AD, 10 with amnestic MCI (a-MCI, 49 with multiple cognitive domain MCI (mcd-MCI, and 46 controls. There was a significant linear increase in A2AR mRNA levels and A2AR density from mcd-MCI to a-MCI, with intermediate levels being found in AD. The IL-10 AA genotype frequency was 67% in a-MCI, 46% in AD, 35% in mcd-MCI, and 20% in controls. These data suggest that the assessment of the IL-10 genotype and the expression of A2AR in PBMCs may be a valuable means of differentiating between a-MCI and mcd-MCI.

  3. Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

    Science.gov (United States)

    Tuder, R M; Karasek, M A; Bensch, K G

    1990-02-01

    The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

  4. Epigalloccatechin-3-gallate Inhibits Ocular Neovascularization and Vascular Permeability in Human Retinal Pigment Epithelial and Human Retinal Microvascular Endothelial Cells via Suppression of MMP-9 and VEGF Activation

    Directory of Open Access Journals (Sweden)

    Hak Sung Lee

    2014-08-01

    Full Text Available Epigalloccatechin-3-gallate (EGCG is the main polyphenol component of green tea (leaves of Camellia sinensis. EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs and vascular endothelial growth factor (VEGF play a key role in the processes of extracellular matrix (ECM remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA and tumor necrosis factor alpha (TNF-α in human retinal pigment epithelial cells (HRPECs. EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2 by inhibiting generation of reactive oxygen species (ROS. EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs. Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31 on corneal neovascularization (CNV induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.

  5. Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration.

    Science.gov (United States)

    Zhu, DanHong; Sreekumar, Parameswaran G; Hinton, David R; Kannan, Ram

    2010-03-31

    Ceramide and its metabolic derivatives are important modulators of cellular apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degeneration. The aim of this study was to identify the expression and regulation of key enzymes of the ceramide pathway in retinal pigment epithelial (RPE) cells. RT-PCR was used to screen the enzymes involved in ceramide metabolism that are expressed in RPE. Over-expression of neutral sphingomyelinase-2 (SMPD3) or sphingosine kinase 1 (Sphk1) in ARPE-19 cells was achieved by transient transfection of SMPD3 or Sphk1 cDNA subcloned into an expression vector. The number of apoptotic or proliferating cells was determined using TUNEL and BrdU assays, respectively. Neutral sphingomyelinase-1, neutral sphingomyelinase-2, acidic ceramidase, ceramide kinase, SphK1 and Sphk2 were expressed in both ARPE-19 and early passage human fetal RPE (fRPE) cells, while alkaline ceramidase 2 was only expressed in fRPE cells. Over-expression of SMPD3 decreased RPE cell proliferation and increased cell apoptosis. The percentage of apoptotic cells increased proportionally with the amount of transfected SMPD3 DNA. Over-expression of SphK1 promoted cell proliferation and protected ARPE-19 cells from ceramide-induced apoptosis. The effect of C(2) ceramide on induction of apoptosis was evaluated in polarized vs. non-polarized RPE cultures; polarization of RPE was associated with much reduced apoptosis in response to ceramide. In conclusion, RPE cells possess the synthetic machinery for the production of ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P). Over-expression of SMPD3 may increase cellular ceramide levels, leading to enhanced cell death and arrested cell proliferation. The selective induction of apoptosis in non-polarized RPE cultures by C(2) ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in

  6. Transport of thiol-conjugates of inorganic mercury in human retinal pigment epithelial cells

    International Nuclear Information System (INIS)

    Bridges, Christy C.; Battle, Jamie R.; Zalups, Rudolfs K.

    2007-01-01

    Inorganic mercury (Hg 2+ ) is a prevalent environmental contaminant to which exposure to can damage rod photoreceptor cells and compromise scotopic vision. The retinal pigment epithelium (RPE) likely plays a role in the ocular toxicity associated with Hg 2+ exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg 2+ to access photoreceptor cells, it must first be taken up by the RPE, possibly by mechanisms involving transporters of essential nutrients. In other epithelia, Hg 2+ , when conjugated to cysteine (Cys) or homocysteine (Hcy), gains access to the intracellular compartment of the target cells via amino acid and organic anion transporters. Accordingly, the purpose of the current study was to test the hypothesis that Cys and Hcy S-conjugates of Hg 2+ utilize amino acid transporters to gain access into RPE cells. Time- and temperature-dependence, saturation kinetics, and substrate-specificity of the transport of Hg 2+ , was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg 2+ : Cys (Cys-S-Hg-S-Cys), Hcy (Hcy-S-Hg-S-Hcy), N-acetylcysteine (NAC-S-Hg-S-NAC) or glutathione (GSH-S-Hg-S-GSH). We discovered that only Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy were taken up by these cells. This transport was Na + -dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B 0,+ and ASC in ARPE-19 cells. Overall, our data suggest that Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy are taken up into ARPE-19 cells by Na-dependent amino acid transporters, possibly systems B 0,+ and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury

  7. TRANSPORT OF THIOL-CONJUGATES OF INORGANIC MERCURY IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS

    Science.gov (United States)

    Bridges, Christy C.; Battle, Jamie R.; Zalups, Rudolfs K.

    2007-01-01

    Inorganic mercury (Hg2+) is a prevalent environmental contaminant to which exposure to can damage rod photoreceptor cells and compromise scotopic vision. The retinal pigment epithelium (RPE) likely plays a role in the ocular toxicity associated with Hg2+ exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg2+ to access photoreceptor cells, it must be first be taken up by the RPE, possibly by mechanisms involving transporters of essential nutrients. In other epithelia, Hg2+, when conjugated to cysteine (Cys) or homocysteine (Hcy), gains access to the intracellular compartment of the target cells via amino acid and organic anion transporters. Accordingly, the purpose of the current study was to test the hypothesis that Cys and Hcy S-conjugates of Hg2+ utilize amino acid transporters to gain access into RPE cells. Time- and temperature-dependence, saturation kinetics, and substrate-specificity of the transport of Hg2+, was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg2+: Cys (Cys-S-Hg-S-Cys), Hcy (Hcy-S-Hg-S-Hcy), N-acetylcysteine (NAC-S-Hg-S-NAC) or glutathione (GSH-S-Hg-S-GSH). We discovered that only Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy were taken up by these cells. This transport was Na+-dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B0,+ and ASC in ARPE-19 cells. Overall, our data suggest that Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy are taken up into ARPE-19 cells by Na-dependent amino acid transporters, possibly systems B0,+ and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury. PMID:17467761

  8. Müller Glia, Vision-Guided Ocular Growth, Retinal Stem Cells, and a Little Serendipity

    Science.gov (United States)

    2011-01-01

    Hypothesis-driven science is expected to result in a continuum of studies and findings along a discrete path. By comparison, serendipity can lead to new directions that branch into different paths. Herein, I describe a diverse series of findings that were motivated by hypotheses, but driven by serendipity. I summarize how investigations into vision-guided ocular growth in the chick eye led to the identification of glucagonergic amacrine cells as key regulators of ocular elongation. Studies designed to assess the impact of the ablation of different types of neurons on vision-guided ocular growth led to the finding of numerous proliferating cells within damaged retinas. These proliferating cells were Müller glia–derived retinal progenitors with a capacity to produce new neurons. Studies designed to investigate Müller glia–derived progenitors led to the identification of a domain of neural stem cells that form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Accelerated ocular growth, caused by visual deprivation, stimulated the proliferation of CMZ progenitors. We formulated a hypothesis that growth-regulating glucagonergic cells may regulate both overall eye size (scleral growth) and the growth of the retina (proliferation of CMZ cells). Subsequent studies identified unusual types of glucagonergic neurons with terminals that ramify within the CMZ; these cells use visual cues to control equatorial ocular growth and the proliferation of CMZ cells. Finally, while studying the signaling pathways that stimulate CMZ and Müller glia–derived progenitors, serendipity led to the discovery of a novel type of glial cell that is scattered across the inner retinal layers. PMID:21960640

  9. Choice of Cell Source in Cell-Based Therapies for Retinal Damage due to Age-Related Macular Degeneration: A Review

    Directory of Open Access Journals (Sweden)

    Sudhakar John

    2013-01-01

    Full Text Available Background. Age-related macular degeneration (AMD is a complex disorder that affects primarily the macula involving the retinal pigment epithelium (RPE but also to a certain extent the photoreceptor layer and the retinal neurons. Cell transplantation is a promising option for AMD and clinical trials are underway using different cell types. Methods. We hypothesize that instead of focusing on a particular cell source for concurrent regeneration of all the retinal layers and also to prevent exhaustive research on an array of cell sources for regeneration of each layer, the choice should depend on, precisely, which layer is damaged. Results. Thus, for a damage limited to the retinal pigment epithelial (RPE layer, the choice we suggest would be RPE cells. When the damage extends to rods and cones, the choice would be bone marrow stem cells and when retinal neurons are involved, relatively immature stem cell populations with an inherent capacity to yield neuronal lineage such as hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells can be tried. Conclusion. This short review will prove to be a valuable guideline for those working on cell therapy for AMD to plan their future directions of research and therapy for this condition.

  10. Investigation of retinal ganglion cells and axons of normal rats using fluorogold retrograde labeling

    International Nuclear Information System (INIS)

    Yin Xiaolei; Ye Jian; Chen Chunlin

    2006-01-01

    To investigate the retinal ganglion cells (RGCs) by means of fluorogold retrograde labeling, RGCs were labeled by injecting the fluorogold bilaterally into the superficial superior colliculus and lateral genicutate nucleus in six adult SD rats. One and two weeks (3 rats in each group) after injecting the fluorogold, RGCs FG-labeled were observed and the number of them were counted. The results showed that after a week mean density of fluorogold-labeled RGCs was 2210 ± 128/mm 2 , and it was 2164 ± 117/mm 2 after two weeks. Our conclusion is fluorogold retrograde labeling could be very useful in the research of RGCs. (authors)

  11. Xenopus laevis Retinal Ganglion Cell Dendritic Arbors Develop Independently of Visual Stimulation

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    Barbara Lom

    2004-01-01

    Full Text Available Newly formed neurons must locate their appropriate target cells and then form synaptic connections with these targets in order to establish a functional nervous system. In the vertebrate retina, retinal ganglion cell (RGC dendrites extend from the cell body and form synapses with nearby amacrine and bipolar cells. RGC axons, however, exit the retina and synapse with the dendrites of midbrain neurons in the optic tectum. We examined how visual stimulation influenced Xenopus RGC dendritic arborization. Neuronal activity is known to be an important factor in shaping dendritic and axonal arborization. Thus, we reared tadpoles in dark and light environments then used rhodamine dextran retrograde labeling to identify RGCs in the retina. When we compared RGC dendritic arbors from tadpoles reared in dark and light environments, we found no morphological differences, suggesting that physiological visual activity did not contribute to the morphological development of Xenopus RGC dendritic arbors.

  12. Adult rat retinal ganglion cells and glia can be printed by piezoelectric inkjet printing

    International Nuclear Information System (INIS)

    Lorber, Barbara; Martin, Keith R; Hsiao, Wen-Kai; Hutchings, Ian M

    2014-01-01

    We have investigated whether inkjet printing technology can be extended to print cells of the adult rat central nervous system (CNS), retinal ganglion cells (RGC) and glia, and the effects on survival and growth of these cells in culture, which is an important step in the development of tissue grafts for regenerative medicine, and may aid in the cure of blindness. We observed that RGC and glia can be successfully printed using a piezoelectric printer. Whilst inkjet printing reduced the cell population due to sedimentation within the printing system, imaging of the printhead nozzle, which is the area where the cells experience the greatest shear stress and rate, confirmed that there was no evidence of destruction or even significant distortion of the cells during jet ejection and drop formation. Importantly, the viability of the cells was not affected by the printing process. When we cultured the same number of printed and non-printed RGC/glial cells, there was no significant difference in cell survival and RGC neurite outgrowth. In addition, use of a glial substrate significantly increased RGC neurite outgrowth, and this effect was retained when the cells had been printed. In conclusion, printing of RGC and glia using a piezoelectric printhead does not adversely affect viability and survival/growth of the cells in culture. Importantly, printed glial cells retain their growth-promoting properties when used as a substrate, opening new avenues for printed CNS grafts in regenerative medicine. (paper)

  13. Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Alice L Yu

    Full Text Available The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE induces cell loss, cellular senescence, and extracellular matrix (ECM synthesis in primary human retinal pigment epithelial (RPE cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J, connective tissue growth factor (CTGF, fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration.

  14. Electric stimulus duration alters network-mediated responses depending on retinal ganglion cell type

    Science.gov (United States)

    Im, Maesoon; Werginz, Paul; Fried, Shelley I.

