Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

The chronic contractile activity model of exercise is useful to study skeletal muscle adaptations to exercise training stimuli in vivo. In particular, this model elicits the phenotypic adaptations of a six week training protocol within seven days. This method can help answer key questions in the muscle physiology field like mitochondrial biogenesis, autophagy, and many other exercise-associated cellular adaptations.

The main advantage of this technique is that it allows for the observation of muscle specific adaptations over a short time course of seven days as compared to other exercise training protocols that require several months. The implications of this technique extend toward therapy of muscle atrophy ages because chronic muscle stimulation has been shown to have an effect on ameliorating aging-related muscle weakness. Generally individuals new to this method will struggle because of difficulty landmarking the common peroneal nerve and attaching electrical coils at either end as these require practice and precision.

Begin by anesthetizing the rat under one to 3%isoflurane inhalation with oxygen and confirm full sedation by checking the response to a hind limb toe pinch and observing respiratory depth and rate. Apply ocular lubricant on eyes to avoid dryness and place the rat on a heating pad at a low to medium setting. Gently shave the left hind limb as well as a strip around the torso from the back of the neck around behind the forelimbs and across the anterior thorax.

Gently wipe shaved areas with iodine and ethyl alcohol to disinfect. With the animal laying on its stomach use a sterile scalpel with a number 10 blade to make a small incision of around 0.5 centimeters on the back of the neck in the center of the shaved region. Then roll the animal onto its right side and make a two to three centimeter cut in the skin of the left hind limb between the dimple of the knee joint and close to the origin of the tail.

Using blunt tipped curved surgical scissors dissect the subcutaneous area to around 3.5 to four centimeters separating the skin from the underlying muscle to make a pocket between the open skin and underlying muscle. Next, use surgical scissors to make a small incision of less than 0.5 centimeters on the biceps femoris muscle ensuring that the tips of the scissors are cutting directly down through the muscle. Then gently open the cut area until the internal muscle groups and the common peroneal nerve are visible.

Use extreme caution to avoid cutting or damaging the nerve. Next prepare 50 to 60 centimeters of PTFE coated fine stainless steel wire and fold it in half. Hook the folded part of the wire into the slit of a 30 centimeter stainless steel rod.

Use the rod to pass the wire through the open pocket of the hind limb up the leg and along the center of the back in an L shape pattern to reach the small incision area at the back of the neck. Fix the window by pulling it open with metal retractors until the size of the window is approximately 1.5 squared centimeters with the peroneal nerve lying in the center of the window. Using a scalpel, carefully strip the ends of the wire by 1.5 centimeters.

Wrap the stripped wire ends around a blunted 21 gauge needle five times to make a coil. Once the coils are properly made release the needle from them. Make a knot at the very end of the coil and suture it on the left hand side of the nerve ensuring that the coil is at 1.5 to 2.5 millimeters from the nerve.

To secure the coil apply two or three additional sutures along the coil. This step requires considerable practice and patience in order to avoid damaging the nerve and underlying muscle and to achieve all consistent stimulation condition across all animals. It's best to have one skilled researcher to complete all surgical procedures during a study.

Apply two or three drops of antibiotic solution and then carefully suture the window using size 5-0 silk. Loosely wind the remaining slack of wire and push into the subcutaneous pocket above the sutured incision of the biceps femoris muscle above the hip. Apply two to three drops of antibiotic solution and close the open skin by stapling.

Next, move the animal to the sternal position and cut the wire loop coming out of the incision at the top of the neck to create two wire ends. Using a scalpel, strip of the ends of the wires by 0.5 centimeters. Cut off any frayed wires.

Slowly push the stripped parts of the wires into the hole of the pin sockets and using a soldering iron, solder the wires on the pin sockets. Pass the pin connected wire ends through a 4.4 centimeter sterile gauze pad. Then pass the wires through the hole in the base of the stimulator box.

Insert the pins into the connection sockets on the CCA unit. Gently put the CCA unit into the chamber using a sticky tack to secure the CCA unit at the bottom of the chamber. Use athletic tape or porous surgical tape to fix the chamber around the shaved torso.

Close the top of the chamber with three layers of taping. And finish by wrapping tape around the sides of the stimulator box to secure the box. Check if the CCA is working by exposing the unit to a single pulse of infrared light emitted by a portable infrared strobe light.

If the CCA is properly working the hind limb muscles will contract in response to the infrared light. Weigh the animals shortly before beginning the CCA procedure to record a baseline measurement to help identify any severe stress or adverse effects by change of body weight after the CCA procedure. On the day of CCA stimulation turn on the CCA by exposing the stimulator unit to a single pulse of infrared light by a portable infrared strobe light and apply three or six hours of 10 hertz CCA stimulation.

Check the stimulation and animal every 30 to 60 minutes. Following the desired CCA period turn off the CCA unit via infrared light exposure. Rats subjected to seven days of CCA for six hours per day display enhanced mitochondrial biogenesis in the stimulated muscle as compared to the unstimulated contralateral hind limb.

This increase in mitochondrial biogenesis is indicated by increased protein expression of PGC-1 alpha. Examination of permeabilized muscle fibers to measure mitochondrial respiratory capacity reveals that CCA resulted in an increase in the maximal respiratory capacity of muscle relative to control muscle. Both subsarcolemmal and intermyofibrillar mitochondrial populations increased following seven days of CCA compared to control unstimulated muscle.

Adaptations to the autophagy and lysosomal systems can also be brought about by CCA. An increase in the protein abundance of Transcription factor EB the principal regulator of lysosomal biogenesis is observed after CCA at all time points. Following this procedure other methods like general subcutaneous or intraperitoneal injections can be performed in order to answer additional questions such as estimation of autophagy flux with colchicine treatment.

After watching this video you should have a good understanding of how to apply the current chronic contractile activity model in order to examine muscle phenotypic changes following endurance training.