Excitability Of Dorsal Root Ganglion Research Articles

Circular RNA-GRIN2B Suppresses Neuropathic Pain by Targeting the NF-κB/SLICK Pathway.

The role of circular RNAs (circRNAs) in neuropathic pain is linked to the fundamental physiological mechanisms involved. However, the exact function of circRNAs in the context of neuropathic pain is still not fully understood. The functional impact of circGRIN2B on the excitability of dorsal root ganglion (DRG) neurons was investigated using siRNA or overexpression technology in conjunction with fluorescence in situ hybridization and whole-cell patch-clamp technology. The therapeutic efficacy of circGRIN2B in treating neuropathic pain was confirmed by assessing the pain threshold in a chronic constrictive injury (CCI) model. The interaction between circGRIN2B and NF-κB was examined through RNA pulldown, RIP, and mass spectrometry assays. CircGRIN2B knockdown significantly affected the action potential discharge frequency and the sodium-dependent potassium current flux (SLICK) in DRG neurons. Furthermore, knockdown of circGRIN2B dramatically reduced the SLICK channel protein and mRNA expression in vivo and in vitro. Our research confirmed the interaction between circGRIN2B and NF-κB. These findings demonstrated that circGRIN2B promotes the transcription of the SLICK gene by binding to NF-κB. In CCI rat models, the overexpression of circGRIN2B has been shown to hinder the progression of neuropathic pain, particularly by reducing mechanical and thermal hyperalgesia. Additionally, this upregulation significantly diminished the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the DRG. Upon reviewing these findings, it was determined that circGRIN2B may mitigate the onset of neuropathic pain by modulating the NF-κB/SLICK pathway.

Differences and Similarities in Spontaneous Activity Between Animal Models of Cancer-Induced Pain and Neuropathic Pain.

BackgroundClinical data on cancer-induced pain (CIP) demonstrate widespread changes in sensory function. It is characterized in humans not only by stimulus-invoked pain, but also by spontaneous pain. In our previous studies in an animal model of CIP, we observed changes in intrinsic membrane properties and excitability of dorsal root ganglion (DRG) sensory neurons corresponding to mechanical allodynia and hyperalgesia, of which abnormal activities of Aβ-fiber sensory neurons are consistent in a rat model of peripheral neuropathic pain (NEP).ObjectiveTo investigate whether there are related peripheral neural mechanisms between the CIP and NEP models of spontaneous pain, we compared the electrophysiological properties of DRG sensory neurons at 2–3 weeks after CIP and NEP model induction.MethodsCIP models were induced with metastasis tumour-1 rat breast cancer cells implanted into the distal epiphysis of the femur. NEP models were induced with a polyethylene cuff implanted around the sciatic nerve. Spontaneous pain in animals is measured by spontaneous foot lifting (SFL). After measurement of SFL, the animals were prepared for electrophysiological recordings of spontaneous activity (SA) in DRG neurons in vivo.ResultsOur data showed that SFL and SA occurred in both models. The proportion of SFL and SA of C-fiber sensory neurons in CIP was more significantly increased than in NEP models. There was no difference in duration of SFL and the rate of SA between the two models. The duration of SFL is related to the rate of SA in C-fiber in both models.ConclusionThus, SFL may result from SA activity in C-fiber neurons in CIP and NEP rats. The differences and similarities in spontaneous pain between CIP and NEP rats is related to the proportion and rate of SA in C-fibers, respectively.

Exploring pharmacological inhibition of Gq/11 as an analgesic strategy.

Misuse of opioids has greatly affected our society. One potential solution is to develop analgesics that act at targets other than opioid receptors. These can be used either as stand-alone therapeutics or to improve the safety profile of opioid drugs. Previous research showed that activation of Gq/11 proteins by G-protein coupled receptors has pro-nociceptive properties, suggesting that blockade of Gq/11 signalling could be beneficial for pain control. The aim of this study was to test this hypothesis pharmacologically by using potent and selective Gq/11 inhibitor YM-254890. We used a series of behavioural assays to evaluate the acute responses of mice to painful thermal stimulation while administering YM-254890 alone and in combination with morphine. We then used electrophysiological recordings to evaluate the effects of YM-254890 on the excitability of dorsal root ganglion (DRG) nociceptor neurons. We found that systemic administration of YM-254890 produced anti-nociceptive effects and also augmented morphine analgesia in both hotplate and tail flick paradigms. However, it also caused substantial inhibition of locomotion, which may limit its therapeutic utility. To circumvent these issues, we explored the local administration of YM-254890. Intrathecal injections of YM-254890 produced lasting analgesia in a tail flick test and greatly augmented the anti-nociceptive effects of morphine without any significant effects on locomotor behaviour. Electrophysiological studies showed that YM-254890 reduced the excitability of DRG nociceptors and augmented their opioid-induced inhibition. These findings indicate that pharmacological inhibition of Gq/11 could be explored as an analgesic strategy.

