Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells
Transcrição
In the vertebrate eye, the cornea – the outermost protective covering- is infected by Pseudomonas aeruginosa, resulting in a corneal inflammatory condition – keratitis.
To establish an ex vivo corneal keratitis model, place a porcine eye corneoscleral button in a Petri dish. This tissue segment consists of the cornea surrounded by remnants of sclera – the fibrous eye covering. Supplement the Petri dish with antibiotic-containing media to eliminate contaminating microbes from the corneal epithelial surface.
Locate the central portion of the cornea. Make superficial horizontal and vertical incisions in the desired pattern piercing the outermost corneal epithelial layer. Transfer the incised tissue, cornea side down, into a specialized hollow mold.
Fill the corneal cavity with liquified agar and allow to solidify, restraining the tissue inside the mold. Invert the mold to expose the wounded surface of the corneal epithelium. Pipette the Pseudomonas suspension into the incised areas of the cornea. Overlay the mold with more growth medium for optimal bacterial growth. Supplement the culture dish with media and incubate.
Bacteria traverse the epithelial incision to invade the stroma. Inside the stroma, bacteria cause the infiltration of immune cells, proteins, and fluid in the cornea resulting in an inflammatory response. The fluid build-up increases the corneal opacity, confirming bacterial keratitis.
