Platelet-rich plasma alleviates neuropathic pain in osteoarthritis by downregulating microglial activation.

BACKGROUND: The development of neuropathic pain (NP) is one of the reasons why the pain is difficult to treat, and microglial activation plays an important role in NP. Recently, platelet-rich plasma (PRP) has emerged as a novel therapeutic method for knee osteoarthritis (KOA). However, it's unclarified whether PRP has analgesic effects on NP induced by KOA and the underlying mechanisms unknown. PURPOSE: To observe the analgesic effects of PRP on NP induced by KOA and explore the potential mechanisms of PRP in alleviating NP. METHODS: KOA was induced in male rats with intra-articular injections of monosodium iodoacetate (MIA) on day 0. The rats received PRP or NS (normal saline) treatment at days 15, 17, and 19 after modeling. The Von Frey and Hargreaves tests were applied to assess the pain-related behaviors at different time points. After euthanizing the rats with deep anesthesia at days 28 and 42, the corresponding tissues were taken for subsequent experiments. The expression of activating transcription factor 3 (ATF3) in dorsal root ganglia (DRG) and ionized-calcium-binding adapter molecule-1(Iba-1) in the spinal dorsal horn (SDH) was detected by immunohistochemical staining. In addition, the knee histological assessment was performed by hematoxylin-eosin (HE) staining. RESULTS: The results indicated that injection of MIA induced mechanical allodynia and thermal hyperalgesia, which could be reversed by PRP treatment. PRP downregulated the expression of ATF3 within the DRG and Iba-1 within the SDH. Furthermore, an inhibitory effect on cartilage degeneration was observed in the MIA + PRP group only on day 28. CONCLUSION: These results indicate that PRP intra-articular injection therapy may be a potential therapeutic agent for relieving NP induced by KOA. This effect could be attributed to downregulation of microglial activation and reduction in nerve injury.

Histone deacetylase as emerging pharmacological therapeutic target for neuropathic pain: From epigenetic to selective drugs.

BACKGROUND: Neuropathic pain remains a formidable challenge for modern medicine. The first-line pharmacological therapies exhibit limited efficacy and unfavorable side effect profiles, highlighting an unmet need for effective therapeutic medications. The past decades have witnessed an explosion in efforts to translate epigenetic concepts into pain therapy and shed light on epigenetics as a promising avenue for pain research. Recently, the aberrant activity of histone deacetylase (HDAC) has emerged as a key mechanism contributing to the development and maintenance of neuropathic pain. AIMS: In this review, we highlight the distinctive role of specific HDAC subtypes in a cell-specific manner in pain nociception, and outline the recent experimental evidence supporting the therapeutic potential of HDACi in neuropathic pain. METHODS: We have summarized studies of HDAC in neuropathic pain in Pubmed. RESULTS: HDACs, widely distributed in the neuronal and non-neuronal cells of the dorsal root ganglion and spinal cord, regulate gene expression by deacetylation of histone or non-histone proteins and involving in increased neuronal excitability and neuroinflammation, thus promoting peripheral and central sensitization. Importantly, pharmacological manipulation of aberrant acetylation using HDAC-targeted inhibitors (HDACi) has shown promising pain-relieving properties in various preclinical models of neuropathic pain. Yet, many of which exhibit low-specificity that may induce off-target toxicities, underscoring the necessity for the development of isoform-selective HDACi in pain management. CONCLUSIONS: Abnormally elevated HDACs promote neuronal excitability and neuroinflammation by epigenetically modulating pivotal gene expression in neuronal and immune cells, contributing to peripheral and central sensitization in the progression of neuropathic pain, and HDACi showed significant efficacy and great potential for alleviating neuropathic pain.

Contribution of mechanoreceptors to spinal cord injury-induced mechanical allodynia.

