Spinal Nerve Ligation Rats Research Articles

Effect and underlying mechanisms of spirocyclopiperazinium salt compound DXL-A-24 in rats following spinal nerve ligation

PurposeNeuropathic pain represents a significant public health problem and its effective management remains a challenge. The present study is designed to evaluate the analgesic effect of the spirocyclopiperazinium salt compound DXL-A-24 in spinal nerve ligation (SNL) model, and further to explore the possible molecular mechanisms. MethodsSNL model was established on rats, and mechanical allodynia and thermal hyperalgesia were estimated with the von Frey and hot plate tests; the expression of CaMKIIα, CREB, JAK2, STAT3 and c-fos was determined by western blotting; the protein level of TNF-α was analysed by ELISA; the mRNA expression of TNF-α and c-fos was detected using qRT-PCR analysis and the receptor blocking test was used for target searching. ResultsAdministration of DXL-A-24 (1, 0.5, 0.25 mg/kg, i.g.) obviously relieved SNL-induced mechanical allodynia and thermal hyperalgesia in rats (P < 0.01), with the percentage of pain threshold elevation (PTE%) was 103 %, 68 % and 47 %, respectively, in mechanical allodynia; the percentage of maximal possible effect (MPE%) was 56 %, 34 % and 21 %, respectively, in thermal hyperalgesia on day 7 after SNL. Pretreatment with peripheral α7 nicotinic or M4 muscarinic receptor antagonist, the effect of DXL-A-24 was completely blocked (P > 0.05). DXL-A-24 significantly reduced the upregulated pCaMKIIα, pCREB, pJAK2, pSTAT3 and TNF-α protein (P < 0.01), which could be blocked by α7 nicotinic receptor or M4 muscarinic receptor antagonist. In addition, administration of DXL-A-24 attenuated the mRNA and protein expression of c-fos and TNF-α mRNA in DRG of SNL rat. We did not observe significant acute toxicity and chronic hepatorenal impairment at effective dose and high dose. ConclusionsWe report firstly that administration of DXL-A-24 displays obvious antineuropathic pain effects in SNL rats. The underlying mechanism may involve the reduction of the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and the suppression of TNF-α and c-fos expression, which may be mediated by activating peripheral α7 nicotinic and M4 muscarinic receptors. This study may provide a new perspective for developing new antineuralgic drug.

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

BackgroundThis study probes into the function and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes loaded with miR-150-5p in mechanical allodynia.MethodsBMSCs were infected with miR-150-5p inhibition lentiviruses to obtain exosomes with low miR-150-5p expression. A L5 spinal nerve ligation (SNL) model was established in rats where exosomes, NOTCH2 overexpression/inhibition plasmids, or microglial cells were intrathecally administered. Hind paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of rats were measured. TUNEL staining was used to measure the apoptotic rate in rat spinal dorsal horn (SDH), ELISA to evaluate pro-inflammatory factor levels, and RT-qPCR, western blotting, and immunohistochemistry to detect miR-150-5p and NOTCH2 expression. Immunofluorescence was used for localizing exosomes and NOTCH2 and detecting the expression of OX42, a maker for microglia. Dual luciferase reporter and RNA pull down assays were performed to validate the putative binding between miR-150-5p and NOTCH2.ResultsNOTCH2 expressed at a high level and miR-150-5p was downregulated in SDH of SNL rats. Exosomes injected were localized in rat SDH. BMSC-exosomes or NOTCH2 downregulation increased PWT and PWL of SNL rats and reduced apoptosis and inflammation in SDH. In contrast, NOTCH2 overexpression aggravated mechanical allodynia and SDH injury. Moreover, inhibiting miR-150-5p in BMSC-exosomes offset the therapeutic effects of BMSC-exosomes. Microglia activation induced mechanical allodynia in wild rats, while intrathecal injection of microglial cells incubated with BMSC-exosomes showed alleviated mechanical allodynia in SNL rats. NOTCH2 was targeted by miR-150-5p.ConclusionBMSC-derived exosomal miR-150-5p alleviates mechanical allodynia by targeting NOTCH2 in microglial cells.

