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Sample records for protects human retinal

  1. Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.

    PubMed

    Carr, Amanda-Jayne; Vugler, Anthony A; Hikita, Sherry T; Lawrence, Jean M; Gias, Carlos; Chen, Li Li; Buchholz, David E; Ahmado, Ahmad; Semo, Ma'ayan; Smart, Matthew J K; Hasan, Shazeen; da Cruz, Lyndon; Johnson, Lincoln V; Clegg, Dennis O; Coffey, Pete J

    2009-12-03

    Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

  2. Lycium barbarum polysaccharides protected human retinal pigment epithelial cells against oxidative stress-induced apoptosis

    PubMed Central

    Liu, Lian; Lao, Wei; Ji, Qing-Shan; Yang, Zhi-Hao; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2015-01-01

    AIM To investigate the protective effect and its mechanism of lycium barbarum polysaccharides (LBP) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells. METHODS ARPE-19 cells, a human retinal pigment epithelial cell lines, were exposed to different concentrations of H2O2 for 24h, then cell viability was measured by Cell Counting Kit-8 (CCK-8) assay to get the properly concentration of H2O2 which can induce half apoptosis of APRE-19. With different concentrations of LBP pretreatment, the ARPE-19 cells were then exposed to appropriate concentration of H2O2, cell apoptosis was detected by flow cytometric analysis. Expression levels of Bcl-2 and Bax were measured by real time quantitative polymerase chain reaction (RT-PCR) technique. RSULTS LBP significantly reduced the H2O2-induced ARPE-19 cells' apoptosis. LBP inhibited the H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax. CONCLUSION LBP could protect ARPE-19 cells from H2O2-induced apoptosis. The Bcl-2 family had relationship with the protective effects of LBP. PMID:25709900

  3. Melissa Officinalis L. Extracts Protect Human Retinal Pigment Epithelial Cells against Oxidative Stress-Induced Apoptosis

    PubMed Central

    Jeung, In Cheul; Jee, Donghyun; Rho, Chang-Rae; Kang, Seungbum

    2016-01-01

    Background: We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells). Methods: ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H2O2). Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry. Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis. The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis. Results: ALS-L1023 clearly reduced H2O2-induced cell apoptosis and intracellular production of ROS. H2O2-induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023. Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells. Conclusions: ALS-L1023 protected human RPE cells against oxidative damage. This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration. PMID:26941573

  4. Retinal remodeling in human retinitis pigmentosa.

    PubMed

    Jones, B W; Pfeiffer, R L; Ferrell, W D; Watt, C B; Marmor, M; Marc, R E

    2016-09-01

    Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.

  5. Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation

    PubMed Central

    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

  6. Retinal oxygen extraction in humans

    NASA Astrophysics Data System (ADS)

    Werkmeister, René M.; Schmidl, Doreen; Aschinger, Gerold; Doblhoff-Dier, Veronika; Palkovits, Stefan; Wirth, Magdalena; Garhöfer, Gerhard; Linsenmeier, Robert A.; Leitgeb, Rainer A.; Schmetterer, Leopold

    2015-10-01

    Adequate function of the retina is dependent on proper oxygen supply. In humans, the inner retina is oxygenated via the retinal circulation. We present a method to calculate total retinal oxygen extraction based on measurement of total retinal blood flow using dual-beam bidirectional Doppler optical coherence tomography and measurement of oxygen saturation by spectrophotometry. These measurements were done on 8 healthy subjects while breathing ambient room air and 100% oxygen. Total retinal blood flow was 44.3 ± 9.0 μl/min during baseline and decreased to 18.7 ± 4.2 μl/min during 100% oxygen breathing (P < 0.001) resulting in a pronounced decrease in retinal oxygen extraction from 2.33 ± 0.51 μl(O2)/min to 0.88 ± 0.14 μl(O2)/min during breathing of 100% oxygen. The method presented in this paper may have significant potential to study oxygen metabolism in hypoxic retinal diseases such as diabetic retinopathy.

  7. Retinal oxygen extraction in humans

    PubMed Central

    Werkmeister, René M.; Schmidl, Doreen; Aschinger, Gerold; Doblhoff-Dier, Veronika; Palkovits, Stefan; Wirth, Magdalena; Garhöfer, Gerhard; Linsenmeier, Robert A.; Leitgeb, Rainer A.; Schmetterer, Leopold

    2015-01-01

    Adequate function of the retina is dependent on proper oxygen supply. In humans, the inner retina is oxygenated via the retinal circulation. We present a method to calculate total retinal oxygen extraction based on measurement of total retinal blood flow using dual-beam bidirectional Doppler optical coherence tomography and measurement of oxygen saturation by spectrophotometry. These measurements were done on 8 healthy subjects while breathing ambient room air and 100% oxygen. Total retinal blood flow was 44.3 ± 9.0 μl/min during baseline and decreased to 18.7 ± 4.2 μl/min during 100% oxygen breathing (P < 0.001) resulting in a pronounced decrease in retinal oxygen extraction from 2.33 ± 0.51 μl(O2)/min to 0.88 ± 0.14 μl(O2)/min during breathing of 100% oxygen. The method presented in this paper may have significant potential to study oxygen metabolism in hypoxic retinal diseases such as diabetic retinopathy. PMID:26503332

  8. Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 mice.

    PubMed

    Sun, Jianan; Mandai, Michiko; Kamao, Hiroyuki; Hashiguchi, Tomoyo; Shikamura, Masayuki; Kawamata, Shin; Sugita, Sunao; Takahashi, Masayo

    2015-05-01

    Retinitis pigmentosa (RP) is a group of visual impairments characterized by progressive rod photoreceptor cell loss due to a genetic background. Pigment epithelium-derived factor (PEDF) predominantly secreted by the retinal pigmented epithelium (RPE) has been reported to protect photoreceptors in retinal degeneration models, including rd1. In addition, clinical trials are currently underway outside Japan using human mesenchymal stromal cells and human neural stem cells to protect photoreceptors in RP and dry age-related macular degeneration, respectively. Thus, this study aimed to investigate the rescue effects of induced pluripotent stem (iPS)-RPE cells in comparison with those types of cells used in clinical trials on photoreceptor degeneration in rd1 mice. Cells were injected into the subretinal space of immune-suppressed 2-week-old rd1 mice. The results demonstrated that human iPS-RPE cells significantly attenuated photoreceptor degeneration on postoperative days (PODs) 14 and 21 and survived longer up to at least 12 weeks after operation than the other two types of graft cells with less immune responses and apoptosis. The mean PEDF concentration in the intraocular fluid in RPE-transplanted eyes was more than 1 µg/ml at PODs 14 and 21, and this may have contributed to the protective effect of RPE transplantation. Our findings suggest that iPS-RPE cells serve as a competent source to delay photoreceptor degeneration through stable survival in degenerating ocular environment and by releasing neuroprotective factors such as PEDF.

  9. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

    PubMed

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-07-15

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

  10. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

    PubMed Central

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-01-01

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite. PMID:25221599

  11. Aerobic Exercise Protects Retinal Function and Structure from Light-Induced Retinal Degeneration

    PubMed Central

    Lawson, Eric C.; Han, Moon K.; Sellers, Jana T.; Chrenek, Micah A.; Hanif, Adam; Gogniat, Marissa A.

    2014-01-01

    Aerobic exercise is a common intervention for rehabilitation of motor, and more recently, cognitive function (Intlekofer and Cotman, 2013; Wood et al., 2012). While the underlying mechanisms are complex, BDNF may mediate much of the beneficial effects of exercise to these neurons (Ploughman et al., 2007; Griffin et al., 2011; Real et al., 2013). We studied the effects of aerobic exercise on retinal neurons undergoing degeneration. We exercised wild-type BALB/c mice on a treadmill (10 m/min for 1 h) for 5 d/week or placed control mice on static treadmills. After 2 weeks of exercise, mice were exposed to either toxic bright light (10,000 lux) for 4 h to induce photoreceptor degeneration or maintenance dim light (25 lux). Bright light caused 75% loss of both retinal function and photoreceptor numbers. However, exercised mice exposed to bright light had 2 times greater retinal function and photoreceptor nuclei than inactive mice exposed to bright light. In addition, exercise increased retinal BDNF protein levels by 20% compared with inactive mice. Systemic injections of a BDNF tropomyosin-receptor-kinase (TrkB) receptor antagonist reduced retinal function and photoreceptor nuclei counts in exercised mice to inactive levels, effectively blocking the protective effects seen with aerobic exercise. The data suggest that aerobic exercise is neuroprotective for retinal degeneration and that this effect is mediated by BDNF signaling. PMID:24523530

  12. Aerobic exercise protects retinal function and structure from light-induced retinal degeneration.

    PubMed

    Lawson, Eric C; Han, Moon K; Sellers, Jana T; Chrenek, Micah A; Hanif, Adam; Gogniat, Marissa A; Boatright, Jeffrey H; Pardue, Machelle T

    2014-02-12

    Aerobic exercise is a common intervention for rehabilitation of motor, and more recently, cognitive function (Intlekofer and Cotman, 2013; Wood et al., 2012). While the underlying mechanisms are complex, BDNF may mediate much of the beneficial effects of exercise to these neurons (Ploughman et al., 2007; Griffin et al., 2011; Real et al., 2013). We studied the effects of aerobic exercise on retinal neurons undergoing degeneration. We exercised wild-type BALB/c mice on a treadmill (10 m/min for 1 h) for 5 d/week or placed control mice on static treadmills. After 2 weeks of exercise, mice were exposed to either toxic bright light (10,000 lux) for 4 h to induce photoreceptor degeneration or maintenance dim light (25 lux). Bright light caused 75% loss of both retinal function and photoreceptor numbers. However, exercised mice exposed to bright light had 2 times greater retinal function and photoreceptor nuclei than inactive mice exposed to bright light. In addition, exercise increased retinal BDNF protein levels by 20% compared with inactive mice. Systemic injections of a BDNF tropomyosin-receptor-kinase (TrkB) receptor antagonist reduced retinal function and photoreceptor nuclei counts in exercised mice to inactive levels, effectively blocking the protective effects seen with aerobic exercise. The data suggest that aerobic exercise is neuroprotective for retinal degeneration and that this effect is mediated by BDNF signaling.

  13. SiC protective coating for photovoltaic retinal prosthesis

    NASA Astrophysics Data System (ADS)

    Lei, Xin; Kane, Sheryl; Cogan, Stuart; Lorach, Henri; Galambos, Ludwig; Huie, Philip; Mathieson, Keith; Kamins, Theodore; Harris, James; Palanker, Daniel

    2016-08-01

    Objective. To evaluate plasma-enhanced, chemically vapor deposited (PECVD) amorphous silicon carbide (α-SiC:H) as a protective coating for retinal prostheses and other implantable devices, and to study their failure mechanisms in vivo. Approach. Retinal prostheses were implanted in rats sub-retinally for up to 1 year. Degradation of implants was characterized by optical and scanning electron microscopy. Dissolution rates of SiC, SiN x and thermal SiO2 were measured in accelerated soaking tests in saline at 87 °C. Defects in SiC films were revealed and analyzed by selectively removing the materials underneath those defects. Main results. At 87 °C SiN x dissolved at 18.3 ± 0.3 nm d-1, while SiO2 grown at high temperature (1000 °C) dissolved at 0.104 ± 0.008 nm d-1. SiC films demonstrated the best stability, with no quantifiable change after 112 d. Defects in thin SiC films appeared primarily over complicated topography and rough surfaces. Significance. SiC coatings demonstrating no erosion in accelerated aging test for 112 d at 87 °C, equivalent to about 10 years in vivo, can offer effective protection of the implants. Photovoltaic retinal prostheses with PECVD SiC coatings exhibited effective protection from erosion during the 4 month follow-up in vivo. The optimal thickness of SiC layers is about 560 nm, as defined by anti-reflective properties and by sufficient coverage to eliminate defects.

  14. Protection of Retina by αB Crystallin in Sodium Iodate Induced Retinal Degeneration

    PubMed Central

    Zhou, Peng; Kannan, Ram; Spee, Christine; Sreekumar, Parameswaran G.; Dou, Guorui; Hinton, David R.

    2014-01-01

    Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator–activator receptor–γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression. PMID:24874187

  15. Inferior retinal light exposure is more effective than superior retinal exposure in suppressing melatonin in humans

    NASA Technical Reports Server (NTRS)

    Glickman, Gena; Hanifin, John P.; Rollag, Mark D.; Wang, Jenny; Cooper, Howard; Brainard, George C.

    2003-01-01

    Illumination of different areas of the human retina elicits differences in acute light-induced suppression of melatonin. The aim of this study was to compare changes in plasma melatonin levels when light exposures of equal illuminance and equal photon dose were administered to superior, inferior, and full retinal fields. Nine healthy subjects participated in the study. Plexiglass eye shields were modified to permit selective exposure of the superior and inferior halves of the retinas of each subject. The Humphrey Visual Field Analyzer was used both to confirm intact full visual fields and to quantify exposure of upper and lower visual fields. On study nights, eyes were dilated, and subjects were exposed to patternless white light for 90 min between 0200 and 0330 under five conditions: (1) full retinal exposure at 200 lux, (2) full retinal exposure at 100 lux, (3) inferior retinal exposure at 200 lux, (4) superior retinal exposure at 200 lux, and (5) a dark-exposed control. Plasma melatonin levels were determined by radioimmunoassay. ANOVA demonstrated a significant effect of exposure condition (F = 5.91, p < 0.005). Post hoc Fisher PLSD tests showed significant (p < 0.05) melatonin suppression of both full retinal exposures as well as the inferior retinal exposure; however, superior retinal exposure was significantly less effective in suppressing melatonin. Furthermore, suppression with superior retinal exposure was not significantly different from that of the dark control condition. The results indicate that the inferior retina contributes more to the light-induced suppression of melatonin than the superior retina at the photon dosages tested in this study. Findings suggest a greater sensitivity or denser distribution of photoreceptors in the inferior retina are involved in light detection for the retinohypothalamic tract of humans.

  16. The Oral Iron Chelator Deferiprone Protects against Iron Overload–Induced Retinal Degeneration

    PubMed Central

    Hadziahmetovic, Majda; Song, Ying; Wolkow, Natalie; Iacovelli, Jared; Grieco, Steven; Lee, Jennifer; Lyubarsky, Arkady; Pratico, Domenico; Connelly, John; Spino, Michael; Harris, Z. Leah

    2011-01-01

    Purpose. Iron-induced oxidative stress may exacerbate age-related macular degeneration (AMD). Ceruloplasmin/Hephaestin double-knockout (DKO) mice with age-dependent retinal iron accumulation and some features of AMD were used to test retinal protection by the oral iron chelator deferiprone (DFP). Methods. Cultured retinal pigment epithelial (ARPE-19) cells and mice were treated with DFP. Transferrin receptor mRNA (Tfrc), an indicator of iron levels, was quantified by qPCR. In mice, retinal oxidative stress was assessed by mass spectrometry, and degeneration by histology and electroretinography. Results. DFP at 60 μM decreased labile iron in ARPE-19 cells, increasing Tfrc and protecting 70% of cells against a lethal dose of H2O2. DFP 1 mg/mL in drinking water increased retinal Tfrc mRNA 2.7-fold after 11 days and also increased transferrin receptor protein. In DKOs, DFP over 8 months decreased retinal iron levels to 72% of untreated mice, diminished retinal oxidative stress to 70% of the untreated level, and markedly ameliorated retinal degeneration. DFP was not retina toxic in wild-type (WT) or DKO mice, as assessed by histology and electroretinography. Conclusions. Oral DFP was not toxic to the mouse retina. It diminished retinal iron levels and oxidative stress and protected DKO mice against iron overload–induced retinal degeneration. Further testing of DFP for retinal disease involving oxidative stress is warranted. PMID:21051716

  17. Protective Effect of Proanthocyanidins from Sea Buckthorn (Hippophae Rhamnoides L.) Seed against Visible Light-Induced Retinal Degeneration in Vivo

    PubMed Central

    Wang, Yong; Zhao, Liang; Huo, Yazhen; Zhou, Feng; Wu, Wei; Lu, Feng; Yang, Xue; Guo, Xiaoxuan; Chen, Peng; Deng, Qianchun; Ji, Baoping

    2016-01-01

    Dietary proanthocyanidins (PACs) as health-protective agents have become an important area of human nutrition research because of their potent bioactivities. We investigated the retinoprotective effects of PACs from sea buckthorn (Hippophae rhamnoides L.) seed against visible light-induced retinal degeneration in vivo. Pigmented rabbits were orally administered sea buckthorn seed PACs (50 and 100 mg/kg/day) for 14 consecutive days of pre-illumination and seven consecutive days of post-illumination. Retinal function was quantified via electroretinography 7 days after light exposure. Retinal damage was evaluated by measuring the thickness of the full-thickness retina and outer nuclear layer 7 days after light exposure. Sea buckthorn seed PACs significantly attenuated the destruction of electroretinograms and maintained the retinal structure. Increased retinal photooxidative damage was expressed by the depletion of glutathione peroxidase and catalase activities, the decrease of total antioxidant capacity level and the increase of malondialdehyde level. Light exposure induced a significant increase of inflammatory cytokines (IL-1β, TNF-α and IL-6) and angiogenesis (VEGF) levels in retina. Light exposure upregulated the expression of pro-apoptotic proteins Bax and caspase-3 and downregulated the expression of anti-apoptotic protein Bcl-2. However, sea buckthorn seed PACs ameliorated these changes induced by light exposure. Sea buckthorn seed PACs mediated the protective effect against light-induced retinal degeneration via antioxidant, anti-inflammatory and antiapoptotic mechanisms. PMID:27144578

  18. Protective Effect of Proanthocyanidins from Sea Buckthorn (Hippophae Rhamnoides L.) Seed against Visible Light-Induced Retinal Degeneration in Vivo.

    PubMed

    Wang, Yong; Zhao, Liang; Huo, Yazhen; Zhou, Feng; Wu, Wei; Lu, Feng; Yang, Xue; Guo, Xiaoxuan; Chen, Peng; Deng, Qianchun; Ji, Baoping

    2016-05-02

    Dietary proanthocyanidins (PACs) as health-protective agents have become an important area of human nutrition research because of their potent bioactivities. We investigated the retinoprotective effects of PACs from sea buckthorn (Hippophae rhamnoides L.) seed against visible light-induced retinal degeneration in vivo. Pigmented rabbits were orally administered sea buckthorn seed PACs (50 and 100 mg/kg/day) for 14 consecutive days of pre-illumination and seven consecutive days of post-illumination. Retinal function was quantified via electroretinography 7 days after light exposure. Retinal damage was evaluated by measuring the thickness of the full-thickness retina and outer nuclear layer 7 days after light exposure. Sea buckthorn seed PACs significantly attenuated the destruction of electroretinograms and maintained the retinal structure. Increased retinal photooxidative damage was expressed by the depletion of glutathione peroxidase and catalase activities, the decrease of total antioxidant capacity level and the increase of malondialdehyde level. Light exposure induced a significant increase of inflammatory cytokines (IL-1β, TNF-α and IL-6) and angiogenesis (VEGF) levels in retina. Light exposure upregulated the expression of pro-apoptotic proteins Bax and caspase-3 and downregulated the expression of anti-apoptotic protein Bcl-2. However, sea buckthorn seed PACs ameliorated these changes induced by light exposure. Sea buckthorn seed PACs mediated the protective effect against light-induced retinal degeneration via antioxidant, anti-inflammatory and antiapoptotic mechanisms.

  19. Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy

    PubMed Central

    Mendel, Thomas A.; Clabough, Erin B. D.; Kao, David S.; Demidova-Rice, Tatiana N.; Durham, Jennifer T.; Zotter, Brendan C.; Seaman, Scott A.; Cronk, Stephen M.; Rakoczy, Elizabeth P.; Katz, Adam J.; Herman, Ira M.; Peirce, Shayn M.; Yates, Paul A.

    2013-01-01

    Background 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. Methodology/Principal Findings 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). Conclusions/Significance 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

  20. Paraoxonase Enzyme Protects Retinal Pigment Epithelium from Chlorpyrifos Insult

    PubMed Central

    Jasna, Jagan Mohan; Anandbabu, Kannadasan; Bharathi, Subramaniam Rajesh; Angayarkanni, Narayanasamy

    2014-01-01

    Retinal pigment epithelium (RPE) provides nourishment and protection to the eye. RPE dysfunction due to oxidative stress and inflammation is one of the major reason for many of the retinal disorders. Organophosphorus pesticides are widely used in the agricultural, industrial and household activities in India. However, their effects on the eye in the context of RPE has not been studied. In this study the defense of the ARPE19 cells exposed to Chlorpyrifos (1 nM to 100 µM) in terms of the enzyme paraoxonase (PON) was studied at 24 hr and 9 days of treatment. Chlorpyrifos was found to induce oxidative stress in the ARPE19 cells as seen by significant increase in ROS and decrease in glutathione (GSH) levels without causing cell death. Tissue resident Paraoxonase 2 (PON2) mRNA expression was elevated with chlorpyrifos exposure. The three enzymatic activities of PON namely, paraoxonase (PONase), arylesterase (PON AREase) and thiolactonase (PON HCTLase) were also found to be significantly altered to detoxify and as an antioxidant defense. Among the transcription factors regulating PON2 expression, SP1 was significantly increased with chlorpyrifos exposure. PON2 expression was found to be crucial as ARPE19 cells showed a significant loss in their ability to withstand oxidative stress when the cells were subjected to chlorpyrifos after silencing PON2 expression. Treatment with N-acetyl cysteine positively regulated the PON 2 expression, thus promoting the antioxidant defense put up by the cells in response to chlorpyrifos. PMID:24979751

  1. Regulation of human retinal blood flow by endothelin-1.

    PubMed

    Polak, Kaija; Luksch, Alexandra; Frank, Barbara; Jandrasits, Kerstin; Polska, Elzbieta; Schmetterer, Leopold

    2003-05-01

    There is evidence from in vitro and animal studies that endothelin is a major regulator of retinal blood flow. We set out to characterize the role of the endothelin-system in the blood flow control of the human retina. Two studies in healthy subjects were performed. The study design was randomized, placebo-controlled, double-masked, balanced, two-way crossover in protocol A and three way-way crossover in protocol B. In protocol A 18 healthy male subjects received intravenous endothelin-1 (ET-1) in a dose of 2.5 ng kg (-1)min(-1) for 30 min or placebo on two different study days and retinal vessel diameters were measured. In protocol B 12 healthy male subjects received ET-1 in stepwise increasing doses of 0, 1.25, 2.5 and 5 ng kg (-1)min(-1) (each infusion step over 20 min) in co-infusion with the specific ET(A)-receptor antagonist BQ123 (60 microg min (-1)) or placebo or BQ123 alone investigating retinal vessel diameters, retinal blood velocity and retinal blood flow. Measurements of retinal vessel size were done with the Zeiss retinal vessel analyzer. Measurements of blood velocities were done with bi-directional laser Doppler velocimetry. From these measurements retinal blood flow was calculated. In protocol A exogenous ET-1 tended to decrease retinal arterial diameter, but this effect was not significant versus placebo. No effect on retinal venous diameter was seen. In protocol B retinal venous blood velocity and retinal blood flow was significantly reduced after administration of exogenous ET-1. These effects were significantly blunted when BQ-123 was co-administered. By contrast, BQ-123 alone had no effect on retinal hemodynamic parameters. Concluding, BQ123 antagonizes the effects of exogenously administered ET-1 on retinal blood flow in healthy subjects. In addition, the results of the present study are compatible with the hypothesis that ET-1 exerts its vasoconstrictor effects in the retina mainly on the microvessels.

  2. Engineering Retina from Human Retinal Progenitors (Cell Lines)

    PubMed Central

    Cao, Yang

    2009-01-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell–cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr2e3, expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription–polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell

  3. Beneficial protective effect of pramipexole on light-induced retinal damage in mice.

    PubMed

    Shibagaki, Keiichi; Okamoto, Kazuyoshi; Katsuta, Osamu; Nakamura, Masatsugu

    2015-10-01

    We investigated the effects of pramipexole, a potent dopamine receptor D2/D3 agonist, on light-induced retinal damage in mice, H2O2-induced retinal pigment epithelium ARPE-19 cell injury in humans, and hydroxyl radical scavenging activity in a cell-free system. Pramipexole (0.1 and 1 mg/kg body weight) was orally administered to mice 1 h before light exposure (5000 lux, 2 h). Electrophysiological and morphologic studies were performed to evaluate the effects of the pramipexole on light-induced retinal damage in mice. Pramipexole significantly prevented the reduction of the a- and b-wave electroretinogram (ERG) amplitudes caused by light exposure in a dose-dependent manner. In parallel, damage to the inner and outer segments (IS/OS) of the photoreceptors, loss of photoreceptor nuclei, and the number of Tdt-mediated dUTP nick-end labeling (TUNEL)-positive cells in the outer nuclear layer (ONL) caused by light exposure were notably ameliorated by pramipexole. Additionally, pramipexole suppressed H2O2-induced ARPE-19 cell death in vitro in a concentration-dependent manner. The effect of pramipexole was significant at concentrations of 10(-6) M or higher. Pramipexole also significantly prevented H2O2-induced activation of caspases-3/7 and the intracellular accumulation of reactive oxygen species (ROS) in a concentration-dependent manner ranging from 10(-5) to 10(-3) M. Furthermore, pramipexole increased the scavenging activity toward a hydroxyl radical generated from H2O2 in a Fenton reaction. Our results suggest that pramipexole protects against light-induced retinal damage as an antioxidant and that it may be a novel and effective therapy for retinal degenerative disorders, such as dry age-related macular degeneration.

  4. Nitric oxide regulates retinal vascular tone in humans.

    PubMed

    Dorner, Guido T; Garhofer, Gerhard; Kiss, Barbara; Polska, Elzbieta; Polak, Kaija; Riva, Charles E; Schmetterer, Leopold

    2003-08-01

    The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.

  5. Protective effect of ascorbate in retinal light damage of rats

    SciTech Connect

    Organisciak, D.T.; Wang, H.M.; Li, Z.Y.; Tso, M.O.

    1985-11-01

    Cyclic light and dark-reared rats were exposed to intense visible light for various periods and then rhodopsin-measured following recovery in darkness for up to 14 days. Animals were injected with ascorbic acid or ascorbate derivatives at various doses prior to light exposure in green Plexiglas chambers. The results show that ascorbic acid administration elevates retinal ascorbate and reduces the loss of rhodopsin and photoreceptor cell nuclei resulting from intense light. When given in comparable doses, L-ascorbic acid, sodium ascorbate, and dehydroascorbate were equally effective in preserving rhodopsin. The ascorbate protective effect in the retina is also dose dependent in both cyclic light and dark-reared rats and exhibits a requirement for the L-stereoisomer of the vitamin. Ascorbic acid is effective when administered before, but not after, light exposure, suggesting that protection from light damage in the retina occurs during the light period. In some experiments, rod outer segments were isolated from rats immediately after light exposure, lipids extracted, and fatty acid composition determined. As judged by the preservation of rod outer segment docosahexaenoic acid in rats given ascorbate, the vitamin may act in an antioxidative fashion by inhibiting oxidation of membrane lipids during intense light.

  6. Phloroglucinol protects retinal pigment epithelium and photoreceptor against all-trans-retinal-induced toxicity and inhibits A2E formation.

    PubMed

    Cia, David; Cubizolle, Aurélie; Crauste, Céline; Jacquemot, Nathalie; Guillou, Laurent; Vigor, Claire; Angebault, Claire; Hamel, Christian P; Vercauteren, Joseph; Brabet, Philippe

    2016-09-01

    Among retinal macular diseases, the juvenile recessive Stargardt disease and the age-related degenerative disease arise from carbonyl and oxidative stresses (COS). Both stresses originate from an accumulation of all-trans-retinal (atRAL) and are involved in bisretinoid formation by condensation of atRAL with phosphatidylethanolamine (carbonyl stress) in the photoreceptor and its transformation into lipofuscin bisretinoids (oxidative stress) in the retinal pigment epithelium (RPE). As atRAL and bisretinoid accumulation contribute to RPE and photoreceptor cell death, our goal is to select powerful chemical inhibitors of COS. Here, we describe that phloroglucinol, a natural phenolic compound having anti-COS properties, protects both rat RPE and mouse photoreceptor primary cultures from atRAL-induced cell death and reduces hydrogen peroxide (H2 O2 )-induced damage in RPE in a dose-dependent manner. Mechanistic analyses demonstrate that the protective effect encompasses decrease in atRAL-induced intracellular reactive oxygen species and free atRAL levels. Moreover, we show that phloroglucinol reacts with atRAL to form a chromene adduct which prevents bisretinoid A2E synthesis in vitro. Taken together, these data show that the protective effect of phloroglucinol correlates with its ability to trap atRAL and to prevent its further transformation into deleterious bisretinoids. Phloroglucinol might be a good basis to develop efficient therapeutic derivatives in the treatment of retinal macular diseases.

  7. Automated spectroscopic imaging of oxygen saturation in human retinal vessels

    NASA Astrophysics Data System (ADS)

    Nakamura, D.; Sueda, S.; Matsuoka, N.; Yoshinaga, Y.; Enaida, H.; Okada, T.; Ishibashi, T.

    2009-02-01

    A new automatic visualization procedure for the oxygen saturation imaging from multi-spectral imaging of human retinal vessels has been proposed. Two-wavelength retinal fundus images at 545 and 560 nm, which were oxygen insensitive and oxygen sensitive, respectively, were captured by CCD cameras simultaneously through a beam splitter and interference filters. We applied a morphological processing technique to presume a distribution of incident light including the vessel parts and an optical density (OD) image of each wavelength image. And the OD ratio (OD560/OD545) image was calculated as a relative indicator of oxygen saturation. Furthermore, processing of line convergence index filter was adopted to identify the retinal vessels. Clear difference between retinal arteries and veins was observed in the automated imaging method. In addition, the decrease of oxygen saturation in the retinal artery without breathing could be monitored by the ODR. This method is possible to be applied to real-time monitoring for oxygen saturation of retinal vessels.

  8. Oligomeric proanthocyanidin protects retinal ganglion cells against oxidative stress-induced apoptosis

    PubMed Central

    Wang, Hui; Zhang, Chanjuan; Lu, Dan; Shu, Xiaoming; Zhu, Lihong; Qi, Renbing; So, Kwok-Fai; Lu, Daxiang; Xu, Ying

    2013-01-01

    The death of retinal ganglion cells is a hallmark of many optic neurodegenerative diseases such as glaucoma and retinopathy. Oxidative stress is one of the major reasons to cause the cell death. Oligomeric proanthocyanidin has many health beneficial effects including antioxidative and neuroprotective actions. Here we tested whether oligomeric proanthocyanidin may protect retinal ganglion cells against oxidative stress induced-apoptosis in vitro. Retinal ganglion cells were treated with hydrogen peroxide with or without oligomeric proanthocyanidin. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that treating retinal ganglion cell line RGC-5 cells with 20 μmol/L oligomeric proanthocyanidin significantly decreased the hydrogen peroxide (H2O2) induced death. Results of flow cytometry and Hoechst staining demonstrated that the death of RGC-5 cells was mainly caused by cell apoptosis. We further found that expression of pro-apoptotic Bax and caspase-3 were significantly decreased while anti-apoptotic Bcl-2 was greatly increased in H2O2 damaged RGC-5 cells with oligomeric proanthocyanidin by western blot assay. Furthermore, in retinal explant culture, the number of surviving retinal ganglion cells in H2O2-damaged retinal ganglion cells with oligomeric proanthocyanidin was significantly increased. Our studies thus demonstrate that oligomeric proanthocyanidin can protect oxidative stress-injured retinal ganglion cells by inhibiting apoptotic process. PMID:25206541

  9. The molecular basis of human retinal and vitreoretinal diseases.

    PubMed

    Berger, Wolfgang; Kloeckener-Gruissem, Barbara; Neidhardt, John

    2010-09-01

    During the last two to three decades, a large body of work has revealed the molecular basis of many human disorders, including retinal and vitreoretinal degenerations and dysfunctions. Although belonging to the group of orphan diseases, they affect probably more than two million people worldwide. Most excitingly, treatment of a particular form of congenital retinal degeneration is now possible. A major advantage for treatment is the unique structure and accessibility of the eye and its different components, including the vitreous and retina. Knowledge of the many different eye diseases affecting retinal structure and function (night and colour blindness, retinitis pigmentosa, cone and cone rod dystrophies, photoreceptor dysfunctions, as well as vitreoretinal traits) is critical for future therapeutic development. We have attempted to present a comprehensive picture of these disorders, including biological, clinical, genetic and molecular information. The structural organization of the review leads the reader through non-syndromic and syndromic forms of (i) rod dominated diseases, (ii) cone dominated diseases, (iii) generalized retinal degenerations and (iv) vitreoretinal disorders, caused by mutations in more than 165 genes. Clinical variability and genetic heterogeneity have an important impact on genetic testing and counselling of affected families. As phenotypes do not always correlate with the respective genotypes, it is of utmost importance that clinicians, geneticists, counsellors, diagnostic laboratories and basic researchers understand the relationships between phenotypic manifestations and specific genes, as well as mutations and pathophysiologic mechanisms. We discuss future perspectives.

  10. Retinal Remodeling and Metabolic Alterations in Human AMD.

    PubMed

    Jones, Bryan W; Pfeiffer, Rebecca L; Ferrell, William D; Watt, Carl B; Tucker, James; Marc, Robert E

    2016-01-01

    Age-related macular degeneration (AMD) is a progressive retinal degeneration resulting in central visual field loss, ultimately causing debilitating blindness. AMD affects 18% of Americans from 65 to 74, 30% older than 74 years of age and is the leading cause of severe vision loss and blindness in Western populations. While many genetic and environmental risk factors are known for AMD, we currently know less about the mechanisms mediating disease progression. The pathways and mechanisms through which genetic and non-genetic risk factors modulate development of AMD pathogenesis remain largely unexplored. Moreover, current treatment for AMD is palliative and limited to wet/exudative forms. Retina is a complex, heterocellular tissue and most retinal cell classes are impacted or altered in AMD. Defining disease and stage-specific cytoarchitectural and metabolic responses in AMD is critical for highlighting targets for intervention. The goal of this article is to illustrate cell types impacted in AMD and demonstrate the implications of those changes, likely beginning in the retinal pigment epithelium (RPE), for remodeling of the the neural retina. Tracking heterocellular responses in disease progression is best achieved with computational molecular phenotyping (CMP), a tool that enables acquisition of a small molecule fingerprint for every cell in the retina. CMP uncovered critical cellular and molecular pathologies (remodeling and reprogramming) in progressive retinal degenerations such as retinitis pigmentosa (RP). We now applied these approaches to normal human and AMD tissues mapping progression of cellular and molecular changes in AMD retinas, including late-stage forms of the disease.

  11. Distributions of elements in the human retinal pigment epithelium.

    PubMed

    Ulshafer, R J; Allen, C B; Rubin, M L

    1990-01-01

    Distributions of elements above the atomic number of sodium were mapped in the retinal pigment epithelia of eight human eyes. X-ray energy spectra and maps were collected from cryofixed, freeze-dried, and epoxy-embedded tissues using energy-dispersive x-ray microanalysis. All eyes had high concentrations of phosphorus in the nuclei of retinal pigment epithelial cells. Melanosomes were rich in sulfur, zinc, calcium, and iron. Lipofuscin and cytoplasm contained only phosphorus and sulfur in detectable amounts. Drusen, when present, contained phosphorus and calcium. Six eyes had a prominent aluminum peak recorded from melanosomes, nuclei, and Bruch's membrane. In one pair of 90-year-old eyes, small, electron-dense deposits surrounded many melanosomes and contained mercury and selenium. Retinal pigment epithelial melanosomes may bind and accumulate metals and other potentially toxic ions over time, preventing them from reaching the neural retina.

  12. Protective Effect of Tang Wang One Decoction on the Retinal Vessels of Diabetic Rats

    PubMed Central

    Kou, Xinyun; Yang, Shufei; Qin, Yali; Yang, Chao; Deng, Tingting; Luo, Dan

    2017-01-01

    Objective. This study aimed to determine the influence of Tang Wang One Decoction (TWOD) on the retinal vessels of diabetic rats. Methods. The hemorheology of diabetic rats was observed. Morphological studies of retinal vessels were conducted using optical microscopy and electron microscopy. Immunological histochemistry assay was used to measure the expression levels of MMP-9, occludin, and claudin-5. Results. Obvious pathological damage was observed in the retinal vessels of diabetic rats. TWOD positively affected the hemorheology and morphology of retinal vessels. The decoction also decreased the expression of MMP-9 and increased the expression of occludin and claudin-5. Conclusions. The results suggest that the retinal protective effects of TWOD might be related to downregulation of MMP-9 and upregulation of occludin and claudin-5. PMID:28367226

  13. Microvascular network topology of the human retinal vessels.

    PubMed

    Schröder, S; Brab, M; Schmid-Schönbein, G W; Reim, M; Schmid-Schönbein, H

    1990-01-01

    A quantitative analysis of blood flow in the human retinal vessels requires a detailed picture of the microvascular network topology. In order to lay the foundation for a quantitative microcirculatory network analysis of the human retina, a novel technique for tissue preparation and network characterization was developed. After injection of hydrogen peroxide into the human bulb, the microvasculature was filled with oxygen produced by endothelial catalase and visualized after embedding in a mixture of cedar oil and gum damar. The vessel topology was documented in the form of photomicrographs, which permitted complete reconstruction of the microvasculature on transparent overlays. By considering the complete capillary system it was possible to divide the retinal network into dichotomous, asymmetric arteriolar and venular trees. The Strahler ordering method, which considers both dichotomous and side branching configurations, was selected and applied to analyze the retinal vascular trees, using the capillaries as the zero order reference vessels. The number of vessel segments was found to be an approximate logarithmic function of the order number, in accordance with Horton's law. Vessel lengths within each order were found to be log-normal distributed, and median lengths for different orders could be approximated by a 2nd degree polynomial curve. Diameters within each order could be approximated by a Gaussian distribution, and the mean values for different orders could be expressed by an exponential curve. These data provide the basis for conductance, pressure and flow computations within the retinal microvessels.

  14. Protective responses to sublytic complement in the retinal pigment epithelium

    PubMed Central

    Tan, Li Xuan; Toops, Kimberly A.; Lakkaraju, Aparna

    2016-01-01

    The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4−/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4−/− mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

  15. Overexpression of Heme Oxygenase-1 in Mesenchymal Stem Cells Augments Their Protection on Retinal Cells In Vitro and Attenuates Retinal Ischemia/Reperfusion Injury In Vivo against Oxidative Stress

    PubMed Central

    Li, Li; Du, GaiPing; Wang, DaJiang; Zhou, Jin; Jiang, Guomin

    2017-01-01

    Retinal ischemia/reperfusion (I/R) injury, involving several ocular diseases, seriously threatens human ocular health, mainly treated by attenuating I/R-induced oxidative stress. Currently, mesenchymal stem cells (MSCs) could restore I/R-injured retina through paracrine secretion. Additionally, heme oxygenase-1 (HO-1) could ameliorate oxidative stress and thus retinal apoptosis, but the expression of HO-1 in MSC is limited. Here, we hypothesized that overexpression of HO-1 in MSC (MSC-HO-1) may significantly improve their retina-protective potentials. The overexpression of HO-1 in MSC was achieved by lentivirus transduction. Then, MSC or MSC-HO-1 was cocultured with retinal ganglion cells (RGC-5) in H2O2-simulated oxidative condition and their protection on RGC-5 was systemically valuated in vitro. Compared with MSC, MSC-HO-1 significantly attenuated H2O2-induced injury of RGC-5, including decrease in cellular ROS level and apoptosis, activation of antiapoptotic proteins p-Akt and Bcl-2, and blockage of proapoptotic proteins cleaved caspase 3 and Bax. In retinal I/R rats model, compared with control MSC, MSC-HO-1-treated retina significantly retrieved its structural thickness, reduced cell apoptosis, markedly attenuated retinal oxidative stress level, and largely regained the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that overexpression of HO-1 provides a promising strategy to enhance the MSC-based therapy for I/R-related retinal injury. PMID:28255307

  16. Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment

    PubMed Central

    Arteni, Ana-Andreea; Fradot, Mathias; Galzerano, Denise; Mendes-Pinto, Maria M.; Sahel, José-Alain; Picaud, Serge; Robert, Bruno; Pascal, Andrew A.

    2015-01-01

    Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health–specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν1 Raman mode. In addition, analysis of the ν4 Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy. PMID:26313550

  17. Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment.

    PubMed

    Arteni, Ana-Andreea; Fradot, Mathias; Galzerano, Denise; Mendes-Pinto, Maria M; Sahel, José-Alain; Picaud, Serge; Robert, Bruno; Pascal, Andrew A

    2015-01-01

    Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health-specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν1 Raman mode. In addition, analysis of the ν4 Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy.

  18. Topography of retinal recovery processes in humans

    PubMed Central

    Mazinani, Babac E; Merx, Elke; Plange, Niklas; Walter, Peter; Roessler, Gernot F

    2014-01-01

    Background The purpose of this study was to examine retinal recovery processes to pographically by the application of three flash sequences with specific interstimulus intervals. Methods Twelve healthy subjects underwent multifocal electroretinography with a light-emitting diode stimulator. Every flash sequence consisted of three flashes with 25 msec between the first and the second flash and 35 msec between the second and the third flash. The interval between the third and the first flash of the next step was 85 msec. The interstimulus interval-dependent amplitude reductions of the multifocal electroretinographic response for these three intervals yielded three data points that were used to determine the complete curve of the recovery kinetics. Results Amplitude reductions were higher with shorter interstimulus intervals. The mean half-life periods of the recovery kinetics for the different concentric rings and all subjects were: ring 1, 29.3±5.9 msec; ring 2, 24.2±6.4 msec; ring 3, 23±4.1 msec; ring 4, 23.1±4.6 msec; and ring 5, 22.3±4.4 msec. The differences between the first and all other rings were statistically significant (P<0.05). Conclusion The kinetics of the amplitude recovery after short interstimulus intervals showed a spatial distribution, with faster recovery toward the macular periphery. PMID:25349472

  19. Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    PubMed Central

    Yan, Bo-jing; Wu, Zhi-zhong; Chong, Wei-hua; Li, Gen-lin

    2016-01-01

    Several studies have investigated the protective functions of brain-derived neurotrophic factor (BDNF) in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19 (ARPE-19) cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases. PMID:28197196

  20. Retinal Remodeling and Metabolic Alterations in Human AMD

    PubMed Central

    Jones, Bryan W.; Pfeiffer, Rebecca L.; Ferrell, William D.; Watt, Carl B.; Tucker, James; Marc, Robert E.

    2016-01-01

    Age-related macular degeneration (AMD) is a progressive retinal degeneration resulting in central visual field loss, ultimately causing debilitating blindness. AMD affects 18% of Americans from 65 to 74, 30% older than 74 years of age and is the leading cause of severe vision loss and blindness in Western populations. While many genetic and environmental risk factors are known for AMD, we currently know less about the mechanisms mediating disease progression. The pathways and mechanisms through which genetic and non-genetic risk factors modulate development of AMD pathogenesis remain largely unexplored. Moreover, current treatment for AMD is palliative and limited to wet/exudative forms. Retina is a complex, heterocellular tissue and most retinal cell classes are impacted or altered in AMD. Defining disease and stage-specific cytoarchitectural and metabolic responses in AMD is critical for highlighting targets for intervention. The goal of this article is to illustrate cell types impacted in AMD and demonstrate the implications of those changes, likely beginning in the retinal pigment epithelium (RPE), for remodeling of the the neural retina. Tracking heterocellular responses in disease progression is best achieved with computational molecular phenotyping (CMP), a tool that enables acquisition of a small molecule fingerprint for every cell in the retina. CMP uncovered critical cellular and molecular pathologies (remodeling and reprogramming) in progressive retinal degenerations such as retinitis pigmentosa (RP). We now applied these approaches to normal human and AMD tissues mapping progression of cellular and molecular changes in AMD retinas, including late-stage forms of the disease. PMID:27199657

  1. miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-κB and TNFα to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose

    PubMed Central

    Ye, Eun-Ah; Steinle, Jena J.

    2016-01-01

    Pathological mechanisms underlying diabetic retinopathy are still not completely understood. Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. A growing body of evidence shows that microRNA (miRNA) play important roles in pathological mechanisms involved in diabetic retinopathy, as well as possessing potential as novel therapeutic targets. The hypothesis of this study was that miR-146a plays a key role in attenuating hyperglycemia-induced inflammatory pathways through reduced TLR4/NF-κB and TNFα signaling in primary human retinal microvascular endothelial cells (REC). We cultured human REC in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC with miRNA mimic (hsa-miR-146a-5p). Our results demonstrate that miR-146a expression was decreased in human REC cultured in high glucose. Overexpression of miR-146a using mimics reduced the levels of TLR4/NF-κB and TNFα in REC cultured in high glucose. Both MyD88-dependent and -independent signaling were decreased by miR-146a overexpression in REC in high glucose conditions. The results suggest that miR-146a is a potential therapeutic target for reducing inflammation in REC through inhibition of TLR4/NF-κB and TNFα. Our study will contribute to understanding of diabetic retinal pathology, as well as providing important clues to develop therapeutics for clinical applications. PMID:26997759

  2. Functional annotation of the human retinal pigment epithelium transcriptome

    PubMed Central

    Booij, Judith C; van Soest, Simone; Swagemakers, Sigrid MA; Essing, Anke HW; Verkerk, Annemieke JMH; van der Spek, Peter J; Gorgels, Theo GMF; Bergen, Arthur AB

    2009-01-01

    Background To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63–78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software. Results In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover. Conclusion Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for

  3. Thioredoxin-like 6 protects retinal cell line from photooxidative damage by upregulating NF-kappaB activity.

    PubMed

    Wang, Xiao Wei; Tan, Bao Zhen; Sun, Miao; Ho, Bow; Ding, Jeak Ling

    2008-08-01

    Apoptosis is the common pathway to photoreceptor cell death in many eye diseases including age-related macular degeneration which affects more than 8 million individuals in the United States alone. RdCVF, a truncated mouse thioredoxin is specifically expressed by rod photoreceptor cells and prevents the apoptosis of cone cells. However the protective mechanism of RdCVF and the implications of its human homologue, thioredoxin-like 6 (TXNL6), on the apoptosis of retinal cells remain unknown. In this study, we examined the function of TXNL6 and investigated its mechanism of protection using a cone photoreceptor cell line, 661W. We found that the photooxidative stress-induced degradation of NF-kappaB proteins is rescued by overexpression of TXNL6, which enabled the NF-kappaB transactivation activity. Furthermore, the overexpression of TXNL6 rescued the photooxidative stress-induced apoptosis of 661W cells. Interestingly, this protective effect was significantly blocked by NF-kappaB specific inhibitors demonstrating that TXNL6 exerts its protective effect against apoptosis via NF-kappaB. Taken together, our study shows that the TXNL6 probably protects retinal cells from photooxidative damage-induced apoptosis via upregulation of NF-kappaB activity. The identification of TXNL6 and the demonstration of its protective mechanism offer new insights into treatment possibilities for photoreceptor cell degradation.

  4. Functional expression of SCL/TAL1 interrupting locus (Stil) protects retinal dopaminergic cells from neurotoxin-induced degeneration.

    PubMed

    Li, Jingling; Li, Ping; Carr, Aprell; Wang, Xiaokai; DeLaPaz, April; Sun, Lei; Lee, Eric; Tomei, Erika; Li, Lei

    2013-01-11

    We previously isolated a dominant mutation, night blindness b (nbb), which causes a late onset of retinal dopaminergic cell degeneration in zebrafish. In this study, we cloned the zebrafish nbb locus. Sequencing results revealed that nbb is a homolog of the vertebrate SCL/TAL1 interrupting locus (Stil). The Stil gene has been shown to play important roles in the regulation of vertebrate embryonic neural development and human cancer cell proliferation. In this study, we demonstrate that functional expression of Stil is also required for neural survival. In zebrafish, decreased expression of Stil resulted in increased toxic susceptibility of retinal dopaminergic cells to 6-hydroxydopamine. Increases in Stil-mediated Shh signaling transduction (i.e. by knocking down the Shh repressor Sufu) prevented dopaminergic cell death induced by neurotoxic insult. The data suggest that the oncogene Stil also plays important roles in neural protection.

  5. Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Hollborn, Margrit; Chen, Rui; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas; Kohen, Leon

    2013-01-01

    Backround Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro. Methodology/Principal Findings Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Conclusion It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of

  6. An experimental comparison of human and bovine rhodopsin provides insight into the molecular basis of retinal disease.

    PubMed

    Morrow, James M; Castiglione, Gianni M; Dungan, Sarah Z; Tang, Portia L; Bhattacharyya, Nihar; Hauser, Frances E; Chang, Belinda S W

    2017-03-30

    Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments. This article is protected by copyright. All rights reserved.

  7. In vivo dynamics of retinal injury and repair in the rhodopsin mutant dog model of human retinitis pigmentosa.

    PubMed

    Cideciyan, Artur V; Jacobson, Samuel G; Aleman, Tomas S; Gu, Danian; Pearce-Kelling, Susan E; Sumaroka, Alexander; Acland, Gregory M; Aguirre, Gustavo D

    2005-04-05

    Genetic and environmental factors modify the severity of human neurodegenerations. Retinal degenerations caused by rhodopsin gene mutations show severity differences within and between families and even within regions of the same eye. Environmental light is thought to contribute to this variation. In the naturally occurring dog model of the human disorder, we found that modest light levels, as used in routine clinical practice, dramatically accelerated the neurodegeneration. Dynamics of acute retinal injury (consisting of abnormal intraretinal light scattering) were visualized in vivo in real time with high-resolution optical imaging. Long term consequences included fast or slow retinal degeneration or repair of injury depending on the dose of light exposure. These experiments provide a platform to study mechanisms of neuronal injury, repair, compensation, and degeneration. The data also argue for a gene-specific clinical trial of light reduction in human rhodopsin disease.

  8. In vivo dynamics of retinal injury and repair in the rhodopsin mutant dog model of human retinitis pigmentosa

    PubMed Central

    Cideciyan, Artur V.; Jacobson, Samuel G.; Aleman, Tomas S.; Gu, Danian; Pearce-Kelling, Susan E.; Sumaroka, Alexander; Acland, Gregory M.; Aguirre, Gustavo D.

    2005-01-01

    Genetic and environmental factors modify the severity of human neurodegenerations. Retinal degenerations caused by rhodopsin gene mutations show severity differences within and between families and even within regions of the same eye. Environmental light is thought to contribute to this variation. In the naturally occurring dog model of the human disorder, we found that modest light levels, as used in routine clinical practice, dramatically accelerated the neurodegeneration. Dynamics of acute retinal injury (consisting of abnormal intraretinal light scattering) were visualized in vivo in real time with high-resolution optical imaging. Long term consequences included fast or slow retinal degeneration or repair of injury depending on the dose of light exposure. These experiments provide a platform to study mechanisms of neuronal injury, repair, compensation, and degeneration. The data also argue for a gene-specific clinical trial of light reduction in human rhodopsin disease. PMID:15784735

  9. Transcriptome analysis and molecular signature of human retinal pigment epithelium

    PubMed Central

    Strunnikova, N.V.; Maminishkis, A.; Barb, J.J.; Wang, F.; Zhi, C.; Sergeev, Y.; Chen, W.; Edwards, A.O.; Stambolian, D.; Abecasis, G.; Swaroop, A.; Munson, P.J.; Miller, S.S.

    2010-01-01

    Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed ‘signature’ genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases. PMID:20360305

  10. Naturally occurring rhodopsin mutation in the dog causes retinal dysfunction and degeneration mimicking human dominant retinitis pigmentosa.

    PubMed

    Kijas, James W; Cideciyan, Artur V; Aleman, Tomas S; Pianta, Michael J; Pearce-Kelling, Susan E; Miller, Brian J; Jacobson, Samuel G; Aguirre, Gustavo D; Acland, Gregory M

    2002-04-30

    Rhodopsin is the G protein-coupled receptor that is activated by light and initiates the transduction cascade leading to night (rod) vision. Naturally occurring pathogenic rhodopsin (RHO) mutations have been previously identified only in humans and are a common cause of dominantly inherited blindness from retinal degeneration. We identified English Mastiff dogs with a naturally occurring dominant retinal degeneration and determined the cause to be a point mutation in the RHO gene (Thr4Arg). Dogs with this mutant allele manifest a retinal phenotype that closely mimics that in humans with RHO mutations. The phenotypic features shared by dog and man include a dramatically slowed time course of recovery of rod photoreceptor function after light exposure and a distinctive topographic pattern to the retinal degeneration. The canine disease offers opportunities to explore the basis of prolonged photoreceptor recovery after light in RHO mutations and determine whether there are links between the dysfunction and apoptotic retinal cell death. The RHO mutant dog also becomes the large animal needed for preclinical trials of therapies for a major subset of human retinopathies.

