Limited versus total epithelial debridement ocular surface injury: Live fluorescence imaging of hemangiogenesis and lymphangiogenesis in Prox1-GFP/Flk1::Myr-mCherry mice

Abstract

Background Immunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo. Methods The eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea+bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated. Results Neither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal+bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal+bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9±0.8%, 7.14±2.4%, 2.29±1%, and 15.05±2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45±1.51%, 4.85±0.95%, 2.95±1.27%, and 4.15±3.85%, respectively. Conclusions Substantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity. General significance Our study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.

Application of corneal injury models in dual fluorescent reporter transgenic mice to understand the roles of the cornea and limbus in angiogenic and lymphangiogenic privilege

“…Previously, studies demonstrated that neither debridement of the central cornea nor bulbar conjunctiva results in significant vessel growth in the mouse cornea, but that of the total cornea, as well as the bulbar conjunctiva plus the total cornea, does 42 . Substantial corneal debridement is required to induce NV in the mouse cornea, and the corneal epithelium may, therefore, be largely responsible for maintaining corneal avascularity.…”

“…The PGFD transgenic mouse used in the present study permits the direct, temporal observation of blood and lymphatic vessels in the eyes, even with subject manipulation. By observing blood and lymphatic vessels simultaneously, we can better understand the processes of angiogenesis and lymphangiogenesis, as well as how they may interact or develop in tandem 41,42 .…”

Cornea-Derived Mesenchymal Stromal Cells Therapeutically Modulate Macrophage Immunophenotype and Angiogenic Function

“…Limbal stem cell deficiency was induced by total corneal epithelial debridement which also creates an inflammatory environment that leads to corneal neovascularization . A 6‐0 nylon suture was pre‐passed through the lids (in preparation for closing the lids afterward).…”

Angiogenesis and lymphangiogenesis in corneal transplantation–A review

“…For many years it was believed that angiogenesis plays the major role in many pathological processes, especially in graft rejection, as corneal lymphangiogenesis is generally invisible with conventional imaging and difficult to appreciate. 36 Through studies using recently discovered lymphatic endothelial cell-specific markers, 19; 49; 193 however, it is now recognized that lymphangiogenesis is equally as important as angiogenesis in graft rejection. Cursiefen and coworkers reported that angiogenesis and lymphangiogenesis may be triggered by an identical pathophysiological process, grow in parallel, but regress over different time scales.…”