Rat Dorsal Root Ganglion Research Articles

STING-IFN-I pathway relieves incision induced acute postoperative pain via inhibiting the neuroinflammation in dorsal root ganglion of rats.

The purpose of this study was to study the effect of STING-IFN-I pathway on incision induced postoperative pain in rats and its possible mechanisms. The pain thresholds were evaluated by measuring the mechanical withdrawal threshold and the thermal withdrawal latency. The satellite glial cell and macrophage of DRG were analyzed. The expression of STING, IFN-a, P-P65, iNOS, TNF-α, IL-1β and IL-6 in DRG was evaluated. The activation of STING-IFN-I pathway can reduce the mechanical hyperalgesia, thermal hyperalgesia, down-regulate the expression of P-P65, iNOS, TNF-α, IL-1β and IL-6, and inhibit the activation of satellite glial cell and macrophage in DRG. The activation of STING-IFN-I pathway can alleviate incision induced acute postoperative pain by inhibiting the activation of satellite glial cell and macrophage, which reducing the corresponding neuroinflammation in DRG.

Members of the CUGBP Elav-like family of RNA-binding proteins are expressed in distinct populations of primary sensory neurons.

Primary sensory dorsal root ganglia (DRG) neurons are diverse, with distinct populations that respond to specific stimuli. Previously, we observed that functionally distinct populations of DRG neurons express mRNA transcript variants with different 3' untranslated regions (3'UTRs). 3'UTRs harbor binding sites for interaction with RNA-binding proteins (RBPs) for transporting mRNAs to subcellular domains, modulating transcript stability, and regulating the rate of translation. In the current study, analysis of publicly available single-cell RNA-sequencing data generated from adult mice revealed that 17 3'UTR-binding RBPs were enriched in specific populations of DRG neurons. This included four members of the CUG triplet repeat (CUGBP) Elav-like family (CELF): CELF2 and CELF4 were enriched in peptidergic, CELF6 in both peptidergic and nonpeptidergic, and CELF3 in tyrosine hydroxylase-expressing neurons. Immunofluorescence studies confirmed that 60% of CELF4+ neurons are small-diameter C fibers and 33% medium-diameter myelinated (likely Aδ) fibers and showed that CELF4 is distributed to peripheral termini. Coexpression analyses using transcriptomic data and immunofluorescence revealed that CELF4 is enriched in nociceptive neurons that express GFRA3, CGRP, and the capsaicin receptor TRPV1. Reanalysis of published transcriptomic data from macaque DRG revealed a highly similar distribution of CELF members, and reanalysis of single-nucleus RNA-sequencing data derived from mouse and rat DRG after sciatic injury revealed differential expression of CELFs in specific populations of sensory neurons. We propose that CELF RBPs may regulate the fate of mRNAs in populations of nociceptors, and may play a role in pain and/or neuronal regeneration following nerve injury.

Zhiqiao Gancao Decoction Ameliorates Hyperalgesia in Lumbar Disc Herniation via the CCL2/CCR2 Signaling Pathway.

The aim of this study was to investigate the effect of Zhiqiao Gancao decoction (ZQGCD) on hyperalgesia in lumbar disc herniation (LDH) and its mechanism. The potential mechanism of ZQGCD's therapeutic effect on LDH was investigated through network pharmacology, which involved screening the targets of eight components that were absorbed into the bloodstream. The effects of CCR2 inhibitors and ZQGCD-containing serum on the excitability of the CCL2/CCR2 signaling pathway and dorsal root ganglion neurons (DRGn) were investigated in vitro. The effects of CCR2 inhibitors and ZQGCD on the expression of the CCL2/CCR2 signaling pathway and ASIC3 in the rat intervertebral disc and dorsal root ganglion (DRG), the degree of disc degeneration, the threshold of foot retreat, and the latency of foot retreat in LDH rats were examined in vivo. The binding affinities and interaction modes between CCR2 and the components absorbed into the blood were analyzed using the AutodockVina 1.2.2 software. Network pharmacology revealed that ZQGCD could treat LDH through a mechanism involving the chemokine signaling pathway. It was observed that the CCR2 inhibitor and ZQGCD-containing serum downregulated CCR2 and ASIC3 expression and decreased cell excitability in DRGn. The CCL2/CCR2 signaling pathway was activated in the degenerated intervertebral disc and DRG of LDH rats, increased the expression of ASIC3, and decreased the mechanical allodynia domain and thermal hyperalgesia domain. However, a CCR2 inhibitor or ZQGCD could ameliorate the above changes in LDH rats. The target proteins, CCL2 and CCR2, exhibited a robust affinity for the eight components that were absorbed into the bloodstream. The CCL2/CCR2 pathway was activated in the intervertebral disc and DRG of LDH rats. This was accompanied by upregulation of ASIC3 expression, increased excitability of DRGn, and the occurrence of hyperalgesia. ZQGCD improves hyperalgesia in LDH rats by inhibiting the CCL2/CCR2 pathway and downregulating ASIC3 expression.

Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program.

The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However, extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of α9 integrin and its activator kindlin-1 (α9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of adult male rat DRG sensory neurons transduced with α9k1, and controls, with and without axotomy of the central branch. Expression of α9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling α9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration. In addition to the program upregulated by α9k1 expression, regeneration in the spinal cord led to expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum (ER), trafficking, and signaling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPSC-derived sensory neurons, validating their causal contributions to sensory regeneration. This CNS regeneration-associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, and Yy1. Signaling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with an additional distinctive program that differs from that involved in PNS regeneration.SIGNIFICANCE STATEMENT Restoration of neurologic function after spinal cord injury has yet to be achieved in human patients. To accomplish this, severed nerve fibers must be made to regenerate. Reconstruction of nerve pathways has not been possible, but recently, a method for stimulating long-distance axon regeneration of sensory fibers in rodents has been developed. This research uses profiling of messenger RNAs in the regenerating sensory neurons to discover which mechanisms are activated. This study shows that the regenerating neurons initiate a novel CNS regeneration program which includes molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum (ER). The study identifies mechanisms that neurons need to activate to regenerate their nerve fibers.

A phase Ib adaptive study of dasatinib for the prevention of oxaliplatin-induced neuropathy in patients with gastrointestinal (GI) cancers receiving FOLFOX chemotherapy with or without bevacizumab.

e15613 Background: Neurotoxicity is one of the most significant and disabling side effects of oxaliplatin (OX) and frequently limits the amount of OX that can be used. OX uptake by organic cation transporter 2 (OCT2) into mouse and rat dorsal root ganglia is a prerequisite for OX-induced peripheral neuropathy (OIPN). Preclinical data in rat and mouse models from our group showed that by inhibiting YES1, pre-treatment with dasatinib (D) inactivated OCT2 and prevented acute and chronic OIPN. A phase I trial is ongoing investigating addition of D prior to OX to prevent OIPN. Methods: This is a Bayesian Phase 1b dose-finding study with adaptive dose selection using efficacy-toxicity trade-offs in patients (pts) with GI cancers who are candidates for mFOLFOX6 ± bevacizumab [NCT04164069]. Pts received chemotherapy on day 1 and 15 schedule, every 28 days. D was administered at one of 2 dose levels – 100mg or 140mg PO, given 24 hours and 30 mins prior to OX on C1D14, C1D15 respectively and repeated on C1D28 and C2D1. During cycle 1, pharmacokinetic and pharmacodynamic data was obtained at baseline, during, and up to 24 hours after D or OX administration. Recommended Phase 2 dose (RP2D) was defined as the lowest intermittent dose of D that increases serum biomarkers of OCT2, including methylnicotinamide (NMN) and creatinine, by ≥2-fold without influencing the clearance of oxaliplatin by ≥20%, toxicity profile was evaluated based on CTCAE v. 5.0. Results: 11 pts were screened. 5 pts enrolled in cohort 1 (D 100 mg) and 2 in cohort 2 (D 140 mg). In this preliminary analysis, 5 patients were evaluated for efficacy biomarkers and 4 pts were evaluated for both efficacy biomarkers and toxicity. There were no DLTs observed in either cohort. 3/5 pts had ≥2-fold increase in AUC of NMN, and 4/5 pts had ≥2-fold increase in AUC of creatinine. 4/5 pts had ≤20% change in the clearance of OX. Conclusions: Addition of D 140 mg to traditional FOLFOX regimen was safe and resulted in increasing levels of biomarkers as indication of inhibition of OCT2 in comparison to baseline for the majority of pts enrolled. Enrollment and analysis are ongoing at D 140mg dose to confirm the RP2D to prevent OPIN in a future randomized phase II study. Clinical trial information: NCT04164069 . [Table: see text]