    2018-06-01

    Objective. To improve the quality of artificial vision that arises from retinal prostheses, it is important to bring electrically-elicited neural activity more in line with the physiological signaling patterns that arise normally in the healthy retina. Our previous study reported that indirect activation produces a closer match to physiological responses in ON retinal ganglion cells (RGCs) than in OFF cells (Im and Fried 2015 J. Physiol. 593 3677-96). This suggests that a preferential activation of ON RGCs would shape the overall retinal response closer to natural signaling. Recently, we found that changes to the rate at which stimulation was delivered could bias responses towards a stronger ON component (Im and Fried 2016a J. Neural Eng. 13 025002), raising the possibility that changes to other stimulus parameters can similarly bias towards stronger ON responses. Here, we explore the effects of changing stimulus duration on the responses in ON and OFF types of brisk transient (BT) and brisk sustained (BS) RGCs. Approach. We used cell-attached patch clamp to record RGC spiking in the isolated rabbit retina. Targeted RGCs were first classified as ON or OFF type by their light responses, and further sub-classified as BT or BS types by their responses to both light and electric stimuli. Spiking in targeted RGCs was recorded in response to electric pulses with durations varying from 5 to100 ms. Stimulus amplitude was adjusted at each duration to hold total charge constant for all experiments. Main results. We found that varying stimulus durations modulated responses differentially for ON versus OFF cells: in ON cells, spike counts decreased significantly with increasing stimulus duration while in OFF cells the changes were more modest. The maximum ratio of ON versus OFF responses occurred at a duration of ~10 ms. The difference in response strength for BT versus BS cells was much larger in ON cells than in OFF cells. Significance. The stimulation rates preferred by

  15. Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

    Directory of Open Access Journals (Sweden)

    Ana V Oliveira

    2013-01-01

    Full Text Available Objective: Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently. Materials and Methods: Chitosan and thiolated chitosan nanoparticles (NPs were prepared in order to obtain a NH3 + :PO4− ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells. Results: In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency. Conclusion: The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery.

  16. Hydrostatic Pressure Does Not Cause Detectable Changes in Survival of Human Retinal Ganglion Cells

    Science.gov (United States)

    Osborne, Andrew; Aldarwesh, Amal; Rhodes, Jeremy D.; Broadway, David C.; Everitt, Claire; Sanderson, Julie

    2015-01-01

    Purpose Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated. Methods A chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (pressure for 24 or 48h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD. Conclusions Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina. PMID:25635827

  17. Characterization of cytochrome c as marker for retinal cell degeneration by uv/vis spectroscopic imaging

    Science.gov (United States)

    Hollmach, Julia; Schweizer, Julia; Steiner, Gerald; Knels, Lilla; Funk, Richard H. W.; Thalheim, Silko; Koch, Edmund

    2011-07-01

    Retinal diseases like age-related macular degeneration have become an important cause of visual loss depending on increasing life expectancy and lifestyle habits. Due to the fact that no satisfying treatment exists, early diagnosis and prevention are the only possibilities to stop the degeneration. The protein cytochrome c (cyt c) is a suitable marker for degeneration processes and apoptosis because it is a part of the respiratory chain and involved in the apoptotic pathway. The determination of the local distribution and oxidative state of cyt c in living cells allows the characterization of cell degeneration processes. Since cyt c exhibits characteristic absorption bands between 400 and 650 nm wavelength, uv/vis in situ spectroscopic imaging was used for its characterization in retinal ganglion cells. The large amount of data, consisting of spatial and spectral information, was processed by multivariate data analysis. The challenge consists in the identification of the molecular information of cyt c. Baseline correction, principle component analysis (PCA) and cluster analysis (CA) were performed in order to identify cyt c within the spectral dataset. The combination of PCA and CA reveals cyt c and its oxidative state. The results demonstrate that uv/vis spectroscopic imaging in conjunction with sophisticated multivariate methods is a suitable tool to characterize cyt c under in situ conditions.

  18. Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

    Science.gov (United States)

    Chen, Mei; Rajapakse, Dinusha; Fraczek, Monika; Luo, Chang; Forrester, John V; Xu, Heping

    2016-06-01

    Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  19. Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.

    Science.gov (United States)

    Yoshikawa, Noriko; Yamada, Shizuo; Takeuchi, Chihiro; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru; Nakamura, Kazuki

    2008-06-01

    Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.

  20. Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

    DEFF Research Database (Denmark)

    Fanelli, Giorgia; Gonzalez-Cordero, Anai; Gardner, Peter J

    2017-01-01

    induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression......, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions....

  1. Optical properties of photoreceptor and retinal pigment epithelium cells investigated with adaptive optics optical coherence tomography

    Science.gov (United States)

    Liu, Zhuolin

    Human vision starts when photoreceptors collect and respond to light. Photoreceptors do not function in isolation though, but share close interdependence with neighboring photoreceptors and underlying retinal pigment epithelium (RPE) cells. These cellular interactions are essential for normal function of the photoreceptor-RPE complex, but methods to assess these in the living human eye are limited. One approach that has gained increased promise is high-resolution retinal imaging that has undergone tremendous technological advances over the last two decades to probe the living retina at the cellular level. Pivotal in these advances has been adaptive optics (AO) and optical coherence tomography (OCT) that together allow unprecedented spatial resolution of retinal structures in all three dimensions. Using these high-resolution systems, cone photoreceptor are now routinely imaged in healthy and diseased retina enabling fundamental structural properties of cones to be studied such as cell spacing, packing arrangement, and alignment. Other important cell properties, however, have remained elusive to investigation as even better imaging performance is required and thus has resulted in an incomplete understanding of how cells in the photoreceptor-RPE complex interact with light. To address this technical bottleneck, we expanded the imaging capability of AO-OCT to detect and quantify more accurately and completely the optical properties of cone photoreceptor and RPE cells at the cellular level in the living human retina. The first objective of this thesis was development of a new AO-OCT method that is more precise and sensitive, thus enabling a more detailed view of the 3D optical signature of the photoreceptor-RPE complex than was previously possible (Chapter 2). Using this new system, the second objective was quantifying the waveguide properties of individual cone photoreceptor inner and outer segments across the macula (Chapter 3). The third objective extended the AO

  2. Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death

    Directory of Open Access Journals (Sweden)

    Rodrigo Meléndez García

    2016-05-01

    Full Text Available The identification of pathways necessary for retinal pigment epithelium (RPE function is fundamental to uncover therapies for blindness. Prolactin (PRL receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19 human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2 expression, which inhibits the TRPM2-mediated intracellular Ca2+ rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr−/− mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

  3. Delayed rectifier K channels contribute to contrast adaptation in mammalian retinal ganglion cells

    Science.gov (United States)

    Weick, Michael; Demb, Jonathan B.

    2011-01-01

    SUMMARY Retinal ganglion cells adapt by reducing their sensitivity during periods of high contrast. Contrast adaptation in the firing response depends on both presynaptic and intrinsic mechanisms. Here, we investigated intrinsic mechanisms for contrast adaptation in OFF Alpha ganglion cells in the in vitro guinea pig retina. Using either visual stimulation or current injection, we show that brief depolarization evoked spiking and suppressed firing during subsequent depolarization. The suppression could be explained by Na channel inactivation, as shown in salamander cells. However, brief hyperpolarization in the physiological range (5–10 mV) also suppressed firing during subsequent depolarization. This suppression was sensitive selectively to blockers of delayed-rectifier K channels (KDR). Somatic membrane patches showed TEA-sensitive KDR currents with activation near −25 mV and removal of inactivation at voltages negative to Vrest. Brief periods of hyperpolarization apparently remove KDR inactivation and thereby increase the channel pool available to suppress excitability during subsequent depolarization. PMID:21745646

  4. Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig

    DEFF Research Database (Denmark)

    Klassen, H; Kiilgaard, Jens Folke; Warfvinge, K

    2012-01-01

    Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig....... Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival...

  5. RBP-Jκ-dependent Notch signaling enhances retinal pigment epithelial cell proliferation in transgenic mice.

    Science.gov (United States)

    Schouwey, K; Aydin, I T; Radtke, F; Beermann, F

    2011-01-20

    The Notch signaling pathway is an ubiquitous cell-cell interaction mechanism, which is essential in controlling processes like cell proliferation, cell fate decision, differentiation or stem cell maintenance. Recent data have shown that Notch signaling is RBP-Jκ-dependent in melanocytes, being required for survival of these pigment cells that are responsible for coloration of the skin and hairs in mammals. In addition, Notch is believed to function as an oncogene in melanoma, whereas it is a tumor suppressor in mouse epidermis. In this study, we addressed the implication of the Notch signaling in the development of another population of pigment cells forming the retinal pigment epithelium (RPE) in mammalian eyes. The constitutive activity of Notch in Tyrp1::NotchIC/° transgenic mice enhanced RPE cell proliferation, and the resulting RPE-derived pigmented tumor severely affected the overall eye structure. This RPE cell proliferation is dependent on the presence of the transcription factor RBP-Jκ, as it is rescued in mice lacking RBP-Jκ in the RPE. In conclusion, Notch signaling in the RPE uses the canonical pathway, which is dependent on the transcription factor RBP-Jκ. In addition, it is of importance for RPE development, and constitutive Notch activity leads to hyperproliferation and benign tumors of these pigment cells.

  6. Lutein Inhibits the Migration of Retinal Pigment Epithelial Cells via Cytosolic and Mitochondrial Akt Pathways (Lutein Inhibits RPE Cells Migration

    Directory of Open Access Journals (Sweden)

    Ching-Chieh Su

    2014-08-01

    Full Text Available During the course of proliferative vitreoretinopathy (PVR, the retinal pigment epithelium (RPE cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

  7. Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

    Science.gov (United States)

    Mittal, Smriti P K; Khole, Swati; Jagadish, Nidhi; Ghosh, Debjani; Gadgil, Vijay; Sinkar, Vilas; Ghaskadbi, Saroj S

    2016-11-01

    Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3β by phosphorylation. Inactivated GSK-3β leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3β, together resulting in activation of HO-1. We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Ganglion cell-inner plexiform layer and retinal nerve fibre layer changes within the macula in retinitis pigmentosa: a spectral domain optical coherence tomography study.

    Science.gov (United States)

    Yoon, Chang Ki; Yu, Hyeong Gon

    2018-03-01

    To investigate how macular ganglion cell-inner plexiform layer (GCIPL) and retinal nerve fibre layer (RNFL) thicknesses within the macula change with retinitis pigmentosa (RP) severity. Spectral domain optical coherence tomography (SD-OCT) was used to examine 177 patients with RP and 177 normal controls. An optical coherence tomography (OCT) line scan was used to grade RP severity. Retinitis pigmentosa (RP) was categorized as more advanced if there was no identifiable inner segment ellipsoid (ISe) band (NISE) and as less advanced if an ISe band could be identified and peripheral loss of ISe was apparent (IISE). Ganglion cell-inner plexiform layer (GCIPL) and RNFL thicknesses were manually measured on OCT images and analysed. Pearson's correlation analyses were used to examine correlations between GCIPL thickness, RNFL thickness, visual acuity (VA) and visual field extent in patients and controls. Ganglion cell-inner plexiform layer (GCIPL) was significantly thicker in IISE than in control eyes (p layer (RNFL) was significantly thicker in eyes with IISE and NISE than in control eyes in both horizontal and vertical meridians (all p layer (GCIPL) thickness showed a weak positive correlation with vision, and RNFL thickness showed a weak negative correlation with vision and visual field extent. Based on these results, the inner retina, including the GCIPL and RNFL, maintains its gross integrity longer than the photoreceptor layer in RP. Additionally, thickening of the inner retina may have some functional implications in patients with RP. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  9. An anti-angiogenic state in mice and humans with retinal photoreceptor cell degeneration

    NARCIS (Netherlands)

    Lahdenranta, J.; Pasqualini, R.; Schlingemann, R. O.; Hagedorn, M.; Stallcup, W. B.; Bucana, C. D.; Sidman, R. L.; Arap, W.

    2001-01-01

    Abnormal angiogenesis accompanies many pathological conditions including cancer, inflammation, and eye diseases. Proliferative retinopathy because of retinal neovascularization is a leading cause of blindness in developed countries. Another major cause of irreversible vision loss is retinitis

  10. Delayed administration of glial cell line-derived neurotrophic factor (GDNF) protects retinal ganglion cells in a pig model of acute retinal ischemia

    DEFF Research Database (Denmark)

    Kyhn, Maria Voss; Klassen, Henry; Johansson, Ulrica Englund

    2009-01-01

    electroretinography (mfERG), quantification of NeuN positive cells and evaluation of the degree of retinal perivasculitis and inflammation 6 weeks after the insult. In the post-injection eyes (days 14, 28 and 42), the ratios of the iN1 and the iP2 amplitudes were 0.10 (95% CI: 0.05-0.15) and 0.09 (95% CI: 0.......04-0.16) in eyes treated with blank microspheres, and 0.24 (95% CI: 0.18-0.32) and 0.23 (95% CI: 0.15-0.33) in eyes treated with GDNF microspheres. These differences were statistically significant (P eyes...... injected with GDNF microspheres compared to eyes injected with blank microspheres. In eyes injected with GDNF microspheres the ganglion cell count was 9.5/field (s.e.m.: 2.1, n = 8), in eyes injected with blank microspheres it was 3.5/field (s.e.m.: 1.2, n = 7). This difference was statistically...

  11. Blue-light filtering alters angiogenic signaling in human retinal pigmented epithelial cells culture model.

    Science.gov (United States)

    Vila, Natalia; Siblini, Aya; Esposito, Evangelina; Bravo-Filho, Vasco; Zoroquiain, Pablo; Aldrees, Sultan; Logan, Patrick; Arias, Lluis; Burnier, Miguel N

    2017-11-02

    Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD. Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines. A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions. Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein

  12. Edaravone suppresses retinal ganglion cell death in a mouse model of normal tension glaucoma

    Science.gov (United States)

    Akaiwa, Kei; Namekata, Kazuhiko; Azuchi, Yuriko; Guo, Xiaoli; Kimura, Atsuko; Harada, Chikako; Mitamura, Yoshinori; Harada, Takayuki

    2017-01-01

    Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino-acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs. Loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP) and exhibits glaucomatous pathology including glutamate neurotoxicity and oxidative stress. In the present study, we found that edaravone, a free radical scavenger that is used for treatment of acute brain infarction and amyotrophic lateral sclerosis (ALS), reduces oxidative stress and prevents RGC death and thinning of the inner retinal layer in EAAC1-deficient (KO) mice. In addition, in vivo electrophysiological analyses demonstrated that visual impairment in EAAC1 KO mice was ameliorated with edaravone treatment, clearly establishing that edaravone beneficially affects both histological and functional aspects of the glaucomatous retina. Our findings raise intriguing possibilities for the management of glaucoma by utilizing a widely prescribed drug for the treatment of acute brain infarction and ALS, edaravone, in combination with conventional treatments to lower IOP. PMID:28703795

  13. Carbamazepine and oxcarbazepine, but not eslicarbazepine, enhance excitatory synaptic transmission onto hippocampal CA1 pyramidal cells through an antagonist action at adenosine A1 receptors.

