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

DEFF Research Database (Denmark)

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

1998-01-01

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

Carrier-Mediated Transport of Nicotine Across the Inner Blood-Retinal Barrier: Involvement of a Novel Organic Cation Transporter Driven by an Outward H(+) Gradient.

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Tega, Yuma; Kubo, Yoshiyuki; Yuzurihara, Chihiro; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

2015-09-01

The present study was carried out to investigate the blood-to-retina transport of nicotine across the inner blood-retinal barrier (BRB). Using the in vivo vascular injection method, the blood-to-retina influx clearance of nicotine across the BRB was determined as 131 μL/(min?g retina), which is much higher than that of a nonpermeable paracellular marker, and blood-to-retina transport of nicotine was inhibited by organic cations such as pyrilamine and verapamil. The nicotine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro model of the inner BRB, exhibited time, temperature, and concentration dependence with a Km of 492 μM. These results suggest the involvement of a carrier-mediated transport process in nicotine transport in the inner BRB. The nicotine uptake by TR-iBRB2 cells was stimulated by an outwardly directed H(+) gradient, and the uptake was significantly inhibited by bulky and hydrophobic cationic drugs, whereas inhibitors of organic cation transporters did not show inhibitory effect. These results suggest that the novel organic cation transport system driven by an outwardly directed H(+) gradient is involved in the blood-to-retina transport of nicotine across the inner BRB. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Effects of simvastatin on CAT-1-mediated arginine transport and NO level under high glucose conditions in conditionally immortalized rat inner blood-retinal barrier cell lines (TR-iBRB).

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Tun, Temdara; Kang, Young-Sook

2017-05-01

Hyperglycemia causes the breakdown of the blood-retinal barrier by impairing endothelial nitric oxide synthase (eNOS) function. Statins have many pleiotropic effects such as improving endothelial barrier permeability and increasing eNOS mRNA stability. The objective of this study was to determine effect of simvastatin on l-arginine transport and NO production under high-glucose conditions in conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB). Changes in l-arginine transport uptake and, expression levels of cationic amino acid transporter 1 (CAT-1) and eNOS mRNA were investigated after pre-treatment with simvastatin and NOS inhibitors (l-NMMA and l-NAME) under high-glucose conditions using TR-iBRB, an in vitro model of iBRB. The NO level released from TR-iBRB cells was examined using Griess reagents. Under high glucose conditions, [ 3 H]l-arginine uptake was decreased in TR-iBRB cells. Simvastatin pretreatment elevated [ 3 H]l-arginine uptake, the expression levels of CAT-1 and eNOS mRNA, and NO production under high-glucose conditions. Moreover, the co-treatment with simvastatin and NOS inhibitors reduced [ 3 H]l-arginine uptake compared to pretreatment with simvastatin alone. Our results suggest that, in the presence of high-glucose levels, increased l-arginine uptake due to simvastatin treatment was associated with increased CAT-1 and eNOS mRNA levels, leading to higher NO production in TR-iBRB cells. Thus, simvastatin might be a good modulator for diabetic retinopathy therapy by increasing of the l-arginine uptake and improving endothelial function in retinal capillary endothelial cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Propranolol transport across the inner blood-retinal barrier: potential involvement of a novel organic cation transporter.

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Kubo, Yoshiyuki; Shimizu, Yoshimi; Kusagawa, Yusuke; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

2013-09-01

The influx transport of propranolol across the inner blood-retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [(3) H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [(3) H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [(3) H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [(3) H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter. Copyright © 2013 Wiley Periodicals, Inc.

Protection of blood retinal barrier and systemic vasculature by insulin-like growth factor binding protein-3.

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Yagna P R Jarajapu

Full Text Available Previously, we showed that insulin growth factor (IGF-1 binding protein-3 (IGFBP-3, independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR. The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1 and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP. IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab. Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs. NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+/calmodulin-dependent protein kinase II (CamKII blockade; however, dephosphorylation of eNOS-Thr(495 was observed. Phosphatidylinositol 3-kinase (PI3K activity and Akt-Ser(473 phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate

Placental Growth Factor Contributes to Micro-Vascular Abnormalization and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

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Kowalczuk, Laura; Touchard, Elodie; Omri, Samy; Jonet, Laurent; Klein, Christophe; Valamanes, Fatemeh; Berdugo, Marianne; Bigey, Pascal; Massin, Pascale; Jeanny, Jean-Claude; Behar-Cohen, Francine

2011-01-01

Objective There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). Materials and Methods pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. Results After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. Conclusion This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease. PMID:21408222

Blood-aqueous Barrier Function in a Patient With Choroideremia

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Muh-Shy Chen

2010-02-01

Full Text Available The purpose was to determine whether there was a breakdown of the blood-aqueous barrier in a patient with choroideremia. A 27-year-old man with typical choroideremia underwent standardized ophthalmo-logical evaluation, including quantitative measurement of aqueous flare intensity, by a laser flare-cell meter. The results showed areas of atrophy of the choriocapillaries and retinal pigment epithelium in the mid-periphery and posterior pole, although not in the macula. Fluorescein angiography showed areas of loss of the choriocapillaries and retinal pigment epithelium. The fovea was spared with a surrounding zone of hy-perfluorescence. Electroretinography showed a subnormal photopic amplitude and extinguished scotopic response. Electrooculography revealed that the light peak/dark trough ratio was reduced. Goldmann perimetry showed constricted peripheral fields. Laser photometry showed an increase in the aqueous flare intensity in both eyes, as compared with normal subjects. We conclude that the function of the blood-aqueous barrier might be affected in patients with choroideremia.

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

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Mingyue Luo

2018-01-01

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

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

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Luo, Mingyue; Chen, Youxin

2018-01-01

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

Blood-retinal barrier glycerol permeability in diabetic macular edema and healthy eyes: estimations from macular volume changes after peroral glycerol

DEFF Research Database (Denmark)

Thornit, Dorte Nellemann; Vinten, Carl Martin; Sander, Birgit

2010-01-01

PURPOSE: To compare the changes in macular volume (MV) between healthy subjects and patients with diabetic macular edema (DME) after an osmotic load and to determine the glycerol permeability (P(gly)) of the blood-retinal barrier (BRB). METHODS: In this unmasked study, 13 patients with DME and 5...

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

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

2017-09-01

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

Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity

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Jiehao Zhou

2010-01-01

Full Text Available We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE. The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP- 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P<.05. Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P<.05 and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.

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

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

2015-01-01

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

Hypoxia-ischemia and retinal ganglion cell damage

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Charanjit Kaur

2008-08-01

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

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

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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.

Transplanting Retinal Cells using Bucky Paper for Support

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

2004-01-01

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

Retinal Macroglial Responses in Health and Disease

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Rosa de Hoz

2016-01-01

Full Text Available 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.

Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice.

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Cheng, Zhuo; Wang, Liping; Qu, Meijie; Liang, Huaibin; Li, Wanlu; Li, Yongfang; Deng, Lidong; Zhang, Zhijun; Yang, Guo-Yuan

2018-05-03

Ischemic stroke induced matrixmetallo-proteinase-9 (MMP-9) upregulation, which increased blood-brain barrier permeability. Studies demonstrated that mesenchymal stem cell therapy protected blood-brain barrier disruption from several cerebrovascular diseases. However, the underlying mechanism was largely unknown. We therefore hypothesized that mesenchymal stem cells reduced blood-brain barrier destruction by inhibiting matrixmetallo-proteinase-9 and it was related to intercellular adhesion molecule-1 (ICAM-1). Adult ICR male mice (n = 118) underwent 90-min middle cerebral artery occlusion and received 2 × 10 5 mesenchymal stem cell transplantation. Neurobehavioral outcome, infarct volume, and blood-brain barrier permeability were measured after ischemia. The relationship between myeloperoxidase (MPO) activity and ICAM-1 release was further determined. We found that intracranial injection of mesenchymal stem cells reduced infarct volume and improved behavioral function in experimental stroke models (p mesenchymal stem cell-treated mice compared to the control group following ischemia (p cells and myeloperoxidase activity were decreased in mesenchymal stem cell-treated mice (p mesenchymal stem cell therapy attenuated blood-brain barrier disruption in mice after ischemia. Mesenchymal stem cells attenuated the upward trend of MMP-9 and potentially via downregulating ICAM-1 in endothelial cells. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway may influence MMP-9 expression of neutrophils and resident cells, and ICAM-1 acted as a key factor in the paracrine actions of mesenchymal stem cell.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

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Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael; Nolan, Daniel J; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M; Prusky, Glen T; Llanos, Pierre; Rabbany, Sina Y; Maminishkis, Arvydas; Miller, Sheldon S; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-05-19

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

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

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

2017-08-18

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

Knockdown of Zebrafish Blood Vessel Epicardial Substance Results in Incomplete Retinal Lamination

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Yu-Ching Wu

2014-01-01

Full Text Available Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL and retinal pigment epithelium (RPE. In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination.

The blood-tendon barrier: identification and characterisation of a novel tissue barrier in tendon blood vessels

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C Lehner

2016-05-01

Full Text Available Tissue barriers function as “gate keepers” between different compartments (usually blood and tissue and are formed by specialised membrane-associated proteins, localising to the apicolateral plasma membrane domain of epithelial and endothelial cells. By sealing the paracellular space, the free diffusion of solutes and molecules across epithelia and endothelia is impeded. Thereby, tissue barriers contribute to the establishment and maintenance of a distinct internal and external environment, which is crucial during organ development and allows maintenance of an organ-specific homeostatic milieu. So far, various epithelial and endothelial tissue barriers have been described, including the blood-brain barrier, the blood-retina barrier, the blood-testis barrier, the blood-placenta barrier, and the cerebrospinal fluid (CSF-brain barrier, which are vital for physiological function and any disturbance of these barriers can result in severe organ damage or even death. Here, we describe the identification of a novel barrier, located in the vascular bed of tendons, which we term the blood-tendon barrier (BTB. By using immunohistochemistry, transmission electron microscopy, and tracer studies we demonstrate the presence of a functional endothelial barrier within tendons restricting the passage of large blood-borne molecules into the surrounding tendon tissue. We further provide in vitro evidence that the BTB potentially contributes to the creation of a distinct internal tissue environment impacting upon the proliferation and differentiation of tendon-resident cells, effects which might be fundamental for the onset of tendon pathologies.

[Blood-brain barrier part III: therapeutic approaches to cross the blood-brain barrier and target the brain].

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Weiss, N; Miller, F; Cazaubon, S; Couraud, P-O

2010-03-01

Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches. Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.

Α-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R.

Science.gov (United States)

Cai, Siwei; Yang, Qianhui; Hou, Mengzhu; Han, Qian; Zhang, Hanyu; Wang, Jiantao; Qi, Chen; Bo, Qiyu; Ru, Yusha; Yang, Wei; Gu, Zhongxiu; Wei, Ruihua; Cao, Yunshan; Li, Xiaorong; Zhang, Yan

2018-01-01

Blood-retinal barrier (BRB) breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy (DR). Hyperglycemia-induced oxidative stress and inflammation are primary pathogenic factors of this severe DR complication. An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage. This study sought to examine the protective effects of α-Melanocyte-stimulating hormone (α-MSH) on early diabetic retina against vascular hyperpermeability, electrophysiological dysfunction, and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the α-MSH's anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells (RF6A). Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes. The rats were intravitreally injected with α-MSH or saline at Week 1 and 3 after hyperglycemia. In another 2 weeks, Evans blue assay, transmission electron microscopy, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to examine the protective effects of α-MSH in diabetic retinas. The expression of pro-inflammatory factors and tight junction at mRNA and protein levels in retinas was analyzed. Finally, the α-MSH's anti-hyperpermeability was confirmed in a high glucose (HG)-treated RF6A cell monolayer transwell culture by transendothelial electrical resistance (TEER) measurement and a fluorescein isothiocyanate-Dextran assay. Universal or specific melanocortin receptor (MCR) blockers were also employed to elucidate the MCR subtype mediating α-MSH's protection. Evans blue assay showed that BRB breakdown and vascular leakage was detected, and rescued by α-MSH both qualitatively and quantitatively in early diabetic retinas; electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in α-MSH-treated diabetic retinas; scotopic ERG suggested

Blood pressure modifies retinal susceptibility to intraocular pressure elevation.

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Zheng He

Full Text Available Primary open angle glaucoma affects more than 67 million people. Elevated intraocular pressure (IOP is a risk factor for glaucoma and may reduce nutrient availability by decreasing ocular perfusion pressure (OPP. An interaction between arterial blood pressure and IOP determines OPP; but the exact contribution that these factors have for retinal function is not fully understood. Here we sought to determine how acute modifications of arterial pressure will affect the susceptibility of neuronal function and blood flow to IOP challenge. Anaesthetized (ketamine:xylazine Long-Evan rats with low (∼60 mmHg, sodium nitroprusside infusion, moderate (∼100 mmHg, saline, or high levels (∼160 mmHg, angiotensin II of mean arterial pressure (MAP, n = 5-10 per group were subjected to IOP challenge (10-120 mmHg, 5 mmHg steps every 3 minutes. Electroretinograms were measured at each IOP step to assess bipolar cell (b-wave and inner retinal function (scotopic threshold response or STR. Ocular blood flow was measured using laser-Doppler flowmetry in groups with similar MAP level and the same IOP challenge protocol. Both b-wave and STR amplitudes decreased with IOP elevation. Retinal function was less susceptible to IOP challenge when MAP was high, whereas the converse was true for low MAP. Consistent with the effects on retinal function, higher IOP was needed to attenuated ocular blood flow in animals with higher MAP. The susceptibility of retinal function to IOP challenge can be ameliorated by acute high BP, and exacerbated by low BP. This is partially mediated by modifications in ocular blood flow.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

Science.gov (United States)

Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

2013-06-18

Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS

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

DEFF Research Database (Denmark)

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

1998-01-01

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

Upregulated inflammatory associated factors and blood-retinal barrier changes in the retina of type 2 diabetes mellitus model

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Rui-Jin Ran

2016-11-01

Full Text Available AIM: To examine the expression of high mobility group box-1 (HMGB-1 and intercellular adhesion molecule-1 (ICAM-1 in the retina and the hippocampal tissues; and further to evaluate the association of these two molecules with the alterations of blood-retinal barrier (BRB and blood-brain barrier (BBB in a rat model of type 2 diabetes. METHODS: The type-2 diabetes mellitus (DM model was established with a high-fat and high-glucose diet combined with streptozotocin (STZ. Sixteen weeks after DM induction, morphological changes of retina and hippocampus were observed with hematoxylin-eosin staining, and alternations of BRB and BBB permeability were measured using Evans blue method. Levels of HMGB-1 and ICAM-1 in retina and hippocampus were detected by Western blot. Serum HMGB-1 levels were determined by enzyme-linked immunosorbent assay (ELISA. RESULTS: A significantly higher serum fasting blood glucose level in DM rats was observed 2wk after STZ injection (P＜0.01. The serum levels of fasting insulin, Insulin resistance homeostatic model assessment (IRHOMA, total cholesterol (TC, total triglycerides (TG and low density lipoprotein cholesterol (LDL-C in the DM rats significantly higher than those in the controls (all P＜0.01. HMGB-1 (0.96±0.03, P＜0.01 and ICAM-1 (0.76±0.12, P＜0.05 levels in the retina in the DM rats were significantly higher than those in the controls. HMGB-1 (0.83±0.13, P＜0.01 and ICAM-1 (1.15±0.08, P＜0.01 levels in the hippocampal tissues in the DM rats were also significantly higher than those in the controls. Sixteen weeks after induction of DM, the BRB permeability to albumin-bound Evans blue dye in the DM rats was significantly higher than that in the controls (P＜0.01. However, there was no difference of BBB permeability between the DM rats and controls. When compared to the controls, hematoxylin and eosin staining showed obvious irregularities in the DM rats. CONCLUSION: BRB permeability increases significantly

Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat.

Science.gov (United States)

Arakaki, Xianghong; McCleary, Paige; Techy, Matthew; Chiang, Jiarong; Kuo, Linus; Fonteh, Alfred N; Armstrong, Brian; Levy, Dan; Harrington, Michael G

2013-03-14

Cerebrospinal fluid (CSF) sodium concentration increases during migraine attacks, and both CSF and vitreous humor sodium increase in the rat migraine model. The Na,K-ATPase is a probable source of these sodium fluxes. Since Na,K-ATPase isoforms have different locations and physiological roles, our objective was to establish which alpha isoforms are present at sites where sodium homeostasis is disrupted. Specific Na,K-ATPase alpha isoforms were identified in rat tissues by immunohistochemistry at the blood-CSF barrier at the choroid plexus, at the blood-CSF-trigeminal barrier at the meninges, at the blood-retina barrier, and at the blood-aqueous barrier at the ciliary body. Calcitonin gene-related peptide (CGRP), occludin, or von Willibrand factor (vWF) were co-localized with Na,K-ATPase to identify trigeminal nociceptor fibers, tight junctions, and capillary endothelial cells respectively. The Na,K-ATPase alpha-2 isoform is located on capillaries and intensely at nociceptive trigeminal nerve fibers at the meningeal blood-CSF-trigeminal barrier. Alpha-1 and -3 are lightly expressed on the trigeminal nerve fibers but not at capillaries. Alpha-2 is expressed at the blood-retina barriers and, with alpha-1, at the ciliary body blood aqueous barrier. Intense apical membrane alpha-1 was associated with moderate cytoplasmic alpha-2 expression at the choroid plexus blood-CSF barrier. Na,K-ATPase alpha isoforms are present at the meningeal, choroid plexus, and retinal barriers. Alpha-2 predominates at the capillary endothelial cells in the meninges and retinal ganglion cell layer.

Quantitative and qualitative retinal microvascular characteristics and blood pressure.

Science.gov (United States)

Cheung, Carol Y; Tay, Wan T; Mitchell, Paul; Wang, Jie J; Hsu, Wynne; Lee, Mong L; Lau, Qiangfeng P; Zhu, Ai L; Klein, Ronald; Saw, Seang M; Wong, Tien Y

2011-07-01

The present study examined the effects of blood pressure on a spectrum of quantitative and qualitative retinal microvascular signs. Retinal photographs from the Singapore Malay Eye Study, a population-based cross-sectional study of 3280 (78.7% response) persons aged 40-80 years, were analyzed. Quantitative changes in the retinal vasculature (branching angle, vascular tortuosity, fractal dimension, and vascular caliber) were measured using a semi-automated computer-based program. Qualitative signs, including focal arteriolar narrowing (FAN), arteriovenous nicking (AVN), opacification of the arteriolar wall (OAW), and retinopathy (e.g., microaneurysms, retinal hemorrhages), were assessed from photographs by trained technicians. After excluding persons with diabetes and ungradable photographs, 1913 persons provided data for this analysis. In multivariable linear regression models controlling for age, sex, BMI, use of antihypertensive medication, and other factors, retinal arteriolar branching asymmetry ratio, arteriolar tortuosity, venular tortuosity, fractal dimension, arteriolar caliber, venular caliber, FAN, AVN, and retinopathy were independently associated with mean arterial blood pressure. In contrast, arteriolar/venular branching angle, venular branching asymmetry ratio and OAW were not related to blood pressure. Retinal arteriolar caliber (sβ = -0.277) and FAN (sβ = 0.170) had the strongest associations with mean arterial blood pressure, and higher blood pressure levels were associated with increasing number of both quantitative and qualitative retinal vascular signs (P trend qualitative retinal vascular signs, with the number of signs increasing with higher blood pressure levels.

In vitro models of the blood-brain barrier

DEFF Research Database (Denmark)

Helms, Hans Christian Cederberg; Abbott, N Joan; Burek, Malgorzata

2016-01-01

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic...... components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug...... transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture...

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

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Kati Juuti-Uusitalo

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

LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

Science.gov (United States)

Pelkonen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Tachikawa, Masanori; Watanabe, Michitoshi; Uchida, Yasuo; Urtti, Arto; Terasaki, Tetsuya

2017-03-06

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na + /K + ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

Lysosomal storage diseases and the blood-brain barrier.

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Begley, David J; Pontikis, Charles C; Scarpa, Maurizio

2008-01-01

The blood-brain barrier becomes a crucial issue in neuronopathic lysosomal storage diseases for three reasons. Firstly, the function of the blood-brain barrier may be compromised in many of the lysosomal storage diseases and this barrier dysfunction may contribute to the neuropathology seen in the diseases and accelerate cell death. Secondly, the substrate reduction therapies, which successfully reduce peripheral lysosomal storage, because of the blood-brain barrier may not have as free an access to brain cells as they do to peripheral cells. And thirdly, enzyme replacement therapy appears to have little access to the central nervous system as the mannose and mannose-6-phosphate receptors involved in their cellular uptake and transport to the lysosome do not appear to be expressed at the adult blood-brain barrier. This review will discuss in detail these issues and their context in the development of new therapeutic strategies.

Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

International Nuclear Information System (INIS)

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

2010-01-01

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

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

Science.gov (United States)

Zhang, Qian; Zhang, Zuo-Ming

2014-04-25

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

Automatic segmentation of blood vessels from retinal fundus images ...

Indian Academy of Sciences (India)

The retinal blood vessels were segmented through color space conversion and color channel .... Retinal blood vessel segmentation was also attempted through multi-scale operators. A few works in this ... fundus camera at 35 degrees field of view. The image ... vessel segmentation is available from two human observers.

Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

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Sharma Kamal

2008-12-01

Full Text Available Abstract Background Effective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells. Methods Magnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging. Results We found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells. Conclusion The therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

An oncological view on the blood-testis barrier

NARCIS (Netherlands)

Bart, J; Groen, HJM; van der Graaf, WTA; Hollema, H; Hendrikse, NH; Vaalburg, W; Sleijfer, DT; de Vries, EGE

The function of the blood-testis barrier is to protect germ cells from harmful influences; thus, it also impedes the delivery of chemotherapeutic drugs to the testis. The barrier has three components: first, a physicochemical barrier consisting of continuous capillaries, Sertoli cells in the tubular

Radiation effects of electromagnetic pulses on mouse blood-testis barrier

International Nuclear Information System (INIS)

Hou Wugang; Zhao Jie; Zhang Yuanqiang

2005-01-01

Radiation effects caused by 100 kV/m and 400 kV/m electromagnetic pulse (EMP) irradiations on mouse blood-testis barrier were studied by means of routine HE staining, Lanthanum traced electron microscope and injection of caudal vein with Evans Blue. The EMP irradiation of different dose rates damaged Sertoli's cell and blood-testis barrier of mouse testis in different levels. Severe injuries were observed with the 400 kV/m irradiation group, with apoptosis and necrosis in a large quantity of the spermatogenic cells, shape and structural changes of the Sertoli's cells, and serious injuries to the blood-testis barrier, one day after the irradiation. The basal compartment separated from the adluminal compartment in most of the VIII stage seminiferous epithelium, and a great number of apoptosis and necrosis spermatogenic cells were released into the cavities. Injuries of blood-testis barrier could be observed 21 days after the 400 kV/m irradiation. The injuries of 100 kV/m irradiation groups were less severe than the 400 kV/m groups, in which the damages to the Sertoli's cells, the seminiferous epithelium and blood-testis barrier recovered to some extent 14 days after the irradiation. The authors conclude that EMP irradiation can damage mouse blood-tests barrier. The injuries, and the time for recovery, are related to EMP power intensity. (authors)

The Drosophila blood-brain barrier: Development and function of a glial endothelium

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Stefanie eLimmer

2014-11-01

Full Text Available The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

The Drosophila blood-brain barrier: development and function of a glial endothelium.

Science.gov (United States)

Limmer, Stefanie; Weiler, Astrid; Volkenhoff, Anne; Babatz, Felix; Klämbt, Christian

2014-01-01

The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

Retinal stem cells and potential cell transplantation treatments

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Tai-Chi Lin

2014-11-01

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

Application of morphological bit planes in retinal blood vessel extraction.

