bromochloroacetic-acid and Retinal-Diseases

This study investigates the similarities and differences between epiretinal membranes in four clinically distinct types of vitreomaculopathy. We propose a hypothesis on the origin of the predominant cell type and its potential role in causing these conditions.. Epiretinal membranes (ERMs) surgically removed from a prospective, consecutive series of vitrectomies for macular pucker associated with an untreated peripheral horseshoe tear (MP), cellophane maculopathy (CM), stage 4 macular hole (MH) and vitreomacular traction syndrome (VMT) were examined by light microscopy and by immunocytochemistry (ICC) using antibodies marking type IV collagen, type II collagen, glial fibrillary acidic protein (GFAP), and low- and high-molecular-weight cytokeratin (MNF116). These specimens were compared with post-mortem control eyes with and without physiological posterior vitreous detachment (PVD). Light microscopy was carried out on 5-microm-thick sections cut from formalin-fixed, paraffin-embedded tissue blocks. Appropriate autoclave or enzyme pre-digestion steps were deployed to retrieve antigens for ICC. No patient had undergone previous vitreoretinal surgery or peripheral retinopexy.. From a series of 38 patients, (13 CM, 8 MP, 16 MH and 1 VMT) a total of 20 specimens contained sufficient tissue for histology and immunocytochemistry. All specimens contained portions of inner limiting membrane (ILM) coated by GFAP-positive cells. Specimens from patients with MP and CM exhibited hyperconvolution of the ILM, which was not found in the specimens from patients with MH or VMT or in the control eyes. Hyperconvolution was associated with increased glial cell density, GFAP staining intensity and duplication of ILM basement membrane. Three cases of ERMs from the MP group contained, in addition, cytokeratin-positive cells. In the control group; post-mortem eyes with PVDs showed patchy staining of the posterior hyaloid membrane for GFAP and type 4 collagen. Post-mortem eyes with attached gel showed weak positivity of the ILM for type 4 collagen, and a monolayer of GFAP-positive cells lined the vitreous aspect of the ILM.. These results indicate that glial cells are fundamentally important in the formation of ERMs found in this group of vitreomaculopathies. The hyperconvolution and duplication of the ILM in CM and MP were striking and distinctive features and suggest a mechanism by which these membranes exert tractional forces on the retina. Post-mortem control eyes contained a similar (but more dispersed) population of GFAP-positive cells in the region of the ILM, suggesting the primary aetiology for CM and MP may originate within the ILM. ERMs from MP cases may, in addition, contain cytokeratin-positive cells, of probable RPE origin.

Topics: Basement Membrane; Biomarkers; Collagen Type II; Collagen Type IV; Epiretinal Membrane; Eye Diseases; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Keratins; Male; Prospective Studies; Retinal Diseases; Vitrectomy; Vitreous Body

Topics: Adult; Amyloidosis; Female; Humans; Immunohistochemistry; Keratins; Lichen Planus; Macula Lutea; Male; Middle Aged; Retinal Diseases; Skin

Visual impairment resulting from retinal, subhyaloid, or vitreous hemorrhages in association with Tersons syndrome is often significant. The most common long-term sequelae that may result in permanent visual deficit is the formation of an epimacular membrane.. This report provides clinicopathologic documentation of epiretinal membrane proliferation secondary to Tersons syndrome. Pars plana vitrectomy was performed in 16 eyes of 11 patients with Tersons syndrome. After removal of vitreous hemorrhage, epimacular membranes were found in 10 eyes (62.5%). The posterior cortical vitreous and the epiretinal tissue were examined histologically.. Immunostainings with glial and retinal pigment epithelial cell markers showed that the majority of cells derived form the glia. Perls staining, specific for iron, showed that the high melanic-like component contained in the histopathologic samples corresponded to degradation of blood products secondary to chronic hemorrhage.. The high risk of epiretinal membrane formation and the toxicity of blood breakdown products over the inner retina worsen the long-term visual prognosis in Tersons syndrome. Early surgery is advocated in such cases.

