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. 2012 Aug 8;32(32):10819-32.
doi: 10.1523/JNEUROSCI.0304-12.2012.

Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury

Shannon D Shields  1 , Xiaoyang Cheng, Nurcan Uçeyler, Claudia Sommer, Sulayman D Dib-Hajj, Stephen G Waxman
Affiliations

Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury

Shannon D Shields et al. J Neurosci. .

Abstract

Marked hypersensitivity to heat and mechanical (pressure) stimuli develop after a burn injury, but the neural mechanisms underlying these effects are poorly understood. In this study, we establish a new mouse model of focal second-degree burn injury to investigate the molecular and cellular basis for burn injury-induced pain. This model features robust injury-induced behavioral effects and tissue-specific altered cytokine profile, but absence of glial activation in spinal dorsal horn. Three voltage-gated sodium channels, Na(v)1.7, Na(v)1.8, and Na(v)1.9, are preferentially expressed in peripheral somatosensory neurons of the dorsal root ganglia (DRGs) and have been implicated in injury-induced neuronal hyperexcitability. Using knock-out mice, we provide evidence that Na(v)1.7 selectively contributes to burn-induced hypersensitivity to heat, but not mechanical, stimuli. After burn model injury, wild-type mice display increased sensitivity to heat stimuli, and a normally non-noxious warm stimulus induces activity-dependent Fos expression in spinal dorsal horn neurons. Strikingly, both effects are absent in Na(v)1.7 conditional knock-out (cKO) mice. Furthermore, burn injury increases density and shifts activation of tetrodotoxin-sensitive currents in a hyperpolarized direction, both pro-excitatory properties, in DRG neurons from wild-type but not Na(v)1.7 cKO mice. We propose that, in sensory neurons damaged by burn injury to the hindpaw, Na(v)1.7 currents contribute to the hyperexcitability of sensory neurons, their communication with postsynaptic spinal pain pathways, and behavioral thresholds to heat stimuli. Our results offer insights into the molecular and cellular mechanisms of modality-specific pain signaling, and suggest Na(v)1.7-blocking drugs may be effective in burn patients.

