Name: Video: Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury
Uploaded: 2023-07-07T12:10:00
Duration: 3 min 18 s
Description: 315 Views. Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury

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

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  

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Research

JoVE Journal

Biology

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

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.