Name: induction of acute skeletal muscle regeneration by cardiotoxin injection
Uploaded: 2017-01-01T13:00:00
Description: Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

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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			<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>
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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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Research

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Developmental Biology

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 ...