Terminal H-reflex Measurements in Mice

The H-reflex allows electrophysiological validation of the integrity of neuro circuits. We present a terminal protocol to quantify the H-reflex directly at the nerves of the forepaw in mice. Different to conventional rather nerve-unspecific transcutaneous recordings, the results are less variable between mice and measurement days.

Similar to H-reflex recordings in patients, as precise quantification allows electrophysiological characterization in disease models, e.g. spinal cord injury and stroke. The protocol requires solid experimental skills in working with surgery equipment in mice.

If you are unsure how to separate nerves from connective tissue and blood vessels, practice first. Demonstrating the procedure will be by Frederique Wieters, a Ph.D student from my laboratory. To begin, place the adult anesthetized 8-to 16-weeks-old C57 black/6J mouse on the covered heating pad and wait until the mouse is calm, breathing is stable, and reflexes are absent.

Turn the mouse on its back. Then, fix the forepaws with tape. Position the tape such that the measuring electrodes are easily inserted into the forepaws.

To measure the temperature of a mouse insert a rectal probe and fix it with tape. Apply eye ointment to prevent the eyes from drying out. Remove the fur carefully with a pair of fine, rounded scissors while lifting the skin with tweezers.

Subsequently, make a one-centimeter incision in the skin along the ventroposterior axis of the forepaw with a pair of fine, rounded scissors. Carefully remove the connective tissue and expose the underneath muscle and nerve. Then remove the exposed pectoralis profundus muscle using forceps to assess the median nerve.

Remove small amounts of blood and tissue fluid with soft tissue paper. Next, carefully cut the breast or axillary muscle from the top to bottom to expose the nerve bundle. Free the nerve bundle from the connective and muscle tissue over a length of approximately 1.5 centimeters.

Separate the ulnar and median nerve of the forepaw carefully using a bent glass pipette. The upper nerve is the ulnar nerve and the lower is the median nerve. Arrange the stimulator hook electrodes in parallel at a distance of 0.5 to one millimeter and use a micromanipulator to position the double hook close to the nerve.

Use the glass hook to lift the ulnar nerve onto the stimulation hook electrodes. Then, pull back the electrode with the nerve and separate it from the other nerves. Place the electrodes along the long axis of the paw to reduce the crosstalk between the muscles.

Superficially dry the electrode hooks attached to the nerve and apply petroleum jelly using a syringe to provide electrical insulation from the adjacent tissue. To measure H-reflex, position the electromyography electrodes intramuscularly in the forepaw. Then, position the reference electrode subcutaneously in the hind limb, held by a miniature alligator clip.

When the stimulator is switched on, observe a successful stimulation as tiny twitches of the forepaw. The minimum stimulation current to elicit the M-wave and tiny visible twitches in the forepaw are between 10 to 50 microamperes. Perform the nerve stimulation 15 times with 0.2-milliseconds-long pulses each.

With pauses of two minutes between the sets of stimuli, the frequency is increased from 0.1, 0.5, 1.0, 2.0, to 5 hertz. An illustration of the recording set up in pathways to measure the Hoffman reflex and muscle response is shown here. The ratio between H-and M-wave amplitude was evaluated.

The stimulus and respective stimulation artifact were set at zero milliseconds, followed by the direct M-wave and the subsequent smaller peak representing the H-wave. The M-wave occurs approximately two milliseconds after stimulation, and the H-wave after six to eight milliseconds due to the longer transit time through the spinal cord. Next to the separation of the nerves, the application of petroleum jelly to the electrode and also between the two hooks are critical.

This is a terminal experiment. In our lab, this technique provided a new gold standard reference to the transcutaneous H-reflex recordings.