Science.gov (United States)

Fraz, M M; Basit, A; Barman, S A

2013-04-01

The appearance of the retinal blood vessels is an important diagnostic indicator of various clinical disorders of the eye and the body. Retinal blood vessels have been shown to provide evidence in terms of change in diameter, branching angles, or tortuosity, as a result of ophthalmic disease. This paper reports the development for an automated method for segmentation of blood vessels in retinal images. A unique combination of methods for retinal blood vessel skeleton detection and multidirectional morphological bit plane slicing is presented to extract the blood vessels from the color retinal images. The skeleton of main vessels is extracted by the application of directional differential operators and then evaluation of combination of derivative signs and average derivative values. Mathematical morphology has been materialized as a proficient technique for quantifying the retinal vasculature in ocular fundus images. A multidirectional top-hat operator with rotating structuring elements is used to emphasize the vessels in a particular direction, and information is extracted using bit plane slicing. An iterative region growing method is applied to integrate the main skeleton and the images resulting from bit plane slicing of vessel direction-dependent morphological filters. The approach is tested on two publicly available databases DRIVE and STARE. Average accuracy achieved by the proposed method is 0.9423 for both the databases with significant values of sensitivity and specificity also; the algorithm outperforms the second human observer in terms of precision of segmented vessel tree.

Experimental model for research on the blood-ocular barrier

Energy Technology Data Exchange (ETDEWEB)

Kim, Hak Jin; Jea, Seung Youn; Park, Jae Sung; Jung, Yeon Joo [Pusan National University, Pusan (Korea, Republic of); Kim, Yong Woo [Inje University, Kimhae (Korea, Republic of); Park, Byung Rae [Catholic University, Seoul (Korea, Republic of)

2006-03-15

The eyeball has 2 blood-ocular barriers, i.e, the blood-retinal and blood-aqueous barriers. The purpose of this study was to evaluate if triolein emulsion could disrupt the barriers, and we wanted to suggest as an experimental model for future blood-ocular barrier studies. The triolein emulsion was made of 0.1 ml triolein and 20 ml normal saline, and this was infused into the carotid artery of ten cats (the experimental group). As a control group, only normal saline was infused in another ten cats. Precontrast and postcontrast T1-weighted MR images were obtained at 30 minutes and 3 hours after embolization in both groups. The signal intensities were evaluate qualitatively and quantitatively in the anterior and posterior chambers and also in the vitreus fluid. Statistical analysis was performed by employing the Kruskal Wallist test, Dunn's Multiple Comparison test and the wilcoxon signed rank test. In the control group, no contrast enhancement was demonstrated in the anterior or posterior chamber or in the vitreus fluid of the ipsilateral or contralateral eyeball on the 30 minutes MR images. The anterior chambers of the ipsilateral and contralateral eyeballs revealed delayed contrast enhancement on the 3 hour MR images. In the experimental group, the 30 minute-postembolization MR images were not different from those of the control group. The 30 minute-postembolization MR images demonstrated delayed contrast enhancement in the anterior chamber of the ipsilateral and contralateral eyeballs and in the posterior chamber of the ipsilateral eyeball. The delayed contrast enhancement of the posterior chamber of the ipsilateral eyeball was statistically significant ({rho} < 0.05). The present study demonstrated significant contrast enhancement in the posterior chamber with infusion of the triolein emulsion, and this can serve as a model for blood-aqueous barrier studies.

Experimental model for research on the blood-ocular barrier

International Nuclear Information System (INIS)

Kim, Hak Jin; Jea, Seung Youn; Park, Jae Sung; Jung, Yeon Joo; Kim, Yong Woo; Park, Byung Rae

2006-01-01

The eyeball has 2 blood-ocular barriers, i.e, the blood-retinal and blood-aqueous barriers. The purpose of this study was to evaluate if triolein emulsion could disrupt the barriers, and we wanted to suggest as an experimental model for future blood-ocular barrier studies. The triolein emulsion was made of 0.1 ml triolein and 20 ml normal saline, and this was infused into the carotid artery of ten cats (the experimental group). As a control group, only normal saline was infused in another ten cats. Precontrast and postcontrast T1-weighted MR images were obtained at 30 minutes and 3 hours after embolization in both groups. The signal intensities were evaluate qualitatively and quantitatively in the anterior and posterior chambers and also in the vitreus fluid. Statistical analysis was performed by employing the Kruskal Wallist test, Dunn's Multiple Comparison test and the wilcoxon signed rank test. In the control group, no contrast enhancement was demonstrated in the anterior or posterior chamber or in the vitreus fluid of the ipsilateral or contralateral eyeball on the 30 minutes MR images. The anterior chambers of the ipsilateral and contralateral eyeballs revealed delayed contrast enhancement on the 3 hour MR images. In the experimental group, the 30 minute-postembolization MR images were not different from those of the control group. The 30 minute-postembolization MR images demonstrated delayed contrast enhancement in the anterior chamber of the ipsilateral and contralateral eyeballs and in the posterior chamber of the ipsilateral eyeball. The delayed contrast enhancement of the posterior chamber of the ipsilateral eyeball was statistically significant (ρ < 0.05). The present study demonstrated significant contrast enhancement in the posterior chamber with infusion of the triolein emulsion, and this can serve as a model for blood-aqueous barrier studies

Advances in Retinal Stem Cell Biology

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Andrea S Viczian

2013-01-01

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

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

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

2012-04-10

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

Relationship between retinal blood flow and arterial oxygen.

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Cheng, Richard W; Yusof, Firdaus; Tsui, Edmund; Jong, Monica; Duffin, James; Flanagan, John G; Fisher, Joseph A; Hudson, Chris

2016-02-01

Vascular reactivity, the response of the vessels to a vasoactive stimulus such as hypoxia and hyperoxia, can be used to assess the vascular range of adjustment in which the vessels are able to compensate for changes in PO2. Previous studies in the retina have not accurately quantified retinal vascular responses and precisely targeted multiple PaO2 stimuli at the same time as controlling the level of carbon dioxide, thus precluding them from modelling the relationship between retinal blood flow and oxygen. The present study modelled the relationship between retinal blood flow and PaO2, showing them to be a combined linear and hyperbolic function. This model demonstrates that the resting tonus of the vessels is at the mid-point and that they have great vascular range of adjustment, compensating for decreases in oxygen above a PETCO2 of 32-37 mmHg but being limited below this threshold. Retinal blood flow (RBF) increases in response to a reduction in oxygen (hypoxia) but decreases in response to increased oxygen (hyperoxia). However, the relationship between blood flow and the arterial partial pressure of oxygen has not been quantified and modelled in the retina, particularly in the vascular reserve and resting tonus of the vessels. The present study aimed to determine the limitations of the retinal vasculature by modelling the relationship between RBF and oxygen. Retinal vascular responses were measured in 13 subjects for eight different blood gas conditions, with the end-tidal partial pressure of oxygen (PETCO2) ranging from 40-500 mmHg. Retinal vascular response measurements were repeated twice; using the Canon laser blood flowmeter (Canon Inc., Tokyo, Japan) during the first visit and using Doppler spectral domain optical coherence tomography during the second visit. We determined that the relationship between RBF and PaO2 can be modelled as a combination of hyperbolic and linear functions. We concluded that RBF compensated for decreases in arterial oxygen content

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

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Park, Susanna S

2016-04-01

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

The Blood-Brain Barrier: An Engineering Perspective

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Andrew eWong

2013-08-01

Full Text Available It has been more than 100 years since Paul Ehrlich reported that various water-soluble dyes injected into the circulation did not enter the brain. Since Ehrlich’s first experiments, only a small number of molecules, such as alcohol and caffeine have been found to cross the blood-brain barrier, and it remains the major roadblock to treatment of many central nervous system diseases. At the same time, many central nervous system diseases are associated with disruption of the blood-brain barrier that can lead to changes in permeability, modulation of immune cell transport, and trafficking of pathogens into the brain. Therefore advances in our understanding of the structure and function of the blood-brain barrier are key to advances in treatment of a wide range of central nervous system diseases. Over the past 10 years it has become recognized that the blood-brain barrier is a complex dynamic system that involves biomechanical and biochemical signaling between the vascular system and the brain. Here we reconstruct the structure, function, and transport properties of the blood-brain barrier from an engineering perspective. New insight into the physics of the blood-brain barrier could ultimately lead to clinical advances in the treatment of central nervous system diseases.

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

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Sanie-Jahromi Fatemeh

2012-04-01

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

Glutamate Transporters in the Blood-Brain Barrier

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Helms, Hans Christian Cederberg; Nielsen, Carsten Uhd; Waagepetersen, Helle S

2017-01-01

concentration of L-glutamate causes excitotoxicity. A tight control of the brain interstitial fluid L-glutamate levels is therefore imperative, in order to maintain optimal neurotransmission and to avoid such excitotoxicity. The blood-brain barrier, i.e., the endothelial lining of the brain capillaries...... cells. The mechanisms underlying transendothelial L-glutamate transport are however still not well understood. The present chapter summarizes the current knowledge on blood-brain barrier L-glutamate transporters and the suggested pathways for the brain-to-blood L-glutamate efflux......., regulates the exchange of nutrients, gases, and metabolic waste products between plasma and brain interstitial fluid. It has been suggested that brain capillary endothelial cells could play an important role in L-glutamate homeostasis by mediating brain-to-blood L-glutamate efflux. Both in vitro and in vivo...

Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

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Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

2014-02-01

Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

Straight versus tortuous retinal arteries in relation to blood pressure and genetics

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Taarnhøj, N C B B; Munch, I C; Sander, B

2008-01-01

dizygotic same-sex healthy twin pairs, aged 20 to 46 years, who were characterised by determination of retinal vessel diameters, arterial blood pressure, blood glucose, body mass index, smoking habits and retinal arterial tortuosity, using a three-level grading scale (straight, wavy, tortuous). Heritability......BACKGROUND/AIMS: To assess the relative influence of genetic and environmental factors on retinal arterial tortuosity and the association between tortuosity and various health indices in healthy young to middle-aged persons. METHODS: This cross-sectional study included 57 monozygotic and 52...... accounting for the remaining 18% (CI(95 )8, 36%). Increasing values of mean arterial blood pressure and body mass index were both associated with decreasing levels of retinal arterial tortuosity. CONCLUSION: There was a large variation in tortuosity of retinal arteries in these healthy subjects...

A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

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Romeo Cecchelli

Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

Heritability of retinal vessel diameters and blood pressure

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Taarnhøj, Nina C B B; Larsen, Michael; Sander, Birgit

2006-01-01

PURPOSE: To assess the relative influence of genetic and environmental effects on retinal vessel diameters and blood pressure in healthy adults, as well as the possible genetic connection between these two characteristics. METHODS: In 55 monozygotic and 50 dizygotic same-sex healthy twin pairs......%-80%) for CRAE, 83% (95% CI: 73%-89%) for CRVE, and 61% (95% CI: 44%-73%) for mean arterial blood pressure (MABP). Retinal artery diameter decreased with increasing age and increasing arterial blood pressure. Mean vessel diameters in the population were 165.8 +/- 14.9 microm for CRAE, 246.2 +/- 17.7 microm...... for CRVE, and 0.67 +/- 0.05 microm for AVR. No significant influence on artery or vein diameters was found for gender, smoking, body mass index (BMI), total cholesterol, fasting blood glucose, or 2-hour oral glucose tolerance test values. CONCLUSIONS: In healthy young adults with normal blood pressure...

Barrier mechanisms in the Drosophila blood-brain barrier

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Samantha Jane Hindle

2014-12-01

Full Text Available The invertebrate blood-brain barrier field is growing at a rapid pace and, in recent years, studies have shown a physiologic and molecular complexity that has begun to rival its vertebrate counterpart. Novel mechanisms of paracellular barrier maintenance through GPCR signaling were the first demonstrations of the complex adaptive mechanisms of barrier physiology. Building upon this work, the integrity of the invertebrate blood-brain barrier has recently been shown to require coordinated function of all layers of the compound barrier structure, analogous to signaling between the layers of the vertebrate neurovascular unit. These findings strengthen the notion that many blood-brain barrier mechanisms are conserved between vertebrates and invertebrates, and suggest that novel findings in invertebrate model organisms will have a significant impact on the understanding of vertebrate BBB functions. In this vein, important roles in coordinating localized and systemic signaling to dictate organism development and growth are beginning to show how the blood-brain barrier can govern whole animal physiologies. This includes novel functions of blood-brain barrier gap junctions in orchestrating synchronized neuroblast proliferation, and of blood-brain barrier secreted antagonists of insulin receptor signaling. These advancements and others are pushing the field forward in exciting new directions. In this review, we provide a synopsis of invertebrate blood-brain barrier anatomy and physiology, with a focus on insights from the past 5 years, and highlight important areas for future study.

Na+/K+-ATPase α1 identified as an abundant protein in the blood-labyrinth barrier that plays an essential role in the barrier integrity.

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Yue Yang

2011-01-01

Full Text Available The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+/K(+-ATPase α1 (ATP1A1 with protein kinase C eta (PKCη and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma.The results presented here provide a novel method for

Meningeal mast cells affect early T cell central nervous system infiltration and blood-brain barrier integrity through TNF: a role for neutrophil recruitment?

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Sayed, Blayne A; Christy, Alison L; Walker, Margaret E; Brown, Melissa A

2010-06-15

Mast cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis, a rodent model of the human demyelinating disease multiple sclerosis. Yet their site and mode of action is unknown. In both diseases, myelin-specific T cells are initially activated in peripheral lymphoid organs. However, for disease to occur, these cells must enter the immunologically privileged CNS through a breach in the relatively impermeable blood-brain barrier. In this study, we demonstrate that a dense population of resident mast cells in the meninges, structures surrounding the brain and spinal cord, regulate basal CNS barrier function, facilitating initial T cell CNS entry. Through the expression of TNF, mast cells recruit an early wave of neutrophils to the CNS. We propose that neutrophils in turn promote the blood-brain barrier breach and together with T cells lead to further inflammatory cell influx and myelin damage. These findings provide specific targets for intervention in multiple sclerosis as well as other immune-mediated CNS diseases.

[The blood-brain barrier and drug delivery in the central nervous system].

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Loch-Neckel, Gecioni; Koepp, Janice

2010-08-01

To provide an updated view of the difficulties due to barriers and strategies used to allow the release of drugs in the central nervous system. The difficulty for the treatment of many diseases of the central nervous system, through the use of intra-venous drugs, is due to the presence of barriers that prevent the release of the same: the blood-brain barrier, blood-cerebro-spinal fluid barrier and the blood-arachnoid barrier. The blood-brain barrier is the main barrier for the transport of drugs in the brain that also acts as a immunologic and metabolic barrier. The endothelial cells of the blood-brain barrier are connected to a junction complex through the interaction of transmembrane proteins that protrude from de inside to the outside, forming a connection between the endothelial cells. The transport of substances to the brain depends on the mechanisms of transport present in the barrier and the diffusion of these compounds also depends on the physicochemical characteristics of the molecule. Some diseases alter the permeability of the blood-brain barrier and thus the passage of drugs. Strategies such as the use of methods for drug delivery in the brain have been investigated. Further details regarding the mechanisms of transport across the blood-brain barrier and the changes in neuropathology would provide important information about the etiology of diseases and lead to better therapeutic strategies.

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

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

2016-12-01

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

Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension.

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Trost, A; Motloch, K; Bruckner, D; Schroedl, F; Bogner, B; Kaser-Eichberger, A; Runge, C; Strohmaier, C; Klein, B; Aigner, L; Reitsamer, H A

2015-07-01

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology

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

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Hak Sung Lee

2014-08-01

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

High glucose alters retinal astrocytes phenotype through increased production of inflammatory cytokines and oxidative stress.

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Eui Seok Shin

Full Text Available Astrocytes are macroglial cells that have a crucial role in development of the retinal vasculature and maintenance of the blood-retina-barrier (BRB. Diabetes affects the physiology and function of retinal vascular cells including astrocytes (AC leading to breakdown of BRB. However, the detailed cellular mechanisms leading to retinal AC dysfunction under high glucose conditions remain unclear. Here we show that high glucose conditions did not induce the apoptosis of retinal AC, but instead increased their rate of DNA synthesis and adhesion to extracellular matrix proteins. These alterations were associated with changes in intracellular signaling pathways involved in cell survival, migration and proliferation. High glucose conditions also affected the expression of inflammatory cytokines in retinal AC, activated NF-κB, and prevented their network formation on Matrigel. In addition, we showed that the attenuation of retinal AC migration under high glucose conditions, and capillary morphogenesis of retinal endothelial cells on Matrigel, was mediated through increased oxidative stress. Antioxidant proteins including heme oxygenase-1 and peroxiredoxin-2 levels were also increased in retinal AC under high glucose conditions through nuclear localization of transcription factor nuclear factor-erythroid 2-related factor-2. Together our results demonstrated that high glucose conditions alter the function of retinal AC by increased production of inflammatory cytokines and oxidative stress with significant impact on their proliferation, adhesion, and migration.

Intraocular pressure, blood pressure, and retinal blood flow autoregulation: a mathematical model to clarify their relationship and clinical relevance.

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Guidoboni, Giovanna; Harris, Alon; Cassani, Simone; Arciero, Julia; Siesky, Brent; Amireskandari, Annahita; Tobe, Leslie; Egan, Patrick; Januleviciene, Ingrida; Park, Joshua

2014-05-29

This study investigates the relationship between intraocular pressure (IOP) and retinal hemodynamics and predicts how arterial blood pressure (BP) and blood flow autoregulation (AR) influence this relationship. A mathematical model is developed to simulate blood flow in the central retinal vessels and retinal microvasculature as current flowing through a network of resistances and capacitances. Variable resistances describe active and passive diameter changes due to AR and IOP. The model is validated by using clinically measured values of retinal blood flow and velocity. The model simulations for six theoretical patients with high, normal, and low BP (HBP-, NBP-, LBP-) and functional or absent AR (-wAR, -woAR) are compared with clinical data. The model predicts that NBPwAR and HBPwAR patients can regulate retinal blood flow (RBF) as IOP varies between 15 and 23 mm Hg and between 23 and 29 mm Hg, respectively, whereas LBPwAR patients do not adequately regulate blood flow if IOP is 15 mm Hg or higher. Hemodynamic alterations would be noticeable only if IOP changes occur outside of the regulating range, which, most importantly, depend on BP. The model predictions are consistent with clinical data for IOP reduction via surgery and medications and for cases of induced IOP elevation. The theoretical model results suggest that the ability of IOP to induce noticeable changes in retinal hemodynamics depends on the levels of BP and AR of the individual. These predictions might help to explain the inconsistencies found in the clinical literature concerning the relationship between IOP and retinal hemodynamics. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences

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Redzic Zoran

2011-01-01

Full Text Available Abstract Efficient processing of information by the central nervous system (CNS represents an important evolutionary advantage. Thus, homeostatic mechanisms have developed that provide appropriate circumstances for neuronal signaling, including a highly controlled and stable microenvironment. To provide such a milieu for neurons, extracellular fluids of the CNS are separated from the changeable environment of blood at three major interfaces: at the brain capillaries by the blood-brain barrier (BBB, which is localized at the level of the endothelial cells and separates brain interstitial fluid (ISF from blood; at the epithelial layer of four choroid plexuses, the blood-cerebrospinal fluid (CSF barrier (BCSFB, which separates CSF from the CP ISF, and at the arachnoid barrier. The two barriers that represent the largest interface between blood and brain extracellular fluids, the BBB and the BCSFB, prevent the free paracellular diffusion of polar molecules by complex morphological features, including tight junctions (TJs that interconnect the endothelial and epithelial cells, respectively. The first part of this review focuses on the molecular biology of TJs and adherens junctions in the brain capillary endothelial cells and in the CP epithelial cells. However, normal function of the CNS depends on a constant supply of essential molecules, like glucose and amino acids from the blood, exchange of electrolytes between brain extracellular fluids and blood, as well as on efficient removal of metabolic waste products and excess neurotransmitters from the brain ISF. Therefore, a number of specific transport proteins are expressed in brain capillary endothelial cells and CP epithelial cells that provide transport of nutrients and ions into the CNS and removal of waste products and ions from the CSF. The second part of this review concentrates on the molecular biology of various solute carrier (SLC transport proteins at those two barriers and underlines

Fingolimod promotes blood-nerve barrier properties in vitro.

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Nishihara, Hideaki; Maeda, Toshihiko; Sano, Yasuteru; Ueno, Maho; Okamoto, Nana; Takeshita, Yukio; Shimizu, Fumitaka; Koga, Michiaki; Kanda, Takashi

2018-04-01

The main effect of fingolimod is thought to be functional antagonism of lymphocytic S1P1 receptors and the prevention of lymphocyte egress from lymphoid tissues, thereby reducing lymphocyte infiltration into the nervous system. However, a growing number of reports suggest that fingolimod also has a direct effect on several cell types in the nervous system. Although we previously reported that fingolimod enhances blood-brain barrier (BBB) functions, there have been no investigations regarding the blood-nerve barrier (BNB). In this study, we examine how fingolimod affects the BNB. An immortalized human peripheral nerve microvascular endothelial cell line (HPnMEC) was used to evaluate BNB barrier properties. We examined tight junction proteins and barrier functions of HPnMECs in conditioned medium with or without fingolimod-phosphate and blood sera from patients with typical chronic inflammatory demyelinating polyneuropathy (CIDP). Incubation with fingolimod-phosphate increased levels of claudin-5 mRNA and protein as well as TEER values in HPnMECs. Conversely, typical CIDP sera decreased claudin-5 mRNA/protein levels and TEER values in HPnMECs; however, pretreatment with fingolimod-phosphate inhibited the effects of the typical CIDP sera. Fingolimod-phosphate directly modifies the BNB and enhances barrier properties. This mechanism may be a viable therapeutic target for CIDP, and fingolimod may be useful in patients with typical CIDP who have severe barrier disruption.

Retinal Cell Degeneration in Animal Models

Directory of Open Access Journals (Sweden)

Masayuki Niwa

2016-01-01

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

Stem Cell Therapies in Retinal Disorders

Directory of Open Access Journals (Sweden)

Aakriti Garg

2017-02-01

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

Müller stem cell dependent retinal regeneration.

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

2017-01-01

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

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

Science.gov (United States)

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

2015-11-09

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

Automatic detection of blood vessels in retinal images for diabetic retinopathy diagnosis.

Science.gov (United States)

Raja, D Siva Sundhara; Vasuki, S

2015-01-01

Diabetic retinopathy (DR) is a leading cause of vision loss in diabetic patients. DR is mainly caused due to the damage of retinal blood vessels in the diabetic patients. It is essential to detect and segment the retinal blood vessels for DR detection and diagnosis, which prevents earlier vision loss in diabetic patients. The computer aided automatic detection and segmentation of blood vessels through the elimination of optic disc (OD) region in retina are proposed in this paper. The OD region is segmented using anisotropic diffusion filter and subsequentially the retinal blood vessels are detected using mathematical binary morphological operations. The proposed methodology is tested on two different publicly available datasets and achieved 93.99% sensitivity, 98.37% specificity, 98.08% accuracy in DRIVE dataset and 93.6% sensitivity, 98.96% specificity, and 95.94% accuracy in STARE dataset, respectively.

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

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

2011-09-01

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

Modeling Group B Streptococcus and Blood-Brain Barrier Interaction by Using Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells

OpenAIRE

Kim, Brandon J.; Bee, Olivia B.; McDonagh, Maura A.; Stebbins, Matthew J.; Palecek, Sean P.; Doran, Kelly S.; Shusta, Eric V.

2017-01-01

ABSTRACT Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs after bacteria interact with and penetrate the blood-brain barrier (BBB). The BBB is comprised of highly specialized brain microvascular endothelial cells (BMECs) that function to separate the circulation from the CNS and act as a formidable barrier for toxins and pathogens. Certain bacteria, such as Streptococcus agalactiae (group B Streptococcus [GBS]), possess the ability to interact with a...

Retinal oximetry during treatment of retinal vein occlusion by ranibizumab in patients with high blood pressure and dyslipidemia.

