DRG Sensory Neurons Research Articles

Aryl hydrocarbon receptor restricts axon regeneration of DRG neurons in response to injury.

Injured neurons sense environmental cues to balance neural protection and axon regeneration, but the mechanisms are unclear. Here, we unveil aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, as a molecular sensor and key regulator of acute stress response at the expense of axon regeneration. We demonstrate responsiveness of DRG sensory neurons to AhR signaling, which functions to inhibit axon regeneration. Conditional Ahr deletion in neurons accelerates axon regeneration after sciatic nerve injury. Ahr deletion partially mimics the conditioning lesion in priming DRG to initiate axonogenesis gene programs; upon peripheral axotomy, Ahr ablation suppresses inflammation and stress signaling while augmenting pro-growth pathways. Moreover, comparative transcriptomics revealed signaling interactions between AhR and HIF-1α, two structurally related bHLH-PAS α units that share the dimerization partner Arnt/HIF-1β. Functional assays showed that the growth advantage of AhR-deficient DRG neurons requires HIF-1α; but in the absence of Arnt, DRG neurons can still mount a regenerative response. We further unveil a link between bHLH-PAS transcription factors and DNA hydroxymethylation in response to peripheral axotomy, while RNA-seq of DRG neurons and neuronal single cell RNA-seq analysis revealed a link of AhR regulon to RNA regulation and integrated stress response (ISR). Altogether, AhR activation favors stress coping and inflammation at the expense of axon regeneration; targeting AhR has the potential to enhance nerve repair.

Quercetin/polyethyleneimine modified gold nanoconjugates inhibit apoptosis and ROS production induced by hydrogen peroxide in DRG sensory neurons

The basis of most neurological syndromes is the accumulation of free radical molecules. Quercetin is a polyphenolic bioflavonoid molecule and it has a very strong antioxidant effect by maintaining oxidative balance. There are many difficulties in the clinical use of quercetin due to its hydrophobic structure, low solubility, instability, poor oral bioavailability, and limited tissue-barrier penetration. Its synergistic use in complex with gold nanoparticles (AuNPs) could overcome these problems. AuNPs have recently emerged as an attractive candidate for delivery applications of various biomolecules and drugs. The aim of this study was to synthesize two different sized gold nanoparticles (AuNP20 and AuNP50) modified with polyethyleneimine (PEI) and quercetin, evaluate their potential neuroprotective effects on the in vitro oxidative stress model using DRG primary sensory neurons. It was shown that the antioxidant and anti-apoptotic ability of the bioflavonoid was preserved after exposure to the designed quercetin modified AuNPs. The PEI surface coating increased the stability and biocompatibility of the AuNPs in both sizes. It also potentially enables additional surface functionalization. This study indicates that designed nanoparticles (AuNP-Q-PEI) with different sizes could be a useful potential platform for the treatment of neurodegenerative syndromes or cancer diseases.

Disease-modifying rdHSV-CA8* non-opioid analgesic gene therapy treats chronic osteoarthritis pain by activating Kv7 voltage-gated potassium channels.

