NLRP10 ablation alleviates neuropathic pain by inhibiting excessive NIX/LC3-dependent mitophagy in the spinal cord.

In this article, we present our previous research, which highlighted adenosine triphosphate (ATP) as the cause of neuropathic pain during the acute phase of neuromyelitis optica spectrum disorder (NMOSD). In NMOSD pathology, damage-associated molecular patterns (DAMPs), including ATP, are released from damaged astrocytes, triggering the activation of innate immune cells. ATP is a central mediator of acute pain in NMOSD. We delve into the mechanisms of ATP in peripheral neuropathic pain, drawing comparisons with our findings in NMOSD. Additionally, we address the intricacies of chronic pain associated with NMOSD. Neuromyelitis optica spectrum disorder and neuropathic pain: NMOSD is an inflammatory disease characterized by optic neuritis and long cord transverse myelitis. It was classified as a form of multiple sclerosis (MS), that is, optic spinal MS, before autoantibody discovery. The presence of autoantibodies against astrocytes in the sera of patients was recognized, and the antigen on astrocyte surfaces was identified to be aquaporin-4 (AQP4). Subsequently, the pathogenicity of the AQP4 antibody was revealed, and the concept of NMOSD was established. Physicians treat NMOSD patients with steroid pulse and plasma exchange in the acute phase, and oral steroids and immunosuppressants in the chronic phase to prevent a relapse. Recently, various biological antibody drugs such as eculizumab, satralizumab, and inebilizumab have become available. However, the treatment for residual symptoms is insufficient. Neuropathic pain is a residual symptom that is difficult to treat and significantly reduces quality of life. More than 70% of NMOSD patients experience neuropathic pain, which is severe and often difficult to control, even with more than two analgesics or opioids (Ayzenberg et al., 2021). Although the clinical characteristics of neuropathic pain in NMOSD are known to be severe and drug-resistant, the underlying mechanisms are poorly understood. However, little research has been conducted on NMOSD pain. In this article, we introduce our research on the role of ATP in acute NMOSD pain. NMOSD pathology and innate immunity: NMOSD is characterized by inflammatory demyelinating lesions with necrotic changes in the optic nerve and spinal cord. In the acute stage, neutrophils and eosinophils are prominent, and noticeable necrotic changes are observed with thickening and hyalinization of the vessel walls. Extensive AQP4 disappearance and astrocyte degeneration are observed in acute phase lesions, which are observed around vessels where immunoglobulin and activated complement are deposited. Pathogenic AQP4 antibodies, which are essential in the pathogenesis of NMOSD, bind to the astrocyte surface and enhance the migration of neutrophils and macrophages, triggering antibody-dependent cellular cytotoxicity and complement recruitment, inducing astrocyte damage via complement-dependent cellular cytotoxicity mechanism. Astrocytic damage leads to secondary neuronal loss. The high complement activation potential of pathogenic AQP4 autoantibodies and neutrophil NETosis contribute to more severe tissue destruction compared to MS. In addition, microglia activation around the lesion and the infiltration of certain macrophages are reported, with monocytic cells secreting inflammatory cytokines such as interleukin-1β (IL-1β). Activation of microglia is also believed to be a key factor in neuropathic pain, and the nature of NMOSD pathology is presumed to be an essential factor in causing NMOSD neuropathic pain. Damage-associated molecular patterns and diseases: When cells undergo necrosis, they release DAMPs, which are intracellular molecules with a high capacity for immunostimulatory effects. DAMPs are paired with pathogen-associated molecular patterns such as lipopolysaccharides (LPS) in the cell walls of gram-negative bacilli. Lipopolysaccharide binds to toll-like receptor 4 on monocytes activating NOD-like receptor pyrin domain-containing protein 3 inflammasome pathway and inducing the release of proinflammatory cytokines such as IL-1β and interleukin-18 (IL-18). In contrast, DAMPs are self-molecules released because of necrosis due to ischemia, trauma, or autoimmunity. They also trigger monocytic cells and induce the release of inflammatory cytokines, leading to tissue damage. Gout attacks are caused by the reaction of monocytes with uric acid crystals in the joint cavity. In cerebral ischemic stroke lesions, dead cells secrete DAMPs such as ATP and high mobility group box 1 (HMGB1), stimulating monocytes and triggering auto-inflammation, worsening the infarction pathology (Shichita et al., 2023). In the cerebrospinal fluid of NMOSD patients, elevated levels of HMGB1, mitochondrial deoxyribonucleic acid (DNA), and ATP have been reported. This may be due to their release from the damaged astrocytes (Uzawa et al., 2013; Yamashita et al., 2018; Ishikura et al., 2021). Therefore, DAMPs may affect the central nervous system and cause neuroinflammation in NMOSD patients. In particular, the role of ATP in neuropathic pain has been the subject of basic research, and its detailed mechanisms have been elucidated. Peripheral neuropathic pain and ATP: Peripheral neuropathic pain can be caused by various diseases including diabetes, autoimmune diseases, traumatic bone compression, infections, and malignancies. In a model of peripheral neuropathic pain, known as spared nerve injury (SNI), microglia are activated in response to peripheral neuropathy and extracellular ATP stimulation, causing the production and release of cytokines and neurotrophic factors and inducing dorsal horn neuronal excitation. Thus, the detailed mechanism of peripheral neuropathic pain has been elucidated (Inoue and Tsuda, 2018). In the SNI model, ATP released from injured nerves and astrocytes triggers the activation of spinal cord microglia and upregulates the expression of interferon regulatory factor 8, which subsequently upregulates the expression of interferon regulatory factor 5 and a purinergic receptor P2RX4. ATP binding to P2X4R on activated microglia stimulates the synthesis and release of brain-derived neurotrophic factor (BDNF). BDNF in turn, downregulates the neuronal chloride transporter KCC2, leading to changes in the transmembrane anion gradient in a subpopulation of neurons in the posterior horn layer I. Several studies have shed light on these underlying mechanisms. Another mechanism involves the release of proinflammatory cytokines by microglia. The activation of nuclear factor-κB through P2RX7 receptors, another type of purinergic receptor on microglia, triggers the NOD-like receptor pyrin domain-containing protein 3 inflammasomes, promoting the release of IL-1β. IL-1β acts on spinal cord dorsal horn neurons and contributes to neuropathic pain. ATP causes neuropathic pain in the NMOSD rat model acute phase: Several DAMPs have been reported to be elevated in NMOSD cerebrospinal fluid. Among these DAMPs, ATP, a key molecule in peripheral neuropathic pain, has been identified. In this context, we hypothesized that ATP released from astrocytes induces neuropathic pain in NMOSD. To investigate this