  11. An improved method of isolating fetal human retinal pigment epithelium.

    PubMed

    Castillo, B V; Little, C W; del Cerro, C; del Cerro, M

    1995-08-01

    The purpose of this study was to develop an improved method of isolating fetal human retinal pigment epithelium (RPE) for tissue culture or transplantation. Fetal human eyes ranging from 8 to 20 wks of gestation were collected and stored in Optisol solution. Under a dissecting microscope, an incision was made behind the ora serrata and extended circumferentially to remove the anterior segment. The vitreous was withdrawn, and the neural retina was carefully detached from the RPE. The sclera then was teased away from the choroid-RPE. The choroid-RPE was treated with 2% dispase in DMEM + 20 mM HEPES at 37 degrees C for 25 min. While still in dispase, the RPE was separated from the choroid using a pair of fine tipped jeweler's forceps under dark-field. An intact sheet of RPE could be separated from the choroid after treatment with dispase. No choroidal contamination was present as determined by light microscopy or cell culture. In vitro, the isolated RPE cells demonstrated classic cobblestone phenotype and expressed cytokeratin. This technique provides an easy and reliable method for isolating pure sheets of fetal human RPE. It also allows utilization of the neural retina of the same eye for other purposes, as the neural retina is not exposed to the enzymatic digestion. These features make this method especially useful for RPE and retinal transplantation; such an application is already underway.

  12. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  13. Selenium Protects Retinal Cells from Cisplatin-Induced Alterations in Carbohydrate Residues

    PubMed Central

    Akşit, Dilek; Yazıcı, Alper; Akşit, Hasan; Sarı, Esin S.; Yay, Arzu; Yıldız, Onur; Kılıç, Adil; Ermiş, Sıtkı S.; Seyrek, Kamil

    2016-01-01

    Background: Investigate alterations in the expression and localization of carbohydrate units in rat retinal cells exposed to cisplatin toxicity. Aims: The aim of the study was to evaluate putative protective effects of selenium on retinal cells subjected to cisplatin. Study Design: Animal experiment. Methods: Eighteen healthy Wistar rats were divided into three equal groups: 1. Control, 2. Cisplatin and 3. Cisplatin+selenium groups. After anesthesia, the right eye of each rat was enucleated. Results: Histochemically, retinal cells of control groups reacted with α-2,3-bound sialic acid-specific Maackia amurensis lectin (MAA) strongly, while cisplatin reduced the staining intensity for MAA. However, selenium administration alleviated the reducing effect of cisplatin on the binding sites for MAA in retinal cells. The staining intensity for N-acetylgalactosamine (GalNAc residues) specific Griffonia simplicifolia-1 (GSL–1) was relatively slight in control animals and cisplatin reduced this slight staining for GSL-1 further. Selenium administration mitigated the reducing effect of cisplatin on the binding sites for GSL-1. A diffuse staining for N-acetylglucosamine (GlcNAc) specific wheat germ agglutinin (WGA) was observed throughout the retina of the control animals. In particular, cells localized in the inner plexiform and photoreceptor layers are reacted strongly with WGA. Compared to the control animals, binding sites for WGA in the retina of rats given cisplatin were remarkably decreased. However, the retinal cells of rats given selenium reacted strongly with WGA. Conclusion: Cisplatin reduces α-2,3-bound sialic acid, GlcNAc and GalNAc residues in certain retinal cells. However, selenium alleviates the reducing effect of cisplatin on carbohydrate residues in retinal cells. PMID:27606141

  14. Identical mutation in a novel retinal gene causes progressive rod-cone degeneration in dogs and retinitis pigmentosa in humans.

    PubMed

    Zangerl, Barbara; Goldstein, Orly; Philp, Alisdair R; Lindauer, Sarah J P; Pearce-Kelling, Susan E; Mullins, Robert F; Graphodatsky, Alexander S; Ripoll, Daniel; Felix, Jeanette S; Stone, Edwin M; Acland, Gregory M; Aguirre, Gustavo D

    2006-11-01

    Progressive rod-cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and a 53-aa protein in the mouse; the first 24 aa, coded for by exon 1, are highly conserved in 14 vertebrate species. A homozygous mutation (TGC --> TAC) in the second codon shows complete concordance with the disorder in 18 different dog breeds/breed varieties tested. The same homozygous mutation was identified in a human patient from Bangladesh with autosomal recessive RP. Expression studies support the predominant expression of this gene in the retina, with equal expression in the retinal pigment epithelium, photoreceptor, and ganglion cell layers. This study provides strong evidence that a mutation in the novel gene PRCD is the cause of autosomal recessive retinal degeneration in both dogs and humans.

  15. Office for Human Research Protections

    MedlinePlus

    ... A A A Print Share Office for Human Research Protections The Office for Human Research Protections (OHRP) provides leadership in the protection of ... welfare, and wellbeing of human subjects involved in research conducted or supported by the U.S. Department of ...

  16. Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration

    PubMed Central

    Shirai, Hiroshi; Mandai, Michiko; Matsushita, Keizo; Kuwahara, Atsushi; Yonemura, Shigenobu; Nakano, Tokushige; Assawachananont, Juthaporn; Kimura, Toru; Saito, Koichi; Terasaki, Hiroko; Eiraku, Mototsugu; Sasai, Yoshiki; Takahashi, Masayo

    2016-01-01

    Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host–graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications. PMID:26699487

  17. αB-Crystallin Protects Retinal Tissue during Staphylococcus aureus- Induced Endophthalmitis▿

    PubMed Central

    Whiston, Emily A.; Sugi, Norito; Kamradt, Merideth C.; Sack, Coralynn; Heimer, Susan R.; Engelbert, Michael; Wawrousek, Eric F.; Gilmore, Michael S.; Ksander, Bruce R.; Gregory, Meredith S.

    2008-01-01

    Bacterial infections of the eye highlight a dilemma that is central to all immune-privileged sites. On the one hand, immune privilege limits inflammation to prevent bystander destruction of normal tissue and loss of vision. On the other hand, bacterial infections require a robust inflammatory response for rapid clearance of the pathogen. We demonstrate that the retina handles this dilemma, in part, by activation of a protective heat shock protein. During Staphylococcus aureus-induced endophthalmitis, the small heat shock protein αB-crystallin is upregulated in the retina and prevents apoptosis during immune clearance of the bacteria. In the absence of αB-crystallin, mice display increased retinal apoptosis and retinal damage. We found that S. aureus produces a protease capable of cleaving αB-crystallin to a form that coincides with increased retinal apoptosis and tissue destruction. We conclude that αB-crystallin is important in protecting sensitive retinal tissue during destructive inflammation that occurs during bacterial endophthalmitis. PMID:18227158

  18. Protective effect of basic fibroblast growth factor on retinal injury induced by argon laser photocoagulation

    NASA Astrophysics Data System (ADS)

    Chen, P.; Zhang, C. P.; San, Q.; Wang, C. Z.; Yang, Z. F.; Kang, H. X.; Qian, H. W.

    2010-12-01

    Laser photocoagulation treatment is often complicated by a side effect of visual impairment, which is caused by the unavoidable laser-induced retinal destruction. At present no specific is found to cure this retinopathy. The aim of this study was to observe the neuroprotective effect of bFGF on laser-induced retinal injury. Chinchilla rabbits were divided into three groups and argon laser lesions were created in the retinas. Then bFGF or dexamethasone, a widely used ophthalmic preparation, or saline was given severally by retrobulbar injection. The retinal lesions were evaluated histologically and morphometrically, and visual function was examined by ERG. The results showed that bFGF administration better preserved morphology of retinal photoreceptors and significantly diminished the area of the lesions. Furthermore, bFGF promoted the restoration of the ERG b-wave amplitude. In rabbits treated with dexamethasone, however, the lesions showed almost no ameliorative changes. This is the first study to investigate the potential role of bFGF as a remedial agent in laser photocoagulation treatment. These findings suggest that bFGF has significant neuroprotective properties in the retina and this type of neuroprotection may be of clinical significance in reducing iatrogenic laser-induced retinal injuries in humans.

  19. Rod photoreceptors protect from cone degeneration-induced retinal remodeling and restore visual responses in zebrafish

    PubMed Central

    Saade, Carole J.; Alvarez-Delfin, Karen; Fadool, James M.

    2013-01-01

    Humans are largely dependent upon cone-mediated vision. However, death or dysfunction of rods, the predominant photoreceptor subtype, results in secondary loss of cones, remodeling of retinal circuitry and blindness. The changes in circuitry may contribute to the vision deficit and undermine attempts at restoring sight. We exploit zebrafish larvae as a genetic model to specifically characterize changes associated with photoreceptor degenerations in a cone-dominated retina. Photoreceptors form synapses with two types of second order neurons, bipolar cells and horizontal cells. Using cell-specific reporter gene expression and immunolabeling for postsynaptic glutamate receptors, significant remodeling is observed following cone degeneration in the pde6cw59 larval retina but not rod degeneration in the Xops:mCFPq13 line. In adults, rods and cones are present in approximately equal numbers, and in pde6cw59 mutants glutamate receptor expression and synaptic structures in the outer plexiform layer are preserved, and visual responses are gained in these once-blind fish. We propose that the abundance of rods in the adult protects the retina from cone degeneration-induced remodeling. We test this hypothesis by genetically manipulating the number of rods in larvae. We show that an increased number and uniform distribution of rods in lor/tbx2bp22bbtl or six7 morpholino-injected larvae protect from pde6cw59-induced secondary changes. The observations that remodeling is a common consequence of photoreceptor death across species, and that in zebrafish a small number of surviving photoreceptors afford protection from degeneration-induced changes provides a model for systematic analysis of factors that slow or even prevent the secondary deteriorations associated with neural degenerative disease. PMID:23365220

  20. High-speed adaptive optics line scan confocal retinal imaging for human eye

    PubMed Central

    Wang, Xiaolin; Zhang, Yuhua

    2017-01-01

    Purpose Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. Methods A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye’s optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. Results The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. Conclusions We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss. PMID:28257458

  1. Curcumin Protects Retinal Cells from Light- and Oxidant Stress-induced Cell Death

    PubMed Central

    Mandal, Md Nawajes A.; Patlolla, Jagan M.R.; Zheng, Lixin; Agbaga, Martin-Paul; Tran, Julie-Thu A.; Wicker, Lea; Kasus-Jacobi, Anne; Elliott, Michael H.; Rao, Chinthalapally V.; Anderson, Robert E.

    2009-01-01

    Age-related macular degeneration (AMD) is a complex disease that has potential involvement of inflammatory and oxidative stress-related pathways in its pathogenesis. In search of effective therapeutic agents, we tested curcumin, a naturally-occurring compound with known anti-inflammatory and anti-oxidative properties, in rat model of light induced retinal degeneration (LIRD) and in retina derived cell lines. We hypothesized that any compound effective against LIRD, which involves significant oxidative stress and inflammation, would be a candidate for further characterization for its potential application in AMD. We observed significant retinal neuroprotection in rats fed diets supplemented with curcumin (0.2% in diet) for 2 weeks. The mechanism of retinal protection from LIRD by curcumin involves inhibition of NF-κB activation and down-regulation of cellular inflammatory genes. When tested on retina-derived cell lines (661W and ARPE-19), pre-treatment of curcumin protected these cells from H2O2-induced cell death by up-regulating cellular protective enzymes, such as HO-1, thioredoxin. Since, curcumin with its pleiotropic activities can modulate the expression and activation of many cellular regulatory proteins such as NF-κB, AKT, NRF2 and growth factors, which in turn inhibit cellular inflammatory responses and protect cells; we speculate that curcumin would be an effective nutraceutical compound for preventive and augmentative therapy of AMD. PMID:19121385

  2. Comparison between PACAP- and enriched environment-induced retinal protection in MSG-treated newborn rats.

    PubMed

    Kiss, Peter; Atlasz, Tamas; Szabadfi, Krisztina; Horvath, Gabor; Griecs, Monika; Farkas, Jozsef; Matkovits, Attila; Toth, Gabor; Lubics, Andrea; Tamas, Andrea; Gabriel, Robert; Reglodi, Dora

    2011-01-10

    Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors occur throughout the nervous system, including the retina. PACAP exerts diverse actions in the eye: it influences ocular blood flow, contraction of the ciliary muscle, and has retinoprotective effects. This effect has been proven in different models of retinal degeneration. We have previously shown that PACAP protects against monosodium-glutamate (MSG)-induced damage in neonatal rats. The beneficial effects of enriched environment, another neuroprotective strategy, have long been known. Environmental enrichment has been shown to decrease different neuronal injuries. It also influences the development of the visual system. We have recently demonstrated that significant neuroprotection can be achieved in MSG-induced retinal degeneration in animals kept in an enriched environment. Combination of neuroprotective strategies often results in increased protection. Therefore, the aim of the present study was to compare the two neuroprotective strategies alone and in combination therapy. We found that both PACAP and environmental enrichment led to a similar degree of retinal protection, but the two treatments together did not lead to increased protection: their effects were not additive.

  3. Multimodal Delivery of Isogenic Mesenchymal Stem Cells Yields Synergistic Protection from Retinal Degeneration and Vision Loss.

    PubMed

    Bakondi, Benjamin; Girman, Sergey; Lu, Bin; Wang, Shaomei

    2017-02-01

    We previously demonstrated that subretinal injection (SRI) of isogenic mesenchymal stem cells (MSCs) reduced the severity of retinal degeneration in Royal College of Surgeons rats in a focal manner. In contrast, intravenous MSC infusion (MSC(IV) ) produced panoptic retinal rescue. By combining these treatments, we now show that MSC(IV) supplementation potentiates the MSC(SRI) -mediated rescue of photoreceptors and visual function. Electrophysiological recording from superior colliculi revealed 3.9-fold lower luminance threshold responses (LTRs) and 22% larger functional rescue area from combined treatment compared with MSC(SRI) alone. MSC(IV) supplementation of sham (saline) injection also improved LTRs 3.4-fold and enlarged rescue areas by 27% compared with saline alone. We confirmed the involvement of MSC chemotaxis for vision rescue by modulating C-X-C chemokine receptor 4 activity before MSC(IV) but without increased retinal homing. Rather, circulating platelets and lymphocytes were reduced 3 and 7 days after MSC(IV) , respectively. We demonstrated MSC(SRI) -mediated paracrine support of vision rescue by SRI of concentrated MSC-conditioned medium and assessed function by electroretinography and optokinetic response. MSC-secreted peptides increased retinal pigment epithelium (RPE) metabolic activity and clearance of photoreceptor outer segments ex vivo, which was partially abrogated by antibody blockade of trophic factors in concentrated MSC-conditioned medium, or their cognate receptors on RPE. These data support multimodal mechanisms for MSC-mediated retinal protection that differ by administration route and synergize when combined. Thus, using MSC(IV) as adjuvant therapy might improve cell therapies for retinal dystrophy and warrants further translational evaluation. Stem Cells Translational Medicine 2017;6:444-457.

  4. Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice.

    PubMed

    Sakamoto, Kenji; Murakami, Yuta; Sawada, Shohei; Ushikubo, Hiroko; Mori, Asami; Nakahara, Tsutomu; Ishii, Kunio

    2016-11-15

    Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca(2+) influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo. The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp(1)]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp(1)]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4H-pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo.

  5. [Study of blue light induced DNA damage of retinal pigment epithelium(RPE) cells and the protection of vitamin C].

    PubMed

    Zhou, Jian Wei; Ren, Guo Liang; Zhang, Xiao Ming; Zhu, Xi; Lin, Hai Yan; Zhou, Ji Lin

    2003-10-01

    To evaluate protection of vitamin C on blue light-induced DNA damage of human retinal pigment epithelium (RPE) cells. The cultured RPE cells were divided into 3 groups: Control group (no blue light exposure), blue light exposure group (blue light exposure for 20 minutes) and blue light exposure + vitamin C group (blue light exposure + 100 mumol/L vitamin C). Travigen's comet assay kit and Euclid comet assay software were used to assay the DNA damage levels. The DNA percentage in the tail of electrophoretogram in the three groups were 18.44%, 54.42% and 32.43% respectively (p < 0.01). Tail moments were 8.2, 48.3, and 18.4 respectively (p < 0.01). Blue light could induce DNA damage to RPE cells but vitamin C could protect the RPE cells from the blue light-induced DNA damage.

  6. Office for Human Research Protections

    MedlinePlus

    ... the Secretary’s Advisory Committee on Human Research Protections. International Learn how OHRP promotes ... Recent Announcements "Institutional Review Board (IRB) Written Procedures: Guidance for Institutions and ...

  7. Retinal blood flow during hyperoxia in humans revisited: concerted results using different measurement techniques.

    PubMed

    Kiss, Barbara; Polska, Elzbieta; Dorner, Guido; Polak, Kaija; Findl, Oliver; Mayrl, Gabriele Fuchsjäger; Eichler, Hans-Georg; Wolzt, Michael; Schmetterer, Leopold

    2002-07-01

    Retinal vasculature shows pronounced vasoconstriction in response to hyperoxia, which appears to be related to the constant oxygen demand of the retina. However, the exact amount of blood flow reduction and the exact time course of this phenomenon are still a matter of debate. We set out to investigate the retinal response to hyperoxia using innovative techniques for the assessment of retinal hemodynamics. In a total of 48 healthy volunteers we studied the effect of 100% O(2) breathing on retinal blood flow using two methods. Red blood cell movement in larger retinal veins was quantified with combined laser Doppler velocimetry and retinal vessel size measurement. Retinal white blood cell movement was quantified with the blue field entoptic technique. The time course of retinal vasoconstriction in response to hyperoxia was assessed by continuous vessel size determination using the Zeiss retinal vessel analyzer. The response to hyperoxia as measured with combined laser Doppler velocimetry and vessel size measurement was almost twice as high as that observed with the blue field technique. Vasoconstriction in response to 100% O(2) breathing occurred within the first 5 min and no counterregulatory or adaptive mechanisms were observed. Based on these results we hypothesize that hyperoxia-induced vasoconstriction differentially affects red and white blood cell movement in the human retina. This hypothesis is based on the complex interactions between red and white blood cells in microcirculation, which have been described in detail for other vascular beds.

  8. Hydroxycinnamic acids in Crepidiastrum denticulatum protect oxidative stress-induced retinal damage.

    PubMed

    Ahn, Hong Ryul; Lee, Hee Ju; Kim, Kyung-A; Kim, Chul Young; Nho, Chu Won; Jang, Holim; Pan, Cheol-Ho; Lee, Chang Yong; Jung, Sang Hoon

    2014-02-12

    We investigated the effects of an ethanol extract of C. denticulatum (EECD) in a mouse model of glaucoma established by optic nerve crush (ONC), and found that EECD significantly protected against retinal ganglion cell (RGC) death caused by ONC. Furthermore, EECD effectively protected against N-methyl-d-aspartate-induced damage to the rat retinas. In vitro, EECD attenuated transformed retinal ganglion cell (RGC-5) death and significantly blunted the up-regulation of apoptotic proteins and mRNA level induced by 1-buthionine-(S,R)-sulfoximine combined with glutamate, reduced reactive oxygen species production by radical species, and inhibited lipid peroxidation. The major EECD components were found to be chicoric acid and 3,5-dicaffeoylquinic acid (3,5-DCQA) that have shown beneficial effects on retinal degeneration both in vitro and in vivo studies. Thus, EECD could be used as a natural neuroprotective agent for glaucoma, and chicoric acid and 3,5-DCQA as mark compounds for the development of functional food.

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

    SciTech Connect

    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{sub 60}(OH){sub 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{sub 60}(OH){sub 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 muM. Exposure to an 8.5 J.cm{sup -2} dose of visible light in the presence of > 5 muM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 muM 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 PHI = 0.05 in D{sub 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.

  10. miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells.

    PubMed

    Cheng, Li-Bo; Li, Ke-Ran; Yi, Nan; Li, Xiu-Miao; Wang, Feng; Xue, Bo; Pan, Ying-Shun; Yao, Jin; Jiang, Qin; Wu, Zhi-Feng

    2017-01-04

    Activation of NF-E2-related factor 2 (Nrf2) signaling could protect cells from ultra violet (UV) radiation. We aim to provoke Nrf2 activation via downregulating its inhibitor Keap1 by microRNA-141 ("miR-141"). In both human retinal pigment epithelium cells (RPEs) and retinal ganglion cells (RGCs), forced-expression of miR-141 downregulated Keap1, causing Nrf2 stabilization, accumulation and nuclear translocation, which led to transcription of multiple antioxidant-responsive element (ARE) genes (HO1, NOQ1 and GCLC). Further, UV-induced reactive oxygen species (ROS) production and cell death were significantly attenuated in miR-141-expressing RPEs and RGCs. On the other hand, depletion of miR-141 via expressing its inhibitor antagomiR-141 led to Keap1 upregulation and Nrf2 degradation, which aggravated UV-induced death of RPEs and RGCs. Significantly, Nrf2 shRNA knockdown almost abolished miR-141-mediated cytoprotection against UV in RPEs. These results demonstrate that miR-141 targets Keap1 to activate Nrf2 signaling, which protects RPEs and RGCs from UV radiation.

  11. Protective effects of catalase on retinal ischemia/reperfusion injury in rats.

    PubMed

    Chen, Baihua; Tang, Luosheng

    2011-11-01

    Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100mmHg for 1h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6h, 24h, 72 h and 5d after injury. Hydrogen peroxide (H(2)O(2)), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H(2)O(2), MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p<0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be

  12. Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival

    PubMed Central

    Guo, Caiwei; Sun, Yu; Liao, Tiffany; Beattie, Ursula; López, Francisco J.; Chen, Dong Feng; Lashkari, Kameran

    2015-01-01

    We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma. PMID:25923430

  13. Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration

    PubMed Central

    Wilson, Brooks; Zhao, Liangliang; Bhuyan, Rupak; Bandyopadhyay, Mausumi; Lyubarsky, Arkady; Yu, Chen; Li, Yafeng; Kanu, Levi; Miwa, Takashi; Song, Wen-Chao; Finnemann, Silvia C.; Rohrer, Bärbel; Dunaief, Joshua L.

    2016-01-01

    Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO. PMID:27893831

  14. Dietary Supplement Enriched in Antioxidants and Omega-3 Protects from Progressive Light-Induced Retinal Degeneration

    PubMed Central

    Ramchani-Ben Othman, Khaoula; Cercy, Christine; Amri, Mohamed; Doly, Michel; Ranchon-Cole, Isabelle

    2015-01-01

    In the present study, we have evaluated one of the dietary supplements enriched with antioxidants and fish oil used in clinical care for patient with age-related macular degeneration. Rats were orally fed by a gastric canula daily with 0.2 ml of water or dietary supplement until they were sacrificed. After one week of treatment, animals were either sacrificed for lipid analysis in plasma and retina, or used for evaluation of rod-response recovery by electroretinography (ERG) followed by their sacrifice to measure rhodopsin content, or used for progressive light-induced retinal degeneration (PLIRD). For PLIRD, animals were transferred to bright cyclic light for one week. Retinal damage was quantified by ERG, histology and detection of apoptotic nuclei. Animals kept in dim-cyclic-light were processed in parallel. PLIRD induced a thinning of the outer nuclear layer and a reduction of the b-wave amplitude of the ERG in the water group. Retinal structure and function were preserved in supplemented animals. Supplement induced a significant increase in omega-3 fatty acids in plasma by 168% for eicosapentaenoic acid (EPA), 142% for docosapentaenoic acid (DPA) and 19% for docosahexaenoic acid (DHA) and a decrease in the omega-6 fatty acids, DPA by 28%. In the retina, supplement induced significant reduction of linolenic acid by 67% and an increase in EPA and DPA by 80% and 72%, respectively, associated with significant decrease in omega-6 DPA by 42%. Supplement did not affect rhodopsin content or rod-response recovery. The present data indicate that supplement rapidly modified the fatty acid content and induced an accumulation of EPA in the retina without affecting rhodopsin content or recovery. In addition, it protected the retina from oxidative stress induced by light. Therefore, this supplement might be beneficial to slow down progression of certain retinal degeneration. PMID:26042773

  15. Retinal and post-retinal contributions to the quantum efficiency of the human eye revealed by electrical neuroimaging.

    PubMed

    Manasseh, Gibran; de Balthasar, Chloe; Sanguinetti, Bruno; Pomarico, Enrico; Gisin, Nicolas; de Peralta, Rolando Grave; Andino, Sara L Gonzalez

    2013-01-01

    The retina is one of the best known quantum detectors with rods able to reliably respond to single photons. However, estimates on the number of photons eliciting conscious perception, based on signal detection theory, are systematically above these values after discounting by retinal losses. One possibility is that there is a trade-off between the limited motor resources available to living systems and the excellent reliability of the visual photoreceptors. On this view, the limits to sensory thresholds are not set by the individual reliability of the receptors within each sensory modality (as often assumed) but rather by the limited central processing and motor resources available to process the constant inflow of sensory information. To investigate this issue, we reproduced the classical experiment from Hetch aimed to determine the sensory threshold in human vision. We combined a careful physical control of the stimulus parameters with high temporal/spatial resolution recordings of EEG signals and behavioral variables over a relatively large sample of subjects (12). Contrarily to the idea that the limits to visual sensitivity are fully set by the statistical fluctuations in photon absorption on retinal photoreceptors we observed that the state of ongoing neural oscillations before any photon impinges the retina helps to determine if the responses of photoreceptors have access to central conscious processing. Our results suggest that motivational and attentional off-retinal mechanisms play a major role in reducing the QE efficiency of the human visual system when compared to the efficiency of isolated retinal photoreceptors. Yet, this mechanism might subserve adaptive behavior by enhancing the overall multisensory efficiency of the whole system composed by diverse reliable sensory modalities.

  16. Retinal and post-retinal contributions to the quantum efficiency of the human eye revealed by electrical neuroimaging

    PubMed Central

    Manasseh, Gibran; de Balthasar, Chloe; Sanguinetti, Bruno; Pomarico, Enrico; Gisin, Nicolas; de Peralta, Rolando Grave; Andino, Sara L. Gonzalez

    2013-01-01

    The retina is one of the best known quantum detectors with rods able to reliably respond to single photons. However, estimates on the number of photons eliciting conscious perception, based on signal detection theory, are systematically above these values after discounting by retinal losses. One possibility is that there is a trade-off between the limited motor resources available to living systems and the excellent reliability of the visual photoreceptors. On this view, the limits to sensory thresholds are not set by the individual reliability of the receptors within each sensory modality (as often assumed) but rather by the limited central processing and motor resources available to process the constant inflow of sensory information. To investigate this issue, we reproduced the classical experiment from Hetch aimed to determine the sensory threshold in human vision. We combined a careful physical control of the stimulus parameters with high temporal/spatial resolution recordings of EEG signals and behavioral variables over a relatively large sample of subjects (12). Contrarily to the idea that the limits to visual sensitivity are fully set by the statistical fluctuations in photon absorption on retinal photoreceptors we observed that the state of ongoing neural oscillations before any photon impinges the retina helps to determine if the responses of photoreceptors have access to central conscious processing. Our results suggest that motivational and attentional off-retinal mechanisms play a major role in reducing the QE efficiency of the human visual system when compared to the efficiency of isolated retinal photoreceptors. Yet, this mechanism might subserve adaptive behavior by enhancing the overall multisensory efficiency of the whole system composed by diverse reliable sensory modalities. PMID:24302913

  17. Human organotypic retinal cultures (HORCs) as a chronic experimental model for investigation of retinal ganglion cell degeneration.

    PubMed

    Osborne, Andrew; Hopes, Marina; Wright, Phillip; Broadway, David C; Sanderson, Julie

    2016-02-01

    There is a growing need for models of human diseases that utilise native, donated human tissue in order to model disease processes and develop novel therapeutic strategies. In this paper we assessed the suitability of adult human retinal explants as a potential model of chronic retinal ganglion cell (RGC) degeneration. Our results confirmed that RGC markers commonly used in rodent studies (NeuN, βIII Tubulin and Thy-1) were appropriate for labelling human RGCs and followed the expected differential expression patterns across, as well as throughout, the macular and para-macular regions of the retina. Furthermore, we showed that neither donor age nor post-mortem time (within 24 h) significantly affected the initial expression levels of RGC markers. In addition, the feasibility of using human post mortem donor tissue as a long-term model of RGC degeneration was determined with RGC protein being detectable up to 4 weeks in culture with an associated decline in RGC mRNA and significant, progressive, apoptotic labelling of NeuN(+) cells. Differences in RGC apoptosis might have been influenced by medium compositions indicating that media constituents could play a role in supporting axotomised RGCs. We propose that using ex vivo human explants may prove to be a useful model for testing the effectiveness of neuroprotective strategies.

  18. Protective Effect of Total Flavones from Hippophae rhamnoides L. against Visible Light-Induced Retinal Degeneration in Pigmented Rabbits.

    PubMed

    Wang, Yong; Huang, Fenghong; Zhao, Liang; Zhang, Di; Wang, Ou; Guo, Xiaoxuan; Lu, Feng; Yang, Xue; Ji, Baoping; Deng, Qianchun

    2016-01-13

    Sea buckthorn (Hippophae rhamnoides L.) flavones have been used as candidate functional food ingredients because of their bioactivities, such as treating cardiovascular disorders, lowering plasma cholesterol level, and regulating immune function. However, the protective effects of sea buckthorn flavones against retinal degeneration remain unclear to date. This study investigated the protective effects of total flavones from H. rhamnoides (TFH) against visible light-induced retinal damage and explored the related mechanisms in pigmented rabbits. Rabbits were treated with TFH (250 and 500 mg/kg) for 2 weeks pre-illumination and 1 week post-illumination until sacrifice. Retinal function was quantified by performing electroretinography 1 day before and 1, 3, and 7 days after light exposure (18000 lx for 2 h). Retinal degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) and performing the TUNEL assay 7 days after light exposure. Enzyme-linked immunosorbent assay, Western blot analysis, and immunohistochemistry were used to explore the antioxidant, anti-inflammatory, and anti-apoptotic mechanisms of TFH during visible light-induced retinal degeneration. Light exposure produced a degenerative effect primarily on the ONL, inner nuclear layer (INL), and ganglion cell layer (GCL). TFH significantly attenuated the destruction of electroretinograms caused by light damage, maintained ONL thickness, and decreased the number of TUNEL-positive cells in the INL and GCL. TFH ameliorated the retinal oxidative stress (GSH-Px, CAT, T-AOC, and MDA), inflammation (IL-1β and IL-6), angiogenesis (VEGF), and apoptosis (Bax, Bcl2, and caspase-3) induced by light exposure. Therefore, TFH exhibited protective effects against light-induced retinal degeneration by increasing the antioxidant defense mechanisms, suppressing pro-inflammatory and angiogenic cytokines, and inhibiting retinal cell apoptosis.

  19. Infrared imaging of sub-retinal structures in the human ocular fundus.

    PubMed

    Elsner, A E; Burns, S A; Weiter, J J; Delori, F C

    1996-01-01

    The interaction of infrared light with the human ocular fundus, particularly sub-retinal structures, was studied in vivo. Visible and infra-red wavelengths and a scanning laser ophthalmoscope were used to acquire digital images of the human fundus. The contrast and reflectance of selected retinal and sub-retinal features were computed for a series of wavelengths or modes of imaging. Near infrared light provides better visibility than visible light for sub-retinal features. Sub-retinal deposits appear light and thickened; the optic nerve head, retinal vessels, and choroidal vessels appear dark. Contrast and visibility of features increases with increasing wavelength from 795 to 895 nm. Optimizing the mode of imaging improves the visibility of some structures. This new quantitative basis for near infrared imaging techniques can be applied to a wide range of imaging modalities for the study of pathophysiology and treatment in diseases affecting the retinal pigment epithelium and Bruch's membrane, such as age-related macular degeneration.

  20. A method and technical equipment for an acute human trial to evaluate retinal implant technology

    NASA Astrophysics Data System (ADS)

    Hornig, Ralf; Laube, Thomas; Walter, Peter; Velikay-Parel, Michaela; Bornfeld, Norbert; Feucht, Matthias; Akguel, Harun; Rössler, Gernot; Alteheld, Nils; Lütke Notarp, Dietmar; Wyatt, John; Richard, Gisbert

    2005-03-01

    This paper reports on methods and technical equipment to investigate the epiretinal stimulation of the retina in blind human subjects in acute trials. Current is applied to the retina through a thin, flexible microcontact film (microelectrode array) with electrode diameters ranging from 50 to 360 µm. The film is mounted in a custom-designed surgical tool that is hand-held by the surgeon during stimulation. The eventual goal of the work is the development of a chronically implantable retinal prosthesis to restore a useful level of vision to patients who are blind with outer retinal degenerations, specifically retinitis pigmentosa and macular degeneration.

  1. Compound 49b Regulates ZO-1 and Occludin Levels in Human Retinal Endothelial Cells and in Mouse Retinal Vasculature

    PubMed Central

    Jiang, Youde; Liu, Li; Steinle, Jena J.

    2017-01-01

    Purpose To investigate whether Epac1 is key to Compound 49b's regulation of zonula occluden 1 (ZO-1) and occludin levels in human retinal endothelial cells (REC) and in an Epac1 vascular-specific conditional knockout mouse retina. Methods Primary REC were grown in normal (5 mM) or high glucose (25 mM). Some cells were treated with a novel β-adrenergic receptor agonist, Compound 49b. Additional dishes were treated with Epac1 siRNA or Compound 49b + Epac1 siRNA. Protein levels of ZO-1, occludin, VEGF, and protein kinase C zeta (PKCz) were measured by Western blotting. Cell permeability was measured in REC grown in normal or high glucose and treated with Compound 49b, a specific Epac 1 agonist (8-CPT-2′-O-Me-cAMP), or VEGF. Epac1 floxed and cdh5-Cre mice were bred to generate Epac1 knockout mice in vascular endothelial cells. Immunofluorescence was done on retinal flatmounts from the floxed and Cre-Lox mice for occludin and ZO-1. Western blotting was also done for both proteins in whole retinal lysates from the mice. Results High glucose significantly reduced ZO-1 and occludin protein levels, which was associated with reduced cell adhesion. Compound 49b increased endothelial cell barrier protein levels through active Epac1. Knockout of Epac1 in vascular endothelial cells substantially reduced ZO-1 and occludin staining in retinal flatmounts, as well as protein levels. Conclusions Compound 49b increased ZO-1 and occludin protein levels, likely leading to decreased permeability. PMID:28114578

  2. Lithium chloride protects retinal neurocytes from nutrient deprivation by promoting DNA non-homologous end-joining

    SciTech Connect

    Zhuang Jing; Li Fan; Liu Xuan; Liu Zhiping; Lin Jianxian; Ge Yihong; Kaminski, Joseph M.; Summers, James Bradley; Wang Zhichong; Ge Jian Yu Keming

    2009-03-13

    Lithium chloride is a therapeutic agent for treatment of bipolar affective disorders. Increasing numbers of studies have indicated that lithium has neuroprotective effects. However, the molecular mechanisms underlying the actions of lithium have not been fully elucidated. This study aimed to investigate whether lithium chloride produces neuroprotective function by improving DNA repair pathway in retinal neurocyte. In vitro, the primary cultured retinal neurocytes (85.7% are MAP-2 positive cells) were treated with lithium chloride, then cultured with serum-free media to simulate the nutrient deprived state resulting from ischemic insult. The neurite outgrowth of the cultured cells increased significantly in a dose-dependent manner when exposed to different levels of lithium chloride. Genomic DNA electrophoresis demonstrated greater DNA integrity of retinal neurocytes when treated with lithium chloride as compared to the control. Moreover, mRNA and protein levels of Ligase IV (involved in DNA non-homologous end-joining (NHEJ) pathway) in retinal neurocytes increased with lithium chloride. The end joining activity assay was performed to determine the role of lithium on NHEJ in the presence of extract from retinal neurocytes. The rejoining levels in retinal neurocytes treated with lithium were significantly increased as compared to the control. Furthermore, XRCC4, the Ligase IV partner, and the transcriptional factor, CREB and CTCF, were up-regulated in retinal cells after treating with 1.0 mM lithium chloride. Therefore, our data suggest that lithium chloride protects the retinal neural cells from nutrient deprivation in vitro, which may be similar to the mechanism of cell death in glaucoma. The improvement in DNA repair pathway involving in Ligase IV might have an important role in lithium neuroprotection. This study provides new insights into the neural protective mechanisms of lithium chloride.

  3. Regional variation in human retinal vessel oxygen saturation.

    PubMed

    Shahidi, A M; Patel, S R; Flanagan, J G; Hudson, C

    2013-08-01

    The purpose of this study was to investigate regional differences in oxygen saturation of blood in first degree retinal vessels using a novel non-flash hyperspectral retinal camera (Photon etc Inc). Nine healthy individuals (mean age 24.4 ± 3.6 yrs, 5 males) were imaged at 548, 569, 586, 600, 605 and 610 nm wavelengths. Optical density values were extracted with the aid of Image-J software for blood oxygen saturation (SO2) determination. Arteriolar and venular SO2 were measured at three locations (ranging 1-3 optic nerve head radii) from the disc margin along the vessels in the superior and inferior temporal quadrants. Retinal SO2 was significantly higher in the superior temporal arteriole and venule as compared to the inferior temporal vessels (p = 0.033 and p = 0.032 for arterioles and venules, respectively). SO2 was not significantly different between the three measurement sites for any of the given vessels imaged (p > 0.05). In conclusion, greater SO2 values were found in the superior temporal first degree retinal arterioles and venules in young healthy individuals than in the equivalent inferior vessels. However, there were no detectable differences in retinal SO2 along each of the major vessels, a finding that is consistent with the concept of these vessels not contributing primarily to gas exchange. Moreover, the SO2 was consistently higher in the arterioles than in the equivalent venules (p < 0.0001).

  4. The Protective Role of PAC1-Receptor Agonist Maxadilan in BCCAO-Induced Retinal Degeneration.

    PubMed

    Vaczy, A; Reglodi, D; Somoskeoy, T; Kovacs, K; Lokos, E; Szabo, E; Tamas, A; Atlasz, T

    2016-10-01

    A number of studies have proven that pituitary adenylate cyclase activating polypeptide (PACAP) is protective in neurodegenerative diseases. Permanent bilateral common carotid artery occlusion (BCCAO) causes severe degeneration in the rat retina. In our previous studies, protective effects were observed with PACAP1-38, PACAP1-27, and VIP but not with their related peptides, glucagon, or secretin in BCCAO. All three PACAP receptors (PAC1, VPAC1, VPAC2) appear in the retina. Molecular and immunohistochemical analysis demonstrated that the retinoprotective effects are most probably mainly mediated by the PAC1 receptor. The aim of the present study was to investigate the retinoprotective effects of a selective PAC1-receptor agonist maxadilan in BCCAO-induced retinopathy. Wistar rats were used in the experiment. After performing BCCAO, the right eye was treated with intravitreal maxadilan (0.1 or 1 μM), while the left eye was injected with vehicle. Sham-operated rats received the same treatment. Two weeks after the operation, retinas were processed for standard morphometric and molecular analysis. Intravitreal injection of 0.1 or 1 μM maxadilan caused significant protection in the thickness of most retinal layers and the number of cells in the GCL compared to the BCCAO-operated eyes. In addition, 1 μM maxadilan application was more effective than 0.1 μM maxadilan treatment in the ONL, INL, IPL, and the entire retina (OLM-ILM). Maxadilan treatment significantly decreased cytokine expression (CINC-1, IL-1α, and L-selectin) in ischemia. In summary, our histological and molecular analysis showed that maxadilan, a selective PAC1 receptor agonist, has a protective role in BCCAO-induced retinal degeneration, further supporting the role of PAC1 receptor conveying the retinoprotective effects of PACAP.

  5. Progressive outer retinal necrosis: manifestation of human immunodeficiency virus infection.

    PubMed

    Lo, Phey Feng; Lim, Rongxuan; Antonakis, Serafeim N; Almeida, Goncalo C

    2015-05-06

    We present the case of a 54-year-old man who developed progressive outer retinal necrosis (PORN) as an initial manifestation of HIV infection without any significant risk factors for infection with HIV. PORN is usually found as a manifestation of known AIDS late in the disease. Our patient presented with transient visual loss followed by decrease in visual acuity and facial rash. Subsequent investigation revealed anterior chamber tap positive for varicella zoster virus (VZV), as well as HIV positivity, with an initial CD4 count of 48 cells/µL. Systemic and intravitreal antivirals against VZV, and highly active antiretroviral therapy against HIV were started, which halted further progression of retinal necrosis. This case highlights the importance of suspecting PORN where there is a rapidly progressive retinitis, and also testing the patient for HIV, so appropriate treatment can be started.

  6. Investigation of the Protective Effects of Taurine against Alloxan-Induced Diabetic Retinal Changes via Electroretinogram and Retinal Histology with New Zealand White Rabbits

    PubMed Central

    Yeh, Shang-Min; Chen, Yi-Chen; Lin, Shiun-Long

    2014-01-01

    The purpose of this study was to investigate the protective role of orally administered taurine against diabetic retinal changes via electroretinogram (ERG) and retinal histology on rabbits. Rabbits were randomly assigned into groups: Group I (vehicle administration only); Group II (diabetes: induced by 100 mg/kg alloxan injection); Group III (diabetes and fed with 200 mg/kg taurine); and Group IV (diabetes and fed with 400 mg/kg taurine). The body weight and blood glucose levels of the rabbits were monitored weekly. The ERG was measured on weeks 5 and 15. Retinal histology was analyzed in the end of the experiment. Results revealed that a taurine supplement significantly ameliorates the alloxan-induced hyperglycemia and protects the retina from electrophysiological changes. Group II showed a significant (P < 0.05) change in the mean scotopic b-wave amplitude when compared to that of Group I, whereas the diabetic rabbits treated with taurine (Group III and IV) were analogous to Group I. Histologically, the amount of Bipolar and Müller cells showed no difference (P > 0.05) between all groups and when compared with those of Group I. Our study provides solid evidences that taurine possesses an antidiabetic activity, reduced loss of body weight, and less electrophysiological changes of the diabetic retina. PMID:25298779

  7. Politics and Human Welfare: Retinitis Pigmentosa Patients in South Africa.

    ERIC Educational Resources Information Center

    McKendrick, B. W.; Leketi, M.

    1990-01-01

    The study found that apartheid impacted the sociopsychological and physical circumstances of 12 African and 11 White people with retinitis pigmentosa in South Africa. Findings are discussed in terms of onset of condition, effects on subjects' lives, knowledge of social services, and needs unmet by existing services. (JDD)

  8. Characterization of single-file flow through human retinal parafoveal capillaries using an adaptive optics scanning laser ophthalmoscope

    PubMed Central

    Tam, Johnny; Tiruveedhula, Pavan; Roorda, Austin

    2011-01-01

    Adaptive Optics Scanning Laser Ophthalmoscopy was used to noninvasively acquire videos of single-file flow through live human retinal parafoveal capillaries. Videos were analyzed offline to investigate capillary flow dynamics. Certain capillaries accounted for a clear majority of leukocyte traffic (Leukocyte-Preferred-Paths, LPPs), while other capillaries primarily featured plasma gap flow (Plasma-Gap-Capillaries, PGCs). LPPs may serve as a protective mechanism to prevent inactivated leukocytes from entering exchange capillaries, and PGCs may serve as relief valves to minimize flow disruption due to the presence of a leukocyte in a neighboring LPP. PMID:21483603

  9. Protective Effects of Melatonin on Retinal Inflammation and Oxidative Stress in Experimental Diabetic Retinopathy

    PubMed Central

    Jiang, Tingting; Cai, Jiyang; Fan, Jiawen; Zhang, Xiaozhe

    2016-01-01

    Oxidative stress and inflammation are important pathogenic factors contributing to the etiology of diabetic retinopathy (DR). Melatonin is an endogenous hormone that exhibits a variety of biological effects including antioxidant and anti-inflammatory functions. The goals of this study were to determine whether melatonin could ameliorate retinal injury and to explore the potential mechanisms. Diabetes was induced by a single intraperitoneal (i.p.) injection of STZ (60 mg/kg) in Sprague-Dawley rats. Melatonin (10 mg kg−1 daily, i.p.) was administered from the induction of diabetes and continued for up to 12 weeks, after which the animals were sacrificed and retinal samples were collected. The retina of diabetic rats showed depletion of glutathione and downregulation of glutamate cysteine ligase (GCL). Melatonin significantly upregulated GCL by retaining Nrf2 in the nucleus and stimulating Akt phosphorylation. The production of proinflammatory cytokines and proteins, including interleukin 1β, TNF-α, and inducible nitric oxide synthase (iNOS), was inhibited by melatonin through the NF-κB pathway. At 12 weeks, melatonin prevented the significant decrease in the ERG a- and b-wave amplitudes under the diabetic condition. Our results suggest potent protective functions of melatonin in diabetic retinopathy. In addition to being a direct antioxidant, melatonin can exert receptor-mediated signaling effects to attenuate inflammation and oxidative stress of the retina. PMID:27143993

  10. Nerve growth factor protects against palmitic acid-induced injury in retinal ganglion cells

    PubMed Central

    Yan, Pan-shi; Tang, Shu; Zhang, Hai-feng; Guo, Yuan-yuan; Zeng, Zhi-wen; Wen, Qiang

    2016-01-01

    Accumulating evidence supports an important role for nerve growth factor (NGF) in diabetic retinopathy. We hypothesized that NGF has a protective effect on rat retinal ganglion RGC-5 cells injured by palmitic acid (PA), a metabolic factor implicated in the development of diabetes and its complications. Our results show that PA exposure caused apoptosis of RGC-5 cells, while NGF protected against PA insult in a concentration-dependent manner. Additionally, NGF significantly attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in RGC-5 cells. Pathway inhibitor tests showed that the protective effect of NGF was completely reversed by LY294002 (PI3K inhibitor), Akt VIII inhibitor, and PD98059 (ERK1/2 inhibitor). Western blot analysis revealed that NGF induced the phosphorylation of Akt/FoxO1 and ERK1/2 and reversed the PA-evoked reduction in the levels of these proteins. These results indicate that NGF protects RGC-5 cells against PA-induced injury through anti-oxidation and inhibition of apoptosis by modulation of the PI3K/Akt and ERK1/2 signaling pathways. PMID:28123432

  11. Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

    PubMed Central

    Flores-Bellver, M; Bonet-Ponce, L; Barcia, J M; Garcia-Verdugo, J M; Martinez-Gil, N; Saez-Atienzar, S; Sancho-Pelluz, J; Jordan, J; Galindo, M F; Romero, F J

    2014-01-01

    Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases. PMID:25032851

  12. CNTF Gene Therapy Confers Lifelong Neuroprotection in a Mouse Model of Human Retinitis Pigmentosa

    PubMed Central

    Lipinski, Daniel M; Barnard, Alun R; Singh, Mandeep S; Martin, Chris; Lee, Edward J; Davies, Wayne I L; MacLaren, Robert E

    2015-01-01

    The long-term outcome of neuroprotection as a therapeutic strategy for preventing cell death in neurodegenerative disorders remains unknown, primarily due to slow disease progression and the inherent difficulty of assessing neuronal survival in vivo. Employing a murine model of retinal disease, we demonstrate that ciliary neurotrophic factor (CNTF) confers life-long protection against photoreceptor degeneration. Repetitive retinal imaging allowed the survival of intrinsically fluorescent cone photoreceptors to be quantified in vivo. Imaging of the visual cortex and assessment of visually-evoked behavioral responses demonstrated that surviving cones retain function and signal correctly to the brain. The mechanisms underlying CNTF-mediated neuroprotection were explored through transcriptome analysis, revealing widespread upregulation of proteolysis inhibitors, which may prevent cellular/extracellular matrix degradation and complement activation in neurodegenerative diseases. These findings provide insights into potential novel therapeutic avenues for diseases such as retinitis pigmentosa and amyotrophic lateral sclerosis, for which CNTF has been evaluated unsuccessfully in clinical trials. PMID:25896245

  13. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    PubMed Central

    Han, Ming-lei; Liu, Guo-hua; Guo, Jin; Yu, Shu-juan; Huang, Jing

    2016-01-01

    Retinal ganglion cell (RGC) degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB)-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H2O2)-induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H2O2. Western blot assay showed that in H2O2 -damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H2O2 -induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H2O2 -induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway. PMID:27127489

  14. First-in-Human Trial of a Novel Suprachoroidal Retinal Prosthesis

    PubMed Central

    Ayton, Lauren N.; Blamey, Peter J.; Guymer, Robyn H.; Luu, Chi D.; Nayagam, David A. X.; Sinclair, Nicholas C.; Shivdasani, Mohit N.; Yeoh, Jonathan; McCombe, Mark F.; Briggs, Robert J.; Opie, Nicholas L.; Villalobos, Joel; Dimitrov, Peter N.; Varsamidis, Mary; Petoe, Matthew A.; McCarthy, Chris D.; Walker, Janine G.; Barnes, Nick; Burkitt, Anthony N.; Williams, Chris E.; Shepherd, Robert K.; Allen, Penelope J.

    2014-01-01

    Retinal visual prostheses (“bionic eyes”) have the potential to restore vision to blind or profoundly vision-impaired patients. The medical bionic technology used to design, manufacture and implant such prostheses is still in its relative infancy, with various technologies and surgical approaches being evaluated. We hypothesised that a suprachoroidal implant location (between the sclera and choroid of the eye) would provide significant surgical and safety benefits for patients, allowing them to maintain preoperative residual vision as well as gaining prosthetic vision input from the device. This report details the first-in-human Phase 1 trial to investigate the use of retinal implants in the suprachoroidal space in three human subjects with end-stage retinitis pigmentosa. The success of the suprachoroidal surgical approach and its associated safety benefits, coupled with twelve-month post-operative efficacy data, holds promise for the field of vision restoration. Trial Registration Clinicaltrials.gov NCT01603576 PMID:25521292

  15. Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling.

    PubMed

    Roche, Sarah L; Wyse-Jackson, Alice C; Gómez-Vicente, Violeta; Lax, Pedro; Ruiz-Lopez, Ana M; Byrne, Ashleigh M; Cuenca, Nicolás; Cotter, Thomas G

    2016-01-01

    Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue 'Norgestrel' is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel's neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising

  16. Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

    PubMed Central

    Gómez-Vicente, Violeta; Lax, Pedro; Ruiz-Lopez, Ana M.; Byrne, Ashleigh M.; Cuenca, Nicolás; Cotter, Thomas G.

    2016-01-01

    Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a

  17. A compensatory mechanism protects retinal mitochondria from initial insult in diabetic retinopathy.

    PubMed

    Santos, Julia M; Tewari, Shikha; Kowluru, Renu A

    2012-11-01

    In the pathogenesis of diabetic retinopathy, an increase in retinal oxidative stress precedes mitochondrial dysfunction and capillary cell apoptosis. This study is designed to understand the mechanism responsible for the protection of mitochondria damage in the early stages of diabetic retinopathy. After 15 days-12 months of streptozotocin-induced diabetes in rats, retina was analyzed for mitochondria DNA (mtDNA) damage by extended length PCR. DNA repair enzyme and replication machinery were quantified in the mitochondria, and the binding of mitochondrial transcriptional factor A (TFAM) with mtDNA was analyzed by ChIP. Key parameters were confirmed in the retinal endothelial cells incubated in 20mM glucose for 6-96h. Although reactive oxygen species (ROS) were increased within 15 days of diabetes, mtDNA damage was observed at 6 months of diabetes. After 15 days of diabetes DNA repair/replication enzymes were significantly increased in the mitochondria, but at 2 months, their mitochondrial accumulation started to come down, and mtDNA copy number and binding of TFAM with mtDNA became significantly elevated. However, at 6 months of diabetes, the repair/replication machinery became subnormal and mtDNA copy number significantly decreased. A similar temporal relationship was observed in endothelial cells exposed to high glucose. Thus, in the early stages of diabetes, increased mtDNA biogenesis and repair compensates for the ROS-induced damage, but, with sustained insult, this mechanism is overwhelmed, and mtDNA and electron transport chain (ETC) are damaged. The compromised ETC propagates a vicious cycle of ROS and the dysfunctional mitochondria fuels loss of capillary cells by initiating their apoptosis.

