Chronic Constriction Injury Of Sciatic Nerve Research Articles

Dissociation between the anti-allodynic effects of fingolimod (FTY720) and desensitization of S1P1 receptor-mediated G-protein activation in a mouse model of sciatic nerve injury

Sphingosine-1-phosphate (S1P) receptor (S1PR) agonists, such as fingolimod (FTY720), alleviate nociception in preclinical pain models by either activation (agonism) or inhibition (functional antagonism) of S1PR type-1 (S1PR1). However, the dose-dependence and temporal relationship between reversal of nociception and modulation of S1PR1 signaling has not been systematically investigated. This study examined the relationship between FTY720-induced antinociception and S1PR1 adaptation using a sciatic nerve chronic constriction injury (CCI) model of neuropathic pain in male and female C57Bl/6J mice. Daily injections of FTY720 for 14 days dose-dependently reversed CCI-induced mechanical allodynia without tolerance development, and concomitantly resulted in a dose-dependent reduction of G-protein activation by the S1PR1-selective agonist SEW2871 in the lumbar spinal cord and brain. These findings indicate FTY720-induced desensitization of S1PR1 signaling coincides with its anti-allodynic effects. Consistent with this finding, a single injection of FTY720 reversed mechanical allodynia while concomitantly producing partial desensitization of S1PR1-stimulated G-protein activation in the CNS. However, mechanical allodynia returned 24-hr post injection, despite S1PR1 desensitization at that time, demonstrating a dissociation between these measures. Furthermore, CCI surgery led to elevations of sphingolipid metabolites, including S1P, which were unaffected by daily FTY720 administration, suggesting FTY720 reversed mechanical allodynia by targeting S1PR1 rather than sphingolipid metabolism. Supporting this hypothesis, acute administration of the S1PR1-selective agonist CYM-5442 mimicked the anti-allodynic effect of FTY720. In contrast, the S1PR1-selective antagonist NIBR-0213 prevented the anti-allodynic effect of FTY720, but NIBR-0213 given alone did not affect nociception. These results indicate that FTY720 alleviates CCI-induced allodynia through a mechanism distinct from functional antagonism.

Exploring the role of Yuxuebi tablet in neuropathic pain with the method of similarity research of drug pharmacological effects based on unsupervised machine learning.

Having multiple pharmacological effects is a characteristic of Traditional Chinese Medicine (TCM). Currently, there is a lack of suitable methods to explore and discover modern diseases suitable for TCM treatment using this characteristic. Unsupervised machine learning technology is an efficient strategy to predict the pharmacological activity of drugs. This study takes Yuxuebi Tablet (YXB) as the research object. Using the unsupervised machine learning technology of drug cell functional fingerprint similarity research, the potential pharmacological effects of YXB were discovered and verified. LC-MS combined with the in vitro intestinal absorption method was used to identify components of YXB that could be absorbed by the intestinal tract of rats. Unsupervised learning hierarchical clustering was used to calculate the degree of similarity of cellular functional fingerprints between these components and 121 marketed Western drugs whose indications are diseases and symptoms that YXB is commonly used to treat. Then, based on the Library of Integrated Network-based Cellular Signatures database, pathway analysis was performed for selected Western drugs with high similarity in cellular functional fingerprints with the components of YXB to discover the potential pharmacological effects of YXB, which were validated by animal experiments. We identified 40 intestinally absorbed components of YXB. Through predictive studies, we found that they have pharmacological effects very similar to non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. In addition, we found that they have very similar pharmacological effects to anti-neuropathic pain medications (such as gabapentin, duloxetine, and pethidine) and may inhibit the NF-κB signaling pathway and biological processes related to pain perception. Therefore, YXB may have an antinociceptive effect on neuropathic pain. Finally, we demonstrated that YXB significantly reduced neuropathic pain in a rat model of sciatic nerve chronic constriction injury (CCI). Transcriptome analysis further revealed that YXB regulates the expression of multiple genes involved in nerve injury repair, signal transduction, ion channels, and inflammatory response, with key regulatory targets including Sgk1, Sst, Isl1, and Shh. This study successfully identified and confirmed the previously unknown pharmacological activity of YXB against neuropathic pain through unsupervised learning prediction and experimental verification.

