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James Nicholson, Dana Morzaev, Shirel Weiss, Edith Hochhauser, Nitza Goldenberg-Cohen; Fluorescent vascular imaging shows that TLR4 knockout reduces neovascularization and corneal scar after mouse corneal burn injury. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3248.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammation after corneal injury leads to corneal neovascularization and loss of clarity. TLR4 knock out (TLR4-/-) mice are resistant to inflammation. The aim of this study was to determine whether TLR4-/- mice are protected from corneal neovascularization following chemical injury, as compared to wild type (WT) mice.
Corneal injury was created using a chemical burn model (75% silver nitrate, 25% potassium nitrate) in TLR4-/- mice (total n=21) and WT mice (n=30). At 6, 8, 10, and 35 days post-injury, groups of mice were analyzed using slit lamp photography, fluorescein angiography (FA), perfusion with fluorescent vascular filling and fluorescence imaging, and measurement of the neovascular and burn area in flat mount corneas. Immunohistochemistry was performed on the same corneas to detect inflammatory and angiogenic changes using CD31, CD45, and VEGF as markers.
Neovascularization was seen in all corneas in vivo by slit lamp examination and FA, as well as post-mortem by fluorescent gelatin perfusion following chemical burn induction. The maximal area of neovascularization occurred on day 8 and was smaller in TLR4-/- animals. TLR4 knockout significantly reduced the area of neovascularization on days 6 & 8 (day 6 TLR4-/- 33.3±4.2% vs. WT 46.9±7.5% p<0.03, day 8 TLR4-/- 36.6±1.1% vs. WT. 52.2±6.4%, n=3-5 for each, p<0.01). There were no differences between the TLR4-/- and WT mice at 2 or 35 days. Corneal burn area as a fraction of corneal area measured via slit lamp photography was not significantly different 10 days post-injury in WT vs. TLR4-/-(WT 38.8±9.8%, n=4 vs. TLR4-/- 39.0±2.4%, n=3). Immunostaining revealed angiogenesis and inflammatory infiltration to the damaged corneas. Maximal staining for CD45+ inflammatory cells and VEGF was found on day 8. The inflammatory reaction was more prominent in the WT injured cornea compared to TLR4-/-.
TLR4 expression appears to promote corneal injury after chemical burn in mouse by being permissive for corneal neovascularization without producing a larger corneal burn area relative to TLR4-/- animals.
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