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Mario Crespo-Moral, Laura Soriano-Romani, Antonio López-García, Yolanda Diebold; An ex vivo model using porcine eyeballs for wound healing studies. Invest. Ophthalmol. Vis. Sci. 2017;58(8):164.
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There is a need to improve experimental 3D models to deepen the knowlegde of wound healing physiopathology. Ex vivo models can be the closer approach to the in vivo situation. Adequate levels of thrombospondin (TSP)-1 and transforming growth factor (TGF)-β are pivotal in the wound healing process. Thus, our aim was to use an ex vivo porcine model of wound healing and to study its response after TSP-1 and TGF-β exposure.
Porcine eyeballs (n = 30), obtained from a local slaughterhouse, were washed and desinfected in povidone iodade. Then, anterior surfaces were isolated and 4% agar aded in the inner face to maintain the normal curvature of the eye. Wounds in central cornea were done using 1-hepthanol (n=15) or a crescent knife (n=15). Pictures of fluorescein-stained wounds were taken to determine wound size after 24, 48, 72, and 96h. Then, corneas were processed for histology analysis. Micrographs of hematoxylin-eosin and TUNEL-stained corneal tissue sections (5 µm) were analyzed to characterize the wound and identify apoptotic cells. Mechanically-induced wounds (n=12) were then treated with human TSP-1, anti-TSP-1 antibody, TGF-β, or PBS (control) for 24h. Wound size was evaluated with fluorescein solution at 0, 24, 48, and 72 h after the treatments. The ratio wounded area/total corneal surface was calculated using ImageJ software. Results showed % of wound healing (wounded area x 100/total corneal area) relative to the baseline (wounded area 24h after treatment=100 %). Data (mean±SEM) were analized using the Student’s t test.
At 24h, mechanically-induced wounds affected to the 15±3; % of the corneal area whereas 1-hepthanol-induced wounds affected to the 20±8; %. Both ways of wounding only damaged epithelial cell layers at times 0 and 24h, but showed TUNEL positive cells in the central stromal area under the wound after 48 and 72h. Mechanically-induced wounds showed a mean wound size of 16±4; %. Control and TSP-1-treated wounds closed after 72h. However, TGF-β-treated wound size was higher at 48 and 72h than that of control and TSP-1-treated wounds. Anti-TSP-1-treated wound size increased at each time point and never reached fully closure.
We have optimized an ex vivo wound healing porcine model on which the effect of TSP-1 and TGF-β was tested. This ex vivo short-term (3-5 days) model of wound healing may serve as a research tool to test potential therapeutic molecules.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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