This work was supported by the Association Fran&#231;aise contre les Myopathies with the Translamuscle program. The authors thank Dr. Perla Reyes-Fernandez and Dr. Matthew Borok for their careful reading of the manuscript.

Experiments were performed in accordance with the French and European Community legislation (license number 11-00010).
1. Tragacanth gum preparation
<ol>
	<li>In a glass beaker, dissolve 6 g of tragacanth powder in 100 mL of deionized water. Cover with foil and leave for at least 3 h. Occasionally stir manually with a metal spatula as the mixture rapidly becomes viscous.</li>
	<li>Leave the tragacanth mixture at 60 &#176;C overnight in a water bath or in the oven. Carefully seal the beaker to avoid drying. Stir at least once before aliquoting.</li>
	<li>Aliquot and store at 4 &#176;C for up to 2 weeks.</li>
</ol>
2. Muscle collection
NOTE: For this experiment, a 4-week-old male mdx mouse was sacrificed by cervical dislocation. This procedure does not require anaesthesia and is a humane killing method in accordance with local legislation.
<ol>
	<li>Dissection of the tibialis anterior (TA) muscle
	<ol>
		<li>After shaving the mouse leg, lay the mouse on its back and pin the feet on a corkboard with needles. Using precision scissors (or a scalpel) and forceps, remove the leg skin from the foot up to the knee to expose the entire length of the tibia and TA.</li>
		<li>With scissors, make an incision between the tibia and TA. This will facilitate separation of the TA from the bone and provide an easy grip to the epimysium. Using precision forceps, carefully remove the epimysium layer from the surface of the TA. 
		NOTE: From this step, the TA may be more susceptible to drying, which should be avoided.</li>
		<li>At the ankle region, isolate the TA tendon from other tendons with forceps. Gently pull up the TA tendon to isolate it from the tibia and surrounding muscles.</li>
		<li>When the TA belly is entirely separated, hold the tendon up so that only the proximal part of the TA muscle is attached to the knee. Gently sever the proximal tendon as close as possible to the knee bone.</li>
		<li>Place the TA in a gauze that is lightly dampened with saline to avoid drying before freezing the muscle.</li>
	</ol>
	</li>
	<li>Pre-cool 70&#8722;100 mL of isopentane in a plastic or polytetrafluoroethylene beaker by partially dipping it into liquid nitrogen. Allow it to cool until the isopentane at the bottom of the beaker becomes a white solid.</li>
	<li>On a circular piece of cork (20 mm x 11 mm x 8 mm), place ~0.5 mL of tragacanth gum. Carefully pre-label the other side of the cork so that the biopsy can be properly identified after the freezing step.</li>
	<li>Embed the TA into the gum with forceps, distal tendon up. To allow appropriate cross sections of the muscle, hold the muscle with its axis perpendicular to the cork surface. The quality of the cryosections will improve if the biopsy is not completely embedded into the gum. Allow for at least half of the TA (tendon side) to be uncovered by the gum.</li>
