We thank the Animal House and the Integrated Microscopy Facilities of IGB-CNR. This work has benefited from research funding from the European Community's Seventh Framework Programme in the project ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue, grant agreement number 241440), the Italian Ministry of Education-University-Research (MIUR-PRIN2 010-2011) to G.M. and S.B. and PON Cluster IRMI to G.M., and the CARIPLO foundation to G.M. and S.B.

All experiments were conducted in strict accordance with the institutional guidelines for animal research and approved by the Department of Public Health, Animal Health, Nutrition and Food Safety of the Italian Ministry of Health in accordance with the law on animal experimentation.&#160;Cervical dislocation procedures may vary from institution to institution based on IACUC or its equivalent requirements.
1. Cardiotoxin Injection in the Tibialis Anterior Muscle
<ol>
	<li>Prepare a 10 &#956;M working solution of cardiotoxin (CTX) by diluting the cardiotoxin stock solution in sterile Phosphate-Buffered Saline (PBS) or in water before starting the procedure. 
	Note: CTX is soluble in water at 1 mg/mL. Prepare a 70 &#956;M stock solution. Aliquot and store at -20 &#176;C. CTX used for this protocol is a mixture of cardiotoxins having a molecular weight of approximately 7,000 Da. 
	CAUTION: It is suggested to wear double gloves, a hygiene mask, and safety glasses (alternatively to safety glasses, it is recommended to work under a hood) and to be very careful to avoid any contact with the solution, even if diluted.</li>
	<li>Anesthetize the mice by intraperitoneal injection of ketamine and xylazine (80 mg/kg and 10 mg/kg of body mass) or other approved available anesthetic.</li>
	<li>With the mouse face up, spray the hind limbs with 70% ethanol. To visualize the exact location of the Tibialis Anterior (TA) muscle, with the help of a scalpel, cut the hair away at the anterior part of the lower leg, under the knee. 
	Note: The TA muscle arises from the proximal body of the tibia, going down lateral to the bone and ending at the ankle. Its long tendon extends into the foot across the ankle (see Figure 1A).</li>
	<li>Draw ~ 100 &#956;L of the solution into the syringe by pulling the plunger back slowly.</li>
	<li>Remove air bubbles, holding the syringe upright (with the needle upward). Gently pull the plunger down and tap the side of the syringe to help any air bubbles attached to the inner part of the tube come upwards. Slowly press the plunger to release large air bubbles. A few drops of liquid will come out the needle. At this point, make sure to pick the liquid up again into the tube and not disperse it, given the toxicity of cardiotoxin.</li>
	<li>Withdraw the solution into the syringe until the desired dose is reached. Inject 20 &#956;L of CTX solution for each TA muscle.</li>
	<li>Insert the needle in the center of the TA by following the position of the Tibia bone as guidance, from the tendon towards the knee. Since the TA is a thin tissue, perform the injection without going too deep (insert the needle 2/3 mm deep, at approximately a 10&#176; to 20&#176; angle ), and avoid going beyond the muscle itself (Figure 1A).</li>
	<li>Do not pull out the needle immediately after the injection, but wait 2 - 3 s to prevent leakage of CTX drops from the muscle.</li>
	<li>After the procedure, place the cage on a heated plate (at 37 &#176;C) until the mice are awake, since the anesthetic and the ethanol used reduce body temperature.</li>
</ol>
2. Tibialis Anterior Isolation
Note: Muscles can be isolated at different time points after cardiotoxin injection according to experimental requirements.
<ol>
	<li>Sacrifice the mouse by cervical dislocation.</li>
	<li>Spray the hindlimbs with 70% ethanol. During the procedure, the animal can be fixed to a support to hold it down.</li>
	<li>Cut the skin at the level of the proximal tendon (above the foot), insert one of the blades of the scissors under the skin, and cut the skin upwards, in the direction of the knee. With the help of forceps, pull up the skin and expose the muscle entirely. Carefully remove all of the skin and the hair from the area of interest.</li>