DUSP8/TAK1 signaling mediates neuropathic pain through regulating neuroinflammation and neuron death in a spinal nerve ligation (SNL) rat model

Nerve injury-induced neuropathic pain is a type of chronic pain associated with neuroinflammatory response and neuronal death; however the underlying molecular mechanisms are still unclear. Dual-specificity phosphatase 8 (DUSP8) can mediate numerous cellular events, but whether it’s involved in neuropathic pain is unknown. In the study, we found that spinal nerve ligation (SNL) operation on rats significantly decreased DUSP8 expression levels in ipsilateral spinal cord (ISC) tissues. Consistently, lipopolysaccharide (LPS) exposure also reduced DUSP8 in murine microglial cells. Adeno-associated virus (AAV)-mediated DUSP8 over-expression was found to considerably ameliorate SNL-induced neuropathic pain in rats. Additionally, neuronal death in the ISC tissues was also attenuated by AAV-DUSP8 following SNL surgery. Moreover, SNL-triggered neuroinflammation and microglial activation were also mitigated upon DUSP8 over-expression by suppressing nuclear factor κB (NF-κB) signaling, which were validated in LPS-exposed microglial cells. Importantly, our in vitro experiments indicated that inflammatory response in microglial cells contributed to neuron death, and such effect could also be ameliorated by DUSP8 over-expression. Notably, we found that DUSP8 directly interacted with transforming growth factor β activated kinase-1 (TAK1) in microglial cells. Both SNL and LPS led to the activation of TAK1/p38/JNK1/2 signaling, whereas being strongly abolished by DUSP8. Intriguingly, TAK1 blockage significantly diminished LPS-induced inflammation and neuron death, whereas being accelerated by DUSP8 knockdown, further indicating that DUSP8-ameliorated neuropathic pain was largely TAK1-dependent. Together, all our findings revealed that DUSP8/TAK1 signaling may be a potential target for neuropathic pain alleviation.

Synergistic action between a synthetic cannabinoid compound and tramadol in neuropathic pain rats.

In the present study the interaction of cannabinoid, PhAR-DBH-Me [(R, Z)-18-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-18-oxooctadec-9-en-7-ylphenyl-acetate] and tramadol in two neuropathy models, as well as their possible toxic effects, was analyzed. The anti-allodynic effect of PhAR-DBH-Me, tramadol, or their combination, were evaluated in neuropathic rats. Furthermore, the effective dose 35 (as the 35 % of the anti allodynic effect) was calculated from the maximum effect of each drug. Moreover, the isobolographic analysis was performed to determine the type of interaction between the drugs. A plasma acute toxicity study was carried out to assess the hepatic, renal, and heart functions after an individual or combined administration of the drugs, as well as histology using the hematoxylin-eosin or Masson-trichome method. PhAR-DBH-Me, tramadol, and their combination produced an antiallodynic effect on spinal nerve ligation (SNL) and cisplatin-induced neuropathic pain in rats. Moreover, PhAR-DBH-Me and tramadol combination showed a synergistic interaction in neuropathic pain rats induced by SNL but not for cisplatin-induced neuropathy. On the other hand, changes in renal and hepatic functions were not observed. Likewise, analysis of liver, kidney and heart histology showed no alterations compared with controls. Results show that the combination of PhAR-DBH-Me and tramadol attenuates the allodynia in SNL rats; the acute toxicology analysis suggests that this combination could be considered safe in administered doses.

Targeting spinal microglia with fexofenadine-loaded nanoparticles prolongs pain relief in a rat model of neuropathic pain

Targeting microglial activation is emerging as a clinically promising drug target for neuropathic pain treatment. Fexofenadine, a histamine receptor 1 antagonist, is a clinical drug for the management of allergic reactions as well as pain and inflammation. However, the effect of fexofenadine on microglial activation and pain behaviors remains elucidated. Here, we investigated nanomedicinal approach that targets more preferentially microglia and long-term analgesics. Fexofenadine significantly abolished histamine-induced microglial activation. The fexofenadine-encapsulated poly(lactic-co-glycolic acid) nanoparticles (Fexo NPs) injection reduced the pain sensitivity of spinal nerve ligation rats in a dose-dependent manner. This alleviation was sustained for 4 days, whereas the effective period by direct fexofenadine injection was 3 h. Moreover, Fexo NPs inhibited microglial activation, inflammatory signaling, cytokine release, and a macrophage phenotype shift towards the alternative activated state in the spinal cord. These results show that Fexo NPs exhibit drug repositioning promise as a long-term treatment modality for neuropathic pain.

