Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

Our aim is to induce a precise and localized alkaline injury into the cornea and limbal area in a mouse model. We developed a novel Punch-Trephine method and studied how effective this method is in creating a corneal and limbal stem cell deficiency. Advanced techniques in the field of corneal opacity treatment and corneal regeneration, including limbal stem cell transplantation, exosome transplantation, and mitochondrial transplantation, are based on studies that investigated corneal responses to various injuries as well as treatments.

Therefore, it is critical to have a standard, accurate, and reliable injury model among researchers. Several techniques and animal models have been on plan to induce various corneal injuries and investigate various aspects of the corneal wound healing process. The alkali bar model is a well-established injury model, which is performed by applying sodium hydroxide directly to the corneal surface, or by using blood filter paper.

We developed a novel Punch-Trephine method to create a mouse model of limbal stem cell deficiency accordingly and reliably, overcome the limitations of previous techniques and minimizing the risk of insufficient injury to the limbal area, or additional injury to the fornix and conjunctiva. This technique has several advantages, effective chemical injury to the entire corneal surface and limbus in mice, localized and well-circumscribed injury to the cornea, application of any liquid for a predetermined duration, induction of various corneal injury sizes with suitable biopsy punches. It's also feasible for rat and rabbit injury models.

Begin by preparing a biopsy punch of 3.5 millimeter diameter. Mark a point on the punch shaft five millimeters distal from the edge. Secure the punch and use a two-speed rotary tool to sever the marked distal section of the shaft.

Cut the shaft at a depth of 3.5 millimeters, leaving the final 1.5 millimeters attached, and bend the tip to 90 degrees. Next, prepare PBS in 900 milliliters of distilled water, and adjust the pH to 7.4. Then, bring the solution to a final volume of one liter by adding distilled water.

Prepare a 4%paraformaldehyde solution by dissolving two grams of paraformaldehyde in 45 milliliters of PBS under a chemical hood, and heat it to 65 degrees Celsius while adjusting the pH to 7.4. Once the paraformaldehyde is fully dissolved, adjust the final volume of the solution to 50 milliliters. Weigh the mouse to determine the correct volume of the injectable anesthetic cocktail for administration.

Once anesthesia is administered, position the mouse on the surgical table, adhering to standard rodent surgery principles. Then, place a five millimeter high pillow under the rodent's head in the lateral decubitus position. Thoroughly dry the ocular surface with a surgical eye spear, and neatly trim the eyelashes.

Adjust the surgical microscope for an optimal view of the anesthetized mouse, and set the timer to 30 seconds. Then, examine the limbal area circumference using the surgical microscope while keeping the eyelids of the mouse wide open using the thumb and index fingers. Now carefully hold the sterilized Punch-Trephine parallel to the eye's axis without applying downward pressure.

Avoid rotating the instrument and maintain the Punch-Trephine's axis parallel to the globe's axis. Ask the surgical assistant to place three drops of sodium hydroxide solution into the Punch-Trephine's hole. After 30 seconds, cleanse the cornea and fornix using five milliliters of PBS.

Then, use a universal pH indicator paper to ensure a pH value between seven and 7.5 on the corneal surface of the injured eye. After the procedure, wash, dry, and sanitize the Punch-Trephine and the surgical table with 70%ethanol. Examine the eyes of an anesthetized mouse under a slit lamp bio microscope, and use a camera to capture images.

Then, apply 0.1%fluorescent eyedrops and soak any excess fluorescent liquid with a cotton applicator. Evaluate the possible presence of corneal epithelial defects utilizing the cobalt blue filter. And take photographs.

Afterward, apply the triple antibiotic ophthalmic ointment over the damaged ocular surface. Enucleate the eye while preserving the medial caruncle and the entire palpebral conjunctiva. Under a surgical microscope, delicately dissect the junction between the caruncle and skin.

Using tooth forceps, retract the caruncle and help guide the surgical scissors beneath the palpebral conjunctiva, heading towards its junction with the tarsal plate. Cut the conjunctiva along the adhesion line towards the lateral canthus. Then, turn the surgical scissors to the junction of the conjunctiva and inferior tarsal plate at the subconjunctival plane.

Now, retract the superior and inferior eyelids from the nasal side using the thumb and index fingers. While retracting, guide the tip of the curved tip tweezer behind the protruded lacrimal gland, moving toward the optic nerve. Firmly grasp the optic nerve and extract the globe.

Later, rinse the globe with PBS and transfer it into the fixation solution. The wound healing process of the mouse eye after corneal and limbal alkali injury in a mouse model is shown. Corneal edema is prominent on days zero and two, whereas fibrosis is more evident during the second week post-injury.

The epithelial defect healed by conjunctival epithelial cell migration in a centripetal pattern in 12 to 14 days. However, 50%of the injured eyes developed persistent epithelial defects at the end of the second week.