Small-sized Dorsal Root Ganglion Neurons Research Articles

Multi-target modulation of ion channels underlying the analgesic effects of α-mangostin in dorsal root ganglion neurons

Backgroundα-Mangostin is a xanthone isolated from the pericarps of mangosteen fruit with, and has analgesic properties. Although the effects suggest an interaction of α-mangostin with ion channels in the nociceptive neurons, electrophysiological investigation of the underlying mechanism has not been performed. HypothesisWe hypothesized that α-Mangostin exerts its analgesic effects by modulating the activity of various ion channels in dorsal root ganglion (DRG) neurons. MethodsWe performed a whole-cell patch clamp study using mouse DRG neurons, HEK293T cells overexpressing targeted ion channels, and ND7/23 cells. Molecular docking (MD) and in silico absorption, distribution, metabolism, and excretion (ADME) analyses were conducted to obtain further insights into the binding sites and pharmacokinetics, respectively. ResultsApplication of α-mangostin (1–3 µM) hyperpolarized the resting membrane potential (RMP) of small-sized DRG neurons by increasing background K+ conductance and thereby inhibited action potential generation. At micromolar levels, α-mangostin activates TREK-1, TREK-2, or TRAAK, members of the two-pore domain K+ channel (K2P) family known to be involved in RMP formation in DRG neurons. Furthermore, capsaicin-induced TRPV1 currents were potently inhibited by α-mangostin (0.43 ± 0.27 µM), and partly suppressed tetrodotoxin-sensitive voltage-gated Na+ channel (NaV) currents. MD simulation revealed that multiple oxygen atoms in α-mangostin may form stable hydrogen bonds with TREKs, TRAAK, TRPV1, and NaV channels. In silico ADME tests suggested that α-mangostin may satisfy the drug-likeness properties without penetrating the blood–brain barrier. ConclusionThe analgesic properties of α-mangostin might be mediated by the multi-target modulation of ion channels, including TREK/TRAAK activation, TRPV1 inhibition, and reduction of the tetrodotoxin-sensitive NaV current. The findings suggest that the phytochemical can be a multi-ion channel-targeting drug and an alternative drug for effective pain management.

Up-regulation of oxytocin receptors on peripheral sensory neuronsmediates analgesia in chemotherapy-induced neuropathic pain.

Chemotherapy-induced neuropathic pain (CINP) currently has limited effective treatment. Although the roles of oxytocin (OXT) and the oxytocin receptor (OXTR) in central analgesia have been well documented, the expression and function of OXTR in the peripheral nervous system remain unclear. Here, we evaluated the peripheral antinociceptive profiles of OXTR in CINP. Paclitaxel (PTX) was used to establish CINP. Quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry were used to observe OXTR expression in dorsal root ganglia (DRG). The antinociceptive effects of OXT were assessed by hot-plate and von Frey tests. Whole-cell patch clamp was performed to record sodium currents, excitability of DRG neurons, and excitatory synapse transmission. Expression of OXTR in DRG neurons was enhanced significantly after PTX treatment. Activation of OXTR exhibited antinociceptive effects, by decreasing the hyperexcitability of DRG neurons in PTX-treated mice. Additionally, OXTR activation up-regulated the phosphorylation of protein kinase C (pPKC) and, in turn, impaired voltage-gated sodium currents, particularly the voltage-gated sodium channel 1.7 (NaV 1.7) current, that plays an indispensable role in PTX-induced neuropathic pain. OXT suppressed excitatory transmission in the spinal dorsal horn as well as excitatory inputs from primary afferents in PTX-treated mice. The OXTR in small-sized DRG neurons is up-regulated in CINP and its activation relieved CINP by inhibiting the neural excitability by impairment of NaV 1.7 currents via pPKC. Our results suggest that OXTR on peripheral sensory neurons is a potential therapeutic target to relieve CINP.

Sevoflurane modulation of tetrodotoxin-resistant Na+ channels in small-sized dorsal root ganglion neurons of rats.

