Neuropathic Pain Model Research Articles

Discovery of a Novel Multitarget Analgesic Through an In Vivo-Guided Approach

Background: Pain is a complex condition influenced by peripheral, central, immune, and psychological factors. Multitarget approaches offer a more effective and safer alternative to single-target analgesics by enhancing efficacy, reducing side effects, and minimizing tolerance. This study aimed to identify a novel multitarget analgesic with improved pharmacological properties. Methods: An in vivo-guided screening approach was used to discover a new analgesic compound. Compound 29, derived from a novel scaffold inspired by opiranserin and vilazodone pharmacophores, was identified through analog screening in the formalin test. Its efficacy was further evaluated in the spinal nerve ligation (SNL) model of neuropathic pain. Mechanistic studies explored its interaction with neurotransmitter transporters and receptors, while pharmacokinetic and safety assessments were conducted to determine its stability, brain penetration, and potential toxicity. Results: Compound 29 demonstrated high potency in the formalin test, with an ED50 of 0.78 mg/kg in the second phase and a concentration-dependent effect in the first phase. In the SNL model, it produced dose-dependent analgesic effects, increasing withdrawal thresholds by 24% and 45% maximum possible effect (MPE) at 50 and 100 mg/kg, respectively. Mechanistic studies revealed strong triple uptake inhibition, particularly at dopamine (DAT) and serotonin (SERT) transporters, alongside high-affinity 5-HT2A receptor antagonism. Pharmacokinetic analysis indicated enhanced stability and blood–brain barrier permeability. In vitro studies confirmed its nontoxicity to HT-22 cells but revealed potential hERG inhibition and strong CYP3A4 inhibition. Conclusions: Compound 29 is a promising multitarget analgesic with potent efficacy and favorable pharmacokinetics. Ongoing optimization efforts aim to mitigate side effects and enhance its therapeutic profile for clinical application.

Evidence for glial reactivity using positron-emission tomography imaging of translocator Protein-18 kD [TSPO] in both sham and nerve-injured rats in a preclinical model of orofacial neuropathic pain.

Chronic neuropathic pain is a debilitating condition that results from damage to the nervous system. Current treatments are largely ineffective, with limited understanding of the underlying mechanisms hindering development of effective treatments. Preclinical models of neuropathic pain have revealed that non-neural changes are important for the development of neuropathic pain, although these data are derived almost exclusively from post-mortem histological analyses. Although these static snapshots have provided valuable data, they cannot provide insights into non-neural cell changes that could be also assessed in human patients with chronic pain. In this study we used translocator protein 18kDa (TSPO) PET imaging with [18F]PBR06 to visualise in-vivo, the activity of macrophages and microglia in a rodent preclinical model of trigeminal neuropathic pain. Using chronic constriction injury of the infraorbital nerve (ION-CCI) we compared temporal changes in TSPO binding in male rats, prior to, and up to 28days after ION-CCI compared with both sham-injured and naïve counterparts. Unexpectedly, we found significant increases in TSPO signal in both ION-CCI and sham-injured rats within the trigeminal ganglion, spinal trigeminal nucleus and paratrigeminal nucleus during the initial phase following surgery and/or nerve injury. This increased TSPO binding returned to control levels by day 28. Qualitative histological appraisal of macrophage accumulation and glial reactivity in both ION-CCI and sham-injured rats indicated macrophage accumulation in the trigeminal ganglion and microglial reactivity in the brainstem trigeminal complex. These findings show, glial changes in the peripheral nerve and brain in both nerve-injured and sham-injured rats in a preclinical model of neuropathic pain which provides a platform for translation into human patients.

Histamine H3 receptor blockade alleviates neuropathic pain through the regulation of glial cells activation.