Activation of GPR55 alleviates neuropathic pain and chronic inflammation.

Neuropathic pain (NP) significantly impacts the quality of life due to its prolonged duration and lack of effective treatment. Recent findings suggest that targeting neuroinflammation is a promising approach for treating NP. G protein-coupled receptor 55 (GPR55), a member of the GPCR family, plays an important role in neuroinflammatory regulation. CID16020046, a GPR55 agonist, possesses promising anti-neuroinflammatory effects. Herein, the therapeutic effect of CID16020046 on NP was investigated in an NP rat model. The NP model was established using the unilateral sciatic nerve chronic constriction injury (CCI) assay. Both sham and CCI rats were intraperitoneally administered with 20mg/kg CID16020046. NP was assessed using paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). First, we showed that GPR55 was downregulated in the spinal dorsal horn of CCI rats. After CCI rats were treated with CID16020046, the values of PWT and PWL were increased, indicating their effect on pain relief. The treated rats had attenuated release of inflammatory cytokines in the spinal cord, decreased spinal malondialdehyde (MDA) levels, and increased spinal glutathione peroxidase (GSH-PX) activity. Additionally, the increased levels of phosphorylated nuclear factor (NF)-κB p65 in CCI rats were significantly alleviated by CID16020046 treatment. Mechanistically, we showed that CID16020046 significantly suppressed the activation of the Janus kinase (JAK2)/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in the spinal cord of CCI-treated rats. However, Colivelin TFA (a STAT3 agonist) abolished the effect of CID16020046 on JAK2/STAT3 activation. In conclusion, our data demonstrate that the activation of GPR55 by CID16020046 alleviates NP and neuroinflammation in CCI rats by mediating the JAK2/STAT3 pathway.

Interleukin-35 alleviates neuropathic pain and induces an anti-inflammatory shift in spinal microglia in nerve-injured male mice

Immune cells are critical in promoting neuroinflammation and neuropathic pain and in facilitating pain resolution, depending on their inflammatory and immunoregulatory cytokine response. Interleukin (IL)-35, secreted by regulatory immune cells, is a member of the IL-12 family with a potent immunosuppressive function. In this study, we investigated the effects of IL-35 on pain behaviors, spinal microglia phenotype following peripheral nerve injury, and in vitro microglial cultures in male and female mice. Intrathecal recombinant IL-35 treatment alleviated mechanical pain hypersensitivity prominently in male mice, with only a modest effect in female mice after sciatic nerve chronic constriction injury (CCI). IL-35 treatment resulted in sex-specific microglial changes following CCI, reducing inflammatory microglial markers and upregulating anti-inflammatory markers in male mice. Spatial transcriptomic analysis revealed that IL-35 suppressed microglial complement activation in the superficial dorsal horn in male mice after CCI. Moreover, in vitro studies showed that IL-35 treatment of cultured inflammatory microglia mitigated their hypertrophied morphology, increased their cell motility, and decreased their phagocytic activity, indicating a phenotypic shift towards homeostatic microglia. Further, IL-35 altered microglial cytokines/chemokines in vitro, suppressing the release of IL-9 and monocyte-chemoattractant protein-1 and increasing IL-10 in the supernatant of male microglial cultures. Our findings indicate that treatment with IL-35 modulates spinal microglia and alleviates neuropathic pain in male mice, suggesting IL-35 as a potential sex-specific targeted immunomodulatory treatment for neuropathic pain.

6-Methoxyflavone antagonizes chronic constriction injury and diabetes associated neuropathic nociception expression