    Science.gov (United States)

    Booker, Sam A; Pires, Nuno; Cobb, Stuart; Soares-da-Silva, Patrício; Vida, Imre

    2015-06-01

    This study assessed the anticonvulsant and seizure generation effects of carbamazepine (CBZ), oxcarbazepine (OXC) and eslicarbazepine (S-Lic) in wild-type mice. Electrophysiological recordings were made to discriminate potential cellular and synaptic mechanisms underlying anti- and pro-epileptic actions. The anticonvulsant and pro-convulsant effects were evaluated in the MES, the 6-Hz and the Irwin tests. Whole-cell patch-clamp recordings were used to investigate the effects on fast excitatory and inhibitory synaptic transmission in hippocampal area CA1. The safety window for CBZ, OXC and eslicarbazepine (ED50 value against the MES test and the dose that produces grade 5 convulsions in all mice), was 6.3, 6.0 and 12.5, respectively. At high concentrations the three drugs reduced synaptic transmission. CBZ and OXC enhanced excitatory postsynaptic currents (EPSCs) at low, therapeutically-relevant concentrations. These effects were associated with no change in inhibitory postsynaptic currents (IPSCs) resulting in altered balance between excitation and inhibition. S-Lic had no effect on EPSC or IPSC amplitudes over the same concentration range. The CBZ mediated enhancement of EPSCs was blocked by DPCPX, a selective antagonist, and occluded by CCPA, a selective agonist of the adenosine A1 receptor. Furthermore, reduction of endogenous adenosine by application of the enzyme adenosine deaminase also abolished the CBZ- and OXC-induced increase of EPSCs, indicating that the two drugs act as antagonists at native adenosine receptors. In conclusion, CBZ and OXC possess pro-epileptic actions at clinically-relevant concentrations through the enhancement of excitatory synaptic transmission. S-Lic by comparison has no such effect on synaptic transmission, explaining its lack of seizure exacerbation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

    Directory of Open Access Journals (Sweden)

    Freya M Mowat

    2010-06-01

    Full Text Available Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

  15. Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage.

    Science.gov (United States)

    de Korte, Dirk; Kleine, Mya; Korsten, Herbert G H; Verhoeven, Arthur J

    2008-06-01

    Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.

  16. Adenovirus-Mediated Delivery of Catalase to Retinal Pigment Epithelial Cells Protects Neighboring Photoreceptors from Photo-Oxidative Stress

    OpenAIRE

    Rex, T.S.; Tsui, I.; Hahn, P.; Maguire, A.M.; Duan, D.; Bennett, J.; Dunaief, J.L.

    2004-01-01

    Oxidative stress is involved in the pathogenesis of many diseases. Overexpression of antioxidant enzymes by gene therapy may protect tissues from oxidative damage. Because the reactive oxygen species hydrogen peroxide can diffuse across cell membranes, we hypothesized that overexpression of the antioxidant catalase within certain cells might protect neighboring cells. To test this hypothesis, we transduced retinal pigment epithelial (RPE) cells in vitro and in vivo with adenovirus carrying th...

  17. Effect of essential fatty acids on glucose-induced cytotoxicity to retinal vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Shen Junhui

    2012-07-01

    Full Text Available Abstract Background Diabetic retinopathy is a major complication of dysregulated hyperglycemia. Retinal vascular endothelial cell dysfunction is an early event in the pathogenesis of diabetic retinopathy. Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by docosahexaenoic acid (DHA, 22:6 ω-3 and eicosapentaenoic acid (EPA, 20:5 ω-3. The influence of dietary omega-3 PUFA on brain zinc metabolism has been previously implied. Zn2+ is essential for the activity of Δ6 desaturase as a co-factor that, in turn, converts essential fatty acids to their respective long chain metabolites. Whether essential fatty acids (EFAs α-linolenic acid and linoleic acid have similar beneficial effect remains poorly understood. Methods RF/6A cells were treated with different concentrations of high glucose, α-linolenic acid and linoleic acid and Zn2+. The alterations in mitochondrial succinate dehydrogenase enzyme activity, cell membrane fluidity, reactive oxygen species generation, SOD enzyme and vascular endothelial growth factor (VEGF secretion were evaluated. Results Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by both linoleic acid (LA and α-linolenic acid (ALA, while the saturated fatty acid, palmitic acid was ineffective. A dose–response study with ALA showed that the activity of the mitochondrial succinate dehydrogenase enzyme was suppressed at all concentrations of glucose tested to a significant degree. High glucose enhanced fluorescence polarization and microviscocity reverted to normal by treatment with Zn2+ and ALA. ALA was more potent that Zn2+. Increased level of high glucose caused slightly increased ROS generation that correlated with corresponding decrease in SOD activity. ALA suppressed ROS generation to a significant degree in a dose dependent fashion and raised SOD activity significantly. ALA suppressed

  18. Comparison of Effects on Gene Expression Activity of Low-Molecular-Weight Lychee Fruit Polyphenol (Oligonol®, Adenosine, and Minoxidil in Human Dermal Papilla Cells

    Directory of Open Access Journals (Sweden)

    Koji Wakame

    2017-06-01

    Full Text Available Background: Oligonol® (OLG is a functional food product and ingredient for cosmetics derived from a lychee fruit polyphenol. It has been reported to act on the skin as an anti-inflammatory and prevent UVB-induced skin damage. Aim: In this study, with the aim of exploring new functionalities of OLG on the scalp, we investigated the effect of OLG on human dermal papilla cells by comparing with adenosine and minoxidil at the genetic level. Method: OLG, adenosine, and minoxidil were applied to human dermal papilla cell lines for 24 h, after which VEGF, FGF-7, WNT5a, and WNT10a mRNA expressions were measured by real-time PCR analysis. Additionally, using DNA microarrays, we investigated the effect on 205 inflammation-related genes. Result: Consequently, in human dermal papilla cell lines, FGF-7 and WNT10a mRNA expression were observed in 100 µg/mL OLG-supplemented cells. The results of the DNA microarray analysis showed that 10 genes were suppressed by OLG. Conclusions: OLG may be expected to affect function of human dermal papilla cell by regulating the expression of genes related to cell proliferation and inflammation.

  19. Hop/STI1 modulates retinal proliferation and cell death independent of PrPC

    International Nuclear Information System (INIS)

    Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.; Linden, Rafael; Chiarini, Luciana B.

    2007-01-01

    Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP C ). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP C dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 (α-STI1) blocked both ganglion cell and NBL cell death independent of PrP C . cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while α-STI1 increased proliferation in the developing retina, both independent of PrP C . We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP C

  20. Modeling retinal degeneration using patient-specific induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Zi-Bing Jin

    Full Text Available Retinitis pigmentosa (RP is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

  1. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, S.S.; Lyerly, D.P. (Environmental Protection Agency, Research Triangle Park, NC (USA))

    1989-12-01

    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with (35S)methionine and (3H)fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure.

  2. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    International Nuclear Information System (INIS)

    Padilla, S.S.; Lyerly, D.P.

    1989-01-01

    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with [35S]methionine and [3H]fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure

  3. Virally delivered, constitutively active NFκB improves survival of injured retinal ganglion cells.

    Science.gov (United States)

    Dvoriantchikova, Galina; Pappas, Steve; Luo, Xueting; Ribeiro, Marcio; Danek, Dagmara; Pelaez, Daniel; Park, Kevin K; Ivanov, Dmitry

    2016-12-01

    As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno-associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2-p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P-value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P-value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  4. Influence of intracellular adenosine-triphosphate concentration of yeast cells on survival following X-irradiation

    International Nuclear Information System (INIS)

    Reinhard, R.D.; Pohlit, W.

    1975-01-01

    The effect of D-glucose, 2-deoxy-D-glucose and starvation in buffer on the ATP-concentration of yeast cells has been studied. In both the wild-type and a respiratory-deficient mutant strain 2-deoxy-D-glucose decreases the value for ATP, while it is enhanced by glucose only in the mutant strain. Populations with different ATP-concentrations have been irradiated. The results suggest that ATP may be an essential factor in the system that determines the length of the shoulder of the dose effect curves. (orig.) [de

  5. Autologous fibrin glue as an encapsulating scaffold for delivery of retinal progenitor cells

    Directory of Open Access Journals (Sweden)

    Tamer Anwar Esmail Ahmed

    2015-02-01

    Full Text Available The retina is a highly sophisticated piece of the neural machinery that begins the translation of incoming light signals into meaningful visual information. Several degenerative diseases of the retina are characterized by photoreceptor loss and eventually lead to irreversible blindness. Regenerative medicine, using tissue engineering-based constructs to deliver progenitor cells or photoreceptors along with supporting carrier matrix is a promising approach for restoration of structure and function. Fresh fibrin glue (FG produced by the CryoSeal®FS system in combination with mouse retinal progenitor cells (RPCs were evaluated in this study. In vitro expanded RPCs isolated from postnatal mouse retina were encapsulated into FG and cultured in the presence of the protease inhibitor, tranexamic acid. Encapsulation of RPCs into FG did not show adverse effects on cell proliferation or cell survival. RPCs exhibited fibroblast-like morphology concomitantly with attachment to the encapsulating FG surface. They expressed α7 and β3 integrin subunits that could mediate attachment to fibrin matrix via an RGD independent mechanism. The three dimensional environment and the attachment surface provided by FG was associated with a rapid downregulation of the progenitor marker SOX2 and enhanced the expression of the differentiation markers CRX and recoverin. However, the in vitro culture conditions did not promote full differentiation into mature photoreceptors. Nevertheless, we have shown that autologous fibrin, when fabricated into a scaffold for RPCs for delivery to the retina, provides the cells with external cues that could potentially improve the differentiation events. Hence, transient encapsulation of RPCs into FG could be a valid and potential treatment strategy to promote retinal regeneration following degenerative diseases. However, further optimization is necessary to maximize the outcomes in terms of mature photoreceptors.

  6. Glutamate modulation of GABA transport in retinal horizontal cells of the skate

    Science.gov (United States)

    Kreitzer, Matthew A; Andersen, Kristen A; Malchow, Robert Paul

    2003-01-01

    Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA-elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole-cell voltage-clamp techniques. GABA (1 mm) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 μm) and SKF89976-A (100 μm), but was unaffected by 100 μm picrotoxin. Prior application of 100 μm glutamate significantly reduced the GABA-elicited current. Glutamate depressed the GABA dose-response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA-elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither elicited a current nor modified the GABA-induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA-elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 mm) and thapsigargin (2 nm), agents known to alter intracellular calcium levels, also reduced the GABA-elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium-dependent process, and imply a close physical relationship between calcium-permeable glutamate receptors and GABA transporters in these cells. PMID:12562999

  7. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  8. Progranulin, a major secreted protein of mouse adipose-derived stem cells, inhibits light-induced retinal degeneration.

    Science.gov (United States)

    Tsuruma, Kazuhiro; Yamauchi, Mika; Sugitani, Sou; Otsuka, Tomohiro; Ohno, Yuta; Nagahara, Yuki; Ikegame, Yuka; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

    2014-01-01

    Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

  9. In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.

    Science.gov (United States)

    Rezanejad, Habib; Soheili, Zahra-Soheila; Haddad, Farhang; Matin, Maryam M; Samiei, Shahram; Manafi, Ali; Ahmadieh, Hamid

    2014-04-01

    The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

  10. Effects of Antipsychotic Drugs Haloperidol and Clozapine on Visual Responses of Retinal Ganglion Cells in a Rat Model of Retinitis Pigmentosa.

    Science.gov (United States)

    Jensen, Ralph J

    2016-12-01

    In the P23H rat model of retinitis pigmentosa, the dopamine D2 receptor antagonists sulpiride and eticlopride appear to improve visual responses of retinal ganglion cells (RGCs) by increasing light sensitivity of RGCs and transforming abnormal, long-latency ON-center RGCs into OFF-center cells. Antipsychotic drugs are believed to mediate their therapeutic benefits by blocking D2 receptors. This investigation was conducted to test whether haloperidol (a typical antipsychotic drug) and clozapine (an atypical antipsychotic drug) could similarly alter the light responses of RGCs in the P23H rat retina. Extracellular recordings were made from RGCs in isolated P23H rat retinas. Responses of RGCs to flashes of light were evaluated before and during bath application of a drug. Both haloperidol and clozapine increased light sensitivity of RGCs on average by ∼0.3 log unit. For those ON-center RGCs that exhibit an abnormally long-latency response to the onset of a small spot of light, both haloperidol and clozapine brought out a short-latency OFF response and markedly reduced the long-latency ON response. The selective serotonin 5-HT2A antagonist MDL 100907 had similar effects on RGCs. The effects of haloperidol on light responses of RGCs can be explained by its D2 receptor antagonism. The effects of clozapine on light responses of RGCs on the other hand may largely be due to its 5-HT2A receptor antagonism. Overall, the results suggest that antipsychotic drugs may be useful in improving vision in patients with retinitis pigmentosa.

