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. 2004 May 19;24(20):4832-9.
doi: 10.1523/JNEUROSCI.0300-04.2004.

Altered sodium channel expression in second-order spinal sensory neurons contributes to pain after peripheral nerve injury

Bryan C Hains  1 , Carl Y Saab, Joshua P Klein, Matthew J Craner, Stephen G Waxman
Affiliations

Altered sodium channel expression in second-order spinal sensory neurons contributes to pain after peripheral nerve injury

Bryan C Hains et al. J Neurosci. .

Abstract

Peripheral nerve injury is known to upregulate the rapidly repriming Na(v)1.3 sodium channel within first-order spinal sensory neurons. In this study, we hypothesized that (1) after peripheral nerve injury, second-order dorsal horn neurons abnormally express Na(v)1.3, which (2) contributes to the responsiveness of these dorsal horn neurons and to pain-related behaviors. To test these hypotheses, adult rats underwent chronic constriction injury (CCI) of the sciatic nerve. Ten days after CCI, allodynia and hyperalgesia were evident. In situ hybridization, quantitative reverse transcription-PCR, and immunocytochemical analysis revealed upregulation of Na(v)1.3 in dorsal horn nociceptive neurons but not in astrocytes or microglia, and unit recordings demonstrated hyperresponsiveness of dorsal horn sensory neurons. Intrathecal antisense oligodeoxynucleotides targeting Na(v)1.3 decreased the expression of Na(v)1.3 mRNA and protein, reduced the hyperresponsiveness of dorsal horn neurons, and attenuated pain-related behaviors after CCI, all of which returned after cessation of antisense delivery. These results demonstrate for the first time that sodium channel expression is altered within higher-order spinal sensory neurons after peripheral nerve injury and suggest a link between misexpression of the Na(v)1.3 sodium channel and central mechanisms that contribute to neuropathic pain after peripheral nerve injury.

