With this method we can estimate the degeneration of the axons as well as the demyelination in the peripheral nerves of mouse models of neurodegeneration. The main advantage of this technique is that this minimally invasive method is feasible for repeated measurements only one did not follow up of the multiple nerves in the same animals. The sensitivity of this method enables early detection of axon loss in the modulation even before notable more defects can be recorded thus allowing early detection of these defects.
To begin this procedure, anesthetize the mouse with isoflurane oxygen inhalation. Confirm adequate anesthesia by applying mild pressure to the hind limb walking pad to check the absence of a pain withdrawal reflex. Then place a nose cone on the animal's face to maintain anesthesia.
Ensure that the nose cone does not block the airway and the animal is breathing steadily. Next, maintain the animal's body temperature at 37 degrees Celsius using a thermostatic heating plate. To measure CMAP on the hind limb, position the mouse in the prone position.
Extend the hind limb and attach the paw on the work surface using adhesive tape. Then place five millimeters of the 27 gauge stimulating electrodes subcutaneously on both sides of the sciatic nodge without puncturing the underlying muscles with two centimeters between the electrodes. Similarly place the recording electrode subcutaneously above and parallel to the gastrocnemius muscle.
Afterward place the reference electrode subcutaneously next to the Achilles tendon in a 30 degree angle and leave two to five millimeters of the needle under the skin. Place the ground electrode subcutaneously on the side of the mouse in a similar manner as the stimulating electrodes. To measure CMAP on the forelimbs, position the mouse on the heading pad in the supine position and use adhesive tape to extend both forelimbs on the sides of the body.
Next, place five millimeters of the stimulating electrode subcutaneously without puncturing the underlying muscles on both sides of the forelimb to align with brachial plexus nerve. Then place the recording electrode subcutaneously above the biceps brachii muscle. Subsequently, place three millimeters of the reference electrode at a 30 degree angle on the walking pads and insert the ground electrode subcutaneously on the side of the mouse.
Electrodes are in close proximity of each other in this setup. Prevent electrodes from touching each other as this distorts the recording. Stimulate all axons using one pulse per second with 0.1 milliseconds stimulus duration.
To acquire data start the stimulation by pushing the recurrent stimulus button in the controller unit and turn the intensity controller knob to increase the stimulus. To reach super maximal stimuli apply increasing stimuli by turning the intensity controller knob until the amplitude of the CMAP response ceases to increase. From there further increase the stimulus by 20%to ensure that the CMAP amplitude has reached its maximal response.
End the stimulation by pushing the recurrent stimulus button again. Use the marker tool to indicate the following points in the recording. Initiation of the response, maximum positive peak, and maximum negative peak.
Initiation of the stimulus is determined automatically by the software. Determine the latency as a delay fro the initiation of the stimulus to the initiation of the response. Use the latency to evaluate demyelination in the axons.
Measure the amplitude from the maximum negative to maximum positive peak and use the magnitude of the amplitude to correlate the number of functional axons. Since the exact placement of the electrodes can affect the outcome value of the recording, replace the electrodes and measure the same nerve for three times using super maximal stimulus to ensure that the largest response is obtained. Use the average of the recordings.
After the measurements remove all the electrodes and leave the mouse to recover on the heating plate or under an infrared lamp for approximately two to five minutes until it has regained sufficient consciousness. Do not leave the mouse unattended and in the company of other mice until it has fully recovered from anesthesia. The C61 PMP22 mice over expressing three to four copies of the human PMP22 and heterozygote mice recapitulate a very mild CMT1A disease phenotype with mild demyelination and reduced CMAPs but with no visible phenotype.
In one and a half to two years of age of C61 PMP22 transgenic mice the CMAP amplitudes are reduced and latencies are prolonged both in the hind limbs and forelimbs. Amplitude was decreased both in hind and forelimbs in transgenic mice, and latency was prolonged in all limbs in CMT transgenic mice, and even subtle change in forelimbs was detected with this measurement. Requirement for stimulus intensity was increased in C61 PMP22 mice, which resembles the detected phenotype in CMT1A patients.
Once mastered CMAP recording can be done for both hind and forelimbs in 15 minutes when performed properly. The presented technique provides novel possibilities of the characterization of mouse models of neurodegenerative disorders such as amyotrophic lat sclerosis and Charcot-Marie-Tooth disease. Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders.
Similar recordings are used in clinical settings emphasizing the translational potential of this method.
