Upregulation In Dorsal Root Ganglion Research Articles

Extracellular signal-regulated kinase phosphorylation enhancement and NaV1.7 sodium channel upregulation in rat dorsal root ganglia neurons contribute to resiniferatoxin-induced neuropathic pain: The efficacy and mechanism of pulsed radiofrequency therapy.

Pulsed radiofrequency (PRF) therapy is one of the most common treatment options for neuropathic pain, albeit the underlying mechanism has not been hitherto elucidated. In this study, we investigated the efficacy and mechanism of PRF therapy on resiniferatoxin (RTX)-induced mechanical allodynia, which has been used as a model of postherpetic neuralgia (PHN). Adult male rats were intraperitoneally injected with a vehicle or RTX. Furthermore, PRF current was applied on a unilateral sciatic nerve in all RTX-treated rats. On both ipsilateral and contralateral sides, the paw mechanical withdrawal thresholds were examined and L4-6 dorsal root ganglia (DRG) were harvested. In the DRG of rats with RTX-induced mechanical allodynia, NaV1.7, a voltage-gated Na+ channel, was upregulated following the enhancement of extracellular signal-regulated kinase phosphorylation. Early PRF therapy, which was applied 1 week after RTX exposure, suppressed this NaV1.7 upregulation and showed an anti-allodynic effect; however, late PRF therapy, which was applied after 5 weeks of RTX exposure, failed to inhibit allodynia. Interestingly, late PRF therapy became effective after daily tramadol administration for 7 days, starting from 2 weeks after RTX exposure. Both early PRF therapy and late PRF therapy combined with early tramadol treatment suppressed NaV1.7 upregulation in the DRG of rats with RTX-induced mechanical allodynia. Therefore, NaV1.7 upregulation in DRG is related to the development of RTX-induced neuropathic pain; moreover, PRF therapy may be effective in the clinical management of patients with PHN via NaV1.7 upregulation inhibition.

Participation of transient receptor potential vanilloid 1 in the analgesic effect of duloxetine for paclitaxel induced peripheral neuropathic pain

Painful peripheral neuropathy is a common dose-limiting side effect of chemotherapeutic paclitaxel (PTX) treatment. The American Society of Clinical Oncology (ASCO) recommends duloxetine (DUL) as a promising treatment alternative for chemotherapy-induced peripheral neuropathic pain. However, this recommendation lacks a robust theoretical basis and supporting data. To elucidate the involvement of transient receptor potential vanilloid 1 (TRPV1) in the analgesic effect of DUL for PTX-induced neuropathic pain, TRPV1 expression in the lumbar dorsal root ganglion (DRG) and spinal cord was evaluated following intraperitoneal administration of PTX (2 mg/kg/day) for four alternate days in rats. Western blot and immunohistochemistry suggested that a cumulative dosage of PTX (8 mg/kg) upregulated TRPV1 expression in the lumbar DRG and spinal dorsal horn (SDH) at day 14 post treatment. TRPV1 upregulation in the DRG was mainly expressed in calcitonin gene-related peptide (CGRP) and IB-4 positive small-size sensory neurons. Additionally, PTX increased CGRP and substance P (SP) expression in the DRG and SDH, induced SDH microglia and astrocyte activation, and upregulated spinal tumor necrosis factor-α (TNF-α) but not IL-1β or IL-10 expression. Behavioral detection showed that PTX-related mechanical and thermal hyperalgesia was significantly inhibited by consecutive administration of DUL 20 mg/kg/day greater than 10 mg/kg/day for 5 days starting at day 10 post PTX injection. Furthermore, DUL (20 mg/kg/day) for 5 days markedly ameliorated PTX-induced TRPV1, CGRP, and SP upregulation in the DRG and SDH, and mitigated PTX-induced spinal cord glia activation and TNF-α expression. Moreover, the pharmacological blockade of TRPV1 resulted in an analgesic effect on PTX-induced hyperalgesia. Collectively, these results suggest that DUL alleviates PTX-induced peripheral neuropathic pain by suppressing TRPV1 upregulation in the lumbar DRG and SDH, which is followed by a reduction in CGRP and SP release, as well as spinal glia activation and TNF-α expression.

Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

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.

Electroacupuncture alleviates diabetic neuropathic pain in rats by suppressing P2X3 receptor expression in dorsal root ganglia

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats’ body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α β-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α β-me ATP blocks EA’s analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.

High Estrogen Level Modifies Postoperative Hyperalgesia via GPR30 and MMP-9 in Dorsal Root Ganglia Neurons.

The cycling of sex hormones is one of the factors affecting pain in females, and the mechanisms are not fully understood. G-protein coupled estrogen receptor 30 (GPR30) is the estrogen receptor known to be involved in mechanical hyperalgesia. Studies have demonstrated that matrix metalloproteinase-9 (MMP-9) is a critical component in peripheral/central nervous system hypersensitivity and neuroinflammation, both of which participate in hyperalgesia. Here, ovariectomized rats were treated with low or high dose estrogen replacement, and then plantar incisions were made. Subsequently, mechanical allodynia was evaluated by determining the paw withdrawal mechanical threshold before and after the incision. In rats with incisions, high estrogen levels induced postoperative hyperalgesia and upregulation of GPR30 and MMP-9 in dorsal root ganglia (DRGs). MMP-9 was expressed primarily in DRG neurons co-expressing GPR30, and led to the activation of IL-1β. After intrathecal injection of the GPR30 agonist G1, female rats with low estrogen and plantar incisions continued to exhibit significant hyperalgesia until 48h post-incision. In high estrogen level rats with plantar incisions, intrathecal injection of GPR30 antagonist G15 significantly attenuated postoperative hyperalgesia. Intraperitoneal injection of N-acetyl-cysteine, a source of cysteine that prevents the oxidation of cysteine residues on MMP-9, significantly relieved high estrogen-induced postoperative hyperalgesia via suppression of MMP-9 and IL-1β activation in DRGs. These results demonstrate that high estrogen level in rats with incisions elicit GPR30 and MMP-9 upregulation in DRGs and subsequently activate IL-1β, leading to induced postoperative hyperalgesia.

Dynorphin and Enkephalin Opioid Peptides and Transcripts in Spinal Cord and Dorsal Root Ganglion During Peripheral Inflammatory Hyperalgesia and Allodynia

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PerspectiveThis is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

Inhibition of DRG-TRPV1 upregulation in myocardial ischemia contributes to exogenous cardioprotection

Myocardium ischemia-reperfusion injury (IRI) is the major cause of postoperative cardiac dysfunction. While intrathecal morphine preconditioning (ITMP) can reduce IRI in animals, the molecular processes underlying IRI and ITMP remain elusive. Transient receptor potential vanilloid type 1 (TRPV1) is highly expressed in cardiac sensory neurons and has a crucial role in detecting myocardial ischemia. This study aimed to determine the role of up-regulated dorsal root ganglion (DRG)-TRPV1 in IRI and whether its inhibition contributes to ITMP-induced cardioprotection. Animal model of IRI was established by left coronary artery occlusion (30 min) and reperfusion (2 h) in rats. Intrathecal intubation was prepared for morphine preconditioning, TRPV1-shRNA or selective TRPV1 antagonist administration. After IRI, both protein and phosphorylation levels of TRPV1 were significantly increased, and the immunofluorescence intensity of TRPV1 was increased and colocalized with μ-opioid receptors in DRG. Intrathecal pre-administration of either TRPV1-shRNA or TRPV1 antagonist significantly reduced myocardial injury and the upregulation of TRPV1 in DRG induced by IRI. Simultaneously, ITMP significantly suppressed TRPV1 protein expression and phosphorylation in DRG, as well as the heart infarct size and arrhythmia score caused by IRI. The suppression of TRPV1 elevation and activation by ITMP were reversed by intrathecal injection of the selective μ receptor antagonist. Furthermore, IRI elevated DRG cAMP, while intrathecal administration of the selective cAMP-PKA inhibitor reduced myocardial injury. Finally, we showed that activation of opioid receptor by morphine inhibited PKA activator-induced TRPV1 channel activity at the cellular level. These findings suggest that the elevation and activation of TRPV1 in DRG during myocardial ischemia-reperfusion might be responsible for cardiac injury. ITMP exerts cardioprotection by inhibiting DRG-TRPV1 activity via modulation cAMP. Therefore, inhibition of TRPV1 upregulation in DRG might be used as a novel therapeutic mechanism for myocardium ischemia-reperfusion injury.