Neuropathic pain is a disorder affecting the somatosensory nervous system. However, this condition is also characterized by significant neuroinflammation, primarily involving CNS-resident non-neuronal cells. A promising target for developing new analgesics is histamine H3 receptor (H3R); thus, we aimed to determine the influence of a novel H3R antagonist/inverse agonist, E-98 (1-(7-(4-chlorophenoxy)heptyl)-3-methylpiperidine), on pain symptoms and glia activation in model of neuropathic pain in male mice (chronic constriction injury to the sciatic nerve). We investigated the effects of single intraperitoneal (i.p.) (1, 5, 10, 20 mg/kg) and intrathecal (i.th.) (10, 30, 60 µg/5 µL) E-98-injections on mechanical (von Frey) and thermal (cold plate, tail flick) stimuli. The effect of chronic E-98 (10 mg/kg, i.p.) treatment and its influence on glia activation within the lumbar spinal cord was investigated. The anti-inflammatory properties of E-98 (10 µM) were screened in primary microglial and astroglial cell cultures. We assessed the presence of H3R within the spinal cord of control and neuropathic animals and in cell cultures. E-98 attenuated nociceptive responses in a dose- and time-dependent manner, and this effect is correlated with reduced microglia and increased astroglia activation. In vitro studies showed a decreased pro-inflammatory IL-6 level in both cell cultures. We observed co-localization of H3R with spinal neurons, microglia, and astrocytes and in primary glial cell cultures. We suggest that an analgesic effect of E-98 is partially due to the modulation of glial activation. We explore a new mechanism of H3R antagonists/inverse agonists analgesic action, bringing the potential benefits in pain management strategies.

What do ultrasound vocalizations really mean in rats with different origins of pain?

This study is to assess how 22 kHz and 50 kHz spontaneous ultrasound vocalization (USV) calls would be affected by different origins of pain so as to validate the use of USV in pain studies. Five well-established rat models of pain were used to evaluate various parameters of spontaneous 22 kHz and 50 kHz calls in adult male rats in terms of both acute and chronic or inflammatory and neuropathic or somatic and visceral origins. The effects of local lidocaine blockade of the injection site and intraperitoneal administration of antidepressant (amitriptyline) and anticonvulsant (gabapentin) were examined as well in typical inflammatory and neuropathic pain models, respectively. The major new gains were as follows: (1) naive rats staying alone and engaging dyadic social interaction with a naive or a conspecific in pain emitted similar power and amounts of both 22 kHz and 50 kHz spontaneous USV calls; however, rats suffering from various origins of pain emitted significantly less USV calls of both 22 kHz and 50 kHz in terms of both number and time. (2) Local blockade of the injury sites of inflammatory pain could reverse the impaired emission of both 22 kHz and 50 kHz spontaneous calls, so did the treatment of the rats with neuropathic pain by amitriptyline and gabapentin. Emissions of both 22 kHz and 50 kHz spontaneous calls were impaired by acute and chronic pain conditions regardless of inflammatory and neuropathic or somatic and visceral origins.

Effects of Biased Analogues of the Kappa Opioid Receptor Agonist, U50,488, in Preclinical Models of Pain and Side Effects

Kappa opioid receptor (KOR) agonists have well-established antinociceptive effects. However, many KOR agonists have negative side effects, which limit their therapeutic potential. Some researchers have suggested that the development of biased agonists that preferentially stimulate KOR G-protein pathways over β-arrestin pathways may yield drugs with fewer adverse side effects. This was investigated in the current study. We describe the synthesis and characterization of three U50,488 analogues, 1, 2, and 3. We evaluated the acute and chronic antinociceptive effects of these compounds in mice using the warm-water tail flick assay and in a paclitaxel-induced neuropathic pain model. Side effects were investigated using open-field, passive wire hang, rotarod, elevated zero maze, conditioned place aversion, and whole-body plethysmography, with some tests being conducted in KOR or β-arrestin2 knock out mice. All compounds were highly potent, full agonists of the KOR, with varying signaling biases in vitro. In the warm-water tail withdrawal assay, these agonists were ~10 times more potent than U50,488, but not more efficacious. All KOR agonists reversed paclitaxel-induced neuropathic pain, without tolerance. Compound 3 showed no significant side effects on any test. Signaling bias did not correlate with the antinociceptive or side effects of any compounds and knockout of β-arrestin2 had no effect on U50,488-induced sedation or motor incoordination. These findings highlight the therapeutic potential of 3, with its lack of side effects typically associated with KOR agonists, and also suggest that G-protein signaling bias is a poor predictor of KOR agonist-induced side effects.

