Dorsal Root Ganglion Cells Research Articles

The peripheral Atf3 + neuronal population is responsible for nerve regeneration at the early stage of nerve injury revealed by single-cell RNA sequencing.

Peripheral nerve injury (PNI) can transform primary somatosensory neurons to a regenerative state. However, the details of the transcriptomic changes associated with the nerve regeneration of somatosensory neurons remain unclear. In this study, single-cell RNA sequencing (scRNA-seq) is conducted on mouse dorsal root ganglion (DRG) cells after the early stage of nerve injury on day 3 after chronic constriction injury (CCI). We observe that a novel CCI-induced neuronal population (CIP) emerge and express high levels of activating transcription factor ( Atf3), a neuronal injury marker. CIP neurons highly express regeneration-associated genes (RAGs) and are enriched in regeneration-related gene ontology (GO) terms, suggesting that these neurons can constitute a pro-regenerative population. Moreover, intercellular communication networks show that CIP neurons closely communicate with satellite glial cells (SGCs) and specifically transmit strong Fgf3- Fgfr1 signaling to SGCs, which could initiate regeneration-associated transcriptional changes in SGCs. We also confirm that regenerative progress occurs at the early stage of nerve injury because immunohistochemistry shows that the expression of ATF3 is significantly increased beginning at 3 days post-CCI and decreased at 1 month post-CCI. Our bioinformatics analysis at single-cell resolution advances the knowledge of regenerative dynamic transcriptional changes in DRG cells after injury and the underlying molecular mechanisms involved.

Optimized primary dorsal root ganglion cell culture protocol for reliable K+ current patch-clamp recordings

DRG primary neuron cultures, derived from rodents, closely mimic properties of sensory neurons in vivo and are highly useful for studying pain and neurological disorders. These cultures are pivotal in patch clamp electrophysiology for sensory neuron properties analysis. A detailed, replicable protocol in scientific research ensures experiment accuracy and reproducibility. This paper provides comprehensive details for replicating the protocol and achieving consistent results in primary DRG cell culture as used for patch clamp recordings. We outlined a comprehensive protocol for establishing primary DRG cell culture, optimized for improved gigaseal formation in whole-cell patch-clamp recordings. Additionally, we conducted a simulation study focused on recording macroscopic K+ channels. The findings established an optimized novel protocol that works reliably for whole-cell patch clamp recordings and data analysis using primary DRG cells prepared as described in this publication. The details for the protocol in the literature are dispersed across various publications, making it challenging to find a comprehensive summary in one source. This study confirms, for the first time, the efficacy of using fewer protocol steps, which reduces stress and variability in obtaining suitable cells for patch clamp recordings compared to existing methods in the literature. Given the challenges posed by the dissociation process of primary DRG cells and the importance of comprehensive method documentation in the literature, the protocol presented provides improved and consistent applications of primary DRG cell culture in patch clamp recordings

A microfluidic model of the first sensory synapse for analgesic target discovery.

The synaptic connections between dorsal root ganglia (DRG) and dorsal horn (DH) neurons are a crucial relay point for the transmission of painful stimuli. To delineate how synaptic plasticity may modulate the excitability of DH neurons, we have devised a microfluidic co-culture model that recapitulates the first sensory synapse using postnatal mouse sensory neurons. We show that DRG-DH co-cultures characterize salient features of the in vivo physiology of sensory neurons. Immunocytcochemical experiments of the cultured DH neurons show a co-localization of MAP2 with VGLUT2 and of MAP2 with Synapsin 1, corroborating the glutamatergic identity of the DH neurons and further suggesting the formation of active synapses in this neuronal set. Fluorometric imaging experiments demonstrate the elicitation of calcium responses in DH neurons following the stimulation of DRG cell bodies or axons. Selective NMDA and AMPA receptor blockade appreciably silences DH neuron responses, suggesting that glutamatergic signaling is maintained in vitro. Last, a surrogate model of peripheral nerve injury is introduced in the form of an axotomy, which results in elevated and prolonged calcium responses of DH neurons. Overall, the microfluidic mouse co-cultures provide a method advancement in the study of periphery-to-center pain signaling, where the potential of utilizing the platform for drug target identification is underscored.

