CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3-5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis.

Melatonin attenuated chronic visceral pain by reducing Nav1.8 expression and nociceptive neuronal sensitization.

BACKGROUND: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, and its specific pathogenesis is still unclear. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized in a rat model of visceral hypersensitivity induced by neonatal colonic inflammation (NCI). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for melatonin (MT) in sensitization of sodium channels in NCI rats. METHODS: Colorectal distention (CRD) in adult male rats as a measure of visceral hypersensitivity. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. Western blot and Immunofluorescence were employed to detect changes in expression of Nav1.8 and MT2. RESULTS: The results showed that rats exhibited visceral hypersensitivity after NCI treatment. Intrathecal application of melatonin significantly increased the threshold of CRD in NCI rats with a dose-dependent manner, but has no role in the control group. Whole-cell patch clamp recording showed that melatonin remarkably decreased the excitability and the density of TTX-R sodium channel in DRG neurons from NCI rats. The expression of MT2 receptor at the protein level was markedly lower in NCI rats. 8MP, an agonist of MT2 receptor, enhanced the distention threshold in NCI rats. Application of 8MP reversed the enhanced hypersensitivity of DRG neurons from NCI rats. 8MP also reduced TTX-R sodium current density and modulated dynamics of TTX-R sodium current activation. CONCLUSIONS: These data suggest that sensitization of sodium channels of colon DRG neurons in NCI rats is most likely mediated by MT2 receptor, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

The Antidiabetic Drug Metformin Regulates Voltage-Gated Sodium Channel NaV1.7 via the Ubiquitin-Ligase NEDD4-2.

The antidiabetic drug metformin has been shown to reduce pain hypersensitivity in preclinical models of chronic pain and in neuropathic pain in humans. Multiple intracellular pathways have been described as metformin targets. Among them, metformin is an activator of the adenosine 5'-monophosphate protein kinase that can in turn modulate the activity of the E3 ubiquitin ligase NEDD4-2 and thus post-translational expression of voltage-gated sodium channels (NaVs). In this study, we found that the bulk of the effect of metformin on Na1.7 is dependent on NEDD4-2. In HEK cells, the expression of NaV1.7 at the membrane fraction, obtained by a biotinylation approach, is only reduced by metformin when cotransfected with NEDD4-2. Similarly, in voltage-clamp recordings, metformin significantly reduced NaV1.7 current density when cotransfected with NEDD4-2. In mouse dorsal root ganglion (DRG) neurons, without changing the biophysical properties of NaV1.7, metformin significantly decreased NaV1.7 current densities, but not in Nedd4L knock-out mice (SNS-Nedd4L-/-). In addition, metformin induced a significant reduction in NEDD4-2 phosphorylation at the serine-328 residue in DRG neurons, an inhibitory phosphorylation site of NEDD4-2. In current-clamp recordings, metformin reduced the number of action potentials elicited by DRG neurons from Nedd4Lfl/fl , with a partial decrease also present in SNS-Nedd4L-/- mice, suggesting that metformin can also change neuronal excitability in an NEDD4-2-independent manner. We suggest that NEDD4-2 is a critical player for the effect of metformin on the excitability of nociceptive neurons; this action may contribute to the relief of neuropathic pain.

Optogenetic Inhibition of Nav1.8 Expressing Corneal Afferents Reduces Persistent Dry Eye Pain.