	<li>Quickly dip the cork with the embedded muscle into the unfrozen, cold isopentane layer. Note that the cork will float in the liquid isopentane. Hold the sample upside down as the muscle needs to be dipped into isopentane. Leave the samples in the isopentane for around 2 min to allow complete freezing. 
	NOTE: From this stage, the TA must remain frozen until cryosection. Store the muscle in dry ice before long term storage in a -80 &#176;C freezer.</li>
</ol>
3. Cryosectioning
<ol>
	<li>Set the cryostat temperature at -25 &#176;C. Keep the object temperature at around -20 &#176;C. Fix the object in the cryostat using optimal cutting temperature (OCT) compound. Trim the muscle until reaching the muscle belly then cut 7&#8722;10 &#181;m sections. 
	NOTE: The stabilization of the object temperature requires at least 10 min.</li>
	<li>Keep glass slides used for collecting cryosections at room temperature (RT). Collect at least two sections on each slide. The muscle sections will automatically stick and thaw at the contact of the warm glass slide. Remove the slide and keep it at RT.</li>
	<li>Allow sections to dry at least 20 min at RT in a ventilated environment.</li>
	<li>Store the cryosections on glass slides at -80 &#176;C until use.</li>
</ol>
4. Immunolabelling
<ol>
	<li>Thaw slides at RT for at least 15 min in a ventilated environment.</li>
	<li>Delineate the sections area with a hydrophobic pen. Allow the hydrophobic barrier to dry.</li>
	<li>Fix the tissue with 2% paraformaldehyde for 10 min in a humid chamber.</li>
	<li>Wash the sections with phosphate buffered saline (PBS) 2x for 5 min each.</li>
	<li>Block the muscle sections with 10% goat serum diluted in PBS for 1 h at RT in a humid chamber.</li>
	<li>Incubate the sections for 2 h at RT (or overnight at 4 &#176;C) in a humid chamber with the primary antibodies diluted in 5% goat serum. For simple IgG uptake labelling in myofibers, only incubate sections with rabbit antibody to mouse pan-laminin. 
	NOTE: Other antigens of the extracellular matrix can be targeted as long as they provide clear labelling of the surrounding extracellular matrix of the myofibers. Markers for muscle regeneration, inflammation, or other parameters can be combined as long as the antibodies are not raised in a mouse host. The microscope used for the analysis should include a supplementary fluorescence color.</li>
	<li>Wash the sections with PBS 3x for 5 min each.</li>
	<li>Incubate the sections with red fluorescent goat polyclonal secondary antibody to rabbit IgG H&#38;L and green fluorescent goat polyclonal secondary antibody to mouse IgG H&#38;L. Incubate at RT for 45 min in a humid chamber.</li>
	<li>Wash with PBS 3x for 10 min each.</li>
	<li>Mount the sections in fluorescent mounting medium containing 100 ng/mL 4&#8242;,6-diamidino-2-phenylindole (DAPI) and cover each slide with a coverslip.</li>
	<li>Allow drying overnight at 4 &#176;C before imaging.</li>
</ol>

