Skin keratinocyte-derived SIRT1 and BDNF modulate mechanical allodynia in mouse models of diabetic neuropathy.

Diabetic neuropathy is a debilitating disorder characterized by spontaneous and mechanical allodynia. The role of skin mechanoreceptors in the development of mechanical allodynia is unclear. We discovered that mice with diabetic neuropathy had decreased sirtuin 1 (SIRT1) deacetylase activity in foot skin, leading to reduced expression of brain-derived neurotrophic factor (BDNF) and subsequent loss of innervation in Meissner corpuscles, a mechanoreceptor expressing the BDNF receptor TrkB. When SIRT1 was depleted from skin, the mechanical allodynia worsened in diabetic neuropathy mice, likely due to retrograde degeneration of the Meissner-corpuscle innervating Aß axons and aberrant formation of Meissner corpuscles which may have increased the mechanosensitivity. The same phenomenon was also noted in skin-keratinocyte specific BDNF knockout mice. Furthermore, overexpression of SIRT1 in skin induced Meissner corpuscle reinnervation and regeneration, resulting in significant improvement of diabetic mechanical allodynia. Overall, the findings suggested that skin-derived SIRT1 and BDNF function in the same pathway in skin sensory apparatus regeneration and highlighted the potential of developing topical SIRT1-activating compounds as a novel treatment for diabetic mechanical allodynia.

Dysfunctional astrocyte glutamate uptake in the hypothalamic paraventricular nucleus contributes to visceral pain and anxiety-like behavior in mice with chronic pancreatitis.

Abdominal visceral pain is a predominant symptom in patients with chronic pancreatitis (CP); however, the underlying mechanism of pain in CP remains elusive. We hypothesized that astrocytes in the hypothalamic paraventricular nucleus (PVH) contribute to CP pain pathogenesis. A mouse model of CP was established by repeated intraperitoneal administration of caerulein to induce abdominal visceral pain. Abdominal mechanical stimulation, open field and elevated plus maze tests were performed to assess visceral pain and anxiety-like behavior. Fiber photometry, brain slice Ca2+ imaging, electrophysiology, and immunohistochemistry were used to investigate the underlying mechanisms. Mice with CP displayed long-term abdominal mechanical allodynia and comorbid anxiety, which was accompanied by astrocyte glial fibrillary acidic protein reactivity, elevated Ca2+ signaling, and astroglial glutamate transporter-1 (GLT-1) deficits in the PVH. Specifically, reducing astrocyte Ca2+ signaling in the PVH via chemogenetics significantly rescued GLT-1 deficits and alleviated mechanical allodynia and anxiety in mice with CP. Furthermore, we found that GLT-1 deficits directly contributed to the hyperexcitability of VGLUT2PVH neurons in mice with CP, and that pharmacological activation of GLT-1 alleviated the hyperexcitability of VGLUT2PVH neurons, abdominal visceral pain, and anxiety in these mice. Taken together, our data suggest that dysfunctional astrocyte glutamate uptake in the PVH contributes to visceral pain and anxiety in mice with CP, highlighting GLT-1 as a potential therapeutic target for chronic pain in patients experiencing CP.

Exploring altered oscillatory activity in the anterior cingulate cortex after nerve injury: Insights into mechanisms of neuropathic allodynia.

