SPARC Levels Modulate the Capacity of Mitomycin to Inhibit the Proliferation of Human Tenon's Capsule Fibroblasts.

PURPOSE: To evaluate the role of SPARC in the antiproliferation effect of MMC on human Tenon's fibroblasts (HTF). METHOD: Sixteen PACG patients aged 59 ± 10 years (31-72 years), including 6 males and 10 females, were recruited. Tenon tissue was harvested during filtering surgery. Cell density was evaluated after MMC application with different concentrations and application times, by which the optimized MMC application modality was determined. MMC, si-SPARC, or SPARC protein was used when needed to evaluate the cell densities under different conditions, by which the role of SPARC in MMC-mediated antifibrotic process was identified. RESULTS: Considering that the cell densities, as well as SPARC expression on mRNA and protein levels, are relatively stable when the MMC concentration is higher than 0.02% and exposure time longer than 90 s, we chose the MMC application pattern with 0.02% and 90 s as an optimized pattern for the downstream work. Compared to control, the si-SPARC and MMC downregulated the SPARC protein by 91% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (. CONCLUSION: This study demonstrates that in HTF, (1) MMC downregulates the expression of SPARC in protein and mRNA levels; (2) SPARC depletion has synergistic effect on the antifibrotic effect of MMC; and (3) reactive oxygen species are the possible mediator in the antifibrotic effect of MMC and si-SPARC.

Role of corneal epithelium in riboflavin/ultraviolet-A mediated corneal cross-linking treatment in rabbit eyes.

PURPOSE: To evaluate the role of corneal epithelium in riboflavin/ultraviolet-A (UVA) mediated corneal collagen cross-linking treatment. METHODS: Fifty New Zealand rabbits were divided into 5 groups: UVA treatment with or without corneal epithelium, UVA+riboflavin treatment with or without corneal epithelium, and control without any treatment. All rabbits were sacrificed after irradiation and subsequently 4 mm × 10 mm corneal strips were harvested for biomechanical evaluation. RESULTS: UVA irradiation alone did not enhance the maximal stress and Young's modulus of corneal specimens with (3.15 ± 0.56 mpa, 1.00 ± 0.09 mpa) or without (3.53 ± 0.85 mpa, 0.94 ± 0.21 mpa) the corneal epithelium, compared to specimens in the control group (4.30 ± 0.68 mpa, 1.03 ± 0.24 mpa). However, UVA irradiation combined with riboflavin significantly increased the maximal stress and Young's modulus of corneal specimens with (5.27 ± 1.09 mpa, 1.23 ± 0.23 mpa, P < 0.05) or without (7.16 ± 1.88 mpa, 1.42 ± 0.16 mpa, P < 0.05) corneal epithelium when compared to the control group. The maximal stress and Young's modulus of cornea in UVA+riboflavin and "epithelium-off" group were 35.9% and 15.4% higher compared to the UVA+riboflavin and "epithelium-on" group, respectively (P < 0.05). CONCLUSIONS: Our study shows that UVA+riboflavin treatment significantly affects the biomechanical properties of the cornea with and without epithelial removal. However, corneas without epithelium seem to benefit more compared to corneas with the epithelium.

Comparison of different methods of glycerol preservation for deep anterior lamellar keratoplasty eligible corneas.

PURPOSE: To compare different methods of glycerol-preserved corneas intended for deep anterior lamellar keratoplasty (DALK). METHODS: We analyzed transparency, transmittance, thickness, biomechanics, morphology, and antigenicity of donor corneas preserved by four different glycerol-based methods (n = 6 per group) for 3 months, as follows: tissues in anhydrous glycerol without aluminosilicate molecular sieves at room temperature (GRT); tissues in anhydrous glycerol with aluminosilicate molecular sieves at room temperature (SRT); tissues in anhydrous glycerol without aluminosilicate molecular sieves at -78°C (G78); and tissues in anhydrous glycerol without aluminosilicate molecular sieves at -20°C (G20). RESULTS: Slit lamp images and transmittance curves obtained by spectrophotometer show that the G78 cornea was the most transparent tissue. Stress-strain behavior indicated that corneas in the G78 group were the most pliable, and SRT corneas were the stiffest. Electron microscopy analysis indicated that corneal cytoarchitecture and keratocyte integrity was destroyed in all glycerol-preserved corneas. Disorganized stromal collagen fibers were evident in groups stored at RT. Especially in SRT corneas, parallelism was lost, fibrils were extremely tortuous and discontinuous, and widespread fibril degeneration could be found. Antigenicity of tissue, assessed via immunohistochemistry for CD45-positive cells, HLA-ABC and HLA-DR, was lowered after glycerol preservation relative to fresh cornea tissues, and immunoreactivity was located mainly on corneal epithelium and limbus rather than stroma. CONCLUSIONS: Anhydrous glycerol preservation without molecular sieves in a -78°C freezer was the best method to obtain DALK-eligible tissues that were both transparent and pliable.

Effect of glucose on the stress-strain behavior of ex-vivo rabbit cornea.

The biomechanical changes in rabbit cornea preserved in storage media with different glucose concentrations are experimentally assessed. Two groups of eight fresh rabbit corneas were preserved for 10 days in storage medium Optisol-GS with glucose concentrations of 14 and 28 mM, respectively. Eight additional corneas preserved, glucose-free, in the same medium served as the control group. All specimens were tested under inflation conditions up to 45 mmHg posterior pressure, and the pressure-deformation data obtained experimentally were analyzed using shell theory to derive the stress-strain behavior. Comparisons were held between the three specimen groups in order to determine the effect of glucose concentration on corneal biomechanical behavior and thickness. After storage, the mean central corneal thickness in the control, low-glucose and high-glucose groups underwent statistically significant increases of 38.7 ± 11.3%, 45.4 ± 7.6% and 50.6 ± 8.6%, respectively. The corneas also demonstrated consistent stiffness increases with higher glucose concentrations. The tangent modulus values determined at different pressure levels between 10 and 40 mmHg underwent statistically significant increases with glucose level (P < 0.05). Compared to the control group, other specimens had higher tangent modulus by 17-20% on average with low glucose and 30-37% with high-glucose concentration. The results of the study indicate that the influence of the high-glucose level commonly experienced in diabetes on the biomechanical stiffness of the cornea should be considered in clinical management and in understanding corneal ectasia, glaucoma and the response to refractive surgery.