Science.gov (United States)

Keilani, C; Halalchi, A; Wakpi Djeugue, D; Regis, A; Abada, S

2016-12-01

In the present study, we examined retinal vascular oxygen saturation in patients with retinal vein occlusion (RVO), high blood pressure (HBP) and dyslipidemia, before and during intravitreal vascular endothelial growth factor (VEGF) injection (ranibizumab). We retrospectively reviewed the medical records of six patients with visual acuity (VA) reduced by macular edema (ME) secondary to RVO with HBP and dyslipidemia, who underwent intravitreal anti-VEGF injection between October 2014 and February 2015 in the department of ophthalmology of François-Quesnay Hospital at Mantes-la-Jolie (France). The main inclusion criterion was the presence of RVO with ME and decreased VA. The primary endpoint was improvement of retinal venous oxygen saturation in patients with RVO before and 3 months after intravitreal ranibizumab injection. Secondary outcomes were improvement of retinal arterial oxygen saturation, improvement of best-corrected visual acuity (BCVA) on the Early Treatment Diabetic Retinopathy Study (ETDRS) scale, regression of ME measured by the central macular thickness (CMT) in nm and studying the correlation between blood pressure (BP) and retinal venous oxygen saturation before and after ranibizumab. Six eyes of six patients were included. Before treatment, the mean (standard deviation [SD]) of the retinal venous saturation (%) was 38.1±14.2. Three months after the injections, the mean (SD) of the retinal venous saturation (%) increased statistically significantly 49.2±11 (P=0.03). In this study, retinal venous oxygen saturation in patients with RVO, HBP and dyslipidemia was partially normalized during intravitreal ranibizumab treatment. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Improvement of retinal blood vessel detection using morphological component analysis.

Science.gov (United States)

Imani, Elaheh; Javidi, Malihe; Pourreza, Hamid-Reza

2015-03-01

Detection and quantitative measurement of variations in the retinal blood vessels can help diagnose several diseases including diabetic retinopathy. Intrinsic characteristics of abnormal retinal images make blood vessel detection difficult. The major problem with traditional vessel segmentation algorithms is producing false positive vessels in the presence of diabetic retinopathy lesions. To overcome this problem, a novel scheme for extracting retinal blood vessels based on morphological component analysis (MCA) algorithm is presented in this paper. MCA was developed based on sparse representation of signals. This algorithm assumes that each signal is a linear combination of several morphologically distinct components. In the proposed method, the MCA algorithm with appropriate transforms is adopted to separate vessels and lesions from each other. Afterwards, the Morlet Wavelet Transform is applied to enhance the retinal vessels. The final vessel map is obtained by adaptive thresholding. The performance of the proposed method is measured on the publicly available DRIVE and STARE datasets and compared with several state-of-the-art methods. An accuracy of 0.9523 and 0.9590 has been respectively achieved on the DRIVE and STARE datasets, which are not only greater than most methods, but are also superior to the second human observer's performance. The results show that the proposed method can achieve improved detection in abnormal retinal images and decrease false positive vessels in pathological regions compared to other methods. Also, the robustness of the method in the presence of noise is shown via experimental result. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

DEFF Research Database (Denmark)

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

2017-01-01

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

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

DEFF Research Database (Denmark)

Rönnbäck, Cecilia; Grønskov, Karen; Larsen, Michael

2014-01-01

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

Segmentation of retinal blood vessels for detection of diabetic retinopathy: A review

Directory of Open Access Journals (Sweden)

Rezty Amalia Aras

2016-05-01

Full Text Available Diabetic detinopathy (DR is effect of diabetes mellitus to the human vision that is the major cause of blindness. Early diagnosis of DR is an important requirement in diabetes treatment. Retinal fundus image is commonly used to observe the diabetic retinopathy symptoms. It can present retinal features such as blood vessel and also capture the pathologies which may lead to DR. Blood vessel is one of retinal features which can show the retina pathologies. It can be extracted from retinal image by image processing with following stages: pre-processing, segmentation, and post-processing. This paper contains a review of public retinal image dataset and several methods from various conducted researches. All discussed methods are applicable to each researcher cases. There is no further analysis to conclude the best method which can be used for general cases. However, we suggest morphological and multiscale method that gives the best accuracy in segmentation.

The influence of venous blood flow on the retinal ganglion cell complex in patients with primary open angle glaucoma

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N. I. Kurysheva

2014-07-01

Full Text Available Purpose: To study the influence of venous blood flow on the ganglion cell complex (GCC in patients with preperimetric and perimetric open angle glaucoma.Methods: 74 patients were included in the research. 59 eyes and 62 eyes were diagnosed with preperimetric and perimetric open angle glaucoma respectively. The mean age was 56.5±10.5 years. 22 (12 female and 10 male healthy individuals constituted the control group. The ganglion cell complex and retinal nerve fibre layer were evaluated with the help of optical coherence tomography (RTVue-100 OCT, Optovue, Inc., Fremont, CA. Ocular blood flow was measured by Color Doppler Imaging (multifunctional VOLUSON 730 ProSystem. The statistical analysis included correlation between GCC and RNFL thickness in both glaucoma groups.Results: The results showed a statistically significant reduction of venous blood flow velocity in both glaucoma groups compared to the control group. No difference in venous blood flow parameters between two glaucoma groups was found, except resistance index, which was higher in perimetric group in comparison to preperimetric group. A correlation was also obtained between venous blood flow parameters and GCC and RNFL thickness in both glaucoma groups.Conclusion: Early GCC damage in glaucoma might occur due to venous blood flow reduction. This fact may be of great value in understanding glaucoma pathogenesis and search for novel treatment options.

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

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

2009-07-01

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

Single cell transcriptome profiling of developing chick retinal cells.

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

2017-08-15

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

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

Science.gov (United States)

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

2006-08-01

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

Targeted liposomes for drug delivery across the blood-brain barrier

NARCIS (Netherlands)

van Rooy, I.

2011-01-01

Our brain is protected by the blood-brain barrier (BBB). This barrier is formed by specialized endothelial cells of the brain vasculature and prevents toxic substances from entering the brain. The downside of this barrier is that many drugs that have been developed to cure brain diseases cannot

Layer-specific blood-flow MRI of retinitis pigmentosa in RCS rats☆

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Li, Guang; Garza, Bryan De La; Shih, Yen-Yu I.; Muir, Eric R.; Duong, Timothy Q.

2013-01-01

The Royal College of Surgeons (RCS) rat is an established animal model of retinitis pigmentosa, a family of inherited retinal diseases which starts with loss of peripheral vision and progresses to eventual blindness. Blood flow (BF), an important physiological parameter, is intricately coupled to metabolic function under normal physiological conditions and is perturbed in many neurological and retinal diseases. This study reports non-invasive high-resolution MRI (44 × 44 × 600 μm) to image quantitative retinal and choroidal BF and layer-specific retinal thicknesses in RCS rat retinas at different stages of retinal degeneration compared with age-matched controls. The unique ability to separate retinal and choroidal BF was made possible by the depth-resolved MRI technique. RBF decreased with progressive retinal degeneration, but ChBF did not change in RCS rats up to post-natal day 90. We concluded that choroidal and retinal circulations have different susceptibility to progressive retinal degeneration in RCS rats. Layer-specific retinal thickness became progressively thinner and was corroborated by histological analysis in the same animals. MRI can detect progressive anatomical and BF changes during retinal degeneration with laminar resolution. PMID:22721720

Layer-specific blood-flow MRI of retinitis pigmentosa in RCS rats.

Science.gov (United States)

Li, Guang; De La Garza, Bryan; Shih, Yen-Yu I; Muir, Eric R; Duong, Timothy Q

2012-08-01

The Royal College of Surgeons (RCS) rat is an established animal model of retinitis pigmentosa, a family of inherited retinal diseases which starts with loss of peripheral vision and progresses to eventual blindness. Blood flow (BF), an important physiological parameter, is intricately coupled to metabolic function under normal physiological conditions and is perturbed in many neurological and retinal diseases. This study reports non-invasive high-resolution MRI (44 × 44 × 600 μm) to image quantitative retinal and choroidal BF and layer-specific retinal thicknesses in RCS rat retinas at different stages of retinal degeneration compared with age-matched controls. The unique ability to separate retinal and choroidal BF was made possible by the depth-resolved MRI technique. RBF decreased with progressive retinal degeneration, but ChBF did not change in RCS rats up to post-natal day 90. We concluded that choroidal and retinal circulations have different susceptibility to progressive retinal degeneration in RCS rats. Layer-specific retinal thickness became progressively thinner and was corroborated by histological analysis in the same animals. MRI can detect progressive anatomical and BF changes during retinal degeneration with laminar resolution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Non-invasive imaging of retinal blood flow in myeloproliferative neoplasms

DEFF Research Database (Denmark)

Willerslev, Anne; Hansen, Mathias M; Klefter, Oliver Niels

2017-01-01

PURPOSE: To study the circulation in the retinal vessels in patients with blood dyscrasia due to myeloproliferative neoplasms using non-invasive retinal imaging. METHODS: Prospective consecutive case series of seven treatment-naïve patients with chronic myeloid leukaemia (n = 2), polycythemia vera...... present at baseline in patients with chronic myeloid leukaemia and were replaced by normal patterns at follow-up. Retinopathy, in the form of cotton-wool spots and retinal haemorrhages, was found at presentation in the two patients with chronic myeloid leukaemia and in one patient with polycythemia vera...

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

Science.gov (United States)

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

2015-01-01

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

Caspase-14 Expression Impairs Retinal Pigment Epithelium Barrier Function: Potential Role in Diabetic Macular Edema

Directory of Open Access Journals (Sweden)

Selina Beasley

2014-01-01

Full Text Available We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR. Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose on caspase-14 expression in human RPE (ARPE-19 cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose. We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER. These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

3-D segmentation of retinal blood vessels in spectral-domain OCT volumes of the optic nerve head

Science.gov (United States)

Lee, Kyungmoo; Abràmoff, Michael D.; Niemeijer, Meindert; Garvin, Mona K.; Sonka, Milan

2010-03-01

Segmentation of retinal blood vessels can provide important information for detecting and tracking retinal vascular diseases including diabetic retinopathy, arterial hypertension, arteriosclerosis and retinopathy of prematurity (ROP). Many studies on 2-D segmentation of retinal blood vessels from a variety of medical images have been performed. However, 3-D segmentation of retinal blood vessels from spectral-domain optical coherence tomography (OCT) volumes, which is capable of providing geometrically accurate vessel models, to the best of our knowledge, has not been previously studied. The purpose of this study is to develop and evaluate a method that can automatically detect 3-D retinal blood vessels from spectral-domain OCT scans centered on the optic nerve head (ONH). The proposed method utilized a fast multiscale 3-D graph search to segment retinal surfaces as well as a triangular mesh-based 3-D graph search to detect retinal blood vessels. An experiment on 30 ONH-centered OCT scans (15 right eye scans and 15 left eye scans) from 15 subjects was performed, and the mean unsigned error in 3-D of the computer segmentations compared with the independent standard obtained from a retinal specialist was 3.4 +/- 2.5 voxels (0.10 +/- 0.07 mm).

Erythropoietin protects the retinal pigment epithelial barrier against ...

African Journals Online (AJOL)

O2-induced hyperpermeability. H Zhang, Y Gong, X Wu, Y Shi, L Yin, Y Qiu. Abstract. Erythropoietin (EPO) is not limited to hematopoiesis; it may act as a protective cytokine. In this study, the retinal pigment epithelial (RPE) cell viability, cell ...

Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

DEFF Research Database (Denmark)

Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp

Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming...... pathways across the barrier in ischemic and postischemic brain endothelium is important for developing new medical therapies capable to exploit the barrier changes occurring during/after ischemia to permeate in the brain and treat this devastating disease. Materials and Methods - Primary cultures...... the wall of brain capillaries. The restrictive nature of the BBB is due to the tight junctions (TJs), which seal the intercellular clefts, limiting the paracellular diffusion, efflux transporters, which extrude xenobiotics, and metabolizing enzymes, which may break down or convert molecules during...

C - reactive protein and chitinase 3-like protein 1 as biomarkers of spatial redistribution of retinal blood vessels on digital retinal photography in patients with diabetic retinopathy

Directory of Open Access Journals (Sweden)

Sonja Predrag Cekic

2014-08-01

Full Text Available The aim of the study was to investegate the correlation between the levels of CRP and YKL-40 in blood samples with morphometric parameters of retinal blood vessels in patients with diabetic retinopathy.Blood laboratory examination of 90 patients included the measurement of glycemia, HbA1C, total cholesterol, LDL-C, HDL-C, triglycerides and CRP. Levels of YKL-40 were detected and measured in serum by ELISA (Micro VueYKL-40 EIA Kit, Quidel Corporation, San Diego, USA.Morphmetric analysis was performed with ImageJ software (http://rsbweb.nih.gov/ij/ for digital retinal photography. We measured the number, diameter of retinal blood vessels in five different parts concentric to the optic disc. Differences between the morphometric parameters and the blood test analysis results were evaluated using the Student’s t – test. One Way ANOVA was used to establish the significance of differences.CRP and YKL-40 levels were moderately higher in the group of patients with severe diabetic retinopathy. Levels of YKL-40 correlated positively with diameter and negatively with number of retinal blood vessels. The average number of the blood vessels per retinal zone was significantly higher in the group of patients with mild non-proliferative diabetic retinopathy than in the group with severe form in the optic disc and all five retinal zones. The average outer diameter of the evaluated retinal zones and optic disc vessels was significantly higher in the group with severe compared to the group with mild diabetic retinopathy.Morphological analysis of the retinal vessels on digital fundus photography and correlation with YKL-40 may be valuable for the follow-up of diabetic retinopathy.

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

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Maryam Parvini

2013-10-01

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

Retinal stem cells and regeneration of vision system.

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Yip, Henry K

2014-01-01

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

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

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

2018-04-01

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

C-reactive protein and chitinase 3-like protein 1 as biomarkers of spatial redistribution of retinal blood vessels on digital retinal photography in patients with diabetic retinopathy.

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Cekić, Sonja; Cvetković, Tatjana; Jovanović, Ivan; Jovanović, Predrag; Pesić, Milica; Stanković Babić, Gordana; Milenković, Svetislav; Risimić, Dijana

2014-08-20

The aim of the study was to investigate the correlation between the levels of C-reactive protein (CRP) and chitinase 3-like protein 1 (YKL-40) in blood samples with morpohometric parameters of retinal blood vessels in patients with diabetic retinopathy. Blood laboratory examination of 90 patients included the measurement of glycemia, HbA1C, total cholesterol, LDL-C, HDL-C, triglycerides and CRP. Levels of YKL-40 were detected and measured in serum by ELISA (Micro VueYKL-40 EIA Kit, Quidel Corporation, San Diego, USA). YKL-40 correlated positively with diameter and negatively with number of retinal blood vessels. The average number of the blood vessels per retinal zone was significantly higher in the group of patients with mild non-proliferative diabetic retinopathy than in the group with severe form in the optic disc and all five retinal zones. The average outer diameter of the evaluated retinal zones and optic disc vessels was significantly higher in the group with severe compared to the group with mild diabetic retinopathy. Morphological analysis of the retinal vessels on digital fundus photography and correlation with YKL-40 may be valuable for the follow-up of diabetic retinopathy.

Association of ABO blood groups and Rh factor with retinal and choroidal thickness.

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Teberik, Kuddusi; Eski, Mehmet Tahir

2018-06-01

To evaluate if ABO blood group and Rh factor have an effect on retinal and choroidal thickness. This study was designed prospectively. Retinal nerve fiber layer, retinal, and choroidal thicknesses were measured with spectral-domain optical coherence tomography. Retinal and choroidal thickness measurements (one subfoveal, three temporal, and three nasal) were obtained at 500-μm intervals up to 1500 μm with the caliper system. In this study, 109 male and 151 female, 260 individuals in total were included. There were 125 subjects in group A, 29 in group B, 34 in group AB, and 72 in group O. Rh factor was positive in 194 subjects and negative in 66. There was no significant difference between the groups regarding age (p = 0.667). The groups did not show any statistical difference in retinal nerve fiber layer thickness. There was significant difference found for mean retinal thickness at temporal 1000 μm when four groups were compared (p = 0.037). No statistically significant difference was detected for the remaining retinal and choroidal sectoral regions. The groups did not statistically significantly differ concerning Rh factor (p > 0.05). Although we found a significant difference in retinal thickness in the temporal retina between group B with group A and group O, we suggest that both blood group and Rh factor have no effect on retinal and choroidal thickness.

Gliomas and the vascular fragility of the blood brain barrier

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Luiz Gustavo eDubois

2014-12-01

Full Text Available Astrocytes, members of the glial family, interact through the exchange of soluble factors or by directly contacting neurons and other brain cells, such as microglia and endothelial cells. Astrocytic projections interact with vessels and act as additional elements of the Blood Brain Barrier (BBB. By mechanisms not fully understood, astrocytes can undergo oncogenic transformation and give rise to gliomas. The tumors take advantage of the BBB to ensure survival and continuous growth. A glioma can develop into a very aggressive tumor, the glioblastoma (GBM, characterized by a highly heterogeneous cell population (including tumor stem cells, extensive proliferation and migration. Nevertheless, gliomas can also give rise to slow growing tumors and in both cases, the afflux of blood, via BBB is crucial. Glioma cells migrate to different regions of the brain guided by the extension of blood vessels, colonizing the healthy adjacent tissue. In the clinical context, GBM can lead to tumor-derived seizures, which represent a challenge to patients and clinicians, since drugs used for its treatment must be able to cross the BBB. Uncontrolled and fast growth also leads to the disruption of the chimeric and fragile vessels in the tumor mass resulting in peritumoral edema. Although hormonal therapy is currently used to control the edema, it is not always efficient. In this review we comment the points cited above, considering the importance of the blood brain barrier and the concerns that arise when this barrier is affected.

Novel Localization of Peripherin 2, the Photoreceptor-Specific Retinal Degeneration Slow Protein, in Retinal Pigment Epithelium

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Patrizia B. Uhl

2015-01-01

Full Text Available Retinal pigment epithelium (RPE builds the outer blood-retinal barrier of the eye. Since one typical feature of the autoimmune disease, equine recurrent uveitis (ERU, is the breakdown of this barrier, we recently performed comparative analysis of healthy and uveitic RPE. We identified for the first time peripherin 2, which is responsible for visual perception and retina development, to be localized in RPE. The purpose of this study was therefore to validate our findings by characterizing the expression patterns of peripherin 2 in RPE and retina. We also investigated whether peripherin 2 expression changes in ERU and if it is expressed by the RPE itself. Via immunohistochemistry, significant downregulation of peripherin 2 in uveitic RPE compared to the control was detectable, but there was no difference in healthy and uveitic retina. A further interesting finding was the clear distinction between peripherin 2 and the phagocytosis marker, rhodopsin, in healthy RPE. In conclusion, changes in the expression pattern of peripherin 2 selectively affect RPE, but not retina, in ERU. Moreover, peripherin 2 is clearly detectable in healthy RPE due to both phagocytosis and the expression by the RPE cells themselves. Our novel findings are very promising for better understanding the molecular mechanisms taking place on RPE in uveitis.

Altered Antioxidant-Oxidant Status in the Aqueous Humor and Peripheral Blood of Patients with Retinitis Pigmentosa

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Martínez-Fernández de la Cámara, Cristina; Salom, David; Sequedo, Ma Dolores; Hervás, David; Marín-Lambíes, Cristina; Aller, Elena; Jaijo, Teresa; Díaz-LLopis, Manuel; Millán, José María; Rodrigo, Regina

2013-01-01

Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa. PMID:24069283

Imidazole-4-acetic acid, a new lead structure for interaction with the taurine transporter in outer blood-retinal barrier cells

DEFF Research Database (Denmark)

Valembois, Sophie Annick N; Krall, Jacob; Frølund, Bente

2017-01-01

therapeutic approach. The taurine transporter (TAUT) plays a key role in the retinal transport of GABA and has been previously suggested to display a higher functional activity in the retina compared to the brain. TAUT would therefore stand as a suitable target for the selective delivery of ρ GABAAR ligands...... by testing their ability to inhibit the TAUT-mediated influx of taurine in ARPE-19 cells. Results showed that taurine influx was seven-fold higher when the ARPE-19 cells were cultured under hyperosmotic conditions and was demonstrated to display saturable kinetics (Km = 27.7 ± 2.2 μM and Jmax = 24.2 ± 0.......6 pmol/cm2·min). Furthermore, the taurine influx was significantly inhibited in a concentration-dependent manner by GABA and imidazole-4-acetic acid (IAA), which is a naturally occurring metabolite of histamine. These compounds display similar Ki values of 644.2 μM and 658.6 μM, respectively. Moreover...

A thresholding based technique to extract retinal blood vessels from fundus images

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Jyotiprava Dash

2017-12-01

Full Text Available Retinal imaging has become the significant tool among all the medical imaging technology, due to its capability to extract many data which is linked to various eye diseases. So, the accurate extraction of blood vessel is necessary that helps the eye care specialists and ophthalmologist to identify the diseases at the early stages. In this paper, we have proposed a computerized technique for extraction of blood vessels from fundus images. The process is conducted in three phases: (i pre-processing where the image is enhanced using contrast limited adaptive histogram equalization and median filter, (ii segmentation using mean-C thresholding to extract retinal blood vessels, (iii post-processing where morphological cleaning operation is used to remove isolated pixels. The performance of the proposed method is tested on and experimental results show that our method achieve an accuracies of 0.955 and 0.954 on Digital retinal images for vessel extraction (DRIVE and Child heart and health study in England (CHASE_DB1 databases respectively.

Liraglutide attenuates the migration of retinal pericytes induced by advanced glycation end products.

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Lin, Wen-Jian; Ma, Xue-Fei; Hao, Ming; Zhou, Huan-Ran; Yu, Xin-Yang; Shao, Ning; Gao, Xin-Yuan; Kuang, Hong-Yu

2018-07-01

Retinal pericyte migration represents a novel mechanism of pericyte loss in diabetic retinopathy (DR), which plays a crucial role in the early impairment of the blood-retinal barrier (BRB). Glucagon-like peptide-1 (GLP-1) has been shown to protect the diabetic retina in the early stage of DR; however, the relationship between GLP-1 and retinal pericytes has not been discussed. In this study, advanced glycation end products (AGEs) significantly increased the migration of primary bovine retinal pericytes without influencing cell viability. AGEs also significantly enhanced phosphatidylinositol 3-kinase (PI3K)/Akt activation, and changed the expressions of migration-related proteins, including phosphorylated focal adhesion kinase (p-FAK), matrix metalloproteinase (MMP)-2 and vinculin. PI3K inhibition significantly attenuated the AGEs-induced migration of retinal pericytes and reversed the overexpression of MMP-2. Glucagon-like peptide-1 receptor (Glp1r) was expressed in retinal pericytes, and liraglutide, a GLP-1 analog, significantly attenuated the migration of pericytes by Glp1r and reversed the changes in p-Akt/Akt, p-FAK/FAK, vinculin and MMP-2 levels induced by AGEs, indicating that the protective effect of liraglutide was associated with the PI3K/Akt pathway. These results provided new insights into the mechanism underlying retinal pericyte migration. The early use of liraglutide exerts a potential bebefical effect on regulating pericyte migration, which might contribute to mechanisms that maintain the integrity of vascular barrier and delay the development of DR. Copyright © 2018 Elsevier Inc. All rights reserved.

Stem cell therapy for retinal diseases

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

2015-01-01

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

Improvement of retinal blood vessel detection by spur removal and Gaussian matched filtering compensation

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Xiao, Di; Vignarajan, Janardhan; An, Dong; Tay-Kearney, Mei-Ling; Kanagasingam, Yogi

2016-03-01

Retinal photography is a non-invasive and well-accepted clinical diagnosis of ocular diseases. Qualitative and quantitative assessment of retinal images is crucial in ocular diseases related clinical application. In this paper, we proposed approaches for improving the quality of blood vessel detection based on our initial blood vessel detection methods. A blood vessel spur pruning method has been developed for removing the blood vessel spurs both on vessel medial lines and binary vessel masks, which are caused by artifacts and side-effect of Gaussian matched vessel enhancement. A Gaussian matched filtering compensation method has been developed for removing incorrect vessel branches in the areas of low illumination. The proposed approaches were applied and tested on the color fundus images from one publicly available database and our diabetic retinopathy screening dataset. A preliminary result has demonstrated the robustness and good performance of the proposed approaches and their potential application for improving retinal blood vessel detection.