Human dorsal root ganglia are either preserved or completely lost after deafferentation by brachial plexus injury

BackgroundPlexus injury results in lifelong suffering from flaccid paralysis, sensory loss, and intractable pain. For this clinical problem, regenerative medicine concepts set high expectations. However, it is largely unknown how dorsal root ganglia (DRG) are affected by accidental deafferentation. MethodsHere, we phenotyped DRG of a clinically and MRI-characterised cohort of 13 patients with plexus injury. Avulsed DRG were collected during reconstructive nerve surgery. For control, we used DRG from forensic autopsy. The cellular composition of the DRG was analysed in histopathological slices with multicolour high-resolution immunohistochemistry, tile microscopy, and deep-learning-based bioimage analysis. We then sequenced the bulk RNA of corresponding DRG slices. ResultsIn about half of the patients we found loss of the typical DRG units consisting of neurones and satellite glial cells. The DRG cells were replaced by mesodermal/connective tissue. In the remaining patients, the cellular units were well preserved. Preoperative plexus MRI neurography was not able to distinguish the two types. Patients with ‘neuronal preservation’ had less maximum pain than patients with ‘neuronal loss’. Arm function improved after nerve reconstruction, but severe pain persisted. Transcriptome analysis of preserved DRGs revealed expression of subtype-specific sensory neurone marker genes, but downregulation of neuronal attributes. Furthermore, they showed signs of ongoing inflammation and connective tissue remodelling. ConclusionsPatients with plexus injury separate into two groups with either neuronal preservation or neuronal loss. The former could benefit from anti-inflammatory therapy. For the latter, studies should explore mechanisms of neuronal loss especially for regenerative approaches. Clinical trial registrationDRKS00017266.

Dorsal root ganglion inflammation by oxaliplatin toxicity: DPEP1 as possible target for peripheral neuropathy prevention

BackgroundPeripheral neuropathy (PN) constitutes a dose-limiting side effect of oxaliplatin chemotherapy that often compromises the efficacy of antineoplastic treatments. Sensory neurons damage in dorsal root ganglia (DRG) are the cellular substrate of PN complex molecular origin. Dehydropeptidase-1 (DPEP1) inhibitors have shown to avoid platin-induced nephrotoxicity without compromising its anticancer efficiency. The objective of this study was to describe DPEP1 expression in rat DRG in health and in early stages of oxaliplatin toxicity. To this end, we produced and characterized anti-DPEP1 polyclonal antibodies and used them to define the expression, and cellular and subcellular localization of DPEP1 by immunohistochemical confocal microscopy studies in healthy controls and short term (six days) oxaliplatin treated rats.ResultsDPEP1 is expressed mostly in neurons and in glia, and to a lesser extent in endothelial cells. Rats undergoing oxaliplatin treatment developed allodynia. TNF-\U0001d6fc expression in DRG revealed a pattern of focal and at different intensity levels of neural cell inflammatory damage, accompanied by slight variations in DPEP1 expression in endothelial cells and in nuclei of neurons.ConclusionsDPEP1 is expressed in neurons, glia and endothelial cells of DRG. Oxaliplatin caused allodynia in rats and increased TNF-α expression in DRG neurons. The expression of DPEP1 in neurons and other cells of DRG suggest this protein as a novel strategic molecular target in the prevention of oxaliplatin-induced acute neurotoxicity.

TUMOR-INFILTRATING NOCICEPTOR NEURONS PROMOTE IMMUNOSUPPRESSION.

