Protective Effect of TLR4 Ablation against Corneal Neovascularization following Chemical Burn in a Mouse Model.

Purpose: To determine whether Toll-like receptor 4 knockout protects mice from corneal neovascularization following chemical injury compared to wild-type (WT) mice. Methods: A chemical burn (75% silver nitrate, 25% potassium nitrate) was created under anesthesia in the central right cornea of 32 WT and 31 Toll-like receptor 4 knockout mice. Corneal neovascularization was evaluated at 3, 4, 6, 8, 10, and 35 days after injury using digital photography, fluorescein angiography, gelatin perfusion with fluorescence vascular imaging, immunofluorescence staining, and molecular analysis. Results: There was no significant between-group difference in relative corneal burn area at 10 days after injury (39.0 +/- 2.4% vs. 38.8 +/- 9.8%, respectively). Neovascularization was detected in all corneas in vivo and perfusion was detected by fluorescence vascular imaging, reaching maximum area on day 10. The relative area of neovascularization was significantly smaller in the knockout than the WT mice on days 6 (33.3 +/- 4.2% vs. 46.8 +/- 7.4%, respectively, p = 0.005) and 8 (36.6 +/- 1.1% vs. 52.2 +/- 6.4%, respectively, p = 0.027), although neovascularization was intensive in both groups. In line with the immunostaining findings of angiogenesis and inflammatory infiltration of damaged corneas, molecular analysis (performed on day 3) revealed elevated expression levels of angiogenesis-related genes (vascular endothelial growth factor, VEGFR2, VEGFR1) and inflammation-related genes (CD45 and TGF beta 1) in the WT mice. The knockout mice had higher TNF-alpha expression than the WT mice. Conclusion: In a mouse corneal chemical burn model, lack of Toll-like receptor 4 expression did not completely inhibit angiogenesis, but did have a relative effect to reduce neovascularization as compared to the WT.