Dexmedetomidine and Ketamine Attenuated Neuropathic Pain Related Behaviors via STING Pathway to Induce ER-Phagy.

Our previous work indicated that ER-phagy level had altered in spinal nerve ligation (SNL) rats. In this study, we investigated whether dexmedetomidine or ketamine exhibits anti-anxiety or anti-nociceptive effects via modulation of the spinal STING/TBK pathway to alter ER-phagy in SNL rats. We evaluated the analgesic and anti-anxiety effects of ketamine and dexmedetomidine in SNL rats. 2’3’-cGAMP (a STING pathway agonist) was administrated to investigate whether enhanced spinal STING pathway activation could inhibit dexmedetomidine or ketamine treatment effects in SNL rats. Analgesic effects were assessed with the mechanical withdrawal threshold (MWT) and anti-anxiety effects were measured via an open field test (OFT). Protein expression levels were evaluated by immunoblotting. Distribution and cellular localization of Grp78 (ER stress marker) were evaluated by confocal immunofluorescence. SNL induced mechanical hypersensitivity and anxiety in rats; dexmedetomidine and ketamine both provided analgesia and anti-anxiety effects in SNL rats. Furthermore, the STING pathway was involved in the modulation of ER stress and ER-phagy in SNL rats and dexmedetomidine and ketamine alleviated ER stress by inhibiting STING pathway to enhance ER-phagy. Thus, both ketamine and dexmedetomidine provided anti-anxiety and anti-nociceptive effects by alleviating ER stress through the inhibition of the STING/TBK pathway to modulate spinal ER-phagy in SNL rats.

The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain.

BackgroundNeuropathic pain (NP) is known to be highly correlated with microglial polarization, of which the regulatory mechanism remains to be elucidated. Here, the aim of this study is to further investigate the relationship between bone morphogenetic protein 4 (BMP4) and microglial polarization in the process of NP.MethodsFirstly, normal adult rats received intrathecal BMP4 administration to assess BMP4ʹs effect on microglial polarization. Secondly, a BMP4 antagonist – Noggin – was applied to a rat NP model achieved by L5 spinal nerve ligation (SNL) to investigate whether antagonizing BMP4 signaling could alleviate allodynia by reversing the imbalance of the M1/M2 polarization ratio. In both experiments, Von-Frey filaments were used to test the changes in the paw withdrawal threshold (PWT), and Western blotting, immuno-fluorescence, PCR and flow cytometry were further performed to investigate microglial activity and the expression patterns of M1 and M2 markers, respectively.ResultsFirstly, BMP4 administration induced a significant PWT decrease and microglial activation in normal rats; Western blotting, PCR and flow cytometry further revealed that M1 markers including CD16, MHCII, and TNF-α showed a marked elevation after BMP4 application; while M2 markers, such as Arg-1, CD204 and IL-4, peaked at an early stage (P1 or P4) and then fell to the Sham level on P7, leading to a persistent imbalance of the M1/M2 ratio throughout the 1st week. Secondly, Noggin treatment significantly relieved allodynia and microglial activation in SNL rats. Moreover, Noggin persistently downregulated the M1 marker levels and simultaneously induced a late-stage elevation of M2 markers expressions, thereby reversing the imbalance of the M1/M2 polarization ratio.ConclusionOur results indicate that BMP4 has the ability to induce microglial polarization. Antagonizing BMP4 signaling can relieve pain behavior via mitigating microglial activation and reversing the imbalance of the M1/M2 polarization ratio in the process of NP.

Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain

Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord.