Purpose: The aim of the present study was to investigate the contribution of Nav1.8 expressing corneal afferent neurons to the presence of ongoing pain in lacrimal gland excision (LGE)-induced dry eye. Methods: The proton pump archaerhodopsin-3/eGFP (ArchT/eGFP) was conditionally expressed in corneal afferents using Nav1.8-cre mice. Dry eye was produced by unilateral LGE. Real time place preference was assessed using a three-chamber apparatus. A neutral, unlit center chamber was flanked by one illuminated with a control light and one illuminated with an ArchT activating light. For real-time preference, animals were placed in the neutral chamber and tracked over five 10-minute sessions, with the lights turned on during the second and fourth sessions. In other studies, movement was tracked over three 10-minute sessions (the lights turned on only during the second session), with animals tested once per day over the course of 4 days. A local anesthetic was used to examine the role of ongoing corneal afferent activity in producing place preference. Results: The corneal afferent nerves and trigeminal ganglion cell bodies showed a robust eGFP signal in Nav1.8-cre;ArchT/eGFP mice. After LGE, Nav1.8-cre;ArchT/eGFP mice demonstrated a preference for the ArchT activating light paired chamber. Preference was prevented with pre-application to the cornea of a local anesthetic. Nav1.8-cre;ArchT/eGFP mice with sham surgery and LGE wild-type control mice did not develop preference. Conclusions: Results indicate LGE-induced persistent, ongoing pain, driven by Nav1.8 expressing corneal afferents. Inhibition of these neurons represents a potential strategy for treating ongoing dry eye-induced pain.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

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.

Neuroendocrine Mechanisms Governing Sex Differences in Hyperalgesic Priming Involve Prolactin Receptor Sensory Neuron Signaling.

Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE2 as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE2 hypersensitivity was more persistent in females. This difference in PGE2 response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female, but not male, mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling.SIGNIFICANCE STATEMENT Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that prolactin receptor expression in Nav1.8+ neurons was necessary for hyperalgesic priming in female, but not male, mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

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.

Enhancement by TNF-α of TTX-resistant NaV current in muscle sensory neurons after femoral artery occlusion.

Femoral artery occlusion in rats has been used to study human peripheral artery disease (PAD). Using this animal model, a recent study suggests that increases in levels of tumor necrosis factor-α (TNF-α) and its receptor lead to exaggerated responses of sympathetic nervous activity and arterial blood pressure as metabolically sensitive muscle afferents are activated. Note that voltage-dependent Na+ subtype NaV1.8 channels (NaV1.8) are predominately present in chemically sensitive thin fiber sensory nerves. The purpose of this study was to examine the role played by TNF-α in regulating activity of NaV1.8 currents in muscle dorsal root ganglion (DRG) neurons of rats with PAD induced by femoral artery occlusion. DRG neurons from control and occluded limbs of rats were labeled by injecting the fluorescent tracer DiI into the hindlimb muscles 5 days before the experiments. A voltage patch-clamp mode was used to examine TTX-resistant (TTX-R) NaV currents. Results were as follows: 72 h of femoral artery occlusion increased peak amplitude of TTX-R [1,922 ± 139 pA in occlusion (n = 11 DRG neurons) vs. 1,178 ± 39 pA in control (n = 10), means ± SE; P < 0.001 between the 2 groups] and NaV1.8 currents [1,461 ± 116 pA in occlusion (n = 11) and 766 ± 48 pA in control (n = 10); P < 0.001 between groups] in muscle DRG neurons. TNF-α exposure amplified TTX-R and NaV1.8 currents in DRG neurons of occluded muscles in a dose-dependent manner. Notably, the amplification of TTX-R and NaV1.8 currents induced by TNF-α was attenuated in DRG neurons with preincubation with respective inhibitors of the intracellular signaling pathways p38-MAPK, JNK, and ERK. In conclusion, our data suggest that NaV1.8 is engaged in the role of TNF-α in amplifying muscle afferent inputs as the hindlimb muscles are ischemic; p38-MAPK, JNK, and ERK pathways are likely necessary to mediate the effects of TNF-α.

Upregulation of transcription factor 4 downregulates NaV1.8 expression in DRG neurons and prevents the development of rat inflammatory and neuropathic hypersensitivity.

