Reduction of TRPV1 expression on neurons due to downregulation of P2X7R in neonatal rat dorsal root ganglion satellite glial cells under co-culture conditions.

BACKGROUND INFORMATION: The purinergic ligand-gated ion channel 7 receptor (P2X7R) is an ATP-gated ion channel that transmits extracellular signals and induces corresponding biological effects, transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that maintains normal physiological functions; numerous studies showed that P2X7R and TRPV1 are associated with inflammatory reactions. RESULTS: The effect of P2X7R knockdown in satellite glial cells (SGCs) on neuronal TRPV1 expression under high glucose and high free fat (HGHF) environment was investigated. P2X7 short hairpin RNA (shRNA) was utilized to downregulate P2X7R in SGCs, and treated and untreated SGCs were co-cultured with neuronal cell lines. The expression levels of inflammatory factors and signaling pathways in SGCs and neurons were measured using Western blot analysis, RT-qPCR, immunofluorescence, and enzyme-linked immunosorbent assays. Results suggested that P2X7 shRNA reduced the expression levels of P2X7R protein and mRNA in SGCs surrounding DRG neurons and downregulated the release of tumor necrosis factor-alpha and interleukin-1 beta via the Ca2+/p38 MAPK/NF-κB pathway. Additionally, the downregulation of P2X7R might decrease TRPV1 expression in neurons via the Ca2+/PKC-ɛ/p38 MAPK pathway. CONCLUSIONS: Reducing P2X7R expression in SCGs in an HGHF environment could decrease neuronal TRPV1 expression via the Ca2+/PKC-ɛ/p38 MAPK pathway.

Sciatic nerve stimulation alleviates neuropathic pain and associated neuroinflammation in the dorsal root ganglia in a rodent model.

BACKGROUND: Satellite glial cells (SGCs) in the dorsal root ganglia (DRG) play a pivotal role in the formation of neuropathic pain (NP). Sciatic nerve stimulation (SNS) neuromodulation was reported to alleviate NP and reduce neuroinflammation. However, the mechanisms underlying SNS in the DRG remain unclear. This study aimed to elucidate the mechanism of electric stimulation in reducing NP, focusing on the DRG. METHODS: L5 nerve root ligation (NRL) NP rat model was studied. Ipsilateral SNS performed 1 day after NRL. Behavioral tests were performed to assess pain phenotypes. NanoString Ncounter technology was used to explore the differentially expressed genes and cellular pathways. Activated SGCs were characterized in vivo and in vitro. The histochemical alterations of SGCs, macrophages, and neurons in DRG were examined in vivo on post-injury day 8. RESULTS: NRL induced NP behaviors including decreased pain threshold and latency on von Frey and Hargreaves tests. We found that following nerve injury, SGCs were hyperactivated, neurotoxic and had increased expression of NP-related ion channels including TRPA1, Cx43, and SGC-neuron gap junctions. Mechanistically, nerve injury induced reciprocal activation of SGCs and M1 macrophages via cytokines including IL-6, CCL3, and TNF-α mediated by the HIF-1α-NF-κB pathways. SNS suppressed SGC hyperactivation, reduced the expression of NP-related ion channels, and induced M2 macrophage polarization, thereby alleviating NP and associated neuroinflammation in the DRG. CONCLUSIONS: NRL induced hyperactivation of SGCs, which had increased expression of NP-related ion channels. Reciprocal activation of SGCs and M1 macrophages surrounding the primary sensory neurons was mediated by the HIF-1α and NF-κB pathways. SNS suppressed SGC hyperactivation and skewed M1 macrophage towards M2. Our findings establish SGC activation as a crucial pathomechanism in the gliopathic alterations in NP, which can be modulated by SNS neuromodulation.

Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain.

BACKGROUND: The channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood. METHODS: Wild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund's adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and ß-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. ß-galactosidase (ß-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice. RESULTS: Global or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/ß-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/ß-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of ß-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG. CONCLUSIONS: The present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

Annulus Fibrosus Injury Induces Acute Neuroinflammation and Chronic Glial Response in Dorsal Root Ganglion and Spinal Cord-An In Vivo Rat Discogenic Pain Model.

