| Objective:To evaluate the changes of MMP-2 expression and cellular biomechanical properties of rabbit scleral fibroblasts, using riboflavin and ultraviolet A (UVA) collagen cross-linking (CXL) of two modes. The potential retina damage will also be analyzed.Methods:24 New Zealand white rabbits were randomly divided into A, B two groups, the left eyes were chosen as the experiment group, and the right ones for control. After anesthesia, the bulbar conjunctiva and fascia between the medial and lateral rectus of left eyes were separated. After a 5-minutes dropping of 0.1% 5-phosphate riboflavin, an 370nm ultraviolet lamp was used for the irradiation. A group were applied irradiation of 3.0mW/cm2 irradiance and 30min duration; while 10mW/cm2 and 9 min for B group.1 week after CXL, the full-field electroretinogram (ERG) was performed.60 days after CXL, the rabbits were executed, and scleral fibroblasts were extracted and cultured from the CXL-treated sclera area and corresponding parts of control sclera. Immuno-cytochemical method was applied to identify scleral fibroblasts. Cellular biomechanical properties were tested using micropipette aspiration technique and the MMP-2 protein expression were detected by western blot technique. SPSS 17.0 system was use for statistical analysis. P<0.05 in T-test was defined as statistically significance.Result:1 The influence of the different irradiance and duration for the full-field electroretinogram (ERG)The ERG results showed that there was no significant difference (P>0.05) in a, b-wave amplitude and latencies time in dark-adapted 3.0 and light-adapted 3.0 in all groups.2. Immuno-cytochemical identification of scleral fibroblastsThe immuno-cytochemical staining results showed:Vimentin staining showed positive result for the brown positive reaction product was clearly visible in the cytoplasm; Desmin, keratin and S-100 staining were all negative, which proved that the obtained cells were scleral fibroblasts.3 Micropipette aspiration on scleral fibroblastsMicropipette aspiration results showed:compared with the control group, the Young’s modulus and apparent viscosity of scleral fibroblasts were increased after CXL(P<0.05), but there was no significant difference between two groups of different illumination mode(P> 0.05).4 MMP-2 expression of scleral fibroblasts after CXLThe western blot results of MMP-2 protein expressions showed: fibroblasts of left eyes in A group and group B showed significantly higher expression of MMP-2than fibroblasts of two groups. Meanwhile, for two different irradiation modes, the MMP-2 protein expression in A CXL group was also significantly higher than that of B group. There was no significant difference between two control groups.The western blot images were scanned and calculated by image analysis software. The statistical results showed that MMP-2 expression in CXL fibroblasts of A and B group was 1.8-fold and 1.3-fold of that in right eye of each group, respectively, which were both have statistical significance (t-test, P=0.001,0.032). Meanwhile the MMP-2 expression level of A CXL group is significant higher than that of B group(t-test, P=0.041). But there was no statistical difference between the two control groups(t-test, P>0.05).Conclusion:1 The Riboflavin-UVA scleral CXL conducted in two different modes both caused no significant side-effects on the retina, so both modes showed relatively high safety.2 Compared with the control group, both illumination modes could increase cell biomechanical properties of scleral fibroblasts significantly. But no significant difference was showed between the two CXL groups..3 Two irradiation modes could both significantly increased the MMP-2 expression in sclera fibroblasts. For the CXL groups, there was a significant difference between A and B groups, which may illuminate that the effects of two different irradiation methods on scleral extracellular matrix were to a certain extent different. |
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