Volatile anesthetics are widely used for general anesthesia during surgical operations. Voltage-gated Na+ channels expressed in central neurons are major targets for volatile anesthetics; but it is unclear whether these drugs modulate native tetrodotoxin-resistant (TTX-R) Na+ channels, which are involved in the development and maintenance of inflammatory pain. In this study, we examined the effects of sevoflurane on TTX-R Na+ currents (INa) in acutely isolated rat dorsal root ganglion neurons, using a whole-cell patch-clamp technique. Sevoflurane slightly potentiated the peak amplitude of transient TTX-R INa but more potently inhibited slow voltage-ramp-induced persistent INa in a concentration-dependent manner. Sevoflurane (0.86 ± 0.02 mM) (1) slightly shifted the steady-state fast inactivation relationship to hyperpolarizing ranges without affecting the voltage-activation relationship, (2) reduced the extent of use-dependent inhibition of Na+ channels, (3) accelerated the onset of inactivation and (4) delayed the recovery from inactivation of TTX-R Na+ channels. Thus, sevoflurane has diverse effects on TTX-R Na+ channels expressed in nociceptive neurons. The present results suggest that the inhibition of persistent INa and the modulation of the voltage dependence and inactivation might be, at least in part, responsible for the analgesic effects elicited by sevoflurane.

Discovery of aryl sulfonamide-selective Nav1.7 inhibitors with a highly hydrophobic ethanoanthracene core

Nav1.7 channels are mainly distributed in the peripheral nervous system. Blockade of Nav1.7 channels with small-molecule inhibitors in humans might provide pain relief without affecting the central nervous system. Based on the facts that many reported Nav1.7-selective inhibitors contain aryl sulfonamide fragments, as well as a tricyclic antidepressant, maprotiline, has been found to inhibit Nav1.7 channels, we designed and synthesized a series of compounds with ethanoanthracene and aryl sulfonamide moieties. Their inhibitory activity on sodium channels were detected with electrophysiological techniques. We found that compound 10o potently inhibited Nav1.7 channels stably expressed in HEK293 cells (IC50 = 0.64 ± 0.30 nmol/L) and displayed a high Nav1.7/Nav1.5 selectivity. In mouse small-sized dorsal root ganglion neurons, compound 10o (10, 100 nmol/L) dose-dependently decreased the sodium currents and dramatically suppressed depolarizing current-elicited neuronal discharge. Preliminary in vivo experiments showed that compound 10o possessed good analgesic activity: in a mouse visceral pain model, administration of compound 10o (30−100 mg/kg, i.p.) effectively and dose-dependently suppressed acetic acid-induced writhing.

NLRP2 inflammasome in dorsal root ganglion as a novel molecular platform that produces inflammatory pain hypersensitivity.

Inflammatory pain hypersensitivity is associated with activation of primary afferent neurons. This study investigated the existence of the inflammasome in dorsal root ganglion (DRG) and the functional significance in the development of inflammatory pain hypersensitivity. Tissue inflammation was induced in male C57BL/6 mice with complete Freund's adjuvant (CFA) or ceramide injection into the hind paw. Behavioral testing was performed to investigate inflammation-induced pain hypersensitivity. Ipsilateral L5 DRGs were obtained for analysis. Expression of nucleotide oligomerization domain-like receptors (NLRs) was analyzed with real-time PCR. Cleaved interleukin (IL)-1β and NLRP2 expression was investigated with immunohistochemistry and western blotting. Caspase 1 activity was also measured. A caspase 1 inhibitor and NLRP2 siRNA were intrathecally administered to inhibit NLRP2 inflammasome signaling in DRG. Cleaved IL-1β expression was significantly increased after CFA injection in small-sized DRG neurons. The amount of cleaved IL-1β and caspase 1 activity were also increased. Among several NLRs, NLRP2 mRNA was significantly increased in DRG after CFA injection. NLRP2 was expressed in small-sized DRG neurons. Intrathecal injection of a caspase 1 inhibitor or NLRP2 siRNA reduced CFA-induced pain hypersensitivity and cleaved IL-1β expression in DRG. Induction of cleaved IL-1β and NLRP2 in DRG neurons was similarly observed after ceramide injection. NLRP2 siRNA inhibited ceramide-induced pain hypersensitivity. These results confirmed the existence of NLRP2 inflammasome in DRG neurons. Activation of the NLRP2 inflammasome leads to activation of DRG neurons and subsequent development of pain hypersensitivity in various types of tissue inflammation.