  18. Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma

    PubMed Central

    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 < 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. Conclusions The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes

  19. Tetramethylpyrazine nitrone protects retinal ganglion cells against N-methyl-d-aspartate-induced excitotoxicity.

    PubMed

    Luo, Xiaopeng; Yu, Yankun; Xiang, Zongqin; Wu, Huisu; Ramakrishna, Seeram; Wang, Yuqiang; So, Kwok-Fai; Zhang, Zaijun; Xu, Ying

    2017-02-03

    Adding a free radical-scavenging nitrone moiety on tetramethylpyrazine, we have previously synthesized a chemical named 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (tetramethylpyrazine nitrone, or TBN) and proved its neuroprotective effect but with limited understanding of its mechanism. Here we ask if TBN protects retinal ganglion cells (RGCs) against excitotoxicity induced by NMDA and explore the underlying mechanism. NMDA was intravitreally injected to induce RGC injury in rats, followed by daily intraperitoneal administrations of TBN. Measurements of TBN concentration at different times after intraperitoneal administration showed that more than 200 μM TBN reached the aqueous humor quickly. Then RGCs' survival was evaluated by quantifying Brn3-positive cells, and retinal functions were examined by electroretinogram and visual behaviors. TBN significantly increased the survival of RGCs after NMDA insult, recovered the amplitude of photopic negative responses to flash, and restored the visual behavior. Furthermore, TBN inhibited the apoptotic process, as indicated by the elevated ratios of cleaved caspase-3/caspase-3 and of Bax/Bcl-2, and decreased the level of reactive oxygen species. Moreover, TBN reduced RGC's calcium overload induced by NMDA or by KCl. Whole-cell patch recording from RGCs further showed that TBN slightly but significantly inhibited L-type calcium channels, but had little effect on T-type calcium channel or NMDA-, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)-induced current. Thus our data indicate that TBN alleviates NMDA-elicited injury of rat RGCs both morphologically and functionally, possibly by inhibiting the L-type calcium channel thus reducing Ca(2+) overload and by directly scavenging free radicals. Therefore, TBN may be a novel candidate for treating excitotoxicity-related visual disorders such as glaucoma.

  20. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

    PubMed Central

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.

    2015-01-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  1. RPE65: role in the visual cycle, human retinal disease, and gene therapy.

    PubMed

    Cai, Xue; Conley, Shannon M; Naash, Muna I

    2009-06-01

    RPE65 is an isomerohydrolase expressed in retinal pigment epithelium. It is critical for the regeneration of the visual pigment necessary for both rod and cone-mediated vision. Mutations in human RPE65 cause Leber's congenital amaurosis and other forms of autosomal recessive retinitis pigmentosa which are associated with early-onset blindness. Several RPE65 animal models including two different mouse models and a naturally occurring canine model have been thoroughly characterized to determine the mechanisms that underlie RPE65 associated retinal dystrophies. More recently, substantial effort has gone into designing gene therapies for these diseases. Based on several encouraging reports from animal models, at least three clinical trials are currently underway for the treatment of LCA using modified AAV vectors carrying the RPE65 cDNA and have reported positive preliminary results.

  2. RPE65: Role in the visual cycle, human retinal disease, and gene therapy

    PubMed Central

    Cai, Xue; Conley, Shannon M.; Naash, Muna I.

    2010-01-01

    RPE65 is an isomerohydrolase expressed in retinal pigment epithelium. It is critical for the regeneration of the visual pigment necessary for both rod and cone-mediated vision. Mutations in human RPE65 cause Leber’s congenital amaurosis and other forms of autosomal recessive retinitis pigmentosa which are associated with early-onset blindness. Several RPE65 animal models including two different mouse models and a naturally occurring canine model have been thoroughly characterized to determine the mechanisms that underlie RPE65 associated retinal dystrophies. More recently, substantial effort has gone into designing gene therapies for these diseases. Based on several encouraging reports from animal models, at least three clinical trials are currently underway for the treatment of LCA using modified AAV vectors carrying the RPE65 cDNA and have reported positive preliminary results. PMID:19373675

  3. Human conjunctival microvasculature assessed with a retinal function imager (RFI)

    PubMed Central

    Jiang, Hong; Ye, Yufeng; DeBuc, Delia Cabrera; Lam, Byron L; Rundek, Tatjana; Tao, Aizhu; Shao, Yilei; Wang, Jianhua

    2012-01-01

    The conjunctival and cerebral vasculatures share similar embryological origins, with similar structural and physiological characteristics. Tracking the conjunctival microvasculature may provide useful information for predicting the onset, progression and prognosis of both systemic and central nervous system (CNS) vascular diseases. The bulbar conjunctival vasculature was imaged using a retinal function imager (RFI, Optical Imaging Ltd, Rehovot, Israel). Hemoglobin in red blood cells was used as an intrinsic motion-contrast agent in the generation of detailed noninvasive capillary-perfusion maps (nCPMs) and the calculation of the blood flow velocity. Five healthy subjects were imaged under normal conditions and again under the stress condition of wearing a contact lens. The retina was also imaged in one eye of one subject for comparison. The nCPMs showed the conjunctival microvasculature in exquisite detail, which appeared as clear as the retinal nCPMs. The blood flow velocities in the temporal conjunctival microvasculature were 0.86 ± 0.08 (mean ± SD, mm/s) for the bare eye and 0.99 ± 0.11 mm/s with contact lens wear. It is feasible to use RFI for imaging the conjunctival vasculature. PMID:23084966

  4. Retinal images in the human eye with implanted intraocular lens

    NASA Astrophysics Data System (ADS)

    Zając, Marek; Siedlecki, Damian; Nowak, Jerzy

    2007-04-01

    A typical proceeding in cataract is based on the removal of opaque crystalline lens and inserting in its place the artificial intraocular lens (IOL). The quality of retinal image after such procedure depends, among others, on the parameters of the IOL, so the design of the implanted lens is of great importance. An appropriate choice of the IOL material, especially in relation to its biocompatibility, is often considered. However the parameter, which is often omitted during the IOL design is its chromatic aberration. In particular lack of its adequacy to the chromatic aberration of a crystalline lens may cause problems. In order to fit better chromatic aberration of the eye with implanted IOL to that of the healthy eye we propose a hybrid - refractive-diffractive IOL. It can be designed in such way that the total longitudinal chromatic aberration of an eye with implanted IOL equals the total longitudinal chromatic aberration of a healthy eye. In this study we compare the retinal image quality calculated numerically on the basis of the well known Liou-Brennan eye model with typical IOL implanted with that obtained if the IOL is done as hybrid (refractive-diffractive) design.

  5. Detection of Anatomic Structures in Human Retinal Imagery

    SciTech Connect

    Tobin Jr, Kenneth William; Chaum, Edward; Muthusamy Govindasamy, Vijaya Priya; Karnowski, Thomas Paul

    2007-01-01

    The widespread availability of electronic imaging devices throughout the medical community is leading to a growing body of research on image processing and analysis to diagnose retinal disease such as diabetic retinopathy (DR). Productive computer-based screening of large, at-risk populations at low cost requires robust, automated image analysis. In this paper we present results for the automatic detection of the optic nerve and localization of the macula using digital red-free fundus photography. Our method relies on the accurate segmentation of the vasculature of the retina followed by the determination of spatial features describing the density,average thickness, and average orientation of the vasculature in relation to the position of the optic nerve. Localization of the macula follows using knowledge of the optic nerve location to detect the horizontal raphe of the retina using a geometric model of the vasculature. We report 90.4% detection performance for the optic nerve and 92.5% localization performance for the macula for red-free fundus images representing a population of 345 images corresponding to 269 patients with 18 different pathologies associated with DR and other common retinal diseases such as age-related macular degeneration.

  6. Protective effect of magnesium acetyltaurate against NMDA-induced retinal damage involves restoration of minerals and trace elements homeostasis.

    PubMed

    Jafri, Azliana Jusnida Ahmad; Arfuzir, Natasha Najwa Nor; Lambuk, Lidawani; Iezhitsa, Igor; Agarwal, Renu; Agarwal, Puneet; Razali, Norhafiza; Krasilnikova, Anna; Kharitonova, Maria; Demidov, Vasily; Serebryansky, Evgeny; Skalny, Anatoly; Spasov, Alexander; Yusof, Ahmad Pauzi Md; Ismail, Nafeeza Mohd

    2017-01-01

    .0001) compared to vehicle injected group. This was accompanied with significant increase of Ca/Mg and Na/K ratios, 3 and 1.27 times respectively, compared to control group. The trace elements such as Cu, Fe and Zn also showed a significant increase amounting to 3.3 (p<0.001), 2.3 (p<0.0001) and 3 (p<0.0001) times respectively compared to control group. Se was increased by 60% (p<0.005). Pre-treatment with MgAT abolished effect of NMDA on minerals and trace elements more effectively than co- and post-treatment. Similar observations were made for retinal oxidative stress, retinal morphology and retinal cell apoptosis. In conclusion, current study demonstrated the protective effect of MgAT against NMDA-induced oxidative stress and retinal cell apoptosis. This effect of MgAT was associated with restoration of retinal concentrations of minerals and trace elements. Further studies are warranted to explore the precise molecular targets of MgAT. Nevertheless, MgAT seems a potential candidate in the management of diseases involving NMDA-induced excitotoxicity.

  7. Inner Retinal Oxygen Extraction Fraction in Response to Light Flicker Stimulation in Humans

    PubMed Central

    Felder, Anthony E.; Wanek, Justin; Blair, Norman P.; Shahidi, Mahnaz

    2015-01-01

    Purpose Light flicker has been shown to stimulate retinal neural activity, increase blood flow, and alter inner retinal oxygen metabolism (MO2) and delivery (DO2). The purpose of the study was to determine the change in MO2 relative to DO2 due to light flicker stimulation in humans, as assessed by the inner retinal oxygen extraction fraction (OEF). Methods An optical imaging system, based on a modified slit lamp biomicroscope, was developed for simultaneous measurements of retinal vascular diameter (D) and oxygen saturation (SO2). Retinal images were acquired in 20 healthy subjects before and during light flicker stimulation. Arterial and venous D (DA and DV) and SO2 (SO2A and SO2V) were quantified within a circumpapillary region. Oxygen extraction fraction was defined as the ratio of MO2 to DO2 and was calculated as (SO2A − SO2V)/SO2A. Reproducibility of measurements was assessed. Results Coefficients of variation and intraclass correlation coefficients of repeated measurements were <5% and ≥0.83, respectively. During light flicker stimulation, DA, DV , and SO2V significantly increased (P ≤ 0.004). Oxygen extraction fraction was 0.37 ± 0.08 before light flicker and significantly decreased to 0.31 ± 0.07 during light flicker (P = 0.001). Conclusions Oxygen extraction fraction before and during light flicker stimulation is reported in human subjects for the first time. Oxygen extraction fraction decreased during light flicker stimulation, indicating the change in DO2 exceeded that of MO2. This technology is potentially useful for the detection of changes in OEF response to light flicker in physiological and pathological retinal conditions. PMID:26469748

  8. Protective effects of the compounds isolated from the seed of Psoralea corylifolia on oxidative stress-induced retinal damage

    SciTech Connect

    Kim, Kyung-A; Shim, Sang Hee; Ahn, Hong Ryul; Jung, Sang Hoon

    2013-06-01

    The mechanism underlying glaucoma remains controversial, but apoptosis caused by increased levels of reactive oxygen species (ROS) is thought to play a role in its pathogenesis. We investigated the effects of compounds isolated from Psoralea corylifolia on oxidative stress-induced cell death in vitro and in vivo. Transformed retinal ganglion cells (RGC-5) were treated with L-buthione-(S,R)-sulfoximine (BSO) and glutamate in the presence or with pre-treatment with compound 6, bakuchiol isolated from P. corylifolia. We observed reduced cell death in cells pre-treated with bakuchiol. Moreover, bakuchiol inhibited the oxidative stress-induced decrease of mitochondrial membrane potential (MMP, ΔΨm). Furthermore, while intracellular Ca{sup 2+} was high in RGC-5 cells after exposure to oxidative stress, bakuchiol reduced these levels. In an in vivo study, in which rat retinal damage was induced by intravitreal injection of N-methyl-D-aspartate (NMDA), bakuchiol markedly reduced translocation of AIF and release of cytochrome c, and inhibited up-regulation of cleaved caspase-3, cleaved caspase-9, and cleaved PARP. The survival rate of retinal ganglion cells (RGCs) 7 days after optic nerve crush (ONC) in mice was significantly decreased; however, bakuchiol attenuated the loss of RGCs. Moreover, bakuchiol attenuated ONC-induced up-regulation of apoptotic proteins, including cleaved PARP, cleaved caspase-3, and cleaved caspase-9. Bakuchiol also significantly inhibited translocation of mitochondrial AIF into the nuclear fraction and release of mitochondrial cytochrome c into the cytosol. These results demonstrate that bakuchiol isolated from P. corylifolia has protective effects against oxidative stress-induced retinal damage, and may be considered as an agent for treating or preventing retinal degeneration. - Highlights: • Psoralea corylifolia have neuroprotective effects in vitro and in vivo. • Bakuchiol attenuated the increase of apoptotic proteins induced by oxidative

  9. Research on the liquid crystal adaptive optics system for human retinal imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Tong, Shoufeng; Song, Yansong; Zhao, Xin

    2013-12-01

    The blood vessels only in Human eye retinal can be observed directly. Many diseases that are not obvious in their early symptom can be diagnosed through observing the changes of distal micro blood vessel. In order to obtain the high resolution human retinal images,an adaptive optical system for correcting the aberration of the human eye was designed by using the Shack-Hartmann wavefront sensor and the Liquid Crystal Spatial Light Modulator(LCLSM) .For a subject eye with 8m-1 (8D)myopia, the wavefront error is reduced to 0.084 λ PV and 0.12 λRMS after adaptive optics(AO) correction ,which has reached diffraction limit.The results show that the LCLSM based AO system has the ability of correcting the aberration of the human eye efficiently,and making the blurred photoreceptor cell to clearly image on a CCD camera.

  10. Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 12-Hours Post-Exposure to 532 nm, 120 ps Pulsed Laser Light

    DTIC Science & Technology

    2004-04-01

    years or younger, either sex, with no mitigating ocular or retinal pathology such as glaucoma, diabetic retinopathy, retinitis pigmentosa , etc. Donor: The...USAFA TR 2004-01 Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 12-hours Post-Exposure to 532 nm, 120 ps Pulsed...TR 2004-01 This article, "Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 12-hours Post-Exposure to 532 nm, 120 ps

  11. Generation of highly enriched populations of optic vesicle-like retinal cells from human pluripotent stem cells.

    PubMed

    Ohlemacher, Sarah K; Iglesias, Clara L; Sridhar, Akshayalakshmi; Gamm, David M; Meyer, Jason S

    2015-02-02

    The protocol outlined below is used to differentiate human pluripotent stem cells (hPSCs) into retinal cell types through a process that faithfully recapitulates the stepwise progression observed in vivo. From pluripotency, cells are differentiated to a primitive anterior neural fate, followed by progression into two distinct populations of retinal progenitors and forebrain progenitors, each of which can be manually separated and purified. The hPSC-derived retinal progenitors are found to self-organize into three-dimensional optic vesicle-like structures, with each aggregate possessing the ability to differentiate into all major retinal cell types. The ability to faithfully recapitulate the stepwise in vivo development in a three-dimensional cell culture system allows for the study of mechanisms underlying human retinogenesis. Furthermore, this methodology allows for the study of retinal dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually patient-specific therapies.

  12. Serum Response Factor Protects Retinal Ganglion Cells Against High-Glucose Damage.

    PubMed

    Cao, Yan; Wang, Liang; Zhao, Junhong; Zhang, Hongbing; Tian, Ying; Liang, Houcheng; Ma, Qiang

    2016-06-01

    Serum response factor (SRF), which encodes the MADS-box family of related proteins, is a common transcription factor related to the expression of genes associated with cell survival. However, SRF's role in retinal ganglion cells (RGCs) after high-glucose injury remains unclear. In this study, we investigate the protective role of SRF after high-glucose injury and its underlying mechanism. The in vitro RGC model subjected to high glucose was established by employing a 50 mmol/L glucose culture environment. As detected by real-time quantitative PCR and Western blot, SRF was significantly upregulated in RGCs treated with high glucose. Overexpression of SRF significantly promoted survival among RGCs exposed to high glucose and inhibited RGC apoptosis. Knockdown of SRF exerted an inverse effect. Moreover, SRF upregulation enhanced expression of an antioxidant protein, nuclear factor erythroid 2-related factor (Nrf2), via control of the Fos-related antigen 1 (Fra-1). SRF upregulation also affected RGC survival after high-glucose treatment. Our findings showed that overexpression of SRF promoted survival of RGCs after high-glucose injury by regulating Fra-1 and Nrf2.

  13. Protective effects of PF-4708671 against N-methyl-d-aspartic acid-induced retinal damage in rats.

    PubMed

    Hayashi, Ikumi; Aoki, Yuto; Ushikubo, Hiroko; Asano, Daiki; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2016-12-01

    We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-d-aspartic acid (NMDA)-induced retinal damage in rats. Rapamycin inhibits mTOR activity, thereby preventing the phosphorylation of ribosomal protein S6, which is a downstream target of S6 kinase. Therefore, we aimed to determine whether PF-4708671, an inhibitor of S6 kinase, protects against NMDA-induced retinal injury. Intravitreal injection of NMDA (200 nmol/eye) caused cell loss in the ganglion cell layer and neuroinflammatory responses, such as an increase in the number of CD45-positive leukocytes and Iba1-positive microglia. Surprisingly, simultaneous injection of PF-4708671 (50 nmol/eye) with NMDA significantly attenuated these responses without affecting phosphorylated S6 levels. These results suggest that PF-4708671 and rapamycin likely protect against NMDA-induced retinal damage via distinct pathways. The neuroprotective effect of PF-4708671 is unlikely to be associated with inhibition of the S6 kinase, even though PF-4708671 is reported to be a S6 kinase inhibitor.

  14. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    PubMed Central

    Youn, Hyun-Yi; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. Results The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusions The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the

  15. Erythropoietin (EPO) protects against high glucose-induced apoptosis in retinal ganglional cells.

    PubMed

    Wang, Yunxiao; Zhang, Hui; Liu, Yanping; Li, Ping; Cao, Zhihong; Cao, Yu

    2015-03-01

    The aim of this study was to investigate the protective effect and mechanism of EPO on the apoptosis induced by high levels of glucose in retinal ganglial cells (RGCs). High glucose-induced apoptosis model was established in RGCs isolated from SD rats (1-3 days old) and identified with Thy1.1 mAb and MAP-2 pAb. The apoptosis was determined by Hochest assay. The levels of ROS were quantitated by staining the cells with dichloro-dihydro-fluorescein diacetate (DCFH-DA) and measure by flow cytometry. The SOD, GSH-Px, CAT activities, and levels of T-AOC and MDA were determined by ELISA. Change in mitochondrial membrane potential (Δψm) was also assessed by flow cytometry, and expressions of Bcl-2, Bax, caspase-3, caspase-9, and cytochrome C were assessed by western blotting. The RGCs treated with high glucose levels exhibited significantly increased apoptotic rate and concentrations of ROS and MDA. Pretreatment of the cells with EPO caused a significant blockade of the high glucose-induced increase in ROS and MDA levels and apoptotic rate. EPO also increased the activities of SOD, GSH-Px, and CAT, and recovered the levels of T-AOC levels. As a consequence, the mitochondrial membrane potential was improved and Cyt c release into the cytoplasm was prevented which led to significantly suppressed up-regulation of Bax reducing the Bax/Bcl-2 ratio. The expressions of caspase-3 and caspase-9 induced by high glucose exposure were also ameliorated in the RGCs treated with EPO. The protective effect of EPO against apoptosis was mediated through its antioxidant action. Thus, it blocked the generation of pro-apoptotic proteins and apoptotic degeneration of the RGCs by preventing the mitochondrial damage.

  16. CO2-induced ion and fluid transport in human retinal pigment epithelium.

    PubMed

    Adijanto, Jeffrey; Banzon, Tina; Jalickee, Stephen; Wang, Nam S; Miller, Sheldon S

    2009-06-01

    In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 +/- 0.05 to 7.14 +/- 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase-mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 +/- 3.3 to 24.0 +/- 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 +/- 1.6 to 6.7 +/- 2.3 microl x cm(-2) x hr(-1) (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H(2)O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

  17. Molecular mechanisms of retinal ganglion cell degeneration in glaucoma and future prospects for cell body and axonal protection

    PubMed Central

    Munemasa, Yasunari; Kitaoka, Yasushi

    2013-01-01

    Glaucoma, which affects more than 70 million people worldwide, is a heterogeneous group of disorders with a resultant common denominator; optic neuropathy, eventually leading to irreversible blindness. The clinical manifestations of primary open-angle glaucoma (POAG), the most common subtype of glaucoma, include excavation of the optic disc and progressive loss of visual field. Axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies are observed in glaucoma, in which the reduction of intraocular pressure (IOP) is known to slow progression of the disease. A pattern of localized retinal nerve fiber layer (RNFL) defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. The mechanisms of degeneration of neuronal cell bodies and their axons may differ. In this review, we addressed the molecular mechanisms of cell body death and axonal degeneration in glaucoma and proposed axonal protection in addition to cell body protection. The concept of axonal protection may become a new therapeutic strategy to prevent further axonal degeneration or revive dying axons in patients with preperimetric glaucoma. Further study will be needed to clarify whether the combination therapy of axonal protection and cell body protection will have greater protective effects in early or progressive glaucomatous optic neuropathy (GON). PMID:23316132

  18. Nuclear receptor NR2E3 gene mutations distort human retinal laminar architecture and cause an unusual degeneration.

    PubMed

    Jacobson, Samuel G; Sumaroka, Alexander; Aleman, Tomas S; Cideciyan, Artur V; Schwartz, Sharon B; Roman, Alejandro J; McInnes, Roderick R; Sheffield, Val C; Stone, Edwin M; Swaroop, Anand; Wright, Alan F

    2004-09-01

    Mutations in the nuclear receptor gene, NR2E3, cause a disorder of human retinal photoreceptor development characterized by hyperfunction and excess of the minority S (short wavelength or blue) cone photoreceptor type, but near absence of function of the majority rod receptor. NR2E3 disease can also progress to blindness. How the human retina accommodates mis-specified types and numbers of neurons and advances to retinal degeneration are unknown. We studied the retinal organization in vivo of patients with NR2E3 mutations. Early human NR2E3 disease with S cone hyperfunction showed thickened retinal layers within an otherwise normally structured retina. With visual loss, however, lamination was coarse and there was a strikingly thick and bulging appearance to the retina, localized to an annulus encircling the central fovea. This pattern was not found in other retinal degenerations. The abnormal laminar retinal architecture of early NR2E3 disease may be due in part to larger cells with an S cone phenotype in place of rods that failed to differentiate. The later-stage dysplastic appearance suggests a previously unrecognized proliferative response in human retinal degeneration.

  19. RPGR-Associated Retinal Degeneration in Human X-Linked RP and a Murine Model

    PubMed Central

    Huang, Wei Chieh; Wright, Alan F.; Roman, Alejandro J.; Cideciyan, Artur V.; Manson, Forbes D.; Gewaily, Dina Y.; Schwartz, Sharon B.; Sadigh, Sam; Limberis, Maria P.; Bell, Peter; Wilson, James M.; Swaroop, Anand; Jacobson, Samuel G.

    2012-01-01

    Purpose. We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model. Methods. XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5–72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset. Results. Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration. Conclusions. RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy. PMID:22807293

  20. Cortical reorganization after long-term adaptation to retinal lesions in humans.

    PubMed

    Chung, Susana T L

    2013-11-13

    Single-unit recordings demonstrated that the adult mammalian visual cortex is capable of reorganizing after induced retinal lesions. In humans, whether the adult cortex is capable of reorganizing has only been studied using functional magnetic resonance imaging, with equivocal results. Here, we exploited the phenomenon of visual crowding, a major limitation on object recognition, to show that, in humans with long-standing retinal (macular) lesions that afflict the fovea and thus use their peripheral vision exclusively, the signature properties of crowding are distinctly different from those of the normal periphery. Crowding refers to the inability to recognize objects when the object spacing is smaller than the critical spacing. Critical spacing depends only on the retinal location of the object, scales linearly with its distance from the fovea, and is approximately two times larger in the radial than the tangential direction with respect to the fovea, thus demonstrating the signature radial-tangential anisotropy of the crowding zone. Using retinal imaging combined with behavioral measurements, we mapped out the crowding zone at the precise peripheral retinal locations adopted by individuals with macular lesions as the new visual reference loci. At these loci, the critical spacings are substantially smaller along the radial direction than expected based on the normal periphery, resulting in a lower scaling of critical spacing with the eccentricity of the peripheral locus and a loss in the signature radial-tangential anisotropy of the crowding zone. These results imply a fundamental difference in the substrate of cortical processing in object recognition following long-term adaptation to macular lesions.

  1. Adaptive optics OCT using 1060nm swept source and dual deformable lenses for human retinal imaging

    NASA Astrophysics Data System (ADS)

    Jian, Yifan; Lee, Sujin; Cua, Michelle; Miao, Dongkai; Bonora, Stefano; Zawadzki, Robert J.; Sarunic, Marinko V.

    2016-03-01

    Adaptive optics concepts have been applied to the advancement of biological imaging and microscopy. In particular, AO has also been very successfully applied to cellular resolution imaging of the retina, enabling visualization of the characteristic mosaic patterns of the outer retinal layers using flood illumination fundus photography, Scanning Laser Ophthalmoscopy (SLO), and Optical Coherence Tomography (OCT). Despite the high quality of the in vivo images, there has been a limited uptake of AO imaging into the clinical environment. The high resolution afforded by AO comes at the price of limited field of view and specialized equipment. The implementation of a typical adaptive optics imaging system results in a relatively large and complex optical setup. The wavefront measurement is commonly performed using a Hartmann-Shack Wavefront Sensor (HS-WFS) placed at an image plane that is optically conjugated to the eye's pupil. The deformable mirror is also placed at a conjugate plane, relaying the wavefront corrections to the pupil. Due to the sensitivity of the HS-WFS to back-reflections, the imaging system is commonly constructed from spherical mirrors. In this project, we present a novel adaptive optics OCT retinal imaging system with significant potential to overcome many of the barriers to integration with a clinical environment. We describe in detail the implementation of a compact lens based wavefront sensorless adaptive optics (WSAO) 1060nm swept source OCT human retinal imaging system with dual deformable lenses, and present retinal images acquired in vivo from research volunteers.

  2. Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo.

    PubMed

    Fontaine, Valérie; Monteiro, Elodie; Brazhnikova, Elena; Lesage, Laëtitia; Balducci, Christine; Guibout, Louis; Feraille, Laurence; Elena, Pierre-Paul; Sahel, José-Alain; Veillet, Stanislas; Lafont, René

    2016-01-01

    The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9'-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9'-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.

  3. Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo

    PubMed Central

    Monteiro, Elodie; Brazhnikova, Elena; Lesage, Laëtitia; Balducci, Christine; Guibout, Louis; Feraille, Laurence; Elena, Pierre-Paul; Sahel, José-Alain; Veillet, Stanislas; Lafont, René

    2016-01-01

    The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial. PMID:27992460

  4. Does the adult human ciliary body epithelium contain "true" retinal stem cells?

    PubMed

    Frøen, Rebecca; Johnsen, Erik O; Nicolaissen, Bjørn; Facskó, Andrea; Petrovski, Goran; Moe, Morten C

    2013-01-01

    Recent reports of retinal stem cells being present in several locations of the adult eye have sparked great hopes that they may be used to treat the millions of people worldwide who suffer from blindness as a result of retinal disease or injury. A population of proliferative cells derived from the ciliary body epithelium (CE) has been considered one of the prime stem cell candidates, and as such they have received much attention in recent years. However, the true nature of these cells in the adult human eye has still not been fully elucidated, and the stem cell claim has become increasingly controversial in light of new and conflicting reports. In this paper, we will try to answer the question of whether the available evidence is strong enough for the research community to conclude that the adult human CE indeed harbors stem cells.

  5. Degradation in the degree of polarization in human retinal nerve fiber layer

    NASA Astrophysics Data System (ADS)

    Yin, Biwei; Wang, Bingqing; Rylander, Henry G.; Milner, Thomas E.

    2014-01-01

    Using a fiber-based swept-source (SS) polarization-sensitive optical coherence tomography (PS-OCT) system, we investigate the degree of polarization (DOP) of light backscattered from the retinal nerve fiber layer (RNFL) in normal human subjects. Algorithms for processing data were developed to analyze the deviation in phase retardation and intensity of backscattered light in directions parallel and perpendicular to the nerve fiber axis (fast and slow axes of RNFL). Considering superior, inferior, and nasal quadrants, we observe the strongest degradation in the DOP with increasing RNFL depth in the temporal quadrant. Retinal ganglion cell axons in normal human subjects are known to have the smallest diameter in the temporal quadrant, and the greater degradation observed in the DOP suggests that higher polarimetric noise may be associated with neural structure in the temporal RNFL. The association between depth degradation in the DOP and RNFL structural properties may broaden the utility of PS-OCT as a functional imaging technique.

  6. Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

    PubMed Central

    Castilho, Áurea F.; Aveleira, Célia A.; Leal, Ermelindo C.; Simões, Núria F.; Fernandes, Carolina R.; Meirinhos, Rita I.; Baptista, Filipa I.; Ambrósio, António F.

    2012-01-01

    Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H2O2). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H2O2 and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H2O2 and NOC-18. In conclusion, HO-1

  7. Potential suppression of the high glucose and insulin-induced retinal neovascularization by Sirtuin 3 in the human retinal endothelial cells.

    PubMed

    Mao, Xin-Bang; You, Zhi-Peng; Wu, Chen; Huang, Jun

    2017-01-08

    Retinal neovascularization generally play roles in the formation of various severe eye diseases, such as age-related macular degeneration and diabetic retinopathy. The regulation of neovascularization-related pathways by Sirtuin 3 (Sirt3), a major mitochondrial NAD(+)-dependent deacetylase, give us a cue that Sirt3 may participate in the retinal neovascularization. However, the mechanism remains unclear. Here, we established a retinal neovascularization model by using human retinal endothelial cells (HRECs) under the induction of high glucose and insulin (HGI). With this model, Sirt3-expressing lentivirus was constructed and then used to investigate the effect of Sirt3 overexpression on the expression of migration-, neovascularization- and autophagy-related genes. After the treatment of HGI on HRECs, the mRNA and protein levels of migration-related genes, including matrix metalloproteinase-2 (MMP-2) and MMP-9, were significantly upregulated. Meanwhile, angiogenesis-related genes, including vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor-1 (IGF-1) were promoted at both mRNA and protein levels. However, HGI had no clear effect on the mRNA and protein levels of microtubule associated protein 1 light chain 3 (LC3), an autophagy-related gene. When Sirt3 was overexpressed by lentivirus infection after HGI, the upregulation of MMP-2, MMP-9, VEGF, HIF-1α, and IGF-1 were suppressed at both transcription and translation levels. At the same time, LC3 mRNA and LC3-II protein increased. These results suggest that Sirt3 may inhibit retinal neovascularization by regulating the migration-, neovascularization- and autophagy-related factors expression. Thus we argue that Sirt3 may be a potential candidate drug for curing various eye diseases induced by retinal neovascularization.

  8. Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation

    PubMed Central

    Aguirre, Geoffrey K; Komáromy, András M; Cideciyan, Artur V; Brainard, David H; Aleman, Tomas S; Roman, Alejandro J; Avants, Brian B; Gee, James C; Korczykowski, Marc; Hauswirth, William W; Acland, Gregory M; Aguirre, Gustavo D; Aguirre, Geoffrey K

    2007-01-01

    Background RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA). Methods and Findings RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3

  9. A new approach to optic disc detection in human retinal images using the firefly algorithm.

    PubMed

    Rahebi, Javad; Hardalaç, Fırat

    2016-03-01

    There are various methods and algorithms to detect the optic discs in retinal images. In recent years, much attention has been given to the utilization of the intelligent algorithms. In this paper, we present a new automated method of optic disc detection in human retinal images using the firefly algorithm. The firefly intelligent algorithm is an emerging intelligent algorithm that was inspired by the social behavior of fireflies. The population in this algorithm includes the fireflies, each of which has a specific rate of lighting or fitness. In this method, the insects are compared two by two, and the less attractive insects can be observed to move toward the more attractive insects. Finally, one of the insects is selected as the most attractive, and this insect presents the optimum response to the problem in question. Here, we used the light intensity of the pixels of the retinal image pixels instead of firefly lightings. The movement of these insects due to local fluctuations produces different light intensity values in the images. Because the optic disc is the brightest area in the retinal images, all of the insects move toward brightest area and thus specify the location of the optic disc in the image. The results of implementation show that proposed algorithm could acquire an accuracy rate of 100 % in DRIVE dataset, 95 % in STARE dataset, and 94.38 % in DiaRetDB1 dataset. The results of implementation reveal high capability and accuracy of proposed algorithm in the detection of the optic disc from retinal images. Also, recorded required time for the detection of the optic disc in these images is 2.13 s for DRIVE dataset, 2.81 s for STARE dataset, and 3.52 s for DiaRetDB1 dataset accordingly. These time values are average value.

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

    PubMed Central

    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

    Purpose 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. Methods 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. Results 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. Conclusions 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. PMID:26107378

  11. Combined transplantation of human mesenchymal stem cells and human retinal progenitor cells into the subretinal space of RCS rats.

    PubMed

    Qu, Linghui; Gao, Lixiong; Xu, Haiwei; Duan, Ping; Zeng, Yuxiao; Liu, Yong; Yin, Zheng Qin

    2017-03-15

    Retinitis pigmentosa (RP) is one of hereditary retinal diseases characterized by the loss of photoreceptors. Cell transplantation has been clinically applied to treat RP patients. Human retinal progenitor cells (HRPCs) and human bone marrow-derived mesenchymal stem cells (HBMSCs) are the two commonly and practically used stem cells for transplantation. Since combined transplantation could be a promising way to integrate the advantages of both stem cell types, we transplanted HRPCs and HBMSCs into the subretinal space (SRS) of Royal College of Surgeons (RCS) rats. We report that HRPCs/HBMSCs combined transplantation maintains the electroretinogram results much better than HRPCs or HBMSCs single transplantations. The thickness of outer nuclear layer also presented a better outcome in the combined transplantation. Importantly, grafted cells in the combination migrated better, both longitudinally and latitudinally, than single transplantation. The photoreceptor differentiation of grafted cells in the retina of RCS rats receiving combined transplantation also showed a higher ratio than single transplantation. Finally, activation of microglia and the gliosis of Müller cells were more effectively suppressed in combined transplantation, indicating better immunomodulatory and anti-gliosis effects. Taken together, combining the transplantation of HRPCs and HBMSCs is a more effective strategy in stem cell-based therapy for retinal degenerative diseases.

  12. Human iPSC derived disease model of MERTK-associated retinitis pigmentosa

    PubMed Central

    Lukovic, Dunja; Artero Castro, Ana; Delgado, Ana Belen Garcia; Bernal, María de los Angeles Martín; Luna Pelaez, Noelia; Díez Lloret, Andrea; Perez Espejo, Rocío; Kamenarova, Kunka; Fernández Sánchez, Laura; Cuenca, Nicolás; Cortón, Marta; Avila Fernandez, Almudena; Sorkio, Anni; Skottman, Heli; Ayuso, Carmen; Erceg, Slaven; Bhattacharya, Shomi S.

    2015-01-01

    Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches. PMID:26263531

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

    PubMed

    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.

  14. Analysis of normal human retinal vascular network architecture using multifractal geometry

    PubMed Central

    Ţălu, Ştefan; Stach, Sebastian; Călugăru, Dan Mihai; Lupaşcu, Carmen Alina; Nicoară, Simona Delia

    2017-01-01

    AIM To apply the multifractal analysis method as a quantitative approach to a comprehensive description of the microvascular network architecture of the normal human retina. METHODS Fifty volunteers were enrolled in this study in the Ophthalmological Clinic of Cluj-Napoca, Romania, between January 2012 and January 2014. A set of 100 segmented and skeletonised human retinal images, corresponding to normal states of the retina were studied. An automatic unsupervised method for retinal vessel segmentation was applied before multifractal analysis. The multifractal analysis of digital retinal images was made with computer algorithms, applying the standard box-counting method. Statistical analyses were performed using the GraphPad InStat software. RESULTS The architecture of normal human retinal microvascular network was able to be described using the multifractal geometry. The average of generalized dimensions (Dq) for q=0, 1, 2, the width of the multifractal spectrum (Δα=αmax − αmin) and the spectrum arms' heights difference (|Δf|) of the normal images were expressed as mean±standard deviation (SD): for segmented versions, D0=1.7014±0.0057; D1=1.6507±0.0058; D2=1.5772±0.0059; Δα=0.92441±0.0085; |Δf|= 0.1453±0.0051; for skeletonised versions, D0=1.6303±0.0051; D1=1.6012±0.0059; D2=1.5531±0.0058; Δα=0.65032±0.0162; |Δf|= 0.0238±0.0161. The average of generalized dimensions (Dq) for q=0, 1, 2, the width of the multifractal spectrum (Δα) and the spectrum arms' heights difference (|Δf|) of the segmented versions was slightly greater than the skeletonised versions. CONCLUSION The multifractal analysis of fundus photographs may be used as a quantitative parameter for the evaluation of the complex three-dimensional structure of the retinal microvasculature as a potential marker for early detection of topological changes associated with retinal diseases. PMID:28393036

  15. Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

    PubMed Central

    Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

    2016-01-01

    Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

  16. Retinitis Pigmentosa

    MedlinePlus

    ... Action You are here Home › Retinal Diseases Listen Retinitis Pigmentosa What is retinitis pigmentosa? What are the symptoms? ... is available? What treatment is available? What is retinitis pigmentosa? Retinitis pigmentosa, also known as RP, refers to ...

  17. Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

    PubMed

    Beltran, William A; Cideciyan, Artur V; Lewin, Alfred S; Iwabe, Simone; Khanna, Hemant; Sumaroka, Alexander; Chiodo, Vince A; Fajardo, Diego S; Román, Alejandro J; Deng, Wen-Tao; Swider, Malgorzata; Alemán, Tomas S; Boye, Sanford L; Genini, Sem; Swaroop, Anand; Hauswirth, William W; Jacobson, Samuel G; Aguirre, Gustavo D

    2012-02-07

    Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

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

    PubMed

    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.

  19. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    PubMed

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

  20. Ginsenoside Rg-1 protects retinal pigment epithelium (RPE) cells from cobalt chloride (CoCl2) and hypoxia assaults.

    PubMed

    Li, Ke-Ran; Zhang, Zhi-Qing; Yao, Jin; Zhao, Yu-Xia; Duan, Jing; Cao, Cong; Jiang, Qin

    2013-01-01

    Severe retinal ischemia causes persistent visual impairments in eye diseases. Retinal pigment epithelium (RPE) cells are located near the choroidal capillaries, and are easily affected by ischemic or hypoxia. Ginsenoside Rg-1 has shown significant neuroprotective effects. This study was performed to test the cytoprotective effect of ginsenoside Rg-1 in RPE cells against hypoxia and cobalt chloride (CoCl2) assaults, and to understand the underlying mechanisms. We found that Rg-1 pre-administration significantly inhibited CoCl2- and hypoxia-induced RPE cell death and apoptosis. Reactive oxygen specisis (ROS)-dependent p38 and c-Jun NH(2)-terminal kinases (JNK) MAPK activation was required for CoCl2-induced RPE cell death, and Rg-1 pre-treatment significantly inhibited ROS production and following p38/JNK activation. Further, CoCl2 suppressed pro-survival mTOR complex 1 (mTORC1) activation in RPE cells through activating of AMP-activated protein kinase (AMPK), while Rg-1 restored mTORC1 activity through inhibiting AMPK activation. CoCl2-induced AMPK activation was also dependent on ROS production, and anti-oxidant N-acetylcysteine (NAC) prevented AMPK activation and RPE cell death by CoCl2. Our results indicated that Rg-1 could be further investigated as a novel cell-protective agent for retinal ischemia.

  1. Nuclear Factor (Erythroid-Derived)-Related Factor 2-Associated Retinal Pigment Epithelial Cell Protection under Blue Light-Induced Oxidative Stress

    PubMed Central

    Kataoka, Keiko; Kimoto, Reona; Hwang, Shiang-Jyi; Nagasaka, Yosuke; Tsunekawa, Taichi; Nonobe, Norie; Ito, Yasuki; Terasaki, Hiroko

    2016-01-01

    Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage. Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type and Nrf2 knockout (Nrf2−/−) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed on NRF2 mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells. Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure induced NRF2 mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells from Nrf2−/− mice than from wild-type mice. Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress. PMID:27774118

  2. Two-photon polymerization for production of human iPSC-derived retinal cell grafts.

    PubMed

    Worthington, Kristan S; Wiley, Luke A; Kaalberg, Emily E; Collins, Malia M; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2017-03-25

    Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials' inability to guide cell alignment. In this work, we used two-photon polymerization to create 180 μm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1 mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3 µm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25 μm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases.

  3. Human retinal imaging using visible-light optical coherence tomography guided by scanning laser ophthalmoscopy

    PubMed Central

    Yi, Ji; Chen, Siyu; Shu, Xiao; Fawzi, Amani A.; Zhang, Hao F.

    2015-01-01

    We achieved human retinal imaging using visible-light optical coherence tomography (vis-OCT) guided by an integrated scanning laser ophthalmoscopy (SLO). We adapted a spectral domain OCT configuration and used a supercontinuum laser as the illumating source. The center wavelength was 564 nm and the bandwidth was 115 nm, which provided a 0.97 µm axial resolution measured in air. We characterized the sensitivity to be 86 dB with 226 µW incidence power on the pupil. We also integrated an SLO that shared the same optical path of the vis-OCT sample arm for alignment purposes. We demonstrated the retinal imaging from both systems centered at the fovea and optic nerve head with 20° × 20° and 10° × 10° field of view. We observed similar anatomical structures in vis-OCT and NIR-OCT. The contrast appeared different from vis-OCT to NIR-OCT, including slightly weaker signal from intra-retinal layers, and increased visibility and contrast of anatomical layers in the outer retina. PMID:26504622

  4. Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

    PubMed

    Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

    2016-11-03

    Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H₂O₂-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H₂O₂-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

  5. Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1)

    PubMed Central

    Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

    2016-01-01

    Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage. PMID:27827892

  6. Zerumbone, a Phytochemical of Subtropical Ginger, Protects against Hyperglycemia-Induced Retinal Damage in Experimental Diabetic Rats

    PubMed Central

    Tzeng, Thing-Fong; Liou, Shorong-Shii; Tzeng, Yu-Cheng; Liu, I-Min

    2016-01-01

    Diabetic retinopathy (DR), the most ordinary and specific microvascular complication of diabetes, is a disease of the retina. Zerumbone (ZER) is a monocyclic sesquiterpene compound, and based on reports, it is the predominant bioactive compound from the rhizomes of Zingiber zerumbet. The aim of the current study is to evaluate the protective effect of zerumbone against DR in streptozotocin (STZ)-induced diabetic rats. STZ-diabetic rats were treated with ZER (40 mg/kg) once a day orally for 8 weeks. ZER administration significantly (p < 0.05) lowered the levels of plasma glucose (32.5% ± 5.7% lower) and glycosylated hemoglobin (29.2% ± 3.4% lower) in STZ-diabetic rats. Retinal histopathological observations indicated that disarrangement and reduction in thickness of retinal layers were reversed in ZER-treated diabetic rats. ZER downregulated both the elevated levels of advanced glycosylated end products (AGEs) and the higher levels of the receptors for AGEs (RAGE) in retinas of diabetic rats. What’s more, ZER significantly (p < 0.05) ameliorated diabetes-induced upregulation of tumor necrosis factor-α, interleukin (IL)-1 and IL-6. ZER also attenuated overexpression of vascular endothelial growth factor and intercellular adhesion molecule-1, and suppressed activation of nuclear factor (NF)-κB and apoptosis in the retinas of STZ-diabetic rats. Our results suggest ZER possesses retinal protective effects, which might be associated with the blockade of the AGEs/RAGE/NF-κB pathway and its anti-inflammatory activity. PMID:27463726

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27116668

  8. The protective effects of bilberry and lingonberry extracts against UV light-induced retinal photoreceptor cell damage in vitro.

    PubMed

    Ogawa, Kenjirou; Tsuruma, Kazuhiro; Tanaka, Junji; Kakino, Mamoru; Kobayashi, Saori; Shimazawa, Masamitsu; Hara, Hideaki

    2013-10-30

    Bilberry extract (B-ext) and lingonberry extract (L-ext) are currently used as health supplements. We investigated the protective mechanisms of the B-ext and L-ext against ultraviolet A (UVA)-induced retinal photoreceptor cell damage. Cultured murine photoreceptor (661W) cells were exposed to UVA following treatment with B-ext and L-ext and their main constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin). B-ext, L-ext, and constituents improved cell viability and suppressed ROS generation. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) were analyzed by Western blotting. B-ext and cyanidin inhibited phosphorylation of p38 MAPK, and B-ext also inhibited phosphorylation of JNK by UVA. L-ext, trans-resveratrol, and procyanidin alleviated the reduction of phosphorylated Akt levels by UVA. Finally, a cotreatment with B-ext and L-ext showed an additive effect on cell viability. Our findings suggest that both B-ext and L-ext endow protective effects against UVA-induced retinal damage.

  9. Human retinal pigment epithelial lysis of extracellular matrix: functional urokinase plasminogen activator receptor, collagenase, and elastase.

    PubMed Central

    Elner, Susan G

    2002-01-01

    PURPOSE: To show (1) human retinal pigment epithelial (HRPE) expression of functional urokinase plasminogen activator receptor (uPAR; CD87), (2) HRPE secretion of collagenase and elastase, (3) uPAR-dependent HRPE migration, and (4) uPAR expression in diseased human retinal tissue. METHODS: Immunohistochemistry for uPAR was performed on cultured HRPE cells and in sections of human retina. Double-immunofluorescent staining of live human RPE cells with anti-CR3 antibody (CD11b) was performed to demonstrate the physical proximity of this beta 2 integrin with uPAR and determine whether associations were dependent on RPE confluence and polarity. Extracellular proteolysis by HRPE uPAR was evaluated using fluorescent bodipy-BSA and assessed for specificity by plasminogen activator inhibitor-1 (PAI-1) inhibition. The effect of interleukin-1 beta (IL-1 beta) on uPAR expression was assessed. Collagenase and elastase secretion by unstimulated and IL-1-stimulated HRPE cells was measured by 3H-labelled collagen and elastin cleavage. HRPE-associated collagenase was also assessed by cleavage of fluorescent DQ-collagen and inhibited by phenanthroline. Using an extracellular matrix assay, the roles of uPAR and collagenase in HRPE migration were assessed. RESULTS: Immunoreactive uPAR was detected on cultured HRPE cells and increased by IL-1. On elongated, live HRPE cells, uPAR dissociated from CD11b (CR3) and translocated to anterior poles of migrating cells. Extracellular proteolysis was concentrated at sites of uPAR expression and specifically inhibited by PAI-1. Cultured HRPE cells secreted substantial, functional collagenase and elastase. IL-1 upregulated uPAR, collagenase, and elastase activities. Specific inhibition of uPAR, and to a lesser degree collagenase, reduced HRPE migration in matrix/gel assays. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue. CONCLUSIONS: HRPE cells express functional u

  10. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin; Kannan, Rangaramanujam M.

    2015-02-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform.Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE

  11. [Aging and retinal vascular diseases].

    PubMed

    Takagi, Hitoshi

    2007-03-01

    Ocular vascular diseases such as diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration, whose population increases along with aging, have become leading causes of severe visual disturbance. Macular edema and serous retinal detachment are associated with abnormal vascular leakage and tractional retinal detachment, and neovascular glaucoma is caused by retinal neovascularization. Such ocular vascular diseases are caused by vascular cell aging and vascular damage associated with lifestyle-related diseases including diabetes mellitus, hypertension, hyperlipidemia, and obesity. In the present study, we investigated molecular mechanisms in such vascular deficiencies using vascular cell biology methodology, and we propose novel strategies for the treatment of such vascular diseases. Along with aging, oxidative stress and physical stress, such as mechanical stretch, continuously and directly insult vascular cells. Such stress induces apoptosis by intracellular signaling through stress kinases in cultured retinal vascular cells. Inhibition of such stress kinases could be an effective treatment to protect the vascular cells against age-related damage. In a retinal vascular developmental model, pericyte loss causes pathology mimicking macular edema and proliferative diabetic retinopathy. Angiopoietin 1 (Ang 1) secreted by pericytes suppresses oxidative stress-induced intracellular signaling through stress kinases linked to cell apoptosis and normalizes such retinal pathology. This suggests that the paracrine action of Ang 1 in the pericytes is necessary to sustain normal retinal vasculature, and that Ang 1-triggered intracellular signaling is useful for the treatment of vascular cell pathology associated with pericyte loss. In diabetic retinopathy and retinal vein occlusion, retinal vessels regress along with retinal vascular cell apoptosis, and the retina becomes ischemic followed by pathological retinal neovascularization. VEGF has been

  12. IGFBP-3 inhibits TNF-α production and TNFR-2 signaling to protect against retinal endothelial cell apoptosis.

    PubMed

    Zhang, Qiuhua; Steinle, Jena J

    2014-09-01

    In models of diabetic retinopathy, insulin-like growth factor binding protein-3 (IGFBP-3) protects against tumor necrosis factors-alpha (TNF-α)-mediated apoptosis of retinal microvascular endothelial cells (REC), but the underlying mechanisms are unclear. Our current findings suggest that at least two discrete but complimentary pathways contribute to the protective effects of IGFBP-3; 1) IGFBP-3 directly activates the c-Jun kinase/tissue inhibitor of metalloproteinase-3/TNF-α converting enzyme (c-Jun/TIMP-3/TACE), pathway, which in turn inhibits TNF-α production; 2) IGFBP-3 acts through the IGFBP-3 receptor, low-density lipoprotein receptor-related protein 1 (LRP1), to inhibit signaling of TNF-α receptor 2 (TNFR2). Combined, these two IGFBP-3 pathways substantially reduce REC apoptosis and offer potential targets for the treatment of diabetic retinopathy.

  13. Establishment of a blue light damage model of human retinal pigment epithelial cells in vitro.

    PubMed

    Su, G; Cai, S J; Gong, X; Wang, L L; Li, H H; Wang, L M

    2016-06-24

    To establish a blue-light damage model of human retinal pigment epithelium (RPE). Fourth-generation human RPE cells were randomly divided into two groups. In group A, cells were exposed to blue light (2000 ± 500 lux) for 0 (control), 3, 6, 9, and 12 h, and cell culture was stopped after 12 h. In group B, cells were exposed to blue light at the same intensity and time periods, but cell culture was stopped after 24 h. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to determine the most suitable illuminating time with apoptotic index. Flow cytometry was used to determine apoptotic ratio of RPEs. In group A, the apoptotic index of cells that received 6, 9 and 12 h of blue light was higher than that of control. The apoptotic index of cells receiving 9 and 12 h was higher than that of 6 h (P = 0.000). In group B, the apoptotic index and RPE cell apoptosis ratio of cells exposed to 6, 9 and 12 h of blue light were higher than that of 3 h (P = 0.000); and cells receiving 9 and 12 h had higher values than that of 6 h. This study demonstrated that the best conditions to establish a blue light damage model of human retinal pigment epithelial cells in vitro are 2000 ± 500 lux light intensity for 6 h, with 24 h of cell culture post-exposure.

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

    PubMed

    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.

  15. Enhanced functional integration of human photoreceptor precursors into human and rodent retina in an ex vivo retinal explant model system.

