Purpose Collagen crosslinking using ultraviolet-A (UVA) -irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone. Methods Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm 2 and with UVA alone between 2 and 9 mW/cm 2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypanblue and Yopro-fluorescence staining. Results An abrupt cytotoxic irradiance level was found at 0.5 mW/cm 2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/ cm 2 after UVA-irradiation alone. Conclusions A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm 2 , which is reached in the clinical setting in human corneas down to a depth of 300 mm using the standard surface UVA-irradiance of 3 mW/cm 2 .

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 µM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm² for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm²) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm² after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm² with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 µm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.

Purpose: The transplantation of cryopreserved human amniotic membrane has been introduced recently for the reconstruction of the ocular surface. However, in some diseases the transplant usually dissolves rather quickly and early detachment may occur. Therefore, we tried to stabilize the amniotic transplant by applying glutaraldehyde for collagen cross-linking of the membrane. Methods: 18 human amnions were prepared. 4 × 4 cm pieces of amnion were treated with 0.1% glutaraldehyde solution for 30 min. Biomechanical force-elongation measurements were performed and resistance to enzymatic digestion by 0.1% collagenase solution was tested and compared to cryopreserved and untreated fresh amnion. 8 patients with various ocular surface defects were treated with cross-linked amnion and compared to 5 patients with cryopreserved amnion. Results: The force of the amnion cross-linked with glutaraldehyde at 2.5 mm elongation was increased statistically significant by 175% versus fresh amnion and 76.8% versus the cryopreserved amnion. Glutaraldehyde-treated membranes were virtually completely resistant to enzymatic digestion, while fresh and cryopreserved amnions were dissolved completely by day 7. In patients, the cross-linked membrane was preserved for up to 90 days without any signs of dissolution of the membrane and good transparency. Conclusions: Collagen cross-linking using glutaraldehyde leads to a significant increase in the biomechanical strength and enzymatic resistance of amnion, better transparency and less wrinkling. The cross-linked membrane does not dissolve for months and is well suited for the surface reconstruction of the cornea.

The tube-like pattern of SMA immunoreactivity demonstrates the presence of contractile elements within the pericyte processes of the rat retina. Thus, pericytes may act as a regulation element within the retinal microcirculation. Our results further suggest that CA inhibitors are able to decrease pH in the extracellular space; however, the pH within the cells increases. The increase in capillary diameter is concomitant with these pH changes. Thus, we may conclude that CA inhibitors can relax pericytes and might improve the retinal blood supply.