  11. Effects of 5 Thio-D-Glucose on cellular adenosine triphosphate levels and deoxyribonucleic acid rejoining in hypoxic and aerobic Chinese hamster cells

    International Nuclear Information System (INIS)

    Nagle, W.A.; Moss, A.J. Jr.; Roberts, H.G. Jr.; Baker, M.L.

    1980-01-01

    Intracellular adenosine triphosphate (ATP) levels were measured in both hypoxic and aerobic cultures of V79 Chinese hamster cells treated with 5-thio-D-glucose (5-SH-D-Glc). This glucose analog, a known inhibitor of D-glucose transport and metabolism, reduced ATP in cell cultures allowed to become hypoxic by cell metabolism, but not in aerobic cultures treated similarly. Cells depleted of ATP were unable to rejoin x-ray induced deoxyribonucleic acid (DNA) strand breaks as measured by the alkaline sucrose gradient sedimentation technique. The inference for radiation therapy is that inhibition of glucose metabolism selectively depletes energy reserves in hypoxic cells, rendering these cells more radiosensitive and leading to a more effective tumor treatment

  12. Structure and function of the interphotoreceptor matrix surrounding retinal photoreceptor cells.

    Science.gov (United States)

    Ishikawa, Makoto; Sawada, Yu; Yoshitomi, Takeshi

    2015-04-01

    The interphotoreceptor matrix (IPM) is a highly organized structure with interconnected domains surrounding cone and rod photoreceptor cells and extends throughout the subretinal space. Based on known roles of the extracellular matrix in other tissues, the IPM is thought to have several prominent functions including serving as a receptor for growth factors, regulating retinoid transport, participating in cytoskeletal organization in surrounding cells, and regulation of oxygen and nutrient transport. In addition, a number of studies suggest that the IPM also may play a significant role in the etiology of retinal degenerative disorders. In this review, we describe the present knowledge concerning the structure and function of the IPM under physiological and pathological conditions. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Behavior of a Spontaneously Arising Human Retinal Pigment Epithelial Cell Line Cultivated on Thin Alginate Film.

    Science.gov (United States)

    Najafabadi, Hoda Shams; Soheili, Zahra-Soheila; Ganji, Shahla Mohammad

    2015-01-01

    A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide. Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle's-medium-and-Ham's-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks. The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features. Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures.

  14. Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells

    Directory of Open Access Journals (Sweden)

    L.D. Loopuijt

    2007-10-01

    Full Text Available To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37 on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 µm ("thin" or "thick" dendrites, respectively. Three cells (4.5% were bistratified, having thick dendrites, and the others (95.5% were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40% and 2 groups with inner (50-100% stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat ß-cells, whereas one group of cells with thick dendrites includes cells that resemble cat a-cells.

  15. Schwann Cell-Mediated Preservation of Vision in Retinal Degenerative Diseases via the Reduction of Oxidative Stress: A Possible Mechanism.

    Science.gov (United States)

    Mahmoudzadeh, Raziyeh; Heidari-Keshel, Saeed; Lashay, Alireza

    2016-01-01

    After injury to the central nervous system (CNS), regeneration is often inadequate, except in the case of remyelination. This remyelination capacity of the CNS is a good example of a stem/precursor cell-mediated renewal process. Schwann cells have been found to act as remyelinating agents in the peripheral nervous system (PNS), but several studies have highlighted their potential role in remyelination in the CNS too. Schwann cells are able to protect and support retinal cells by secreting growth factors such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and basic fibroblast growth factor. Retinal degenerative diseases can be highly debilitating, and they are a major concern in countries with an ageing populations. One of the leading causes of permanent loss of vision in the West is a retinal degenerative disease known as age-related macular degeneration (AMD). In the United States, nearly 1.75 million people over the age of 40 have advanced AMD, and it is estimated that this number will increase to approximately 3 million people by 2020. One of the most common pathways involved in the initiation and development of retinal diseases is the oxidative stress pathway. In patients with diabetes, Schwann cells have been shown to be able to secrete large amounts of antioxidant enzymes that protect the PNS from the oxidative stress that results from fluctuations in blood glucose levels. This antioxidant ability may be involved in the mechanism by which Schwann cells are able to promote reconstruction in the CNS, especially in individuals with retinal injuries and degenerative diseases.

  16. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  17. Trimetazidine protects retinal ganglion cells from acute glaucoma via the Nrf2/Ho-1 pathway.

    Science.gov (United States)

    Wan, Peixing; Su, Wenru; Zhang, Yingying; Li, Zhidong; Deng, Caibin; Zhuo, Yehong

    2017-09-15

    Acute glaucoma is one of the leading causes of irreversible vision impairment characterized by the rapid elevation of intraocular pressure (IOP) and consequent retinal ganglion cell (RGC) death. Oxidative stress and neuroinflammation have been considered critical for the pathogenesis of RGC death in acute glaucoma. Trimetazidine (TMZ), an anti-ischemic drug, possesses antioxidative and anti-inflammatory properties, contributing to its therapeutic potential in tissue damage. However, the role of TMZ in acute glaucoma and the underlying molecular mechanisms remain elusive. Here, we report that treatment with TMZ significantly attenuated retinal damage and RGC death in mice with acute glaucoma, with a significant decrease in reactive oxygen species (ROS) and inflammatory cytokine production in the retina. Furthermore, TMZ treatment directly decreased ROS production and rebalanced the intracellular redox state, thus contributing to the survival of RGCs in vitro TMZ treatment also reduced the production of inflammatory cytokines in vitro Mechanistically, the TMZ-mediated inhibition of apoptosis and inflammatory cytokine production in RGCs occurred via the regulation of the nuclear factor erythroid 2-related factor 2/heme oxygenase 1/caspase-8 pathway. Moreover, the TMZ-mediated neuroprotection in acute glaucoma was abrogated when an HO-1 inhibitor, SnPP, was used. Our findings identify potential mechanisms of RGC apoptosis and propose a novel therapeutic agent, TMZ, which exerts a precise neuroprotective effect against acute glaucoma. © 2017 The Author(s).

  18. Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma

    Science.gov (United States)

    Pitha, Ian F.; Nguyen, Cathy; Steinhart, Matthew R.; Nguyen, Thao D.; Pease, Mary Ellen; Oglesby, Ericka N.; Berlinicke, Cynthia A.; Mitchell, Katherine L.; Kim, Jessica; Jefferys, Joan J.

    2015-01-01

    Purpose To determine if oral losartan treatment decreases the retinal ganglion cell (RGC) death caused by experimental intraocular pressure (IOP) elevation in mice. Methods We produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by β-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry. Results Losartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13), while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p glaucoma eyes (p = 0.007). Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP. Conclusions The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at the optic nerve head. PMID:26505191

  19. Image quality of the cat eye measured during retinal ganglion cell experiments.

    Science.gov (United States)

    Bonds, A B; Enroth-Cugell, C; Pinto, L H

    1972-01-01

    1. The modulation transfer function (MTF) of the dioptrics of fifteen cat eyes was determined. The aerial image, formed by the eye of a standard object (a 0.5-1.0 degrees annulus), was photographed. The transmission of the film negative was measured with a scanning microdensitometer to yield the light distribution within the aerial image. Correcting for the double passage, this experimentally determined light distribution and the known object light distribution were used to obtain the MTF, applying Fourier methods. Each MTF was used to calculate the light distribution within the retinal image of stimuli of various geometry used in experiments on retinal ganglion cells in the same eye.2. When the eye was equipped with an artificial pupil of the same size as that used in the neurophysiological experiments (4.0-4.8 mm diam.) the MTF had fallen to 0.5 at 2.43 c/deg. When the pupil was removed the MTF had fallen to 0.5 at a much lower spatial frequency (1.0 c/deg). This shows that even when one uses an artificial pupil too large to provide optimal image quality there is a vast improvement over using no pupil.3. These image quality measurements were prompted by the need to know the actual stimulus image in experiments on the functional organization of the receptive field, a need exemplified in this paper by a few specific physiological results. The full neurophysiological results appear in the next two papers.

  20. Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

    DEFF Research Database (Denmark)

    Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

    2018-01-01

    PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

  1. Light-induced retinal injury enhanced neurotrophins secretion and neurotrophic effect of mesenchymal stem cells in vitro

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2013-04-01

    Full Text Available PURPOSE: To investigate neurotrophins expression and neurotrophic effect change in mesenchymal stem cells (MSCs under different types of stimulation. METHODS: Rats were exposed in 10,000 lux white light to develop light-induced retinal injury. Supernatants of homogenized retina (SHR, either from normal or light-injured retina, were used to stimulate MSCs. Quantitative real time for polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA were conducted for analysis the expression change in basic fibroblast growth factor (bFGF, brain-derived neurotrophic factor (BDNF and ciliary neurotrophic factor (CNTF in MSCs after stimulation. Conditioned medium from SHR-stimulated MSCs and control MSCs were collected for evaluation their effect on retinal explants. RESULTS: Supernatants of homogenized retina from light-injured rats significantly promoted neurotrophins secretion from MSCs (p<0.01. Conditioned medium from mesenchymal stem cells stimulated by light-injured SHR significantly reduced DNA fragmentation (p<0.01, up-regulated bcl-2 (p<0.01 and down-regulated bax (p<0.01 in retinal explants, displaying enhanced protective effect. CONCLUSIONS: Light-induced retinal injury is able to enhance neurotrophins secretion from mesenchymal stem cells and promote the neurotrophic effect of mesenchymal stem cells.

  2. Differential behavioral outcomes following neonatal versus fetal human retinal pigment epithelial cell striatal implants in parkinsonian rats

    DEFF Research Database (Denmark)

    Russ, Kaspar; Flores, Joseph; Brudek, Tomasz

    2017-01-01

    Following the failure of a Phase II clinical study evaluating human retinal pigment epithelial (hRPE) cell implants as a potential treatment option for Parkinson's disease, speculation has centered on implant function and survival as possible contributors to the therapeutic outcomes. We recently ...

  3. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas.

    Science.gov (United States)

    Chintalapudi, Sumana R; Djenderedjian, Levon; Stiemke, Andrew B; Steinle, Jena J; Jablonski, Monica M; Morales-Tirado, Vanessa M

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

  4. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    Science.gov (United States)

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. PMID:27242509

  5. Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors

    Directory of Open Access Journals (Sweden)

    Tyler DiStefano

    2018-01-01

    Full Text Available Pluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25 reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

  6. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    OpenAIRE

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes ...

  7. Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes.

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    Dierk Wittig

    Full Text Available BACKGROUND: Tunneling nanotubes (TNTs may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. CONCLUSIONS AND SIGNIFICANCE: Our observations indicate that

  8. Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

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    Jordan J Toutounchian

    Full Text Available Radiation-induced damage to the retina triggers leukostasis, retinal endothelial cell (REC death, and subsequent hypoxia. Resultant ischemia leads to visual loss and compensatory retinal neovascularization (RNV. Using human RECs, we demonstrated that radiation induced leukocyte adhesion through mechanisms involving p38MAPK, p53, and ICAM-1 activation. Additional phenotypic changes included p38MAPK-dependent tyrosine phosphorylation of the focal adhesion scaffolding protein, paxillin (Tyr118. The quinic acid derivative KZ-41 lessened leukocyte adhesion and paxillin-dependent proliferation via inhibition of p38MAPK-p53-ICAM-1 signaling. Using the murine oxygen-induced retinopathy (OIR model, we examined the effect of KZ-41 on pathologic RNV. Daily ocular application of a KZ-41-loaded nanoemulsion significantly reduced both the avascular and neovascular areas in harvested retinal flat mounts when compared to the contralateral eye receiving vehicle alone. Our data highlight the potential benefit of KZ-41 in reducing both the retinal ischemia and neovascularization provoked by genotoxic insults. Further research into how quinic acid derivatives target and mitigate inflammation is needed to fully appreciate their therapeutic potential for the treatment of inflammatory retinal vasculopathies.

  9. Subpopulations of Bone Marrow Mesenchymal Stem Cells Exhibit Differential Effects in Delaying Retinal Degeneration.

    Science.gov (United States)

    Li, P; Tian, H; Li, Z; Wang, L; Gao, F; Ou, Q; Lian, C; Li, W; Jin, C; Zhang, J; Xu, J-Y; Wang, J; Zhang, J; Wang, F; Lu, L; Xu, G-T

    2016-01-01

    Bone marrow mesenchymal stem cells (BMSCs) have a therapeutic role in retinal degeneration (RD). However, heterogeneity of BMSCs may be associated with differential therapeutic effects in RD. In order to confirm this hypothesis, two subsets of rat BMSCs, termed rBMSC1 and rBMSC2, were obtained, characterized and functionally evaluated in the treatment of RD of Royal College of Surgeons (RCS) rats. Both subpopulations expressed mesenchymal stem cells (MSC) markers CD29 and CD90, but were negative for hemacyte antigen CD11b and CD45 expression. In comparison with rBMSC2, rBMSC1 showed higher rate of proliferation, stronger colony formation, and increased adipogenic potential, whereas rBMSC2 exhibited higher osteogenic potential. Microarray analysis showed differential gene expression patterns between rBMSC1 and rBMSC2, including functions related to proliferation, differentiation, immunoregulation, stem cell maintenance and division, survival and antiapoptosis. After subretinal transplantation in RCS rats, rBMSC1 showed stronger rescue effect than rBMSC2, including increased b-wave amplitude, restored retinal nuclear layer thickness, and decreased number of apoptotic photoreceptors, whereas the rescue function of rBMSC2 was essentially not better than the control. Histological analysis also demonstrated that rBMSC1 possessed a higher survival rate than rBMSC2 in subretinal space. In addition, treatment of basic fibroblast growth factor, an accompanying event in subretinal injection, triggered more robust increase in secretion of growth factors by rBMSC1 as compared to rBMSC2. Taken together, these results have suggested that the different therapeutic functions of BMSC subpopulations are attributed to their distinct survival capabilities and paracrine functions. The underlying mechanisms responsible for the different functions of BMSC subpopulation may lead to a new strategy for the treatment of RD.