Suppressing PKC-dependent membrane P2X3 receptor upregulation in dorsal root ganglia mediated electroacupuncture analgesia in rat painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a common and troublesome diabetes complication. Protein kinase C (PKC)-mediated dorsal root ganglia (DRG) P2X3 receptor upregulation is one important mechanism underlying PDN. Accumulating evidence demonstrated that electroacupuncture (EA) at low frequency could effectively attenuate neuropathic pain. Our previous study showed that 2-Hz EA could relieve pain well in PDN. The study aimed to investigate whether 2-Hz EA relieves pain in PDN through suppressing PKC-mediated DRG P2X3 receptor upregulation. A 7-week feeding of high-fat and high-sugar diet plus a single injection of streptozotocin (STZ) in a dose of 35 mg/kg after a 5-week feeding of the diet successfully induced type 2 PDN in rats as revealed by the elevated body weight, fasting blood glucose, fasting insulin and insulin resistance, and the reduced paw withdrawal threshold (PWT), as well as the destructive ultrastructural change of sciatic nerve. DRG plasma membrane P2X3 receptor level and DRG PKC expression were elevated. Two-hertz EA failed to improve peripheral neuropathy; however, it reduced PWT, DRG plasma membrane P2X3 receptor level, and DRG PKC expression in PDN rats. Intraperitoneal administration of P2X3 receptor agonist αβ-meATP or PKC activator phorbol 12-myristate 13-acetate (PMA) blocked 2-Hz EA analgesia. Furthermore, PMA administration increased DRG plasma membrane P2X3 receptor level in PDN rats subject to 2-Hz EA treatment. These findings together indicated that the analgesic effect of EA in PDN is mediated by suppressing PKC-dependent membrane P2X3 upregulation in DRG. EA at low frequency is a valuable approach for PDN control.

Puerarin ameliorates allodynia and hyperalgesia in rats with peripheral nerve injury.

Puerarin is a major active ingredient of the traditional Chinese plant medicine, Radix Puerariae, and commonly used in the treatment of myocardial and cerebral ischemia. However, the effects of puerarin on neuropathic pain are still unclear. In this study, a neuropathic pain animal model was created by partial sciatic nerve ligation. Puerarin (30 or 60 mg/kg) was intraperitoneally injected once a day for 7 days. Mechanical allodynia and thermal hyperalgesia were examined at 1 day after model establishment. Mechanical threshold and paw withdrawal latency markedly increased in a dose-dependent manner in puerarin-treated rats, especially at 7 days after model establishment. At 7 days after model establishment, quantitative real-time reverse transcriptase-polymerase chain reaction results showed that puerarin administration reversed mRNA expression of transient receptor potential vanilloid 1 (Trpv1) and transient receptor potential ankyrin 1 (Trpa1) in a dose-dependent manner in dorsal root ganglion neurons after peripheral nerve injury. These results suggest that puerarin dose-dependently ameliorates neuropathic pain by suppressing Trpv1 and Trpa1 up-regulation in dorsal root ganglion of neuropathic pain rats.

Regulation of transient receptor potential cation channel subfamily V1 protein synthesis by the phosphoinositide 3-kinase/Akt pathway in colonic hypersensitivity

The transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as the capsaicin receptor or vanilloid receptor 1 (VR1), is expressed in nociceptive neurons in the dorsal root ganglia (DRG) and participates in the transmission of pain. The present study investigated the underlying molecular mechanisms by which TRPV1 was regulated by nerve growth factor (NGF) signaling pathways in colonic hypersensitivity in response to colitis. We found that during colitis TRPV1 protein levels were significantly increased in specifically labeled colonic afferent neurons in both L1 and S1 DRGs. TRPV1 protein up-regulation in DRG was also enhanced by NGF treatment. We then found that TRPV1 protein up-regulation in DRG was regulated by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway both in vivo and in vitro. Suppression of endogenous PI3K/Akt activity during colitis or NGF treatment with a specific PI3K inhibitor LY294002 reduced TRPV1 protein production in DRG neurons, and also reduced colitis-evoked TRPV1-mediated visceral hypersensitivity tested by hyper-responsiveness to colorectal distention (CRD) and von Frey filament stimulation of abdomen. Further studies showed that TRPV1 mRNA levels in the DRG were not regulated by either colitis or NGF. We then found that an up-regulation of the protein synthesis pathway was involved by which both colitis and NGF caused a PI3K-dependent increase in the phosphorylation level of eukaryotic translation initiation factor 4E-binding protein (4E-BP)1. These results suggest a novel mechanism in colonic hypersensitivity which involves PI3K/Akt-mediated TRPV1 protein, not mRNA, up-regulation in primary afferent neurons, likely through activation of the protein synthesis pathways.

Intrathecal administration of TRPA1 antagonists attenuate cyclophosphamide-induced cystitis in rats with hyper-reflexia micturition.

BackgroundThe activation of TRPA1 channel is implicated in hyper-reflexic micturition similar to overactive bladder. In this study, we aimed to investigate the effects of blocking TRPA1 via intrathecal administration of antagonists on the afferent pathways of micturition in rats with cystitis.MethodsThe cystitis was induced by intraperitoneal cyclophosphamide administration. Cystometry was performed in control and cystitis rats, following the intrathecal injection of the TRPA1 antagonists HC-030031 and A-967079. Real-time PCR, agarose gel electrophoresis, western blotting and immunohistochemistry were used to investigate the levels of TRPA1 mRNA or protein in the bladder mucosa and L6-S1 dorsal root ganglia (DRG).ResultsEdema, submucosal hemorrhaging, stiffness and adhesion were noted during removal of the inflamed bladder. The expression of TRPA1 mRNA and protein was higher in the cystitis group in both the mucosa and DRG, but the difference was significant in the DRG (P < 0.05). Intrathecal administration of HC-030031 and A-967079 decreased the micturition reflex in the cystitis group. A 50 μg dose of HC-030031 increased the intercontraction interval (ICI) to 183 % of the no-treatment value (P < 0.05) and decreased the non-voiding contraction (N-VC) to 60 % of control (P < 0.01). Similarly, the treatment with 3 μg A-967079 increased the ICI to 142 % of the control value (P < 0.05) and decreased the N-VC to 77 % of control (P < 0.05). The effects of both antagonists weakened approximately 2 h after injection.ConclusionsThe TRPA1 had a pronounced upregulation in DRG but more slight in mucosa in rat cystitis. The blockade of neuronal activation of TRPA1 by intrathecal administration of antagonists could decrease afferent nerve activities and attenuated detrusor overactivity induced by inflammation.

Early increasing-intensity treadmill exercise reduces neuropathic pain by preventing nociceptor collateral sprouting and disruption of chloride cotransporters homeostasis after peripheral nerve injury.

Activity treatments, such as treadmill exercise, are used to improve functional recovery after nerve injury, parallel to an increase in neurotrophin levels. However, despite their role in neuronal survival and regeneration, neurotrophins may cause neuronal hyperexcitability that triggers neuropathic pain. In this work, we demonstrate that an early increasing-intensity treadmill exercise (iTR), performed during the first week (iTR1) or during the first 2 weeks (iTR2) after section and suture repair of the rat sciatic nerve, significantly reduced the hyperalgesia developing rapidly in the saphenous nerve territory and later in the sciatic nerve territory after regeneration. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in sensory neurons and spinal cord was reduced in parallel. iTR prevented the extension of collateral sprouts of saphenous nociceptive calcitonin gene-related peptide fibers within the adjacent denervated skin and reduced NGF expression in the same skin and in the L3 dorsal root ganglia (DRG). Injury also induced Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) upregulation in DRG, and K⁺-Cl⁻ cotransporter 2 (KCC2) downregulation in lumbar spinal cord dorsal horn. iTR normalized NKCC1 and boosted KCC2 expression, together with a significant reduction of microgliosis in L3-L5 dorsal horn, and a reduction of BDNF expression in microglia at 1 to 2 weeks postinjury. These data demonstrate that specific activity protocols, such as iTR, can modulate neurotrophins expression after peripheral nerve injury and prevent neuropathic pain by blocking early mechanisms of sensitization such as collateral sprouting and NKCC1/KCC2 disregulation.