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 μM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Reduction of SIRT1-Mediated Epigenetic Upregulation of Nav1.7 Contributes to Oxaliplatin-Induced Neuropathic Pain

BACKGROUND: Clinically, neuropathic pain is a severe side effect of oxaliplatin chemotherapy, which usually leads to dose reduction or cessation of treatment. Due to the unawareness of detailed mechanisms of oxaliplatin-induced neuropathic pain, it is difficult to develop an effective therapy and limits its clinical use. OBJECTIVES: The aim of the present study was to identify the role of sirtuin 1 (SIRT1) reduction in epigenetic regulation of the expression of voltage-gated sodium channels 1.7 (Nav1.7) in the dorsal root ganglion (DRG) during oxaliplatin-induced neuropathic pain. STUDY DESIGN: Controlled animal study. SETTING: University laboratory. METHODS: The von Frey test was performed to evaluate pain behavior in rats. Real-time quantitative polymerase chain reaction, western blotting, electrophysiological recording, chromatin immunoprecipitation, and small interfering RNA (siRNA) were used to illustrate the mechanisms. RESULTS: In the present study, we found that both the activity and expression of SIRT1 were significantly decreased in rat DRG following oxaliplatin treatment. The activator of SIRT1, resveratrol, not only increased the activity and expression of SIRT1, but also attenuated the mechanical allodynia following oxaliplatin treatment. In addition, local knockdown of SIRT1 by intrathecal injection of SIRT1 siRNA caused mechanical allodynia in naive rats. Besides, oxaliplatin treatment enhanced the action potential firing frequency of DRG neurons and the expression of Nav1.7 in DRG and activation of SIRT1 by resveratrol reversed this effect. Furthermore, blocking Nav1.7 by ProTx II (a selective Nav1.7 channel blocker) reversed oxaliplatin-induced mechanical allodynia. In addition, histone H3 hyperacetylation at the Nav1.7 promoter in DRG of rats following oxaliplatin treatment was significantly suppressed by activation of SIRT1 with resveratrol. Moreover, both the expression of Nav1.7 and histone H3 acetylation at the Nav1.7 promoter were upregulated in the DRG by local knockdown of SIRT1 with SIRT1 siRNA in naive rats. LIMITATIONS: More underlying mechanism(s) of SIRT1 reduction after oxaliplatin treatment needs to be explored in future research. CONCLUSIONS: These findings suggest that reduction of SIRT1-mediated epigenetic upregulation of Nav1.7 in the DRG contributes to the development of oxaliplatin-induced neuropathic pain in rats. The intrathecal drug delivery treatment of activating SIRT1 might be a novel therapeutic option for oxaliplatin-induced neuropathic pain. KEY WORDS: SIRT1, neuropathic pain, dorsal root ganglion, NAV1.7 voltage-gated sodium channel, oxaliplatin

Peripheral sensory neurons and non-neuronal cells express functional Piezo1 channels.

Here, we present evidence showing Piezo1 protein expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in ∼60% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons. Piezo1-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by non-specific Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yoda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity induced by Yoda1. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury appeared associated with activation of Piezo1 in PSNs and peripheral glial cells.

Argentatin C Analogues with Potential Antinociceptive Activity and Other Triterpenoid Constituents from the Aerial Parts of Parthenium incanum.

