Dorsal Root Ganglion Neurons Research Articles

MiR-374 family is a key regulator of chronic primary pain onset.

Chronic primary pain conditions (CPPCs) are linked to catecholamine activation of peripheral adrenergic receptors. Yet, catecholamine-dependent epigenetic mechanisms, such as microRNA (miRNA) regulation of mRNA transcripts, remain largely unknown. We sought to identify RNA species correlated with case status in 3 pain cohorts, to validate RNAs found to be dysregulated in a mouse model of CPPC onset, and to directly test the role of adrenergic receptors in miRNA regulation. Furthermore, we tested antinociceptive effects of miR-374 overexpression. We used RNA-seq and quantitative reverse transcription polymerase chain reaction to measure RNA expression in 3 pain cohorts. Next, we validated identified RNAs with quantitative reverse transcription polymerase chain reaction in a mouse model of CPPC onset, measuring expression in plasma, peripheral (adipose, muscle, dorsal root ganglia [DRG]), and central (spinal cord) tissues. Then, we stimulated adrenergic receptors in primary adipocyte and DRG cultures to directly test regulation of microRNAs by adrenergic signaling. Furthermore, we used in vitro calcium imaging to measure the antinociceptive effects of miR-374 overexpression. We found that one miRNA family, miR-374, was downregulated in the plasma of individuals with temporomandibular disorder, fibromyalgia syndrome, or widespread pain following a motor vehicle collision. miR-374 was also downregulated in plasma, white adipose tissue, and spinal cord from mice with multisite mechanical sensitivity. miR-374 downregulation in plasma and spinal cord was female specific. Norepinephrine stimulation of primary adipocytes, but not DRG, led to decreased miR-374 expression. Furthermore, we identified tissue-specific and sex-specific changes in the expression of predicted miR-374 mRNA targets, including known (HIF1A, NUMB, TGFBR2) and new (ATXN7, CRK-II) pain targets. Finally, we demonstrated that miR-374 overexpression in DRG neurons reduced capsaicin-induced nociceptor activity. Downregulation of miR-374 occurs between adrenergic receptor activation and mechanical hypersensitivity, and its adipocyte source implicates adipose signaling in nociception. Further study of miR-374 may inform therapeutic strategies for the millions worldwide who experience CPPCs.

Protective role of TRPV1+ nociceptive neurons communication to macrophages against T. cruzi infection in mice

Chagas’ disease is a life-threatening condition caused by Trypanosoma cruzi. Patients with chronic disease may develop gastrointestinal, neurological, or associated neuro-digestive dysfunctions. CNS invasion by T. cruzi can occur in the acute phase, and its presence in the brain and cerebrospinal fluid was reported. T. cruzi induces nociceptor neuron activation and pain. Nociceptive neurons and macrophage interact in diseases, and this neuroimmune communication has a pivotal role in disease outcome. We investigated, the role of TRPV1+ neurons in experimental T. cruzi infection in mice. T. cruzi forms were observed in the DRG and spinal cord in early stages of acute infection, and intrathecal administration of T. cruzi antigens into spinal cord induced pain. Trpv1 mRNA expression was increased in the DRG of infected mice and targeting TRPV1 reduced T. cruzi-induced pain. TRPV1 nociceptor ablation increased blood parasitemia while TRPV1 knockout mice presented 50% mortality upon infection in a normally non-lethal model. TRPV1 knockout also worsened clinical outcomes (hepatomegaly and megacecum), increased plasmatic Th2 cytokines and nitrite in cardiac tissue, and reduced heart leukocyte infiltration. Conditioned media of capsaicin-stimulated DRG neurons decreased macrophage internalization of T. cruzi, and CGRP receptor antagonism in infected mice reduced pain, increased early parasitemia and promoted 18% mortality. This indicates that soluble mediators released upon nociceptor activation such as CGRP increase macrophage ability to control disease outcome. These data unveil TRPV1+ neurons release CGRP to limit macrophage internalization of T. cruzi, triggering protective mechanisms against T. cruzi infection.