hypothesis, we established a rat spinal cord AQP4 antibody injection model and demonstrated that ATP released from astrocytes causes acute phase pain (Ishikura et al., 2021). We administered recombinant AQP4 antibody derived from the cerebrospinal fluid plasmablasts of NMOSD patients and control IgG to the spinal cord at the level of the 10th thoracic vertebra in Lewis rats. The von Frey test revealed significant mechanical pain hypersensitivity in the AQP4 antibody group, but only during the acute phase, compared to the Control IgG group. Transcriptome analysis of the spinal cord demonstrated increased expression of several ATP receptor genes, including P2RX4, in the AQP4 antibody-treated group compared to that in the Control IgG group. Furthermore, we demonstrated that administration of TNP-ATP, a receptor inhibitor of P2RX4, alleviated neuropathic pain in an NMOSD rat model. Additionally, we confirmed that administration of AQP4 antibody to human embryonic kidney (HEK293) cells with forced expression of AQP4 and to primary rat astrocytes resulted in complement-dependent release of ATP. Furthermore, our findings indicate that NMOSD patients in the acute phase have remarkably higher ATP levels in their cerebrospinal fluid compared to individuals with MS in the acute phase or other neurological conditions. Notably, we also observed an increase in ATP levels among NMOSD patients in the chronic phase. The transcriptome and quantitative polymerase chain reaction results also showed elevated levels of IL-1β, a typical cytokine gene released by monocytic cells when stimulated by DAMPs, in the spinal cord of rats treated with AQP4 compared to the control IgG-treated group. Previous studies have established that in models of SNI, ATP released from injured nerves interacts with activated microglia expressing ATP receptors, such as P2RX4, and triggers the release of neurotrophic factors such as BDNF and IL-1β via P2RX7. However, there are reports that IL-1β release is partially stimulated by P2RX4 in spinal cord injury models (de Rivero Vaccari et al., 2012). We speculate that the effectiveness of ATP receptors may vary from model to model and that the P2RX4-IL-1β axis may have been stronger in our NMOSD model. Puregenic receptors and downstream cytokines are known to overlap. P2RX4 downstream cytokines may vary in different models, with IL-1β being associated with central pain conditions like SCI and NMOSD, and BDNF with peripheral pain. Our results suggest that in NMOSD, ATP released due to the binding of AQP4 antibodies to astrocytes may be intercepted by activated microglia, leading to the subsequent release of IL-1β, contributing to the development of neuropathic pain (Figure 1).Figure 1: NMOSD acute pain and ATP, compared with peripheral neuropathic pain.In acute NMOSD pain, AQP4 antibodies bind to astrocytes, cause cell necrosis with complement and neutrophils, and promote the release of ATP as DAMPs, which bind to P2RX4 in activated microglia. In peripheral neuropathic pain, ATP released from injured nerves and astrocytes binds to P2RX7 in activated microglia. In both pathological conditions, microglial mediators including IL-1β released from activated microglia, promote neuroexcitation of SDH neurons and cause neuropathic pain. Ab: Antibody; AQP4: aquaporin-4; ATP: adenosine triphosphate; DAMPs: damage-associated molecular patterns; DRG: dorsal root ganglion; IL: interleukin; NMOSD: neuromyelitis optica spectrum disorder; P2RX4, P2RX7: purinergic receptors; SDH: superficial dorsal horn.NMOSD chronic pain and treatment: Our results indicated that ATP plays a key role in acute pain in NMOSD. Although many NMOSD patients experience chronic pain, there has been almost no basic research that elucidates chronic pain in NMOSD. There is only one publication that reports the administration of an anti-repulsive guidance molecule (RGMa) antibody to an NMOSD animal model, which prevents neutrophil infiltration from the acute phase and can prevent chronic pain (Iwamoto et al., 2022). NMOSD lesions predominantly occur in regions characterized by high AQP4 expression, including the hypothalamus, optic nerve, ventral medulla around the third and fourth ventricles, and the central canal of the spinal cord. The descending pain inhibitory system begins in various areas of the brainstem, particularly in the periaqueductal gray matter of the midbrain and rostral ventromedial medulla, and descends via the dorsolateral cord to all levels of the spinal cord. The pain inhibitory pathway is often involved in NMOSD lesions. Therefore, lesions in NMOSD may inhibit the descending pain inhibitory system. Furthermore, AQP4 loss in astrocytes has been noted in the first layer of the cortex in NMOSD brain autopsies (Kawachi and Lassmann, 2017). It is speculated that astrocyte dysfunction is associated with chronic pain. In the intact central nervous system, AQP4 is co-expressed with the excitatory amino acid transporter 2 and facilitates glutamate uptake into astrocytes. Glutamate, an excitatory neurotransmitter, is converted to glutamine, a precursor of amino acid neurotransmitters, within astrocytes. This glutamine is then released from astrocytes and taken up by neurons by active transport. In GABAergic neurons, glutamine is hydrolyzed to glutamate, which is then partially decarboxylated to gamma-aminobutyric acid (GABA), replenishing the synaptic neurotransmitter pool. Astrocyte death interrupts the glutamine-glutamate-GABA pathway, possibly leading to a delicate balance between the excitation and inhibition of the nociceptive pathway. Elevated extracellular glutamate concentration also affects the vulnerable inhibitory alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, as well as GABA neurons. Astrocytes also release endogenous cannabinoid 2-arachidonoylglycerol, which potently activates GABAergic inhibition (Bradl et al., 2014). A recent study has revealed a link between allodynia symptoms in SNI models and the formation of pain nerve circuit spines in the sensory cortex, a process regulated by astrocytes. We speculate that astrocytopathy in the sensory cortex, a region not typically predisposed to NMOSD lesions, may also be associated with enhanced or reduced NMOSD chronic pain (Takeda et al., 2022). These are the current opinions on the mechanisms of NMOSD chronic pain, and it is unclear if and how the release of DAMPs, such as ATP, is related to NMOSD chronic pain. However, in the SNI model, the autonomous secretion of ATP by excited neurons via nucleotide transporters, such as the vesicular nucleotide transporter, is the maintenance mechanism of chronic pain, which is a potential target of pain treatment (Kato et al., 2022). The release of ATP as a DAMP possibly triggers NMOSD chronic pain. Nevertheless, more basic research on NMOSD chronic pain is required. This perspective has highlighted the role of ATP in NMOSD acute pain, in comparison to that in PNI pain. We hope that advanced basic research will discover new insights into the pathogenesis, revealing further NMOSD pain mechanisms including NMOSD chronic pain. We are grateful to Dr. Makoto Kinoshita (Osaka University, Japan) for proposing the concept of ATP-induced NMOSD acute pain. C-Editors: Zhao M, Sun Y, Qiu Y; T-Editor: Jia Y