Topics: Adolescent; Adult; Cell Membrane; Child, Preschool; Female; Fundus Oculi; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Neuroglia; Retina; Retinal Diseases; S100 Proteins; Subarachnoid Hemorrhage; Syndrome; Vitrectomy; Vitreous Hemorrhage

Proliferative vitreoretinopathy may be associated with taut subretinal membranes (SRMs) that prevent retinal reattachment. Light microscopic and immunohistochemical procedures were used to identify the cell composition of 15 surgically excised SRMs. Anti glial fibrillary acidic protein (anti-GFAP) and anti S-100 protein stainings were used to examine the distribution of glial cells, and antikeratin staining was used to investigate the distribution of epithelial cells. Our findings confirm the presence of retinal pigment epithelium (RPE) and glial cells in human SRMs. A semiquantitative evaluation of the immunohistochemical studies demonstrated the prevailing role of cells derived from the RPE (48.4%). The glial component of the SRMs appeared to be smaller than in epiretinal membranes, but was not negligible (30.8%). A high incidence (26.6%) of damaged external retina adhered to the SRMs was also observed.

Topics: Adolescent; Adult; Child; Eye Diseases; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Pigment Epithelium of Eye; Retinal Diseases; S100 Proteins; Vitreous Body

Immunotoxins directed against a membrane marker of cell proliferation, transferrin receptor, were investigated to inhibit the growth of retinal pigment epithelial (RPE) cells in proliferative vitreoretinopathy (PVR). We undertook an immunocytological study in specimens of vitreous, subretinal fluid, and epiretinal membranes from patients with PVR to address the expression of transferrin receptor by proliferating pigment epithelial cells during the course of PVR and in normal human ocular structures. Thirty four specimens of vitreous and subretinal fluid, as well as seven epiretinal membranes, were immunocytologically examined using monoclonal antibodies to transferrin receptor. They showed a strong expression of this marker by a large majority of the cells in these two periretinal fluids (mean percentages 80 and 91% in vitreous and subretinal fluid, respectively). In contrast, only a few cells within epiretinal membranes were found to express transferrin receptor. In normal human eye sections conjunctival and corneal epithelial cells, subcapsular epithelium of the lens strongly expressed transferrin receptor, whereas RPE cells remained negative to antitransferrin receptor antibodies. A few iris or ciliary pigment epithelial cells reacted weakly. Thus, this study shows that most intravitreal and subretinal fluid proliferating cells strongly express transferrin receptor on their surface. Also confirmed is that immunotoxins to this membrane antigen could constitute potentially useful therapeutic agents in PVR.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Cell Membrane; Conjunctiva; Epithelium; Exudates and Transudates; Eye Diseases; Fluorescent Antibody Technique; Humans; Keratins; Lens, Crystalline; Middle Aged; Pigment Epithelium of Eye; Receptors, Transferrin; Retinal Detachment; Retinal Diseases; Vitreous Body

The identification of cells comprising epiretinal membranes is difficult because of the phenotypic changes that occur. Examination of intermediate filament protein content by immunocytochemical analysis can help to identify some cells with altered ultrastructure but is not always definitive because altered expression of intermediate filament proteins can also occur. To examine this issue further, the authors utilized a postembedding immunocytochemical technique with epiretinal membranes in which they were able to double label for keratin, a useful marker for identifying retinal pigment epithelial cells, and glial fibrillary acidic protein (GFAP), a useful marker for identifying glial cells. Nine of ten idiopathic epiretinal membranes contained cells that labeled for GFAP and not keratin. Two of these membranes also contained cells that labeled only for keratin and one membrane contained cells that simultaneously labeled for both GFAP and keratin. Other types of epiretinal membranes had an equal participation by cells that expressed only GFAP or keratin (12 of 17 membranes contained cells positive for keratin; 13 of 17 contained cells positive for GFAP). Ten of 17 nonidiopathic membranes contained cells simultaneously expressing GFAP and keratin, although they comprised only a minor subpopulation of the total number of cells present. These findings demonstrate that keratin and GFAP are not mutually exclusive intermediate filament proteins in cells of epiretinal membranes and that, although each may provide a helpful adjunct for cell type identification, neither is an absolutely specific marker.