Neuropathy is a disturbance of function or a pathological change in nerves causing poor health and quality of life. A proportion of chronic pain patients in the community suffer persistent neuropathic pain symptoms because current drug therapies may be suboptimal so there is a need for new therapeutic modalities. This study investigated the neuroprotective flavonoid, 6-methoxyflavone (6MF), as a potential therapeutic agent and gabapentin as the standard comparator, against neuropathic models. Thus, neuropathic-like states were induced in Sprague-Dawley rats using sciatic nerve chronic constriction injury (CCI) mononeuropathy and systemic administration of streptozotocin (STZ) to induce polyneuropathy. Subsequent behaviors reflecting allodynia, hyperalgesia, and vulvodynia were assessed and any possible motoric side-effects were evaluated including locomotor activity, as well as rotarod discoordination and gait disruption. 6MF (25–75 mg/kg) antagonized neuropathic-like nociceptive behaviors including static- (pressure) and dynamic- (light brushing) hindpaw allodynia plus heat/cold and pressure hyperalgesia in the CCI and STZ models. 6MF also reduced static and dynamic components of vulvodynia in the STZ induced polyneuropathy model. Additionally, 6MF reversed CCI and STZ suppression of locomotor activity and rotarod discoordination, suggesting a beneficial activity on motor side effects, in contrast to gabapentin. Hence, 6MF possesses anti-neuropathic-like activity not only against different nociceptive modalities but also impairment of motoric side effects.

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

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. 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. 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. 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.

The impact of sciatic nerve injury and social interactions testing on glucocorticoid receptor expression in catecholaminergic medullary cell populations

Paradoxically, while acute pain leads to transiently elevated corticosterone, chronic pain does not result in persistently elevated corticosterone. In the sciatic nerve chronic constriction injury (CCI) model of chronic pain, we have shown that the same nerve injury produces a range of behavioural outcomes, each associated with distinctive adaptations to the HPA-axis to achieve stable plasma corticosterone levels. We also demonstrated that CRF and GR expression in the paraventricular hypothalamus (PVH) was increased in rats that showed persistent changes to their social behaviours during Resident-Intruder testing (‘Persistent Effect’ rats) when compared to rats that showed no behavioural changes (‘No Effect’ rats). In this study, we investigated whether these changes were driven in part by altered sensitivity of the brainstem catecholaminergic pathways (known to regulate the PVH) to glucocorticoids. GR expression in adrenergic (C1,C2) and noradrenergic (A1,A2) cells was determined using immunohistochemistry in behaviourally tested CCI rats and in uninjured controls. We found no differences between Persistent Effect and No Effect rats in (1) the glucocorticoid sensitivity of these cells, or (2) the numbers of adrenergic and noradrenergic cells in each region. However, we discovered an overall reduction in GR expression in the non-catecholaminergic cells of these regions in both experimental groups when compared to uninjured controls, most likely attributable to the repeated Resident-Intruder testing. Taken together, these data suggest strongly that brainstem mechanisms are unlikely to play a key role in the rebalancing of the HPA-axis triggered by CCI, increasing the probability that these changes are driven by supra-hypothalamic regions.

Astrocyte senescence-like response related to peripheral nerve injury-induced neuropathic pain

BackgroundPeripheral nerve damage causes neuroinflammation, which plays a critical role in establishing and maintaining neuropathic pain (NeP). The mechanisms contributing to neuroinflammation remain poorly elucidated, and pharmacological strategies for NeP are limited. Thus, in this study, we planned to explore the possible link between astrocyte senescence and NeP disorders following chronic sciatic nerve injury.MethodsAn NeP animal model was established by inducing chronic constrictive injury (CCI) to the sciatic nerve in adult rats. A senolytic drug combination of dasatinib and quercetin was gavaged daily from the first postoperative day until the end of the study. Paw mechanical withdrawal threshold (PMWT) and paw thermal withdrawal latency (PTWL) were evaluated to assess behaviors in response to pain in the experimental rats. Senescence-associated β-galactosidase staining, western blot analysis, and immunofluorescence were applied to examine the levels of proinflammatory factors and severity of the senescence-like response in the spinal cord. Lipopolysaccharide (LPS) was administered to induce senescence of spinal astrocytes in primary cultures in vitro, to explore the potential impacts of senescence on the secretion of proinflammatory factors. Furthermore, single-cell RNA sequencing (scRNA-seq) was conducted to identify senescence-related molecular responses in spinal astrocytes under neuropathic pain.ResultsFollowing sciatic nerve CCI, rats exhibited reduced PMWT and PTWL, increased levels of spinal proinflammatory factors, and an enhanced degree of senescence in spinal astrocytes. Treatment with dasatinib and quercetin effectively attenuated spinal neuroinflammation and mitigated the hypersensitivities of the rats subjected to sciatic nerve CCI. Mechanistically, the dasatinib-quercetin combination reversed senescence in LPS-stimulated primary cultured astrocytes and decreased the levels of proinflammatory factors. The scRNA-seq data revealed four potential senescence-related genes in the spinal astrocyte population, and the expression of clusterin (CLU) protein was validated via in vitro experiments.ConclusionThe findings indicate the potential role of astrocyte senescence in neuroinflammation following peripheral nerve injury, and suggest that targeting CLU activation in astrocytes might provide a novel therapeutic strategy to treat NeP.Graphical abstract

LncRNA Kcnq1ot1relieves neuropathic pain through downregulation of Myd88.