The voltage sodium channel 1.8 (NaV1.8) in the dorsal root ganglion (DRG) neurons contributes to the initiation and development of chronic inflammatory and neuropathic pain. However, an effective intervention on NaV1.8 remains to be studied in pre-clinical research and clinical trials. In this study, we aimed to investigate whether transcription factor 4 (TCF4) overexpression represses NaV1.8 expression in DRG neurons, thus preventing the development of chronic pain. Using chromatin immunoprecipitation (CHIP), we verified the interaction of TCF4 and sodium voltage-gated channel alpha subunit 10A (SCN10A) enhancer in HEK293 cells and rat DRG neurons. Using a dual luciferase reporter assay, we confirmed the transcriptional inhibition of TCF4 on SCN10A promoter in vitro. To investigate the regulation of TCF4 on Nav1.8, we then upregulated TCF4 expression by intrathecally delivering an overexpression of recombinant adeno-associated virus (rAAV) in the Complete Freund's adjuvant (CFA)-induced inflammatory pain model and spared nerve injury (SNI)-induced neuropathic pain model. By using a quantitative polymerase chain reaction (qPCR), western blot, and immunostaining, we evaluated NaV1.8 expression after a noxious stimulation and the application of the TCF4 overexpression virus. We showed that the intrathecal delivery of TCF4 overexpression virus significantly repressed the increase of NaV1.8 and prevented the development of hyperalgesia in rats. Moreover, we confirmed the efficient role of an overexpressed TCF4 in preventing the CFA- and SNI-induced neuronal hyperexcitability by calcium imaging. Our results suggest that attenuating the dysregulation of NaV1.8 by targeting TCF4 may be a novel therapeutic strategy for chronic inflammatory and neuropathic pain.

Activation of human macrophage sodium channels regulates RNA processing to increase expression of the DNA repair protein PPP1R10.

Prior work demonstrated that a splice variant of SCN5A, a voltage-gated sodium channel gene, acts as a cytoplasmic sensor for viral dsRNA in human macrophages. Expression of this channel also polarizes macrophages to an anti-inflammatory phenotype in vitro and in vivo. Here we utilized global expression analysis of splice variants to identify novel channel-dependent signaling mechanisms. Pharmacological activation of voltage-gated sodium channels in human macrophages, but not treatment with cytoplasmic poly I:C, was associated with splicing of a retained intron in transcripts of PPP1R10, a regulator of phosphatase activity and DNA repair. Microarray analysis also demonstrated expression of a novel sodium channel splice variant, human macrophage SCN10A, that contains a similar exon deletion as SCN5A. SCN10A localizes to cytoplasmic and nuclear vesicles in human macrophages. Simultaneous expression of human macrophage SCN5A and SCN10A was required to decrease expression of the retained intron and increase protein expression of PPP1R10. Channel activation also increased protein expression of the splicing factor EFTUD2, and knockdown of EFTUD2 prevented channel dependent splicing of the retained PPP1R10 intron. Knockdown of the SCN5A and SCN10A variants in human macrophages reduced the severity of dsDNA breaks induced by treatment with bleomycin and type 1 interferon. These results suggested that human macrophage SCN5A and SCN10A variants mediate an innate immune signaling pathway that limits DNA damage through increased expression of PPP1R10. The functional significance of this pathway is that it may prevent cytotoxicity during inflammatory responses.

Pyrethroid neurotoxicity studies with bifenthrin indicate a mixed Type I/II mode of action.

BACKGROUND: Bifenthrin is usually considered a Type I pyrethroid, because it lacks an α-CN group present in Type II pyrethroids, but some previous studies suggest a mixed Type I/II mode-of-action. Results are presented for bifenthrin in a rat developmental neurotoxicity (DNT) study along with effects on Na currents in human VGSC subtypes. Molecular modeling comparisons were also made for bifenthrin and other pyrethroids. RESULTS: In a rat DNT study, bifenthrin produced tremors and clonic convulsions in dams and pups and slightly reduced acoustic startle response amplitude, and increased Tmax, at PND20 in females. Similar blood levels of bifenthrin were measured in dams and pups at each dose level i.e. no concentration in pups. In human VGSC experiments, using the Nav1.8 subtype, bifenthrin's effects on inactivation were slight, as for Type II pyrethroids, but without large prolongation of the tail current (deactivation) seen with Type II. Molecular modeling of bifenthrin indicates that the o-Me group may occupy a similar space to the α-CN group of cypermethrin and fenpropathrin. CONCLUSION: In a DNT study and on human Nav1.8 tail currents bifenthrin showed Type I and II effects, similar to some published studies. Overall, bifenthrin acts as a mixed Type I/II pyrethroid. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Loss-of-function of Nav1.8/D1639N linked to human pain can be rescued by lidocaine.

