This article describes the procedure for excising the corneoscleral button from a porcine eye. It outlines the necessary steps to ensure the eye is properly prepared and the corneoscleral button is isolated for further assays.
The anterior portion of the eye is covered by the cornea - a transparent layer that allows the transmission of light rays. The cornea extends into the sclera - a white protective tissue around the eye. The part of the eye that consists of the cornea surrounded by a thin ring of the sclera is the corneoscleral button.
To excise the corneoscleral button, place an intact porcine eye on a Petri dish. Remove the conjunctiva - a mucilaginous covering around the sclera - along with the surrounding extraocular muscles to access the scleral cover. Treat the eye with a suitable disinfectant solution to remove any contaminating microorganisms from the eye's surface.
Wash off the disinfectant with a buffer solution. Transfer the disinfected eye to a fresh culture dish. Make an incision on the sclera near the corneal boundary. Using this opening, make a complete circumferential cut to divide the eye into two fragments.
Visualize the interior side of the frontal segment to locate the uveal coat attached to the sclera. This tissue consists of pigmented iris, a ring-shaped ciliary body, and a portion of the vascular choroid layer. Detach the uvea to visualize the corneoscleral layer. Store the button in a sterile buffer solution for further assays.
Use sterile forceps to transfer the eyeball to a Petri dish. Remove the conjunctiva and muscle tissue around the eyeball using a scalpel blade # 15 and forceps. Then, while holding the optic nerve with the forceps, gently lift the eyeball, and transfer it to a half-liter jar filled with sterile PBS.
Once all eyeballs are cleared of surrounding tissue, use sterile forceps to move them to another half-liter jar filled with 3% povidone-iodine in PBS. After the eyeballs have been in the jar for one minute, transfer them to a third jar containing sterile PBS.
Place an eyeball on a clean Petri dish and use the forceps to hold it steady. With a # 10A scalpel blade, make a cut near the cornea. Use scissors to excise the cornea, leaving about 3 millimeters of sclera surrounding it. Ensure the sharp end of the scissors is in the supra-choroidal space and does not pierce the iris.
Hold the corneoscleral button with forceps, and use another pair of pointed-end forceps to gently separate the uveal tissue. Then, lift the corneoscleral button to separate it from the remainder of the eyeball. Briefly rinse the button in a 1.5% solution of povidone-iodine in PBS. Then, place the button in sterile PBS in a 12-well plate.
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