Distribution of ascorbate in the anterior bovine eye.
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PURPOSE: To analyze the ascorbate distribution in the anterior eye wall to better understand the functional significance of this compound in the eye. METHOD: Ascorbic acid was determined by high-performance liquid chromatography using an LC-10 system (Shimadzu, Kyoto, Japan). Bovine eye samples were used. RESULTS: The highest ascorbate concentration was observed in the corneal epithelium, with significantly higher values in the central (1.56 mg/g) than in the peripheral (1.39 mg/g) area. The ascorbate content was similar in the corneal stroma (0.22 mg/g), the Descemet's membrane (DM)/endothelium (0.22 mg/g), and the aqueous humor (0.21 mg/ml). By comparison, the sclera (0.15 mg/g) and the conjunctiva (0.11 mg/g) showed lower values, as did the lacrimal gland (0.09 mg/g) and the serum (0.0008 mg/ml). CONCLUSIONS: (1) Peak ascorbate concentration was observed in the central corneal epithelium covering the pupillary area. This is compatible with the idea that the ascorbate may act as an UV filter shielding internal eye structures from radiation damage. (2) The ascorbate concentration in the corneal stroma and DM/endothelium was as high as in the aqueous humor, and it is suggested that the aqueous humor plays a key role in the distribution of ascorbate to the anterior eye wall. (+info)
Extracellular matrix components in retrocorneal fibrous membrane in comparison to corneal endothelium and Descemet's membrane.
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PURPOSE: To investigate the extracellular matrix macromolecules found in Descemet's membrane and in retrocorneal fibrous membrane (RCFM), and to examine whether the corneal endothelium has the capacity to produce both basement and non-basement membrane phenotypes. METHODS: Rabbit corneas with and without RCFM were analyzed by immunofluorescence using antibodies to 8 different collagens (basement membrane collagens: types IV and VIII; fibrillar collagens: types I and III; interfibrillar collagens: type VI and two spliced variant forms of type XII and one anchoring fiber: type VII), proteoglycans (perlecan and decorin), (beta)ig-h3 and laminin-1. RESULTS: Normal corneal endothelium stains positively for all of the tested collagen types except type VII collagen. On the other hand, Descemet's membrane reacts positively only to the type IV collagen antibody. When non-collagenous components in normal cornea were examined, corneal endothelium stained positively for perlecan, decorin, (beta)ig-h3 and laminin, whereas Descemet's membrane staining for these proteins was negative. When collagenous components of RCFM were examined, RCFM stained positively for all of the tested collagen types except type IV collagen. When non-collagenous components of RCFM were examined, RCFM demonstrated a strong positive staining with decorin, (beta)ig-h3 and laminin, while perlecan staining was weak. CONCLUSIONS: These observations suggest that corneal endothelium is able to produce both basement membrane phenotypes and non-basement membrane, fibrillar phenotypes. This in vivo study confirms our in vitro model of endothelial mesenchymal transformation, in which corneal endothelial cells are transformed to fibroblasts that are responsible for fibrosis. (+info)
Hereditary corneal dystrophy in the Manx cat: a preliminary report.
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A progressive, apparently inherited corneal dystrophy is described in an inbred line of Manx cats. Initial changes in the cornea are seen at four months of age and characterized by anterior stromal edema. Progressive worsening of the condition produces severe bullous keratopathy with eventual breakdown of both epithelium and stroma. Light microscopic and ultrastructural studies in the advanced disease state revealed marked edema of the corneal stroma, disintegration of collagen material, and the formation of epithelial bullae. Ultrastructural evidence shows a normal endothelium to be present. The pathogenesis of this corneal dystrophy is not clear and further studies are underway. (+info)
Screening human donor corneas during organ culture for the presence of guttae.
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AIMS: To detect the presence of guttae by means of light microscopy during organ culture and to evaluate the influence of the presence of guttae in the donor tissue on transplantation outcome. METHODS: Donor corneas were investigated for the presence of guttae by means of light microscopy at the end of organ culture. Recipient corneal buttons from patients with severe Fuchs' dystrophy and donor corneas with advanced guttae were first studied by light microscopy and subsequently by transmission electron microscopy. Lastly, 168 consecutive donor corneas were evaluated for the presence of guttae and issued for transplantation. RESULTS: Corneal specimens with Fuchs' dystrophy displayed numerous round highly reflecting guttae at the level of the corneal endothelium. Donor corneas with advanced guttae showed less numerous guttae. Among 168 organ cultured donor corneas issued for transplantation, low density guttae were found in 43 (25.6%) corneas. The endothelial cell density and figure coefficient were significantly lower and organ culture time was significantly higher in the cornea guttata group than in the control group. The presence of grouped guttae significantly decreased the adjusted graft survival. The incidence of postoperative stage 3 cornea guttata was significantly higher when grouped guttae were found (5/6) than when no guttae or scattered guttae were found (8/101). CONCLUSION: Cornea guttata can be detected during organ culture by means of light microscopy. It is associated with a decrease in endothelial cell figure coefficient and cell density. The presence of grouped guttae is associated with poorer graft survival and more frequent stage 3 cornea guttata in the graft after transplantation. (+info)
Ultrastructural morphology and expression of proteoglycans, betaig-h3, tenascin-C, fibrillin-1, and fibronectin in bullous keratopathy.
