Studies on corneal wound healing. Effect of fucose on iodine vapor-burnt rabbit corneas.

Corneal wound healing often leads to the development of scar tissue with loss of transparency. Reconstitution of transparent corneal stroma depends on the regulation of the biosynthetic activities of postlesional keratocytes and also to a large extent on the limitation of matrix degradation, attributed essentially to the upregulation of matrix metalloproteases and especially MMP-9. Using a standardized method for the production of reproducible corneal lesions by burning with iodine vapors, we could show that the local application of 0.5 mg/ml L-fucose reduced significantly MMP-9 upregulation and accelerated the recovery of the epithelial layer of the cornea. The iodine vapor used in the experiments produces a rapid loss of epithelium with no or slight effect below the basement membrane. A relatively rapid regrowth of epithelium was observed. The speed of this reepithelialization was stimulated by the local application of fucose. At 48 h after burn, there was a difference between fucose-treated and control corneas (epithelial thickness was about 50 mum for fucose-treated corneas and 37 microm for control corneas). Culture media of in vivo fucose-treated corneas showed an important decrease of MMP-9 activity (-51%, n = 6, p < 0.01). It appears that the in vivo fucose treatment reduced the MMP-9 activity released in the media. This effect is significant 24 h after iodine vapor burn. In order to study the effect of fucose on normal corneas, it was added to rabbit as well as human cornea explant cultures, and the production and release of MMP-9 was determined by zymography. Fucose at a concentration of 0.5 mg/ml produced a 70% decrease of MMP-9 activity released in the medium by corneal explant cultures. Other mono- and oligosaccharides were also tested. Besides lactose, fucose-rich oligosaccharides also produced significant inhibition. Galactose, melibiose, mannose and glucose were inactive. These results justify the use of fucose for the local treatment of corneal wounds.

Confocal microscopic examination of trabecular meshwork removed during ab externo trabeculectomy.

AIMS: The aim of the ab externo trabeculectomy (AET) is to remove the external portion of the trabecular meshwork (ETM) responsible for the main aqueous outflow resistance in glaucoma patients, with no opening of the anterior chamber. ETM characteristics were evaluated with a confocal microscope. METHODS: A prospective comparative observational case series was performed in 60 consecutive medically treated patients with primary open angle glaucoma and eight postmortem normal donors' eyes that underwent AET. Once deroofing the Schlemm' s canal (SC), a deeper dissection led to removal of a coherent membrane (ETM) which allowed satisfactory aqueous egress through the remaining intact internal trabecular meshwork (TM) layers. After fixation with acetone and immunostaining with anti-vimentin antibody, ETM were analysed with a confocal microscope. RESULTS: Glaucomatous ETM (mean thickness: 29.5 (7.6) micro m) were characterised by a severe paucicellularity compared with the controls (respectively 37.3 (9.7) cells/area and 167.5 (24.9) cells/area, p<10(-4)). ETM analysis showed involvement of both cribriform and corneoscleral layers. ETM cell density was significantly decreased in case of preoperative fluorometholone instillation. CONCLUSION: Paucicellularity of glaucomatous TM is confirmed by this original technique. Structural characteristics of the ETM, whose removal allows satisfactory aqueous egress, suggest that aqueous outflow resistance not only involves inner wall of SC and juxtacanalicular meshwork but also corneoscleral trabecular layers.

Inhibition of cell proliferation and fibronectin biosynthesis by Na ascorbate.

BACKGROUND: The importance of ascorbate on the production of extracellular matrix proteins (as elastin and collagens) is now well documented, but no studies have been published concerning its effects on fibronectin biosynthesis. Fibronectin is important for cell attachment and for proliferation. MATERIALS AND METHODS: The effects of Na ascorbate were investigated on cell attachment, proliferation, viability and fibronectin biosynthesis by human skin fibroblasts in vitro. Proliferation was followed by the monitoring of [(3)H]-thymidine incorporation; viability by the MTT-test, cell adherence by counting adherent and nonadherent cells and fibronectin biosynthesis by immunoprecipitation of biosynthetically labelled fibronectin. RESULTS: In the presence of ascorbate, the fibroblasts showed a biphasic growth pattern. At 500 microM ascorbate, [(3)H]-thymidine incorporation was stimulated by 15% as compared to the controls. Higher concentrations gradually decreased proliferation up to 36% of the control value at 5 mM. These effects of ascorbate on DNA synthesis were followed to > 1.25 mM by a strong inhibition, cytotoxic effect and cell death. The non-adherent cell count increased to 10% of the total population at 2.5 mM and to 31% at 5.0 mM ascorbate.Increasing concentrations of ascorbate resulted in a dose-dependent decrease of fibronectin biosynthesis, both in the culture supernates and cell extracts. This inhibition mainly concerned cell membrane-associated fibronectin.Superoxide-dismutase or catalase could inhibit Na ascorbate-induced cytotoxicity and partially re-establish fibronectin biosynthesis. Desferrioxamine, ergothionein and vitamin E were inefficient. CONCLUSIONS: Our results indicate that ascorbate decreases fibronectin biosynthesis of cultured human skin fibroblasts, thereby producing cell detachment and decreased proliferation. This effect is mainly mediated by the reactive oxygen species and can be inhibited by superoxide-dismutase and catalase.