Mutations in voltage-gated sodium channels are associated with altered pain perception in humans. Most of these mutations studied to date present with a direct and intuitive link between the altered electrophysiological function of the channel and the phenotype of the patient. In this study, we characterize a variant of Nav1.8, D1639N, which has been previously identified in a patient suffering from the chronic pain syndrome "small fiber neuropathy". Using a heterologous expression system and patch-clamp analysis, we show that Nav1.8/D1639N reduces current density without altering biophysical gating properties of Nav1.8. Therefore, the D1639N variant causes a loss-of-function of the Nav1.8 sodium channel in a patient suffering from chronic pain. Using immunocytochemistry and biochemical approaches, we show that Nav1.8/D1639N impairs trafficking of the channel to the cell membrane. Neither co-expression of ß1 or ß3 subunit, nor overnight incubation at 27 °C rescued current density of the D1639N variant. On the other hand, overnight incubation with lidocaine fully restored current density of Nav1.8/D1639N most likely by overcoming the trafficking defect, whereas phenytoin failed to do so. Since lidocaine rescues the loss-of-function of Nav1.8/D1639N, it may offer a future therapeutic option for the patient carrying this variant. These results demonstrate that the D1639N variant, identified in a patient suffering from chronic pain, causes loss-of-function of the channel due to impaired cell surface trafficking and that this trafficking defect can be rescued by lidocaine.

Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain.

Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specific contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, inflammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, significantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma. Together, NaV1.6 in NaV1.8-positive neurons does not influence pain thresholds under normal or pathological conditions, but NaV1.6 in large NaV1.8-negative DRG neurons plays an important role in neuropathic pain.

Role of ASIC3, Nav1.7 and Nav1.8 in electroacupuncture-induced analgesia in a mouse model of fibromyalgia pain.

BACKGROUND: The mechanisms underlying fibromyalgia (FM) pain are not understood. The US Food and Drug Administration has recommended three drugs for treating FM-namely, pregabalin, duloxetine and milnacipran; however, these medications are associated with severe side effects. OBJECTIVE: To create a mouse model of FM pain using dual injections of acidic saline to cause mechanical hyperalgesia and test whether ASIC3, Nav1.7 and Nav1.8 are involved in this process and whether electroacupuncture (EA) can reverse these phenomena. METHODS: The FM model was established by injecting acidic saline twice into 40 ICR mice. The mice were assigned to subgroups (n=8 each) treated with different EA frequencies (2, 15 and 50 Hz). ASIC3, Nav1.7 and Nav1.8 expression levels were measured by Western blotting and immunohistochemistry. RESULTS: Significant mechanical hyperalgesia was induced on day 8 in FM mice, which was reversed by 2, 15 and 50 Hz EA. ASIC3, Nav1.7 and Nav1.8 protein levels increased significantly in both the dorsal root ganglion and in the spinal cord of FM model mice. These changes were further attenuated by 2, 15 and 50 Hz EA. CONCLUSION: Reduced nociceptive ASIC3, Nav1.7 and Nav1.8 proteins are involved in the preventive effects of EA against FM, and this series of molecules may represent targets for FM treatment.

Effects of SCN9A gene modification on Na+ channel and the expression of nerve growth factor in a rat model of diarrhea­predominant irritable bowel syndrome.