Resolvin D1 Alleviates Mechanical Allodynia via ALX/FPR2 Receptor Targeted Nod-like Receptor Protein 3/Extracellular Signal-Related Kinase Signaling in a Neuropathic Pain Model

The current study aimed to investigate the role and underlying mechanism of Resolvin D1 (RvD1) alleviating spinal nerve ligation (SNL)-induced neuropathic pain (NP) and its interplay with regulatory cascades of Nod-like Receptor Protein 3 (NLRP3) inflammasome. Sprague-Dawley male rat models of SNL-stimulated NP were established, which were pre-treated with different doses of RvD1, WRW4 (ALX/FPR2 inhibitor) or U0126 (ERK inhibitor) for three successive days following the operation. Pain behavior was assessed by measuring changes in the mechanical sensitivity of the hind paws during an observation period of seven consecutive days. The spinal cord (SC) and dorsal root ganglions (DRGs) tissues were collected on postoperative day 7. Immunohistochemistry (IHC) and Western blot were performed to determine the expression levels of NLRP3 inflammasome complex, ALX/FPR2 receptor and extracellular signal-related kinase (ERK). The pro-inflammatory mediators (IL-1β and IL-18) were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that RvD1 could alleviate mechanical allodynia significantly in the SNL-induced NP rat models. Also, RvD1 inhibited the expression of p-ERK, the NLRP3 inflammasomes complex and its corresponding downstream pro-inflammatory mediators which were significantly enhanced in the SC and DRGs of the rat SNL models. While these changes were partially reversed by pre-administration of WRW4 and further strengthened by co-treated with U0126. Our results suggest that RvD1 dependent on ALX/FPR2 may have an analgesic and anti-inflammatory influence on SNL-induced NP driven by inhibiting NLRP3 inflammasome via ERK signaling pathway. These data also provide strong support for the recent modulation of neuro-inflammatory priming and highlight the potential for specialized pro-resolving mediators (SPMs) as novel therapeutic avenues for NP.

MicroRNA-489-3p attenuates neuropathic allodynia by regulating oncoprotein DEK/TET1-dependent epigenetic modification in the dorsal horn

Originally characterized as an oncoprotein overexpressed in many forms of cancer that participates in numerous cellular pathways, DEK has since been well described regarding the regulation of epigenetic markers and transcription factors in neurons. However, its role in neuropathic allodynia processes remain elusive and intriguingly complex. Here, we show that DEK, which is induced in spinal dorsal horn neurons after spinal nerve ligation (SNL), is regulated by miR-489-3p. Moreover, SNL-induced decrease in miR-489-3p expression increased the expression of DEK, which recruited TET1 to the promoter fragments of the Bdnf, Grm5, and Stat3 genes, thereby enhancing their transcription in the dorsal horn. Remarkably, these effects were also induced by intrathecally administering naïve animals with miR-489-3p inhibitor, which could be inhibited by knockdown of TET1 siRNA or DEK siRNA. Conversely, delivery of intrathecal miR-489-3p-mimic into SNL rats attenuated allodynia behavior and reversed protein expression coupled to the promoter segments in the dorsal horn. Thus, a spinal miR-489-3p/DEK/TET1 transcriptional axis may contribute to neuropathic allodynia. These results may provide a new target for treating neuropathic allodynia.

Effect of surgical damage to spinal nerve on dorsal-root ganglion gene expressions: Comprehensive analysis of differentially expressed genes

BackgroundNeuropathic pain can cause significant physical and economic burden, and there are no effective long-term treatments. We conducted a bioinformatics analysis to identify mechanisms to determine strategies for more effective treatments of neuropathic pain. MethodGSE24982 and GSE63442 microarray datasets were extracted from the Gene Expression Omnibus database to analyze transcriptome differences of neuropathic pain in the dorsal root ganglions (DRGs). We filtered the differentially expressed genes (DEGs) in the two datasets and conducted Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the shared DEGs. The Protein–Protein Interaction network was used to determine the hub genes, which were verified in the GSE30691 dataset. miRDB and miRWalk Databases were used to predict potential miRNA of the selected DEGs. We made the spinal nerve ligation (SNL) rat model and qPCR was used to verify the differential expression of hub genes. ResultsA total of 182 overlapped DEGs were found between GSE24982 and GSE63442 datasets. The GO and KEGG analysis showed that the selected DEGs were enriched in infection, transmembrane transport of ion channels, and synaptic transmission. We identified seven hub genes (Atf3, Aif1, Ctss, Gfap, Scg2, Jun, and Vgf). qPCR verified the expression differences of the hub genes in the DRGs after SNL model. Predicted miRNA targeting each selected hub genes were identified. ConclusionsSeven hub genes related to the pathogenesis of neuropathic pain and potential targeting miRNA were identified, expanding understanding of the mechanism of neuropathic pain and facilitating treatment development.