Nociceptor neurons impact tumor immunity. Removing nociceptor neurons reduced myeloid-derived suppressor cell (MDSCs) tumor infiltration in mouse models of head and neck carcinoma and melanoma. Carcinoma-released small extracellular vesicles (sEVs) attract nociceptive nerves to tumors. sEV-deficient tumors fail to develop in mice lacking nociceptor neurons. Exposure of dorsal root ganglia (DRG) neurons to cancer sEVs elevated expression of Substance P, IL-6 and injury-related neuronal markers while treatment with cancer sEVs and cytotoxic CD8 T-cells induced an immunosuppressive state (increased exhaustion ligands and cytokines). Cancer patient sEVs enhanced DRG responses to capsaicin, indicating increased nociceptor sensitivity. Conditioned media from DRG and cancer cell co-cultures promoted expression of MDSC markers in primary bone marrow cells while DRG conditioned media together with cancer sEVs induced checkpoint expression on T-cells. Our findings indicate that nociceptor neurons facilitate CD8+ T cell exhaustion and enhance MDSC infiltration. Targeting nociceptor-released IL-6 emerges as a novel strategy to disrupt harmful neuro-immune interactions in cancer and enhance anti-tumor immunity.

Relief of pain in mice by an antibody with high affinity for cell adhesion molecule 1 on nerves

AimsCell adhesion molecule 1 (CADM1) is a member of the immunoglobulin superfamily and is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has proven useful as a drug delivery vector for CADM1-expressing cells in vitro. When injected subcutaneously into mice, whether 3E1 accumulates on nerve fibers and serves as an analgesic was examined. Main methodsInjected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca2+ fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves. Key findingsSubcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells. Significance3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.

Siponimod treatment response shows partial BDNF dependency in multiple sclerosis models

So far, only a small number of medications are effective in progressive multiple sclerosis (MS). The sphingosine-1-phosphate-receptor (S1PR)-1,5 modulator siponimod, licensed for progressive MS, is acting both on peripheral immune cells and in the central nervous system (CNS). So far it remains elusive, whether those effects are related to the neurotrophin brain derived neurotrophic factor (BDNF). We hypothesized that BDNF in immune cells might be a prerequisite to reduce disease activity in experimental autoimmune encephalomyelitis (EAE) and prevent neurotoxicity. MOG35–55 immunized wild type (WT) and BDNF knock-out (BDNFko) mice were treated with siponimod or vehicle and scored daily in a blinded manner. Immune cell phenotyping was performed via flow cytometry. Immune cell infiltration and demyelination of spinal cord were assessed using immunohistochemistry. In vitro, effects on neurotoxicity and mRNA regulation were investigated using dorsal root ganglion cells incubated with EAE splenocyte supernatant. Siponimod led to a dose-dependent reduction of EAE scores in chronic WT EAE. Using a suboptimal dosage of 0.45 µg/day, siponimod reduced clinical signs of EAE independent of BDNF-expression in immune cells in accordance with reduced infiltration and demyelination. Th and Tc cells in secondary lymphoid organs were dose-dependently reduced, paralleled with an increase of regulatory T cells. In vitro, neuronal viability trended towards a deterioration after incubation with EAE supernatant; siponimod showed a slight rescue effect following treatment of WT splenocytes. Neuronal gene expression for CCL2 and CX3CL1 was elevated after incubation with EAE supernatant, which was reversed after siponimod treatment for WT, but not for BNDFko. Apoptosis markers and alternative death pathways were not affected. Siponimod exerts both anti-inflammatory and neuroprotective effects, partially related to BDNF-expression. This might in part explain effectiveness during progression in MS and could be a target for therapy.

Non-integrin laminin receptor (LamR) plays a role in axonal outgrowth from chicken DRG via modulating the Akt and Erk signaling.

37/67kDa laminin receptor (LamR)/ribosomal protein SA exhibits dual function as both a ribosomal protein and cell surface receptor for laminin. LamR influences critical cellular processes such as invasion, adhesion, and migration when acting as a receptor. Despite the acknowledged importance of LamR/67LR in various cellular processes, its contribution to the peripheral nervous system development is obscure. Thus, this study investigated the biological activity of LamR in peripheral axonal outgrowth in the presence of laminin-1 or Ile-Lys-Val-Ala-Val (IKVAV) peptide, whose important role in dorsal root ganglia (DRG) axonal outgrowth we recently showed. Unexpectedly, we did not observe LamR on the surface of DRG cells or in a conditioned medium, suggesting its intracellular action in the negative regulation of DRG axonal outgrowth. Using C-terminus LamR-targeting IgG, we demonstrated the role of LamR in that process, which is independent of the presence of Schwann cell precursors (SCPs) and is mediated by extracellular signal-regulated kinase (Erk) and Protein kinase B (Akt1/2/3) signaling pathways. Additionally, we show that the action of LamR towards laminin-1-dependent axonal outgrowth is unmasked only when the activity of integrin β1 is perturbed. We believe that modulation of LamR activity provides the basis for its use for inhibiting axon growth as a potential therapeutic agent for regulating abnormal or excessive neurite growth during neurodevelopmental diseases or pathological nerve regeneration.