TRPV1 Channel Contributes to the Behavioral Hypersensitivity in a Rat Model of Complex Regional Pain Syndrome Type 1.

Complex regional pain syndrome type 1 (CRPS-I) is a debilitating pain condition that significantly affects life quality of patients. It remains a clinically challenging condition and the mechanisms of CRPS-I have not been fully elucidated. Here, we investigated the involvement of TRPV1, a non-selective cation channel important for integrating various painful stimuli, in an animal model of CRPS-I. A rat model of chronic post-ischemia pain (CPIP) was established to mimic CRPS-I. TRPV1 expression was significantly increased in hind paw tissue and small to medium-sized dorsal root ganglion (DRG) neurons of CPIP rats. CPIP rats showed increased TRPV1 current density and capsaicin responding rate in small-sized nociceptive DRG neurons. Local pharmacological blockage of TRPV1 with the specific antagonist AMG9810, at a dosage that does not produce hyperthermia or affect thermal perception or locomotor activity, effectively attenuated thermal and mechanical hypersensitivity in bilateral hind paws of CPIP rats and reduced the hyperexcitability of DRG neurons induced by CPIP. CPIP rats showed bilateral spinal astrocyte and microglia activations, which were significantly attenuated by AMG9810 treatment. These findings identified an important role of TRPV1 in mediating thermal and mechanical hypersensitivity in a CRPS-I animal model and further suggest local pharmacological blocking TRPV1 may represent an effective approach to ameliorate CRPS-I.

(207) Oxaliplatin Depolarizes Isolectin B4-Negative Dorsal Root Ganglion Neurons to Initiate Neuropathic Pain in Mouse

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting neurotoxicity caused by chemotherapy drugs. CIPN is frequently associated with neuropathic pain that significantly impairs the quality of life and work capability of cancer patients. Use of oxaliplatin for colorectal cancer is associated with a characteristic cold-sensitive pain in addition to mechanical allodynia. The objectives of this work were to study 1) effects of oxaliplatin on membrane excitability in Isoletcin B4-positive and –negative (IB4+ and IB4-) subpopulations of dorsal root ganglion (DRG), and 2) targeting oxaliplatin-induced membrane hyperexcitability to relief neuropathic pain. The effects of oxaliplatin on membrane excitability were examined by whole-cell patch clamping in small sized DRG neurons dissociated from adult C57BL/6 mice in vitro. Pain and motor behaviors were tested in mice treated with oxaliplatin. Cold, heat, mechanical sensitivity, and motor function were assessed by acetone spray, Hargreaves, Von-Frey, and Rotarod tests, respectively. In vitro experiments found that oxaliplatin selectively caused membrane depolarization in IB4-, but not IB4+ neurons. Oxaliplatin also reduced threshold for action potentials (APs), and increased firing numbers of APs in IB4- neurons. The oxaliplatin-induced membrane depolarization and hyperexcitability was completely reversed by retigabine, an FDA approved anti-epileptic drug and a KCNQ/M channel opener. In vivo, oxaliplatin induced cold and mechanical hypersensitivity while it did not change heat sensitivity or motor function. Co-treatment of retigabine inhibited oxaliplatin-induced cold and mechanical hypersensitivity without changing heat sensitivity or motor function. These results suggest that oxaliplatin initiates neuropathic pain through depolarizing IB4- neurons and targeting membrane depolarization can prevent oxaliplatin-induce neuropathic pain.