The efficacy of duloxetine depends on spinal cholinergic plasticity in neuropathic pain model rats.

Antidepressants, such as duloxetine, are widely used to treat chronic pain, including neuropathic pain; however, their efficacy is unsatisfactory. In our previous studies, we showed that in a spinal nerve ligation (SNL) rat model, the descending noradrenergic inhibitory system, which involves in the anti-hypersensitivity mechanism of antidepressants, decrease its activity over time following peripheral nerve injury. In this study, we hypothesized that the analgesic effects of duloxetine may diminish following the attenuation of the descending noradrenergic inhibitory system. The analgesic effects of duloxetine in SNL model rats at the early (SNL2W) and chronic (SNL6W) phases following spinal nerve ligation were compared. Male Sprague-Dawley rats were randomly assigned to the SNL2W or SNL6W groups and used to evaluate the anti-allodynic effects of duloxetine using the von Frey filament test. The anti-allodynic effects of duloxetine at a dose of 10 mg/kg were lower in SNL6W rats than in SNL2W rats. Basal noradrenaline concentrations in rat spinal dorsal horns were higher in the SNL6W group than in the SNL2W group, and there was no difference in the increase in spinal noradrenaline concentrations between the 2 groups following duloxetine administration. In addition, we found that duloxetine-induced acetylcholine (ACh) release and choline acetyltransferase (ChAT) expression in the spinal dorsal horn decreased in SNL6W rats. At a dose of 30 mg/kg, duloxetine showed anti-allodynic effects even in SNL6W rats and induced ACh release in the spinal cord. Furthermore, these anti-allodynic effects were completely inhibited by intrathecal atropine (muscarinic antagonist) administration. Moreover, 5 daily intraperitoneal injections of the TrkB agonist, 7,8-dihydroxyflavone (5 mg/kg), not only restored ChAT expression, but also decreased the anti-allodynic effects of duloxetine. These findings suggest that the attenuation of the anti-allodynic effects of duloxetine at the chronic phase of SNL may be due to impaired spinal acetylcholine-mediated analgesia. In addition, the activation of BDNF-TrkB signaling may be beneficial in reversing this impairment.

Electroacupuncture suppresses spinal nerve ligation-induced neuropathic pain via regulation of synaptic plasticity through upregulation of basic fibroblast growth factor expression.

Improving synaptic plasticity is a good way to alleviate neuropathic pain. Electroacupuncture (EA) is currently used worldwide to treat this disease, but its specific mechanisms of action need further investigation. Evidence has suggested that basic fibroblast growth factor (bFGF) plays an important role in promoting nerve regeneration and can promote the expression of vascular endothelial growth factor (VEGF). In this study, we examined the effects of EA on synaptic plasticity and its underlying mechanism. A spinal nerve ligation (SNL) rat model was established. NSC37204 (a specific inhibitor of bFGF) was used to determine the relationship between bFGF and putative EA-mediated improvements in synaptic plasticity. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed to evaluate hyperalgesia in rats with SNL. Tissue morphology was detected by hematoxylin-eosin (HE) and Nissl staining, while neural plasticity and its molecular mechanisms were examined by Western blotting, quantitative real-time polymerase chain reaction (qPCR), dual-label immunohistochemistry and transmission electron microscopy. We found that EA improved synaptic plasticity, consistent with higher levels of expression of bFGF and VEGF. Contrary to the beneficial effects of EA, NSC37204 promoted synaptic reconstruction. Furthermore, EA-induced improvements in the neurobehavioral state and improved synaptic plasticity were blocked by NSC37204, consistent with lower expression levels of bFGF and VEGF. These findings indicate that EA suppresses SNL-induced neuropathic pain by improving synaptic plasticity via upregulation of bFGF expression.

