May 31, 2019 Conference
Edgar Espana, University of South Florida
Murine eyes were harvested from WT C57 mice and Col12a1 deficient mice and de-epithelialized using 35% ethanol. In the first experiment, 0.136% riboflavin in 25% dextran solution was applied to P60 eyes every 5 minutes for 35 minutes while exposed to UVA light at 3 mW/cm2. In the second, P60 eyes were soaked in balanced salt solution for 30 minutes. In the third, eyes at P210 were soaked in balanced salt solution. All eyes were fixed and imaged with transmission electron microscopy. Fibrils in the anterior stroma were then quantified using ImageJ software as the number of fibrils per 0.04 ?m2 area.
12 images from 1 to 2 eyes were analyzed per experimental group. With riboflavin and UVA light, the average number of fibrils per 0.04 um area in WT C57 was 21.50 versus 9.83 in Col12a1 deficient mice (p<0.05). When compared to de-epithelialized control eyes: 16.4 versus 21.50 (p<0.05) and 20.00 versus 9.83 (p<0.05). WT C57 versus Col12a1 deficient eyes at P60 hydrated in balanced salt solution were similar, 11.83 versus 11.25 (p = 0.48), despite each showing change from un-hydrated controls, 14.58 versus 11.83 (p<0.05), 17.83 versus 11.25 (p<0.05). Likewise WT C57 versus Col12a1 deficient eyes at P210 hydrated in balance salt solution showed similar amounts, 13.0 versus 13.17 (p=0.84).
This preliminary data suggests collagen XII may affect fibril spacing after riboflavin and UVA treatment, but its impact on fibril organization during hydration appears minimal. Repeat trials are needed. Future studies with uniform epithelial removal and alternative analysis may better reveal the role of collagen XII in collagen cross-linking.