Animal models of cisplatin-induced neuropathic pain.

Cisplatin chemotherapy has been used as the main treatment for different types of cancer. However, cisplatin chemotherapy-induced peripheral neuropathic pain (CIPNP) seriously affects the treatment process and quality of life of patients. In addition, it impacts the underlying mechanism and prevention and treatment strategies, indicating that drug selection and efficacy evaluation need to be further investigated. Furthermore, an animal model that is more consistent with the pathological mechanism needs to be developed. In this study, we describe and discuss the methods of developing and detecting CIPNP models in rats and mice induced by cisplatin chemotherapy. The aim was to improve the modeling rate and develop animal models that are more consistent with the developmental pattern of the disease. In addition, the study provides ideal reference animal models for clinical research and drug discovery and development.

Sprouting sympathetic fibres release CXCL16 and norepinephrine to synergistically mediate sensory neuronal hyperexcitability in a rodent model of neuropathic pain.

Chronic neuropathic pain generally has a poor response to treatment with conventional drugs. Sympathectomy can alleviate neuropathic pain in some patients, suggesting that abnormal sympathetic-somatosensory signaling interactions might underlie some forms of neuropathic pain. The molecular mechanisms underlying sympathetic-somatosensory interactions in neuropathic pain remain obscure. Lumbar sympathectomy was performed in spared nerve injury (SNI) mice or rats, and the up-down method was used to measure the mechanical paw withdrawal threshold. Dorsal root ganglia (DRG) injection and perfusion were used to deliver virus or drugs. Methylated RNA immunoprecipitation sequencing, RNA-sequencing, and immunoelectron microscopy were used to identify neurotransmitters. We found that sprouting tyrosine hydroxylase-positive sympathetic fibres in DRG mediated the maintenance of mechanical allodynia after SNI (day 28, P<0.001). We further found that SNI significantly increased the N6-methyladenosine level of CXCL16 messenger RNA (day 28, P<0.001), which was attributable to the reduced N6-methyladenosine demethylase fat mass and obesity-associated protein (P=0.002) and increased interaction with YTHDF1 (P=0.013) in the sympathetic ganglion. Enhanced expression of CXCL16 in the sympathetic ganglia can lead to increases release into the DRG and act synergistically with norepinephrine from sympathetic terminals to enhance DRG neuronal excitability. Norepinephrine and CXCL16 co-released from sympathetic nerve terminals in the DRG synergistically contribute to maintenance of neuropathic pain in a rodent model.

Proteomic analysis of spinal dorsal horn in prior exercise protection against neuropathic pain