CXCL10 Enhances Acid-Sensing Ion Channel Currents in Rat Dorsal Root.

Both CXCL10/CXCR3 and acid-sensing ion channels (ASICs) are expressed in nociceptive sensory neurons and participate in various pain processes, but it is still unclear whether there is a link between them. Herein, we report that CXCL10 enhances the electrophysiological activity of ASICs in rat dorsal root ganglia (DRG) neurons. A brief (10min) application of CXCL10 increased acid-evoked ASIC currents in a concentration-dependent manner. CXCL10 increased the maximum response of ASICs to acidic stimuli without changing their sensitivity. CXCL10 enhanced ASIC currents in DRG cells through CXCR3, as this enhancement was completely blocked by AMG487, a selective CXCR3 antagonist. CXCL10 also increased ASIC3 currents in CHO cells coexpressing ASIC3 and CXCR3 but not in cells expressing ASIC3 alone. The CXCL10-mediated increase in ASIC currents was prevented by the application of either the G protein inhibitor GDP-β-S or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 but not by the ERK inhibitor U0126 or the JNK inhibitor SP600125. Moreover, CXCL10 increased the number of action potentials triggered by acidic stimuli via CXCR3. CXCL10 dose-dependently exacerbated acid-induced nociceptive behavior in rats through peripheral CXCR3. These results indicated that CXCL10/CXCR3 signaling enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats via a p38 MAPK-dependent pathway, revealing a novel mechanism underlying pain. CXCL10/CXCR3 signaling may be an effective target in the treatment of pain associated with tissue acidification.

63 Developing a 3D in Vitro Schwann Cell Model of Myelination for Peripheral Nerve Injury

Abstract Aim This study investigated the effectiveness of fabricating engineered neural tissue (EngNT) to develop a 3D in vitro model of myelination for peripheral nerve injury. Additionally, the effect of metformin and sulindac sulfide in nerve regeneration was evaluated by assessing neurite growth in both monolayer and 3D co-culture systems. Method Neuronal cells (NG108-15 and SH-SY5Y cell lines) were treated with metformin for 72 hrs in an Incucyte® live-cell analysis system. EngNT cellular collagen gels were fabricated using Schwann cells (SCL4.1/F7), SCL4.1/F7:NG108-15 (1:1) cells or harvested rat dorsal root ganglion cells. NG108–15 cells were co-cultured with SCL4.1/F7 cellular gels. 3D EngNT co-cultures were treated with metformin and sulindac sulfide for 72 hrs. EngNT cellular gels were analysed with transmission electron microscopy (TEM). Results TEM evaluation of EngNT co-cultures demonstrated the enfoldment of neurones by Schwann cells resulting in cell-cell contacts and myelination was identified. NG108-15 and SH-SY5Y cells exhibited significant neurite growth with 100 μM metformin being the optimal dose (151.4 ± 3 μM and 108.0 ± 12 μM respectively). Increased neurite growth of NG108-15 neurones was demonstrated with 100 μM (179.3 ± 2 μM) metformin treatment in 3D EngNT. Additionally, significant neurite growth was demonstrated following treatment with 100 μM (203.9 ± 32 μM) sulindac sulfide. Conclusions A 3D EngNT in vitro co-culture model of Schwann cell myelination was developed illustrating the cell-cell interaction between Schwann cells and neurones and the formation of myelin in the peripheral nervous system. Metformin elicits significant positive results on neurite growth in vitro highlighting its therapeutic potential in PNI.