HC-HA/PTX3 from Human Amniotic Membrane Induced Differential Gene Expressions in DRG Neurons: Insights into the Modulation of Pain

Background: The biologics derived from human amniotic membranes (AMs) demonstrate potential pain-inhibitory effects in clinical settings. However, the molecular basis underlying this therapeutic effect remains elusive. HC-HA/PTX3 is a unique water-soluble regenerative matrix that is purified from human AMs. We examined whether HC-HA/PTX3 can modulate the gene networks and transcriptional signatures in the dorsal root ganglia (DRG) neurons transmitting peripheral sensory inputs to the spinal cord. Methods: We conducted bulk RNA-sequencing (RNA-seq) of mouse DRG neurons after treating them with HC-HA/PTX3 (15 µg/mL) for 10 min and 24 h in culture. Differential gene expression analysis was performed using the limma package, and Gene Ontology (GO) and protein–protein interaction (PPI) analyses were conducted to identify the networks of pain-related genes. Western blotting and in vitro calcium imaging were used to examine the protein levels and signaling of pro-opiomelanocortin (POMC) in DRG neurons. Results: Compared to the vehicle-treated group, 24 h treatment with HC-HA/PTX3 induced 2047 differentially expressed genes (DEGs), which were centered on the ATPase activity, receptor–ligand activity, and extracellular matrix pathways. Importantly, PPI analysis revealed that over 50 of these DEGs are closely related to pain and analgesia. Notably, HC-HA/PTX3 increased the expression and signaling pathway of POMC, which may affect opioid analgesia. Conclusions: HC-HA/PTX3 induced profound changes in the gene expression in DRG neurons, centered around various neurochemical mechanisms associated with pain modulation. Our findings suggest that HC-HA/PTX3 may be an important biological active component in human AMs that partly underlies its pain inhibitory effect, presenting a new strategy for pain treatment.

Membrane lipid nanodomains modulate HCN pacemaker channels in nociceptor DRG neurons.

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.

The epidermal growth factor receptor inhibitor gefitinib enhances in vitro and in vivo sensory axon regeneration and functional recovery following transection in a mouse median nerve injury model.

The epidermal growth factor receptor (EGFR; ErbB1), a membrane bound receptor tyrosine kinase, is hypothesized to have an inhibitory influence on peripheral nerve regeneration. This study examines the impact of EGFR inhibition on nerve regeneration using the commercially available small molecule inhibitor gefitinib. In vitro assays included neurite outgrowth of cultured dorsal root ganglion (DRG) neurons from adult C57Bl/6 wildtype mice on immobilized chondroitin sulfate proteoglycans (CSPG). Following forelimb median nerve injury, EGFR expression, number of regenerated neurons (using retrograde labeling) and myelination of motor and sensory neurons were compared between mice that received either gefitinib or vehicle. Functional recovery was assessed using grip strength. EGFR expression on DRG and spinal motor neurons was confirmed. Gefitinib significantly increased neurite outgrowth in medium sized (30-50 μm) DRG neurons, resulting in longer neurites (183 ± 36 μm) compared with CSPG alone (49 ± 9 μm). After median nerve injury, significantly greater numbers of sensory neurons (638 ± 112 vs. 301 ± 81), but not motor neurons (31 ± 12 vs. 42 ± 13) regenerated in animals treated with gefitinib compared with controls. Regenerated axons in gefitinib treated animals displayed significantly greater diameter and increased g-ratio compared with controls. Grip strength recovered more quickly in animals receiving gefitinib compared with controls (27.6 vs. 19.1 g 18 days post-injury). This study provides data supporting the role of EGFR as a negative regulator of sensory but not motor neuron regeneration. Further, it demonstrates versatile potential uses of existing pharmaceuticals.

Piezo1 Regulates Stiffness-Dependent DRG Axon Regeneration via Modifying Cytoskeletal Dynamics.

