Antifibrotic effects of tocotrienols on human Tenon's fibroblasts

Antifibrotic effects of α- (40, 60, 80, 100, and 120 μM), γ- (10, 20, 30, and 40 μM) and δ-tocotrienol (10, 20, 30, and 40 μM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7 days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100 μg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. Results All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for α-tocotrienol 80 μM with 36.7% and at day 7 for α-tocotrienol 80 μM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80 μM for α- and above 30 μM for γ- and δ-tocotrienol. The highest collagen synthesis inhibition has been found with 80 µM α-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 µM α- and 30 µM γ-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. Conclusion In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon’s fibroblasts and therefore may have the potential as an anti-scarring agent in filtrating glaucoma surge

Lens epithelial cell apoptosis and intracellular Ca(2+) increase in the presence of xanthurenic acid

BACKGROUND: Xanthurenic acid is an endogenous product of tryptophan degradation by indoleamine 2,3-dioxygenase (IDO). We have previously reported that IDO is present in mammalian lenses, and xanthurenic acid is accumulated in the lenses with aging. Here, we studied the involvement of xanthurenic acid in the human lens epithelial cell physiology. METHODS: Human lens epithelial cells primary cultures were used. Control cells, and cells in the presence of xanthurenic acid grow in the dark. Western blot analysis and immunofluorescence studies were performed. RESULTS: In the presence of xanthurenic acid human lens epithelial cells undergo apoptosis-like cell death. In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 μM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown. In the same condition caspase-3 activation, and DNA fragmentation was observed. At low (5 to 10 μM) of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release. At higher concentrations xanthurenic acid (20 μM and 40 μM) damaged mitochondria were observed in the perinuclear region, and nuclear DNA cleavage was observed. We observed an induction of calpain Lp 82 and an increase of free Ca(2+) in the cells in a xanthurenic acid concentration-dependent manner. CONCLUSIONS: The results show that xanthurenic acid accumulation in human lens epithelial cells disturbs the normal cell physiology and leads to a cascade of pathological events. Xanthurenic acid induces calpain Lp82 and caspases in the cells growing in the dark and can be involved in senile cataract development

Efficacy of Amniotic Membrane Transplantation for the Treatment of Corneal Ulcers.

PURPOSE To evaluate the outcome of amniotic membrane transplantation (AMTX) as a treatment for corneal ulcers. METHODS Patients treated with AMTX for refractory corneal ulcers between 2012 and 2017 were evaluated in a retrospective analysis. Primary outcome measure was complete reepithelialization. RESULTS A total of 149 patients were included (mean age 68 ± 18 years). The mean duration between ulcer onset and AMTX was 42 ± 46 days. The longest time between ulcer diagnosis and AMTX was found in bacterial ulcers and the shortest time to AMTX in eyes with trauma/chemical burns (mean 65 ± 15 days and 14 ± 4 days, respectively). In 70% of the patients, a single AMTX procedure was sufficient to achieve epithelial closure (21% <1 month, 40% within 1 -3 months, and 9% within 3-6 months). Treatment failure was observed in 30% of all patients, and most of them underwent further interventions. Highest closure rates were found in bacterial ulcers, herpetic ulcers, and neurotrophic ulcers (80%, 85%, and 93%, respectively), whereas the lowest reepithelialization rates were found in ulcers after corneal surgery and ulcers associated with rheumatic disease (52% and 57%, respectively). CONCLUSIONS AMTX is a valuable treatment option to achieve corneal epithelial wound healing in cases refractory to conventional treatment. Success rates differ depending on the etiology of ulcer

Keratoconus Progression After Corneal Cross-Linking in Eyes With Preoperative Maximum Keratometry Values of 58 Diopters and Steeper.

PURPOSE To evaluate the effectiveness of cross-linking (CXL) in treating keratoconus eyes with Kmax values ≥58.0 D. METHODS Retrospective analysis of outcomes of standard Dresden epi-off CXL in progressive keratoconus with preoperative Kmax ≥58.0 Diopters (D). Inclusion criteria were Kmax ≥58.0 D and minimum follow-up of 1 year. Corneal topography and tomography were performed preoperatively and at 1 and 2 years. Sixty-one eyes of 56 patients with mean age of 24.9 ± 8.6 years (mean ± SD, range 12-57 years) had 1-year follow-up. Fifty of these eyes had 2-year follow-up. The definition of progression was an increase in Kmax of ≥1.0 D over 1 year. RESULTS Mean Kmax was 63.9 ± 6.1 D (mean ± SD, range 58.2-87.0 D) preoperatively (n = 61) and 62.9 ± 5.9 D (range 54.6-82.5 D) after 1 year. This represented a significant decrease in steepness (P = 0.0029). Mean pachymetry decreased significantly from 433.7 ± 44.8 μm preoperatively to 423.0 ± 41.8 μm (P = 0.001) at 1 year. Progression occurred in 14 of the 61 eyes (23%) at 1 year, and 5 (8.2%) steepened more than 2.0 D. In the group with 2-year follow-up, mean Kmax was 63.0 ± 5.0 D (range 58.2-87 D) before CXL and decreased to 61.5 ± 4.8 D (range 53.6-78.3 D) at 2 years (P = 0.001). Nine of the 50 eyes (18%) showed an increase of Kmax of ≥ 1 D. CONCLUSIONS The incidence of progression (23% at 1 and 18% at 2 years, respectively) is considerably higher than in previously reported results of CXL in eyes with mean Kmax ≥58.0 D. To the best of our knowledge, this study represents the largest number of such steep corneas analyzed with respect to long-term progression after CXL