	<li>Eliminate the fascia by slowly pinching or cutting (with thin-tip tweezers or with fine micro dissecting scissors) the extreme periphery of the muscle on the opposite side of the tibia (Figure 1B). Once broken, gently remove the fascia with the help of thin-tip tweezers; avoid damaging the underlying muscle. 
	Note: As with other muscles and organs, the TA is covered by fascia (deep fascia of the leg), a thin sheet of connective tissue that lies deep under the skin and that needs to be removed to easily isolate the underlying muscle.</li>
	<li>Visualize the distal TA tendon and the Extensor Digitorum tendon above the foot, which are juxtaposed. The tendon of the Extensor Digitorum Longus (EDL) muscle is located slightly below the TA tendon. Tendons of adult mice are visible to the naked eye.</li>
	<li>Remove the TA muscle following one of the two following techniques:
	<ol>
		<li>Cut both tendons with fine micro dissecting scissors and pull them towards the proximal side, holding the tendons with forceps. Separate the two tendons, pulling them gently in opposite directions to separate the muscles.</li>
		<li>With the help of thin-tip tweezers, separate the distal TA tendon from the Extensor Digitorum tendon by passing the tip below the tendon itself (Figure 1C, upper picture). Gently slide the tweezers below the TA to separate it from the underlying muscles (Figure 1C, bottom picture).</li>
		<li>Hold the tweezers under the muscle to keep it slightly raised, cut the TA tendon, and gently pull it upwards until only the area below the knee is attached (Figure 1D, left picture). Cut the TA below the knee, following the edge of the muscle with the scissors (Figure 1D, right picture). 
		Note: If the muscle remains slightly attached to the sides, use fine micro dissecting scissors to help detach it. 
		Note: If the forceps do not easily fit below the TA, it is possible that the fascia has not been completely removed, and it could still be visible on top of the muscle. In this case, completely remove the fascia before proceeding.</li>
	</ol>
	</li>
</ol>
3. Fresh Frozen Muscle Technique
<ol>
	<li>Place a small amount of tragacanth gum or a similar adhesive freezing compound (e.g., Optimal Cutting Temperature (OCT) compound) on a slice of cork. Label the cork&#39;s slice on the reverse side before freezing, if necessary (Figure 2A).</li>
	<li>In order to obtain transverse sections of the muscle, sink the TA into the tragacanth gum by inserting the distal tendon and leaving about 3/4 of the muscle outside, making sure to have the muscle in a perpendicular position with respect to the cork (Figure 2A).</li>
	<li>Fill an aluminum can (alternatively, a metal cup or a glass beaker), with isopentane. Suspend the can of isopentane in a Dewar containing liquid nitrogen, not allowing the liquid nitrogen to enter into the can. 
	Note: Isopentane needs to reach the proper temperature (from -150 to -160 &#176;C) for optimal freezing of the tissue. The correct temperature is reached when some white solid particles form at the bottom of the can and the isopentane becomes slightly viscous. 
	Note: Do not freeze the sample before the formation of the solid particles, as it can lead to freezing artifacts. On the other hand, if the isopentane becomes completely solid, leave it at room temperature until it is thawed, making sure that some white particles are still present.</li>
	<li>Using Mixter forceps, rapidly dip the cork with the muscle into the isopentane, keeping the muscle positioned downwards, and make sure to release the cork when it is completely immersed in the liquid. In this way, while the specimen floats, only the reverse side of the cork should be visible in the liquid.</li>
	<li>Leave the sample in the isopentane for 1 min.</li>
	<li>Using Mixter forceps (or similar forceps), take the muscle and immerse it in liquid nitrogen for at least 2 min.</li>
	<li>Wrap the specimens individually in labeled aluminum foil and immediately place them in dry ice (-70 &#176;C), or directly store them in a -80 &#176;C freezer. 