ABSTRACT: Evidence from previous studies supports the concept that spinal cord injury (SCI)-induced neuropathic pain (NP) has its neural roots in the peripheral nervous system. There is uncertainty about how and to which degree mechanoreceptors contribute. Sensorimotor activation-based interventions (eg, treadmill training) have been shown to reduce NP after experimental SCI, suggesting transmission of pain-alleviating signals through mechanoreceptors. The aim of the present study was to understand the contribution of mechanoreceptors with respect to mechanical allodynia in a moderate mouse contusion SCI model. After genetic ablation of tropomyosin receptor kinase B expressing mechanoreceptors before SCI, mechanical allodynia was reduced. The identical genetic ablation after SCI did not yield any change in pain behavior. Peptidergic nociceptor sprouting into lamina III/IV below injury level as a consequence of SCI was not altered by either mechanoreceptor ablation. However, skin-nerve preparations of contusion SCI mice 7 days after injury yielded hyperexcitability in nociceptors, not in mechanoreceptors, which makes a substantial direct contribution of mechanoreceptors to NP maintenance unlikely. Complementing animal data, quantitative sensory testing in human SCI subjects indicated reduced mechanical pain thresholds, whereas the mechanical detection threshold was not altered. Taken together, early mechanoreceptor ablation modulates pain behavior, most likely through indirect mechanisms. Hyperexcitable nociceptors seem to be the main drivers of SCI-induced NP. Future studies need to focus on injury-derived factors triggering early-onset nociceptor hyperexcitability, which could serve as targets for more effective therapeutic interventions.

Implication of system xc- in neuroinflammation during the onset and maintenance of neuropathic pain.

BACKGROUND: Despite the high prevalence of neuropathic pain, treating this neurological disease remains challenging, given the limited efficacy and numerous side effects associated with current therapies. The complexity in patient management is largely attributed to an incomplete understanding of the underlying pathological mechanisms. Central sensitization, that refers to the adaptation of the central nervous system to persistent inflammation and heightened excitatory transmission within pain pathways, stands as a significant contributor to persistent pain. Considering the role of the cystine/glutamate exchanger (also designated as system xc-) in modulating glutamate transmission and in supporting neuroinflammatory responses, we investigated the contribution of this exchanger in the development of neuropathic pain. METHODS: We examined the implication of system xc- by evaluating changes in the expression/activity of this exchanger in the dorsal spinal cord of mice after unilateral partial sciatic nerve ligation. In this surgical model of neuropathic pain, we also examined the consequence of the genetic suppression of system xc- (using mice lacking the system xc- specific subunit xCT) or its pharmacological manipulation (using the pharmacological inhibitor sulfasalazine) on the pain-associated behavioral responses. Finally, we assessed the glial activation and the inflammatory response in the spinal cord by measuring mRNA and protein levels of GFAP and selected M1 and M2 microglial markers. RESULTS: The sciatic nerve lesion was found to upregulate system xc- at the spinal level. The genetic deletion of xCT attenuated both the amplitude and the duration of the pain sensitization after nerve surgery, as evidenced by reduced responses to mechanical and thermal stimuli, and this was accompanied by reduced glial activation. Consistently, pharmacological inhibition of system xc- had an analgesic effect in lesioned mice. CONCLUSION: Together, these observations provide evidence for a role of system xc- in the biochemical processes underlying central sensitization. We propose that the reduced hypersensitivity observed in the transgenic mice lacking xCT or in sulfasalazine-treated mice is mediated by a reduced gliosis in the lumbar spinal cord and/or a shift in microglial M1/M2 polarization towards an anti-inflammatory phenotype in the absence of system xc-. These findings suggest that drugs targeting system xc- could contribute to prevent or reduce neuropathic pain.

Unraveling the Connection: Pain and Endoplasmic Reticulum Stress.

Pain is a complex and multifaceted experience. Recent research has increasingly focused on the role of endoplasmic reticulum (ER) stress in the induction and modulation of pain. The ER is an essential organelle for cells and plays a key role in protein folding and calcium dynamics. Various pathological conditions, such as ischemia, hypoxia, toxic substances, and increased protein production, may disturb protein folding, causing an increase in misfolding proteins in the ER. Such an overload of the folding process leads to ER stress and causes the unfolded protein response (UPR), which increases folding capacity in the ER. Uncompensated ER stress impairs intracellular signaling and cell function, resulting in various diseases, such as diabetes and degenerative neurological diseases. ER stress may be a critical universal mechanism underlying human diseases. Pain sensations involve the central as well as peripheral nervous systems. Several preclinical studies indicate that ER stress in the nervous system is enhanced in various painful states, especially in neuropathic pain conditions. The purpose of this narrative review is to uncover the intricate relationship between ER stress and pain, exploring molecular pathways, implications for various pain conditions, and potential therapeutic strategies.

