A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

The overall goal of this procedure is to create a reproducible, sustainable, and persistent mouse model of limbal stem cell deficiency. This simple technique provides a consistent animal model of limbal stem cell deficiency, without inducing unwanted injury to the eye, which is difficult to achieve with the standard chemical injury models. This model is useful for studying the disease process in long-term at cellular and molecular level.

And for testing or comparing the efficacy of novel treatments. After anesthetizing the mouse, pinch the toe to ensure adequate depth of anesthesia. Inspect the eye under a slit lamp to verify the absence of any previous corneal injury.

Instill a drop of 1%tetracaine hydrochloride onto the eye, and wait for 60 seconds. Next, gently apply 5%povidone iodine on the eye and the hair around it. After one minute, wipe off the drop or spread it onto the hair around the eye to prevent the hair from interfering with the tip of the tool.

Prepare a sterile rotating burr and a pair of sterile bent tweezers, and keep them on a sterile sheet during the procedure. Now, stabilize the eye by grabbing the lids from the nasal side using the bent tweezers, and gently proctozing the eye. Be careful not to apply too much pressure.

Under a surgical microscope, shave around the limbal area twice using the sterile rotating burr. Remove the whole corneal epithelium from limbus to limbus with the rotating burr in a circular fashion, and work with the lateral side of its tip. To avoid stroma injury, do not re-brush corneal parts from which the epithelium has been removed.

Do not apply any pressure on the eye. Clean the brush tip as required. Apply a drop of sterile PBS on the cornea, and wipe away any detached epithelial sheets with a soft cleaning tissue.

Then, shave the remaining epithelium, if any. Pay attention to the mouse tail to ensure adequate depth of anesthesia during the surgery. Apply a drop of Flourescein sodium onto the cornea.

Clean the cornea after 30 seconds, and examine it under a microscope with a cobalt blue filter to verify the complete removal of the epithelium. After the procedure, place the animal in its cage on a heating blanket and observe it for 30-45 minutes until complete recovery from anesthesia. In the meantime, apply 1%Erythromycin Ophthalmic ointment to the eye and continue the application of the antibiotic daily for a week.

In addition, inject a 0.1mg/kg of Buprenorphine subcutaneously immediately, and 12 hours after the procedure, and continue the injection twice a day for two days. This is the cornea phenotype one month after the procedure. You can see extensive neovascularization which is a feature of limbal stem cell deficiency.

A normal cornea expresses Cytokeratin 12 but is devoid of Cytokeratin 8 or goblet cells. In limbal stem cell deficiency, Cytokeratin 8 and goblet cells appear on the cornea, while Cytokeratin 12 is lost. Once mastered, this technique will result in corneal neovascularization that will cover almost the entire mouse corneal surface in one month, and is stable up to three months.

It is important to avoid re-brushing the corneal parts that the epithelium has already been removed. This can cause undesired stroma inflammation and opacification. Following development of limbal stem cell deficiency, experimental treatment methods can be tested and compared.

Alternatively, treatments can be started shortly after the injury. This video displayed how to effectively remove the limbal and corneal epithelium to induce limbal stem cell deficiency in mouse. With some modifications, it can be performed on other animals as well.