Long non-coding RNAs (lncRNAs) have already been documented to become the therapeutic targets for neuropathic pain. Here, this work focused on exploring the specific mechanism underlying Kcnq1 overlapping transcript 1 (kcnq1ot1) in neuropathic pain. Sciatic nerve chronic constriction injury (CCI) in vivo and LPS-stimulated microglia BV2 cell injury in vitro were adopted to construct neuropathic pain models. Expressions of kcnq1ot1, MyD88, and microglia activation marker Iba-1 were measured. In this study, we carried out fluorescence in-situ Hybridization (FISH) and immunofluorescence for examining Kcnq1ot1 localization within microglial cells in mouse spinal dorsal horn. Subsequently, we evaluated binding between Kcnq1ot1 and Myd88, together with the expressions of IL-1β, IL-6, TNF-α, and Myd88 ubiquitination. Kcnq1ot1 levels decreased within CCI mice and LPS-induced BV2 cells. According to the results of FISH and immunofluorescence, Kcnq1ot1 is located in microglia. Overexpression of Kcnq1ot1 suppressed Iba-1, IL-1β, IL-6 together with TNF-α expression. RNA pull-down and RIP assay confirmed that Kcnq1ot1 bound to Myd88. In addition, Kcnq1ot1 overexpression promoted the degradation, enhanced the ubiquitination, and reduced protein level of Myd88. Overexpression of Myd88 eliminated the effects of Kcnq1ot1 overexpression on Iba-1level and production of pro-inflammatory cytokines. Further in vivo results revealed that increased Kcnq1ot1 level alleviated neuropathic pain and myelinated nerve fiber injury of CCI mice. Kcnq1ot1 downregulated Myd88 protein expression by binding to Myd88 and promoting its ubiquitination, which in turn suppressed microglia activation, pro-inflammatory cytokine production, and relieved neuropathic pain.

Downregulation of ciRNA-Kat6b in dorsal spinal horn is required for neuropathic pain by regulating Kcnk1 in miRNA-26a-dependent manner.

Nerve injury-induced maladaptive changes in gene expression in the spinal neurons are essential for neuropathic pain genesis. Circular RNAs (ciRNA) are emerging as key regulators of gene expression. Here, we identified a nervous-system-tissues-specific ciRNA-Kat6 with conservation in humans and mice. We aimed to investigate whether and how spinal dorsal horn ciRNA-Kat6b participates in neuropathic pain. Unilateral sciatic nerve chronic constrictive injury (CCI) surgery was used to prepare the neuropathic pain model. The differentially expressed ciRNAs were obtained by RNA-Sequencing. The identification of nervous-system-tissues specificity of ciRNA-Kat6b and the measurement of ciRNA-Kat6b and microRNA-26a (miRNA-26a) expression level were carried out by quantitative RT-PCR. The ciRNA-Kat6b that targets miRNA-26a and miRNA-26a that targets Kcnk1 were predicted by bioinformatics analysis and verified by in vitro luciferase reports test and in vivo experiments including Western-blot, immunofluorescence, and RNA-RNA immunoprecipitation. The correlation between neuropathic pain and ciRNA-Kat6b, miRNA-26a, or Kcnk1 was examined by the hypersensitivity response to heat and mechanical stimulus. Peripheral nerve injury downregulated ciRNA-Kat6b in the dorsal spinal horn of male mice. Rescuing this downregulation blocked nerve injury-induced increase of miRNA-26a, reversed the miRNA-26a-triggered decrease of potassium channel Kcnk1, a key neuropathic pain player, in the dorsal horn, and alleviates CCI-induced pain hypersensitivities. On the contrary, mimicking this downregulation increased the miRNA-26a level and decreased Kcnk1 in the spinal cord, resulting in neuropathic pain-like syndrome in naïve mice. Mechanistically, the downregulation of ciRNA-Kat6b reduced the accounts of miRNA-26a binding to ciRNA-Kat6b, and elevated the binding accounts of miRNA-26a to the 3' untranslated region of Kcnk1 mRNA and degeneration of Kcnk1 mRNA, triggering in the reduction of KCNK1 protein in the dorsal horn of neuropathic pain mice. The ciRNA-Kat6b/miRNA-26a/Kcnk1 pathway in dorsal horn neurons regulates the development and maintenance of neuropathic pain, ciRNA-Kat6b may be a potential new target for analgesic and treatment strategies.