Despite medical interventions, the regenerative capacity of the peripheral nervous system is limited. Dorsal root ganglion (DRG) neurons possess the capacity to detect mechanical signals from their microenvironment, but the impact and mechanism by which these signals regulate axon regrowth and even regeneration in DRG neurons remain unclear. In this study, DRG neurons from newborn rats are cultured on substrates with varying degrees of stiffness in vitro to investigate the role of mechanical signals in axon regrowth. The findings reveal that substrate stiffness plays a crucial role in regulating axon regrowth, with an optimal stiffness required for this process. In addition, the data demonstrate that Piezo1, a mechanosensitive cation channel, detects substrate stiffness at the growth cone and regulates axon regrowth through activating downstream Ca2+-CaMKII-FAK-actin cascade signaling pathway. Interestingly, knocking down Piezo1 in adult rat DRG neurons leads to enhanced axon regeneration and accelerated recovery of sensory function after sciatic nerve injury. Overall, these findings contribute to the understanding of the role of mechanical signals in axon regeneration and highlight microenvironmental stiffness as a promising therapeutic target for repairing nerve injuries.

Pharmacologically enabling the degradation of NaV1.8 channels to reduce neuropathic pain.

In phase II clinical trials, NaV1.8 channels were identified as viable targets to treat acute pain. Results were modest, however, and NaV1.8 pore blockers must be given systemically, potentially leading to adverse effects, especially during prolonged use. A local, long-lasting approach is desirable, yet local anesthetics are neither specific nor long-lasting. In lieu of a pore blocker approach, we show a pharmacological method targeting the scaffolding and degradation of NaV1.8 channels, which attenuated neuropathic pain behavior in mice. NaV1.8 channels interact with the WW domain-containing scaffold protein called Magi-1. WW domains are typically found in ubiquitin ligases, and NaV1.8 channels are susceptible to degradation by ubiquitin ligases. Here, we show NaV1.8 and MAGI-1 colocalized in human tissues. We demonstrate that a lipidated peptide derived from the NaV1.8 WW binding domain, at sub-micromolar concentrations, inhibited rodent dorsal root ganglion neuronal firing. The peptide reduced NaV1.8 channel immunoreactivity and tetrodotoxin-resistant currents in human dorsal root ganglion neurons. We found that the lipidated peptide attenuated neuropathic pain behaviors in mice for multiple weeks after a single injection. Our results reveal that the NaV1.8-targeted lipidated peptide provides local and sustained analgesia, serving as a viable alternative to NaV1.8 pore blockers.

Primary sensory neuron-derived miR-let-7b underlies stress-elicited psoriasis

Psoriasis, a chronic autoimmune skin condition with significant global morbidity, badly impairs patients’ quality of life. Stress has been identified as a prominent trigger for psoriasis, and effectively management of stress can ameliorate its pathological manifestations. However, the precise mechanisms by which stress influences psoriasis remain elusive. In this study, we found that mice subjected to chronic social defeat stress (CSDS) exhibit severer imiquimod (IMQ)-induced psoriasis with increased epidermal scaling, epidermal hyperplasia, number of epidermal ridges, itch, and skin inflammation than control mice. Mechanistic study reveals that CSDS leads to an elevated release of miR-let-7b, an endogenous ligand of Toll-like receptor 7 (TLR7), from the peripheral terminal of dorsal root ganglia (DRG) neurons into the skin. This process can stimulate skin-resident macrophages to release cytokines (such as IL-6 and TNF-a) and chemokines (such as MCP-1), subsequently promoting the recruitment of additional macrophages into the skin. Notably, the specific blockade of miR-let-7b in DRG neurons effectively relieve stress-induced exacerbations of psoriasis. Furthermore, intradermal injection of synthetic miR-let-7b can induce a psoriasis-like phenotype in wildtype mice, a phenomenon that can be countered by the application of a TLR7 antagonist. Additionally, microfluidic chamber coculture assays demonstrated that miR-let-7b released by DRG neurons activates macrophages via TLR7 expressed on these immune cells. Totally, this study found that stress-induced upregulation and release of miR-let-7b from DRG neurons stimulates macrophages to secrete more inflammatory cytokines and chemokines, thereby exacerbating skin inflammation and the psoriatic phenotype. These findings provide a potential therapeutic strategy targeting the miR-let-7b/TLR7 pathway to alleviate stress-induced exacerbation of psoriasis.

