collection, fixation, and mounting of caudal penduncle from zebrafish after cryoinjury

Caudal Peduncle Cryoinjury in Adult Zebrafish

In vertebrates, damaged parts of various tissues undergo homeostatic renewal and restoration during the lifespan. This capacity for renewal and restoration typically depends on the presence of competent stem cells or the proliferative capacity of the mature cells1,2. Skeletal muscle comprises post-mitotic myofibers, which are associated with local stem cells, called satellite cells3,4,5,6. Thus, this tissue contains cellular sources for the efficient sealing of areas of interrupted continuity or for the repair of minor wounds. However, larger volumetric losses in mammalian skeletal muscle are often followed by non-regenerative repair, such as fibrosis7. Animal models could provide new insights into the biological mechanisms that promote the regeneration of extensively damaged organs.
The zebrafish is a well-established model organism with a high regenerative capacity. Adult zebrafish can regenerate an amputated part of their caudal fin or the resected apex of the cardiac ventricle8,9,10,11. In addition, a cryoinjury method has previously been applied to study fin and heart regeneration in zebrafish12,13,14,15. In the case of inner organs, the cryoinjury method has the advantage of inducing cell death without disrupting the organ integrity, thus mimicking physiological conditions16,17. Tissue debris is disintegrated by natural clearance during wound healing, followed by the reparative processes. However, whether this method could be applied to skeletal muscle remains to be established.
In fish, the lateral musculature enables the side-to-side bending of the trunk during swimming18. The skeletal muscles are organized into metameric units, called myomeres, which are separated by connective tissue5,19. Zebrafish can regenerate their muscle after minor tissue disruptions, such as those caused by laser ablation or a stab wound20,21,22,23,24, but whether whole myomeres can regenerate after extensive injury remains unknown. This gap in knowledge is probably due to the lack of a suitable injury model. This protocol establishes a new approach to induce extensive injury of the skeletal muscle, spanning multiple myomeres. The described cryoinjury method is based on the rapid freezing and thawing of the myofibers with a precooled stainless-steel instrument. Despite the extensive damage, the well-being of the fish was not severely impaired. Entire myomeres could be restored, and, thus, this work provides a new model system for studying the mechanisms of musculature regeneration in adult zebrafish.

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

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 cryo injury method for studying how zebrafish restore their heart and fitness. 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 cryo injury 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 cryo injury, 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 deciphering the molecular mechanisms that guide the myogenesis program to reconstruct complex musculature in adult zebrafish.

Watch this Scientific Journal Video about Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury at JoVE.com

Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury  

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 deionized 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 microcentrifuge 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 microcentrifuge 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 4 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 as 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 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 trichrome staining composed of Aniline Blue, Acid Fuschin, and Orange-G. The damaged areas were determined by the absence of orange staining.At 4 and 7 dpci, the transverse section displayed extensively degenerated skeletal muscle in the cryoinjured flank of the body. Immunofluorescence analysis showed that, at 4 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 7 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-actin staining, indicating efficient skeletal muscle restoration.

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307 Views.  University of Fribourg. 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 deionized 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 int...

Video: Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury  

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

This protocol describes a cryoinjury model to induce profound damage of several caudal myomeres in adult zebrafish. This method provides a new approach for studying skeletal muscle regeneration after severe loss of tissue in non-mammalian vertebrates.

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the ...

Performing Muscle Cryoinjury on a Zebrafish

For performing the myomere 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 opercular 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-injury 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 5 DPCI, normal behavior was still persistent indicating the cryoinjury procedure did not severely affect the wellbeing of the animals.