While neural oscillations play a critical role in sensory perception, it remains unclear how these rhythms function under conditions of neuropathic allodynia. Recent studies demonstrated that the anterior cingulate cortex (ACC) is associated with the affective-aversive component of pain, and plasticity changes in this region are closely linked to abnormal allodynic sensations. Here, to study the mechanisms of allodynia, we recorded local field potentials (LFPs) in the bilateral ACC of awake-behaving rats and compared the spectral power and center frequency of brain oscillations between healthy and CCI (chronic constriction injury) induced neuropathic pain conditions. Our results indicated that activation of the ACC occurs bilaterally in the presence of neuropathic pain, similar to the healthy condition. Furthermore, CCI affects both spontaneous and stimulus-induced activity of ACC neurons. Specifically, we observed an increase in spontaneous beta activity after nerve injury compared to the healthy condition. By stimulating operated or unoperated paws, we found more intense event-related desynchronization (ERD) responses in the theta, alpha, and beta frequency bands and faster alpha center frequency after CCI compared to before CCI. Although the behavioral manifestation of allodynia was more pronounced in the operated paw than the unoperated paw following CCI, there was no significant difference in the center frequency and ERD responses observed in the ACC between stimulation of the operated and unoperated limbs. Our findings offer evidence supporting the notion that aberrancies in ACC oscillations may contribute to the maintenance and development of neuropathic allodynia.

TNFR1/p38αMAPK signaling in Nex + supraspinal neurons regulates estrogen-dependent chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP). Previously, we have shown that estrogen modulates sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP. The estrogen-dependent role of TNFR1-mediated supraspinal neuronal circuitry in CNP remains unknown. In this study, we interrogated the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that mechanical hypersensitivity induced by chronic constriction injury (CCI) decreases over time in males, but not in females. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38MAPK and NF-κB activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed a similar recovery from acute pain in male mice following CCI when p38αMAPK was knocked out of supraspinal Nex + neurons (NexCreERT2::p38αMAPKf/f), while chronic pain developed in female mice. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lends therapeutic relief to females following CCI comparable to the response evaluated in male mice. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

Attenuation of Streptozotocin-Induced Diabetic Neuropathic Allodynia by Flavone Derivative Through Modulation of GABA-ergic Mechanisms and Endogenous Biomarkers.

Diabetic neuropathic pain is one of the most devasting disorders of peripheral nervous system. The loss of GABAergic inhibition is associated with the development of painful diabetic neuropathy. The current study evaluated the potential of 3-Hydroxy-2-methoxy-6-methyl flavone (3-OH-2'MeO6MF), to ameliorate peripheral neuropathic pain using an STZ-induced hyperglycemia rat model. The pain threshold was assessed by tail flick, cold, mechanical allodynia, and formalin test on days 0, 14, 21, and 28 after STZ administration accompanied by evaluation of several biochemical parameters. Administration of 3-OH-2'-MeO6MF (1,10, 30, and 100 mg/kg, i.p) significantly enhanced the tail withdrawal threshold in tail-flick and tail cold allodynia tests. 3-OH-2'-MeO6MF also increased the paw withdrawal threshold in mechanical allodynia and decreased paw licking time in the formalin test. Additionally, 3-OH-2'-MeO6MF also attenuated the increase in concentrations of myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), nitrite, TNF-α, and IL 6 along with increases in glutathione (GSH). Pretreatment of pentylenetetrazole (PTZ) (40 mg/kg, i.p.) abolished the antinociceptive effect of 3-OH-2'-MeO6MF in mechanical allodynia. Besides, the STZ-induced alterations in the GABA concentration and GABA transaminase activity attenuated by 3-OH-2'-MeO6MF treatment suggest GABAergic mechanisms. Molecular docking also authenticates the involvement of α2ß2γ2L GABA-A receptors and GABA-T enzyme in the antinociceptive activities of 3-OH-2'-MeO6MF.

A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior.

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aß fibers. However, the mechanism by which Aß fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP+) and show that specific ablation or silencing of AAV-NpyP+ SDH interneurons converted touch-sensing Aß fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP+ neurons received excitatory inputs from Aß fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP+ neurons exhibited deeper resting membrane potentials, and their excitation by Aß fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP+ neurons in nerve-injured rats reversed Aß fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aß fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats.

BACKGROUND: GABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms. METHODS: Wistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened. RESULTS: Unlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats. CONCLUSION: Our study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine.