Characterization of temperature-sensitive leak K+ currents and expression of TRAAK, TREK-1, and TREK2 channels in dorsal root ganglion neurons of rats

Leak K+ currents are mediated by two-pore domain K+ (K2P) channels and are involved in controlling neuronal excitability. Of 15 members of K2P channels cloned so far, TRAAK, TREK-1, and TREK-2 are temperature sensitive. In the present study, we show that strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels was present mainly in small-sized dorsal root ganglion (DRG) neurons of rats. The percentages of neurons with strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels were 27, 23, and 20%, respectively. Patch-clamp recordings were performed to examine isolated leak K+ currents on acutely dissociated small-sized rat DRG neurons at room temperature of 22 °C, cool temperature of 14 °C and warm temperature of 30 °C. In majority of small-sized DRG neurons recorded (76%), large leak K+ currents were observed at 22 °C and were inhibited at 14 °C and potentiated at 30 °C, suggesting the presence of temperature-sensitive K2P channels in these neurons. In a small population (18%) of small-sized DRG neurons, cool temperature of 14 °C evoked a conductance which was consistent with TRPM8 channel activation in cold-sensing DRG neurons. In these DRG neurons, leak K+ currents were very small at 22 °C and were not potentiated at 30 °C, suggesting that few temperature-sensitive K2P channels was present in cold-sensing DRG neurons. For DRG neurons with temperature-sensitive leak K+ currents, riluzole, norfluoxetine and prostaglandin F2α (PGE2α) inhibited the leak K+ currents at both 30 °C and 22 °C degree, and did not have inhibitory effects at 14 °C. Collectively, the observed temperature-sensitive leak K+ currents are consistent with the expression of temperature-sensitive K2P channels in small-sized DRG neurons.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Oxaliplatin-induced changes in expression of transient receptor potential channels in the dorsal root ganglion as a neuropathic mechanism for cold hypersensitivity

Transient receptor potential (TRP) receptors are involved in the development of chemotherapy-induced peripheral neuropathic pain, which is a common side effect of selected chemotherapeutic agents such as oxaliplatin. However, the precise contribution of TRPs to this condition remains unknown. Cold hypersensitivity is the hallmark of oxaliplatin-induced neuropathy, so we used a preclinical model of oxaliplatin-induced cold hypersensitivity in rats to determine the effects of oxaliplatin on TRP channels. To this end, immunohistochemistry was used to examine TRP vanilloid 1 (TRPV1), TRP ankyrin 1 (TRPA1), and TRP melastatin 8 (TRPM8) expression in the rat dorsal root ganglion (DRG) after 4days of oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that oxaliplatin significantly increased acute cold hypersensitivity after 4days of treatment. Double-staining immunohistochemistry showed that 4days after oxaliplatin treatment, there was increased co-expression of TRPA1 and TRPV1 in isolectin B4-positive small-sized DRG neurons, as well as a significant increase in the co-localization of TRPM8 and neurofilament 200 in medium-sized DRG neurons. In addition, in situ hybridization revealed that TRPV1 protein was co-expressed with TRPA1 mRNA on day 4 after oxaliplatin administration. Thus, at an early stage following oxaliplatin treatment there is an increased expression of TRPA1 and TRPV1 in small-sized DRG neurons and of TRPM8 in medium-sized DRG neurons. Collectively, these changes may contribute to the development of oxaliplatin-induced peripheral neuropathic pain.

Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain

Nociceptors are a particular subtype of dorsal root ganglion (DRG) neurons that detect noxious stimuli and elicit pain. Although recent efforts have been made to reveal the molecular profile of nociceptors in normal conditions, little is known about how this profile changes in pathological conditions. In this study we exploited laser capture microdissection to specifically collect individual injured and non-injured nociceptive DRG neurons and to define their gene profiling in rat spared nerve injury (SNI) model of neuropathic pain. We found minimal transcriptional changes in non-injured neurons at 7 days after SNI. In contrast, several novel transcripts were altered in injured nociceptors, and the global signature of these LCM-captured neurons differed markedly from that the gene expression patterns found previously using whole DRG tissue following SNI. Pathway analysis of the transcriptomic profile of the injured nociceptors revealed oxidative stress as a key biological process. We validated the increase of caspase-6 (CASP6) in small-sized DRG neurons and its functional role in SNI- and paclitaxel-induced neuropathic pain. Our results demonstrate that the identification of gene regulation in a specific population of DRG neurons (e.g., nociceptors) is an effective strategy to reveal new mechanisms and therapeutic targets for neuropathic pain from different origins.