    PubMed

    Yanai, Anat; Laver, Christopher R J; Gregory-Evans, Cheryl Y; Liu, Ran R; Gregory-Evans, Kevin

    2015-06-01

    Retinal disease is the major cause of irreversible blindness in developed countries. Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is a promising and widely applicable approach for the treatment of these blinding conditions. Previously, it has been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is low. The factors that inhibit PPC engraftment remain largely unknown, in part, because so many adverse factors could be at play during in vivo experiments. To advance our knowledge in overcoming potential adverse effects and optimize PPC transplantation, we have developed a novel ex vivo system. Harvested neural retina was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Explants cocultured with hESC-derived RPE maintained normal gross morphology and viability for up to 2 weeks, whereas the explants cultured on ARPE19 and RPE-J failed by 7 days. Furthermore, the proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when cocultured with hESC-derived RPE (20% and 10%, respectively), than when cocultured with ARPE19 (only 6% and 2%, respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs cocultured with hESC-derived RPE increased to ∼30% and 15%, respectively. These data demonstrate the utility of an ex vivo model system to define factors, such as TSP-1, which could influence integration efficiency in future in vivo experiments in models of retinal degeneration.

  16. Chromatic aberration correction of the human eye for retinal imaging in the near infrared

    NASA Astrophysics Data System (ADS)

    Fernández, Enrique J.; Unterhuber, Angelika; Považay, Boris; Hermann, Boris; Artal, Pablo; Drexler, Woflgang

    2006-06-01

    An achromatizing lens has been designed for the human eye in the near infrared range, from 700 to 900 nm, for retinal imaging purposes. Analysis of the performance of the lens, including tolerance to misalignments, has been mathematically accomplished by using an existing eye model. The calculations have shown a virtually perfect correction of the ocular longitudinal chromatic aberration, while still keeping a high optical quality. Ocular aberrations in five subjects have been measured with and without the achromatizing lens by using a Hartmann-Shack wavefront sensor and a broad bandwidth femtosecond Ti:sapphire laser in the spectral range of interest with a set of interference filters, studying the benefits and limits in the use of the achromatizing lens. Ocular longitudinal chromatic aberration has been experimentally demonstrated to be fully corrected by the proposed lens, with no induction of any other parasitic aberration. The practical implementation of the achromatizing lens for Ophthalmoscopy, specifically for optical coherence tomography where the use of polychromatic light sources in the near infrared portion of the spectrum is mandatory, has been considered. The potential benefits of using this lens in combination with adaptive optics to achieve a full aberration correction of the human eye for retinal imaging have also been discussed.

  17. Structural and functional human retinal imaging with a fiber-based visible light OCT ophthalmoscope

    PubMed Central

    Chong, Shau Poh; Bernucci, Marcel; Radhakrishnan, Harsha; Srinivasan, Vivek J.

    2016-01-01

    The design of a multi-functional fiber-based Optical Coherence Tomography (OCT) system for human retinal imaging with < 2 micron axial resolution in tissue is described. A detailed noise characterization of two supercontinuum light sources with different pulse repetition rates is presented. The higher repetition rate and lower noise source is found to enable a sensitivity of 96 dB with 0.15 mW light power at the cornea and a 98 microsecond exposure time. Using a broadband (560 ± 50 nm), 90/10, fused single-mode fiber coupler designed for visible wavelengths, the sample arm is integrated into an ophthalmoscope platform, similar to current clinical OCT systems. To demonstrate the instrument’s range of operation, in vivo structural retinal imaging is also shown at 0.15 mW exposure with 10,000 and 70,000 axial scans per second (the latter comparable to commercial OCT systems), and at 0.03 mW exposure and 10,000 axial scans per second (below maximum permissible continuous exposure levels). Lastly, in vivo spectroscopic imaging of anatomy, saturation, and hemoglobin content in the human retina is also demonstrated. PMID:28101421

  18. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

    PubMed

    Milenkovic, Andrea; Brandl, Caroline; Milenkovic, Vladimir M; Jendryke, Thomas; Sirianant, Lalida; Wanitchakool, Potchanart; Zimmermann, Stephanie; Reiff, Charlotte M; Horling, Franziska; Schrewe, Heinrich; Schreiber, Rainer; Kunzelmann, Karl; Wetzel, Christian H; Weber, Bernhard H F

    2015-05-19

    In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

  19. Degradation in the degree of polarization in human retinal nerve fiber layer

    PubMed Central

    Yin, Biwei; Wang, Bingqing; Rylander, Henry G.; Milner, Thomas E.

    2014-01-01

    Abstract. Using a fiber-based swept-source (SS) polarization-sensitive optical coherence tomography (PS-OCT) system, we investigate the degree of polarization (DOP) of light backscattered from the retinal nerve fiber layer (RNFL) in normal human subjects. Algorithms for processing data were developed to analyze the deviation in phase retardation and intensity of backscattered light in directions parallel and perpendicular to the nerve fiber axis (fast and slow axes of RNFL). Considering superior, inferior, and nasal quadrants, we observe the strongest degradation in the DOP with increasing RNFL depth in the temporal quadrant. Retinal ganglion cell axons in normal human subjects are known to have the smallest diameter in the temporal quadrant, and the greater degradation observed in the DOP suggests that higher polarimetric noise may be associated with neural structure in the temporal RNFL. The association between depth degradation in the DOP and RNFL structural properties may broaden the utility of PS-OCT as a functional imaging technique. PMID:24390374

  20. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells†

    PubMed Central

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin

    2016-01-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform. PMID:25213606

  1. Chromatic aberration correction of the human eye for retinal imaging in the near infrared.

    PubMed

    Fernández, Enrique J; Unterhuber, Angelika; Povazay, Boris; Hermann, Boris; Artal, Pablo; Drexler, Woflgang

    2006-06-26

    An achromatizing lens has been designed for the human eye in the near infrared range, from 700 to 900 nm, for retinal imaging purposes. Analysis of the performance of the lens, including tolerance to misalignments, has been mathematically accomplished by using an existing eye model. The calculations have shown a virtually perfect correction of the ocular longitudinal chromatic aberration, while still keeping a high optical quality. Ocular aberrations in five subjects have been measured with and without the achromatizing lens by using a Hartmann-Shack wavefront sensor and a broad bandwidth femtosecond Ti:sapphire laser in the spectral range of interest with a set of interference filters, studying the benefits and limits in the use of the achromatizing lens. Ocular longitudinal chromatic aberration has been experimentally demonstrated to be fully corrected by the proposed lens, with no induction of any other parasitic aberration. The practical implementation of the achromatizing lens for Ophthalmoscopy, specifically for optical coherence tomography where the use of polychromatic light sources in the near infrared portion of the spectrum is mandatory, has been considered. The potential benefits of using this lens in combination with adaptive optics to achieve a full aberration correction of the human eye for retinal imaging have also been discussed.

  2. Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

    NASA Astrophysics Data System (ADS)

    Schori, Hadas; Kipnis, Jonathan; Yoles, Eti; Woldemussie, Elizabeth; Ruiz, Guadalupe; Wheeler, Larry A.; Schwartz, Michal

    2001-03-01

    Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P <0.05), but not when they were immunized 48h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8%±6.8% to 4.3%±1.6%, without affecting the intraocular pressure

  3. Protective effects of triptolide on retinal ganglion cells in a rat model of chronic glaucoma

    PubMed Central

    Yang, Fan; Wang, Dongmei; Wu, Lingling; Li, Ying

    2015-01-01

    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. PMID:26604697

  4. Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium

    PubMed Central

    Ach, Thomas; Huisingh, Carrie; McGwin, Gerald; Messinger, Jeffrey D.; Zhang, Tianjiao; Bentley, Mark J.; Gutierrez, Danielle B.; Ablonczy, Zsolt; Smith, R. Theodore; Sloan, Kenneth R.; Curcio, Christine A.

    2014-01-01

    Purpose. Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. Methods. Retinal pigment epithelium–Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. Results. Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. Conclusions. Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LF's role in AMD. PMID:25034602

  5. Experimental Study of the Biological Properties of Human Embryonic Stem Cell–Derived Retinal Progenitor Cells

    PubMed Central

    Shao, Jingzhi; Zhou, Peng-Yi; Peng, Guang-Hua

    2017-01-01

    Retinal degenerative diseases are among the leading causes of blindness worldwide, and cell replacement is considered as a promising therapeutic. However, the resources of seed cells are scarce. To further explore this type of therapy, we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore, we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats’ outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks, resulting in beneficial effects on the morphology of the host retina, and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function. PMID:28205557

  6. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

    PubMed Central

    Kambhampati, Siva P.; Mishra, Manoj K.; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A.; Kannan, Rangaramanujam M.

    2016-01-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (~21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ~100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  7. Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells.

    PubMed Central

    Navanglone, A; Rispoli, G; Gabellini, N; Carafoli, E

    1997-01-01

    The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity. Images FIGURE 1 PMID:9199770

  8. The human fetal retinal pigment epithelium: A target tissue for thyroid hormones.

    PubMed

    Duncan, K G; Bailey, K R; Baxter, J D; Schwartz, D M

    1999-01-01

    Thyroid hormone (T(3)) has previously been shown to regulate visual function in experimental animals and humans. To determine if T(3) exerts direct effects on retinal function, cultured human fetal retinal pigment epithelial (RPE) cells were tested for the presence of thyroid hormone receptors (TRs) and T(3) responses. Using TR-isoform-specific reverse-transcriptase polymerase chain reaction techniques, mRNA was detected for alpha1, alpha2 and beta1 TR isoforms. Immunohistochemistry using a polyclonal antibody that simultaneously recognizes alpha1, alpha2 and beta1 TRs showed nuclear staining of the fetal RPE. Specific binding of (125)I-T(3) to RPE cell nuclear extracts was detected, and Scatchard analysis revealed a K(d) of 110 pM. To determine if RPE cells can respond to T(3), hyaluronic acid (HA) levels in cell culture media were measured after 2, 4 or 6 days of growth in medium containing 10(-7) M T(3). T(3) inhibited accumulation of HA in the cell culture medium of RPE cells. This effect was not evident at 2 days, but at 4 days there was 42.8% less HA in cell culture medium of RPE cells grown in 10(-7) M T(3) (p < 0.01, t test). The effect persisted through 6 days, when there was 46.3% less HA in cell culture medium of RPE cells grown in 10(-7) M T(3) (p < 0.001, t test). The data indicate that human fetal RPE cells are a direct target for thyroid hormones.

  9. SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

    PubMed Central

    Cao, Guo-fan; Cao, Cong; Jiang, Qin

    2016-01-01

    Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis. PMID:27517753

  10. SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling.

    PubMed

    Gong, Yi-Qing; Huang, Wei; Li, Ke-Ran; Liu, Yuan-Yuan; Cao, Guo-Fan; Cao, Cong; Jiang, Qin

    2016-09-13

    Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis.

  11. Three-dimensional pointwise comparison of human retinal optical property at 845 and 1060 nm using optical frequency domain imaging.

    PubMed

    Chen, Yueli; Burnes, Daina L; de Bruin, Martijn; Mujat, Mircea; de Boer, Johannes F

    2009-01-01

    To compare the optical properties of the human retina, 3-D volumetric images of the same eye are acquired with two nearly identical optical coherence tomography (OCT) systems at center wavelengths of 845 and 1060 nm using optical frequency domain imaging (OFDI). To characterize the contrast of individual tissue layers in the retina at these two wavelengths, the 3-D volumetric data sets are carefully spatially matched. The relative scattering intensities from different layers such as the nerve fiber, photoreceptor, pigment epithelium, and choroid are measured and a quantitative comparison is presented. OCT retinal imaging at 1060 nm is found to have a significantly better depth penetration but a reduced contrast between the retinal nerve fiber, the ganglion cell, and the inner plexiform layers compared to the OCT retinal imaging at 845 nm.

  12. Three-dimensional pointwise comparison of human retinal optical property at 845 and 1060 nm using optical frequency domain imaging

    NASA Astrophysics Data System (ADS)

    Chen, Yueli; Burnes, Daina L.; de Bruin, Martijn; Mujat, Mircea; de Boer, Johannes F.

    2009-03-01

    To compare the optical properties of the human retina, 3-D volumetric images of the same eye are acquired with two nearly identical optical coherence tomography (OCT) systems at center wavelengths of 845 and 1060 nm using optical frequency domain imaging (OFDI). To characterize the contrast of individual tissue layers in the retina at these two wavelengths, the 3-D volumetric data sets are carefully spatially matched. The relative scattering intensities from different layers such as the nerve fiber, photoreceptor, pigment epithelium, and choroid are measured and a quantitative comparison is presented. OCT retinal imaging at 1060 nm is found to have a significantly better depth penetration but a reduced contrast between the retinal nerve fiber, the ganglion cell, and the inner plexiform layers compared to the OCT retinal imaging at 845 nm.

  13. MicroRNA-29 regulates high-glucose-induced apoptosis in human retinal pigment epithelial cells through PTEN.

    PubMed

    Lin, Xiaohui; Zhou, Xiyuan; Liu, Danning; Yun, Lixia; Zhang, Lina; Chen, Xiaohai; Chai, Qinghe; Li, Langen

    2016-04-01

    Hyperglycemia or high-glucose (HG)-induced apoptosis in human retinal pigment epithelial (RPE) cells is a characteristic process in diabetic retinopathy. In our study, we examined whether microRNA-29 (miR-29) may regulate HG-induced RPE cell apoptosis. Human RPE cell line, ARPE-19 cells, was treated with various high concentration of glucose in vitro. HG-induced RPE cell apoptosis was examined by terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and miR-29 gene expression by quantitative RT-PCR (qRT-PCR). miR-29 was then downregulated in RPE cells, and its effect on HG-induced apoptosis was examined by TUNEL assay and western blot assay on caspase-7 protein. Association of miR-29 on its downstream target, PTEN, in HG-induced RPE cell apoptosis was evaluated by dual-luciferase assay and qRT-PCR. PTEN was silenced in RPE cells. The effects of PTEN downregulation on miR-29-mediated HG-induced RPE cell apoptosis were also examined by TUNEL and western blot assays. HG induced significant apoptosis in RPE cells in a dose-dependent manner. miR-29 was upregulated by HG in RPE cells. miR-29 downregulation protected HG-induced apoptosis and reduced the production of caspase-7 protein in RPE cells. PTEN was shown to be directly downregulated by HG and then upregulated by miR-29 downregulation in RPE cells. Small interfering RNA (siRNA)-mediated PTEN downregulation reversed the protective effect of miR-29 downregulation on HG-induced RPE cell apoptosis. This study demonstrates that miR-29, through inverse association of PTEN, plays an important role in the process of HG-induced apoptosis in RPE cells.

  14. Spatial and Spectral Characterization of Human Retinal Pigment Epithelium Fluorophore Families by Ex Vivo Hyperspectral Autofluorescence Imaging

    PubMed Central

    Ben Ami, Tal; Tong, Yuehong; Bhuiyan, Alauddin; Huisingh, Carrie; Ablonczy, Zsolt; Ach, Thomas; Curcio, Christine A.; Smith, R. Theodore

    2016-01-01

    Purpose Discovery of candidate spectra for abundant fluorophore families in human retinal pigment epithelium (RPE) by ex vivo hyperspectral imaging. Methods Hyperspectral autofluorescence emission images were captured between 420 and 720 nm (10-nm intervals), at two excitation bands (436–460, 480–510 nm), from three locations (fovea, perifovea, near-periphery) in 20 normal RPE/Bruch's membrane (BrM) flatmounts. Mathematical factorization extracted a BrM spectrum (S0) and abundant lipofuscin/melanolipofuscin (LF/ML) spectra of RPE origin (S1, S2, S3) from each tissue. Results Smooth spectra S1 to S3, with perinuclear localization consistent with LF/ML at all three retinal locations and both excitations in 14 eyes (84 datasets), were included in the analysis. The mean peak emissions of S0, S1, and S2 at λex 436 nm were, respectively, 495 ± 14, 535 ± 17, and 576 ± 20 nm. S3 was generally trimodal, with peaks at either 580, 620, or 650 nm (peak mode, 650 nm). At λex 480 nm, S0, S1, and S2 were red-shifted to 526 ± 9, 553 ± 10, and 588 ± 23 nm, and S3 was again trimodal (peak mode, 620 nm). S1 often split into two spectra, S1A and S1B. S3 strongly colocalized with melanin. There were no significant differences across age, sex, or retinal location. Conclusions There appear to be at least three families of abundant RPE fluorophores that are ubiquitous across age, retinal location, and sex in this sample of healthy eyes. Further molecular characterization by imaging mass spectrometry and localization via super-resolution microscopy should elucidate normal and abnormal RPE physiology involving fluorophores. Translational Relevance Our results help establish hyperspectral autofluorescence imaging of the human retinal pigment epithelium as a useful tool for investigating retinal health and disease. PMID:27226929

  15. Regulation of plasminogen activation by TGF-β in cultured human retinal endothelial cells

    PubMed Central

    Wileman, S.; Booth, N.; Moore, N.; Redmill, B.; Forrester, J.; Knott, R.

    2000-01-01

    BACKGROUND/AIMS—Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor β (TGF-β) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-β in retinal angiogenesis in the context of diabetes mellitus.
METHODS—Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-β (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay.
RESULTS—Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-β resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-β did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05).
CONCLUSIONS—These data demonstrate that TGF-β increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

 PMID:10729302

  16. [An updated review of methods for human retinal oximetry measurements and current applications].

    PubMed

    Ben-Zion, Itay; Harris, Alon; Weizman, Yosi; Ehrlich, Rita; Rechtman, Ehud

    2008-10-01

    The concept of retinal oximetry is based on physical properties that have been recognized since the 18th century. Attempts to non-invasively quantify the oxygen saturation of blood within the retinal vasculature date back to the 1950's. There are different techniques in existence for the measurement of retinal oxygenation, the leading ones are: photographic, digital, spectroscopy and the pulse methods. Each method has its advantages and disadvantages. Current data from studies on retinal oximetry is presented, for both the healthy retina and in diseases such as glaucoma, diabetic retinopathy and age-related macular degeneration. It is clear that a thorough understanding of retinal oxygen tension is vital to our understanding of normal retinal physiology and the pathophysiology of degenerative eye diseases.

  17. Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes

    PubMed Central

    Duarte, Diego A.; Papadimitriou, Alexandros; Gilbert, Richard E.; Thai, Kerri; Zhang, Yanling; Rosales, Mariana A. B.; Lopes de Faria, José B.; Lopes de Faria, Jacqueline M.

    2016-01-01

    Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR. PMID:26836609

  18. Increased production of omega-3 fatty acids protects retinal ganglion cells after optic nerve injury in mice.

    PubMed

    Peng, Shanshan; Shi, Zhe; Su, Huanxing; So, Kwok-Fai; Cui, Qi

    2016-07-01

    Injury to the central nervous system causes progressive degeneration of injured axons, leading to loss of the neuronal bodies. Neuronal survival after injury is a prerequisite for successful regeneration of injured axons. In this study, we investigated the effects of increased production of omega-3 fatty acids and elevation of cAMP on retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) crush injury in adult mice. We found that increased production of omega-3 fatty acids in mice enhanced RGC survival, but not axonal regeneration, over a period of 3 weeks after ON injury. cAMP elevation promoted RGC survival in wild type mice, but no significant difference in cell survival was seen in mice over-producing omega-3 fatty acids and receiving intravitreal injections of CPT-cAMP, suggesting that cAMP elevation protects RGCs after injury but does not potentiate the actions of the omega-3 fatty acids. The observed omega-3 fatty acid-mediated neuroprotection is likely achieved partially through ERK1/2 signaling as inhibition of this pathway by PD98059 hindered, but did not completely block, RGC protection. Our study thus enhances our current understanding of neural repair after CNS injury, including the visual system.

  19. Protective Effects of Panax notoginseng Saponins against High Glucose-Induced Oxidative Injury in Rat Retinal Capillary Endothelial Cells

    PubMed Central

    Fan, Yue; Qiao, Yuan; Huang, Jianmei

    2016-01-01

    Diabetic retinopathy, a leading cause of visual loss and blindness, is characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for diabetic retinopathy and is associated with increased oxidative stress in the retina. In this study, we investigated the potential protective effects of Panax notoginseng Saponins (PNS) in retinal capillary endothelial cells (RCECs) exposed to high glucose conditions. We found a pronounced increase in cell viability in rat RCECs incubated with both PNS and high glucose (30 mM) for 48 h or 72 h. The increased viability was accompanied by reduced intracellular hydrogen peroxide (H2O2) and superoxide (O2−), decreased mitochondrial reactive oxygen species (ROS), and lowered malondialdehyde (MDA) levels. PNS also increased the activities of total superoxide dismutase (SOD), MnSOD, catalase (CAT), and glutathione peroxidase (GSH-PX). The glutathione (GSH) content also increased after PNS treatment. Furthermore, PNS reduced NADPH oxidase 4 (Nox4) expression. These results indicate that PNS exerts a protective effect against high glucose-induced injury in RCECs, which may be partially attributed to its antioxidative function. PMID:27019662

  20. Ginsenoside Rb1 protects rat retinal ganglion cells against hypoxia and oxidative stress.

    PubMed

    Liu, Zhaochun; Chen, Juying; Huang, Wendong; Zeng, Zhi; Yang, Yongfei; Zhu, Banghao

    2013-11-01

    The current study was designed to investigate the effect of ginsenoside Rb1 (Rb1) on apoptosis induced by hypoxia and oxidative stress in a retinal ganglion cell line (RGC-5). The underlying mechanism was also investigated. RGC-5 cells were pretreated with 10 µmol/l Rb1 for 24 h and exposed to 400 µmol/l cobalt chloride (CoCl2) for 48 h or 600 µmol/l H2O2 for 24 h. The percentage of cells actively undergoing apoptosis was determined by flow cytometry with Annexin V/propidium iodide (PI) double staining. The expression of caspases was determined using western blot analysis. CoCl2 and H2O2 treatments significantly increased the apoptotic percentages to 24.5 and 21.63%, respectively. Pretreatment of Rb1 reduced the total apoptotic percentages to 15.12 and 12.03%, respectively. The expression of cleaved caspase-3, -9 and -8 was increased in the CoCl2-treated group, however, caspase-3 was not increased in the H2O2-treated group. Pretreatment of Rb1 reduced the expression of cleaved caspase-3 and -9 in the CoCl2-treated group, but reduced only cleaved caspase-9 in the H2O2-treated group. These results suggest that Rb1 may prevent RGC-5 cells from apoptosis against hypoxia and oxidative stress via the mitochondrial pathway.

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

    PubMed

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

    2017-01-04

    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 promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC-RPE and performs better than often used biodegradable poly(d,l-lactide) (PDLLA) film. Human ESC-RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE-specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC-RPE monolayer in serum-free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC-RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Imaging human retinal pigment epithelium cells using adaptive optics optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Liu, Zhuolin; Kocaoglu, Omer P.; Turner, Timothy L.; Miller, Donald T.

    2016-03-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, but are often compromised in ageing and major ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, and while biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. We present a novel method based on optical coherence tomography (OCT) equipped with adaptive optics (AO) that overcomes the associated technical obstacles. The method takes advantage of the 3D resolution of AO-OCT, but more critically sub-cellular segmentation and registration that permit organelle motility to be used as a novel contrast mechanism. With this method, we successfully visualized RPE cells and characterized their 3D reflectance profile in every subject and retinal location (3° and 7° temporal to the fovea) imaged to date. We have quantified RPE packing geometry in terms of cell density, cone-to-RPE ratio, and number of nearest neighbors using Voronoi and power spectra analyses. RPE cell density (cells/mm2) showed no significant difference between 3° (4,892+/-691) and 7° (4,780+/-354). In contrast, cone-to- RPE ratio was significantly higher at 3° (3.88+/-0.52:1) than 7° (2.31+/- 0.23:1). Voronoi analysis also showed most RPE cells have six nearest neighbors, which was significantly larger than the next two most prevalent associations: five and seven. Averaged across the five subjects, prevalence of cells with six neighbors was 51.4+/-3.58% at 3°, and 54.58+/-3.01% at 7°. These results are consistent with histology and in vivo studies using other imaging modalities.

  3. Effects of light-emitting diode radiations on human retinal pigment epithelial cells in vitro.

    PubMed

    Chamorro, Eva; Bonnin-Arias, Cristina; Pérez-Carrasco, María Jesús; Muñoz de Luna, Javier; Vázquez, Daniel; Sánchez-Ramos, Celia

    2013-01-01

    Human visual system is exposed to high levels of natural and artificial lights of different spectra and intensities along lifetime. Light-emitting diodes (LEDs) are the basic lighting components in screens of PCs, phones and TV sets; hence it is so important to know the implications of LED radiations on the human visual system. The aim of this study was to investigate the effect of LEDs radiations on human retinal pigment epithelial cells (HRPEpiC). They were exposed to three light-darkness (12 h/12 h) cycles, using blue-468 nm, green-525 nm, red-616 nm and white light. Cellular viability of HRPEpiC was evaluated by labeling all nuclei with DAPI; Production of reactive oxygen species (ROS) was determined by H2DCFDA staining; mitochondrial membrane potential was quantified by TMRM staining; DNA damage was determined by H2AX histone activation, and apoptosis was evaluated by caspases-3,-7 activation. It is shown that LED radiations decrease 75-99% cellular viability, and increase 66-89% cellular apoptosis. They also increase ROS production and DNA damage. Fluorescence intensity of apoptosis was 3.7% in nonirradiated cells and 88.8%, 86.1%, 83.9% and 65.5% in cells exposed to white, blue, green or red light, respectively. This study indicates three light-darkness (12 h/12 h) cycles of exposure to LED lighting affect in vitro HRPEpiC.

  4. Dissociation of retinal and headcentric disparity signals in dorsal human cortex

    PubMed Central

    Arnoldussen, David M.; Goossens, Jeroen; van Den Berg, Albert V.

    2015-01-01

    Recent fMRI studies have shown fusion of visual motion and disparity signals for shape perception (Ban et al., 2012), and unmasking camouflaged surfaces (Rokers et al., 2009), but no such interaction is known for typical dorsal motion pathway tasks, like grasping and navigation. Here, we investigate human speed perception of forward motion and its representation in the human motion network. We observe strong interaction in medial (V3ab, V6) and lateral motion areas (MT+), which differ significantly. Whereas the retinal disparity dominates the binocular contribution to the BOLD activity in the anterior part of area MT+, headcentric disparity modulation of the BOLD response dominates in area V3ab and V6. This suggests that medial motion areas not only represent rotational speed of the head (Arnoldussen et al., 2011), but also translational speed of the head relative to the scene. Interestingly, a strong response to vergence eye movements was found in area V1, which showed a dependency on visual direction, just like vertical-size disparity. This is the first report of a vertical-size disparity correlate in human striate cortex. PMID:25759642

  5. Course for undergraduate students: analysis of the retinal image quality of a human eye model

    NASA Astrophysics Data System (ADS)

    del Mar Pérez, Maria; Yebra, Ana; Fernández-Oliveras, Alicia; Ghinea, Razvan; Ionescu, Ana M.; Cardona, Juan C.

    2014-07-01

    In teaching of Vision Physics or Physiological Optics, the knowledge and analysis of the aberration that the human eye presents are of great interest, since this information allows a proper evaluation of the quality of the retinal image. The objective of the present work is that the students acquire the required competencies which will allow them to evaluate the optical quality of the human visual system for emmetropic and ammetropic eye, both with and without the optical compensation. For this purpose, an optical system corresponding to the Navarro-Escudero eye model, which allows calculating and evaluating the aberration of this eye model in different ammetropic conditions, was developed employing the OSLO LT software. The optical quality of the visual system will be assessed through determinations of the third and fifth order aberration coefficients, the impact diagram, wavefront analysis, calculation of the Point Spread Function and the Modulation Transfer Function for ammetropic individuals, with myopia or hyperopia, both with or without the optical compensation. This course is expected to be of great interest for student of Optics and Optometry Sciences, last courses of Physics or medical sciences related with human vision.

  6. Protective effect of alpha-melanocyte-stimulating hormone (α-MSH) on the recovery of ischemia/reperfusion (I/R)-induced retinal damage in a rat model.

    PubMed

    Varga, Balazs; Gesztelyi, Rudolf; Bombicz, Mariann; Haines, David; Szabo, Adrienn Monika; Kemeny-Beke, Adam; Antal, Miklos; Vecsernyes, Miklos; Juhasz, Bela; Tosaki, Arpad

    2013-07-01

    The present study demonstrates capacity of α-MSH to augment recovery from ischemia/reperfusion (I/R)-induced retinal damage in vivo and correlation of its protective effects with expression of heme oxygenase-1 (HO-1). Used techniques include ocular ischemia and reperfusion, electroretinography, histology, electron microscopy, and molecular-biological techniques. The results demonstrate the α-MSH-mediated inhibition of I/R-induced functional deterioration of the retina. Outcomes suggest that the protective effects of α-MSH occur mainly through HO-1-dependent pathways but HO-1-independent mechanisms may also contribute to protection. The observation that post-ischemic treatment with α-MSH exhibits therapeutic efficacy in the same range as pre-ischemic treatment, is a novel result. This outcome suggests a highly conserved protective role for α-MSH as a major stress response mechanism--and offers the possibility for development of novel therapeutic strategies utilizing this hormone, in particular in treatment of conditions resulting from I/R injury, such as deterioration of retinal microcirculation. The merit of the study lies in the fact that I/R injury contribute significantly to the severity of retinopathies. However, currently there are no evidence-based treatments for retinal I/R injury available for clinical use. Our finding suggests that α-MSH may have a very wide range of uses in the prevention of I/R-mediated pathologies.

  7. Retinitis pigmentosa

    PubMed Central

    Hamel, Christian

    2006-01-01

    Retinitis pigmentosa (RP) is an inherited retinal dystrophy caused by the loss of photoreceptors and characterized by retinal pigment deposits visible on fundus examination. Prevalence of non syndromic RP is approximately 1/4,000. The most common form of RP is a rod-cone dystrophy, in which the first symptom is night blindness, followed by the progressive loss in the peripheral visual field in daylight, and eventually leading to blindness after several decades. Some extreme cases may have a rapid evolution over two decades or a slow progression that never leads to blindness. In some cases, the clinical presentation is a cone-rod dystrophy, in which the decrease in visual acuity predominates over the visual field loss. RP is usually non syndromic but there are also many syndromic forms, the most frequent being Usher syndrome. To date, 45 causative genes/loci have been identified in non syndromic RP (for the autosomal dominant, autosomal recessive, X-linked, and digenic forms). Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in the fundus, hypovolted electroretinogram traces, and progressive worsening of these signs. Molecular diagnosis can be made for some genes, but is not usually performed due to the tremendous genetic heterogeneity of the disease. Genetic counseling is always advised. Currently, there is no therapy that stops the evolution of the disease or restores the vision, so the visual prognosis is poor. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection and vitaminotherapy, treating the complications (cataract and macular edema), and helping patients to cope with the social and psychological impact of blindness. However, new therapeutic strategies are emerging from intensive research (gene therapy, neuroprotection, retinal prosthesis). PMID:17032466

  8. Protection of Human Subjects: Proposed Policy.

    ERIC Educational Resources Information Center

    Federal Register, 1974

    1974-01-01

    In the Federal Register of May 30, 1974, regulations were published as Part 46 of Title 45 of the Code of Federal Regulations providing generally for the protection of human subjects involved in research, development, or related activities supported by Department of Health, Education, and Welfare grants or contracts. This notice of proposed…

  9. Retinal oximetry in patients with ischaemic retinal diseases.

    PubMed

    Rilvén, Sandra; Torp, Thomas Lee; Grauslund, Jakob

    2017-03-01

    The retinal oximeter is a new tool for non-invasive measurement of retinal oxygen saturation in humans. Several studies have investigated the associations between retinal oxygen saturation and retinal diseases. In the present systematic review, we examine whether there are associations between retinal oxygen saturation and retinal ischaemic diseases. We used PubMed and Embase to search for retinal oxygen saturation and retinal ischaemic diseases. Three separate searches identified a total of 79 publications. After two levels of manual screening, 10 studies were included: six about diabetic retinopathy (DR) and four about retinal vein occlusion. No studies about retinal artery occlusion were included. In diabetes, all studies found that increases in retinal venous oxygen saturation (rvSatO2 ) were associated with present as well as increasing levels of DR. Four of six studies also found increased retinal arterial oxygen saturation (raSatO2 ) in patients with DR. In patients with central retinal vein occlusion (CRVO), all studies found that rvSatO2 was reduced, but raSatO2 remained unchanged. Branch retinal vein occlusion was not associated with changes in retinal oxygen saturation, but this was based on a single study. In conclusion, DR is associated with increased rvSatO2 and might also be related to increased raSatO2 . Central retinal vein occlusion (CRVO) is correlated with increased rvSatO2 but unrelated to raSatO2 . Prospective studies are needed to expand these findings. These would tell whether retinal oximetry could be a potential tool for screening or a biomarker of treatment outcome in patients with ischaemic retinal diseases.

  10. Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways

    PubMed Central

    Fu, Pingping; Wu, Qiang; Hu, Jianyan; Li, Tingting; Gao, Fengjuan

    2016-01-01

    Retinal ganglion cells (RGCs) consume large quantities of energy to convert light information into a neuronal signal, which makes them highly susceptible to hypoxic injury. This study aimed to investigate the potential protection by baclofen, a GABAB receptor agonist of RGCs against hypoxia-induced apoptosis. Cobalt chloride (CoCl2) was applied to mimic hypoxia. Primary rat RGCs were subjected to CoCl2 with or without baclofen treatment, and RNA interference techniques were used to knock down the GABAB2 gene in the primary RGCs. The viability and apoptosis of RGCs were assessed using cell viability and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, Hoechst staining, and flow cytometry. The expression of cleaved caspase-3, bcl-2, bax, Akt, phospho-Akt, protein kinase RNA (PKR)-like ER kinase (PERK), phospho-PERK, eIF2α, phospho-eIF2α, ATF-4 and CCAAT/enhancer-binding protein homologous protein (CHOP) were measured using western blotting. GABAB2 mRNA expression was determined using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Our study revealed that CoCl2 significantly induced RGC apoptosis and that baclofen reversed these effects. CoCl2-induced reduction of Akt activity was also reversed by baclofen. Baclofen prevented the activation of the PERK pathway and the increase in CHOP expression induced by CoCl2. Knockdown of GABAB2 and the inactivation of the Akt pathway by inhibitors reduced the protective effect of baclofen on CoCl2-treated RGCs. Taken together, these results demonstrate that baclofen protects RGCs from CoCl2-induced apoptosis by increasing Akt activity and by suppressing the PERK pathway and CHOP activation. PMID:27867349

  11. Human milk glycoproteins protect infants against human pathogens.

    PubMed

    Liu, Bo; Newburg, David S

    2013-08-01

    Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease.

  12. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    PubMed Central

    Liu, Bo

    2013-01-01

    Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

  13. Retinal iron homeostasis in health and disease

    PubMed Central

    Song, Delu; Dunaief, Joshua L.

    2013-01-01

    Iron is essential for life, but excess iron can be toxic. As a potent free radical creator, iron generates hydroxyl radicals leading to significant oxidative stress. Since iron is not excreted from the body, it accumulates with age in tissues, including the retina, predisposing to age-related oxidative insult. Both hereditary and acquired retinal diseases are associated with increased iron levels. For example, retinal degenerations have been found in hereditary iron overload disorders, like aceruloplasminemia, Friedreich's ataxia, and pantothenate kinase-associated neurodegeneration. Similarly, mice with targeted mutation of the iron exporter ceruloplasmin and its homolog hephaestin showed age-related retinal iron accumulation and retinal degeneration with features resembling human age-related macular degeneration (AMD). Post mortem AMD eyes have increased levels of iron in retina compared to age-matched healthy donors. Iron accumulation in AMD is likely to result, in part, from inflammation, hypoxia, and oxidative stress, all of which can cause iron dysregulation. Fortunately, it has been demonstrated by in vitro and in vivo studies that iron in the retinal pigment epithelium (RPE) and retina is chelatable. Iron chelation protects photoreceptors and retinal pigment epithelial cells (RPE) in a variety of mouse models. This has therapeutic potential for diminishing iron-induced oxidative damage to prevent or treat AMD. PMID:23825457

  14. Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium

    PubMed Central

    Kinnick, Tyson R.; Stanton, J. Brett; Johnson, Adiv A.; Lynch, Ronald M.; Marmorstein, Lihua Y.

    2015-01-01

    Purpose Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. Methods Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. Results Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control

  15. Modeling human retinal development with patient-specific iPS cells reveals multiple roles for VSX2

    PubMed Central

    Phillips, M. Joseph; Perez, Enio T.; Martin, Jessica M.; Reshel, Samantha T.; Wallace, Kyle A.; Capowski, Elizabeth E.; Singh, Ruchira; Wright, Lynda S.; Clark, Eric M.; Barney, Patrick M.; Stewart, Ron; Dickerson, Sarah J.; Miller, Michael J.; Percin, E. Ferda; Thomson, James A.; Gamm, David M.

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions hiPSCs form optic vesicle-like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC-derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, Visual System Homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC-OVs displayed a significant growth deficit compared to control hiPSC-OVs, as well as increased production of retinal pigmented epithelium (RPE) at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC-OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC-OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wildtype VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC-OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2-mediated gene regulation. Our results establish hiPSC-OVs as versatile model systems to study retinal development at stages not previously accessible in humans, and support the bona fide nature of hiPSC-OV-derived retinal progeny. PMID:24532057

  16. Modeling human retinal development with patient-specific induced pluripotent stem cells reveals multiple roles for visual system homeobox 2.

    PubMed

    Phillips, M Joseph; Perez, Enio T; Martin, Jessica M; Reshel, Samantha T; Wallace, Kyle A; Capowski, Elizabeth E; Singh, Ruchira; Wright, Lynda S; Clark, Eric M; Barney, Patrick M; Stewart, Ron; Dickerson, Sarah J; Miller, Michael J; Percin, E Ferda; Thomson, James A; Gamm, David M

    2014-06-01

    Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle-like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC-derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC-OVs displayed a significant growth deficit compared to control hiPSC-OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC-OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC-OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild-type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC-OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2-mediated gene regulation. Our results establish hiPSC-OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC-OV-derived retinal progeny.

  17. Human subjects research handbook: Protecting human research subjects. Second edition

    SciTech Connect

    1996-01-30

    This handbook serves as a guide to understanding and implementing the Federal regulations and US DOE Orders established to protect human research subjects. Material in this handbook is directed towards new and continuing institutional review board (IRB) members, researchers, institutional administrators, DOE officials, and others who may be involved or interested in human subjects research. It offers comprehensive overview of the various requirements, procedures, and issues relating to human subject research today.

  18. The influences of purple sweet potato anthocyanin on the growth characteristics of human retinal pigment epithelial cells

    PubMed Central

    Sun, Min; Lu, Xiaoling; Hao, Lei; Wu, Tao; Zhao, Huanjiao; Wang, Chao

    2015-01-01

    Background Anthocyanins have been proven to be beneficial to the eyes. However, information is scarce about the effects of purple sweet potato (Ipomoea batatas, L.) anthocyanin (PSPA), a class of anthocyanins derived from purple sweet potato roots, on visual health. Objective The aim of this study was to investigate whether PSPA could have influences on the growth characteristics (cellular morphology, survival, and proliferation) of human retinal pigment epithelial (RPE) cells, which perform essential functions for the visual process. Methods The RPE cell line D407 was used in the present study. The cytotoxicity of PSPA was assessed by MTT assay. Then, cellular morphology, viability, cell cycle, Ki67expression, and PI3K/MAPK activation of RPE cells treated with PSPA were determined. Results PSPA exhibited dose-dependent promotion of RPE cell proliferation at concentrations ranging from 10 to 1,000 µg/ml. RPE cells treated with PSPA demonstrated a predominantly polygonal morphology in a mosaic arrangement, and colony-like cells displayed numerous short apical microvilli and typical ultrastructure. PSPA treatment also resulted in a better platform growing status, statistically higher viability, an increase in the S-phase, and more Ki67+ cells. However, neither pAkt nor pERK were detected in either group. Conclusions We found that PSPA maintained high cell viability, boosted DNA synthesis, and preserved a high percentage of continuously cycling cells to promote cell survival and division without changing cell morphology. This paper lays the foundation for further research about the damage-protective activities of PSPA on RPE cells or human vision. PMID:26070791

  19. Are Pharmaceutical Patents Protected By Human Rights?

    PubMed Central

    Millum, Joseph

    2016-01-01

    The International Bill of Rights enshrines a right to health, which includes a right to access essential medicines. This right frequently appears to conflict with the intellectual property regime that governs pharmaceutical patents. However, there is also a human right that protects creative works, including scientific productions. Does this right support intellectual property protections, even when they may negatively affect health? This article examines the recent attempt by the Committee on Economic, Social and Cultural Rights to resolve this issue and argues that it fails. This is problematic because it means defenders of the present patent regime can continue using human rights documents to support their position. I offer a new framework for resolving the problem by examining the values that underlie human rights. PMID:18974405

  20. Intracellular Signalling in Retinal Ischemia

    DTIC Science & Technology

    1990-07-01

    36) However, vascularization of the RPE is not known to occur in human diseases of photoreceptor degeneration, such as retinitis pigmentosa ...A.C. (1986) Retinitis pigmentosa and retinal neovascularization. Ophthalmology 91, 1599- 1603. Figure la: Control rat retina, 8 weeks of age, central...TITLE (Include Security Classification) Intracellular Signalling in Retinal Ischemia 12. PERSONAL AUTHOR(S) Burns, Margaret Sue; Bellhorn, Roy William

  1. Potential Role of Exercise in Retinal Health.

    PubMed

    Pardue, Machelle T; Chrenek, Micah A; Schmidt, Robin H; Nickerson, John M; Boatright, Jeffrey H

    2015-01-01

    For many patients suffering vision loss due to retinal degeneration, the potential exists for therapeutic intervention to halt or delay disease progression. Proposed molecular, pharmacological, and surgical treatments are expensive and complicated. Finding low-cost interventions to sustain vision and thereby quality of life is vitally important. This chapter reviews findings from animal model and human subject studies indicating that physical exercise has direct, beneficial effects on regions of the central nervous system and is protective against neurodegenerative disease, including recent data from animal models showing similar effects for retina and vision. Potential local and systemic mechanistic pathways for exercise-induced retinal neuroprotection are discussed.

  2. Electric impedance of human embryonic stem cell-derived retinal pigment epithelium.

    PubMed

    Onnela, Niina; Savolainen, Virpi; Juuti-Uusitalo, Kati; Vaajasaari, Hanna; Skottman, Heli; Hyttinen, Jari

    2012-02-01

    The barrier properties of epithelium are conventionally defined by transepithelial resistance (TER). TER provides information about the tightness of the epithelium. Electrical impedance spectroscopy (EIS) provides additional information regarding cell membrane properties, such as changes in electric capacitance and possible parallel or serial pathways that may correlate with the morphology of the cell layer. This study presents EIS of retinal pigment epithelial (RPE) cell model of the putative RPE differentiated from human embryonic stem cells (hESC-RPE). The generally utilized RPE cell model, ARPE-19, was used as immature control. The measured EIS was analyzed by fitting an equivalent electrical circuit model describing the resistive and capacitive properties of the RPE. Our results indicated that TER of hESC-RPE cells was close to the values of human RPE presented in the literature. This provides evidence that the stem cell-derived RPE in vitro can reach high-barrier function. Furthermore, hESC-RPE cells produced impedance spectra that can be modeled by the equivalent circuit of one time constant. ARPE-19 cells produced low-barrier properties, that is, an impedance spectra that suggested poor maturation of ARPE-19 cells. To conclude, EIS could give us means for non-invasively estimating the functionality and maturation of differentiated-RPE cells.

  3. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line

    PubMed Central

    Sluch, Valentin M.; Davis, Chung-ha O.; Ranganathan, Vinod; Kerr, Justin M.; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A.; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S.; Mao, Hai-Quan; Zack, Donald J.

    2015-01-01

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation. PMID:26563826

  4. Differential expression of TYRP1 in adult human retinal pigment epithelium and uveal melanoma cells

    PubMed Central

    QIU, CHUN; LI, PENG; BI, JIANJUN; WU, QING; LU, LINNA; QIAN, GUANXIANG; JIA, RENBING; JIA, RONG

    2016-01-01

    Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM. PMID:27073483

  5. A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells.

    PubMed

    Xiang, Ping; Wu, Kun-Chao; Zhu, Ying; Xiang, Lue; Li, Chong; Chen, Deng-Long; Chen, Feng; Xu, Guotong; Wang, Aijun; Li, Min; Jin, Zi-Bing

    2014-12-01

    Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3-5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.

  6. Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells.

    PubMed

    Varga, E V; Stropova, D; Kim, T; Wang, M; Roeske, W R; Yamamura, H I

    2000-01-01

    Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.

  7. Reciprocal protective effects of all-trans retinol and alpha-tocopherol during lipid peroxidation in retinal membranes.

    PubMed

    Tesoriere, L; Bongiorno, A; Re, R; Livrea, M A

    1995-09-01

    Interactions between vitamin A and vitamin E in suppressing lipid peroxidation were observed in bovine retinal membrane preparations submitted to peroxidative injury by the water soluble azo initiator 2,2'-azobis(2-amidino-propane) hydrochloride (AAPH). Incorporation of 0.75 nmol mg prot(-1) all-trans retinol, an amount comparable with that of the endogenous alpha-tocopherol, significantly elongated the induction time preceding the release of TBA-reactive lipid peroxidation products, and reduced the consumption rate of the endogenous alpha-tocopherol. On the other hand, all-trans retinol was not able to induce any delay to the onset of lipid peroxidation when incorporated in membranes deprived of endogenous alpha-tocopherol by exposure to UV light, although TBARS produced within 60 min decreased slightly. Consumption of all-trans retinol during peroxidation was more rapid when all-trans retinol was incorporated in membranes deprived of alpha-tocopherol than in native membranes. These data suggest that reciprocal protective effects between vitamin A and vitamin E may strongly contribute to the defence of membranes against oxidative stress.

  8. Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells

    PubMed Central

    Fuma, Shinichiro; Murase, Hiromi; Kuse, Yoshiki; Tsuruma, Kazuhiro; Shimazawa, Masamitsu

    2015-01-01

    Purpose Photobiomodulation is the treatment with light in the far-red to near-infrared region of the spectrum and has been reported to have beneficial effects in various animal models of disease, including an age-related macular degeneration (AMD) mouse model. Previous reports have suggested that phagocytosis is reduced by age-related increased oxidative stress in AMD. Therefore, we investigated whether photobiomodulation improves phagocytosis caused by oxidative stress in the human retinal pigment epithelial (ARPE-19) cell line. Methods ARPE-19 cells and human primary retinal pigment epithelium (hRPE) cells were incubated and irradiated with near-infrared light (670 nm LED light, 2,500 lx, twice a day, 250 s/per time) for 4 d. Next, hydrogen peroxide (H2O2) and photoreceptor outer segments (POS) labeled using a pH-sensitive fluorescent dye were added to the cell culture, and phagocytosis was evaluated by measuring the fluorescence intensity. Furthermore, cell death was observed by double staining with Hoechst33342 and propidium iodide after photobiomodulation. CM-H2DCFDA, JC-1 dye, and CCK-8 were added to the cell culture to investigate the reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell viability, respectively. We also investigated the expression of phagocytosis-related proteins, such as focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK). Results Oxidative stress inhibited phagocytosis, and photobiomodulation increased the oxidative stress-induced hypoactivity of phagocytosis in ARPE-19 cells and hRPE cells. Furthermore, H2O2 and photobiomodulation did not affect cell death in this experimental condition. Photobiomodulation reduced ROS production but did not affect cell viability or mitochondrial membrane potential. The expression of phosphorylated MerTK increased, but phosphorylated FAK was not affected by photobiomodulation. Conclusions These findings indicate that near-infrared light photobiomodulation (670 nm) may

  9. Pharmacotherapies for Retinal Detachment.

    PubMed

    Wubben, Thomas J; Besirli, Cagri G; Zacks, David N

    2016-07-01

    Retinal detachment is an important cause of visual loss. Currently, surgical techniques, including vitrectomy, scleral buckle, and pneumatic retinopexy, are the only means to repair retinal detachment and restore vision. However, surgical failure rates may be as high as 20%, and visual outcomes continue to vary secondary to multiple processes, including postoperative cystoid macular edema, epiretinal membrane formation, macular folds, and, ultimately, photoreceptor death. Therefore, pharmacotherapies are being sought to aid the success rates of modern surgical techniques and reduce or slow the degeneration of photoreceptors during retinal detachment. This review discusses potential therapeutic avenues that aid in retinal reattachment, reduce the rate of retinal redetachment by limiting proliferative vitreoretinopathy, and protect against photoreceptor cell death.

  10. Development of Lead Hammerhead Ribozyme Candidates against Human Rod Opsin mRNA for Retinal Degeneration Therapy

    PubMed Central

    Abdelmaksoud, Heba E.; Yau, Edwin H.; Zuker, Michael; Sullivan, Jack M.

    2011-01-01

    To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p < 0.05). Successful lead candidates (hhRz CUC↓ 266, hhRz CUC↓ 1411, hhRz AUA↓ 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48 hr paradigm of testing. These results validate a rigorous computational

  11. Protective action of nipradilol mediated through S-nitrosylation of Keap1 and HO-1 induction in retinal ganglion cells.

    PubMed

    Koriyama, Yoshiki; Kamiya, Marie; Takadera, Tsuneo; Arai, Kunizo; Sugitani, Kayo; Ogai, Kazuhiro; Kato, Satoru

    2012-12-01

    Nipradilol (Nip), which has α1- and β-adrenoceptor antagonist and nitric oxide (NO)-donating properties, has clinically been used as an anti-glaucomatous agent in Japan. NO mediates cellular signaling pathways that regulate physiological functions. The major signaling mechanisms mediated by NO are cGMP-dependent signaling and protein S-nitrosylation-dependent signalings. Nip has been described as having neuroprotective effects through cGMP-dependent pathway in retinal ganglion cells (RGCs). However, the effect seems to be partial. On the other hand, whether Nip can prevent cell death through S-nitrosylation is not yet clarified. In this study, we therefore focused on the neuroprotective mechanism of Nip through S-nitrosylation. Nip showed a dramatic neuroprotective effect against oxidative stress-induced death of RGC-5 cells. However, denitro-nipradilol, which does not have NO-donating properties, was not protective against oxidative stress. Furthermore, an NO scavenger significantly reversed the protective action of Nip against oxidative stress. In addition, we demonstrated that α1- or β-adrenoceptor antagonists (prazosin or timolol) did not show any neuroprotective effect against oxidative stress in RGC-5 cells. We also demonstrated that Nip induced the expression of the NO-dependent antioxidant enzyme, heme oxygenase-1 (HO-1). S-nitrosylation of Kelch-like ECH-associated protein by Nip was shown to contribute to the translocation of NF-E2-related factor 2 to the nucleus, and triggered transcriptional activation of HO-1. Furthermore, RGC death and levels of 4-hydroxy-2-nonenal (4HNE) were increased after optic nerve injury in vivo. Pretreatment with Nip significantly suppressed RGC death and accumulation of 4HNE after injury through an HO-1 activity-dependent mechanism. These data demonstrate a novel neuroprotective action of Nip against oxidative stress-induced RGC death in vitro and in vivo.

  12. Murine Ccl2/Cx3cr1 Deficiency Results in Retinal Lesions Mimicking Human Age-Related Macular Degeneration

    PubMed Central

    Tuo, Jingsheng; Bojanowski, Christine M.; Zhou, Min; Shen, Defen; Ross, Robert J.; Rosenberg, Kevin I.; Cameron, D. Joshua; Yin, Chunyue; Kowalak, Jeffrey A.; Zhuang, Zhengping; Zhang, Kang; Chan, Chi-Chao

    2007-01-01

    Purpose Senescent Ccl2-/- mice are reported to develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are also found to be associated with AMD. The authors generated Ccl2-/-/Cx3cr1-/- mice to establish a more characteristic and reproducible AMD model. Methods Single Ccl2- and Cx3cr1-deficient mice were crossbred to obtain Ccl2-/-/Cx3cr1-/- mice. Funduscopy, histopathology, retinal A2E quantification, proteomics, RT-PCR gene expression assay, immunochemistry, and Western blotting were used to examine the retina and to evaluate gene expression within the retinal tissue. Results By 6 weeks of age, all Ccl2-/-/Cx3cr1-/- mice developed AMD-like retinal lesions, including drusen, retinal pigment epithelium alteration, and photoreceptor degeneration. Furthermore, choroidal neovascularization occurred in 15% of the mice. These degenerative lesions progressed with age. A2E, a major lipofuscin fluorophore that accumulated during AMD progression, was significantly higher in the Ccl2-/-/Cx3cr1-/- retina than in the wild-type retina. Complement cofactor was higher in the Ccl2-/-/Cx3cr1-/- RPE. Proteomics data indicated that four proteins were differentially expressed in Ccl2-/-/Cx3cr1-/- retina compared with control. One of these proteins, ERp29, an endoplasmic reticulum protein, functions as an escort chaperone and in protein folding. Conclusions The authors concluded that Ccl2-/-/Cx3cr1-/- mice develop a broad spectrum of AMD abnormalities with early onset and high penetrance. These observations implicate certain chemokines and endoplasmic reticulum proteins in AMD pathogenesis. Similar to the mechanism of neurodegeneration caused by dysfunction of endoplasmic reticulum proteins, decreased chaperoning may cause misfolded protein accumulation, leading to drusen formation and retinal degeneration. PMID:17652758

  13. 48 CFR 1552.223-70 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Protection of human... 1552.223-70 Protection of human subjects. As prescribed in 1523.303-70, insert the following contract clause when the contract involves human test subjects. Protection of Human Subjects (APR 1984) (a)...

