The influence of Nav1.9 channels on intestinal hyperpathia and dysmotility.

The Nav1.9 channel is a voltage-gated sodium channel. It plays a vital role in the generation of pain and the formation of neuronal hyperexcitability after inflammation. It is highly expressed in small diameter neurons of dorsal root ganglions and Dogiel II neurons in enteric nervous system. The small diameter neurons in dorsal root ganglions are the primary sensory neurons of pain conduction. Nav1.9 channels also participate in regulating intestinal motility. Functional enhancements of Nav1.9 channels to a certain extent lead to hyperexcitability of small diameter dorsal root ganglion neurons. The hyperexcitability of the neurons can cause visceral hyperalgesia. Intestinofugal afferent neurons and intrinsic primary afferent neurons in enteric nervous system belong to Dogiel type II neurons. Their excitability can also be regulated by Nav1.9 channels. The hyperexcitability of intestinofugal afferent neurons abnormally activate entero-enteric inhibitory reflexes. The hyperexcitability of intrinsic primary afferent neurons disturb peristaltic waves by abnormally activating peristaltic reflexes. This review discusses the role of Nav1.9 channels in intestinal hyperpathia and dysmotility.

Role of NaV 1.9 in inflammatory mediator-induced activation of mouse airway vagal C-fibres.

The influence of NaV 1.9 on inflammatory mediator-induced activation of airway vagal nodose C-fibres was evaluated by comparing responses in wild-type versus NaV 1.9-/- mice. A single-cell RT-PCR analysis indicated that virtually all nodose C-fibre neurons expressed NaV 1.9 (SCN11A) mRNA. Using extracellular electrophysiological recordings in an isolated vagally innervated mouse trachea-lung preparation, it was noted that mediators acting via G protein-coupled receptors (PAR2), or ionotropic receptors (P2×3) were 70-85% less effective in evoking action potential discharge in the absence of NaV 1.9. However, there was no difference in action potential discharge between wild-type and NaV 1.9-/- when the stimulus was a rapid punctate mechanical stimulus. An analysis of the passive and active properties of isolated nodose neurons revealed no difference between neurons from wild-type and NaV 1.9-/- mice, with the exception of a modest difference in the duration of the afterhyperpolarization. There was also no difference in the amount of current required to evoke action potentials (rheobase) or the action potential voltage threshold. The inward current evoked by the chemical mediator by a P2×3 agonist was the same in wild-type versus NaV 1.9-/- neurons. However, the current was sufficient to evoke action potential only in the wild-type neurons. The data support the speculation that NaV 1.9 could be an attractive therapeutic target for inflammatory airway disease by selectively inhibiting inflammatory mediator-associated vagal C-fibre activation. KEY POINTS: Inflammatory mediators were much less effective in activating the terminals of vagal airway C-fibres in mice lacking NaV 1.9. The active and passive properties of nodose neurons were the same between wild-type neurons and NaV 1.9-/- neurons. Nerves lacking NaV 1.9 responded, normally, with action potential discharge to rapid punctate mechanical stimulation of the terminals or the rapid stimulation of the cell bodies with inward current injections. NaV 1.9 channels could be an attractive target to selectively inhibit vagal nociceptive C-fibre activation evoked by inflammatory mediators without blocking the nerves' responses to the potentially hazardous stimuli associated with aspiration.

NaV1.9 current in muscle afferent neurons is enhanced by substances released during muscle activity.

Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NaV) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NaV currents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NaV1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NaV current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NaV1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NaV1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NaV1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NaV1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

Transcription factor mesenchyme homeobox protein 2 (MEOX2) modulates nociceptor function.