Exploring the effect of Tuina on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation based on NR2B/PSD-95 pathway

ObjectiveTo investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation.MethodsFifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention.ResultsCompared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn.ConclusionTuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.

Analgesic Effect of Perineural Injection of BoNT/A on Neuropathic Pain Induced by Chronic Constriction Injury of Sciatic Nerve in Rats.

This study was designed to investigate the analgesic effect of perineural injection of BoNT/A on neuropathic pain induced by sciatic nerve chronic constriction injury (CCI) and possible mechanisms. SD rats were randomly divided into Sham group, CCI group and BoNT/A group. Paw mechanical withdrawal threshold (pMWT) and paw thermal withdrawal latency (pTWL) of each group were detected at different time points after surgery. The expression of myelin markers, autophagy markers and NLRP3 inflammasome-related molecules in injured sciatic nerves were examined at 12days after surgery. Moreover, C-fiber evoked potential in spinal dorsal horn was recorded. The expression of SNAP-25, neuroinflammation and synaptic plasticity in spinal dorsal horn of each group were examined. Then rats treated with BoNT/A were randomly divided into DMSO group and Wnt agonist group to further explore the regulatory effect of BoNT/A on Wnt pathway. We found that pMWT and pTWL of ipsilateral paw were significantly decreased in CCI group compared with Sham group, which could be improved by perineural injection of BoNT/A at days 7, 9 and 12 after surgery. The peripheral analgesic mechanisms of perineural injection of BoNT/A might be related to the protective effect on myelin sheath by inhibiting NLRP3 inflammasome and promoting autophagy flow, while the central analgesic mechanisms might be associated with inhibition of neuroinflammation and synaptic plasticity in spinal dorsal horn due to inhibiting SNAP-25 and Wnt pathway. As a new route of administration, perineural injection of BoNT/A can relieve CCI induced neuropathic pain probablyvia both peripheral andcentral analgesic mechanisms.

Transcription factor ETS proto-oncogene 1 contributes to neuropathic pain by regulating histone deacetylase 1 in primary afferent neurons.

Nerve injury can induce aberrant changes in ion channels, enzymes, and cytokines/chemokines in the dorsal root ganglia (DRGs); these changes are due to or at least partly governed by transcription factors that contribute to the genesis of neuropathic pain. However, the involvement of transcription factors in neuropathic pain is poorly understood. In this study, we report that transcription factor (TF) ETS proto-oncogene 1 (ETS1) is required for the initiation and development of neuropathic pain. Sciatic nerve chronic constrictive injury (CCI, a clinical neuropathic pain model) increases ETS1 expression in the injured male mouse DRG. Blocking this upregulation alleviated CCI-induced mechanical allodynia and thermal hyperalgesia, with no apparent effect on locomotor function. Mimicking this upregulation results in the genesis of nociception hypersensitivity; mechanistically, nerve injury-induced ETS1 upregulation promotes the expression of histone deacetylase 1 (HDAC1, a key initiator of pain) via enhancing its binding activity to the HDAC1 promotor, leading to the elevation of spinal central sensitization, as evidenced by increased expression of p-ERK1/2 and GFAP in the dorsal spinal horn. It appears that the ETS1/HDAC1 axis in DRG may have a critical role in the development and maintenance of neuropathic pain, and ETS1 is a potential therapeutic target in neuropathic pain.

New insights into the analgesic properties of the XCL1/XCR1 and XCL1/ITGA9 axes modulation under neuropathic pain conditions - evidence from animal studies.