Chronic pain is common in our population, and most of these patients are inadequately treated, making the development of safer analgesics a high priority. Knee osteoarthritis (OA) is a primary cause of chronic pain and disability worldwide, and lower extremity OA is a major contributor to loss of quality-adjusted life-years. In this study we tested the hypothesis that a novel JDNI8 replication-defective herpes simplex-1 viral vector (rdHSV) incorporating a modified carbonic anhydrase-8 transgene (CA8*) produces analgesia and treats monoiodoacetate-induced (MIA) chronic knee pain due to OA. We observed transduction of lumbar DRG sensory neurons with these viral constructs (vHCA8*) (~40% of advillin-positive cells and ~ 50% of TrkA-positive cells colocalized with V5-positive cells) using the intra-articular (IA) knee joint (KJ) route of administration. vHCA8* inhibited chronic mechanical OA knee pain induced by MIA was dose- and time-dependent. Mechanical thresholds returned to Baseline by D17 after IA KJ vHCA8* treatment, and exceeded Baseline (analgesia) through D65, whereas negative controls failed to reach Baseline responses. Weight-bearing and automated voluntary wheel running were improved by vHCA8*, but not negative controls. Kv7 voltage-gated potassium channel-specific inhibitor XE-991 reversed vHCA8*-induced analgesia. Using IHC, IA KJ of vHCA8* activated DRG Kv7 channels via dephosphorylation, but negative controls failed to impact Kv7 channels. XE-991 stimulated Kv7.2-7.5 and Kv7.3 phosphorylation using western blotting of differentiated SH-SY5Y cells, which was inhibited by vHCA8* but not by negative controls. The observed prolonged dose-dependent therapeutic effects of IA KJ administration of vHCA8* on MIA-induced chronic KJ pain due to OA is consistent with the specific activation of Kv7 channels in small DRG sensory neurons. Together, these data demonstrate for the first-time local IA KJ administration of vHCA8* produces opioid-independent analgesia in this MIA-induced OA chronic pain model, supporting further therapeutic development.

Sonic hedgehog restrains the ubiquitin-dependent degradation of SP1 to inhibit neuronal/glial senescence associated phenotypes in chemotherapy-induced peripheral neuropathy via the TRIM25-CXCL13 axis

IntroductionChemotherapy-induced peripheral neuropathy (CIPN) is a common complication that affects an increasing number of cancer survivors. However, the current treatment options for CIPN are limited. Paclitaxel (PTX) is a widely used chemotherapeutic drug that induces senescence in cancer cells. While previous studies have demonstrated that Sonic hedgehog (Shh) can counteract cellular dysfunction during aging, its role in CIPN remains unknown. ObjectivesHerein, the aim of this study was to investigate whether Shh activation could inhibits neuronal/glial senescence and alleviates CIPN. MethodsWe treated ND7/23 neuronal cells and RSC96 Schwann cells with two selective Shh activators (purmorphamine [PUR] and smoothened agonist [SAG]) in the presence of PTX. Additionally, we utilized a CIPN mouse model induced by PTX injection. To assess cellular senescence, we performed a senescence-associated β-galactosidase (SA-β-gal) assay, measured reactive oxygen species (ROS) levels, and examined the expression of P16, P21, and γH2AX. To understand the underlying mechanisms, we conducted ubiquitin assays, LC-MS/MS, H&E staining, and assessed protein expression through Western blotting and immunofluorescence staining. ResultsIn vitro, we observed that Shh activation significantly alleviated the senescence-related decline in multiple functions included SA-β-gal activity, expression of P16 and P21, cell viability, and ROS accumulation in DRG sensory neurons and Schwann cells after PTX exposure. Furthermore, our in vivo experiments demonstrated that Shh activation significantly reduced axonal degeneration, demyelination, and improved nerve conduction. Mechanistically, we discovered that PTX reduced the protein level of SP1, which was ubiquitinated by the E3 ligase TRIM25 at the lysine 694 (K694), leading to increased CXCL13 expression, and we found that Shh activation inhibited PTX-induced neuronal/glial senescence and CIPN through the TRIM25-SP1-CXCL13 axis. ConclusionThese findings provide evidence for the role of PTX-induced senescence in DRG sensory neurons and Schwann cells, suggesting that Shh could be a potential therapeutic target for CIPN.