  14. 48 CFR 1352.235-70 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Protection of human... Protection of human subjects. As prescribed in 48 CFR 1335.006(a), insert the following provision: Protection of Human Subjects (APR 2010) (a) Research involving human subjects is not permitted under this...

  15. 48 CFR 1552.223-70 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Protection of human... 1552.223-70 Protection of human subjects. As prescribed in 1523.303-70, insert the following contract clause when the contract involves human test subjects. Protection of Human Subjects (APR 1984) (a)...

  16. 48 CFR 1352.235-70 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Protection of human... Protection of human subjects. As prescribed in 48 CFR 1335.006(a), insert the following provision: Protection of Human Subjects (APR 2010) (a) Research involving human subjects is not permitted under this...

  17. 48 CFR 1552.223-70 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Protection of human... 1552.223-70 Protection of human subjects. As prescribed in 1523.303-70, insert the following contract clause when the contract involves human test subjects. Protection of Human Subjects (APR 1984) (a)...

  18. 48 CFR 1352.235-70 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Protection of human... Protection of human subjects. As prescribed in 48 CFR 1335.006(a), insert the following provision: Protection of Human Subjects (APR 2010) (a) Research involving human subjects is not permitted under this...

  19. Cell-Deposited Matrix Improves Retinal Pigment Epithelium Survival on Aged Submacular Human Bruch's Membrane

    PubMed Central

    Sugino, Ilene K.; Gullapalli, Vamsi K.; Sun, Qian; Wang, Jianqiu; Nunes, Celia F.; Cheewatrakoolpong, Noounanong; Johnson, Adam C.; Degner, Benjamin C.; Hua, Jianyuan; Liu, Tong; Chen, Wei; Li, Hong

    2011-01-01

    Purpose. To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. Methods. Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. Results. The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. Conclusions. Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes. PMID:21398292

  20. A new immunodeficient pigmented retinal degenerate rat strain to study transplantation of human cells without immunosuppression

    PubMed Central

    Seiler, Magdalene J.; Aramant, Robert B.; Jones, Melissa K.; Ferguson, Dave L.; Bryda, Elizabeth C.

    2015-01-01

    Purpose The goal of this study was to develop an immunodeficient rat model of retinal degeneration (RD nude rats) that will not reject transplanted human cells. Methods SD-Tg(S334ter)3Lav females homozygous for a mutated mouse rhodopsin transgene were mated with NTac:NIH-Whn (NIH nude) males homozygous for the Foxn1rnu allele. Through selective breeding, a new stock, SD-Foxn1 Tg(S334ter)3Lav (RD nude) was generated such that all animals were homozygous for the Foxn1rnu allele and either homo- or hemizygous for the S334ter transgene. PCR-based assays for both the Foxn1rnu mutation and the S334ter transgene were developed for accurate genotyping. Immunodeficiency was tested by transplanting sheets of hESC-derived neural progenitor cells to the subretinal space of RD nude rats, and, as a control, NIH nude rats. Rats were killed between 8 and 184 days after surgery, and eye sections were analyzed for human, neuronal, and glial markers. Results After transplantation to RD nude and to NIH nude rats, hESC-derived neural progenitor cells differentiated to neuronal and glial cells, and migrated extensively from the transplant sheets throughout the host retina. Migration was more extensive in RD nude than in NIH nude rats. Already 8 days after transplantation, donor neuronal processes were found in the host inner plexiform layer. In addition, host glial cells extended processes into the transplants. The host retina showed the same photoreceptor degeneration pattern as in the immunocompetent SD-Tg(S334ter)3Lav rats. Recipients survived well after surgery. Conclusions This new rat model is useful for testing the effect of human cell transplantation on the restoration of vision without interference of immunosuppression. PMID:24817311

  1. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    PubMed Central

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-01-01

    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. PMID:27246808

  2. Human cadaver retina model for retinal heating during corneal surgery with a femtosecond laser

    NASA Astrophysics Data System (ADS)

    Sun, Hui; Fan, Zhongwei; Yun, Jin; Zhao, Tianzhuo; Yan, Ying; Kurtz, Ron M.; Juhasz, Tibor

    2014-02-01

    Femtosecond lasers are widely used in everyday clinical procedures to perform minimally invasive corneal refractive surgery. The intralase femtosecond laser (AMO Corp. Santa Ana, CA) is a common example of such a laser. In the present study a numerical simulation was developed to quantify the temperature rise in the retina during femtosecond intracorneal surgery. Also, ex-vivo retinal heating due to laser irradiation was measured with an infrared thermal camera (Fluke Corp. Everett, WA) as a validation of the simulation. A computer simulation was developed using Comsol Multiphysics to calculate the temperature rise in the cadaver retina during femtosecond laser corneal surgery. The simulation showed a temperature rise of less than 0.3 degrees for realistic pulse energies for the various repetition rates. Human cadaver retinas were irradiated with a 150 kHz Intralase femtosecond laser and the temperature rise was measured withan infrared thermal camera. Thermal camera measurements are in agreement with the simulation. During routine femtosecond laser corneal surgery with normal clinical parameters, the temperature rise is well beneath the threshold for retina damage. The simulation predictions are in agreement with thermal measurements providing a level of experimental validation.

  3. Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

    PubMed

    Zhang, Pingbo; Kirby, David; Dufresne, Craig; Chen, Yan; Turner, Randi; Ferri, Sara; Edward, Deepak P; Van Eyk, Jennifer E; Semba, Richard D

    2016-04-01

    The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

  4. Plasticity of the human visual system after retinal gene therapy in patients with Leber's congenital amaurosis.

    PubMed

    Ashtari, Manzar; Zhang, Hui; Cook, Philip A; Cyckowski, Laura L; Shindler, Kenneth S; Marshall, Kathleen A; Aravand, Puya; Vossough, Arastoo; Gee, James C; Maguire, Albert M; Baker, Chris I; Bennett, Jean

    2015-07-15

    Much of our knowledge of the mechanisms underlying plasticity in the visual cortex in response to visual impairment, vision restoration, and environmental interactions comes from animal studies. We evaluated human brain plasticity in a group of patients with Leber's congenital amaurosis (LCA), who regained vision through gene therapy. Using non-invasive multimodal neuroimaging methods, we demonstrated that reversing blindness with gene therapy promoted long-term structural plasticity in the visual pathways emanating from the treated retina of LCA patients. The data revealed improvements and normalization along the visual fibers corresponding to the site of retinal injection of the gene therapy vector carrying the therapeutic gene in the treated eye compared to the visual pathway for the untreated eye of LCA patients. After gene therapy, the primary visual pathways (for example, geniculostriate fibers) in the treated retina were similar to those of sighted control subjects, whereas the primary visual pathways of the untreated retina continued to deteriorate. Our results suggest that visual experience, enhanced by gene therapy, may be responsible for the reorganization and maturation of synaptic connectivity in the visual pathways of the treated eye in LCA patients. The interactions between the eye and the brain enabled improved and sustained long-term visual function in patients with LCA after gene therapy.

  5. Geldanamycin increases 4-hydroxynonenal (HNE)-induced cell death in human retinal pigment epithelial cells.

    PubMed

    Kaarniranta, Kai; Ryhänen, Tuomas; Karjalainen, Hannu M; Lammi, Mikko J; Suuronen, Tiina; Huhtala, Anne; Kontkanen, Matti; Teräsvirta, Markku; Uusitalo, Hannu; Salminen, Antero

    Development of age-related macular degeneration (AMD) is associated with functional abnormalities and cell death in retinal pigment epithelial (RPE) cells attributable to oxidative stress. To minimize the adverse effects of oxidative stress, cells activate their defence systems, e.g., via increased expression of heat shock protein (Hsp), activation of stress sensitive AP-1 and NF-kappaB transcription factors. In this study, we examined the accumulation of Hsp70 protein, activation of AP-1 and NF-kappaB transcription factors in human ARPE-19 cells subjected to a 4-hydroxynonenal (HNE)-induced oxidative stress. In addition, the influence of Hsp90 inhibitor geldanamycin (GA) was studied in HNE-treated cells. Mitochondrial metabolic activity and apoptosis were determined to evaluate cell death in the ARPE-19 cells. The ARPE-19 cells showed increased accumulation of Hsp70 protein before of the cytotoxic hallmarks appearing in response to HNE. In contrast, increased DNA-binding activities of AP-1 or NF-kappaB transcription factors were not seen under HNE insults. Interestingly, GA significantly increased cell death in the HNE-treated cells, which was involved in caspase-3 independent apoptosis. This study reveals that the Hsps have an important role in the cytoprotection of RPE cells subjected to HNE-derived oxidative stress.

  6. Monocarboxylate transporter mediated uptake of moxifloxacin on human retinal pigmented epithelium cells

    PubMed Central

    Barot, Megha; Gokulgandhi, Mitan R.; Agrahari, Vibhuti; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Objectives This work was aim to determine in vitro interaction of moxifloxacin with monocarboxylate transporter (MCT) using a human retinal pigment epithelium cells (ARPE-19). Methods In vitro moxifloxacin uptakes were performed at 37°C across ARPE-19 cells. Concentration-dependent uptake of moxifloxacin was performed to delineate moxifloxacin kinetics with MCT. Effects of MCT substrates, MCT inhibitors, pH and metabolic inhibitors on moxifloxacin uptake were conducted to delineate mechanism of moxifloxacin influx via MCT. Key findings Moxifloxacin uptake was found to exhibit saturable kinetics (Km = 1.56 ± 0.32 μM and Vmax = 0.58 ± 0.16 μM/min/mg protein). Higher uptake of moxifloxacin was observed at acidic pH. MCT substrates such as salisylic acid, ofloxacin and L-lactic acid significantly inhibited the uptake of moxifloxacin. Furthermore, moxifloxacin uptake was significantly reduced in the presence of metabolic and MCT inhibitors. Overall, this study demonstrated an interaction of moxifloxacin with Na+ and H+-coupled transporter, most likely MCT1. Conclusions Apart from the lipophilicity, we anticipate that lowest vitreal half-life of intravitreal moxifloxacin compared with other fluoroquinolones may be due to its interaction with MCT. This information might be crucial in clinical settings and can be further explored to improve vitreous half-life and therapeutic efficacy of moxifloxacin. PMID:24102496

  7. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    NASA Astrophysics Data System (ADS)

    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.

  8. Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

    SciTech Connect

    Lee, Sang Yull; Jo, Hong Jae; Kim, Kang Mi; Song, Ju Dong; Chung, Hun Taeg; Park, Young Chul

    2008-01-25

    Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.

  9. Utilization of scanning laser ophthalmoscopy in laser-induced bilateral human retinal nerve fiber layer damage

    NASA Astrophysics Data System (ADS)

    Zwick, Harry; Gagliano, Donald A.; Ruiz, S.; Stuck, Bruce E.

    1995-05-01

    In this paper, we describe a military laser accident case where bilateral Q-switched laser exposure resulted in bilateral macular damage with immediate visual acuity loss in one eye (OS) and delayed visual acuity loss in the other exposed eye (OD), where retinal damage appeared more parafoveal. At 6 weeks post exposure, OS had recovered to 20/17 and OD had dropped to 20/100 Snellen activity. Retinal nerve fiber damage was observed in both eyes at this time. Contrast sensitivity measurements made in OS were suppressed across all spatial frequencies, even though Snellen acuity measured in the normal range. More severe high spatial frequency loss in contrast was measured in the right eye as well as low spatial frequency loss. Both OS and OD revealed a parafoveal preferred retinal locus with scanning laser ophthalmoscopy contrast sensitivity measurements, suggesting parafoveal retinal compensatory processes.

  10. Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Reichenbach, Andreas; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1

  11. Role of the Retinal Vascular Endothelial Cell in Ocular Disease

    PubMed Central

    Bharadwaj, Arpita S.; Appukuttan, Binoy; Wilmarth, Phillip A.; Pan, Yuzhen; Stempel, Andrew J.; Chipps, Timothy J.; Benedetti, Eric E.; Zamora, David O.; Choi, Dongseok; David, Larry L.; Smith, Justine R.

    2012-01-01

    Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell. PMID:22982179

  12. Gene therapy for retinitis pigmentosa caused by MFRP mutations: human phenotype and preliminary proof of concept.

    PubMed

    Dinculescu, Astra; Estreicher, Jackie; Zenteno, Juan C; Aleman, Tomas S; Schwartz, Sharon B; Huang, Wei Chieh; Roman, Alejandro J; Sumaroka, Alexander; Li, Qiuhong; Deng, Wen-Tao; Min, Seok-Hong; Chiodo, Vince A; Neeley, Andy; Liu, Xuan; Shu, Xinhua; Matias-Florentino, Margarita; Buentello-Volante, Beatriz; Boye, Sanford L; Cideciyan, Artur V; Hauswirth, William W; Jacobson, Samuel G

    2012-04-01

    Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.

  13. Gene Therapy for Retinitis Pigmentosa Caused by MFRP Mutations: Human Phenotype and Preliminary Proof of Concept

    PubMed Central

    Dinculescu, Astra; Estreicher, Jackie; Zenteno, Juan C.; Aleman, Tomas S.; Schwartz, Sharon B.; Huang, Wei Chieh; Roman, Alejandro J.; Sumaroka, Alexander; Li, Qiuhong; Deng, Wen-Tao; Min, Seok-Hong; Chiodo, Vince A.; Neeley, Andy; Liu, Xuan; Shu, Xinhua; Matias-Florentino, Margarita; Buentello-Volante, Beatriz; Boye, Sanford L.; Cideciyan, Artur V.

    2011-01-01

    Abstract Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy. PMID:22142163

  14. An overview of Leber congenital amaurosis: a model to understand human retinal development.

    PubMed

    Koenekoop, Robert K

    2004-01-01

    Leber congenital amaurosis is a congenital retinal dystrophy described almost 150 years ago. Today, Leber congenital amaurosis is proving instrumental in our understanding of the molecular events that determine normal and aberrant retinal development. Six genes have been shown to be mutated in Leber congenital amaurosis, and they participate in a wide variety of retinal pathways: retinoid metabolism (RPE65), phototransduction (GUCY2D), photoreceptor outer segment development (CRX), disk morphogenesis (RPGRIP1), zonula adherens formation (CRB1), and cell-cycle progression (AIPL1). Longitudinal studies of visual performance show that most Leber congenital amaurosis patients remain stable, some deteriorate, and rare cases exhibit improvements. Histopathological analyses reveal that most cases have extensive degenerative retinal changes, some have an entirely normal retinal architecture, whereas others have primitive, poorly developed retinas. Animal models of Leber congenital amaurosis have greatly added to understanding the impact of the genetic defects on retinal cell death, and response to rescue. Gene therapy for RPE65 deficient dogs partially restored sight, and provides the first real hope of treatment for this devastating blinding condition.

  15. Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells

    PubMed Central

    Ikarashi, Rina; Akechi, Honami; Kanda, Yuzuki; Ahmad, Alsawaf; Takeuchi, Kouhei; Morioka, Eri; Sugiyama, Takashi; Ebisawa, Takashi; Ikeda, Masaaki; Ikeda, Masayuki

    2017-01-01

    Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks. PMID:28276525

  16. The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium

    PubMed Central

    Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B.; Weinberger, Dov; Sredni, Benjamin

    2016-01-01

    Purpose Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch’s membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Methods Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Results Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Conclusions Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases. PMID:27293373

  17. Protected areas as frontiers for human migration.

    PubMed

    Zommers, Zinta; MacDonald, David W

    2012-06-01

    Causes of human population growth near protected areas have been much debated. We conducted 821 interviews in 16 villages around Budongo Forest Reserve, Masindi district, Uganda, to explore the causes of human migration to protected areas and to identify differences in forest use between migrant and nonmigrant communities. We asked subjects for information about birthplace, migration, household assets, household activities, and forest use. Interview subjects were categorized as nonmigrants (born in one of the interview villages), socioeconomic migrants (chose to emigrate for economic or social reasons) from within Masindi district (i.e., local migrants) and from outside the Masindi district (i.e., regional migrants), or forced migrants (i.e., refugees or internally displaced individuals who emigrated as a result of conflict, human rights abuses, or natural disaster). Only 198 respondents were born in interview villages, indicating high rates of migration between 1998 and 2008. Migrants were drawn to Budongo Forest because they thought land was available (268 individuals) or had family in the area (161 individuals). A greater number of regional migrants settled in villages near Lake Albert than did forced and local migrants. Migration category was also associated with differences in sources of livelihood. Of forced migrants 40.5% earned wages through labor, whereas 25.5% of local and 14.5% of regional migrants engaged in wage labor. Migrant groups appeared to have different effects on the environment. Of respondents that hunted, 72.7% were regional migrants. Principal component analyses indicated households of regional migrants were more likely to be associated with deforestation. Our results revealed gaps in current models of human population growth around protected areas. By highlighting the importance of social networks and livelihood choices, our results contribute to a more nuanced understanding of causes of migration and of the environmental effects of

  18. Functional and morphological analysis of the subretinal injection of human retinal progenitor cells under Cyclosporin A treatment

    PubMed Central

    Huang, Rui; Baranov, Petr; Lai, Kunbei; Zhang, Xinmei; Ge,, Jian

    2014-01-01

    Purpose The purpose of this study is to evaluate the functional and morphological changes in subretinal xenografts of human retinal progenitor cells (hRPCs) in B6 mice treated with Cyclosporin A (CsA; 210 mg/l in drinking water). Methods The hRPCs from human fetal eyes were isolated and expanded for transplantation. These cells, with green fluorescent protein (GFP) at 11 passages, were transplanted into the subretinal space in B6 mice. A combination of invasive and noninvasive approaches was used to analyze the structural and functional consequences of the subretinal injection of the hRPCs. The process of change was monitored using spectral domain optical coherence tomography (SDOCT), histology, and electroretinography (ERG) at 3 days, 1 week, and 3 weeks after transplantation. Cell counts were used to evaluate the survival rate with a confocal microscope. ERGs were performed to evaluate the physiologic changes, and the structural changes were evaluated using SDOCT and histological examination. Results The results of the histological examination showed that the hRPCs gained a better survival rate in the mice treated with CsA. The SDOCT showed that the bleb size of the retinal detachment was significantly decreased, and the retinal reattachment was nearly complete by 3 weeks. The ERG response amplitudes in the CsA group were less decreased after the injection, when compared with the control group, in the dark-adapted and light-adapted conditions. However, the cone-mediated function in both groups was less affected by the transplantation after 3 weeks than the rod-mediated function. Conclusions Although significant functional and structural recovery was observed after the subretinal injection of the hRPCs, the effectiveness of CsA in xenotransplantation may be a novel and potential approach for increasing retinal progenitor cell survival. PMID:25352736

  19. αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium

    PubMed Central

    Roggia, Murilo F.; Ueta, Takashi

    2015-01-01

    Purpose To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by photoreceptor outer segments (POS) and its effects on retinal pigment epithelium (RPE). Methods PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK), and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK) were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined. Results POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch’s membrane, and poor choriocapillaris vasculature. Conclusions The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway. PMID:26244551

  20. Viral-mediated RdCVF and RdCVFL expression protects cone and rod photoreceptors in retinal degeneration

    PubMed Central

    Byrne, Leah C.; Dalkara, Deniz; Luna, Gabriel; Fisher, Steven K.; Clérin, Emmanuelle; Sahel, Jose-Alain; Léveillard, Thierry; Flannery, John G.

    2014-01-01

    Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy. We used adeno-associated virus (AAV) to express these isoforms in mice and found that both systemic and intravitreal injection of engineered AAV vectors resulted in RdCVF and RdCVFL expression in the eye. Systemic delivery of AAV92YF vectors in neonates resulted in earlier onset of RdCVF and RdCVFL expression compared with that observed with intraocular injection using the same vectors at P14. We also evaluated the efficacy of intravitreal injection using a recently developed photoreceptor-transducing AAV variant (7m8) at P14. Systemic administration of AAV92YF-RdCVF improved cone function and delayed cone loss, while AAV92YF-RdCVFL increased rhodopsin mRNA and reduced oxidative stress by-products. Intravitreal 7m8-RdCVF slowed the rate of cone cell death and increased the amplitude of the photopic electroretinogram. Together, these results indicate different functions for Nxnl1 isoforms in the retina and suggest that RdCVF gene therapy has potential for treating retinal degenerative disease. PMID:25415434

  1. Protections for Subjects in Human Research with Pesticides

    EPA Pesticide Factsheets

    All pesticide research using human subjects must meet our strict protective standards before we would consider using them in evaluating pesticides. EPA's regulation “Protections for Subjects in Human Research” was promulgated in 2006 and amended in 2013.

  2. Human retinal development in an in situ whole eye culture system.

    PubMed

    Engelsberg, Karl; Ghosh, Fredrik

    2011-01-01

    Phenotypic characterization of human retinogenesis may be facilitated by use of the tissue culture system paradigm. Traditionally, most culture protocols involve isolation of retinal tissue and/or cells, imposing various degrees of trauma, which in many cases leads to abnormal development. In this paper, we present a novel culture technique using whole embryonic eyes to investigate whether the retina in situ can develop normally in vitro. All procedures were carried out in accordance with the Declaration of Helsinki. Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion. A total of 19 eyes were enucleated. The ages of the embryonic retinas were 6-7.5 weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 17 eyes were placed on culture plates and divided into 3 groups kept for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the specimens were processed for hematoxylin and eosin staining, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; rod bipolar cells) and vimentin (Müller cells) were used. TUNEL was used to detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. Specimens kept 7-14 DIV had a similar appearance, while 28-day specimens consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured retinas displayed completely normal lamination without rosettes or double folds. Pyknotic cells were found at the inner margin of the retinas, and the proportion of these cells increased with time in vitro. TUNEL staining revealed a few scattered cells in 7-DIV specimens, and the amount of stained cells in the inner part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin labeling showed cells

  3. Retinitis Pigmentosa.

    ERIC Educational Resources Information Center

    Carr, Ronald E.

    1979-01-01

    The author describes the etiology of retinitis pigmentosa, a visual dysfunction which results from progressive loss of the retinal photoreceptors. Sections address signs and symptoms, ancillary findings, heredity, clinical diagnosis, therapy, and research. (SBH)

  4. Retinitis pigmentosa

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/001029.htm Retinitis pigmentosa To use the sharing features on this page, please enable JavaScript. Retinitis pigmentosa is an eye disease in which there is ...

  5. 48 CFR 1252.223-72 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Traffic Safety Administration (NHTSA) policies and procedures for the protection of human subjects... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Protection of human....223-72 Protection of human subjects. As prescribed in (TAR) 48 CFR 1223.7000(b), insert the...

  6. 34 CFR 75.681 - Protection of human research subjects.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 1 2013-07-01 2013-07-01 false Protection of human research subjects. 75.681 Section... Conditions Must Be Met by a Grantee? Other Requirements for Certain Projects § 75.681 Protection of human research subjects. If a grantee uses a human subject in a research project, the grantee shall protect...

  7. 48 CFR 1252.223-72 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Protection of human....223-72 Protection of human subjects. As prescribed in (TAR) 48 CFR 1223.7000(b), insert the following clause: Protection of Human Subjects (APR 2005) The Contractor shall comply with the National...

  8. 34 CFR 75.681 - Protection of human research subjects.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Protection of human research subjects. 75.681 Section... Conditions Must Be Met by a Grantee? Other Requirements for Certain Projects § 75.681 Protection of human research subjects. If a grantee uses a human subject in a research project, the grantee shall protect...

  9. 34 CFR 75.681 - Protection of human research subjects.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Protection of human research subjects. 75.681 Section... Conditions Must Be Met by a Grantee? Other Requirements for Certain Projects § 75.681 Protection of human research subjects. If a grantee uses a human subject in a research project, the grantee shall protect...

  10. 34 CFR 75.681 - Protection of human research subjects.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Protection of human research subjects. 75.681 Section... Conditions Must Be Met by a Grantee? Other Requirements for Certain Projects § 75.681 Protection of human research subjects. If a grantee uses a human subject in a research project, the grantee shall protect...

  11. 34 CFR 75.681 - Protection of human research subjects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Protection of human research subjects. 75.681 Section... Conditions Must Be Met by a Grantee? Other Requirements for Certain Projects § 75.681 Protection of human research subjects. If a grantee uses a human subject in a research project, the grantee shall protect...

  12. 48 CFR 1252.223-72 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Protection of human....223-72 Protection of human subjects. As prescribed in (TAR) 48 CFR 1223.7000(b), insert the following clause: Protection of Human Subjects (APR 2005) The Contractor shall comply with the National...

  13. 48 CFR 252.235-7004 - Protection of Human Subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 3 2011-10-01 2011-10-01 false Protection of Human... of Provisions And Clauses 252.235-7004 Protection of Human Subjects. As prescribed in 235.072(e), use the following clause: Protection of Human Subjects (JUL 2009) (a) Definitions. As used in this...

  14. 48 CFR 252.235-7004 - Protection of Human Subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Protection of Human... of Provisions And Clauses 252.235-7004 Protection of Human Subjects. As prescribed in 235.072(e), use the following clause: Protection of Human Subjects (JUL 2009) (a) Definitions. As used in this...

  15. 48 CFR 1252.223-72 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Protection of human....223-72 Protection of human subjects. As prescribed in (TAR) 48 CFR 1223.7000(b), insert the following clause: Protection of Human Subjects (APR 2005) The Contractor shall comply with the National...

  16. 76 FR 54408 - Human Subjects Research Protections: Enhancing Protections for Research Subjects and Reducing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-01

    ... Menikoff, M.D., J.D., Office for Human Research Protections (OHRP), Department of Health and Human Services... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND... Parts 50 and 56 Human Subjects Research Protections: Enhancing Protections for Research Subjects...

  17. Intercellular Ca(2+) wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation.

    PubMed

    Abu Khamidakh, A E; Juuti-Uusitalo, K; Larsson, K; Skottman, H; Hyttinen, J

    2013-03-01

    Ca(2+) signaling is vitally important in cellular physiological processes and various drugs also affect Ca(2+) signaling. Thus, knowledge of Ca(2+) dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca(2+) communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca(2+) wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) in the monolayer were tracked with fluorescence microscopy imaging using Ca(2+)-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca(2+), after depletion of intracellular Ca(2+) stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca(2+)](i) increase, and percentage of responsive cells were calculated to quantitatively characterize Ca(2+) wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca(2+)](i), which propagated to neighboring cells in a wave-like manner. Ca(2+) wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca(2+) reduced [Ca(2+)](i) increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca(2+) stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca(2+)](i) increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca(2+) wave propagation. The P2-receptor blocker suramin reduced the percentage

  18. The transplantation of human fetal neuroretinal cells in advanced retinitis pigmentosa patients: results of a long-term safety study.

    PubMed

    Das, T; del Cerro, M; Jalali, S; Rao, V S; Gullapalli, V K; Little, C; Loreto, D A; Sharma, S; Sreedharan, A; del Cerro, C; Rao, G N

    1999-05-01

    The purpose of this study was to determine the long-term safety of transplanting human fetal neuroretinal cells (14 to 18 week gestational age) into a series of patients with advanced retinitis pigmentosa (RP). After obtaining informed consent, both hosts and mothers of donors were screened for transmissible diseases. Pre- and postoperative clinical exams, visual acuity, electroretinograms, and fluorescein angiograms were performed and visual field testing was attempted in each case. Surgically, an anterior approach through pars plana ciliaris was used. A retinotomy was performed in the paramacular area and a two-function cannula was introduced into the subretinal space to deliver a suspension of donor cells. The cell suspension carried approximately 4000 cells/microl; the volume injected did not exceed 150 microl. The patients were examined for periods ranging from 12 to 40 months posttransplantation. To date, no evidence of inflammation, infection, or overt rejection of the graft was noted in the host eye, neither was any change observed in the contralateral, unoperated eye. In conclusion, neuroretinal cells were injected into the subretinal space of 14 patients with advanced RP with no clinical appearance of detrimental effects at the time of surgery or up to 40 months postinjection except in 1 patient who developed retinal detachment. This sets the stage for a phase II clinical trial to determine the possible beneficial effects of this procedure in patients blinded by degenerative retinal disease.

  19. Prevention of VEGF-induced growth and tube formation in human retinal endothelial cell by aldose reductase inhibition

    PubMed Central

    Yadav, Umesh CS; Srivastava, SK; Ramana, KV

    2012-01-01

    Objective Since diabetes-induced vascular endothelial growth factor (VEGF) is implicated in retinal angiogenesis, we aimed to examine the role of aldose reductase (AR) in VEGF–induced human retinal endothelial cell (HREC) growth and tube formation. Materials and Methods HREC were stimulated with VEGF and cell-growth was determined by MTT assay. AR inhibitor, fidarestat, to block the enzyme activity and AR siRNA to ablate AR gene expression in HREC were used to investigate the role of AR in neovascularization using cell-migration and tube formation assays. Various signaling intermediates and angiogenesis markers were assessed by Western blot analysis. Immuno-histochemical analysis of diabetic rat eyes was performed to examine VEGF expression in the retinal layer. Results Stimulation of primary HREC with VEGF caused increased cell growth and migration, and AR inhibition with fidarestat or ablation with siRNA significantly prevented it. VEGF-induced tube formation in HREC was also significantly prevented by fidarestat. Treatment of HREC with VEGF also increased the expression of VCAM, AR, and phosphorylation and activation of Akt and p38-MAP kinase, which were prevented by fidarestat. VEGF-induced expression of VEGFRII in HREC was also prevented by AR inhibition or ablation. Conclusions Our results indicate that inhibition of AR in HREC prevents tube formation by inhibiting the VEGF-induced activation of the Akt and p38-MAPK pathway and suggest a mediatory role of AR in ocular neovascularization generally implicated in retinopathy and AMD. PMID:22658411

  20. Geldanamycin and its analog induce cytotoxicity in cultured human retinal pigment epithelial cells.

    PubMed

    Wu, Wen-Chuan; Wu, Meng-Hsien; Chang, Yo-Chen; Hsieh, Ming-Chu; Wu, Horng-Jiun; Cheng, Kai-Chun; Lai, Yu-Hung; Kao, Ying-Hsien

    2010-08-01

    Geldanamycin (GA), a benzoquinone ansamycin, was originally isolated as a natural product with anti-fungal activity. GA and its analogs, including 17-allylamino-demethoxy geldanamycin (17-AAG), are also known to block the function of a molecular chaperone, heat shock protein 90 (Hsp90). In light of their anti-tumor properties through direct cytotoxicity and anti-angiogenicity, GA has been previously demonstrated to suppress hypoxia-induced VEGF production in retinal pigment epithelium (RPE) cells, implicating its applicability in treating intraocular neovascularization. This study aimed at investigating the effectiveness of Hsp90 inhibitor treatment in suppressing proliferation of cultured human RPE cells and elucidating its underlying mechanism. Cultured RPE cells were treated with GA or 17-AAG and subjected for cell proliferation assay and cell cycle analysis. Expression of apoptotic regulators and survival signaling activity were monitored by Western blotting. The results showed that both GA and 17-AAG significantly inhibited RPE cell proliferation at micromolar levels. Treatment with GA and 17-AAG led to growth arrests in G1 and S phases, increased sub-G1 hypodipoid cell population, induced apoptotic cell death, and upregulated P53 and P21 expression, although the drug-induced Bcl-2 upregulation cannot prevent cell death. Additionally, GA and 17-AAG significantly suppressed constitutive contents of phosphorylated ERK1/2 and total Akt proteins, and completely abrogated wortmannin-sensitized Akt phosphorylation. In conclusion, GA and 17-AAG inhibit RPE cell proliferation and induce cytotoxicity, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. They might potentially constitute a therapeutic agent for ocular disorders with RPE over proliferation, such as proliferative vitreoretinopathy.

  1. A five-primary photostimulator suitable for studying intrinsically photosensitive retinal ganglion cell functions in humans

    PubMed Central

    Cao, Dingcai; Nicandro, Nathaniel; Barrionuevo, Pablo A.

    2015-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) can respond to light directly through self-contained photopigment, melanopsin. IpRGCs also receive synaptic inputs from rods and cones. Thus, studying ipRGC functions requires a novel photostimulating method that can account for all of the photoreceptor inputs. Here, we introduced an inexpensive LED-based five-primary photostimulator that can control the excitations of rods, S-, M-, L-cones, and melanopsin-containing ipRGCs in humans at constant background photoreceptor excitation levels, a critical requirement for studying the adaptation behavior of ipRGCs with rod, cone, or melanopsin input. We described the theory and technical aspects (including optics, electronics, software, and calibration) of the five-primary photostimulator. Then we presented two preliminary studies using the photostimulator we have implemented to measure melanopsin-mediated pupil responses and temporal contrast sensitivity function (TCSF). The results showed that the S-cone input to pupil responses was antagonistic to the L-, M- or melanopsin inputs, consistent with an S-OFF and (L + M)-ON response property of primate ipRGCs (Dacey et al., 2005). In addition, the melanopsin-mediated TCSF had a distinctive pattern compared with L + M or S-cone mediated TCSF. Other than controlling individual photoreceptor excitation independently, the five-primary photostimulator has the flexibility in presenting stimuli modulating any combination of photoreceptor excitations, which allows researchers to study the mechanisms by which ipRGCs combine various photoreceptor inputs. PMID:25624466

  2. Rimonabant, a selective cannabinoid1 receptor antagonist, protects against light-induced retinal degeneration in vitro and in vivo.

    PubMed

    Imamura, Tomoyo; Tsuruma, Kazuhiro; Inoue, Yuki; Otsuka, Tomohiro; Ohno, Yuta; Ogami, Shiho; Yamane, Shinsaku; Shimazawa, Masamitsu; Hara, Hideaki

    2017-03-16

    The endocannabinoid system is involved in some neurodegenerative diseases such as Alzheimer's disease. An endogenous constellation of proteins related to cannabinoid1 receptor signaling, including free fatty acids, diacylglycerol lipase, and N-acylethanolamine-hydrolyzing acid amidase, are localized in the murine retina. Moreover, the expression levels of endogenous agonists of cannabinoid receptors are changed in the vitreous fluid. However, the role of the endocannabinoid system in the retina, particularly in the light-induced photoreceptor degeneration, remains unknown. Therefore, we investigated involvement of the cannabinoid1 receptor in light-induced retinal degeneration using in vitro and in vivo models. To evaluate the effect of cannabinoid1 receptors in light irradiation-induced cell death, the mouse retinal cone-cell line (661W) was treated with a cannabinoid1 receptor antagonist, rimonabant. Time-dependent changes of expression and localization of retinal cannabinoid1 receptors were measured using Western blot and immunostaining. Retinal damage was induced in mice by exposure to light, followed by intravitreal injection of rimonabant. Electroretinograms and histologic analyses were performed. Rimonabant suppressed light-induced photoreceptor cell death. Cannabinoid1 receptor expression was upregulated by light exposure. Treatment with rimonabant improved both a- and b-wave amplitudes and the thickness of the outer nuclear layer. These results suggest that the cannabinoid1 receptor is involved in light-induced retinal degeneration and it may represent a therapeutic target in the light-induced photoreceptor degeneration related diseases.

  3. Retinal Detachment

    MedlinePlus

    ... men more than women and whites more than African Americans. A retinal detachment is also more likely to occur in people who Are extremely nearsighted Have had a retinal detachment in the other eye Have a family history of retinal detachment Have had cataract surgery Have ...

  4. Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

    PubMed

    Sorkio, Anni; Porter, Patrick J; Juuti-Uusitalo, Kati; Meenan, Brian J; Skottman, Heli; Burke, George A

    2015-09-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.

  5. Novel Serum Proteomic Signatures in a Non-Human Primate Model of Retinal Injury

    DTIC Science & Technology

    2011-01-01

    photoreceptors [50] and PGK1 deficiency has been implicated in one case of retinitis pigmentosa [51]. It is likely this enzyme is abundant in metabolically...DB=tr 2 3 2 1 0.234633 0.218250 Q4G3V7 KIAA0476 (Fragment) OS=Macaca mulatta PE=2 SV=1 DB=tr 2 3 2 1 0.234633 0.218250 Q4G413 Retinitis pigmentosa 1...4.070046 0.142449 Q4G413 Retinitis pigmentosa 1 (Fragment) OS=Macaca mulatta GN=RP1 PE=2 SV=1 DB=tr 5 6 1 4 4.070046 0.142449 Q6IYG8 NADH-ubiquinone

  6. A c-myc antisense oligonucleotide inhibits human retinal pigment epithelial cell proliferation.

    PubMed

    Capeáns, C; Piñeiro, A; Domínguez, F; Loidi, L; Buceta, M; Carneiro, C; Garcia-Caballero, T; Sanchez-Salorio, M

    1998-05-01

    The purpose of this work was to investigate if MYC-dependent intracellular mitogenic pathway is active in cultures of human retinal pigment epithelial (hRPE) cells and whether myc antisense phosphorotioate oligonucleotides (c-myc-AS-ODN) are useful tools for inhibiting the proliferation of hRPE cells. Cultures of hRPE cells were established from adult human corneal donors. These cells were positively stained for cytokeratins and vimentin. Myc mRNA expression was determined by Northern blot analysis and it was determined by means of immunofluorescence if MYC was expressed. C-myc-AS-ODN effect on cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine into cellular DNA. Cell number was estimated by using a tetrazolium bromide based colorimetric method. Human RPE cells in culture expressed MYC and myc mRNA as well as prothymosin alpha mRNA--a gene whose transcription is under MYC control--indicating that MYC-dependent intracellular mitogenic pathway is active in these cells. In accordance with this, we found that blocking the expression of myc by the addition of c-myc-AS-ODN to the culture medium inhibited hRPE cell proliferation. The effect of the c-myc-AS-ODN was found to be sequence specific (the use of a control oligonucleotide with the same sequence but in an opposite direction had no effect) and dose-dependent (4 microM was the lowest effective dose tested). By using RT-PCR we found that the c-myc-AS-ODN inhibition of cell proliferation was related to a diminution in c-myc mRNA expression, and by immunofluorescence we detected a diminution in c-MYC protein staining in RPE cells after 48 hr of treatment with c-myc-AS-ODN. Furthermore, growth inhibition remained for at least 5 days after addition of a single dose of the c-myc-AS-ODN to the culture. We conclude that hRPE cell proliferation is under MYC control. Blocking the expression of myc by c-myc-AS-ODN inhibited hRPE cell proliferation. These findings establish a rationale

  7. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

    PubMed Central

    Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

    2016-01-01

    Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases. PMID:27009841

  8. In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy

    PubMed Central

    Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

    2016-01-01

    Purpose The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. Methods A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. Results The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Conclusions Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells. PMID:27564519

  9. α-Aminoadipic acid protects against retinal disruption through attenuating Müller cell gliosis in a rat model of acute ocular hypertension

    PubMed Central

    Wang, Xiaolei; Su, Jier; Ding, Jingwen; Han, Song; Ma, Wei; Luo, Hong; Hughes, Guy; Meng, Zhaoyang; Yin, Yi; Wang, Yanling; Li, Junfa

    2016-01-01

    Objective Ocular hypertension is an important risk factor for glaucoma. The purpose of this study was to investigate the gliotoxic effects of α-aminoadipic acid (AAA) in a rat model of AOH and its underlying mechanisms. Materials and methods In the rat model of acute ocular hypertension (AOH), intraocular pressure was increased to 110 mmHg for 60 minutes. Animals were divided into four groups: sham operation (Ctrl), AOH, AOH + phosphate-buffered saline (PBS), and AOH + AAA. Cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay, and retinal ganglion cells (RGCs) immunostained with Thy-1 were counted. Müller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein. Tumor necrosis factor-α (TNF-α) was examined using Western blot. Results In the rat model of AOH, cell apoptosis was induced in the ganglion cell layer and the number of RGCs was decreased. Müller cell gliosis in the retinas of rats was induced, and retinal protein levels of TNF-α were increased. Intravitreal treatment of AAA versus PBS control attenuated these retinal abnormalities to show protective effects in the rat model of AOH. Conclusion In the retinas of the rat model of AOH, AAA treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of RGCs, likely through the attenuation of Müller cell gliosis and suppression of TNF-α induction. Our observations suggest that AAA might be a potential therapeutic target in glaucoma. PMID:27799744

  10. RTP801 immunoreactivity in retinal ganglion cells and its down-regulation in cultured cells protect them from light and cobalt chloride.

    PubMed

    del Olmo-Aguado, Susana; Núñez-Álvarez, Claudia; Ji, Dan; Manso, Alberto García; Osborne, Neville N

    2013-09-01

    RTP801, a stress-related protein, is activated by adverse environmental conditions and inhibits the activity of mammalian target of rapamycin (mTOR) in promoting oxidative stress-dependent cell death. RTP801 exists both in the mammalian retina and the lens of the eye. Here, we observed RTP801 immunoreactivity in some retinal ganglion cells. Intravitreal injection of cobalt chloride (CoCl2) to mimick hypoxia influenced retinal GFAP (glial fibrillary acidic protein) and heme oxygenase-1 (HO-1) levels, but did not affect RTP801 immunoreactivity or mRNA content relative to GAPDH. However, RTP801 mRNA was elevated when compared with Brn3a mRNA, suggesting that RTP801 is activated in stressed Brn3a retinal ganglion cells. In cultures of RGC-5 cells, RTP801 immunoreactivity was located in the cytoplasm and partly present in the mitochondria. An insult of blue light or CoCl2 increased RTP801 expression, which was accompanied by cell death. However, in cultures where RTP801 mRNA was down-regulated, the negative influence of blue light and CoCl2 was blunted. Rapamycin nullified the CoCl2-induced up-regulation of RTP801 and attenuated cell death. Moreover, rapamycin was non-toxic to RGC-5 cells, even at a high concentration (10μM). The protective effect of rapamycin on RGC-5 cells caused by the inhibition of RTP801 suggests that rapamycin might attenuate retinal ganglion cell death in situ, as in glaucoma.

  11. 75 FR 59264 - Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-27

    ... HUMAN SERVICES Secretary's Advisory Committee on Human Research Protections AGENCY: Department of Health and Human Services, Office of the Secretary, Office of the Assistant Secretary for Health. ACTION..., notice is hereby given that the Secretary's Advisory Committee on Human Research Protections...

  12. 77 FR 37408 - Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-21

    ... HUMAN SERVICES Secretary's Advisory Committee on Human Research Protections AGENCY: Office of the Assistant Secretary for Health, Office of the Secretary, Department of Health and Human Services. ACTION..., notice is hereby given that the Secretary's Advisory Committee on Human Research Protections...

  13. 77 FR 58383 - Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-20

    ... HUMAN SERVICES Secretary's Advisory Committee on Human Research Protections AGENCY: Office of the Assistant Secretary for Health, Office of the Secretary, Department of Health and Human Services. ACTION..., notice is hereby given that the Secretary's Advisory Committee on Human Research Protections...

  14. Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

    PubMed Central

    Delyfer, Marie-Noëlle; Aït-Ali, Najate; Camara, Hawa; Clérin, Emmanuelle; Korobelnik, Jean-François; Sahel, José-Alain; Léveillard, Thierry

    2013-01-01

    Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD. PMID:23979175

  15. Antiangiogenic Effect of (±)-Haloperidol Metabolite II Valproate Ester [(±)-MRJF22] in Human Microvascular Retinal Endothelial Cells.

    PubMed

    Olivieri, Melania; Amata, Emanuele; Vinciguerra, Shila; Fiorito, Jole; Giurdanella, Giovanni; Drago, Filippo; Caporarello, Nunzia; Prezzavento, Orazio; Arena, Emanuela; Salerno, Loredana; Rescifina, Antonio; Lupo, Gabriella; Anfuso, Carmelina Daniela; Marrazzo, Agostino

    2016-11-10

    (±)-MRJF22 [(±)-2], a novel prodrug of haloperidol metabolite II (sigma-1 receptor antagonist/sigma-2 receptor agonist ligand) obtained by conjugation to valproic acid (histone deacetylase inhibitor) via an ester bond, exhibits antiangiogenic activity, being able to reduce human retinal endothelial cell (HREC) viability in a comparable manner to bevacizumab. Moreover, (±)-2 was able to significantly reduce viable cells count, endothelial cell migration, and tube formation in vascular endothelial growth factor A (VEGF-A) stimulated HREC cultures.

  16. Effects of Histone Deacetylase Inhibitor (Valproic Acid) on the Expression of Hypoxia-inducible Factor-1 Alpha in Human Retinal Müller Cells

    PubMed Central

    Kim, Young Jun; Park, Sang Jun; Kim, Na Rae

    2017-01-01

    Purpose To evaluate the effects of valproic acid (VPA), a histone deacetylase inhibitor (HDACI), on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in human retinal Müller cells under hypoxic conditions. Methods Chemical hypoxia was induced in human retinal Müller cells (MIO-M1) by treatment with increasing concentrations of cobalt(II) chloride (CoCl2). Müller cells were also treated with a set concentration of CoCl2, along with various concentrations of VPA. The expression of HIF-1α and VEGF in the treated Müller cells was determined by enzyme-linked immunosorbent assay. Results Exposure of human retinal Müller cells to increasing concentrations of CoCl2 produced a dose-dependent increase in HIF-1α expression. The addition of increasing concentrations of VPA lead to a dose-dependent decrease in expression of HIF-1α and VEGF in Müller cells exposed to a set concentration of CoCl2. Conclusions HDACI VPA downregulated the expressions of HIF-1α and VEGF in human retinal Müller cells under hypoxic conditions. Using HDACI to target HIF-1α expression in Müller cells could be a new therapeutic strategy for the treatment of retinal vascular diseases. PMID:28243027

  17. Human organic anion transporting polypeptide 1A2 (OATP1A2) mediates cellular uptake of all-trans-retinol in human retinal pigmented epithelial cells

    PubMed Central

    Chan, Ting; Zhu, Ling; Madigan, Michele C; Wang, Ke; Shen, Weiyong; Gillies, Mark C; Zhou, Fanfan

    2015-01-01

    Background and Purpose Vision depends on retinoid exchange between the retinal pigment epithelium (RPE) and photoreceptors. Defects in any step of the canonical visual cycle can lead to retinal degenerations. All-trans-retinol (atROL) plays an important role in visual signal transduction. However, how atROL enters human RPE from the apical membrane remains unclear. This study investigated the role of human organic anion transporting polypeptide 1A2 (OATP1A2) in atROL uptake in human RPE. Experimental Approach Immunoblotting and immunostaining elucidated the expression and localization of OATP1A2 in human RPE. Transporter functional studies were conducted to assess the interaction of OATP1A2 with atROL. Key Results Our study revealed OATP1A2 is expressed in human RPE, mainly at the apical membrane. Our data also indicated atROL inhibited the uptake of the typical OATP1A2 substrate, oestrone-3-sulfate (E3S), in over-expressing cells. Studies on the uptake of 3H-atROL in these over-expressing cells revealed atROL is a substrate of OATP1A2. We confirmed these findings in human primary RPE cells. The transport of E3S and atROL was significantly reduced in human primary RPE cells with OATP1A2 siRNA silencing. Conclusion and Implications Our data provides the first evidence of OATP1A2 expression in human RPE and more importantly, its novel role in the cellular uptake of atROL, which might be essential to the proper functioning of the canonical visual cycle. Our findings contribute to the understanding of the molecular mechanisms involved in retinoid transport between the RPE and photoreceptors and provide novel insights into potential pharmaceutical interventions for visual cycle disruption associated with retinal degenerations. PMID:25560245

  18. Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol

    PubMed Central

    Wang, Wen-Der; Hsu, Hwei-Jan; Li, Yi-Fang; Wu, Chang-Yi

    2017-01-01

    Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf), and PBZ-treated embryos (2–72 hpf) were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of gnat1 (rod photoreceptor marker), gnat2 (cone photoreceptor marker), aldehyde dehydrogenases (encoding key enzymes for RA synthesis), and phospho-histone H3 (an M-phase marker) in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division. PMID:28085063

  19. A simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe.

    PubMed

    Parekh, Mohit; Ferrari, Stefano; Di Iorio, Enzo; Barbaro, Vanessa; Camposampiero, Davide; Karali, Marianthi; Ponzin, Diego; Salvalaio, Gianni

    2012-06-12

    Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them

  20. Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium

    PubMed Central

    Ilmarinen, Tanja; Hiidenmaa, Hanna; Kööbi, Peeter; Nymark, Soile; Sorkio, Anni; Wang, Jing-Huan; Stanzel, Boris V.; Thieltges, Fabian; Alajuuma, Päivi; Oksala, Olli; Kataja, Marko; Uusitalo, Hannu; Skottman, Heli

    2015-01-01

    In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation. PMID:26606532

  1. Inflammatory Cytokines Induce Expression of Chemokines by Human Retinal Cells: Role in Chemokine Receptor Mediated Age-related Macular Degeneration.

    PubMed

    Nagineni, Chandrasekharam N; Kommineni, Vijay K; Ganjbaksh, Nader; Nagineni, Krishnasai K; Hooks, John J; Detrick, Barbara

    2015-11-01

    Chemokine reeptor-3 (CCR-3) was shown to be associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). AMD is a vision threatening retinal disease that affects the aging population world-wide. Retinal pigment epithelium and choroid in the posterior part of the retina are the key tissues targeted in the pathogenesis of CNV in AMD. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast (HCHF) cells, prepared from aged adult human donor eyes, to evaluate the expression of major CCR-3 ligands, CCL-5, CCL -7, CCL-11,CCL-24 and CCL-26. Microarray analysis of gene expression in HRPE cells treated with inflammatory cytokine mix (ICM= IFN-γ+TNF-α+IL-1β) revealed 75 and 23-fold increase in CCL-5 and CCL-7 respectively, but not CCL-11, CCL-24 and CCL-26. Chemokine secretion studies of the production of CCL5 and CCL7 by HRPE corroborated with the gene expression analysis data. When the HRPE cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent manner. Similar to the gene expression data, the ICM did not enhance HRPE production of CCL-11, CCL-24 and CCL-26. CCL-11 and CCL-26 were increased with IL-4 treatment and this HRPE production was augmented in the presence of TNF-α and IL1β. When HCHF cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent fashion. IL-4 induced low levels of CCL-11 and CCL-26 in HCHF and this production was significantly enhanced by TNF-α. Under these conditions, neither HRPE nor HCHF were demonstrated to produce CCL-24. These data demonstrate that chronic inflammation triggers CCL-5 and CCL-7 release by HRPE and HCHF and the subsequent interactions with CCR3 may participate in pathologic processes in AMD.

  2. Calibration-free measurement of the oxygen saturation in human retinal vessels

    NASA Astrophysics Data System (ADS)

    Schweitzer, Dietrich; Leistritz, Lutz; Hammer, Martin; Scibor, Mateusz; Bartsch, Ulrich; Strobel, Juergen

    1995-05-01

    of a piglet measured by reflectometry with the oxygen saturation of the left ventricular and the venous blood measured by a laboratory hemoximeter. First results of the measurement of the oxygen saturation in human retinal vessels are demonstrated.

  3. Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells.