Neuropathic pain (NP) is a complex and prevalent chronic pain condition that affects millions of individuals worldwide. Previous studies have shown that prior exercise protects against NP caused by nerve injury. However, the underlying mechanisms of this protective effect remain to be uncovered. Therefore, the purpose of this study is to investigate how prior exercise affects protein expression in NP model rats and thus gain comprehensive insights into the molecular mechanisms involved. To achieve this objective, 6-week-old male Sprague–Dawley rats were randomly assigned into three groups, named as chronic constriction injury (CCI) of the sciatic nerve, CCI with prior 6-week swimming training (CCI_Ex), and sham operated (Sham). The CCI_Ex group underwent 6 weeks of swimming training before CCI surgery, while the CCI and sham groups had no intervention. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were used as the main observation indicators to evaluate the behavioral changes associated with pain. Tissues from the spinal dorsal horn of the rats in the three groups were collected at 4 weeks after operation. LC–MS/MS proteomic analysis based on the label-free approach was used to detect protein profiles, and volcano plots, Venn diagrams, and clustering heatmaps were used to identify differentially expressed proteins (DEPs). Gene Ontology (GO) annotations, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and protein–protein interaction networks were employed to explore the biological importance of DEPs. At 14, 21, and 28 days following CCI, CCI rats with prior exercise showed a significant increase in the MWT and TWL of the injured lateral hind paw compared with those without exercise. A total of 122 proteins with significant changes in abundance were detected after CCI surgery, and 55 proteins were detected in the comparison between the CCI_Ex and CCI groups. GO and KEGG enrichment analysis revealed that oxygen transport capacity and the complement and coagulation cascades may be the critical mechanism by which prior exercise protects against NP. Serpina1, DHX9, and Alb are the key proteins in this process and warrant further attention, as confirmed by the results of Western blot analysis. In conclusion, this study provides new evidence that active physical activity can accelerate the relief of hyperalgesia after NP. Proteomic analyses revealed the potential target proteins and pathways for this process, offering valuable data resources and new insights into the pathogenesis and therapeutic targets of NP.

Sex-Specific Contrasting Role of BECLIN-1 Protein in Pain Hypersensitivity and Anxiety-Like Behaviors.

Chronic pain is a debilitative disease affecting one in five adults globally and is a major risk factor for anxiety ( Goldberg and McGee, 2011; Lurie, 2018). Given the current dearth of available treatments for both individuals living with chronic pain and mental illnesses, there is a critical need for research into the molecular mechanisms involved in order to discover novel treatment targets. Cellular homeostasis is crucial for normal bodily functions, and investigations of this process may provide better understanding of the mechanisms driving the development of chronic pain. Using the spared nerve injury (SNI) model of neuropathic pain, we found contrasting roles for BECLIN-1 in the development of pain hypersensitivity and anxiety-like behaviors in a sex-dependent manner. Remarkably, we found that male SNI mice with impaired BECLIN-1 function demonstrated heightened mechanical and thermal hypersensitivity compared with male wild-type SNI mice, while female SNI mice with impaired BECLIN-1 function demonstrated similar thresholds to the female wild-type SNI mice. We also found that disruptions of BECLIN-1 prevented SNI-induced increases in anxiety-like behaviors in male mice. Our data thus indicate that BECLIN-1 is differentially involved in the nociceptive and emotional components of chronic pain in male but not female mice.

Discovery of Novel Nav1.7-Selective Inhibitors with the 1H-Indole-3-Propionamide Scaffold for Effective Pain Relief.

Nav1.7 is considered a promising target for developing next-generation analgesic drugs, given its critical role in human pain pathologies. Although most reported inhibitors with strong invitro activity and high selectivity share the aryl sulfonamide scaffold, they failed to demonstrate marked clinical efficacy. Therefore, exploring new Nav1.7-selective antagonists is quite urgent to the development of next-generation analgesic drugs. Here, we report a highly effective 1H-indole-3-propionamide inhibitor, WN2, identified through an integrated drug discovery strategy. Notably, the structure of WN2 is quite different from previously reported aryl sulfonamide inhibitors. Molecular dynamics simulations and experimental findings reveal that the R configuration of WN2 (WN2-R) is the preferred form (IC50 = 24.7 ± 9.4 nM) within the VSDIV pocket of Nav1.7. WN2-R exhibits impressive analgesic effects in acute and chronic inflammatory pain, as well as neuropathic pain models in mice. Additionally, it displays favorable subtype selectivity and positive drug safety in acute toxicity studies. Pharmacokinetic studies indicate that WN2-R has high bioavailability (F = 20.29%), highlighting its considerable potential for drug development. Our study establishes WN2-R as a novel Nav1.7-selective inhibitor with a unique structural scaffold, offering a promising candidate for the next generation of analgesic drugs.