Mesenchyme homeobox protein 2 (MEOX2) is a transcription factor involved in mesoderm differentiation, including development of bones, muscles, vasculature and dermatomes. We have previously identified dysregulation of MEOX2 in fibroblasts from Congenital Insensitivity to Pain patients, and confirmed that btn, the Drosophila homologue of MEOX2, plays a role in nocifensive responses to noxious heat stimuli. To determine the importance of MEOX2 in the mammalian peripheral nervous system, we used a Meox2 heterozygous (Meox2+/- ) mouse model to characterise its function in the sensory nervous system, and more specifically, in nociception. MEOX2 is expressed in the mouse dorsal root ganglia (DRG) and spinal cord, and localises in the nuclei of a subset of sensory neurons. Functional studies of the mouse model, including behavioural, cellular and electrophysiological analyses, showed altered nociception encompassing impaired action potential initiation upon depolarisation. Mechanistically, we noted decreased expression of Scn9a and Scn11a genes encoding Nav 1.7 and Nav 1.9 voltage-gated sodium channels respectively, that are crucial in subthreshold amplification and action potential initiation in nociceptors. Further transcriptomic analyses of Meox2+/- DRG revealed downregulation of a specific subset of genes including those previously associated with pain perception, such as PENK and NPY. Based on these observations, we propose a novel role of MEOX2 in primary afferent nociceptor neurons for the maintenance of a transcriptional programme required for proper perception of acute and inflammatory noxious stimuli.

Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with NaV1.9.

ABSTRACT: Human NaV1.9 (hNaV1.9), encoded by SCN11A, is preferentially expressed in nociceptors, and its mutations have been linked to pain disorders. NaV1.9 could be a promising drug target for pain relief. However, the modulation of NaV1.9 activity has remained elusive. Here, we identified a new candidate NaV1.9-interacting partner, protein arginine methyltransferase 7 (PRMT7). Whole-cell voltage-clamp recordings showed that coelectroporation of human SCN11A and PRMT7 in dorsal root ganglion (DRG) neurons of Scn11a-/- mice increased the hNaV1.9 current density. By contrast, a PRMT7 inhibitor (DS-437) reduced mNaV1.9 currents in Scn11a+/+ mice. Using the reporter molecule CD4, we observed an increased distribution of hLoop1 on the cell surface of PRMT7-overexpressing HKE293T cells. Furthermore, we found that PRMT7 mainly binds to residues 563 to 566 within the first intracellular loop of hNaV1.9 (hLoop1) and methylates hLoop1 at arginine residue 519. Moreover, overexpression of PRMT7 increased the number of action potential fired in DRG neurons of Scn11a+/+ mice but not Scn11a-/- mice. However, DS-437 significantly inhibited the action potential frequency of DRG neurons and relieved pain hypersensitivity in Scn11aA796G/A796G mice. In summary, our observations revealed that PRMT7 modulates neuronal excitability by regulating NaV1.9 currents, which may provide a potential method for pain treatment.

Contributions of NaV1.8 and NaV1.9 to excitability in human induced pluripotent stem-cell derived somatosensory neurons.

The inhibition of voltage-gated sodium (NaV) channels in somatosensory neurons presents a promising novel modality for the treatment of pain. However, the precise contribution of these channels to neuronal excitability, the cellular correlate of pain, is unknown; previous studies using genetic knockout models or pharmacologic block of NaV channels have identified general roles for distinct sodium channel isoforms, but have never quantified their exact contributions to these processes. To address this deficit, we have utilized dynamic clamp electrophysiology to precisely tune in varying levels of NaV1.8 and NaV1.9 currents into induced pluripotent stem cell-derived sensory neurons (iPSC-SNs), allowing us to quantify how graded changes in these currents affect different parameters of neuronal excitability and electrogenesis. We quantify and report direct relationships between NaV1.8 current density and action potential half-width, overshoot, and repetitive firing. We additionally quantify the effect varying NaV1.9 current densities have on neuronal membrane potential and rheobase. Furthermore, we examined the simultaneous interplay between NaV1.8 and NaV1.9 on neuronal excitability. Finally, we show that minor biophysical changes in the gating of NaV1.8 can render human iPSC-SNs hyperexcitable, in a first-of-its-kind investigation of a gain-of-function NaV1.8 mutation in a human neuronal background.

A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing.

Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG-hCG, IAS-hCG and eCG-IAS-hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS-hCG and eCG-IAS-hCG methods. However, low fertilisation rates (36.3-38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG-hCG and IAS-hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.

Bioinformatic Analysis of Prognostic and Immune-Related Genes in Pancreatic Cancer.

