Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.
This model can help answer key questions in the field of muscle contraction function. Such as the mechanism of force production, during the disease progression. The main advantage of this technique is that this procedure is a non-invasive, which can be performed on the same animal over different time points, to monitor disease progression and treatment.
Visual demonstration of this method is critical, as alteration to any of these steps may lead to increase variability in your results. Therefore, practice may allow you to obtain more consistent results. To begin this procedure, set up the mouse knee clamp on the platform, as well as the mouse footplate on the transducer.
Then turn on the heating platform to 37 degrees Celsius. Open the dynamic muscle control software on the desktop. On the setup drop down list, select instant stem, and change the run time parameters to 120 seconds.
In the window labeled autosave base, input the name of the autosave file location. Click the check box to the left of the autosave base window, and change it to enable autosave. At the top of the DMC control screen, click sequencer, which will open a new pop-up window.
Select open sequence and select the protocol to be used. Then click load sequence and close window. To prepare the animal for experiment, remove the hair on the right leg of the mouse with electric hair clippers.
Place the animal in a supine position on the heated platform and clean the right leg with 70%alcohol and iodine. At this point, adjust the Isoflurane to 2%with oxygen flow at one liter per minute. Place the foot onto the footplate and secure it using medical tape.
Then apply the conductive gel to the skin where electrodes will be placed. Next, clamp the knee to stabilize the leg during the procedure. Use the knobs on the platform to position its hind limb in order to form a 90 degree angle at the ankle.
Once the mouse is secured on the platform, position the electrodes under the skin of the right leg, one near the head of the fibula, and the other electrode more distally on the lateral side of the leg. The electrodes are positioned careful in order to achieve sufficient stimulation of the Peroneal nerve. Also this position can be adjusted to reach a stable peak, visualized on the screen.
Once these steps are achieved, adjust the high power bi-phase stimulator to obtain a stimulation of the Peroneal nerve that results in maximum dorsiflexion torque. During stimulation, turn the transducer clockwise to yield negative values, which are important to ensure that the electrodes are stimulating only the dorsi flexor muscles by Peroneal nerve. Once this step is achieved, stabilize the electrodes using a clamp, in order to prevent any movement during the procedure.
Achieving the negative peak slowly and gradually is important to verify that the electrodes are placed in the correct position, and the voltage is sufficient to achieve the maximum force. Subsequently, stop the instant stem on the software. On the main screen, turn on the button labeled start sequence"to start the previous setup sequence.
Once the force measurements are finished, remove the electrodes, remove the foot tape, and release the knee clamp. Turn off the Isoflurane and maintain oxygen delivery for a few minutes to aid the animal's recovery. Once the mouse starts moving and regains consciousness, return it to the cage.
This graph shows the representative force frequency curve data from a mouse across time, where the procedure was repeated once a week until five time points were completed. These observations have shown consistent force production values throughout the time points. This procedure has also shown to be consistent between different mice, as indicated in the representative area, under the curve of the FFC stimulation, over five different observations in six mice tested once a week.
After its development, this technique allows researchers in the field of neuromuscular diseases to investigate temporal changes in muscle function in mirroring models of disease.