P176 TYK2 inhibition attenuates the stimulation of sensory neurons with hyper-IL-6

Abstract Background Abdominal pain and diarrhoea are a cause of significant morbidity for people with colitis. Recent single cell RNA sequencing of sensory neurons innervating the gut highlights a likely role for sensory nerves in the pathophysiology of these symptoms based on the marked expression of cytokine receptors from the IL-6 receptor family in gut projecting sensory neurons. In keeping with the reported downstream signalling pathway for these receptors marked co-expression of TYK2 transcripts were also found along with IL-6 signal transducer (GP130) in colonic nociceptors characterised by expression of the capsaicin receptor TRPV1. These observations suggest that TYK2 inhibitors may ameliorate symptoms of pain and diarrhoea in people with colitis by preventing neurogenically mediated gut contractility and nociceptor activation. Methods TYK2 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to the hyper-IL-6 fusion protein of the soluble IL-6 receptor and IL-6, which transactivates the GP130 receptor subunit to mimic IL-6 family receptor signalling following pre-treatment with vehicle, the TYK2 inhibitor Deucravacitinib (100nM) or the pan-JAK inhibitor Pyridone-6 (2 μM). Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of hyper-IL-6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons which was significantly attenuated by pre-treatment with the TYK2 specific inhibitor Deucravacitinib (p<0.01) or the pan JAK inhibitor Pyridone-6 (p = <0.05). Conclusion Findings demonstrate that inhibition of TYK2 inhibitor prevents sensory neuron activation in response to IL-6 trans-signalling. This data suggests that TYK2 inhibitors may have utility for the treatment of abdominal pain in people with colitis.

P045 MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1

Abstract Background Abdominal pain is a cause of significant morbidity for people with colitis. We have previously shown that biopsy samples from people with Crohn’s disease (CD) are pro-nociceptive suggesting that peripheral nociceptor activation is a cause of pain in colitis. Furthermore, these effects were correlated with biopsy expression of macrophage metalloproteinase (MMP12), which was shown to stimulate mouse and human colonic nociceptors highlighting the potential utility of MMP12 inhibitors to act as visceral analgesics. Following on from this work we sought to determine the contribution of proteinase-activated receptor 1 (PAR1) to MMP12 mediated nociceptor activation. Methods MMP12 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to MMP12 or the PAR1 receptor agonists TRAP6 following pre-treatment with vehicle, the MMP12 inhibitor MMP408 or the PAR1 antagonist SCH79797. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. MMP12 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and of the isolated colorectum (ex-vivo) perfused with carbogenated Krebs solution, and responses determined following application with MMP12 alone or in the presence of respective MMP12 or PAR1 inhibitors. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of MMP12 or TRAP6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons classified as nociceptors by their co-sensitivity to capsaicin. These effects were significantly attenuated by pre-treatment with respective MMP12 or PAR1 inhibitors, with the response to MMP12 also being inhibited by PAR1 antagonist pre-treatment. Consistent with this pattern of nociceptor stimulation, the application of MMP12 also a robust activation of colonic afferents that was abolished by inhibition of MMP12 or PAR1 activity. Conclusion Findings demonstrate that MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1.

Inhibition of Nonsense-Mediated Decay Induces Nociceptive Sensitization through Activation of the Integrated Stress Response.

RNA stability is meticulously controlled. Here, we sought to determine whether an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of ∼10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine DRG sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons, including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask whether suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-α phosphorylation and reduced the abundance of the eIF2-α phosphatase constitutive repressor of eIF2-α phosphorylation. Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small-molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called nonsense-mediated decay (NMD). Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.

Ultrasound mediated control of neurons and immune cells

Ultrasound holds great promise for realizing noncontact stimulation and control of cell function. Here, we will present our ongoing efforts in controlling cell responses using two distinct approaches. The first is based on engineered cytotoxic immune cells with thermal switches, where closed-loop controlled MRgFUS-hyperthermia (20-mins at 42 °C) is employed to drive the local production of key transgenes and potentiate anti-tumor responses in brain tumors. The responses are assessed using bioluminescence, cell viability, and cell cytotoxicity assays. The second is based on US pulse sequences designed to promote mechanical effects on cultured cells with the goal to decode US neuromodulation. For our investigations, we employed a high throughput ultrasonic platform designed to monitor and locally control sound and vibration in combination DRG sensory neurons. The later are the most sensitive neuronal type to mechanical stretch and present an excellent experimental system to examine and identify mechanosensitive ion channels sensing sonication-induced membrane stretch. Cell responses are quantified and analyzed for different pulse durations and amplitude, cell culture conditions, and degassing protocols. Together, our findings show that ultrasound thermal and mechanical effects can be employed to control the function of different cell types, albeit cells with thermal switches provide more robust responses.