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

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

Resolvin D1 attenuates mechanical allodynia after burn injury: Involvement of spinal glia, p38 mitogen-activated protein kinase, and brain-derived neurotrophic factor/tropomyosin-related kinase B signaling.

Resolvin D1 (RvD1) suppresses inflammatory, postoperative, and neuropathic pain. The present study assessed the roles and mechanisms of RvD1 in mechanical allodynia after burn injury. A rat model of burn injury was established for analyses, and RvD1 was injected intraperitoneally. Pain behavior and the expression levels of spinal dorsal horn Iba-1 (microglia marker), GFAP (astrocyte marker), p-p38 mitogen-activated protein kinase (MAPK), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase B (TrkB) were detected by behavioral and immunocytochemical assays. The results showed that RvD1 attenuated mechanical allodynia after burn injury, prevented microglial and astroglial activation, and downregulated p-p38 MAPK in microglia and BDNF/TrkB following burn injury. Similarly, inhibition of p38 MAPK and BDNF/TrkB signaling attenuated mechanical allodynia after burn injury. In addition, inhibition of p38 MAPK prevented spinal microglial activation and downregulated BDNF/TrkB following burn injury. Furthermore, inhibition of BDNF/TrkB signaling prevented spinal microglial activation and downregulated p-p38 MAPK within spinal microglia. Taken together, this study demonstrated that RvD1 might attenuate mechanical allodynia after burn injury by inhibiting spinal cord glial activation, microglial p38 MAPK, and BDNF/TrkB signaling in the spinal dorsal horn.

HSV-1 infection-induced herpetic neuralgia involves a CCL5/CCR5-mediated inflammation mechanism.

Herpetic-related neuralgia (HN) caused by varicella-zoster virus (VZV) infection is one of the most typical and common neuropathic pain in the clinic. However, the potential mechanisms and therapeutic approaches for the prevention and treatment of HN are still unclear. This study aims to provide a comprehensive understanding of the molecular mechanisms and potential therapeutic targets of HN. We used an HSV-1 infection-induced HN mouse model and screened the differentially expressed genes (DEGs) in the DRG and spinal cord using an RNAseq technique. Moreover, bioinformatics methods were used to figure out the signaling pathways and expression regulation patterns of the DEGs enriched. In addition, quantitative real-time RT-PCR and western blot were carried out to further confirm the expression of DEGs. HSV-1 inoculation in mice resulted in mechanical allodynia, thermal hyperalgesia, and cold allodynia, following the infection of HSV-1 in both DRG and spinal cord. Besides, HSV-1 inoculation induced an up-regulation of ATF3, CGRP, and GAL in DRG and activation of astrocytes and microglia in the spinal cord. Moreover, 639 genes were upregulated, 249 genes were downregulated in DRG, whereas 534 genes were upregulated and 12 genes were downregulated in the spinal cord of mice 7 days after HSV-1 inoculation. GO and KEGG enrichment analysis suggested that immune responses and cytokine-cytokine receptor interaction are involved in DRG and spinal cord neurons in mice after HSV-1 infection. In addition, CCL5 and its receptor CCR5 were significantly upregulated in DRG and spinal cord upon HSV-1 infection in mice. And blockade of CCR5 exhibited a significant analgesic effect and suppressed the upregulation of inflammatory cytokines in DRG and spinal cord induced by HSV-1 infection in mice. HSV-1 infection-induced allodynia and hyperalgesia in mice through dysregulation of immune response and cytokine-cytokine receptor interaction mechanism. Blockade of CCR5 alleviated allodynia and hyperalgesia probably through the suppression of inflammatory cytokines. Therefore, CCR5 could be a therapeutic target for the alleviation of HSV-1 infection-induced HN.

