Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury

Abstract

Spinal cord injury (SCI) can result in hyperexcitability of dorsal horn neurons and central neuropathic pain. We hypothesized that these phenomena are consequences, in part, of dysregulated expression of voltage-gated sodium channels. Because the rapidly repriming TTX-sensitive sodium channel Nav1.3 has been implicated in peripheral neuropathic pain, we investigated its role in central neuropathic pain after SCI. In this study, adult male Sprague Dawley rats underwent T9 spinal contusion injury. Four weeks after injury when extracellular recordings demonstrated hyperexcitability of L3-L5 dorsal horn multireceptive nociceptive neurons, and when pain-related behaviors were evident, quantitative RT-PCR, in situ hybridization, and immunocytochemistry revealed an upregulation of Nav1.3 in dorsal horn nociceptive neurons. Intrathecal administration of antisense oligodeoxynucleotides (ODNs) targeting Nav1.3 resulted in decreased expression of Nav1.3 mRNA and protein, reduced hyperexcitability of multireceptive dorsal horn neurons, and attenuated mechanical allodynia and thermal hyperalgesia after SCI. Expression of Nav1.3 protein and hyperexcitability in dorsal horn neurons as well as pain-related behaviors returned after cessation of antisense delivery. Responses to normally noxious stimuli and motor function were unchanged in SCI animals administered Nav1.3 antisense, and administration of mismatch ODNs had no effect. These results demonstrate for the first time that Nav1.3 is upregulated in second-order dorsal horn sensory neurons after nervous system injury, showing that SCI can trigger changes in sodium channel expression, and suggest a functional link between Nav1.3 expression and neuronal hyperexcitability associated with central neuropathic pain.

Figure 1.

Behavioral changes after T9 spinal contusion SCI (n = 10 animals per group). Mean ± SD. BBB open-field locomotor rating scores (A) show spontaneous partial recovery of motor function after SCI, permitting nociceptive testing. Mean ± SD von Frey filament (VF) threshold values to hindlimb paw withdrawal (PWD) (B) showed that by 28 d after SCI, withdrawal thresholds significantly (*p < 0.05) decreased compared with sham-operated controls, indicating the development of mechanical allodynia. Mean ± SD paw withdrawal latency (seconds) to radiant thermal stimuli for hindlimbs (C) after SCI demonstrates significant (*p < 0.05) development of thermal hyperalgesia by 28 d after injury. In sham-operated animals, no differences in paw withdrawal latency were observed.

Figure 2.

In situ hybridization for Na_v1.3 shows very light signal within the gray matter of the lumbar dorsal horn of sham-operated animals (A). In contrast, 28 d after SCI, Na_v1.3 hybridization signal was more intense and widely distributed throughout dorsal horn lamina I-V neurons (B). Higher-magnification images (B, inset boxes) of individual cells demonstrate neuronal morphologies and cytoplasmic staining. No labeling was observed in afferent fibers, dendritic or axonal arborizations, or white matter. Quantitative RT-PCR amplification (C) showed a significant (*p < 0.05) increase in lumbar Na_v1.3 mRNA in the SCI group (n = 10 animals) when compared with sham-operated controls (CTRL) (n = 10 animals). Scale bar: A, B, 300 μm; insets, 10 μm.

Figure 3.

Immunocytochemical localization of Na_v1.3 protein within lumbar dorsal horn in sham-operated controls (A) revealed very few Na_v1.3 immunopositive profiles within superficial and deep laminas. Twenty-eight days after SCI (B), positively labeled cell profiles were observed throughout dorsal horn lamina I-V neurons. In these sections, small neurons within laminas I-II and larger neurons within laminas III-V were strongly labeled. Localization of immunopositive profiles from a number of sections (n = 4) plotted on a representative schematic of the lumbar spinal cord (C, D) show that after SCI, the number of profiles was increased within all laminas. The distribution of immunopositive cells was fairly homogenous and is not confined to specific laminas. Scale bar, 300 μm. CTRL, Controls.

Figure 4.

After SCI, colocalization experiments demonstrate that upregulation of Na_v1.3 took place in lumbar dorsal horn nociceptive neurons. Na_v1.3 (A) was not found to colocalize with OX-42 (B), a marker for microglia (C, merged image). Similarly, Na_v1.3 (D) was not found to colocalize with GFAP (E, F), a marker for astrocytes. Na_v1.3 (G) did not colocalize with CGRP (H, I), present within primary afferent terminals. Colocalization was observed between Na_v1.3 (J) and NK1R (K, L), which is present within second-order nociceptive neurons within laminas I-V. Colocalization was observed in neurons in superficial laminas I-II as well as in larger neurons in deeper laminas IV-V. Scale bar, 300 μm.

Figure 5.

Representative peristimulus time histograms (spikes per 1 sec bin) of multireceptive neurons recorded extracellularly from L3-L5 in sham-operated control animals (A) and in animals 28 d after SCI (B) in response to various innocuous and noxious peripheral stimuli: brush (BR), press (PR), pinch (PI), increasing intensity von Frey filaments (0.39, 1.01, and 20.8 gm), and thermal (47°C). Single-unit waveforms are also shown to the right. Compared with controls, injured animals demonstrated evoked hyperexcitability to all peripheral stimuli. Spontaneous background activity was unchanged from control rates in injured animals. Quantification (C) of mean ± SD spontaneous and evoked discharge rates of neurons sampled from sham-operated controls (n = 15 cells) and SCI (n = 15 cells) groups demonstrated significantly (*p < 0.05) increased evoked activity to all peripheral stimuli after injury. No significant changes in background activity were detected.

