Anterior Eye Segment Dissection: A Method to Isolate Cornea Bearing Anterior Segment from Porcine Eye

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissection of tissue

1. Prepare the porcine tissue using non-sterile dissection workspace.
   1. Procure enucleated porcine eyes from a local abattoir, animal studies, or vendor. Maintain the eyes on ice submerged in PBS during delivery and use immediately upon receiving.
   2. Cut away the extraorbital tissue and trim the conjunctiva leaving only the corneoscleral shell and optic nerve. Perform the dissection under non-sterile conditions with large surgical scissors and forceps in a gross dissection tool pack.
   3. Place the eyes back into fresh PBS on ice until all eyes required for experimental setup have been preliminary/gross dissected.
   4. Submerge the eyes in 10% betadine solution for 2 min in closed containers and transfer aseptically to BSC II cabinet for microdissection (cabinet #1). Perform all the subsequent works under sterile conditions to minimize contaminations during setup.
2. Sterilely dissect anterior segments.
   1. After 2 min in betadine solution, transfer the eyes into three serial washes of sterile 1x Antibiotic-Antimycotic-PBS (AA-PBS) to remove excess betadine solution from the ocular surface while maintaining sterility of the ocular tissue. After three washes, maintain the tissue in AA-PBS until further use.
   2. Hemisect the eye using a razor blade/scalpel and curved scissors. Place the eye on an AA-PBS-soaked gauze and create an incision with a sterile razor blade or scalpel near the equator of the eye (60/40 split with 40 on the anterior side). Using curved surgical scissors, hemisect the eye to isolate the anterior eye (corneal half).
      NOTE: The cut around the anterior segment needs to be continuous to prevent jagged, rough edges in the sclera that will create fluid leaks after setup in the organ culture.
   3. Use microscissors as a shovel to scoop vitreous humor from the anterior segment. Remove the lens from the anterior segment using microscissors. Leave the anterior segments in AA-PBS until further dissection steps.
      NOTE: All the eyes that will be dissected can be held at this step and one by one taken through the remainder of the dissection process.
   4. With a dissecting microscope, cut back iris to iris root gradually, radially until trabecular meshwork (TM) is visible. The TM is a pigmented tissue that comprises fibers circumferentially oriented around the corneoscleral shell. Careful cuts into the iris toward the iris root will expose the depth of the TM under the tissue.
   5. Cut 360° around the iris at the same depth as the initial cut into the tissue to expose the entire TM region. Clean up any remaining residual iris covering TM as is necessary.
   6. Trim the ciliary body remnants posterior to the TM, leaving only a thin band of tissue posterior to the TM region (approximately 1 mm).
   7. Place the dissected anterior segment (AS) in media until further setup in anterior segment organ culture (ASOC) in dish assembly BSC II cabinet (cabinet #2).
      NOTE: All eyes can be held at this point prior to ASOC setup if a single user is performing dissection and organ culture dish assembly.

Materials

Name	Company	Catalog Number	Comments
Anti-Anti	Gibco	15240-096
Betadine	Fisher Scientific	NC1696484
Cotton swabs	Puritan	25-8061WC
Fine forceps	World Precision Instruments	15914
Gauze	Covidien	8044
Large forceps	World Precision Instruments	500365
Large surgical scissors	World Precision Instruments	503261
Medium toothed forceps	World Precision Instruments	501217
PBS	Gibco	10010-023
Petri dish (100 mm)	Fisher	FB0875713
Razor blade	Fisher	12-640
Sterile 100 mL beakers with lids	VWR	15704-092
Vannas scissors	World Precision Instruments	WP5070