Inhibition of sodium conductance by cannabigerol contributes to a reduction of dorsal root ganglion neuron excitability.

Cannabigerol (CBG), a non-psychotropic phytocannabinoid and a precursor of ∆9 -tetrahydrocannabinol and cannabidiol, has been suggested to act as an analgesic. A previous study reported that CBG (10μM) blocks voltage-gated sodium (Nav ) currents in CNS neurons, although the underlying mechanism is not well understood. Genetic and functional studies have validated Nav 1.7 channels as an opportune target for analgesic drug development. The effects of CBG on Nav 1.7 channels, which may contribute to its analgesic properties, have not been previously investigated. To determine the effects of CBG on Nav channels, we used stably transfected HEK cells and primary dorsal root ganglion (DRG) neurons to characterize compound effects using experimental and computational techniques. These included patch-clamp, multielectrode array, and action potential modelling. CBG is a ~10-fold state-dependent Nav channel inhibitor (KI -KR : ~2-20 μM) with an average Hill-slope of ~2. We determined that, at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation. However, as the concentration is increased, CBG also induces a hyperpolarizing shift in the half-voltage of inactivation. Our modelling and multielectrode array recordings suggest that CBG attenuates DRG excitability. Inhibition of Nav 1.7 channels in DRG neurons may underlie CBG-induced neuronal hypoexcitability. As most Nav 1.7 channels are inactivated at the resting membrane potential of DRG neurons, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav 1.7 channels, compared with other Nav channels (via Gmax block).

Sex-specific role for serotonin 5-HT2A receptor in modulation of opioid-induced antinociception and reward in mice

Opioids are among the most effective analgesics and the mainstay of pain management. However, concerns about safety and abuse liability have challenged their widespread use by the medical community. Opioid-sparing therapies include drugs that in combination with opioids have the ability to enhance analgesia while decreasing opioid requirement as well as their side effects. Sex differences in antinociceptive responses to opioids have received increasing attention in recent years. However, the molecular mechanisms underlying sex differences related to opioid-sparing adjuncts remain largely unexplored. Using warm water tail-withdrawal as a mouse model of acute thermal nociception, our data suggest that adjunctive administration of the serotonin 5-HT2A receptor (5-HT2AR) antagonist volinanserin dose-dependently enhanced potency of the opioid analgesic oxycodone in male, but not female, mice. This antinociceptive-like response induced by oxycodone was also augmented in 5-HT2AR knockout (5-HT2AR−/−) male, but not female mice; an effect that was reversed by Cre-loxP-mediated selective expression of 5-HT2AR in dorsal root ganglion (DRG) neurons of 5-HT2AR−/− littermates. Pharmacological inhibition with volinanserin or genetic deletion in 5-HT2AR−/− animals potentiated the ability of oxycodone to reduce DRG excitability in male mice. Adjunctive volinanserin did not affect oxycodone-induced conditioned place preference (CPP), whereas it reduced oxycodone-induced locomotor sensitization in male and female mice. Together, these results suggest that adjunctive volinanserin augments opioid-induced antinociception, but not abuse-related behavior, through a sex-specific signaling crosstalk mechanism that requires 5-HT2AR expression in mouse DRG neurons. Ultimately, our results may pave the way for the clinical evaluation of volinanserin as a potential sex-specific opioid adjuvant.

Protein kinase G signaling pathway is involved in sympathetically maintained pain by modulating ATP-sensitive potassium channels

BackgroundSympathetically maintained pain (SMP) involves an increased excitability of dorsal root ganglion (DRG) neurons to sympathetic nerve stimulation and circulating norepinephrine. The current treatment of SMP has limited efficacy, and...

