Post-transcriptional Regulation of Voltage-Gated Sodium Channel 1.8 (Nav1.8): A Key Underlying Mechanism of Neuropathic Pain Pathogenesis

Neuropathic pain is a chronic disease that commonly arises following injury to the peripheral nervous system. With an estimated prevalence level of 1.5% in the general population, the current treatment results of peripheral neuropathy remain unsatisfactory. Previous reports revealed that axonal up-regulation of the Nav1.8 tetrodotoxin-resistant (TTX-R) sodium channel contributed to aberrant activity in primary afferent neurons, causing neuropathic symptoms after sciatic nerve entrapment (SNE). However, the mechanism underlying this local accumulation of Nav1.8 has yet to be elucidated. In keeping with previous findings, we hypothesize that abnormal regulation of Nav1.8 gene transcription and/or post-transcription are involved in peripheral neuropathic pain pathogenesis. Objectives: This study was designed to elucidate the mechanistic role, including transcription and post-transcription, of Nav1.8 in painful neuropathy. Methods: To determine a bona fide level of Nav1.8 gene transcription in rat dorsal root ganglia (DRG) somata, we used real-time Polymerase chain reaction (PCR) analysis to determine the expression of Nav1.8 heterogeneous nuclear RNA (hnRNA), a transcriptional marker. To address the role of Nav1.8 mRNA in painful neuropathy, RNA interference (RNAi) gene silencing vectors encoding Nav1.8 short-hairpin RNA (shRNA) were peripherally transferred to sciatic nerves of SNE rats and behavioral modulation was quantified. Results: 1) Unexpectedly, a decrease of Nav1.8 hnRNA was observed in ipsilateral L4/L5 DRG of SNE rats (P=0.03, paired t-test), suggesting that up-regulation of Nav1.8 functional expression in the injured nerve was not under transcriptional but perhaps post-transcriptional control. 2) One highly specific and efficient Nav1.8 shRNA (6009-6027) was generated out of four target sites (1203-1233, 3407-3425, 6009-6027 & 6033-6051) based on in vitro assays using lentiviral transduction in rat cultured DRG neurons. For further post-transcriptional investigation, non-viral in vivo transfection of Nav1.8 shRNA or Random shRNA to DRG was achieved by subcutaneous injection of cationized gelatin/plasmid DNA polyplexes into rat ipsilateral hindpaws. 3) Behavioral examination after the first injection revealed a significant decrease (P<0.05, Repeated measure ANOVA) in SNE-induced mechanical allodynia and thermal hyperalgesia at 4 days after Nav1.8 shRNA injections, which lasted for 5 days. A second injection after pain alleviation subsided revealed delayed but reproducible pain alleviation effects. 4) Real-time PCR and immunohistochemical analyses of the sciatic nerve ipsilateral to Nav1.8 shRNA injection revealed a significant down-regulation of Nav1.8 mRNA and Nav1.8 protein, respectively. 5) Acutely dissociated DRG culture from SNE rats in Boyden chamber system, which isolated DRG and axonal compartments, revealed a significant up-regulation of Nav1.8 mRNA in the injured axonal compartment. Conclusions: These data indicate that Nav1.8 post-transcriptional regulation plays a plausible role in peripheral neuropathy and that it can be successfully targeted by RNAi against Nav1.8 mRNA to attenuate neuropathic pain symptoms.