Effect of Pregabalin and Gabapentin on Nociceptive Behaviors Induced by Spinal Nerve Ligation

Pain is defined as an unpleasant sensory and emotional experience, associated with actual or potential tissue damage. According to its neurobiological mechanism, pain is classified into nociceptive, inflammatory, dysfunctional, and neuropathic. Neuropathic pain (NP) is caused by a lesion or disease of the somatosensory nervous system. Both pregabalin and gabapentin are pharmaceuticals used as validation drugs in experimental models of NP. Pregabalin was shown to produce significant antihyperalgesic and antiallodynic effects. Gabapentin is used as a reference compound for new analgesics and reduces tactile allodynia in rats. The aim of this work is to evaluate pregabalin and gabapentin effects on nociceptive behaviors induced by spinal nerve ligation (SNL). Female Wistar rats of 140 - 160 g were used, divided into five groups: Naive, SHAM, SNL rats treated with saline solution, SNL rats treated with pregabalin 30 mg/kg p.o., SNL rats treated with gabapentin 300 mg/kg p.o. Nociceptive behaviors were determined by the up and down method. In the establishment of SNL-induced allodynic behavior, a reduction in paw withdrawal threshold was observed in the time course, which was present from day 1 and it was maintained for 28 days post-ligation. With the administration of pregabalin and gabapentin, anti-allodynic behavior was observed in the time course and in the areas under the curve (AUC) of the time course of anti-allodynic behavior, significant difference was observed between pregabalin, and gabapentin groups compared to vehicle with a value of p < 0.0001. The results showed pregabalin and gabapentin induce an antinociceptive effect in rats subjected to SNL.

Akt/Aquaporin-4 Signaling Aggravates Neuropathic Pain by Activating Astrocytes after Spinal Nerve Ligation in Rats

Aquaporins (AQPs) play critical physiological roles in water balance in the central nervous system (CNS). Aquaporin-4 (AQP4), the principal aquaporin expressed in the CNS, has been implicated in the processing of sensory and pain transmission. Akt signaling is also involved in pain mediation, such as neuroinflammatory pain and bone cancer pain. Previously, we found that expression of AQP4 and p-Akt was altered in the rat spinal cord after spinal nerve ligation (SNL). Here, we further investigated the effects of the AQP4 and Akt pathways in the spinal dorsal horn (SDH) on the pathogenesis of neuropathic pain (NP). Spinal AQP4 was significantly upregulated after SNL and was primarily expressed in astrocytes in the SDH. Inhibition of AQP4 with TGN-020 attenuated the development and maintenance of NP by inhibiting glial activation and anti-neuroinflammatory mechanisms. Moreover, inhibition of AQP4 suppressed astrocyte activation both in the SDH and in primary cultures. Similar to AQP4, we found that p-Akt was also significantly elevated after SNL. Inhibition of Akt with MK2206 suppressed AQP4 upregulation and astrocyte activation both in vivo and in vitro. Furthermore, Akt blockade with MK2206 alleviated NP in the early and late phases after SNL. These results elucidate the mechanisms involved in the roles of Akt/AQP4 signaling in the development and maintenance of NP. AQP4 is likely to be a novel therapeutic target for NP management.

A Bioinformatics Study of Differentially Expressed Genes in Microarrays of Dorsal Root Ganglia from Rat Models of Neuropathic Pain.

