Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

This protocol is one of the first studies that enables scientists to perform dosage-adjusted resistance training in mice similar to how it's performed in humans. The main advantage of this technique is that it allows investigators to precisely adjust resistance against which the mouse's anterior tibial muscles have to work during resistance training. Furthermore, since muscle contractions are eccentrically biased, there is a lower chance of contraction-induced muscle injury.

Our dosage-adjusted resistance training technique can be incorporated into basic and preclinical studies in mice to develop interventions for the improvement or maintenance of muscle mass and strength across a diverse array of disease conditions. The most challenging aspect of the protocol is to precisely stimulate the fibular branch of the sciatic nerve, which innervates the anterior tibial muscles. To reduce inaccurate electrical stimulation, adjust the electrode position and stimulation amplitude until maximum twitch torque is recorded by a dynamometer.

To begin, provide thermal support to the mouse by using an isothermal gel heating pad and place a heat lamp 1 meter above the mouse. To prepare the skin for DART or ISOM, remove the fur from the left hind limb by applying a depilatory cream. After 2 minutes, clean the leg with wipes soaked in distilled water to remove fur and all residual cream from the skin.

Disinfect the skin using a povidone iodine scrubbing solution and 70%ethanol, then use a clean cotton swab to apply a protectant over the eyes and the depilated skin to prevent drying. Next, apply 5%lidocaine cream over the tibia to numb the area. Pass a 1/2-inch 26 gauge sterile hypodermic needle through the widest part of the proximal portion of the tibial bone.

Once the stabilizing pin is secured, hold the needle with a sterile hemostat and bend the plastic portion until it breaks off, then lay the mouse in a supine position. Ensure that the mouse is still securely connected to the nose cone to maintain anesthesia. With a pair of sterile tipped tweezers, feed the tibial pin into a metal alligator clip, such that the end of the tibial pin are held by the alligator clamp.

Move the adjustable arm of the alligator clamp to ensure that the foot of the mouse is placed on the footplate of the DART device. Strap the foot of the mouse onto the DART device footplate with adhesive laboratory tape. Ensure that the foot is placed at a 90-degree angle in relation to the long axis of the mouse tibial bone.

Place an 18 gauge 1.5=inch-long hypodermic needle through the pre-drilled holes on the protractor of the DART device to create a plantarflexion stop, then rest the footplate on the plantarflexion stop. To optimize the electrode placement, place a bipolar, transcutaneous, and NMES electrode on the inferolateral aspect of the mouse knee joint. Use a laboratory electrical stimulator to apply single pulses of 1 hertz to stimulate the fibular branch of the sciatic nerve.

Observe the tibialis anterior, or TA belly muscle and tendon for evidence of electrically-elicited twitch contractions. Now, strap the suture to the dynamometer floor plate, then optimize the amplitude of the voltage output from the NMES stimulator so that NMES is confined to the common fibular nerve and TA muscle. To set the stimulator to produce repeated pulse trains, adjust the dials for pulse frequency to 125 hertz, train duration to 500 milliseconds, and trains per second to 1.

Turn on the toggle switch for repeating pulse trains. Set the stimulator to produce pulse trains that are 500 milliseconds in duration interspersed with 500 millisecond rest between pulse trains. Move the plantarflexion stop to the hole on the protractor that corresponds to 160 degrees on the long axis of the tibia.

In DART, for the TA muscle to work concentrically, apply resistance by hanging a suitable weight such as 5 grams with a non-elastic silk suture tied to the DART device footplate. Adjust the resistance by applying 50%weight of the 1-repetition maximum. Ensure that the foot pulls through at least half of the available active range of dorsiflexion.

Perform a single bout of DART training that involves 1 set of 10 repetitions of concentric contractions and 2-minute rest between the sets. For ISOM training, place the foot of the mouse at 160 degrees to the long axis of the tibia. Maintain the static position by taping the silk suture to the footplate of the robotic dynamometer.

Perform a single bout of ISOM training that involves 4 sets of 10 repetitions to isometric contractions and 2-minute rest between sets. As post-procedural care for mice, take precautions to maintain the hygiene of the exercised hind limb and reduced needle site pain. Histological changes in the TA muscle were studied after 3 days of DART or ISOM training.

Hematoxylin and eosin staining indicated that the extent of muscle damage was low in both DART and ISOM groups, but muscle damage was slightly more obvious in the ISOM group. It is important to specifically stimulate contractions in the anterior tibial muscles and precisely adjust the resistance against which these muscles have to work during dosage-adjusted resistance training. Following this procedure, investigators will be able to assess exercise tolerance in the form of susceptibility to fatigue and injury from repeated muscle contractions against resistance.

This technique can be incorporated in a wide variety of basic and preclinical research applications, such as studies on mouse models, of neuromuscular diseases like muscular dystrophies and mouse models of sports injuries.