The Potential Antinociceptive Effect and Mechanism of Cannabis sativa L. Extract on Paclitaxel-Induced Neuropathic Pain in Rats Uncovered by Multi-Omics Analysis.

Cannabis sativa L. (hemp) is a herbaceous plant rich in cannabinoids with a long history of use in pain treatment. The most well-characterized cannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC), garnered much attention in chemotherapy-induced peripheral neuropathy (CIPN) treatment. However, few studies have investigated the biological benefits and mechanism of hemp extract on CIPN. In the present study, hemp extract (JG) rich in cannabinoids was extracted by supercritical fluid carbon dioxide extraction (SFCE). The antinociceptive efficacy was evaluated using a paclitaxel-induced peripheral neuropathy (PIPN) rat model based on behavioral tests. Further omics-based approaches were applied to explore the potential mechanisms. The results showed that JG decreased mechanical allodynia, thermal hyperalgesia, and inflammatory cytokines in PIPN rats significantly. Transcriptome analysis identified seven key genes significantly regulated by JG in PIPN model rats, mainly related to the neuroactive ligand-receptor interaction pathway, PPAR signaling pathway, and cAMP signaling pathway. In metabolomic analysis, a total of 39 significantly altered metabolites were identified, mainly correlated with pentose and glucuronate interconversions and the glycerophospholipid metabolism pathway. Gut microbiota analysis suggested that increased community Lachnoclostridium and Lachnospiraceae_UCG-006 in PIPN rats can be reversed significantly by JG. In conclusion, hemp extract exhibited antinociceptive effects on PIPN. The analgesic mechanism was probably related to the regulation of inflammation, neuroactive ligand-receptor interaction pathway, sphingolipid metabolism, etc. This study provides novel insights into the functional interactions of Cannabis sativa L. extract on PIPN.

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

AIMS: This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. METHODS: Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. RESULTS: Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. In vitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. In vivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1ß, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. CONCLUSION: Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

Transcriptomic analysis of differentially alternative splicing patterns in mice with inflammatory and neuropathic pain.

Although the molecular mechanisms of chronic pain have been extensively studied, a global picture of alternatively spliced genes and events in the peripheral and central nervous systems of chronic pain is poorly understood. The current study analyzed the changing pattern of alternative splicing (AS) in mouse brain, dorsal root ganglion, and spinal cord tissue under inflammatory and neuropathic pain. In total, we identified 6495 differentially alternatively spliced (DAS) genes. The molecular functions of shared DAS genes between these two models are mainly enriched in calcium signaling pathways, synapse organization, axon regeneration, and neurodegeneration disease. Additionally, we identified 509 DAS in differentially expressed genes (DEGs) shared by these two models, accounting for a small proportion of total DEGs. Our findings supported the hypothesis that the AS has an independent regulation pattern different from transcriptional regulation. Taken together, these findings indicate that AS is one of the important molecular mechanisms of chronic pain in mammals. This study presents a global description of AS profile changes in the full path of neuropathic and inflammatory pain models, providing new insights into the underlying mechanisms of chronic pain and guiding genomic clinical diagnosis methods and rational medication.

LncRNA-PCat19 acts as a ceRNA of miR-378a-3p to facilitate microglia activation and accelerate chronic neuropathic pain in rats by promoting KDM3A-mediated BDNF demethylation.

The pathogenesis of neuropathic pain (NP) is complex, and there are various pathological processes. Previous studies have suggested that lncRNA PCAT19 is abnormally expressed in NP conduction and affects the occurrence and development of pain. The aim of this study is to analyze the role and mechanism of PCAT19 in NP induced by chronic compressive nerve injury (CCI) in mice. In this study, C57BL/6 mice were applied to establish the CCI model. sh-PCAT19 was intrathecally injected once a day for 5 consecutive days from the second day after surgery. We discovered that PCat19 level was gradually up-regulated with the passage of modeling time. Downregulation of Iba-1-positive expression, M1/M2 ratio of microglia, and pro-inflammatory factors in the spinal cords of CCI-mice after PCat19 knock-downed was observed. Mechanically, the expression of miR-378a-3p was negatively correlated with KDM3A and PCat19. Deletion of KDM3A prevented H3K9me2 demethylation of BDNF promoter and suppressed BDNF expression. Further, KDM3A promotes CCI-induced neuroinflammation and microglia activation by mediating Brain-derived neurotrophic factor (BDNF) demethylation. Together, the results suggest that PCat19 may be involved in the development of NP and that PCat19 shRNA injection can attenuate microglia-induced neuroinflammation by blocking KDM3A-mediated demethylation of BDNF and BDNF release.