MicroRNAs downregulated in neuropathic pain regulate MeCP2 and BDNF related to pain sensitivity

Nerve injury induces chronic pain and dysregulation of microRNAs in dorsal root ganglia (DRG). Several downregulated microRNAs are predicted to target Mecp2. MECP2 mutations cause Rett syndrome and these patients report decreased pain perception. We confirmed MeCP2 upregulation in DRG following nerve injury and repression of MeCP2 by miRNAs in vitro. MeCP2 regulates brain-derived neurotrophic factor (BDNF) and downregulation of MeCP2 by microRNAs decreased Bdnf in vitro. MeCP2 T158A mice exhibited reduced mechanical sensitivity and Mecp2-null and MeCP2 T158A mice have decreased Bdnf in DRG. MeCP2-mediated regulation of Bdnf in the DRG could contribute to altered pain sensitivity.

In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis

The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis.

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice

IntroductionEndothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice.MethodsGene expression was examined in lumbar dorsal root ganglia two days after induction of antigen-induced arthritis (AIA) using mRNA microarray analysis. Effects of drug treatment were determined by repeated assessment of joint swelling, pain-related behavior, and histopathological manifestations during AIA.ResultsDaily oral administration of the mixed ETA and ETB endothelin receptor antagonist bosentan significantly attenuated knee joint swelling and inflammation to an extent that was comparable to dexamethasone. In addition, bosentan reduced inflammatory mechanical hyperalgesia. Chronic bosentan administration also inhibited joint swelling and protected against inflammation and joint destruction during AIA flare-up reactions. In contrast, the ETA-selective antagonist ambrisentan failed to promote any detectable antiinflammatory or antinociceptive activity.ConclusionsThus, the present study reveals a pivotal role for the endothelin system in the development of arthritis and arthritic pain. We show that endothelin receptor antagonists can effectively control inflammation, pain and joint destruction during the course of arthritis. Our findings suggest that the antiinflammatory and antinociceptive effects of bosentan are predominantly mediated via the ETB receptor.

Profiling of dynamically changed gene expression in dorsal root ganglia post peripheral nerve injury and a critical role of injury-induced glial fibrillary acetic protein in maintenance of pain behaviors

To explore cellular changes in sensory neurons after nerve injury and to identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post-array validation indicated that there was a temporal correlation between injury-induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender-matched wild-type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury-induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury-induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may also play a critical, but yet identified, role in the maintenance of neuropathic pain states.

NF-κB Mediated Enhancement of Potassium Currents by the Chemokine CXCL1/Growth Related Oncogene in Small Diameter Rat Sensory Neurons

BackgroundInflammatory processes play important roles in both neuropathic and inflammatory pain states, but the effects of inflammation per se within the sensory ganglia are not well understood. The cytokine growth-related oncogene (GRO/KC; CXCL1) shows strong, rapid upregulation in dorsal root ganglion (DRG) in both nerve injury and inflammatory pain models. We examined the direct effects of GRO/KC on small diameter DRG neurons, which are predominantly nociceptive. Whole cell voltage clamp technique was used to measure voltage-activated potassium (K) currents in acutely cultured adult rat small diameter sensory neurons. Fluorescently labeled isolectin B4 (IB4) was used to classify cells as IB4-positive or IB4-negative.ResultsIn IB4-negative neurons, voltage-activated K current densities of both transient and sustained components were increased after overnight incubation with GRO/KC (1.5 nM), without marked changes in voltage dependence or kinetics. The average values for the slow and fast decay time constants at 20 mV were unchanged by GRO/KC. The amplitude of the fast inactivating component increased significantly with no large shifts in the voltage dependence of inactivation. The increase in K currents was completely blocked by co-incubation with protein synthesis inhibitor cycloheximide (CHX) or NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) or quinazoline (6-Amino-4-(4-phenoxypheny lethylamino;QNZ). In contrast, the voltage-activated K current of IB4-positive neurons was unchanged by GRO/KC. GRO/KC incubation caused no significant changes in the expression level of eight selected voltage-gated K channel genes in quantitative PCR analysis.ConclusionThe results suggest that GRO/KC has important effects in inflammatory processes via its direct actions on sensory neurons, and that activation of NF-κB is involved in the GRO/KC-induced enhancement of K currents.

Alterations in N-methyl-d-aspartate receptor subunits in primary sensory neurons following acid-induced esophagitis in cats

The excitatory amino acid glutamate plays an important role in the development of neuronal sensitization and the ionotropic N-methyl-d-aspartate receptor (NMDAR) is one of the major receptors involved. The objective of this study was to use a cat model of gastroesophageal reflux disease (GERD) to investigate the expression of the NR1 and NR2A subunits of NMDAR in the vagal and spinal afferent fibers innervating the esophagus. Two groups of cats (Acid-7D and PBS-7D) received 0.1 N HCl (pH 1.2) or 0.1 M PBS (pH 7.4) infusion in the esophagus (1 ml/min for 30 min/day for 7 days), respectively. NR1 splice variants (both NH(2) and COOH terminals) and NR2A in the thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophagus were evaluated by RT-PCR, Western blot, and immunohistochemistry. Acid produced marked inflammation and a significant increase in eosinophil peroxidase and myeloperoxidase contents compared with PBS-infused esophagus. The NR1-4 splice variant gene exhibited a significant upregulation in DRGs and esophagus after acid infusion. In DRGs, NGs, and esophagus, acid infusion resulted in significant upregulation of NR1 and downregulation of NR2A subunit gene expression. A significant increase in NR1 polypeptide expression was observed in DRGs and NGs from Acid-7D compared with control. In conclusion, long-term acid infusion in the cat esophagus resulted in ulcerative esophagitis and differential expressions of NR1 and NR2A subunits. It is possible that these changes may in part contribute to esophageal hypersensitivity observed in reflux esophagitis.

The Chemokine CXCL1/Growth Related Oncogene Increases Sodium Currents and Neuronal Excitability in Small Diameter Sensory Neurons

BackgroundAltered Na+ channel expression, enhanced excitability, and spontaneous activity occur in nerve-injury and inflammatory models of pathological pain, through poorly understood mechanisms. The cytokine GRO/KC (growth related oncogene; CXCL1) shows strong, rapid upregulation in dorsal root ganglion in both nerve injury and inflammatory models. Neurons and glia express its receptor (CXCR2). CXCL1 has well-known effects on immune cells, but little is known about its direct effects on neurons.ResultsWe report that GRO/KC incubation (1.5 nM, overnight) caused marked upregulation of Na+ currents in acutely isolated small diameter rat (adult) sensory neurons in vitro. In both IB4-positive and IB4-negative sensory neurons, TTX-resistant and TTX-sensitive currents increased 2- to 4 fold, without altered voltage dependence or kinetic changes. These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide. Amplification of cDNA from the neuronal cultures showed that 3 Na channel isoforms were predominant both before and after GRO/KC treatment (Nav 1.1, 1.7, and 1.8). TTX-sensitive isoforms 1.1 and 1.7 significantly increased 2 – 3 fold after GRO/KC incubation, while 1.8 showed a trend towards increased expression. Current clamp experiments showed that GRO/KC caused a marked increase in excitability, including resting potential depolarization, decreased rheobase, and lower action potential threshold. Neurons acquired a striking ability to fire repetitively; IB4-positive cells also showed marked broadening of action potentials. Immunohistochemical labelling confirmed that the CXCR2 receptor was present in most neurons both in dissociated cells and in DRG sections, as previously shown for neurons in the CNS.ConclusionMany studies on the role of chemokines in pain conditions have focused on their rapid and indirect effects on neurons, via release of inflammatory mediators from immune and glial cells. Our study suggests that GRO/KC may also have important pro-nociceptive effects via its direct actions on sensory neurons, and may induce long-term changes that involve protein synthesis.