corneoscleral button isolation: a surgical procedure to obtain corneoscleral button from porcine eye

Corneoscleral Button Isolation: A Surgical Procedure to Obtain Corneoscleral Button from Porcine Eye

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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JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Sterilization
<ol>
	<li>CRITICAL STEP: Disinfect all forceps and scissors by soaking for 1 h in 5% (v/v) solution of Distel in distilled water, clean with a brush, rinse with tap water and sterilize in an oven at 185 &#176;C for a minimum of 2 h.</li>
	<li>Sterilize all other glassware and reagents by autoclaving at 121 &#176;C for 15 minutes or prepare reagents according to the manufacturer&#39;s instructions. Carry out the following procedures in a class II microbiology safety cabinet.</li>
</ol>
2. Sample collection
<ol>
	<li>Collection of porcine eyes
	<ol>
		<li>Large white landrace sows, a cross with a Hampshire boar was used. The animals were stunned with an electric current and the eyes were enucleated 2 h later in the abattoir.</li>
		<li>CRITICAL STEP: Once enucleated, transfer the eyes to the lab in a sterile phosphate buffered saline (PBS) solution to prevent them from drying out and process them immediately upon arrival.</li>
	</ol>
	</li>
</ol>
3. Preparation of the corneoscleral button
<ol>
	<li>Use sterile forceps to hold the tissue surrounding the eyeball and transfer it to a Petri dish. Remove the conjunctiva and muscle tissue around the eyeball on a Petri dish using scalpel blade no. 15 and forceps.</li>
	<li>Gently lift the eyeball while holding the optic nerve with forceps and transfer to a 0.5 L jar filled with sterile PBS.</li>
	<li>Once all eyes are cleared of surrounding tissue, move them using sterile forceps to another 0.5 L jar filled with 3% (v/v) povidone iodine in PBS and leave for 1 min.</li>
	<li>Transfer eyeballs to another 0.5 L jar with sterile PBS.</li>
	<li>Use forceps to hold the eye still on a Petri dish and make a cut near the cornea with a scalpel blade no 10A.</li>
	<li>CRITICAL STEP: Hold the edge of the cut and use scissors to excise the cornea leaving about 3 mm of sclera surrounding the cornea. Ensure the sharp end of scissors does not pierce the iris or the choroidal tissue and is in the supra-choroidal space.</li>
	<li>Hold the corneoscleral button with forceps and use another pair of pointed end forceps to gently separate the uveal tissue.</li>
	<li>Lift the corneoscleral button from remaining globe and briefly rinse it in 1.5% (v/v) povidone iodine solution in PBS in a 12 well plate.</li>
	<li>Place the corneoscleral button into another 12 well plate filled with sterile PBS.</li>
</ol>

<table><tbody><tr><td>Cellstar 12 well plate</td><td>Greiner Bio-One</td><td>665180</td><td></td></tr><tr><td>Distel</td><td>Fisher Scientific</td><td>12899357</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>15307805</td><td></td></tr><tr><td>PBS</td><td>SLS</td><td>P4417</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>12664785</td><td></td></tr><tr><td>Povidone iodine</td><td>Weldricks pharmacy</td><td>2122828</td><td></td></tr><tr><td>Scalpel blade number 15</td><td>Fisher Scientific</td><td>O305</td><td></td></tr><tr><td>Scalpel Swann Morton</td><td>Fisher Scientific</td><td>11849002</td><td></td></tr></tbody></table>

Source: Okurowska, K., et al. <a href="https://www.jove.com/v/61156">Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.</a> J. Vis. Exp. (2020).
In this video, we show a procedure to surgically isolate the corneoscleral button to generate an ex vivo porcine cornea model. The model is helpful for histological, pathological, and pharmacological studies due to its similarity to the human eye.

Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp. (2020).
...

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&#39;s surface.&#160;
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.&#160; Detach the uvea to visualize the corneoscleral layer. Store the button in a sterile buffer solution for further assays.&#160;

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Corneoscleral Button Isolation: A Surgical Procedure to Obtain Corneoscleral Button from Porcine Eye

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Corneoscleral Button Isolation: A Surgical Procedure to Obtain Corneoscleral Button from Porcine Eye