Chronic painful intervertebral disc (IVD) degeneration (i.e., discogenic pain) is a major source of global disability needing improved knowledge on multiple-tissue interactions and how they progress in order improve treatment strategies. This study used an in vivo rat annulus fibrosus (AF) injury-driven discogenic pain model to investigate the acute and chronic changes in IVD degeneration and spinal inflammation, as well as sensitization, inflammation, and remodeling in dorsal root ganglion (DRG) and spinal cord (SC) dorsal horn. AF injury induced moderate IVD degeneration with acute and broad spinal inflammation that progressed to DRG to SC changes within days and weeks, respectively. Specifically, AF injury elevated macrophages in the spine (CD68) and DRGs (Iba1) that peaked at 3 days post-injury, and increased microglia (Iba1) in SC that peaked at 2 weeks post-injury. AF injury also triggered glial responses with elevated GFAP in DRGs and SC at least 8 weeks post-injury. Spinal CD68 and SC neuropeptide Substance P both remained elevated at 8 weeks, suggesting that slow and incomplete IVD healing provides a chronic source of inflammation with continued SC sensitization. We conclude that AF injury-driven IVD degeneration induces acute spinal, DRG, and SC inflammatory crosstalk with sustained glial responses in both DRGs and SC, leading to chronic SC sensitization and neural plasticity. The known association of these markers with neuropathic pain suggests that therapeutic strategies for discogenic pain need to target both spinal and nervous systems, with early strategies managing acute inflammatory processes, and late strategies targeting chronic IVD inflammation, SC sensitization, and remodeling.

Store-operated calcium entry in the satellite glial cells of rat sympathetic ganglia.

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca2+ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca2+ entry (SOCE) which is critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca2+ stores were depleted in the absence of extracellular Ca2+, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca2+ entry channels. Intracellular Ca2+ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca2+ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

The Role of Glial Cells in Different Phases of Migraine: Lessons from Preclinical Studies.

Migraine is a complex and debilitating neurological disease that affects 15% of the population worldwide. It is defined by the presence of recurrent severe attacks of disabling headache accompanied by other debilitating neurological symptoms. Important advancements have linked the trigeminovascular system and the neuropeptide calcitonin gene-related peptide to migraine pathophysiology, but the mechanisms underlying its pathogenesis and chronification remain unknown. Glial cells are essential for the correct development and functioning of the nervous system and, due to its implication in neurological diseases, have been hypothesised to have a role in migraine. Here we provide a narrative review of the role of glia in different phases of migraine through the analysis of preclinical studies. Current evidence shows that astrocytes and microglia are involved in the initiation and propagation of cortical spreading depolarization, the neurophysiological correlate of migraine aura. Furthermore, satellite glial cells within the trigeminal ganglia are implicated in the initiation and maintenance of orofacial pain, suggesting a role in the headache phase of migraine. Moreover, microglia in the trigeminocervical complex are involved in central sensitization, suggesting a role in chronic migraine. Taken altogether, glial cells have emerged as key players in migraine pathogenesis and chronification and future therapeutic strategies could be focused on targeting them to reduce the burden of migraine.

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

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

A systematic literature review on the role of glial cells in the pathomechanisms of migraine.