Nociceptive neuronal Fc-gamma receptor I is involved in IgG immune complex induced pain in the rat

Antigen-specific immune diseases such as rheumatoid arthritis are often accompanied by pain and hyperalgesia. Our previous studies have demonstrated that Fc-gamma-receptor type I (FcγRI) is expressed in a subpopulation of rat dorsal root ganglion (DRG) neurons and can be directly activated by IgG immune complex (IgG-IC). In this study we investigated whether neuronal FcγRI contributes to antigen-specific pain in the naïve and rheumatoid arthritis model rats. In vitro calcium imaging and whole-cell patch clamp recordings in dissociated DRG neurons revealed that only the small-, but not medium- or large-sized DRG neurons responded to IgG-IC. Accordingly, in vivo electrophysiological recordings showed that intradermal injection of IgG-IC into the peripheral receptive field could sensitize only the C- (but not A-) type sensory neurons and evoke action potential discharges. Pain-related behavioral tests showed that intradermal injection of IgG-IC dose-dependently produced mechanical and thermal hyperalgesia in the hindpaw of rats. These behavioral effects could be alleviated by localized administration of non-specific IgG or an FcγRI antibody, but not by mast cell stabilizer or histamine antagonist. In a rat model of antigen-induced arthritis (AIA) produced by methylated bovine serum albumin, FcγRI were found upregulated exclusively in the small-sized DRG neurons. In vitro calcium imaging revealed that significantly more small-sized DRG neurons responded to IgG-IC in the AIA rats, although there was no significant difference between the AIA and control rats in the magnitude of calcium changes in the DRG neurons. Moreover, in vivo electrophysiological recordings showed that C-nociceptive neurons in the AIA rats exhibited a greater incidence of action potential discharges and stronger responses to mechanical stimuli after IgG-IC was injected to the receptive fields. These results suggest that FcγRI expressed in the peripheral nociceptors might be directly activated by IgG-IC and contribute to antigen-specific pain in pathological conditions.

Mechanical sensitivity and electrophysiological properties of acutely dissociated dorsal root ganglion neurons of rats

Primary afferent fibers use mechanically activated (MA) currents to transduce innocuous and noxious mechanical stimuli. However, it is largely unknown about the differences in MA currents between the afferents for sensing innocuous and noxious stimuli. In the present study, we used dorsal root ganglion (DRG) neurons acutely dissociated from rats and studied their MA currents and also their intrinsic membrane properties. Recorded from small-sized DRG neurons, we found that most of these neurons were mechanically sensitive (MS) showing MA currents. The MS neurons could be classified into nociceptive-like mechanically sensitive (Noci-MS) and non-nociceptive-like mechanically sensitive (nonNoci-MS) neurons based on their action potential shapes. Noci-MS neurons responded to mechanical stimulation with three types of MA currents, rapidly adapting (RA), intermediately adapting (IA), and slowly adapting (SA) currents. In contrast, almost all nonNoci-MS neurons showed RA current type in response to mechanical stimulation. Mechanical thresholds had a broad range for both nonNoci-MS and Noci-MS neurons, and the thresholds were not significantly different between them. However, MA current densities were significantly smaller in Noci-MS than in nonNoci-MS neurons. Noci-MS and nonNoci-MS neurons also showed significant differences in their electrophysiological properties including action potential (AP) thresholds and AP firing patterns. These differences may contribute to the differential sensory encoding for innocuous and noxious mechanical stimuli.