Dynamic contour tonometry in keratoconus and postkeratoplasty eyes

PURPOSE: To compare the performance of dynamic contour tonometry (DCT) and Goldmann applanation tonometry (GAT) in measuring intraocular pressure in eyes with irregular corneas. METHODS: GAT and DCT measures were taken in 30 keratoconus and 29 postkeratoplasty eyes of 35 patients after pachymetry and corneal topography. Regression and correlation analyses were performed between both tonometry methods and between tonometry methods and corneal parameters. Bland-Altman plots were constructed. RESULTS: DCT values were significantly higher than GAT values in both study groups: +4.1 +/- 2.3 mm Hg (mean +/- SD) in keratoconus and +3.1 +/- 2.5 mm Hg after keratoplasty. In contrast to DCT, GAT values were significantly higher in postkeratoplasty eyes than in keratoconus. The correlation between the 2 tonometry methods was moderate in keratoconus (Kendall correlation coefficient, tau = 0.34) as well in postkeratoplasty eyes (tau = 0.66). The +/-1.96 SD span of the DCT-GAT differences showed a considerable range: -0.42 to +8.70 mm Hg in keratoconus and -1.87 to +7.98 mm Hg in postkeratoplasty eyes. In the keratoconus group, neither DCT nor GAT correlated significantly with any of the corneal parameters. In the postkeratoplasty group, both DCT and GAT measures showed a moderate positive correlation with corneal steepness, but only DCT had a significant negative correlation with the central corneal thickness (tau = -0.33). CONCLUSIONS: DCT measured significantly higher intraocular pressures than GAT in keratoconus and postkeratoplasty eyes. DCT and GAT measures varied considerably, and DCT was not less dependent on biomechanical properties of irregular corneas than GAT

Riboflavin Concentrations at the Endothelium During Corneal Cross-Linking in Humans.

Purpose To determine the riboflavin concentration in the posterior corneal stroma, Descemet's membrane, and endothelium prior to UV irradiation in corneal cross-linking (CXL) in humans. Methods Five human deepithelialized cadaver corneas were mounted into artificial anterior chambers. After the establishment of stable physiological hydration, two-photon imaging with a certified multiphoton tomograph was used to determine fluorescence intensity and second harmonic generation signals from collagen throughout each cornea by optical sectioning, with a step size of 2.5 μm. Afterward, 0.1% riboflavin solution was applied to the anterior corneal surface, similar to the standard CXL protocol. To determine the absolute riboflavin concentration immediately before UV irradiation, corneas were measured by two-photon imaging just at the end of the riboflavin imbibition and after riboflavin saturation. Results The topical application of 0.1% riboflavin results in a riboflavin concentration that decreases to 0.035% in the posterior stroma. Inside Descemet's membrane and endothelium, the concentration drops further to only approximately 0.015% at the endothelial level. Local riboflavin distribution indicates a predominantly paracellular passive diffusion of riboflavin into the anterior chamber. Conclusion The experimentally determined riboflavin concentration of 0.015% at the endothelium shows a substantial discrepancy of a factor of 1.7 to the previously theoretically calculated 0.025%. A lower riboflavin concentration at the endothelium may enable higher radiant exposures and further improve the efficacy of CXL

Oxygen kinetics during corneal crosslinking with and without supplementary oxygen.

PURPOSE To measure and simulate oxygen kinetics during corneal crosslinking (CXL) at different irradiances with and without supplementary oxygen. DESIGN Experimental, laboratory study. METHODS In de-epithelialized porcine eyes, a femtosecond-laser generated tunnel was used to place a fiber-probe in corneal depths of 100, 200 and 300μm to measure the local oxygen concentration. After riboflavin imbibition, the corneas were irradiated at 3, 9, 18 and 30mW/cm2 while the oxygen concentration was measured. All experiments were performed under normoxic (21%) and hyperoxic (>95%) conditions. The obtained data were used to identify parameters of a numerical model for oxygen consumption and diffusion. RESULTS The equilibrium stromal oxygen concentration under atmospheric oxygen at 3mW/cm2 was 2.3% in 100μm decreasing to <1% in 300μm. With 9, 18 and 30mW/cm2, no oxygen was available in 200μm respectively 100μm or deeper. Using a hyperoxic environment, the concentration was 50% using 3mW/cm2 in 100μm, decreasing to 40% in 300μm. At 9mW/cm2 the concentrations were 5%, 3% and 1% in 100, 200 and 300μm, respectively. Using 18 and 30mW/cm2 all oxygen was depleted at 100μm, however, oxygen half-lives were longer at 18mW/cm2 than at 30mW/cm2. The oxygen model was able to reproduce the experiments and indicated an exponential decay with increasing distance to the anterior surface. CONCLUSION Supplementary oxygen increases the oxygen-availability during CXL. At higher irradiances, supplementary oxygen is beneficial and eliminates the bottleneck of oxygen allowing a potentially more efficient crosslinking. The calibrated numerical model can quantify the spatial oxygen concentration related to different scenarios such as irradiance or environmental oxygen concentration

Keratitis resulting from Thielavia subthermophila Mouchacca

PURPOSE: The purpose of this study was to report the first case of fungal keratitis resulting from Thielavia sp. METHODS: We conducted a retrospective chart review. RESULTS: A 10-year old girl presented 2 weeks after ocular plant injury with pain and corneal stromal infiltration with central ulceration and ill-defined margins. Cultures of corneal scrapings and biopsy sequence analysis of the ribosomal internal transcribed spacer region isolated Thielavia subthermophila Mouchacca. Clinically, the organism appeared to respond to topical amphotericin B and oral voriconazole. Best-corrected visual acuity at last follow-up visit counted 0.5. CONCLUSIONS: A rare case of Thielavia sp. keratitis was successfully treated with topical amphotericin B and oral voriconazole. Newly developed molecular diagnostic tools contribute to the recognition of a widening spectrum of emerging fungal pathogens capable of causing serious ocular infections