Mitochondria play an essential role in pathophysiology of both inflammatory and neuropathic pain (NP), but the mechanisms are not yet clear. Dynamin-related protein 1 (Drp1) is broadly expressed in the central nervous system and plays a role in the induction of mitochondrial fission process. Spared nerve injury (SNI), due to the dysfunction of the neurons within the spinal dorsal horn (SDH), is the most common NP model. We explored the neuroprotective role of Drp1 within SDH in SNI. SNI mice showed pain behavior and anxiety-like behavior, which was associated with elevation of Drp1, as well as increased density of mitochondria in SDH. Ultrastructural analysis showed SNI induced damaged mitochondria into smaller perimeter and area, tending to be circular. Characteristics of vacuole in the mitochondria further showed SNI induced the increased number of vacuole, widened vac-perimeter and vac-area. Stable overexpression of Drp1 via AAV under the control of the Drp1 promoter by intraspinal injection (Drp1 OE) attenuated abnormal gait and alleviated pain hypersensitivity of SNI mice. Mitochondrial ultrastructure analysis showed that the increased density of mitochondria induced by SNI was recovered by Drp1 OE which, however, did not change mitochondrial morphology and vacuole parameters within SDH. Contrary to Drp1 OE, down-regulation of Drp1 in the SDH by AAV-Drp1 shRNA (Drp1 RNAi) did not alter painful behavior induced by SNI. Ultrastructural analysis showed the treatment by combination of SNI and Drp1 RNAi (SNI + Drp1 RNAi) amplified the damages of mitochondria with the decreased distribution density, increased perimeter and area, as well as larger circularity tending to be more circular. Vacuole data showed SNI + Drp1 RNAi increased vacuole density, perimeter and area within the SDH mitochondria. Our results illustrate that mitochondria within the SDH are sensitive to NP, and targeted mitochondrial Drp1 overexpression attenuates pain hypersensitivity. Drp1 offers a novel therapeutic target for pain treatment.

