This study focuses on the selective damage of corneal endothelial cells (CECs) in porcine eyes using laser irradiation. The methodology aims to create an ex vivo model for further research on corneal injuries and treatments.
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.
After obtaining freshly enucleated porcine eyes from the local abattoir, place the samples in 4 degrees Celsius, full medium. Use scissors to remove the extracellular tissues, and soak the eyes in 5% povidone-iodine ophthalmic solution for five minutes. Next, place the disinfected samples in sterile PBS at room temperature.
Using a spectral-domain optical coherence tomography device, screen the eyes for major anterior segment pathologies, such as corneal scarring, edema, and other opacities. After screening, position the eyes in front of a slit-lamp unit equipped with a Nd:YAG laser with a wavelength of 1064 nanometers, and a focal spot diameter of 10 micrometers in air. Select the 12X magnification, and deflect the illumination to visualize the individual corneal layers.
After setting the pulse energy and focus point to the appropriate parameters for the selective ablation of the corneal endothelial cells, apply several laser shots to the tissue. Then, under a dissecting microscope, place a clear cornea paracentesis close to the limbus, and inject viscoelastic to stabilize the interior chamber. Then, use an 8-millimeter trephine to excise the laser-treated central cornea, and place the isolated cornea into one well of a 12-well plate, endothelial side up. When all of the corneas have been collected, add 3 milliliters of full medium to each sample well, and incubate the specimens for up to three days at 37 degrees Celsius.
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