Laser speckle imaging of rat retinal blood flow with hybrid temporal and spatial analysis method

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Cheng, Haiying; Yan, Yumei; Duong, Timothy Q.

2009-02-01

Noninvasive monitoring of blood flow in retinal circulation will reveal the progression and treatment of ocular disorders, such as diabetic retinopathy, age-related macular degeneration and glaucoma. A non-invasive and direct BF measurement technique with high spatial-temporal resolution is needed for retinal imaging. Laser speckle imaging (LSI) is such a method. Currently, there are two analysis methods for LSI: spatial statistics LSI (SS-LSI) and temporal statistical LSI (TS-LSI). Comparing these two analysis methods, SS-LSI has higher signal to noise ratio (SNR) and TSLSI is less susceptible to artifacts from stationary speckle. We proposed a hybrid temporal and spatial analysis method (HTS-LSI) to measure the retinal blood flow. Gas challenge experiment was performed and images were analyzed by HTS-LSI. Results showed that HTS-LSI can not only remove the stationary speckle but also increase the SNR. Under 100% O2, retinal BF decreased by 20-30%. This was consistent with the results observed with laser Doppler technique. As retinal blood flow is a critical physiological parameter and its perturbation has been implicated in the early stages of many retinal diseases, HTS-LSI will be an efficient method in early detection of retina diseases.

Scintigraphic assessment of vascularity and blood-tissue barrier of human brain tumours

International Nuclear Information System (INIS)

Front, D.

1978-01-01

Assessment of vascularity and blood-tissue barrier was performed by sequential scintigraphy in 43 patients with brain tumours. The blood-tumour barrier was evaluated by use of sup(99m)Tc-pertechnetate, and vascularity using sup(99m)Tc-labelled red blood cells. Three groups of tumours were found: tumours with low vascularity and permeable barrier, tumours with high vascularity and permeable barrier, and tumours with low vascularity and relatively impermeable barrier. The first group indicates that when vessels are permeable, there may be a rapid penetration of large amounts of pertechnetate into the tumour even when vascularity is not increased. In the other two groups penetration of pertechnetate into the tumour is affected by vascularity, as it determines the total area where passage of the radiopharmaceutical takes place. It is suggested that the permeability of the blood-tumour barrier and the amount of vascularity may have an effect on the success of chemotherapy in brain tumours. (author)

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

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

2013-06-01

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

Endothelial progenitor cells physiology and metabolic plasticity in brain angiogenesis and blood-brain barrier modeling

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Natalia Malinovskaya

2016-12-01

Full Text Available Currently, there is a considerable interest to the assessment of blood-brain barrier (BBB development as a part of cerebral angiogenesis developmental program. Embryonic and adult angiogenesis in the brain is governed by the coordinated activity of endothelial progenitor cells, brain microvascular endothelial cells, and non-endothelial cells contributing to the establishment of the BBB (pericytes, astrocytes, neurons. Metabolic and functional plasticity of endothelial progenitor cells controls their timely recruitment, precise homing to the brain microvessels, and efficient support of brain angiogenesis. Deciphering endothelial progenitor cells physiology would provide novel engineering approaches to establish adequate microfluidically-supported BBB models and brain microphysiological systems for translational studies.

Müller cells and retinal axons can be primary targets in experimental neuromyelitis optica spectrum disorder.

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Zeka, Bleranda; Lassmann, Hans; Bradl, Monika

2017-01-01

Recent work from our laboratory, using different models of experimental neuromyelitis optica spectrum disorder (NMOSD), has led to a number of observations that might be highly relevant for NMOSD patients. For example: (i) in the presence of neuromyelitis optica immunoglobulin G, astrocyte-destructive lesions can be initiated by CD4+ T cells when these cells recognize aquaporin 4 (AQP4), but also when they recognize other antigens of the central nervous system. The only important prerequisite is that the T cells have to be activated within the central nervous system by "their" specific antigen. Recently activated CD4+ T cells with yet unknown antigen specificity are also found in human NMOSD lesions. (ii) The normal immune repertoire might contain AQP4-specific T cells, which are highly encephalitogenic on activation. (iii) The retina might be a primary target of AQP4-specific T cells and neuromyelitis optica immunoglobulin G: AQP4-specific T cells alone are sufficient to cause retinitis with low-grade axonal pathology in the retinal nerve fiber/ganglionic cell layer. A thinning of these layers is also observed in NMOSD patients, where it is thought to be a consequence of optic neuritis. Neuromyelitis optica immunoglobulin G might target cellular processes of Müller cells and cause their loss of AQP4 reactivity, when AQP4-specific T cells open the blood-retina barrier in the outer plexiform layer. Patchy loss of AQP4 reactivity on Müller cells of NMOSD patients has been recently described. Cumulatively, our findings in experimental NMOSD suggest that both CD4+ T cell and antibody responses directed against AQP4 might play an important role in the pathogenesis of tissue destruction seen in NMOSD.

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

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Kim Chan

2007-10-01

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

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

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

2007-01-01

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

Retinal hemodynamic oxygen reactivity assessed by perfusion velocity, blood oximetry and vessel diameter measurements

DEFF Research Database (Denmark)

Klefter, Oliver Niels; Lauritsen, Anne Øberg; Larsen, Michael

2015-01-01

PURPOSE: To test the oxygen reactivity of a fundus photographic method of measuring macular perfusion velocity and to integrate macular perfusion velocities with measurements of retinal vessel diameters and blood oxygen saturation. METHODS: Sixteen eyes in 16 healthy volunteers were studied at two...... is a valid method for assessing macular perfusion. Results were consistent with previous observations of hyperoxic blood flow reduction using blue field entoptic and laser Doppler velocimetry. Retinal perfusion seemed to be regulated around individual set points according to blood glucose levels. Multimodal...

Automatic segmentation of blood vessels from retinal fundus images ...

Indian Academy of Sciences (India)

The retinal blood vessels were segmented through color space conversion and color channel extraction, image pre-processing, Gabor filtering, image postprocessing, feature construction through application of principal component analysis, k-means clustering and first level classification using Naïve–Bayes classification ...

The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis

International Nuclear Information System (INIS)

Perides, George; Zhuge, Yuzheng; Lin, Tina; Stins, Monique F; Bronson, Roderick T; Wu, Julian K

2006-01-01

Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model. Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system. Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg -/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg -/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner. Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain

Efficacy and Safety of Human Retinal Progenitor Cells

Science.gov (United States)

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

2016-01-01

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

You Shall Not Pass – Tight junctions of the Blood Brain Barrier.

Directory of Open Access Journals (Sweden)

Hans-Christian eBauer

2014-12-01

Full Text Available Tissue barriers restricting the free diffusion of substances between the central nervous system and the systemic circulation are of great medical interest. Excessive leakage of blood-borne molecules into the parenchyma and the concomitant fluctuations in the microenvironment following a transient breakdown of the blood-brain barrier (BBB during ischemic/hypoxic conditions or due to an autoimmune disease are detrimental to the physiology of nervous tissue.On the other hand, the treatment of neurological disorders is often hampered as only minimal amounts of therapeutic agents are able to penetrate a functional BBB or blood cerebrospinal fluid barrier. At the basis of the BBB are, next to an elaborate transporting machinery, intimate cell-cell contacts (tight junctions creating not only a paracellular diffusion constraint but also enabling the vectorial transport across cell monolayers.More recent findings indicate that functional barriers are already established during development, protecting the fetal brain. As an understanding of the biogenesis of TJs might reveal the underlying mechanisms of barrier formation during ontogenic development numerous in vitro systems have been developed to study the assembly and disassembly of TJs. In addition, monitoring the stage-specific expression of TJ proteins during development has brought much insight into the developmental tightening of tissue barriers. Further, over the last two decades a detailed molecular map of tight junctions has emerged.TJs not only represent a cell-cell adhesion structure, but integrate various signaling pathways, thereby directly or indirectly impacting upon processes such as cell proliferation, cytoskeletal rearrangement, and transcriptional control.This review will provide a brief overview on the establishment of the BBB during embryonic development in mammals and a detailed description of the ultrastructure, biogenesis, and molecular composition of epithelial and endothelial

Associations of blood pressure variability and retinal arteriolar diameter in participants with type 2 diabetes.

Science.gov (United States)

Veloudi, Panagiota; Blizzard, Leigh; Srikanth, Velandai K; McCartney, Paul; Lukoshkova, Elena V; Hughes, Alun D; Head, Geoffrey A; Sharman, James E

2016-07-01

Blood pressure variability is associated with macrovascular complications and stroke, but its association with the microcirculation in type II diabetes has not been assessed. This study aimed to determine the relationship between blood pressure variability indices and retinal arteriolar diameter in non-diabetic and type II diabetes participants. Digitized retinal images were analysed to quantify arteriolar diameters in 35 non-diabetic (aged 52 ± 11 years; 49% male) and 28 type II diabetes (aged 61 ± 9 years; 50% male) participants. Blood pressure variability was derived from 24-h ambulatory blood pressure. Arteriolar diameter was positively associated with daytime rate of systolic blood pressure variation (p = 0.04) among type II diabetes participants and negatively among non-diabetics (p = 0.008; interaction p = 0.001). This finding was maintained after adjusting for age, sex, body mass index and mean daytime systolic blood pressure. These findings suggest that the blood pressure variability-related mechanisms underlying retinal vascular disease may differ between people with and without type II diabetes. © The Author(s) 2016.

Influencing factors affecting the retinal blood vessel morphology in patients with diabetes mellitus

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Xiao-Lu Kong

2017-03-01

Full Text Available AIM: To analyze the influencing factors affecting retinal blood vessel morphology in patients with diabetes mellitus. METHODS: Totally 312 patients with type 2 diabetes mellitus in our hospital from January 2012 to September 2016 were selected as study subjects. The patients were examined by fundus photography and related laboratory. As grouping factors in the patients'age, sex, disease duration, smoking, drinking, hypertension, hyperlipidemia or diabetic nephropathy, we compared the incidence of retinal vascular changes in different groups. The meaningful factors were introduced into the Logistic regression equation again. Independent risk factors for retinal vascular changes in patients with diabetes mellitus were screened out. RESULTS:In 312 cases of patients with type 2 diabetes mellitus,169 cases were accompanied with retinal vascular abnormalities, and 143 cases were not associated with retinal vascular abnormalities. Univariate analysis showed that age, duration of disease, hypertension, hyperlipidemia or diabetes nephropathy were significantly correlated with retinal vascular morphological changes(PP>0.05. Retinal vascular abnormalities were used as the dependent variable, and the above mentioned factors were grouped as independent variables. By Logistic stepwise regression analysis showed that the course of disease, patients with hypertension or diabetic nephropathy were the independent risk factors of abnormal retinal vascular morphology(PCONCLUSION: The independent risk factors for the occurrence of retinal vascular changes in patients with diabetes mellitus are increased course of disease, hypertension or diabetic nephropathy. Early diagnosis and intervention, to take measures and control blood pressure, reduce kidney damage can reduce the incidence of diabetic retinopathy, and macrovascular disease caused by diabetes, the incidence of adverse cardiovascular and cerebrovascular events.

In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

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Yukari Komuta

2016-06-01

Full Text Available Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

The in vitro blood-brain barrier model under OGD condition

DEFF Research Database (Denmark)

Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp

Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming...... the wall of brain capillaries. The restrictive nature of the BBB is due to the presence of tight junctions, which seal the paracellular space, a low number of endocytotic vesicles and the presence of efflux transporters, resulting in a very tight layer. Ischemic insult and the subsequent reperfusion...... of therapies to treat this devastating disease. Materials and Methods - Primary cultures of endothelial cells from bovine brain microvessels were cocultured with rat astrocytes in transwell inserts. At day 11, cells were treated with 4h of OGD by changing the culture medium with glucose-free medium...

Erythropoietin protects the retinal pigment epithelial barrier against ...

African Journals Online (AJOL)

zhanghongmei

2011-05-09

May 9, 2011 ... Results showed that, EPO increased the viability of H2O2-treated RPE cells, the disruption of .... in RPE damage that occurs in AMD and other retinal diseases of aging ..... However, preventing the cellular effects of ROIs in the.

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

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Holan, Vladimir; Hermankova, Barbora; Kossl, Jan

2017-09-01

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

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

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Wagner, Sylvia; Zensi, Anja; Wien, Sascha L; Tschickardt, Sabrina E; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U; Kreuter, Jörg; von Briesen, Hagen

2012-01-01

The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

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Sylvia Wagner

Full Text Available BACKGROUND: The blood-brain barrier (BBB represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. CONCLUSIONS/SIGNIFICANCE: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

Blood Vessel Extraction in Color Retinal Fundus Images with Enhancement Filtering and Unsupervised Classification

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Zafer Yavuz

2017-01-01

Full Text Available Retinal blood vessels have a significant role in the diagnosis and treatment of various retinal diseases such as diabetic retinopathy, glaucoma, arteriosclerosis, and hypertension. For this reason, retinal vasculature extraction is important in order to help specialists for the diagnosis and treatment of systematic diseases. In this paper, a novel approach is developed to extract retinal blood vessel network. Our method comprises four stages: (1 preprocessing stage in order to prepare dataset for segmentation; (2 an enhancement procedure including Gabor, Frangi, and Gauss filters obtained separately before a top-hat transform; (3 a hard and soft clustering stage which includes K-means and Fuzzy C-means (FCM in order to get binary vessel map; and (4 a postprocessing step which removes falsely segmented isolated regions. The method is tested on color retinal images obtained from STARE and DRIVE databases which are available online. As a result, Gabor filter followed by K-means clustering method achieves 95.94% and 95.71% of accuracy for STARE and DRIVE databases, respectively, which are acceptable for diagnosis systems.

[The blood-brain barrier in ageing persons].

Science.gov (United States)

Haaning, Nina; Damsgaard, Else Marie; Moos, Torben

2018-03-26

Brain capillary endothelial cells (BECs) form the ultra-tight blood-brain barrier (BBB). The permeability of the BBB increases with increasing age and neurovascular and neurodegenerative diseases. Major defects of the BBB can be initiated by increased permeability to plasma proteins in small arteriosclerotic arteries and release of proteins from degenerating neurons into the brain extracellular space. These proteins deposit in perivascular spaces, and subsequently negatively influence the BECs leading to decreased expression of barrier proteins. Detection of BBB defects by the use of non-invasive techniques is relevant for clinical use in settings with advanced age and severe brain disorders.

Retinitis pigmentosa-associated cystoid macular oedema: pathogenesis and avenues of intervention

Science.gov (United States)

Strong, S; Liew, G; Michaelides, M

2017-01-01

Hereditary retinal diseases are now the leading cause of blindness certification in the working age population (age 16–64 years) in England and Wales, of which retinitis pigmentosa (RP) is the most common disorder. RP may be complicated by cystoid macular oedema (CMO), causing a reduction of central vision. The underlying pathogenesis of RP-associated CMO (RP-CMO) remains uncertain, however, several mechanisms have been proposed, including: (1) breakdown of the blood-retinal barrier, (2) failure (or dysfunction) of the pumping mechanism in the retinal pigment epithelial, (3) Müller cell oedema and dysfunction, (4) antiretinal antibodies and (5) vitreous traction. There are limited data on efficacy of treatments for RP-CMO. Treatments attempted to date include oral and topical carbonic anhydrase inhibitors, oral, topical, intravitreal and periocular steroids, topical non-steroidal anti-inflammatory medications, photocoagulation, vitrectomy with internal limiting membrane peel, oral lutein and intravitreal antivascular endothelial growth factor injections. This review summarises the evidence supporting these treatment modalities. Successful management of RP-CMO should aim to improve both quality and quantity of vision in the short term and may also slow central vision loss over time. PMID:27913439

Engineering an in vitro air-blood barrier by 3D bioprinting

Science.gov (United States)

Horváth, Lenke; Umehara, Yuki; Jud, Corinne; Blank, Fabian; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing. PMID:25609567

Measuring retinal blood flow in rats using Doppler optical coherence tomography without knowing eyeball axial length

International Nuclear Information System (INIS)

Liu, Wenzhong; Yi, Ji; Chen, Siyu; Jiao, Shuliang; Zhang, Hao F.

2015-01-01

Purpose: Doppler optical coherence tomography (OCT) is widely used for measuring retinal blood flow. Existing Doppler OCT methods require the eyeball axial length, in which empirical values are usually used. However, variations in the axial length can create a bias unaccounted for in the retinal blood flow measurement. The authors plan to develop a Doppler OCT method that can measure the total retinal blood flow rate without requiring the eyeball axial length. Methods: The authors measured the retinal blood flow rate using a dual-ring scanning protocol. The small and large scanning rings entered the eye at different incident angles (small ring: 4°; large ring: 6°), focused on different locations on the retina, and detected the projected velocities/phase shifts along the probing beams. The authors calculated the ratio of the projected velocities between the two rings, and then used this ratio to estimate absolute flow velocity. The authors tested this method in both Intralipid phantoms and in vivo rats. Results: In the Intralipid flow phantom experiments, the preset and measured flow rates were consistent with the coefficient of determination as 0.97. Linear fitting between preset and measured flow rates determined the fitting slope as 1.07 and the intercept as −0.28. In in vivo rat experiments, the measured average total retinal blood flow was 7.02 ± 0.31μl/min among four wild-type rats. The authors’ measured flow rates were consistent with results in the literature. Conclusions: By using a dual-ring scanning protocol with carefully controlled incident angle difference between the two scanning rings in Doppler OCT, the authors demonstrated that it is feasible to measure the absolute retinal blood flow without knowing the eyeball axial length

Measuring retinal blood flow in rats using Doppler optical coherence tomography without knowing eyeball axial length.

Science.gov (United States)

Liu, Wenzhong; Yi, Ji; Chen, Siyu; Jiao, Shuliang; Zhang, Hao F

2015-09-01

Doppler optical coherence tomography (OCT) is widely used for measuring retinal blood flow. Existing Doppler OCT methods require the eyeball axial length, in which empirical values are usually used. However, variations in the axial length can create a bias unaccounted for in the retinal blood flow measurement. The authors plan to develop a Doppler OCT method that can measure the total retinal blood flow rate without requiring the eyeball axial length. The authors measured the retinal blood flow rate using a dual-ring scanning protocol. The small and large scanning rings entered the eye at different incident angles (small ring: 4°; large ring: 6°), focused on different locations on the retina, and detected the projected velocities/phase shifts along the probing beams. The authors calculated the ratio of the projected velocities between the two rings, and then used this ratio to estimate absolute flow velocity. The authors tested this method in both Intralipid phantoms and in vivo rats. In the Intralipid flow phantom experiments, the preset and measured flow rates were consistent with the coefficient of determination as 0.97. Linear fitting between preset and measured flow rates determined the fitting slope as 1.07 and the intercept as -0.28. In in vivo rat experiments, the measured average total retinal blood flow was 7.02 ± 0.31 μl/min among four wild-type rats. The authors' measured flow rates were consistent with results in the literature. By using a dual-ring scanning protocol with carefully controlled incident angle difference between the two scanning rings in Doppler OCT, the authors demonstrated that it is feasible to measure the absolute retinal blood flow without knowing the eyeball axial length.

Measuring retinal blood flow in rats using Doppler optical coherence tomography without knowing eyeball axial length

Energy Technology Data Exchange (ETDEWEB)

Liu, Wenzhong; Yi, Ji; Chen, Siyu [Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Jiao, Shuliang [Department of Biomedical Engineering, Florida International University, Miami, Florida 33174 (United States); Zhang, Hao F., E-mail: hfzhang@northwestern.edu [Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208 and Department of Ophthalmology, Northwestern University, Chicago, Illinois 60611 (United States)

2015-09-15

Purpose: Doppler optical coherence tomography (OCT) is widely used for measuring retinal blood flow. Existing Doppler OCT methods require the eyeball axial length, in which empirical values are usually used. However, variations in the axial length can create a bias unaccounted for in the retinal blood flow measurement. The authors plan to develop a Doppler OCT method that can measure the total retinal blood flow rate without requiring the eyeball axial length. Methods: The authors measured the retinal blood flow rate using a dual-ring scanning protocol. The small and large scanning rings entered the eye at different incident angles (small ring: 4°; large ring: 6°), focused on different locations on the retina, and detected the projected velocities/phase shifts along the probing beams. The authors calculated the ratio of the projected velocities between the two rings, and then used this ratio to estimate absolute flow velocity. The authors tested this method in both Intralipid phantoms and in vivo rats. Results: In the Intralipid flow phantom experiments, the preset and measured flow rates were consistent with the coefficient of determination as 0.97. Linear fitting between preset and measured flow rates determined the fitting slope as 1.07 and the intercept as −0.28. In in vivo rat experiments, the measured average total retinal blood flow was 7.02 ± 0.31μl/min among four wild-type rats. The authors’ measured flow rates were consistent with results in the literature. Conclusions: By using a dual-ring scanning protocol with carefully controlled incident angle difference between the two scanning rings in Doppler OCT, the authors demonstrated that it is feasible to measure the absolute retinal blood flow without knowing the eyeball axial length.

Retinal progenitor cell xenografts to the pig retina

DEFF Research Database (Denmark)

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

2005-01-01

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

Sulodexide prevents activation of the PLA2/COX-2/VEGF inflammatory pathway in human retinal endothelial cells by blocking the effect of AGE/RAGE.

Science.gov (United States)

Giurdanella, Giovanni; Lazzara, Francesca; Caporarello, Nunzia; Lupo, Gabriella; Anfuso, Carmelina Daniela; Eandi, Chiara M; Leggio, Gian Marco; Drago, Filippo; Bucolo, Claudio; Salomone, Salvatore

2017-10-15

Diabetic retinopathy is characterized by the breakdown of endothelial blood-retinal barrier. We tested the hypothesis that sulodexide (SDX), a highly purified glycosaminoglycan composed of 80% iduronylglycosaminoglycan sulfate and 20% dermatan sulfate, protects human retinal endothelial cells (HREC) from high glucose (HG)-induced damage, through the suppression of inflammatory ERK/cPLA2/COX-2/PGE 2 pathway, by blocking the effect of advanced glycation end-products (AGEs). HREC were treated with HG (25mM) or AGEs (glycated-BSA, 2mg/ml) for 48h, with or without SDX (60μg/ml) or aflibercept (AFL, 40μg/ml), a VEGF-trap. SDX protected HREC from HG-induced damage (MTT and LDH release) and preserved their blood-retinal barrier-like properties (Trans Endothelial Electrical Resistance and junction proteins, claudin-5, VE-cadherin and occludin, immunofluorescence and immunoblot) as well as their angiogenic potential (Tube Formation Assay). Both HG and AGEs increased phosphoERK and phospho-cPLA 2 , an effect counteracted by SDX and, less efficiently, by AFL. Both HG and exogenous VEGF (80ng/ml) increased PGE 2 release, an effect partially reverted by SDX for HG and by AFL for VEGF. Analysis of NFκB activity revealed that HG increased the abundance of p65 in the nuclear fraction (nuclear translocation), an effect entirely reverted by SDX, but only partially by AFL. SDX, AFL and SDX+AFL protected HREC even when added 24h after HG. These data show that SDX protects HREC from HG damage and suggest that it counteracts the activation of ERK/cPLA2/COX-2/PGE 2 pathway by reducing AGE-related signaling and downstream NFκB activity. This mechanism, partially distinct from VEGF blockade, may contribute to the therapeutic effect of SDX. Copyright © 2017 Elsevier Inc. All rights reserved.

Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

Science.gov (United States)

Shenoy, Anitha K; Lu, Jianrong

2016-10-01

Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

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Richard Longeras

2012-01-01

Full Text Available Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs. Pigment epithelium-derived factor (PEDF contains an N-terminal 34-amino acid peptide (PEDF-34 that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS. OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

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

Science.gov (United States)

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

2015-01-01

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

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

Directory of Open Access Journals (Sweden)

Laura eFernández-Sánchez

2015-12-01

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

Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

Science.gov (United States)

Chapouly, Candice; Tadesse Argaw, Azeb; Horng, Sam; Castro, Kamilah; Zhang, Jingya; Asp, Linnea; Loo, Hannah; Laitman, Benjamin M; Mariani, John N; Straus Farber, Rebecca; Zaslavsky, Elena; Nudelman, German; Raine, Cedric S; John, Gareth R

2015-06-01

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an

Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

International Nuclear Information System (INIS)

MacDonald, P.N.; Ong, D.E.; Bok, D.

1990-01-01

Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases.

Science.gov (United States)

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

2017-07-28

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

Live-cell imaging: new avenues to investigate retinal regeneration

Directory of Open Access Journals (Sweden)

Manuela Lahne

2017-01-01

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

Tryps and trips: cell trafficking across the 100-year-old blood-brain barrier.

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Bentivoglio, Marina; Kristensson, Krister

2014-06-01

One hundred years ago, Edwin E. Goldmann discovered the blood-brain barrier (BBB) using trypan dyes. These dyes were developed and named by Paul Ehrlich during his search for drugs to kill African trypanosomes (extracellular parasites that cause sleeping sickness) while sparing host cells. For Ehrlich, this was the first strategy based on the 'chemotherapy' concept he had introduced. The discovery of the BBB revealed, however, the difficulties in drug delivery to the brain. Mechanisms by which parasites enter, dwell, and exit the brain currently provide novel views on cell trafficking across the BBB. These mechanisms also highlight the role of pericytes and endocytosis regulation in BBB functioning and in disrupted BBB gating, which may be involved in the pathogenesis of neurodegeneration. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

An approach to localize the retinal blood vessels using bit planes and centerline detection.

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Fraz, M M; Barman, S A; Remagnino, P; Hoppe, A; Basit, A; Uyyanonvara, B; Rudnicka, A R; Owen, C G

2012-11-01

The change in morphology, diameter, branching pattern or tortuosity of retinal blood vessels is an important indicator of various clinical disorders of the eye and the body. This paper reports an automated method for segmentation of blood vessels in retinal images. A unique combination of techniques for vessel centerlines detection and morphological bit plane slicing is presented to extract the blood vessel tree from the retinal images. The centerlines are extracted by using the first order derivative of a Gaussian filter in four orientations and then evaluation of derivative signs and average derivative values is performed. Mathematical morphology has emerged as a proficient technique for quantifying the blood vessels in the retina. The shape and orientation map of blood vessels is obtained by applying a multidirectional morphological top-hat operator with a linear structuring element followed by bit plane slicing of the vessel enhanced grayscale image. The centerlines are combined with these maps to obtain the segmented vessel tree. The methodology is tested on three publicly available databases DRIVE, STARE and MESSIDOR. The results demonstrate that the performance of the proposed algorithm is comparable with state of the art techniques in terms of accuracy, sensitivity and specificity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Genetic determinants of hyaloid and retinal vasculature in zebrafish

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Hyde David R

2007-10-01

Full Text Available Abstract Background The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. Results We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO, subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. Conclusion Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

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Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

2018-01-01

PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

Advances in Bone Marrow Stem Cell Therapy for Retinal Dysfunction

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

2016-01-01

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

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

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

2016-04-01

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

HIV-1 impairs human retinal pigment epithelial barrier function: possible association with the pathogenesis of HIV-associated retinopathy.

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Tan, Suiyi; Duan, Heng; Xun, Tianrong; Ci, Wei; Qiu, Jiayin; Yu, Fei; Zhao, Xuyan; Wu, Linxuan; Li, Lin; Lu, Lu; Jiang, Shibo; Liu, Shuwen

2014-07-01

The breakdown of human retinal pigment epithelial (HRPE) barrier is considered as the etiology of retinopathy, which affects the quality of life of HIV/AIDS patients. Here we demonstrate that HIV-1 could directly impair HRPE barrier function, which leads to the translocation of HIV-1 and bacteria. HRPE cells (D407) were grown to form polarized, confluent monolayers and treated with different HIV-1 infectious clones. A significant increase of monolayer permeability, as measured by trans-epithelial electrical resistance (TEER) and apical-basolateral movements of sodium fluorescein, was observed. Disrupted tightness of HRPE barrier was associated with the downregulation of several tight junction proteins in D407 cells, including ZO-1, Occludin, Claudin-1, Claudin-2, Claudin-3, Claudin-4, and Claudin-5, after exposure to HIV-1, without affecting the viability of cells. HIV-1 gp120 was shown to participate in the alteration of barrier properties, as evidenced by decreased TEER and weakened expression of tight junction proteins in D407 monolayers after exposure to pseudotyped HIV-1, UV-inactivated HIV-1, and free gp120, but not to an envelope (Env)-defective mutant of HIV. Furthermore, exposure to HIV-1 particles could induce the release of pro-inflammatory cytokines in D407, including IL-6 and MCP-1, both of which downregulated the expression of ZO-1 in the HRPE barrier. Disrupted HRPE monolayer allowed translocation of HIV-1 and bacteria across the epithelium. Overall, these findings suggest that HIV-1 may exploit its Env glycoprotein to induce an inflammatory state in HRPE cells, which could result in impairment of HRPE monolayer integrity, allowing virus and bacteria existing in ocular fluids to cross the epithelium and penetrate the HRPE barrier. Our study highlights the role of HIV-1 in the pathogenesis of HIV/AIDS-related retinopathy and suggests potential therapeutic targets for this ocular complication.

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

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

2017-03-15

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

Initial Attempts of Development and Characterization of an In Vitro Blood Brain Barrier Model Derived from Human Pluripotent Stem Cells

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Goldeman, Charlotte; Saaby, Lasse; Hall, Vanessa Jane

The human blood brain barrier has yet to be successfully replicated as an in vitro model. One of the more promising approaches has been to develop an in vitro model derived from human pluripotent stem cells. However, as promising as this model may be, a successful replication of the differentiation...... method on different kinds of pluripotent stem cell lines have yet to be accomplished. We try to approach the promising method as described by Stebbins et al. (2015) to differentiate human pluripotent stem cells into brain like endothelial cells (BECs). Five different human pluripotent stem cell lines...... configurations (mono culture, non-contact co-culture and contact co-culture) with primary rat astrocytes to induce barrier-like properties. Endothelial cell media supplemented with retinoic acid were then applied to the cells to ensure selective expansion of BECs. The different culture configurations were...

Gene therapy for inherited retinal and optic nerve degenerations.

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Moore, Nicholas A; Morral, Nuria; Ciulla, Thomas A; Bracha, Peter

2018-01-01

The eye is a target for investigational gene therapy due to the monogenic nature of many inherited retinal and optic nerve degenerations (IRD), its accessibility, tight blood-ocular barrier, the ability to non-invasively monitor for functional and anatomic outcomes, as well as its relative immune privileged state.Vectors currently used in IRD clinical trials include adeno-associated virus (AAV), small single-stranded DNA viruses, and lentivirus, RNA viruses of the retrovirus family. Both can transduce non-dividing cells, but AAV are non-integrating, while lentivirus integrate into the host cell genome, and have a larger transgene capacity. Areas covered: This review covers Leber's congenital amaurosis, choroideremia, retinitis pigmentosa, Usher syndrome, Stargardt disease, Leber's hereditary optic neuropathy, Achromatopsia, and X-linked retinoschisis. Expert opinion: Despite great potential, gene therapy for IRD raises many questions, including the potential for less invasive intravitreal versus subretinal delivery, efficacy, safety, and longevity of response, as well as acceptance of novel study endpoints by regulatory bodies, patients, clinicians, and payers. Also, ultimate adoption of gene therapy for IRD will require widespread genetic screening to identify and diagnose patients based on genotype instead of phenotype.

THE MODULATORY ROLE OF TAURINE IN RETINAL GANGLION CELLS

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

2017-01-01

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

Stem cells in clinical trials for treatment of retinal degeneration.

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Klassen, Henry

2016-01-01

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

Limitations of the hCMEC/D3 cell line as a model for Aβ clearance by the human blood-brain barrier.

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Biemans, Elisanne A L M; Jäkel, Lieke; de Waal, Robert M W; Kuiperij, H Bea; Verbeek, Marcel M

2017-07-01

Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid-β (Aβ) at the cerebrovasculature due to decreased clearance at the blood-brain barrier (BBB). However, the exact mechanism of Aβ clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Aβ clearance across the BBB, with an emphasis on brain-to-blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Aβ was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Aβ. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Aβ clearance. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Zika Virus Infects Human Sertoli Cells and Modulates the Integrity of the In Vitro Blood-Testis Barrier Model.

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Siemann, David N; Strange, Daniel P; Maharaj, Payal N; Shi, Pei-Yong; Verma, Saguna

2017-11-15

Confirmed reports of Zika virus (ZIKV) in human seminal fluid for months after the clearance of viremia suggest the ability of ZIKV to establish persistent infection in the seminiferous tubules, an immune-privileged site in the testis protected by the blood-testis barrier, also called the Sertoli cell (SC) barrier (SCB). However, cellular targets of ZIKV in human testis and mechanisms by which the virus enters seminiferous tubules remain unclear. We demonstrate that primary human SCs were highly susceptible to ZIKV compared to the closely related dengue virus and induced the expression of alpha interferon (IFN-α), key cytokines, and cell adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1] and intracellular adhesion molecule 1 [ICAM-1]). Furthermore, using an in vitro SCB model, we show that ZIKV was released on the adluminal side of the SCB model with a higher efficiency than in the blood-brain barrier model. ZIKV-infected SCs exhibited enhanced adhesion of leukocytes that correlated with decreases in SCB integrity. ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin, and JAM-A; however, exposure of SCs to inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of the ZO-1 protein, which correlated with increased SCB permeability. Taken together, our data suggest that infection of SCs may be one of the crucial steps by which ZIKV gains access to the site of spermatozoon development and identify SCs as a therapeutic target to clear testicular infections. The SCB model opens up opportunities to assess interactions of SCs with other testicular cells and to test the ability of anti-ZIKV drugs to cross the barrier. IMPORTANCE Recent outbreaks of ZIKV, a neglected mosquito-borne flavivirus, have identified sexual transmission as a new route of disease spread, which has not been reported for other flaviviruses. To be able to sexually transmit for months after the clearance of

T-Lymphocytes Traffic into the Brain across the Blood-CSF Barrier: Evidence Using a Reconstituted Choroid Plexus Epithelium.

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Strazielle, Nathalie; Creidy, Rita; Malcus, Christophe; Boucraut, José; Ghersi-Egea, Jean-François

2016-01-01

An emerging concept of normal brain immune surveillance proposes that recently and moderately activated central memory T lymphocytes enter the central nervous system (CNS) directly into the cerebrospinal fluid (CSF) via the choroid plexus. Within the CSF space, T cells inspect the CNS environment for cognate antigens. This gate of entry into the CNS could also prevail at the initial stage of neuroinflammatory processes. To actually demonstrate T cell migration across the choroidal epithelium forming the blood-CSF barrier, an in vitro model of the rat blood-CSF barrier was established in an "inverse" configuration that enables cell transmigration studies in the basolateral to apical, i.e. blood/stroma to CSF direction. Structural barrier features were evaluated by immunocytochemical analysis of tight junction proteins, functional barrier properties were assessed by measuring the monolayer permeability to sucrose and the active efflux transport of organic anions. The migratory behaviour of activated T cells across the choroidal epithelium was analysed in the presence and absence of chemokines. The migration pathway was examined by confocal microscopy. The inverse rat BCSFB model reproduces the continuous distribution of tight junction proteins at cell margins, the restricted paracellular permeability, and polarized active transport mechanisms, which all contribute to the barrier phenotype in vivo. Using this model, we present experimental evidence of T cell migration across the choroidal epithelium. Cell migration appears to occur via a paracellular route without disrupting the restrictive barrier properties of the epithelial interface. Apical chemokine addition strongly stimulates T cell migration across the choroidal epithelium. The present data provide evidence for the controlled migration of T cells across the blood-CSF barrier into brain. They further indicate that this recruitment route is sensitive to CSF-borne chemokines, extending the relevance of this

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

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Awais Javed

2017-12-01

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

A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

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Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

2003-10-01

A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

Melanopsin retinal ganglion cell loss in Alzheimer's disease

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

2015-01-01

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

Compact Laser Doppler Flowmeter (LDF Fundus Camera for the Assessment of Retinal Blood Perfusion in Small Animals.

Directory of Open Access Journals (Sweden)

Marielle Mentek

Full Text Available Noninvasive techniques for ocular blood perfusion assessment are of crucial importance for exploring microvascular alterations related to systemic and ocular diseases. However, few techniques adapted to rodents are available and most are invasive or not specifically focused on the optic nerve head (ONH, choroid or retinal circulation. Here we present the results obtained with a new rodent-adapted compact fundus camera based on laser Doppler flowmetry (LDF.A confocal miniature flowmeter was fixed to a specially designed 3D rotating mechanical arm and adjusted on a rodent stereotaxic table in order to accurately point the laser beam at the retinal region of interest. The linearity of the LDF measurements was assessed using a rotating Teflon wheel and a flow of microspheres in a glass capillary. In vivo reproducibility was assessed in Wistar rats with repeated measurements (inter-session and inter-day of retinal arteries and ONH blood velocity in six and ten rats, respectively. These parameters were also recorded during an acute intraocular pressure increase to 150 mmHg and after heart arrest (n = 5 rats.The perfusion measurements showed perfect linearity between LDF velocity and Teflon wheel or microsphere speed. Intraclass correlation coefficients for retinal arteries and ONH velocity (0.82 and 0.86, respectively indicated strong inter-session repeatability and stability. Inter-day reproducibility was good (0.79 and 0.7, respectively. Upon ocular blood flow cessation, the retinal artery velocity signal substantially decreased, whereas the ONH signal did not significantly vary, suggesting that it could mostly be attributed to tissue light scattering.We have demonstrated that, while not adapted for ONH blood perfusion assessment, this device allows pertinent, stable and repeatable measurements of retinal blood perfusion in rats.

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

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

2014-11-01

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

Does Physical Fitness Buffer the Relationship between Psychosocial Stress, Retinal Vessel Diameters, and Blood Pressure among Primary Schoolchildren?

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Endes, Katharina; Herrmann, Christian; Colledge, Flora; Brand, Serge; Donath, Lars; Faude, Oliver; Pühse, Uwe; Hanssen, Henner; Zahner, Lukas

2016-01-01

Background. Strong evidence exists showing that psychosocial stress plays an important part in the development of cardiovascular diseases. Because physical inactivity is associated with less favourable retinal vessel diameter and blood pressure profiles, this study explores whether physical fitness is able to buffer the negative effects of psychosocial stress on retinal vessel diameters and blood pressure in young children. Methods. 325 primary schoolchildren (51% girls, Mage = 7.28 years) took part in this cross-sectional research project. Retinal arteriolar diameters, retinal venular diameters, arteriolar to venular ratio, and systolic and diastolic blood pressure were assessed in all children. Interactions terms between physical fitness (performance in the 20 m shuttle run test) and four indicators of psychosocial stress (parental reports of critical life events, family, peer and school stress) were tested in a series of hierarchical regression analyses. Results. Critical life events and family, peer, and school-related stress were only weakly associated with retinal vessel diameters and blood pressure. No support was found for a stress-buffering effect of physical fitness. Conclusion. More research is needed with different age groups to find out if and from what age physical fitness can protect against arteriolar vessel narrowing and the occurrence of other cardiovascular disease risk factors. PMID:27795958

Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease

DEFF Research Database (Denmark)

Fox, Ira J; Daley, George Q; Goldman, Steven A

2014-01-01

Pluripotent stem cells (PSCs) directed to various cell fates holds promise as source material for treating numerous disorders. The availability of precisely differentiated PSC-derived cells will dramatically affect blood component and hematopoietic stem cell therapies and should facilitate......, and industry is critical for generating new stem cell-based therapies....... treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Although an unlimited supply of specific cell types is needed, other barriers must be overcome. This review of the state of cell therapies highlights important challenges. Successful...

Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy

NARCIS (Netherlands)

Martens, Thomas F.; Remaut, Katrien; Deschout, Hendrik; Engbersen, Johan F J; Hennink, Wim E.; Van Steenbergen, Mies J.; Demeester, Jo; De Smedt, Stefaan C.; Braeckmans, Kevin

2015-01-01

Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or

Delivery of Biologics Across the Blood-Brain Barrier Through Nanoencapsulation

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Bruun, Jonas

is a polymeric micelle made from an anionic triblock copolymer and was intended for delivery of drugs to the central nervous system (CNS), which is protected by the largely impermeable blood-brain barrier (BBB). In order to target the nanocarrier to the brain endothelial cells and obtain receptor...... of the reporter protein. One of the great challenges for drug delivery by nanocarriers is the dilemma of designing a particle that is highly stable whit no cellular interaction while in the blood stream but has a high uptake and efficient drug release in the diseased cells. As a solution to this dilemma...

Biosensor Technology Reveals the Disruption of the Endothelial Barrier Function and the Subsequent Death of Blood Brain Barrier Endothelial Cells to Sodium Azide and Its Gaseous Products.

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Kho, Dan T; Johnson, Rebecca H; O'Carroll, Simon J; Angel, Catherine E; Graham, E Scott

2017-09-21

Herein we demonstrate the sensitive nature of human blood-brain barrier (BBB) endothelial cells to sodium azide and its gaseous product. Sodium azide is known to be acutely cytotoxic at low millimolar concentrations, hence its use as a biological preservative (e.g., in antibodies). Loss of barrier integrity was noticed in experiments using Electric Cell-substrate Impedance Sensing (ECIS) biosensor technology, to measure endothelial barrier integrity continuously in real-time. Initially the effect of sodium azide was observed as an artefact where it was present in antibodies being employed in neutralisation experiments. This was confirmed where antibody clones that were azide-free did not mediate loss of barrier function. A delayed loss of barrier function in neighbouring wells implied the influence of a liberated gaseous product. ECIS technology demonstrated that the BBB endothelial cells had a lower level of direct sensitivity to sodium azide of ~3 µM. Evidence of gaseous toxicity was consistently observed at 30 µM and above, with disrupted barrier function and cell death in neighbouring wells. We highlight the ability of this cellular biosensor technology to reveal both the direct and gaseous toxicity mediated by sodium azide. The sensitivity and temporal dimension of ECIS technology was instrumental in these observations. These findings have substantial implications for the wide use of sodium azide in biological reagents, raising issues of their application in live-cell assays and with regard to the protection of the user. This research also has wider relevance highlighting the sensitivity of brain endothelial cells to a known mitochondrial disruptor. It is logical to hypothesise that BBB endothelial dysfunction due to mitochondrial dys-regulation could have an important but underappreciated role in a range of neurological diseases.

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

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

2017-09-01

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

Optimization of the Ultrasound-Induced Blood-Brain Barrier Opening

OpenAIRE

Konofagou, Elisa E.

2012-01-01

Current treatments of neurological and neurodegenerative diseases are limited due to the lack of a truly non-invasive, transient, and regionally selective brain drug delivery method. The brain is particularly difficult to deliver drugs to because of the blood-brain barrier (BBB). The impermeability of the BBB is due to the tight junctions connecting adjacent endothelial cells and highly regulatory transport systems of the endothelial cell membranes. The main function of the BBB is ion and vol...

E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

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Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

2017-10-02

Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

Mitochondrial expression and activity of P-glycoprotein under oxidative stress in outer blood-retinal barrier

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Yue-Hong Zhang

2017-07-01

Full Text Available AIM: To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein (P-gp in mitochondria of D407 cells. METHODS: D407 cells were exposed to different ranges of concentrations of H2O2. The mitochondrial location of P-gp in the cells subjected to oxidative stress was detected by confocal analysis. Expression of P-gp in isolated mitochondria was assessed by Western blot. The pump activity of P-gp was evaluated by performing the efflux study on isolated mitochondria with Rhodamine 123 (Rho-123 alone and in the presence of P-gp inhibitor (Tariquidar using flow cytometry analysis. The cells were pretreated with 10 mmol/L N-acetylcysteine (NAC for 30min before exposing to H2O2, and analyzed the mitochondrial extracts by Western blot and flow cytometry. RESULTS: P-gp was co-localized in the mitochondria by confocal laser scanning microscopy, and it was also detected in the mitochondria of D407 cells using Western blot. Exposure to increasing concentrations of H2O2 led to gradually increased expression and location of P-gp in the mitochondria of cells. Rho-123 efflux assay showed higher uptake of Rho-123 on isolated mitochondria in the presence of Tariquidar both in normal and oxidative stress state. H2O2 up-regulated P-gp in D407 cells, which could be reversed by NAC treatment. CONCLUSION: H2O2 could up-regulate the functional expression of P-gp in mitochondria of D407 cells, while antioxidants might suppress oxidative-stress-induced over-expression of functional P-gp. It is indicative that limiting the mitochondrial P-gp transport in retinal pigment epithelium cells would be to improve the effect of mitochondria-targeted antioxidant therapy in age-related macular degeneration-like retinopathy.

Defense at the border : the blood-brain barrier versus bacterial foreigners

NARCIS (Netherlands)

van Sorge, Nina M.; Doran, Kelly S.

Bacterial meningitis is among the top ten causes of infectious disease-related deaths worldwide, with up to half of the survivors left with permanent neurological sequelae. The blood-brain barrier (BBB), composed mainly of specialized brain microvascular endothelial cells, maintains biochemical

In-vivo imaging of blood flow in human retinal vessels using color Doppler optical coherence tomography

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Yazdanfar, Siavash; Rollins, Andrew M.; Izatt, Joseph A.

1999-04-01

Quantification of retinal blood flow may lead to a better understanding of the progression and treatment of several ocular disorders, including diabetic retinopathy, age- related macular degeneration, and glaucoma. Current techniques, such as fluorescein angiography and laser Doppler velocimetry are limited, failing to provide sufficient information to the clinician. Color Doppler optical coherence tomography (CDOCT) is a novel technique using coherent heterodyne detection for simultaneous cross- sectional imaging of tissue microstructure and blood flow. This technique is capable of high spatial and velocity resolution imaging in highly scattering media. We implemented CDOCT for retinal blood flow mapping in human subjects. No dilation of the pupil was necessary. CDOCT is demonstrated for determining bidirectional flow in sub- 100micrometers diameter vessels in the retina. Additionally, we calculated Doppler broadening using the variance of depth- resolved spectra to identify regions with large velocity gradients within the Xenopus heart. This technique may be useful in quantifying local tissue perfusion in highly vascular retinal tissue.

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

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

2017-01-01

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

Molecular Responses of Human Retinal Cells to Infection with Dengue Virus

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Jillian M. Carr

2017-01-01

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

Schwann Cell-Mediated Preservation of Vision in Retinal Degenerative Diseases via the Reduction of Oxidative Stress: A Possible Mechanism.

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Mahmoudzadeh, Raziyeh; Heidari-Keshel, Saeed; Lashay, Alireza

2016-01-01

After injury to the central nervous system (CNS), regeneration is often inadequate, except in the case of remyelination. This remyelination capacity of the CNS is a good example of a stem/precursor cell-mediated renewal process. Schwann cells have been found to act as remyelinating agents in the peripheral nervous system (PNS), but several studies have highlighted their potential role in remyelination in the CNS too. Schwann cells are able to protect and support retinal cells by secreting growth factors such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and basic fibroblast growth factor. Retinal degenerative diseases can be highly debilitating, and they are a major concern in countries with an ageing populations. One of the leading causes of permanent loss of vision in the West is a retinal degenerative disease known as age-related macular degeneration (AMD). In the United States, nearly 1.75 million people over the age of 40 have advanced AMD, and it is estimated that this number will increase to approximately 3 million people by 2020. One of the most common pathways involved in the initiation and development of retinal diseases is the oxidative stress pathway. In patients with diabetes, Schwann cells have been shown to be able to secrete large amounts of antioxidant enzymes that protect the PNS from the oxidative stress that results from fluctuations in blood glucose levels. This antioxidant ability may be involved in the mechanism by which Schwann cells are able to promote reconstruction in the CNS, especially in individuals with retinal injuries and degenerative diseases.

