Activation of peripheral nerve fibers by electrical stimulation in the sole of the foot

Abstract

Background: Human nociceptive withdrawal reflexes (NWR) can be evoked by electrical stimulation applied to the sole of the foot. However, elicitation of NWRs is highly site dependent, and NWRs are especially difficult to elicit at the heel. The aim of the present study was to investigate potential peripheral mechanisms for any site dependent differences in reflex thresholds.

Results: The first part of the study investigated the neural innervation in different sites of the sole of the foot using two different staining techniques. 1) Staining for the Nav1.7 antigen (small nociceptive fibers) and 2) the Sihler whole nerve technique (myelinated part of the nerve). No differences in innervation densities were found across the sole of the foot using the two staining techniques: Nav1.7 immunochemistry (small nociceptive fibers (1-way ANOVA, NS)) and the Sihler's method (myelinated nerve fibers (1-way ANOVA, NS)). However, the results indicate that there are no nociceptive intraepidermal nerve fibers (IENFs) innervating the heel.Secondly, mathematical modeling was used to investigate to what degree differences in skin thicknesses affect the activation thresholds of Aδ and Aβ fibers in the sole of the foot. The modeling comprised finite element analysis of the volume conduction combined with a passive model of the activation of branching cutaneous nerve fibers. The model included three different sites in the sole of the foot (forefoot, arch and heel) and three different electrode sizes (diameters: 9.1, 12.9, and 18.3 mm). For each of the 9 combinations of site and electrode size, a total of 3000 Aβ fibers and 300 Aδ fibers was modeled. The computer simulation of the effects of skin thicknesses and innervation densities on thresholds of modeled Aδ and Aβ fibers did not reveal differences in pain and perception thresholds across the foot sole as have been observed experimentally. Instead a lack of IENFs at the heel decreased the electrical activation thresholds compared to models including IENFs.

Conclusions: The nerve staining and modeling results do not explain differences in NWR thresholds across the sole of the foot which may suggest that central mechanisms contribute to variation in NWR excitability across the sole of the foot.

Figure 1

Staining of cutaneous nerves across three sites on the sole of the foot using Sihler’s method. Nerve fibers were stained in abundance across all three sites; A – Forefoot, B – Arch, C – Heel. The epidermis in the heel was thicker than in the forefoot and arch. Thus, the identifiable nerves terminated deeper in the heel. The solid line in A-C is the skin surface, the dashed line is the dermo-epidermal junction, and the arrows indicate examples of cutaneous nerve fibers used for quantification. D– Approach used to quantify the number of nerve fibers. The # value indicates how many fibers were counted in each of the five examples. Na_v1.7 immunoreactive nerve fibers were only counted inside the dermal papillary, the grey area in D, while nerves identified using Sihler’s staining were counted inside the dermis over a maximum distance of 1 mm from the dermo-epidermal junction. Scale bars represents 1 millimeter. SC: stratum corneum, Epi: Vital epidermis, Der: Dermis.

Figure 2

Geometry of the finite element model (FEM), and examples of the morphology in the nerve model. A– The model was rotationally symmetric about the z-axis. The dorsum of the foot is the top of the model and the sole is the bottom. The geometry shown is for site 1. B – The location of the three modeled sites on the sole of the foot. C– Examples of the morphology of one Aβ fiber and one Aδ fiber. The fiber morphologies were randomly generated. The nodes of Ranvier are indicated by the filled circles, but the diameters of the fibers are not to scale. Note the larger internode length for the Aβ fiber. D– Detailed view of the morphology of the Aδ nerve plexus and intraepidermal nerve fibers (IENFs). The internode length for the IENFs was reduced to 1 μm to simulate the loss of myelin in the epidermis. Note that the aspect ratio of D was not maintained to improve visualization of the IENFs.

Figure 3

Quantification of the identified nerves from Na_v 1.7 immunoreactivity and Sihler staining. A– Number of intrapapillary nerves per papillae showing Na_V1.7 immunoreactivity. There were significant differences between the sites (1-way ANOVA, p < 0.01, F_(3,170 = 5.174); the heel (post-hoc, p < 0.05) and arch (post-hoc, p < 0.01) had significantly lower nerve fiber densities than the dorsum. There were no statistically significant differences between the sites in the sole of the foot. B– No significant differences in the densities of intradermal nerve fibers were found between the sites stained with Sihler’s method (1-way ANOVA, p=0.994, F_(2,8) = 0.006).

Figure 4

Immunolabeling of cutaneous nerve fibers using Na_v 1.7 antibodies in different skin areas in the sole and dorsum of the foot. The intra epidermal nerve fibers (IENF) were less abundant in the sole (A,C,E) than in the dorsum (G). Intrapapillary nerve fibers (IPNF) were observed at all sites (B,D,F,H). There were several sections where no IENFs were observed in the sole, while IENFs were present in the dorsum in most sections. IENFs were not observed in the heel. Arrowheads indicate examples of IENFs. Arrows indicate examples of IPNFs. SC: stratum corneum, Epi: Vital epidermis, Der: Dermis. The dashed lines indicate the dermo-epidermal junction.

Figure 5

Distribution of extracellular potential (Ve) in the finite element model and corresponding changes in membrane potential (Vm) in the nerve fiber model. An example of both an Aβ fiber (A-B) and an Aδ fiber (D-E) are depicted. B and D are zoomed versions of A and D, respectively. The colored circles in A,B,D,E indicate the nodes of Ranvier in the models, and the colors indicate Vm. The stimulation current used to calculate Ve and Vm was 1 mA. Panels C and F illustrate the locations of the nodes of Ranvier (black dots) that were activated in the models for Aβ and Aδ fibers, respectively. In C it can be seen that most Aβ fibers are activated at nodes which lie inside the dermis. The activation of Aδ fibers (F) occurred in the plexus which is located at a random depth within the most superficial 100 μm of the dermis. SC = stratum corneum, Epi = vital epidermis, Der = dermis. The electrode diameter is 9.1 mm, and r = 0 indicates the vertical symmetry axis in the model. The horizontal black lines indicate the boundaries between the different tissue layers. The color scale for Ve was truncated to clarify the potentials in the vital epidermis and dermis. The model geometries were taken for site 2, the lateral arch (Table 1). Note that the aspect ratios in B and E are not maintained to improve visualization.

Figure 6

Calculated stimulus–response curves for modeled Aβ and Aδ nerve fibers. Generally Aδ fibers (B) had higher thresholds than Aβ fibers (A). Three electrode sizes were modeled and the larger electrodes required larger stimulation currents to activate the nerve fibers. For the Aδ fibers at the heel, the red curves are the stimulus–response functions from models without intraepidermal nerve fibers (IENFs). The lack of IENFs decreased the activation threshold. The grey areas depict experimental values of perception and pain threshold (mean ± 95% confidence intervals). Perception thresholds are depicted with the Aβ fibers and the pain thresholds are depicted with Aδ fibers. Original data taken from [30], using an electrode diameter of 8 mm. Simulated pulse durations were set to 1 ms.