A Mouse Model for Corneal Neovascularization by Alkali Burn

We are trying to develop a highly reproducible model for inducing corneal neovascularization and to identify the most relevant histological and immunostaining methods to monitor therapies to prevent and reduce neovascularization. The drawbacks of corneal alkali burn models are difficulties in controlling the area and severity of alkali burn, variation of corneal neovascularization, and unintentional burn of edges and tissues due to excess alkali solution. Corneal neovascularization is a sight-threatening condition.

The pathogenesis of this condition is poorly understood. This model will be used to gain new insight into the condition and to identify relevant therapeutic targets. Compared to the corneal pocket angiogenesis model, alkali burn models are relatively straightforward to create and can also be used to study corneal inflammation, fibrosis, and epithelial proliferation.

These models are also more closely related to clinical chemical burns than corneal suture models of angiogenesis. We will use alkali burn cornea as a disease model to investigate novel anti-angiogenic therapy to treat cornea neovascularization and opacity. To begin, inject meloxicam subcutaneously into the six to eight weeks old C57 Black 6J male mouse 30 minutes before the procedure for pain relief.

After anesthetizing the mouse, pinch the toes and confirm the absence of the reflex. Apply one drop of 0.5%proparacaine on the corneal surface of one and a drop of artificial tears on the other eye. Next, using a two-millimeter biopsy punch, punch out Whatman filter paper discs.

Add two microliters of one normal sodium hydroxide to a clean Petri dish, and place the filter paper disc on the sodium hydroxide drop, allowing it to soak up for 15 seconds. Using forceps, pick up the filter paper, and apply it to the proparacaine-treated eye at the center of the cornea for 30 seconds. Wash the eye by flushing with 20 milliliters of sterile saline in a syringe.

Wipe the excess saline gently from the eyes and the surrounding area using disposable soft wipes. Place the mouse in a recovery cage on a warm heating pad until ambulatory. On day 10 after the burn, capture OCT images of the anterior segment of the eyes as a volume scan using HRA+OCT mode with a 30 degree field of view and 100%IR intensity.

The corneal neovascularization sprouted from the limbus vessels toward the corneal center in the alkali-burned mouse eye, but not in the healthy eye. The alkali burn group demonstrated significantly elevated neovascularization and opacity scores compared to the control group. Immunostaining of corneas for blood and lymph vessels showed significantly higher densities of blood vessels and lymph vessels after 10 days in the burn group compared to the control group.

The thickness of the cornea imaged and quantified using OCT was significantly higher in the burn group.