Four new triterpenes, 25-dehydroxy-25-methoxyargentatin C (1), 20S-hydroxyargentatin C (2), 20S-hydroxyisoargentatin C (3), and 24-epi-argentatin C (4), together with 10 known triterpenes (5-14) were isolated from the aerial parts of Parthenium incanum. The structures of 1-4 were elucidated by detailed analysis of their spectroscopic data, and the known compounds 5-14 were identified by comparison of their spectroscopic data with those reported. Since argentatin C (11) was found to exhibit antinociceptive activity by decreasing the excitability of rat and macaque dorsal root ganglia (DRG) neurons, 11 and its new analogues 1-4 were evaluated for their ability to decrease the excitability of rat DRG neurons. Of the argentatin C analogues tested, 25-dehydroxy-25-methoxyargentatin C (1) and 24-epi-argentatin C (4) decreased neuronal excitability in a manner comparable to 11. Preliminary structure-activity relationships for the action potential-reducing effects of argentatin C (11) and its analogues 1-4, and their predicted binding sites in pain-relevant voltage-gated sodium and calcium channels (VGSCs and VGCCs) in DRG neurons are presented.

Transcriptome analysis reveals dysregulation of inflammatory and neuronal function in dorsal root ganglion of paclitaxel-induced peripheral neuropathy rats.

Chemotherapy-induced peripheral neuropathy (CIPN) is the most common side-effect of anti-cancer therapy. To date, there are no clinically effective analgesics that could prevent and treat CIPN. However, the exact pathogenesis of CIPN is still unclear. In the present study, we use the paclitaxel-induced peripheral neuropathy (PIPN) model, aiming to better understand the transcriptomic level of the Dorsal root ganglia (DRG) neurons in rats with PIPN. mRNA from each DRG sample was reverse transcribed to cDNA and sequenced using next-generation high throughput sequencing technology. Quantitative RT-PCR verification was used to confirm the identified Differentially expressed genes (DEGs) in the DRG of PIPN rats. RNAseq results have identified 384 DEGs (adjusted P-value < 0.05; fold change ≥ 2) in the DRG of rats 14days after paclitaxel injection in total, including 97 up-regulated genes, and 287 down-regulated genes. GO analysis revealed that these DEGs were majorly involved in neuropeptide activity, chemokine receptor activity, defense response, and inflammatory response. Kyoto Encyclopedia of Gene and Genomes analysis showed that neuroactive ligand-receptor interaction and cytokine-cytokine receptor interaction were involved in sensory neurons of rats with PIPN. Besides, comparison analysis identified that 11 DEGs in the PIPN model are shared with either inflammatory pain (Ces1d, Cfd, Retn, and Fam150b) or neuropathic pain (Atf3, Csrp3, Ecel1, Gal, Sprr1a, Tgm1, and Vip). Quantitative RT-PCR results also confirmed the validation of the RNAseq data. These results suggested that neuroactive ligand-receptor interaction and cytokine-cytokine receptor interaction are majorly involved in sensory neurons of rats with PIPN. Immune, inflammatory responses and neuron functional changes are the major pathogenesis of PIPN. Paclitaxel-induced peripheral neuropathy has shared characteristics with both inflammatory pain and neuropathic pain.

MiR-140 and miR-200 regulate the migratory heterogeneity of location-specific Schwann cell population.

Schwann cells are functional cells in nerve regeneration and are commonly used as seed cells in tissue engineering. Enhanced Schwann cell migration capacity improves recovery effects, and thus, the identification of Schwann cells with greater migration ability is of great importance. In the present study, we examined the biological activities of Schwann cells collected from rat sciatic nerves (SN) and dorsal root ganglia (DRG). Observations from transwell migration assay and wound healing assay demonstrate that DRG Schwann cells migrate at a faster speed as compared with SN Schwann cells. Sequencing and bioinformatics suggest that differentially expressed genes between SN and DRG Schwann cells are associated with cell motility and migration. miR-140 and miR-200, two microRNAs (miRNAs) that are highly expressed in SN Schwann cells negatively influence Schwann cell migration and thus may be key regulators of Schwann cell phenotype. Igsf10, Plxna2, and Lcp1 are screened as candidate downstream targets of miR-140 and miR-200 based on bioinformatic analysis and their expression correlation with miRNAs. Our comparative analysis displays the unique characteristics of Schwann cells in different anatomical localizations and demonstrates that DRG Schwann cells are suitable seed cells for tissue engineering and regenerative medicine.