Oxidative stress induced by NOX2 contributes to neuropathic pain via plasma membrane translocation of PKC\u03b5 in rat dorsal root ganglion neurons

BackgroundNicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-induced oxidative stress, including the production of reactive oxygen species (ROS) and hydrogen peroxide, plays a pivotal role in neuropathic pain. Although the activation and plasma membrane translocation of protein kinase C (PKC) isoforms in dorsal root ganglion (DRG) neurons have been implicated in multiple pain models, the interactions between NOX2-induced oxidative stress and PKC remain unknown.MethodsA spared nerve injury (SNI) model was established in adult male rats. Pharmacologic intervention and AAV-shRNA were applied locally to DRGs. Pain behavior was evaluated by Von Frey tests. Western blotting and immunohistochemistry were performed to examine the underlying mechanisms. The excitability of DRG neurons was recorded by whole-cell patch clamping.ResultsSNI induced persistent NOX2 upregulation in DRGs for up to 2 weeks and increased the excitability of DRG neurons, and these effects were suppressed by local application of gp91-tat (a NOX2-blocking peptide) or NOX2-shRNA to DRGs. Of note, the SNI-induced upregulated expression of PKCε but not PKC was decreased by gp91-tat in DRGs. Mechanical allodynia and DRG excitability were increased by ψεRACK (a PKCε activator) and reduced by εV1-2 (a PKCε-specific inhibitor). Importantly, εV1-2 failed to inhibit SNI-induced NOX2 upregulation. Moreover, the SNI-induced increase in PKCε protein expression in both the plasma membrane and cytosol in DRGs was attenuated by gp91-tat pretreatment, and the enhanced translocation of PKCε was recapitulated by H2O2 administration. SNI-induced upregulation of PKCε was blunted by phenyl-N-tert-butylnitrone (PBN, an ROS scavenger) and the hydrogen peroxide catalyst catalase. Furthermore, εV1-2 attenuated the mechanical allodynia induced by H2O2ConclusionsNOX2-induced oxidative stress promotes the sensitization of DRGs and persistent pain by increasing the plasma membrane translocation of PKCε.

Fibroblast growth factor 13 stabilizes microtubules to promote Na+ channel function in nociceptive DRG neurons and modulates inflammatory pain

IntroductionFibroblast growth factor homologous factors (FHFs), among other fibroblast growth factors, are increasingly found to be important regulators of ion channel functions. Although FHFs have been link to several neuronal diseases and arrhythmia, its role in inflammatory pain still remains unclear. ObjectivesThis study aimed to investigate the role and mechanism of FGF13 in inflammatory pain. MethodsFgf13 conditional knockout mice were generated and CFA-induced chronic inflammatory pain model was established to measure the pain threshold. Immunostaining, western blot and quantitative real-time reverse transcription PCR (qRT-PCR) were performed to detect the expression of FGF13 in CFA-induced inflammatory pain. Whole-cell patch clamp recording was used to record the action potential firing properties and sodium currents of DRG neurons. ResultsConditional knockout of Fgf13 in dorsal root ganglion (DRG) neurons (Fgf13-/Y) led to attenuated pain responses induced by complete Freund's adjuvant (CFA). FGF13 was expressed predominantly in small-diameter DRG neurons. CFA treatment resulted in an increased expression of FGF13 proteins as well as an increased excitability in nociceptive DRG neurons which was inhibited when FGF13 was absent. The role of FGF13 in neuronal excitability of DRG was linked to its modulation of voltage-gated Na+ channels mediated by microtubules. Overexpression of FGF13, but not FGF13 mutant which lacks the ability to bind and stabilize microtubules, rescued the decreased neuronal excitability and Na+ current density in DRG neurons of Fgf13-/Y mice. ConclusionThis study revealed that FGF13 could stabilize microtubules to modulate sodium channel function in DRG neurons and modulate inflammatory pain. This study provides a novel mechanism for FGF13 modulation of sodium channel function and suggests that FGF13 might be a novel target for inflammatory pain treatment.

