This protocol is significant because it is an excellent model for studying corneal inflammation and its contribution to wound healing under normal and pathological conditions. The main advantage of this technique is that it creates a precise and reproducible epithelial wound without breaching the basement membrane. This model of corneal wounding is straightforward and quick to perform.
Demonstrating the procedure will be Prince Akowuah, a PhD student from my laboratory. To begin, prepare a 1%fluorescein solution by dissolving 10 milligrams of sodium fluorescein salt in one milliliter of sterile saline or PBS. Next, for anesthesia, prepare 10 milliliters of a ketamine-xylazine cocktail by mixing two milliliters of ketamine, one milliliter of xylazine, and seven milliliters of sterile PBS.
After anesthetizing an 8-to 12-week-old C57 black 6 wild-type mouse, evaluate the depth of anesthesia by assessing the pedal reflex after toe pinch. To create the epithelial corneal wound, place the anesthetized mouse under a dissection microscope. Wound the right or left eye only and maintain consistency with the eye being wounded when moving from mouse to mouse.
Keep the eye wide open by holding the eyelids with the thumb and index finger. Then, using a two-millimeter-diameter sterile trephine, demarcate the center of the cornea and gently twirl the trephine to make an impression on the corneal epithelium. Do not apply excessive pressure, as this can result in corneal perforation.
Next, holding the sterile blunt golf club spud at an approximately 45-degree angle from the cornea's surface, debride the epithelium by careful and continuous scraping within the demarcated area using the spud. Do not apply excessive force and maintain hydration using sterile PBS if necessary. For monitoring wound closure and re-epithelization, pipette 1 to 1.5 microliters of 1%fluorescein solution onto the wounded surface and image the cornea using a digital microscope with a blue light source.
Image the wounded cornea at specific times after wounding. Take images within the first minute of fluorescein solution addition to avoid spreading of the solution to the surrounding epithelium, which can lead to an overestimation of the wound size. Next, using an image analysis software, trace the wound area and express the wound area at each time point as a percentage of the original wound area at zero hour.
A transmission electron micrograph of the corneal wound demonstrates that the epithelium basement membrane remains intact even after the injury. In 8-to 12-week-old wild-type mice, wound closure is complete 24 hours after wounding. Inflammatory response to wounding is characterized by cell imaging at the limbus.
Shown here is the limbal vasculature of an unwounded cornea with extra-vascular neutrophils, and that of a wounded cornea with extravascular platelets and neutrophils 30 hours after abrasion. To successfully reproduce this protocol, the same strain in each range of mouse must be used, and the wound size and depth must be created as described.
