Primary Dorsal Root Ganglion Neurons Research Articles

Dipeptidyl Peptidase (DPP)-4 Inhibitors and Pituitary Adenylate Cyclase-Activating Polypeptide, a DPP-4 Substrate, Extend Neurite Outgrowth of Mouse Dorsal Root Ganglia Neurons: A Promising Approach in Diabetic Polyneuropathy Treatment.

Individuals suffering from diabetic polyneuropathy (DPN) experience debilitating symptoms such as pain, paranesthesia, and sensory disturbances, prompting a quest for effective treatments. Dipeptidyl-peptidase (DPP)-4 inhibitors, recognized for their potential in ameliorating DPN, have sparked interest, yet the precise mechanism underlying their neurotrophic impact on the peripheral nerve system (PNS) remains elusive. Our study delves into the neurotrophic effects of DPP-4 inhibitors, including Diprotin A, linagliptin, and sitagliptin, alongside pituitary adenylate cyclase-activating polypeptide (PACAP), Neuropeptide Y (NPY), and Stromal cell-derived factor (SDF)-1a-known DPP-4 substrates with neurotrophic properties. Utilizing primary culture dorsal root ganglia (DRG) neurons, we meticulously evaluated neurite outgrowth in response to these agents. Remarkably, all DPP-4 inhibitors and PACAP demonstrated a significant elongation of neurite length in DRG neurons (PACAP 0.1 μM: 2221 ± 466 μm, control: 1379 ± 420, p < 0.0001), underscoring their potential in nerve regeneration. Conversely, NPY and SDF-1a failed to induce neurite elongation, accentuating the unique neurotrophic properties of DPP-4 inhibition and PACAP. Our findings suggest that the upregulation of PACAP, facilitated by DPP-4 inhibition, plays a pivotal role in promoting neurite elongation within the PNS, presenting a promising avenue for the development of novel DPN therapies with enhanced neurodegenerative capabilities.

Brain-derived neurotrophic factor contributes to activity-induced muscle pain in male but not female mice

Activity-induced muscle pain increases interleukin-1β (IL-1β) release from muscle macrophages and the development of hyperalgesia is prevented by blockade of IL-1β in muscle. Brain derived neurotrophic factor (BDNF) is released from sensory neurons in response to IL-1β and mediates both inflammatory and neuropathic pain. Thus, we hypothesize that in activity-induced pain, fatigue metabolites combined with IL-1β activate sensory neurons to increase BDNF release, peripherally in muscle and centrally in the spinal dorsal horn, to produce hyperalgesia. We tested the effect of intrathecal or intramuscular injection of BDNF-Tropomyosin receptor kinase B (TrkB) inhibitors, ANA-12 or TrkB-Fc, on development of activity-induced pain. Both inhibitors prevented the hyperalgesia when given before or 24hr after induction of the model in male but not female mice. BDNF messenger ribonucleic acid (mRNA) and protein were significantly increased in dorsal root ganglion (DRG) 24hr after induction of the model in both male and female mice. Blockade of IL-1β in muscle had no effect on the increased BNDF mRNA observed in the activity-induced pain model, while IL-1β applied to cultured DRG significantly induced BDNF expression, suggesting IL-1β is sufficient but not necessary to induce BNDF. Thus, fatigue metabolites, combined with IL-1β, upregulate BDNF in primary DRG neurons in both male and female mice, but contribute to activity-induced pain only in males.

Implantable and transcutaneous photobiomodulation promote neuroregeneration and recovery of lost function after spinal cord injury.