A novel approach to completely alleviate peripheral neuropathic pain in human patients: insights from preclinical data.

Neuropathic pain is a pervasive health concern worldwide, posing significant challenges to both clinicians and neuroscientists. While acute pain serves as a warning signal for potential tissue damage, neuropathic pain represents a chronic pathological condition resulting from injury or disease affecting sensory pathways of the nervous system. Neuropathic pain is characterized by long-lasting ipsilateral hyperalgesia (increased sensitivity to pain), allodynia (pain sensation in response to stimuli that are not normally painful), and spontaneous unprovoked pain. Current treatments for neuropathic pain are generally inadequate, and prevention remains elusive. In this review, we provide an overview of current treatments, their limitations, and a discussion on the potential of capsaicin and its analog, resiniferatoxin (RTX), for complete alleviation of nerve injury-induced neuropathic pain. In an animal model of neuropathic pain where the fifth lumbar (L5) spinal nerve is unilaterally ligated and cut, resulting in ipsilateral hyperalgesia, allodynia, and spontaneous pain akin to human neuropathic pain. The application of capsaicin or RTX to the adjacent uninjured L3 and L4 nerves completely alleviated and prevented mechanical and thermal hyperalgesia following the L5 nerve injury. The effects of this treatment were specific to unmyelinated fibers (responsible for pain sensation), while thick myelinated nerve fibers (responsible for touch and mechanoreceptor sensations) remained intact. Here, we propose to translate these promising preclinical results into effective therapeutic interventions in humans by direct application of capsaicin or RTX to adjacent uninjured nerves in patients who suffer from neuropathic pain due to peripheral nerve injury, following surgical interventions, diabetic neuropathy, trauma, vertebral disc herniation, nerve entrapment, ischemia, postherpetic lesion, and spinal cord injury.

Cannabis sativa L. Extract Increases COX-1, COX-2 and TNF-α in the Hippocampus of Rats with Neuropathic Pain.

Inflammation is the critical component of neuropathic pain; therefore, this study aimed to assess the potential anti-inflammatory effects of Cannabis sativa L. extracts in a vincristine-induced model of neuropathic pain. The effects of different doses (5.0-40.0 mg/kg) of two Cannabis sativa L. extracts (B and D) on COX-1, COX-2, TNF-α, and NF-κB mRNA and protein levels were examined in the rat hippocampus, cerebral cortex, and blood lymphocytes. There were statistically significant differences in COX-1, COX-2, and TNF-α mRNA and protein expression in the hippocampus and cerebral cortex, with significant differences in COX-2 and TNF-α in the lymphocytes. Extract D dose-dependently increased COX-1 mRNA and protein in the hippocampus and cortex. In contrast, Extract B dose-dependently increased COX-1 mRNA and decreased COX-2 mRNA (in a dose of 7.5 mg/kg) and TNF-α protein levels in the cortex. Cannabis sativa L. extracts significantly influenced the expression of inflammatory genes and proteins, with effects varying based on dose and tissue type. The increased expression of COX-1, COX-2, and TNF-α (in comparison to groups receiving NaCl, vincristine, and gabapentin) in the rat hippocampus and COX-1 in the cerebral cortex suggests that Cannabis may have a pro-inflammatory effect. Due to species specificity, the results of our research based on rats require confirmation in humans. However, Cannabis sativa should be recommended with caution for treating pain with an inflammatory component.

N-Palmitoylethanolamide enhances antinociceptive effect of tramadol in neuropathic rats.