Perineural treatment with anti-TNF-α antibody ameliorates persistent allodynia and edema in novel mouse models with complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is an intractable chronic pain syndrome with various signs and symptoms including allodynia/hyperalgesia, edema, swelling, and skin abnormalities. However, a definitive therapeutic treatment for CRPS has not been established. In CRPS patients, inflammatory cytokines such as TNF-α and IL-1ß have been shown to increase in affected areas, suggesting that these molecules may be potential therapeutic targets for CRPS. Here, we first created a novel CRPS mouse model (CRPS-II-like) via sciatic nerve injury and cast immobilization, which was characterized by mechanical allodynia, local edema, and skin abnormalities, to evaluate the pathophysiology and pharmacotherapy of CRPS. When an anti-TNF-α antibody was consecutively administered near the injured sciatic nerve of CRPS model mice, persistent allodynia and CRPS-related signs in the ipsilateral hindpaw were markedly attenuated to control levels. Perineural administration of anti-TNF-α antibody also suppressed the upregulation of inflammatory cytokines as well as the activation of macrophages and Schwann cells in the injured sciatic nerve. These findings indicate that persistent allodynia and CRPS-related signs in CRPS models are primarily associated with TNF-α-mediated immune responses in injured peripheral nerves, suggesting that perineural treatment with anti-TNF-α antibody might be therapeutically useful.

Quantitative Dynamic Allodynograph-A Standardized Measure for Testing Dynamic Mechanical Allodynia in Chronic Limb Pain.

BACKGROUND: Dynamic mechanical allodynia (DMA) is both a symptom and a central sensitization sign, yet no standardized method for quantifying the DMA area has been reported. This study aimed to establish psychometric properties for Quantitative Dynamic Allodynography (QDA), a newly developed protocol measuring the DMA area as a percentage of the body surface. METHODS: Seventy-eight patients aged 18-65 diagnosed with chronic complex regional pain syndrome (CRPS) participated in this study. Test-retest reliability was conducted twice, one week apart (N = 20), and inter-rater (N = 3) reliability was conducted on 10 participants. Disease severity (CRPS Severity Score, CSS), pain intensity (VAS), and quality of life (SF-36) measures were utilized to test construct validity. RESULTS: High inter-rater reliability (intraclass correlation coefficient (ICC) = 0.96, p < 0.001) and test-retest reliability (r = 0.98, p < 0.001) were found. Furthermore, the QDA score was found to be correlated with the CSS (r = 0.47, p < 0.001), VAS (r = 0.37, p < 0.001), and the SF-36 physical health total (r = -0.47, p < 0.001) scores. CONCLUSION: The QDA is the first developed reliable and valid protocol for measuring DMA in a clinical setting and may be used as a diagnostic and prognostic measure in clinics and in research, advancing the pain precision medicine approach.

Microglia and p38 MAPK Inhibitors Suppress Development of Mechanical Allodynia in Both Sexes in a Mouse Model of Antiretroviral-Induced Neuropathic Pain.

Microglia activation in the spinal cord play a major role in the pathogenesis of neuropathic pain. The p38 mitogen-activated protein kinase (MAPK) regulates microglia activation. Previously, 2',3'-dideoxycytidine (ddC), a nucleoside reverse transcriptase inhibitor (NRTI), was found to induce mechanical allodynia and microglia activation in the spinal cords of male and female mice. In this study, we investigated the role of spinal microglia and p38 MAPK signaling in the development of mechanical allodynia using immunofluorescence staining and treatment with microglia and p38 MAPK inhibitors in both sexes. Male and female mice (BALB/c strain) treated intraperitoneally once daily with ddC 25 mg/kg for five consecutive days developed mechanical allodynia, assessed using the dynamic plantar aesthesiometer. Treatment with ddC increased microglia markers CD11b and ionized calcium-binding adapter molecule 1 (Iba1) staining intensity in male mice, while only CD11b was increased in female mice. Both sexes had increased phosphorylated p38 MAPK staining intensity. The administration of minocycline, an inhibitor of microglia activation, and adezmapimod, a selective p38 MAPK inhibitor, suppressed mechanical allodynia in both sexes at day 7 after ddC treatment. Therefore, microglia activation and p38 MAPK signaling are important for the development of antiretroviral drug-induced mechanical allodynia.