Spinal cord injury (SCI) is a cause of profound and irreversible damage, with no effective therapy to promote functional recovery. Photobiomodulation (PBM) may provide a viable therapeutic approach using red or near-infrared light to promote recovery after SCI by mitigating neuroinflammation and preventing neuronal apoptosis. Our current study aimed to optimize PBM dose regimens and develop and validate the efficacy of an invasive PBM delivery paradigm for SCI. Dose optimization studies were performed using a serum withdrawal model of injury in cultures of primary adult rat dorsal root ganglion neurons (DRGN). Implantable and transcutaneous PBM delivery protocols were developed and validated using cadaveric modeling. The efficacy of PBM in promoting recovery after SCI in vivo was studied in a dorsal column crush injury model of SCI in adult rats. Optimal neuroprotection in vitro was achieved between 4 and 22 mW/cm2. 11 mW/cm2 for 1 min per day (0.66 J/cm2) increased cell viability by 45% over 5 days (p <0.0001), increasing neurite outgrowth by 25% (p <0.01). A method for invasive application of PBM was developed using a diffusion-tipped optogenetics fiber optic. Delivery methods for PBM were developed and validated for both invasive (iPBM) and noninvasive (transcutaneous) (tcPBM) application. iPBM and tcPBM (24 mW/cm2 at spinal cord, 1 min per day (1.44 J/cm2) up to 7 days) increased activation of regeneration-associated protein at 3 days after SCI, increasing GAP43+ axons in DRGN from 18.0% (control) to 41.4% ± 10.5 (iPBM) and 45.8% ± 3.4 (tcPBM) (p <0.05). This corresponded to significant improvements at 6 weeks post-injury in functional locomotor and sensory function recovery (p <0.01), axonal regeneration (p <0.01), and reduced lesion size (p <0.01). Our results demonstrated that PBM achieved a significant therapeutic benefit after SCI, either using iPBM or tcPBM application and can potentially be developed for clinical use in SCI patients.

Abstract 2886: Osteoclast and neuronal cross-talk as a mechanism for chest-wall pain caused by thoracic radiosurgery for lung cancer

Abstract Radiation therapy (RT)-induced chest wall pain (CWP) is a severe complication after thoracic stereotactic body radiation therapy (SBRT) for lung tumors; with increased survivorship, ~30% of patients develop severe CWP. The causes of CWP are unknown, but could involve: 1] damage to intercostal peripheral nerves; 2] bone loss from affected osteoclast activity, or 3] both (secreted factors from activated osteoclasts can affect nerves). This study tested the potential for radiation to affect osteoclasts and/or sensory neurons to induce pain. Mature osteoclasts differentiated from RAW264.7 cells plated on Osteoassay surfaces were exposed to an acute 10 Gy Cs137 γ-rays and bone resorption was assessed. We collected conditioned media (CM) from these cells at 48h and applied to cultured primary mouse dorsal root ganglia neurons (irradiated and non-irradiated neuron), assessing neurite growth and pain-associated marker (CGRP, SP) expression through immunohistochemistry and RT-qPCR. Risedronate (50µM), an anti-resorptive agent, was administered pre-radiation to inhibit osteoclast activity. Unpaired t-tests were utilized for statistical analyses (α=0.05). Osteoclast resorption activity was increased by 48h after irradiation. These irradiated, activated osteoclasts exhibited increased mRNA expression of differentiation (RANK) and activity (CTSK, MMP9) markers. Neurons treated with CM from irradiated osteoclasts exhibited elevated levels of CGRP (+38.5%) and SP (+29.5%) compared to cells treated with CM from non-irradiated cells. Consistently, in co-cultures of osteoclasts and neurons exposed to radiation, irradiated neurons exhibited significantly increased neuropeptide levels. Risedronate not only reduced osteoclast activity but also inhibited neuropeptide expression in irradiated neurons within these co-cultures. Moreover, neuropeptide levels were similar between directly irradiated neurons and non-irradiated neurons. However, irradiated neurons exposed to CM from either irradiated or non-irradiated osteoclasts showed a marked 4-fold increase in neuropeptide levels vs neurons that were non-irradiated and then received osteoclast CM. Our findings indicate that radiation activates osteoclasts and leads to direct bone loss in vitro and signaling between irradiated osteoclasts and non-irradiated neurons increases pain responses. Importantly, this response is enhanced when neurons were also previously irradiated. During SBRT, intercostal nerves may be influenced by adjacent irradiated osteoclasts, increasing expression of pain-related neuropeptides as the osteoclasts break down bone, thus enhancing the risk of CWP as treatment progresses across time. Understanding these interactions could reveal potential targets for preventing CWP, allowing higher tumor radiation doses with less toxicity. Support: The Milton Raben Foundation. Citation Format: Sun Park, Megan Peters, Joseph Moore, Kaitlyn Reno, Michael Farris, Christopher Peters, Jeffery Willey. Osteoclast and neuronal cross-talk as a mechanism for chest-wall pain caused by thoracic radiosurgery for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2886.