Modulation of G-protein activation, calcium currents and opioid receptor phosphorylation by the pH-dependent antinociceptive agonist NFEPP.

N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide is a newly-designed pain killer selectively activating G-protein-coupled mu-opioid receptors (MOR) in acidic injured tissues, and therefore devoid of central side effects which are typically elicited at normal pH values in healthy tissues. However, the neuronal mechanisms underlying NFEPP's antinociceptive effects were not examined in detail so far. Voltage-dependent Ca2+ channels (VDCCs) in nociceptive neurons play a major role in the generation and inhibition of pain. In this study, we focused on the effects of NFEPP on calcium currents in rat dorsal root ganglion (DRG) neurons. The inhibitory role of the G-protein subunits Gi/o and Gβγ on VDCCs was investigated using the blockers pertussis toxin and gallein, respectively. GTPγS binding, calcium signals and MOR phosphorylation were also investigated. All experiments were performed at acidic and normal pH values using NFEPP in comparison to the conventional opioid agonist fentanyl. At low pH, NFEPP produced more efficient G-protein activation in transfected HEK293 cells and significantly reduced VDCCs in depolarized DRG neurons. The latter effect was mediated by Gβγ subunits, and NFEPP-mediated MOR phosphorylation was pH-dependent. Fentanyl's responses were not affected by pH changes. Our data indicate that NFEPP-induced MOR signaling is more effective at low pH and that the inhibition of calcium channels in DRG neurons underlies NFEPP's antinociceptive actions.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats

BackgroundBone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. MethodsWalker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). ResultsThe PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. ConclusionThese results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

Simple methods of dissection protocols for the rapid isolation of rat dorsal root ganglia under the non-microscopic condition

The dorsal root ganglia (DRG) neurons have long been studied to advance the understanding of sensory nerve function and growth. DRG neurons are also involved in determining the fundamental mechanisms of neurodegenerative and painful disorders of the peripheral nervous system. To explore the simple methods of rapid and accurate removal of DRG in anesthetized rats with the naked eye without destroying the spinal column or blood circulation. Male Wistar rats were fixed on the operating table, lumbar DRG was removed, and sufficient DRG neurons were harvested without microscopic equipment. At the same time, the procedure was accomplished without disruption to breathing and circulation. These approaches reduce the time required for DRG collection, thereby improving efficiency, and increasing the chances of generating healthy primary DRG cultures and reproducible experiments using DRG tissue.

Promising application of a novel biomaterial, light chain of silk fibroin combined with NT3, in repairment of rat sciatic nerve defect injury

Autologous nerve transplantation is the gold standard for treating peripheral nerve defects, but it is associated with defects such as insufficient donor and secondary injury. Artificial nerve guidance conduits (NGCs) are now considered promising alternatives for bridging long nerve gaps, although exploring new biomaterials to construct NGCs remains challenging. Silk fibroin (SF) has good biocompatibility and can self-assemble in aqueous solutions. However, the lack of proximal neurotrophic factors after nerve injury is a major concern, leading to incomplete nerve regeneration. In this study, NT-3, a neurotrophin that promotes neuronal survival and differentiation, was bound to the light chain of silk fibroin (FIBL) in two ways: one was directly bound to FIBL (FIBL-NT3) and the other was a polypeptides-linker (FIBL-Linker-NT3). The design aimed to take advantage of silk fiber's character of self-assembly of heavy-light chains and test whether a flexible linker with NT3 molecule is easy to be a NT3 dimer, the active form. In vitro studies indicated that FIBL-Linker-NT3 combined with SF membranes promoted axon growth in adult rat dorsal root ganglion (DRG) neurons. Then we tested if FIBL-Linker-NT3 could self-assemble with the SF heavy chain (SFH). DTT (Dithiothreitol) was used to break the disulfide bonds between the SF light and heavy chains, and the light-chain protein was removed via dialysis. SFH was assembled using FIBL-Linker-NT3, as evidenced by the western blotting results that showed a high molecular band corresponding to SFH-FIBL-Linker-NT3. Chitosan scaffolds have been identified to provide a suitable microenvironment, so a chitosan/SF-FIBL-Linker-NT3 conduit was also constructed. Nerve transplantation of this conduit was evaluated in vivo in a rat sciatic nerve defect model. Immunohistochemical assays showed that the chitosan/SF-FIBL-Linker-NT3 group was superior to the chitosan/PBS, SF, PBS + FIBL-Linker-NT3 groups in nerve regeneration. In addition, the chitosan/SF-FIBL-Linker-NT3 conduit-transplanted group exhibited better recovery in terms of neurite length, sciatic functional index value, sensitivity to heat, time on the rotarod, wet weight ratio, cross-sectional area, compound muscle action potential, number of myelin layers, and myelin thickness in the nerve. Taking together, our study identified that FIBL-Linker-NT3 could promote axonal growth and regeneration in vivo and in vitro and is a promising candidate biomaterial for artificial NGCs.