Deep RNA sequencing of human dorsal root ganglion neurons reveals somatosensory mechanisms.

Deep RNA sequencing of human dorsal root ganglion neurons reveals somatosensory mechanisms.

Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

BackgroundAbnormal sensory perception, particularly pain insensitivity (PAI), is a typical symptom of autism spectrum disorder (ASD). Despite the role of myelin metabolism in the regulation of pain perception, the mechanisms underlying ASD-related PAI remain unclear.MethodsThe pain-associated gene sphingosine-1-phosphate receptor 1 (S1PR1) was identified in ASD samples through bioinformatics analysis. Its expression in the dorsal root ganglion (DRG) tissues of BTBR ASD model mice was validated using RNA-seq, western blot, RT-qPCR, and immunofluorescence. Pain thresholds were assessed using the von Frey and Hargreaves tests. Patch-clamp techniques measured KCNQ/M channel activity and neuronal action potentials. The expression of S1PR1, KCNQ/M, mitogen-activated protein kinase (MAPK), and cyclic AMP/protein kinase A (cAMP/PKA) signaling proteins was analyzed before and after inhibiting the S1P-S1PR1-KCNQ/M pathway via western blot and RT-qPCR.ResultsThrough integrated transcriptomic analysis of ASD samples, we identified the upregulated gene S1PR1, which is associated with sphingolipid metabolism and linked to pain perception, and confirmed its role in the BTBR mouse model of ASD. This mechanism involves the regulation of KCNQ/M channels in DRG neurons. The enhanced activity of KCNQ/M channels and the decreased action potentials in small and medium DRG neurons were correlated with PAI in a BTBR mouse model of ASD. Inhibition of the S1P/S1PR1 pathway rescued baseline insensitivity to pain by suppressing KCNQ/M channels in DRG neurons, mediated through the MAPK and cAMP/PKA pathways. Investigating the modulation and underlying mechanisms of the non-opioid pathway involving S1PR1 will provide new insights into clinical targeted interventions for PAI in ASD.ConclusionsS1PR1 may contribute to PAI in the PNS in ASD. The mechanism involves KCNQ/M channels and the MAPK and cAMP/PKA signaling pathways. Targeting S1PR1 in the PNS could offer novel therapeutic strategies for the intervention of pain dysesthesias in individuals with ASD.

Nerve injury inhibits Oprd1 and Cnr1 transcription through REST in primary sensory neurons

The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of four opioid receptor genes (Oprm1, Oprd1, Oprl1 and Oprk1) and the cannabinoid CB1 receptor (Cnr1) gene in the DRG regulate nociception. In this study, we determined the role of REST in controlling their expression in the DRG induced by spared nerve injury (SNI). SNI induced chronic pain hypersensitivity in wild-type mice and was accompanied by increased levels of Rest transcript and protein. Transcriptomic analyses of wild-type mouse DRGs suggested that SNI upregulates the expression of Rest transcripts and downregulates the transcripts of all four opioid receptor genes and the Cnr1 gene. Quantitative reverse transcription polymerase chain reaction analyses of these tissues validated these results. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated the presence of REST at these promoters. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in mouse DRG. Our results suggest that nerve injury represses the transcription of at least the Oprd1 and Cnr1 genes via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain. Thus, inhibiting REST activity could potentially reduce chronic neuropathic pain and augment opioid/cannabinoid analgesic actions by increasing the transcription of Oprd1 and Cnr1 genes in DRG neurons.

Leveraging deep single-soma RNA sequencing to explore the neural basis of human somatosensation.