An All-Optical Electrophysiology Screening Platform to Identify Nav Channel Modulators as Pain Therapeutics

More than 100M people in the US suffer from chronic pain. Current treatments, including opioids and non-steroidal inflammatory agents, have severe limitations for chronic treatment due to tolerance and dose-limiting toxicities. To develop novel pain therapeutics, we have created an all-optical electrophysiology (Optopatch) screening platform using engineered optogenetic proteins. Blue and red light can be used to stimulate and record bioelectrical activity, respectively. For target-based screening application, we demonstrate Optopatch measurements in engineered excitable HEK cells (spiking HEK cells) with heterologous expression of Nav channels targets implicated in pain transmission, including Nav1.7, Nav1.8 and Nav1.9. The Nav channel Optopatch assays can distinguish compounds with different working mechanisms (state-dependent versus state independent block). To achieve a throughput of 10,000 compounds/day, we developed a next generation kinetic plate reader (SWARM) capable of recording 24 wells simultaneously. We have executed a 14,000-compound screen on Nav1.7 spiking HEK cells and identified novel Nav1.7 inhibitors with sub-micromolar potency. To further qualify candidate compounds, we have developed proprietary secondary assays for hit prioritization, which combined the Optopatch platform with an in vitro model of chronic pain, in which dorsal root ganglion (DRG) sensory neurons are exposed to a mixture of inflammatory mediators. This assay leverages our custom Firefly microscope to make highly-parallelized measurements of DRG excitability, where ∼100 neurons can be measured in parallel with single-cell precision and ms-temporal resolution. The newly identified hits are benchmarked against Nav1.7 inhibitors in Phase II trial (PF-05089771 and vixotrigine) and selected hits demonstrate more favorable features in our secondary assay. Interestingly, we found that highly selective Nav1.7 cannot fully reverse the inflammatory mediator-induced DRG hyperexcitability; we dissect Nav-subtype contributions to hyperexcitability using control pharmacology and protein knockdown with antisense oligonucleotides (ASOs).

KLHL1 Controls CaV3.2 Expression in DRG Neurons and Mechanical Sensitivity to Pain.

Dorsal root ganglion (DRG) neurons process pain signaling through specialized nociceptors located in their peripheral endings. It has long been established low voltage-activated (LVA) CaV3.2 calcium channels control neuronal excitability during sensory perception in these neurons. Silencing CaV3.2 activity with antisense RNA or genetic ablation results in anti-nociceptive, anti-hyperalgesic and anti-allodynic effects. CaV3.2 channels are regulated by many proteins (Weiss and Zamponi, 2017), including KLHL1, a neuronal actin-binding protein that stabilizes channel activity by recycling it back to the plasma membrane through the recycling endosome. We explored whether manipulation of KLHL1 levels and thereby function as a CaV3.2 modifier can modulate DRG excitability and mechanical pain transmission or sensitivity to pain. We first assessed the mechanical sensitivity threshold and DRG properties in the KLHL1 KO mouse model. KO DRG neurons exhibited smaller T-type current density compared to WT without significant changes in voltage dependence, as expected in the absence of its modulator. Western blot analysis confirmed CaV3.2 but not CaV3.1, CaV3.3, CaV2.1, or CaV2.2 protein levels were significantly decreased; and reduced neuron excitability and decreased pain sensitivity were also found in the KLHL1 KO model. Analogously, transient down-regulation of KLHL1 levels in WT mice with viral delivery of anti-KLHL1 shRNA also resulted in decreased pain sensitivity. These two experimental approaches confirm KLHL1 as a physiological modulator of excitability and pain sensitivity, providing a novel target to control peripheral pain.

Neonatal Maternal Deprivation Followed by Adult Stress Enhances Adrenergic Signaling to Advance Visceral Hypersensitivity

The pathophysiology of visceral pain in patients with irritable bowel syndrome remains largely unknown. Our previous study showed that neonatal maternal deprivation (NMD) does not induce visceral hypersensitivity at the age of 6 weeks in rats. The aim of this study was to determine whether NMD followed by adult stress at the age of 6 weeks induces visceral pain in rats and to investigate the roles of adrenergic signaling in visceral pain. Here we showed that NMD rats exhibited visceral hypersensitivity 6 h and 24 h after the termination of adult multiple stressors (AMSs). The plasma level of norepinephrine was significantly increased in NMD rats after AMSs. Whole-cell patch-clamp recording showed that the excitability of dorsal root ganglion (DRG) neurons from NMD rats with AMSs was remarkably increased. The expression of β2 adrenergic receptors at the protein and mRNA levels was markedly higher in NMD rats with AMSs than in rats with NMD alone. Inhibition of β2 adrenergic receptors with propranolol or butoxamine enhanced the colorectal distention threshold and application of butoxamine also reversed the enhanced hypersensitivity of DRG neurons. Overall, our data demonstrate that AMS induces visceral hypersensitivity in NMD rats, in part due to enhanced NE-β2 adrenergic signaling in DRGs.