Activation of 5-HT1A Receptors Normalizes the Overexpression of Presynaptic 5-HT1A Receptors and Alleviates Diabetic Neuropathic Pain.

Neuropathic pain is a well-documented phenomenon in experimental and clinical diabetes; however, current treatment is unsatisfactory. Serotoninergic-containing neurons are key components of the descending autoinhibitory pathway, and a decrease in their activity may contribute at least in part to diabetic neuropathic pain (DNP). A streptozotocin (STZ)-treated rat was used as a model for type 1 diabetes mellitus (T1DM). Pain transmission was evaluated using well-established nociceptive-based techniques, including the Hargreaves apparatus, cold plate and dynamic plantar aesthesiometer. Using qRT-PCR, Western blotting, immunohistochemistry, and HPLC-based techniques, we also measured in the central nervous system and peripheral nervous system of diabetic animals the expression and localization of 5-HT1A receptors (5-HT1AR), levels of key enzymes involved in the synthesis and degradation of tryptophan and 5-HT, including tryptophan hydroxylase-2 (Tph-2), tryptophan 2,3-dioxygenase (Tdo), indoleamine 2,3-dioxygenase 1 (Ido1) and Ido2. Moreover, spinal concentrations of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA, a metabolite of 5-HT) and quinolinic acid (QA, a metabolite of tryptophan) were also quantified. Diabetic rats developed thermal hyperalgesia and cold/mechanical allodynia, and these behavioral abnormalities appear to be associated with the upregulation in the levels of expression of critical molecules related to the serotoninergic nervous system, including presynaptic 5-HT1AR and the enzymes Tph-2, Tdo, Ido1 and Ido2. Interestingly, the level of postsynaptic 5-HT1AR remains unaltered in STZ-induced T1DM. Chronic treatment of diabetic animals with 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT), a selective 5-HT1AR agonist, downregulated the upregulation of neuronal presynaptic 5-HT1AR, increased spinal release of 5-HT (↑ 5-HIAA/5-HT) and reduced the concentration of QA, decreased mRNA expression of Tdo, Ido1 and Ido2, arrested neuronal degeneration and ameliorated pain-related behavior as exemplified by thermal hyperalgesia and cold/mechanical allodynia. These data show that 8-OH-DPAT alleviates DNP and other components of the serotoninergic system, including the ratio of 5-HIAA/5-HT and 5-HT1AR, and could be a useful therapeutic agent for managing DNP.

Extracellular ATP and cAMP signaling promote Piezo2-dependent mechanical allodynia after trigeminal nerve compression injury.

Trigeminal neuralgia (TN) is a type of severe paroxysmal neuropathic pain commonly triggered by mild mechanical stimulation in the orofacial area. Piezo2, a mechanically gated ion channel that mediates tactile allodynia in neuropathic pain, can be potentiated by a cyclic adenosine monophosphate (cAMP)-dependent signaling pathway that involves the exchange protein directly activated by cAMP 1 (Epac1). To study whether Piezo2-mediated mechanotransduction contributes to peripheral sensitization in a rat model of TN after trigeminal nerve compression injury, the expression of Piezo2 and activation of cAMP signal-related molecules in the trigeminal ganglion (TG) were detected. Changes in purinergic P2 receptors in the TG were also studied by RNA-seq. The expression of Piezo2, cAMP, and Epac1 in the TG of the TN animals increased after chronic compression of the trigeminal nerve root (CCT) for 21 days, but Piezo2 knockdown by shRNA in the TG attenuated orofacial mechanical allodynia. Purinergic P2 receptors P2X4, P2X7, P2Y1, and P2Y2 were significantly up-regulated after CCT injury. In vitro, Piezo2 expression in TG neurons was significantly increased by exogenous adenosine 5'-triphosphate (ATP) and Ca2+ ionophore ionomycin. ATP pre-treated TG neurons displayed elevated [Ca2+ ]i and faster increase in responding to blockage of Na+ /Ca2+ exchanger by KB-R7943. Furthermore, mechanical stimulation of cultured TG neurons led to sustained elevation in [Ca2+ ]i in ATP pre-treated TG neurons, which is much less in naïve TG neurons, or is significantly reduced by Piezo2 inhibitor GsMTx4. These results indicated a pivotal role of Piezo2 in peripheral mechanical allodynia in the rat CCT model. Extracellular ATP, Ca2+ influx, and the cAMP-to-Epac1 signaling pathway synergistically contribute to the pathogenesis and the persistence of mechanical allodynia.