BackgroundNeuropathic pain is a significant complication of nerve injury. This study aimed to conduct bioinformatics analysis of differentially expressed genes (DEGs) in microarrays of dorsal root ganglia (DRG) from rat models of neuropathic pain, based on 4 GEO datasets: GSE15041, GSE38038, GSE2884, and GSE24982.Material/MethodsWe retrieved the 4 microarray datasets, which were generated using DRG samples collected in the early and late stages after spinal nerve ligation in rats. The common DEGs (co-DEGs) were identified and then subjected to Gene Ontology, pathway enrichment, and Protein–protein interaction network analyses. Drugs targeting the identified hub genes were analyzed using the Drug Gene Interaction Database.ResultsWe identified 75 early-stage co-DEGs, which were enriched in chromosome segregation and protein catabolic processes, cytosol and extracellular exosome components, and ATP binding function and metabolic pathways. We identified 29 late-stage co-DEGs, which were enriched in protein tetramerization and drug responses, extracellular and membrane raft components, and protein homodimerization and binding functions and calcium signaling pathways. We also identified several hub genes, including Snap25 (synaptosome-associated protein of 25 kDa), Vamp2 (vesicle associated membrane protein 2), and Sf3b1 (splicing factor 3b subunit 1), the first 2 of which can be targeted by botulinum toxin derivatives. SNAP25 plays a role in synaptogenesis and the exocytotic release of neurotransmitters, and VAMP2 participates in neurotransmitter release at a step between docking and fusion.ConclusionsThe present study reveals new mechanisms of neuropathic pain and provides key genes, including SNAP25 and VAMP2, for future studies.

Spinal γ-Aminobutyric Acid Interneuron Plasticity Is Involved in the Reduced Analgesic Effects of Morphine on Neuropathic Pain

Systemic administration of morphine increases serotonin (5-HT) in the spinal dorsal horn (SDH), which attenuates the analgesic effects of morphine on neuropathic pain through spinal 5-HT3 receptors. We hypothesized that dysfunction of the descending serotonergic system, including the periaqueductal gray (PAG), contributes to attenuate the efficacy of morphine on neuropathic pain through spinal 5-HT3 receptors and GABA neurons. Morphine (100 ng) injected into the PAG produced analgesic effects in normal rats, but not in spinal nerve ligation (SNL) rats. In vivo microdialysis showed that PAG morphine increased the SDH 5-HT concentration in both groups. Intrathecal injection of the 5-HT3 receptor antagonist ondansetron and the GABAA receptor antagonist bicuculline attenuated the analgesic effects of PAG morphine in normal rats, but increased the effects in SNL rats. The increased analgesic effect of PAG morphine induced by bicuculline was reversed by pretreatment with the tropomyosin receptor kinase B (TrkB) antagonist K252a. Activation of spinal 5-HT3 receptors by 2-methyl-5-HT increased the GABA concentration in both groups. Morphine activates GABAergic interneurons in the SDH by activating descending serotonergic neurons. Functional changes in GABAA receptors from inhibitory to facilitatory through the activation of TrkB receptors may contribute to the attenuated efficacy of morphine against neuropathic pain. PerspectiveAlthough morphine provides strong analgesia against acute pain, it has limited efficacy against neuropathic pain. This article demonstrates that functional changes in GABAA receptors in the spinal dorsal horn after nerve injury might strongly contribute to the attenuation of opioid-induced analgesia for neuropathic pain.

PCC-0105002, a novel small molecule inhibitor of PSD95-nNOS protein-protein interactions, attenuates neuropathic pain and corrects motor disorder associated with neuropathic pain model

In view of postsynaptic density 95kDA (PSD95) tethers neuronal NO synthase (nNOS) to N-methyl-d-aspartate receptor (NMDAR), the PSD95-nNOS complex represents a therapeutic target of neuropathic pain. This study therefore sought to explore the ability of PCC-0105002, a novel PSD95-nNOS small molecule inhibitor, to alter pain sensitivity in rodent neuropathic pain models. Firstly, the IC50 of PCC-0105002 for PSD95 and NOS1 binding activity was determined using an Alpha Screen assay kit. Then, we examined the effects of PCC-0105002 in the mouse formalin test and in the rat spinal nerve ligation (SNL) model, and explored the ability of PCC-0105002 to mediate analgesia and to effect motor coordination in a rota-rod test. Moreover, the mechanisms whereby PCC-0105002 mediates analgesia was explored via western blotting, Golgi staining, and co-immunoprecipitation experiments in dorsal horn. The outcomes indicated that PCC-0105002 exhibited dose-dependent attenuation of phase II pain-associated behaviors in the formalin test. The result indicated that PCC-0105002 disrupted the PSD95-nNOS interaction with IC50 of 1.408 μM. In the SNL model, PCC-0105002 suppressed mechanical allodynia, thermal hyperalgesia, and abnormal dorsal horn wide dynamic range neuron discharge. PCC-0105002 mediated an analgesic effect comparable to that of MK-801, while it was better able to enhance motor coordination as compared with MK-801. Moreover, PCC-0105002 altered signaling downstream of NMDAR and thus functionally and structurally attenuating synaptic plasticity through respective regulation of the NR2B/GluR1/CaMKIIα and Rac1/RhoA pathways. These findings suggest that the novel PSD95-nNOS inhibitor PCC-0105002 is an effective agent for alleviating neuropathic pain, and that it produces fewer motor coordination-associated side effects than do NMDAR antagonists.