The versatility of somatosensation arises from heterogeneous dorsal root ganglion (DRG) neurons. However, soma transcriptomes of individual human (h)DRG neurons-critical information to decipher their functions-are lacking due to technical difficulties. In this study, we isolated somata from individual hDRG neurons and conducted deep RNA sequencing (RNA-seq) to detect, on average, over 9,000 unique genes per neuron, and we identified 16 neuronal types. These results were corroborated and validated by spatial transcriptomics and RNAscope in situ hybridization. Cross-species analyses revealed divergence among potential pain-sensing neurons and the likely existence of human-specific neuronal types. Molecular-profile-informed microneurography recordings revealed temperature-sensing properties across human sensory afferent types. In summary, by employing single-soma deep RNA-seq and spatial transcriptomics, we generated an hDRG neuron atlas, which provides insights into human somatosensory physiology and serves as a foundation for translational work.

Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System.

Sensory disturbances and central nervous system symptoms are important in patients with Minamata disease. In the peripheral nervous system of these patients, motor nerves are not strongly injured, whereas sensory nerves are predominantly affected. In this study, we investigated the mechanisms underlying the sensory-predominant impairment of the peripheral nervous system caused by methylmercury. We found that the types of cell death in rat dorsal root ganglion (DRG) neurons caused by methylmercury included apoptosis, necrosis, and necroptosis. Methylmercury induced apoptosis in cultured rat DRG neurons but not in anterior horn neurons or Schwann cells. Additionally, methylmercury activated both caspase 8 and caspase 3 in DRG neurons. It increased the expression of tumor necrosis factor (TNF) receptor-1 and the phosphorylation of receptor-interacting protein kinase 3 (RIP3) and mixed-lineage kinase domain-like pseudokinase (MLKL). The expression of TNF-α was increased in macrophage-like RAW264.7 cells by methylmercury. The increase was suggested to be mediated by the NF-κB pathway. Moreover, methylmercury induced neurological symptoms, evaluated by a hindlimb extension response, were significantly less severe in TNF-α knockout mice. Based on these results and our previous studies, we propose the following hypothesis regarding the pathogenesis of sensory nerve-predominant damage by methylmercury: First, methylmercury accumulates within sensory nerve neurons and initiates cell death mechanisms, such as apoptosis, on a small scale. Second, cell death triggers the infiltration of macrophages into the sensory fibers. Third, the macrophages are stimulated by methylmercury and secrete TNF-α through the NF-κB pathway. Fourth, TNF-α induces cell death mechanisms, including necrosis, apoptosis through the caspase 8/3 pathway, and necroptosis through the TNFR1-RIP1-RIP3-MLKL pathway, activated by methylmercury in sensory neurons. Consequently, methylmercury exhibits potent cytotoxicity specific to the DRG/sensory nerve cells in the peripheral nervous system. This chain of events caused by methylmercury may contribute to sensory disturbances in patients with Minamata disease.

MiR-30c-5p Gain and Loss of Function Modulate Sciatic Nerve Injury-Induced Nucleolar Stress Response in Dorsal Root Ganglia Neurons.

Neuropathic pain is a prevalent and debilitating chronic syndrome that is often resistant to treatment. It frequently arises as a consequence of damage to first-order nociceptive neurons in the lumbar dorsal root ganglia (DRG), with chromatolysis being the primary neuropathological response following sciatic nerve injury (SNI). Nevertheless, the function of miRNAs in modulating this chromatolytic response in the context of neuropathic pain remains unexplored. Our previous research demonstrated that the intracisternal administration of a miR-30c mimic accelerates the development of neuropathic pain, whereas the inhibition of miR-30c prevents pain onset and reverses established allodynia. In the present study, we sought to elucidate the role of miR-30c-5p in the pathogenesis of neuropathic pain, with a particular focus on its impact on DRG neurons following SNI. The organisation and ultrastructural changes in DRG neurons, particularly in the protein synthesis machinery, nucleolus, and Cajal bodies (CBs), were analysed. The results demonstrated that the administration of a miR-30c-5p mimic exacerbates chromatolytic damage and nucleolar stress and induces CB depletion in DRG neurons following SNI, whereas the administration of a miR-30c-5p inhibitor alleviates these effects. We proposed that three essential cellular responses-nucleolar stress, CB depletion, and chromatolysis-are the pathological mechanisms in stressed DRG neurons underlying neuropathic pain. Moreover, miR-30c-5p inhibition has a neuroprotective effect by reducing the stress response in DRG neurons, which supports its potential as a therapeutic target for neuropathic pain management. This study emphasises the importance of miR-30c-5p in neuropathic pain pathogenesis and supports further exploration of miRNA-based treatments.