Bone marrow mesenchymal stem cell-derived exosomes shuttling miR-150-5p alleviates mechanical allodynia in rats by targeting NOTCH2 in microglia.

BACKGROUND: This study probes into the function and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes loaded with miR-150-5p in mechanical allodynia. METHODS: BMSCs were infected with miR-150-5p inhibition lentiviruses to obtain exosomes with low miR-150-5p expression. A L5 spinal nerve ligation (SNL) model was established in rats where exosomes, NOTCH2 overexpression/inhibition plasmids, or microglial cells were intrathecally administered. Hind paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of rats were measured. TUNEL staining was used to measure the apoptotic rate in rat spinal dorsal horn (SDH), ELISA to evaluate pro-inflammatory factor levels, and RT-qPCR, western blotting, and immunohistochemistry to detect miR-150-5p and NOTCH2 expression. Immunofluorescence was used for localizing exosomes and NOTCH2 and detecting the expression of OX42, a maker for microglia. Dual luciferase reporter and RNA pull down assays were performed to validate the putative binding between miR-150-5p and NOTCH2. RESULTS: NOTCH2 expressed at a high level and miR-150-5p was downregulated in SDH of SNL rats. Exosomes injected were localized in rat SDH. BMSC-exosomes or NOTCH2 downregulation increased PWT and PWL of SNL rats and reduced apoptosis and inflammation in SDH. In contrast, NOTCH2 overexpression aggravated mechanical allodynia and SDH injury. Moreover, inhibiting miR-150-5p in BMSC-exosomes offset the therapeutic effects of BMSC-exosomes. Microglia activation induced mechanical allodynia in wild rats, while intrathecal injection of microglial cells incubated with BMSC-exosomes showed alleviated mechanical allodynia in SNL rats. NOTCH2 was targeted by miR-150-5p. CONCLUSION: BMSC-derived exosomal miR-150-5p alleviates mechanical allodynia by targeting NOTCH2 in microglial cells.

Bone marrow-derived mesenchymal stem cells alleviate paclitaxel-induced mechanical allodynia in rats.

Anticancer drug paclitaxel (PTX) frequently causes painful peripheral neuropathy; however, no medication has been shown to be effective in the treatment of this debilitating side effect. We aimed to investigate the efficacy of two different doses of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) on PTX-induced mechanical allodynia and spinal cytokine levels and their localization to target tissues such as the spinal cord and sciatic nerve. After the development of mechanical allodynia with repeated PTX administration, two different doses of rat BM-MSCs, low or high (1 × 106 -5 × 106 ), were transplanted into rats and the evaluation continued for 30 days. Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-10 levels in spinal cord samples of animals were analyzed by enzyme-linked immunosorbent assay. PTX-induced mechanical allodynia was relieved significantly 15 days after the transplantation of high-dose of BM-MSCs. Both MSCs doses were effective in alleviating allodynia, but the onset of effect was earlier with the high dose. High-dose of BM-MSCs significantly decreased spinal IL-1ß and TNF-α levels compared to the PTX group. Fluorescent dye-labeled BM-MSCs were observed much more frequently in the sciatic nerve and spinal cord samples of the high-dose BM-MSCs transplanted group than in the low-dose group animals. In conclusion, we found that the antiallodynic effects of BM-MSCs appeared earlier when high-dose of cells were administered. We think that other mechanisms may play a role in the effects of MSCs, besides localization to damaged tissues and reducing spinal inflammatory cytokine levels. We show that BM-MSCs can be a novel approach in PTX-induced mechanical allodynia.

