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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EDL muscle isolation and collagenase digestion
<ol>
	<li>Spray all equipment with 70% ethanol to avoid contamination.</li>
	<li>Sacrifice the mouse in accordance with the national regulations for animal experimentation.</li>
	<li>Spray the hindlimbs of the mouse with 70% ethanol. Use hardened fine curved scissors (24 mm cutting edge) and fine forceps (Dumont 7, curved or straight) to remove the skin and to expose the underlying muscles. Avoid any contact of the underlying muscles with fur (increases the risk of contamination).</li>
	<li>Remove the surrounding fascia with fine curved forceps without damaging the underlying muscles (Figure 1A). Close the forceps to avoid bending.</li>
	<li>Use curved forceps to expose the distal tendons of the TA (tibialis anterior) and EDL muscle. To remove the TA, grab the distal TA tendon with forceps and cut with fine Vannas spring scissors (5 mm cutting edge, 0.35 mm tip diameter). While holding the TA at the tendon, pull it towards the knee and cut the muscle close to the knee (Figure 1B), the EDL muscle is now exposed. 
	NOTE: Make sure that only the TA tendon is grabbed in this step, otherwise the underlying EDL might get damaged. When cutting off the TA muscle make sure that the tendons at the knee can be seen easily afterwards.</li>
	<li>Lift the distal EDL tendon with fine curved forceps and cut with fine Vannas spring scissors (Figure 1C). Expose the proximal EDL tendon by carefully pulling the EDL towards the knee. Cut the proximal tendon with fine Vannas spring scissors. Transfer the EDL muscle to the 37&#176;C preheated 2.5 mL of collagenase digestion solution, in the 15 mL reaction tube. The collagenase solution is prepared as according to this procedure:
	<ol>
		<li>Prepare collagenase digestion solution by dissolving 0.2% (w/v) collagenase type 1 (from&#160;Clostridium histolyticum) in DMEM (Dulbecco&#39;s modified Eagle&#39;s medium with 4.5 g/L glucose and sodium pyruvate), filter through 0.22 &#181;m filter. (Figure 1D). 
		NOTE: Make a small incision at the outer knee connective tissue to fully expose the proximal EDL tendon. Make sure to only grab the tendons and not to stretch the EDL too much.</li>
	</ol>
	</li>
	<li>Repeat steps 1.3. to 1.6 with the second EDL. Add both EDL muscles to the same 15 mL reaction tube filled with 2.5 mL collagenase digestion solution.</li>
	<li>Incubate the EDL muscles in the reaction tube at 37&#176;C in a circulating water bath. 
	NOTE: Incubation time depends on several factors, such as collagenase activity, age of the mouse and amount of fibrotic tissue. The typical incubation time for EDL muscles of adult mice (2-6 months of age) is 1-1.5 h and for aged mice (18 months of age) 1.5-2 h.</li>
	<li>To avoid excessive digestion of the muscles, check the muscles during the digestion time. Stop the digestion when muscles loosen up and single myofibers are visible (Figure 1E). Transfer muscles carefully with the large bore pipette to the first well of the prewarmed 12-well plate containing myofiber isolation medium (Figure 1G), which is prepared as follows:
	<ol>
		<li>Supplement DMEM (Dulbecco&#39;s modified Eagle&#39;s medium with 4.5 g/L glucose and sodium pyruvate) with 20% FBS (fetal bovine serum), filter through 0.22 &#181;m filter. Add medium to the pre-coated isolation plates (12-well plate, 2-4 mL isolation medium per well) approximately 30 min before the isolation and equilibrate in a humidified 37&#176;C incubator with 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Myofiber dissociation 
<ol>
	<li>For the following steps use a stereo binocular microscope, preferentially equipped with a heating plate (37 &#176;C). Use the large bore pipette to flush the muscles with warm isolation medium until single myofibers are released. Dissociate the muscles with the large bore pipette until the desired number of myofibers are floating freely in the solution. 
	NOTE: Avoid excessive trituration to reduce the risk of damaged myofibers. Using a heating plate for myofiber dissociation is strongly advised since temperature drops during the isolation process, which will result in myofiber death.</li>
</ol>