Brain natriuretic peptide suppresses pain induced by BmK I, a sodium channel-specific modulator, in rats

BackgroundA previous study found that brain natriuretic peptide (BNP) inhibited inflammatory pain via activating its receptor natriuretic peptide receptor A (NPRA) in nociceptive sensory neurons. A recent study found that functional NPRA is expressed in almost all the trigeminal ganglion (TG) neurons at membrane level suggesting a potentially important role for BNP in migraine pathophysiology.MethodsAn inflammatory pain model was produced by subcutaneous injection of BmK I, a sodium channel-specific modulator from venom of Chinese scorpion Buthus martensi Karsch. Quantitative PCR, Western Blot, and immunohistochemistry were used to detect mRNA and protein expression of BNP and NPRA in dorsal root ganglion (DRG) and dorsal horn of spinal cord. Whole-cell patch clamping experiments were conducted to record large-conductance Ca2+-activated K+ (BKCa) currents of membrane excitability of DRG neurons. Spontaneous and evoked pain behaviors were examined.ResultsThe mRNA and protein expression of BNP and NPRA was up-regulated in DRG and dorsal horn of spinal cord after BmK I injection. The BNP and NPRA was preferentially expressed in small-sized DRG neurons among which BNP was expressed in both CGRP-positive and IB4-positive neurons while NPRA was preferentially expressed in CGRP-positive neurons. BNP increased the open probability of BKCa channels and suppressed the membrane excitability of small-sized DRG neurons. Intrathecal injection of BNP significantly inhibited BmK-induced pain behaviors including both spontaneous and evoked pain behaviors.ConclusionsThese results suggested that BNP might play an important role as an endogenous pain reliever in BmK I-induced inflammatory pain condition. It is also suggested that BNP might play a similar role in other pathophysiological pain conditions including migraine.

Colocalization of insulin-like growth factor-1 receptor and T type Cav3.2 channel in dorsal root ganglia in chronic inflammatory pain mouse model.

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays important roles in the nervous system. Increasing evidence supports that IGF-1 contributes to pain hypersensitivity through its insulin-like growth factor-1 receptor (IGF-1R) by activating IGF-1R/Akt or MAPK signaling pathways, whereas T-type Cav3.2 channel can facilitate and amplify pain signals originating from the sensory periphery. A recent study showed that activated IGF-1R can increase T-type Cav3.2 channel currents and further activate the G protein-dependent PKCα pathway to contribute to inflammatory pain sensitivity. However, the colocalization of IGF-1R and Cav3.2 in mouse dorsal root ganglion (DRG) under chronic inflammatory pain conditions remains elusive. In this study, we investigated changes in the expression of IGF-1R and the Cav3.2 channel, and their colocalization in mouse DRGs in chronic inflammatory pain condition (induced by complete Freund's adjuvant intraplanter injection) using real-time RT-PCR and immunohistochemistry approaches to confirm that Cav3.2 channel can mediate pain facilitation following IGF-1/IGF-1R signaling. We found that IGF-1R was expressed extensively in DRG neurons including small-, medium-, and large-sized neurons, whereas Cav3.2 channel was expressed exclusively in small-sized DRG neurons of naive mice. Expression of Cav3.2, but not IGF-1R, and colocalization of Cav3.2 and IGF-1R were increased in lumbar (L)4-L6 primary sensory neurons in DRGs of mice in chronic inflammatory pain. Moreover, the increased colocalization of IGF-1R and Cav3.2 is exclusively localized in small- and medium-sized primary sensory neurons. Our findings provided morphological evidence that T-type Cav3.2 channel, at least partially, mediates the pain facilitation of IGF-1/IGF-1R signaling in chronic inflammatory pain condition.

SDF1–CXCR4 signaling contributes to persistent pain and hypersensitivity via regulating excitability of primary nociceptive neurons: involvement of ERK-dependent Nav1.8 up-regulation