Markers for blood-brain barrier integrity

DEFF Research Database (Denmark)

Saunders, Norman R; Dziegielewska, Katarzyna M; Møllgård, Kjeld

2015-01-01

In recent years there has been a resurgence of interest in brain barriers and various roles their intrinsic mechanisms may play in neurological disorders. Such studies require suitable models and markers to demonstrate integrity and functional changes at the interfaces between blood, brain......, and cerebrospinal fluid. Studies of brain barrier mechanisms and measurements of plasma volume using dyes have a long-standing history, dating back to the late nineteenth-century. Their use in blood-brain barrier studies continues in spite of their known serious limitations in in vivo applications. These were well...... known when first introduced, but seem to have been forgotten since. Understanding these limitations is important because Evans blue is still the most commonly used marker of brain barrier integrity and those using it seem oblivious to problems arising from its in vivo application. The introduction...

Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

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Mónica Díaz-Coránguez

Full Text Available Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

Science.gov (United States)

Díaz-Coránguez, Mónica; Segovia, José; López-Ornelas, Adolfo; Puerta-Guardo, Henry; Ludert, Juan; Chávez, Bibiana; Meraz-Cruz, Noemi; González-Mariscal, Lorenza

2013-01-01

Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-05-14

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

Pharmacokinetics and In Vitro Blood-Brain Barrier Screening of the Plant-Derived Alkaloid Tryptanthrin.

Science.gov (United States)

Jähne, Evelyn A; Eigenmann, Daniela E; Sampath, Chethan; Butterweck, Veronika; Culot, Maxime; Cecchelli, Roméo; Gosselet, Fabien; Walter, Fruzsina R; Deli, Mária A; Smieško, Martin; Hamburger, Matthias; Oufir, Mouhssin

2016-07-01

The indolo[2,1-b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxygenase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2 mg/kg bw i. v.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux. Georg Thieme Verlag KG Stuttgart · New York.

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

Science.gov (United States)

Williams, D L

2017-02-01

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

Design and validation of a microfluidic device for blood-brain barrier monitoring and transport studies

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Ugolini, Giovanni Stefano; Occhetta, Paola; Saccani, Alessandra; Re, Francesca; Krol, Silke; Rasponi, Marco; Redaelli, Alberto

2018-04-01

In vitro blood-brain barrier models are highly relevant for drug screening and drug development studies, due to the challenging task of understanding the transport mechanism of drug molecules through the blood-brain barrier towards the brain tissue. In this respect, microfluidics holds potential for providing microsystems that require low amounts of cells and reagent and can be potentially multiplexed for increasing the ease and throughput of the drug screening process. We here describe the design, development and validation of a microfluidic device for endothelial blood-brain barrier cell transport studies. The device comprises of two microstructured layers (top culture chamber and bottom collection chamber) sandwiching a porous membrane for the cell culture. Microstructured layers include two pairs of physical electrodes, embedded into the device layers by geometrically defined guiding channels with computationally optimized positions. These electrodes allow the use of commercial electrical measurement systems for monitoring trans-endothelial electrical resistance (TEER). We employed the designed device for performing preliminary assessment of endothelial barrier formation with murine brain endothelial cells (Br-bEnd5). Results demonstrate that cellular junctional complexes effectively form in the cultures (expression of VE-Cadherin and ZO-1) and that the TEER monitoring systems effectively detects an increase of resistance of the cultured cell layers indicative of tight junction formation. Finally, we validate the use of the described microsystem for drug transport studies demonstrating that Br-bEnd5 cells significantly hinder the transport of molecules (40 kDa and 4 kDa dextran) from the top culture chamber to the bottom collection chamber.

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

Science.gov (United States)

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

2017-05-10

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

Blood-brain barrier transport of drugs for the treatment of brain diseases.

Science.gov (United States)

Gabathuler, Reinhard

2009-06-01

The central nervous system is a sanctuary protected by barriers that regulate brain homeostasis and control the transport of endogenous compounds into the brain. The blood-brain barrier, formed by endothelial cells of the brain capillaries, restricts access to brain cells allowing entry only to amino acids, glucose and hormones needed for normal brain cell function and metabolism. This very tight regulation of brain cell access is essential for the survival of neurons which do not have a significant capacity to regenerate, but also prevents therapeutic compounds, small and large, from reaching the brain. As a result, various strategies are being developed to enhance access of drugs to the brain parenchyma at therapeutically meaningful concentrations to effectively manage disease.

Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity.

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Montserrat Torres-Oliva

2018-01-01

Full Text Available Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells.

Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

Science.gov (United States)

Torres-Oliva, Montserrat; Schneider, Julia; Wiegleb, Gordon

2018-01-01

Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells. PMID:29360820

Alteration of blood-brain barrier integrity by retroviral infection.

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Philippe V Afonso

2008-11-01

Full Text Available The blood-brain barrier (BBB, which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans, both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

NUTRITION AND VASCULAR SUPPLY OF RETINAL GANGLION CELLS DURING HUMAN DEVELOPMENT

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Paul eRutkowski

2016-04-01

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

Effects of intracarotid ioxaglate on the normal blood-brain barrier

International Nuclear Information System (INIS)

Wilcox, J.; Sage, M.R.

1985-01-01

Using two different models, the effect on the blood-brain barrier of intracarotid injections of sodium/meglumine ioxaglate at similar iodine concentrations (280 mgI/ml) was investigated. In both models the degree of blood-brain barrier damage was assessed visually using Evans' Blue stain. Quantitative assessment of blood-brain barrier disruption was made by contrast enhancement as measured by CT of the dog brain, and by 99m Tc-pertechnetate uptake by the brain in the rabbit model. No Evans' Blue staining was observed in any study using the canine/CT model. Slight staining was observed in two studies with ioxaglate using the rabbit/pertechnetate model. Statistical analysis of results from the canine/CT model did not detect any damage to the blood-brain barrier with either ioxaglate or saline control studies (P>0.1). However, in the rabbit/pertechnetate model a slight increase in disruption of the blood-brain barrier was observed with ioxaglate compared with control studies, but this was only significant at the 0.1 level. The results suggest that the rabbit/pertechnetate model is a more sensitive measure of blood-brain barrier disruption than the canine/CT model. This study also demonstrates that blood-brain barrier disruption following intracarotid injection of ioxaglate is minimal. (orig.)

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

Science.gov (United States)

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

2016-01-01

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

Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model

DEFF Research Database (Denmark)

Helms, Hans CC; Aldana, Blanca I; Groth, Simon

2017-01-01

The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood-brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co...... brain to blood via the concerted action of abluminal and luminal transport proteins, but the total brain clearance is highly dependent on metabolism in astrocytes and endothelial cells followed by transport of metabolites....

The blood pressure-induced diameter response of retinal arterioles decreases with increasing diabetic maculopathy

DEFF Research Database (Denmark)

Frederiksen, Christian Alcaraz; Jeppesen, Peter; Knudsen, Søren Tang

2006-01-01

A consisted of normal individuals and groups B-D consisted of type 2 diabetic patients matched for diabetes duration, age, and gender, and characterized by: Group B no retinopathy, Group C mild retinopathy, Group D maculopathy not requiring laser treatment. The diameter changes of a large retinal arteriole......+/-4.5 microm), and Group C (253+/-4.4 microm), but was significantly (p=0.006) increased in Group D (279+/-5.3 microm). CONCLUSIONS: The diameter response was reduced in type 2 diabetic patients with retinopathy, whereas retinal thickness was increased in patients with diabetic maculopathy. This suggests......BACKGROUND: The aim of the study was to compare the diameter response of retinal arterioles and retinal thickness in patients with different stages of diabetic maculopathy during an increase in the arterial blood pressure. METHODS: Four groups each consisting of 19 individuals were studied. Group...

New algorithm for detecting smaller retinal blood vessels in fundus images

Science.gov (United States)

LeAnder, Robert; Bidari, Praveen I.; Mohammed, Tauseef A.; Das, Moumita; Umbaugh, Scott E.

2010-03-01

About 4.1 million Americans suffer from diabetic retinopathy. To help automatically diagnose various stages of the disease, a new blood-vessel-segmentation algorithm based on spatial high-pass filtering was developed to automatically segment blood vessels, including the smaller ones, with low noise. Methods: Image database: Forty, 584 x 565-pixel images were collected from the DRIVE image database. Preprocessing: Green-band extraction was used to obtain better contrast, which facilitated better visualization of retinal blood vessels. A spatial highpass filter of mask-size 11 was applied. A histogram stretch was performed to enhance contrast. A median filter was applied to mitigate noise. At this point, the gray-scale image was converted to a binary image using a binary thresholding operation. Then, a NOT operation was performed by gray-level value inversion between 0 and 255. Postprocessing: The resulting image was AND-ed with its corresponding ring mask to remove the outer-ring (lens-edge) artifact. At this point, the above algorithm steps had extracted most of the major and minor vessels, with some intersections and bifurcations missing. Vessel segments were reintegrated using the Hough transform. Results: After applying the Hough transform, both the average peak SNR and the RMS error improved by 10%. Pratt's Figure of Merit (PFM) was decreased by 6%. Those averages were better than [1] by 10-30%. Conclusions: The new algorithm successfully preserved the details of smaller blood vessels and should prove successful as a segmentation step for automatically identifying diseases that affect retinal blood vessels.

Real-Time Imaging of Retinal Ganglion Cell Apoptosis

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Timothy E. Yap

2018-06-01

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

Veratridine increases the survival of retinal ganglion cells in vitro

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S.P.F. Pereira

1997-12-01

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

Protection of the blood-brain barrier by hypercapnia during acute hypertension

International Nuclear Information System (INIS)

Baumbach, G.L.; Mayhan, W.G.; Heistad, D.D.

1986-01-01

The purpose of this study was to examine effects of hypercapnia on susceptibility of the blood-brain barrier to disruption during acute hypertension. Two methods were used to test the hypothesis that cerebral vasodilation during hypercapnia increases disruption of the blood-brain barrier. First, permeability of the blood-brain barrier was measured in anesthetized cats with 125 I-labeled serum albumin. Severe hypertension markedly increased permeability of the blood-brain barrier during normocapnia, but not during hypercapnia. The protective effect of hypercapnia was not dependent on sympathetic nerves. Second, in anesthetized rats, permeability of the barrier was quantitated by clearance of fluorescent dextran. Disruption of the blood-brain barrier during hypertension was decreased by hypercapnia. Because disruption of the blood-brain barrier occurred primarily in pial venules, the authors also measured pial venular diameter and pressure. Acute hypertension increased pial venular pressure and diameter in normocapnic rats. Hypercapnia alone increased pial venular pressure and pial venular diameter, and acute hypertension during hypercapnia further increased venular pressure. The magnitude of increase in pial venular pressure during acute hypertension was significantly less in hypercapnic than in normocapnic rats. They conclude that hypercapnia protects the blood-brain barrier. Possible mechanisms of this effect include attenuation of the incremental increase in pial venular pressure by hypercapnia or a direct effect on the blood-brain barrier not related to venous pressure

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

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Jeffery Glen

2008-05-01

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

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

DEFF Research Database (Denmark)

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

2012-01-01

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

Blood brain barrier and brain tissue injury by Gd-DTPA in uremia-induced rabbits

International Nuclear Information System (INIS)

Choi, Sun Seob; Huh, Ki Yeong; Han, Jin Yeong; Lee, Yong Chul; Eun, Choong Gi; Yang, Yeong Il

1996-01-01

An experimental study was carried out to evaluate the morphological changes in the blood brain barrier and neighbouring brain tissue caused by Gd-DTPA in uremia-induced rabbits. Bilateral renal arteries and veins of ten rabbits were ligated. Gd-DTPA(0.2mmol/kg) was intravenously injected into seven rabbits immediately after ligation. After MRI, they were sacrificed 2 or 3 days after ligation in order to observe light and electron microscopic changes in the blood brain barrier and brain tissue. MRI findings were normal, except for enhancement of the superior and inferior sagittal sinuses on T1 weighted images in uremia-induced rabbits injected with Gd-DTPA. On light microscopic examination, these rabbits showed perivascular edema and glial fibrillary acidic protein expression: electron microscopic examination showed separation of tight junctions of endothelial cells, duplication/rarefaction of basal lamina, increased lysosomes of neurons with neuronal death, demyelination of myelin, and extravasation of red blood cells. Uremia-induced rabbits injected with Gd-DTPA showed more severe changes than those without Gd-DTPA injection. Injuries to the blood brain barrier and neighbouring brain tissue were aggravated by Gd-DTPA administration in uremia-induced rabbits. These findings appear to be associated with the neurotoxicity of Gd-DTPA

Versatile functional roles of horizontal cells in the retinal circuit.

Science.gov (United States)

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

2017-07-17

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

Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

Science.gov (United States)

Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

2015-04-15

The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

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Torres P.M.M.

1998-01-01

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

Cellular mechanisms of IL-17-induced blood-brain barrier disruption.

Science.gov (United States)

Huppert, Jula; Closhen, Dorothea; Croxford, Andrew; White, Robin; Kulig, Paulina; Pietrowski, Eweline; Bechmann, Ingo; Becher, Burkhard; Luhmann, Heiko J; Waisman, Ari; Kuhlmann, Christoph R W

2010-04-01

Recently T-helper 17 (Th17) cells were demonstrated to disrupt the blood-brain barrier (BBB) by the action of IL-17A. The aim of the present study was to examine the mechanisms that underlie IL-17A-induced BBB breakdown. Barrier integrity was analyzed in the murine brain endothelial cell line bEnd.3 by measuring the electrical resistance values using electrical call impedance sensing technology. Furthermore, in-cell Western blots, fluorescence imaging, and monocyte adhesion and transendothelial migration assays were performed. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. IL-17A induced NADPH oxidase- or xanthine oxidase-dependent reactive oxygen species (ROS) production. The resulting oxidative stress activated the endothelial contractile machinery, which was accompanied by a down-regulation of the tight junction molecule occludin. Blocking either ROS formation or myosin light chain phosphorylation or applying IL-17A-neutralizing antibodies prevented IL-17A-induced BBB disruption. Treatment of mice with EAE using ML-7, an inhibitor of the myosin light chain kinase, resulted in less BBB disruption at the spinal cord and less infiltration of lymphocytes via the BBB and subsequently reduced the clinical characteristics of EAE. These observations indicate that IL-17A accounts for a crucial step in the development of EAE by impairing the integrity of the BBB, involving augmented production of ROS.-Huppert, J., Closhen, D., Croxford, A., White, R., Kulig, P., Pietrowski, E., Bechmann, I., Becher, B., Luhmann, H. J., Waisman, A., Kuhlmann, C. R. W. Cellular mechanisms of IL-17-induced blood-brain barrier disruption.

Hormones and the blood-brain barrier.

Science.gov (United States)

Hampl, Richard; Bičíková, Marie; Sosvorová, Lucie

2015-03-01

Hormones exert many actions in the brain, and brain cells are also hormonally active. To reach their targets in brain structures, hormones must overcome the blood-brain barrier (BBB). The BBB is a unique device selecting desired/undesired molecules to reach or leave the brain, and it is composed of endothelial cells forming the brain vasculature. These cells differ from other endothelial cells in their almost impermeable tight junctions and in possessing several membrane structures such as receptors, transporters, and metabolically active molecules, ensuring their selection function. The main ways how compounds pass through the BBB are briefly outlined in this review. The main part concerns the transport of major classes of hormones: steroids, including neurosteroids, thyroid hormones, insulin, and other peptide hormones regulating energy homeostasis, growth hormone, and also various cytokines. Peptide transporters mediating the saturable transport of individual classes of hormones are reviewed. The last paragraph provides examples of how hormones affect the permeability and function of the BBB either at the level of tight junctions or by various transporters.

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

Science.gov (United States)

Yuan, Jing; Yu, Jian-Xiong

2016-05-01

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

[Neurological disorders and the blood-brain barrier. Strategies and limitations for drug delivery to the brain].

Science.gov (United States)

Domínguez, Alazne; Álvarez, Antonia; Suárez-Merino, Blanca; Goñi-de-Cerio, Felipe

2014-03-01

The incidence in the central nervous system diseases has increased with a growing elderly population. Unfortunately, conventional treatments used to treat the mentioned diseases are frequently ineffective due to the presence of the blood brain barrier. To illustrate the blood-brain barrier properties that limit drug transport into the brain and the main strategies employed to treat neurologic disorders. The blood-brain barrier is mainly composed of a specialized microvascular endothelium and of glial cells. It constitutes a valuable tool to separate the central nervous system from the rest of the body. Nevertheless, it also represents an obstacle to the delivery of therapeutic drugs to the brain. To be effective, drugs must reach their target in the brain. On one hand, therapeutic agents could be designed to be able to cross the blood brain barrier. On the other hand, drug delivery systems could be employed to facilitate the therapeutic agents' entry into the central nervous system. In vivo models of neurological diseases, in addition to in vitro models of the blood brain barrier, have been widely employed for the evaluation of drugs utilized to treat central nervous system diseases.

Apelin is a novel angiogenic factor in retinal endothelial cells

International Nuclear Information System (INIS)

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

2004-01-01

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

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

International Nuclear Information System (INIS)

Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M.

1991-01-01

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate ∼ 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26

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

Science.gov (United States)

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

2014-01-01

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

Neutrophil-Mediated Delivery of Therapeutic Nanoparticles across Blood Vessel Barrier for Treatment of Inflammation and Infection

OpenAIRE

Chu, Dafeng; Gao, Jin; Wang, Zhenjia

2015-01-01

Endothelial cells form a monolayer in lumen of blood vessels presenting a great barrier for delivery of therapeutic nanoparticles (NPs) into extravascular tissues where most diseases occur, such as inflammation disorders and infection. Here, we report a strategy for delivering therapeutic NPs across this blood vessel barrier by nanoparticle in situ hitchhiking activated neutrophils. Using intravital microscopy of TNF-α-induced inflammation of mouse cremaster venules and a mouse model of acute...

Barriers and motivators to blood and cord blood donations in young African-American women.

Science.gov (United States)

Grossman, Brenda; Watkins, Andre R; Fleming, Faye; Debaun, Michael R

2005-03-01

The primary aim of this study was to assess potential barriers and motivators to blood and cord blood donation among African-American women. A telephone survey of African-American women, ages 18-30 years, in the St. Louis metropolitan area was performed. The survey was administered by trained telemarketing personnel using a Computer-Assisted Direct Interview (CADI) system. One hundred sixty-two women were surveyed. Common barriers to blood donation were inconvenience of donor sites (19%), fear of needles (16%), and too much time required to donate (15%). Potential motivators were increasing awareness of need for blood (43%), increasing the number of convenient donor locations (19%), and encouragement by spiritual leaders to have blood drives at their church (17%). Lack of awareness was the only identified barrier to cord blood donation. Most women surveyed (88%) indicated that they definitely or probably would donate cord blood. Strategies to increase the proportion of African-American blood and cord blood donations may include educating potential donors about the process and benefits of donation to particular patient populations and engaging church leadership in supporting blood and cord blood donations.

Retinal Electrophysiology Is a Viable Preclinical Biomarker for Drug Penetrance into the Central Nervous System

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Jason Charng

2016-01-01

Full Text Available Objective. To examine whether retinal electrophysiology is a useful surrogate marker of drug penetrance into the central nervous system (CNS. Materials and Methods. Brain and retinal electrophysiology were assessed with full-field visually evoked potentials and electroretinograms in conscious and anaesthetised rats following systemic or local administrations of centrally penetrant (muscimol or nonpenetrant (isoguvacine compounds. Results. Local injections into the eye/brain bypassed the blood neural barriers and produced changes in retinal/brain responses for both drugs. In conscious animals, systemic administration of muscimol resulted in retinal and brain biopotential changes, whereas systemic delivery of isoguvacine did not. General anaesthesia confounded these outcomes. Conclusions. Retinal electrophysiology, when recorded in conscious animals, shows promise as a viable biomarker of drug penetration into the CNS. In contrast, when conducted under anaesthetised conditions confounds can be induced in both cortical and retinal electrophysiological recordings.

The circadian response of intrinsically photosensitive retinal ganglion cells.

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Andrew J Zele

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

Blood-CNS Barrier Impairment in ALS Patients versus an Animal Model

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Svitlana eGarbuzova-Davis

2014-02-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a severe neurodegenerative disease with a compli-cated and poorly understood pathogenesis. Recently, alterations in the blood-Central Nervous System barrier (B-CNS-B have been recognized as a key factor possibly aggravating motor neuron damage. The majority of findings on ALS microvascular pathology have been deter-mined in mutant SOD1 rodent models, identifying barrier damage during disease develop-ment which might similarly occur in familial ALS patients carrying the SOD1 mutation. However, our knowledge of B-CNS-B competence in sporadic ALS (SALS has been limited. We recently showed structural and functional impairment in postmortem gray and white mat-ter microvessels of medulla and spinal cord tissue from SALS patients, suggesting pervasive barrier damage. Although numerous signs of barrier impairment (endothelial cell degenera-tion, capillary leakage, perivascular edema, downregulation of tight junction proteins, and microhemorrhages are indicated in both mutant SOD1 animal models of ALS and SALS pa-tients, other pathogenic barrier alterations have as yet only been identified in SALS patients. Pericyte degeneration, perivascular collagen IV expansion, and white matter capillary abnor-malities in SALS patients are significant barrier related pathologies yet to be noted in ALS SOD1 animal models. In the current review, these important differences in blood-CNS barrier damage between ALS patients and animal models, which may signify altered barrier transport mechanisms, are discussed. Understanding discrepancies in barrier condition between ALS patients and animal models may be crucial for developing effective therapies.

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

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Jing Xia

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

Troxler Fading, Eye Movements, and Retinal Ganglion Cell Properties

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Romain Bachy

2014-12-01

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

In vivo imaging of the retinal pigment epithelial cells

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Morgan, Jessica Ijams Wolfing

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

Endogenous retinal neural stem cell reprogramming for neuronal regeneration

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Romain Madelaine

2017-01-01

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

Timing the Generation of Distinct Retinal Cells by Homeobox Proteins

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

2006-01-01

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

Timing the generation of distinct retinal cells by homeobox proteins.

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Sarah Decembrini

2006-09-01

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

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

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Thomas A Mendel

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

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

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2016-12-01

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

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

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

2015-01-28

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

Scutellaria barbata attenuates diabetic retinopathy by preventing retinal inflammation and the decreased expression of tight junction protein

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Xi-Yu Mei

2017-06-01

Full Text Available AIM: To observe the attenuation of ethanol extract of Herba Scutellaria barbata (SE against diabetic retinopathy (DR and its engaged mechanism. METHODS: C57BL/6J mice were intraperitoneally injected with streptozotocin (STZ, 55 mg/kg for 5 consecutive days to induce diabetes. The diabetic mice were orally given with SE (100, 200 mg/kg for 1mo at 1mo after STZ injection. Blood-retinal barrier (BRB breakdown was detected by using Evans blue permeation assay. Real-time polymerase chain reaction (RT-PCR, Western blot and immunofluorescence staining were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA was used to detect serum contents of tumor necrosis factor-α (TNF-α and interleukin (IL-1β. RESULTS: SE (100, 200 mg/kg reversed the breakdown of BRB in STZ-induced diabetic mice. The decreased expression of retinal claudin-1 and claudin-19, which are both tight junction (TJ proteins, was reversed by SE. SE decreased the increased serum contents and retinal mRNA expression of TNF-α and IL-1β. SE also decreased the increased retinal expression of intercellular cell adhesion molecule-1 (ICAM-1. SE reduced the increased phosphorylation of nuclear factor kappa B (NFκB p65 and its subsequent nuclear translocation in retinas from STZ-induced diabetic mice. Results of Western blot and retinal immunofluorescence staining of ionized calcium-binding adapter molecule 1 (Iba1 demonstrated that SE abrogated the activation of microglia cells in STZ-induced diabetic mice. CONCLUSION: SE attenuates the development of DR by inhibiting retinal inflammation and restoring the decreased expression of TJ proteins including claudin-1 and claudin-19.