<table><tbody><tr><td>Collagenase type 1</td><td>Sigma</td><td>C0130</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium with 4.5&amp;nbsp;g/l glucose and sodium pyruvate)</td><td>GibCo</td><td>41966029</td><td>Cell culture medium</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10270-106</td><td>Fetal bovine serum</td></tr></tbody></table>

myofiber isolation: a technique to obtain single myofibers from harvested mouse extensor digitorum longus (edl) muscle

Source: H&#252;ettner, S.S. et al. <a href="https://www.jove.com/v/62257">Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo.</a> J. Vis. Exp. (2021)
This video demonstrates the isolation of single myofibres from harvested murine Extensor Digitorum Longus (EDL) muscle. The isolated myofibres can be cultured to study myofiber-associated muscle stem cell activity.

<img alt="Figure 1" src="/files/ftp_upload/20814/20814_Figure1.jpg" /> 
Figure 1. Mouse EDL dissection and single myofiber isolation.&#160; (A) The skin of the mouse hindlimb is removed and the muscle surrounding fascia is pulled away to expose the TA (tibialis anterior) muscle. (B) The TA muscle is removed to expose the EDL (extensor digitorum longus) muscle, marked by the white arrow. (C) The EDL muscle is dissected by cutting its tendons. (D) Two EDL muscles are collagenase digested. (E) Appearance of collagenase digested muscle with visible single myofibers loosening up from the tissue core. (F) Large and small-bore Pasteur pipette with scale. (G) The EDL muscles (marked by black arrows) are physically triturated using a large bore Pasteur pipette. (H) Single intact myofibers are thin and shiny and can be individually collected for culture and analysis. (I) Hypercontracted and dead myofibers are unsuitable for culture and analysis. The microscopic images of 2H and 2I were captured with a microscope using a N-Achroplan 5x objective. Scale bars are 200 &#181;m.

Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) Muscle

Source: H&#252;ettner, S.S. et al. Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo. J. Vis. Exp. (2021)
...

Begin with a euthanized mouse in the supine position. Sterilize its hindlimb. Surgically remove the skin and fascia&#8212;a thin casing of connective tissue beneath the skin&#8212;to expose the underlying hindlimb muscles.
Locate the tibialis anterior or TA muscle on the lateral side of the tibia. Holding the TA distal tendon, detach the muscle from the underlying tissue. Cut the muscle close to the knee to excise the TA muscle, revealing the underlying extensor digitorum longus or EDL muscle located in the anterior compartment of the hindlimb.
Incise the distal EDL tendon and pull the EDL muscle towards the knee. Cut the proximal EDL tendon to excise the EDL muscle. Transfer the harvested muscle to a tube with prewarmed media containing collagenase enzymes and incubate for the appropriate duration. Collagenases break down the connective tissue surrounding each muscle fiber or myofiber within the muscle. This digestion step loosens the myofibres from the inner layer of the muscle.
Following incubation, carefully transfer the digested muscle to a multi-well plate containing prewarmed media to increase the survival rate of myofibres. Transfer the plate to the heated stage of a stereo microscope. Now, gently triturate the digested muscle to dissociate and release the individual myofibers into the solution.
The isolated EDL myofibres can be used for further downstream analysis.

myofiber-isolation-technique-to-obtain-single-myofibers-from

Research

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

1.8K vistas. Fuente: Hüettner, S.S. et al. Ensayo de cultivo de miofibras únicas para la evaluación de la funcionalidad de las células madre musculares adultas ex vivo. J. Vis. Exp. (2021) Este video muestra el aislamiento de miofibras individuales del músculo extensor largo de los dedos murino (EDL) recolectado. Las miofibras aisladas se pueden cultivar para estudiar la actividad de las células madre musculares asociadas a las miofibras. 

Video: Aislamiento de miofibras: una técnica para obtener miofibras individuales a partir de músculo extensor largo de los dedos (EDL) de ratón - Concepto

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.

La inducción de la regeneración muscular esquelético aguda por inyección Cardiotoxin

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Investigación

JoVE Journal

Biología del Desarrollo

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.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

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

Biología

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Aislamiento de miofiber único y cultura de un modelo murino de distrofia muscular de Emery-Dreifuss en el desarrollo post-Natal temprano

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) Muscle

Transcripción

Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) Muscle