<ol><li>Carpenter, S., Karpati, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Duchenne+muscular+dystrophy%3A+plasma+membrane+loss+initiates+muscle+cell+necrosis+unless+it+is+repaired%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Duchenne muscular dystrophy: plasma membrane loss initiates muscle cell necrosis unless it is repaired.</a> Brain. 102 (1), 147-161 (1979).</li>
<li>Cornelio, F., Dones, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+fiber+degeneration+and+necrosis+in+muscular+dystrophy+and+other+muscle+diseases%3A+cytochemical+and+immunocytochemical+data%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Muscle fiber degeneration and necrosis in muscular dystrophy and other muscle diseases: cytochemical and immunocytochemical data.</a> Annals of Neurology. 16 (6), 694-701 (1984).</li>
<li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes%5BTitle%5D)+AND+%22Cell+Death+and+Differerentiation%22%5BJournal%5D)">Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.</a> Cell Death and Differerentiation. 16 (8), 1093-1107 (2009).</li>
<li>Morgan, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Necroptosis+mediates+myofiber+death+in+dystrophin-deficient+mice%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Necroptosis mediates myofiber death in dystrophin-deficient mice.</a> Nature Communications. 9 (1), 3655(2018).</li>
<li>Moens, P., Baatsen, P. H., Marechal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+susceptibility+of+EDL+muscles+from+mdx+mice+to+damage+induced+by+contractions+with+stretch%5BTitle%5D)+AND+%22Journal+of+Muscle+Research+and+Cell+Motility%22%5BJournal%5D)">Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch.</a> Journal of Muscle Research and Cell Motility. 14 (4), 446-451 (1993).</li>
<li>Tidball, J. G., Villalta, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+interactions+between+muscle+and+the+immune+system+during+muscle+regeneration%5BTitle%5D)+AND+%22American+Journal+of+Physiology+Regulatory+Integrative+and+Comparative+Physiology%22%5BJournal%5D)">Regulatory interactions between muscle and the immune system during muscle regeneration.</a> American Journal of Physiology Regulatory Integrative and Comparative Physiology. 298 (5), R1173-R1187 (2010).</li>
<li>Riederer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slowing+down+differentiation+of+engrafted+human+myoblasts+into+immunodeficient+mice+correlates+with+increased+proliferation+and+migration%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration.</a> Molecular Therapy. 20 (1), 146-154 (2012).</li>
<li>Arnold, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+monocytes+recruited+after+skeletal+muscle+injury+switch+into+antiinflammatory+macrophages+to+support+myogenesis%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.</a> Journal of Experimental Medicine. 204 (5), 1057-1069 (2007).</li>
<li>Bencze, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proinflammatory+macrophages+enhance+the+regenerative+capacity+of+human+myoblasts+by+modifying+their+kinetics+of+proliferation+and+differentiation%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.</a> Molecular Therapy. 20 (11), 2168-2179 (2012).</li>
<li>Moat, S. J., Bradley, D. M., Salmon, R., Clarke, A., Hartley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newborn+bloodspot+screening+for+Duchenne+muscular+dystrophy%3A+21+years+experience+in+Wales+%28UK%29%5BTitle%5D)+AND+%22European+Journal+of+Human+Genetics%22%5BJournal%5D)">Newborn bloodspot screening for Duchenne muscular dystrophy: 21 years experience in Wales (UK).</a> European Journal of Human Genetics. 21 (10), 1049-1053 (2013).</li>
<li>Serrano, A. L., Munoz-Canoves, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+and+dysregulation+of+fibrosis+in+skeletal+muscle%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Regulation and dysregulation of fibrosis in skeletal muscle.</a> Experimental Cell Research. 316 (18), 3050-3058 (2010).</li>
<li>Rosenberg, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune-mediated+pathology+in+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Immune-mediated pathology in Duchenne muscular dystrophy.</a> Science Translational Medicine. 7 (299), (2015).</li>
<li>Kobayashi, Y. M., Rader, E. P., Crawford, R. W., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endpoint+measures+in+the+mdx+mouse+relevant+for+muscular+dystrophy+pre-clinical+studies%5BTitle%5D)+AND+%22Neuromuscular+Disorders%22%5BJournal%5D)">Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies.</a> Neuromuscular Disorders. 22 (1), 34-42 (2012).</li>
<li>Saunders, N. R., Dziegielewska, K. M., Mollgard, K., Habgood, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Markers+for+blood-brain+barrier+integrity%3A+how+appropriate+is+Evans+blue+in+the+twenty-first+century+and+what+are+the+alternatives%3F+Frontiers+in+Neuroscience%5BTitle%5D)+AND+%229%22%5BJournal%5D)">Markers for blood-brain barrier integrity: how appropriate is Evans blue in the twenty-first century and what are the alternatives? Frontiers in Neuroscience.</a> 9. 385, (2015).</li>
<li>Straub, V., Rafael, J. A., Chamberlain, J. S., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+for+muscular+dystrophy+show+different+patterns+of+sarcolemmal+disruption%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.</a> Journal of Cell Biology. 139 (2), 375-385 (1997).</li>
<li>Pastoret, C., Sebille, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+differences+in+regeneration+of+dystrophic+%28mdx%29+and+normal+muscle+in+the+mouse%5BTitle%5D)+AND+%22Muscle+and+Nerve%22%5BJournal%5D)">Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse.</a> Muscle and Nerve. 18 (10), 1147-1154 (1995).</li>
<li>Villalta, S. A., Nguyen, H. X., Deng, B., Gotoh, T., Tidball, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shifts+in+macrophage+phenotypes+and+macrophage+competition+for+arginine+metabolism+affect+the+severity+of+muscle+pathology+in+muscular+dystrophy%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.</a> Human Molecular Genetics. 18 (3), 482-496 (2009).</li>
</ol>