Changes in VGLUT2 expression and function in pain-related supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model

Mechanical allodynia can be evoked by punctate pressure contact with the skin (punctate mechanical allodynia) and dynamic contact stimulation induced by gentle touching of the skin (dynamic mechanical allodynia). Dynamic allodynia is insensitive to morphine treatment and is transmitted through the spinal dorsal horn by a specific neuronal pathway, which is different from that for punctate allodynia, leading to difficulties in clinical treatment. K+-Cl− cotransporter-2 (KCC2) is one of the major determinants of inhibitory efficiency, and the inhibitory system in the spinal cord is important in the regulation of neuropathic pain. The aim of the current study was to determine whether neuronal KCC2 is involved in the induction of dynamic allodynia and to identify underlying spinal mechanisms involved in this process. Dynamic and punctate allodynia were assessed using either von Frey filaments or a paint brush in a spared nerve injury (SNI) mouse model. Our study discovered that the downregulated neuronal membrane KCC2 (mKCC2) in the spinal dorsal horn of SNI mice is closely associated with SNI-induced dynamic allodynia, as the prevention of KCC2 downregulation significantly suppressed the induction of dynamic allodynia. The over activation of microglia in the spinal dorsal horn after SNI was at least one of the triggers in SNI-induced mKCC2 reduction and dynamic allodynia, as these effects were blocked by the inhibition of microglial activation. Finally, the BDNF-TrkB pathway mediated by activated microglial affected SNI-induced dynamic allodynia through neuronal KCC2 downregulation. Overall, our findings revealed that activation of microglia through the BDNF-TrkB pathway affected neuronal KCC2 downregulation, contributing to dynamic allodynia induction in an SNI mouse model.

Activating the vlPAG-LC neural pathway alleviates neuropathic pain and comorbid anxiety-like behaviors through distinct projections.