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Figures

Figure 1.
Figure 1.
A mouse model of burn injury-induced pain produces peripheral edema and focal hypersensitivity to heat and mechanical stimuli. A, Response thresholds to radiant heat stimuli (Hargreaves' test) are strongly decreased on the ipsilateral side in burn model animals and unchanged in sham-treated animals. Heat hypersensitivity is present at the earliest time point assessed and resolves within 2 weeks. (Repeated-measures ANOVA, p < 0.05; n = 12 mice/group.) B, In the contralateral hindpaw, no significant difference in response threshold to the heat stimulus was noted between burn model and sham-treated animals at any time point. C, Response thresholds to mechanical stimuli in the von Frey test are strongly decreased on the ipsilateral side in burn model animals and unchanged in sham-treated animals. Mechanical hypersensitivity resolves within 3 weeks. (Repeated-measures ANOVA, p < 0.05; n = 12 mice/group.) D, In the contralateral hindpaw, no significant difference in response threshold to the mechanical stimuli was noted between burn model and sham-treated animals at any time point. E, A prominent edema develops in burn model animals, as measured by an increased paw diameter. Paw swelling is resolved within ∼2 weeks of burn injury in this model. (Repeated-measures ANOVA, p < 0.05; n = 12 mice/group.) *p <0.05.
Figure 2.
Figure 2.
The nerve injury-related transcription factor ATF-3 is induced in DRG neurons after burn injury to the hindpaw. ATF-3 (A–C, red, top) is expressed in some DRG neurons after burn injury to the hindpaw, including those that express markers of various subtypes of sensory neurons (A–C, green, middle). The bottom image in each panel (A–C) is a merged view. Arrows highlight double-labeled neurons; arrowheads indicate single-labeled neurons. Scale bars, 100 μm. A, Some ATF-3-positive cells express NF, a marker of neurons with myelinated axons (Aβ and Aδ afferents). B, Some ATF-3-positive cells express CGRP, a marker that labels peptidergic C-nociceptors and some A-fiber afferents. C, Some ATF-3-positive cells bind IB4, a marker of nonpeptidergic C-nociceptors. D–F, Size distribution of sensory neurons in which ATF-3 is induced after hindpaw heat injury in lumbar DRGs L4 (D), L5 (E), and L6 (F). Quantification is provided in Table 1.
Figure 3.
Figure 3.
Cytokine gene regulation along the pain–sensory neuraxis at 24 h following burn model injury. The pro-inflammatory cytokines TNF, IL-1β, and IL-6, and the anti-inflammatory cytokine IL-10 were tracked by qRT-PCR in various tissues. The qRT-PCR data are illustrated as box-and-whisker plots giving the median, the upper 75% and lower 25% percentiles, and the minimum and maximum values. A, The expression of TNF (p < 0.01), IL-1β (p < 0.01), IL-6 (p < 0.01), and IL-10 (p < 0.01) were increased in the hindpaw skin of burn model animals on the ipsilateral side compared with controls. B, The expression of TNF (p < 0.01), IL-1β (p < 0.001), and IL-10 (p < 0.01) was increased in the sciatic nerves of burn model animals on the ipsilateral side compared with controls. C, The expression of IL-6 (p < 0.01) was increased in lumbar DRGs of burn model animals on the ipsilateral side compared with controls. D, The expression of IL-6 (p < 0.001) and IL-10 (p < 0.05) were decreased in lumbar spinal cord of burn model animals on the contralateral side compared with controls. E, Gene expression of the investigated cytokines did not change in the thalamus. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4.
Figure 4.
Nav1.7 cKO mice do not become hypersensitive to heat stimuli in the burn model or the CFA model of inflammatory pain. Filled symbols indicate control littermates, open symbols indicate Nav1.7 cKO mice (squares), Nav1.8 KO mice (diamonds), or Nav1.9 KO mice (triangles). A, Nav1.7 cKO mice do not develop reduced withdrawal thresholds to heat stimuli after burn model injury, unlike control littermates (repeated-measures ANOVA, p < 0.05; n = 16 Nav1.7 cKO mice, 14 control [floxed] littermates). However, reduced withdrawal thresholds to mechanical stimuli develop in Nav1.7 cKO mice, indistinguishably from controls. Nav1.8 KO and Nav1.9 KO have normal pain-like responses to both heat and mechanical modalities after burn model injury (n = 9 Nav1.8 KO, 8 Nav1.8 wild type; n = 7 Nav1.9 KO, 7 Nav1.9 wild type). B, Similar results were found in the CFA model of inflammatory pain. Nav1.7 cKO mice do not develop reduced withdrawal thresholds to heat stimuli in the CFA model, unlike control littermates (repeated-measures ANOVA, p < 0.05; n = 8 Nav1.7 cKO mice, 7 control [floxed] littermates). However, reduced withdrawal thresholds to mechanical stimuli develop in Nav1.7 cKO mice, indistinguishably from controls. Nav1.8 KO and Nav1.9 KO have normal pain-like responses to both heat and mechanical modalities in the CFA model (n = 8 Nav1.8 KO, 7 Nav1.8 wild type; n = 9 Nav1.9 KO, 9 Nav1.9 wild type).
Figure 5.
Figure 5.
Fos induction in dorsal horn neurons by a non-noxious warm stimulus occurs after burn injury in control and Nav1.8 KO mice, but not Nav1.7 cKO mice. A–H, Representative photomicrographs of spinal cord dorsal horn tissue from sham-treated or burn model mice showing Fos-positive profiles under control conditions (no stimulation, A, C, E, G) or after immersion of the affected hindpaw in a 40°C water bath (B, D, F, H). Genotypes and treatment groups are indicated on the left. I, Quantification of Fos-positive profiles per section. The warm stimulus did not induce Fos in sham-treated mice, thus we argue that 40°C is not normally a noxious stimulus in uninjured mice. However, in mice that had previously undergone burn model injury, immersion of the paw in 40°C water induces Fos in spinal laminae I-II. This effect is still present in mice lacking Nav1.8, but is abolished in Nav1.7 cKO mice. J–Q, Representative images of spinal cord dorsal horn tissue from sham-treated or burn model mice showing Fos-positive profiles after walking on a rotating beam (contralateral to sham/burn-injured hindpaw, J, L, N, P; ipsilateral to sham/burn-injured hindpaw, K, M, O, Q). Genotypes and treatment groups are indicated on the left. R, Quantification of Fos-positive profiles per section. Walking on a rotating beam induced Fos expression only in lamina III and deeper laminae in sham-treated mice and on the side contralateral to burn injury, and is thus considered a non-noxious mechanical stimulus. However, in mice that had previously undergone burn model injury, walking on the rotating beam induces Fos in laminae I-II of the ipsilateral dorsal horn as well (denoted by brackets). This is true in mice of all genotypes tested. *p < 0.05.
Figure 6.
Figure 6.
Burn model injury increases TTX-S current density and enhances activation of TTX-S channels in DRG neurons of wild-type but not Nav1.7 cKO mice. A–D, Control mice; E–H, Nav1.7 cKO mice. A, Representative voltage-dependent inward currents from Nav1.8 channels (left), total sodium channels (middle), and TTX-S channels (right, obtained by subtracting Nav1.8 currents from total sodium currents) in DRG neurons isolated from control mice that underwent sham procedure (top) or burn model injury (bottom). B, Burn model injury has no effect on Nav1.8 activation, but shifts the activation curve of TTX-S channels by ∼−3 mV in DRG neurons from control mice. C, There is no significant difference in the mean values of Nav1.8 current density in DRG neurons isolated from control mice with sham treatment or burn model injury. D, DRG neurons isolated from mice that had undergone burn model injury produce larger TTX-S currents than neurons from sham-treated animals. E, Representative voltage-dependent inward currents of Nav1.8 channels (left), total sodium channels (middle), and TTX-S channels (right, obtained by subtracting Nav1.8 currents from total sodium currents) in DRG neurons isolated from Nav1.7 cKO mice that underwent sham procedure (top) or burn model injury (bottom). F, Burn model injury has no effect on voltage-dependent activation of Nav1.8 or TTX-S channels in DRG neurons from Nav1.7 cKO mice. G, There is no significant difference in the mean values of Nav1.8 current density in DRG neurons isolated from Nav1.7 cKO mice with sham treatment or burn model injury. H, There is no significant difference in TTX-S current density between DRG neurons from sham-treated or burn model Nav1.7 cKO mice. *p <0.05.
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