Ursolic acid molecules dock MAPK1 to modulate gut microbiota diversity to reduce neuropathic pain.

To investigate the efficacy of Ursolic acid in alleviating neuropathic pain in rats with spinal nerve ligation (SNL), the SNL rat model was surgically induced. Different concentrations of Ursolic acid and manipulated target mitogen-activated protein kinase 1 (MAPK1) were administered to the SNL rats. Fecal samples were collected from each group of rats for 16S rDNA analysis to examine the impact of gut microbiota. Molecular docking experiments were conducted to assess the binding energy between Ursolic acid and MAPK1. In vivo studies were carried out to evaluate the expression of inflammatory factors and signaling pathways in spinal cord and colon tissues. Ursolic acid was found to have a beneficial effect on pain reduction in rats by increasing plantar withdrawal latency (PWL) and paw withdrawal threshold (PWT). Comparing the Ursolic acid group with the control group revealed notable differences in the distribution of Staphylococcus, Allobaculum, Clostridium, Blautia, Bifidobacterium, and Prevotella species. Network pharmacology analysis identified MAPK1 and intercellular adhesion molecule-1 (ICAM1) as common targets for Ursolic acid, SNL, and neuropathic pain. Binding sites between Ursolic acid and these targets were identified. Additionally, immunofluorescent staining showed a decrease in GFAP and IBA1 intensity in the spinal cord along with an increase in NeuN following Ursolic acid treatment. Overexpression of MAPK1 in SNL rats led to an increase in inflammatory factors and a decrease in PWL and PWT. Furthermore, MAPK1 counteracted the pain-relieving effects of Ursolic acid in SNL rats. Ursolic acid was found to alleviate neuropathic pain in SNL rats by targeting MAPK1 and influencing gut microbiota homeostasis.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Schwann cell-derived extracellular vesicles promote memory impairment associated with chronic neuropathic pain.

BACKGROUND: The pathogenesis of memory impairment, a common complication of chronic neuropathic pain (CNP), has not been fully elucidated. Schwann cell (SC)-derived extracellular vesicles (EVs) contribute to remote organ injury. Here, we showed that SC-EVs may mediate pathological communication between SCs and hippocampal neurons in the context of CNP. METHODS: We used an adeno-associated virus harboring the SC-specific promoter Mpz and expressing the CD63-GFP gene to track SC-EVs transport. microRNA (miRNA) expression profiles of EVs and gain-of-function and loss-of-function regulatory experiments revealed that miR-142-5p was the main cargo of SC-EVs. Next, luciferase reporter gene and phenotyping experiments confirmed the direct targets of miR-142-5p. RESULTS: The contents and granule sizes of plasma EVs were significantly greater in rats with chronic sciatic nerve constriction injury (CCI)than in sham rats. Administration of the EV biogenesis inhibitor GW4869 ameliorated memory impairment in CCI rats and reversed CCI-associated dendritic spine damage. Notably, during CCI stress, SC-EVs could be transferred into the brain through the circulation and accumulate in the hippocampal CA1-CA3 regions. miR-142-5p was the main cargo wrapped in SC-EVs and mediated the development of CCI-associated memory impairment. Furthermore, α-actinin-4 (ACTN4), ELAV-like protein 4 (ELAVL4) and ubiquitin-specific peptidase 9 X-linked (USP9X) were demonstrated to be important downstream target genes for miR-142-5p-mediated regulation of dendritic spine damage in hippocampal neurons from CCI rats. CONCLUSION: Together, these findings suggest that SCs-EVs and/or their cargo miR-142-5p may be potential therapeutic targets for memory impairment associated with CNP.

Combining fecal 16 S rRNA sequencing and spinal cord metabolomics analysis to explain the modulatory effect of PPARα on neuropathic pain.