Phytic Acid Maintains Peripheral Neuron Integrity and Enhances Survivability against Platinum-Induced Degeneration via Reducing Reactive Oxygen Species and Enhancing Mitochondrial Membrane Potential.

Phytic acid (PA) has been reported to possess anti-inflammatory and antioxidant properties that are critical for neuroprotection in neuronal disorders. This raises the question of whether PA can effectively protect sensory neurons against chemotherapy-induced peripheral neuropathy (CIPN). Peripheral neuropathy is a dose-limiting side effect of chemotherapy treatment often characterized by severe and abnormal pain in hands and feet resulting from peripheral nerve degeneration. Currently, there are no effective treatments available that can prevent or cure peripheral neuropathies other than symptomatic management. Herein, we aim to demonstrate the neuroprotective effects of PA against the neurodegeneration induced by the chemotherapeutics cisplatin (CDDP) and oxaliplatin. Further aims of this study are to provide the proposed mechanism of PA-mediated neuroprotection. The neuronal protection and survivability against CDDP were characterized by axon length measurements and cell body counting of the dorsal root ganglia (DRG) neurons. A cellular phenotype study was conducted microscopically. Intracellular reactive oxygen species (ROS) was estimated by fluorogenic probe dichlorofluorescein. Likewise, mitochondrial membrane potential (MMP) was assessed by fluorescent MitoTracker Orange CMTMRos. Similarly, the mitochondria-localized superoxide anion radical in response to CDDP with and without PA was evaluated. The culture of primary DRG neurons with CDDP reduced axon length and overall neuronal survival. However, cotreatment with PA demonstrated that axons were completely protected and showed increased stability up to the 45-day test duration, which is comparable to samples treated with PA alone and control. Notably, PA treatment scavenged the mitochondria-specific superoxide radicals and overall intracellular ROS that were largely induced by CDDP and simultaneously restored MMP. These results are credited to the underlying neuroprotection of PA in a platinum-treated condition. The results also exhibited that PA had a synergistic anticancer effect with CDDP in ovarian cancer in vitro models. For the first time, PA's potency against CDDP-induced PN is demonstrated systematically. The overall findings of this study suggest the application of PA in CIPN prevention and therapeutic purposes.

Cholinergic Receptor Muscarinic 1 Co-Localized with Mitochondria in Cultured Dorsal Root Ganglion Neurons, and Its Deletion Disrupted Mitochondrial Ultrastructure in Peripheral Neurons: Implications in Alzheimer's Disease.

Loss of Cholinergic Receptor Muscarinic 1 (CHRM1) has been linked to the pathogenesis of Alzheimer's disease (AD). Our recent study found significantly lower CHRM1 protein levels in AD patient cortices, linked to reduced survival. Furthermore, using knockout mice (Chrm1-/-) we demonstrated that deletion of Chrm1 alters cortical mitochondrial structure and function, directly establishing a connection between its loss and mitochondrial dysfunction in the context of AD. While CHRM1's role in the brain has been extensively investigated, its impact on peripheral neurons in AD remains a crucial area of research, especially considering reported declines in peripheral nerve conduction among AD patients. The objective was to characterize Chrm1 localization and mitochondrial deficits in Chrm1-/- dorsal root ganglion (DRG) neurons. Recombinant proteins tagged with Green or Red Fluorescent Protein (GFP/RFP) were transiently expressed to investigate the localization of Chrm1 and mitochondria, as well as mitochondrial movement in the neurites of cultured primary mouse DRG neurons, using confocal time-lapse live cell imaging. Transmission electron microscopy was performed to examine the ultrastructure of mitochondria in both wild-type and Chrm1-/- DRGs. Fluorescence imaging revealed colocalization and comigration of N-terminal GFP-tagged Chrm1 and mitochondrial localization signal peptide-tagged RFP-labelled mitochondria in the DRGs neurons. A spectrum of mitochondrial structural abnormalities, including disruption and loss of cristae was observed in 87% neurons in Chrm1-/- DRGs. This study suggests that Chrm1 may be localized in the neuronal mitochondria and loss of Chrm1 in peripheral neurons causes sever mitochondrial structural aberrations resembling AD pathology.

