Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

The overall goal of this procedure is to obtain a mouse model of skeletal muscle acute injury to study the regeneration process in vivo, from the very early stages to the end of the process. Mouse models of muscle acute injury is a powerful tool to investigate many aspects of the regeneration process. The main advantage of this technique is the high reproducibility of all the steps involved.

To begin this procedure, apply a few drops of 70%ethanol onto the the hind limb of an anesthetized mouse. To visualize the exact location of the tibialis anterior muscle, remove the hair at the anterior part of the lower leg with a scalpel. To inject the cardiotoxin solution, draw 100 microliters of it into the syringe.

Hold the syringe upright and remove the air bubbles. Then, insert the needle in the center of TA from the tendon towards the knee, following the position of the tibia bone as guidance. Perform the injection without going too deep, and avoid going beyond the muscle itself.

Wait two to three seconds before pulling out the needle to prevent leakage of cardiotoxin from the muscle. Apply a few drops of 70%ethanol onto the hind limb of the euthanized animal. Cut the skin at the level of the proximal tendon, then insert one of the blades of the scissors under the skin and cut the skin in the direction of the knee.

Pull up the skin to expose the muscle entirely. Carefully remove all the skin and the hair from the area of interest. After that, eliminate the fascia by slowly pinching or cutting the extreme periphery of the muscle.

Once broken, gently remove the fascia with the help of the thin-tipped tweezers, avoiding damaging the underlying muscle. Visualize the distal tibialis anterior tendon above the foot, then separate the distal tibialis anterior tendon from the extensor digitorum tendon by passing the tweezers’tip below the tendon. Gently slide the tweezers below the tibialis anterior to separate it from the underlying muscle.

Hold the tweezers under the muscle to keep it slightly raised. Cut the tibialis anterior tendon and gently pull it upwards until only the area below the knee is attached. Subsequently, cut the TA below the knee, following the edge of the muscle with the scissors.

Place a small amount of tragacanth gum on a slice of cork. Insert the distal tendon of the TA into the tragacanth gum, and leave about 3/4ths of the muscle outside, making sure to have the muscle in a perpendicular position with respect to the cork. After that, fill an aluminum can with isopentane.

Suspend the can of isopentane in a dewar containing liquid nitrogen. When isopentane reaches the proper temperature, white solid particles form at the bottom of the can. Rapidly dip the cork with the muscle into isopentane, keeping the muscle position downwards.

While the specimen floats, only the reverse side of the cork should be visible in the liquid. Leave the sample in isopentane for one minute. After that, immerse the muscle in liquid nitrogen for at least two minutes.

Then, wrap the specimens individually in labeled aluminum foil and immediately place them in dry ice. During the procedure, it is extremely important to maintain the correct temperature and to prevent the specimen from thawing. In this procedure, place the specimen stub, the blade, and the specimen in the cryostat chamber, allowing them to equilibrate for at least 30 minutes.

Next, place a small amount of freezing compound on the specimen stub, and immerse the cork with the specimen in the OCT compound before the freezing compound solidifies. Place the blade in the blade holder. Place the specimen in its proper holder.

Cut the sections at a thickness of 10 micrometers. Then, place the sections on a polarized slide. After sectioning, store the slides at 80 degrees Celsius.

In a chemical hood, completely air dry the frozen slides with sections for 10 to 15 minutes at room temperature. Then, lodge the slides in a staining tray. Stain the sections for one minute with hematoxylin.

After that, lay the staining trough under a fairly weak tap water jet for 10 minutes to wash out the hematoxylin. Following that, counterstain the sections with eosin solution for one minute, then rinse them with water to eliminate excessive eosin solution. Subsequently, dehydrate the samples in a graded ethanol series at 50%followed by 70%Afterward, rapidly immerse the samples in 95%ethanol, followed by two changes in 100%ethanol.

Clear the samples in two changes of xylene, each for at least five minutes. To mount the slides, apply a few drops of the xylene-based mounting medium on each slide, and cover it with a cover slip. Keep the slides in the fume hood for a few hours, or overnight.

Shown here is the experimental scheme of the acute skeletal muscle injury. Here are the representative pictures of hematoxylin-and eosin-stained sections of cardiotoxin-treated muscles at the indicated days after injury. A couple of necrotic fibers, which is enclosed by the black continuous line, are likely invaded by the inflammatory cells.

The dashed line marks an area of the infiltrating mononucleated cells. A single immature myofiber with centrally located nucleus is indicated by the black circle. Here are the large regenerated eosinophilic myofibers, with the centrally located nuclei that are marked by the black circles.

This graph shows the average of the centrally-nucleated myofiber size values in TA muscle sections. And this graph shows the myofiber cross-sectional area distribution at 6, 15, and 30 days after cardiotoxin injection. Following this procedure, skeletal muscle sections can be used to perform different histological and immunofluorescence protocols to identify specific features of the muscle tissue.

After watching this video, you should have a good understanding of how to investigate different aspects of skeletal muscle regeneration process that follow acute injury.