We thank V. Zimmermann for fish care, as well as Dr. Thomas Bise, Dr. Catherine Pfefferli, and Lea Gigon for the initiation of this project and their preliminary results. This work was supported by the Swiss National Science Foundation, grant number 310030_208170.

This study was conducted in agreement with all the relevant ethical regulations. The zebrafish were bred, raised, and maintained in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines25. The animal housing and all experimental procedures were approved by the cantonal veterinary office of Fribourg, Switzerland.
1. Equipment and setup
<ol>
	<li>Arrange the production of a stainless steel cryoprobe in a technical workshop that can manufacture instruments for research. 
	NOTE: Provide a design of the instrument with the specific dimensions (Figure 1A).</li>
	<li>To avoid frostbite while handling the probe during the procedure, insert the handle into a pipette tip, and wrap it with tape.</li>
	<li>Prepare a beaker for the anesthesia working solution, a spoon for handling the fish, a moist sponge, and a tank with system water to let the fish recover after the procedure.</li>
	<li>Prepare the working solution freshly before every experiment. To prepare the working solution, add 4 mL of Tricaine stock solution to 100 mL of system water in a beaker. 
	NOTE: The anesthesia stock solution consists of 4 g of Tricaine dissolved in 980 mL of distilled water. After adjusting the pH to 7 with 1 M Tris-HCl, pH 9, fill up the solution to 1 L with distilled water. The stock solution is light-sensitive and should be stored at 4 &#730;C in an amber bottle.</li>
</ol>
2. Muscle cryoinjury procedure
<ol>
	<li>Begin the procedure by immersing the probe in liquid nitrogen for a minimum of 3 min. Wet the sponge in system water, and place it on a flat surface.</li>
	<li>Transfer a single adult zebrafish into the Tricaine working solution, and confirm its unresponsiveness by touching the fish gently with the spoon after 1 min or 2 min. If the fish is still reactive, wait a bit longer.</li>
	<li>Place the anesthetized fish onto the wet sponge. Locate the caudal peduncle posterior to the anal fin and anterior to the caudal fin.</li>
	<li>Remove the cryoprobe from the liquid nitrogen. Shake the probe gently to ensure no residual liquid nitrogen remains on the tip.</li>
	<li>Place the edge of the spatula perpendicularly to the body on the caudal peduncle (Figure 1C). Keep the probe in this position for 6 s without applying pressure (Figure 1D). 
	NOTE: The weight of the cryoprobe is sufficient to ensure contact between the instrument and the tissue.</li>
	<li>Release the cryoprobe from the tissue, and transfer the fish into the tank with system water.</li>
	<li>Monitor the fish while it resumes breathing and swimming after waking up from the anesthesia. If opercular movements do not occur after 30 s, stimulate the fish by pipetting system water into the gills until the animal initiates respiration by itself. 
	NOTE: The fish should resume swimming within a few minutes in the recovery tank. 
	&#8203;NOTE: In the following days, the fish can be filmed to monitor their swimming activity (Video 1 and Video 2). Before the video recording, transfer the control and injured fish into a translucent mating tank, and let them habituate for at least 1 min.</li>
</ol>
3. Caudal peduncle collection and fixation
<ol>
	<li>Prepare 2 mL of 4% formalin or other fixative that is suitable for the subsequent assays, a Petri dish, forceps, and surgical scissors.</li>
	<li>Euthanize the fish at a selected time point after the cryoinjury according to the legal permission given by the regional veterinary office. 
	NOTE: Euthanasia is performed by an overdose of tricaine solution (300 mg/L) for approximately 10 min. The loss of gill movement and the tail fin pinch reflex confirms death. The time points of euthanasia should be selected according to the phase of regeneration: for example, from 1 day post-cryoinjury (dpci) to 3 dpci for wound clearance; from 3 dpci to 10 dpci for the initiation of muscle regeneration; from 10 dpci to 30 dpci for progressing regeneration; and after 30 dpci for the completion of tissue restoration. Thus, to assess the different steps and biological processes of muscle degeneration and regeneration, groups of fish need to be euthanized at various time points after cryoinjury.</li>
	<li>Place the euthanized fish in a Petri dish containing deionized water. Use scissors to perform a cut through the body anterior and posterior to the caudal peduncle (Figure 1E). Let the tissue bleed out in the deionized water of the Petri dish.</li>
	<li>Collect the caudal peduncle, and transfer it with forceps into the prepared fixative solution in a microcentrifuge tube. 
	&#8203;NOTE: Carefully invert the tubes several times, and keep them overnight at 4 &#730;C.</li>
</ol>
4. Mounting the caudal peduncle
<ol>
	<li>Wash the fixed tissue in 1x PBS for 10 min on a rocker. Then, transfer it into a 2 mL microcentrifuge tube with pre-cooled 30% sucrose in deionized water at 4 &#730;C, and gently invert the tube several times. Leave the microcentrifuge tubes for a minimum of 24 h at 4 &#730;C in an upright position. 
	NOTE: The caudal peduncle will float on top of the sucrose solution and begin to sink as the tissue dehydrates.</li>
	<li>Fill an embedding mold with a 5 mm layer of O.C.T. mounting medium. Use forceps to adjust the caudal peduncle in the medium. Place it at the bottom of the mold, and adjust its orientation for either transversal or coronal sections (Figure 1F).</li>
	<li>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 O.C.T. completely freezes over. Store the mold for a minimum of 1 h at &#8722;80 &#730;C. 
	&#8203;NOTE: Under these conditions, the tissues can be stored for many months.</li>
</ol>
5. Cutting the sections with a cryostat
<ol>
	<li>Set up a cryostat with a cutting thickness of 25 &#181;m. Adjust the chamber temperature to &#8722;26&#730;C, the specimen temperature to &#8722;24 &#730;C, and the cutting angle to 12&#730;.</li>
	<li>Place the frozen block with the specimen in the cryostat, and fix its orientation in order to cut parallel to the bottom of the block. Cut until you reach the specimen, and then trim the block to ease the sample collection.</li>
	<li>Prepare six adhesion slides. Label the slides with consecutive numbers in order to prepare replicates of the sample.</li>
	<li>Start cutting, and collect the tissue sections on the labeled adhesion slides. Arrange the sections on the slides as per the further experimental demands.</li>
	<li>Let the slides dry for 1 h at room temperature. Store them in closed boxes at &#8722;20 &#176;C. 
	NOTE: The slides can be safely stored in this condition for up to 1 year.</li>
</ol>

Caudal Peduncle Cryoinjury in Adult Zebrafish

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The authors have nothing to disclose.

The zebrafish provides an anamniote vertebrate model organism to study the mechanisms of muscle regeneration. Most of the existing injury methods, such as laser ablation or stab wounding, result in relatively minor tissue disruption20,21,22,23. Major resections have been conducted on extraocular muscle26. However, this surgical approach would probably be less appropriate for the lateral musculature due to the health hazards of cutting the body wall. To avoid such invasive procedures, this protocol describes a milder form of injury that, nevertheless, results in profound damage to the caudal peduncle. This approach relies on a superficial manipulation that allows for the very precise targeting of a few myomeres on one side of the body. The strengths of the cryoinjury model lie in its reproducibility and ability to produce extensive muscle degeneration; based on these strengths, this model provides a new path to study how the body reacts to significant muscle loss.
The application of extreme cold leads to a thermal shock, which destroys the plasma membrane and organelles in the affected muscle tissue27. As a result, the injured myofibers undergo &#34;accidental&#34; cell death28. Consequently, the damaged tissue can be resorbed by natural mechanisms of wound clearance. Zebrafish tolerate the cryoinjury procedure well, as the survival rate in this study was nearly 100%, given the precooled probe was correctly positioned on the body for the exact duration. However, if the wound is too extensive (e.g., if too much pressure is applied or the duration of cryoinjury is too long), the fish may display aberrant swimming movements shortly after the procedure, and the animal should be euthanized as a humane endpoint. For other fish species, the exposure time to the cryoprobe should be adjusted according to the size of the body.
After cryoinjury, the fish can resume their swimming activity without any symptoms of abnormal movement. However, cryoinjured fish swim less dynamically than control fish, which indicates some mild impairments. Further quantification of the fish behavior at different time points after cryoinjury will be necessary to determine temporal changes in the swimming performance.
The effect of the cryoinjury method on other non-muscle tissues of the caudal peduncle remains to be elucidated. Obviously, the outer-most body layer (i.e., the skin) is damaged by the procedure. In this context, the cryoinjury method can provide a new strategy to study wound healing, scale regeneration, and the restoration of the pigmentation pattern. Furthermore, the vasculature and innervation of the myomeres could also be affected by cryoinjury, and these topics require further investigation.
The cryoinjury model has been previously used to investigate zebrafish heart regeneration13,14,15,29. This method showed some advantages compared to the ventricular resection method10 due to the transient deposition of a collagen-rich scar, which better mimics the infarct healing response in humans30. Remarkably, zebrafish can regenerate their heart after multiple cryoinjuries31. Interestingly, cryoinjury has also been applied to the zebrafish fin, resulting in histolytic processes12. As opposed to the classical fin amputation, the remaining cryoinjured stump contains a distorted margin with a mixture of dead material and healthy cells. Studies with both zebrafish organs, the heart and the fin, have revealed the powerful capacity of zebrafish to restore their original functional components even after extensive tissue damage. Whether the cryoinjured skeletal muscle activates an interplay between reparative and regenerative processes warrants future studies.

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.

<table><tbody><tr><td>&lt;strong&gt;Program&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health (NIH)</td><td></td><td></td></tr><tr><td>Photoshop Version 23.5.3</td><td>Adobe</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Material/ Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>35/10 mm Petri Dish</td><td>Greiner Bio-one</td><td>Item No.: 627102</td><td></td></tr><tr><td>Camera</td><td>Sony</td><td>/</td><td>HDR-PJ410</td></tr><tr><td>Cryostat</td><td>Histcom</td><td>HRA C50</td><td></td></tr><tr><td>Formaldehyde ~36%</td><td>Sigma-Aldrich</td><td>47630</td><td></td></tr><tr><td>Macro 50 mm f/2.8 EX DG lens</td><td>Sigma</td><td>/</td><td>Discontinued lense</td></tr><tr><td>Peel-A-Way Embedding Truncated Molds T8</td><td>Polyscience, Inc.</td><td>18985</td><td></td></tr><tr><td>Slides Superfrost Plus</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Sponge</td><td>any</td><td>any</td><td>flat sponge, c.a. 7cm x 3 cm x 1 cm</td></tr><tr><td>Stainless steel cryoprobe</td><td>Custom-made</td><td>/</td><td>specifics in the article</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84100</td><td></td></tr><tr><td>Surgical scissors</td><td>Any</td><td>/</td><td></td></tr><tr><td>TCS SP2</td><td>Leica</td><td>/</td><td>Discontinoued product</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Tricaine (Anestethic)</td><td>Sigma</td><td>E10521</td><td></td></tr><tr><td>&lt;strong&gt;Dyes and Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dapi</td><td>Sigma</td><td>10236276001</td><td>Concentration: 1/2000</td></tr><tr><td>Phalloidin-Atto-565 (F-actin)</td><td>Sigma</td><td>94072</td><td>Concentration: 1 / 500</td></tr><tr><td>Tropomyosin (TPM1)</td><td>DHSB</td><td>CH1</td><td>Concentration: 1 / 50</td></tr><tr><td>&lt;strong&gt;Recipies/Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS</td><td>123 mM NaCl</td><td>Sigma</td><td></td></tr><tr><td></td><td>2.7 mM KCl</td><td>Sigma</td><td></td></tr><tr><td></td><td>10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td></td></tr><tr><td></td><td>1.8 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td></td></tr><tr><td>AFOG solution</td><td>3 g Fuchsin</td><td>Fisher Scientific</td><td></td></tr><tr><td></td><td>2 g Orange G</td><td>Sigma</td><td></td></tr><tr><td></td><td>1 g Anilin blue</td><td>Fulka AG</td><td></td></tr><tr><td></td><td>200 ml acifidied distilled H&lt;sub&gt;2&lt;/sub&gt;O (pH 1.1)</td><td></td><td></td></tr></tbody></table>

a cryoinjury model for studying skeletal muscle regeneration of the caudal peduncle in adult zebrafish

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.

Monitoring of the fish after cryoinjury 
To determine the effect of myomere cryoinjury on animals, a video recording of control and cryoinjured fish at 1 days post-cryoinjury (dpci) and 5 dpci was performed. Each group contained five fish. At 1 dpci, the cryoinjured fish were swimming less actively, but they did not display any abnormal movements, such as swirling, convolution, or reduced equilibrium (Video 1). In the husbandry system, their position in the tank and food intake were similar to those of the uninjured fish. Normal behavior persisted throughout the following days, as exemplified by the video at 5 dpci (Video 2). In conclusion, the cryoinjury procedure of the caudal peduncle did not severely affect the well-being of the animals.
Histological analysis of the caudal peduncle sections 
To assess the extent of the injury, the time point of 4 dpci was selected, as this is when the myofiber debris has been completely resorbed in the wound. To analyze the effects of cryoinjury along the dorso-ventral and anterior-posterior axes of the body, two groups of fish were used (i.e., coronal and transversal sections of the caudal peduncle, respectively) (Figure 1F).
The sections were analyzed by trichrome staining composed of Aniline Blue, Acid Fuchsin, and Orange G (AFOG). Using this combination of reagents, intact muscles were shown in orange, the spinal cord in dark red, and the collagenous matrix in blue. To determine the number of damaged myomeres, which are the metameric units of the fish musculature, a series of sections were analyzed (Figure 2). Myomere boundaries, called myocommata, were identified by collagen deposition, as detected by blue coloration. The damaged areas were determined by the absence of orange staining. A closer examination of specimens with evident myocommata revealed that approximately four consecutive myomeres were damaged, as inferred from the lack of orange staining (n, number of fish = 4; Figure 3A,A&#39;). The uninjured side of the same fish served as the internal reference.
To examine the depth of the wound perpendicular to the body axis, transversal sections were prepared using zebrafish at 4 dpci and 7 dpci. The latter time point corresponds to the activation of the myogenic program and, thus, the onset of muscle regeneration. AFOG staining of these specimens displayed an extensive lack of orange staining in the cryoinjured flank of the body, demarcating the zone of degenerated skeletal muscle (Figure 3B,C). At 4 dpci and 7 dpci, the wound area spanned tissues from the skin toward the vertical septum. This demonstrates that the cryoinjury method deeply targeted one lateral half of the caudal peduncle, which remained devoid of functional muscle for 7 days after the procedure. Taken together, four myomeres were profoundly damaged on one side of the caudal peduncle.
Immunofluorescence analysis of the transversal sections 
To assess the dynamics of muscle regeneration, experimental groups of fish were euthanized at 4 dpci, 7 dpci, 10 dpci, and 30 dpci. The transversal sections of the caudal peduncle were labeled by multicolor fluorescence staining using Phalloidin (which binds to filamentous actin [F-actin]), Tropomyosin-1 antibody, which detects a sarcomere protein, and DAPI, which labels nuclei. At all the time points, the uninjured half of the body provided an internal control; both F-actin and Tropomyosin 1 were strongly detected in the uninjured control parts, indicating undamaged tissue (Figure 4).
At 4 dpci, the injured side of the caudal peduncle contained abundant DAPI-positive cells, but little to no F-actin and Tropomyosin 1 immunofluorescence was observed, indicating the wound zone with degenerated muscles (Figure 4A-B&#39;). At 7 dpci, Tropomyosin 1 and F-actin could be detected in a part of the wound close to the vertical body midline (Figure 4C-D&#39;). This expression pattern demarcates the position where the formation of new myofibers begins in the caudal peduncle. At 10 dpci, both muscle markers expanded toward the surface of the body, suggesting progressing regeneration of the skeletal muscle (Figure 4E-F&#39;). At 30 dpci, both sides of the body displayed a similar distribution of F-actin staining (Figure 4G-H&#39;). This finding indicates that the skeletal muscle was efficiently restored after the cryoinjury of the caudal peduncle.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65491/65491fig01.jpg" /> 
Figure 1: Experimental setup for myomere cryoinjury. (A) Dimensions of the custom-manufactured cryoprobe made of stainless steel. The distal part of the instrument consists of a spatula with a concave edge at the depth of 1 mm to account for the curvature of the zebrafish body. The middle part of the tool comprises a cylinder that functions as a weight and a reservoir to maintain the low temperature of the spatula during the procedure. The proximal end of the instrument is in the form of a thin metal handle. (B,C) Anesthetized adult fish on a moist sponge with the cryoprobe on the caudal peduncle. The probe was at room temperature. (B) The margin of the probe is placed horizontally in the vicinity of the caudal peduncle to display the relative size between the fish and the tool. (C) For cryoinjury, the tip of the tool is positioned perpendicular to the fish. (D) Schematic illustration of the cryoinjury procedure from the ventral side of the fish to show the manipulations in a comprehensive manner. The cryoprobe was precooled in liquid nitrogen and immediately placed on one side of the fish for 6 s. (E) At a specific time point after cryoinjury, the fish were euthanized, and their caudal peduncles were collected for fixation. (F) The fixed material was histologically processed and sectioned along the coronal or transversal planes. <a href="https://www.jove.com/files/ftp_upload/65491/65491fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65491/65491fig02.jpg" /> 
Figure 2: Histological analysis of damaged myomeres in the caudal peduncle from the dorsal to the ventral position of the body. AFOG staining of a series of coronal sections at 4 days post-cryoinjury (dpci). The sections are from the dorsal toward the ventral side, as indicated on the top of the first and the last panel. The sections are non-adjacent, with an interval of approximately 150 &#181;m between them. The uninjured muscle is detected by orange staining of the muscle, whereas the injured tissue lacks this staining and appears grayish (area encircled with a dashed line). Collagen-containing tissues, such as the skin, are stained in blue. The spinal cord appears as a rod-like structure and is stained in red. Number of fish, n = 4. <a href="https://www.jove.com/files/ftp_upload/65491/65491fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65491/65491fig03.jpg" /> 
Figure 3: Assessment of injury depth in the caudal peduncle using AFOG staining. (A,A&#39;) The coronal section is at the level of the spinal cord (a red-stained horizontal rod). The bottom image shows a magnified area encompassed with a frame in the upper image. The sequential myomere boundaries appear as collagenous stripes (blue) positioned obliquely to the spinal cord (red arrows in the magnified image A&#39;).(B,C) The cross-sections display the uninjured flank with orange-stained muscles and the cryoinjured flank with grayish staining. The damaged area is encircled with a black dashed line. The vertical septum (depicted with a red dashed line) subdivides the body into the control and cryoinjured sides. Number of fish, n = 4 per time point. <a href="https://www.jove.com/files/ftp_upload/65491/65491fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65491/65491fig04.jpg" /> 
Figure 4: Immunofluorescent detection of muscle proteins after cryoinjury. Fluorescence staining of cross-sections at 4 dpci, 7 dpci, 10 dpci, and 30 dpci, as labeled on the left side and the top of the panels (A-B&#39;). At 4 dpci, the injured tissue (encircled with the dashed line) is DAPI-positive (blue) but devoid of Phalloidin staining (green) or Tropomyosin-1 immunoreactivity (red), suggesting degeneration of the muscle fibers after cryoinjury. (C-D&#39;) At 7 dpci, both muscle markers progressively emerge in the wounded area, indicating the regenerative process. Tropomyosin-1 appears more intense than F-actin in the newly formed fibers. (E-F&#39;) At 10 dpci, the injury zone is filled with new myofibers that display a higher intensity of Tropomyosin-1 immunoreactivity in comparison to F-actin. (G-H&#39;) At 30 dpci, a similar pattern of myofibers is detected in both sides of the body. The frames in panels A, C, E, and H encompass the areas that are magnified in the adjacent images to the right. Dermal scales, emanating fluorescence outside of the myomere, were erased from the images using Adobe Photoshop.&#160;Number of fish, n = 4 per time point. <a href="https://www.jove.com/files/ftp_upload/65491/65491fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish at JoVE.com

Author Spotlight: A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle 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.

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

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

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Biology

2.0K Views.  University of Fribourg. 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. 

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

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Developmental Biology

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Medicine

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Neuroscience

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

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

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...