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>

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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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+interactions+between+muscle+and+the+immune+system+during+muscle+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slowing+down+differentiation+of+engrafted+human+myoblasts+into+immunodeficient+mice+correlates+with+increased+proliferation+and+migration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+monocytes+recruited+after+skeletal+muscle+injury+switch+into+antiinflammatory+macrophages+to+support+myogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinflammatory+macrophages+enhance+the+regenerative+capacity+of+human+myoblasts+by+modifying+their+kinetics+of+proliferation+and+differentiation.">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/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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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 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. ...

immunolabelling-myofiber-degeneration-in-muscle-biopsies

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Biology

8.6K Views.  Institut Mondor de Recherche Biomédicale. 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.

Video: Immunolabelling Myofiber Degeneration in Muscle Biopsies

Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) procedure. In the past, we standardized a model consisting of latex glove containing fresh cattle liver for practicing FNAB. However, this model is difficult to organize and prepare on short notice, with the procurement of fresh cattle liver being the most challenging aspect. Handling of liver with contamination-related problems is also a significant draw back. In addition, the glove material leaks after a few needle passes, with resulting mess.
We have established a novel simple method of embedding a small piece of sausage or banana in a commercially available silicone rubber caulk. This model allows the retention of vacuum seal and aspiration of material from the embedded specimen, resembling an actual FNAB procedure on clinical mass lesions.
The aspirated material in the needle hub can be processed similar to the specimens procured during an actual FNAB procedure, facilitating additional proficiency in smear preparation and staining.

Practicing of fine needle aspiration biopsies (FNAB) by trainees is relatively challenging, due to the lack of an easily available, appropriate lesion. Preparation of an AV phantom lesion for practicing the FNAB procedure and mastering proficiency is relatively easy.

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.
A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.
In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.

This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy.

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...