Our objective is to develop an in vitro model of the anterior region of the eye which will replace a significant number of procedures currently carried out on live animal eyes world wide. We have carried out preliminary work on a model which demonstrates that this proposal is feasible. In our proposed model bovine corneal-scleral buttons will be clamped in a specially designed chamber via the sclera outside the limbus. The epithelium will be exposed to air and its anterior surface automatically irrigated with a solution replicating the tear film. The endothelial layer will be perfused separately with media under normal intraocular pressure and at physiological flow rates. Critical parameters such as pH, flow rate, dissolved oxygen; temperature etc will be monitored and regulated through a series of sensors and an electronic control unit. We will rigorously evaluate the model over a period of 30 days. This evaluation will include all cell types in the cornea and the response of our model to epithelial debridement, corneal transplantation and toxicological agents. We will monitor corneal transparency, thickness and permeability. We will monitor cell viability parameters to assess the epithelial keratocytes and endothelium in the in vitro model. We will use light, confocal, scanning and transmission electron microscopy to evaluate the morphology and ultrastructure of the in vitro model. The results from the model will be compared with normal in vivo bovine cornea and published in vivo studies on both human and animal corneas, several decades of which are available in the literature. We plan to validate the model for use both in basic corneal research and for commercial applications including toxicological testing through the European Commission for Validation of Alternative Methods for the Chemicals and Cosmetics Legislation. We anticipate that this model replace 20% of current tests carried out on the anterior region of the eye, in the fields of basic corneal research and commercial toxicological testing.
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