Recent studies have indicated the involvement of chemokine-C-motif ligand 1 (XCL1) in nociceptive transmission; however, the participation of its two receptors, canonical chemokine-C-motif receptor 1 (XCR1) and integrin alpha-9 (ITGA9), recently recognized as a second receptor, has not been clarified to date. The aim was to explore by which of these receptors XCL1 reveals its pronociceptive properties and how the XCL1-XCR1 and XCL1-ITGA9 axes blockade/neutralization influence on pain-related behavior and opioid analgesia in the model of neuropathic pain. In our studies we used Albino Swiss mice which were exposed to the unilateral sciatic nerve chronic constriction injury (CCI) as a neuropathic pain model. Animals received single intrathecal (i.t.) injection of XCL1, XCL1 neutralizing antibodies, antagonist of XCR1 (vMIP-II) and neutralizing antibodies of ITGA9 (YA4), using lumbar puncture technique. Additionally we performed i.t. co-administration of abovementioned neutralizing antibodies and antagonists with single dose of morphine/buprenorphine. To assess pain-related behavior the von Frey and cold plate tests were used. To measure mRNA and protein level the RT-qPCR and Western Blot/Elisa/immunofluorescence techniques were performed, respectively. Statistical analysis was conducted using ANOVA with a Bonferroni correction. Presented studies have shown time-dependent upregulation of the mRNA and/or protein expression of XCL1 in the spinal cord after nerve injury as measured on day 1, 4, 7, 14, and 35. Our immunofluorescence study showed that XCL1 is released by astroglial cells located in the spinal cord, despite the neural localization of its receptors. Our results also provided the first evidence that the blockade/neutralization of both receptors, XCR1 and ITGA9, reversed hypersensitivity after intrathecal XCL1 administration in naive mice; however, neutralization of ITGA9 was more effective. In addition, the results proved that the XCL1 neutralizing antibody and, similarly, the blockade of XCR1 and neutralization of ITGA9 diminished thermal and mechanical hypersensitivity in nerve injury-exposed mice after 7 days. Additionally, neutralization of XCL1 improves morphine analgesia. Moreover, blockade of XCR1 positively influences buprenorphine effectiveness, and neutralization of ITGA9 enhances not only buprenorphine but also morphine analgesia. Therefore, blockade of the XCL1-ITGA9 interaction may serve as an innovative strategy for the polypharmacotherapy of neuropathic pain in combination with opioids.

Chronic constriction injury-induced changes in circular RNA expression profiling of the dorsal root ganglion in a rat model of neuropathic pain

BackgroundThe pathogenesis of neuropathic pain (NP) has not been fully elucidated. Gene changes in dorsal root ganglia (DRG) may contribute to the development of NP. Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that form covalently closed loop structures and are crucial for genetic and epigenetic regulation. However, little is known about circRNA changes in DRG neurons after peripheral nerve injury.MethodsA sciatic nerve chronic constriction injury (CCI) model was established to induce neuropathic pain. We performed genome-wide circRNA analysis of four paired dorsal root ganglion (DRG) samples (L4–L5) from CCI and negative control (NC) rats using next-generation sequencing technology. The differentially expressed circRNAs (DEcircRNAs) were identified by differential expression analysis, and the expression profile of circRNAs was validated by quantitative PCR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to predict the function of DEcircRNAs.ResultsA total of 374 DEcircRNAs were identified between CCI and NC rats using circRNA high-throughput sequencing. Among them, 290 were upregulated and 84 were downregulated in the CCI group. The expression levels of nine DEcircRNAs were validated by qPCR. Functional annotation analysis showed that the DEcircRNAs were mainly enriched in pathways and functions, including ‘dopaminergic synapse,’ ‘renin secretion,’ ‘mitogen-activated protein kinase signaling pathway,’ and ‘neurogenesis.’ Competing endogenous RNA analysis showed that the top 50 circRNAs exhibited interactions with four pain-related microRNAs (miRNAs). Circ:chr2:33950934–33955969 was the largest node in the circRNA–miRNA interaction network.ConclusionsPeripheral nerve injury-induced neuropathic pain led to changes in the comprehensive expression profile of circRNAs in the DRG of rats. DEcircRNAs may advance our understanding of the molecular mechanisms underlying neuropathic pain.