APP Genetic Deficiency Alters Intracellular Ca2+ Homeostasis and Delays Axonal Degeneration in Dorsal Root Ganglion Sensory Neurons.

The activation of self-destructive cellular programs helps sculpt the nervous system during development, but the molecular mechanisms used are not fully understood. Prior studies have investigated the role of the APP in the developmental degeneration of sensory neurons with contradictory results. In this work, we sought to elucidate the impact of APP deletion in the development of the sensory nervous system in vivo and in vitro. Our in vivo data show an increase in the number of sciatic nerve axons in adult male and female APP-null mice, consistent with the hypothesis that APP plays a pro-degenerative role in the development of peripheral axons. In vitro, we show that genetic deletion of APP delays axonal degeneration triggered by nerve growth factor deprivation, indicating that APP does play a pro-degenerative role. Interestingly, APP depletion does not affect caspase-3 levels but significantly attenuates the rise of axoplasmic Ca2+ that occurs during degeneration. We examined intracellular Ca2+ mechanisms that could be involved and found that APP-null DRG neurons had increased Ca2+ levels within the endoplasmic reticulum and enhanced store-operated Ca2+ entry. We also observed that DRG axons lacking APP have more mitochondria than their WT counterparts, but these display a lower mitochondrial membrane potential. Finally, we present evidence that APP deficiency causes an increase in mitochondrial Ca2+ buffering capacity. Our results support the hypothesis that APP plays a pro-degenerative role in the developmental degeneration of DRG sensory neurons, and unveil the importance of APP in the regulation of calcium signaling in sensory neurons.SIGNIFICANCE STATEMENT The nervous system goes through a phase of pruning and programmed neuronal cell death during development to reach maturity. In such context, the role played by the APP in the peripheral nervous system has been controversial, ranging from pro-survival to pro-degenerative. Here we present evidence in vivo and in vitro supporting the pro-degenerative role of APP, demonstrating the ability of APP to alter intracellular Ca2+ homeostasis and mitochondria, critical players of programmed cell death. This work provides a better understanding of the physiological function of APP and its implication in developmental neuronal death in the nervous system.

Conventional Dorsal Root Ganglion Stimulation in an Experimental Model of Painful Diabetic Peripheral Neuropathy: A Quantitative Immunocytochemical Analysis of Intracellular γ-Aminobutyric Acid in Dorsal Root Ganglion Neurons

ABSTRACTBackground and ObjectiveThe sensory cell somata in the DRG contain all equipment necessary for extensive GABAergic signaling and are able to release GABA upon depolarization. With this study, we hypothesize that pain relief induced by conventional dorsal root ganglion stimulation (Con‐DRGS) in animals with experimental painful diabetic peripheral neuropathy is related to the release of GABA from DRG neurons. With use of quantitative immunocytochemistry, we hypothesize DRGS to result in a decreased intensity of intracellular GABA‐immunostaining in DRG somata.Materials and MethodsFemale Sprague‐Dawley rats (n = 31) were injected with streptozotocin (STZ) in order to induce Diabetes Mellitus. Animals that developed neuropathic pain after four weeks (Von Frey) were implanted with a unilateral DRGS device at L4 (n = 14). Animals were then stimulated for 30 min with Con‐DRGS (20 Hz, pulse width = 0.2 msec, amplitude = 67% of motor threshold, n = 8) or Sham‐DRGS (n = 6), while pain behavior (von Frey) was measured. DRGs were then collected and immunostained for GABA, and a relation to size of sensory cell soma diameter (small: 12–26 μm, assumed to be C‐fiber related sensory neurons; medium: 26–40 μm, assumed to be Aδ related sensory neurons; and large: 40–54 μm, assumed to be Aβ related sensory neurons) was made.ResultsDRGS treated animals showed significant reductions in STZ‐induced mechanical hypersensitivity. No significant differences in GABA immunostaining intensity per sensory neuron cell soma type (small‐, medium‐, or large‐sized) were noted in DRGs of stimulated (Con‐DRGS) animals versus Sham animals. No differences in GABA immunostaining intensity per sensory cell soma type in ipsi‐ as compared to contralateral DRGs were observed.ConclusionCon‐DRGS does not affect the average intracellular GABA immunofluorescence staining intensity in DRG sensory neurons of those animals which showed significant pain reduction. Similarly, no soma size related changes in intracellular GABA immunofluorescence were observed following Con‐DRGS.