Fibroblast growth factor-21: preserving cell viability in diabetic neuropathy through the PI3K/AKT cellular pathway

Aim : This study aims to investigate the potential neuroprotective effects of fibroblast growth factor-21 (FGF21) on dorsal root ganglion (DRG) neurons under high glucose (HG) conditions, mimicking diabetic neuropathy. Specifically, we hypothesize that FGF21 enhances cell viability and reduces glucose-induced neuronal death via the activation of the phosphatidyl-inositol-3-kinase (PI3K)/AKT signaling pathway. Materials and Methods: Primary DRG neurons were cultured from laboratory models and exposed to HG concentrations to simulate diabetic conditions. Various concentrations of FGF21 were administered to the DRG neurons. Cell viability was assessed using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), a widely used enzymatic method for determining cellular metabolic activity. The involvement of the PI3K /AKT signaling pathway in mediating the effects of FGF21 was also examined through biochemical assays and pathway inhibitors. Results: Administration of FGF21 to DRG neurons exposed to HG conditions significantly protected cell viability and reduced glucose-induced neuronal death (p &lt; 0.05). The protective effects of FGF21 were found to be dose-dependent, with higher concentrations showing more pronounced benefits. Furthermore, the activation of the PI3K /AKT signaling pathway was confirmed to play a crucial role in the neuroprotective mechanism of FGF21, as inhibition of this pathway attenuated the protective effects. Conclusion: This study demonstrates for the first time that FGF21 has a neuroprotective effect on DRG neuron survival in a diabetic neuropathy model. By activating the PI3K /AKT signaling pathway, FGF21 helps maintain cell viability and reduces glucose-related neuronal death. These findings provide a promising basis for the development of new therapeutic strategies for the treatment of diabetic neuropathy, leveraging the neuroprotective properties of FGF21.

A Live Imaging Assay for Regenerating Peripheral Neurons.

The cell intrinsic mechanisms directing peripheral nerve regeneration have remained largely understudied, thus limiting our understanding of these processes and constraining the advancement of novel clinical therapeutics. The use of primary adult rat dorsal root ganglion (DRG) neurons cultured in vitro is well established. Despite this, these cells can be challenging to culture and have so far not been amenable to robust transfection or live-cell imaging. The ability to transfect these cells with fluorescent plasmid constructs to label subcellular structures, combined with high resolution time-lapse imaging has the potential to provide invaluable insight into how peripheral neurons coordinate their regenerative response, and which specific cellular structures are involved in this process. Here we describe a protocol that facilitates transfection and subsequent live-imaging of adult rat DRG neurons.

Highly specific σ2R/TMEM97 ligand FEM-1689 alleviates neuropathic pain and inhibits the integrated stress response.

The sigma 2 receptor (σ2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ2R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal antinociceptive effect is approximately 24 h following dosing. We sought to understand this unique antineuropathic pain effect by addressing two key questions: do these σ2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout mice for Tmem97, we find that a σ2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce antinociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a σ2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.

Mitochondrial transplantation attenuates traumatic neuropathic pain, neuroinflammation, and apoptosis in rats with nerve root ligation.

Traumatic neuropathic pain (TNP) is caused by traumatic damage to the somatosensory system and induces the presentation of allodynia and hyperalgesia. Mitochondrial dysfunction, neuroinflammation, and apoptosis are hallmarks in the pathogenesis of TNP. Recently, mitochondria-based therapy has emerged as a potential therapeutic intervention for diseases related to mitochondrial dysfunction. However, the therapeutic effectiveness of mitochondrial transplantation (MT) on TNP has rarely been investigated. Here, we validated the efficacy of MT in treating TNP. Both in vivo and in vitro TNP models by conducting an L5 spinal nerve ligation in rats and exposing the primary dorsal root ganglion (DRG) neurons to capsaicin, respectively, were applied in this study. The MT was operated by administrating 100 µg of soleus-derived allogeneic mitochondria into the ipsilateral L5 DRG in vivo and the culture medium in vitro. Results showed that the viable transplanted mitochondria migrated into the rats' spinal cord and sciatic nerve. MT alleviated the nerve ligation-induced mechanical and thermal pain hypersensitivity. The nerve ligation-induced glial activation and the expression of pro-inflammatory cytokines and apoptotic markers in the spinal cord were also repressed by MT. Consistently, exogenous mitochondria reversed the capsaicin-induced reduction of mitochondrial membrane potential and expression of pro-inflammatory cytokines and apoptotic markers in the primary DRG neurons in vitro. Our findings suggest that MT mitigates the spinal nerve ligation-induced apoptosis and neuroinflammation, potentially playing a role in providing neuroprotection against TNP.

