Our research focuses on novel approaches to regenerating the retinal pigment epithelium. Dysfunction of the retinal pigment epithelium is thought to play a central role in the pathophysiology of many eye diseases, such as macular degeneration. So regenerating this layer of cells is attractive from a therapeutic standpoint.
Visualizing the structural anatomy of the retina via immunohistochemistry has wide applications in retinal research. In our laboratory, we found obtaining useful results from rabbit eyes difficult due to tissue artifacts like retinal attachments and retinal folds. Despite these challenges, there are very few protocols existing that describe successful tissue processing of rabbit retina.
This protocol makes several important modifications to standard tissue processing techniques that result in excellent tissue sections of the rabbit retina. This study may allow researchers to obtain better results from their research that involves the use of immunohistochemistry in rabbit models. After transconjunctival enucleation of the rabbit eye, immediately place in a 50 milliliters of 4%paraformaldehyde and PBS on ice.
After 15 minutes, transfer the eye to a 100-millimeter dissection dish and fill the dish with PBS to prevent the eye from drying out. Under a dissection microscope, using a surgical blade, start making an incision one millimeter anterior to the limbus, keeping it parallel to the iris plane. Insert curved scissors into the initial incision and continue cutting circumferentially, maintaining a one millimeter distance from the limbus.
Then remove the cornea with forceps and gently wipe away PBS with a laboratory wipe. Place the cornea in 30%sucrose solution at room temperature for cryoprotection. Make an initial cut with curved scissors through the iris.
Then cut circumferentially around the eye, similar to the cornea removal, to separate the iris from the rest of the tissue. Remove the iris with forceps and gently wipe away excess PBS with a laboratory wipe. Place the iris in 30%sucrose solution at room temperature for cryoprotection.
After removing the cornea and iris, place the eye at 50 milliliters of 4%paraformaldehyde and put it on a shaker for gentle agitation. Leave the eye in 4%paraformaldehyde at four degrees Celsius overnight. The next day, place the eye in a dissection dish and fill the dish with PBS.
Under the dissection microscope, use forceps to carefully grasp the anterior lens capsule. Then carefully insert the scissors into the posterior chamber, orienting the blades posteriorly along the lens. Make an initial cut through the lens zonules with curved scissors.
Continue making small cuts in a circumferential pattern through the lens zonules. To confirm lens removal, gently grasp the anterior lens capsule with forceps and attempt to lift it. Upon separation, place the lens in 30%sucrose solution at room temperature for cryoprotection.
Place the eye in 50 milliliters of 30%sucrose in PBS at four degrees Celsius for 24 to 48 hours for cryoprotection. To expedite cryoprotection, replace the initial sucrose solution with a fresh one after eight to 12 hours. After cryoprotection of the rabbit eye is complete, transfer it from a 30%sucrose solution to a 100-millimeter dissection dish.
Under a dissecting microscope, carefully flip the eye to remove excess sucrose solution from the inner part of the eye. Using a wipe, gently wipe away excess sucrose solution from the outer part of the eye. Fill a disposable embedding mold with optimal cutting temperature or OCT compound to a depth of 0.5 to one centimeter.
Place the eye in the embedding mold facing upward and slowly fill the inner part of the eye with OCT compound, avoiding bubble formation. Once the eye is submerged in the OCT compound and does not touch the inner surfaces of the mold, wrap the embedding mold in paraffin and store it at four degrees Celsius overnight. The next day, place the embedding mold in a dissection dish.
If possible, gently remove the OCT compound from the inside of the eye with a wipe. Transfer the eye to a new embedding mold filled with fresh OCT compound. Then fill the mold as previously done to ensure the final embedding medium is free of debris or imperfections.
Label the mold for orientation purposes. Position the eye upward in the cryo mold so the OCT compound lines the inner retina. Use the optic nerve head as a landmark for determining the superior part of the eye.
Place the labeled embedding mold on dry ice, ensuring that the eye is not touching the inner surfaces of the mold or exposed to air. Mount the block on the cryostat with the superior part of the eye facing up and the inferior part down with the remaining cornea and iris facing to the right or left. Position a fresh blade parallel to the vertical axis of the eye to section it.
After cutting the sections and drying overnight, place the slide in a freezer at 80 degrees Celsius. The fluorescence images of a whole retinal cryosection of rabbit eye demonstrated a uniform pigmented retinal pigment epithelium layer without significant detachment from the overlying photoreceptor layer. A brightfield image of the retinal section further demonstrates intact retinal morphology of all layers.
