Dynamic aspects of corneal lymphatic vessels after keratoplasty in rats

Abstract

To study the development of lymphatic vessels after keratoplasty and to explore the molecular mechanisms of corneal lymphangiogenesis in transplanted corneas. Experimental research. The development of corneal lymphangiogenesis was examined by LYVE-1 immunohistochemistry and whole mount immunofluorescence 1, 3, 7, 10, 14, 30 and 60 days after corneal transplantation, then lymphatic vessels counting (LVC)was evaluated. The expression of vascular endothelial growth factor-C (VEGF-C) in transplanted corneas was examined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and real time-PCR at same time. In addition, the inflammatory index (IF) was recorded at each time point. The association of VEGF-C and IF with LVC in transplanted corneas was examined. Analysis of the significance of differences between two groups was performed using paired Student's t-test. Pearson's analysis was used to analyze the correlation between VEGF-C, IF and LVC. Corneal lymphangiogenesis occurred in the stroma with LVC (1.8 ± 0.3) on Day 3, then developed and reached the peak with LVC (9.1 ± 1.5) on Day 14 after corneal transplantation. Both VEGF-C protein and mRNA up-regulated dramatically in rat transplanted corneas. The immunoreactivity reached the peak on the 3(rd) day and the 14(th) day after keratoplasty. Compared with the expression of VEGF-C mRNA (1.62 ± 0.08 copies/g) on Day 3, the expression of VEGF-C mRNA (2.48 ± 0.03 copies/g) significantly increased 14 days after the transplantation (t = 4.296, P = 0.02). LVC was strongly and positively correlated with IF (r = 0.55, P = 0.003) and the expression of VEGF-C mRNA (r = 0.51, P = 0.003). Corneal lymphangiogenesis correlates closely with corneal inflammation. The increased expression of VEGF-C in the cornea may be one of the important molecular mechanisms in the occurrence of corneal lymphangiogenesis after keratoplasty.