Development of an In Vitro Assessment Method for Chemotherapy-Induced Peripheral Neuropathy (CIPN) by Integrating a Microphysiological System (MPS) with Morphological Deep Learning of Soma and Axonal Images.

Several anticancer drugs used in cancer therapy induce chemotherapy-induced peripheral neuropathy (CIPN), leading to dose reduction or therapy cessation. Consequently, there is a demand for an in vitro assessment method to predict CIPN and mechanisms of action (MoA) in drug candidate compounds. In this study, a method assessing the toxic effects of anticancer drugs on soma and axons using deep learning image analysis is developed, culturing primary rat dorsal root ganglion neurons with a microphysiological system (MPS) that separates soma from neural processes and training two artificial intelligence (AI) models on soma and axonal area images. Exposing the control compound DMSO, negative compound sucrose, and known CIPN-causing drugs (paclitaxel, vincristine, oxaliplatin, suramin, bortezomib) for 24 h, results show the somatic area-learning AI detected significant cytotoxicity for paclitaxel (* p < 0.05) and oxaliplatin (* p < 0.05). In addition, axonal area-learning AI detected significant axonopathy with paclitaxel (* p < 0.05) and vincristine (* p < 0.05). Combining these models, we detected significant toxicity in all CIPN-causing drugs (** p < 0.01) and could classify anticancer drugs based on their different MoA on neurons, suggesting that the combination of MPS-based culture segregating soma and axonal areas and AI image analysis of each area provides an effective evaluation method to predict CIPN from low concentrations and infer the MoA.

Zhenxin Anshen formula ameliorates atopic der-matitis-like skin dysfunction in mice and regulation of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 in Neural pathways.

To investigate the efficacy of Zhenxin Anshen formula (, ZXAS) on atopic dermatitis (AD) by transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) signalling pathway in mice and . AD-like lesions were induced by 1-chloro-2,4-dinitrobenzene (DNCB) to the shaved dorsal skin of BALB/c mice. BALB/c mice were divided into five groups: normal control, model control, cetirizine, low-, medium-, and high-dose of ZXAS. After ZXAS in-tervention, the skin lesions and blood samples were collected for hematoxylin and eosin-stained and measuring the concentrations of inflammatory cytokines. Immun-oglobulin E (IgE), interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin (TSLP) were de-tected by Enzyme-linked immunosorbent assay (ELISA). The spinal cords were collected for measuring the expression of gastrin-releasing peptide receptor (GRPR), TRPV1, and TRPA1 by using immunohistochemistry, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. In addition, 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, ELISA, and Western blotting were conducted for analysis of primary dorsal root ganglia (DRG) neurons . ZXAS treatment improved DNCB-induced AD-like lesions through reducing dermatitis score, number of scratching and epidermal thickness, accompanied by the de-creased IgE and Th2 inflammatory cytokines. ZXAS also supressed the mRNA and protein expression of GRPR, TRPV1, and TRPA1 in the spinal cord. The medicated sera of ZXAS decreased capsaicin-induced Ca influx and downregulated the expression of TRPV1, TRPA1, and phospholipase C in DRG neurons. The therapeutic effect of ZXAS on AD may be related to the regulation of TRPV1 and TRPA1 and inhibition of Ca2+ signals in neurons.

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.