Background: The pathomechanisms underlying migraine are intricate and remain largely unclear. Initially regarded as a neuronal disorder, migraine research primarily concentrated on understanding the pathophysiological changes within neurons. However, recent advances have revealed the significant involvement of neuroinflammation and the neuro-glio-vascular interplay in migraine pathogenesis. Methods: A systematic search was conducted in PubMed, Scopus, and Web of Science databases from their inception until November 2022. The retrieved results underwent a screening process based on title and abstract, and the full texts of the remaining papers were thoroughly assessed for eligibility. Only studies that met the predetermined inclusion criteria were included in the review. Results: Fifty-nine studies, consisting of 6 human studies and 53 animal studies, met the inclusion criteria. Among the 6 human studies, 2 focused on genetic analyses, while the remaining studies employed functional imaging, serum analyses and clinical trials. Regarding the 53 animal studies investigating glial cells in migraine, 19 of them explored the role of satellite glial cells and/or Schwann cells in the trigeminal ganglion and/or trigeminal nerve. Additionally, 17 studies highlighted the significance of microglia and/or astrocytes in the trigeminal nucleus caudalis, particularly in relation to central sensitization during migraine chronification. Furthermore, 17 studies examined the involvement of astrocytes and/or microglia in the cortex. Conclusion: Glial cells, including astrocytes, microglia, satellite glial cells and Schwann cells in the central and peripheral nervous system, participate both in the development as well as chronic progression of migraine in disease-associated regions such as the trigeminovascular system, trigeminal nucleus caudalis and cortex, among other brain regions.

Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia.

Activation of neurons and glial cells in the dorsal root ganglion is one of the key mechanisms for the development of hyperalgesia. The aim of the present study was to examine the role of neuroglial activity in the development of opioid-induced hyperalgesia. Male rats were treated with morphine daily for 3 days. The resultant phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the dorsal root ganglion was analyzed by immunohistochemistry and Western blotting. Pain hypersensitivity was analyzed using behavioral studies. The amount of cytokine expression in the dorsal root ganglion was also analyzed. Repeated morphine treatment induced hyperalgesia and marked induction of phosphorylated ERK1/2 in the neurons and satellite glial cells on day 3. An opioid receptor antagonist, toll like receptor-4 inhibitor, MAP/ERK kinase (MEK) inhibitor and gap junction inhibitor inhibited morphine-induced hyperalgesia and ERK1/2 phosphorylation. Morphine treatment induced alteration of cytokine expression, which was inhibited by the opioid receptor antagonist, toll like receptor-4 inhibitor, MEK inhibitor and gap junction inhibitor. Dexamethasone inhibited morphine-induced hyperalgesia and ERK1/2 phosphorylation after morphine treatment. The peripherally restricted opioid receptor antagonist, methylnaltrexone, inhibited hyperalgesia and ERK1/2 phosphorylation. Morphine activates ERK1/2 in neurons and satellite glial cells in the dorsal root ganglion via the opioid receptor and toll like receptor-4. ERK1/2 phosphorylation is gap junction-dependent and is associated with the alteration of cytokine expression. Inhibition of neuroinflammation by activation of neurons and glia might be a promising target to prevent opioid-induced hyperalgesia.

Central role of purinergic receptors with inflammation in neuropathic pain-related macrophage-SGC-neuron triad.

Adenosine triphosphate (ATP) acts on P2 purinergic receptors as an extracellular signaling molecule. P2 purinergic receptors include P2X ionotropic receptors and P2Y metabotropic receptors. Satellite glial cells (SGCs) and macrophages express P2X and P2Y receptors. Inflammatory cytokines and pro-nociceptive mediators are released by activated macrophages and SGCs, which can act on neurons to promote excitability and firing. In the primary sensory ganglia, in response to signals of injury, SGCs and macrophages accumulate around primary sensory neurons, forming a macrophage-SGC-neuron triad. In addition to affecting the pathological alterations of inflammation-related neuropathic pain, inflammatory cytokines and pro-nociceptive mediators are released by the action of ATP on P2X and P2Y receptors in macrophages and SGCs. Macrophages and SGCs work together to enhance and prolong neuropathic pain. The macrophage-SGC-neuron triad communicates with each other through ATP and other inflammatory mediators and maintains and promotes the initiation and development of inflammation related-neuropathic pain. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

Integrative Analysis Reveals the Expression Pattern of SOX9 in Satellite Glial Cells after Sciatic Nerve Injury.

