Name: immunolabelling myofiber degeneration in muscle biopsies
Uploaded: 2019-12-05T12:30:00
Description: Watch this Scientific Journal Video about Immunolabelling Myofiber Degeneration in Muscle Biopsies at JoVE.com

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 t...

<ol>
	<li>Carpenter, S., Karpati, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duchenne+muscular+dystrophy:+plasma+membrane+loss+initiates+muscle+cell+necrosis+unless+it+is+repaired.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+fiber+degeneration+and+necrosis+in+muscular+dystrophy+and+other+muscle+diseases:+cytochemical+and+immunocytochemical+data.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+mediates+myofiber+death+in+dystrophin-deficient+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+susceptibility+of+EDL+muscles+from+mdx+mice+to+damage+induced+by+contractions+with+stretch.">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. 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J., Bradley, D. M., Salmon, R., Clarke, A., Hartley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newborn+bloodspot+screening+for+Duchenne+muscular+dystrophy:+21+years+experience+in+Wales+(UK).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+dysregulation+of+fibrosis+in+skeletal+muscle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-mediated+pathology+in+Duchenne+muscular+dystrophy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endpoint+measures+in+the+mdx+mouse+relevant+for+muscular+dystrophy+pre-clinical+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+blood-brain+barrier+integrity:+how+appropriate+is+Evans+blue+in+the+twenty-first+century+and+what+are+the+alternatives?+Frontiers+in+Neuroscience.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+muscular+dystrophy+show+different+patterns+of+sarcolemmal+disruption.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+differences+in+regeneration+of+dystrophic+(mdx)+and+normal+muscle+in+the+mouse.">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Circular cork disks</td>
			<td>Pyramid Innovation</td>
			<td>R30001-E</td>
			<td>Don&#39;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&#176;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&#176;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. ...

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