Pancreatic cancer (PC) is a malignant tumor with poor prognosis. The poor effect of surgery and chemotherapy makes the research of immunotherapy target molecules significant. Therefore, identifying the new molecular targets of PC is important for patients. In our study, we systematically analyzed molecular correlates of pancreatic cancer by bioinformatic analysis. We characterized differentially expressed analysis based on the TCGA pancreatic cancer dataset. Then, univariate Cox regression was employed to screen out overall survival- (OS-) related DEGs. Based on these genes, we established a risk signature by the multivariate Cox regression model. The ICGC cohort and GSE62452 cohort were used to validate the reliability of the risk signature. The impact of T lymphocyte-related genes from risk signature was confirmed in PC. Here, we observed the correlation between the T lymphocyte-related genes and the expression level of targeted therapy. We established a five-mRNA (LY6D, ANLN, ZNF488, MYEOV, and SCN11A) prognostic risk signature. Next, we identified ANLN and MYEOV that were associated with T lymphocyte infiltrations (P < 0.05). High ANLN and MYEOV expression levels had a poorer prognosis in decreased T lymphocyte subgroup in PC. Correlation analysis between ANLN and MYEOV and immunomodulators showed that ANLN and MYEOV may have potential value in pancreatic cancer immunotherapy.

The insecticide deltamethrin enhances sodium channel slow inactivation of human Nav1.9, Nav1.8 and Nav1.7.

The insecticide deltamethrin of the pyrethroid class mainly targets voltage-gated sodium channels (Navs). Deltamethrin prolongs the opening of Navs by slowing down fast inactivation and deactivation. Pyrethroids are supposedly safe for humans, however, they have also been linked to the gulf-war syndrome, a neuropathic pain condition that can develop following exposure to certain chemicals. Inherited neuropathic pain conditions have been linked to mutations in the Nav subtypes Nav1.7, Nav1.8, and Nav1.9. Here, we examined the effect of deltamethrin on the human isoforms Nav1.7, Nav1.8, and Nav1.9_C4 (chimera containing the C-terminus of rat Nav1.4) heterologously expressed in HEK293T and ND7/23 cells using whole-cell patch-clamp electrophysiology. For all three Nav subtypes, we observed increased persistent and tail currents that are typical for Nav channels modified by deltamethrin. The most surprising finding was an enhanced slow inactivation induced by deltamethrin in all three Nav subtypes. An enhanced slow inactivation is contrary to the prolonged opening caused by pyrethroids and has not been described for deltamethrin or any other pyrethroid before. Furthermore, we found that the fraction of deltamethrin-modified channels increased use-dependently. However, for Nav1.8, the use-dependent potentiation occurred only when the holding potential was increased to -90 mV, a potential at which the tail currents decay more slowly. This indicates that use-dependent modification is due to an accumulation of tail currents. In summary, our findings support a novel mechanism whereby deltamethrin enhances slow inactivation of voltage-gated sodium channels, which may, depending on the cellular resting membrane potential, reduce neuronal excitability and counteract the well-described pyrethroid effects of prolonging channel opening.

N58A Exerts Analgesic Effect on Trigeminal Neuralgia by Regulating the MAPK Pathway and Tetrodotoxin-Resistant Sodium Channel.

The primary studies have shown that scorpion analgesic peptide N58A has a significant effect on voltage-gated sodium channels (VGSCs) and plays an important role in neuropathic pain. The purpose of this study was to investigate the analgesic effect of N58A on trigeminal neuralgia (TN) and its possible mechanism. The results showed that N58A could significantly increase the threshold of mechanical pain and thermal pain and inhibit the spontaneous asymmetric scratching behavior of rats. Western blotting results showed that N58A could significantly reduce the protein phosphorylation level of ERK1/2, P38, JNK, and ERK5/CREB pathways and the expression of Nav1.8 and Nav1.9 proteins in a dose-dependent manner. The changes in current and kinetic characteristics of Nav1.8 and Nav1.9 channels in TG neurons were detected by the whole-cell patch clamp technique. The results showed that N58A significantly decreased the current density of Nav1.8 and Nav1.9 in model rats, and shifted the activation curve to hyperpolarization and the inactivation curve to depolarization. In conclusion, the analgesic effect of N58A on the chronic constriction injury of the infraorbital (IoN-CCI) model rats may be closely related to the regulation of the MAPK pathway and Nav1.8 and Nav1.9 sodium channels.