Self-assembled innervated vasculature-on-a-chip to study nociception

Nociceptor sensory neurons play a key role in eliciting pain. An active crosstalk between nociceptor neurons and the vascular system at the molecular and cellular level is required to sense and respond to noxious stimuli. Besides nociception, interaction between nociceptor neurons and vasculature also contributes to neurogenesis and angiogenesis. In vitro models of innervated vasculature can greatly help delineate these roles while facilitating disease modeling and drug screening. Herein, we report the development of a microfluidic-assisted tissue model of nociception in the presence of microvasculature. The self-assembled innervated microvasculature was engineered using endothelial cells and primary dorsal root ganglion (DRG) neurons. The sensory neurons and the endothelial cells displayed distinct morphologies in presence of each other. The neurons exhibited an elevated response to capsaicin in the presence of vasculature. Concomitantly, increased transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor expression was observed in the DRG neurons in presence of vascularization. Finally, we demonstrated the applicability of this platform for modeling nociception associated with tissue acidosis. While not demonstrated here, this platform could also serve as a tool to study pain resulting from vascular disorders while also paving the way towards the development of innervated microphysiological models.

Inhibition of NHE1 transport activity and gene transcription in DRG neurons in oxaliplatin-induced painful peripheral neurotoxicity

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.

A novel high throughput combined voltage-clamp/current-clamp analysis of single primary neurons.

The patch-clamp technique is the gold-standard methodology for biophysical investigations of channels and receptors studied by voltage-clamp, and for analysis of excitability of cells, such as neurons studied via current-clamp. However, the throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for assessments, such as drug screening, has been primarily used to study channels and receptors expressed in heterologous expression systems. In this study, we introduce a novel approach to automated high throughput patch-clamping that substantially enhances high throughput analysis of excitable cells at the channel and cellular level. As a proof-of-concept, we apply this approach to investigate the detailed biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables high throughput, unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of primary neurons. Furthermore, our approach eliminates the need for culturing of cells on coverslips and provides the ability to perform both voltage- and current-clamp recordings on the same neuron. This approach can be used for many applications, including both physiological and pharmacological analyses of primary DRG neurons.

NaV1.7 Channel Blocker [Ala5, Phe6, Leu26, Arg28]GpTx-1 Attenuates CFA-induced Inflammatory Hypersensitivity in Rats via Endogenous Enkephalin Mechanism

Venom-derived NaV1.7 channel blockers have promising prospects in pain management. The 34-residue tarantula peptide GpTx-1 is a potent NaV1.7 channel blocker. Its powerful analog [Ala5, Phe6, Leu26, Arg28]GpTx-1 (GpTx-1-71) displayed excellent NaV1.7 selectivity and analgesic properties in mice. The current study aimed to elucidate the anti-hyperalgesic activities of GpTx-1-71 in inflammatory pain and reveal the underlying mechanisms. Our results demonstrated that intrathecal and intraplantar injections of GpTx-1-71 dose-dependently attenuated CFA-induced inflammatory hypersensitivity in rats. Moreover, GpTx-1-71-induced anti-hyperalgesia was significantly reduced by opioid receptor antagonists and the enkephalin antibody and diminished in proenkephalin (Penk) gene knockout animals. Consistently, GpTx-1-71 treatment increased the enkephalin level in the spinal dorsal horn and promoted the Penk transcription and enkephalin release in primary dorsal root ganglion (DRG) neurons, wherein sodium played a crucial role in these processes. Mass spectrometry analysis revealed that GpTx-1-71 mainly promoted the secretion of Met-enkephalin but not Leu-enkephalin from DRG neurons. In addition, the combination of subtherapeutic Met-enkephalin and GpTx-1-71 produced synergistic anti-hyperalgesia in CFA-induced inflammatory hypersensitivity. These findings suggest that the endogenous enkephalin pathway is essential for GpTx-1-71-induced spinal and peripheral analgesia in inflammatory pain. PerspectiveThis article presents a possible pharmacological mechanism underlying NaV1.7 blocker-induced analgesia in inflammatory pain, which helps us to better understand and develop venom-based painkillers for incurable pain.

