Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Ex vivo and in vivo animal models were set up for studying mechanical and chemical corneal injury. The technique provides an easily built-up platform for researchers to study mechanical and chemical injury of the cornea. To create a murine corneal epithelial wound, mark the central cornea of the anesthetized mouse using a skin biopsy punch to confirm a well-circumscribed and well-measurable area of the wound.

Gently indent the punch over the central cornea to leave a circular mark Debride the corneal epithelial down to the Bowman's layer utilizing a hand-held corneal rust ring remover with a 0.5 millimeter burr ensuring not to damage the Bowman's layer. Remove the residual loose tissues in the wound margin with corneal forceps. Confirm the area of debridement with fluorescent staining by putting a drop of normal saline onto a fluorescein paper to dissolve fluorescein and then placing the fluorescein-containing drop onto murine epithelial defect to visualize it under cobalt blue light.

Proceed with ex vivo culture of the murine corneal abrasion wound model by gently pressing at the superior and inferior orbital rims of euthanized mice to push the eyeball out. Introduce the tip of the closed corneal scissors into the retrobulbar space along the inferior orbital wall ensuring not to penetrate the eyeball. Hold the eyeball steady with 0.3-millimeter corneal forceps and then cut the optic nerve and periorbital soft tissue with corneal scissors to isolate the eyeball.

For ex vivo culturing of murine eyeballs, prepare a 48-well plate with melted wax inside the well and wait for solidification, then with the tip of conjunctiva forceps, create a round hole on solidified wax's surface to accommodate the eyeballs. Place the harvested eyeballs directly onto the 48-well plate with wax-covered bottoms and sidewalls to establish stabilization. Culture the eyeballs with DMEM containing 1%fetal bovine serum with or without antibiotics depending on the purpose of the study.

Immerse the ocular surface with the culture medium without causing the eyeball to float and document the course of wound healing by fluorescein staining and collecting photographs with a digital camera under cobalt blue light. To induce an alkaline burn injury, place a circular filter paper with a diameter of 8 millimeters in a Petri dish. Using a dropper, add 0.5 normal sodium hydroxide into the Petri dish to soak the filter papers and drain the excess solution from the filter paper before placing them onto the anesthetized rabbit cornea.

After opening the eyelids with a lid speculum, ensure that the rabbit nictitating membrane is not interfering with the insertion of filter paper and place the alkali-soaked filter paper onto the central cornea for 30 seconds. Remove the filter paper and rinse the ocular surface with 10 milliliters of normal saline to wash out alkali material. To complete the corneal defect, debride the corneal epithelium within the opacified area down to the Bowman's membrane using a corneal rust ring remover.

Confirm the debridement area with fluorescein staining under the cobalt blue light and remove residual corneal epithelium using corneal forceps. To secure wound condition with tarsorrhaphy, confirm that the nictitating membrane smoothly covers the ocular surface and corneal epithelial defect at the nasal side. Perform a temporary tarsorrhaphy with or without topical agents using a 6-0 suture to protect the ocular surface and to prevent the rabbit from scratching it, ensure that the suture for tarsorrhaphy is at 3 to 4 millimeters from the upper and lower lid margins with four to five ties and longer knots to prevent the rabbit from breaking the sutures.

In the mouse corneal epithelium's ex vivo wound healing model, the mildly depressed central corneal area with positive fluorescein stain was observed in the central two millimeters after the in vivo debridement of the mouse corneal epithelium. The ex vivo culture of the murine eyeballs fixed onto a wax-coated 48-well culture plate was examined and documented daily within a 48-well culture plate under a stereo microscope. A day after debriding the murine corneal epithelium, one circular fluorescein-stained epithelial defect of 2 millimeters in diameter was revealed in digital photographs obtained under cobalt blue light.

After creating an alkali injury to the rabbit corneal epithelium, positive fluorescein staining was observed with or without cobalt blue light over the central cornea with a clear and complete circular margin. The corneal epithelial wound re-epithelialization with pannus ingrowth was observed from the limbus. The most important steps are procedures to create smooth and even epithelial defects on mouse and rabbit corneal surfaces.

New medical and surgical therapeutics can be tested on these platforms. The effect of re-epithelialization can be monitored after the procedures. Corneal neovascularization and opacity after injury in this protocol would be an unmet need for further research.