BACKGROUND: Several complex cellular and gene regulatory processes are involved in peripheral nerve repair. This study uses bioinformatics to analyze the differentially expressed genes (DEGs) in the satellite glial cells of mice following sciatic nerve injury. METHODS: R software screens differentially expressed genes, and the WebGestalt functional enrichment analysis tool conducts Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis. The Search Tool for the Retrieval of Interacting Genes/Proteins constructs protein interaction networks, and the cytoHubba plug-in in the Cytoscape software predicts core genes. Subsequently, the sciatic nerve injury model of mice was established and the dorsal root ganglion satellite glial cells were isolated and cultured. Satellite glial cells-related markers were verified by immunofluorescence staining. Real-time polymerase chain reaction assay and Western blotting assay were used to detect the mRNA and protein expression of Sox9 in satellite glial cells. RESULTS: A total of 991 DEGs were screened, of which 383 were upregulated, and 508 were downregulated. The GO analysis revealed the processes of biosynthesis, negative regulation of cell development, PDZ domain binding, and other biological processes were enriched in DEGs. According to the KEGG pathway analysis, DEGs are primarily involved in steroid biosynthesis, hedgehog signaling pathway, terpenoid backbone biosynthesis, American lateral skeleton, and melanoma pathways. According to various cytoHubba algorithms, the common core genes in the protein-protein interaction network are Atf3, Mmp2, and Sox9. Among these, Sox9 was reported to be involved in the central nervous system and the generation and development of astrocytes and could mediate the transformation between neurogenic and glial cells. The experimental results showed that satellite glial cell marker GS were co-labeled with Sox9; stem cell characteristic markers Nestin and p75NTR were labeled satellite glial cells. The mRNA and protein expression of Sox9 in satellite glial cells were increased after sciatic nerve injury. CONCLUSIONS: In this study, bioinformatics was used to analyze the DEGs of satellite glial cells after sciatic nerve injury, and transcription factors related to satellite glial cells were screened, among which Sox9 may be associated with the fate of satellite glial cells.

Satellite glial cells drive the transition from acute to chronic pain in a rat model of hyperalgesic priming.

Chronic pain is one of the most common clinical syndromes affecting patients' quality of life. Regulating the transition from acute to chronic pain is a novel therapeutic strategy for chronic pain that presents a major clinical challenge. However, the mechanism underlying pain transitions remains poorly understood. A rat hyperalgesic priming (HP) model, which mimics pain transition, was established decades ago. Here, this HP model and RNA sequencing (RNA-seq) were used to study the potential role of neuroinflammation in pain transition. In this study, HP model rats developed prolonged hyperalgesia in the hind paw after carrageenan (Car) and PGE2 injection, accompanied by obvious satellite glial cell (SGC) activation in the dorsal root ganglion (DRG), as indicated by upregulation of GFAP. RNA-Seq identified a total of differentially expressed genes in the ipsilateral DRG in HP model rats. The expression of several representative genes was confirmed by real-time quantitative PCR (qPCR). Functional analysis of the differentially expressed genes indicated that genes related to the inflammatory and neuroinflammatory response showed the most significant changes in expression. We further found that the expression of the chemokine CXCL1 was significantly upregulated in the rat DRG. Pharmacological blockade of CXCL1 reduced protein kinase C epsilon overproduction as well as hyperalgesia in HP rats but did not prevent the upregulation of GFAP in the DRG. These results reveal that neuroinflammatory responses are involved in pain transition and may be the source of chronic pain. The chemokine CXCL1 in the DRG is a pivotal contributor to chronic pain and pain transition in HP model rats. Thus, our study provides a putative novel target for the development of effective therapeutics to prevent pain transition.

Single-cell transcriptomic profile of satellite glial cells in trigeminal ganglion.

