Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

All experiments were conducted in strict accordance with the institutional guidelines for animal research and approved by the Department of Public Health, Animal Health, Nutrition and Food Safety of the Italian Ministry of Health in accordance with the law on animal experimentation. Cervical dislocation procedures may vary from institution to institution based on IACUC or its equivalent requirements.

1. Cardiotoxin Injection in the Tibialis Anterior Muscle

1. Prepare a 10 μM working solution of cardiotoxin (CTX) by diluting the cardiotoxin stock solution in sterile Phosphate-Buffered Saline (PBS) or in water before starting the procedure.
   Note: CTX is soluble in water at 1 mg/mL. Prepare a 70 μM stock solution. Aliquot and store at -20 °C. CTX used for this protocol is a mixture of cardiotoxins having a molecular weight of approximately 7,000 Da.
   CAUTION: It is suggested to wear double gloves, a hygiene mask, and safety glasses (alternatively to safety glasses, it is recommended to work under a hood) and to be very careful to avoid any contact with the solution, even if diluted.
2. Anesthetize the mice by intraperitoneal injection of ketamine and xylazine (80 mg/kg and 10 mg/kg of body mass) or other approved available anesthetic.
3. With the mouse face up, spray the hind limbs with 70% ethanol. To visualize the exact location of the Tibialis Anterior (TA) muscle, with the help of a scalpel, cut the hair away at the anterior part of the lower leg, under the knee.
   Note: The TA muscle arises from the proximal body of the tibia, going down lateral to the bone and ending at the ankle. Its long tendon extends into the foot across the ankle (see Figure 1A).
4. Draw ~ 100 μL of the solution into the syringe by pulling the plunger back slowly.
5. Remove air bubbles, holding the syringe upright (with the needle upward). Gently pull the plunger down and tap the side of the syringe to help any air bubbles attached to the inner part of the tube come upwards. Slowly press the plunger to release large air bubbles. A few drops of liquid will come out the needle. At this point, make sure to pick the liquid up again into the tube and not disperse it, given the toxicity of cardiotoxin.
6. Withdraw the solution into the syringe until the desired dose is reached. Inject 20 μL of CTX solution for each TA muscle.
7. Insert the needle in the center of the TA by following the position of the Tibia bone as guidance, from the tendon towards the knee. Since the TA is a thin tissue, perform the injection without going too deep (insert the needle 2/3 mm deep, at approximately a 10° to 20° angle ), and avoid going beyond the muscle itself (Figure 1A).
8. Do not pull out the needle immediately after the injection, but wait 2 – 3 s to prevent leakage of CTX drops from the muscle.
9. After the procedure, place the cage on a heated plate (at 37 °C) until the mice are awake, since the anesthetic and the ethanol used reduce body temperature.

2. Tibialis Anterior Isolation

Note: Muscles can be isolated at different time points after cardiotoxin injection according to experimental requirements.

1. Sacrifice the mouse by cervical dislocation.
2. Spray the hindlimbs with 70% ethanol. During the procedure, the animal can be fixed to a support to hold it down.
3. Cut the skin at the level of the proximal tendon (above the foot), insert one of the blades of the scissors under the skin, and cut the skin upwards, in the direction of the knee. With the help of forceps, pull up the skin and expose the muscle entirely. Carefully remove all of the skin and the hair from the area of interest.
4. Eliminate the fascia by slowly pinching or cutting (with thin-tip tweezers or with fine micro dissecting scissors) the extreme periphery of the muscle on the opposite side of the tibia (Figure 1B). Once broken, gently remove the fascia with the help of thin-tip tweezers; avoid damaging the underlying muscle.
   Note: As with other muscles and organs, the TA is covered by fascia (deep fascia of the leg), a thin sheet of connective tissue that lies deep under the skin and that needs to be removed to easily isolate the underlying muscle.
5. Visualize the distal TA tendon and the Extensor Digitorum tendon above the foot, which are juxtaposed. The tendon of the Extensor Digitorum Longus (EDL) muscle is located slightly below the TA tendon. Tendons of adult mice are visible to the naked eye.
6. Remove the TA muscle following one of the two following techniques:
   1. Cut both tendons with fine micro dissecting scissors and pull them towards the proximal side, holding the tendons with forceps. Separate the two tendons, pulling them gently in opposite directions to separate the muscles.
   2. With the help of thin-tip tweezers, separate the distal TA tendon from the Extensor Digitorum tendon by passing the tip below the tendon itself (Figure 1C, upper picture). Gently slide the tweezers below the TA to separate it from the underlying muscles (Figure 1C, bottom picture).
   3. Hold the tweezers under the muscle to keep it slightly raised, cut the TA tendon, and gently pull it upwards until only the area below the knee is attached (Figure 1D, left picture). Cut the TA below the knee, following the edge of the muscle with the scissors (Figure 1D, right picture).
      Note: If the muscle remains slightly attached to the sides, use fine micro dissecting scissors to help detach it.
      Note: If the forceps do not easily fit below the TA, it is possible that the fascia has not been completely removed, and it could still be visible on top of the muscle. In this case, completely remove the fascia before proceeding.

