This method can help answering key questions relating to the cellular and molecular mechanisms involved during the corneal wound healing. An advantage of this technique is that it reflects the situations in emergency room in case of corneal insert, meaning abrasion or scratch on the eye surface. I first had the idea for this method when I was studying corneal epithelial progenitor cells, and their behavior in a situation that challenges the homeostatic maintenance of the cornea.
Visual demonstration of this method is critical, because performing the corneal abrasion is difficult, and it requires practice. First, prepare a 0.1%Florecine solution for visualizing the abrasion under cobalt blue light by measuring 10 milligrams of Florecine salt with a fine scale. Then, add the salt to 10 milliliters of Fosfate buffered saline solution.
Protect the Florecine solution from light and shake for five minutes. Sterile filter the solution into an eyedropper bottle, and protect from light. The Florecine solution can be stored at four degrees Celsius for two to three days.
Clean the ocular burr tip before and after use. First, with PBS, and then, with 70%Ethanol. Begin by placing the anesthetized mouse onto a paper towel on a warming plate heated to 37 degrees Celsius.
Check the level of anesthesia by administering a tail pinch and a toe pinch. The level of anesthesia is good if the tail reflex is absent but the toe reflex is present. Rotate the base of the burr to turn on the vibration.
Open the eye to be abraded by holding the eyelids apart with fingers. Then, firmly touch the burr to the cornea, and move the instrument back and forth and sideways on the ocular surface without lifting the burr. The burr vibration will perform the scratch.
Do not press, shake, or tear the cornea. In the central cornea, 20 abrading movements on the surface are sufficient to induce the wound. Making the corneal abrasion is challenging and requires practice.
Make sure to pay attention to correct pressure, and try to perform the abrasion in only one attempt. Administer one drop of Flourecine solution to the abraded eye from the dropper bottle. Then, wash the eye once with 0.9%percent saline to reduce background of the Florecine.
Absorb the excess liquid with a soft wipe, and clean the eye lashes if needed. To image the abrasion, position a table lamp with an attached cobalt blue pen light just above the mouse eye. The button of the pen light can be taped down to keep the light on when working alone.
Focus an SLR camera on the eye in room light, then turn off all other lights except the cobalt blue pen light. When illuminated with the cobalt blue pen light, the abraded region fluoresces green due to Florecine molecules trapped in the uneven surface. Use the SLR camera to take pictures of the eye.
After imaging the eye, return the mouse to the warmed plate. Place a drop of antibacterial Fucidin eye ointment into both abraded and non-abraded eyes to moisturize and keep the ocular surface clean from bacteria after anesthesia. Observe the mouse on the heated plate for the five to 20 minute, post-anesthetic, wake up period.
When it becomes mobile, single house the mouse in a clean cage. After euthanizing the animal using an approved method, collect the eyeball by first cutting and opening in the skin to the side of the eye with dissection scissors. Then, place the scissors under the eyeball, and cut the ocular nerve to pump the eyeball out of the orbit.
Place the eye in a dish containing PBS. Use a 26 gage needle to make a hole on the back of the eye on the retinal side, to allow free penetration of solutions inside the eye. Transfer the eye to a two milliliter tube containing PBS.
Then, remove the PBS, add four percent paraformaldehyde, and fix the eye at four degrees Celsius for four to five hours. This image shows a mouse eye immediately after abrasion. The Florecine signal marks the abraded region in bright green, where as all other regions in the epithelium remain dark.
The dashed line marks the borders of the wound. The image shows the uninjured bilateral eye that serves as a control. The white spot is the reflection from the camera.
18 hours after abrasion, the corneal epithelium has largely closed over the exposed region with only a small abrasion remaining. At 72 hours post abrasion, no green signal is visible. This indicates that the corneal epithelium was fully re-epitheliulized by 72 hours after the abrasion.
The abrasion is focused on the corneal epithelium, shown here in the intact eye, so the deeper layers of the cornea remained intact. Immediately after abrasion, H and E staining reveals the edge of the abrasion, indicated by this asterix as a narrow, a-cellular ledge, that is continuous from a regular four to five cell layer thick corneal epithelium. At 18 hours post injury, the healing process is active, and re-epithelialization is ongoing.
This is suggested by the appearance of a leading edge of one to two epithelial cell layers that covers the exposed region. The surface appears fully re-epithelialized by 72 hours, as the migrating fronts cover the wound, and all the epithelial layers are again present. Once mastered, this technique can be done in two hours if it's performed properly by two researchers.
While planning and attempting this procedure, it is important to remember to follow your local animal welfare guidelines, and keep a good and sterile laboratory practice. Following this protocol, other methods like linis tracing can be performed to answer additional questions on corneal wound healing, re-epithelialization, and recovery of tissue homeostasis in the cornea.