BackgroundExisting remedial approaches for relieving neuropathic pain (NPP) are challenging and open the way for alternative therapeutic measures such as electroacupuncture (EA). The mechanism underlying the antinociceptive effects of repeated EA sessions, particularly concerning the regulation of the Adora3 receptor and its associated enzymes, has remained elusive.MethodsThis study used a mouse model of spared nerve injury (SNI) to explore the cumulative analgesic effects of repeated EA at ST36 (Zusanli) and its impact on Adora3 regulation in the spinal cord dorsal horn (SCDH). Forty‐eight male mice underwent SNI surgery for induction of neuropathic pain and were randomly assigned to the SNI, SNI + 2EA, SNI + 4EA, and SNI + 7EA groups. Spinal cord (L4–L6) was sampled for immunofluorescence, adenosine (ADO) detection and for molecular investigations following repeated EA treatment.ResultsFollowing spared nerve injury (SNI), there was a significant decrease in mechanical withdrawal thresholds (PWTs) and thermal nociceptive withdrawal latency (TWL) in the ipsilateral hind paw on the third day post‐surgery, while the contralateral hind paw PWTs showed no significant changes. On subsequent EA treatments, the SNI + EA groups led to a significant increase in pain thresholds (p < 0.05). Repeated EA sessions in SNI mice upregulated Adenosine A3 (Adora3) and cluster of differentiation‐73 (CD73) expression while downregulating adenosine deaminase (ADA) and enhancing neuronal instigation in the SCDH. Colocalization analysis of Neun‐treated cells revealed increased Adora3 expression, particularly in the SNI + 7EA group.ConclusionsIn conclusion, cumulative electroacupuncture treatment reduced neuropathic pain by regulating Adora3 and CD73 expression, inhibiting ADA and most likely increasing neuronal activation in the SCDH. This study offers a promising therapeutic option for managing neuropathic pain, paving the way for further research.

Exercise has been proven to be an efficient intervention in attenuating neuropathic pain. However, the underlying mechanisms that drive exercise analgesia remain unknown. In this study, we aimed to examine the role of complement component 3 (C3) in neuropathic pain and whether antinociceptive effects are produced by exercise via regulating C3 in mice. In this study, using a spared nerve injury (SNI)-induced neuropathic pain mice model, C57BL/6J mice were divided into 3 groups: Sham mice, SNI mice, and SNI + Exercise (Ex) mice with 30-minute low-intensity aerobic treadmill running (10 m/min, no inclination). Paw withdrawal threshold; thermal withdrawal latency; and glial fibrillary acidic protein, C3, tumor necrosis factor-α, and interlukin-1β expression in the spinal cord were monitored. C3 knockout (KO) mice were further used to verify the role of C3 in neuropathic pain. von Frey test, acetone test, and CatWalk gait analysis revealed that treadmill exercise for 4 weeks reversed pain behaviors. In addition, exercise reduced astrocyte reactivity (SNI mean = 14.5, 95% confidence interval [CI], 12.7-16.3; SNI + Ex mean = 10.3, 95% CI, 8.77-11.9, P = .0003 SNI + Ex versus SNI) and inflammatory responses in the spinal cord after SNI. Moreover, it suppressed the SNI-induced upregulation of C3 expression in the spinal cord (SNI mean = 5.46, 95% CI, 3.39-7.53; SNI + Ex mean = 2.41, 95% CI, 1.42-3.41, P = .0054 SNI + Ex versus SNI in Western blot). C3 deficiency reduced SNI-induced pain and spinal astrocyte reactivity (wild type mean = 7.96, 95% CI, 6.80-9.13; C3 KO mean = 5.98, 95% CI, 5.14-6.82, P = .0052 C3 KO versus wild type). Intrathecal injection of recombinant C3 (rC3) was sufficient to produce mechanical (rC3-Ex mean = 0.77, 95% CI, 0.15-1.39; rC3 mean = 0.18, 95% CI, -0.04 to 0.41, P = .0168 rC3-Ex versus rC3) and cold (rC3-Ex mean = 1.08, 95% CI, 0.40-1.77; rC3 mean = 3.46, 95% CI, 1.45-5.47, P = .0025 rC3-Ex versus rC3) allodynia in mice. Importantly, exercise training relieved C3-induced mechanical and cold allodynia, and the analgesic effect of exercise was attenuated by a subeffective dose of intrathecal injection of C3. Overall, these results suggest that exercise suppresses neuropathic pain by regulating astroglial C3 expression and function, thereby providing a rationale for the analgesic effect of exercise as an acceptable alternative approach for treating neuropathic pain.

