Modeling Retinal Degeneration and Regeneration in Zebrafish Using a Focal Laser Injury

Begin with an anesthetized adult zebrafish.

Transfer the fish to a silicone pin holder and apply a layer of synthetic polymer to lubricate the eye.

Next, position a laser lens in front of the eye to focus a laser-aiming beam on the retina.

Direct the laser onto the eye of the zebrafish, inducing injury to the retina.

The laser induces thermal damage and the death of retinal photoreceptor cells, initiating a degenerative process that progresses over days and extends from the retinal pigmented epithelium to the outer plexiform layer.

In response, Muller glia (MG) cells undergo reactive gliosis, becoming hypertrophic and proliferative.

These cells then de-differentiate into stem cell progenitors.

The progenitor cells gradually migrate to the injury site and, guided by spatial and temporal cues from the microenvironment, differentiate into mature retinal cell types over several days, thereby facilitating the regeneration of the retinal structure.

Prepare a stock solution of anesthetic by dissolving 400 milligrams of tricaine powder in 97.9 milliliters of tank water and 2.1 milliliters of one molar TBS. Adjust to pH 7.0 with one molar tris at pH 9. To make the working solution, dilute the tricaine stock solution 1 to 25 in tank water, and transfer 50 milliliters to a Petri dish. Then place the zebrafish into the anesthetic solution for 2 to 5 minutes, until they become immobile and do not respond to external stimuli.

Transfer each fish by hand to a custom-made silicone pin holder for laser treatment.

Appropriate anesthesia is critical for the well-being of the animal and the success of procedure. Therefore, freshly prepared tricaine solution is pivotal, and the time out of the water should not far exceed 10 minutes.

Set up the output power of the 532-nanometer diode laser to 70 milliwatts, the pulse duration to 100 milliseconds, and the aerial diameter to 50 microns. Then, apply 1 to 2 drops of 2% hydroxypropyl methylcellulose to the eye.

When applying methylcell to the eye, ensure that the viscous solution doesn't go on the gills.

Next, use a 2-millimeter fundus laser lens to focus the laser aiming beam on the retina. Place four laser spots around the optic nerve on the left eye, and use the right, untreated eye as internal control.