低侵襲の筋肉 (MIME) の埋め込み - ドナー細胞の筋を容易にする新しい実験手法

The overall goal of this technique is to implant a small segment of donor muscle tissue into a host muscle, in order to promote donor-cell-mediated myogenesis. This method can help answer key questions in the regenerative muscle biology field, such as, can muscle tissue harvested post-mortem promote donor-cell-mediated myogenesis in a host muscle. The main advantage of this technique is that it is minimally invasive.

The implications of this technique extend toward therapy for non-lethal muscular dystrophies because a combination of muscle embedding, exercise and immunotherapy might be able to rejuvenate the regenerative potential of certain affected muscles. As a physical therapist and a biomedical engineer, I believe the minimally invasive nature of the MIME procedure is key because it provides a source of myogenic cells, along with a natural tissue scaffold without excessively disrupting host muscle structure and function. Demonstrating the procedure will be Miss Morium Begam, a research assistant in my laboratory.

To begin, euthanize 12 to 16 week old C57 black six donor mice as described in the text protocol. Use a pair of surgical scissors to open the skin over the anterior surface of the hind leg. After finding the TA muscle, remove it to visualize the EDL muscle located behind the TA muscle.

Slide the tip of a pair of forceps behind the muscle and pull gently to see if the toes extend, confirming that the muscle is the EDL. After identifying the EDL muscle, use approximately 10 centimeter long segments of 4-0 silk to tie guiding sutures onto the distal and proximal tendons. Make two double knots in the sutures of the proximal and distal tendons of the muscle.

Cut the distal EDL tendon and reflect the muscle. Then, cut the proximal tendon to release the muscle. Place the collected EDL muscles in a petri dish filled with mouse ringer solution.

Anesthetize the eight to 10 week old male NSG-GFP host mouse, according to the text protocol. Apply petroleum jelly to the eyes to prevent dryness. To prepare the skin, apply depilatory cream over the anterior aspect of the left hind leg and leave it on for two minutes.

Using PBS soaked wipes, remove the depilatory cream along with the fur. To clean the skin, alternate scrubbing with three wipes of povidone iodine solution and 70%ethanol. To create a needle tract in the host TA muscle, pass a surgical needle through the center of the muscle, along the muscle's long axis.

Make sure to pass the needle in a cephalo-caudal direction, along the length of the TA muscle and through the center of the muscle belly. After the needle insertion, pass the guiding sutures from one end of the donor tissue, through the lumen of the surgical needle in a caudo-cephalic direction. Retract the needle from the host muscle in a caudo-cephalic direction, to guide the donor tissue through the needle tract.

Properly place the donor EDL muscle tissue within the host TA muscle, use the guiding sutures at the caudal and cephalic ends of the donor muscle. After the embedding of the donor tissue, cut off the guiding sutures and make any adjustments with fine tipped forceps. Finally, seal the needle wounds in the muscle tissue by closing the wound dent with forceps and applying veterinary tissue adhesive with the tip of a 27 gauge surgical needle.

After MIME, inject 50 to 60 microliters of 1.2%Barium Chloride into the host TA muscle at three sites along the length of the muscle, proximal, middle and distal one third of the muscle belly. After the injection, clean the leg of the host mouse with ethanol and protect the skin by applying a thin layer of petroleum jelly. Place the mouse in a solitary recovery cage without bedding.

An isothermal gel heating pad should be placed under half of the cage to provide thermal support to the host mouse while still allowing the mouse to move to the unheated half. After completed recovery, return the mouse to its original cage. Use a pair of surgical scissors to open the skin over the anterior surface of the leg.

After locating the TA muscle, cut its distal tendon and reflect the muscle. Cut the proximal muscle tendon to release the TA muscle. Collect both the left or treated and the right or control TA muscles.

Weigh the harvested muscles by placing them on weigh paper and a weighing scale. For cryoprotection, dip the muscles briefly in mineral oil and then blot off the excess oil. To snap freeze the muscles, place them on aluminum foil and rapidly immerse them in a dual flask filled with liquid nitrogen.

Transfer the frozen samples to labeled cryogenic vials. Precise embedding of the donor tissue within the host muscle, is critical for the success of MIME. After three days, the donor EDL muscle remains implanted in the host TA muscle with needle marks still visible on the host muscle.

Additionally, the cross sections of the donor muscles, do not show green fluorescence. Whereas, the cross section of the host muscles do. In the cross sections of the implanted muscles, there is a clear border between the GFP positive husk muscle fibers and the GFP negative donor muscle fibers.

14 days after the treatment, all muscle fibers and the untreated muscles are GFP positive. But not in the treated host muscles. Many of these GFP negative fibers are shown to be viable with red desmin labeling.

This demonstrates that MIME leads to donor-cell derived myogenesis. There are chimeric muscle fibers present in the TA muscle. Detected by their low to moderate fluorescence, likely arising from the fusion of the host and donor myogenic cells.

Their presence across the entire diameter of the host muscle, suggests that donor myogenic cells can migrate several hundred microns within the muscle. Once mastered, this technique can be done in 15 to 20 minutes, if performed properly. After watching this video, you should have a good understanding of how to use the MIME technique to embed a segment of donor muscle into a host muscle, in order to facilitate donor-cell-mediated myogenesis.

The MIME technique paves the way for us to study whether or not, cadaveric human muscle can promote donor-cell-mediated myogenesis in a host mouse. It also helps assess if neuromuscular electrical stimulation is capable of enhancing donor-cell-mediated myogenesis. This method can also be applied to other systems such as, generation of animal models of human diseases by embedding biopsy human muscle into host animals and to test if muscle tissue mimetics containing myogenic cells can promote myogenesis.