Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Current disease models for corneal endothelial cell loss focus on the destruction of the endothelium and more likely represent the final stages of the disease when corneal transplantation is inevitable. New treatments using stem cells or gene therapy, however, are now available that could be more useful for the early stages of the disease, the models for which are lacking. Noninvasive intraocular surgery by photodisruption using an Nd:YAG laser has become a routine procedure for ophthalmologists.

After obtaining freshly enucleated porcine eyes from the local abattoir, place the samples in four 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 slip lamp unit equipped with an Nd:YAG laser with a wavelength of 1, 064 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 anterior chamber. Then use an eight 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 three millimeters of full medium to each sample well and incubate the specimens for up to three days at 37 degrees Celsius. At the end of the incubation, replace the medium in each well with methanol-free 4%paraformaldehyde in Sorensen's buffer for a 20-minute incubation at room temperature. At the end of the incubation, place the fixed samples in 20%sucrose in PBS for about one hour until the corneas sink.

Transfer the samples into 30%sucrose in PBS overnight before embedding the tissues in optimal cutting temperature for storage at minus 80 degrees Celsius. Use a cryostat set to minus 27 degrees Celsius to obtain 10 micrometer thick sections of the frozen tissues collecting each section on a microscope slide within one minute of it being acquired. Then store the slides at minus 80 degrees Celsius until staining.

For hematoxylin and eosin staining, air dry the frozen sections for several minutes to remove moisture before staining with filtered 0.1%Mayer's hematoxylin for 10 minutes in a 50 milliliter tube. At the end of the incubation, rinse the slides in double distilled water for five minutes in a cuvette. Next, dip the slides in 0.5%eosin 10 times, followed by dip rinsing in double distilled water until the eosin stops streaking.

Dip the rinsed slides 10 times in 50%ethanol and 10 times in 70%ethanol. After the last 70%ethanol dip, equilibrate the sections in 95%ethanol for 30 seconds, followed by 60 seconds in 100%ethanol before several dips in xylene. Then mount the specimens with coverslip before obtaining images on a light microscope.

Two photon and light microscopy images should be independently evaluated as no damage, too much damage, or correct amount of damage. Based on these assessments, a heat map can then be calculated to select the right constellation of laser parameters for selective ablation of the corneal endothelial cells with minimal damage to the surrounding tissue. Previous analysis has determined that the focal point of the laser must be at least 0.15 millimeters behind the corneal endothelium for the lowest pulse energy tested.

For pulse energies higher than 2.9 millijoules, the longest focal distance tested is still too close to the endothelium. Various cytoprotective agents can be tested while the excised specimens are in culture. If proven successful, they can be added to the irrigation solution for treatment.