3. Fresh Frozen Muscle Technique

1. Place a small amount of tragacanth gum or a similar adhesive freezing compound (e.g., Optimal Cutting Temperature (OCT) compound) on a slice of cork. Label the cork's slice on the reverse side before freezing, if necessary (Figure 2A).
2. In order to obtain transverse sections of the muscle, sink the TA into the tragacanth gum by inserting the distal tendon and leaving about 3/4 of the muscle outside, making sure to have the muscle in a perpendicular position with respect to the cork (Figure 2A).
3. Fill an aluminum can (alternatively, a metal cup or a glass beaker), with isopentane. Suspend the can of isopentane in a Dewar containing liquid nitrogen, not allowing the liquid nitrogen to enter into the can.
   Note: Isopentane needs to reach the proper temperature (from -150 to -160 °C) for optimal freezing of the tissue. The correct temperature is reached when some white solid particles form at the bottom of the can and the isopentane becomes slightly viscous.
   Note: Do not freeze the sample before the formation of the solid particles, as it can lead to freezing artifacts. On the other hand, if the isopentane becomes completely solid, leave it at room temperature until it is thawed, making sure that some white particles are still present.
4. Using Mixter forceps, rapidly dip the cork with the muscle into the isopentane, keeping the muscle positioned downwards, and make sure to release the cork when it is completely immersed in the liquid. In this way, while the specimen floats, only the reverse side of the cork should be visible in the liquid.
5. Leave the sample in the isopentane for 1 min.
6. Using Mixter forceps (or similar forceps), take the muscle and immerse it in liquid nitrogen for at least 2 min.
7. Wrap the specimens individually in labeled aluminum foil and immediately place them in dry ice (-70 °C), or directly store them in a -80 °C freezer.
   Note: Throughout the procedure, from steps 3.4 to 3.7, it is extremely important to maintain the correct temperature and to prevent the specimens from thawing (e.g., during the transfer to the -80 °C freezer). The rising temperature results in uncontrolled thawing and refreezing of the water microcrystals that are present within the tissue and eventually causes the formation of artifacts or the breakdown of muscle fibers (visualized on the tissue sections as large white holes in the center of the fiber).

4. Cryostat Sectioning of Frozen Muscles

1. Set the cryostat chamber temperature to -20 to -22 °C.
2. Put the specimen stub, the blade, and the specimen in the cryostat chamber, allowing them to equilibrate for at least 30 min.
3. Place a small amount of freezing compound (e.g., OCT compound) on the specimen stub and immerse the cork with the specimen before the freezing compound solidifies. In the meantime, place the blade in the blade holder.
4. Align the specimen and the blade using the provided screws to orient the specimen holder with the blade holder (Figure 2B).
5. Set the section thickness to 10 μm. Proceed to cut the tissue. For each turn of the drive wheel, the specimen holder advances a controlled distance towards the blade.
6. Place the sections on a polarized slide (8 to 10 section per slide) (Figure 2C).
7. After sectioning, store the slides at -80 °C.
   Note: The specimen can be stored in a -80 °C freezer and reused for further sectioning, if properly handled and stored.

5. Routine Histological Staining (Hematoxylin & Eosin Stains)

Note: Several histological stains can be performed on muscle sections according to the analysis. A routine histological stain for morphological and morphometric analysis is the Hematoxylin & Eosin (H&E) bichromic stain. The hematoxylin stains the nuclei a deep purple. Nuclear staining is counterstained with eosin (pink/red), which stains eosinophilic structures, such as the myofibers in the cytoplasm.

1. Completely air-dry frozen slides with sections for 10 – 15 min at room temperature, lodge the slides in a staining tray, and put the tray in a staining trough.
2. Stain for 1 min with hematoxylin.
3. Lay the staining trough under a fairly weak tap water jet for 10 min to wash out the hematoxylin.
4. Immerse the slides for 5 min in 95% ethanol, if an alcoholic solution of eosin will be used (skip this step in case of an aqueous eosin solution).
5. Counterstain for 1 min with eosin solution.
6. Rinse with water to eliminate excessive eosin solution.
   Note: Optimal timing of staining with either hematoxylin or eosin should be defined for each new batch.
7. Dehydrate the samples in a graded ethanol series: 50% and 70% (a few seconds each). Rapidly immerse in 95% ethanol, followed by 2 changes in 100% ethanol, 5 min each.
8. Clear in 2 changes of xylene, each 5 min or longer. This step must be carried out under a chemical hood.
9. Mount the slides with a xylene-based mounting medium, applying a few drops of mounting medium on the slide and covering with a coverslip. The mounting medium can also be applied to the coverslip and the coverslip put on the slide.
   1. Avoid forming air bubbles between the slide and the coverslip. To eliminate air bubbles, keep the slide vertical (after mounting) and squeeze out the excess mounting medium and air bubbles by gently pressing on the coverslip with blunt forceps (or a similar blunt tool).
10. Keep the slides under the hood for a few hours or overnight to let the xylene evaporate completely before proceeding to the analysis.
11. Perform the morphological and morphometric analysis of H&E-stained regenerating muscle sections by using non-overlapping serial images of at least one whole muscle section per mouse. Capture images with a bright-field microscope, with 20X magnification.
    Note: A minimum number of 5 mice is recommended to obtain statistical significance of the measurements.
    Note: Image analysis software is available for this purpose, such as the free download software ImageJ (http://imagej.nih.gov/ij/).