Satellite glial cells (SGCs) play an important role in regulating the function of trigeminal ganglion (TG) neurons. Multiple mediators are involved in the bidirectional communication between SGCs and neurons in different physiological and pathological states. However, molecular insights into the transcript characteristics of SGCs are limited. Moreover, little is known about the heterogeneity of SGCs in TG, and a more in-depth understanding of the interactions between SGCs and neuron subtypes is needed. Here we show the single-cell RNA sequencing (scRNA-seq) profile of SGCs in TG under physiological conditions. Our results demonstrate TG includes nine types of cell clusters, such as neurons, SGCs, myeloid Schwann cells (mSCs), non-myeloid Schwann cells (nmSCs), immune cells, etc., and the corresponding markers are also presented. We reveal the signature gene expression of SGCs, mSCs and nmSCs in the TG, and analyze the ligand-receptor pairs between neuron subtypes and SGCs in the TG. In the heterogeneity analysis of SGCs, four SGCs subtypes are identified, including subtypes enriched for genes associated with extracellular matrix organization, immediate early genes, interferon beta, and cell adhesion molecules, respectively. Our data suggest the molecular characteristics, heterogeneity of SGCs, and bidirectional interactions between SGCs and neurons, providing a valuable resource for studying SGCs in the TG.

Specific Cellular Effects of Low Bortezomib Concentrations on Purified Cultures of Schwann Cells, Satellite Glial Cells, Macrophages, and Dorsal Root Ganglion Neurons.

Peripheral neuropathy is one of the major adverse effects that limit the clinical application of bortezomib (BTZ). However, the underlying mechanisms of BTZ-induced peripheral neuropathy (BIPN) remain elusive. To examine cell types potentially involved in the development of BIPN, we used four purified cultures of cells of the peripheral nervous system: Schwann cells (SCs), satellite glial cells (SGCs), macrophages, and dorsal root ganglion (DRG) neurons. Administration of a low BTZ concentration (5 nM; similar to concentrations in clinical use) caused dedifferentiation of cultured SCs, returning mature SCs to an immature state. In cultured SGCs, BTZ increased glial fibrillary acidic protein (GFAP) levels without inducing the release of inflammatory cytokines or chemokines. In macrophages, BTZ caused little inflammatory response. Finally, in DRG neurons, BTZ strongly suppressed the expression levels of sensor and transducer ion channels without affecting cell morphology. Taken together, low concentrations of BTZ can cause SC dedifferentiation (i.e., demyelination), increased GFAP level in SGC, and decreased expression levels of sensor and transducer ion channels in DRG neurons (i.e., numbness feeling). Thus, we have reported, for the first time, specific effects of BTZ on peripheral nervous system cells, thereby contributing to a better understanding of the initiating mechanism of BIPN.

Neohesperidin Alleviates the Neuropathic Pain Behavior of Rats by Downregulating the P2X4 Receptor.

Neuropathic pain (NP) is a type of chronic pain affecting 6-8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1ß. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain.

Introduction: Satellite glial cells (SGCs) that envelop the cell bodies of neurons in sensory ganglia have been shown to both release glutamate, and be activated by glutamate in the context of nociceptive signaling. However, little is known about the subpopulations of SGCs that are activated following nerve injury and whether glutamate mechanisms in the SGCs are involved in the pathologic pain. Methods: To address this issue, we used light and electron microscopic immunohistochemistry to examine the change in the glutamate levels in the SGCs and the structural relationship between neighboring neurons in the trigeminal ganglion (TG) in a rat model of craniofacial neuropathic pain, CCI-ION. Results: Administration of ionomycin, ATP and Bz-ATP induced an increase of extracellular glutamate concentration in cultured trigeminal SGCs, indicating a release of glutamate from SGCs. The level of glutamate immunostaining in the SGCs that envelop neurons of all sizes in the TG was significantly higher in rats with CCI-ION than in control rats, suggesting that SGCs enveloping nociceptive as well as non-nociceptive mechanosensitive neurons are activated following nerve injury, and that the glutamate release from SGCs increases in pathologic pain state. Close appositions between substance-P (SP)-immunopositive (+) or calcitonin gene-related peptide (CGRP)+, likely nociceptive neurons, between Piezo1+, likely non-nociceptive, mechanosensitive neurons and SP+ or CGRP+ neurons, and between SGCs of neighboring neurons were frequently observed. Discussion: These findings suggest that glutamate in the trigeminal SGCs that envelop all types of neurons may play a role in the mechanisms of neuropathic pain, possibly via paracrine signaling.

