An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats.

Purpose: Since adult rats are used in pre-clinical studies, and due to the necessity of investigating the side-effects of drugs on RPE cells in vitro, there is a great need for primary RPE cells from these animals. The aim of this study was to develop a reproducible and quantifiable method of isolation, culture, and maintenance of adult rat RPE cells. Moreover, potential differences between RPE cells from albino versus pigmented rats were also investigated.

Effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.

Purpose: It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.

A new kind of labyrinth-like capillary is responsible for leakage from human choroidal neovascular endothelium, as investigated by high-resolution electron microscopy.

Purpose: This study reports the clinicopathologic findings of leaky sites in pathological vessels after submacular removal of choroidal neovascular membranes (CNV). As the site that causes fluid exudation from neovascular vessels is unknown, specific attention was focused on the formation of fenestrations, cellular junctions, and morphologic alteration which can cause endothelial leakage.

Methods: Choroidal neovascular membranes of 15 patients who underwent submacular surgery for CNV were investigated.

Towards a quantitative OCT image analysis.

Background: Optical coherence tomography (OCT) is an invaluable diagnostic tool for the detection and follow-up of retinal pathology in patients and experimental disease models. However, as morphological structures and layering in health as well as their alterations in disease are complex, segmentation procedures have not yet reached a satisfactory level of performance. Therefore, raw images and qualitative data are commonly used in clinical and scientific reports.

Choriocapillaris breakdown precedes retinal degeneration in age-related macular degeneration.

This work presents a combined light and electron microscopical approach to investigate the initial breakdown of the retinal pigment epithelium (RPE) and choriocapillaris (CC) in age-related macular degeneration (AMD). Perimacular sections of 12 dry and wet AMD eyes (82 ± 15 years) and 7 age-matched controls (75 ± 10 years) without retinal pathology were investigated. Disease progression was classified into 5 stages of retinal degeneration to investigate the concurrent CC breakdown.

Different effects of intravitreally injected ranibizumab and aflibercept on retinal and choroidal tissues of monkey eyes.

Background: Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may cause unexpected consequences in retinal and choroidal vessels, the effects of intravitreal ranibizumab and aflibercept on monkey eyes were investigated.

Methods: Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of aflibercept.

In vitro induction of protein complexes between bevacizumab, VEGF-A¹⁶⁵ and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

Purpose: By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin.

Effects of bevacizumab in retina and choroid after intravitreal injection into monkey eyes.

Objective: Due to its low price, bevacizumab, which binds vascular endothelial growth factor, is currently used off-label for the treatment of over 50 different eye diseases and has been adopted worldwide despite the absence of serious preclinical data. This study examines the effects of intravitreal bevacizumab on monkey eyes with particular focus on choroidal and retinal vessels.

Methods: Cynomolgus monkeys received an intravitreal injection of 1.

[Compensable occupational disease and cancer: how to improve the recognition rate? "Cursus Laboris" Project in Midi-Pyrenees: feasibility study].

Five to ten percent of cancers are of occupational origin but only 0.5% of cancers are compensable occupational diseases recognized in 2001. The project "Curriculum Laboris" was launched in Midi-Pyrenees with the objective to establish an organization to improve the identification, reporting and recognition of occupational cancers.

Formation of immune complexes and thrombotic microangiopathy after intravitreal injection of bevacizumab in the primate eye.

Background: In this study, the effect of intravitreal injection of bevacizumab on choroidal blood vessels was examined in primate eyes.

Methods: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab.

A low zinc diet leads to loss of Zn in melanosomes of the RPE but not in melanosomes of the choroidal melanocytes.

Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composition of melanosomes of the RPE and choroidal melanocytes and RPE lipofuscin granules were investigated using Analytical Electron Microscopy (AEM). In controls the Zn mole fraction of melanosomes was 0.05 at% (RPE) and 0.

Lipofuscin can be eliminated from the retinal pigment epithelium of monkeys.

Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin.

Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats.

Background: Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet.

RGMA and neogenin protein expression are influenced by lens injury following optic nerve crush in the rat retina.

Background: The death and the failure of neurons to regenerate their axons after lesion of the central nervous system in mammals, as in the case of spinal cord injury and optic nerve trauma, remain a challenge. In this study, we focused on the repulsive guidance molecule A (RGMA) and its receptor neogenin. Since it was reported that RGMA+ cells accumulate in lesioned areas after spinal cord injury, brain trauma, and optic nerve crush, and curiously, anti-apoptotic effects of RGMA were also described, we investigated the role of RGMA and neogenin in the retina after optic nerve crush (ONC).

Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 μm), highly porous (Ø 1 μm spaced 3 μm), gelatin-coated polyimide (PI) membrane.

Different spatial and temporal protein expressions of repulsive guidance molecule a and neogenin in the rat optic nerve after optic nerve crush with and without lens injury.

The failure of lesioned mammalian CNS neurons to regenerate their axons remains a challenge. Evidence is emerging that repulsive proteins contribute to this failure. The repulsive guidance molecule A (RGMA) induces growth cone collapse in vitro, accumulates in the scar after spinal cord injury, and is up-regulated in glaucoma.

Loss of retinal function in aged DBA/2J mice - New insights into retinal neurodegeneration.

The DBA/2J mouse is a common animal model of glaucoma. The intraocular pressure increases with age, and retinal ganglion cells (RGC) degenerate, usually starting at an age of approximately six months. In this study, we used two-year-old DBA/2J mice presuming an end-point of RGC degeneration.

Ultrastructural analysis of the pigment dispersion syndrome in DBA/2J mice.

Purpose: To characterise ocular pigment abnormalities associated with iris atrophy in DBA/2J mice as a model for human pigment dispersion syndrome.

Methods: Immunohistochemistry, electron and light microscopy were performed to examine the eyes of DBA/2J mice ranging in age from 2.5 to 18 months old.

A reproducible and quantifiable model of choroidal neovascularization induced by VEGF A165 after subretinal adenoviral gene transfer in the rabbit.

Purpose: To determine the effects of the vascular endothelial growth factor (VEGF)-A(165) delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye. To combine different detection methods of VEGF-A(165) overexpression-induced neovascularization in the rabbit.

UV-A induced oxidative stress is more prominent in naturally pigmented aged human RPE cells compared to non-pigmented human RPE cells independent of zinc treatment.

To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation.

Bevacizumab (avastin) does not harm retinal function after intravitreal injection as shown by electroretinography in adult mice.

Purpose: Scavenging of VEGF by specific antibodies is a promising way to treat ocular conditions connected with neovascularization. Intravitreal injections of Avastin (bevacizumab) are performed frequently as a treatment of such conditions. In this study, the authors examine whether the retinal function in wild-type mice is affected by an intravitreal injection of Avastin.

Melanin precursor 5,6-dihydroxyindol: protective effects and cytotoxicity on retinal cells in vitro and in vivo.

5,6-dihydroxyindole (DHI) is a melanin pigment precursor with antioxidant properties. In the light of a report about cytotoxicity of DHI, the aim of this study was to assess possible toxic effects of DHI on cells related to the eye, such as human ARPE-19 cells and mouse retinal explants. Moreover, DHI was tested on its effects on retinal function in vivo using electroretinography.

Gene expression of the repulsive guidance molecules/neogenin in the developing and mature mouse visual system: C57BL/6J vs. the glaucoma model DBA/2J.

We used in-situ hybridization to analyze the expression patterns of three known members (a, b and c) of the RGM ("repulsive guidance molecule") gene family and of the RGMa receptor neogenin in a glaucoma mouse model (DBA/2J strain) and the C57BL/6J strain, which served as a control. In order to understand the role of the RGMs and neogenin in glaucoma, we characterized their expression patterns in the developing and mature mouse retina and in the optic nerve. In all investigated stages from post-natal day (P) 0 to 15 months (M) RGMa, RGMb and neogenin expression was detected in the ganglion cell layer (GCL).

Purkinje cell survival in organotypic cultures: implication of Rho and its downstream effector ROCK.

Organotypic cultures of postnatal day 1 (P1) to P7 mouse cerebella are well-established models for studying cell survival. In the present work, we investigate the involvement of the Rho/ROCK intracellular pathway in Purkinje cell survival by using organotypic cultures of P3 Swiss mice. Specific inhibitors of Rho or ROCK were applied at different concentrations to the slice cultures, which were maintained for 5 days in vitro.

Penetration of bevacizumab through the retina after intravitreal injection in the monkey.

Purpose: The penetration of intravitreally injected bevacizumab in its commercial formulation (Avastin; Roche, Grenzach, Germany) through the retina was studied, to determine whether a full-length antibody would be able to penetrate the retina as easily as an antibody fragment.

Methods: Six cynomolgus monkeys (Macaca fascicularis) were used in this study. Two compositions of intravitreal injection into the right eyes were performed: one with commercial Avastin (group 1, four animals) and the other one with commercial Avastin labeled with 125I (group 2, one animal).