Topics: Eye Diseases; Glial Fibrillary Acidic Protein; Humans; Keratins; Membranes; Retina; Retinal Detachment; Retinal Diseases; Staining and Labeling; Vitreous Body

The pathogenesis of proliferative vitreoretinopathy (PVR) membranes remains poorly understood. We have studied the presence of acidic fibroblast growth factor (aFGF), a potent mitogen for many cells, within these membranes. We have used affinity purified monospecific anti-aFGF polyclonal antibodies, in conjunction with highly sensitive immunofluorescence techniques. The labelling was exclusively localized to cell bodies and was absent from the extracellular matrix. Double labelling techniques revealed that all cytokeratin positive cells (probably pigmented epithelial cells) and macrophages contained aFGF-like immunoreactivity, whilst glial cells were unlabelled. Appropriate controls indicated the specificity of the antibodies. Hence, the presence of this mitogenic molecule within certain cell types constituting PVR membranes may contribute to the pathogenesis.

Topics: Animals; Cell Membrane; Extracellular Matrix; Eye Diseases; Fibroblast Growth Factor 1; Fluorescent Antibody Technique; Humans; Keratins; Pigment Epithelium of Eye; Rabbits; Retinal Diseases; Vitreous Body

Cell growth in proliferative vitreoretinopathy (PVR) is activated by naturally occurring mitogenic substances; thus, growth factors seem to play an important role in PVR. Cell activation by growth factors requires the presence of specific cell-bound receptors by which the mitogenic signal is transmitted. Using the indirect immunoperoxidase method, we investigated frozen sections of 15 epiretinal membranes in PVR for the presence of epidermal growth factor receptor (EGFR), which is commonly utilized by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This receptor could be localized only in one epiretinal membrane. The receptor-bearing cells stained positively for vimentin, cytokeratin, and macrophage marker Ki-M7, indicating that they most probably correspond to transformed macrophages. They stained negatively for desmin and glial fibrillary acidic protein. The cells were embedded in a fibronectin-positive intercellular matrix and were in an actively proliferating state as demonstrated by positive staining for the nuclear proliferation marker Ki-67. We propose that EGFR might be expressed only in certain as yet undefined stages of PVR.

Topics: Autoantigens; Cell Cycle; Cell Membrane; ErbB Receptors; Eye Diseases; Humans; Immunoenzyme Techniques; Keratins; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Retinal Diseases; Vimentin; Vitreous Body

Electron-immunocytochemical staining for three intermediate filament (IF) proteins, keratin (K), glial fibrillary acidic protein (GFAP), and vimentin (V), and for the macrophage marker, EBM/11 (E), was performed on epiretinal membranes obtained during vitrectomies performed for proliferative vitreoretinopathy (PVR), postdetachment macular puckers (PDMPs), idiopathic macular puckers (IMPs), or macular puckers associated with other disease processes. The ultrastructural and immunocytochemical characteristics of the cells were compared. Unstained cells outnumbered stained cells for each of the markers in almost all membranes. Six cell types, based on ultrastructure, were found in the majority of epiretinal membranes: 1) polarized cells with microvilli on the free border and foot processes anchoring them to extracellular matrix that consistently stained negative for all of the immunocytochemical markers; 2) spindle-shaped fibroblastlike cells that were generally negative for all markers, but rarely positive for V; 3) large undifferentiated cells with large, lightly stained nuclei and little cytoplasm that frequently expressed one of the intermediate filament (IF) proteins; 4) poorly differentiated cells that contained numerous mitochondria and frequently expressed one of the IF proteins; 5) undifferentiated, pigment-laden cells that rarely stained for any of the above IF proteins, but occasionally showed K or V positivity in a portion of the cell, suggesting that they may be losing or acquiring these proteins, and that rarely expressed GFAP; and 6) small, round, mononuclear cells with short processes that were sometimes, but not always, positive for E and that were consistently negative for K, V, and GFAP. In addition to these morphologic types, transitional cells demonstrating features of two or more of the above cell types were seen, suggesting that phenotypic changes between the various cell types can occur. The amount of extracellular matrix in epiretinal membranes showed a correlation with disease process (PVR greater than PDMP greater than IMP), and a negative correlation with the percentage of cells expressing a highly differentiated polarized morphology and with the percentage of cells staining for IF proteins. These data suggest that both cell morphology and IF protein expression may be dependent in part on microenvironment and that neither alone can be used to identify unequivocally the derivation of particular cells found in epiretinal membranes. T