The authors have nothing to disclose.

Myofiber necrosis is a common consequence of traumatic exercise in normal muscles. It is well-compensated by a powerful regenerative capacity of the local muscle progenitors. However, in several muscle conditions such as in MDs, the regenerative capacity of satellite cells is compromised by chronic myonecrosis and excessive fibrosis. Recent findings show that muscle fibres can die by necroptosis, a regulated form of necrosis. More specifically, the inhibition of necroptosis may become a new therapeutic strategy for DMD t...

Striated skeletal muscle mainly consists of muscle fibres (myofibers), which are responsible for the characteristic voluntary contractile function. These cells are multinucleated, post-mitotic structures that support mechanical stress occurring during contraction. Structural stability of the myofiber membrane (sarcolemma) and its extracellular matrix are crucial for tissue homeostasis. Satellite cells comprise the main muscle progenitor population in mature skeletal muscle and exist in a quiescent state in healthy muscles. Following myofiber death, muscle regeneration is supported by satellite cells following a myogenic program that involves satellite cell activation, proliferation, differentiation, and fusion to ultimately form new multinucleated myofibers.
Myofiber demise can occur in multiple muscle conditions, including mechanical trauma, ischemia-reperfusion injuries, or muscular dystrophies, and it is associated with the necrotic morphology of dead cells1,2. Necrotic death is characterized by the rapid permeability of the plasma membrane and release of cell content in the extracellular compartment3. It can result from either an unregulated process involving no proper cell signalling (i.e., accidental necrosis), or an orchestrated intracellular pathway (i.e., regulated necrosis). In myofibers, both regulated4 and unregulated5 processes can lead to necrosis. A typical consequence of myonecrosis is the release of damage-associated molecule patterns, activating a powerful inflammatory response6. The presence of macrophages is observed at around 48 h and 72 h following injury7. Besides their role in the clearance of necrotic debris, they are also important in muscle regeneration8,9.
Muscular dystrophies (MDs) are a heterogeneous group of pathologies which often result from a defect in the sarcolemma structure. Duchenne muscular dystrophy (DMD) is a juvenile X-linked disease affecting approximately 1 out of every 3,500 male births worldwide10, and it is caused by the absence of dystrophin expression at the sarcolemma. Chronic degeneration of the muscle tissue in DMD boys leads to extreme muscle weakness and early mortality. Inflammation resulting from necrotic death enhances cytotoxicity, and promotes muscle fibrosis and the loss of muscle function11,12. Treatments currently in clinical trials targeting the roots of muscle degenerative disorders, such as gene therapy, are expected to alleviate myonecrosis. Simple techniques to accurately quantify muscle degeneration are therefore needed.
Several methods are routinely used to monitor myofiber loss in vivo. The measurement of the enzymatic activity of creatine kinase (CK) in the blood allows reliable quantification of ongoing necrosis in muscle and heart tissues. In situ, the haematoxylin and eosin (H&#38;E) staining is the most popular method currently used in diagnosis to assess degeneration-regeneration remodelling. However, the molecular basis of the H&#38;E labelling of dead cells remains unclear. Furthermore, color modifications suggesting myofiber death in H&#38;E staining are relatively subtle and do not facilitate reliable and reproducible quantification. Methods revealing DNA fragmentation, such as the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), imperfectly label necrotic death3. They are also poorly adapted to monitor the death of syncytial cells such as myofibers. The injection of vital dyes, such as Evan&#39;s blue dye (EBD), represents a useful alternative for assessing myofibers that have lost the integrity of sarcolemma, but is not necessarily convenient in some experimental protocols. For instance, the presence of EBD in blood samples can affect results of CK measurements, a colorimetric assay. Furthermore, intracellular uptake of EBD makes co-immunolabelling challenging. Therefore, an alternative method allowing direct labelling of myofibers undergoing necrosis is of interest.
The mechanism of action of vital dyes relies on the loss of plasma membrane integrity in necrotic myofibers and the passive uptake of the injected dye. Similarly, necrotic myofibers uptake blood proteins such as albumin, for which EBD has a strong affinity13,14, immunoglobulin G (IgG), and IgM15. The abnormal presence of blood proteins within myofibers therefore represents convenient markers for myonecrosis in situ. Staining these proteins can be an alternative for the use of vital dyes.
By using IgG uptake as a marker of myonecrosis in situ, this protocol is used to assess muscle degeneration in the tibialis anterior (TA) of mdx dystrophin-deficient mice. This method presents significant advantages over alternative techniques: 1) it is reproducible and simple in its execution; 2) it does not require any animal treatment prior to muscle collection, such as the injection of circulating vital dyes, and 3) as any conventional immunolabelling, it is compatible with co-labelling.