TET1-TRPV4 Signaling Contributes to Bone Cancer Pain in Rats

Bone cancer pain (BCP) is excruciating for cancer patients, with limited clinical treatment options and significant side effects, due to the complex and unclear pathogenesis of bone cancer pain. Peripheral sensitization in dorsal root ganglion (DRG) neurons is a recognized cellular mechanism for bone cancer pain. The pathological mechanism of chronic pain is increasingly being affected by epigenetic mechanisms. In this study, we unbiasedly showed that the DNA hydroxymethylase ten-eleven translocation 1 (TET1) expression was significantly increased in the L4-6 DRG of BCP rats and ten-eleven translocation 2 (TET2) expression did not change significantly. Notably, TET1 inhibition by intrathecal injection of Bobcat339 (a TET1 inhibitor) effectively relieved mechanical hyperalgesia in BCP rats. Peripheral sensitization in chronic pain relies on the activation and overexpression of ion channels on neurons. Here, we demonstrated that TRPV4, one of the transient receptor potential ion channel family members, was significantly elevated in the L4-6 DRG of BCP rats. In addition, TRPV4 inhibition by intrathecal injection of HC067047 (a TRPV4 inhibitor) also significantly attenuated mechanical hyperalgesia in BCP rats. Interestingly, we found that TET1 inhibition downregulated TRPV4 expression in the L4-6 DRG of BCP rats. As a result, these findings suggested that TET1 may contribute to bone cancer pain by upregulating TRPV4 expression in the L4-6 DRG of BCP rats and that TET1 or TRPV4 may become therapeutic targets for bone cancer pain.

Endogenous Glycoprotein GPM6a Is Involved in Neurite Outgrowth in Rat Dorsal Root Ganglion Neurons.

The peripheral nervous system (PNS) has a unique ability for self-repair. Dorsal root ganglion (DRG) neurons regulate the expression of different molecules, such as neurotrophins and their receptors, to promote axon regeneration after injury. However, the molecular players driving axonal regrowth need to be better defined. The membrane glycoprotein GPM6a has been described to contribute to neuronal development and structural plasticity in central-nervous-system neurons. Recent evidence indicates that GPM6a interacts with molecules from the PNS, although its role in DRG neurons remains unknown. Here, we characterized the expression of GPM6a in embryonic and adult DRGs by combining analysis of public RNA-seq datasets with immunochemical approaches utilizing cultures of rat DRG explants and dissociated neuronal cells. M6a was detected on the cell surfaces of DRG neurons throughout development. Moreover, GPM6a was required for DRG neurite elongation in vitro. In summary, we provide evidence on GPM6a being present in DRG neurons for the first time. Data from our functional experiments support the idea that GPM6a could contribute to axon regeneration in the PNS.

Antagonism of TRPV4 channels partially reduces mechanotransduction in rat skeletal muscle afferents.