	Note: Throughout the procedure, from steps 3.4 to 3.7, it is extremely important to maintain the correct temperature and to prevent the specimens from thawing (e.g., during the transfer to the -80 &#176;C freezer). The rising temperature results in uncontrolled thawing and refreezing of the water microcrystals that are present within the tissue and eventually causes the formation of artifacts or the breakdown of muscle fibers (visualized on the tissue sections as large white holes in the center of the fiber).</li>
</ol>
4. Cryostat Sectioning of Frozen Muscles
<ol>
	<li>Set the cryostat chamber temperature to -20 to -22 &#176;C.</li>
	<li>Put the specimen stub, the blade, and the specimen in the cryostat chamber, allowing them to equilibrate for at least 30 min.</li>
	<li>Place a small amount of freezing compound (e.g., OCT compound) on the specimen stub and immerse the cork with the specimen before the freezing compound solidifies. In the meantime, place the blade in the blade holder.</li>
	<li>Align the specimen and the blade using the provided screws to orient the specimen holder with the blade holder (Figure 2B).</li>
	<li>Set the section thickness to 10 &#956;m. Proceed to cut the tissue. For each turn of the drive wheel, the specimen holder advances a controlled distance towards the blade.</li>
	<li>Place the sections on a polarized slide (8 to 10 section per slide) (Figure 2C).</li>
	<li>After sectioning, store the slides at -80 &#176;C. 
	Note: The specimen can be stored in a -80 &#176;C freezer and reused for further sectioning, if properly handled and stored.</li>
</ol>
5. Routine Histological Staining (Hematoxylin &#38; Eosin Stains)
Note: Several histological stains can be performed on muscle sections according to the analysis. A routine histological stain for morphological and morphometric analysis is the Hematoxylin &#38; Eosin (H&#38;E) bichromic stain. The hematoxylin stains the nuclei a deep purple. Nuclear staining is counterstained with eosin (pink/red), which stains eosinophilic structures, such as the myofibers in the cytoplasm.
<ol>
	<li>Completely air-dry frozen slides with sections for 10 - 15 min at room temperature, lodge the slides in a staining tray, and put the tray in a staining trough.</li>
	<li>Stain for 1 min with hematoxylin.</li>
	<li>Lay the staining trough under a fairly weak tap water jet for 10 min to wash out the hematoxylin.</li>
	<li>Immerse the slides for 5 min in 95% ethanol, if an alcoholic solution of eosin will be used (skip this step in case of an aqueous eosin solution).</li>
	<li>Counterstain for 1 min with eosin solution.</li>
	<li>Rinse with water to eliminate excessive eosin solution. 
	Note: Optimal timing of staining with either hematoxylin or eosin should be defined for each new batch.</li>
	<li>Dehydrate the samples in a graded ethanol series: 50% and 70% (a few seconds each). Rapidly immerse in 95% ethanol, followed by 2 changes in 100% ethanol, 5 min each.</li>
	<li>Clear in 2 changes of xylene, each 5 min or longer. This step must be carried out under a chemical hood.</li>
	<li>Mount the slides with a xylene-based mounting medium, applying a few drops of mounting medium on the slide and covering with a coverslip. The mounting medium can also be applied to the coverslip and the coverslip put on the slide.
	<ol>
		<li>Avoid forming air bubbles between the slide and the coverslip. To eliminate air bubbles, keep the slide vertical (after mounting) and squeeze out the excess mounting medium and air bubbles by gently pressing on the coverslip with blunt forceps (or a similar blunt tool).</li>
	</ol>
	</li>
	<li>Keep the slides under the hood for a few hours or overnight to let the xylene evaporate completely before proceeding to the analysis.</li>
	<li>Perform the morphological and morphometric analysis of H&#38;E-stained regenerating muscle sections by using non-overlapping serial images of at least one whole muscle section per mouse. Capture images with a bright-field microscope, with 20X magnification. 
	Note: A minimum number of 5 mice is recommended to obtain statistical significance of the measurements. 
	Note: Image analysis software is available for this purpose, such as the free download software ImageJ (http://imagej.nih.gov/ij/).</li>
</ol>

