Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.

This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.

The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

As most of the epithelia are in contact with the external environment, they are prone to physical injury, making them well suited for the study of wound healing processes. Among the well-studied tissues, the cornea is an extremely useful model in the investigation of the cellular and molecular aspects of wound healing. As a transparent external surface, it provides physical protection to the eye and is the first element to focus the light onto the retina. While the structure and cell composition of the retina differ between species¹, these elements of the cornea are generally similar in all camera-type eyes, regardless of species.

All experiments were approved by the national animal experiment board.

1. Preparations

1. Prepare the tricaine stock solution used for anesthesia²⁶ in advance (0.4% stock solution used in this protocol). Use gloves and keep the materials in a fume hood whenever possible.
   1. For 50 mL of a 0.4% solution, weigh 200 mg of tricaine powder into a 50 mL tube. Dissolve the powder in approximately 45 mL of double-distilled water.
   2. Ad.......

This study describes a method using an ophthalmic burr in zebrafish corneal wound healing experiments. The method is modified from previous studies on mice, where the burr was shown to remove the epithelial cell layers efficiently¹³. The challenges in zebrafish corneal wounding include the relatively small size of the eye, and in the case of time-consuming experiments, the need to maintain a constant water flow through the gills (as described by Xu and colleagues²⁸). The ma.......

Corneal physical injuries are the most common cause of ophthalmology patient visits to the hospital. Therefore, it is important to establish relevant models for the study of different aspects of corneal pathophysiology. So far, the mouse is the most commonly used model for the study of corneal wound healing. However, adding eyedrops on murine wounded eyes to validate the impact of specific drugs on corneal wound healing can be difficult. In this respect, the zebrafish model is particularly useful for the pharmacological .......

The authors thank Pertti Panula for the access to the Zebrafish unit and Henri Koivula for the guidance and help with the zebrafish experiments. This research was supported by the Academy of Finland, the Jane and Aatos Erkko Foundation, the Finnish Cultural Foundation, and the ATIP-Avenir Program. Imaging was performed at the Electron Microscopy unit and the Light Microscopy Unit, Institute of Biotechnology, supported by HiLIFE and Biocenter Finland.