Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

Stab wound injury is a mechanical injury method that induces nonspecific cell ablation. This method facilitates the evaluation of RGC proliferation and differentiation after brain injury. Needle-mediated stab wound injury is a simple, efficiently-implemented method to induce brain injury.

This method can be applied to many experimental samples using a standard set of tools. The regenerative capacity of zebrafish was illustrated using the stab wound injury model. New insights into molecular mechanism regulating neuronal regeneration contribute to regenerative therapy in the central nervous system.

Begin by placing an anesthetized adult zebrafish or medaka on a styrofoam tray. Fix the fish upright between two 30-gauge needles inserted vertically into the styrofoam. Hold the fish body to prevent the fish head from moving, and then vertically insert a 30-gauge needle through the skull into the medial region of one of the optic tectum spheres.

Next, transfer the injured fish into a fish tank with fresh fish facility water. Once the fish have fully recovered from the anesthesia, move the tank to the breeding system. Put paper towels on the styrofoam tray to place the anesthetized fish.

Then hold the fish body in place by vertically inserting two 30-gauge needles on either side of the anal fin. Once the fish is placed, make a ventral incision from the anus to the heart. Keep the heart visible by inserting two 30-gauge needles on each side.

Then use the tip of the forceps to remove the silver epithelial layer. Insert the 30-gauge needle into the ventricle, keeping the bevel up, and make an incision into the atrium using the forceps. Next, push PBS into the atrium by pressing down on the syringe to flush the blood from the atrium.

When the blood is drained and the gills turn white, stop the PBS perfusion. Later, remove the needles fixing the fish body in place, and fix the fish ventral side down. After cutting the spinal cord and removing the skull from the optic tectum and telencephalon, cut the optic nerves and dissect the brain.

Next, transfer the brains into 1.5-milliliter tubes with one milliliter of 4%paraformaldehyde in PBS, to fix overnight at four degrees Celsius. After washing the fixed brains three times in PBS for five minutes each, transfer the brains into 1.5-milliliter tubes with one milliliter of 30%weight by volume sucrose in PBS as a cryoprotectant, and incubate them overnight at four degrees Celsius. Prepare the embedding compound by combining 30%sucrose with 30 milliliters of optimal cutting temperature, or OCT, compound, and store the compound at four degrees Celsius.

To cool the cryomold once, incubate an aluminum block overnight at minus 80 degrees Celsius, and put the pre-cooled block in a styrofoam box with a lid to prevent warming. For coronal sections, embed the dissected brain in a cryomold by adjusting the orientation using forceps under the microscope. Next, place the cryomold on the pre-cooled aluminum block in the styrofoam box, and allow the OCT compound to freeze and become white.

As the compound freezes, apply a small circle of OCT compound on a specimen disc before mounting the cryoblock on the specimen disc. By placing the specimen disc in the cryostat, freeze the OCT compound. Then place the specimen disc on the specimen head in the cryostat.

After setting the orientation of the cryoblock, trim the OCT to remove any extra regions. Cut 14-micrometer thick serial sections through the whole optic tectum using a cryostat, and store the slides at minus 25 degrees Celsius. The radial glial cell, or RGC, proliferation in the brain tissue of zebrafish was evaluated.

Most RGCs were proliferating cell antigen negative in the contralateral uninjured hemisphere. At two days post-injury, RGC proliferation was induced on the injured side of the medaka tectum. After the brain injury, cell lineage and RGC differentiation were evaluated with bromodeoxyuridine, or BrdU, labeling.

The representative analysis shows newborn neurons at seven days post-injury in the injured zebrafish and medaka. One of the critical steps in stab wound injuries is manual needle insertion. A consistent injury is necessary for creating reproducible results.

Fluorescent dyes are effective as wound markers. The extent of fluorescent dye diffusion indicates the location and depth of the stab wound injury. This technique is potentially adapted to other small fish species, and adaptation to medaka allows for comparisons of regenerative capacity.