Figure 6.

Schematic of injury and ODN delivery paradigm [i.t. ODN (CY3)] (A) showing relative locations of T9 contusion SCI, and intrathecally delivered AS or MM in relation to the lumbar enlargement (LE). A schematic cross section of the lumbar dorsal horn is shown illustrating field of view in C. After a one-time injection of Cy3-labeled AS (45 μg/5 μl of aCSF followed by 10 μl of aCSF flush), Cy3 fluorescence is detectable to a depth of 800 μm below the dorsal surface of the spinal cord (B) and can be seen to penetrate as far as lamina V (C). Higher-magnification image of cells exhibiting neuronal profiles (C, boxes) shows strong uptake of Cy3-labeled AS into cells with neuronal morphologies (D′, D″) after a single injection. Scale bar: A, C, 300 μm; D′, D″, 10 μm.

Figure 7.

Behavioral changes after T9 spinal contusion injury and intrathecal administration (days 31-34, shaded) of either MM or AS sequences generated against Na_v1.3 (n = 10 animals per group, except for SC + AS/WD where n = 6). The dotted line in each panel indicates sham-operated control thresholds. Mean ± SD. BBB open-field locomotor scores (A) did not change during the course of ODN administration or after cessation of ODN injections, in any groups. One day after initiation of ODN administration, mean ± SD von Frey mechanical (VF) thresholds (B) began to increase in AS-treated animals, eventually rising to 18 gm by day 4. A significant (*p < 0.05) difference when compared with SCI and SCI-MM groups. Cessation of AS administration resulted in restoration of mechanical allodynia. By 2 d of ODN administration, mean ± SD paw withdrawal (PWD) latency (C) to radiant thermal stimuli increased significantly (*p < 0.05) in AS-treated animals, compared with SCI and SCI + MM groups. In these ODN-treated animals, latencies were not significantly different from sham-operated control latencies. Cessation of AS administration resulted in restoration of thermal hyperalgesia.

Figure 8.

In situ hybridization for Na_v1.3 after SCI and administration of MM or AS sequences generated against Na_v1.3. In animals treated with MM for 4 d (A), widely distributed punctate staining was observed throughout the lumbar dorsal horn in small and large cells exhibiting neuronal morphologies, similar to untreated SCI (Fig. 2 B). In contrast, in AS-treated animals (B), Na_v1.3 hybridization signal was markedly decreased in both intensity and distribution throughout the lumbar dorsal horn. Quantitative RT-PCR amplification (C) showed a significant (*p < 0.05) decrease in lumbar Na_v1.3 mRNA in the SCI + AS group (n = 10 animals) when compared with SCI + MM animals (n = 10). Dotted line indicates mRNA levels in SCI group. Scale bar, 300 μm.

Figure 9.

Immunocytochemical localization of Na_v1.3 protein within lumbar dorsal horn in SCI animals after administration of MM or AS sequences against Na_v1.3. In animals that received MM injections (A), Na_v1.3-immunopositive cell profiles were widely distributed throughout the lumbar dorsal horn, similar to untreated SCI (Fig. 3B). These neurons were of both small and large diameter. In contrast, in animals receiving AS injections (B), the number of immunopositive cell profiles was markedly decreased within the dorsal horn. A decrease in distribution was most notable in lamina I-III neurons. After cessation of AS administration, (C), the number Na_v1.3-immunopositive cell profiles was increased. Localization of immunopositive profiles from a number of sections (n = 4) plotted on a representative schematic of the lumbar spinal cord (D-F) shows that in AS-treated animals, but not after interruption of AS administration, the number of profiles was decreased within all laminas. Scale bar, 300 μm.

Figure 10.

Representative peristimulus time histograms (spikes per 1 sec bin) of multireceptive neurons recorded extracellularly from L3-L5 in animals after administration of MM (A) or AS (B) sequences generated against Na_v1.3 after SCI. Records show evoked activity in response to various innocuous and noxious peripheral stimuli: brush (BR), press (PR), pinch (PI), increasing intensity von Frey filaments (0.39, 1.01, and 20.8 gm), and thermal (47°C). Single-unit waveforms are also shown. In MM-receiving animals (A), evoked hyperexcitability was demonstrated in response to all peripherally applied stimuli. In contrast, in animals receiving AS injections (B), evoked activity was decreased to all stimuli except PI. In a separate group of SCI + AS animals 5 d after cessation of AS administration (SCI + AS/WD) (C), cells were hyperexcitable. Spontaneous background activity was not significantly different in either group. Quantification (D) of mean ± SD spontaneous and evoked discharge rates of neurons sampled from MM- or AS-receiving animals (n = 11-15 cells sampled per group) demonstrated significantly (*p < 0.05) reduced evoked activity in AS-receiving animals only.