Fibronectin in the vitreous body--distribution and possible functional role.

The presence of fibronectin in the bovine vitreous was demonstrated by immunohistochemical procedures which showed a uniform coating of the vitreous collagen network. A fractional extraction of bovine vitreous was carried out in order to determine the distribution of fibronectin and glycosaminoglycans as related to collagen fibers. About half of total fibronectin could be extracted with aqueous buffers with increasing concentrations of KCl, part of fibronectin remained however strongly associated with the insoluble collagen network even after a final extraction with 4 M urea and 0.05 M DTT. Total extractable fibronectin was of the order of 76 micrograms per vitreous, corresponding to approximately 0.17 nM fibronectin. Total quantity of GAG-s determined as uronic acid were of the order of 2200 micrograms/vitreous corresponding approximately to 4400 micrograms disaccharide units that is to about 11 nM disaccharide units of GAG per vitreous. The persistence of fibronectin, strongly associated with the collagen fibers even after repeated KCl and urea-DTT extractions was confirmed using immuno-gold labelling of vitreous collagen fibers. Gold particle density on the collagen fibers increased with the molarity of KCl used for the extractions. These findings suggest that KCl mainly removed fiber associated components probably GAG-s, which hindered the immune recognition of fiber-bound fibronectin. The strong association of fibronectin with vitreous collagen suggested a modified model for vitreous structure taking in account the binding of fibronectin both by collagen and GAG-s.

Mineralocorticoid hormone signaling regulates the 'epithelial sodium channel' in fibroblasts from human cornea.

We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na(+)-K(+)-ATPase in the basolateral membrane.

Dibutyryl cyclic adenosine monophosphate and forskolin alter the paracellular pathway in cultured corneal endothelial cells.

PURPOSE: This study describes the effects of the cyclic adenosine monophosphate (cAMP) pathway on the tight junctional barrier of the corneal endothelium, which plays a critical role in maintaining the corneal stroma in an underhydrated, transparent state. METHODS: Subcultured bovine corneal endothelial cells grown on filters were used to study the effects of dibutyryl-cAMP and forskolin on transendothelial electrical resistance and [3H]inulin flux. The tight junction-associated protein ZO-1 (zonula occludens protein-1) and F-actin were visualized by indirect immunofluorescence, and the ultrastructural organization of junctional complexes was studied by freeze-fracture electron microscopy. RESULTS: Cells formed a continuous monolayer of closely apposed hexagonal-type cells separated by a discontinuous belt of tight junctions with a transendothelial electrical resistance of 20.8 +/- 0.6 omega.cm2. Dibutyryl-cAMP (10(-4) M) and forskolin (10(-5) M) increased cell cAMP, significantly decreased the transendothelial resistance by 54% and 43%, respectively, and increased the flux of [3H]inulin from the apical to the basal side of the cells by 56% and 40%, respectively. Both agents also induced condensation of F-actin at the cell borders without any marked changes in the immunostaining of ZO-1 that delineated cell peripheries. However, freeze-fracture studies showed that dibutyryl-cAMP and forskolin induced dispersion of the tight junction network. CONCLUSIONS: These data suggest that activation of the cAMP-dependent pathway, leading to structural changes of the tight junctional network, may modulate the passive fluxes mediated by the paracellular pathway of the corneal endothelial barrier.

Swelling evaluation of "Scyliorhinus Canicula L." cornea.

The cornea of "Scyliorhinus Canicula L.," and Elasmobranch fish species, is generally considered to be a nonedematic tissue. We reinvestigated swelling capacities of these corneas by determining ultrastructural morphological parameters such as diameters, interfibrillar distances, and number of fibers per surface following several immersions of 25 micron sections of stroma in 0.15 M NaCl solution. Absorbed solution in corneal extracellular matrix induced changes in the above-mentioned morphological parameters. The generated changes are more important in Scyliorhinus Canicula L. than in mammalian stromas that have been studied. Elasmobranch corneas, however, remain transparent because new morphological parameters of the swelled tissue are still compatible with physical requirements of light transmission. Swelling impact on mammalian corneas, although weaker, generates morphological parameters that will diffuse light and diminish tissue transparency. Sutural fibers, the most original anatomical Elasmobranch species characteristic, are supposed to serve as mechanical binding of collagen network fibers. We observed that sutural fibers do swell by themselves and could additionally canalize solutions in stroma.

Induced corneal swelling: an experimental model for investigation of collagen and interfibrillar substance relationships.

We report on the evolution of ultrastructural modifications in bovine and rabbit corneal sections after in vitro swelling. The number of stromal fibers decreases with time during swelling. The fact that swelling occurs more rapidly in the center of a lamellae than near the interlamellar spaces indicates that there is a heterogeneous distribution of interfibrillar matrix components and that corneal inhibition mechanisms vary according to the number of lamellaes. The appearance during swelling of periodic striations of collagen fibers and of an irregular network constituted of filaments and granules in the interfibrillar spaces suggests that corneal collagen fibers are covered by a pleated proteoglycan-glycoportein substance that is unrolled during swelling. The filaments seem to be linked to collagen via dense granules located on fibers. The distance between the granules is 643 A. A definite correspondence between the distribution of these linking granules and the periodic striations of collagen cannot be determined. Other granules of undetermined chemical nature seem to link only filaments in the interfibrillar space.