Search Results (28)

The mineralocorticoid receptor (MR), traditionally associated with renal function, has also been identified in various extrarenal tissues, including the heart, brain, and dorsal root ganglion (DRG) neurons in rodents. Previous studies suggest a role for the MR in modulating peripheral nociception, with MR activation in rat DRG neurons by its endogenous ligand, aldosterone. This study aimed to determine whether MR, its protective enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), its endogenous ligand aldosterone, and the aldosterone-synthesizing enzyme CYP11B2 are expressed in human DRG neurons and whether they colocalize with key pain-associated signaling molecules as potential targets for genomic regulation. To this end, we performed mRNA transcript profiling and immunofluorescence confocal microscopy on human and rat DRG tissues. We detected mRNA transcripts for MR, 11β-HSD2, and CYP11B2 in human DRG, alongside transcripts for key thermosensitive and nociceptive markers such as TRPV1, the TTX-resistant sodium channel Nav1.8, and the neuropeptides CGRP and substance P (Tac1). Immunofluorescence analysis revealed substantial colocalization of MR with 11β-HSD2 and CGRP, a marker of unmyelinated C-fibers and thinly myelinated Aδ-fibers, in human DRG. MR immunoreactivity was primarily restricted to small- and medium-diameter neurons, with lower expression in large neurons (>70 µm). Similarly, aldosterone colocalized with CYP11B2 and MR with nociceptive markers including TRPV1, Nav1.8, and TrkA in human DRG. Importantly, functional studies demonstrated that prolonged intrathecal inhibition of aldosterone synthesis within rat DRG neurons, using an aldosterone synthase inhibitor significantly downregulated pain-associated molecules and led to sustained attenuation of inflammation-induced hyperalgesia. Together, these findings identify a conserved peripheral MR signaling axis in humans and highlight its potential as a novel target for pain modulation therapies. Full article

Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale studies. Therefore, standardized in vitro systems are needed to facilitate efficient and reliable functional analyses of genes identified in such studies. Here, we describe a practical and efficient method for oligodendrocyte precursor cell (OPC) isolation from mouse brains on postnatal day 6–8 and a gene delivery method for the isolated OPCs. By modifying the magnetic-activated cell sorting (MACS) procedure with reduced processing volumes, we simplified OPC isolation, allowing simultaneous handling of multiple samples and improving workflow efficiency. We also optimized electroporation parameters to achieve robust transfection efficiency with minimal cell death. Transfected OPCs are suitable for both monoculture-based differentiation assays and co-culture with dorsal root ganglion (DRG) explants, in which they reliably differentiate into mature oligodendrocytes and myelinate along the axons. This system enables stable and reproducible in vitro analysis of oligodendrocyte function, supports investigations into both intrinsic differentiation and neuron–glia interactions, and provides a powerful platform for oligodendrocyte research with efficient and timely gene manipulation. Full article

The presence of dichotomizing neurons in the dorsal root ganglia (DRG) of cattle, innervating both the reticulum and the withers, may indicate a pre-spinal convergence of visceral and cutaneous sensory information, i.e., that the DRG primary sensory neurons may elaborate the sensory information coming from two different anatomical areas before reaching the secondary sensory neurons within the spinal cord. This anatomical feature could be the underlying basis for the cutaneous allodynia observed in traumatic reticuloperitonitis, also known as the “Kalchschmidt pain test”. The aim of the study was to identify the DRG primary sensory neurons innervating the reticulum and the withers by using two different retrograde fluorescent tracers, Fast Blue (FB, affinity for cytoplasm) and Diamidino Yellow (DY, affinity for nucleus). In two anesthetized calves, FB and DY were injected into the reticulum and skin of the withers, respectively. At the end of the experimental period, the calves were deeply anesthetized and then euthanatized. The thoracic (T1–T8) DRG were collected and processed to obtain cryosections which were examined on a fluorescent microscope. A large number of neurons localized, especially in the T7 DRG, presented nuclei labeled with DY. On the contrary, only a few neurons localized exclusively in T6 and T7 DRG presented the cytoplasm labeled with FB. No neurons displayed FB and DY simultaneously within the cytoplasm and nucleus, respectively. The absence of double-labeled DRG neurons suggests that the convergence of visceral and somatic sensory inputs underlying the Kalchschmidt pain response likely does not occur at the level of individual DRG neurons. Rather, it may involve higher-order integrative centers, possibly including vagal pathways and brainstem nuclei which integrate the afferent information to coordinate respiratory movements of the diaphragm, intercostal muscles, and larynx. Although limited by the sample size, this case study provides a neuroanatomical basis for further investigation into central mechanisms of referred visceral pain in cattle. Full article