The efficacy of opioids in the treatment of chronic pain is limited; however, the adverse effects they produce are considerable. N-palmitoylethanolamide (PEA), a bioactive lipid mediator with structural similarities to endocannabinoids, has exhibited notable anti-inflammatory and analgesic effects in preclinical models. The objective of this study was to investigate the antinociceptive properties, motor coordination (MC), and constipation effects of tramadol and PEA in combination within a neuropathic pain model. The antinociceptive effects of tramadol (TRA) and PEA in various combination ratios were assessed using a CCI model of neuropathic pain in 126 male Wistar rats, divided into 21 groups: vehicles, GBP (1.0-177.78 mg/kg), TRA (1.0-31.62 mg/kg), PEA (0.0316-10 mg/kg), or TRA (1.0-10 mg/kg) with PEA (0.0316-1.0 mg/kg), all compounds were administered orally. The results of the dose-response analyses indicated that PEA was approximately five and eightfold more potent than tramadol in producing anti-hyperalgesic and anti-allodynic effects, respectively. The results of the surface of synergistic interaction (SSI) analysis indicated that the combination of TRA 10 mg/kg + PEA 0.0316 mg/kg exhibited the most pronounced anti-hyperalgesic effects and the most favorable anti-allodynic outcomes among the various combinations. No significant differences in MC or constipation were observed between the vehicle and optimal combination group. The results of this study demonstrate that sub-antinociceptive doses of tramadol, when combined with PEA, significantly enhance the antinociceptive efficacy of tramadol without the induction of typical opioid-associated side effects. These findings propose a novel approach to the treatment of pain.

Emotional Impairments in Animal Models of Traumatic Neuropathic Pain: Where Do We Stand?

Emotional Impairments in Animal Models of Traumatic Neuropathic Pain: Where Do We Stand?

Modulating Anxiety-Like Behaviors in Neuropathic Pain: Role of Anterior Cingulate Cortex Astrocytes Activation.

The comorbidity of anxiety-like symptoms in neuropathic pain (NP) is a significant yet often overlooked health concern. Anxiety sufferers may have a lower tolerance for pain, but which is difficult to treat. Accumulating evidence suggests a strong link between astrocytes and the manifestation of NP with concurrent anxiety-like behaviors. And the anterior cingulate cortex (ACC) has emerged as a key player in pain modulation and related emotional processing. However, the complex mechanisms that astrocytes in ACC influence anxiety behavior in mouse models of NP remain largely unexplored. Utilizing the traditional spared nerve injury (SNI) surgical model, we employed chemogenetic approaches, immunofluorescence, and western blot to investigate the functional significance and interactive dynamics between ACC astrocytes and excitatory neurons. Our results revealed that SNI surgery induces NP and delayed anxiety-like behaviors, accompanied by increased astrocyte activity in the ACC. Chemogenetic manipulation demonstrated that inhibiting astrocytes alleviates anxiety symptoms, while activating them exacerbates anxiety-like behaviors, affecting local excitatory neurons and synapse density. Direct manipulation of ACC excitatory neurons also significantly impacted anxiety-like behaviors. Our results highlight the pivotal role of ACC astrocytes in modulating anxiety-like behavior, suggesting a novel therapeutic strategy for anxiety associated with NP by targeting astrocyte function.

Cross-modal cortical circuit for sound sensitivity in neuropathic pain.

Hyperacusis, exaggerated sensitivity to sound, frequently accompanies chronic pain in humans, suggesting interactions between different sensory systems in the brain. However, the neural mechanisms underlying this comorbidity remain largely unexplored. In this study, behavioral tests measuring sound-evoked pupil dilation and reaction times to lick water following auditory stimuli showed hyperacusis-like behaviors in neuropathic pain model mice. Through viral tracing, fiber photometry, and multi-electrode recordings, we identified glutamatergic projections from primary somatosensory cortex (S1HLGlu) to the auditory cortex (ACx) that participate in amplifying sound-evoked neuronal activity following spared nerve injury in the hindlimb. Chemo- or optogenetic manipulation and electrophysiology recordings confirmed that the S1HLGlu → ACx pathway is essential for this heightened response to sound. Specifically, activating this pathway intensified glutamatergic neuronal activity in the ACx and induced hyperacusis-like behaviors, while chemogenetic suppression reversed these effects in neuropathic pain model mice. These findings illustrate the mechanism by which central gain increases in the ACx of neuropathic pain mice, improving our understanding of cross-modal sensory system interactions and suggesting circuit pathway targets for developing interventions to treat pain-associated hyperacusis in clinic.