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

P2X7 and P2Y1 receptors in DRG mediate electroacupuncture to inhibit peripheral sensitization in rats with IBS visceral pain.

Although multiple purinergic receptors mediate the analgesic effects of acupuncture, it remains unclear whether there is mutual interaction between purinergic receptors to jointly mediate the electroacupuncture inhibition of peripheral sensitization in visceral pain. Visceral hypersensitivity was induced by intracolonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rat. The antinociception effect of electroacupuncture on visceral pain was evaluated by morphology, behaviors, neuroelectrophysiology and molecular biology techniques. After labeling the colon-related primary sensory neurons with neural retrograde tracer and employing neuropharmacology, neuroelectrophysiology, and molecular biotechnology, the mechanisms of P2X7R, P2Y1R, and P2X3R in colon-related dorsal root ganglion (DRG) neurons alleviating visceral hypersensitivity of irritable bowel syndrome (IBS) by electroacupuncture at Zusanli and Sanyinjiao acupoints.were elucidated from the perspective of peripheral sensitization. Electroacupuncture significantly inhibited TNBS-induced colonic hypersensitivity in rats with IBS, and Satellite Glial Cells (SGCs) in DRG were found to be involved in electroacupuncture-mediated regulation of the electrophysiological properties of neurons. P2X7R was found to play a pain-inducing role in IBS visceral hypersensitivity by affecting P2X3R, and electroacupuncture exerted an analgesic effect by inhibiting P2X7R activation. P2Y1R was found to play an analgesic role in the process of visceral pain, mediating electroacupuncture to relieve visceral hypersensitivity. P2Y1R relieved visceral pain by inhibiting P2X3R in neurons associated with nociception, with P2X7R identified as upstream of P2Y1R up-regulation by electroacupuncture. Our study suggests that the P2X7R → P2Y1R → P2X3R inhibitory pathway in DRG mediates the inhibition of peripheral sensitization by electroacupuncture in rats with IBS visceral hypersensitivity.

Exosomes From Muscle-Derived Stem Cells Repair Peripheral Nerve Injury by Inhibiting Ferroptosis via the Keap1-Nrf2-Ho-1 Axis.

Currently, the clinical outcomes of peripheral nerve injuries are suboptimal, highlighting the urgent need to understand the mechanisms of nerve injury to enhance treatment strategies. Muscle-derived stem cells (MDSCs) are a diverse group of multipotent cells that hold promise for peripheral nerve regeneration due to their strong antioxidant and regenerative properties. Our research has revealed that severe ferroptosis occurs in the sciatic nerve and ipsilateral dorsal root ganglion following sciatic nerve injury. Interestingly, we have observed that MDSC-derived exosomes effectively suppress cell ferroptosis and enhance cell viability in Schwann cells and dorsal root ganglion cells. Treatment with exosomes led to increased expression of BDNF and P62 in Schwann cells, decreased expression of Keap1, Nrf2, and HO-1 in Schwann cells, and upregulated dorsal root ganglion cells. Rats treated with exosomes exhibited improvements in sciatic nerve function, sensitivity to stimuli, and reduced muscle atrophy, indicating a positive impact on post-injury recovery. In conclusion, our findings demonstrate the occurrence of ferroptosis in the sciatic nerve and dorsal root ganglion post-injury, with MDSC exosomes offering a potential therapeutic strategy by inhibiting ferroptosis, activating the Keap1-Nrf2-HO-1 pathway, and optimizing the post-injury repair environment.

Nerve Growth Factor Signaling Modulates the Expression of Glutaminase in Dorsal Root Ganglion Neurons during Peripheral Inflammation.

Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.