A Low-Frequency Pulsed Magnetic Field Reduces Neuropathic Pain by Regulating NaV1.8 and NaV1.9 Sodium Channels at the Transcriptional Level in Diabetic Rats.

Low-frequency pulsed magnetic field (LF-PMF) application is a non-invasive, easy, and inexpensive treatment method in pain management. However, the molecular mechanism underlying the effect of LF-PMF on pain is not fully understood. Considering the obvious dysregulations of gene expression observed in certain types of voltage-gated sodium channels (VGSCs) in pain conditions, the present study tested the hypothesis that LF-PMF shows its pain-relieving effect by regulating genes that code VGSCs proteins. Five experimental rat groups (Control, Streptozotocin-induced experimental painful diabetic neuropathy (PDN), PDN Sham, PDN 10 Hz PMF, and PDN 30 Hz PMF) were established. After the pain formation in PDN groups, the magnetic field groups were exposed to 10/30 Hz, 1.5 mT PMF for 4 weeks, an hour daily. Progression of pain was evaluated using behavioral pain tests during the entire experimental processes. After the end of PMF treatment, SCN9A (NaV1.7 ), SCN10A (NaV1.8 ), SCN11A (NaV1.9 ), and SCN3A (NaV1.3 ) gene expression level changes were determined by analyzing real-time polymerase chain reaction results. We found that 10 Hz PMF application was more effective than 30 Hz on pain management. In addition, NaV1.7 and NaV1.3 transcriptions were upregulated while NaV1.8 and NaV1.9 were downregulated in painful conditions. Notably, the downregulated expression of the genes encoding NaV1.8 and NaV1.9 were re-regulated and increased to control level by 10 Hz PMF application. Consequently, it may be deduced that 10 Hz PMF application reduces pain by modulating certain VGSCs at the transcriptional level. © 2021 Bioelectromagnetics Society.

SCN11A gene deletion causes sensorineural hearing loss by impairing the ribbon synapses and auditory nerves.

BACKGROUND: The SCN11A gene, encoded Nav1.9 TTX resistant sodium channels, is a main effector in peripheral inflammation related pain in nociceptive neurons. The role of SCN11A gene in the auditory system has not been well characterized. We therefore examined the expression of SCN11A in the murine cochlea, the morphological and physiological features of Nav1.9 knockout (KO) ICR mice. RESULTS: Nav1.9 expression was found in the primary afferent endings beneath the inner hair cells (IHCs). The relative quantitative expression of Nav1.9 mRNA in modiolus of wild-type (WT) mice remains unchanged from P0 to P60. The number of presynaptic CtBP2 puncta in Nav1.9 KO mice was significantly lower than WT. In addition, the number of SGNs in Nav1.9 KO mice was also less than WT in the basal turn, but not in the apical and middle turns. There was no lesion in the somas and stereocilia of hair cells in Nav1.9 KO mice. Furthermore, Nav1.9 KO mice showed higher and progressive elevated ABR threshold at 16 kHz, and a significant increase in CAP thresholds. CONCLUSIONS: These data suggest a role of Nav1.9 in regulating the function of ribbon synapses and the auditory nerves. The impairment induced by Nav1.9 gene deletion mimics the characters of cochlear synaptopathy.

Pain Severity Correlates With Biopsy-Mediated Colonic Afferent Activation But Not Psychological Scores in Patients With IBS-D.