Establishing an Electrophysiological Recording Platform for Epidural Spinal Cord Stimulation in Neuropathic Pain Rats.

Spinal cord stimulation (SCS) is pivotal in treating chronic intractable pain. To elucidate the mechanism of action among conventional and current novel types of SCSs, a stable and reliable electrophysiology model in the consensus animals to mimic human SCS treatment is essential. We have recently developed a new in vivo implantable pulsed-ultrahigh-frequency (pUHF) SCS platform for conducting behavioral and electrophysiological studies in rats. However, some technical details were not fully understood. This study comprehensively analyzed methodology and technical challenges and pitfalls encountered during the development and implementation of this model. Employing a newly developed pUHF-SCS (±3 V, 2Hz pulses with 500-kHz biphasic radiofrequency sinewaves), we assessed analgesic effect and changes of evoked local field potentials (eLFPs) in the bilateral primary somatosensory and anterior cingulate cortices in the rats with or without spared nerve injury (SNI) using the platform. The placement of stimulating needle electrodes in the hind paw was examined and optimized for functionality. SNI enhanced the C component of eLFPs in bilateral cortexes elicited by stimulating the contralateral but not the ipsilateral lateral aspect of the hind paw. Repeated pUHF-SCS significantly reversed SNI-induced paw hypersensitivity and reduced C-component enhancement. An impedance test can determine an optimal SCS electrode function: an SCS discharge threshold of 100-400μA for cortical activation or a motor threshold of 150-600μA for the hind limbs. Impedance increased due to growth of fibrotic tissue but stabilized after post-implantation day 12. We presented a reliable electrophysiological platform for SCS application in rat neuropathic pain model and demonstrated potent analgesic effects of pUHF-SCS. All device implantations or pUHF-SCS per se did not cause evident spinal cord damage. This safe and stable platform provides an in vivo rat model for SCS investigation of mechanisms of action and device innovation.

The Aqueous Extract of Armadillidium vulgare Latreille Alleviates Neuropathic Pain via Inhibiting Neuron-Astrocyte Crosstalk Mediated by the IL-12-IFN-γ-IFNGR-CXCL10 Pathway

Ethnopharmacological relevanceArmadillidium vulgare Latreille (AV), the dried body of pillbug, was originally described in Shennong’s Classic of Materia Medica. As a common analgesic in animal-based traditional Chinese medicine, it is mainly used to relieve pain, promoting diuresis, relieving fatigue and so on. Our work demonstrated that AV could alleviate various types of acute and chronic pain including neuropathic pain (NP). And transcriptome sequencing analysis revealed that AV could suppress CXCL10 to alleviate NP, however, the upstream mechanisms governing CXCL10 synthesis remain vague.Aim of the study: The research’s goal was to identify the mechanism via which AV regulates CXCL10 to ameliorate NP. Materials and methodsChronic constriction injury (CCI) to the sciatic nerve was used to induce the NP model 14 days following surgery. To identify cell signaling pathways, various approaches were used, including transcriptome sequencing, western blotting, immunofluorescence, as well as ELISA. The in vitro assay involved the cultivation of neuron PC12 cells and astrocyte C6 cells. ResultsBoth in vivo and in vitro results demonstrated that IL-12/IL-18 enhanced IFN-γ production in spinal neurons, which acted on IFN-γ receptors on neurons and astrocytes to upregulate CXCL10 expression in these cells, illustrating the pivotal role of IL-12 in the crosstalk between neurons and astrocytes. The role of IL-12 in pain regulation was elucidated for the first time within the nervous system. Additionally, its synergistic interaction with IL-18 on the downstream IFN-γ-CXCL10 pathway dramatically altered the activation of neurons and astrocytes. And AV could suppress CXCL10 to alleviate NP by mediating the IL-12-IFN-γ-IFNGR signaling pathway. ConclusionsWe explored a new target for NP by regulating neuron-astrocyte crosstalk and provided a theoretical basis for AV in clinical use.