    PubMed

    Szurman, Peter; Kaczmarek, Radoslaw; Spitzer, Martin S; Jaissle, Gesine B; Decker, Patrice; Grisanti, Salvatore; Henke-Fahle, Sigrid; Aisenbrey, Sabine; Bartz-Schmidt, Karl U

    2006-09-01

    The aim of the study was to evaluate the antiproliferative and cytotoxic properties of triamcinolone acetonide (TA) on human retinal pigment epithelium cells (ARPE19) and the role of epicellular crystalline deposits. Monolayer cultures of ARPE19 cells were used. Purified or unpurified crystalline TA suspension (0.01-1.0 mg/ml) or the vehicle alone (benzyl alcohol, 0.025%-0.00025%), diluted in culture medium, were added to the cells that were either grown on cell culture dishes covered by a protecting membrane filter insert or without a filter. After 1, 3, 5 and 7 days mitochondrial activity was measured using the MTT assay and the morphology assessed microscopically. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays ARPE19 cells were grown to confluence and then cultured in a serum-deficient medium to ensure a static milieu. Annexin V-FITC and propidium iodide co-staining was performed and analyzed by flow cytometry. Exposure to TA without direct cellular contact showed a moderate antiproliferative activity resulting in a dose-dependent suppression of DNA synthesis (maximum 42.7%), but not a cytotoxic effect. In contrast, adherent deposits of crystalline TA particles on top of the cell layer caused a rapid-progressive and dose-dependent cell death preceded by an early phosphatidylserine externalization to the outer leaflet of the plasma membrane. Within a healthy, confluent cell layer the number of viable cells decreased by 14.2, 20.8 and 68.8%, respectively, after one day of direct exposure. Exposure to the vehicle alone caused only a slight growth inhibitory effect in a proliferating cell layer, but early signs of cell death were detected even at the lowest concentration tested. In conclusion, the effect of the vehicle is less pronounced than formerly assumed, but not negligible, thus indicating a beneficial effect of purification. While non-adherent TA, if purified, appears to be safe in clinically

  4. Possible protective role of the ABCA4 gene c.1268A>G missense variant in Stargardt disease and syndromic retinitis pigmentosa in a Sicilian family: Preliminary data.

    PubMed

    D'Angelo, Rosalia; Donato, Luigi; Venza, Isabella; Scimone, Concetta; Aragona, Pasquale; Sidoti, Antonina

    2017-04-01

    In the wide horizon of ophthalmologically rare diseases among retinitis pigmentosa forms, Stargardt disease has gradually assumed a significant role due to its heterogeneity. In the present study, we aimed to support one of two opposite hypotheses concerning the causative or protective role of heterozygous c.1268A>G missense variant of the ABCA4 gene in Stargardt disease and in syndromic retinitis pigmentosa. This study was based on a family consisting of three members: proband, age 54, with high myopia, myopic chorioretinitis and retinal dystrophy; wife, age 65, with mild symptoms; daughter, age 29, asymptomatic. After genetic counseling, ABCA4 and RP1 gene analysis was performed. The results highlighted an important genetic picture. The proband was found to carry two variant RP1 SNPs, rs2293869 (c.2953A>T) and rs61739567 (c.6098G>A), and, a wild-type condition for four RP1 polymorphisms, rs444772 (c.2623G>A) and three SNPs in the 'hot-spot' region, exon 4. The proband's wife, instead, showed an opposite condition compared to her husband: a homozygous mutated condition for the first four SNPs analyzed, while the last two were wild-type. Regarding the ABCA4 gene, the proband evidenced a wild-type condition. Furthermore, the wife showed a heterozygous condition of ABCA4 rs3112831 (c.1268A>G). As expected, the daughter presented heterozygosity for all variants of both genes. In conclusion, even though the c.1268A>G missense variant of the ABCA4 gene has often been reported as causative of disease, and in other cases protective of disease, in our family case, the variant appears to reduce or delay the risk of onset of Stargardt disease.

  5. Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina.

    PubMed

    Aparicio, J G; Hopp, H; Choi, A; Mandayam Comar, J; Liao, V C; Harutyunyan, N; Lee, T C

    2016-11-17

    Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated value for human disease study, drug screening, and therapeutic applications; however, their full potential remains underdeveloped. To characterize RGCs in human embryonic stem cell (hESC) derived retinal organoids we examined RGC markers and surface antigen expression and made comparisons to human fetal retina. RGCs in both tissues exhibited CD184 and CD171 expression and distinct expression patterns of the RGC markers BRN3 and RBPMS. The retinal progenitor cells (RPCs) of retinal organoids expressed CD184, consistent with its expression in the neuroblastic layer in fetal retina. In retinal organoids CD184 expression was enhanced in RGC competent RPCs and high CD184 expression was retained on post-mitotic RGC precursors; CD171 was detected on maturing RGCs. The differential expression timing of CD184 and CD171 permits identification and enrichment of RGCs from retinal organoids at differing maturation states from committed progenitors to differentiating neurons. These observations will facilitate molecular characterization of PSC-derived RGCs during differentiation, critical knowledge for establishing the veracity of these in vitro produced cells. Furthermore, observations made in the retinal organoid model closely parallel those in human fetal retina further validating use of retinal organoid to model early retinal development.

  6. Lycium barbarum (Goji Berry) extracts and its taurine component inhibit PPAR-γ-dependent gene transcription in human retinal pigment epithelial cells: Possible implications for diabetic retinopathy treatment.

    PubMed

    Song, M K; Salam, N K; Roufogalis, Basil D; Huang, T H W

    2011-11-01

    The peroxisome proliferator activated receptor-γ (PPAR-γ) is involved in the pathogenesis of diabetic retinopathy. Diabetic retinopathy is a preventable microvascular diabetic complication that damages human retinal pigment epithelial cells. Taurine is abundant in the fruit of Lycium barbarum (Goji Berry), and is reportedly beneficial for diabetic retinopathy. However, the mechanism of its action is unknown. Hence, we have investigated the mechanism of action of an extract from L. barbarum on a model of diabetic retinopathy, the retinal ARPE-19 cell line, and identified the receptor function of taurine, an active component of L. barbarum (Goji Berry) extract, which is potentially responsible for the protective effect on diabetic retinopathy. We demonstrate for the first time that L. barbarum extract and its taurine component dose-dependently enhance PPAR-γ luciferase activity in HEK293 cell line transfected with PPAR-γ reporter gene. This activity was significantly decreased by a selective PPAR-γ antagonist GW9662. Moreover, L. barbarum extract and taurine dose-dependently enhanced the expression of PPAR-γ mRNA and protein. In an inflammation model where ARPE-19 cells were exposed to high glucose L. barbarum extract and taurine down-regulated the mRNA of pro-inflammatory mediators encoding MMP-9, fibronectin and the protein expression of COX-2 and iNOS proteins. The predicted binding mode of taurine in the PPAR-γ ligand binding site mimics key electrostatic interactions seen with known PPAR-γ agonists. We conclude that PPAR-γ activation by L. barbarum extract is associated with its taurine content and may explain at least in part its use in diabetic retinopathy progression.

  7. 48 CFR 3424.170 - Protection of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Protection of human... Individual Privacy 3424.170 Protection of human subjects. In this subsection, “Research” means a systematic... generalizable knowledge. (34 CFR 97.102(d)) Research is considered to involve human subjects when a...

  8. 45 CFR 63.31 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 1 2012-10-01 2012-10-01 false Protection of human subjects. 63.31 Section 63.31 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION GRANT PROGRAMS ADMINISTERED... Protection of human subjects. All grants made pursuant to this part are subject to the specific provisions...

  9. 48 CFR 352.270-4 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 4 2012-10-01 2012-10-01 false Protection of human subjects. 352.270-4 Section 352.270-4 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES... Protection of human subjects. (a) As prescribed in 370.303(a), the Contracting Officer shall insert...

  10. 45 CFR 63.31 - Protection of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 1 2014-10-01 2014-10-01 false Protection of human subjects. 63.31 Section 63.31 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION GRANT PROGRAMS ADMINISTERED... Protection of human subjects. All grants made pursuant to this part are subject to the specific provisions...

  11. 48 CFR 352.270-4 - Protection of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 4 2014-10-01 2014-10-01 false Protection of human subjects. 352.270-4 Section 352.270-4 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES... Protection of human subjects. (a) As prescribed in 370.303(a), the Contracting Officer shall insert...

  12. 48 CFR 352.270-4 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 4 2013-10-01 2013-10-01 false Protection of human subjects. 352.270-4 Section 352.270-4 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES... Protection of human subjects. (a) As prescribed in 370.303(a), the Contracting Officer shall insert...

  13. 45 CFR 63.31 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 45 Public Welfare 1 2013-10-01 2013-10-01 false Protection of human subjects. 63.31 Section 63.31 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION GRANT PROGRAMS ADMINISTERED... Protection of human subjects. All grants made pursuant to this part are subject to the specific provisions...

  14. 48 CFR 3424.170 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Protection of human... Individual Privacy 3424.170 Protection of human subjects. In this subsection, “Research” means a systematic... generalizable knowledge. (34 CFR 97.102(d)) Research is considered to involve human subjects when a...

  15. 48 CFR 3424.170 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Protection of human... Individual Privacy 3424.170 Protection of human subjects. In this subsection, “Research” means a systematic... generalizable knowledge. (34 CFR 97.102(d)) Research is considered to involve human subjects when a...

  16. 75 FR 37813 - Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-30

    ... HUMAN SERVICES Secretary's Advisory Committee on Human Research Protections AGENCY: Department of Health... hereby given that the Secretary's Advisory Committee on Human Research Protections (SACHRP) will hold its... agenda will be posted on the SACHRP Web site at: http://www.hhs.gov/ohrp/sachrp/index.html . DATES:...

  17. 48 CFR 352.270-4 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 4 2011-10-01 2011-10-01 false Protection of human subjects. 352.270-4 Section 352.270-4 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES... Protection of human subjects. (a) As prescribed in 370.303(a), the Contracting Officer shall insert...

  18. 45 CFR 63.31 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Protection of human subjects. 63.31 Section 63.31 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION GRANT PROGRAMS ADMINISTERED... Protection of human subjects. All grants made pursuant to this part are subject to the specific provisions...

  19. 48 CFR 352.270-4 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Protection of human subjects. 352.270-4 Section 352.270-4 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES... Protection of human subjects. (a) As prescribed in 370.303(a), the Contracting Officer shall insert...

  20. 48 CFR 1523.303-70 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Protection of human... Hazardous Material and Material Safety Data 1523.303-70 Protection of human subjects. Contracting Officers shall insert the contract clause at 1552.223-70 when the contract involves human test subjects....

  1. 48 CFR 3424.170 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 7 2011-10-01 2011-10-01 false Protection of human... Individual Privacy 3424.170 Protection of human subjects. In this subsection, “Research” means a systematic... generalizable knowledge. (34 CFR 97.102(d)) Research is considered to involve human subjects when a...

  2. 45 CFR 63.31 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Protection of human subjects. 63.31 Section 63.31 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION GRANT PROGRAMS ADMINISTERED... Protection of human subjects. All grants made pursuant to this part are subject to the specific provisions...

  3. 48 CFR 1523.303-70 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Protection of human... Hazardous Material and Material Safety Data 1523.303-70 Protection of human subjects. Contracting Officers shall insert the contract clause at 1552.223-70 when the contract involves human test subjects....

  4. Learning about Retinitis Pigmentosa

    MedlinePlus

    ... genetic terms used on this page Learning About Retinitis Pigmentosa What is retinitis pigmentosa? What are the symptoms ... Pigmentosa Additional Resources for Retinitis Pigmentosa What is retinitis pigmentosa? Retinitis pigmentosa (RP) is the name given to ...

  5. Cue-dependent memory-based smooth-pursuit in normal human subjects: importance of extra-retinal mechanisms for initial pursuit.

    PubMed

    Ito, Norie; Barnes, Graham R; Fukushima, Junko; Fukushima, Kikuro; Warabi, Tateo

    2013-08-01

    Using a cue-dependent memory-based smooth-pursuit task previously applied to monkeys, we examined the effects of visual motion-memory on smooth-pursuit eye movements in normal human subjects and compared the results with those of the trained monkeys. These results were also compared with those during simple ramp-pursuit that did not require visual motion-memory. During memory-based pursuit, all subjects exhibited virtually no errors in either pursuit-direction or go/no-go selection. Tracking eye movements of humans and monkeys were similar in the two tasks, but tracking eye movements were different between the two tasks; latencies of the pursuit and corrective saccades were prolonged, initial pursuit eye velocity and acceleration were lower, peak velocities were lower, and time to reach peak velocities lengthened during memory-based pursuit. These characteristics were similar to anticipatory pursuit initiated by extra-retinal components during the initial extinction task of Barnes and Collins (J Neurophysiol 100:1135-1146, 2008b). We suggest that the differences between the two tasks reflect differences between the contribution of extra-retinal and retinal components. This interpretation is supported by two further studies: (1) during popping out of the correct spot to enhance retinal image-motion inputs during memory-based pursuit, pursuit eye velocities approached those during simple ramp-pursuit, and (2) during initial blanking of spot motion during memory-based pursuit, pursuit components appeared in the correct direction. Our results showed the importance of extra-retinal mechanisms for initial pursuit during memory-based pursuit, which include priming effects and extra-retinal drive components. Comparison with monkey studies on neuronal responses and model analysis suggested possible pathways for the extra-retinal mechanisms.

  6. United States Federal Guidance on Witness Protection in Human Trafficking

    DTIC Science & Technology

    2015-06-12

    UNITED STATES FEDERAL GUIDANCE ON WITNESS PROTECTION IN HUMAN TRAFFICKING A thesis presented to the Faculty of the U.S. Army...JUN 2015 4. TITLE AND SUBTITLE United States Federal Guidance on Witness Protection in Human Trafficking 5a. CONTRACT NUMBER 5b. GRANT NUMBER...United States needs overarching federal guidance on witness protection for human trafficking victims/witnesses in order to enhance their safety and

  7. Pathogenic Mutations in Retinitis Pigmentosa 2 Predominantly Result in Loss of RP2 Protein Stability in Human and Zebrafish.

    PubMed

    Liu, Fei; Qin, Yayun; Yu, Shanshan; Soares, Dinesh C; Yang, Lifang; Weng, Jun; Li, Chang; Gao, Meng; Lu, Zhaojing; Hu, Xuebin; Liu, Xiliang; Jiang, Tao; Liu, Jing Y; Shu, Xinhua; Tang, Zhaohui; Liu, Mugen

    2017-02-16

    Mutations in retinitis pigmentosa 2 (RP2) account for 10-20% of X-linked retinitis pigmentosa (RP) cases. The encoded RP2 protein is implicated in ciliary trafficking of myristoylated and prenylated proteins in photoreceptor cells. To date, over 70 mutations in RP2 have been identified. How these mutations disrupt the function of RP2 is not fully understood. Here, we report a novel in-frame 12-bp deletion (c.357_368del, p.Pro120_Gly123del) in zebrafish rp2 The mutant zebrafish shows reduced rod phototransduction proteins and progressive retinal degeneration. Interestingly, the protein level of mutant Rp2 is almost undetectable, while its mRNA level is near normal, indicating a possible post-translational effect of the mutation. Consistent with this hypothesis, the equivalent 12-bp deletion in human RP2 markedly impairs RP2 protein stability and reduces its protein level. Furthermore, we found that a majority of the RP2 pathogenic mutations (including missense, single-residue deletion and C-terminal truncation mutations) severely destabilize the RP2 protein. The destabilized RP2 mutant proteins are degraded via the proteasome pathway, resulting in dramatically decreased protein levels. The remaining non-destabilizing mutations T87I, R118H/G/L/C, E138G and R211H/L are suggested to impair the interaction between RP2 and its protein partners (such as ARL3) or with as yet unknown partners. By utilizing a combination of in silico, in vitro and in vivo approaches, our work comprehensively indicates that loss of RP2 protein structural stability is the predominating pathogenic consequence for most RP2 mutations. Our study also reveals a role of the C-terminal domain of RP2 in maintaining the overall protein stability.

  8. Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution

    PubMed Central

    Starnes, Austin C; Huisingh, Carrie; McGwin, Gerald; Sloan, Kenneth R; Ablonczy, Zsolt; Smith, R. Theodore; Curcio, Christine A; Ach, Thomas

    2016-01-01

    Background The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Methods Nineteen human RPE-flatmounts (9≤51years, 10>80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. Results A total of 11403 RPE cells at 200 locations were analyzed: 94.66 % mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. Conclusion This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies. PMID:26923500

  9. Inactivity of human β,β-carotene-9′,10′-dioxygenase (BCO2) underlies retinal accumulation of the human macular carotenoid pigment

    PubMed Central

    Li, Binxing; Vachali, Preejith P.; Gorusupudi, Aruna; Shen, Zhengqing; Sharifzadeh, Hassan; Besch, Brian M.; Nelson, Kelly; Horvath, Madeleine M.; Frederick, Jeanne M.; Baehr, Wolfgang; Bernstein, Paul S.

    2014-01-01

    The macula of the primate retina uniquely concentrates high amounts of the xanthophyll carotenoids lutein, zeaxanthin, and meso-zeaxanthin, but the underlying biochemical mechanisms for this spatial- and species-specific localization have not been fully elucidated. For example, despite abundant retinal levels in mice and primates of a binding protein for zeaxanthin and meso-zeaxanthin, the pi isoform of glutathione S-transferase (GSTP1), only human and monkey retinas naturally contain detectable levels of these carotenoids. We therefore investigated whether or not differences in expression, localization, and activity between mouse and primate carotenoid metabolic enzymes could account for this species-specific difference in retinal accumulation. We focused on β,β-carotene-9′,10′-dioxygenase (BCO2, also known as BCDO2), the only known mammalian xanthophyll cleavage enzyme. RT-PCR, Western blot analysis, and immunohistochemistry (IHC) confirmed that BCO2 is expressed in both mouse and primate retinas. Cotransfection of expression plasmids of human or mouse BCO2 into Escherichia coli strains engineered to produce zeaxanthin demonstrated that only mouse BCO2 is an active zeaxanthin cleavage enzyme. Surface plasmon resonance (SPR) binding studies showed that the binding affinities between human BCO2 and lutein, zeaxanthin, and meso-zeaxanthin are 10- to 40-fold weaker than those for mouse BCO2, implying that ineffective capture of carotenoids by human BCO2 prevents cleavage of xanthophyll carotenoids. Moreover, BCO2 knockout mice, unlike WT mice, accumulate zeaxanthin in their retinas. Our results provide a novel explanation for how primates uniquely concentrate xanthophyll carotenoids at high levels in retinal tissue. PMID:24982131

  10. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells.

    PubMed

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  11. Retinal remodeling.

    PubMed

    Jones, B W; Kondo, M; Terasaki, H; Lin, Y; McCall, M; Marc, R E

    2012-07-01

    Retinal photoreceptor degeneration takes many forms. Mutations in rhodopsin genes or disorders of the retinal pigment epithelium, defects in the adenosine triphosphate binding cassette transporter, ABCR gene defects, receptor tyrosine kinase defects, ciliopathies and transport defects, defects in both transducin and arrestin, defects in rod cyclic guanosine 3',5'-monophosphate phosphodiesterase, peripherin defects, defects in metabotropic glutamate receptors, synthetic enzymatic defects, defects in genes associated with signaling, and many more can all result in retinal degenerative disease like retinitis pigmentosa (RP) or RP-like disorders. Age-related macular degeneration (AMD) and AMD-like disorders are possibly due to a constellation of potential gene targets and gene/gene interactions, while other defects result in diabetic retinopathy or glaucoma. However, all of these insults as well as traumatic insults to the retina result in retinal remodeling. Retinal remodeling is a universal finding subsequent to retinal degenerative disease that results in deafferentation of the neural retina from photoreceptor input as downstream neuronal elements respond to loss of input with negative plasticity. This negative plasticity is not passive in the face of photoreceptor degeneration, with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels involving all cell classes in the retina, including neurons and glia. Retinal remodeling has direct implications for the rescue of vision loss through bionic or biological approaches, as circuit revision in the retina corrupts any potential surrogate photoreceptor input to a remnant neural retina. However, there are a number of potential opportunities for intervention that are revealed through the study of retinal remodeling, including therapies that are designed to slow down photoreceptor loss, interventions that are designed to limit or arrest remodeling events, and

  12. Lack of Correlation Between the Spatial Distribution of A2E and Lipofuscin Fluorescence in the Human Retinal Pigment Epithelium

    PubMed Central

    Ablonczy, Zsolt; Higbee, Daniel; Anderson, David M.; Dahrouj, Mohammad; Grey, Angus C.; Gutierrez, Danielle; Koutalos, Yiannis; Schey, Kevin L.; Hanneken, Anne; Crouch, Rosalie K.

    2013-01-01

    Purpose. The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. Methods. Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. Results. Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. Conclusions. This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging. PMID:23847313

  13. 48 CFR 252.235-7004 - Protection of Human Subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... requirements of 32 CFR Part 219, as well as the terms of the assurance, which the Human Research Protection Official determines to be appropriate for the research supported by the Department of Defense (DoD) component (32 CFR 219.103). (2) Human Research Protection Official (HRPO) means the individual designated...

  14. 48 CFR 252.235-7004 - Protection of Human Subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... requirements of 32 CFR Part 219, as well as the terms of the assurance, which the Human Research Protection Official determines to be appropriate for the research supported by the Department of Defense (DoD) component (32 CFR 219.103). (2) Human Research Protection Official (HRPO) means the individual designated...

  15. 48 CFR 252.235-7004 - Protection of Human Subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... requirements of 32 CFR Part 219, as well as the terms of the assurance, which the Human Research Protection Official determines to be appropriate for the research supported by the Department of Defense (DoD) component (32 CFR 219.103). (2) Human Research Protection Official (HRPO) means the individual designated...

  16. Protection of Human Subjects: A Primer for the New Administrator.

    ERIC Educational Resources Information Center

    Steinert, Bruce W.

    2002-01-01

    Discusses clinical trials in general, protecting human research subjects, and the role the research administrator can play in facilitating the process. The discussion is not intended to be a thorough treatise on clinical trials, but is an overview of new and lesser known human protection issues that are better handled prospectively. (EV)

  17. Long-Term Protection of Retinal Ganglion Cells and Visual Function by Brain-Derived Neurotrophic Factor in Mice With Ocular Hypertension

    PubMed Central

    Feng, Liang; Chen, Hui; Yi, Ji; Troy, John B.; Zhang, Hao F.; Liu, Xiaorong

    2016-01-01

    Purpose Glaucoma, frequently associated with elevated intraocular pressure (IOP), is characterized by progressive retinal ganglion cell (RGC) death and vision loss. Brain-derived neurotrophic factor (BDNF) has been studied as a candidate for neuroprotection in rodent models of experimental glaucoma, yet it remains to be determined whether BDNF exerts long-term protection for subtype RGCs and vision against chronic IOP elevation. Methods We induced modest and sustained IOP elevation by laser illumination and microbead injection in mice. Using a tamoxifen-induced Cre recombinase system, BDNF was upregulated in the mouse retina when sustained IOP elevation was induced. We then examined whether overexpression of BDNF protected RGCs and vision during the period of ocular hypertension. Given that BDNF modulates axon growth and dendritic formation in a subtype-dependent manner, we tested whether BDNF protects RGC dendritic structure against the hypertensive insult also in a subtype-dependent manner. Results Sustained IOP elevation was induced and lasted up to 6 months. Overexpression of BDNF delayed progressive RGC and axon loss in hypertensive eyes. Brain-derived neurotrophic factor overexpression also helped to preserve acuity against the chronic hypertensive insult. We classified RGCs into ON and ON–OFF subtypes based on their dendritic lamination pattern in the inner plexiform layer and found that BDNF prevented ON–RGC dendritic degeneration in mice with sustained ocular hypertension. Conclusions Our data demonstrated that BDNF can protect the dendritic fields of ON RGCs and reduce RGC and vision loss in mice with sustained ocular hypertension. PMID:27421068

  18. Neutron effects in humans: protection considerations

    SciTech Connect

    Fry, R.J.M.

    1985-01-01

    Committee I of the International Commission on Radiological Protection has recommended that the Quality Factor for neutrons should be changed from 10 to 20. This article is an interesting recount of the tale of Q from the viewpoint of an observer which illustrates many of the problems that the selection of protection standards pose. 32 refs., 5 tabs.

  19. Retinoids and Retinal Diseases

    PubMed Central

    Kiser, Philip D.; Palczewski, Krzysztof

    2016-01-01

    Recent progress in molecular understanding of the retinoid cycle in mammalian retina stems from painstaking biochemical reconstitution studies supported by natural or engineered animal models with known genetic lesions and studies of humans with specific genetic blinding diseases. Structural and membrane biology have been used to detect critical retinal enzymes and proteins and their substrates and ligands, placing them in a cellular context. These studies have been supplemented by analytical chemistry methods that have identified small molecules by their spectral characteristics, often in conjunction with the evaluation of models of animal retinal disease. It is from this background that rational therapeutic interventions to correct genetic defects or environmental insults are identified. Thus, most presently accepted modulators of the retinoid cycle already have demonstrated promising results in animal models of retinal degeneration. These encouraging signs indicate that some human blinding diseases can be alleviated by pharmacological interventions. PMID:27917399

  20. In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter

    PubMed Central

    Luz-Madrigal, Agustín; Clapp, Carmen; Aranda, Jorge; Vaca, Luis

    2007-01-01

    Background Endothelial cells are a target for gene therapy because they are implicated in a number of vascular diseases. Recombinant baculovirus have emerged as novel gene delivery vectors. However, there is no information available concerning the use of endothelial-specific promoters in the context of the baculovirus genome. In the present study, we have generated a recombinant baculovirus containing the human flt-1 promoter (BacFLT-GFP) driving the expression of the green fluorescent protein. Transcriptional gene targeting was analyzed in vitro in different mammalian cell lines and in vivo in adult rat retinal vasculature. Results BacFLT-GFP evoked the highest levels of expression in the endothelial cell line BUVEC-E6E7-1, similar to those reached by recombinant baculovirus carrying the CMV promoter (112% relative to BacCMV-GFP, n = 4). Interestingly, BacFLT-GFP directed high levels of expression in rat glioma C6 and in human glioblastoma CH235 cells (34.78% and 47.86% relative to BacCMV-GFP, respectively). Histone deacetylase inhibitors such as butyrate or trichostatin A enhanced the transcriptional activity of both BacCMV-GFP and BacFLT-GFP. Thus, in this study histone deacetylation appears to be a central mechanism for the silencing of baculovirus, independently of the promoter utilized. In vivo transcriptional targeting was demonstrated in adult rat retinal vasculature by intravitreal delivery of BacFLT-GFP and immunohistochemical staining with von Willebrand factor (vWF). Analysis by fluorescence microscopy and deconvolved three-dimensional confocal microscopy of retinal whole mounts obtained after 3 days of baculovirus injection showed that most GFP-expressing cells localized to the inner limiting membrane (ILM) and ganglion cell layer (GCL) and colocalize with vWF (70%, n = 10) in blood vessels, confirming the endothelial phenotype of the transduced cells. Conclusion Taken together, our results indicate that the restricted expression in endothelial cells

  1. Effects of increasing numbers of phagocytic inclusions on human retinal pigment epithelial cells in culture: a model for aging.

    PubMed Central

    Boulton, M; Marshall, J

    1986-01-01

    Cultures of human retinal pigment epithelial cells have been challenged with a number of biological (lipofuscin, melanin, and rod outer segments) and non-biological (latex microspheres) particles at a variety of concentrations. The particles were chosen to include examples of both degradable and non-degradable systems. A range of morphological changes were observed by phase contrast microscopy, and these became more atypical with increasing concentration. At the highest concentration cells had ingested so many particles that many had died and others had ruptured. The time course of these changes indicated a relationship between cellular lytic activity and the capacity of the particle to degrade. The potential of this system as a model for studying senescence is discussed. Images PMID:3790481

  2. Overexpression of Brain-Derived Neurotrophic Factor Protects Large Retinal Ganglion Cells After Optic Nerve Crush in Mice

    PubMed Central

    Chen, Hui; Liang, Peiji; Troy, John B.

    2017-01-01

    Abstract Brain-derived neurotrophic factor (BDNF), a neurotrophin essential for neuron survival and function, plays an important role in neuroprotection during neurodegenerative diseases. In this study, we examined whether a modest increase of retinal BDNF promotes retinal ganglion cell (RGC) survival after acute injury of the optic nerve in mice. We adopted an inducible Cre-recombinase transgenic system to up-regulate BDNF in the mouse retina and then examined RGC survival after optic nerve crush by in vivo imaging. We focused on one subtype of RGC with large soma expressing yellow fluorescent protein transgene that accounts for ∼11% of the total SMI-32–positive RGCs. The median survival time of this subgroup of SMI-32 cells was 1 week after nerve injury in control mice but 2 weeks when BDNF was up-regulated. Interestingly, we found that the survival time for RGCs taken as a whole was 2 weeks, suggesting that these large-soma RGCs are especially vulnerable to optic nerve crush injury. We also studied changes in axon number using confocal imaging, confirming first the progressive loss reported previously for wild-type mice and demonstrating that BDNF up-regulation extended axon survival. Together, our results demonstrate that the time course of RGC loss induced by optic nerve injury is type specific and that overexpression of BDNF prolongs the survival of one subgroup of SMI-32–positive RGCs. PMID:28101532

  3. PINK1 and Parkin cooperatively protect neurons against constitutively active TRP channel-induced retinal degeneration in Drosophila.

    PubMed

    Huang, Z; Ren, S; Jiang, Y; Wang, T

    2016-04-07

    Calcium has an important role in regulating numerous cellular activities. However, extremely high levels of intracellular calcium can lead to neurotoxicity, a process commonly associated with degenerative diseases. Despite the clear role of calcium cytotoxicity in mediating neuronal cell death in this context, the pathological mechanisms remain controversial. We used a well-established Drosophila model of retinal degeneration, which involves the constitutively active TRP(P365) channels, to study calcium-induced neurotoxicity. We found that the disruption of mitochondrial function was associated with the degenerative process. Further, increasing autophagy flux prevented cell death in Trp(P365) mutant flies, and this depended on the PINK1/Parkin pathway. In addition, the retinal degeneration process was also suppressed by the coexpression of PINK1 and Parkin. Our results provide genetic evidence that mitochondrial dysfunction has a key role in the pathology of cellular calcium neurotoxicity. In addition, the results demonstrated that maintaining mitochondrial homeostasis via PINK1/Parkin-dependent mitochondrial quality control can potentially alleviate cell death in a wide range of neurodegenerative diseases.

  4. Research resource: nuclear receptor atlas of human retinal pigment epithelial cells: potential relevance to age-related macular degeneration.

    PubMed

    Dwyer, Mary A; Kazmin, Dmitri; Hu, Peng; McDonnell, Donald P; Malek, Goldis

    2011-02-01

    Retinal pigment epithelial (RPE) cells play a vital role in retinal physiology by forming the outer blood-retina barrier and supporting photoreceptor function. Retinopathies including age-related macular degeneration (AMD) involve physiological and pathological changes in the epithelium, severely impairing the retina and effecting vision. Nuclear receptors (NRs), including peroxisome proliferator-activated receptor and liver X receptor, have been identified as key regulators of physiological pathways such as lipid metabolic dysregulation and inflammation, pathways that may also be involved in development of AMD. However, the expression levels of NRs in RPE cells have yet to be systematically surveyed. Furthermore, cell culture lines are widely used to study the biology of RPE cells, without knowledge of the differences or similarities in NR expression and activity between these in vitro models and in vivo RPE. Using quantitative real-time PCR, we assessed the expression patterns of all 48 members of the NR family plus aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in human RPE cells. We profiled freshly isolated cells from donor eyes (in vivo), a spontaneously arising human cell line (in vitro), and primary cell culture lines (in vitro) to determine the extent to which NR expression in the cultured cell lines reflects that of in vivo. To evaluate the validity of using cell culture models for investigating NR receptor biology, we determined transcriptional activity and target gene expression of several moderately and highly expressed NRs in vitro. Finally, we identified a subset of NRs that may play an important role in pathobiology of AMD.

  5. Tunicamycin-induced Endoplasmic Reticulum Stress Upregulates the Expression of Pentraxin 3 in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Hwang, Narae; Kwon, Min-Young; Cha, Jae Bong; Chung, Su Wol

    2016-01-01

    Purpose To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. Methods PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. Results The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. Conclusions These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated. PMID:27980366

  6. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    PubMed Central

    Bennis, Anna; Gorgels, Theo G. M. F.; ten Brink, Jacoline B.; van der Spek, Peter J.; Bossers, Koen; Heine, Vivi M.; Bergen, Arthur A.

    2015-01-01

    Background The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE. Methods We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE. Results We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier. Conclusion These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular

  7. Retinal Prosthesis

    PubMed Central

    Weiland, James D.; Humayun, Mark S.

    2015-01-01

    Retinal prosthesis have been translated from the laboratory to the clinical over the past two decades. Currently, two devices have regulatory approval for the treatment of retinitis pigmentosa. These devices provide partial sight restoration and patients use this improved vision in their everyday lives. Improved mobility and object detection are some of the more notable findings from the clinical trials. However, significant vision restoration will require both better technology and improved understanding of the interaction between electrical stimulation and the retina. This paper reviews the recent clinical trials, highlights technology breakthroughs that will contribute to next generation of retinal prostheses. PMID:24710817

  8. Combined effect of wavelength and polarization in double-pass retinal images in the human eye.

    PubMed

    Bueno, Juan M; Pérez, Guillermo M

    2010-11-23

    A polychromatic double-pass setup was developed to study the effects of wavelength and polarization on retinal image quality. The results show that the central part of the images was similar for all wavelengths (543, 633 and 780 nm) and polarization states. However, the image tails increased significantly when using infrared light for all the polarization states used. For the set of subjects involved in the study, ocular diattenuation presented individual differences, however significant changes were not found across the different wavelengths. Moreover the Stokes vectors providing the maximum intensity transmittance varied across subjects and corresponded to elliptically polarized light. These non-negligible diattenuation effects might affect the performance of clinical devices which only take into account ocular birefringence.

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

    SciTech Connect

    Bridges, Christy C. . E-mail: bridges_cc@mercer.edu; Battle, Jamie R.; Zalups, Rudolfs K.

    2007-06-01

    Inorganic mercury (Hg{sup 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{sup 2+} exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg{sup 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{sup 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{sup 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{sup 2+}, was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg{sup 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{sup +}-dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B{sup 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{sup 0,+} and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury.

  10. Retinal Detachment

    MedlinePlus

    ... immediately. Treatment How is retinal detachment treated? Small holes and tears are treated with laser surgery or ... laser surgery tiny burns are made around the hole to “weld” the retina back into place. Cryopexy ...

  11. Retinal Disorders

    MedlinePlus

    ... be serious enough to cause blindness. Examples are Macular degeneration - a disease that destroys your sharp, central vision Diabetic eye disease Retinal detachment - a medical emergency, when the retina is ... children. Macular pucker - scar tissue on the macula Macular hole - ...

  12. Glycoconjugates in human milk: protecting infants from disease.

    PubMed

    Peterson, Robyn; Cheah, Wai Yuen; Grinyer, Jasmine; Packer, Nicolle

    2013-12-01

    Breastfeeding is known to have many health benefits for a newborn. Not only does human milk provide an excellent source of nutrition, it also contains components that protect against infection from a wide range of pathogens. Some of the protective properties of human milk can be attributed to the immunoglobulins. Yet, there is another level of defense provided by the "sweet" protective agents that human milk contains, including free oligosaccharides, glycoproteins and glycolipids. Sugar epitopes in human milk are similar to the glycan receptors that serve as pathogen adhesion sites in the human gastrointestinal tract and other epithelial cell surfaces; hence, the milk glycans can competitively bind to and remove the disease-causing microorganisms before they cause infection. The protective value of free oligosaccharides in human milk has been well researched and documented. Human milk glycoconjugates have received less attention but appear to play an equally important role. Here, we bring together the breadth of research that has focused on the protective mechanisms of human milk glycoconjugates, with a particular focus on the glycan moieties that may play a role in disease prevention. In addition, human milk glycoconjugates are compared with bovine milk glycoconjugates in terms of their health benefits for the human infant.

  13. 34 CFR 76.681 - Protection of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Protection of human subjects. 76.681 Section 76.681 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must... of human subjects. If a State or a subgrantee uses a human subject in a research project, the...

  14. 34 CFR 76.681 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 1 2013-07-01 2013-07-01 false Protection of human subjects. 76.681 Section 76.681 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must... of human subjects. If a State or a subgrantee uses a human subject in a research project, the...

  15. 34 CFR 76.681 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Protection of human subjects. 76.681 Section 76.681 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must... of human subjects. If a State or a subgrantee uses a human subject in a research project, the...

  16. 34 CFR 76.681 - Protection of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Protection of human subjects. 76.681 Section 76.681 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must... of human subjects. If a State or a subgrantee uses a human subject in a research project, the...

  17. 34 CFR 76.681 - Protection of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Protection of human subjects. 76.681 Section 76.681 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must... of human subjects. If a State or a subgrantee uses a human subject in a research project, the...

  18. Pathway to Retinal Oximetry

    PubMed Central

    Beach, James

    2014-01-01

    Events and discoveries in oxygen monitoring over the past two centuries are presented as the background from which oximetry of the human retina evolved. Achievements and the people behind them are discussed, showing parallels between the work in tissue measurements and later in the eye. Developments in the two-wavelength technique for oxygen saturation measurements in retinal vessels are shown to exploit the forms of imaging technology available over time. The last section provides a short summary of the recent research in retinal diseases using vessel oximetry. PMID:25237591

  19. Pathway to Retinal Oximetry.

    PubMed

    Beach, James

    2014-09-01

    Events and discoveries in oxygen monitoring over the past two centuries are presented as the background from which oximetry of the human retina evolved. Achievements and the people behind them are discussed, showing parallels between the work in tissue measurements and later in the eye. Developments in the two-wavelength technique for oxygen saturation measurements in retinal vessels are shown to exploit the forms of imaging technology available over time. The last section provides a short summary of the recent research in retinal diseases using vessel oximetry.

  20. Misfolded Proteins and Retinal Dystrophies

    PubMed Central

    Lin, Jonathan H.; LaVail, Matthew M.

    2010-01-01

    Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death by activating the Unfolded Protein Response, a set of conserved intracellular signaling pathways that detect protein misfolding within the endoplasmic reticulum and control protective and proapoptotic signal transduction pathways. Here, we review the misfolded proteins associated with select types of retinitis pigmentosa, Stargadt-like macular degeneration, and Doyne Honeycomb Retinal Dystrophy and discuss the role that endoplasmic reticulum stress and UPR signaling play in their pathogenesis. Last, we review new therapies for these diseases based on preventing protein misfolding in the retina. PMID:20238009

  1. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

    SciTech Connect

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  2. Evidence of Flicker-Induced Functional Hyperaemia in the Smallest Vessels of the Human Retinal Blood Supply

    PubMed Central

    Bui, Bang V.

    2016-01-01

    Regional changes in blood flow are initiated within neural tissue to help fuel local differences in neural activity. Classically, this response was thought to arise only in larger arterioles and venules. However, recently, it has been proposed that a) the smallest vessels of the circulation make a comparable contribution, and b) the response should be localised intermittently along such vessels, due to the known distribution of contractile mural cells. To assess these hypotheses in human neural tissue in vivo, we imaged the retinal microvasculature (diameters 3–28 μm) non-invasively, using adaptive optics, before and after delivery of focal (360 μm) patches of flickering visible light. Our results demonstrated a definite average response in 35% of all vessel segments analysed. In these responding vessels, the magnitude of proportional dilation (mean ± SEM for pre-capillary arterioles 13 ± 5%, capillaries 31 ± 8%, and post-capillary venules 10 ± 3%) is generally far greater than the magnitudes we and others have measured in the larger retinal vessels, supporting proposition a) above. The dilations observed in venules were unexpected based on previous animal work, and may be attributed either to differences in stimulus or species. Response heterogeneity across the network was high; responses were also heterogeneous along individual vessels (45% of vessel segments showed demonstrable locality in their response). These observations support proposition b) above. We also observed a definite average constriction across 7% of vessel segments (mean ± SEM constriction for capillaries -16 ± 3.2%, and post-capillary venules -18 ± 12%), which paints a picture of dynamic redistribution of flow throughout the smallest vessel networks in the retina in response to local, stimulus-driven metabolic demand. PMID:27617960

  3. Bioelectronic retinal prosthesis

    NASA Astrophysics Data System (ADS)

    Weiland, James D.

    2016-05-01

    Retinal prosthesis have been translated to clinical use over the past two decades. Currently, two devices have regulatory approval for the treatment of retinitis pigmentosa and one device is in clinical trials for treatment of age-related macular degeneration. These devices provide partial sight restoration and patients use this improved vision in their everyday lives to navigate and to detect large objects. However, significant vision restoration will require both better technology and improved understanding of the interaction between electrical stimulation and the retina. In particular, current retinal prostheses do not provide peripheral visions due to technical and surgical limitations, thus limiting the effectiveness of the treatment. This paper reviews recent results from human implant patients and presents technical approaches for peripheral vision.

  4. Photoreceptor Rescue by an Abbreviated Human RPGR Gene in a Murine Model of X-linked Retinitis Pigmentosa

    PubMed Central

    Pawlyk, Basil S.; Adamian, Michael; Sun, Xun; Bulgakov, Oleg V.; Shu, Xinhua; Smith, Alexander J.; Berson, Eliot L.; Ali, Robin R.; Khani, Shahrokh; F.Wright, Alan; Sandberg, Michael A.; Li, Tiansen

    2015-01-01

    The X-linked RP3 gene codes for the ciliary protein RPGR and accounts for over 10% of inherited retinal degenerations. The critical RPGR-ORF15 splice variant contains a highly repetitive purine-rich linker region that renders it unstable and difficult to adapt for gene therapy. To test the hypothesis that the precise length of the linker region is not critical for function, we evaluated whether AAV-mediated replacement gene therapy with a human ORF15 variant containing in-frame shortening of the linker region could reconstitute RPGR function in vivo. We delivered human RPGR-ORF15 replacement genes with deletion of most (314-codons, “short form”) or 1/3 (126-codons, “long form”) of the linker region to Rpgr null mice. Human RPGR-ORF15 expression was detected post-treatment with both forms of ORF15 transgenes. However, only the long form correctly localized to the connecting cilia and led to significant functional and morphological rescue of rods and cones. Thus the highly repetitive region of RPGR is functionally important but that moderate shortening of its length, which confers the advantage of added stability, preserves its function. These findings provide a theoretical basis for optimizing replacement gene design in clinical trials for X-linked RP3. PMID:26348595

  5. 48 CFR 1552.223-70 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Contractor shall protect the rights and welfare of human subjects in accordance with the procedures specified... Contractor shall bear full responsibility for the proper and safe performance of all work and...

  6. 48 CFR 1552.223-70 - Protection of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Contractor shall protect the rights and welfare of human subjects in accordance with the procedures specified... Contractor shall bear full responsibility for the proper and safe performance of all work and...

  7. Phobos and Deimos: Planetary Protection Knowledge Gaps for Human Missions

    NASA Astrophysics Data System (ADS)

    Lee, P.; Lorber, K.

    2015-03-01

    Phobos and Deimos, Mars’ two moons, are associated with significant planetary protection knowledge gaps for human missions, that may be filled by a low cost robotic reconnaissance mission focused on elucidating their origin and volatile content.

  8. 48 CFR 1523.303-70 - Protection of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Protection of human subjects. 1523.303-70 Section 1523.303-70 Federal Acquisition Regulations System ENVIRONMENTAL PROTECTION AGENCY SOCIOECONOMIC PROGRAMS ENVIRONMENTAL, CONSERVATION, OCCUPATIONAL SAFETY, AND DRUG-FREE...

  9. 48 CFR 1523.303-70 - Protection of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Protection of human subjects. 1523.303-70 Section 1523.303-70 Federal Acquisition Regulations System ENVIRONMENTAL PROTECTION AGENCY SOCIOECONOMIC PROGRAMS ENVIRONMENTAL, CONSERVATION, OCCUPATIONAL SAFETY, AND DRUG-FREE...

  10. 48 CFR 1523.303-70 - Protection of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Protection of human subjects. 1523.303-70 Section 1523.303-70 Federal Acquisition Regulations System ENVIRONMENTAL PROTECTION AGENCY SOCIOECONOMIC PROGRAMS ENVIRONMENTAL, CONSERVATION, OCCUPATIONAL SAFETY, AND DRUG-FREE...

  11. Method and system for the diagnosis of disease using retinal image content and an archive of diagnosed human patient data

    DOEpatents

    Tobin, Kenneth W; Karnowski, Thomas P; Chaum, Edward

    2013-08-06

    A method for diagnosing diseases having retinal manifestations including retinal pathologies includes the steps of providing a CBIR system including an archive of stored digital retinal photography images and diagnosed patient data corresponding to the retinal photography images, the stored images each indexed in a CBIR database using a plurality of feature vectors, the feature vectors corresponding to distinct descriptive characteristics of the stored images. A query image of the retina of a patient is obtained. Using image processing, regions or structures in the query image are identified. The regions or structures are then described using the plurality of feature vectors. At least one relevant stored image from the archive based on similarity to the regions or structures is retrieved, and an eye disease or a disease having retinal manifestations in the patient is diagnosed based on the diagnosed patient data associated with the relevant stored image(s).

  12. Federal Policy for the Protection of Human Subjects. Final rule.

    PubMed

    2017-01-19

    The departments and agencies listed in this document announce revisions to modernize, strengthen, and make more effective the Federal Policy for the Protection of Human Subjects that was originally promulgated as a Common Rule in 1991. This final rule is intended to better protect human subjects involved in research, while facilitating valuable research and reducing burden, delay, and ambiguity for investigators. These revisions are an effort to modernize, simplify, and enhance the current system of oversight.

  13. Optimization of In Vivo Confocal Autofluorescence Imaging of the Ocular Fundus in Mice and Its Application to Models of Human Retinal Degeneration

    PubMed Central

    Issa, Peter Charbel; Singh, Mandeep S.; Lipinski, Daniel M.; Chong, Ngaihang V.; Delori, François C.; Barnard, Alun R.; MacLaren, Robert E.

    2012-01-01

    Purpose. To investigate the feasibility and to identify sources of experimental variability of quantitative and qualitative fundus autofluorescence (AF) assessment in mice. Methods. Blue (488 nm) and near-infrared (790 nm) fundus AF imaging was performed in various mouse strains and disease models (129S2, C57Bl/6, Abca4−/−, C3H-Pde6brd1/rd1, Rho−/−, and BALB/c mice) using a commercially available scanning laser ophthalmoscope. Gray-level analysis was used to explore factors influencing fundus AF measurements. Results. A contact lens avoided cataract development and resulted in consistent fundus AF recordings. Fundus illumination and magnification were sensitive to changes of the camera position. Standardized adjustment of the recorded confocal plane and consideration of the pupil area allowed reproducible recording of fundus AF from the retinal pigment epithelium with an intersession coefficient of repeatability of ±22%. Photopigment bleaching occurred during the first 1.5 seconds of exposure to 488 nm blue light (∼10 mW/cm2), resulting in an increase of fundus AF. In addition, there was a slight decrease in fundus AF during prolonged blue light exposure. Fundus AF at 488 nm was low in animals with an absence of a normal visual cycle, and high in BALB/c and Abca4−/− mice. Degenerative alterations in Pde6brd1/rd1 and Rho−/− were reminiscent of findings in human retinal disease. Conclusions. Investigation of retinal phenotypes in mice is possible in vivo using standardized fundus AF imaging. Correlation with postmortem analysis is likely to lead to further understanding of human disease phenotypes and of retinal degenerations in general. Fundus AF imaging may be useful as an outcome measure in preclinical trials, such as for monitoring effects aimed at lowering lipofuscin accumulation in the retinal pigment epithelium. PMID:22169101

  14. Age-Related Susceptibility to Apoptosis in Human Retinal Pigment Epithelial Cells Is Triggered by Disruption of p53–Mdm2 Association

    PubMed Central

    Bhattacharya, Sujoy; Chaum, Edward; Johnson, Dianna A.; Johnson, Leonard R.

    2012-01-01

    Purpose. Relatively little is known about the contribution of p53/Mdm2 pathway in apoptosis of retinal pigment epithelial (RPE) cells or its possible link to dysfunction of aging RPE or to related blinding disorders such as age-related macular degeneration (AMD). Methods. Age-associated changes in p53 activation were evaluated in primary RPE cultures from human donor eyes of various ages. Apoptosis was evaluated by activation of caspases and DNA fragmentation. Gene-specific small interfering RNA was used to knock down expression of p53. Results. We observed that the basal rate of p53-dependent apoptosis increased in an age-dependent manner in human RPE. The age-dependent increase in apoptosis was linked to alterations in several aspects of the p53 pathway. p53 phosphorylation Ser15 was increased through the stimulation of ATM-Ser1981. p53 acetylation Lys379 was increased through the inhibition of SIRT1/2. These two posttranslational modifications of p53 blocked the sequestration of p53 by Mdm2, thus resulting in an increase in free p53 and of p53 stimulation of apoptosis through increased expression of PUMA (p53 upregulated modulator of apoptosis) and activation of caspase-3. Aged RPE also had reduced expression of antiapoptotic Bcl-2, which contributed to the increase in apoptosis. Of particular interest in these studies was that pharmacologic treatments to block p53 phosphorylation, acetylation, or expression were able to protect RPE cells from apoptosis. Conclusions. Our studies suggest that aging in the RPE leads to alterations of specific checkpoints in the apoptotic pathway, which may represent important molecular targets for the treatment of RPE-related aging disorders such as AMD. PMID:23139272

  15. Thermal elevation in the human eye and head due to the operation of a retinal prosthesis.

    PubMed

    Gosalia, Keyoor; Weiland, James; Humayun, Mark; Lazzi, Gianluca

    2004-08-01

    An explicit finite-difference time-domain formulation of the bio-heat equation is employed with a three-dimensional head eye model to evaluate the temperature increase in the eye and surrounding head tissues due to the operation of the implanted stimulator IC chip of a retinal prosthesis designed to restore partial vision to the blind. As a first step, a validation of the thermal model and method used is carried out by comparison with in vivo measurements of intraocular heating performed in the eyes of dogs. Induced temperature increase in the eye and surrounding tissues is then estimated for several different operational conditions of the implanted chip. In the vitreous cavity, temperature elevation of 0.26 degrees C is observed after 26 min for a chip dissipating 12.4 mW when positioned in the mid-vitreous cavity while it is 0.16 degrees C when the chip is positioned in the anterior portion between the eye's ciliary muscles. Corresponding temperature rises observed on chip are 0.82 degrees C for both the positions of the chip. A comprehensive account of temperature elevations in different tissues under different operational conditions is presented.

  16. Retinal nerve fiber bundle tracing and analysis in human eye by polarization sensitive OCT.

    PubMed

    Sugita, Mitsuro; Pircher, Michael; Zotter, Stefan; Baumann, Bernhard; Roberts, Philipp; Makihira, Tomoyuki; Tomatsu, Nobuhiro; Sato, Makoto; Vass, Clemens; Hitzenberger, Christoph K

    2015-03-01

    We present a new semi-automatic processing method for retinal nerve fiber bundle tracing based on polarization sensitive optical coherence tomography (PS-OCT) data sets. The method for tracing is based on a nerve fiber orientation map that covers the fovea and optic nerve head (ONH) regions. In order to generate the orientation map, two types of information are used: optic axis orientation based on polarization data, and complementary information obtained from nerve fiber layer (NFL) local thickness variation to reveal fiber bundle structures around the fovea. The corresponding two orientation maps are fused into a combined fiber orientation map. En face maps of NFL retardation, thickness, and unit-depth-retardation (UDR, equivalent to birefringence) are transformed into "along-trace" maps by using the obtained traces of the nerve fiber bundles. The method is demonstrated in the eyes of healthy volunteers, and as an example of further analyses utilizing this method, maps illustrating the gradients of NFL retardation, thickness, and UDR are demonstrated.

  17. Inhibition of DNA methyltransferase or histone deacetylase protects retinal pigment epithelial cells from DNA damage induced by oxidative stress by the stimulation of antioxidant enzymes.

    PubMed

    Tokarz, Paulina; Kaarniranta, Kai; Blasiak, Janusz

    2016-04-05

    Epigenetic modifications influence DNA damage response (DDR). In this study we explored the role of DNA methylation and histone acetylation in DDR in cells challenged with acute or chronic oxidative stress. We used retinal pigment epithelial cells (ARPE-19), which natively are exposed to oxidative stress due to permanent exposure to light and high blood flow. We employed a DNA methyltransferase inhibitor - RG108 (RG), or a histone deacetylase inhibitor - valproic acid (VA). ARPE-19 cells were exposed to tert-butyl hydroperoxide, an acute oxidative stress inducer, or glucose oxidase, which slowly liberates low-doses of hydrogen peroxide in the presence of glucose, creating chronic conditions. VA and RG reduced level of intracellular reactive oxygen species and DNA damage in ARPE-19 cells in normal condition and in oxidative stress. This protective effect of VA and RG was associated with the up-regulated expression of antioxidant enzyme genes: CAT, GPx1, GPx4, SOD1 and SOD2. RG decreased the number of cells in G2/M checkpoint in response to chronic oxidative stress. Neither RG nor VA changed the DNA repair or apoptosis induced by oxidative stress. Therefore, certain epigenetic manipulations may protect ARPE-19 cells from detrimental effects of oxidative stress by modulation of antioxidative enzyme gene expression, which may be further explored in pharmacological studies on oxidative stress-related eye diseases.