  10. The Role of Bloom Index of Gelatin on the Interaction with Retinal Pigment Epithelial Cells

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    Jui Yang Lai

    2009-08-01

    Full Text Available Biocompatible materials are of considerable interest in the development of cell/drug delivery carriers for therapeutic applications. This paper investigates the effects of the Bloom index of gelatin on its interaction with retinal pigment epithelial (RPE cells. Following two days of culture of ARPE-19 cells with gelatin samples G75-100, G175, and G300, the in vitro biocompatibility was determined by cell proliferation and viability assays, and glutamate uptake measurements, as well as cytokine expression analyses. The mitochondrial dehydrogenase activity in the G300 groups was significantly lower than that of G75-100 and G175 groups. The Live/Dead assays also showed that the gelatin samples G300 induced mild cytotoxicity. In comparison with the treatment of gelatins with low Bloom index, the exposure to high Bloom strength gelatins markedly reduced the glutamate uptake capacity of ARPE-19 cells. One possible explanation for these observations is that the presence of gelatin samples G300 with high viscosity in the medium may affect the nutrient availability to cultured cells. The analyses of pro-inflammatory cytokine IL-6 expression at both mRNA and protein levels showed that the gelatins with low Bloom index caused less cellular inflammatory reaction and had more acceptable biocompatibility than their high Bloom strength counterparts. These findings suggest that the Bloom index gives influence on cellular responses to gelatin materials.

  11. Bone Marrow–Derived Cells Home to and Regenerate Retinal Pigment Epithelium after Injury

    Science.gov (United States)

    Harris, Jeffrey R.; Brown, Gary A. J.; Jorgensen, Marda; Kaushal, Shalesh; Ellis, E. Ann; Grant, Maria B.; Scott, Edward W.

    2013-01-01

    Purpose To determine whether hematopoietic stem and progenitor cells (HSCs/HPCs) can home to and regenerate the retinal pigment epithelium (RPE) after induced injury. Methods Enriched HSCs/HPCs from green fluorescent protein (gfp) transgenic mice were transplanted into irradiated recipient mice to track bone marrow–derived cells. Physical damage was induced by breaching Bruch’s membrane and inducing vascular endothelial growth factor A (VEGFa) expression to promote neovascularization. RPE damage was also induced by sodium iodate injection (40 mg/kg) into wild-type or albino C57Bl/6 mice. Cell morphology, gfp expression, the presence of the Y chromosome, and the presence of melanosomes were used to determine whether the injured RPE was being repaired by the donor bone marrow. Results Injury to the RPE recruits HSC/HPC–derived cells to incorporate into the RPE layer and differentiate into an RPE phenotype. A portion of the HSCs/HPCs adopt RPE morphology, express melanosomes, and integrate into the RPE without cell fusion. Conclusions HSCs/HPCs can migrate to the RPE layer after physical or chemical injury and regenerate a portion of the damaged cell layer. PMID:16639022

  12. Increased proliferation of late-born retinal progenitor cells by gestational lead exposure delays rod and bipolar cell differentiation.

    Science.gov (United States)

    Chaney, Shawnta Y; Mukherjee, Shradha; Giddabasappa, Anand; Rueda, Elda M; Hamilton, W Ryan; Johnson, Jerry E; Fox, Donald A

    2016-01-01

    Studies of neuronal development in the retina often examine the stages of proliferation, differentiation, and synaptic development, albeit independently. Our goal was to determine if a known neurotoxicant insult to a population of retinal progenitor cells (RPCs) would affect their eventual differentiation and synaptic development. To that end, we used our previously published human equivalent murine model of low-level gestational lead exposure (GLE). Children and animals with GLE exhibit increased scotopic electroretinogram a- and b-waves. Adult mice with GLE exhibit an increased number of late-born RPCs, a prolonged period of RPC proliferation, and an increased number of late-born rod photoreceptors and rod and cone bipolar cells (BCs), with no change in the number of late-born Müller glial cells or early-born neurons. The specific aims of this study were to determine whether increased and prolonged RPC proliferation alters the spatiotemporal differentiation and synaptic development of rods and BCs in early postnatal GLE retinas compared to control retinas. C57BL/6N mouse pups were exposed to lead acetate via drinking water throughout gestation and until postnatal day 10, which is equivalent to the human gestation period for retinal neurogenesis. RT-qPCR, immunohistochemical analysis, and western blots of well-characterized, cell-specific genes and proteins were performed at embryonic and early postnatal ages to assess rod and cone photoreceptor differentiation, rod and BC differentiation and synaptic development, and Müller glial cell differentiation. Real-time quantitative PCR (RT-qPCR) with the rod-specific transcription factors Nrl , Nr2e3 , and Crx and the rod-specific functional gene Rho , along with central retinal confocal studies with anti-recoverin and anti-rhodopsin antibodies, revealed a two-day delay in the differentiation of rod photoreceptors in GLE retinas. Rhodopsin immunoblots supported this conclusion. No changes in glutamine synthetase gene

  13. Quantitative and Topographical Analysis of the Losses of Cone Photoreceptors and Retinal Ganglion Cells Under Taurine Depletion.

    Science.gov (United States)

    Hadj-Saïd, Wahiba; Froger, Nicolas; Ivkovic, Ivana; Jiménez-López, Manuel; Dubus, Élisabeth; Dégardin-Chicaud, Julie; Simonutti, Manuel; Quénol, César; Neveux, Nathalie; Villegas-Pérez, María Paz; Agudo-Barriuso, Marta; Vidal-Sanz, Manuel; Sahel, Jose-Alain; Picaud, Serge; García-Ayuso, Diego

    2016-09-01

    Taurine depletion is known to induce photoreceptor degeneration and was recently found to also trigger retinal ganglion cell (RGC) loss similar to the retinal toxicity of vigabatrin. Our objective was to study the topographical loss of RGCs and cone photoreceptors, with a distinction between the two cone types (S- and L- cones) in an animal model of induced taurine depletion. We used the taurine transporter (Tau-T) inhibitor, guanidoethane sulfonate (GES), to induce taurine depletion at a concentration of 1% in the drinking water. Spectral-domain optical coherence tomography (SD-OCT) and electroretinograms (ERG) were performed on animals after 2 months of GES treatment administered through the drinking water. Retinas were dissected as wholemounts and immunodetection of Brn3a (RGC), S-opsin (S-cones), and L-opsin (L-cones) was performed. The number of Brn3a+ RGCs, and L- and S-opsin+ cones was automatically quantified and their retinal distribution studied using isodensity maps. The treatment resulted in a significant reduction in plasma taurine levels and a profound dysfunction of visual performance as shown by ERG recordings. Optical coherence tomography analysis revealed that the retina was thinner in the taurine-depleted group. S-opsin+cones were more affected (36%) than L-opsin+cones (27%) with greater cone cell loss in the dorsal area whereas RGC loss (12%) was uniformly distributed. This study confirms that taurine depletion causes RGC and cone loss. Electroretinograms results show that taurine depletion induces retinal dysfunction in photoreceptors and in the inner retina. It establishes a gradient of cell loss depending on the cell type from S-opsin+cones, L-opsin+cones, to RGCs. The greater cell loss in the dorsal retina and of the S-cone population may underline different cellular mechanisms of cellular degeneration and suggests that S-cones may be more sensitive to light-induced retinal toxicity enhanced by the taurine depletion.

  14. Assessment of Safety and Functional Efficacy of Stem Cell-Based Therapeutic Approaches Using Retinal Degenerative Animal Models

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    Tai-Chi Lin

    2017-01-01

    Full Text Available Dysfunction and death of retinal pigment epithelium (RPE and or photoreceptors can lead to irreversible vision loss. The eye represents an ideal microenvironment for stem cell-based therapy. It is considered an “immune privileged” site, and the number of cells needed for therapy is relatively low for the area of focused vision (macula. Further, surgical placement of stem cell-derived grafts (RPE, retinal progenitors, and photoreceptor precursors into the vitreous cavity or subretinal space has been well established. For preclinical tests, assessments of stem cell-derived graft survival and functionality are conducted in animal models by various noninvasive approaches and imaging modalities. In vivo experiments conducted in animal models based on replacing photoreceptors and/or RPE cells have shown survival and functionality of the transplanted cells, rescue of the host retina, and improvement of visual function. Based on the positive results obtained from these animal experiments, human clinical trials are being initiated. Despite such progress in stem cell research, ethical, regulatory, safety, and technical difficulties still remain a challenge for the transformation of this technique into a standard clinical approach. In this review, the current status of preclinical safety and efficacy studies for retinal cell replacement therapies conducted in animal models will be discussed.

  15. Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

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    Yu Zhu

    Full Text Available A goal in human embryonic stem cell (hESC research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

  16. Vitreous humor and albumin augment the proliferation of cultured retinal precursor cells

    DEFF Research Database (Denmark)

    Yang, Jing; Klassen, Henry; Pries, Mette

    2008-01-01

    concentrations of vitreous fluid supplementation was quantified by using a (3)H-thymidine incorporation assay. Active components of vitreous fluid were partially characterized by gel filtration chromatography (GFC) and UV spectral analysis. The effect of each vitreous fraction on proliferation was determined...... Da, consistent with ascorbic acid. Ascorbic acid was confirmed in vitreous fluid by UV spectral analysis. Growth-augmenting activity was present in higher molecular mass vitreous fractions, consistent with protein components. Albumin, the major protein in vitreous fluid, was found to augment...... proliferation. Because vitreous-associated augmentation of retinal precursor proliferation remains an epidermal growth factor-dependent phenomenon, the proliferative status of transplanted cells in the vitreous cavity is likely determined by a combination of factors. (c) 2008 Wiley-Liss, Inc....

  17. Synchronized Firings in Retinal Ganglion Cells in Response to Natural Stimulation

    International Nuclear Information System (INIS)

    Zhang Ying-Ying; Xiao Lei; Liu Wen-Zhong; Gong Hai-Qing; Liang Pei-Ji

    2011-01-01

    The response of synchronously firing groups of population retinal ganglion cells (RGCs) to natural movies (NMs) and pseudo-random white-noise checker-board flickering (CB, as control) are investigated using an information-theoretic algorithm. The main results are: (1) the population RGCs tend to fire in synchrony far more frequently than expected by chance during both NM and CB stimulation; (2) more synchronous groups could be formed and each group contains more neurons under NM than CB stimulation; (3) the individual neurons also participate in more groups and have more distinct partners in NM than CB stimulation. All these results suggest that the synchronized firings in RGCs are more extensive and diverse, which may account for more effective information processing in representing the natural visual environment. (cross-disciplinary physics and related areas of science and technology)

  18. The oxysterol 27-hydroxycholesterol increases β-amyloid and oxidative stress in retinal pigment epithelial cells

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    Dasari Bhanu

    2010-09-01

    Full Text Available Abstract Background Alzheimer's disease (AD and age-related macular degeneration (AMD share several pathological features including β-amyloid (Aβ peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD. Methods ARPE-19 cells were treated with 0, 10 or 25 μM 27-OHC for 24 hours. Levels of Aβ peptide, mitochondrial and endoplasmic reticulum (ER stress markers, Ca2+ homeostasis, glutathione depletion, reactive oxygen species (ROS generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays. Results 27-OHC dose-dependently increased Aβ peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP, reduced mitochondrial membrane potential, triggered Ca2+ dyshomeostasis, increased levels of the nuclear factor κB (NFκB and heme-oxygenase 1 (HO-1, two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death. Conclusions The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aβ accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for

  19. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    Science.gov (United States)

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  20. Retinal astrocytes pretreated with NOD2 and TLR2 ligands activate uveitogenic T cells.

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    Guomin Jiang

    Full Text Available On entering the tissues, infiltrating autoreactive T cells must be reactivated locally to gain pathogenic activity. We have previously reported that, when activated by Toll-like receptor 3 (TLR3 and TLR4 ligands, retinal astrocytes (RACs are able to function as antigen-presenting cells to re-activate uveitogenic T cells and allow responder T cells to induce uveitis in mice. In the present study, we found that, although the triggering of TLR2 or nucleotide-binding oligomerization domain receptor 2 (NOD2 alone did not activate RACs, their combined triggering induced RACs with the phenotypes required to efficiently re-activate interphotoreceptor retinoid-binding protein (IRBP-specific T cells. The synergistic effect of TLR2 and NOD2 ligands on RAC activation might be explained by the observations that bacterial lipoprotein (BLP, a TLR2 ligand was able to upregulate NOD2 expression and the combination of BLP and muramyldipeptide (MDP, a NOD2 ligand enhanced the expression of RICK (Rip2, the signaling molecule of NOD2. Moreover, the synergistic effect of MDP and BLP on RACs was lost when the RACs were derived from NOD2 knockout mice or were pre-treated with Rip2 antagonist. Thus, our data suggest that exogenous or endogenous molecules acting on both TLR2 and NOD2 on RACs might have an enhancing effect on susceptibility to autoimmune uveitis.