Palmitoylethanolamide Mitigates Paclitaxel Toxicity in Primary Dorsal Root Ganglion Neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of several chemotherapeutic agents, such as Paclitaxel. The main symptoms of CIPN are pain and numbness in the hands and feet. Paclitaxel is believed to accumulate in the dorsal root ganglia and free nerve endings. Novel therapeutic agents might help to mitigate or prevent Paclitaxel toxicity on dorsal root ganglion (DRG) neurons. Thus, we used primary DRG neurons as a model to investigate the potential neuroprotective effects of the endocannabinoid-like substance, palmitoylethanolamide (PEA). DRG neurons were isolated from cervical to sacral segments of spinal nerves of Wister rats (6-8 weeks old). After isolation and purification of neuronal cell populations, different concentrations of Paclitaxel (0.01-10 µM) or PEA (0.1-10 µM) or their combination were tested on cell viability by MTT assay at 24 h, 48, and 72 h post-treatment. Furthermore, morphometric analyses of neurite length and soma size for DRG neurons were performed. Adverse Paclitaxel effects on cell viability were apparent at 72 h post-treatment whereas Paclitaxel significantly reduced the neurite length in a concentration-dependent manner nearly at all investigated time points. However, Paclitaxel significantly increased the size of neuronal cell bodies at all time windows. These phenotypic effects were significantly reduced in neurons additionally treated with PEA, indicating the neuroprotective effect of PEA. PEA alone led to a significant increase in neuron viability regardless of PEA concentrations, apparent improvements in neurite outgrowth as well as a significant decrease in soma size of neurons at different investigated time points. Taken together, PEA showed promising protective effects against Paclitaxel-related toxicity on DRG neurons.

Sex differences in the inflammatory response of the mouse DRG and its connection to pain in experimental autoimmune encephalomyelitis

Multiple Sclerosis (MS) is an autoimmune disease with notable sex differences. Women are not only more likely to develop MS but are also more likely than men to experience neuropathic pain in the disease. It has been postulated that neuropathic pain in MS can originate in the peripheral nervous system at the level of the dorsal root ganglia (DRG), which houses primary pain sensing neurons (nociceptors). These nociceptors become hyperexcitable in response to inflammation, leading to peripheral sensitization and eventually central sensitization, which maintains pain long-term. The mouse model experimental autoimmune encephalomyelitis (EAE) is a good model for human MS as it replicates classic MS symptoms including pain. Using EAE mice as well as naïve primary mouse DRG neurons cultured in vitro, we sought to characterize sex differences, specifically in peripheral sensory neurons. We found sex differences in the inflammatory profile of the EAE DRG, and in the TNFα downstream signaling pathways activated intracellularly in cultured nociceptors. We also found increased cell death with TNFα treatment. Given that TNFα signaling has been shown to initiate intrinsic apoptosis through mitochondrial disruption, this led us to investigate sex differences in the mitochondria’s response to TNFα. Our results demonstrate that male sensory neurons are more sensitive to mitochondrial stress, making them prone to neuronal injury. In contrast, female sensory neurons appear to be more resistant to mitochondrial stress and exhibit an inflammatory and regenerative phenotype that may underlie greater nociceptor hyperexcitability and pain. Understanding these sex differences at the level of the primary sensory neuron is an important first step in our eventual goal of developing sex-specific treatments to halt pain development in the periphery before central sensitization is established.

Effects of Photodynamic Therapy on Nav1.7 Expression in Spinal Dorsal Root Ganglion Neurons.

The aim of this study was to examine the effects of photodynamic therapy (PDT) on the expression of Nav1.7 in spinal dorsal root ganglion (DRG) neurons. The primary DRG neurons from newborn SD rats were cultured. The cells were identified by neuron-specific enolase immunofluorescence staining. DRG neurons were divided into four groups: control group, photosensitizer group, laser group, and PDT group. The cell viability was detected by a cell counting kit-8 (CCK8) assay. qRT-PCR and Western blotting were used to determine the mRNA and protein expression levels of Nav1.7 in DRG neurons. The purity of the cultured primary DRG neurons was greater than 90%. Compared with the control group, no significant change was found in the cell viability of the photosensitizer group, while the viability in the laser group and the PDT group was significantly reduced. The mRNA and protein expression levels of Nav1.7 were significantly greater in the laser group and the PDT group than in the control group. At the same time, the mRNA and protein expression levels of Nav1.7 were greater in the laser group than in the PDT group. Both laser and PDT could upregulate the expression of Nav1.7 in DRG neurons, and the promoting effect might be related to the pain induced by clinical treatment. This study provides a research basis for the use of laser and PDT to treat pain. A better understanding of the relationship between Nav1.7 and PDT can help clinicians better manage PDT-related pain.