Inhibition and potentiation of the mechanoreflex following pharmacological modulation of TRPC6 in male rats

We determined the role played by the transient receptor potential canonical 6 (TRPC6) in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. Quadruple-labelled immunohistochemistry were used in dorsal root ganglion cells to identify the cells that innervate the triceps surae muscles (DiI), the cells that express TRPC6 and the cells that are (Nf200) or are not myelinated (peripherin) (n=4). In addition, static contraction of triceps surae muscles was evoked by electrically stimulating the left tibial nerve before and after injecting into the superficial epigastric artery the TRPC6 antagonists, BI-749327 (n = 11; 12μg.kg−1) or SAR7334 (n = 11; 7μg.kg−1), or the TRPC6 positive modulator, C20 (n = 11; 18μg.kg−1). Similar experiments were conducted while the triceps surae muscles were passively stretched (n = 8 to 12), a maneuver that isolates the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded throughout the experiments. The neurons innervating the triceps-surae muscles were positive to TRPC6 antigen and co-expressed the neurofilament 200kDa. These cells were not positive for peripherin. While the TRPC6 blockers decreased the pressor response to static contraction (-32 to -42%; P < 0.05) and passive stretch (-35 to 52%; P < 0.05), the positive modulator increased both of these responses (55 to 65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05) whereas C20 increased these responses to stretch only (55 to 235%; P < 0.05). All of these results were not replicated when the drugs were injected intravenously or when the vehicle of the drug was injected into the superficial epigastric artery. Collectively, our results showed that TRPC6 play a key role in evoking the mechanical component of the exercise pressor reflex in male rats. Supported by NIH grants HL 161160, HL 156594 and HL 156513. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analgesic candidate adenosine A 3 receptors are expressed by perineuronal peripheral macrophages in human dorsal root ganglion and spinal cord microglia.

Adenosine receptors are a family of purinergic G protein-coupled receptors that are widely distributed in bodily organs and in the peripheral and central nervous systems. Recently, antihyperalgesic actions have been suggested for the adenosine A 3 receptor, and its agonists have been proposed as new neuropathic pain treatments. We hypothesized that these receptors may be expressed in nociceptive primary afferent neurons. However, RNA sequencing across species, eg, rat, mouse, dog, and human, suggests that dorsal root ganglion (DRG) expression of ADORA3 is inconsistent. In rat and mouse, Adora3 shows very weak to no expression in DRG, whereas it is well expressed in human DRG. However, the cell types in human DRG that express ADORA3 have not been delineated. An examination of DRG cell types using in situ hybridization clearly detected ADORA3 transcripts in peripheral macrophages that are in close apposition to the neuronal perikarya but not in peripheral sensory neurons. By contrast, ADORA1 was found primarily in neurons, where it is broadly expressed at low levels. These results suggest that a more complex or indirect mechanism involving modulation of macrophage and/or microglial cells may underlie the potential analgesic action of adenosine A 3 receptor agonism.

Generalized modality responses in primary sensory neurons of awake mice during the development of neuropathic pain.

Peripheral sensory neurons serve as the initial responders to the external environment. How these neurons react to different sensory stimuli, such as mechanical or thermal forces applied to the skin, remains unclear. Using in vivo two-photon Ca2+ imaging in the lumbar 4 dorsal root ganglion (DRG) of awake Thy1.2-GCaMP6s mice, we assessed neuronal responses to various mechanical (punctate or dynamic) and thermal forces (heat or cold) sequentially applied to the paw plantar surface. Our data indicate that in normal awake male mice, approximately 14 and 38% of DRG neurons respond to either single or multiple modalities of stimulation. Anesthesia substantially reduces the number of responsive neurons but does not alter the ratio of cells exhibiting single-modal responses versus multi-modal responses. Following peripheral nerve injury, DRG cells exhibit a more than 5.1-fold increase in spontaneous neuronal activity and a 1.5-fold increase in sensory stimulus-evoked activity. As neuropathic pain resulting from nerve injury progresses, the polymodal nature of sensory neurons intensifies. The polymodal population increases from 39.1 to 56.9%, while the modality-specific population decreases from 14.7 to 5.0% within a period of 5 days. Our study underscores polymodality as a significant characteristic of primary sensory neurons, which becomes more pronounced during the development of neuropathic pain.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Peripheral nerve-derived Sema3A promotes osteogenic differentiation of mesenchymal stem cells through the Wnt/β-catenin/Nrp1 positive feedback loop.

Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. Invitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/β-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation invivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/β-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis invivo and invitro.