  1. Stanniocalcin-1 protects retinal ganglion cells by inhibiting apoptosis and oxidative damage.

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    Sang Jin Kim

    Full Text Available Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs, a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1 can prevent loss of RGCs in the rat retina with optic nerve transection (ONT and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.

  2. Meis1 regulates Foxn4 expression during retinal progenitor cell differentiation

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    Mohammed M. Islam

    2013-09-01

    The transcription factor forkhead box N4 (Foxn4 is a key regulator in a variety of biological processes during development. In particular, Foxn4 plays an essential role in the genesis of horizontal and amacrine neurons from neural progenitors in the vertebrate retina. Although the functions of Foxn4 have been well established, the transcriptional regulation of Foxn4 expression during progenitor cell differentiation remains unclear. Here, we report that an evolutionarily conserved 129 bp noncoding DNA fragment (Foxn4CR4.2 or CR4.2, located ∼26 kb upstream of Foxn4 transcription start site, functions as a cis-element for Foxn4 regulation. CR4.2 directs gene expression in Foxn4-positive cells, primarily in progenitors, differentiating horizontal and amacrine cells. We further determined that the gene regulatory activity of CR4.2 is modulated by Meis1 binding motif, which is bound and activated by Meis1 transcription factor. Deletion of the Meis1 binding motif or knockdown of Meis1 expression abolishes the gene regulatory activity of CR4.2. In addition, knockdown of Meis1 expression diminishes the endogenous Foxn4 expression and affects cell lineage development. Together, we demonstrate that CR4.2 and its interacting Meis1 transcription factor play important roles in regulating Foxn4 expression during early retinogenesis. These findings provide new insights into molecular mechanisms that govern gene regulation in retinal progenitors and specific cell lineage development.

  3. RBX2 maintains final retinal cell position in a DAB1-dependent and -independent fashion.

    Science.gov (United States)

    Fairchild, Corinne L; Hino, Keiko; Han, Jisoo S; Miltner, Adam M; Peinado Allina, Gabriel; Brown, Caileigh E; Burns, Marie E; La Torre, Anna; Simó, Sergi

    2018-02-02

    The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function. © 2018. Published by The Company of Biologists Ltd.

  4. Mef2d is essential for the maturation and integrity of retinal photoreceptor and bipolar cells.

    Science.gov (United States)

    Omori, Yoshihiro; Kitamura, Tamiki; Yoshida, Satoyo; Kuwahara, Ryusuke; Chaya, Taro; Irie, Shoichi; Furukawa, Takahisa

    2015-05-01

    Mef2 transcription factors play a crucial role in cardiac and skeletal muscle differentiation. We found that Mef2d is highly expressed in the mouse retina and its loss causes photoreceptor degeneration similar to that observed in human retinitis pigmentosa patients. Electroretinograms (ERGs) were severely impaired in Mef2d-/- mice. Immunohistochemistry showed that photoreceptor and bipolar cell synapse protein levels severely decreased in the Mef2d-/- retina. Expression profiling by microarray analysis showed that Mef2d is required for the expression of various genes in photoreceptor and bipolar cells, including cone arrestin, Guca1b, Pde6h and Cacna1s, which encode outer segment and synapse proteins. We also observed that Mef2d synergistically activates the cone arrestin (Arr3) promoter with Crx, suggesting that functional cooperation between Mef2d and Crx is important for photoreceptor cell gene regulation. Taken together, our results show that Mef2d is essential for photoreceptor and bipolar cell gene expression, either independently or cooperatively with Crx. © 2015 Institution for Protein Research. Genes to Cells published by Wiley Publishing Asia Pty Ltd and the Molecular Biology Society of Japan.

  5. Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

    Science.gov (United States)

    Zhao, Wen-Jie; Zhang, Min; Miao, Yanying; Yang, Xiong-Li; Wang, Zhongfeng

    2010-07-15

    In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner

  6. Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

    Directory of Open Access Journals (Sweden)

    Alena Z Minton

    Full Text Available Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1 is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B receptors in the retina, mainly in retinal ganglion cells (RGCs, nerve fiber layer (NFL, and also in the inner plexiform layer (IPL and inner nuclear layer (INL. To determine the role of ET(B receptors in neurodegeneration, Wistar-Kyoto wild type (WT and ET(B receptor-deficient (KO rats were subjected to retrograde labeling with Fluoro-Gold (FG, following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

  7. Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma.

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    Harry A Quigley

    Full Text Available To determine if oral losartan treatment decreases the retinal ganglion cell (RGC death caused by experimental intraocular pressure (IOP elevation in mice.We produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by β-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry.Losartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13, while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p < 0.0001. The lower RGC loss with losartan was significantly less than the loss with spironolactone or enalapril (regression model p = 0.001; drug treatment group term p = 0.01. Both losartan and enalapril significantly lowered blood pressure (p< 0.001, but losartan was protective, while enalapril led to worse than water-treated RGC loss. RGC loss after crush injury was unaffected by losartan treatment (difference from control p = 0.9. Survival of RGC in cell culture was not prolonged by sartan treatment. Axonal transport blockade after 3 day IOP elevations was less in losartan-treated than in control glaucoma eyes (p = 0.007. Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP.The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at

  8. Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.

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    Omori, Yoshihiro; Araki, Fumiyuki; Chaya, Taro; Kajimura, Naoko; Irie, Shoichi; Terada, Koji; Muranishi, Yuki; Tsujii, Toshinori; Ueno, Shinji; Koyasu, Toshiyuki; Tamaki, Yasuhiro; Kondo, Mineo; Amano, Shiro; Furukawa, Takahisa

    2012-05-02

    Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.

  9. AMP is an adenosine A1 receptor agonist.

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    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

    2012-02-17

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  10. Retinal dendritic cell recruitment, but not function, was inhibited in MyD88 and TRIF deficient mice.

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    Heuss, Neal D; Pierson, Mark J; Montaniel, Kim Ramil C; McPherson, Scott W; Lehmann, Ute; Hussong, Stacy A; Ferrington, Deborah A; Low, Walter C; Gregerson, Dale S

    2014-08-13

    Immune system cells are known to affect loss of neurons due to injury or disease. Recruitment of immune cells following retinal/CNS injury has been shown to affect the health and survival of neurons in several models. We detected close, physical contact between dendritic cells and retinal ganglion cells following an optic nerve crush, and sought to understand the underlying mechanisms. CD11c-DTR/GFP mice producing a chimeric protein of diphtheria toxin receptor (DTR) and GFP from a transgenic CD11c promoter were used in conjunction with mice deficient in MyD88 and/or TRIF. Retinal ganglion cell injury was induced by an optic nerve crush, and the resulting interactions of the GFPhi cells and retinal ganglion cells were examined. Recruitment of GFPhi dendritic cells to the retina was significantly compromised in MyD88 and TRIF knockout mice. GFPhi dendritic cells played a significant role in clearing fluorescent-labeled retinal ganglion cells post-injury in the CD11c-DTR/GFP mice. In the TRIF and MyD88 deficient mice, the resting level of GFPhi dendritic cells was lower, and their influx was reduced following the optic nerve crush injury. The reduction in GFPhi dendritic cell numbers led to their replacement in the uptake of fluorescent-labeled debris by GFPlo microglia/macrophages. Depletion of GFPhi dendritic cells by treatment with diphtheria toxin also led to their displacement by GFPlo microglia/macrophages, which then assumed close contact with the injured neurons. The contribution of recruited cells to the injury response was substantial, and regulated by MyD88 and TRIF. However, the presence of these adaptor proteins was not required for interaction with neurons, or the phagocytosis of debris. The data suggested a two-niche model in which resident microglia were maintained at a constant level post-optic nerve crush, while the injury-stimulated recruitment of dendritic cells and macrophages led to their transient appearance in numbers equivalent to or greater

  11. Noninvasive near infrared autofluorescence imaging of retinal pigment epithelial cells in the human retina using adaptive optics.

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    Liu, Tao; Jung, HaeWon; Liu, Jianfei; Droettboom, Michael; Tam, Johnny

    2017-10-01

    The retinal pigment epithelial (RPE) cells contain intrinsic fluorophores that can be visualized using infrared autofluorescence (IRAF). Although IRAF is routinely utilized in the clinic for visualizing retinal health and disease, currently, it is not possible to discern cellular details using IRAF due to limits in resolution. We demonstrate that the combination of adaptive optics (AO) with IRAF (AO-IRAF) enables higher-resolution imaging of the IRAF signal, revealing the RPE mosaic in the living human eye. Quantitative analysis of visualized RPE cells in 10 healthy subjects across various eccentricities demonstrates the possibility for in vivo density measurements of RPE cells, which range from 6505 to 5388 cells/mm 2 for the areas measured (peaking at the fovea). We also identified cone photoreceptors in relation to underlying RPE cells, and found that RPE cells support on average up to 18.74 cone photoreceptors in the fovea down to an average of 1.03 cone photoreceptors per RPE cell at an eccentricity of 6 mm. Clinical application of AO-IRAF to a patient with retinitis pigmentosa illustrates the potential for AO-IRAF imaging to become a valuable complementary approach to the current landscape of high resolution imaging modalities.

  12. Melanopsin expressing human retinal ganglion cells: Subtypes, distribution, and intraretinal connectivity.

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    Hannibal, Jens; Christiansen, Anders Tolstrup; Heegaard, Steffen; Fahrenkrug, Jan; Kiilgaard, Jens Folke

    2017-06-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N- and C-terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1 (GDM1)." Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin-ir (0.63-0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin-ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin-ir RGCs, which most likely are involved in different functions. © 2017 Wiley Periodicals, Inc.

  13. Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments.

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    Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

    2016-10-01

    Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation

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    Hytti, Maria; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Kauppinen, Anu

    2015-01-01

    Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD. PMID:26619957

  15. Effects of mechanical stress and vitreous samples in retinal pigment epithelial cells

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    Takahashi, Eri, E-mail: eritakahashi@fc.kuh.kumamoto-u.ac.jp; Fukushima, Ayako; Haga, Akira; Inomata, Yasuya; Ito, Yasuhiro; Fukushima, Mikiko; Tanihara, Hidenobu

    2016-02-12

    In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid. We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples. In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis.

  16. Increased Number of Circulating CD8/CD26 T Cells in the Blood of Duchenne Muscular Dystrophy Patients Is Associated with Augmented Binding of Adenosine Deaminase and Higher Muscular Strength Scores

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    Jonathan H. Soslow

    2017-12-01

    Full Text Available Duchenne muscular dystrophy (DMD is an X-linked disorder that leads to cardiac and skeletal myopathy. The complex immune activation in boys with DMD is incompletely understood. To better understand the contribution of the immune system into the progression of DMD, we performed a systematic characterization of immune cell subpopulations obtained from peripheral blood of DMD subjects and control donors. We found that the number of CD8 cells expressing CD26 (also known as adenosine deaminase complexing protein 2 was increased in DMD subjects compared to control. No differences, however, were found in the levels of circulating factors associated with pro-inflammatory activation of CD8/CD26 cells, such as tumor necrosis factor-α (TNFα, granzyme B, and interferon-γ (IFNγ. The number of CD8/CD26 cells correlated directly with quantitative muscle testing (QMT in DMD subjects. Since CD26 mediates binding of adenosine deaminase (ADA to the T cell surface, we tested ADA-binding capacity of CD8/CD26 cells and the activity of bound ADA. We found that mononuclear cells (MNC obtained from DMD subjects with an increased number of CD8/CD26 T cells had a greater capacity to bind ADA. In addition, these MNC demonstrated increased hydrolytic deamination of adenosine to inosine. Altogether, our data demonstrated that (1 an increased number of circulating CD8/CD26 T cells is associated with preservation of muscle strength in DMD subjects, and (2 CD8/CD26 T cells from DMD subjects mediated degradation of adenosine by adenosine deaminase. These results support a role for T cells in slowing the decline in skeletal muscle function, and a need for further investigation into contribution of CD8/CD26 T cells in the regulation of chronic inflammation associated with DMD.

  17. Patient-specific induced pluripotent stem cells to evaluate the pathophysiology of TRNT1-associated Retinitis pigmentosa

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    Tasneem P. Sharma

    2017-05-01

    Full Text Available Retinitis pigmentosa (RP is a heterogeneous group of monogenic disorders characterized by progressive death of the light-sensing photoreceptor cells of the outer neural retina. We recently identified novel hypomorphic mutations in the tRNA Nucleotidyl Transferase, CCA-Adding 1 (TRNT1 gene that cause early-onset RP. To model this disease in vitro, we generated patient-specific iPSCs and iPSC-derived retinal organoids from dermal fibroblasts of patients with molecularly confirmed TRNT1-associated RP. Pluripotency was confirmed using rt-PCR, immunocytochemistry, and a TaqMan Scorecard Assay. Mutations in TRNT1 caused reduced levels of full-length TRNT1 protein and expression of a truncated smaller protein in both patient-specific iPSCs and iPSC-derived retinal organoids. Patient-specific iPSCs and iPSC-derived retinal organoids exhibited a deficit in autophagy, as evidenced by aberrant accumulation of LC3-II and elevated levels of oxidative stress. Autologous stem cell-based disease modeling will provide a platform for testing multiple avenues of treatment in patients suffering from TRNT1-associated RP.

