Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

The innermost layer of the porcine cornea - the transparent avascular front eye portion - consists of corneal endothelial cells, or CECs, supported by the basement membrane - the Descemet's membrane, or DM. CECs maintain corneal transparency and optimal vision. Any damage to them could affect vision.

For selective CEC damage, begin with a freshly harvested porcine eye in a suitable medium. Remove the surrounding tissues. Treat the eye with an appropriate disinfectant to eliminate surface microbial contamination. Following saline wash, optically examine the anterior eye to rule out any abnormalities before laser irradiation.

Secure the eye in a customized holding apparatus in front of a slit-lamp unit equipped with a laser of a specific wavelength. Visualize the individual corneal layers. With appropriate laser energy and focus point position, direct the laser pulses to the corneal endothelial layer. The focused short-duration, high-power pulses, cause selective CEC rupture from the exposed surface while avoiding heat diffusion, thereby protecting the DM or stromal tissue.

Creating an entry port close to the limbus - the corneoscleral junction, inject a viscoelastic solution into the anterior chamber containing the cornea to stabilize it and protect the remaining CECs. Excise and transfer the laser-treated corneal segment into a media-containing multi-well plate, CEC side facing upward. The ex vivo corneal endothelium injury model can be used for further studies.