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AIMS: To investigate the ultrastructural localisation of proteoglycans (PG), betaig-h3 (keratoepithelin), tenascin-C (TN-C)), fibrillin, and fibronectin in bullous keratopathy (BK) corneas. METHODS: Five corneas from cases of pseudophakic bullous keratopathy (BK) were examined by electron microscopy. PG were demonstrated using cuprolinic blue, and the proteins betaig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-betaig-h3, mouse anti-TN-C (BC10 and TN2), mouse anti-fibrillin-1 (MAB2502), mouse anti-fibrillin (MAB1919), and rabbit anti-fibronectin by using a standard immunogold technique. RESULTS: Epithelial cells contained numerous vacuoles. Epithelial folds and large, electron lucent subepithelial bullae were present. Basal lamina was thickened and traversed by disrupted anchoring filaments. In the stroma, interfibrillar collagen spacing was increased and abnormally large PG were present. Descemet's membrane (DM) contained lucent spaces in which there were small filaments. Keratocyte and endothelial cells contained melanin granules. A posterior collagenous layer (PCL) contained numerous microfilaments and wide spacing collagen fibres with a periodicity of 100 nm. Large quantities of abnormal PG were observed at the endothelial face of the PCL. Very strong labelling with betaig-h3 antibody was observed in the basement membrane, Bowman's layer, stroma, DM, and PCL, but not in keratocytes and endothelial cells. Strong labelling with BC10 and TN2 was seen below the epithelium, in electron lucent spaces where the hemidesmosomes were absent, in the fibrotic pannus, in parts of Bowman's layer, the stroma, and Descemet's membrane. Labelling with BC10 was stronger and more evenly distributed than with TN2. Fibrillin-1 (MAB2502) and fibrillin (MAB1919) labelling was similar to TN-C labelling. Fibrillin (MAB1919) labelling was stronger than fibrillin-1 (MAB2502) labelling. CONCLUSIONS: Immunoelectron microscopy showed precise labelling of proteins at both the cellular and the subcellular level. Expression of proteins betaig-h3, TN-C, fibrillin, and fibronectin was highly increased compared with normal cornea. In the oedematous stroma, increased collagen fibril separation may facilitate a wider distribution of some soluble proteins, such as betaig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response. (+info)
Deep lamellar keratoplasty with lyophilised tissue in the management of keratoconus.
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AIMS: Data are presented on the use of deep lamellar keratoplasty (DLK) using lyophilised donor corneal tissue, in the management of patients with keratoconus (KC). METHOD: The results of DLK on 44 eyes (42 patients) are reported. The mean patient age was 29.8 years (range 10-56). Mean follow up was 25 months (range 6-100). In seven patients with mental handicap or severe mental illness, the collection of acuity and refractive data was limited. RESULTS: Perforation of Descemet's membrane (DM) occurred in nine cases (20%). A double anterior chamber formed in five cases, which resolved spontaneously in three patients. Persistent epithelial defects occurred in two cases, one of which necessitated replacement of the graft. The median postoperative uncorrected visual acuity was 6/36. The median corrected postoperative acuity was 6/9. Those with more than 1 year of follow up (n=25) had a significantly better acuity (p=0.015). This group achieved 6/12 or better in 80% (n=20) and 6/6 or better in 40% (n=10). The mean postoperative spherical error was +0.28 (SD 3.49) dioptres (D). The mean refractive cylinder was 3.85 (1.87) D. CONCLUSION: This detailed retrospective study of DLK for the treatment of patients with KC, with an average follow up of 2 years, highlights the advantages and disadvantages of this technique. (+info)
In situ immunohistochemical study of Bcl-2 and heat shock proteins in human corneal endothelial cells during corneal storage.
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AIM: To investigate the expression of Bcl-2 and heat shock proteins (HSPs), which are known to increase cell survival, in human corneal endothelial cells (HCECs) of corneas stored in organ culture. METHODS: 32 paired corneas were randomly assigned to either a short or a long storage time. The flat mounts of endothelium were examined after immunostaining with monoclonal antibodies to Bcl-2 and HSP 27, 60, 70, and 90. RESULTS: HCECs expressed generally all the proteins studied. Bcl-2 expression was weaker in the long stored corneas (p=0.035). There was no relation between immunostaining, age, sex, or death to culture time. Frequently some Descemet membranes carried negative cells preferentially located in folds and exhibiting morphological changes consistent with swelling cells corresponding to early stages of apoptosis. CONCLUSION: Expression of these cytoprotective proteins reflects the high level of HCEC resistance to stresses induced by organ culture. The decreased immunostaining of Bcl-2 in the long storage group could act in cellular loss currently observed with storage time. The negativity of Bcl-2 and HSP labelling in corneal folding may be related to apoptosis. (+info)
Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment.
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PURPOSE: To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea. METHODS: Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced. RESULTS: Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment. CONCLUSIONS: Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea. (+info)