Mechanical distortion of working skeletal muscle induces sympathoexcitation via thin fibre afferents, a reflex response known as the skeletal muscle mechanoreflex. However, to date, the receptor ion channels responsible for mechanotransduction in skeletal muscle remain largely undetermined. Transient receptor potential vanilloid 4 (TRPV4) is known to sense mechanical stimuli such as shear stress or osmotic pressure in various organs. It is hypothesized that TRPV4 in thin-fibre primary afferents innervating skeletal muscle is involved in mechanotransduction. Fluorescence immunostaining revealed that 20.1 ± 10.1% of TRPV4 positive neurons were small dorsal root ganglion (DRG) neurons that were DiI-labelled, and among them 9.5 ± 6.1% of TRPV4 co-localized with the C-fibre marker peripherin. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons demonstrated that mechanically activated current amplitude was significantly attenuated after the application of the TRPV4 antagonist HC067047 compared to control (P=0.004). Such reductions were also observed in single-fibre recordings from a muscle-nerve ex vivo preparation where HC067047significantly decreased afferent discharge to mechanical stimulation (P=0.007). Likewise, in an in vivo decerebrate rat preparation, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch of hindlimb muscle were significantly reduced by intra-arterial injection of HC067047 (ΔRSNA: P=0.019, ΔMAP: P=0.002). The findings suggest that TRPV4 plays an important role in mechanotransduction contributing to the cardiovascular responses evoked by the skeletal muscle mechanoreflex during exercise. KEY POINTS: Although a mechanical stimulus to skeletal muscle reflexively activates the sympathetic nervous system, the receptors responsible for mechanotransduction in skeletal muscle thin fibre afferents have not been fully identified. Evidence suggests that TRPV4 is a mechanosensitive channel that plays an important role in mechanotransduction within various organs. Immunocytochemical staining demonstrates that TRPV4 is expressed in group IV skeletal muscle afferents. In addition, we show that the TRPV4 antagonist HC067047 decreases the responsiveness of thin fibre afferents to mechanical stimulation at the muscle tissue level as well as at the level of dorsal root ganglion neurons. Moreover, we demonstrate that intra-arterial HC067047 injection attenuates the sympathetic and pressor responses to passive muscle stretch in decerebrate rats. These data suggest that antagonism of TRPV4 attenuates mechanotransduction in skeletal muscle afferents. The present study demonstrates a probable physiological role for TRPV4 in the regulation of mechanical sensation in somatosensory thin fibre muscle afferents.

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

Apitoxin alleviates methyl mercury-induced peripheral neurotoxicity in male rats by regulating dorsal root ganglia neuronal degeneration and oxidative stress

Methylmercury (MeHg) toxicity is associated with extensive neuronal degeneration of dorsal root ganglia (DRG). This study aimed to assess the ameliorative effect of bee venom (BV) on methyl mercury chloride (MeHgCl)-induced peripheral neurotoxicity using DRGs in rats. Forty-eight adult male Sprague Dawley rats were allocated into four equal groups: G I: control (gavaged MilliQ water 1 ml/rat), G II: subcutaneously injected with BV (0.5 mg/kg b.wt), G III: gavaged MeHgCl (6.7 mg/kg b.wt), and G IV: received MeHgCl+BV. Dosing was done five times/week for 2 weeks. Ataxic behavior and visual impairments were significantly increased, whereas the movement behavior and motility gait were suppressed in the MeHgCl group. MeHgCl significantly decreased total antioxidant capacity (TAC) in DRG and significantly decreased the serum levels of glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β) levels were significantly elevated, whereas interleukin 10 (IL-10) levels were significantly decreased in the MeHgCl group compared with the control group. DRGs of the MeHgCl-exposed rats showed pyknotic shrunken neurons with perineural vacuolations, demyelination of nerve axons, and proliferation of the satellite cells. MeHgCl significantly induced a higher positive index ratio of Iba-1, SOX10, neurofilament, pan-neuron, and vimentin immunostaining in the DRG. BV administration significantly mitigated the MeHgCl-induced alterations in oxidative stress-related indices. BV modified the immunostaining of Iba-1, SOX10, neurofilament, pan-neuron, and vimentin-positive index ratio in the DRG of the MeHgCl group. Our findings acknowledged that BV could enhance in vivo neuroprotective effects against MeHgCl-induced DRGs damage in male rats.