MOTS-c, a recently discovered mitochondrial-derived peptide, plays an important role in many physiological and pathological functions via adenosine monophosphate-activated protein kinase (AMPK) activation. Numerous studies have demonstrated that AMPK is an emerging target for the modulation of neuropathic pain. Meanwhile, microglia-activation-evoked neuroinflammation is known to contribute to the development and progression of neuropathic pain. MOTS-c is also known to inhibit microglia activation, chemokine and cytokine expression, and innate immune responses. Accordingly, in this study, we evaluated the effects of MOTS-c on neuropathic pain and investigated the putative underlying mechanisms. We found that MOTS-c levels in plasma and spinal dorsal horn were significantly lower in mice with spared nerve injury (SNI)-induced neuropathic pain than in control animals. Accordingly, MOTS-c treatment produced pronounced dose-dependent antinociceptive effects in SNI mice; however, these effects were blocked by dorsomorphin, an AMPK inhibitor, but not naloxone, a nonselective opioid receptor antagonist. Moreover, intrathecal (i.t.) injection of MOTS-c significantly enhanced AMPKα1/2 phosphorylation in the lumbar spinal cord of SNI mice. MOTS-c also significantly inhibited proinflammatory cytokine production and microglia activation in the spinal cord. The antinociceptive effects of MOTS-c were retained even when microglia activation in the spinal cord was inhibited by minocycline pretreatment, indicating that spinal cord microglia are dispensable for the antiallodynic effects of MOTS-c. In the spinal dorsal horn, MOTS-c treatment inhibited c-Fos expression and oxidative damage mainly in neurons rather than microglia. Finally, in contrast to morphine, i.t. administration of MOTS-c resulted in limited side effects relating to antinociceptive tolerance, gastrointestinal transit inhibition, locomotor function, and motor coordination. Collectively, the present study is the first to provide evidence that MOTS-c may be a promising therapeutic target for neuropathic pain.

Astrocytic activation in the spinal dorsal horn contributes to the central sensitization of neuropathic pain. Bone morphogenetic protein (BMP) 10, one of the BMPs highly expressed in the central nervous system, has been demonstrated to have an accelerated effect on astrocytic activation. This study aimed to investigate the functional effects of BMP10 on the activation of astrocytes in the spinal dorsal horn of animal model of neuropathic pain and to explore potential mechanisms involved in this process. A neuropathic pain mice model was established using the spared nerve injury (SNI). Western blot analysis was performed to detect the expressional levels of BMP10, activin receptor-like receptor 2 (ALK2), Smad1/5/8, phosphorylated Smad1/5/8, and glial fibrillary acidic protein (GFAP). Immunofluorescence staining was used to detect BMP10, ALK2, and GFAP distribution and expression. The behavioral changes in mice were evaluated using paw withdrawal threshold (PWT), thermal withdrawal latency (TWL), and open field test (OFT). The BMP10 siRNA, Smad1 siRNA, BMP10 peptide, and ALK2-IN-2 (ALK2 inhibitor) were intrathecally administrated to mice. A model of lipopolysaccharide (LPS)-stimulated astrocytes was established to investigate the effect of Smad1. The transfection efficiency of siRNAs was detected by western blot and qRT-PCR analysis. BMP10 levels were increased in the L4-6 ipsilateral spinal dorsal horn of SNI mice and particularly elevated in astrocytes. Consistently, GFAP and phosphorylated Smad1/5/8 were upregulated in the L4-6 ipsilateral spinal dorsal horn after SNI, indicating the activation of astrocytes and Smad1/5/8 signaling. An intrathecal injection of BMP10 siRNA abrogated pain hypersensitivity and astrocytic activation in SNI mice. In addition, intrathecal administration of BMP10 peptide evoked pain hypersensitivity and astrocytic activation in normal mice, and this action was reversed by inhibiting the ALK2. Furthermore, targeting Smad1 in vitro with the help of siRNA inhibited the activation of astrocytes induced by LPS. Finally, targeting Smad1 abrogated BMP10-induced hypersensitivity and activation of astrocytes. These findings indicate that the BMP10/ALK2/Smad1/5/8 axis plays a key role in pain hypersensitivity after peripheral nerve injury, which indicates its stimulative ability toward astrocytes.