INTRODUCTION: Despite heterogeneity, an increased prevalence of psychological comorbidity and an altered pronociceptive gut microenvironment have repeatedly emerged as causative pathophysiology in patients with irritable bowel syndrome (IBS). Our aim was to study these phenomena by comparing gut-related symptoms, psychological scores, and biopsy samples generated from a detailed diarrhea-predominant IBS patient (IBS-D) cohort before their entry into a previously reported clinical trial. METHODS: Data were generated from 42 patients with IBS-D who completed a daily 2-week bowel symptom diary, the Hospital Anxiety and Depression score, and the Patient Health Questionnaire-12 Somatic Symptom score and underwent unprepared flexible sigmoidoscopy. Sigmoid mucosal biopsies were separately evaluated using immunohistochemistry and culture supernatants to determine cellularity, mediator levels, and ability to stimulate colonic afferent activity. RESULTS: Pain severity scores significantly correlated with the daily duration of pain (r = 0.67, P < 0.00001), urgency (r = 0.57, P < 0.0005), and bloating (r = 0.39, P < 0.05), but not with psychological symptom scores for anxiety, depression, or somatization. Furthermore, pain severity scores from individual patients with IBS-D were significantly correlated (r = 0.40, P < 0.008) with stimulation of colonic afferent activation mediated by their biopsy supernatant, but not with biopsy cell counts nor measured mediator levels. DISCUSSION: Peripheral pronociceptive changes in the bowel seem more important than psychological factors in determining pain severity within a tightly phenotyped cohort of patients with IBS-D. No individual mediator was identified as the cause of this pronociceptive change, suggesting that nerve targeting therapeutic approaches may be more successful than mediator-driven approaches for the treatment of pain in IBS-D.

MicroRNA-96 is required to prevent allodynia by repressing voltage-gated sodium channels in spinal cord.

Voltage-gated sodium channels (Navs) 1.7, 1.8, and 1.9 are predominately expressed in peripheral sensory neurons and are critical for action potential propagation in nociceptors. Unexpectedly, we found that expression of SCN9A, SCN10A, SCN11A, and SCN2A, the alpha subunit of Nav1.7, Nav1.8, Nav1.9 and Nav1.2, respectively, are up-regulated in spinal dorsal horn (SDH) neurons of miR-96 knockout mice. These mice also have de-repression of CACNA2D1/2 in DRG and display thermal and mechanical allodynia that could be attenuated by intrathecal or intraperitoneal injection of Nav1.7 or Nav1.8 blockers or Gabapentin. Moreover, Gad2::CreERT2 conditional miR-96 knockout mice phenocopied global knockout mice, implicating inhibitory neurons; nerve injury induced significant loss of miR-96 in SDH GABAergic and Glutamatergic neurons in mice which negatively correlated to up-regulation of Nav1.7, Nav1.8, Nav1.9 and Scn2a, this dis-regulation of miR-96 and Navs in SDH neurons contributed to neuropathic pain which can be alleviated by intrathecal injection of Nav1.7 or Nav1.8 blockers. In conclusion, miR-96 is required to avoid allodynia through limiting the expression of VGCCs and Navs in DRG and Navs in SDH in naïve and nerve injury-induced neuropathic pain mice. Our findings suggest that central nervous system penetrating Nav1.7 and Nav1.8 blockers may be efficacious for pain relief.

Modulations of Nav1.8 and Nav1.9 Channels in Monosodium Urate-Induced Gouty Arthritis in Mice.

The aim of the present study was to observe the changes of TTX-R, Nav1.8, and Nav1.9 Na+ currents in MSU-induced gouty arthritis mice, and to explore the possibility of Nav1.8 and Nav1.9 channels as potential targets for gout pain treatment. Acute gouty arthritis was induced by monosodium urate (MSU) in mice. Swelling degree was evaluated by measuring the circumference of the ankle joint. Mechanical allodynia was assessed by applying the electronic von Frey. Na+ currents were recorded by patch-clamp techniques in acute isolated dorsal root ganglion (DRG) neurons. MSU treatment significantly increased the swelling degree of ankle joint and decreased the mechanical pain threshold. The amplitude of TTX-R Na+ current was significantly increased and reached its peak on the 4th day after injection of MSU. For TTX-R Na+ channel subunits, Nav1.8 current density was significantly increased, but Nav1.9 current density was markedly decreased after MSU treatment. MSU treatment shifted the steady-state activation curves of TTX-R Na+ channel, Nav1.8 and Nav1.9 channels, and the inactivation curves of TTX-R Na+ channel and Nav1.8 channels to the depolarizing direction, but did not affect the inactivation curve of Nav1.9 channel. Compared with the normal group, the recovery of Nav1.8 channel was faster, while that of Nav1.9 channel was slower. The recovery of TTX-R Na+ channel remained unchanged after MSU treatment. Additionally, MSU treatment increased DRG neurons excitability by reducing action potential threshold. Nav1.8 channel, not Nav1.9 channel, may be involved in MSU-induced gout pain by increasing nerve excitability.

