Corneal Collagen Cross-Linking Pretreatment Mitigates Injury-Induced Inflammation, Hemangiogenesis and Lymphangiogenesis In Vivo

PURPOSE: The purpose of this study was to evaluate if corneal collagen cross-linking (CXL) pretreatment dampens suture-induced hemangiogenesis and lymphangiogenesis driven by inflammation. METHODS: Four weeks after CXL pretreatment, suture emplacement was performed in rats. The time dependent effects were compared of this procedure in three groups: (1) suture-induced neovascularization (SNV group); (2) CXL treatment prior to suture-induced neovascularization (CXL + SNV group); (3) Normal control (NC group). Serial morphometric measurements evaluated suture-induced hemangiogenesis and lymphangiogenesis. CD45 and CD68 immunofluorescent staining pattern changes determined immune cell activation, stromal leucocyte, and macrophage infiltration. The real-time quantitative polymerase chain reaction (RT-qPCR) determined angiogenic and lymphangiogenic gene expression level changes. Western blots evaluated protein expression levels of vascular endothelial cell CD31 and lymphatic vessel endothelial hyaluronan receptor (LYVE-1). RESULTS: On days 7 and 14 after suture emplacement, the rises in angiogenesis, lymphangiogenesis, CD45+ and CD68+ cell infiltration were less in the CXL pretreated (CXL + SNV) group than in the untreated (SNV) group. Angiogenic and lymphangiogenic mRNA levels and CD31 and LYVE-1 protein and proinflammatory cytokines were also suppressed, confirming that CXL pretreatment improved the wound healing response. CONCLUSIONS: CXL pretreatment inhibits injury-induced angiogenesis and lymphangiogenesis. These reductions suggest that prior CXL therapy decrease ocular inflammation reactivated by secondary trauma. TRANSLATIONAL RELEVANCE: CXL pretreatment induces increases in stromal stiffness which in turn reduces trauma or microbial driven increases in inflammation, angiogenesis, and lymphangiogenesis. These beneficial effects suggest that this novel procedure may improve therapeutic management of trauma-induced corneal disease in a clinical setting.