BACKGROUND: Existing evidence suggests that the composition of the gut microbiota is associated with neuropathic pain (NP), but the mechanistic link is elusive. Peroxisome proliferator-activated receptor α (PPARα) has been shown to be a pharmacological target for the treatment of metabolic disorders, and its expression is also involved in inflammatory regulation. The aim of this study was to investigate the important modulatory effects of PPARα on gut microbiota and spinal cord metabolites in mice subjected to chronic constriction injury. METHODS: We analyzed fecal microbiota and spinal cord metabolic alterations in mice from the sham, CCI, GW7647 (PPARα agonist) and GW6471 (PPARα antagonist) groups by 16 S rRNA amplicon sequencing and untargeted metabolomics analysis. On this basis, the intestinal microbiota and metabolites that were significantly altered between treatment groups were analyzed in a combined multiomics analysis. We also investigated the effect of PPARα on the polarization fractionation of spinal microglia. RESULTS: PPARα agonist significantly reduce paw withdrawal threshold and paw withdrawal thermal latency, while PPARα antagonist significantly increase paw withdrawal threshold and paw withdrawal thermal latency. 16 S rRNA gene sequencing showed that intraperitoneal injection of GW7647 or GW6471 significantly altered the abundance, homogeneity and composition of the gut microbiome. Analysis of the spinal cord metabolome showed that the levels of spinal cord metabolites were shifted after exposure to GW7647 or GW6471. Alterations in the composition of gut microbiota were significantly associated with the abundance of various spinal cord metabolites. The abundance of Licheniformes showed a significant positive correlation with nicotinamide, benzimidazole, eicosanoids, and pyridine abundance. Immunofluorescence results showed that intraperitoneal injection of GW7647 or GW6471 altered microglial activation and polarization levels. CONCLUSION: Our study shows that PPARα can promote M2-type microglia polarization, as well as alter gut microbiota and metabolites in CCI mice. This study enhances our understanding of the mechanism of PPARα in the treatment of neuropathic pain.

[Interventional effect and inflammatory regulation mechanism of dihydroartemisinin combined with pregabalin on neuropathic pain mice].

This study aims to clarify the effects of dihydroartemisinin(DHA) combined with pregabalin(PGB) on neuropathic pain(NP) in mice and explore the neuroinflammatory regulatory mechanism. NP mice model was established using spinal nerve ligation, whereas the sham group exposed the spinal nerve without ligation. The mice were randomly divided into sham group, model group, PGB groups of low, medium, and high doses(PGB-L, PGB-M, and PGB-H, with 22, 45, and 91 mg·kg~(-1)), DHA group(16 mg·kg~(-1)), and DHA combined with PGB groups of low, medium, and high doses(DHA + PGB-L, DHA + PGB-M, and DHA + PGB-H). Administration by gavage 18 days after modeling. Von Frey and cold plate were used to detect mechanical pain threshold and cold pain sensitivity in mice. The tail suspension test and forced swimming test were used to investigate depressive behavior, and the open field test was used to estimate anxiety behavior. The Morris water maze was used to evaluate cognitive function. Liquid suspension chip technology was used to quantitatively analyze immune inflammation-related factors. Immunofluorescence was used to detect the expression of CC chemokine ligand 3(CCL3) and transmembrane protein 119(TMEM119). The results showed that compared with the sham group, the mechanical pain and cold pain sensitivity thresholds of the model group were significantly reduced, and the struggle time was significantly increased in the tail suspension test and forced swimming test. The activity time in the central area was significantly reduced in the open field test. The residence time in the second/fourth quadrant was significantly longer than that in other quadrants, and the latency time of platform climbing significantly increased after platform withdrawal in the Morris water maze experiment. The expression of CCL3 was significantly increased; the number of TMEM119 positive cells and the cell body area were significantly increased. Compared with the model group, the DHA + PGB-M group showed a significant increase in mechanical pain and cold pain sensitivity thresholds, as well as a significant increase in struggle time in the tail suspension test and forced swimming test. The activity time in the central area of the open field test was significantly reduced. The residence time in the second/fourth quadrant was significantly shorter than that in other quadrants, and the latency time of platform climbing after platform withdrawal was significantly reduced. Compared with the PGB-M group, the mechanical pain threshold of D14-17 in the DHA + PGB-M group was significantly increased, and the struggle time during forced swimming was significantly increased. The residence time in the second/fourth quadrant of the Morris water maze was significantly shorter than that in other quadrants. Compared with the model group, the expression of CCL3, the number of TMEM119 positive cells, and the cell body area in the DHA + PGB-M group were significantly decreased. This study indicates that DHA + PGB can enhance the analgesic effect of PGB on NP mice, break through the limitations of PGB tolerance, and make up for the shortcomings of PGB in antidepressant and cognitive improvement. Its mechanism may be related to regulating neuroinflammation by inhibiting the activation of microglial cells and expression of CCL3.