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

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

2015-06-01

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

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

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

2016-01-01

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

Expression and deposition of basement membrane proteins by brain capillary endothelial cells in a primary murine model of the blood-brain barrier

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Thomsen, Maj Schneider; Birkelund, Svend; Larsen, Annette Burkhart

2016-01-01

The blood-brain barrier (BBB) represents the interface between the blood and the brain parenchyma and consists of endothelial cells which are tightly sealed together by tight junction proteins. The endothelial cells are in addition supported by pericytes, which are embedded in the vascular basement...... of the present study was to create four different in vitro constructs of the murine BBB to characterise if the expression and secretion of basement membrane proteins by the murine brain capillary endothelial cells (mBCECs) was affected by co-culturing with pericytes, mixed glial cells, or both. Primary m......BCECs and pericytes were isolated from brains of adult mice. Mixed glial cells were prepared from cerebral cortices of newborn mice. The mBCECs were grown as mono-culture, or co-cultured with pericytes, mixed glial cells, or both. To study the expression of basement membrane proteins RT-qPCR, mass spectrometry...

The Role of Gene Therapy in the Treatment of Retinal Diseases: A Review.

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Campa, C; Gallenga, C E; Bolletta, E; Perri, P

2017-01-01

Gene therapy represents the therapeutic delivery of nucleic acid polymers into patient cells with the aim of treating an underlying disease. Over the past 2 decades this new therapy has made substantial progress owing to better understanding of the pathobiologic basis of various diseases coupled with growth of gene transfer biotechnologies. The eye, in particular, represents a suitable target for such therapy due to the immune privilege provided by the blood-ocular barrier, the ability to directly visualize, access and locally treat the cells and the minimal amount of vector needed given the size of this organ. It is not surprising therefore that several clinical trials are now ongoing in this field. The purpose of this review was to provide an update on gene therapy for retinal diseases, discussing differences in treatment strategies, vector designs and surgical techniques. Research was performed on PubMed, ClinicalTrials.gov, and Home Genetic Reference. We additionally utilized the internet database for genetics of retinal diseases, the portal for rare diseases and orphan drugs and the NCBI database Online Mendelian Inheritance in Man. No restriction was applied on the language of publications. We present the available results of current active clinical trials for inherited retinal disease such as Leber's congenital amaurosis type 2, choroideremia, Stargardt disease, achromatopsia and juvenile X-linked retinoschisis. We also illustrate a new approach of this therapy for the treatment of much more common ocular diseases such as age-related macular degeneration and diabetic retinopathy. Gene therapy represents an emerging and promising therapeutic approach for the treatment not only of rare inherited retinal diseases but also much more common retinal pathologies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

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Jasmin Balmer

2015-07-01

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

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

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Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

1994-08-01

trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.

The blood-brain barrier in migraine treatment

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Edvinsson, L; Tfelt-Hansen, P

2008-01-01

Salient aspects of the anatomy and function of the blood-barrier barrier (BBB) are reviewed in relation to migraine pathophysiology and treatment. The main function of the BBB is to limit the access of circulating substances to the neuropile. Smaller lipophilic substances have some access...

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

DEFF Research Database (Denmark)

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

2005-01-01

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

Imaging blood-brain barrier dysfunction as a biomarker for epileptogenesis.

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Bar-Klein, Guy; Lublinsky, Svetlana; Kamintsky, Lyn; Noyman, Iris; Veksler, Ronel; Dalipaj, Hotjensa; Senatorov, Vladimir V; Swissa, Evyatar; Rosenbach, Dror; Elazary, Netta; Milikovsky, Dan Z; Milk, Nadav; Kassirer, Michael; Rosman, Yossi; Serlin, Yonatan; Eisenkraft, Arik; Chassidim, Yoash; Parmet, Yisrael; Kaufer, Daniela; Friedman, Alon

2017-06-01

A biomarker that will enable the identification of patients at high-risk for developing post-injury epilepsy is critically required. Microvascular pathology and related blood-brain barrier dysfunction and neuroinflammation were shown to be associated with epileptogenesis after injury. Here we used prospective, longitudinal magnetic resonance imaging to quantitatively follow blood-brain barrier pathology in rats following status epilepticus, late electrocorticography to identify epileptic animals and post-mortem immunohistochemistry to confirm blood-brain barrier dysfunction and neuroinflammation. Finally, to test the pharmacodynamic relevance of the proposed biomarker, two anti-epileptogenic interventions were used; isoflurane anaesthesia and losartan. Our results show that early blood-brain barrier pathology in the piriform network is a sensitive and specific predictor (area under the curve of 0.96, P brain barrier pathology as a clinically relevant predictive, diagnostic and pharmaco!dynamics biomarker for acquired epilepsy. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Microfluidic organ-on-chip technology for blood-brain barrier research.

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van der Helm, Marinke W; van der Meer, Andries D; Eijkel, Jan C T; van den Berg, Albert; Segerink, Loes I

2016-01-01

Organs-on-chips are a new class of microengineered laboratory models that combine several of the advantages of current in vivo and in vitro models. In this review, we summarize the advances that have been made in the development of organ-on-chip models of the blood-brain barrier (BBBs-on-chips) and the challenges that are still ahead. The BBB is formed by specialized endothelial cells and separates blood from brain tissue. It protects the brain from harmful compounds from the blood and provides homeostasis for optimal neuronal function [corrected]. Studying BBB function and dysfunction is important for drug development and biomedical research. Microfluidic BBBs-on-chips enable real-time study of (human) cells in an engineered physiological microenvironment, for example incorporating small geometries and fluid flow as well as sensors. Examples of BBBs-on-chips in literature already show the potential of more realistic microenvironments and the study of organ-level functions. A key challenge in the field of BBB-on-chip development is the current lack of standardized quantification of parameters such as barrier permeability and shear stress. This limits the potential for direct comparison of the performance of different BBB-on-chip models to each other and existing models. We give recommendations for further standardization in model characterization and conclude that the rapidly emerging field of BBB-on-chip models holds great promise for further studies in BBB biology and drug development.

A Morphological Hessian Based Approach for Retinal Blood Vessels Segmentation and Denoising Using Region Based Otsu Thresholding.

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Khan BahadarKhan

Full Text Available Diabetic Retinopathy (DR harm retinal blood vessels in the eye causing visual deficiency. The appearance and structure of blood vessels in retinal images play an essential part in the diagnoses of an eye sicknesses. We proposed a less computational unsupervised automated technique with promising results for detection of retinal vasculature by using morphological hessian based approach and region based Otsu thresholding. Contrast Limited Adaptive Histogram Equalization (CLAHE and morphological filters have been used for enhancement and to remove low frequency noise or geometrical objects, respectively. The hessian matrix and eigenvalues approach used has been in a modified form at two different scales to extract wide and thin vessel enhanced images separately. Otsu thresholding has been further applied in a novel way to classify vessel and non-vessel pixels from both enhanced images. Finally, postprocessing steps has been used to eliminate the unwanted region/segment, non-vessel pixels, disease abnormalities and noise, to obtain a final segmented image. The proposed technique has been analyzed on the openly accessible DRIVE (Digital Retinal Images for Vessel Extraction and STARE (STructured Analysis of the REtina databases along with the ground truth data that has been precisely marked by the experts.

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

NARCIS (Netherlands)

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

2017-01-01

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

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

NARCIS (Netherlands)

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

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

Role of the Blood-Brain Barrier in the Formation of Brain Metastases

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István A. Krizbai

2013-01-01

Full Text Available The majority of brain metastases originate from lung cancer, breast cancer and malignant melanoma. In order to reach the brain, parenchyma metastatic cells have to transmigrate through the endothelial cell layer of brain capillaries, which forms the morphological basis of the blood-brain barrier (BBB. The BBB has a dual role in brain metastasis formation: it forms a tight barrier protecting the central nervous system from entering cancer cells, but it is also actively involved in protecting metastatic cells during extravasation and proliferation in the brain. The mechanisms of interaction of cancer cells and cerebral endothelial cells are largely uncharacterized. Here, we provide a comprehensive review on our current knowledge about the role of junctional and adhesion molecules, soluble factors, proteolytic enzymes and signaling pathways mediating the attachment of tumor cells to brain endothelial cells and the transendothelial migration of metastatic cells. Since brain metastases represent a great therapeutic challenge, it is indispensable to understand the mechanisms of the interaction of tumor cells with the BBB in order to find targets of prevention of brain metastasis formation.

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

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Sudhakar John

2013-01-01

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

The role of NgR-Rhoa-Rock signal pathway in retinal ganglion cell apoptosis of early diabetic rats

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Yun-Jie Fu

2014-09-01

Full Text Available AIM: To study the function and mechanism of the NgR-Rhoa-Rock signal pathways which exists in the retinal ganglion cells apoptosis in diabetes mellitus(DMrats. METHODS: Some healthy SD rats were operated by means of single intraperitoneal injection of 1% streptozotocin based on the standard of 50mg/kg wight, after that the blood sugar value was greater than 16.7mmol/L as DM model, then randomly divided into 3 groups, each group was 10 rats. In addition to take 10 healthy SD rats as control group. Four groups of rats were bilaterally eyeball intravitreal injection in turn with NgR-siRNA virus 10μL(siRNA group, NgR-siRNA virus diluted 10μL(DM group, NgR-siRNA virus-negative-control solution 10μL(siRNA blank group, NgR-siRNA virus diluted 10μL(normal control group, and fed normally. During that time, some life indexes like blood glucose, body mass, etc. were measured and recorded. After 12wk, the expression of NgR and Rhoa, HE staining, and TUNNEL staining were detected by Western blot analysis. RESULTS: Western blot analysis: compared with normal control group, the expression of NgR and Rhoa in DM group and siRNA blank group increased significantly(PP>0.05; compared with DM group and siRNA blank group, the expression of those proteins significantly lowered in siRNA group. HE staining: compared with normal control group, some extent ganglion cells arranged disorder, irregular shape, spacing not consistent were all found in three groups of model rats; compared with DM group and siRNA blank group, there was some improvement in siRNA group of ganglion cells about the order and shape size. TUNEL staining: compared with normal control group, there were retinal ganglion cells apoptosis in all of three groups of model rats. Compared with DM group and siRNA blank group, the number of retinal ganglion cells apoptotic cells was less, and the shape of cells had improved significantly in siRNA group. CONCLUSION: In the DM phase, the expression of NgR and

Inter-arm Blood Pressure Difference and its Relationship with Retinal Microvascular Calibres in Young Individuals: The African-PREDICT Study.

Science.gov (United States)

Strauss, Michél; Smith, Wayne; Schutte, Aletta E

2016-08-01

Bilateral systolic blood pressure (SBP) differences > 10mmHg is a common finding in clinical practice. Such BP differences in older individuals are associated with peripheral vascular disease, linked to microvascular dysfunction. Investigating retinal vessel calibres could provide insight into systemic microvascular function and may predict cardiovascular outcomes. Therefore we investigated the link between inter-arm systolic blood pressure differences (IASBPD) and the retinal microvasculature to determine the usefulness of IASBPD as an early marker of microvascular changes. In this cross-sectional study, we used data from 403 apparently healthy participants (20-30 years) (42% men; 49% black) taking part in the African-PREDICT study. Participants underwent retinal vessel imaging, anthropometric measurements and blood sampling. Brachial BP was measured sequentially in both arms to determine the mean IASBPD. Participants were stratified into two groups with an IASBPD difference in characteristics being a higher right arm SBP in the latter group (p=0.005). We found no association between IASBPD and retinal vessel calibres in any group. Less than 2% of the variance in IASBPD was explained by potential risk factors, with only SBP associating independently with IASBPD (β=115; p=0.039). In a young population an increased IASBPD is not related to retinal vessel diameters suggesting that it does not reflect early microvascular alterations. Copyright © 2016 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.

Development of the blood-brain barrier: a historical point of view.

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Ribatti, Domenico; Nico, Beatrice; Crivellato, Enrico; Artico, Marco

2006-01-01

Although there has been considerable controversy since the observation by Ehrlich more than 100 years ago that the brain did not take up dyes from the vascular system, the concept of an endothelial blood-brain barrier (BBB) was confirmed by the unequivocal demonstration that the passage of molecules from blood to brain and vice versa was prevented by endothelial tight junctions (TJs). There are three major functions implicated in the term "BBB": protection of the brain from the blood milieu, selective transport, and metabolism or modification of blood- or brain-borne substances. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of complex TJs and a number of specific transport and enzyme systems that regulate molecular traffic across the endothelial cells. The development of the BBB is a complex process that leads to endothelial cells with unique permeability characteristics due to high electrical resistance and the expression of specific transporters and metabolic pathways. This review article summarizes the historical background underlying our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB. (c) 2006 Wiley-Liss, Inc.

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier.

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Shu Yang

Full Text Available To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123 uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.

Restraint stress-induced morphological changes at the blood-brain barrier in adult rats

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Petra eSántha

2016-01-01

Full Text Available Stress is well known to contribute to the development of both neurological and psychiatric diseases. While the role of the blood-brain barrier is increasingly recognised in the development of neurodegenerative disorders, such as Alzheimer’s disease, dysfunction of the blood-brain barrier has been linked to stress-related psychiatric diseases only recently. In the present study the effects of restraint stress with different duration (1, 3 and 21 days were investigated on the morphology of the blood-brain barrier in male adult Wistar rats. Frontal cortex and hippocampus sections were immunostained for markers of brain endothelial cells (claudin-5, occludin and glucose transporter-1 and astroglia (GFAP. Staining pattern and intensity were visualized by confocal microscopy and evaluated by several types of image analysis. The ultrastructure of brain capillaries was investigated by electron microscopy. Morphological changes and intensity alterations in brain endothelial tight junction proteins claudin-5 and occludin were induced by stress. Following restraint stress significant increases in the fluorescence intensity of glucose transporter-1 were detected in brain endothelial cells in the frontal cortex and hippocampus. Significant reductions in GFAP fluorescence intensity were observed in the frontal cortex in all stress groups. As observed by electron microscopy, one-day acute stress induced morphological changes indicating damage in capillary endothelial cells in both brain regions. After 21 days of stress thicker and irregular capillary basal membranes in the hippocampus and edema in astrocytes in both regions were seen. These findings indicate that stress exerts time-dependent changes in the staining pattern of tight junction proteins occludin, claudin-5 and glucose transporter-1 at the level of brain capillaries and in the ultrastructure of brain endothelial cells and astroglial endfeet, which may contribute to neurodegenerative processes

Retinal venous blood carbon monoxide response to bright light in male pigs: A preliminary study.

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Oren, Dan A; Duda, Magdalena; Kozioł, Katarzyna; Romerowicz-Misielak, Maria; Koziorowska, Anna; Sołek, Przemysław; Nowak, Sławomir; Kulpa, Magdalena; Koziorowski, Marek

2017-03-01

The physical mechanism by which light is absorbed in the eye and has antidepressant and energizing effects in Seasonal Affective Disorder and other forms of psychiatric major depression is of scientific interest. This study was designed to explore one specific aspect of a proposed humoral phototransduction mechanism, namely that carbon monoxide (CO) levels increase in retinal venous blood in response to bright light. Eleven mature male pigs approximately six months of age were kept for 7days in darkness and fasted for 12h prior to surgery. Following mild sedation, anesthesia was induced. Silastic catheters were inserted into the dorsal nasal vein through the angular vein of the eye to reach the ophthalmic sinus, from which venous blood outflowing from the eye area was collected. The animals were exposed to 5000lx of fluorescent-generated white light. CO levels in the blood were analyzed by gas chromatography before and after 80min of light exposure. At baseline, mean CO levels in the retinal venous blood were 0.43±0.05(SE)nmol/ml. After bright light, mean CO levels increased to 0.54±0.06nmol/ml (two-tailed t-test plight exposure raises carbon monoxide levels in ophthalmic venous blood. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

2010-01-01

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

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

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

2011-11-01

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

Potential Pathways for CNS Drug Delivery Across the Blood-Cerebrospinal Fluid Barrier

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Strazielle, Nathalie; Ghersi-Egea, Jean-Fran?ois

2016-01-01

The blood-brain interfaces restrict the cerebral bioavailability of pharmacological compounds. Various drug delivery strategies have been developed to improve drug penetration into the brain. Most strategies target the microvascular endothelium forming the blood-brain barrier proper. Targeting the blood-cerebrospinal fluid (CSF) barrier formed by the epithelium of the choroid plexuses in addition to the blood-brain barrier may offer added-value for the treatment of central nervous system dise...

Molecular targets in radiation-induced blood-brain barrier disruption

International Nuclear Information System (INIS)

Nordal, Robert A.; Wong, C. Shun

2005-01-01

Disruption of the blood-brain barrier (BBB) is a key feature of radiation injury to the central nervous system. Studies suggest that endothelial cell apoptosis, gene expression changes, and alteration of the microenvironment are important in initiation and progression of injury. Although substantial effort has been directed at understanding the impact of radiation on endothelial cells and oligodendrocytes, growing evidence suggests that other cell types, including astrocytes, are important in responses that include induced gene expression and microenvironmental changes. Endothelial apoptosis is important in early BBB disruption. Hypoxia and oxidative stress in the later period that precedes tissue damage might lead to astrocytic responses that impact cell survival and cell interactions. Cell death, gene expression changes, and a toxic microenvironment can be viewed as interacting elements in a model of radiation-induced disruption of the BBB. These processes implicate particular genes and proteins as targets in potential strategies for neuroprotection

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

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

2016-04-01

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

Retinal blood vessel extraction using tunable bandpass filter and fuzzy conditional entropy.

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Sil Kar, Sudeshna; Maity, Santi P

2016-09-01

Extraction of blood vessels on retinal images plays a significant role for screening of different opthalmologic diseases. However, accurate extraction of the entire and individual type of vessel silhouette from the noisy images with poorly illuminated background is a complicated task. To this aim, an integrated system design platform is suggested in this work for vessel extraction using a sequential bandpass filter followed by fuzzy conditional entropy maximization on matched filter response. At first noise is eliminated from the image under consideration through curvelet based denoising. To include the fine details and the relatively less thick vessel structures, the image is passed through a bank of sequential bandpass filter structure optimized for contrast enhancement. Fuzzy conditional entropy on matched filter response is then maximized to find the set of multiple optimal thresholds to extract the different types of vessel silhouettes from the background. Differential Evolution algorithm is used to determine the optimal gain in bandpass filter and the combination of the fuzzy parameters. Using the multiple thresholds, retinal image is classified as the thick, the medium and the thin vessels including neovascularization. Performance evaluated on different publicly available retinal image databases shows that the proposed method is very efficient in identifying the diverse types of vessels. Proposed method is also efficient in extracting the abnormal and the thin blood vessels in pathological retinal images. The average values of true positive rate, false positive rate and accuracy offered by the method is 76.32%, 1.99% and 96.28%, respectively for the DRIVE database and 72.82%, 2.6% and 96.16%, respectively for the STARE database. Simulation results demonstrate that the proposed method outperforms the existing methods in detecting the various types of vessels and the neovascularization structures. The combination of curvelet transform and tunable bandpass

Atomistic modeling of the structural components of the blood-brain barrier

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Glukhova, O. E.; Grishina, O. A.; Slepchenkov, M. M.

2015-03-01

Blood-brain barrier, which is a barrage system between the brain and blood vessels, plays a key role in the "isolation" of the brain of unnecessary information, and reduce the "noise" in the interneuron communication. It is known that the barrier function of the BBB strictly depends on the initial state of the organism and changes significantly with age and, especially in developing the "vascular accidents". Disclosure mechanisms of regulation of the barrier function will develop new ways to deliver neurotrophic drugs to the brain in the newborn. The aim of this work is the construction of atomistic models of structural components of the blood-brain barrier to reveal the mechanisms of regulation of the barrier function.

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

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

2016-10-15

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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

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

2013-01-01

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

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

DEFF Research Database (Denmark)

Wiencke, Anne Katrine

2001-01-01

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

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

DEFF Research Database (Denmark)

Wiencke, Anne Katrine

2001-01-01

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

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

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Aaron H Fronk

2016-07-01

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

The Protective Effects of Lycium Barbarum Polysaccharides on Transient Retinal Ischemia

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Di Yang

2011-05-01

Full Text Available Retinal ischemia/reperfusion (I/R injury leads to irreversible neuronal death, glial activation, retinal swelling and oxidative stress. It is a common feature in various ocular diseases, such as glaucoma, diabetic retinopathy and amaurosis fugax. In the present study, we aimed to evaluate the effects of Lycium Barbarum Polysaccharides (LBP in a murine retinal I/R model. Mice were orally treated with either vehicle (PBS or LBP (1mg/kg daily for 1 week before induction of retinal ischemia. Retinae were collected after 2 hours ischemia and 22 hours reperfusion. Paraffin-embedded sections were prepared for immunohistochemical analyses. Significantly fewer viable cells were found in vehicle-treated retinae comparing to LBP group. This finding was further confirmed by TUNEL assay where significantly fewer apoptotic cells were identified in LBP-treated retinae. Additionally, retinal swelling induced by retinal I/R injury in the vehicle-treated group was not observed in LBP-treated group. Moreover, intense GFAP immunoreactivity and IgG extravasation were observed in vehicle-treated group but not in LBP treated group. The results showed that pre-treatment with LBP was protective in retinal I/R injury via reducing neuronal death, apoptosis, retinal swelling, GFAP activation and blood vessel leakage. LBP may be used as a preventive agent for retinal ischemia diseases.

The Protective Effects of Lycium Barbarum Polysaccharides on Transient Retinal Ischemia

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Yang, Di; Li, Suk-Yee; Yeung, Chung-Man; Yu, Wing-Yan; Chang, Raymond Chuen-Chung; So, Kwok-Fai; Wong, David; Lo, Amy C. Y.

2011-01-01

Retinal ischemia/reperfusion (I/R) injury leads to irreversible neuronal death, glial activation, retinal swelling and oxidative stress. It is a common feature in various ocular diseases, such as glaucoma, diabetic retinopathy and amaurosis fugax. In the present study, we aimed to evaluate the effects of Lycium Barbarum Polysaccharides (LBP) in a murine retinal I/R model. Mice were orally treated with either vehicle (PBS) or LBP (1mg/kg) daily for 1 week before induction of retinal ischemia. Retinae were collected after 2 hours ischemia and 22 hours reperfusion. Paraffin-embedded sections were prepared for immunohistochemical analyses. Significantly fewer viable cells were found in vehicle-treated retinae comparing to LBP group. This finding was further confirmed by TUNEL assay where significantly fewer apoptotic cells were identified in LBP-treated retinae. Additionally, retinal swelling induced by retinal I/R injury in the vehicle-treated group was not observed in LBP-treated group. Moreover, intense GFAP immunoreactivity and IgG extravasation were observed in vehicle-treated group but not in LBP treated group. The results showed that pre-treatment with LBP was protective in retinal I/R injury via reducing neuronal death, apoptosis, retinal swelling, GFAP activation and blood vessel leakage. LBP may be used as a preventive agent for retinal ischemia diseases.