Silencing P2X7R Alleviates Diabetic Neuropathic Pain Involving TRPV1 via PKCε/P38MAPK/NF-κB Signaling Pathway in Rats.

Transient receptor potential vanillic acid 1 (TRPV1) is an ion channel activated by heat and inflammatory factors involved in the development of various types of pain. The P2X7 receptor is in the P2X family and is associated with pain mediated by satellite glial cells. There might be some connection between the P2X7 receptor and TRPV1 in neuropathic pain in diabetic rats. A type 2 diabetic neuropathic pain rat model was induced using high glucose and high-fat diet for 4 weeks and low-dose streptozocin (35 mg/kg) intraperitoneal injection to destroy islet B cells. Male Sprague Dawley rats were administrated by intrathecal injection of P2X7 shRNA and p38 inhibitor, and we recorded abnormal mechanical and thermal pain and nociceptive hyperalgesia. One week later, the dorsal root ganglia from the L4-L6 segment of the spinal cord were harvested for subsequent experiments. We measured pro-inflammatory cytokines, examined the relationship between TRPV1 on neurons and P2X7 receptor on satellite glial cells by measuring protein and transcription levels of P2X7 receptor and TRPV1, and measured protein expression in the PKCε/P38 MAPK/NF-κB signaling pathway after intrathecal injection. P2X7 shRNA and p38 inhibitor relieved hyperalgesia in diabetic neuropathic pain rats and modulated inflammatory factors in vivo. P2X7 shRNA and P38 inhibitors significantly reduced TRPV1 expression by downregulating the PKCε/P38 MAPK/NF-κB signaling pathway and inflammatory factors in dorsal root ganglia. Intrathecal injection of P2X7 shRNA alleviates nociceptive reactions in rats with diabetic neuropathic pain involving TRPV1 via PKCε/P38 MAPK/NF-κB signaling pathway.

Loss of the chromatin remodeler CHD7 impacts glial cells and myelination in the mouse cochlear spiral ganglion.

CHARGE syndrome is a multiple anomaly developmental disorder characterized by a variety of sensory deficits, including sensorineural hearing loss of unknown etiology. Most cases of CHARGE are caused by heterozygous pathogenic variants in CHD7, the gene encoding Chromodomain DNA-binding Protein 7 (CHD7), a chromatin remodeler important for the development of neurons and glial cells. Previous studies in the Chd7Gt/+ mouse model of CHARGE syndrome showed substantial neuron loss in the early stages of the developing inner ear that are compensated for by mid-gestation. In this study, we sought to determine if early developmental delays caused by Chd7 haploinsufficiency affect neurons, glial cells, and inner hair cell innervation in the mature cochlea. Analysis of auditory brainstem response recordings in Chd7Gt/+ adult animals showed elevated thresholds at 4 kHz and 16 kHz, but no differences in ABR Wave I peak latency or amplitude compared to wild type controls. Proportions of neurons in the Chd7Gt/+ adult spiral ganglion and densities of nerve projections from the spiral ganglion to the organ of Corti were not significantly different from wild type controls. Inner hair cell synapse formation also appeared unaffected in mature Chd7Gt/+ cochleae. However, histological analysis of adult Chd7Gt/+ cochleae revealed diminished satellite glial cells and hypermyelinated Type I spiral ganglion axons. We characterized the expression of CHD7 in developing inner ear glia and found CHD7 to be expressed during a tight window of inner ear development at the Schwann cell precursor stage at E9.5. While cochlear neurons appear to differentiate normally in the setting of Chd7 haploinsufficiency, our results suggest an important role for CHD7 in glial cells in the inner ear. This study highlights the dynamic nature of CHD7 activity during inner ear development in mice and contributes to understanding CHARGE syndrome pathology.

SEPT9 Upregulation in Satellite Glial Cells Associated with Diabetic Polyneuropathy in a Type 2 Diabetes-like Rat Model.

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.

BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo.

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.