<ol>
	<li>Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells.">Muscle satellite cells.</a> Int J Biochem Cell Biol. 35 (8), 1151-1156 (2003).</li><li>Roca, I., Requena, J., Edel, M. J., Alvarez-Palomo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+Precursors+from+iPS+Cells+for+Skeletal+Muscle+Cell+Replacement+Therapy.">Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy.</a> J Clin Med. 4 (2), 243-259 (2015).</li><li>Cheung, T. H., Rando, T. 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The authors have nothing to disclose.

Here, we describe a protocol to induce acute injury in skeletal muscle (i.e., the intramuscular injection of CTX). It is widely used as a powerful tool to study the dynamics of skeletal muscle regeneration in vivo. CTX injection induces the degeneration of muscle fibers, which is caused by the depolarization of the sarcolemma and the contraction of the fibers12, and triggers the cascade of events that leads to muscle regeneration. Skeletal muscles are dissected at desired time points after th...

Mammalian adult skeletal muscles are formed by groups of fascicules of multinucleated muscle cells (myofibers) that are specialized for contraction. Each myofiber is an elongated syncytium, surrounded by the sarcolemma (plasmatic membrane) and containing myofibrils, which are made up of regularly and repeatedly organized contractile proteins (actin and myosin filaments). In adult life and in resting conditions, skeletal muscles have a very low turnover of their myonuclei1; indeed, the myonuclei, which are located at the periphery of the myofiber, under the sarcolemma, are arrested in the G0 phase of the cell cycle and are unable to proliferate1,2.
Skeletal muscles have the peculiar ability to regenerate following damage, reaching homeostasis after several events of tissue remodeling that are tightly related to each other. After an acute injury or trauma, degeneration is induced, followed by regeneration processes that involve different cell populations, including a resident population of muscle cells, the satellite cells (SCs). Indeed, in the absence of any environmental stimuli, the satellite cells are in a quiescent state and are located in a specialized niche between the sarcolemma and the basal lamina3,4. Following an injury or disease, SCs become activated, proliferate, migrate to the damaged areas, and eventually differentiate, giving rise to newly forming myofibers5. Activated SCs establish cross-talk with different cell populations, mainly inflammatory cells, which are recruited in the site of trauma6-8. This cross-talk allows the cells to follow a regulated paradigm by which molecular signals drive structural modifications, eventually leading to homeostasis9. Besides SCs, inflammatory and interstitial cells, angiogenic processes, and re-innervation events are also involved, acting in a coordinated manner to repair this highly organized and specialized structure.
There is great interest in studying different aspects of skeletal muscle regeneration, not only to understand the physiology of the muscle, but also to improve therapeutic strategies that require deeper knowledge of the whole process. Several experimental approaches are currently available to study the identity and function of the different cell populations, the signaling pathways, and the molecular mechanisms involved. Mouse models of acute injury represent a powerful tool to investigate many aspects of this process. Different commonly used techniques to induce acute muscle damage allow researchers to follow the regeneration process in vivo, from the very early stages to the end of the process. This protocol describes the steps from the intramuscular injection of snake venom-derived cardiotoxin (CTX), which induces myolysis and triggers the regeneration process, up to the analysis of tissue samples. Following CTX injection, mice can be sacrificed at different time points depending on experimental requirements, and the skeletal muscles can be dissected and processed for further analysis. Finally, we describe the staining protocol of tissue sections to perform morphological observations and basic quantitative analyses. This protocol allows for the study of acute skeletal muscle regeneration in vivo in a highly reproducible manner10.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cardiotoxin from&#160;Naja mossambica mossambica</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">C9759</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe For Insulin BD Micro-Fine+ Needle 30 G X 8 mm - Da 0,3 ml</td>
			<td style="width:199px;">BD</td>
			<td style="width:119px;">324826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Tragacanth Gum</td>
			<td style="width:199px;">MP BIOMEDICALS,LLC</td>
			<td style="width:119px;">104792</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylbutane (Isopentane)</td>
			<td>SIGMA ALDRICH</td>
			<td style="width:119px;">78-78-4.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">OCT Killik Solution For Inclusion Cryostat</td>
			<td>Bio-optica</td>
			<td>&#160;05-9801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Feather Microtome Blade S35</td>
			<td style="width:199px;">Bio-optica</td>
			<td style="width:119px;">&#160;01-S35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Glass Slide Superfrost Plus</td>
			<td style="width:199px;">Menzel-Gl&#228;ser</td>
			<td style="width:119px;">09-OPLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dumon #5 Mirror Finish Forceps&#160;</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">11251-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scissors Straight Sharp/Sharp</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">15024-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scissors Noyes Straight</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">15012-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Fine Iris Scissors Straight Sharp/Sharp 10,5 Cm</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">14094-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Eukitt</td>
			<td style="width:199px;">Bio-optica</td>
			<td style="width:119px;">09-00100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Slide Coverslip</td>
			<td style="width:199px;">BIOSIGMA</td>
			<td style="width:119px;">VBS651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Xylene</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">214736</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 100%</td>
			<td>sigma-Aldrich</td>
			<td>02860-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Hematoxyline</td>
			<td style="width:199px;">J.T. BAKER</td>
			<td style="width:119px;">3873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Eosin</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">HT110116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cryostat</td>
			<td style="width:199px;">LEICA</td>
			<td style="width:119px;">CM3050 S</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of acute skeletal muscle regeneration by cardiotoxin injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

H&#38;E staining allows for the evaluation of the morphology of the regeneration process at specific time points during skeletal muscle regeneration. Figure 3 shows the time course analysis performed on injured TA muscles of wild type mice. Muscles have been isolated at 3, 7, 15, and 30 days after CTX injection, as schematized in Figure 3A. Representative pictures of H&#38;E-stained transverse sections show the dynamics of skeletal muscle repair over time...

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Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

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JoVE Journal

Developmental Biology

21.8K Views.  Institute of Genetics and Biophysics "A. Buzzati Traverso", CNR. This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Video: Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

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 ...

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...