772 Terminal arborizations of itch-sensing neurons exhibit large receptive field in the skin and regionally specific organization in the spinal cord

Itch sensation is initiated by the activation of a specific subset of sensory neurons that project their peripheral and central axons to the skin and spinal cord respectively. Different subtypes of sensory neurons exhibit distinct terminal arborization subserving their functions. Sensory arborization of itch-sensing neurons has never been observed due to the lack of proper molecular genetic tool. Here we have genetically labeled a small subset of DRG sensory neurons using a newly generated MrgprC11CreER mouse line.

The cellular basis of protease-activated receptor 2-evoked mechanical and affective pain.

Protease-activated receptor 2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. Although PAR2-evoked pain has been attributed to sensory neuron expression, RNA-sequencing experiments show ambiguous F2rl1 mRNA detection. Moreover, many pharmacological tools for PAR2 are nonspecific, acting also on the Mas-related GPCR family (Mrg) that are highly enriched in sensory neurons. We sought to clarify the cellular basis of PAR2-evoked pain. We developed a PAR2-conditional knockout mouse and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist-evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, are dependent on PAR2 expression in sensory neurons that project to the hind paw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of mostly small-diameter sensory neurons that coexpress the Nppb and IL31ra genes. This cell population has been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings show that a discrete population of DRG sensory neurons mediate PAR2-evoked pain.

A Cold-Sensing Receptor Encoded by a Glutamate Receptor Gene.

In search of the molecular identities of cold-sensing receptors, we carried out an unbiased genetic screen for cold-sensing mutants in C.elegans and isolated a mutant allele of glr-3 gene that encodes a kainate-type glutamate receptor. While glutamate receptors are best known to transmit chemical synaptic signals in the CNS, we show that GLR-3 senses cold in the peripheral sensory neuron ASER to trigger cold-avoidance behavior. GLR-3 transmits cold signals via G protein signaling independently of its glutamate-gated channel function, suggesting GLR-3 asa metabotropic cold receptor. The vertebrate GLR-3 homolog GluK2 from zebrafish, mouse, and human can all function as a cold receptor in heterologous systems. Mouse DRG sensory neurons express GluK2, and GluK2 knockdown in these neurons suppresses their sensitivity to cold but not cool temperatures. Our study identifies an evolutionarily conserved cold receptor, revealing that a central chemical receptor unexpectedly functions as a thermal receptor in the periphery.

