An Epithelial Abrasion Model for Studying Corneal Wound Healing

Summary

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

Abstract

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Introduction

The cornea is the transparent anterior one-third of the eye. The cornea serves several functions including protecting the inner structures of the eye and forming a structural barrier that protects the eye against infections¹. More importantly, the cornea is critical for vision, providing about two-thirds of the refractive power of the eye^2,3. Crucial to the role of the cornea in vision is its transparency. However, due to its outward position, the cornea is exposed to a wide variety of injuries on a day-to-day basis that can lead to disruption of its barrier function, loss of transparen....

Protocol

All animal protocols were approved by the Institutional Animal Care and Use Committees at the University of Houston and Baylor College of Medicine. The guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in vision and ophthalmic research were followed in handling and using the mice.

1. Preparation

1. Preparation of fluorescein solution
   1. Prepare 1% fluorescein solution by dissolving 10 mg of .......

Discussion

The purpose of this methods paper was to describe a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud. This murine model has been used to study corneal inflammation and its contribution to wound healing. This type of model can be used to study corneal wound healing mechanisms under normal physiological conditions and in pathologies^17,28,29,

Materials

Name	Company	Catalog Number	Comments
Anti-CD31 antibody	BD Bioscience, Pharmingen	550274
Anti-CD41 antibody	BD Bioscience, Pharmingen	553847
Anti-Ly6G antibody	BD Bioscience, Pharmingen	551459
Bovine serum albumin (BSA)	ThermoFisher scientific	B14
C57BL/6 mice	Jackson Laboratories	664
DAPI	Sigma Aldrich	D8417
DeltaVision wide-field deconvolution fluorescence microscope	GE Life Sciences
Dissecting microscope	Leica microsystems
Electronic Toploading Balances (Weighing scale)	Fisher Scientific
Ethanol	ThermoFisher scientific	T038181000CS
Golf-club spud	Stephens instruments	S2-1135
Iris curve scissors	Fisher Scientific	31212
Isoflurane	Patterson veterinary	07-893-1389
Ketamine	Patterson veterinary	07-890-8598
Phospate buffered saline (PBS)	ThermoFisher scientific	AM9624
Sodium fluorescein salt	Sigma Aldrich	46970
Surgical blade (scapel blade)	Fine Science tools	10022-00
Trephine	Integra Miltex	33-31
TritonX -100	Fisher Scientific	50-295-34
Forcep	Fine Science tools	11923-13
Xylazine	Patterson veterinary	07-808-1947