Neuropathic pain has long-term consequences in affective and cognitive disturbances, suggesting the involvement of supraspinal mechanisms. In this study, we used the spared nerve injury (SNI) model to characterize the development of sensory and aversive components of neuropathic pain and to determine their electrophysiological impact across prefrontal cortex and limbic regions. Moreover, we evaluated the regulation of several genes involved in immune response and inflammation triggered by SNI. We showed that SNI led to sensorial hypersensitivity (cold and mechanical stimuli) and depressive-like behavior lasting 12 months after nerve injury. Of interest, changes in nonemotional cognitive tasks (novel object recognition and Y maze) showed in 1-month SNI mice were not evident normal in the 12-month SNI animals. In vivo electrophysiology revealed an impaired long-term potentiation at prefrontal cortex-nucleus accumbens core pathway in both the 1-month and 12-month SNI mice. On the other hand, a reduced neural activity was recorded in the lateral entorhinal cortex-dentate gyrus pathway in the 1-month SNI mice, but not in the 12-month SNI mice. Finally, we observed the upregulation of specific genes involved in immune response in the hippocampus of 1-month SNI mice, but not in the 12-month SNI mice, suggesting a neuroinflammatory response that may contribute to the SNI phenotype. These data suggest that distinct brain circuits may drive the psychiatric components of neuropathic pain and pave the way for better investigation of the long-term consequences of peripheral nerve injury for which most of the available drugs are to date unsatisfactory.

PurposePyroptosis, a programmed inflammatory cell death mechanism, plays a significant role in neuropathic pain (NP) pathogenesis. However, the specific pyroptosis-related genes (PRGs) driving NP development remain poorly understood. This study employs systematic approaches to identify and validate PRGs, aiming to delineate their mechanistic contributions to NP progression.MethodsTo elucidate pyroptosis-related genes (PRGs) in neuropathic pain pathogenesis, we first performed integrated bioinformatics analysis of the GSE236754 dataset, revealing differentially expressed PRGs in the spinal cord dorsal horn of spared nerve injury (SNI) rats. Subsequent functional enrichment analyses coupled with protein-protein interaction network construction delineated pathway convergences among identified PRGs. Experimental validation utilizing SNI rat model, Western blot and immunofluorescence quantification confirmed protein expression patterns, and immunofluorescence mapping determined cellular localization collectively. Statistical analyses via ANOVA method.ResultsBioinformatics screening identified 11 candidate PRGs in the SNI model, particularly highlighting Nlrc4 and Nlrp3 as the most upregulated targets. Gene Ontology (GO) analysis demonstrated significant enrichment in three domains, pyroptosis regulation, inflammasome complex assembly, and cysteine-type endopeptidase activity associated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis specifically identified the “NOD-like receptor signaling pathway” as significantly enriched. Gene Set Enrichment Analysis (GSEA) further corroborated these findings. Behavioral quantification showed progressive mechanical hypersensitivity, with mechanical pain and cold pain reaching maximal sensitivity at day 7 post-injury (p<0.001). Western blot detected synchronized elevation of NLRP3 and NLRC4 inflammasome components and downstream effectors across the observation window (all p<0.05). Immunofluorescence analysis demonstrated a time-dependent increase in the expression of GSDMD-N, the pyroptotic-executing protein, a trend consistent with the findings from behavioral tests and Western blot analysis (p<0.05). And cellular localization analysis revealed neuron-predominant accumulation of GSDMD-N.ConclusionWe identified 11 potential biomarkers for NP. Our data conclusively show that SNI induced NLRP3/NLRC4 inflammasome activation and neuronal pyroptosis in the rat spinal cord.