  18. Dose-Dependent Protective Effect of Lithium Chloride on Retinal Ganglion Cells Is Interrelated with an Upregulated Intraretinal BDNF after Optic Nerve Transection in Adult Rats

    PubMed Central

    Wu, Ming-Mei; Zhu, Ting-Ting; Wang, Peng; Kuang, Fang; Hao, Ding-Jun; You, Si-Wei; Li, Yao-Yu

    2014-01-01

    Neuroprotection of lithium for axotomized retinal ganglion cells (RGCs) is attributed to upregulated intraretinal Bcl-2. As lithium also upregulates brain-derived neurotrophic factor (BDNF) which can rescue axotomized RGCs, it is hypothesized that lithium could protect RGCs through BDNF. This study investigated this hypothesis and a possible relationship between the dose and protection of lithium. All adult experimental rats received daily intraperitoneal injections of lithium chloride (LiCl) at 30, 60 or 85 mg/kg·bw until they were euthanized 2, 7 or 14 days after left intraorbital optic nerve (ON) transection. Our results revealed that RGC densities promoted and declined with increased dose of LiCl and the highest RGC densities were always in the 60 mg/kg·bw LiCl group at both 7 and 14 day points. Similar promotion and decline in the mRNA and protein levels of intraretinal BDNF were also found at the 14 day point, while such BDNF levels increased in the 30 mg/kg·bw LiCl group but peaked in the 60 and 85 mg/kg·bw LiCl groups at the 7 day point. These findings suggested that lithium can delay the death of axotomized RGCs in a dose-dependent manner within a certain period after ON injury and such beneficial effect is interrelated with an upregulated level of intraretinal BDNF. PMID:25100168

  19. Reversal of the Caspase-Dependent Apoptotic Cytotoxicity Pathway by Taurine from Lycium barbarum (Goji Berry) in Human Retinal Pigment Epithelial Cells: Potential Benefit in Diabetic Retinopathy.

    PubMed

    Song, M K; Roufogalis, B D; Huang, T H W

    2012-01-01

    Diabetic retinopathy is a preventable microvascular diabetic complication and a leading cause of vision loss. Retinal pigment epithelial cell apoptosis is an early event in diabetic retinopathy. Taurine is reportedly beneficial for diabetic retinopathy and is abundant in the fruit of Lycium barbarum (LB). We have investigated the effect of pure taurine and an extract of LB rich in taurine on a model of diabetic retinopathy, the retinal ARPE-19 cell line exposed to high glucose. We demonstrate for the first time that LB extract and the active ligand, taurine, dose dependently enhance cell viability following high glucose treatment in the ARPE-19 retinal epithelial cell line. This cytoprotective effect was associated with the attenuation of high glucose-induced apoptosis, which was shown by characteristic morphological staining and the dose-dependent decrease in the number of apoptotic cells, determined by flow cytometry. Moreover, we have shown that LB extract and taurine dose dependently downregulate caspase-3 protein expression and the enzymatic activity of caspase-3. We conclude that taurine, a major component of LB, and the LB extract, have a cytoprotective effect against glucose exposure in a human retinal epithelial cell line and may provide useful approaches to delaying diabetic retinopathy progression.

  20. Reversal of the Caspase-Dependent Apoptotic Cytotoxicity Pathway by Taurine from Lycium barbarum (Goji Berry) in Human Retinal Pigment Epithelial Cells: Potential Benefit in Diabetic Retinopathy

    PubMed Central

    Song, M. K.; Roufogalis, B. D.; Huang, T. H. W.

    2012-01-01

    Diabetic retinopathy is a preventable microvascular diabetic complication and a leading cause of vision loss. Retinal pigment epithelial cell apoptosis is an early event in diabetic retinopathy. Taurine is reportedly beneficial for diabetic retinopathy and is abundant in the fruit of Lycium barbarum (LB). We have investigated the effect of pure taurine and an extract of LB rich in taurine on a model of diabetic retinopathy, the retinal ARPE-19 cell line exposed to high glucose. We demonstrate for the first time that LB extract and the active ligand, taurine, dose dependently enhance cell viability following high glucose treatment in the ARPE-19 retinal epithelial cell line. This cytoprotective effect was associated with the attenuation of high glucose-induced apoptosis, which was shown by characteristic morphological staining and the dose-dependent decrease in the number of apoptotic cells, determined by flow cytometry. Moreover, we have shown that LB extract and taurine dose dependently downregulate caspase-3 protein expression and the enzymatic activity of caspase-3. We conclude that taurine, a major component of LB, and the LB extract, have a cytoprotective effect against glucose exposure in a human retinal epithelial cell line and may provide useful approaches to delaying diabetic retinopathy progression. PMID:22567031

  1. DKK1 inhibits proliferation and migration in human retinal pigment epithelial cells via the Wnt/β-catenin signaling pathway.

    PubMed

    Zhou, Jinzi; Jiang, Jian; Wang, Shuhong; Xia, Xiaobo

    2016-08-01

    Retinal pigment epithelial (RPE) cells play important roles in diabetic retinopathy (DR). Dickkopf 1 (DKK1) has been reported to be important in the regulation of cell proliferation and migration. However, there are few previous studies regarding DKK1 in RPE cells. Therefore, in the present study, we investigated the effect of DKK1 on the proliferation and migration of human RPE cells, and the signaling mechanisms underlying these effects. The results showed that the overexpression of DKK1 significantly inhibited the proliferation and migration of ARPE-19 cells. In addition, overexpression of DKK1 markedly inhibited the expression of β-catenin and cyclin D1 in ARPE-19 cells. Collectively, the present findings suggest that the overexpression of DKK1 inhibited the proliferation and migration of RPE cells by suppressing the Wnt/β-catenin signaling pathway. Therefore, DKK1 are able to augment the growth of human RPE, and further studies are warranted to investigate the effects of DKK1 effects on DR.

  2. Dietary Compound Chrysin Inhibits Retinal Neovascularization with Abnormal Capillaries in db/db Mice

    PubMed Central

    Kang, Min-Kyung; Park, Sin-Hye; Kim, Yun-Ho; Lee, Eun-Jung; Antika, Lucia Dwi; Kim, Dong Yeon; Choi, Yean-Jung; Kang, Young-Hee

    2016-01-01

    Diabetic retinopathy (DR) develops in a significant proportion of patients with chronic diabetes, characterized by retinal macular edema and abnormal retinal vessel outgrowth leading to vision loss. Chrysin, a naturally-occurring flavonoid found in herb and honeycomb, has anti-inflammatory, antioxidant, and anti-cancer properties. This study sought to determine the protective effects of chrysin on retinal neovascularization with abnormal vessels and blood-retinal barrier (BRB) breakdown in 33 mM glucose-exposed human retinal endothelial cells and in db/db mouse eyes. High glucose caused retinal endothelial apoptotic injury, which was inhibited by submicromolar chrysin. This compound diminished the enhanced induction of HIF-1α, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (VEGFR2) in high glucose-exposed retinal endothelial cells. Consistently, oral administration of 10 mg/kg chrysin reduced the induction of these proteins in db/db mouse eye tissues. In addition, chrysin restored the decrement of VE-cadherin and ZO-1 junction proteins and PECAM-1 in hyperglycemia-stimulated retinal endothelial cells and diabetic mouse retina, possibly maintaining tight cell-cell interactions of endothelial cells and pericytes. Anti-apoptotic chrysin reduced the up-regulation of Ang-1, Ang-2, and Tie-2 crucial to retinal capillary occlusion and BRB permeability. Furthermore, orally treating chrysin inhibited acellular capillary formation, neovascularization, and vascular leakage observed in diabetic retinas. These observations demonstrate, for the first time, that chrysin had a capability to encumber diabetes-associated retinal neovascularization with microvascular abnormalities and BRB breakdown. PMID:27918469

  3. Does biodiversity protect humans against infectious disease?

    PubMed

    Wood, Chelsea L; Lafferty, Kevin D; DeLeo, Giulio; Young, Hillary S; Hudson, Peter J; Kuris, Armand M

    2014-04-01

    Control of human infectious disease has been promoted as a valuable ecosystem service arising from the conservation of biodiversity. There are two commonly discussed mechanisms by which biodiversity loss could increase rates of infectious disease in a landscape. First, loss of competitors or predators could facilitate an increase in the abundance of competent reservoir hosts. Second, biodiversity loss could disproportionately affect non-competent, or less competent reservoir hosts, which would otherwise interfere with pathogen transmission to human populations by, for example, wasting the bites of infected vectors. A negative association between biodiversity and disease risk, sometimes called the "dilution effect hypothesis," has been supported for a few disease agents, suggests an exciting win-win outcome for the environment and society, and has become a pervasive topic in the disease ecology literature. Case studies have been assembled to argue that the dilution effect is general across disease agents. Less touted are examples in which elevated biodiversity does not affect or increases infectious disease risk for pathogens of public health concern. In order to assess the likely generality of the dilution effect, we review the association between biodiversity and public health across a broad variety of human disease agents. Overall, we hypothesize that conditions for the dilution effect are unlikely to be met for most important diseases of humans. Biodiversity probably has little net effect on most human infectious diseases but, when it does have an effect, observation and basic logic suggest that biodiversity will be more likely to increase than to decrease infectious disease risk.

  4. Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

    PubMed Central

    Vainio, Iina; Abu Khamidakh, Amna; Paci, Michelangelo; Skottman, Heli; Juuti-Uusitalo, Kati; Hyttinen, Jari; Nymark, Soile

    2015-01-01

    Objective Computational models of calcium (Ca2+) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer. Methods We combined six essential Ca2+ signaling components into a model: stretch-sensitive Ca2+ channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked. Results Our model was able to explain Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance. Conclusions The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions. PMID:26070134

  5. Retine revisited.

    PubMed

    Douglas, D E

    2002-10-01

    Retine, so named by Albert Szent-Györgyi, an inhibitor of the growth of transplanted malignant tumours in animals, is present in all mammalian tissues and in urine. Its inhibitory activity was extensively investigated by Szent-Györgyi, but its exact chemical identity was not determined. Details of the reported physical and chemical properties of retine and its ubiquitous occurrence identify it as being identical to a complex mixture of lipid 2,4-diketones of similar ubiquitous occurrence. This lipid mixture has been extensively studied, and individual members have been synthesized.

  6. Hereditary Retinal Dystrophy.

    PubMed

    Hohman, Thomas C

    2016-12-30

    As our understanding of the genetic basis for inherited retinal disease has expanded, gene therapy has advanced into clinical development. When the gene mutations associated with inherited retinal dystrophies were identified, it became possible to create animal models in which individual gene were altered to match the human mutations. The retina of these animals were then characterized to assess whether the mutated genes produced retinal phenotypes characteristic of disease-affected patients. Following the identification of a subpopulation of patients with the affected gene and the development of techniques for the viral gene transduction of retinal cells, it has become possible to deliver a copy of the normal gene into the retinal sites of the mutated genes. When this was performed in animal models of monogenic diseases, at an early stage of retinal degeneration when the affected cells remained viable, successful gene augmentation corrected the structural and functional lesions characteristic of the specific diseases in the areas of the retina that were successfully transduced. These studies provided the essential proof-of-concept needed to advance monogenic gene therapies into clinic development; these therapies include treatments for: Leber's congenital amaurosis type 2, caused by mutations to RPE65, retinoid isomerohydrolase; choroideremia, caused by mutations to REP1, Rab escort protein 1; autosomal recessive Stargardt disease, caused by mutations to ABCA4, the photoreceptor-specific ATP-binding transporter; Usher 1B disease caused by mutations to MYO7A, myosin heavy chain 7; X-linked juvenile retinoschisis caused by mutations to RS1, retinoschisin; autosomal recessive retinitis pigmentosa caused by mutations to MERTK, the proto-oncogene tyrosine-protein kinase MER; Leber's hereditary optic neuropathy caused by mutations to ND4, mitochondrial nicotinamide adenine dinucleotide ubiquinone oxidoreductase (complex I) subunit 4 and achromatopsia, caused by

  7. Synthetic and endogenous cannabinoids protect retinal neurons from AMPA excitotoxicity in vivo, via activation of CB1 receptors: Involvement of PI3K/Akt and MEK/ERK signaling pathways.

    PubMed

    Kokona, Despina; Thermos, Kyriaki

    2015-07-01

    Cannabinoids have been suggested to protect retinal ganglion cells in different models of toxicity, but their effects on other retinal neurons are poorly known. We investigated the neuroprotective actions of the endocannabinoid N-arachidonoyl ethanolamine (Anandamide/AEA) and the synthetic cannabinoids R1-Methanandamide (MethAEA) and HU-210, in an in vivo retinal model of AMPA excitotoxicity, and the mechanisms involved in the neuroprotection. Sprague-Dawley rats were intravitreally injected with PBS or AMPA in the absence or presence of the cannabinoid agonists. Brain nitric oxide synthase (bNOS) and choline acetyltransferase (ChAT) immunoreactivity (IR), as well as TUNEL staining, assessed the AMPA-induced retinal amacrine cell loss and the dose-dependent neuroprotection afforded by cannabinoids. The CB1 receptor selective antagonist AM251 and the PI3K/Akt inhibitor wortmannin reversed the cannabinoid-induced neuroprotection, suggesting the involvement of CB1 receptors and the PI3K/Akt pathway in cannabinoids' actions. Experiments with the CB2 agonist JWH015 and [(3)H]CP55940 radioligand binding suggested that the CB2 receptor is not involved in the neuroprotection. AEA and HU-210 induced phosphorylation of Akt but only AEA induced phosphorylation of ERK1/2 kinases, as revealed by western blot analysis. To investigate the role of caspase-3 in the AMPA-induced cell death, the caspase-3 inhibitor Z-DEVD-FMK was co-injected with AMPA. Z-DEVD-FMK had no effect on AMPA excitotoxicity. Moreover, no difference was observed in the phosphorylation of SAPK/JNK kinases between PBS- and AMPA-treated retinas. These results suggest that endogenous and synthetic cannabinoids protect retinal amacrine neurons from AMPA excitotoxicity in vivo via a mechanism involving the CB1 receptors, and the PI3K/Akt and/or MEK/ERK1/2 signaling pathways.

  8. Logarithmic intensity and speckle-based motion contrast methods for human retinal vasculature visualization using swept source optical coherence tomography

    PubMed Central

    Motaghiannezam, Reza; Fraser, Scott

    2012-01-01

    We formulate a theory to show that the statistics of OCT signal amplitude and intensity are highly dependent on the sample reflectivity strength, motion, and noise power. Our theoretical and experimental results depict the lack of speckle amplitude and intensity contrasts to differentiate regions of motion from static areas. Two logarithmic intensity-based contrasts, logarithmic intensity variance (LOGIV) and differential logarithmic intensity variance (DLOGIV), are proposed for serving as surrogate markers for motion with enhanced sensitivity. Our findings demonstrate a good agreement between the theoretical and experimental results for logarithmic intensity-based contrasts. Logarithmic intensity-based motion and speckle-based contrast methods are validated and compared for in vivo human retinal vasculature visualization using high-speed swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was identified as regions of motion by creating LOGIV and DLOGIV tomograms: multiple B-scans were collected of individual slices through the retina and the variance of logarithmic intensities and differences of logarithmic intensities were calculated. Both methods captured the small vessels and the meshwork of capillaries associated with the inner retina in en face images over 4 mm2 in a normal subject. PMID:22435098

  9. A Simple and Scalable Process for the Differentiation of Retinal Pigment Epithelium From Human Pluripotent Stem Cells

    PubMed Central

    Maruotti, Julien; Wahlin, Karl; Gorrell, David; Bhutto, Imran; Lutty, Gerard

    2013-01-01

    Age-related macular degeneration (AMD), the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries, is associated with the dysfunction and death of the retinal pigment epithelial (RPE) cells. As a result, there has been significant interest in developing RPE culture systems both to study AMD disease mechanisms and to provide substrate for possible cell-based therapies. Because of their indefinite self-renewal, human pluripotent stem cells (hPSCs) have the potential to provide an unlimited supply of RPE-like cells. However, most protocols developed to date for deriving RPE cells from hPSCs involve time- and labor-consuming manual steps, which hinder their use in biomedical applications requiring large amounts of differentiated cells. Here, we describe a simple and scalable protocol for the generation of RPE cells from hPSCs that is less labor-intensive. After amplification by clonal propagation using a myosin inhibitor, differentiation was induced in monolayers of hPSCs, and the resulting RPE cells were purified by two rounds of whole-dish single-cell passage. This approach yields highly pure populations of functional hPSC-derived RPE cells that display many characteristics of native RPE cells, including proper pigmentation and morphology, cell type-specific marker expression, polarized membrane and vascular endothelial growth factor secretion, and phagocytic activity. This work represents a step toward mass production of RPE cells from hPSCs. PMID:23585288

  10. The influence of hypotonicity on large-conductance calcium-activated potassium channels in human retinal pigment epithelial cells.

    PubMed

    Sheu, Shwu-Jiuan; Wu, Sheng-Nan; Hu, Dan-Ning; Chen, Jane-Fane

    2004-12-01

    The aim of this study was to characterize the effects of hypotonicity on the activity of large-conductance Ca(2+)-activated K+ (BK(Ca)) channels in human retinal pigment epithelial (RPE R-50) cells. Effects of hypotonicity on ion currents were investigated with the aid of the patch-clamp technique. A regulatory volume decrease in response to a hypotonic solution (200 mOsm/L) was observed that could be blunted by paxilline. In whole-cell current recordings, a hypotonic solution (200 mOsm/L) reversibly increased the amplitude of K+ outward currents (I(K)). The increase of I(K) could be reversed by iberiotoxin (200 nM), paxilline (1 microM), or tetrandrine (5 microM), but not by glibenclamide (10 microM), disulphonic acid (DIDS) (100 microM), or dequalinium dichloride (10 microM). In RPE R-50 cells pretreated with thapsigargin, aristolochic acid, or pertussis toxin, the increased amplitude of I(K) in response to hypotonicity was unaltered. In cell-attached patches, an increase in BK(Ca)-channel activity was observed during hypotonicity-induced cell swelling. The enhanced channel activity elicited under this condition was mainly mediated by an increase in the number of long-lived openings. These findings support the evidence for the coupling of volume swelling to the functional activity of BK(Ca) channels.

  11. Plasticity of the human visual system after retinal gene therapy in patients with Leber’s congenital amaurosis

    PubMed Central

    Ashtari, Manzar; Zhang, Hui; Cook, Philip A.; Cyckowski, Laura L.; Shindler, Kenneth S.; Marshall, Kathleen A.; Aravand, Puya; Vossough, Arastoo; Gee, James C.; Maguire, Albert M.; Baker, Chris I.; Bennett, Jean

    2015-01-01

    Much of our knowledge of the mechanisms underlying plasticity in the visual cortex in response to visual impairment, vision restoration, and environmental interactions comes from animal studies. We evaluated human brain plasticity in a group of patients with Leber’s congenital amaurosis (LCA), who regained vision through gene therapy. Using non-invasive multimodal neuroimaging methods, we demonstrated that reversing blindness with gene therapy promoted long-term structural plasticity in the visual pathways emanating from the treated retina of LCA patients. The data revealed improvements and normalization along the visual fibers corresponding to the site of retinal injection of the gene therapy vector carrying the therapeutic gene in the treated eye compared to the visual pathway for the untreated eye of LCA patients. After gene therapy, the primary visual pathways (for example, geniculostriate fibers) in the treated retina were similar to those of sighted control subjects, whereas the primary visual pathways of the untreated retina continued to deteriorate. Our results suggest that visual experience, enhanced by gene therapy, may be responsible for the reorganization and maturation of synaptic connectivity in the visual pathways of the treated eye in LCA patients. The interactions between the eye and the brain enabled improved and sustained long-term visual function in patients with LCA after gene therapy. PMID:26180100

  12. Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter

    PubMed Central

    1992-01-01

    We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (Al-Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198). To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOT1 by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence. Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-Wintersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen. PMID:1334963

  13. High glucose-induced barrier impairment of human retinal pigment epithelium is ameliorated by treatment with Goji berry extracts through modulation of cAMP levels.

    PubMed

    Pavan, Barbara; Capuzzo, Antonio; Forlani, Giuseppe

    2014-03-01

    Human retinal pigment epithelium cells were used to investigate the mechanisms underlying blood-retinal barrier disruption under conditions of chronic hyperglycemia. The treatment with 25 mM glucose caused a rapid drop in the transepithelial electrical resistance (TEER), which was reversed by the addition of either a methanolic extract from Goji (Lycium barbarum L.) berries or its main component, taurine. Intracellular cAMP levels increased concurrently with the high glucose-induced TEER decrease, and were correlated to an increased activity of the cytosolic isoform of the enzyme adenylyl cyclase. The treatment with plant extract or taurine restored control levels. Data are discussed in view of a possible prevention approach for diabetic retinopathy.

  14. Reimagining Human Research Protections for 21st Century Science

    PubMed Central

    Bietz, Matthew; Bae, Deborah; Bigby, Barbara; Devereaux, Mary; Fowler, James; Waldo, Ann; Weibel, Nadir; Patrick, Kevin; Klemmer, Scott; Melichar, Lori

    2016-01-01

    Background Evolving research practices and new forms of research enabled by technological advances require a redesigned research oversight system that respects and protects human research participants. Objective Our objective was to generate creative ideas for redesigning our current human research oversight system. Methods A total of 11 researchers and institutional review board (IRB) professionals participated in a January 2015 design thinking workshop to develop ideas for redesigning the IRB system. Results Ideas in 5 major domains were generated. The areas of focus were (1) improving the consent form and process, (2) empowering researchers to protect their participants, (3) creating a system to learn from mistakes, (4) improving IRB efficiency, and (5) facilitating review of research that leverages technological advances. Conclusions We describe the impetus for and results of a design thinking workshop to reimagine a human research protections system that is responsive to 21st century science. PMID:28007687

  15. The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells

    PubMed Central

    Plafker, Kendra S.; Farjo, Krysten M.; Wiechmann, Allan F.; Plafker, Scott M.

    2008-01-01

    Purpose Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. Methods UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27Kip1 were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for β-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. Results UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27Kip1, and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. Conclusions UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation. PMID:18614808

  16. Beneficial Effects of Berberine on Oxidized LDL-Induced Cytotoxicity to Human Retinal Müller Cells

    PubMed Central

    Fu, Dongxu; Yu, Jeremy Y.; Connell, Anna R.; Yang, Shihe; Hookham, Michelle B.; McLeese, Rebecca; Lyons, Timothy J.

    2016-01-01

    Purpose Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal Müller cells. We now explore pathogenic effects of modified LDL on Müller cells, and the efficacy of berberine in mitigating this cytotoxicity. Methods Confluent human Müller cells were exposed to in vitro–modified ‘highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/without pretreatment with berberine (5 μM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 μM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-α), and glial cell activation (glial fibrillary acidic protein). Results Native-LDL had no effect on cultured human Müller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). Conclusions Berberine inhibits modified LDL-induced Müller cell injury by activating

  17. Ectoine as a promising protective agent in humans and animals.

    PubMed

    Bownik, Adam; Stępniewska, Zofia

    2016-12-01

    Ectoine is a compatible water molecule-binding solute (osmoprotectant) produced by several bacterial species in response to osmotic stress and unfavourable environmental conditions. This amino acid derivative can accumulate inside cells at high concentrations without interfering with natural processes and can protect the cell against radiation or osmotic stress. This brief review presents the current state of knowledge about the effects of ectoine on animals and focuses on its practical use for enzyme stabilisation, human skin protection, anti-inflammatory treatment, inhibitory effects in neurodegenerative diseases, and other therapeutic potential in human or veterinary medicine.

  18. Planetary Protection Knowledge Gaps for Human Extraterrestrial Missions: Workshop Report

    NASA Technical Reports Server (NTRS)

    Race, Margaret S. (Editor); Johnson, James E. (Editor); Spry, James A. (Editor); Siegel, Bette; Conley, Catharine A.

    2015-01-01

    This report on Planetary Protection Knowledge Gaps for Human Extraterrestrial Missions summarizes the presentations, deliberations and findings of a workshop at NASA Ames Research Center, March 24-26, 2015, which was attended by more than 100 participants representing a diverse mix of science, engineering, technology, and policy areas. The main objective of the three-day workshop was to identify specific knowledge gaps that need to be addressed to make incremental progress towards the development of NASA Procedural Requirements (NPRs) for Planetary Protection during human missions to Mars.

  19. Federal policy for the protection of human subjects. Final rule.

    PubMed

    1991-06-18

    This document sets forth a common Federal Policy for the Protection of Human Subjects (Model Policy) accepted by the Office of Science and Technology Policy and promulgated in regulation by each of the listed Departments and Agencies. A Proposed Federal Policy for the Protection of Human Subjects published November 10, 1988 (53 FR 45661) has been revised in response to public comments. The Policy as revised is now set forth as a common final rule. For related documents, see other sections of this Federal Register part.

  20. Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: A potential role for reducing UVB light-induced retinal damage

    SciTech Connect

    Li, Chao-Peng; Yao, Jin; Tao, Zhi-Fu; Li, Xiu-Miao; Jiang, Qin Yan, Biao

    2013-09-06

    Highlights: •UVB irradiation induces RPE autophagy. •EGCG treatment represses UVB-mediated autophagy. •EGCG regulates UVB-mediated autophagy through mTOR signaling pathway. •EGCG sensitizes RPE cells to UVB-induced damage in an autophagy-dependent manner. -- Abstract: Autophagy is an intracellular catabolic process involved in protein and organelle degradation via the lysosomal pathway that has been linked in the pathogenesis of age-related macular degeneration (AMD). UVB irradiation-mediated degeneration of the macular retinal pigment epithelial (RPE) cells is an important hallmark of AMD, which is along with the change in RPE autophagy. Thus, pharmacological manipulation of RPE autophagy may offer an alternative therapeutic target in AMD. Here, we found that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, plays a regulatory role in UVB irradiation-induced autophagy in RPE cells. UVB irradiation results in a marked increase in the amount of LC3-II protein in a dose-dependent manner. EGCG administration leads to a significant reduction in the formation of LC3-II and autophagosomes. mTOR signaling activation is required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation is significantly impaired by rapamycin administration. Moreover, EGCG significantly alleviates the toxic effects of UVB irradiation on RPE cells in an autophagy-dependent manner. Collectively, our study reveals a novel role of EGCG in RPE autophagy. EGCG may be exploited as a potential therapeutic reagent for the treatment of pathological conditions associated with abnormal autophagy.

  1. Tempol protects human lymphocytes from genotoxicity induced by cisplatin

    PubMed Central

    Khabour, Omar F; Alzoubi, Karem H; Mfady, Doa’a S; Alasseiri, Mohammed; Hasheesh, Taghrid F

    2014-01-01

    The use of cisplatin in treatments of human malignancies is limited by its side effects that include DNA damage and the subsequent risk of developing secondary cancer. In this study, we examined the possible protective effect of Tempol against DNA damage induced by cisplatin in human lymphocytes using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Cisplatin induced significant elevation in the frequencies of CAs and SCEs in cultured human lymphocytes (P < 0.01). Treatment of lymphocytes with Tempol significantly lowered CAs and SCEs induced by cisplatin. Tempol alone did not affect spontaneous levels of SCEs and CAs observed in the control group (P > 0.05). In conclusion, Tempol protects human lymphocytes against genotoxicity induced by the anticancer drug cisplatin. PMID:24955171

  2. Adenosine Deaminase-2–Induced Hyperpermeability in Human Retinal Vascular Endothelial Cells Is Suppressed by MicroRNA-146b-3p

    PubMed Central

    Samra, Yara A.; Saleh, Heba M.; Hussein, Khaled A.; Elsherbiny, Nehal M.; Ibrahim, Ahmed S.; Elmasry, Khaled; Fulzele, Sadanand; El-Shishtawy, Mamdouh M.; Eissa, Laila A.; Al-Shabrawey, Mohamed; Liou, Gregory I.

    2017-01-01

    Purpose We recently demonstrated that adenosine deaminase-2 (ADA2) contributes to diabetic retinopathy (DR) via up-regulating the production of inflammatory cytokines in macrophages. Also, microRNA (miR)-146b-3p has the ability to inhibit ADA2. The goal of this study was to investigate the potential role of ADA2 and therapeutic benefit of miR-146b-3p in retinal inflammation and endothelial barrier dysfunction during diabetes. Methods Adenosine deaminase-2 activity was determined by colorimetric method in diabetic human vitreous. Human monocyte cell line U937 was differentiated into macrophages and then treated with amadori glycated albumin (AGA), and conditioned medium (CM) was used to assess the changes in ADA2 activity and TNF-α and IL-6 levels by ELISA. Also, macrophages were transfected with miR-146b-3p before treatment with AGA. Permeability of human retinal endothelial cells (hRECs) was assessed by electric cell-substrate impedance sensing (ECIS) after treatment with macrophage CM. Zonula occludens (ZO)-1 was examined by immuno-fluorescence in hRECs. Leukocyte adhesion was assessed in hRECs by measuring myeloperoxidase (MPO) activity and intercellular adhesion molecule-1 (ICAM-1) expression. Results Adenosine deaminase-2 activity was significantly increased in diabetic human vitreous. ADA2 activity and TNF-α and IL-6 levels were significantly increased in human macrophages by AGA treatment. Amadori glycated albumin–treated macrophage CM significantly increased hREC permeability, disrupted ZO-1 pattern, and increased leukocyte adhesion to hRECs through up-regulating ICAM-1. All these changes were reversed by miR-146b-3p. Conclusions Adenosine deaminase-2 is implicated in breakdown of the blood–retinal barrier (BRB) in DR through macrophages-derived cytokines. Therefore, inhibition of ADA2 by miR-146b-3p might be a useful tool to preserve BRB function in DR. PMID:28170537

  3. Retinal Angiogenesis Effects of TGF-β1 and Paracrine Factors Secreted From Human Placental Stem Cells in Response to a Pathological Environment.

    PubMed

    Kim, Kyung-Sul; Park, Ji-Min; Kong, TaeHo; Kim, Chul; Bae, Sang-Hun; Kim, Han Wool; Moon, Jisook

    2016-01-01

    Abnormal angiogenesis is a primary cause of many eye diseases, including diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Mesenchymal stem cells (MSCs) are currently being investigated as a treatment for several such retinal diseases based on their neuroprotective and angiogenic potentials. In this study, we evaluated the role of systemically injected human placental amniotic membrane-derived MSCs (AMSCs) on pathological neovascularization of proliferative retinopathy. We determined that AMSCs secrete higher levels of transforming growth factor-β (TGF-β1) than other MSCs, and the secreted TGF-β1 directly suppresses the proliferation of endothelial cells under pathological conditions in vitro. Moreover, in a mouse model of oxygen-induced retinopathy, intraperitoneally injected AMSCs migrated into the retina and suppressed excessive neovascularization of the vasculature via expression of TGF-β1, and the antineovascular effect of AMSCs was blocked by treatment with TGF-β1 siRNA. These findings are the first to demonstrate that TGF-β1 secreted from AMSCs is one of the key factors to suppress retinal neovascularization in proliferative retinopathy and further elucidate the therapeutic function of AMSCs for the treatment of retinal neovascular diseases.

  4. P2X7-pannexin-1 and amyloid β-induced oxysterol input in human retinal cell: Role in age-related macular degeneration?

    PubMed

    Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Leguillier, Teddy; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

    2016-08-01

    Age-related macular degeneration (AMD) is the most common cause of severe vision loss worldwide. Amyloid β involvement in degenerative diseases such as AMD is well known and its toxicity has been related to P2X7 receptor-pannexin-1. Recently, oxysterols (oxidized derivatives of cholesterol) have been implicated in AMD pathogenesis. The aim of our study was to highlight amyloid β/oxysterols relationship and to describe P2X7 receptor-pannexin-1 role in oxysterols toxicity. Using retinal epithelial cells, we first quantified sterols levels after amyloid β incubation and second we investigated the cytotoxic effects induced by oxysterols. For the first time, our results showed that amyloid β induced oxysterols formation in human retinal pigmented epithelial cells. We showed that oxysterol toxicity is mediated by P2X7 receptor activation. This activation was dependent on pannexin-1 with 25-hydroxycholesterol whereas P2X7 receptor signaling pathway was pannexin-1-independent for 7-ketocholesterol. Taken together our data suggest a pivotal role of P2X7 receptor-pannexin-1 in oxysterols toxicity in retinal cells which could be an important target to develop new treatments for AMD.

  5. Protective effects of intraperitoneal vitamin C, aprotinin and melatonin administration on retinal edema during experimental uveitis in the guinea pig.

    PubMed

    Kükner, A Sahap; Kükner, Aysel; Naziroğlu, Mustafa; Colakoğlu, Neriman; Celebi, Serdal; Yilmaz, Turgut; Aydemir, Orhan

    2004-01-01

    A considerable amount of clinical and experimental evidence exists suggesting the involvement of reactive oxygen substances (ROS) in the aetiology of uveitis. The activated phagocytic system of polymorphonuclear leucocytes in uveitis is involved in the generation of ROS. In addition to their direct free radical scavenging action, aprotinin, melatonin and vitamin C are known to protect against oedema formation and can preserve plasma membrane fluidity and free radical production. Histological changes in the retina that occur during uveitis are not well explained. The purpose of this study was to determine whether vitamin C, aprotinin and melatonin can protect the retina from damage accompanying experimental uveitis (EU). Thirty adult male guinea pigs were divided into five groups of six animals each. The first group was used as control. The right eyes of groups 2, 3, 4 and 5 received an intravitreal injection of bovine serum albumin for induction of experimental uveitis. At the same time and also on the consecutive third day, groups 3, 4 and 5 received intraperitoneal injections of vitamin C (ascorbic acid, 100 mg kg(-1) body wt), aprotinin (20,000 kIU kg(-1) body wt) and melatonin (10 mg kg(-1) body wt), respectively. The animals were killed on the sixth day. The average thickness of the retina and inner plexiform layer for each eye was measured in sagittal section near the optic nerve and expressed in microns. The thickness of the retina and inner plexiform layer in the control group was significantly (p < 0.01) lower than in the group EU as compared with the group EU plus vitamin C, group EU plus aprotinin, group EU plus melatonin (p < 0.05). The thicknesses of the retina and inner plexiform layer in group EU plus vitamin C, group EU plus aprotinin and group EU plus melatonin were significantly (p < 0.01) lower than that in the group EU. The difference in thickness of the retina and inner plexiform layer among the groups 3, 4 and 5 was not significant (p > 0

  6. ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell–Derived Retinal Pigmented Epithelium

    PubMed Central

    Croze, Roxanne H.; Thi, William J.; Clegg, Dennis O.

    2016-01-01

    Purpose Nonexudative (dry) age-related macular degeneration (AMD), a leading cause of blindness in the elderly, is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy, which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However, the factors regulating RPE responses to AMD-associated lesions are not well understood. Here, we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell–derived RPE (hESC-RPE) attachment, proliferation, and wound closure. Methods H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment, and proliferation and cell size within an in vitro scratch assay were examined. Results Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation, and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain, suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. Conclusions ROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. Translational Relevance Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. PMID:27917311

  7. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells.

    PubMed

    Long, Qin; Cao, Xiaoguang; Bian, Ailing; Li, Ying

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  8. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis. PMID:27747237

  9. [Vitro study on gene transfection efficiency of hyaluronic acid modified core-shell liponanoparticles in human retinal pigment epithelium cells].

    PubMed

    Zhao, Ya-Nan; Gan, Li; Wang, Jing; Chen, Xi; Jia, Zheng; Gan, Yong; Liu, Jian-Ping

    2014-05-01

    The aim of this study is to prepare hyaluronic acid (HA) modified core-shell liponanoparticles (pHA-LCS-NPs) as gene delivery system and investigate its gene transfection efficiency in human retinal pigment epithelium (ARPE-19) cells in vitro. The pHA-LCS-NPs was prepared by firstly hydrating dry lipid film with CS-NPs suspension to get LCS-NPs, then modifying the lipid bilayer with HA by amidation reaction between HA and dioleoyl phosphatidylethanolamine (DOPE). Its morphology, particle size and zeta potential were investigated. XTT assay was used to evaluate the cell safety of different vectors in vitro. The gene transfection efficiency of pHA-LCS-NPs modified with different contents of HA was investigated in ARPE-19 cells with green fluorescent protein (pEGFP) as the reporter gene. The results showed that the obtained pHA-LCS-NPs exhibited a clear core-shell structure with the average particles size of (214.9 +/- 7.2) nm and zeta potential of (-35 +/- 3.7) mV. The 24 h cumulative release of gene from pHA-LCS-NPs was less than 30%. After 48 h incubation, gene transfection efficiency of pHA-LCS-NPs/pEGFP was 1.81 times and 3.75 times higher than that of CS-NPs/pEGFP and naked pEGFP, respectively. Also no obvious cytotoxicity was observed on pHA-LCS-NPs. It suggested that the pHA-LCS-NPs might be promising non-viral gene delivery systems with high efficiency and low cytotoxicity.

  10. TNF-α mediates PKCδ/JNK1/2/c-Jun-dependent monocyte adhesion via ICAM-1 induction in human retinal pigment epithelial cells.

    PubMed

    Lee, I-Ta; Liu, Shiau-Wen; Chi, Pei-Ling; Lin, Chih-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

    2015-01-01

    Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases.

  11. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    SciTech Connect

    Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  12. Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2.

    PubMed

    Mueller, Brett H; Park, Yong; Ma, Hai-Ying; Dibas, Adnan; Ellis, Dorette Z; Clark, Abbot F; Yorio, Thomas

    2014-11-01

    Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p < 0.001) and inhibited pERK1/2 expression by 65% (p < 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p < 0.001) and pERK1/2 expression was increased by 1.6 fold (p < 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of

  13. Effects of the vegetable polyphenols epigallocatechin-3-gallate, luteolin, apigenin, myricetin, quercetin, and cyanidin in primary cultures of human retinal pigment epithelial cells

    PubMed Central

    Chen, Rui; Grosche, Antje; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2014-01-01

    Purpose Vegetable polyphenols (bioflavonoids) have been suggested to represent promising drugs for treating cancer and retinal diseases. We compared the effects of various bioflavonoids (epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were obtained from several donors within 48 h of death. Secretion of vascular endothelial growth factor (VEGF) was determined with enzyme-linked immunosorbent assay. Messenger ribonucleic acid levels were determined with real-time reverse transcription polymerase chain reaction. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. The number of viable cells was determined by Trypan Blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation enzyme-linked immunosorbent assay. The phosphorylation level of signaling proteins was revealed by western blotting. Results With the exception of EGCG, all flavonoids tested decreased dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. EGCG inhibited the secretion of VEGF evoked by CoCl2-induced hypoxia. The gene expression of VEGF was reduced by myricetin at low concentrations and elevated at higher concentrations. Luteolin, apigenin, myricetin, and quercetin induced significant decreases in the cell viability at higher concentration, by triggering cellular necrosis. Cyanidin reduced the rate of RPE cell necrosis. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. Most flavonoids tested diminished the phosphorylation levels of extracellular signal-regulated kinases 1/2 and Akt

  14. Retinal blood vessels extraction using probabilistic modelling.

    PubMed

    Kaba, Djibril; Wang, Chuang; Li, Yongmin; Salazar-Gonzalez, Ana; Liu, Xiaohui; Serag, Ahmed

    2014-01-01

    The analysis of retinal blood vessels plays an important role in detecting and treating retinal diseases. In this review, we present an automated method to segment blood vessels of fundus retinal image. The proposed method could be used to support a non-intrusive diagnosis in modern ophthalmology for early detection of retinal diseases, treatment evaluation or clinical study. This study combines the bias correction and an adaptive histogram equalisation to enhance the appearance of the blood vessels. Then the blood vessels are extracted using probabilistic modelling that is optimised by the expectation maximisation algorithm. The method is evaluated on fundus retinal images of STARE and DRIVE datasets. The experimental results are compared with some recently published methods of retinal blood vessels segmentation. The experimental results show that our method achieved the best overall performance and it is comparable to the performance of human experts.

  15. 78 FR 36783 - Meeting of the Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-19

    ... HUMAN SERVICES Meeting of the Secretary's Advisory Committee on Human Research Protections AGENCY.... Appendix 2, notice is hereby given that the Secretary's Advisory Committee on Human Research Protections... meeting agenda will be posted on the SACHRP Web site at:...

  16. Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells

    PubMed Central

    Wang, Weiping; Brautigan, David L

    2008-01-01

    Background Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood. Results Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium. Conclusion The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium. PMID:19036150

  17. Retinal Macroglial Responses in Health and Disease

    PubMed Central

    de Hoz, Rosa; Rojas, Blanca; Ramírez, Ana I.; Salazar, Juan J.; Gallego, Beatriz I.; Triviño, Alberto; Ramírez, José M.

    2016-01-01

    Due to their permanent and close proximity to neurons, glial cells perform essential tasks for the normal physiology of the retina. Astrocytes and Müller cells (retinal macroglia) provide physical support to neurons and supplement them with several metabolites and growth factors. Macroglia are involved in maintaining the homeostasis of extracellular ions and neurotransmitters, are essential for information processing in neural circuits, participate in retinal glucose metabolism and in removing metabolic waste products, regulate local blood flow, induce the blood-retinal barrier (BRB), play fundamental roles in local immune response, and protect neurons from oxidative damage. In response to polyetiological insults, glia cells react with a process called reactive gliosis, seeking to maintain retinal homeostasis. When malfunctioning, macroglial cells can become primary pathogenic elements. A reactive gliosis has been described in different retinal pathologies, including age-related macular degeneration (AMD), diabetes, glaucoma, retinal detachment, or retinitis pigmentosa. A better understanding of the dual, neuroprotective, or cytotoxic effect of macroglial involvement in retinal pathologies would help in treating the physiopathology of these diseases. The extensive participation of the macroglia in retinal diseases points to these cells as innovative targets for new drug therapies. PMID:27294114

  18. Accreditation of human research protection program: An Indian perspective.

    PubMed

    Bairy, K L; Pereira, Pratibha

    2012-04-01

    With the increasing number of clinical trials being placed in India, it is the collective responsibility of the Investigator sites, Government, Ethics Committees, and Sponsors to ensure that the trial subjects are protected from risks these studies can have, that subjects are duly compensated, and credible data generated. Most importantly, each institution/hospital should have a strong Human Research Protection Program to safe guard the trial subjects. In order to look at research with a comprehensive objective approach, there is a need for a formal auditing and review system by a recognized body. As of now, only the sponsors are monitoring/auditing their respective trials; however, there is an increasing need to perform a more detailed review and assessment of processes of the institution and the Ethics Committee. This challenge can be addressed by going for accreditation by a reputed association that encompasses-the institutions, the ethics committees, and researcher/research staff. Starting their journey for the accreditation process in late 2010, Kasturba Medical College and Hospital [KMC], Manipal, and Manipal Hospital Bangalore [MHB] received full Association for the Accreditation of Human Research Protection Programs (AAHRPP) accreditation in Dec 2011-a first in India. This article delves into the steps involved in applying for AAHRPP accreditation from an Indian Perspective, the challenges, advantages, and testimonials from the two hospitals on the application experience and how the accreditation has improved the Human Research Protection Program at these hospitals.

  19. Planetary protection issues in advance of human exploration of Mars

    NASA Technical Reports Server (NTRS)

    Mckay, Christopher P.; Davis, Wanda L.

    1989-01-01

    The major planetary quarantine issues associated with human exploration of Mars, which is viewed as being more likely to harbor indigenous life than is the moon, are discussed. Special attention is given to the environmental impact of human missions to Mars due to contamination and mechanical disturbances of the local environment, the contamination issues associated with the return of humans, and the planetary quarantine strategy for a human base. It is emphasized that, in addition to the question of indigenous life, there may be some concern of returning to earth the earth microorganisms that have spent some time in the Martian environment. It is suggested that, due to the fact that a robot system can be subjected to more stringent controls and protective treatments than a mission involving humans, a robotic sample return mission can help to eliminate many planetary-quarantine concerns about returning samples.

  20. Issues around radiological protection of the environment and its integration with protection of humans: promoting debate on the way forward.

    PubMed

    Brownless, G P

    2007-12-01

    This paper explores issues to consider around integrating direct, explicit protection of the environment into the current system of radiological protection, which is focused on the protection of humans. Many issues around environmental radiological protection have been discussed, and ready-to-use toolboxes have been constructed for assessing harm to non-human biota, but it is not clear how (or even if) these should be fitted into the current system of protection. Starting from the position that the current approach to protecting the environment (namely that it follows from adequately protecting humans) is generally effective, this paper considers how explicit radiological protection of the environment can be integrated with the current system, through developing a 'worked example' of how this could be done and highlighting issues peculiar to protection of the environment. The aim of the paper is to promote debate on this topic, with the ultimate aim of ensuring that any changes to the system are consensual and robust.

  1. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    SciTech Connect

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  2. Planetary protection issues linked to human missions to Mars

    NASA Astrophysics Data System (ADS)

    Debus, A.

    According to United Nations Treaties and handled presently by the Committee of Space Research COSPAR the exploration of the Solar System has to comply with planetary protection requirements The goal of planetary protection is to protect celestial bodies from terrestrial contamination and also to protect the Earth environment from an eventual biocontamination carried by return samples or by space systems returning to the Earth Mars is presently one of the main target at exobiology point of view and a lot of missions are operating on travel or scheduled for its exploration Some of them include payload dedicated to the search of life or traces of life and one of the goals of these missions is also to prepare sample return missions with the ultimate objective to walk on Mars Robotic missions to Mars have to comply with planetary protection specifications well known presently and planetary protection programs are implemented with a very good reliability taking into account an experience of 40 years now For sample return missions a set of stringent requirements have been approved by the COSPAR and technical challenges have now to be won in order to preserve Earth biosphere from an eventual contamination risk Sending astronauts on Mars will gather all these constraints added with the human dimension of the mission The fact that the astronauts are huge contamination sources for Mars and that they are also potential carrier of a contamination risk back to Earth add also ethical considerations to be considered For the preparation of a such

  3. Planetary Protection Issues in the Human Exploration of Mars

    NASA Technical Reports Server (NTRS)

    Criswell, Marvin E.; Race, M. S.; Rummel, J. D.; Baker, A.

    2005-01-01

    This workshop report, long delayed, is the first 21st century contribution to what will likely be a series of reports examining the effects of human exploration on the overall scientific study of Mars. The considerations of human-associated microbial contamination were last studied in a 1990 workshop ("Planetary Protection Issues and Future Mars Missions," NASA CP-10086, 1991), but the timing of that workshop allowed neither a careful examination of the full range of issues, nor an appreciation for the Mars that has been revealed by the Mars Global Surveyor and Mars Pathfinder missions. Future workshops will also have the advantage of Mars Odyssey, the Mars Exploration Rover missions, and ESA's Mars Express, but the Pingree Park workshop reported here had both the NCR's (1992) concern that "Missions carrying humans to Mars will contaminate the planet" and over a decade of careful study of human exploration objectives to guide them and to reconcile. A daunting challenge, and one that is not going to be simple (as the working title of this meeting, "When Ecologies Collide?" might suggest), it is clear that the planetary protection issues will have to be addressed to enable human explorers to safely and competently extend out knowledge about Mars, and its potential as a home for life whether martian or human.

  4. Modelling the optical response of human retinal photoreceptors to plane wave illumination with the finite integration technique

    NASA Astrophysics Data System (ADS)

    Akhlagh Moayed, Alireza; Dang, Shannon; Ramahi, Omar M.; Bizheva, Kostadinka K.

    2009-02-01

    The early stages of ocular diseases such as Diabetic Retinopathy are manifested by morphological changes in retinal tissue occurring on cellular level. Therefore, a number of ophthalmic diseases can be diagnosed at an early stage by detecting spatial and temporal variations in the scattering profile of retinal tissue. It was recently demonstrated that, OCT can be used to probe the functional response of retinal photoreceptors to external light stimulation [1]-[3]. fUHROCT measures localized differential changes in the retina reflectivity over time resulting from external light stimulation of the retina. Currently the origins of the observed reflectivity changes are not well understood. However, due to the complex nature of retinal physiology using purely experimental approaches in this case is problematic. For example fUHROCT is sensitive to small changes in the refractive index of biological tissue which as demonstrated previously, can result from a number of processes such as membrane hyperpolarization, osmotic swelling, metabolic changes, etc. In this paper, we present a computational model of interaction between photoreceptor cells and optical plane wave based on the Finite Integration Technique (FIT).

  5. Loss of the metalloprotease ADAM9 leads to cone-rod dystrophy in humans and retinal degeneration in mice.

    PubMed

    Parry, David A; Toomes, Carmel; Bida, Lina; Danciger, Michael; Towns, Katherine V; McKibbin, Martin; Jacobson, Samuel G; Logan, Clare V; Ali, Manir; Bond, Jacquelyn; Chance, Rebecca; Swendeman, Steven; Daniele, Lauren L; Springell, Kelly; Adams, Matthew; Johnson, Colin A; Booth, Adam P; Jafri, Hussain; Rashid, Yasmin; Banin, Eyal; Strom, Tim M; Farber, Debora B; Sharon, Dror; Blobel, Carl P; Pugh, Edward N; Pierce, Eric A; Inglehearn, Chris F

    2009-05-01

    Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.

  6. Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice

    PubMed Central

    Parry, David A.; Toomes, Carmel; Bida, Lina; Danciger, Michael; Towns, Katherine V.; McKibbin, Martin; Jacobson, Samuel G.; Logan, Clare V.; Ali, Manir; Bond, Jacquelyn; Chance, Rebecca; Swendeman, Steven; Daniele, Lauren L.; Springell, Kelly; Adams, Matthew; Johnson, Colin A.; Booth, Adam P.; Jafri, Hussain; Rashid, Yasmin; Banin, Eyal; Strom, Tim M.; Farber, Debora B.; Sharon, Dror; Blobel, Carl P.; Pugh, Edward N.; Pierce, Eric A.; Inglehearn, Chris F.

    2009-01-01

    Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction. PMID:19409519

  7. Modulation of Abnormal Metabolic Brain Networks by Experimental Therapies in a Nonhuman Primate Model of Parkinson Disease: An Application to Human Retinal Pigment Epithelial Cell Implantation.

    PubMed

    Peng, Shichun; Ma, Yilong; Flores, Joseph; Cornfeldt, Michael; Mitrovic, Branka; Eidelberg, David; Doudet, Doris J

    2016-10-01

    Abnormal covariance pattern of regional metabolism associated with Parkinson disease (PD) is modulated by dopaminergic pharmacotherapy. Using high-resolution (18)F-FDG PET and network analysis, we previously derived and validated a parkinsonism-related metabolic pattern (PRP) in nonhuman primate models of PD. It is currently not known whether this network is modulated by experimental therapeutics. In this study, we examined changes in network activity by striatal implantation of human levodopa-producing retinal pigment epithelial (hRPE) cells in parkinsonian macaques and evaluated the reproducibility of network activity in a small test-retest study.

  8. Planetary protection issues related to human missions to Mars

    NASA Astrophysics Data System (ADS)

    Debus, A.; Arnould, J.