CCR1 enhances SUMOylation of DGCR8 by up-regulating ERK phosphorylation to promote spinal nerve ligation-induced neuropathic pain.

Neuropathic pain is a somatosensory nervous system dysfunction that remains a threatening health problem globally. Recent studies have highlighted the involvement of C-C motif chemokine receptor 1 (CCR1) in neuropathic pain. Herein, the current study set out to explore the modulatory role of CCR1 in spinal nerve ligation (SNL)-induced neuropathic pain and its underlying molecular mechanism. First, it was found that CCR1 was highly expressed in spinal cord tissues and microglial cells of SNL rats. On the other hand, CCR1 knockdown attenuated nerve pain in SNL rats and repressed microglial cell activation in SNL rats and also in the LPS-induced microglial cell model of nerve injury, as evidenced by elevated microglial cell markers OX-42 and IL-1β, IL-6 and TNF-α. Mechanistically, CCR1 enhanced small ubiquitin-like modifier 1 (SUMO1) modification of DiGeorge syndrome critical region gene 8 (DGCR8) in LPS-treated microglial cells by phosphorylating ERK. Moreover, CCR1 silencing brought about elevations in mechanical withdrawal threshold and thermal withdrawal latency. To conclude, our findings indicated that CCR1 enhanced the modification of DGCR8 by SUMO1 through phosphorylation of ERK, thereby promoting the activation and inflammatory response of spinal cord microglial cells and increasing the sensitivity of SNL rats to pain. Thus, this study offers a promising therapeutic target for the management of neuropathic pain.

Effect of intrathecal administration of exogenous noggin on neuropathic pain.

To explore the effect of intrathecal administration of exogenous noggin (NOG) on the pain behavior in the neuropathic pain (NP) rats through L5 spinal nerve ligation (SNL), and to examine the regulative role of NOG in astrocyte activation, inflammatory cytokines and downstream signals. A total of 40 adult male Sprague Dawley (SD) rats were randomly divided into 3 groups: a control group (n=10), a SNL group (SNL+intrathecal injection of artificial cerebrospinal fluid, n=15), and a SNL+NOG group (SNL+intrathecal injection of recombinant NOG protein, n=15). Von-Frey filaments were used to test the changes of paw withdrawal threshold (PWT) at Day 1 before operation, and Day 1, Day 4, Day 7 and Day 14 after operation in each group. Immunofluorescence was used to observe the activation of astrocyte located in the dorsal horn of spinal cord in the 3 groups. Western blotting was conducted to detect the expression levels of glial fibrillary acidic protein (GFAP), interleukin-6 (IL-6), signal transducer and activator of transcription (STAT3) and phosphorylation STAT3 (p-STAT3). Compared with the control group, the PWT in the SNL group was markedly decreased at each time point, together with the increase in GFAP, IL-6 and the ratio of p-STAT3/STAT3 (all P<0.05). Meanwhile, compared with the SNL group, the PWT in the lumbar swelling of spinal cord in the SNL+NOG group was elevated at Day 4 and lasted to Day 14 (P<0.05), accompanied by the decrease in GFAP, IL-6 and the ratio of p-STAT3/STAT3 (all P<0.05). The intrathecal administration of NOG may alleviate NP in the SNL rats through inhibiting astrocyte activation and down-regulating the STAT3 signal pathway.