A COMPARATIVE STUDY ON THE IMPACT OF RIBOFLAVIN-UVA CROSS LINKING AND ROSE BENGAL GREEN LIGHT ON CORNEAL NEOVASCULARIZATION IN RAT EYES

To evaluate the comparative effects of riboflavin-UVA cross-linking (CXL) and rose bengal-green light CXL on corneal neovascularization in rat eyes.

Animal experiment

40 Wistar albino rats were included. Corneal neovascularization was induced by sutur placement into the cornea of each eye of the animals. On the 3rd day, the animals were randomly divided into 4 groups: Control, riboflavin-UVA CXL, rose bengal-green light CXL, green light group. CXL and green light applications were performed on the 3rd day and each corneas were evaluated on days 0-3-7-14. Anterior segment optical coherence tomography (AS-OCT) and anterior segment photographs were taken at each visits. Half of the animals from each group were sacrificed on day 7, while the half were sacrificed on day 14. The right corneas of the animals were removed and examined for VEGF, LYVE-1, CD68 immunohistochemistry, haematoxylin-eosin staining and TUNEL analysis, while the left corneas were analysed for VEGF and CD68 levels by ELISA method for quantitative analysis.

Vascularized area in the riboflavin-UVA CXL group was significantly lower compared to other groups on 7 and 14th days (p=0,002, p<0,001, respectively). Vascularized area was also significantly lower in the rose bengal-green light CXL group compared to the green light group receiving on day 7 (p<0,001). In ELISA VEGF analysis, while the level was lower in all groups on day 7 compared to the control, the level was lower in the riboflavin-UVA CXL group on day 14 compared to the control (p<0,001, p=0,047, respectively). In VEGF immunohistochemistry examination, although the score was lower in the riboflavin-UVA CXL group on the 7th and 14th days compared to the control, the difference between them and the green light group was significant (p=0,024, p=0,015, respectively). Although VEGF and LYVE-1 immunohistochemistry staining was lower in the rose bengal-green light CXL group at both time points, the difference was not significant (p>0,005). When inflammation findings were evaluated, ELISA CD68 level was significantly lower in rose bengal-green light CXL group on day 14 compared to control (p=0,017), while CD68 immunohistochemistry staining was significantly lower in both CXL groups on both days 7 and 14 (p=0,002, p=0,002, respectively). Leukocyte counts with haematoxylin-eosin staining were similarly lower in the CXL groups on the 7th and 14th days (p=0,003, p=0,002, respectively). While keratocyte counts were lower in the CXL groups on the 7th and 14th days (p<0,001, p<0,001, respectively), apoptotic index assessed by TUNEL analysis was highest in the riboflavin-UVA CXL group compared to the other groups, then higher in the rose bengal-green light CXL group compared to the control and green light groups (p<0,001, p<0,001, respectively).

Both cross-linking methods suppressed corneal inflammation and were effective on haemangiogenesis and lymphangiogenesis; however, riboflavin-UVA CXL was more effective than rose bengal green light CXL.