bromochloroacetic-acid and Retinal-Degeneration

To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats.. Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected.. In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments.. Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.

Topics: Adenoviridae; Animals; Bone Marrow Cells; Cell Differentiation; Cell Survival; Coculture Techniques; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Genetic Vectors; Green Fluorescent Proteins; Humans; Keratins; Male; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Middle Aged; Nerve Growth Factors; Phosphoproteins; Pigment Epithelium of Eye; Rats; Rats, Mutant Strains; Rats, Wistar; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Serpins; Stromal Cells; Zonula Occludens-1 Protein

To examine whether and how the retinal distribution of the chondroitin sulfate proteoglycan neurocan is affected after photoreceptor cell loss and whether it correlates with the multiple secondary cellular changes that accompany the photoreceptor degeneration.. Retinas from normal rats (Sprague-Dawley; postnatal days [P]0-P70), RCS rats with dystrophic retinas (P0-P300), RCS-rdy(+) congenic rats with nondystrophic retinas (P0-202), and rhodopsin mutant rats, P23H (P0-P257) and S334ter (P0-P220), were processed for immunohistochemistry using a polyclonal antibody to rat neurocan.. The overall distribution of neurocan was similar in all retinas examined. Neurocan immunostaining was detected over the nerve fiber layer, the plexiform layers, the photoreceptor outer segments region, and the ciliary epithelium. With age, labeling throughout the plexiform layers decreased continuously. In RCS rats however, conspicuous labeling was also seen in association with retinal vessels, from P15 onward.. Accumulation of neurocan in association with the retinal vasculature does not correlate with photoreceptor cell loss, because it was not observed in the rhodopsin mutant rats. During the earliest stages of the disease, accumulation of debris in the subretinal space in RCS rats may be sufficient per se to initiate a cascade of metabolic changes that result in accumulation of neurocan. With time, the neurocan accumulated perivascularly may, by interaction with other matrix molecules, modulate at least some of the vascular alterations observed in this animal model.

Topics: Animals; Blotting, Western; Chondroitin Sulfate Proteoglycans; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Keratins; Lectins, C-Type; Male; Nerve Fibers; Nerve Tissue Proteins; Neurocan; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Retinal Vessels

The aim of this study was to investigate the possible rescue effect of subretinal iris pigment epithelial (IPE) cell transplantation in Royal College of Surgeons (RCS) rats by light and electron microscopic histology.. IPE cells were harvested from 20- to 26-day-old Long-Evans rats and were directly trans planted transsclerally into the subretinal space of 32 16- to 20-day-old RCS rats using a 32-gauge Hamilton syringe. Specimens of transplanted eyes were embedded for electron microscopy after 8 weeks. Specimens from the iris and retinal pigment epithelium (RPE) of Long-Evans rats and RPE from RCS rats without surgical treatment were also embedded. Sham surgery was also performed in 8 eyes.. The IPE cells transplanted into the subretinal space were localized between host RPE and retina, had round cell shapes without polar organization, and contained phagosomes resulting from rod outer segment (ROS) uptake. The underlying host RPE cells were heavily pigmented. RPE cells from RCS rats revealed fragmentation of endoplasmic reticulum, which distinguishes them ultrastructurally from pigment epithelial cells of Long-Evans rats. Ultrastructural alterations were observed in the cytoplasm of transplanted cells. Melanin granules in the IPE cells were found in large vacuoles, which also contained phagosomes originating from ROS uptake. In 13 eyes, 1 to 4 rows and 5 to 8 rows of saved photoreceptors were detected facing transplanted IPE cells in 6 (46%) and 4 (31%) eyes, respectively, 2 months after surgery. However, in 10 (53%) and 7 (37%) of 19 eyes, 1 to 4 rows and 5 to 8 rows, respectively, were also found at sites without IPE cells in the plane of section. ROS directed toward transplanted IPE cells were seen in one case, but these rods were shortened and disorganized. At most sites between transplanted cells and inner segments of photoreceptors, outer segments and cellular debris were absent. In eyes without transplanted cells no photoreceptor cells were alive at the age of 2 months. After sham surgery 6 (75%) eyes had 1 to 4 rows and 2 (25%) 5 to 8 rows of photoreceptors.. Transplanted IPE cells can take up and degrade ROS in vivo in RCS rats. Uptake of ROS alters the morphology of pigment granules in transplanted IPE cells. Pigmentation is an uncertain marker for identifying transplanted pigment cells. IPE transplants are not as good as RPE transplants in rescuing photoreceptors. However, there is a significant difference between transplanted eyes and nontreated eyes. The rescue effect of IPE cells was not significantly different from that of sham surgery.

Topics: Animals; Cell Separation; Cell Survival; Cell Transplantation; Cells, Cultured; Fluorescent Antibody Technique, Indirect; Graft Survival; Iris; Keratins; Melanosomes; Phagocytosis; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Rats; Rats, Long-Evans; Rats, Mutant Strains; Retinal Degeneration; Rod Cell Outer Segment

In the Royal College of Surgeons rat with inherited retinal dystrophy, vascularization of the retinal pigment epithelium (RPE) is preceded by migration and proliferation of Müller cell processes in the subretinal space where they contact the RPE. Later, RPE cells envelope subretinal vessels which have lost their perivascular Müller cell sheath. To characterize RPE cell changes and interactions in relation to glial and vascular transformations in retinal dystrophy, we used immunocytochemical techniques and antibodies against cytokeratin (CK) and glial fibrillary acidic protein (GFAP). Prior to the proliferation of Müller cell processes in the dystrophic retina, CK filaments in RPE cells formed a circumferential meshwork with intense cytoplasmic and perinuclear labeling as in control RPE cells. Following entry of Müller cell processes into the membranous debris zone and formation of RPE-Müller cell contact, RPE cells became pleomorphic and extended prominent apical processes in the debris zone. Some CK-reactive RPE cells detached from Bruch's membrane and migrated into the debris zone. Electron microscopic study showed extensive areas of close RPE-Müller cell contact at this time. Obvious junctional specializations of the plasma membranes were not seen but prominent tubulo-vesicular profiles occupied the cytoplasm of altered RPE and Müller cell processes. Following RPE vascularization, hypertrophic CK-positive cells surrounded blood vessels and accompanied them into the inner retina. Electron microscopic analysis showed that RPE-associated vessels were fenestrated and devoid of their perivascular glial sheath. Apparent proliferation of RPE cells and redistribution of CK filaments were observed. Our study shows that RPE cell alterations accompany Müller cell and vascular changes which result in altered RPE-Müller cell and RPE-endothelial cell relationships in the dystrophic rat retina. The altered relationships among RPE, Müller and endothelial cells may result in increased cellular interaction and promote proliferation and transformation of all three cells types in diseased retinas.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Cell Movement; Immunohistochemistry; Keratins; Microscopy, Electron; Neovascularization, Pathologic; Pigment Epithelium of Eye; Rats; Rats, Mutant Strains; Retinal Degeneration

Topics: Chromatography; Chromatography, Gel; Keratins; Rare Diseases; Retinal Degeneration

Topics: Epidermis; Histological Techniques; Humans; Keratins; Retinal Degeneration; Skin