$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animals
- Maintain TgBAC (gfap:gfap-GFP) zebrafish 167 (AB) strain aged between 6 - 9 months under standard conditions in water with a temperature of 26.5 °C and a 14/10 h light/dark cycle.
2. Reversible Systemic Anesthesia
- Prepare the stock solution of ethyl 3-aminobenzoate methanesulfonate salt (tricaine) by dissolving 400 mg of tricaine powder in 97.9 mL of tank water and 2.1 mL of 1 M of Tris buffered saline (TBS). Adjust to pH 7.0 with 1 M Tris (pH 9) and store at 4 °C in the dark up to one month.
NOTE: Tricaine should be prepared in water like the natural living conditions of the animal, preferably original tank water should be used.
- Dilute the stock solution to 1:25 in tank water and use immediately.
- Place the zebrafish into a 10 cm Petri dish containing 50 mL of anesthesia solution until they become immobile and do not respond to external stimuli (approximately 2 - 5 min, depending on weight and age).
- Transfer each fish by hand to a custom-made silicone pin holder for laser treatment (Figure 1A).
CAUTION! The fish can remain anesthetized outside of the tank for up to 10 min only.
- To reverse anesthesia after the treatment and/or imaging, place the zebrafish in container containing tank water.
- To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water.
3. Laser Focal Injury on Retina
NOTE: A 532 nm diode laser is used to create focal light damage on the retina of the zebrafish. The experimental set-up of the laser enables the establishment of a reproducible focal retinal injury in adult zebrafish.
- Set up the output power of the laser: 70 mW; aerial diameter: 50 µm; pulse duration: 100 ms.
CAUTION! The use of laser light requires appropriate personal protection and labelling of the area.
- Apply 1 - 2 drops of 2% hydroxypropylmethylcellulose topically in the eye before treatment and use a 2.0 mm fundus laser lens to focus the laser-aiming beam on the retina.
CAUTION! hydroxypropylmethylcellulose drops are viscous and may cause problems in breathing if it goes on the gills.
- Place four laser spots around the optic nerve on the left eye and use the right, untreated eye as internal control.
