isolation-of-primary-mouse-retinal-pigmented-epithelium-cells

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Oakland University

William Beaumont School of Medicine

Ischemic damage results in irreversible ganglion cell loss in the retina. While the mechanisms underlying ischemia-induced ganglion cell loss are not clearly understood, we have recently reported that ischemia, induced by optic nerve ligation, results in increased nerve fiber layer-associated matrix metalloproteinase-9 (MMP-9) induction and loss of ganglion cells from the retina. This study was conducted to determine the cellular source of MMP-9 using antibodies against MMP-9 and various cell types in the inner retina. The results presented in this study show that optic nerve ligation leads to induction of MMP-9 and activation of astrocytes. Double labeling studies using antibodies against MMP-9 and GFAP showed a greater overlap of MMP-9 with GFAP, compared to antibodies against glutamine synthetase and MMP-9 which showed no overlapping, suggesting that activated astrocytes contribute to MMP-9 expression in the retina. Further, double labeling studies using antibodies against von Willebrand factor and MMP-9 or Mac-1 and MMP-9 showed no overlapping of MMP-9 with any antibodies indicating that endothelial cells and microglial cells are not the principal source of MMP-9 in the retina following optic nerve ligation.

Optic nerve ligation leads to astrocyte-associated matrix metalloproteinase-9 induction in the mouse retina.

Excitotoxicity has been proposed to play a pivotal role in retinal damage, but the mechanisms that underlie retinal damage are not clearly understood. In this study, the role of matrix metalloproteinases in excitotoxin-mediated retinal damage was investigated.

Kainic acid-mediated upregulation of matrix metalloproteinase-9 promotes retinal degeneration.

Naturally occurring mutations of the beta subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (beta-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod beta-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type beta-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction. Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2-P42 rd mice were exposed for 20 hr to 2.5 x 10(8) plaque forming units (pfu) ml(-1) of adenoviral vector with a beta-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for beta-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7-P8, then decreased at subsequent ages; little transduction could be found after P17. In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells.

Adenoviral-mediated gene transfer to retinal explants during development and degeneration.

Membrane depolarization and subsequent synaptic release of l-glutamate have been implicated in ischemic retinal damage. However, the mechanisms that lead to ischemia-induced retinal damage are poorly understood. In this study, KCl, a classic membrane depolarizing agent, was injected into the vitreous humor, and the role of matrix metalloproteinase (MMP)-9 in KCl-induced retinal damage was investigated.

Intravitreous injection of a membrane depolarization agent causes retinal degeneration via matrix metalloproteinase-9.

Increased levels of extracellular l-glutamate have been suggested to play a role in retinal damage in a number of blinding diseases such as glaucoma and diabetic retinopathy. Although glutamate can cause retinal damage in part by hyperstimulating its receptors ("excitotoxicity"), the downstream events that lead to retinal damage are poorly understood. In this study, we injected kainic acid (KA), a glutamate receptor agonist that specifically hyperstimulates non-NMDA-type receptors, into the vitreous humor of CD-1 mice and have investigated the role of plasminogen activators (PAs) [tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)] in excitotoxicity-induced retinal damage. Injection of KA into the vitreous humor led to an up-regulation in tPA and an induction in uPA activity in the retina and this was associated with activation of zymogen plasminogen to active plasmin. Immunocytochemical analysis indicated that retinal ganglion cells (RGCs), constitutively express tPA and release it into the extracellular space upon KA injection. Immunocytochemical analysis also indicated an increase in uPA in the nerve fiber layer after KA injection that was absent in the control retinas. These events were associated with apoptotic death of cells initially in the ganglion cell layer and subsequently in the inner and outer nuclear layer, associated with loss of RGCs and amacrine cells. These phenomena were inhibited when recombinant plasminogen activator inhibitor (rPAI-1) or tPA-STOP were injected into the vitreous humor with KA, whereas a plasmin inhibitor, alpha-2-antiplasmin, failed to attenuate KA-induced retinal damage. Taken together, these results suggest that inhibition of plasminogen activators might attenuate retinal damage in blinding retinal diseases in which hyperstimulation of glutamate receptors is implicated as a causative factor to retinal damage.

Plasminogen activators promote excitotoxicity-induced retinal damage.

Para comparar la eficacia de la transducci&#xF3;n de un vector lentiviral en la retina de los ratones normales y ratones degenerativas de retina (DR) despu&#xE9;s de la inyecci&#xF3;n subretiniana en diferentes edades posnatales.

Eficacia de la transducci&#xF3;n lentiviral durante el desarrollo en ratones normales y Rd

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To compare the transduction efficiency of a lentiviral vector in the retina of normal mice and retinal degenerative (rd) mice following subretinal injection at various postnatal ages.

Efficiency of lentiviral transduction during development in normal and rd mice.

Pour comparer l&#x26;#39;efficacit&#xE9; de la transduction d&#x26;#39;un vecteur lentiviral dans la r&#xE9;tine de souris normales et r&#xE9;tiniennes d&#xE9;g&#xE9;n&#xE9;ratives (e) des souris apr&#xE8;s l&#x26;#39;injection sous-r&#xE9;tinienne &#xE0; diff&#xE9;rents &#xE2;ges postnataux.