Background: A large number of patients suffer from neuropathic pain, and systemic therapy often remains ineffective while inducing severe side effects. Topical therapy with the TRPV1-agonist capsaicin is an established alternative, and the identification of co-therapeutics that modulate TRPV1 may be a promising approach to reduce the dose of capsaicin while maintaining efficacy. Here, we aimed to determine if the antidepressant amitriptyline displays properties rendering it a potential co-therapeutic agent. Methods: We performed patch clamp and calcium imaging experiments on HEK293T cells expressing human (h) TRPV1 as well as on dorsal root ganglion (DRG) neurons from adult mice. Results: Amitriptyline induced an increase in intracellular calcium in both HEK293T and mouse DRG neurons expressing TRPV1. Patch clamp experiments revealed a concentration-dependent activation of hTRPV1 by amitriptyline that was also evident in cell-free inside-out patches. When hTRPV1 was fully activated by capsaicin, amitriptyline induced concentration-dependent and partly reversible current inhibition. In contrast, amitriptyline potentiated small responses to capsaicin, heat and protons. We also found that amitriptyline desensitized hTRPV1 to capsaicin. This effect was reduced by the intracellular application of the strong calcium chelator BAPTA. Furthermore, the non-desensitizing mutant hTRPV1-Y672K displayed a reduced amitriptyline-induced desensitization. Conclusions: Our data showed that amitriptyline can activate, sensitize, desensitize and even inhibit TRPV1. Together with its property as a strong local anesthetic, our data suggest that amitriptyline may be a promising adjunct to topical capsaicin. Full article

Background: Peripheral nerves have a certain regenerative ability, but their repair and regeneration after injury is a complex process, usually involving a large number of genes and proteins. In a previous study, we analyzed the gene expression profile in rats after sciatic nerve injury and found significant changes in transforming growth factor-beta 1 (TGF-β1) expression, suggesting that TGF-β1 may be involved in the process of nerve regeneration after injury. Methods: In this study, we first detected the time-course expression and localization of TGF-β1 in dorsal root ganglion (DRG) tissues in a rat sciatic nerve transection model via RT-qPCR. Secondly, we investigated the bioactive roles of TGF-β1 in primary cultured DRG neuron cells through a CCK8 assay, TUNEL assay, and immunofluorescence staining. Thirdly, we explored the neuroprotective roles of TGF-β1 in an in vivo model of sciatic nerve regeneration through morphological observation, behavioral, and electrophysiological tests, and a molecular biological measure. Results: We found that TGF-β1 expression was increased after injury and mainly located in the cytoplasm and nuclei of neuron cells in the DRG. TGF-β1 may regulate the viability, apoptosis, and neurite outgrowth of primary DRG neuron cells. In our in vivo model of sciatic nerve regeneration, TGF-β1 improved nerve regeneration and neuronal function recovery after sciatic nerve injury, alleviated the inflammatory response, and relieved neuropathic pain via the TGF-β1/smad2 pathway. Conclusions: This study provides an experimental and theoretical basis for using TGF-β1 as a neuroprotective agent after peripheral nerve injury in clinical practice in the future. Full article

In vitro experiments performed on dissociated dorsal root ganglion (DRG) neurons suggest the involvement of the hyperpolarization-activated cation current (I_h) in enhancing neuronal excitability, potentially contributing to neuropathic pain. However, the more confirmative in vivo information about how nerve injury interacts with I_h is lacking. In this study, I_h was recorded in vivo using the dynamic single-electrode voltage clamp (dSEVC) technique on L5 DRG neurons of normal rats and those seven days after spinal nerve axotomy (SNA). Compared to normal rats, SNA unexpectedly inhibited the activity of I_h channels on A-fiber DRG neurons: (a) the I_h current magnitude, density, and conductance were consistently diminished; and (b) the I_h activation velocity was slowed and the voltage for I_h activation was hyperpolarized. The half-activation voltage (V_0.5) exhibited a negative shift, and the time constant for I_h activation was prolonged across all test potentials, indicating the reduced availability of I_h after SNA. To further investigate the mechanisms of SNA on I_h, the underlying HCN channels and the correlated mRNA were quantified and compared. The mRNA expression level of HCN1-4 was uniformly enhanced after SNA, which might have contributed to the increased cytoplasmic HCN1 intensity observed in both medium- and large-sized DRG neurons. This finding contradicted the functional reduction of I_h after SNA. Surprisingly, the HCN labeling pattern was altered after SNA: the labeling area of HCN1 and HCN2 at the membranous ring region of the axotomized large neurons became significantly thinner or absent. We concluded that the diminished ring immunoreactivity for HCN1 and HCN2 correlated with a reduced availability of I_h channels, elucidating the observed decrease in I_h in axotomized A-fiber neurons. Full article

