Among our SCN9A+ patient cohorts, only 4 patients reported clinical improvement with mexiletine, accompanied by a 1- or 2- point reduction in Faces pain rating scale. These patients were studied using nerve excitability tests before or after medication, and the results are shown in Figures 4C and D; there is a reduction in SDTC in both motor and sensory axons while other excitability measures were not altered (motor SDTC: pre-mexiletine, 0.46 ± 0.03 ms vs on mexiletine, 0.42 ± 0.04 ms; sensory SDTC: pre-mexiletine, 0.61 ± 0.00 ms vs on mexiletine, 0.51 ± 0.08 ms). These results are in agreement with previous reports on the effects of mexiletine on excitability parameters.15,40,41,50,73
A mathematical model of the human sensory axon was used to assist in the interpretation of the changes measured in clinical nerve excitability in patients with EM. Membrane hyperpolarization best explained the differences between the EM SCN9A+ and EMSCN9A− patients and the sensory controls. Specifically, hyperpolarization of the resting membrane potential by 2.8 mV accounted for 89.4% of the discrepancy between the EMSCN9A+ and sensory control data. Similarly, a 1.6-mV hyperpolarization of resting membrane potential accounted for 83.6% of the discrepancy between the EMSCN9A− patients and sensory controls. Internodal shortening was also included in the modelling and reduced the discrepancy by 25%. It was not considered the most plausible explanation for the findings. The model did not show any further improvement with additional changes in Na+ channel expression or the fraction of Na+ channels operating in a persistent mode. The effect of temperature on nerve excitability changes in EM is shown in Figure 5. The 5° warming in 1 subject was modelled well by a 2-mV depolarization of resting membrane potential from the EM model.
This is the first study of primary EM using threshold tracking techniques to evaluate peripheral motor and sensory axonal function and the excitability changes related to temperature and mexiletine treatment. The techniques applied in this study allowed noninvasive in vivo assessment of the biophysical properties of axonal membranes, although Nav1.7 is not known to be expressed on large alpha-myelinated motor fibres and may only be expressed in some of these sensory fibres.3,24 Accordingly, this approach differs from previous electrophysiological studies of EM, which are focused on the short-term effects on single cells by voltage and current clamp directly or microneurography at the site of abnormal Nav1.7 expression.60 Notably, there were significant differences in sensory and motor excitability parameters between EM patients with and without SCN9A pathogenic variants compared with the healthy controls in this study. These findings reveal alterations in peripheral axonal membrane potential and conductances and reflect systemic abnormalities in EM. A paradoxical pattern of changes in Na+ channel-dependent parameters with heating was evident in SCN9A mutant patients in comparison to normal controls. There was variable and partial clinical benefit or change in neurophysiological parameters with pain medications, revealing challenges in therapy.
Significant reductions in sensory axonal excitability were seen in both groups of EM patients, with a “fanning out” of responses during TE, increases in threshold and rheobase, similar to the effects established with membrane hyperpolarization.42 A similar but lesser pattern of changes occurred in motor nerves of patients with EM.35 While these changes are subclinical, in part related to Nav1.7 not being expressed in these neurons, they are important in comprehensively understanding EM pathophysiology and systemic effects. In the same way, subclinical changes in axonal membrane function have been demonstrated with temperature changes or maturation paralleling alterations in conduction velocity and in many neuropathies.28,44,51,61 Associated with greater membrane hyperpolarization in EMSCN9A+ patients, there was also evidence of reduction in slow K+ currents, with decreased subexcitability, S2 accommodation, TE undershoot, and greater superexcitability. The mathematical modelling suggests that these changes are secondary to the changes in membrane potential, as approximately 35% of slow K+ channels are open at resting membrane potential.66
Altered ion channel expression or gating properties may provide an alternative explanation for alterations in membrane potential.67 While this was not supported by changes in multiple excitability parameters or mathematical modelling, this may be complicated by interneuronal heterogeneity in the dynamics of Na+ conductances that reflect differences in the structure of the underlying channel molecule in the neuronal membrane.65 Alterations in passive cable properties (myelin thickness and internodal length) may also change membrane potential or ion channel conductances, for example, shorter internodes in regenerated nerve and an increased number of nodes provide a greater intra-axonal Na+ load to drive the Na+/K+ pump.58 This concept is unlikely as latency and conduction velocities were maintained and mathematical modeling did not support this as the most plausible explanation.