Efficacit&#xE9; de la transduction Lentiviral cours de d&#xE9;veloppement chez des souris normales et Rd

Die Transduktion Effizienz des Lentiviralen Vektor in der Netzhaut von normalen M&auml;usen und retinale degenerative (rd) M&auml;usen nach Subretinale Injektion an verschiedenen postnatale Alters zu vergleichen.

Effizienz der Lentivirale Transduktion w&auml;hrend der Entwicklung Normal und rd-M&auml;usen.

Per confrontare l&#39;efficienza di trasduzione di un vettore lentivirale nella retina di topi normali e degenerativa della retina (rd) topi dopo iniezione alle varie et&agrave; postnatale.

Efficienza di trasduzione lentivirale durante lo sviluppo in topi normali e rd.

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To investigate the effects of norrin, a nonconventional ligand for Wingless-Int (Wnt)-beta-catenin signaling pathway, on protease-mediated death of transformed rat retinal ganglion cells (RGC-5).

Norrin attenuates protease-mediated death of transformed retinal ganglion cells.

Intravitreal bevacizumab (BV) (Avastin, Genentech Inc., South San Francisco, CA) is frequently used for the treatment of age-related macular degeneration. Previous studies have demonstrated full-thickness retinal penetration. Intravitreal recombinant microplasmin (MP) has been shown to successfully induce a posterior vitreous detachment (PVD) and vitreous liquefaction in animals. It has been suggested that a PVD may alter the retinal penetration of molecules in the vitreous cavity. The aim of this study was to compare BV retinal penetration in rabbit eyes with and without an MP-induced PVD.

Posterior vitreous detachment with microplasmin alters the retinal penetration of intravitreal bevacizumab (Avastin) in rabbit eyes.

Wnt-signaling has been implicated in retinal development. The aim of this study was to investigate the possibility of improving retinal vasculature in an animal model of retinopathy by activating Wnt-signaling.

Retinal vascular rescue of oxygen-induced retinopathy in mice by norrin.

Inhibition of VEGF is widely used in patients to control neovascularization and decrease vascular permeability. To date, the effect of VEGF inhibition has not been evaluated in the developing retina such as that seen in premature infants. The goal of this study was to address the effect of anti-VEGF treatment on retinal development of a mouse model of retinopathy.

Effects of anti-VEGF treatment on the recovery of the developing retina following oxygen-induced retinopathy.

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and bipolar cells. 

Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons.

Treatment of a mouse model of oxygen-induced retinopathy (OIR) with recombinant human Norrin (Norrie Disease Protein, gene: NDP) accelerates regrowth of the microvasculature into central ischemic regions of the neural retina, which are generated after treatment with 75% oxygen. While this reduces the average duration and severity of ischemia overall, we do not know if this accelerated recovery of the microvasculature results in any significant survival of retinal ganglion cells (RGCs). The purpose of this study was to investigate ganglion cell survival with and without the intravitreal injection of Norrin in the murine model of oxygen induced retinopathy (OIR), using two strains of mice: C57BL/6J and Thy1-YFP mice. Intravitreal injections of Norrin or vehicle were done after five days of exposure to 75% oxygen from ages P7 to P12. The C57BL/J mice were followed by Spectral-Domain Optical Coherence Tomography (SD-OCT), and the average nerve fiber layer (NFL) and inner-plexiform layer (IPL) thicknesses were measured at twenty-four locations per retina at P42. Additionally, some C57BL/J retinas were flat mounted and immunostained for the RGC marker, Brn3a, to compare the population density of surviving retinal ganglion cells. Using homozygous Thy1-YFP mice, single intrinsically fluorescent RGCs were imaged in live animals with a Micron-III imaging system at ages P21, 28 and P42. The relative percentage of YFP-fluorescent RGCs with dendritic arbors were compared. At age P42, the NFL was thicker in Norrin-injected OIR eyes, 14.4 μm, compared to Vehicle-injected OIR eyes, 13.3 μm (p = 0.01). In the superior retina, the average thickness of the IPL was greater in Norrin-injected OIR eyes, 37.7 μm, compared to Vehicle-injected OIR eyes, 34.6 μm (p = 0.04). Retinas from Norrin injected OIR mice had significantly more surviving RGCs (p = 0.03) than vehicle-injected mice. Based upon NFL thickness and counts of RGCs, we conclude that Norrin treatment, early in the ischemic phase, increased the relative population density of surviving RGCs in the central retinas of OIR mice.

Norrin treatment improves ganglion cell survival in an oxygen-induced retinopathy model of retinal ischemia.

The data presented in this article are related to the research paper entitled "Norrin treatment improves ganglion cell survival in an oxygen-induced model of retinal ischemia" (Dailey et al., 2017) [1] This article describes treatment with the human Norrin protein, an atypical Wnt-protein, to improve the survival of retinal ganglion cells in a murine model of Oxygen-Induced Retinopathy (OIR). That study utilized Optical coherence tomography (OCT) to visualize retinal layers at high resolution , and to quantify changes to nerve fiber layer thickness. Organization of the laminar structure of other retinal layers in this model , were not known because of uncertainties regarding potential artifacts during the processing of tissue for traditional histology. The OCT image data provided here shows researchers the retinal laminar structural features that exist  in this popular mouse OIR model. Traditional H&E stained retinal tissue sections are also provided here for comparison.

Mei Cheng

Eye Research Institute

Oakland University

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Mei Cheng

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Eye Research Institute

Oakland University

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Eye Research Institute