Satellite glial cells (SGCs) are the main type of glial cells in sensory ganglia. Animal studies have shown that these cells play essential roles in both normal and disease states. In a large number of pain models, SGCs were activated and contributed to the pain behavior. Much less is known about SGCs in humans, but there is emerging recognition that SGCs in humans are altered in a variety of clinical states. The available data show that human SGCs share some essential features with SGCs in rodents, but many differences do exist. SGCs in DRG from patients suffering from common painful diseases, such as rheumatoid arthritis and fibromyalgia, may contribute to the pain phenotype. It was found that immunoglobulins G (IgG) from fibromyalgia patients can induce pain-like behavior in mice. Moreover, these IgGs bind preferentially to SGCs and activate them, which can sensitize the sensory neurons, causing nociception. In other human diseases, the evidence is not as direct as in fibromyalgia, but it has been found that an antibody from a patient with rheumatoid arthritis binds to mouse SGCs, which leads to the release of pronociceptive factors from them. Herpes zoster is another painful disease, and it appears that the zoster virus resides in SGCs, which acquire an abnormal morphology and may participate in the infection and pain generation. More work needs to be undertaken on SGCs in humans, and this review points to several promising avenues for better understanding disease mechanisms and developing effective pain therapies. Full article

Twenty-four adult molly fish (Poecilia sphenops, Valenciennes 1846) were collected to study the morphology and distribution of ganglia using histological, immunohistochemical, and electron microscopy and focusing on their relation to the immune cells. The ganglia were classified spatially into cranial and spinal, and functionally into sensory and autonomic. Spinal ganglia (dorsal root ganglia, DRG) contained large close ganglionic cells, enclosed by satellite cells, as well as bundles of both myelinated and non-myelinated nerve fibers. There are glial cells, immune cells and telocytes close to the ganglion. In addition, oligodendrocytes were closely related to myelinated axons. Glial fibrillary acidic protein (GFAP) expression was confined to the glia cells and the nerve fibers in the cervical ganglia next to the gills, and surprisingly, in the large ganglionic cells of the DRG. The vestibular ganglia were large, connected to the hind brain, and contained numerous neurons packed in columns. The cervical ganglia were large and observed around the pseudobranch, head kidney, and thymus. Their neurons are randomly distributed, and nerve fibers are peripherally situated. CD3-positive T-lymphocytes, dendritic cells, and CD68-positive macrophages were in close contact with the ganglia. Furthermore, the ganglia around the head kidney showed positive Iba1-expressing cells. Most ganglion cells and nerve fibers in the DRG, autonomic, and vestibular ganglia showed moderate to strong S-100 immunoreactivity. The enteric glia, CD68-expressing macrophages, and acetylcholine (Ach)-expressing neurons were observed along the muscular layer of the intestinal wall. In conclusion, different ganglia of molly fish displayed direct communication with immune cells which support and maintain healthy ganglionic cells. Full article

Chikungunya virus is an arthropod-borne infectious agent that causes Chikungunya fever disease. About 90% of the infected patients experience intense polyarthralgia, affecting mainly the extremities but also the large joints such as the knees. Chronic disease symptoms persist for months, even after clearance of the virus from the blood. Envelope proteins stimulate the immune response against the Chikungunya virus, becoming an important therapeutic target. We inactivated the Chikungunya virus (iCHIKV) and produced recombinant E2 (rE2) protein and three different types of anti-rE2 monoclonal antibodies. Using these tools, we observed that iCHIKV and rE2 protein induced mechanical hyperalgesia (electronic aesthesiometer test) and thermal hyperalgesia (Hargreaves test) in mice. These behavioral results were accompanied by the activation of dorsal root ganglia (DRG) neurons in mice, as observed by calcium influx. Treatment with three different types of anti-rE2 monoclonal antibodies and absence or blockade (AMG-9810 treatment) of transient receptor potential vanilloid 1 (TRPV1) channel diminished mechanical and thermal hyperalgesia in mice. iCHIKV and rE2 activated TRPV1+ mouse DRG neurons in vitro, demonstrating their ability to activate nociceptor sensory neurons directly. Therefore, our mouse data demonstrate that targeting E2 CHIKV protein with monoclonal antibodies and inhibiting TRPV1 channels are reasonable strategies to control CHIKV pain. Full article