Response profile in a rat model of exercise-induced hypoalgesia is associated with duloxetine, pregabalin and diclofenac effect on constriction-induced neuropathy.

Exercise is a known trigger of the inhibitory pain modulation system and its analgesic effect is termed exercise-induced hypoalgesia (EIH). Previous studies have demonstrated that rats with deficient analgesic response following exercise develop more significant hypersensitivity following nerve injury compared to rats with substantial analgesic response following exercise. A rat model of EIH as an indicator of the pain inhibitory system's efficiency was used to explore the association between EIH profiles and the effect of pharmacotherapy on rat's neuropathic pain. EIH profiles were assessed by evaluating paw responses to mechanical stimuli before and after exercise on a rotating rod. Rats with a reduction of ≤33% in responses were classified as low EIH and those with ≥67% as high EIH. Low and high EIH rats underwent sciatic nerve chronic constriction injury (CCI). Paw responses to mechanical stimuli were measured at baseline, following CCI, and after treatment with diclofenac, duloxetine or pregabalin. In a different group of low and high EIH rats, EIH was measured before and following treatment with the same medications. Low EIH rats developed more significant hypersensitivity following CCI. Duloxetine and pregabalin successfully reduced hypersensitivity, although significantly more so in low EIH rats. Diclofenac had limited effects, and only on low EIH rats. Four days of duloxetine administration transformed low EIH rats' profiles to high EIH. The findings of this study suggest that EIH profiles in rats can not only predict the development of hypersensitivity following injury but may also support targeted pharmacological treatment. Exercise is a known trigger of the inhibitory pain modulation. Rats with deficient analgesic response following exercise develop more significant hypersensitivity following nerve injury. Pain modulation profiles in rats can also support targeted pharmacological treatment; rats with deficient analgesic response following exercise benefit more from treatment with duloxetine and gabapentin. Treatment with duloxetine can improve pain modulation profile.

Analgesic effects of Terminalia chebula extract are mediated by the suppression of the protein expression of nerve growth factor and nuclear factor-κB in the brain and oxidative markers following neuropathic pain in rats.

Due to the complications related to the use of the current pharmacological approach for the alleviation of neuropathic pain, searching for effective compound with fewer complications is a requirement of the present era. It is well known that the pathophysiological mechanism of neuropathic pain is related to excessive inflammation in the nervous system. Hence, the present study focuses on whether the potential analgesic effects of Terminalia chebula (TC) extract are mediated by the changes in the protein expression of nerve growth factor (NGF) and nuclear factor-kappa B (NF-κB) in the brain in a rat model of sciatic nerve chronic constriction injury (CCI). Neuropathic pain was induced by the left sciatic nerve CCI. Male Wistar rats were assigned to three groups: sham, CCI, and CCI + TC (40mg/kg). Animals received either normal saline (1 mL) or the aqueous-alcoholic extract of TC (40mg/kg) for 30 days via gavage needles once a day. Cold allodynia and anxiety-like behaviors were examined one day before CCI surgery (day - 1), as well as days 2, 7, 14, and 30 following CCI. We also assessed the effects of the TC extract oxidative stress markers on day 30 following CCI. Moreover, a western blot analysis was performed on day 30 following CCI to evaluate the effects of the TC extract on the protein expression of NGF and NF-κB in the brain. Oral gavage of the TC extract significantly decreased cold allodynia on days 2 and 14 following CCI. Additionally, the CCI model of chronic pain significantly increased the protein expression of NGF and NF-κB in the brain on day 30 following CCI. Furthermore, the TC extract significantly decreased the protein expression of NGF and NF-κB in the brain. The TC extract also significantly increased the brain glutathione (GSH) content and decreased the malondialdehyde (MDA) content. It is suggested that the analgesic effects of the TC extract are mediated by the suppression of brain NGF, NF-κB, and by its antioxidant activity in the brain following neuropathic pain in rats.

Transcutaneous auricular vagal nerve stimulation releases extrapineal melatonin and reduces thermal hypersensitivity in Zucker diabetic fatty rats.