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Figures

Figure 1.
Figure 1.
A, In situ hybridization for Nav1.3 transcripts revealed no signal in the dorsal horn gray matter of sham-operated animals. However, 10 d after CCI of the sciatic nerve, Nav1.3 signal is detectable in cells exhibiting neuronal profiles within the superficial and deep dorsal horn, mostly in the medial region where afferent fibers terminate, on the ipsilateral side (B). Under high magnification, cells (B, inset) demonstrate multipolar neuronal morphologies and cytoplasmic staining. No labeling was observed in primary afferent fibers or white matter after CCI. C, Signal was significantly (*p < 0.05) increased in neurons expressing Nav1.3 after CCI on the ipsilateral side. D, Quantitative RT-PCR amplification showed a significant (*+p < 0.05) increase in ipsilateral DRG (183% compared with the contralateral DRG) and ipsilateral dorsal horn [158% compared with the contralateral side of the lumber (LE) cord] Nav1.3 mRNA in CCI animals. Dotted lines indicate control levels in C and D. No changes were observed in the dorsal horn contralateral to CCI. Upregulation of Nav1.3 mRNA was observed in the ipsilateral DRG (F) but not in the contralateral DRG (E). Scale bars: (in A) A, B, 300 μm; B, inset, 10 μm; (in F) E, F, 50 μm. CONT, Contralateral; IPSI, ipsilateral.
Figure 2.
Figure 2.
Immunocytochemical localization of Nav1.3 (red channel) and substance-P receptor (NK1R, green channel) protein within the ipsilateral and contralateral dorsal horn 10 d after CCI. A, On the contralateral side, very few Nav1.3 immunopositive profiles were detectable, whereas NK1R signal was present in cells exhibiting neuronal morphologies throughout all dorsal horn laminas. Inset, High-magnification of double-labeled neuron. B, On the side ipsilateral to CCI, Nav1.3 and NK1R positively labeled cell profiles were observed throughout laminas I–V. Merged images show colocalization of Nav1.3 and NK1R (yellow) within ipsilateral dorsal horn after CCI (B). C, In reactions for which primary antibody (16153) was omitted, no Nav1.3 signal was detectable. Increased signal for Nav1.3 was observed in the ipsilateral DRG (E) compared with the contralateral side of CCI animals (D). Nav1.3 AS reduced Nav1.3 but not NK1R signal in the ipsilateral dorsal horn after CCI (data not shown) (Table 1). Scale bar, 300 μm. CONT, Contralateral; IPSI, ipsilateral.
Figure 3.
Figure 3.
Colocalization experiments demonstrate that after CCI, upregulation of Nav1.3 did not take place within glial cells. Nav1.3 (A) was not found to colocalize with OX-42 (B), a marker for microglia (C, MERGE). Nav1.3 (D) was not found to colocalize with GFAP (E), a marker for astrocytes (F, MERGE). Scale bar, 300 μm.
Figure 4.
Figure 4.
Representative peristimulus time histograms (spikes/1 sec bin) of dorsal horn multireceptive neurons recorded extracellularly from L3–L5 in response to BR, PR, and PI stimulation to mapped receptive fields on the hindpaw. Unit waveforms are also shown. Records are shown for sham-operated animals (A) and for animals 10 d after CCI (B). Compared with controls, injured animals demonstrated increased spontaneous activity and evoked hyperresponsiveness to all peripheral stimuli.
Figure 7.
Figure 7.
After 4 d of intrathecal administration, beginning 11 d after CCI, Nav1.3 MM had no effect on spontaneous or evoked activity (A), but Nav1.3 AS reduced both spontaneous activity and evoked responses to BR, PR, and PI stimulation after CCI (B). Unit waveforms are also shown. C, Quantification of mean ± SD spontaneous and evoked discharge rates of neurons sampled from sham-operated CCI plus 1.3 MM and CCI plus 1.3 AS groups demonstrated significantly decreased evoked activity to all peripheral stimuli after injury in the AS group (*p < 0.05; CCI plus 1.3 MM vs CCI plus 1.3 AS).
Figure 5.
Figure 5.
A single injection (5 μl) of Cy3-labeled AS ODN diffused to a depth of 800–900 μm [through the dorsolateral funiculus (DLF) and into laminas I–V] in spinal parenchyma and was taken up by cells exhibiting a neuronal morphology (A). Cy3-labeled AS was unable to penetrate into the DRG neurons after intrathecal injection (B). Neither MM (C) nor AS (D) administration was effective in reducing Nav1.3 mRNA expression in ipsilateral DRG neurons after CCI. Scale bars: (in A) A, B, 300μm; (in D) C, D, 50 μm. p, Proximal; d, distal to intrathecal space.
Figure 6.
Figure 6.
After 4 d of intrathecal administration of Nav1.3 AS or MM oligodeoxynucleotides, beginning 11 d after CCI, Nav1.3 in situ hybridization signal intensity and distribution were unchanged in the MM group (A), whereas in AS-receiving animals, Nav1.3 signal was markedly reduced (B). C, Quantitative RT-PCR amplification showed a significant (*p < 0.05) decrease in ipsilateral dorsal horn Nav1.3 mRNA in CCI animals receiving Nav1.3 AS compared with animals receiving MM. The dotted line indicates mRNA levels in sham-operated animals. Scale bar, 300 μm. CONT, Contralateral; IPSI, ipsilateral.
Figure 8.
Figure 8.
Pain-related behaviors after CCI and effects of administration of Nav1.3 MM or AS, for 4 d, beginning 11 d after CCI. A, Mean ± SD von Frey (VF) filament threshold values, displayed as the difference between ipsilateral and contralateral paw thresholds, show that by 10 d after CCI, paw-withdrawal thresholds were significantly decreased on the ipsilateral side compared with the contralateral side and sham-operated controls. Intrathecal administration (indicated by gray shading) of Nav1.3 MM had no effect on mechanical thresholds, whereas Nav1.3 AS resulted in a significant (*p < 0.05) reduction in mechanical allodynia. After cessation of AS administration, mechanical thresholds returned to levels consistent with CCI. B, Mean ± SD paw-withdrawal latency (in seconds) to radiant thermal stimuli after CCI demonstrates decreased latencies and development of thermal hyperalgesia ipsilaterally by 10 d after CCI. In sham-operated animals, no differences in paw-withdrawal latency were observed. Administration of Nav1.3 MM had no effect on withdrawal latency, whereas administration of Nav1.3 AS resulted in withdrawal latencies that were significantly (*p < 0.05) increased in the ipsilateral paw. Cessation of AS administration resulted in restoration of thermal hyperalgesia.
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