A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish

Zebrafish models can help us understand the natural mechanisms of efficient organ regeneration. Our laboratory has previously established a cryoinjury method for studying how zebrafish restore their heart and fins. With this new protocol, we aim to expand our research into another organ, namely the musculature.

We have yet to understand why organ regeneration is more efficient in zebrafish than in humans. This protocol provides a promising approach to studying the successful restoration of musculature, despite a relatively large wound size in the fish body. This cryoinjury model has at least three advantages.

Firstly, the musculature is damaged rapidly and without disrupting the body's integrity. Secondly, the wound is reproducible and cleared by natural healing. And finally, fish can swim normally after cryoinjury, suggesting a mild impact of this procedure on the animals.

Our findings introduced the zebrafish caudal peduncle into the field of regenerative biology. This research path will contribute to the deciphering the molecular mechanisms that guide the myogenesis program to reconstruct complex musculature in adult zebrafish. For performing the myo-mere cryoinjury on zebrafish, use a custom manufactured stainless steel cryoprobe.

To avoid frostbite while handling the probe during the procedure, insert the probe handle into a pipette tip and secure it by wrapping it with tape. Before beginning with the cryoinjury procedure, prepare a beaker for the anesthesia working solution, a spoon for handling the fish, a moist sponge, and a tank with system water for post procedure recovery of the fish. Prepare the anesthesia stock solution following the steps shown on the screen.

Then add four milliliters of the prepared stock solution to 100 milliliters of system water in a beaker to prepare the tricaine working solution. To begin the cryoinjury procedure, immerse the probe in liquid nitrogen for a minimum of three minutes. Wet the sponge in system water and place it on a flat surface.

Then, one to two minutes after transferring a single adult zebrafish into the tricaine working solution, confirm its unresponsiveness by touching the fish gently with the spoon. Place the anesthetized fish onto the wet sponge. Locate the caudal peduncle posterior to the anal fin and anterior to the caudal fin.

At this stage, remove the cryoprobe from the liquid nitrogen. Shake the probe gently to ensure no residual liquid nitrogen remains on the tip. Place the edge of the spatula perpendicular to the body on the caudal peduncle.

Keep the probe in this position for six seconds without applying pressure before releasing the cryoprobe from the tissue, then transfer the fish into the system water in the recovery tank. Monitor the fish while it resumes breathing and swimming after waking up from the anesthesia. If auricular movements do not occur after 30 seconds, stimulate the fish by pipetting system water into the gills until the animal initiates respiration by itself.

Video recording of control and cryoinjured fish at one day post cryoinjury or DPCI, revealed that although the cryoinjured fish swam less actively, they did not display any abnormal movements such as swirling, convolution, or reduced equilibrium. In the husbandry system, their position in the tank and food intake was similar to those of the uninjured fish. As observed at five DPCI, normal behavior was still persistent, indicating the cryoinjury procedure did not severely affect the wellbeing of the animals.

To begin, prepare two milliliters of 4%formalin or any other appropriate fixative. Also keep a petri dish, forceps, and surgical scissors ready before starting the procedure. Place the properly euthanized cryoinjured fish in a petri dish containing the ionized water.

Using scissors, perform a cut through the body, anterior and posterior to the caudal peduncle. Let the tissue bleed out in the deionized water inside the petri dish. Using forceps, collect and transfer the caudal peduncle into the prepared fixative solution in a micro centrifuge tube.

Carefully invert the tube several times. Before mounting, wash the fixed tissue in PBS for 10 minutes on a rocker. Then transfer it into a two milliliter micro centrifuge tube containing a solution of 30%sucrose in deionized water, pre-cooled to four degrees Celsius, and gently invert the tube several times.

Leave the tube upright at four degrees Celsius for at least 24 hours. Fill an embedding mold with a five millimeter layer of OCT mounting medium. Using forceps, place the caudal peduncle at the bottom of the mold, adjust its orientation for either transversal or coronal sections.

Let the medium freeze in a box of dry ice. As soon the tissue is stabilized in the desired position, fill up the rest of the mold before the OCT completely freezes over. Store the mold at minus 80 degrees Celsius for at least one hour before sectioning the tissue using a cryostat.

The extent of cryoinjury at different days post cryoinjury or DPCI was analyzed in the caudal peduncle sections using tri-chrome staining, composed of anolon blue, acid fixin, and orange G.The damaged areas were determined by the absence of orange staining. At four and seven DPCI, the transverse section displayed extensively degenerated skeletal muscle in the cryoinjured flank of the body. Immunofluorescence analysis showed that at four DPCI, the injured side of the caudal peduncle contained abundant DAPI positive cells, but little to no F-actin and tropomyosin 1, indicating degenerated muscles.

At seven DPCI, tropomyosin 1 and F-actin could be detected in the wound close to the vertical body midline, indicating the start of new myofiber formation. At 30 DPCI, both sides of the body displayed a similar distribution of f-acton staining, indicating efficient skeletal muscle restoration.