Selective Blockade of HCN1/HCN2 Channels as a Potential Pharmacological Strategy Against Pain

A prominent role of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels has been suggested based on their expression and (dys)function in dorsal root ganglion (DRG) neurons, being likely involved in peripheral nociception. Using HCN blockers as antinociceptive drugs is prevented by the widespread distribution of these channels. However, tissue-specific expression of HCN isoforms varies significantly, HCN1 and HCN2 being considered as major players in DRG excitability. We characterized the pharmacological effect of a novel compound, MEL55A, able to block selectively HCN1/HCN2 isoforms, on DRG neuron excitability in-vitro and for its antiallodynic properties in-vivo. HEK293 cells expressing HCN1, HCN2, or HCN4 isoforms were used to verify drug selectivity. The pharmacological profile of MEL55A was tested on mouse DRG neurons by patch-clamp recordings, and in-vivo in oxaliplatin-induced neuropathy by means of thermal hypersensitivity. Results were compared to the non-isoform-selective drug, ivabradine. MEL55A showed a marked preference toward HCN1 and HCN2 isoforms expressed in HEK293, with respect to HCN4. In cultured DRG, MEL55A reduced Ih amplitude, both in basic conditions and after stimulation by forskolin, and cell excitability, its effect being quantitatively similar to that observed with ivabradine. MEL55A was able to relieve chemotherapy-induced neuropathic pain. In conclusion, selective blockade of HCN1/HCN2 channels, over HCN4 isoform, was able to modulate electrophysiological properties of DRG neurons similarly to that reported for classical Ih blockers, ivabradine, resulting in a pain-relieving activity. The availability of small molecules with selectivity toward HCN channel isoforms involved in nociception might represent a safe and effective strategy against chronic pain.

Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Horn.

Presynaptic voltage-gated K+ (Kv) channels in dorsal root ganglion (DRG) neurons are thought to regulate nociceptive synaptic transmission in the spinal dorsal horn. However, the Kv channel subtypes responsible for this critical role have not been identified. The Kv3.4 channel is particularly important because it is robustly expressed in DRG nociceptors, where it regulates action potential (AP) duration. Furthermore, Kv3.4 dysfunction is implicated in the pathophysiology of neuropathic pain in multiple pain models. We hypothesized that, through their ability to modulate AP repolarization, Kv3.4 channels in DRG nociceptors help to regulate nociceptive synaptic transmission. To test this hypothesis, we investigated Kv3.4 immunoreactivity (IR) in the rat cervical superficial dorsal horn (sDH) in both sexes and implemented an intact spinal cord preparation to investigate glutamatergic synaptic currents from second order neurons in the sDH under conditions that selectively inhibit the Kv3.4 current. We found presynaptic Kv3.4 IR in peptidergic and nonpeptidergic nociceptive fibers of the sDH. The Kv3.4 channel is hypersensitive to 4-aminopyridine and tetraethylammonium (TEA). Accordingly, 50 μm 4-aminopyridine and 500 μm TEA significantly prolong the AP, slow the maximum rate of repolarization in small-diameter DRG neurons, and potentiate monosynaptic excitatory postsynaptic currents (EPSCs) in dorsal horn laminae I and II through a presynaptic mechanism. In contrast, highly specific inhibitors of BK, Kv7, and Kv1 channels are less effective modulators of the AP and have little to no effect on EPSCs. The results strongly suggest that presynaptic Kv3.4 channels are major regulators of nociceptive synaptic transmission in the spinal cord.SIGNIFICANCE STATEMENT Intractable neuropathic pain can result from disease or traumatic injury and many studies have been conducted to determine the underlying pathophysiological changes. Voltage-gated ion channels, including the K+ channel Kv3.4, are dysregulated in multiple pain models. Kv3.4 channels are ubiquitously expressed in the dorsal root ganglion (DRG), where they are major regulators of DRG excitability. However, little is known about the ionic mechanisms that regulate nociceptive synaptic transmission at the level of the first synapse in the spinal cord, which is critical to pain transmission in both intact and pathological states. Here, we show that Kv3.4 channels have a significant impact on glutamatergic synaptic transmission in the dorsal horn, further illuminating its potential as a molecular pain therapeutic target.

Tanshinone IIA Improves Painful Diabetic Neuropathy by Suppressing the Expression and Activity of Voltage-Gated Sodium Channel in Rat Dorsal Root Ganglia.