<table><tbody><tr><td>Circular cork disks</td><td>Pyramid Innovation</td><td>R30001-E</td><td>Don&#039;t forget to clearly label the cork so that the the ID of the sample can be determined after freezing</td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050 sn34</td><td>Muscle cryosectionning should be performed between -20 and -25&amp;deg;C. Thickness:7-10 micrometers.</td></tr><tr><td>Dakopen</td><td>Dako</td><td>S2002</td><td>A hydrophobic barrier around the muscle sections. It prevents the dispertion of medium during incubation</td></tr><tr><td>Forceps</td><td>FST</td><td>91117-10</td><td>/</td></tr><tr><td>Goat anti-Mouse IgG (H+L) antibody, Alexa Fluor Plus 488</td><td>ThermoFischer Scientific</td><td>A-11029</td><td>Dilution: 1/500</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) antibody Alexa Fluor Plus 594</td><td>ThermoFischer Scientific</td><td>A32740</td><td>Dilution: 1/500</td></tr><tr><td>Goat serum</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td>At the blocking step, use 10% dilution in PBS. For antibodies incubation, use 5% dilution in PBS</td></tr><tr><td>Isopentane</td><td>Sigma Aldrich</td><td>78-78-4</td><td>Freezing medium. Should be cooled down in a beaker placed in liquid nitrogen.</td></tr><tr><td>mdx mouse</td><td>Jackson Laboratory</td><td>C57BL/10ScSn-Dmdmdx/J</td><td>Mdx mice are mutated for the dystrophin gene. From three weeks of age, muscles are characterized by chronic degeneration</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Imager.D1</td><td>/</td></tr><tr><td>OCT</td><td>Cellpath</td><td>KMA-0100-00A</td><td>Embedding matrix</td></tr><tr><td>PFA (Paraformaldehyde)</td><td>ThermoFischer Scientific</td><td>28908</td><td>Used for fixing cryosection (2% or 4% PFA can be used)</td></tr><tr><td>PBS</td><td>Eurobio</td><td>CS3PBS00-01</td><td>Dilution medium for immunolabeling</td></tr><tr><td>Precision scisors</td><td>FST</td><td>fst 14001-12 and fst 14001-14</td><td>Used for the muscle collection</td></tr><tr><td>Rabbit antibody to mouse pan-Laminin</td><td>Sigma Aldrich</td><td>L9393</td><td>Dilution: 1/1000</td></tr><tr><td>Tragacanth</td><td>Sigma Aldrich</td><td>G1128</td><td>Aliquots to keep at +4&amp;deg;C</td></tr></tbody></table>

immunolabelling myofiber degeneration in muscle biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Myofibers are surrounded by a laminin-containing extracellular matrix. Red staining delimits myofibers periphery and allows for their identification. IgG is shown in green. Nuclei are stained with DAPI and are found blue under the microscope. However, nuclei are shown in white here (Figure 1). A weak IgG immunoreactivity is expected in the extracellular compartment, which can be increased in case of inflammation. Green staining within the myofibers reflects t...

Watch this Scientific Journal Video about Immunolabelling Myofiber Degeneration in Muscle Biopsies at JoVE.com

Immunolabelling Myofiber Degeneration in Muscle Biopsies

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

immunolabelling-myofiber-degeneration-in-muscle-biopsies

Research

JoVE Journal

Biology

8.7K Views.  Institut Mondor de Recherche Biomédicale. Myofiber necrose is kenmerkend voor meerdere neuromusculaire aandoeningen en speelt een centrale rol bij pathogenese. Een specifieke, betrouwbare methode voor het beoordelen van spierdegeneratie is belangrijk voor diagnose en translationeel onderzoek. Deze uiterst aanpasbare techniek maakt het mogelijk om meerdere antigeens als dezelfde tijd in dezelfde sectie binnen die myofibers te labelen.Het is mogelijk met vitale kleurstoffen. H&E kleuring is een empirische methode die niet aanpas...

Video: Immunolabelling Myofiber Degeneration in Muscle Biopsies

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 ...

Developmental Biology

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.

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 ...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

Biochemistry

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Medicine

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

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

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

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

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

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Neuroscience

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

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