Keypoint

At the 12th week post-transplantation, a fully functional electromyography machine performed neural electrophysiological evaluation on six rats in each group (Keypoint; Medtronic). Briefly, exposing the nerve graft along the original incision, the stimulating electrode was placed on the proximal end of the graft and the recording electrode was placed inside the abdomen of the gastrocnemius muscle, and then the compound muscle action potentials (CMAPs) were induced and recorded. Meanwhile, CMAPs of the contralateral normal sciatic nerve also need to be recorded. The parameters of electrical stimulation were 3.0 mA and 1 Hz. The recovery of nerve conduction function was evaluated by the peak amplitude and latency of CMAPs.

Nerve conduction studies were performed only on visit 1 by a senior consultant neurophysiologist. Nerve conduction studies of the common peroneal (including F wave studies) and sural nerves in the right leg were performed with a Medtronic Keypoint device (Medtronic, Minneapolis, MN, USA). Nerve conduction studies were performed once at the baseline visit for all patients. Sural antidromic sensory action potentials of <5 μV amplitude and <40 m/s conduction velocity, and common peroneal nerve (compound muscle action potential from extensor digitorum brevis) values <3 mV amplitude, <40 m/s conduction velocity, were considered abnormal (35 (link)).

Electrophysiological studies and readouts were done and validated at Neurofit laboratories. Rats or mice were anaesthetised by 2.5–3% isoflurane-air mixture. CMAP was recorded by needle electrodes placed into the intrinsic foot muscles of the plantar surface (Keypoint electromyography, Medtronic, France). Subcutaneous monopolar needle electrodes (Medtronic, 9013R0312) were used for both stimulation and recording. 12.8 mA square wave pulses of 0.2 ms duration were used to stimulate sciatic nerve. For the CMT1A rats (after 8 months of treatment, 9 months of age), the latencies of CMAPs elicited by stimulation at both proximal (hip) and distal (hock) sites were measured. Motor Nerve Conduction Velocity (MNCV) was estimated by the time interval between two stimulation sites latencies relative to distance between these sites (with leg fully extended) [5 (link),17 (link)]. For the CD-1 crushed mice (42 days of treatment), the right sciatic nerve (ipsilateral) was stimulated with single pulse applied at the sciatic notch. CMAP was recorded by needle electrodes placed at the gastrocnemius muscle. The amplitude of the action potential was determined 30 days after the crush.

Nerve conduction and sympathetic skin response (SSR) studies were performed only at baseline to characterise the neuropathy, on a Medtronic Keypoint electromyogram system (Medtronic, Minneapolis, MN, USA) (3 (link)). Antidromic sensory action potentials for the sural and superficial peroneal nerves and orthodromic sensory action potentials for the medial and lateral plantar, median, and ulnar nerves on both sides were recorded. For the sural, superficial peroneal, medial plantar, and ulnar nerves, action potentials of <5 μV amplitude were considered abnormal, while for the lateral plantar and median nerves, amplitudes of <2.5 and <10 μV were considered abnormal, respectively. Sensory conduction velocities of <50 and 40 m/s were considered abnormal for upper and lower limb nerves, respectively. The nerve compound action potentials were recorded using standard described techniques, and the issue of skin resistance was mitigated by: i) carrying out pre-recording skin preparation identically in all subjects, and ii) by using the identical type of recording electrodes in all subjects. These were also the methods employed while collecting normative data from our control subjects. SSRs were recorded from both palms and both soles in response to a sudden unexpected tactile stimulus and a sudden inspiratory gasp. An absent SSR was considered abnormal.