  18. Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate

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    Molina, Anthony J A; Verzi, Michael P; Birnbaum, Andrea D; Yamoah, Ebenezer N; Hammar, Katherine; Smith, Peter J S; Malchow, Robert Paul

    2004-01-01

    Self-referencing H+-selective microelectrodes were used to measure extracellular H+ fluxes from horizontal cells isolated from the skate retina. A standing H+ flux was detected from quiescent cells, indicating a higher concentration of free hydrogen ions near the extracellular surface of the cell as compared to the surrounding solution. The standing H+ flux was reduced by removal of extracellular sodium or application of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), suggesting activity of a Na+–H+ exchanger. Glutamate decreased H+ flux, lowering the concentration of free hydrogen ions around the cell. AMPA/kainate receptor agonists mimicked the response, and the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) eliminated the effects of glutamate and kainate. Metabotropic glutamate agonists were without effect. Glutamate-induced alterations in H+ flux required extracellular calcium, and were abolished when cells were bathed in an alkaline Ringer solution. Increasing intracellular calcium by photolysis of the caged calcium compound NP-EGTA also altered extracellular H+ flux. Immunocytochemical localization of the plasmalemma Ca2+–H+-ATPase (PMCA pump) revealed intense labelling within the outer plexiform layer and on isolated horizontal cells. Our results suggest that glutamate modulation of H+ flux arises from calcium entry into cells with subsequent activation of the plasmalemma Ca2+–H+-ATPase. These neurotransmitter-induced changes in extracellular pH have the potential to play a modulatory role in synaptic processing in the outer retina. However, our findings argue against the hypothesis that hydrogen ions released by horizontal cells normally act as the inhibitory feedback neurotransmitter onto photoreceptor synaptic terminals to create the surround portion of the centre-surround receptive fields of retinal neurones. PMID:15272044

  19. Protective effects of triptolide on retinal ganglion cells in a rat model of chronic glaucoma

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    Yang F

    2015-11-01

    Full Text Available Fan Yang, Dongmei Wang, Lingling Wu, Ying Li Ophthalmology Department, Peking University Third Hospital, Beijing, People’s Republic of China Purpose: To study the effects of triptolide, a Chinese herb extract, on retinal ganglion cells (RGCs in a rat model of chronic glaucoma.Methods: Eighty Wistar rats were randomly divided into triptolide group (n=40 and normal saline (NS group (n=40. Angle photocoagulation was used to establish the model of glaucoma, with right eye as laser treated eye and left eye as control eye. Triptolide group received triptolide intraperitoneally daily, while NS group received NS. Intraocular pressure (IOP, anti-CD11b immunofluorescent stain in retina and optic nerve, RGCs count with Nissel stain and microglia count with anti-CD11b immunofluorescence stain in retina flat mounts, retinal tumor necrosis factor (TNF-α mRNA detection by reverse transcription–polymerase chain reaction, and double immunofluorescent labeling with anti-TNF-α and anti-CD11b in retinal frozen section were performed.Results: Mean IOP of the laser treated eyes significantly increased 3 weeks after photocoagulation (P<0.05, with no statistical difference between the two groups (P>0.05. RGCs survival in the laser treated eyes was significantly improved in the triptolide group than the NS group (P<0.05. Microglia count in superficial retina of the laser treated eyes was significantly less in the triptolide group (30.40±4.90 than the NS group (35.06±7.59 (P<0.05. TNF-α mRNA expression in the retina of the laser treated eyes in the triptolide group decreased by 60% compared with that in the NS group (P<0.01. The double immunofluorescent labeling showed that TNF-α was mainly distributed around the microglia.Conclusion: Triptolide improved RGCs survival in this rat model of chronic glaucoma, which did not depend on IOP decrease but might be exerted by inhibiting microglia activities and reducing TNF-α secretion. Keywords: glaucoma, triptolide

  20. The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue

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    Richard J. Davis

    2017-07-01

    Full Text Available Age-related macular degeneration (AMD is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC-derived RPE cells (RPESC-RPE preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

  1. Role of transglutaminase 2 in A1 adenosine receptor- and β2-adrenoceptor-mediated pharmacological pre- and post-conditioning against hypoxia-reoxygenation-induced cell death in H9c2 cells.

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    Vyas, Falguni S; Nelson, Carl P; Dickenson, John M

    2018-01-15

    Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A 1 adenosine receptor and β 2 -adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A 1 adenosine receptor and β 2 -adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O 2 ) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N 6 -cyclopentyladenosine (CPA; A 1 adenosine receptor agonist), formoterol (β 2 -adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (G i/o -protein inhibitor), DPCPX (A 1 adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A 1 adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β 2 -adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A 1 adenosine receptor and β 2 -adrenoceptor

  2. Retinal Pigmented Epithelial Cells Obtained from Human Induced Pluripotent Stem Cells Possess Functional Visual Cycle Enzymes in Vitro and in Vivo*

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    Maeda, Tadao; Lee, Mee Jee; Palczewska, Grazyna; Marsili, Stefania; Tesar, Paul J.; Palczewski, Krzysztof; Takahashi, Masayo; Maeda, Akiko

    2013-01-01

    Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat−/− and Rpe65−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat−/− and Rpe65−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases. PMID:24129572

  3. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress

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    Wang, Kaijun [Eye Center, The 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou (China); Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou (China); Jiang, Yiqian [The First People Hospital of Xiaoshan, Hangzhou (China); Wang, Wei; Ma, Jian [Eye Center, The 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou (China); Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou (China); Chen, Min, E-mail: eyedrchenminzj@163.com [Eye Center, The 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou (China); Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou (China)

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H{sub 2}O{sub 2}) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H{sub 2}O{sub 2}-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H{sub 2}O{sub 2} were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H{sub 2}O{sub 2}. Reversely, escin was more potent against H{sub 2}O{sub 2} damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H{sub 2}O{sub 2} was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.

  4. Dominant inheritance of retinal ganglion cell resistance to optic nerve crush in mice

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    Schlamp Cassandra L

    2007-03-01

    Full Text Available Abstract Background Several neurodegenerative diseases are influenced by complex genetics that affect an individual's susceptibility, disease severity, and rate of progression. One such disease is glaucoma, a chronic neurodegenerative condition of the eye that targets and stimulates apoptosis of CNS neurons called retinal ganglion cells. Since ganglion cell death is intrinsic, it is reasonable that the genes that control this process may contribute to the complex genetics that affect ganglion cell susceptibility to disease. To determine if genetic background influences susceptibility to optic nerve damage, leading to ganglion cell death, we performed optic nerve crush on 15 different inbred lines of mice and measured ganglion cell loss. Resistant and susceptible strains were used in a reciprocal breeding strategy to examine the inheritance pattern of the resistance phenotype. Because earlier studies had implicated Bax as a susceptibility allele for ganglion cell death in the chronic neurodegenerative disease glaucoma, we conducted allelic segregation analysis and mRNA quantification to assess this gene as a candidate for the cell death phenotype. Results Inbred lines showed varying levels of susceptibility to optic nerve crush. DBA/2J mice were most resistant and BALB/cByJ mice were most susceptible. F1 mice from these lines inherited the DBA/2J phenotype, while N2 backcross mice exhibited the BALB/cByJ phenotype. F2 mice exhibited an intermediate phenotype. A Wright Formula calculation suggested as few as 2 dominant loci were linked to the resistance phenotype, which was corroborated by a Punnett Square analysis of the distribution of the mean phenotype in each cross. The levels of latent Bax mRNA were the same in both lines, and Bax alleles did not segregate with phenotype in N2 and F2 mice. Conclusion Inbred mice show different levels of resistance to optic nerve crush. The resistance phenotype is heritable in a dominant fashion involving

  5. The patterning of retinal horizontal cells: normalizing the regularity index enhances the detection of genomic linkage

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    Patrick W. Keeley

    2014-10-01

    Full Text Available Retinal neurons are often arranged as non-random distributions called mosaics, as their somata minimize proximity to neighboring cells of the same type. The horizontal cells serve as an example of such a mosaic, but little is known about the developmental mechanisms that underlie their patterning. To identify genes involved in this process, we have used three different spatial statistics to assess the patterning of the horizontal cell mosaic across a panel of genetically distinct recombinant inbred strains. To avoid the confounding effect cell density, which varies two-fold across these different strains, we computed the real/random regularity ratio, expressing the regularity of a mosaic relative to a randomly distributed simulation of similarly sized cells. To test whether this latter statistic better reflects the variation in biological processes that contribute to horizontal cell spacing, we subsequently compared the genetic linkage for each of these two traits, the regularity index and the real/random regularity ratio, each computed from the distribution of nearest neighbor (NN distances and from the Voronoi domain (VD areas. Finally, we compared each of these analyses with another index of patterning, the packing factor. Variation in the regularity indexes, as well as their real/random regularity ratios, and the packing factor, mapped quantitative trait loci (QTL to the distal ends of Chromosomes 1 and 14. For the NN and VD analyses, we found that the degree of linkage was greater when using the real/random regularity ratio rather than the respective regularity index. Using informatic resources, we narrow the list of prospective genes positioned at these two intervals to a small collection of six genes that warrant further investigation to determine their potential role in shaping the patterning of the horizontal cell mosaic.

  6. Reversal of retinal and optic disc ischemia in a patient with sickle cell trait and glaucoma secondary to traumatic hyphema.

    Science.gov (United States)

    Wax, M B; Ridley, M E; Magargal, L E

    1982-07-01

    A 14-year-old black boy with sickle cell trait, who sustained a traumatic hyphema, developed moderately elevated intraocular pressure that failed to respond to carbonic anhydrase inhibitors and osmotic agents. On the tenth postinjury day, a sudden increased cupping of the optic disc and partial central retinal artery obstruction caused painless loss of vision. Reversal of the cupping, the retinal ischemia, and the intraocular pressure was documented following anterior chamber paracentesis, and visual acuity returned to 6/6. Pathophysiology of the posterior ischemia is discussed. This case documents the potentially debilitating course of traumatic hyphema in "benign" sickle cell trait and its avoidance with proper management. The authors endorse recent suggestions for careful observation of any sickle cell patient with traumatic hyphema, and recommend anterior chamber paracentesis, supplemental oxygen, and avoidance of osmotic agents, if secondary glaucoma develops following the initial trauma.

  7. Hydrostatic pressure does not cause detectable changes in survival of human retinal ganglion cells.

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    Andrew Osborne

    Full Text Available Elevated intraocular pressure (IOP is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP. The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC survival in the human retina was investigated.A chamber was designed to expose cells to increased HP (constant and fluctuating. Accurate pressure control (10-100 mmHg was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs from donor eyes (<24 h post mortem were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD. Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1 and RGC number by immunohistochemistry (NeuN. Activated p38 and JNK were detected by Western blot.Exposure of HORCs to constant (60 mmHg or fluctuating (10-100 mmHg; 1 cycle/min pressure for 24 or 48 h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1 or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24 h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100 mmHg; 1 cycle/min for 15, 30, 60 and 90 min durations, whereas OGD (3 h increased activation of p38 and JNK, remaining elevated for 90 min post-OGD.Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.

  8. Interspike Interval Based Filtering of Directional Selective Retinal Ganglion Cells Spike Trains

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    Aurel Vasile Martiniuc

    2012-01-01

    Full Text Available The information regarding visual stimulus is encoded in spike trains at the output of retina by retinal ganglion cells (RGCs. Among these, the directional selective cells (DSRGC are signaling the direction of stimulus motion. DSRGCs' spike trains show accentuated periods of short interspike intervals (ISIs framed by periods of isolated spikes. Here we use two types of visual stimulus, white noise and drifting bars, and show that short ISI spikes of DSRGCs spike trains are more often correlated to their preferred stimulus feature (that is, the direction of stimulus motion and carry more information than longer ISI spikes. Firstly, our results show that correlation between stimulus and recorded neuronal response is best at short ISI spiking activity and decrease as ISI becomes larger. We then used grating bars stimulus and found that as ISI becomes shorter the directional selectivity is better and information rates are higher. Interestingly, for the less encountered type of DSRGC, known as ON-DSRGC, short ISI distribution and information rates revealed consistent differences when compared with the other directional selective cell type, the ON-OFF DSRGC. However, these findings suggest that ISI-based temporal filtering integrates a mechanism for visual information processing at the output of retina toward higher stages within early visual system.

  9. Stem Cell Ophthalmology Treatment Study (SCOTS for retinal and optic nerve diseases: a preliminary report

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    Jeffrey N Weiss

    2015-01-01

    Full Text Available In this report, we present the results of a single patient with optic neuropathy treated within the Stem Cell Ophthalmology Treatment Study (SCOTS. SCOTS is an Institutional Review Board approved clinical trial and is the largest ophthalmology stem cell study registered at the National Institutes of Health to date- www.clinicaltrials.gov Identifier NCT 01920867. SCOTS utilizes autologous bone marrow-derived stem cells in the treatment of optic nerve and retinal diseases. Pre- and post-treatment comprehensive eye exams were independently performed at the Wilmer Eye Institute at the Johns Hopkins Hospital, USA. A 27 year old female patient had lost vision approximately 5 years prior to enrollment in SCOTS. Pre-treatment best-corrected visual acuity at the Wilmer Eye Institute was 20/800 Right Eye (OD and 20/4,000 Left Eye (OS. Four months following treatment in SCOTS, the central visual acuity had improved to 20/100 OD and 20/40 OS.

  10. Autophagy in retinal ganglion cells in a rhesus monkey chronic hypertensive glaucoma model.

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    Shuifeng Deng

    Full Text Available Primary open angle glaucoma (POAG is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs. In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi and late or degraded autophagic vacuoles (AVd accumulated in the ganglion cell layer (GCL and in the inner plexiform layer (IPL as determined by transmission electron microscopy (TEM analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1 and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.