(211) Deep Pain Mechanisms in Sickle Cell Disease

Sickle Cell Disease (SCD), an autosomal recessive red blood cell disorder, affects more than 100,000 individuals in the US. Deep pain that appears to stem from muscle and bones is a major complaint of patients with SCD and very little is known about putative mechanisms that cause this deep pain. To assess this pain, we used the Townes HbSS (SS) transgenic mouse model of SCD to perform behavioral assays including evoked withdrawal responses to gastrocnemius muscle pinch and the inverted screen hang, a potential measure of muscle or bone pain. For the muscle pinch assay, SS mice exhibited lower withdrawal thresholds than Townes AA controls. Furthermore, there was a sex difference between SS males and SS females in their withdrawal thresholds where females had higher sensitivity and greater pain-like behaviors than males. For the inverted screen hang assay, SS mice had significantly lower total hang time and more time on 3 paws or less compared to AA controls. Together these data suggest that these mice may experience greater deep pain. Because the TRPV1 channel has been shown to contribute to mechanical hypersensitivity in SCD mice, we used the selective and potent TRPV1 inhibitor A-425619 in behavioral assays. Intraperitoneal injection of A-425619 (100µmol/kg) partially reversed the sensitized muscle pinch withdrawal thresholds in both sexes of SS SCD mice, with a greater effect on males than in females. To further assess mechanisms underlying the sensitization in SCD mice, calcium imaging of lumbar DRG sensory neurons was performed to begin to investigate whether there is increased sensitization of sensory neurons to endogenous metabolites that could act on TRPV1, ASIC3, and P2X channels. In conclusion, these data suggest that the Townes SS mouse model of SCD has increased deep pain sensitivity compared to controls and this sensitivity appears to be partly mediated by TRPV1.

Parathyroid hormone-related peptide activates and modulates TRPV1 channel in human DRG neurons.

Parathyroid hormone-related peptide (PTHrP) is associated with advanced tumor growth and metastasis, especially in breast, prostate and myeloma cancers that metastasize to bones, resulting in debilitating chronic pain conditions. Our recent studies revealed that the receptor for PTHrP, PTH1R, is expressed in mouse DRG sensory neurons, and its activation leads to flow-activation and modulation of TRPV1 channel function, resulting in peripheral heat and mechanical hypersensitivity. In order to verify the translatability of our findings in rodents to humans, we explored whether this signalling axis operates in primary human DRG sensory neurons. Analysis of gene expression data from recently reported RNA deep sequencing experiments performed on mouse and human DRGs reveals that PTH1R is expressed in DRG and tibial nerve. Furthermore, exposure of cultured human DRG neurons to PTHrP leads to slow-sustained activation of TRPV1 and modulation of capsaicin-induced channel activation. Both activation and modulation of TRPV1 by PTHrP were dependent on PKC activity. Our findings suggest that functional PTHrP/PTH1R-TRPV1 signalling exists in human DRG neurons, which could contribute to local nociceptor excitation in the vicinity of metastatic bone tumor microenvironment.

Interactions of peripheral endothelin-1 and nerve growth factor as contributors to persistent cutaneous pain.

Endothelin-1 (ET-1) and Nerve Growth Factor (NGF) are proteins, released from cancer-ridden tissues, which cause spontaneous pain and hypersensitivity to noxious stimuli. Here we examined the electrophysiological and behavioral effects of these two agents for evidence of their interactions. Individual small-medium cultured DRG sensory neurons responded to both ET-1 (50 nM, n=6) and NGF (100 ng/ml, n=4), with increased numbers of action potentials and decreased slow K(+) currents; pre-exposure to ET-1 potentiated NGF´s actions, but not vice versa. Behaviorally, single intraplantar (i.pl.) injection of low doses of ET-1 (20 pmol) or NGF (100 ng), did not increase hindpaw tactile or thermal sensitivity, but their simultaneous injections sensitized the paw to both modalities. Daily i.pl. injections of low ET-1 doses in male rats caused tactile sensitization after 21 days, and enabled further tactile and thermal sensitization from low dose NGF, in ipsilateral and contralateral hindpaws. Single injections of 100 ng NGF, without changing the paw's tactile sensitivity by itself, acutely sensitized the ipsilateral paw to subsequent injections of low ET-1. The sensitization from repeated low ET-1 dosing and the cross-sensitization between NGF and ET-1 were both significantly greater in female than in male rats. These findings reveal a synergistic interaction between cutaneously administered low doses of NGF and ET-1, which could contribute to cancer-related pain.

