Upregulation of the sodium channel NaVβ4 subunit and its contributions to mechanical hypersensitivity and neuronal hyperexcitability in a rat model of radicular pain induced by local dorsal root ganglion inflammation

Abstract

High frequency spontaneous firing in myelinated sensory neurons plays a key role in initiating pain behaviors in several different models, including the radicular pain model in which the rat lumbar dorsal root ganglia (DRG) are locally inflamed. The sodium channel isoform NaV1.6 contributes to pain behaviors and spontaneous activity in this model. Among all the isoforms in adult DRG, NaV1.6 is the main carrier of TTX-sensitive resurgent Na currents that allow high-frequency firing. Resurgent currents flow after a depolarization or action potential, as a blocking particle exits the pore. In most neurons the regulatory β4 subunit is potentially the endogenous blocker. We used in vivo siRNA mediated knockdown of NaVβ4 to examine its role in the DRG inflammation model. NaVβ4 but not control siRNA almost completely blocked mechanical hypersensitivity induced by DRG inflammation. Microelectrode recordings in isolated whole DRGs showed that NaVβ4 siRNA blocked the inflammation-induced increase in spontaneous activity of Aβ neurons, and reduced repetitive firing and other measures of excitability. NaVβ4 was preferentially expressed in larger diameter cells; DRG inflammation increased its expression and this was reversed by NaVβ4 siRNA, based on immunohistochemistry and Western blotting. NaVβ4 siRNA also reduced immunohistochemical NaV1.6 expression. Patch clamp recordings of TTX-sensitive Na currents in acutely cultured medium diameter DRG neurons showed that DRG inflammation increased transient and especially resurgent current; effects blocked by NaVβ4 siRNA. NaVβ4 may represent a more specific target for pain conditions that depend on myelinated neurons expressing NaV1.6.

Localized Sympathectomy Reduces Mechanical Hypersensitivity by Restoring Normal Immune Homeostasis in Rat Models of Inflammatory Pain

“…For experiments in which DRGs were collected for protein isolation or electrophysiological experiments, both L4 and L5 were inflamed. This method of DRG inflammation is less invasive than our originally published description of this model (Xie et al, 2006), which involved drilling a small hole into the bone overlying the DRG.…”

“…Approximately 1 mm of gray ramus was further removed to make a gap and slow regeneration. Both L4 and L5 gray rami were cut in all mSYMPX surgeries, even though only the L5 DRG was inflamed in LID surgeries, because our previous study showed that there was possible spread of the zymosan solution into the ipsilateral L4 spinal nerve (Xie et al, 2006). Sham controls received similar exposure of the spinal nerves, but the gray rami were not cut.…”

“…When observed, responses to acetone or light brush strokes consisted of several rapid flicks of the paw and/or licking and shaking of the paw; walking movements were not scored as positive responses. Hypersensitivity to thermal (heat) stimuli was not examined because we have previously observed that this is little affected by LID (Xie et al, 2006).…”

“…Cells were classified by conduction velocity (stimulation of attached dorsal root) as follows: Ͻ1.2 m/s, C; Ն7.5 m/s, A␤; between 1.2 and 7.5 m/s, A␦ (Stebbing et al, 1999). Excitability parameters were analyzed as described previously (Xie et al, 2015). Data from recorded cells were pooled for each group of rats.…”

“…In preclinical back pain models, signs of inflammation include invasion of the DRG by immune cells, activation of the satellite glia, upregulation of pro-inflammatory cytokines, and downregulation of anti-inflammatory cytokines (Kawakami et al, 1996;Murata et al, 2006b;Xie et al, 2006;Sasaki et al, 2007;Amaya et al, 2009;Otoshi et al, 2010;Huang et al, 2011;Watanabe et al, 2011;de Souza Grava et al, 2012). However, DRG inflammation also plays a role in neuropathic pain models that involve axon damage or transection, especially during the early phase.…”