BackgroundPain is one critical hallmark of inflammatory responses. A large number of studies have demonstrated that stromal cell-derived factor 1 (SDF1, also named as CXCL12) and its cognate receptor C-X-C chemokine receptor type 4 (CXCR4) play an important role in immune reaction and inflammatory processes. However, whether and how SDF1–CXCR4 signaling is involved in inflammatory pain remains unclear.MethodsUnder the intraplantar (i.pl.) bee venom (BV) injection-induced persistent inflammatory pain state, the changes of SDF1 and CXCR4 expression and cellular localization in the rat dorsal root ganglion (DRG) were detected by immunofluorescent staining. The role of SDF1 and CXCR4 in the hyperexcitability of primary nociceptor neurons was assessed by electrophysiological recording. Western blot analysis was used to quantify the DRG Nav1.8 and phosphorylation of ERK (pERK) expression. Behavioral tests were conducted to evaluate the roles of CXCR4 as well as extracellular signal-regulated kinase (ERK) and Nav1.8 in the BV-induced persistent pain and hypersensitivity.ResultsWe showed that both SDF1 and CXCR4 were dramatically up-regulated in the DRG in i.pl. BV-induced inflammatory pain model. Double immunofluorescent staining showed that CXCR4 was localized in all sizes (large, medium, and small) of DRG neuronal soma, while SDF1 was exclusively expressed in satellite glial cells (SGCs). Electrophysiological recording showed that bath application with AMD3100, a potent and selective CXCR4 inhibitor, could reverse the hyperexcitability of medium- and small-sized DRG neurons harvested from rats following i.pl. BV injection. Furthermore, we demonstrated that the BV-induced ERK activation and Nav1.8 up-regulation in the DRG could be blocked by pre-antagonism against CXCR4 in the periphery with AMD3100 as well as by blockade of ERK activation by intrathecal (i.t.) or intraplantar (i.pl.) U0126. At behavioral level, the BV-induced persistent spontaneous pain as well as primary mechanical and thermal hypersensitivity could also be significantly suppressed by blocking CXCR4 and Nav1.8 in the periphery as well as by inhibition of ERK activation at the DRG level.ConclusionsThe present results suggest that peripheral inflammatory pain state can trigger over release of SDF1 from the activated SGCs in the DRG by which SGC-neuronal cross-talk is mediated by SDF1–CXCR4 coupling that result in subsequent ERK-dependent Nav1.8 up-regulation, leading to hyperexcitability of tonic type of the primary nociceptor cells and development and maintenance of persistent spontaneous pain and hypersensitivity.

Effects of curcumin on TTX-R sodium currents of dorsal root ganglion neurons in type 2 diabetic rats with diabetic neuropathic pain

Type 2 diabetic mellitus (T2DM) has reached pandemic status and shows no signs of abatement. Diabetic neuropathic pain (DNP) is generally considered to be one of the most common complications of T2DM, which is also recognized as one of the most difficult types of pain to treat. As one kind of peripheral neuropathic pain, DNP manifests typical chronic neuralgia symptoms, including hyperalgesia, allodynia, autotomy, and so on. The injured dorsal root ganglion (DRG) is considered as the first stage of the sensory pathway impairment, whose neurons display increased frequency of action potential generation and increased spontaneous activities. These are mainly due to the changed properties of voltage-gated sodium channels (VGSCs) and the increased sodium currents, especially TTX-R sodium currents. Curcumin, one of the most important phytochemicals from turmeric, has been demonstrated to effectively prevent and/or ameliorate diabetic mellitus and its complications including DNP. The present study demonstrates that the TTX-R sodium currents of small-sized DRG neurons isolated from DNP rats are significantly increased. Such abnormality can be efficaciously ameliorated by curcumin.

Reduced expression of pain mediators and pain sensitivity in amyloid precursor protein over-expressing CRND8 transgenic mice