C-X-C motif chemokine ligand 1 and its receptor C-X-C motif chemokine receptor 2 in trigeminal ganglion contribute to nerve injury-induced orofacial mechanical allodynia.

BACKGROUND: Orofacial ectopic pain induced by trigeminal nerve injury is a serious complication of dental treatment. C-X-C motif chemokine ligand 1 (CXCL1) and its primary receptor C-X-C motif chemokine receptor 2 (CXCR2) contribute to the development and maintenance of neuropathic pain in the spinal nervous system, but their roles in trigeminal neuropathic sensation are still poorly understood. OBJECTIVES: This study aimed to investigate the exact role of CXCL1 and CXCR2 in the regulation of orofacial ectopic mechanical allodynia and their potential downstream mechanisms in the trigeminal ganglion (TG). METHODS: The head withdrawal threshold (HWT) of C57BL/6 mice was evaluated after inferior alveolar nerve (IAN) transection (IANX). Then, the distribution and expression of CXCL1 and CXCR2, and their potential downstream mechanisms in the TG were further measured using immunohistochemistry, real-time reverse transcription-quantitative polymerase chain reaction and Western blotting. Moreover, the effect of SB225002 (an inhibitor of CXCR2) on mechanical allodynia was examined. The data were analysed using the Student's t test and a analysis of variance (ANOVA). RESULTS: IANX triggered persistent (>21 days) mechanical allodynia and upregulation of CXCL1 and CXCR2 in the TG. In addition, exogenous CXCL1 also lowered the HWT, which was alleviated by CXCR2 and protein kinase C (PKC) antagonists (p < .05). In addition, IANX increased the phosphorylated PKC (p-PKC) levels and decreased the expression of voltage-gated potassium channels (Kv), and these effects were reversed by inhibition of CXCR2 (p < .05). CONCLUSION: Our results demonstrated that CXCR2 participated in orofacial ectopic mechanical allodynia via downregulation of Kv1.4 and Kv1.1 through the PKC signalling pathway. This mechanism may be a potential target in developing a treatment strategy for ectopic orofacial pain.

TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury.

Trichinella Infection Ameliorated Vincristine-Induced Neuroinflammation in Mice.

Vincristine (VCR) is a chemotherapeutic agent widely used in treatment of malignancies. However, VCR has a limitation in use since it commonly causes a painful neuropathy (VCR-induced peripheral neuropathy, VIPN). Inflammatory cytokines secreted by immune cells such as macrophages can exacerbate allodynia and hyperalgesia, because inhibiting the inflammatory response is a treatment target for VIPN. In this study, we investigated whether Trichinella spiralis, a widely studied helminth for its immunomodulatory abilities, can alleviate VCR-induced allodynia. Von Frey test showed that T. spiralis infection improved mechanical allodynia at 10 days after VCR injection. We further observed whether the difference was due to mitigated axon degeneration, but no significant difference between the groups in axonal degeneration in sciatic nerves and intra-epidermal nerve fibers was found. Conversely, we observed that number of infiltrated macrophages was decreased in the sciatic nerves of the T. spiralis infected mice. Moreover, treatment of T. spiralis excretory-secretory products caused peritoneal macrophages to secrete decreased level of IL-1ß. This study suggests that T. spiralis can relieve VCR-induced mechanical allodynia by suppressing neuroinflammation and that application of controllable degree of helminth may prove beneficial for VIPN treatment.