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
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	<li>Prepare a 10 &#956;M working solution of ...

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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.

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	<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...

Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

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 ...

The overall goal of this procedure is to obtain a mouse model of skeletal muscle acute injury to study the regeneration process in vivo, from the very early stages to the end of the process. Mouse models of muscle acute injury is a powerful tool to investigate many aspects of the regeneration process. The main advantage of this technique is the high reproducibility of all the steps involved.To begin this procedure, apply a few drops of 70%ethanol onto the the hind limb of an anesthetized mouse. To visualize the exact location of the tibialis anterior muscle, remove the hair at the anterior part of the lower leg with a scalpel. To inject the cardiotoxin solution, draw 100 microliters of it into the syringe.Hold the syringe upright and remove the air bubbles. Then, insert the needle in the center of TA from the tendon towards the knee, following the position of the tibia bone as guidance. Perform the injection without going too deep, and avoid going beyond the muscle itself.Wait two to three seconds before pulling out the needle to prevent leakage of cardiotoxin from the muscle. Apply a few drops of 70%ethanol onto the hind limb of the euthanized animal. Cut the skin at the level of the proximal tendon, then insert one of the blades of the scissors under the skin and cut the skin in the direction of the knee.Pull up the skin to expose the muscle entirely. Carefully remove all the skin and the hair from the area of interest. After that, eliminate the fascia by slowly pinching or cutting the extreme periphery of the muscle.Once broken, gently remove the fascia with the help of the thin-tipped tweezers, avoiding damaging the underlying muscle. Visualize the distal tibialis anterior tendon above the foot, then separate the distal tibialis anterior tendon from the extensor digitorum tendon by passing the tweezers'tip below the tendon. Gently slide the tweezers below the tibialis anterior to separate it from the underlying muscle.Hold the tweezers under the muscle to keep it slightly raised. Cut the tibialis anterior tendon and gently pull it upwards until only the area below the knee is attached. Subsequently, cut the TA below the knee, following the edge of the muscle with the scissors.Place a small amount of tragacanth gum on a slice of cork. Insert the distal tendon of the TA into the tragacanth gum, and leave about 3/4ths of the muscle outside, making sure to have the muscle in a perpendicular position with respect to the cork. After that, fill an aluminum can with isopentane.Suspend the can of isopentane in a dewar containing liquid nitrogen. When isopentane reaches the proper temperature, white solid particles form at the bottom of the can. Rapidly dip the cork with the muscle into isopentane, keeping the muscle position downwards.While the specimen floats, only the reverse side of the cork should be visible in the liquid. Leave the sample in isopentane for one minute. After that, immerse the muscle in liquid nitrogen for at least two minutes.Then, wrap the specimens individually in labeled aluminum foil and immediately place them in dry ice. During the procedure, it is extremely important to maintain the correct temperature and to prevent the specimen from thawing. In this procedure, place the specimen stub, the blade, and the specimen in the cryostat chamber, allowing them to equilibrate for at least 30 minutes.Next, place a small amount of freezing compound on the specimen stub, and immerse the cork with the specimen in the OCT compound before the freezing compound solidifies. Place the blade in the blade holder. Place the specimen in its proper holder.Cut the sections at a thickness of 10 micrometers. Then, place the sections on a polarized slide. After sectioning, store the slides at 80 degrees Celsius.In a chemical hood, completely air dry the frozen slides with sections for 10 to 15 minutes at room temperature. Then, lodge the slides in a staining tray. Stain the sections for one minute with hematoxylin.After that, lay the staining trough under a fairly weak tap water jet for 10 minutes to wash out the hematoxylin. Following that, counterstain the sections with eosin solution for one minute, then rinse them with water to eliminate excessive eosin solution. Subsequently, dehydrate the samples in a graded ethanol series at 50%followed by 70%Afterward, rapidly immerse the samples in 95%ethanol, followed by two changes in 100%ethanol.Clear the samples in two changes of xylene, each for at least five minutes. To mount the slides, apply a few drops of the xylene-based mounting medium on each slide, and cover it with a cover slip. Keep the slides in the fume hood for a few hours, or overnight.Shown here is the experimental scheme of the acute skeletal muscle injury. Here are the representative pictures of hematoxylin-and eosin-stained sections of cardiotoxin-treated muscles at the indicated days after injury. A couple of necrotic fibers, which is enclosed by the black continuous line, are likely invaded by the inflammatory cells.The dashed line marks an area of the infiltrating mononucleated cells. A single immature myofiber with centrally located nucleus is indicated by the black circle. Here are the large regenerated eosinophilic myofibers, with the centrally located nuclei that are marked by the black circles.This graph shows the average of the centrally-nucleated myofiber size values in TA muscle sections. And this graph shows the myofiber cross-sectional area distribution at 6, 15, and 30 days after cardiotoxin injection. Following this procedure, skeletal muscle sections can be used to perform different histological and immunofluorescence protocols to identify specific features of the muscle tissue.After watching this video, you should have a good understanding of how to investigate different aspects of skeletal muscle regeneration process that follow acute injury.

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21.5K vistas.  Institute of Genetics and Biophysics "A. Buzzati Traverso", CNR. The overall goal of this procedure is to obtain a mouse model of skeletal muscle acute injury to study the regeneration process in vivo, from the very early stages to the end of the process. Mouse models of muscle acute injury is a powerful tool to investigate many aspects of the regeneration process. The main advantage of this technique is the high reproducibility of all the steps involved.To begin this procedure, apply a few drops of 70%ethanol onto the the hind limb of an anesthetiz...