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

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

2013-04-01

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

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

Science.gov (United States)

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

2009-11-06

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

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

Science.gov (United States)

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

2018-02-12

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

Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

Science.gov (United States)

Li, Nan; Mruk, Dolores D; Mok, Ka-Wai; Li, Michelle W M; Wong, Chris K C; Lee, Will M; Han, Daishu; Silvestrini, Bruno; Cheng, C Yan

2016-04-01

Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to

Nampt/PBEF/visfatin serum levels: a new biomarker for retinal blood vessel occlusions

Directory of Open Access Journals (Sweden)

Kaja S

2015-04-01

Full Text Available Simon Kaja,1,* Anna A Shah,1,* Shamim A Haji,1,* Krishna B Patel,1 Yuliya Naumchuk,1 Alexander Zabaneh,1 Bryan C Gerdes,1 Nancy Kunjukunju,1 Nelson R Sabates,1 Michael A Cassell,1 Ron K Lord,1 Kevin P Pikey,1 Abraham Poulose,1 Peter Koulen1,21Vision Research Center, Department of Ophthalmology, 2Department of Basic Medical Science, School of Medicine, University of Missouri – Kansas City, Kansas City, MO, USA*These authors contributed equally to this workAbstract: The main objective of the study was to quantify serum levels of nicotinamide phosphoribosyltransferase (Nampt/pre-B-Cell colony-enhancing factor 1/visfatin in subjects with a history of retinal vascular occlusions (RVOs, disease conditions characterized by pronounced ischemia, and metabolic energy deficits. A case–control study of 18 subjects with a history of RVO as well as six healthy volunteers is presented. Serum Nampt levels were quantified using a commercially available enzyme-linked immunosorbent assay kit. Serum Nampt levels were 79% lower in patients with a history of RVO compared with that in healthy volunteers (P<0.05. There was no statistically significant difference among the types of RVOs, specifically branch retinal vein occlusions (n=7, central retinal vein occlusions (n=5, hemiretinal vein occlusions (n=3, and central retinal artery occlusions (n=3; P=0.69. Further studies are needed to establish the temporal kinetics of Nampt expression and to determine whether Nampt may represent a novel biomarker to identify at-risk populations, or whether it is a druggable target with the potential to ameliorate the long-term complications associated with the condition, ie, macular edema, macular ischemia, neovascularization, and permanent loss of vision.Keywords: Nampt, PBEF, visfatin, nicotinamide phosphoribosyltransferase, pre-B-cell colony-enhancing factor, retinal artery occlusion, retinal vein occlusion, biomarker, retina, vasculature

Cerebrospinal fluid aquaporin-4-immunoglobulin G disrupts blood brain barrier

DEFF Research Database (Denmark)

Asgari, Nasrin; Berg, Carsten Tue; Mørch, Marlene Thorsen

2015-01-01

associated with blood-borne horseradish peroxidase leakage indicating blood-brain barrier breakdown. The cerebrospinal fluid aquaporin-4-immunoglobulin G therefore distributes widely in brain to initiate astrocytopathy and blood-brain barrier breakdown....... was evaluated. A distinct distribution pattern of aquaporin-4-immunoglobulin G deposition was observed in the subarachnoid and subpial spaces where vessels penetrate the brain parenchyma, via a paravascular route with intraparenchymal perivascular deposition. Perivascular astrocyte-destructive lesions were...

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS.

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Svitlana Garbuzova-Davis

2007-11-01

Full Text Available The blood-brain barrier (BBB, blood-spinal cord barrier (BSCB, and blood-cerebrospinal fluid barrier (BCSFB control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.Evans Blue (EB dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.

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

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

2011-11-15

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

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

International Nuclear Information System (INIS)

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

2011-01-01

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

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

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Budd A Tucker

2011-04-01

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

Retinal biometrics based on Iterative Closest Point algorithm.

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Hatanaka, Yuji; Tajima, Mikiya; Kawasaki, Ryo; Saito, Koko; Ogohara, Kazunori; Muramatsu, Chisako; Sunayama, Wataru; Fujita, Hiroshi

2017-07-01

The pattern of blood vessels in the eye is unique to each person because it rarely changes over time. Therefore, it is well known that retinal blood vessels are useful for biometrics. This paper describes a biometrics method using the Jaccard similarity coefficient (JSC) based on blood vessel regions in retinal image pairs. The retinal image pairs were rough matched by the center of their optic discs. Moreover, the image pairs were aligned using the Iterative Closest Point algorithm based on detailed blood vessel skeletons. For registration, perspective transform was applied to the retinal images. Finally, the pairs were classified as either correct or incorrect using the JSC of the blood vessel region in the image pairs. The proposed method was applied to temporal retinal images, which were obtained in 2009 (695 images) and 2013 (87 images). The 87 images acquired in 2013 were all from persons already examined in 2009. The accuracy of the proposed method reached 100%.

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

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

2016-01-01

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

Cytomegalovirus glycoprotein B genotyping in ocular fluids and blood of AIDS patients with cytomegalovirus retinitis

NARCIS (Netherlands)

Peek, R.; Verbraak, F.; Bruinenberg, M.; van der Lelij, A.; van den Horn, G.; Kijlstra, A.

1998-01-01

To determine the frequency of cytomegalovirus glycoprotein B (gB) genotypes in clinical samples of ocular fluids of patients with acquired immune deficiency syndrome (AIDS) who have cytomegalovirus retinitis and to compare these with the cytomegalovirus gB genotype in paired peripheral blood

Cultured cells of the blood-brain barrier from apolipoprotein B-100 transgenic mice: effects of oxidized low-density lipoprotein treatment.

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Lénárt, Nikolett; Walter, Fruzsina R; Bocsik, Alexandra; Sántha, Petra; Tóth, Melinda E; Harazin, András; Tóth, Andrea E; Vizler, Csaba; Török, Zsolt; Pilbat, Ana-Maria; Vígh, László; Puskás, László G; Sántha, Miklós; Deli, Mária A

2015-07-17

The apolipoprotein B-100 (ApoB-100) transgenic mouse line is a model of human atherosclerosis. Latest findings suggest the importance of ApoB-100 in the development of neurodegenerative diseases and microvascular/perivascular localization of ApoB-100 protein was demonstrated in the cerebral cortex of ApoB-100 transgenic mice. The aim of the study was to characterize cultured brain endothelial cells, pericytes and glial cells from wild-type and ApoB-100 transgenic mice and to study the effect of oxidized low-density lipoprotein (oxLDL) on these cells. Morphology of cells isolated from brains of wild type and ApoB-100 transgenic mice was characterized by immunohistochemistry and the intensity of immunolabeling was quantified by image analysis. Toxicity of oxLDL treatment was monitored by real-time impedance measurement and lactate dehydrogenase release. Reactive oxygen species and nitric oxide production, barrier permeability in triple co-culture blood-brain barrier model and membrane fluidity were also determined after low-density lipoprotein (LDL) or oxLDL treatment. The presence of ApoB-100 was confirmed in brain endothelial cells, while no morphological change was observed between wild type and transgenic cells. Oxidized but not native LDL exerted dose-dependent toxicity in all three cell types, induced barrier dysfunction and increased reactive oxygen species (ROS) production in both genotypes. A partial protection from oxLDL toxicity was seen in brain endothelial and glial cells from ApoB-100 transgenic mice. Increased membrane rigidity was measured in brain endothelial cells from ApoB-100 transgenic mice and in LDL or oxLDL treated wild type cells. The morphological and functional properties of cultured brain endothelial cells, pericytes and glial cells from ApoB-100 transgenic mice were characterized and compared to wild type cells for the first time. The membrane fluidity changes in ApoB-100 transgenic cells related to brain microvasculature indicate

Alterations in blood-brain barrier function following acute hypertension: comparison of the blood-to-brain transfer of horseradish peroxidase with that of alpha-aminisobutyric acid

International Nuclear Information System (INIS)

Ellison, M.D.B.

1985-01-01

The blood-brain barrier (BBB) selectively restricts the blood-to-brain passage of many solutes owing to unique properties of cerebrovascular endothelial cell membranes. To date, experimental study of the BBB has been accomplished primarily through the use of two different methodological approaches. Morphological studies have mostly employed large molecular weight (MW) tracers to detect morphological alterations underlying increased permeability. Physiological studies, employing smaller, more physiologic tracers have successfully described, quantitatively, certain functional aspects of blood-to-brain transfer. The current work attempts to merge these two approaches and to consider barrier function/dysfunction from both a morphological and a functional perspective. Specifically, the study compares in rats, following acute hypertension, the cerebrovascular passage of 14 C-alpha-aminoisobutyric acid (AIB) and that of horseradish peroxidase (HRP). The blood-to-brain passage of AIB and HRP were compared following acute hypertension, with regard to both the distributions of the tracer extravasation patterns and the magnitude of tracer extravasation. The results of this study suggest that traditional morphological barrier studies alone do not reveal all aspects of altered barrier status and that multiple mechanisms underlying increased BBB permeability may operate simultaneously during BBB dysfunction

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

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

2017-01-01

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

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

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Lorena Olivares-González

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

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

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Girish K. Srivastava

2014-01-01

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

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

Science.gov (United States)

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

2017-10-01

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

In Vitro Modeling of Blood-Brain Barrier with Human iPSC-Derived Endothelial Cells, Pericytes, Neurons, and Astrocytes via Notch Signaling

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Kohei Yamamizu

2017-03-01

Full Text Available The blood-brain barrier (BBB is composed of four cell populations, brain endothelial cells (BECs, pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A and efflux transporters (ABCA1, BCRP, PGP, MRP5, and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

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

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Chi-Hsien Peng

2010-06-01

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

Sorting Tubules Regulate Blood-Brain Barrier Transcytosis

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Roberto Villaseñor

2017-12-01

Full Text Available Transcytosis across the blood-brain barrier (BBB regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

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

Science.gov (United States)

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

2018-02-21

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

Ocular hemodynamics in patients with rhegmatogenous retinal detachment

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N. H. Zavgorodnya

2014-10-01

Full Text Available Aim. In case of retinal detachment atrophic processes lead to irreversible loss of functions within 4–6 days, it happens on underlying low ocular blood flow. In order to evaluate the degree of violation of regional hemodynamics in patients with retinal detachment two groups of patients were examined: the main group (52 patients with rhegmatogenous retinal detachment and the control group (24 myopic patients with lattice form of peripheral chorioretinal dystrophy. Methods and results. Doppler and reography results had been compared, significant decrease of blood flow in patients with retinal detachment was found. No differences between affected and fellow eye in these patients, close negative correlation between the level of ocular blood flow and the degree of myopia in the control group. Conclusion. This demonstrates the feasibility of actions to improve regional blood flow in patients operated on for retinal detachment.

Blood-brain barrier-on-a-chip: Microphysiological systems that capture the complexity of the blood-central nervous system interface.

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Phan, Duc Tt; Bender, R Hugh F; Andrejecsk, Jillian W; Sobrino, Agua; Hachey, Stephanie J; George, Steven C; Hughes, Christopher Cw

2017-11-01

The blood-brain barrier is a dynamic and highly organized structure that strictly regulates the molecules allowed to cross the brain vasculature into the central nervous system. The blood-brain barrier pathology has been associated with a number of central nervous system diseases, including vascular malformations, stroke/vascular dementia, Alzheimer's disease, multiple sclerosis, and various neurological tumors including glioblastoma multiforme. There is a compelling need for representative models of this critical interface. Current research relies heavily on animal models (mostly mice) or on two-dimensional (2D) in vitro models, neither of which fully capture the complexities of the human blood-brain barrier. Physiological differences between humans and mice make translation to the clinic problematic, while monolayer cultures cannot capture the inherently three-dimensional (3D) nature of the blood-brain barrier, which includes close association of the abluminal side of the endothelium with astrocyte foot-processes and pericytes. Here we discuss the central nervous system diseases associated with blood-brain barrier pathology, recent advances in the development of novel 3D blood-brain barrier -on-a-chip systems that better mimic the physiological complexity and structure of human blood-brain barrier, and provide an outlook on how these blood-brain barrier-on-a-chip systems can be used for central nervous system disease modeling. Impact statement The field of microphysiological systems is rapidly evolving as new technologies are introduced and our understanding of organ physiology develops. In this review, we focus on Blood-Brain Barrier (BBB) models, with a particular emphasis on how they relate to neurological disorders such as Alzheimer's disease, multiple sclerosis, stroke, cancer, and vascular malformations. We emphasize the importance of capturing the three-dimensional nature of the brain and the unique architecture of the BBB - something that until recently

Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

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Álcio Coutinho de Paula

2015-04-01

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

The long noncoding RNA RNCR2 directs mouse retinal cell specification

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Blackshaw Seth

2010-05-01

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

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

Directory of Open Access Journals (Sweden)

Robert Cantrup

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

Retinal ganglion cell topography and spatial resolving power in penguins.

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

2012-01-01

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

Iron uptake and transport at the blood-brain barrier

DEFF Research Database (Denmark)

Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben

The mechanism by which iron is transported across the blood-brain barrier (BBB) remains controversial, and in this study we aimed to further clarify mechanisms by which iron is transported into the brain. We analyzed and compared the mRNA and protein expression of a variety of proteins involved...... in the transport of iron (transferrin receptor, divalent metal transporter I (DMT1), steap 2, steap 3, ceruloplasmin, hephaestin and ferroportin) in both primary rat brain capillary endothelial cells (BCEC) and immortalized rat brain capillary endothelial cell line (RBE4) grown in co-culture with defined polarity....... The mRNA expression of the iron-related molecules was also investigated in isolated brain capillaries from iron deficiency, iron reversible and normal rats. We also performed iron transport studies to analyze the routes by which iron is transported through the brain capillary endothelial cells: i) We...

Fibronectin changes in eosinophilic meningitis with blood-CSF barrier disruption.

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Shyu, Ling-Yuh; Hu, Ming-E; Chou, Chun-Hui; Chen, Ke-Min; Chiu, Ping-Sung; Lai, Shih-Chan

2015-01-01

Fibronectin, which is present at relatively low levels in healthy central nervous systems (CNS), shows increased levels in meningitis. In this study, fibronectin processing was correlated with the increased permeability of the blood-cerebrospinal fluid (CSF) barrier as well as with the formation of eosinophil infiltrates in angiostrongyliasis meningitis. The immunohistochemistry results show matrix metalloproteinase-9 (MMP-9) is localized in the choroid plexus epithelium. Coimmunoprecipitation demonstrated fibronectin strongly binds MMP-9. Furthermore, treatment with the MMP-9 inhibitor GM6001 significantly inhibited fibronectin processing, reduced the blood-CSF barrier permeability, and decreased the eosinophil counts. The decreased fibronectin processing in CSF implies decreased cellular invasion of the subarachnoid space across the blood-CSF barrier. Therefore, increased fibronectin processing may be associated with barrier disruption and participate in the extravasation and migration of eosinophils into the CNS during experimental parasitic infection. Copyright © 2015 Elsevier Inc. All rights reserved.

Spectrophotometric retinal oximetry in pigs

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Traustason, Sindri; Kiilgaard, Jens Folke; Karlsson, Robert

2013-01-01

PURPOSE: To assess the validity of spectrophotometric retinal oximetry, by comparison to blood gas analysis and intra-vitreal measurements of partial pressure of oxygen (pO2). METHODS: Female domestic pigs were used for all experiments (n=8). Oxygen fraction in inspired air was changed using...... a mixture of room air, pure oxygen and pure nitrogen, ranging from 5% to 100% oxygen. Femoral arterial blood gas analysis and retinal oximetry was performed at each level of inspiratory oxygen fraction. Retinal oximetry was performed using a commercial instrument, the Oxymap Retinal Oximeter T1 (Oxymap ehf...... arterial oxygen saturation and the optical density ratio over retinal arteries revealed an approximately linear relationship (R(2) = 0.74, p = 3.4 x 10(-9)). In order to test the validity of applying the arterial calibration to veins, we compared non-invasive oximetry measurements to invasive pO2...

Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

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

2012-01-01

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

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

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Georg, Birgitte; Ghelli, Anna; Giordano, Carla

2017-01-01

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

Protein kinase C in porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

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Gesslein, Bodil; Gustafsson, Lotta; Wackenfors, Angelica

2009-01-01

Identification of the intracellular signal-transduction pathways activated in retinal ischemia may be important in revealing novel pharmacological targets. To date, most studies have focused on identifying neuroprotective agents. The retinal blood vessels are key organs in circulatory failure, an...

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

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

2015-09-01

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

Propitious Therapeutic Modulators to Prevent Blood-Spinal Cord Barrier Disruption in Spinal Cord Injury.

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Kumar, Hemant; Ropper, Alexander E; Lee, Soo-Hong; Han, Inbo

2017-07-01

The blood-spinal cord barrier (BSCB) is a specialized protective barrier that regulates the movement of molecules between blood vessels and the spinal cord parenchyma. Analogous to the blood-brain barrier (BBB), the BSCB plays a crucial role in maintaining the homeostasis and internal environmental stability of the central nervous system (CNS). After spinal cord injury (SCI), BSCB disruption leads to inflammatory cell invasion such as neutrophils and macrophages, contributing to permanent neurological disability. In this review, we focus on the major proteins mediating the BSCB disruption or BSCB repair after SCI. This review is composed of three parts. Section 1. SCI and the BSCB of the review describes critical events involved in the pathophysiology of SCI and their correlation with BSCB integrity/disruption. Section 2. Major proteins involved in BSCB disruption in SCI focuses on the actions of matrix metalloproteinases (MMPs), tumor necrosis factor alpha (TNF-α), heme oxygenase-1 (HO-1), angiopoietins (Angs), bradykinin, nitric oxide (NO), and endothelins (ETs) in BSCB disruption and repair. Section 3. Therapeutic approaches discusses the major therapeutic compounds utilized to date for the prevention of BSCB disruption in animal model of SCI through modulation of several proteins.

Transplantation of in vitro cultured endothelial progenitor cells repairs the blood-brain barrier and improves cognitive function of APP/PS1 transgenic AD mice.

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Zhang, Shishuang; Zhi, Yongle; Li, Fei; Huang, Shan; Gao, Huabin; Han, Zhaoli; Ge, Xintong; Li, Dai; Chen, Fanglian; Kong, Xiaodong; Lei, Ping

2018-04-15

To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. It is well-known that excessive deposition of Aβ in the brain is a crucial part of the pathogenesis of AD. In recent years, the AD neurovascular unit hypothesis has attracted much attention. Impairment of the blood-brain barrier (BBB) leads to abnormal amyloid-β (Aβ) transport, and chronic cerebral hypoperfusion causes Aβ deposition throughout the onset and progression of AD. Endothelial progenitor cells (EPCs) are the universal cells for repairing blood vessels. Our previous studies have shown that a reduced number of EPCs in the peripheral blood results in cerebral vascular repair disorder, cerebral hypoperfusion and neurodegeneration, which might be related to the cognitive dysfunction of AD patients. This study was designed to confirm whether EPCs transplantation could repair the blood-brain barrier, stimulate angiogenesis and reduce Aβ deposition in AD. The expression of ZO-1, Occludin and Claudin-5 was up-regulated in APP/PS1 transgenic mice after hippocampal transplantation of EPCs. Consistent with previous studies, EPC transplants also increased the microvessel density. We observed that Aβ senile plaque deposition was decreased and hippocampal cell apoptosis was reduced after EPCs transplantation. The Morris water maze test showed that spatial learning and memory functions were significantly improved in mice transplanted with EPCs. Consequently, EPCs could up-regulate the expression of tight junction proteins, repair BBB tight junction function, stimulate angiogenesis, promote Aβ clearance, and decrease neuronal loss, ultimately improve cognitive function. Taken together, these data demonstrate EPCs may play an important role in the therapeutic implications for vascular dysfunction in AD. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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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.

Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa

NARCIS (Netherlands)

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

2014-01-01

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

Barrier mechanisms in the Drosophila blood-brain barrier.

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Hindle, Samantha J; Bainton, Roland J

2014-01-01

The invertebrate blood-brain barrier (BBB) field is growing at a rapid pace and, in recent years, studies have shown a physiologic and molecular complexity that has begun to rival its vertebrate counterpart. Novel mechanisms of paracellular barrier maintenance through G-protein coupled receptor signaling were the first demonstrations of the complex adaptive mechanisms of barrier physiology. Building upon this work, the integrity of the invertebrate BBB has recently been shown to require coordinated function of all layers of the compound barrier structure, analogous to signaling between the layers of the vertebrate neurovascular unit. These findings strengthen the notion that many BBB mechanisms are conserved between vertebrates and invertebrates, and suggest that novel findings in invertebrate model organisms will have a significant impact on the understanding of vertebrate BBB functions. In this vein, important roles in coordinating localized and systemic signaling to dictate organism development and growth are beginning to show how the BBB can govern whole animal physiologies. This includes novel functions of BBB gap junctions in orchestrating synchronized neuroblast proliferation, and of BBB secreted antagonists of insulin receptor signaling. These advancements and others are pushing the field forward in exciting new directions. In this review, we provide a synopsis of invertebrate BBB anatomy and physiology, with a focus on insights from the past 5 years, and highlight important areas for future study.

G protein-coupled receptor 91 signaling in diabetic retinopathy and hypoxic retinal diseases.

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Hu, Jianyan; Li, Tingting; Du, Xinhua; Wu, Qiang; Le, Yun-Zheng

2017-10-01

G protein-coupled receptor 91 (GPR91) is a succinate-specific receptor and activation of GPR91 could initiate a complex signal transduction cascade and upregulate inflammatory and pro-angiogenic cytokines. In the retina, GPR91 is predominately expressed in ganglion cells, a major cellular entity involved in the pathogenesis of diabetic retinopathy (DR) and other hypoxic retinal diseases. During the development of DR and retinopathy of prematurity (ROP), chronic hypoxia causes an increase in the levels of local succinate. Succinate-mediated GPR91 activation upregulates vascular endothelial growth factor (VEGF) through ERK1/2-C/EBP β (c-Fos) and/or ERK1/2-COX-2/PGE2 signaling pathways, which in turn, leads to the breakdown of blood-retina barriers in these disorders. In this review, we will have a brief introduction of GPR91 and its biological functions and a more detailed discussion about the role and mechanisms of GPR91 in DR and ROP. A better understanding of GPR91 regulation may be of great significance in identifying new biomarkers and drug targets for the prediction and treatment of DR, ROP, and hypoxic retinal diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

2010-01-08

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

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

International Nuclear Information System (INIS)

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

2010-01-01

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

BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

Science.gov (United States)

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

2015-01-01

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

The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes

OpenAIRE

DeSalvo, Michael K.; Hindle, Samantha J.; Rusan, Zeid M.; Orng, Souvinh; Eddison, Mark; Halliwill, Kyle; Bainton, Roland J.

2014-01-01

Central nervous system (CNS) function is dependent on the stringent regulation of metabolites, drugs, cells, and pathogens exposed to the CNS space. Cellular blood-brain barrier (BBB) structures are highly specific checkpoints governing entry and exit of all small molecules to and from the brain interstitial space, but the precise mechanisms that regulate the BBB are not well understood. In addition, the BBB has long been a challenging obstacle to the pharmacologic treatment of CNS diseases; ...

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

Directory of Open Access Journals (Sweden)

Kosuke Fujita

2017-06-01

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

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

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

2017-12-01

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

Long-term effects of short-term retinal bleb detachments in rabbits.

Science.gov (United States)

Ivert, Lena; Kjeldbye, Hild; Gouras, Peter

2002-03-01

To examine the effects of saline-induced bleb detachments in rabbit retina. Retinal bleb detachments were produced by the injection of 50 microl of balanced salt solution (BSS) into the subretinal space of one eye of each of six rabbits using a glass pipette with a flat tip, 50 microm in diameter. The retina was examined by biomicroscopy, scanning laser ophthalmoscopy (SLO), auto-fluorescence and simultaneous fluorescein and indocyanine green (ICG) angiography. Histological examination was carried out at 1, 2, 3 and 4 months after surgery. All rabbits showed leakage of fluorescein for at least a day after detachment, but within 1 month the leakage ceased. ICG staining developed gradually at the level of the RPE or Bruch's membrane near sites of previous staining. Lipofuscin fluorescence also developed gradually around areas of staining. Histology revealed the source of the excessive lipofuscin to be in the RPE layer, especially in cells migrating away from Bruch's membrane. Short-term bleb detachments cause a transient breakdown in the blood-retinal barrier, long-term ICG staining at or deep to the RPE layer, hyperlipofuscinosis and migration of the RPE. The abnormal lipofuscin accumulation is apparent on fluorescence ophthalmoscopy and can be confused with markers such as green fluorescent protein.

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