We deliberately chose to test the median nerve at the wrist, remote from the ongoing clinical symptomatology, to avoid possible secondary changes associated with EM (eg, cutaneous thickening or swelling) that may have influenced nerve excitability testing and thereby ensuring changes were constitutional.
The most striking result was the significant increases in SDTC in EMSCN9A+ patients, an indirect measure of the activity of nodal persistent Na+ current with heating,9 the opposite of controls.36 Similar to controls, increases of skin temperature reduced superexcitability and shortened accommodation to depolarizing current, related to activation of slow K+ channels during hyperthermia. Heating typically triggers acute EM symptoms, such that the excitability changes with increased temperature are likely to measure acute modulations in membrane potential and conductances. Mathematical modelling indicated that the acute excitability changes with heating in patients with EM correspond to axonal ischaemic depolarisation and supporting the concept of vascular dysregulation.42 This will produce an intra-axonal Na+ load to chronically drive the Na+/K+ pump. Recent in vitro studies have demonstrated that heating selectively increases excitability in gain-of-function mutant Nav1.7 channels, in comparison to wild-type Nav1.7 channels,.12,82 Together with increases in skin metabolism and oxygen consumption, the resultant neurovascular changes would exacerbate tissue hypoxia and worsen cutaneous arteriovenous shunting in EM, creating a vicious cycle to evoke the heat hyperalgesia.59 It is possible that these paradoxical changes may also be caused by structural membrane changes as a function of temperature, disturbing the temperature dependence of Na+ channel conductance and gating kinetics.68 Tissue oedema from skin hyperaemia and release of inflammatory peptides may also influence the membrane structure and Na+ channel activity. Importantly, increases in persistent Na+ conductances in peripheral axons have been linked to a number of common painful neuropathies,17,52,53 participating in the generation of ectopic neuropathic symptoms.
Concerning the episodic excruciating pain in patients with EM, a frequently used treatment of this disorder are the sodium channel blockers such as lidocaine and mexiletine. This nonselective partial block of voltage-gated Na+ channels targets segment 6 of domain IV of the alpha subunit.63 Clinical response varies and suggests that genotype may be important.12,81 In agreement with the previous study by Kuwabara et al.,50 the effect of mexiletine on axonal excitability in EMSCN9A+ patients demonstrates small reductions in SDTC. Even so, SDTC remained increased such that it may be expected to have an ongoing part in neuropathic symptoms. There is an urgent clinical need to develop more effective Na+ channel blockers for the treatment of neuropathic pain, particularly the selective and potent Nav1.7 channel blockers, with initial results showing promise in EM.12
The variability of pain between and within subjects poses challenges in assessing response to treatment and highlights the need to develop sensitive and relevant biomarkers to facilitate establishing novel disease modifying therapies. Recently, molecular modelling, thermodynamic analysis, and functional profiling were able to predict the responsiveness to treatment and may provide a platform for personalized medicine in the future with a preclinical assessment of potential efficacy.32
Excitability studies are now being used widely in the clinical investigation of patients with neurological disease to provide functional information about peripheral nerves, with changes in measurements of excitability being interpreted in terms of direct and/or remote effects of altered ion channel function.47 These have provided novel insights into disease mechanisms and treatment strategies.26,27,29,49,54,57,73 This study advances this possibility by exploring the variations in excitability properties along with temperature in patients with EM. Specific indices of nerve excitability responded very differently to increases in temperature and these effects can be related to the indirect behaviour of mutant Nav1.7 channels systemically, producing a vicious cycle of pain. These preliminary findings support the use of excitability techniques as a biomarker to assess abnormalities of axonal function and monitor changes with the development of novel therapies in vivo.
M.-J. Lee was supported by the grants from National Taiwan University Hospital (NTUH 105-003153) and the Ministry of Science and Technology, Taiwan, R.O.C. (MOST 104-2314-B-002-061).
M. A. Farrar and M.-J. Lee contributed equally to this article.
M.-J. Lee thank the assistance from the second and third common laboratory, National Taiwan University Hospital, Taipei, Taiwan.
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