The skin secretion of tree frogs contains a vast array of bioactive chemicals for repelling predators, but their structural and functional diversity is not fully understood. Paxilline (PAX), a compound synthesized by Penicillium paxilli, has been known as a specific antagonist of large conductance Ca²⁺-activated K⁺ Channels (BK_Ca). Here, we report the presence of PAX in the secretions of tree frogs (Hyla japonica) and that this compound has a novel function of inhibiting the potassium channel subfamily K member 18 (KCNK18) channels of their predators. The PAX-induced KCNK18 inhibition is sufficient to evoke Ca²⁺ influx in charybdotoxin-insensitive DRG neurons of rats. By forming π-π stacking interactions, four phenylalanines located in the central pore of KCNK18 stabilize PAX to block the ion permeation. For PAX-mediated toxicity, our results from animal assays suggest that the inhibition of KCNK18 likely acts synergistically with that of BK_Ca to elicit tingling and buzzing sensations in predators or competitors. These results not only show the molecular mechanism of PAX-KCNK18 interaction, but also provide insights into the defensive effects of the enriched PAX. Full article

Despite the worldwide prevalence and severe complications of type 2 diabetes mellitus (T2DM), the pathophysiological mechanisms underlying the development of diabetic polyneuropathy (DPN) are poorly understood. Beyond strict control of glucose levels, clinical trials for reversing DPN have largely failed. Therefore, understanding the pathophysiological and molecular mechanisms underlying DPN is crucial. Accordingly, this study explored biochemical and neuropathological deficits in a rat model of T2DM induced through high-fat diet (HFD) feeding along with two low-dose streptozotocin (STZ) injections; the deficits were explored through serum lipid, neurobehavioral, neurophysiology, neuropathology, and immunohistochemistry examinations. Our HFD/STZ protocol induced (1) mechanical hyperalgesia and depression-like behaviors, (2) loss of intraepidermal nerve fibers (IENFs) and reduced axonal diameters in sural nerves, and (3) decreased compound muscle action potential. In addition to hyperglycemia, which was correlated with the degree of mechanical hyperalgesia and loss of IENFs, we observed that hypertriglyceridemia was the most dominant deficit in the lipid profiles of the diabetic rats. In particular, SEPT9, the fourth component of the cytoskeleton, increased in the satellite glial cells (SGCs) of the dorsal root ganglia (DRG) in the T2DM-like rats. The number of SEPT9(+) SGCs/DRG was correlated with serum glucose levels and mechanical thresholds. Our findings indicate the putative molecular mechanism underlying DPN, which presumably involves the interaction of SGCs and DRG neurons; nevertheless, further functional research is warranted to clarify the role of SEPT9 in DPN. Full article

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3’-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability. Full article

Peripheral nerve injury involves divergent alterations within dorsal root ganglia (DRG) neurons sensitized by persistent inflammation. Thymic stromal lymphopoietin (TSLP) production is crucial in the development of chronic inflammatory responses. Herein, we investigate the changes of TSLP expression in rats’ DRG neurons between injured and uninjured sides in the same rat. Linalyl acetate (LA) was served as a TSLP inhibitor and given intraperitoneally. Rats were assigned to be group of chronic constriction injury (CCI) of the sciatic nerve and the group of CCI of the sciatic nerve administrated with LA. Over 14 days, the rats were measured for paw withdrawal thresholds. DRGs were collected to assess morphological changes via immunofluorescence study. After receiving CCI, the rats rapidly developed mechanical hyperalgesia. TSLP expression at DRG, on the ipsilateral injured side, was consistent with changes in pain behaviors. TSLP appeared in nerve fibers with both small diameters and large diameters. Additionally, TSLP was expressed mostly in transient receptor potential vanilloid-1 (TRPV1)-positive nociceptive neurons. Administration with LA can attenuate the pain behaviors and expression of TSLP in DRG neurons, and in apoptotic neurons at the injured side, but not in the contra-lateral uninjured side. Overall, these results imply that altered expressions of TSLP in nociceptive DRG neurons contributed to mechanical hyperalgesia in a CCI rat model. Full article

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions. Full article

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established. Full article