Analgesic Effects of Topical Amitriptyline in Patients With Chemotherapy-Induced Peripheral Neuropathy: Mechanistic Insights From Studies in Mice.

Oral amitriptyline hydrochloride (amitriptyline) is ineffective against some forms of chronic pain and is often associated with dose-limiting adverse events. We evaluated the potential effectiveness of high-dose topical amitriptyline in a preliminary case series of chemotherapy-induced peripheral neuropathy patients and investigated whether local or systemic adverse events associated with the use of amitriptyline were present in these patients. We also investigated the mechanism of action of topically administered amitriptyline in mice. Our case series suggested that topical 10% amitriptyline treatment was associated with pain relief in chemotherapy-induced peripheral neuropathy patients, without the side effects associated with systemic absorption. Topical amitriptyline significantly increased mechanical withdrawal thresholds when applied to the hind paw of mice, and inhibited the firing responses of C-, Aß- and Aδ-type peripheral nerve fibers in ex vivo skin-saphenous nerve preparations. Whole-cell patch-clamp recordings on cultured sensory neurons revealed that amitriptyline was a potent inhibitor of the main voltage-gated sodium channels (Nav1.7, Nav1.8, and Nav1.9) found in nociceptors. Calcium imaging showed that amitriptyline activated the transient receptor potential cation channel, TRPA1. Our case series indicated that high-dose 10% topical amitriptyline could alleviate neuropathic pain without adverse local or systemic effects. This analgesic action appeared to be mediated through local inhibition of voltage-gated sodium channels. PERSPECTIVE: Our preliminary case series suggested that topical amitriptyline could provide effective pain relief for chemotherapy-induced peripheral neuropathy patients without any systemic or local adverse events. Investigation of the mechanism of this analgesic action in mice revealed that this activity was mediated through local inhibition of nociceptor Nav channels.

Mechanical allodynia triggered by cold exposure in mice with the Scn11a p.R222S mutation: a novel model of drug therapy for neuropathic pain related to NaV1.9.

Mutations within the SCN11A gene which encodes the voltage-gated sodium channel NaV1.9 mainly expressed in small fiber sensory neurons have been associated with neuropathic disorders; however, suitable medications have not been fully investigated. To develop drug therapies against NaV1.9-related neuropathic pain, we aimed to establish a novel model using mice carrying the Scn11a p.R222S mutation initially identified in patients with familial episodic limb pain that is characterized by paroxysmal pain induced by fatigue or bad weather conditions. We investigated the influence of cold exposure (4 °C, overnight) on the behavioral and biochemical phenotypes of Scn11a p.R222S mutant (R222S) and wild type C57BL/6N (WT) mice. We also tested the effects of acetaminophen (125, 250 mg/kg, perorally, p.o.) and traditional Japanese medicine, goshajinkigan (0.5 or 1.0 g/kg, p.o.), which are analgesic drugs prescribed to patients with neuropathic pain, in this model of cold-induced mechanical allodynia in R222S mice.Cold-exposed R222S mice exhibited enhanced mechanical allodynia and thermal hypersensitivity compared with WT mice. The decrease of the mechanical withdrawal threshold in R222S mice was reversible 24 h after housing at room temperature. There was no significant change in the levels of interleukin-1ß, interleukin-6, tumor necrosis factor-α, or interferon-γ in the plasma or spinal cords of WT and R222S mice after cold exposure. Both acetaminophen (250 mg/kg) and goshajinkigan (1.0 g/kg) significantly attenuated mechanical allodynia in R222S mice. The model of cold-induced mechanical allodynia in mice with the Scn11a p.R222S mutation is novel and useful for evaluating analgesic drugs for intractable neuropathies related to NaV1.9.

