Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

We are investigating cornea innate immunity in diabetic wound healing. Specifically, we aim to understand why diabetic patients are more susceptible to bacterial infection and what causes delayed wound healing associated with hyperglycemia. We identified that the balance between IL-1 beta and IL-1 receptor antagonist, as well as TGF beta one and TGF beta three, is important for corneal wound healing.

This could lead to new treatment for diabetic keratopathy. Our model mimic human delayed wound healing in diabetic patients. This allows us to study programmed cell death pathway and develop targeted therapies.

To begin, take porcine eyes placed in a one liter beaker. Hold an eyeball with tweezers and remove the extraocular tissues with scissors in a sterile Petri plate. For epithelium wounding, hold the eyeball using sterilized lint-free wipes and mark the center of the cornea with a six millimeter trephine.

Then, using a small scalpel or corner-blunted soft razor blade, gently scrape the epithelial cells within the trephine-marked circle. Remove all cell debris while ensuring the basement membrane remains intact. Afterward, clean the wound area with cotton swabs.

Using a scalpel and scissors, dissect the eyeball by cutting along the corneal scleral rims. Then, rinse the corneas in a sterilized 500 milliliter beaker containing PBS. Next, place the excised corneas upside down into a sterile mold.

Fill the endothelial corneal cavity with a minimum essential medium, or MEM, containing 1%agarose maintained at 48 degrees Celsius. Now, invert and transfer the corneas to a 35 millimeter dish. Add two milliliters of MEM, with or without the testing agent, dropwise to the surface of the central cornea, ensuring the limbal conjunctiva region is covered while leaving the epithelium exposed to air.

Then, place the culture dishes in a humidified 5%carbon dioxide incubator at 37 degrees Celsius. At 40 hours post-wound, stain the wounded cultured corneas with Richardson's staining solution. Wash the stained corneas with PBS before photographing them.

Then, use the same size trephine to mark the original wound, and using a small scalpel. scrape the epithelial cells within the marked circles. Remove the collected cells and transfer them into a precooled centrifuge tube.

The remaining wound area was significantly larger in corneas treated with nepafenac 0.1%compared to those treated with bromfenac 0.09%and ketorolac 0.45%Corneas treated with LL-37 at 0.2 and 0.5 micrograms per milliliter showed significantly accelerated wound healing under high glucose conditions compared to untreated corneas.