Neuropathic pain (NP) resulting from nerve damage shows diurnal fluctuation of intensity in patients, indicating circadian regulation. However, mechanisms linking NP and circadian regulation remain unclear. This study aimed to investigate time-dependent transcriptomic changes during a 24-hour period using a spared nerve injury (SNI) mouse model of NP. Pain-related behaviours were assessed at baseline and on days 7, 14, and 21 after SNI and control sham surgeries in C57BL/6JRJ mice. Spinal cord (SC) and periaqueductal gray (PAG) were collected 4-hourly over 24h upon completion of behavioural testing. RNA sequencing revealed 111 up- and 21 downregulated differentially expressed genes (DEGs) in the SC, and 35 up- and 33 downregulated DEGs in the PAG, across all six time points. The large majority of DEGs, 245 in the SC and 191 in the PAG, are involved in regulation of immunity. Among the top expressed genes, five DEGs in the SC, Atf3, Anxa10, Gpr151, Cxcl10, Sprr1a, and two DEGs in the PAG, Igf2 and Wnt6, were previously reported to regulate pain. Circadian analysis using CircaCompare identified 383 SC transcripts and 261 PAG transcripts with altered rhythmicity. Variability of gene expression during circadian day was increased in the SC and decreased in the PAG from the SNI mice. These findings suggest that NP disrupts the circadian expression of rhythmic transcripts in the SC and PAG, potentially revealing new targets for chronotherapy of NP.

Chronic pain is often accompanied by memory deficits. Previous studies have shown that peripheral nerve injury produces both neuropathic pain and memory deficits and induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn (SDH) but inhibits LTP in hippocampus. The opposite changes in synaptic plasticity may contribute to chronic pain and memory deficits, respectively. However, the structural and molecular bases of these alterations of synaptic plasticity are unclear. Here, we show that the complexity of excitatory synaptic connectivity and brain-derived neurotrophic factor (BDNF) expression are enhanced in SDH but reduced in the hippocampus in neuropathic pain and the opposite changes depend on tumor necrosis factor-alpha/tumor necrosis factor receptor 1 signaling and microglial activation. The region-dependent synaptic alterations may underlie chronic neuropathic pain and memory deficits induced by peripheral nerve injury.

Clinical studies show that chronic pain is accompanied by memory deficits and reduction in hippocampal volume. Experimental studies show that spared nerve injury (SNI) of the sciatic nerve induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, but impairs LTP in the hippocampus. The opposite changes may contribute to neuropathic pain and memory deficits, respectively. However, the cellular and molecular mechanisms underlying the functional synaptic changes are unclear. Here, we show that the dendrite lengths and spine densities are reduced significantly in hippocampal CA1 pyramidal neurons, but increased in spinal neurokinin-1-positive neurons in mice after SNI, indicating that the excitatory synaptic connectivity is reduced in hippocampus but enhanced in spinal dorsal horn in this neuropathic pain model. Mechanistically, tumor necrosis factor-alpha (TNF-α) is upregulated in bilateral hippocampus and in ipsilateral spinal dorsal horn, whereas brain-derived neurotrophic factor (BDNF) is decreased in the hippocampus but increased in the ipsilateral spinal dorsal horn after SNI. Importantly, the SNI-induced opposite changes in synaptic connectivity and BDNF expression are prevented by genetic deletion of TNF receptor 1 <i>in vivo</i> and are mimicked by TNF-α in cultured slices. Furthermore, SNI activated microglia in both spinal dorsal horn and hippocampus; pharmacological inhibition or genetic ablation of microglia prevented the region-dependent synaptic changes, neuropathic pain, and memory deficits induced by SNI. The data suggest that neuropathic pain involves different structural synaptic alterations in spinal and hippocampal neurons that are mediated by overproduction of TNF-α and microglial activation and may underlie chronic pain and memory deficits. <b>SIGNIFICANCE STATEMENT</b> Chronic pain is often accompanied by memory deficits. Previous studies have shown that peripheral nerve injury produces both neuropathic pain and memory deficits and induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn (SDH) but inhibits LTP in hippocampus. The opposite changes in synaptic plasticity may contribute to chronic pain and memory deficits, respectively. However, the structural and molecular bases of these alterations of synaptic plasticity are unclear. Here, we show that the complexity of excitatory synaptic connectivity and brain-derived neurotrophic factor (BDNF) expression are enhanced in SDH but reduced in the hippocampus in neuropathic pain and the opposite changes depend on tumor necrosis factor-alpha/tumor necrosis factor receptor 1 signaling and microglial activation. The region-dependent synaptic alterations may underlie chronic neuropathic pain and memory deficits induced by peripheral nerve injury.

Crocetin attenuates spared nerve injury-induced neuropathic pain in mice