The analgesic effect and mechanism of the active components screening from Corydalis yanhusuo by P2X3 receptors

Ethnopharmacological relevanceCavidine (CAV) is the main bioactive ingredient of Corydalis ternata f. yanhusuo (Y.H.Chou & Chun C.Hsu) Y.C.Zhu, which is a traditional Chinese herbal containing a variety of uses such as analgesic, anticancer, and anti-inflammatory properties. Aim of the studyThe goal is to screen Corydalis yanhusuo for anti-central sensitization active components and investigate and clarify the pharmacological mechanism and therapeutic efficacy of the active ingredient CAV in the treatment of chronic pain. Material and methodsFirst, cell membrane immobilized chromatography was used to screen the bioactive ingredients in Corydalis yanhusuo. Spare nerve injury (SNI) model and complete Freund's adjuvant (CFA) mice model were constructed to identify the analgesic effect of CAV. RNA-seq and bioinformatics analyses were used to explore the potential targets of CAV in CFA mice and SNI mice. HE staining was used to observe the infiltration of inflammatory cells in the dorsal root ganglion (DRG) and spinal cord(SC) of CFA mice and SNI mice. WB and qPCR were used to detect the level of inflammatory factors TNF-α, IL-1β, and IL-6 in DRG and SC of mice. SNI and CFA mice were used to study the effect and mechanism of CAV on microglial activation. Results9 potential active ingredients were screened out from Corydalis yanhusuo that can regulate P2X3 receptors. CAV showed good analgesic effects, increased the mechanical pain and thermal pain thresholds of CFA mice and SNI mice, inhibited the expression of DRG and SC inflammatory factors, downregulated IBA-1, and inhibited microglial activation. Further in vivo and in vitro experiments showed that CAV significantly inhibited the expression of P2X3 receptors and the activation of its downstream MAPK pathway in DRG neurons and SC. ConclusionThis study is the first to indicate that CAV exerts an analgesic effect by inhibiting microglia activation via the P2X3 signaling pathway axis, providing the clinical utility of CAV in chronic pain therapy.

Histone modifications and Sp1 promote GPR160 expression in bone cancer pain within rodent models.

Bone cancer pain (BCP) affects ~70% of patients in advanced stages, primarily due to bone metastasis, presenting a substantial therapeutic challenge. Here, we profile orphan G protein-coupled receptors in the dorsal root ganglia (DRG) following tumor infiltration, and observe a notable increase in GPR160 expression. Elevated Gpr160 mRNA and protein levels persist from postoperative day 6 for over 18 days in the affected DRG, predominantly in small-diameter C-fiber type neurons specific to the tibia. Targeted interventions, including DRG microinjection of siRNA or AAV delivery, mitigate mechanical allodynia, cold, and heat hyperalgesia induced by the tumor. Tumor infiltration increases DRG neuron excitability in wild-type mice, but not in Gpr160 gene knockout mice. Tumor infiltration results in reduced H3K27me3 and increased H3K27ac modifications, enhanced binding of the transcription activator Sp1 to the Gpr160 gene promoter region, and induction of GPR160 expression. Modulating histone-modifying enzymes effectively alleviated pain behavior. Our study delineates a novel mechanism wherein elevated Sp1 levels facilitate Gpr160 gene transcription in nociceptive DRG neurons during BCP in rodents.

Early life cisplatin exposure induces nerve growth factor mediated neuroinflammation and chemotherapy induced neuropathic pain.