Changes in SLITRK1 Level in the Amygdala Mediate Chronic Neuropathic Pain-Induced Anxio-Depressive Behaviors in Mice.

BACKGROUND: Comorbid chronic neuropathic pain (NPP) and anxio-depressive disorders (ADD) have become a serious global public-health problem. The SLIT and NTRK-like 1 (SLITRK1) protein is important for synaptic remodeling and is highly expressed in the amygdala, an important brain region involved in various emotional behaviors. We examined whether SLITRK1 protein in the amygdala participates in NPP and comorbid ADD. METHODS: A chronic NPP mouse model was constructed by L5 spinal nerve ligation; changes in chronic pain and ADD-like behaviors were measured in behavioral tests. Changes in SLITRK1 protein and excitatory synaptic functional proteins in the amygdala were measured by immunofluorescence and Western blot. Adeno-associated virus was transfected into excitatory synaptic neurons in the amygdala to up-regulate the expression of SLITRK1. RESULTS: Chronic NPP-related ADD-like behavior was successfully produced in mice by L5 ligation. We found that chronic NPP and related ADD decreased amygdalar expression of SLITRK1 and proteins important for excitatory synaptic function, including Homer1, postsynaptic density protein 95 (PSD95), and synaptophysin. Virally-mediated SLITRK1 overexpression in the amygdala produced a significant easing of chronic NPP and ADD, and restored the expression levels of Homer1, PSD95, and synaptophysin. CONCLUSION: Our findings indicated that SLITRK1 in the amygdala plays an important role in chronic pain and related ADD, and may prove to be a potential therapeutic target for chronic NPP-ADD comorbidity.

Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.

CXCL13/CXCR5 promote chronic postsurgical pain and astrocyte activation in rats by targeting NLRP3.

Chronic postsurgical pain (CPSP) with high incidence negatively impacts the quality of life. X-C motif chemokine 13 (CXCL13) has been associated with postsurgery inflammation and exacerbates neuropathic pain in patients with CPSP. This study was aimed to illustrate the relationship between CXCL13 and nod-like receptor protein-3 (NLRP3), which is also involved in CPSP. A CPSP model was constructed by skin/muscle incision and retraction (SMIR) in right medial thigh, and the rats were divided into three groups: Sham, SMIR, and SMIR + anti-CXCL13 (intrathecally injected with anti-CXCL13 antibody). Then, the paw withdrawal threshold (PWT) score of rats was recorded. Primary rat astrocytes were isolated and treated with recombinant protein CXCL13 with or without NLRP3 inhibitor INF39. The expressions of CXCL13, CXCR5, IL-1ß, IL-18, GFAP, NLRP3, and Caspase-1 p20 were detected by real-time quantitative reverse transcription PCR, western blot, ELISA, immunocytochemistry, and immunofluorescence analyses. The anti-CXCL13 antibody alleviated SMIR-induced decreased PWT and increased expression of GFAP, CXCL13, CXCR5, NLRP3, and Caspase-1 p20 in spinal cord tissues. The production of IL-1ß, IL-18, and expression of CXCL13, CXCR5, GFAP, NLRP3, and Caspase-1 p20 were increased in recombinant protein CXCL13-treated primary rat astrocytes in a dose-dependent manner. Treatment with NLRP3 inhibitor INF39 inhibited the function of recombinant protein CXCL13 in primary rat astrocytes. The CXCL13/CXCR5 signaling could promote neuropathic pain, astrocytes activation, and NLRP3 inflammasome activation in CPSP model rats by targeting NLRP3. NLRP3 may be a potential target for the management of CPSP.

A novel microglia-targeting strategy based on nanoparticle-mediated delivery of miR-26a-5p for long-lasting analgesia in chronic pain.