Gain-of-function mutation in SCN11A causes itch and affects neurogenic inflammation and muscle function in Scn11a+/L799P mice.

Mutations in the genes encoding for voltage-gated sodium channels cause profound sensory disturbances and other symptoms dependent on the distribution of a particular channel subtype in different organs. Humans with the gain-of-function mutation p.Leu811Pro in SCN11A (encoding for the voltage-gated Nav1.9 channel) exhibit congenital insensitivity to pain, pruritus, self-inflicted injuries, slow healing wounds, muscle weakness, Charcot-like arthropathies, and intestinal dysmotility. As already shown, knock-in mice (Scn11a+/L799P) carrying the orthologous mutation p.Leu799Pro replicate reduced pain sensitivity and show frequent tissue lesions. In the present study we explored whether Scn11a+/L799P mice develop also pruritus, muscle weakness, and changes in gastrointestinal transit time. Furthermore, we analyzed morphological and functional differences in nerves, skeletal muscle, joints and small intestine from Scn11a+/L799P and Scn11a+/+ wild type mice. Compared to Scn11a+/+ mice, Scn11a+/L799P mice showed enhanced scratching bouts before skin lesions developed, indicating pruritus. Scn11a+/L799P mice exhibited reduced grip strength, but no disturbances in motor coordination. Skeletal muscle fiber types and joint architecture were unaltered in Scn11a+/L799P mice. Their gastrointestinal transit time was unaltered. The small intestine from Scn11a+/L799P showed a small shift towards less frequent peristaltic movements. Similar proportions of lumbar dorsal root ganglion neurons from Scn11a+/L799P and Scn11a+/+ mice were calcitonin gene-related peptide (CGRP-) positive, but isolated sciatic nerves from Scn11a+/L799P mice exhibited a significant reduction of the capsaicin-evoked release of CGRP indicating reduced neurogenic inflammation. These data indicate important Nav1.9 channel functions in several organs in both humans and mice. They support the pathophysiological relevance of increased basal activity of Nav1.9 channels for sensory abnormalities (pain and itch) and suggest resulting malfunctions of the motor system and of the gastrointestinal tract. Scn11a+/L799P mice are suitable to investigate the role of Nav1.9, and to explore the pathophysiological changes and mechanisms which develop as a consequence of Nav1.9 hyperactivity.

Painful and painless mutations of SCN9A and SCN11A voltage-gated sodium channels.

Chronic pain is a global problem affecting up to 20% of the world's population and has a significant economic, social and personal cost to society. Sensory neurons of the dorsal root ganglia (DRG) detect noxious stimuli and transmit this sensory information to regions of the central nervous system (CNS) where activity is perceived as pain. DRG neurons express multiple voltage-gated sodium channels that underlie their excitability. Research over the last 20 years has provided valuable insights into the critical roles that two channels, NaV1.7 and NaV1.9, play in pain signalling in man. Gain of function mutations in NaV1.7 cause painful conditions while loss of function mutations cause complete insensitivity to pain. Only gain of function mutations have been reported for NaV1.9. However, while most NaV1.9 mutations lead to painful conditions, a few are reported to cause insensitivity to pain. The critical roles these channels play in pain along with their low expression in the CNS and heart muscle suggest they are valid targets for novel analgesic drugs.

Spider venom-derived peptide induces hyperalgesia in Nav1.7 knockout mice by activating Nav1.9 channels.

The sodium channels Nav1.7, Nav1.8 and Nav1.9 are critical for pain perception in peripheral nociceptors. Loss of function of Nav1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of Kv4.2, restores nociception in Nav1.7 knockout (Nav1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Nav1.7 and activates Nav1.9 but does not affect Nav1.8. This toxin produces pain in wild-type (WT) and Nav1.7-KO mice, and attenuates nociception in Nav1.9-KO mice, but has no effect in Nav1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Nav1.9 activation and offers pharmacological insight into the relationship of the three Nav channels in pain signalling.