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Child; Extracellular Matrix; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Male; Membranes; Middle Aged; Pigment Epithelium of Eye; Retina; Retinal Diseases; Vimentin; Vitrectomy

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.

Topics: Antibodies, Monoclonal; Colchicine; Cytoskeleton; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Intermediate Filaments; Keratins; Pigment Epithelium of Eye; Retinal Diseases; Vimentin

Culture of cells from subretinal fluid (SRF) was performed using 29 SRF samples obtained at retinal reattachment surgery. Proliferating cells were found in 58.6% of the samples studied. The cells were of retinal pigment epithelial (RPE) origin, as evidenced by their brown pigmentation in primary culture and their positive immunostaining for cytokeratins 8/18. The age of the patients did not affect the proliferative capacity of the cells. Proliferating cells were present in all samples from eyes with proliferative vitreoretinopathy (PVR) of grade C1 or more. In primary culture the cells had a fibroblast-like morphology, resembling that of ordinary RPE cells exposed to the vitreous. We conclude that the SRF of many patients with PVR contains viable proliferating RPE cells and that SRF offers a new source of RPE cells for studies on the pathogenesis of PVR.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cell Division; Cell Separation; Cells, Cultured; Extracellular Space; Female; Fibronectins; Humans; Keratins; Male; Microscopy, Fluorescence; Middle Aged; Pigment Epithelium of Eye; Retinal Detachment; Retinal Diseases

To determine the cellular composition of subretinal membranes in proliferative vitreoretinopathy, surgically excised membranes were studied using electron microscopic and immunohistochemical procedures. Light microscopic immunohistochemistry was found to be superior to conventional electron microscopy as a means of evaluating the relative contributions of retinal pigment epithelium and glia to subretinal membranes. The cells of subretinal membranes chiefly comprised retinal pigment epithelium while glial cells were either absent or contributed only a minor component. It is proposed that the clinical properties of subretinal membranes in proliferative vitreoretinopathy derive from their retinal pigment epithelial cell element.

Topics: Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Membranes; Retinal Diseases

Intermediate filament proteins in cultured cells from epiretinal membranes of 5 cases of proliferative vitreoretinopathy and subretinal strands of 2 cases were immunohistologically characterized. Double staining technique after colcemid treatment revealed that either keratin-positive or vimentin-positive cells were found with different ratios depending on the cases. Both positive and both negative cells were also observed. Desmin and glial fibrillary acidic protein were totally negative.

Topics: Adult; Aged; Female; Fluorescent Antibody Technique; Humans; Keratins; Male; Middle Aged; Pigment Epithelium of Eye; Protein Precursors; Retina; Retinal Diseases; Vimentin; Vitreous Body

Topics: Adult; Cells, Cultured; Eye Diseases; Female; Fluorescent Antibody Technique; Humans; Keratins; Retina; Retinal Diseases; Vimentin; Vitreous Body