Our protocol can be useful in the systems and mechanism. And the right information shows the effect of physical stress and massage. Our local cyclic completion system is much more simple.
The conventional methods aren't required for local insertion. Apply surgical tape to the bilateral hind limbs in the supine position with the knee joints extended and the ankle joints plantar flexed. Place an aluminum wire on the trunk at the L4-5 spine level, and coil the wire in a spiral configuration around the hind limbs with five millimeter gaps between each turn of the spiral.
To minimize the possibility of escape from the wiring, manually adjust the wire configuration to immobilize the hip joints at a 90 degree position of abduction. Then return the mouse to it's home cage with monitoring until full recovery. To measure the intramuscular gastrocnemius muscle pressure place the anesthetized mouse in the prone position and shave, depilate, and semi-sterilize the skin of the posterior calf.
Use a scalpel to make a two millimeter incision in the disinfected skin. And insert a 20 gauge indwelling needle into the gastrocnemius muscle at a 150 to 170 degree angle to the skin surface. Next, using the plastic sheath of the needle as a guide, place a blood pressure telemeter sensor in the mid belly of gastrocnemius muscle.
Enclose the skin with a 4-O nylon suture. Then, use several different cylindrical unit weights to apply local cylindrical compression to the calf muscle for one minute. And monitor the intramuscular pressure using the appropriate software for biological signal analysis.
For local cyclical compression treatment disengage the anesthetized mouse from the hind limb wiring. And place the animal in the prone position. Adjust the hind limbs so that the knee joints are extended and the ankle joints are plantar flexed with the calves facing upward.
And vertically move a cylindrical weight unit, covered with a cushioned pad, over the calf muscle at one hertz for 30 minutes per day for seven days. After each daily bout of compression rewire the mouse hind limbs before returning the animal to its home cage. At the end of the experiment depilate the posterior calf surface and make a skin incision to allow separation of the gastrocnemius muscles from the tibiofibular bone.
Quickly freeze the harvested muscle tissue in an optimal cutting temperature compound solution. And use a cryostat to obtain sections of the gastrocnemius muscles on glass slides. For immunohistochemical labeling thaw and dehydrate the sections by air drying at room temperature before using a hydrophobic pen to draw a ring around the tissue sections on each slide.
Next, place the slides in humidity chamber. And add 100 microliters of blocking buffer to each side. After 30 minutes at room temperature rinse the slides with two 5 minute washes in PBS-T, before labeling the sections with 100 microliters of the primary antibody of interest overnight at room temperature.
The next morning wash the slides 3 times in PBS-T for 5 minutes per wash. And label the samples with an appropriate secondary antibody for 1 hour at room temperature, protected from light. At the end of the incubation, wash the slides 3 times in fresh PBS-T as demonstrated.
And stain the nuclei with 100 microliters of DAPI for three minutes at room temperature in the dark. Then, wash the slide three more times. And mount the samples with a compatible mounting medium and cover slips.
For histomorphometric analysis, place the slides on the stage of a fluorescence microscope and view the samples under a 20-X subjective in the appropriate filters. Then, in the image analysis software measure the cross sectional area of each myofiber and count the number of cells expressing the experimental markers of interest. The cross sectional area of gastrocnemius myofiber are significantly decreased by hind limb immobilization.
Further, immunofluorescent staining analysis reveals that cells expressing MCP-1 and TNF-alpha are significantly increased in the gastrocnemius muscle tissues of immobilized hind limbs. Together, with the increase in cells positively stained with F4/80, hind limb immobilization appears to instigate calf muscle atrophy, involving local inflammatory responses, including macrophage accumulation. Among the several different local cyclical compression magnitudes tested by changing the weight of the cylindrical unit, the compression corresponding to 50 milliliters of mercury intermuscular pressure waves, appears to most efficiently alleviate the immobilization induced decrease in myofiber cross sectional area and to increase in the macrophage accumulation in gastrocnemius muscles.
A 66 gram local cyclical compression, applied at 1 hertz for 30 minutes per day for 7 days, significantly alleviates the observed immobilization induced decreases in the myofiber cross sectional area of the gastrocnemius muscles. More, local cyclical compression partially tempers immobilization induced decreases in the contracting force of the triceps surae muscles. In addition, local cyclical compression tempers the increase in F4/80 positive, TNF-alpha positive, and MCP-1 one positive cells in the gastrocnemius muscle tissue of immobilized hind limbs.
Our approach will provide a scientific basis for muscle rise in the patient and our potential strategy for preventive procedure. The rat's found the limited of physical inactivity. Using our system, we have started assessing the most patients that's muscles lack mechanic in the patient.
Moderate local industrial way to do it makes a perfect immobilization induced muscle atrophy.