Accumulating evidence supports the notion that microglia play versatile roles in different chronic pain conditions. However, therapeutic strategies of chronic pain by targeting microglia remain largely overlooked. This study seeks to develop a miRNA-loaded nano-delivery system by targeting microglia, which could provide a decent and long-lasting analgesia for chronic pain. Surface aminated mesoporous silica nanoparticles were adopted to load miR-26a-5p, a potent analgesic miRNA, by electrostatic adsorption, which can avoid miR-26a-5p is rapidly released and degraded. Then, targeting peptide MG1 was modified on the surface of aminated mesoporous silica particles for microglia targeting. In peripheral nerve injury induced neuropathic pain model, a satisfactory anti-allodynia effect with about 6 weeks pain-relief duration were achieved through targeting microglia strategy, which decreased microglia activation and inflammation by Wnt5a, a non-canonical Wnt pathway. In inflammatory pain and chemotherapy induced peripheral neuropathic pain, microglia targeting strategy also exhibited more efficient analgesia and longer pain-relief duration than others. Overall, we developed a microglia-targeting nano-delivery system, which facilitates precisely miR-26a-5p delivery to enhance analgesic effect and duration for several chronic pain conditions.

Post-treatment with maropitant reduces oxidative stress, endoplasmic reticulum stress and neuroinflammation on peripheral nerve injury in rats.

OBJECTIVE: To determine the effective dose and therapeutic potential of maropitant using through expression of mediators of oxidative stress, inflammatory and of the unfolded protein response (UPR) (bio) markers on spinal cord using a model of neuropathic pain induced through chronic constriction injury (CCI) in rats. STUDY DESIGN: Randomized, blinded, prospective experimental study. ANIMALS: 98 male Wistar rats. METHODS: Rats were anesthetized with sevoflurane and after CCI, they were randomly assigned to the following groups that received: vehicle, 3, 6, 15, 30 e 50 mg/kg/24q of maropitant. The effect on inflammatory mediators (IL10, TNFα), oxidative stress (GPx, CAT, SOD), microglial (IBA-1) and neuronal (NeuN, TACR1) markers was evaluated though immunohistochemistry and expression levels of markers of hypoxia (HIF1α, Nrf2), antioxidant enzymes (Catalse, Sod1 and GPx1), and endoplasmic reticulum stress mediators (GRP78, CHOP and PERK) through qRT-PCR. RESULTS: Intraperitoneal injection (IP) of maropitant inhibited nociception with ID50 values of 4,1 mg/kg (5,85-19,36) in a neuropathic pain model through CCI. A dose of 30 mg/kg/24q was significantly effective in reducing mechanical allodynia 1 to 4h after treatment with nociception inhibition (145,83%). A reduction in the expression of hypoxia factors (HIF1α, Nrf2) was observed, along with an increase in antioxidant activity (CAT, SOD and GPX). Additionally, there was a reduction in inflammatory markes (IL10, TNFα), microglial (IBA-1), and neuronal markers (NeuN, TACR1). CONCLUSION AND CLINICAL RELEVANCE: These findings demonstrate that the determined dose, administered daily for seven days, had an antinociceptive effect, as well as anti-inflammatory and antioxidant activity.

Electroacupuncture suppresses neuronal ferroptosis to relieve chronic neuropathic pain.

Growing evidence supports the analgesic efficacy of electroacupuncture (EA) in managing chronic neuropathic pain (NP) in both patients and NP models induced by peripheral nerve injury. However, the underlying mechanisms remain incompletely understood. Ferroptosis, a novel form of programmed cell death, has been found to be activated during NP development, while EA has shown potential in promoting neurological recovery following acute cerebral injury by targeting ferroptosis. In this study, to investigate the detailed mechanism underlying EA intervention on NP, male Sprague-Dawley rats with chronic constriction injury (CCI)-induced NP model received EA treatment at acupoints ST36 and GV20 for 14 days. Results demonstrated that EA effectively attenuated CCI-induced pain hypersensitivity and mitigated neuron damage and loss in the spinal cord of NP rats. Moreover, EA reversed the oxidative stress-mediated spinal ferroptosis phenotype by upregulating reduced expression of xCT, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH1) and superoxide dismutase (SOD) levels, and downregulating increased expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), malondialdehyde levels and iron overload. Furthermore, EA increased the immunofluorescence co-staining of GPX4 in neurons cells of the spinal cord of CCI rats. Mechanistic analysis unveiled that the inhibition of antioxidant pathway of Nrf2 signalling via its specific inhibitor, ML385, significantly countered EA's protective effect against neuronal ferroptosis in NP rats while marginally diminishing its analgesic effect. These findings suggest that EA treatment at acupoints ST36 and GV20 may protect against NP by inhibiting neuronal ferroptosis in the spinal cord, partially through the activation of Nrf2 signalling.