  11. Loss of Melanopsin-Expressing Retinal Ganglion Cells in Severely Staged Glaucoma Patients

    DEFF Research Database (Denmark)

    Obara, Elisabeth Anne; Hannibal, Jens; Heegaard, Steffen

    2016-01-01

    Purpose: Multiple studies have shown overwhelming evidence supporting the impairment of melanopsin function due to glaucoma. However, few studies have been carried out in humans analyzing the histology of melanopsin-expressing retinal ganglion cells (mRGCs) in retinas with glaucoma. The aim...... of this study was to analyze the pattern of expression of mRGCs relative to RGCs in the normal retina and retinas harboring varying stages of glaucoma. Methods: Paraffin-embedded human donor eyes with glaucoma (n = 11) and age-matched controls (n = 10) were obtained from Department of Pathology at Rigshospital...... difference was observed in mRGC expression in the normal retinas and mild-staged retinas with glaucoma; the densities of mRGCs were 3.08 ± 0.47 and 3.00 ± 0.13 cell counts/mm2, respectively. However, the severely staged retinas with glaucoma showed a significant loss in mRGCs density, 1.09 ± 0.35 cell counts...

  12. Delayed-rectifier K channels contribute to contrast adaptation in mammalian retinal ganglion cells.

    Science.gov (United States)

    Weick, Michael; Demb, Jonathan B

    2011-07-14

    Retinal ganglion cells adapt by reducing their sensitivity during periods of high contrast. Contrast adaptation in the firing response depends on both presynaptic and intrinsic mechanisms. Here, we investigated intrinsic mechanisms for contrast adaptation in OFF Alpha ganglion cells in the in vitro guinea pig retina. Using either visual stimulation or current injection, we show that brief depolarization evoked spiking and suppressed firing during subsequent depolarization. The suppression could be explained by Na channel inactivation, as shown in salamander cells. However, brief hyperpolarization in the physiological range (5-10 mV) also suppressed firing during subsequent depolarization. This suppression was selectively sensitive to blockers of delayed-rectifier K channels (K(DR)). In somatic membrane patches, we observed tetraethylammonium-sensitive K(DR) currents that activated near -25 mV. Recovery from inactivation occurred at potentials hyperpolarized to V(rest). Brief periods of hyperpolarization apparently remove K(DR) inactivation and thereby increase the channel pool available to suppress excitability during subsequent depolarization. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry.

    Science.gov (United States)

    Chintalapudi, Sumana R; Patel, Need N; Goldsmith, Zachary K; Djenderedjian, Levon; Wang, Xiang Di; Marion, Tony N; Jablonski, Monica M; Morales-Tirado, Vanessa M

    2017-07-05

    Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

  14. Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins.

    Science.gov (United States)

    Thomas, Sara E; Harrison, Earl H

    2016-10-01

    The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  15. Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors.

    Science.gov (United States)

    Iwamoto, K; Mata, D; Linn, D M; Linn, C L

    2013-05-01

    Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 μM ACh, 100 μM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double

  16. Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

    Science.gov (United States)

    la Marca, Giancarlo; Canessa, Clementina; Giocaliere, Elisa; Romano, Francesca; Duse, Marzia; Malvagia, Sabrina; Lippi, Francesca; Funghini, Silvia; Bianchi, Leila; Della Bona, Maria Luisa; Valleriani, Claudia; Ombrone, Daniela; Moriondo, Maria; Villanelli, Fabio; Speckmann, Carsten; Adams, Stuart; Gaspar, Bobby H; Hershfield, Michael; Santisteban, Ines; Fairbanks, Lynette; Ragusa, Giovanni; Resti, Massimo; de Martino, Maurizio; Guerrini, Renzo; Azzari, Chiara

    2013-06-01

    Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 μmol/L (normal value, <1.5 μmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 μmol/L (normal value, <0.07 μmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  17. Effects of melatonin and its receptor antagonist on retinal pigment epithelial cells against hydrogen peroxide damage

    Science.gov (United States)

    Rosen, Richard B.; Hu, Dan-Ning; Chen, Min; McCormick, Steven A.; Walsh, Joseph

    2012-01-01

    Purpose Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H2O2 damage. Methods Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H2O2. Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate. Results H2O2 at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H2O2. Cell viability of RPE cells pretreated with 10−10, 10−8, 10−6, and 10−4 M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H2O2 alone (all p<0.05). Using cells cultured without H2O2 as the control, cell viability of cells treated with H2O2 after pretreatment with 10−10-10−4 M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H2O2. Luzindole completely blocked melatonin’s protective effects at low concentrations of melatonin (10−10-10−8 M) but not at high concentrations (10−6-10−4 M). Conclusions Melatonin has a partial protective effect on RPE cells against H2O2 damage across a wide range of concentrations (10−10-10−4 M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10−10

  18. Transport of protons and lactate in cultured human fetal retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Cour, Morten la; Ming Lui, Ge

    2000-01-01

    Electron microscopy, intracellular pH, monocarboxylate transport, pigment epithelium of eye, proton-lactate cotransport, retinal metabolism, sodium/proton exchange......Electron microscopy, intracellular pH, monocarboxylate transport, pigment epithelium of eye, proton-lactate cotransport, retinal metabolism, sodium/proton exchange...

  19. Myo-inositol uptake by cultured calf retinal pigment epithelial cells: regulation by glucose

    International Nuclear Information System (INIS)

    Khatami, M.; Rockey, J.H.

    1986-01-01

    Confluent primary (P-1) or subcultured passage 2 or 3 (P-2, P-3) calf retinal pigment epithelial cells (RPE) were incubated with [ 3 H]-myo-inositol (MI, 100-200 μM) in balanced salt solution (BSS), for 5 to 60 min at 37 0 C. MI uptake into RPE (P-2, 5 days old) was saturable with K/sub m/ of 147 μM and V/sub max/ of 5.5 pmole/min/μg DNA. P-1 or P-2 incubated with 10 μM MI for 40 min accumulated MI against a concentration gradient ([MI]in/[MI]out > 20). Replacement of 150 mM NaCl in BSS by 150 mM choline-Cl reduced the uptake of MI by 87%. MI uptake was inhibited (39%) when cells were incubated in BSS in the absence of Ca Cl 2 . Transport of MI into RPE incubated in the presence of phloridzin, ouabain or 2,4-dinitrophenol (1 mM each) for 10 min was inhibited by 65, 37 and 21%, respectively. α-D-Glucose (20 mM) in the incubation media inhibited MI uptake into primary (or P-2) cultured RPE by 30 or 43% when cells were incubated for 10 or 60 min, respectively. The ability of RPE cells, grown in the presence of 50 mM glucose for 15-25 days, to concentrate MI (40 μM) was reduced up to 41%. Cultured RPE cells accumulated myo-inositol by an active transport system, sensitive to ouabain, DNP and phloridzin. High glucose in the incubation media or in the growth media inhibited the uptake of MI into calf RPE cells

  20. Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage

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    Kumar Anil

    2010-10-01

    Full Text Available Abstract Background The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6 plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis. Results We have shown that RGCs exposed to hypoxia (1% or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM, revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6. Conclusion Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.

  1. DNA repair synthesis in rat retinal ganglion cells treated with chemical carcinogens or ultraviolet light in vitro, with special reference to aging and repair level

    International Nuclear Information System (INIS)

    Ishikawa, T.; Takayama, S.; Kitagawa, T.

    1978-01-01

    A system in which the retinal tissues of noninbred Wistar rats were used in combination with autoradiography was developed for measurement of DNA repair synthesis in ganglion cells of the central nervous system. Retinal tissues in short-term organ culture were treated with various carcinogens plus tritiated thymidine ([methyl -3 H]dThd) or were irradiated with uv light and then treated with [methyl -3 H]dThd. Preliminary study with retinal tissues from rats at various ages revealed no age-associated changes in the levels of unscheduled DNA synthesis in ganglion cells

  2. Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts

    International Nuclear Information System (INIS)

    Kaplan, J.H.; Forbush, B. III; Hoffman, J.F.

    1978-01-01

    2-Nitrobenzyl phosphate and 1-(2-nitro)phenylethyl phosphate have been synthesized and demonstrated to be suitable as photolabile sources of inorganic phosphate. The same protecting groups were attached to the terminal phosphate of adenosine 5'-triphosphate. These caged ATP compounds released adenosine 5'-triphosphate on illumination at 340 nm in aqueous solution and P 3 -1-(2-nitro)phenylethyl-ATP gave about a 70 percent yield in under 30 s. The unphotolyzed caged ATP was neither a substrate nor inhibitor of purified renal Na,K-ATPase (EC 3.61.3). Following photolysis in the presence of the enzyme, the liberated ATP was hydrolyzed but at an inhibited rate. The photo-dependent inhibition could be eliminated by prior addition of glutathione or bisulfite to the irradiated solution. Caged ATP was incorporated into resealed human erythrocyte ghosts prepared from red blood cells depleted of internal energy stores. While the NA : K pump was unable to use incorporated caged ATP as a substrate, the ATP liberated by photolysis activated the pump as evidenced by measurements of K-dependent, ouabain-sensitive Na efflux. Thus the caged ATP can be used as a stable source of ATP unmetabolizable by intracellular ATPases until the ATP is released following photolytic irradiation

  3. Effect of Extracellular Zinc Chelator on Rat Retinal Ganglion Cell Number, and Taurine and Zinc Transporters in These Cells

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    Asarí Márquez García

    2017-05-01

    Full Text Available Zinc deficiency in humans causes decreased antioxidants in the retina and is related with abnormal darkness adaptation, cataracts, blindness, and macular degeneration. There is little information about the effects of zinc on the taurine system in mammalian retinal cells. Therefore, we studied the effect of zinc on the taurine transporter (TAUT and zinc transporters (ZnT-1 and 3 using the extracellular zinc chelator, diethylenetriaminepentaacetic acid (DTPA by fluorescence immunocytochemistry and immunohistochemistry in the ganglion cells (CG and cell layers of the retina of rats. Three days after administration of DTPA (10µM primary antibodies and secondary antibodies conjugated with rhodamine or fluorescein isothiocyanate (FITC were used as required. For immunocytochemical labeling approximately three hundred cells per condition were counted. For immunohistochemical labeling, the fluorescence intensity was measured as integrated optical density (DOI in four areas for each layer of tissue. DTPA produced a decrease of 32 % and 29 % in GC of the total cells labeled with antibody against glycoprotein Thy 1.1 and γ-synuclein, respectively. It also produced a significant decrease in TAUT localization in 27 and 28 % compared to controls. DTPA produced a decrease in the localization of ZnT-1 and ZnT-3 in the retina layers (ganglion cells, GCC and the outer and inner plexiform, CEP and CIP. The study of these molecules in the retina is relevant to understanding the interactions of taurine and zinc in this structure.

  4. Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases.

    Science.gov (United States)

    Wiley, Luke A; Burnight, Erin R; Songstad, Allison E; Drack, Arlene V; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2015-01-01

    Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement. Copyright © 2014. Published by Elsevier Ltd.

  5. Reciprocal actions of microRNA-9 and TLX in the proliferation and differentiation of retinal progenitor cells.

    Science.gov (United States)

    Hu, Yamin; Luo, Min; Ni, Ni; Den, Yuan; Xia, Jing; Chen, Junzhao; Ji, Jing; Zhou, Xiaojian; Fan, Xianqun; Gu, Ping

    2014-11-15

    Recent research has demonstrated critical roles of a number of microRNAs (miRNAs) in stem cell proliferation and differentiation. miRNA-9 (miR-9) is a brain-enriched miRNA. Whether miR-9 has a role in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. In this study, we show that miR-9 plays an important role in RPC fate determination. The expression of miR-9 was inversely correlated with that of the nuclear receptor TLX, which is an essential regulator of neural stem cell self-renewal. Overexpression of miR-9 downregulated the TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, and the effect of miR-9 overexpression on RPC proliferation and differentiation was inhibited by the TLX overexpression; knockdown of miR-9 resulted in increased TLX expression as well as enhanced proliferation of RPCs. Furthermore, inhibition of endogenous TLX by small interfering RNA suppressed RPC proliferation and promoted RPCs to differentiate into retinal neuronal and glial cells. These results suggest that miR-9 and TLX form a feedback regulatory loop to coordinate the proliferation and differentiation of retinal progenitors.

  6. Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

    International Nuclear Information System (INIS)

    Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun; Shan, Kun; Yang, Hong; Yan, Biao; Jiang, Qin

    2015-01-01

    Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative and anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.

  7. Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

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    Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun; Shan, Kun; Yang, Hong [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China); Yan, Biao, E-mail: yanbiao1982@hotmail.com [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China); Jiang, Qin, E-mail: jiangqin710@126.com [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China)

    2015-10-02

    Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative and anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.

  8. Heterogenic final cell cycle by chicken retinal Lim1 horizontal progenitor cells leads to heteroploid cells with a remaining replicated genome.

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    Shahrzad Shirazi Fard

    Full Text Available Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.

  9. Edaravone, an ROS Scavenger, Ameliorates Photoreceptor Cell Death after Experimental Retinal Detachment

    Science.gov (United States)

    Roh, Mi In; Murakami, Yusuke; Thanos, Aristomenis; Miller, Joan W.

    2011-01-01

    Purpose. To investigate whether edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, would be neuroprotective against photoreceptor cell death in a rat model of retinal detachment (RD). Methods. RD was induced in adult Brown Norway rats by subretinal injection of sodium hyaluronate. Edaravone (3, 5, or 10 mg/kg) or physiologic saline was administered intraperitoneally once a day until death on day 3 or 5. Oxidative stress in the retina was assessed by 4-hydroxynonenal staining or ELISA for protein carbonyl content. Photoreceptor death was assessed by TUNEL and measurement of the outer nuclear layer thickness. Western blot analysis and caspase activity assays were pe