Chemotherapy-induced neuropathic pain (CINP) is a common adverse health related comorbidity that manifests later in life in paediatric cancer patients. Current analgesia is ineffective, aligning closely with our lack of understanding of CINP. The aim of this study was to investigate how cisplatin induces nerve growth factor mediated neuroinflammation and nociceptor sensitisation. In a rodent model of cisplatin induced survivorship pain, cisplatin induced a neuroinflammatory environment in the dorsal root ganglia (DRG) demonstrated by nerve growth factor (NGF) positive macrophages infiltrating into the DRG. Cisplatin treated CD11b/F480 positive macrophages expressed more NGF compared to vehicle treated. Primary DRG sensory neuronal cultures demonstrated enhanced NGF-dependent TRPV1 mediated nociceptor activity after cisplatin treatment. Increased nociceptor activity was also observed when cultured DRG neurons were treated with conditioned media from cisplatin activated macrophages. Elevated nociceptor activity was dose-dependently inhibited by a neutralising NGF antibody. Intraperitoneal administration of a NGF neutralising antibody reduced cisplatin-induced mechanical hypersensitivity and aberrant nociceptor intraepidermal nerve fibre density. These findings identify that a monocyte/macrophage driven NGF/TrkA pathway is a novel analgesic target for adult survivors of childhood cancer.

Toll-Like Receptor 4 (TLR4) Promotes DRG Regeneration and Repair after Sciatic Nerve Injury via the ERK-NF-kB Pathway.

Previously, we found that the expression of Toll-like receptor 4 (TLR4) is altered after sciatic nerve injury, and its differential expression plays a key role in recovery. However, the mechanisms by which TLR4 affects neuronal function in the dorsal root ganglion (DRG) have not been completely evaluated. The objective is to determine TLR4 expression in DRG tissues after sciatic neural injury and exploring the effects of TLR4 knockdown and overexpression in the DRG on neuronal function and nerve regeneration in rats in vivo and in vitro. We established a model of nerve injury and utilized molecular biology and cell biology experiments to explore the molecular mechanisms by which TLR4 in the DRG affects sciatic nerve restoration and regeneration after injury. Verified the localization of TLR4 in DRG neurons. Investigated pathways that related to apoptosis or nerve regeneration by which TLR4 regulates the function of DRG neurons. TLR4 expression was upregulated in the DRG tissues of rats after sciatic nerve injury. TLR4 overexpression promoted axon regeneration and inhibited apoptosis in DRG neurons. TLR4 promoted the regeneration of axons and the recovery of motor and sensory functions in the sciatic nerve after injury in vivo, and the data showed that TLR4 may regulate the function of DRG neurons and promote nerve repair and regeneration through the ERK and NF-κB signaling pathways in vivo and ex vivo. The study suggests that TLR4 may regulate the function of DRG neurons and promote nerve regeneration by affecting the ERK and NF-κB signaling pathways.

Elucidation of Medusozoan (Jellyfish) Venom Constituent Activities Using Constellation Pharmacology.

Within the phylum Cnidaria, sea anemones (class Anthozoa) express a rich diversity of ion-channel peptide modulators with biomedical applications, but corollary discoveries from jellyfish (subphylum Medusozoa) are lacking. To bridge this gap, bioactivities of previously unexplored proteinaceous and small molecular weight (~15 kDa to 5 kDa) venom components were assessed in a mouse dorsal root ganglia (DRG) high-content calcium-imaging assay, known as constellation pharmacology. While the addition of crude venom led to nonspecific cell death and Fura-2 signal leakage due to pore-forming activity, purified small molecular weight fractions of venom demonstrated three main, concentration-dependent and reversible effects on defined heterogeneous cell types found in the primary cultures of mouse DRG. These three phenotypic responses are herein referred to as phenotype A, B and C: excitatory amplification (A) or inhibition (B) of KCl-induced calcium signals, and test compound-induced disturbances to baseline calcium levels (C). Most notably, certain Alatina alata venom fractions showed phenotype A effects in all DRG neurons; Physalia physalis and Chironex fleckeri fractions predominantly showed phenotype B effects in small- and medium-diameter neurons. Finally, specific Physalia physalis and Alatina alata venom components induced direct excitatory responses (phenotype C) in glial cells. These findings demonstrate a diversity of neuroactive compounds in jellyfish venom potentially targeting a constellation of ion channels and ligand-gated receptors with broad physiological implications.

Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-β (TGF-β) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.