Effect of a novel TRPV1 antagonist on capsaicin nociceptors

Effect of a novel TRPV1 antagonist on capsaicin nociceptors

Enhanced axonal regeneration of the injured sciatic nerve by administration of Buyang Huanwu decoction

Ethnopharmacological relevanceBuyang Huanwu decoction (BYHWD) has been used in the traditional Chinese medicine for the treatment of cardiovascular and neurological symptoms, and recent experimental studies have begun to provide evidence showing its protective effects on neural cells. Yet, its function for the regenerative responses of axons in the peripheral nerve after injury is not known. Aim of the studyThe primary objective of the present study was to explore that BYHWD is involved in growth-promoting activity of the peripheral nerve axons after injury. We further examined whether the effect of BYHWD exerted directly on regrowing axons or Schwann cells. Materials and methodsSciatic nerves in rats were given crush injury, and BYHWD was injected by oral administration. Sciatic nerves or DRG tissues were prepared for immunofluorescence staining and western blot analysis. Levels of axonal regeneration were quantified by retrograde tracing technique. Cultured DRG sensory neurons and Schwann cells were prepared from rats and used to examine the effects of BYHWD on the neurite outgrowth. Behavioral analysis on functional recovery after nerve injury was assessed in mice by pin prick test, adhesive removal test, and toe-spreading reflex. ResultsImmunofluorescence and retrograde tracing analyses showed that the distal extension of the sciatic nerve axons was significantly improved by BYHWD treatment. Levels of axonal growth-associated protein GAP-43 were upregulated by BYHWD treatment in the sciatic nerve after injury and in the neurites of cultured DRG neurons. In vivo administration of BYHWD in rats upregulated the induction level of cell division cycle 2 (Cdc2) and its phosphorylation of vimentin in Schwann cells from injured sciatic nerve. Coculture of DRG neurons with Schwann cells prepared from preinjured sciatic nerves in animals administered with BYHWD led to the enhancement in neurite outgrowth. Behavioral tests in mice given sciatic nerve injury showed a significant improvement in sensorimotor activity by BYHWD administration. ConclusionsOur results suggest that BYHWD administration into animals given sciatic nerve injury facilitates axonal regeneration by acting on both the axons undergoing regeneration and neighboring Schwann cells and improves functional recovery.

Morphology and intrinsic excitability of regenerating sensory and motor neurons grown on a line micropattern.

Axonal regeneration is one of the greatest challenges in severe injuries of peripheral nerve. To provide the bridge needed for regeneration, biological or synthetic tubular nerve constructs with aligned architecture have been developed. A key point for improving axonal regeneration is assessing the effects of substrate geometry on neuronal behavior. In the present study, we used an extracellular matrix-micropatterned substrate comprising 3 µm wide lines aimed to physically mimic the in vivo longitudinal axonal growth of mice peripheral sensory and motor neurons. Adult sensory neurons or embryonic motoneurons were seeded and processed for morphological and electrical activity analyses after two days in vitro. We show that micropattern-guided sensory neurons grow one or two axons without secondary branching. Motoneurons polarity was kept on micropattern with a long axon and small dendrites. The micro-patterned substrate maintains the growth promoting effects of conditioning injury and demonstrates, for the first time, that neurite initiation and extension could be differentially regulated by conditioning injury among DRG sensory neuron subpopulations. The micro-patterned substrate impacts the excitability of sensory neurons and promotes the apparition of firing action potentials characteristic for a subclass of mechanosensitive neurons. The line pattern is quite relevant for assessing the regenerative and developmental growth of sensory and motoneurons and offers a unique model for the analysis of the impact of geometry on the expression and the activity of mechanosensitive channels in DRG sensory neurons.

M2 Receptors Exert Analgesic Action on DRG Sensory Neurons by Negatively Modulating VR1 Activity

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments.