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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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myogenic+precursor+cell+that+could+contribute+to+regeneration+in+zebrafish+and+its+similarity+to+the+satellite+cell.">A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell.</a> The FEBS Journal. 280 (17), 4074-4088 (2013).</li><li>Corona, B. T., Wenke, J. C., Ward, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+volumetric+muscle+loss+injury.">Pathophysiology of volumetric muscle loss injury.</a> Cells Tissues Organs. 202 (3-4), 180-188 (2016).</li><li>Pfefferli, C., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+of+fin+regeneration+in+zebrafish.">The art of fin regeneration in zebrafish.</a> Regeneration. 2 (2), 72-83 (2015).</li><li>Sehring, I. M., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+understanding+fin+regeneration+in+zebrafish.">Recent advancements in understanding fin regeneration in zebrafish.</a> WIREs Developmental Biology. 9 (1), 367 (2020).</li><li>Poss, K. D., Wilson, L. G., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration+in+zebrafish.">Heart regeneration in zebrafish.</a> Science. 298 (5601), 2188-2190 (2002).</li><li>Sanz-Morej&#243;n, A., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+zebrafish+cardiac+regeneration.">Recent insights into zebrafish cardiac regeneration.</a> Current Opinion in Genetics &amp; Development. 64, 37-43 (2020).</li><li>Chassot, B., Pury, D., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+fin+regeneration+after+cryoinjury-induced+tissue+damage.">Zebrafish fin regeneration after cryoinjury-induced tissue damage.</a> Biology Open. 5 (6), 819-828 (2016).</li><li>Chablais, F., Veit, J., Rainer, G., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+heart+regenerates+after+cryoinjury-induced+myocardial+infarction.">The zebrafish heart regenerates after cryoinjury-induced myocardial infarction.</a> BMC Developmental Biology. 11, 21 (2011).</li><li>Schnabel, K., Wu, C. C., Kurth, T., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+cryoinjury+induced+necrotic+heart+lesions+in+zebrafish+is+associated+with+epicardial+activation+and+cardiomyocyte+proliferation.">Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation.</a> PLoS One. 6 (4), e18503 (2011).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138 (9), 1663-1674 (2011).</li><li>Ryan, R., Moyse, B. R., Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+regeneration-Looking+beyond+cardiomyocytes+to+a+complex+microenvironment.">Zebrafish cardiac regeneration-Looking beyond cardiomyocytes to a complex microenvironment.</a> Histochemistry and Cell Biology. 154 (5), 533-548 (2020).</li><li>Ja&#378;wi&#324;ska, A., Sallin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+versus+scarring+in+vertebrate+appendages+and+heart.">Regeneration versus scarring in vertebrate appendages and heart.</a> Journal of Pathology. 238 (2), 233-246 (2016).</li><li>Alexander, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+muscle+fibres+in+the+myomeres+of+fishes.">The orientation of muscle fibres in the myomeres of fishes.</a> Journal of the Marine Biological Association of the United Kingdom. 49, 163-290 (1969).</li><li>Morin-Kensicki, E. M., Melancon, E., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+relationship+between+somites+and+vertebral+column+in+zebrafish.">Segmental relationship between somites and vertebral column in zebrafish.</a> Development. 129 (16), 3851-3860 (2002).</li><li>Berberoglu, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite-like+cells+contribute+to+pax7-dependent+skeletal+muscle+repair+in+adult+zebrafish.">Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish.</a> Developmental Biology. 424 (2), 162-180 (2017).</li><li>Montandon, M., Currie, P. D., Ruparelia, A. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Program</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>Material/ Equipment</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>Dyes and Antibodies</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>Recipies/Solutions</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 Na2HPO4</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1.8 mM KH2PO4</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 H2O (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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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  

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...