Painful diabetic neuropathy (PDN) is one of the intractable complications of diabetes mellitus, which manifest as exaggerated pain perception. Previous studies showed that Tanshinone IIA (TIIA), one of the major bioactive extracts of Salvia miltiorrhiza Bunge, have obvious analgesic effect on different types of pain process, and the underlying analgesic mechanisms are not fully understood. The present study combined the behavioral, electrophysiological and biochemical methods to elucidate the analgesic mechanism of TIIA, using streptozotocin (STZ)-induced PDN rat models. Intraperitoneal injection (i.p.) of TIIA for 3 weeks in PDN rats significantly improved mechanical allodynia and thermal hyperalgesia. Patch clamp recordings showed that the excitability of dorsal root ganglion (DRG) nociceptive neuron was increased in diabetic state, and TIIA treatment effectively recovered the subnormality, which was achieved by preventing augments of both Tetrodotoxin-sensitive (TTX-resistant) and Tetrodotoxin-sensitive (TTX-S) sodium currents. Further, the protein expressions of voltage-gated sodium channels (VGSCs) α-subunits Nav1.3, Nav1.7 and Nav1.9 increased in DRG of diabetic rats and were normalized by TIIA application. In conclusion, this study provides evidence that the TIIA attenuated PDN by effecting VGSCs activities and expressions, indicating that the TIIA could be a promising agent for PDN treatment.

Protein Kinase C Mediates the Corticosterone-induced Sensitization of Dorsal Root Ganglion Neurons Innervating the Rat Stomach.

Background/AimsGastric hypersensitivity contributes to abdominal pain in patients with functional dyspepsia. Recent studies showed that hormones induced by stress are correlated with visceral hypersensitivity. However, the precise mechanisms underlying gastric hypersensitivity remain largely unknown. The aim of the present study was designed to investigate the roles of corticosterone (CORT) on excitability of dorsal root ganglion (DRG) neurons innervating the stomach.MethodsDRG neurons innervating the stomach were labeled by DiI injection into the stomach wall. Patch clamp recordings were employed to examine neural excitability and voltage-gated sodium channel currents. Electromyograph technique was used to determine the responses of neck muscles to gastric distension.ResultsIncubation of acutely isolated DRG neurons with CORT significantly depolarized action potential threshold and enhanced the number of action potentials induced by current stimulation of the neuron. Under voltage-clamp mode, incubation of CORT enhanced voltage-gated sodium current density of the recorded neurons. Pre-incubation of GF109203X, an inhibitor of protein kinase C, blocked the CORT-induced hyperexcitability and potentiation of sodium currents. However, pre-incubation of H-89, an inhibitor of protein kinase A, did not alter the sodium current density. More importantly, intraperitoneal injection of CORT produced gastric hypersensitivity of healthy rats, which was blocked by pre-administration of GF109203X but not H-89.ConclusionsOur data strongly suggest that CORT rapidly enhanced neuronal excitability and sodium channel functions, which is most likely mediated by protein kinase C but not protein kinase A signaling pathway in DRG neurons innervating the stomach, thus underlying the gastric hypersensitivity induced by CORT injection.

Rat model of cancer-induced bone pain: changes in nonnociceptive sensory neurons in vivo.

Clinical data on cancer-induced bone pain (CIBP) suggest extensive changes in sensory function. In a previous investigation of an animal model of CIBP, we have observed that changes in intrinsic membrane properties and excitability of dorsal root ganglion (DRG) nociceptive neurons correspond to mechanical allodynia and hyperalgesia. To investigate the mechanisms underlying changes in nonnociceptive sensory neurons in this model, we have compared the electrophysiological properties of primary nonnociceptive sensory neurons at <1 and >2 weeks after CIBP model induction with properties in sham control animals. Copenhagen rats were injected with 106 MAT-LyLu rat prostate cancer cells into the distal femur epiphysis to generate a model of CIBP. After von Frey tactile measurement of mechanical withdrawal thresholds, the animals were prepared for acute electrophysiological recordings of mechanically sensitive neurons in the DRG in vivo. The mechanical withdrawal threshold progressively decreased in CIBP model rats. At <1 week after model induction, there were no changes observed in nonnociceptive Aβ-fiber DRG neurons between CIBP model rats and sham rats. However, at >2 weeks, the Aβ-fiber low-threshold mechanoreceptors (LTMs) in CIBP model rats exhibited a slowing of the dynamics of action potential (AP) genesis, including wider AP duration and lower AP amplitude compared with sham rats. Furthermore, enhanced excitability of Aβ-fiber LTM neurons was observed as an excitatory discharge in response to intracellular injection of depolarizing current into the soma. After induction of the CIBP model, Aβ-fiber LTMs at >2 weeks but not <1 week had undergone changes in electrophysiological properties. Importantly, changes observed are consistent with observations in models of peripheral neuropathy. Thus, Aβ-fiber nonnociceptive primary sensory neurons might be involved in the peripheral sensitization and tumor-induced tactile hypersensitivity in CIBP.