Type 2 diabetes (T2D) is the most common comorbidity of COVID-19, and both are related to the lack of circulating melatonin. In addition, chronic pain is a common sequela of both COVID-19 and T2D. Using a neuropathic pain model produced by sciatic nerve chronic constriction injury in Zucker diabetic fatty rats, a verified preclinical genetic T2D neuropathy animal model, this study aimed to show that transcutaneous auricular vagal nerve stimulation (taVNS) could elevate plasma melatonin concentration, upregulate the expression of melatonin receptors (MTRs) in the amygdala, and relieve peripheral neuropathic pain. Furthermore, taVNS would restore melatonin levels and relieve pain even in pinealectomized rats. On the contrary, intraperitoneally injected luzindole, a melatonin receptor antagonist, would attenuate the antinociceptive effects of taVNS. In conclusion, the mechanism of the therapeutic effect of taVNS on chronic pain involves the release of extrapineal melatonin and the positive regulation of the expression of central MTRs. This beneficial efficacy should be considered during COVID-19 rehabilitation in individuals with diabetes.

Co-Combination of Pregabalin and Withaniacoagulans-Extract-Loaded Topical Gel Alleviates Allodynia and Hyperalgesia in the Chronic Sciatic Nerve Constriction Injury for Neuropathic Pain in Animal Model.

The current study reports the fabrication of co-combination gel using Pregabalin and Withania coagulans fruit extract to validate its effectiveness for neuropathic pain in chronic constriction injury (CCI) rat models. Three topical gels were prepared using Carbopol 934 through a pseudo-ternary phase diagram incorporating the Pregabalin (2.5%), Withania coagulans extract (2%), and co-combination of both Pregabalin (2.5%) and Withania coagulans extract (2%). Gels were characterized. FTIR showed a successful polymeric network of the gel without any interaction. The drug distribution at the molecular level was confirmed by XRD. The AFM images topographically indicated the rough surface of gels with a size range from 0.25 to 330 nm. DSC showed the disappearance of sharp peaks of the drug and extract, showing successful incorporation into the polymeric network of gels. The in vitro drug release of co-combination gel was 73% over 48 h. The mechanism of drug release by combination gel was Higuchi+ fickian with values of n (0.282) and R2 (0.947). An in vivo study for pain assessment via four methods: (i) heat hyperalgesia, (ii) cold allodynia, (iii) mechano-hyperalgesia, and (iv) dynamic mechano-allodynia, confirmed that topical treatment with co-combination gel reduced the pain significantly as indicated by the p value: R1 (p < 0.001), R2 (p < 0.001), R3 (p < 0.015), and R4 (p < 0.0344). The significance order was R2 (****) > R1 (***) > R3 (**) > R4 (*) > R5 (ns).

Preventive Intrathecal Injection of Bupivacaine Alleviated Microglia Activation and Neuropathic Pain in a Rat Model of Chronic Constriction Injury

Spinal microglia are crucial to neuronal hyper-excitability and pain hypersensitivity. The local anesthetic bupivacaine is commonly used for both peripheral and spinal anesthesia. The pain-relief effects resulting from the peripheral and systemic administration of bupivacaine have been previously reported. In this study, the preventive effects of intrathecal bupivacaine administration against neuropathic pain were revealed in a rat model of sciatic nerve chronic constriction injury (CCI). Using a CCI rat model, pain hypersensitivity, characterized by mechanical allodynia and thermal hyperalgesia, correlated well with microglia M1 polarization, activation and pro-inflammatory cytokine expression in both spinal cord dorsal horns and sciatic nerves. Bupivacaine attenuated pain behaviors and inflammatory alternations. We further identified that the Interferon Regulatory Factor 5 (IRF5)/P2X Purinoceptor 4 (P2X4R) and High Mobility Group Box 1 (HMGB1)/Toll-Like Receptor 4 (TLR4)/NF-κB inflammatory axes may each play pivotal roles in the acquisition of microglia M1 polarization and pro-inflammatory cytokine expression under CCI insult. The relief of pain paralleled with the suppression of microglia M1 polarization, elevation of microglia M2 polarization, and inhibition of IRF5/P2X4R and HMGB1/TLR4/NF-κB in both the spinal cord dorsal horns and sciatic nerve. Our findings provide molecular and biochemical evidence for the anti-neuropathic effect of preventive bupivacaine.