β-Amyloid (Aβ) peptides are derived from the sequential cleavage of the amyloid precursor protein (APP). They are enriched in plaques present in Alzheimer’s brains and thus play important roles in the pathogenesis of this disease. APP is also known to be expressed in the neurons of dorsal root ganglion (DRG) and contributes to neuronal survival and axonal growth during development. However, whether APP and Aβ peptides are involved in nociception and pathological pain states is mostly unknown. In the present study, we have used behavioral, biochemical and morphological approaches to address this issue in both adult rats and APP over-expressing CRND8 transgenic mice. We observed that the Aβ peptide (17–24) was predominantly expressed in small-sized DRG neurons of rats. Following intraplantar (i.pl.) injection of complete Freud’s adjuvant (CFA), the levels of APP and Aβ peptides were significantly reduced in the ipsilateral lumbar 4–6 rat DRG. In 3-, 12- and 24-month-old CRND8 mice, pain sensitivity in response to heat and mechanical stimulation was significantly dampened compared to their age-matched wild-type littermates. In parallel with reduced pain sensitivity, the expression of pain mediators such as substance P, calcitonin gene-related peptide and transient receptor potential vanilloid-1 was significantly reduced in L4–6 DRG of CRND8 mice. Although i.pl. injection of CFA induced a rather similar pattern of inflammatory pain in 3-month-old CRND8 mice and their wild-type littermates, recovery from inflammatory pain seemed faster in 12-month-old CRND8 mice than wild-type mice. These findings suggest that APP and Aβ peptides suppress both nociception and inflammatory pain and are likely involved in blunt pain perception of Alzheimer’s patients in clinical settings.

Activation of Tetrodotoxin-Resistant Sodium Channel NaV1.9 in Rat Primary Sensory Neurons Contributes to Melittin-Induced Pain Behavior

Tetrodotoxin-resistant (TTX-R) sodium channels NaV1.8 and NaV1.9 in dorsal root ganglion (DRG) neurons play important roles in pathological pain. We recently reported that melittin, the major toxin of whole bee venom, induced action potential firings in DRG neurons even in the presence of a high concentration (500nM) of TTX, indicating the contribution of TTX-R sodium channels. This hypothesis is fully investigated in the present study. After subcutaneous injection of melittin, NaV1.8 and NaV1.9 significantly upregulate mRNA and protein expressions, and related sodium currents also increase. Double immunohistochemical results show that NaV1.8-positive neurons are mainly medium- and small-sized, whereas NaV1.9-positive ones are only small-sized. Antisense oligodeoxynucleotides (AS ODNs) targeting NaV1.8 and NaV1.9 are used to evaluate functional significance of the increased expressions of TTX-R sodium channels. Behavioral tests demonstrate that AS ODN targeting NaV1.9, but not NaV1.8, reverses melittin-induced heat hypersensitivity. Neither NaV1.8 AS ODN nor NaV1.9 AS ODN affects melittin-induced mechanical hypersensitivity. These results provide previously unknown evidence that upregulation of NaV1.9, but not NaV1.8, in small-sized DRG neurons contributes to melittin-induced heat hypersensitivity. Furthermore, melittin-induced biological effect indicates a potential strategy to study properties of TTX-R sodium channels.

Suppression of KCNQ/M (Kv7) potassium channels in dorsal root ganglion neurons contributes to the development of bone cancer pain in a rat model

Bone cancer pain has a strong impact on the quality of life of patients, but is difficult to treat. Better understanding of the pathogenic mechanisms underlying bone cancer pain will likely lead to the development of more effective treatments. In the present study, we investigated whether inhibition of KCNQ/M channels contributed to the hyperexcitability of primary sensory neurons and to the pathogenesis of bone cancer pain. By using a rat model of bone cancer pain based on intratibial injection of MRMT-1 tumour cells, we documented a prominent decrease in expression of KCNQ2 and KCNQ3 proteins and a reduction of M-current density in small-sized dorsal root ganglia (DRG) neurons, which were associated with enhanced excitability of these DRG neurons and the hyperalgesic behaviours in bone cancer rats. Coincidently, we found that inhibition of KCNQ/M channels with XE-991 caused a robust increase in the excitability of small-sized DRG neurons and produced an obvious mechanical allodynia in normal rats. On the contrary, activation of the KCNQ/M channels with retigabine not only inhibited the hyperexcitability of these small DRG neurons, but also alleviated mechanical allodynia and thermal hyperalgesia in bone cancer rats, and all of these effects of retigabine could be blocked by KCNQ/M-channel antagonist XE-991. These results suggest that repression of KCNQ/M channels leads to the hyperexcitability of primary sensory neurons, which in turn causes bone cancer pain. Thus, suppression of KCNQ/M channels in primary DRG neurons plays a crucial role in the development of bone cancer pain.