    2008-09-01

    In accordance with the United Nations Outer Space Treaties [United Nations, Agreement Governing the Activities of States on the Moon and Other Celestial Bodies, UN doc A/RES/34/68, resolution 38/68 of December 1979], currently maintained and promulgated by the Committee on Space Research [COSPAR Planetary Protection Panel, Planetary Protection Policy accepted by the COSPAR Council and Bureau, 20 October 2002, amended 24 March 2005, http://www.cosparhq.org/scistr/PPPolicy.htm], missions exploring the Solar system must meet planetary protection requirements. Planetary protection aims to protect celestial bodies from terrestrial contamination and to protect the Earth environment from potential biological contamination carried by returned samples or space systems that have been in contact with an extraterrestrial environment. From an exobiology perspective, Mars is one of the major targets, and several missions are currently in operation, in transit, or scheduled for its exploration. Some of them include payloads dedicated to the detection of life or traces of life. The next step, over the coming years, will be to return samples from Mars to Earth, with a view to increasing our knowledge in preparation for the first manned mission that is likely to take place within the next few decades. Robotic missions to Mars shall meet planetary protection specifications, currently well documented, and planetary protection programs are implemented in a very reliable manner given that experience in the field spans some 40 years. With regards to sample return missions, a set of stringent requirements has been approved by COSPAR [COSPAR Planetary Protection Panel, Planetary Protection Policy accepted by the COSPAR Council and Bureau, 20 October 2002, amended 24 March 2005, http://www.cosparhq.org/scistr/PPPolicy.htm], and technical challenges must now be overcome in order to preserve the Earth’s biosphere from any eventual contamination risk. In addition to the human dimension of

  9. Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin.

    PubMed

    Bray, A F; Cevallos, R R; Gazarian, K; Lamas, M

    2014-11-07

    Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain

  10. Imaging individual neurons in the retinal ganglion cell layer of the living eye

    PubMed Central

    Rossi, Ethan A.; Granger, Charles E.; Yang, Qiang; Saito, Kenichi; Schwarz, Christina; Walters, Sarah; Nozato, Koji; Zhang, Jie; Kawakami, Tomoaki; Fischer, William; Latchney, Lisa R.; Hunter, Jennifer J.; Chung, Mina M.; Williams, David R.

    2017-01-01

    Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain. PMID:28049835

  11. Vasovagal syncope in humans and protective reactions in animals.

    PubMed

    Blanc, Jean-Jacques; Alboni, Paolo; Benditt, David G

    2015-03-01

    Vasovagal syncope (VVS) is not known to occur in animals, although other similar reflex responses are common. This review examines the possible relation of these latter presumably protective reflexes in animals to VVS in humans. The goal is to provide practitioners, and ultimately their patients, a meaningful understanding of the origins and appropriate management of this unpredictable affliction. This report utilized review of computer databases (e.g. PubMed) addressing VVS pathophysiology and origins, spontaneous transient loss of consciousness in animals, and comparative physiology. We also examined articles cited in the publications obtained by computer search and others suggested by colleagues. Articles were chosen based on those providing original observations and/or suggestions of novel mechanisms. In animals self-preservation is directed towards protection of the body through an escalation of behaviours depending on severity and proximity to danger. In humans self-preservation is directed not only to protection of the body, but also to protection of the brain's functional integrity. By virtue of loss of postural tone, the faint causes the body to assume a gravitationally neutral position, thereby offering a better chance of restoring brain blood supply and preserving brain function. Vasovagal syncope may seem to be a disadvantageous evolutionary adaptation. However, it is a reversible condition, that while exposing risk of injury and embarrassment, ultimately favours brain self-preservation in potentially threatening circumstances.

  12. Texture Descriptors Ensembles Enable Image-Based Classification of Maturation of Human Stem Cell-Derived Retinal Pigmented Epithelium

    PubMed Central

    Caetano dos Santos, Florentino Luciano; Skottman, Heli; Juuti-Uusitalo, Kati; Hyttinen, Jari

    2016-01-01

    Aims A fast, non-invasive and observer-independent method to analyze the homogeneity and maturity of human pluripotent stem cell (hPSC) derived retinal pigment epithelial (RPE) cells is warranted to assess the suitability of hPSC-RPE cells for implantation or in vitro use. The aim of this work was to develop and validate methods to create ensembles of state-of-the-art texture descriptors and to provide a robust classification tool to separate three different maturation stages of RPE cells by using phase contrast microscopy images. The same methods were also validated on a wide variety of biological image classification problems, such as histological or virus image classification. Methods For image classification we used different texture descriptors, descriptor ensembles and preprocessing techniques. Also, three new methods were tested. The first approach was an ensemble of preprocessing methods, to create an additional set of images. The second was the region-based approach, where saliency detection and wavelet decomposition divide each image in two different regions, from which features were extracted through different descriptors. The third method was an ensemble of Binarized Statistical Image Features, based on different sizes and thresholds. A Support Vector Machine (SVM) was trained for each descriptor histogram and the set of SVMs combined by sum rule. The accuracy of the computer vision tool was verified in classifying the hPSC-RPE cell maturation level. Dataset and Results The RPE dataset contains 1862 subwindows from 195 phase contrast images. The final descriptor ensemble outperformed the most recent stand-alone texture descriptors, obtaining, for the RPE dataset, an area under ROC curve (AUC) of 86.49% with the 10-fold cross validation and 91.98% with the leave-one-image-out protocol. The generality of the three proposed approaches was ascertained with 10 more biological image datasets, obtaining an average AUC greater than 97%. Conclusions Here we

  13. Efficient delivery of NF-κB siRNA to human retinal pigment epithelial cells with hyperbranched cationic polysaccharide derivative-based nanoparticles.

    PubMed

    Liu, Zhenzhen; Gong, Haijun; Zeng, Rui; Liang, Xuan; Zhang, Li-Ming; Yang, Liqun; Lan, Yuqing

    2015-01-01

    A hyperbranched cationic polysaccharide derivative-mediated small interfering (si)RNA interference strategy was proposed to inhibit nuclear transcription factor-kappa B (NF-κB) activation in human retinal pigment epithelial (hRPE) cells for the gene therapy of diabetic retinopathy. Two hyperbranched cationic polysaccharide derivatives containing the same amount of cationic residues, but with different branching structures and molecular weights, including 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) and amylopectin (DMAPA-Amp) derivatives, were developed for the efficient delivery of NF-κB siRNA into hRPE cells. The DMAPA-Glyp derivative showed lower toxicity against hRPE cells. Furthermore, the DMAPA-Glyp derivative more readily condensed siRNA and then formed the nanoparticles attributed to its higher branching architecture when compared to the DMAPA-Amp derivative. Both DMAPA-Glyp/siRNA and DMAPA-Amp/siRNA nanoparticles were able to protect siRNA from degradation by nuclease in 25% fetal bovine serum. The particle sizes of the DMAPA-Glyp/siRNA nanoparticles (70-120 nm) were smaller than those of the DMAPA-Amp/siRNA nanoparticles (130-180 nm) due to the higher branching architecture and lower molecular weight of the DMAPA-Glyp derivative. In addition, the zeta potentials of the DMAPA-Glyp/siRNA nanoparticles were higher than those of the DMAPA-Glyp/siRNA nanoparticles. As a result, siRNA was much more efficiently transferred into hRPE cells using the DMAPA-Glyp/siRNA nanoparticles rather than the DMAPA-Amp/siRNA nanoparticles. This led to significantly high levels of suppression on the expression levels of NF-κB p65 messenger RNA and protein in the cells transfected with DMAPA-Glyp/siRNA nanoparticles. This work provides a potential approach to promote hyperbranched polysaccharide derivatives as nonviral siRNA vectors for the inhibition of NF-κB activation in hRPE cells.

  14. Efficient delivery of NF-κB siRNA to human retinal pigment epithelial cells with hyperbranched cationic polysaccharide derivative-based nanoparticles

    PubMed Central

    Liu, Zhenzhen; Gong, Haijun; Zeng, Rui; Liang, Xuan; Zhang, Li-Ming; Yang, Liqun; Lan, Yuqing

    2015-01-01

    A hyperbranched cationic polysaccharide derivative-mediated small interfering (si)RNA interference strategy was proposed to inhibit nuclear transcription factor-kappa B (NF-κB) activation in human retinal pigment epithelial (hRPE) cells for the gene therapy of diabetic retinopathy. Two hyperbranched cationic polysaccharide derivatives containing the same amount of cationic residues, but with different branching structures and molecular weights, including 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) and amylopectin (DMAPA-Amp) derivatives, were developed for the efficient delivery of NF-κB siRNA into hRPE cells. The DMAPA-Glyp derivative showed lower toxicity against hRPE cells. Furthermore, the DMAPA-Glyp derivative more readily condensed siRNA and then formed the nanoparticles attributed to its higher branching architecture when compared to the DMAPA-Amp derivative. Both DMAPA-Glyp/siRNA and DMAPA-Amp/siRNA nanoparticles were able to protect siRNA from degradation by nuclease in 25% fetal bovine serum. The particle sizes of the DMAPA-Glyp/siRNA nanoparticles (70–120 nm) were smaller than those of the DMAPA-Amp/siRNA nanoparticles (130–180 nm) due to the higher branching architecture and lower molecular weight of the DMAPA-Glyp derivative. In addition, the zeta potentials of the DMAPA-Glyp/siRNA nanoparticles were higher than those of the DMAPA-Glyp/siRNA nanoparticles. As a result, siRNA was much more efficiently transferred into hRPE cells using the DMAPA-Glyp/siRNA nanoparticles rather than the DMAPA-Amp/siRNA nanoparticles. This led to significantly high levels of suppression on the expression levels of NF-κB p65 messenger RNA and protein in the cells transfected with DMAPA-Glyp/siRNA nanoparticles. This work provides a potential approach to promote hyperbranched polysaccharide derivatives as nonviral siRNA vectors for the inhibition of NF-κB activation in hRPE cells. PMID:25897219

  15. Basic Science Research and the Protection of Human Research Participants

    NASA Astrophysics Data System (ADS)

    Eiseman, Elisa

    2001-03-01

    Technological advances in basic biological research have been instrumental in recent biomedical discoveries, such as in the understanding and treatment of cancer, HIV/AIDS, and heart disease. However, many of these advances also raise several new ethical challenges. For example, genetic research may pose no physical risk beyond that of obtaining the initial blood sample, yet it can pose significant psychological and economic risks to research participants, such as stigmatization, discrimination in insurance and employment, invasion of privacy, or breach of confidentiality. These harms may occur even when investigators do not directly interact with the person whose DNA they are studying. Moreover, this type of basic research also raises broader questions, such as what is the definition of a human subject, and what kinds of expertise do Institutional Review Boards (IRBs) need to review the increasingly diverse types of research made possible by these advances in technology. The National Bioethics Advisory Commission (NBAC), a presidentially appointed federal advisory committee, has addressed these and other ethical, scientific and policy issues that arise in basic science research involving human participants. Two of its six reports, in particular, have proposed recommendations in this regard. "Research Involving Human Biological Materials: Ethical and Policy Guidance" addresses the basic research use of human tissues, cells and DNA and the protection of human participants in this type of research. In "Ethical and Policy Issues in the Oversight of Human Research" NBAC proposes a definition of research involving human participants that would apply to all scientific disciplines, including physical, biological, and social sciences, as well as the humanities and related professions, such as business and law. Both of these reports make it clear that the protection of research participants is key to conducting ethically sound research. By ensuring that all participants in

  16. Retinoids for treatment of retinal diseases.

    PubMed

    Palczewski, Krzysztof

    2010-06-01

    Knowledge about retinal photoreceptor signal transduction and the visual cycle required for normal eyesight has increased exponentially over the past decade. Substantial progress in human genetics has facilitated the identification of candidate genes and complex networks underlying inherited retinal diseases. Natural mutations in animal models that mimic human diseases have been characterized and advanced genetic manipulation can now be used to generate small mammalian models of human retinal diseases. Pharmacological repair of defective visual processes in animal models not only validates their involvement in vision, but also provides great promise for the development of improved therapies for millions who are progressing towards blindness or are almost completely robbed of their eyesight.

  17. Retinoids for Treatment of Retinal Diseases

    PubMed Central

    Palczewski, Krzysztof

    2010-01-01

    Knowledge about retinal photoreceptor signal transduction and the visual cycle required for normal eyesight has expanded exponentially over the past decade. Substantial progress in human genetics has allowed identification of candidate genes and complex networks underlying inherited retinal diseases. Natural mutations in animal models that mimic human diseases have been characterized and advanced genetic manipulation now permits generation of small mammalian models of human retinal diseases. Pharmacological repair of defective visual processes in animal models not only validates their involvement in vision but also provides great promise for developing improved therapies for the millions that are progressing towards blindness or are almost completely robbed of eyesight. PMID:20435355

  18. Protective effect of Aster tataricus extract on retinal damage on the virtue of its antioxidant and anti-inflammatory effect in diabetic rat.

    PubMed

    Du, Hao; Zhang, Meng; Yao, Kejun; Hu, Zhitao

    2017-03-02

    Effect of Aster tataricus (AT) was estimated on the retinal injury in diabetic rats by its antioxidant and anti-inflammatory activity. Streptozotocin (STZ) was used to induce diabetes at a dose of 60mg/kg, i.p. and blood glucose was estimated to confirm the diabetic rats. All the animals were separated in to 5 different groups (n=10) such as control, diabetic retinopathy (DR) receives saline solution, and AT treated group receives AT (100, 200 and 400mg/kg) for the duration of 8 week. After treatment protocol period blood glucose and HbA1c% was estimated in the blood sample of diabetic rats. Retinal tissue was isolated for the fundus photography and retinal vessel diameter, retinal vascular permeability and leukocytosis were estimated. Moreover in the retinal tissue homogenate oxidative stress parameters such as superoxide dismutase (SOD), glutathione peroxidase (GSH) and catalase (CAT) and concentration of cytokines (TNFα, IL10) was estimated. Result of the study suggested that root extract of AT contain rich amount of polyphenol in it which significantly reduces the body weight and concentration of glucose in blood in diabetic rats. Fundus photography suggested that AT extract attenuates the structure and functional abnormalities that develops due to diabetes. Retinal leukocytosis and vascular permeability was significantly decreases in AT treated group than DR group. There was significant increase in the activity of GSH, CAT and SOD in AT treated group than DR group. Moreover AT also attenuates the altered concentration of TNFα, IL10 and NF-κB in the retina of STZ induced diabetic rat. Thus present study concludes that root extract of AT effectively manages the diabetic retinopathy by controlling the blood glucose and also by attenuating the altered oxidative stresss and inflammatory mediators such as TNFα, IL10 and NF-κB in the retina of STZ induced diabetic rat.

  19. Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 24-Hours Post-Exposure to 532 nm, 3.0 ns Pulsed Laser Light and 1064 nm, 170 ps Pulsed Laser Light 12-Hours Post-Exposure: Results Compendium

    DTIC Science & Technology

    2004-06-01

    Laser Light and 1064 nm, 170 ps Pulsed Laser Light 12-hours Post-Exposure: Results Compendium John W. Obringer Martin D. Johnson Laser and Optics...Explanted Human Retinal Pigment Epithelial Cells 12-hours Post-Exposure to 532 nm, 3.0 ns Pulsed Laser Light and 1064 nm, 170 ps Pulsed Laser Lightl2-hours...Explanted Human Retinal Pigment Epithelial USAFA F05611-02-P-0471 Cells 24-Hours Post-Exposure to 532 nm, 3.0 ns Pulsed Laser-Light and 1064nm, 170 ps Pulsed

  20. Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice.

    PubMed

    Li, Xiangming; Huang, Jing; Zhang, Min; Funakoshi, Ryota; Sheetij, Dutta; Spaccapelo, Roberta; Crisanti, Andrea; Nussenzweig, Victor; Nussenzweig, Ruth S; Tsuji, Moriya

    2016-08-31

    A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

  1. Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases

    PubMed Central

    Malik, Deepika; Hsu, Tiffany; Falatoonzadeh, Payam; Cáceres-del-Carpio, Javier; Tarek, Mohamed; Chwa, Marilyn; Atilano, Shari R.; Ramirez, Claudio; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin; Kenney, M. Cristina

    2014-01-01

    Background It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. Methodology/Principal Findings Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. Conclusion/Significance In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be

  2. Missed retinal breaks in rhegmatogenous retinal detachment

    PubMed Central

    Takkar, Brijesh; Azad, Shorya; Shashni, Adarsh; Pujari, Amar; Bhatia, Indrish; Azad, Rajvardhan

    2016-01-01

    AIM To evaluate the causes and associations of missed retinal breaks (MRBs) and posterior vitreous detachment (PVD) in patients with rhegmatogenous retinal detachment (RRD). METHODS Case sheets of patients undergoing vitreo retinal surgery for RRD at a tertiary eye care centre were evaluated retrospectively. Out of the 378 records screened, 253 were included for analysis of MRBs and 191 patients were included for analysis of PVD, depending on the inclusion criteria. Features of RRD and retinal breaks noted on examination were compared to the status of MRBs and PVD detected during surgery for possible associations. RESULTS Overall, 27% patients had MRBs. Retinal holes were commonly missed in patients with lattice degeneration while missed retinal tears were associated with presence of complete PVD. Patients operated for cataract surgery were significantly associated with MRBs (P=0.033) with the odds of missing a retinal break being 1.91 as compared to patients with natural lens. Advanced proliferative vitreo retinopathy (PVR) and retinal bullae were the most common reasons for missing a retinal break during examination. PVD was present in 52% of the cases and was wrongly assessed in 16%. Retinal bullae, pseudophakia/aphakia, myopia, and horse shoe retinal tears were strongly associated with presence of PVD. Traumatic RRDs were rarely associated with PVD. CONCLUSION Pseudophakic patients, and patients with retinal bullae or advanced PVR should be carefully screened for MRBs. Though Weiss ring is a good indicator of PVD, it may still be over diagnosed in some cases. PVD is associated with retinal bullae and pseudophakia, and inversely with traumatic RRD. PMID:27990367

  3. Retinitis pigmentosa in southern Africa.

    PubMed

    Greenberg, J; Bartmann, L; Ramesar, R; Beighton, P

    1993-11-01

    Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal disorders which are a common cause of genetic blindness. The relative frequencies of the different forms of RP in South Africa, as determined from the register at the DNA banking centre for RP at the Department of Human Genetics, University of Cape Town, are presented and discussed. Of the 125 families analysed, 29 (23%) showed autosomal dominant, 33 (27%) autosomal recessive and 3 (3%) X-linked inheritance. In 10 families the pedigree data were insufficient to allow accurate genetic subtyping and a further 50 patients were sporadic without a family history of RP or other syndromic features which would allow categorization.

  4. Gentiana asclepiadea protects human cells against oxidation DNA lesions.

    PubMed

    Hudecová, Alexandra; Hašplová, Katarína; Miadoková, Eva; Magdolenová, Zuzana; Rinna, Alessandra; Collins, Andrew R; Gálová, Eliška; Vaculčíková, Dagmar; Gregáň, Fridrich; Dušinská, Mária

    2012-03-01

    The objectives of this study were to examine whether the methanolic and aqueous extracts from the haulm and flower of Gentiana asclepiadea exhibited free radical scavenging and protective (antigenotoxic) effect against DNA oxidation induced by H(2)O(2) in human lymphocytes and human embryonic kidney cells (HEK 293). All four extracts exhibited high scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at concentrations 2.5 and 25 mg ml(-1). The level of DNA damage was measured using the alkaline version of single-cell gel electrophoresis (comet assay). Challenge with H(2)O(2) shows that the pre-treatment of the cells with non-genotoxic doses of Gentiana extracts protected human DNA-either eliminated or significantly reduced H(2)O(2) induced DNA damage. The genotoxic activity of H(2)O(2) was most effectively decreased after 30 min of pre-incubation with 0.05 mg ml(-1) (range, 93.5%-96.3% of reduction in lymphocytes) and 0.25 mg ml(-1) (range, 59.5%-71.4% and 52.7%-66.4% of reduction in lymphocytes and HEK 293 cells, respectively) of G. asclepiadea extracts. These results suggest that the tested G. asclepiadea extracts could be considered as an effective natural antioxidant source.

  5. 48 CFR 1352.235-72 - Protection of human subjects-institutional approval.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Protection of human... Clauses 1352.235-72 Protection of human subjects—institutional approval. As prescribed in 48 CFR 1335.006(c), insert the following clause: Protection of Human Subjects—Institutional Approval (APR 2010)...

  6. 48 CFR 1352.235-71 - Protection of human subjects-exemption.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Protection of human....235-71 Protection of human subjects—exemption. As prescribed in 48 CFR 1335.006(b), insert the following clause: Protection of Human Subjects (APR 2010) (a) Contractor has satisfied the requirements...

  7. 48 CFR 1352.235-72 - Protection of human subjects-institutional approval.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Protection of human... Clauses 1352.235-72 Protection of human subjects—institutional approval. As prescribed in 48 CFR 1335.006(c), insert the following clause: Protection of Human Subjects—Institutional Approval (APR 2010)...

  8. 48 CFR 1352.235-71 - Protection of human subjects-exemption.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Protection of human....235-71 Protection of human subjects—exemption. As prescribed in 48 CFR 1335.006(b), insert the following clause: Protection of Human Subjects (APR 2010) (a) Contractor has satisfied the requirements...

  9. 48 CFR 1352.235-71 - Protection of human subjects-exemption.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Protection of human....235-71 Protection of human subjects—exemption. As prescribed in 48 CFR 1335.006(b), insert the following clause: Protection of Human Subjects (APR 2010) (a) Contractor has satisfied the requirements...

  10. 48 CFR 1352.235-72 - Protection of human subjects-institutional approval.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Protection of human... Clauses 1352.235-72 Protection of human subjects—institutional approval. As prescribed in 48 CFR 1335.006(c), insert the following clause: Protection of Human Subjects—Institutional Approval (APR 2010)...

  11. 48 CFR 1352.235-71 - Protection of human subjects-exemption.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Protection of human....235-71 Protection of human subjects—exemption. As prescribed in 48 CFR 1335.006(b), insert the following clause: Protection of Human Subjects (APR 2010) (a) Contractor has satisfied the requirements...

  12. 48 CFR 1352.235-72 - Protection of human subjects-institutional approval.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Protection of human... Clauses 1352.235-72 Protection of human subjects—institutional approval. As prescribed in 48 CFR 1335.006(c), insert the following clause: Protection of Human Subjects—Institutional Approval (APR 2010)...

  13. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  14. Enhanced Ca(2+) response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines.

    PubMed

    Catalioto, Rose-Marie; Valenti, Claudio; Maggi, Carlo Alberto; Giuliani, Sandro

    2015-09-15

    Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1β enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.

  15. Potential for autoimmune pathogenesis of Rift Valley Fever virus retinitis.

    PubMed

    Newman-Gerhardt, Shoshana; Muiruri, Samuel; Muchiri, Eric; Peters, Clarence J; Morrill, John; Lucas, Alexander H; King, Charles H; Kazura, James; LaBeaud, Angelle Desiree

    2013-09-01

    Rift Valley Fever (RVF) is a significant threat to human health because it can progress to retinitis, encephalitis, and hemorrhagic fever. The timing of onset of Rift Valley Fever virus (RVFV) retinitis suggests an autoimmune origin. To determine whether RVFV retinitis is associated with increased levels of IgG against retinal tissue, we measured and compared levels of IgG against healthy human eye tissue by immunohistochemical analysis. We found that serum samples from RVFV-exposed Kenyans with retinitis (n = 8) were slightly more likely to have antibodies against retinal tissue than control populations, but the correlation was not statistically significant. Further investigation into the possible immune pathogenesis of RVFV retinitis could lead to improved therapies to prevent or treat this severe complication.

  16. Potential for Autoimmune Pathogenesis of Rift Valley Fever Virus Retinitis

    PubMed Central

    Newman-Gerhardt, Shoshana; Muiruri, Samuel; Muchiri, Eric; Peters, Clarence J.; Morrill, John; Lucas, Alexander H.; King, Charles H.; Kazura, James; LaBeaud, Angelle Desiree

    2013-01-01

    Rift Valley Fever (RVF) is a significant threat to human health because it can progress to retinitis, encephalitis, and hemorrhagic fever. The timing of onset of Rift Valley Fever virus (RVFV) retinitis suggests an autoimmune origin. To determine whether RVFV retinitis is associated with increased levels of IgG against retinal tissue, we measured and compared levels of IgG against healthy human eye tissue by immunohistochemical analysis. We found that serum samples from RVFV-exposed Kenyans with retinitis (n = 8) were slightly more likely to have antibodies against retinal tissue than control populations, but the correlation was not statistically significant. Further investigation into the possible immune pathogenesis of RVFV retinitis could lead to improved therapies to prevent or treat this severe complication. PMID:23918215

  17. Oximetry of retinal capillaries by multicomponent analysis.

    PubMed

    Furukawa, Hiromitsu; Arimoto, Hidenobu; Shirai, Tomohiro; Ooto, Sotaro; Hangai, Masanori; Yoshimura, Nagahisa

    2012-08-01

    Retinal oximetry of capillaries was performed for early detection of retinal vascular abnormalities, which are caused predominantly by complications of systemic circulatory diseases. As the conventional method for determining absorbance is not applicable to capillaries, multicomponent analysis was used to estimate the absorbance spectra of the retinal blood vessels. In this analysis, the capillary spectrum was classified as intermediate between those of the retinal arteries and veins, enabling relative estimation of oxygen saturation in the capillaries. This method could be useful for early recognition of disturbances in the peripheral circulation. Furthermore, a spectroscopic ophthalmoscope system based on the proposed method was developed to examine the human retina. A clinical trial of this system demonstrated that oximetry of the retinal capillaries may be an improvement over the present diagnosis for patients of malignant hypertension.

  18. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    PubMed

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-03-04

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

  19. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  20. Retinal Optical Coherence Tomography Imaging

    NASA Astrophysics Data System (ADS)

    Drexler, Wolfgang; Fujimoto, James G.

    The eye is essentially transparent, transmitting light with only minimal optical attenuation and scattering providing easy optical access to the anterior segment as well as the retina. For this reason, ophthalmic and especially retinal imaging has been not only the first but also most successful clinical application for optical coherence tomography (OCT). This chapter focuses on the development of OCT technology for retinal imaging. OCT has significantly improved the potential for early diagnosis, understanding of retinal disease pathogenesis, as well as monitoring disease progression and response to therapy. Development of ultrabroad bandwidth light sources and high-speed detection techniques has enabled significant improvements in ophthalmic OCT imaging performance, demonstrating the potential of three-dimensional, ultrahigh-resolution OCT (UHR OCT) to perform noninvasive optical biopsy of the living human retina, i.e., the in vivo visualization of microstructural, intraretinal morphology in situ approaching the resolution of conventional histopathology. Significant improvements in axial resolution and speed not only enable three-dimensional rendering of retinal volumes but also high-definition, two-dimensional tomograms, topographic thickness maps of all major intraretinal layers, as well as volumetric quantification of pathologic intraretinal changes. These advances in OCT technology have also been successfully applied in several animal models of retinal pathologies. The development of light sources emitting at alternative wavelengths, e.g., around #1,060 nm, not only enabled three-dimensional OCT imaging with enhanced choroidal visualization but also improved OCT performance in cataract patients due to reduced scattering losses in this wavelength region. Adaptive optics using deformable mirror technology, with unique high stroke to correct higher-order ocular aberrations, with specially designed optics to compensate chromatic aberration of the human eye, in

  1. Multifaceted pathways protect human skin from UV radiation.

    PubMed

    Natarajan, Vivek T; Ganju, Parul; Ramkumar, Amrita; Grover, Ritika; Gokhale, Rajesh S

    2014-07-01

    The recurrent interaction of skin with sunlight is an intrinsic constituent of human life, and exhibits both beneficial and detrimental effects. The apparent robust architectural framework of skin conceals remarkable mechanisms that operate at the interface between the surface and environment. In this Review, we discuss three distinct protective mechanisms and response pathways that safeguard skin from deleterious effects of ultraviolet (UV) radiation. The unique stratified epithelial architecture of human skin along with the antioxidant-response pathways constitutes the important defense mechanisms against UV radiation. The intricate pigmentary system and its intersection with the immune-system cytokine axis delicately balance tissue homeostasis. We discuss the relationship among these networks in the context of an unusual depigmenting disorder, vitiligo. The elaborate tunable mechanisms, elegant multilayered architecture and evolutionary selection pressures involved in skin and sunlight interaction makes this a compelling model to understand biological complexity.

  2. Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium

    PubMed Central

    Ablonczy, Zsolt; Higbee, Daniel; Grey, Angus C.; Koutalos, Yiannis; Schey, Kevin L.; Crouch, Rosalie K.

    2013-01-01

    The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE. PMID:23969078

  3. Characterisation of the canine rod-cone dysplasia type one gene (rod photoreceptor cGMP phosphodiesterase beta subunit (PDEB)) - a model for human retinitis pigmentosa

    SciTech Connect

    Clements, P.J.M.; Gregory, C.Y.; Petersen-Jones, S.M.

    1994-09-01

    Rod-cone dysplasia type one (rod-1) is an early onset, autosomal recessive retinal dystrophy segregating in the Irish setter breed. It is a model for certain forms of human autosomal recessive retinitis pigmentosa (arRP) caused by mutations in the same gene, PDEB. We confirmed the codon 807 Trp to Stop mutation and were the first to show cosegregation of the mutant allele with disease in a pedigree. We believe that this currently represents the best animal model available for some aspects of arRP, since canine tissues are relatively easy to access compared to human and yet the canine eye is of comparable size, unlike that of the rd mouse. This facilitates therapeutic intervention particularly at the subretinal level. In order to more fully investigate this model we have been characterizing the PDEB gene in the normal dog. Using PCR we have partially mapped the intron/exon structure, demonstrating a very high degree of evolutionary conservation with the mouse and human genes. RT-PCR has been used to reveal expression in a variety of neural and non-neural tissues. A PCR product spanning exons 19 to 22 (which also contains the site for the rcd-1 mutation) is detected in retina but also in tissues such as visual cortex, cerebral cortex, cerebellum, lateral geniculate nucleus, adrenal gland, lung, kidney and ovary. All of these tissues gave a negative result with primers for rds/peripherin, a gene which is expressed in rods and cones. This raises interesting questions about the regulation of PDEB transcripts which is initially being investigated by Northern analysis. In addition, anchored PCR techniques have generated upstream genomic sequences and we are currently mapping the 5{prime} extent of the mRNA transcript in the retina. This will facilitate the analysis of potential upstream promoter elements involved in directing expression.

  4. Planetary protection issues and human exploration of Mars

    NASA Technical Reports Server (NTRS)

    Devincenzi, D. L.

    1991-01-01

    A key feature of the Space Exploration Initiative involves human missions to Mars. The report describing the initiative cites the search for life on Mars, extant or extinct, as one of the five science themes for such an endeavor. Because of this, concerns for planetary protection (PP) have arisen of two fronts: (1) forward contamination of Mars by spacecraft-borne terrestrial microbes which could interfere with exobiological analyses; and (2) back contamination of Earth by species that may be present in returned Mars samples. The United States is also signatory to an international treaty designed to protect Earth and planets from harmful cross-contamination during exploration. Therefore, it is timely to assess the necessity for, and impact of, PP procedures on the mission set comprising the human exploration of Mars. The ground-rules adopted at a recent workshop which addressed PP questions of this type are presented. In addition, the workshop produced several recommendations for dealing with forward and back contamination concerns for non-scientific perspectives, including public relations, legal, regulatory, international, and environmental.

  5. 78 FR 12061 - Meeting of the Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-21

    ... HUMAN SERVICES Meeting of the Secretary's Advisory Committee on Human Research Protections AGENCY.... Appendix 2, notice is hereby given that the Secretary's Advisory Committee on Human Research Protections... 20201. FOR FURTHER INFORMATION CONTACT: Jerry Menikoff, M.D., J.D., Director, Office for Human...

  6. 78 FR 56233 - Meeting of the Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-12

    ... HUMAN SERVICES Meeting of the Secretary's Advisory Committee on Human Research Protections AGENCY.... Appendix 2, notice is hereby given that the Secretary's Advisory Committee on Human Research Protections... FURTHER INFORMATION CONTACT: Jerry Menikoff, M.D., J.D., Director, Office for Human Research...

  7. 78 FR 12664 - Human Subject Protection; Acceptance of Data From Clinical Studies for Medical Devices

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-25

    ... HUMAN SERVICES Food and Drug Administration 21 CFR Parts 807, 812, and 814 RIN 0910-AG48 Human Subject... ensure the protection of human subjects and the quality and integrity of data obtained from these studies... the protection of human subjects and the quality and integrity of data obtained from these...

  8. 78 FR 10538 - Protections for Subjects in Human Research Involving Pesticides

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-14

    ... AGENCY 40 CFR Part 26 RIN 2070-AJ76 Protections for Subjects in Human Research Involving Pesticides... tailored amendments to the portions of its rules for the protection of human subjects of research applying... human subjects and to persons who submit the results of human research with pesticides to EPA....

  9. Generation of Functional Human Retinal Ganglion Cells with Target Specificity from Pluripotent Stem Cells by Chemically Defined Recapitulation of Developmental Mechanism.

    PubMed

    Teotia, Pooja; Chopra, Divyan A; Dravid, Shashank Manohar; Van Hook, Matthew J; Qiu, Fang; Morrison, John; Rizzino, Angie; Ahmad, Iqbal

    2017-03-01

    Glaucoma is a complex group of diseases wherein a selective degeneration of retinal ganglion cells (RGCs) lead to irreversible loss of vision. A comprehensive approach to glaucomatous RGC degeneration may include stem cells to functionally replace dead neurons through transplantation and understand RGCs vulnerability using a disease in a dish stem cell model. Both approaches require the directed generation of stable, functional, and target-specific RGCs from renewable sources of cells, that is, the embryonic stem cells and induced pluripotent stem cells. Here, we demonstrate a rapid and safe, stage-specific, chemically defined protocol that selectively generates RGCs across species, including human, by recapitulating the developmental mechanism. The de novo generated RGCs from pluripotent cells are similar to native RGCs at the molecular, biochemical, functional levels. They also express axon guidance molecules, and discriminate between specific and nonspecific targets, and are nontumorigenic. Stem Cells 2017;35:572-585.

  10. Differential phase-contrast, swept-source optical coherence tomography at 1060 nm for in vivo human retinal and choroidal vasculature visualization

    NASA Astrophysics Data System (ADS)

    Motaghiannezam, S. M. Reza; Koos, David; Fraser, Scott E.

    2012-02-01

    Human retinal and choroidal vasculature was visualized by a differential phase-contrast (DPC) method using high-speed, swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was recognized as regions of motion by creating differential phase-variance (DPV) tomograms: multiple B-scans of individual slices through the retina were collected and the variance of the phase differences was calculated. DPV captured the small vessels and the meshwork of capillaries associated with the inner retina in en-face images over 4 mm2. The swept-source laser at 1060 nm offered the needed phase sensitivity to perform DPV and generated en-face images that capture motion in the inner choroidal layer exceeding the capabilities of previous spectrometer-based instruments. In comparison with the power Doppler phase-shift method, DPV provided better visualization of the foveal avascular zone in en-face images.

  11. Analysis by NASA's VESGEN Software of Retinal Blood Vessels in Human Subjects Undergoing Head-Down Tilt During 70-Day Bed Rest

    NASA Technical Reports Server (NTRS)

    Vyas, Ruchi J.; Murray, Matthew C.; Predovic, Marina; Lim, Shiyin; Askin, Kayleigh N.; Vizzeri, Gianmarco; Taibbi, Giovanni; Mason, Sara Stroble; Zanello, Susana B.; Young, Millenia; Parsons-Wingerter, Patricia

    2017-01-01

    Significant risks for visual impairment associated with increased intracranial pressure (VIIP) are incurred by microgravity spaceflight, especially long-duration missions [1]. We hypothesize that microgravity-induced fluid shifts result in pathological changes within blood vessels of the retina that precede development of visual and other ocular impairments. Potential contributions of retinal vascular remodeling to VIIP etiology are therefore being investigated for two studies in 30deg infrared (IR) Heidelberg Spectralis(Registered Trademark) images with NASA's innovative VESsel GENeration Analysis (VESGEN) software [2,3]. The retrospective studies include: (1) before, during and after (pre, mid and post) 6º head-down tilt (HDT) in human subjects during 70 days of bed rest, and (2) before and after missions to the International Space Station (ISS) by U.S. crew members. Results for both studies are almost complete. A preliminary example for HDT is described below.

  12. Ursodeoxycholic Acid Attenuates Endoplasmic Reticulum Stress-Related Retinal Pericyte Loss in Streptozotocin-Induced Diabetic Mice

    PubMed Central

    Choi, Jeong A.; Koh, Jae-Young

    2017-01-01

    Loss of pericytes, an early hallmark of diabetic retinopathy (DR), results in breakdown of the blood-retinal barrier. Endoplasmic reticulum (ER) stress may be involved in this process. The purpose of this study was to examine the effects of ursodeoxycholic acid (UDCA), a known ameliorator of ER stress, on pericyte loss in DR of streptozotocin- (STZ-) induced diabetic mice. To assess the extent of DR, the integrity of retinal vessels and density of retinal capillaries in STZ-induced diabetic mice were evaluated. Additionally, induction of ER stress and the unfolded protein response (UPR) were assessed in diabetic mice and human retinal pericytes exposed to advanced glycation end products (AGE) or modified low-density lipoprotein (mLDL). Fluorescein dye leakage during angiography and retinal capillary density were improved in UDCA-treated diabetic mice, compared to the nontreated diabetic group. Among the UPR markers, those involved in the protein kinase-like ER kinase (PERK) pathway were increased, while UDCA attenuated UPR in STZ-induced diabetic mice as well as AGE- or mLDL-exposed retinal pericytes in culture. Consequently, vascular integrity was improved and pericyte loss reduced in the retina of STZ-induced diabetic mice. Our findings suggest that UDCA might be effective in protecting against DR. PMID:28127564

  13. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    PubMed Central

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J.

    2011-01-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082

  14. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    PubMed

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J

    2011-10-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol's effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  15. Retinal Nerve Fiber and Optic Disc Morphology in Patients with Human Immunodeficiency Virus Using the Heidelberg Retina Tomography 3

    PubMed Central

    Bartsch, Dirk-Uwe; Kozak, Igor; Grant, Igor; Knudsen, Victoria L.; Weinreb, Robert N.; Lee, Byung Ro; Freeman, William R.

    2015-01-01

    Purpose To use novel confocal scanning ophthalmoscopy technology to test hypothesis that HIV-seropositive patients without history of retinitis with a history of a low CD4 count are more likely to have damage to their retinal nerve fiber layer (RNFL) when compared to patients with high CD4 count. In addition, we compared optic disc morphologic changes with glaucoma. Design Cross-sectional study. Participants and Controls 171 patients were divided into four groups. The control group consisted of 40 eyes of 20 HIV-seronegative patients. The second group consisted of 80 eyes of 41 HIV-positive patients whose CD4 cell count never dropped below 100 (1.0 x 109/L). The third group consisted of 44 eyes of 26 HIV-positive patients with a history of low CD4 counts <100. Fourth group consisted of 79 eyes of 79 patients with confirmed glaucoma who served as positive controls. Testing Confocal scanning laser ophthalmoscopy was performed with the Heidelberg Retina Tomograph (HRT3) and data were analyzed with HRT3, software (Heyex version 1.5.10.0). Main Outcome Measures Disc area, cup area, cup volume, rim volume, mean cup depth, maximum cup depth, cup-to-disc ration, mean RNFL thickness, and RNFL cross-sectional area. Results Analysis of the global optic nerve and cup parameters showed no difference in disk area among the four groups. There was also no difference in cup, rim volume, mean cup depth, or maximum cup depth among the first three groups but they were all different from glaucoma group. The RNFL was thinner in glaucoma and both HIV-positive groups compared to HIV-seronegative subjects. The cross sectional RNFL area was thinner in both high and low CD4 HIV-positive groups compared to HIV-seronegative group in the nasal and temporal/inferior sectors, respectively. Glaucoma group showed thinning in all sectors. Conclusions HIV retinopathy results in retinal nerve fiber layer loss without structural optic nerve supportive tissue change. RNFL damage may occur early in HIV

  16. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  17. Immune correlates of protection in human invasive aspergillosis.

    PubMed

    Camargo, Jose F; Husain, Shahid

    2014-08-15

    Protective immunity against Aspergillus depends on a highly coordinated interaction between the innate and adaptive arms of the immune system. Fungal recognition via pattern recognition receptors, such as pentraxin 3, dectin-1, and Toll-like receptors, leads to complement activation, phagocytosis, and killing of ingested fungi. Aspergillus-specific T-helper 1 and 17 cells produce cytokines such as interferon γ and interleukin 17, which facilitate macrophage activation and neutrophil recruitment, respectively. Genetic (or drug-induced) defects in components of these networks of antifungal immunity result in increased risk of invasive aspergillosis after chemotherapy or transplantation. We review the most important genetic, immunological, and pharmacological factors that influence human susceptibility to Aspergillus and discuss the potential role of immune biomarkers in risk stratification strategies that facilitate individualized antifungal therapy/prophylaxis in immunocompromised hosts.

  18. Screening Diabetic Retinopathy Through Color Retinal Images

    NASA Astrophysics Data System (ADS)

    Li, Qin; Jin, Xue-Min; Gao, Quan-Xue; You, Jane; Bhattacharya, Prabir

    Diabetic Retinopathy (DR) is a common complication of diabetes that damages the eye's retina. Recognition DR as early as possible is very important to protect patients' vision. We propose a method for screening DR and distin-guishing Prolifetive Diabetic Retinopathy (PDR) from Non-Prolifetive Retino-pathy (NPDR) automatatically through color retinal images. This method evaluates the severity of DR by analyzing the appearnce of bright lesions and retinal vessel patterns. The bright lesions are extracted through morphlogical re-consturction. After that, the retinal vessels are automatically extracted using multiscale matched filters. Then the vessel patterns are analyzed by extracting the vessel net density. The experimental results domonstrate that it is a effective solution to screen DR and distinguish PDR from NPDR by only using color retinal images.

  19. Human threat management systems: self-protection and disease avoidance.

    PubMed

    Neuberg, Steven L; Kenrick, Douglas T; Schaller, Mark

    2011-03-01

    Humans likely evolved precautionary systems designed to minimize the threats to reproductive fitness posed by highly interdependent ultrasociality. A review of research on the self-protection and disease avoidance systems reveals that each system is functionally distinct and domain-specific: each is attuned to different cues; engages different emotions, inferences, and behavioral inclinations; and is rooted in somewhat different neurobiological substrates. These systems share important features, however. Each system is functionally coherent, in that perceptual, affective, cognitive, and behavioral processes work in concert to reduce fitness costs of potential threats. Each system is biased in a risk-averse manner, erring toward precautionary responses even when available cues only heuristically imply threat. And each system is functionally flexible, being highly sensitive to specific ecological and dispositional cues that signal greater vulnerability to the relevant threat. These features characterize a general template useful for understanding not only the self-protection and disease avoidance systems, but also a broader set of evolved, domain-specific precautionary systems.

  20. Human Threat Management Systems: Self-Protection and Disease Avoidance

    PubMed Central

    Neuberg, Steven L.; Kenrick, Douglas T.; Schaller, Mark

    2010-01-01

    Humans likely evolved precautionary systems designed to minimize the threats to reproductive fitness posed by highly interdependent ultrasociality. A review of research on the self-protection and disease avoidance systems reveals that each system is functionally distinct and domain-specific: Each is attuned to different cues; engages different emotions, inferences, and behavioral inclinations; and is rooted in somewhat different neurobiological substrates. These systems share important features, however. Each system is functionally coherent, in that perceptual, affective, cognitive, and behavioral processes work in concert to reduce fitness costs of potential threats. Each system is biased in a risk-averse manner, erring toward precautionary responses even when available cues only heuristically imply threat. And each system is functionally flexible, being highly sensitive to specific ecological and dispositional cues that signal greater vulnerability to the relevant threat. These features characterize a general template useful for understanding not only the self-protection and disease avoidance systems, but also a broader set of evolved, domain-specific precautionary systems. PMID:20833199

  1. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    SciTech Connect

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E.

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  2. Does biodiversity protect humans against infectious disease? Reply

    USGS Publications Warehouse

    Wood, Chelsea L.; Lafferty, Kevin D.; DeLeo, Giulio; Young, Hillary S.; Hudson, Peter J.; Kuris, Armand M.

    2016-01-01

    The dilution effect is the sort of idea that everyone wants to be true. If nature protects humans against infectious disease, imagine the implications: nature's value could be tallied in terms of human suffering avoided. This makes a potent argument for conservation, convincing even to those who would otherwise be disinclined to support conservation initiatives. The appeal of the dilution effect has been recognized by others: “the desire to make the case for conservation has led to broad claims regarding the benefits of nature conservation for human health” (Bauch et al. 2015). Randolph and Dobson (2012) were among the first to critique these claims, making the case that promotion of conservation to reduce Lyme disease risk, although well intentioned, was flawed. Along with Randolph and Dobson's critique, there have been several calls for a more nuanced scientific assessment of the relationship between biodiversity and disease transmission (Dunn 2010, Salkeld et al. 2013, Wood and Lafferty 2013, Young et al. 2013). In response, supporters of the dilution effect have instead increased the scope of their generalizations with review papers, press releases, and, like Levi et al. (2015), letters. These responses have been successful; it is not uncommon to read papers that repeat the assertion that biodiversity generally interferes with disease transmission and that conservation will therefore generally benefit human health. Here, we explain how Levi et al. (2015) and other, similar commentaries use selective interpretation and shifting definitions to argue for the generality of the dilution effect hypothesis.

  3. Temporal Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells at 0.5, 1.0, 3.0, 6.0, 12 and 24 Hours Post-Exposure to 1064 nm, 3.6 ns Pulsed Laser Light

    DTIC Science & Technology

    2005-05-01

    USAFA TR 2005-05 Temporal Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells at 0.5, 1.0, 3.0, 6.0, 12 and 24 Hours...AIR FORCE ACADEMY COLORADO 80840 20050630 417 USAFA TR 2005-05 This article, "Temporal Differential Gene Expression in Explanted Human Retinal ...Differential Gene Expression in Explanted Human Retinal Pigment USAFA F05611-02-P-0471 Epithelial Cells at 0.5, 1.0, 3.0, 6.0, 12 and 24-Hours Post-Exposure

  4. Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration

    PubMed Central

    Jones, Melissa K.; Lu, Bin; Saghizadeh, Mehrnoosh

    2016-01-01

    Purpose Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell–based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function. Furthermore, neural progenitor cells are currently in clinical trials for treating age-related macular degeneration; however, the molecular mechanisms of stem cell–based therapies are largely unknown. This is the first study to analyze gene expression changes in the retina of RCS rats following subretinal injection of hNPCs using high-throughput sequencing. Methods RNA-seq data of retinas from RCS rats injected with hNPCs (RCShNPCs) were compared to sham surgery in RCS (RCSsham) and wild-type Long Evans (LEsham) rats. Differential gene expression patterns were determined with in silico analysis and confirmed with qRT-PCR. Function, biologic, cellular component, and pathway analyses were performed on differentially expressed genes and investigated with immunofluorescent staining experiments. Results Analysis of the gene expression data sets identified 1,215 genes that were differentially expressed between RCSsham and LEsham samples. Additionally, 283 genes were differentially expressed between the RCShNPCs and RCSsham samples. Comparison of these two gene sets identified 68 genes with inverse expression (termed rescue genes), including Pdc, Rp1, and Cdc42ep5. Functional, biologic, and cellular component analyses indicate that the immune response is enhanced in RCSsham. Pathway analysis of the differential expression gene sets identified three affected pathways in RCShNPCs, which all play roles in phagocytosis signaling. Immunofluorescent

  5. Ligation of CD40 in Human Müller Cells Induces P2X7 Receptor–Dependent Death of Retinal Endothelial Cells

    PubMed Central

    Portillo, Jose-Andres C.; Lopez Corcino, Yalitza; Dubyak, George R.; Kern, Timothy S.; Matsuyama, Shigemi; Subauste, Carlos S.

    2016-01-01

    Purpose Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox. Methods Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry. Results Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7–mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner. Conclusions Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration. PMID:27893093

  6. Programming Retinal Stem Cells into Cone Photoreceptors

    DTIC Science & Technology

    2015-12-01

    to program human stem cells directly into cones. Using RNA -seq, we identified several genes that are upregulated in advance of the earliest...reverse vision loss. 15. SUBJECT TERMS Cone photoreceptor, retina, retinal stem cell, Otx2, Onecut1, Blimp1, RNA -seq., transcription factors, and...1 Keywords: 1. Cone photoreceptor 2. Retina 3. Retinal stem cell 4. Otx2 5. Onecut1 6. Blimp1 7. RNA -seq. 8. Transcription factors 9

  7. Retinal vessel oximetry: toward absolute calibration

    NASA Astrophysics Data System (ADS)

    Smith, Matthew H.; Denninghoff, Kurt R.; Lompado, Arthur; Hillman, Lloyd W.

    2000-06-01

    Accurately measuring the oxygen saturation of blood within retinal arteries and veins has proven to be a deceptively difficult task. Despite the excellent optical accessibility of the vessels and a wide range of reported instrumentation, we are unaware of any measurement technique that has proven to be calibrated across wide ranges of vessel diameter and fundus pigmentation. We present an overview of our retinal oximetry technique, present the results of an in vitro calibration experiment, and present preliminary human data.

  8. Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and ziv-aflibercept on human retinal pigment epithelium cells in culture

    PubMed Central

    Malik, Deepika; Tarek, Mohamed; Caceres del Carpio, Javier; Ramirez, Claudio; Boyer, David; Kenney, M Cristina; Kuppermann, Baruch D

    2014-01-01

    Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Methods Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Results Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumab-treated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mitochondrial membrane potential was seen at 1×, 2× and 10× concentrations of bevacizumab (86.53%, 74.38%, 66.67%; p<0.01) and ziv-aflibercept (73.50%, 64.83% and 49.65% p<0.01) suggestive of early apoptosis at lower doses, including the clinical doses. Conclusions At clinical doses, neither ranibizumab nor aflibercept produced evidence of mitochondrial toxicity or cell death. However, bevacizumab and ziv-aflibercept showed mild mitochondrial toxicity at clinically relevant doses. PMID:24836865

  9. The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells.

    PubMed

    Johansson, Ida; Monsen, Vivi Talstad; Pettersen, Kristine; Mildenberger, Jennifer; Misund, Kristine; Kaarniranta, Kai; Schønberg, Svanhild; Bjørkøy, Geir

    2015-01-01

    Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD.

  10. 3,3'-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB pathways in human retinal pigment epithelial cells under chemical hypoxic conditions.

    PubMed

    Park, Hongzoo; Lee, Dae-Sung; Yim, Mi-Jin; Choi, Yung Hyun; Park, Saegwang; Seo, Su-Kil; Choi, Jung Sik; Jang, Won Hee; Yea, Sung Su; Park, Won Sun; Lee, Chang-Min; Jung, Won-Kyo; Choi, Il-Whan

    2015-07-01

    Oxidative stress in the retinal pigment epithelium (RPE) can lead to the pathological causes of age-related macular degeneration (AMD). Hypoxia induces oxidative damage in retinal pigment epithelial cells (RPE cells). In this study, we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the expression of vascular endothelial growth factor (VEGF) under hypoxic conditions, as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells) were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The production of VEGF was measured by enzyme-linked immunosorbent assay. The translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) was determined by western blot analysis. The binding activity of HIF-1α and NF-κB was analyzed by electrophoretic mobility shift assay. The phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured by western blot analysis. The levels of mitochondrial reactive oxygen species (ROS) were detected by fluorescence microplate assay. The results revealed that DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19 cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were also attenuated by treatment with DIM. In addition, DIM inhibited the CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken together, the findings of our study demonstrate that DIM inhibits the CoCl2-induced production of VEGF by suppressing mitochondrial ROS production, thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.

  11. 10 CFR 63.321 - Individual protection standard for human intrusion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Individual protection standard for human intrusion. 63.321... Standards Human Intrusion Standard § 63.321 Individual protection standard for human intrusion. (a) DOE must determine the earliest time after disposal that the waste package would degrade sufficiently that a...

  12. 10 CFR 63.321 - Individual protection standard for human intrusion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Individual protection standard for human intrusion. 63.321... Standards Human Intrusion Standard § 63.321 Individual protection standard for human intrusion. (a) DOE must determine the earliest time after disposal that the waste package would degrade sufficiently that a...

  13. 10 CFR 63.321 - Individual protection standard for human intrusion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Individual protection standard for human intrusion. 63.321... Standards Human Intrusion Standard § 63.321 Individual protection standard for human intrusion. (a) DOE must determine the earliest time after disposal that the waste package would degrade sufficiently that a...

  14. 76 FR 58006 - Meeting of the Secretary's Advisory Committee on Human Research Protections

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-19

    ... HUMAN SERVICES Meeting of the Secretary's Advisory Committee on Human Research Protections AGENCY.... Appendix 2, notice is hereby given that the Secretary's Advisory Committee on Human Research Protections... SACHRP and the meeting agenda will be posted on th