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 β subunits (β1–β3), 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 β1 subunits and TNFR1 contribute to increased nociceptor excitability and hyperalgesia in the TNFtg mice.

Exogenous brain-derived neurotrophic factor relieves pain symptoms of diabetic rats by reducing excitability of dorsal root ganglion neurons

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes lacking of effective treatments. Enhanced excitability of dorsal root ganglion (DRG) neuron plays a crucial role in the progression of diabetic neuropathic hyperalgesia. Brain-derived neurotrophic factor (BDNF) is known as a neuromodulator of nociception, but whether and how BDNF modulates the excitability of DRG neurons in the development of DPN remain to be clarified. This study investigated the role of exogenous BDNF and its high-affinity tropomyosin receptor kinase B (TrkB) in rats with streptozotocin-induced diabetic neuropathic pain. The results showed that continued intrathecal administration of BDNF to diabetic rats dramatically alleviated mechanical and thermal hyperalgesia, as well as inhibited hyperexcitability of DRG neurons. These effects were blocked by pretreatment with TrkB Fc (a synthetic fusion protein consisting of the extracellular ligand-binding domain of the TrkB receptor). The expression of BDNF and TrkB was upregulated in the DRG of diabetic rats. Intrathecal administration of BDNF did not affect this upregulation. These data provide novel information that exogenous BDNF relieved pain symptoms of diabetic rats by reducing hyperexcitability of DRG neurons and might be the potential treatment of painful diabetic neuropathy.

Docosahexaenoic acid inhibits mechanical allodynia and thermal hyperalgesia in diabetic rats by decreasing the excitability of DRG neurons

Diabetes mellitus is a common metabolic disease in human beings with characteristic symptoms of hyperglycemia, chronic inflammation and insulin resistance. One of the most common complications of early-onset diabetes mellitus is peripheral diabetic neuropathy, which is manifested either by loss of nociception or by allodynia and hyperalgesia. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown the potential of anti-inflammation and modulating neuron excitability. The present study investigated the effects of docosahexaenoic acid (DHA) on the excitability of dorsal root ganglion (DRG) neurons in streptozotocin (STZ)-induced diabetes rats. The effects of DHA on the allodynia and hyperalgesia of diabetic rats were also evaluated. Dietary DHA supplementation effectively attenuated both allodynia and hyperalgesia induced by STZ injection. DHA supplementation decreased the excitability of DRG neurons by decreasing the sodium currents and increasing potassium currents, which may contribute to the effect of alleviating allodynia and hyperalgesia in diabetic rats. The results suggested that DHA might be useful as an adjuvant therapy for the prevention and treatment of painful diabetic neuropathy.

EGb761 Ameliorates Neuropathic Pain by Scavenging Reactive Oxygen Species

Background: Neuropathic pain is a well-known type of chronic pain caused by damage to the nervous system. Until recently, researchers have found that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during neuropathic pain. Methods: In this study, we investigated the antinociceptive efficacy of Ginkgo biloba extract (EGb761) in chronic constriction injury (CCI) model of neuropathic pain of rats. To explore the underlying mechanisms, the effects of EGb761 on the excitability of dorsal root ganglion (DRG) neurons and activation of JNK in DRG were explored. Results: We showed that systemic administration of EGb761 inhibited the behavioral responses of neuropathic pain and found that EGb761 treatment could inhibit the H₂O₂-induced depolarization in the acutely dissociated DRG neurons. In addition, we found that EGb761 treatment could inhibit the expression of p-JNK in DRG. Conclusion: Taken together, our results suggest that administration of EGb761 can ameliorate neuropathic pain, and further indicate that JNK, which is activated by both exogenous and endogenous ROS, might be the mechanism underlying the effects of EGb761 on CCI neuropathic pain.