Electroacupuncture Alleviates Neuropathic Pain and Negative Emotion in Mice by Regulating Gut Microbiota.

Neuropathic pain (NP) is a prevalent chronic condition frequently accompanied by adverse emotional states. Previous research has demonstrated the clinical efficacy of electroacupuncture (EA) in mitigating neuropathic pain and its associated mood disorders. Recent studies have underscored a correlation between gut microbiota and both NP and negative emotional states. Nevertheless, the relationship between the modulation of gut microbiota by EA and the amelioration of NP remains inadequately understood. Mice were randomly assigned to one of the three groups: the Control (Con) group, the EA group, and the Chronic Constrictive Injury (CCI) group (n = 12 each). Starting from the 8th day post-CCI induction, the EA group underwent EA treatment once every two days, for a total of 20 sessions. To investigate the impact of gut microbiota on CCI mice, we employed a variety of methods, including various behavioral tests and 16S ribosomal DNA (rDNA) sequencing. The results indicated that EA significantly ameliorated mechanical allodynia and emotional dysfunction induced by CCI in mice. Analysis through 16S rDNA sequencing revealed that the gut microbiota of NP model mice exhibited a marked increase in diversity. However, EA could partially reverse changes in the diversity of gut microbiota. The abundance of Alloprevotella, A2, Roseburia, Muribaculum, Ruminiclostridium, and Rikenella was increased, and the abundance levels of Bacteroides were decreased at the genus level in CCI mice. Following EA treatment, the relative abundance of Alistipes, A2, Roseburia, and Rikenella was decreased, whereas the relative abundance of Alloprevotella and Parabacteroides was increased in EA group when compared with the CCI group. These findings suggested that EA exerted a significant therapeutic effect on NP, potentially through modulation of the gut microbiota.

The diagnostic value and molecular mechanisms of LncRNA ZFAS1 in neuropathic pain.

Long non-coding RNA (lncRNA) has been playing an increasingly significant role in neuropathic pain (NP). This study aimed to investigate the clinical significance and mechanism of LncRNA ZNFX1 antisense RNA 1 (ZFAS1) in NP. 92 patients with NP and 85 healthy controls were enrolled, and a rat NP model was constructed by chronic constrictive injury (CCI). LPS-induced microglia BV2 cells were used to construct an in vitro cellular model. RT-qPCR analysis of the mRNA levels of ZFAS1, miR-421, and Iba-1 (markers of microglia activation). Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were used to assess mechanosensitive and thermal nociceptive allergic responses. ELISA assay for pro-inflammatory factors and anti-inflammatory factors expression. ROC assay for the diagnostic value of ZFAS1. Validation of the targeting between ZFAS1 and miR-421 by dual luciferase reporter assay. ZFAS1 significantly increased while miR-421 significantly decreased in individuals with NP, in a rat model of CCI, and in LPS-induced microglial cells. Functionally, miR-421 directly targeted ZFAS1. ZFAS1 levels could significantly differentiate between NP patients and control (AUC=0.910). Low expression of ZFAS1 significantly alleviated PWL and PWT in CCI rats. Elevated neuro-proinflammatory factors and decreased anti-inflammatory factors in CCI rats were significantly reversed by low expression of ZFAS1, but this is partially weakened by low expression of miR-421. Moreover, silencing ZFAS1 hindered the upregulation of Iba-1 expression induced by LPS, which was rescued significantly by miR-421. Elevated ZFAS1 is a potential bio-diagnostic marker for NP. Inhibition of ZFAS1 may alleviate NP progression by inhibiting microglia activation and neuro-inflammatory responses.