A Rabbit Corneal Endothelial Dysfunction Model Using Endothelial-Mesenchymal Transformed Cells (original) (raw)

Unlike humans, rabbit corneal endothelial wounds are known to spontaneously heal. The current study was aimed to develop a new rabbit bullous keratopathy model using corneal endothelial cells that were induced to undergo endothelial-mesenchymal transformation (EMT). EMT was induced in rabbit corneal endothelial cells (RCECs) by culturing with TGFβ and basic FGF Supplemented Medium. The corneal endothelia in recipient rabbits were mechanically scraped from the corneal endothelial surface inside an 8 mm mark. Then, a suspension of EMT-induced RCECs (EMT-RCECs) was injected into the anterior chamber. Eyes injected with freshly isolated RCECs (Fresh RCECs group) and eyes that were scraped without injection of cells (Scrape group) were used as controls. Immediately following operation, subepithelial and stromal edema was observed with increased central corneal thickness and corneal opacity in all groups. In the EMT-RCECs group, bullous keratopathy persisted for 42 days up to the end of the study. In the Fresh-RCECs and Scrape groups, corneal transparency and thickness recovered by 7 days after treatment and was maintained up to 42 days. The activated fibroblast marker, α-SMA, was observed spanning from corneal endothelium to corneal stroma in the EMT-RCECs group. Interestingly, α-SMA was upregulated in the Scrape-group as well. In all groups, there was no damage to other intraocular structures, and intraocular pressure was normal throughout the observation period. Transplanting a fresh donor cornea effectively treated corneal edema due to bullous keratopathy. This model is a promising tool for pre-clinical trials in the development of new therapies against corneal endothelial dysfunction. Corneal transparency is maintained by the corneal endothelium through the barrier function of tight junctions and by Na + /K +-ATPase pumps located along the lateral cell walls 1. Corneal endothelial dysfunction causes irreversible loss of corneal transparency, and is a major cause of visual impairment and corneal blindness. Although efforts to develop pharmaceutical agents to treat corneal endothelial dysfunction demonstrate promising results 2-5 , corneal transplantation is still the standard treatment of choice. In order to develop new therapies for corneal endothelial disease, an animal model that resembles human corneal dysfunction is required. Several investigators have studied corneal endothelial dysfunction models using cryo-injury 6-8 , ultrasonic emulsification 9 , Nd:YAG laser 10 , chemical injury (intracameral injection of povidone-iodine 11 , benzalkonium bromide 12 and NaOH 13), mechanical scraping of corneal endothelium 14 or stripping of Descemet membrane with corneal endothelium 15-18. Animal species used in these models include the rabbit 6-8,10,11,13-15 , mouse 12 , rat 19 , cat 7 , pig 20-22 and monkey 9,23,24. However, the reliability and stability of small animal models are less than ideal, and monkeys are very expensive. The rabbit model is the most popular because of low cost and a similar corneal diameter compared to humans 25. Unfortunately, unlike humans, rabbit corneal endothelial cells (RCECs) proliferate in vivo and wounds tend to heal spontaneously 7. In addition, previous animal models have limitations such as increased intraocular pressure (IOP) or glaucoma, anterior chamber inflammation and tissue damage. Also, models that involve acute damage to the central cornea to create a defect in the corneal endothelium do not reflect the