The aim of the present study was to identify whether the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene modification is a potential treatment for diarrhea­predominant irritable bowel syndrome (D­IBS), via regulating the Na+ channel and the expression of nerve growth factor (NGF). The recombinant adenovirus vector of the SCN9A gene was established, and rat colon cells were isolated for SCN9A gene modification. All subjects were divided into four groups: i) The SCN9A­modified (D­IBS rat model implanted with SCN9A­modified colon cells), ii) negative control (NC; D­IBS rat model implanted with colon cells without SCN9A gene modification), iii) blank (D­IBS rat model without any treatment) and iv) normal (normal rats without any treatment). Western blotting and reverse transcription­quantitative polymerase chain reaction were used to detect the protein and mRNA expression levels of SCN9A, NGF and voltage gated sodium channels (Nav)1.8 and Nav1.9 in rat colon tissues. Compared with the normal group, the rats in the SCN9A, NC and blank groups had significantly elevated mRNA and protein expression levels of NGF, SCN9A, Nav1.8 and Nav1.9. The rats in the SCN9A group demonstrated significantly increased mRNA and protein expression levels of NGF, SCN9A, Nav1.8 and Nav1.9 compared with the NC group and the blank group (all P<0.05). SCN9A gene modification can promote the expression of Nav1.8 and Nav1.9 channels, in addition to NGF which may provide a novel therapeutic basis for treating of D­IBS.

Protein kinase A regulates inflammatory pain sensitization by modulating HCN2 channel activity in nociceptive sensory neurons.

Several studies implicated cyclic adenosine monophosphate (cAMP) as an important second messenger for regulating nociceptor sensitization, but downstream targets of this signaling pathway which contribute to neuronal plasticity are not well understood. We used a Cre/loxP-based strategy to disable the function of either HCN2 or PKA selectively in a subset of peripheral nociceptive neurons and analyzed the nociceptive responses in both transgenic lines. A near-complete lack of sensitization was observed in both mutant strains when peripheral inflammation was induced by an intradermal injection of 8br-cAMP. The lack of HCN2 as well as the inhibition of PKA eliminated the cAMP-mediated increase of calcium transients in dorsal root ganglion neurons. Facilitation of Ih via cAMP, a hallmark of the Ih current, was abolished in neurons without PKA activity. Collectively, these results show a significant contribution of both genes to inflammatory pain and suggest that PKA-dependent activation of HCN2 underlies cAMP-triggered neuronal sensitization.

Chronic exposure to tumor necrosis factor in vivo induces hyperalgesia, upregulates sodium channel gene expression and alters the cellular electrophysiology of dorsal root ganglion neurons.

The goal of these studies was to investigate the links between chronic exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF), hyperalgesia and the excitability of dorsal root ganglion (DRG) sensory neurons. We employed transgenic mice that constitutively express TNF (TNFtg mice), a well-established model of chronic systemic inflammation. At 6 months of age, TNFtg mice demonstrated increased sensitivity to both mechanical and thermal heat stimulation relative to aged-matched wild-type controls. These increases in stimulus-evoked behaviors are consistent with nociceptor sensitization to normal physiological stimulation. The mechanisms underlying nociceptor sensitization were investigated using single-cell analysis to quantitatively compare gene expression in small-diameter (<30µm) DRG neurons. This analysis revealed the upregulation of mRNA encoding for tetrodotoxin-resistant (TTX-R) sodium (Na+) channels (Nav1.8, Nav1.9), Na+ channel ß subunits (ß1-ß3), TNF receptor 1 (TNFR1) and p38α mitogen-activated protein kinase in neurons of TNFtg mice. Whole-cell electrophysiology demonstrated a corresponding increase in TTX-R Na+ current density, hyperpolarizing shifts in activation and steady-state inactivation, and slower recovery from inactivation in the TNFtg neurons. Increased overlap of activation and inactivation in the TNFtg neurons produces inward Na+ currents at voltages near the resting membrane potential of sensory neurons (i.e. window currents). The combination of increased Na+ current amplitude, hyperpolarized shifts in Na+ channel activation and increased window current predicts a reduction in the action potential threshold and increased firing of small-diameter DRG neurons. Together, these data suggest that increases in the expression of Nav1.8 channels, regulatory ß1 subunits and TNFR1 contribute to increased nociceptor excitability and hyperalgesia in the TNFtg mice.